1 | ¢ - iy 5 SERVICE | Toxicological | A @ | é 5 Be TRE A : ; rvs | Profile Bo a CHLOROFORM Draft for Public Comment IB (Update) l ; 2. ~e, 3 : | omment Period Ends: February 20, 1996 TE) lap 13g OF HEALTH & HUMAN SERVICES Public Health Service : Agency for Toxic Substances and Disease Registry = PURLIC HEALTH LIBRARY Seis LIBRARY | 1 URVERYITY CF 3 7 a 2 14 DRAFT TOXICOLOGICAL PROFILE FOR CHLOROFORM Prepared by: Research Triangle Institute Under Contract No. 205-93-0606 Prepared for: U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES Public Health Service Agency for Toxic Substances and Disease Registry August 1995 ***DRAFT FOR PUBLIC COMMENT*** < CHLOROFORM a vw EL At Je DISCLAIMER The use of company or product name(s) is for identification only and does not imply endorsement by the Agency for Toxic Substances and Disease Registry. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM ii UPDATE STATEMENT A Toxicological Profile for chloroform was released in April 1993. This edition supersedes any previously released draft or final profile. Toxicological profiles are revised and republished as necessary, but no less than once every three years. For information regarding the update status of previously released profiles, contact ATSDR at: Agency for Toxic Substances and Disease Registry Division of Toxicology/Toxicology Information Branch 1600 Clifton Road NE, E-29 Atlanta, Georgia 30333 ***DRAFT FOR PUBLIC COMMENT*** CX FOREWORD This toxicological profile is prepared in accordance with guidelines developed by ATSDR and EPA. The original guidelines were published in the Federal Register on April 17, 1987. Each profile will be revised and republished as necessary. The ATSDR toxicological profile succinctly characterizes the toxicologic and adverse health effects information for the hazardous substance being described. Each profile identifies and reviews the key literature (that has been peer-reviewed) that describes a hazardous substance’s toxicologic properties. Other pertinent literature is also presented, but described in less detail than the key studies. The profile is not intended to be an exhaustive document; however, more comprehensive sources of specialty information are referenced. Each toxicological profile begins with a public health statement, that describes in nontechnical language, a substance’s relevant toxicological properties. Following the public health statement is information concerning levels of significant human exposure and, where known, significant health effects. The adequacy of information to determine a substance’s health effects is described in a health effects summary. Data needs that are of significance to protect public health will be identified by ATSDR and EPA. The focus of the profiles is on health and toxicologic information; therefore, we have included this information in the beginning of the document. Each profile must include the following: (A) The examination, summary, and interpretation of available toxicologic information and epidemiologic evaluations on a hazardous substance in order to ascertain the levels of significant human exposure for the substance and the associated acute, subacute, and chronic health effects. (B) A determination of whether adequate information on the health effects of each substance is available or in the process of development to determine levels of exposure that present a significant risk to human health of acute, subacute, and chronic health effects. (C) Where appropriate, identification of toxicologic testing needed to identify the types or levels of exposure that may present significant risk of adverse health effects in humans. The principal audiences for the toxicological profiles are health professionals at the federal, state, and local levels, interested private sector organizations and groups, and members of the public. We plan to revise these documents in response to public comments and as additional data become available. Therefore, we encourage comments that will make the toxicological profile series of the greatest use. Comments should be sent to: Agency for Toxic Substances and Disease Registry Division of Toxicology Mail Stop E-29 Atlanta, Georgia 30333 vi Foreword The toxicological profiles are developed in response to the Superfund Amendments and Reauthorization Act (SARA) of 1986 (Public Law 99-499) which amended the Comprehensive Environmental Response, Compensation, and Liability Act of 1980 (CERCLA or Superfund). This public law directed the Agency for Toxic Substances and Disease Registry (ATSDR) to prepare toxicological profiles for hazardous substances most commonly found at facilities on the CERCLA National Priorities List and that pose the most significant potential threat to human health, as determined by ATSDR and the Environmental Protection Agency (EPA). The availability of the revised priority list of 275 hazardous substances was announced in the Federal Register on February 28, 1994 (59 FR 9486). For prior versions of the list of substances, see Federal Register notices dated April 17, 1987 (52 FR 12866); October 20, 1988 (53 FR 41280); October 26, 1989 (54 FR 43619); October 17, 1990 (55 FR 42067); and October 17, 1991 (56 FR 52166); and October 28, 1992 (57 FR 48801). Section 104(i)(3) of CERCLA, as amended, directs the Administrator of ATSDR to prepare a toxicological profile for each substance on the list. This profile reflects our assessment of all relevant toxicologic testing and information that has been peer reviewed. It has been reviewed by scientists from ATSDR, the Centers for Disease Control and Prevention (CDC), and other federal agencies. It has also been reviewed by a panel of nongovernment peer reviewers and is being made available for public review. Final responsibility for the contents and views expressed in this toxicological profile resides with ATSDR. Oh Sh David Satcher, M.D., Ph.D. Administrator Agency for Toxic Substances and Disease Registry CHLOROFORM vii CONTRIBUTORS CHEMICAL MANAGER(S)/AUTHORS(S): Selene Chou, Ph.D. ATSDR, Division of Toxicology, Atlanta, GA Wayne Spoo, D.V.M. Research Triangle Institute, Research Triangle Park, NC THE PROFILE HAS UNDERGONE THE FOLLOWING ATSDR INTERNAL REVIEWS: 1. Green Border Review. Green Border review assures consistency with ATSDR policy. 2. Health Effects Review. The Health Effects Review Committee examines the health effects chapter of each profile for consistency and accuracy in interpreting health effects and classifying end points. 3. Minimal Risk Level Review. The Minimal Risk Level Workgroup considers issues relevant to substance-specific minimal risk levels (MRLs), reviews the health effects database of each profile, and makes recommendations for derivation of MRLs. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM ix PEER REVIEW A peer review panel was assembled for chloroform. The panel consisted of the following members: 1. Dr. Richard Bull, Associate Professor of Toxicology and Pharmacology, College of Pharmacy, Washington State University, Pulman, Washington; 2. Dr. Derek Hodgson, Vice President for Research, University of Wyoming, Laramie, Wyoming; and 3. Dr. Nancy Reiches, Private Consultant, Columbus, Ohio. These experts collectively have knowledge of chloroform’s physical and chemical properties, toxico- kinetics, key health end points, mechanisms of action, human and animal exposure, and quantification of risk to humans. All reviewers were selected in conformity with the conditions for peer review specified in Section 104(i)(13) of the Comprehensive Environmental Response, Compensation, and Liability Act, as amended. Scientists from the Agency for Toxic Substances and Disease Registry (ATSDR) have reviewed the peer reviewers’ comments and determined which comments will be included in the profile. A listing of the peer reviewers’ comments not incorporated in the profile, with a brief explanation of the rationale for their exclusion, exists as part of the administrative record for this compound. A list of databases reviewed and a list of unpublished documents cited are also included in the administrative record. The citation of the peer review panel should not be understood to imply its approval of the profile’s final content. The responsibility for the content of this profile lies with the ATSDR. **DRAFT FOR PUBLIC COMMENT*** CHLOROFORM XI CONTENTS FOREWORD ....0vicetsuvsrnnscasssnssasssssssnsasssasasrnsssasanssnnsse Vv CONTRIBUTORS .....ovvevsrnensarsnsrmnnasssssnrassssasssnstasavanrnnscnn vii PEER REVIEW ...vvitvaeaneanansnsssmesasnnssnsesssnssrarssunnassrs eos ix LISTOF FIGURES . .. cvintsmarsramesasaisnissn-orssnsadasdisusananassesas Xv LISTOF TABLES ....cvivurvrirrnrnnronsasnosrossssasssssacoune rons xvii 1. PUBLIC HEALTH STATEMENT .......... remem 1 1.1 WHATIS CHLOROFORM? . .. ities ames mes seen rn 2 1 12 WHAT HAPPENS TO CHLOROFORM WHEN IT ENTERS THE ENVIRONMENT? .. 2 1.3 HOW MIGHT I BE EXPOSED TO CHLOROFORM? ove eee eee 3 1.4 HOW CAN CHLOROFORM ENTER AND LEAVE MY BODY? ................ 4 1.5 HOW CAN CHLOROFORM AFFECT MY HEALTH? .... eens 4 1.6 1S THERE A MEDICAL TEST TO DETERMINE WHETHER 1 HAVE BEEN EXPOSED TO CHLOROFORM? ieee es me 6 1.7 WHAT RECOMMENDATIONS HAS THE FEDERAL GOVERNMENT MADE TO PROTECT HUMAN HEALTH? ..... iiss mee 6 1.8 WHERE CAN I GET MORE INFORMATION? oon ee 8 2 HEALTH EFFECTS . cite iii nasser meee 9 71 INTRODUCTION iit rit ninr sce vsr maa ssa 9 22 DISCUSSION OF HEALTH EFFECTS BY ROUTE OF EXPOSURE .............. 9 2.2.1 Inhalation EXPOSUIE . . ...cvovvrv river reraunea ranean nnn 11 22.1.1 Death «oo 11 2212 Systemic Effects .......... 0 nai 12 22.1.3 Immunological and Lymphoreticular BHeCtS ... cis uinmesr wn rms 29 2.2.14 Neurological Effects ............crvvnrrrrrnnern 30 2.2.1.5 Reproductive Effects «o.oo 31 22.1.6 Developmental Effects ..... o.oo 32 27217 GenotoxiC Effects ....... uve 32 AGA CANCET «vv vnsinsnmninsrscnsmas sR onmssessasnnsiiassesns 33 222 ORIEXPOSUIE . ...ooivv rr ren semen n esas ns rss 33 D9) PEMA sess vnsne sms Bienes mr ek HEE FE rE swe 23 2222 Systemic Effects ...........cvnvrneunnnrr ae 35 2223 Immunological and Lymphoreticular Effects ......ooveenn.n 72 2.224 Neurological Effects ......... nnn 72 2.225 Reproductive Effects ....... o.oo 73 222.6 Developmental Effects ....... ovine 74 7927 GenotoxiCc Effects ........0 rr 75 D238 CAMEL «ove vvensrssssoonnnssessnssassssvnrnssonnsns 75 2.23 Dermal EXPOSUTE «o.oo vein eens meee 78 D973] Dettll co. onernvnnisesrssssmtvaasnsnasbiansrnsrnnesss 78 2232 Systemic Effects .... o.oo 78 ~**DRAFT FOR PUBLIC COMMENT""" CHLOROFORM xii 3 2.2.33 Immunological and Lymphoreticular Effects . . ........... .. . 80 2.234 Neurological Effects .................. ..... . .. 80 2.2.35 Reproductive Effects ................. ... . ... ... 80 2.2.3.6 Developmental Effects ....................... ... 80 2.237 Genotoxic Effects ................... 81 2238 Cancer ............i 81 23 TOXICOKINETICS .............................. ............. 81 23.1 Absorption... 81 23.1.1 Inhalation Exposure ................. .. ... .. . .. ... 81 23.1.2 Oral Exposure ....................0ouiiii 82 23.1.3 Dermal Exposure ..................... ... .. ... 83 232 Distribution ooo 84 2.3.2.1 Inhalation Exposure ..................... . ... ... 84 2322 Oral Exposure .................................. 85 2323 Dermal Exposure ..................... 85 23.3 Metabolism ........ LL 86 2.34 Elimination and Excretion ................... 89 2.34.1 Inhalation Exposure ................ ... .. ..... .. 89 23.42 Oral Exposure .......................... ....... 90 2343 Dermal Exposure . ..................... .. .. ..... 90 2.3.5 Physiologically Based Pharmacokinetic (PBPK)/Pharmacodynamic (PD) Models 90 2.3.5.1 Summary of PBPK/PD Models .................... .. . 92 2.3.5.2 Chloroform PBPK Model Comparison . ........ ... ..... . 94 2.3.53 Discussion of Chloroform Models . . . . . HE Hit 3 Ade mm ee wing 95 24 MECHANISMS OF ACTION .......................... .......... 105 24.1 Pharmacokinetic Mechanisms. ................. 105 2.42 Mechanisms of Toxicity ....................... .... .... 107 2.4.3 Animal-to-Human Extrapolations ................... . .. _. .. _ 108 2.5 RELEVANCE TO PUBLIC HEALTH ................... ......... 109 2.6 BIOMARKERS OF EXPOSURE AND EFFECT ...................... 130 2.6.1 Biomarkers Used to Identify or Quantify Exposure to Chloroform .......... 131 2.6.2 Biomarkers Used to Characterize Effects Caused by Chloroform ........... 133 2.7 INTERACTIONS WITH OTHER CHEMICALS .................... 133 2.8 POPULATIONS THAT ARE UNUSUALLY SUSCEPTIBLE .. . . ......... 135 2.9. METHODS FOR REDUCING TOXIC EFFECTS ..................... 136 29.1 Reducing Peak Absorption Following Exposure . ............ .. .. . 137 2.9.2 Reducing Body Burden .................... .. . .. 137 2.9.3 Interfering with the Mechanism of Action for Toxic Effects .......... . 137 2.10 ADEQUACY OF THE DATABASE ................... ........... 139 2.10.1 Existing Information on Health Effects of Chloroform . .... ........ .. 140 2.10.2 Identification of Data Needs .......................... ..._ 140 2.10.3 Ongoing Studies .................... 148 CHEMICAL AND PHYSICAL INFORMATION . ................ ......... 151 3.1 CHEMICAL IDENTITY ............................. .......... 151 3.2 PHYSICAL AND CHEMICAL PROPERTIES ............ ........... 151 "**DRAFT FOR PUBLIC COMMENT*** CHLOROFORM xiii 4. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL ....civnuvainmsmesnsnns 155 41 PRODUCTION ..iucveenssniiatguimssvnsnsnsonssossiiertmssnnnasss, 155 42 IMPORT/EXPORT . otitis nese 157 43 USE cv uusinsistisssnmrnarus stat snatastseswn a sss SRsuzennsre. 159 4A DISPOSAL ..vrrsrrsssssncnstssipsssniscssvsnnsasimsegssssnnsnnay 160 5 POTENTIAL FOR HUMAN EXPOSURE . . ...... ieee 161 51 OVERVIEW oii iiittintee sauna snaneaaaa arses 161 52 RELEASES TO THE ENVIRONMENT ............. hein 162 5.2.1 AIL tt ee ee ee 162 5.2.2 WVALET « «ov tts ve sense sassos sna san nae 166 B93 SOI vvuvversssteatasnsattsaana rasa teresa aaa 167 53 ENVIRONMENTAL FATE . . .... ooo 168 5.3.1 Transport and Partitioning . . .......... cern .... 168 5.32 Transformation and Degradation ................ 170 ERY Alu .vssnmcanmcmasnmend sass sassmecosvvsa¥ igs times 170 E370 WHEL oo von ree as an I RE rms snr na me ARAL EE IHRE EY ee 170 5323 Sediment and Soil . .. 172 54 LEVELS MONITORED OR ESTIMATED IN THE ENVIRONMENT ............ 173 5.4.1 FN I 173 54.2 WALCT © «ov oe ee ee eee 174 54.3 Sediment and SOIl . «oe 176 544 Other Environmental Media... .. «oir 177 55 GENERAL POPULATION AND OCCUPATIONAL EXPOSURE ............... 178 56 POPULATIONS WITH POTENTIALLY HIGH EXPOSURES .................. 180 5.7 ADEQUACY OF THE DATABASE .............. cumin s 180 5.7.1 Identification of Data Needs . ... o.oo 181 572 Ongoing SWAIES ..... ovine 183 6. ANALYTICAL METHODS . . ... cities renee 187 6.1 BIOLOGICAL SAMPLES ........cvt ir rntrcnrnsrnenassnsens nave 187 6.2 ENVIRONMENTAL SAMPLES ............ ieee 190 6.3 ADEQUACY OF THE DATABASE ............cothrnuninenrrne een 197 6.3.1 Identification of Data Needs . . ... «orn 197 632 Ongoing SPIES oicnronsnnsnitmstusspursttn cesses abiscsunes 199 7. REGULATIONS AND ADVISORIES... .... ieee ee 201 8 REFERENCES .... i ttvutrnensstessssaansssssnss senses aasnnssccveens 215 O. GUOSSARY ov vvenviSosunssasatimss nr i 4s0besuatpasncmmsnusbdiosins ns 257 APPENDICES A. MINIMAL RISK LEVEL (MRL) WORKSHEETS .............. coven A-1 B. USERS GUIDE ....cc.otssniuiienimn-mernstBesBesnssnsemenns nushdsns B-1 C. ACRONYMS, ABBREVIATIONS, AND SYMBOLS... C-1 **DRAFT FOR PUBLIC COMMENT*** CHLOROFORM Xv 2-5 2-6 5-1 LIST OF FIGURES Levels of Significant Exposure to Chloroform—Inhalation ..................coooe nn 20 Levels of Significant Exposure to Chloroform—Oral cone 53 Metabolic Pathways of Chloroform Biotransformation .................cooee. 87 Conceptual Representation of a Physiologically Based Pharmacokinetic (PBPK) Model for Hypothetical Chemical Substance . ..............ooonennree 93 Parameters used in the Corley PBPK Model . .......... vee 96 Existing Information on Health Effects of Chloroform . ...... «oo. 141 Frequency of NPL Sites With Chloroform Contamination . . . . «ovo ete 163 **DRAFT FOR PUBLIC COMMENT"** CHLOROFORM XVii 2-1 2-2 2-3 2-6 3-1 3-2 4-1 4-2 6-1 6-2 7-1 LIST OF TABLES Levels of Significant Exposure to Chloroform—Inhalation . ............ 13 Levels of Significant Exposure to Chloroform—Oral cove 36 Levels of Significant Exposure to Chloroform—Dermal . .......... 79 Parameters Used in the Corley PBPK Model ..... oii 97 Genotoxicity of Chloroform In Vivo... o.oo 125 Genotoxicity of Chloroform In Vitro ..........oooenne reer 126 Chemical Identity of Chloroform . ..... one ww x in mh # 152 Physical and Chemical Properties of Chloroform . . oo oe eee 153 US. Production of ChIOIOfOIM . ..... covet rnenusnasorrnnarssnsror scans 156 Facilities That Manufacture or Process Chloroform. ..........eveeeee eee 158 Releases to the Environment From Facilities That Manufacture or Process Chloroform .... 164 Analytical Methods for Determining Chloroform in Biological Samples . .............. 188 Analytical Methods for Determining Chloroform in Environmental Samples . ........... 191 Regulations and Guidelines Applicable to Chloroform... . «ove vie 203 **DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 1 1. PUBLIC HEALTH STATEMENT This public health statement tells you about chloroform and the effects of exposure. The Environmental Protection Agency (EPA) has identified 1,416 hazardous waste sites as the most serious in the nation. These sites make up the National Priorities List (NPL) and are targeted for long-term federal cleanup. Chloroform has been found in at least 719 NPL sites, including 7 in Puerto Rico. However, it’s unknown how many NPL sites have been evaluated for this substance. As EPA looks at more sites, the sites with chloroform may increase. This is important because exposure to this substance may harm you and because these sites may be sources of exposure. When a substance is released from a large area, such as an industrial plant, or from a container, such as a drum or bottle, it enters the environment. This release does not always lead to exposure. You can be exposed to a substarice only when you come in contact with it by breathing, eating, touching, or drinking. If you are exposed to chloroform, many factors determine whether you’ll be harmed. These factors include the dose (how much), the duration (how long), and how you come in contact with it. You must also consider the other chemicals you're exposed to and your age, sex, diet, family traits, lifestyle, and state of health. 1.1 WHAT IS CHLOROFORM? Chloroform is also known as trichloromethane, methane chloride, or methyltrichloride. It is a colorless liquid with a pleasant, non-irritating odor and a slightly sweet taste. Most of the chloroform found in the environment comes from industry. It will only burn when it reaches very high temperatures. Chloroform was one of the first inhaled anesthetics to be used during surgery, but it is not used for anesthesia today. Nearly all the chloroform made in the United DRAFT FOR PUBLIC COMMENT"*** CHLOROFORM 2 1. PUBLIC HEALTH STATEMENT States today is used to make other chemicals, but some is sold or traded to other countries. We also import chloroform. Chloroform enters the environment from chemical companies, paper mills, waste water from sewage treatment plants, and drinking water that contains chlorine. Chloroform can enter the air directly from factories that make or use it, and by evaporating from water and soil that contain it. It can enter water and soil when waste water that contains chlorine is released into water or soil. It may enter water and soil from spills and by leaks from storage and waste sites. In addition to its industrial production and use, small amounts of chloroform are formed as an unwanted product during the process of adding chlorine to water. Chlorine is added to most drinking water and many waste waters to destroy bacteria. There are many ways for chloroform to enter the environment, so small amounts of it are likely to be found almost everywhere. You will find more information about what chloroform is, how it is used, and where it comes from in Chapters 3 and 4. 1.2 WHAT HAPPENS TO CHLOROFORM WHEN IT ENTERS THE ENVIRONMENT? Chloroform evaporates very quickly when exposed to air. Chloroform also dissolves easily in water, but does not stick to the soil very well. This means that it can travel down through soil to ground water where it can enter a water supply. Chloroform lasts for a long time in both the air and in the ground water. Most chloroform in the air eventually breaks down, but this process is slow. The breakdown products in air include phosgene, which is more toxic than chloroform, and hydrogen chloride, which is also toxic. Some chloroform may break down in soil. Chloroform does not appear to build up in great amounts in plants and animals, but we may find some small amounts of chloroform in foods. You will find more information about where chloroform comes from, how it behaves, and how long it remains in the environment in Chapter 5. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 3 1. PUBLIC HEALTH STATEMENT 1.3 HOW MIGHT | BE EXPOSED TO CHLOROFORM? You are probably exposed to small amounts of chloroform by drinking water and beverages (such as soft drinks) made using water that contains it. You can also get chloroform in your body by eating food, by breathing air, and by skin contact with water that contains it. You are most likely to be exposed to chloroform by drinking water and breathing indoor or outdoor air containing it. The amount of chloroform normally expected to be present in air ranges from 0.02 to 0.05 parts of chloroform per billion parts of air (ppb) and from 2 to 44 ppb in treated drinking water. However, in some places, chloroform concentrations may be higher than 44 ppb. It is estimated that the concentration of chloroform in surface water is 0.1 ppb, the concentration in untreated ground water is 0.1 ppb, and the amount in soil is 0.1 ppb. Even though these levels seem low, much higher levels have been recorded. As much as 610 ppb was found in air at a municipal landfill and up to 88 ppb was found in treated municipal drinking water. Drinking water derived from well water near a hazardous waste site contained 1,900 ppb, and ground water taken near a hazardous waste site also contained 1,900 ppb. Surface water containing 394 ppb has been found, and more than 0.13 ppb has been found in soil at hazardous waste sites. Chloroform has been found in the air from all areas of the United States and in nearly all of the public drinking water supplies. We do not know how many areas have surface water, ground water, or soil that contains chloroform. The average amount of chloroform that you might be exposed to on a typical day by breathing air in various places ranges from 2 to 5 micrograms per day (pg/day) in rural areas, 6 to 200 pg/day in cities, and 80 to 2,200 pg/day in areas near major sources of the chemical. The estimated amount of chloroform you probably are exposed to in drinking water ranges from 4 to 88 pg/day. We cannot estimate the amounts that you may be exposed to by eating food and by coming into contact with water that has chloroform in it. Swimming in swimming pools allows chloroform to be absorbed through a person’s skin. People who work at or near chemical plants and factories that make or use chloroform can be exposed to higher than normal amounts of chloroform. Higher exposures might occur in workers at drinking water treatment plants, waste water treatment plants, and paper and pulp mills. People who **DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 4 1. PUBLIC HEALTH STATEMENT operate waste-burning equipment may also be exposed to higher than normal levels. The National Institute for Occupational Safety and Health (NIOSH) estimated that 95,778 individuals in the United States have had the potential for occupational exposure to chloroform. You will find more information about how you can be exposed to chloroform in Chapter 5. 1.4 HOW CAN CHLOROFORM ENTER AND LEAVE MY BODY? Chloroform can enter your body if you breathe air, eat food, or drink water that contains chloroform. Chloroform easily enters your body through the skin. Therefore, chloroform may also enter your body if you take a bath or shower in water containing chloroform. In addition, you can breathe in chloroform if the shower water is hot enough for chloroform to evaporate. Studies in people and in animals show that after you breathe air or eat food that has chloroform in it, the chloroform can quickly enter your bloodstream from your lungs or intestines. Inside your body, chloroform is carried by the blood to all parts of your body, such as your liver and kidneys. Chloroform usually collects in body fat. Some of the chloroform that enters your body leaves unchanged in the air that you breathe out, and some chloroform in your body is broken down into other chemicals. These chemicals are known as breakdown products, and some of them can attach to other chemicals inside the cells of your body and may cause harmful effects if they collect in high enough amounts in your body. Some of the breakdown products also leave the body in the air you breathe out. Only a small amount of the breakdown products leaves the body in the urine and stool. You can find more information about the behavior of chloroform in the body in Chapter 2. 1.5 HOW CAN CHLOROFORM AFFECT MY HEALTH? To protect the public from the harmful effects of toxic chemicals and to find ways to treat people who have been harmed, scientists use many tests. ***DRAFT FOR PUBLIC COMMENT *** CHLOROFORM 1. PUBLIC HEALTH STATEMENT One way to see if a chemical will hurt people is to learn how the chemical is absorbed, used, and released by the body; for some chemicals, animal testing may be necessary. Animal testing may also be used to identify health effects such as cancer or birth defects. Without laboratory animals, scientists would lose a basic method to get information needed to make wise decisions to protect public health. Scientists have the responsibility to treat research animals with care and compassion. Laws today protect the welfare of research animals, and scientists must comply with strict animal care guidelines. In humans, chloroform affects the central nervous system (brain), liver, and kidneys after a person breaths air or drinks liquids that contain large amounts of chloroform. Chloroform was used as an anesthetic during surgery for many years before its harmful effects on the liver and kidneys were recognized. Breathing about 900 parts of chloroform in a million parts of air (900 ppm or 900,000 ppb) for a short time causes fatigue, dizziness, and headache. If you breathe air, eat food, or drink water that has small amounts of chloroform, over a long period of time the chloroform may damage your liver and kidneys. Large amounts of chloroform can cause sores when the chloroform touches your skin. We do not know whether chloroform causes harmful reproductive effects or birth defects in people. Miscarriages occurred in rats and mice that breathed smaller amounts of chloroform during pregnancy and in rats that ate chloroform during pregnancy. Abnormal sperm were found in mice that breathed small amounts of chloroform for a few days. Offspring of rats and mice that breathed chloroform during pregnancy had birth defects. Results of studies of people who drank chlorinated water showed a possible link between the chloroform in chlorinated water and the occurrence of cancer of the colon and urinary bladder. Cancer of the liver and kidneys developed in rats and mice that ate food or drank water for a long time that had large amounts of chloroform in it. We do not know whether liver and kidney cancer would develop in people after long-term exposure to chloroform in drinking water. Based on animal studies, the Department of Health and Human Services has determined that chloroform may reasonably be anticipated to be a carcinogen (a substance that causes cancer). The International Agency for Research on Cancer has determined that ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 6 1. PUBLIC HEALTH STATEMENT chloroform is possibly carcinogenic to humans. The EPA has determined that chloroform is a probable human carcinogen. You can find a more complete discussion about how chloroform affects your health in Chapter 2. 1.6 IS THERE A MEDICAL TEST TO DETERMINE WHETHER | HAVE BEEN EXPOSED TO CHLOROFORM? Although we can measure the amount of chloroform in the air that you breathe out, and in the blood, urine, and body tissues, we have no reliable test to determine how much chloroform you have been exposed to or whether you will experience any harmful health effects. The measurement of chloroform in body fluids and tissues may help to determine if you have come into contact with large amounts of chloroform. However, these tests are useful only a short time after you are exposed to chloroform because it leaves the body quickly. Because it is a breakdown product of other chemicals (chlorinated hydrocarbons), chloroform in your body might also indicate that you have come into contact with those other chemicals. Therefore, small amounts of chloroform in the body may indicate exposure to these other chemicals and may not indicate low chloroform levels in the environment. From blood tests to determine the amount of liver enzymes, we can tell whether the liver has been damaged, but we cannot tell whether the liver damage was caused by chloroform. 1.7 WHAT RECOMMENDATIONS HAS THE FEDERAL GOVERNMENT MADE TO PROTECT HUMAN HEALTH? The federal government develops regulations and recommendations to protect public health. Regulations can be enforced by law. Federal agencies that develop regulations for toxic substances include EPA, the Occupational Safety and Health Administration (OSHA), and the Food and Drug Administration (FDA). Recommendations provide valuable guidelines to protect public health but cannot be enforced by law. Federal organizations that develop “**DRAFT FOR PUBLIC COMMENT *** CHLOROFORM 1. PUBLIC HEALTH STATEMENT recommendations for toxic substances include the Agency for Toxic Substances and Disease Registry (ATSDR) and the National Institute for Occupational Safety and Health (NIOSH. Regulations and recommendations can be expressed in not-to-exceed levels in air, water, soil, or food that are usually based on levels that affect animals, then they are adjusted to help protect people. Sometimes these not-to-exceed levels differ among federal organizations because of different exposure times (an 8-hour workday or a 24-hour day), the use of different animal studies, or other factors. Recommendations and regulations are also periodically updated as more information becomes available. For the most current information, check with the federal agency or organization that provides it. Some regulations and recommendations for chloroform include the following: The EPA sets rules for the amount of chloroform allowed in water. The EPA limit for total trihalomethanes, a class of chemicals that includes chloroform, in drinking water is 100 micrograms per liter (ug/L, 1 pg/L = 1 ppb in water). Furthermore, EPA requires that spills of 10 pounds or more of chloroform into the environment be reported to the National Response Center. OSHA sets the levels of chloroform allowed in workplace air in the United States. A permissible occupational exposure limit is 2 ppm in air during an 8-hour workday, 40-hour workweek. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 8 1. PUBLIC HEALTH STATEMENT 1.8 WHERE CAN | GET MORE INFORMATION? If you have any more questions or concerns, please contact your community or state health or environmental quality department or: Agency for Toxic Substances and Disease Registry Division of Toxicology 1600 Clifton Road NE, Mailstop E-29 Atlanta, GA 30333 * Information line and technical assistance Phone: (404) 639-6000 Fax: (404) 639-6315 or 6324 ATSDR can also tell you the location of occupational and environmental health clinics. These clinics specialize in recognizing, evaluating, and treating illnesses resulting from exposure to hazardous substances. * To order toxicological profiles, contact: National Technical Information Service 5285 Port Royal Road Springfield, VA 22161 Phone: (800) 553-6847 or (703) 487-4650 ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 9 2. HEALTH EFFECTS 2.1 INTRODUCTION The primary purpose of this chapter is to provide public health officials, physicians, toxicologists, and other interested individuals and groups with an overall perspective of the toxicology of chloroform. It contains descriptions and evaluations of toxicological studies and epidemiological investigations and provides conclusions, where possible, on the relevance of toxicity and toxicokinetic data to public health. A glossary and list of acronyms, abbreviations, and symbols can be found at the end of this profile. 2.2 DISCUSSION OF HEALTH EFFECTS BY ROUTE OF EXPOSURE To help public health professionals and others address the needs of persons living or working near hazardous waste sites, the information in this section is organized first by route of exposure—inhalation, oral, and dermal; and then by health effect—death, systemic, immunological, neurological, reproductive, developmental, genotoxic, and carcinogenic effects. These data are discussed in terms of three exposure periods—acute (14 days or less), intermediate (15-364 days), and chronic (365 days or more). Levels of significant exposure for each route and duration are presented in tables and illustrated in figures. The points in the figures showing no-observed-adverse-effect levels (NOAELSs) or lowest- observed-adverse-effect levels (LOAELSs) reflect the actual doses (levels of exposure) used in the studies. LOAELS have been classified into "less serious" or "serious" effects. "Serious" effects are those that evoke failure in a biological system and can lead to morbidity or mortality (e.g., acute respiratory distress or death). "Less serious” effects are those that are not expected to cause significant dysfunction or death, or those whose significance to the organism is not entirely clear. ATSDR acknowledges that a considerable amount of judgment may be required in establishing whether an end point should be classified as a NOAEL, "less serious” LOAEL, or "serious" LOAEL, and that in some cases, there will be insufficient data to decide whether the effect is indicative of significant dysfunction. However, the Agency has established guidelines and policies that are used to classify these end points. ATSDR believes that there is sufficient merit in this approach to warrant an attempt ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 10 2. HEALTH EFFECTS at distinguishing between "less serious" and "serious" effects. The distinction between "less serious" effects and "serious" effects is considered to be important because it helps the users of the profiles to identify levels of exposure at which major health effects start to appear. LOAELs or NOAELSs should also help in determining whether or not the effects vary with dose and/or duration, and place into perspective the possible significance of these effects to human health. The significance of the exposure levels shown in the Levels of Significant Exposure (LSE) tables and figures may differ depending on the user’s perspective. Public health officials and others concerned with appropriate actions to take at hazardous waste sites may want information on levels of exposure associated with more subtle effects in humans or animals (LOAEL) or exposure levels below which no adverse effects (NOAELSs) have been observed. Estimates of levels posing minimal risk to humans (Minimal Risk Levels or MRLs) may be of interest to health professionals and citizens alike. Levels of exposure associated with the carcinogenic effects (Cancer Effect Levels, CELs) of chloroform are indicated in Figure 2-2. Because cancer effects could occur at lower exposure levels, Figures 2-1 and 2-2 also show a range for the upper bound of estimated excess risks, ranging from a risk of 1 in 10,000 to 1 in 10,000,000 (10 to 107), as developed by EPA. Estimates of exposure levels posing minimal risk to humans (Minimal Risk Levels or MRLs) have been made for chloroform. An MRL is defined as an estimate of daily human exposure to a substance that is likely to be without an appreciable risk of adverse effects (noncarcinogenic) over a specified duration of exposure. MRLs are derived when reliable and sufficient data exist to identify the target organ(s) of effect or the most sensitive health effect(s) for a specific duration within a given route of exposure. MRLs are based on noncancerous health effects only and do not consider carcinogenic effects. MRLs can be derived for acute, intermediate, and chronic duration exposures for inhalation and oral routes. Appropriate methodology does not exist to develop MRLs for dermal exposure. Although methods have been established to derive these levels (Barnes and Dourson 1988; EPA 1990b), uncertainties are associated with these techniques. Furthermore, ATSDR acknowledges additional uncertainties inherent in the application of the procedures to derive less than lifetime MRLs. As an example, acute inhalation MRLs may not be protective for health effects that are delayed in development or are acquired following repeated acute insults, such as hypersensitivity reactions, ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 11 2. HEALTH EFFECTS asthma, or chronic bronchitis. As these kinds of health effects data become available and methods to assess levels of significant human exposure improve, these MRLs will be revised. A User’s Guide has been provided at the end of this profile (see Appendix A). This guide should aid in the interpretation of the tables and figures for Levels of Significant Exposure and the MRLs. 2.2.1 Inhalation Exposure Most of the data regarding inhalation exposure to chloroform in humans were obtained from clinical reports describing health effects in patients under anesthesia. In some instances, the results may have been confounded by the concurrent administration of other drugs with chloroform or by artificial respiration of patients under chloroform anesthesia. Furthermore, most of the studies did not provide any information regarding actual exposure levels for observed effects. Nonetheless, chloroform- induced effects in humans are supported by those observed in animals under experimental conditions. The human studies cited in the profile provide qualitative information on chloroform toxicity in humans. 2.2.1.1 Death Information on the exposure levels of chloroform leading to death in humans was obtained from clinical reports of patients exposed to chloroform as a method of anesthesia. It should be noted that when examining the ability of chloroform to cause death, these clinical reports may be misleading, in that many of these patients had pre-existing health conditions that may have contributed to the cause of death and that chloroform toxicity may not have been the only factor involved in the death of the patient. Older clinical reports suggested that concentrations of =40,000 ppm, if continued for several minutes, may be an overdose (Featherstone 1947). When a cohort of 1,502 patients, ranging in age from 1 to 80 years, exposed under anesthesia to less than 22,500 ppm chloroform was evaluated, no indication of increased mortality was found (Whitaker and Jones 1965). In most patients, the anesthesia did not last longer than 30 minutes; however, a few received chloroform for more than 2 hours. Several studies reported deaths in women after childbirth when chloroform anesthesia had been used (Royston 1924; Townsend 1939). No levels of actual exposure were provided in either study. Death was caused by acute hepatotoxicity. Prolonged labor with starvation, dehydration, and exhaustion contributed to the chloroform-induced hepatotoxicity. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 12 2. HEALTH EFFECTS Levels of acute exposure resulting in animal deaths are generally lower than those reported for human patients under anesthesia; however, the exposure durations are generally longer in the animal studies. An inhalation LCs, (lethal concentration, 50% kill) of 9,770 ppm for a 4-hour exposure was reported for female rats (Lundberg et al. 1986). However, exposure to 8,000 ppm for 4 hours was lethal to albino rats (Smyth et al. 1962). Male mice appear to be more sensitive than female mice. Following exposure to 1,024 ppm for 1-3 hours, 15 of 18 male mice died within 11 days; however, most of the female mice similarly exposed survived for several months (Deringer et al. 1953). Male mice that died had kidney and liver damage, while females did not. An exposure as low as 692 ppm for 1-3 hours resulted in the death of 3 of 6 male mice within 8 days. When exposed to 4,500 ppm chloroform for 9 hours, 10 of 20 female mice died (Gehring 1968). Increased mortality was observed in male rats exposed to 85 ppm chloroform for 6 months (Torkelson et al. 1976). The deaths were attributed to interstitial pneumonia. Rats of either sex exposed to 50 ppm survived. Exposure to 85 ppm for 6 months did not increase mortality in rabbits and guinea pigs. Similarly, no deaths were reported in dogs exposed to 25 ppm chloroform for the same time period. The LCs, and all reliable LOAEL values for death in each species and duration category are recorded in Table 2-1 and plotted in Figure 2-1. 2.2.1.2 Systemic Effects No studies were located regarding musculoskeletal, dermal, or ocular effects in humans or animals after inhalation exposure to chloroform. The highest NOAEL values and all reliable LOAEL values for each systemic effect in each species and duration category are recorded in Table 2-1 and plotted in Figure 2-1. Respiratory Effects. Changes in respiratory rate were observed in patients exposed to chloroform via anesthesia (exposure less than 22,500 ppm) (Whitaker and Jones 1965). Increased respiratory rates were observed in 44% of 1,502 patients who were exposed to light chloroform anesthesia. Respiratory rates were depressed, however, during deep and prolonged anesthesia when chloroform concentrations did not exceed 2.25%. No other studies were located regarding respiratory effects in humans after inhalation exposure to chloroform. “**DRAFT FOR PUBLIC COMMENT*** +=INIWWOD O118Nd HOH 1d4vHQ... TABLE 2-1. Levels of Significant Exposure to Chloroform - Inhalation a Exposure/ LOAEL Keyto Species/ duration/ NOAEL Less serious Serious figure (strain) frequency System (ppm) (ppm) (ppm) Reference ACUTE EXPOSURE Death 1 Rat 4 hr 9770 F (LC50) Lundberg et al. (Sprague- 1986 Dawley) 2 Rat 4 hr 8000 (5/6 died) Smyth et al. 1962 (Albino) 3 Mouse 1-3 hr 692 M (3/6 died) Deringer et al. (C3H) 1953 4 Mouse 9 hr 4500 F (10/20 died) Gehring 1968 (Swiss- Webster) Systemic 5 Human 113 min Cardio 8000 (arrhythmia) Smith et al. 1973 Gastro 8000 (vomiting) Hemato 8000 (increased prothrombin time) Hepatic 8000 (increased sulfobromophthalein sodium retention) 6 Human 0.5-2 hr Resp 22,500 (changes in respiratory Whitaker and rate) Jones 1965 Cardio 22,500 (cardiac arrhythmia, bradycardia) Gastro 22,500 (vomiting) 7 Rat 10d Bd Wt 30 F (18% decreased weight 100 F (24% decreased weight Baeder and (Wistar) Gd 7-16 gain of dams) gain of dams) Hofmann 1988 7 hr/d S103443 H1TV3H 2 WHO40HOTHO €} + INIWWOD O1718Nd HOS 14vHQ... TABLE 2-1. Levels of Significant Exposure to Chloroform - Inhalation (continued) a Exposure/ LOAEL Key to Species/ duration/ NOAEL Less serious Serious figure (strain) frequency System (ppm) (ppm) (ppm) Reference 8 Rat 8 hr Hepatic 50 M (elevated liver Ikatsu and (Wistar) triglycerides and liver Nakajima 1992 GSH) 9 Rat 7d Resp 3M 10 M (epithelial goblet cell Larson et al. 1994c (Fischer- 344) 6 hr/d hyperplasia and degeneration of Bowman's glands in olfactory mucosa) Hepatic 100 M 300 M (swelling and mild centrilobular vacuolation) Renal 10 M 30 M (increased number of S-phase nuclei for tubule cells in the cortex) Bd Wt 3M 10 M (decreased weight gain) 10 Rat 4 hr Hepatic 76 F 153 F (SDH-enzyme levels Lundberg et al. (Sprague- increased) 1986 Dawley) 11 Rat 7d Resp 3M 10 M (degeneration of Mery et al. 1994 (Fischer- 344) 6 hr/d Bowman's gland; new bone formation; increased number of S-phase nuclei) 12 Rat 8d Bd Wt 2200 F 4100 F (60% decreased maternal Newell and Dilley (Sprague- Gd 7-14 body weight gain) 1978 Dawley) 1 hr/d 13 Mouse 2 hr Hepatic 246 (fatty changes) Culliford and (CBA) Hewitt 1957 Renal 246 M (tubular necrosis in males) S103443 H1TV3H 2 WHO40HOTHO 14 ++ LNIWNOD O118Nd JO4 13vHQa... TABLE 2-1. Levels of Significant Exposure to Chloroform - Inhalation (continued) a Exposure/ LOAEL Key to Species/ duration/ NOAEL Less serious Serious figure (strain) frequency System (ppm) (ppm) (ppm) Reference 14 Mouse 1-3 hr Hepatic 942 M (liver necrosis in males Deringer et al. (C3H) that died) 1953 Renal 692 M (tubular necrosis in males that died) 15 Mouse 9 hr Hepatic 4500 (increased SGPT activity) Gehring 1968 (Swiss- Webster) 16 Mouse 4 hr Hepatic 100 (fatty changes) Kylin et al. 1963 (NS) 17 Mouse 7d Resp 300 F Larson et al. 1994c (B6C3F1) 6 hr/d Hepatic 3b F 10 (mild to moderate 100 F (centrilobular hepatocyte vacuolar changes in necrosis and severe centrilobular diffuse vacuolar hepatocytes) degeneration of midzonal and periportal hepatocytes) Renal 100 F 300 (proximal tubules lined by regenerating epithelium) Bd Wt 30 F 100 (weight loss) 18 Mouse 7d Resp 3 F 10 F (increased number of Mery et al. 1994 (B6C3F1) 6 hr/d S-phase nuclei) Bd Wt 30 F 100 (decreased body weight) 19 Mouse 8d Hepatic 100 (increased SGPT activity) Murray et al. 1979 (CF1) Gd 1-7, Gd 6-15, or Gd Bg Wt 100 (decreased weight gain 8-15 of dams) 7 hr/d S103443 H1TV3H ¢ WHO40HOTHO St «x LNJWWOO O1718Nd HOS 14vHQ... TABLE 2-1. Levels of Significant Exposure to Chloroform - Inhalation (continued) a Exposure/ LOAEL Key to Specles/ duration/ NOAEL Less serious Serious figure (strain) frequency System (ppm) (ppm) (ppm) Reference Neurological 20 Human 3 min 920 (dizziness, vertigo) Lehman and Hasegawa 1910 21 Human 113 min 8000 (narcosis) Smith et al. 1973 22 Human 0.5-2 hr 22,500 (narcosis) Whitaker and Jones 1965 23 Mouse 0.5-2 hr 2500 3100 (slight narcosis) Lehmann and (NS) Flury 1943 24 Cat 5-93 min 7,200 (disturbed equilibrium) Lehmann and (NS) Flury 1943 Reproductive 25 Rat 10d 30 F (empty implantations in Baeder and (Wistar) Gd 7-16 2/20 dams) Hofmann 1988 7 hr/d 26 Rat 10d 100 F 300 F (73% decreased Schwetz et al. (Sprague- Gd 6-15 conception rate) 1974 Dawley) 7 hr/d 300 F (fetal resorptions) 27 Mouse 5d 400 M (24-27% increased Land et al. 1979 (C57B1/C3H) 4 hr/d abnormal sperm) 28 Mouse 8d 100 F (30-48% decreased ability Murray et al. 1979 (CF1) Gd 1-7, Gd to maintain pregnancy) 6-15, or Gd 8-15 7 hr/d S103443 H1TV3H 2 WHO40HOTHO 9 ++ LINIWWOO O118Nd HOS 14VHQA... TABLE 2-1. Levels of Significant Exposure to Chloroform - Inhalation (continued) a Exposure/ LOAEL Key to Species/ duration/ NOAEL Less serious Serious figure (strain) frequency System (ppm) (ppm) (ppm) Reference Developmental 29 Rat 10d 30 (slight growth retardation) Baeder and (Wistar) Gd 7-16 Hofmann 1988 7 hr/d 30 Rat 10d 30 (delayed ossification and 100 (missing ribs; acaudate Schwetz et al. (Sprague- Gd 6-15 wavy ribs) fetuses with imperforate 1974 Dawley) 7 hr/d anus) 31 Mouse 8d 100 (cleft palate, decreased Murray et al. 1979 (CF1) Gd 1-7, Gd ossification) 6-15, or Gd 8-15 . 7 hr/d INTERMEDIATE EXPOSURE Death 32 Rat 6 mo 85 M (increased mortality (6/10) Torkelson et al. (NS) 5 d/wk 1976 7 hr/d Systemic 33 Human 1-6 mo Gastro 14 (vomiting) Phoon et al. 1983 Hepatic 14°¢ (toxic hepatitis) 34 Rat 6 mo Resp 50 M 85 M (interstitial pneumonia) Torkelson et al. (NS) 5 d/wk 1976 7 hr/d Hemato 85 Hepatic 25 M (degenerative changes) Renal 25 M (cloudy swelling) Bd Wt 25 M 50 M (decreased body weight in males) S103443 H1TV3H ¢ WHO40HOTHO LL +: LNJWWOD 2178Nd HOH 14VvHQ... TABLE 2-1. Levels of Significant Exposure to Chloroform - Inhalation (continued) a Exposure/ LOAEL Key to Species/ duration/ NOAEL Less serious Serious figure (strain) frequency System (ppm) (ppm) (ppm) Reference 35 Dog 6 mo Hemato 25 Torkelson et al. (NS) 5 d/wk 1976 7 hr/d Hepatic 25 Renal 25 F (cloudy swelling of tubular epithelium) 36 Rabbit 6 mo Resp 25 F (interstitial pneumonia) Torkelson et al. (NS) 5 d/wk 1976 7 hrid Hemato 85 Hepatic 25 (centrilobular granular degeneration and necrosis) Renal 25 (interstitial nephritis) 37 Gn pig 6 mo Hemato 85 Torkelson et al. (NS) 5 d/wk 1976 7 hr/d Hepatic 25 (centrilobular granular degeneration) Renal 25 (tubular and interstitial nephritis) CHRONIC EXPOSURE Systemic 38 Human 1-4 yr Hepatic 2d (hepatomegaly) Bomski et al. 1967 39 Human 10-24 mo Hepatic 71 F Challen et al. 1958 Gastro 22 F (nausea) 40 Human 3-10 yr Gastro 77 F (nausea) Challen et al. 1958 Hepatic 237 F S103443 H1V3H 2 WHO40HOTHO 8 +» INSWWOD 0118Nd "HO4 14VYdQd... TABLE 2-1. Levels of Significant Exposure to Chloroform - Inhalation (continued) . Exposure/ LOAEL Keyto Species/ duration/ NOAEL Less serious Serious figure (strain) frequency System (mg/m3) (mg/m3) (mg/m3) Reference 41 Human 1-15 yr Hepatic 29.5 (elevated serum Li et al. 1993 prealbumin and transferrin) Renal 13.5 Neurological 42 Human 3-10 yr 77 F (exhaustion, irritability, Challen et al. 1958 depression, lack of concentration) 43 Human 10-24 mo 22 F (exhaustion) Challen et al. 1958 44 Human 1-15 yr 13.5 (dizziness, fatigue, Lietal 1993 somnolence, insomnia, increased dreaming, hypomnesia, anorexia, and palpitations) a The number corresponds to entries in Figure 2-1. b : : Used to derive an acute inhalation minimal risk level (MRL) of 1 ppm; concentration was converted to a human equivalent concentration and divided by an uncertainty factor of 30 (3 for extrapolation from animals to humans and 10 for human variability). c Used to derive an intermediate MRL of 0.05 ppm; concentration is divided by an uncertainty factor of 100 (10 for use of a LOAEL and 10 for human variability). d Used to derive a chronic MRL of 0.02 ppm; concentration is divided by an uncertainty factor of 100 (10 for use of a LOAEL and 10 for human variability). Bd Wt = body weight; Cardio = cardiovascular; d = day(s); F = female; Gastro = gastrointestinal; Gd = gestational day; Gn pig = guinea pig; GSH = glutathione; Hemato = hematological; LC50 = lethal concentration, 50% kill: LOAEL = lowest-observed- adverse-effect level; M = male; min = minute(s); NOAEL = no-observed-adverse-effect level, NS = not specified; (occup) = occupational; Resp = respiratory; SDH = sorbitol dehydrogenase; SGPT = serum glutamic pyruvic transaminase; wk = week; yr = year(s) S103443 H1TV3H ¢ WHO404H0 THO 61 -:INIWWOD 0118Nd HOH 14vHd... FIGURE 2-1. Levels of Significant Exposure to Chloroform — Inhalation Acute (< 14 days) Systemic NY LQ \ \ 4 > ys NFS 8

