N rats and B6C3F1 mice

FUNDAMENTAL

AND

APPLIED

TOXICOLOGY

5, 1128-

1136 ( 1985)

Toxicity and Carcinogenicity of Chlorodibromomethane Fischer 344/N Rats and B6C3F1 Mice’ JUNE K. DUNNICK,*

in

JOSEPH K. HASEMAN, HERMAN S. LILJA, AND STUART WYAND

National Toxicology Program, National Institute of Environmental Health Sciences, Research TriangIe Park, North Carolina 27709, and EG&G Mason Research Institute, Worchester, Massachusetts

Toxicity and Carcinogenicity of Chlorodibromomethane in Fischer 344/N Rats and B6C3FI Mice. DUNNICK, J. K., HASEMAN, J. K., LIWA, H. S., AND WYAND, S. (1985). Fundam. Appl. Toxicol. 5, 1128- 1136. Chlorodibromomethane, a trihalomethane found in water supplies after chlorination, was administered by gavage in corn oil to male and female Fischer 344/N rats and B6C3FI mice in toxicity studies of 13 weeks and 2 years duration. Doses used in the lfweek study were 0, 15,30,60, 125, and 250 mg/kg in rats and mice. At 250 mg/kg, hepatic and renal toxicity was produced in male and female rats and male mice, and mortality was increased in male and female rats. In the chronic study, male and female rats were administered the chemical at 0,40, and 80 mg/kg. No adverse effects on survival in treated rats were observed. Male rats receiving 80 mg/kg had reduced body weight gains relative to controls. Fatty change and cytoplasmic changes were seen in the liver of treated male and female rats. The male and female mice in the chronic study received doses of 0, 50, and 100 mgjkg of chemical. A dosing accident rendered the number of lowdose male mice inadequate for statistical analysis. High-dose male mice had reduced survival relative to controls. Survival was similar in dosed and control female mice. Dosed male and highdose female mice had reduced body weight relative to controls. Non-neoplastic hepatic lesions were seen in treated male mice (necrosis and hepatocytomegaly) and in treated female mice (calcification and fatty change); nephrosis was seen in treated male mice. The incidence of hepatocellular adenoma and carcinoma (combined) was increased in treated female mice, but only marginally so in treated male mice. Chlorodibromomethane, like chloroform, is toxic to liver and kidneys, and like chloroform induces hepatocellular tumors in mice. o 19115 sociay or foxicology.

Chlorodibromomethane is one of the four common trihalomethanes formed after chlorination of water supplies; the other common trihalomethanes are bromoform, chloroform, and bromodichloromethane. The presence of the trihalomethanes in drinking water was first reported in 1974 (Bellar et al., 1974; Rook, 1974). Chloroform has caused hepatic and renal neoplasms in rodents (Eschenbrenner and Miller, 1945; NCI, 1976; Roe et al., 1979). Epidemiologic studies to determine the relationship between trihalomethanes in drinking ’ Single copies of the full technical report (TR245) are available without cost from the NTP Public Information Office, P.O. Box 12233, Research Triangle Park, N.C. * To whom requests for reprints should be addressed. 0272-0590/85 $3.00 Copyright 0 1985 by the Society of Toxicology. All rights of reproduction in any form reserved.

water and cancer in humans are ongoing (IARC, 1982; NAS, 1980). The Environmental Protection Agency established a maximum contamination for total trihalomethanes of 0.1 mg/liter for community water systems serving populations greater than 10,000 persons (US EPA, 1979, 1983) in order to reduce exposure to potential carcinogenic substances. Chlorodibromomethane, like chloroform, adversely affected the liver and kidneys of rodents (Bowman et al., 1978; Chu et al., 1980, 1982; Condie et al., 1982; Munson et al., 1982), but the carcinogenic potential of chlorodibromomethane has not been previously investigated. The studies described in this paper were undertaken to determine the chronic toxicity and potential carcinogenicity of chlo-

1128

TOXICITV

1129

OF CHLORODIBROMOMETHANE

rodibromomethane in Fischer 344/N rats and B6C3F1 mice, and to gather additional information on the relative toxicity and carcinogenicity of the trihalomethanes. The oral route of administration was chosen for this study because human exposure is primarily oral. In order to reach concentrations of maximum exposure it was necessary to administer the chemical in corn oil because the chemical at high concentrations was insoluble in water. Maximal exposure concentrations were chosen to increase the sensitivity of the assay which uses a relatively small number of animals to help predict carcinogenic potential to humans (Office of Science and Technology Policy, 1984; NTP, 1984b).

