Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, N-(hydroxyethyl) derivs.

Endpoint summary

Additional information

Evaluation of genetic toxicity of Tris (2-hydroxyethyl) tallow diaminopropane, CAS 90367-27-4 (recently redefined as Amines, N-(C16-18 (even numbered) and C18-unsatd. alkyl) trimethylenedi-, ethoxylated (NLP), CAS 1290049-56-7), also referred to as Tallow-diamine3EO:

For this endpoint partial read-across is applied to Coco-diamine3EO (Tris (2-hydroxyethyl) Cocoalkyl diaminopropane, CAS 90367-21-8). Data from Coco-diamine3EO is considered to represent a worst case situation for Tallow-diamine3EO, as both substances have the same molecular structure, thus showing the exact same basic chemical reactivity. But due to its overall smaller alkyl chain length, the solubility and potential to pass cell-membranes of Coco-diamine3EO is expected to be higher than that of Tallow-diamine3EO. Therefore, any inherent genotoxic hazards are more likely to be expressed when testing with Coco-diamine3EO than with Tallow-diamine3EO. (See also separate document in support to read-across)

 

The following studies are available Tallow-diamine3EO and Coco-diamine3EOfor the assessment of genetic toxicity. Al tests are performed to current OECD/EU protocols and in compliance to GLP.

 

Tallow-diamine3EOwas tested in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system. All controls showed adequate responses. The test material was tested up to the toxic limit. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

 

Coco-diamine3EO was also tested in this Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat), and did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA, both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

 

Coco-diamine3EO was assessed for its potential to induce gene mutations at the Thymidine kinase (TK) locus using mouse lymphoma L5178Y. The test was performed in two independent experiments in the absence and presence of S9-mix. In both the presence and absence of S9-mix,Coco-diamine3EO did not induce a significant increase in the mutation frequency.

 

Coco-diamine3EO was further evaluated forpossible clastogenicity and aneugenicityin ani n vitro micronucleus assay in cultured peripheral human lymphocytes (with independent repeat) in the presence and absence of a metabolic activation system. The study was performedaccording to OECD 487 and in compliance to GLP. The results of the positive and negative controls indicate that the conditions of the test were adequate.

Coco-diamine3EOinduces the formation of micronuclei in human lymphocytes in the absence of S9 metabolic activation after a short exposure period. The results onlyreach statically significance at one concentration.In contrast, in the presence of S9-mix and in the absence of S9-mix at a prolonged exposure period no such genotoxic effect was found. Based on the criteria that a statistically significant increase would be evaluated as positive, even in the absence of a dose response relation, the report concludes to a positive genotoxic result forCoco-diamine3EO.

 

A similar micronucleus assaywas done on Tallow-diamine3EO under the same test conditions. The results of this study show a statistically significant increase in the number of binucleated cells with micronuclei at an intermediate concentration at prolonged exposure duration only. Although this increase is not dose dependent, the number of binucleated cells with micronuclei is above the historical control data range. The report therefore concludes that Tallow-diamine3EO is equivocal in thein vitro micronucleus study.

 

When comparing the MN studies on Coco-diamine3EO closely to the Tallow-diamine3EO, the following observations can be made:

- Both studies basically showed the same picture:A small increase in micronuclei can be observed at toxic dose levels without needing metabolic activation.

- The coco-derivative of ethoxylated diamine is somewhat better water soluble, and no precipitation has been observed.

- Contrary to the tallow, shows the coco substance a somewhat higher toxicity with longer exposure duration.

- Similar to tallow, there are no differences between assays with or without metabolic activation with respect to cytotoxicity. (Which corresponds with the notion that these substances do not undergo important phase-one metabolism). Also with S9 it looks like some increase of micronuclei is visible for Coco-diamine3EO, but only without S9 it reaches a statically significance at one concentration. This could be because 30 and 40µg/ml were possibly too toxic (also lack of mononucleated cells to score and at 40 even also of binucleated cells), and the 25 µg/ml that showed the increase without metabolic activation was not evaluated in the presence of S9.

- The small increase of micronuclei at toxic levels is similar in Coco as for Tallow, and again not systematically observed. In this case in the short exposure period, but not in the long exposure assay as for Tallow.

- The effects are only seen in combination of toxicity. In the slides evaluated as positive, there were not enough mononucleated cells available for scoring, indicating an extreme loss of cells. If this cell loss was due to toxicity, this could be indication for excessive toxicity (> 50% cell death is generally considered as excessive)

- For both Coco and Tallow-diamine3EO there is no dose-relationship observed. In case of absence of dose relation, a substance can still be considered positive in case the increase is biologically relevant. This should be interpreted for the evaluation where only the highest dose which is not too toxic shows an really significant increase which is not just passing the level of statistical significance or the limit seen in historical controls. In both cases it can be questioned whether this criteria of biological significance is really reached: response are marginal, and not consistent, and at levels that show (possible too high) toxicity, and not showing clear dose-response relation.

 

In conclusion, both studies evaluating the possible clastogenicity and aneugenicity properties of Coco-diamine3EO and Tallow-diamine3EO in anin vitro micronucleus assay in cultured peripheral human lymphocytes show consistent results that can, on overall be characterised as inconsistent, possibly weakly positive responses at cytotoxic levels.

 

 

The overall evaluation concludes that microbiological mutagenicity and mammalian mutagenicity studies show negative results, whereas the results from in vitro micronucleus assays in human lymphocytes can only be described as equivocal.

These obtained results are consistent, and do not lead to a need for classification.

Also further studies are not expected to improve upon the available data, and are therefore not proposed.

Justification for selection of genetic toxicity endpoint

For each endpoint bacterial mutagenicity, mammalian mutagenicity and mammalian clastogenicity one or more GLP compliant studies are available on the basis of testing with Tallow-diamine3EO itself or from cross-reading from Coco-diamine3EO. The evaluation is based on the weight of evidence from all available data.

Short description of key information:

The overall evaluation concludes that microbiological mutagenicity and mammalian mutagenicity studies show clear negative results, whereas the results from in vitro micronucleus assays in human lymphocytes are equivocal.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The overall evaluation concludes that microbiological mutagenicity and mammalian mutagenicity studies show negative results, whereas the results from in vitro micronucleus assays in human lymphocytes can only be described as equivocal. Overall conclusion is therefore inconclusive.

These results obtained results are consistent, and do not lead to a need for classification for genotoxicity.

Also further studies are not expected to improve upon the available data, and are therefore not proposed.