Mouse lymphoma thymidine kinase locus gene mutation assay: International Workshop on Genotoxicity Test Procedures Workgroup report

Abstract

The Mouse Lymphoma Assay (MLA) Workgroup addressed and reached consensus on a number of issues. Discussion focused on five areas: (1) acceptable assay versions; (2) cytotoxicity measure; (3) 24‐hr treatment; (4) microwell colony counting and sizing; and (5) data acceptability/statistical analysis. Although the International Conference on Harmonisation (ICH) indicated a preference for the microwell over the soft agar method, all of the workgroup members agreed that both versions of the MLA are equally acceptable. The workgroup agreed that it is desirable for both assay versions to use the same measure of cytotoxicity to define the acceptable and required concentration range. Currently, laboratories using the microwell version use the relative survival (RS) determined by cloning immediately after the treatment. Laboratories using the soft agar method do not obtain an RS but use the relative total growth (RTG), a combination of the relative suspension growth (RSG) during the expression period and the relative cloning efficiency determined at the time of mutant selection. The workgroup agreed to investigate the RSG, the RS, and the RTG and to develop further guidance. In the interim, the workgroup reached consensus that the RTG be used as the standard measure of cytotoxicity. The ICH recommended a 24‐hr treatment in the absence of S9 when negative results are obtained with short (3–4 hr) treatments. The workgroup agreed to retain this requirement but acknowledged that more data are needed prior to making final recommendations concerning the need for and the specific protocol for the 24‐hr treatment. Environ. Mol. Mutagen. 35:185–190, 2000 Published 2000 Wiley‐Liss, Inc.

The spectral karyotype of L5178Y TK+/− mouse lymphoma cells clone 3.7.2C and factors affecting mutant frequency at the thymidine kinase (tk) locus in the microtitre mouse lymphoma assay

“…For example, what is the effect on mutant frequency of dilution of slow growing small tk 2/2 colony mutant over time in culture? Small colony mutants are considered to be formed from deletions of chromosome 11 extending beyond the TK gene yielding a slower growing phenotype [Moore et al, 1985[Moore et al, , 2007. Furthermore, the cell karyotype may also be expected to change over time, especially for a transformed mammalian cell line.…”

Interdisciplinary review for correlation between the plant origin capsaicinoids, non-steroidal antiinflammatory drugs, gastrointestinal mucosal damage and prevention in animals and human beings

“…Thymidine kinase proficient cells are sensitive to TFT, which causes the inhibition of cellular metabolism and halts further cell division. Thus the mutant cells are able to proliferate in the presence of TFT, whereas normal cells which contain thymidine kinase are not (Moore et al 2003).…”

“…The lowest positive concentration in the absence of S9 was 65 lg/mL. Although criteria for the determination of positive response in the lymphoma assay remained controversial according to the criteria used by the laboratory that conducted in these studies (Clive et al 1979;Moore et al 2003).These results can be interpreted as week toxicity. With longer exposures (about 24 h), higher capsaicin concentrations were too toxic for analysis and the mutant fractions were not significantly different from the controls at the usable concentrations.…”

Evaluation of U. S. National Toxicology Program (NTP) mouse lymphoma assay data using International Workshop on Genotoxicity Tests (IWGT) and the Organization for Economic Co‐Operation and Development (OECD) criteria

“…The IWGT held six meetings and reached consensus on several important aspects for conducting the assay and interpretation of the data. This working group recommended individual experiment acceptance criteria for both the microwell and soft‐agar versions of the MLA and developed an approach for interpreting data to provide more biologically relevant results (Moore et al , , , , , ). For the agar version of the MLA, a response is considered positive if the induced mutant frequency meets or exceeds the global evaluation factor (GEF) of 90 × 10 −6 ; for the microwell version of the MLA the GEF is 126 × 10 −6 .…”

“…Because the NTP database represents a large portion of the data and is readily available online, we chose to review those data. We utilized the significant modifications for data evaluation that were developed in recent years under the auspice of IWGT (Moore et al , , , , ) and included in OECD TG490 (OECD ), for our re‐evaluation. This manuscript presents the re‐evaluation results.…”