Small-colony Mutants Research Articles

Molecular analysis of mutations at the tk locus of L5178Y mouse-lymphoma cells induced by ethyl methanesulphonate and mitomycin C

Mutations at the tk locus of mouse-lymphoma L5178Y cells were induced by treatment with ethyl methanesulphonate (EMS) primarily a point mutagen and mitomycin C (MMC), a potent clastogen. Mutant colony size was distinctly bimodal with 35% of spontaneous mutants growing as small colonies and 65% large. The proportion of small colonies increased only slightly to 41% in EMS-treated cultures but to 64% after MMC treatment. Mutations were analysed by Southern and Northern blotting. Digestion of DNA with the restriction enzyme, Nco I, revealed that many mutants had lost a 6.3-kb fragment which constituted the loss of the entire tk gene. Almost all of the EMS-induced large-colony mutants analysed ( 9 10 ) retained the tk + allele suggesting the presence of an intragenic mutation. Of the small-colony mutants, half ( 6 12 ) had a lost the tk + gene and presumably other genes affecting growth and half retained the tk + allele suggesting point mutations in both the tk gene and other sites in the genome affecting growth. A very different spectrum of mutation was induced with MMC. Only 1 12 of the large-colony mutants were due to intragenic mutation, the remaining large-colony mutants having lost the tk + allele while all the small-colony mutants had lost the tk + gene presumably with the deletion extending to genes essential for normal growth. Northern blot analysis showed no changes in the size of tk transcript in any mutants. Alterations in the amount of tk mRNA were not detectable since all mutants produced an mRNA of similar size and amount, which may indicate the production of an abnormal mRNA from the tk − allele. Unlike cell-mutation assays that use hemizygous loci (such as hprt +/0) for detecting potential chemical carcinogens, the mouse-lymphoma tk +/− assay allows the recovery of both intragenic and intergenic mutations thus enabling the detection of both point mutagens such as EMS and potent clastogens like MMC.

An evaluation of 6 chromosomal mutagens in the AS52/XPRT mutation assay utilizing suspension culture and soft agar cloning

The AS52/XPRT mutation assay was examined for sensitivity in the detection of chromosomal mutagens. 6 compounds identified as chromosomal mutagens in the mouse lymphoma assay (MLA) were tested for mutagenicity in AS52 cells using suspension treatment and soft agar cloning. The 6 compounds were benzo[ a]pyrene (BAP), 2-acetylaminofluorene (2AAF), methylmethanesulfonate (MMS), methyl acrylate (MCR), benzoin (BZ), and p-aminophenol (AM). BAP, 2AAF and MMS were mutagenic. The mutagenic responses for BAP, 2AAF and MMS included small colony mutants which have been shown to correlate with chromosomal mutation in the MLA. Colonies ranged from approximately 0.2 to 1.5 mm in size. The mutant frequency (MF) for the AS52 cells treated with BAP and 2AAF exceeded that previously reported for the MLA by 2-fold. In contrast, the MF for AS52 cells treated with MMS was one-third that reported in the MLA. The MF obtained in AS52 cells exceeded that reported for the CHO/HGPRT mutation assay for all 3 compounds. MCR, which produces almost entirely small colonies in the MLA, was negative in AS52 cells as were the MLA chromosomal mutagens AM and BZ. However, AM and BZ have only been reported mutagenic in the MLA. Both are considered nongenotoxic and noncarcinogenic. The results with the latter 3 compounds suggest that the AS52 assay is not as sensitive as the MLA for the detection of compounds identified as chromosomal mutagens.

Lysogeny in Bradyrhizobium japonicum and Its Effect on Soybean Nodulation

Rhizobiophage V, isolated from soil in the vicinity of soybean roots, was strongly lytic on Bradyrhizobium japonicum 123B (USDA 123) but only mildly lytic on strain L4-4, a chemically induced small-colony mutant of 123. Numerous bacteriophage-resistant variants were isolated from L4-4 infected with phage V; two were studied in detail and shown to be lysogenic. The two, L4-4 (V5) and L4-4 (V12), are the first reported examples of temperate-phage infection in B. japonicum. Phage V and its derivative phages V5 and V12 were closely related on the basis of common sensitivity to 0.01 M sodium citrate and phage V antiserum, phage immunity tests, and apparently identical morphology when examined by electron microscopy. However, the three phages differed in host range and in virulence. Lysogens L4-4 (V5) and L4-4 (V12) were immune to infection by phages V and V5 but not to infection by V12. Southern hybridization analysis confirmed the incorporation of phage V into the genomes of strains L4-4(V5) and L4-4(V12) and also demonstrated the incorporation of phage V into the genome of a phage V-resistant derivative of USDA 123 designated 123 (V2). None of the three lysogens, L4-4(V5), L4-4(V12), or 123B(V2), was able to nodulate soybean plants. However, Southern hybridization profile data indicated that phage V had not incorporated into any of the known B. japonicum nodulation genes.

