'OMy copy E p A HA #5 NITROCELLULOSE Health Advisory Office of Drinking Water U.S. Environmental Protection Agency Washington, DC 20460 CO CO PREFACE 1 This report was prepared in accordance with the Memorandum of L’ndtrscand-r.g between the Department of che Army, Deputy for Environment, Safety and Occupational Health (OASA(IiL)) and the U.S. Environmental Protection Agency (EPA), Office of Drinking Water (ODW), Criteria and Standards Division, for che purpose of developing drinking water Health Advisories (HAs) for selected environmental contaminants, as requested by che Army. Health Advisories provide specific advice on the levels of contaminants in drinking water at which adverse health effects would not be anticipated and which include a margin of safety so as to protect che most sensitive member of che population at risk. A Health Advisory provides health effects guidelines and analytical methods and recommends treatment techniques on a case-by-case basis. These advisories are normally prepared for One-day, 10-day, Longer-term and Lifetime exposure periods where available toxicological data permit. These advisories do not condone the presence of contaminants in drinking water; nor are they legally enforceable standards. They are noe Issued as official regulations, and they may or may not lead to che issuance of national standards or Maximum Contaminant Levels (MCLs). This report is the product of the foregoing process. Available toxicological data, as provided by che Army, on the munitions chemical, nitrocellulose (NC), have been revleved, and the relevant findings are presented in this report in a manner so as to allow for an evaluation of the data without continued reference to che primary documents. Significant chemical and physical properties of NC as well as potential sources of exposure art provided as a means of identifying che subject chemical. Data on the pharmacokinetic properties, although somewhat limited in scope, are summarized. All available toxicological data, including short-term, longer-term and lifetime feeding studies in three species, as well as the results of reproductive and genetic toxicology studies, have been reviewed, and those effects deemed relevant to the ingestion of NC are summarized. Results of immunologic and carcinogenic studies are also Included. This report also includes a section describing state-of-the-ert methods of analyses tor 1C In drinking water and includes essential treatment techniques approprlato for NC removal froa an affected water supply, should the levels of NC in che drinking water reach a level considered unpalatable due to taste, clarity or similar Indicators. This report has been submitted to a critical review by the E?A to include a panel of Health Effects Branch (HE3) toxicologists. Their comments, es appropriate, have been incorporated Into this report. A companion document, 'Tata Deficiencies/Problem Areas and Recommendations r> e r. c \ for Additional Sara 3ase Develop report under Appendix 2. C ^ Nitrocellulose" is me.udeo I would like to express my thanks to Dr. John Glenr.on, Life Systems, Ir.c., who afforded valuable coordination and logistical assistance. I also thank Dr. Janet Normandy, Ms. Lori Gordon and Dr. William Hartley who provided the extensive technical skills required for the preparation of this report. I am grateful to the following members of the EPA Tox-Review Panel, Dr. Kenneth Bailey, Dr. Ambika Bathija, Dr. Bill Marcus and Mr. Bruce Mintz, who took time to review this report and to provide their invaluable input. Finally, I would like to thank Dr. Edward Ohanian, Chief, Health Effects 3ranch, Dr. Joseph Cotruvo, Director Criteria and Standards Division, and Dr. Penelope Fenner-Crisp, Manager, Health Advisory Program, for providing me wich th opportunity and encouragement to be a part of this project. The preparation of this Advisory was funded in part by Interagency Agreement (IAG) 35PP5869 between the U.S. EPA and the U.S. Army Medical Research and Development Command (USAMRDC). This IAG was conducted with the technical support of the U.S. Army Biomedical Research and Development Laboratory (USABRDl). Krishan Khanna, Project Officer Office of Drinking Water I. INTRODUCTION .. I-i II. GENERAL INFORMATION. 11-1 III. SOURCES OF EXPOSURE. Ill-1 IV. PHARMACOKINETICS. IV-L V. HEALTH effects. V-l A. Short-Tar* Expoaura . V-l 1. Primary Skin and Eya Irritation. v-l B. Longar-Tarm Expoaura. V-l 1. Thirtaan-W««k Studiaa. V-l 2. Lifatiaa Expoaura. V-3 C. Ganatlc Toxicology. V-7 D. Carclnoganiclty. V-3 E. Raproductlva Effacta. V-9 F. Taratoganicity. V-10 G. Immunologic Effacta . 7-10 VI. HEALTH ADVISORY DEVELOPMENT. VI-1 VII. ANALYSIS. VII-1 VIII. TREATMENT.VIII-l IX. CONCLUSIONS AND RECOMMENDATIONS. IX-1 X. REFERENCES. X-L LIST OF TABLES Tabla II-l Ganaral Chamical and Phyalcal Propartlaa of Calluloaa . I1-2 Trinierat* h» er.dix Al Calculation Methods . A A2 Data Deiiciencies/Problem Areas and Recommendations for Additional Data Base Development for Nitrocellulose . a j The Health Advisory (HA) Program, sponsored by the Office of Drinking Water (ODW), provides information on the health effects, analytical methodology and treatment technology that would be useful in dealing with the contamination of drinking water. Health Advisories describe nonregulatory concentrations of drinking water contaminants at which adverse health effects would not be anticipated to occur over specific exposure durations. Health Advisories contain a margin of safety to protect sensitive members of the population. Health Advisories serve as informal technical guidance to assist Federal, State and local officials responsible for protecting public health when emergency spills or contamination situations occur. They are not to be construed as legally enforceable Federal standards. The Advisories are subject to change as nev Information becomes available. Health Advisories are developed for One-day, Ten-day, Longer-term (approximately 7 years, or 101 of an individual's lifetime) apd Lifetime exposures based on data describing noncarclnogenic end points of toxicity. Health Advisories do not quantitatively incorporate any potential carcinogenic risk from such exposure. For those substances that are knovn or probable human carcinogens, according to the Agency classification scheme (Group A or B), Lifetime HAs are not recommended. The chemical concentration values for Group A or B carcinogens are correlated with carcinogenic risk estimates by employing a cancer potency (unit risk) value together with assumptions for lifetime exposure and the consumption of drinking water. The cancer unit risk is usually derived from the linear multistage model with 9SZ upper confidence limits. This provides a low-dose estimate of cancer risk to humans that Is considered unlikely to pose a carcinogenic risk in excess of the stated values. Excess cancer risk estimates may also be calculated using the One-hit, Velbull, Logit and Problt models. There is no current understanding of the biological mechanisms Involved in cancer to suggest that any one of these models is able to predict risk more accurately than another. Because each model is based upon differing assumptions, the estimates that are derived can differ by several orders of magnitude. 