Standardized cell sources and recommendations for good cell culture practices in genotoxicity testing.

Good cell culture practice and characterization of the cell lines used are of critical importance in in vitro genotoxicity testing. The objective of this initiative was to make continuously available stocks of the characterized isolates of the most frequently used mammalian cell lines in genotoxicity testing anywhere in the world ('IVGT' cell lines). This project was organized under the auspices of the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Project Committee on the Relevance and Follow-up of Positive Results in In Vitro Genetic Toxicity (IVGT) Testing. First, cell isolates were identified that are as close as possible to the isolate described in the initial publications reporting their use in genotoxicity testing. The depositors of these cell lines managed their characterization and their expansion for preparing continuously available stocks of these cells that are stored at the European Collection of Cell Cultures (ECACC, UK) and the Japanese Collection of Research Bioresources (JCRB, Japan). This publication describes how the four 'IVGT' cell lines, i.e. L5178Y TK+/- 3.7.2C, TK6, CHO-WBL and CHL/IU, were prepared for deposit at the ECACC and JCRB cell banks. Recommendations for handling these cell lines and monitoring their characteristics are also described. The growth characteristics of these cell lines (growth rates and cell cycles), their identity (karyotypes and genetic status) and ranges of background frequencies of select endpoints are also reported to help in the routine practice of genotoxicity testing using these cell lines.

A refined protocol for conducting the low pH 6.7 Syrian hamster embryo (SHE) cell transformation assay.

The Syrian hamster embryo (SHE) cell transformation assay evaluates the potential of chemicals to induce morphological transformation in karyotypically normal primary cells. Induction of transformation has been shown to correlate well with the carcinogenicity of many compounds in the rodent bioassay. Historically the assay has not received wide-spread use due to technical difficulty. An improved protocol for a low pH 6.7 assay was developed by LeBoeuf et al. [R.A. LeBoeuf, G.A. Kerckaert, M.J. Aardema, D.P. Gibson, R. Brauninger, R.J. Isfort, Mutat. Res., 356 (1996) 85-127], that greatly reduced many of the technical difficulties associated with the SHE assay. The purpose of this paper is to describe the most current execution of the pH 6.70 protocol including protocol refinements made since the publication of a comprehensive protocol for this assay in Kerckaert et al. [G.A. Kerckaert, R.J. Isfort, G.J. Carr, M.J. Aardema, Mutat. Res., 356 (1996) 65-84].

Characterization of p53 in Chinese hamster cell lines CHO-K1, CHO-WBL, and CHL: implications for genotoxicity testing.

Since the p53 gene function is critical to how a cell responds to DNA damage, we investigated the p53 status in Chinese hamster cell lines commonly used in genotoxicity tests for cytogenetic damage around the world. These included: Chinese hamster ovary K1 (CHO-K1), Chinese hamster ovary WBL (CHO-WBL), and Chinese hamster lung (CHL) cells. The results of DNA sequencing, protein analysis, and cell cycle analysis demonstrate that the CHO-K1 and CHO-WBL cell lines have mutant p53 sequence [a mutation in codon 211 in exon 6 resulting in a change from Thr (ACA) to Lys (AAA)], mutant protein (high spontaneous levels that are non-inducible after X-irradiation), and mutant function (lack of G1 checkpoint). Interestingly, the CHL cell line has a completely wild-type p53 DNA sequence. However, the CHL cells have an abnormally high spontaneous level of wild-type p53 protein expression that is not inducible after X-irradiation, yet there is some evidence of G1 delay after irradiation. The protein data suggests that p53 in CHL cells is not being regulated normally, and thus is probably not functioning normally. The mechanism leading to this abnormal regulation of p53 in CHL cells clearly does not involve mutation in the p53 gene. Overall, the CHL cell line may be similar to the CHO cell lines, in that they all appear to have abnormal p53 function. Further work is needed to determine whether the presence of spontaneously high levels of wild-type p53 in CHL cells results in a difference in response to DNA damage (quantitatively or qualitatively) compared to the p53 mutant CHO cell lines.

Aneuploidy: a report of an ECETOC task force.

