Dichloroacetic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genotoxicity/mutagenicity of DCA has been investigated in an extensive number of studies. The preponderance of in vitro studies are negative, with only a few equivocal or positive results in the bacterial and gene mutation studies. Studies in vivo are mixed, with internally inconsistent results between studies and between endpoints. The difference in results does not appear to be clearly related to differences in exposure levels. An increased frequency of mutations was found in the Big Blue transgenic mouse in the Lac I loci after 60 weeks, whereas this was not observed after 4 and 10 weeks. This time-response pattern suggests that the mutational events might be secondary to toxicological changes in the liver rather than a direct genotoxic effect, since a direct effect would be expected to be time-independent. The results indicate that a large cumulative dose (due to the 60-week exposure period) is necessary to increase mutations in this in vivo system.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Although some details are missing, the study is consdired to be reliable, relevant and adequate.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

mutagenicity: 0, 0.333, 0.667, 1.00, 3.33, and 5.00 mg DCA/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: , 2-aminoanthracene

Remarks:

with S9: 2.5 µg/plate

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9: TA98 1.0 µg/plate

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9:TA100 and TA1535 2.0 µg/plate

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

without S9: TA 1537 2.0 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in 2 layer plates

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: at least in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Background bacterial lawn was evaluated for evidence of cytotoxicity. Dose-ranging studies demonstrated no cytotoxic effects of DCA up to 5 mg per plate using Salmonella strains TA100.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

Sodium dichloroacetate tested negative for genotoxicity with and without metabolic activation under the conditions of this test.

Executive summary:

Sodium dichloroacetate (DCA) pharmaceutical grade was tested for genotoxicity in a bacterial reverse mutation assay in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 with and without metabolic activation (S9).

Dose-ranging studies demonstrated no cytotoxic effects of DCA up to 5 mg per plate using Salmonella strains TA100. The mutagenicity testing itself was done at 0, 0.333, 0.667, 1.00, 3.33, and 5.00 mg DCA or with the positive controls per plate.

In all cases, there was no evidence for a mutagenic effect of DCA. The vehicle-treated and positive control plates gave predicted results.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

In vitro testing:

There have been multiple studies investigating the hypothesis that DCA or Dichloroacetate is a genotoxic agent. Results from in vitro studies indicate that DCA is only genotoxic at high doses or after long durations. Most of the in vitro tests are negative or equivocal, either in the presence or absence of metabolic activation.

A key study for bacterial mutation was the Ames assay reported by Fox et al., 1996 (Endpoints 07.06.01_02 and 07.06.01_03). Dichloroacetate was evaluated respectively in E.Coli strain WP2uvrA and Salmonella strains TA98, 100, 1535, 1537, in the presence and absence of S9 activation. There was no evidence for a mutagenic effect of DCA up to 5000 µg/plate.  

Supporting studies for bacterial mutation were also available:

• DeMarini et al., 1994 (Endpoints 07.06.01_01 and 07.06.01_15) reported that DCA may increase prophage 8 induction in E. coli, however EPA reported that this was not confirmed by other laboratories and required DCA concentrations in the mM range to achieve significant increase. It is possible that the form of DCA affected the study results. The increased revertants may be the result of low pH resulting from the use of the free acid in the vapor phase or differences in the membrane transport of the non-ionized acid.

• Herbert et al., 1980 and EPA 2003 (Endpoints 07.06.01_04 and 07.06.01_05) reported an equivocal increase in revertants in strains TA98 and TA1538 when exposed to 1-10 µg/plate DCA (salt) both in the presence and absence of metabolic activation; all other strains gave negative results. The increase (1.4- to 1.7-fold) did not reach the limit (2- to 3-fold) that most laboratories would typically require for the compound to be identified as mutagenic and the dose response trend reached statistical significance in both the absence and presence of S9.

