Julie Clements

The Mouse Lymphoma Assay

In this paper, the current status of the protocol for the Mouse Lymphoma Assay is discussed. A brief history describes the events leading to current protocol recommendations. Areas for further development such as cytotoxicity, 24-h treatments, acceptability criteria and statistical analysis are also considered. Recent guidelines are reviewed, and consensus issues from the Mouse Lymphoma workgroup assembled as part of the International Workshop on Genotoxicity Test Procedures (IWGTP) are included. There are two versions of the assay - soft agar and microwell - and both will be discussed. For assay procedures, the emphasis will be on a typical microwell protocol but an attempt will be made to highlight protocol variations between laboratories and between the microwell and agar versions of the assay.

Mouse Lymphoma thymidine kinase locus gene mutation assay : International Workshop on Genotoxicity Test Procedures Workgroup Report

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.

A method for assessment of the genotoxicity of mainstream cigarette-smoke by use of the bacterial reverse-mutation assay and an aerosol-based exposure system.

To date there are no widely accepted methods for the toxicological testing of complex gaseous mixtures and aerosols, such as cigarette smoke, although some modifications to the standard regulatory methods have been developed and used. Historically, routine testing of cigarettes has primarily focused on the particulate fraction of cigarette smoke. However, this fraction may not accurately reflect the full toxicity and mutagenicity of the smoke aerosol as a whole, which contains semi-volatiles and short-lived products of combustion. In this study we have used a modified version of the bacterial reverse-mutation (Ames) assay for the testing of mainstream smoke generated from 3R4F reference cigarettes with a Vitrocell(®) VC 10 exposure system. This method has been evaluated in four strains of Salmonella typhimurium (TA98, TA100, YG1024 and YG1042) and one strain of Escherichia coli (WP2 uvrA pKM101) in the absence and presence of a metabolic activation system. Following exposure at four concentrations of diluted mainstream cigarette-smoke, concentration-related and reproducible increases in the number of revertants were observed in all four Salmonella strains. E. coli strain WP2 uvrA pKM101 was unresponsive at the four concentrations tested. To quantify the exposure dose and to enable biological response to be plotted as a function of deposited mass, quartz-crystal microbalances were included in situ in the smoke-exposure set-up. This methodology was further assessed by comparing the responses of strain YG1042 to mainstream cigarette-smoke on a second VC 10 Smoking Robot. In summary, the Ames assay can be successfully modified to assess the toxicological impact of mainstream cigarette-smoke.

Suitable top concentration for tests with mammalian cells: mouse lymphoma assay workgroup.

The Mouse Lymphoma Expert Workgroup of the International Workshop for Genotoxicity Tests (IWGT) met in Basel, Switzerland in August of 2009. The Workgroup (WG) was tasked with discussing the appropriate top concentration for non-pharmaceuticals that would be required for the conduct of the mouse lymphoma assay (MLA) when sufficient cytotoxicity [to between 10 and 20% relative total growth (RTG)] has not been attained. The WG approached this task by (1) enumerating the various regulatory decisions/use for MLA data, (2) discussing the appropriate assays to which MLA data and assay performance should be compared and (3) discussing all the proposals put forth concerning the top concentration for non-pharmaceuticals. In addition, one of the members presented a summary of a re-evaluation of the National Toxicology Program MLA data using the IWGT harmonized guidance that was underway as a separate (non IWGT) activity, being conducted by two members of the Expert WG. The WG was asked to vote on each of the various proposals for top concentration for when cytotoxicity is not concentration limiting. While there was general agreement that the top concentration for non-pharmaceuticals should be re-evaluated and likely lowered from the current recommended levels, there was no agreement on a specific new recommendation.

Recommendations for spacing of test chemical concentrations in the mouse lymphoma tk mutation assay (MLA)

Recent test guidelines for the mouse lymphoma tk mutation assay (MLA) have highlighted the need to achieve 80-90% reduction in cell survival for a valid, robust assay with toxic chemicals. For many pharmaceuticals, under new ICH recommendations, this may be the only in vitro mammalian cell test that is performed. It was important to discover, therefore, how critical it is to achieve 80-90% toxicity, and how best to select the number and spacing of test concentrations to fulfil this requirement. We analysed data from 121 positive chemicals, provided by nine industrial and commercial laboratories, and found that for 17 chemicals (14%), the response profiles were so steep that using a conventional 2-fold dilution series of test concentrations would have failed to identify the active range (> 90% toxicity at one concentration, and no significant mutation at 50% of this dose), and positive responses would have been missed. Analysis of genotoxicity results in other test systems with these 17 chemicals revealed no differences in overall response profiles from the 104 chemicals that exhibited less steep MLA responses. The MLA results were therefore deemed to be equally biologically relevant. From this analysis, it is recommended that concentration spacing in the MLA needs to be closer than that obtained with a 2-fold dilution series, and a dilution factor where each concentration is 0.75 or 0.8 of the one above is recommended to identify the active range of positive mutagens.