Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

The mutagenic effect of the test chemical was studied in Salmonella typhimurium stains TA1535, TA100, TA1537, TA1538 and TA98. The mutagenic assay was performed by plate incorporation method with and without metabolic activation i.e. S9 mix prepared from liver fraction of Aroclor pretreated rats (Aroclor 1254). Five doses of each substance dissolved in DMSO were tested (up to 3.6 mg/plate) in all five tester strains and plates were incubated for 48 hr. Concurrent positive control chemicals were also nincluded in the study. The test substance was tested at least twice. As seen by the results, the test chemical did not induce gene mutation in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538 or TA98 in the presence or absence of a S9 metabolic activation system. Hence, the test chemical is not likely to classify as a gene mutant.

In vitro mammalian chromosome aberration study:

The test chemical is non-clastogenic in the mammalian cell line used at the highest tested concentration 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.

In vitro mammalian cell gene mutation assay:

The test chemical did not induce gene mutation in the mammalian cell line used in the presence and absence of S9 metabolic activation system and hence it is not likley to classify as a gene mutant in vitro.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
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
Justification for type of information:
Data is from peer reviewed publication
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
The mutagenic potential of the test chemical was determned by using Salmonella typhimurium TA1535, TA100, TA1537, TA1538 and TA 98.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
other: TA 1535, TA 1537, TA 98, TA1538 and TA 100
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
S9 mixture from liver fraction of rat pretreated with Aroclor 1254
Test concentrations with justification for top dose:
Up to 3600 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubility of test substance in DMSO solvent.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: In agar (plate incorporation)

DURATION
- Preincubation period: No data
- Exposure duration: 48 hrs
- 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: Two

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
Rationale for test conditions:
No data
Evaluation criteria:
Dose dependent increase in the number of revertants.
Statistics:
With regard to the Ames tests, results that met the following additional criteria were regarded as positive ('+"): a reproducible, dose-related and at least two-fold elevation of the spontaneous revertant frequency. Agents producing reproducible, dose-related and significant but less than two-fold elevations were classified as marginally mutagenic under the experimental conditions
Species / strain:
other: TA 1535, TA 1537, TA 98n TA1538 and TA 100
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:
No data available
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce gene mutation in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, TA98 in the presence and absence of a S9 metabolic activation system and hence it is not likely to classify as a gene mutant.
Executive summary:

The mutagenic effect of the test chemical was studied in Salmonella typhimurium stains TA1535, TA100, TA1537, TA1538 and TA98. The mutagenic assay was performed by plate incorporation method with and without metabolic activation i.e. S9 mix prepared from liver fraction of Aroclor pretreated rats (Aroclor 1254). Five doses of each substance dissolved in DMSO were tested (up to 3.6 mg/plate) in all five tester strains and plates were incubated for 48 hr. Concurrent positive control chemicals were also nincluded in the study. The test substance was tested at least twice. As seen by the results, the test chemical did not induce gene mutation in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538 or TA98 in the presence or absence of a S9 metabolic activation system. Hence, the test chemical is not likely to classify as a gene mutant.

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:
according 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:
lymphocytes: human peripheral blood lymphocytes
Remarks:
1
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: 25-30 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
Species / strain / cell type:
other: Chinese hamster lung (CHL/IU) cells
Remarks:
Short term treatment / 2
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
other: Chinese hamster lung (CHL/IU) cells
Remarks:
Continuous treatment / 3
Details on mammalian cell type (if applicable):
No data
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:
1. 0.00 (NC), 0.00025 (T1), 0.0005 (T2) and 0.001 (T3) mg/mL
2. Continuous treatment: 0, 61.4, 76.8, 96.0 and 120 µg/mL
Short term treatment: 0, 76.8, 96.0 and 120 µg/mL without S9 mix and at 0, 16.1, 20.1 and 25.2 µg/mL with S9 mix
Vehicle / solvent:
1./2. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
1
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
2
Details on test system and experimental conditions:
1. METHOD 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 24 hrs (without metabolic activation system)
- Expression time (cells in growth medium): Phase 1: 20 hrs (with and without metabolic activation system)
Phase 2: 20 hrs (with metabolic activation system)
- Selection time (if incubation with a selection agent):No data
- Fixation time (start of exposure up to fixation or harvest of cells): 24 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: Cytotoxicity was assessed at the concentrations of 0.00 (NC), 0.00025 (T7), 0.0005 (T8) and 0.001 (T9) mg/mL of culture media.