Genetic toxicology of acrylic acid

Acrylic acid was tested for gene mutations in the in vitro CHO/HGPRT assay, for chromosome aberrations in CHO cells in culture, and for potential to induce unscheduled DNA synthesis in rat hepatocytes in culture. In vivo assays performed included the Drosophila sex-linked recessive lethal assay by both the feeding and injection routes, the in vivo cytogenetic assay in rat bone marrow cells after both a 1-day and 5-day oral dosing regimen, and a dominant lethal assay in mice by both an acute and 5-day dosing regimen. All results were negative (non-mutagenic) except for the in vitro chromosome aberration assay. This latter result is consistent with the previously reported possible clastogenic activity suggested by the results of the mouse lymphoma L5178Y TK locus assay in which a predominance of small-colony mutants was observed (Moore et al., Environmental and Molecular Mutagenesis 1988, 11, 49–63). The rapid clearance of acrylic acid in animals and the weight of evidence of genetic toxicity testing, including negative in vivo data in both somatic and germ cells, indicate a lack of genetic toxicity of acrylic acid in vivo.

Mutagenicity of 2-amino- N6-hydroxyadenine (AHA) at three loci in L5178Y/ tk± mouse lymphoma cells: molecular and preliminary cytogenetic characterizations of AHA-induced tk−/− mutants

2-Amino- N 6-hydroxyadenine (AHA) is a remarkably efficient and specific inducer of point mutations in Neurospora, with few or no larger scale events being detected (de Serres et al., 1985). In the present studies, AHA is shown to be a potent point mutagen at the tk ±, hprt + and Na +/ K + ATPase loci in L5178 Y/ tk ±−3.7.2C mouse lymphoma cells. Both large and small colony tk −/− mutants were analyzed at the molecular level and a preliminary assessment was made of small colony mutant karyotypes (230 bands/haploid metaphase cell; large colony mutants typically have normal karyotypes and were not analyzed). AHA induced greatly delayed (7–9 cell doublings) cytotoxicity, suggestive of a mutational mechanism (e.g., base-pair substitution) requiring DNA replection prior to its phenotypic expression. Approximately one-third of the tk −/− mutants formed small colonies, a phenotype which is typically associated with alterations to chromosome 11b, the site of the functional tk b allele in the parental cells. However, banded karyotypes have provided convincing evidence for alterations to chromosome 11b in only 2 of the 7 small colony mutants analyzed. Southern blot analysis showed that 78% ( 21 27 ) of these small colony mutants have retained the Nco-1 6.3-kb band, which is diagnostic of the tk b allele. This makes AHA unique among the mutagens examined so far in inducing small colony mutants without inducing large losses of tk b DNA. Although a dose-dependent increase in the proportion of small colony mutants was noted, no significant dose-dependent differences were seen at the molecular level in the relatively few mutants analyzed. The majority of AHA-induced tk −/− mutants formed large colonies. Southern blot analysis showed that 86% ( 25 29 ) of these had retained the Nco-1 6.3-kb band which is diagnostic of the tk b allele. It is concluded that AHA induces primarily micromutations (< 100 base pairs), probably through a base-pair substitution mechanism, at the tk, hprt and Na +/ K + ATPase loci in this system, with some larger scale damage (kilobases of DNA at the molecular level; chromosome 11b damage at the cytogenetic level) also occuring.

Stability of tk −/− mutants of L5178Y mouse lymphoma cells in Fischer's medium

The phenotypic stability of over 2000 large- and small-colony trifluorothymidine-resistant (TFT res) variants of L5178 Y/ tk +/− -3.7.2C cells has been examined. All except 4 of 488 spontaneously arising small-colony variants analyzed (0.8%) retained the TFT res phenotype when rechallenged with TFT after growth for several generations in its absence. All of 558 spontaneous large-colony variants, and 440 small-colony or 487 large-colony variants arising from 13 different mutagens showed similar stability. These results attest to the completeness of TFT selection in the mouse-lymphoma assay when used at 1 μg/ml in Fischer's medium supplemented with heat-inactivated serum and, together with previous cytogenetic and molecular studies, justify considering essentially all such TFT res variants as stable mutants. The implications of these results for those versions of the mouse lymphoma assay that fail to optimize the recovery and scoring of small-colony mutants is discussed.

