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NTP REPORT ON THE TOXICITY STUDIES OF 1,2-DICHLOROETHANE (ETHYLENE DICHLORIDE) IN F344/N RATS, SPRAGUE DAWLEY RATS, OSBORNE-MENDEL RATS, AND B6C3Ff MICE (DRINKING WATER AND GAVAGE STUDIES) NATIONAL TOXICOLOGY PROGRAM P.O. Box 12233 Research Triangle Park, NC 27709 January 1991 NTP TOX 4 NIH Publication No. 91-3123 U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES Public Health Service National Institutes of Health SL 068429 FOREWORD The National Toxicology Program (NTP) is made up of four charter agencies of the U.S. Department of Health and Human Services (DHHS): the National Cancer Institute (NCI), National Institutes of Health; the National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health; the National Center for Toxicological Research (NCTR), Food and Drug Administration; and the National Institute for Occupational Safety and Health (NIOSH), Centers for Disease Control. The NTP coordinates the relevant programs, staff, and resources from these Public Health Service agencies relating to basic and applied research and to biological assay development and validation. The NTP develops, evaluates, and disseminates scientific information about potentially toxic and hazardous chemicals. This knowledge is used for protecting the health of the American people and for the primary prevention ofdisease. The studies described in this Technical Report were performed under the direction of the NIEHS and were conducted in compliance with NTP chemical health and safety requirements and must meet or exceed all applicable Federal, state, and local health and safety regulations. Animal care and use were in accordance with the Public Health Service Policy on Humane Care and Use of Animals. These studies are designed and conducted to characterize and evaluate the toxicologic potential of se lected chemicals in laboratory animals. Chemicals selected for NTP toxicology studies are chosen pri marily on the bases of human exposure, level of production, and chemical structure. Anyone who is aware of related ongoing or published studies not mentioned in this report, or of any errors in this report, is encouraged to make this information known to the NTP. Comments and ques tions should be directed to Dr. J.R. Bucher, NIEHS, P.O. Box 12333, Research Triangle Park, NC 27709(919-541-4532). These NTP Toxicity Study Reports are available for sale from the National Technical Information Service, U.S. Department of Commerce, 5285 Port Royal Road, Springfield, VA 22161 (703-487-4650). Single copies of this Toxicity Study Report are available without charge while supplies last from the NTP Public Information Office, NIEHS, P.O. Box 12233, Research Triangle Park, NC 27709 (919541-3991). r r C < C 1,2-Dichloroethane, NTP TOX 4 l ( SL 068430 k TOXICITY STUDIES OF 1,2-DICHLOROETHANE (ETHYLENE DICHLORIDE) (CAS NO. 107-06-2) IN F344/N RATS, SPRAGUE DAWLEY RATS, OSBORNE-MENDEL RATS, AND B6C3Fi MICE (DRINKING WATER AND GAVAGE STUDIES) D. Morgan, Ph.D., Study Scientist NATIONAL TOXICOLOGY PROGRAM P.O. Box 12233 Research Triangle Park, NC 27709 January 1991 NTP TOX 4 NIH Publication No. 91-3123 U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES Public Health Service National Institutes of Health These studies were supported in part by funds from the Comprehensive Envi ronmental Response, Compensation, and Liability Act trust fund by interagency agreement with the Agency for Toxic Substances and Disease Registry, U.S. Public Health Service. SL 068431 CONTENTS PAGE ABSTRACT .................................................................................................................................................................................... CONTRIBUTORS ......................................................................................................................................................................... PEER REVIEW PANEL ............................................................................................................................................................... SUMMARY OF PEER REVIEW COMMENTS ..................................................................................................................... 3 * 5 6 I. INTRODUCTION ............................................................................................................................................................ 7 D. MATERIALS AND METHODS ................................................................................................................................ 14 HI. RESULTS.......................................................................................................................................................................... 18 RATS........................................................................................................................................................................ MICE ........................................................................................................................................................................ 18 28 IV. DISCUSSION AND CONCLUSIONS ........................................................................................................................ 32 V. REFERENCES ............................................................................................................................................................... 34 APPENDIX: ORGAN WEIGHT. HEMATOLOGIC, AND SERUM CHEMICAL DATA IN THE THIRTEEN-WEEK STUDIES OF 1,2-DICHLOROETHANE ........................................................................................... 39 1,2-Dichloroethane, N'TP TOX 4 2 SL 068432 HH H -- C-- C -- H II Cl Cl 1,2-DICHLOROETHANE CAS No. 107-06-2 C2H4CI2 Molecular weight 98.97 Synonyms: Ethylene dichloride; 1,2-bichloroethane; a,p-dichloroethane; sym-dichloroethane; ethylene chloride; glycol dichloride Trade Names: Freon 150; Brocide; Dutch liquid; Dutch oil ABSTRACT Thirteen-week studies were conducted to investigate potential differences in rat strain susceptibility to 1,2-dichloroethane toxicity. F344/N rats, Sprague Dawley rats, Osborne-Mendel rats, and B6C3Fi mice (10 animals of each sex) were exposed to 1,2-dichloroethane in drinking water at 0, 500, 1,000, 2,000,4,000, or 8,000 ppm for 13 weeks. In addition, groups of 10 F344/N rats of each sex were admin istered 1,2-dichloroethane in corn oil by gavage to compare toxicity resulting from bolus administra tion with that of continuous exposure in drinking water. Gavage doses of 1,2-dichloroethane were within the range of daily doses resulting from exposure in drinking water. No compound-related deaths occurred in any of the rat strains exposed to 1,2-dichloroethane in drinking water. Weight gain depression was common in each sex of all three rat strains in the 4,000and 8,000-ppm groups throughout the studies. Water consumption was decreased by 50%-60% with increasing dose for all exposed male and female rats regardless of strain. Kidney and liver weights were increased in dosed rats of all three strains. No chemical-related lesions were observed except for a dose-related incidence of renal tubular regeneration in female F344/N rats. Nine of 10 female mice exposed to 8,000 ppm 1,2-dichloroethane in drinking water died before the end of the study. Mean body weights of males at 500 ppm or more and females at 1,000 ppm or more were lower than those of controls throughout most of the studies. Kidney weights were significantly in creased for dosed males and females. Renal tubular cell regeneration was seen in males at 8,000 ppm; at 4,000 ppm, minimal regeneration was present in 8/10 male mice. All male F344/N rats that received 240 or 480 mg/kg and 9/10 females that received 300 mg/kg 1,2dichloroethane by gavage died before the end of the studies. Mean body weights of the highest dose males and females were lower than those of vehicle controls throughout the studies. Liver and kidney weights were increased for dosed males and females; however, no compound-related lesions were ob served. Necrosis of the cerebellum, hyperplasia, inflammation, and mineralization of the forestom ach, and necrosis of the thymus were seen in animals that died or were killed in moribund condition. Rat strain differences in susceptibility to 1,2-dichloroethane toxicity were not apparent at the drink ing water concentrations used in these studies; only female F344/N rats exhibited mild chemicalrelated renal lesions. Male B6C3Fi mice appeared to be more susceptible than rats to toxicity of 1,2dichloroethane administered in drinking water; renal tubule regeneration was observed in male mice in the 4,000- and 8,000-ppm groups. The higher toxicity in mice was likely due to higher water con sumption, resulting in up to tenfold higher doses to mice than to rats. 1,2-Dichloroethane adminis tered in drinking water resulted in less toxicity to F344/N rats than administration of similar doses by gavage. 1,2-Dichloroethane, NTP TOX 4 3 SL 068433 CONTRIBUTORS The NTP Report on the Toxicity Studies of 1,2-Dichloroethane is based on the various 13-week stud ies of 1,2-dichloroethane that began in November 1985 and ended in November 1986 at EG&G Mason Research Institute (Worcester, MA). National Toxicology Program (Evaluated Experiment, Interpreted Results, and Reported Findings) D. Morgan, Ph.D., Study Scientist John R. Bucher, Ph.D. Michael Elwell, D.V.M., Ph.D. Joel Leininger, D.V.M., Ph.D. B.A. Schwetz, D.V.M., Ph.D. James K. Selkirk, Ph.D. NTP Pathology Working Group aluated Slides and Prepared Pathology Report on 7/21/88) John Seely, D.Y i. (Chair) (PATHCO, Inc.) Michael Elwell, D.V.M., Ph.D. (NTP) Jerry Hardisty, D.V.M. (Experimental Pathology Laboratories, Inc.) Margarita McDonald, D. V.M., Ph.D. (NTP) Satoru Motooka, D.V.M. (Eisai Pharmaceutical, Japan) Suzanne Neuenschwander, D.V.M. Experimental Pathology Laboratories, Inc. Brian Short, D.V.M. (Chemical Industry Institute ofToxicology) Principal Contributors at EG&G Mason Research Institute (Conducted Studies and Evaluated Tissues) Herman S. Lilja, Ph.D. A.S. Krishna Murthy, Ph.D. Principal Contributors at Experimental Pathology Laboratories, Inc. (Provided Pathology Quality Assurance) Jerry Hardisty, D.V.M. Suzanne Neuenschwander, D.V.M. Principal Contributors at Analytical Sciences, Inc. (Contractor for Statistical Analysis) Steven Seilkop, M.S. Janet Teague, M.S. Principal Contributors at Carltech Associates, Inc. (Contractor for Technical Report Preparation) William D. Theriault, Ph.D. Abigail C. Jacobs, Ph.D. John Warner, M.S. Naomi Levy, B.A. 4 1,2-Dichloroethane, NTP TOX 4 SL 068434 PEER REVIEW PANEL The members of the Peer Review Panel who evaluated the draft report on the Toxicity Studies on 1,2dichloroethane on June 27, 1989, are listed below. Panel members serve as independent scientists, not as representatives of any institution, company, or governmental agency. In this capacity, Panel members have four major responsibilities: (a) to ascertain that all relevant literature data have been adequately cited and interpreted, (b) to determine if the design and conditions of the NTP studies were appropriate, (c) to ensure that the Technical Report presents the experimental results and con clusions fully and clearly, and (d) to judge the significance of the experimental results by scientific criteria. National Toxicology Program Board of Scientific Counselors Technical Reports Review Subcommittee Robert A. Scala, Ph.D. (Chair) Senior Scientific Advisor, Medicine and Environmental Health Department Research and Environmental Health Division, Exxon Corporation East Millstone, NJ Michael A. Gallo, Ph.D. Associate Professor, Director of Toxicology Department of Environmental and Community Medicine, UMDNJ - Robert Wood Johnson Medical School, Piscataway, NJ Frederica Perera, Dr. P.H. Division of Environmental Sciences School of Public Health Columbia University New York, NY Ad Hoc Subcommittee Panel of Experts John Ashby, Ph.D. Imperial Chemical Industries, PLC Central Toxicology Laboratory Alderley Park, England William Lijinsky, Ph.D. Director, Chemical Carcinogenesis Frederick Cancer Research Facility Frederick, MD Robert H. Garman, D.V.M. Bushy Run Laboratories Export, PA Consultants in Veterinary Pathology Murrysville, PA Lois Swirsky Gold, Ph.D. University of California Lawrence Berkeley Laboratory Berkeley, CA Curtis D. Klaassen, Ph.D. (Principal Reviewer) Professor, Department of Pharmacology and Toxicology University of Kansas Medical Center Kansas City, KS Barbara McKnight, Ph.D. Assistant Professor, Department of Biostatistics, University of Washington Seattle, WA Franklin E. Mirer, Ph.D. Director, Health and Safety Department International Union, United Auto Workers, Detroit, MI Paul M. Newberne, D.V.M., Ph.D. Professor, Mallory Institute of Pathology Boston, MA James A. Popp, D.V.M., Ph.D. (Principal Reviewer) Head, Department of Experimental Pathology and Toxicology Chemical Industry Institute of Toxicology Research Triangle Park, NC 1,2-Dichloroethane, NTP TOX 4 5 SL 068435 SUMMARY OF PEER REVIEW COMMENTS ON THE TOXICITY STUDIES OF 1,2-DICHLOROETHANE On June 27, 1989, the draft Technical Report on the toxicity studies of 1,2-dichloroethane received public review by the National Toxicology Program Board of Scientific Counselors' Technical Reports Review Subcommittee and associated Panel of Experts. The review meeting was held at the National Institute of Environmental Health Sciences, Research Triangle Park, NC. Dr. D.L. Morgan, NIEHS, introduced the short-term toxicity studies of 1,2-dichloroethane by review ing the rationale, experimental design, and results. Dr. Klaassen, a principal reviewer, commented that stating the rationale for the studies earlier in the Report, especially in the Abstract, would be helpful. Dr. Morgan agreed. Dr. Popp, a second princir reviewer, said that the Resort was clearly written and ocaquately presents the background ar urrent studies. He inquired ; to the rationale for a separa - group of animals for evaluation of cunical pathology parameters. Dr. Morgan replied that this -vas done because of uncertainty about the effects of bleeding on animal response to the chemical. Dr. Mirer observed that if the comparative route studies were aimed at determining if there was saturation of metabolic mechanisms, the question was not answered. He said that this could be more directly addressed by an absorption and distribution study. Dr. Gold said that newer human exposure data should be available. Dr. J. Haartz, National Institute for Occupational Safety and Health, said that newer exposure data were available. Dr. L. Zeise, California Department of Health Services, suggested that it would be helpful to include discussion of how the delivered dose was calculated in the drinking water studies. Dr. Bucher said that this information would be included in the Report and in future reports of drinking water studies (see Table 15, page 31). Dr. Scala said that seeing no objections, the Panel would accept the Technical Report with the modifications as discussed. 