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

2. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 3 days
- Exposure duration:
Short term treatment: 6 hrs
Continuous treatment: 24 hrs
- Expression time (cells in growth medium):
Short term treatment: 6 hrs
Continuous treatment: 24 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: No data

NUMBER OF CELLS EVALUATED: 200 metaphase cells/dose (100/plate) for structural aberrations (i.e. gap, ctb, csb, cte, cse and other abnormalities) and for polyploid

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: yes, cytotoxicity was measured as inhibition of cell growth

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

OTHER: No data
Rationale for test conditions:
No data
Evaluation criteria:
1. 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.

2. The final result of each test was decided for structural aberrations or polyploid cells, separately as follows:negative (-): if the frequency of aberrant cells was less than 5%,inconclusive (±): in greater than 5% but less than 10%,and positive (+): if 10% or more.
Statistics:
1. Statistical significance at the p < 0.05 was evaluated by means of the non-parametric Mann-Whitney test
2. No data
Species / strain:
lymphocytes: Human perpheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
In the cytotoxicity experiment III the highest test concentration 0.001 (T9) mg/ mL of culture media show 41.8 % reduction in absence of metabolic activation and 42.18% 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
Species / strain:
other: Chinese hamster lung (CHL/IU) cells
Remarks:
Short term treatment / 2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
cytotoxicity (more than 50% inhibition of cell growth) was shown at 120 µg/mL and above under the absence of metabolic activation and at 25.2 µg/mL and above under the presence of metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
other: Chinese hamster lung (CHL/IU) cells
Remarks:
Long term treatment / 2
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
cytotoxicity (more than 50% inhibition of cell growth) was shown at 120 µg/mL and above under the absence of metabolic activation and at 25.2 µg/mL and above under the presence of metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
1. 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 no precipitation observed at 0.0625 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. Three test concentrations (0.00025, 0.0005 and 0.001 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. The procedure for conducting cytotoxicity was the same as main experiment phase I up to the scoring of the mitotic index, except slide coding.

Before conducting the chromosomal aberration study, Methyl-2-napthyl ether (CAS no. 93-04-9) was evaluated for cytotoxicity both in the absence and presence of metabolic activation system (1%). Cytotoxicity was assessed at the concentrations of 0.016 (T1), 0.0312 (T2) and 0.0625 (T3) mg/mL at initial cytotoxicity experiment (cytotxicity experiment I). All the tested concentrations at intial cytotoxicity experiment were cytotoxic. A second cytotoxicity experiment (cytotoxicity experiment II) was conducted with 0.002 (T4), 0.004 (T5) and 0.008 (T6) mg/mL of culture media. In second cytotoxicity experiment all tested concentrations were cytotoxic.

Hence one more cytotoxicity experiment (cytotoxic experiment III) was conducted with further lower concentrations of 0.00025 (T7), 0.0005 (T8) and 0.001 (T9) mg/mL of culture media. In the absence of S9 mix, the mean mitotic index observed was 10.03 (NC), 9.95 (VC), 8.69 (T7), 6.54 (T8), 5.79 (T9) and 8.54 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.05 (NC), 9.94 (VC), 8.84 (T7), 6.55 (T8), 5.74 (T9) and 8.55 (PC).

In the cytotoxicity experiment III the highest test concentration 0.001 (T9) mg/ mL of culture media show 41.8 % reduction in absence of metabolic activation and 42.18% in the presence of metabolic activation indicates slight cytotoxicity of test item. Hence 0.001 was selected as highest concentaration 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 0.00025, 0.0005 and 0.001 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

2. No data
Remarks on result:
other: No mutagenic potential

1. Cytotoxicity results:

                

Before conducting the chromosomal aberration study, the test chemical was evaluated for cytotoxicity both in the absence and presence of metabolic activation system (1%). Cytotoxicity was assessed at the concentrations of 0.016 (T1), 0.0312 (T2) and 0.0625 (T3) mg/mL at initial cytotoxicity experiment (cytotxicityexperiment I). All the tested concentrations atintialcytotoxicity experiment were cytotoxic. A second cytotoxicity experiment (cytotoxicity experiment II) was conducted with 0.002 (T4), 0.004 (T5) and 0.008 (T6) mg/mL of culture media. In second cytotoxicity experiment all tested concentrations were cytotoxic.

