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Genetic toxicity in vitro

In an Ames test according to OECD TG 471 evidence of mutagenic activity of Desmodur VP.PU 60WF14 (TDI Biuret) was seen (Herbold, 2010). On Salmonella typhimurium strains TA 98, TA 102 and TA 1537 a biologically relevant increase was found in the mutant count compared to the corresponding negative control. Positive response was found only with S9 mix. In the absence of a metabolic activation system no mutagenic activity was found with the strains TA 98, TA 100, TA 102, TA 1535 and TA 1537. Based on these results, Desmodur VP.PU 60WF14 is considered to be mutagenic in the Ames test with metabolic activation.

 

In another test on point mutagenic effects Desmodur VP.PU 60WF14 (TDI Biuret) was investigated at the hypoxanthine-guanine phosphoribosyl transferase locus (HPRT test) in Chinese hamster V79 cells according to OECD TG 476 (Entian, 2010). Without and with S9 mix Desmodur VP.PU 60WF14 induced no decreases in survival to treatment or in relative population growth. However, precipitation of the test substance in the culture medium was observed at 32 µg/ml and above. Without and with S9 mix there was no biologically relevant increase in mutant frequency above that of the negative controls. Based on these results, Desmodur VP.PU 60WF14 is considered to be non-mutagenic in the V79/HPRT Forward Mutation Assay, both with and without metabolic activation.

 

The clastogenic potential of Desmodur VP.PU 60WF14 (TDI Biuret) was evaluated in a chromosome aberration test on Chinese hamster V79 cells in the presence and absence of S9 mix according to OECD TG 473 (D'Acquisto, 2010). Without and with S9 mix cytotoxic effects were not observed up to 60 µg/ml after 4 hours treatment and up to 48 µg/ml after 18 hours treatment. Precipitation in the medium did not occur. None of the cultures treated with Desmodur VP.PU 60WF14 in the absence and in the presence of S9 mix showed biologically relevant or statistically significant increased numbers of aberrant metaphases. Based on this test, Desmodur VP.PU 60WF14 is considered not to be clastogenic for mammalian cells in vitro.

 

Genetic toxicity in vivo

Since no in vivo data on genetic toxicity are available for TDI Biuret, such data have been 'read across' from TDI (justification of read-across see below).

A number of in vivo genotoxicity studies have been carried out with TDI. A slight increase in numbers of micronucleated erythrocytes was measured in a non-GLP micronucleus assay in rats exposed to TDI via inhalation (Loeser 1983; Owen, 1980 ). As the increase was not significant, occurred at only one dose level and because of the probably hyperthermia caused by the treatment the result was not considered to be biologically significant. Negative results were obtained with mice in the same study using similar exposures. Negative results have also been seen in a well conducted micronucleus assay in mice using inhalation route of exposure (Mackay, 1992) and an unscheduled DNA synthesis assay examining effects in liver and lungs in rats after acute and sub-acute inhalation exposures to TDI (Benford and Riley, 1988). Commercial grade TDI was also inactive in inducing sister chromatid exchanges and micronuclei in lung cells after intratracheal instillation in rats (Whong et al, 1991, cited in Zeiger 2005). Studies examining DNA adduct formation have produced mixed results and are inconclusive as to their relevance to human exposures.

Conclusion

In summary, weight of scientific evidence supports the conclusion that TDI Biuret is not mutagenic or clastogenic. In an Amest test TDI Biuret showed positive results in three Salmonella strains after metabolic activation, whereas no indications for a mutagenic potential were seen in a HPRT test in mammalian cells with and without metabolic activation. The negative result of an in vitro chromosome aberration test with TDI Biuret was confirmed by several micronucleus tests in rats and mice with inhalation exposure of TDI.

Justification of read-across from supporting substance (2,4-/2,6-TDI to TDI Biuret)

The 80:20 mixture of 2,4-/2,6-TDI (CAS No. 26471-62-5) is the monomeric component of the oligomeric TDI Biuret. The examination of the material balance of Desmodur VP.PU 60WF14 (TDI Biuret) yielded amounts of 42 % 2,4-TDI, 13.7 % 2,6-TDI and ca. 44 % TDI Biuret (Currenta, 2009). Thus, TDI Biuret contains ca. 56 % of a 80:20 mixture of 2,4-/2,6-TDI.

 

With regard to the toxicological comparability of TDI Biuret and 2,4-/2,6-TDI acute inhalation toxicity studies in rats revealed 4-hour LC50 values (aerosol) of 112 mg/m3 for TDI Biuret (based on sum of TDI isomers) and 107 mg/m3 for 2,4-/2,6-TDI (Folkerts, 2010). All qualitative cornerstones of TDI-induced respiratory tract injury were essentially identical. This included the typical delayed-onset mortality, likely as a result of a bronchiolitis obliterans. Of note is the over-proportional presence of TDI vapor relative to the TDI Biuret after inhalation exposure. This is consistent with the higher vapor pressure of TDI. In summary, the similarities of LC50s in the presence of TDI Biuret up to analytically verified breathing zone concentrations of 112 mg TDI Biuret/m3 demonstrates that the inhalation toxicity of TDI per se isnot affected to any appreciable extent by the presence of TDI Biuret aerosol. This means, modulating factors due to physicochemical interactions (partitioning of the vapor phase with the liquid aerosol phase) were not apparent as this would have lead to a more immediate onset of mortality (immediate acute lung edema rather that delayed bronchiolitis obliterans). Overall, these data demonstrate that the acute inhalation toxicity of TDI Biuret is negligible relative to TDI and any dependence of acute hazards on specific use patterns (vapor vs. aerosol) cannot be envisaged(expert opinion of Prof. J. Pauluhn: Desmodur VP.PU 60 WF14 (TDI Biuret): Comparison of acute inhalation toxicities of TDI Biuret and TDI, dated Sep. 3, 2010; complete expert opinion attached in IUCLID chapter 7 “Endpoint summary: Toxicological information”).

In addition, the toxicity profiles of TDI Biuret and 2,4-/2,6-TDI also show a high degree of consistency regarding the endpoints acute oral toxicity, skin irritation, eye irritation, skin sensitization and genotoxicity in vitro.

Therefore, based on all available data the test results obtained for 2,4-/2,6-TDI can be transferred to TDI Biuret and based on such a read-across further testing of TDI Biuret is not required. This approach is in accordance with Annex XI, section 1.5 of the REACH Regulation (EC) No 1907/2006. 


Endpoint Conclusion:

Justification for classification or non-classification

According to CLP Regulation (EC) No 1272/2008 the classification of 2,4-/2,6-TDI (CAS No 26471-62-5) was considered for the classification of TDI Biuret since TDI Biuret contains >= 50 % of 2,4-/2,6-TDI.

 

Genetic toxicity (in vitro and in vivo)

2,4-/2,6-TDI is not classified under Annex I of Directive 67/548/EEC. According to Annex I of CLP Regulation (EC) No 1272/2008 no classification is required for genetic toxicity. The negative results of a HPRT test and a chromosome aberration test on Chinese hamster V79 cells with Desmodur VP.PU 60WF14 (TDI Biuret) confirm the non-classification.