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Genetic Toxicology
Battery of Assays

Inotiv provides all genetic toxicology tests needed for global regulatory compliance

Inotiv

A standard (harmonized) battery of genetic toxicology assays is an integral part of many global regulatory guidelines. Inotiv offers the most comprehensive portfolio of assays and study designs to meet all regulations for product types requiring genetic toxicology testing.

Assay categories:

Bacterial Reverse Mutation Assay (Ames Assay)

The Ames Test (Bacterial Reverse Mutation test) provides a simple, straight-forward measure of the mutagenicity of small molecule formulations of pharmaceuticals, chemicals, cosmetics, flavors and fragrances, consumer products, veterinary products, impurities, and more.

Bacterial-Reverse-Mutation-Assay

Exposure Methods: The plate incorporation method combines the test article with bacteria in top agar just prior to plating whereas the preincubation method co-incubates the test article with the bacteria before mixing with top agar and plating. The preincubation method is considered the more sensitive method for the detection of mutagens, especially for certain classes of chemicals.

At Inotiv, we offer a comprehensive array of methods, designs, and options for Ames tests, including:

    • Ames Plate Incorporation Method
    • Ames Preincubation Method
    • Ames Treat and Plate Method (for compounds containing histidine or tryptophan)
    • Enhanced Ames Test (EAT) for nitrosamine qualification
    • Prival modification of the Ames test for azo dyes
    • Ames for medical device biocompatibility testing per ISO 10993-3

Comparative Evaluation of Different Plating Methodologies and Sources of Induced S9 Metabolic Activation on the Mutagenic Potential of N-Nitrosodipropylamine Using the Ames Test

OECD Test Guideline 471

AMES Assay FAQ:

What is the Ames assay?

The Ames assay is a reverse mutation process that exposes histidine or tryptophan deficient auxotrophs to mutagens causing reversions to histidine and tryptophan proficient prototrophs (wild-type bacteria). The reversion process yields growth of revertant colonies on agar plates that are enumerated during evaluation.

What tester strains are typically used?

The standard Ames assay utilizes five strains of bacteria. Usually, four strains of Salmonella typhimurium (TA98, TA100, TA1535, and TA1537) and one strain of Escherichia coli (WP2 uvrA or WP2 uvrA (pKM101)). Salmonella tester strain TA102 can also be used in place of E.coli.

What are options for early screening?

Screening can be performed using a minimum of two Salmonella tester strains (TA98 and TA100). These two strains provide information for base-pair and frameshift mutations and have high concordance with the GLP assay. Screening can also be performed using up to all five tester strains as are used in the GLP assay. We also offer miniaturized versions of the assay performed in multi-well plates which require less test compound.

What is the next step if the Ames test is positive?

There are several options for a follow-up to a positive Ames test, including the Pig-a assay, transgenic rodent mutation, and duplex sequencing.

What is the Enhanced Ames Test (EAT)?

Regulatory agencies such as U.S. Food and Drug Administration (FDA), the European Medicines Agency (EMA), and others have provided updates to their guidance as related to testing of Nitrosamine Drug Substance-Related Impurities (NDSRIs). The Enhanced Ames Test provides test conditions that are considered optimal for determining the mutagenic potential of this class of compounds. In addition to use of specific tester strains and use of the preincubation methodology, the test conditions call for the use of both induced rat and hamster liver S9 at higher concentrations than routinely used in an Ames assay, and the inclusion of two additional N-nitrosamine positive controls. FDA EAT guidance and EU EAT guidance.

In Vitro Cytogenetics (In Vitro Micronucleus and Chromosome Aberration Assays)

in-vitro-cytogenetics

The in vitro micronucleus assay measures micronucleus formation in vitro, using human TK6 Cells, Human Peripheral Blood Lymphocytes (HPBL), Chinese Hamster Ovary (CHO), or other cell lines. Studies are performed with (+S9) and without (-S9) metabolic activation and include measurement of cytotoxicity by various methods (population doubling, cytokinesis blocked proliferation index (CBPI), Relative Viability Cell Count (RVCC) dependent on the cell type utilized.