2&0 © \ \ ; SO RX ( ppm ) & ot & L & S & KL & FF & FF ¥ ¥ Qf J < <° 100,000 — A A A A 6 6 6 23 10,000 |— B ® ne A A A A A ® 5 5 5 5 > 21 23m 4c am 1 O 1,000 |— e oY om 12r 12r A Sn 9r m 0 20 2 17m Og >" ats 8 20g" dD 17m18m & 100 [__ Q D,Tm13m@ PO 0 O © eo © O 16m19m a» ® 19m 26r 28m 30r 31m ¢] 10r 9 7r OO ® 8 y @'8mam “or 0 ‘ dD 10 — 200 ® Q 7r 29r 30r gr 11r18m 2 17r or 17r O ur 5 OO O 0 1p 18m L or 01 Key . Rat BW LD50,LCS0 So 0.01 7 Mouse @ LOAEL for serious effects (animals) ; Mma risk level for effects A ee t th h Rabbit (P LOAEL for less serious effects (animals) ES er han cancer d Dog (OO NOAEL (animals) 0.001 — g Guinea Pig A LOAEL for serious effect (humans) The number next to each point c Cal A LOAEL for less serious effect (humans) corresponds to entries in 0.0001 | A NOAEL (humans) Tablsi2-1. S103443 H1TV3H 2 WHO40HOTHO 02 +: INIWWOO 0118Nd HOH 14vHd... FIGURE 2-1. Levels of Significant Exposure to Chloroform — Inhalation (continued) Intermediate (15 - 364 days) Systemic \ & N : > S&F o> o > 8 © © \ Q (ppm) x S &£ & <2 S © oad & NS QQ x QO 1,000 — 100 |— & oO OO 32r 34r 34r 36h 37g O 34r 34r O POD DODD O 36h A 35d A 34r 35d 36h37g 34r 35d 36h37g 34r 10 33 33 1 1 1 Key v |r Rat BW cso | = | m Mouse @ LOAEL for serious effects (animals) | Minimal risk level for effects h Rabbit LOAEL for less serious effects (animals) J yer than cancer d Dog O NOAEL (animals) c Cat A LOAEL for serious effect (humans) . . . A ) The number next to each point g Guinea Pig LOAEL for less serious effect (humans) corresponds to entries in A NOAEL (humans) Table 2-1. 0.1 \— © CEL, cancer effect level S103443 H1TV3H ¢ WHO40HO THO 12 ««INIWWOD OI18Nd HOH 14VYHQA... Figure 2-1. Levels of Significant Exposure to Chloroform - Inhalation (continued) Chronic ( >365 days) Systemic ?> Ss > & © S . L & & & & (ppm) od JF & 2 & 1000 r 100 | % 2 A A A 40 A 42 10 F A 41 A 39 A 43 A 41 44 A 1 F 38 I I | 01 } 0.01 f+ Key r rat B 1 0.001 } m mouse LCso } 1 Minimal risk level 10 h rabbit @ LOAEL for serious effect (animals) for effects other Esimalen I h d dog Q LOAEL for less serious effect (animals) + "2" “2"%" Upper-bound 0.0001 g guineapig O NOAEL (animals) ho 10% puman Cancer . €e number next to eacl IS evels A LOAEL for less serious effect (NUMans) point corresponds to entries A LOAEL for less serious effect (humans) in Table 2-1. 0.00001 } 10 * Doses represent the lowest dose tested per study that produced a tumorigenic response and do not imply the existence of a threshold for the cancer end point. 0.000001 + 107 S103443 H1IV3H 2 WHO40HOTHO 22 CHLOROFORM 23 2. HEALTH EFFECTS In some animal species, the lung may be a target organ when inhalation exposure to chloroform is of intermediate duration. Interstitial pneumonitis was observed in male rats exposed to 85 ppm and in rabbits exposed to 25 ppm chloroform for 6 months (Torkelson et al. 1976). The NOAEL was 50 ppm for rats and 25 ppm for rabbits. No respiratory changes were reported in guinea pigs and dogs exposed to 85 and 25 ppm chloroform, respectively. Larson et al. (1994c) investigated the ability of chloroform vapors to produce toxicity and regenerative cell proliferation in the liver and kidneys of female B6C3F, mice and male Fischer 344 rats, respectively. Nasal passages were also examined for toxic response. Groups of 5 animals were exposed to 0, 1, 3, 10, 30, 100, or 300 ppm chloroform via inhalation for 6 hours/day for 7 consecutive days. Actual exposure concentrations measured for mice were 0, 1.2, 3.0, 10.0, 29.5, 101, and 288 ppm and for rats were 0, 1.5, 3.1, 10.4, 29.3, 100, and 271 ppm. Necropsies were performed on day 8. Animals were administered bromodeoxyuridine (BrdU) via implanted osmotic pump for the last 3.5 days to quantitate S-phase cell proliferation using a labeling index (LI) method. No histopathological lesions were observed in the nasal passages of female mice at any exposure concentration. In the nasal passages of rats, chloroform concentrations of 10 ppm and above induced histopathological changes that exhibited a clear concentration-related response. These lesions consisted of respiratory epithelial goblet cell hyperplasia and degeneration of Bowman's glands in olfactory mucosa with an associated osseous hyperplasia of the endo and ectoturbinates in the periphery of the ethmoid region. Chloroform may also induce site specific changes in the nasal region of female B6C3F, mice and male Fischer 344 rats (Mery et al. 1994). Animals were exposed to 1, 3, 10, 30, 100, and 300 ppm chloroform for 6 hours/day for 7 days to determine the nasal cavity site-specific lesions and the occurrence of cell induction/proliferation associated with varying concentrations of chloroform. In male rats, the respiratory epithelium of the nasopharyngeal meatus exhibited an increase in the size of goblet cells at 100 and 300 ppm chloroform, in addition to an increase in both neutral and acidic mucopolysaccharides. Affected epithelium was up to twice its normal thickness. New bone formation within the nasal region was prominently seen at 10 ppm and above, and followed a concentration response curve. At 30 and 100 ppm, new osseous spicules were present at the beginning of the first endoturbinate, while at 300 ppm, the width of the new bone was almost doubled compared to controls. The Bowman's glands were markedly reduced in size. Cytochrome P-450-2El staining was most prominent in the cytoplasm of olfactory epithelial sustentacular cells and in the acinar cells of “**DRAFT FOR PUBLIC COMMENT"*** CHLOROFORM 24 2. HEALTH EFFECTS Bowman’s glands in control animals. In general, increasing the chloroform concentration tended to decrease the amount of P-450 staining in exposed animals. Exposure to chloroform resulted in a dramatic increase in the number of S-phase nuclei, with the proliferative response confined to activated periosteal cells, including both osteogenic (round) and preosteogenic (spindle) cells. The proximal and central regions of the first endoturbinate had the highest increase of cell proliferation. Interestingly, the only detectable treatment-related histologic change observed in female mice was a slight indication of new bone growth in the proximal part of the first endoturbinate in one mouse exposed to 300 ppm chloroform. The S-phase response was observed at chloroform concentrations of 10 ppm and higher. If similar nasal cavity changes occur in humans, the sense of smell could potentially be altered. Cardiovascular Effects. Epidemiology studies indicate that chloroform causes cardiac effects in patients under anesthesia. In a cohort of 1,502 patients (exposure less than 22,500 ppm), dose-related bradycardia developed in 8% of the cases, and cardiac arrhythmia developed in 1.3% of the cases (Whitaker and Jones 1965). Hypotension was observed in 27% of the patients and was related to the duration of the anesthesia and to pretreatment with thiopentone. Chloroform anesthesia (exposure 8,000-10,000 ppm) caused arrhythmia (nodal rhythm, first degree atrio-ventricular block, or complete heart block) in 50% of the cases from the cohort of 58 patients and hypotension in 12% (Smith et al. 1973). No studies were located regarding cardiovascular effects in animals after inhalation exposure to chloroform. Gastrointestinal Effects. Nausea and vomiting were frequently observed side effects in humans exposed to chloroform via anesthesia (exposure 8,000-22,500 ppm) (Royston 1924; Smith et al. 1973: Townsend 1939; Whitaker and Jones 1965). Nausea and vomiting were observed in male and female workers exposed solely to 14-400 ppm chloroform for 1-6 months (Phoon et al. 1983). Similarly, gastrointestinal symptoms (nausea, dry mouth, and fullness of the stomach) were reported in female workers occupationally exposed to 22-71 ppm chloroform for 10-24 months and 77-237 ppm chloroform for 3-10 years (Challen et al. 1958). No studies were located regarding gastrointestinal effects in animals after inhalation exposure to chloroform. “**DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 25 2. HEALTH EFFECTS Hematological Effects. The hematological system does not appear to be a significant target after inhalation exposure to chloroform. Except for increased prothrombin time in some individuals after anesthesia exposure to 8,000 ppm, no hematological effects were observed in humans after inhalation exposure to chloroform (Smith et al. 1973). This effect reflects the hepatotoxicity of chloroform because prothrombin is formed in the liver. No hematological effects were observed in rats, rabbits, and guinea pigs exposed to 85 ppm chloroform or in dogs exposed to 25 ppm chloroform for intermediate durations (Torkelson et al. 1976). Hepatic Effects. Chloroform-induced hepatotoxicity is one of the major toxic effects observed in both humans and animals after inhalation exposure. Increased sulfobromophthalein retention was observed in some patients exposed to chloroform via anesthesia (exposure 8,000-10,000 ppm), indicating impaired liver function (Smith et al. 1973). Serum transaminase, cholesterol, total bilirubin, and alkaline phosphatase levels were not affected. Several earlier studies report acute hepatic necrosis in women exposed to chloroform via anesthesia (exact exposure not provided) during childbirth (Lunt 1953; Royston 1924; Townsend 1939). The effects observed in the women included jaundice, liver enlargement and tenderness, delirium, coma, and death. Centrilobular necrosis was found at autopsy in those who died. Workers exposed to 14-400 ppm chloroform for 1-6 months developed toxic hepatitis and other effects including jaundice, nausea, and vomiting, without fever (Phoon et al. 1983). The workers were originally diagnosed with viral hepatitis; however, in light of epidemiology data, the diagnosis was changed to toxic hepatitis. No clinical evidence of liver injury was observed in workers exposed to as much as 71 and 237 ppm chloroform for intermediate and chronic durations, respectively; however, liver function was not well characterized (Challen et al. 1958). In contrast, toxic hepatitis (with hepatomegaly, enhanced serum glutamic pyruvic transaminase [SGPT] and serum glutamic oxaloacetic transaminase [SGOT] activities, and hypergammaglobulinemia) was observed in workers exposed to 2-205 ppm chloroform (Bomski et al. 1967). Co-exposure to trace amounts of other solvents was also detected, however. An intermediate-duration inhalation MRL of 0.05 ppm was derived from the LOAEL of 14 ppm from the data presented by Phoon et al. (1983); a chronic- duration inhalation MRL of 0.02 ppm was derived from the LOAEL of 2 ppm from the data presented by Bomski et al. (1967). **DRAFT FOR PUBLIC COMMENT"*** CHLOROFORM 26 2. HEALTH EFFECTS A study by Aiking et al. (1994) examined the possible hepatotoxicity of chloroform exposure in competitive swimmers who trained in indoor chlorinated swimming pools compared to those who trained in outdoor chlorinated swimming pools. The actual amount of chloroform inhaled was not determined; however, the mean concentration of chloroform was determined to be 24.0 pg/L in the indoor pools and 18.4 ug/L in the outdoor pools. Mean blood chloroform concentration in the indoor pool swimmers was found to be 0.89 ug/L, while the control group and the outdoor pool swimmers had blood chloroform concentrations of less than 0.5 ug/L, suggesting that the indoor swimmers had higher chloroform blood concentrations because the chloroform did not have the opportunity to be removed by environmental air currents (resulting in higher exposure dose) as it did in an outdoor pool environment. No significant differences in liver enzyme function was seen between any of the groups. Chloroform-induced hepatotoxicity in various animal species has been reported in several studies. No changes in SGPT activity were observed in rats exposed to 300 ppm chloroform during gestation days 6-15 (Schwetz et al. 1974). In contrast, serum sorbitol dehydrogenase (SDH) activity was increased in rats exposed to 153 ppm and above for 4 hours (Lundberg et al. 1986), and SGPT levels were increased in mice exposed to 100 ppm, 7 hour/day for 8 days during various stages of pregnancy (Murray et al. 1979) and 4,500 ppm for 9 hours (Gehring 1968). These increased enzyme levels in serum indicate hepatocellular necrosis. Fatty changes were observed microscopically in male and female mice after acute exposure to chloroform concentrations >100 ppm (Culliford and Hewitt 1957; Kylin et al. 1963). Liver necrosis was observed in female rats exposed to 4,885 ppm chloroform for 4 hours (Lundberg et al. 1986) and in male mice that died after acute exposure to 692-1,106 ppm chloroform, but not in those that survived and were terminated after a 12-month recovery period (Deringer et al. 1953), indicating that the liver damage was reversible. Centrilobular granular degeneration was observed in rats, rabbits, and guinea pigs exposed to 25 ppm chloroform for 6 months, but not in dogs exposed to 25 ppm for the same time period (Torkelson et al. 1976); however, these pathological findings were not observed in the 50 ppm exposure group of rabbits and guinea pigs, or in the 85 ppm exposure group of guinea pigs. Although the liver effects in rabbits and guinea pigs were not dose-related, the small number of surviving animals in the higher exposure group may have biased the results of the study and may not fully describe the pathological effects of chloroform at the higher dose. Larson et al. (1994c¢) investigated the ability of chloroform vapors to produce toxicity and regenerative cell proliferation in the liver and kidneys of female B6C3F, mice and male Fischer 344 rats, ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 27 2. HEALTH EFFECTS respectively. Groups of 5 animals were exposed to 0, 1, 3, 10, 30, 100, or 300 ppm chloroform via inhalation for 6 hours/day for 7 consecutive days. Actual exposure concentrations measured for mice were 0, 1.2, 3.0, 10.0, 29.5, 101, and 288 ppm and for rats were 0, 1.5, 3.1, 10.4, 29.3, 100, and 271 ppm. Necropsies were performed on day 8. Animals were administered BrdU via implanted osmotic pump for the last 3.5 days in order to measure S-phase cell proliferation using a LI method. Female mice exposed to 100 or 300 ppm exhibited centrilobular hepatocyte necrosis and severe diffuse vacuolar degeneration of midzonal and periportal hepatocytes, while exposure to 10 or 30 ppm resulted in mild to moderate vacuolar changes in centrilobular hepatocytes. Specifically, decreased eosinophilia of the centrilobular and midzonal hepatocyte cytoplasm relative to periportal hepatocytes was observed at 30 ppm. Livers of mice in the 1 and 3 ppm groups did not differ from controls. Slight, dose- related increases in the hepatocyte LIs were observed in the 10 and 30 ppm dose groups, while the LI was increased more than 30-fold in the 100 and 300 ppm groups. Relative liver weights were increased in a dose-dependent manner at exposures of 3 ppm and above. Livers from mice exposed to 100 or 300 ppm were enlarged and pale. In male rats, swelling and mild centrilobular vacuolation was observed only in the livers of rats exposed to 300 ppm. Necrosis was minimal and confined to individual hepatocytes immediately adjacent to the central vein; livers were dark red and congested. The hepatocyte LI in rats were increased only at 100 and 300 ppm, 3- and 7-fold over controls, respectively. An acute-duration inhalation MRL of 1 ppm was based on the NOAEL of 3 ppm for hepatic effects in mice. Renal Effects. Few studies regarding kidney toxicity effects in humans after inhalation exposure to chloroform were found. One report was obtained from case reports of death among women exposed to chloroform via anesthesia during childbirth (Royston 1924). The fatty degeneration of kidneys observed at autopsy indicated chloroform-induced damage. A study by Aiking et al. (1994) examined the possible renal toxicity of chloroform exposure in competitive swimmers who trained in indoor and outdoor chlorinated swimming pools in the Netherlands. Although no significant differences in liver enzyme function was seen between any of the groups, the study did determine that B-2-microglobulin was elevated in the indoor pool swimmers (after controlling for possible age bias using multiple regression analysis), suggesting some degree of renal damage due to higher inhaled air concentrations of chloroform present in the air of indoor swimming pools. In animals, the kidney is a target organ of inhalation exposure to chloroform. Larson et al. 1994c studied the ability of chloroform vapors to produce toxicity and regenerative cell proliferation in the **DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 28 2. HEALTH EFFECTS liver and kidneys of female B6C3F, mice and male Fischer 344 rats, respectively. Groups of 5 animals were exposed to 0, 1, 3, 10, 30, 100, or 300 ppm chloroform via inhalation for 6 hours/day for 7 consecutive days. Actual exposure concentrations measured for mice were 0, 1.2, 3.0, 10.0, 29.5, 101, and 288 ppm and for rats were 0, 1.5, 3.1, 10.4, 29.3, 100, and 271 ppm. Necropsies were performed on day 8. Animals were administered BrdU via implanted osmotic pump for the last 3.5 days to examine cell proliferation as the percentage of cells in S-phase. The kidneys of mice were affected only at the 300 ppm exposure, with approximately half of the proximal tubules lined by regenerating epithelium and an increased LI of tubule cells of 8-fold over controls. In the kidneys of male rats exposed to 300 ppm, about 25-50% of the proximal tubules were lined by regenerating epithelium. The LI for tubule cells in the cortex was increased at 30 ppm and above. Tubular necrosis was observed in male mice after acute exposure to chloroform concentrations 2246 ppm (Culliford and Hewitt 1957; Deringer et al. 1953). Tubular calcifications were observed in mice that survived the exposure and were terminated after a 12-month recovery period. In a study of intermediate duration, increased kidney weight (both sexes) and cloudy swelling (males) were observed in rats exposed to chloroform concentrations >25 ppm chloroform (Torkelson et al. 1976). Results were not consistent in rabbits and guinea pigs under the same exposure conditions. Cloudy swelling and tubular and interstitial nephritis were observed in groups of rats exposed to 25 ppm chloroform, but not in groups exposed to 50 ppm. The results in rabbits and guinea pigs, however, may be biased due to the low survival rate at the higher exposure level. Body Weight Effects. No studies were located regarding body weight effects in humans after inhalation exposure to chloroform. Larson et al. (1994c) noted that in female B6C3F, mice and male Fischer 344 rats exposed to 0, 1, 3, 10, 30, 100, or 300 ppm chloroform via inhalation for 6 hours/day for 7 consecutive days that body weight gains were significantly decreased relative to controls in mice exposed to 100 and 300 ppm (1% weight loss at 100 and 300 ppm). Body weight gain was significantly decreased in a concentration-dependent manner in rats exposed to 10 ppm of chloroform and above (2% weight loss at 300 ppm; weight gains of 9-12% at 10-100 ppm, as compared to 18% weight gain by controls). A dose-dependent reduction in feed consumption, resulting in decreased body weight gain, was observed in pregnant female rats exposed to 30 ppm chloroform (7 hours/day for 10 days) and above ***DRAFT FOR PUBLIC COMMENT *** CHLOROFORM 29 2. HEALTH EFFECTS during gestation (Baeder and Hofmann 1988). Newell and Dilley (1978) report that average fetal body weights in Sprague-Dawley rats decreased when the chloroform concentration reached 4,100 ppm when exposed for 1 hour/day during gestation days 7-14. Decreased body weights in dams were also noted. Similarly, decreased body weight was observed in pregnant mice exposed to 100 ppm chloroform during gestation (Murray et al. 1979). Decreased body weight was reported in male rats exposed to chloroform at 300 ppm for 6 hours/day for 7 days; however, no discernable decrease in body weight was noted at concentrations from 1 to 100 ppm. No decreases in body weight were noted in female mice exposed to identical concentrations and durations of chloroform (Mery et al. 1994). Decreased body weight also occurred in male rats exposed to 50 ppm for 6 months (Torkelson et al. 1976). Other Systemic Effects. No studies were located regarding other systemic effects in humans after inhalation exposure to chloroform. Gearhart et al. (1993) studied the interactions of chloroform exposure with body temperature in mice. Male mice were exposed to chloroform concentrations up to 5,500 ppm chloroform for 6 hours and their core body temperature monitored. The largest decrease in core body temperature was observed in the 5,500 ppm exposure group, followed by the 2,000, 800, and 100 ppm groups. There was no significant decrease in in vitro cytochrome P-450 activity at any temperature tested. The data collected were used to develop a PBPK model, which is discussed in more detail in Section 2.3.5 of this profile. 2.2.1.3 Immunological and Lymphoreticular Effects No studies were located regarding immunological effects in humans after inhalation exposure to chloroform. Information on the immunotoxicity of chloroform is limited to one study on effects of chloroform on host resistance in CD-1 mice. A single exposure to 10.6 ppm chloroform for 3 hours did not increase the mortality rate after streptococcal challenge and did not alter the ability of alveolar macrophages to destroy bacteria in these mice (Aranyi et al. 1986). After repeated chloroform exposure (3 hours/day for 5 days), the mortality rate significantly increased, but the bactericidal activity of macrophages was not suppressed compared to control animals. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 30 2. HEALTH EFFECTS 2.2.1.4 Neurological Effects The central nervous system is a major target for chloroform toxicity in humans and in animals. Chloroform was once widely used as an anesthetic during surgery in humans, but is not currently used as a surgical inhalant anesthetic in modern-day medical practice. Levels of 8,000-30,000 ppm were used to induce anesthesia (Featherstone 1947: Smith et al. 1973; Whitaker and Jones 1965). Concentrations of =40,000 ppm, if continued for several minutes, could result in death (Featherstone 1947). To induce anesthesia, increasing the concentration of chloroform gradually to 25,000 or 30,000 ppm during the first 2 or 3 minutes with maintenance at much lower levels was recommended. Concentrations <1,500 ppm are insufficient to induce anesthesia; concentration of 1,500-2,000 ppm cause light anesthesia (Goodman and Gilman 1980). Dizziness and vertigo were observed in humans after exposure to 920 ppm chloroform for 3 minutes; headache and slight intoxication were observed at higher concentrations (Lehmann and Hasegawa 1910). Exhaustion was reported in 10 women exposed to >22 ppm chloroform during intermediate- duration occupational exposure (Challen et al. 1958). Chronic exposure to chloroform concentrations 277 ppm caused exhaustion, lack of concentration, depression, or irritability in 9 of 10 occupationally exposed women. A case report of an individual addicted to chloroform inhalation for =12 years reported psychotic episodes, hallucinations and delusions, and convulsions (Heilbrunn et al. 1945). Withdrawal symptoms, consisting of pronounced ataxia and dysarthria, occurred following an abrupt discontinuation of chloroform use. Moderate, unspecified, degenerative changes were observed in the ganglion cells in the putamen and the cerebellum at autopsy. Death resulted from an unrelated disease. A study of 61 workers exposed for 1-15 years (average 7.8 years) attempted to delineate a possible exposure-effect relationship and to determine the toxicity of chloroform after long-term exposures at a low concentrations in factories in China (Li et al. 1993). Concentrations of chloroform ranged from 0.87 to 28.9 ppm. Dizziness, fatigue, somnolence, insomnia, increased dreaming, hypomnesia, anorexia, and palpitations were significantly elevated in these individuals. Depression, anger, and fatigue were also reported to be significantly elevated. Significant changes were found in neurologic testings of Simple Visual Reaction Time, Digital Symbol Substitution, Digit Span, Benten Retention and Aim Pursuiting in some workers. A limitation of this study was that the exposed group, based on information indicating where the exposed groups originated, indicated that these individuals probably ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 31 2. HEALTH EFFECTS inhaled much more than just chloroform (i.e., other solvents, drugs, pesticides, etc.) and all the effects attributed to chloroform may be attributable to other chemicals in addition to chloroform. Evidence of central nervous system toxicity in animals includes disturbed equilibrium in cats exposed to 7,200 ppm chloroform for 5 minutes, deep narcosis in cats exposed to 21,500 ppm for 13 minutes, deep narcosis in mice exposed to 4,000 ppm for 30 minutes, slight narcosis in mice exposed to 3,100 ppm for 1 hour, and no obvious effects in mice exposed to 2,500 ppm for 2 hours (Lehmann and Flury 1943; Sax 1979). Memory retrieval was affected in mice exposed to chloroform via anesthesia (concentration not specified) (Valzelli et al. 1988). The amnesic effect was not long-lasting. The highest NOAEL value and all reliable LOAEL values for neurological effects in each species and duration category are recorded in Table 2-1 and plotted in Figure 2-1. 2.2.1.5 Reproductive Effects No studies were located regarding reproductive effects in humans after inhalation exposure to chloroform. Several studies indicate that inhalation exposure to chloroform may cause reproductive effects in animals. Rats exposed to chloroform during gestation had decreased conception rates after exposure (0 300 ppm, but not after exposure (0 100 ppm (Baeder and Hofmann 1988; Schwetz et al. 1974). Similarly, a decreased ability to maintain pregnancy, characterized by an increased number of fetal resorptions and decreased conception rates, was observed in mice exposed to 100 ppm chloroform (Murray et al. 1979). In addition to the reproductive effects described above, a significant increase in the percentage of abnormal sperm was observed in mice exposed to 400 ppm chloroform for 5 days (Land et al. 1979, 1981). The highest NOAEL value and all reliable LOAEL values for reproductive effects in each species in the acute-duration category are recorded in Table 2-1 and plotted in Figure 2-1. **DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 32 2. HEALTH EFFECTS 2.2.1.6 Developmental Effects No studies were located regarding developmental effects in humans after inhalation exposure to chloroform. Chloroform-induced fetotoxicity and teratogenicity were observed in experimental animals. The offspring of rats exposed during gestation had decreased fetal crown-rump length, delayed ossification (at 30 ppm), acaudate fetuses with imperforate anus and missing ribs (at 100 ppm), and decreased fetal body weight and increased fetal resorptions (at 300 ppm) (Schwetz et al. 1974). Slight growth retardation of live fetuses at 30 ppm was observed in rats exposed during gestation; no major teratogenic effects were observed (Baeder and Hofmann 1988). The offspring of mice exposed to 100 ppm chloroform during gestation had increased incidences of cleft palate, decreased ossification, and decreased fetal crown-rump length (Murray et al. 1979). The observed malformations occurred in the fetuses that were exposed during organogenesis (days 8-15). Increased resorptions were observed in dams exposed during gestation days 1-7. In another study using relatively higher doses of chloroform, female Sprague-Dawley rats were exposed to 0, 950, 2,200, or 4,100 ppm chloroform 8 days during gestations days 7-14, for 1 hour/day during gestation. The number of resorptions was enhanced in the highest exposure group only (45% percent resorptions), with no adverse effects noted in the 2,200 ppm and lower doses. The average fetal weight was decreased at the highest dose. No gross teratologic effects or anomalies in ossification were observed in the offspring of exposed dams (Newell and Dilley 1978). All reliable LOAEL values for developmental effects in each species in the acute-duration category are recorded in Table 2-1 and plotted in Figure 2-1. 2.2.1.7 Genotoxic Effects No studies were located regarding genotoxic effects in humans after inhalation exposure to chloroform. Inhalation exposure to 400 ppm chloroform for 5 days increased the percentage of abnormal sperm in mice (Land et al. 1979, 1981). Other genotoxicity studies are discussed in Section 2.5. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 33 2. HEALTH EFFECTS 2.2.1.8 Cancer No studies were located regarding cancer in humans or animals after inhalation exposure to chloroform. Studies in animals indicate that oral exposure to chloroform causes cancer (see Section 2.2.2.8). Because chloroform is carcinogenic in animals exposed orally and because chloroform has identical toxicological end points following oral or inhalation exposure, EPA (1985a) derived a q,* for inhalation exposure to chloroform based on mouse liver tumor data from a chronic gavage study (NCI 1976). EPA considered the NCI (1976) study to be appropriate to use in the inhalation risk estimate because there are no inhalation cancer bioassays and no pharmacokinetic data to contraindicate the use of gavage data (IRIS 1995). The geometric mean of the estimates for male and female mice in the NCI (1976) study, 8x10” (mg/kg/day), was recommended as the inhalation g,* for chloroform. EPA (1985a) combined the estimates for both data sets because the data for males includes observations at a lower dose, which appear to be consistent with the female data. Expressed in terms of air concentration, the q,* is equal to 23x10” (ug/m’)" or 1.1x10* (ppb). The air concentrations associated with individual, lifetime upper-bound risks of 10* to 107 are 4.3x10” to 43x10 mg/m’ (8.8x10* to 8.8x10”7 ppm), assuming that a 70-kg human breathes 20 m’® air/day. The 10 to 107 levels are indicated in Figure 2-1. 2.2.2 Oral Exposure 2.2.2.1 Death Information regarding mortality in humans after oral exposure to chloroform is limited. A man died of severe hepatic injury 9 days after reportedly drinking =6 ounces of chloroform (3,755 mg/kg) (Piersol et al. 1933). He was admitted to a hospital, in a deep coma, within 15 minutes of ingestion. This man was also noted to be a long-time user of chloroform in his occupation and a heavy drinker, suggesting that damage inflicted by previous use of chloroform and alcohol over a long period of time may have been contributing factors in his death. In contrast, a patient who ingested 4 ounces (=2,500 mg/kg) recovered from toxic hepatitis (Schroeder 1965). The recovery may have been due to **DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 34 2. HEALTH EFFECTS better therapeutic handling of the case. A fatal dose may be as low as 10 mL (14.8 grams) or 212 mg/kg (Schroeder 1965). Oral LD, (lethal dose, 50% kill) values in animals vary somewhat. Acute LD, values of 2,000 mg/kg chloroform (Torkelson et al. 1976) and 2,180 mg/kg chloroform (Smyth et al. 1962) were reported for rats. LDj, values in male rats varied with age: 446 mg/kg for 14-day-olds, 1,337 mg/kg for young adults, and 1,188 mg/kg for old adults (Kimura et al. 1971). LDy, values were different for male rats (908 mg/kg/day) and female rats (1,117 mg/kg/day) (Chu et al. 1982b). Similarly, the LD, for male mice was lower (1,120 mg/kg) than for female mice (1,400 mg/kg) (Bowman et al. 1978). In general, young adult males had lower survival rates. In another study, an acute oral LD, value of 1,100 mg/kg/day was reported for male and female mice (Jones et al. 1958). Decreased survival rates were also observed in male mice exposed to 250 mg/kg/day chloroform for 14 days, but not in mice exposed to 100 mg/kg/day (Gulati et al. 1988). Female mice, however, survived 500 mg/kg/day chloroform treatment. Pregnant animals may be more susceptible to chloroform lethality. Increased mortality was observed in pregnant rats exposed to 516 mg/kg/day. Rabbits exposed to 63, 100, 159, 251, and 398 mg/kg/day chloroform during gestation days 6-18 had increasing rates of mortality as the dose of chloroform increased (Thompson et al. 1974). There was a high rate of mortality in rats exposed to 83 mg/kg/day chloroform in drinking water for 90 days and during a 90-day observation period. Histopathological examination revealed atrophy of the liver and extensive squamous debris in the esophagus and gastric cardia. These changes suggested to the authors that the rats had died of starvation. Mortality was not increased in the 45 mg/kg/day group (Chu et al. 1982a). The vehicle and mode of administration may influence the lethality of chloroform in mice. In 90-day studies in mice, no effect on mortality was observed in groups treated by gavage with doses up to 250 mg/kg/day chloroform in oil (Munson et al. 1982) or with 435 mg/kg/day in drinking water (Jorgenson and Rushbrook 1980). The maximum tolerated dose of chloroform in drinking water was calculated as 257 mg/kg/day for mice (Klaunig et al. 1986). Survival was affected in mice exposed by gavage to 400 mg/kg/day chloroform in oil for 60 days, but not in those exposed to 100 mg/kg (Balster and Borzelleca 1982). Exposure to 150 mg/kg/day chloroform in toothpaste by gavage for 6 weeks caused death in 8 of 10 male mice (Roe et al. 1979). In contrast, no death occurred in mice exposed to 149 mg/kg/day chloroform in oil for 30 days; there was an increased incidence of death in males exposed to 297 mg/kg/day (Eschenbrenner and Miller ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 35 2. HEALTH EFFECTS 19452). No deaths occurred in dogs exposed to 120 mg/kg/day chloroform in toothpaste capsules for 12-18 weeks (Heywood et al. 1979). Decreased survival was observed in rats exposed by gavage to concentrations 290 mg/kg/day chloroform in oil for 78 weeks and in female mice exposed to 477 mg/kg/day, but not in male mice exposed to 277 mg/kg/day TWA (time-weighted average) during the same time period (NCI 1976). In addition, no increase in compound-related mortality was observed in mice exposed by gavage to 60 mg/kg/day chloroform in toothpaste (Roe et al. 1979) in rats or mice exposed to =160 mg/kg/day chloroform in drinking water for chronic durations (Jorgenson et al. 1985; Klaunig et al. 1986). Similarly, mortality was not affected in dogs exposed to 30 mg/kg/day chloroform in toothpaste capsules for 7.5 years (Heywood et al. 1979). The LD, and all reliable LOAEL values for death in each species and duration category are recorded in Table 2-2 and plotted in Figure 2-2. 2.2.2.2 Systemic Effects No studies were located regarding ocular effects in humans or animals after oral exposure to chloroform. The other systemic effects of oral exposure to chloroform are discussed below. The highest NOAEL values and all reliable LOAEL values for each effect in each species and duration category are recorded in Table 2-2 and plotted in Figure 2-2. Respiratory Effects. Information regarding respiratory effects in humans after oral exposure to chloroform is limited. Upper respiratory tract obstruction due to muscular relaxation was observed in a patient who accidentally ingested =2,500 mg/kg chloroform (Schroeder 1965). Congested lungs and scattered patches of pneumonic consolidation were found at autopsy in a man who committed suicide by drinking =6 ounces (3,755 mg/kg) of chloroform (Piersol et al. 1933). The respiratory system does not appear to be a target of chloroform-induced toxicity in animals after oral exposure. No treatment-related histopathological changes were found in the lungs of rats exposed to 160 mg/kg/day or mice exposed to 435 mg/kg/day chloroform in drinking water in a 90-day study (Jorgenson and Rushbrook 1980), in mice exposed by gavage to 41 mg/kg/day chloroform in oil for 105 days (Gulati et al. 1988), or in mice exposed to 257 mg/kg/day in drinking water for 1 year ***DRAFT FOR PUBLIC COMMENT*** ++ LN3WIWOD O118Nd HOS L4vHQA... TABLE 2-2. Levels of Significant Exposure to Chloroform - Oral Key to’ figure Exposure/ Duration/ Species/ prequency (Strain) (Specific Route) System ACUTE EXPOSURE Death Human once Rat once (Sprague- (GO) Dawley) Rat once (Sprague- (G) Dawley) Rat once (Wistar) (G) Rat 10d (Sprague- ~~ Gd 6-15 Dawley) 1-2x/d (GO) Rat once (NS) (G) Mouse once (ICR Swiss) (GO) Mouse 14d (CD-1) 1x/d (GO) Mouse once (Swiss) (GO) Serious (mg/kg/day) 602 (fatal dose) 908 M (LD50) 1117 F (LD50) 1337 M (LD50 for young adults) 1188 M (LD50 for old adults) 446 M (LD5O0 for 14-day olds) 2180 F (LD50) 516 F (4/6 died) 2000 M (LD50) 1120 M (LD50) 1400 F (LD50) 250 M (5/8 died) 1100 (LD50) Reference Schroeder 1965 Chu et al. 1982b Kimura et al. 1971 Smyth et al. 1962 Thompson et al. 1974 Torkelson et al. 1976 Bowman et al. 1978 Gulati et al. 1988 Jones et al. 1958 S103443 H1IV3H 2 WHO40HOTHO 9€ +:INIWWOO O1N8Nd HOS L4VHQ... TABLE 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ Duration/ LOAEL Key to" Specles/ Frequency NOAEL Less Serious Serious figure (Strain) (specific Route) System (mg/kg/day) (mg/kg/day) (mg/kg/day) Reference 10 Rabbit 13d 100 F (3/5 died) Thompson et al. (Dutch Belted) Gd 6-18 1974 2x/d (GO) Systemic 11 Human once Resp 2503 M (respiratory tract Schroeder 1965 (IN) obstruction) Cardio 2500 M (arrhythmia) Gastro 2500 M (vomiting) Musc/skel 2500 M (muscle relaxation) Hepatic 2500 M (jaundice and toxic hepatitis) Renal 2500 M (oliguria) 12 Rat once Hemato 546 (reduced hemoglobin and Chu et al. 1982b (Sprague- (GO) hematocrit by 10-12%) Dawley) Renal 546 F (increased kidney weight) 13 Rat once Hepatic 34 M (elevated SDH, ALT and Larson et al. 1993 (Fischer- 344) (GO) AST; scattered necrotic foci) Renal 34 M (renal proximal tubule necrosis) Bd Wt 477M 14 Rat 4d Hepatic 10M 34 M (slight to mild Larson et al. 1995 (Fischer- 344) 1x/d centrilobular sinusoidal (GO) leukostasis) Renal 10M 34 M (degeneration of renal proximal tubules) Bd Wt 90M 180 M (decreased body weight gain) S103443 H1TV3H 2 WHO404O THO LE ++ LNIWWOD O1N8Nd HO 14vHd... TABLE 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ 5 . Duration/ LOAEL Key to Se Clos Frequency NOAEL Less Serious Serious figure (Strain) (specific Route) System (mg/kg/day) (mg/kg/day) (mg/kg/day) Reference 15 Rat 4d Hepatic 33.2M 68.1 M (mild hepatocyte Larson et al. 1995 (Fischer- 344) 1x/d vacuolation) w) Renal 57.5M Bd Wt 57.5 M (decreased body weight gain) 16 Rat 10d Hemato 100 F (decreased hemoglobin Ruddick et al. 1983 (Sprague- ~~ Gd 6-15 and hematocrit) Dawley) 1x/d (GO) Hepatic 100 F (increased liver weight) Renal 200 F 400 F (increased kidney weight) Bd Wt 100 F (32% decreased body weight gain) 17 Rat 10d Gastro 516 F (gastric erosions) Thompson et al. (Sprague- Gd 6-15 1974 Dawley) 1-2x/d (GO) Hepatic 516 F (acute toxic hepatitis) Renal 516 F (acute toxic nephrosis) Bd Wt 79F 126 F (decreased body weight gain) 18 Rat 10d Dermal 50 F 126 F (alopecia) Thompson et al. (Sprague- Gd 6-15 1974 Dawley) 2x/d (GO) Bd Wt 20 F 50 F (decreased maternal body weight gain) 19 Mouse 14d Dermal 50 100 (rough hair coat) Gulati et al. 1988 (CD-1) 1x/d (GO) Bd Wt 100 M 250 M (12% weight loss) 500 M (32% weight loss) 20 Mouse once Hepatic 35 (midzonal fatty changes) 350 (centrilobular necrosis) Jones et al. 1958 (Swiss- (GO) Webster) S103443 H1V3H 2 WHOJO0HOTHO 8€ +.LNIWNOD 2118Nd HO4 14vHd... TABLE 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ Duration/ LOAEL Keyto' SPecies/ Frequency NOAEL Less Serious Serious figure (Strain) (gpecific Route) System (mg/kg/day) (mg/kg/day) (mg/kg/day) Reference 21 Mouse once Hepatic 34 F 238 F (small randomly Larson et al. 1993 (B6C3F1) (GO) scattered foci of . hepatocyte necrosis) 22 Mouse 4d Hepatic 238 F (centrilobular vacuolar Larson et al. 1994b (B6C3F1) 1x/d degeneration; increased (GO) hepatic cell proliferation) 23 Mouse 4d Hepatic 260 F 53 F (pale pink tinctorial Larson et al. 1994b (B6C3F1) (W) changes in centrilobular hepatocytes) Bd Wt 81 F (approx. 20% decreased body weight) 24 Mouse once Hepatic 59.2 M 199 M (increased SGPT) Moore et al. 1982 (CFLP- (G) Swiss) Renal 59.2 M 199 M (increased thymidine 199 M (tubular necrosis) uptake) 25 Mouse once Hepatic 65.6 M 273 M (increased thymidine Moore et al. 1982 (CFLP Swiss) (GO) uptake, increased SGOT) Renal 17.3 M 65.6 M (tubular necrosis) 26 Mouse 14d Hemato 250 Munson et al. 1982 (CD-1) 1x/d (GO) Hepatic 125 250 (increased SGPT and SGOT levels) Bd Wt 125M 250 M (16% decreased body weight) 27 Mouse 5 d/wk Bd Wt 263 F Pereira 1994 (B6C3F1) 5or12d (GO) S103443 H1TV3aH ¢ WHO40HO THO 6€ «x LN3IWWNOD O118Nd HOH 13VHQA... TABLE 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ LOAEL Duration/ Keyto' Specles/ Frequency NOAEL Less Serious Serious figure (Strain) (gpecific Route) System (mgng/day) (mg/kg/day) (mg/kg/day) Reference 28 Mouse 7 d/wk Bd Wt 67.1 F (decreased body weight) Pereira 1994 (86C3F1) ~~ 5ori12d (W) 29 Rabbit 13d Gastro 20 F (diarrhea) Thompson et al. (Dutch Belted) Gd 6-18 1974 bin Bd Wt 35F 50 F (decreased maternal 30 31 32 33 35 36 Immunological/ Lymphoreticular Rat once (Sprague- (GO) Dawley) Mouse 14d (CD-1) xd (GO) Neurological Human once Mouse 14d (ICR) 1x/d (GO) Mouse once (ICR) (GO) Mouse 14d (CD-1) 1x/d (GO) Mouse once (Swiss) (GO) 765 F 31.1 M 100M body weight gain) 1071 F (reduced lymphocytes) 50 (suppressed humoral immunity) 2503 M (deep coma) 484 M (calculated ED50 for motor performance) 250 M (hunched posture, inactivity) 350 (calculated ED5O0 for narcosis) Chu et al. 1982b Munson et al. 1982 Schroeder 1965 Balster and Borzelleca 1982 Balster and Borzelleca 1982 Gulati et al. 1988 Jones et al. 1958 S103443 H1TV3H 2 WHO40HOTHO oy ++INIWWOD O1718Nd HOH 14vHd... TABLE 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ . Duration/ LOAeE Keyto" Specles/ Frequency NOAEL Less Serious Serious figure (Strain) (go qcific Route) System (mg/kg/day) (mg/kg/day) (mg/kg/day) Reference 37 Mouse 10d 10M 30 M (taste aversion) Landauer et al. (CD-1) 1x/d 1982 (GO) Reproductive 38 Rat 10d 300 F 316 F (increased resorptions) Thompson et al. (Sprague- Gd 6-15 1974 Dawley) 1-2x/d (GO) 39 Rabbit 13d 25 F 63 F (abortion) Thompson et al. (Dutch Belted) Gd 6-18 1974 2x/d 100 F (no.viable concepti) (GO) Developmental 40 Rat 10d 200 400 (19% decreased fetal Ruddick et al. 1983 (Sprague- Gd 6-15 weight) Dawley) 1x/d (GO) 41 Rat 10d 300 316 (decreased fetal weight) Thompson et al. (Sprague- Gd 6-15 1974 Dawley) 1-2x/d (GO) 42 Rat 10d 50 126 (decreased fetal weight) Thompson et al. (Sprague- Gd 6-15 1974 Dawley) 2x/d (GO) 43 Rabbit 13d 100 Thompson et al. (Dutch Belted) Gd 6-18 1974 2x/d (GO) S103443 H1TV3H 2 WHO40HOTHO Ly «INJWWOO O1N8Nd HOH 14vHa... TABLE 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ Duration/ LOAEL a Key to Species Frequency NOAEL Less Serious Serious figure (Strain) gpecific Route) System (mg/kg/day) (mg/kg/day) (mg/kg/day) Reference 44 45 46 47 48 INTERMEDIATE EXPOSURE Death Rat (Sprague- Dawley) Mouse (Schofield) Systemic Rat (Sprague- Dawley) Rat (Sprague- Dawley) Rat (Osborne- Mendel) 90d WwW) 6 wk 6 d/wk (G) 90d Hemato 149.8 WwW) Bd Wt 44.9 28d Hemato 22.8 M W) Hepatic 192.9 M 90d Resp 160M (WwW) Gastro 160M Hemato 160M Hepatic 160 M Renal 160M Bd Wt 81M 142.2 (high mortality during exposure and during recovery period) 150 M (8/10 died) 142.2 (25% decreased body weight gain) 192.9 M (decreased neutrophils) 160 M (11-17% decreased body weight) Chu et al. 1982a Roe et al. 1979 Chu et al. 1982a Chu et al. 1982b Jorgenson and Rushbrook 1980 S103443 H1TV3H 2 WHO40HOTHO cy +LNIAWWOO 0118Nd JO L4vHQa... TABLE 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ Drations LOAEL Key to’ Species/ Frequency NOAEL Less Serious Serious figure (Strain) (gpecific Route) System (mgng/day) (mg/kg/day) (mg/kg/day) Reference 49 Rat 3 wk Hepatic 90M 180 M (degeneration of Larson et al. 1995 (Fischer- 344) 5 d/wk centrilobular 1x/d hepatocytes) (GO) Renal 90M 180 M (progressive degeneration of the proximal tubules) Bd Wt 34M 90 M (decreased body weight gain) 50 Rat 3 wk Hepatic 62.3 M 106 M (mild hepatocyte Larson et al. 1995 (Fischer- 344) 7 d/wk vacuolation) 1x/d Renal 6M 17.4 M (increased numbers of Ww) focal areas of regenerating renal proximal tubular epithelium and cell proliferation) Bd Wt 32M 62.3 M (decreased body weight gain) 51 Rat 13 wk Hemato 150 410 (increased cellular Palmer et al. 1979 (Sprague- 7 d/wk proliferation in bone Dawley) 1x/d marrow) @G) Hepatic 30 150 (increased relative liver 410 (fatty changes, necrosis) weight) Renal 30 150 (increased relative kidney weight) 52 Mouse 90d Hepatic 60 (fatty changes) 270 (cirrhosis) Bull et al. 1986 (B6C3F1) 1x/d (GO) Bd Wt 130M 270 M (15% decreased body weight) S103443 H1TV3H ¢ WHO40HOTHO ey «:LNIWWOD 2118Nd HOH 14vHQd... TABLE 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ Duration/ LOAEL Key to" Species Frequency NOAEL Less Serious Serious figure (Strain) ghecific Route) System (mg/kg/day) (mg/ig/day) (mg/kg/day) Reference 53 Mouse 30d Hepatic 297 594 (cirrhosis) Eschenbrenner and (Strain A) 1x/d Miller 1945a (GO) 54 Mouse 105d Resp 41 Gulati et al. 1988 (CD-1) 1x/d (GO) Hepatic 16 F 41 F (increased liver weight and hepatocellular degeneration) Renal 41 55 Mouse 90d Resp 435 F Jorgenson and Gastro 435 F Hemato 435 F Hepatic 32F 64 F (fatty changes) Renal 435 F 56 Mouse 52 wk Resp 257M Klaunig et al. 1986 (B6C3F1) 7 diwk Ww) Hepatic 86 M (focal necrosis) Renal 86 M (tubular necrosis) Bd Wt 86 M (15% decreased body weight gain) 57 Mouse 3 wk Hepatic 34 F (vacuolation of the Larson et al. 1994b (B6C3F1) 5 d/wk centrilobular and 1x/d midzonal hepatocytes; (GO) increased ALT and SDH) Renal 477 F 58 Mouse 3 wk Hepatic 82 F (increased liver weight) Larson et al. 1994b (B6C3F1) 7 d/wk Ww) Renal 329 F S103443 H1TV3H 2 WHO40HO THO vy ««LNIWWOO 0118Nd HO4 1dVHA... TABLE 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ LOAEL a Duration/ Key to’ Species/ Frequency NOAEL Less Serious Serious figure (Strain) (goecific Route) System (mg/kg/day) (mg/kg/day) (mg/kg/day) Reference 59 Mouse 90 d Hepatic 50 (hydropic degeneration) Munson et al. 1982 (CD-1) 1x/d (GO) Renal 50 (chronic inflammation of lymphocytes) Bd Wt 250 60 Mouse 5 d/wk Bd Wt 263 F Pereira 1994 (B6C3F1) 330r159d (GO) 61 Mouse 7 d/wk Bd Wt 231 F Pereira 1994 (B6C3F1) 33o0r 159d Ww) 62 Dog 6 wk Hepatic 15¢ 30 (significantly increased Heywood et al. (Beagle) 6 d/wk SGPT activity) 1979 1x/d © Immunological/ Lymphoreticular 63 Mouse 90d 50 (depressed humoral Munson et al. 1982 (CD-1) 1x/d immunity) (GO) Neurological 64 Mouse 90d 31.1 M Balster and (ICR) 1x/d Borzelleca 1982 (GO) 65 Mouse 60 d 100 M (operant behavior Balster and (ICR) 1x/d affected) Borzelleca 1982 (GO) WHOJOHOTHO S103443 H1TV3H 2 ig == INIWWOD 0118Nd HOH 14VHQ... TABLE 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ Duration/ LOAEL Key to’ Sp os Frequency NOAEL Less Serious Serious figure (Strain) (Specific Route) ~~ System (mg/kg/day) (mg/kg/day) (mg/kg/day) Reference Reproductive 66 Rat 90d 160 M Jorgenson and (Osborne- (W) Rushbrook 1980 Mendel) 67 Rat 13 wk 150 410 (gonadal atrophy) Palmer et al. 1979 (Sprague- 7 d/wk Dawley) 1x/d (@) 68 Mouse 105d 41 Gulati et al. 1988 (CD-1) 1x/d (GO) Developmental 69 Mouse 6-10 wk 31.1 Burkhalter and (ICR) 1x/d Balster 1979 (GO) 70 Mouse 105d 41 M (increased epididymal Gulati et al. 1988 (CD-1) 1x/d weights, degeneration of (GO) epididymal epithelium in F1) 71 Mouse 105d 41 F (increased liver weight Gulati et al. 1988 (CD-1) 1x/d and hepatocellular (GO) degeneration in F1 females) Cancer 72 Mouse 30d 594 (CEL: hepatomas) Eschenbrenner and (Strain A) 1x/d Miller 1945a (GO) S103443 H1TV3H 2 WHO404HOTHO 9 «:LNIWWOO 0118Nd HOH 14VHQ... TABLE 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ } Duration/ LOAEL Keyto" SPecies/ Frequency NOAEL Less Serious Serious figure (Strain) (goecific Route) System (mg/kg/day) (mg/kg/day) (mg/kg/day) Reference CHRONIC EXPOSURE Death 73 Rat 78 wk 90 M (decreased survival) NCI 1976 (Osborne- 5 d/wk Mendel) 1x/d (GO) 100 F 74 Mouse 78 wk 477 F (decreased survival) NCI 1976 (B6C3F1) 5 d/wk 1x/d (GO) Systemic 75 Human 1-5yr Hepatic 1 De Salva et al. 1975 Renal 1 76 Human 10 yr Hemato 21 M (decreased erythrocytes) Wallace 1950 1x/d Hepatic 21 M (increased (IN) sulfobromophthalein sodium retention) Renal 21 M (albuminuria) 77 Rat 104 wk Renal 160 M Jorgenson et al. (Osborne- 7 d/wk 1985 Mendel) (W) Bd Wt 38M 81 M (decreased body weight) S103443 H1TV3H ¢ WHO404d0OTHO Ly «+LNIWWOD O118Nd HO 14VHQ... TABLE 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ Duration/ LOAEL Keyto' Specles/ Frequency NOAEL Less Serious Serious figure (Strain) (specific Route) System (mg/kg/day) (mg/kg/day) (mg/kg/day) Reference 78 Rat 78 wk Resp 200 F NCI 1976 (Osborne- 5 d/wk Mendel) 1x/d (GO) Cardio 200 F Gastro 200 F Hemato 200 F Musc/skel 200 F Hepatic 100 F 200 F (necrosis of hepatic parenchyma) 180M Renal 200 F Bd Wt 90 M (15% decreased weight gain) 79 Rat 180 wk Hepatic 200 (adenofibrosis) Tumasonis et al. (Wistar) 7d/wk 1985, 1987 (W) Bd Wt 200 M (50% decreased body weight gain) 80 Mouse 104 wk Bd Wt 130 F 263 F (decreased body weight) Jorgenson et al. (B6C3F1) 7 d/wk 1985 (W) S103443 HITV3H 2 WHO40HOTHO 8v «= INIWWNOD O1NaNd "HOH 14vHA... TABLE 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Ex ation’ LOAEL Key to’ Specles/ Frequency NOAEL Less Serious Serious figure (Strain) (gpecific Route) System (mgng/day) (mg/kg/day) (mg/kg/day) Reference 81 Mouse 78 wk Resp 238 F (pulmonary inflammation) NCI 1976 (B6C3F1) 5 d/wk xd Cardio 238 F (cardiac thrombosis) (G0) Gastro 477 F Hemato 477 F Musc/skel 477 F Hepatic 138 M (nodular hyperplasia of the liver) 238 F Renal 477 F Bd Wt 477 F 82 Mouse 80 wk Resp 60 Roe etal. 1979 (Ich) 6 d/wk @G) Hepatic 17 (fatty degeneration) Renal 60 Bd Wt 60 83 Dog 7.5yr Cardio 30 Heywood et al. (Beagle) 6 d/wk 1979 © Hemato 30 Hepatic 15d (increased SGPT activity) Renal 15 30 (fatty changes) Neurological 84 Rat 78 wk 200 F NCI 1976 (Osborne- 5 d/wk Mendel) 1x/d (GO) S103443 H1TV3H ¢ WHO40HOTHO 6 =: INIFWWOO O118Nd HOH 14vHQ... TABLE 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ Duration/ LOAEL Keyto' Species/ Frequency NOAEL Less Serious Serious figure (Strain) (gpecific Route) System (mg/kg/day) (mg/kg/day) (mg/kg/day) Reference 85 Mouse 78 wk 477 F NCI 1976 (B6C3F1) 5 d/wk 1x/d (GO) 86 Mouse 80 wk 60 Roe etal. 1979 (Icy 6 d/wk (G) Reproductive 87 Rat 78 wk 200 F NCI 1976 (Osborne- 5 d/wk Mendel) 1x/d (GO) 180M 88 Mouse 78 wk 477 F NCI 1976 (B6C3F1) 5 d/wk wd 277M (GO) 89 Dog 75yr 30 Heywood et al. (Beagle) 6 d/wk 1979 (©) Cancer 90 Rat 78 wk 90 M (kidney tubular cell Dunnick and (Osbome- 5 d/wk neoplasms in 4/50) Melnick 1993 Mendel) (GO) 200 F (kidney tubular cell neoplasms in 2/48) 91 Rat 104 wk 160 M (CEL:tubular cell adenoma) Jorgenson et al. (Osborne- 7 d/wk 1985 Mendel) (W) S103443 H1TV3H 2 WHO40HOTHO 0S «x LNSWWOO 2118Nd HOH 14vH0... TABLE 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ } Duration/ LOAEL Key to” Species! Frequency NOAEL Less Serious Serious figure (Strain) (Specific Route) ~~ System (mg/kg/day) (mg/kg/day) (mg/kg/day) Reference 92 Rat 78 wk 90 M (CEL:tubular cell adenoma NCI 1976 (Osborne- 5 d/wk and carcinoma) Mendel) 1x/d (GO) 93 Rat 180 wk 200 F (CEL:hepatic neoplastic Tumasonis et al. (Wistar) 7 d/wk nodules) 1985, 1987 (Ww) 94 Mouse 78 wk 138 M (hepatocellular adenomas ~~ Dunnick and (B6C3F1) 5 d/wk or carcinomas in 18/50 Melnick 1993 (GO) mice) 238 F (hepatocellular adenomas or carcinomas in 36/45 mice) 95 Mouse 78 wk 138 M (CEL:hepatocellular NCI 1976 (B6C3F1) 5 d/wk carcinoma) 1x/d 238 F (GO) S103443 H1TV3H 2 WHO40HO THO 1S =+INIWWOD 0118Nd "Od 14vHd... TABLE 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Exposure/ Duration/ . LoAg! Keyto' Species/ Frequency NOAEL Less Serious Serious figure (Strain) (Specific Route) ~~ System (mg/kg/day) (mg/kg/day) (mg/kg/day) Reference 96 Mouse 80 wk 60 M (CEL:epithelial tumors of ~~ Roe etal. 1979 (Icy 6 d/wk the Kidney) (G) ®The number corresponds to entries in Figure 2-2. PUsed to derive an acute oral minimal risk level (MRL) of 0.3 mg/kg/day; dose divided by an uncertainty factor of 100 (10 for extrapolation from animals to humans and 10 for human variability). Used to derive an intermediate oral MRL of 0.1 mg/kg/day; dose adjusted for intermittent exposure and divided by an uncertainty factor of 100 (10 for extrapolation from animals to humans and 10 for human variability). 9dUsed to derive an chronic oral MRL of 0.01 mg/kg/day; dose adjusted for intermittent exposure and divided by an uncertainty factor of 1,000 (10 for use of a LOAEL, 10 for extrapolation from animals to humans, and 10 for human variability). Bd Wt = body weight; (C) = capsule; Cardio = cardiovascular; CEL = cancer effect level; d = day(s); ED, = effective dose for a given effect in 50% of animals; F = female; F, = first filial generation; (G) = gavage; Gastro = gastrointestinal; Gd = gestational day; (GO) = gavage in oil; Hemato = hematological; (IN) = ingestion; LD,, = lethal dose, 50% kill; LOAEL = lowest-observed-adverse-effect level; M = male; min = minute(s); Musc/skel = musculoskeletal; NOAEL= no-observed-adverse-effect level, NS = not specified; Resp = respiratory; SDH = sorbitol dehydrogenase; SGPT = serum glutamic pyruvic transaminase; (W) = drinking water; x = time(s); wk = week; yr = year(s) S103443 H1TV3H ¢ WHO40HOTHO 2s «»LNIWWOD 0118Nd HOH 14VH0... FIGURE 2-2. Levels of Significant Exposure to Chloroform - Oral Acute (< 14 days) Systemic N < > 2 2 > NY 3 -O Q SOF S oF © 0 & o o /kg/d F&F SNF © (mg/kg/day) & LF & 3 > ® YC XP 2 WO EE & 10,000 — —————— — — —_— A A A A A A 1,000 — _HEg gy 111 11 11 A B 4 617m om 1 2 ® dm ® 22 ® > Hs @ 7 12r 17r @ 20 120 yg 17 24m 100 |— 3r 8m @ > 0 39 20m @ 25m pgr26m 141 157 16¢ 10h 14r 16r 151 161 23m 24m OS 24m PO 24m 25m O O S 14 m 10 — ® ‘299 O O h@ 2m 29h 13r20m 21m 2m 13r 25m | 1 SX Key 0.1 [we | LD50 r Bat @ LOAEL for serious effects (animals) I Minimal risk level for effects 0.01 |— m Mouse (| OAEL for less serious effects (animals) | other than cancer i — h Rabbit ~ NOAEL (animals) d Dog A LOAEL for serious effect (humans) 0.001 — A LOAEL for less serious effect (humans) Thenumber nest Dench point corresponds to entries in /\ NOAEL (humans) Table 2-2. 0.0001 L— & CEL, cancer effect level S103443 H1TV3H 2 WHO4040THO £5 «+LNIWWOD O118Nd HOH 14vdQ... FIGURE 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Acute (<14 days) Systemic N DoS N OTN > 8 Se & Si & > oe 3 QO & > QQ St © ¥ 3 (mg/kg/day) & RN & & RN © © Q S NS Ww Q¥ od 10,000 1,000 d 4 ® a» O 3or 14r 27m 30m 30r 18r my PPO am 9 Oe 2060 100 do ry > Yom O 6m Sem 387 38r Q 401 gr 41r dD 0 O17 4 26m 42 19m 16r m QO 35m 43h 7c @ 14r23m > OO 2 0 0 18r 19m O8r 151 O 29h 31m 0 a» gon a2r 10 % 29h 33m O 37m 39h 37m 1 0.1 Key ne ms | . ; Minimal risk level for effect 0.01 m Mouse @ LOAEL for serious effects (animals) ; I pi refiects h Rabbit ( LOAEL for less serious effects (animals) Sot d Dog OO NOAEL (animals) 0.001 A LOAEL for serious effect (humans) The number next to each point A LOAEL for less serious effect (humans) corresponds to entries in A NOAEL (humans) Fabis2-2 0.0001 €@ CEL cancer effect level S103443 H1TV3H 2 WHO40HOTHO vs -INIWWOD 01N8Nd HOS 14vdQ... (mg/kg/day) 1,000 100 10 0.1 pce FIGURE 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) LOAEL for serious effect (humans) LOAEL for less serious effect (humans) NOAEL (humans) JONN J Intermediate (15 - 364 Days) Systemic N\ > ON > Ns Q & ~O SS x0 & Oo? { NN S < © © \ Q > SN > & Nid & S Q & o RS ¥ & Q ° o a 53m Qn Sh ) Q 51 any O som 60m Q TD 00 aren d 3" Sn do 00h 52m 53m ® 0 go 000% 200 TR0 %o 29 nh Gn 44r 45m ’ 461 8r 51r S51r 49r O 48r 51r O () 46r 48r 50r > 0 49 @ ao OO 0 a» 56m 58m 56m 48r 50r 56m O 55m dD O @ 52m 59m O 59m OO 54m 0 54m O J ® OO 54m 46r Sir 55m __ 57m 62d 51r (J O a7r O O 507 49r 501 54m 62d | Ke | O y | 50r | Rat LD50 | LOAEL for serious effects (animals) m Mouse I h Rabbit LOAEL for less serious effects (animals) ! I NOAEL (animals) | d Dog , | I | I | % CEL, cancer effect level ~ , Minimal risk level for effects The number next to each point I other than cancer corresponds to entries in | i WY, Table 2-2. S103443 H1IV3aH ¢ WHO40HOTHO SS + INIWWOD 01N8Nd HOH 14vHad... FIGURE 2-2. Levels of Significant Exposure to Chloroform - Oral (continued) Intermediate (15-364 Days) 2 N 30 > ® OX LO Q o SOL PFS { (mg/kg/day) SE © © & & EE QP ® WD 1,000 — ® 72m 67r QO 100 |— do 66r 67r 65m dD 63m O > 0 oO 68m O 70m 71m 64m 69m 10 |— Key Bl LD50 r Rat @ LOAEL for serious effects (animals) ! Minimal risk level for effects 1 m Mouse (D LOAEL for less serious effects (animals) ; other than cancer h Rabbit O NOAEL (animals) Nert d Dog A LOAEL for serious effect (humans) A LOAEL for less serious effect (humans) The number next to each point corresponds to entries in AA NOAEL (humans) Table 2-2. 0.1 $ CEL, cancer effect level * Doses represent the lowest dose tested per study that produced a tumorigenic response and do not imply the exsistence of a threshold for the cancer end point. S1O03443 HLTV3H 2 WHO40HOTHO 9s +. INSWWOO 2118Nd "YO4 14vdQ... FIGURE 2-2. Levels of Significant Exposure to Chloroform - Oral Chronic (> 365 days ) System \ > S © ¥ 5° &° 3 Na I) 5 Res ae CoN > ON > (mg/kg/day) A S° & oF &° o Ne @ Pd 0° ° & 1,000 °° oF ® © CW Nail Qe © Ww al & oO 81m 0 ® & Sn 2 81m > o 2 88m 74 m 780 790 Oo 2 95m 00 oO O o} ° Q * 100 2 81m 78" gim 78r 78r 81 ® oe 770 780 79r eS ® gar 87r * ou ® ® ® 78r 81m é @ 9 & 94m 3 @ 78 90r gp San oO oO 770 82m 82m 82m 86m 0 2 § 0 77 10 Ee A 83d A O A 83a ' 91d 76 76 82m 83d Q | 83d | I | | 1 A A 75 | 75 ¥ | Key | \ 0.1 B LD50 r Rat @ LOAEL for serious effects (animals) m Mouse (PD LOAEL for less serious effects (animals) 10 - h Rabbit O NOAEL (animals) d Dog A LOAEL for serious effect (humans) The umber next io A LOAEL for less serious effect (humans) Estimated Upper- each point AA NOAEL (humans) 10°_| Bo und Hy n ancer Ris 0.001 corresponds to ff \ | entries in Table 2-2. ¢ GEL, cancerelisctisve Levels | Minimal risk level for effects other | than cancer Y -6 10 0.0001 * Doses represent the lowest dose tested per study that produced a tumorigenic response and do not imply the exsistence of a threshold for the cancer end point. -7 0.00001 10 — S103443 H1TV3H ¢ WHOJO0HO THO LS CHLOROFORM 58 2. HEALTH EFFECTS (Klaunig et al. 1986). Following chronic exposure, no histopathological changes were observed in rats exposed by gavage to 200 mg/kg/day (TWA) chloroform in oil (NCI 1976). Respiratory disease was observed in all chloroform exposed groups of rats (>15 mg/kg/day); however, no histopathological changes were observed in a 60 mg/kg/day exposure group during another experiment by the same investigators (Palmer et al. 1979). No histopathological changes were observed in the lungs of mice exposed by gavage to 477 mg/kg/day (TWA) chloroform in oil for 78 weeks (NCI 1976) or to 60 mg/kg/day in toothpaste for 80 weeks (Roe et al. 1979). Cardiovascular Effects. Information regarding cardiovascular effects after oral exposure to chloroform is limited to case report studies. On admission to the hospital, the blood pressure was 140/90 mm Hg and pulse was 70 beats per minute in a patient who accidentally ingested =2,500 mg/kg chloroform (Schroeder 1965). Electrocardiography showed occasional extrasystoles and a slight S-T segment depression. The patient recovered with no persistent cardiovascular change. In another individual, blood pressure was 100/40 mm Hg and pulse was 108 beats per minute after ingestion of an unknown quantity of chloroform and alcohol (Storms 1973). Co-exposure with alcohol was involved in this case. The cardiovascular system was not a target for chloroform toxicity in animal studies. No histopathological changes were observed in rats and mice chronically exposed by gavage to 200 and 477 mg/kg/day (TWA) chloroform, respectively. In low-dose female mice (but not high-dose female or in any exposed males), cardiac thrombosis was observed (NCI 1976). Similarly, no cardiovascular changes were observed in dogs exposed to 30 mg/kg/day chloroform in toothpaste capsules for 7.5 years (Heywood et al. 1979). Gastrointestinal Effects. Retrosternal soreness, pain on swallowing, and gastric distress with vomiting were reported in cases of intentional and accidental ingestion of chloroform (Piersol et al. 1933; Schroeder 1965). At autopsy, congestion with patchy necrosis of the mucosa was observed in the stomach and duodenum of a man who died after drinking =3,755 mg/kg chloroform (Piersol et al. 1933). The colonic mucosa was edematous, and the rectosigmoid junction was hemorrhagic. A 16-year-old female who ingested an unknown of amount of chloroform arrived at a hospital semiconscious and with repeated vomiting. She was treated with gastric lavage, antacids, intravenous glucose and antiemetics. She had apparently recovered and was released. Seven days later, she ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 59 2. HEALTH EFFECTS presented with hepatomegaly, slightly depressed hemoglobin, and an abnormal liver sonogram, but no gastric side-effects (Hakim et al. 1992). The effects of chronic oral exposure to chloroform, as a by-product of the chlorination of drinking water, were evaluated in three epidemiology studies (Alavanja et al. 1978; Cantor et al. 1978; Young et al. 1981). The association between the incidence of gastrointestinal cancer in humans and the chlorination of drinking water is discussed in Section 2.2.2.8. Gastrointestinal irritation has been observed in some animals after oral exposure to chloroform. Gastric erosions were observed in pregnant rats gavaged with 516 mg/kg chloroform in oil during gestation (Thompson et al. 1974). Rabbits exposed by gavage to 20 mg/kg/day chloroform in oil during gestation had diarrhea; no histological results were provided. In a 90-day drinking water study, no histopathological changes were observed in rats and mice exposed to 160 and 435 mg/kg/day chloroform, respectively (Jorgenson and Rushbrook 1980). Vomiting was observed in dogs exposed to 30 mg/kg/day chloroform in toothpaste capsules for 12-18 weeks (Heywood et al. 1979). In a chronic exposure study, no histopathological changes in gastrointestinal tissue were observed in rats and mice exposed by gavage to 200 and 477 mg/kg/day (TWA) chloroform, respectively (NCI 1976). Hematological Effects. The only information regarding hematological effects in humans after chronic oral exposure to chloroform was reported in a case study. Decreased erythrocytes and hemoglobin were observed in a subject who ingested =21 mg/kg/day chloroform in a cough medicine for 10 years (Wallace 1950). Hematological effects have been observed in some animals after oral exposure to chloroform. Hemoglobin and hematocrit decreased in male and female rats after a single oral dose of 546 mg/kg chloroform in oil (Chu et al. 1982b) and in female rats exposed to 100 mg/kg/day chloroform during gestation (Ruddick et al. 1983). However, no hematological changes were observed in mice exposed to 250 mg/kg/day for 14 days (Munson et al. 1982). In an intermediate-duration study, decreased neutrophils were observed in rats exposed to 193 mg/kg/day in drinking water (Chu et al. 1982b); however, no hematological changes were observed in rats and mice exposed to 160 and 435 mg/kg/day chloroform, respectively, for 90 days in drinking water (Jorgenson and Rushbrook 1980). Increased cellular proliferation in the bone marrow was observed in rats exposed by gavage for 13 weeks to 410 mg/kg chloroform in toothpaste (Palmer et al. 1979). No hematological changes were observed, ***DRAFT FOR PUBLIC COMMENT"** CHLOROFORM 60 2. HEALTH EFFECTS however, in rats similarly exposed to 165 and 60 mg/kg/day chloroform by gavage for 52 and 80 weeks, respectively. Moreover, no histopathological changes in hematopoietic tissues were observed in rats and mice after chronic exposure to 200 and 477 mg/kg/day (TWA) chloroform in oil, respectively (NCI 1976). No hematological effects were observed in dogs exposed to 30 mg/kg/day chloroform in toothpaste in capsules for 7.5 years (Heywood et al. 1979). Musculoskeletal Effects. Muscular relaxation of the jaw caused upper respiratory obstruction in a man who accidentally ingested 2,500 mg/kg chloroform (Schroeder 1965), reflecting the neurological effects of chloroform exposure. No histopathological changes were observed in the musculoskeletal system of rats and mice after chronic gavage exposure to 200 and 477 mg/kg/day (TWA) chloroform in oil, respectively (NCI 1976). Hepatic Effects. The liver is a primary target of chloroform toxicity in humans, with some evidence that suggests that the damage may be reversible (Wallace 1950). Hepatic injury occurred in patients within 1-3 days following chloroform ingestion (Piersol et al. 1933; Schroeder 1965; Storms 1973). Jaundice and liver enlargement and tenderness developed in all patients. The clinical observations were supported by blood biochemistry results with increased SGOT, SGPT, and lactate dehydrogenase (LDH) activities and increased bilirubin levels. At autopsy, fatty degeneration and extensive centrilobular necrosis were observed in one fatal case (Piersol et al. 1933). A 16-year-old female who ingested an unknown of amount of chloroform and arrived at a hospital semiconscious and with repeated vomiting was reported by Hakim et al. (1992). She was treated with gastric lavage, antacids, intravenous glucose and antiemetics. She had apparently recovered and was released. Seven days later, she presented with hepatomegaly, slightly depressed hemoglobin, and an abnormal liver sonogram, suggesting toxic hepatic disease due to chloroform toxicosis. A 33-year-old female had injected herself intravenously with 0.5 mL of chloroform and then became unconscious. When she awoke approximately 12 hours later, she then drank another 120 mL of chloroform. She was treated with hyperbaric oxygen, cimetidine (to inhibit cytochrome P-450 and inhibit formation of phosgene) and N-acetylcystine (to replenish glutathione stores). Liver serum enzymes alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and LDH were elevated in a pattern that suggested liver cell necrosis. Generally, these enzymes were noted to peak ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 61 2. HEALTH EFFECTS by day 4 and decrease by day 11. Total bilirubin and direct bilirubin did not change appreciably. GGT (gamma glutamyltransferase, also known as gamma glutamyl transpeptidase), alpha-feto protein and retinol binding protein showed increases between 6 and 8 days after ingestion, but still within normal ranges for humans (Rao et al. 1993). Increased sulfobromophthalein retention indicated impaired liver function in an individual who ingested 21 mg/kg/day chloroform in a cough medicine for 10 years (Wallace 1950). The changes reversed to normal after exposure was discontinued. Biochemical tests indicate that liver function in male and female humans was not affected by the use of mouthwash providing 0.96 mg/kg/day chloroform for <5 years (De Salva et al. 1975). The liver is also a target organ for chloroform toxicity in animals. In acute studies, hepatitis was observed in pregnant rats exposed by gavage to 516 mg/kg/day chloroform in oil (Thompson et al. 1974), while increased maternal liver weight without any histopathological changes was observed in pregnant rats similarly exposed to 100 mg/kg/day chloroform (Ruddick et al. 1983). Increased serum levels of transaminases, indicative of liver necrosis, were observed in mice treated with a single gavage dose of 199 mg/kg chloroform in toothpaste, 273 mg/kg in oil (Moore et al. 1982), or 250 mg/kg/day in oil for 14 days (Munson et al. 1982). Centrilobular necrosis of the liver with massive fatty changes was also observed in mice after a single dose of 350 mg/kg chloroform in oil (Jones et al. 1958). At a dose of 35 mg/kg, minimal lesions consisting of midzonal fatty changes were observed