Differential locus sensitivity to mutation induction by ionizing radiations of different LETs in Chinese hamster ovary K1 cells

Mutation induction after exposures to 250 kVp X-rays, alpha-particles from the radon daughter 212Bi, and fission-spectrum neutrons from the JANUS reactor was studied in Chinese hamster ovary (CHO) K1 cells and in CHO-10T5, a K1 derivative containing the bacterial gene xanthine-guanine phosphoribosyl transferase (gpt). Mutation induction was analyzed at three genetic loci: the gpt locus, the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus, and the thymidine kinase (tk) locus. After X-irradiation, mutants were induced at the tk loci at approximately 8-9 times the rate of mutant induction at the hprt locus, and the rate of mutant induction at the gpt locus was 8-10 times greater than that at the hprt locus. Neutron and alpha-radiation were more effective mutagenic agents. Mutant frequencies were approximately 4- to 6-fold higher than for X-rays at the hprt and gpt loci and greater than 12-fold greater than X-rays at the tk locus. The greater sensitivity of the tk locus to mutation induction by ionizing radiation (especially neutron and alpha-particle radiation) compared to the hprt locus is likely to be due to the recovery of an additional class of mutants, possibly ones containing larger-sized mutational events. Approximately half of the X-ray-induced tk-1- mutants were small-colony mutants, and 75% of the alpha- and neutron-induced tk-1- mutants were small-colony mutants. The increase in the proportion of small-colony mutants seen with increasing radiation linear energy transfer (LET) suggests that the radiation quality influenced the type of mutation recovered at this locus. There is probably a different reason for the hypersensitivity of the gpt locus because the frequency of gpt mutants, compared to the hprt locus, was independent of radiation quality. Therefore, the LET dependence of mutant induction is gene specific and not necessarily related to the size of deletion recoverable.

Comparison of chromosome aberration frequency and small-colony TK-deficient mutant frequency in L5178Y/TK+/− -3.7.2C mouse lymphoma cells

The L5178Y/TK(+/-)-3.7.2C mouse lymphoma assay is used to quantitate the induction of thymidine kinase (TK)-deficient mutants. The mutants detected in the assay form colonies that can be distinguished as large or small. The induction of small-colony mutants has been associated with the induction of chromosome mutations. In the present paper, we compare the analysis of induced small-colony TK mutants with gross aberration analysis (the more classical approach to analyzing chromosomal damage). Data are presented for 34 mutagens. As expected, we find that while the induction of gross aberrations and the induction of small-colony TK mutants is correlated, there is no simple mathematical relationship between the two endpoints. The two markers evaluate different subpopulations of chromosome mutations. While either endpoint can be used to detect chromosomal mutations, it should be remembered that the small-colony TK mutants represent genetic events which are compatible with cell viability. Only those alterations compatible with cell viability are a significant risk for human carcinogenicity or mutagenicity.

Molecular aspects of chemical mutagenesis in L5178Y/tk+/− mouse lymphoma cells

Southern blot analyses were performed on DNA from at least 10 large and 10 small colony thymidine kinase-deficient (tk -/-) mutants induced by each of 10 mutagens [2-amino-N6-hydroxyadenine (AHA), ethyl methanesulfonate (EMS), methyl methanesulfonate, 2-acetylaminofluorene, methotrexate, caffeine, methapyrilene, 4-(9-acridinylamino)-methanesulfo-m-anisidide, hycanthone methanesulfonate and procarbazine]. Two molecular mutant genotypes were recognized upon digestion with NcoI and subsequent probing with a 1.1 kb cDNA insert from plasmid pMtk 4: (i) no detectable alteration, and (ii) the absence of the functional tkb allele as indicated by the absence of the 6.3 kb fragment. In combination with the previously established chromosomal nature of most small colony tk -/- mutants, this permitted the classification of these 10 mutagens according to the relative proportions of each of four classes of genetic damage they induced. AHA and EMS gave mutational spectra consistent with their point mutational effects in other systems. The other eight mutagens induced mostly small colony mutants, most of which had lost the entire original tkb allele. Methotrexate induced high frequencies of large colony mutants at the tk locus, most of which lacked the tkb allele, although it is weakly or non-mutagenic at the hemizygous hprt locus in these same cells. At least three of these mutagens-methotrexate, caffeine, methapyrilene (and possibly procarbazine)--lack structural alerts for DNA reactivity, implying a major class of non-DNA primary targets for mutagenicity in mammalian cells that interact secondarily with the chromosome. These results are discussed in relation to the known differences in sensitivity among various short-term tests for genotoxicity.