6 1,2-Dichloroethane, NTP TOX 4 SL 068436 H--C-- C --H II Cl Cl 1,2-DICHLOROETHANE CAS No. 107-06-2 C2H4CI2 Molecular weight 98.97 Synonyms: Ethylene dichloride; 1,2-bichloroethane; Q,P-dichloroethane; sym-dichloroethane; ethylene chloride; glycol dichloride Trade Names: Freon 150; Brocide; Dutch liquid; Dutch oil I. INTRODUCTION Physical and Chemical Properties 1,2-Dichloroethane (ethylene dichloride) is a low molecular weight, chlorinated, aliphatic hydro carbon. It is a clear, colorless, oily liquid with a chloroform-like odor (Patterson et al., 1976). Other physical and chemical properties are shown in Table 1. catalyst (Drury and Hammons, 1979). The an nual production of 13 billion pounds (6 billion kg) in 1986 (USITC, 1987) makes 1,2-dichloroethane one of the largest volume synthetic chemicals produced in the United States. World capacity production of 1,2-dichloroethane was estimated to be 51 billion pounds (23 billion kg) in 1980 (Gold, 1980). Production and Use 1,2-Dichloroethane is produced commercially either by the vapor- or liquid-phase reaction of chlorine with ethylene in the presence of 1,2-dibromoethane or a metal chloride catalyst or by reaction of ethylene with oxygen and hydrogen chloride in the presence of a copper(II) chloride About 85% of the 1,2-dichloroethane produced in the United States is used in the synthesis of vi nyl chloride, and 2%-4% is used in the produc tion of other chemicals, such as 1,1,1-trichloroethane, trichloroethylene, tetrachloroethylene, vinylidine chloride, and ethyleneamines (IARC, 1979). 1,2-Dichloroethane is used as a lead scav enger in gasoline (IARC, 1979); in 1976, about TABLE 1. SOME CHEMICAL AND PHYSICAL PROPERTIES OF U-DICHLOROETHANE (a) Melting point Boiling point Water solubility Log n-octanol/water partition coefficient Relative density Vapor pressure Flash point Flammability limits Conversion factor -35*C 83* C 8.69 g/liter at 20` C 1.48 1.23 at 20* C 8.53 kPa (64 mm mercury) at 20* C 13* C (closed cup) 0.25-0.64 g/liter, 6%-16% by volume 1 ppminair a 4.05 rag/m3 (at 25 C and 760 mm mercury) (a)tPCS<1987) 7 1,2-Dichloroethane, NTP TOX 4 SL 068437 92 million kg of 1,2-dichloroethane was used in the United States for this purpose. About 0.1% of 1,2-dichloroethane produced in the United States in 1977 was used in fumigants for grain, upholstery, and carpets and as a solvent for metal degreasing (Gold, 1980). Exposure The greatest potential for human exposure to 1,2-dichloroethane occurs in the industrial set ting, where an estimated 80,000 workers could be at risk (NIOSH, 1989 unpublished data). The primary contact with 1,2-dichloroethane in the workplace results from its use as a solvent. 1,2Dichloroethane concentrations ranging from 40 to 800 mg/m3 (Cetnarowicz, 1959) have been de tected in industrial settings (IPCS, 1987). In a U.S. antiknock-agent blending plant, the maxi mum exposure concentration measured was 8.9 mg/m3 (Jacobs, 1980). Nonoccupational exposure to 1,2-dichloroethane can occur by inhalation of contaminated air. Singh et al. (1983) estimated the exposure to 1,2dichloroethane from urban air in the United States to be between 8 and 140 pg/day. Near production sites in the United States, an esti mated 12.5 million people were exposed to 1,2dichloroethane at an average annual concentra tion of up to 40 pg/m3 (Elfers, 1979; Kellam and Dusetzina, 1980). Nonoccupational exposure to 1,2-dichloroethane can also occur by consumption of contaminated water. The National Organics Reconnaissance Survey (Symons et al., 1975) measured 1,2-di chloroethane concentrations of 0-6 pg/liter in finished drinking water in 26 of 80 U.S. cities sampled. Ewing et al. (1977) detected levels of 1,2-dichloroethane greater than 1 pg/liter in sur face water from 53 of 204 heavily industrialized U.S. sites. Letkiewicz et al. (1982) estimated that 1,2-dichloroethane levels in all groundwater and surface water systems in the United States are below 10 pg/liter and that most are below 1.0 pg/liter. Daily intake of 1,2-dichloro ethane from drinking water containing 10 pg/ liter was estimated to be 0.29 pg/kg for a 70-kg adult. Symons et al. (1975) observed 1,2-dichloro ethane more frequently in finished water than in untreated water, suggesting that contamina tion may occur during water chlorination (IPCS, 1987). Production of 1,2-dichloroethane by wa ter chlorination has been suggested by others (Versar, 1975; Seufert et al., 1980); however, in dustrial discharges to surface water and leach ing of solid wastes are considered the primary causes of 1,2-dichloroethane contamination in drinking water (Letkiewicz et al., 1982). Absorption and Distribution 1.2- Dichloroethane is rapidly absorbed into the blood of rodents after dermal (Tsuruta, 1975; Jakobson et al., 1982), oral (Sopikov and Gor shunova, 1979; Reitz et al., 1982), or inhalation (Spreafico et al., 1980; Reitz et al., 1982) expo sure. Spreafico et al, (1980) observed that 1,2-di chloroethane administered to rats by gavage at doses of 25, 50, or 150 mg/kg was rapidly ab sorbed, with peak levels in the blood occurring within 20 minutes. Similarly, Reitz et al. (1980, 1982) found that [1*C]1,2-dichloroethane admin istered to rats by gavage (150 mg/kg) was com pletely absorbed. After administration by gavage, 1,2-dichloro ethane was found to accumulate most rapidly in the liver, with peak levels attained within 10 minutes of administration (Spreafico et al., 1980). Levels of 1,2-dichloroethane in the lung appeared to be in equilibration with levels in blood. Accumulation in epididymal adipose tis sue was slower, with peak levels occurring 45-60 minutes after administration; however, these levels were significantly higher than those in blood. In the same study, Spreafico et al. (1980) com pared 1,2-dichloroethane distribution in rats ex posed by inhalation (250 ppm for 6 hours) or gavage (50 mg/kg). These doses resulted in com parable peak concentrations of 1,2-dichloroethane in blood. After inhalation exposure, peak 1.2- dichloroethane concentrations were higher than after oral exposure in the lung and adipose tissues and lower in the liver. 1,2-Dichloroethane concentrations in the spleen, kidney, and brain were similar to concentrations in blood af ter administration by either route. During inha lation exposure of rats, equilibrium between blood and tissues (adipose, liver, and lung) was 1,2-Dichloroethane, NTP TOX 4 8 SL 068438 established alter 2 hours of exposure to 50 ppm 1.2- dichloroethane and alter 3 hours at 250 ppm. In similar studies, Reitz et al. (1980, 1982) in vestigated the distribution of radioactivity in tissues alter oral (150 mg/kg by gavage) and inhalation (150 ppm for 6 hours) exposure to [14C]l,2-dichloroethane. During inhalation ex posure, equilibration of 1,2-dichloroethane be tween blood and tissues required 2-3 hours. Tar get tissues (forestomach, liver, spleen) that developed neoplasms in rats exposed to 1,2-dichloroethane by gavage (NCI, 1978), as well as nontarget tissues (kidney, lung, stomach, and remaining carcass homogenate), were surveyed. No striking differences were seen in the distri bution of radioactivity in target and nontarget tissues when evaluated 48 hours after oral or in halation exposure. Levels of radioactivity were consistently about two times higher in tissues from animals exposed by gavage than in tissues from animals exposed by inhalation. 1.2- Dichloroethane crosses the placental barrier and has been detected in the fetus. Alter inha lation exposure of pregnant rats at 1,000 mg/m3 for 4 hours per day, 1,2-dichloroethane was found to accumulate in the placental and fetal tissues over a period of 7 days (Vosovaya, 1977). Withey and Karpinski (1985) also demonstrated that inhalation exposure of pregnant rats re sulted in dose-dependent accumulation of 1,2-dichloroethane in the fetus. Urusova (1953) re ported that 1,2-dichloroethane accumulated in human breast milk (5.4-6.4 mg/liter) during oc cupational exposure. Metabolism 1,2-Dichloroethane has been shown to be me tabolized extensively via two principal pathways involving microsomal cytochrome P450 and cy tosolic glutathione-S-transferase (GST) with reduced glutathione (GSH) (Figure 1). The cyto chrome P450-catalyzed metabolism of 1,2-di chloroethane results in an unstable ge/n-chlorohydrin intermediate that rapidly eliminates hydrochloric acid to form 2-chloroacetaldehyde, followed by oxidation to chloroacetic acid or re duction to 2-chloroethanol (Guengerich et al., 1980; IPCS, 1987). These intermediates may undergo further reaction with GSH and appear as nontoxic urinary metabolites. The GST-dependent metabolic pathways of 1,2dichloroethane do not occur to any extent with the other chlorinated ethanes (Anders and Jakobson, 1985). This pathway involves the direct reaction of 1,2-dichloroethane with GSH to form S-(2-chloroethyl)glutathione, which is nonenzymatically converted to a glutathione episulfonium ion that can undergo several fates (IPCS, 1987). Reaction with water results in the formation of S-(hydroxyethyl)glutathione, and reaction with GSH produces ethene bisglutathione. These reaction products undergo further metabolism to nontoxic urinary metabolites. However, the episulfonium ion is a putative al kylating agent that can also form adducts with protein, RNA, and DNA (Inskeep et al., 1986). This pathway is considered to be the major in vivo route for DNA damage by 1,2-dichloro ethane (Guengerich et al., 1980; Rannug, 1980; Sundheimer et al., 1982; Inskeep et al., 1986; IPCS, 1987). Excretion 1,2-Dichloroethane is excreted rapidly by rats and mice, regardless of the route of exposure. Approximately 89% or more of 1,2-dichloro ethane administered to mice by intraperitoneal injection was excreted within 24 hours (YUner, 1971) or within 48 hours by mice receiving the chemical orally (Mitoma et al., 1985) and by rats exposed by gavage or inhalation (Reitz et al., 1982; Mitoma et al., 1985). Excretion of 1,2dichloroethane or its metabolites occurs primari ly in exhaled air and in urine in rats and mice exposed by various routes (Davidson et al., 1982; IPCS, 1987). Yllner (1971) found that up to 42% of the 1,2-dichloroethane given to mice by intra peritoneal injection was recovered unchanged in the exhaled air. The percentage of unmetabo lized 1,2-dichloroethane exhaled was greater at higher doses than at lower doses, indicating a limited capacity for metabolism. Similarly, in rats, 29% of an oral dose of 1,2-dichloroethane (150 mg/kg) and 1.8% of a lower dose adminis tered by inhalation (150 ppm for 6 hours) were recovered unchanged in the breath (Reitz et al., 1982). Toxicity in Humans Data on the effects of 1,2-dichloroethane in humans are limited to reports of accidental 9 1,2-Dichloroethane, NTP TOX 4 SL 068439 CHj = CHj ethene *. GSSG.HCI GS \? S-<2-Formylmethyl)glutathione Glutathione episulfonium ion ^/T -+ GS^VOH 5-(2-Hydroxyethyl)glutathione GSM Protein DNA ANA Cellular macromolecular adducts GS.V HO,/jV'YOH O H NHj O S-CarbOxymethyl-L-cysteinylglycine S,5'-Ethene bisglutathione 1 i S,5'-Ethene bis-i-cysteine DipeendHC HjO O HOVsYH NH2 O Acetyl Coa N-Acetyltranfferate S-Carboxymethyl-c-cysteme Yy HNCCHj o II 0 N-Acetyl-S-carboxymethyl-L-cysteme Thiodiaceticacid (thiodiglycolic acid) FIGURE I. PROPOSED PATHWAYS FOR 1,2-DICHLOROETHANE METABOLISM (from IPCS, 1987) 1,2-Dichloroethane, NTP TOX 4 10 SL 68440 exposures, and many of these are concerned with mixed chemical exposures. Short-term inhala tion exposure to 1,2-dichloroethane at high con centrations initially affects the central nervous system. Signs and symptoms include headache, dizziness, weakness, muscle spasms, cyanosis, hypotonia, vomiting, epigastric pain, and diar rhea. Unconsciousness and death may follow. Irritation and inflammation of the respiratory tract result in symptoms of cough and rales. Bronchial inflammation and respiratory insuffi ciency due to central nervous system depression may result in cyanosis (Kozik, 1957; Cetnarowicz, 1959; USEPA, 1985; IPCS, 1987). Changes in heart rhythm, probably secondary to cardiac sensitization to catecholamines, were reported (Suveev and Babichenko, 1969). Short-term oral exposure of humans to 1,2-dichloroethane produces effects similar to, but more pronounced than, those after short-term inhalation exposure. In addition, ocular effects such as dilation or constriction of the pupils, im pairment of eye reflexes (Weiss, 1957; Troisi and Cavallazzi, 1961), conjunctivitis (Menschick, 1957), and corneal opacity (Weiss, 1957) have been reported after oral exposure to 1,2-dichloro ethane. Toxicity in Animals The effects of short-term (4-9 months) inhalation exposure to 1,2-dichloroethane were investi gated in several studies in a number of labora tory animal species (Heppel et al., 1946; Spencer et al., 1951; Hofmann et al., 1971). Of the spe cies studied, rats and mice appear to be the most sensitive to the toxic effects of 1,2-dichloro ethane. The no-observed-adverse-effect level for short-term exposure (4-9 months) of rats in three investigations is about 100 ppm (IPCS, 1987). The oral LD$o for 1,2-dichloroethane was 413 (female) or 489 (male) mg/kg body weight in CD-1 mice (Munson et al., 1982), 680-850 mg/kg in rats (McCollister et al., 1956; Larionov and Kokarovtseva, 1976), and 2,500 mg/kg in dogs (Barsoum and Saad, 1934). Spreafico et al. (1980) investigated the effects of long-term 1,2-dichloroethane inhalation expo sure on clinical chemistry indices of Sprague Dawley rats. Three-month-old rats of each sex