MATERIALS

AND

METHODS

Animals. Male and female Fischer 344/N rats and B6C3Fr mice were obtained from Charles River Breeding Laboratories (Portage, Mich.). The animals were assigned to groups using a table of random numbers and were housed by sex,five per cage,in polycarbonate cagescovered with fiber filters and were provided with Betta Chips as bedding (Agway, Northboro, Mass.). Tap water and Wayne Lab Blox feed were available ud libitum. The animals were maintained in a room that was kept at 17.8-26.7”C with IO-12 air changes per hour, and a 12-hr fluorescent light cycle. In the 13-week study rats and mice were placed on study at 6-7 weeks of age and in the 2-year chronic study rats and mice were placed on study at 8 weeks of age. All animals were checked twice daily. Moribund animals were killed for necropsy. Animal body weights were taken once a week in the 13-week study, once a week for the first 12 weeks of the chronic study, and once a month thereafter.

TABLE 1 HISTOPATHOLCGIC

LESIONS

IN RATS IN THE 1 ~-WEEK

STUDIES

he bWk4) Vehicfe control

15

30

Male Liver Vacuolar change” Bile duct hyperplasia Central necrosis

4110 O/IO O/l0

7110 O/IO l/IO”

8/10 O/IO o/10

Kidney Toxic nephropathy

O/IO

NE’

Salivary gland Inllammation Squamous metaplasia

Of10 Of10

Female Liver Vacuolar change” Bile duct hypetplasia Central necrosis

60

125

250

O/IO Of10

lO/lOd o/10 o/10

NE

NE

o/10

8/10d

NE NE

NE NE

NE NE

o/10

O/IO

5110d 9/10d

l/10 l/10 O/IO

NE NE NE

NE NE NE

NE NE NE

Of10 Of10 Of10

9/9d 6/9d 7/9d

Kidney Toxic nephropathy

o/10

NE

NE

NE

o/10

9/9d

Salivary gland Inflammation Squamous metaplasia

O/IO Of10

NE NE

NE NE

NE NE

o/10 o/10

5/8d 6/8d

0 Interpreted as fatty metamorphosis. b Focal necrosis. c Not examined. dp -z 0.05 vs vehicle control.

10/lod

10/lod l/10

8/10d

DUNNICK

1130

Chemical and dose mixtures. Chlorodibromomethane (CAS No. 12448-l) was obtained from Freeman Industries (Tuckahoe, N.Y.; Lots F122277 and F810626). Acid impurities were removed with 1 M sodium carbonate treatment. The infrared and nuclear magnetic spectra agreed with literature values as did the boiling point, density, and refractive index. Gas chromatography/mass spectrometry were used to determine impurities, and based on these analyses, the chemical was 98% pure (NTP, 1984a). Dose mixtures of chlorodibromomethane in corn oil were prepared fresh each week and administered to rats and mice by gavage at 5 ml/kg body wt. Analyses of the dose mixtures were performed throughout the study, and the sample concentrations were within -tlO% of the targeted concentrations, with the exception of the dose mixture prepared for low-dose male and female mice at Week 58 of the chronic study (NTP, 1984a). The Week 58 mixture was seven times the targeted concentration and was used for 2 days. Thirteen-week study design. The 13-week studies were conducted to evaluate the toxicity of chlorodibromomethane and to set doses for the chronic studies. Groups of 10 rats and 10 mice of each sex were administered chlorodibromomethane at 0, 15, 30, 60, 125, or 250 mg/kg body wt in corn oil by gavage (5 days/week for 13 weeks). The control group received corn oil alone. At the conclusion of the 13-week study, all survivors were killed by carbon dioxide inhalation. Two-year chronic study design. The 2-year chronic studies were conducted to evaluate the toxicity and carcinogenicity of chlorodibromomethane. Groups of 50 rats and 50 mice of each sex were administered chlorodibromomethane in corn oil by gavage 5 days per week. The rat dose groups were 0,40, or 80 mg/kg body wt administered for 104 weeks and the mouse dose groups wereO,