Aneuploidy plays a significant role in adverse human health conditions including birth defects, pregnancy wastage and cancer. Although there is clear evidence of chemically induced aneuploidy in experimental systems, to date there are insufficient data to determine with certainty if chemically induced aneuploidy contributes to human disease. However, since there is no reason to assume that chemically induced aneuploidy will not occur in human beings, it is prudent to address the aneugenic potential of chemicals in the safety assessment process. A wide range of methods has been described for the detection of chemically induced aneuploidy including subcellular systems, tests with fungi, plants and Drosophila as well as in vitro mammalian systems and in vivo mammalian somatic and germ cell assays. However, none of these methods is sufficiently validated or widely used in routine screening. Underlying the efforts to develop aneuploidy-specific assays is the presumption that current genetic toxicology tests do not detected chemicals that have aneuploidy-inducing potential. To address this, we have critically evaluated data from standard genetic toxicology assays for 16 known or suspected aneugens. The conclusions from the review are listed below. 1. At present there are only nine chemicals that can be classified as definitive aneugens, as determined by positive results in in vivo rodent assays. 2. As expected, the majority of definitive and suspected aneugens are negative in the bacterial mutation assay. 3. The majority of definitive aneugens evaluated induce polyploidy in vitro. With few exception, they also induced structural chromosome aberrations in vitro. 4. All of the definitive aneugens that have been sufficiently tested induce micronuclei in rodent bone marrow cells in vivo. A number of these chemicals also induced structural chromosome aberrations in vivo. 5. There is no evidence for a unique germ cell aneugen, that is a chemical that induces aneuploidy in germ cells and not in somatic cells. Furthermore, an analysis of several databases indicates the proportion of chemicals which induce polyploidy and not chromosome aberrations in vitro is low. Based on these conclusions, the following recommendations are made: for screening purposes, a standard genotoxicity test battery (including an in vitro cytogenetic assay with an assessment of polyploidy and clastogenicity at the same harvest time) should be performed; in the absence of polyploidy induction in vitro no further evaluation of aneuploidy-inducing potential is needed; if polyploidy is observed, in vitro follow-up testing to investigate further the aneuploidy-inducing potential should be conducted; such follow-up testing will generally start with the conduct of a standard in vivo somatic cell micronucleus assay; if the in vivo somatic cell micronucleus assay is negative, with adequate evidence of exposure of the bone marrow to the test compound, no further testing of aneuploidy-inducing potential is needed; if the in vivo somatic cell micronucleus assay is positive, further information on mechanisms of micronucleus induction can be obtained by using kinetochore/centromeric staining in vitro and/or in vivo; an assessment of potential germ cell aneuploidy activity may then be considered; aneuploidy induction which does not involve the direct interaction of a chemical or its metabolite(s) with DNA is expected to have a threshold. This must be considered in the risk assessment of such chemicals; this is not addressed by current risk assessment guidelines.

Induction of micronuclei in Syrian hamster embryo cells: comparison to results in the SHE cell transformation assay for National Toxicology Program test chemicals.

Sixteen chemicals currently being tested in National Toxicology Program (NTP) carcinogenicity studies were evaluated in the Syrian hamster embryo (SHE) cell in vitro micronucleus assay. Results from these studies were compared to the results from the SHE cell transformation assay for the same chemicals The overall concordance between induction of micronuclei and transformation of SHE cells was 56%, which is far lower that the 93% concordance between these two tests reported previously by Fritzenschaf et al. (1993; Mutation Res. 319, 47-53). The difference between our results appears to be due to differences in the types of chemicals in the two studies. Overall, there is good agreement between the SHE cell micronucleus and transformation assays for mutagenic chemicals, but, as our study highlights, the SHE cell transformation assay has the added utility of detecting nonmutagenic carcinogens. The utility of a multi-endpoint assessment in SHE cells for carcinogen screening is discussed.

Aneuploidy and consistent structural chromosome changes associated with transformation of Syrian hamster embryo cells.

