Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: gene mutation
Type of information:
experimental study planned
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
cyclohex-1,2-ylenediamine


CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION

- Available GLP studies
No in vivo studies with the registered substance are available
- Available non-GLP studies
No in vivo studies with the registered substance are available
- Historical human data
No data available
- (Q)SAR
The mutagenic potential of the test substance was screened using the OECD QSAR Toolbox v4.4. The following profilers were checked, including all metabolites predicted by the simulators ‘in vivo Rat metabolism’ and ‘Rat liver S9 metabolism’ (No empirical metabolism data is available):
• DNA binding by OASIS: negative
• DNA binding by OECD: negative
• Carcinogenicity (genotox and nongenotox) alerts by ISS: negative
• DNA alerts for AMES, CA and MNT by OASIS: negative
• in vitro mutagenicity (Ames test) alerts by ISS: negative
• in vivo mutagenicity (Micronucleus) alerts by ISS: positive for the structural alert ‘H acceptor-path3-H acceptor’. It should be noted that this alert is associated with the possibility that the structure allows non-covalent binding to DNA via any two hydrogen bond-accepting atoms attached to two adjacent atoms. This represents a very unspecific structural alert for mutagenicity.
- In vitro methods
In a recent Ames assay (Guideline and GLP conform) with the registered substance the strains TA 100 and TA 98 tested positive for in vitro mutagenicity in the pre-incubation experiments. These strains, as well as all remaining strains were negative in the plate incorporation experiment.
In an older Ames assay, the substance tested negative. This assay was performed to the standards of its time, however it does not meet current standards because one of the required strains is missing. The two positive strains from the recent evaluation (TA 100 and TA98) were among the tested strains, however only the plate incorporation assay was performed in this study.
A recently performed MLA (Guideline and GLP conform) showed no mutagenic activity in mammalian cells.
An in vitro chromosome aberration test was performed in 2007 and gave a negative result. This is a high quality GLP study which meets current standards.

- Grouping and read-across
Hexamethylenediamine, a structurally similar amine, was tested for chromosomal aberration in vivo (similar to OECD 475, under GLP). No chromosomal aberrations were observed. In micronucleus assay (comparable to OECD 474, under GLP) the Chloride salt of hexamethylenediamine was tested. No increase of micronucleus formation was observed.
Albeit the negative outcome, these assays do not address the same mechanism of genotoxicity which is indicated by mutation in salmonella strains TA100 and TA98 (base substitutions and frameshift mutations). Additionally, available in vitro data for the mutagenicity of hexamethylenediamine is negative, which questions the validity as a read across source for the registered substance.
No in vivo data on other structurally sufficiently similar substances is available.
The available in vivo mutagenicity studies are therefore not sufficient to close the data gap by read-across.


- Weight of evidence
The positive result in the Ames test in the strains TA98 and TA100 occurred only in the pre-incubation assay and not in the plate incorporation. The mutation frequency was only hardly above the thresholds set in the criteria for a positive response. All other available data from Ames tests with similar substances (two amine groups on a C6 backbone with no other functional groups) uniformly gave a negative result.
Altogether, the recent Ames test on cyclohex-1,2-ylenediamine does not give rise to a particularly high level of concern and a negative outcome of the proposed in vivo testing is expected. Yet, no sufficiently convincing case can be made to disregard the positive in vitro result. According to Regulation (EC) No 1907/2006 (REACH) Annex VII and VIII, section 8.4, a positive in vitro genotoxicity test mandates the proposal of appropriate in vivo testing to ECHA.

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:

Waiving according to column 2 is not applicable to the registered substance.
FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
Based on the recommendations given in the Endpoint specific guidance, Chapter R.7a (Version 6.0, July 2017) for substances that appear preferentially to induce gene mutations, the transgenic rodent (TGR) assay, the rat liver UDS assay or the in vivo comet assay may be used to address this data gap.
The guidance states further, that ‘the choice of any of these three assays can be justified only if it can be demonstrated that the tissue(s) studied in the assay is (are) sufficiently exposed to the test substance…. This information can be derived from toxicokinetic data or, in case no toxicokinetic data are available, from the observation of treatment-related effects in the organ of interest.’
No toxicokinetic data is available which allows deducing target organs for possible mutagenicity. In a combined repeated dose and reproduction / developmental screening study by oral gavage of the registered substance, macro- and microscopic changes were noted in the liver (pale discoloration, hepatocellular vacuolization). Other target organs with minor findings were the lungs (alveolar inflammation), adrenal glands (vacuolization of the zona fasiculata) and the kidneys (weight change in conjunction with basophilia). Based on these effects, a systemic availability of the test substance can be assumed with sufficient certainty. The affected targets can be directly addressed with the in vivo comet assay (e.g. liver).
Taken together, an in vivo comet assay (OECD Test Guideline 489) fulfils the testing requirements and is proposed as follow-up in vivo test.
Pursuant the 3R principle, joined testing with an already commissioned oral 90-day study was considered. However, consultation with the testing laboratory revealed that a joined test design that would result in a lower total number of animals required was not possible.

Data source

Materials and methods

Test guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Test material

Constituent 1
Chemical structure
Reference substance name:
Cyclohex-1,2-ylenediamine
EC Number:
211-776-7
EC Name:
Cyclohex-1,2-ylenediamine
Cas Number:
694-83-7
Molecular formula:
C6H14N2
IUPAC Name:
cyclohexane-1,2-diamine
Test material form:
liquid

Results and discussion

Applicant's summary and conclusion