ET AL. 50, or 100 mg/kg body wt administered for 105 weeks. The control groups received corn oil alone. At the conclusion of the 2-year dosing period, animals were kept for 1 to 2 weeks of observation without dosing and killed by carbon dioxide inhalation. Pathology. Necropsy was performed on all animals found dead or killed at the end of the 13-week and chronic studies unless precluded by autolysis or cannibalism. Tissues were preserved in 10% neutral buffered Formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The following tissueswere examined microscopically: grossly observed tissue masses,abnormal regional lymph nodes, mandibular and mesenteric lymph nodes, mammary gland, salivary gland, bone marrow, costochondral junction, thymus, larynx, trachea, lungs and bronchi, heart, thyroids, parathyroids, esophagus, stomach, colon, duodenum, jejunum, ileum, liver, gall bladder (mice), pancreas, spleen, kidneys, adrenals, urinary bladder, brain, pituitary, testes/prostate/seminal vesicles,or ovaries/ uterus. In the 13-week studies, all control and highdose animal tissues were examined microscopically, and when chemically related organ toxicity was found, these target tissues were examined in the lower dose groups to a noeffect level. In the 2-year chronic study, tissues from all dose groups and controls were examined microscopically. Statistical analysis for chronic studies. Differences in survival were analyzed by life table methods (Cox, 1972). Dose-response trends for weight gain were assessedby the multiple comparison procedures of Williams ( 197 1) and Dunnett (1955). For the analysis of tumor incidence data, three different procedures were used to assessdose-response trends and to make pairwise comparisons between dose groups and controls: (1) life table analysis (appropriate for fatal tumors), (2) Peto’s incidental tumor test (appropriate for tumors merely observed at necropsy in animals

TABLE 2 HISTOPATHOL~GIC

LESIONS IN MALE MICE IN THE 1 ~-WEEK

STUDIES

Dose (mg/kg)

Liver Vacuolar change” Multifocal necrosis Kidney Toxic nephropathy Mineralization without nephropathy

Vehicle control

15

30

60

125

250

o/10 o/10

NEb NE

NE NE

NE NE

o/10 o/10

5110’ l/lo=

o/10

NE

NE

NE

o/10

S/lOC

o/10

NE

NE

NE

o/10

l/l0

’ Interpreted as fatty metamorphosis. b Not examined. ‘p < 0.05 vs vehicle control.

TOXICITY

OF CHLORODIBROMOMETHANE

dying from an unrelated cause) (Pet0 et aI., 1980), and (3) Fisher’s exact tests (for pairwise comparisons) and Cochran-Armitage trend tests based on the overall proportion of tumor-bearing animals (Gart et al., 1979). Except where noted, results reported as “significant” are significant by all three procedures. All p values for the tumor incidence analyses are one sided.

RESULTS Thirteen- Week Studies Fischer 344/N ruts. In the 13-week rat studies, only l/ 10 males and l/ 10 females receiving 250 mg/kg survived, with most deaths occurring during Weeks 8-10 (NTP, 1984a); no other mortality was observed. Males receiving 125 mg/kg showed a significantly (p < 0.05) reduced body weight gain relative to controls. At 250 mg/kg, chlorodibromomethane produced lesions in the kidneys, liver, and salivary glands of male and female rats (Table 1). Intracytoplasmic clear vacuoles in hepatocytes, interpreted as severe fatty change, were found in the livers of 1O/ 10 male and 9/9 female rats that received 250 mg/kg. This change was also seen in other groups of dosed male-rats and in 4/ 10 control male rats. Hepatocellular centrilobular necrosis was observed at 250 mg/kg in male (8/ 10) and female (7/9) rats. Toxic cortical nephropathy characterized by tubular cell degeneration and regeneration and tubular cast formation was found in male (S/10) and female (9/9) rats at 250 mg/kg. Lesions of the salivary gland, characterized as acute inflammation and squamous metaplasia were seen in male and female rats at 250 mgfkg. B6C3Fj mice. In the 13-week mouse studies, no compound-related mortality was observed. Males and females receiving 250 mgf kg showed significantly (p < 0.05) reduced body weight gain relative to controls. At a dose of 250 mg/kg, chlorodibromomethane produced vacuolar change in the liver (interpreted as fatty change) in 5/ 10 male mice, and toxic nephropathy in 5/10 male mice. The lesions in the kidneys and liver were not seen in female mice (Table 2).

