In vitro Genotox

Bacterial Reverse Mutation Test (Ames test) using Salmonella typhimurium and E. coli WP2uvrA

It detects mutations which revert mutations originally present in the tester strains and which restore the functional capability of the bacteria to synthesise an essential amino acid. The revertant bacteria are detected by their ability to grow in the absence of the amino acid required by the parent tester strain. The tester strains used for the bacterial reverse mutation test are the histidine auxotrophic strains of Salmonella typhimurium TA98 and TA1537 (Frame shift mutation), TA100, TA102 and TA1535 (Base pair substitution) and tryptophan auxotrophic strain of Escherichia coli WP2uvrA (Base pair substitution). The bacterial reverse mutation test is rapid, inexpensive and relatively easy to perform. JRF has expertise and huge historical data base to conduct study by using different methods and customize protocol based on chemical properties of the test item. JRF has conducted more than 1000 of GLP studies as per different regulatory guidelines such as OECD, OCSPP (EPA), EC, ICH and JMAFF. JRF offers Ames test following three different types of exposure method:

• Plate incorporation
• Pre-incubation
• Treat and Plate Method

In vitro Mammalian Chromosome Aberration Test using Human Peripheral Blood Lymphocytes or Chinese Hamster Ovary - K1 Cell Line
The in vitro chromosomal aberration (CA) test detects structural aberrations and may give an indication for numerical chromosome aberrations (polyploidy) in cultured mammalian cells caused by the test chemical. At JRF, we conduct in vitro chromosomal aberration test using either CHO-K1 cell line or human peripheral blood lymphocytes. Cytotoxicity is evaluated by assessing inhibition of mitotic index. Cells in metaphase are analysed for the presence of chromosomal aberrations both in the absence and presence of S9 activations. JRF has adopted all the revised OECD guideline changes (OECD, 2014). As per recent OECD recommendations, 150 metaphases are scored per culture and 300 metaphase per dose concentration. JRFs standard study plan comprises both short exposure both in the absence and presence of S9 and continuous exposure in the absence of S9. JRF has conducted more than 200 of GLP studies as per different regulatory guidelines such as OECD, OCSPP (EPA), EC, ICH and JMAFF.

In vitro Mammalian Cell Gene Mutation Test (HPRT assay) using Chinese Hamster Ovary-K1 cell line
Cells deficient in Hypoxanthine-guanine Phosphoribosyl Transferase (HGPRT or HPRT), due to mutation, are resistant to the cytotoxic effects of the purine analogue (6-thioguanine). HPRT proficient cells are sensitive to 6-thioguanine which causes the inhibition of cellular metabolism and halts further cell division.
The assay can detect a wide range of chemicals capable of causing DNA damage that leads to gene mutation. The test follows a very similar methodology to the thymidine kinase (TK) mouse lymphoma assay (MLA), and both are included in the guidelines for mammalian gene mutation tests (OECD (1997). The HPRT methodology is such that mutations which destroy the functionality of the HPRT gene are detected by positive selection using a toxic analogue, and HPRT −mutants are seen as viable colonies. JRF has conducted more than 100 GLP studies as per different regulatory guidelines such as OECD, OCSPP (EPA) and EC.    

In vitro micronucleus Test Using Human Peripheral Blood Lymphocytes and CHO-K1 cell line

JRF Global’s top-notch genotox team is proud to offer the in vitro micronucleus test as a preferred alternative to the in vitro chromosomal aberration test.

Genotoxic carcinogenic compounds change normal cell behavior by damaging DNA. One of the mechanisms for DNA damage is attributed to incorrect chromosomal replication. During normal cell division, an equal number of replicated chromosomes are present in the nuclei of both daughter cells. In the presence of certain carcinogenic compounds, however, the replication process is disrupted and a piece of the replicated chromosome breaks away and is not incorporated in either of the daughter nuclei. Such a chromosome may then go on to form a micronucleus.

The in vitro micronucleus test identifies genotoxic, potentially carcinogens by detecting both clastogens and aneugens. It delivers rapid multi-parametric assessment with the benefits being small amounts of the test compound requirements, and is easier to observe than the Chromosomal Aberration Tests. This test complements other genotoxicity assays, to provide a stronger mechanistic insight into the genotoxic potential of the test compound.

JRF Global offers the study from our state-of-the-art facilities in conjunction with the 3Rs of animal research.

Services available at following locations:

• JRF India, Gujarat, INDIA, India