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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay

Based on the data summarized and applying the weight of evidence approach, the test chemical did not induce gene mutation in the Salmonella typhimurium strains used in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro

In vitro micronucleus assay

Based on the data summarized and applying the weight of evidence approach, the test chemical did not induce gene mutation in the presence and absence of S9 metabolic activation system to Chinese hamster V79 cells and hence it is not likely to be mutagenic in vitro.

In vitro mammalian cell gene mutation assay

Based on the available studies and applying the weight of evidence approach, it can be concluded that the given test chemical did not induce mutation in mammalian cell line in the presence and absence of metabolic activation and hence it is not likely to classify as a gene mutant in vitro 

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
experimental data of read across substances
Justification for type of information:
Data for the target chemical is summarized based on the structurally similar read across chemicals
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
other: Refer below principle
Principles of method if other than guideline:
WoE report is based on three gene mutation in vitro toxicity studies as-
1., 2. The Ames Salmonella typhimurium mutagenicity test was conducted for the test chemicals
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium, other: TA98, TA100, TA102, and TA1535
Remarks:
RA 1
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
S9 metabolic activation system
Test concentrations with justification for top dose:
2,3. 5–5000 μg/plate
Vehicle / solvent:
2, 3.. No data
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
sodium azide
mitomycin C
Remarks:
RA 1
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
mitomycin C
other: 2-aminofluorene for all the strains with S9 mix
Remarks:
RA 2
Details on test system and experimental conditions:
1 and 2. METHOD OF APPLICATION: in agar (plate incorporation) with preincubation modification

DURATION
- Preincubation period: No data available
- Exposure duration: 48 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available

STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Yes, the cytotoxicity study were carried out by accounting for the microcolonies
(histidine auxotroph) in the background lawn. The sparse or absent growth indicated the toxic nature of dyes toward the tester strains.

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available

OTHER: No data available
Rationale for test conditions:
No data
Evaluation criteria:
1 and 2. A dose-related increase (at least 2-fold) in revertant colonies was used to define a statistically significant mutagenic response.
Statistics:
No data
Species / strain:
S. typhimurium, other: TA98, TA100, TA102, and TA1535
Remarks:
RA 1
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
≥ 500 μg
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA98, TA100, TA102, and TA1535
Remarks:
RA 2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
≥ 500 μg
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
No data
Remarks on result:
other: No mutagenic potential
Conclusions:
Based on the data summarized and applying the weight of evidence approach, the test chemical did not induce gene mutation in the Salmonella typhimurium strains used in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.
Executive summary:

Data available for the test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

Ames mutagenicity test was conducted for two test chemical to evaluate its genotoxic effects when exposed to Salmonella typhimurium strains TA98, TA100, TA102, and TA1535 with dose concentration of 5–5000 µg/plate in plate incorporation assay. The plates were incubated for 48 h. The doses of test chemical, together with the appropriate concurrent positive controls, were tested in triplicate on each tester strain with and without S9 metabolic activation. A dose-related increase (at least 2-fold) in revertant colonies was used to define a statistically significant mutagenic response. Both the test chemicals did not induce gene mutation in the Salmonella typhimuriumTA98, TA100, TA102, and TA1535 both in the presence and absence of S9 activation system and hence the chemical is not likely to be a gene mutant.

Based on the data summarized and applying the weight of evidence approach, the test chemical did not induce gene mutation in the Salmonella typhimurium strains used in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Weight of evidence approach based on various test chemicals
Justification for type of information:
Weight of evidence approach based on various test chemicals
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
other: Weight of evidence approach based on various test chemicals
Principles of method if other than guideline:
Weight of evidence approach based on various test chemicals
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Remarks:
STUDY 5,6
Metabolic activation:
with and without
Metabolic activation system:
5. Liver S9 fraction from phenobarbital/β-naphthoflavone-induced rats was used as exogenous metabolic activation system.
6. Liver S9 fraction from phenobarbital/β-naphthoflavone-induced rats was used as exogenous metabolic activation system.
Test concentrations with justification for top dose:
5. experiment IA: 128.1, 256.3, 2050.0 and 4100.0 μg/ml without S9-mix ; experiment IA: 128.1, 256.3, 1025.0 and 2050.0 μg/ml with S9 -mix ; experiment IB: 31.3, 62.5, and 125.0 without S9-mix ; experiment IIA: 128.1, 256.3 and 512.5 μg/ml without S9-mix ; experiment IIA: 128.1, 256.3, 512.5 and 1025.0 μg/ml with S9-mix ; experiment IIB: 100.0, 150.0, 200.0, 250.0, 300.0 and 350.0 μg/ml without S9-mix ; experiment IIB: 100.0, 200.0, 400.0, 600.0 and 800.0 μg/ml with S9- mix.
6. Experiment I- without S9-mix: 1000, 1500, 2000 μg/ml; with S9-mix: 1000, 1200, 1400 μg/ml, Experiment II- without S9-mix: 1000, 1500, 2000 μg/ml.
Vehicle / solvent:
6. deionised water was used as vehicle
7. The test article was dissolved in Dulbecco’s Modified Eagle Medium (DMEM).
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
deionsed water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
not specified
Remarks:
study 5
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
The test article was dissolved in Dulbecco’s Modified Eagle Medium (DMEM).
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
mitomycin C
other: CYCLOPHOSPHAMIDE MONOHYDRATE
Remarks:
study 6
Details on test system and experimental conditions:
5.NUMBER OF REPLICATIONS:
- Number of cultures per concentration - duplicate
- Number of independent experiments - 2 independent experiments

