Toxicology of haloacetonitriles.

Haloacetonitriles are by-products of water chlorination and may form in vivo from the reaction of residual chlorine with endogenous compounds such as amino acids. Dibromoacetonitrile (DBAN) was negative in selected mutagenic assays; dichloroacetonitrile (DCAN) was mutagenic in S. typhimurium, but not in S. cerevisiae. Both DBAN and DCAN may be carcinogenic. There is a paucity of basic toxicological data for these compounds. The studies described were conducted to determine the acute, subacute, and subchronic toxicity of DBAN and DCAN. The acute oral LD50 values (mg/kg) in mice and rats are: DBAN, mice: 289 (M), 303 (F); DBAN, rats: 245 (M), 361 (F); DCAN, mice: 270 (M), 279 (F); DCAN, rats: 339 (M), 330 (F). Death was preceded by slowed respiration, depressed activity, prostration, and coma. There were no apparent compound-related gross pathological effects. DBAN (in corn oil) was administered by gavage to male and female CD rats for 14 or 90 days at levels of 23, 45, 90, and 180 mg/kg/day or 6, 23, and 45 mg/kg/day, respectively. Mortality was 100% at 180 mg/kg and 40% (M) and 20% (F) at 90 mg/kg/day. Compound-related mortality was 10% (M) and 5% (F) at 45 mg/kg and 0% (M) and 10% (F) at 23 mg/kg during the 90-day study. No consistent, significant, adverse compound-related effects on any of the parameters evaluated were evident. Possible target organs might be spleen, thymus, and liver. The no-observed adverse-effect level (NOAEL) for 14 days was 45 mg/kg/day and for 90 days was 23 mg/kg/day. DCAN (in corn oil) was administered by gavage to male and female CD rats for 14 or 90 days at levels of 12, 23, 45, and 90 mg/kg/day or 8, 33, and 65 mg/kg/day, respectively. There were no deaths during the 14-day study. Compound-related mortality was 50% (M) and 25% (F) at 65 mg/kg, 10% (M) and 5% (F) at 33 mg/kg, and 5% (M) and 0% (F) at 8 mg/kg during the 90-day study. Body weights were significantly lower at 90 and 65 mg/kg/day; weight and ratios of spleen and gonads and cholesterol levels were significantly lower at 90 mg/kg/day. No consistent, significant adverse compound-related effects on any of the parameters evaluated were evident. The NOAEL for 14 days was 45 mg/kg/day and for 90 days was 8 mg/kg/day.

Exposure to DHAN may not be limited to consumption of drinking water. For example, Trehy and Bieber (7) cite two proposed uses of DCAN: as an insecticide for grains (10) and as a biological growth inhibitor in cooling towers (11). * Concern about the potential adverse health effects associated with DHAN was heightened by data from Simmon et al. (12) showing that DCAN tested positive in the Salmonella typhimurium TA 100 assay (but did not increase mitotic recombination in Saccharomyces cerevisiae). Bull (13) and Meier et al. (14) extended these findings and reported that DCAN tested positive in three Salmonella tester strains (TA 98, TA 100, and TA 1535), but the relatively high cytotoxicity of DBAN compromised the in vitro mutagenicity testing (13,14). DBAN tested positive, however, in a SENCAR mouse skin initiation-phorbol promotion model, which generated inconclusive data on DCAN (13). On the other hand, Kraybill (15,16) identified DCAN as a mutagen or suspected mutagen in drinking water in the United States.
A review of the literature failed to identify toxicity data for DHAN. Since data were lacking on these compounds and they were of interest to the U.S. Environmental Protection Agency (EPA) because of their presence in drinking water, the following studies were undertaken as a joint effort to provide the needed toxicological information.

Necropsy
The rats were weighed and then anesthetized with ether; blood was collected by cardiac puncture into 3.0% sodium citrate (1:10 citrate to blood) for the hematology studies and into uncitrated tubes for the serum chemistries. Gross pathological examination was performed, followed by removal and weighing of selected organs. All tissues were preserved in 10% neutral buffered formalin for subsequent histopathological examination.

Statistical Evaluation
All data were subjected to an analysis of variance and test for homogeneity, as well as a Dunnett's t-test, and nonhomogenous data were subjected to a Wilcoxon rank sum test. Those values which differed from the vehicle group at p -0.05 were considered significant.

Results
Acute Oral Toxicity of DBAN and DCAN Acute oral toxicity data are summarized in Table  5.The LD50 values were calculated using the method of Litchfield and Wilcoxon (19). Ataxia, depressed respiration, depressed activity, and coma preceded death. No consistent, compound-related, gross pathological effects were observed at necropsy. Hematology A Coulter counter (Model Z81) was used to determine leukocyte, erythrocyte, and platelet numbers. Microhematocrits were determined, and hemoglobin was assayed as cyanomethemoglobin. Leukocyte differentials were evaluated by the classic Wright's Giemsa staining procedure. Prothrombin times and plasma fibrinogen levels were determined using reagents from Dade Diagnostics, Inc. (Miami, FL).

