Dichloroacetic acid was nominated for study by the United States Environmental Protection Agency (EPA) and by the National Institute of Environmental Health Sciences because of its widespread occurrence in drinking water as a by-product of water disinfection using chlorination. It was also nominated because dichloroacetic acid is the most studied representative of the class of haloacetic acids and has been shown to cause liver tumors in both rats and mice. Haloacetic acids are second only to trihalomethanes as a family of disinfection by-products found in many drinking water supplies. Dichloroacetic acid is one of several disinfection by-products being evaluated to determine whether genetically modified mouse models can serve as a more rapid and cost-effective means of evaluating and ranking potential hazards of disinfection by-products.
The NTP has explored the use of genetically altered mouse models as adjuncts to 2-year rodent cancer assays. These models may prove to be more rapid, use fewer animals, and provide some mechanistic insights into neoplastic responses. As part of the evaluation of new mouse cancer screening models, dichloroacetic acid was tested for potential toxicity and carcinogenicity in two relatively well-studied models, the Tg.AC hemizygous strain and the p53 haploinsufficient strain. Male and female Tg.AC hemizygous and p53 haploinsufficient mice were exposed to dichloroacetic acid in the drinking water (greater than 98% pure) for 26 or 39 weeks. Genetic toxicology studies were conducted in Salmonella typhimurium strains TA98, TA100, and TA1535 and in mouse peripheral blood erythrocytes.
26- and 39-Week Dermal Studies in Tg.AC Hemizygous Mice
Groups of 15 male and 15 female Tg.AC hemizygous mice were administered 0, 31.25, 125, or 500 mg dichloroacetic acid/kg body weight 5 days per week for 26 weeks with additional groups of 10 males and 10 females continued on treatment for 39 weeks. Survival of dosed males and females was similar to that of the vehicle control groups for both studies. Mean body weights of dosed males and females in the 26-week study were similar to those of the vehicle control groups. Mean body weights of dosed males in the 39-week study were similar to those of the vehicle control groups. Mean body weights of the 500 mg/kg females were greater than those of the vehicle controls in the 39-week study. The absolute liver weights were increased by greater than 50% compared to the vehicle controls for the 500 mg/kg males and females in both studies. At the site of application, the incidences of squamous cell papilloma were significantly increased in 500 mg/kg males and females at 39 weeks. In addition, one 125 mg/kg male, two 500 mg/kg males, and two 500 mg/kg females had squamous cell papillomas at 26 weeks. The incidences of epidermal hyperplasia and hyperkeratosis were significantly increased at the site of application in the 125 and 500 mg/kg males and females at 26 weeks. At 39 weeks, the incidence of epidermal hyperkeratosis was increased in the 31.25 mg/kg males, but in females, increased epidermal hyperkeratosis and hyperplasia occurred only in the 500 mg/kg group. There was a modest increase in pulmonary adenomas at 39 weeks that may have been related to the dichloroacetic acid exposure in males and females exposed to 125 or 500 mg/kg. In both studies, there was a dose-related increase in the mean severity of hepatocyte cytoplasmic vacuolization in males and females, and the incidence of nephropathy was increased in 500 mg/kg males.
26- and 41-Week Drinking Water Studies in Tg.AC Hemizygous Mice
Groups of 15 male and 15 female Tg.AC hemizygous mice were exposed to drinking water containing 0, 500, 1,000, or 2,000 mg/L dichloroacetic acid for 26 weeks with additional groups of 10 males and 10 females exposed for 41 weeks. The equivalent average daily doses were approximately 75, 145, and 235 mg dichloroacetic acid/kg body weight to males and approximately 100, 185, and 285 mg/kg to females. Survival of exposed males was similar in both studies. In the females, survival was decreased in the 26-week but not the 41-week study. While there was some variability, the mean body weights of mice exposed to dichloroacetic acid tended to be similar to those of the control groups. In the 41-week study, mean body weights of exposed males and females tended to be less than those of the control groups. Water consumption by males and females exposed to 1,000 and 2,000 mg/L was less than that by the controls throughout both studies. The incidences and/or severity of hepatocyte cytoplasmic vacuolization were increased in males and females in both studies. The incidence of pulmonary adenoma was increased in the male mice exposed to 1,000 mg/L dichloroacetic acid for 41 weeks. Two pulmonary adenomas were found in the 2,000 mg/L females at 41 weeks. At 26 weeks, a pulmonary carcinoma was found in one 1,000 mg/L male, one 500 mg/L female, and one 2,000 mg/L female.