in mice. Similar results were reported by Larson et al. (1993) in male rats in order to identify target tissues for the acute effects of chloroform in rats and mice and to establish the time-course of chloroform-induced histopathologic and proliferative responses. Rats were given 34, 180, or 477 mg/kg once in corn oil by gavage and sacrificed 24 hours after administration (acute study). In a related time-course study (which focused on histologic changes in tissues over time), rats received 180 mg/kg chloroform in corn oil by gavage and were sacrificed at 0.5, 1, 2, 4, and 8 days after treatment, or received 477 mg/kg in corn oil by gavage and were sacrificed either 1 or 2 days after administration. In the acute study, gross liver to body weight ratios were unaffected at all doses. Histologically, chloroform caused hepatic injury, in a dose-related manner, producing morphologic changes generally limited to the centrilobular hepatocytes. Liver enzymes (SDH, ALT and AST) were slightly elevated above controls in the 34 and 180 mg/kg group, but significantly higher in the 477 mg/kg group for all three ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 62 2. HEALTH EFFECTS enzymes. In the time-course study, 1 day after dosing, about 50% of the hepatocytes adjacent to the central veins were degenerated or necrotic in the 180 mg/kg treatment group. Larger vessels had perivascular edema, influx of neutrophils and eosinophils. Only scattered hepatocyte necrosis was seen by day 2 after treatment. By eight days however, the livers were not histologically different from controls. In a similar study, mice were administered 34, 238, or 477 mg/kg once in corn oil by gavage and sacrificed 24 hours after administration (acute study) or 350 mg/kg chloroform in corn oil by gavage and sacrificed at 0.5, 1, 2, 4, and 8 days after treatment (time-course study). Livers of female mice were much more sensitive than the kidneys to the toxic effects of chloroform. In the acute study, livers of mice receiving 34 mg/kg chloroform were not histologically different from controls; however, those treated with 238 mg/kg had few small randomly scattered foci of hepatocyte necrosis. Livers from the 477 mg/kg group had centrilobular coagulative necrosis of 50% of the lobule. In the time- course study, a significant increase in liver weights and liver to body-weight ratios was observed in mice at 2 and 4 days after treatment with the 350 mg/kg dose of chloroform. At 12 hours after treatment, mice had marked swelling of the centrilobular hepatocytes, affecting about 50% of the lobule. One day after treatment, the hepatocytes adjacent to the central vein were necrotic. Two days after chloroform treatment, centrilobular sinusoids were dilated with inflammatory cells associated with centrilobular necrosis. At eight days after treatment, the livers from the treated mice were not histologically different from those of control animals. Serum liver enzymes (SDH and ALT) were elevated in the groups sacrificed at 0.5, 1, 2, and 4 days after treatment, but not in controls or in those animals sacrificed 8 days after treatment. Differences in chloroform toxicity have been noted in female mice when chloroform was administered in different vehicles (Larson et al. 1994b). Mice were treated orally with 3, 10, 34, 90, 238, or 477 mg/kg/day of chloroform in corn oil for 4 days. Chloroform treatment resulted in significant increases in liver weights of mice at the 238 and 477 mg/kg/day dose levels. Mice treated with 238 mg/kg chloroform had moderate centrilobular vacuolar degeneration of hepatocytes and scattered centrilobular and subcapsular hepatocyte necrosis. At the 477 mg/kg dose, severe centrilobular coagulative necrosis with small number of inflammatory cells in the necrotic areas. Dose dependent increases in both ALT and SDH were also observed. At daily doses of 90 mg/kg or less, no increase in hepatic cell proliferation was noted. Dose-dependent increases in hepatic cell proliferation and cells observed to be in S-phase occurred in the 238 and 477 mg/kg/day doses. For mice dosed with 16, 26, ***DRAFT FOR PUBLIC COMMENT *** CHLOROFORM 63 2. HEALTH EFFECTS 53, 81, or 105 mg/kg/day in the drinking water, serum ALT or SDH were not different from controls at any dose. In the 53, 81, and 105 mg/kg/day treatment groups, the livers had changes that were characterized by pale cytoplasmic eosinophilic staining of centrilobular hepatocytes compared to the periportal hepatocytes and controls. Livers from mice treated with 26 mg/kg/day chloroform or less failed to showed significant histologic changes when compared to controls. Cell proliferations in the liver were not found at any dose or duration. An acute oral MRL of 0.3 mg/kg/day was calculated using the 26.4 mg/kg/day NOAEL based on the hepatic effects in these animals from this study. Another study by Larson et al. (1995) examined the dose response relationships for the induction of cytolethality and regenerative cell proliferation in the livers of male Fischer 344 rats given chloroform by gavage. Groups of 12 rats were administered oral doses of 0, 3, 10, 34, 90, and 180 mg/kg/day chloroform in corn oil by gavage for 4 days. BrdU was administered via an implanted osmotic pump to label cells in S-phase. Cells having incorporated BrdU were visualized in tissue sections immunohistochemically and the LI evaluated as the percentage of S-phase cells. Necropsies and histopathological examinations were performed at death. The relative liver weights were increased at doses of 10 mg/kg/day and above at 4 days post-treatment. Rats treated with 34, 90, or 180 mg/kg/day by gavage for 4 days had mild to moderate degeneration of centrilobular hepatocytes. The livers of rats given 90 mg/kg/day for 4 days had a slight increase in centrilobular pallor and necrosis of hepatocytes surrounding the central vein; the remaining central and some mid-zonal hepatocytes were swollen and displayed a cytoplasmic granularity. In the 180 mg/kg/day dose group, the livers had scattered individual cell necrosis throughout the central and midzonal regions. The cytoplasm of the centrilobular hepatocytes were pale, eosinophilic and mildly vacuolated. Dose- dependent increases in both ALT and SDH were observed at 4 days in the 90 and 180 mg/kg/day dose groups in the 180 mg/kg/day dose group only. A dose-dependent increase in LI was seen in rat liver after 4 days of treatment with 90 and 180 mg/kg/day by gavage. Larson et al. (1995) also examined the toxicological effects of chloroform administered in the drinking water in rats. Groups of 12 rats were administered chloroform ad libitum in drinking water at concentrations of 0, 60, 200, 400, 900, and 1,800 ppm for 4 days. BrdU was administered via an implanted osmotic pump to label cells in S-phase. Cells having incorporated BrdU were visualized in tissue sections immunohistochemically and the LI evaluated as the percentage of S-phase cells. Necropsies and histopathological examinations were performed at death. Average daily doses of chloroform ingested from drinking water were: 0, 6.6, 19.3, 33.2, 68.1, and 57.5 mg/kg/day for 0, 60, **DRAFT FOR PUBLIC COMMENT"** CHLOROFORM 64 2. HEALTH EFFECTS 200, 400, 900, and 1,800 ppm concentration levels, respectively. Only mild hepatocyte vacuolation was observed in rats given 900 or 1,800 ppm in water for 4 days; no increase in the hepatic LI was observed at any time point. Liver effects in animals have been reported in numerous oral studies of intermediate duration. Larson et al. (1994b) exposed female mice to 3, 10, 34, 90, 238, and 477 mg/kg/day of chloroform in corn oil via gavage for 5 days per week for 3 weeks. Chloroform treatment resulted in significant increases in liver weights of mice at the 90, 238, and 477 mg/kg/day doses. Doses of 34 mg/kg/day resulted in pale cytoplasmic eosinophilia of the centrilobular hepatocytes and mild vacuolation of the centrilobular and midzonal hepatocytes relative the periportal hepatocytes and livers from control mice. At the 238 mg/kg/day dose, the livers were characterized by a severe centrilobular hepatocyte necrosis. At 477 mg/kg/day, the central zone of the liver was populated by degenerate vacuolated hepatocytes and regenerating hepatocytes with markedly basophilic cytoplasm and small round nuclei with clumped chromatin and prominent nucleoli. Significant dose-dependent increases in ALT and SDH were observed at doses of 34 mg/kg/day and greater. Cell proliferation was markedly increased in the liver at the 238 and 477 mg/kg/day doses. Mice dosed with 16, 43, 82, 184, or 329 mg/kg/day of chloroform in the drinking water for 7 days a week for 3 weeks resulted in no histological changes in livers at all doses studied. Liver weights were significantly increased at 82, 184, and 329 mg/kg/day. Another study by Larson et al. (1995) examined the dose response relationships for the induction of cytolethality and regenerative cell proliferation in the livers of male Fischer 344 rats given chloroform by gavage. Groups of 12 rats were administered oral doses of 0, 3, 10, 34, 90, and 180 mg/kg/day chloroform in corn oil by gavage for 5 days per week for 3 weeks. BrdU was administered via an implanted osmotic pump to label cells in S-phase. Cells having incorporated BrdU were visualized in tissue sections immunohistochemically and the LI evaluated as the percentage of S-phase cells. Necropsies and histopathological examinations were performed at death. The relative liver weights were increased at doses of 90 mg/kg/day and greater at 3 weeks. After 3 weeks of exposure, livers of rats in the 34 or 90 mg/kg/day dose groups did not differ from controls. In the 180 mg/kg/day dose group, effects were similar to those seen at 4 days after exposure. Dose-dependent increases in both ALT and SDH were observed after 3 weeks in the 180 mg/kg/day dose group only. Larson et al. (1995) also examined the toxicological effects of chloroform administered in the drinking water in rats. Groups of 12 rats were administered chloroform ad libitum in drinking water at ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 65 2. HEALTH EFFECTS concentrations of 0, 60, 200, 400, 900, and 1,800 ppm for 7 days/week for 3 weeks. BrdU was administered via an implanted osmotic pump to label cells in S-phase. Cells having incorporated BrdU were visualized in tissue sections immunohistochemically and the LI evaluated as the percentage of S-phase cells. Necropsies and histopathological examinations were performed at death. Average daily doses of chloroform ingested from drinking water were: 0, 6.0, 17.4, 32.0, 62.3, and 106 mg/kg/day for 3 weeks exposure for 0, 60, 200, 400, 900, and 1,800 ppm concentration levels, respectively. Only mild hepatocyte vacuolation was observed in rats given 900 or 1,800 ppm in water for 3 weeks. No increase in the hepatic LI was observed at any time point. Fatty changes, necrosis, increased liver wig, and hyperplasia have been observed in rats exposed to >150 mg/kg/day chloroform in a toothpaste vehicle via gavage for 90 days (Palmer et al. 1979). An increased incidence of sporadic, mild, reversible, liver changes occurred in rats exposed to chloroform in the drinking water at doses of 0.64-150 mg/kg/day for 90 days, but the incidences were not significantly higher than the incidences in controls (Chu et al. 1982a). The effect- and no-effect-levels in the study are clearly defined. Fatty and hydropic changes, necrosis, and cirrhosis were observed in mice treated by gavage with 250 mg/kg/day chloroform in oil for 90 days (Bull et al. 1986; Munson et al. 1982) or 86 mg/kg/day in drinking water for 1 year (Klaunig et al. 1986). In contrast, centrilobular fatty changes observed in mice at 64 mg/kg/day chloroform in drinking water for 90 days appeared to be reversible (Jorgenson and Rushbrook 1980), and no liver effects were found in mice treated with >50 mg/kg/day chloroform in aqueous vehicles (Bull et al. 1986). In addition, hepatocellular degeneration was induced in F, females in a 2-generation study in which mice were treated by gavage with 41 mg/kg/day chloroform in oil (Gulati et al. 1988). Significantly increased (p<0.05) SGPT activity occurred in dogs beginning at 6 weeks of exposure to chloroform in toothpaste in capsules at a dose of 30 mg/kg/day in a 7.5-year study (Heywood et al. 1979). SGPT activity was not increased at 15 mg/kg/day until week 130. Therefore, 15 mg/kg/day was the NOAEL for intermediate-duration exposure. This NOAEL was used to derive an intermediate-duration oral MRL of 0.1 mg/kg/day, as described in footnote "c" in Table 2-2. In chronic exposure studies, liver effects have been observed in rats, mice, and dogs after oral exposure to chloroform. Necrosis was observed in female rats treated by gavage with 200 mg/kg/day chloroform in oil for 78 weeks (NCI 1976). Nodular hyperplasia occurred in all groups of male and female mice similarly treated at 138 mg/kg/day. Fibrosis of the liver was observed in both sexes of rats exposed to 200 mg/kg/day chloroform in the drinking water for <180 weeks (Tumasonis et al. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 66 2. HEALTH EFFECTS 1985, 1987). Increased SGPT was observed in dogs given chloroform in toothpaste capsules for 7.5 years (Heywood et al. 1979). The lowest oral dose administered to animals in chronic studies was 15 mg/kg/day, which increased SGPT in dogs. This LOAEL was used to derive a chronic oral MRL of 0.01 mg/kg/day, as described in footnote "d" of Table 2-2. Renal Effects. The kidney is also a major target of chloroform-induced toxicity in humans. Oliguria was observed 1 day after the ingestion of =3,755 or 2,500 mg/kg chloroform (Piersol et al. 1933; Schroeder 1965). Increased blood urea nitrogen and creatinine levels also indicated renal injury. Albuminuria and casts were detected in the urine. Histopathological examination at autopsy revealed epithelial swelling and hyaline and fatty degeneration in the convoluted tubules of kidneys in one fatal case of oral exposure to chloroform (Piersol et al. 1933). Numerous hyaline and granular casts and the presence of albumin were observed in the urine of one subject who ingested 21 mg/kg/day chloroform in cough medicine for 10 years (Wallace 1950).. The urinalysis results reversed to normal after discontinuation of chloroform exposure. No indications of renal effects were observed in humans who ingested estimated doses of 0.34-2.46 mg/kg/day chloroform in mouthwash for 5 years (De Salva et al. 1975). The renal toxicity of chloroform in animals has been reported in many studies of acute duration. Lipsky et al. (1993) studied groups of male Fischer 344 rats gavaged with either 90 or 180 mg/kg/day of chloroform in corn oil or water for 5 days per week for 4 weeks. Rats exposed to chloroform by gavage in corn oil displayed acute cell injury and necrosis, primarily in the epithelial cells lining the S2 segment of the proximal tubule, with some apparent damage/necrosis occurring in the S1 segment as well. This injury was present in all rats exposed to the 180 mg/kg/day dose and in less than half of the animals exposed to the 90 mg/kg/day dose. There was also a dose-dependent increase in the total BrdU labeling of nuclei in renal cells of the chloroform-treated oil-gavaged animals compared to controls. The largest increase in DNA BrdU labeling was in the cells of the S2 segment. The 90 mg/kg/day dose of chloroform also showed an increase in DNA labeling in the S3 segment, but not for the 180 mg/kg/day dose of chloroform. Animals exposed to chloroform in water showed minimal histopathologic alterations in the kidneys. Mild injury and necrosis was seen in cells of the S2 segment in | of 6 animals in the 180 mg/kg/day group, while none were seen in the 90 mg/kg/day dose group. Little to no change in DNA labelling of renal cells was seen in the water-gavaged rats. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 67 2. HEALTH EFFECTS A study by Larson et al. (1993) studied the effects of dose and time after chloroform administration on the renal toxicology of chloroform in male rats. Rats were given 34, 180, or 477 mg/kg once in corn oil by gavage and sacrificed 24 hours after administration (acute study). In a related time-course study (which focused on histologic changes in tissues over time), rats received 180 mg/kg chloroform in corn oil by gavage and were sacrificed at 0.5, 1, 2, 4, and 8 days after treatment; others received 477 mg/kg in corn oil by gavage and were sacrificed either 1 or 2 days after administration. Histologically, chloroform caused extensive renal damage, and to a much lesser extent, hepatic injury, in a dose-related manner. One day after treatment with a single dose of chloroform of 34 mg/kg or greater, the kidneys of male rats developed tubular necrosis that was restricted to the proximal convoluted tubules. The severity of these lesions occurred in a dose-dependent manner. Rats given 34 mg/kg had scattered necrotic tubules affecting less than 10% of the midcortical nephrons. In the 180 mg/kg group, 25% of the proximal convoluted tubules were necrotic. Nearly all segments of the proximal tubules had necrosis in the rats receiving 477 mg/kg chloroform. Despite extensive renal injury, increases in blood urea nitrogen (BUN) or in urinary protein or glucose were not observed. In the time-course study, the kidneys, after 12 hours of treatment, had a diffuse granularity of cytoplasm of the epithelium lining of the proximal convoluted tubules in the 180 mg/kg group. Damage was severe after 1 day, and after 2 days, 100% of the proximal tubules were lined by necrotic epithelium. After 8 days, the kidneys had returned to normal appearance. No increases in BUN or urinary protein or glucose were noted at any time after treatment. Another study by Larson et al. (1995) examined the dose response relationships for the induction of cytolethality and regenerative cell proliferation in the kidneys of male Fischer 344 rats given chloroform by gavage. Groups of 12 rats were administered oral doses of 0, 3, 10, 34, 90, and 180 mg/kg/day chloroform in corn oil by gavage for 4 days. BrdU was administered via an implanted osmotic pump to label cells in S-phase. Cells having incorporated BrdU were visualized in tissue sections immunohistochemically and the LI evaluated as the percentage of S-phase cells. Necropsies and histopathological examinations were performed at death. Rats treated with 34, 90, or 180 mg/kg/day by gavage for 4 days had mild to moderate degeneration of renal proximal tubules and centrilobular hepatocytes. After 4 days of dosing with 34 mg/kg/day, the proximal convoluted tubule epithelial cells had increased numbers and prominence of apical cytoplasmic vacuoles. Likewise, rats given 90 mg/kg/day for 4 days displayed swelling and vacuolation of 25-50% of the proximal tubules. Progressive degeneration of the proximal tubules was observed in rats exposed to 180 mg/kg/day. At 4 days, swollen and vacuolated cytoplasm in approximately 10-20% of proximal tubule epithelium ***DRAFT FOR PUBLIC COMMENT"** CHLOROFORM 68 2. HEALTH EFFECTS was observed. LI were increased in the kidney cortex only in the rats treated with 180 mg/kg/day for 4 days. Larson et al. (1995) also examined the toxicological effects of chloroform administered in the drinking water in rats. Groups of 12 rats were administered chloroform ad libitum in drinking water at concentrations of 0, 60, 200, 400, 900, and 1,800 ppm for 4 days. BrdU was administered via an implanted osmotic pump to label cells in S-phase. Cells having incorporated BrdU were visualized in tissue sections immunohistochemically and the LI evaluated as the percentage of S-phase cells. Necropsies and histopathological examinations were performed at death. Average daily doses of chloroform ingested from drinking water were: 0, 6.6, 19.3, 33.2, 68.1, and 57.5 mg/kg/day for 4 days exposure for the 0, 60, 200, 400, 900, and 1,800 ppm concentration levels, respectively. When chloroform was administered in the drinking water, no microscopic alterations were seen in the kidneys after 4 days of treatment. The overall renal LI was not increased at any dose. Acute toxic nephrosis was observed in female rats exposed to 516 mg/kg/day chloroform during gestation, with maternal lesions characterized by tubular swelling, hydropic fatty degeneration and necrosis (Thompson et al. 1974). Increased kidney weight was observed in female rats after a single gavage dose of 546 mg/kg chloroform (Chu et al. 1982b). Similarly, rats exposed to 400 mg/kg/day by gavage during gestation had increased kidney weight (Ruddick et al. 1983). No increase in kidney weight was found in the rats treated with 200 mg/kg/day during gestation. Renal necrosis in convoluted tubules was observed in male mice after a single dose of 199 mg/kg chloroform in toothpaste or 65.6 mg/kg chloroform in oil (Moore et al. 1982). In intermediate-duration studies, mice appeared to be more sensitive than rats to the nephrotoxic effects of chloroform. Rats exposed to 193 mg/kg/day for 28 days (Chu et al. 1982b) or to 160 mg/kg/day chloroform for 90 days (Jorgenson and Rushbrook 1980) in drinking water had no kidney effects. Increased relative kidney weight was observed in rats exposed by gavage to 150 mg/kg/day for 13 weeks, but not in rats exposed to 30 mg/kg/day (Palmer et al. 1979). Chronic inflammatory changes were observed in the kidneys of mice exposed to 50 mg/kg/day chloroform (dissolved in an emulsion prepared with emulphor in water) by gavage (Munson et al. 1982); however, no changes were observed in mice exposed to 41 mg/kg/day by gavage (Gulati et al. 1988) or in mice exposed to 435 mg/kg/day chloroform in drinking water (Jorgenson and Rushbrook 1980). ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 69 2. HEALTH EFFECTS Nonetheless, exposure to 86 mg/kg/day in drinking water for 1 year caused tubular necrosis in mice (Klaunig et al. 1986). Larson et al. (1994b) exposed female mice to 3, 10, 34, 90, 238, and 477 mg/kg/day of chloroform in corn oil via gavage for 5 days per week for 3 weeks. Mice were also dosed with 16, 43, 82, 184, or 329 mg/kg/day of chloroform in the drinking water for 7 day per week for 3 weeks. In both studies, no increases in cell proliferation were noted and no significant changes in renal histopathology were reported. Another study by Larson et al. (1995) examined the dose response relationships for the induction of cytolethality and regenerative cell proliferation in the kidneys of male Fischer 344 rats given chloroform by gavage. Groups of 12 rats were administered oral doses of 0, 3, 10, 34, 90, and 180 mg/kg/day chloroform for 5 days/week for 3 weeks. BrdU was administered via an implanted osmotic pump to label cells in S-phase. Cells having incorporated BrdU were visualized in tissue sections immunohistochemically and the LI evaluated as the percentage of S-phase cells. Necropsies and histopathological examinations were performed at death. Relative kidney weights were increased after 3 weeks in the 180 mg/kg/day dose group only. Rats treated with 34, 90, or 180 mg/kg/day by gavage for 4 days had mild to moderate degeneration of renal proximal tubules and centrilobular hepatocytes. These alterations were absent or slight after 3 weeks of treatment, except at the highest dose level. After 4 days of dosing with 34 mg/kg/day, the proximal convoluted tubule epithelial cells had increased numbers and prominence of apical cytoplasmic vacuoles, but these changes were not observed at 3 weeks. Likewise, rats given 90 mg/kg/day for 4 days displayed swelling and vacuolation of 25-50% of the proximal tubules, at 3 weeks only 1 of 3 rats had vacuolated and degenerated epithelium. Progressive degeneration of the proximal tubules was observed in rats exposed to 180 mg/kg/day. At 4 days, swollen and vacuolated cytoplasm in approximately 10-20% of proximal tubule epithelium was observed, while at 3 weeks the percentage was 25-50%. At 3 weeks, scattered tubules also had mineral concretions that appeared subepithelial. LI was increased in the kidney cortex only in the rats treated with 180 mg/kg/day for 4 days. Larson et al. (1995) also examined the toxicological effects of chloroform administered in the drinking water in rats. Groups of 12 rats were administered chloroform ad libitum in drinking water at concentrations of 0, 60, 200, 400, 900, and 1,800 ppm for 7 days/week for 3 weeks. BrdU was administered via an implanted osmotic pump to label cells in S-phase. Cells having incorporated ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 70 2. HEALTH EFFECTS BrdU were visualized in tissue sections immunohistochemically and the LI evaluated as the percentage of S-phase cells. Necropsies and histopathological examinations were performed at death. Average daily doses of chloroform ingested from drinking water were 0, 6.0, 17.4, 32.0, 62.3, and 106 mg/kg/day for 3 weeks exposure for 0, 60, 200, 400, 900, and 1,800 ppm concentration levels, respectively. As a general observation, rats treated for 3 weeks with 200 ppm chloroform and greater had slightly increased numbers of focal areas of regenerating renal proximal tubular epithelium and cell proliferation than were noted in controls, but no clear dose response relationship was evident. The ‘overall renal LI was not increased at any dose or time point. In chronic oral studies, no definite renal effects were observed in rats exposed to <200 mg/kg/day or mice exposed to <477 mg/kg/day (TWA) (Jorgenson et al. 1985; NCI 1976; Roe et al. 1979). In dogs, however, fat deposition in renal glomeruli was observed at a dose of 30 mg/kg/day chloroform for 7.5 years, but not at 15 mg/kg/day (Heywood et al. 1979). Dermal Effects. No studies were located regarding dermal effects in humans after oral exposure to chloroform. Alopecia was observed in pregnant rats exposed to 126 mg/kg/day chloroform in oil (Thompson et al. 1974). Rough coats were observed in mice exposed to 100 mg/kg/day chloroform in oil for 14 days (Gulati et al. 1988). Ocular Effects. Only one reference was located that discussed the ocular effects of chloroform after oral ingestion. Li et al. (1994) examined the effects of chloroform administered in drinking water to guinea pigs with cedar pollen-induced allergic conjunctivitis, prepared by passive cutaneous anaphylaxis. Groups of 5 male Hartley guinea pigs were given drinking water with chloroform concentrations of 0.01, 0.1, 1.0, 10, 100, or 1,000 ppm 48 hours before applying an antigen eye drop (starting on the 8th day after antiserum administration). One control group was not administered chloroform and another control group was not administered the antiserum (chloroform alone) for every dose level. The light absorption rate of Evans blue extracted from conjunctiva was used as an index of the relative intensity of allergic conjunctivitis. In a separate experiment, using the dose level which caused the most intense aggravating effect in the above testing, groups of 3 male guinea pigs were given 1 ppm chloroform in ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 71 2. HEALTH EFFECTS drinking water for 48 hours and the residual effect on the allergic conjunctivitis was examined. Animals were examined immediately after, and 1, 2, 4, 7, and 14 days after exposure; antigen eye drops were applied 10 days after the antiserum administration. Water intake was monitored and blood chloroform concentrations were measured. At 0.1 ppm chloroform, significant aggravation of allergic conjunctivitis was observed. Allergic conjunctivitis was most intensely aggravated at 1 ppm chloroform. At higher doses (10 and 100 ppm) the aggravation was still noticeable, yet less significant. At 1,000 ppm chloroform, the aggravating effect was not present. Blood chloroform concentrations increased as the concentration in drinking water increased from 0.01 to 1,000 ppm. Body Weight Effects. No studies were located regarding body weight effects in humans after oral exposure to chloroform. Several studies were located regarding body weight changes in animals after oral exposure to chloroform; however, the effect of chloroform on body weight is variable and depends somewhat on the dose and dosing method. A dose-related decrease in body weight gain was observed in rats exposed to 100 mg/kg/day and in rabbits exposed to 50 mg/kg/day chloroform by gavage in oil during gestation (Ruddick et al. 1983; Thompson et al. 1974). In addition, decreased body weight was observed in male mice after acute exposure to 250 mg/kg/day chloroform by gavage in oil (Gulati et al. 1988; Munson et al. 1982). Others have reported similar reductions in body weight after oral dosing with chloroform (Davis and Berndt 1992; Larson et al. 1994b, 1995; Reddy et al. 1992). Dose-related decreases in body weight or body weight gain were observed in rats exposed to >83 mg/kg/day in water (Jorgenson and Rushbrook 1980) or in oil (NCI 1976) and in mice (Roe et al. 1979) in studies of intermediate duration. Similar effects were found in rats exposed to >60 mg/kg/day regardless of the vehicle (Jorgenson et al. 1985; Larson 1995; NCI 1976; Palmer et al. 1979; Tumasonis et al. 1985) and mice exposed to 263 mg/kg/day in water (Jorgenson et al. 1985) in studies of chronic exposure; taste aversion may have been a complicating factor in these studies. In contrast, no effect on body weight was observed in mice treated with 477 mg/kg/day by gavage in oil (NCI 1976) or dogs treated with 30 mg/kg/day chloroform (Heywood et al. 1979) in studies of chronic duration. Food and/or water consumption were decreased in chloroform exposed animals in some studies (Chu et al. 1982a; Jorgenson et al. 1985), but others reported fluctuating food intake unrelated to chloroform exposure (Palmer et al. 1979) or no significantly depressed food consumption at the lowest LOAEL level for body weight effects (Thompson et al. 1974). The effects chloroform has on ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 72 2. HEALTH EFFECTS changes in body weight and water consumption when administered orally in different vehicles and varying doses have also been reported in female mice (Pereira 1994). 2.2.2.3 Immunological and Lymphoreticular Effects No studies were located regarding immunological effects in humans after oral exposure to chloroform. Information regarding immunological effects in animals after oral exposure to chloroform is limited to three studies. Reduced lymphocyte counts were observed in female rats after a single gavage dose of 1,071 mg/kg chloroform (Chu et al. 1982b); no effects were observed in the 765 mg/kg group. Humoral immunity, defined as antibody-forming cells (AFC)/spleen x 100,000, was depressed in both sexes of mice after oral dosing with 50 mg/kg/day chloroform for 14 days (Munson et al. 1982). In contrast, hemagglutination titer was not significantly influenced, and no changes in cell-mediated immunity were recorded. Similar results were obtained in a 90-day experiment (Munson et al. 1982). Depressed humoral immunity was observed in mice exposed to 50 mg/kg/day chloroform. Cell- mediated immunity (delayed type hypersensitivity) was affected in the high-dose (250 mg/kg/day) group of females. The chloroform-induced changes were more marked in the 14-day study than in the 90-day study. The highest NOAEL value and all reliable LOAEL values for immunological effects in each species and duration category are recorded in Table 2-2 and plotted in Figure 2-2. 2.2.2.4 Neurological Effects The data regarding neurological effects in humans after oral exposure to chloroform were obtained from clinical case reports. Deep coma occurred immediately after exposure to 2,500 or 3,755 mg/kg in all cases of intentional or accidental ingestion of chloroform (Piersol et al. 1933; Schroeder 1965; Storms 1973). All reflexes were abolished, and pupil size varied. All patients survived the first coma and became fully conscious; however, one patient died in coma several days later due to extensive liver necrosis (Piersol et al. 1933). Mild cerebellar damage (instability of gait, intentional tremor) was observed in one patient, but reversed to normal in two weeks (Storms 1973). “**DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 73 2. HEALTH EFFECTS The central nervous system in animals is a target of chloroform toxicity after oral exposure to chloroform. High, single doses of chloroform caused ataxia, incoordination, and anesthesia in mice (Balster and Borzelleca 1982; Bowman et al. 1978). The calculated EDs, for motor performance was 484 mg/kg chloroform (Balster and Borzelleca 1982). The effects disappeared within 90 minutes postexposure. A minimal narcotic dose for 50% of the treated mice was calculated to be 350 mg/kg (Jones et al. 1958). Hunched posture and inactivity were observed in male mice exposed by gavage to 250 mg/kg chloroform in oil for 14 days (Gulati et al. 1988). No effects were observed after exposure to 100 mg/kg day. Hemorrhaging in the brain was observed during gross pathological examinations of mice that died under chloroform anesthesia following doses >500 mg/kg/day (Bowman et al. 1978). Lower concentrations of chloroform induced taste aversion to a saccharin solution in mice exposed by gavage for 10 days to 30 mg/kg/day in oil, but not in mice exposed to 10 mg/kg/day (Landauer et al. 1982). No signs of behavioral toxicity were observed in mice exposed to 31.1 mg/kg/day chloroform for 14 days, or 10 or 13 weeks, or in mice exposed to 100 mg/kg for 30 days (Balster and Borzelleca 1982). Operant behavior in mice was affected after exposure to 100 mg/kg/day for 60 days (Balster and Borzelleca 1982). The most severe effects were observed early in the experiment; partial tolerance was observed later. No histopathological changes were observed in the brains of rats after chronic exposure to 200 mg/kg/day, in the brains of mice after chronic exposure to 477 mg/kg/day (NCI 1976), or in the brains of mice after chronic exposure to 60 mg/kg/day (Roe et al. 1979). The highest NOAEL values and all reliable LOAEL values for neurological effects in each species and duration category are recorded in Table 2-2 and plotted in Figure 2-2. 2.2.2.5 Reproductive Effects No studies were located regarding reproductive effects in humans after oral exposure to chloroform. In animals, increased resorptions were observed in rats exposed to 316 mg/kg/day chloroform during gestation, but not in those exposed to 300 mg/kg/day (Thompson et al. 1974). Furthermore, abortions (not otherwise specified) were observed in rabbits exposed to 63 mg/kg/day chloroform during gestation (Thompson et al. 1974). No histopathological changes were observed in the testes of rats exposed to 160 mg/kg/day chloroform in drinking water for intermediate durations (Jorgenson and Rushbrook 1980). Gonadal atrophy was observed in both sexes of rats treated by gavage with 410 mg/kg/day chloroform in toothpaste (but not with 150 mg/kg/day) (Palmer et al. 1979). In a ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 74 2. HEALTH EFFECTS 2-generation reproductive study, chloroform exposure did not affect the fertility in either generation (Gulati et al. 1988). No histopathological changes were observed in the reproductive organs of male and female rats and mice chronically exposed to 200 and 477 mg/kg/day chloroform via gavage (NCI 1976). Similarly, no changes were observed in dogs chronically exposed to 30 mg/kg chloroform (Heywood et al. 1979). The highest NOAEL values and all reliable LOAEL values for reproductive effects in each species and duration category are recorded in Table 2-2 and plotted in Figure 2-2. 2.2.2.6 Developmental Effects One study by Kramer et al. (1992) was located regarding developmental effects in humans after oral exposure to chloroform. The study was conducted to determine whether water supplies containing relatively high levels of chloroform and other trihalomethanes within the state of Iowa are associated with low birth weight, prematurity, or intrauterine growth retardation. Subjects selected include 159 low birth weight infants, 342 premature infants, and 187 growth-retarded infants; however, case definitions were not mutually exclusive. Infants studied were divided into three groups: those that lived in areas where the water supply had undetectable amounts of chloroform, those that lived in areas where the water supply had 1-9 pg/L chloroform, and those that lived in areas where the water supply had more than 10 pg/L. The estimated relative risk of low birth weight associated with drinking water sources having chloroform levels of greater than or equal to 10 pg/L was 30% higher than the risk for sources with undetectable levels of chloroform. Prematurity was not associated with chloroform/trihalomethane exposure. The estimated relative risk of intrauterine growth retardation associated with drinking water supplies with chloroform concentration of >10 pg/L. was 80% more than the risk for those sources with undetectable levels of chloroform. Sources with intermediate chloroform levels (1-9 pg/L) had an elevated risk of 30%. The authors concluded that there is an increased risk of intrauterine growth retardation associated with higher concentrations of waterborne chloroform and dichlorobromomethane; however, it also should be noted that other organic halides (haloacetic acids and haloacetonitriles) are also associated with developmental effects. No teratological effects or skeletal anomalies in rats or rabbits after oral exposure to chloroform were reported in developmental studies (Ruddick et al. 1983; Thompson et al. 1974). Decreased fetal weight was observed in the offspring of rats exposed by gavage to 400 mg/kg/day chloroform during “**DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 75 2. HEALTH EFFECTS gestation, but not in those exposed to 200 mg/kg/day (Ruddick et al. 1983). In a preliminary dose- finding study, decreased fetal weight and increased resorptions were observed in rats exposed to 316 mg/kg/day chloroform during gestation (Thompson et al. 1974). In the principal study, reduced birth weight of the offspring was reported in the 126 mg/kg/day group; no effects were observed in the 50 mg/kg/day exposure group. Similarly, increased resorptions were observed in rabbits exposed to 100 mg/kg/day during gestation. No behavioral effects were observed in the offspring of the F, generation mice treated for 6-10 weeks with 31.1 mg/kg chloroform (Burkhalter and Balster 1979). In a 2-generation reproductive study, increased epididymal weights and degeneration of epididymal ductal epithelium were observed in mice in the F, generation dosed with 41 mg/kg/day in oil (Gulati et al. 1988). The production and viability of sperm was not affected, however. The highest NOAEL values and all reliable LOAEL values for developmental effects in each species and duration category are recorded in Table 2-2 and plotted in Figure 2-2. 2.2.2.7 Genotoxic Effects No studies were located regarding genotoxic effects in humans after oral exposure to chloroform. Unscheduled DNA synthesis (UDS) in hepatocytes was not increased in rats exposed to chloroform at gavage doses >400 mg/kg in oil (Mirsalis et al. 1982). Exposure to 200 mg/kg/day chloroform in oil by gavage for 4 days increased sister chromatid exchange frequency in bone marrow cells of mice (Morimoto and Koizumi 1983). Other genotoxicity studies are discussed in Section 2.5. 2.2.2.8 Cancer Epidemiology studies suggest an association between cancer in humans and consumption of chlorinated drinking water, but the results are not conclusive (Alavanja et al. 1978; Cantor et al. 1978; Young et al. 1981). Such an association implicates chloroform because chloroform is a known animal carcinogen (see below) and the predominant trihalomethane in chlorinated drinking water (see Chapter 5); however, it is important to note that some of the many chemicals produced in the process of water chlorination are highly mutagenic and carcinogenic. Although attempts were made to control for various demographic variables in all of these studies (e.g., social class, ethnic group, marital status, ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 76 2. HEALTH EFFECTS occupation, urban or rural, etc.), many confounding effects remained unaccounted for, most notably the likelihood that numerous chemicals other than chloroform were present in the drinking water, as stated above. Furthermore, the studies differed regarding the type of cancer associated with consumption of chlorinated water. Bladder cancer was reported to have the strongest association with chlorinated water in one study (Cantor et al. 1978), but only colon cancer had an elevated odds ratio (3.6) in another study (Young et al. 1981). Overall, the human data are insufficient to support any conclusion regarding the carcinogenic potential of chloroform in humans. Chloroform is carcinogenic in animals after oral exposure at intermediate durations. An increased incidence of hepatomas was observed in mice exposed by gavage for 30 days to 594 mg/kg/day chloroform in oil, but not in mice exposed to 297 mg/kg/day (Eschenbrenner and Miller 1945a). An 8-week exposure to 1,800 mg/kg/day chloroform in oil by gavage did not induce lung tumors in mice (Stoner et al. 1986). Chloroform acted as a promoter rather than an initiator of preneoplastic foci in a rat liver bioassay (Deml and Oesterle 1985). In addition, no increase in tumors was found in mice exposed to 257 mg/kg/day chloroform in drinking water for 52 weeks (Klaunig et al. 1986). In addition, Reddy et al. (1992) dosed male rats with 14, 25, 52, and 98 mg/kg/day of chloroform in the drinking water for 12 weeks. The study conclusively showed that chloroform, at the doses administered and routes studied in the rat, reduced the number of pre-neoplastic enzyme-altered foci (gamma-glutamyltranspeptidase-positive and glutathione S-transferase-positive) in the liver of male rats after induction of foci with diethylnitrosamine in a dose-related fashion. The exact mechanism behind this effect was not determined. Chloroform was found to be carcinogenic in several chronic animal studies of oral exposure. A chronic study performed by Dunnick and Melnick (1993) demonstrated the incidence of liver and kidney tumors in male rats given 0, 90, and 180 mg/kg/day and in female rats given 0, 100, and 200 mg/kg/day via gavage, for 5 days a week for 78 weeks and sacrificed at 111 weeks after dosing. No hepatocellular or large intestine neoplasms were noted for male or female rats. Kidney tubular cell neoplasms did not occur in controls, but neoplasms were observed at 90 mg/kg/day (4/50) and at 180 mg/kg/day (12/50) in males. Kidney tubular cell neoplasms did not occur in controls or at 100 mg/kg/day, but did occur at 200 mg/kg/day (2/48) in females. In a similar study using male mice dosed at 0, 138, and 277 mg/kg/day and female mice dosed with 0, 238, and 477 mg/kg/day (both sexes dosed for 5 days per week for 78 weeks), no kidney tubular cell ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 77 2. HEALTH EFFECTS neoplasms or large intestine neoplasms were reported in either sex of mice (Dunnick and Melnick 1993). Hepatocellular neoplasms were recorded as 1 of 18 in control mice, 18 of 50 in the 138 mg/kg/day dose group, and 44 of 45 mice in the 277 mg/kg/day dose group in males. Hepatocellular neoplasms were recorded as 0 of 20 in control mice, 36 of 45 in the 238 mg/kg/day dose group, and 39 of 41 mice in the 477 mg/kg/day dose group in females. The incidence of hepatic neoplastic nodules was increased in female Wistar rats chronically exposed to 200 mg/kg/day chloroform in drinking water, and lymphosarcoma was increased in males (Tumasonis et al. 1987). An increased incidence of tubular cell adenoma and carcinoma was observed in the kidneys of Osborne-Mendel rats chronically exposed to 160 mg/kg/day chloroform in drinking water, but not in those exposed to 81 mg/kg/day (Jorgenson et al. 1985). Similarly, renal tumors (tubular ceil adenoma and carcinoma) were observed in male Osborne-Mendel rats after a 78-week exposure to 90 mg/kg/day chloroform by gavage (NCI 1976). In contrast, no increase in the incidence of tumors was observed in Sprague-Dawley rats exposed by gavage to 60 and 165 mg/kg/day chloroform in toothpaste for 80 and 52 weeks, respectively (Palmer et al. 1979). Hepatocellular carcinoma was observed in all groups of B6C3F, mice exposed to gavage doses 2138 mg/kg/day chloroform in oil for 78 weeks (NCI 1976). An increased incidence of kidney tumors was observed in ICI mice chronically exposed to 60 mg/kg/day chloroform by gavage, but not in those exposed to 17 mg/kg/day (Roe et al. 1979). Under the same experimental conditions, chloroform exposure had no effect on the frequency of tumors in C57Bl, CBA, and CF-1 mice. Moreover, no increase in tumor incidence was observed in B6C3F, mice exposed to 263 mg/kg/day chloroform in drinking water for 2 years (Jorgenson et al. 1985). Cancer was not observed in dogs exposed to 30 mg/kg/day chloroform in toothpaste capsules for 7.5 years (Heywood et al. 1979). The CELs (cancer effect levels) are recorded in Table 2-2 and plotted in Figure 2-2. EPA (IRIS 1995) selected the study by Jorgenson et al. (1985) as the basis for the q,” for oral exposure to chloroform because administration via drinking water better approximates oral exposure in humans than does administration in corn oil by gavage as used in the NCI (1976) study. Based on the incidence of renal tumors in male Osborne-Mendel rats, the q,” was calculated to be 6.1x10° (mg/kg/day). The oral doses associated with individual lifetime upper-bound risks of 10° to 107 are 1.6x107 to 1.6x10° mg/kg/day, respectively, and are plotted in Figure 2-2. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 78 2. HEALTH EFFECTS 2.2.3 Dermal Exposure 2.2.3.1 Death No studies were located regarding death in humans after dermal exposure to chloroform. No deaths resulted from dermal exposure of rabbits exposed to doses of up to 3,980 mg/kg chloroform for 24 hours (Torkelson et al. 1976). 2.2.3.2 Systemic Effects No studies were located regarding respiratory, cardiovascular, gastrointestinal, hematological, musculoskeletal, or ocular effects in humans or animals after dermal exposure to chloroform. Hepatic Effects. No studies were located regarding hepatic effects in humans after dermal exposure to chloroform. No hepatic effects were observed in rabbits when 3,980 mg/kg chloroform was applied to the abdominal skin for 24 hours (Torkelson et al. 1976). The NOAEL for hepatic effects is recorded in Table 2-3. Renal Effects. No studies were located regarding renal effects in humans after dermal exposure to chloroform. Renal degenerative tubular changes were observed in rabbits when 1,000 mg/kg chloroform was applied to the abdominal skin for 24 hours (Table 2-3) (Torkelson et al. 1976). Dermal Effects. Completely destroyed stratum corneum was observed in the skin of two young volunteers exposed to chloroform for 15 minutes on 6 consecutive days (Malten et al. 1968). Milder changes were observed in two older individuals. Chloroform was applied in a glass cylinder (exact exposure was not specified). ***DRAFT FOR PUBLIC COMMENT*** «x LNIWWOO 01718Nd HO4 14VvHd... TABLE 2-3. Levels of Significant Exposure to Chloroform - Dermal Exposure/ LOAEL Duration/ Species/ Frequency/ NOAEL Less Serlous Serious Ref (Strain) (specific Route) >Y*'™ (mg/kg) (mg/kg) (mg/kg) eference ACUTE EXPOSURE Systemic Rabbit 24 hr Dermal 0.01mL M (slight skin irritation) Smyth et al. 1962 (New Zealand) Rabbit 24 hr Hepatic 3980 Torkelson et al. (NS) 1976 Renal 1000 (degenerative tubular changes) Derm 1000 (necrosis) Bd Wt 1000 (weight loss) hr = hour(s); LOAEL = lowest-observed-adverse-effect-level; NOAEL = no-observed-adverse-effect-level; NS = not specified S103443 H1TV3H 2 WHO304OTHO 6L CHLOROFORM 80 2. HEALTH EFFECTS A clinical study of 21 females and 21 males used to determine the efficacy of using aspirin dissolved in chloroform which was then applied topically to patients infected with herpes zoster and post- therapeutic neuralgia with painful skin lesions has been reported. When an aspirin/chloroform combination (approximately 43.3 mg/mL) was applied to all painful lesions, patients reported a diminishment of pain at the affected application site(s) within 1-5 minutes after application. Maximum relief was achieved about 20-30 minutes after application and lasted 2-4 hours for most patients. Aspirin dissolved in water and chloroform application alone failed to produce pain relief. The only reported side-effect was an occasional burning sensation on the skin as the chloroform evaporated from the skin surface; however, the possible impact on other major body organs (liver, kidney, etc.) was not investigated (King 1993). Application of 0.01 mL chloroform for 24 hours to the skin of rabbits caused only slight irritation (Smyth et al. 1962). Skin necrosis was observed in rabbits dermally exposed to 1,000 mg/kg chloroform for 24 hours (Torkelson et al. 1976). These LOAEL values are recorded in Table 2-3. Body Weight Effects. No studies were located regarding body weight effects in humans after dermal exposure to chloroform. Dermal exposure to 1,000 mg/kg chloroform for 24 hours caused weight loss in rabbits (Table 2-3) (Torkelson et al. 1976). No studies were located regarding the following health effects in humans or animals after dermal exposure to chloroform: 2.2.3.3 Immunological and Lymphoreticular Effects 2.2.3.4 Neurological Effects 2.2.3.5 Reproductive Effects 2.2.3.6 Developmental Effects ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 81 2. HEALTH EFFECTS 2.2.3.7 Genotoxic Effects Genotoxicity studies are discussed in Section 2.5. 2.2.3.8 Cancer No studies were located regarding cancer in humans or animals after dermal exposure to chloroform. 2.3 TOXICOKINETICS Overview Sufficient information exists on the absorption, distribution, metabolism, and excretion of chloroform, with most information on the pharmacokinetics being derived from animal data. Generally, chloroform is absorbed easily into the blood from the lungs after inhalation exposures. Following oral exposure, peak blood levels are achieved within 5-6 minutes, depending on the dosing vehicle and dosing frequency used. The chemical properties of chloroform also permit percutaneous absorption without difficulty. After absorption, chloroform has been reported to distribute to adipose tissues, brain, liver, kidneys, blood, adrenals, and embryonic neural tissues. Higher levels of chloroform can be found in the renal cortex of male animals than in female animals, a finding apparently mediated by the presence of testosterone. Approximately 50% of a dose of chloroform is eventually metabolized to carbon dioxide in humans; however, an intermediate toxic metabolite, phosgene, is formed in the process in the liver. Chloroform undergoes metabolism primarily in the liver and may undergo covalent binding to both lipid and microsomal protein. Chloroform is excreted from the body either unchanged by pulmonary desorption or in the form of carbon dioxide, with small amounts of either detectable in the urine and feces. 2.3.1 Absorption 2.3.1.1 Inhalation Exposure Chloroform absorption depends on the concentration in inhaled air, the duration of exposure, the blood/air partition coefficient, the solubility in various tissues, and the state of physical activity which ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 82 2. HEALTH EFFECTS influences the ventilation rate and cardiac output. Pulmonary absorption of chloroform is also influenced by total body weight and total fat content, with uptake and storage in adipose tissue increasing with excess body weight and obesity. In inhalation exposures, the arterial blood concentration of chloroform is directly proportional to the concentration in inhaled air. At anesthetic concentrations (8,000-10,000 ppm), steady-state arterial blood concentrations of chloroform were 0.07-0.165 mg/mL (Smith et al. 1973). Total body equilibrium with inspired chloroform concentration required at least two hours in normal humans at resting ventilation and cardiac output (Smith et al. 1973). The amount of chloroform absorbed and exhaled from the body in alveolar air from male and female swimmers in indoor swimming pools in Italy was measured by Aggazzotti et al. (1993). Alveolar air samples were collected from both swimmers and observers present in indoor chlorinated swimming pools. Of all the nonexposed subjects, 47% had chloroform concentrations below the detection limit of the assay, and the remainder of this control group had low concentrations (75.39 nmol/m’) of chloroform present in their alveolar air. Of all persons in the indoor swimming pools (swimmers and observers), alveolar concentrations of chloroform were significantly higher (median=695.02 nmol/m?). No differences were found between males and females in any exposure group. No studies were located regarding absorption in animals after inhalation exposure to chloroform. Evidence that chloroform is absorbed after inhalation exposure is provided in toxicity studies (see Section 2.2.1), but the rate and extent cannot be determined from the toxicity data. 2.3.1.2 Oral Exposure A case report of a 33-year-old female (weight not reported) who had injected herself intravenously with 0.5 mL of chloroform, became unconscious, and when she awoke approximately 12 hours later, drank another 120 mL of chloroform. Plasma chloroform levels were determined 18 hours after ingestion by gas chromatography and showed a blood chloroform level of 0.66 mg/dL. Subsequent serum samples were analyzed for chloroform content and were reported to have been less than this level, steadily declining over time (Rao et al. 1993). ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 83 2. HEALTH EFFECTS Absorption of an oral dose of “C-labeled chloroform (0.5 grams in a gelatin capsule) was rapid in volunteers, reaching peak blood levels in 1 hour (Fry et al. 1972). Almost 100% of the dose was absorbed from the gastrointestinal tract. Experiments in mice, rats, and monkeys indicate that oral doses (60 mg/kg) of “C-labeled chloroform in olive oil were almost completely absorbed as indicated by a 93-98% recovery of radioactivity in expired air, urine, and carcass (Brown et al. 1974a; Taylor et al. 1974). Absorption in mice and monkeys was rapid; the peak blood levels were reached 1 hour after oral administration of 60 mg/kg chloroform in olive oil. Intestinal absorption of chloroform in either water or corn oil administered intragastrically to rats was rapid with both vehicles, but the rate and extent of absorption varied greatly (Withey et al. 1983). The peak concentration of chloroform in blood was 39.3 pg/mL when administered in water and 5.9 pg/mL when administered in corn oil. The greater degree of absorption following administration in water can be explained by the faster partitioning of a lipophilic compound such as chloroform with mucosal lipids from an aqueous vehicle. Peak blood concentrations were reached somewhat more rapidly with the water vehicle (5.6 minutes as opposed to 6.0 minutes for corn oil). The uptake from a corn oil solution was more complex (pulsed) than from aqueous solution. A possible explanation for this behavior is that the chloroform in corn oil was broken up into immiscible globules, some of which did not come into contact with the gastric mucosa. Another possible explanation was that intragastric motility may have separated the doses into aliquots that were differentially absorbed from the gastrointestinal tract. The differences in chloroform toxicity based on the vehicle has been recently reported elsewhere (Larson et al. 1994b, 1995) 2.3.1.3 Dermal Exposure No experimental studies were located regarding dermal absorption of chloroform in humans. According to dermal absorption studies with solvents other than chloroform, the absorption of such solvents in guinea pigs is more rapid than the metabolism or pulmonary excretion (Jakobson et al. 1982). A dermal absorption rate of 329 nmol/minute/cm? (+ 60 nmol/minute/cm) was calculated for the shaved abdominal skin of mice (Tsuruta 1975). This is equivalent to a human absorption rate of 29.7 mg/minute, assuming that a pair of hands are immersed in liquid chloroform (Tsuruta 1975). The ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 84 2. HEALTH EFFECTS calculation was based on the assumptions that the rate of chloroform penetration is uniform for all kinds of skin and that the total surface area of a pair of human hands is 800 cm’. 2.3.2 Distribution 2.3.2.1 Inhalation Exposure Chloroform is lipid soluble and readily passes through cell membranes, causing narcosis at high concentrations. Blood chloroform concentrations during anesthesia (presumed concentrations 8,000-10,000 ppm) were 0.07-0.165 mg/mL in 10 patients (Smith et al. 1973). An arterial chloroform concentration of 0.24 mg/mL during anesthesia corresponded to the following partition coefficients: blood/gas = 8, blood/vessel rich compartment = 1.9, blood/muscle compartment = 1.9, blood/fat compartment = 31, blood/vessel poor compartment = 1, and blood/liver = 2 (Feingold and Holaday 1977). Recently, partition coefficients were calculated based on results in mice and rats, and in human tissues in vitro for humans: blood/air = 7.4, liver/air = 17.0, kidney/air = 11, and fat/air = 280 (Corley et al. 1990). The chloroform levels in 7 patients who died after excessive administration during anesthesia were: brain, 372-480 mg/kg; lungs, 355-485 mg/kg; and liver, 190-275 mg/kg (Gettler and Blume 1931). The chloroform levels in patients under anesthesia who died from other causes were: brain, 120-182 mg/kg; lungs, 92-145 mg/kg; and liver, 65-88 mg/kg tissue wet weight. After whole-body autoradiography to study the distribution of “C-labeled chloroform in mice, most of the radioactivity was found in fat immediately after exposure, while the concentration of radioactivity in the liver increased during the postanesthetic period, most likely due to covalent binding to lipid and protein in the liver (Cohen and Hood 1969). Partition coefficients (tissue/air) for mice and rats were 21.3 and 20.8 for blood; 19.1 and 21.1 for liver; 11.0 and 11.0 for kidney; and 242 and 203 for fat, respectively (Corley et al. 1990). Arterial levels of chloroform in mongrel dogs reached 0.35-0.40 mg/mL by the time animals were in deep anesthesia (Chenoweth et al. 1962). Chloroform concentrations in the inhaled stream were not measured, however. After 2.5 hours of deep anesthesia, there were 392 mg/kg chloroform in brain tissue, 1,305 mg/kg in adrenals, 2,820 mg/kg in omental fat, and 290 mg/kg in the liver. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 85 2. HEALTH EFFECTS Radioactivity from “C-labeled chloroform was detected in the placenta and fetuses of mice immediately after inhalation exposure (Danielsson et al. 1986). In early gestation, accumulation of radioactivity was observed in the embryonic neural tissues, while the respiratory epithelium was more involved in chloroform metabolism in the late fetal period. Due to its lipophilic character, chloroform accumulates to a greater extent in tissues of high lipid content. As shown by the results presented above, the relative concentrations of chloroform in various tissues decreased as follows: adipose tissue > brain > liver > kidney > blood. 