Mammalian Mutation Assays
HPRT Test and Mouse Lymphoma Assay
(In Vitro Mammalian Cell Gene Mutation Assays)
HPRT Assay
The HPRT in vitro mammalian cell gene mutation test (OECD 476) is a genetic toxicology assay that measures induction of gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus. Typically the HPRT test is performed using Chinese hamster (CHO) cells and can identify a range of genetic alterations including base pair mutations, frameshift mutations, and small deletions or insertions.
The experimental design of the HPRT assay at Inotiv includes the exposure of replicate cell cultures to a range of test article concentrations, with cytotoxicity assessed in triplicate cultures per concentration, and four independent cultures per concentration designated for mutant selection. Testing is conducted in the presence (+S9) and absence (–S9) of metabolic activation to assess both direct and metabolically mediated mutagenic potential.
The HPRT assay can be used as a follow-up test to gain deeper understanding from a positive Ames assay and is referenced specifically as part of the draft FDA guidance on follow-up tests for Ames positive results. The HPRT assay is currently also used as a secondary assay in the testing of nitrosamine impurities and is an invaluable tool for mutagenicity assessment in situations where the Ames test is less effective, such as with antibiotics or with lipid nanoparticles (LNP).
Mouse Lymphoma Assay
The Mouse Lymphoma assay (MLA) (OECD 490) is a forward mutation genetic toxicology assay that measures induction of mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells. The MLA can effectively detect gene mutations as well as structural chromosomal damage caused by a test substance. This assay is part of the ICH S2(R1) guidance as a part of Option 1 of the genetox test battery.
The MLA is conducted using suspension cultures. A range of test article concentrations are evaluated across replicate cultures per dose level, including triplicate cultures for cytotoxicity and mutant selection evaluation. The assay is performed with (+S9) and without (-S9) metabolic activation for short term and extended exposure. Inotiv utilizes the soft agar method for mutant selection. Colony sizing is performed for vehicle and positive controls and for mutagenic test articles.
Per ICH S2(R1) guidance document, the MLA is considered equivalent to the in vitro chromosome aberration assay and in vitro micronucleus assay and are interchangeable in the standard testing battery for pharmaceuticals.
In Vitro Mammalian Cell Gene Mutation Assay FAQ:
Difference between HPRT test and Mouse Lymphoma test?
The HPRT gene mutation assay and the mouse lymphoma assay (MLA) are both mammalian gene mutation assays that can detect a wide range of chemicals that cause DNA damage and gene mutation. However, the HPRT assay is more specific for point mutation-inducing chemicals than the MLA. The HPRT assay is generally more specific for point mutation-inducing agents, whereas the MLA can detect a broader spectrum of genotoxic effects including large deletions and chromosomal damage.
When is the mammalian mutation test run?
Mutagenicity testing evaluates the potential of a chemical to induce genetic mutations. The mammalian mutation assay can be run to confirm mutagenic activity such as a positive Ames test and to evaluate mutagenicity where the Ames assay may be unsuitable, such as for the mutagenicity assessment of liquid nanoparticles (LNPs) and antibiotics. Additionally, the MLA test can be run as an in vitro option within the ICH S2(R1) standard battery of assays.
Does mammalian mutation replace the Ames assay?
Typically not, as a standard regulatory test battery should include 2 or 3 validated tests with at least one test on bacteria and one test on mammalian cell cultures.
What is follow-up to a positive mammalian mutation assay?
The follow-up to a positive mammalian mutation assay is a Pig-a mutation assay and/or a transgenic rodent mutation assay.
Explore other genetic toxicology assays:
| Mutation | Cytogenetic Damage | DNA Damage |
|---|---|---|
| Ames assay | In Vitro Micronucleus assay | Comet assay |
| HPRT assay | Chromosome Aberration assay | |
| Mouse Lymphoma assay | In Vivo Micronucleus assay | |
| Pig-a assay |
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