1-1 ZVMtJd. INFOiWAT N As described by Sullivan et al. (19“8), nitrocellulose, or cellulose trinitrate, is a non-volatile, fibrous, cotton-like, white solid (specific gravitv 1.66) consisting of chains of beta, I to 4 linked glucoside units in which :r.e hydroxyl groups of the glucose subunits have reacted to fora nitrate esters. The cheaical formula is approximately [C (ONO^)3 ]^; the molecular weight depends on chain length, and the. degree of polymerization'(DP) varies appreciably. Cotton litters and wood pulp used for the preparation of military grades of N’C have a 2? of approximately 1,000 to 1,500. The fully nitrated form of NC has a nitrogen concent of 14.14 X and a formula weight of 297.14. This degree of nitration is difficult to accomplish (Department of the Army Technical Manual TM9- 1300-214, 1967). Nitrocellulose is extremely flammable and has a flash point of 12.3 9 C. The melting point range is 160* to 170*C — also its autoignition temperature. "Guncotton", military grade cellulose nitrate, contains 13.52 nitrogen and is the most highly nitrated form. Theoretically, mononitrated, dinitrated, and trinicraced cellulose contain 6.82 N, 11.12 N, and 14.12 N, respectively. "Guncotton" is essentially fully nitrated and, therefore, can be considered to be a crude cellulose trinitrate contaminated by traces of less completely nitrated esters (Sullivan et al., 1978). The general properties of cellulose trinitrate arc summarized in Table II-l. The nitrate esters, in general, are soluble in esters, aldehydes, and ketones. The more completely nitrated a cellulose is, however, the smaller the range of materials in which it is soluble. The less nitrated forms of cellulose nitrate are also very soluble in methanol, benzene, toluene, and mixtures of ether and ethanol. These compounds (pyroxylin: 82 to 122 N and pyrocellulose: 12.52 N) are forms of collodion and are used chiefly as lacquers, ink bases, and filter membranes, and in veterinary medicine for wound closure. Cellulose trinitrate is insoluble in water, ethanol, ethyl ether, and benzene but completely miscible In acetone, methylethyl ketone, tetrahydrofuran, as well as nitrobenzene and ethyl-, butyl-, and amylacetates (Sullivan et al., 1978). Nitrocellulose it resistant to biological degradation and is a persistent compound in the environment. It has been suggested by Sullivan et al. (1978) that cellulose triacetate and cellulose trinitrate are resistant to enzymatic attack. These authors, further stated that although direct biodegradation of nitrocellulose does not nee to be feasible, alkaline hydrolysis yields material that can be decomposed by microbial activity. Nitrocellulose, therefore, .an be characterized as being persistent in the envlronaent unless cheslcally altered. Based on an extensive volume of literature dealing with the adsorptive capacity of nitrocellulose for biologically important macromolecules, Sullivan et al. (1978) have indicated that nitrocellulose strongly adsorbs and concentrates DNA and various RNA molecules from solution, and adsorbs proteins and polypeptides under various conditions. Proteins with an uncoiled structure or molecular weight greater than l(r are strongly bound. Denatured proteins are also strongly adsorbed, especially under acidie conditions. II-l CAS Number L f :ZL1UL0SE TRINITRATE*' Names Molecular weight Empirical formula Structure 9004-70-0 Cellulose trinitrate; nitrocellulose; guncotton Varies with chain length [C 6 H 7°2 C0NO 2 ) 3 I n Color Physical state Specific gravity Percent nitrogen carbon oxygen hydrogen Solubility characteristics Melting poiafc. Whitish Fibrous solid 1.66 14.l b/ 24.3 59.3 2.4 Insolusble in water* ethanol, benzene and most other solvents. Soluble in 25 parts of 1:3 alcohol:ether; soluble in acetone in all proportions; soluble in aethylethyl ketone, nitrobenzene, tetrahydbofuran, and ethyl, butyl and aaylscetate. 160-170* C Autoignltlo* temperature 160-170* C Flash point 12.7* C b/ References: Sullivan et al., 1978; Havley. 1981; RTECS, 1986. Approximate, depends on processing and chain length. II-2 Nitrocellulose (NC) is a principal ingredient of propellants, smokeless powder, rocket fuel, ball powder, mortar increments, and some explosives (Rosenblatt et al., 1973). It is produced for military use at selected Army ammunition plants (AAP) by treating cotton linters or wood pulp with mixed nitric and sulfuric acids at 30*C. The resulting slurry is centrifuged to remove most c the acid, treated with several changes of boiling water, washed with a heavy stream of water, and finally screened to remove most of the water (Department of the Army Technical Manual TM9- l 300-214, 1967). Production requires 16 to 22 gallons of process water per pound of NC produced. Most of this water is discharged and contains, in addition to NC, 0.7 to 1.0 pound of sulfuric acid ar.d 0.3 to 0.4 pound of nitric acid per pound of NC produced, thus resulting in a low pH (Rosenblatt et al., 1973; Helton, 1976). Nitrocellulose fines are found in production wastewater because of settling pit overflow and some escape after flowing through the waste acid neutralization process lines (Huff et al., 1975). Helton (1976) analyzed samples of NC fines from wastewaters of an AAP and found that more chan 99X of the particulate material was military grade NC with an average nitrogen content of 12.9Z and particle size greater chan 5 microns. Sullivan at al. (1978) indicated chat these data suggested that suspended solids below 0.8 microns contained significant portions of nonnlcrated cellulose and other materials while chose greater than 44 microns were mostly NC. III-l There art currently no available data to suggest chat nitrocellulose car. re absorbed fro* any route. A study in rats has shown chat NC appears to terrain within cht gastrointestinal tract until it is excreced in che feces. Ocher available data are inconclusive. Ellis et al. (1976) studied the absorption, distribution, and excretion of 1 "“e-nitrocellulose (prepared from cotton grown in the presence of D-glucose-L’L-‘ "*C and nitrated by standard procedures) in two male Charles River CD rats. Each rat was fasted overnight before being given NC orally either in an aqueous suspension or in a suspenaion of 0.2Z methyl cellulose-0. 