1132

DUNNICK

Two- Year Chronic Studies Fischer 344/N rats. Survival in all dosed groups of male and female rats was comparable to survival in the corresponding control groups. Mean body weight of high-dose male rats was lower than that of the vehicle control after Week 20. Mean body weight of the lowdose male rats was similar to the corresponding control group throughout the chronic study. Mean body weights of high- and low-dose female rats were greater than those of the controls during the first year of the study and similar thereafter (Table 3). Dose-related non-neoplastic lesions were seen in the liver of male and female rats (Table 4) including fatty change and “ground glass” cytoplasmic change. The ground glass cytoplasmic change microscopically was pink staining, lightly stippled cytoplasm in slightly enlarged heptocytes; the nucleus was usually eccentric. Sometimes, there was no hepatic cell enlargement, and the ground glass change appeared as pink-staining hyaline droplets in the cytoplasm. The incidence of liver basophilic cytoplasm change was decreased in dosed male and female rats. A dose-related decrease was observed in the incidence of fibroadenomas of the mammary

ET AL.

gland [vehicle control, 18/50 (36%); low dose, 12/50 (24%); high dose, 4/50 (8%)] and a decreased incidence of endometrial stromal polyps of the uterus [vehicle control, 14/50 (28%); low dose, 8/50 (16%); and high dose, 5/50 ( IO%)]. No statistically significant increases in tumor incidence were observed in male or female rats. B6C3Fl mice. Survival of low- and highdose male mice was less than the corresponding control groups. An overdosing accident at Week 58 killed 35 lowdose male mice, and this group was considered inadequate for tumor analysis. Survival in the groups of lowand high-dose female mice was comparable to that of the control group. Mean body weights of low- and high-dose male mice and highdose female mice were lower than the mean weights of the corresponding control groups throughout most of the study (Table 5). Compound-related lesion&were seen in the livers of male and female mice (Table 6). Nonneoplastic lesions of the liver in male mice included focal necrosis and hepatocytomegaly. The incidence of hepatocellular carcinomas was increased, but not the incidence of hepatocellular adenomas, in high-dose male mice. The combined incidence of hepatocellular adenoma or hepatocellular carcinoma in

TABLE 4 DOSE-RELATED

LIVER TOXICITY

IN FBCHER

344/N

RATS IN THE ~-YEAR

SIIJDES be

bw&t)

Vehicle control

40

80

Males Liver Fatty metamorphosis Ground glass cytoplasmic changes Basophilic cytoplasmic change

21150 (54%) S/50 (16%) 29/50 (58%)

47150 (94%)6 22150 (44%) b 7/50 (1496)b

49150 (98%)b 34/50 (68%)b 8/50 (16%)b

Females Liver Fatty metamorphosis Ground glass cytoplasmic changes Basophilic cytoplasmic change

12/50 (24%) o/50 (0%) 47/50 (94%)

23150 (46%)” l/50 (2%) 26/50 (52%)b

so/so (loo%)b 12/50 (24%)* 18/50 (36%)b

‘p < 0.05 relative to controls. bp i 0.0 1 relative to controls.