To gain a better understanding of the role of specific numerical and structural chromosome changes in the multistage process of transformation of Syrian hamster embryo (SHE) cells, we analyzed seven benzo(a)pyrene (BP)-induced immortal SHE cell lines, and one spontaneously immortalized cell line. In addition, we analyzed chromosome changes in early passage tumor-derived cell lines induced by injection of four immortalized cell lines into neonate hamsters. Of particular interest was the observation of a deletion in the short arm of chromosome 2 in four of the seven BP-immortalized cell lines. Other types of alterations in chromosome 2 were observed in two other cell lines. Loss of one copy of chromosome 16 was also observed in more than 90 to 100% of the cells in three of seven BP-immortalized cell lines. In contrast, the only chromosome alteration seen in the spontaneously immortalized cell line was a deletion in the short arm of chromosome 20. Genetic instability, as indicated by increased numerical or structural chromosome changes, was observed in all tumor-derived cell lines compared to the immortal cell line from which they originated. These results, along with previous reports in the literature, suggest that alterations in specific chromosomes, like chromosome 2, may be involved in transformation of SHE cells.

Evaluation of lacI mutation in germ cells and micronuclei in peripheral blood after treatment of male lacI transgenic mice with ethylnitrosourea, isopropylmethane sulfonate or methylmethane sulfonate.

Male C57B1/6 lacI transgenic mice were used to evaluate germ cell mutagenesis in vivo as part of a collaborative study. Groups of 10 mice were administered single intraperitoneal doses of ethylnitrosourea (ENU; 150 mg/kg), isopropyl methanesulfonate (IPMS; 200 mg/kg), methyl methanesulfonate (MMS; 40 mg/kg) or vehicle. Epididymal spermatozoa and testes were recovered 3 days later and DNA isolated subsequently from epididymal spermatozoa and seminiferous tubules were analyzed for lacI mutations. The mutant frequency in seminiferous tubules (average +/- SEM) increased significantly compared with untreated controls (7.2 +/- 0.7 x 10(-5) following treatment with ENU (11.7 +/- 0.8 x 10(-5), p = 0.003) or with IPMS (9.6 +/- 0.5 x 10(-5), p = 0.018) but not following treatment with MMS (8.1 +/- 0.8 x 10(-5), p = 0.213). Group mutant frequencies were not determined for epididymal spermatozoa from MMS- or IPMS-treated mice because of poor DNA recoveries. As another indicator of the genotoxicity of these alkylating agents, the frequencies of micronuclei were determined in the peripheral blood 48 h after carcinogen administration in the same transgenic mice. The micronuclei frequencies were elevated significantly (p < 0.05) by each treatment (IPMS: 1.0%; MMS: 0.94%) compared to vehicle controls (0.3%). In a separate experiment, 40 mg/kg ENU was previously found to increase the frequency of micronuclei in peripheral blood of lacI transgenic mice 48 h after treatment (3.2%; Gibson et al., 1995). These results demonstrate that the lacI transgenic mouse male germ cells are sensitive to some, but not all, mutagens under the conditions used in this experiment. Investigation of other experimental designs would offer additional perspective on the usefulness of this transgenic model for routine mutagenicity testing in germ cells.

Thiabendazole-induced cytogenetic abnormalities in mouse oocytes.

Of the various classes of human genetic disorders, aneuploidy is the most prevalent. Besides its association with maternal age and its predominant origin during maternal meiosis I, little is known about the etiology of aneuploidy. Although various classes of chemicals have been shown to induce aneuploidy in experimental systems, there is no definitive evidence for the role of chemically induced aneuploidy and adverse human health effects, particularly germ cell effects. Thus, it is important to understand the potential of chemicals for inducing aneuploidy in germ cells. There are conflicting data in the literature about the ability of thiabendazole (TBZ) to induce aneuploidy; therefore, we investigated the potential of TBZ for inducing aneuploidy in oocytes. Superovulated ICR female mice were administered 0, 50, 100, or 150 mg/kg TBZ by intraperitoneal injection. The frequencies and percentages of hyperploid oocytes were 0/472 (0), 2/410 (0.5), 6/ 478 (1.3), and 3/427 (0.7) for control, 50, 100, and 150 mg/kg TBZ, respectively. The difference between controls and the 100 mg/kg dose was statistically significant. Also, the proportions of ovulatory mice and the number of oocytes collected per ovulatory female were reduced in the TBZ groups relative to controls. Based on these results, we conclude that TBZ induces a small, but significant increase in the frequency of aneuploid oocytes at toxic doses that also impair ovulation.

