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Administrative data

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Additional information

Genetic toxicity

Reverse mutation assays

Bis(2,6-diisopropylphenyl)carbodiimide was investigated with an reverse mutation assay in the Salmonella typhimurium strains TA1535, TA1537, TA100, TA 98 and TA 102 with and without metabolic activation (rat S9 mix comprised 10% S9 fraction, 70% cofactor solution and 20% 0.15 M KCI) by Herbold in 2009 according to TG OECD 471 and according to good laboratory principles. The test substance was initially investigated using the Salmonella/microsome plate incorporation test for point mutagenic effects in doses of up to and including 5000 µg per plate. The independent repeat was performed as preincubation for 20 minutes at 37°C. Other conditions remained unchanged. Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. Substance precipitation occurred at the dose 5000 µg per plate. Nevertheless, all doses could be used for assessment purposes. Evidence of mutagenic activity of the compound was not seen. Evaluation of individual dose groups, with respect to relevant assessment parameters (dose effect, reproducibility) revealed no biologically relevant variations from the respective negative controls. The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, mitomycin C, cumene hydroperoxide and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls (demonstrating the system's high sensitivity). Despite this sensitivity, no indications of mutagenic effects of the test substance could be found at assessable doses of up to 5000 µg per plate in any of the Salmonella typhimurium strains used. Therefore, 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.

 

Additionally bis(2,6-diisopropylphenyl)carbodiimide was tested in an reverse mutation assay in the Salmonella typhimurium strains TA1535, TA1537, TA100, TA 98 and in Escherichia coli (strain WP2 uvr A) with and without metabolic activation (rat liver homogenate metabolizing system: 10% liver S9 in standard co-factors) in 2001 (Thompson, 2001, according to TG OECD 471). The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. An oily precipitate was observed at and above 500 µg/plate, this did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains. Therefore, 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.

 

The test substance was investigated using the Salmonella/microsome plate incorporation test for point mutagenic effects in doses of up to and including 10,000 µg per plate on five Salmonella typhimurium mutants (Haworth, 1994, similar to TG OECD 471). These comprised the five histidine-auxotrophic Salmonella strains TA 98, TA 100, TA 1535, TA 1537 and TA1538. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 10,000 µg/plate. A precipitate was observed at and above 500 µg/plate, this did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains. Therefore, the test substance was considered to be non-mutagenic without and with S9 mix of Aroclor induced rat liver microsomes in the plate incorporation in the Salmonella/microsome test.

In all tests the substance was used up to precipitating concentrations (up to 5000 and 10,000 µg per plate). Even though the strains investigated in two of the available Ames-tests differ in part from the strains required according to OECD 471, the third test was conducted with the strains required in OECD 471. It can be seen that none of the strains used showed any signs of mutagenic potential of bis(2,6 -diisopropylphenyl)carbodiimide.

All other available tests revealed, that the active ingredient: bis(2,6-diisopropylphenyl)carbodiimide did not show any mutagenic activity towards the Salmonella or Escherichia species tested. Therefore the substance is considered to be non-mutagenic without and with activation.

 

Gene mutation assay

Bis(2,6-diisopropylphenyl)carbodiimide was investigated using the gene mutation assay in Chinese hamster V79 cells (V79 / HPRT) for its ability to induce gene mutations (Wollny, 2011). The experiments were conducted according to GLP and the OECD 476. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest applied concentration in the pre-tests on toxicity (3200 µg/mL) was equal to approximately 10 mM. The concentration range of the main experiments was limited by cytotoxic effects of the test item. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. Relevant cytotoxic effects defined as a reduction of the relative cloning efficiency or the relative cell density at the first sub-cultivation following treatment to values below 50% in both parallel cultures were noted in the first experiment at 1.5 µg/mL and above with and without metabolic activation. In the second experiment cytotoxic effects occurred at 0.19 µg/mL and above without metabolic activation and at 3.0 µg/mL and above with metabolic activation. The recommended cytotoxic range of approximately 10 – 20% of the respective solvent control cell cultures was covered with and without metabolic activation. No relevant and reproducible increase in mutant colony numbers/10E6 cells was observed in the main experiments up to the maximum concentration. The mutation frequency remained well within the historical range of solvent controls. The induction factor did not reach or exceed the threshold of three times the mutation frequency of the solvent control. No significant dose dependent trend of the mutation frequency was determined in any of the experimental groups. In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 7.0 up to 20.2 mutants per 10E6 cells; the range of the groups treated with the test item was from 3.1 up to 33.2 mutants per 10E6 cells. EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, bis(2,6-diisopropylphenyl)carbodiimide is considered to be non-mutagenic in this HPRT assay.