Serum Enzyme Chemistry
Serum enzyme levels and calcium and phosphorus concentrations were determined by using an Abbott Bichromatic Analyzer and diagnostic chemistry kits from  Figures 1 and 2, show that the depression in body weight gain was dose-dependent. There was 100% mortality at 180 mg/kg/day; all males were dead by day 4 and all females by day 7. At 90 mg/ kg/day, 40% of males and 20% of females were dead by day 14. No significant, consistent, compound-related and dose-dependent adverse effects were apparent in any of the hematological or urinary parameters measured, although a trend toward higher values for hemoglobin, total red blood count (RBC) and white blood count (WBC) and fibrinogen was observed in all treated animals (Tables 6 and 7). Additionally, no consistent, significant, compound-related adverse effects were observed in any of the serum chemistry parameters measured, which are summarized in Tables 6 and 7. Organ weights and ratios are summarized in Tables 8   and 9. No remarkable findings were observed at necropsy. Spleen and thymus in males and liver, lungs, and thymus in females were affected only at the highest dose. The significance of these effects in the absence of appropriate biochemical findings is unclear at this time.
90-Day Subchronic Study. The body weight data, summarized in Figures 3 and 4, show that body weight gain was significantly depressed only in males at the highest dose tested (45 mg/kg/day), confirming the apparent greater sensitivity of the male rat to the effects aBy gavage; corn oil vehicle. bMethod of Litchfield and Wilcoxon (19); LD50 ± confidence limits. 10 [10][11][12][13][14][15]. Additionally, no significant, consistent, compound-related and dose-dependent adverse effects were observed in the serum chemistry parameters measured, which are summarized in Tables Tables 20 and 21. No remarkable findings were observed at necropsy, although most organ weights and ratios were significantly lower at the   Tables 22-27, revealed few significant, consistent, compound-related and dose-dependent adverse effects. These effects included a lowering of cholesterol at the highest dose tested (65 mg/kg/day) and elevated levels of serum glutamic-pyruvic transaminase (SGPT) suggesting possible liver involvement. The significance of decreases in the activity of selected enzymes is unclear. DBAN   Organ weights and ratios are summarized in Tables 28 and 29. No remarkable findings were observed at necropsy, although most organ weights and ratios were lower in males at 65 mg/kg, and livers appeared to be larger in treated females. The apparent NOAEL for DCAN, based on these 90-day data, was determined to be 8 mg/kg/day. Pereira et al. (20) studied the metabolism and excretion of haloacetonitriles (HAN) and reported that 7.7 to 12.8% of orally administered DBAN and DCAN was converted to thiocyanate and excreted in the urine. They also reported that some orally administered HAN aAll data expressed as mean ± SEM. *Significantly different from vehicle control (p G 0.05). Table 17. Organ weights and ratios at termination of subchronic study of female CD rats exposed to dibromoacetonitrile by gavage. 170 ± 10 170 ± 10 160 ± 10 170 ± 10 % body weight 0.06 ± 0.00 0.06 ± 0.00 0.06 ± 0.00 0.05 ± 0.00 0.06 ± 0.00 % brain weight 8.9 ± 0.4 9.1 ± 0.5 9.2 ± 0.5 8.9 ± 0.5 9.4 ± 0.6 'All data expressed as mean ± SEM. aSignificantly different from vehicle control at (p S 0.05).

Discussion
will inhibit hepatic dimethylnitrosamine demethylase (DMN-DM) activity. They concluded that HAN are converted to highly toxic metabolites. Daniel et al. (21) reported that HAN are direct-acting alkylating agents and could elicit liver and/or other organ toxicity, including carcinogenesis. These findings and those of Sim- . Weekly body weights of female CD rats exposed to dibromoacetonitrile for 13 weeks.
------TOXICOLOGY OF-HALOACETONITRILES  and long-term testing, as well as pharmacokinetic and pharmacodynamic studies, are indicated.
Acute toxicity data for DBAN and DCAN are com-mon et al. (12), Bull (13), and Meier et al. (14) suggest that the HAN may be biologically reactive, directly or by conversion to toxic products. Thorough short-term    pared with data for acetonitrile and acrylonitrile in Table 30. The toxicity of the DHAN appears to be intermediate between that of acetonitrile and that of acrylonitrile. The rat and mouse appear to be equally sensitive to the acute effects of DCAN; however, the male rat appears to be more sensitive to DBAN than the female rat.
The data obtained following 14-and 90-day repeated oral administration of DBAN or DCAN to rats revealed a dose-dependent decrease in weight gain. Weight gain is a sensitive indicator of the general health status of the animal, and the decrease suggests that the body is responding to the toxic effects of DBAN and DCAN.
The nature of this reaction is not evident, although it is unlikely that it is caused by chronic cyanide intoxication. The biochemical data failed to identify specific target organs. Organ weight and ratio data suggest that the thymus, liver, spleen, and gonads may be possible target organs. No consistent, compound-related and dose-dependent adverse effects were noted except the effect on body weight. The NOAEL for DBAN was determined to be 23 mg/kg/day; for DCAN, the NOAEL was identified as 8 mg/kg/day.
Insight into possible mechanisms of toxicity and significance of the data awaits further studies. For example, gavage administration using a corn oil vehicle  does not simulate human exposure conditions. This issue is currently being addressed by the National Toxicology Program. Limited water solubility and lack of acceptance by rats in taste aversion studies necessitates the use of a solvent to assess effects at high doses. Limit studies (solubility limit in water) should be considered.
The histopathological evaluation of major organs (tissues) from exposed animals should provide some infor-