Genotoxicity and subchronic toxicity studies with heated olestra.

Olestra is a class of sucrose-fatty acid polyesters intended for use as a non-caloric replacement of edible oil. Genotoxicity and subchronic toxicity studies were conducted to determine whether olestra could form genotoxic or toxic breakdown products during simulated commercial use. Heated olestra was prepared for these studies by batch-frying potato slices in olestra at 177-185 degrees C for 25-32 hr over 5-7 days. Genotoxicity of this previously heated olestra was assessed in four standard in vitro assays: (1) Salmonella mutagenesis (Ames test); (2) forward mutagenesis of mouse lymphoma cells at the thymidine kinase locus; (3) unscheduled DNA synthesis in rat hepatocytes; and (4) clastogenicity in cultured Chinese hamster ovary cells. These tests were conducted with previously heated olestra at concentrations up to at least 5 mg/ml both in the absence of exogenous bioactivation and, for assays (1), (2) and (4) with added liver microsomal (S-9) activation. The Ames and mouse lymphoma assays were performed with olestra (10 mg/ml and 23 mg/litre, respectively) either alone or emulsified with the non-toxic, non-ionic surfactant Pluronics F68, both in the presence and absence of metabolic activation. To test for clastogenicity in vivo, rats were administered previously heated olestra by gavage at 5 g/kg per day for up to 5 days and bone marrow cells were examined for chromosomal aberrations. Heated olestra lacked genotoxic activity detectable by the aforementioned assays. Heated olestra was fed to Fischer 344 rats at up to 10% of the diet (w/w) for 91 days. Evaluation of survival, food consumption, feed efficiency, physical condition, body weight, organ weight, haematological and clinical chemistry parameters, and histomorphology revealed no adverse effects attributable to ingestion of heated olestra at exposure levels in excess of those anticipated for human consumption. It is concluded that olestra used as a deep-frying medium conveys no genotoxic or toxic hazard at anticipated levels of human consumption.

The low pH Syrian hamster embryo (SHE) cell transformation assay: a revitalized role in carcinogen prediction.

A series of publications of the results of National Toxicology Program (NTP) studies (Tennant et al. (1987) Science, 236, 933-941; Haseman et al. (1990) J. Am. Stat. Assoc., 85, 964-971; Shelby et al. (1993) Environ. Mol. Mutagen., 21, 160-179) show that the commonly used short-term genotoxicity tests are less predictive of rodent carcinogenicity than once thought. These results have fueled a great deal of debate in the field of genetic toxicology regarding appropriate strategies for assessing the potential carcinogenicity of chemicals. The debate has continued in the recent discussion of harmonized genotoxicity test strategies (Ashby (1993) Mutation Res., 298, 291-295 and Ashby (1994) 308, 113-114; Madle (1993) Mutation Res., 300, 73-76 and Madle (1994) 308, 111-112; Zeiger (1994) Mutation Res., 304, 309-314) since the underlying problem still has not been resolved. The underlying problem is the fact that the current short-term genotoxicity tests in any combination do not provide both the necessary high sensitivity and high specificity needed for accurate rodent carcinogen detection. In this discussion, we describe the utility of the newly revised Syrian hamster embryo (SHE) cell transformation assay alone and in combination with the Salmonella mutation assay for improved accuracy of screening of rodent carcinogens relative to standard short-term genotoxicity tests. The accompanying papers provide details of improved methodologies for the conduct of the SHE cell transformation assay and an extensive review of the databases which support our conclusion that the SHE cell transformation assay provides an improved prediction of rodent bioassay results relative to other in vitro genotoxicity test batteries.

A comprehensive protocol for conducting the Syrian hamster embryo cell transformation assay at pH 6.70.

Studies from our laboratory have demonstrated several advantages of conducting the Syrian hamster embryo (SHE) cell transformation assay at pH 6.70 compared to that done historically at higher pH values (7.10-7.35). These include reduction of the influence of SHE cell isolates and fetal bovine serum lot variability on the assay, an increase in the frequency of chemically induced morphological transformation (MT) compared to controls, and an increased ease in scoring the MT phenotype. The purpose of this paper is to report a comprehensive protocol for conduct of the pH 6.70 SHE transformation assay including experimental procedures, a description of criteria for an acceptable assay and statistical procedures for establishing treatment-related effects. We have also identified several assay parameters in addition to pH which can affect transformation frequencies, particularly the critical role colony number per plate can have on transformation frequency. Control of this parameter, for which details are provided, can greatly increase the reproducibility and predictive value of the assay.

The pH 6.7 Syrian hamster embryo cell transformation assay for assessing the carcinogenic potential of chemicals.

Cell transformation models have been established for studying the cellular and molecular basis of the neoplastic process. Transformation models have also been utilized extensively for studying mechanisms of chemical carcinogenesis and, to a lesser degree, screening chemicals for their carcinogenic potential. Complexities associated with the conduct of cell transformation assays have been a significant factor in discouraging broad use of this approach despite their reported good predictivity for carcinogenicity. We previously reported that many of the experimental difficulties with the Syrian hamster embryo (SHE) cell transformation assay could be reduced or eliminated by culturing these cells at pH 6.7 culture conditions compared to the historically used pH 7.1-7.3. We and others have shown that morphological transformation (MT), the earliest recognizable phenotype in the multi-step transformation process and the endpoint used in the standard assay to indicate a chemical's transforming activity, represents a pre-neoplastic stage in this model system. In the collaborative study reported here, in which approx. 50% of the chemicals were tested under code in one laboratory (Hazelton) and the other 50% evaluated by several investigators in the second laboratory (P & G), we have evaluated 56 chemicals (30 carcinogens, 18 non-carcinogens, 8 of inconclusive carcinogenic activity) in the SHE cell transformation assay conducted at pH 6.7 culture conditions with a standardized, Good Laboratory Practices-quality protocol. An overall concordance of 85% (41/48) between SHE cell transformation and rodent bioassay results was observed with assay sensitivity of 87% (26/30) and specificity of 83% (15/18), respectively. The assay exhibited a sensitivity of 78% (14/18) for Salmonella assay negative carcinogens, supporting its value for detecting non-mutagenic carcinogens. For maximum assay sensitivity, two exposure durations were required, namely a 24-h exposure and a 7-day exposure assay. Depending on the duration of chemical treatment required to induce transformation, insight into the mechanism of transformation induction may also be gained. Based on the data reported here, as well as the larger historical dataset reviewed by Isfort et al. (1996), we conclude that the SHE cell transformation assay provides an improved method for screening chemicals for carcinogenicity relative to current standard genotoxicity assays.

Establishment of immortalized alveolar type II epithelial cell lines from adult rats.

We developed methodology to isolate and culture rat alveolar Type II cells under conditions that preserved their proliferative capacity, and applied lipofection to introduce an immortalizing gene into the cells. Briefly, the alveolar Type II cells were isolated from male F344 rats using airway perfusion with a pronase solution followed by incubation for 30 min at 37 degrees C. Cells obtained by pronase digestion were predominantly epithelial in morphology and were positive for Papanicolaou and alkaline phosphatase staining. These cells could be maintained on an extracellular matrix of fibronectin and Type IV collagen in a low serum, insulin-supplemented Ham's F12 growth medium for four to five passages. Rat alveolar epithelial cells obtained by this method were transformed with the SV40-T antigen gene and two immortalized cell lines (RLE-6T and RLE-6TN) were obtained. The RLE-6T line exhibits positive nuclear immunostaining for the SV40-T antigen and the RLE-6TN line does not. PCR analysis of genomic DNA from the RLE-6T and RLE-6TN cells demonstrated the T-antigen gene was present only in the RLE-6T line indicating the RLE-6TN line is likely derived from a spontaneous transformant. After more than 50 population doublings, the RLE-6T cells stained positive for cytokeratin, possessed alkaline phosphatase activity, and contained lipid-containing inclusion bodies (phosphine 3R staining); all characteristics of alveolar Type II cells. The RLE-6TN cells exhibited similar characteristics except they did not express alkaline phosphatase activity. Early passage RLE-6T and 6TN cells showed a near diploid chromosome number.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of aneuploidy-inducing carcinogens in the Syrian hamster embryo (SHE) cell transformation assay.