• Watanabe et al, 1996 (Endpoint 07.06.01_08) reported that DCA was negative with wand without metabolic activation in an interlaboratory study to compare the specific spectrum of response to chemicals among four strains Salmonella typhimurium TA102 and TA2638 and Escherichia coli WP2/pKM101and WP2 uvrA/pKM101.

Finally, following studies were disregarded as they were not particular for DCA:

• Meier et al., 1985 (Endpoint 07.06.01_06) reported 19 chlorinated organic compounds that were identified and quantified in ether extracts of chlorinated humic acid solutions. Ten of these compounds, including chlorinated propanones and chlorinated propenals, were direct- acting mutagens in the Salmonella/microsome mutagenicity assay. DCA was reported to be not mutagenic.

• Waskell, 1978 (Endpoint 07.06.01_7) examined DCA as a metabolite of methyxoflurane in Salmonella strains TA98, TA100, TS24, TA2322, TA1950 in the presence and absence of S9 activation. Weak mutagenesis was found, however after recrystallization mutagenic activity could no longer be detected.

• Voogd et al, 1972 (Endpoint 07.06.01_13) studied mutagenic properties of Dichlorvos. The results of a few tests with some related products, e.g. dichloroacetic acid were also given. DCA tested negative in this test.

• Ono et al, 1991 (Endpoint 07.06.01_16) studied the genotoxicities of 37 commercial chemicals in the umu-test. DCA tested positive in this test, with and without S9 activation

 

A key study for gene mutation was the Mouse lymphoma assay in L5178Y cells reported by Fox et al. 1996 (Endpoint 07.06.01_11). At all doses studied (125-5000 µg/mL), Dichloroacetate tested negative for mutagenicity with and without metabolic activation, while concurrent positive and negative controls gave predicted results in all assays. There was no evidence for cytotoxicity up to 5000 µg/mL. Supporting studies for gene mutation or DNA strand breaks/repair were also available:

• Fassio et al, 1994 (Endpoint 07.06.01_09) reported that DCA did nor induce any significant increase of gene mutation (HGPRT- frequency in V79 Chinese hamster lung cells) up to the dosage level of 600 and 1000 µg/mL, both in the presence and the absence of hepatic microsomal enzymes, respectively in two independent experiments. 

• Harrington-Brock et al. 1998 (Endpoint 07.06.01_10) reported that DCA induces mutations at the thymidine kinase locus, as well as gross chromosomal aberrations in L5178Y mouse lymphoma cells in vitro, but the concentrations required to induce these effects were in the mM range.

• Waskell, 1978 (Endpoint 07.06.01_12) reported DNA repair was negative in the absence of S9 activation on the normal hisG and DNA-repair deficient bacteria, TS24, TA2322, TA1950. 

A key study for in vitro Chromosomal aberration was the study in Chinese hamster ovary cells reported by Fox et al. 1996 and EPA 2003 (Endpoint 07.06.01_14). Sodium Dichloroacetate pharmaceutical grade was tested for chromosomal aberrations in Chinese hamster ovary cells at concentrations of 500, 1250, 2500, and 3750 µg DCA /mL. Neither the 10.0-hr nor the 20.0-hr harvests demonstrated any dose-related trends or other evidence of a mutagenic potential for DCA, with and without S9 activation.

In vivo testing:

Based on the equivocal findings in the in vitro mutagenicity studies and positive findings in the carcinogenicity studies, in vivo genotoxicity studies were performed to confirm or exclude a genotoxic carcinogenic potential.

A key in vivo micronucleus assay was performed by Fox et al. 1996 (Endpoint 07.06.02_01), in which Sprague-Dawley rats intravenously dosed at 275, 550, and 1100 mg/kg Sodium Dichloroacetate for 3 days did not show increased number of micronuclei of bone marrow cells.Supporting studies for micronucleus formation were also available:

•   Fuscoe et al, 1996 (Endpoints 07.06.02_02 and 07.06.02_03) showed a small but statistically significant increase in micronucleated polychromatic erythrocytes in mice after exposure via the drinking water to DCA at 3.5 g/L (corresponding with 665 mg/kg bw day) for 9 and 28 days, respectively. Coadministration of the antioxidant vitamin E did not affect the ability of DCA to induce this damage, indicating that the small induction of MN by DCA was probably not due to oxidative damage.