were exposed to 0, 5, 10, 50, or 250 ppm for 7 hours per day, 5 days per week for 3, 6, or 18 months. The highest exposure concentration was reduced to 150 ppm after several weeks be cause of high mortality. An additional group of 14-month-old rats was exposed for 12 months at the same 1,2-dichloroethane concentrations. In the older rats, changes were detected in serum aspartate aminotransferase, serum alanine aminotransferase, and y-glutamyl transpepti dase activity and in serum uric acid, blood urea nitrogen, and serum cholesterol concentrations after exposure for 12 months. These effects were not observed after the 3-month-old animals were exposed for 3,6, or 18 months. Administration of 1,2-dichloroethane to rats by gavage, five times per week for 2 weeks at doses of 150 mg/kg or less, had no effect on organ or body weights, histology, clinical chemistry, or hematology (Van Esch et al., 1977; Reitz et al., 1982). When rats were administered 30 or 90 mg/kg 1,2-dichloroethane by gavage, 5 days per week for 13 weeks, decreased weight gain was observed (Van Esch et al., 1977). Relative kid ney weights of rats of each sex and relative brain and liver weights of females receiving 90 mg/kg 1,2-dichloroethane by gavage were increased. Histology and clinical chemistry were normal. Six of six rats died after receiving 300 mg/kg 1,2dichloroethane by gavage for 5 days; fatty de generation of liver and an increase in liver tri glycerides were observed (Van Esch et al., 1977). Alumot et al. (1976) observed increased total liv er fat and triglycerides in rats after ingestion of approximately 100 mg/kg 1,2-dichloroethane per day in feed for 7 weeks. In a long-term study, rats were administered feed that had been fumi gated with 1,2-dichloroethane, resulting in doses of 0, 11-17, or 23-25 mg/kg per day. After expo sure for 2 years, no adverse effects were observed on growth, survival, or serum composition. I mmunotoxicity Immunosuppression was observed in rabbits ex posed to 1,2-dichloroethane at 100 mg/m3 for 3 hours per day, 6 days per week for 7.5-8 months (Shmuter, 1977). Production of antibodies against typhoid vaccine was reduced by 80% in exposed animals, and a concomitant twofold 11 1,2-Dichloroethane, NTP TOX 4 SL 068441 increase in Forsman sheep erythrocyte anti bodies was observed. Munson et al. (1982) reported a 30% reduction in leukocyte counts in CD-1 mice administered 49 mg/kg 1,2-dichloroethane by gavage for 14 days. The number of antibody-forming cells in the spleen was decreased by 25% and 40% in mice receiving 4.9 and 49 mg/kg by gavage, respec tively. No effects were observed on cell-medi ated immunity in a second group of mice receiv ing 3, 24, or 189 mg/kg 1,2-dichloroethane in drinking water for 13 weeks. Teratology and Reproductive Toxicology Administration of 1,2-dichloroethane either by inhalation (Rao et al., 1980), in drinking water (Lane et al., 1982), or in formulated diets (Alumot et al., 1976) did not affect fertility, nor did it induce embryotoxic, fetotoxic, or teratogenic ef fects in several species. Vosovaya (1977) ob served a possible adverse effect of 1,2-dichloro ethane on reproduction after female rats were exposed to 1,2-dichloroethane by inhalation at 15 mg/m3 for 4 hours per day, 6 days per week for 4 months before mating. During this period, the length of the estrous cycle increased. The rats were then mated and the exposure con tinued. Total embryonal mortality was in creased, and preimplantation losses were about five times greater in exposed rats than in con trols. In another study (Vosovaya, 1974), female rats were exposed to 57 10 mg/m3 for 4 hours per day, 6 days per week for 6 or 9 months. The fertility mated females and the weight of new born rats were reduced, and perinatal mortality was increased. Genetic Toxicology 1,2-Dichloroethane has been shown to be muta genic in a variety of in vitro tests. It induced DNA damage in Escherichia coli (Brem et al., 1974; Rosenkranz, 1977) and gene mutations in Salmonella (McCann et al., 1975; Bignami et al., 1977; Rosenkranz, 1977; Simmon et al., 1977; NTP unpublished data). 1,2-Dichloroethane has also been shown to induce sex-linked recessive lethal mutations in Drosophila (Shakarnis, 1969; King et al., 1979; Kramers and Bissumbhar, 1983) and gene mutations in mammalian lymphoblastoid cells (Crespi et al., 1985), Additional effects observed in mammali an cells in vitro include induction of sister chro matid exchanges and chromosomal aberrations in Chinese hamster ovary cells (NTP unpub lished data). Although mutagenic in vitro, 1,2-dichloroethane has demonstrated no genotoxic activity in mam malian cells in vivo, as shown by results from a limited number of studies. Analysis of periph eral blood smears obtained from the 13-week study animals showed no increase in micronucleated erythrocytes (NTP unpublished data), and bone marrow micronucleus studies in mice that received one or two intraperitoneal injec tions c: 1,2-dichloroethane were also negative (King . al., 1979; Jenssen and Ramel, 1980). Carcinogenicity The potential carcinogenicity of 1,2-dichloro ethane was investigated in a number of studies in which 1,2-dichloroethane was administered to rats and mice by various routes. The results of studies evaluating the carcinogenicity of 1,2dichloroethane are conflicting. The National Cancer Institute carcinogenesis studies of 1,2-dichloroethane conducted in Osborne-Mendel rats and B6C3Fj mice via gavage in corn oil indicated that 1,2-dichloroethane caused squamous cell carcinomas of the forestomach, hemangiosarcomas, and subcutaneous tissue fibromas in male rats and mammary gland adenocarcinomas in female rats. Alveolar/bronchiolar adenomas were observed in ex posed male and female B6C3Fi mice, and mam mary adenocarcinomas and endometrial tumors were observed in female mice (NCI, 1978). How ever, results of inhalation studies in Sprague Dawley rats and Swiss mice were negative (Maltoni et al., 1980). Attempts to reconcile the results of these two conflicting reports have cen tered around the purity of the study chemical, strain and route differences, contamination of the animal room with known carcinogens, and other technical considerations (Maltoni et al., 1980). Although most confounding factors can be excluded, species and route differences re main the most likely reasons for the contradic tory findings. 1,2-Dichloroethane, NTP TOX 4 12 SL 068442 Pharmacokinetic data showing more rapidly at tained and sustained levels of 1,2-dichloroethane in blood of Osborne-Mendel rats after oral exposure, as opposed to inhalation of 1,2-dichloroethane at comparable doses, correlated with greater DNA alkylation after oral exposure (Reitz et al., 1982). A comparable route-specific genotoxic effect was reported by Storer et al. (1984), who showed significant hepatic DNA damage in mice after short-term oral or intraperitoneal administration but not with compara ble inhalation exposure to 1,2-dichloroethane. Van Duuren et al. (1979) gave female Swiss mice dermal applications of 42 or 126 mg 1,2-dichloroethane in acetone, three times per week for 440594 days; an increased incidence of lung papil lomas was detected in mice given 126 mg. Another group of female mice received one appli cation of 1,2-dichloroethane, followed 2 weeks later by application of phorbol myristate acetate in acetone three times per week for 428-576 days. Although 1,2-dichloroethane was found to induce a significant increase in the incidences of benign lung papillomas, it did not initiate skin neoplasms. Klaunig et al. (1986) investigated the effect of 1,2-dichloroethane on the incidences of liver and lung neoplasms in male B6C3Fi mice according to a two-stage initiation/promotion protocol. Mice received 10 mg/liter diethylnitrosamine in drinking water for 4 weeks and then 835 or 2,500 mg/liter 1,2-dichloroethane in drinking water for 52 weeks. Neither the incidences of lung or liver neoplasms nor the number of neo plasms per mouse were affected in mice receiv ing 1,2-dichloroethane alone or after initiation with diethylnitrosamine. Theiss et al. (1977) conducted a pulmonary tu mor bioassay with 1,2-dichloroethane adminis tered to A/St mice by intraperitoneal injection. Doses were 20, 40, or 100 mg/kg, three times per week for 24 weeks. The number of lung ade nomas per mouse increased with dose; however, the number of adenomas was not significantly greater than that in controls. Study Rationale 1.2- Dichloroethane was included in the first group of 24 priority chemicals for toxicologic evaluation by the National Toxicology Program (NTP) as part of an interagency agreement be tween the NTP and the Agency for Toxic Sub stances and Disease Registry. Drinking water may be an important source of human exposure to 1,2-dichloroethane because of contamination from industrial discharge and because of leach ing from dump sites into surface water and groundwater. An adequate study of 1,2-dichlo roethane toxicity and carcinogenicity using oral, nonbolus (i.e., formulated drinking water mix tures or feed) administration has not been conducted. Conflicting results in earlier studies of 1,2-di chloroethane may have been due to differences in routes of administration and/or rat strains (Hooper et al., 1980). Potential differences in toxicity resulting from bolus or continuous ad ministration were investigated by administer ing 1,2-dichloroethane to F344/N rats by gavage or in drinking water; potential differences in rat strain susceptibility to 1,2-dichloroethane toxic ity were investigated in F344/N, OsborneMendel, and Sprague Dawley rats administered 1.2- dichloroethane in drinking water. 13 1,2-Dichloroethane, NTP TOX 4 SL 068443 II. MATERIALS AND ME" JDS Procurement and Characterization of 1,2-Dichloroethane 1,2-Dichloroethane was obtained in one lot from B.F. Goodrich Chemicals Group (Cleveland, OH). Purity and identity analyses were con ducted at Midwest Research Institute (MRI) (Kansas City, MO). MRI reports on the analyses performed in support of the 1,2-dichloroethane studies are on file at the National Institute of Environmental Health Sciences. The study material was identified as 1,2-dichlo roethane by infrared, ultraviolet/visible, and nu clear magnetic resonance spectroscopy; the puri ty was determined to be greater than 99% by elemental analysis, Karl Fischer water analysis, potentiometric titration in methanol with 0.01 N aqueous sodium hydroxide to determine free acid content, and gas chromatography. The stability of the chemical during the toxicolo gy studies was monitored by gas chromatogra phy. No deterioration of the 1,2-dichloroethane was seen over the course of the studies. Preparation and Characterization of Dose Formulations in Corn Oil and in Drinking Water The appropriate amounts of 1,2-dichloroethane and corn oil were mixed (w/v) to give the desired concentra:. .is for the gavage studies. Stability studies of i,2-dichloroethane in corn oil (approx imately 10 mg/ml), using gas chromatography, established that the solutions were stable for at least 3 weeks when stored in the dark at room temperature. Solutions maintained under simu lated animal-room conditions (open to air and light for 3 hours) had a chemical loss of approxi mately 4%. During the studies, dose formula tions were stored for no longer than 3 weeks at approximately 4 C in serum vials. Three complete sets of corn oil formulations were analyzed over the course of the 13-week studies, and all were within specifications (10% of the target concentration) (Table 2). The analysis of the formulations remaining after dosing was completed gave results that were in reasonable agreement with those from samples taken immediately after mixing, indicating no loss of chemical during dose administration. Two referee analyses confirmed the results ob tained by the study laboratory. For the drinking water formulations, the ap propriate amounts of 1,2-dichloroethane and deionized water were mixed (v/v) to give the de sired concentrations. Stability studies of 1,2dichloroethane in water (approximately 5 mg/ml), using gas chromatographic analysis of methylene chloride extracts of the water TABLE 2. RESULTS OF ANALYSIS OF CORN OIL FORMULATIONS IN THE THIRTEENWEEK GAVAGE STUDIES OF 1,2-DICHLOROETHANE Target Concentration (mg/g) Determined Concentration (a) (mg/g) 3.9 6.5 8.1 13.3 16.1 26.5 32.0 52.3 63.5 103.4 3.8 0.05 6.5 0.23 7.8 0.19 12.9 0.31 15.6 0.55 25.2 0.43 31.1 1.14 (b) 51.3 0.49 * 62.3 0.59 (c) 103.2 (a) Mean standard deviation for three determinations unless otherwise specified; for each determination, all samples analyzed in duplicate. (b) Results for two determinations (c) Results for a single determination 1,2-Dichloroethane, NTP TOX 4 14 SL 068444 solutions, established that the solutions were stable for at least 3 weeks in the dark at 5 C in sealed bottles. 