Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in vitro:

Ames test:

The test chemical did not induce mutation in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

In vitro mammalian chromosome aberration study:

The test chemical is not mutagenic in the mammalian cell line used both in the presence nd in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Experimenta data from various test chemicals
Justification for type of information:
Data for the target chemical is summarized based on data from various test chemicals
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
WoE for the target CAS is summarized based on data from various test chemicals
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Remarks:
2
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
other: TA98 and TA100
Remarks:
3
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 100
Remarks:
4
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:
Liver S-9 fractions were routinely prepared from male Sprague-Dawley rats and male Syrian hamsters that were injected, ip, with Aroclor 1254 (200 mg/ml in corn oil) at 500 mg/kg.
Test concentrations with justification for top dose:
2. 0, 3.3, 10, 33, 100, 333, 1000 or 3333 µg/plate
3. 0, 9.85, 19.7, 39.3, 78.5, 157, 313, 625, 1250, 2500 or 5000 µmoles/plate
4. 0, 0.01, 0.1, 1.0, 2.5, 5.0 or 10.0µmoles/plate
Vehicle / solvent:
2. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The chemical was soluble in DMSO

3. - Vehicle(s)/solvent(s) used: 100% ethanol
- Justification for choice of solvent/vehicle:The test chemical was soluble in 100% ethanol

4. No data
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2-Aminoanthracene (2-AA), 4-Nitro-o-phenylenediamine (NOPD)
Remarks:
2
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
100% ethanol
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
other: 2- aminoanthracene (with S9)
Remarks:
3
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Remarks:
4
Details on test system and experimental conditions:
2. METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

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

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: No data

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

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

OTHER: No data

3. METHOD OF APPLICATION: in agar (plate incorporation)

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

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

NUMBER OF REPLICATIONS:Triplicate

NUMBER OF CELLS EVALUATED:No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:Yes, bacterial growth was observed

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

OTHER: No data

4. METHOD OF APPLICATION: in agar (plate incorporation)

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

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

NUMBER OF REPLICATIONS:Triplicate

NUMBER OF CELLS EVALUATED:No data

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

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

OTHER:No data
Rationale for test conditions:
No data
Evaluation criteria:
2. A dose related increase in the number of revertants was noted whether it be twofold over background or not

3. To be considered mutagenic, a test article treatment had to induce at least twice the number of revertants/plate in atleast 1 tester strain compared to those induced in the appropriate vehicle control and exhibit an increasing number of revertants/plate with increasing test article dosage

4. A dose related increase in the number of revertants was noted whether it be twofold over background or not
Statistics:
No data
Species / strain:
other: TA1535, TA1537, TA98, and TA100
Remarks:
2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
other: TA98 and TA100
Remarks:
3
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 1250 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Remarks:
4
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
2. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To select the dose range for the mutagenesis assay, the test chemical was checked for toxicity to TA100 up to a concentration of 10 mg/plate or the limit of solubility, both in the presence and absence of S-9 mix. One or more parameters were used as an indication of toxicity: viability on complete medium and reduced numbers of revertant colonies per plate and/or thinning or absence of the bacterial lawn. If toxicity was not apparent in the preliminary toxicity determination, the highest dose tested was 10 mg/plate; otherwise the upper limit of solubility was used. If toxicity was observed, the doses of test chemical were chosen so that the high dose exhibited some degree of toxicity. Occasionally, in the earlier tests, the high dose was greater than 10 mg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

3. No data

4. No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce mutation in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.
Executive summary:

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

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed by the preincubation protocol using Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100 both in the presence and absence of S9 metabolic activation system. Preincubation was carried at 37°C for 20 mins followed by exposure period of 48 hrs at dose levels of 0, 3.3, 10, 33, 100, 333, 1000 or 3333µg/plate. DMSO was used as solvent control and concurrent positive control chemicals were included in the study. A dose related increase in the number of revertants was noted whether it be twofold over background or not. The test chemical did not induce mutation in the Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100 both in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

In anothet study, gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed by the plate incorporation method using Salmonella typhimurium strains TA100 both in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in 100% ethanol and doses selected for the study were 0, 9.85, 19.7, 39.3, 78.5, 157, 313, 625, 1250, 2500 or 5000 µg/plate. Concurrent solvent control and positive chemical was also included in the study. 2-Methylisoborneol did not induce mutation in the Salmonella typhimurium strains TA98 and TA100 both in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

