Mutagenicity studies on ketone solvents: methyl ethyl ketone, methyl isobutyl ketone, and isophorone

3 ketone solvents (methyl ethyl ketone (MEK), methyl isobutyl ketone (MiBK), and isophorone) were tested for potential genotoxicity. The assays of MEK and MiBK included the Salmonella/microsome (Ames) assay, L5178Y/TK+/- mouse lymphoma (ML) assay, BALB/3T3 cell transformation (CT) assay, unscheduled DNA synthesis (UDS) assay, and micronucleus (MN) assay. Only the ML, UDS, and MN assays were conducted on samples of isophorone. No genotoxicity was found for MEK or isophorone. The presence of a marginal response only at the highest, cytotoxic concentration tested in the ML assay, the lack of reproducibility in the CT assay, and clearly negative results in the Ames assay, UDS and MN assays, suggest that MiBK is unlikely to be genotoxic in mammalian systems.     

Assessment of genotoxic potential of ethylenediamine: in vitro and in vivo studies

Ethylenediamine (EDA) was evaluated for potential genotoxic activity using a battery in vitro and in vivo mammalian tests. The tests employed were the Chinese hamster ovary (CHO) gene mutation assay, the sister-chromatid exchange (SCE) test with CHO cells, unscheduled DNA synthesis (UDS) assays with primary rat hepatocytes and a dominant lethal study with Fischer 344 rats. EDA did not produce a positive, dose-related, mutagenic effect in either the CHO mutation assay or in the SCE test when evaluated both with and without the addition of a rat-liver S9 activation system. With hepatocytes, no positive effects of EDA upon UDS values were noted in 2 separate studies using either a scintillation counting procedure or an autoradiographic method to determine UDS activity. In a dominant lethal study, male rats fed for 23 weeks with dietary levels of EDA X 2HCl of 0, 0.05, 0.15 or 0.50 g/kg/day, and mated with 1 virgin female/week for 3 consecutive weeks, showed no dose-related or statistically significant effects upon fertility, total number of implantations/female, or the number of living and dead implants per female; marked effects upon the incidence of dominant lethal mutations were noted in the positive control group injected intraperitoneally with one dose of 0.25 mg/kg triethylenemelamine. We conclude that EDA was not genotoxic in the in vitro and in vivo mammalian test systems employed.     

Evaluation of the in vitro genetic toxicity of 4-(2, 4-dichlorophenoxy)butyric acid

The herbicide 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB) is principally used in the USA on peanuts, soybeans and alfalfa. In Europe, it is used on undersown spring barley and grassland (with clover). The genetic toxicity in vitro of the dimethylamine salt of 2,4-DB was examined by employing a range of end points including gene mutation in bacteria (Ames test) and mammalian cell cultures (CHO/HGPRT assay), cytogenetic abnormalities in mammalian cells (CHO/chromosomal aberration assay), and induction of DNA damage and repair in rat hepatocytes. There were no indications of genotoxic potential for 2,4-DB in the first three of these assays. One of the two criteria for a positive response in the UDS assay was exceeded but the increases did not exceed the second criteria for a positive response. The test material was therefore evaluated as weakly active in this assay. The weight of the evidence clearly indicates that 2, 4-DB is not genotoxic to mammals and are consistent with the reported lack of carcinogenic potential for 2,4-DB in both mice and rats.     

Evaluation of the genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) in a battery of in vitro and in vivo genotoxicity assays

The genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) was evaluated in a battery of in vitro and in vivo genotoxicity assays. These comprised a bacterial reverse-mutation assay (Ames test), an in vitro human lymphocyte chromosome-aberration assay, an in vivo mouse bone-marrow micronucleus assay and an in vivo rat-liver UDS assay. Maximum test concentrations in in vitro assays were determined by the TTX limit of solubility in the formulation vehicle (0.02% acetic acid solution). In the Ames test, TTX was tested at concentrations of up to 200 microg/plate. In the chromosome-aberration assay human lymphocytes were exposed to TTX at concentrations of up to 50 microg/ml for 3 and 20 h in the absence of S9, and for 3h in the presence of S9. For the in vivo assays, maximum tested dose levels were determined by the acute lethal toxicity of TTX after subcutaneous administration. In the mouse micronucleus assay TTX dose levels of 2, 4 and 8 microg/kg were administered to male and female animals, and bone-marrow samples taken 24 and 48 h (high-dose animals only) after administration. In the UDS assay, male rats were given TTX on two occasions with a 14-h interval at dose levels of 2.4 and 8 microg/kg, the last dose being administered 2h before liver perfusion and hepatocyte culturing. Relevant vehicle and positive control cultures and animals were included in all assays. TTX was clearly shown to lack in vitro or in vivo genotoxic activity in the assays conducted in this study. The results suggest that administration of TTX as a therapeutic analgesic agent would not pose a genotoxic risk to patients.     

Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens III. Appropriate follow-up testing in vivo

There has been much discussion in recent years regarding the most appropriate follow-up testing in vivo when positive results are obtained in vitro but the in vivo micronucleus (MN) test (traditionally the most widely-used test) is negative. Not all rodent carcinogens give positive results in the micronucleus test, and so it has been common practice to include a second in vivo assay such as the unscheduled DNA synthesis (UDS) test. This has proved useful but is usually limited to analysis of rodent (usually rat) liver. With the increased evaluation and use of other in vivo assays, e.g. for transgenic mutations (TG) and DNA damage (Comet assay) it was important to investigate their usefulness. We therefore examined the published in vivo UDS, TG and Comet-assay results for 67 carcinogens that were negative or equivocal in the micronucleus test. Between 30 and 41 chemicals were evaluated in each of the three in vivo tests, with some overlap. In general, the UDS test was disappointing and gave positive results with <20% of these carcinogens, some of which induced tumours in rat liver and produced DNA adducts in vivo. The TG assay gave positive responses with >50% of the carcinogens, but the Comet assay detected almost 90% of the micronucleus-negative or equivocal carcinogens. This pattern of results was virtually unchanged when the in vitro profile (gene mutagen or clastogen) was taken into account. High sensitivity (ability to detect carcinogens as positive) is only really useful when the specificity (ability to give negative results with non-carcinogens) is also high. Based on small numbers of publications with non-carcinogens, the TG and Comet assays gave negative results with non-carcinogens on 69 and 78% of occasions, respectively. Although further evaluation of the Comet and TG assays, particularly with non-carcinogens, is needed, these data suggest that they both should play a more prominent role in regulatory testing strategies than the UDS test.     

Lack of DNA damage induction by okadaic acid, a marine toxin, in the CHO-Hprt and the in vitro UDS assays

Okadaic acid (OA) is a marine toxin produced by dinoflagellates and responsible for human intoxications. OA is a specific inhibitor of serine/threonine protein phosphatases PP1 and PP2A and a potent tumor promoter in mouse skin and rat glandular stomach. In a previous study, we demonstrated that OA induced aneuploidy in CHO-K1 cells using the cytokinesis-block micronucleus (CBMN) assay coupled to FISH and concluded that OA was not a direct mutagen. As some previous in vitro mutagenicity studies had given positive results with OA, we decided to perform two additional in vitro mutagenicity assays in accordance with the OECD guidelines: (i) the CHO/Hprt test, which provides end points about locus-specific gene mutation; (ii) the in vitro unscheduled DNA synthesis (UDS) assay in rat hepatocytes, which measures [(3)H]thymidine incorporation into DNA undergoing excision repair. In the CHO/Hprt assay, there was no significant increase in the number of mutants for doses ranging from 5 to 5000 nM in the presence or absence of rat liver S9 fraction. In the in vitro UDS assay, OA did not induce primary DNA damages in rat hepatocytes following 18 h exposure at concentrations between 1.32 and 100 nM. As OA could affect the DNA repair systems via the inhibition of protein phosphatases, its effects on the repair kinetic of 2AAF-induced DNA damage were also investigated with the UDS assay. The results showed that OA did not interact with the DNA-repair process involved in in vitro UDS in rat hepatocytes. We concluded that OA failed to induce direct DNA damage but acted principally by altering the chromosome number, which could contribute to its carcinogenic effect.     

Absence of genotoxic activity of penbutolol in bacterial and mammalian cell screening systems

The genotoxic potential of the beta-adrenergic blocker penbutolol was assessed using the Ames and HGPRT tests, unscheduled DNA synthesis (UDS) and alkaline elution assays. In the Ames test, penbutolol was tested for cytotoxicity and genotoxic activity in concentration ranges of 0.8-500 micrograms/plate and 0.1-125 micrograms/ml in the HGPRT, UDS and alkaline elution assays. In the Ames test penbutolol showed significant toxicity above 500 micrograms/plate. In the mammalian cells (V79) used for the HGPRT test and A459 cells used for alkaline elution and UDS assays, penbutolol was cytotoxic at concentrations above 30 micrograms/ml. In another series of experiments, male Wistar rats were treated i.p. with penbutolol (1, 10 and 100 mg/kg) and after 2 h liver nuclei were isolated and formation of single DNA-strand breaks was measured. The results of the present study demonstrate the absence of genotoxic activity of penbutolol in the 5 strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA1538) and in the strain of Escherichia coli WP2 uvrA in the presence or absence of metabolic activation. In V79 cells, penbutolol showed no mutagenic effects at the HGPRT locus in the presence or absence of metabolic activation. Additionally, no significant incorporation of [3H]thymidine into the DNA in the UDS test or formation of DNA-strand breaks in the alkaline elution assay was detected in the non-toxic concentration range of penbutolol with or without metabolic activation. Furthermore, penbutolol did not cause DNA damage in liver nuclei isolated from penbutolol-treated rats.     

Genotoxicity assessment of ethylenediamine dinitrate (EDDN) and diethylenetriamine trinitrate (DETN)

Ethylenediamine dinitrate (EDDN) and diethylenetriamine trinitrate (DETN) are relatively insensitive explosive compounds that are being explored as safe alternatives to other more sensitive compounds. When used in combination with other high explosives they are an improvement and may provide additional safety during storage and use. The genetic toxicity of these compounds was evaluated to predict the potential adverse human health effects from exposure by using a standard genetic toxicity test battery which included: a gene mutation test in bacteria (Ames), an in vitro Chinese Hamster Ovary (CHO) cell chromosome aberration test and an in vivo mouse micronucleus test. The results of the Ames test showed that EDDN increased the mean number of revertants per plate with strain TA100, without activation, at 5000μg/plate compared to the solvent control, which indicated a positive result. No positive results were observed with the other tester strains with or without activation in Salmonella typhimurium strains TA98, TA1535, TA1537, and Escherichia coli strain WP2 uvrA. DETN was negative for all Salmonella tester strains and E. coli up to 5000μg/plate both with and without metabolic activation. The CHO cell chromosome aberration assay was performed using EDDN and DETN at concentrations up to 5000μg/mL. The results indicate that these compounds did not induce structural chromosomal aberrations at all tested concentrations in CHO cells, with or without metabolic activation. EDDN and DETN, when tested in vivo in the CD-1 mouse at doses up to 2000mg/kg, did not induce any significant increase in the number of micronuclei in bone marrow erythrocytes. These studies demonstrate that EDDN is mutagenic in one strain of Salmonella (TA100) but was negative in other strains, for in vitro induction of chromosomal aberrations in CHO cells, and for micronuclei in the in vivo mouse micronucleus assay. DETN was not genotoxic in all in vitro and in vivo tests. These results show the in vitro and in vivo genotoxicity potential of these chemicals.     

A comparative study on the metabolic activation of 3,4-benzo[a]pyrene to mutagens by aorta smooth muscle cells of rat and rabbit

The mutagenic activation of benzo[a]pyrene (BaP) after exposure to aorta smooth muscle cells of different origin was examined. Three test systems with different genetic endpoints--sister-chromatid exchange (SCE), gene mutation at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus and unscheduled DNA synthesis (UDS)--were used. Treatment of rat and rabbit aorta smooth muscle cells with BaP (1-6 micrograms/ml) resulted in a significant increase of SCEs, HGPRT mutations and UDS. So smooth muscle cells are capable of converting BaP to metabolites with a DNA-damaging action. In order to investigate the relation between the formation of mutagenic BaP metabolites and the susceptibility to atherosclerosis we compared the mutagenic potential of BaP using aorta smooth muscle cells of different species (rat, rabbit) and locations (thoracic and abdominal aorta). Rabbits and abdominal aortas are more susceptible to atherosclerosis than rats and thoracic aortas. The SCE, HGPRT and UDS assays revealed that smooth muscle cells of different origin possessed the same metabolic potential towards BaP. There was no correlation between the mutagenic potency of BaP metabolites and the susceptibility to atherosclerosis. As smooth muscle cells have a low metabolic capacity towards BaP, probably other factors in addition to the metabolic capacity of smooth muscle cells are responsible for species and tissue differences in susceptibility to atherosclerosis.     

In vitro genotoxic evaluation of conventional bleached and biobleached softwood pulp mill effluents

The effluents of pulp and paper mills contain about 300 different chemical compounds; many of them are mutagens and clastogens. Genotoxic studies have shown that chlorination stage liquors are significantly more genotoxic, in the Ames Salmonella assay, than the other process of lignin extraction, and that lyophilized effluents are genotoxic in cultured mammalian cells. Since these effluents from conventional bleaching stages are genotoxic, Chilean industries are interested in changing this process to a less toxic one, such as biobleaching using enzymes. In this study, we tested the in vitro genotoxicity of two types of effluents: an effluent obtained from a conventional radiata pine kraft-bleaching process (effluent D) and one derived from a biobleaching process with hemicellulase (effluent B). Both effluents were tested without any concentration or purification steps in the Ames Salmonella assay (TA100) and in the micronuclei (MN) and sister chromatid exchange (SCE) tests in CHO cells. The results showed that neither effluent induced base pair substitution mutations in the Ames Salmonella assay, and neither increased the micronucleus frequency in CHO cells. But, both increased the SCE frequencies in CHO cells, showing that this assay is more sensitive than the other ones, and that the two effluents contained chemical compounds in amounts enough to induce in vitro genotoxicity measured by the SCE induction.     

Ames assays and unscheduled DNA synthesis assays on 2, 4-dichlorophenoxyacetic acid and its derivatives.

2,4-dichlorophenoxyacetic acid and several of its derivatives (collectively known as 2,4-D) are herbicides used to control a wide variety of broadleaf and woody plants. The genetic toxicity in vitro of 2,4-D and seven of its salts and esters were examined by employing gene mutation in bacteria (Ames test) and induction of DNA damage and repair in rat hepatocytes. In addition, an in vivo unscheduled DNA synthesis (UDS) assay was performed on 2,4-D. There were no indications of genotoxic potential for 2,4-D acid, or any of its derivatives, in these assays. These results are consistent with the reported lack of carcinogenic potential for 2,4-D in both mice and rats.     

Genotoxicity of o-aminoazotoluene (AAT) determined by the Ames test, the in vitro chromosomal aberration test, and the transgenic mouse gene mutation assay

o-Aminoazotoluene (AAT) has been evaluated as a possible human carcinogen (Class 2B) by the International Agency for Research on Cancer (IARC). The Ames test found it to be mutagenic in the presence of a metabolic activation system, whereas it has little clastogenicity either in vitro or in vivo in the chromosomal aberration assay. AAT is also carcinogenic in the lung or liver of mice and rats given long-term administrations. Therefore, metabolites generated in the liver etc. may have gene mutation activity, and carcinogenesis would occur. We examined the mutagenicity of AAT in a gene mutation assay, using lacZ transgenic mice (MutaMice) and a positive selection method. AAT showed positive results for organs with metabolic functions, such as liver and colon and other organs. Positive results were also seen in an Ames test in the presence of metabolic activation and negative results seen in a chromosomal aberration test. Therefore, AAT had the potential to cause gene mutation in the presence of metabolic activation systems in vitro and the same reaction was confirmed in vivo with organs with metabolic function, such as liver and colon, but little clastogenicity in vitro or in vivo. Thus, metabolites with gene mutation activity may be responsible for the carcinogenicity of AAT. The transgenic mouse mutation assay proved to be useful for concurrent assessment of in vivo mutagenicity in multiple organs and to supplement the standard in vivo genotoxicity tests, such as the micronucleus assay which is limited to bone marrow as the only target organ.     