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment:
The treatment period in the main test was 4 h in experiment I (without and with S9-mix) and in experiment II (with S9-mix) or 20 h in experiment II (without S9-mix).
- Harvest time after the end of treatment (sampling/recovery times):
Harvest time was 24 h or 48 h (experiment II with S9-mix only) after the beginning of culture.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method - For assessment of cytotoxicity a XTT test was additionally carried out in parallel to the main micronucleus test.
6. METHOD OF APPLICATION: No data available

DURATION
- Preincubation period: No data available
- Exposure duration:
Experiment I: 4 hr
Experiment II: 24 hr

- Expression time (cells in growth medium): No data available
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: In each experimental group two parallel cultures were set up.

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available

OTHER: A pre-test on cytotoxicity (MTT assay) was performed in order to determine the toxicity of the test item, the solubility during exposure and changes in osmolarity and pH value at experimental conditions. The concentrations used in the main study were based on this pretest.
Evaluation criteria:
5. Per culture 1000 cells were scored for micronuclei.
6. Per culture 1000 cells were scored for micronuclei.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
5.Micronucleus test in mammalian cells:
In all experiments clear toxic effects indicated by reduced cell numbers below 40% of control were observed at least at the highest concentrations scored after treatment with the test chemical except in experiment IB in the absence of S9-mix. In experiment IA, in the absence of S9-mix, a statistically significant but non-dose-related increase in the rate of micronucleated cells was observed at the lowest and highest dose.
The values of highest dose were at the laboratory’s control data range (0.0 – 1.8% micronucleated cells). Concerning the lowest dose, in the confirmatory experiment IB this finding was not confirmed. Consequently, the positive finding was considered not biologically relevant. In experiment IA, in the presence of S9-mix no biologically relevant increase in the percentage of micronucleated cells was observed after treatment with the test item.
In experiment IIA, in the absence and the presence of S9-mix, a statistically significant increase in the number of micronucleated cells exceeding the range of the historical control data was observed at the highest doses (512.5 and 1025 μg/ml, respectively). These concentrations were strongly cytotoxic indicated as by cell numbers of 7.9% and 12.9% of control, respectively.
Due to the steep dose-toxicity curve a repeat experiment, designated experiment IIB, was performed with narrower dilution steps to prove if the genotoxicity observed could have been an artefact induced by general test item toxicity. In the absence of S9-mix, at a cytotoxic level of about 40% of control the number of micronucleated cells (2.05% and 2.00%) slightly exceeded the historical control data range (0.0 – 1.8% micronucleated cells). Therefore, the test item was regarded as non-genotoxic in the absence of metabolic activation.
In the presence of S9-mix, at cytotoxic test item levels and associated with precipitation from doses equal or exceeding 200 μg/ml, the number of micronucleated cells (2.68% and 2.33%) slightly exceeded the range of the historical control data (0.0 – 1.8% micronucleated cells). Due to the high value of the respective solvent control (1.80% micronucleated cells), these two slight increases have to be regarded as biologically irrelevant.
The observations of experiment IIA in the absence and the presence of metabolic activation were not confirmed in the repeat experiment IIB with narrower dilution steps. Therefore, it has to be considered that the findings in both parts of experiment IIA were artefacts induced by general test item toxicity.
6. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data available
- Effects of osmolality: No data available
- Evaporation from medium: No data available
- Water solubility: No data available
- Precipitation: No signs of precipitation were observed at the end of incubation.
- Other confounding effects: No decrease in the number of attached cells was observed at the end of incubation of both tests.

Test chemical did not induce an increase in the number of cells with micronuclei either with or without metabolic activation (4h incubation) in the first test or in the second test after a continuous treatment time of 24 h without S9-mix.

RANGE-FINDING/SCREENING STUDIES:

COMPARISON WITH HISTORICAL CONTROL DATA: The micronucleus frequency of negative controls was partly below the range of the historical data. These slightly low values did not impair the outcome of the study as the functionality of the test system was demonstrated by the corresponding positive controls. These induced the expected increase in micronucleus frequency, with and without metabolic activation,respectively, thus demonstrating the sensitivity of the test system used for the endpoints investigated in this study.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Determination of the cytokinesis-block proliferation index (CBPI) showed a substantial cytotoxic effect at and above 2000 μg/ml in the first test (with and without metabolic activation) and 1400 μg/ml (without metabolic activation) in the second test.
Remarks on result:
other: not mutagenic
Conclusions:
Based on the available studies and applying the weight of evidence approach, it can be concluded that the given test chemical did not induce mutation in mammalian cell line in the presence and absence of metabolic activation and hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

