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EC number: 437-760-1 | CAS number: -
- Ames Test (OECD 471, GLP, K, rel. 1): non mutagenic up to cytotoxic concentration in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2uvrA.
- L5178Y/MLA Mammalian Cell Gene Mutation Assay (OECD 476, GLP, K, rel. 1): non mutagenic up to cytotoxic concentration.
- Human lymphocytes chromosome aberration test (OECD 473, GLP, K, rel. 1): non clastogenic up to cytotoxic concentration.
See the attached document for information on tables of results
In a reverse gene mutation assay performed according to the OECD test guideline No. 471/EU Method B.13/14 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli WP2 uvr A were exposed to the test material diluted in DMSO at the following concentrations:
Preliminary toxicity test: 10, 100, 500, 1000, 2500 and 5000 µg/plate in TA 98, TA 100 and WP2 uvrA strains, with and without S9 mix, using direct plate incorporation method
Mutagenicity tests without S9 mix (direct plate incorporation method):
- 93.75, 187.5, 375, 750 and 1500 µg/plate: for any tester strains in the first experiment as well as for the WP2 uvrA strain in the second experiment,
- 46.875, 93.75, 187.5, 375 and 750 µg/plate: for the TA 1535 and TA 100 strains in the second experiment,
- 23.438, 46.875, 93.75, 187.5 and 375 µg/plate: for the TA 1537 and TA 98 strains in the second experiment.
Mutagenicity tests with S9 mix:
- 93.75, 187.5, 375, 750 and 1500 µg/plate: for all tester strains in the first experiment (direct plate incorporation method) as well as for the WP2 uvrA strain in the second experiment (pre-incubation method),
- 46.875, 93.75, 187.5, 375 and 750 µg/plate: for Salmonella typhimurium strains in the second experiment (pre-incubation method).
Vehicle and positive control groups were also included in mutagenicity tests.
No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels. Experiment without S9 mix: A slight to strong toxicity was induced in Salmonella typhimurium strains mainly at dose-levels ≥ 750 µg/plate. In the WP2 uvrA strain, a slight to moderate toxicity was noted at 1500 µg/plate. The test substance did not induce any noteworthy increase in the number of revertants in any of the five strains.
Experiment with S9 mix: In the first experiment, a slight to marked toxicity was generally noted at 1500 µg/plate (direct plate incorporation method). In the second experiment (preincubation method), a slight to marked toxicity was generally induced at dose-levels ≥ 375 µg/plate. The test substance did not induce any noteworthy increase in the number of revertants in any of the five strains.
The vehicle control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
Under the test condition, the test material is not mutagenic with and without metabolic activation to S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli WP2 uvr A.
This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.
In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, L5178Y tk+/-(3.7.2C) mouse lymphoma cells were exposed to test item at the following concentrations:
Preliminary toxicity test: 3.44, 6.89, 13.77, 27.54, 55.08, 110.16, 220.33, 440.65, 881.3, 1762.6 µg/mL
- Without S9 mix, Test 1 (3 hours): 25, 50, 100, 200, 210, 220, 230 240, 250 µg/mL
- With S9 mix, Test 1 (3 hours): 25, 100, 200, 250, 300, 350, 400, 450 µg/mL
- Without S9 mix, Test 2 (24 hours): 5, 10, 15, 20, 30, 50, 100, 200, 250 µg/mL
Vehicle and positive control groups were also included in each mutation test. Metabolic activation system used in this test was 5 % (v/v) S9 mix; S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with Aroclor 1254.
In the preliminary toxicity test, toxicity was observed following a 3 hour exposure to test item in both the absence and presence of S9 mix, and following a 24 hour exposure in the absence of S9 mix. At concentrations from 3.44 to 1762.6 μg/mL, relative suspension growth was reduced from 117% to 0%, from 101% to 0% and from 87% to 0% respectively. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess concentrations which span the complete toxicity range of approximately 10% to 100% relative total growth (RTG). In the first and second main tests in the absence and presence of S9 mix, the maximum concentration test item plated for determination of mutant frequency was 250 μg/mL, where RTG was reduced to 20% relative to the concurrent solvent control. There were no increases in induced mutation frequency (i.e. the mean mutant frequency of test item minus the mean concurrent solvent control mutant frequency) that exceeded the Global Evaluation at any of the concentrations tested within acceptable levels of toxicity. In all tests the concurrent solvent and positive control were within acceptable ranges.
Under the test conditions, test item is not considered as mutagenic at the tk locus of L5178Y mouse lymphoma cells in the presence and absence of metabolic activation.
