Amines, N-(3-aminopropyl)-N'-C12-18-alkyltrimethylenedi-, reaction products with N-C12-18-alkyltrimethylenediamines and formic acid

Endpoint summary

Additional information

GENETIC TOXICITY IN VITRO

1- Gene mutation in bacteria:

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

A preliminary toxicity test was performed to define the dose-levels of the test substance to be used for the mutagenicity study. 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 50 µg/plate in all strains tested with a total absence of bacterial growth. The immediately lower dose of 15 µg/plate induced only a slight toxicity. Under these conditions, the dose of 100 µg/plate was retained as the maximum dose tested for the first mutagenicity assay in the absence of metabolic activation in all strains TA1535.

In the preliminary assays with metabolic activation, the test substance also demonstrated a potent toxicity from 5000 µg/plate to 500 µg/platein all strains tested with a total absence of bacterial growth. The immediately lower dose of 150 µg/plate induced only a slight toxicity in all strains tested.

Under these conditions, the dose of 300 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. Nevertheless,

in the first assay in the presence of S9, the number of revertants observed for the positive control of strainTA102 was slightly lower than the lowest limit for historical values. However, the positive reference compound induced a statistically and biologically significant increase in the number of revertants compared to the control with a ratio of 2.8. The study was therefore considered valid.

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.

During the first assay, a statistically significant increase in the number of revertants was found in strain TA100 at the intermediate dose of 1.5 µg/plate without metabolic activation, and at the two highest doses of 50 and 150 µg/plate with metabolic activation. However, the biological threshold (ratio 2) was not reached, as the calculated ratio was 1.4 in all cases. Moreover, the effect observed without metabolic activation was neither dose-related nor reproduced in the second assay. Concerning the effect noted with metabolic activation, an increase was indeed observed in the second assay (performed with the preincubation protocol) but (i) it was not statistically significant and (ii) the ratio was not higher than 1.3. 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:

The objective of the study performed by Sire (2010c) was to evaluate the potential of the test substance to induce chromosome aberrations in cultured human lymphocytes.The study was performed according to the international guidelines (OECD 473 and Commission Directive No. B10) and in compliance with the principles of Good Laboratory Practice.

 The test item was tested in two independent experiments, both with and without a liver metabolizing system (S9 mix), obtained from rats previously treated with Aroclor 1254.The highest dose-level for treatment in the first experiment was selected on the basis of pH, osmolality and solubility. For selection of the dose-levels for the second experiment, any toxicity indicated by the reduction of mitotic index (MI) in the first experiment was also taken into account.The test substance was dissolved in dimethylsulfoxide (DMSO).Mitomycin C and cyclophosphamide were used as positive controls without and with S9 mix respectively.

 In the first experiment, lymphocyte cultures were exposed to the test or control items (with or without S9 mix) for 3 hours then rinsed. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles.The second experiment was performed as follows:without S9 mix, cells were exposed continuously to the test or control items until harvest and with S9 mix, cells were exposed to the test or control items for 3 hours and then rinsed.Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later, respectively.

 One and a half hours before harvest, each culture was treated with a colcemid solution (10 µg/mL) to block cells at the metaphase-stage of mitosis. After hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa. All the slides were coded for scoring.

  At the dose-level of 5000 µg/mL, no precipitate was observed in the culture medium; the pH was approximately 8.5 (7.4 for the vehicle control) and the osmolality was equal to 347 mOsm/kg H₂O (446 mOsm/kg for the vehicle control). At the dose-level of 1667 µg/mL, the pH and the osmolality values were equivalent to those of the vehicle control culture.

 With a treatment volume of 55 µL/5.5 mL culture medium, a first treatment was performed for the first experiment using the treatment levels ranging from 0.03 to 5000 µg/mL. With and without S9 mix, a severe decrease in the mitotic index was noted at dose-levels ≥ 62 µg/mL (82‑100% decrease). In the absence of a dose-level inducing a decrease in mitotic index greater than 50% but non-severely toxic, the metaphase analysis was not undertaken and another treatment was performed using closer ranges of dose-levels, as follows: 2.5, 5, 10, 20, 30, 40, 50 and 60 µg/mL with S9 mix and 5, 10, 20, 30, 40, 50, 60 and 80 µg/mL without S9 mix.