 

Chromosome aberration test

The clastogenic potential of bis(2,6-diisopropylphenyl)carbodiimide was evaluated in a chromosome aberration test in vitro (Ciethier, 2011). Initially Chinese hamster V79 cells were exposed in the absence of S9 mix for 4 hours to concentrations of 0.045, 0.09, 0.18, 0.26 and 0.34 µg/mL of bis(2,6-diisopropylphenyl)carbodiimide. Cultures of all concentrations were harvested 18 hours after the beginning of the treatment. In addition, cells treated with 0.18, 0.26 and 0.34 µg/mL were harvested 30 hours after the beginning of the treatment. In the presence of S9 mix cells were exposed for 4 hours to concentrations of 0.3, 0.6, 1.2, 1.8 and 2.4 µg/mL of bis(2,6-diisopropylphenyl)carbodiimide. Cultures of all concentrations were harvested 18 hours after the beginning of the treatment. In addition, cells treated with 1.2, 1.8 and 2.4 µg/mL were harvested 30 hours after the beginning of the treatment. Without S9 mix an additional experiment was performed using continuous treatment for 18 hours, harvested at the same time, and bis(2,6-diisopropylphenyl)carbodiimide-concentrations of 0.015, 0.03, 0.06, 0.08 and 0.1 µg/mL. Based on the scarce amount of analysable meta-phases in the 0.26 µg/mL and 0.34 µg/mL-concentrations without S9 mix and the 2.4 µg/mL-concentration with S9 mix exposed for 4 hours and based on the absence of analysable metaphases in the continuous treatment bis(2,6-diisopropylphenyl)carbodiimide-concentrations were selected for reading of metaphases.

Without S9 mix cytotoxic effects were observed at 0.18 g/mL and above after 4 hours treatment and at 0.06 µg/mL and above after 18 hours treatment. With S9 mix cytotoxic effects were observed at 1.2 µg/mL and above. Therefore, concentrations of 0.045, 0.09 and 0.18 µg/mL (4 hours treatment) and 0.015, 0.03 and 0.06 µg/mL (18 hours treatment) were chosen for reading in the absence of S9 mix. In the presence of S9 mix 0.6, 1.2 and 1.8 g/mL were employed. All of these cultures harvested 18 hours after the beginning of the treatment were included. In addition, cultures treated in the absence of S9 mix with 0.18 mg/mL and harvested 30 hours after the beginning of the treatment were used. The same was true for cultures treated in the presence of S9 mix with 1.8 µg/mL.

None of the cultures treated with bis(2,6-diisopropylphenyl)carbodiimide in the absence and in the presence of S9 mix for 4 hours showed biologically relevant or statistically significant increased numbers of aberrant metaphases. None of the cultures treated with the test item in the absence of S9 mix for 18 hours showed biologically relevant increased numbers of aberrant metaphases.

The positive controls Mitomycin C and Cyclophosphamide induced clastogenic effects and demonstrated the sensitivity of the test system and the activity of the used S9 mix. Based on this test, bis(2,6-diisopropylphenyl)carbodiimide is considered not to be clastogenic for mammalian cells in vitro.

 


Justification for selection of genetic toxicity endpoint
No study was selected, since all in vitro studies were negative.

Short description of key information:
1) Genetic toxicity (2009), GLP, OECD 471, Ames-Test, up to 5000 µg, with and without metabolic activation, negative
2) Genetic toxicity (2001), GLP, OECD 471, Ames-Test, up to 5000 µg, with and without metabolic activation, negative
3) Genetic toxicity (1984), similar to OECD 471, Ames-Test, up to 10,000 µg, with and without metabolic activation, negative
4) Genetic toxicity (2011), GLP, OECD 476, V79 / HPRT test, with and without metabolic activation, negative
5) Genetic toxicity (2011), GLP, OECD 473, chromosome aberration, V79 cells, with and without metabolic activation, negative

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on these study results, bis(2,6-diisopropylphenyl)carbodiimide is considered to be non-mutagenic and therefore classification is not warranted according to the criteria of the Regulation (EC) No. 1272/2008.