•   Fuscoe et al, 1996 (Endpoint 07.06.02_04) also administered DCA for 10, 26 and 31 weeks via the drinking water at 3.5 g/L (corresponding with 665 mg/kg bw day). At each time point, slight but significant increases in normochromic erythrocytes were observed while micronucleated polychromatic erythrocytes slightly increased in a dose-dependent manner, but did not reach statistical significance.

A key study to exclude the genotoxic mechanism for carcinogenicity was performed by Leavitt et al. 1997 (Endpoint 07.06.02_09), who exposed transgenic Big Blue mice to 1 or 3.5 g/L DCA (approximate doses of 190 or 665 mg/kg-day) in their drinking water for 60 weeks. Neither concentration of DCA induced an increased frequency of mutations in the Lac I loci after 4 and 40 weeks, however, at 60 weeks, both concentrations of DCA induced a significantly elevated. Increased mutation frequencies at the 1 and 3.5 g/l concentrations were 1.3 and 2.3 versus control, respectively. This time-response pattern suggests that the mutational events might be secondary to toxicological changes in the liver rather than a direct genotoxic effect, since a direct effect would be expected to be time-independent. The results indicate that a large cumulative dose (due to the 60-week exposure period) is necessary to increase mutations in this in vivo system. Following supporting (mechanistic) studies were done to clear out the carcinogenic mechanism:

• Sanchez & Bull, 1990 (Endpoint 07.06.02_05) administered DCA at concentrations of 0, 300, 1000 or 2000 mg/L in the drinking water to male B6C3F1 and male and female Swiss-Webster mice for up to 14 days. Marked increase in liver weights after 14 days of treatment and local necrosis in both B6C3F1 and Swiss-Webster mice were observed, as well as a significant increase in the labeling index of hepatocytes. Significant increases in [3H] thymidine were observed in the livers of DCA- and TCA- treated animals after 5 days of treatment. These data support the hypothesis that the tumorigenic effect of DCA is strongly influenced by necrosis and reparative hyperplasia.

• Nelson & Bull, 1988 (Endpoints 07.06.02_06 and 07.06.02_07) showed that metabolites of TCE (including DCA) induced strand breaks in hepatic DNA in a dose-dependent manner in rats and mice, respectively. These were observed at doses that produced no observable hepatotoxic effects as measured by serum aspartate aminotransferase and alanine aminotransferase levels.

• Nelson et al, 1989 (Endpoint 07.06.02_08) showed significantly increased strand breaks in DNA at 1, 2, and 4 h post-treatment single DCA administration in male mice, however no evidence for an increase in peroxisomal β-oxidation was produced up to 24 h after administration. In a separate experiment, mice were treated with DCA or TCA for 10 days, showing an increase in liver weight and increased peroxisomal β-oxidation in liver homogenates. Peroxisomes induced by DCA treatment frequently lacked nucleoid cores (lacking peroxisomal enzymes). Repeated doses of DCA producef multifocal, subcapsular necrotic regions, and a marked hypertrophic response in the liver. This indicates that peroxisomal proliferation does not contribute to the induction of DNA strand breaks.

Justification for selection of genetic toxicity endpoint

Key study for bacterial reverse mutation, but also cross-reference for gene mutation and chromosomal aberration in vitro and genetic toxicity (micronucleus assay) in vivo.

Justification for classification or non-classification

Based on the results and according to the EC criteria for classification and labelling requirements for dangerous substances and preparations (Guidelines in Commission Directive 93/21/EEC) and CLP regulation (EC No. 1272/2008 of 16 December 2008), dichloroacetic acid is not classified and has no obligatory labelling requirement for genotoxicity.