TOXICITJ

1133

3F CHLORODIBROMOMETHANE

high-dose male mice was significantly increased over that of the control group by the life table test, but not by the incidental tumor test. Hepatic toxicity in female mice was characterized by an increase in fatty change and calcification of the liver. The incidence of hepatocellular adenoma and the incidence of hepatocellular adenoma and carcinoma (combined) was increased in female mice. Chlorodibromomethane was toxic to the kidney of male mice causing nephrosis [vehicle control, O/50 (0%); high dose, 37/50 (74%)], but a similar change was not seen in the kidneys of treated female mice. Chlorodibromomethane administration was associated with a decrease in the incidence of malignant lymphoma in male mice [vehicle control, 9/50 (18%); high dose, O/50 (O%), p < 0.011. DISCUSSION In these studies chlorodibromomethane was toxic for the liver and kidneys. In the 13-week studies, toxicity to the liver and kidneys was seen in male and female rats and male mice at the highest dose of 250 mg/kg. After 2 years of treatment, non-neoplastic hepatic lesions were present in male and female rats and mice, and non-neoplastic renal lesions were present in male mice. In the 2-year study, chlorodibromomethane administration was associated with an increased incidence of hepatocellular adenomas and an increased incidence of combined hepatocellular adenomas or carcinomas in female mice. Evidence of carcinogenicity for male mice was equivocal as only the incidence of hepatocellular carcinomas was increased. The incidence of combined hepatocellular adenomas and carcinomas was only marginally increased. No evidence of carcinogenicity was found in male or female rats receiving chlorodibromomethane. Historically, the incidences of hepatocellular adenomas or carcinomas in corn oil gavage control male B6C3Fi mice are 67/199 (33.7%; SD = 9.4%; range 26-46%) for this contract laboratory and 357/1091 (32.7%; SD = 9.6%;

1134

DUNNICK

ET AL.

TABLE 6 DOSE-RELATED

LIVER AND KIDNEY

Males Liver Non-neoplastic lesions Hepatocytomegaly Necrosis Fatty metamorphosis Neoplastic lesions Adenoma Carcinoma Adenoma or carcinoma Kidney Nephrosis Females Liver Non-neoplastic lesions Calcification Fatty metamorphosis Neoplastic lesions Adenoma Carcinoma Adenoma or carcinoma

TOXICITY

IN B6C3F,

MICE IN THE ~-YEAR

STUDIES

Vehicle control

50

100

o/50 (0%) 2/50 (4%) 13/50 (26%)

-a -

12/50 (24%)’ 9150 ( 18pb 20/50 (40%)

14/50 (28%) 1o/50 (20%) 23150 (46%)

-

10/50 (20%) 19/50 (38%)b 27f50

Of50 (0%)

-

37f50 (74%)C

o/49 (0%) 21 f49 (43%)’

7f50 (14%)’ 28/50 (56%)=

4149 (8%)

I l/50 (22%)b 8/50 (16%)

Of 50 (0%) 7f50 (14%) 2f50 (4%) 4f50 (8%)

6f49 (12%)

6/50 (12%)

10/49 (20%)

(54%)d

19/50 (38%)’

’ Male low-dose group was inadequate for statistical analysis. bp < 0.05 relative to controls. ’ p < 0.0 1 relative to controls. *p < 0.01 (life table analysis); p = 0.065 (incidental tumor test) relative to controls.

range 14-50%) for all NTP laboratories. For female mice the corresponding rates are 17/ 198 (8.6%; SD = 5.5%; range 2-14%) for this contract laboratory and 74/1092 (6.8%; SD = 3.6%; range 2- 14%) for all NTP laboratories. The control incidences of hepatocellular tumors observed in this study were near the up per end of the historical control range, while the corresponding incidence in high-dose female mice far exceeded the maximum historical control value (Table 6). In the current study, treated male mice had an increased incidence of hepatic neoplasms and a decreased incidence of malignant lymphoma relative to controls. This inverse as-

sociation between hepatic and hematopoietic system tumors has been reported by several investigators for other strains of rats and mice (Haseman, 1983; Wahrendorf, 1983; Young and Gries, 1984), and the underlying mechanism of action for this association is unknown. However, a decrease in malignant lymphoma was not observed for female mice [controls, lo/50 (20%); low dose, 15/49 (3 1%); high dose, 1 l/50 (22%)], the group having the greatest increase in hepatic neoplasms. The mechanisms for the hepatic and renal toxicity and the hepatic carcinogenicity of chlorodibromomethane are not known. The similarities between the structure and toxicity