Data available for the test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

In vitro micronucleus test was carried out using Chinese hamster V79 cells as per OECD 487 and OECD 473 (in vitro chromosomal aberration test). The test concentrations used were as follows: experiment IA: 128.1, 256.3, 2050.0 and 4100.0 μg/ml without S9-mix ; experiment IA: 128.1, 256.3, 1025.0 and 2050.0 μg/ml with S9 -mix ; experiment IB: 31.3, 62.5, and 125.0 without S9-mix ; experiment IIA: 128.1, 256.3 and 512.5 μg/ml without S9-mix ; experiment IIA: 128.1, 256.3, 512.5 and 1025.0 μg/ml with S9-mix ; experiment IIB: 100.0, 150.0, 200.0, 250.0, 300.0 and 350.0 μg/ml without S9-mix ; experiment IIB: 100.0, 200.0, 400.0, 600.0 and 800.0 μg/ml with S9- mix. Deionised water was used as a vehicle. Liver S9 fraction from phenobarbital/β-naphthoflavone-induced rats was used as exogenous metabolic activation system. A pretest on cell growth inhibition (XTT assay) with 4 h treatment was performed in order to determine the toxicity of test substance, the solubility during exposure and thus the test concentrations for the main micronucleus test. On the basis of pre-test (range finding study) and the occurrence of precipitation of test chemical, 4100 μg/ml (≈ 10 mM the prescribed maximum concentration) was chosen as top concentration in experiment IA. To corroborate the data of this experiment in the absence of S9-mix, a confirmatory experiment (experiment IB) was performed with a top dose of 500 μg/ml. Dose selection in experiment IIA was influenced by the test chemical toxicity and precipitation observed in experiment I. Due to the steep dose toxicity curve, a repeat experiment (experiment IIB) was performed with narrower dilution steps to prove if genotoxicity observed at highly toxic concentrations far below the 40% of control level was an artificial finding. The treatment period in the main test was 4 h in experiment I (without and with S9-mix) and in experiment II (with S9-mix) or 20 h in experiment II (without S9-mix). Harvest time was 24 h or 48 h (experiment II with S9-mix only) after the beginning of culture. For assessment of cytotoxicity a XTT test was additionally carried out in parallel to the main micronucleus test. In all experiments clear toxic effects indicated by reduced cell numbers below 40% of control were observed at least at the highest concentrations scored after treatment with the test chemical except in experiment IB in the absence of S9-mix. In experiment IA, in the absence of S9-mix, a statistically significant but non-dose-related increase in the rate of micronucleated cells was observed at the lowest and highest dose. The values of highest dose were at the laboratory’s control data range (0.0 – 1.8% micronucleated cells). Concerning the lowest dose, in the confirmatory experiment IB this finding was not confirmed. Consequently, the positive finding was considered not biologically relevant. In experiment IA, in the presence of S9-mix no biologically relevant increase in the percentage of micronucleated cells was observed after treatment with the test item. In experiment IIA, in the absence and the presence of S9-mix, a statistically significant increase in the number of micronucleated cells exceeding the range of the historical control data was observed at the highest doses (512.5 and 1025 μg/ml, respectively). These concentrations were strongly cytotoxic indicated as by cell numbers of 7.9% and 12.9% of control, respectively. Due to the steep dose-toxicity curve a repeat experiment, designated experiment IIB, was performed with narrower dilution steps to prove if the genotoxicity observed could have been an artefact induced by general test item toxicity. In the absence of S9-mix, at a cytotoxic level of about 40% of control the number of micronucleated cells (2.05% and 2.00%) slightly exceeded the historical control data range (0.0 – 1.8% micronucleated cells). Therefore, the test item was regarded as non-genotoxic in the absence of metabolic activation. In the presence of S9-mix, at cytotoxic test item levels and associated with precipitation from doses equal or exceeding 200 μg/ml, the number of micronucleated cells (2.68% and 2.33%) slightly exceeded the range of the historical control data (0.0 – 1.8% micronucleated cells). Due to the high value of the respective solvent control (1.80% micronucleated cells), these two slight increases have to be regarded as biologically irrelevant. The observations of experiment IIA in the absence and the presence of metabolic activation were not confirmed in the repeat experiment IIB with narrower dilution steps. Therefore, it has to be considered that the findings in both parts of experiment IIA were artefacts induced by general test item toxicity. Under the experimental conditions, the test chemical did not induce an increase in micronucleated cells and thus, the test was considered to be negative.

