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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

negative with and witout metabolic activation (in vitro gene mutation in bacteria)

negative with and witout metabolic activation (in vitro chromosome aberration in mammalian cells)

negative with and witout metabolic activation (in vitro mammalian cell gene mutation)

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

in vitro gene mutation in bacteria

The potential of the test item, to induce reverse mutation in Salmonella typhimurium was evaluated in one study on the substance and in two studies on a similar substance.

The strains were exposed to the substance with and without metabolic activation.

In all three studies, the tested substance did not induce and reverse mutations to S.typhimurium and therefore the test is negative both with and without metabolic activation.

 

in vitro chromosome aberration in mammalian cells

The potential of the similar substance, to induce structural chromosome aberrations in human lymphocytes in vitro was assessed according to OECD guideline no. 473 and EC guideline no B10 and in compliance with the Principles of Good Laboratory Practice.  The test item (dissolved in water for injections) was tested in two independent experiments, both with and without a liver metabolizing system (S9 mix). In the first experiment, lymphocyte cultures were exposed to the test or control items (with or without S9 mix) for 3 hours then rinsed. Cells were harvested 20 hours after the beginning of treatment.

The second experiment was performed as follows:

-  without S9 mix, cells were exposed continuously to the test or control items until harvest,

- with S9 mix, cells were exposed to the test or control items for 3 hours and then rinsed.

Cells were harvested 20 hours and 44 hours after the beginning of treatment. Three hours before harvest, each culture was treated with a Colcemid® solution to block cells at the metaphase-stage of mitosis. After hypotonic treatment, the cells were fixed, spread on glass slides and stained with Giemsa. All the slides were coded for scoring. The negative control was the vehicle (Water for injections).

 The dose-levels selected for the first experiment were 39.06, 78.13, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/ml (doses expressed in terms of active ingredient i.e the three forms of sodium isopropylnaphtalenesulphonate: mono, di and tri => equivalent to 51.9, 103.9, 207.9, 415.6, 831.25, 1662.5, 3325 and 6650 µg/ml in terms of registered substance), both with and without S9 mix.

Based on the cytotoxicity observed in the first experiment, the dose-levels selected for the second experiment were as follows:

- 31.3, 62.5, 125, 250, 500 and 1000 µg/ml (i.e. 41.6, 83.1, 166.3, 332.5, 665 and 1330 µg/ml in terms of registered substance), without S9 mix,

- 62.5, 125, 250, 375, 500 and 750 µg/ml (i.e.83.1, 166.3, 332.5, 498.75, 665 and 997.5 µg/ml in terms of registered substance), with S9 mix.

 

Experiments without S9 mix

Cytotoxicity

Following the 3-hour treatment, a moderate to severe toxicity was observed at dose-levels ≥ 625 µg/ml, as shown by a 48-100% decrease in the mitotic index.

Following the 20-hour treatment, a severe toxicity was observed at dose-levels ≥ 500 µg/ml, as shown by a 82-100% decrease in the mitotic index.

Following the 44-hour treatment, a severe toxicity was observed at dose-levels ≥ 500 µg/ml, as shown by a 99-100% decrease in the mitotic index.

Metaphase analysis

The dose-levels selected for metaphase analysis were as follows:

.            156.3, 312.5 and 625 µg/ml for the 3-hour treatment, the latter inducing a 48 % decreasein themitotic index,

.            62.5, 125 and 250 µg/ml for the 20-hour treatment, higher dose-levels being too cytotoxic,

.            250 µg/ml for the 44-hour treatment, this dose inducing a 17 % decrease in the mitotic index and higher dose-levels being too cytotoxic.

 

No significant increase in the frequency of cells with structural chromosomal aberrations was noted after 3-, 20- as well as 44-hour treatments.

 

Experiments with S9 mix

Cytotoxicity

At the 20-hour harvest time in the first experiment, a slight to severe toxicity was observed at dose-levels ≥ 156.3 µg/ml, as shown by a 32-100 % decrease in the mitotic index.

At the 20-hour harvest time in the second experiment, a moderate to severe toxicity was observed at dose-levels ≥ 250 µg/ml, as shown by a 54-100 % decrease in the mitotic index.

At the 44-hour harvest time (second experiment), a marked to severe toxicity was observed at dose-levels ≥ 375 µg/ml, as shown by a 69-100 % decrease in the mitotic index.

