Table 2 Criteria for the assay-specific evaluation of the validated in vitro mammalian cell genotoxicity assays. Obligatory criteria are presented in italics. The non-obligatory criteria were applied to evaluate the significance of the acceptable test results

 1. Adequate dose range: Doses with no or low cytotoxicity and moderate cytotoxicity should be included. For non-toxic materials a maximum dose of ~ 250 µg/ml is considered adequate

 2. If the result is positive only at highly cytotoxic doses (close to 50–60% cytotoxicity for micronucleus test or 10–20% survival for gene mutation tests) the conclusions of the study must be reevaluated

 3. Cellular uptake should be confirmed

Mammalian cell micronucleus (MN) test

 4. Background MN frequency must be provided

 5. The concurrent positive controls must elicit a statistically significant increase in MN frequency

 6. Concurrent cytotoxicity assessment must be performed as described in OECD TG487 (with Cyt-B CBPI or RI; without Cyt-B RPD or RICC)

 7. A minimum of 2000 cells have been scored per concentration. A minimum of two replicates or independent experiments have been performed

 8. If Cytochalasin B is used, it is added ≥ 6 h after starting the exposure to allow uninhibited cellular uptake. Tests with positive results are accepted regardless of this criterion

 9. There are no major problems in the study design (e.g. less than 3 doses with up to 50–60% cytotoxicity, only cytotoxic doses tested, harvesting schedule does not fall within 1.5–2 cell cycles, treatment time less than 3 h, inappropriate solvent without vehicle control, too high solvent concentration)

 10. Treatment schedule: The cells have completed at least one cell cycle in the presence of the NM so that NMs taken up by the cells may come into direct contact with the DNA when the nuclear membrane breaks down during mitosis. Tests with positive results are accepted regardless of this criterion

Mammalian chromosomal aberration (CA) test

 11. Background CA level must be provided

 12. Positive control induces a statistically significant increase in CAs

 13. Concurrent cytotoxicity assessment is performed as described by the OECD guideline (RPD or RICC, MI is acceptable for primary lymphocytes)

 14. A minimum of 300 metaphases per concentration (unless clearly positive response) are analyzed. A minimum of two replicates or independent experiments are performed

 15. There are no major problems in the study design (e.g., less than 3 doses with up to 50–60% cytotoxicity, only cytotoxic doses tested, clearly inadequate treatment time, harvesting schedule does not fall within 1.5 cell cycles, inappropriate solvent without vehicle control, too high solvent concentration)

 16. Treatment schedule: The cells have completed at least one cell cycle in the presence of the NM so that NMs taken up by the cells may come into direct contact with the DNA when the nuclear membrane breaks down during mitosis. Tests with positive results are accepted regardless of this criterion

Mammalian cell Hprt, Xprt, and Tk gene mutation tests and mouse lymphoma assay (MLA)

 17. Negative control mutation frequency must be reported

 18. The concurrent positive controls must elicit a statistically significant increase in mutant frequency

 19. Concurrent cytotoxicity evaluation must be performed with appropriate cytotoxicity parameter, as described by OECD TGs (RS for Hprt/Xprt/Tk; RTG for MLA)

 20. A minimum of two replicates or independent experiments are performed

 21. There are no major problems in the study design (e.g., less than 4 doses above 10–20% survival, only cytotoxic doses tested, treatment time less than 3 h, inadequate phenotypic expression time—for MLA 2 days, for Tk 3–4 days, for Hprt/Xprt a minimum of 7–9 days, inappropriate solvent without vehicle control, too high solvent concentration)

 22. Treatment schedule: The cells have completed at least one cell cycle in the presence of the NM so that NMs taken up by the cells may come into direct contact with the DNA when the nuclear membrane breaks down during mitosis. Tests with positive results are accepted regardless of this criterion