Ethyltriphenylfosfonium bromide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

The test chemical was found to be non-mutagenic as it did not induce (point) gene mutations in the histidine operon by base-pair changes or frameshifts, in the presence or absence of S9 metabolic activation system, in any of the five tester strains of Salmonella typhimurium (TA1537, TA1535, TA98, TA100 and TA102). Hence, the test chemical is not likely to be classified as a gene mutant as per the criteria mentioned in the CLP regulation.

In vitro mammalian cell gene mutation assay:

The test chemical was found to be non-mutagenic in the in vitro mammalian chromosomal aberration study performed using human peripheral blood lymphocytes both in presence (1% and 2%) and in the absence of S9 metabolic activation. Hence it is not likely to be classified as a gene mutant as per the criteria mentioned in CLP regulation.

In vitro mammalian cell gene mutation assay:

The test chemical does not give rise to gene mutations when CHO cells are exposed to the test chemicalin vitroat 0, 0.5, 1.0, 2.5 or 5.0 mM for 3 hrs, in the presence or absence of metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 20, 2018 to September 10, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from experimental study report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

The experiment was performed to assess the potential of the test item to induce gene mutations in the Bacterial reverse mutation assay using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The experiment was performed as a plate incorporation assay, Negative results were obtained in this experiment; hence the second experiment was performed as a pre-incubation assay. .

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine Operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Not applicable

Cytokinesis block (if used):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Arochlor induced S9 was procured from Defence Research and Development Organization.
- method of preparation of S9 mix : Appropriate quantity of S9 supernatant was mixed with S9 cofactor solution which contains D-glucose-6-phosphate 0.8 g, β-NADP 1.75 g, MgCl2 1.0 g, KCl 1.35g, Na2HPO4 6.4 g, NaH2PO4.H2O 1.4 g in 500 ml of distilled water
- concentration or volume of S9 mix and S9 in the final culture medium : 10% v/v
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No data

Test concentrations with justification for top dose:

0.0 (NC), 0.005, 0.016, 0.050, 0.158 and 0.501 mg/plate doses were selected based on the result obtained from pre-experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: The test item was found to be soluble in distilled water at 50 mg/ml.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-Nitro-o-phenylenediamine

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : Triplicate
- Number of independent experiments : The test chemical was tested in two independent experiments (Trial I and Trial II). Trial I: plate incorporation method and Trial II: preincubation method.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 60 min at 37°C
- Exposure duration/duration of treatment: 48 hours at 37°C for both Trial I and Trial II
- Harvest time after the end of treatment (sampling/recovery times): No data

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Reduction in revertant count and inhibition of background lawn
- Any supplementary information relevant to cytotoxicity: The pre-experiment was performed with TA 98 and TA 100 strain of Salmonella typhimurium and eight concentrations spaced by half log intervals in triplicates. 5 mg/plate were selected as the highest dose in the pre-experiment based on the solubility and precipitation test, Concentration 0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment study.

METHODS FOR MEASUREMENTS OF GENOTOXICIY

- OTHER: No data

Rationale for test conditions:

No data available.

Evaluation criteria:

A test item was considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding vehicle/solvent control was observed.
A dose-dependent increase was considered biologically relevant if the threshold exceeded at more than one concentration.
An increase exceeding the threshold of biological significance at only one concentration was judged as biologically relevant if it was reproduced in an independent second experiment.
A dose-dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative control such an increase was not considered biologically relevant.

Statistics:

Microsoft Office Excel based calculations were used for descriptive statistical analysis.

Species / strain:

S. typhimurium, other: TA 1535, TA1537, TA98, TA100, TA102

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No data
- Data on osmolality: No data
- Possibility of evaporation from medium: No data
- Water solubility: Test item was found to be soluble in water at 50 mg/ml .
- Precipitation and time of the determination: Slight precipitation was observed which was assumed to be non interfering with the scoring. Therefore, 5 mg/plate was selected as the highest concentration for pre-experiment.
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES (if applicable): The pre-experiment was performed with TA 98 and TA 100 strain of Salmonella typhimurium and eight concentrations spaced by half log intervals in triplicates. 5 mg/plate were selected as the highest dose in the pre-experiment based on the solubility and precipitation test. 0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate.

In TA 98 and TA 100 there was no reduction in colony count but reduction in background lawn was observed in treated concentrations 5 (T8) and 1.582 (T7) mg/plate and no reduction in backreduction in colony count as well as in background lawn in treated concentration 0.501 (T6) mg/plate - 0.002 (T1) mg/plate both in the absence and in the presence of metabolic activation.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The positive controls showed an unequivocal increase in revertant counts with all the five tester strains and the respective controls used. And the spontaneous reversion rates in the negative and positive controls were within the range of in house historical data

Ames test:
- Signs of toxicity : No substantial increase in Revertant colony count was observed
- Individual plate counts : Please refer the attached table
- Mean number of revertant colonies per plate and standard deviation : Please refer the table attached in any other information on result section.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Please refer the table attached in remark section.
- Negative (solvent/vehicle) historical control data: Please refer the table attached in remark section.

