N-benzyl-N-C16-18 (even numbered)-alkyl-N-methyl-C16-18 (even numbered)-alkyl-1-aminium chloride

Endpoint summary

Additional information

GENETIC TOXICITY IN VITRO

1- Gene mutation in bacteria:

In the study of Nesslani (2002), the potential of the test substance to induce reverse mutation in bacteria was assessed using five strains of Salmonella typhimurium according to the OECD guideline 471.The study was conducted in compliance with the principles of Good Laboratory Practice.

A preliminary toxicity test was performed to define the dose-levels of the test substance to be used for the mutagenicity study (all doses were expressed in pure substance).

The test item was then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C).After 48 hours of incubation at 37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

 The preliminary assays without metabolic activation showed that the test substance demonstrated a potent toxicity from 5000 to 500 µg/plate in all strains tested with a total absence of bacterial growth. The immediately lower dose of 150 µg/plate also induced a strong toxicity with a total absence of growth for strains TA1537, TA100 and TA102. In strains TA1535 and TA98, the examination of the background lawn revealed a slight and a moderate level of toxicity, respectively. Under these conditions, the dose of 150 µg/plate was retained as the maximum dose tested for the first mutagenicity assay in the absence of metabolic activation in strain TA1535. In strain TA98 and in all other strains, the top dose was 100 and 50 µg/plate, respectively.

In the preliminary assays with metabolic activation, the test substance also demonstrated a potent toxicity from 5000 µg/plate (total absence of bacterial lawn in all strains) to 500 µg/plate (total absence of bacterial lawn in strain TA102 and a strong decrease of the bacterial growth in all other strains tested). The immediately lower dose of 150 µg/plate also induced a moderate but acceptable toxicity in all strains tested.

Under these conditions, the dose of 150 µg/plate was retained as the maximum dose tested for the first mutagenicity assay in the presence of metabolic activation in all strains.

In the main experiment, the number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

No precipitate was observed in the petri plates when scoring the revertants at all dose-levels. Without metabolic activation, toxicity was observed at dose level of 50 µg /plate and above for TA 1535, TA 1537 and TA 102 strains. In TA 98 and TA 100 strains, toxicity was noted at 15 µg /plate and above. With metabolic activation, cytotoxicity was limited to the highest dose level (150 µg/ plate) except for TA 1537 which exhibited toxicity till100 µg/plate.

In the two independent assays, no significant increase in the mean number of revertants was noted in the five Salmonella typhimurium strains tested in the presence of the test substance neither with nor without metabolic activation, except in the second assay in strain TA100. At the lowest dose tested without metabolic activation (0.5 µg /plate), a weak increase of the number of revertants was observed (1.3 fold the solvent control). This effect was neither dose-related nor reproducible. Therefore, this increase could not be attributed to a mutagenic effect.

Under these experimental conditions the test substance did not show any mutagenic activity in the bacterial reverse mutation test withSalmonella typhimurium.

2- Chromosomal aberrations in mammalian cells:

In the Hoechst AG study (1989), the structural analogue 'Quaternary ammonium compounds, bis(hydrogenated tallow alkyl)dimethyl, chlorides' (DHTDMAC)

was examined for mutagenic activity in V79 Chinese hamster cells( see the toxicological information for The justification for read across The induction of chromosome aberrations after in vitro treatment was investigated in the presence and absence of a fraction of liver homogenate for metabolic activation (S9-mix).

 A preliminary cytotoxicity experiment was performed in order to select appropriate dose levels for the mutagenicity study. The test substance produced a significant cytotoxic effect (reduction of plating efficiency) without metabolic activation from 50 µg/ml up to a concentration of 200 µg/ml which is the limit of solubility (200 µg/ml). A cytotoxic effect was also observed with metabolic activation from 100 ug/ml up to the limit of solubility. For mutagenicity testing, two independent cell cultures with and without metabolic activation (S9-mix) were used.

 For main experiment dose levels of 4, 20 and 40 ug/ml in the absence of metabolic activation and in the presence of S9-mix metabolic activation dose levels of 5, 25 and 50 ug/ml were used.

The test compound did not induce a significant increase in the number of metaphases with aberrations at any preparation time and dose level of the test substance.

A cytotoxic effect (reduction of mitotic index) of the compound was observed 18 and 28 h after treatment at 40 ug/ml without metabolic activation in the main experiments. Marked increases in the rate of chromosome aberrations were obtained with the positive control substance indicating the sensitivity of the assay.

In conclusion, the test substance does not induce chromosome aberrations in V79 Chinese hamster cells, neither in the presence nor in the absence of a metabolic activation system, under the experimental conditions described.

3- Gene mutation in mammalian cells:

In the Sarlang study (2010), the potential for the test substance to induce mutations at the TK locus, was investigated in L5178Y mouse lymphoma cells according to OECD guideline 476 and Good Laboratory Practice. The test substance was tested in two independent experiments, both with and without metabolic activation. Approximately 0.5 x 10⁶(3-hour treatment) or 0.15 x 10⁶(24-hour treatment) cells/mL in 20 mL culture medium with 5% horse serum were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%), at 37°C. Since the test item was toxic in the preliminary test, the choice of the highest dose-level for the main experiments was based on the level of toxicity (decrease in Adj. RTG), according to the criteria specified in the international guidelines.

 In the experiments without metabolic activation,the selected dose-levels were as follows:

.  2.34, 4.69, 9.38, 18.75, 37.5 and 75 µg/mL for the first experiment (3-hour treatment),

.  1.56, 3.13, 6.25, 12.5, 25 and 50 µg/mL for the second experiment (24‑hour treatment).

Cytotoxicity was observed. Following the 3-hour treatment, a marked to severe toxicity was induced at dose-levels ≥ 37.5 µg/mL, as shown by a 61-100% decrease in Adj. RTG. Following the 24-hour treatment, a marked to severe toxicity was induced at dose‑levels ≥ 6.25 µg/mL, as shown by a 80-100% decrease in Adj. RTG.

No noteworthy increase in the mutation frequency was noted in comparison to the vehicle control following the 3-hour or the 24-hour treatments.

In the experiments with metabolic activation,the selected dose-levels were as follows:

.  4.69, 9.38, 18.75, 37.5, 75 and 150 µg/mL for the first experiment,

. 2.34, 4.69, 9.38, 18.75, 37.5 and 75 µg/mL for the second experiment.

 In the first experiment, a slight to strong precipitate was noted in the culture medium at the end of the 3-hour treatment at dose-levels ≥ 4.69 µg/mL.

Cytotoxicity was observed. In the first experiment, a severe toxicity was induced at dose-levels ≥ 75 µg/mL, as shown by a 88-100% decrease in Adj. RTG. In the second experiment, a moderate to severe toxicity was induced at dose-levels ≥ 37.5 µg/mL, as shown by a 49-100% decrease in Adj. RTG.

In either experiment, no noteworthy increase in the mutation frequency was noted in comparison to the vehicle control.

Short description of key information:
The genetic toxicity of the test substance was assessed in 3 in vitro studies conducted in compliance with the principles of Good Laboratory Practices:
- a gene mutation assay on Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 (OECD guideline 471)
- an in vitro chromosomal aberration assay in V79 Chinese Hamster cells on the structural analogue 'Quaternary ammonium compounds, bis(hydrogenated tallow alkyl)dimethyl, chlorides' (DHTDMAC) (OECD guideline 473)
- an in vitro gene mutation assay in mouse lymphoma L5178Y cells (OECD guideline 476)

In all three studies negative results were reported in the presence and absence of metabolic activation.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

According to the criteria laid down in EU regulation (EC) n° 1272/2008 (CLP) and the EU directive 67/548/EEC, the substance is not classified for genetic toxicity as all in vitro mutagenicity assays are negative.