ICH recommended standard genotoxicity test combination
Combination one
a.Ames
b. In vitro chromosome aberration test or in vitro micronucleus test or in vitro mouse lymphoma tk test, one of these three tests can be selected.
c. In vivo micronucleus test or in vivo bone marrow cell chromosome aberration test; taking peripheral blood lymphocytes for cytogenetic analysis after animal administration is acceptable, but not commonly used.
Combination two
a.Ames
b. Two different tissues are used for in vivo genotoxicity assessment, usually including in vivo micronucleus test and another in vivo test. The second intra-individual test usually chooses to assess the DNA strand breaks in the animal’s liver.
Ames is the most relevant in vitro test for carcinogen detection.
Bacterial reverse mutation (Ames) test
• Used to detect genetic mutations caused by DNA damage. By detecting the ability of the test substance to cause mutations in certain specially constructed mutants of the test strain, that is, causing the strain to mutate from histidine/tryptophan-dependent to prototrophic type, it is judged whether the test substance is a mutagen.
• The test should be conducted simultaneously with and without s9 metabolic activation system.
• ※ Adding trace amounts of histidine or tryptophan to the agar medium where the bacteria are cultivated allows the bacteria to grow a few hours before, manifesting as background moss. When the added histidine/tryptophan is exhausted, only the mutated bacteria can continue to grow into visible mutant colonies.
Different types of Ames tests
GLP test uses standard Ames test.
The tests commonly used in the early stage of drug screening are Mini-Ames test, Micro-Ames test, AmesII test, etc., which is characterized by low dosage.
The Mini/Micro Ames test usually selects 2 strains (ie, TA98 and TA100, which can detect 98% mutagen). In order to eliminate the possibility of another 2%, the same 5 strains as the standard Ames test can be selected.
The AmesII test uses strain TA98 and mixed strains TA7001 to 7006. After pre-cultivation and serial dilution of the bacteria and the test substance, they are cultured at 30°C overnight, and then inoculated with each selective reagent 5-FU and pH sensitive agent bromocresol On the purple 384 well, the degree of yellowing of the culture medium was detected by an ultraviolet spectrophotometer to judge the mutagenicity of the test substance.
Ames test flow
Test strain (Ames Test System)
At least 5 strains should be selected
• TA1535
• TA1537 or TA97 or TA97a
• TA98
• TA100
• WP2 uvrA or WP2 uvrA (pKM101) or TA102
Each strain has different mutations in the operon encoding histidine/tryptophan biosynthesis.
More laboratories abroad choose WP2, while domestically use TA102.
Test strain genotype and mutation type
Test strain genotype and detection sensitivity
The presence of Rfa membrane mutations increases the permeability of cell membranes to macromolecular chemicals
The excision repair function of uvrB-strains is defective, and the strains cannot excise DNA adducts. Yes, these strains are sensitive to the mutagenicity and bacterial toxicity of a large number of mutagen.
The plasmid pKM101 contains the muc+ gene, which participates in the DNA repair pathway induced by DNA damage, while giving bacteria resistance to the mutagenicity of the mutagen while increasing the susceptibility to mutation. Strains containing plasmid pKM101 had a higher number of spontaneously reverted mutant colonies.
TA1537, TA97, and TA97a all contain cytosine repeats and are located in the mutation-sensitive sites within the corresponding histidine target sites. They are similarly sensitive to frameshift mutagens that cause base deletions in these frameshift hotspots.
TA98 and TA100 are sensitive to most mutagenic agents (data shows that 98% of these two strains tested positive for mutagen, so only these two strains can be detected in the preliminary screening test.)
Hydroxyanthraquinones can only be detected by strain TA1537
Oxidizing mutants and DNA cross-linking agents hydrazine can only be detected by strains TA102, WP2urA, WP2uvrA (pKM101) containing AT site mutations
Both strains TA1535 and TA100 have a fever-base substitution mutation at the hisG46 gene locus, the only difference is that TA100 contains the pKM101 plasmid, thereby increasing mutation sensitivity. However, the expert team showed through a large amoun types of Ames tests t of data that about 5% of the 659 known mutagens can only be detected by TA1535, not TA100.
Choice of solvent
• Water: water-soluble compounds
• Dimethyl sulfoxide (DMSO): No water-soluble compound preferred
• Ethanol
• Acetone
• Infrequently used vehicles: set up a parallel negative control