1,2-Dichloroethane solutions maintained under simulated animal-room condi tions (clear glass drinking water bottles under normal room light) had losses of 1,2-dichloroethane of 13%, 22%, and 27% after 1, 2, and 3 days, respectively. Because of concerns about the sta bility of dose formulations during the toxicology studies, drinking water formulations were stored in sealed bottles for no longer than 3 weeks and drinking water bottles were changed at the end of each day. Three complete sets of drinking water formula tions were analyzed over the course of the 13week studies. Four of the 16 formulations were out of specifications (varied by more than 10% from the target concentration), with values ranging from -12% to - 33% of target (Table 3). Samples that were out of specifications were restirred and reanalyzed and were then found to be within specifications. Two referee analyses con firmed the results obtained by the study labora tory. The analysis of formulations remaining in the drinking water bottles after 24 hours in the animal cages showed that the concentrations of the formulations had decreased an average of 29% (with values ranging from --13% to --53%) of target concentrations. Fresh drinking water mixtures were placed in the cages at the end of each day; thus, animals were exposed at concen trations ranging between the initial concentra tion and the concentration found at the end of 24 hours. Thirteen-Week Study Design Groups of 20 male rats and 10 female rats of each strain and 10 mice of each sex were exposed to drinking water containing 0, 500, 1,000, 2,000,4,000, or 8,000 ppm 1,2-dichloroethane for 13 weeks. Groups of 10 or 20 male F344/N rats were administered 0, 30, 60, 120, 240, or 480 mg/kg 1,2-dichloroethane in corn oil by gavage 5 days per week. Groups of 10 female F344/N rats were administered 0, 18, 37, 75, 150, or 300 mg/kg in corn oil by gavage on the same schedule. The male and female F344/N rats, Sprague Dawley rats, Osborne-Mendel rats, and B6C3Fj (C57BL/6N, female X C3H/HeN MTV", male) mice used in these studies were produced under barrier conditions at Taconic Farms (Sprague Dawley rats), Frederick Cancer Research Facili ty (B6C3Fi mice and F344/N rats), or CAMM Research Institute (Osborne-Mendel rats). Ani mals were progeny of defined microfiora-associated parents that were transferred from isola tors to barrier-maintained rooms. Animals were shipped to the study laboratory at 4 weeks of age. The rats were quarantined at the study lab oratory for 11-14 days and mice for 12-14 days. All animals were placed on study at approxi mately 6 weeks of age. Hematologic and serum chemical analyses were performed on days 3, 7, 14, and 45 and at the terminal kill on groups of 10 male rats of each strain that received 0,2,000,4,000, or 8,000 ppm TABLE 3. RESULTS OF ANALYSIS OF DRINKING WATER FORMULATIONS IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF U DICHLOROETHANE Target Concentration (ppm) Determined Concentration (a) (ppm) 500 1.000 2,000 4,000 8,000 (b)462 897 1,767 3,640 7,190 10 153 338 546 148 (a) Mean standard deviation for the determination ofthree formulations unless otherwise specified; for each determination, all analyses performed in triplicate. (b) Four formulations were analyzed. 15 1,2-Dichloroethane, NTP TOX 4 SL 068445 1,2-dichloroethane in drinking water and on groups of 10 male F344/N rats that were admin istered 0, 120, 240, or 480 mg/kg 1,2-dichloroethane in corn oil by gavage. A separate group of animals was used for evaluation of hemato logic and serum chemical parameters at 3, 7,14, and 45 days because the effects of bleeding on the animals' response to 1,2-dichloroethane ex posure is not known. The core group animals were bled at the terminal kill for clinical pathol ogy evaluation at 90 days. Blood ( 1.2 ml) was drawn from the tail of each animal and analyzed for erythrocyte and leukocyte counts, hemo globin, hematocrit, mean cell volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration; a qualitative evalu ation of number and morphology of platelets, leukocytes, number of reticulocytes, and eryth rocyte morphology was performed. Serum sam ples were analyzed for sorbitol dehydrogenase, creatine kinase, alanine aminotransferase, alka line phosphatase, and blood urea nitrogen. Rats used for clinical pathology evaluations were killed without necropsy, and their tissues were not saved. Animals found moribund and those surviving to the end of the studies were humanely killed. A necropsy was performed on all animals not used in hematologic and serum chemical studies. In some instances, a particular organ was autolyzed or lost; thus, the number of animals from whvi particular organs or tissues were exam ine croscopically varies and is not necessari ly ; to the number of animals that were plac n study. Tissues examined are listed in Tab Organs and tissues were examined for gross lesions. Tissues were preserved in 10% neutral buffered formalin and routinely processed for preparation of histologic sections for microscopic examination. Tissues and groups examined are listed in Table 4. The liver, right kidney, brain, heart, thymus, lung, and right testis were weighed. Upon completion of the histologic evaluation by the laboratory pathologist, slides, paraffin blocks, and residual wet tissues were sent to the National Toxicology Program Archives for in ventory, slide/block match, and wet tissue audit. The slides, individual animal data records, and pathology tables were sent to an independent pathology laboratory where quality assessment was performed, and the results were reviewed and evaluated by the NTP Pathology Working Group (PWG). The target organs reviewed by the PWG were the forestomach, brain, kidney, and thymus for F344/N rats dosed by gavage and the kidney for all rat strains and B6C3Fj mice receiving formulated drinking water. The final diagnoses represent a consensus of contractor pathologists and the PWG. Details of these re view procedures have been described by Maronpot and Boorman (1982) and Boorman et al. (1985). Statistical Methods The analysis of organ weight, hematologic, and serum chemistry data was carried out by using the nonparametric multiple comparison proce dures of Dunn (1964) or Shirley (1977). Jonckheere's test (Jonckheere, 1954) was used to eval uate the significance of dose-response trends and to determine whether Dunn's or Shirley's test was more appropriate for pairwise comparisons. The incidences of nonneoplastic lesions were as sessed by the Fisher exact test. Dose Selection The solubility of 1,2-dichloroethane in water was the limiting factor in setting the high con centration for drinking water studies. The max imum solubility of 1,2-dichloroethane in water is about 9,000 ppm. Gavage doses were selected to be within the range of doses (in milligrams per kilogram per day) ingested by rats exposed to formulated drinking water. Quality Assurance The studies of 1,2-dichloroethane were per formed in compliance with Good Laboratory Practices and regulations (21 CFR 58). The Quality Assurance Unit of EG&G Mason Re search Institute performed audits and inspec tions of protocols, procedures, data, and reports throughout the conduct of the studies. The oper ations of the Quality Assurance Unit were mon itored by the NTP, including a site visit during the period of study performance. 1,2-Dichloroethane, NTP TOX 4 16 SD 068446 TABLE 4. EXPERIMENTAL DESIGN AND MATERIALS AND METHODS IN THE THIRTEEN-WEEK STUDIES OF U-DICHLOROETHANE Drinking Water Studies Gavage Studies Strain and Species F344/N rats, Osborne-Mendel rats, Sprague Dawley rats, and B6C3Fi mice F344/N rats Study Laboratory EG&G Mason Research Institute EG&G Mason Research Institute Size of Study Groups 10 or 20 males and 10 females of each strain and species 10 or 20 males and 10 females Doses 0,500,1,000,2,000,4,000, or 8,000 ppm 1,2-dichloroethane in drinking water Male-0,30,60,120,240, or 480 mg/kg 1,2-dichloroethane in corn oil by gavage; female-0,18,37,75,150, or 300 mg/kg; dose vol-5 ml/kg Method of Animal Distribution Animals distributed to weight classes and then assigned to cages by one table of random numbers and to groups by another table of random numbers Same as drinking water studies Diet NIH 07 Rat and Mouse Ration (Zeigler Bros., Inc., Gardners, PA); available ad libitum Same as drinking water studies Animal Room Environment F344/N rats-temp: 68*-72* F; hum: 38%-56%; Sprague Dawley rate-temp: 66*-73* F; hum: 37%-53%; Osborne-Mendel rats-temp: 8*-73* F; hum: 35%-S3%; B6C3Fl mice-temp: 68*-77* F; hum: 38%-56%; fluores cent light 12 h/d for all animals Temp~70*-74* F; hum-24%-64%; fluorescent light 12 h/d Age When Placed on Study 6 wk 6 wk Duration of Dosing 13 wk, dosed until necropsy 5 d/wfc for 13 wk, dosed at least 2 consecutive days before necropsy Type and Frequency of Observation Observed 2 x d; weighed initially and 1 X wk thereafter Observed 2 x d; weighed initially and 1 X wk thereafter Necropsy, Histologic Examinations, and Supplemental Studies Necropsy performed on all mice and on all rats not used in Necropsy performed on all rats not used in the serial hematologic the serial hematologic and serum chemical studies; the and serum chemical studies; the following tissues examined following tissues examined histologically for all control histologically for all vehicle control and high dose animals, and high dose animals and for female mice receiving 4,000 males receiving 120 or 240 mg/kg, and females receiving 150 ppm: adrenal glands, brain, esophagus, eyes (if grossly mg/kg: adrenal glanda, brain, esophagus, eyes (if grossly ab abnormal), gallbladder (mice), gross lesions and tissue normal), gross lesions and tissue masses and regional lymph masses and regional lymph nodes, heart, kidneys, large nodes, heart, kidneys, large intestine, liver, lungs and mainstem intestine, liver, lungs and mainstem bronchi, mammary bronchi, mammary gland, mandibular and mesenteric lymph gland, mandibular and mesenteric lymph nodes, nasal nodes, nasal cavity and turbinates, ovaries, pancreas, parathy cavity and turbinates, ovaries, pancreas, parathyroids, roids, pharynx (if grossly abnormal), pituitary gland, preputial pharynx (if grossly abnormal), pituitary gland, preputial or clitoral glands (rats), prostate, salivary glands, skin, small or clitoral glands (rats), prostate, salivary glands, skin, intestine, spinal cord and sciatic nerve (if neurologic signs small intestine, spinal cord and sciatic nerve (if neuro present), spleen, sternebrae or femur or vertebrae including logic signs present), spleen, sternebrae or femur or verte marrow, stomach, testes/epididymis/seminal vesicles, thymus, brae including marrow, stomach, testes/epididymis/ thyroid gland, trachea, urinary bladder, and uterus. Hematolog seminal vesicles, thymus, thyroid gland, trachea, urinary ic and serum chemical analyses performed on groups of 10 male bladder, and uterus. Hematologic and serum chemical rats at d 3,7,14, and 45 and at terminal kill. Organ weights analyses performed on groups of 10 male rats ofeach obtained at necropsy strain at d 3,7,14, and 45 and at terminal kill. Organ weights obtained at necropsy 17 1,2-Dichloroethane, NTPTOX4 SL 068447 III. RESULTS THIRTEEN-WEEK STUDIES IN RATS Drinking Water Studies F344/N Rats: No deaths of F344/N rats occurred during the studies (Table 5). Mean body weights of males exposed to 4,000 ppm or more and of fe male' exposed to 8,000 ppm were lower than tho? controls throughout the studies (Fig ure /Vater consumption at the higher concentrati was about 60% that by controls. The in crease i erythrocyte counts, mild decreases in mean 11 volume, and the mild increases in blood urea nitrogen in the high dose male rats are all indicative of animal dehydration (Table A3). The decrease in mean cell volume (hematocrit/erythrocytes) may be related to dehydra tion resulting in an increase in serum osmolarity, with a subsequent loss of water from and shrinkage of the erythrocytes. The absolute and relative kidney weights and relative liver weights were increased for dosed males and fe males (Tables 6 and 7). No compound-related clinical signs were observed. Renal tubular re generation was observed in all dosed and control male rats and consisted of one or more foci of basophilic-staining tubules lined by closely packed tubular epithelium in the cortex or outer medulla of the kidney. The lesion was minimal to mild and occurred in 9/10 rats in each group. No difference in severity was seen between groups. The incidence of renal tubular regenera tion in females, however, was dose related and was observed in 9/10 at 8,000 ppm, 3/10 at 4,000 ppm, 2/10 at 2,000 ppm, 1/10 at 1,000 ppm, 0/10 at 500 ppm, and in 0/10 controls. This lesion was of minimal severity in all affected rats. No le sions attributable to 1,2-dichloroethane were ob served in the liver. TABLE 5. SURVIVAL, MEAN BODY WEIGHTS, AND WATER CONSUMPTION OF F344/N RATS IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF 1,2-DICHLOROETHANE Concentration Survival (ppm) (a) Mean Body Weights (grama) Initial Final Change (b) (c) Final Weight Relative to Controls (percent) Water Consumption (d) MALE 0 500 1,000 2,000 4,000 8,000 FEMALE 0 500 1,000 2,000 4,000 8,000 10/10 10/10 10/10 10/10 10/10 10/10 134 2 133 2 133 2 132 2 134 1 133 2 358 4 359 7 358 5 358 3 329 3 302 4 + 223 3 + 226 6 + 225 5 + 226 3 +195 3 +168 4 10/10 10/10 10/10 10/10 10/10 10/10 109 2 108 1 108 1 108 2 105 3 106 1 202 2 204 3 207 2 199 3 195 1 187 2 + 93 2 + 96 2 + 99 1 + 92 1 +90 3 + 81 2 100 100 100 92 84 101 102 99 97 93 25 24 21 18 15 14 19 18 16 14 12 11 (a) Number surviving/number initially in group (b) Initial group mean body weight standard error of the mean. (c) Mean body weight change ofthe group standard error of the mean (d) Grams per animal per day; not corrected for spillage. 1,2-Dichloroethane, NTPTOX 4 18 SL 068448 390- 100- CN1L 2000 900 0 4000 70 mcixs on stuoy * 1000 13 IS FIGURE 2. GROWTH CURVES FOR F344/N RATS IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF U-DICHLOROETHANE 19 1,2-Dichl roethane, NTPTOX 4 SL 06844g TABLE . ORGAN WEIGHT DATA FOR MALE RATS IN THE THIRTEENU-DICHLOROETHANE <) STUDIES OF Study/Strain/Organ Dose or Concentration Drinking waur itudiM F344/N Control Body weight (peas) 380 12.0 Kidney Absolute Relative Liver Absolute Relative 1,932 48 3.4 0,16 15,430 660 42.9 2.17 Sprague Dawiey Body weight'srrem 449 n.o Kidney Absolut Relativ- Liver Absolute Relative 1,871 74 4.2 0.14 18,480 790 41.1 1.03 S00 ppm 354 6,9 1,345 38 3.8 0.08 16.500 540 46$ 0.95 446 7,9 1.943 4.4 20.080 45 0 l 5 1,000 ppm 35$ 4.5 2,000 ppm 355 2.8 Ml.433 28 "4.0 0.09 16.960 570 47 7 1.37 "1,523 15 **4.3 0.04 17,840 250 "50 2 0.49 431 7.0 432 11.3 1,954 58 *4.5 008 18,810 `>70 43 6 : 75 1,856 74 4.3 0.11 20,100 1 790 "46.5 i.ii 4,000 ppi "327 2.8 8,000 ppm "300 4.3 "1,451 18 "4.4 0.06 16,050 330 49.1 0.79 "1,377 22 "4,6 0.07 14,760 t 340 *49 2 + 0.85 436 7,9 *414 9.2 2,000 52 4.6 0.11 19,970 490 "45.9 0.82 2.008 55 **4*.9 0.11 19.230 560 *46.5 1.20 0sboneMaudeJ Body weight (grams) 421 25 3 477 13.1 465 17.2 Kidney Absolute Relative Liver Absolute Relative (b) 1,506 36 lb) 3,7 0.28 <b) 16,230 810 (b)39.2 2.01 1.600 41 3.4 0.09 17,830 610 37.4 0.85 **1,751 40 38 0,14 **21,080 840 *45.4 0.90 Garage study F344/N Body weight (grams) Vehicle Control 339 4.8 30 mg/kg 353 6.7 Kidney Absolute Relative Liver Absolute Relative 1,324 t 29 3.9 0.06 17.000 440 30.2 0.87 *1,441 26 4.1 0.10 (b) 17,960 $10 (b> 50.9 0.97 433 14.0 1,656 59 3.8 t 0,09 19,310 800 *44.6 1.24 80 mg/kg 354 9.0 **1,800 54 "4.5 t 0.08 18,270 540 $1.7 0.92 393 u.a 380 11.3 1.613 t 44 "4,1 *. 0.13 15.190 510 38.8 1.45 1,507 68 *4,0 t 0.18 15.900 800 41.9 1.59 110 af/kf 341 8.1 **1,653 47 "4,9 0.07 (b) 19.400 960 "(b) 57 4 0.83 (a) Mrq standard error id milligrams (absolute) or milligram* per gram irelative) for groups of 10 animals unless otherwise specified: F values vs. the controls by Dunn's test (Dunn, 1904) or Shirley's test (Shirley, 1977). fbi Nine animals were weighed. *P<0.05 **P<Q.0l 1,2-Dichloroethane, NTP TOX 4 20 SL 068450 TABLE 7. ORGAN WEIGHT DATA FOR FEMALE RATS IN THE THIRTEEN-WEEK STUDIES OF U-DICHLOROETHANE (a) Study/Strain/Organ Dose or Concentration Drinking water studies F344/N Control Body weight (grams) 194 2.4 Kidney Absolute Relative Liver Absolute Relative 739 26 3.8 0.13 6,829 154 35.3 0.85 Sprague Dawley Body weight (grams) 271 5.5 Kidney Absolute Relative Liver Absolute Relative 1,030 36 3.8 0.11 11,140 350 41.2 1.07 500 pp< 199 2.9 1,000 ppm 213 t 10.1 *814 16 4.1 0.07 7,268 t 179 36.6 0.60 **885 16 4.2 0.17 "7,627 177 36.3 1,57 2,000 ppm 196 t 2.4 **845 t 17 **4.3 t 0.07 7.278 165 37,2 0.75 283 7,8 287 t 6.4 271 4,5 *1,160 t 27 *4.1 0.09 11,890 530 42.0 1.49 *1,221 28 *4.3 0.13 12,200 680 42,7 2 60 *1,211 33 "4.5 0.11 10.990 310 40.6 t 1.32 4,000 ppm 193 1.3 **932 15 *4.8 t 0.09 7,551 171 **39.2 t 0.94 265 6.6 "1,208 50 "4.6 0.16 11,500 370 43.5 1.37 8,000 ppm 185 t 2.3 *923 15 *5.0 1 0.04 7,134 147 **38 5 0.61 256 4.8 "1,342 16 "5.2 0.10 (b) 11,950 450 (b) 46.6 t 1.41 Oiborn*-Mnd*l Body weight (grams) 274 t 99 Kidney Absolute Relative Liver Absolute Relative 894 t 28 3.3 Q.U 10.390 450 37.9 1.04 279 + 5.6 **1.017 1 15 *3.7 i 006 11.580 t 360 41,5 t 0.96 271 - 4.7 *1,041 22 **3.9 0.06 10,810 t 230 40.0 0 81 256 6,5 *1,020 U "4.0 t 0.16 10,390 430 41.0 t 2.39 270 6.6 "1,096 37 **4,1 0.14 10,750 300 39.8 t 0.73 266 11.2 "1,094 33 "4.2 + 0.26 10,100 410 38.6 2.49 Givifi ttndjr F344/N Vehicle Control 18 mg/kg 37 (/kg 75 mg/kg 150 mg/kg Body weight (grams) 190 1.9 190 2.5 194 3.3 197 2,7 192 1.9 Kidney Absolute Rdaciv, Liver Absolute Relative 800 + 16 4.2 0.08 7,345 120 38.7 0.54 717 70 3.8 0,37 *8,000 201 42.1 0.87 798 20 4.1 0.09 *7,920 191 *40.8 0.61 *898 23 *4.6 0.06 "8,577 197 *43.6 0.69 "984 9 "5.1 0.08 *9,775 151 *51.0 t 1.08 <a) Mud standard error id milligrams (absolute) or milligrams par gram (relative) for groups of 10 animals unless otherwise specified: P vain** vs. the controls by Dunn's test (Dunn, 1964) or Shirley's test (Shirley. 1977). (b) Nine animals were weighed, P<0.Q5 "P<Q,0l 21 1,2-Dichloroethane, NTP TOX 4 SL 068451 Sprague Dawley Rats: All Sprague Dawley rats lived to the end of the studies (Table 8). Mean body weights of males and females exposed to 4,000 ppm or more were lower than those of con trols throughout the studies (Figure 3). Water consumption by the three highest dose groups was about half that by controls for males and was less than half that by controls for females. Mild increases in erythrocyte counts, hemoglo bin, hematocrit, and blood urea nitrogen at days 3 and 7 in dosed male rats are evidence of mild animal dehydration (Table A6). The absolute and relative kidney weights for dosed females, relative kidney weights for dosed males, and the relative liver weights for dosed males and fe males were significantly increased (see Tables 6 and 7). No compound-related clinical signs were observed. Tubular regeneration occurred in the kidney of males and females in all dosed and control groups; the severity and incidence did not differ between groups. No lesions in the liv er were attributed to 1,2-dichloroethane admin istration. TABLE 8. SURVIVAL. MEAN BODY WEIGHTS, AND WATER CONSUMPTION OF SPRAGUE DAWLEY RATS IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF 1,2-DICHLOROETHANE Concentration Survival (ppm) (a) Mean Body Weights (grams) Initial Final Change (b) (c) Final Weight Relative to Controls (percent) Water Consumption (d> MALE 0 500 1,000 2,000 4,000 8,000 FEMALE 0 500 1,000 2,000 4,000 8,000 10/10 10/10 10/10 10/10 10/10 10/10 170 2 169 2 169 2 169 2 168 2 169 + 3 457 11 452 7 439 6 436 12 440 8 418 9 +288 10 +283 7 +270 6 + 267 12 + 272 7 + 248 7 10/10 10/10 10/10 10/10 10/10 10/10 139 2 144 2 143 2 143 2 141 2 135 2 281 291 290 276 270 257 6 8 5 5 7 5 + 141 + 147 + 147 + 133 + 128 + 123 5 8 4 4 6 4 99 96 95 96 91 104 103 98 96 91 43 37 30 25 21 19 44 33 23 18 16 13 (a) Number surviving/number initially in group (b) Initial group mean body weight standard error ofthe mean (c) Mean body weight change ofthe group standard error of the mean (d) Grams per animal per day; not corrected for spillage. 1,2-Dichloroethane, NTP TOX 4 22 SL 068452 FIGURE 3. GROWTH CURVES FOR SPRAGUE DAWLEY RATS IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF 1,2-DICHLOROETHANE 23 1,2-DichIoroethane, NTPTOX 4 SL O68453 Osborne-Mendel Rats: No compound-related deaths occurred in Osborne-Mendel rats (Table 9). Mean body weights of males exposed to 2,000 ppm or more and of females exposed to 1,000 ppm or more were lower than those of controls throughout the studies (Figure 4). Water con sumption by the three highest dose groups was half or less than half that by controls. The in creases in erythrocyte counts, hematocrit, and hemoglobin (day 3) and the decrease in mean cell volume in dosed male rats are evidence of animal dehydration (Table A9). The absolute and relative kidney weights were increased for dosed females, and the relative liver weights were increased for males receiving 1,000 or -,000 ppm (see Tables 6 and 7). No compoundrelated clinical signs were observed. Renal tu bular regeneration was seen in all dosed and control groups of each sex; although the inci dences were increased in rats administered the higher doses of 1,2-dichloroethane, the increases were not clearly dose related and the severity was not different between groups. Gavage Studi s All male F344/N rats that received 240 or 480 mg/kg and 9/10 females that received 300 mg/kg died before the end of the studies (Table 10). Mean body weights of males at 480 mg/kg and of females at 300 mg/kg were lower than those of vehicle controls throughout the studies (Figure 5). The mean body weight for one cage of female vehicle controls was decreased at week 9, possi bly due to not receiving water. Compound-relat ed clinical signs included tremors, salivation, emaciation, abnormal postures, ruffled fur. and dyspnea in males at 240 mg/kg and in feir s at 300 mg/kg. The absolute and relative ey and liver weights were increased for dosec aes and females (see Tables 6 and 7). Hyperplasia, inflammation, and mineralization were seen in the mucosa of the forestomach in animals that died or were killed in a moribund condition (Table 11). Foci ofepithelial necrosis were some times seen with hyperplasia and inflammation. Necrosis of the cerebellum and of the thymus TABLE 9. SURVIVAL, MEAN BODY WEIGHTS, AND WATER CONSUMPTION OF OSBORNE-MENDEL RATS IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF 1,2-DICHLOROETHANE Concentration Survival (ppm) (a) Mean Bodv Weights (grains) Initial Final Change (b) (c) Final Weight Relative to Controls (percent) Water Consumption (d) MALE 0 500 1,000 2,000 4,000 8.000 FEMALE 0 500 1,000 2,000 4,000 8,000 <e)9/10 10/10 10/10 10/10 10/10 10/10 172 3 171 - 4 17' 3 16 1 17. . 3 171 3 452 15 482 13 468 17 435 14 399 12 382 11 -281 16 311 14 298 18 266 14 227 + 14 -211 12 10/10 10/10 10/10 10/10 10/10 10/10 138 3 139 3 138 + 3 137 3 136 2 138 2 278 12 277 6 275 5 261 4 275 7 258 t 5 + 140 12 +137 5 + 138 3 + 124 3 + 139 t 5 + 121 4 107 104 96 88 85 100 99 94 99 93 42 35 28 22 19 17 43 34 26 23 22 18 (a) Number surviving/number initially in group (b) Initial group mean body weight standard error of the mean. Subsequent calculations are based on animals surviving to the end ofthe study. (c) Mean body weight change ofthe survivors standard error ofthe mean (d) Grama per animal per day; not corrected for spillage. (e) Week ofdeath: 7 1,2- Dichloroethane, NTP TOX 4 24 SL 068454 FIGURE 4. GROWTH CURVES FOR OSBORNE-MENDEL RATS IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF 1,2-DICHLOROETHANE 25 1,2-Dichloroethane, -VTP TOX 4 SL 068455 TABLE 10. SURVIVAL AND MEAN BODY WEIGHTS OF F344/N F S IN THE THIRTEEN-WEEK GAVAGE STUDIES OF 1,2-DICHLC THANE Dose (mg/kg) Survival (a) ______ Mean Body Weights (grarFinal Weight Relative Initial (b) Final Change (c) to Vehicle Controls (percent) MALE 0 30 60 120 240 480 FEMALE 0 18 37 75 150 300 10/10 10/10 10/10 10/10 (d)0/10 If) 0/10 10/10 10/10 10/10 10/10 10/10 (g) 1/10 118 4 119 5 120 4 120 4 118 4 117 4 104 2 102 2 102 2 104 2 104 2 101 + 2 333 + 4 346 5 349 9 338 9 <e) <e) 193 2 193 2 197 3 199 3 194 3 177 + 215 6 + 226 4 + 229 9 + 218 7 (e) (e) + 89 3 +91 2 + 95 3 + 95 2 + 90 3 + 76 104 105 102 (e) (e) 100 102 103 101 92 (a) Number surviving/number initially in group < b) Initial group mean body weight standard error ofthe mean. Subsequent calculations are based on animals surviving to the end ofthe study. (c) Mean body weight change ofthe survivors standard error ofthe mean (d) Week ofdeath: 1.1.5.5.6.7.8.8,9.11 (e) No data are reported due to 100% mortality in this group. (f) Week ofdeath: all 1 (g) Week ofdeath: 1,1,2,2,2,3,5,11,13 TABLE 11. NUMBERS OF F344/N RATS WITH SELECTED LESIONS IN THE THIRTEEN-WEEK GAVAGE STUDIES OF U-DICHLOROETHANE (a) Site/Lesion Group MALE Forestomach Hyperplasia Mineralization Inflammation Cerebellum Necrosis Thymus Necrosis FEMALE Forestomach Hyperplasia Mineralization Inflammation Cerebellum Necrosis Thymus Necrosis Vehicle Control 0 0 0 0 0 Vehicle Control 0 0 0 0 0 120 mg/kg 1 0 1 0 0 75 mg/kg -* -- - "* 240 mg/kg 5 3 *5 3 4 150 mg/kg 0 0 0 0 0 480 mg/kg 2 2 3 0 10 300 mg/kg 3 1 1 3 5 (a)Tena als were examined microscopically in each group. *P<0.05 ehicle controls **P < 0.0 i vehicle controls 1,2-Dichloroethane, NTP TOX 4 26 SL 068456 FIGURE 5. GROWTH CURVES FOR F344/N RATS ADMINISTERED 1.2-DICHLOROETHANE IN CORN OIL BY GAVAGE FOR THIRTEEN WEEKS 27 1,2-Dichloroethane, NTP TOX 4 SL 068457 was also observed. Necrosis in the cerebellum was mainly in the granular layer of the lateral folia, and mineralization was also present in the areas of necrosis in a few animals. Renal tubu lar regeneration in vehicle control and dosed groups of males or females did not differ in inci dence or severity. THIRTEEN-WEEK STUDIES IN MICE Drinking Water Studies: Nine of 10 female mice exposed to 8,000 ppm died before the end of the studies (Table 12). Mean body weights of males exposed to 500 ppm or more and of females exposed to 1,000 ppm or more were lower than those of controls throughout most of the studies (Figure 6). Water consumption varied greatly from wek to week, but overall water consumption by dosed and control groups appeared to be similar. The absolute and relative kidney and liver weights were significantly increased for dosed males and females (Table 13). No compound-related clini cal signs were observed. Compound-related le sions were seen in the kidney of male mice and were most prominent at the highest concentra tion (Table 14). At 8,000 ppm, a minimal-tomoderate tubular cell regeneration consisting of foci of basophilic-staining tubular epithelium was seen in the cortex of the kidney. Karyomegaly in the tubular epithelium, particularly in areas of regeneration, was characterized by nu clei that were slightly enlarged and more vari able in size than in controls. Protein casts were present in the lumen of a few tubules and were sometimes associated with tubular dilatation. In addition, foci of mineralization were present in the renal papilla at the highest dose. At 4,000 ppm, minimal tubular cell regeneration was present in 8/10 male mice; a similar change was present in only one or two mice per group at the lower doses. TABLE 12. SURVIVAL, MEAN BODY WEIGHTS, AND WATER CONSUMPTION OF MICE IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF 1,2-DICHLOROETHANE Concentration Survival (ppm) (a) Mean Body Weights (grama) Initial Final Change (b) (c) Final Weight Relative to Controls (percent) Water Consumption (d) MALE 0 500 1,000 2,000 4,000 8,000 FEMALE 0 500 1,000 2.000 4,000 8,000 10/10 10/10 10/10 10/10 10/10 10/10 21.2 0.2 20.5 0.4 21.1 0.4 20.8 0.4 20.3 0.2 20.5 0.3 31.4 0.6 28.9 0.6 29.3 0.5 29.4 0.8 28.6 0.7 25.9 0.7 +10.2 0.4 + 8.4 0.4 + 8.2 0.6 +8.6 0.7 + 8.3 0.6 + 5.4 0.8 10/10 10/10 10/10 10/10 10/10 (e) 1/10 17.1 0.2 17.8 0.3 16.9 0.2 16.9 0.3 17.1 0.3 17.2 0.4 25.9 0.6 24.7 0.5 23.2 0.6 23.7 0.5 23.8 0.6 23.4 +8.8 0.5 + 6.9 0.4 +6.3 0.5 +6.8 0.4 +6.7 0.5 + 4.7 92.0 93.3 93.6 91.1 82.5 95.4 89.6 91.5 91.9 90.3 13.1 12.3 11.3 9.8 16.6 12.2 8.1 10.4 13.0 12.0 12.7 12.5 (a) Number surviving/number initially in group (b) Initial group mean body weight standard error ofthe mean. Subsequent calculations are based on animals surviving to the end ofthe study. (c) Mean body weight change of the survivors standard error ofthe mean (d) Grams per animal per day; average of determinations from week 2 to week 13; not corrected for spillage. (e) Week ofdeath: 1,1,5,5,9,10,10,11.13 1,2-Dichloroethane, NTP TOX 4 28 SL 068458 FIGURE 6. GROWTH CURVES FOR MICE IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF U-DICHLOROETHANE 29 l,2-Dichlorothane, NTP TOX 4 SL 068459 TABLE 13. ORGAN WEIGHT DATA FOR MICE IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF U-DICHLOROETHANE (> Organ Control 500 ppm 1,000 ppm 2,000 ppm 4,000 ppm 8,000 ppm MALE Number weighed 10 Body weight (grams) 30.0 0.73 9 28.0 0.81 10 28.4 + 0.47 10 29.0 0.79 9 10 28.3 + 0.68 **25.4 0.65 Kidney Absolute Relative Liver Absolute Relative 305 7 10.2 0.22 301 8 *323 7 **358 6 **385 9 **379 12 10.8 0.12 **11.4 0.12 **12.4 + 0.33 **13.8 0.40 **15.0 0.54 1,455 55 1,490 42 1,519 55 1,571 56 1,628 + 54 *1,598 78 48.5 1.06 ***5*3.6 0.91 **53.4 '..18 **54.3 1.46 **57.6 1.10 **62.8 2.13 FEMALE Number weighed 10 8 9 10 Body weight (grams) 24.0 0.59 23.7 + 0.52 22.5 - 54 22.8 0.57 23.2 + 0.57 (b) 1 23.0 Kidney Absolute Relative Liver Absolute Relative 191 4 225 6 *211 5 *212 7 *215 7 8.0 0.23 9.4 + 0.21 **9.4 + 0.17 **9.3 0.24 **9.3 + 0.22 1,258 + 39 1,258 52 1,263 34 1,314 56 1,383 + 29 52.5 0.85 51.5 + 0.95 *56.0 + 0.67 *56.1 + 1.18 59.7 1.01 217 9.4 1,391 60.5 (a) Mean standard error in milligrams (absolute) or milligrams per gram (relative) unless otherwise specified; P values vs. the controls by Dunn's test (Dunn, 1904) or Shirley's test (Shirley, 1977). (b) Not included in statistical analysis P<0.05 **P<0.01 TABLE 14. NUMBERS OF MICE WITH