− −3.7.2C mouse lymphoma cells

Mutation Research 413 Ž1998. 265–276

Mutagenicity of three disinfection by-products: di- and trichloroacetic acid and chloral hydrate in L5178YrTKqry y3.7.2C mouse lymphoma cells Karen Harrington-Brock ) , Carolyn L. Doerr, Martha M. Moore EnÕironmental Carcinogenesis DiÕision, National Health and EnÕironmental Effects Research Laboratory, U.S. EnÕironmental Protection Agency, Research Triangle Park, NC 27709, USA Received 16 July 1997; revised 20 January 1998; accepted 22 January 1998

Abstract The disinfection of water, required to make it safe for human consumption, leads to the presence of halogenated organic compounds. Three of these carcinogenic ‘disinfection by-products’, dichloroacetic acid ŽDCA., trichloroacetic acid ŽTCA. and chloral hydrate ŽCH. have been widely evaluated for their potential toxicity. The mechanismŽs. by which they exert their activity and the steps in the etiology of the cancers that they induce are important pieces of information that are required to develop valid biologically-based quantitative models for risk assessment. Determining whether these chemicals induce tumors by genotoxic or nongenotoxic mechanisms Žor a combination of both. is key to this evaluation. We evaluated these three chemicals for their potential to induce micronuclei and aberrations as well as mutations in L5178YrTKqry y3.7.2C mouse lymphoma cells. TCA was mutagenic Žonly with S9 activation. and is one of the least potent mutagens that we have evaluated. Likewise, CH was a very weak mutagen. DCA was weakly mutagenic, with a potency Žno. of induced mutantsrm g of chemical. similar to Žbut less than. ethylmethanesulfonate ŽEMS., a classic mutagen. When our information is combined with that from other studies, it seems reasonable to postulate that mutational events are involved in the etiology of the observed mouse liver tumors induced by DCA at drinking water doses of 0.5 to 3.5 grl, and perhaps chloral hydrate at a drinking water dose of 1 grl. The weight-of-evidence for TCA suggest that it is less likely to be a mutagenic carcinogen. However, given the fact that DCA is a weak mutagen in the present and all of the published studies, it seems unlikely that it would be mutagenic Žor possibly carcinogenic. at the levels seen in finished drinking water. q 1998 Elsevier Science B.V. Keywords: Chlorination by-product; Chromosome aberration; Gene mutation; Mouse lymphoma cell; Mutagen in drinking water; DCA; TCA; CH; Thymidine kinase locus

1. Introduction

) Corresponding author. Genetic and Cellular Toxicology Branch, Environmental Carcinogenesis Division, MD-68, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711, USA. Tel.: q1-919-541-3933; fax: q1-919-541-0694.

Drinking water treatment is necessary to remove harmful impurities including pathogenic microorganisms and hazardous chemicals. Chemical oxidation and disinfection are two of the widely used treatment processes that use chlorine as an oxidant and a

1383-5718r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 8 . 0 0 0 2 6 - 6

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primary chemical disinfectant, respectively. Chloroacetic acids and chloroacetaldehydes are formed when the chlorine reacts with humic acids and other natural organic compounds in surface water w1–3x. The production of disinfection by-products and the demonstrated mutagenic and carcinogenic activities in microbial and animal systems have prompted concern for the health of exposed human populations. This study examined the mutagenicity in mammalian cells of two haloacetic acids; di- and trichloroacetic acid, and one chlorinated aldehyde, chloral hydrate. In addition to their status as disinfection by-products, all three compounds are metabolites of trichloroethylene ŽTCE. w4–7x, a known rodent carcinogen and frequent water contaminant w8– 11x. All three compounds are rodent carcinogens when given in drinking water at relatively high Ž0.5 to 3.5 grl. concentrations: dichloroacetic acid ŽDCA. has been demonstrated to be a hepatocellular carcinogen for both the male F344 rat and the B6C3F1 mouse w12 – 14 x. Trichloroacetic acid Ž TCA . w13,15,16x and chloral hydrate ŽCH. w17,18x induced hepatocarcinogenicity in male B6C3F1 mice. The genotoxicity results for DCA and TCA in a variety of in vivo and in vitro systems have been less conclusive than the carcinogenicity data. Both generally have been regarded as nongenotoxic due to either negative, weak or seemingly conflicting responses w19–25x. However, more recent studies have provided data indicating that DCA, but not TCA, has the potential to induce genotoxic damage. DCA, but not TCA, was reported to be weakly mutagenic with and without exogenous activation in strain TA 100 in the Ames Salmonella assay, to induce lambda prophage in Escherichia coli w26x and to produce a low frequency of micronuclei in mouse bone marrow cells w27,28x. DCA-treated animals showed a mutational spectrum at codon 61 in the H-ras gene of mouse liver tumors that was different from the background spectra w29,30x. The mutational spectra of the H-ras gene of mouse liver tumors from animals treated with TCA appeared to be the same as that found in the spontaneously occurring tumors w29x. However, the number of tumors analyzed from the TCA-treated animals was very small. Historically, the major evidence that TCA might be genotoxic was reported by Bhunya and Behera w31x, who found