Molecular dissection of mutations at the heterozygous thymidine kinase locus in mouse lymphoma cells.

The mouse lymphoma L5178Y TK+/- 3.7.2C cell line allows quantitation of induced TK(+/-)----TK-/- mutations at the heterozygous thymidine kinase (Tk) locus. TK-/- mutant colonies show a bimodal size distribution, reflecting a difference in the growth rates of the two size classes that is hypothesized to result from different degrees of genetic damage. The two homologous chromosomes 11 containing the alleles of the Tk gene in L5178Y 3.7.2C TK+/- cells are distinguishable at the cytogenetic level. We find, in addition, that the two alleles are distinguishable at the molecular level because of an Nco I restriction fragment length polymorphism at the 3' end of the gene. In a set of 51 large-colony and 48 small-colony TK-/- mutants induced by ionizing radiation or by chemical mutagens, we find that 78, including all except one of the small-colony mutants, have lost the Tk+ allele and that some of these have two to four copies of the remaining Tk- allele. Nineteen of the large-colony TK-/- mutants that do not show Tk+ allele loss show no other structural changes detectable at the level of Southern blot analysis. One shows a partial deletion. The variety of mutagenic lesions recorded at the heterozygous Tk locus may be representative of events observed in human malignancy and may include a wider range of mutagenic events than can be observed at hemizygous test loci.

High-resolution cytogenetic analysis of L5178Y TK +/− 3.7.2C cells: variation in chromosome 11 breakpoints among small-colony TJ −/− mutants

Since the finding that the mouse lymphoma L5178 TK +/− → TK −/− forward mutational assay system can detect and distinguish a range of genetic lesions, including large chromosomal aberrations and smaller, perhaps point mutational events, the chromosomal of these lesions at the highest possible level of band resolution has become increasingly important. We have developed an acridine orange/colcemid/hypotonic treatment for TK −/− mutants to provide high-resolution chromosomes with ocer 500 G-bands for breakpoint analysis. Using such high-resolution procedures, we find that independently induced small-colony mutants show rearrangements in the distal portion of chromosoem 11, with breakpoints occuring between bands B3 and E1.2. This finding of a range of chromosomal breakpoints in different TK −/− mutants complements recent molecular analysis of mutants and is consistent with the hypothesis that chromosomal lesions in small-colony mutants may affect a large portion of the genome in the vicinity of the tk-1 gene.

Synergism of mutant frequencies in the mouse lymphoma cell mutagenicity assay by binary mixtures of methyl methanesulfonate and ethyl methanesulfonate

The effect of mixed mutagen exposures on the rate and type of induced mutants was studied in the L5178Y/TK +/− → TK −/− mouse lymphoma cell mutagenicity assay. In this assay, exposure to ethyl methanesulfonate (EMS) results in more mutants that form large colonies than small colonies. Exposure to methyl methanesulfonate (MMS) results in more mutants that form small colonies than large colonies. Other reports in the literature suggest that large colony TK −/− mutants appear to result from small-scale, perhaps single-gene mutations, and that small-colony TK −/− mutants appear to be associated with chromosomal mutations. Treating cells for 4 h with simple, 2-component mixtures containing 6.45 μg/ml MMS and either 261, 392, 560 or 712 μg/ml EMS resulted in synergism of mutants at each mixture level. The frequencies of total mutants were synergized 12,20,35 and 72%, respectively, in mixed exposures with graded doses of EMS, above the sums of the mixture components. Small colony mutants were synergized to a greater extent than large colony mutants. The frequencies of small colony mutants in mixed exposures were increased 31, 54, 73 and 123%, respectively, while the frequencies of large colony mutants were increased −7, −6, 11 and 39%. Statistical analyses provide strong evidence of synergism (within the limits of the assay) for total and small-colony mutants at all doses of EMS tested, and for large-colony mutants above 400 μg/ml EMS. Similar magnitudes of synergism resulted when other constant levels of MMS (4.30 or 8.60 μg/ml) were mixed with the same graded doses of EMS. The degree of synergism was dependent on EMS concentration but not on MMS concentration.

