Reaction mass of ethoxylated N-oleyl-1,3-propanediamine, hydrofluorides of and ethoxylated N-palmityl-1,3-propanediamine, hydrofluorides of and ethoxylated N-stearyl-1,3-propanediamine, hydrofluorides of and olaflur

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance Olaflur was examined in a Reverse Mutation Assay using Bacteria (AMES) (two independent studies), an in vitro test of the clastogenic activity in cultured Human peripheral lymphocytes and in an in vitro Mammalian Cell Gene Mutation Test in mouse lymphoma L5178Y/TK+/- cells. The resulting data were supported by the results obtained with Oleaflur, a structure analogue of Olaflur, in an in vitro Gene Mutation Assay (HPRT Test) in Mammalian cells (V79), as well as in chromosomal aberration test in Human peripheral lymphocytes. Neither Olaflur nor its structure analogue Oleaflur have to be considered as mutagenic based on the results obtained in these in vitro studies.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Olaflur was investigated in two independent micronucleus assays in vivo. Both tests also demonstrate the lack of any mutagenic effect in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro

Key Studies

- Reverse Mutation Assay Using Bacteria (Leuschner, J., 2002)

Olaflur was examined up to 31.6 ug/plate (causing severe bacteriotoxic effect) in the five Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535, TA 1537 in two independent experiments, each carried out with and without metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). In conclusion, under the present test conditions Olaflur caused no mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the plate pre-incubation test each carried out with and without metabolic activation. The study was conducted according to the OECD Guideline 471 and the EU guideline B.13/14.

This result is therefore supported by an additional Ames-Test which was performed in 1981 by Fouillet and Gutty at Battelle, Geneva Research Centres, Switzerland (see below "Supporting Studies")

- In vitro assessment of the clastogenic activity of Olaflur in cultured Human peripheral lymphocytes (Leuschner, J.,2002)

The test samples of Olaflur were assayed in an in vitro cytogenetic study using human lymphocyte cultures both in the presence and absence of metabolic activation by rat liver post-mitochondrial fraction (S9 mix) from Aroclor-1254 induced animals. Aqua ad iniectabilia served as the vehicle.

The test was carried out employing 2 exposure times without S9 mix: 4 and 24 hours, and 1 exposure time with S9 mix: 4 hours. Harvesting time was 25 hours after start of exposure. The incubation procedure took place in the dark. The study was conducted in duplicate. The following concentrations were tested:

1.25, 2.5, 5, 10 ug Olaflur/mL without metabolic activation (4 h, 24 h exposure)

2.5, 5, 10, 20 ug Olaflur/mL with metabolic activation (4 h exposure)

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with Olaflur at concentrations of 1.25, 2.5, 5 of 10 ug Olaflur/mL in the absence of metabolic activation ranged from 1.5 % to 3.0 % in the two independent experiments with blood from different donors.

The top concentration of 10 ug Olaflur/mL (mitotic index: 0.13) caused a pronounced cytotoxicity after the 24 h exposure, exceeding the toxicity range recommended in the respective guidelines for 24 h exposure.

Only at the pronounced cytotoxic concentration of 10 µg Olaflur/mL medium a marginal, though not significant, increase was noted in the number of aberrations to 5.4 %, exceeding slightly the toxicity control range.

It is known that high cytotoxicity causes artefacts in form of aberrations in in vitro chromosomal tests. Hence, the increase at the concentration of 10 ug Olaflur/mL medium at 24 h only and obtained at very high cytotoxicity is considered as artefact and not test substance-related.

All other results obtained are considered to be within the normal range of the negative control where a mean incidence of chromosomal aberrations (excluding gaps) of 1.5 or 1.0 % was observed after a 4 h or 24 h exposure.

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with Olaflur (2.5, 5, 10 or 20 ug Olaflur/mL) in the presence of metabolic activation ranged from 0.0 % to 3.0 % in the two independent experiments.

The concentrations of 10 or 20 ug Olaflur/mL (mitotic indices: 0.82 and 0.14, respectively) caused a pronounced cytotoxicity after the 4 h exposure. The results obtained are considered to be within the normal range of the negative control where the incidence of chromosomal aberrations (excluding gaps) ranged from 0.0 % to 5.0 % for the last 30 experiments. In the same test, mitomycin C and cyclophosphamide induced significant damage.

- In vitro Mammalian Cell Gene Mutation Test (BioReliance, 2018)

An in vitro mammalian cell gene mutation test was performed in mouse lymphoma L5178Y/TK+/- cells according to OECD guideline 490. The purpose of this study was to evaluate the genotoxic potential of the test article based on quantitation of forward mutations at the thymidine kinase locus of L5178Y mouse lymphoma cells and the sizing of the resulting colonies. Olaflur was tested in the presence and absence of metabolic activation (Aroclor 1254-induced rat liver S9) at the following concentrations and exposure times:

4-hour treatment with S9: 0.53, 1.05, 2.1, 4.2, 5.63, 7.5 and 10 µg/mL

4-hour treatment without S9: 0.13, 0.27, 0.53, 1.05, 1.41, 1.88 and 2.5 µg/mL

24-hour treatment without S9: 0.07, 0.13, 0.27, 0.53, 0.70, 0.94 and 1.25 µg/mL

No visible precipitate was observed at the beginning or end of treatment. The test article did not have an adverse impact on the pH of the cultures (pH 7.5 at the top dose). Cultures treated at concentrations of 0.53, 1.05, 2.1, 4.2, and 5.63 µg/mL (4-hour treatment with S9), 0.27, 0.53, 1.05, 1.41 and 1.88 µg/mL (4-hour treatment without S9) and 0.07, 0.13, 0.27, 0.53, 0.70 and 0.94 µg/mL (one replicate culture of 0.94 µg/mL were not cloned due to excessive toxicity for cloning) (24-hour treatment without S9) exhibited 12 to 102%, 12 to 94% and 13 to 77% relative suspension growth (RSG), respectively, and were cloned. All other cultures were not cloned as there were sufficient number of higher concentrations available for cloning or the cultures were excessively toxic to clone. Relative total growth of the cloned cultures ranged from 10 to 107% (4‑hour treatment with S9), 11 to 114% (4-hour treatment without S9) and 12 to 67% (24‑hour treatment without S9). No increases in induced mutant frequency ≥90 mutants/10^-6 clonable cells were observed under any treatment condition. There was a statistically significant dose dependent decrease in trend observed with 4 hour treatment condition without activation. However, this is not biologically relevant as the trend was a dose dependent decrease.

Trifluorothymidine-resistant colonies for the positive and vehicle control cultures, were sized according to diameter over a range from approximately 0.2 to 1.1 mm. The colony sizing for the MMS and DMBA positive controls yielded the expected increase in small colonies (verifying the adequacy of the methods used to detect small colony mutants) and large colonies. All positive and vehicle control values were within acceptable ranges, and all criteria for a valid assay were met. 

Under the conditions of the assay described in this report, Olaflur was concluded to be negative for the induction of forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro L5178Y/TK+/-mouse lymphoma assay.

Supporting Studies

- Mutagenicity study of Oleaflur in Mammalian cells (V79) in the in vitro Gene Mutation Assay (HPRT Test) (Leuschner, J. 2002)

Test samples of Oleaflur were assayed in a gene mutation assay in cultured mammalian cells (V79, genetic marker HPRT) both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced animals (up to cytotoxic concentrations).

Under the present test conditions Oleaflur tested up to cytotoxic concentrations in the experiments without and with metabolic activation was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.

- Report on a study of the mutagenic potential of Amine Fluoride 297 (Ames test) (Fouillet, X., et al., 1981)

The substance Amine fluoride 297 (Batch F-0785) was tested with the Salmonella typhimurium strains TA 1535, 1537, TA 98 and TA 100 at concentrations from 0.2 to 2000 ug /plate both in the presence and in the absence of metabolic activation. No mutagenic effect was observed.

- In vitro assessment of the clastogenic activity of Oleaflur in cultured Human peripheral lymphocytes (Leuschner, J., 1997)

Under the present test conditions Oleaflur tested up to a cytotoxic concentration of 40 ug/mL medium in the absence and presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of mutagenic properties with respect to chromosomal or chromatid damage. In the same test, mitomycin C and cyclophosphamide induced significant damage.

Genetic toxicity in vivo

Key study

- Micronucleus Test in vivo (Leuschner, J., 2002):

Olaflur was assayed in an in vivo bone marrow micronucleus test in the mouse for the detection of damage to the chromosomes or the mitotic apparatus using the maximum tolerated dose level. Doses of 100, 300 and 1000 mg Olaflur/kg b.w. p.o. were administered to groups of five male and five female mice each killed 24 and 48 h after dose administration. The maximum tolerated dose of 1000 mg Olaflur/ kg b.w. p.o. resulted in no increase in the incidence of micronucleated polychromatic erythrocytes (PCE). No increase was noted in the lower dose levels, either. The positive control, Cyclophosphamide (27 mg/kg b.w. i.p.) resulted in a significant increase to 10.5 micronuclei per 1000 PCEs. The ratio of polychromatic to normochromatic erythrocytes was not influenced.

Supporting study

- Micronucleus Test in vivo (Fouillet, X. et al., 1981):

Olaflur (Amine fluoride 297) was administered orally to mice at concentrations of 2 x 50 and 2 x 200 mg/kg bw.

No noticeable differences in bone marrow nucleated cells were observed between animals treated with Amine fluoride 297 (low and high dose) and those animals receiving only water. In addition, the ratios in all three groups of polychromatic to normochromatic erythrocytes were similar.

There was no significant increase (p> 0.05) in animals exposed to the low dose (50 mg/kg bw) and to the high dose (200 mg/kg bw) of Amine Fluoride 297. A positive control (Thio-TEPA) administered at the concentration of 2 x 40 mg/kg showed pronounced evidence of mutagenicity.

Amine Fluoride 297 does not present a mutagenic effect with dose-relationship in the micronucleus test using mice treated with two oral administrations at 50 and 200 mg/kg bw.

Justification for classification or non-classification

Based on the results of the of the in vitro as well as the in vivo genetic toxicity studies, Olaflur was not classified as genotoxic according to Regulation (EC) No. 1272/2008 (CLP).