TCA positive in three cytogenetic in vivo assays Žbone marrow chromosomal aberrations, micronucleus and sperm head abnormality test.. More recent results cast doubt on these data and suggest that TCA is not genotoxic w28x. The genotoxicity of CH has been demonstrated in a variety of test systems, as recently reviewed by Salmon et al. w32x. Its aneugenic properties have been shown in yeast w33x, Aspergillus w34,35x, Chinese hamster cells w36x, mouse spermatogonia w37,38x and human lymphocytes w39x. It is mutagenic in Salmonella TA 100 w40,41x and fungi w34,42x, positive for spermatid micronucleus induction in the mouse w43x and has been reported to induce sister chromatid exchanges in human lymphocytes w44x. Since the potential for DCA, TCA or CH to induce DNA damage has not been clearly evaluated in mammalian cells, we have undertaken this study. The mouse lymphoma assay was selected because of its ability to detect a broad spectrum of genetic damage, a characteristic which makes it the recommended in vitro mammalian gene mutation assay for EPA regulatory submissions w45x.

2. Materials and methods 2.1. Chemicals DCA ŽCAS no. 79-43-6., TCA ŽCAS no. 76-03-9., TCA, sodium salt ŽCAS no. 650-51-1., and methylmethanesulfonate ŽMMS. ŽCAS no. 66-27-3. were obtained from Aldrich Chemical ŽMilwaukee, WI.. CH ŽCAS no. 302-17-0., pluronic, bromodeoxyuridine ŽBrdUrd., cytochalasin B ŽCYB., and trifluorothymidine ŽTFT. were purchased from Sigma ŽSt. Louis, MO.. DCA and TCA were dissolved in cell culture medium and CH and MMS were dissolved in saline. Colcemid, Fischer’s Medium for Leukemic Cells of Mice, horse serum, penicillin–streptomycin, sodium bicarbonate and sodium pyruvate were purchased from GIBCO ŽGrand Island, NY.. The BBL agar used for cloning was obtained from Becton Dickinson ŽCockeysville, MD. and the S9 was obtained from Sitek Research Laboratories ŽRockville, MD.. The stains used and their sources were: acridine orange, Polysciences ŽWarrington, PA.; Gurr Giemsa Improved R66, Biomedical Specialties ŽSanta

K. Harrington-Brock et al.r Mutation Research 413 (1998) 265–276

Monica, CA., and Hoechst no. 33258, Cal Biochem.-Behring ŽSan Diego, CA.. All chloroacetic acid stocks were prepared from the free acid form except when TCA was evaluated with S9, then the sodium salt form was used to maintain neutral pH levels. For DCA and TCA, the pH of the culture medium was measured at the beginning and end of the treatment period for selected dosed points. 2.2. Cell culture and treatment The TKqry y3.7.2C heterozygote of the L5178Y mouse lymphoma cell line was used. Cells were maintained in culture using Fischer’s Medium for Leukemic Cells of Mice supplemented with 10% horse serum, sodium pyruvate, pluronic, penicillin,

