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Regulatory Submission Assays

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

Although guidelines do vary somewhat by governing body (US, EU, Japan, etc.) and by product type (Pharmaceuticals, Food Additives, Veterinary Medicine, Medical Devices, Industrial and Agricultural Chemicals, Consumer Products and Flavors and Fragrances), BioReliance provides a portfolio and consultation on the myriad of assays available to meet these regulations and product types. All assays conform to OECD guidelines (when applicable) and are GLP-compliant.

Assay Categories:

Bacterial Mutation Assays

  • Ames Test

In Vitro Mammalian Cell Gene Mutation Assays

  • L5178 TK+/- Mouse Lymphoma Assay (MLA)
  • CHO/HPRT Assay

In Vitro Cytogenetics

  • Chromosomal Aberration Assays
    • Options: HPBL, CHO, Rat Lymphocytes
  • Micronucleus Assay
    • Cells: TK6, HPBL, CHO
    • Analysis: Microscopy, FISH and CREST, Flow Cytometry

*Also available in EpiDerm™ 3D Reconconstructed Human Skin (RSMN Assay)

DNA Damage and Repair

  • Unscheduled DNA Synthesis (UDS)
  • Sister Chromatid Exchange (SCE)

Morphological Transformation

  • BALB/3T3 Transformation
  • Syrian Hamster Embryo (SHE) Cell Transformation

In Vivo Cytogenetics

  • Chromosomal Aberration Assays
    • Options: Mouse, Rat
  • Micronucleus Assay
    • Options: Species: Mouse, Rat
      Analysis: Microscopy, FISH and CREST, Flow Cytometry

Bacterial Mutation Assays

Ames Test for Genotoxicity

The Ames Test (Bacterial Reverse Mutation test) is required by regulatory agencies such as the
FDA, EPA, EMEA, EU, etc. prior to IND, chemical, or other submission. It provides a simple,
straight-forward measure of the mutagenicity or genotoxicity of small molecule formulations -
pharmaceuticals, cosmetics, flavors & fragrances, consumer products, and more.

At BioReliance, we are the exclusive owners of the original tester strains developed in Bruce
Ames’ laboratory and thus offer the most comprehensive array of methods, designs and options
for Ames Tests, including:

  • Ames Plate Incorporation Method
  • Ames Preincubation Method
  • Ames Treat and Plate Method

Consult with one of our genotoxicity experts to determine the toxicology assays that are best for
your product needs or use the search tool to the right to browse our Ames Test offerings.

Ames Test Basics

The Ames assay measures the direct mutation potential of a test substance using bacterial
cells that carry point mutations in genes required to synthesize the amino acid histidine.
Typically Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and Escherichia
coli WP2 are used, but others are acceptable under OECD guidelines. These mutated strains
fail to grow on histidine-deficient media. The basic concept is to incubate a substance with the
bacteria and score for the “rescue” of the bacteria allowing it to grow in the histidine-deficient
media, because the point mutations have been reversed by the test substance. The mutagenicity
of the test substance is proportional to the number of bacteria colonies observed. A 2-fold
increase over baseline bacterial growth qualifies a biologic or drug metabolite as genotoxic
according to FDA guidelines.

The strains of bacteria used are selected for drug-permeability, making the Ames Test an ideal
assay to identify potential genotoxic effects.

Micronucleus Assay (Micronucleus Test)

The micronucleus assay (micronucleus test) provides a simple straightforward measure of clastogenic and aneugenic agents resulting in chromosomal damage. We provide micronucleus assays for small molecule formulations of industrial chemicals, consumer products, agrochemicals, pharmaceuticals, and more.

At BioReliance, we offer a full battery of micronucleus tests and analysis options, including:

  • In vitro micronucleus tests
  • In vivo micronucleus tests
  • Micronucleus evaluation by Flow Cytometry
  • Micronucleus evaluation by microscopy

Consult with one of our genotoxicty experts to determine the toxicology assays that are best for your product testing needs or use the search tool to the right to browse our micronucleus offerings.

Micronucleus assay basics
The micronucleus assay examines the formation of small membrane bound DNA fragments or micronuclei in the cytoplasm of interphase cells caused by a tested agent. Micronuclei can originate from chromosome fragments lacking a centromere or whole chromosomes that are unable to migrate with the rest of the chromosomes during cell division. This test can be performed in both in vitro cultured cells and in vivo in mice and rats.

BioReliance routinely tests the following cell types and has many designs to fit your needs:

  • CHO (Chinese Hamster Ovary)
  • HPBL (Human Peripheral Blood Lymphocytes)
  • TK6

In vivo micronucleus tests include:

  • Rat or Mouse Bone Marrow
  • Rat or Mouse Peripheral Blood
  • Medical Device testing

In the in vitro assay, cell cultures are grown until chromosomal damage leads to the formation of micronuclei in binucleated or multinucleated interphase cells. Stained interphase cells are then examined microscopically for the presence of micronuclei. At least 1000 binucleated cells per duplicate cell culture are scored to assess the frequency of cells containing micronuclei. The cells also are classified as mononucleates, binucleates or multinucleates to estimate the proliferation index as a measure of genotoxicity. If a substance induces a concentration-related increase or a reproducible increase in the number of cells containing micronuclei, it is called positive. A positive result indicates that the test substance induces chromosome aberration or damage to the cell division apparatus. All micronucleus assays are performed with both positive and negative controls to ensure assay validity.

The in vivo micronucleus assay is a short-term cytogenetic assay for detecting agents that induce chromosomal breakage or spindle malfunction. Femoral bone marrow or peripheral blood is microscopically evaluated for the presence of polychromatic erythrocytes containing micronuclei. A clastogenic effect may be seen if micronuclei are formed during cell division as a result of chromosome breakage or malfunctioning of the mitotic spindle. In the case of chromosome breakage, acentric chromosome fragments may not be included in the nuclei of daughter cells forming single or multiple micronuclei in the cytoplasm. An aneugenic effect is seen if the test material interferes with the mitotic spindle apparatus; non-disjunction or lagging chromosome at anaphase may not be included in the nuclei of daughter cells forming single or multiple micronuclei in cytoplasm of the new cells. The incidence of micronucleated PCEs serves as an indicator of test article genotoxicity.

BioReliance has performed thousands of Micronucleus Assays for genotoxicity testing. Contact an expert today for your genotoxicity testing needs.

TK6 Micronucleus Assay

Recent research has identified a problem with gene tox assays using rodent cell lines, as they are shown to be p53-deficient. Assays using rodent cell lines such as CHO have shown a high rate of positive results, thus poorly predicting in vivo genotoxicity assays and rodent carcinogenicity. The use of TK6 cells in these assays has improved performance, because TK6 cells are derived from a human B lymphoblastoid cell line.

TK6 cells are:

  • Of human origin
  • p53-competent
  • Karyotypically stable
  • Less subject to donor-related variability that occurs occasionally with HPBL (human peripheral blood lymphocytes)

The TK6 micronucleus assay is ideal for use as a follow up assay to explore mechanism of action (aneugenic or clastogenic) from an initial genotoxicity result. In a TK6 micronucleus assay, either FISH or CREST techniques may be used without the need to interrogate additional cells beyond the basic assay design.

The TK6 Micronucleus assay:

  • Provides a reduction in the percentage of non-relevant positive results compared to p53-mutated cell lines
  • Eliminates the use of rodent cell lines
  • Lessens donor variability
  • Offers a more rapid turnaround time and more straightforward scoring than the in vitro Chromosome Aberration Assay

BioReliance has performed thousands of Micronucleus Assays for genotoxicity testing. Contact an expert today for your genotoxicity testing needs.

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