Hence one more cytotoxicity experiment (cytotoxic experiment III) was conducted with further lower concentrations of 0.00025 (T7), 0.0005 (T8) and 0.001 (T9) mg/mL of culture media. In the absence of S9 mix, the mean mitotic index observed was 10.03 (NC), 9.95 (VC), 8.69 (T7), 6.54 (T8), 5.79 (T9) and 8.54 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.05 (NC), 9.94 (VC), 8.84 (T7), 6.55 (T8), 5.74 (T9) and 8.55 (PC).

In the cytotoxicity experiment III the highest test concentration 0.001 (T9) mg/ mL of culture media show 41.8 % reduction in absence of metabolic activation and 42.18% in the presence of metabolic activation indicates slight cytotoxicity of test item. Hence0.001 mg/mL was selected as highest concentaration 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 0.00025, 0.0005 and 0.001 mg/mL. The main study was performed in two independent phases;

Phase 1 results:           

In the experiment, the cultures were exposed to Methyl-2-napthyl ether (CAS no. 93-04-9) for a short period of time (4 h) both in the absence and in the presence of metabolic activation system (1%). The mean percentage of aberrant cells was 0.333 (NC), 0.667 (VC), 0.667 (T1), 0.667 (T2), 0.667 (T3) and 10.333 (PC) in the absence of metabolic activation and 0.667 (NC), 0.667 (VC), 0.667 (T1), 0.6676 (T2), 0.667 (T3) and 10.000 (PC) in the presence of metabolic activation at the concentration of 0.00 (NC), 0.00 (VC), 0.00025 (T1), 0.0005 (T2) and 0.001 (T3) mg/mL and positive controls, respectively.

Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamidemonohydrate at the concentration of30 µg/mL in the presence of metabolic activation (1%) causedsignificant increase in percent aberrant cells.Even though the analysis did not reveal any statistical significance, the increase was biologically significant.

During thetreatment with test item in the absence and presence of S9 mix, there was noreduction in mitotic index observed at the tested concentrations. The observed mean mitotic indexin the absence of metabolic activation were 10.02, 9.93, 8.58, 6.69, 5.39 and 8.48 andin the presence ofmetabolic activation were 10.02, 10.03, 8.73, 6.33, 5.68 and 8.62 for NC, VC, T1, T2, T3 and PC concentrations respectively.

Phase 2 results:

            

The phase II experiment was performed to confirm the negative results obtained in the absence and in the presence of metabolic activation in Phase I. In the Phase II, test item concentrations used were 0.00025 (T1), 0.0005 (T2) and 0.001 (T3) mg/mL culture both in presence and in absence of metabolic activation (2%). The duration of exposure to the test item in presence of metabolic activation system was 4 hours and in absence of metabolic activation the duration of exposure was 24 hours. The mean percent aberrant cells were 0.667 (NC), 0.667 (VC), 0.667 (T1), 0.667 (T2), 0.667 (T3) and 9.667 (PC) in the absence of metabolic activation and 0.667 (NC), 0.333 (VC), 0.333 (T1), 0.667 (T2), 0.667 (T3) and 10.000 (PC) in the presence of metabolic activation at the concentration of 0.00 (NC), 0.00 (VC), 0.00025 (T1), 0.0005 (T2) and 0.001 (T3) mg/mL of culture and positive control, respectively.

Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamidemonohydrate at the concentration of30 µg/mL in the presence of metabolic activation (2%) causedsignificant increase in percent aberrant cells.Though the analysis did not reveal any statistical significance, the increase was biologically significant.

The increased frequency of aberrations observed in the concurrent positive control groups (Phase I and II) demonstrated the sensitivity of the test system, suitability of the methods and conditions employed in the experiment.

