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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

In vitro bacterial reverse gene mutation assay for the test chemical was studied using S. typhimurium and Escherichia coli WP2 uvrA strains. The mutagenicity assay was conducted as described by Ames et al. with slight modifications. Ames test was performed using Salmonella typhimurium strains TA100, TA1535, TA98, TA1537 and TA1538 and Escherichia coli WP2 uvrA strains in the presence and absence of S9 metabolic activation system at dose level of 0, 250, 750, 1500 or 3000 µg/plate. For Salmonella strains, the assays without S9 were performed by the plate-incorporation method and the assays with S9 were conducted by the pre-incubation method described by Yahagi et al. (1975). The pre-incubation method is useful in detecting weak mutagenicity in samples (Yahagi et al., 1977). Histidine-independent colonies were scored after incubation at 37°C for 48-72 h. For E. coli strains, the assay was performed in the same manner as with the Salmonella assay except that the supplement of 0.1 µmole histidine plus 0.1 mole biotin in the soft agar was replaced with a supplement of 0.1 µmole of tryptophan. Tryptophan-independent revertant colonies were scored with E. coli. No mutagenicity was noted at the tested dose levels. Based on the observations made, the test chemical was negative in Ames test carried out using Salmonella typhimurium strains TA100, TA1535, TA98, TA1537, TA1538 as well in Escherichia coli WP2 uvr A in the presence and absence of S9 metabolic activation. Hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian chromosome aberration study:

The test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

In vitro Mammalian cell gene mutation assay:

The mouse lymphoma assay was performed to evaluate the mutagenic and clastogenic potential of the test material. The assay was performed using mouse lymphoma L5178Y TK+/- 3.7.C cells in the presence and absence of S9 metabolic activation system. The test chemical was tested at concentrations ranging from 3-15 µg/mL (Without S9: 0, 3, 6.8, 11, 11, 15 or 15 µg/mL; With S9: 0, 3, 3, 6.8, 6.8, 11, 11, 15 or 15 µg/mL) both with and without metabolic activation system. The mutagenicity assay was performed according to the procedure described by Clive and Spector. A total of 1.2 × 107 cells in duplicate cultures were exposed to the test chemical, positive control, and solvent control for 4 h at 37 ± 1 °C, washed twice with growth medium, and maintained at 37 ± 1 °C for 48 h in log-phase growth to allow recovery and mutant expression. Cells in the cultures were adjusted to 3×105/mL at 24 h intervals. They were then cloned (1×106 cells/plate for mutant selection and 200 cells/plate for viable count determinations) in soft agar medium containing Fischer’s medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% pluronic F-68, and 0.23% granulated agar. Resistance to trifluorothymidine (TFT) was determined by adding TFT (final concentration, 3 µg/mL) to the cloning medium for mutant selection. The 100×stock solution of TFT in saline was stored at -70 °C and was thawed immediately before use. Plates were incubated at 37 ± 1 °C in 5% CO2 in air for 10-12 days and then counted with an Artek automated colony counter or ProtoCol colony counter. Only colonies larger than∼0.2 mm in diameter were counted. Mutant frequencies were expressed as mutants per 106 surviving cells. Although there are several different methods for evaluating mouse lymphoma data, results from this study were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Doubling of the mutant frequency was reported as representing a positive effect. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response. The size of mutant mouse lymphoma colonies was also determined using an Artek 982 colony counter/sizer or the ProtoCol colony counter. An internal discriminator was set to step sequentially to exclude increasingly larger colonies in approximate increments of 0.1 mm in colony diameter. The size range used was from∼0.2 to 1.1 mm. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls. The doses of chemical selected for testing were within the range yielding approximately 0-90% cytotoxicity. Based on the details of the study, the test substance did not induce gene mutation in the mouse lymphoma L5178Y TK+/- 3.7.C cells in the presence and absence of S9 metabolic activation system and hence it not likely to be mutagenic in L5178Y TK +/- mouse lymphoma assay.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
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 journal.
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
In vitro bacterial reverse gene mutation assay for test chemical was studied in S. typhimurium and Escherichia coli strains. The mutagenicity assay was conducted as described by Ames et al. with slight modifications. All tester strains were examined periodically for the markers indicated by Ames et al.



GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine for Salmonella strains and tryptophan for E. coli strains
Species / strain / cell type:
S. typhimurium, other: TA100, TA1535, TA98, TA1537, TA1538
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: S9 mix (500 µM) consisted of 100µL of S9, 50 µmoles sodium phosphate buffer (pH 7.4), 4 µmoles MgCl2, 16.5 µmoles KCl, 2.5 µmoles G-6-P, 2 µmoles NADH and 2 µmoles NADPH.
- source of S9 : PCB-treated male Sprague-Dawley rats
- method of preparation of S9 mix : No data
- concentration or volume of S9 mix and S9 in the final culture medium : No data
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No data
Test concentrations with justification for top dose:
0, 250, 750, 1500 or 3000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: No data available
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
9-aminoacridine
2-nitrofluorene
sodium azide
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION:
For Salmonella strains:
The assays without S9 were performed by the plate-incorporation method.
The assays with S9 were conducted by the pre-incubation method described by Yahagi et al. (1975). The pre-incubation method is useful in detecting weak mutagenicity in samples (Yahagi et al., 1977).. Histidine-independent colonies were scored after incubation at 37°C for 48-72 h.

For E. coli strains:
The assay was performed in the same manner as with the Salmonella assay except that the supplement of 0.1 µmole histidine plus 0.1 mole biotin in the soft agar was replaced with a supplement of 0.1 µmole of tryptophan. Tryptophan-independent revertant colonies were scored with E. coli.

DURATION
- Preincubation period: 37°C
- Exposure duration: 48-72 h.


Rationale for test conditions:
No data available
Evaluation criteria:
A bacterial cell line was observed for biologically relevant increase in the number of revertants. Revertants per plate represent average values from 3 to 5 replications.
Statistics:
No data available
Species / strain:
S. typhimurium, other: TA100, TA1535, TA98, TA1537, TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
All test compounds showed killing on bacteria at the highest doses used.
Remarks on result:
other: No mutagenic potential

Table: RESULTS OF MUTAGENICITY TEST OF THE TEST CHEMICAL


Chemical

Dose (µg/plate)

Revertants/plate (mean±S.D.)

Salmonella typhimuriumstrains

Escherichia coli

Without S9

 

DMSO

50µL

99±6

8±2

24±4

4±2

15±3

55±6

Positive control

 

769±54

266±34

660±30

822±88

301±33

452±68

Test chemical

250

98±5

10±4

28±1

3±1

9±1

50±9

750

81±6

5±1

24±1

3±1

10±2

57±6

1500

81±13

7±1

22±8

5±0

14±0

46±3

3000

24±13

2±1

20±8

2±2

6±1

33±6

With S9

 

 

 

 

 

 

 

DMSO

50µL

106±9

10±3

32±4

7±2

21±4

59±5

Positive control

 

431±92

158±45

180±7

125±37

154±17

753±95

Test chemical

250

87±1

7±2

31±3

9±2

24±8

58±6

 

750

130±3

10±1

27±3

7±2

18±4

62±3

 

1500

123±12

9±2

24±3

8±3

21±3

42±4

 

3000

72±11

12±2

21±2

4±2

11±0

39±8

Conclusions:
The test chemical was negative in Ames test carried out using Salmonella typhimurium strains TA100, TA1535, TA98, TA1537, TA1538 as well in Escherichia coli WP2 uvr A in the presence and absence of S9 metabolic activation. Hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

In vitro bacterial reverse gene mutation assay for test chemical was studied using S. typhimurium and Escherichia coli WP2 uvrA strains. The mutagenicity assay was conducted as described by Ames et al. with slight modifications. Ames test was performed using Salmonella typhimurium strains TA100, TA1535, TA98, TA1537 and TA1538 and Escherichia coli WP2 uvrA strains in the presence and absence of S9 metabolic activation system at dose level of 0, 250, 750, 1500 or 3000 µg/plate. For Salmonella strains, the assays without S9 were performed by the plate-incorporation method and the assays with S9 were conducted by the pre-incubation method described by Yahagi et al. (1975). The pre-incubation method is useful in detecting weak mutagenicity in samples (Yahagi et al., 1977). Histidine-independent colonies were scored after incubation at 37°C for 48-72 h. For E. coli strains, the assay was performed in the same manner as with the Salmonella assay except that the supplement of 0.1 µmole histidine plus 0.1 mole biotin in the soft agar was replaced with a supplement of 0.1 µmole of tryptophan. Tryptophan-independent revertant colonies were scored with E. coli. No mutagenicity was noted at the tested dose levels. Based on the observations made, the test chemical was negative in Ames test carried out using Salmonella typhimurium strains TA100, TA1535, TA98, TA1537, TA1538 as well in Escherichia coli WP2 uvr A in the presence and absence of S9 metabolic activation. Hence it 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 CAS 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:
in vitro mammalian chromosome aberration test
Target gene:
No data
Species / strain / cell type:
lymphocytes: human peripheral blood lymphocytes
Remarks:
5
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: 28-35 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:
Chinese hamster Ovary (CHO)
Remarks:
6
Details on mammalian cell type (if applicable):
- Name of test material (as cited in study report): Cinnamyl alcohol
- IUPAC name : 3-Phenylprop-2-en-1-ol
- Molecular Formula: C9H10O
- Molecular Weight: 134.177 g/mol
- Smiles: c1(ccccc1)/C=C/CO
- InChI: 1S/C9H10O/c10-8-4-7-9-5-2-1-3-6-9/h1-7,10H,8H2/b7-4+
- Substance type: Organic
- Physical state: Crystalline solid
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
5. Type and composition of metabolic activation system: Aroclor 1254 induced S9
- source of S9 : No data
- method of preparation of S9 mix : An appropriate quantity of S9 supernantant was thawed and mixed with S9 cofactor solution to result in final concentration in the S9 mix of approximately 1% v/v for Phase I of experiment and 2% v/v for Phase II of experiment

Cofactor solution contained following quantity of chemicals in 500 mL of RO water
D-glucose-6-phosphate 0.80g
MgCl2 1.00g
KCl 1.35 g
Na2HPO4 6.40g
M=NaH2PO4. H2O 1.40 g
NADP 1.75g

S9 mix was prepared freshly
- concentration or volume of S9 mix and S9 in the final culture medium : approximately 1% v/v for Phase I of experiment and 2% v/v for Phase II of experiment
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No data

6. - Type and composition of metabolic activation system: The S9 mix consisted of 15 µl/ml liver homoge nate (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.
- source of S9 : Sprague-Dawley rats
- method of preparation of S9 mix : Sprague-Dawley rats, induced with Aroclor 1254
- concentration or volume of S9 mix and S9 in the final culture medium : 15 µl/ml liver homogenate
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No data
Test concentrations with justification for top dose:
5. 0.00, 0.125, 0.25 or 0.5 mg/mL
6. Without S9: 160-1600 µg/ ml With S9: 500-5000 µg/ ml
Vehicle / solvent:
5. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

6. Water, dimethyl sulfoxide (DMSO), ethanol, or Acetone (in the order of preference)
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:
5
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Triethylenemelamine
Remarks:
6
Details on test system and experimental conditions:
5. 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: 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°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°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°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 (0.5, 1.0 or 2.0 mg/mL of culture media) based on solubility, precipitation and pH test of the test item was tested. Cytotoxicity was determined by reduction in the mitotic index in comparison to the 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

6. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: With S9: 2 hrs Without S9: Apprx. 8.5-9 hrs
- Expression time (cells in growth medium): 8.5-9 hrs
- Selection time (if incubation with a selection agent): after 18-26 hrs
- Fixation time (start of exposure up to fixation or harvest of cells): 8-12 hr after the beginning of treatment.

SELECTION AGENT (mutation assays): Giemsa stain
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available
NUMBER OF REPLICATIONS: No data available
NUMBER OF CELLS EVALUATED: 100 cells
DETERMINATION OF CYTOTOXICITY - Method: No data available

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: No data available
- Other: No data available
OTHER: No data available
Rationale for test conditions:
No data
Evaluation criteria:
5. 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.

6. Chromosomal aberrations were noted; Cells were selected for scoring on the basis of good mo rphology and completeness of karyotype (21 ± 2 chromosomes).

Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized chromosomes). Gaps and endo-reduplications were recorded but were not included in the totals. Aberrations were not scored in polyploidy cells but metaphases w ith 19-23 chromosomes were used (the modal number being 21).
Statistics:
5. Statistical significance at the p < 0.05 was evaluated by means of the non-parametric Mann-Whitney test

6. For chromosome aberrations, 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 was used. The P values were adjusted 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.
Species / strain:
lymphocytes: Human peripheral blood lymphocytes
Remarks:
5
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
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
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
5. 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°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 0.0 (NC), 0.0 (VC), 0.5 (T1), 1.0 (T2) and 2.0 (T3) mg/mL of culture media. Cytotoxicity was observed in treated concentrations of 1 (T2) and 2 (T3) mg/mL both in the presence and absence of metabolic activation (1%). In the absence of S9 mix, the mean mitotic index observed was 10.05 (NC), 9.93 (VC), 6.44 (T1), 4.17 (T2), 2.67 (T3) and 8.20 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.19 (NC), 9.92 (VC), 6.94 (T1), 4.35 (T2), 2.92 (T3) and 8.49 (PC). In the cytotoxicity experiment the highest test concentration 2 (T3) and 1 (T2) mg/ mL of treated culture media showed more than 50% reduction in the mitotic index when compared to the respective vehicle control both th the presence and in absence of metabolic activation confirms the cytotoxic effects. Hence 0.5 mg/mL was selected as highest concentration for main study considering the selection of test concentrations upto cytotoxicity. T

Hence the concentrations selected for the main study are 0.00, 0.125, 0.25 or 0.5 mg/mL. The main study was performed in two independent phases - Phase I and Phase II

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

6. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data available
- Effects of osmolality: No data available
- Evaporation from medium: No data available
- Water solubility: No data available
- Precipitation: No data available
- Other confounding effects: No data available

RANGE-FINDING/SCREENING STUDIES: Doses were chosen for the aberration test based on a preliminary test of cell survival 24 hr after treatment. Doses were based on observations of cell confluence and mitotic cell availability in the SCE test.
COMPARISON WITH HISTORICAL CONTROL DATA: No data available
ADDITIONAL INFORMATION ON CYTOTOXICITY: No data available
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce chromosome aberrations in the mammalian cell line or lymphocytes used in th e presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.
Executive summary:

Data available from 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 study was conducted to determine the chromosomal aberration induction potential of the test chemical in human peripheral blood lymphocyte cultures. The method followed was 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 28-35 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 DMSO and used at dose level of 0, 0.00, 0.125, 0.25 or 0.5 mg/mL in the presence and absence of S9 metabolic activation system in phase I and phase II. 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 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. Each tube 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°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°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 °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°C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37°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. The test chemical is not mutagenic at the highest tested concentration of 0.5 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.

In another study, In vitro mammalian chromosome aberration test was performed to evaluate the mutagenic nature of the test chemical. Cloned Chinese hamster ovary cells (CHO-W-B1) were cultured in Mc-Coy’s 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics. Tests were carried out with and without an in vitro metabolic activation system (S9 mix). Cells were exposed to the test chemical at doses of 160-1600µg/mL without S9 and 500-5000 µg/mL with S9 for 2 hr in the presence of S9 or throughout the incubation period without S9. If little or no delay was found, the cell harvest time for the aberration test was 8-12 hr after the beginning of treatment. Cells were collected by mitotic shake-off. Slides were stained with Giemsa and coded, and 100 cells were scored from each of the three highest dose groups having sufficient metaphases for analysis and from positive (triethylenemelamine, mitomycin C, or cyclophosphamide) and solvent controls. 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 endoreduplications were recorded but were not included in the totals. The test chemical did not induce chromosome aberrations in the Chinese hamster ovary cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

Based on the details of the study, the test chemical did not induce chromosome aberrations in the mammalian cell line or lymphocytes used in th e presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

Endpoint:
in vitro gene mutation study in mammalian cells
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:
The mouse lymphoma assay was performed to evaluate the mutagenic and clastogenic potential of the test material.
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
Thymidine Kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Remarks:
TK+/- 3.7.C
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: No data
- source of S9 : Aroclor 1254-induced male Sprague-Dawley rats
- method of preparation of S9 mix : S9 mix was prepared according to the procedure of Clive et al.
- concentration or volume of S9 mix and S9 in the final culture medium : No data
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability) : No data
Test concentrations with justification for top dose:
0 µg/mL, 3-15 µg/mL

Without S9: 0, 3, 6.8, 11, 11, 15 or 15 µg/mL
With S9: 0, 3, 3, 6.8, 6.8, 11, 11, 15 or 15 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: no data available
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
not specified
Details on test system and experimental conditions:
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 12000000 cells in duplicate
- Test substance added was added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: No data
- Exposure duration/duration of treatment: 4 hrs
- Harvest time after the end of treatment (sampling/recovery times): 48 h in log-phase growth


FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 10-12 days
- Selection time (if incubation with a selective agent): 10-12 days
- Fixation time (start of exposure up to fixation or harvest of cells): No data
- Method used: agar
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure. : trifluorothymidine (TFT) at final concentration, 3 µg/mL
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 1000000 cells/plate for mutant selection and 200 cells/plate for viable count determinations. They were then cloned in soft agar medium containing Fischer’s medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% pluronic F-68, and 0.23% granulated agar. Resistance to trifluorothymidine (TFT) was determined by adding TFT (final concentration, 3 µg/mL) to the cloning medium for mutant selection. The 100×stock solution of TFT in saline was stored at -70 °C and was thawed immediately before use. Plates were incubated at 37 ± 1 °C in 5% CO2 in air for 10-12 days and then counted with an Artek automated colony counter or ProtoCol colony counter. Mutant frequencies were expressed as mutants per 106 surviving cells. Results from this study were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Doubling of the mutant frequency was previously reported as representing a positive effect. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response.
- Criteria for small (slow growing) and large (fast growing) colonies: An internal discriminator was set to step sequentially to exclude increasingly larger colonies in approximate increments of 0.1 mm in colony diameter. The size range used was from ∼0.2 to 1.1 mm.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other: Cell growth
- Any supplementary information relevant to cytotoxicity: Cells at a concentration of 6 × 105/mL (6 × 106 cells total) were exposed for 4 h to a range of concentrations from 0.0005 to 10000 µg/mL. The cells were then washed, resuspended in growth medium, and incubated at 37 ± 1 °C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls. The doses of chemical selected for testing were within the range yielding approximately 0-90% cytotoxicity. For each assay, there were 2-4 solvent controls, a positive control of ethyl methylsulfonate at 4.7×10-6 M (or methyl methanesulfonate at 10-20 µg/ mL) for the test without metabolic activation, and a positive control of 3-methylcholanthrene at 1.86 × 10-5 M (or dimethylbenz[a]anthracene at 0.5-4 µg/mL) for the test with metabolic activation.
Rationale for test conditions:
No data
Evaluation criteria:
Results from this study were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Doubling of the mutant frequency was previously reported as representing a positive effect (28). Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response.
Statistics:
No data
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
TK+/- 3.7.C
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
No data
Remarks on result:
other: No mutagenic potential

Table: Mutagenicity data for the test chemical

Solvent

Non-Activated Cultures

 

S9-Activated Cultures

 

Dose (ug/mL)

Average TFT

Average VC

Mut Freq

RTG

Dose (ug/mL)

Average TFT

Average VC

Mut Freq

RTG

DMSO

3 to 15

 

3

43

194

0.4

93

3

82

194

0.8

86

6.8

55

200

0.6

83

64

195

0.7

82

11

45

---

---

---

6.8

86

187

0.9

77

43

179

0.5

68

70

202

0.7

80

15

48

192

0.5

53

11

61

205

0.6

73

62

163

0.8

38

74

194

0.8

71

Solvent

49

192

0.5

15

33

137

0.5

4

Positive

366

91

8

22

Solvent

70

219

0.6

 

Positive

207

179

2.3

79

Conclusions:
The test substance did not induce gene mutation in the mouse lymphoma L5178Y TK+/- 3.7.C cells in the presence and absence of S9 metabolic activation system and hence it not likely to be mutagenic in L5178Y TK +/- mouse lymphoma assay.
Executive summary:

The mouse lymphoma assay was performed to evaluate the mutagenic and clastogenic potential of the test material. The assay was performed mouse lymphoma L5178Y TK+/- 3.7.C cells in the presence and absence of S9 metabolic activation system. The test chemical was tested at concentrations ranging from 3-15 µg/mL (Without S9: 0, 3, 6.8, 11, 11, 15 or 15 µg/mL; With S9: 0, 3, 3, 6.8, 6.8, 11, 11, 15 or 15 µg/mL) both with and without metabolic activation system. The mutagenicity assay was performed according to the procedure described by Clive and Spector. A total of 1.2 × 107 cells in duplicate cultures were exposed to the test chemical, positive control, and solvent control for 4 h at 37 ± 1 °C, washed twice with growth medium, and maintained at 37 ± 1 °C for 48 h in log-phase growth to allow recovery and mutant expression. Cells in the cultures were adjusted to 3×105/mL at 24 h intervals. They were then cloned (1×106 cells/plate for mutant selection and 200 cells/plate for viable count determinations) in soft agar medium containing Fischer’s medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% pluronic F-68, and 0.23% granulated agar. Resistance to trifluorothymidine (TFT) was determined by adding TFT (final concentration, 3 µg/mL) to the cloning medium for mutant selection. The 100×stock solution of TFT in saline was stored at -70 °C and was thawed immediately before use. Plates were incubated at 37 ± 1 °C in 5% CO 2 in air for 10-12 days and then counted with an Artek automated colony counter or ProtoCol colony counter. Only colonies larger than ∼0.2 mm in diameter were counted. Mutant frequencies were expressed as mutants per 106 surviving cells. Although there are several different methods for evaluating mouse lymphoma data, results from this study were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Doubling of the mutant frequency was reported as representing a positive effect. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response. The size of mutant mouse lymphoma colonies was also determined using an Artek 982 colony counter/sizer or the ProtoCol colony counter. An internal discriminator was set to step sequentially to exclude increasingly larger colonies in approximate increments of 0.1 mm in colony diameter. The size range used was from ∼0.2 to 1.1 mm. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls. The doses of chemical selected for testing were within the range yielding approximately 0-90% cytotoxicity. Based on the details of the study, the test substance did not induce gene mutation in the mouse lymphoma L5178Y TK+/- 3.7.C cells in the presence and absence of S9 metabolic activation system and hence it not likely to be mutagenic in L5178Y TK +/- mouse lymphoma assay.

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 from various sources for the target chemical and the closely related test chemicals was reviewed to determine the mutagenic nature of the given test chemical. The studies are as mentioned below:

Ames assay:

In vitro bacterial reverse gene mutation assay for test chemical was studied using S. typhimurium and Escherichia coli WP2 uvrA strains. The mutagenicity assay was conducted as described by Ames et al. with slight modifications. Ames test was performed using Salmonella typhimurium strains TA100, TA1535, TA98, TA1537 and TA1538 and Escherichia coli WP2 uvrA strains in the presence and absence of S9 metabolic activation system at dose level of 0, 250, 750, 1500 or 3000 µg/plate. For Salmonella strains, the assays without S9 were performed by the plate-incorporation method and the assays with S9 were conducted by the pre-incubation method described by Yahagi et al. (1975). The pre-incubation method is useful in detecting weak mutagenicity in samples (Yahagi et al., 1977). Histidine-independent colonies were scored after incubation at 37°C for 48-72 h. ForE. colistrains, the assay was performed in the same manner as with theSalmonellaassay except that the supplement of 0.1 µmole histidine plus 0.1 mole biotin in the soft agar was replaced with a supplement of 0.1 µmole of tryptophan. Tryptophan-independent revertant colonies were scored withE. coli.No mutagenicity was noted at the tested dose levels. Based on the observations made, the test chemical was negative in Ames test carried out usingSalmonella typhimuriumstrains TA100, TA1535, TA98, TA1537, TA1538 as well inEscherichia coliWP2 uvr A in the presence and absence of S9 metabolic activation. Hence it is not likely to classify as a gene mutant in vitro.

In another publication, Bacterial gene mutation assay was performed to evaluate the mutagenic potential of the test material. The test chemical was non mutagenic at concentrations up to 500–4000 µg/plate inE. colistrain WP2 uvrA (trp-) and hence, according to CLP criteria, it can be concluded that the test chemical was non mutagenic in nature.

The above details are further supported by data from closely related read across analogue. Ames assay as per OECD 471 was performed to investigate the potential of the test chemical to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.0(VC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC), 0.0(VC),  0.005, 0.016, 0.050, 0.158 and 0.501 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the test chemical at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. The spontaneous reversion rates in the negative, vehicle and positive controls are within the range of our historical data. The positive controls used for various strains showed a distinct in­crease in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method. In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.

In vitro mammalian chromosome aberration study:

Data available from 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 study was conducted to determine the chromosomal aberration induction potential of the test chemical in human peripheral blood lymphocyte cultures. The method followed was 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 28-35 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 DMSO and used at dose level of 0, 0.00, 0.125, 0.25 or 0.5 mg/mL in the presence and absence of S9 metabolic activation system in phase I and phase II. 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 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. Each tube 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°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°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 °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°C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37°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. The test chemical is not mutagenic at the highest tested concentration of 0.5 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.

 

In another study, an in vitro mammalian chromosome aberration test was performed to evaluate the mutagenic nature of the test chemical. Cloned Chinese hamster ovary cells (CHO-W-B1) were cultured in Mc-Coy’s 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics. Tests were carried out with and without an in vitro metabolic activation system (S9 mix). Cells were exposed to the test chemical at doses of 160-1600µg/mL without S9 and 500-5000 µg/mL with S9 for 2 hr in the presence of S9 or throughout the incubation period without S9. If little or no delay was found, the cell harvest time for the aberration test was 8-12 hr after the beginning of treatment. Cells were collected by mitotic shake-off. Slides were stained with Giemsa and coded, and 100 cells were scored from each of the three highest dose groups having sufficient metaphases for analysis and from positive (triethylenemelamine, mitomycin C, or cyclophosphamide) and solvent controls. 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 endoreduplications were recorded but were not included in the totals. The test chemical did not induce chromosome aberrations in the Chinese hamster ovary cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

 

Based on the details of the study, the test chemical did not induce chromosome aberrations in the mammalian cell line or lymphocytes used in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

 

In vitro Mammalian cell gene mutation assay:

The mouse lymphoma assay was performed to evaluate the mutagenic and clastogenic potential of the test material. The assay was performed using mouse lymphoma L5178Y TK+/- 3.7.C cells in the presence and absence of S9 metabolic activation system. The test chemical was tested at concentrations ranging from 3-15 µg/mL (Without S9: 0, 3, 6.8, 11, 11, 15 or 15 µg/mL; With S9: 0, 3, 3, 6.8, 6.8, 11, 11, 15 or 15 µg/mL) both with and without metabolic activation system. The mutagenicity assay was performed according to the procedure described by Clive and Spector. A total of 1.2 × 107 cells in duplicate cultures were exposed to the test chemical, positive control, and solvent control for 4 h at 37 ± 1 °C, washed twice with growth medium, and maintained at 37 ± 1 °C for 48 h in log-phase growth to allow recovery and mutant expression. Cells in the cultures were adjusted to 3×105/mL at 24 h intervals. They were then cloned (1×106 cells/plate for mutant selection and 200 cells/plate for viable count determinations) in soft agar medium containing Fischer’s medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% pluronic F-68, and 0.23% granulated agar. Resistance to trifluorothymidine (TFT) was determined by adding TFT (final concentration, 3 µg/mL) to the cloning medium for mutant selection. The 100×stock solution of TFT in saline was stored at -70 °C and was thawed immediately before use. Plates were incubated at 37 ± 1 °C in 5% CO 2 in air for 10-12 days and then counted with an Artek automated colony counter or ProtoCol colony counter. Only colonies larger than∼0.2 mm in diameter were counted. Mutant frequencies were expressed as mutants per 106 surviving cells. Although there are several different methods for evaluating mouse lymphoma data, results from this study were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Doubling of the mutant frequency was reported as representing a positive effect. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response. The size of mutant mouse lymphoma colonies was also determined using an Artek 982 colony counter/sizer or the ProtoCol colony counter. An internal discriminator was set to step sequentially to exclude increasingly larger colonies in approximate increments of 0.1 mm in colony diameter. The size range used was from∼0.2 to 1.1 mm. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls. The doses of chemical selected for testing were within the range yielding approximately 0-90% cytotoxicity. Based on the details of the study, the test substance did not induce gene mutation in the mouse lymphoma L5178Y TK+/- 3.7.C cells in the presence and absence of S9 metabolic activation system and hence it not likely to be mutagenic in L5178Y TK +/- mouse lymphoma assay.

 

The non-mutagenic nature of the test chemical is further supported by data from various closely related test chemicals as mentioned-

 

An in vitro mammalian cell gene mutation assay was performed to evaluate the possible antimutagenic activity of the test chemical in Chinese hamster V79 cell line. The effect of the test chemical on the induction of HGPRT mutations in V79 cells by known mutagens. Cells were properly maintained in Dulbecco's MEM supplemented with 5% fetal calf serum (FCS), 100 U/ml penicillin G and 100 µg/ml streptomycin sulfate. Prior to test chemical exposure, HGPRT-mutants were induced by methyl methanesulfonate (MMS),N-nitroso-N-methylurea (MNU), ethyl methanesulfonate (EMS) and UV light. The test chemical was tested upto a concentration of 50 or 100 µM. Cells were seeded at a density of 2.5 x 106in 25-cm2tissue culture flasks or for UV treatment in 60-mm petri dishes. Approximately 18 h later, the medium was removed and the cells were treated with UV light (12 j/m2), or with MMS (2 mM), EMS (30 mM) or MNU (1 mM) for 1 h. After treatment, the cells were washed twice with HBSS to remove mutagens and the incubation was continued with fresh medium containing or not test chemcical (50 or 100 mM) for 2 or 4 h. In some experiments, the test chemical was added to cells together with chemical mutagens. Then, cells were washed, trypsinized and 2.5 x 105cells/dish were seeded in eight 100-mm petri dishes containing 20 ml of medium. The survival was measured by seeding 102cells in three 60-mm petri dishes containing 5 ml of fresh medium. Cultures were maintained for 7 days expression time and then reseeded at a density of 5 × 105cells/100-mm dish, taking four dishes for each condition. When the cells were attached, 6TG was added. Also 100 cells/60-mm dish containing growth medium were seeded in triplicate to test the viability. All colonies were stained and counted after 10 days. The mutation frequency was calculated as mutants/ 106viable cells. At the test chemical concentrations tested, the test chemical itself did not induce any modification of spontaneous mutation frequency or cell survival. Based on the details of the study, the test chemical did not induce gene mutation in the Chinese hamster V79 cells and the test chemical is therefore regarded as not mutagenic in vitro.

In vitro Mammalian Cell Gene Mutation Test was carried out in compliance with the OECD Guideline No. 476, adopted by the council on 29 July 2016. The test chemical was evaluated in mammalian cell gene mutation assay to determine its ability to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosome in the genome of Chinese hamster ovary (CHO) cells. Based upon the preliminary tests conducted to assess the solubility/precipitation and cytotoxicity of the test chemical, the CHO cells were exposed to the test item in duplicate cultures at the doses of 31.25, 15.625, 7.8125 and 3.90625 µg/mL of culture medium, in the absence and presence of metabolic activation system (S9). Liver S9 fraction prepared from sodium phenobarbitone and b- naphthoflavone-induced Wistar rats was used in the study. Dimethyl sulphoxide was used as a vehicle. The target cells were exposed to the test compound for 3 hours at 37 ± 1oC, with approximately 5% CO2 supply. The cells were sub-cultured immediately to determine cytotoxicity as relative survival (RS) and to initiate the phenotypic expression period.The culture flasks were incubated at 37 ± 1oC with approximately 5% CO2 supply during experimental periods. Concurrent vehicle and positive control groups were also included in the experiment, as specified by the test guideline. The cultures were sub-cultured during the expression period at suitable intervals. After phenotypic expression, the plates were incubated at 37 ± 1oC with approximately 5% CO2 supply for 11 days. After incubation, the medium was discarded, and the plates were stained and observed for the clones. Relative survivaland mutant frequency were calculated for all treatment, vehicle control, and positive control groups. The relative survival (RS) was used as the measure of treatment-related cytotoxicity. The RS (relative survival) for cultures treated withthe test chemical with and without metabolic activation system (S9) indicated that the test chemical induces 10 t 20% relative survival at the concentration of 31.25µg/mL. The % relative survival (% RS) for the cloned cultures ranged from 18.0% to71.8% for cultures treated without metabolic activation system and 19.0% to 70.9% for cultures treated with metabolic activation system at test item concentrations of31.25, 15.625, 7.8125 and 3.90625µg/mL in the treatment medium. The results of the present study indicate that there was no significant difference in the mutant frequencies of cultures treated withthe test chemical as compared to the vehicle control cultures, in the absence or presence of metabolic activation. An increase in the mutant frequency of concurrent positive controls demonstrated the sensitivity of the assay in the absence and presence of metabolic activation. Under the test conditions described in the study, it is concluded that the test chemical is non-mutagenic in 'In Vitro Mammalian cell gene mutation test' using the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosome in the genome of Chinese hamster ovary (CHO) cells and hence it is not likely to classify as a "Gene mutant in vitro".

 

In another gene mutation study, 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. A preliminary dose-finding study was conducted prior to the main study. A range of different test chemical concentrations were tested in 96-well plates and analyzed by two commonly used assays, i.e. the colorimetric assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the bicinchoninic acid (BCA) assay to assess cell viability and protein concentration, respectively. From the basis of the results from the MTT and BCA assays, test concentrations of the test chemical was chosen to be included in the gene toxicity test. In the genotoxicity test, the test chemical was administered to CHO cells for 3 hrs at the dose levels of 0, 0.5, 1.0, 2.5 or 5.0 mM and in the absenceor presenceof exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activationand 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. The results showed indication of gene mutations occurring in the positive controls ENUand 7,12-dimethylbenz(a) anthracenewhile no other treatment gave rise to gene toxicity. Two very diffuse colonies were seen in one well out of four at 2.5 mM in the absence with 4% S9 liver microsomal fraction. These diffuse colonies are not regarded to be relevant since the 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. No cytotoxic effects were observed when CHO cells were exposed to the test chemical 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 exposed to the test chemical at ≤ 5.0 mM for 3 hrs or more, and the test chemical does not induce cytotoxic effects at concentrations of ≤ 5.0 mM. The test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM did not show any evidence of gene toxicity when CHO cells were exposed to the test chemical in the absence and presence of S9 metabolic activation system and hence it is not likely to classify for gene mutation in vitro.

 

Other studies:

The influence of the test chemical on sister-chromatid exchanges (SCEs) induced by mitomycin C was investigated in cultured Chinese hamster CHO K-1 cells. The test chemical in DMSO was tested at dose levels of 0, 1.0, 3.3, 10, 33.3 or 100 uM. CHO K-1 cells were seeded at a density of 0.5-1.0 X 10 6 cells/100-mm dish. All treatments were done with cells in the log-phase. The cells were exposed to MMC for 21 h, and washed twice with Hanks' balanced salt solution. Monolayers without medium were treated with X-rays or UV using an X-ray machine with an adjusted dosimeter, or a 10-W germicidal lamp with a UV dosimeter. Pulse treatment with the test chemical for 3 h was done in synchronized CHO K-1 cells. Following the cultivation in the presence of BrdUrd for 1 cell cycle (21 h), the cells were treated with 10 uM TNI6 which has a colchicine-like effect for 20 h. Mitotic cells were collected by mitotic shake-off, seeded at a density of 0.5-1.0 x 105 ceils/60-mm dish, irradiated with UV at 1.2 J/m 2 or X-rays at 450 rad, and cultured in fresh medium containing BrdUrd. At the end of the pulse treatment. The cells were washed twice with Hanks' balanced salt solution and recovered in medium with BrdUrd. Since the cell cycle was not prolonged in cells treated with UV (1.2 J/m 2) or X-rays (450 rad), mitotic preparations were made 21 h after mitotic shake-off. The cells were treated with colchicine for 30 rain. Fifty metaphases per culture were analyzed for SCEs. No effects on the cell cycle and spontaneous SCEs by the test chemical were observed. Based on the details available, the test chemical did not induce sister chromatids in Chinese hamster CHO K-1 cells and hence the test chemical is not likely to classify as a gene mutant in vitro.

Bacillus subtilis recombination assay was also performed to evaluate the mutagenic potential of the test material. The study was performed using B. subtilis M45 (rec-) & H17 (rec+) strains in the absence of S9 metabolic activation system. In Bacillus subtilis recombination assay, the test chemical at dose of 21 µg/disk was not mutagenic in B. subtilis M45 (rec-) & H17 (rec+) without metabolic activation and hence it is not likely to classify as a gene mutant in vitro.

 

Based on the data available for the target chemical and its closely related test chemicals, the target chemical Cinnamyl alcohol (CAS 104-54-1) is likely to be “Non-mutagenic”. The structurally and functionally closely related test chemicals support the non-mutagenic classification in vitro. Hence the test chemical is “Not classified” for gene mutation end point as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available for the target chemical and its closely related test chemicals, the target chemical Cinnamyl alcohol (CAS 104-54-1) is likely to be “Non-mutagenic”. Also, the structurally and functionally closely related test chemicals support the non-mutagenic classification in vitro. Hence the test chemical is “Not classified” for gene mutation end point as per the criteria mentioned in CLP regulation.