Micronucleus Assay Options:

  • Cells: TK6, HPBL, CHO-WBL, CHO-K1, V79, CHL, A375, EpiDermTM Reconstructed Skin
  • Analysis: Microscopy or Flow Cytometry

 

Mechanism of Action (MOA) Determination: FISH and CREST

fish-crest-labelingFISH and CREST labeling investigate the mode of action for a test article that is positive in a micronucleus assay. FISH and CREST labeling identify whether a micronucleus contains a whole chromosome(s), as a result of an aneugenic mechanism, or a chromosome fragment(s) as a result of a clastogenic mechanism. An aneugenic mechanism may be useful in identifying a threshold, which is very important in the overall risk assessment of a compound. The micronucleus assay is ideal for use as a follow up assay to explore mode of action (aneugenic or clastogenic) from an initial genotoxicity result.

OECD TG 487
Revised OECD TG 487 Blog
Validation of A375 Cell Line for the In Vitro Micronucleus Assay

Picture6

The chromosome aberration assay measures both structural and numerical abnormalities in chromosomes in vitro. Studies are performed with (+S9) and without (-S9) metabolic activation and includes measurement of cytotoxicity either by mitotic inhibition or cell growth inhibition (utilizing relative increase in cell counts).

  • Chromosomal Aberration Assay
  • Cell Option:
    • Human Peripheral Blood Lymphocyte (HPBL)
    • Chinese Hamster Ovary Cells (CHO-WBL), (CHO-K1)

OECD Test Guideline 473

 

In Vitro FAQ:

Difference between chromosome aberration and in vitro MN?

Chromosome aberration assay provides more comprehensive information regarding the structural damage (breaks, gaps, interchanges between chromatid and chromosome) and high-level information on numerical changes (endo-reduplication, centromeric split, polyploidy) in chromosomes. The micronucleus assay provides quantitative damage in terms of micronucleus induction. A follow up testing using either FISH or CREST is required to understand the mechanism of micronucleus formation.

Which cell lines are preferred to use for in vitro assays?

HPBL Micronucleus assay, HPBL chromosome aberration assay, and TK6 micronucleus assay should be preferred over the rodent based cell lines (CHO-WBL, CHO-K1, V79, CHL cells) since they are human based test system with functional p53 genes.

Microscopic vs flow analysis?

Both modalities are valid and acceptable methods of analysis. Microscopic analysis is qualitative data based on expert visual observation of the slides that allows the confirmatory review of specimens on slides as needed. Flow cytometer based MN evaluation is performed using larger sample sizes and provides quick, reliable, and reproducible objective data using validated flow cytometers.

When to use FISH/CREST?

When micronucleus induction is positive in either microscopy or flow cytometry, a mechanistic study using FISH/CREST is recommended to be performed for risk assessment to identify Mode of Action.

IN VITRO MAMMALIAN CELL GENE MUTATION ASSAYS

mammalian-cell-gene-mutation-assays

The HPRT assay measures induction of mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene in Chinese hamster (CHO) cells. Preliminary dose-finding study and mutation assay is performed with (+S9) and without (-S9) metabolic activation. The assay design includes cloning multiple test article concentrations in triplicate plates per replicate culture for cytotoxicity and five plates per replicate culture for mutant selection.

OECD Testing Guideline 476

The Mouse Lymphoma assay measures induction of mutations at the thymidine kinase (TK) gene in L5178Y mouse lymphoma cells. Preliminary toxicity assay and mutation assays are performed with (+S9) and without (-S9) metabolic activation. The mutation assay includes cloning multiple test article concentrations in three replicate plates per dose for cytotoxicity and mutant selection. Colony sizing is performed for vehicle and positive controls and for mutagenic test articles. Soft agar for mutant selection.

OECD Testing Guideline 490

 

Mammalian Mutation FAQ:

Difference between HPRT and Mouse Lymphoma?

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 MLA can detect a broader spectrum of genotoxic effects than the HPRT assay.

When is the mammalian mutation run?

Mutagenicity testing evaluates the potential of a chemical to possibly cause genetic mutations. The mammalian mutation assay is run to confirm mutagenic activity such as an Ames positive or large colony MLA, and also to evaluate mutagenicity where the Ames assay may not be appropriate such as the mutagenicity assessment of liquid nanoparticles (LNPs) and antibiotics.

Does mammalian mutation replace the Ames assay?

No, a standard test battery suggests for in vitro genotoxicity tests, recommended to be suitable for regulatory purposes, 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 a transgenic rodent mutation assay.