Do trifluorothymidine-resistant mutants of L5178Y mouse lymphoma cells re-express thymidine kinase activity following 5-azacytidine treatment?

TFT is an effective selective agent for TK-deficient mutants of L5178Y TK +/− -3.7.2C mouse lymphoma cells. Mutants can be classified by colony size into small colonies (many of which show readily observable chromosome abnormalities associated with chromosome 11 — the location of the TK gene) and large colonies (which may represent events affecting only the expression of the TK gene). The precise nature of the induced damage causing the loss of the TK-enzyme activity for both mutant type is not known and is currently under investigation. The hypomethylating agent 5-azacytidine can be utilized to investigate the possibility that mutants might be the result of a suppressed rather than an altered TK gene. Mutant cell lines are treated with 5-azacytidine and then evaluated for re-expression of the TK enzyme as measured by resistance to THMG. In these studies, 11 mutants have been evaluated. None of the 11, including 10 small-colony mutants (6 with chromosome 11 translocations) and 1 large-colony mutant, show a high conversion to TK competency following 5-azacytidine treatment.

Methapyrilene is a genotoxic carcinogen: Studies on methapyrilene and pyrilamine in the L5178Y/TK +/− mouse lymphoma assay

Methapyrilene (MP), a sedating antihistamine, is a potent rat hepatocarcinogen which has been thought to be non-genotoxic on the basis of the negative results in a small number of short-term mutagenicity tests. The present studies show that MP is a moderately active mutagen in the L5178Y/TK +/− → TK −/− mouse lymphoma assay (MLA) in the presence of Aroclor-induced rat-liver S9, and that it induces predominantly small-colony thymidine kinase-deficient (TK −/−) mutants of demonstrated chromosomal origin. 10 of 12 small colony TK −/− mutants analyzed by banded karyotype (230-band level of resolution) show aberrations to chromosome 11b, the known location of the single functional TK gene in these cells. The observed aberrations from nine of the mutants included insertions, deletions and translocations while the tenth mutant had highly rearranged, multiple copies of chromosome 11 segments. By varying the concentrations of the S9 protein and cofactors it was shown that our standard S9 composition was close to optimum for activating MP to a mutagen. The activity and stability of various lots of S9 prepared in-house or purchased from a contract laboratory revealed significant differences. The ability of 2 lots of in-house S9 to activate a standard concentration of MP increased rapidly over the first 4 weeks of liquid nitrogen storage then declined slowly over the next 16 weeks. Three separate lots of purchased S9 were essentially inactive for the first 2 weeks of liquid nitrogen storage then increased in activity thereafter; these were the only occasions in which MP was not mutagenic in our hands. The mutagenic activity of pyrilamine (PYR), a structurally related antihistamine which is far less carcinogenic in rats, but easily detected in short-term tests as being genotoxic, was also investigated in the MLA. PYR was slightly less mutagenic than MP over a comparable range of concentrations, and also induced predominantly small-colony mutants. These studies fail to adequately explain the great carcinogenic differences between these two compounds, but are consistent with the potent hepatocarcinogenicity of MP resulting through a mutagenic mechanism.

Mutagenicity and clastogenicity of adriamycin in L5178Y/TK +/−-3.7.2C mouse lymphoma cells

Adriamycin was found to be both mutagenic and clastogenic to L5178Y/TK +/−-3.7.2C mouse lymphoma cells. A dose of only 5 ng/ml (survival = 62% or 67%) gave an induced TK mutant frequency of 307 or 296 per 10 6 survivors in two separate experiments. This dose was also clastogenic, inducing 20 chromosome aberrations/100 cells analyzed. The majority of the mutants were small-colony mutants, indicating that adriamycin likely acts primarily by a clastogenic mechanism.

Mutagenicity and clastogenicity of acrylamide in L5178Y mouse lymphoma cells.

Acrylamide was tested without exogenous activation in L5178Y/TK+/- -3.7.2C cells for mutation at the thymidine kinase locus and for clastogenicity. Acrylamide gave a positive induced mutagenic response (approximately 70 mutants/10(6) survivors) when tested at 600-650 micrograms/ml. The highest dose tested (850 micrograms/ml) resulted in an induced mutant frequency of approximately 380 mutants/10(6) survivors (survival = 13%). Acrylamide induced almost exclusively small-colony mutants, indicating that it might be acting by a clastogenic mechanism. As predicted, acrylamide was clastogenic, inducing both chromatid and chromosome breaks and rearrangements. A clearly positive clastogenic response was observed at both the 750 micrograms/ml and 850 micrograms/ml doses, which showed 16 and 64 aberrations per 100 cells, respectively (background = 3 aberrations per 100 cells). These studies indicate that the L5178Y/TK+/- mouse lymphoma assay can detect some chromosomal mutagens (clastogens) that show little activity in other single gene mutation assays, the CHO/HPRT and Salmonella.