Remarks on result:

other: No mutagenic potential

TABLE 4 - MEAN REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIAL-I)

Dose (mg/plate)	In the Presence (+S9) of Metabolic Activation
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	6.33	0.58	16.33	0.58	23.67	2.52	122.67	3.51	274.67	10.26
T1 (0.005)	4.67	0.58	10.67	1.53	21.00	1.00	107.67	2.08	274.67	6.11
T2 (0.016)	4.33	0.58	11.33	1.53	19.33	1.53	111.33	2.08	222.67	9.02
T3 (0.050)	5.00	1.00	13.00	1.00	21.67	0.58	112.00	3.61	231.00	6.24
T4 (0.158)	5.67	0.58	12.33	1.53	21.33	1.53	118.00	200	246.67	9.02
T5 (0.501)	5.33	0.58	15.67	2.08	23.00	1.00	118.67	2.52	272.00	7.21
PC	180.33	9.87	493.33	46.70	1186.67	33.31	1464.00	32.00	1309.33	51.43

Dose (mg/plate)	In the Absence (-S9) of Metabolic Activation
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	7.67	0.58	15.67	0.58	23.00	2.00	120.67	4.04	275.33	12.06
T1 (0.005)	5.00	1.00	12.33	3.21	16.00	1.00	104.67	2.08	218.00	4.00
T2 (0.016)	4.33	0.58	10.33	1.53	19.00	1.00	107.00	3.00	228.00	10.58
T3 (0.050)	5.67	0.58	11.67	1.15	20.33	1.53	112.00	3.61	246.33	5.51
T4 (0.158)	5.33	0.58	13.33	1.53	20.67	0.58	115.33	4.73	264.00	9.17
T5 (0.501)	6.00	0.00	14.67	0.58	19.00	2.65	113.33	3.51	262.00	10.58
PC	171.33	10.21	1192.67	49.00	986.67	20.13	1269.33	24.44	1717.33	142.29

NC= Negative Control, T =Test concentration (T5: Highest, T1: Lowest), SD= Standard deviation

PC= Positive control                                                                         2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA98, TA100,        
2-Aminoanthracene [10μg/plate]:TA 102,                                              Sodium azide [10μg/plate]: TA 1535, TA 100,                                            

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate],    Methyl methanesulfonate [4μl/plate]: TA 102.

TABLE 5 - MEAN REVERTANT COUNT IN PRE-INCUBATION METHOD (TRIAL-II)

Dose (mg/plate)	In the Presence (+S9) of Metabolic Activation
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	7.33	0.58	16.00	1.00	26.33	0.58	120.33	1.53	265.33	7.77
T1 (0.005)	4.67	0.58	11.67	2.08	21.33	0.58	102.67	1.53	229.33	9.07
T2 (0.016)	4.00	0.00	12.00	1.00	24.00	1.00	110.67	2.08	237.33	8.14
T3 (0.050)	5.33	1.53	13.67	1.53	25.00	1.00	115.33	2.08	244.33	7.37
T4 (0.158)	6.33	0.58	13.33	1.53	23.67	2.52	120.00	1.00	261.67	6.43
T5 (0.501)	5.67	0.58	15.67	0.58	25.67	0.58	117.00	1.00	259.33	7.23
PC	181.00	11.14	419.33	37.75	1325.33	48.88	1490.67	24.44	1469.33	51.43

 

Dose (mg/plate)	In the Absence (-S9) of Metabolic Activation
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	6.67	0.58	15.67	0.58	27.33	1.53	119.33	3.79	264.33	10.41
T1 (0.005)	4.33	0.58	10.33	1.53	20.33	2.08	104.33	3.06	220.33	4.93
T2 (0.016)	5.00	1.00	12.67	1.53	21.00	3.46	116.00	2.00	215.67	3.06
T3 (0.050)	4.67	0.58	11.00	1.00	24.33	0.58	109.00	8.19	225.67	7.23
T4 (0.158)	5.67	0.58	13.00	1.00	23.00	2.00	117.67	2.08	236.33	2.52
T5 (0.501)	6.33	0.58	15.00	1.00	26.67	0.58	116.67	3.21	251.00	3.00
PC	174.00	9.17	1224.00	32.00	930.67	38.02	1122.67	44.06	1624.00	48.00

NC= Negative Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard deviation

PC= Positive control                                                                    2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA98, TA100,        
2-Aminoanthracene [10μg/plate]:TA 102,                                              Sodium azide [10μg/plate]: TA 1535, TA 100,                                            

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate],   Methyl methanesulfonate [4μl/plate]: TA 102.

 

Conclusions:

The test chemical did not induce gene mutations either by base pair changes or frameshift in the genome of the Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, TA 102 both in presence or in absence of metabolic activation system. Hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Executive summary:

The study was performed to investigate the potential of the test chemical to induce gene mutations in comparison to negative control according to plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, TA102. The study was conducted as per OECD 471, Adopted July 21, 1997. The assay was performed in two independent experiments both with and without liver microsomal activation, each concentration, including the negative and positive controls was tested in triplicates. Based on the solubility and precipitation test result eight different concentrations 0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment. The pre-experiment was performed with TA100 and TA98 strain of salmonella typhimurium. In TA98 and TA100 there was no reduction in colony count but reduction in background lawn was observed in treated concentrations 5 (T8) and 1.582 (T7) mg/plate and no reduction in colony count as well as in background lawn in treated concentration 0.501 (T6) mg/plate – 0.002 (T1) mg/plate both in absence and in presence of metabolic activation. Based on the results, 0.501 mg/plate was selected as the highest dose for the main study trials both in the absence as well as in the presence of metabolic activation. Trial-I was performed with five concentration of test item along with the negative and concurrent positive control with the remaining three strains i.e. TA 1537, TA1535, TA 102 by plate incorporation method. For TA 98 and TA 100 revertant colony count were directly incorporated in the Trial-I from the pre-experiment up to the five concentrations [T2 (0.005 mg/plate) to T6 (0.501 mg/plate)]. Test item concentration 0.0 (NC), 0.005, 0.016, 0.050, 0.158 and 0.501mg/plate, were prepared with half log interval both in presence and in absence of metabolic activation. The plates were treated and incubated at 37±2⁰C for 48 hours. No increase in the revertant count in any of the five strains was reported at any of the test concentrations. Positive controls resulted in an unequivocal response in all the five tester strains compared to respective control used. Trial II was performed independently with all the five tester strains along with the negative and positive controls by pre-incubation method for the confirmation of the Trial-I results. The concentration 0.0 (NC), 0.005, 0.016, 0.050, 0.158 and 0.501 mg/plate were used both in the absence (-S9) as well as in the presence of metabolic activation (+S9). The concentration of positive controls used was same as used in the plate incorporation assay. In Trial II, the test item, negative and positive controls were pre-incubated along with 500µl of metabolic activation mix (+S9)/Buffer (-S9) and 100µl of bacterial culture for 60 minutes at 37±2^oC in an incubator. After pre-incubation, 2 mL of top agar was mixed with the pre-incubation mixture and poured on minimal glucose agar plates. The treaed plates were incubated for 48 hours in an incubator. No substantial increase in the revertant count was observed in any of the five tester strains pre-incubated with the test item. The positive controls showed an unequivocal increase in revertant counts with all the five tester strains and the respective controls used, hence confirming the non-mutagenic activity of the test item. Based on the results of this study, it is concluded that the test chemical was found to be non-mutagenic as it did not induce (point) gene mutations in the histidine operon by base-pair changes or frameshifts, in the presence or the absence of metabolic activation system, in any of the five tester strains of Salmonella typhimurium (TA1537, TA1535, TA98, TA100 and TA102). Hence, the test chemical is not likely to be classified as a gene mutant as per the criteria mentioned in the CLP regulation.