4Z Tween 80 (MC-TW80) at a volume of 1 ml/100 g* (presumably bodyvelght; about 20,000 dptn/ml). After dosing, each rat was placed immediately In a "Roth-Delmar" metabolism chamber. Expired C0 ? , feces, and urine were each collected separately in the apparatus. To ensure that sufficient radioactivity was administered, the dosing was repeated dally for 4 days. Twenty-four hours after the last dose, the animals were anesthetized and aortic blood was collected. Liver, spleen, brain, kidneys, lungs and thigh muscle along with the stomach, small intestine, cecum, large intestine and their contents were removed and each assayed for radioactivity via liquid scintillation spectrometry. After repeated oral doses of radiolabeled nitrocellulose, no detectable radioactivity was found in any tissue or body fluid. Radioactivity was recovered only in the various components of the gastrointestinal (GI) tract plus contents and in the feces (percentage dpm recovered not specified). From these results, the authors concluded that the nitrocellulose molecule was not absorbed by the rat. Ellis et al. (1980) conducted a mass-balance metabolism study of nitrocellulose using a dog fed 90 g (mg/kg dose could not be determined from data provided) of wet nitrocellulose (27.9 g dry weight). After 24, 48, 72 and 96 hours, the feces were collected and analyzed by the method of Sellg (1961) which involves reduction of nitro groups and titration to a colorimetric endpoint. During the first'48-hour period, 9.S g (dry weight) of NC ware recovered representing a recovery of 34Z (g recovered/g fed, dry weight). No further nitrocellulose vs* identified in lster fecsl samples. We can draw no useful generallistlea* fro* these results since they could indicate either that the method of analysis was not particularly effective, that some nitrocellulose may have bee* absorbed fro* the digestive tract of this dog, or that NC was denitrated by gut bacteria. IV-1 ILLINOIS STATE WATER SURVEY LIBRARY COPY Currently available studies on the health effects of nltrocellulc chat it is not "toxic" unless ingested in enormous quantities such as total diet. In those cases, death from intestinal impaction may occur does in animals fed the same dietary levels of non-nitrated cotton lin se saigas - ' I o : : - just as te r s . a A. Short-Term Exposure The acute oral toxicity of nitrocellulose is very low in mice and rats as evidenced by LD '§ of more chan 5,000 mg/kg in both species. No acute toxic::;/ data of nitrocellulose ere provided in the 1986 edition of the Registry of Toxic Substances (RTECS, 1986). Lee ec al. (1975) used male and female Charles River rats and male and female albino Swiss mice (apparently 10/sex/dose; number and range of doses not clearly specified) to determine the acute oral LD of nitrocellulose. Each dose of nitrocellulose, suspended in water at a final concentration of 5X NC (dry vtight basis), was divided in half and given 30 minutes apart due to the large volume necessary to administer a dose of 5,000 mg/kg (the authors did not indicate the volume administered). Test animals were fasted for at least 16 hours prior to oral dosing via gastric Intubation. After treatment, the survivors were observed daily for 14 days for delayed mortality or signs of toxicity. The LD was calculated by a computer program based on the method of maximum likelihood of Finney (1971). The LD^’s for nitrocellulose in male and female rets and in male and female mice were greater than 5,000 mg/kg. No toxic signs were observed in animals receiving the highest dose of nitrocellulose. Two of 10 male mice given 5,000 mg/kg died without any apparent gross lesions. No other animals died at any dose level. I. Primary Skin and Eye Irritation Using the modified Draize method for skin and eye irritation, a 331 concentration of NC in water was applied directly to the eye or shaved skin of the rabbit and evaluated at 24 and 72 hours (Lea at al., 1975). This concentration of NC was not irritating to either rabbit skin or eye. B. Longer-Term Exposure 1. Thirteen-Week Studies No adverse effects that can be related to the chemical characteristics of nitrocellulose vers identified in the available thirteen-week studies in dogs, rats, and mice (Ellis et al., 1976). Intestinal blockage was the cause of death in high-dose mice (10X NC) as veil as in control mice fed the same amount of cotton llnters (10Z of total dlat). V-i Ellis et al. ( 1 9 76 ) conducted thir teen-week studies c: the oral nitrocellulose using dogs, rats and nice. In the studies with dogs. 13 ycu-.g healthy beagle dogs (7.2 to 13.6 kg) were divided into five groups c: two males and two females. Three groups were administered NC as 1%, 3Z, or 10* of their feed as dry weight. (Table Al-1, Appendix Al, describes method used to calculate doses. Using the authors’ data for body weight at week 4 and average intake data for weeks 1 through 4, these doses were calculated to correspond to approximately 513, 1,900 and 6,890 tng/kg/day, respectively, for males and 610, 2,976 and 3,185 mg/kg/day, respectively, for females.) The fourth group received a mixture of 1CT (approximately 6,181 and 8,627 mg/kg/day, males and females, respectively, calculated as above) of cotton cellulose linters and served as a cotton control to determine if any observed effects were due to the passage of non-nutritive bulk through the gastrointestinal tract. The fifth group served as an uncreated control. The tests Included hematology, clinical blood chemistry and bromo- sulfophthalein (BSP) retention tests. At termination, the dogs were sacrificed and examined for