Genotoxicity of γ-irradiation in L5178Y mouse lymphoma cells

The ability of γ-irradiation to induce gene mutation at the thymidine kinase locus and gross chromosome aberrations in L5178Y TK +/− 3.7.2C mouse lymphoma cells was evaluated. Positive results were obtained for both end-points. The majority of mutants were found to be small-colony mutants which correlated with the induction of gross chromosome aberrations.

Genotoxicity studies of benz[ l]aceanthrylene

The genotoxicity of the cyclopenta-fused polycyclic aromatic hydrocarbon, benz[ l]aceanthrylene (B[ l]A), was evaluated in vitro using the L5178Y TK + − mouse lymphoma assay and in vivo using the mouse peripheral blood lymphocyte (PBL) culture system. The mutagenicity and sister chromatid exchange (SCE) inducing potential of B[ l]A was then compared to that of benzo[ a]pyrene (B[ a]P). B[ l]A appeared to be slightly less mutagenic than B[ a]P at the TK locus, and each compound produced both small and large colony mutants indicating that they are clastogenic as well as mutagenic. Gross chromosome aberration analysis of treated L5178Y TK + − mouse lymphoma cells confirmed the clastogenicity of B[ l]A in vitro. In the mouse PBL system, after administration by gavage, B[ l]A was more cytotoxic and produced a sharper elevation in SCE frequency than B[ a]P.

Chromosome 11 aberrations in small colony L5178Y TKPsu−/− mutants early in their clonal history

We have developed a cytogenetic technique that allows observation of chromosome rearrangements associated with TK −/− mutagenesis of the L5178Y/TK +/−3.7.2C cell line early in mutant clonal history. For a series of mutagenic treatments we show that the major proportion (93%) of small-colony (σ) mutants studied have chromosome 11 rearrangements (the chromosome containing the thymidine kinase gene) while large-colony (λ) mutants do not have detectable chromosome rearrangements. In addition, we find among the chromosome abnormalities in σ mutants a significant proportion (34%) with dicentric chromosomes involving chromosome 11. These potentially unstable chromosome rearrangements may help to explain the karyotypic instability and heterogeneity among chromosome 11 aberrations previously noted in σ mutants when they are analyzed later in their clonal history.

Preclinical toxicology studies with acyclovir: Genetic toxicity tests

Acyclovir (ACV), an antiviral drug active in the treatment of oral and genital Herpes infections, has been evaluated for mutagenic and carcinogenic potential in a battery of in vitro and in vivo short-term assays. Negative results were obtained in the following in vitro tests: Ames Salmonella, plate incorporation and preincubation modification assays; E. coli polA +/polA − DNA repair; yeast ( S. cerevisiae D4) gene conversion; Chinese hamster ovary cells (HGPRT, APRT loci and ouabain-resistance marker); L5178Y mouse lymphoma cells (HGPRT locus and ouabain-resistance marker); and C3H/10T1/2 mouse fibroblast neoplastic transformation assay. All except the last assay were performed in the presence and absence of an exogenous metabolic activation system. ACV was positive at high concentrations × exposure times in the absence of exogenous metabolic activation in the following in vitro systems and at the indicated concentrations: BALB/c-3T3 neoplastic transformation (50 μg/mL, 72 h exposure); human lymphocyte cytogenetics (250–500 μg/mL, 48 h exposure); and L5178Y mouse lymphoma cells (TK locus, 400–2400 μg/mL, 4 h exposure; predominantly small colony mutants of chromosomal origin produced). No effects were seen in vivo (mouse dominant lethal assay; rat and Chinese hamster bone marrow cytogenetics) at up to maximum tolerated doses (MTD). An unusual clastogenic effect was seen in Chinese hamsters at 5 times the MTD. Overall, positive effects were seen only at either high concentrations (≥250 μg/mL in vitro or plasma levels) or prolonged exposure (72 hr in the BALB/c-3T3 neoplastic transformation assay). These studies support the view that ACV is a chromosomal mutagen, i.e., one which causes multi-locus damage but not single gene effects. The significance of these results for the genetic risk of ACV to man is discussed.