26- and 41-Week Drinking Water Studies in p53 Haploinsufficient Mice
Groups of 15 male and 15 female p53 haploinsufficient mice were exposed to drinking water containing 0, 500, 1,000, or 2,000 mg/L dichloroacetic acid for 26 weeks with additional groups of 10 males and 10 females exposed for 41 weeks. The equivalent average daily doses were approximately 45, 80, and 145 mg/kg to males and approximately 75, 145, and 220 mg/kg to females. Survival of all exposed groups was similar to that of the control groups in both studies. Mean body weights of 1,000 and 2,000 mg/L males and females were generally less than those of the control groups throughout most of both studies; mean body weights of 500 mg/L males and females were less than those of the controls for much of the 41-week study. Water consumption by 1,000 and 2,000 mg/L males and females was less than that by the control groups throughout both studies. The incidences and/or severities of hepatocyte cytoplasmic vacuolization were increased in males in the 26-week study and females in both studies.
Genetic Toxicology
Dichloroacetic acid was mutagenic in Salmonella typhimurium strains TA100 and TA1535 in tests conducted in the absence of S9 liver activation enzymes; no increase in mutations was observed in either strain in the presence of rat or hamster liver S9. Dichloroacetic acid was not mutagenic in S. typhimurium strain TA98 with or without S9. Dichloroacetic acid was also tested for micronucleus induction in peripheral blood erythrocytes of male and female Tg.AC hemizygous and p53 haploinsufficient mice treated by drinking water or dermal application for 26 weeks. No induction of micronuclei was seen in Tg.AC hemizygous mice treated by either route or in the p53 haploinsufficient mice, which were exposed only by the drinking water route. In another study, analysis of peripheral blood samples for frequency of micronucleated erythrocytes in male and female B6C3F1 mice exposed to dichloroacetic acid in drinking water for 3 months revealed no alteration in micronucleus frequencies in male mice; a small increase seen in females was judged to be equivocal.
Conclusions
Under the conditions of these drinking water studies, there was no evidence of carcinogenic activity of dichloroacetic acid in male or female p53 haploinsufficient mice exposed to 0, 500, 1,000, or 2,000 mg/L for 26 or 41 weeks. The incidences and/or severities of cytoplasmic vacuolization of the hepatocyte were increased in males and females exposed to dichloroacetic acid for 26 or 41 weeks.
Under the conditions of these dermal studies, there were increased incidences of squamous cell papillomas at the site of application in male and female Tg.AC hemizygous mice exposed to 500 mg/kg for 39 weeks. There were dose-related increased incidences of epidermal hyperkeratosis and hyperplasia at the site of application in both male and female mice exposed to dichloroacetic acid for 26 or 39 weeks.
Under the conditions of these drinking water studies, there was an increase in the incidence of alveolar/bronchiolar adenoma in male Tg.AC hemizygous mice exposed to 1,000 mg/L for 41 weeks. There were a few bronchiolar/alveolar carcinomas in males and females exposed to dichloroacetic acid in the drinking water for 26 weeks and a few bronchiolar/alveolar adenomas in females exposed to dichloroacetic acid in the drinking water for 41 weeks.
There were increased incidences and/or severities of cytoplasmic vacuolization of the hepatocyte in male and female Tg.AC hemizygous mice exposed to dichloroacetic acid in the drinking water study for 26 or 41 weeks.
The marginally increased incidences of pulmonary adenomas and/or carcinomas compared to the unexposed groups found in both the dermal and drinking water studies at 39 or 41 weeks were considered to be related to dichloroacetic acid exposure.