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The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/ apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.

The Alamar Blue (AB) assay is widely used to investigate cytotoxicity, cell proliferation and cellular metabolic activity within different fields of toxicology. The use of the assay with nanomaterials (NMs) entails specific aspects including the potential interference of NMs with the test. The procedure of the AB assay applied for testing NMs is described in detail and step-by-step, from NM preparation, cell exposure, inclusion of interference controls, to the analysis and interpretation of the results. Provided that the proper procedure is followed, and relevant controls are included, the AB assay is a reliable and high throughput test to evaluate the cytotoxicity/proliferation/metabolic response of cells exposed to NMs.

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Cell transformation assays (CTAs) are in vitro carcinogenicity tests measuring morphological transformation of cells either as transformed colonies (SHE cells) or foci (C3H/10T1/2 and BALB/c 3T3 including Bhas 42 cells) derived from a single cell. CTAs such as Bhas 42 CTA can detect both genotoxic and nongenotoxic carcinogens. When used as an initiation assay to test tumor-initiating activity, cells at low density are treated with a test chemical for 3 days, whereas a promotion assay to test for tumor-promoting activity, near-confl uent cells are treated with a test chemical for a period of 10 days. The Bhas 42 CTA has advantages compared with BALB/c 3T3 and other CTAs due to its simplicity, higher sensitivity, less time needed for assay performance, and robustness (exemplifi ed by its adaptation to a high-throughput method). The Bhas 42 CTA has been validated together with other CTAs and recommended for development of an OECD guideline. The assay has already been applied in testing various chemical and physical agents including particles and nanomaterials. Protocols for both 6 and 96-well plate formats of initiation and promotion assays are described in detail.