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Nanomaterials (NMs) present unique challenges in safety evaluation. An international working group, the Genetic Toxicology Technical Committee of the International Life Sciences Institute's Health and Environmental Sciences Institute, has addressed issues related to the genotoxicity assessment of NMs. A critical review of published data has been followed by recommendations on methods alterations and best practices for the standard genotoxicity assays: bacterial reverse mutation (Ames); in vitro mammalian assays for mutations, chromosomal aberrations, micronucleus induction, or DNA strand breaks (comet); and in vivo assays for genetic damage (micronucleus, comet and transgenic mutation assays). The analysis found a great diversity of tests and systems used for in vitro assays; many did not meet criteria for a valid test, and/or did not use validated cells and methods in the Organization for Economic Co-operation and Development Test Guidelines, and so these results could not be interpreted. In vivo assays were less common but better performed. It was not possible to develop conclusions on test system agreement, NM activity, or mechanism of action. However, the limited responses observed for most NMs were consistent with indirect genotoxic effects, rather than direct interaction of NMs with DNA. We propose a revised genotoxicity test battery for NMs that includes in vitro mammalian cell mutagenicity and clastogenicity assessments; in vivo assessments would be added only if warranted by information on specific organ exposure or sequestration of NMs. The bacterial assays are generally uninformative for NMs due to limited particle uptake and possible lack of mechanistic relevance, and are thus omitted in our recommended test battery for NM assessment. Recommendations include NM characterization in the test medium, verification of uptake into target cells, and limited assay-specific methods alterations to avoid interference with uptake or endpoint analysis. These recommendations are summarized in a Roadmap guideline for testing.

We have developed a system to study mutations that affect xanthine-guanine phosphoribosyltransferase gene (gpt) expression in hypoxanthine-guanine phosphoribosyltransferase-deficient CHO cells that have been transformed by the plasmid vector pSV2gpt. One isolated transformant, designated AS52, carries a single copy of the Escherichia coli gpt gene stably integrated into the high-molecular-weight DNA and expresses the bacterial gene for the enzyme xanthine-guanine phosphoribosyltransferase. Mutants deficient in this enzyme can be induced in the AS52 cell line by a variety of mutagens, and spontaneous or induced mutants can be selected for resistance to 6-thioguanine (Tgr). Two Tgr clones derived from the AS52 line were analyzed by Southern blot hybridization and were found to contain deletions involving at least a portion of the gpt gene. Because of the small size and stability of the integrated pSV2gpt plasmid, and the well-defined selection protocol for mutant isolation, the AS52 line offers promise as a system suitable for the study of mutation at the molecular level in CHO cells.We have developed a system to study mutations that affect xanthine-guanine phosphoribosyltransferase (XGPRT) gene (gpt) expression in hypoxanthine-guanine phosphoribosyltransferase-deficient (HGPRT-) CHO cells which have been transformed by the plasmid vector pSV2gpt (10, 11). pSV2gpt carries the Escherichia coli gpt gene and through the use of the simian virus 40 early promoter expresses the bacterial gene for XGPRT in mammalian cells. One isolated CHO transformant, designated AS52, carries a single copy of the gpt gene stably integrated into the high-molecularweight DNA. Bacterial XGPRT and mammalian HGPRT are functionally analogous except that under physiological conditions XGPRT catalyzes the formation of xanthine 5'-monophosphate from xanthine, whereas HGPRT does not. Selection protocols utilizing the purine analog 6-thioguanine for the study of mutation at the hgprt locus in CHO cells (12) can be used to select for mutations at the single gpt gene in the pSV2gpt-transformed CHO line. In this study, we show that mutation induction at the gpt locus in pSV2gpt-transformed CHO cells can be quantified and that specific deletion mutations which affect gpt gene function are the basis for the observed 6-thioguanine-resistant (Tg') phenotype in two mutants derived from this transformed line.The parental line, CHO-K1-BH4 (hgprt+), has been previously described (6). The X3/5 line (HGPRT-), which served as the transformation host for the pSV2gpt gene transfer experiments, was derived from the CHO-K1-BH4 line after an X-ray irradiation of 500 rads and selection in 10 ,uM 6-thioguanine; several lines of evidence indicate that it carries a stable, nonrevertible mutation at the hgprt locus (R. Machanoff, M.S. thesis, University of Tennessee, Knoxville, Tenn., 1982 supplemented with 5% heat-inactivated and dialyzed fetal bovine serum (F12FCM5) at 5% C02, 37°C, and 100%o humidity.The AS52 line is a gpt transformant of X3/5 which carries a single ...

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).

A collaborative international trial was conducted to evaluate the reproducibility and transferability of an in vivo mutation assay based on the enumeration of CD59-negative rat erythrocytes, a phenotype that is indicative of Pig-a gene mutation. Fourteen laboratories participated in this study, where anti-CD59-PE, SYTO 13 dye, and flow cytometry were used to determine the frequency of CD59-negative erythrocytes (RBC(CD59-)) and CD59-negative reticulocytes (RET(CD59-)). To provide samples with a range of mutant phenotype cell frequencies, male rats were exposed to N-ethyl-N-nitrosourea (ENU) via oral gavage for three consecutive days (Days 1-3). Each laboratory studied 0, 20, and 40 mg ENU/kg/day (n = 5 per group). Three sites also evaluated 4 mg/kg/day. At a minimum, blood samples were collected three times: predosing and on Days 15 and 30. Blood samples were processed according to a standardized sample processing and data acquisition protocol, and three endpoints were measured: %reticulocytes, frequency of RET(CD59-) , and frequency of RBC(CD59-) . The methodology was found to be reproducible, as the analysis of technical replicates resulted in experimental coefficients of variation that approached theoretical values. Good transferability was evident from the similar kinetics and magnitude of the dose-related responses that were observed among different laboratories. Concordance correlation coefficients showed a high level of agreement between the reference site and the test sites (range: 0.87-0.99). Collectively, these data demonstrate that with adequate training of personnel, flow cytometric analysis is capable of reliably enumerating mutant phenotype erythrocytes, thereby providing a robust in vivo mutation assay that is readily transferable across laboratories.

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