In an in vitro chromosome aberration test performed according to OECD Guideline 473 and in compliance with GLP, cultured human lymphocytes were exposed to test item at the following concentrations:
Preliminary Toxicity Test (Cell Growth Inhibition Test):
0, 6.88, 13.75, 27.5, 55, 110, 220, 440, 880 and 1760 μg/mL; 4-hour exposure time with and without metabolic activation followed by a 20-hour recovery period, and a continuous exposure of 24 hours without metabolic activation
EXPERIMENT 1: 4 hour treatment followed by 24 hour harvest after the start of treatment
without S9: 0, 25, 50, 100, 200, 300 and 400 μg/mL
with S9 (2%): 0, 25, 50, 100, 200, 300 and 400 μg/mL
without S9: 0, 6.25, 12.5, 25, 50, 100 and 150 μg/mL; 24 hour treatment followed by harvest at the end of treatment
with S9 (1%): 0, 50, 100, 200, 240, 300 and 360 μg/mL; 4 hour treatment followed by 24 hour harvest after the start of treatment
Mitotic activity was arrested by addition of colcemid at 0.1 µg/mL for each culture, two hours before the harvest. The cells were then treated with a hypotonic solution, fixed, stained and examined for mitotic indices and chromosomal aberrations. Vehicle and positive controls were also included in this test.
In Experiment 1, the highest evaluable concentration was 300 µg/mL based on toxicity and a lack of metaphases suitable for scoring at the highest test concentration of 400 µg/mL. The results of the mitotic indices (MI) from the cultures after their respective treatments show that 15% and 23% growth inhibition was achieved with and without S9 at 300 µg/mL, respectively. In Experiment 2, there was an increase in toxicity in the presence of S9 compared to Experiment 1. The MI from the cultures after their respective treatments confirmed 56% growth inhibition was achieved at 150 µg/mL in the absence of S9 and 65% growth inhibition was observed at 300 µg/mL in the presence of S9 and there were metaphases suitable for scoring at these dose levels. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that exceeded or was approximately 50% mitotic inhibition, depending on the exposure group. All vehicle (solvent) control groups had frequencies of cells with aberrations within the range expected for normal human lymphocytes. The positive control substances, mitomycin C and cyclophosphamide gave the expected statistically significant increases in the number of cells with structural chromosome aberrations, which demonstrates the sensitivity of the test system.
Under the test conditions, the test item was considered to be non-clastogenic to human lymphocytes in vitro.
Table 7.6/1: Summary of genotoxicity tests
Test / Guideline
K, rel. 1
E.coli WP2 uvrA
Up to cytotoxic concentration
-S9 : non mutagenic
+S9 : non mutagenic
L5178YTK+/-/MLA test (OECD 476)
L5178Y tk+/-(3.7.2C) mouse lymphoma cells
-S9 : non clastogenic
+S9 : non clastogenic
Gene mutation Assays (Tests n° 1 -2):
- A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD test guideline No 471 with the substance (See Table 1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose, either in the presence or absence of metabolic activation. The substance does not induce gene mutations in bacteria under the test condition whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The substance is therefore considered as non-mutagenic according to the Ames test.
- Inability to produce gene mutation was confirmed in mammal cells using an in vitro gene mutation assay in L5178Y tk+/-(3.7.2C) mouse lymphoma cells (L5178Y TK+/- /MLA test) (Test n°2). None of the dose levels up to the cytotoxicity limit, either in the presence or absence of metabolic activation, induced significant mutant frequency increases in the initial or repeat experiments whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases. Therefore, the substance is considered as negative for inducing gene mutations at the TK locus in L5178Y mouse lymphoma cells under activation and non-activation conditions used in this assay. This result confirms the results of the Ames test and extends the non-mutagenic effect of the substance to mammalian cells.
Chromosomal aberration (Test n°3)
The clastogenic potential of the test material was determined using an in vitro chromosome aberration test in Human lymphocytes, which measures the potential of a substance to increase the incidence of structural chromosome aberrations in cultured Human lymphocytes.
None of the dose levels up to the cytotoxicity limit, either with or without metabolic activation, induced significant increases in the frequency of cells with aberrations in either of two experiments. The substance does not induce structural aberrations in the chromosomes of Human lymphocytes under activation and non-activation conditions, whereas both positive control chemicals (with and without metabolic activation) induced significant increases in the frequency of aberrant cells. Therefore, the substance is considered as negative for inducing chromosomal mutations in Human lymphocytes in vitro under activation and non-activation conditions used in this assay.
The test material has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008.
Based on the available data, no additional classification is proposed regarding germ cell mutagenicity according to the Annex I of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
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