treatment-related gross lesions and organs were weighed. Various tissues were processed for microscopic examination of lesions. Feeding NC to dogs at up to 10X of their diets or cotton linters at IQZ for 13 weeks did not cause any adverse effects. Dogs fed 10Z NC or linters ate about 15Z more than the others, indicating the test materials were merely non-nutritive bulk. All dogs, including the untreated and cotton controls, shoved some variations in body weight, peripheral blood elements and various clinical chemistry tests but all data were reported to be within normal limits. Nitrocellulose was not reported to cause any treatment-related gross or microscopic lesions. In thirteen-week studies with rats, Ellis et al. (1976) used 40 male and 40 female young healthy CD rats divided into five groups, each consisting of eight males and eight females. Three groups of rats were fed NC at LZ, 3Z, or I0Z in the feed as dry weight. The fourth group, the cotton controls, was fed cotton linters as 10Z of the feed. (Ref. Table Al-i: Using the authors' data for the body weights averaged from weeks 0, 4, 8 and 13 and the averige Intake over weeks l through 13, these doses correspond to approximately 667 and 2,366 mg/kg/day for IZ and 3Z, respectively, in the males and 820 and 2,673 mg/kg/day, respectively, in the females. Measured Intakes for the rats fed 10Z NC or 10Z cotton linters in the diet were more than double those of the untreated control rats. The authors indicated thee at 10Z NC or 10Z cotton linters in the diet, enormous mounds of white fluffy material were scattered all around the cage. Therefore, actual Intake of NC or eotton linters at 10Z in the diet cannot be calculated with any degree of accuracy.) The fifth group, the untreated controls, was given the powdered standard rodent chow without NC. Experimental procedures were similar to chose used with dogs (Ellis et al., 1976) except that BSP retention tests were not performed. V-2 Nitrocellulose and the cocron linters ac 1C" in che feed apparently a - :i - is a non-nucricivt bulk ingredient vhich Che racs acrenpced co remove. These ra:= gained less weight chan did the uncreated control racs. The authors concluded chat chis was because chey did not absorb enough of the nutritive portion c: the feed. Rats fed che low or middle levels of NC apparently received enough nutritional intake and gained weight comparably co the uncreated controls. Nitrocellulose administration did not result in any significant changes in peripheral blood elements or clinical blood chemistry or any apparent gross or microscopic lesions. Observed changes in che absolute organ weights of the liver, kidney and spleen of male rats fed 10Z NC or cotton linters were statistically significant when compared to untreated controls. These changes were attributed by the authors to depressed body weight gain* as evidenced by comparable organ weight/body weight ratios. In the thirteen-week studies with mice, Ellis et al. (1976) used 40 male and 40 female young healthy albino Swiss mice divided into five groups, each consisting of eight males and eight females. The treated groups were fed the same diets as prepared for the rats: LZ, 3Z, or 10Z of NC as dry weight in feed. Control mice received either the powdered standard rodent chow alone (as uncreated controls), or 10Z of cotton linters (as cotton controls). (Ref. Table A1 - L : Using the authors' data for the body weights averaged from weeks 0, 4, 8 and 13, and che average intake for weeks l through 13, these doses correspond to approximately 1,690 and 5,062 mg/kg/day for 1Z and 3Z, respectively, in males and 1,741 and 7,000 mg/kg/day, respectively, in females. As in che rat study previously described, che actual Intake of 10Z NC or 10Z cotton linters in che diet cannot be calculated with any degree of accuracy due to excessive scattering of che fibers.) The procedures used were similar to chose described for racs except chat clinical blood chemistry tests were not performed in mice. 91ood samples for terminal hematology were collected by heart puncture under ether anesthesia. Mice fed che lov, middle or high levels of NC showed no apparent adverse effects from che chemical nature of this compound. In the first few weeks of the study, there were weight losses and deaths, apparently due to the fibrous nature of che substance, in mice fed the high level (10Z) of NC as well as in mice fed 10Z of cotton linters. The authors concluded chat deaths were due to che blocking of the lover part of the gastrointestinal tract by masses of che fibers, particularly la tha regions where water Is removed from the chyme. Additional animals fro» a chronic study (same shipment and identical levels of NC) were added to this 13-veek study to compensate for the early losses. No adverse effects or changes were observed In peripheral blood elements nor were any treatment-related gross or microscopic lesions reported by the authors. 2. Lifetime Exposure Long-term studies conducted In dogs, rats and alee Indicated a dose-related Increase In total feed consumption and decreases In weight gain In hlgh-dose V-3 .1 .. SC-treated rats and nice. Histopathoic created for up to 24 months shoved r.atu treatment-related) ltsioas in the thyro several organ* in nice (kidneys, adrena causing early deaths due to intestinal gic evaluations o: ccgs, :a:s rally occurring (as opposed to id and lungs in dogs, the lungs Is, liver and intestines). Adve impaction occurred in high-dose in r r se nice a a Ellis «t al. (1980) studied the effects of NC administered orally at dose levels up to 10* NC in feed for dogs, rats and mice for 12 and 24 months. Three groups of beagle dogs, six of each sex per group, were administered NC at II, 31 or 10Z in the feed calculated on a dry weight basis for up to 24 months. (Ref. Table Al-1: Using the authors' data for the average body weight of 2 dogs/sex at 24 months and the average consumption [mean of 22 monthly measurements], these doses correspond to approximately 311, 1,013 and 4,070 mg/kg/day, respectively, for males and 344, 1,034 and 4,376 mg/kg/day, respectively, for females). Two additional control groups were run concurrently for each sex and were either untreated or received 101 cotton linters (cellulose linters; approximately 2,388 and 3,874 mg/kg/day, males and feme les, respectively, when calculated as above) in their diets. No toxic