Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

Ames test:

Polypropoxylated p-nonylphenol-formaldehyde-diethanolamine, Mannich base was examined for mutagenic activity in the bacterial reverse mutation test (GLP compliant and according to OECD guideline 471) using the histidine-requiring Salmonella typhimuriumstrains TA1535, TA1537, TA98, TA100 and the tryptophan-requiring Escherichia coli strain WP2uvrA, in the absence and presence of S9-mix (TOXI-COOP Zrt. 2013). In the initial mutation test (plate incorperation test) the following concentrations were tested:1000, 316, 100, 31.6, 10, 3.16 and 1 μg/plate based on the preliminary range finding test.Because of the observed cytotoxic effect of the test item (in the Salmonella typhimurium strains) the above concentrations were modified in the Confirmatory Mutation Test (Pre Incubation Test). The following concentrations were examined in the Salmonella typhimurium strains: -S9 Mix: 100; 31.6; 10; 3.16; 1; 0.32 and 0.1 μg/plate, +S9 Mix: 1000; 316; 100; 31.6; 10; 3.16 and 1 μg/plate. No inhibition was obtained in the Initial Mutation Test in the case of Escherichia coli WP2 uvrA. Therefore, the following modified concentration range was examined in the Confirmatory Mutation Test for the E. Coli strain: 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate. In the Initial Mutation Test in Escherichia coli WP2 uvrA the examined concentration range did not fulfill the guideline criterion as the highest examined concentration level was 1000 μg/plate and was not unequivocally a cytotoxic concentration, therefore a short completion of this experimental part was necessary (Completed Plate Incorporation Test). In the Completed Plate Incorporation Test, the concentrations were: 5000; 2500; 1000 and 316 μg/plate. No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with test substance at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. The positive controls showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains. The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Chromosome aberration test:

The potential of Reaction product of p-nonylphenol, formaldehyde and diethanolamine, propoxylate to induce chromosome aberrations was investigated in a GLP compliant study performed according to OECD guideline 473 (TOXI-COOP Zrt. 2013). The test item was dissolved in DMSO and tested using Chinese Hamster lung cells (V79). Firstly a cytotoxicity test was conducted to determine the doses for the main experiments. Two main experiments were performed. In experiment A, an exposure time of 3 hours was tested both with (7, 20, 40, 60 and 70 μg/mL) and without (5, 20, 30, 40 and 50 μg/mL) metabolic activation. In experiment B in which the exposure time without metabolic activation was 20 hours (harvest time of either 20 or 28 hours) and with metabolic activation 3 hours (harvest time 28 hours after treatment). The following concentrations were tested: without S9 -mix: 5, 10, 12.5, 15, 17.5 μg/mL (harvest time 20 hours after treatment); without S9 -mix: 5, 10, 12.5, 15, 17.5 μg/mL (harvest time 28 hours after treatment); with S9 -mix: 7, 20, 40, 60 and 70 μg/mL. Following exposure and expression time cells were exposed to selection agent Colchicine (0.2 μg/mL) 2 -2.5 hours prior to harvesting. Following harvesting, cells were treated with fixative for ca. 10 min. before being placed on slides and stained. Chromosome aberration frequencies were then scored for at least 200 well-spread metaphase cells. In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations without gap, either in the absence or in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and concurrent negative control groups and no dose-response relationships were noted. In Experiment B, the frequency of the cells with structural chromosome aberrations without gaps did not show significant alterations compared to the concurrent control, when the test substance was examined up to cytotoxic concentrations without S9-mix over a prolonged treatment period of 20 hours and 20 and 28 hour sampling times. Further, a three-hour treatment and 28 -hour sampling time with the test substance up to the cytotoxic concentration in the presence of S9 -mix did not cause an increase in the number of cells with structural chromosome aberrations without gaps. In conclusion, Reaction product of p-nonylphenol, formaldehyde and diethanolamine, propoxylated

tested up to cytotoxic concentrations, both with and without metabolic activation, did not induce structural chromosome aberrations in this test in Chinese Hamster lung cells.

Mammalian cell gene mutation test:

The potential of Reaction product of p-nonylphenol, formaldehyde and diethanolamine, propoxylated cause gene mutation and/or chromosome damage was investigated in a GLP compliant study performed according to OECD guideline 476. The test item was dissolved in DMSO and tested using mouse lymphoma L5178Y TK+/- 3.7.2 C cells. Firstly a cytotoxicity test was conducted to determine the doses for the main experiments. Two main experiments were performed. In assay A, an exposure time of 3 hours was tested both without (1, 2.5, 5, 7.5, 10, 12.5, 15 μg/mL) and with (5, 10, 15, 20, 25, 30 μg/mL) metabolic activation. In assay 2 in which the exposure time without metabolic activation was 24 hours and with metabolic activation 3 hours the following concentrations were tested: without S9 -mix: 1, 2, 3, 4, 5, 6 μg/mL; with S9 -mix: 5, 10, 15, 20, 25, 30 μg/mL. After the treatment the cell cultures were washed, re-suspended, the cell densities determined and adjusted to 2E5 cells/mL. The cells were transferred to flasks for growth through the expression period (for approximately 2 days) and diluted to be plated for survival. At the end of the expression period cells were allowed to grow and form colonies for approximately 2 weeks in culturing plates with and without selective agent (TFT) for determination of mutations and viability. In the main assays, the relative harmonised survival, the relative total growth of the cells, the viability (colony-forming ability at the end of the 2 day expression period following the treatment) and the potential mutagenicity (5-trifluorothymidine resistance) were determined. The performed assays fulfilled the validity criteria with regard to the negative control and positive control treatments as well as the number of analysable concentration levels (at least four). The cytotoxicity results showed that the concentration range covered a range from maximum to little or no toxicity in the presence and also in the absence of exogenous metabolic activation, based on the harmonised relative survival data. In the performed assays the obtained mutation frequencies (in absence and also in the presence of exogenous metabolic activation) did not show dose-related tendencies, remained far below the relevant GEF thresholds for positive call and remained in the validity criterion range of the negative vehicle control cultures. The mutation frequencies were not statistically significantly different (Dunnett’s Test, α = 0.05) from that of the corresponding vehicle control throughout the study. Therefore it can be concluded that under the conditions of this study, the test item did not induce gene mutations in presence and absence of metabolic activation in the cultured mammalian cells used.


Short description of key information:
The test substance is not mutagenic in the bacterial reverse mutation test (GLP-compliant, OECD 471) and in the mammalian gene mutation assay (GLP-compliant, OECD 476) in the presence and absence of metabolic activation. The test substance is not clastogenic in the chromosome aberration test (GLP-compliant, OECD 473) in the presence and absence of metabolic activation.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available genotoxicity studies, the test substance does not need to be classified for genotoxicity according to the Directive 67/548/EEC and according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008