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Genetic toxicity in vitro

Description of key information
The test compound did not cause mutations in bacteria (Ciba-Geigy, 1981) and in mammalian cell culture (Harlan, 2013). Data obtained with a structural analogue did not reveal any potential for clastogenic effects in mammalian cells (RCC, 2005).
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 09, 2012 - November 12, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. QA statement)
Remarks:
Harlan Cytotest Cell Research GmbH (Harlan CCR), In den Leppsteinswiesen 19, 64380 Rossdorf, Germany
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
The V79 cell line (supplied by Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of Harlan CCR allowing the repeated use of the same cell culture batch in experiments. Before freezing, the level of spontaneous mutants was depressed by treatment with HAT-medium. Each batch is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.
- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts supplemented with 10 % foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1 %).
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
See any other information on materials and methods incl. tables.
Vehicle / solvent:
On the day of the experiment (immediately before treatment), the test item was dissolved in ethanol. The solvent was chosen based on solubility properties and its relative non-toxicity to the cell cultures. The final concentration of ethanol in the culture medium was 0.5 % (v/v).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
- Expression/fixation time: Three or four days after treatment 1.5x10^6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5x10^5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability.
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 7-10 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution.

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: The study was performed in two independent experiments, using identical experimental procedures.

NUMBER OF CELLS EVALUATED: The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).
Evaluation criteria:
Acceptability of the Assay
The gene mutation assay is considered acceptable if it meets the following criteria:
The numbers of mutant colonies per 10^6 cells found in the solvent controls falls within the laboratory historical control data.
The positive control substances should produce a significant increase in mutant colony frequencies.
The cloning efficiency II (absolute value) of the solvent controls should exceed 50 %.
The data of this study comply with the above mentioned criteria.

Evaluation of Results
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.

A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory's historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS GENOTOXICITY:
No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration. The mutant frequency remained well within the historical range of solvent controls.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A p-value below 0.05 was detected in culture I of the first experiment with metabolic activation and in culture II of the second experiment without metabolic activation. These trends however, were judged as biologically irrelevant as the mutation frequency remained well within the range of the historical solvent controls.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 8.8 up to 25.0 mutants per 10^6 cells; the range of the groups treated with the test item was from 3.8 up to 35.3 mutants per 10^6 cells.

EMS (150 μg/mL) and DMBA (1.1 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

RANGE-FINDING/SCREENING STUDIES:
The range finding pre-experiment was performed using a concentration range of 39.1 to 5000 μg/mL according to the current OECD guideline 476 to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. Relevant cytotoxic effects were observed at 78.1 μg/mL and above after 4 hours treatment without metabolic activation and at 156.3 μg/mL with metabolic activation. Following 24 hours treatment the cell growth was completely inhibited down to the lowest concentration.

The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Phase separation was observed at 625 μg/mL and above in the presence of metabolic activation following 4 hours treatment and at 312.5 mg/mL and above in the absence of metabolic activation following 24 hours treatment.
Based on the results of the pre-experiment, the individual concentrations of the main experiments were selected. A series of concentrations generally spaced by a factor of 2 was applied. Narrower spacing was used at high concentrations with metabolic activation to cover the onset of cytotoxicity more closely.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative cloning efficiency I and/or a relative cell density below 50% in both cultures were observed in the first experiment at 78.0 μg/mL with metabolic activation. In the second experiment cytotoxic effects as described above occurred at 10.0 μg/mL without metabolic activation. The recommended cytotoxic range of approximately 10-20% relative cloning efficiency I or relative cell density was covered without metabolic activation. In the presence of metabolic activation moderate cytotoxic effects were noted in the first experiment with metabolic activation at the highest analysable concentration of 78 μg/mL with metabolic activation. Exceedingly severe cytotoxic effects occurred at the next higher, phase separating concentration of 156 μg/mL. In the second experiment with metabolic activation turbidity observed at the maximum concentration of 120 μg/mL indicated that the limit of solubility was reached.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Experimental Result:

concentration (µg/ml) PST S9 Mix relative cloning efficiency I (%) relative cell density (%) relative cloning efficiency II (%) mutant colonies / 106cells induction factor relative cloning efficiency I (%) relative cell density (%) relative cloning efficiency II (%) mutant colonies / 106cells induction factor
Experiment I / 4h treatment culture I culture II
solvent control (ethanol) - 100.0 100.0 100.0 8.8 1.0 100.0 100.0 100.0 25.0 1.0
positive control (EMS) 150.0 - 101.7 122.4 92.9 98.5 11.2 85.8 146.3 74.5 157.8 6.3
test item 0.16 - 110.4 culture was not continued# 95.1 culture was not continued#
test item 0.31 - 110.2 culture was not continued# 87.9 culture was not continued#
test item 0.63 - 119.0 117.1 83.0 14.7 1.7 87.9 102.1 128.4 14.4 0.6
test item 1.3 - 111.1 119.7 88.1 7.9 0.9 97.6 112.3 111.7 25.2 1.0
test item 2.5 - 114.4 97.7 77.6 19.5 2.2 98.2 72.9 123.4 10.1 0.4
test item 5.0 - 122.6 100.2 86.2 20.1 2.3 100.0 52.6 124.7 14.6 0.6
test item 10.0 - 96.7 56.5 78.8 19.5 2.2 66.8 45.2 99.2 25.6 1.0
solvent control (ethanol) + 100.0 100.0 100.0 18.0 1.0 100.0 100.0 100.0 22.7 1.0
positive control (DMBA) 1.1 + 62.8 91.9 41.4 1579.1 87.7 59.3 104.1 73.7 1200.1 52.9
test item 9.8 + 102.2 106.8 84.3 15.5 0.9 98.4 93.5 89.1 22.0 1.0
test item 19.5 + 97.0 123.0 94.6 17.8 1.0 92.2 93.5 101.9 15.7 0.7
test item 39.0 + 99.7 113.1 98.4 19.3 1.1 96.8 85.8 82.0 23.2 1.0
test item 78.0 + 44.1 68.1 70.2 25.7 1.4 54.0 41.5 56.4 35.3 1.6
test item 156.0 PS + 0.0 culture was not continued## 0.0 culture was not continued##
test item 234.0 + 0.0 culture was not continued## 0.0 culture was not continued##
Experiment II / 24h treatment
solvent control (ethanol) - 100.0 100.0 100.0 12.5 1.0 100.0 100.0 100.0 14.3 1.0
positive control (EMS) 150.0 - 89.7 113.2 91.1 364.3 29.1 89.7 96.8 72.9 368.9 25.9
test item 0.31 - 98.8 culture was not continued# 101.3 culture was not continued#
test item 0.63 - 100.6 114.7 76.7 21.8 1.7 98.2 88.5 124.6 7.8 0.5
test item 1.3 - 98.9 109.6 178.7 3.8 0.3 98.5 83.5 95.2 7.0 0.5
test item 2.5 - 95.8 118.0 89.8 25.2 2.0 96.1 100.1 130.8 9.4 0.7
test item 5.0 - 77.6 107.9 122.0 17.6 1.4 72.9 75.8 79.1 11.4 0.8
test item 10.0 - 21.1 18.3 87.8 29.2 2.3 23.8 15.2 82.0 26.6 1.9
test item 20.0 - 0.0 culture was not continued## 0.0 culture was not continued##
Experiment II / 4h treatment
solvent control (ethanol) + 100.0 100.0 100.0 14.5 1.0 100.0 100.0 100.0 14.7 1.0
positive control (DMBA) 1.1 + 83.9 80.9 87.3 544.1 37.6 79.3 78.5 71.8 577.2 39.3
test item 5.0 + 100.4 culture was not continued# 92.6 culture was not continued#
test item 10.0 + 101.1 86.0 103.7 15.0 1.0 89.6 93.6 91.5 18.5 1.3
test item 20.0 + 99.6 88.9 100.8 17.0 1.2 86.3 100.0 88.2 18.8 1.3
test item 40.0 + 93.8 78.5 108.7 15.6 1.1 93.5 108.0 54.8 20.5 1.4
test item 80.0 + 94.9 79.2 101.2 18.1 1.3 94.3 103.4 69.9 32.8 2.2
test item 120.0 T + 94.9 75.6 113.8 16.6 1.1 88.2 100.0 70.4 24.3 1.7

PS = Phase Separation visible at the end of treatment

T = Turbidity

# culture was not continued as a minimum of only four concentrations is required

## culture was not continued due to exceedingly severe cytotoxic effects

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Genetic toxicity in bacterial cells

In a reverse gene mutation assay performed according to a Japanese guideline similar and equivalent to the OECD Test guideline 471, Salmonella typhimurium strains TA 100, TA 1535, TA 1537, TA 1538 and TA 98 and E. coli WP2 uvrA were exposed to the test substance (no data on purity) in DMSO at concentrations of 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 500, 1000 and 5000 µg/plate in the presence and absence of mammalian metabolic activation in a plate incorporation test. The test substance was tested up to precipitating and cytotoxic concentrations. Only one experiment with duplicates was performed. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in this test (Ciba Geigy Ltd. 1981).

Genetic toxicity in mammalian cells

A GLP-compliant mammalian cell mutagenicity test according to OECD guideline 476 was performed to investigate the potential of the test article to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The test item was dissolved in ethanol. The concentration range of the main experiments was limited by cytotoxic effects and the solubility of the test item in aqueous medium. The tested concentrations ranged from 0.63 to 120 µg/ml. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. The mutant frequency remained well within the historical range of solvent controls. Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay (Harlan, 2013).

Chromosomal damage in mammalian cells

Clastogenicity of the test article is assessed by read across approach to a structurally related compound (CAS 866625-93-6), another lubricant substance with a methyl-benzotriazole basic structure possessing similar structural and physico-chemical properties as the substance of concern. A similar cleavage process is suggested as for the test substance, leading to methyl-benzotriazole as the main metabolite.

The test article dissolved in ethanol was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments (OECD 473, GLP). In each experimental group two parallel cultures were set up. The highest applied concentration in the pre-test on toxicity (3300 µg/mL; approx. 10 mM) was chosen with regard to the molecular weight with respect to the current OECD Guideline 473. Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation. The chosen treatment concentrations ranged from 0.39 to 100 µg/ml. Toxic effects indicated by reduced cell numbers of about and below 50 % of control were observed in all experimental parts, except in the absence of S9 mix in Experiment II at 28 hrs preparation interval and in the presence of S9 mix in Experiment I at 18 hrs preparation interval. However, in these experimental parts, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, the test article is considered to be non-clastogenic in this chromosome aberration test with and without S9 mix when tested up to cytotoxic test item concentrations.


Justification for selection of genetic toxicity endpoint
The most recent guideline study was selected, although several key studies are provided to cover mutagenicity.

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available experimental test data is reliable and suitable for the purpose of classification under Directive 67/548/EEC. Based on the present data, classification for genotoxicity is not warranted under Directive 67/548/EEC.

 

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the present data, classification for genotoxicity is not warranted under Regulation (EC) No.1272/2008.