reaction product of toluenediisocyanate ( reaction mass of isomers: 65 % 2,4- and 35 % 2,6-diisocyanate) and aniline (molarratio 1:2)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

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: Reaction mass of aniline and m-tolylidene diisocyanate (also known as A054)

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies: A054: Reverse Mutation Assay 'Ames Test' using Salmonella typhimurium and Escherichia coli, Envigo Research Limited, Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD, UK, study number: XW05CL. GLP compliant; positive in the presence of S9 mammalian metabolic activation system.

A054: Reverse Mutation Assay 'Ames Test' using Salmonella typhimurium and Escherichia coli, Envigo Research Limited, Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD, UK, study number: HN22JS. GLP compliant; positive in the presence of S9 mammalian metabolic activation system.

These studies were conducted on two separately-prepared batches of the registered substance. No substance-specific in vivo studies were identified.

- Available non-GLP studies: No substance-specific data were identified.

- Historical human data: No substance-specific data were identified.
- (Q)SAR: The results of QSAR modelling have limited applicability for this type of UVCB
- In vitro methods: The two in vitro tests in bacteria that were conducted as the requirement for Annex VII registration for this endpoint (see List of ‘Available GLP studies’ above) gave positive results. Further in vitro testing in mammalian cells for this endpoint is considered unnecessary in view of the existing positive in vitro results.
- Weight of evidence: Reaction mass of aniline and m-tolylidene diisocyanate was found to exhibit mutagenic activity in two in vitro bacterial reverse mutation assays using four strains of Salmonella typhimuriam (TA 1535, 1537, TA98 and TA 100) and one strain of Escherichia coli (WP2 uvra) in the presence of S9 only. No in vivo genotoxicity studies were identified for this substance.
- Grouping and read-across: Although Reaction mass of aniline and m-tolylidene diisocyanate is part of a category for REACH registration and read-across for a number of endpoints, no other member of the category gave a similar positive mutagenic response in bacteria when subjected to testing. Since Reaction mass of aniline and m-tolylidene diisocyanate showed a positive response in two separate studies, it is not considered appropriate to read across the negative results in other category substances.
- Substance-tailored exposure driven testing [if applicable]:N/A
- Approaches in addition to above [if applicable]: N/A
- Other reasons [if applicable] :N/A

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:
Since Reaction mass of aniline and m-tolylidene diisocyanate is not classified for mutagenicity or carcinogenicity, there are no appropriate waivers for genetic toxicity testing. There is no evidence that the positive responses in the bacterial reversion assays are the result of unique interaction or specific metabolism between the bacteria and the test substance. Annex VII Column 2 of the REACH Regulation states that in the case of an in vitro gene mutation study in bacteria “further mutagenicity studies shall be considered in case of a positive result”, and “REACH Annex VII substances for which only a bacterial gene mutation test has been conducted and for which the result is positive should be studied further, according to the requirements of Annex VIII”. Annex VIII Column 2 then states that “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”. The Integrated Testing Strategy in ECHA guidance document Chapter R7a (ECHA 2017) indicates that in relation to Annexes IX and X “if there is a positive result in any of the in vitro studies from Annex VII or VIII and there are no appropriate results available from an in vivo study already, an appropriate in vivo somatic cell genotoxicity study should be proposed”. No in vivo studies on Reaction mass of aniline and m-tolylidene diisocyanate have been identified. The observation of positive responses in two bacterial reversion assays necessitates the consideration of in vivo testing. If the results of the proposed in vivo test are negative, then an in vitro assay for structural and numerical chromosomal aberrations will be performed.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed: In order to assess genotoxicity potential in vivo, an alkaline comet assay (OECD TG 489, 2016) is proposed. The purpose of the comet assay is to identify substances that cause DNA damage. Under alkaline conditions (>pH 13), the comet assay can detect single and double stranded breaks, resulting, for example, from direct interactions with DNA, alkali labile sites or as a consequence of transient DNA strand breaks resulting from DNA excision repair. These strand breaks may be repaired, resulting in no persistent effect, may be lethal to the cell, or may be fixed into a mutation resulting in a permanent viable change. They may also lead to chromosomal damage which is also associated with many human diseases including cancer (OECD 2016). The test will be conducted on rats via the oral route. If appropriate, a single sex (male) will be used. The substance showed no evidence of toxicity at the maximum treatment level in an acute oral toxicity study (2000 mg/kg bw). Somatic cells will be sampled and analysed from two tissues: liver (due to first pass metabolism exposure, the influence of S9 metabolism in the in vitro study, and the fact that it is a frequent site for carcinogenesis), and glandular stomach (site of contact tissue). Samples of duodenum will also be taken and the need for analysis of this tissue will be determined by the results from the first two tissues.

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

(2016)

Deviations:

not applicable

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

430-960-1

EC Name:

-

Molecular formula:

C21H20N4O2

IUPAC Name:

Reaction mass of aniline and m-tolylidene diisocyanate

Test material form:

solid

Test animals

Sex:

male

Administration / exposure

Route of administration:

oral: unspecified