RENAL LESIONS IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF 1,2-DICHLOROETHANE (a) Lesion Control 300 ppm 1,000 ppm 2,000 ppm 4,000 ppm 8,000 ppm MALE Tubular regeneration 0 1 2 2 Karyomegaly 0000 Dilatation 0000 Protein casts 0000 Mineralization 0000 FEMALE Tubular regeneration 0 0 0 0 '8 *9 0 *10 0 *5 0 **8 0 *5 10 (a) Ten mice were examined microscopically in each group. *P<0.05 vs. controls **P<0.01 vs. controls 1,2-Dichloroethane, NTP TOX 4 30 SL 068460 Daily intake doses, on a milligram per kilogram body weight basis, were estimated for rats ad ministered 1,2-dichloroethane in drinking water or by gavage (Table 15). For the drinking water studies, these estimates were obtained by divid ing the mean water consumption over the 13week studies by the mean of the initial and final body weights. All rat strains received approxi mately the same dose of 1,2-dichloroethane in drinking water; female Osborne-Mendel rats received a slightly higher dose than male Osborne-Mendel rats. Because mice typically consume more water than rats on a milligram per kilogram body weight basis, they received considerably higher doses of 1,2-dichloroethane than rats in the drinking water studies. Admin istration of 8,000 ppm 1,2-dichloroethane in drinking water resulted in up to eightfold higher doses in mice than in in rats. TABLE 15. ESTIMATED DAILY DOSES OF 1.2-DICHLOROETHANE ADMINISTERED BY GAVAGE OR IN DRINKING WATER IN THE THIRTEEN-WEEK STUDIES F344/N Gavage Dose (mg/kg/day) Concentration in Drinking Water <ppm) Estimated Intake Drinking Water Dose (a) F344/N Sprague Dawley Osborne-Mendel B6C3Fi Mice MALE 30 60 120 240 480 FEMALE 18 37 75 150 300 500 1,000 2,000 4,000 8,000 500 1,000 2.000 4,000 8,000 49 60 86 99 147 165 259 276 515 518 58 76 102 106 182 172 320 311 601 531 54 249 88 448 146 781 266 2,710 492 4,207 82 244 126 647 213 1,182 428 2,478 727 4.926 (a) Milligrams per kilogram per day based on the mean ofthe initial and final body weights for ten animals 31 1,2-Dichloroethane, NTP TOX 4 SL 068461 IV. DISCUSSION AND CONCLUSIONS 1.2- DichIoroethane administered at up to 8,000 ppm in drinking water for 13 weeks caused few adverse effects in F344/N, Sprague Dawley, and Osborne-Mendel rats. No deaths occurred in ex posed rats, and body weight changes were simi lar for all three rat strains of each sex. The high dose level of 1,2-dichloroethane (8,000 ppm) was selected based on limitations in the solubility and palatability of the chemical in drinking wa ter. The maximum solubility of 1,2-dichloro ethane in water is about 9,000 ppm (Torkelson and Rowe, 1981). Weight gain depression was common in males and females in the two higher dose groups throughout the studies and was likely caused by dehydration due to poor palatability of the for mulated drinking water. Water consumption decreased substantially with increasing dose for all exposed male and female rats, regardless of strain. The decrease in water intake, which was as much as 60% at the highest dose in male and female Osborne-Mendel rats, indicates that the dose received by all exposed animals was less than the target dose; however, because water in take was reduced at most exposure levels, equiv alent exposure did not occur at different dose levels within a strain. The estimated daily intake of 1,2-dichloroethane was similar for each rat strain at each dose level. Rats administered drinking water containing 8,000 ppm 1,2-dichloroethane received an esti mated intake of about 500-725 mg/kg per day. This estimated daily intake is close to the report ed oral LD50 for 1,2-dichloroethane adminis tered by gavage (680-850 mg/kg) (McCollister et al., 1956); however, intake of this dose over 24 hours rather than as a bolus resulted in little toxicity. 1.2- Dichloroethane toxicity administered by gavage or in formulated drinking water was compared in F344/N rats. Gavage doses were calculated to be approximately equivalent (in milligrams per kilogram) to the range of expo sures resulting from the formulated water mix tures. The F344/N rats were more sensitive to 1,2-dichloroethane administered by gavage than in drinking water, as evidenced by the fact that all males receiving 240 and 480 mg/kg and 9/10 females receiving 300 mg/kg died before the end of the studies. Necrosis of the cerebellum, observed in the brains of three males receiving 240 mg/kg and three females receiving 300 mg/kg, appeared to be related to 1,2-dichloroethane administration. Morphologic alterations in cells of the cerebel lum, parenchymous changes in the brain and spinal cord, and hyperemia and hemorrhage of the brain have been observed in humans who died of acute oral poisoning by 1,2-dichloro ethane (Hueper and Smith, 1935; Lochhead and Close, 1951). Hyperplasia, inflammation, and mineralization of the forestomach were observed in eight male and three female F344/N rats dosed by gavage which died or were killed in a moribund condi tion. Although forestomach lesions were chemi cal related, they were not considered life threat ening. However, hyperplasia of the forestomach epithelium after 13 weeks of exposure may be of significance, since long-term administration of 1,2-dichloroethane by gavage has been shown to cause neoplasms of the forestomach in OsborneMendel rats (NCI, 1978). Thymic necrosis in four mid dose and all high dose males and in five high dose females was at tributed to stress in animals that died or were killed in a moribund condition. Administration of bolus doses of 1,2-dichloroeth ane by gavage may result in saturation of 1,2dichloroethane elimination and increased levels of 1,2-dichloroethane in the blood (Reitz et al., 1982). Exposure at lower concentrations of 1,2dichloroethane over the course of the day (in drinking water or by inhalation) would result in lower peak blood levels and a lower area under the curve (the integral of the 1,2-dichloroethane concentration in blood as a function of time) and the chemical could be rapidly eliminated, even when the total daily dose was equal to the 1,2-Dichloroethane, NTP TOX 4 32 SL 068462 amount administered by gavage (Reitz et al., 1982). This mechanism may explain the greater toxicity for F344/N rats of 1,2-dichloroethane ad ministered by gavage compared with that after drinking water exposure. Based on the significant organ weight changes in rats receiving the chemical by either the drinking water or gavage routes, the liver and kidney appear to be target organs for 1,2-di chloroethane. Liver weights were usually in creased in rats of all strain, sex, and dose com binations. The kidney was also increased in weight and was significantly increased more fre quently than the liver. Despite increases of 10%-2Q% in kidney and liver weights, no histo logic changes could be clearly attributed to 1,2dichloroethane, except perhaps for renal tubular epithelium regeneration in female F344/N rats. Serum chemistry data were not indicative of liv er or kidney injury. Increased blood urea nitro gen was attributed to dehydration. Regenerative lesions of the rat kidney are com monly seen and are associated with chronic pro gressive nephropathy, which occurs in most strains of albino rats. The incidence and severi ty of progressive nephropathy are sex depend ent; in general, male rats are more susceptible than females, with the earliest lesions appearing at about 3 months of age (Goldstein et al., 1988). Rats were 4.5 months old at the end of the cur rent studies. Renal tubular epithelial regenera tion was present in many dosed and control ani mals of all strains; however, only female F344/N rats exposed to 1,2-dichloroethane in drinking water had a higher incidence of kidney lesions than controls. The degree of severity was not in creased, however, and was minimal even in the highest dose group. Administration of up to 8,000 ppm 1,2-dichloro ethane in drinking water resulted in greater tox icity to B6C3Fj mice than to rats. Nine of 10 female mice exposed to 8,000 ppm 1,2-dichloro ethane died before the end of the study. The esti mated daily intake of 1,2-dichloroethane in mice (with no corrections made for spillage) adminis tered 8,000 ppm 1,2-dichloroethane was approxi mately 4,200 mg/kg in males and 4,900 mg/kg in females. These intake levels are approximately tenfold greater than the reported LD50 of 1,2-di chloroethane administered by gavage (489 mg/kg for male mice and 413 mg/kg for female mice) (Munson et al., 1982). The estimated daily intake of 1,2-dichloroethane was considerably higher for mice than for rats receiving the same concentrations in drinking water. Mice typical ly consume more water than rats on a milligram per kilogram body weight basis, and palatability did not reduce water consumption by mice. Based on organ weight changes, the target or gans for male and female B6C3Fj mice exposed to 1,2-dichloroethane in drinking water were the liver and kidney. However, histopathologic changes were limited to protein casts, minerali zation, karyomegaly, and regeneration in the re nal tubules of male mice. The regenerative le sions were similar to those observed in rats; however, such lesions are generally less common in mice than in rats. Although significant in creases were observed in kidney weights of most exposed female mice, regeneration was detected in only one mouse. Long-term studies have shown that 1,2-dichloro ethane administered by gavage causes neo plasms in the mammary gland, endometrium, and lungs (but not in the kidney) in B6C3F! mice (NCI, 1978); inhalation exposure of Swiss mice resulted in no carcinogenic effects (Maltoni et al., 1980). The differing results of the two long-term studies have been attributed to a dif ference in responsiveness in the test strains and to the different routes of administration (Hooper et al., 1980). The results from a short-term study on B6C3F1 mice indicated that 1,2-dichloroethane is ca pable of inducing single-strand breaks and/or alkali-labile lesions in hepatic DNA when ad ministered by intraperitoneal injection or by ga vage, but not after inhalation exposure to com parable doses (Storer et al., 1984); this suggests that the liver is more likely to be a target organ when 1,2-dichloroethane is administered orally or parenterally than when administered by in halation. The current drinking water studies in B6C3Fi mice demonstrated increases in liver weights in mice receiving drinking water con taining 1,2-dichloroethane, although histologic lesions were not observed. In addition, lesions 33 1,2-Dichloroethane, NTP TOX 4 SL 068463 were observed in the kidney, which had not pre viously been identified as a target organ in mice. 1.2- Dichloroethane administered at up to 8,000 ppm in drinking water for 13 weeks was relative ly nontoxic for F344/N, Sprague Dawley, and Osborne-Mendel rats. Administration of the same drinking water concentrations of 1,2-dichloroethane to B6C3Fi mice resulted in greater toxicity; 9/10 female mice exposed to 8,000 ppm 1.2- dichloroethane died before the end of the study. The estimated daily intake (milligram per kilogram per day) of 1,2-dichloroethane in mice was about eightfold greater than in rats. 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Toxi col. 30:257-265. 1,2-Dichloroethane, NTP TOX 4 38 SL 068468 APPENDIX ORGAN WEIGHT, HEMATOLOGIC, AND SERUM CHEMICAL DATA IN THE THIRTEEN-WEEK STUDIES OF 1,2-DICHLOROETHANE TABLE A1 TABLE A2 TABLE A3 TABLE A4 TABLE AS TABLE A6 TABLE A7 TABLE AS TABLE A9 TABLE A10 TABLE All TABLE A12 TABLE A13 TABLE A14 PAGE ABSOLUTE ORGAN WEIGHTS FOR F344/N RATS IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF 1,2-DICHLOROETHANE 40 ORGAN WEIGHT TO BODY WEIGHT RATIOS FOR F344/N RATS IN THE THIRTEENWEEK DRINKING WATER STUDIES OF 1,2-DICHLOROETHANE 40 HEMATOLOGIC AND SERUM CHEMICAL DATA FOR MALE F344/N RATS IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF 1,2-DICHLOROETHANE 41 ABSOLUTE ORGAN WEIGHTS FOR SPRAGUE DAWLEY RATS IN THE THIRTEENWEEK DRINKING WATER STUDIES OF 1,2-DICHLOROETHANE 43 ORGAN WEIGHT TO BODY WEIGHT RATIOS FOR SPRAGUE DAWLEY RATS IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF 1,2-DICHLOROETHANE 43 HEMATOLOGIC AND SERUM CHEMICAL DATA FOR MALE SPRAGUE DAWLEY RATS IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF 1,2-DICHLOROETHANE 44 ABSOLUTE ORGAN WEIGHTS FOR OSBORNE-MENDEL RATS IN THE THIRTEENWEEK DRINKING WATER STUDIES OF U-DICHLOROETHANE 46 ORGAN WEIGHT TO BODY WEIGHT RATIOS FOR OSBORNE-MENDEL RATS IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF 1,2-DICHLOROETHANE 47 HEMATOLOGIC AND SERUM CHEMICAL DATA FOR MALE OSBORNE-MENDEL RATS IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF 1,2-DICHLOROETHANE 48 ABSOLUTE ORGAN WEIGHTS FOR F344/N RATS IN THE THIRTEEN-WEEK GAVAGE STUDIES OF 1,2-DICHLOROETHANE 50 ORGAN WEIGHT TO BODY WEIGHT RATIOS FOR F344/N RATS IN THE THIRTEENWEEK GAVAGE STUDIES OF 1,2-DICHLOROETHANE 50 HEMATOLOGIC AND SERUM CHEMICAL DATA FOR MALE F344/N RATS IN THE THIRTEEN-WEEK GAVAGE STUDIES OF 1,2-DICHLOROETHANE 51 ABSOLUTE ORGAN WEIGHTS FOR B6C3Fy MICE IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF 1,2-DICHLOROETHANE 53 ORGAN WEIGHT TO BODY WEIGHT RATIOS FOR B6C3Fy MICE IN THE THIRTEENWEEK DRINKING WATER STUDIES OF 1,2-DICHLOROETHANE 54 39 1,2-Dichloroethane, NTP TOX 4 SL 068469 TABLE Al. ABSOLUTE ORGAN WEIGHTS FOR F344/N RATS IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF U-DICHLOROETHANE (a) Control 500 ppm 1,000 ppm 2,000 ppm 4,000 ppm 8,000 ppm MALE Brain 1,959 Heart 1,044 Right kidney 1,232 Liver 15,450 Lung 1,731 Right testis 1,462 Thymus 285 23 24 48 + 660 41 + 15 14 1,927 + 30 1,077 23 1,345 38 16,500 540 1,864 74 1,460 33 304 17 FEMALE Brain 1,796 + 16 Heart 633 + 17 Right kidney 739 26 Liver 6,829 154 Lung 1,203 t 35 Thymus 242 9 1,817 20 654 12 814 t 16 7.268 179 1,488 169 247 7 1,958 1,062 **1,433 16,960 (b) 1,824 1,467 287 + 24 1,954 18 1,078 28 **1.523 570 *17,840 + 97 1,770 19 1,462 8 302 + 21 1,930 24 *991 + 15 ***1*,451 250 61 24 15 16,050 1,634 1,476 307 30 1,908 + 9 **927 + 18 **1,377 330 14,760 72 19 21 1,632 1,422 258 28 + 12 + 22 340 + 47 30 13 1,786 28 665 9 **885 16 **7,627 177 1,353 126 242 13 1.772 667 * '5 7 1 17 + 13 t 17 165 61 16 1,801 + 648 + *932 16 8 15 *7,551 171 1,243 35 236 13 1,773 37 643 4- 12 **923 15 7,134 147 1,224 50 234 x 12 (a) Mean standard error in milligrams for groups of 10 animals unless otherwise specified; P values vs. the controls by Dunn's test (Dunn, 1964) or Shirley's test (Shirley. 