signs related to NC Intake were seen in any of the dogs at any time. Folloving 12 months of treatment without a recovery period, one male and one female per treatment group were sacrificed. Histopathological examination revealed a variety of "minimal to mild lesions" in all the dogs examined and moderate thyroid hyperplasia in a control and a high dose female and moderate thymus involution in a cotton control male. These lesions Included microcalculi in the medula of the kidney, parafollicular cell hyperplasia of the thyroid, hemosiderosis of the spleen and axillary lymph nodes and involution of the thymus. The lesions were considered by the authors to be typical of those naturally occurring in dogs of the age tested and were, therefore, not considered treatment-related. Since there were no effects related to NC treatment, histologic data from the lov and middle dose dogs were not evaluated, and the necropsy on the dogs in the recovery group (one/sex/trcatmenc group) was omitted. No dose-related variations were reported for routine hematologic parameters nor in fasting blood glucose* serum transaminases (SCOT* SGPT), cholesterol, alkaline phosphatase and blood urea nitrogen (BUN). The body weights, absolute and relative orgam weight* were comparable to unexposed controls or cotton controls (10Z linters). However* there was a dose-related Increase in feed consumption. Similatly* dogs (2/sex/treatment group) administered NC (up to 101) for 24 months (without a recovery period) showed what the authors considered naturally occurring histopathologic lesions. These lesions Included pituitary cysts, mild to severe chromic lymphocytic thyroiditis* pleural fibrosis, muscular hypertrophy of bronchioles* mild to moderate granulomatous pneumonia* renal calcinosis, extramedullary hematopoiesis of the spleen and hemosiderosis of the lymph nodes. The absolute and relative organ weights had soma variations between individual dogs, but no apparent differences between dogs in different treatment groups. V-4 Because :."ere vers no observable effaces : necropsy on che dogs in che recovery group :n :ia; (-/sex/ r.g .;c at It, 3Z or 101 as dry weight in the feed for up to 24 months. (Ref. Table a 1-L: These levels correspond to approximately 1,814 and 4,866 mg/kg/day at It and 3T, respectively, for males and 1,767 and 6,056 mg/kg/day, respectively, for females. Intake for the 10Z level could not be calculated.) Two additional groups were run concurrently for each sex and either were untreated or received 101 cotton linters. At the end of the treatment period, the control and treated mice were sacrificed and tissues were examined for occurrence of tumors in various organ systems. The authors reported the occurrence of bronchioalveolar carcinomas in control animals, but none were observed in mice administered 10Z NC for 24 months. The absence of such tumors was statistically significant in males but not in females (P • 0.004 for males, P • 0.29 for females, P • 0.002 for the combined sexes, by exact analysis of the contingency table). However, the authors concluded that the difference probably represents natural variation and is toxlcologically meaningless. The slides from other dosage groups were not examined. E. Reproductive Effects Three-generation studies in rats Indicated chat NC administered alone in the diet at dose levels up to I0Z did not adversely affect reproduction. However, the Increased inert bulk of I0Z fibers (NC or linters) may reduce the lactation index and pup weight. Ellis et al. (1980) administered nitrocellulose in the diet of rats at It, 3Z or I0Z in a three-generation study. (Ref. Table Al-i: These doses correspond to approximately 350 and 1,280 mg/kg/day for 1Z and 3Z, respectively, in males and 373 and 1,422 mg/kg/day, respectively, for females. The actual Intake for the I0Z dose level cannot be calculated.) The initial groups of rats used as the parental (F ) generation# vara started at tha same time as the chronic study. Rats of each subsequent group, parents and offspring, received tha same control or NC-containing diets a# their original F^ generation. First mating occurred when rats were & months of age. Following treatment, all offspring vers examined at birth for gross physical abnormalities and the numbers of live and dead pups of each litter were recorded. Survival and body weights were recorded at 0, 4 and 21 days. Reproductive performance for each parental and offspring generation was determined* The authors reported that the mean body weights at the time of first matings for males of all parental generations given diets containing 10Z NC were significantly reduced when compared to males given untreated control diet. This reduction in body weight was also significant in males and females fed cotton control (10Z linters) diets. V-9 Thera were r.o indications that the treatments adversely e::e::ed :.-e fertility of the males or females in the mating or pregnancy ratics. For t.na F_ generation, most of these parameters actually suggested chat either 10Z ,\'C or T cotton linters in the diet increased the fertility of rats given these treatr.en: However, chi* effect appears to be caused by the decreased fertility of the control F. females. The fertility of the F females was similar to that of concro Q- —-' O Is cor groups given IZ and 3Z NC (approximately 373 and 1,422 mg/kg/day) This decreased fertility in the F^ control and low dose groups was attributed by the authors to both the age at time of first mating (8 months for all F parental generations) and body weights greater than that expected to give optimal reproductive performance. No treatment-related effects were apparent at any dose or in any generation on litter size, llvebom Index* birth weight* viability index, or the ratio of males to total offspring. Significant reductions in the lactation index and the weight of pups at weaning occurred with 10Z NC and 10Z cotton llnter controls Reductions in these parameters were observed chiefly with the through F litters for both groups but were not observed with the subsequent litters. "The authors attributed these reductions during lactation to relatively large amounts of inert bulk in a diet of 10Z fibers (NC or linters). F. Teratogenicity Ellis et al, (1980) did not report conducting teratology studies. G. Immunologic Effects Ellis et al. (1976) studied the potential adverse effects