In Vivo Micronucleus

Micronucleus Assay Options:

  • Species: Mouse, Rat
  • Analysis: Microscopy or Flow Cytometry
  • Blood samples or bone marrow smears prepared at other laboratories and shipped to Inotiv for micronucleus frequency assessment

The in vivo micronucleus assay is a short-term cytogenetic assay for detecting agents that induce chromosomal breakage or spindle malfunction. Mice or rats are used to detect micronuclei in polychromatic erythrocytes (immature reticulocytes) in the bone marrow or peripheral blood. Femoral bone marrow or peripheral blood is evaluated either microscopically or by flow cytometry for the presence of polychromatic erythrocytes containing micronuclei.

The typical study design is to use three test article dose levels, concurrent vehicle, and positive controls with two days of dose administration, followed by one collection time point, using six animals/sex/dose level. Plasma is collected for demonstration of bone marrow exposure to the test article. Dose selection is based on a dose range finding test. The assay can be conducted with a single sex if there is no difference in toxicity between the sexes in the dose range finding study.

The micronucleus assay can also be integrated into repeat dose toxicity testing. Inotiv accepts blood samples stabilized in EDTA or bone marrow smears prepared at other laboratories for micronucleus frequency assessment.

Mechanism of Action (MoA) Determination: CREST Antibody Labeling

Labeling bone marrow slides with a CREST (Calcinosis, Raynaud's phenomenon, Esophageal involvement, Sclerodactyly, Telangiectasia) anti-centromere antibody investigates the mode of action of a test article that is positive in a micronucleus assay. CREST labeling determines whether a micronucleus contains a whole chromosome(s), an indication of an aneugenic mechanism, or chromosome fragment(s), an indication of a clastogenic mechanism. An aneugenic mechanism may allow a threshold for genotoxic damage, which is very important in the overall risk assessment of a compound. The micronucleus assay is ideal for use as a follow up assay to explore MoA (aneugenic or clastogenic) of an initial genotoxicity result. Slides containing bone marrow depleted of nucleated cells can be prepared for CREST antibody staining using the same animals used in the standard micronucleus assay design.

 

In Vivo MN FAQ:

Which species is best to use for MN?

The most commonly used species are rats and mice. Species selection is often based on test material availability, existing exposure information, and previous experience with the test substance in repeat dose toxicity studies.

What are pros and cons of flow cytometric vs microscopic analysis?

Microscopic analysis utilizes a fluorescent nucleic acid dye to distinguish immature erythrocytes from mature erythrocytes (also known as reticulocytes) by color and to identify brightly stained micronuclei in these cells, typically, 4,000 polychromatic (immature) erythrocytes are scored per animal. Generally, microscopy is used for assessing micronuclei formation directly in bone marrow and this assay format has been in use for many decades.

Flow cytometry utilizes antibodies to differentially label surface proteins to distinguish populations of immature erythrocytes from mature erythrocytes and platelets in peripheral blood; a DNA stain is used to gate out nucleated cells (leukocytes) and to identify micronuclei in erythrocytes. Typically, 20,000 polychromatic erythrocytes are scored per animal. Flow cytometric analysis provides a more rapid and robust means to enumerate micronucleated cells without potential for scorer bias. Evaluation of micronucleated erythrocytes in blood serves as a surrogate measurement for induction of micronuclei in bone marrow and permits multiple time points to be assessed using the same animals. Both analysis formats are accepted by regulatory agencies.

Do I need to collect plasma for bioanalysis?

Proof of bone marrow exposure is required to support a negative result in the micronucleus assay. In the absence of clinical signs indicative of systemic exposure, a statistical change in the immature to mature erythrocyte ratio, or preexisting data demonstrating bone marrow bioavailability using the same species and administration route, direct detection of test material in the bone marrow or plasma is necessary. No demonstration of bone marrow exposure is required when the test material is administered intravenously.

Which dose routes are available?

Test material is most frequently administered by oral gavage, but intravenous, subcutaneous, intramuscular, and nasal instillation administration routes can be used. Intraperitoneal injection is not recommended without good justification. Administration via diet and drinking water are typically reserved for longer dosing regimens.


Speak to a GLP Genetic Toxicology expert to learn more about how we can help move your product development program forward.

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Genetic Toxicology Battery of Assays