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: II. Mutation in Chinese hamster ovary, V79 Chinese hamster lung and L5178Y mouse lymphoma cells

Laboratory protocols and guidelines have been developed for the performance of point mutation assays using Chinese hamster ovary (CHO) cells, V79 cells, and L5178Y mouse lymphoma cells. Since only minor differences in the treatment of CHO and V79 cells exist, these two assays could be combined in one procedural guideline. A second protocol was developed for the mouse lymphoma assay in order to incorporate concerns and methods specific to that cell type and genetic locus. The protocols were based primarily on current laboratory practices as determined by responses to a detailed questionnaire completed by North-American and European governmental, university and contract laboratories involved with in vitro mutation testing. This report identifies those modifications to previously described methodologies which are being used on a regular basis, provides recommendations, and also serves to clarify confusing or inconsistent practices.     

Genotoxicity of aloeemodin in vitro and in vivo

The present in vitro and in vivo experiments were undertaken to clarify the genotoxic potential of the hydroxyanthrachinone aloeemodin which can be found in different plant derived products for therapy of constipation. The results demonstrate that aloeemodin is able to induce mutagenic effects in vitro. Positive results were obtained in the chromosomal aberration assay with CHO cells, as well as in the Salmonella reverse mutation assay (frameshift mutations in strains TA 1537, TA 1538 and TA 98). No mutagenic potential of aloeemodin, however, was observed in the gene mutation assay with mammalian cells in vitro (HPRT assay in V79 cells). Each assay was performed in the presence and absence of an extrinsic metabolic activation system (S9-mix). In in vivo studies (micronucleus assay in bone marrow cells of NMRI mice; chromosome aberration assay in bone marrow cells of Wistar rats; mouse spot test [DBA/2J × NMRI]) no indication of a mutagenic activity of aloeemodin was found. Information about a possible reaction of aloeemodin with DNA was derived from an in vivo UDS assay. Hepatocytes of aloeemodin-treated male Wistar rats did not show DNA damage via repair synthesis. All these data suggest that aloeemodin is able to interact with DNA under certain in vitro conditions. However, in vivo the results that were negative did not indicate a genotoxic potential. Therefore, it may be assumed that a genotoxic risk for man might be unlikely.     

In vivo mutagenicity studies with iodinated glycerol azeo

Previous studies have shown that iodinated glycerol azeo is positive in a number of in vitro mutagenicity assays including the Ames assay (TA100; TA1535), mouse lymphoma assay, Chinese hamster ovary (cytogenetic) assay and in one in vivo study, the sex-linked-recessive-lethal assay in Drosophila. Prior studies have also shown that the drug is negative in the mouse micronucleus assay. We now report that the drug is also negative for mutagenic activity in a number of other in vivo tests. Single intraperitoneal doses of 25, 125 and 250 mg/kg were without effect in the rat bone marrow chromosomal aberration assay. Single oral doses of 30, 75, 150 and 300 were negative in the rat hepatocyte DNA-repair assay. Single intraperitoneal doses of 30 and 100 mg/kg were without effect in the sister chromatid exchange (SCE) assay in the mouse. Statistically significant effects were seen at 200 and 300 mg/kg in the initial SCE assay and at 300 and 350 mg/kg in the confirmatory SCE assay. The rationale for considering the SCE results to be anomalous and thus not relative to the overall safety evaluation of the drug is presented.     