 

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 July 2018 to 04 December 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from experimental study report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Principles of method if other than guideline:

This in vitro assay was performed to assess the potential of test chemical to induce structural / numerical chromosomal aberrations in one experiment (Phase I). The induction of cytogenetic damage in human lymphocytes was assessed with and without metabolic activation. Due to the negative result in Phase I, a second experiment (Phase II) was performed.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

lymphocytes:

Remarks:

Human Peripheral Blood Lymphocyte

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Human Blood
- Suitability of cells: No data

For lymphocytes:
- Sex, age and number of blood donors: 21 to 27 years age range
- Whether whole blood or separated lymphocytes were used: Seperated Lymphocytes
- Whether blood from different donors were pooled or not: No data

Cytokinesis block (if used):

No data available

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : S9 was procured from Defence Research and Development Establishment, Nagpur
- method of preparation of S9 mix : Appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution, which contains 0.80 g of D-glucose-6-phosphate, 1.00 g of MgCl2, 1.35 g of KCl, 6.40 g of Na2HPO4, 1.40 g of NaH2PO4.H2O, 1.75 g of β-NADP in 500 mL of RO water. During the experiment, S9 mix was freshly prepared.
- concentration or volume of S9 mix and S9 in the final culture medium : 1% v/v for phase I experiment and 2% v/v for phase II experiment
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No data

Test concentrations with justification for top dose:

Test concentrations with justification for top dose: 0, 0.25, 0.5 and 1 mg/mL (both in presence and in absence of metabolic activation system) were selected as the doses for the main study on the basis of results obtained from the preliminary cytotoxicity test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was found to be soluble in DMSO at 200 mg/mL

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Negative Control: Distilled water Vehicle control: DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: Cyclophosphamide monohydrate

Details on test system and experimental conditions:

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure. : Colcemide (0.3 μg/mL) was added 3 hour prior to harvesting and kept under incubation at 37 ± 2 °C

- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The slides were dried on the slide warmer and labelled. Two slides were made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX. All slides, including those of positive, vehicle and negative controls, were independently coded before microscopic analysis.

- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 1000 cells per slides
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): 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. To describe a cytotoxic effect the mitotic index (% cells in mitosis) were determined.
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Reduction in mitotic index in comparison with vehicle control.
- 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 and 2 mg/mL of culture media) based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with vehicle control.


- OTHER: No data

Rationale for test conditions:

No data available

Evaluation criteria:

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 vehicle 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 vehicle control
- f there is no dose-related increase
- All results are inside the historical vehicle control range
Statistical significance was confirmed by means of the non-parametric Mann-Whitney Test. However, both biological and statistical significance were considered together.
If the above-mentioned criteria for the test item were not clearly met, the classification with regard to the historical data and the biological relevance was discussed and/or a confirmatory experiment was performed.

Statistics:

Statistical significance at the p < 0.05 was evaluated by means of the non-parametric Mann-Whitney test.

Species / strain:

lymphocytes:

Remarks:

Human Peripheral Blood Lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH of test item in culture medium was assessed at 0 h and 4 h after incubation at 37 ± 2 °C. No significant change in pH was observed at 0 h and 4 h when compared with negative controls.
- Data on osmolality: No data
- Possibility of evaporation from medium: No data
- Water solubility: No data
- Precipitation and time of the determination: Since, Precipitation was not observed at 2 mg/ml it was taken as high concentration for the treatment in cytotoxicity experiment.
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES (if applicable): To evaluate the toxicity of test item, cytotoxicity was performed both in presence and in absence of metabolic activation system (1%). 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 the treated concentrations of 2 (T3) mg/mL both, in the absence and in the presence of metabolic activation (1%). In the absence of S9 mix, the mean mitotic index observed was 10.12 (NC), 9.94 (VC), 8.39 (T1), 6.59 (T2), 4.85 (T3) and 8.40 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.10 (NC), 9.94 (VC), 8.24(T1), 6.40 (T2), 4.75 (T3) and 8.49 (PC). In the cytotoxicity experiment, the test concentration 2 (T3) mg/L of culture media showed more than 50% reduction in mitotic index when compared to the respective vehicle control both in the presence or absence of metaboli activation. Hence these concentrations was not selected for the main study due to extensive cytotoxicity evoked.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The increased frequency of aberrations observed in the concurrent positive control groups (Phase I and II)
Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For lymphocytres in primary cultures: mitotic index (MI) : In the absence of S9 mix, the mean mitotic index observed was 10.05 (NC), 9.93 (VC), 5.43, (T1), 419 (T2), 3.58 (T3) and 8.20 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.195 (NC), 9.92 (VC), 5.49 (T1), 4.47 (T2), 3.03 (T3) and 8.49 (PC).
o For cell lines: relative population doubling (RPD), relative Increase in cell count (RICC), number of cells treated and cells harvested for each culture, information on cell cycle length, doubling time or proliferation index. No data
- Genotoxicity results (for both cell lines and lymphocytes)
o Definition for chromosome aberrations, including gaps : No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Negative (solvent/vehicle) historical control data: Please refer the table attached in remark section (Table 5)