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and streptomycin according to the procedures described by Turner et al. w46x. Cells were maintained in logarithmic growth. For treatment, cells were centrifuged and suspended at a concentration of 0.6 = 10 6 cellsrml in culture medium supplemented with 3% horse serum. 6 = 10 6 cells Žin 10 ml of medium. were placed in 50 ml polystyrene tubes, test chemicals were added, tubes were gassed with 5% CO 2 in air and sealed for the duration of the treatment. In lieu of treating replicate cultures, this laboratory performs multiple independent experiments. The cell culture tubes were placed on a roller drum and incubated at 378C. At the end of a 4 h treatment period, the cell cultures were centrifuged, washed twice with fresh medium, resuspended in fresh medium, and placed on a roller drum in a 378C

Fig. 1. Cytotoxic and mutagenic effects of DCA, TCA ŽqS9. and CH in L5178YrTKqry mouse lymphoma cells. ŽTop panels. Cytotoxicity, Žbottom panels. total Tk mutant frequency ŽB. and small- Ž'. and large- Žv . colony TK mutant frequencies after cells were treated for 4 h with either DCA, TCA ŽqS9. or CH. Results are taken from one representative experiment.

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incubator. Cells were maintained in log-phase growth for a 2-day expression period and then cloned in medium containing 0.22% BBL agar with TFT for selection Ž1 m grml. and without TFT for determination of viability. After 10–13 days incubation at 378C, colonies were counted and the colony-size distribution determined using an Artek model 880 automatic colony counter modified with a 10-turn potentiometer w47x. Relative survival values were calculated according to the method described by Clive and Spector w48x and include both a measure of the growth in the suspension and cloning phases of the assay. 2.3. Cytogenetic analysis Following cell treatment, 10 m M BrdUrd was added to those cultures to be used for chromosome aberration analysis. The cells were incubated for 14 to 15 h with colcemid Ž0.1 m grml. present for the last 2 h. Slides were prepared and stained using the fluorescence-plus-Giemsa method w49,50x and coded. For each concentration, 100 metaphase spreads Ž46 " 2. were analyzed for aberrations. Aberrations were classified as chromatid breaks, deletions, and fragments Žgrouped as chromatid type breaks.; triradials, quadriradials, and complex rearrangements Žchromatid type rearrangements.; chromosome breaks, deletions, fragments, and minutes Žchromosome type breaks.; and dicentrics, rings and translocations Žchromosome type rearrangements.. Chromatid and chromosome gaps were recorded but not included as aberrations. Any metaphase with a chromosome count greater than or less than 46 but within the metaphase selection criteria Ž46 " 2. was scored as aneuploid. For the micronucleus analysis, cultures were treated with 3.0 m grml CYB w51,52x and harvested 12 or 13 h after the addition of CYB. This time was

selected to be as close to the harvest time for chromosome aberration analysis as technically feasible. Slides were made from cultures corresponding to those used for the aberration analysis. One thousand binucleated cells were scored for each treatment. Only cells containing two separate, well-defined nuclei totally surrounded by cytoplasm were analyzed w53x. 3. Results DCA was a weak direct-acting mutagen in mouse lymphoma cells. In Fig. 1, the survival curves and the Tk mutant frequency curves Žincluding the small and large colony frequencies. from one representative experiment are shown. For DCA, a dose-related cytotoxic and mutagenic ŽFig. 1. effect was observed at concentrations between 100 and 800 m grml. These results were duplicated in a second experiment ŽTable 1.. Colony sizing analysis was performed at selected concentrations across the dose range and small- and large-colony mutant frequencies were calculated. Most of the mutagenic activity of DCA at the Tk locus was due to the production of small-colony Tk mutants ŽFig. 1 and Table 1.. The results of the cytogenetic analysis performed on cultures treated with concentrations of 0, 600, and 800 m grml of DCA are shown in Table 2. A clearly positive induction of aberrations was observed at b o th c o n c e n tr a tio n s Ž b a c k g r o u n d s 8 aberrationsr100 cells, 600 m grml s 22 aberrationsr 100 cells and 800 m gr m l s 26 aberrationsr100 cells.. There was no significant increase in MN or aneuploidy frequencies. At 600 m grml we observed a doubling of the MN frequency over the negative control Žbackgrounds 5 and 600 m grml s 11., but this frequency does not represent a doubling of the historic mean for all

Notes to Table 1: pH measurements taken at the beginning of treatment. b Plating efficiency is based on plating 600 cells. c Mutant count is the total number of mutants per 3,000,000 cells plated in TFT selection. d Mutant frequency is expressed as per 10 6 surviving cells. e Percent survival is calculated according to the method of Clive and Spector Ž1975. w48x. f Total mutant frequency divided into smallrlarge ŽSrL. colony mutant frequencies. g EMS or BŽ a.P were used as positive controls. h Colony sizing was only performed on selected cultures. a