of NC exposure on the Immunologic response based on serum concentration of immunoglobulin (IgE) measured by the immunodiffusion technique of Manclni et al. (1964). The administration of 10Z NC in the feed to dogs or rats (this dietary level can be calculated, as described under Thirteen-Week Studies, to correspond to an average of approximately 7,690 mg/kg/day for dogs but could not be calculated for rats due to excessive scattering of fibers) for up to 13 weeks did not alter their serum concentrations of V-10 health advisory development Nitrocellulose, at dos«s averaging 4,300 mg/kg/day in dogs and in excess o: approximately 1,400 mg/kg/day in racs and 6,000 mg/kg/day in mice, was not ccxcc when fed for up to 24 months. The actual intakes in mg/kg/day in racs and mice fed 10Z NC could not be determined with any degree of accuracy due to visible scattering of the fibers around the cage. Assuming, however, that these animals consumed only twice the amount of NC as chose receiving the 3Z diet, a reasonably conservative estimate, intake would be, at a minimum, approximately 2,300 mg/kg/day in rats and 12,000 mg/kg/day in mice. The only treatment-related effects in these high-dose, long-term feeding studies were early weight loss in racs and mice and subsequent lower average body weights in the mature rat, hyperemia with edema of the ears, eyelids, genitalia, feet and call apparently due to physical-mechanical effects of the fibers and death due to intestinal impaction in mice fed the 10Z diets (NC and cotton linters). The weight effect was attributed to the non-nutritive bulk of the fibrous diet and was confirmed by similar findings in control animals fed I0Z cotton linters (cellulose linters), the material that is nitrated to form NC. This lower body weight, however, is not necessarily an adverse effect as the rodents fed the I0Z diets were characterized by less fat, not less lean body mass, when compared to untreated animals of the same species. This condition can, however, ba detrimental if there la a very high body demand for nutrients, as during pregnancy and lactation. This condition occurred, to soma degree, in the early litters of the three-generation reproduction study in rats, as evidenced by a decreased lactation index and weight at weaning, but not in later generations, possibly indicating adaptation. The hyperemia with edema chat occurred in mice was not life-threatening, resolved spontaneously and was of unknown cause. As this effect occurred in mice fed either 10Z NC or cotton linters and was accompanied by continuous scratching, a direct physical effect due to irritation from contact with the fibers seems likely. Tha deaths dua to intestinal impaction wars attributed to the relatively large size of tha fibers in relation to the size of the lumen of the intestines, allowing tha masses formed by the fibers to completely obstruct the gut. This effect occurred is mice fed both the 10Z NC and eotton linters. Unresolved is a spate of of unknown cause that occurred at 9 months in both the I0Z NC and cotton listers groups but was at a higher level in the NC group. This may be due, in part* to the chemical nature of NC. In 13-week feeding studies at the same levels, intakes averaged 7,700 mg/kg/day in dogs and were in excess of approximately 2,700 mg/kg/day in racs and 7,000 mg/kg/day in mice. Calculation of the 10Z levels to approximates, as in the long-term study, would yield velues, at a minimum* of 5*400 ag/kg/dey in racs and 14,000 mg/kg/day in mice. There was no evidence of toxicity. The Increased food VI-1 • do 3 consumption* decreased weight the I--xonth study, occurred -inters and was attributed to gain pattern in rats and at the LC.T level in anir.als the non-nutritive bulk of fed the either fibers. ar .1 ^ ^ Death due to intestinal inpaction also occurred in the high-dosed nice in this study and vas.attributed to the physical nature of the fibers. Since the created animals, like humans, cannot digest cellulose, passage of these fibers through the digestive tract would be expected. This non-digestion of the fibrous diet wes confirmed in absorption studies. After repeated oral doses (once daily for four days) of radiolabelled nitrocellulose to rats, no detectable radioactivity vas found in any tissue or body fluid but was recovered only from various componsnts of ths GI tract, contents snd feces. Nitrocellulose vss not mutsgsnic in tlthsr bsctsrial blossssys nor did it producs chromosomt abnormalities in mammalian cells sftsr in vivo exposure. It did not significantly affect tumor incidence in dogs, rets and mice and produced only soms adverse effects in rats with high nutrient requirements during pre-gnancy and lactation. In view of ths non-toxic nature of NC sc ell doses studied and its failure to be digested and absorbed in the species tested, htslch advisory (HA) values for 1-Dsy, 10-Days and Longer-term appear to be unnscssssry. It seems probable that, due to the fibrous nature of ths substance snd its insolubility in water, clarity or turbidity of ths water would be the only guideline necessary. VI-2 V 0 AN/ S ' w * Rosenblatt al. ( 1973) in summar iz ir.g literature on che analysis of N'C indicated chat all analytical procedures probably vould begin with collection of NC fibers from the water on filters. Weighing che filter is cited as a method to roughly estimate NC, but its accuracy vould be limited by che presence of inert solids. Ocher methods cited Include: o Ferrous-ticanous titration o Ferrous sulfate titration o Liberation of NO^ gss o Analysis of NH^ after reduction by Devarda's alloy o Transnitration of salicylate or citrate followed by ferrous-titanous titration o Chromous chloride-ferric ammonium sulfate micro-determination o Zinc dust reduction of the nitrate ester o Hydrolytic liberation of nitrite ion in acetone ? Of the detection methods listed above, the last one is considered to be che most effective for detecting low levels of NC in the environment (Sullivan et al. f 1978; Rosenblatt et al., 1973). It is a colorimetric method based on hydrolytic liberation of nitrite by OH from acetone solutions of nitrate esters. The resulting NO^ is then dlaaotlzed with either alpha-(naphthyl)-ethylenedlamlne hydrochloride or alpha-naphthylamlne and the absorbance of the solution determined at 520 to 530 nm. The reaction is noe specific for NC; however, the insolubility of this compound in water allows quantitative separation from NO^ , NO , and other soluble nitrate esters or nitrocompounds in mixed wastewater by filtration or dialysis. Barkley and Rosenblatt (1978) adapted this procedure to the Technlcon Autoanalyser. The procedure involves aspiration of a stirred NC suspension, dialysis against 9Z saline, and hydrolysis with 5N NaOH at 70* C for 10 minutes to release nitrite ion. Sulfanilic acid is diazotized by che nitrite ion at low pH. The resulting diazoniua salt is coupled with che N—( 1 -naphthyl)ethylenediamine, and the color produced is measured at 520 nm. The limit of detectability ia 0.4 mg/L of nitrocellulose. Sulllvsft et el.