Weight of evidence approach using a TK gene mutation assay with human TK6 cells for follow-up of positive results in Ames tests: a collaborative study by MMS/JEMS

Abstract

Background Conflicting results between bacterial mutagenicity tests (the Ames test) and mammalian carcinogenicity tests might be due to species differences in metabolism, genome structure, and DNA repair systems. Mutagenicity assays using human cells are thought to be an advantage as follow-up studies for positive results in Ames tests. In this collaborative study, a thymidine kinase gene mutation study (TK6 assay) using human lymphoblastoid TK6 cells, established in OECD TG490, was used to examine 10 chemicals that have conflicting results in mutagenicity studies (a positive Ames test and a negative result in rodent carcinogenicity studies). Results Two of 10 test substances were negative in the overall judgment (20% effective as a follow-up test). Three of these eight positive substances were negative after the short-term treatment and positive after the 24 h treatment, despite identical treatment conditions without S9. A toxicoproteomic analysis of TK6 cells treated with 4-nitroanthranilic acid was thus used to aid the interpretation of the test results. This analysis using differentially expressed proteins after the 24 h treatment indicated that in vitro specific oxidative stress is involved in false positive response in the TK6 assay. Conclusions The usefulness of the TK6 assay, by current methods that have not been combined with new technologies such as proteomics, was found to be limited as a follow-up test, although it still may help to reduce some false positive results (20%) in Ames tests. Thus, the combination analysis with toxicoproteomics may be useful for interpreting false positive results raised by 24 h specific reactions in the assay, resulting in the more reduction (> 20%) of false positives in Ames test.

Genotoxicity of oxidative hair dye precursors: A systematic review

“…PTD tested positive in all in vitro assays identified in this search, 28,30 however in the thymidine kinase gene mutation study (TK6 assay) using human lymphoblastoid TK6 cells, which express human metabolic enzymes, no increase in mutation frequency was observed after 4-h treatment in the absence or presence of S9 metabolic activation nor after 24-h treatment without S9. 44 PPD was positive in one in vitro micronucleus assay in cultured human lymphocytes, 29 and showed increased micronucleus frequencies in mice erythrocytes following high dose acute oral exposure in vivo 31 Another indicator of genotoxicity, namely DNA breaks, were investigated in four in vitro studies 37,38,39,30 by an alkaline comet assay, and significantly higher DNA damage scores after PPD and PTD exposure was noted in all of them. The in vitro comet assay is considered to provide complementary information on genotoxicity and mechanism of action of chemicals, but it is not a standard battery test for mutagenic, clastogenic and aneugenic potential, and consequently has not been implemented into official regulatory testing guidelines.…”

Detection and analysis of chemical-induced chromosomal damage for public health: integrating new approach methodologies and non-animal methods

“…As a meta-analysis identified 32 genomic signatures (gene expression) of in vivo genotoxicity as general signatures to discriminate between genotoxic and non-genotoxic chemicals and compare in vivo outcomes (Auerbach, 2016), these in vivo signatures can be useful to compare the results from in vitro methods and interpret misleading positives. Other omics technologies such as toxicoproteomics have been employed to interpret positive results in genotoxicity evaluations on cell lines, and protein markers of oxidative stress were identified to discriminate misleading positives in continuous treatment conditions (Yasui et al, 2021). Thus, these new technologies could discriminate partly genotoxic substances from misleading positives based on the underlying molecular mechanisms.…”

A fast and reliable method for monitoring genomic instability in the model organism Caenorhabditis elegans

“…In both cases, in vitro and in vivo models (mostly rodents) are used and guidelines recommend a test battery starting with testing for gene mutation in bacteria, followed by in vitro assays using mammalian cell lines, before recommending an in vivo test system (Committee 2011; EMA/CHMP/ICH 2011; Phillips and Arlt 2009). Although these tests are routinely used, they present crucial limitations (i. e. lack of xenobiotic metabolism and bacteria-specific reactions (Yasui et al 2021), use of tumor cells), which affect the usefulness of the assays to predict the genotoxic potential of a substance in vivo. With the emergence of a stronger awareness of animal welfare in scientific experiments, classic and well-established in vivo studies are increasingly attempted to be replaced by equally meaningful tests, which follow the 3R (refine, reduce, replace) principle (Doke and Dhawale 2015;Freires et al 2017).…”