In yet another study, gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed by the plate incorporation method using Salmonella typhimurium strains TA100 both in the absence of S9 metabolic activation system. The doses selected for the study were 0, 0.01, 0.1, 1.0, 2.5, 5.0 or 10.0 µmoles/plate. Concurrent solvent control chemical was also included in the study. The test chemical was toxic to the bacterial strain but it did not induce mutation in the Salmonella typhimurium strains TA100 both in the absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Based on the data available, the test chemical did not induce mutation in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
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 from various test chemicals
Justification for type of information:
Data for the target chemical is summarized based on data from various test chemicals
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Principles of method if other than guideline:
WoE for the target CAS is summarized based on data from various test chemicals
GLP compliance:
not specified
Type of assay:
other: In vitro mammalian chromosome aberration assay
Target gene:
No data
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
CHO-W-B1 / 6
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy’s 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics
- Properly maintained: No data available
- Periodically checked for Mycoplasma contamination: No data available
- Periodically checked for karyotype stability: No data available
- Periodically "cleansed" against high spontaneous background: No data available
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
lymphocytes: human peripheral blood lymphocytes
Remarks:
7
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Human blood
- Suitability of cells: No data
- Cell cycle length, doubling time or proliferation index:
- Sex, age and number of blood donors if applicable:Age: 27-32 years age
- Whether whole blood or separated lymphocytes were used if applicable: Separated lymphocytes were used
- Number of passages if applicable: No data
- Methods for maintenance in cell culture if applicable: No data
- Modal number of chromosomes: No data
- Normal (negative control) cell cycle time: No data

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Blood cultures were set up in medium containing RPMI-1640, Fetal Bovine Serum, Phytohaemagglutinin, Heparin solution, Whole Blood and Antibiotic Solution
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically 'cleansed' against high spontaneous background: No data
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
The S9 mix consisted of 15 µl/ml liver homogenate (from male Sprague-Dawley rats, induced with Aroclor 1254), 2.4 mg/ml NADP, and 4.5 mg/ml isocitric acid in serum-free medium.
Test concentrations with justification for top dose:
6. 479-663 µg/mL (in the absence of S9) and 630-810 µg/mL (in the presence of S9)
7. 0.00, 0.004, 0.008 and 0.016 mg/mL
Vehicle / solvent:
6. - Vehicle(s)/solvent(s) used: The chemical was dissolved immediately before use in water, dimethyl sulfoxide (DMSO), ethanol, or acetone, in that order of preference. Details are not available
- Justification for choice of solvent/vehicle: No data available

7. - Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The test chemical was soluble in ethanol
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
triethylenemelamine
cyclophosphamide
mitomycin C
Remarks:
6
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
7
Details on test system and experimental conditions:
6. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period:No data
- Exposure duration: Cells were exposed to the test chemical for 2 hr in the presence of S9 or throughout the incubation period without S9.
- Expression time (cells in growth medium):18-26 hrs during the delayed harvest time
- Selection time (if incubation with a selection agent):No data
- Fixation time (start of exposure up to fixation or harvest of cells): The cell harvest time for the aberration test was 8-12 hr after the beginning of treatment. This yielded cells in their first mitosis. Depending on the amount of delay seen in the SCE test, later harvest times, eg, 18-26 hr, were used to allow delayed cells to reach mitosis.

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

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED:100 cells were scored from each of the three highest dose groups having sufficient metaphases for analysis

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

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

OTHER: No data

7. OF APPLICATION: in medium
- Cell density at seeding (if applicable): A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks

DURATION
- Preincubation period: No data
- Exposure duration: Phase 1: 4 hrs (with and without metabolic activation system)
Phase 2: 4 hrs (with metabolic activation system) and 22-25 hrs (without metabolic activation system)
- Expression time: 16-21 hrs (with and without metabolic activation system- Phase I and II)
- Selection time (if incubation with a selection agent):No data
- Fixation time (start of exposure up to fixation or harvest of cells): 21-25 hrs

