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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial gene mutation (OECD 471): negative

Cytogenicity/chromosome aberration in mammalian cells (OECD 473): negative

Gene mutation in mammalian cells (OECD 476): negative

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

Genetic toxicity in vivo

Description of key information

Erythrocyte micronucleus study in mice (OECD 474): negative

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

Additional information

There are reliable in vitro and in vivo genotoxicity studies for the test substance available.

 

In vitro:

- Gene mutation in bacteria:

A GLP guideline study according to OECD Guideline 471 is available with the test material (M-000357-01-3). Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 were treated with the test material for 48 h using the Ames plate incorporation and the preincubation method at concentrations ranging from 16 to 5000 µg/plate, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The test material was suspended in the solvent dimethylsulfoxide. The negative control, in both the plate incorporation and the pre-incubation study, was a solvent control. The positive control agents used in this assay were sodium azide (TA 1535), nitrofurantoin (TA 100), 4-nitro-1,2-phenylene diamine (TA 1537 and TA 98), mitomycin C (TA 102 for plate incorporation), and cumene hydroperoxide (TA 102 for pre-incubation) for the incubations without S9, and 2-aminoanthracene for the incubations with S9 for all strains and all incubation methods. In a preliminary cytotoxicity study using the plate incorporation method with and without S9 in the same strains and at the same concentrations as in the main study there was no effect on bacterial growth at concentrations of up to and including 158 µg/plate, although growth of TA 1535, TA 100, and TA 1537 was decreased at concentrations of 500 µg/plate and above. In TA 98 and TA 102, bacterial growth was only decreased at concentrations of 1581 µg/plate and above. Precipitation of the test substance was not reported at any concentration. In the main study using the preincubation method there was no cytotoxicity of the test material. The background revertant levels reported are in line with both historical data from the laboratory, and with that reported by other laboratories using these bacterial strains. The incidence of positive control induced revertants is also consistent with both historical data and public data. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. Neither the plate incorporation nor the preincubation trials produced any indication of mutagenicity in any strain, either with or without metabolic activation.

The test material was evaluated as not mutagenic under the conditions of this test.

 

- Cytogenicity / chromosome aberration in mammalian cells

A GLP guideline study performed according to OECD Guideline 473 is available with the test material (M-076036-01-2). The study was performed to investigate the potential of the test material to induce clastogenicity in V79 Chinese hamster lung cells. Cells were treated with the test material both with and without the addition of a rat liver homogenate metabolising system (S9 in standard co-factors). The S9 fraction used for the metabolic activation was prepared from livers of male rats induced with Aroclor 1254. The test material was suspended in the solvent dimethylsulfoxide. The concentrations in the main study were from 500 to 2500 µg/mL for the 4-hour incubation cultures, and from 200 to 1000 µg/mL for the 18-hour incubation periods. The 18-hour incubations, which were conducted without S9, were harvested immediately after the end of the incubation period. The 4-hour incubations were conducted either with or without S9. At the end of the 4-hour incubation period, the medium with the test substane was replaced with fresh medium, and then cells were harvested after a total time of either 18 h or 30 h. In all cases, colcemid was added to each flask two hours prior to the end of the incubation period. The positive controls used were mitomycin C in the absence and cyclophosphamide in the presence of metabolic activation. Duplicate cultures were tested for every concentration of test substance and for the negative and positive controls. From each culture, the mitotic index was determined by counting 1000 cells / culture and noting the number of mitotic and non-mitotic cells. At each concentration of the test substance, 100 metaphases from each of two parallel cultures were examined, and structural chromosome damage was assessed on both the chromatid and the chromosome level. At high concentrations mitotic indices were decreased in both the absence and presence of S9. The survival index was decreased in the 4-hour incubation both with and without S9, but not in the 18-hour incubation without S9. There were no biologically relevant increases in the incidence of metaphases with aberrations in any culture examined, either in the presence or the absence of S9. The incidence of metaphases with aberrations was statistically significantly increased at a concentration of 2500 µg/mL after 4 h-incubation and harvest time of 30 h, however this increase was considered not to be biologically relevant as it was within the historical control data for that endpoint.