Remarks on result:

other: No mutagenic potential

1. MITOTIC INDEX - CYTOTOXICITY TEST

Treatment	R	Mitotic Index (%)
		In the Absence of Metabolic Activation (-S9)				In the Presence of Metabolic Activation (1% S9)
		Mitotic Index	Mean	SD	Percent Reduction	Mitotic Index	Mean	SD	Percent Reduction
NC	R1	9.96	10.12	0.23	-	10.20	10.10	0.15	-
	R2	10.29				9.99
VC (DMSO)	R1	10.00	9.94	0.08	-	9.88	9.94	0.08	-
	R2	9.88				9.99
T1 (0.5 mg/mL)	R1	8.30	8.39	0.13	15.58	8.10	8.24	0.20	17.05
	R2	8.48				8.38
T2 (1.0 mg/mL)	R1	6.69	6.59	0.14	33.67	6.50	6.40	0.15	35.61
	R2	6.49				6.29
T3 (2.0 mg/mL)	R1	5.00	4.85	0.22	51.23	4.50	4.75	0.35	52.21
	R2	4.70				5.00
PC	R1	8.29	8.40	0.15	15.54	8.38	8.49	0.15	14.57
	R2	8.50				8.59

Key: R = Replicate,NC = Negative control, VC = Vehicle control,PC = Positive control, SD = Standard Deviation

2. SUMMARY OF MITOTIC INDEX

Treatment	Mitotic Index (%)
	Phase I
	In the Absence of Metabolic Activation (-S9)		In the Presence of Metabolic Activation (1% S9)
	Mean	SD	Mean	SD
NC	10.18	0.15	10.13	0.05
VC (DMSO)	9.90	0.14	9.95	0.08
T1 (0.25 mg/mL)	8.88	0.16	9.03	0.22
T2 (0.5 mg/mL)	7.88	0.16	8.34	0.19
T3 (1.0 mg/mL)	6.64	0.21	6.78	0.29
PC	8.40	0.14	8.49	0.26

Treatment	Mitotic Index (%)
	Phase II
	In the Absence of Metabolic Activation (-S9)		In the Presence of Metabolic Activation (2% S9)
	Mean	SD	Mean	SD
NC	10.07	0.13	10.00	0.15
VC (DMSO)	9.94	0.05	9.94	0.06
T1 (0.25 mg/mL)	8.69	0.28	8.83	0.20
T2 (0.5 mg/mL)	7.84	0.20	8.10	0.14
T3 (1.0 mg/mL)	6.04	0.22	6.39	0.14
PC	8.75	0.08	8.64	0.22

Key:  NC = Negative control, VC = Vehicle controlPC = Positive control, MI = Mitotic Index, -S9 = In the absence of metabolic activation, +S9 = In the presence of metabolic activation

3. Summary of Percent aberrant Cells

Treatment	Percent Aberrant Cells
	Phase I
	In the Absence of Metabolic Activation (-S9)		In the Presence of Metabolic Activation (1% S9)
	Mean	SD	Mean	SD
NC	0.333	0.471	0.333	0.471
VC (DMSO)	0.333	0.471	0.333	0.471
T1 (0.25 mg/mL)	0.333	0.471	0.333	0.471
T2 (0.5 mg/mL)	0.333	0.471	0.667	0.000
T3 (1.0 mg/mL)	0.667	0.000	0.333	0.471
PC	10.000	0.943	10.333	0.471

Treatment	Percent Aberrant Cells
	Phase II
	In the Absence of Metabolic Activation (-S9)		In the Presence of Metabolic Activation (2% S9)
	Mean	SD	Mean	SD
NC	0.333	0.471	0.333	0.471
VC (DMSO)	0.333	0.471	0.333	0.471
T1 (0.25 mg/mL)	0.333	0.471	0.333	0.471
T2 (0.5 mg/mL)	0.333	0.471	0.333	0.471
T3 (1.0 mg/mL)	0.667	0.000	0.667	0.000
PC	10.000	0.943	10.333	0.471

Key: NC = Negative Control, VC = Vehicle control, SD = Standard Deviation, PC = Positive Control

Conclusions:

In Vitro Mammalian Cell Gene Mutation Test of the test chemical was carried out in compliance with the OECD Guideline No. 473 (29 July 2016). Under the conditions described in this study, it is concluded that the test chemical is non-mutagenic when tested in an In vitro mammalian chromosomal aberration test using Human Peripheral Blood Lymphocyte in the presence and absence of S9 metabolic activation system and hence is likely to be non-mutagenic in vitro.

Executive summary:

The in vitro mammalian chromosomal aberration study was conducted to determine the chromosomal aberration induction potential of test chemical in human peripheral blood lymphocyte cultures. The methods followed were as per OECD guideline No. 473, adopted on 29th July 2016. 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 hours mitogenic stimulation. The cells were treated with metaphase arresting substance (colcemid) 3 hours prior to harvesting and stained. The slides were analysed with the aid of microscope and a minimum number of 1000 of cells per culture were counted and number of metaphases were recorded in different fields to determine the mitotic index. The number of aberrant cells with aberrations were recorded (150 cells per slide) to calculate percent aberrant cells. Based on the solubility and precipitation test, Dimethyl sulfoxide (DMSO) was selected as the vehicle for treatment. The pH of test item in culture medium was assessed at 0 hour and 4 hours after incubation at 37°C. No significant change in pH was observed at 0 hour and 4 hours when compared with negative control. Hence, 2.0 mg/mL was selected as the highest concentration for cytotoxicity test. Before conducting the chromosomal aberration study, test chemical was evaluated for cytotoxicity both in the absence and presence of metabolic activation system (1%). Cytotoxicity was assessed at the concentrations of 0.0 (NC), 0.0 (VC), 0.5 (T1), 1.0 (T2) and 2.0 (T3) mg/mL of culture media. Cytotoxicity was observed in the treated concentration of 2.0 (T3) mg/mL both in the absence and in the presence of metabolic activation (1%). In the absence of S9 mix, the mean mitotic index observed was 10.12 (NC), 9.94 (VC), 8.39(T1), 6.59 (T2), 4.85 (T3) and 8.40 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.10 (NC), 9.94(VC), 8.24 (T1), 6.40 (T2), 4.75 (T3) and 8.49 (PC). The test concentration 2.0 (T3) mg/ mL showed more than 50% reduction in the mitotic index in treated culture both in the presence or absence of metabolic activation. Hence the concentration [0.25, 0.5 and 1.0 mg/mL] were selected for the main study. Hence, 1.0 mg/mL of culture media was selected as the highest concentration for main study both in the presence and in the absence of metabolic activation. The main study was performed in two independent phases: In Phase I experiment, the cultures were exposed to the test chemical for a short period of time (4 hours) both in the absence and in the presence of metabolic activation system (1%).The mean percentage of aberrant cells was 0.333 (NC), 0.333 (VC), 0.333 (T1), 0.333 (T2), 0.667 (T3) and 10.000 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (VC), 0.333 (T1), 0.667 (T2), 0.333 (T3), 10.333 (PC) and 10.333 (PC) in the presence of metabolic activation at the concentration of 0.0 (NC), 0.0 (VC) 0.25 (T1), 0.5 (T2) and 1.0 (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 Cyclophosphamide monohydrate at the concentration of 30 μg/mL in the presence of metabolic activation (1%) caused significant increases in percent aberrant cells. Even though the analysis did not reveal any statistical significance, the increases were biologically significant. During the treatment with the test item both in the absence and presence of S9 mix, there was no reduction in mitotic index observed at the tested concentrations. The observed mean mitotic index in the absence of metabolic activation were 10.18, 9.90, 8.88, 7.88, 6.64 and 8.40 and in the presence of metabolic activation were 10.13, 9.95, 9.03, 8.34, 6.78 and 8.49 for 0.0 (NC), 0.0 (VC) 0.25 (T1), 0.5 (T2) and 1.0 (T3) mg/mL and 30 μg/mL (PC) concentration. 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 absence of metabolic activation, test items concentrations used were 0.0 (NC), 0.0 (VC) 0.25 (T1), 0.5 (T2) and 1.0 (T3) mg/mL and 30μg/mL (PC). 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 was 24 hours. The mean percent aberrant cells were 0.333 (NC), 0.333 (VC) 0.333 (T1), 0.333(T2), 0.667 (T3) and 10.000 (PC) in the absence of metabolic activation and 0.333 (NC),0.333 (VC),0.333 (T1), 0.333 (T2), 0.667 (T3) and 10.333 (PC) in the presence of metabolic activation at the concentration of 0.0 (NC), 0.0 (VC) 0.25 (T1), 0.5 (T2) and 1.0 (T3) mg/mL of culture and positive control. Treatment with Ethyl methanesulfonate at the concentration of 600μg/mL in the absence of metabolic activation and Cyclophosphamide monohydrate at the concentration of 30μg/mL in the presence of metabolic activation (2%) caused significant increases in percent aberrant cells .Though the analysis did not reveal any statistical significance, the increases were biologically significant. The increased frequency of aberrations observed in the concurrent positive control groups (Phase I and II) validated the sensitivity of the test system, suitability of the methods and conditions employed in the experiment. Treatment with test item both 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 index in the absence of metabolic activation were 10.07, 9.94, 8.69, 7.84, 6.04 and 8.75 and in the presence of metabolic activation were 10.00, 9.94, 8.83, 8.10, 6.39and 8.64 for 0.0 (NC), 0.0 (VC) 0.25 (T1), 0.5 (T2) and 1.0 (T3) and 30μg/mL PC concentrations, respectively.

Hence from the above data it can be concluded that, the test chemical was found to be not mutagenic for chromosomal aberration in human peripheral blood lymphocyte both in presence (1% and 2%) and in the absence of metabolic activation under the specified conditions. Hence it is not likely to be classified as a gene mutant as per the criteria mentioned in CLP regulation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 04, 2014 to March 19, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from experimental study report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

The purpose of this study was to assess toxic and genotoxic effects of the test chemical on Chinese Hamster Ovary (CHO) cells by using several different in vitro-based assays, including genotoxicity tests based on the OECD Guideline No. 476 “In Vitro Mammalian Cell Gene Mutation Test”.

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot.

This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.

The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.

HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.

Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- 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

Cytokinesis block (if used):

No data available.

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:

0, 0.5, 1, 2.5 or 5 mM

Vehicle / solvent:

Vehicle(s)/solvent(s) used: Phosphate-buffered saline (PBS)
Justification for choice of solvent/ vehicle: Ethyltriphenylphosphonium bromide was easily dissolved in PBS.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Phosphate-buffered saline (PBS)

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylnitrosurea

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium with pre-incubation

DURATION
- Pre-incubation: 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: 7 days
- Selection time: 14 days
- Fixation time: 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
- Method: 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
- Determination of polyploidy: Not applicable
- Determination of endoreplication: Not applicable
- Other: Not applicable

Rationale for test conditions:

No data available.

Evaluation criteria:

The assay is used for the detection of forward gene mutations in the HPRT locus on the X chromosomes of hypodiploid (modal No. 20) CHO cells.

Statistics:

No data available

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

not valid

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Please refer overall attachment and remark section.

Remarks on result:

other: No mutagenic potential.

Table 1A.Effect of test chemical exposure on gene toxicity in CHO cells. After being exposed to the test chemical for 3 hrs, cells was washed with sterile PBS and then incubated for 7 days at 37°C, 5% CO₂. After 7 days, cells were re-seeded in new 6-well plates in the absence or presence of 10mM TG as a selection agent and returned to the incubator for 14 days at 37°C, 5% CO₂. On day 15, all 6-well plates were stained with crystal violet and the number of colonies were counted manually. The results are presented as the total number of colonies found in the number of independent wells analyzed (e.g. 0 colonies in 4 wells will give 0/4) (n = 2 samples from 2 independent cultures).

 

	With S9		Without S9
	with TG	without TG	with TG	without TG
Neg. control	0/4	449/4	0/4	348/4
Pos. control	0/4	471/4	20/4	464/4
0.5 mM	0/4	329/4	0/4	301/4
1.0 mM	0/4	267/4	0/4	232/4
2.5 mM	0/4	230/4	0/4	406/4
5.0 mM	0/4	232/4	0/4	215/4

 

 

 

Table 1B.Mutation frequency in CHO cells after 3 hrs of exposure to test chemical in the absence or presence of 4% S9 liver microsomal fraction. N/A, no colonies present in the samples selected with TG, i.e. no mutation frequency could be determined.

 

	With S9	Without S9
Neg. control	N/A	N/A
Pos. control	N/A	3.39x10^-4
0.5 mM	N/A	N/A
1.0 mM	N/A	N/A
2.5 mM	N/A	N/A
5.0 mM	N/A	N/A

 

Conclusions:

The test chemical in the concentration of 0, 0.5, 1, 2.5 or 5 mM did not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence or absence of metabolic activation system and hence it is not likely to cause gene mutation in vitro.

Executive summary:

The study was performed to assess toxic and genotoxic effects of the test chemical on Chinese Hamster Ovary (CHO) cells by using several different in vitro-based assays, including genotoxicity tests based on the OECD Guideline No. 476 “In Vitro Mammalian Cell Gene Mutation Test”. 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. All tests of the preliminary dose-finding study was completed without S9-induced metabolic activation. The test chemical was added to the cells in the test concentrations (0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0 or 5.0 mM) and the MTT and BCA assays were used to evaluate the chemical effects on cell viability and total protein concentration, respectively. The test chemical was added to each applicable well, and in the absence or presence of S9 liver microsomal fraction, to give a final concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM. Negative controls, solvent/vehicle controls and positive control substance(s) were also included in each experiment. The results show evidence of cytotoxicity when CHO cells are exposed to the test chemical in the concentration of 5.0 mM in the presence of S9-induced metabolic activation, whereas exposure to 0.5 or 1 mM does not induce cytotoxicity. In addition, only a very mild cytotoxicity was shown at a concentration of 2.5 mM. Thus, the results indicate that the induced cytotoxicity may be induced by an alternative cellular route, e.g. impaired mitochondrial respiration, since the test chemical does not give rise to gene mutation(s) at 0, 0.5, 1.0, 2.5 or 5.0 mM. In similarity the evidence of cytotoxicity when CHO cells are exposed to the test chemical in the concentration of 5 mM in the absence of S9-induced metabolic activation, while exposure to 0.5, 1.0 or 2.5 mM does not induce cytotoxicity. Thus, we conclude that the test chemical does not induce cytotoxic effects at concentrations < 2.5 mM. Based on the results from the MTT and BCA assays, revised test concentrations of the chemical may be chosen to be included in the gene toxicity test. Test chemical concentrations at 0.1 mM and higher showed a clear evidence of being cytotoxic when analyzed with MTT. Since cytotoxicity was observed in the MTT assay from 0.1 mM, further testing concentrations were revised and adapted to have test concentrations of 0, 0.5, 1.0, 2.5 and 5.0 mM when cells should be exposed to the test chemical for 3 hrs.

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 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. At time of exposure, the test chemical was added in the absence or presence of S9 liver microsomal fraction. The test chemical was added to each applicable well to give a final concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM. Negative controls, solvent/vehicle controls and positive control substance(s) were also included in each experiment. pH and osmolality was not determined in the gene mutation test.

The results showed a clear indication of gene mutations only in the positive control ENU. No other treatment showed evidence of gene mutations. The results also showed evidence of cytotoxicity when CHO cells were exposed to the test chemical in the concentration of 5.0 mM and in the absence or presence of S9-induced metabolic activation, whereas exposure to 0.5 or 1 mM does not induce cytotoxicity. A very mild cytotoxicity was shown at a concentration of 2.5 mM in the presence of S9-induced metabolic activation. Thus, the results indicate that the induced cytotoxicity may be induced by an alternative cellular route, e.g. impaired mitochondrial respiration. Since, the test chemical does not give rise to gene mutation(s) when CHO cells are exposed in vitro to the test chemical at 0, 0.5, 1.0, 2.5 or 5.0 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, 0.5, 1.0, 2.5 or 5.0 mM for 3 hrs, in the presence or absence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

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

 

AMES test:

The study was performed to investigate the potential of the test chemical to induce gene mutations in comparison to negative control according to plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, TA102. The study was conducted as per OECD 471, Adopted July 21, 1997. The assay was performed in two independent experiments both with and without liver microsomal activation, each concentration, including the negative and positive controls was tested in triplicates. Based on the solubility and precipitation test result eight different concentrations 0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment. The pre-experiment was performed with TA100 and TA98 strain of salmonella typhimurium. In TA98 and TA100 there was no reduction in colony count but reduction in background lawn was observed in treated concentrations 5 (T8) and 1.582 (T7) mg/plate and no reduction in colony count as well as in background lawn in treated concentration 0.501 (T6) mg/plate – 0.002 (T1) mg/plate both in absence and in presence of metabolic activation. Based on the results, 0.501 mg/plate was selected as the highest dose for the main study trials both in the absence as well as in the presence of metabolic activation. Trial-I was performed with five concentration of test item along with the negative and concurrent positive control with the remaining three strains i.e. TA 1537, TA1535, TA 102 by plate incorporation method. For TA 98 and TA 100 revertant colony count were directly incorporated in the Trial-I from the pre-experiment up to the five concentrations [T2 (0.005 mg/plate) to T6 (0.501 mg/plate)]. Test item concentration 0.0 (NC), 0.005, 0.016, 0.050, 0.158 and 0.501mg/plate, were prepared with half log interval both in presence and in absence of metabolic activation. The plates were treated and incubated at 37±2⁰C for 48 hours. No increase in the revertant count in any of the five strains was reported at any of the test concentrations. Positive controls resulted in an unequivocal response in all the five tester strains compared to respective control used. Trial II was performed independently with all the five tester strains along with the negative and positive controls by pre-incubation method for the confirmation of the Trial-I results. The concentration 0.0 (NC), 0.005, 0.016, 0.050, 0.158 and 0.501 mg/plate were used both in the absence (-S9) as well as in the presence of metabolic activation (+S9). The concentration of positive controls used was same as used in the plate incorporation assay. In Trial II, the test item, negative and positive controls were pre-incubated along with 500µl of metabolic activation mix (+S9)/Buffer (-S9) and 100µl of bacterial culture for 60 minutes at 37±2^oC in an incubator. After pre-incubation, 2 mL of top agar was mixed with the pre-incubation mixture and poured on minimal glucose agar plates. The treaed plates were incubated for 48 hours in an incubator. No substantial increase in the revertant count was observed in any of the five tester strains pre-incubated with the test item. The positive controls showed an unequivocal increase in revertant counts with all the five tester strains and the respective controls used, hence confirming the non-mutagenic activity of the test item. Based on the results of this study, it is concluded that the test chemical was found to be non-mutagenic as it did not induce (point) gene mutations in the histidine operon by base-pair changes or frameshifts, in the presence or the absence of metabolic activation system, in any of the five tester strains of Salmonella typhimurium (TA1537, TA1535, TA98, TA100 and TA102). Hence, the test chemical is not likely to be classified as a gene mutant as per the criteria mentioned in the CLP regulation.

 

In vitro Mammalian chromosomal aberration test:

The in vitro mammalian chromosomal aberration study was conducted to determine the chromosomal aberration induction potential of test chemical in human peripheral blood lymphocyte cultures. The methods followed were as per OECD guideline No. 473, adopted on 29th July 2016. 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 hours mitogenic stimulation. The cells were treated with metaphase arresting substance (colcemid) 3 hours prior to harvesting and stained. The slides were analysed with the aid of microscope and a minimum number of 1000 of cells per culture were counted and number of metaphases were recorded in different fields to determine the mitotic index. The number of aberrant cells with aberrations were recorded (150 cells per slide) to calculate percent aberrant cells. Based on the solubility and precipitation test, Dimethyl sulfoxide (DMSO) was selected as the vehicle for treatment. The pH of test item in culture medium was assessed at 0 hour and 4 hours after incubation at 37°C. No significant change in pH was observed at 0 hour and 4 hours when compared with negative control. Hence, 2.0 mg/mL was selected as the highest concentration for cytotoxicity test. Before conducting the chromosomal aberration study, test chemical was evaluated for cytotoxicity both in the absence and presence of metabolic activation system (1%). Cytotoxicity was assessed at the concentrations of 0.0 (NC), 0.0 (VC), 0.5 (T1), 1.0 (T2) and 2.0 (T3) mg/mL of culture media. Cytotoxicity was observed in the treated concentration of 2.0 (T3) mg/mL both in the absence and in the presence of metabolic activation (1%). In the absence of S9 mix, the mean mitotic index observed was 10.12 (NC), 9.94 (VC), 8.39(T1), 6.59 (T2), 4.85 (T3) and 8.40 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.10 (NC), 9.94(VC), 8.24 (T1), 6.40 (T2), 4.75 (T3) and 8.49 (PC). The test concentration 2.0 (T3) mg/ mL showed more than 50% reduction in the mitotic index in treated culture both in the presence or absence of metabolic activation. Hence the concentration [0.25, 0.5 and 1.0 mg/mL] were selected for the main study. Hence, 1.0 mg/mL of culture media was selected as the highest concentration for main study both in the presence and in the absence of metabolic activation. The main study was performed in two independent phases: In Phase I experiment, the cultures were exposed to the test chemical for a short period of time (4 hours) both in the absence and in the presence of metabolic activation system (1%).The mean percentage of aberrant cells was 0.333 (NC), 0.333 (VC), 0.333 (T1), 0.333 (T2), 0.667 (T3) and 10.000 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (VC), 0.333 (T1), 0.667 (T2), 0.333 (T3), 10.333 (PC) and 10.333 (PC) in the presence of metabolic activation at the concentration of 0.0 (NC), 0.0 (VC) 0.25 (T1), 0.5 (T2) and 1.0 (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 Cyclophosphamide monohydrate at the concentration of 30 μg/mL in the presence of metabolic activation (1%) caused significant increases in percent aberrant cells. Even though the analysis did not reveal any statistical significance, the increases were biologically significant. During the treatment with the test item both in the absence and presence of S9 mix, there was no reduction in mitotic index observed at the tested concentrations. The observed mean mitotic index in the absence of metabolic activation were 10.18, 9.90, 8.88, 7.88, 6.64 and 8.40 and in the presence of metabolic activation were 10.13, 9.95, 9.03, 8.34, 6.78 and 8.49 for 0.0 (NC), 0.0 (VC) 0.25 (T1), 0.5 (T2) and 1.0 (T3) mg/mL and 30 μg/mL (PC) concentration. 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 absence of metabolic activation, test items concentrations used were 0.0 (NC), 0.0 (VC) 0.25 (T1), 0.5 (T2) and 1.0 (T3) mg/mL and 30μg/mL (PC). 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 was 24 hours. The mean percent aberrant cells were 0.333 (NC), 0.333 (VC) 0.333 (T1), 0.333(T2), 0.667 (T3) and 10.000 (PC) in the absence of metabolic activation and 0.333 (NC),0.333 (VC),0.333 (T1), 0.333 (T2), 0.667 (T3) and 10.333 (PC) in the presence of metabolic activation at the concentration of 0.0 (NC), 0.0 (VC) 0.25 (T1), 0.5 (T2) and 1.0 (T3) mg/mL of culture and positive control. Treatment with Ethyl methanesulfonate at the concentration of 600μg/mL in the absence of metabolic activation and Cyclophosphamide monohydrate at the concentration of 30μg/mL in the presence of metabolic activation (2%) caused significant increases in percent aberrant cells .Though the analysis did not reveal any statistical significance, the increases were biologically significant. The increased frequency of aberrations observed in the concurrent positive control groups (Phase I and II) validated the sensitivity of the test system, suitability of the methods and conditions employed in the experiment. Treatment with test item both 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 index in the absence of metabolic activation were 10.07, 9.94, 8.69, 7.84, 6.04 and 8.75 and in the presence of metabolic activation were 10.00, 9.94, 8.83, 8.10, 6.39and 8.64 for 0.0 (NC), 0.0 (VC) 0.25 (T1), 0.5 (T2) and 1.0 (T3) and 30μg/mL PC concentrations, respectively.

Hence from the above data it can be concluded that, the test chemical was found to be non-mutagenic in the in vitro mammalian chromosomal aberration study performed using human peripheral blood lymphocytes both in presence (1% and 2%) and in the absence of metabolic activation under the specified conditions. Hence it is not likely to be classified as a gene mutant as per the criteria mentioned in CLP regulation.

In vitro Mammalian cell gene mutation assay:

The study was performed to assess toxic and genotoxic effects of the test chemical on Chinese Hamster Ovary (CHO) cells by using several differentin vitro-based assays, including genotoxicity tests based on the OECD Guideline No. 476 “In VitroMammalian Cell Gene Mutation Test”. 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. All tests of the preliminary dose-finding study was completed without S9-induced metabolic activation. The test chemical was added to the cells in the test concentrations (0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0 or 5.0 mM) and the MTT and BCA assays were used to evaluate the chemical effects on cell viability and total protein concentration, respectively. The test chemical was added to each applicable well, and in the absence or presence of S9 liver microsomal fraction, to give a final concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM. Negative controls, solvent/vehicle controls and positive control substance(s) were also included in each experiment. The results show evidence of cytotoxicity when CHO cells are exposed to the test chemical in the concentration of 5.0 mM in the presence of S9-induced metabolic activation, whereas exposure to 0.5 or 1 mM does not induce cytotoxicity. In addition, only a very mild cytotoxicity was shown at a concentration of 2.5 mM. Thus, the results indicate that the induced cytotoxicity may be induced by an alternative cellular route, e.g. impaired mitochondrial respiration, since the test chemical does not give rise to gene mutation(s) at 0, 0.5, 1.0, 2.5 or 5.0 mM. In similarity the evidence of cytotoxicity when CHO cells are exposed to the test chemical in the concentration of 5 mM in the absence of S9-induced metabolic activation, while exposure to 0.5, 1.0 or 2.5 mM does not induce cytotoxicity. Thus, we conclude that the test chemical does not induce cytotoxic effects at concentrations < 2.5 mM. Based on the results from the MTT and BCA assays, revised test concentrations of the chemical may be chosen to be included in the gene toxicity test. Test chemical concentrations at 0.1 mM and higher showed a clear evidence of being cytotoxic when analyzed with MTT. Since cytotoxicity was observed in the MTT assay from 0.1 mM, further testing concentrations were revised and adapted to have test concentrations of 0, 0.5, 1.0, 2.5 and 5.0 mM when cells should be exposed to the test chemical for 3 hrs. 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 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. At time of exposure, the test chemical was added in the absence or presence of S9 liver microsomal fraction. The test chemical was added to each applicable well to give a final concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM. Negative controls, solvent/vehicle controls and positive control substance(s) were also included in each experiment. pH and osmolality was not determined in the gene mutation test. The results showed a clear indication of gene mutations only in the positive control ENU. No other treatment showed evidence of gene mutations. The results also showed evidence of cytotoxicity when CHO cells were exposed to the test chemical in the concentration of 5.0 mM and in the absence or presence of S9-induced metabolic activation, whereas exposure to 0.5 or 1 mM does not induce cytotoxicity. A very mild cytotoxicity was shown at a concentration of 2.5 mM in the presence of S9-induced metabolic activation. Thus, the results indicate that the induced cytotoxicity may be induced by an alternative cellular route, e.g. impaired mitochondrial respiration. Since, the test chemical does not give rise to gene mutation(s) when CHO cells are exposedin vitroto the test chemical at 0, 0.5, 1.0, 2.5 or 5.0 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, 0.5, 1.0, 2.5 or 5.0 mM for 3 hrs, in the presence or absence of metabolic activation.

Based on the experimental data available for the target chemical, it can be concluded that the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro.

Justification for classification or non-classification

Based on the experimental data available for the target chemical, it can be concluded that the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro.