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Key value for chemical safety assessment

Additional information

No data on the registered substance itself was available, but data were available on an analogue substance, cocamidopropylhydroxysultaine

which contains alkylamidopropylhydroxysultaine with carbon chain from C8 to C18. The main component of Cocamidopropylhydroxysultaine C8-18 is lauramidopropylhydroxysultaine. Justification for the read-across is documented in a separate document attached in Iuclid Section 13.

Cocamidopropyl hydroxysultaine was assessed for its genotoxic potential in in vitro (Ames test, chromosome aberration test, mouse lymphoma assay) and in vivo (bone marrow micronucleus test) studies.

 

In vitro:

 

Cocamidopropyl hydroxysultaine was tested for its mutagenic potential in vitro in a Klimisch score 2 bacterial reverse mutation (Ames) test (1997), used as a key study. In this study, the test substance as a 50% aqueous solution, was tested in Salmonella typhimurium TA1535, TA1537, TA1538, TA98 and TA100 strains. The bacterial strains were exposed to a range of concentrations up to 20 µL of test solution per plate, using the direct incorporation method both in the presence or absence of an exogenous metabolic activation system. No significant increase in the mean number of revertants over the respective vehicle controls was observed in any of the bacterial strains tested, either in the presence or in the absence of metabolic activation, up to 0.2 or 0.6 µL test solution/plate depending on the strain. Cyotoxic effects were observed at higher dose levels. Therefore, under the conditions of this assay, Cocamidopropyl hydroxysultaine, as a 50% aqueous solution, was not mutagenic up to cytotoxic concentrations.

 

In compliance with REACH Annex VIII requirements and based on negative results in both other in vitro tests, Cocamidopropyl hydroxysultaine was further tested for its mutagenic potential in vitro in a Klimisch score 1 mammalian gene mutation assay (2012), used also as a key study. In this study, the test substance as a 36.2% aqueous solution, was assessed in the L5178Y Tk +/- mouse lymphoma cell line in the presence and the absence of mammalian metabolic activation (S9 mix). The cytotoxicity of the test substance was determined in a preliminary study up to 5000 µg/mL. Two independent experiments were then performed. Cells were exposed to the test substance without metabolic activation for 3 hours in the experiment 1 and 24 hours in the experiment 2. In the condition with metabolic activation, cells were exposed to the test substance for 3 hours in both experiments. Under the experimental conditions of this study, the test item, Cocamidopropyl hydroxysultaine, did not show any mutagenic activity in the mouse lymphoma assay, up to 200μg/mL in the presence of a rat metabolizing system (3-hour treatment) or up to 75μg/mL (3-hour treatment) or 50μg/mL (24-hour treatment) in the absence of a rat metabolizing system. In conclusion, the test item was considered not to be mutagenic to L5178Y cells.

 

In compliance with REACH Annex VII requirements, Cocamidopropyl hydroxysultaine was also tested for its clastogenic potential in vitro in a Klimisch score 1 chromosome aberration test (2012), used as a key study. In this study, the test substance as a 36.2% aqueous solution, was assessed in cultured human lymphocytes. The dose-levels used for treatments were up to 5000 µg/mL for the first experiment, both with and without S9 mix. As cytotoxicity was observed, dose ranges were lowered in the second experiment from 9.38, to 600 µg/mL without S9 mix, and from 9.4 to 300 µg/mL with S9 mix. Without metabolic activation, cells were exposed to the test substance for 3 (exp 1), 20 or 44h (exp 2) whereas with metabolic activation the treatment period was of 3 hours in both experiments. Cells were harvested 20 or 44 h after the beginning of the experiment, corresponding to approximately 1.5 normal cell cycles and 24 hours later. Cytotoxicity of the test substance was assessed by the mitotic index. The highest tested dose level for metaphase analysis induced around 50% cytotoxicity. Under the test conditions, Cocamidopropyl hydroxysultaine did not induce structural chromosome aberrations in cultured human lymphocytes with and without metabolic activation at any treatment time.

In the second experiment only, increases in the numerical aberrations were noted when compared to the vehicle control cultures, without any clear evidence of a dose-response relationship or consistency between cell cultures. These numerical aberrations exclusively consisted of polyploidy. To explore further these numerical aberrations, a bone marrow micronucleus assay was performed in order to assess the aneugenic potential of Cocamidopropyl hydroxysultaine in vivo. Indeed, micronucleus induction may be the result of clastogenic or aneugenic activity.

