In vitro genotoxicity testing needs to include tests in both bacterial and mammalian cells, and be able to detect gene mutations, chromosomal damage and aneuploidy. This may be achieved by a combination of the Ames test (detects gene mutations) and the in vitro micronucleus test (MNvit), since the latter detects both chromosomal aberrations and aneuploidy. In this paper we therefore present an analysis of an existing database of rodent carcinogens and a new database of in vivo genotoxins in terms of the in vitro genotoxicity tests needed to detect their in vivo activity. Published in vitro data from at least one test system (most were from the Ames test) were available for 557 carcinogens and 405 in vivo genotoxins. Because there are fewer publications on the MNvit than for other mammalian cell tests, and because the concordance between the MNvit and the in vitro chromosomal aberration (CAvit) test is so high for clastogenic activity, positive results in the CAvit test were taken as indicative of a positive result in the MNvit where there were no, or only inadequate data for the latter. Also, because Hprt and Tk loci both detect gene-mutation activity, a positive Hprt test was taken as indicative of a mouse-lymphoma Tk assay (MLA)-positive, where there were no data for the latter. Almost all of the 962 rodent carcinogens and in vivo genotoxins were detected by an in vitro battery comprising Ames+MNvit. An additional 11 carcinogens and six in vivo genotoxins would apparently be detected by the MLA, but many of these had not been tested in the MNvit or CAvit tests. Only four chemicals emerge as potentially being more readily detected in MLA than in Ames+MNvit--benzyl acetate, toluene, morphine and thiabendazole--and none of these are convincing cases to argue for the inclusion of the MLA in addition to Ames+MNvit. Thus, there is no convincing evidence that any genotoxic rodent carcinogens or in vivo genotoxins would remain undetected in an in vitro test battery consisting of Ames+MNvit.

A core in vitro genotoxicity battery comprising the Ames test plus the in vitro micronucleus test is sufficient to detect rodent carcinogens and in vivo genotoxins.

Since the publication of the International Programme on Chemical Safety (IPCS) Harmonized Scheme for Mutagenicity Testing, there have been a number of publications addressing test strategies for mutagenicity. Safety assessments of substances with regard to genotoxicity are generally based on a combination of tests to assess effects on three major end points of genetic damage associated with human disease: gene mutation, clastogenicity and aneuploidy. It is now clear from the results of international collaborative studies and the large databases that are currently available for the assays evaluated that no single assay can detect all genotoxic substances. The World Health Organization therefore decided to update the IPCS Harmonized Scheme for Mutagenicity Testing as part of the IPCS project on the Harmonization of Approaches to the Assessment of Risk from Exposure to Chemicals. The approach presented in this paper focuses on the identification of mutagens and genotoxic carcinogens. Selection of appropriate in vitro and in vivo tests as well as a strategy for germ cell testing are described.

/pdf/mutagenicity-testing-for-chemical-risk-assessment-update-of-4299r4lh47.pdf

Mutagenicity testing for chemical risk assessment: update of the WHO/IPCS Harmonized Scheme.

ICH guideline S2 (R1) on genotoxicity testing and data interpretation for pharmaceuticals intended for human

Chromosomal translocations (CTs) are hallmark mutations of hematopoietic malignancy that result in the deregulated expression of oncogenes or the generation of novel fusion genes. The polymerase chain reaction (PCR) can be used to detect illegitimate recombinations of genomic DNA sequences as a more sensitive assay than cytogenetics for determining the presence of CTs. Both direct DNA-PCR and reverse transcriptase-PCR were used to examine healthy individuals for lymphoma- and leukemia-associated CTs. Two oncogene-activating CTs [t(14;18)(q32;q21) and t(8;14)(q24;q32)] and one fusion-gene CT [t(2;5)(p23;q35)] from lymphomas and five fusion-gene CTs from leukemia [t(9;22)(q34;q11), t(4;11)(q21;q23), t(15;17)(q22;q11), t(12;21)(p13;q22), t(8;21)(q22;q22)] were detected in such studies. The biological implication is that CTs associated with malignant tumors may also be found in cells that are not neoplastic. CTs are characteristic attributes of neoplastic clones but are by themselves insufficient to cause malignant transformation. A better understanding of the special biology of non-neoplastic CT-bearing cells will provide insight into their putative role as tumor precursors. Prospective epidemiological studies are needed to determine whether such cells in healthy individuals may, in some instances, become clonogenic founders of lymphoma or leukemia.

Lymphoma- and leukemia-associated chromosomal translocations in healthy individuals.