This is supported by the results of another in vitro micronucleus test carried out using Chinese hamster V79 cells to determine the mutagenic potential of the test chemical. The study was performed according to OECD 487 Guidelines. Chinese Hamster V79 cells were used to examine the potential of the test chemical to induce micronuclei in the presence and absence of phenobarbital and β-naphthoflavone stimulated rat liver S9- mix. A pre-test on cytotoxicity (MTT assay) was performed in order to determine the toxicity of the test item, the solubility during exposure and changes in osmolarity and pH value at experimental conditions. The concentrations used in the main study were based on this pretest. In each experimental group two parallel cultures were set up. The test article was dissolved in Dulbecco’s Modified Eagle Medium (DMEM). The concentrations used for the main test were as follows: Experiment I- without S9-mix: 1000, 1500, 2000 μg/ml; with S9-mix: 1000, 1200, 1400 μg/ml, Experiment II- without S9-mix: 1000, 1500, 2000 μg/ml. The duration of treatment and exposure was: Experiment I: 4h treatment + 18h incubation and Experiment II: 24h treatment. Mitomycin C (- S9-mix), Cyclophosphamide monohydrate (+ S9-mix) were used as positive controls. Per culture 1000 cells were scored for micronuclei.No signs of precipitation were observed at the end of incubation. No decrease in the number of attached cells was observed at the end of incubation of both tests. Determination of the cytokinesis-block proliferation index (CBPI) showed a substantial cytotoxic effect at and above 2000 μg/ml in the first test (with and without metabolic activation) and 1400 μg/ml (without metabolic activation) in the second test. The micronucleus frequency of negative controls was partly below the range of the historical data. These slightly low values did not impair the outcome of the study as the functionality of the test system was demonstrated by the corresponding positive controls. These induced the expected increase in micronucleus frequency, with and without metabolic activation, respectively, thus demonstrating the sensitivity of the test system used for the endpoints investigated in this study.The test chemical did not induce an increase in the number of cells with micronuclei either with or without metabolic activation (4h incubation) in the first test or in the second test after a continuous treatment time of 24 h without S9-mix.Under the experimental conditions reported, the test chemical did not induce micronuclei in Chinese hamster V79 cells in the absence and in the presence of metabolic activation, and was considered to be not clastogenic and/or aneugenic in vitro.

Based on the data summarized and applying the weight of evidence approach, the test chemical did not induce gene mutation in the presence and absence of S9 metabolic activation system to Chinese hamster V79 cells and hence it is not likely to be mutagenic in vitro.

 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Weight of evidence report based on various test chemicals
Justification for type of information:
Weight of evidence report based on various test chemicals
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
other: Weight of evidence report based on various test chemicals
Principles of method if other than guideline:
Weight of evidence report based on various test chemicals
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
8. tk locus
9. tk locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Remarks:
study 8
Details on mammalian cell type (if applicable):
- Type and identity of media: The cells were grown in Fischer’s medium for leukemic cells of mice supplemented with 10% horse serum and 0.02% pluronic F-68.
- Properly maintained: No data available
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: No data available
- Periodically "cleansed" against high spontaneous background: No data available
Species / strain / cell type:
mouse lymphoma L5178Y cells
Remarks:
study 9
Cytokinesis block (if used):
no data available
Metabolic activation:
with and without
Metabolic activation system:
8. Liver S9 fraction was prepared from Aroclor 1254-induced male Sprague- Dawley rats.
9. The assay was performed in the presence and absence of phenobarbital and β-naphthoflavone stimulated rat liver S9-mix.
Test concentrations with justification for top dose:
8. 0.001-6 µg/mL
9. Experiment I
without S9-mix: 118.8, 237.5, 475.0, 712.5, 950.0 μg/ml
with S9-mix: 118.8, 237.5, 475.0, 712.5, 950.0 μg/ml

Experiment II
without S9-mix: 59.4, 118.8, 237.5, 475.0, 950.0 μg/ml

Experiment IIA:
without S9-mix: 400, 500, 600, 700 μg/ml
Vehicle / solvent:
8. DMSO
9. water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
cyclophosphamide
ethylmethanesulphonate
methylmethanesulfonate
Remarks:
study 8
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Remarks:
study 9
Details on test system and experimental conditions:
8. METHOD OF APPLICATION: in medium;
- Cell density at seeding (if applicable): 6000000 cells

DURATION
- Preincubation period: No data available
- Exposure duration: 4 h
- Expression time (cells in growth medium):48 h
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: 1 X 106 cells/plate for mutant selection and 200
cells/plate for viable count determinations

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: The rate of cell growth was determined for each of the treated cultures

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available

OTHER: No data available
9.METHOD OF APPLICATION: No data available
DURATION
- Preincubation period: No data available
- Exposure duration:
Experiment I: 4 h treatment without and with S9-mix
Experiment II and IIA: 24 h treatment without S9-mix

- Expression time (cells in growth medium): No data available
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available
NUMBER OF REPLICATIONS: 3 independent experiments, using 2 parallel cultures each.
NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available

OTHER: No data available
Evaluation criteria:
8. Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Doubling of the mutant frequency was previously reported as representing a positive effect. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response.
Statistics:
8. The calculations for CE, RTG, and mutant frequency (MF) have been described and were performed by computer at NIEHS. Statistical analyses were performed by computer for both the MF trend and comparisons between each dose level and the solvent controls in each experiment.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
8. STUDY RESULTS: The results in all the assays with test chemical were negative
9. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data available
- Effects of osmolality: No data available
- Evaporation from medium: No data available
- Water solubility: No data available
- Precipitation: No precipitation was observed by the naked eye in any of the experiments performed.
- Other confounding effects: No relevant and reproducible increase of the mutant frequency was observed in the first experiment with and without metabolic activation. In the second experiment, performed solely without metabolic activation, the number of mutant colonies/106 cells exceeded the range of the historical control data at 237.5 μg/ml (culture II), and at 475 μg/ml (both cultures) and there was a concentration related increase except at the highest concentration, where the decline in mutant frequency could be due to strong toxicity. In culture two of the second experiment the mutant frequency was increased 2.2 times compared to the corresponding solvent control and the induced mutant frequency was 163 x 10-6 at 475 μg/ml, which is above the recommended increase for a positive result. In culture I there was a minor increase (1.5 times compared to the negative control and induction of 75 mutants) and no concentration response relationship.