Genotoxicity tests with 6-acetyl-1,1,2,4,4,7-hexamethyltetraline and 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-gamma-2-benzopyr an

6-Acetyl-1,1,2,4,4,7-hexamethyltetraline (AHTN) and 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-gamma-2-ben zopyran (HHCB), synthetic fragrance ingredients, were evaluated for potential genotoxicity in a battery of short-term tests. Salmonella typhimurium/Escherichia coli plate incorporation and liquid preincubation assays were conducted on AHTN using tester strains TA97, TA98, TA100, TA102, TA1535, TA1537 and WP2 uvrA +/- S9 activation at doses from 8 to 5000 micrograms/plate. The plate incorporation mutagenicity assay was conducted on HHCB using tester strains TA98, TA100, TA1535, TA1537, TA1538 and WP2 uvrA +/- S9 activation at doses from 10 to 5000 micrograms/plate. An in vitro cytogenetics assay in Chinese hamster ovary (CHO) cells was conducted with AHTN and HHCB at three concentrations each with +/- S9 activation. In the non-activated study, the exposure/harvest periods were 4/20-, 20/20- and 44/44-h. In the S9 activated study, the exposure/harvest periods were 4/20- and 4/44-h. In vitro unscheduled DNA synthesis (UDS) assays were conducted in primary rat hepatocytes at concentrations between 0.15 and 50 micrograms/ml for AHTN and HHCB. In vivo mouse micronucleus assays were conducted with high doses of 1600 mg AHTN/kg and of 1500 mg HHCB/kg in corn oil. No positive responses were observed in any of the tests with HHCB. With AHTN, no positive responses were observed except for cells with structural aberrations in the in vitro cytogenetics assay in CHO cells with S9 activation at the treatment/harvest time of 4/20 h. In initial studies with AHTN, the high dose of 7.8 micrograms/ml showed 0.5% aberrant cells, with the mitotic index at 41% relative to vehicle control and cell growth inhibition in the range of 25-50%. Thus the genotoxicity findings with AHTN were limited to this one positive response; all other genotoxicity tests with AHTN were considered as negative. In particular, the negative finding in the in vivo assay supports AHTN as not likely to be mutagenic in mammalian systems. These considerations, along with other negative published data, lead to the conclusion that both AHTN and HHCB do not have significant potential to act as genotoxic carcinogens.     

A review of the genotoxicity of ethylbenzene

Ethylbenzene is an important industrial chemical that has recently been classified as a possible human carcinogen (IARC class 2B). It induces tumours in rats and mice, but neither the relevance of these tumours to humans nor their mechanism of induction is clear. Considering the carcinogenic potential of ethylbenzene, it is of interest to determine whether there is sufficient data to characterize its mode of action as either genotoxic or non-genotoxic. A review of the currently available genotoxicity data is assessed. Ethylbenzene is not a bacterial mutagen, does not induce gene conversion or mutations in yeast and does not induce sister chromatid exchanges in CHO cells. Ethylbenzene is not clastogenic in CHO or rat liver cell lines but was reported to induce micronuclei in SHE cells in vitro. No evidence for genotoxicity has been seen in humans exposed to relatively high levels of ethylbenzene. Mouse lymphoma gene mutation studies produced a mixed series of responses that have proved difficult to interpret. An increase in morphological transformation of SHE cells was also found. Results from a more relevant series of in vivo genotoxicity studies, including acute and sub-chronic micronucleus tests and the mouse liver UDS assay, indicate a lack of in vivo genotoxic activity. The composite set of results from both in vitro and in vivo tests known to assess direct damage to DNA have been predominantly negative in the absence of excessive toxicity. The available data from the standard battery of genotoxicity assays do not support a genotoxic mechanism for ethylbenzene-induced kidney, liver or lung tumors in rats and mice.     

Mutagenicity of an antitumor protein, neocarzinostatin, in Escherichia coli.

An assay is described for the measurement of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells utilizing resistance to 6-thioguanine (TG). Optimal selection conditions are defined for such parameters as phenotypic expression time prior to selection, and TG concentration and cell density which permits maximum mutant recovery. The nature of the TG-resistant mutants is characterized by several physiological and biochemical methods. The data demonstrate that more than 98% of the mutant clones isolated by this selection procedure contain altered HGPRTase activity. The CHO/HGPRT system thus shows the specificity necessary for a specific gene locus mutational assay.     

A review of the genotoxicity of marketed pharmaceuticals

Information in the 1999 Physician's Desk Reference as well as from the peer-reviewed published literature was used to evaluate the genotoxicity of marketed pharmaceuticals. This survey is a compendium of genotoxicity information and a means to gain perspective on the inherent genotoxicity of structurally diverse pharmaceuticals. Data from 467 marketed drugs were collected. Excluded from analysis were anti-cancer drugs and nucleosides, which are expected to be genotoxic, steroids, biologicals and peptide-based drugs. Of the 467 drugs, 115 had no published gene-tox data. This group was comprised largely of acutely administered drugs such as antibiotics, antifungals, antihistamines decongestants and anesthetics. The remaining 352 had at least one standard gene-tox assay result. Of these, 101 compounds (28.7%) had at least one positive assay result in the pre-ICH/OECD standard four-test battery (bacterial mutagenesis, in vitro cytogenetics, mouse lymphoma assay (MLA), in vivo cytogenetics). Per assay type, the percentage of positive compounds was: bacterial mutagenesis test, 27/323 (8.3%); in vitro cytogenetics 55/222 (24.8%); MLA 24/96 (25%); in vivo cytogenetics 29/252 (11.5%). Of the supplemental genetic toxicology test findings reported, the sister chromatid exchange (SCE) assay had the largest percentage of positives 17/39 (43.5%) and mammalian mutagenesis assays (excluding MLA) had the lowest percentage of positives 2/91 (2.2%). The predictive value of genetic toxicology findings for 2-year bioassay outcomes is difficult to assess since carcinogenicity can occur via non-genotoxic mechanisms. Nevertheless, the following survey findings were made: 201 drugs had both gene-tox data and rodent carcinogenicity data. Of these, 124 were negative and 77 were equivocal or positive for carcinogenicity in at least 1 gender/1 species. Of the 124 non-carcinogens, 100 had no positive gene-tox findings. Of the remaining 24, 19 were positive in in vitro cytogenetics assays. Among the 77 compounds that exhibited equivocal or positive effects in carcinogenesis studies, 26 were positive in gene-tox assays and 51 were negative. Of the 51 negatives, 47 had multiple negative gene-tox assay results suggesting that these are probably non-genotoxic carcinogens. Statistical analyses suggested that no combination of gene-tox assays provided a higher predictivity of rodent carcinogenesis than the bacterial mutagenicity test itself.     

Genetic toxicology evaluation of commercial beers, III. SCE, chromosome aberrations, and forward mutation (HGPRT) of commercial beer products in CHO cells.

Concentrated organic residues extracted from 5 blended aliquots of commercial beers were evaluated for their ability to induce sister chromatid exchange (SCE), chromosomal aberrations and forward mutation in Chinese hamster ovary (CHO) cells. Each extract was prepared by blending 4 commercial beers of similar ingredients and brewing method, passing the beer pool over XAD-2 resin, extracting the resin and concentrating the extract. Studies were performed both with and without metabolic activation using variable amounts of reconstituted residues from 225-fold concentrates of the blended samples. CHO cultures were treated with 0.75 microliters/ml through 10.0 microliters/ml of the concentrates in the SCE assays, 1.0 microliters/ml through 10.0 microliters/ml of the extracts in the aberration assays and 2.5 microliters/ml up to 20 microliters/ml for forward mutation assays. In preliminary screening for SCE as an indicator of potential DNA damage, a significant increase was observed for 3 of 5 concentrated samples; however, no increase in SCE was induced by any of the 5 samples when S9 was added as a source of exogenous metabolic activation. More definitive tests for induction of genetic events, i.e., chromosome aberrations and forward HGPRT mutations, were negative for all 5 extracts whether or not S9 mix was present. Since SCE were not induced in tests with metabolic activation and since there was no concordant aberration or point mutation induction, the preliminary indication of potential DNA damage shown by elevated SCE under conditions without metabolic activation appears to have little biological significance.