For the second experiment, the treatment-levels were as follows: 1.25, 2.5, 5, 10, 20, 30 and 40 µg/mL without S9 mix and 2.5, 5, 10, 20, 25, 30 and 40 µg/mL with S9 mix.

 The frequency of cells with structural chromosome aberrations of the vehicle and positive controls was as specified in acceptance criteria. The study was therefore considered as valid.

 Experiments without S9 mix

Cytotoxicity:

Following the 3-hour treatment, a moderate to severe decrease in the mitotic index was noted at dose-levels ≥ 30 µg/mL (40-100% decrease).

Following the 20-hour treatment, a marked to severe decrease in the mitotic index was noted at dose-levels ≥ 20 µg/mL (65-100% decrease).

Following the 44-hour treatment, a moderate to marked decrease in the mitotic index was noted at dose-levels ≥ 20 µg/mL (41-78% decrease).

 Metaphase analysis:

The dose-levels selected for metaphase analysis were as follows:

.           20, 30 and 40 µg/mL, for the 3-hour treatment, the latter inducing 69% decrease in the mitotic index,

.           5, 10 and 20 µg/mL, for the 20-hour treatment, the latter inducing 65% decrease in the mitotic index,

.           20 µg/mL, for the 44-hour treatment, this dose inducing 41% decrease in the mitotic index, but higher dose-level being too cytotoxic.

 No significant increase in the frequency of cells with structural chromosomal aberrations was noted after 3-, 20- as well as 44-hour treatments.

 Experiments with S9 mix

Cytotoxicity:

At the 20-hour harvest time, a slight to severe decrease in the mitotic index was noted at dose‑levels ≥ 20 µg/mL (28-100% decrease).

At the 44-hour harvest time, a slight to severe decrease in the mitotic index was noted at dose‑levels ≥ 5 µg/mL (30-100% decrease).

Metaphase analysis:

The dose-levels selected for metaphase analysis were as follows:

.           10, 20 and 30 µg/mL for the first experiment, the latter inducing 72% decrease in the mitotic index,

.           2.5, 5 and 10 µg/mL for the second experiment, the latter inducing 21% decrease in the mitotic index but higher dose-levels being too cytotoxic,

.           20 µg/mL for the 44-hour harvest time, this dose-level inducing 68% decrease in the mitotic index.

 No significant increase in the frequency of cells with structural chromosomal aberrations was noted in either experiment and at either harvest time.

Therefore, the test substance did not induce chromosome aberrations in cultured human lymphocytes.

3- Gene mutation in mammalian cells:

In the second study of Sire (2010d), the potential for the test substance to induce mutations at the TK locus, was investigated inL5178Y mouse lymphoma cells according to OECD guideline 476 and Good Laboratory Practices.

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:

 -0.63, 1.25, 2.5, 5, 7.5, 10 and 15 µg/mL for the first experiment (3-hour treatment),

- 0.16, 0.31, 0.63, 1.25, 2.5, 5 and 10 µg/mL for the second experiment (24‑hour treatment).

 Cytotoxicity was observed. Following the 3-hour treatment, a slight to severe toxicity was noted at dose-levels ≥ 5 µg/mL, as shown by 39-100% decrease in Adj. RTG. Following the 24-hour treatment, a slight to severe toxicity was noted at dose-levels ≥ 0.63 µg/mL, as shown by 34-100% 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:

 - 2.5, 5, 10, 15, 20, 35 and 50 µg/mL for the first experiment,

- 0.63, 1.25, 2.5, 5, 10, 20 and 40 µg/mL for the second experiment.

Cytotoxicity was observed. In the first and second experiments,a slight to severe toxicity was noted at dose-levels ≥ 10 µg/mL, as shown by 28-100% decrease in Adj. RTG.

 No noteworthy increase in the mutation frequency was observed in either experiment.

In conclusion, the substance did not show any mutagenic activity in the mouse lymphoma assay.

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 in cultured Human lymphocytes (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.