As evidenced by the recent report of the Commission of the European Communities (CEEC) project (Detection of Aneugenic Chemicals-CEEC project, 1993), there currently is a great deal of effort towards developing and validating assays to detect aneuploidy-inducing chemicals. In this report, we describe the utility of the Syrian hamster embryo (SHE) cell transformation assay for detecting carcinogens with known or suspected aneuploidy-inducing activity. The following carcinogens were tested: asbestos, benomyl, cadmium chloride, chloral hydrate, diethylstilbestrol dipropionate, and griseofulvin. Thiabendazole, a noncarcinogen, was also tested. Chemicals of unknown or inconclusive carcinogenicity data, colcemid, diazepam, econazole nitrate, and pyrimethamine were also evaluated. All of the above chemicals except thiabendazole induced a significant increase in morphological transformation (MT) in SHE cells. Based on these results as well as those published in the literature previously, the SHE cell transformation assay appears to have utility for detecting carcinogens with known or suspected aneuploidy-inducing ability.

Assessment of the in vivo genotoxicity of 2-hydroxy 4-methoxybenzophenone.

The genotoxic potential of 2-hydroxy 4-methoxy-benzophenone (benzophenone-3, Bz-3), a commonly used sunscreen, has been evaluated previously with in vitro systems. Data from Salmonella studies (with and without activation) have been predominantly negative, but two reports have shown weakly positive results in a single bacterial strain under conditions of metabolic activation. In addition, Bz-3 has been reported to induce chromosome aberrations and equivocal results for sister chromatid exchange in Chinese hamster ovary (CHO) cells. We used the Drosophila somatic mutation and recombination test (SMART) and in vivo cytogenetics in rat bone marrow to define the potential for in vivo expression of this in vitro activity. For the SMART assay, larva from a mating of "multiple wing hair" (mwh) females with heterozygous "flare" (flr) males were exposed to 0, 3000, or 3500 ppm Bz-3 or 25 ppm dimethylnitrosamine (DMN, positive control) for 72 hr. A recombination between the mwh and flr genes produces twin wing spots, while events such as deletions produce single spots. None of the Bz-3-treated larva produced flies with significantly more single or multiple wing spots than controls. In contrast, DMN-treated larva produced flies with significantly more single or multiple wing spots than controls. The in vivo cytogenetic assay in rat bone marrow cells was conducted to evaluate the clastogenicity of Bz-3. Sprague-Dawley rats were treated by oral gavage with a single administration of 0.0, 0.5, 1.67, or 5 gm/kg Bz-3 or a single dose of 5 gm/kg/day Bz-3 for 5 consecutive days. Cyclophosphamide (CP) was the positive control and was administered at 20 mg/kg with both treatment regimens. Colchicine growth-arrested bone marrow cells were collected 8 and 12 hr after the single treatment and 12hr after the last daily treatment. Under either treatment protocol none of the Bz-3 concentrations caused any significant increase in chromosomal aberrations. Results from these two studies strongly support the conclusion that Bz-3 is not genotoxic in vivo.

Benomyl-induced aneuploidy in mouse oocytes.

Benomyl (methyl-1-[butylcarbamoyl]-2-benzimidazole carbamate) is a plant fungicide which acts by inhibiting tubulin polymerization. It is also a carcinogen and a teratogen. A substantial number of reports consistently show that benomyl inhibits cellular division and induces numerical chromosome changes in somatic cells, while results from standard genotoxicity assays which measure mutations and structural chromosome aberrations are conflicting. To define further the genotoxic effects of benomyl and to demonstrate the utility of the in vivo mouse oocyte assay for detecting chemicals which induce numerical chromosome changes, we investigated the ability of benomyl to induce numerical and structural chromosome aberrations in mouse oocytes. Superovulated female ICR mice were administered benomyl by oral gavage and oocytes were collected 17 h later. The proportions (and percentages) of hyperploid oocytes were 1/309(0.3), 6/155(3.9), 38/229(16.6), 46/130(35.4), 60/215 (27.9), 42/143(29.4) for control, 500, 1000, 1500, 1750 and 2000 mg/kg respectively. No increase in structural aberrations was observed. These results demonstrate that benomyl specifically induces numerical chromosome changes in mouse oocytes.

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.