To verify this minor increase a repeat experiment was performed using a rather narrow concentration range. The toxic range of 10 – 20% of survival was covered but no relevant increase of the mutant frequency was observed in any of both cultures. Therefore, the minor effects noted in experiment II were judged as irreproducible fluctuations with no biological relevance.

RANGE-FINDING/SCREENING STUDIES: No data available

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the first two main experiments the test item induced strong toxic effects in both parallel cultures at 950 μg/ml. In the first experiment the relative total growth (RTG) was below 10% except for culture one without S9-mix, at the second highest concentration (712.5 μg/ml) RTG was between 30 and 60%. In the second experiment RTG was 8.8% and 6.5% at the highest concentration and 43% and 40% at the second highest concentration. In the third experiment the RTG, at the highest tested concentration, was 11.5% and 13.6% in the two cultures respectively.
Remarks on result:
other: not mutagenic
Conclusions:
Based on the available studies and applying the weight of evidence approach, it can be concluded that the given test chemical did not induce mutation in mammalian cell line in the presence and absence of metabolic activation and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

L5178Y TK +/- mouse lymphoma assay was conducted to determine the mutagenic potential of the test chemical.L5178Y TK +/- mouse lymphoma cells were used for the study. The cells were grown in Fischer's medium for leukemic cells of mice supplemented with 10% horse serum, antibiotics (50 U penicillin/mi and 50 microgram streptomycin/ml), and 0.02% Pluronic F-68. All serum lots were pre-screened for their ability to support optimal growth. The cells were checked for the presence of mycoplasma by agar block isolation and Hoechst staining before and after cryopreservation. The toxicity of each chemical was first determined both with and without S9 prepared from Aroclor-1254-induced male Fischer 344 rats. Cells at a concentration of 6 × 105/ml were exposed for 4 h to a range of concentrations from 0.0 to 10000 microgram/ml or the limit of solubility. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls. The doses of chemical selected for testing were within the range yielding approximately 0-90% cytotoxicity. For each assay there was a solvent control, a positive control for the test without metabolic activation and for the test with metabolic activation. The maximum solvent concentration was 1% for organic solvents and 10% for water. These levels had no effects on cell growth or spontaneous mutation frequency. The mutagenicity assay was performed according to the procedure described by Clive and Spector (1975). Cells in duplicate cultures were exposed to the test chemical, positive control, and solvent control for 4 h at 37°C; washed twice with growth medium; and maintained at 37 °C for 48 h in log phase growth to allow recovery and mutant expression. The cultures were adjusted to 0.3 × 106 cells/ml at 24-h intervals. They were then cloned in soft agar medium containing Fischer's medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% Pluronic F-68 and 0.35% Noble agar. Resistance to trifluorothymidine (TFT) was determined by adding 3 microgram/ml TFT to one set of plates. The 100 × stock solution of TFT in saline was stored at -70°C and thawed immediately before use. Plates were incubated at 37°C in 5% CO2 in air for 12 days, and then counted with an automatic colony counter. Mutant frequencies were expressed as mutants per 104 surviving cells. In general, a response was considered positive if there was a dose-related increase in the mutant frequency above the spontaneous control frequency, with a 2-fold increase at more than 1 dose and relative total growth greater than 10%.The results in all the assays with test chemical were negative. Hence, the test chemical can be considered to be non mutagenic to L5178Y TK +/- mouse lymphoma cells.

