Assessment of the performance of the Ames II™ assay: a collaborative study with 19 coded compounds

doi:10.1016/j.mrgentox.2003.12.001

Mutation Research/Genetic Toxicology and Environmental Mutagenesis

Volume 558, Issues 1–2, 14 March 2004, Pages 181-197

https://doi.org/10.1016/j.mrgentox.2003.12.001 Get rights and content

Abstract

Nineteen coded chemicals were tested in an international collaborative study for their mutagenic activity. The assay system employed was the Ames II Mutagenicity Assay, using the tester strains TA98 and TAMix (TA7001–7006). The test compounds were selected from a published study with a large data set from the standard Ames plate-incorporation test. The following test compounds including matched pairs were investigated: cyclophoshamide, 2-naphthylamine, benzo(a)pyrene, pyrene, 2-acetylaminofluorene, 4,4′-methylene-bis(2-chloroaniline), 9,10-dimethylanthracene, anthracene, 4-nitroquinoline-N-oxide, diphenylnitrosamine, urethane, isopropyl-N(3-chlorophenyl)carbamate, benzidine, 3,3′-5,5′-tetramethylbenzidine, azoxybenzene, 3-aminotriazole, diethylstilbestrol, sucrose and methionine. The results of both assay systems were compared, and the inter-laboratory consistency of the Ames II test was assessed. Of the eight mutagens selected, six were correctly identified with the Ames II assay by all laboratories, one compound was judged positive by five of six investigators and one by four of six laboratories. All seven non-mutagenic samples were consistently negative in the Ames II assay. Of the four chemicals that gave inconsistent results in the traditional Ames test, three were uniformly classified as either positive or negative in the present study, whereas one compound gave equivocal results. A comparison of the test outcome of the different investigators resulted in an inter-laboratory consistency of 89.5%.

Owing to the high concordance between the two test systems, and the low inter-laboratory variability in the Ames II assay results, the Ames II is an effective screening alternative to the standard Ames test, requiring less test material and labor.

Introduction

The value of the Salmonella mutagenicity assay has been clearly confirmed as a suitable primary test for the detection of potential mutagens and carcinogens, and since the mid-seventies the Ames assay [1], [2] is used routinely as a screening assay to predict animal carcinogens.

The Ames II assay is a liquid microtiter modification of the Ames test and consists of the ‘strains’ TAMix and TA98. TAMix is a mixture of the Salmonella typhimurium strains TA7001, TA7002, TA7003, TA7004, TA7005 and TA7006 [3]. The genetic complementation among the six TA700x strains (where x=1, 2, 3, 4, 5, and 6) is low enough such they may be combined in a single assay to facilitate screening for mutagens. The strains in TAMix (base-pair substitutions) are like TA98 (frameshift mutation), histidine auxotrophs and mutagenesis will cause reversion to histidine prototrophy. Like the traditional strains, the genetic background of the TA700x series of strains has been modified to improve the sensitivity of their reversion by many classes of compound. The uvrB gene that is involved in excision repair has been deleted to allow lesions in the DNA to accumulate. The selection pressure to mutate or revert is facilitated so that less compound is needed to see an effect. The galE503 mutation reduces the effectiveness of epimerase responsible for the inter-conversion of UDP-galactose and UDP-glucose. This inter-conversion is necessary for the synthesis of a complete cell wall, thus the point mutation in the epimerase allows a higher permeability of larger compounds into the cell and gives a population of cells which have a ‘rough’ phenotype (rfa). The tester strains carry the plasmid pKM101, which has the umuDC homologues, mucA/B and the β-lactamase gene that confers ampicillin resistance. These gene products increase the cell’s ability to perform mutagenic lesion bypass repair during DNA replication.

This study had two goals: (1) to corroborate the use of the Ames II test as a suitable alternative screening assay [4], [5] to the traditional Ames plate-incorporation method, and (2) to test the Ames II assay system for its reproducibility among different laboratories. The 19 compounds included in this study were selected on the basis of traditional Ames data published as a report of the International Collaborative Program for the Evaluation of Short-Term Tests for Carcinogens (ICPESTTC study) [6]. The chemicals selected were either Ames-positive, -negative or equivocal: among the compounds that were positive in the traditional Ames assay, weak and strong mutagens were chosen, and the necessity of metabolic activation (S9 mix) for a positive response as well as the target site (frameshift mutation versus base-pair substitution) were considered. The equivocal chemicals that were chosen gave either inconsistent results in the ICPESTTC study or are known to be difficult to detect in bacterial mutagenesis assays. Although the discrimination between carcinogens and non-carcinogens played a secondary role in the present study, some chemical ‘pairs’ (carcinogens and their non-carcinogenic analogs) were included.

The 19 chemicals (Table 2) were coded at random before being distributed among nine independent laboratories, which allowed an opportunity for an inter-laboratory comparison of the Ames II system. Each compound was tested by 4–6 different investigators. The following companies participated in this study: Aventis Pharma Deutschland GmbH (Hattersheim, DE), BASF AG (Ludwigshafen, DE), Boehringer Ingelheim (Biberach, DE), Johnson&Johnson Pharmaceutical Research&Development (Beerse, BE), Novartis Consumer Health (Nyon, CH), Schering AG (Berlin, DE), Servier Group (Orléans-Gidy, FR), Federal Environmental Agency (Bad Elster, DE) and Xenometrix by Endotell GmbH (Allschwil, CH).

Section snippets

Bacterial strains

The Ames II test was performed with S. typhimurium TA98 and TAMix [3]. TAMix consists of the strains TA7001–7006 in equal proportions and was treated as if it were an individual strain. The tester strains are characterized in Table 1.

Freshly thawed frozen strains of 10 μl were inoculated in 10 ml of growth medium (Xenometrix by Endotell GmbH) and the cultures were grown overnight (12–17 h) at 37 °C in an environmental shaker at 250 rpm in the presence of 50 μg/ml ampicillin (Xenometrix by Endotell

Results and discussion

The results for the positive and equivocal test chemicals generated in the present study are shown in Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9, Fig. 10, Fig. 11, Fig. 12. The figures represent the raw data obtained by the different laboratories. For representation reasons, the y-axis of the strong mutagens (maximum 48 positive wells) differ from those of the weak mutagenic and equivocal compounds. Clearly negative results are not shown graphically. The robotic

Conclusions

The present Ames II study revealed an overall agreement of 84.2% (16 of 19 compounds, Fig. 13a–p) with the standard Ames results of the ICPESTTC study [6]. All eight mutagenic chemicals that were selected from the ICPESTTC report (Fig. 12a–h) were also positive in the Ames II test, except cyclophosphamide (h) which was found to be positive in 4 of 6 laboratories in the present study. All seven Ames-negative compounds were also clearly negative in the Ames II test (Fig. 13i and k–p).

Disagreement

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Mutation Research/Genetic Toxicology and Environmental Mutagenesis

Abstract

Introduction

Section snippets

Bacterial strains

Results and discussion

Conclusions

References (7)

Revised methods for the Salmonella mutagenicity test

Mutation Res.

Comparison of responses of base-specific Salmonella tester strains with the traditional strains for identifying mutagens: the result of a validation study

Mutation Res.

Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test

Mutation Res.

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Assessment of the performance of the Ames MPF™ assay: A multicenter collaborative study with six coded chemicals

The ecotoxicity and mutagenicity of fire water runoff from small-scale furnishing materials fire tests

Inoculum cell count influences separation efficiency and variance in Ames plate incorporation and Ames RAMOS test

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