Ferrate(4-), hexakis(cyano-C)-, methylated 4-[(4-aminophenyl)(4-imino-2,5-cyclohexadien-1-ylidene)methyl]benzenamine copper(2+) salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Assay:

Ames assay 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 the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. It was 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 and hence it is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.

In vitro chromosomal aberration study

This study was conducted to determine the chromosomal aberration induction potential of the test chemical in human peripheral blood lymphocyte cultures. The methods followed were as per OECD guideline No. 473, adopted on 29thJuly 2016 “ In Vitro Mammalian Chromosome Aberration Test. The experiment was conducted using human peripheral blood lymphocytes.The test chemical is non-clastogenic at the highest tested concentration of 0.062 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 vitro mammalian cell gene mutation assay

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the test chemical when administered to Chinese Hamster Ovary (CHO) cells. The study was performed as per OECD 476 Guidelines.Based on the results of the current study, it was concluded that the test chemical give rise to gene mutations when exposed to the test chemical at concentrations of ≥ 0.05 mM for 3 hrs or more. Also, the test chemical does not give rise to cytotoxicity in CHO cells at concentrations of ≤ 0.05 mM. Hence, the test chemical can be considered to be non mutagenic to CHO cells.

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:

16 October 2020 to 12 November 2020

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 study was performed to investigate the potential of test chemical to induce gene mutations in comparison to vehicle control according to the plate incorporation method (Trial I) and the pre-incubation method (Trial II) both in presence and in absence of metabolic activation system, using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

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):

Nnot applicable

Cytokinesis block (if used):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Aroclor 1254-induced S9 metabolic activation system.
- source of S9 : Aroclor 1254-induced S9 was procured from the Defence Research and Development Organization
- method of preparation of S9 mix : An appropriate quantity of S9 supernatant is thawed and mixed with S9 cofactor solution to result in a final concentration of approximately 10 % v/v in the S9 mix. Cofactor solution contains the following quantity of chemicals in 500 mL of Distilled Water.
D-glucose-6-phosphate 0.8 g
β-NADP 1.75 g
MgCl2 1.0 g
KCl 1.35 g
Na2HPO4.H2O 6.4 g
NaH2PO4.H2O 1.4 g
During the experiment, the S9 mix was prepared freshly and used.

- concentration or volume of S9 mix and S9 in the final culture medium : 10 % v/v in the S9 mix
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No data available.

Test concentrations with justification for top dose:

Based on the results of pre-experiment, the following doses were selected for the main study Trial I and Trial II:
0.0 (NC), 0.0(VC), 0.004, 0.012, 0.038, 0.119 and 0.376 mg/plate, both in the absence (-S9) and presence of metabolic activation (+S9).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was found to be soluble at 50 mg/mL to give the final treatment concentration of 5 mg/plate. Hence, DMSO was chosen as a solvent for the study.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Negative Control: Distilled Water Solvent control: DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-Nitro-o-phenylenediamine [Without metabolic activation (For Strains TA 1537, TA 98)] 2-Aminoanthracene [With metabolic activation (For Strains TA 1535, TA 1537, TA 98, TA 100 and TA 102)]

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 was performed according to plate incorporation method and Trial II was performed according to the pre-incubation method.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Not applicable
- Test substance added in agar (plate incorporation Trial I); preincubation (Trial II)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: For 60 minutes at 37 °C
- Exposure duration/duration of treatment: For 48 hours at 37 °C.
- Harvest time after the end of treatment (sampling/recovery times): No data

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: Bacerial background growth inhibition
- Any supplementary information relevant to cytotoxicity: No data

METHODS FOR MEASUREMENTS OF GENOTOXICIY : For each strain and dose level, including the controls, three plates (triplicate) were used. The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µL Test solution at each dose level, negative control, vehicle control, and reference mutagen solution (positive control),
500 µL S9 mix (for the test with metabolic activation) or S9 mix substitution buffer (for the test without metabolic activation),
100 µL Bacterial suspension,
2000 µL Overlay agar
In the pre-incubation assay, 100 µL test solution, 500 µL S9 mix and S9 mix substitution buffer and 100 µL bacterial suspensions were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation, 2.0 mL overlay agar (47 °C) was added to each tube. The mixture was poured on minimal agar plates. After solidification, the plates were incubated in an inverted position for 48 hours at 37 °C

- 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 of biological significance was 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 above the threshold of biological significance was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative and vehicle control, the increase was not considered biologically relevant.

Statistics:

Microsoft Office Excel-based calculation was used for descriptive statistical analysis.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 0.376 mg/plate (T6): reduction in colony count and moderate inhibition of back ground lawn was observed both in the absence (-S9) and in presence (+S9) of metabolic activation.

