Registration Dossier

Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study planned
Study period:
2018
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out:
Zinc, bis(O,O-bis(1-methylethyl) phosphorodithioato-.kappa.S)bis(cyclohexanamine)-, (T-4)- (CAS 52585-16-7)

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies:
A reliable in vitro gene mutation study in bacteria according to OECD TG 471 and GLP is available (Envigo CSR, 2016). The test substance (CAS 52585-16-7) was tested at concentrations up to 5000 µg/plate in 2 independent experiments, including the plate incorporation and preincubation method. Precipitates were visible at the two highest doses tested. Vehicle and positive controls were valid and lay within or marginally above the range of historical control data. Based on the results, the test substance (CAS 52585-16-7) did not induce an increase in reversions in the S. typhimurium strains TA 98, 100, 1535 and 1537, and in the E.coli strain WP2 uvrA with or without metabolic activation. Therefore, Zinc, bis(O,O-bis(1-methylethyl) phosphorodithioato-.kappa.S)bis(cyclohexanamine)-, (T-4)- (CAS 52585-16-7) can be considered as non-mutagenic in bacterial cells.
The clastogenic activity of Zinc, bis(O,O-bis(1-methylethyl) phosphorodithioato-.kappa.S)bis(cyclohexanamine)-, (T-4)- (CAS 52585-16-7) was investigated in an in vitro mammalian cell micronucleus test in cultured peripheral human lymphocytes according to OECD guideline 487 and GLP (Envigo CSR, 2016). In the first experiment, the test substance was tested in the absence and in the presence of metabolic activation for a 4-h exposure time. In the second experiment, the test substance was tested for a 20-h treatment period in the absence of S9 mix. In Experiment I, a statistical significant increase (1.05%) in micronucleated cells at the lowest evaluated concentration (39.79 μg/mL) in the presence of S9 mix and a statistical significant increase (1.10%) in micronucleated cells at the highest evaluated concentration (213.2 μg/mL) in the absence of S9 mix was observed. However, both values can be declared as biologically irrelevant, because they are well within the range of the historical laboratory control data (0.02 – 1.15% in the absence and 0.08 – 1.20% in the presence of S9 mix). In Experiment II in the absence of S9 mix a statistical significant increase (1.65%) in micronucleated cells was observed at the highest evaluated concentration (177.8 μg/mL). The value is outside of the 95% control limit of the laboratory historical control data (0.05 – 1.05%). Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei. In conclusion, it can be stated that under the experimental conditions reported, the test substance induced micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, Zinc, bis(O,O-bis(1-methylethyl) phosphorodithioato-.kappa.S)bis (cyclohexanamine)-,(T-4)- is considered to be clastogenic in the in vitro micronucleus test, when tested up to precipitating or the highest evaluable concentrations.
- Available non-GLP studies: not available
- Historical human data: not available
- (Q)SAR: not applicable
- In vitro methods: Both in vitro studies are described under “available GLP-studies”.
- Weight of evidence: not available
- Grouping and read-across: not available

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- According to Reach Regulation (EC) 2006/1907, Annex VIII, 8.4.3, Column 2, appropriate in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VIII.
FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed: An in vitro Mammalian Cell Micronucleus Test conducted according to OECD 487 with peripheral cultured human lymphocytes revealed a clastogenic potential for the registered substance. Therefore, additional in vivo data are needed, and the alkaline single-cell gel electrophoresis assay (Comet Assay according to OECD TG 489) is considered the most appropriate follow up test, to confirm or not the positive results of the in vitro test. In fact, the detection of DNA strand breaks is the principle of this method. These DNA strand breaks may result from direct interactions with DNA, alkali labile sites or as a consequence of incomplete excision repair. Therefore, the alkaline comet assay recognizes primary DNA damage that would lead to gene mutations and/or chromosome aberrations, but also detects DNA damage that may be effectively repaired or lead to cell death. The comet assay can be applied to almost every tissue of an animal from which single cell or nuclei suspensions can be made, including specific site of contact tissues.

Data source

Materials and methods

Results and discussion

Applicant's summary and conclusion