1977). (b) Lungs of nine animals were weighed. *P<0.05 *P<0.01 TABLE A2. ORGAN WEIGHT TO BODY WEIGHT RATIOS FOR F344/N RATS IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF 1,2-DICHLOROETHANE (a) Control 500 ppm 1,000 ppm 2,000 ppm 4,000 ppm 8,000 ppm MALE Body weight (grams) Brain Heart Right kidney Liver Lung Right testis Thymus FEMALE Body weight (grams) Brain Heart Right kidney Liver Lung Thymus 363 + 12.0 5.4 0.15 2.9 0.08 3.4 0.16 42.9 + 2.17 4.8 0.18 4.1 0.11 0.8 0.05 354 6.9 355 4.5 355 2.8 327 2.8 300 4.3 5.5 0.11 3.0 0.06 3.8 0.08 46.5 0.95 5.3 0.23 4.1 0.05 0.9 0.04 5.5 0.07 3.0 0.04 *4.0 0.09 47.7 + 1.37 (b) 5.2 + 0.29 4.1 0.07 0.8 0.02 5.5 0.08 3.0 0.07 **4.3 0.04 **50.2 + 0.49 5.0 0.17 4.1 + 0.05 0.9 0.05 5.9 0.06 3.0 0.03 **4.4 0.06 *49.1 0.79 5.0 0.21 **4.5 0.05 0.9 0.07 **6.4 0.11 3.1 0.03 *4.6 0.07 49.2 0.85 5.5 0.18 **4.7 0.08 0.9 0.04 194 2.4 9.3 0.15 3.3 0.09 3.8 0.13 35.3 0.85 6.2 0.15 1.3 0.04 199 + 2.9 9.2 0.13 3.3 0.05 4.1 0.07 36.6 0.60 7.5 0.82 1.2 0.04 213 + 10.1 8.5 0.30 3.2 0.12 *4.2 + 0.17 36.3 1.57 6.4 0.60 1.2 0.09 196 + 2.4 193 1.3 185 2.3 9.0 0.07 3.4 0.05 *4.3 t 0.07 37.2 0.75 6.0 0.27 1.1 0.08 9.4 + 0.09 3.4 + 0.05 4.8 0.09 *39.2 0.94 6.5 0.17 1.2 0.07 9.6 0.14 *3.5 0.05 *5.0 0.04 *38.5 0.61 6.6 0.23 1.3 0.06 (a) Mean standard error in milligrams per gram for groups of 10 animals unless otherwise specified; P values vs. the controls by Dunn's test (Dunn, 1964) or Shirley's test (Shirley, 1977) (b) Lungs of nine animals were weighed. *P<0.05 **P<0.01 1,2-Dichloroethane, NTP TOX 4 40 SL 068470 TABLE A3. HEMATOLOGIC AND SERUM CHEMICAL DATA FOR MALE F344/N RATS IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF 1,2-DICHLOROETHANE (a) Analysis Day Control 2,000 ppm 4,000 ppm 8,000 ppm Number examined (b) Leukocytes (1,000/pl) 3 7 14 45 90 Hematocrit (percent) 3 7 14 45 90 Hemoglobin (g/dl) 3 7 14 45 90 Mean corpuscular hemoglobin (pg) 3 7 14 45 90 Mean cell hemoglobin concentration (g/dl) 3 7 14 45 90 Mean cell volume (p3) 3 7 14 45 90 Platelets (1,000/pl) 3 7 14 45 90 Erythrocytes (108/pl> 3 7 14 45 90 Alkaline phosphatase (lU/liter) 3 7 14 45 90 Alanine aminotransferase (lU/liter) 3 7 14 45 90 8 20.7 + 4.28 (e) 7.4 0.96 6.9 0.61 7.7 0.48 7.2 0.35 40.7 1.18 (e)41.2 0.71 43.5 0.50 45.4 + 1.03 46.5 0.37 13.7 + 1.13 (e) 14.8 0.17 15.0 0.12 17.0 + 0.15 16.8 0.07 21.5 1.24 (e>22.5 0.29 22.2 0.18 20.0 0.41 18.9 0.16 33.4 2.28 (e)36.0 0.43 34.4 0.24 37.6 0.85 36.2 + 0.33 65.4 2.26 <e>62.6 0.87 64.5 0.53 53.1 0.64 52.0 0.57 <e>979 58.3 <e>861 34.8 836 33.6 (e)540 25.7 471 24.9 6.3 0.36 <e)6.6 0.11 6.8 0.08 8.6 0.17 8.9 0.10 638 20.2 690 + 44.2 631 + 21.5 330 12.5 290 5.7 <f) 50.0 4.40 (e) 38.7 1.74 (e)37.6 2.00 <e)48.3 3.24 (e)73.1 6.07 10 (015.1 5.64 8.9 1.18 6.6 0.34 8.1 0.21 8.8 0.82 (c)40.1 0.56 41.8 0.52 43.8 0.38 45.5 1.04 46.3 0.43 (c) 14.3 0.20 14.9 0.12 15.2 0.10 17.1 0.13 16.7 0.08 (022.6 + 0.68 22.0 0.45 21.5 t 0.35 19.8 0.58 18.4 0.24 (035.7 0.49 35.7 0.27 34.7 0.28 37.9 0.93 36.0 0.37 <063.3 1.89 62.0 + 1.00 *62.1 0.85 52.5 0.58 50.9 0.23 (0 943 53.0 831 27.4 775 25.6 550 17.8 488 24.2 (0 6.4 0.23 6.8 + 0.14 7.1 0.12 8.7 0.22 9.1 + 0.10 (e)614 41.7 *(0 590 10.7 561 + 19.8 316 11.9 263 8.0 <g)41.6 1.08 (e) 37.6 1.29 (g) 35.8 2.31 47.8 3.40 (061.0 3.87 10 <d>24.9 6.89 (0 7.9 0.54 6.4 0.30 7.6 + 0.19 7.6 0.24 (d)40.5 0.61 *(c) 44.1 0.87 44.3 0.33 47.1 0.29 46.5 + 0.44 10 *6.9 0.47 7.2 0.39 6.9 0.27 7.0 0.36 6.9 0.27 **44.7 0.66 41.5 0.38 43.5 0.51 46.8 0.70 46.9 0.51 (d) 14.0 0.23 (c) 15.5 0.28 15.3 0.14 17.4 0.07 16.7 0.12 *15.4 0.17 15.1 0.19 15.2 0.19 16.8 0.07 16.8 0.07 (d) 22.4 0.54 (c) 22.0 0.25 21.5 0.26 19.6 0.19 *18.2 0.10 *20.7 + 0.25 **21.2 0.12 **20.7 0.14 *18.8 + 0.27 **18.1 0.12 (d) 34.6 0.43 (0 35.1 0.25 34.5 0,29 36.8 0.25 36.0 0.27 34.5 0.32 36.5 0.27 35.0 0.24 36.0 0.46 35.8 0.31 <d)65.0 2.06 (0 62.7 0.97 62.4 0.92 53.3 0.30 *50.6 0.34 60.0 0.33 **57.9 0.35 **59.4 0.31 52.3 0.33 *50.3 0.37 (e) 1,019 57.2 (0 821 27.5 738 37.1 543 7.6 488 17.2 (0 872 18.7 *738 20.8 **706 24.3 544 6.6 508 19.2 (d)6.3 0.24 (07.0 0.16 7.1 0.11 8.9 0.07 9.2 0.08 **7.4 0.13 **7.2 0.09 **7.3 0.07 *8.9 0.14 *9.3 0.07 (d)614 24.3 **(0 553 21.5 594 30.5 329 7.4 278 9.0 609 + 19.5 **(0562 32.1 (0 557 19.1 338 17.1 285 9.6 *(h)36.8 2.43 *(f) 40.0 3.29 <d)36.9 2.86 (e) 32.0 1.36 <d)35.5 1.35 Id) 36-8 + 1.62 (043.1 2.40 48.7 3.32 **(e)54.0 3.73 **(e)54.3 2.20 41 1,2-Dichloroethane, NTP TOX 4 SL 068471 TABLE A3. HEMATOLOGIC AND SERUM CHEMICAL DATA FOR MALE F344/N RATS IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF 1,2-DICHLOROETHANE (Continued) Analysis Day Blood urea nitrogen (mg/dl) 3 7 14 45 90 Creatine kinase (lU/liter) 3 7 14 45 90 Sorbitol dehydrogenase (IU/liter) 3 7 14 45 90 Control 2.000 ppm 4.000 ppm 8.000 ppm (i) 14.0 0.00 (h) 17.3 2.17 (e) 19.4 + 1.46 *(c) 24.7 1.76 (e) 16.4 + 0.90 **(d) 23.4 + 1.51 <d)25.1 2.16 27.9 1.54 (d) 20.8 + 0.88 20.5 0.67 (0 986 225 587 125 381 53 562 49 341 33 (0605 96 (0 598 42 400 28 478 + 49 351 39 (e)6.6 0.65 (e)8.6 0.48 (g)9.2 0.97 10.3 0.82 (e)22.0 9.75 <e>7,4 1.19 (0 9.9 + 0.77 (0 8.7 0.24 (d) 11.9 1.61 12.0 1.14 *tf)26.8 2.00 tg> 19.2 1.85 *<0 25.1 1.40 **(e)30.9 4.23 **(c) 24.7 1.44 *<n 2i.8 1.97 (e)25.4 2.08 21.3 + 0.60 25,7 t 1.37 21.3 0.90 695 86 803 118 351 34 424 33 315 21 718 129 (0 504 39 374 48 441 48 320 12 (f) 11.7 4.94 (e> 9.1 0.60 (d)9.4 0.32 (e) 11.3 0.84 10.0 0.56 <e)8.7 0.52 (d) 12.3 2.84 *10.2 + 0.33 *13.5 1.15 10.4 1.16 (a) Mean standard error; P values vs. the controls by Dunn's test (Dunn, 1964) or Shirley's test (Shirley, 1977), (b) Unless otherwise specified (c) Nine animals were examined. (d) Eight animals were examined. (e) Seven animals were examined. (f) Six animals were examined. (g) Five animals were examined. (h) Four animals were examined. (i) Three animals were examined. *P<0.05 **P<0.01 1,2-Dichloroethane, NTP TOX 4 42 SL 068472 TABLE A4. ABSOLUTE ORGAN WEIGHTS FOR SPRAGUE DAWLEY RATS IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF 1,2-DICHLOROETHANE (a) Control 500 ppm 1,000 ppm 2,000 ppm 4,000 ppm 8,000 ppm MALE Brain 2,089 37 2,139 41 2,125 + 23 2,117 22 2,105 25 Heart 1,847 85 1,729 41 *1,623 54 *1,597 + 54 **1,579 55 Right kidney Liver 1,871 18,480 + 74 790 1,943 20,080 + 59 590 1,954 18,810 58 570 1,856 20,100 74 790 2,000 19,970 + 52 490 Lung 2,468 83 2,728 + 161 2,407 + 127 2,558 95 2,342 68 Right testis Thymus 1,821 t 48 493 32 1,728 All + 53 25 1.843 448 54 32 1,756 474 + 53 39 1,704 + 468 35 33 2,103 19 **1,566 55 2,008 55 19,230 560 2,220 96 1.825 34 485 34 FEMALE Brain 1,975 29 1,975 + 36 2,005 31 1,963 19 1,913 + 29 1.956 34 Heart 1,069 26 1,072 + 30 1,084 24 1,061 32 1,041 27 1.085 36 Right kidney 1,030 36 *1,160 + 27 *1,221 28 **1,211 + 33 **1.208 + 50 1,342 t 16 Liver 11,140 350 11,890 530 12,200 680 10,990 310 11,500 370 (b) 11,950 450 Lung 1,929 t 89 1,988 114 1,861 65 1,993 109 1,915 99 1,941 135 Thymus 364 23 395 36 337 t 17 365 23 359 30 326 23 (a) Mean standard error in milligrams for groups of 10 animals unless otherwise specified; P values vs. the controls by Dunn's test (Dunn, 1964) or Shirley's test (Shirley, 1977). (b) Nine livers were weighed. *P<0.06 P<0.01 TABLE A5. ORGAN WEIGHT TO BODY WEIGHT RATIOS FOR SPRAGUE DAWLEY RATS IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF 1,2-DICHLOROETHANE (a) Control 500 ppm 1,000 ppm 2,000 ppm 4,000 ppm 8,000 ppm* * ** MALE Body weight (grams) Brain Heart Right kidney Liver Lung Right testis Thymus FEMALE Body weight (grams) Brain Heart Right kidney Liver Lung Thymus 449 + 11.0 4.7 0.16 4.1 + 0.20 4.2 0.14 41.1 1.03 5.5 0.18 4.1 0.14 1.1 0.07 446 7.9 4.8 0.10 3.9 0.08 4.4 t 0.11 45.0 1.15 6.1 0.33 3.9 0.11 1.1 0.06 431 7.0 432 11.3 436 7.9 4.9 t 0.09 3.8 0.11 4.5 i 0.08 43.6 0.75 5.6 0.29 4.3 + 0.12 1.1 0.08 4.9 + 0.17 3.7 0.09 4.3 0.11 **46.5 1.11 5.9 0.19 4.1 + 0.17 1.1 0.07 4.9 0.12 *3.6 0.13 4.6 + 0.11 **45.9 0.82 5.4 0.18 3.9 0.10 1.1 0.07 *414 9.2 5.1 0.10 3.8 0.11 4.9 0.11 **46.5 1.20 5.4 0.20 4.4 0.15 1.2 0.08 271 5.5 7.3 0.13 4.0 + 0.08 3.8 0.11 41.2 1.07 7.1 0.29 1.4 0.09 283 7.8 7.0 0.16 3.8 0.11 *4.1 + 0.09 42.0 1.49 7.1 + 0.42 1.4 0.13 287 6.4 7.0 + 0.16 3.8 0.11 *4.3 + 0.13 42.7 + 2.60 6.5 t 0.24 1.2 0.07 271 + 4.5 7.3 0.10 3.9 0.12 **4.5 + 0.11 40.6 1.32 7.3 0.34 1.4 0.09 265 6.6 256 4.8 7.2 + 0.17 7.7 0.19 3.9 0.09 4.2 0.09 **4.6 0.16 *5.2 0.10 43.5 1.37 (b) 46.6 1.41 7.2 0.36 7.6 0.53 1.4 0.10 1.3 0.08 (a) Mean standard error in milligrams per gram for groups of 10 animals unless otherwise specified; P values vs. the controls by Dunn's test (Dunn, 1964) or Shirley's test (Shirley, 1977). (b) Nine livers were weighed. *P<0.05 **P<0.01 43 1,2-Dichloroethane, NTP TOX 4 SL 068473 TABLE A6. HEMATOLOGIC AND SERUM CHEMICAL DATA FOR MALE SPRAGUE DAWLEY RATS IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF 1,2-DICHLOROETHANE (a) Analysis Day Control 2,000 ppm 4,000 ppm 8,000 ppm Leukocytes (1,000/pl) 3 7 14 45 90 Hematocrit (percent) 3 7 14 45 90 Hemoglobin (g/dl) 3 7 14 45 90 Mean corpuscular hemoglobin (pg) 3 7 14 45 90 Mean corpuscular hemoglobin 3 concentration (g/dl) 7 14 45 90 Mean cell volume (p3) 3 7 14 45 90 Platelets (1,000/pl) 3 7 14 45 90 Erythrocytes (106/pl> 3 7 14 45 90 Alkaline phosphatase (IU/liter) 3 7 14 45 90 Alanine aminotransferase (IU/liter) 3 7 14 45 90 10.6 0.71 (O15.0 1.31 11.7 + 0.86 9.8 0.59 9.1 0.24 38.1 0.76 (c) 41.5 0.39 47.0 0.55 47.1 0.68 48.0 0.73 13.6 t 0.18 14.2 + 0.16 5.3 0.11 7.0 0.13 l7.0 0.24 24.5 0.57 (0 23.3 0.28 22.9 t 0.21 20.8 0.22 19.4 0.19 35.7 0.63 (0 34.1 0.21 32.6 0.40 36.0 0.38 35.5 0.32 68.5 0.65 (c) 68.4 1.09 70.6 + 1.28 57.7 + 0.88 54.7 0.62 976 47.0 (b) 1,183 40.1 (d)990 + 46.6 755 27.1 758 16.7 5.6 + 0.13 (c)6.1 0.11 6.7 + 0.07 8.2 0.12 8.8 0.18 477 + 13.3 420 17.9 406 29.9 245 t 13.9 291 15.3 (0 47.4 4.09 42.2 3.21 (d) 39.7 + 2.22 50.4 3.50 50.7 3.11 10.8 + 0.74 *ld) 12.2 0.68 (b) 11.3 + 0.80 9.7 0.28 9.2 0.39 18.5 4.16 (c)12.6 0.92 (b) 10.8 0.46 10.2 0.61 9.3 0.68 (b) 10.1 0,27 *(b) 12.9 1.60 (e) 12.1 1.00 *8.3 0.35 8.6 t 0.51 "42.3 0.74 40.7 0.80 "(b) 44.2 + 0.70 (d) 43.2 0.93 "(c) 43.6 0.34 (b) 43.3 0.62 <b)47.7 1.20 (b) 46.4 0.70 (e)47.0 0.80 47.3 0.88 46.9 0.37 47.5 0.42 49.2 + 0.29 48.8 0.51 47.9 0.49 "14.3 0.15 13.8 0.17 "(b) 15.1 0.21 (d) 14.6 0.23 "(c) 14.8 0.09 (b) 14.6 0.21 (b) 15.6 0.16 (b) 15.1 0.10 (e) 15.5 0.24 17.0 0.26 16.9 0.14 16.8 0.13 17.3 0.12 17.0 0.12 16.8 0.15 *22.7 0.32 <d)23.0 + 0.28 (b)22.0 0.39 20.8 0.23 18.8 0.23 23.2 0.35 (c) 23.0 0.33 (b)22.8 0.18 20.8 0.12 18.9 0.17 (b) 22.8 0.26 (b)22.4 0.42 <e>22.7 0.64 *20.2 0.17 18.7 0.24 33.9 0.43 (d)33.8 0.27 <b>32.7 0.76 35.9 0.23 35.1 0.26 34.0 0.31 (0 34.0 0.25 <b)32.7 0.39 36.1 0.28 35.0 0.27 (b)34.0 0.12 (b)33.7 0.20 (e)33.0 0.66 35.3 t 0.22 35.1 0.24 67.0 + 0.70 <d)68.2 + 0.97 (b) 67.4 1.51 58.1 0.59 53.6 t 0.76 68.2 0.93 (0 68.0 1.13 (b)70.0 1.22 57.8 0.25 54.2 0.47 (b)66.9 0.85 (b)66.8 t 1.29 <e>69.3 2.59 57.1 0.57 53.3 0.83 1,060 89.0 "949 + 49.7 838 36.4 775 t 28.4 699 27.4 1 080 37.6 '7 44.3 31.7 17.6 21.8 (b) 1,031 47.9 "(d) 946 65.3 (b)904 + 58.4 (d)758 21.3 742 t 23.7 "6.3 0.12 <d> 6.3 0.10 (b) 7.1 0.12 8.2 0.14 9.2 0.13 '6.0 0.16 (c) 6.4 + 0.08 (b)6.6 0.07 8.1 0.06 9.0 0.11 (b) 6.6 0.15 *(b)6.5 0.19 (e)6.8 0.25 8.3 0.07 9.0 0.14 430 14.5 428 24.5 416 12.1 228 8.0 235 17.7 (b) 49.0 2.46 (b)42.0 2.52 <d) 41,1 1.59 (039.0 1.68 45.7 2.13 441 19.5 (d)403 35.3 456 + 22.8 241 20.5 233 11.7 (0 41.9 2.01 (d) 37.6 1.84 <d> 40.3 2.12 <d> 43.3 1.76 (b>47.5 + 3.48 (d) 390 34.7 (d)439 20.7 (d)423 20.0 258 19.8 253 30.8 (c) 38.6 2.03 (d)37.2 + 4.24 (d) 37.9 2.45 51.0 4.94 (d>45.3 t 3.63 1,2-Dichloroethane, NTP TOX 4 44 SL 068474 TABLE A. HEMATOLOGIC AND SERUM CHEMICAL DATA FOR MALE SPRAGUE DAWLEY RATS IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF 1,2-DICHLOROETHANE (Continued) Analysis Day Control 2,000 ppm 4,000 ppm 8,000 ppm Blood urea nitrogen (mg/dl) Creatine kinase (IU/liter) Sorbitol dehydrogenase (IU/liter) 3 7 14 45 90 3 7 14 45 90 3 7 14 45 90 (d) 21.7 + 0.75 18.6 t 1.74 (b) 23.4 + 1.07 16.6 1.33 22.4 1.21 808 69 891 115 (d)742 80 829 45 818 73 (b) 25.6 1.85 *(d) 28.4 + 3.56 (d)26.3 1.22 (d) 22.3 0.53 20.6 + 1.06 (d) 21.2 + 1.74 (d) 26.3 2.82 (c) 25.4 3.14 (d) 19.3 t 1.63 22.4 t 1.51 (d) 27.2 + 2.18 (b) 26.4 + 3.61 (b) 28.6 2.82 22.1 1.69 23.9 1.77 1,035 119 1,079 101 (d)889 72 1,220 234 1,098 + 98 1,008 t 82 989 112 1,057 + 127 1,026 t 100 959 t 98 (d) 1,186 163 (d)898 69 (d)822 49 863 98 739 46 9.9 1.37 (b) 7.1 0.44 <d)9.8 0.49 11.5 + 1.52 5.8 1.04 (d) 10.1 1.09 (d)7.6 + 0.24 10.9 + 1.04 9.2 t 0.36 7.0 0.78 8.7 t 0.40 (c) 8.6 t 0.84 10.5 0.34 10.6 0.45 7.4 1.27 (011.0 1.35 (d) 8.3 0.47 (b) 11.8 0.75 13.3 2.16 7.9 1.05 (a) Mean standard error for groups of 10 animals unless otherwise specified; P values vs. the controls by Dunn's test (Dunn, 1964) or Shirley's test (Shirley, 1977). (b) Eight animals were examined. (c) Seven animals were examined. (d) Nine animals were examined. (e) Six animals were examined. (f) Four animals were examined. P<0.05 **P<0.01 45 1,2-Dichloroethane, NTPTOX 4 SL O68475 TABLE A7. ABSOLUTE ORGAN WEIGHTS FOR OSBORNE-MENDEL RATS IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF 1,2-DICHLOROETHANE (a) Control 500 ppm 1,000 ppm 2,000 ppm 4,000 ppm 8,000 ppm MALE Number weighed (b) 9 10 10 10 10 Brain Heart 2,056 33 1,498 74 2,106 25 1,526 48 2,089 + 1,605 36 70 1,995 t 1,386 37 62 1,991 + 1,289 57 53 Right kidney 1,506 36 1,600 41 *1,751 + 40 1,656 59 1,613 44 Liver 16,230 + 810 17,830 + 610 **21,080 840 19,310 800 15,190 510 Lung 1,821 80 (c) 1.946 118 2,075 61 2,074 123 1,960 83 Right testis 1,725 59 Thymus 314 19 1,655 64 305 28 1,747 323 + 40 19 1,725 314 + 82 22 1.635 + 326 53 22 10 1,982 + 50 1,295 59 1,507 + 68 15,900 800 1,717 53 1,631 38 333 27 FEMALE Number weighed 10 Brain 1,936 + 37 Heart 1,012 40 Right kidney 894 + 28 Liver 10,390 450 Lung 1,532 68 Thymus 304 14 10 1.996 + 26 1,051 28 *1,017 15 11,580 360 1,612 69 319 25 10 10 10 1,956 22 1,907 23 1,965 30 1,022 62 939 22 980 34 **1,041 22 **1,020 24 **1,096 t 37 10,810 230 10,390 430 10,750 300 1,629 81 1,497 79 1,565 57 278 40 309 34 341 25 10 1.933 33 909 22 **1,094 33 10,100 410 1,571 51 258 16 (a) Mean standard error in milligrams unless otherwise specified; P values vs. the controls by Dunn's test (Dunn, 1964) or Shirley's test (Shirley, 