Treatment with test item in the absence and presence of S9 mix, there was no reduction in mitotic index was observed at the tested concentrations. The observed mean mitotic indexin the absence of metabolic activation were 9.99, 10.01, 8.12, 6.78, 5.59 and 8.48 andin the presence ofmetabolic activation were 9.97, 9.94, 8.67, 6.68, 5.54 and 8.62 for NC, VC, T1, T2, T3 and PC concentrations respectively.

Conclusions:
The test chemical is non-clastogenic in the mammalian cell line used at the highest tested concentration 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.
Executive summary:

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

This 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 29thJuly 2016 “ In Vitro Mammalian Chromosome Aberration Test. The experiment was conducted using human peripheral blood lymphocytes. Blood was drawn from a healthy volunteer, by venous puncture using heparinised syringe. 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 DMSO and used at dose level of 0, 0.00025, 0.0005 and 0.001 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 (0.5, 1 and 2mg/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 negative control. The medium of the proliferatingblood 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, pluverization, 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. The test chemical is non-clastogenic at the highest tested concentration of 0.001mg/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.

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical using Chinese hamster lung cells (CHL/IU). The test chemical was dissolved in DMSO and used at dose levels of 0, 76.8, 96.0 and 120µg/mL without S9 mix and at 0, 16.1, 20.1 and 25.2µg/mL with S9 mix for the short-term treatment, and at 0, 61.4, 76.8, 96.0 and 120µg/mL for continuous treatment. In either test condition, no increase in structural aberrations or polyploidy was observed, although cytotoxicity (more than 50% inhibition of cell growth) was shown at 120µg/mL and above under the absence of metabolic activation and at 25.2µg/mL and above under the presence of metabolic activation. The positive controls were effective for induction of chromosome aberrations. The test chemical did not induce chromosome aberration in Chinese hamster lung (CHL/IU) cells in the short term treatment (with and without) and continuous treatment (without) and hence it is not likely to classify as a gene mutant in vitro.

Based on the observations made, the test chemical is non-clastogenic in the mammalian cell line used at the highest tested concentration 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.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
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:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation 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:
mammalian cell gene mutation assay
Target gene:
1. HGPRT
2. TK
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
1
Details on mammalian cell type (if applicable):
- Cell line used: Chinese Hamster Ovary (CHO) cells
- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable
Additional strain / cell type characteristics:
other: Hypodiploid, modal No. 20
Species / strain / cell type:
mouse lymphoma L5178Y cells
Remarks:
2
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
1. 0, 0.1, 0.25, 0.5 or 1 mM
2. 75 -1200 ug/mL
Vehicle / solvent:
1. Vehicle(s)/solvent(s) used: Ethanol
Justification for choice of solvent/ vehicle: Methyl 2-napthyl ether was easily dissolved in ethanol.
Untreated negative controls:
yes
Remarks:
1
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
N-ethyl-N-nitrosourea (ENU) was the positive control substance in the tests done without S9
Untreated negative controls:
not specified
Remarks:
2
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
1. METHOD OF APPLICATION: In medium with pre-incubation

DURATION
- Preincubation period: One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 7 days (harvest of cells)

SELECTION AGENT (mutation assays): 6-thioguanine (TG)
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Crystal violet

NUMBER OF REPLICATIONS: A minimum of 2 replicates per dose concentration including negative and positive control.

NUMBER OF CELLS EVALUATED: 5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated.

DETERMINATION OF CYTOTOXICITY
- Cytotoxicity test
After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.

OTHER EXAMINATIONS: Not applicable

2. METHOD OF APPLICATION: In medium

DURATION
- Preincubation period: No data
- Exposure duration: 34 or 24 hrs without S9 and 4 hrs with S9
- 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: Duplicate

DETERMINATION OF CYTOTOXICITY
- Cytotoxicity test: No data

OTHER EXAMINATIONS: No data
Rationale for test conditions:
No data
Evaluation criteria:
The cell line was observed for gene mutation at the loci
Statistics:
No data
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
1
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not valid
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce gene mutation in the mammalian cell line used in the presence and absence of S9 metabolic activation system and hence it is not likley 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:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the test chemical when administered to Chinese Hamster Ovary (CHO) cells. In the genotoxicity test, the test chemical was administered to CHO cells for 3 hrs at the dose levels of 0, 0.1, 0.25, 0.5 or 1.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such asN-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. The positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity. One very diffuse colony was seen in one well out of four at the concentration of 0.5 mM and in the absence with 4% S9 liver microsomal fraction, and two very diffuse colonies were detected in one well out of four at the concentration of 0.1 mM and in the presence with 4% S9 liver microsomal fraction. These diffuse colonies are not regarded to be relevant since the three spots were only mildly colored by crystal violet, thus indicating that it were small clusters of apoptotic cells taking their last breath instead of cells surviving the TG-selection. This is further supported by the results of the higher tested concentrations of methyl 2-napthyl ether, i.e. these concentrations did not show any evidence of diffuse or clear colonies present. When the mutation frequency was determined, a frequency of 5.35 x 10-4was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction. Since no other tested concentration of methyl 2-napthyl ether in the absence or presence of S9 liver microsomal fraction resulted in colonies, we conclude that methyl 2-napthyl ether does not give rise to gene mutations when CHO cells are exposed in vitro to the test chemical at 0, 0.1, 0.25, 0.5 or 1.0 mM for 3 hrs. Based on the results of the current study, it is concluded that the test chemical does not give rise to gene mutations when CHO cells are exposed to the test chemical in vitro at 0, 0.1, 0.25, 0.5 or 1.0 mM for 3 hrs, in the presence or absence of metabolic activation.

In another study, mouse lymphoma thymidine kinase assay was performed to determine the mutagenic nature of the test chemical. The study was performed using mouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system. The test chemical was studied up to cytotoxic concentrations (dose range 75 -1200 ug/mL) in two separate experiments with two replicates each. Exposure duration was 4 or 24 hrs without S9 and 4 hrs with S9. The test was performed according to OECD Guideline 476 (GLP study). There was no indication of a mutagenic activity at the thymidine kinase locus in mouse lymphoma L5178Y cells. Based on the observations made, the test chemical did not induce gene mutation in mouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Based on the observations made, the test chemical did not induce gene mutation in the mammalian cell line used in the presence and absence of S9 metabolic activation system and hence it is not likley to classify as a gene mutant in vitro.

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

Genetic toxicity in vivo

Description of key information

Gene mutation toxicity was performed to determine the mutagenic nature of the test chemical in vivo. Micronucleus assay was performed using bone marrow smears of male and female NMRI mice. The test chemical was dissolved in olive oil and used at dose levels of 0, 340, 681 or 876 mg/Kg. The chemical was given intraperitoneally during the 24 hrs study period. The mice were killed and bone-marrow smears were prepared 30 hr after treatment. The smears were stained according to the method of Schmid (1976) and were observed for genetic effects in polychromated erthrocytes. As seen by the results, the test chemical did not produce genetic effects in the micronucleus assay performed in NMR1 mice. Hence, it is not likely to classify as a gene mutant in vivo.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
Micronucleus assay was performed in NMRI mice to determine the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
other: Micronucleus assay in mice
Species:
mouse
Strain:
NMRI
Details on species / strain selection:
No data available
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: lvanovas GmbH, Kisslegg
- Age at study initiation: 10 to 14 week old
- Weight at study initiation: No data
- Assigned to test groups randomly: No data
- Fasting period before study: No data
- Housing: No data
- Diet (e.g. ad libitum): standard chow (Altromin) ad libitum
- Water (e.g. ad libitum): Water ad libitum
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%):No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): No data

IN-LIFE DATES: From: To: No data
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: Olive oil
- Justification for choice of solvent/vehicle: The chemical was soluble in olive oil
- Concentration of test material in vehicle: 0, 340, 681 or 876 mg/Kg
- Amount of vehicle (if gavage or dermal): No data
- Type and concentration of dispersant aid (if powder): No data
- Lot/batch no. (if required): No data
- Purity: No data
Details on exposure:
No data
Duration of treatment / exposure:
0 and 24 hours
Frequency of treatment:
Once
Post exposure period:
6 hours
Remarks:
0, 340, 681 or 876 mg/Kg
No. of animals per sex per dose:
Total: 16
0 mg/Kg: 4
405 mg/Kg: 4
809 mg/Kg: 4
1213 mg/Kg: 4
Control animals:
yes, concurrent vehicle
Positive control(s):
No data available
Tissues and cell types examined:
Bone marrow micronucleus were examined.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: No data