(1978) outlined two methods for determining NC in sediment. The first method involves solvent extraction of the dried sediment in acetone or ethylacetstm* The procedure is sensitive to as little as 0.5 mg/kg of nlcrace ester; however* it is not specific for NC because it also extracts other organic nitrate esters and nitrocompounds. The second method Involves an initial acetone extraction to remove the nitrate ester. Nitrate is then determined colorimetrically since it will oxidize ferrous iron to ferric iron after creating che extract with acetic acid, ferrous sulfate in sulfuric acid, and sodium sulfite. Absorbance of the resulting yellow color is determined at 500 nm. This procedure, however, lacks sensitivity since the minimum detectable concentration in a 10 g sediment sample is 140 mg/kg. Vll-i Wastewater from NC production facilities is neutralized, then seeded, centrifuged, and/or screened to recover NC fibers (Sullivan et al., 1979). Centrifugation leads to more efficient and constant recovery due to the high specific gravity of NC (Rosenblatt et al., 1973). The US Army Natick Laboratories (Massachusetts) has developed a chemical and microbiological process for the degradation of NC in wastewater (Rosenblatt et al., 1973). The process involves membrane ultrafderation of the wastewater to concentrate the suspended NC. A 200-fold concentration, to a 3Z to 5Z NC suspension, was desired; however, only a 10-fold concentration, to 0.2Z NC, has been obtained by this process. The suspension is then treated with 3Z N’aOH at 9C a to 93* C for 20 minutes to yield a soluble material containing little nitrate ester. After acid neutralization, nitrate ester content of the filtrate Is determined by IR analysis. An extract of the filter is similarly analyzed to detect any undlssolved nitrate ester, presumably NC. The neutralized solution is mixed with domestic waste and fermented anaerobically to denitrify thus producing gaseous nitrogen as a product. Methanol could be added as a nutrient in this step. The next step is an aerobic activated sludge treatment and finally, a second denitrification, again using methanol nutrient. The final product was reported to be nitrocellulose free. VIII-1 :x. V ^ v ,v :i:s:oss and ?.zc:mmz'Cat::n3 3ased on available toxicity data and the chemical and physical properties :: the compound* NC is apparently non-toxic to dogs, rats and mice and is not digested nor absorbed in these species. These data, along with the relative insolubility of NC in water, suggest that Health Advisory values for NC in drinking water are unnecessary. The physical characteristics of the drinking water as they relate to turbidity, clarity, taste and similar indicators of palatabllity appear to be the only guidelines necessary. A companion report, "Data Deficiences/Problem Areas and Recommendations for Additional Data Base Development For Nitrocellulose" (Appendix 2) summarizes the scope and adequacy of existing data reviewed for this HA and delineates those areas where additional data, if any, are deemed necessary. IX-1 x. REFERENCES 3arkley, J.J. and Rosenblatt, D.H., 1973. Automated nitrocellulose analysis. t\S. Army Medical Bioengineering Research and Development Laboratory Technical Report 7807, AD A067081, Fort Detrick, MD. Department of the Army Technical Manual TM9-1300—214 , 7-7. 1967. Departaents of the Army and the Air Force. Military Explosives. Nitrocellulose. Ellis, H.V., III et al., 1976. Mammalian toxicity of munitions compounds Phase II: Effects of multiple dosei Part IV: Nitrocellulose. Report No. 5. Midwest Research Institute, Kansas City, MO, Contract No. DAMD-17-74-C-4073, AD AQ620 L6. Ellis, H.V., III et al., 1978. Mammalian toxicity of munitions compounds Phase I: Acute oral toxicity, primary skin and eye irritation, dermal sensitization, disposition and metabolism, and Ames tests of additional compounds. Report No. 6, Midwest Research Institute, Kansas City, MO, Contract No. DAMD-l7-74-C-4073, AD A069333. Ellis, H.V., III et al., 1980. Mammalian toxicity of munitions compounds Phase III: Effects of life-time exposure Part III: Nitrocellulose. Report No. 9. Midwest Research Institute, Kansas City, MO, Contract No. DAMD-17-74-C-4073. Finney, D.J., 1971. Problt Analysis, Cambridge University Press. Hawley, G.G., 1981. The condensed chemical dictionary. Tenth edition. Van Nostrand-Relnhold. Helton, D.O., 1976. Chemical and physical characterization of nitrocellulose fines. Special Report. Midwest Research Institute, Kansas City, MO, Contract No. DAMD-17-74-C-4073. Huff, 8.L. et al., 1975. Aquatic field surveys at Radford, Holston, Volunteer, and Milan Army ammunition plants. Volume I-Radford. Final Report. Wapora, Inc., Washington, D.C., Contract No* DAMD-17-74-C-4138. Lee, C.C. et al. , 1975* Mammalian toxicity of munition compounds: Phase I. Acute oral toxlelt^* primary skin and eye Irritation, dermal sensitization, and dispositionand metabolism* Report No. 1* Midvest Research Institute, Kansas City, MO,. Contract No. DAMD-17-74-C-4073, AD B01U30L. Manclnl, 6* et al., 1964* Immunochemical quantitation of antigens by single radial Immunodiffusion* Immunochemistry, 2:233* Rosenblatt, D.B. et al., 1973* Munitions production products of potential concern as waterborne pollutants-Phase I. U.S. Army Medical Environmental Engineering Research Unit Report No. 73-07, Edgevood Arsenal, MD. X-i " 3 RTIC5 , 1986. Registry of Toxic Effaces of Chemical Substances, National for Occupational Safety and Health (NIOSH). National Library of Medicine File. n. me Selig, W. , 1961. Microdetermination of aromatic nitro compounds, nitrocellulose, and cyclic nitramines. AEC Report l'CRL-6639 : 20-28. Sullivan, J.H. et al., 1978. A