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Table 1 Mutagenicity of dichloroacetic acid, trichloroacetic acid and chloral hydrate Concentration Ž m grml. wpHxa Dichloroacetic acid, experiment 1 0 w7.2x 100 150 w7.2x 600 w6.6x 800 w6.1x 1000 w6.1x MMS g 13 Dichloroacetic acid, experiment 2 0 w7.2x 200 300 400 w6.8x 500 w6.7x 600 w6.4x 700 w6.3x 800 w6.3x 900 MMS g 13 Trichloroacetic acid, experiment 1 0 1500 1975 2000 2025 2050 2100 2125 2150 MMS g 13 Trichloroacetic acid, experiment 2 0 w7.1x 750 w6.8x 1000 w6.6x 1500 1900 1950 w6.5x 1975 w6.5x MMS g 13

Percent plating efficiency b

Total mutant count c

Mutant frequency Ž=10y6 . d

Percent survivale

Mutant frequency ŽSrL. f

92 99 75 83 58 35

237 289 337 968 1309 1631

86 97 150 389 752 1553

100 100 96 42 28 5

60r26 h 116r34 303r86 620r132 h

33

803

811

22

669r142

90 92 97 87 72 72 66 31 28

270 431 591 925 968 1294 1494 1512 1609

100 156 203 354 448 599 755 1626 1915

100 97 98 53 46 38 27 7 5

67r33 h 156r47 283r71 358r90 495r104 651r104 h h

23

721

1053

15

841r212

97 85 55 41 59 47 37 43 44

209 206 216 238 214 261 187 223 322

72 81 131 193 121 185 168 173 244

100 47 h 13 11 14 10 3 8 7

12

410

1139

4

59 63 65 71 33 41 49

188 136 129 207 169 208 206

106 73 66 97 171 170 140

100 90 100 79 11 10 6

62r44 h 40r26 51r46 h 137r33 h

16

232

483

13

374r109

192 311 302 294

70 105 109 118

100 54 28 19

Trichloroacetic acid qS9, experiment 1 0 92 2000 99 2500 92 2750 83

25r47 h h 106r87 h 93r92 h h 152r92 861r278

43r27 57r48 69r40 81r37

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Table 1 Žcontinued. Concentration Ž m grml. wpHxa

Percent plating efficiency b

Trichloroacetic acid q S9, experiment 1 3000 83 3250 87 BŽ a.P g 3 79 4 57

Chloral hydrate, experiment 2 0 600 1000 1100 1200 1250 1300 1350 1400 1450 1500 1550 1600 MMS g 15

Mutant frequency Ž=10y6 . d

Percent survivale

Mutant frequency ŽSrL. f

485 431

195 165

13 14

128r67 108r57

953 1144

402 669

51 11

295r107 537r132

183 220 269 290 408 478 555 350 459 610

71 91 104 101 155 202 253 172 232 303

100 71 56 51 28 15 8 18 8 8

46r25 h h h 97r58 116r86 h 106r66 h h

1045 1283

447 737

49 15

h 599r138

63 59 42 44 31 38 35 25

130 107 186 163 140 216 230 327

69 60 148 123 151 189 219 436

100 96 53 42 23 28 19 7

h h h h h h h h

15

228

507

15

h

96 73 62 40 55 46 35 36 31 42 46 49 26

278 303 403 380 513 529 445 485 464 512 545 479 675

97 138 217 317 311 383 424 449 502 406 395 326 865

100 43 21 12 18 11 7 6 5 8 9 7 4

73r24 94r44 170r47 259r58 269r42 328r55 h h h h h h h

30

787

874

12

758r116

Trichloroacetic acid qS9, experiment 2 0 86 1500 81 1750 86 2000 96 2250 88 2500 79 2600 73 2800 68 3000 66 3400 67 BŽ a.P g 3 78 4 58 Chloral hydrate, experiment 1 0 350 600 900 950 1000 1250 1500 MMS g 13

Total mutant count c

0 4 3 1 5 7 0 1250 1300

CH

AB, aberrations; total aberrations per 100 cells scored; b MN, micronuclei; total micronuclei per 1000 cells scored; c MF, mutant frequency; total TK mutant frequency for concurrently treated culture; d Survival calculated according to the method of Clive and Spector Ž1975..

