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Genetic toxicity in vitro:

The mutagenic potential of Desmodur MT (MDI MT) was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of S9 mix according to OECD TG 471 (Herbold, 2007). Evidence of mutagenic activity was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed. Based on this test, the test substance was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.

Remarks on genetic toxicity in vitro studies with MDI

Aromatic diisocyanates are virtually insoluble in water and require an solvent to ensure homogeneous dispersion in in vitro genotoxicity assays. Dimethylsulphoxide (DMSO) has been used routinely as the solvent of choice for such assays. The validity of using DMSO as a solvent was queried by Gahlman et al (JETOC,1993) when it was found there is a chemical conversion of MDI to MDA in the solvent which could explain a number of positive responses recorded in some genotoxicity assays in vitro. A detailed evaluation of the stability of MDI in DMSO by Herbold et al (1998) and Seel et al (1999) showed that there was a rapid breakdown of MDI in DMSO with less than 40% of the initial amount remaining after 15 minutes with almost complete breakdown within 2 hours. A HPLC examination of the breakdown products showed the presence of MDA which is known to produce positive responses in various in vitro genotoxicity assays including mutations in Salmonella typhimurium. To determine if the positive results seen in in vitro genotoxicity assays when MDI was dissolved in DMSO were in fact a consequence of the chemical break down of MDI to MDA Herbold et al (1998) and Seel et al (1999) undertook a series of mutagenic investigations using dry ethyleneglycol dimethylether (EGDE) as the organic solvent as investigations indicated MDI was stable in this solvent with no formation of MDA.

The studies with Salmonella typhimurium showed quite clearly the absence of any mutagenic response when MDI was dissolved in EGDE. In contrast when MDI was dissolved in DMSO clear positive effects were seen consistent with generation of MDA. Based on such evaluation it was concluded that the results of in vitro genotoxicity studies undertaken using solvents such as DMSO must be treated with caution as any positive response may very well be an artifact of the testing conditions caused by the breakdown of the isocyanate into the mutagenic amine. Based on these observations the use of results from in vitro tests in aqueous cell systems are problematic because of interaction with the test system components. Those in vitro studies not addressing problems of hydrolysis of the substance (which represent the majority of in vitro investigations) are considered to be invalid, and not useful for determining the genotoxic potential of MDI. Because of these technical problems conducting mammalian cell gene mutation assays in vitro is challenging and interpretation of data problematic, thus an assessment relies on data from in vivo studies.

Genetic toxicity in vivo - read-across with other MDI substances:

A number of in vivo genotoxicity studies have been undertaken using MDI. A mouse micronucleus study has been reported in summary form by JETOC (1982). In summary MDI was dissolved in dry DMSO, mixed with corn oil and administered to mice by intra-peritoneal injection at doses of 32, 80 or 200 mg/kg (six male mice per treatment group). The mice were killed 24 hours following final treatment and incidence of polychromatic and normochromatic erythrocytes with micronuclei evaluated. There was no difference in incidence of micronuclei between animals treated with MDI and the untreated control group.

In another bone marrow micronucleus study conducted by Zhong and Siegel, (2000) Brown Norway rats were exposed to MDI by aerosol one hour a day for three weeks and then left for 7 days before being killed and the bone marrow examined for micronuclei. The authors reported an increase in micronuclei in the MDI treated animals as compared to controls. There are however several methodological problems with this study including: the test was not carried out according to standard protocol, lack of historical data in Brown Norway rat, lack of positive control group, treatment will have produced hyperthermia in the animals which is associated with increases in micronuclei, the exposure regimen employed a final 7 day holding period after final treatment prior to examination of the bone n marrow and the erythrocyte cell cycle is such that after 7 days holding period any polychromatic erythrocytes at treatment will have disappeared. As a result of these flaws in the study the data are impossible to interpret. To fulfill the need for an acceptable in vivo micronucleus study on MDI, Pauluhn and Gollapudi (2001) conducted a guideline, GLP, inhalation rat micronucleus study which also included the procedures used by Zhong and Siegel (1999). The results of this study indicate that aerosolised, inhaled MDI at concentrations as high as 118 mg/m³ air, (a concentration high enough to produce portal-of-entry specific toxic effects, including statistically significantly increased lung weights), failed to induce formation of micronuclei and cytogenetic damage in vivo. Other in vivo studies investigating relevant endpoints, such as DNA-adduct formation, have not demonstrated any significant binding to DNA in epidermis or olfactory tissues following topical or inhalation exposure to MDI in experimental rodents.

Some human exposure studies have reported possible alterations in DNA status, but these data were obtained with uncertain methodologies and the results are not easy to interpret.

Overall the data on genotoxicity show:

• Weight of scientific evidence supports the conclusion that MDI is not mutagenic or genotoxic

• As MDI is unstable in solvents such as DMSO and degrades rapidly to MDA results from the majority of in vitro genotoxicity test are unsuitable for assessing the genotoxic potential of MDI

• In vivo micronuclei studies in mouse and rat using contemporary protocol design and conducted according to GLP are negative • MDI shows no capacity to form DNA adducts

Short description of key information:
Desmodur MT (MDI MT) shows no mutagenic potential in the Salmonella/microsome test. In vivo micronucleus tests with MDI in mouse and rat were negative. MDI shows no capacity to form DNA adducts.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

According to CLP Regulation (EC) No.1272/2008 the non-classification of 4,4'-MDI (CAS No.101-68-8) was considered for the non-classification of MDI MT (CAS No.147993-65-5):

4,4'-MDI was not classified as a germ cell mutagen by DSD or GHS (there is conclusive data but it is not sufficient for classification). MDI MT was not mutagenic in the Ames test.