2.3.2.2 Oral Exposure No studies were located regarding distribution in humans after oral exposure to chloroform. High concentrations of radioactivity were observed in body fat and livers of rats, mice, and squirrel monkeys given oral doses of 60 mg/kg “C-labeled chloroform (Brown et al. 1974a). The maximum levels of radioactivity in the blood appeared within 1 hour and were 3 ug equivalents chloroform/mL for mice and 10 pg equivalents chloroform/mL for monkeys, which represented =0.35% and 1%, respectively, of the total radioactivity. In monkeys, bile concentrations peaked within 6 hours. The distribution of radioactively labeled chloroform was studied in three strains of mice (Taylor et al. 1974). No strain-related differences were observed; however, higher levels of radioactivity were found in the renal cortex of males and in the liver of females. The renal binding of radioactive metabolites may have been altered by variations in the testosterone levels as a result of hormonal pretreatment in females or castration in males. Sex-linked differences in chloroform distribution were not observed in rats or monkeys (Brown et al. 1974a). Chloroform accumulates in the adipose tissue of rats after oral exposure of intermediate duration (Pfaffenberger et al. 1980). 2.3.2.3 Dermal Exposure No studies were located regarding distribution in humans or animals after dermal exposure to chloroform. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 86 2. HEALTH EFFECTS 2.3.3 Metabolism The metabolism of chloroform is well understood. Approximately 50% of an oral dose of 0.5 grams of chloroform was metabolized to carbon dioxide in humans (Fry et al. 1972). Metabolism was dose- dependent, decreasing with higher exposure. A first pass effect was observed after oral exposure (Chiou 1975). Approximately 38% of the dose was converted in the liver, and <17% was exhaled unchanged from the lungs before reaching the systemic circulation. On the basis of pharmacokinetic results obtained in rats and mice exposed to chloroform by inhalation, and of enzymatic studies in human tissues in vitro, in vivo metabolic rate constants (VC = 15.7 mg/hour/kg, K,, = 0.448 mg/L) were defined for humans (Corley et al. 1990). The metabolic activation of chloroform to its toxic intermediate, phosgene, was slower in humans than in rodents. Metabolic pathways of chloroform biotransformation are shown in Figure 2-3. Metabolism studies indicated that chloroform was, in part, exhaled from the lungs or was converted by oxidative dehydrochlorination of its carbon-hydrogen bond to form phosgene (Pohl et al. 1981; Stevens and Anders 1981). This reaction was mediated by cytochrome P-450 and was observed in the liver and kidneys (Branchflower et al. 1984; Smith et al. 1984). Phosgene may react with two molecules of glutathione (GSH) to form diglutathionyl dithiocarbonate, which is further metabolized in the kidneys, or it may react with other cellular elements and induce cytotoxicity (Pohl and Gillette 1984). [In vitro studies indicate that phosgene and other reactive chloroform metabolites bind to lipids and proteins of the endoplasmatic reticulum proximate to the cytochrome P-450 (Sipes et al. 1977; Wolf et al. 1977). The metabolism of chloroform to reactive metabolites occurs not only in microsomes but alsé in nuclear preparations (Gomez and Castro 1980). Covalent binding of chloroform to lipids can occur under anaerobic and aerobic conditions, while binding to the protein occurs only under aerobic conditions (Testai et al. 1987). It was further demonstrated that chloroform can induce lipid peroxidation and inactivation of cytochrome P-450 in rat liver microsomes under anaerobic conditions (De Groot and Noll 1989). This mechanism may also contribute to chloroform-induced hepatotoxicity in rats, although phosgene and other active metabolites are primarily responsible. Covalent binding of chloroform metabolites to microsomal protein in vitro was intensified by microsomal enzyme inducers and prevented by glutathione (Brown et al. 1974b). It was proposed that the reaction of chloroform metabolites with glutathione may act as a detoxifying mechanism. When glutathione is depleted, however, the metabolites react with microsomal protein, and may cause necrosis. This is supported by observations that chloroform doses that caused liver glutathione depletion produced liver necrosis ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 87 2. HEALTH EFFECTS Figure 2-3. Metabolic Pathways of Chloroform Biotransformation MAJOR AEROBIC PATHWAY P450 Q H-CCl, —» [HOCCl)) NADPH Microsomes HCl ACCEPTOR PROTEIN | HO CO «= 0=CCl, ————» 2HCl+CO, _ PHOSGENE <5 ot — CO.H Ee Ss, CELLULAR & < i, Ch PROTEIN BINDING s NH 2-OXOTHIAZOLIDINE GLUTATHIONE NL oe 4-CARBOXYLIC ACID CONJUGATES? " o MINOR ANAEROBIC PATHWAY PHOSPHOLIPID BINDING A | CHI, | P450 ANAEROBIC ™ | NADPH CHCl, > BCHCI] REDUCED MICROSOMES | HO CO + 2HCI ***DRAFT FOR PUBLIC COMMENT"** CHLOROFORM 88 2. HEALTH EFFECTS (Docks and Krishna 1976). Furthermore, chloroform has been found to be more hepatotoxic in fasted animals, possibly due to decreased glutathione content in the liver (Brown et al. 1974b: Docks and Krishna 1976). This may explain the clinical finding of severe acute hepatotoxicity in women exposed to chloroform via anesthesia during prolonged parturition. Evidence that chloroform is metabolized at its carbon-hydrogen bond is provided by experiments using the deuterated derivative of chloroform (Branchflower et al. 1984; McCarty et al. 1979; Pohl et al. 1980a). Deuterated chloroform was one- half to one-third as cytotoxic as chloroform, and its conversion to phosgene was much slower. The results confirmed that the toxicity of chloroform is primarily due to its metabolites. A recent in vitro study of mice hepatic microsomes indicated that a reductive pathway may also play an important role in chloroform hepatotoxicity (Testai et al. 1990). It was demonstrated that radical chloroform metabolites bind to macromolecules (proteins, lipids) and the process can be inhibited by reduced glutathione. The final product of the aerobic metabolic pathway of chloroform is carbon dioxide (Brown et al. 1974a; Fry et al. 1972). This carbon dioxide is mostly eliminated through the lungs, but some is incorporated into endogenous metabolites and excreted as bicarbonate, urea, methionine, and other amino acids (Brown et al. 1974a). Chloride ions are an end product of chloroform metabolism found in the urine (Van Dyke et al. 1964). Carbon monoxide was a minor product of the anaerobic metabolism of chloroform in vitro (Ahmed et al. 1977) and in vivo in rats (Anders et al. 1978). A sex-related difference in chloroform metabolism was observed in mice (Taylor et al. 1974). Chloroform accumulated and metabolized in the renal cortex of males to a greater extent than in females; the results may have been influenced by testosterone levels. This effect was not observed in any other species. Interspecies differences in the rate of chloroform conversion were observed in mice, rats, and squirrel monkeys, with species differences in metabolism being highly dose dependant. The conversion of chloroform to carbon dioxide was highest in mice (85%) and lowest in squirrel monkeys (28%) (Brown et al. 1974a). Similarly, chloroform metabolism was calculated to be slower in humans than in rodents. Therefore, it was estimated that the exposure to equivalent concentrations of chloroform would lead to a much lower delivered dose in humans (Corley et al. 1990). ***DRAFT FOR PUBLIC COMMENT *** CHLOROFORM 89 2. HEALTH EFFECTS A study by Gearhart et al. (1993) was conducted to determine the interactions of chloroform exposure with body temperature, gas uptake, and tissue solubility in mice as possible explanations for the difficulty in fitting a PBPK model to chloroform gas uptake data to derive in vivo metabolic constants. Male mice were exposed to air concentrations of 100, 800, 2,000, or 5,500 ppm chloroform for 6 hours and their core body temperatures monitored frequently over the exposure period. After exposure, blood, liver, thigh muscle. and fat tissues were removed for tissue/air and tissue/blood partition coefficient analysis at 3 temperatures (25, 31, and 37 °C). For all tissues, tissue/air partition coefficients exhibited temperature-dependent decreases with increasing temperature. The rate of decrease was greatest for the blood/air partition coefficient. Average body temperatures for each exposure group decreased as the exposure concentrations increased. Temperature dependent decreases in core body temperature were hypothesized to decrease overall metabolism of chloroform in mice. The data collected were also used to develop a PBPK model for chloroform disposition. 2.3.4 Elimination and Excretion 2.3.4.1 Inhalation Exposure Chloroform was detected in the exhaled air of volunteers exposed to a normal environment, to heavy automobile traffic, or to air in a dry cleaning establishment (Gordon et al. 1988). Higher chloroform levels in the breath corresponded to higher exposure levels. The calculated biological half-time for chloroform was 7.9 hours. Excretion of radioactivity in mice and rats was monitored for 48 hours following exposure to 14C_labeled chloroform (Corley et al. 1990). In general, 92-99% of the total radioactivity was recovered in mice, and 58-98% was recovered in rats; percent recovery decreased with increasing exposure. With increasing concentration, mice exhaled 80-85% of the total radioactivity recovered as 14C_ labeled carbon dioxide, 0.4-8% as “C-labeled chloroform, and 8-11% and 0.6-1.4% as urinary and fecal metabolites, respectively. Rats exhaled 48-85% of the total radioactivity as C-labeled carbon dioxide, 242% as “C-labeled chloroform, and 811% and 0.1-0.6% in the urine and feces, respectively. A 4-fold increase in exposure concentration was followed by a 50- and 20-fold increase in the amount of exhaled, unmetabolized chloroform in mice and rats, respectively. ***DRAFT FOR PUBLIC COMMENT"** CHLOROFORM 90 2. HEALTH EFFECTS 2.3.4.2 Oral Exposure Following a single, oral exposure, most of the 0.5 grams of radioactively labeled chloroform administered to volunteers was exhaled during the first 8 hours after exposure (Fry et al. 1972). A slower rate of pulmonary excretion was observed during the first 8 hours in volunteers who had more adipose tissue than the other volunteers. Up to 68.3% of the dose was excreted unchanged, and up to 50.6% was excreted as carbon dioxide. A positive correlation was made between pulmonary excretion and blood concentration. Less than 1% of the radioactivity was detected in the urine. Approximately 80% of a single dose of 60 mg/kg '“C-labeled chloroform was converted within 24 hours to “C-labeled carbon dioxide in mice (Brown et al. 1974a: Taylor et al. 1974), while only =~66% of the dose was converted to “C-labeled carbon dioxide in rats (Brown et al. 1974a). Eight hours after administration of 100-150 mg/kg of “C-labeled chloroform, 49.6% and 6.5% of radioactivity was converted to carbon dioxide, 26.1% and 64.8% was expired as unmetabolized parent compound, and 4.9% and 3.6% was detected in the urine in mice and rats, respectively (Mink et al. 1986). These results indicate that mice metabolize high doses of chloroform to a greater degree than rats do. Only 18% of a chloroform dose was metabolized to “C-labeled carbon dioxide in monkeys, and =79% was detected as unchanged parent compound or toluene soluble metabolites (Brown et al. 1974a). Within 48 hours after exposure, =2%, 8%, and 3% of the administered radioactivity was detected in the urine and feces of monkeys, rats, and mice, respectively. 2.3.4.3 Dermal Exposure No studies were located regarding excretion in humans or animals after dermal exposure to chloroform. 2.3.5 Physiologically Based Pharmacokinetic (PBPK)/Pharmacodynamic (PD) Models Physiologically based pharmacokinetic (PBPK) models use mathematical descriptions of the uptake and disposition of chemical substances to quantitatively describe the relationships among critical biological processes (Krishnan et al. 1994). PBPK models are also called biologically based tissue dosimetry models. PBPK models are increasingly used in risk assessments, primarily to predict the concentration of potentially toxic moieties of a chemical that will be delivered to any given target “**DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 91 2. HEALTH EFFECTS tissue following various combinations of route, dose level, and test species (Clewell and Andersen 1985). Physiologically based pharmacodynamic (PBPD) models use mathematical descriptions of the dose-response function to quantitatively describe the relationship between target tissue dose and toxic end points. PBPK/PD models refine our understanding of complex quantitative dose behaviors by helping to delineate and characterize the relationships between: (1) the external/exposure concentration and target tissue dose of the toxic moiety, and (2) the target tissue dose and observed responses (Andersen et al. 1987; Andersen and Krishnan 1994). These models are biologically and mechanistically based and can be used to extrapolate the pharmacokinetic behavior of chemical substances from high to low dose, from route to route, between species, and between subpopulations within a species. The biologically basis of PBPK models results in more meaningful extrapolations than those generated with the more conventional use of uncertainty factors. The PBPK model for a chemical substance is developed in four interconnected steps: (1) model representation, (2) model parametrization, (3) model simulation, and (4) model validation (Krishnan and Andersen 1994). In the early 1990s, validated PBPK models were developed for a number of toxicologically important chemical substances, both volatile and nonvolatile (Krishnan and Andersen 1994; Leung 1993). PBPK models for a particular chemical substance require estimates of the chemical substance-specific physicochemical parameters, and species-specific physiological and biological parameters. The numerical estimates of these model parameters are incorporated within a set of differential and algebraic equations that describe the pharmacokinetic processes. Solving these differential and algebraic equations provides the predictions of tissue dose. Computers then provide process simulations based on these solutions. The structure and mathematical expressions used in PBPK models significantly simplify the true complexities of biological systems. This simplification, however, is desirable if the uptake and disposition of the chemical substance(s) is adequately described because data are often unavailable for many biological processes and using a simplified scheme reduces the magnitude of cumulative uncertainty. The adequacy of the model is therefore of great importance and thus the importance of model validation. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 92 2. HEALTH EFFECTS PBPK models improve the pharmacokinetic extrapolation aspects of the risk assessment process, which seeks to identify the maximal (i.e., safe) levels for human exposure to chemical substances (Andersen and Krishnan 1994). PBPK models provide a scientifically-sound means to predict the target tissue dose of chemicals in humans who are exposed to environmental levels (for example, levels that might occur at hazardous waste sites) based upon the results of studies where doses were higher or were administered in different species. Figure 2-4 shows a conceptualized representation of a PBPK model. If PBPK models for chloroform exist, the overall results and individual models are discussed in this section in terms of their use in risk assessment, tissue dosimetry, and dose, route, and species extrapolations. 2.3.5.1 Summary of PBPK/PD Models Several rodent and human models have been used to predict the absorption (oral, inhalation, and dermal) from water and air, distribution, metabolism, and excretion of chloroform. In a PBPK model that used simulations with mice, rats, and humans (Corley et al. 1990), the tissue delivered dose from equivalent concentrations of chloroform was highest in the mouse, followed by rats and then humans. The authors suggest that this behavior is predicted by the model because of the lower relative rates of metabolism, ventilation, and cardiac output (per kg of body weight) in the larger species. Assuming that equivalent target doses produce equivalent toxicities in target tissues, the relative sensitivities of the three species used in the study (mouse > rat > human) predicted by the model under identical exposure conditions are quite different from the relative sensitivity to chloroform assumed by the "uncertainty factor." In a PBPK/PD model based closely on the Corley model, Reitz et al. (1990) described a pharmacodynamic end point (cytotoxicity) in the livers of chloroform-exposed animals produced by phosgene, the reactive metabolite of chloroform. The Rietz model estimated that higher concentrations of chloroform than the proposed EPA estimates could be tolerated in the air and drinking water of humans without a significant increase in the incidence of liver carcinogenesis. In gas uptake experiments, Gearhart et al. (1993) demonstrated a dose-dependent decrease in core body temperature with increased inhaled concentrations of chloroform. The decrease in body temperature ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 93 2. HEALTH EFFECTS Figure 2-4. Conceptual Representation of a Physiologically Based Pharmacokinetic (PBPK) Model for a Hypothetical Chemical Substance Inhaled chemical ncozm< O0O0Orw ----=* Exhaled chemical les ion wn JY Urine Ingestion Lungs : Liver « ) Ll] A Vmax Km Gl |] T Tract £ Fat < B A Slowly L perfused tissues : B Richly ’ L perfused 0 tissues 0 D Kidney « Skin A + ----Chemical in air Source: adapted from Krisnan et al. 1992 contacting skin Note: This is a conceptual representation of a physiologically based pharmacokinetic (PBPK) model for a hypothetical chemical substance. The chemical substance is shown to be absorbed via the skin, by inhalation, or by ingestion, metabolized in the liver, and excreted in the urine or by exhalation. ***DRAFT FOR PUBLIC COMMENT"** CHLOROFORM 94 2. HEALTH EFFECTS could account for decreased in vivo chloroform metabolism, partition coefficients, pulmonary ventilation, and cardiac output rates in mice. Chinery and Gleason (1993) used a shower model for chloroform-contaminated water to predict breath concentration (as a quantifiable function of tissue dose) and actual absorbed dose from a measured water supply concentration following exposure while showering. The model's predictions demonstrated that dose information based only on dermal absorption (without considering an inhalation component) may underestimate actual dose to target organs in dosimetric assessment for chloroform in water supplies during shower. The model also predicted a steady-state stratum corneum permeability of chloroform in human skin in the range of 0.16-3.6 cm/hour with the most likely value being 0.2 c/hour. The authors suggest that the results predicted by this model could be used to estimate household exposures to chloroform or other exposures which include dermal absorption. Another chloroform shower model (McKone 1993) demonstrated that chloroform in shower water had an average effective dermal permeability between 0.16 and 0.42 cm/hour for a 10-minute shower. The model predicted that the ratio of chloroform dermally absorbed in the shower (relative to chloroform- contaminated water concentration) ranged between 0.25 and 0.66 mg per mg/L. In addition, the McKone model demonstrated that chloroform metabolism by the liver was not linear across all dermal/ inhalation exposure concentrations and became nonlinear at higher (60-100 mg/L) dose concentrations. 2.3.5.2 Chloroform PBPK Model Comparison Five chloroform PBPK models that describe the disposition of chloroform in animals and humans have been identified from the recent open literature (early 19805-1994). Based on the information presented in these five models, there appears to be sufficient evidence to suggest that PBPK models for chloroform are fairly refined and have a strong potential for use in human risk assessments. The PBPK model developed by Corley et al. (1990) has provided a basic model for the fate of chloroform in humans and laboratory animals. Using this model as a template, other more sophisticated and refined models have been developed that can be used in human risk assessment work. The models of Corley et al. (1990) and Reitz et al. (1990) have described several aspects of chloroform metabolism and disposition in laboratory animals and humans; however, they do not address the dermal routes of exposure. The models of McKone (1993) and Chinery and Gleason (1993) address both the inhalation and dermal exposure routes in humans The Chinery and Gleason model uses a three compartment skin ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 95 2. HEALTH EFFECTS component which may more accurately reflect the flux of chloroform through the skin after dermal only or dermal plus inhalation exposure scenarios, while the McKone model uses a single compartment within the skin to describe chloroform flux. Further discussion of each model and its application in human risk assessments is presented below. 2.3.5.3 Discussion of Chloroform Models The Corley Model The Corley model (Corley et al. 1990) was the first chloroform PBPK model to describe and ultimately predict the fate of chloroform in several species (including humans) under a variety of exposure conditions. Many subsequent PBPK models for chloroform (Chinery and Gleason 1993; McKone 1993) are based on the Corley model. The Corley model has been used for cancer risk assessment (Reitz et al. 1990). Risk Assessment. This model successfully described the disposition of chloroform in rats, mice and humans following various exposure scenarios and developed dose surrogates more closely related to toxicity response. With regard to target tissue dosimetry, the Corley model predicts the relative order of susceptibility to chloroform toxicity consequent to binding to macromolecules (MMB) to be mouse > rat > human. Linking the pharmacokinetic parameters of this model to the pharmacodynamic cancer model of Reitz et al. (1990) provides a biologically based risk assessment model for chloroform. Description of the Model. The Corley chloroform PBPK model was based on an earlier PBPK model developed by Ramsey and Andersen (1984) to describe the disposition of styrene exposure in rats, mice, and humans. A schematic representation of the Corley model (taken from Corley et al. 1990) is shown in Figure 2-5 with oral, inhalation, and intraperitoneal routes represented. The dermal route of exposure is not represented in this model; however, others have modified the Corley model to include this route of exposure (see below). Liver and kidney are represented as separate compartments since both are target organs for chloroform. The physiologic, biochemical constants and partition coefficients required for the model are shown in Table 2-4. Physiologic constants (organ weight, blood flows, etc) were similar to those used by ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 2. HEALTH EFFECTS Figure 2-5. Parameters Used in the Corley PBPK Model QQ » | ALVEOLARSPACE | — @ Ly CX \ J ( ~ —— SN A LUNG BLOOD CA \ J < QR QR CVR RAPIDLY PERFUSED + L J ( ~ 2 -B lf ee CVS SLOWLY PERFUSED 1 \ wy, QF QF 5 F— rs] CVE FAT | > i KIDNEY -— AMK Ki, VK Kiss RK rym L OL ka «QL wtf] CVL LIVER cx Ip k,S AML Ko Via, Kiosss Kresym Oral Physiological model used to describe the pharmacokinetics of chloroform in rats, mice, and humans during inhalation, oral, and intraperitoneal exposures. AMK = amount metabolized in kidney; AML = amount metabolized in liver ***DRAFT FOR PUBLIC COMMENT*** 96 CHLOROFORM 2. HEALTH EFFECTS 97 Table 2-4. Parameters Used in the Corley PBPK Model PARAMETERS MOUSE RAT HUMAN Weights (kg): Body 0.02858 0.230 70.0 Percentage of Body Weight Liver 5.86 2.53 3.14 Kidney 1.70 0.71 0.44 Fat 6.00 6.30 23.10 Rapidly perfused tissues 3.30 4.39 3.27 Slcwly perfused tissues 74.14 77.07 61.05 Flows (L/hr/kg) Alveolar ventilation 2.01 5.06 347.9 Cardiac output 2.01 5.06 347.9 Percentage of Cardiac Output Liver 25.0 25.0 25.0 Kidney 25.0 25.0 25.0 Fat 2.0 5.0 5.0 Rapidly perfused tissues 29.0 26.0 26.0 Slowly perfused tissues 19.0 19.0 19.0 Partition Coefficients Blood/air 21.3 20.8 7.43 Liver/air 19.1 21.4 17.0 Kidney/air 11.0 11.0 11.0 Fat/air 242 203 280 Rapidly perfused/air 19.1 21.1 17.0 Slowly perfused/air 13.0 13.9 12.0 Metabolic and macromolecular binding constants Vmax C (mg/hr/kg) 22.8 6.8 15.7 Kn, (mg/L) 0.352 0.543 0.448 Kioss (Mg) 5.72x10™ 0 0 Kresyn (N71) 0.125 0 0 A (kidney/liver) 0.153 0.052 0.033 fMMB (hr'"), liver 0.003 0.00104 0.00202 fMMB (hr), kidney 0.010 0.0086 0.00931 Gavage Absorption Rate Constants ks (hr'"), corn oil 0.6 0.6 0.6 Kos (hr), water 5.0 5.0 5.0 k, (hr!) Intraperitoneal Injection Absorption Rate Constant 1.0 1.0 1.0 ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 98 2. HEALTH EFFECTS Andersen et al. (1987) or were taken from other literature sources. Tissue and blood partition coefficients were determined in tissues by vial equilibration techniques in the rat and human, with extrapolated values used for the mouse. All metabolism of chloroform was assumed to occur only in the liver and kidneys through a single metabolic pathway (mixed function oxidase) that followed simple Michaelis-Menten kinetic parameters. Metabolic rate constants were obtained from the gas uptake experiments. Human metabolic rate constants were obtained from in vitro human microsomal fractions of liver and kidney samples using '““CHCI, as the substrate. Binding of chloroform metabolites (phosgene) to macromolecules was assumed to occur in bioactivating tissues (liver and kidney) in a non-enzymatic, nonspecific, and dose-independent fashion. Macromolecular binding constants for the liver and kidney were estimated from in vivo MMB data obtained from rats and mice exposed to "““CHCL, via inhalation. The gas uptake data for rats were well-described using a single Michaelis-Menten equation to describe metabolism. For the mouse inhalation studies, a simple Michaelis-Menten equation failed to adequately describe the chloroform-metabolizing capacity based on the data collected and model constants. The authors suspected that, following the administration of chloroform (particularly at higher concentrations), destruction of microsomal enzymes and subsequent resynthesis of microsomal enzymes was important in the mouse. This phenomenon has been documented in phenobarbital- induced but not naive rats. To account for this phenomenon, a first-order rate constant for the loss and subsequent regeneration of metabolic capacity was incorporated into the model for mice only. The model also provided a good description of the in vivo levels of MMB in both rats and mice, with good agreement between observed and predicted values. Validation of the Model. The Corley model was validated using chloroform data sets from oral (Brown et al. 1974a) and intraperitoneal (Ilett et al. 1973) routes of administration and from human pharmacokinetic studies (Fry et al. 1972). Metabolic rate constants obtained from the gas uptake experiments were validated by modeling the disposition of radiolabeled chloroform in mice and rats following inhalation of chloroform at much lower doses. For the oral data set, the model accurately predicted the total amounts of chloroform metabolized for both rats and mice. Target Tissues. The model provided excellent predictions of MMB in both the target tissues of chloroform (liver and kidney) after intraperitoneal administration in mice (rat data was not generated). ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 99 2. HEALTH EFFECTS The model adequately predicted the amount of unchanged material exhaled at infinite time and the total amount metabolized by groups of male and female humans of widely varying age and weight. Species Extrapolation. The Corley model used species-specific information to outline the model parameters; little extrapolation of information among mice, rats and humans was required. Certain parameters previously reported in the scientific literature were assumed, however, such as body weight, percentage of body weight, and percentage of blood from the heart (i.e., percentage of cardiac output of body organs, see Table 2-4). High-Dose to Low-Dose Extrapolation. The Corley model was designed to facilitate extrapolations from high doses (similar to those used for chronic rodent studies) to low doses that humans may potentially be exposed to at home or in the workplace. Interroute Extrapolation. The Corley model used three routes of administration, intraperitoneal, oral and inhalation, in rats and mice to describe the disposition of chloroform. This data was validated for humans by comparing the model output using the animal data with actual human data from human oral chloroform pharmacokinetic studies. Using the human pharmacokinetic constants from the in vitro studies conducted by Corley, the model made adequate predictions of the amount of chloroform metabolized and exhaled in both males and females. The Reitz Model Risk Assessment. This model demonstrated that, compared to the existing quantitative EPA risk estimates for chloroform (EPA 1985a), much higher concentrations of chloroform could be tolerated in the air and drinking water of humans without a significant increase in the incidence of liver carcinogenesis. For example, the authors report that the EPA’s estimate of a 10° risk specific dose (RSD) for continuous air inhalation of chloroform is 0.089 ppb. For humans consuming 2 liters of water per day containing chloroform, the RSD is 4.3 ug/L. Dose-surrogates, a more sophisticated and more accurate measure of target tissue dose derived from measuring a pharmacodynamic effect, were used. Corresponding RSDs from the Reitz model were closer to 2,200-3,200 ppb for air and 33,000 pg/L for water, depending on whether a safety factor or a linearized multistage method was used. The Reitz analysis suggests that the use of dose-surrogates, which are more intimately associated with the cancer response, represent a refined approach to cancer risk assessment in humans. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 100 2. HEALTH EFFECTS Description of the Model. The Reitz PBPK model was largely based on the Corley et al. (1990) model, but differed in the use of a pharmacodynamic end point, cytotoxicity in the livers of chloroform-exposed animals produced by phosgene (the reactive metabolite of chloroform). The Reitz model focused on the liver as the target organ for chloroform, hence the kidney compartment toxicity was not addressed. The kidney compartment was combined with the rapidly-perfused tissue group. The Reitz model used two types of dose measurement, referred to as dose surrogates. One type of dose surrogate used was covalent binding to macromolecules (average daily macromolecular binding, AVEMMB), a rate independent parameter. The second type of dose surrogate was cytotoxicity (PTDEAD), a rate dependent parameter that measured cell death (by histopathological analysis and thymidine uptake) due to the formation of reactive chloroform metabolites (i.e., phosgene). Model calculations of PTDEAD were based on several assumptions: that liver cells have a finite capability for repairing damage caused by CHCl, metabolites; that liver cells differ from cell to cell in their capabilities to repair this damage; and that induction of cytotoxicity in liver cells does not occur instantaneously. Validation of the Model. The model simulations of PTDEAD were compared with two experimental measures of cytotoxicity: (1) the percentage of nonviable cells observed microscopically in mice gavaged with solutions of chloroform in corn oil, and (2) the rate of incorporation of thymidine into normal DNA during compensatory cell replication (CCR). CCR was measured following exposure of mice to chloroform vapor for 5-6 hours. Model predictions were in good agreement (within 10%) with observed percentages of dead liver cells evaluated microscopically. Agreement between predicted and observed values of cell killing based on CCR was less satisfactory. Target Tissues. The Reitz model describes the metabolism of chloroform and the induction of cytotoxicity in liver tissue following exposure by inhalation, drinking water, and gavage routes using rat and mouse data. Species Extrapolation. The Reitz model used the same species and physiologic parameters that the Corley model utilized (average body weights, organ percentage of body weight, blood flow, etc.) for model predictions. See Table 2-4 for these parameters. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 101 2. HEALTH EFFECTS High-Dose to Low-Dose Extrapolation. The Reitz model was designed to facilitate extrapolations from high doses (similar to those used for chronic rodent studies) to low doses that humans may potentially be exposed to at home or in the workplace. Interroute Extrapolation. Inhalation and oral routes of administration were examined in the Reitz model; however, interroute extrapolations were not specifically addressed in the Reitz model. The Gearhart Model Risk Assessment. The Gearhart model provided strong evidence that temperature changes play an important role in predicting chloroform metabolism in mice and also provided a testable hypothesis for the lack of fit of the Corley model prediction with respect to the mouse data. These data strengthen the Corley model and its implications for human risk assessment (see the Corley model description above). Description of the Model. Gearhart et al. (1993) developed a PBPK model that described the effects of decreased core body temperature on the analysis of chloroform metabolic data. Experimental data showed that when male B6C3F, mice were exposed for 6 hours to chloroform vapor concentrations of 100-5,500 ppm, a dose-dependent drop in core body temperature occurred, with the least amount of temperature drop occurring at the 100 ppm concentration and the most dramatic drop in temperature occurring at the 5,500-ppm level. The Gearhart model incorporated a model previously used by Ramsey and Andersen (1984) (the same model and parameters the Corley model was based on) in conjunction with a separate model reflecting changes in body core temperature to drive equations accounting for changes in partition coefficients, cardiac output, minute ventilation volumes, and rate of chloroform metabolism. The model predicted that the V,,, for chloroform metabolism without correcting for core temperature effects was 14.2 mg/hour/kg (2/3 of that reported in the Corley model) and the K,, was 0.25 mg/L. Without body temperature corrections, the model underpredicted the rate of metabolism at the 5,500 ppm vapor concentration. Addition of a first-order kinetic rate constant (kf=1.86 hour) to account for liver metabolism of chloroform at high doses of chloroform did provide a small improvement in model predictions at 5,500 ppm, but was still considered inadequate for predicting metabolism at high concentrations. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 102 2. HEALTH EFFECTS Validation of the Model. The Gearhart model was not validated against a comparable data set. Corrections for the temperature effects (V,,, increased to 15.1 mg/hour/kg) and inclusion of a first- max order metabolism correction equation provided an accurate prediction of chloroform metabolism across all concentrations tested. Target Tissues. The liver was the target tissue for this model. Species Extrapolation. No species extrapolation was specifically addressed by the Gearhart model. High-Dose to Low-Dose Extrapolation. No high-low dose extrapolation was specifically addressed by the Gearhart model. Interroute Extrapolation. No interroute extrapolation was specifically addressed by the Gearhart model. The Chinery-Gleason Model Risk Assessment. The Chinery-Gleason model has the greatest potential for use in estimating exposures to chloroform in a household environment as well as for occupational exposures that result from dermal exposure. Description of the Model. The Chinery and Gleason (1993) PBPK model is a combination of the Corley et al. (1990) model and other existing models that includes a multicompartment skin component similar to that of Shatkin and Szejnwald-Brown (1991). This compartment is used to simulate penetration of chloroform into the skin while showering for 10 minutes with water containing chloroform. The skin module for this new model assumed a physiologic skin compartment consisting of three linear compartments: (1) the dilute aqueous solution compartment; (2) the stratum corneum (the primary barrier to the absorption of most chemicals, including chloroform); and (3) the viable epidermis. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 103 2. HEALTH EFFECTS Validation of the Model. The model was validated using published data on experimentally derived exhaled breath concentrations of chloroform following exposure in a shower stall (Jo et al. 1990a). Target Tissues. Based on this data set, the Chinery-Gleason model predicted the stratum corneum permeability coefficient for chloroform to be 0.2 cm/hour (range = 0.6 and 2.2) and the estimated ratio of the dermally and inhaled absorbed doses to be 0.75 (range = 0.6 and 2.2) cm/hour. This new model showed that a simple steady-state model can be used to predict the degree of dermal absorption for chloroform. It was also shown that the model would be useful in predicting the concentrations of chloroform in shower air and in the exhaled breath of individuals exposed both dermally and by inhalation routes while showering with water containing low amounts (20 pg/L) of chloroform. At this concentration, the model predicted a dermal absorption dose of 0.0047 mg and inhalation of 0.0062 mg. In addition, the model also demonstrated that as the concentration of chloroform rises due to increases in chloroform vapor, the absorbed inhalation dose increases faster and becomes larger than the absorbed dermal dose. Species Extrapolation. No species extrapolation was specifically addressed by the this model. High-Dose to Low-Dose Extrapolation. No high-low dose extrapolation was specifically addressed by this model. Interroute Extrapolation. The Chinery-Gleason model examined two routes of exposure, inhalation-only exposure and inhalation/dermal exposure. The model was useful in predicting the concentration of chloroform in shower air and in the exhaled breath of individuals exposed by the dermal and inhalation routes. The McKone Model Risk Assessment. The McKone model has some use in human chloroform risk assessments, in that the model defined the relationship between the dermal and inhalation exposure to measures of dose and the amounts that can be metabolized by the liver by each route. The model also provided information about the inhalation and dermal exposure concentrations at which chloroform metabolism becomes non-linear in humans. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 104 2. HEALTH EFFECTS Description of the Model. The PBPK model of McKone (1993) addressed potential exposure to chloroform by the inhalation and dermal routes. McKone revised existing shower-compartment, dermal uptake and PBPK models to produce a revised PBPK model for simulating chloroform breath levels in persons exposed in showers by the inhalation route only and by the inhalation and dermal routes combined. Parameters used by this model were taken primarily from two main sources, Jo et al. (1990a) and Corley et al. (1990). The model was also used to assess the relationship of dermal and inhalation exposure to metabolized dose in the liver, as well as to determine the tap-water concentrations at which hepatic metabolism of dermal and inhalation doses of chloroform become non-linear. This information is especially useful for risk assessment on persons exposed to a wide range of chloroform concentrations. Experimentally measured ratios of chloroform concentrations in air and breath to tap water concentration (Jo et al. 1990a) were compared with the model predictions. Validation of the Model. The McKone model used one data set to evaluate the model results (Jo et al. 1990a). The McKone model results were also compared to other existing chloroform models, with an in-depth discussion of similarities an differences between those models. Target Tissues. The skin and lung were the target tissues studied in this model. Based on the information presented, the McKone model is appropriate for simulating chloroform breath levels in persons exposed in showers by both exposure routes. A major difference between the McKone model and the Chinery-Gleason model is that the McKone model assumes the skin to be a one compartment organ, whereas the Chinery-Gleason assumed three compartments within the skin. The McKone model indicated that the ratio of chloroform dermally absorbed in the shower to the concentration in tap water ranges from 0.25 to 0.66 mg/L, and that chloroform can effectively permeate through the skin at a rate of 0.16-0.42 cm/hour during a 10-minute shower. Species Extrapolation. The human was the only species addressed by the McKone model. No extrapolation between species was addressed in this model. High-Dose to Low-Dose Extrapolation. For tap-water concentrations below 100 mg/L, the model predicted a linear relationship between potential dose (i.e., amounts present in the drinking water, inhaled in a shower, or skin surface contact) and the cumulative metabolized dose. At tap-water ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 105 2. HEALTH EFFECTS concentrations greater than 100 mg/dL for inhalation-only showers and 60 mg/L or normal showers, however, the relationship was no longer linear and modifications to this model may be required. Interroute Extrapolation. The dermal and inhalation routes were addressed in this model. The McKone model did not specifically address interroute extrapolations for chloroform. 2.4 MECHANISMS OF ACTION 2.4.1 Pharmacokinetic Mechanisms Absorption. In humans and laboratory animals, chloroform is generally absorbed quickly. Primarily because of its high blood/air partition coefficient, it passes with some ease through most tissue and cellular barriers in the body. Chloroform can be absorbed by inhalation and ingestion, and by dermal routes of exposure. Inhalation studies were performed by Corley et al. (1990) on groups of mice exposed to various concentrations of chloroform for 6 hours and sacrificed 48 hours after the last exposure. Chloroform absorption by the lungs varied by concentration and was generally 34-46%. An earlier study by Von Oettingen (1964) found that when dogs were exposed to 15,000 ppm chloroform, the concentration of chloroform in the blood rose quickly and leveled off, apparently establishing a steady-state concentration in the blood at 80-100 minutes after inhalation exposures began. The average steady-state concentration in the blood was 0.4 mg/mL. Less information is available on the absorption of chloroform by inhalation in humans. Humans exposed to 10,000 ppm of chloroform during surgical anesthesia showed a rapid absorption of chloroform detected in arterial blood samples, with peak concentrations occurring within 2 hours after initiation of anesthesia. The average arterial blood concentration of chloroform was reported to be about 0.1 mg/mL (Smith et al. 1973). “chloroform orally demonstrated that absorption was practically Rats and mice exposed to 60 mg/kg complete within 48 hours for mice and within 96 hours in rats. Peak blood levels occurred within 1 hour after the oral dose (Brown et al. 1974a). Humans dosed orally with 0.5 grams of “chloroform delivered as a capsule containing olive oil showed near complete absorption of chloroform within 8 hours after administration. Peak blood levels generally occurred at approximately 1 hour after dosing, with "*“chloroform concentrations in blood ranging from 1 to 5 pg/mL (Fry et al. 1972). ***NRAFT FOR PLIRI ICC COMMENT *** CHLOROFORM 106 2. HEALTH EFFECTS Chloroform can also permeate the stratum corneum of rabbit skin (Torkelson et al. 1976) and mouse skin (Tsuruta 1975). Percutaneous absorption of chloroform across mouse skin was calculated to be approximately 38 pg/min/cm’, indicating that the dermal absorption of chloroform occurs fairly rapidly in mice. No reliable studies report the percutaneous absorption of chloroform in humans; however, a few clinical reports indicate that chloroform is used as a vehicle for drug delivery (King 1993). Distribution. Chloroform in mice, once absorbed, is widely distributed to most organs and tissues, specifically the liver, kidney, lungs, spleen, body fat, muscle, and nervous tissue, as reported by Cohen and Hood (1969) and Bergman (1979). Significant accumulations were noted 48 hours after inhalation exposure in the central nervous system, particularly in the cerebellar cortex, spinal nerves, and meninges. When administered orally (Brown et al. 1974a), rats and squirrel monkeys showed significant accumulations of 1“Cchloroform in the brain, lung, muscle, and kidney in both species, with an unusual accumulation of chloroform in the gall bladder of the monkey. When administered orally to mice, similar accumulations of chloroform occurred in the liver, kidney, lung, muscle, blood, intestines, and gall bladder (Taylor et al. 1974). Little current information on the distribution of chloroform in humans was available for review. Chloroform tends to accumulate to a significantly higher degree in the kidneys of male mice than in those of female mice given equivalent doses, which leads to a higher degree of chloroform nephrotoxicity in male mice. The sex differences seen with the renal cortical accumulation of chloroform can be halted if chloroform is administered to castrated males; the sex difference can be reversed if chloroform is administered to females pretreated with testosterone prior to dosing with chloroform. This difference in chloroform accumulation is obviously dependent on the presence of testosterone and is very consistent with a body of evidence that indicates chloroform is more nephrotoxic to male mice than to female mice (Ilett et al. 1973; Pohl et al. 1984; Smith et al. 1973). Although this sex-related toxic effect is known to occur in mice, it is not known at present if a similar effect occurs in humans. Excretion. Chloroform is largely excreted either in the parent form or as the end metabolite (CO,) in the bodies of both laboratory animals and humans. Corley et al. (1990) demonstrated that mice exposed to 10 or 89 ppm of chloroform by inhalation excreted 99% of the chloroform body burden as CO, in exhaled air. As the chloroform concentrations in the air rose however, the amount of chloroform metabolized to CO, decreased and the amount of unchanged chloroform rose in the ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 107 2. HEALTH EFFECTS exhaled air, indicating that chloroforrn metabolism in mice is a saturable process. Rats exposed in a similar manner to 93, 356, and 1,041 ppm chloroform excreted 2, 20, and 42.5%, respectively, of the total body burden of chloroform as unchanged parent compound, indicating that chloroform is metabolized to CO, in rats, but to a lesser degree than in mice. In humans, Fry et al. (1972) administered 500 mg of chloroform orally in olive oil in capsular form and found that 17-67% of the total dose of chloroform was exhaled as unchanged parent compound, and that the extent of pulmonary elimination of chloroform was governed inversely by the amount of adipose tissue on the individual ingesting the chloroform. The study also found that most of the chloroform tended to be exhaled between 40 minutes and 2 hours after dosing, which coincided with peak blood levels of chloroform produced at approximately 1 hour after dosing. Chloroform in humans tends to be eliminated in a biphasic manner. After ingesting 500 mg of chloroform orally, an initial (ct) half-life in the blood of 9-21 minutes was reported, with the second (B) half-life ranging from 86 to 96 hours. 2.4.2 Mechanisms of Toxicity Chloroform is widely distributed to many tissues of the body in both laboratory animals and humans; however, many studies have demonstrated that chloroform does not tend to accumulate in the body for extended periods. Chloroform may accumulate to some degree in the body fat stores; however, it quickly partitions out the fat and is excreted by the normal routes and mechanisms. The liver (primary) and kidneys (secondary) are considered to be the target organs for chloroform toxicity in both humans and laboratory animals. Thus, humans (and animals) with existing hepatic or renal disease who are exposed to chloroform, particularly by the oral or inhalation routes, are more likely to be at risk to the toxic effects of chloroform. Reproductive/developmental effects due to chloroform’s presence in the drinking water of both humans and laboratory animals has been reported, thereby placing women of child-bearing age at a potentially higher risk of reproductive organ anomalies than those women past menopausal age. Chloroform is largely metabolized in many tissues (particularly the liver and kidney) to carbon dioxide (CO,) in humans and animals (Brown et al. 1974a; Corley et al. 1990; Fry et al. 1972). Chloroform metabolism is catalyzed by cytochrome P-450, initiating an oxidative cleavage of the C-H bond ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 108 2. HEALTH EFFECTS producing trichloromethanol. Trichloromethanol is unstable and is rapidly transformed to phosgene (COCl,). Phosgene may react with water to form CO,, which can be exhaled by the lung or excreted in the urine as carbonate or bicarbonate, and hydrochloric acid. Phosgene can also react with other molecules such as cysteine, deplete hepatic glutathione (Docks and Krishna 1976; Pohl et al. 1981) and form adducts with microsomal proteins (Corley et al. 1990). Chloroform toxicity can be attributed to the presence of both the parent compound and the formation of phosgene in most instances of toxicosis. High doses of inhaled chloroform have been reported to cause death (due to respiratory depression), ataxia, narcosis, and central nervous system depression, and are due to the direct effects of the parent compound. Lower doses of chloroform in the air, feed, or water, or administered by gavage, with variable exposure times, may induce toxicity due to the presence of the parent compound or to production of phosgene during metabolism. Parent compound or metabolite may be responsible for hepatocellular damage, resulting in the ultimate elevation of hepatic enzymes (SGPT, SGOT, GGT, etc.) and cell damage/necrosis. The accumulation of chloroform in the renal cortex of mice with the subsequent metabolism to phosgene most likely contributes to the renal toxicity of chloroform seen in male mice. Tubular necrosis, calcification, nephritis, increased kidney weight, alterations in Na/K excretion, and other cellular anomalies were observed in response to one or both of these toxicants. Although the sex-related nephrotoxic effect is know to occur in mice, it is not known at present if a similar effect occurs in humans or other laboratory animals. 2.4.3 Animal-to-Human Extrapolations Many laboratory animal models have been used to describe the toxicity and pharmacology of chloroform. By far, the most commonly used laboratory animal species are the rat and mouse models. Generally, the pharmacokinetic and toxicokinetic data gathered from rats and mice compare favorably with the limited information available from human studies. PBPK models have been developed using pharmacokinetic and toxicokinetic data for use in risk assessment work for the human. The models are discussed in depth in section 2.3.5. As mentioned previously, male mice have a sex-related (and possibly species-related) tendency to develop severe renal disease when exposed to chloroform, particularly by the inhalation and oral exposure routes. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 109 2. HEALTH EFFECTS 2.5 RELEVANCE TO PUBLIC HEALTH Overview. Data are available regarding health effects in humans and animals after inhalation, oral, and dermal exposure to chloroform; however, data regarding dermal exposure are quite limited. Chloroform was used as a general anesthetic, pain reliever, and antispasmodic for more than a century before its toxic effects were fully recognized. High levels of chloroform in the air are found specifically in highly industrialized areas. Exposure of the general population to chloroform can also occur via the drinking water as a result of the chlorination process. Occupational exposure is another source of inhalation and/or dermal exposure for humans. Most of the presented information regarding chloroform toxicity following inhalation exposure in humans was obtained from clinical reports of patients undergoing anesthesia. In some instances, the results in these studies may have been confounded by unreported data, intake of other drugs or by artificial respiration during anesthesia. The target organs of chloroform toxicity in humans and animals are the central nervous system, liver, and kidneys. There is a great deal of similarity between chloroform-induced effects following inhalation and oral exposure. No studies were located regarding reproductive effects in humans after exposure to chloroform, and only one study was located regarding the developmental effects of chloroform in humans. Animal studies indicate that chloroform can cross the placenta and cause fetotoxic and teratogenic effects. Chloroform exposure has also caused increased resorptions in animals. Epidemiology studies suggest a possible risk of colon and bladder cancer in humans that is associated with chloroform in drinking water. In animals, chloroform was carcinogenic after oral exposure. Minimal Risk Levels for Chloroform. Inhalation MRLs. * An MRL of 1 ppm has been derived for acute-duration inhalation exposure (14 days or less) to chloroform. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 110 2. HEALTH EFFECTS The MRL was based on a hepatic NOAEL of 3 ppm chloroform administered for 6 hours/day for 7 consecutive days to mice (Larson et al. 1994c). Female mice exposed to 100 or 300 ppm exhibited centrilobular hepatocyte necrosis and severe diffuse vacuolar degeneration of midzonal and periportal hepatocytes, while exposure to 10 or 30 ppm resulted in mild to moderate vacuolar changes in centrilobular hepatocytes. Decreased eosinophilia of the centrilobular and midzonal hepatocyte cytoplasm relative to periportal hepatocytes was observed at 30 ppm. Livers of mice in the 1 and 3 ppm groups did not differ significantly from control animals and were considered to be NOAELSs for liver effects. Reports regarding chloroform hepatotoxicity in animals are numerous (Larson et al. 1993, 1994a, 1994b, 1994c). Liver damage has been reported in several other studies, and was usually indicated by liver biochemical/enzyme alterations in rats (Lundberg et al. 1986) and mice (Gehring 1968; Murray et al. 1979) after acute inhalation exposure. Fatty changes (Culliford and Hewitt 1957; Kylin et al. 1963) and liver necrosis (Deringer et al. 1953) were observed histologically in mice after acute inhalation exposure. Histological findings indicative of liver toxicity were also observed other laboratory animals following inhalation exposure of intermediate duration, but the findings were not dose-related (Torkelson et al. 1976). Fatty infiltration was observed in mice given a single gavage dose of 35 mg/kg/day chloroform in oil (Jones et al. 1958). No toxic effects on the livers of mice occurred after a single dose of 17.3 or 59.2 mg/kg chloroform in oil, but increased SGPT occurred at 199 mg/kg (Moore et al. 1982). The 17.3 mg/kg dose was also a NOAEL for kidney effects, but tubular necrosis occurred at 65.6 mg/kg/day. « An MRL of 0.05 ppm has been derived for intermediate-duration inhalation exposure (15 days to 364 days) to chloroform. The MRL was based on a LOAEL of 14 ppm in workers exposed to concentrations of chloroform of up to 400 ppm for less than 6 months (Phoon et al. 1983). Vomiting and toxic hepatitis were noted to occur at an inhaled chloroform concentration of 14 ppm. Alterations in liver functions have been reported in several studies in both humans and animals, and is discussed in more detail in the chronic- duration inhalation MRL section immediately below. « An MRL of 0.02 ppm has been derived for chronic-duration inhalation exposure (365 days or more) to chloroform. ***DRAFT FOR PUBLIC COMMENT"** CHLOROFORM 111 2. HEALTH EFFECTS The MRL was based on a LOAEL of 2 ppm in workers exposed to concentrations of chloroform ranging from 2 to 205 ppm for 1-4 years (Bomski et al. 1967). Hepatomegaly was found in 25% of chloroform exposed workers. Toxic hepatitis was found in 5.6% of the liver enlargement cases. Hepatosteatosis (fatty liver) was detected in 20.6% of liver-enlargement cases. Chloroform exposed workers had a higher frequency of jaundice over the years than the control group. Alterations in liver functions have been reported in several studies in both humans and animals. In humans, impaired liver function was indicated by increased sulfobromophthalein retention in some patients exposed to chloroform via anesthesia (Smith et al. 1973), in addition to acute toxic hepatitis developing after childbirth in several women exposed to chloroform via anesthesia (Lunt 1953; Royston 1924; Townsend 1939). Toxic hepatic disease, characterized by hepatomegaly and abnormal liver sonograms as late as seven days after an unknown amount of oral chloroform, has been reported (Hakim et al. 1992). Elevated liver enzymes and changes in GGT, alpha-feto protein and retinol binding protein were reported in a female who injected herself intravenously and also consumed chloroform orally during a 12-hour period (Rao et al. 1993). In contrast, no clinical evidence of liver toxicity was found in another study among chloroform workers exposed to <237 ppm (Challen et al. 1958). Liver damage was induced by chronic use of a cough medicine containing chloroform (Wallace 1950), but not by chronic exposure to chloroform in mouthwash (De Salva et al. 1975). Oral MRLs. e An MRL of 0.3 mg/kg/day has been derived for acute-duration oral exposure (14 days or less) to chloroform. The MRL was based on a NOAEL of 26.4 mg/kg/day for 4 days for hepatic effects from a study by Larson et al. (1994b). A study performed by Moore et al. (1982) found renal effects in CFLP Swiss mice dosed at greater than 17.3 mg/kg/day. No renal effects were found at the 17.3 mg/kg/day dose in that study. A study by Davis and Berndt (1992) also found renal effects induced by chloroform, but the doses were much higher (520 mg/kg/day) and were also classified as less serious LOAELSs. Another study by Larson et al. (1993) found both hepatic (elevated SDH, ALT and AST, hepatocyte necrosis) and renal (proximal tubule necrosis) lesions in Fischer 344 rats and hepatic lesions only in B6C3F, mice induced by chloroform administered at 34 mg/kg/day once by gavage in oil. Lesions in the Larson et al. (1993) study were ranked as less serious LOAELSs. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 112 2. HEALTH EFFECTS * An MRL of 0.1 mg/kg/day has been derived for intermediate-duration oral exposure (15-364 days) to chloroform. This MRL is based on a NOAEL of 15 mg/kg/day for hepatic effects in dogs dosed with chloroform in a capsule 1 time per day, 6 days per week for 6 weeks in a study by Heywood et al. (1979). Clinical chemistry parameters showed significantly increase SGPT in the 30 mg/kg/day group beginning at 6 weeks. SGPT activity was not increased in the 15 mg/kg/day group until week 130. Liver effects in animals have been reported in numerous oral studies of intermediate duration. Fatty changes, necrosis, increased liver weight, and hyperplasia have been observed in rats exposed to 2150 mg/kg/day chloroform in drinking water for 90 days (Palmer et al. 1979). An increased incidence of sporadic, mild, reversible liver changes occurred in mice exposed to chloroform in drinking water at doses of 0.3-114 mg/kg/day for 90 days, but the incidences were not significantly higher than the incidences in controls (Chu et al. 1982a). Fatty and hydropic changes, necrosis, and cirrhosis were observed in mice treated by gavage with 250 mg/kg/day chloroform in oil for 90 days (Bull et al. 1986; Munson et al. 1982) or at 86 mg/kg/day in drinking water for 1 year (Klaunig et al. 1986). In contrast, centrilobular fatty changes observed in mice at 64 mg/kg/day chloroform in drinking water for 90 days appeared to be reversible (Jorgenson and Rushbrook 1980), and no liver effects were found in mice treated with 250 mg/kg/day in aqueous vehicles (Bull et al. 1986). In addition, hepatocellular degeneration was induced in F, females in a 2-generation study in which mice were treated by gavage with 41 mg/kg/day chloroform in oil (Gulati et al. 1988). * An MRL of 0.01 mg/kg/day has been derived for chronic-duration oral exposure (365 days or more) to chloroform. This MRL is based on a NOAEL of 15 mg/kg/day for hepatic effects in dogs dosed with chloroform 6 days per week for 7.5 years in a study by Heywood et al. (1979). SGPT activity was not increased in the 15 mg/kg/day group until week 130, providing the LOAEL on which this MRL was based. Numerous chronic oral studies examined hepatic and renal end points as well as neurological and cancer effects. Serious effects occurred at higher doses; 15 mg/kg/day was the lowest dose used in available animals studies. A NOAEL of 2.46 mg/kg/day for liver and kidney effects (SGPT, SGOT, BUN and SAP) was found in humans who used a dentifrice containing 0.34% or a mouthwash containing 0.43% chloroform for 1-5 years (DeSalva et al. 1974). ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 113 2. HEALTH EFFECTS Death. Chloroform levels of =40,000 ppm cause death in patients under chloroform anesthesia (Featherstone 1947; Whitaker and Jones 1965). Death is usually due to severe respiratory depression/ failure or disturbances in cardiac rhythm. Accidental or intentional ingestion of large doses of chloroform may lead to death (Piersol et al. 1933). Death in humans after oral exposure to chloroform is usually caused by respiratory obstruction by the tongue due to jaw relaxation, central respiratory paralysis, acute cardiac failure, or severe hepatic injury (Piersol et al. 1933; Schroeder 1965). The levels of chloroform exposure that cause death in animals are usually lower than those administered to patients to induce anesthesia; however, the duration of exposure in animals is generally longer. Following acute exposure to high concentrations of chloroform, all male mice died; however, most females survived the exposure for several months (Deringer et al. 1953). Survival was associated with lower testosterone levels, as suggested by the higher mortality rate in non-castrated adult males. This conclusion is supported by similar observations of higher survival rates in female rats, compared to male rats, after intermediate-duration exposure to chloroform (Torkelson et al. 1976). In regard to LC, values in rats, survival rates were highest among females and lowest among young adult males. The correlation between mortality rates and male hormone levels is evident. Increased mortality was also observed in rats and mice after oral exposure of intermediate and chronic duration (Balster and Borzelleca 1982; Chu et al. 1982a; Jorgenson et al. 1985; Klaunig et al. 1986; NCI 1976; Palmer et al. 1979; Roe et al. 1979). Deaths were caused by starvation, toxic liver and kidney effects, and tumors. Deaths after dermal exposures in either humans or laboratory animals have not been reported. Chloroform concentrations in air and drinking water in the general environment or near hazardous waste sites are not likely to be high enough to cause death in humans after acute exposure. Whether chronic exposure to low levels of chloroform in the environment, drinking water, or hazardous wastes could shorten the life span of humans is not currently known. Currently available epidemiologic findings about the chronic exposure to chloroform are inconsistent at best which, in large part, may be due to study design issues. Systemic Effects. Respiratory Effects. The respiratory failure observed in patients under chloroform anesthesia was probably due to a direct effect of chloroform on the respiratory center of the central nervous system. A decline of the systolic pressure in the cerebral vessels may also contribute to respiratory failure, as ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 114 2. HEALTH EFFECTS demonstrated in animals: when respiration had stopped under chloroform anesthesia, the animals (species not specified) breathed again if positioned head down (Featherstone 1947). Upper respiratory tract obstruction can occur in patients after inhalation exposure to chloroform via anesthesia (Featherstone 1947) and after chloroform ingestion (Schroeder 1965). Few autopsy reports were located in the literature. Hemorrhage into the lungs, without any signs of consolidation, was reported in a case study involving death after inhalation exposure (Royston 1924); however, congested lungs with pneumonic consolidation were observed in a man who died after drinking chloroform (Piersol et al. 1933). In addition to lower respiratory tract effects, chloroform has been demonstrated to induce site-specific changes in the nasal region of rats and mice after inhalation exposure. Increased sizes of goblet cells and nasal epithelium, changes in the proliferation rates of cells and changes in biochemical parameters (especially cytochrome P-450-2E1) have been reported (Mery et al. 1994), indicating that chloroform can adversely affect the upper as well as the lower respiratory tract at low concentrations. Interstitial pneumonitis was observed in male rats and rabbits after inhalation exposure to 85 or 50 ppm chloroform, respectively, for 6 months (Torkelson et al. 1976). In most oral studies, no exposure-related histopathological changes were observed in the lungs of exposed animals (Gulati et al. 1988; Jorgenson and Rushbrook 1980; NCI 1976; Palmer et al. 1979; Roe et al. 1979). Respiratory effects are more likely to occur after inhalation exposure to high concentrations of chloroform. It has been demonstrated that chloroform has a destructive influence on the pulmonary surfactant (Enhorning et al. 1986). This effect is probably due to the solubility of phospholipids in the surfactant monolayer and can cause collapse of the respiratory bronchiole due to the sudden increase in inhalation tension. Immediate death after chloroform inhalation may be due principally to this effect in the lungs (Fagan et al. 1977). It is unlikely that exposure levels of chloroform in the general environment or at hazardous waste sites would be high enough to cause these severe respiratory effects. Cardiovascular Effects. Chloroform induces cardiac arrhythmia in patients exposed to chloroform via anesthesia (Smith et al. 1973; Whitaker and Jones 1965). Similarly, heart effects were observed upon electrocardiography of an individual who accidentally ingested chloroform (Schroeder 1965). Hypotension was observed in 12-27% of patients exposed to chloroform via anesthesia (Smith et al. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 115 2. HEALTH EFFECTS 1973; Whitaker and Jones 1965) and also was observed in a patient who ingested chloroform (Storms 1973). No studies were located regarding cardiovascular effects in animals after inhalation exposure to chloroform. No histopathological changes were observed in the heart of rats, mice, (NCI 1976) or dogs (Heywood et al. 1979) chronically exposed to chloroform; however, cardiovascular function was not assessed in these studies. It has been demonstrated in an in vitro study on heart-lung preparations of guinea pigs that chloroform may cause a permanent contractile failure of the heart (Doring 1975). The effect is due to structural damage of the transverse tubular system and is accompanied by increased storage of adenosine triphosphate (ATP) and phosphocreatine. The in vitro induction of changes showed that contractile failure is a direct effect on the cardiovascular system rather than an indirect cardiovascular effect on the central nervous system. This mechanism may operate in humans exposed to high vapor concentrations such as those used in anesthesia or in humans exposed to high oral doses from accidental or intentional ingestion. It is unlikely, however, that concentrations of chloroform in the environment would be high enough to cause overt cardiovascular effects. Cytotoxicity of chloroform (1,000 ppm) in male Sprague-Dawley rat cardiac myocytes has been examined in vitro. Cell viability was measured using the criterion of Trypan blue exclusion as well as counting the number of rod and spherical cells in the media. Creatinine phosphokinase (CPK) leakage was measured as an indirect measurement of heart cell function. Myocytes treated with chloroform showed statistically significant decreases in cell viability and significant decreases in rod-shaped cells compared to controls. Significant increases in enzyme leakage of CPK from myocytes were noted (El-Shenawy and Abdel-Rahman 1993b). The effects of dye transfer between cardiac myocytes exposed to various concentrations of chloroform has also been examined (Toraason et al. 1992). Gastrointestinal Effects. Nausea and vomiting were not only frequently observed side effects in patients exposed to chloroform via anesthesia (Hakim et al. 1992; Royston 1924; Smith et al. 1973; Townsend 1939; Whitaker and Jones 1965), but also occurred in humans exposed to lower chloroform concentrations (22-237 ppm) in occupational settings (Challen et al. 1958; Phoon et al. 1983). Vomiting, gastric distress, and pain were observed in individuals who intentionally or accidentally ingested high doses of chloroform (Piersol et al. 1933; Schroeder 1965; Storms 1973). ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 116 2. HEALTH EFFECTS No studies were located regarding gastrointestinal effects in animals after inhalation exposure to chloroform. Vomiting in dogs (Heywood et al. 1979) and gastric erosions in rats (Thompson et al. 1974) were observed in oral studies of intermediate duration. These results indicate that severe gastrointestinal irritation in humans and animals is due to direct damage of the gastrointestinal mucosa caused by ingesting high concentrations of chloroform (Piersol et al. 1933; Schroeder 1965; Thompson et al. 1974). Following anesthesia, vomiting and nausea may be due to possible gastric irritation which is provoked by swallowing chloroform gas during induction of narcosis or, more frequently, may be due to the hepatic and renal toxicity of chloroform (Featherstone 1947). Nausea and vomiting experienced by occupationally exposed individuals is also probably secondary to liver toxicity. Since toxic hepatitis may occur at occupational levels as low as 2 ppm (Bomski et al. 1967), it is possible that levels of chloroform in the air at hazardous waste sites may be high enough to cause some liver effects with secondary gastrointestinal effects, if exposure is prolonged. Hematological Effects. Information regarding hematological effects in humans exposed to chloroform is limited. Increased prothrombin time was observed in some patients, following exposure to chloroform via anesthesia (Smith et al. 1973). This effect, however, reflects chloroform hepatotoxicity, because prothrombin is formed in the liver. Decreased erythrocytes and hemoglobin were observed in a patient who was chronically exposed to chloroform in a cough medicine (Wallace 1950). No hematological effects were observed in rats, rabbits, guinea pigs, or dogs after inhalation exposure to chloroform for intermediate durations (Torkelson et al. 1976). Studies report conflicting results regarding hematological effects in animals after oral exposure to chloroform. No conclusion about hematological effects in humans after exposure to chloroform can be made on the basis of one case study in humans. From the experimental data in animals, it is evident that all hematological effects observed in rats were due to oral exposure of acute, intermediate, or chronic duration. It is possible that the hematological effects observed in rats are transient. Human exposure to chloroform in the environment, drinking water, or at hazardous waste sites is likely to cause few or no hematological effects. *“**DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 117 2. HEALTH EFFECTS Musculoskeletal Effects. Little data is available that examines the effects of chloroform toxicity on the musculoskeletal system; however, it appears that chloroform has few significant toxic effects on this system. Hepatic Effects. The liver is a primary target organ of chloroform toxicity in humans and animals after inhalation and oral exposure, with some evidence that suggests that the damage may be reversible (Wallace 1950). Impaired liver function was indicated by increased sulfobromophthalein retention in some patients exposed to chloroform via anesthesia (Smith et al. 1973). Acute toxic hepatitis developed after childbirth in several women exposed to chloroform via anesthesia (Lunt 1953; Royston 1924: Townsend 1939). Upon autopsy, centrilobular necrosis was observed in the women who died; however, the hepatotoxicity was associated with exhaustion from prolonged delivery, starvation, and dehydration, indicating improper handling of the delivery procedure by an obstetrician. Toxic hepatic disease, characterized by hepatomegaly and abnormal liver sonograms as late as seven days after an unknown amount of oral chloroform, has been reported (Hakim et al. 1992). Elevated liver enzymes and changes in GGT, alpha-feto protein and retinol binding protein were reported in a female who injected herself intravenously and also consumed chloroform orally during a 12-hour period (Rao et al. 1993). During occupational exposure to concentrations ranging from 14 to 400 ppm, chloroform hepatotoxicity was characterized by jaundice (Phoon et al. 1983), hepatomegaly, enhanced SGPT and SGOT activities, and hypergammaglobulinemia following exposure to concentrations ranging from 2 to 205 ppm (Bomski et al. 1967). In contrast, no clinical evidence of liver toxicity was found in another study among chloroform workers exposed to <237 ppm (Challen et al. 1958). Case reports of intentional and accidental ingestion of high doses (22,500 mg/kg) of chloroform indicate severe liver injury (Piersol et al. 1933; Schroeder 1965; Storms 1973). The diagnosis was supported by clinical and biochemical results: fatty degeneration and extensive centrilobular necrosis were observed in one patient who died (Piersol et al. 1933). Liver damage was induced by chronic use of a cough medicine containing chloroform (Wallace 1950), but not by chronic exposure to chloroform in mouthwash (De Salva et al. 1975). An intermediate-duration inhalation MRL of 0.05 ppm was derived from the LOAEL of 14 ppm from the data presented by Phoon et al. (1983); a chronic-duration inhalation MRL of 0.02 ppm was derived from the LOAEL of 2 ppm from the data presented by Bomski et al. (1967). Reports regarding chloroform hepatotoxicity in animals are numerous (Larson et al. 1993, 1994a, 1994b, 1994¢). An acute-duration inhalation MRL of 1 ppm was based on a NOAEL for hepatic effects in mice exposed to 3 ppm chloroform for 6 hours per day for 7 days in a study by Larson et al. (1994c). ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 118 2. HEALTH EFFECTS Liver damage was indicated by biochemical changes in rats (Lundberg et al. 1986) and mice (Gehring 1968; Murray et al. 1986) after acute inhalation exposure. Fatty changes (Culliford and Hewitt 1957: Kylin et al. 1963) and liver necrosis (Deringer et al. 1953) were observed histologically in mice after acute inhalation exposure. Histological findings indicative of liver toxicity were also observed in rabbits and guinea pigs following inhalation exposure of intermediate duration, but the findings were not dose-related (Torkelson et al. 1976). Liver effects have been observed in many species (rats, mice, and dogs) that were tested by the oral route by various methods of administration (gavage or drinking water) and durations (acute, intermediate, or chronic). Observed effects include increased liver weight, increased serum levels of transaminases indicative of liver necrosis, and histological evidence of fatty changes, hydropic changes, necrosis, hyperplasia, cirrhosis, and toxic hepatitis. Two acute oral studies define a LOAEL and a NOAEL for liver effects in mice. Fatty infiltration was observed in mice given a single gavage dose of 35 mg/kg/day chloroform in oil (Jones et al. 1958). No toxic effects on the livers of mice occurred after a single dose of 17.3 or 59.2 mg/kg chloroform in oil, but increased SGPT occurred at 199 mg/kg (Moore et al. 1982). The 17.3 mg/kg dose was also a NOAEL for kidney effects, but tubular necrosis occurred at 65.6 mg/kg/day. In a 7.5-year study in which dogs were administered chloroform in toothpaste, SGPT activity was significantly increased at 30 mg/kg/day beginning at 6 weeks (Heywood et al. 1979). SGPT activity was not increased at 15 mg/kg/day until 130 weeks. Therefore, 15 mg/kg/day was a NOAEL for intermediate-duration exposure and a LOAEL for chronic-duration exposure. The 15 mg/kg/day dose was used to derive MRL values of 0.1 and 0.01 mg/kg/day for intermediate- and chronic-duration oral exposure, respectively. Data regarding chloroform-induced hepatotoxicity were also supported by results obtained after acute intraperitoneal exposure in rats (Bai et al. 1992; Ebel et al. 1987; El-Shenawy and Abdel-Rahman 1993a; Lundberg et al. 1986), mice (Klaassen and Plaa 1966), dogs (Klaassen and Plaa 1967), and gerbils (Ebel et al. 1987). No hepatic effects were observed in rabbits when chloroform was applied to their skin for 24 hours (Torkelson et al. 1976). The toxicity of chloroform on laboratory animal hepatocytes in vitro has been reported (Azri-Meehan et al. 1992, 1994; Bai and Stacey 1993; El-Shenawy and Abdel-Rahman 1993a; Suzuki et al. 1994). As discussed in Section 2.3.3, the mechanism of chloroform-induced liver toxicity may involve metabolism to the reactive intermediate, phosgene, which binds to lipids and proteins of the endoplasmic reticulum, lipid peroxidation, or depletion of glutathione by reactive intermediates. Because liver toxicity has been observed in humans exposed to chloroform levels as low as 2 ppm in ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 119 2. HEALTH EFFECTS the workplace and in several animal species after inhalation and oral exposure, it is possible that liver effects could occur in humans exposed to environmental levels, to levels in drinking water, or to levels found at hazardous waste sites. Endocrine Effects. No reports of chloroform toxicity to endocrine organs have been reported. Renal Effects. Clinical reports indicate that the renal damage observed in women exposed to chloroform via anesthesia during prolonged parturition most likely occurs when chloroform anesthesia is associated with anoxia. Competitive swimmers who swim in indoor pools have been reported to have elevated B-2-microglobin, suggesting some degree of renal damage (Aiking et al. 1994). Case studies of individuals who intentionally or accidentally ingested high doses of chloroform report biochemical changes indicative of kidney damage, as well as fatty degeneration at autopsy (Piersol et al. 1933: Schroeder 1965). Albuminuria and casts were also reported in a case of chronic use of a cough medicine containing chloroform (Wallace 1950); however, no renal effects were observed in individuals chronically exposed to chloroform in a mouthwash (De Salva et al. 1975). Renal effects of chloroform have also been examined (Larson et al. 1994b, 1994¢). Kidney effects in animals after inhalation exposure to chloroform include tubular necrosis, tubular calcification, increased kidney weight, cloudy swelling, and interstitial nephritis. Animal studies regarding renal toxicity after oral exposure are numerous. Effects include acute toxic nephrosis, tubular necrosis, chronic inflammation, and fatty degeneration. The effects of dose and vehicle have been examined (Larson et al. 1993; Lipsky et al. 1993). Mice seem to be more sensitive to chloroform-induced renal toxicity than other experimental animals. Certain strains of male mice are susceptible to chloroform-induced nephrotoxicity, while female mice appear to be somewhat resistant (Culliford and Hewitt 1957; Eschenbrenner and Miller 1945b). Castrated mice were no longer susceptible to the effect, and testosterone treatment increased the severity of kidney damage in females, suggesting the role of hormones in chloroform-induced nephrotoxicity. It has been demonstrated that sensitivity to kidney damage is related to the capacity of the kidney to metabolize chloroform to phosgene (Pohl et al. 1984). The activation of chloroform to its reactive metabolites appeared to be cytochrome P-450-dependent: the covalent binding to microsomal protein required nicotinamide adenine dinucleotide phosphate (NADPH) and oxygen, and could be inhibited by carbon monoxide (Hook and Smith 1985; Smith and Hook 1983, 1984; Smith et ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 120 2. HEALTH EFFECTS al. 1984). Furthermore, administration of chloroform to male mice caused a depletion of renal glutathione, indicating that glutathione can react with reactive intermediates, thereby reducing the extent of the reaction with tissue macromolecules and kidney damage. The renal toxicity of chloroform in rats after intraperitoneal dosing has also been reported (Kroll et al. 1994a, 1994b). It is likely that kidney effects may occur in humans after inhalation or oral exposure to high levels of chloroform; however, it is not known whether such effects would occur at the levels of chloroform found in the environment, in drinking water, or at hazardous waste sites. Dermal Effects. No reports are available on the toxicity of chloroform to skin after inhalation and oral exposures in humans. Stratum corneum damage was reported after a topical exposure of chloroform of 15 minutes duration for 6 consecutive days (Malten et al. 1968). Chloroform was used as a vehicle for the topical application of aspirin for the treatment of painful herpes zoster lesions in male and female humans. The only reported side-effect was an occasional burning sensation to the skin as the chloroform evaporated after application (King 1993). Few reports exist on the dermal effects of chloroform in animals after inhalation or oral exposures. Alopecia has been observed in pregnant rats (Thompson et al. 1974) and in mice (Gulati et al. 1988). Skin irritation and necrosis and been reported in rabbits after topical application of chloroform (Smyth et al. 1962; Torkelson et al. 1976). Ocular Effects. No studies were located regarding the ocular effects of chloroform in humans or animals. Body Weight Effects. Decreased body weight has been observed frequently in animals after inhalation or oral exposure to chloroform, although the degree of body weight changes are somewhat variable and may be linked to taste aversion (in oral studies). The degree of decreased weight gain was often dose-related and was caused by chloroform toxicity. Decreased weight gain generally occurred at exposure levels similar to or lower than those that induced liver and kidney effects in animals. The possibility of effects on body weight in humans exposed to ambient or elevated levels of chloroform cannot be dismissed. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 121 2. HEALTH EFFECTS Immunological and Lymphoreticular Effects. No studies were located regarding immunological effects in humans after inhalation, oral, or dermal exposure to chloroform. Information about immunological effects in animals is limited. After repeated inhalation exposure to chloroform, mortality was increased in mice challenged with streptococcus infection, suggesting increased susceptibility (Aranyi et al. 1986). However, the bacterial activity of alveolar macrophages was not suppressed in this study. After acute oral exposure, reduced lymphocyte counts were observed in rats (Chu et al. 1982b). Furthermore, humoral immunity was depressed in mice exposed to 50 mg/kg/day chloroform for acute or intermediate durations (Munson et al. 1982). In contrast, cell- mediated immunity was influenced only at high chloroform concentrations administered orally for intermediate durations; however, the chloroform-induced immunological changes appeared to be more severe following acute exposure. [n vitro treatment of serum with chloroform resulted in a loss of complement activity (Stefanovic et al. 1987). Immunological effects may result from the ability of chloroform to dissociate antigen-antibody complexes, since it can cause dissociation of certain enzyme- inhibitor complexes (Berger et al. 1983). Every day, humans are exposed to very low levels of chloroform in the environment, mainly via inhalation and oral exposure (Hajimiragha et al. 1986; Peoples et al. 1979; Wallace et al. 1987a, 1989). There is a risk of chloroform exposure at or near hazardous waste sites. Although no evidence that chloroform can cause immunological effects in humans was located in the literature, the possibility remains that these effects may result from exposure to chloroform. Neurological Effects. Neurological effects in humans after acute inhalation exposure to chloroform are well documented because chloroform has been used as an anesthetic for surgery. Inhaled chloroform acts as a depressant on the central nervous system. Chronic inhalation exposure to chloroform resulted in exhaustion, lack of concentration, depression, and irritability in occupationally exposed people (Challen et al. 1958). In a case study, chloroform inhalation for 12 years resulted in psychotic episodes, hallucinations, and convulsions (Heilbrunn et al. 1945). Central nervous system toxicity was observed in humans after oral exposure to chloroform, which suggests that the effects of inhalation and oral exposure are similar. In case reports of patients who intentionally or accidentally ingested several ounces of chloroform, deep coma with abolished reflexes occurred within a few minutes (Piersol et al. 1933; Schroeder 1965; Storms 1973). ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 122 2. HEALTH EFFECTS Inhalation exposure to high chloroform concentrations induced narcosis (Lehmann and Flury 1943; Sax 1979) and reversible impairment of memory retrieval in animals. High, single, oral doses of chloroform caused ataxia, incoordination, anesthesia, and brain hemorrhage in mice (Balster and Borzelleca 1982; Bowman et al. 1978). Behavioral effects were observed at lower oral doses. Chloroform concentrations from 1.5 to 6.0 mmol chloroform were used to determine how chloroform may modify glutamate receptor agonist responses in mouse brain cortical wedges. The two agonists examined were N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA). Responses were determined by measuring electrical responses within the cortical slices. Three mmol of chloroform selectively inhibited AMPA but did not affect NMDA responses. Higher concentrations of chloroform failed to inhibit the AMPA or NMDA content in the wedges (Carla and Moroni 1992). The clinical effects of chloroform toxicity on the central nervous system are well documented. However, the molecular mechanism of action is not well understood. It has been postulated that anesthetics induce their action at a cell-membranal level due to lipid solubility. The lipid-disordering effect of chloroform and other anesthetics on membrane lipids was increased by gangliosides (Harris and Groh 1985), which may explain why the outer leaflet of the lipid bilayer of neuronal membranes, which has a large ganglioside content, is unusually sensitive to anesthetic agents. Anesthetics may affect calcium-dependent potassium conductance in the central nervous system (Caldwell and Harris 1985). The blockage of potassium conductance by chloroform and other anesthetics resulted in depolarization of squid axon (Haydon et al. 1988). The potential for neurological and behavioral effects in humans exposed to chloroform at levels found in the environment, in drinking water, or at hazardous waste sites is not known. Reproductive Effects. No studies were located regarding reproductive effects in humans after inhalation, oral, or dermal exposure to chloroform. Studies indicate that exposure to chloroform causes reproductive effects in animals. Dose-related increases of embryonal resorptions were observed in rats and mice after inhalation or oral exposure to chloroform during gestation. A significant increase in the incidence of abnormal sperm was observed in mice after acute inhalation exposure (Land et al. 1979, 1981), but no sperm-head abnormalities were “**DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 123 2. HEALTH EFFECTS noted in mice after receiving 5 daily intraperitoneal injections of chloroform in concentrations up to 0.25 mg/kg/day in corn oil (Topham 1980). Gonadal atrophy was observed in male and female rats treated by gavage (Palmer et al. 1979). Fertility was not affected in either generation of mice exposed orally to chloroform in a 2-generation study (Gulati et al. 1988). Oral exposure to chloroform did not induce histopathological changes in the reproductive organs of rats exposed for intermediate durations (Jorgenson and Rushbrook 1980) or in rats and mice (NCI 1976) and dogs (Heywood et al. 1979) exposed for chronic durations. It is not known whether chloroform exposure induces reproductive effects in humans. Developmental Effects. One study regarding developmental effects in humans after oral exposure to chloroform has been reported (Kramer et al. 1992). The estimated relative risk of low birth weight associated with drinking water sources having chloroform levels of greater than or equal to 10 ug/L was 30% higher than sources with undetectable levels of chloroform. Prematurity was not associated with chloroform/trihalomethane exposure. The estimated relative risk of intrauterine growth retardation associated with drinking water supplies with chloroform concentrations of >10 pg/L was 80% higher than the risk for sources with undetectable levels of chloroform. Sources with intermediate chloroform levels (1-9 ug/L) had an elevated risk of 30%. There seems to be reasonable evidence to suggest that some correlation with an increased risk of intrauterine growth retardation associated with higher concentrations of waterborne chloroform and dichlorobromomethane does exist. Inhalation exposure to chloroform during gestation induced fetotoxicity and teratogenicity in rats (Schwetz et al. 1974) and mice (Murray et al. 1979). Decreased fetal crown-rump length, decreased ossifications, imperforate anus (rats), and cleft palate (mice) were observed in the offspring of exposed dams. In contrast, fetotoxicity (decreased fetal weight), but not teratogenicity, was observed in rats after oral exposure to chloroform (Ruddick et al. 1983; Thompson et al. 1974). Increased resorptions were observed in rats and rabbits (Thompson et al. 1974). In a 2-generation oral study, degeneration of the epididymal ductal epithelium was observed in mice of the F, generation (Gulati et al. 1988). Due to its chemical nature, chloroform can cross the placenta easily, as demonstrated by its detection in the placenta and fetuses of mice a short time after inhalation exposure (Danielsson et al. 1986). Chloroform may accumulate in the amniotic fluid and fetal tissues. Various developmental effects may result from exposure, depending on the period of in utero exposure. Although no studies have conclusively reported developmental effects in humans, chloroform (or in tandem with other organic halomethanes) may have the potential to cause developmental effects in humans. Whether such effects *+DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 124 2. HEALTH EFFECTS could occur from exposure to levels in the environment, in drinking water, or at hazardous waste sites is not known. Genotoxic Effects. In vivo and in vitro studies of the genotoxic effects of chloroform are summarized in Tables 2-5 and 2-6. Information regarding genotoxic effects after in vivo exposure to chloroform is limited. Acute inhalation exposure to chloroform caused an increase in the percentage of abnormal sperm in mice (Land et al. 1979, 1981). Mice exposed to chloroform by gavage had an increase in sister chromatid exchange frequency in bone marrow cells (Morimoto and Koizumi 1983). In contrast, oral exposure to chloroform did not increase unscheduled DNA synthesis in rat hepatocytes (Mirsalis et al. 1982). Chloroform exposure caused mitotic arrest in grasshopper embryos (Liang et al. 1983) and a nonsignificant increase in the recessive lethals in Drosophila melanogaster (Gocke et al. 1981). In general, most of the assays for chloroform genotoxicity are negative. Therefore, it seems that chloroform is a weak mutagen and that its potential to interact with DNA is low. In in vitro experiments, chloroform did not cause reverse mutations in Salmonella typhimurium (Gocke et al. 1981; San Augustin and Lim-Sylianco 1978; Simmon et al. 1977; Uehleke et al. 1977; Van Abbe et al. 1982; Varma et al. 1988) or in Escherichia coli (Kirkland et al. 1981) with or without metabolic activation. Inconclusive results were obtained in Saccharomyces cerevisiae and Schisto- zosaccharomyces pombe (Callen et al. 1980; De Serres et al. 1981). Chloroform, however, induced aneuploidia in Aspergillus nidulans (Crebelli et al. 1988). Chloroform caused forward mutations in L5178Y mouse lymphoma cells after metabolic activation (Mitchell et al. 1988), but did not cause mutations at 8-azaguanine locus in Chinese hamster lung fibroblasts (Sturrock 1977) or sister chromatid exchange in Chinese hamster ovary cells (White et al. 1979). A study performed in mice examined the ability of chloroform to induce unscheduled DNA synthesis in hepatocytes in vitro from 15-week-old female B6C3F, mice. Chloroform concentrations ranged from 0.01 to 10 mmol. Mice were sacrificed at 2 and 12 hours postdosing to determine if and when UDS began to occur. DimethyInitrosamine, a known inducer of UDS, was used as a positive control and did induce UDS in these hepatic cells. No induction of DNA repair was observed at any concentration of chloroform at either the 2-hour or 12-hour post-treatment groups. All concentrations of chloroform added to the cell cultures of mouse hepatocytes proved to be toxic. The study showed that chloroform is not directly genotoxic in hepatocytes of female mice, either in vivo or in vitro, despite the fact that it is the target organ of chloroform carcinogenesis (Larson et al. 1994a). In human lymphocytes, chloroform did not "DRAFT FOR PUBLIC COMMENT*** + LNIWWOO 01N8Nd HO4 13VHQ... Table 2-5. Genotoxicity of Chloroform In Vivo Species (test system) End point Results Reference Mammalian cells: Mirsalis et al. 1982 Rat hepatocytes Unscheduled DNA synthesis —- Mouse bone marrow Sister chromatid exchange - Morimoto and Koizumi 1983 Mouse Sperm abnormalities + Land et al. 1981 Grasshopper embryo Mitotic arrest + Liang et al. 1983 Drosophila melanogaster Recessive lethals — Gocke et al. 1981 Host-mediated assays: Salmonella typhimurium TA1535 Reverse mutation - San Agustin and Lim-Sylianco 1978 (mouse host-mediated assay) S. typhimurium TA1537 Reverse mutation + San Agustin and Lim-Sylianco 1978 (mouse host-mediated assay) (males only) — = negative result; + = positive result; DNA = Deoxyribonucleic acid S103443 H1TV3H 2 WHOJ0dOTHO Sci =» LN3IWWOO 0178Nd HOH L4VvHA... Table 2-6. Genotoxicity of Chloroform In Vitro Results Species (test system) End point With Without Reference activation activation Prokaryotic organism: Salmonella typhimurium TA98 Reverse mutation - - Gocke et al. 1981 S. typhimurium TA100 Reverse mutation - - Gocke et al. 1981 S. typhimurium TA1535 Reverse mutation - = Gocke et al. 1981 S. typhimurium TA1535 Reverse mutation = - Uehleke et al. 1977 S. typhimurium TA1538 Reverse mutation — wo Uehleke et al. 1977 S. typhimurium TA98 Reverse mutation - - Simmon et al. 1977 S. typhimurium TA100 Reverse mutation - - Simmon et al. 1977 S. typhimurium TA1535 Reverse mutation - - Simmon et al. 1977 S. typhimurium TA1537 Reverse mutation - - Simmon et al. 1977 S. typhimurium TA98 Reverse mutation — - Van Abbe et al. 1982 S. typhimurium TA100 Reverse mutation - - Van Abbe et al. 1982 S. typhimurium TA1535 Reverse mutation - - Van Abbe et al. 1982 S. typhimurium TA1537 Reverse mutation - - Van Abbe et al. 1982 S. typhimurium TA1538 Reverse mutation - - Van Abbe et al. 1982 S. typhimurium TA98 Reverse mutation - (+) Varma et al. 1988 S. typhimurium TA100 Reverse mutation + (+) Varma et al. 1988 S. typhimurium TA1535 Reverse mutation - (+) Varma et al. 1988 S. typhimurium TA1537 Reverse mutation - (+) Varma et al. 1988 S. typhimurium TA98 Reverse mutation Not - San Augustin and Lim-Sylianco 1978 tested S. typhimurium TA1535 Reverse mutation Not - San Augustin and Lim-Sylianco 1978 tested S. typhimurium TA1537 Reverse mutation Not — San Augustin and Lim-Sylianco 1978 tested S103443 H1TV3H 2 WHO404HOTHO 992i +. INFWWOD 01N8Nd JO4 14VHC... Table 2-6. Genotoxicity of Chloroform In Vitro (continued) Results Species (test system) End point With Without Reference activation activation Escherichia coli Reverse mutation - - Kirkland et al. 1981 Aspergillus nidulans Aneuploidia + Not tested Crebelli et al. 1988 Saccharomyces cerevisiae Reverse mutation - (+) De Serres et al. 1981 Schizosaccharomyces pombe Recombinations - (+) Callen et al. 1980 Eukaryotic organisms: Mammalian cells: L5178Y mouse lymphoma cells Forward mutation + - Mitchell et al. 1988 Chinese hamster lung fibroblasts Chinese hamster ovary cells Human lymphocytes Human lymphocytes Human lymphocytes Human lymphocytes Mutation at 8—azaquonine Sister chromatid exchange Unscheduled DNA synthesis Sister chromatid exchange Sister chromatid exchange Chromosome aberrations Sturrock 1977 White et al. 1979 Perocco and Prodi 1981 Morimoto and Koizumi 1983 Kirkland et al. 1981 Kirkland et al. 1981 — = negative result; + = positive result; (+) = weakly positive; DNA = Deoxyribonucleic acid S103443 H1TV3H 2 WHO40"O THO L2) CHLOROFORM 128 2. HEALTH EFFECTS induce unscheduled DNA synthesis (Peroccio and Prodi 1981) and did not increase the frequency of sister chromatid exchange and chromosome aberrations (Kirkland et al. 1981). In contrast, increases in sister chromatid exchange were reported after metabolic activation in another study (Morimoto and Koizumi 1983). Cancer. No studies were available regarding cancer in humans or animals after inhalation exposure to chloroform. Epidemiology studies suggest an association between chronic exposure to chlorinated drinking water sources and increased incidences of colon cancer (Young et al. 1981), and bladder cancer (Cantor et al. 1978). The carcinogenic potential of chloroform has been tested in animal studies. A dose-related increase in the incidence of hepatomas was observed in mice exposed to chloroform for intermediate durations (Eschenbrenner and Miller 1945a). Chronic exposure induced an increased incidence of renal adenoma and carcinoma in rats exposed to chloroform in drinking water (Jorgenson et al. 1985) and in rats treated by gavage with chloroform in oil (NCI 1976). Hepatic neoplastic nodules were observed in female rats, and lymphosarcoma was observed in male rats exposed to chloroform in drinking water (Tumasonis et al. 1987). In addition, hepatocellular carcinoma was observed in B6C3F, mice given chloroform in oil by gavage (NCI 1976), and kidney tumors were observed in ICI mice exposed by gavage to chloroform in toothpaste (Roe et al. 1979). The incidence of liver and kidney tumors in male and female rats given chloroform in a chronic duration study was reported by Dunnick and Melnick (1993). No hepatocellular or large intestine neoplasms were noted for male or female rats. Kidney tubular cell neoplasms were observed at 90 mg/kg/day and at 180 mg/kg/day in male rats. Kidney tubular cell neoplasms did occur in female rats, but only at higher doses of chloroform than those given to the male rats. In a similar study performed in mice, hepatocellular neoplasms were recorded. In a another study by the same author, using male mice dosed with similar amounts of chloroform, no kidney tubular cell neoplasms or large intestine neoplasms were reported in either sex of mice. Hepatocellular neoplasms were recorded in both male and female mice. The data concerning mouse liver tumors are conflicting. In contrast to the increased incidence of liver tumors observed in B6C3F, mice exposed by gavage to chloroform in oil (NCI 1976), no increased incidence of liver tumors was observed in female B6C3F, mice exposed to chloroform in drinking water (Jorgenson et al. 1985). This result is consistent with the absence of liver tumor effects in four other strains of mice exposed by gavage to chloroform in toothpaste (Roe et al. 1979). In a ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 129 2. HEALTH EFFECTS pharmacokinetic study, chloroform was absorbed more slowly and to a lesser extent from corn oil than from water (Withey et al. 1983), suggesting that pharmacokinetic effects are not responsible for the differences in liver tumor responses. Nevertheless, data from historical controls indicate that corn oil alone is not responsible for the increased incidence of liver tumors (Jorgenson et al. 1985). The corn oil vehicle effect on mouse liver tumors may be due to an interaction between the vehicle and chloroform (Bull et al. 1986; Jorgenson et al. 1985), possibly resulting in altered pharmacokinetics. Larson et al. (1994b) demonstrated that female B6C3F, mice developed increased hepatocyte cytotoxicity after gavage dosing in oil with chloroform concentrations of 238 and 477 mg/kg/day for 4 days or 3 weeks (as determined by BrdU-labelling of hepatocytes), but not for the same strain of female mice administered up to 1,800 ppm for 4 days or 3 weeks in the drinking water. Other studies have shown similar results (Jorgenson et al. 1985; NCI 1976). The difference in results are most likely due to the method of dosing and the vehicle used, both having profound effects on the pharmacokinetics of chloroform and hence the degree of hepatotoxicity (and perhaps the renal toxicity in males) that chloroform may induce in these animals. The corn oil vehicle may lead to higher overall tissue concentrations than does the water vehicle, resulting in increased cellular damage to susceptible tissues. In addition, gavage dosing may also significantly contribute to the increased cellular damage. Gavaged animals typically receive a large dose of chloroform all at one time over a period of several days, while the animals in the drinking water studies consume somewhat equal amounts of chloroform; however, it is consumed in small sips throughout the day (Larson et al. 1994b). It seems clear that the design of the gavage studies inherently result in repeated, massive doses of chloroform to the liver (and other susceptible cells) that likely overwhelm the liver defense mechanisms for chloroform detoxification, resulting in hepatotoxicity, cell death, or both. Drinking water studies, however, ultimately expose the liver to continuous, low doses of chloroform, resulting in detoxification, elimination, and few apparent signs of hepatocellular damage. Clearly, further studies that describe the differences in pharmacokinetics between dosing method (gavage as opposed to drinking water) and vehicle effects (oil as opposed to water) need to be performed to correctly estimate human risk to orally consumed chloroform. In the liver bioassay for GGTase positive foci, chloroform had neither an initiating effect nor a promoting effect when administered in drinking water (Herren-Freund and Pereira 1987), but had a **DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 130 2. HEALTH EFFECTS promoting effect of these loci initiated by diethylnitrosamine if given in a corn oil vehicle (Deml and Oesterle 1985); both studies were performed in rats. Moreover, chloroform enhanced the growth of experimentally inoculated tumors in mice (Capel et al. 1979). In contrast, chloroform had an inhibiting effect on the growth of tumors induced by known carcinogens (1,2-dimethylhydrazine and ethylnitrosurea) in rats (Daniel et al. 1989; Herren-Freund and Pereira 1987). In epidemiology studies, chloroform is not identified as the sole or primary cause of excess cancer rates, but it is one of many organic contaminants found in chlorinated drinking water, many of which are considered to have carcinogenic potential. These studies are often flawed by a lack of measured chloroform concentrations in drinking water; lack of data concerning concentrations of other organics; limited information concerning personal drinking water consumption; long latency periods; and effects of migration, making it difficult to quantify exposure. Although human data suggest a possible increased risk of cancer from exposure to chloroform in chlorinated drinking water, the data are too weak to draw a conclusion about the carcinogenic potential of chloroform in humans. Based on the positive carcinogenic response in animals, some authors tried to estimate the risk of liver cancer associated with human exposure to chloroform (Reitz et al. 1990). Based on animal studies, chloroform has been classified as a probable human carcinogen by EPA (IRIS 1995), as a possible human carcinogen by IARC (1987), and as a substance that may reasonably be anticipated to be carcinogenic in humans (NTP 1989). 2.6 BIOMARKERS OF EXPOSURE AND EFFECT Biomarkers are broadly defined as indicators signaling events in biologic systems or samples. They have been classified as markers of exposure, markers of effect, and markers of susceptibility (NAS/NRC 1989). Due to a nascent understanding of the use and interpretation of biomarkers, implementation of biomarkers as tools of exposure in the general population is very limited. A biomarker of exposure is a xenobiotic substance or its metabolite(s), or the product of an interaction between a xenobiotic agent and some target molecule(s) or cell(s) that is measured within a compartment of an organism (NAS/NRC 1989). The preferred biomarkers of exposure are generally the substance itself or substance-specific metabolites in readily obtainable body fluid(s) or excreta. However, several factors ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 131 2. HEALTH EFFECTS can confound the use and interpretation of biomarkers of exposure. The body burden of a substance may be the result of exposures from more than one source. The substance being measured may be a metabolite of another xenobiotic substance (e.g., high urinary levels of phenol can result from exposure to several different aromatic compounds). Depending on the properties of the substance (e.g., biologic half-life) and environmental conditions (e.g., duration and route of exposure), the substance and all of its metabolites may have left the body by the time samples can be taken. It may be difficult to identify individuals exposed to hazardous substances that are commonly found in body tissues and fluids (e.g., essential mineral nutrients such as copper, zinc, and selenium). Biomarkers of exposure to chloroform are discussed in Section 2.6.1. Biomarkers of effect are defined as any measurable biochemical, physiologic, or other alteration within an organism that, depending on magnitude, can be recognized as an established or potential health impairment or disease (NAS/NRC 1989). This definition encompasses biochemical or cellular signals of tissue dysfunction (e.g., increased liver enzyme activity or pathologic changes in female genital epithelial cells), as well as physiologic signs of dysfunction such as increased blood pressure or decreased lung capacity. Note that these markers are often not substance specific. They also may not be directly adverse, but can indicate potential health impairment (e.g., DNA adducts). Biomarkers of effects caused by chloroform are discussed in Section 2.6.2. A biomarker of susceptibility is an indicator of an inherent or acquired limitation of an organism's ability to respond to the challenge of exposure to a specific xenobiotic substance. It can be an intrinsic genetic or other characteristic or a pre-existing disease that results in an increase in absorbed dose, biologically effective dose, or target tissue response. If biomarkers of susceptibility exist, they are discussed in Section 2.7, Populations That Are Unusually Susceptible. 26.1 Biomarkers Used to Identify or Quantify Exposure to Chloroform Chloroform concentrations measured in tissue and/or air samples can not be currently be used as specific biomarkers for chloroform exposure; however, they may indicate exposure to chloroform or other halogenated compounds that have undergone metabolism to chloroform. Methods for measuring chloroform in biological fluids, tissues, and exhaled breath are available; however, there is relatively little quantitative information relating monitored chloroform levels in tissues or fluids to exposure levels. The presence of chloroform or its metabolites in biological fluids and tissues may result from ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 132 2. HEALTH EFFECTS the metabolism of other chlorinated hydrocarbons; thus, elevated tissue levels of chloroform or its metabolites may reflect exposure to other compounds. The relationship between chloroform concentration in inspired air and resulting blood chloroform levels is the most well-defined measure of exposure due to the extensive use of chloroform as a surgical anesthetic. A mean arterial blood concentration of 9.8 mg/dL (range 7-16.6 mg/dL) was observed among 10 patients receiving chloroform anesthesia at an inspired air concentration of 8,000—10,000 ppm (Smith et al. 1973). Monitoring of blood levels in workers experiencing toxic jaundice due to chloroform exposure revealed that when workroom air concentrations were estimated to be >400 ppm, the blood samples of 13 workers with jaundice were 0.10-0.3 pg/100 mL blood (Phoon et al. 1983). In another group of 18 workers with toxic hepatitis, blood samples revealed chloroform in some but not all workers, and workroom air contained 14.4-50.4 ppm on various days. These data suggest an association between increased blood concentrations and increased exposure concentrations, but the blood levels varied too greatly to establish a direct quantitative relationship. ~ Environmental exposure to chloroform in humans probably represents a combination of inhalation exposure (from the air polluted with volatile halogenated hydrocarbons) and oral exposure (from chlorinated water sources), in addition to a dermal absorption route for chloroform from contaminated water sources (from showering or bathing). The chloroform levels detected in human blood varied according to geographical areas. Chloroform levels ranged from 13 to 49 pg/L in serum samples taken from 10 individuals in Florida (Peoples et al. 1979). The level of environmental exposure was not reported. The mean blood chloroform concentration was 1.5 pg/L in blood samples taken from 250 individuals in Louisiana; exposure levels were not reported (Antoine et al. 1986). Chloroform was found in breath samples from large cohorts of people from New Jersey, North Carolina, and North Dakota (Wallace et al. 1987a). The levels of chloroform in breathing zone (personal) air were consistently higher than outdoor concentrations and correlated with chloroform concentrations in the exhaled breath samples. Some activities (visiting the dry cleaners or showering) were associated with increased chloroform breath levels (Wallace et al. 1989; Jo et al. 1990a, 1990b). Chloroform was detected in 7 of 42 samples of human milk collected in 4 geographical areas in the United States (Pellizzari et al. 1982). “**DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 133 2. HEALTH EFFECTS Tissue levels of chloroform obtained at autopsy reflected environmental exposure levels in other studies. The levels ranged from 20 to 49 pg/kg of hexane extractable fat in adipose tissue samples taken from 10 individuals in Florida (Peoples et al. 1979). In 30 autopsy cases in Germany, the adipose tissue contained a mean of 23.4 pg/kg wet tissue; 24.8 pg/kg perinephric fat; 10.8 pg/kg liver tissue; 9.9 pg/kg lung tissue; and 10.0 pg/kg muscle tissue (Alles et al. 1988). The maximum chloroform content increased with age and was not dependent on the volume of fat in the tissues. No correlation has been made between the exact environmental levels of chloroform and the amount of chloroform in the exhaled breath or in the blood. Furthermore, chloroform also can be detected in the breath after exposure to carbon tetrachloride and other chlorinated hydrocarbons (Butler 1961). Therefore, chloroform levels cannot be used as reliable biomarkers of exposure to this chemical. 2.6.2 Biomarkers Used to Characterize Effects Caused by Chloroform The primary targets of chloroform toxicity are the central nervous system, liver, and kidney. The signs and symptoms of central nervous system effects (e.g., dizziness, fatigue, headache) are easily recognized. Monitoring liver and kidney effects induced by exposure to low levels of chloroform requires the testing of organ functions. Liver effects are commonly detected by monitoring for elevated levels of liver enzymes in the serum or testing for sulfobromophthalein retention. Urinalysis and measurements of blood urea nitrogen and B-2-microglobin are used to detect abnormalities in kidney function. Because many toxic chemicals can cause adverse liver and kidney effects, these tests are not specific for chloroform. No specific biomarkers used to characterize effects caused specifically by chloroform were located. For more information on biomarkers for renal and hepatic effects of chemicals see ATSDR/CDC Subcommittee Report on Biological Indicators of Organ Damage (1990) and for information on biomarkers for neurological effects see OTA (1990). 2.7 INTERACTIONS WITH OTHER CHEMICALS Clinical reports of patients who underwent chloroform anesthesia indicated that premedication with morphine caused serious respiratory depression when chloroform was co-administered. Thiopentone ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 134 2. HEALTH EFFECTS (thiopental Na, an ultra-short acting barbiturate anesthetic) was associated with increased incidences of hypotension in chloroform-anesthetized patients (Whitaker and Jones 1965). Several animal studies indicate that chloroform interacts with other chemicals within the organism. The lethal and hepatotoxic effects of chloroform were increased by dicophane (DDT) (McLean 1970) and phenobarbital (a long-acting barbiturate) in rats (Ekstrom et al. 1988; McLean 1970: Scholler 1970). Increased hepatotoxic and nephrotoxic effects were observed after interaction with ketonic solvents and ketonic chemicals in rats (Hewitt and Brown 1984; Hewitt et al. 1990) and in mice (Cianflone et al. 1980; Hewitt et al. 1979). The hepatotoxicity of chloroform was also enhanced by co-exposure to carbon tetrachloride in rats (Harris et al. 1982) and by co-exposure to ethanol in mice (Kutob and Plaa 1962). Furthermore, ethanol pretreatment in rats increased the in vitro metabolism of chloroform (Sato et al. 1981). A mixture of cadmium and chloroform potentiated the cytotoxicity of each in in vitro experiments in rat hepatocytes (Stacey 1987a, 1987b). In contrast, mirex did not increase chloroform toxicity in mice (Hewitt et al. 1979). Disulfiram, an inhibitor of microsomal enzymes, decreases the hepatotoxicity of chloroform (Masuda and Nakayama 1982; Scholler 1970). Diethyldithiocarbamate and carbon disulfide pretreatment also protect against chloroform hepatotoxicity (Gopinath and Ford 1975; Masuda and Nakayama 1982, 1983), presumably by inhibiting microsomal enzymes. In general, chloroform toxicity can be influenced by chemicals that alter microsomal enzyme activity or hepatic glutathione levels. The role that dichloroacetate (DCA) and trichloroacetate (TCA) play in chloroform toxicity was studied in rats (Davis 1992). TCA and DCA are formed in conjunction with chloroform during the chlorination of drinking water; therefore, animals drinking chlorinated water may be exposed to all three compounds simultaneously. It was found that DCA increases the hepatotoxicity and nephrotoxicity of chloroform in rats, that TCA increases the nephrotoxicity of chloroform, and that these effects were gender-specific, occurring mainly in females. The effects of monochloroacetate (MCA) on chloroform toxicity has also been investigated, with the combination (MCA + chloroform) shown to have toxic effects on the liver and kidneys of rats (Davis and Berndt 1992). The effect of chloroform and other organic halides (i.e., dichlorobromomethane) on intrauterine growth retardation has also been explored (Kramer et al. 1992). “**DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 135 2. HEALTH EFFECTS Ikatsu and Nakajima (1992) studied the effect of low-dose inhalation of chloroform with or without co-exposure to carbon tetrachloride on hepatotoxicity when rats were or were not previously exposed to ethanol. Groups of control or ethanol-pretreated rats inhaled 0, 50, or 100 ppm chloroform alone; 0, 25, or 50 ppm chloroform with 5 ppm carbon tetrachloride; or 0, 10, 25, or 50 ppm chloroform with 10 ppm carbon tetrachloride. Exposures to either 50 or 100 ppm of chloroform alone did not significantly change SGOT, SGPT, liver or serum malondialdehyde (MDA) concentrations. In the rats pretreated with ethanol, SGOT and SGPT levels were significantly elevated above control animals at 100 ppm chloroform and SGOT levels were increased at 50 ppm chloroform as well. There was no change in either serum or liver concentrations of MDA in either exposure group. Liver triglycerides and GSH levels were significantly elevated above those of control animals for both exposure levels for animals not pretreated with ethanol; however, overall liver weights were elevated at only 100 ppm chloroform. In rats pretreated with ethanol, there was no significant change in liver triglyceride concentrations at either dose; however, liver GSH and liver weights were significantly elevated above control at both exposure concentrations. In chloroform and carbon tetrachloride treated rats not pretreated with ethanol, elevations in SGOT (10 ppm CCl, + 10 and 50 ppm CHCI,), SGPT (5 ppm CCl, + 50 ppm CHCl,, 10 ppm CCl, + 25 ppm CHCl), and plasma MDA (10 ppm CCI, + 10, 25, and 50 ppm CHCI,) were observed. In chloroform and carbon tetrachloride treated rats pretreated with ethanol, elevations in SGOT (all doses), SGPT (all doses except 5 ppm CCl, + 25 ppm CHCl,), liver MDA (all doses), and plasma MDA (5 and 10 ppm CCI, + 50 ppm CHCl, 10 ppm CCl, + 25 ppm CHCl) were observed. In chloroform and carbon tetrachloride treated rats not pretreated with ethanol, elevations in liver triglyceride (all doses) and GSH (5 ppm CCl, + 50 ppm CHCl, 10 ppm CCl, + 10 and 50 ppm CHCl) were observed. In chloroform and carbon tetrachloride treated rats pretreated with ethanol, elevations in liver triglyceride (all chloroform doses at 10 ppm CCl,) and GSH (all doses except 5 ppm CCl, + 25 ppm CHCI,) were observed. The results suggest that chloroform enhances carbon tetrachloride-induced hepatotoxicity. 