4 5 5 85 383 424 3 4 5 3 4 5 5 10 10 6 14 13 1 2 1 4 3 2

6 2 4 0 5 21 1 15 1 DCA 0 600 800

a

100 11 7

100 42 28 8 8 12 86 397 761 5 11 8 5 11 8 3 19 17 8 22 26 1 0 0

Cells wrAB Total number of AB a Chromosome Break Rearrang. Chromatid Break Rearrang. Compound Ždose. Ž m grml.

Table 2 Clastogenic effects in L5178Y cells after treatment

Total MN b

Cells wrMN

Total MF=10y6c

% Aneuploidy

Survival Ž%. d

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negative controls, which we use as an additional criterion for reporting a positive response w53x. Results of the evaluation of TCA with and without metabolic activation are also summarized in Table 1. When TCA was tested without activation, some concentrations caused a doubling of the background mutant frequency, while in other cultures at similar or higher concentrations, it induced mutant frequencies that were less than twice the background. Thus, without activation, TCA was equivocal. The addition of S9 induced a slight increase in the mutant frequency at concentrations yielding greater than 10% survival. Both small and large colony mutants were induced ŽFig. 1.. This was repeated in a separate experiment, which confirmed the very weak positive response ŽTable 1.. Cytogenetic analysis was not performed on TCA-treated cells due to the weak mutagenic response. CH Žwithout activation. induced concentration-related cytotoxicity and a very weak mutagenic response. Like DCA, most of the mutagenic activity of CH was due to the induction of small colonies ŽFig. 1 and Table 1.. Analysis of CH-treated cells for chromosome aberrations indicated that CH was weakly clastogenic in mammalian cells Žbackground s 6 aberrationsr100 metaphases and 1250 m grml s 14 aberrationsr100 metaphases. ŽTable 2.. There was no increase in the MN ŽTable 2..

4. Discussion Based on limited genotoxic studies, DCA and TCA have generally been regarded as nongenotoxic compounds w21,22,25x. Several nongenotoxic mechanisms such as increased peroxisome proliferation and inappropriate apoptosis have been investigated in an attempt to define the ‘nongenotoxic’ nature of the carcinogenic activity w54,55x. When the present study was initiated, questions still remained concerning the potential genotoxicity of DCA and TCA. During the course of this investigation, Mackay et al. w28x reported that TCA was negative in the mouse bone marrow micronucleus test and cited pH effects as a possible reason for an earlier positive response reported by Bhunya and Behera w31x. It is recognized that in in vitro mammalian assays, positive clastogenic effects and gene

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mutations have been induced by low pH, particularly in the presence of S9 w56,57x. Brusick w57x reported that acetic acid ŽCH 3 COOH. and hydrochloric acid ŽHCl. induced significant increases in chromosome aberrations in Chinese hamster ovary cells when added to the treatment medium in the presence of S9. When acetic acid was used to lower the pH of the treatment medium the following results were observed: pH 5.76 resulted in 4.0% of the cells with aberrations, pH 5.5 resulted in 67% of the cells with aberrations and pH 5.28 resulted in no viable cells. The HCl treatment at pH 5.75 resulted in 2.0% of the cells with aberrations, pH of 5.50 resulted in 0.0% of the cells with aberrations and pH 5.25 resulted in 60% of the cells with aberrations. Significant increases in chromosome aberrations were not observed at these pHs in the nonactivation trials. Cifone et al. w56x treated mouse lymphoma cells

with HCl to evaluate the effect of pH shifts on the mutant frequency at the Tk locus. In the presence of S9, treatment in the pH range of 6.5–6.0 resulted in an increase in the mutant frequency. Relative to the mutant frequency of the control ŽpH 7.0., a 2.6-fold increase in mutant frequency was observed at pH 6.5 and a 8.7-fold increase was observed at pH 6.0. At pHs 6.9 and 6.7, the mutant frequencies were at background responses Žbackgrounds 43, pH 6.9 s 57, pH 6.7 s 60.. The results observed under nonactivation treatment conditions indicate that a borderline increase was observed only at pH 6.3 Ž1.9-fold increase. and treatments below pH 6.3 were lethal w56x. To evaluate any potential pH effects in the present studies, we monitored the pH of the culture medium in two separate DCA experiments Žsee Table 1.. DCA concentrations of 400, 500, and 600 m grml

Fig. 2. Mutant colony size distribution for DCA and MMS, the positive control. RTGs relative total growth; MF s mutant frequency. The numbers above the two-headed arrow are the small and large colony mutant frequencies, respectively.