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 30 hrs

DETAILS OF SLIDE PREPARATION: The smears were stained according to the method of Schmid (1976)

METHOD OF ANALYSIS: increase in the number of micronucleus

OTHER: No data
Evaluation criteria:
The smears were noted for micronucleated polychromatic erythrocytes.
Statistics:
Statistical significance was determined according to the methods of Kastenbaum & Bowman (1970).
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
other: No mutagenic potential
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: No data
- Solubility: No data
- Clinical signs of toxicity in test animals: No data
- Evidence of cytotoxicity in tissue analyzed: No data
- Rationale for exposure: No data
- Harvest times: No data
- High dose with and without activation: No data
- Other: No data


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): No data
- Induction of micronuclei (for Micronucleus assay):Yes
- Ratio of PCE/NCE (for Micronucleus assay): Refer table below
- Appropriateness of dose levels and route: Intra-peritoneal
- Statistical evaluation:No data
Conclusions:
The test chemical did not produce genetic effects in a micronucleus assay performed in NMR1 mice and hence is not likely to classify as a gene mutant in vivo.
Executive summary:

Gene mutation toxicity was performed to determine the mutagenic nature of the test chemical in vivo. Micronucleus assay was performed using bone marrow smears of male and female NMRI mice. The test chemical was dissolved in olive oil and used at dose levels of 0, 340, 681 or 876 mg/Kg. The chemical was given intraperitoneally during the 24 hrs study period. The mice were killed and bone-marrow smears were prepared 30 hr after treatment. The smears were stained according to the method of Schmid (1976) and were observed for genetic effects in polychromated erthrocytes. As seen by the results, the test chemical did not produce genetic effects in the micronucleus assay performed in NMR1 mice. Hence, it is not likely to classify as a gene mutant in vivo.

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

Additional information

Gene mutation in vitro:

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

Ames assay:

The mutagenic effect of the test chemical was studied in Salmonella typhimurium stains TA1535, TA100, TA1537, TA1538 and TA98. The mutagenic assay was performed by plate incorporation method with and without metabolic activation i.e. S9 mix prepared from liver fraction of Aroclor pretreated rats (Aroclor 1254). Five doses of each substance dissolved in DMSO were tested (up to 3.6 mg/plate) in all five tester strains and plates were incubated for 48 hr. Concurrent positive control chemicals were also nincluded in the study. The test substance was tested at least twice. As seen by the results, the test chemical did not induce gene mutation in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538 or TA98 in the presence or absence of a S9 metabolic activation system. Hence, the test chemical is not likely to classify as a gene mutant.

An in vitro genetic toxicity study for the test chemical was performed using Salmonella typhimurium strains TA97 and TA102 in the absence and presence of exogenous metabolic activation. The test chemical was administered to the bacteria at a dose level of0.001-0.1µg/plate being dissolved in DMSO. No mutagenic activity was detected with any of the Salmonella strains tested. Based on the results, the test chemical did not induce a dose-related increase in his+ revertants in the S. typhimurium tester strains TA97 and TA102 in the presence and absence of S9 metabolic activation system. and hence is negative for gene mutation in vitro. Hence, it is not likely to classify as a gene mutant in vitro.

In vitro mammalian chromosome aberration study:

The 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 29thJuly 2016 “In Vitro Mammalian Chromosome Aberration Test. The experiment was conducted using human peripheral blood lymphocytes. Blood was drawn from a healthy volunteer, by venous puncture using heparinised syringe. 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 DMSO and used at dose level of 0, 0.00025, 0.0005 and 0.001 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 (0.5, 1 and 2mg/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 negative control. The medium of the proliferatingblood 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, pluverization, 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. The test chemical is non-clastogenic at the highest tested concentration of 0.001mg/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.