2.8 POPULATIONS THAT ARE UNUSUALLY SUSCEPTIBLE A susceptible population will exhibit a different or enhanced response to chloroform than will most persons exposed to the same level of chloroform in the environment. Reasons may include genetic makeup, age, health and nutritional status, and exposure to other toxic substances (e.g., cigarette smoke). These parameters may result in reduced detoxification or excretion of chloroform, or compromised function of target organs affected by chloroform. Populations who are at greater risk *»**ADACT CAD DI IRL I CORNRAENIT*** CHLOROFORM 136 2. HEALTH EFFECTS due to their unusually high exposure to chloroform are discussed in Section 5.6, Populations With Potentially High Exposure. Since the liver and kidney are the two main organs responsible for chloroform metabolism, individuals who have hepatic or renal impairment may be more susceptible to chloroform toxicity. Drinking water containing higher than acceptable levels of chloroform for extended periods of time may also increase the risk for toxic side-effects. Also, exhaustion and starvation may potentiate chloroform hepatotoxicity, as indicated in some human clinical reports (Royston 1924; Townsend 1939) and in animal studies (Ekstrom et al. 1988; McMartin et al. 1981). Animal studies indicate that male mice and rats may be more susceptible to the lethal and renal effects of chloroform than female mice and rats (Deringer et al. 1953; Torkelson et al. 1976). The greater susceptibility of adult male animals is associated with testosterone levels in the animals (Deringer et al. 1953). Whether or not human males would be more susceptible than human females to the harmful effects of chloroform is not presently known. 2.9. METHODS FOR REDUCING TOXIC EFFECTS This section will describe clinical practice and research concerning methods for reducing toxic effects of exposure to chloroform. However, because some of the treatments discussed may be experimental and unproven, this section should not be used as a guide for treatment of exposures to chloroform. When specific exposures have occurred, poison control centers and medical toxicologists should be consulted for medical advice. The following texts provide specific information about treatment following exposures to chloroform: Ellenhorn, MJ and Barceloux, DG, (eds.) (1988). Medical Toxicology: Diagnosis and Treatment of Human Poisoning. Elsevier Publishing, New York, NY., pp. 972-974. Dreisback, RH, (ed.) (1987). Handbook of Poisoning. Appleton and Lange, Norwalk, CT. Haddad, LM and Winchester, JF, (eds.) (1990). Clinical Management of Poisoning and Drug Overdose. 2nd edition, WB Saunders, Philadelphia, PA. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 137 2. HEALTH EFFECTS Aaron, CK and Howland, MA (eds.) (1994). Goldfrank’s Toxicologic Emergencies. Appleton and Lange, Norwalk, CT. 2.9.1 Reducing Peak Absorption Following Exposure Human exposure to chloroform may occur by inhalation, ingestion, or by dermal contact. General recommendations for reducing absorption of chloroform include removing the exposed individual from the contaminated area and removing the contaminated clothing. If the eyes and skin were exposed, they should be flushed with water. In order to reduce absorption of ingested chloroform, emesis may be considered unless the patient is comatose, is convulsing, or has lost the gag reflex. Controversy exists concerning use of emesis because of the rapid onset of central nervous system depression, the risk of aspiration of stomach contents into the lungs, and the relative ineffectiveness of this method. In comatose patients with absent gag reflexes, an endotracheal intubation may be performed in advance to reduce the risk of aspiration pneumonia. Gastric lavage may also be used. 2.9.2 Reducing Body Burden Chloroform is not stored to any appreciable extent in the human body and is mostly metabolized to phosgene (see Section 2.3); however, some chloroform may be stored in fat depots in the body. The half-life of chloroform in humans has been calculated to be 7.9 hours following inhalation exposure (Gordon et al. 1988). Furthermore, an oral exposure study found most of the chloroform dose being eliminated within 8 hours postexposure (Fry et al. 1972). Hepatic and pulmonary first pass effect was reported in humans (Chiou 1975). Despite a relatively fast clearance of chloroform from the body, toxic effects may develop in exposed individuals. No method is commonly practiced to enhance the elimination of the absorbed dose of chloroform. Although there is evidence that ethanol pretreatment of rats can increase the in vitro metabolism of chloroform (Sato et al. 1981), such treatment would not be recommended (Kutob and Plaa 1962). 2.9.3 Interfering with the Mechanism of Action for Toxic Effects Target organs of chloroform toxicity are the central nervous system, liver, and kidneys (see Section 2.2). Respiratory, cardiovascular, and gastrointestinal toxic effects have also been reported. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 138 2. HEALTH EFFECTS Studies in animals also indicated that chloroform exposure may induce reproductive and developmental effects and cause cancer. Several studies investigated the possible mechanism for chloroform induced toxicity (see Section 2.5). Proposed mechanisms of chloroform toxicity and potential mitigations based on these mechanisms are discussed below. The potential mitigation techniques mentioned are all experimental. One of the possible mechanisms of chloroform toxicity is thought to be linked to its high lipid solubility and its ability to bind covalently to lipids (Testai et al. 1987). For example, neurotoxic and respiratory effects of chloroform may be due to the interaction of chloroform with gangliosides in neuronal membranes (Harris and Groh 1985) and phospholipids in the surfactant monolayer of the lower respiratory tract (Enhorning et al. 1986), respectively. Another proposed reaction of chloroform and lipids would result in the formation of conjugated dienes which are indicative of lipid peroxidation (De Groot and Noll 1989). Some authors reported that conjugated dienes may play a key role in the hepatotoxicity induced by haloalkanes (Comporti 1985; Recknagel et al. 1982). Others, however, argue that lipid peroxidation alone is not responsible for all changes found in the liver following chloroform exposure (Brown et al. 1974b; Lavigne and Marchand 1974). Instead, it was proposed that the mechanism of chloroform-induced liver and kidney toxicity involved metabolism to the reactive intermediate, phosgene, which binds to proteins of the endoplasmatic reticulum (Pohl et al. 1980a, 1980b). The toxicity of chloroform is increased by inducers of cytochrome P-450 such as phenobarbital (Scholler 1970). The involvement of cytochrome P-450 is further supported by the finding that disulfiram (Scholler 1970) and methoxsalen (Letteron et al. 1987), both inhibitors of microsomal enzymes, decreased the liver injury caused by chloroform in rats and mice, respectively. In addition, pretreatment with diethyldithiocarbamate and carbon disulfide protected mice against chloroform hepatotoxicity as indicated by biochemical and histopathological results (Masuda and Nakayama 1982, 1983; Gopinath and Ford 1975). Similarly, pretreatment of mice with methoxsalen (Letteron et al. 1987) and piperonyl butoxide (Kluwe and Hook 1981) reduced the chloroform-induced nephrotoxicity. Further research to determine which isozymes of P-450 are involved in metabolism to the more harmful metabolite, phosgene, as well as which isozymes are involved in enhancing the elimination of chloroform, could lead to the development of strategies designed to selectively inhibit specific P-450 isozymes, and thus reduce the toxic effects of chloroform. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 139 2. HEALTH EFFECTS Administration of chloroform to laboratory animals resulted in the depletion of renal glutathione, indicating that glutathione reacts with reactive intermediates, thus reducing the kidney damage otherwise caused by the reaction of these intermediates with tissue macromolecules (Hook and Smith 1985; Smith and Hook 1983, 1984; Smith et al. 1984). Similarly, chloroform treatment resulted in the depletion of hepatic glutathione and alkylation of macromolecules (Docks and Krishna 1976). Other studies demonstrated that sulfhydryl compounds such as L-cysteine (Bailie et al. 1984) and reduced glutathione (Kluwe and Hook 1981) may provide protection against nephrotoxicity induced by chloroform. The sulfhydryl compound N-acetylcysteine is an effective antidote for poisoning by acetaminophen, which, like chloroform, depletes glutathione and produces toxicity by reactive intermediates. All mitigations of the chloroform-induced toxicity cited above are experimental. Further studies would be needed for implications of any of these methods to humans. 2.10 ADEQUACY OF THE DATABASE Section 104(i)(5) of CERCLA directs the Administrator of ATSDR (in consultation with the Administrator of EPA and agencies and programs of the Public Health Service) to assess whether adequate information on the health effects of chloroform is available. Where adequate information is not available, ATSDR, in conjunction with the National Toxicology Program (NTP), is required to assure the initiation of a program of research designed to determine the health effects (and techniques for developing methods to determine such health effects) of chloroform. The following categories of possible data needs have been identified by a joint team of scientists from ATSDR, NTP, and EPA. They are defined as substance-specific informational needs that if met would reduce or eliminate the uncertainties of human health assessment. This definition should not be interpreted to mean that all data needs discussed in this section must be filled. In the future, the identified data needs will be evaluated and prioritized, and a substance-specific research agenda will be proposed. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 140 2. HEALTH EFFECTS 2.10.1 Existing Information on Health Effects of Chloroform The existing data on health effects of inhalation, oral, and dermal exposure of humans and animals to chloroform are summarized in Figure 2-6. The purpose of this figure is to illustrate the existing information concerning the health effects of chloroform. Each dot in the figure indicates that one or more studies provide information associated with that particular effect. The dot does not necessarily imply anything about the quality of the study or studies, nor should missing information in this figure be interpreted as a "data need." A data need, as defined in ATSDR’s Decision Guide for Identifying Substance-Specific Data Needs Related to Toxicological Profiles (ATSDR 1989), is substance-specific information necessary to conduct comprehensive public health assessments. Generally, ATSDR defines a data gap more broadly as any substance-specific information missing from the scientific literature. As seen from Figure 2-6, information is available regarding death, systemic effects, and neurological effects in humans after inhalation and oral exposure to chloroform. In addition, information is available regarding carcinogenic effects in humans after oral exposure to chlorinated drinking water. Limited information is available regarding dermal effects in humans after exposure to chloroform. Inhalation and oral studies in animals provide data on death, systemic effects after acute- and intermediate-duration exposure, immunological effects, neurological effects, developmental effects, reproductive effects, and genotoxic effects. Information is available regarding systemic effects and carcinogenic effects in animals after oral exposure to chloroform. The carcinogenic effects after oral exposure is inconsistent and not totally conclusive. In addition, data regarding death and acute systemic effects in animals after dermal exposure to chloroform were located in the available literature. 2.10.2 Identification of Data Needs Acute-Duration Exposure. Clinical reports indicate that the central nervous system, cardiovascular system, stomach, liver, and kidneys in humans are target organs of chloroform toxicity afior inhalation and oral exposure to chloroform (Schroeder 1965; Smith et al. 1973; Whitaker and Jones 1965). These findings are supported by results obtained from acute inhalation and oral exposure studies in animals in which target organs identical to those observed in human studies (central nervous system, liver, and kidney) were identified (Culliford and Hewitt 1957; Jones et al. 1958; Lehmann and Flury 1943; Lundberg et al. 1986; Moore et al. 1982). The data are sufficient to derive an MRL for ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 141 2. HEALTH EFFECTS Figure 2-6. Existing Information on Health Effects of Chloroform © & Systemic © K oF ® © &/ 2 & Sy YAS SSS ESSE S/o SL/L RASS L SEES SSL L/S LSS SEL SES SoR SC SE SS IESE T/C LLY Inhalation ® |O ® e Oral ® ® ® ® ® Dermal Human © & i dS Systemic $ £ > oY Q 2 Q S .O XN Q & OSE S/o S/o SEC /° R/S X82 SS SSE SS SE SE LES LSE ES LSS Inhalation . . e909 ss Oral ® ® ® ® ® ® @® ® @ Dermal . Animal ® Existing Studies ***DRAFT FOR PUBLIC COMMENT"** CHLOROFORM 142 2. HEALTH EFFECTS acute oral exposure. An acute inhalation MRL was derived based on a NOAEL for hepatic effects in mice (Larson et al. 1994c). Lethality studies were conducted in rats and mice after acute inhalation exposure (Deringer et al. 1953; Gehring 1968; Lundberg et al. 1986; Smyth et al. 1962). Similarly, lethal doses were identified after single oral exposure in rats and mice (Bowman et al. 1978; Chu et al. 1982b; Jones et al. 1958; Kimura et al. 1971; Smyth et al. 1962). Information regarding dermal effects in humans and animals after exposure to chloroform is limited. Degenerative changes in the kidney tubules of rabbits were reported in one dermal study (Torkelson et al. 1976). Toxicokinetic data regarding dermal exposure are very limited; however, there is evidence that chloroform can be absorbed through the skin (Tsuruta 1975). Due to its lipophilic quality, chloroform is likely to be distributed in the organism, after dermal exposure, in patterns similar to those for inhalation and oral exposure. Information regarding acute dermal exposure in rodents would be useful to identify target organs and threshold levels of chloroform toxicity. Several in vitro skin models are available that would be adequate for describing the absorption of chloroform through the skin and the effects that differing concentrations of chloroform would have on skin histology. Intermediate-Duration Exposure. An occupational study suggests that the liver is a target organ of chloroform toxicity after inhalation exposure of intermediate duration (Phoon et al. 1983). No data were located regarding intermediate-duration oral and dermal exposure in humans. Several studies were located regarding chloroform toxicity in animals after oral exposure (including three 90-day studies in rats, three 90-day studies in mice, and a >6-week oral study in dogs) (Bull et al. 1986; Chu et al. 1982a, 1982b; Heywood et al. 1979; Jorgenson and Rushbrook 1980; Klaunig et al. 1986; Munson et al. 1982; Palmer et al. 1979); fewer data were located regarding inhalation exposure (Torkelson et al. 1976), and no data were located regarding dermal exposure. In animals, the target organs for chloroform toxicity were identified as the central nervous system, liver, and kidneys. An intermediate-duration oral MRL was derived based on liver effects in dogs (Heywood et al. 1979). An intermediate-duration inhalation MRL was derived based on toxic hepatitis which occurred in humans (Phoon et al. 1983). Pharmacokinetic data regarding dermal exposure to chloroform are limited, but it is known that chloroform can be absorbed through the skin. Intermediate-duration dermal studies in animals would provide information about chloroform toxicity via this exposure route. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 143 2. HEALTH EFFECTS The information would be useful for populations living at or near hazardous waste sites, that may be exposed to chloroform for intermediate durations. Chronic-Duration Exposure and Cancer. Information regarding chronic inhalation exposure to chloroform in humans is limited to occupational studies (Bomski et al. 1967; Challen et al. 1958). The liver and central nervous system are target organs of chloroform toxicity. Regarding chronic oral exposure in humans, limited information is available from a case study reporting hematological, hepatic, and renal effects in an individual who used a cough medicine containing chloroform for 10 years (Wallace 1950) and from a follow-up study of individuals who used a mouthwash containing chloroform for <5 years (De Salva et al. 1975). Animal data indicate that the central nervous system, liver, and kidneys are target organs of chloroform toxicity after chronic oral exposure (Heywood et al. 1979; Jorgenson et al. 1985; NCI 1976; Roe et al. 1979; Tumasonis et al. 1985; 1987). The data are sufficient to derive a chronic oral MRL. No studies were located regarding chloroform toxicity in humans and animals after dermal exposure to chloroform and in animals after inhalation exposure to chloroform. Considering the similar pattern of chloroform toxicity after inhalation and oral exposures for acute and intermediate durations, similar target organs in animals after chronic inhalation exposure to chloroform may be predicted. Nonetheless, studies designed to assess the chronic toxicity of chloroform in animals after inhalation and dermal exposure would be useful to establish dose-response relationships. This information is important to humans occupationally exposed or exposed to contaminated air, water, or soil at or near hazardous waste sites. Epidemiology studies suggest a possible association between chloroform in drinking water and cancer risk. Increased incidences of colon and bladder cancer were identified in separate populations exposed to chlorinated water. Studies in rats and mice indicate that oral exposure to chloroform causes cancer (Jorgenson et al. 1985; NCI 1976; Roe et al. 1979; Tumasonis et al. 1985; 1987). No data were located regarding carcinogenicity in humans and animals following inhalation and dermal exposure to chloroform. Nonetheless, pharmacokinetic data indicate similar toxicokinetics of chloroform after inhalation and oral exposure; therefore, similar targets for carcinogenic effects may be predicted. Data were located suggesting different effects of chloroform depending on the vehicle and method of oral administration. Chloroform in corn oil administered by gavage caused an increased incidence of liver tumors (NCI 1976), while administration of the same dose in drinking water did not (Jorgenson et al. 1985). It was demonstrated, however, that chloroform uptake is much slower from the oil vehicle (Withey et al. 1983). Therefore, the higher cancer incidence cannot be explained merely by the levels ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 144 2. HEALTH EFFECTS of chloroform in tissues. Furthermore, chloroform acted as a promoter rather than an initiator of preneoplastic foci in a rat liver bioassay (Deml and Oesterle 1985). In contrast, some studies indicate that chloroform inhibits the growth of tumors induced by known carcinogens (Daniel et al. 1989; Herren-Freund and Pereira 1987). Animal studies also suggest an epigenetic mechanism for the carcinogenicity of chloroform. Because of these differences, further studies on the possible mechanism of chloroform carcinogenicity would be useful. Genotoxicity. Chloroform has been tested for genotoxicity in several in vitro and in vivo experiments. Its potency to induce mutations seems to be weak. No induction of reverse mutations was observed in prokaryotic systems (Gocke et al. 1981, Kirkland et al. 1981; San Augustin and Lim- Sylianco 1978; Simmon et al. 1977; Uehleke et al. 1977; Van Abbe et al. 1982: Varma et al. 1988). Mixed results were obtained in the induction of mutations in human lymphocytes and Chinese hamster cells in vitro (Kirkland et al. 1981; Mitchell et al. 1988; Peroccio and Prodi 1981; White et al. 1979). Nonetheless, an increase in sperm anomalies and sister chromatid exchanges in the bone marrow of rodents was observed after in vivo exposure (Land et al. 1979, 1981; Morimoto and Koizumi 1983). Cytogenetic analysis of peripheral lymphocytes from exposed individuals would provide useful information about the ability of chloroform to induce mutations in humans. Reproductive Toxicity. No information was located regarding reproductive effects in humans exposed to chloroform via any route or in animals exposed by the dermal route. Increased resorptions were observed in rats and mice after inhalation exposure to chloroform during gestation (Murray et al. 1979; Schwetz et al. 1974) and in rats and rabbits after oral exposure (Thompson et al. 1974). In addition to effects in dams, abnormal sperm were found in mice after inhalation exposure (Land et al. 1979, 1981). Furthermore, exposure-related gonadal atrophy was observed in both sexes of rats following oral exposure to chloroform (Palmer et al. 1979). The results suggest that reproductive organs are a target of chloroform toxicity in animals; however, some inhalation and oral studies in animals do not report any effects. More studies assessing the reproductive function in animals would be useful for the purpose of extrapolating the data to human exposure. Developmental Toxicity. Only one study was located regarding developmental effects in humans exposed to chloroform via an oral route. Animal data indicate that chloroform can cross the placenta. Fetotoxicity effects (decreased birth weight, decreased fetal crown-rump length, increased resorptions) and teratogenicity (acaudate fetuses with imperforate anus, cleft palates) were observed in rats and ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 145 2. HEALTH EFFECTS mice after inhalation exposure to chloroform (Murray et al. 1979; Schwetz et al. 1974). Oral exposure to chloroform-induced fetotoxicity in rats and rabbits (Ruddick et al. 1983; Thompson et al. 1974). Degeneration of the epididymal ductal epithelium (not affecting the fertility) was observed in mice in the F, generation in a 2-generation oral reproductive study (Gulati et al. 1988). No information is available regarding the developmental toxicity of chloroform after dermal exposure. More data regarding developmental toxicity both in humans and in experimental animals (especially after oral and dermal exposure) would be useful to identify the possible risk for humans. Immunotoxicity. No data were located regarding immunological effects in humans after inhalation, oral, or dermal exposure to chloroform. The data obtained from animal studies are limited to one inhalation study in mice and three oral studies in rats and mice (Aranyi et al. 1986; Chu et al. 1982b; Munson et al. 1982). Depressed humoral and cell-mediated immunity were detected; however, the chloroform-induced changes were more serious in the acute exposure study than in the intermediate-duration study, indicating that the changes may be transient. Studies regarding skin sensitization with chloroform were not performed. A battery of immune function tests has not been performed in humans or in animals, but would provide helpful information to support or refute the limited evidence for chloroform immunotoxicity. Neurotoxicity. The central nervous system is a target organ for chloroform toxicity in humans after inhalation and oral exposure. The neurotoxic effect is well documented in studies of patients exposed to chloroform via anesthesia (Featherstone 1947; Smith et al. 1973; Whitaker and Jones 1965) or of individuals who intentionally and accidentally ingested the chemical (Piersol et al. 1933; Schroeder 1965; Storms 1973). Lower chloroform doses produced neurological effects during occupational exposure (Challen et al. 1958). Similarly, neurotoxicity is reported in animal studies involving inhalation and oral exposure to chloroform (Bowman et al. 1978; Jones et al. 1958; Lehmann and Flury 1943). A battery of neurobehavioral tests was conducted in mice after oral exposure to chloroform (Balster and Borzelleca 1982). No data were located regarding chloroform neurotoxicity in humans or animals after dermal exposure to chloroform. Animal studies involving dermal exposure to chloroform would be useful for risk assessment of occupational exposure. Continued research on the toxicity of inhaled and dermally absorbed chloroform in humans when exposed to contaminated water sources during showering would also be useful. More information regarding the mechanism of chloroform-induced neurotoxicity and structural alterations produced in the central nervous system would be helpful. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 146 2. HEALTH EFFECTS Epidemiological and Human Dosimetry Studies. Populations may be exposed to chloroform in the workplace, near hazardous waste sites containing chloroform, from chlorinated water, and from various consumer products that contain chloroform. Limited information was obtained from occupational studies reporting central nervous system and liver effects in exposed workers (Bomski et al. 1967; Challen et al. 1958; Phoon et al. 1983). Reliable dosimetry data correlating occupational exposure with signs of toxic effects would be useful. Epidemiology studies suggest an association between chloroform levels in drinking water and colon, rectal, and bladder cancer in humans (Alavanja et al. 1978; Cantor et al. 1978; Young et al. 1981). All of these studies were limited by a lack of attention to important details (e.g., migration, exposure to other carcinogens). Better designed and better conducted epidemiology studies of occupational exposure would be helpful. The information can be useful to populations living near hazardous waste sites where chloroform is present. In addition, further refining of the PBPK/PD models would further advance our understanding of chloroform tissue dosimetry in humans and animals. Biomarkers of Exposure and Effect. Exposure. Methods for detecting chloroform in exhaled breath, blood, urine, and tissues are available. Nevertheless, it is difficult to correlate chloroform levels in biological samples with exposure, because of the volatility and short half-life of chloroform in biological tissues. Several studies monitored chloroform levels in environmentally exposed populations (Antoine et al. 1986; Hajimiragha et al. 1986; Peoples et al. 1979); however, the measured levels probably reflect both inhalation and oral exposure. Moreover, increased tissue levels of chloroform or its metabolites may reflect exposure to other chlorinated hydrocarbons. Studies to better quantitate chloroform exposure would enhance the database. Effect. No biomarkers were identified that are particularly useful in characterizing the effects induced by exposure to chloroform. The target organs of chloroform toxicity are the central nervous system, the liver, and kidneys; however, damage to these organs may result from exposure to other chemicals. More effort to identify subtle biochemical changes to serve as biomarkers of effects of chloroform exposure would be useful in detecting early, subtle signs of chloroform-induced damage. Absorption, Distribution, Metabolism, and Excretion. Human data indicate that chloroform absorption from the lungs is rapid and fairly complete (Smith et al. 1973). The data also indicate that “**DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 147 2. HEALTH EFFECTS absorption after oral exposure is fairly complete for both animals and humans (Brown et al. 1974a; Fry et al. 1972; Taylor et al. 1974). Although there are no experimental data regarding dermal absorption in humans, some data have been extrapolated from mouse studies (Tsuruta 1975). The rate of absorption following oral or inhalation exposure is rapid (within 1-2 hours). Additional studies investigating the rate of dermal absorption would be useful to quantitate dermal absorption and to compare information from oral and inhalation studies. Data are available regarding the distribution of chloroform in humans and animals after inhalation and oral exposure to chloroform (Brown et al. 1974a; Chenoweth et al. 1962; Cohen and Hood 1969; Corley et al. 1990; Danielsson et al. 1986; Feingold and Holaday 1977; Taylor et al. 1974). It appears that distribution following oral exposure is similar to that following inhalation exposure. Another well-conducted animal study focusing on distribution and excretion after dermal exposure would be useful to assess exposure via this route. The metabolic pathway of chloroform is well understood. It appears that both the mode of oral administration and the vehicle affect metabolism. Additional data investigating the mode and vehicle of administration would be useful in order to understand the role of these factors in the mechanism of chloroform’s toxicity. In addition, the fate of the reactive metabolites of chloroform should be monitored. The excretion of chloroform and its metabolites is understood, based on human and animal data derived from oral and inhalation studies (Brown et al. 1974a; Corley et al. 1990; Fry et al. 1972; Taylor et al. 1974). The major route of chloroform elimination is pulmonary, but minor pathways are through enterohepatic circulation, urine, and feces. There are no human or animal data regarding excretion of dermally applied chloroform. Comparative Toxicokinetics. Target organs for chloroform distribution appear to be similar in humans and animals, according to inhalation studies (Corley et al. 1990; Feingold and Holaday 1977). Nonetheless, human and animal studies indicate that there are large interspecies differences in chloroform metabolism and tissue partition coefficients (Brown et al. 1974a; Corley et al. 1990). Marked sex-related differences in tissue distribution and covalent binding to tissue macromolecules in mice also have been observed (Taylor et al. 1974). Excretion data indicate that humans and nonhuman primates excrete chloroform in the breath primarily as unchanged chloroform; mice ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 148 2. HEALTH EFFECTS eliminated almost 80% of an oral chloroform dose as carbon dioxide (Brown et al. 1974a). Thus, toxicokinetic data indicate that it may be difficult to compare the toxicokinetics of chloroform in animals with that in humans. There are a large number of oral studies, relatively few inhalation studies, and almost no dermal studies regarding the toxicokinetics of chloroform. Quantitative toxicokinetic studies in several animal species involving exposure to chloroform via all three routes, especially inhalation and dermal, would help complete the database. Methods for Reducing Toxic Effects. Protective clothing and protective breathing devices may be used to prevent exposure to large amounts of chloroform, although for everyday low exposures to chloroform, these methods are obviously impractical. General procedures such as flushing the skin with water following dermal exposure and emesis or gastric lavage following oral exposure may be used to reduce absorption of chloroform. However, specific medical treatments that prevent absorption of chloroform have not been identified. Such mechanisms might be beneficial because they might be more effective than general procedures and might involve less risk than procedures such as emesis. Ways to enhance elimination of chloroform from the body are not known. Although chloroform is eliminated fairly rapidly, methods to accelerate elimination without producing toxic metabolites would be helpful in reducing toxicity. The mechanism by which chloroform produces toxicity appears to involve metabolism by phenobarbital-inducible isozymes of cytochrome P-450 to phosgene (Pohl et al. 1980a, 1980b). Development of methods to selectively inhibit the P-450 isozymes responsible for this reaction might reduce chloroform toxicity. There is also evidence that glutathione conjugates with reactive products of chloroform metabolism, providing protection from damaging effects (Docks and Krishna 1976; Hook and Smith 1985). Development of a method to maintain high tissue glutathione levels following exposure to chloroform might have a mitigating effect on toxicity. Therefore, although no treatments are currently available to block the toxic action of chloroform or repair damage caused by this chemical, there are indications that further research in this area would enable identification of such treatments. 2.10.3 Ongoing Studies A few ongoing studies involving chloroform have been identified. The effects of volatile anesthetics on the N-methyl-D-aspartate (NMDA) receptor-channel complex are being studied. Specific aims are to determine the effects of several volatile anesthetics (halothane, enflurane, isoflurane, diethyl ether, cyclopropane, nitrous oxide and chloroform) on ligand binding to glutamate binding sites on the “**DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 149 2. HEALTH EFFECTS NMDA receptor complex; to study ligand binding to the glycine modulatory site, glutamate, glycine, divalent cation and spermidine activation of NMDA receptor ion channels; and to examine NMDA- receptor mediated changes in calcium content of rat brain microvesicles (Aronstam 1994). Mechanistic work on the hepatotoxicity and toxigenic sequence will be studied in vitro with suspensions of hepatocytes exposed to carbon tetrachloride and other agents known to alter calcium homeostasis and stimulate phospholipase A, (bromotrichloromethane and chloroform) (Glende 1994). Other research will investigate the toxicity and bioaccumulation of a mixture of sediment contaminants (trichlorethylene, lead, benzene, chloroform, phenol, chromium, and arsenic) in several species of invertebrates and fish. Uptake and depuration will be measured in chironomids (Chironomus riparius), and pharmacokinetic models will be developed to describe bioaccumulation of these sediment contaminants (Clements 1994). Studies by Yang (1994) will evaluate age- and dosing-related changes in pharmacokinetics, biochemical markers, liver cell proliferation, and histopathology in male Fischer 344 rats chronically exposed (up to 2 years) to low levels of a chemical mixture of 7 organic and inorganic groundwater pollutants (including arsenic, benzene, chloroform, chromium, lead, phenol, trichloroethylene). Also, this research will further explore the pharmacokinetic modeling of chemical mixtures and incorporate time-course information on biochemical markers, cell proliferation and histopathology into pharmacokinetic and pharmacodynamic modeling. A study by the Japan Industrial Safety and Health Association (Japan Bioassay Lab) that explores the toxicity of chloroform inhaled over a 2-year period (6 hours/day, 5 days/week) in male and female Fischer 344 rats and BDF, mice is reportedly close to completion (Matsushima 1994). Efforts are also being made to develop a high- efficiency activated carbon granule for drinking water treatment that can remove water contaminants, including chloroform (Mieville 1992). ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 151 3. CHEMICAL AND PHYSICAL INFORMATION 3.1 CHEMICAL IDENTITY Information regarding the chemical identity of chloroform is located in Table 3-1. 3.2 PHYSICAL AND CHEMICAL PROPERTIES Information regarding the physical and chemical properties of chloroform is located in Table 3-2. No information was found regarding rate constants associated with the degradation of chloroform or its reaction with metal ions or other species expected to be found in the environment. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 152 3. CHEMICAL AND PHYSICAL INFORMATION Table 3-1. Chemical Identity of Chloroform Characteristic Information Reference Chemical name Trichloromethane SANSS 1990 Synonym(s) Metheny! chloride, IARC 1979 methane trichloride, methyl trichloride, formyl trichloride Registered trade name(s) Freon 20, R 20, IARC 1979 R 20 refrigerant Chemical formula CHCly Weast 1988 Chemical structure Cl IARC 1979 H—C —2cl Cl Identification numbers: CAS registry 67-66-3 Weast 1988 NIOSH RTECS FS 9100000 HSDB 1994 EPA hazardous waste Uo44 HSDB 1994 OHM/TADS 7216639 HSDB 1994 DOT/UN/NA/IMCO shipping Chloroform; UN 1888 HSDB 1994 HSDB 56 HSDB 1994 NCI C0O2686 HSDB 1994 CAS = Chemical Abstracts Services; DOT/UN/NA/IMCO = Department of Transportation/United Nations/ North America/International Maritime Dangerous Goods Code; EPA = Environmental Protection Agency; HSDB = Hazardous Substances Data Bank; NCI = National Cancer Institute; NIOSH = National Institute for Occupational Safety and Health; OHM/TADS = Oil and Hazardous Materials/Technical Assistance Data System; RTECS = Registry of Toxic Effects of Chemical Substances ***DRAFT FOR PURI IC COMMENT *** CHLOROFORM 153 3. CHEMICAL AND PHYSICAL INFORMATION Table 3-2. Physical and Chemical Properties of Chloroform Property Information Reference Molecular weight 119.38 Deshon 1979 Color Colorless Hawley 1981 Physical state Liquid Deshon 1979 Melting point -63.2 °C Deshon 1979 -64 °C Verschueren 1983 -63.5 °C Weast 1988 Boiling point 61.3 °C Deshon 1979 62 °C Verschueren 1983 61.7 °C Weast 1988 Density: at 20 °C 1.485 glcm® Hawley 1981 Odor Odor threshold: Water Air Solubility: Water at 25 °C Organic solvent(s) Partition coefficients: Log Kow Log Ky. Vapor pressure at 20 °C Henry's law constant: at 20 °C at 24.8 °C Autoignition temperature Flashpoint Flammability limits Conversion factors in air (20 °C) in air (20 °C) Explosive limits Pleasant, ethereal, nonirritating 2.4 ppm (W/V) 85 ppm (V/V) 7.22x10% mg/L 9.3x10% mg/L Miscible with principal organic solvents 1.97 1.65 159 mm Hg 160 mm Hg 3.0103 atm-m%/mol 3.67x1 03 atm-m°/mol >1,000 °C None No data 1 ppm (v/v)=4.96 mg/m® 1 mg/m°=0.20 ppm (V/V) No data Deshon 1979 Amoore and Hautala 1983 Amoore and Hautala 1983 Banerjee et al. 1980 Verschueren 1983 Deshon 1979 Hansch and Leo 1985, Verschueren 1983 Sabljic 1984 Boublik et al. 1984 Verschueren 1983 Nicholson et al. 1984 Gossett 1987 Deshon 1979 Deshon 1979 No data Calculated Calculated No data v/v = volume per volume; w/v = weight per volume ***DRAFT FOR PUBLIC COMMENT"** CHLOROFORM 155 4. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL Chloroform is used primarily in the production of the refrigerant fluorocarbon-22 (chlorodifluoro- methane) and in the production of fluoropolymers. Chloroform has also been used as a solvent, a heat transfer medium in fire extinguishers, an intermediate in the preparation of dyes and pesticides, and other applications highlighted below. Its use as an anesthetic has been largely discontinued. It has limited medical uses in some dental procedures and in the administration of drugs for the treatment of some diseases. 4.1 PRODUCTION The chlorination of methane and the chlorination of methyl chloride produced by the reaction of methanol and hydrogen chloride are the two common methods for commercial chloroform production (Ahlstrom and Steele 1979; Deshon 1979). The methanol process accounts for =92% of the production capacity, while the methane process accounts for only 8%. The Vulcan Materials Co., Wichita, Kansas, is the only known production facility that uses the methane process, which is used for one-third of the plant’s annual production capacity (SRI 1990). One U.S. manufacturer began chloroform production in 1903, but significant commercial production was not reported until 1922 (IARC 1979). Since the early 1980s, chloroform production increased by 20-25%, due primarily to a higher demand for fluorocarbon-22, the major chemical produced from chloroform. The Montreal Protocol established goals for phasing out the use of a variety of ozone- depleting chemicals, including most chlorofluorbarbons (CECs). Fluorocarbon-22 was one of the few fluorocarbons not restricted by the international agreement. Chloroform is used in the manufacture of fluorocarbon-22, and an increase in the production of this refrigerant has led to an increase in the demand for chloroform (CMR 1989). This increase in U.S. production, based on information compiled in the trade journal Chemical & Engineering News (CEN 1994), is summarized in Table 4-1. The manufacturers and sites of major chloroform production facilities listed for 1993 and 1994 (SRI 1993, 1994) include the following: Dow Chemical U.S.A., Freeport, Texas, and Plaquemine, Louisiana; Occidental Petroleum Corp., Belle, West Virginia; and Vulcan Materials Co., Geismar, Louisiana, and Wichita, Kansas. Estimated annual production capacity (SRI 1993) from these facilities **DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 156 4. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL Table 4-1. U.S. Production of Chloroform Year U.S. production U.S. production (in millions of pounds) (in millions of kg) 1983 362 164 1984 405 183 1985 275 124 1986 422 191 1987 462 209 1988 524 237 1989 588 266 1990 484 219 1991 505 229 1992 na na 1993 476 215 Taken from Chemical & Engineering News 1994 ""*DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 157 4. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL as of January 1, 1993, was 460 million pounds (208 million kg). Each of these facilities have production capacity of at least 30 million pounds (13.6 million kg) per year. Table 4-2 lists the facilities in each state that manufacture or process chloroform, the intended use, and the range of maximum amounts of chloroform that are stored on site. The data listed in Table 4-2 are derived from the Toxics Release Inventory (TRI92 1994). Only certain types of facilities were required to report; therefore, this is not an exhaustive list. In some cases, facility names are not available or numeric values for amounts of chloroform produced, stored, transferred, or released are missing. This complicates making comparisons between the TRI listings and information from other information sources. 4.2 IMPORT/EXPORT In 1985, the United States imported 27.6 million pounds (12.5 million kg) of chloroform; 24 million pounds (10.8 million kg) of chloroform were imported into the United States in 1988 (CMR 1989; HSDB 1994). More recent U.S. import figures from the National Trade Data Bank (NTDB 1994) are: U.S. Exports for Year Quantity (kg) 1991 9,460,747 1992 6,038,483 1993 8,467,294 Comparison of these import statistics suggests a slight decrease in chloroform imports from the 1980s into the 1990s. In 1985, 33.5 million pounds (15.2 million kg) of chloroform were exported (HSDB 1994); for 1988, exports of 40 million pounds (18.1 million kg) were estimated (CMR 1989). More recent export figures from the National Trade Data Bank (NTDB 1994) are listed below: U.S. Exports for Year Quantity (kg) 1989 26,756,412 1990 21,897,011 1991 23,709,482 1992 20,131,629 ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 158 4. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL Table 4-2. Facilities That Manufacture or Process Chloroform Range of maximum amounts on site Number of in thousands state® facilities of pounds b Activities and uses® AK 2 0-1 1, 5, 6 AL 1" 0-100 1, 5,6, 12, 3 AR 5 0-1000 1, 5,6, 7 AZ 1 0-1 1, 5 CA 4 0-1000 1,5,7, 13 co 1 10-100 1" cT 1 100-1000 1 FL 6 0-1 1, 5 GA 6 0-10 1, 5, 6 10 1 1-10 1, 6 IL 2 10-1000 1" IN 3 10-100 1, 3 KS 3 100-50000 1, 3,4, 7, 13 KY 6 0-10000 1, 3,5,6,7, 10 LA 15 0-50000 1,2,3,4,5,6,7, 13 MD 1 0-1 1. 5 ME 7 0-1 1, 5, 6 MI 7 0-10000 1,5,6,7, 1,13 MN 3 0-100 1, 5, 8, 12 MO 2 1-100 8, 1 MS 2 0-10 1, 5, 6 MT 1 0-1 %.5 NC 5 0-100 1.5.6, 1 NH 1 0-1 1, S NJ 5 0-1000 1,5, 7,10, 11 NY 3 0-10 5. OH S 0-100 1, 5, 10, 1 0K 1 0-0 1, 5 OR 4 0-10 1,5, 1 PA 7 0-100 1, 5, 1 PR 4 0-1000 1" SC 5 0-10 1, 5, 8, 10 ™ 2 0-1 1,8 ™@ 16 0-50000 1,4,5,6,7, 12, 13 VA 4 0-1000 1, 5, 11 vT 1 100-1000 8 WA 1" 0-10 1, 5, 6 WI 13 0-100 1, 5,7, 11, 13 wv 4 10-10000 1, 4,5, 9,10, 11, 13 3Post office state abbreviations used ®Data in TRI are maximum amounts on-site at each facility CActivities/Uses 1. Produce 5. As a by-product 9. As a product component 13. Ancillary or other 2. Import 6. As an impurity 10. For repackaging uses 3. For on-site use/processing 7. As a reactant 11. As a chemical processing aid 4. For sale/distribution 8. As a formulation component 12. As a manufacturing aid ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 159 4. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL These statistics suggest that while exports declined during the late 1980s to early 1990s, recent export levels are presently comparable to those for the mid-1980s. 4.3 USE The major use for chloroform (CMR 1989) is in the manufacture of the refrigerant fluorocarbon-22 (chlorodifluoromethane). Chemical plants that manufacture significant amounts of fluorocarbon-22 are operated by Allied-Signal Inc. in Baton Rouge, Louisiana, and El Segundo, California; and E.I. du Pont de Nemours and Company in Louisville, Kentucky, and Montague, Michigan (SRI 1990; TRI92 1994). Chloroform has been used in the past as a solvent or an extraction solvent for fats, oils, greases, resins, lacquers, rubber, alkaloids, gums, waxes, gutta-percha, penicillin, vitamins, flavors, floor polishes, and adhesives in artificial silk manufacture. It is also used as a dry cleaning spot remover, in fire extinguishers, as an intermediate in the manufacture of dyes and pesticides, and as a fumigant (Deshon 1979; Windholz 1983). Chloroform was previously used as an anesthetic, but it has been replaced by safer and more versatile materials (Deshon 1979). The U.S. FDA banned chloroform use in drug, cosmetic, and food packaging products in 1976 (Windholz 1983). This ruling did not include drug products that contain chloroform in residual amounts resulting from its use as a processing solvent in manufacturing or its presence as a by-product from the synthesis of drug ingredients (IARC 1979). Chloroform can still apparently be used as a local anesthetic and solvent in certain dental endodontic (gutta-percha root canal) surgery procedures (McDonald and Vire 1992). Topically applied aspirin-chloroform mixtures are also used to relieve pain from severe cases of herpes zoster (shingles) or post-therapeutic neuralgia (King 1993). The most common chloroform exposure opportunities are related less to any commercially produced form of the chemical than to chloroform generated when organic materials come in contact with chlorinated oxidants (e.g., chlorine or hypochlorous acid) widely used to purify water or remove pathogens from waste materials (see Chapter 5). Chlorinated oxidants at NPL sites can also react with organics to release chloroform. Microbial biodegradation processes may also generate chloroform from the breakdown of other chlorinated aliphatic compounds. ***DRAFT FOR PUBLIC COMMENT"*** CHLOROFORM 160 4. PRODUCTION, IMPORT/EXPORT, USE, AND DISPOSAL 4.4 DISPOSAL According to the 1992 Toxics Release Inventory (TRI), the amount of chloroform released to land is only a small fraction of the total amount of chloroform released to the environment by facilities that produce and process the chemical (see Section 5.2.3) (TRI92 1994). In addition, transfer of chloroform to off-site locations and discharges to publicly-owned treatment works appear to be relatively minor, with the exception of a handful of facilities. The TRI data should be used with caution since only certain types of facilities are required to report. This is not an exhaustive list. Chloroform has been identified as a hazardous waste by EPA, and disposal of this waste is regulated under the Federal Resource Conservation and Recovery Act (RCRA) (EPA 1988a, 1989b). Specific information regarding federal regulations on chloroform disposal on land is available in the Code of Federal Regulations (EPA 1988a, 1989b). Ultimate disposal of chloroform, preferably mixed with another combustible fuel, can be accomplished by controlled incineration. Complete combustion must be ensured to prevent phosgene formation, and an acid scrubber should be used to remove the haloacids produced. Chloroform also is a potential candidate for liquid injection incineration. Because chloroform has been used in some pesticides, the disposal of containers for these pesticides may be relevant. Combustible containers from organic or many metallo-organic pesticides could be disposed of in pesticide incinerators or in specified landfill sites. Noncombustible containers could be disposed of in a designated landfill or recycled (HSDB 1994). No data were located regarding the approximate amounts of chloroform disposal. Chapter 7 provides more details on federal or state regulations governing the disposal of chloroform. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 161 5. POTENTIAL FOR HUMAN EXPOSURE 5.1 OVERVIEW Chloroform is both a synthetic and naturally occurring compound, although anthropogenic sources are responsible for most of the chloroform in the environment. Chloroform is released into the environment as a result of its manufacture and use; its formation in the chlorination of drinking water, municipal and industrial waste water, and swimming pool and spa water; and from other water treatment processes involving chlorination. Most of the chloroform released into the environment will eventually be released into the atmosphere. Volatilization from water and soil is a major source of chloroform release to the atmosphere. Volatilization half-lives from water for a model shallow river and pond were 3.5 and 44 hours, respectively. In the atmosphere, chloroform may be transported long distances before ultimately being degraded by indirect photochemical reactions (half-disappearance time of =80 days) with such free radicals as hydroxyl. The compound has been detected in ambient air in locations that are remote from anthropogenic sources. Chemical hydrolysis is not a significant removal process. While microbial biodegradation can take place, such reactions are generally possible only at fairly low concentration levels due to chloroform’s toxicity. Because of its low soil adsorption and slight but significant water solubility, chloroform will readily leach from soil into groundwater. In groundwater, chloroform is expected to persist for a long time. The general population is exposed to chloroform by ingesting water and food, inhaling contaminated air, and possibly through dermal contact with chloroform-containing water. Generalizations can be made concerning the chloroform concentrations in the environment. Background air concentrations appear to be in the sub-ppb range, but certain urban, indoor, and source-dominated areas appear to have elevated concentrations when compared to background concentrations. Drinking water levels as high as 311 ppb have been reported in public water supplies, although most of the reported concentrations ranged between 2 and 44 ppb. Levels in drinking water derived from groundwater contaminated with leachate from landfills and hazardous waste sites can be much higher. Except for a few special surveys, regular testing for chloroform or other trihalomethanes (THMs) has focused on larger community water treatment systems serving at least 10,000 people. No information was located regarding the concentrations found in ambient soil. Chloroform has also been detected in the ppb range in certain foods. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 162 5. POTENTIAL FOR HUMAN EXPOSURE Occupational exposure to higher than background levels of chloroform can be expected to occur in various occupations, although very little quantitative exposure data were located. Populations with the highest potential exposures appear to be workers employed in or persons living near industries and facilities that manufacture or use chloroform, operators and individuals who live near municipal and industrial waste water treatment plants and incinerators, and paper and pulp plants, and persons who derive their drinking water from groundwater sources contaminated with leachate from hazardous waste sites. Chloroform has been identified in at least 686 of 1,416 hazardous wastes sites that have been proposed for inclusion on the EPA National Priorities List (NPL) (HazDat 1995). However, the number of sites evaluated for chloroform is not known. Figure 5-1 shows the distribution of sites in the continental United States; there are 719 such sites. In addition, there are 7 sites in the Commonwealth of Puerto (not shown in Figure 5-1). 5.2 RELEASES TO THE ENVIRONMENT 5.2.1 Air According to the Toxic Chemical Release Inventory (TRI), in 1992, releases of chloroform to the air from 182 large processing facilities were 8,413,971 kg (17,034,926 pounds) (TRI92 1994). The releases of chloroform to air from facilities that manufactured and processed it in the United States during 1992 are reported in Table 5-1 (TRI92 1994). In 1992, total releases to all environmental media were reported as 8,776,153 kg (17,768,200 pounds). Therefore, releases to the air constitute over 95% of the releases to the total environment documented in the TRI. The TRI data should be used with caution because only certain types of facilities are required to report. This is not a exhaustive list. Current comprehensive quantitative data or estimates of chloroform releases to the atmosphere are lacking. Direct release to the atmosphere is expected to occur during the manufacture, loading, and transport of chloroform (EPA 1985a, 1985b). Indirect chloroform releases have resulted from its use in the manufacture of fluorocarbon-22, fluoropolymers, pharmaceuticals, ethylene dichloride, dyes, and fumigants (Deshon 1979; EPA 1985a, 1985b; HSDB 1994: Windholz 1983). Chloroform releases result from its formation and subsequent volatilization from chlorinated waters including drinking ***DRAFT FOR PUBLIC COMMENT*** .+»LNIWWOO 0118Nd HO4 14vHQ... FIGURE 5-1. FREQUENCY OF NPL SITES WITH CHLOROFORM CONTAMINATION * 3HNSOdX3 NVANH HO TVILN3LOd 'S FREQUENCY BEFFH 1 TO 10 SITES BEER 11 TO 32 SITES 50 TO 56 SITES BE “0 SITES Derived from HazDat 19985 WHO404HO THO £91 ««INJWWOD 0178Nd HOH 14vHA... Table 5-1. Releases to the Environment from Facilities that Manufacture or Process Chloroform Range of reported amounts released in pounds per year® Number of Underground Total POTW off-site State” facilities Air Veter Land injection envirorment® transfer waste transfer AK 2 3460000-379050 28000-53200 0 0 388000-432250 0 0 AL n 13219-630000 0-18000 0-5000 0 13219-648000 0 0-92142 AR S 1-166000 0-3600 0-5 0 1-166530 0 0-1907 AZ 1 45569 0 0 0 45569 0 0 CA 4 0-42700 0-48000 0 0 57-90700 0 0 co 1 500 0 0 0 500 250 74098 cr 1 2400 20800 0 0 23200 250 35700 FL é 26005-226100 0-11200 0-90 0 26005-237300 0-63000 0-300 GA 6 29300-470000 600-15000 0-920 0 30050-474270 0 0-414 10 1 159000 2950 5 0 161955 0 0 I 2 260-4000 0 0 0 260-4000 0-99 16637-224072 IN 3 120-10000 0-5 0 0 120- 10005 0 5-48800 KS 3 14-73245 0 0 0-50154 14-123399 0 0-11124 KY 6 796-172000 0-8200 0-250 0 796- 180450 0-550 0-23765 LA 15 10-263000 0-15600 0-895 0-13 26-263250 0 0-6000 MD 1 17000 0 0 0 17000 30000 0 ME 7 32000-425000 980-6420 0-240 0 33600-431420 0 0-734 L)| 7 32-44000 0-580 0-85 0 32-44610 0-18000 0-12250 | 3 16600-53000 0-4200 0-29 0 20829-53000 0-18540 0-800 MO 2 11450-99000 0 0 0 11450-99000 250-4000 117600- 160000 MS 2 39200-247000 3100-12000 19-21 0 42319-259021 0 0 MT 1 77300 800 0 0 78100 0 0 NC S 10000- 1040000 0-40000 0-250 0 11000- 1080000 0-39000 0-1100 NH 1 4e773 96 19 0 44888 0 0 NJ 5 70-20000 0-630 0 0 70-20630 0-15000 0-61669 NY 3 1615-36000 0-1500 0-14 0 1615-37500 0-15 0-29435 OH 5 123-74000 0-1500 0 0 123-75500 0-800 52- 19000 3HNSOdX3 NVWNH HO4 TVILNILOd 'S WHO40HO THO vol wax LINJGNNOD VI idl Id 80d 137 0Usss Table 5-1. Releases to the Environment from Facilities that Manufacture or Process Chloroform (continued) Range of reported amounts released in pounds per year" Number of Underground Total POTW off-site state” focilities Afr Water Land injection envi rorment® transfer waste transfer oK 1 102400 250 250 0 102900 0 0 OR 4 1000- 154740 0-5400 0-137 0 1005-160277 0-100000 0-17500 PA 7 9300-295000 0-4283 0-9 0 9303 - 296005 0-67200 0-15600 PR 4 0-35410 0 0 0 0-35410 0-62300 458-151487 SC 5 250-381095 0-3550 0 0 250-384645 0 0-250 ™ 2 38000-42131 1900-6527 0-175 0 39900-48833 0 0-3 ™ 16 5-650000 0-5300 0-17000 0-73 5-650000 0-100000 0-1397459 VA 4 43423-310000 20-4687 0-58 0 48115-310100 0 0 v1 1 1 0 0 0 1 0 383600 WA 1" 9000-331000 600- 104020 0-5 0 22200-435020 0 0-1 }] 13 500- 185800 0-4900 0-250 0 500- 186050 0-13910 0-42000 w 4 5502-22508 0-624 0 0 5502-23062 0-1 0-541 Source: TRI92 1994 aData in TRI are maximum amounts released by each facility post office state abbreviations used POTW = Publically owned treatment works “The sum of all releases of the chemical to air, land, water, and underground injection wells by a given facility 34NSOdX3 NVYIWNH HOH TVILN3L1Od 'S WHO404HO0 THO S91 CHLOROFORM 166 5. POTENTIAL FOR HUMAN EXPOSURE water, municipal and industrial waste waters, process waters and effluent from the bleaching of pulp in pulp and paper mills, cooling tower water, and swimming pool and whirlpool spa water (Benoit and Jackson 1987; EPA 1985a, 1985b; Hoigne and Bader 1988). Increased release rates of the chloroform in waters can be expected from chloroform-containing waters that are heated (e.g., water used for cooking, showers, swimming pools, and spas). Aeration and use of groundwater contaminated with chloroform are a potential source of emission to the atmosphere (Crume et al. 1990). Chloroform is released as a result of hazardous and municipal waste treatment processes. The chloroform released may have initially been present in the waste or possibly formed during chlorination treatment (Corsi et al. 1987; EPA 1990b: Namkung and Rittmann 1987). Releases may also occur from hazardous waste sites and sanitary landfills where chloroform was disposed, and from municipal and hazardous waste incinerators that burn chloroform-containing wastes or produce chloroform during the combustion process (LaRegina et al. 1986; Travis et al. 1986). In the past, minor releases may have resulted from the use of consumer products (e.g., certain air deodorizers and cleaning products) that contained chloroform as a component or residual product (Bayer et al. 1988; Wallace et al. 1987a). Chloroform is widely used in laboratory work as an extractant. It is also still used in certain medical procedures, such as dental root canal surgeries (McDonald and Vire 1992), and in combination with aspirin as an experimental treatment for serious cases of herpes zoster (King 1993). These medical uses are extremely limited and would contribute very minor amounts of chloroform as releases to the air. 5.2.2 Water In 1992, releases of chloroform to the water from as many as 167 large processing facilities were 323,250 kg (654,452 pounds) (TRI92 1994). The releases of chloroform to water from facilities that manufactured and processed it in the United States during 1992 are reported in Table 5-1 (TRI92 1994). In 1992, total releases to all environmental media were reported as 8,776,153 kg (17,768,200 pounds). The 1992 TRI data indicate that only a small fraction of the chloroform released to the environment is released to water. The TRI data should be used with caution because only certain types of facilities are required to report. This is not a exhaustive list. Current, more comprehensive quantitative data or estimates of chloroform releases to natural waters are lacking. Direct release to water is expected via waste waters generated during chloroform manufacture ***DRAFT FOR PUBLIC COMMENT *** CHLOROFORM 167 5. POTENTIAL FOR HUMAN EXPOSURE and its use in the manufacture of other chemicals and materials (EPA 1985a). Direct discharge sources are expected to be relatively minor contributors to total chloroform emissions to water relative to the formation of chloroform resulting from the chlorination of drinking water or chlorination to eliminate pathogens in discharged wastes or other process waters (EPA 1985a). Since chlorination to disinfect water supplies is nearly universal, chloroform contamination resulting from chlorination will also be widespread (see discussion on levels monitored or estimated in water in Section 5.4.2). Other chloroform emission sources tend to be relatively isolated point sources. Chlorination of municipal and industrial waste waters at municipal and industrial waste water treatment plants, process waters and effluent from the bleaching of pulp in pulp and paper mills, cooling tower water, and swimming pool and whirlpool spa water will also result in chloroform formation (Benoit and Jackson 1987; EPA 1985a, 1985b, 1990a; Hoigne and Bader 1988). The use of modern treatment facilities may reduce the amounts of chloroform released to environmental waters. This has been demonstrated at a modern kraft pulp mill (Paasivirta et al. 1988); however, much of the chloroform removed from the waste water may be released to the atmosphere by volatilization. Release of chloroform to groundwater has resulted from improper disposal of chloroform-containing waste at hazardous waste sites (Clark et al. 1982; Dewalle and Chian 1981; Harris et al. 1984; Sawhney 1989). An additional minor source of water contamination may be atmospheric rainout since chloroform has been found in rainwater (Kawamura and Kaplan 1983). Other sources of chloroform release to surface water include breweries, thermal combustion of plastics, reaction of dissolved chlorine with sediment and other materials in water, biological production by marine algae, and the reaction of chlorinated pollutants with humic materials in natural waters (EPA 1985a). 