TOXICITY

1135

OF CHLORODIBROMOMETHANE

of chloroform and chlorodibromomethane suggest that similar mechanisms may be involved. Chlorodibromomethane was mutagenic to Salmonella typhimurium TAlOO when tested in desiccators, a procedure used to decrease the loss of volatile compounds (Simmon et al., 1977; Simmon and Tardiff, 1977). This compound was positive also in several in vivo mutagenicity test systems (Davidson et al., 1982). The extent to which the mutagenic properties of this chemical were the cause of the neoplastic response cannot be determined on the basis of the data currently available. Both chlorodibromomethane and chloroform (NCI, 1976) caused hepatic tumors in mice but not in rats, while only chloroform increased the incidence of renal neoplasms in male rats. The incidence of hepatic tumors in male and female mice in the chloroform study was higher than the incidence for this tumor in the chlorodibromomethane study. The greater carcinogenic response of chloroform might be explained on the basis of the higher dose of chloroform administered (e.g., in female mice an average dose of 238 and 477 mg/kg in the low- and high-dose groups, respectively). The chlorodibromomethane studies corroborate previous findings with chloroform that in rodents trihalomethanes are hepatic and renal toxicants, and after long-term exposure, cause hepatic tumors in mice. Chlorodibromomethane is found in water supplies in the presence of other organic compounds (Kool et al., 1983; Hauser and Bromberg, 1980), some of which (e.g., acetone) may potentiate the hepatotoxicity of the chemical (Hewitt et al., 1983). Chlorodibromomethane contamination of water supplies may be a problem especially in debilitated populations (Kroneld and Reunanen, 1983) and ongoing epidemiology studies should help in defining any potential risk in humans. REFERENCES BELLAR, T. A., LICHTENBERG, J., AND KRONER, R. C. (1974). Occurrence of organohalides in chlorinated

drinking waters. J. Amer. Water Works Assoc. 66,703706. BOWMAN, F. J., B~RZELLECA, J. F., AND MUNSON, A. E. (1978). Toxicity of some halomethanes in mice. Toxicol. Appl. Pharrnacol. 4l, 2 13-225. CHU, I., SECOURS,V., MARINO, I., AND VILLENEUVE, D. C. ( 1980). Acute toxicity of four trihalomethanes in male and female rats. Toxicol. Appl. Pharmacol. 52, 35 l-353. CHU, I., VILLENEUVE, D. C., SECOURS,V. E., AND BECKING, G. C. (1982). Trihalomethanes. II. Reversibility of toxicological changes produced by chloroform, bromodichloromethane, chlorodibromomethane and bromoform in rats. J. Environ. Sci. Health Part B 17(3), 225-240. CONDIE, L. W., SMALLWOOD, C. L., AND LAURIE, R. D. (1982). Comparative renal and hepatotoxicity of halomethanes: Bromodichloromethane, bromoform, chloroform, dibromomethane, and methylene chloride. Drug

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Toxicol.

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Cox, D. (1972). Regression models and life tables. R. Stat. Sac. J. B 34, 187-220. DAVIDSON, I. W. F., SUMNER, D. D., AND PARKER, J. L. (1982). Chloroform: A review of its metabolism, teratogenic, mutagenic, and carcinogenic potential. Drug Chem. Toxicol. 5, l-87. DUNNETT, W. (1955). A multiple comparison procedure for comparing several treatments with a control. J. Amer. Stat. Assoc. 50, 1096-l 121. ESCHENBRENNER,A. B., AND MILLER, E. (1945). Induction of hepatomas in mice by repeated oral administration of chloroform with observation on sex differences. J. Natl. Cancer Inst. 5,25 l-255. GART, J., CHU, K., AND TARONE, R. (1979). Statistical issues in interpretation of chronic bioassay tests for carcinogenicity. J. Natl. Cancer Inst. 62, 957-974. HASEMAN, J. K. (1983). Patterns of tumor incidence in two-year cancer bioassay feeding studies in Fischer 344 rats. Fundam. Appl. Toxicol. 3, 1-9. HAUSER, T. R., AND BROMBERG, S. M. (1980). EPA’s monitoring program at Love Canal 1980. Environ. Monit.

