Fatty acids, C16-18 and C18-unsatd., reaction products with diethylenetriamine, di-Me sulfate-quaternized

Endpoint summary

Additional information

In vitro data from bacterial reverse mutation assays (Ames Test) are available for partially unsaturated IQAC, DMS quaternised (palm oil fatty acids) (CAS-no. 92201-88-2), partially unsaturated IQAC, DMS quaternised (tallow fatty acids) (CAS-no. 68122-86-1) and for oleic-acid based IQAC, DES quaternised (CAS-no. 67846.14-4) as a read-across substance. In addition data are available from a mammalian cell gene mutation assay (L 5178Y/ TK Mouse Lymphoma assay) with the read-across substance oleic-acid based IQAC, DMS quaternised (CAS-no. 72749-55-3), a chromosome aberration test in Chinese Hamster Ovary Cells based on partially unsaturated IQAC, DMS quaternised (tallow fatty acids; CAS-no. 68122-86-1) and a chromosome aberration test in human lymphocytes with partially unsaturated IQAC, DMS quaternised (palm oil fatty acids; CAS-no. 98219-51-3).

Bacterial reverse mutagenicity test

In a reverse gene mutation assay in bacteria, strains TA 100, TA 1535, TA 1537, TA 98 and TA 100 of S. typhimurium and WP2 uvr A pKM 101 of E. coli were exposed to partially unsaturated IQAC, DMS quaternised (palm oil fatty acids based; a.i. 76.9 %) diluted in water at concentrations of 125 to 2500 µg per plate in the presence and absence of mammalian metabolic activation (co-incubation).

Significant bacteriotoxic effects were not observed in the main study up to and including the highest concentration tested of 2500 µl/plate. In the range finding study the test substance reduced the survival of S. typhimurium TA 100 at a concentration of 2500 µg per plate to 0.6 % of the control value. For E. coli WP2 uvr A pKM 101 only slight decreases in plating efficiency were observed. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

Partially unsaturated IQAC, DMS quaternised (tallow fatty acids), 75 %) did not reveal gene mutations by base pair changes or frame shifts in the DNA of the bacterial strains used in the reverse gene mutation assay (according to OECD Guideline 471, 1984 and US EPA-TSCA Guidelines: strains TA 100, TA 1535, TA 1537, TA 1538 and TA 98 of S. typhimurium at concentrations of 0.050 to 8.0 µl/plate, corresponding to 6000 µg/plate based on active ingredient at the highest dose, in the presence and absence of mammalian metabolic activation as a plate incorporation test). The test material exhibited varying degrees of toxicity with all the strains at higher doses in the non-activation and activation assays.

Supporting data of a mutagenicity test are available for a reverse gene mutation assay in bacteria according to the OECD Guideline No. 471 (1997) with a read-across substance. In this test Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the Escherichia coli strain WP2 uvrA were exposed to the oleic-acid based IQAC, DES quaternised, (CAS-no. 67846-14-4; 99 % solid matter, diluted with acetone) at concentrations of 5, 15, 44, 62, 133, 185, 400, 556, 1667 and 5000 µg/plate in the presence and absence of mammalian metabolic activation.

The oleic-acid based IQAC, DES quaternised was tested up to cytotoxic concentrations (400 µg/plate). The positive control treatments in both the non-activation and S9 activation assays induced large increases in the revertant numbers with all the indicator strains, which demonstrated the effectiveness of the S9 activation system and the ability of the test system to detect known mutagens.

No substantial increases in the revertant colony numbers of any of the five test strains were detected at any dose level of the test item either with or without metabolic activation in both independently performed experiments.

Mouse Lymphoma Assay for mutagenic activity in mammalian cells

In a mammalian gene mutation assay,according to OECD guideline 476, (1997) and EU method B.17 (2000), L5178 Y (mouse lymphoma thymidine kinase locus) cells cultured in vitro were exposed to the read-across substance oleic-acid based IQAC, DMS quaternised (CAS-no. 72749-55-4; 98 % a.i.) at the following concentrations:

Experiment I

- with metabolic activation: 2.50, 5.00, 7.50, 10.0, 12.5, 15.0, 20.0, 40.0 and 60.0 µg/ml

- without metabolic activation: 1.00, 3.00, 5.00, 10.0, 12.0, 16.0, 18.0, 20.0, 25.0 and 30.0 µg/ml

Experiment II

- with metabolic activation: 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0 and 55.0 µg/ml

- without metabolic activation: 0.50, 1.00, 2.00, 4.00, 6.00, 8.00, 10.0, 12.0 and 16.0 µg/ml

Oleic-acid based IQAC, DMS quaternised was tested up to cytotoxic concentrations (≥16.0 µg/ml without metabolic activation and ≥ 40.0 µg/ ml with metabolic activation). The positive controls induced the appropriate responses. There was no evidence of induced mutant colonies over background.

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item oleic-acid based IQAC, DMS quaternised is considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y. 