This is supported by another study where mouse lymphoma cell line L5178Y was used to examine the potential of the test chemical to induce mutations at the thymidine kinase locus. The study was performed according to OECD 476 Guidelines. The assay was performed in the presence and absence of phenobarbital and β-naphthoflavone stimulated rat liver S9-mix. The assay was performed in three independent experiments, using two parallel cultures each. Experiment I was performed with and without S9-mix and a treatment period of 4 hours. Experiment II was performed solely without metabolic activation and a treatment period of 24 hours. An additional experiment IIA without metabolic activation was performed (24 hour treatment) to verify a dose related increase of the mutant frequency observed in the second experiment. The test substance was dissolved in deionised water and appropriate negative and positive controls were included. Methyl methane sulfonate (- S9-mix), Cyclophosphamide (+ S9-mix) were used as positive controls. The test concentrations were as follows: Experiment I- without S9-mix: 118.8, 237.5, 475.0, 712.5, 950.0 μg/ml; with S9-mix: 118.8, 237.5, 475.0, 712.5, 950.0 μg/ml; Experiment II- without S9-mix: 59.4, 118.8, 237.5, 475.0, 950.0 μg/ml; Experiment IIA: without S9-mix: 400, 500, 600, 700 μg/ml. No precipitation was observed by the naked eye in any of the experiments performed. In the first two main experiments the test item induced strong toxic effects in both parallel cultures at 950 μg/ml. In the first experiment the relative total growth (RTG) was below 10% except for culture one without S9-mix, at the second highest concentration (712.5 μg/ml) RTG was between 30 and 60%. In the second experiment RTG was 8.8% and 6.5% at the highest concentration and 43% and 40% at the second highest concentration. In the third experiment the RTG, at the highest tested concentration, was 11.5% and 13.6% in the two cultures respectively. No relevant and reproducible increase of the mutant frequency was observed in the first experiment with and without metabolic activation. In the second experiment, performed solely without metabolic activation, the number of mutant colonies/106cells exceeded the range of the historical control data at 237.5 μg/ml (culture II), and at 475 μg/ml (both cultures) and there was a concentration related increase except at the highest concentration, where the decline in mutant frequency could be due to strong toxicity. In culture two of the second experiment the mutant frequency was increased 2.2 times compared to the corresponding solvent control and the induced mutant frequency was 163x 10-6 at 475 μg/ml, which was above the recommended increase for a positive result. In culture I there was a minor increase (1.5 times compared to the negative control and induction of 75 mutants) and no concentration response relationship. To verify this minor increase a repeat experiment was performed using a rather narrow concentration range. The toxic range of 10 – 20% of survival was covered but no relevant increase of the mutant frequency was observed in any of both cultures. Therefore, the minor effects noted in experiment II were judged as irreproducible fluctuations with no biological relevance. Under the experimental conditions reported, the test chemical did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore the test chemical was considered to be not mutagenic in this mouse lymphoma assay.

Based on the available studies and applying the weight of evidence approach, it can be concluded that the given test chemical did not induce mutation in mammalian cell line in the presence and absence of metabolic activation and hence it is not likely to classify as a gene mutant in vitro.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames Assay

Data available for the test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

Ames mutagenicity test was conducted for two test chemical to evaluate its genotoxic effects when exposed to Salmonella typhimurium strains TA98, TA100, TA102, and TA1535 with dose concentration of 5–5000 µg/plate in plate incorporation assay. The plates were incubated for 48 h. The doses of test chemical, together with the appropriate concurrent positive controls, were tested in triplicate on each tester strain with and without S9 metabolic activation. A dose-related increase (at least 2-fold) in revertant colonies was used to define a statistically significant mutagenic response. Both the test chemicals did not induce gene mutation in the Salmonella typhimuriumTA98, TA100, TA102, and TA1535 both in the presence and absence of S9 activation system and hence the chemical is not likely to be a gene mutant.

Based on the data summarized and applying the weight of evidence approach, the test chemical did not induce gene mutation in the Salmonella typhimurium strains used in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

In vitro micronucleus test

Data available for the test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

In vitro micronucleus test was carried out using Chinese hamster V79 cells as per OECD 487 and OECD 473 (in vitro chromosomal aberration test). The test concentrations used were as follows: experiment IA: 128.1, 256.3, 2050.0 and 4100.0 μg/ml without S9-mix ; experiment IA: 128.1, 256.3, 1025.0 and 2050.0 μg/ml with S9 -mix ; experiment IB: 31.3, 62.5, and 125.0 without S9-mix ; experiment IIA: 128.1, 256.3 and 512.5 μg/ml without S9-mix ; experiment IIA: 128.1, 256.3, 512.5 and 1025.0 μg/ml with S9-mix ; experiment IIB: 100.0, 150.0, 200.0, 250.0, 300.0 and 350.0 μg/ml without S9-mix ; experiment IIB: 100.0, 200.0, 400.0, 600.0 and 800.0 μg/ml with S9- mix. Deionised water was used as a vehicle. Liver S9 fraction from phenobarbital/β-naphthoflavone-induced rats was used as exogenous metabolic activation system. A pretest on cell growth inhibition (XTT assay) with 4 h treatment was performed in order to determine the toxicity of test substance, the solubility during exposure and thus the test concentrations for the main micronucleus test. On the basis of pre-test (range finding study) and the occurrence of precipitation of test chemical, 4100 μg/ml (≈ 10 mM the prescribed maximum concentration) was chosen as top concentration in experiment IA. To corroborate the data of this experiment in the absence of S9-mix, a confirmatory experiment (experiment IB) was performed with a top dose of 500 μg/ml. Dose selection in experiment IIA was influenced by the test chemical toxicity and precipitation observed in experiment I. Due to the steep dose toxicity curve, a repeat experiment (experiment IIB) was performed with narrower dilution steps to prove if genotoxicity observed at highly toxic concentrations far below the 40% of control level was an artificial finding. The treatment period in the main test was 4 h in experiment I (without and with S9-mix) and in experiment II (with S9-mix) or 20 h in experiment II (without S9-mix). Harvest time was 24 h or 48 h (experiment II with S9-mix only) after the beginning of culture. For assessment of cytotoxicity a XTT test was additionally carried out in parallel to the main micronucleus test. In all experiments clear toxic effects indicated by reduced cell numbers below 40% of control were observed at least at the highest concentrations scored after treatment with the test chemical except in experiment IB in the absence of S9-mix. In experiment IA, in the absence of S9-mix, a statistically significant but non-dose-related increase in the rate of micronucleated cells was observed at the lowest and highest dose. The values of highest dose were at the laboratory’s control data range (0.0 – 1.8% micronucleated cells). Concerning the lowest dose, in the confirmatory experiment IB this finding was not confirmed. Consequently, the positive finding was considered not biologically relevant. In experiment IA, in the presence of S9-mix no biologically relevant increase in the percentage of micronucleated cells was observed after treatment with the test item. In experiment IIA, in the absence and the presence of S9-mix, a statistically significant increase in the number of micronucleated cells exceeding the range of the historical control data was observed at the highest doses (512.5 and 1025 μg/ml, respectively). These concentrations were strongly cytotoxic indicated as by cell numbers of 7.9% and 12.9% of control, respectively. Due to the steep dose-toxicity curve a repeat experiment, designated experiment IIB, was performed with narrower dilution steps to prove if the genotoxicity observed could have been an artefact induced by general test item toxicity. In the absence of S9-mix, at a cytotoxic level of about 40% of control the number of micronucleated cells (2.05% and 2.00%) slightly exceeded the historical control data range (0.0 – 1.8% micronucleated cells). Therefore, the test item was regarded as non-genotoxic in the absence of metabolic activation. In the presence of S9-mix, at cytotoxic test item levels and associated with precipitation from doses equal or exceeding 200 μg/ml, the number of micronucleated cells (2.68% and 2.33%) slightly exceeded the range of the historical control data (0.0 – 1.8% micronucleated cells). Due to the high value of the respective solvent control (1.80% micronucleated cells), these two slight increases have to be regarded as biologically irrelevant. The observations of experiment IIA in the absence and the presence of metabolic activation were not confirmed in the repeat experiment IIB with narrower dilution steps. Therefore, it has to be considered that the findings in both parts of experiment IIA were artefacts induced by general test item toxicity. Under the experimental conditions, the test chemical did not induce an increase in micronucleated cells and thus, the test was considered to be negative.

