Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

Bacterial reverse mutagenicity test

In a reverse gene mutation assay in bacteria, strains TA 100, TA 1535, TA 1537, TA 98 and TA 100 ofS. typhimuriumand WP2 uvr A pkM 101 ofE. coliwere exposed to the read-across substance partially unsaturated IQAC, DMS quaternised (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 partially unsaturated IQAC, DMS quaternised reduced the survival at a concentration of 2500 µg per plate to 0.6 % of the control value.There was no evidence of induced mutant colonies over backgroundin both independently performed experiments.

 

Supporting information is available from a reverse gene mutation assay in bacteria according to the OECD Guideline No. 471 (1997). 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, (100 % active matter (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 oleic-acid based IQAC, DMS quaternised (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. Furthermore there were no indications of clastogenic effects and/or chromosomal aberrations in the study, based on evaluation of colony sizing (ratio of small vs. large colonies).

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), 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 testitem. 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.

 

Further information is available from a mammalian chromosome aberration test performed according to OECD Guideline 473 (1997). Human lymphocyte cultures were exposed to partially unsaturated IQAC, DMS quaternised (no solvent), (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.

Partially unsaturated IQAC, DMS quaternised (no solvent) was tested 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 of Chromosome Aberration induced over background.

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.

 

Read-across

The toxicological profile for systemic endpoints is expected to be similar for target substance oleic-acid based IQAC, DMS quaternised when compared to the source substance partially unsaturated IQAC, DMS quaternised. Read-across is expected to be reliable.

This read-across is based on the hypothesis that source and target substances, both Imidazolium quaternary ammonium compounds (IQACs)have similar toxicological properties, based on the followingunderlying identical generic structure. There are only minor differences in carbon chain length (C16 and C18) and degree of saturation of the fatty acid moiety present in the structure of source and target substance.

This prediction is supported by toxicological data on the substances themselves. Associated Robust study summaries are provided in the Technical Dossier.

 

Detailed information on substance identity of source and target substance can be found in the “Justification for read-across - toxicological information”, provided in a separate document in chapter 13 of Technical Dossier and attached to the endpoint summary.

Read-across within the endpoint genetic toxicity is considered an appropriate adaptation to meet the standard information requirements according to REACH Regulation. 

 

Overview on available genotoxicity data:

 

 

Endpoint

Source substance

 

Partially unsaturated IQAC, DMS quaternised

Supporting source substance

Oleic-acid based IQAC, DES quaternised

Target substance

 

Oleic-acid based IQAC, DMS quaternised

In vitrogene mutation study in bacteria

negative

in S. typhimurium and E. coliwith and without metabolic activation

negative

inS. typhimuriumandE. coliwith and without metabolic activation

 

Mutagenicity;In vitrogene mutation study in mammalian cells

 

 

negative

inMouse lymphoma L5178Ycells with and without metabolic activation

no indicationof clastogenic effects and/or chromosomal aberrations based on colony sizing.

In vitrocytogenicity study in mammalian cells

negative

inChinese hamster Ovary (CHO)with and without metabolic activation

 

 

negative

inHuman lymphocyteswith and without metabolic activation

 

 

  

The structural similarities between the source and the target substances are the basis for the read-across hypothesis. Adequate, reliable and available scientific information indicates that the source and target substances have similar physicochemical properties and toxicity profiles and thus support the read-across hypothesis. For detailed information refer to the separate document “Justification for read-across - toxicological information”, provided in chapter 13 of Technical Dossier.

Moreover for bacterial reverse mutation testing additional supporting information is available from oleic-acid based IQAC, DES quaternised. This substance is quaternised with diethylamin instead of dimethlyamin which is used for source and target substance. In essence the structure is nearly identical to source and target substance and therefore the toxicological profiles are expected to be comparable.

This assumption is proven by the same low oral toxicity for the oleic-acid based IQAC, DES quaternised, as shown for source and target substance. The oral LD50 in rats was determined to be > 2000 mg/kg bw.

 

Mammalian gene mutation was evaluated in L5178 Y (mouse lymphoma thymidine kinase locus) cells with the target substance (oleic-acid based IQAC, DMS quaternised). The substance was considered to be non-mutagenic in this study. Furthermore there were no indications of clastogenic effects and/or chromosomal aberrations in the study, based on evaluation of colony sizing (ratio of small/large colonies).

 

This negative genotoxic profile is proven by further genetic toxicity studies conducted with source substance (partially unsaturated IQAC, DMS quaternised) and supported by data from oleic-acid based IQAC, DES quaternised, evaluating point mutations in bacteria and chromosomal aberrations inChinese hamster Ovary (CHO) and human lymphocytes.

 

In conclusion there were no indications of genotoxic properties in the three in-vitro test systems and thus there is no evidence for genotoxicity of the evaluated group of IQACs.

 

 

 


Justification for selection of genetic toxicity endpoint
No single key study has been selected since all available studies were negative.

Short description of key information:
In vitro data from a bacterial reverse mutation assay (Ames Test) are available with partially unsaturated IQAC, DMS quaternised and oleic-acid based IQAC, DES quaternised as a read-across substance. In addition data are available from a mammalian cell gene mutation assay (L 5178Y/ TK Mouse Lymphoma assay) with oleic-acid based IQAC, DMS quaternised and a chromosome aberration test in Chinese Hamster Ovary Cells based on the read-across partially unsaturated IQAC, DMS quaternised.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The oleic-acid based IQAC, DMS quaternised is considered to have no genotoxic properties as shown in the Ames-Tests, mouse lymphoma assay and chromosome aberration study.

Therefore, the oleic-acid based 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.