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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An in vitro genetic toxicity study is available, conducted with Reaction mass of aniline and m-tolylidene diisocyanate. In the bacterial reverse mutation assay (Ames test) (Thompson, 2017), the test item was considered to have induced a weak but reproducible increase in the revertant colony frequency of a single strain of Salmonella typhimurium (TA98) in the presence of S9-mix only, at higher concentrations, where precipitation was also observed. No increases in the frequency of revertant colonies were recorded for any of the other bacterial strains, at any concentration of the test item, either with or without metabolic activation (S9-mix). The test item was considered to be weakly mutagenic to a single strain of Salmonella typhimurium (in the presence of S9-mix only) at the upper concentration levels under the conditions of this test. A repeat Ames test was commissioned with the substance Reaction mass of aniline and m-tolylidene diisocyanate, in order to determine if the same result would be observed.

In the repeat study (Thompson 2018a), conducted using a different batch of test item compared to the original 2017 study, the test item induced a statistically significant, dose-related and reproducible increase in the frequency of revertant colonies in the Salmonella typhimurium strains TA98 and TA100 at the upper dose levels in the presence of metabolic activation (S9 -mix) only. Therefore, Reaction mass of aniline and m-tolylidene diisocyanate was concluded to be mutagenic on the basis of this test result. It is of note that as was observed in the Thompson 2017 study, precipitation was also observed at and above 500 µg/plate.

An additional Ames test was also conducted with the second category member, Reaction product of m-tolylidene diisocyanate and cyclohexylamine, in order to determine if the same effects are observed with this test item. The Ames test conducted with Reaction product of m-tolylidene diisocyanate and cyclohexylamine (Thompson 2018b), concluded that there was no visible reduction in the growth of the bacterial background at any dose level, for the experiments with and without metabolic activation. There were also no biologically relevant increases in the frequency of revertant colonies for any bacterial strains, with any dosage, with and without metabolic activation, in both experiment 1 and 2. Therefore Reaction product of m-tolylidene diisocyanate and cyclohexylamine was considered to be non-mutagenic.

Based on the information generated from these studies, it can therefore be concluded that Reaction product of m-tolylidene diisocyanate and cyclohexylamine is not mutagenic and no further investigations of this substance are required. However, Reaction mass of aniline and m-tolylidene diisocyanate is considered to be mutagenic based on the results of the two Ames tests. 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”. Therefore, based on the REACH regulation, a testing proposal is submitted for Reaction mass of aniline and m-tolylidene diisocyanate for further in vivo genotoxicity studies, beyond the requirement of Annex VII, to further investigate the positive results observed in the two Ames tests (Thompson (2017) and Thompson (2018a)).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

PU TDI-II substances all contain the same core structure, with cyclic groups (aromatic or aliphatic) attached. PU TDI-II structures are therefore similar between all category members, and organisms will be exposed to very similar compounds. Organisms would be exposed to common structures, only differing by the cyclic groups attached to the core structure. In the body, there may be metabolism of the PU TDI-II structures, however due to the structural similarity of the parent compounds any metabolites are also likely to be similar. 

Justification for classification or non-classification

An in vitro genetic toxicity study has been conducted on one substance in the TDI-II category. In the bacterial reverse mutation assay (Ames tests) (Thompson, 2017) The test item was considered to have induced a weak but reproducible increase in the revertant colony frequency of a single strain of Salmonella typhimurium (TA98) in the presence of S9-mix only, at higher concentrations, where precipitation was also observed. No increases in the frequency of revertant colonies were recorded for any of the other bacterial strains, at any concentration of the test item, either with or without metabolic activation (S9-mix). The test item was considered to be weakly mutagenic to a single strain of Salmonella typhimurium (in the presence of S9-mix only) at the upper concentration levels under the conditions of this test. A repeat study is being performed on this substance, and an additional study is being conducted on the second category member, to clarify the positive result observed in the initial study.

 

Classification for mutagenicity is not based on the results of a single in vitro assay. Further Ames tests have been commissioned on this substance and a second member of the category to clarify the initial results. Depending on the results of the additional studies further investigations will be conducted as required.