Table 1: Major in vitro and in vivo testing models for mutation.

Test model

Test guidance (Adopted)

Design (taken from the OECD test guidance)

Bacterial reverse mutation test

OECD TG 471 [30] - in vitro

Bacterial cell suspensions are exposed to the test substance (liquid or solid) in the presence and in the absence of an exogenous metabolic activation system. At least five different analysable concentrations of the test substance should be used. The recommended maximum test concentration for soluble non-cytotoxic substances is 5 mg/plate or 5 ml/plate. There are two methods: the plate incorporation method and the preincubation method. For both techniques, after two or three days of incubation at 37 °C, revertant colonies are counted and compared to the number of spontaneous revertant colonies on solvent control plates.

Mammalian cell gene mutation test

OECD TG 476 [72] - in vitro

Genetic endpoints measure mutation at hypoxanthine-guanine phosphoribosyl transferase (HPRT), and at a transgene of xanthineguanine phosphoribosyl transferase (XPRT). The HPRT and XPRT mutation tests detect different spectra of genetic events.

Cells in suspension or monolayer culture are exposed to, at least four analysable concentrations of the test substance, both with and without metabolic activation. They are subcultured to determine cytotoxicity and to allow phenotypic expression prior to mutant selection. Mutant frequency is determined by seeding known numbers of cells in medium containing the selective agent to detect mutant cells, and in medium without selective agent to determine the cloning efficiency (viability). After a suitable incubation time, colonies are counted.

OECD TG 490 [73] - in vitro

This TG includes two distinct in vitro mammalian gene mutation assays requiring two specific TK heterozygous cells lines: L5178Y TK ± 3.7.2 C cells for the mouse lymphoma assay (MLA) and TK6 TK ± cells for the TK6 assay. Genetic events detected using the TK locus include both gene mutations and chromosomal events. Cells in suspension or monolayer culture are exposed to, at least four analysable concentrations of the test substance, both with and without metabolic activation. They are subcultured to determine cytotoxicity and to allow phenotypic expression prior to mutant selection. Cytotoxicity is usually determined by measuring the relative cloning efficiency (survival) or relative total growth of the cultures after the treatment period. The treated cultures are maintained in growth medium for a sufficient period of time, characteristic of each selected locus and cell type, to allow near-optimal phenotypic expression of induced mutations. Mutant frequency is determined by seeding known numbers of cells in medium containing the selective agent to detect mutant cells, and in medium without selective agent to determine the cloning efficiency (viability). After a suitable incubation time, colonies are counted.

Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays

OECD TG 488 [74] - in vivo

This TG detects gene mutations in transgenic rats or mice that contain multiple copies of chromosomally integrated plasmid or phage shuttles. The transgenes contain reporter genes for the detection of various types of mutations induced by test substances. Administration is done 28 consecutive days followed by a 3-day recovery period, during which unrepaired DNA lesions are fixed into stable mutations. Genomic DNA is isolated from the tissue (s), and mutations are scored by recovering the transgene and analysing the phenotype of the reporter gene in a bacterial host deficient for the reporter gene. Mutant frequency is calculated by dividing the number of plaques/plasmids containing mutations in the transgene by the total number of plaques/plasmids recovered from the same DNA sample.