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Testing (IWGT), comprised of experts from Japan, Europe, and the United States, met on August 29, 2003, in Aberdeen, Scotland, United Kingdom. This meeting of the MLA Workgroup was devoted to reaching a consensus on the appropriate approach to data evaluation and on acceptance criteria for both the positive and negative/vehicle controls. The Workgroup reached consensus on the acceptance criteria for both the agar and microwell versions of the MLA. Recommendations include acceptable ranges for mutant frequency, cloning efficiency, and suspension growth of the negative/vehicle controls and on criteria to define an acceptable positive control response. The recommendation for the determination of a positive/negative test chemical response includes both the requirement that the response exceeds a defined value [the global evaluation factor (GEF)] and that there also be a positive dose-response (evaluated by an appropriate statistical method).

Mouse lymphoma thymidine kinase gene mutation assay: Follow‐up meeting of the international workshop on Genotoxicity testing—Aberdeen, Scotland, 2003—Assay acceptance criteria, positive controls, and data evaluation

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Test Procedures held a second harmonization meeting just prior to the U.S. Environmental Mutagen Society Meeting in New Orleans, LA, in April 2000. The discussion focused on several important aspects of the MLA, including: 1) cytotoxicity measures and their determination, 2) use of a 24-hr treatment, 3) the ability of the assay to detect aneugens, and 4) concentration selection. Prior to the meeting the group developed Microsoft Excel Workbooks for data entry. Ten laboratories entered their data into the workbooks (primarily as coded chemicals). The Excel Workbooks were used to facilitate data analysis by generating an extensive set of graphs that were evaluated by the meeting participants. Based on the Workgroup's previous agreement that a single cytotoxicity measure should be established for both the microwell and soft agar versions of the assay, the Workgroup analyzed the submitted data and unanimously agreed that the relative total growth (RTG) should be used as the cytotoxicity measure for concentration selection and data evaluation. The Workgroup also agreed that the various cytotoxicity measures should be calculated using the same methods regardless of whether the soft agar or microwell version of the assay was used. In the absence of sufficient data to make a definitive determination, the Workgroup continued to endorse the International Committee on Harmonization recommendation for the use of 24-hr treatment and made some specific 24-hr treatment protocol recommendations. The Workgroup recognized the ability of the MLA to detect at least some aneugens and also developed general guidance and requirements for appropriate concentration selection.

Mouse lymphoma thymidine kinase gene mutation assay: follow-up International Workshop on Genotoxicity Test Procedures, New Orleans, Louisiana, April 2000.

V(D)J recombinase is normally involved in the highly regulated rearrangement of immunoglobulin and T-cell-receptor gene segments (in B and T cells, respectively) to form functional antibody genes and T-cell-receptor genes. Occasionally, this tightly controlled process acts on inappropriate places in the genome and results in deletions and translocations. Some of these illegitimate V(D)J recombinase-mediated events have been implicated in the genetic changes associated with several forms of leukemia and lymphoid malignancy. We have developed a sensitive, specific polymerase chain reaction (PCR)-based assay to quantify such events in the peripheral blood cells of humans. This assay detects a V(D)J recombinase-mediated deletion in the hprt gene, which codes for a housekeeping enzyme and is not implicated in cancer development. Alterations in this gene serve as a surrogate indicator for these illegitimate events, which may be occurring throughout the genome. The assay involves a heminested PCR with two sets of primers. Multiple replicates of genomic DNA (each representing 4 × 105 cells) are amplified with specific primers under conditions in which a single copy will give a detectable PCR product. Poisson statistics are then used to estimate the deletion mutant frequency. The frequency of cells with the hprt deletion among 20 healthy young adults ranged from < 1.3 × 10−7 to 4.1 × 10−7 and was compared with the frequency of t(14; 18) previously determined in these same individuals. No correlation was found between the frequencies of these two measures of genomic rearrangement. The DNA sequences of the deletion junctions were determined and provided evidence for multiple independent mutations in some individuals. This assay may serve as a biomarker for the level of illegitimate V(D)J recombination occurring in peripheral blood cells of humans. Environ. Mol. Mutagen. 29:28–35, 1997 © l997 Wiley-Liss, Inc.

Quantification of hprt gene deletions mediated by illegitimate V(D)J recombination in peripheral blood cells of humans.

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Mouse lymphoma thymidine kinase gene mutation assay: follow-up International Workshop on Genotoxicity Test Procedures, New Orleans, Louisiana, April 2000.

Quantification of hprt gene deletions mediated by illegitimate V(D)J recombination in peripheral blood cells of humans.