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. |