Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study planned
Study period:
The study will be performed in 2020 or in the following years depending on ECHA decision.
Justification for type of information:
Testing proposal Mammalian Erythrocyte Micronucleus Test

TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out:
3-((5-(3-Acetoxy-2,2-dimethylpropylideneamino)-1,3,3-trimethylcyclohexyl)methylimino)-2,2-dimethylpropyl acetate

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies: There are no GLP studies available for this substance covering the endpoint of gene mutation in vivo
- Available non-GLP studies: There are no non-GLP studies available for this substance covering the endpoint of gene mutation in vivo
- Historical human data: There are no historical human data available on gene mutation for the substance.
- (Q)SAR: There is no valid (Q)SAR model available to address gene mutation and or chromosome aberration in vivo and which outcome would be reliable enough to rule out the concern raised from a positive in vitro Chromosome Aberration test (ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R 7a: Endpoint specific guidance, 2017)
- In vitro methods: Three in vitro tests in regards to genotoxicity were conducted with the registered substance:
- Bacterial reverse mutation assay (Ames): negative
- Gene mutation study in mammalian cells (HPRT): negative
- Chromosome Aberration study in mammalian cells: positive

The test item indicated a clastogenic potential under the conditions of a chromosome aberration assay in vitro. Based on ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R 7a (2017) the follow-up procedure on a positive chromosome aberration assay in vitro is to conduct a chromosome aberration study in vivo such as a mammalian erythrocyte micronucleus test in vivo (OECD 474).

- Weight of evidence: There is no data available which is sufficient for a weight of evidence approach.
- Grouping and read-across: There are no substances which apply for read across addressing gene mutation in vivo in this case.

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:

The substance is fully registered according to REACH Annex VIII. Therefore, three in vitro tests on mutation in bacteria, mammalian cells as well as chromosome aberration are legally required. Moreover, according to REACH Annex VIII Section 8.4 “Further mutagenicity studies shall be considered in case of a positive result”. Thus, the registrant considers it his duty to conduct a respective mammalian erythrocyte micronucleus test in vivo.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
Details on study design / methodology proposed:

Based on the available in vitro data, mammalian erythrocyte micronucleus test in vivo according to OECD Test Guideline 474 is proposed to assess the clastogenic properties of the test substance in vivo. This study is considered to be the appropriate test system to investigate clastogenic potential of bioavailable substances, i. e. substances or their metabolites (hydrolysis products) that become readily systemically available upon ingestion. Furthermore, historical control data are available for various tissues. The test substance is proposed to be administered orally by gavage to mice. Doses will be based on data available for acute as well as repeated oral toxicity studies.
Based on the results of the acute and repeated oral toxicity studies and taken the OECD requirements into consideration, a maximum dose of 2000 mg/kg bw/day, and two additional doses (separated by a factor of 2 to 4) are proposed at a single administration.
It is further proposed to assess the erythrocytes of the bone marrow, rather than peripheral blood, since there is more experience and historical control data available for this approach which makes it more reliable.
Six animals per dose or control group are proposed to reach a minimum of five analysable animals for one sex according to the Guideline. Since the available data did not demonstrate relevant differences between males and females, the use of males only is proposed.

Data source

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Type of assay:
mammalian erythrocyte micronucleus test

Test animals

Species:
rat

Administration / exposure

Route of administration:
oral: gavage

Results and discussion

Applicant's summary and conclusion