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa stain in phosphate buffer

NUMBER OF REPLICATIONS: No data

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cultures were incubated at 37 ± 2 °C for duration (exposure period) as mentioned. For Phase I, after incubation cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in Phosphate Buffer Saline (PBS). The washing procedure was repeated once again. After washing the cells were re-suspended in complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37 ± 2 °C for 1.5 normal cell cycle lengths (22 - 25 hours). The cultures were harvested at the end of incubation of 24 hours after treatment. Before 3 hours of harvesting, 240 µL of colcemid (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each of the culture tube, and kept under incubation at 37 ± 2 °C. The cultures were harvested 24 hours after beginning of treatment by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded and the cells were re-suspended in 7 mL of freshly prepared, pre-warmed (37 ± 2 °C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37 ± 2 °C for 30 minutes in water bath. After hypotonic treatment, the culture was centrifuged and supernatant was removed. After that 5 mL of freshly prepared, chilled Carnoy’s fixative (3:1 methanol: acetic acid solution) was added and left for 5 min. The cells were collected by centrifugation and washed twice with Carnoy’s fixative. After the final centrifugation, the supernatant was removed completely, and the cell pellet resuspended in 0.5 mL of Carnoy’s fixative. The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The labelled slides were dried over a slide warmer at 50°C and labelled. At least one slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant.

NUMBER OF CELLS EVALUATED: A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Mitotic index
- Any supplementary information relevant to cytotoxicity: To evaluate the toxicity of the test item a cytotoxicity assay was performed both in the presence and absence of metabolic activation system. 3 test concentrations per cytotoxicity experiment were selected. Since there was cytotoxicity observed in the experiment I (0.25 (T1), 0.5 (T2) and 1.0 (T3) mg/mL) and II (0.031 (T4), 0.0625 (T5) and 0.125 (T6) mg/mL), experiment III is conducted with further lower concentrations (0.004 (T7), 0.008 (T8) and 0.016 (T9) mg/mL of culture media) based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with vehicle control.

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data
Rationale for test conditions:
No data
Evaluation criteria:
6. All types of aberrations were recorded separately, but for data analysis they were grouped into categories of “simple” (breaks and terminal deletions), “complex” (exchanges and rearrangements), “other” (includes pulverized chromosomes), and “total. ” Gaps and endo-reduplications were recorded but were not included in the totals. We did not score aberrations in polyploidy cells but used metaphases with 19-23 chromosomes (the modal number being 21).

7. A test item can be classified as clastogenic if:
 At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
 If the increase is dose-related
 Any of the results are outside the historical negative control range
A test item can be classified as non – clastogenic if:
 None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
 If there is no dose-related increase
 All results are within the historical negative control range
Statistical significance was confirmed by means of the non-parametric Mann Whitney Test. However, both biological and statistical significance should be considered together.

If the above mentioned criteria for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Statistics:
6. Linear regression analysis of the percentage of cells with aberrations vs the log-dose was used as the test for trend. To examine absolute increases over control levels at each dose, a binomial sampling assumption (as opposed to Poisson) was used, and the test was that described by Margolin et al. The P values were adjusted by Dunnett’s method to take into account the multiple dose comparisons. For data analysis, we used the “total” aberration category, and the criterion for a positive response was that the
adjusted P value be < 0.05.

7. Statistical significance at the p < 0.05 was evaluated by means of the non-parametric Mann-Whitney test
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
CHO-W-B1 / 6
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
lymphocytes: Human perpheral blood lymphocytes
Remarks:
7
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
In the cytotoxicity experiment III the highest test concentration 0.016 (T9) mg/ mL of culture media show 47.16 % reduction in absence of metabolic activation and 48.69% in the presence of metabolic activation indicates slight cytotoxicity of test item.
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
6. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: Yes, Precipitation occurred at 663 µg/mL in the absence of S9 metabolic activation system and at all doses in the presence of S9 metabolic activation system
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: Dose selection was based on a preliminary growth inhibition test in which cells that excluded trypan blue were counted 24 hr after treatment. The top
doses selected for the cytogenetics assays were those estimated to reduce growth by
50%.This approach was subsequently modified such that toxicity estimates were made from observations of cell monolayer confluence and mitotic activity in the same cultures used for analysis of SCEs or aberrations. In some cases, test chemical precipitate was observed at the higher dose levels. Dose selection for repeat trials involved a range of doses based on observations from the first trial.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

7. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of test item in culture medium was assessed at 0 h and 4 h after incubation at 37 ± 2 °C. Significant change in pH was not observed at 0 h and 4 h when compared with negative controls.
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: There was slight precipitation observed at 1 mg/mL concentration.
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item a cytotoxicity assay was performed both in the presence and absence of metabolic activation system. Cytotoxicity was assessed at the concentrations of 0.25 (T1), 0.5 (T2) and 1.0 (T3) mg/mL at initial cytotoxicity experiment (Experiment I). All the tested concentrations at intial cytotoxicity experiment were cytotoxic. A second cytotoxicity experiment (Experiment II) was conducted with 0.031 (T4), 0.0625 (T5) and 0.125 (T6) mg/mL of culture media. In second cytotoxicity experiment all tested concentrations were cytotoxic.

Hence one more cytotoxicity experiment (Experiment III) was conducted with further lower concentrations of 0.004 (T7), 0.008 (T8) and 0.016 (T9) mg/mL of culture media. In the absence of S9 mix, the mean mitotic index observed was 10.04 (NC), 9.83 (VC), 8.40 (T7), 6.43 (T8), 5.19 (T9) and 8.53 (PC). In the presence of S9 mix, the mean mitotic index observed was 9.98 (NC), 9.94 (VC), 8.24 (T7), 6.63 (T8), 5.10 (T9) and 8.54 (PC).

In the cytotoxicity experiment III the highest test concentration 0.016 (T9) mg/ mL of culture media show 47.16 % reduction in absence of metabolic activation and 48.69% in the presence of metabolic activation indicates slight cytotoxicity of test item. Hence 0.016 was selected as highest concentration for main study considering the selection of test concentrations upto cytotoxicity. The mitotic index when compared to the respective vehicle control both in the presence or absence of metabolic activation.

Hence the concentrations selected for the main study are 0.004, 0.008 and 0.016 mg/mL. The main study was performed in two independent phases

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: Please refer table remarks section

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical is not mutagenic in the mammalian cell line used both in the presence nd in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.
Executive summary:

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

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The test chemical was studied at a dose level of 479-663µg/mL (in the absence of S9) and 630-810µg/mL (in the presence of S9) using Chinese hamster ovary cells (CHO-W-B1).Cells were exposed to the test chemical for 2 hr in the presence of S9 or throughout the incubation period without S9.100 cells were scored from each of the three highest dose groups having sufficient metaphases for analysis.All types of aberrations were recorded separately, but for data analysis they were grouped into categories of “simple” (breaks and terminal deletions), “complex” (exchanges and rearrangements), “other” (includes pulverized chromosomes), and “total”. Gaps and endo-reduplications were recorded but were not included in the totals. Polyploid cells were not scored but used metaphases with 19-23 chromosomes (the modal number being 21). Precipitation occurred at 663µg/mL in the absence of S9 metabolic activation system and at all doses in the presence of S9 metabolic activation system. Based on the results noted, thetest chemical did notinduce chromosome aberrations in the Chinese hamster ovary cells (CHO-W-B1) in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Another study was conducted to determine the chromosomal aberration induction potential of  the test chemical in human peripheral blood lymphocyte cultures. The methods followed were as per OECD guideline No. 473, adopted on 29th July 2016 “In Vitro Mammalian Chromosome Aberration Test. Blood samples were obtained by vein puncture using syringe from healthy donor (non smoker, non alcoholic) not receiving medication for at least 3 months and being in the range of 27-32 years age. Samples were collected in heparinized vials. The experiment was performed both in the presence and in the absence of metabolic activation system after 48 h mitogenic stimulation. The test chemical was dissolved in ethanol and used at dose level of 0, 0.004, 0.008 and 0.016 mg/mL mg/mL in the presence and absence of S9 metabolic activation system in phase 1 and phase 2. Phase I of experiment was performed by short term treatment method both in the presence and absence of metabolic activation system(1%). Phase II of experiment was performed by short term treatment as well as long term treatment method. Long term treatment was performed in absence of metabolic activation to confirm the negative results obtained in the absence of metabolic activation in Phase I. Short term treatment method was performed with increased metabolic activation (2%) condition to confirm the negative results obtained in the presence of metabolic activation in Phase I. The doses for the main study were based on the cytotoxicity study conducted both in the presence and absence of metabolic activation system. 3 test concentrations (0.004, 0.008 and 0.016 mg/mL {cytotoxicity experiment 3} of culture media) based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with negative control. The medium of the proliferating blood culture was removed by centrifugation at 1500 rpm for 10 minutes. The cells were suspended in plain medium (medium without serum) mixed with S9 mix (Phase I - 1 % and Phase II - 2 % v/v) and in complete media mixed with phosphate buffer for the treatment in presence and in absence of metabolic activation system respectively. A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks. Each tube/flask according to treatment groups was identified. Negative control tubes were treated with 80 µL of RPMI media and treatment group were treated with 80 µL of respective test item stock solution. The cultures were incubated at 37 ± 2 °C for duration (exposure period). For Phase I, after incubation cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in Phosphate Buffer Saline (PBS). The washing procedure was repeated once again. After washing the cells were re-suspended in complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37 ± 2 °C for 1.5 normal cell cycle lengths (22 - 25 hours). The cultures were harvested at the end of incubation of 24 hours after treatment. Before 3 hours of harvesting, 240 µL of colcemid (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each of the culture tube, and kept under incubation at 37 ± 2 °C. The cultures were harvested 24 hours after beginning of treatment by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded and the cells were re-suspended in 7 mL of freshly prepared, pre-warmed (37 ± 2 °C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37 ± 2 °C for 30 minutes in water bath. After hypotonic treatment, the culture was centrifuged and supernatant was removed. After that 5 mL of freshly prepared, chilled Carnoy’s fixative (3:1 methanol: acetic acid solution) was added and left for 5 min. The cells were collected by centrifugation and washed twice with Carnoy’s fixative. After the final centrifugation, the supernatant was removed completely, and the cell pellet resuspended in 0.5 mL of Carnoy’s fixative. The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The slides were dried over a slide warmer and labelled. At least two slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation. 300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pulverization, polyploidy (including endoreduplication) and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. Only metaphases with 46± 2 centromere regions were included in the analysis. Based on the observations made, the test chemical is at the highest tested concentration of 0.016 mg/ml both in the presence (1% and 2%) and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Based on the data avaialble, the test chemical is not mutagenic in the mammalian cell line used both in the presence nd in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutation in vitro:

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

Ames assay:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed by the preincubation protocol using Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100 both in the presence and absence of S9 metabolic activation system. Preincubation was carried at 37°C for 20 mins followed by exposure period of 48 hrs at dose levels of 0, 3.3, 10, 33, 100, 333, 1000 or 3333µg/plate. DMSO was used as solvent control and concurrent positive control chemicals were included in the study. A dose related increase in the number of revertants was noted whether it be twofold over background or not. The test chemical did not induce mutation in the Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100 both in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

In anothet study, gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed by the plate incorporation method using Salmonella typhimurium strains TA100 both in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in 100% ethanol and doses selected for the study were 0, 9.85, 19.7, 39.3, 78.5, 157, 313, 625, 1250, 2500 or 5000 µg/plate. Concurrent solvent control and positive chemical was also included in the study. 2-Methylisoborneol did not induce mutation in the Salmonella typhimurium strains TA98 and TA100 both in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

In yet another study, gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed by the plate incorporation method using Salmonella typhimurium strains TA100 both in the absence of S9 metabolic activation system. The doses selected for the study were 0, 0.01, 0.1, 1.0, 2.5, 5.0 or 10.0 µmoles/plate. Concurrent solvent control chemical was also included in the study. The test chemical was toxic to the bacterial strain but it did not induce mutation in the Salmonella typhimurium strains TA100 both in the absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Based on the data available, the test chemical did not induce mutation in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

In vitro mammalian chromosome aberration study:

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The test chemical was studied at a dose level of 479-663µg/mL (in the absence of S9) and 630-810µg/mL (in the presence of S9) using Chinese hamster ovary cells (CHO-W-B1).Cells were exposed to the test chemical for 2 hr in the presence of S9 or throughout the incubation period without S9.100 cells were scored from each of the three highest dose groups having sufficient metaphases for analysis.All types of aberrations were recorded separately, but for data analysis they were grouped into categories of “simple” (breaks and terminal deletions), “complex” (exchanges and rearrangements), “other” (includes pulverized chromosomes), and “total”. Gaps and endo-reduplications were recorded but were not included in the totals. Polyploid cells were not scored but used metaphases with 19-23 chromosomes (the modal number being 21). Precipitation occurred at 663µg/mL in the absence of S9 metabolic activation system and at all doses in the presence of S9 metabolic activation system. Based on the results noted, thetest chemical did notinduce chromosome aberrations in the Chinese hamster ovary cells (CHO-W-B1) in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Another study was conducted to determine the chromosomal aberration induction potential of  the test chemical in human peripheral blood lymphocyte cultures. The methods followed were as per OECD guideline No. 473, adopted on 29th July 2016 “In Vitro Mammalian Chromosome Aberration Test. Blood samples were obtained by vein puncture using syringe from healthy donor (non smoker, non alcoholic) not receiving medication for at least 3 months and being in the range of 27-32 years age. Samples were collected in heparinized vials. The experiment was performed both in the presence and in the absence of metabolic activation system after 48 h mitogenic stimulation. The test chemical was dissolved in ethanol and used at dose level of 0, 0.004, 0.008 and 0.016 mg/mL mg/mL in the presence and absence of S9 metabolic activation system in phase 1 and phase 2. Phase I of experiment was performed by short term treatment method both in the presence and absence of metabolic activation system(1%). Phase II of experiment was performed by short term treatment as well as long term treatment method. Long term treatment was performed in absence of metabolic activation to confirm the negative results obtained in the absence of metabolic activation in Phase I. Short term treatment method was performed with increased metabolic activation (2%) condition to confirm the negative results obtained in the presence of metabolic activation in Phase I. The doses for the main study were based on the cytotoxicity study conducted both in the presence and absence of metabolic activation system. 3 test concentrations (0.004, 0.008 and 0.016 mg/mL {cytotoxicity experiment 3} of culture media) based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with negative control. The medium of the proliferating blood culture was removed by centrifugation at 1500 rpm for 10 minutes. The cells were suspended in plain medium (medium without serum) mixed with S9 mix (Phase I - 1 % and Phase II - 2 % v/v) and in complete media mixed with phosphate buffer for the treatment in presence and in absence of metabolic activation system respectively. A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks. Each tube/flask according to treatment groups was identified. Negative control tubes were treated with 80 µL of RPMI media and treatment group were treated with 80 µL of respective test item stock solution. The cultures were incubated at 37 ± 2 °C for duration (exposure period). For Phase I, after incubation cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in Phosphate Buffer Saline (PBS). The washing procedure was repeated once again. After washing the cells were re-suspended in complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37 ± 2 °C for 1.5 normal cell cycle lengths (22 - 25 hours). The cultures were harvested at the end of incubation of 24 hours after treatment. Before 3 hours of harvesting, 240 µL of colcemid (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each of the culture tube, and kept under incubation at 37 ± 2 °C. The cultures were harvested 24 hours after beginning of treatment by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded and the cells were re-suspended in 7 mL of freshly prepared, pre-warmed (37 ± 2 °C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37 ± 2 °C for 30 minutes in water bath. After hypotonic treatment, the culture was centrifuged and supernatant was removed. After that 5 mL of freshly prepared, chilled Carnoy’s fixative (3:1 methanol: acetic acid solution) was added and left for 5 min. The cells were collected by centrifugation and washed twice with Carnoy’s fixative. After the final centrifugation, the supernatant was removed completely, and the cell pellet resuspended in 0.5 mL of Carnoy’s fixative. The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The slides were dried over a slide warmer and labelled. At least two slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation. 300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pulverization, polyploidy (including endoreduplication) and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. Only metaphases with 46± 2 centromere regions were included in the analysis. Based on the observations made, the test chemical is at the highest tested concentration of 0.016 mg/ml both in the presence (1% and 2%) and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Based on the data available, the test chemical is not mutagenic in the mammalian cell line used both in the presence nd in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

On the basis of data available and applying the weight of evidence approach, the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro.

 

Justification for classification or non-classification

On the basis of data available and applying the weight of evidence approach, the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro.