N,N'-dibutyl-N,N'-bis(1,2,2,6,6-pentamethylpiperidin-4-yl)-6-(pyrrolidin-1-yl)-1,3,5-triazine-2,4-diamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli at concentrations of 33 μg - 5 000 μg/plate (SPT) 3.3 μg - 1 000 μg/plate (PIT), in a reverse mutation assay. Precipitation of the test substance was found from about 1 000 μg/plate onward depending on the strain and test conditions. A bacteriotoxic effect was observed depending on the strain and test conditions from about 33 μg/plate onward. A biologically relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

The test substance was assessed for its potential to induce structural chromosome aberrations and/or changes in the number of chromosomes in V79 cells in vitro both with and without the addition of liver S9 mix from induced rats. According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following doses were tested.

Main Experiment

4-hour exposure, 18-hour sampling time, without S9 mix 0; 0.08; 0.16; 0.31; 0.63; 1.25; 2.5; 5; 10 μg/mL

4-hour exposure, 18-hour sampling time, with S9 mix 0; 0.08; 0.16; 0.31; 0.63; 1.25; 2.5; 5; 10 μg/mL

Cytotoxicity indicated by clearly reduced cell numbers or mitotic rates was observed at least at the highest applied test substance concentration in both experimental parts of this study. On the basis of the results of the present study, the test substance caused a clear, statistically significant and biologically relevant increase in the number of structurally aberrant metaphases either without S9 mix or after adding a metabolizing system. Thus, under the experimental conditions described, the test item is considered to have a chromosome-damaging effect under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

The substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Three independent experiments were carried out with and/or without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted.

In this study, in the 1st Experiment in the absence of metabolic activation and in the 1st and 2nd Experiment in the presence of metabolic activation the highest concentrations tested were clearly cytotoxic. In all experimental parts, the highest concentrations evaluated for gene mutations showed strong precipitation in culture medium. Two single statistically significant increased mutant frequencies as well as a positive trend in mutant colonies in the 1st Experiment in the presence of S9 mix were considered as not biological relevant. Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in three experiments performed independently of each other.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test substance was assessed for its potential to induce clastogenicity or aneugenic activity in NMRI mice using the micronucleus test method. For this purpose, the test substance, suspended in DMSO/corn oil, was administered once orally to male animals at dose levels of 500 mg/kg, 1 000 mg/kg and 2 000 mg/kg body weight. The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 2 000 mg/kg body weight and in the vehicle controls. In the test groups of 1 000 mg/kg and 500 mg/kg body weight and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2 000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2 000 polychromatic erythrocytes were also recorded.

An inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected at the top dose of 2 000 mg/kg body weight at 48-hour sacrifice interval. The single statistical significant increase in the number of micronucleated polychromatic erythrocytes after treatment with 2 000 mg/kg body weight at 48-hour sacrifice interval is based on the low value of the respective vehicle control group, and therefore, it has to be regarded as biologically irrelevant.

Thus, under the experimental conditions chosen here, the test substance has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells of NMRI mice in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)