This is supported by the results of another in vitro micronucleus test carried out using Chinese hamster V79 cells to determine the mutagenic potential of the test chemical. The study was performed according to OECD 487 Guidelines. Chinese Hamster V79 cells were used to examine the potential of the test chemical to induce micronuclei in the presence and absence of phenobarbital and β-naphthoflavone stimulated rat liver S9- mix. A pre-test on cytotoxicity (MTT assay) was performed in order to determine the toxicity of the test item, the solubility during exposure and changes in osmolarity and pH value at experimental conditions. The concentrations used in the main study were based on this pretest. In each experimental group two parallel cultures were set up. The test article was dissolved in Dulbecco’s Modified Eagle Medium (DMEM). The concentrations used for the main test were as follows: Experiment I- without S9-mix: 1000, 1500, 2000 μg/ml; with S9-mix: 1000, 1200, 1400 μg/ml, Experiment II- without S9-mix: 1000, 1500, 2000 μg/ml. The duration of treatment and exposure was: Experiment I: 4h treatment + 18h incubation and Experiment II: 24h treatment. Mitomycin C (- S9-mix), Cyclophosphamide monohydrate (+ S9-mix) were used as positive controls.Per culture 1000 cells were scored for micronuclei.No signs of precipitation were observed at the end of incubation. No decrease in the number of attached cells was observed at the end of incubation of both tests. Determination of the cytokinesis-block proliferation index (CBPI) showed a substantial cytotoxic effect at and above 2000 μg/ml in the first test (with and without metabolic activation) and 1400 μg/ml (without metabolic activation) in the second test. The micronucleus frequency of negative controls was partly below the range of the historical data. These slightly low values did not impair the outcome of the study as the functionality of the test system was demonstrated by the corresponding positive controls. These induced the expected increase in micronucleus frequency, with and without metabolic activation, respectively, thus demonstrating the sensitivity of the test system used for the endpoints investigated in this study.The test chemical did not induce an increase in the number of cells with micronuclei either with or without metabolic activation (4h incubation) in the first test or in the second test after a continuous treatment time of 24 h without S9-mix.Under the experimental conditions reported, the test chemical did not induce micronuclei in Chinese hamster V79 cells in the absence and in the presence of metabolic activation, and was considered to be not clastogenic and/or aneugenic in vitro.

Based on the data summarized and applying the weight of evidence approach, the test chemical did not induce gene mutation in the presence and absence of S9 metabolic activation system to Chinese hamster V79 cells and hence it is not likely to be mutagenic in vitro.

 

In vitro mammalian cell gene mutation assay

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

L5178Y TK +/- mouse lymphoma assay was conducted to determine the mutagenic potential of the test chemical.L5178Y TK +/- mouse lymphoma cells were used for the study. The cells were grown in Fischer's medium for leukemic cells of mice supplemented with 10% horse serum, antibiotics (50 U penicillin/mi and 50 microgram streptomycin/ml), and 0.02% Pluronic F-68. All serum lots were pre-screened for their ability to support optimal growth. The cells were checked for the presence of mycoplasma by agar block isolation and Hoechst staining before and after cryopreservation. The toxicity of each chemical was first determined both with and without S9 prepared from Aroclor-1254-induced male Fischer 344 rats. Cells at a concentration of 6 × 105/ml were exposed for 4 h to a range of concentrations from 0.0 to 10000 microgram/ml or the limit of solubility. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls. The doses of chemical selected for testing were within the range yielding approximately 0-90% cytotoxicity. For each assay there was a solvent control, a positive control for the test without metabolic activation and for the test with metabolic activation. The maximum solvent concentration was 1% for organic solvents and 10% for water. These levels had no effects on cell growth or spontaneous mutation frequency. The mutagenicity assay was performed according to the procedure described by Clive and Spector (1975). Cells in duplicate cultures were exposed to the test chemical, positive control, and solvent control for 4 h at 37°C; washed twice with growth medium; and maintained at 37 °C for 48 h in log phase growth to allow recovery and mutant expression. The cultures were adjusted to 0.3 × 106 cells/ml at 24-h intervals. They were then cloned in soft agar medium containing Fischer's medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% Pluronic F-68 and 0.35% Noble agar. Resistance to trifluorothymidine (TFT) was determined by adding 3 microgram/ml TFT to one set of plates. The 100 × stock solution of TFT in saline was stored at -70°C and thawed immediately before use. Plates were incubated at 37°C in 5% CO2 in air for 12 days, and then counted with an automatic colony counter. Mutant frequencies were expressed as mutants per 104 surviving cells. In general, a response was considered positive if there was a dose-related increase in the mutant frequency above the spontaneous control frequency, with a 2-fold increase at more than 1 dose and relative total growth greater than 10%.The results in all the assays with test chemical were negative. Hence, the test chemical can be considered to be non mutagenic to L5178Y TK +/- mouse lymphoma cells.

This is supported by another study where mouse lymphoma cell line L5178Y was used to examine the potential of the test chemical to induce mutations at the thymidine kinase locus. The study was performed according to OECD 476 Guidelines. The assay was performed in the presence and absence of phenobarbital and β-naphthoflavone stimulated rat liver S9-mix. The assay was performed in three independent experiments, using two parallel cultures each. Experiment I was performed with and without S9-mix and a treatment period of 4 hours. Experiment II was performed solely without metabolic activation and a treatment period of 24 hours. An additional experiment IIA without metabolic activation was performed (24 hour treatment) to verify a dose related increase of the mutant frequency observed in the second experiment. The test substance was dissolved in deionised water and appropriate negative and positive controls were included. Methyl methane sulfonate (- S9-mix), Cyclophosphamide (+ S9-mix) were used as positive controls. The test concentrations were as follows: Experiment I- without S9-mix: 118.8, 237.5, 475.0, 712.5, 950.0 μg/ml; with S9-mix: 118.8, 237.5, 475.0, 712.5, 950.0 μg/ml; Experiment II- without S9-mix: 59.4, 118.8, 237.5, 475.0, 950.0 μg/ml; Experiment IIA: without S9-mix: 400, 500, 600, 700 μg/ml. No precipitation was observed by the naked eye in any of the experiments performed. In the first two main experiments the test item induced strong toxic effects in both parallel cultures at 950 μg/ml. In the first experiment the relative total growth (RTG) was below 10% except for culture one without S9-mix, at the second highest concentration (712.5 μg/ml) RTG was between 30 and 60%. In the second experiment RTG was 8.8% and 6.5% at the highest concentration and 43% and 40% at the second highest concentration. In the third experiment the RTG, at the highest tested concentration, was 11.5% and 13.6% in the two cultures respectively. No relevant and reproducible increase of the mutant frequency was observed in the first experiment with and without metabolic activation. In the second experiment, performed solely without metabolic activation, the number of mutant colonies/106cells exceeded the range of the historical control data at 237.5 μg/ml (culture II), and at 475 μg/ml (both cultures) and there was a concentration related increase except at the highest concentration, where the decline in mutant frequency could be due to strong toxicity. In culture two of the second experiment the mutant frequency was increased 2.2 times compared to the corresponding solvent control and the induced mutant frequency was 163x 10-6 at 475 μg/ml, which was above the recommended increase for a positive result. In culture I there was a minor increase (1.5 times compared to the negative control and induction of 75 mutants) and no concentration response relationship. To verify this minor increase a repeat experiment was performed using a rather narrow concentration range. The toxic range of 10 – 20% of survival was covered but no relevant increase of the mutant frequency was observed in any of both cultures. Therefore, the minor effects noted in experiment II were judged as irreproducible fluctuations with no biological relevance. Under the experimental conditions reported, the test chemical did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore the test chemical was considered to be not mutagenic in this mouse lymphoma assay.

Based on the available studies and applying the weight of evidence approach, it can be concluded that the given test chemical did not induce mutation in mammalian cell line in the presence and absence of metabolic activation and hence it is not likely to classify as a gene mutant in vitro.

Justification for classification or non-classification

Based on the available studies the test chemical can be considered to be non-mutagenic in nature. Hence, it can be classified under the category "Not Classified" as per CLP Regulation.