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical using Chinese hamster lung cells (CHL/IU). The test chemical was dissolved in DMSO and used at dose levels of 0, 76.8, 96.0 and 120µg/mL without S9 mix and at 0, 16.1, 20.1 and 25.2µg/mL with S9 mix for the short-term treatment, and at 0, 61.4, 76.8, 96.0 and 120µg/mL for continuous treatment. In either test condition, no increase in structural aberrations or polyploidy was observed, although cytotoxicity (more than 50% inhibition of cell growth) was shown at 120µg/mL and above under the absence of metabolic activation and at 25.2µg/mL and above under the presence of metabolic activation. The positive controls were effective for induction of chromosome aberrations. The test chemical did not induce chromosome aberration in Chinese hamster lung (CHL/IU) cells in the short term treatment (with and without) and continuous treatment (without) and hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian cell gene mutation assay:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the test chemical when administered to Chinese Hamster Ovary (CHO) cells. In the genotoxicity test, the test chemical was administered to CHO cells for 3 hrs at the dose levels of 0, 0.1, 0.25, 0.5 or 1.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such asN-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. The positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity. One very diffuse colony was seen in one well out of four at the concentration of 0.5 mM and in the absence with 4% S9 liver microsomal fraction, and two very diffuse colonies were detected in one well out of four at the concentration of 0.1 mM and in the presence with 4% S9 liver microsomal fraction. These diffuse colonies are not regarded to be relevant since the three spots were only mildly colored by crystal violet, thus indicating that it were small clusters of apoptotic cells taking their last breath instead of cells surviving the TG-selection. This is further supported by the results of the higher tested concentrations of methyl 2-napthyl ether, i.e. these concentrations did not show any evidence of diffuse or clear colonies present. When the mutation frequency was determined, a frequency of 5.35 x 10-4was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction. Since no other tested concentration of methyl 2-napthyl ether in the absence or presence of S9 liver microsomal fraction resulted in colonies, we conclude that methyl 2-napthyl ether does not give rise to gene mutations when CHO cells are exposedin vitroto the test chemical at 0, 0.1, 0.25, 0.5 or 1.0 mM for 3 hrs. Based on the results of the current study, it is concluded that the test chemical does not give rise to gene mutations when CHO cells are exposed to the test chemicalin vitroat 0, 0.1, 0.25, 0.5 or 1.0 mM for 3 hrs, in the presence or absence of metabolic activation.

In another study, mouse lymphoma thymidine kinase assay was performed to determine the mutagenic nature of the test chemical. The study was performed using mouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system. The test chemical was studied up to cytotoxic concentrations (dose range 75 -1200 ug/mL) in two separate experiments with two replicates each. Exposure duration was 4 or 24 hrs without S9 and 4 hrs with S9. The test was performed according to OECD Guideline 476 (GLP study). There was no indication of a mutagenic activity at the thymidine kinase locus in mouse lymphoma L5178Y cells. Based on the observations made, the test chemical did not induce gene mutation in mouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Gene mutation in vivo:

Gene mutation toxicity was performed to determine the mutagenic nature of the test chemical in vivo. Micronucleus assay was performed using bone marrow smears of male and female NMRI mice. The test chemical was dissolved in olive oil and used at dose levels of 0, 340, 681 or 876 mg/Kg. The chemical was given intraperitoneally during the 24 hrs study period. The mice were killed and bone-marrow smears were prepared 30 hr after treatment. The smears were stained according to the method of Schmid (1976) and were observed for genetic effects in polychromated erthrocytes. As seen by the results, the test chemical did not produce genetic effects in the micronucleus assay performed in NMR1 mice. Hence, it is not likely to classify as a gene mutant in vivo.

In the same study, a Basc test on Drosophila melanogaster was performed to study the gene mutation ability of the test chemical. Solutions or emulsions of test substances of 12 mM was fed to the flies prepared in 5% saccharose with the addition of 2% ethanol and 2% Tween 80. Based on the results, the test chemical did not induce SLRL mutation in vivo in Drosophila melanogaster. Hence, the test chemical is not likely to classify as a gene mutant in vivo.

Based on the data available, the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available, the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.