The test material was evaluated as not clastogenic under the conditions of this test.

 

- Gene mutation in mammalian cells:

A GLP guideline study performed according to OECD Guideline 476 is available with the test material (M-084536-02-2). The study was performed to investigate the ability of the test material to induce reverse mutations at the HPRT locus in V79 Chinese hamster lung cells. Cells were treated with the test material both with and without the addition of a rat liver homogenate metabolising system (S9 in standard co-factors). The S9 used for metabolic activation in this study was prepared from the livers of male rats induced by administration of Aroclor 1254. The test material was suspended in the solvent dimethylsulfoxide. In a preliminary cytotoxicity test treatment concentrations of the test material ranged from 18.8 to 2400 µg/mL followed by a 5 h-incubation and further 6 to 8 days without the test material to allow colony growth. Osmolality in the test medium did not change at any concentration, but the pH of the culture medium changed at 312.5 µg/mL and above, and thus concentrations used in the definitive tests ranged from 30 to 960 µg/ml. Cells were treated with the test substance for 5 h, replated and incubated for 6 days for cytotoxicity determination. For mutagenicity cells were additionally incubated with selective medium containing 6-thioguanidine for 6 to 8 days before colonies were counted. The positive control agents were ethylmethanesulfonate (EMS) at a final concentration of 900 µg/mL in the absence of metabolic activation and dimethylbenzanthracene (DMBA) in DMSO at a final concentration of 20 µg/mL in the presence of metabolic activation.

The positive control substances produced clear biologically and statistically significant increases in mutation frequency. In the absence of S9, relative population growth was decreased in a concentration-related manner. There was no increase in mutation frequency related to dose, and all findings were within the historical control frequency. This finding was therefore considered to be not an indication of mutagenicity of the test compound. In the presence of S9, there were no relevant increases in mutagenic frequencies, while relative population growth was decreased.

The test material was evaluated as not mutagenic under the conditions of this test.

 

In vivo:

- Cytogenicity / micronucleus test in mice:          

A GLP guideline study performed according to OECD Guideline 474 is available with the test material (M-110250-01-2). The study was performed to assess the potential of the test material to induce clastogenicity when administered to mice. The test material was suspended in 0.5% aqueous Cremophor and administered to groups of 5 male and 5 female Hsd/Win:NMRI mice by two intraperitoneal injections separated by 24 h. Based on a preliminary toxicity study, the doses used were 125, 250, and 500 mg/kg bw for males and 0, 250, 500, and 1000 mg/kg bw for females. An additional 5 replacement animals were administered the test substance by intraperitoneal injection at the top doses of 500 (males) or 1000 mg/kg bw (females). The positive control substance cyclophosphamide was dissolved in deionized water and administered to a further group of 5 male and 5 female mice by intraperitoneal injection at 20 mg/kg bw (one injection only). The mice were sacrificed 24 h after the second injection.Bone marrow was removed from one femur of each animal and slides were prepared and stained. From each animal, 2000 polychromatic erythrocytes (PCE) were counted, and the number of micronucleated PCE was determined. Additionally, the number of normochromatic erythrocytes (NCE) per 2000 PCE was counted.

In males, two intraperitoneal injections at 125, 250, and 500 mg/kg bw produced a number of clinical signs of systemic exposure, including apathy, roughened fur, weight loss, staggering gait, spasm, twitching, stretching of the body, decreased body temperature, and difficulty in breathing. In females, 8/10 treated females at 1000 mg/kg bw died during the test period. At 250, 500, and 1000 mg/kg bw, clinical signs of systemic toxicity in females included apathy, roughened fur, weight loss, spasm, stretching of the body, difficulty in breathing, and slitted eyes. As there were no differences between males and females in the toxicity of the test material only slides from the males were read while slides from the females were archived unread. The ratio of PCE to NCE was decreased in a generally dose-related manner in males, demonstrating relevant systemic exposure to the test agent. There was no increase in micronucleated PCE or NCE at any dose. The positive control, cyclophosphamide, induced a marked increase in micronucleated PCE.

The test material was evaluated as not clastogenic under the conditions of this test.

Justification for classification or non-classification

The available data on genetic toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.