Genetic and in vitro toxicology studies

This guideline focuses on animal free approach for chemical hazard identification, classification and labelling. This test guideline addresses the human health endpoint skin corrosion. It makes use of reconstructed human epidermis (RhE) (obtained from human derived non-transformed epidermal keratinocytes) which closely mimics the properties of the upper layer of the human skin, i.e. the epidermis. The guideline aims at topically exposing test item to the skin tissues (3-D RhE) for 3 to 60 minutes. The RhE test method is based on the principle that corrosive chemicals can penetrate the stratum corneum by diffusion or erosion and are cytotoxic to the cells in the underlying layers. Cell viability is measured by enzymatic conversion of the dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, thiazolyl blue tetrazolium bromide]; into a blue formazan salt that is quantitatively measured after extraction from tissues.

This test guideline addresses the use of reconstructed human epidermis (RhE) (obtained from human derived non-transformed epidermal keratinocytes) which demonstrates original human epiderm structure. The guideline aims at exposing the skin tissues with the test item topically for 4 hours. Cell viability is measured by enzymatic conversion of the dye MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, thiazolyl blue tetrazolium bromide]; into a blue formazan salt that is quantitatively measured after extraction from tissues. Irritant chemicals are identified by their ability to decrease cell viability below defined threshold levels.

The Salmonella typhimurium strains (TA97A, TA98, TA100, TA102, and TA1535) have been employed for Ames mutagenicity or bacterial reverse mutation test. They have been chosen based on their sensitivity to mutations. The tester strains due to a mutation in their gene sequence requires histidine for growth. In general, the test item at defined concentrations is mixed with tester strains with minimum amount of histidine present, which is insufficient for normal growth of bacteria. If, the strains underwent a reverse mutation, the visible bacterial colonies will grow on agar plate. The test is performed both with and without S9 activation system.

The CHO cells having a confluency of 50- 60 % of cells are exposed to different concentrations of test item both in the presence as well as absence of metabolic activation system. The metabolic activation system comprises of S9 homogenate, NADP and isocitrate. The cells are observed for cytotoxicity before the harvest. After the time completion of exposure and expression, colcemid is used to arrest cells in the metaphase stage. Thereafter, the cells are removed and treated with hypotonic solution and fixative. The cells are fixed on microscopic slides and observed for chromosomal aberrations after staining with Giemsa stain.

The In vitro mammalian cell gene mutation test can be used to detect the potential of a chemical to induce gene mutations. The hypoxanthine phosphoribosyl transferase (Hprt) test detects mutations in the endogeneous hypoxanthine-guanine phosphoribosyl transferase gene (Hprt in rodent cells, HPRT in human cells); more specifically, base pair mutations, frameshift mutations and small deletions and insertions. To estimate the mutagenic potential of a test item, treated cells are placed under selective pressure so that only mutant cells can survive. Lack of HPRT activity results in resistance to the toxic analogue 6-thioguanine (6TG). Thus, mutant cells are able to proliferate in the presence of TG, whereas normal cells, which contain HPRT, are not. The most used cells for the HPRT test include the CHO, CHL and V79 lines of Chinese hamster cells, L5178Y mouse lymphoma cells, and TK6 human lymphoblastoid cells.

This guideline focuses on animal free approach for chemical hazard identification, classification and labelling. This guideline is based upon use of reconstructed human cornea like epithelium (RhCE) obtained from human derived non-transformed epidermal keratinocytes, human immortalized corneal epithelial cells, or primary culture human corneal epithelial cells. The test item is applied topically to a minimum of two RhCE tissue constructs, and tissue viability is measured following exposure and a post-treatment incubation period. Cell viability is measured by enzymatic conversion of the dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide]; into a blue formazan salt that is quantitatively measured after extraction from tissues.