N,N-bis(3-aminopropyl)methylamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bis-(3-aminopropyl)-methylamin was found to be not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay (Ames test) and HPRT test. Based on the results of an in vitro mammalian cell micronucleus test, the test substance was considered to be clastogenic. The test substance showed no mutagenic activity in an in vivo micronucleus test.

Additional information

Ames test

The substance Bis-(3-aminopropyl)-methylamin 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 (TA 1535, TA 100, TA 1537, TA 98) and Escherichia coli (WP2 uvrA) in a reverse mutation assay according to OECD 471/472 guideline. The reverse mutation assay was performed in two ways by the standard plate assay (SPT) and the preincubation test (PIT) both with and without metabolic activation (Aroclor-induced rat liver S-9 mix). The bacteria cells were exposed to 100 µg -7500 µg/plate of the test substance in the SPT and to 4 µg - 2500 µg/plate in the PIT. No precipitation of the test substance was found and a bacteriotoxic effect was observed under all test conditions. An increase in the number of his+ or trp+ revertants was not observed in the SPT or in the PIT either without S-9 mix or after the addition of a metabolizing system. Therefore the test substance was not considered to be mutagenic in this reverse mutation assay under the study experimental conditions.

In vitro micronucleus assay

The substance N,N-Bis(3-aminopropyl)methylamine was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity) both in the absence and the presence of a metabolizing system. According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the test substance did not exhibit any pronounced toxicity up to the highest recommended dose of approx. 10 mM (1 500.0 μg/mL). Thus, the following doses were tested:

1st experiment

4 hours exposure; 24 hours harvest time; without S9 mix:

0; 46.9; 93.8; 187.5; 375; 750; 1 500 μg/mL

4 hours exposure, 24 hours harvest time, with S9 mix:

0; 46.9; 93.8; 187.5; 375; 750; 1 500 μg/mL

2nd experiment

4 hours exposure, 24 hours harvest time, without S9 mix

0; 500; 750; 1 000; 1 250; 1 500 μg/mL

A sample of at least 1 000 cells for each culture were analyzed for micronuclei, i.e. 2 000 cells for each test group. The negative controls gave frequencies of micronucleated cells within the historical negative control data range for V79 cells. Both positive control substances, EMS and cyclophosphamide,led to the expected increase in the number of cells containing micronuclei.

In this study, no cytotoxicity indicated by reduced relative increase in cell count (RICC) or proliferation index (PI) was observed up to the highest required test substance concentration.

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 cells containing micronuclei without S9 mix in two experiments carried out independently of each other.

Thus, under the experimental conditions described, N,N-Bis(3-aminopropyl)methylamine is considered to have a chromosome-damaging (clastogenic) effect or does induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence of metabolic activation.

HPRT

The substance N,N-Bis(3-aminopropyl)methylamine 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. Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation) at concentrations up to 1500 µg/ml. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and MCA, led to the expected increase in the frequencies of HPRT forward mutations. In this study in the presence of metabolic activation no cytotoxicity was observed up to the highest required concentration evaluated for gene mutations. Besides, in the 1st and 2nd Experiment in the absence of metabolic activation the highest concentrations tested for gene mutations were cytotoxic. Based on the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in two experiments performed independently of each other.

In vivo MNT

According to the results of an in vivo micronucleus test, there are no biologically relevant differences in the frequency of erythrocytes containing micronuclei either between the vehicle control groups and the three dose groups (250 mg/kg, 500 mg/kg and 1 000 mg/kg) or between the two sacrifice intervals (24 and 48 hours). The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) or large micronuclei (d ≥ D/4) did not clearly deviate from the vehicle control values at any of the sacrifice intervals and was within the historical vehicle control data range.

Based on the pretest, 1 000 mg/kg body weight was defined as maximum tolerated dose (MTD) due to deaths observed at 2 000 mg/kg body weight. In the main experiment one animal exposed to 1 000 mg/kg body weight died before reaching the intended 48-hour sacrifice interval.

Due to the unexpected lethality of a single animal in the top dose group at 48-hour sacrifice interval only four animals were investigated for cytogenetic damage. However, the 24-hour sacrifice interval is the major interval for the detection of genotoxic effects in case of lacking indication of reduced erythrocyte maturation by test substance treatment, as demonstrated in this study. Therefore, the data of the delayed 48-hour sacrifice interval are of minor relevance. Thus, this deviation has no detrimental impact on the validity and the outcome of this study.

Besides, at delayed 48-hour sacrifice interval at 1 000 mg/kg body weight a statistically significant value (1.8‰ micronucleated PCEs) compared to the respective vehicle control value (0.6‰ micronucleated PCEs) was obtained. However, this observation occurred due to the low rate of micronucleated cells in the concurrent vehicle control group. The micronucleus rate of this dose group was clearly within our historical vehicle control data range and, therefore, this finding has to be regarded as biologically irrelevant.

In this study, after single oral administration of the vehicle deionized water the ratio of PCEs/NCEs in the vehicle control animals at both sacrifice intervals was within the normal range for the animal strain selected. Besides the number of cells containing micronuclei in these vehicle control animals was within the range of the historical vehicle control data for PCEs.

In addition, both positive control substances, cyclophosphamide and vincristine sulfate, induced a statistically significant increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above.

Justification for classification or non-classification

Based on the available data, the classification criteria according to Regulation (EC) No 1272/2008 (CLP) are not met.