Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

Genetic toxicity in vitro

Ames test:

Non micronized ETH50 was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay (CIT 26390MMT, 2003).

STRAINS: TA 1535, TA 100, TA 1537, TA 98, TA 102 and E. coli WP2 uvrA. CONCENTRATIONS: 62.5, 125, 250, 500 and 1000 μg/plate. TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats). SOLUBILITY: A moderate to marked precipitate was observed when scoring the revertants mainly at concentrations above 500 µg/plate.

TOXICITY: No toxicity was noted towards all the strains used, both with and without S9 mix. MUTAGENICITY: The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the six strains tested. CONCLUSION: Thus, under the experimental conditions of this study, non-micronized ETH50 was not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of mammalian metabolic activation as compared with concurrent vehicle controls.

Mouse Lymphoma Assay (MLA):

The potential of non-micronized ETH50 to induce mutations at the TK (thymidine kinase) locus in L5178Y mouse lymphoma cells was evaluated in a study performed according to OECD TG 476 in compliance with GLP (CIT 28644MLY, 2005). The test item was tested in two independent experiments, with (3 hour treatment) and without (3 and 24 hour treatment) a metabolic activation system. Since the test item was poorly soluble and non-toxic in the preliminary assay, the highest dose-level used for treatment in the main test was limited by precipitate in the culture medium. No noteworthy toxicity was noted at any dose-level, in either experiments. The test item did not induce any significant increase in the mutation frequency in either experiments. Under the experimental conditions of this study, non-micronized ETH50 did not show any mutagenic activity in the mouse lymphoma assay.

Chromosomal Aberration test (CA):

Non -micronized ETH50 was tested in an in vitro chromosomal aberration test in human lymphocytes (CIT 28643MLH, 2005). The study was performed following the OECD 473 guideline under GLP. The test item was tested in two independent experiments, both with and without a liver metabolizing system (S9 mix), obtained from rats previously treated with Aroclor 1254. The highest concentration for treatment in the first experiment was selected on the basis of pH, osmolality and solubility. For selection of the concentrations for the second experiment, any toxicity indicated by the reduction of mitotic index (MI) in the first experiment was also taken into account. Final concentrations selected were 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250 and 500 μg/mL for the first experiment with and without S9 mix, and 15.6, 31.3, 62.5, 125, 250 and 500 μg/mL for the second experiment with and without S9 mix. The duration of the treatment was 3, 20 or 44 hours (short or continuous treatment method). Both the positive control substances mitomycin C and cyclophosphamid induced a sufficiently high frequency of chromosomal structural aberrations. Non-micronized ETH50 did not induce a higher frequency of chromosomal aberration for both the continuous treatment method and short term treatment method with or without metabolic activation.

Genetic toxicity in vivo

The substance ETH50 registered represents the form of pure (bulk) non-nano material.  As such, however, it is not usable for its intended use in cosmetic products. In order to transform it into suitable cosmetic ingredient, ETH50 has to be micronized in a water-mill. As a consequence of the extreme hydrophobicity of substance, surfactants need to be added to introduce it in the water phase. After milling, rheology modifiers prevent the nano-particles from agglomerating and stabilize the resulting preparation. Thus, the substance is marketed in a 50 % mixture together with different components; it is not available as pure material in nanoform. The key studies for genotoxity in vivo were performed with the bulk (non-nano) form of ETH50 (relating to the substance registered) and a formulation containing micronized ETH50 (relating to the marketed mixture for the use in cosmetics).


Unscheduled DNA Synthesis test (UDS):

Micronized (nano-form) and non-micronized ETH50 was investigated at a dose level of 1000 mg/kg bw (micronized only) and 2000 mg/kg bw and at two expression times (2 -4 hours and 12 -16 hours) in the in vivo unscheduled DNA Synthesis (UDS) test in hepatocytes from 3 male Fischer rats in a study performed in accordance with OECD TG 486 under GLP (Institut Pasteur FSR-IPL 070101, 2008). Neither micronized nor non-micronized ETH50 did reveal any genotoxic activity under the test conditions and did not induce unscheduled DNA synthesis in hepatocytes.


Micronucleus test (MNT):

The ability of micronized (nano-form) and non-micronized ETH50 to cause chromosomal damage in vivo was investigated in a OECD 474 micronucleus test (CIT 32753MAS, 2008). Dose-levels for treatment were selected on the basis of a preliminary toxicity test. Groups of 5 male and female Swiss Ico: OF1 (IOPS Caw) mice were dosed twice intraperitoneally with vehicle only or with 500 mg/kg bw/ day (micronized only), 1000 mg/kg bw/ day (micronized only) and 2000 mg/kg bw/day. Cyclophosphamide served as positive control (oral, 50 mg/kg bw). The animals of the treated and vehicle control groups were killed 24 hours after the last treatment.

For all test item treated groups, the mean values of MPE were equivalent to those of the vehicle control group. The PE/NE ratio was significantly lower when compared to that of the vehicle control group, in the group of females treated with non-micronized Tinosorb A2B.

Under the experimental conditions chosen, micronized and non-micronized ETH 50 did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells.

Overall, ETH50 regardless of its particle size, is not clastogenic or aneuploidic in mice under conditions of this study.

Justification for classification or non-classification

The present data on genetic toxicity do not fulfill the criteria laid down in 67/548/EEC and regulation (EU) 1272/2008 and therefore, a non-classification is warranted.