K. Harrington-Brock et al.r Mutation Research 413 (1998) 265–276

resulted in pH values of 6.8, 6.7, and 6.4, respectively. These DCA concentrations induced a clearly positive response for chromosome aberrations and gene mutations at pH values that have previously been reported to be negative under nonactivation treatment conditions. Thus, based on the observed pH values, the mutagenic and cytogenetic effects observed in these experiments appear to have been induced by the chemical, rather than pH effects. These studies indicate that DCA is capable of inducing a dose-dependent increase in mutant frequency and causes chromosomal breakage. Representative histograms of colony sizings for DCA and MMS Žour positive control. are shown in Fig. 2. This Figure illustrates the strikingly similar pattern of small- and large-colony Tk mutants induced by both

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DCA and MMS, a well known clastogen. Although the magnitude of the induced mutant frequency is approximately the same with DCA and MMS, the mutagenic potency of DCA is much lower. That is, MMS induces this mutant frequency at 13 m grml Ž118 m M. while DCA requires 800 m grml Ž6204 m M. to give approximately the same response. Treatment of the cell cultures with TCA at concentrations of 1950 and 1975 m grml both resulted in pH values of 6.5. When TCA was evaluated with S9 activation the salt form of the acid was used and no pH change was detected. Our results demonstrate that the genotoxic effects of DCA and TCA are measurably different. TCA was considerably less cytotoxic than DCA, requiring concentrations of 1950 and 2050 m grml Žin two

Fig. 3. Concentration–response curves comparing the mutagenic potency of DCA, TCA ŽqS9. and CH to that of EMS at the tk locus of mouse lymphoma cells. The cytotoxicity is shown in the top panel and the Tk mutant frequency in the bottom panel.

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separate experiments. to produce a relative total growth ŽRTG. of 10%, whereas 800 m grml of DCA resulted in 7% RTG. DCA was a direct-acting mutagen at the Tk locus; TCA was equivocal without activation and only weakly positive with exogenous activation. Just as the mechanism for inducing hepatocellular carcinoma does not appear to be the same for DCA and TCA w13x, we conclude from our investigations that the genotoxic potentials of DCA and TCA are also not the same. Our data for CH were consistent with previous published results showing CH to be genotoxic. The response was, however, quite weak. We concluded that CH is cytotoxic, mutagenic and clastogenic to mammalian cells in vitro. Interestingly, in our studies, CH was not positive for micronucleus induction and did not induce aneuploidy. It is not clear why CH Ža known aneuploidy inducing agent. would not induce micronuclei in mouse lymphoma cells. To put the mutagenic potency of these three chemicals into perspective, TCA Žwith S9 activation. was one of the least potent compounds that we have tested in the assay. Chloral hydrate was also a very weak mutagen. DCA was the most potent of the three and is similar in potency to EMS, a standard mutagen. Fig. 3 shows the relative mutagenicity of these three compounds compared to EMS. Our demonstration that DCA has the potential to be mutagenic combined with the in vivo micronucleus data of Fuscoe et al. w27x and a recent study using the Big Blue Mouse w58x supports the hypothesis that mutation induction is involved in the etiology of the observed DCA-induced mouse liver tumors. The weight-of-evidence indicates that it is unlikely that mutation induction is involved in the etiology of TCA tumor induction in mice. The mechanismŽs. for chloral hydrate induced tumors are less clear. It is quite weak as an in vitro mutagen. Although these studies demonstrate that all three compounds are mutagenic in mammalian cells in vitro and, therefore, have the potential to induce DNA mutation in somatic cells in vivo, it is reasonable to postulate that DCA would have the greatest potential as an in vivo mutagen. However, in the context of evaluating whether DCA might induce tumors by a mutational mechanism in humans exposed to DCA in their drinking water, the weak

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