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HEWI~, W. R., BROWN, E. M., AND PLAA, G. L. (1983). Acetone-induced potent&ion of trihalomethane toxicity in male rats. Toxicol. Lett. 16,285-290. International Agency for Research on Cancer (IARC) (1982). IARC Monographs on the evaluation of the carcinogenic risk of chemicals to humans: Chemicals, industrial prcccsscs,and industries associated with cancer in humans. IARC Monograph Supplement 4,87-88. KOOL, H. J., VAN KRFJIL, C. F., AND ZOETEMAN, B. C. J. (1983). Toxicclogy assessmentof organic compounds in drinking water. Crit. Rev. Environ. Control 12,307357. KRONELD, R., AND REUNANEN, M. (1983). Volatile halocarbons in tap water as a problem in haemodialysis therapy. Clin. Chim. Acta 129, 141-149. MuN~~N, A. E., SUN, L. E., SANDERS,V. M., KAUFFMAN,

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DUNNICK

B. M., WHITE, K. L., PAGE, D. Cl., BARNES, D. W., AND BORZELLECA, J. F. (1982). Toxicology of organic drinking water contaminants: Trichloromethane, bromodichloromethane, and trichloromethane. Environ. Health Perspect. 46, 117-126. National Academy of Sciences (NAS) (1980). Drinking Water and Health, Vol. 3. National Academy Press, Washington, D.C. National Cancer Institute (NCI) (1976). NCI Report on the Carcinogenesis Bioassay ofChloroform, Natl. Tech. Inf. Serv. PB264018, NIH, Washington, D.C. National Toxicology Program (NTP) (1984a). NTP Technical Report on the Toxicology and Carcinogenesis Studies of Chlorodibromomethane, DHHS Publication (NIH) 84-2583, Research Triangle Park, N.C. National Toxicology Program (NTP) (1984b). Report oj the NTP Ad Hoc Panel on Chemical Carcinogenesis Testing and Evaluation, pp. 165-l 88. Board of Scientific Counselors, NIEHS, U.S. Dept. Health & Human Services, Research Triangle Park, N.C. Office of Science and Technology Policy Executive Office of the President. (1984). Chemical carcinogens: Review of the science and its associated principles. Fed. Regist. 49,21594-21661. PETO, R., PIKE, M., DAY, N., GRAY, R., LEE, P., PARISH, S., PETO, J., RICHARD, S., AND WAHRENDORF, J. ( 1980). Guidelines for simple, sensitive, significant tests for carcinogenic effectsin long-term animal experiments. In IARC Monographs on the Long-Term and ShortTerm Screening Assaysfor Carcinogens: A Critical Appraisal, Supplement 2, p. 3 I I. ROE, F. J. C., PALMER, A. K., WORDEN, A. N., AND VAN ABBE, N. J. (1979). Safetyevaluation oftoothpaste con-

ET AL. taining chloroform. I. Long term studies in mice. J. Environ. Pathol. Toxicol. 2, 799-8 19. ROOK, J. J. (1974). Formation of haloforms during chlorination of natural waters. Water Treatment Exam. 23, 234-243. SIMMON, V. E., KAUHANEN, K., AND TARDIFF, R. Cl. (1977). Mutagenic activity of chemicals identified in drinking water. Dev. Toxicol. Environ. Sci. 2,249-258. SIMMON, V. E., AND TARDIFF, R. G. (1977). Mutagenic activity of halogenated compounds found in chlorinated drinking water. In Water Chlorination: Environmental Impact and Health Eficts (W. Jolley, H. Gorchev, and D. Hamilton, eds.), Vol. 2, pp. 417-461. Ann Arbor, Mich. U.S. Environmental Protection Agency (1979). National interim primary drinking water regulations: control of trihalomethanes in drinking water: final rules. Fed. Regist. 44(23 I), 68624-68705 (Nov 29). U.S. Environmental Protection Agency (1983). National interim primary drinking water regulations; trihalomethanes. Fed. Regist. 48(40), 8406-8414 (Feb 28). WAHRENDORF, J. (1983). Simultaneous analysis of different tumor types in a long-term study with scheduled sacrifices. .I. Natl. Cancer Inst. 70,915-921. WILLIAMS, D. A. (197 1). A test for differences between treatment means when several dose levels are compared with a zero dose control. Biometrics 27, 103- 117. YOUNG, S. S., AND GRCES,C. L. (1984). Exploration of the negative correlation between proliferative hepatocellular lesions and lymphoma in rats and mice: Estab lishment and implications. Fundam. Appl. Toxicol. 4, 632-640.