5.2.3 Soil In 1992, releases of chloroform to the land from as many as 137 large processing facilities were 14,117 kg (28,582 pounds) (TRI92 1994). In 1992, total releases to all environmental media were reported as 8,776,153 kg (17,768,200 pounds). The releases of chloroform to soil from facilities that manufactured and processed it in the United States during 1992 are reported in Table 5-1 (TRI92 1994). The 1992 TRI data indicate that only a very small fraction of the chloroform released to the environment is released to land. The TRI data should be used with caution since only certain types of facilities are required to report. This is not an exhaustive list. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 168 5. POTENTIAL FOR HUMAN EXPOSURE Current comprehensive quantitative data or estimates of chloroform releases to soil are lacking. Chloroform release to soil has occurred at hazardous waste sites containing improperly disposed wastes where chloroform has leached through soil to groundwater (Clark et al. 1982; Dewalle and Chian 1981; Harris et al. 1984; Sawhney 1989). Land disposal of sludge from municipal and industrial waste water treatment plants may also result in chloroform release to soil (EPA 1990a). Direct land disposal of chloroform-containing wastes may have occurred in the past, but land disposal of chloroform wastes is currently subject to restrictive regulations (EPA 1988a, 1989b). An additional minor source of soil contamination may be atmospheric rainout since chloroform has been found in rainwater (Kawamura and Kaplan 1983). Chloroform has been used as a carrier or solvent for some pesticides (HSDB 1994). It is still used as a carrier for at least one pesticide formulation with dichlorvos as the active ingredient (Petrelli et al. 1993). This could have resulted in releases of chloroform to the land. It is impossible to quantify the magnitude of such releases, and the chloroform could be expected to be transported to either the atmosphere through volatilization or, if dissolved in water, carried into surface waters or groundwater. 5.3 ENVIRONMENTAL FATE 5.3.1 Transport and Partitioning Based upon a vapor pressure of 159 mm Hg at 20 °C, chloroform is expected to exist almost entirely in the vapor phase in the atmosphere (Boublik et al. 1984; Eisenreich et al. 1981). Large amounts of chloroform in the atmosphere may be removed by wet deposition since chloroform has significant solubility in water. This is confirmed by its detection in rainwater (Kawamura and Kaplan 1983). Most of the chloroform removed in precipitation, however, is likely to reenter the atmosphere by volatilization. Trace amounts of chloroform have been documented in air samples from remote, often relatively pristine, areas of the world (Class and Ballschmidter 1986). Since chloroform is relatively nonreactive in the atmosphere, long-range transport within the atmosphere is possible. The detections in remote areas may also mean that the chloroform is produced as the result of more localized transformation processes, possibly including the reaction of naturally generated chlorinated oxidants with organic matter. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 169 5. POTENTIAL FOR HUMAN EXPOSURE The dominant fate process for chloroform in surface waters is volatilization. Chloroform present in surface water is expected to rapidly volatilize to the atmosphere. An experimental half-disappearance range of 18-25 minutes has been measured for volatilization of chloroform from a I-ppm solution with a depth of 6.5 cm that was stirred with a shallow pitch propeller at 200 rpm at 25 °C under still air (=0.2 mph air currents) (Dilling 1977; Dilling et al. 1975). Using the Henry’s law constant, a half- life of 3.5 hours was calculated for volatilization from a model river I meter deep flowing at 1 meter/second, with a wind velocity of 3 m/second, and neglecting adsorption to sediment (Lyman et al. 1982). A half-life of 44 hours was estimated for volatilization from a model pond using EXAMS (1988). ) of 1.65), Based on a measured soil organic carbon sorption coefficient (K_.) of 45 (or a log (K oc oc chloroform is not expected to adsorb significantly to sediment or suspended organic matter in surface water (Sabljic 1984). This prediction is supported by sediment monitoring data that indicate that this compound has not been detected, or was detected at very low concentrations, in sediment samples (Bean et al. 1985; Ferrario et al. 1985; Helz and Hsu 1978). Little or no chloroform concentration was observed on peat moss, clay, dolomite limestone, or sand added to water (Dilling et al. 1975). Chloroform slightly adsorbed to aquifer solids in laboratory studies utilizing different amounts of two different aquifer materials with K . values ranging from 63.4 to 398. The authors reported higher adsorption with increasing organic content of the solids (Uchrin and Mangels 1986). K values ranging from 45 to 80 in soil have been experimentally determined for chloroform (Sabljic 1984; Wilson et al. 1981). Chloroform does not appear to bioconcentrate in higher aquatic organisms based upon measured bioconcentration factors (BCF) of 6 and 8 for bluegill sunfish (Lepomis macrochirus) (Barrows et al. 1980; Veith et al. 1980). A BCF of 690 experimentally determined for the bioconcentration of chloroform in the green algae Selenastrum capricornutum suggests that the compound has a slight tendency to concentrate in aquatic plants (Mailhot 1987). No data regarding the biomagnification potential of chloroform were found. Based upon the observed BCF, however, significant biomagnification of chloroform is apparently unlikely. In soil, the dominant transport mechanism for chloroform near the surface will probably be volatilization because of its high volatility and low soil adsorption. Volatization rates seem relatively constant over a wide variety of soil types (Park et al. 1988). In other laboratory studies, 75% of the **NRAFT FOR PLIRI IC COMMFENT*** CHLOROFORM 170 5. POTENTIAL FOR HUMAN EXPOSURE chloroform initially present in water volatilized when applied to a fine sandy soil, and 54% of the chloroform volatilized from a soil column during a percolation study utilizing a sandy soil (Piwoni et al. 1986; Wilson et al. 1981). All or nearly all of the remaining chloroform travelled through the soil because of its low adsorption onto soil. Another laboratory study of 15 common volatile or semivolatile organic chemicals reported a disappearance half-life for chloroform of 4.1 days, which assumed first-order kinetic decay (Anderson et al. 1991). The leaching potential of chloroform is further confirmed by the detection of chloroform in groundwater, especially at hazardous waste sites (Clark et al. 1982; Dewalle and Chian 1981; Harris et al. 1984; Sawhney 1989). 5.3.2 Transformation and Degradation 5.3.2.1 Air The vapor-phase reaction of chloroform with photochemically generated hydroxyl radicals is the dominant degradation process in the atmosphere. The rate constant for this process at 25 °C has been experimentally determined as 1.0x10™"* cm*/molecule-second, which corresponds to a half-life of ~80 days based upon a 12-hour sunlit day in a typical atmosphere containing 1x10° hydroxyl radicals/cm® (Hampson 1980; Singh et al. 1981). Breakdown products from reaction with hydroxyl radicals probably include phosgene and hydrogen chloride (Atkinson 1985). Chloroform is more reactive in photochemical smog conditions where the approximate half-life is 11 days (Dimitriades and Joshi 1977). Direct photolysis of chloroform will not be a significant degradation process in the atmosphere. Chloroform solutions sealed in quartz tubes and exposed to sunlight for one year degraded at almost the same rate as solutions in sealed tubes stored in the dark, which indicated that very little or no photodegradation of the compound had occurred (Dilling et al. 1975). This is expected because chloroform does not show significant light absorbance at wavelengths >290 nm (Hubrich and Stuhl 1980). 5.3.2.2 Water Hydrolysis will not be a significant degradation process in water based upon rate constants experimentally determined at 25 °C that correspond to half-lives ranging from 1,850 to 3,650 years at pH 7, and from 25 to 37 years at pH 9 (Jeffers et al. 1989; Mabey and Mill 1978). Direct photolysis of chloroform will not be a significant degradation process in surface waters because, as noted above, ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 171 5. POTENTIAL FOR HUMAN EXPOSURE the compound does not absorb light at wavelengths >290 nm (Hubrich and Stuhl 1980). The reaction rate of chloroform with hydrated electrons photochemically produced from dissolved organic matter has been predicted to correspond to a near-surface half-life of =44 days based upon an experimentally determined rate constant and a hydrated electron concentration of 1.2x10""7 mol of hydrolyzed electrons/L (Zepp et al. 1987). This latter process is probably too slow to effectively compete with volatilization as a removal process from surface waters. Biological degradation of chloroform has been studied primarily with an eye to batch process operation at waste water treatment plants or remediation possibilities at hazardous waste disposal sites. Above certain dosage levels, chloroform becomes toxic to anaerobic and aerobic microorganisms. This is especially noticeable for plants that use anaerobic digestion systems, where sustained inputs with chloroform concentrations approaching 100 mg/L can all but eliminate methanogenic (methane- fermenting) bacteria (Rhee and Speece 1992). Other studies have shown appreciable inhibition of methanogenesis with levels of chloroform in the range of 1 mg/L (Hickey et al. 1987). Other chlorinated hydrocarbons, and particularly such common 2-carbon chlorinated aliphatics as trichloro- ethylene (TCE), can similarly inhibit bacteria found in sewage sludges (Long et al. 1993; Rhee and Speece 1992). Similar inhibition effects can be the result of heavy metal toxics, zinc being particularly stressful to methanogenic bacteria (van Beelen et al. 1994; van Vlaardingen and van Beelen 1992). Studies of actual natural waters or waste waters, where it is difficult to control the levels of specific chemicals or preclude inputs of other toxicants, yield a wide variety of results on the efficiencies of chloroform biodegradation. For instance, little or no degradation was observed during 25 weeks in aqueous aerobic screening tests utilizing primary sewage effluent inocula (Bouwer et al. 1981a). No chloroform degradation was observed in aerobic biofilm column studies (Bouwer et al. 1981b). Significant degradation of chloroform (46-49% loss in 7 days, at least some of which was apparently due to volatilization) in aerobic screening tests utilizing settled domestic waste water as inoculum was reported (Tabak et al. 1981). Under the proper conditions, chloroform appears to be much more susceptible to anaerobic biodegradation. Degradation of chloroform under anaerobic conditions was more rapid at lower chloroform concentrations (81% and 99% degradation after 2 and 16 weeks, respectively, at 16 ppb); a more gradual degradation was observed at higher concentrations (25% and 78% degradation after 2 and 16 weeks, respectively, at 157 ppb) (Bouwer et al. 1981a). No degradation was observed, however, when chloroform was incubated with aquifer material under anaerobic conditions for 27 weeks (Wilson et al. 1981). ***NRAFT FOR PLIRI ICC COMMENT *** CHLOROFORM 172 5. POTENTIAL FOR HUMAN EXPOSURE In the absence of toxicity from other chlorinated hydrocarbons or heavy metals, and where chloroform concentrations can be held below approximately 100 ppb, both aerobic and anaerobic bacteria can biodegrade chloroform, with removal rates well over 80% in a period of 10 days (Long et al. 1993). Deviations from these ideal conditions can lead to lower removal efficiencies. These biodegradation reactions generally lead to the mineralization of the chloroform to chlorides and carbon dioxide (Bouwer and McCarty 1983; Rhee and Speece 1992). One study, however, documents the production of the toxicant methylene chloride (dichloromethane) from the breakdown of chloroform-containing wastes in a mixed culture of bacteria from sewage sludge (Rhee and Speece 1992 citing results from work at Tyndall AFB, Florida). Caution should be exercised in making generalizations without site- specific evidence, however, since commercial grades of chloroform will often contain methylene chloride as an impurity (HSDB 1994). In waters containing mixtures of different chlorinated aliphatics, biodegradation may produce new chloroform, at least as a temporary by-product, the breakdown of carbon tetrachloride into chloroform having been confirmed in laboratory studies (Long et al. 1993; Picardal et al. 1993). 5.3.2.3 Sediment and Soil Little information was located regarding the degradation of chloroform in soil. Based upon data for degradation in water, chemical degradation in soil is not expected to be significant. The available soil data suggest that chloroform biodegradation rates in soil may vary, depending upon conditions. In soil column studies, the chloroform present in the influent secondary waste water appeared to pass through the column nearly unchanged even though some of the other organic compounds present were apparently biodegraded, which indicated that the waste water was not too toxic to the microorganisms in the soil (Bouwer et al. 1981b). In contrast to these studies, significant degradation of chloroform (33% removed in 6 days) was observed in fine sandy soil in sealed bottles; however, the chloroform may have been co-metabolized by methylotropic bacteria already present in the soil. The aerobic degradation was even faster in methane-enriched soil (Henson et al. 1988). Such bio-oxidation of chloroform was also observed under methanogenic conditions in batch experiments using an inoculum derived from activated sludge and in a continuous-flow laboratory scale column, using a methanogenic fixed film derived from primary sewage effluent (Bouwer and McCarty 1983). Overall, biodegradation in soil is not expected to compete with the predicted rapid rate of volatilization from soil (Park et al. 1988). As with biodegradation in water, concentrations of chloroform above certain threshold levels ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 173 5. POTENTIAL FOR HUMAN EXPOSURE may inhibit many bacteria, especially methane-fermenting bacteria under anaerobic or near-anaerobic conditions (Hickey et al. 1987). 5.4 LEVELS MONITORED OR ESTIMATED IN THE ENVIRONMENT 5.4.1 Air Data from the most recent study located (1982-85 air samples) reported that the background level of chloroform concentrations over the northern Atlantic ocean ranges from 2x10” to 5x10” ppm (Class and Ballschmidter 1986). This range does not differ significantly from the range reported for 1976-79 (1.4-4x10” ppm) and the range reported from the 1987 update of the National Ambient Volatile Organic Compounds Database (NAVOCDB), which was 2x10” ppm (Brodzinsky and Singh 1982; EPA 1988b; Singh 1977; Singh et al. 1979). The maximum and background levels found in seven U.S. cities between 1980 and 1981 were 5.1x10” and 2x10” ppm, respectively (Singh et al. 1982). Average atmospheric levels in U.S. cities ranged from 2x10” to 2x10” ppm between 1980 and 1981. The median concentration reported between 1977 and 1980 was 7.2x10” ppm, and the median reported in the 1987 update of the NAVOCDB was 6x10” ppm (Brodzinsky and Singh 1982; EPA 1988b; Singh et al. 1981, 1982; Wallace et al. 1986a, 1986b, 1988). The median concentration for source-dominated areas in the United States is 8.2x10* ppm for data reported between 1977 and 1980, and this figure does not differ significantly from the 5.1x10* ppm values reported in the 1987 update of the NAVOCDB (Brodzinsky and Singh 1982; EPA 1988b). Certain source-dominated areas contained much higher chloroform levels. The ambient air concentrations outside homes in Love Canal, New York, in 1978, ranged from 2x10 to 2.2x10? ppm, and the maximum concentration found in ambient air at 20 California municipal landfills was 0.61 ppm (Barkley et al. 1980; Wood and Porter 1987). Concentrations ranging from 2.9x10™ to 6x10° ppm were found in air samples taken from 5 hazardous waste sites in New Jersey (LaRegina et al. 1986). Ambient air samples measured near a hazardous waste landfill contained NIOSH 1987 desorption with carbon disulfide method 1003) sample Ambient air Adsorption onto Tenax; thermal GC/FID or ECD No data No data Parsons and and stacks desorption Mitzner 1975 Air Collection in flask; transfer to GC GC/ECD 1.5 ng/m?3 No data Bureau Air (phosgene) by syringe Phosgene collection in an impinger containing a solution of aniline (forming carbanilide); evaporation of solvent; dissolution of residue in acetonitrile HPLC/UV (EPA Method TOG) 0.1 ppb (v/v) 100 at 3 ppb (v/v); 15% RSD International Technique des Solvants Chlores 1976 EPA 1988n SAOHL3W TVOILATVYNY 9 WHO404HO THO 161 = INIJWWOO O1N8Nd HOH 14vHd... Table 6-2. Analytical Methods for Determining Chloroform in Environmental Samples (continued) Sample Analytical detection Sample matrix ~~ Preparation method method limit Percent recovery Reference Air (phosgene) Phosgene collection onto HPLC/UV 0.005 ppm (w/v) 100 over range Rando et al. Chromosorb coated with 0.02-1 ppm (w/v) 1993 1-(2-pyridyl)-piperazine; elution of derivative with acetonitrile Air (phosgene) Passage of air through a tube GC/ECD 7 ppt (v/v) No data Bachmann filled with magnesium and Polzer perchlorate (for water removal); 1989 cryogenic trapping Air, water Solid phase microextraction GC/ECD 0.9 ppb (v/v) gas No data Chai et al. (from air, water, or headspace phase; 30 ng/L 1993 over water) (30 ppt, w/v) liquid phase Drinking water Direct injection or purge-and-trap GC/ECD, 1 ng/L (1 ppb, wiv 103-126 at 35-70 pg/L Nicholson et pre-concentration GC/Hall direct); 0.1 ug/L (direct); 91-106 at al. 1977 (0.1 ppb, w/v with 35-70 pg/L (purge-and- purge-and-trap) trap) Drinking water ~~ Purge-and-trap pre- GC/MS 0.1 pg/L (0.1 ppb, No data Coleman et al. concentration; thermal w/v) 1976 desorption Drinking water Direct injection GC/MS 0.1 pg/L (0.1 ppb, No data Fujii 1977 w/v) Seawater and Solvent extraction with pentane; GC/ECD 80 ng/L (0.08 ppb, No data Bureau freshwater extract dried with sodium sulfate w/v) International Technique des Solvants Chlores 1976 SAOHL3W IVOILATIVNY 9 WHO40HOTHO 261 .-LNIWWOD O1N8Nd "HO L4vHQ... Table 6-2. Analytical Methods for Determining Chloroform in Environmental Samples (continued) Sample Analytical detection Sample matrix ~~ Preparation method method limit Percent recovery Reference Water Permeation through a silicon GC/FID 74.0 ug/L (74 ppb, No data Blanchard and polycarbonate membrane into an W/V) Hardy 1986 inert gas stream and into GC port Drinking water Acidification and dechlorination; GC/ECD or 0.002 ug/L 100 (13% RSD) at EPA 1990g extraction with methyl-t-butyl GC/MS (EPA (0.002 ppm, w/v) 0.005 ng/L ether; direct injection of extract Method 551) Drinking water, Purge-and-trap pre- GC/MS < 0.1 pg/L (reagent 105 (3% RSD) narrow Greenberg et waste water concentration; thermal (Standard water) using narrow bore column al. 1992 desorption Method 6210) bore capillary column Tap water Solvent extraction HRGC/ECD with No data No data Kroneld 1986 HRGC/MS confirmation Water Solvent extraction HRGC/ECD 1 ng/L (ppb, w/v) 100.3 at 50 pg/L Reunanen and Kroneld 1982 Drinking water ~~ Purge-and-trap pre-concentration = GC with PID 0.02 pg/L 98 (2.5% RSD) Ho 1989 onto Tenax/ silica/charcoal; and Hall in thermal desorption series Finished Purge-and-trap pre-concentration ~GC/Hall (EPA No data 0.90C+3.44 where C = EPA 1991a drinking/ raw onto Tenax/ method 502.1) true concentration (ug/L) source water silica/charcoal;thermal desorption Finished Purge-and-trap pre-concentration GC with PID 0.10 pg/L (0.1 ppb, 98 (2% RSD) EPA 1991b drinking/ raw onto Tenax/ silica/charcoal; and HSD in w/v) source water thermal desorption series (EPA method 502.2) SAOHL3IW TVOILATVYNY 9 WHO40HO THO £61 «+INIWWOD O118Nd HOH 14vHAa... Table 6-2. Analytical Methods for Determining Chloroform in Environmental Samples (continued) Sample Analytical detection Sample matrix ~~ Preparation method method limit Percent recovery Reference Finished Purge-and-trap pre-concentration ~~ Subambient 0.2 pg/L (0.2 ppb, 103 at 1.0 pg/L EPA 1991c drinking/ raw onto Tenax/ silica/charcoal; programmable W/V) source water thermal desorption HRGC/MS (EPA method 524.1) Finished Purge-and-trap pre-concentration Cryofocusing 0.03 ug/L (0.03 ppb, 90 (6.1% RSD) at EPA 1992a drinking/ raw onto Tenax’ silica/charcoal; (wide or narrow w/v) with wide bore 0.5-10 pg/L (wide bore); source water thermal desorption bore); column; 0.02 ug/L 95 (3.2% RSD) at HRGC/MS (EPA (0.02 ppb, w/v) with 0.1 ug/L with narrow method 524.2) narrow bore column bore column Groundwater, Direct injection of head-space GC/HSD (EPA 0.5 ug/L (ppb, w/v) Water: 0.93C -0.39 EPA 1986a liquid, and gas (EPA method 5020) or method 8010) for groundwater, where C = true solid matrices purge-and-trap pre-concentration 0.5 ug/g (ppm, w/w) concentration in pg/L. and thermal desorption (EPA for low-level soil, method 5030) 500 pg/L (ppb, w/v) for water-miscible liquid waste, 1,250 ug/g (ppm, w/w) for soil, sludge, and non-water- miscible waste Waste water Purge-and-trap pre- GC/HSD or MS 0.05 pg/L (for HSD); 102 at 0.44-50 pg/L (for EPA 19982a Solid and liquid waste, soil concentration; thermal desorption Dispersion in glycol; purge-and- trap pre-concentration onto Tenax’ silica/charcoal; thermal desorption (EPA methods 601 and 624) GC/ECD and FID in series 1.6 ng/L (for MS) No data HSD); 101 at 10-100 pg/L (for MS) 105 at 5 pg/L Lopez-Avila et al. 1987 SAOHL3W TVOILATVYNY 9 WHO40HOTHO p61 .«LNIWWOO 0118Nd HOH 14vHQA... Table 6-2. Analytical Methods for Determining Chloroform in Environmental Samples (continued) Sample Analytical detection Sample matrix ~~ Preparation method method limit Percent recovery Reference Sediment Extraction into methanol; GC/FID/ECD 0.1 ug/g (0.1 ppm, 99 (4% RSD) at Amaral et al. dilution with water; purge-and- W/W) 0.8 ng/mL (0.8 ppm, 1994 trap pre-concentration w/v) Bulk oils Purge-and-trap pre-concentration GC/MS 1 ppb (W/V) 88 (6.7% RSD) at Thompson (with deuterated internal 93 ppb 1994 standards) onto Tenax; thermal desorption; Food Extraction of composited, table- GC-ECD/Hall 5 ng/g (ECD); 5 ng/g 15-161 Daft 1988a, ready food with isooctane or (Hall) 1989 acetone-isooctane; micro-Florisil clean-up (if fat content 21-70%); direct injection into GC Volatile food Direct injection of head-space GC/ECD or MS 4.2 ng/kg (4.2 pb, No data Entz et al. components gas w/w in beverages); No data 1982 12.5 pg/kg (12.5 ppb, w/w in dairy No data products); 18 ng/kg No data (meats); 28 ng/kg (fats/oils) ECD = electron capture detector; EPA = Environmental Protection Agency; FID = flame ionization detector; GC = gas chromatography; Hall = Hall electrolytic conductivity detector; HPLC/UV = high performance liquid chromatography with ultraviolet absorbance detection; HRGC = high-resolution gas chromatography; HSD = halogen-specific electrolytic conductivity detector; MS = mass spectrometry; NIOSH = National Institute for Occupational Safety and Health; OSHA = Occupational Safety and Health Administration; PID = photo-ionization detector; RSD = relative standard deviation; v/v = volume/volume; w/v = weight/volume SAOHL3IW TVOILATYNY 9 WHOH40HO THD S61 CHLOROFORM 196 6. ANALYTICAL METHODS The most common method for the determination of chloroform levels in water, sediment, soil, and foods is the purging of the vapor from the sample, or its suspension in a solvent with an inert gas and trapping (purge-and-trap) the desorbed vapors onto a sorbent trap (EPA 1991a, 1991b, 1991c, 1992a; Greenberg et al. 1992; Ho 1989; Lopez-Avila et al. 1987). Solid phase microextraction is a method that combines the ease of headspace analysis with some of the concentration benefits of purge-and-trap (Chai et al. 1993). Subsequent thermal desorption is used for the quantification of chloroform concentrations. Solvent extraction is also used in a number of methods (Amaral et al. 1994; Daft 1988a, 1989; EPA 1990g; Kroneld 1986; Reunanen and Kroneld 1982). No methods were found for phosgene in water, sediment, soil, and foods. All of the methods listed for the analysis of environmental samples use gas chromatography with various detection methods. The two methods that provide the lowest detection limits are halide- specific detectors (e.g., Hall electrolytic conductivity detector or electron capture detector) and the mass spectrometer (EPA 1986a; Ho 1989; Lopez-Avila et al. 1987; Ramus et al. 1984). The advantage of halide specific detectors is they are not only very sensitive, but are also specific for halide compounds. The mass spectrometer, on the other hand, provides additional confirmation of the presence of a compound through its ionization pattern and is desirable when a variety of compounds are required to be identified and quantified. The disadvantage of halide-specific detectors is their inability to detect and quantify nonhalogen compounds, if non-halogenated compounds are of interest also; this can be greatly overcome by using other detectors (e.g., photoionization detector) in series (Lopez-Avila et al. 1987). High-resolution gas chromatography (HRGC) with capillary columns coupled with mass spectrometry (MS) provides better resolution and increased sensitivity for volatile compounds than packed columns. In methods such as EPA method TO14 (EPA 1988i), desorbed compounds are cryogenically trapped onto the head of the capillary column. Such HRGC/MS methods overcome some common problems involved in analyses of excessively complex samples, samples with large ranges of concentrations, and samples that also contain high-boiling compounds (Dreisch and Munson 1983; EPA 1986a). Limits of detection in the sub-parts per billion (ppb) range are routinely possible in both air and liquid/solid matrices. Numerous standard methods exist. The reproducibilities of the methods listed in Table 6-2 are generally acceptable, but will vary, depending on the laboratories doing the analyses. Probable interferences for the methods of analysis include contamination from chloroform vapors in the laboratory. For this reason, it is often recommended that the laboratories doing the analysis should not contain chloroform or any other “**DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 197 6. ANALYTICAL METHODS solvent to be measured in the sample (EPA 1986a). Plastic or rubber system components should be avoided as they can contaminate a sample or result in carryover from one analysis to the next (EPA 1992a). The formation of aerosols and foam during purge-and-trap of liquid samples can contaminate the analytical system, so precautions must be taken (Thompson 1994; Vallejo-Cordoba and Nakai 1993). The use of field blanks is extremely important to correct for chloroform that might have diffused into the sample during shipping and storage (EPA 1986a). Other interferences include those volatile compounds that have similar retention times in the various GC columns used. This problem is often eliminated by analyzing the samples with two different types of GC columns such that the retention times will not be coincidental in both columns. Refer to the references cited in Table 6-2 and the text for specific information regarding reproducibility and potential interferences. 6.3 ADEQUACY OF THE DATABASE Section 104(i)(5) of CERCLA directs the Administrator of ATSDR (in consultation with the Administrator of EPA and agencies and programs of the Public Health Service) to assess whether adequate information on the health effects of chloroform is available. Where adequate information is not available, ATSDR, in conjunction with NTP, is required to assure the initiation of a program of research designed to determine the health effects (and techniques for developing methods to determine such health effects) of chloroform. The following categories of possible data needs have been identified by a joint team of scientists from ATSDR, NTP, and EPA. They are defined as substance-specific informational needs that if met would reduce or eliminate the uncertainties of human health assessment. This definition should not be interpreted to mean that all data needs discussed in this section must be filled. In the future, the identified data needs will be evaluated and prioritized, and a substance-specific research agenda will be proposed. 6.3.1 Identification of Data Needs Methods for Determining Biomarkers of Exposure and Effect. Methods are available for the determination of chloroform in breath (Aggazzotti et al. 1993; Krotoszynski et al. 1979; Phillips and Greenberg 1992), blood (Antoine et al. 1986; Ashley et al. 1992; EPA 1985a; Kroneld 1986; Peoples et al. 1979; Pfaffenberger et al. 1980; Reunanen and Kroneld 1982; Seto et al. 1993; Streete et ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 198 6. ANALYTICAL METHODS al., 1992), and other fluids and tissues such as urine and adipose (EPA 1985a; Peoples et al. 1979; Pfaffenberger et al. 1980; Reunanen and Kroneld 1982; Streete et al. 1992). Sub-parts per billion limits of detection have been shown (e.g., Ashley et al. 1992; Pfaffenberger et al. 1980) and the methods are adequate for the determination of chloroform concentrations in samples from the general population. No biomarker that can be associated quantitatively with chloroform exposure has been identified (see Sections 2.5.1 and 6.1). Although chloroform levels can be determined in biological samples, the relationship between these levels and the exposure levels has not been adequately studied. In one study, the concentrations of chloroform in alveolar air of people attending activities at an indoor swimming pool were found to be proportional to the concentrations in air (Aggazzotti et al. 1993). Such proportionality was observed, in part, because the alveolar air samples were taken soon after exposure termination. Correlations of alveolar air concentrations with exposure air concentrations based on breath samples at unknown postexposure times will be complicated by metabolism and other factors, such as activity, important in the elimination of chloroform from the body (see PBPK discussion, Chapter 2). Furthermore, the presence of chloroform, or a transformation product of chloroform such as phosgene and reaction products of phosgene, in a biological sample may have resulted from the metabolism of another chlorinated hydrocarbon. If a biomarker of exposure for this compound in a human tissue or fluid was available and a correlation between the level of the biomarker and exposure existed, it could be used as an indication of the extent of chloroform exposure. Further information regarding the accuracy of sample recovery for the methods of chloroform analysis would be useful in interpreting monitoring data. No biomarker of effect that can be associated quantitatively and directly to chloroform exposure has been identified (see Section 2.5.2). If biomarkers of effect were available for this compound and a correlation between the level or intensity of the biomarker of effect and the exposure level existed, it could be used as an indication of the levels and extent of chloroform exposure. Methods for Determining Parent Compounds and Degradation Products in Environmental Media. Methods for determining chloroform in the environment are available. These include methods for drinking water (Blanchard and Hardy 1986; EPA 1990g, 1991a, 1991b, 1991c, 1992a; Greenberg et al. 1992; Ho 1989; Kroneld 1986; Nicholson et al. 1977), air (EPA 1988f, 1988g, 1988h, 1988i; NIOSH 1987; OSHA 1979: Parsons and Mitzner 1975), and foods (Daft 1988a, 1989; Entz et al. 1982; Thompson 1994). These three media are of most concern for human exposure. The precision, accuracy, reliability, and specificity of the methods are well-documented and well-suited ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 199 6. ANALYTICAL METHODS for the determination of low levels of chloroform and levels at which health effects occur. For example, the MRL for acute inhalation is 0.02 ppm (weight per volume [w/v] or 0.099 mg/m’) so any method used must have a limit of detection equal to or less than this. The methods of the Bureau International Technique des Solvants Chlores (1976) and Chai et al. (1993) report limits of detection of 1.5 pg/m® and 0.9 ppb volume per volume (v/v), respectively, and are adequate for the measurement of chloroform in air. Although no limits of detection were reported for EPA methods TO2 and TO3 (EPA 1988f, 1988g), recoveries were acceptable for low ppb concentrations of chloroform in air and thus these methods should be applicable to concentrations at the acute inhalation MRL. Similarly, the chronic oral MRL is 0.01 mg/kg/day, which converts to 0.7 mg/day for a 70-kg person. For a 2 L/day water consumption, this translates into a required method limit of detection of 0.35 mg/L. This concentration is easily measured by the methods of EPA (1990g) (limit of detection 0.002 pg/L), Greenberg et al. (1992) (limit of detection 0.1 pg/L), and Chai et al. (1993) (limit of detection 30 ng/L). Assuming a food intake of 2 kg/day, this oral MRL translates to a needed method limit of detection in food of 0.35 mg/kg. The methods of Entz et al. (1982) and Daft (1988a, 1989) provide limits of detection of less than 0.028 mg/kg and are adequate. Method sensitivities are clearly adequate for matrices in which the higher acute oral MRL is of concern. There is not much information regarding the degradation products of chloroform in the environment. Although phosgene can be produced in the environment and its high reactivity suggests that it would not persist, several methods were found for the quantification of phosgene in ambient air (Bichmann and Polzer 1989; EPA 1988n; Rando et al. 1993). No methods were found for phosgene in other environmental- matrices. 6.3.2 Ongoing Studies The Environmental Health Laboratory Sciences Division of the Center for Environmental Health and Injury Control, Centers for Disease Control and Prevention, is developing methods for the analysis of chloroform and other volatile organic compounds in blood. These methods use purge-and-trap methodology, high resolution gas chromatography, and magnetic sector mass spectrometry which gives detection limits in the low ppt range (see Ashley et al. 1992). Researchers at Physical Sciences, Inc. are developing an imaging infrared spectrometer that can rapidly screen field sites to detect the presence of volatile organic compounds, including chloroform, from remote locations (either in the air or on the ground). C.V. King at Westinghouse Electric Corporation/ ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 200 6. ANALYTICAL METHODS Westinghouse Hanford Co. is developing membrane-based methods to recover chloroform and carbon tetrachloride from gas streams. Although not a method per se, information acquired during this study could be applicable to membrane-based sensors for chloroform in air. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 201 7. REGULATIONS AND ADVISORIES The International Agency for Research on Cancer (IARC), national, and state regulations and guidelines regarding human exposure to chloroform are summarized in Table 7-1. Chloroform is regulated by the Clean Water Act Effluent Guidelines for the following industrial point sources: electroplating; organic chemicals; steam electric, asbestos, and timber products processing; paving and roofing; paint and formulating; formulating; gum, wood and carbon black; metal molding, casting, and finishing; coil coating; copper forming; and electrical and electronic components (EPA 1988a). An MRL of 1 ppm has been derived for acute-duration inhalation exposure to chloroform. The MRL is based on a NOAEL of 3 ppm for hepatic effects in mice (Larson et al. 1994c). An MRL of 0.05 ppm has been derived for intermediate-duration exposure to chloroform. The MRL is based on a LOAEL of 14 ppm for toxic hepatitis in workers exposed to up to 400 ppm for less than 6 months (Phoon et al. 1983). An MRL of 0.02 ppm has been derived for chronic-duration exposure to chloroform. The MRL was based on a LOAEL of 2 ppm for hepatic effects in workers exposed to concentrations of chloroform ranging from 2 to 205 ppm for 1-4 years (Bomski et al. 1967). An MRL of 0.3 mg/kg/day has been derived for acute-duration oral exposure to chloroform. The MRL is based on a NOAEL of 26.4 mg/kg/dy for hepatic effects in mice (Larson et al. 1994b). An MRL of 0.1 mg/kg/day has been derived for intermediate-duration oral exposure to chloroform. The MRL is based on a NOAEL of 15 mg/kg/day for liver effects (increased SGPT) in dogs exposed to chloroform in toothpaste for 26 weeks (Heywood et al. 1979). An MRL of 0.01 mg/kg/day has been derived for chronic oral exposure to chloroform based on a LOAEL for liver effects (increased SGPT) in dogs administered 15 mg/kg/day chloroform in toothpaste in capsules for 7.5 years (Heywood et al. 1979). ***DRAFT FOR PLIBL IC COMMENT*** CHLOROFORM 202 7. REGULATIONS AND ADVISORIES The chronic oral reference dose (RfD) for chloroform is also 0.01 mg/kg/day, based on the LOAEL for liver effects in dogs administered 15 mg/kg/day chloroform (Heywood et al. 1979; IRIS 1995). No reference concentration (RfC) exists for the compound. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 203 7. REGULATIONS AND ADVISORIES Table 7-1. Regulations and Guidelines Applicable to Chloroform Agency Description Information References INTERNATIONAL IARC Carcinogenic classification Group 2B? IARC 1987 Guidelines: WHO Drinking water guidelines 30 pg/L WHO 1984 IARC Group (cancer ranking) Limits in Workplace Air (mg/m®) TWA STEL Sittig 1994 Australia 49 —_ Belgium 49 _ Brazil 94 — Switzerland 50 100 Commonwealth of Independent States 250 — Szechoslovakia 10 20 Germany 50 —_— Denmark 10 se Finland 50 100 France 25 250 United Kingdom 9.8 _ Hungary _ 10 Israel 49 — Japan 240 — Poland 50 on Sweden 10 25 Limits in Ambient Air Sittig 1994 Commonwealth of Independent States 30 pg/m® (24 hour) Limits in Water (Domestic/Drinking) Sittig 1994 Canada 350 pg/L Commonwealth of Independent States 60 pg/L NATIONAL Regulations: a. Air: EPA OAQPS Intent to list under Section 112 of Clean Air Yes 40 CFR 61.01 Act EPA 1985d New Source Performance Standards Chemicals Produced at SOCMI Facilities Yes 40 CFR 60.489 EPA 1983b Chemical Affected by Standards for SOCMI Yes 40 CFR 60.667 Distillation Operations EPA 1990e National Emission Standards for Hazardous Air Pollutants Hazardous Air Pollutants Yes 40 CFR 61.01 EA 1985d Proposed Rule: Identification of *More Yes 59 FR 15504 Hazardous" Emission Decreases 40 CFR 63.48 EPA 1994a ***DRAFT FOR PUBLIC COMMENT"** CHLOROFORM 7. REGULATIONS AND ADVISORIES 204 Table 7-1. Regulations and Guidelines Applicable to Chloroform (continued) Agency Description Information References NATIONAL (cont.) Proposed Rule: National Emission Standards Yes 58 FR 62566 for Halogenated Solvent Clearing-Applicability 40 CFR 63.460 EPA 1993a OSHA PEL-TWA 2 ppm (9.78 mg/m?) 29 CFR 1910 OSHA 1989 Permissible Exposure Limit (Ceiling) 240 mg/m? OSHA 1993 Occupational Exposure to Hazardous Yes OSHA 1990 Chemicals in Laboratories b. Water: EPA PQL 0.5 pg/L 40 CFR 264 and 270 EPA 1987a Effluent Guidelines and Standards List of Toxic Pollutants Yes 40 CFR 401.15 EPA 1979b List of Toxic Pollutants Subject to Yes 40 CFR 403, App. B Pretreatment Standards EPA 1981a Definition of Total Toxic Organics (TTO) for Yes 40 CFR 413.02 Electroplating Point Source Category EPA 1981b Bulk Organic Chemicals in Wastewater from Yes 40 CFR 414.70 Sources of Organic Chemicals, Plastics and EPA 1987e Synthetic Fibers Organic Chemicals, Plastics, and Synthetic 40 CFR 414.91 Fibers - Effluent Limitations for Direct EPA 1987f Discharge Point Sources Using End-of-Pipe Biological Treatment One-day maximum 46 pg/L Maximum for monthly average 21 ug/L Organic Chemicals, Plastics, and Synthetic 40 CFR 414.101 Fibers - Effluent Limitations for Direct EPA 1987g Discharge Point Sources That Do Not Use End-of-Pipe Biological Treatment One-day maximum 325 pg/L Maximum for monthly average 111 pg/L Priority Pollutants, Associated With Steam Yes 40 CFR 423, App. A Electric Power Generating Sources EPA 1982b Proposed Rule: Effluent Limitations for Pupl, 58 FR 66078 Paper, and Paperboard - Dissolving Kraft 40 CFR 430 (bleach plant effluent) EPA 1993d 1-Day Max. Monthly Avg. Existing BAT New Source Pretreatment 10.1 g/kkg 7.06 10.1 g/kkg g/kkg 7.07 g/kkg ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 7. REGULATIONS AND ADVISORIES 205 Table 7-1. Regulations and Guidelines Applicable to Chloroform (continued) Agency Description Information References NATIONAL (cont.) Bleached paper grade kraft and soda 5.06 g/kkg 2.01 (bleach plant effluent - existing BAT and g/kkg pretreatment for existing Dissolving sulfite - pretreatment for new 23.2 g/kkg 74.4 sources g/kkg Proposed Rule: Monitoring Requirements for Yes 59 FR 12567 Pulp, Paper, and Paperboard Category 40 CFR 430.02 EPA 1994c Definition of TTO for Metal Finishing Sources Yes 40 CFR 433.11 EPA 1983d Definition of TTO for Metal Molding and Yes 40 CFR 464.21 Casting Sources EPA 1985e Definition of TTO for Ferrous Casting Sources 40 CFR 464.31 EPA 1985f Definition of TTO for Coil Coating Sources Yes 40 CFR 465.02 EPA 1982c EPA ODW National Primary Drinking Water Regulations Definition of Trihalomethanes (THM) Yes 40 CFR 141.2 EPA 1975 Maximum Contaminant Levels for THM 0.10 mg/L 40 CFR 141.12 EPA 1991f Method of Analysis for THM Yes 40 CFR 141, App. C EPA 1979a Monitoring for Inorganic and Organic Yes 40 CFR 141.40 Chemicals EPA 1987d Proposed Rule: Total Trihalomethane Yes 58 FR 65622 Sampling and Analysis 40 CFR 141.30 EPA 1993c Proposed Rule: Maximum Contaminant Yes 59 FR 38668 Levels for Total THM 40 CFR 141.12 EPA 1994b Proposed Rule: Total THM Sampling, Yes 59 FR 38668 Analytical and Other Requirements 40 CFR 141.30 EPA 1994b Proposed Rule: Best Available Technology for Yes 59 FR 38668 Achieving Compliance with MCLs for Total 40 CFR 141.64 THM EPA 1994b Proposed Rule: Disinfection By-Product Yes 59 FR 38668 Compliance Monitoring for Total THM 40 CFR 141.133 EPA 1994b EPA OW Designation of Hazardous Substances Yes 40 CFR 116.4 EPA 1978 Reportable Quantities for Hazardous 10 pounds 40 CFR 117.3 Substances EPA 1986e ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 7. REGULATIONS AND ADVISORIES 206 Table 7-1. Regulations and Guidelines Applicable to Chloroform (continued) Agency Description Information References NATIONAL (cont.) National Pollutants Charge Elimination Yes 40 CFR 122, App. D System: Permit Application Testing EPA 1983c Requirements Proposed Rule: Application of Part 132 Yes 58 FR 20802 Requirements to Great Lake States and Tribes 40 CFR 132.6 - Pollutants of Initial Focus EPA 1993b Test Procedures for the Analysis of Pollutants Yes 40 CFR 136.3 EPA 1973a Methods of Analysis of Municipal and Yes 40 CFR 136, App. A Industrial Wastewater EPA 1973b c. Other: 40 CFR 122 EPA 1983c EPA OERR RQ (Ruled) 10 pounds 54 CFR 155 EPA 1989c Threshold planning quantity 10,000 pounds EPA 1987b Constituents for Solid Waste Detection Yes 40 CFR 258, App. | Monitoring EPA 1991g Hazardous Constituents Subject to Regulatory ~~ Yes 40 CFR 258, App. Il Requirements EPA 1991h Maximum Concentration Contaminants for the ~~ Yes 40 CFR 261.24 Toxicity Characteristic EPA 1990f Discarded commercial chemical products, off- Yes 40 CFR 261.33 specification species, container residues, and EPA 1990d spill residues Basis for Listing Hazardous Waste Yes 40 CFR 261, App. VII EPA 1981c Proposed Rule: Basis Listing Hazardous Yes 59 FR 9808 Waste 40 CFR 261, App. VII EPA 1994d Hazardous Constituents Yes 40 CFR 261, App. VIII EPA 1988j Groundwater Monitoring List for Hazardous Yes 40 CFR 264, App. IX Waste TSDF EPA 1987h Health Based Limits for Exclusion of Waste- 6x10? 40 CFR 266, App. VII Derived Residues EPA 1991d Potential PICs For Determination of Exclusion Yes 40 CFR 266, App. VIII of Waste-Derived Residues EPA 1991e EPA Land Disposal Restrictions Identification of Waste to Be Evaluated by Yes 40 CFR 268.10 August 8, 1988 ***DRAFT FOR PUBLIC COMMENT"** EPA 1986f CHLOROFORM 7. REGULATIONS AND ADVISORIES 207 Table 7-1. Regulations and Guidelines Applicable to Chloroform (continued) Agency Description Information References NATIONAL (cont.) Constituent Concentrations Which May Not be 40 CFR 268.43 Exceeded for Land Disposal EPA 1988k Waste Code: Waste Non- water waste water F025 0.046 mg/L 6.2 mg/kg F039, K117, U044 0.046 mg/L 5.6 mg/kg Halogenated organic compounds regulated Yes 40 CFR 268, App. lll under 40 CFR 268.32 EPA 1987i Proposed Rule: Treatment Standards for 58 FR 48092 Land Disposed Waste 40 CFR 268.40 & 268.48 EPA 1993e Waste Code: Waste water ~~ Non- waste water D022, KO19, K029, K150, K151, universal 0.046 mg/L 6/9 mg/kg treatment standard F025, KO73 0.046 mg/L 6.2 mg/kg F038, K117, K118, K136, U044 0.046 mg/L 5.6 mg/kg K009, K010 0.1 mg/L 6.0 mg/kg List of Hazardous Substances and Reportable 10 pounds 40 CFR 302.4 Quantities EPA 1989d Proposed Rule: Reportable Quantity Yes 58 FR 54836 Adjustments - Designation of Hazardous 40 CFR 302.4 Waste EPA 1993f Guidelines: a. Air: ACGIH NIOSH Extremely Hazardous Substances and Threshold Planning Quantities Chemicals Subject to Toxic Chemical Release Reporting: Community Right-to-Know Chemicals Subject to the Health and Safety Data Reporting Requirements Under TSCA TLV-TWA Threshold Limit Value for Occupational Exposure STEL (60 minutes) Recommended Exposure Limit for Occupational Exposure (STEL-60 min) Immediately Dangerous to Life & Health 10,000 pounds Yes Yes 10 ppm (49 mg/m?) 49 mg/m® 2 ppm (9.78 mg/m?) 9.78 mg/m? 1,000 ppm ***DRAFT FOR PUBLIC COMMENT*** 40 CFR 355, App. A EPA 1987] 40 CFR 372.65 EPA 1988| 40 CFR 716.120 EPA 1988m ACGIH 1992 ACGIH 1994 NIOSH 1990 NIOSH 1992 NIOSH 1990 CHLOROFORM 7. REGULATIONS AND ADVISORIES 208 TABLE 7-1. Regulations and Guidelines Applicable to Chloroform (continued) Agency Description Information References NATICNAL (cont.) b. Water: EPA 1-d Health Advisory 4 mg/L (child) EPA 1994e 10-d Health Advisory (child & adult) 4 mg/L (child) EPA 1994e Longer-term Health Advisory (child & adult) 0.1 mg/L (child EPA 1994e 0.4 mg/L (adult) Maximum Contaminant Level EPA 1994e Chloroform 0.1 mg/L Total THM (community and non-transcient, 0.080 mg/L 59 FR 38668 non-community water systems) 40 CFR 141.64 Total THM (serve more than 10,000 0.040 mg/L EPA 1994b people) Maximum Contaminant Level Goal zero 59 FR 38668 40 CFR 141.53 EPA 1994b EPA 1994e q'" cancer slope factor (oral exposure) 6.1x10" per mg/(kg/day) IRIS 1995 RfD (oral) 1.10? mg/kg/day) IRIS 1995 (UF 1,000) EPA ODW Individual lifetime cancer risk 10° 60 pg/L IRIS 1995 NAS Suggested No-Adverse-Response Level 24-hour 22 mg/L NAS 1980 7-day 3.2 mg/L c. Non-specific media EPA q," (oral) 6.1x10° (mg/kg/day)! IRIS 1995 q," (inhalation) 8.1x10? (mg/kg/day)! IRIS 1995 [2.3x10%(ug/m?)") RfD (chronic oral) 0.01 mg/kg/day IRIS 1995 Cancer classification B2® IRIS 1995 d. Other EPA Cancer classification B2 IRIS 1995 NIOSH Cancer classification Yes NIOSH 1990 NIOSH 1992 STATE Regulations and Guidelines: a. Air Acceptable ambient air concentrations AZ 1 hour 60 pg/m* NATICH 1992 24 hours 16 pg/m® 1 year 0.043 pug/m® CT 8 hours 250 pg/m’ NATICH 1992 “**DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 7. REGULATIONS AND ADVISORIES Table 7-1. Regulations and Guidelines Applicable to Chloroform (continued) 209 Agency Description Information References STATE (Cont) FL (Ft. 8 hours 0.5 pg/m® NATICH 1992 Lauderdale) FL (Pinellas 8 hours 97.8 pg/m’ Co.) 24 hours 23.5 pg/m® 1 year 0.043 pug/m3 IN 8 hours 48.9 pg/m® NATICH 1992 Annual 0.043 pg/m* IN (Innap) 8 hours 1203 pg/m? KS 1 year 0.0435 pg/m* NATICH 1992 KS-KC Annual 0.0435 pg/m® State of Kentucky 1986 LA Annual 4.3 pg/m3 MA 24 hours 133 pg/m® NATICH 1992 1 year 0.04 pg/m’ ME 1 year 0.043 pg/m* NATICH 1992 MI 1 year 0.40 pg/m® NATICH 1992 NC 1 year 0.0043 mg/m® NATICH 1992 NC 1 year 0.0043 mg/m?® ND BACT NATICH 1992 NV 8 hours 1.19 mg/m® NATICH 1992 NY 1 year 167 pg/m® NATICH 1992 OK 24 hours 97 pugim? NATICH 1992 PA (Phil.) 1 year 120 pg/m3 NATICH 1992 RI 1 year 0.04 pg/m® NATICH 1992 sc 24 hours 250 pg/m? NATICH 1992 TX 30 minutes 98 pg/m® NATICH 1992 1 year 10 pg/m® VA 24 hours 490 pg/m® NATICH 1992 VT 1 year 0.043 pg/m® NATICH 1992 WA/SWEST 1 year 0.043 pg/m® b. Water: Water Quality: Human Health Drinking water quality standards AZ 0.49 pg/L IL 1 pg/L MA 5 ug/L MN 57 ng/L **DRAFT FOR PUBLIC COMMENT"** FSTRAC 1990 CELDs 1994 CHLOROFORM 210 7. REGULATIONS AND ADVISORIES Table 7-1. Regulations and Guidelines Applicable to Chloroform (continued) Agency Description Information References STATE (Cont.) RI 6 pg/L Drinking water standard 100 pg/L 6 ug/L Water Quality: Human Health CELDs 1994 AL Consumption of water & fish = Fish consumption only he AZ Yes Domestic Water Source (DWS) TTHM Fish Consumption (FC) 590 pg/L Full Body Contact (FBC) 230 pg/L Partial Body Contact (PBC) 1400 pg/L CO MCL 0.10 mg/L CT 2.0 ug/L DC Water Quality Criteria C 3,000 mg/L D 0.2 mg/L FL MCL 0.10 mg/L HI Maximum organic contaminant level: Total trihalomethane (TTHM) (TTHM = the sum of the concentration of bromodichloromethane, dibromochloromethane and chloroform) Acute Chronic Fresh water 9,600 pg/L ns Salt water ns ns Fish consumption 5.1 IN Outside of mixing zone 157 ng/L Point of water intake 1.9 pg/L MCL (TTHM) 0.10 mg/L KY Consumption of fish 15.7 ug/L MCL 0.19 pg/L KS MCL (TTHM) 0.10 mg/L LA Drinking water 5.30 pg/L Non-drinking water 70.00 pg/L ME Maximum level (TTHM) 0.10 mg/L NH MCL (TTHM) 0.10 mg/L NM Groundwater levels 0.1 mg/L NY Groundwater effluent standard (max.) 7ug/L ND MCL (TTHM) 0.10 mg/L ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 211 7. REGULATIONS AND ADVISORIES Table 7-1. Regulations and Guidelines Applicable to Chloroform (continued) Agency Description Information References STATE (Cont.) NC MCL (TTHM) 0.10 mg/L Water quality standard for class GS 0.00019 mg/L Groundwater OG Groundwater quality reference level (TTHM) 0.100 mg/L MCL (TTHM) 0.10 mg/L OK Groundwater water quailty criteria 10.0 (no units specified) MCL (TTHM) 0.10 mg/L OH MCL (TTHM) 0.10 mg/L SC MCL (TTHM) 0.10 mg/L VT Class A or B waters 0.19 mg/L Class C water 15.7 mg/L WV MCL (TTHM) 0.10 mg/L wi Human cancer criteria: Public Water Supplier Warm water sport fish communities 1.9 mg/L Cold water communities 1.8 mg/L Great Lakes communities 1.8 mg/L Non-Public Water Supplies Warm water sport fish communities 87 mg/L Cold water communities 31 mg/L Warm water forage and limited fish 380 mg/L communities and limited aquatic life Groundwater Enforcement standard Prevention action limit 6 ug/L WV All water use categories, maximum criteria B1, B2, B3 waters 15.7 pg/L A 0.19 pg/L Water Quality: Aquatic Life CELDs 1994 AZ Acute Criteria for Aquatic and Wildlife Uses Acute Chronic Cold water fishery (A/W) 14,000 pg/L 900 pg/L Warm water fishery (A/W) 14,000 pg/L 900 pg/L Effluent dominated water (A/W) 14,000 pg/L 900 pg/L Ephemeral (A/W) NNS NNS co Aquatic Life Segments Acute 28,900 ng/L Chronic 1,240 pg/L FL MCL for mixing zone pollutants 1.57 mg/L LA Acute Criteria Freshwater 2,890 ug/L-acute 1,445 pg/L-chronic Marine water 8,150 pg/L-acute 4,075 pg/L-chronic OH Outside mixing zone: maximum 1,800 pg/L Inside mixing zone: maximum 3,600 pg/L Cold water mixing zone: 30-day average 79 pg/L +**DRAFT FOR PUBLIC COMMENT"** CHLOROFORM 7. REGULATIONS AND ADVISORIES 212 Table 7-1. Regulations and Guidelines Applicable to Chloroform (continued) Agency Description Information References STATE (Cont.) Groundwater Monitoring CELDs 1994 AL Yes CA Yes CO Yes IL Yes KY Yes LA Yes MN Yes MT Yes NY Yes OH Yes SC Yes TN Yes VA Yes Wi Yes WV Yes Hazardous Constituents CELDs 1994 AL Maxixmum Concentration of Contaminants for 60 mg/L the Toxicity Characteristics CA Concentration in waste; non-RCRA solvent 2.0 mg/kg waste Maximum concentration for the toxicity 6.0 mg/L characteristics CO Maximum concentration for the toxicity 6.0 mg/L characteristics IL Maximum concentration for the toxicity 6.0 mg/L characteristics KY Maximum concentration for the toxicity 6.0 mg/L characteristics LA Maximum concentration for the toxicity 6.0 mg/L characteristics MA Maximum concentration for the toxicity 6.0 mg/L characteristics MD Maximum concentration for the toxicity 6.0 mg/L characteristics ME Maximum concentration for the toxicity Yes characteristics MN Maximum concentration for the toxicity 6.0 mg/L characteristics "**DRAFT FOR PUBLIC COMMENT*** CHLOROFORM Table 7-1. Regulations and Guidelines Applicable to Chloroform (continued) 7. REGULATIONS AND ADVISORIES 213 Agency Description Information References STATE (Cont) MT Maximum concentration for the toxicity 6.0 mg/L characteristics ND Maximum concentration for the toxicity 6.0 mg/L characteristics NH Maximum concentration for the toxicity Yes characteristics NJ Maximum concentration for the toxicity 6.0 mg/L characteristics NY Maximum concentration for the toxicity Yes characteristics PA Maximum concentration for the toxicity 6.0 mg/L characteristics OH Maximum concentration for the toxicity 6.0 mg/L characteristics SC Maximum concentration for the toxicity 6.0 mg/L characteristics TX Maximum leachable concentration 6.0 mg/L VA Maximum concentration for the toxicity 6.0 mg/L characteristics vT Maximum concentration for the toxicity 6.0 mg/L characteristics Wi Maximum concentration for the toxicity 6.0 mg/L characteristics WV Maximum concentration for the toxicity Yes characteristics WY Maximum concentration for the toxicity 6.0 mg/L characteristics 3Group 2B: Possible Human Carcinogen. Group B2: Probable human carcinogen ACGIH = American Conference of Governmental Industrial Hygienists; EPA = Environmental Protection Agency; IARC = International Agency for Research on Cancer; OAQPS = Office of Air Quality Planning and Standards; ODW = Office of Drinking Water; OERR = Office of Emergency and Remedial Response; OSHA = Occupational Safety and Health Administration; PEL = Permissible Exposure Limit; PQL = Permissible Quantity Limit; RfD = Reference Dose; RQ = Reportable Quantity; STEL = Short-Term Exposure Limit; TLV = Threshold Limit Value; TWA = Time-Weighted Average for 8- hour exposure ***DRAFT FOR PUBLIC COMMENT"** CHLOROFORM 215 8. REFERENCES * Aaron CK, Howland MA, eds. 1994. Goldfrank’s toxicologic emergencies. Norwalk, CT: Appleton and Lange. * Abdel-Rahman MA. 1982. The presence of trihalomethanes in soft drinks. J Appl Toxicol 2:165-166. Abrams K, Harvell JD, Shriner D, et al. 1993. Effect of organic solvents on in vitro human skin water barrier function. 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Recent advances in studies on cardiac structure and metabolism, vol. 5: Basic functions of cations in myocardial activity. Baltimore, MD: University Park Press, 395-403. *Dreisback RH, ed. 1987. Handbook of poisoning-1987. Norwalk, CT: Appleton and Lange. *Dreisch FA, Munson TO. 1983. Purge-and-trap analysis using fused silica capillary column gc/ms. J Chromatogr Sci 21:111-118. *Dunnick JK, Melnick RL . 1993. Assessment of the carcinogenic potential of chlorinated water: experimental studies of chlorine, chloramine, and trihalomethanes. J Natl Cancer Inst 85(10):817-822. Ebel RE. 1989. Pyrazole treatment of rats potentiates CCl,- but not CHCl;-hepatotoxicity. Biochem Biophys Res Commun 161:615-618. *Ebel RE. 1990. Cytochrome p-450 and halomethane activation. USDA/CRIS database. July 1990. *Ebel RE, Barlow RL, McGrath EA. 1987. Chloroform hepatotoxicity in the mongolian gerbil. Fundam Appl Toxicol 8:207-216. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 225 8. REFERENCES *Eisenreich SJ, Looney BB, Thornton JD. 1981. Airborne organic contaminants of the Great Lakes ecosystem. Environ Sci Technol 15(1):30-38. Ekstrom T, Stahl A, Sigvardsson K, et al. 1986. Lipid peroxidation in vivo monitored as ethane exhalation and malondialdehyde excretion in urine after oral administration of chloroform. Acta Pharmacol Toxicol 58:289-296. *Ekstrom T, Warholm M, Kronevi T, et al. 1988. Recovery of malondialdehyde in urine as a 2,4-dinitrophenylhydrazine derivative after exposure to chloroform or hydroquinone. Chem Biol Interact 67:25-31. *El-shenawy NS, Abdel-Rahman MS. 1993a. The mechanism of chloroform toxicity in isolated rat hepatocytes. Toxicol Lett 69(1):77-85. *El-shenawy NS, Abdel-Rahman MS. 1993b. Evaluation of chloroform cardiotoxicity utilizing a modified isolated rat cardiac myocytes. Toxicol Lett 69(3):249-256. #Ellenhorn MJ, Barceloux DG, eds. 1988. Medical toxicology: Diagnosis and treatment of human poisoning. New York, NY: Elsevier Publishing, 972-974. *Enhorning G, Potoschnik R, Possmayer F, et al. 1986. Pulmonary surfactant films affected by solvent vapors. Anesth Analg 65:1275-1280. Enosawa S, Nakazawa Y. 1986. Changes in cytochrome P450 molecular species in rat liver in chloroform intoxication. Biochem Pharmacol 35:1555-1560. *Entz, RC, Thomas KW, Diachenko GW. 1982. Residues of volatile halocarbons in foods using headspace gas chromatography. J Agric Food Chem 30:846-849. *EPA. 1973a. U.S. Environmental Protection Agency. Code of Federal Regulations. 40 CFR 136.3. *EPA. 1973b. U.S. Environmental Protection Agency. Code of Federal Regulations. 40 CFR 136 (App. A). *EPA. 1975. U.S. Environmental Protection Agency. Code of Federal Regulations. 40 CFR 141.2. *EPA. 1978. U.S. Environmental Protection Agency. Code of Federal Regulations. 40 CFR 116.4. *EPA. 1979a. U.S. Environmental Protection Agency. Code of Federal Regulations. 40 CFR 141 (App. ©). *EPA. 1979b. U.S. Environmental Protection Agency. Code of Federal Regulations. 40 CFR 401.15. EPA. 1980. Ambient water quality criteria for chloroform. Office of Water Regulations and Standards, Criteria and Standards Division, U.S. Environmental Protection Agency, Washington, DC. *EPA. 198la. U.S. Environmental Protection Agency. Code of Federal Regulations. 40 CFR 403 (App. B). **DRAFT FOR PUBLIC COMMENT"*** CHLOROFORM 226 8. REFERENCES *EPA. 1981b. U.S. Environmental Protection Agency. Code of Federal Regulations. 40 CFR 413.02. *EPA. 1981c. U.S. Environmental Protection Agency. Code of Federal Regulations. 40 CFR 261 (App. VII). *EPA. 1982a. Method Nos. 601 and 625. Test methods. Methods for organic chemical analysis of municipal and industrial wastewater. U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, OH. *EPA. 1982b. U.S. Environmental Protection Agency. Code of Federal Regulations. 40 CER 423 (App. A). *EPA. 1982c. U.S. Environmental Protection Agency. Code of Federal Regulations. 40 CFR 465.02. EPA. 1983a. Chloroform and maleic hydrazide; Determination concluding the rebuttable presumptions against registration and notice of availability of position documents. Federal Register 48:498-501. *EPA. 1983b. U.S. Environmental Protection Agency. Code of Federal Regulations. 40 CFR 60.489. *EPA. 1983c. U.S. Environmental Protection Agency. Code of Federal Regulations. 40 CFR 122 (App. D). *EPA. 1983d. U.S. Environmental Protection Agency. Code of Federal Regulations. 40 CFR 433.11. *EPA. 1985a. U.S. Environmental Protection Agency: Health assessment document for chloroform. Washington, DC: Office of Health and Environmental Assessment. EPA/600/8-84-004F. NTIS PB86-105004/X AB. *EPA. 1985b. U.S. Environmental Protection Agency: Survey of chloroform emission sources. Research Triangle Park, NC: Office of Air Quality. EPA/450/3-85-026. EPA. 1985c. U.S. Environmental Protection Agency: Intent to list chloroform as a hazardous air pollutant. Federal Register 50:39626-39629. *EPA. 1985d. U.S. Environmental Protection Agency. Code of Federal Regulations. 40 CER 61.01. *EPA. 1985e. U.S. Environmental Protection Agency. Code of Federal Regulations. 40 CEFR 464.21. *EPA. 1985f. U.S. Environmental Protection Agency. Code of Federal Regulations. 40 CFR 464.31. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 227 8. REFERENCES EPA. 1985g. Health Assessment Document for Chloroform. Final Report. U.S. Environmental Protection Agency. EPA/600/8-84/004F. National Technical Information Service Publication No. PB86-105004. *EPA. 1986a. Method 8010. Test methods for evaluating solid waste. Volume IB: Laboratory manual physical/chemical methods. SW 846, 3rd ed. U.S. Environmental Protection Agency, Office of Solid Waste, Washington, DC. EPA. 1986b. Evaluation of the potential carcinogenicity of chloroform. 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U.S. Environmental Protection Agency: List (Phase 1) of hazardous constituents for ground-water monitoring; Final rule. 40 CFR Parts 264 and 270. *EPA. 1987b. U.S. Environmental Protection Agency: Extremely hazardous substances list and threshold planning quantities; Emergency planning and release notification requirements. Federal Register 52:13378-13410. *EPA. 1987c. Reportable quantity document for chloroform. Report by Environmental Criteria and Assessment Office, Office of Health and Environmental Assessment, U.S. Environmental Protection Agency, Cincinnati, OH, for the Office of Solid Waste and Emergency Response, U.S. Environmental Protection Agency. *EPA. 1987d. U.S. Environmental Protection Agency. Code of Federal Regulations. 40 CFR 141.40. *EPA. 1987e. U.S. Environmental Protection Agency. Code of Federal Regulations. 40 CFR 414.70. *EPA. 1987f. U.S. Environmental Protection Agency. Code of Federal Regulations. 40 CFR 414.91. *EPA. 1987g. U.S. Environmental Protection Agency. Code of Federal Regulations. 40 CFR 414.101. ***DRAFT FOR PUBLIC COMMENT"** CHLOROFORM 228 8. REFERENCES *EPA. 1987h. U.S. Environmental Protection Agency. Code of Federal Regulations. 40 CER 264 (App. IX). *EPA. 1987i. U.S. Environmental Protection Agency. Code of Federal Regulations. 40 CER 268 (App. III). *EPA. 1987j. U.S. Environmental Protection Agency. Code of Federal Regulations. 40 CFR 355 (App. A). “EPA. 1988a. U.S. Environmental Protection Agency: Land disposal restrictions for third scheduled wastes; final rule. 40 CFR Parts 264-266, 268, and 271. Federal Register 53:31138-31145. “EPA. 1988b. U.S. Environmental Protection Agency: National ambient volatile organic compounds (VOCS) Database update. EPA/600/3-88/010. *EPA. 1988c. Contract Laboratory Program statement of work for organics analysis multi-media multi-component 2/88. EPA. 1988d. Analysis of clean water act effluent guidelines pollutants. 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Environ Sci Technol 21:485-490. Zogorski JS. 1984. Experience in monitoring domestic water sources and process waters for trace organics. J Environ Sci Health A19:233-249. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 257 9. GLOSSARY Acute Exposure—Exposure to a chemical for a duration of 14 days or less, as specified in the Toxicological Profiles. Adsorption Coefficient (K,)—The ratio of the amount of a chemical adsorbed per unit weight of organic carbon in the soil or sediment to the concentration of the chemical in solution at equilibrium. Adsorption Ratio (Kd)—The amount of a chemical adsorbed by a sediment or soil (i.e., the solid phase) divided by the amount of chemical in the solution phase, which is in equilibrium with the solid phase, at a fixed solid/solution ratio. It is generally expressed in micrograms of chemical sorbed per gram of soil or sediment. Bioconcentration Factor (BCF)—The quotient of the concentration of a chemical in aquatic organisms at a specific time or during a discrete time period of exposure divided by the concentration in the surrounding water at the same time or during the same period. Cancer Effect Level (CEL)—The lowest dose of chemical in a study, or group of studies, that produces significant increases in the incidence of cancer (or tumors) between the exposed population and its appropriate control. Carcinogen—A chemical capable of inducing cancer. Ceiling Value—A concentration of a substance that should not be exceeded, even instantaneously. Chronic Exposure—Exposure to a chemical for 365 days or more, as specified in the Toxicological Profiles. Developmental Toxicity—The occurrence of adverse effects on the developing organism that may result from exposure to a chemical prior to conception (either parent), during prenatal development, or postnatally to the time of sexual maturation. Adverse developmental effects may be detected at any point in the life span of the organism. Embryotoxicity and Fetotoxicity—Any toxic effect on the conceptus as a result of prenatal exposure to a chemical; the distinguishing feature between the two terms is the stage of development during which the insult occurred. The terms, as used here, include malformations and variations, altered growth, and in utero death. EPA Health Advisory—An estimate of acceptable drinking water levels for a chemical substance based on health effects information. A health advisory is not a legally enforceable federal standard, but serves as technical guidance to assist federal, state, and local officials. Immediately Dangerous to Life or Health (IDLH)—The maximum environmental concentration of a contaminant from which one could escape within 30 minutes without any escape-impairing symptoms or irreversible health effects. Intermediate Exposure—Exposure to a chemical for a duration of 15-364 days, as specified in the Toxicological Profiles. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 258 9. GLOSSARY Immunologic Toxicity—The occurrence of adverse effects on the immune system that may result from exposure to environmental agents such as chemicals. In Vitro—TIsolated from the living organism and artificially maintained, as in a test tube. In Vivo—Occurring within the living organism. Lethal Concentration, (LC,,)—The lowest concentration of a chemical in air which has been reported to have caused death in humans or animals. Lethal Concentrations, (LCs))—A calculated concentration of a chemical in air to which exposure for a specific length of time is expected to cause death in 50% of a defined experimental animal population. Lethal Dose, (LD, ,)—The lowest dose of a chemical introduced by a route other than inhalation that is expected to have caused death in humans or animals. Lethal Doses, (LDs)—The dose of a chemical which has been calculated to cause death in 50% of a defined experimental animal population. Lethal Time, (LTs)—A calculated period of time within which a specific concentration of a chemical is expected to cause death in 50% of a defined experimental animal population. Lowest-Observed-Adverse-Effect Level (LOAEL)—The lowest dose of chemical in a study, or group of studies, that produces statistically or biologically significant increases in frequency or severity of adverse effects between the exposed population and its appropriate control. Malformations—Permanent structural changes that may adversely affect survival, development, or function. Minimal Risk Level—An estimate of daily human exposure to a dose of a chemical that is likely to be without an appreciable risk of adverse noncancerous effects over a specified duration of exposure. Mutagen—A substance that causes mutations. A mutation is a change in the genetic material in a body cell. Mutations can lead to birth defects, miscarriages, or cancer. Neurotoxicity—The occurrence of adverse effects on the nervous system following exposure to chemical. No-Observed-Adverse-Effect Level (NOAEL)—The dose of chemical at which there were no statistically or biologically significant increases in frequency or severity of adverse effects seen between the exposed population and its appropriate control. Effects may be produced at this dose, but they are not considered to be adverse. Octanol-Water Partition Coefficient (K,,)—The equilibrium ratio of the concentrations of a chemical in n-octanol and water, in dilute solution. Permissible Exposure Limit (PEL)—An allowable exposure level in workplace air averaged over an 8-hour shift. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM 259 9. GLOSSARY q,*—The upper-bound estimate of the low-dose slope of the dose-response curve as determined by the multistage procedure. The q,* can be used to calculate an estimate of carcinogenic potency, the incremental excess cancer risk per unit of exposure (usually pg/L for water, mg/kg/day for food, and pg/m’ for air). Reference Dose (RfD)—An estimate (with uncertainty spanning perhaps an order of magnitude) of the daily exposure of the human population to a potential hazard that is likely to be without risk of deleterious effects during a lifetime. The RfD is operationally derived from the NOAEL (from animal and human studies) by a consistent application of uncertainty factors that reflect various types of data used to estimate RfDs and an additional modifying factor, which is based on a professional judgment of the entire database on the chemical. The RfDs are not applicable to nonthreshold effects such as cancer. Reportable Quantity (RQ)—The quantity of a hazardous substance that is considered reportable under CERCLA. Reportable quantities are (1) 1 pound or greater or (2) for selected substances, an amount established by regulation either under CERCLA or under Sect. 311 of the Clean Water Act. Quantities are measured over a 24-hour period. Reproductive Toxicity—The occurrence of adverse effects on the reproductive system that may result from exposure to a chemical. The toxicity may be directed to the reproductive organs and/or the related endocrine system. The manifestation of such toxicity may be noted as alterations in sexual behavior, fertility, pregnancy outcomes, or modifications in other functions that are dependent on the integrity of this system. Short-Term Exposure Limit (STEL)—The maximum concentration to which workers can be exposed for up to 15 min continually. No more than four excursions are allowed per day, and there must be at least 60 minutes between exposure periods. The daily TLV-TWA may not be exceeded. Target Organ Toxicity—This term covers a broad range of adverse effects on target organs or physiological systems (e.g., renal, cardiovascular) extending from those arising through a single limited exposure to those assumed over a lifetime of exposure to a chemical. Teratogen—A chemical that causes structural defects that affect the development of an organism. Threshold Limit Value (TLV)—A concentration of a substance to which most workers can be exposed without adverse effect. The TLV may be expressed as a TWA, as a STEL, or as a CL. Time-Weighted Average (TWA)—An allowable exposure concentration averaged over a normal 8- hour workday or 40-hour workweek. Toxic Dose (TD,,)—A calculated dose of a chemical, introduced by a route other than inhalation, which is expected to cause a specific toxic effect in 50% of a defined experimental animal population. Uncertainty Factor (UF)—A factor used in operationally deriving the RfD from experimental data. UFs are intended to account for (1) the variation in sensitivity among the members of the human population, (2) the uncertainty in extrapolating animal data to the case of human, (3) the uncertainty in extrapolating from data obtained in a study that is of less than lifetime exposure, and (4) the uncertainty in using LOAEL data rather than NOAEL data. Usually each of these factors is set equal to 10. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM A-1 APPENDIX A MINIMAL RISK LEVEL (MRL) WORKSHEETS Chemical name(s): Chloroform CAS number(s): 000067-66-3 Date: August, 1995 Profile status: For Public Comment Draft Route: [X] Inhalation [ ] Oral Duration: [X] Acute [ |Intermediate [ ] Chronic Key to figure: 17 Species: Mouse MRL: 1.0 [] mg/kg/day [X] ppm [ ] mg/m’ Reference: Larson JL, Wolf DC, Morgan KT, et al. 1994c¢. The toxicity of 1-week exposures (0 inhaled chloroform in female B6C3F1 mice and male F-344 rats. Fund Appl Toxicol 22:431-446. Experimental design: The authors investigated the ability of chloroform vapors to produce toxicity and regenerative cell proliferation in female B6C3F1 mice and male Fischer 344 rats. Groups of 5 animals were exposed to 0, 1, 3, 10, 30, 100, or 300 ppm chloroform via inhalation for 6 hour/day for 7 consecutive days. Actual exposure concentrations measured for mice were 0, 1.2, 3.0, 10.0, 29.5, 101, and 288 ppm and for rats were 0, 1.5, 3.1, 10.4, 29.3, 100, and 271 ppm. Necropsies were performed on day 8. Animals were administered BrdU via implanted osmotic pump for the last 3.5 days. Cell proliferation was quantitated as the percentage of cells in S-phase (labeling index = LI) measured by the immunohistochemical detection of BrdU-labeled nuclei. CHLOROFORM A-2 APPENDIX A Effects noted in study and corresponding doses: Female Mice: 300 ppm: Respiratory NOAEL; proximal tubules of kidney lined by regenerating epithelium (less serious LOAEL). 100 ppm: Renal NOAEL,; centrilobular hepatocyte necrosis and severe diffuse vacuolar degeneration of midzonal and periportal hepatocytes (serious LOAEL); weight loss (less serious LOAEL). 30 ppm: Body weight NOAEL 10 ppm: Mild to moderate vacuolar changes in centrilobular hepatocytes (less serious LOAEL). 3 ppm: Hepatic effects NOAEL Male Rats: 300 ppm: Swelling and mild centrilobular vacuolation of hepatocytes (less serious LOAEL). 100 ppm: Hepatic effects NOAEL. 30 ppm: Increased number of S-phase nuclei for tubule cells in the renal cortex (less serious LOAEL). 10 ppm: Renal effect NOAEL; decreased body weight gain (less serious LOAEL); epithelial goblet cell hyperplasia and degeneration of Bowman's glands in olfactory mucosa (less serious LOAEL). 3 ppm: Body weight gain and respiratory NOAEL. Dose and end point used for MRL derivation: [X] NOAEL [ |] LOAEL: 3 ppm for hepatic effects in mice Uncertainty factors used in MRL derivation: 30 [11 [X]3 [] 10 (for extrapolation from animals to humans) [11 []3 [X] 10 (for human variability) CHLOROFORM A-3 APPENDIX A Was a conversion factor used from ppm in food or water to a mg/body weight dose? If so, explain: No. If an inhalation study in animals, list conversion factors used in determining human equivalent dose: Chloroform exerts critical effects that are extra-respiratory effects, therefore Equation 4-11 in Interim Methods for Development of Inhalation Reference Concentrations (EPA 1990b) was used to calculate the Human Equivalent Concentration (HEC). Animal body weights and alveolar ventilation rates for both mice and humans were obtained from Corley et al. (1990). The HEC was calculated as follows: NOAEL yz; = NOAEL x ([V/BW,I/[Vy/BWy]) NOAEL yg, = 3 ppm x ([2.01/0.02858 kg]/[347.9/70 kgl) NOAEL gc; = 3 ppm x (70.3289/4.97) NOAEL yc, = 42 ppm where: NOAEL zc; = Human Equivalent Concentration of the NOAEL (no-observed-adverse effect level) V,= Mouse alveolar ventilation rate BW,= Mouse body weight V, = Human alveolar ventilation rate BW, = Human body weight The MRL calculation is as follows: MRL = NOAEL gc, / UF MRL = 42 ppm / 30 MRL = 1 ppm Was a conversion used from intermittent to continuous exposure? If so, explain: No. Other additional studies or pertinent information that lend support to this MRL: The Larson et al. (1994¢) study is accompanied by a companion study performed by Mery et al. (1994), which examined the nasal lesions much more closely than this Larson study. The purpose of the Mery et al. study was to determine nasal cavity site-specific lesions and any cell induction/proliferation associated with varying concentrations of chloroform (0, 1, 3, 10, 30, 100, CHLOROFORM A-4 APPENDIX A 300 ppm) inhaled by both rats and mice 6 hours/day for 7 days. Female B6C3F1 mice and male Fischer 344 rats were used. Tissue abnormalities seen grossly, histopathologically, enzymatically (cytochrome P-450 levels), and in cell proliferation (BrdU labeling of cells in the S-phase) were reported for both rats and mice. The respiratory epithelium of the nasopharyngeal meatus exhibited an increase in the size of goblet cells at 100 and 30) ppm chloroform, in addition to an increase in both neutral and acidic mucopolysaccharides. Affected epithelium was up to twice the normal thickness. New bone formation within the nasal region was prominently seen at 10 ppm and above and followed a concentration response curve. At 1 ppm, only one animal showed mild bone enlargement of the first endoturbinate, with no changes seen at 3 ppm. At 10 ppm, minor enlargement was present in all animals. At 30 and 100 ppm, new osseous spicules was present at the beginning of the first endoturbinate, while at 300 ppm, the width of the new bone was almost doubled compared to controls receiving no chloroform, with lesions extending to involve up to 75% of the turbinate in all of the sites studied. Enzymatically, staining for P-450-2E1 was most prominent in the control animals in the cytoplasm of olfactory epithelial sustentacular cells and in the acinar cells of Bowman's glands, and more intense in the superficial cells than in the deep cells. In general, starting at about 3 ppm, increasing the chloroform concentration tended to decrease the amount of P-450 Staining. Exposure to chloroform resulted in a dramatic increase in the number of S-phase nuclei. A clear proportional concentration-related effect was observed, with the proliferative response confined to activated periosteal cells, including both osteogenic (round) and preosteogenic (spindle) cells. The proximal and central regions of the first endoturbinate had the highest increase of cell proliferation, while the distal part had only a moderate response, with this response being statistically significant from controls at concentrations of greater than 10 ppm. Decreased body weight was observed at 300 ppm only (data not provided). In mice, decreased body weight was observed at 100 and 300 ppm (data not provided). The only treatment-related histologic change observed in female mice was a slight indication of new bone growth in the proximal part of the first endoturbinate in one mouse exposed to 300 ppm chloroform. The S-phase response was observed at chloroform concentration of 10 ppm and higher. Agency Contact (Chemical Manager): Selene Chou CHLOROFORM A-5 APPENDIX A MINIMAL RISK LEVEL WORKSHEET Chemical name(s): Chloroform CAS number(s): 000067-66-3 Date: August, 1995 Profile status: For Public Comment Draft Route: [X] Inhalation [ ] Oral Duration: [ ] Acute [X]Intermediate [ ] Chronic Key to figure: 33 Species: Human MRL: 0.05 [ | mg/kg/day [X] ppm [| mg/m’ Reference: Phoon WH, Goh KT, Lee LT, et al. 1983. Toxic jaundice from occupational exposure to chloroform. Med J Malaysia 38:31-34. Experimental design: The study describes outbreaks of toxic hepatitis in workers occupationally exposed to chloroform in two different factories. Mostly women were employed in both places. Effects noted in study and corresponding doses: The workers in the first outbreak were exposed to concentrations up to 400 ppm chloroform in the workplace. No other chemical was involved. Blood chloroform levels in exposed workers ranged from 0.10 to 0.29 mg/100 mL. Workplace concentration levels of chloroform ranged from 14 to 50 ppm in the second outbreak. Vomiting and toxic hepatitis were noted to occur at an inhaled concentration of 14 ppm (less serious LOAEL). All affected workers were exposed to chloroform for less than six months. The patients exhibited anorexia, nausea, vomiting, and jaundice without fever. The subjects had originally been diagnosed with viral hepatitis, however the diagnosis of toxic hepatitis due to chloroform exposure was based upon epidemiological considerations. Dose and end point used for MRL derivation: [ ] NOAEL [X] LOAEL : 14 ppm for hepatic effects Uncertainty factors (UF) used in MRL derivation: 100 [11 [13 [X] 10 (for use of a LOAEL) [11 [13 [X] 10 (for human variability) Modifying factor (MF) used in MRL derivation: [11 [X13 [] 10 (for insufficient diagnostic data to determine the seriousness of hepatotoxic effects) Was a conversion factor used from ppm in food or water to a mg/body weight dose? If so, explain: No. CHLOROFORM A-6 APPENDIX A If an inhalation study in animals, list conversion factors used in determining human equivalent dose: No factors were used to convert to a human equivalent dose, since the data obtained from this study was obtained from human exposures to chloroform. The MRL calculation is as follows: MRL = LOAEL / (UF x MF) MRL = 14 ppm / (100 x 3) MRL = 0.05 ppm Was a conversion used from intermittent to continuous exposure? If so, explain: No. Other additional studies or pertinent information that lend support to this MRL: The study by Bomski et al. (1967) noted similar finding in a group of 68 workers occupationally exposed to chloroform for 1 to 4 years in a pharmaceutical plant. Inhaled chloroform concentrations ranged from 0.01 mg/L to 1 mg/L. Other solvents were reported in the air in trace amounts. Hepatomegaly was found in 25% of chloroform exposed workers. Toxic hepatitis was found in 5.6% of the liver enlargement cases The workers were diagnosed as having hepatosplenomegaly, enhanced serum glutamic pyruvic transaminase (SGPT) and serum glutamic oxaloacetic transaminase (SGOT) activities, and hyper- gammaglobulinemia. Hepatosteatosis (fatty liver) was detected in 20.6% of liver-enlargement cases. Chloroform exposed workers had a higher frequency of jaundice over the years than the control group. Agency Contact (Chemical Manager): Selene Chou CHLOROFORM A-7 APPENDIX A MINIMAL RISK LEVEL WORKSHEET Chemical name(s): Chloroform CAS number(s): 000067-66-3 Date: August, 1995 Profile status: For Public Comment Draft Route: [X] Inhalation | | Oral Duration: [ ] Acute | |Intermediate [X] Chronic Key to figure: 38 Species: Human MRL: 0.02 | | mg/kg/day [X] ppm |} mg/m’ Reference: Bomski H, Sobolewska A, Strakowski A. 1967. [Toxic damage of the liver by chloroform in chemical industry workers.] Int Arch F Gewerbepathologie u. Gewerbehygiene 24:127-134 (German). Experimental design: A group of 68 workers occupationally exposed to chloroform for 1-4 years in a pharmaceutical plant were examined. Doses of inhaled chloroform ranged from 2 to 205 ppm over a 1-4 year-period. Air concentrations of chloroform ranged from 0.01 mg/L to 1 mg/L. Other solvents were reported in the air in trace amounts. Effects noted in study and corresponding doses: A systemic LOAEL (hepatomegaly) of 2 ppm was determined from the data presented in this study. Hepatomegaly was found in 25% of chloroform exposed workers. The results were compared with a group of unexposed controls, and a group of persons who had infectious hepatitis during the last 1-4 years. Toxic hepatitis was found in 5.6% of the liver enlargement cases (the workers were diagnosed as having hepatosplenomegaly, enhanced serum glutamic pyruvic transaminase [SGPT] and serum glutamic oxaloacetic transaminase [SGOT] activities, and hyper- gammaglobulinemia). Hepatosteatosis (fatty liver) was detected in 20.6% of liver-enlargement cases. Functional tests were negative in most of the subjects; a biopsy was not performed in any case. Chloroform exposed workers had a higher frequency of jaundice over the years than the control group. The authors speculated that a viral infection might have been promoted in the chloroform damaged liver. Dose end point used for MRL derivation: [ ] NOAEL [X] LOAEL: 2 ppm for hepatic effects Uncertainty factors used in MRL derivation: 100 [11 [13 [X] 10 (for use of a LOAEL) [11 [13 [X] 10 (for human variability) Was a conversion factor used from ppm in food or water to a mg/body weight dose? If so, explain: No. CHLOROFORM A-8 APPENDIX A If an inhalation study in animals, list conversion factors used in determining human equivalent dose: No factors were used to convert to a human equivalent dose, since the data obtained from this study was obtained from human exposures to chloroform. The MRL calculation is as follows: MRL = LOAEL / (UF) MRL = 2 ppm / (100) MRL = 0.02 ppm Was a conversion used from intermittent to continuous exposure"? If so, explain: No. Other additional studies or pertinent information that lend support to this MRL: Agency Contact (Chemical Manager): Selene Chou CHLOROFORM A-9 APPENDIX A ‘MINIMAL RISK LEVEL WORKSHEET Chemical name(s): Chloroform CAS number(s): 000067-66-3 Date: August, 1995 Profile status: For Public Comment Draft Route: [ ] Inhalation [X] Oral Duration: [X] Acute [ ]Intermediate [ ] Chronic Key to figure: 23 Species: Mouse MRL: 0.3 [X] mg/kg/day []ppm [] mg/m’ Reference: Larson JL, Wolf DC, Butterworth BE. 1994b. Induced cytotoxicity and cell proliferation in the hepatocarcinogenicity of chloroform in female B6C3F1 mice: comparison of administration by gavage in corn oil vs ad libitum in drinking water. Fund Appl Toxicol 22:90-102. Experimental design: This study was designed to identify a relationship between the magnitude and duration of chloroform- induced histopathologic and proliferative responses for female mice dosed with chloroform in the drinking water vs those dosed in corn oil via gavage. Authors placed 0, 60, 200, 400, 900, or 1,800 ppm of chloroform in drinking water; however, due to decreased water intake, the authors’ calculation of consumed chloroform was 0, 16, 26, 53, 81, or 105 mg/kg/day. Effects noted in study and corresponding doses: In the 400, 900, and 1,800 ppm treatment groups, the livers had tinctorial changes characterized by pale cytoplasmic eosinophilic staining of centrilobular hepatocytes compared to the periportal hepatocytes and controls. Livers from mice treated with 200 ppm (26 mg/kg/day actual intake) chloroform or less failed to showed significant histologic changes when compared to controls. Thus the dose of 26 mg/kg/day was considered the NOAEL for hepatic effects in these mice. Chloroform exposure did cause a slight dose dependent decrease in number of cells in S-phase in the kidneys, mainly in the cortex, while there was an increase in these type of cells in the outer medullary region. Decreased body weight was observed at the two highest doses. After 4 days treatment, serum clinical chemistry analyses were not different from controls in either liver alanine aminotransferase (ALT) or sorbitol dehydrogenase (SDH) at any dose. Dose end point used for MRL derivation: [X] NOAEL [ ] LOAEL: 26 mg/kg/day for hepatic effects in mice CHLOROFORM A-10 APPENDIX A Uncertainty factors used in MRL derivation: 100 [11 [13 [] 10 (for use of a LOAEL) [11 [13 [X] 10 (for extrapolation from animals to humans) [11 [13 [X] 10 (for human variability) Was a conversion factor used from ppm in food or water to a mg/body weight dose? If so, explain: Conversion of the ppm concentration of chloroform in the drinking water to a mg/kg/day dose was provided by the authors of the paper. The MRL calculation is as follows: MRL = NOAEL / UF MRL = 26 mg/kg/day / 100 MRL = 0.3 mg/dg/day If an inhalation study in animals, list conversion factors used in determining human equivalent dose: Was a conversion used from intermittent to continuous exposure? If so, explain: Other additional studies or pertinent information that lend support to this MRL: Larson et al. (1995) also studied the dose response relationships for the induction of cytolethality and regenerative cell proliferation in male F-344 rats given chloroform in corn oil by gavage or in the drinking water. Groups of 12 rats were administered oral doses of 0, 3, 10, 34, 90, and 180 mg/kg/day chloroform in corn oil by gavage for 4 days or for 5 days/wk for 3 weeks. Bromodeoxyuridine (BrdU) was administered via an implanted osmotic pump to label cells in S-phase. Statistically significant decreases in body weight gains were observed in the 180 mg/kg/day dose group at 4 days and in the 90 and 180 mg/kg/day dose groups at 3 weeks. At 34 mg/kg/day, slight to mild centrilobular sinusoidal leukostasis was observed after 4 days of exposure. The livers of rats given 90 mg/kg/day for 4 days had a slight increase in centrilobular pallor and necrosis of hepatocytes surrounding the central vein; the remaining central and some mid-zonal hepatocytes were swollen and displayed a cytoplasmic granularity. After 3 weeks of exposure, livers of rats in the 34 or 90 mg/kg/day dose groups did not differ from controls. In the 180 mg/kg/day dose group, the livers of rats after 4 days had scattered individual cell necrosis throughout the central and midzonal regions. The cytoplasm of the centrilobular hepatocytes was pale eosinophilic and mildly vacuolated. In the 180 mg/kg/day dose group, after 3 weeks effects were similar to those seen at 4 days after exposure. Dose-dependent increases in both alanine aminotransferase (ALT) CHLOROFORM A-11 APPENDIX A and sorbitol dehydrogenase (SDH) were observed at 4 days in the 90 and 180 mg/kg/day dose groups and at 3 weeks in the 180 mg/kg/day dose group only. A dose-dependent increase in LI was seen in rat liver after 4 days of treatment with 90 and 180 mg/kg/day by gavage, but the LI remained elevated after 3 weeks of treatment only at the 180 mg/kg/day dose. Interestingly, when chloroform was administered in the drinking water at doses of 0, 60, 200, 400, 900, and 1800 ppm for 4 days, no microscopic alterations were seen in the kidneys after 4 days of treatment. As a general observation, rats treated for 3 weeks with 200 ppm chloroform and greater had slightly increased numbers of focal areas of regenerating renal proximal tubular epithelium and cell proliferation than were noted in controls, but no clear dose response relationship was evident. However, the overall renal LI was not increased at any dose or time point. Similarly, only mild hepatocyte vacuolation was observed in rats given 900 or 1800 ppm in water for 4 days and in rats given 1800 ppm in water for 3 weeks. No increase in the hepatic LI was observed at any time point. When chloroform was administered in the drinking water at doses of 0, 60, 200, 400, 900, and 1800 ppm for 3 weeks, no microscopic alterations were seen in the kidneys after 4 days of treatment. As a general observation, rats treated for 3 weeks with 200 ppm chloroform and greater had slightly increased numbers of focal areas of regenerating renal proximal tubular epithelium and cell proliferation than were noted in controls, but no clear dose response relationship was evident. However, the overall renal LI was not increased at any dose or time point. Similarly, only mild hepatocyte vacuolation was observed in rats given 900 or 1800 ppm in water for 4 days and in rats given 1800 ppm in water for 3 weeks. No increase in the hepatic LI was observed at any time point. The authors noted that these data indicated more severe hepatic and renal toxicity when chloroform is administered by gavage than in the drinking water. Agency Contact (Chemical Manager): Selene Chou CHLOROFORM A-12 APPENDIX A MINIMAL RISK LEVEL WORKSHEET Chemical name(s): Chloroform CAS number(s): 000067-66-3 Date: August, 1995 Profile status: For Public Comment Draft Route: [ ] Inhalation [X] Oral Duration: [ 1 Acute [X]Intermediate [ ] Chronic Key to figure: 62 Species: Dog MRL: 0.1 [X] mg/kg/day []ppm [|] mg/m’ Reference: Heywood R, Sortwell RJ, Noel PRB, et al. 1979. Safety evaluation of toothpaste containing chloroform. III. Long-term study in beagle dogs. J Environ Pathol Toxicol 2:835-851. Experimental design: An intermediate oral exposure MRL of 0.1 mg/kg/day was derived using the study by Heywood et al. (1979). The study was of 7.5 years duration in which 8 male and 8 female Beagle dogs were exposed to chloroform in toothpaste capsules, with doses of 0, 15, and 30 mg/kg/day, 6 days/wk for 6 weeks. Clinical chemistry parameters were monitored at 6 and 13 weeks of exposure and thereafter at intervals of 8-32 weeks. Effects noted in study and corresponding doses: Serum glutamic pyruvic transaminase (SGPT) activity was significantly increased (p<0.05) in the 30 mg/kg/day group beginning at 6 weeks and at every interval thereafter. SGPT activity was not increased in the 15 mg/kg/day group until week 130. Thus, 15 mg/kg/day is the NOAEL for intermediate duration exposure. Dose end point used for MRL derivation: [X] NOAEL [ ] LOAEL : 15 mg/kg/day for hepatic effects Uncertainty factors used in MRL derivation: 100 X] 10 (for extrapolation from animals to humans) (11113 [11 [13 [X] 10 (for human variability) ( [ Was a conversion factor used from ppm in food or water to a mg/body weight dose? If so, explain: No. If an inhalation study in animals, list conversion factors used in determining human equivalent dose: CHLOROFORM A-13 APPENDIX A Was a conversion used from intermittent to continuous exposure? If so, explain: (15 mg/kg/day) x 6/7 days = 12.9 mg/kg/day The MRL calculation is as follows: MRL = NOAEL,,p;, / UF MRL = 12.9 mg/kg/day/ 100 MRL = 0.1 mg/kg/day Other additional studies or pertinent information that lend support to this MRL: Liver effects in animals have been reported in numerous oral studies of intermediate duration. Fatty changes, necrosis, increased liver weight, and hyperplasia have been observed in rats exposed to >150 mg/kg/day chloroform in drinking water for 90 days (Palmer et al. 1979). An increased incidence of sporadic, mild, reversible liver changes occurred in mice exposed to chloroform in drinking water at doses of 0.3-114 mg/kg/day for 90 days, but the incidences were not significantly higher than the incidences in controls (Chu et al. 1982a). Fatty and hydropic changes, necrosis, and cirrhosis were observed in mice treated by gavage with 250 mg/kg/day chloroform in oil for 90 days (Bull et al. 1986; Munson et al. 1982) or at 86 mg/kg/day in drinking water for 1 year (Klaunig et al. 1986). In contrast, centrilobular fatty changes observed in mice at 64 mg/kg/day chloroform in drinking water for 90 days appeared to be reversible (Jorgenson and Rushbrook 1980), and no liver effects were found in mice treated with >50 mg/kg/day in aqueous vehicles (Bull et al. 1986). In addition, hepatocellular degeneration was induced in F, females in a 2-generation study in which mice were treated by gavage with 41 mg/kg/day chloroform in oil (Gulati et al. 1988). Agency Contact (Chemical Manager): Selene Chou CHLOROFORM A-14 APPENDIX A MINIMAL RISK LEVEL WORKSHEET Chemical name(s): Chloroform CAS number(s): 000067-66-3 Date: August, 1995 Profile status: For Public Comment Draft Route: [ ] Inhalation [X] Oral Duration: [ ] Acute [ ]Intermediate [X] Chronic Key to figure: 83 Species: Dog MRL: 0.01 [X] mg/kg/day | | ppm [ | mg/m’ Reference: Heywood R, Sortwell RJ, Noel PRB, et al. 1979. Safety evaluation of toothpaste containing chloroform. III. Long-term study in beagle dogs. J Environ Pathol Toxicol 2:835-851. Experimental design: Eight male and 8 female Beagle dogs were exposed to chloroform in toothpaste capsules. Doses used were 0, 15, and 30 mg/kg/day, 6 days/wk for 7.5 years. Clinical chemistry parameters were monitored at 6 and 13 weeks of exposure and thereafter at intervals of 8-32 weeks for 7.5 years. Effects noted in study and corresponding doses: SGPT activity was significantly increased (p<0.05) in the 30 mg/kg/day group beginning at 6 weeks and at every interval thereafter. SGPT activity was not increased in the 15 mg/kg/day group until week 130. No treatment-related body weight changes were observed in chloroform exposed dogs. No hematological changes were found. Increased SGPT levels, and less distinct elevation of SGOT and SAP seemed to be dose-related. However, the SGPT levels tended to return to normal during the recovery period. Bromsulphalein retention test was performed during the sixth year of the study; no treatment-related abnormality was found. No organ weight changes were found in the exposed groups. No remarkable histopathological differences were observed in dogs regarding the cardiovascular system. Fatty cysts were observed in the liver in all groups; however, in females the incidence seemed to be dose-related (3 of 12,5 of 8, 7 of 8). Fat deposition in renal glomeruli was reportedly higher in the 30 mg/kg/day chloroform group. Dose end point used for MRL derivation: [ 1 NOAEL [X] LOAEL: 15 mg/kg/day for hepatic effects Uncertainty factors used in MRL derivation: 1000 [X] 10 (for use of a LOAEL) [11 []3 [11 [13 [X] 10 (for extrapolation from animals to humans) [11 []3 [X] 10 (for human variability) CHLOROFORM A-15 APPENDIX A Was a conversion factor used from ppm in food or water to a mge/body weight dose? If so, explain: No. If an inhalation study in animals, list conversion factors used in determining human equivalent dose: Was a conversion used from intermittent to continuous exposure? If so, explain: (15 mg/kg/day) x 6/7 days = 12.9 mg/kg/day. The MRL calculation is as follows: MRL = LOAEL py, / UF MRL = 12.9 mg/kg/day / 1000 MRL = 0.01 mg/kg/day Other additional studies or pertinent information that lend support to this MRL: Numerous chronic oral studies examined hepatic and renal end points as well as neurological and cancer effects. Serious effects occurred at higher doses; 15 mg/kg/day was the lowest dose used in available animals studies. A NOAEL of 2.46 mg/kg/day for liver and kidney effects (SGPT, SGOT, BUN and SAP) was found in humans who used a dentifrice containing 0.34% or a mouthwash containing 0.43% chloroform for 1-5 years (DeSalva et al. 1974). Agency Contact (Chemical Manager): Selene Chou CHLOROFORM B-1 APPENDIX B USER’S GUIDE Chapter 1 Public Health Statement This chapter of the profile is a health effects summary written in non-technical language. Its intended audience is the general public especially people living in the vicinity of a hazardous waste site or chemical release. If the Public Health Statement were removed from the rest of the document, it would still communicate to the lay public essential information about the chemical. The major headings in the Public Health Statement are useful to find specific topics of concern. The topics are written in a question and answer format. The answer to each question includes a sentence that will direct the reader to chapters in the profile that will provide more information on the given topic. Chapter 2 Tables and Figures for Levels of Significant Exposure (LSE) Tables (2-1, 2-2, and 2-3) and figures (2-1 and 2-2) are used to summarize health effects and illustrate graphically levels of exposure associated with those effects. These levels cover health effects observed at increasing dose concentrations and durations, differences in response by species, minimal risk levels (MRLs) to humans for noncancer end points, and EPA’s estimated range associated with an upper-bound individual lifetime cancer risk of 1 in 10,000 to 1 in 10,000,000. Use the LSE tables and figures for a quick review of the health effects and to locate data for a specific exposure scenario. The LSE tables and figures should always be used in conjunction with the text. All entries in these tables and figures represent studies that provide reliable, quantitative estimates of No-Observed-Adverse-Effect Levels (NOAELS), Lowest-Observed-Adverse-Effect Levels (LOAELSs), or Cancer Effect Levels (CELs). The legends presented below demonstrate the application of these tables and figures. Representative examples of LSE Table 2-1 and Figure 2-1 are shown. The numbers in the left column of the legends correspond to the numbers in the example table and figure. LEGEND See LSE Table 2-1 (1) Route of Exposure One of the first considerations when reviewing the toxicity of a substance using these tables and figures should be the relevant and appropriate route of exposure. When sufficient data exists, three LSE tables and two LSE figures are presented in the document. The three LSE tables present data on the three principal routes of exposure, i.e., inhalation, oral, and dermal (LSE Table 2-1, 2-2, and 2-3, respectively). LSE figures are limited to the inhalation (LSE Figure 2-1) and oral (LSE Figure 2-2) routes. Not all substances will have data on each route of exposure and will not therefore have all five of the tables and figures. (2) Exposure Period Three exposure periods - acute (less than 15 days), intermediate (15-364 days), and chronic (365 days or more) are presented within each relevant route of exposure. In this example, an inhalation study of intermediate exposure duration is reported. For quick reference to ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM B-2 3) “) ®) (6) (7 ®) 9) (10) (In APPENDIX B health effects occurring from a known length of exposure, locate the applicable exposure period within the LSE table and figure. Health Effect The major categories of health effects included in LSE tables and figures are death, systemic, immunological, neurological, developmental, reproductive, and cancer. NOAELs and LOAELS can be reported in the tables and figures for all effects but cancer. Systemic effects are further defined in the "System" column of the LSE table (see key number 18). Key to Figure Each key number in the LSE table links study information to one or more data points using the same key number in the corresponding LSE figure. In this example, the study represented by key number 18 has been used to derive a NOAEL and a Less Serious LOAEL (also see the 2 "18r" data points in Figure 2-1). Species The test species, whether animal or human, are identified in this column. Section 2.5, "Relevance to Public Health," covers the relevance of animal data to human toxicity and Section 2.3, "Toxicokinetics," contains any available information on comparative toxicokinetics. Although NOAELs and LOAELSs are species specific, the levels are extrapolated to equivalent human doses to derive an MRL. Exposure Frequency/Duration The duration of the study and the weekly and daily exposure regimen are provided in this column. This permits comparison of NOAELs and LOAELSs from different studies. In this case (key number 18), rats were exposed to 1,1,2,2-tetrachloroethane via inhalation for 6 hours per day, 5 days per week, for 3 weeks. For a more complete review of the dosing regimen refer to the appropriate sections of the text or the original reference paper, i.e., Nitschke et al. 1981. System This column further defines the systemic effects. These systems include: respiratory, cardiovascular, gastrointestinal, hematological, musculoskeletal, hepatic, renal, and dermal/ocular. "Other" refers to any systemic effect (e.g., a decrease in body weight) not covered in these systems. In the example of key number 18, 1 systemic effect (respiratory) was investigated. NOAEL A No-Observed-Adverse-Effect Level (NOAEL) is the highest exposure level at which no harmful effects were seen in the organ system studied. Key number 18 reports a NOAEL of 3 ppm for the respiratory system which was used to derive an intermediate exposure, inhalation MRL of 0.005 ppm (see footnote "b"). LOAEL A Lowest-Observed-Adverse-Effect Level (LOAEL) is the lowest dose used in the study that caused a harmful health effect. LOAELs have been classified into "Less Serious" and "Serious" effects. These distinctions help readers identify the levels of exposure at which adverse health effects first appear and the gradation of effects with increasing dose. A brief description of the specific endpoint used to quantify the adverse effect accompanies the LOAEL. The respiratory effect reported in key number 18 (hyperplasia) is a Less serious LOAEL of 10 ppm. MRLs are not derived from Serious LOAELSs. Reference The complete reference citation is given in chapter 8 of the profile. CEL A Cancer Effect Level (CEL) is the lowest exposure level associated with the onset of carcinogenesis in experimental or epidemiologic studies. CELs are always considered serious “**DRAFT FOR PUBLIC COMMENT*** CHLOROFORM B-3 APPENDIX B effects. The LSE tables and figures do not contain NOAELS for cancer, but the text may report doses not causing measurable cancer increases. (12) Footnotes Explanations of abbreviations or reference notes for data in the LSE tables are found in the footnotes. Footnote "b" indicates the NOAEL of 3 ppm in key number 18 was used to derive an MRL of 0.005 ppm. LEGEND See Figure 2-1 LSE figures graphically illustrate the data presented in the corresponding LSE tables. Figures help the reader quickly compare health effects according to exposure concentrations for particular exposure periods. (13) Exposure Period The same exposure periods appear as in the LSE table. In this example, health effects observed within the intermediate and chronic exposure periods are illustrated. (14) Health Effect These are the categories of health effects for which reliable quantitative data exists. The same health effects appear in the LSE table. (15) Levels of Exposure concentrations or doses for each health effect in the LSE tables are graphically displayed in the LSE figures. Exposure concentration or dose is measured on the log scale "y" axis. Inhalation exposure is reported in mg/m’ or ppm and oral exposure is reported in mg/kg/day. (16) NOAEL In this example, 18r NOAEL is the critical endpoint for which an intermediate inhalation exposure MRL is based. As you can see from the LSE figure key, the open-circle symbol indicates to a NOAEL for the test species-rat. The key number 18 corresponds to the entry in the LSE table. The dashed descending arrow indicates the extrapolation from the exposure level of 3 ppm (see entry 18 in the Table) to the MRL of 0.005 ppm (see footnote "b" in the LSE table). (17) CEL Key number 38r is | of 3 studies for which Cancer Effect Levels were derived. The diamond symbol refers to a Cancer Effect Level for the test species-mouse. The number 38 corresponds to the entry in the LSE table. (18) Estimated Upper-Bound Human Cancer Risk Levels This is the range associated with the upper-bound for lifetime cancer risk of 1 in 10,000 to 1 in 10,000,000. These risk levels are derived from the EPA’s Human Health Assessment Group’s upper-bound estimates of the slope of the cancer dose response curve at low dose levels (q,%). (19) Key to LSE Figure The Key explains the abbreviations and symbols used in the figure. ***DRAFT FOR PUBLIC COMMENT"*** «~INIFWWOD O1NEaNd HOH 14vHd... SAMPLE 1” TABLE 2-1. Levels of Significant Exposure to [Chemical x] — Inhalation Exposure LOAEL (effect) Key to frequency/ NOAEL = - figure? Species duration ~~ System (ppm) Less serious (ppm) Serious (ppm) Reference 2 |— INTERMEDIATE EXPOSURE =] [&] [=F] [=] =F [2 ]- Systemic l : 3 l { l l 4 [> 18 Rat 13 wk Resp 3 10 (hyperplasia) Nitschke et al. 5d/wk 1981 6hr/d CHRONIC EXPOSURE mn ss Cancer 1 38 Rat 18 mo 20 (CEL, multiple Wong et al. 1982 5d/wk organs) 7hr/d 39 Rat 89-104 wk 10 (CEL, lung tumors, NTP 1982 5d/wk nasal tumors) 6hr/d 40 Mouse ~~ 79-103 wk 10 (CEL, lung tumors, NTP 1982 5d/wk hemangiosarcomas) 6hr/d * The number corresponds to entries in Figure 2-1. — ° Used to derive an intermediate inhalation Minimal Risk Level (MRL) of 5 x 10° ppm; dose adjusted for intermittent exposure and 12 divided by an uncertainty factor of 100 (10 for extrapolation from animal to humans, 10 for human variability). CEL = cancer effect level; d = days(s); hr = hour(s); LOAEL = lowest-observed-adverse-effect level; mo = month(s observed-adverse-effect level; Resp = respiratory; wk = week(s) ); NOAEL = no- gd XION3ddv WNHO40HOTHO v-d we LINFAWOO 0178Nd HOH 1dVHOux — > Figure 2-1. Levels of Significant Exposure to [Chemical X] — Inhalation Acute Intermediate (<14 days) (15-364 days) Systemic Systemic N N $F S&L oF <> N > > NN > > o Q —> (ppm) & & & & RL & & of &° 10000 1000 O @ 37m Q ® oO 0 @® 30 SI. 10 Or dg 20mD Boom ir @ 3m “TO g18r 22g 21r O 29r 27r 18r 33 22m 34r 10 > r Q@ 18r | 1 | | 104 — I 0.1 I 10-5 Estimated <— Y Upper Bound 0.01 5 Human Cancer Key 10% — Risk Levels 0.001 r Rat @ LOAEL for serious effects (animals) I Minimal risk level for effects 1077 I m Mouse (DP LOAEL for less serious effects (animals) | aie han saneer 0.0001 h Rabbit (OO NOAEL (animals) g Guinea Pig CEL - Cancer Effect Level The number next to each point 0.00001 k Monkey * corresponds to entries in the accompanying table. 0.000001 Co ; * Doses represent the lowest dose tested per study that produced a tumorigenic response and do not imply the existence of a threshold for the cancer end point. 0.0000001 g XIAN3ddY WHO404HOTHO S-9 CHLOROFORM B-6 APPENDIX B Chapter 2 (Section 2.5) Relevance to Public Health The Relevance to Public Health section provides a health effects summary based on evaluations of existing toxicologic, epidemiologic, and toxicokinetic information. This summary is designed to present interpretive, weight-of-evidence discussions for human health end points by addressing the following questions. 1. What effects are known to occur in humans? 2. What effects observed in animals are likely to be of concern to humans? 3. What exposure conditions are likely to be of concern to humans, especially around hazardous waste sites? The section covers end points in the same order they appear within the Discussion of Health Effects by Route of Exposure section, by route (inhalation, oral, dermal) and within route by effect. Human data are presented first, then animal data. Both are organized by duration (acute, intermediate, chronic). In vitro data and data from parenteral routes (intramuscular, intravenous, subcutaneous, etc.) are also considered in this section. If data are located in the scientific literature, a table of genotoxicity information is included. The carcinogenic potential of the profiled substance is qualitatively evaluated, when appropriate, using existing toxicokinetic, genotoxic, and carcinogenic data. ATSDR does not currently assess cancer potency or perform cancer risk assessments. Minimal risk levels (MRLs) for noncancer end points (if derived) and the end points from which they were derived are indicated and discussed. Limitations to existing scientific literature that prevent a satisfactory evaluation of the relevance to public health are identified in the Data Needs section. Interpretation of Minimal Risk Levels Where sufficient toxicologic information is available, we have derived minimal risk levels (MRLs) for inhalation and oral routes of entry at each duration of exposure (acute, intermediate, and chronic). These MRLs are not meant to support regulatory action; but to acquaint health professionals with exposure levels at which adverse health effects are not expected to occur in humans. They should help physicians and public health officials determine the safety of a community living near a chemical emission, given the concentration of a contaminant in air or the estimated daily dose in water. MRLs are based largely on toxicological studies in animals and on reports of human occupational exposure. MRL users should be familiar with the toxicologic information on which the number is based. Chapter 2.5, "Relevance to Public Health," contains basic information known about the substance. Other sections such as 2.7, "Interactions with Other Substances,” and 2.8, "Populations that are Unusually Susceptible" provide important supplemental information. MRL users should also understand the MRL derivation methodology. MRLs are derived using a modified version of the risk assessment methodology the Environmental Protection Agency (EPA) provides (Barnes and Dourson 1988) to determine reference doses for lifetime exposure (RfDs). ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM B-7 APPENDIX B To derive an MRL, ATSDR generally selects the most sensitive endpoint which, in its best judgement, represents the most sensitive human health effect for a given exposure route and duration. ATSDR cannot make this judgement or derive an MRL unless information (quantitative or qualitative) is available for all potential systemic, neurological, and developmental effects. If this information and reliable quantitative data on the chosen endpoint are available, ATSDR derives an MRL using the most sensitive species (when information from multiple species is available) with the highest NOAEL that does not exceed any adverse effect levels. When a NOAEL is not available, a lowest-observed-adverse-effect level (LOAEL) can be used to derive an MRL, and an uncertainty factor (UF) of 10 must be employed. Additional uncertainty factors of 10 must be used both for human variability to protect sensitive subpopulations (people who are most susceptible to the health effects caused by the substance) and for interspecies variability (extrapolation from animals to humans). In deriving an MRL, these individual uncertainty factors are multiplied together. The product is then divided into the inhalation concentration or oral dosage selected from the study. Uncertainty factors used in developing a substance-specific MRL are provided in the footnotes of the LSE Tables. ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM ACGIH ADME atm ATSDR BCF BSC C CDC CEL CERCLA CFR CLP cm CNS d DHEW DHHS DOL ECG EEG EPA EKG F F, FAO FEMA FIFRA fpm ft FR g GC gen HPLC hr IDLH IARC APPENDIX C ACRONYMS, ABBREVIATIONS, AND SYMBOLS American Conference of Governmental Industrial Hygienists Absorption, Distribution, Metabolism, and Excretion atmosphere Agency for Toxic Substances and Disease Registry bioconcentration factor Board of Scientific Counselors Centigrade Centers for Disease Control Cancer Effect Level Comprehensive Environmental Response, Compensation, and Liability Act Code of Federal Regulations Contract Laboratory Program centimeter central nervous system day Department of Health, Education, and Welfare Department of Health and Human Services Department of Labor electrocardiogram electroencephalogram Environmental Protection Agency see ECG Fahrenheit first filial generation Food and Agricultural Organization of the United Nations Federal Emergency Management Agency Federal Insecticide, Fungicide, and Rodenticide Act feet per minute foot Federal Register gram gas chromatography generation high-performance liquid chromatography hour Immediately Dangerous to Life and Health International Agency for Research on Cancer International Labor Organization inch adsorption ratio kilogram metric ton organic carbon partition coefficient octanol-water partition coefficient ***DRAFT FOR PUBLIC COMMENT*** CHLOROFORM mm mm Hg mmol mo mppcf MRL MS NIEHS NIOSH NIOSHTIC ng nm NHANES nmol NOAEL NOES NOHS NPL NRC NTIS NTP OSHA PEL pg pmol PHS PMR ppb ppm ppt RfD RTECS sec SCE SIC SMR APPENDIX C liter liquid chromatography lethal concentration, low lethal concentration, 50% kill lethal dose, low lethal dose, 50% kill lowest-observed-adverse-effect level Levels of Significant Exposure meter milligram minute milliliter millimeter millimeters of mercury millimole month millions of particles per cubic foot Minimal Risk Level mass spectrometry National Institute of Environmental Health Sciences National Institute for Occupational Safety and Health NIOSH’s Computerized Information Retrieval System nanogram nanometer National Health and Nutrition Examination Survey nanomole no-observed-adverse-effect level National Occupational Exposure Survey National Occupational Hazard Survey National Priorities List National Research Council National Technical Information Service National Toxicology Program Occupational Safety and Health Administration permissible exposure limit picogram picomole Public Health Service proportionate mortality ratio parts per billion parts per million parts per trillion recommended exposure limit Reference Dose Registry of Toxic Effects of Chemical Substances second sister chromatid exchange Standard Industrial Classification standard mortality ratio ***DRAFT FOR PUBLIC COMMENT*** C-2 CHLOROFORM STEL STORET TLV TSCA RXR OR KINA NIV YV pm Hg APPENDIX C short term exposure limit STORAGE and RETRIEVAL threshold limit value Toxic Substances Control Act Toxics Release Inventory time-weighted average United States uncertainty factor year World Health Organization week greater than greater than or equal to equal to less than less than or equal to percent alpha beta delta gamma micrometer microgram ***DRAFT FOR PUBLIC COMMENT*** # U.S. GOVERNMENT PRINTING OFFICE:1995-639-414 U.C. 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