summary and evaluation of aquatic environmental data in relation to establishing water quality criteria for munitions unique compounds. Part I. Nitrocellulose. Final Report. U.S. Army Medical Research and Development Command Contract Number DAMD-17-77-C-7027, Water and Air Research, Inc., Gainsville, FI. APPENDIX I CALCULATION METHODS Al-l Calculation of Intake of Test Material (ag/kg/day) Intake - (a)(b)(1000) (LOO)(c) where: a • X NC in diec b ■ intake of feed in grama/day* 1000 ■ conversion faceor. grama to milligrams 100 • conversion factor* Z to grams/100 grams * c * animal weight in kilograms* *Intake/anlaal weight data used may vary for each calculation depending upon information available in report. Text will describe exact conditions for determining these calculations for each experiment. APPENDIX 2 data deficiencies/problem areas and recommendations for ADDITIONAL DATA BASE DEVELOPMENT FOR NITROCELLULOSE t A2-1 INTRODUCTION . A2-3 OBJECTIVES .A2-3 BACKGROUND. A2-3 DISCUSSION .A2-4 CONCLUSIONS/RECOMMENDATIONS . A2-5 REFERENCES. A2-6 l A2-2 r \ N The Office of Drinking Water (ODW), Envtrorasental Protection Agency 'rp A conjunction with the Department of the Army, has reviewed the available d a -a -n nitrocellulose (NC) for the purpose of developing a Health Advisory (HA) use-’"’ •• dealing with contamination :f drinking water, to include "state-of-the-art" information on health effects, analytical methodology and treatment technology. This information is contained in detail in the report entitled "Health Advisory or Nitrocellulose." OBJECTIVES The objective of this document is to provide an evaluation of data deficiencies and/or problem areas encountered in the review process for NC and to make recommendations, as appropriate, for additional data base development, -his document is presented as an independent analysis of the current status of NC toxicology, as related to its possible presence in drinking water, and includes a summary of the background information used in the development of the HA. For greater detail on the toxicology of NC. the original "Health Advisory on Nitrocellulose" should be consulted. BACKGROUND Nitrocellulose is a non-volatile, fibrous, cotton-like, white solid used as a principal ingredient of propellants, smokeless powder, rocket fuel, mortar increments and some explosives (Sullivan ee al., 1978). It is produced for military use at selected Army ammunition plants (AAP), and NC fines are found in production wastewater (Huff et al., 1975). It is resistant to biological degradation and is, therefore, persistent in the environment and deemed a potential, if not known, contaminant in drinking water. The metabolism of NC as studied in rats (Ellis et al., 1976) indicated that the compound remained in the gastrointestinal (GI) tract and was excreted unchanged. > Acute toxicity studies in rats and miee (Lee et al., 1975) indicated chat NC was nontoxic* with values greater than 5,000 ag/kg reported in both species. Thirteen week (Ellis et al., 1976) and 24-aonth (Ellis et al., 1980) feeding studies im three specie# (dogs, rats and alee) gave no evidence of adverse or toxic effect# related to the chemical characteristics of NC; nor was NC found to be carcinogenic. The fibrous physical nature of NC, however, wee the cause of early deaths due to intestinal impaction in alee fed 101 NC or cotton linters in both the 13-week and 24-month studies and was associated with an increased food intake-decreased weight gain pattern in high dosed and cotton control rats and mice. The fibers were also reported to be the cause of a physical irritation of the extremities of alee fed 10Z NC or cotton linters as evidenced by hyperemia and A2-3 edema. A space of deaths of unknown cause occurred in mice after nine mcr.t.-.s feeding NC or cotton linters at IQZ in the diet. The number of deaths was higher in the NC treated mice and may, therefore, be related to the chemical nature of the compound via a mechanism, as yet, undetermined (Ellis et al., 1980 ). Nitrocellulose vas reported to be non-outagenic in various indicator systems (Ellis et al., 1976, 1978, 1980). Three-generation reproduction studies in rats indicated that NC did not adversely effect reproduction but that the non-nutritive bulk of the 10Z diet may cause an adverse effect during periods of high nutritional demand, such as pragnancy or lactation, as evidenced by a decrease in lactation lndax and weight at weaning (Ellis et al., 1980). No caracogenic studlas were raported. Exposure to NC did not affect the immunological response of dogs or rata (Ellis et al., 1976). Mathods of analysis (Barklay and Rosenblatt, 1978) and treatment (Rosenblatt et al., 1973) adequate for detection and removal of NC at levels which might be deemed hazardous to health have been presented in detail in "Health Advisory on Nitrocellulose." The lack of toxicological indicators, along with tha apparent non-ebaorptloa/non-dlgeatlon of tha NC fibers, led to the conclusion chat HA values for NC in drinking water were unnecessary. DISCUSSION Available data on the pharmacokinetics, health effaces, analysis and NC vastavacer treatment have been reviewed. While the available daea on the metabolism of NC are limited in scope, the chemical and physical nature of NC generally supports the finding of the one available study chat NC pasees through tha G1 tract apparently unchanged and unabsorbed. Additional studies would, therefore, be unnecessary. i The availabla studlas on the toxicity of NC lncluda LD. *• in rats and mice and short-ter* (11 week) and longer-term (24 month) studlas in dogs, rats and mica that included assessments for possible carcinogenicity. Three-generation reproductionstudies Is rats, mutagenicity assays in bactarlal and cytogenetic systems antfiamsnologlcal studies in dogs end rats have also bean reported. All studies appear adequaca for uaa in HA development. Further investigation of the causa of tha spats of deaths in nice occurring at nine months in the longer-term feeding study seem warranted but, in view of the otherwise lov toxicity of NC along with ths other availabla data, would not be necessary for HA development. Teratogenic studies vsrs not reported; however, the apparent inability of NC to be absorbed mould preclude its acting as a potential taratogen. Therefore, additional studlas teem unnecessary et this time. A2-4 Primary s'