Chromosomal aberration test in mammalian cells:

In a mammalian cell cytogenetics assay according to OECD Guideline 473 (1983) and US EPA-TSCA Guidelines, CHO cell cultures were exposed with partially unsaturated IQAC, DMS quaternised (tallow fatty acids, CAS-no. 68122-86-1), 75 %) at concentrations of 15.0 to 199 µl/ml with metabolic activation and with 3.74 to 74.8 µl/ml without metabolic activation. Microscopic evaluation of aberrant cells was performed for the concentrations of 49.9, 99.7, 150 and 199 µl/ml (with metabolic activation) and 24.9, 37.4, 49.9, and 74.8 µl/ml, respectively, without metabolic activation.

Cytotoxic effects of the test item were observed with and without metabolic activation; at the highest evaluated concentrations as evident by reduction of the cell monolayer confluency to 40 % in both experiments, without and with metabolic activation.

Neither without metabolic activation nor with metabolic activation a significant increase in chromosomally aberrant cells was observed at the concentrations analysed.

Positive control references Mitomycin C and cyclophosphamide were tested in parallel to the test item. They induced distinct and biologically relevant increases in cells with structural chromosomal aberrations.

There was no evidence of chromosome aberration induced over background in the CHO Chinese hamster cell line by partially unsaturated IQAC, DMS quaternised (tallow fatty acids). Therefore, the test item partially unsaturated IQAC, DMS quaternised (tallow fatty acids), 75 %) is considered to be non-clastogenic. 

In a mammalian chromosome aberration test performed according to OECD Guideline 473 (1997), human lymphocyte cultures were exposed to partially unsaturated IQAC, DMS quaternised, palm-oil based (100% a.i.), suspended in ethanol at concentrations between 11.9 - 3200 µg/mL with metabolic activation and 20.8 - 3200 µg/mL without metabolic activation.

The test was performed up to cytotoxic or precipitating concentrations. 

The following experimental points were microscopically evaluated: 11.9, 20.0, 20.8, 50.0, 125.0, 341.2, 597.1 µg/mL with metabolic activation and 20.8, 36.4, 43.5, 76.2, 133.3, 233.2, 341.2, 597.1 µg/mL without metabolic activation.

In the absence of S9 mix, reduced mitotic indices of about or below 50 % of control were observed at the highest evaluated concentrations. In the presence of S9 mix, concentrations showing clear cytotoxic effects were excluded from scoring for the endpoint cytogenicity.

In Experiment II, in the absence of S9 mix, a single increase in chromosomal aberrations was observed, slightly exceeding the laboratory’s historical control data range, but since the value was not statistically significantly increased these findings were considered as biologically irrelevant. A single statistically significant increase was observed in Experiment II, in the presence of S9 mix, but the value was clearly within the range of the laboratory’s historical control data and thus considered as being without biological relevance.

Positive controls induced the appropriate response. There was no evidence for chromosome aberrations induced over background.

Read-Across justification for other members of the IQAC structural family:

The variance in the relative composition of the aliphatic chain moieties between the partially unsaturated IQAC, DMS quaternised (tallow fatty acids; CAS-no. 68122-86-1), partially unsaturated IQAC, DMS quaternised (palm oil fatty acids; CAS-no. 92201-88-2 & 98219-51-3), oleic acid based IQAC, DMS quaternised (CAS-no. 72749-55-4) and oleic-acid based IQAC, DES quaternised; CAS-no. 67846-14-4) is primarily a reflection of the degree of saturation of the fatty acids side chains which are produced using predominantly natural sources, but has little impact on the overall aliphatic side chain length. With respect to the fully reacted quaternising agent, no significant biological differences have been reported.

Given the structural similarity, significant differences in metabolic patterns are highly unlikely between the three compound classes, and absorption following oral or dermal uptake can be expected to be similarly low in all three cases.

Thus, there does not appear to be any restriction in the extrapolation of the above presented toxicological data generated for homologous members of the IQAC, quaternised structure family for the purpose of the assessment of the toxicological properties of the partially unsaturated IQAC, DMS quaternised.

As there were no indications of genotoxicity in the three in-vitro test systems mandatory for Annex IX substances and reported above, no in-vivo genotoxicity tests have been conducted or proposed.

Short description of key information:
In vitro data from a bacterial reverse mutation assay (Ames Test) are available for partially unsaturated IQAC, DMS quaternised and for oleic-acid based IQAC, DES quaternised (CAS-no. 67846.14-4) as a read-across substance. In addition data are available from a mammalian cell gene mutation assay (L 5178Y/ TK Mouse Lymphoma assay) with the read-across substance oleic-acid based IQAC, DMS quaternised (CAS-no. 72749-55-3), a chromosome aberration test in Chinese Hamster Ovary Cells and a chromosome aberration assay with human lymphocytes based on partially unsaturated IQAC, DMS quaternised. The full set of in vitro tests required by REACH Regulation Annexes VII and VIII are negative; additional testing is not required.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The partially unsaturated IQAC, DMS quaternised is considered to have no genotoxic properties as shown in the Ames-Tests, mouse lymphoma assay and chromosome aberration studies with this and structurally closely related compounds.

Therefore, the partially unsaturated IQAC, DMS quaternised does not need to be classified as “genotoxic” according to Directive 67/548/EEC as well as GHS Regulation EC No 1272/2008. No labelling is required.