Metaphase analysis

The dose-levels selected for metaphase analysis were as follows:

- 78.13, 156.3 and 312.5 µg/mL for the 20-hour harvest time in the first experiment, the latter inducing a 36 % decrease in themitotic index and higher dose-levels being too cytotoxic,

- 62.5, 125 and 250 µg/ml for the 20-hour harvest time in the second experiment, the latter inducing a 54 % decrease in themitotic index,

- 250 µg/ml for the 44-hour harvest time, this dose inducing a 15% decrease in themitotic index and higher dose-levels being too cytotoxic.

No significant increase in the frequency of cells with structural chromosomal aberrations was noted in both experiments and at both harvest times.

The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in the acceptance criteria. The study was therefore considered as valid.

 

in vitro gene mutation in mammalian cells

This study was performed to investigate the potential of the test item, to induce mutations at the TK (Thymidine Kinase) locus in L5178Y TK+/-mouse lymphoma cells. The study was performed according to OECD guideline no. 476 and EC guideline no B17 and in compliance with the Principles of Good Laboratory Practice.

The test item, dissolved in water for injections, was tested in three independent experiments with a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, and two independent experiments without S9 mix. Cultures of 20 ml at 5x 105cells/ml (3-hour treatment) or cultures of 50 ml at 2x 105cells/ml (24-hour treatment) were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2 %). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5 % (3-hour treatment) or 10 % (24-hour treatment) in a 37 °C, 5% CO2 humidified incubator. For the 24-hour treatment, flasks were gently shaken at least once. The negative control was the vehicle (Water for injections).

Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE2). The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.

Since the test item was toxic in the preliminary test, the highest dose-level selected for the main test was 400 µg/ml, on the basis of the level of toxicity, according to the criteria specified in theinternational guidelines (decrease in the Adj. RTG). 

In the experiments without S9 mix, the selected dose-levels were:6.25,12.5, 25, 50, 100, 200 and 400 µg/ml (i.e. 8.3, 16.6, 33.3, 66.5, 133, 266 and 532 µg/ml in terms of registered substance) for the first experiment (3-hour treatment), and 25, 50, 100, 200, 266.7, 333.3 and 400 µg/ml (i.e. 33.3, 66.5, 133, 266, 354.7, 443.3 and 532 µg/ml in terms of registered substance) for the second experiment (24hour treatment).

In the experiments with S9 mix, the selected dose-levels were: 6.25,12.5, 25, 50, 100, 200 and 400 µg/ml (i.e. 8.3, 16.6, 33.3, 66.5, 133, 266 and 532 µg/ml in terms of registered substance) for the first and second experiments, and 25, 50, 100, 200, 250, 300 and 400 µg/ml (i.e. 33.3, 66.5, 133, 266, 332.5, 399 and 532 µg/ml in terms of registered substance)for the third experiment.

Following the 3-hour treatment either with or without S9 mix as well as the 24-hour treatment without S9 mix, no relevant increase in the mutation frequency was noted up to the dose-level of 200 µg/ml or 300 µg/ml in comparison to the vehicle control.

The cloning efficiencies, the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria. The mutation frequencies of the positive controls also met the acceptance criteria of a study. The study was therefore considered as valid.

Justification for classification or non-classification

According to Annex I: 3.5.2.2 ofthe CLP regulation n. 1272/2008 substances are allocated to one of two categories (Category 1A-1B and Category 2):

- Category 1A: The classification in Category 1A is based on positive evidence from human epidemiological studies. Substances to be regarded as if they induce heritable mutations in the germ cells of humans.

- Category 1B: The classification in Category 1B is based on: positive result(s) from in vivo heritable germ cell mutagenicity tests in mammals; positive result(s) from in vivo somatic cell mutagenicity tests in mammals, in combination with some evidence that the substance has potential to cause mutations to germ cells. It is possible to derive this supporting evidence from mutagenicity/genotoxicity tests in germ cells in vivo, or by demonstrating the ability of the substance or its metabolite(s) to interact with the genetic material of germ cells; positive results from tests showing mutagenic effects in the germ cells of humans, without demonstration of transmission to progeny; for example, an increase in the frequency of aneuploidy in sperm cells of exposed people.

- Category 2: Substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans. The classification in Category 2 is based on: positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from: somatic cell mutagenicity tests in vivo, in mammals; or other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays. Substances which are positive in in-vitro mammalian mutagenicity assays, and which also show chemical structure activity relationship to known germ cell mutagens, shall be considered for classification as Category 2 mutagens.

Based on the available testing results and according to the Annex I: 3.5.2.2 (Table 3.5.1) of Regulation EC No. 1272/2008, the substance is not classified for mutagenicity.

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