 

In vivo:

 

As part of the OECD 422 compliant study on the test substance, following daily oral administration (by gavage) to male and female rats from before mating, during mating and, for the females, throughout gestation until day 5 post‑partum (p.p)inclusive,an evaluation of the potential of the test item to induce damage to the chromosomes or the mitotic apparatus in bone marrow cells (increase in the frequency of micronucleated cells) was performed. Three groups of ten male and ten female Sprague-Dawley rats received the test item, Cocamidopropyl hydroxysultaine, as a 36.2% aqueous solution, daily, by oral administration (gavage), over the administration period, at dose levels of 30, 100 or 300 mg/kg/day. An additional group of 10 males and 10 females received the vehicle control, drinking water, under the same experimental conditions. Another group of five males and five females received Cyclophosphamide (CPA) as a single dose of 30 mg/kg on the day preceding scheduled sacrifice, and acted as a positive control group for micronuclei induction. At necropsy, femur was sampled for bone marrow micronucleus analysis. The mean values of the polychromatic/normochromatic erythrocytes (PE/NE) ratios in animals treated with the test item at 30, 100 or 300 mg/kg/day were not statistically significantly different from that of the vehicle control animals. The mean frequencies of micronucleated PE in the three test item treated groups were not found significantly different from that in the vehicle group. Therefore, under the experimental conditions of the study, Cocamidopropyl hydroxysultaine did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells up to 300 mg/kg administered daily for the whole dosing period (i.e., approximately 5 to 6 weeks).

 

Particular case of the numerical abnormalities seen in the Chromosome Aberration assay:

Experiment 1 1 2 2 2 2 1 1 2 2 2 2
Culture 1 2 1 2 1 2 1 2 1 2 1 2
Treatment time (h) 3 3 20 20 44 44 3 3 3 3 3 3
Harvest time (h) 20 20 20 20 44 44 20 20 20 20 44 44
S9 mix - - - - - - + + + + + +
Concentrations (µg/mL)                
0 1 0 0 1 0 0 0 0 0 0 0 0
9.4 - - - - - - - - 2 2 - -
18.8 - - - - - - - - 1 1 - -
37.5 - - - - - - - - 7 11 - -
39.06 - - - - - - 0 0 - - - -
75 - - 3 7 - - - - - - 3 6
78.13 0 0 - - - - 0 1 - - - -
150 - - 1 3 - -    
156.3 1 0 - - - - 0 2 - - - -
300 - - 2 3 7 10 - - - - - -
312.5 1 0 - - - - - - - - - -

As shown in the summary table above, increases in the numerical aberrations when compared to the vehicle controls were noted in the chromosome aberration assay, but:

  • with no consistency between cell cultures (only in the second experiment, not in the first, for the same treatment and harvest times with S9),
  • with no clear evidence of a dose-response relationship (between 75 and 300 µg/mL without S9 for 20 hours of treatment, harvesting at 20 hours; between 37.5 and 156.3 µg/mL with S9 for 3 hours of treatment, harvesting at 20 hours; lower number of numerical abberations at 75 µg/mL than at 37.5 µg/mL without S9 for 3 hours of treatment).

Significant numerical aberrations could be the indication of an aneugenic activity. A weight-of-evidence approach was applied also taking into consideration:

  • the negative results of the Mouse Lymphoma Assay (which is technically able to detect some aneugens),
  • the negative results of the in vivo micronucleus assay, in the bone marrow of rats continuously exposed for 5 to 6 weeks to the test substance (with signs of toxicity indicative of a systemic distribution of the substance and/or its metabolites or degradation products),
  • the absence in the OECD 422 study of histopathological changes, such as atrophy and/or necrosis, in fast-renewal tissues, such as bone marrow or digestive tract, indicative of an inhibitory effect on mitosis,

Based on all these elements, increased numerical aberrations in the chromosome aberration assay were considered toxicologically irrelevant.

 

Conclusion:

 

Based on the results of in vitro (Ames test, chromosome aberration test, mouse lymphoma assay) and in vivo (bone marrow micronucleus test) studies, Cocamidopropyl hydroxysultaine is considered to be devoid of mutagenic, clastogenic or aneugenic properties.


Justification for selection of genetic toxicity endpoint
No study was selected, since all three in vitro studies and the in vivo assay were negative.

Short description of key information:
In vitro tests:
- Ames test: negative in S. typhimurium bacterial strains with and without metabolic activation
- Chromosome aberrations: negative in cultured human lymphocytes with and without metabolic activation for structural chromosome aberrations
- Mouse lymphoma assay: negative with and without metabolic activation

In vivo test:
Bone marrow micronucleus test in rats as part of an OECD 422 repeated dose toxicity study, negative up to 300 mg/kg bw/d (5-6 weeks of exposure)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available experimental data, Cocamidopropyl hydroxysultaine did not induce gene mutations as clear negative results were obtained in the Ames and Mouse Lymphoma assays. The test substance did not induce structural chromosome aberration in vitro in human cultured lymphocytes. No relevant aneugenic activity was observed.

 

Therefore, by analogy with Cocamidopropyl hydroxysultaine, Lauramidopropylhydroxysultaine as a pure substance is considered to be devoid of mutagenic, clastogenic or aneugenic properties and does not need to be classified for genetic toxicity.