Dichloroacetic acid

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

publication

Title:

Assessment of the mutagenicity of dichloroacetic acid in lacI transgenic B6C3F1 mouse liver

Author:

Leavitt SA, DeAngelo AB, George MH and Ross JA.

Year:

1997

Bibliographic source:

Carcinogenesis vol.18 no.11 pp.2101–2106, 1997

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Cromosomal aberration on mouse liver.

GLP compliance:

not specified

Type of assay:

other: Big Blue® transgenic mouse mutagenesis assay

Test material

Test material information

Constituent 1

Reference substance name:

Dichloroacetic acid

EC Number:

201-207-0

EC Name:

Dichloroacetic acid

Cas Number:

79-43-6

Molecular formula:

C2H2Cl2O2

IUPAC Name:

2,2-dichloroacetic acid

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): DCA
- Other: Purchased from Aldrich Chemical Co., Milwaukee, WI

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Stratagene (La Jolla, CA).
- Age at study initiation: 8–9 weeks at time of dosing

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

- Vehicle(s)/solvent(s) used: deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
DCA (CAS no. 79-43-6; Aldrich Chemical Co., Milwaukee, WI) was administered ad libitum in deionized water at doses of either 3.5 or 1.0 g/L for 4, 10 or 60 weeks. The pH was adjusted to 6.8–7.2 by addition of sodium hydroxide. Control mice were given deionized water ad libitum.

Duration of treatment / exposure:

4, 10 or 60 weeks

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
3.5 or 1.0 g/L
Basis:
nominal in water

No. of animals per sex per dose:

Five or six animals were assigned randomly to each treatment group.

Control animals:

yes, concurrent vehicle

Examinations

Tissues and cell types examined:

At each time point animals were killed by asphyxiation with CO2 and cervical dislocation. Livers were removed, quick frozen in liquid nitrogen and stored at –80°C until DNA isolation. Whole livers were homogenized in buffer (1.75 g/L Na2PO4, 8.0 g/L NaCl, 0.2 g/L KH2PO4, 10 mM Na2EDTA, pH 8.0) and stored in aliquots proportional to 100 mg tissue at –80°C. Only visually normal tissue was used in this analysis. Genomic DNA was isolated from homogenized liver aliquots by digestion with proteinase K, followed by phenol/chloroform extraction and ethanol precipitation.

Statistics:

Analyses of the statistical significance of increases in mutant frequency were performed using the generalized Cochran–Armitage test using the COCHARM program written by Troy D. Johnson. Comparison of mutation spectra was performed using the hypergeometric test as implemented in the program HG-PUBL .

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Any other information on results incl. tables

A 2.34-fold increase in mutant frequency was observed in the liver of mice given 3.5 g/L DCA for 60 weeks. The mice receiving 1.0 g/L DCA for the same length of time showed a 1.33-fold increase in mutant frequency relative to the control value. There was no significant increase in mutant frequency relative to the control values for those mice treated for 4 or 10 weeks at either dose level. However, the mean mutant frequency for the control group at 10 weeks was relatively high, largely due to one animal that displayed a mutant frequency of 8.08 x 10^-5. The increases in mutant frequency over time following treatment with DCA at 3.5 g/L exhibit a highly significant trend overall (P= 0.001), with both 10 and 60 week mutant frequencies significantly increased relative to the frequency at 4 weeks (P =0.04 and 0.002 respectively).

The mice treated with 3.5 g/L DCA for 60 weeks was selected for sequence analysis because it showed the highest induced mutant frequency, thereby minimizing the presence of background mutants in the DCA-induced spectrum. The proportions of specific mutation types recovered are significantly different (P= 0.05, hypergeometric test) for control and DCA-treated mice. When the mutation frequency is adjusted for possible ‘clonal expansion’ by subtracting duplicate identical mutations recovered from the same animals, the proportions of mutation classes recovered from control and DCA-exposed mice are still significantly different (P= 0.03, hypergeometric test).

Single base substitutions comprised 78.71% of the background mutations, with 59.57% being transitions and 19.14% being transversions. Seven of the base substitutions resulted in stop codons. The mutation spectrum recovered from control mice had a high frequency of G:C→A:T transitions (53.19%), with about half of these mutations occurring at CpG sites. It has been reported that CpG dinucleotides are preferentially involved in background mutations in this system. Although most mutations recovered from the controls were base substitutions, five frameshifts of either +1 or –1 base were noted. Three deletions of 6, 11 and 300 bases and one complex mutation were also recovered.

In DCA-treated mice, single base substitutions comprised 88.52% of the mutations, with 47.54% being transitions and 40.98% being transversions. Of the 20 G:C→A:T transitions,eight were at CpG sites, implying that these particular mutations were not induced preferentially by DCA treatment. Nine of the base substitutions resulted in the original amino acid being changed to a stop codon.

In contrast to the mutation spectrum obtained from control animals, there was a high frequency of mutations at T:A sites in the DCA-treated animals. The controls had 19.15% of total mutants at T:A sites, whereas the treated had 32.79% of total mutations at these sites. The A:T→T:A transversion at base 1004 is the only mutation of this class occurring in both the control and treated mutation spectra. Other classes of mutations that were recovered from DCA-exposed mice included five frameshifts of –1 or +1 base, a 3 base deletion resulting in

the loss of a tyrosine and a large deletion of 143 bases.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Neither concentrations of DCA induced an increased frequency of mutations in the Lac I loci after 4 and 10 weeks, however after 60 weeks both concentrations of DCA induced a significantly elevated mutational frequency at the Lac I loci. 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.

Executive summary:

In this study, transgenic mice (Big Blue) were exposed to 1 or 3.5 g/L DCA (approximate doses of 190 or 665 mg/kg-day) in their drinking water for 60 weeks. At interim time points (4 and 10 weeks), neither concentration of DCA induced an increased frequency of mutations in the Lac I loci. Neither concentrations of DCA induced an increased frequency of muations in the Lac I loci after 4 and 10 weeks, however at 60 weeks both concentrations of DCA induced a significantly elevated mutational frequency at this loci. Increased mutation frequencies at the 1 and 3.5 g/L concentations 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.