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Additional information

A reverse bacterial gene mutation assay (Ames-Test, plate incorporation assay) according to OECD Guideline 471(1997) with the partially unsaturated TEA-Esterquat was negative up to the limit concentration of 5000 µg/plate with and without mammalian metabolic activation (rat liver S9-mix 10 and 30 %) in S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA (pKM101).

Significant bacteriotoxic effects of varying severity were observed, depending on the test strain and the presence of metabolic activation. Generally, bacteriotoxicity was less pronounced in the presence of metabolic activation, especially at concentrations of 30 % S9-mix. Precipitation was observed at 1600 µg/plate and above. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in any of the tester strains in the presence or absence of mammalian metabolic activation.

Results from other reliable bacterial gene mutation assays are available for the partially unsaturated TEA-Esterquat.

All four studies were performed according to the OECD guideline 471 of 1983. Therefore, the additional strain S. typhimurium TA 102 or E. coli WP2 was not tested as required by the current guideline. Studies were performed with and without mammalian metabolic activation up to the limit concentration of 5000 µg/plate. Usable concentration ranges of the experiments were limited by significant bacteriotoxic effects varying in degree of severity, depending on the test strain and the presence of metabolic activation. Generally, bacteriotoxicity was less pronounced in the presence of metabolic activation. Test article precipitation was observed at higher concentrations.The positive controls induced the appropriate responses in the corresponding strains and activity of the metabolizing system was confirmed.There was no evidence of induced mutant colonies over background, in any of the studies.

Data from a similar study with the fully saturated TEA-Estequat are available. No evidence of biologically significant mutagenic activity of the test item was found in the presence and absence of metabolic activation, up to and including the limit concentration of 5000 µg/plate. Significant bacteriotoxic effects were observed at concentrations of 200µg/plate and above. Test article precipitation was not observed.There was no evidence of induced mutant colonies over background in this study.

 

Additional data from an HPRT mammalian cell gene mutation assay and a mammalian cell cytogenicity assay both with the partially unsaturated TEA-Esterquat are available.

In a mammalian cell gene mutation assay (HPRT locus) according to OECD guideline 476, Chinese hamster lung fibroblasts (V79) cells cultured in vitro were exposed to the partially unsaturated TEA-Esterquat (100 % a.i. of UVCB description) at concentrations up to 200 µg/mL in the absence and up to 1500 µg/mL in the presence of mammalian metabolic activation (rat liver S9). 

The concentration range of the main experiments was limited by the solubility of the test item in aqueous medium and by cytotoxic effects. The assay was performed in two independent experiments, using two parallel cultures each. A treatment period of 4 hours was used for all experiments, with the exception of the second experiment with metabolic activation, were a 24 hour treatment period was selected for the cultures.No substantial and reproducible dose dependent increase of the mutation frequency was observed in both experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase of mutant colonies and, thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

The results of this HPRT assay indicate that, the partially unsaturated TEA-Esterquat did not cause a positive response in the non-activated and S9-activated systems and was assessed to be negative under the conditions of this study.

In a mammalian cell cytogenicity assay according to OECD Guideline 473 1997, V79 cell cultures were exposed to the partially unsaturated TEA-Estequat at concentration ranges of 3.1 – 200 µg/mL without metabolic activation and 4.7 – 600 µg/mL in the presence of mammalian metabolic activation.

In experiment I and II, in the absence and the presence of S9 mix, no biological relevant increase in the number of cells carrying structural chromosome aberrations was observed. However, in the presence of S9 mix two significant (p < 0.05) increases were observed, in experiment I at preparation interval 18 hrs after treatment with 37.5 µg/mL (4 % aberrant cells, exclusive gaps), and in experiment II at preparation interval 28 hrs with 300 µg/mL (4.8 % aberrant cells, exclusive gaps). In addition, a dose related increase in the number of cells carrying structural chromosome aberrations was observed in experiment II with metabolic activation.

A confirmatory experiment III was performed to verify these observations. In the repeated experiment in the presence of S9 mix after

4 hrs treatment at a prolonged 28 hrs preparation interval no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. Although the aberration rates showed a dose related increase, the values were clearly within the historical control data range of the testing laboratory. Finally, the observations of experiment II in the presence of S9 mix were not confirmed in experiment III and therefore they have to be regarded as biologically insignificant.

In all experiments, no biologically relevant increase in the rate of polyploid metaphases was found.

In conclusion it can be stated that under the experimental conditions reported, the test item did not induce structural or numeric chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. The test item is considered to be non-clastogenic in this chromosome aberration test with and without S9 mix when tested up to cytotoxic test item concentrations.

 

In vivo data

In a mouse bone marrow micronucleus assay according to OECD guideline No. 474, 1983, 6 male and 6 female albino mice (CFW1) per group were treated by oral intubation with the partially unsaturated TEA-Esterquat at a dose of 5000 mg/kg bw. Bone marrow cells were harvested at 24, 48 and 72 hours post-treatment.

There were no signs of toxicity as indicated by an enhanced mortality rate. A slight reduction in the ratio of polychromatic to normochromatic erythrocytes were determined in female mice 24 and 48 h after administration, indicating possibly a weak toxic effect to the bone marrow. The partially unsaturated TEA-Esterquat was tested at an adequate dose, based on the results of the range-finding test. The positive control induced the appropriate response.

There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

 

Discussion

Most of the studies were performed with the partially unsaturated TEA-Esterquat. Read-across for the whole group of TEA-Esterquats is justified without restrictions.

The full set of in vitro tests required by REACH Regulation Annexes VII and VIII is covered with the studies. There was no evidence of mutagenic or genotoxic intrinsic properties in any of the performed studies.

Additional data from anin-vivomouse micronucleus Test are available, likewise showing no evidence to cause any chromosomal damage in the bone marrow of mice.

Additional in vitro genotoxicity tests have been conducted with other Esterquats and have been reported in the HERA RAR Esterquats (2009). The following information is taken into account as supportive evidence for any hazard / risk assessment.

A mammalian gene mutation test conducted with an HEQ-based esterquat (CAS-no. 19467-38-0) in the CHO/HRTP Locus Assay in the presence and absence of metabolic activation did not show any evidence for mutation.

Moreover, DEEDMAC (CAS-no. 67846-68-8), neither showed clastogenic activity in the chromosomal aberration test in cultured human lymphocytes with and without S9 mix nor mutagenicity in a forward mutation assay with mouse lymphoma cells. From these results it can be extrapolated that Esterquats as a structural family are devoid of in-vitro mutagenicity / genotoxicity.

 

In conclusion there is no evidence for a genotoxic property for any of the TEA-Esterquats. 

 


Short description of key information:
In vitro data from a total of 6 bacterial reverse mutation assays (Ames Test) are available, five with the read-across substance partially unsaturated
TEA-Esterquat and one with the fully saturated TEA-Esterquat. In addition, data are available from a chromosome aberration test and a mammalian cell gene mutation assay (L 5178Y/ TK Mouse Lymphoma assay) with the read-across substance partially unsaturated TEA-Esterquat.
No mutagenicity data are available for the oleic acid-based TEA-Esterquat.
In addition, an in-vivo micronucleus assay was conducted with the read-across substance partially unsaturated TEA-Esterquat in mice.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

None of the TEA-Esterquats are considered to have genotoxic properties as shown in the Ames-Test, mouse lymphoma assay, chromosome aberration study and in thein-vivoMicronucleus Test.

Therefore, none of the TEA-Esterquats have to be classified as “genotoxic” according to Directive 67/548/EEC as well as GHS Regulation EC No 1272/2008. No labelling is required.