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In vitro study: Ames test

The substance N,N'-Dimethylpropylene urea was tested for mutagenicity in the Ames test and in the Escherichia coli reverse mutation assay according to OECD471 and 472 and GLP using the following strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA. An increase in the number of his+ or trp+ revertants was not observed both in the standard plate test and in the preincubation test either without 5-9 mix or after the addition of a metabolizing system. Furthermore, no bacteriotoxic effect was observed. This result is consistent with a study report by Litton Bionetics from 1982 and a publication by Jung et al. in 1992, showing that the test item did not induce mutagenic transformation of S. typhimurium

TA100, TA98, TA1538, TA1537, TA1535 and S. typhimurium TA 102, respsectively.According to the results of the present data, the test substance NN'-Dimethylpropylene urea is not mutagenic in the Ames test and in the Escherichia coli reverse mutation assay under the experimental conditions chosen here.


In vitro study: chromosome abberation test

An in vitro mammalian cell assay (micronucleus test) was performed according to OECD Guideline 473 and GLP in the Chinese Hamster cell line V79 in two independent experiments. In experiment 1, the exposure period was 4 hrs with and without metabolic activation. Experiment II was performed only without metabolic activation. The exposure period was 18 hrs and 28 hrs. In both experiments, the chromosomes were prepared 18 hrs and 28 hrs after start of treatment with the test article. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. The highest applied concentration in the pre-test on toxicity (1300 µg/mL ~ 10 mM) was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests. Test article concentrations between 15.6 and 1300 µg/mL (with and without S9 mix) were chosen for the assessment of the cytotoxic potential. Neither in the absence nor in the presence of S9 mix clear dose related toxic effects were observed. No test article precipitation and no influence of the test article on the pH value or osmolarity was observed. In the main experiments, test article concentrations within a range of 40.625 - 1300 µg/mL were applied for the investigation of the potential to induce cytogenetic damage. In this study in the absence of S9 mix in experiment II after continuous treatment reduced mitotic indices were observed at interval 18 hrs, whereas reduced cell numbers were observed at interval 28 h. In the presence of S9 mix, no relevant toxicity could be observed up to treatment concentrations of 10 mM. In both independent experiments, neither a significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test article. In addition, no increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

In conclusion, it can be stated that in the study described and under the experimental conditions reported. The test article did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro and can hence be regarded as non-mutagenic.


In vitro study: mammalian gene mutation assay

A GLP guideline study was performed to investigate the potential of N,N'-Dimethyl propylene urea to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster (BASF, 2012). 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 concentration applied in the pre-experiment and in the main experiments (1290 µg/mL) was equal to a molar concentration of about 10 mM. The test item was dissolved in deionised water. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (and DMBA), 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. 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, N,N'-Dimethyl propylene urea is considered to be non-mutagenic in this HPRT assay.


In vivo: Micronucleus assay

Supportingly, an unpublished report by Sandoz LTD assessed the potential of the test item DMPU (Tetrahydro-l.3-dimethyl- 2) to induce micronuclei in vivo. The following doses were administered on the basis of a previous toxicity study: 0.04, 0.125, 0.4 mL/kg bw. 0.4 mL equals 426 mg/kg bw. 4 males and 4 females were administered per dose, 2 males and 2 females of each dose were killed 48 or 72 hrs following administration. Slides were prepared from the bone marrow of femora. 1000 polychromatic erythrocytes of these bone marrow smears were examined per each animal for the presence of micronuclei. The mean number of the micronuclei did not exceed the historical control value of 0.3% in any DMPU dose group or in any time-period. The results did not reveal any statistically significant differences compared to controls. Based on these results it is concluded that DMPU, in contrast to HMPA, which was used as positive control in this assay, has no clastogenic action in the in vivo system.

Three non-GLP studies (Zijlstra and Vogel, 1988; Vogel et al, 1990 and Vogel et al, 1993) performing a gene mutation and chromosome aberration test in Drosophila indicated a potential mutagenic potential of the test item inducing crosslinking. However, the test system is not appropriate but unsuitable with regard to human risks and the obtained data can hence be regarded as not reliable. Taken together, the in vivo data indicate that the test item is non-mutagenic.


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

Short description of key information:
in vitro genotoxicity (OECD 471/472): negative (BASF SE, 1989)
in vitro genotoxicity (OECD 473,chromososme aberration test): negative (BASF SE, Cytotest, 1998)
in vitro genotoxicity (OECD 476, HPRT test): negative (BASF SE, Harlan, 2012)
in vivo genotoxicity ( MN test): negative (Sandoz LTD, 1985)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available results of the in vitro and in vivo genetic toxicity studies, the test substance does not have to be classified and labelled as genotoxic according to Directive 67/548/EEC (DSD) and Regulation (EC) No 1272/2008 (CLP).