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Key value for chemical safety assessment

Additional information

The genotoxic potential of CS-1246 has been examined in a variety of in vitro and in vivo test assays. In vitro assays include bacterial mutation (), mammalian mutation, chromosomal aberration and UDS. Thein vivo assays are mouse micronucleus and in vivo UDS.

For the bacterial mutation assay CS-1246 was tested in five Salmonella tester strains with and without metabolic activation (Haworth, S.R. and Lawlor, T.E., 1982). No evidence mutagenicity was seen at any dose level in any of the tester strains and CS-1246 is considered negative for mutagenicity in this system. The mutagenic potential of CS-1246 was examined in an in vitro mammalian mutation assay by assessing the ability of CS-1246 to induce dose-related and significant increases in the frequency of TK+/- mutants as compared to the negative control values (Linscombe, V.A.et al, 2002) Under the experimental conditions CS-1246, both in the presence and absence of metabolic activation, induced dose-related and reproducible increases in the frequency of TK-/- mutants as compared to the negative control values and was considered to be positive in this in vitro mouse lymphoma (L5178Y TK+/-) forward mutation assay. The in vitro study examining the ability of CS-1246 to induce chromosomal aberrations in CHO cells failed to find any evidence of chromosome aberrations in either the activated or non-activated systems (Paika, I., 1991). There was no evidence of a treatment related increase in aberrations at any dose level in the presence or absence of metabolic activation. The positive controls exhibited an obvious positive response in both systems.The test material is considered negative in the chromosomal aberration test with Chinese hamster ovary cells.

The ability of CS-1246 to induce unscheduled DNA synthesis (UDS) was examined in rat hepatocytes obtained by in situ perfusion of rat livers and then maintaining the cells as monolayers on coverslips in a humidified incubator. Monolayers of hepatocytes were exposed to various concentrations of CS-1246 for 18-20 hours. The cells were then washed in fresh culture medium, the nuclei were swollen by incubating in sodium citrate for 8-10 minutes, fixed in acetic acid and methanol, and dried. The fixed monolayers on the coverslips were mounted on glass slides, dipped in Kodak NTB emulsion, and dried. They were stored for 8 days in a dessicant chamber and subsequently stained with Harris hematoxylin. Positive control cells were treated with 2-acetylaminofluorene (2-AAF) at 2.0 and 10.0 ug/mL while negative controls were untreated
Cells were examined microscopically and the nuclear grains were counted; normally-appearing nuclei were scored, and any occasional nuclei blackened by grains too numerous to count were excluded as cells in which replicative DNA synthesis occurred.Based on the comparison of grain counts in the nuclei of treated and untreated hepatocytes CS-1246 is considered to be negative in the UDS with primary rat hepatocytes (Paika, I., 1991). The presence of UDS in positive control cells confirmed the utility of the experiment.

The ability of SC-1246 to induce micronuclei was examined in mice using doses at 2,000, 1,000 or 500 mg/kg/day (Spencer, P.J., and Gorski, T.A., 2002).There were no significant differences in MN-PCE frequencies between the groups treated with the test material and the negative controls. The adequacy of the experimental conditions for the detection of induced micronuclei was ascertained from the observation of a significant increase in the frequencies of micronucleated polychromatic erythrocytes in the positive control group. Based on these data CS-1246 is considered negative in the in vivo micronucleus assay.

For the in vivo UDS assay rats were dosed with CS-1246 at 1000 and 2000 mg/kg/day (Cifone, M.A., 2002). At two time points post-dosing, (i.e. 2-4 hours and 14-16 hours) the animals were killed and cell cultures were obtained by perfusion of livers in situ. Hepatocyte monolayers were then incubated at 35-37oC in culture medium containing 10 uCi/mL 3H-TdR at 35 to 60 Ci/mmol. Three replicate cultures from each animal were used for the UDS assay, and one was used to assess attachment. Attachment efficiency, an estimate of the number and viability of cells attaching to the dishes, was determined for one culture from each animal. After a labeling period of about 4 hours, labeled cell cultures were washed twice, and then incubated for a further 16 to 20 hours in culture medium after which cell nuclei were swollen with sodium citrate for 8-10 minutes. The cell monolayers were then fixed in acetic acid and ethanol, dipped in Kodak NTB emulsion, dried and stored for 8 days in a dessicant chamber. Slides were then stained and examined microscopically for evidence of unscheduled DNA synthesis. The grain counts in the nuclei of the vehicle controls were within historical control ranges while the positive control, (dimethylnoytrosamine treated cells, showed clear increases in nuclear labeling indicating UDS. Cells from animals treated with CS-1246 showed no evidence of UDS and the material was considered negative at both time points.

 

Overall Assessment of Genotoxic Potential

In summary the overall weight of evidence shows that CS-1246 is not mutagenic.

References:

Cifone, M.A. (2002) In Vivo/In Vitro Unscheduled DNA Synthesis in Rat Primary Hepatocyte Cultures at Two Timepoints with a Dose Rangefinding Assay with BIOBAN CS-1246. Covance Laboratories The Dow Chemical Company Report No: DR-0365-7725-031. GLP, Unpublished.

 

Haworth, S.R. and Lawlor, T.E. (1982) Salmonella/ Mammalian-Microsome Plate Incorporation Mutagenicity Assay (Ames Test). Microbiological Associates,    The Dow Chemical Company Report No: DR-0365-7725-012.GLP, Unpublished

Linscombe, V.A., Schisler, M.R., and Treadway, K.F.  (2002) Evaluation of BIOBAN CS-1246 Antimicrobial in the Mouse Lymphoma (L5178Y TK+/-) Forward Mutation Assay.  The Dow Chemical Company Report No: DR-0365-7725-026  GLP, Unpublished

Paika, I. (1991) Chromosome Aberrations in Chinese Hamster Ovary (CHO) Cells.    

Toxikon Corporation. The Dow Chemical Company Report No: DR-0365-7725-013 GLP, Unpublished

 

Paika,(1991) Unscheduled DNA Synthesis in Rat Liver Primary Cultures.    

Toxikon Corporation. The Dow Chemical Company Report No: DR-0365-7725-014   GLP, Unpublished

Spencer, P.J., and Gorski, T.A. (2002) Evaluation of BIOBAN CS-1246 in the Mouse Bone Marrow Micronucleus Test. The Dow Chemical Company Report No: DR-0365-7725-032. GLP, Unpublished

 


Short description of key information:
The potential for genetic toxicity of the substance was evaluated in various in vitro and in vivo mutagenicity studies. The substance was negative in the Ames test, the chromosome aberration test, the unscheduled DNA synthesis, as evaluated in several in vitro mutagenicity and genotoxicity assays, and positive in a mouse lymphoma genotoxicity assay.

The substance EDHO was negative in the in vivo mouse micronucleus assay and in the in vivo UDS assay, designed to evaluate genotoxic potential in the whole animal.

On the basis of various negative mutagenicity tests results, there is currently no relevant indication that the substance is a germ cell mutagen.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based the available data regarding effects on mutagenicity, classification is not warranted according to EU Directive 67/548/EEC and EU CLP Regulation (EC) No. 1272/2008.