1977). (b I Unless otherwise specified (c) Lungs of nine animals were weighed. PC0.01 1,2-Dichloroethane, NTP TOX 4 46 SL 068476 TABLE AS. ORGAN WEIGHT TO BODY WEIGHT RATIOS FOR OSBORNE-MENDEL RATS IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF 1J-DICHLOROETHANE (a) Control 500 ppm 1,000 ppm 2,000 ppm 4,000 ppm 8,000 ppm MALE Number weighed (b) Body weight (grams) Brain Heart Right kidney Liver Lung Right testis Thymus FEMALE Number weighed Body weight (grams) Brain Heart Right kidney Liver Lung Thymus 9 10 421 25.3 477 13.1 5.0 0.37 3.7 + 0.31 3.7 0.28 39.2 2.01 4.5 0.47 4.2 0.25 0.8 0.05 4.4 0.10 3.2 0.14 3.4 0.09 37.4 0.85 (c)4.1 0.28 3.5 0.15 0.6 0.06 10 465 17.2 4.5 0.17 3.5 0.11 3.8 0.14 *45.4 t 0.90 4.5 0.23 3.8 0.19 0.7 + 0.05 10 433 14.0 4.6 0.10 3.2 0.08 3.8 0.09 44.6 t 1.24 4.8 0.30 4.0 0.21 0.7 0.04 10 393 11.8 5.1 0.09 3.3 0.09 **4.1 0.13 38.8 1.45 5.0 0.19 4.2 0,15 0.8 0.06 10 *380 11.3 5.2 0.14 3.4 0.09 4.0 0.18 41.9 1.59 4.5 0.17 4.3 0.11 0.9 0.06 10 274 9.9 7.1 0.23 3.7 0.11 3.3 0.11 37.9 1.04 5.6 0.20 1.1 0.05 10 10 10 10 10 279 5.6 271 4.7 256 6.5 270 6.6 266 11.2 7.2 0.21 3.8 0.11 *3.7 0.06 41.5 0.96 5.8 0.22 l.l 0.08 7.2 0.11 3.8 0.19 3.9 0.06 40.0 0.81 6.0 0.32 1.0 0.14 7.5 0.21 3.7 0.16 4.0 0.16 41.0 2.39 5.9 0.32 1.2 0.12 7.3 0.14 3.6 0.13 **4.1 0.14 39.8 1 0.73 5.8 0.19 1.3 0.07 7.3 0.25 3.5 0.12 4.2 0.26 38.6 2.49 6.0 0.27 1.0 0.06 (a) Mean standard error in milligrams per gram unless otherwise specified; P values vs. the controls by Dunn's test (Dunn, 1964) or Shirley's test (Shirley, 1977). (b) Unless otherwise specified (c) Lungs of nine animals were weighed. *P<0.06 P<0.01 47 1,2-Dichloroethane, NTP TOX 4 SL 068477 TABLE A9. HEMATOLOGIC AND SERUM CHEMICAL DATA FOR MALE OSBORNEMENDEL RATS IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF 1,2-DICHLOROETHANE (a) Analysis Day Control 2,000 ppm 4,000 ppm 8,000 ppm Leukocytes (1,000/til) Hematocrit (percent) 3 9.7 + 1.06 7 (biu.e 1.16 14 9.7 0.90 4a 9.1 + 0.93 90 8.7 i 0.62 (b) 12.7 3.33 (b>9.5 0.87 8.7 0.42 8.3 0.53 7.2 i 0.52 3 38.9 0.65 *<b) 41.1 1.03 7 <b)41.3 0.45 (b) 43.1 0.99 14 42.7 + 0.92 44.5 0.88 45 46.8 + 0.76 47.4 0.70 90 48.0 0.52 46.8 0.54 11.2 1.18 (b) 11.3 + 1.01 9.4 0.91 8.2 + 0.75 <b)7.6 0.67 10,0 + 10.1 9.9 8.9 8.0 + 0.94 1.23 1.01 0.88 0.50 41.2 (b)43.1 43.3 47.4 (b)47.1 + 0.79 0.74 1.22 0.73 0.57 **45.4 0.68 42.5 0.58 42.5 0.58 47.2 0.62 47.7 + 0.64 Hemoglobin <g/dl) 3 13.5 i 0.16 (b) 13.9 0.46 *14.4 0.23 **15.2 0.24 7 (b) 13.5 + 0,12 (b) 14.1 0.08 (b) 14.2 + 0.18 13.9 0.13 14 14.9 0.16 15.0 + 0.15 15.3 + 0.14 14.9 + 0.09 45 16.6 + 0.16 16.6 0.14 16.7 + 0.09 16.6 0.16 90 16.7 0.14 16.4 0.13 (b) 16.6 + 0.20 16.7 0.16 Mean corpuscular h iglobin (pg) 3 23.2 0.27 7 <b>22.5 0.44 14 23.6 0.30 45 90 20.8 19.0 i+ 0.23 0.19 <b>22.4 0.20 (b)21,5 * 0.27 21.5 0.52 19.8 0.19 18.7 0.31 22.7 0.29 (b)22.0 0.26 *23.2 0.47 *19.9 0.30 (b> 19.0 0.19 **21.9 *21.3 **22.2 *19.5 18.5 + t 0.30 0.38 0.21 0.26 0.19 Mean corpuscular hemoglobin concentration (g/dl> Mean cell volume (p3) Platelets (l ,000/pl) 3 34.7 0.35 7 (b)32.8 0.47 14 34.9 0.51 45 35.4 + 0.28 90 34.8 i 0.17 (b)33.8 0.41 (b) 32.9 0.73 33.9 0.68 35.0 0.43 35.1 0.31 35.0 0.24 (b)33.0 0.50 35.4 0.79 35.3 (b> 35.1 + 0.53 0.27 33.6 0.45 32.7 0.47 35.1 0.37 35.1 0.28 34.9 0.26 3 66.9 + 0.77 (b>66.3 1.03 7 (b) 68.9 1.01 (b) 65.7 1.17 14 67.9 0.60 *63.4 1.01 45 58.5 0.52 *56.4 0.52 90 54.6 0.64 53.5 0.95 64.9 0.74 (b>66.4 + 0.73 **65.6 i 0.43 56.6 0.62 (b) 54.1 + 0.54 65.4 0.40 *65.1 0.69 **63.3 0.72 *55.7 0.52 53.2 0.55 3 969 7 (b) 1,166 14 898 + 45 760 90 702 15.5 52.7 26.9 26.2 35.9 (b) 997 (b) 1,089 867 734 692 + 55.0 74.7 33.6 31.4 28.2 939 (b) 1,049 863 681 fb>787 t 29.2 65.9 30.0 26.0 35.5 *1,106 1,021 874 726 685 30,3 48.3 45.8 38.0 38.7 Erythrocytes (106/pl> 3 5.8 + 0.11 **(b)6.2 0.22 **6.4 0.12 *6.9 0.09 7 (b) 6.0 0.11 **(b) 6.6 0.11 (b) 6.5 + 0.12 **6.5 0.12 14 6.3 0,12 **7.0 0.15 6.6 + 0.18 6.7 0.08 45 8.0 0.12 8.4 0.13 8.4 0.12 **8.5 0.10 90 8.8 i 0.10 8.8 0.12 (b)8.7 0.13 9.0 t 0.12 Alkaline phosphatase (lU/liter) 3 387 34.0 ic)350 37.6 7 365 26.3 370 34.5 14 329 22.9 309 24.9 45 229 17.0 217 25.9 90 180 15.0 183 13.5 339 29.0 314 + 21.2 337 24.9 (b) 351 31.9 292 + 26.9 312 21.1 175 14.3 217 + 21.0 169 + 18.2 161 5.5 Alanine aminotransferase (IU/liter) 3 7 14 45 90 (bl 52.6 49.9 (b>42.8 Id) 52.6 48.0 + + 3.06 1.77 2.76 4.28 3.81 (d)45.1 3.78 (b) 39.6 3.46 35.9 1.68 45.0 + 3.69 *41.0 1.83 *37.5 0.95 (c) 39.5 3.24 (b) 34.9 t 2,10 *33.6 1.97 i(e )40.7 2.58 (e) 38.0 3.30 (c) 45.5 3.81 (b)50.7 6.51 **(b 134.9 2.25 (c) 38.5 2.25 1,2-Dichloroethane, NTP TOX 4 48 5^ TABLE A9. HEMATOLOGIC AND SERUM CHEMICAL DATA FOR MALE OSBORNE-MENDEL RATS IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF 1,2-DICHLOROETHANE (Continued) Analyst* Day Control 2,000 ppm 4,000 ppm 8,000 ppm Blood urea nitrogen (mg/dl) Creatine kinase (lU/liter) Sorbitol dehydrogenase (IU/liter) 3 (O 16.0 0.93 7 18.4 1.59 14 (b) 21.7 1.89 45 (0 25.6 2.45 90 20.3 0.80 3 698 + 97 7 1,019 163 14 691 115 45 497 95 90 484 + 83 3 (b>8.3 0.58 7 8.9 + 0.31 14 12.4 1.03 45 (c)7.6 0.46 90 9.5 1.12 If) 23.3 + 3.92 (0 21.9 + 1.08 *(c)22.6 2.03 20.4 + 2.09 (b) 18.9 1.43 (b) 21.1 2.26 21.0 2.02 <b)20.2 1.64 (0 21.4 2.61 (b) 28.2 + 1.93 27.3 1.51 (b) 31.1 3.61 18.3 0.96 21.2 1.09 21.3 1.63 (b>648 80 1,039 190 743 + 61 557 69 419 48 703 150 847 111 694 129 334 34 443 65 821 + 130 851 121 679 100 493 81 395 50 (08.9 0.77 8.9 0.50 11.8 0.80 (0 8.5 1.38 10.9 1.66 9.8 0.87 (b) 11.0 1.25 8.3 0.78 8.3 0.79 10.5 0.54 11.4 0.67 6.6 0.22 (b) 10.0 1.65 (b)8.0 0.37 (b) 10.3 0.87 (a) Mean standard error for groups of 10 animals unless otherwise specified; P values vs. the controls by Dunn's test (Dunn, 1964) or Shirley's test (Shirley, 1977). (b) Nine animals were examined. (c) Eight animals were examined. (d) Seven animals were examined. (e) Six animals were examined. (f) Four animals were examined. PC0.05 **P<0.01 49 1,2-Dichloroethane, NTP TOX 4 SL 068479 TABLE A10. ABSOLUTE ORGAN WEIGHTS FOR F344/N RATS IN THE THIRTEEN-WEEK GAVAGE STUDIES OF 1J-DICHLOROETHANE (a) Organ Group MALE Brain Heart Right kidney Liver Lung Right testis Thymus FEMALE Brain Heart Right kidney Liver Lung Thymus Vehicle Control 30 mg/kg 60 mg/kg 120 mg/kg 1,997 1,079 1,324 17,000 1,701 1,467 305 27 1,972 27 32 1,095 29 29 *1,441 26 440 (b) 17,960 510 52 1,726 34 26 1.431 43 10 310 22 1,995 1,115 **1,600 18,270 1,760 1,443 345 18 1,958 48 1,126 54 *1,653 540 (b) 19,400 75 1,703 18 1,388 19 296 23 37 47 660 35 33 14 Vehicle Control 18 mg/kg 37 mg/kg 75 mg/kg 150 mg/kg 1,815 16 660 10 800 16 7,345 120 1,178 36 261 16 1,830 679 717 *8,000 1,249 238 14 15 70 201 65 11 1,824 663 798 *7,920 1,210 248 16 12 20 191 32 11 1,826 26 *727 24 *898 23 **8,577 197 1,263 57 228 17 1,816 **737 **98**9,775 1,233 227 17 10 :9 151 40 15 (a) Mean standard error in milligrams for groups of 10 animals unless otherwise specified; P values vs. the vehicle controls by Dunn's test (Dunn, 1964)or Shirley's test(Shirley, 1977). (b) Nine livers were weighed. P<0.05 **P<0.01 TABLE All. ORGAN WEIGHT TO BODY WEIGHT RATIOS FOR F344/N RATS IN THE THIRTEEN-WEEK GAVAGE STUDIES OF 1,2-DICHLOROETHANE (a) Organ Group MALE Body weight (grams) Brain Heart Right kidney Liver Lung Right testis Thymus FEMALE Body weight (grams) Brain Heart Right kidney Liver Lung Thymus Vehicle Control 30 mg/kg 339 4.8 353 6.7 5.9 0.10 3.2 0.09 3.9 0.06 50.2 0.87 5.0 0.15 4.3 0.08 0.9 0.03 5.6 0.12 3.1 0.05 4.1 0.10 (b) 50.9 0.97 4.9 0.10 4.1 0.16 0.9 0.06 Vehicle Control 18 mg/kg 190 1.9 190 2.5 9.6 0.10 3.5 0.04 4.2 0.08 38.7 0.54 6.2 0.19 1.4 0.09 9.6 0.10 3.6 0.08 3.8 0.37 **42.1 0.87 6.6 0.32 1.3 0.06 60 mg/kg 354 9.0 5.7 0.14 3.2 0.08 *4.5 0.08 51.7 0.92 5.0 0.18 4.1 0.08 1.0 0.06 37 mg/kg 194 3.3 9.4 0.12 3.4 0.05 4.1 0.09 40.8 0.61 6.2 0.15 1.3 0.05 120 mg/kg 341 8.1 5.8 0.14 3.3 0.12 **4.9 0.07 *(b) 57.4 0.83 5.0 0.17 4.1 0.12 0.9 0.04 75 mg/kg 197 2.7 9.3 0.10 3.7 0.12 *4.6 0.08 **43.6 0.69 6.4 0.25 1.2 0.08 150 mg/kg 192 1.9 9.5 0.10 **3.8 0.06 **5.1 0.08 *51.0 1.08 6.4 0.22 1.2 0.08 (a) Mean standard error in milligrams per gram for groups of 10 animals unless otherwise specified; P values vs. the vehicle controls by Dunn's test (Dunn, 1964) or Shirley's test (Shirley, 1977). (b) Nine livers were weighed. *P<0,05 **P<0.01 1,2-Dichloroethane, NTP TOX 4 50 SL 068480 TABLE AH. HEMATOLOGIC AND SERUM CHEMICAL DATA FOR MALE F344/N RATS IN THE THIRTEEN-WEEK GAVAGE STUDIES OF U-DICHLOROETHANE (a) Analyst* Day Vehicle Control 120 mg/kg 240 mg/kg Number examined (b) Leukocytes (1 ,000/pl) Hematocrit (percent) Hemoglobin (g/dl) Mean corpuscular hemoglobin (pg) Mean corpuscular hemoglobin concentration (g/dl) Mean cell volume <p3) Erythrocytes (108/pl) Alkaline phosphatase (lU/liter) Alanine aminotransferase (IU/1) Blood urea nitrogen (mg/dl) 3 7 14 45 90 3 7 14 45 90 3 7 14 45 90 3 7 14 45 90 3 7 14 45 90 3 7 14 45 90 3 7 14 45 90 3 7 14 45 90 3 7 14 45 90 3 7 14 46 90 10 18.4 7.55 13.7 2.64 7.4 0.33 8.2 0.32 <d)6.8 0.28 40.7 0.85 41.0 0.47 42.9 0.22 45.6 0.39 (d) 43.1 + 0.41 14.5 0.37 14.1 0.13 15.1 0.07 16.4 0.09 (d) 16.1 0.10 23.5 t 0.61 22.7 + 0.40 22.6 0.29 19.5 0.11 (d) 18.2 + 0.09 35.5 0.51 34.5 + 0.25 35.1 0.11 36.0 + 0.17 (d) 37.2 0.25 66.2 1.60 66.0 1.56 64.6 0.81 54.2 0.33 (d) 49.0 0.24 6.2 0.18 6.2 0.13 6.7 0.11 8.4 0.09 (d)8.8 0.07 (e)740 16.3 (d)618 17.8 (e)594 6.1 394 5.0 (f) 1,101 34.0 <e>51.0 3.48 44.3 + 2.13 (e)40.0 1.15 44.2 1.28 (d)53.6 1.99 (e) 14.4 0.48 <d) 13.3 0.47 <f) 15.8 0.92 16.9 1.09 13.8 0.63 10 9.2 0.70 17.9 3.85 7.8 0.28 8.6 0.17 7.3 0.35 40.4 0.39 39.6 + 0.54 42.9 0.35 45.0 0.33 42.3 0.44 14.1 0.12 13.7 0.13 15.0 0.08 16.1 0.06 15.8 0.11 22.2 0.18 22.6 + 0.56 22.3 + 0.37 19.2 0.23 18.2 + 0.15 35.0 0.34 34.7 0.30 34.9 0.21 35.9 0.27 37.2 0.29 63.8 0.53 65.4 1.56 63.6 1.01 53.7 0.47 48.8 0.25 6.3 0.08 6.1 0.20 6.7 0.14 8.4 0.09 8.7 0.09 (0835 47.9 (d)604 20.8 (d)660 35.7 418 9.5 1,166 46.5 (h)56.2 2.48 (d) 50.6 1.45 51.7 + 4.88 *52.9 1.47 54.8 2.47 (e) 15.3 0.42 12.8 0.53 (d) 15.3 0.91 13.6 0.22 16.6 0.54 29.1 8.98 16.5 3.53 6,7 0.45 (0 8.2 0.45 '* 39.1 + 0.84 41.9 0.40 42.7 0.50 (0 44.5 + 0.22 " 13.5 0.37 14.2 0.13 14.7 0.16 **(c) 15.6 0.07 " 23.5 0.41 23.5 0.55 22.8 0.53 (c) 19.6 0.44 34.3 0.30 33.8 0.16 34.4 0.27 (0 35.2 0.17 ** 68.7 t 1.65 69.5 + 1.82 66.3 1.99 (056.0 1.00 - 5.7 0.24 6.1 0.13 6.5 0.20 (0 8.0 0.19 - (g) 688 35.9 575 31.3 557 25.0 (0393 8.8 - (i) 52.0 4.31 *(i)58.0 t 6.32 (g) 49.8 1.65 (051.3 t 3.67 - (c) 19.7 2.03 12.3 0.71 13.2 0.48 (c) 12.3 1.20 - 51 1,2-DichIoroethane, NTP TOX 4 SL 068481 TABLE A12. HEMATOLOGIC AND SERUM CHEMICAL DATA FOR MALE F344/N RATS IN THE THIRTEEN-WEEK GAVAGE STUDIES OF 1,2-DICHLOROETHANE (C ntinued) Analysis Day Vehicle Control 120 mg/kg 240 mg/kg Creatine kinase (lU/liter) 3 (f)775 111 931 79 7 572 37 <d>629 52 14 (d)373 33 389 25 45 446 24 481 30 90 (d)545 29 543 40 (i)653 + 82 558 + 74 399 40 (c) 334 62 Sorbitol dehydrogenase (lU/liter) 3 (e> 11.0 0.72 *(f) 12.6 0.56 (c) 12.3 0.33 7 13.8 + 0.77 15.3 1.69 14.7 1.52 14 (d) 24.2 3.36 23.0 + 2.98 <g)22.3 + 4.33 45 8.9 0.41 **11.9 0.57 *(010.7 1.67 90 <d)9.4 0.56 10.5 t 0.43 (a) Mean standard error; P values vs. the centre is by Dunn's test (Dunn, 1964) or Shirley's test (Shirley, 1977). (b) Unless otherwise specified (c) Three animals were examinee (d) Nine animals were examined (e) Seven animals were examine (f) Eight animals were examine (g) Four animals were examiner; (h) Six animals were examined. (i) Five animals were examined. *P<0,05 *P<0.01 1,2-Dichloroethane, NTP TOX 4 52 SL 068482 TABLE A13. ABSOLUTE ORGAN WEIGHTS FOR B6C3F. MICE IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF 1,2-DICHLOROETHANE (a) Control 300 ppm 1,000 ppm 2,000 ppm 4,000 ppm 8,000 ppm MALE Number weighed (b) Brain Heart Right kidney Liver Lung Right testis Thymus FEMALE Number weighed (b) Brain Heart Right kidney Liver Lung Thymus 10 459 4 160 7 305 7 1,455 55 230 10 115 2 33 1 9 442 6 163 + 7 301 + 8 1,490 42 236 15 112 5 (033 2 10 10 456 4 171 7 *323 7 1,519 55 244 15 113 + 2 33 5 2 461 4 165 5 358 8 1,571 56 224 11 116 2 34 1 9 455 5 176 4 385 9 1,628 t 54 208 i 8 115 3 (d)36 2 10 443 5 173 7 *379 12 *1,598 78 219 9 108 2 27 2 10 8 10 9 460 6 125 3 191 4 1,258 39 192 8 48 3 475 4 125 + 4 225 6 1,258 + 52 219 + 10 <d)44 1 465 + 8 130 + 3 211 5 1,263 34 214 + 13 45 3 442 10 126 5 212 7 1,314 56 212 10 43 2 10 456 6 133 + 5 215 7 1,383 29 228 + 23 41 1 <e) 1 437 121 217 1,391 190 40 (a) Mean standard error in milligrams; P values vs. the controls by Dunn's test (Dunn, 1964) or Shirley's test (Shirley, 1977). (b) Unless otherwise specified (c) Eight thymuses were weighed. (d) Seven thymuses were weighed. (e) Not included in statistical analysis P<0.05 **P<0.01 53 1,2-Dichloroethane, NTP TOX 4 SL 068483 TABLE AU. ORGAN WEIGHT TO BODY WEIGHT RATIOS FOR B8C3F, MICE IN THE THIRTEEN-WEEK DRINKING WATER STUDIES OF U-DICHLOROETHANE (a) Control 500 ppm 1,000 ppm 2.000 ppm 4,000 ppm 8,000 ppm MALE Number weighed (b) Body weight (grams) Brain Heart Right kidney Liver Lung Right testis Thymus FEMALE Number weighed Body weight (grams) Brain Heart Right kidney Liver Lung Thymus 10 9 10 10 9 10 30.0 0.73 28.0 0.81 28.4 0.47 29.0 0.79 28.3 0.68 **25.4 0.65 15.4 0.30 5.3 0.25 10.2 0.22 48.5 1.06 7.7 0.33 3.9 0.09 1.1 0.03 15.9 0.39 5.9 0.15 10.8 0.12 *53.6 0.91 8.5 0.71 4.1 0.08 Ic)1.2 0.06 16.1 0.33 6.0 0.24 **11.4 0.12 **53.4 1.18 8.6 0.44 4.0 0.10 1.2 0.06 16.0 0.41 5.7 0.19 12.4 0.33 **54.3 1.46 7.7 0.31 4.0 0.09 1.2 0.03 16.1 0.35 6.2 0.14 **13.8 0.40 **57.6 1.10 7.4 0.38 4.1 0.16 (d) 1.3 0.06 **17.5 0.31 **6.9 0.32 **15.0 0.54 **62.8 2.13 8.7 0.39 *4.3 0.12 1.0 0.07 10 24.0 0.59 19,3 0.41 5.2 0.13 8.0 0.23 52.5 0.85 8.0 - 0.34 2.0 0.12 8 23.7 0.52 20.1 0.35 5.2 0.16 **9.4 0.21 51.5 0.95 8.7 0.21 1.9 0.08 10 22.5 0.54 20.7 0.43 5.8 0.18 **9.4 0.17 *56.0 0.67 9.5 0.57 2.0 0.13 9 10 22.8 0.57 23.2 0.57 19.7 0.24 5.6 0.25 **9.3 0.24 56.1 1.18 9.1 0.38 1.9 0.10 19.8 0.65 5.7 0.23 9.3 0.22 **59.7 1.01 9.8 0.94 1.8 0.08 (e)l 23.0 19.0 5.3 9.4 60.5 8.3 1.7 (a) Mean standard error in milligrams per gram unless otherwise specified; P values vs. the controls by Dunn's test (Dunn, 1964) or Shirley's test (Shirley, 1977). (b) Unless otherwise specified (c) Eight thymuses were weighed. (d) Seven thymuses were weighed. (e) Not included in statistical analysis *P<0.05 **P<0.01 1,2-Dichloroethane, NTP TOX 4 54 SL 068484