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: Insoluble in water
- Precipitation and time of the determination: Different amounts of formulated test item preparation (50 mg/mL) were added to overlay agar (top agar) in test tubes to give various test item concentration of (maximum 5 mg/plate) and plated on minimal glucose agar (MGA) plates. Precipitation was noticed at 5 mg/plate concentration which was assumed to interfere with the scoring. At treatment concentration, 3.750 mg/plate slight precipitation was observed, which was assumed to be non-interfering with the scoring.
- 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 100 and TA 98 strain of Salmonella typhimurium and with eight concentrations spaced by (√10) half-log intervals in triplicates. Based on the solubility and precipitation test, 3.750 mg/plate was selected as the highest dose in the pre-experiment. The following doses were selected for the pre-experiment: 0.0 (NC), 0.0(VC), 0.001, 0.004, 0.012, 0.038, 0.119, 0.376, 1.187 and 3.750 mg/plate. In TA 98 and TA 100 tester strains, the complete inhibition of background lawn and the absence of revertant colonies were observed at 3.750 and 1.187 mg/plate (T8-T7) both in the presence and absence of metabolic activation. Whereas, at 0.376 mg/plate (T6) the, reduction in colony count and moderate inhibition of back ground lawn was observed both in the absence (-S9) and presence (+S9) of metabolic activation. No reduction in colony count and diminution of the background lawn were observed at 0.119 – 0.001 mg/plate (T5-T1) neither in the absence (-S9) nor the presence (+S9) of metabolic activation. Based on the results of the pre-experiment, 0.376 mg/plate was selected as the highest dose for the main study trials both in the absence and presence of metabolic activation.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The spontaneous reversion rates in the negative, vehicle, and positive controls were within the range of in-house historical data range. The positive controls showed distinct increases in the number of revertant colonies in all the tester strains both in the presence and absence of metabolic activation, thus confirming the validity of the assay.

Ames test:
- Signs of toxicity : Increase in the revertant colony count
- Individual plate counts : Please refer the the table attached in remark section.
- 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 of Metabolic Activation (+S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	4.00	1.00	12.00	3.00	18.33	4.93	112.67	12.06	241.33	8.33
VC (0.00)	5.67	0.58	16.33	1.53	22.67	4.16	123.33	5.03	265.33	8.33
T1 (0.004)	5.00	1.00	13.67	2.52	19.67	4.51	120.67	7.02	249.33	12.22
T2 (0.012)	5.33	0.58	14.67	1.53	18.67	3.06	116.00	9.85	254.67	14.05
T3 (0.038)	4.33	0.58	15.00	2.00	19.00	3.00	117.33	7.02	244.00	12.00
T4 (0.119)	4.67	0.58	14.00	2.00	21.00	3.61	118.00	5.29	252.00	12.00
T5 (0.376)	0.00	0.00	0.00	0.00	7.33	2.89	28.00	8.19	64.67	10.07
PC	182.67	32.08	416.00	71.11	1234.67	54.45	1221.33	120.36	1472.00	178.12

Dose (mg/plate)	In the Absence of Metabolic Activation (-S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	3.67	0.58	11.33	1.53	16.00	2.00	110.00	11.14	236.00	12.00
VC (0.00)	5.33	1.53	14.00	2.00	20.33	3.51	119.33	7.57	260.00	16.00
T1 (0.004)	4.33	0.58	13.33	1.53	18.33	4.16	118.00	4.00	241.33	20.53
T2 (0.012)	4.67	1.15	12.67	2.52	19.00	4.58	117.33	8.08	237.33	10.07
T3 (0.038)	4.00	1.00	11.67	2.08	17.67	3.79	114.00	8.00	248.00	12.00
T4 (0.119)	5.00	1.00	13.00	2.00	18.67	4.04	115.33	5.03	240.00	8.00
T5 (0.376)	0.00	0.00	0.00	0.00	4.00	1.00	30.33	4.51	62.33	4.93
PC	190.67	32.08	1188.00	127.00	970.67	80.53	1109.33	117.12	1672.00	186.03

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

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100                  

Methyl methanesulfonate [4μl/plate]: TA 102

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]

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

Dose (mg/plate)	Presence of Metabolic Activation (+S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	4.67	0.58	12.33	1.53	18.00	3.00	114.00	8.00	240.00	10.58
VC (0.00)	6.00	1.00	16.00	1.00	24.00	1.73	122.67	11.37	262.67	8.33
T1 (0.004)	5.00	0.00	13.33	2.08	21.00	2.65	116.67	7.02	249.33	8.33
T2 (0.012)	4.67	0.58	14.00	2.00	19.67	3.21	117.33	10.07	246.67	12.22
T3 (0.038)	5.67	0.58	15.00	1.00	18.33	1.53	114.67	8.33	244.00	10.58
T4 (0.119)	5.33	0.58	14.33	2.08	20.00	1.00	115.33	9.45	246.00	15.62
T5 (0.376)	0.00	0.00	0.00	0.00	2.67	1.53	27.00	5.00	68.00	4.00
PC	172.00	24.00	432.00	140.63	1304.00	150.04	1300.00	170.27	1228.00	159.80

Dose (mg/plate)	Absence of Metabolic Activation (-S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	4.00	0.00	11.33	2.08	17.67	2.08	110.67	7.02	236.00	10.58
VC (0.00)	5.67	0.58	14.00	1.00	22.00	2.00	121.33	9.02	252.00	12.00
T1 (0.004)	4.67	0.58	13.00	1.00	19.33	2.08	115.33	10.07	248.00	12.00
T2 (0.012)	5.00	1.00	13.33	2.08	20.67	2.52	114.00	7.21	244.00	10.58
T3 (0.038)	5.33	0.58	12.33	2.08	18.67	1.53	113.33	9.87	245.33	6.11
T4 (0.119)	4.33	0.58	12.67	2.08	19.00	2.00	114.67	8.33	246.67	10.07
T5 (0.376)	0.00	0.00	0.00	0.00	2.67	1.15	22.33	3.21	57.33	8.74
PC	182.67	22.74	1296.00	153.21	736.00	126.05	1248.00	141.48	1384.00	136.47

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

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100

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 in the histidine operon by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system as per the study performed by Ames assay (OECD guideline no. 471).And hence it is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.

Executive summary:

Ames test as per the OECD guideline No. 471 (Adopted: July 21, 1997, Corrected: June 26, 2020) was performed to investigate the potential of test chemical to induce gene mutations in comparison to vehicle control according to the plate incorporation method (Trial I) and the pre-incubation method (Trial II) using the Salmonella typhimurium strains 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, were tested in triplicate. The pre-experiment was performed with TA 100 and TA 98 strain of Salmonella typhimurium and with eight concentrations spaced by (√10) half-log intervals in triplicates. Based on the solubility and precipitation test, 3.750 mg/plate was selected as the highest dose in the pre-experiment. The concentration 0.0 (NC), 0.0(VC), 0.001, 0.004, 0.012, 0.038, 0.119, 0.376, 1.187 and 3.750 mg/plate doses were selected for the pre-experiment. In TA 98 and TA 100 tester strains, the complete inhibition of background lawn and the absence of revertant colonies were observed at 3.750 and 1.187 mg/plate (T8-T7) both in the presence and absence of metabolic activation. Whereas, at 0.376 mg/plate (T6) the reduction in colony count and moderate inhibition of background lawn was observed both in the absence (-S9) and presence (+S9) of metabolic activation. No reduction in colony count and diminution of the background lawn were observed at 0.119 – 0.001 mg/plate (T5-T1) neither in the absence (-S9) nor the presence (+S9) of metabolic activation. Based on the results of the pre-experiment, 0.376 mg/plate was selected as the highest dose for the main study trials both in the absence and presence of metabolic activation.

Trial-I was performed with five concentrations of the test item along with the negative, vehicle, and concurrent positive controls with the remaining three strains, i.e., TA 1537, TA1535, and TA 102 by the plate incorporation method. For TA 98 and TA 100 tester strains, the revertant colony counts were directly incorporated in the Trial-I from the pre-experiment up to the five concentrations [T2 (0.004 mg/plate) to T6 (0.376 mg/plate)]. The 0.0 (NC), 0.0 (VC), 0.004, 0.012, 0.038, 0.119 and 0.376 mg/plate concentrations of test item were prepared using (√10) half-log intervals, both in the absence (-S9) and presence of metabolic activation (+S9). The plates were treated and incubated at 37°C for 48 hours (approximately). No biologically relevant increase in the revertant count in any of the five strains was observed at any concentrations tested.

Trial-II was performed independently with all the five tester strains along with the negative, vehicle, and positive controls by the pre-incubation method for the confirmation of the Trial-I results. The 0.0 (NC), 0.0 (VC), 0.004, 0.012, 0.038, 0.119 and 0.376 mg/plate concentrations of test item were prepared using (√10) half-log intervals,both in the absence (-S9) and the presence of metabolic activation (+S9) for Experiment -I. The concentration of positive controls used was the same as used in the plate incorporation assay. The test item, negative, vehicle and positive controls were pre-incubated along with 500 µL of metabolic activation mix (+S9) and Buffer (-S9) and 100µL of bacterial culture for 60 minutes at 37 °C 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 treated plates were incubated for 48 hours (approximately) in an incubator. No biologically relevant increase in the revertant count was observed in any of the five tester strains pre-incubated with the test item.The positive controls showed distinct increases in the number of revertant colonies in all the tester strains both in the presence and absence of metabolic activation, thus confirming the validity of the assay.The positive controls showed distinct increases in the number of revertant colonies in all the tester strains both in the presence and absence of metabolic activation, thus confirming the validity of the assay.

From the above data it can be concluded that, the test chemical did not induce gene mutations in the histidine operon by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system as per the study performed by Ames assay (OECD guideline no. 471) and hence it is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.

 

 

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

This in vitro assay was performed to assess the potential of the 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: human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Human blood
- Suitability of cells: No data
- Cell cycle length, doubling time or proliferation index:
- Sex, age and number of blood donors if applicable:Age: 25-30 years age
- Whether whole blood or separated lymphocytes were used if applicable: Separated lymphocytes were used
- Number of passages if applicable: No data
- Methods for maintenance in cell culture if applicable: No data
- Modal number of chromosomes: No data
- Normal (negative control) cell cycle time: No data

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Blood cultures were set up in medium containing RPMI-1640, Fetal Bovine Serum, Phytohaemagglutinin, Heparin solution, Whole Blood and Antibiotic Solution
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically 'cleansed' against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

S9 metabolic activation system

Test concentrations with justification for top dose:

0.0 (NC), 0.0 (VC) 0.016 (T1), 0.031 (T2) and 0.062 (T3) mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: No data
- Exposure duration: Phase 1: 4 hrs (with and without metabolic activation system)
Phase 2: 4 hrs (with metabolic activation system) and 24 hrs (without metabolic activation system)
- Expression time (cells in growth medium): Phase 1: 20 hrs (with and without metabolic activation system)
Phase 2: 20 hrs (with metabolic activation system)
- Selection time (if incubation with a selection agent):No data
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hrs

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

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

NUMBER OF REPLICATIONS: No data

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

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

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

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Mitotic index
- Any supplementary information relevant to cytotoxicity: Cytotoxicity was assessed at the concentrations of 0, 0.062, 0.125 and 0.250 mg/mL of culture media

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

- OTHER: No data

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

If the above mentioned criteria for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Statistics:

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

Key result

Species / strain:

lymphocytes: Human perpheral blood lymphocytes

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
- Effects of pH: The pH of test item in culture medium was assessed at 0 h and 4 h after incubation at 37 ± 2 °C. Significant change in pH was not observed at 0 h and 4 h when compared with negative controls.
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: There was no precipitation observed at 0.0625 mg/mL concentration
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item a cytotoxicity assay was performed both in the presence and absence of metabolic activation system. 3 test concentrations (0, 0.062, 0.125 and 0.250 mg/mL of culture media) based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with vehicle control. The procedure for conducting cytotoxicity was the same as main experiment phase I up to the scoring of the mitotic index, except slide coding.On the basis of solubility, precipitation and effect on media pH of the test item, the maximum concentration 0.250 mg/mL was selected as the highest concentration for the treatment in cytotoxicity experiment. An experiment was performed to assess the effect of test item on the pH of media at 0 h and 4 h exposure. The pH of test item in culture medium was assessed after incubation at 37 °C.No Significant change in pH was observed at 0 h and 4 h when compared with negative controls. Based on this the highest dose was selected for cyto-toxicity experiment.Based on the results of cyto-toxicity experiment following doses were selected for the main study:0.016, 0.031 and 0.062 mg/mL culture media.

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

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

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

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data

Remarks on result:

other: No mutagenic potential

CYTOTOXIC EXPERIMENT      

Before conducting the chromosomal aberration study,the test chemical was evaluated for cytotoxicity both in the absence and presence of metabolic activation system (1%). Cytotoxicity was assessed at the concentrations of 0.0 (NC), 0.0 (VC), 0.062 (T1), 0.125 (T2) and 0.250 (T3) mg/mL of culture media. Cytotoxicity was observed in all treated concentration of 0.125 (T2) and 0.250 (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 10.08 (NC), 9.94 (VC), 7.22 (T1), 4.90 (T2), 3.50 (T3) and 8.34 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.10 (NC), 9.88 (VC), 7.56 (T1), 5.10 (T2), 3.68 (T3) and 8.42 (PC).In the cytotoxicity experiment, the highest test concentration 0.250 (T3) and 0.125 (T2) mg/ mLof treated culture mediashowed more than 50% reduction the mitotic index when compared to the respective vehicle control both in the presence or absence of metabolic activation confirms the cytotoxicity effect. Hence these concentration were not selected for the main study. Hence, 0.062 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;

MAIN STUDY

Phase I       

In the experiment, the cultures were exposed to the test chemical for a short period of time (4 h) both in the absence and in the presence of metabolic activation system (1%).The mean percentage of aberrant cells was 0.333 (NC), 0.333 (VC), 0.333 (T1), 0.667 (T2), 0.333 (T3) and 11.333 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (VC), 0.333 (T1), 0.000(T2), 0.333 (T3) and 8.667 (PC)in the presence of metabolic activation at the concentration of 0.0 (NC), 0.0 (VC) 0.016 (T1), 0.031 (T2) and 0.062 (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%) causedsignificant increase in percent aberrant cells.Even though the analysis did not reveal any statistical significance, the increase was biologically significant. During the treatment with test item in the absence and presence of S9 mix, there was noreduction in mitotic index observed at the tested concentrations.The observed mean mitotic indexin the absence of metabolic activation were 10.04, 10.00, 8.84, 7.95, 7.15 and 8.24 andin the presence of metabolic activation were 10.19, 9.89, 8.95, 8.20, 7.44 and 8.55 for0.0 (NC), 0.0 (VC) 0.016 (T1), 0.031 (T2) and 0.062 (T3) mg/mLand 30 µg/mL(PC)concentrations respectively.

 

PHASE II         

The phase II experiment was performed to confirm the negative results obtained in the absence and in the presence of metabolic activation in Phase I. In the Phase II, test item concentrations used were   0.0 (NC), 0.0 (VC) 0.016 (T1), 0.031 (T2) and 0.062 (T3) mg/mLand 30µg/mL(PC)culture both in absence and presence of metabolic activation (2%). The duration of exposure to the test item in presence of metabolic activation system was 4 hours and in absence of metabolic activation the duration of exposure was 24 hours. The mean percent aberrant cells were 0.333 (NC), 0.333 (VC) 0.333 (T1), 0.333 (T2), 0.333 (T3) and 10.667 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (VC), 0.667 (T1), 0.333 (T2), 0.333 (T3) and 10.000 (PC) in the presence of metabolic activation at the concentration of 0.0 (NC), 0.0 (VC) 0.016 (T1), 0.031 (T2) and 0.062 (T3)  mg/mL of culture and positive control, respectively.Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamidemonohydrate at the concentration of 30 µg/mL in the presence of metabolic activation (2%) causedsignificant increase in percent aberrant cells.Though the analysis did not reveal any statistical significance, the increase was biologically significant.The increased frequency of aberrations observed in the concurrent positive control groups (Phase I and II) demonstrated the sensitivity of the test system, suitability of the methods and conditions employed in the experiment.Treatment with test item in the absence and presence of S9 mix, there was noreduction in mitotic index was observed at the tested concentrations. The observed mean mitotic indexin the absence of metabolic activation were 10.13, 9.95, 8.95, 8.24, 7.25 and 8.25 andin the presence ofmetabolic activation were 10.08, 9.99, 8.95, 8.50, 7.63 and 8.60 for 0.0 (NC), 0.0 (VC) 0.016 (T1), 0.031 (T2) and 0.062 (T3) and 30 µg/mL(PC) concentrations respectively.

Conclusions:

The test chemical is non-clastogenic at the highest tested concentration of 0.062 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.

Executive summary:

This study was conducted to determine the chromosomal aberration induction potential of the test chemical in human peripheral blood lymphocyte cultures. The methods followed were as per OECD guideline No. 473, adopted on 29^thJuly 2016 “ In Vitro Mammalian Chromosome Aberration Test. The experiment was conducted using human peripheral blood lymphocytes. Blood was drawn from a healthy volunteer, by venous puncture using heparinised syringe. The experiment was performed both in the presence and in the absence of metabolic activation system after 48 h mitogenic stimulation. The test chemical was dissolved in DMSO and used at dose level of 0.0 (NC), 0.0 (VC) 0.016 (T1), 0.031 (T2) and 0.062 (T3) mg/mL in the presence and absence of S9 metabolic activation system in phase 1 and phase 2. Phase I of experiment was performed by short term treatment method both in the presence and absence of metabolic activation system (1%). Phase II of experiment was performed by short term treatment as well as long term treatment method. Long term treatment was performed in absence of metabolic activation to confirm the negative results obtained in the absence of metabolic activation in Phase I. Short term treatment method was performed with increased metabolic activation (2%) condition to confirm the negative results obtained in the presence of metabolic activation in Phase I. The doses for the main study were based on the cytotoxicity study conducted both in the presence and absence of metabolic activation system. 3 test concentrations (0,0.062, 0.125 and 0.250 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 negative control. The medium of the proliferating blood culture was removed by centrifugation at 1500 rpm for 10 minutes. The cells were suspended in plain medium (medium without serum) mixed with S9 mix (Phase I - 1 % and Phase II - 2 % v/v) and in complete media mixed with phosphate buffer for the treatment in presence and in absence of metabolic activation system respectively. A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks. Each tube/flask according to treatment groups was identified. Negative control tubes were treated with 80 µL of RPMI media and treatment group were treated with 80 µL of respective test item stock solution. The cultures were incubated at 37 ± 2 °C for duration (exposure period). For Phase I, after incubation cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in Phosphate Buffer Saline (PBS). The washing procedure was repeated once again. After washing the cells were re-suspended in complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37 ± 2 °C for 1.5 normal cell cycle lengths (22 - 25 hours). The cultures were harvested at the end of incubation of 24 hours after treatment. Before 3 hours of harvesting, 240 µL of colcemid (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each of the culture tube, and kept under incubation at 37 ± 2 °C. The cultures were harvested 24 hours after beginning of treatment by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded and the cells were re-suspended in 7 mL of freshly prepared, pre-warmed (37 ± 2 °C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37 ± 2 °C for 30 minutes in water bath. After hypotonic treatment, the culture was centrifuged and supernatant was removed. After that 5 mL of freshly prepared, chilled Carnoy’s fixative (3:1 methanol: acetic acid solution) was added and left for 5 min. The cells were collected by centrifugation and washed twice with Carnoy’s fixative. After the final centrifugation, the supernatant was removed completely, and the cell pellet resuspended in 0.5 mL of Carnoy’s fixative. The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The slides were dried over a slide warmer and labelled. At least two slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation. 300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pulverization, polyploidy (including endo-reduplication) and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. Only metaphases with 46± 2 centromere regions were included in the analysis. The test chemical is non-clastogenic at the highest tested concentration of 0.062 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.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

Mammalian cell gene mutation study was performed to determine the mutagenic nature of the test chemical

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

Stability Data:The test chemical was dissolved in 99.5% ethanol and this freshly made stock solution was stored at -20°C until day of testing. On this day, the stock solution was thawed and the appropriate volume of the test chemical was taken for further dilutions in PBS or in cell culture medium (depending on assay used). The stock solution was then immediately returned to the freezer. No changes in chemical stability was observed since the chemical did not precipitate upon thawing.
Storage Condition:At room temperature (RT) in ventilated cabinets.

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (HGPRT)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: European Collection of Cell Cultures (ECACC), Public Health England, Porton Down, Salisbury, United Kingdom
- Suitability of cells:
- Normal cell cycle time (negative control):

For cell lines:
- Absence of Mycoplasma contamination: No detection for mycoplasma was performed
- Number of passages if applicable: A subclone of the parental CHO cell line originated by Puck in 1975. The cells have an absolute requirement for L-proline
- Methods for maintenance in cell culture: Cell Culture Medium:Ham’s F12K (Kaighn’s) Medium including 2 mM Glutamine supplemented with 10% Foetal Bovine Serum (FBS; qualified, EU-approved, South America origin), penicillin (50 U/ml) and streptomycin (50 g/ml). All cell culture medium components were purchased from Gibco®, Life Technologies Corp., Carlsbad, CA, USA.
Medium For Cell Dissociation:Trypsin-EDTA (0.25%) with phenol red (Gibco®, Life Technologies Corp., Carlsbad, CA, USA).
- Periodically checked for karyotype stability: [yes/no] : No detection for karyotype stability was performed
- Periodically ‘cleansed’ of spontaneous mutants: [yes/no]

Additional strain / cell type characteristics:

other: Hypodiploid, modal No. 20

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats (Supplier: Molecular Toxicology Inc. via Trinova Biochem GmbH, Giessen, Germany).

Test concentrations with justification for top dose:

0, 0.005, 0.01, 0.025 or 0.05 mM

Vehicle / solvent:

Vehicle(s)/solvent(s) used: Ethanol
Justification for choice of solvent/ vehicle:
Justification of Ethanol as a solvent and vehicle:
1) The test chemical was not soluble in water or PBS, and therefore was ethanol chosen as a solvent instead.
2) When neither water nor PBS can be used, ethanol is a commonly used instead in biological applications as a solvent to dissolve test chemicals. Previous studies has shown that ethanol is neither genotoxic nor cytotoxicty when used at low to moderate treatment concentrations. Since we use a limit concentration of maximum 1% of ethanol in our studies, ethanol is regarded to be non-toxic, non-genotoxic and thus does not affect our results in the study.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

other: N-ethyl-N-nitrosourea (ENU) (2.5 mM) was used as positive control substance in experiments without metabolic activation

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)
6-Thioguanine (TG; Sigma-Aldrich Sweden AB, Stockholm, Sweden) was dissolved in 0.5% Na2CO3 solution to a stock solution of 2 mM. The solution is then sterile filtered and stored at -20°C.

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

Cells (0.5 x 10-5 per well) were seeded in fresh medium (2 ml per well) in 6 well-plates and were incubated overnight at 37°C, 5% CO2. On the day of exposure, the test chemical added to each applicable well to give a final concentrations of 0, 0.5, 1.0, 5.0 or 10.0 mM and in the absence or 4% (80 l) presence of S9-induced metabolic activation. Negative controls (cell medium), solvent/vehicle controls (PBS) and positive control substances were also included in each experiment. ENU was used as positive control substance in experiments without metabolic activation, while 7,12-dimethylbenz(a) anthracene was used in experiments with metabolic activation. During the exposure period, treated cultures were incubated at 37°C, 5% CO2.
After the 3 hour exposure period, cells were washed several times with sterile PBS to remove the test chemical. Cells were then trypsinized and re-seeded in duplicates from two parallel duplicate cultures into new 6-well plates, containing fresh medium, in the concentration of 0.5 x 10-5 cells per well. The cultures were then incubated for 24 and 48 hrs at 37°C, 5% CO2. The relative total growth and cytotoxicity was evaluated 24 and 48 hrs after seeding by counting in a haemocytometer.
A minimum of 2 replicates per dose concentration, including negative and positive control, were performed for each experiment.

OTHER: PRELIMINARY DOSE-FINDING/TOXICITY TEST

Initial tests of dose-finding was completed without S9-induced metabolic activation. A range of test concentrations (0, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5 or 1.0 mM) was applied at a volume of 20 l per applicable well 24 hrs after seeding to single cultures (4 x 104 cells per well) in fresh medium (180 l per well) in 96-well plates. The cell population (control and treated cells) were assessed 24 and 48 hrs after treatment using 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.
In the MTT assay, 20 l of MTT solution was added to each well and the 96-well plates were returned to the incubator for 1 hour. After 1 hour, the MTT solution was removed by gently pouring out the fluid from the plate onto a paper cloth. Each well was washed gently with two times of 100 l of PBS. The blue formazan product was dissolved by addition of 100 l of 100% DMSO per well. The plates were swirled gently for 10 min. to dissolve the precipitate. Absorbance was monitored at 540 nm using a SpectraMax M2 Microplate Reader and the software SoftMax Pro® (Molecular Devices, Sunnyvale, CA, USA).
After 24 and 48 hrs of treatment, the medium of the 96-well plates destined for analyzing protein content was removed by gently pouring out the fluid from the plate onto a paper cloth. Each well was washed gently with two times of 100 l of PBS before 50 l of Mammalian protein extraction reagent (M-PER) solution was added to each well. The plates were swirled gently overnight before 25 l of the cell lysate were transferred to a new 96-well plate together with 25 l bovine serum albumin (25 l of 5-750 g/ml in M-PER) as a standard and with 25 l of M-PER as a blank. A solution, containing 50 part of BCA Reagent A (containing sodium carbonate, sodium bicarbonate, bicinchoninic acid and sodium tartrate in 0.1 M sodium hydroxide) and 1 part of BCA Reagent B (containing 4% cupric sulfate), was added to each well and the plates were mixed thoroughly on a plate shaker for 30 sec. The plates were then covered and incubated for 30 min. at 37°C. After cooling the plate to RT, absorbance was measured at 562 nm using a Multiskan™ FC Microplate Photometer and the software ScanIt™ (Thermo Fisher Scientific, Waltham, MA, USA.
A minimum of 6 replicates per dose concentration from 3 independent cultures was performed for each experiment. Results obtained by the two cytotoxicity assays were expressed as percentage of controls. From the basis of the results from the MTT and BCA assays, test concentrations of the chemical was chosen to be included in the gene toxicity test

Rationale for test conditions:

No data available

Evaluation criteria:

The number of mutant colonies were counted and compared to the negative and positive controls. The mutation frequency was calculated.

Statistics:

The Standard deviation (± SD) was calculated.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test chemical resulted in cytotoxicity at concentrations of ≥ 0.025 mM after 24 or 48 hrs of treatment. Similar results on protein content were seen for the BCA assay

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES (if applicable): 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, 0.0005, 0.0025, 0.005, 0.025, 0.05, 0.25, 0.5 or 2.5 mM) and the MTT and BCA assays were used to evaluate the chemical effects on cell viability and total protein concentration, respectively. 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. pH and osmolality was not determined in the preliminary dose-finding/toxicity test. Exposure to the test chemical resulted in cytotoxicity at concentrations of ≥ 0.025 mM after 24 or 48 hrs of treatment. Similar results on protein content were seen for the BCA assay. Since cytotoxicity was observed in the MTT and BCA assay after 24 and 48 hrs of treatment in this preliminary dose-finding test, further testing concentrations were revised and adapted to have a maximum test concentration of 0.05 mM for further investigations.

Gene mutation 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, 0.005, 0.01, 0.025 or 0.05 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 positive control ENU showed indication of gene mutations as well as treatment with the test chemical at 0.05 mM in the absence or presence of S9 liver microsomal fraction, while no other treatment gave rise to gene toxicity. When the mutation frequency was determined, a frequency of 3.40 x 10-4 was shown after a 3 hour exposure of ENU and 1.52 x 10-2 for 0.05 mM of the test chemical, both in the absence of S9 liver microsomal fraction. A frequency of -4.33 x 10-4 was also shown after a 3 hour exposure to 0.05 mM of the test chemical in the presence of S9 liver microsomal fraction Thus, it was concluded that the test chemical may give rise to gene mutations at ≥ 0.05 mM after 3 hrs when CHO cells are exposed to the test chemical in the absence and presence of S9 liver microsomal fraction.

Cytotoxicity test
In similarity to the Gene mutation test, 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, 0.005, 0.01, 0.025 or 0.05 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.Treatment with the test chemical did not cause any cytotoxic effects in CHO cells. Thus, it was concluded that the test chemical does not induce cytotoxic effects in the absence or presence of S9 liver microsomal fraction independently of test chemical concentration.

 

Remarks on result:

other: No mutagenic potential

GENE MUTATION TEST

 

Table 1A.Effect of the 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	810/4	0/4	655/4
Pos. control	0/4	780/4	22/4	604/4
0.1 mM	0/4	632/4	0/4	678/4
0.25 mM	0/4	744/4	0/4	702/4
0.5 mM	0/4	316/4	0/4	707/4
1.0 mM	81/4	2/4	118/4	5/4

 

 

Table 1B.Mutation frequency in CHO cells after 3 hrs of exposure to the 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	3.40x10^-4
0.005 mM	N/A	N/A
0.01 mM	N/A	N/A
0.025 mM	N/A	N/A
0.05 mM	-4.33x10^-3	1.52x10^-2

PRELIMINARY DOSE-FINDING/TOXICITY TEST - SEEDING OF CELLS

 

Methods: MTT & BCA

Cell line: CHO

Passage: 8

Day of seeding: September 14, 2015

 

	Flask 1	Flask 2	Flask 3
Number of cells in cell culture flask (cells/ml)	1.95 x 10^6	2.33 x 10^6	2.36 x 10^6
Total number of cells in cell culture flask	8.60 x 10^6	10.48 x 10^6	10.62 x 10^6
Number of cells to be seeded per well (in 96-well plate)	4000	4000	4000
Dilution(s) made to create a cell suspension with 4000 cells per 180ml	0.82 ml cell suspension + 74.18 ml cell medium	0.69 ml cell suspension + 74.31 ml cell medium	0.68 ml cell suspension + 74.32 ml cell medium

 

PRELIMINARY DOSE-FINDING/TOXICITY TEST - EXPOSURE

Methods: MTT & BCA

Concentrations tested: 0 (vehicle control), 0.0005, 0.0025, 0.005, 0.025, 0.05, 0.25, 0.5 or 2.5 mM

Volume added to each well: 20ml

Day of treatment: September 15, 2015

 

Tested substance was added according to the following treatment schedule (with all concentrations in mM):

1 2 3 4 5 6 7 8 9 10 11 12 A Medium only 0 0 0.0005 0.0025 0.005 0.025 0.05 0.25 0.5 2.5 Medium only B Medium only 0 0 0.0005 0.0025 0.005 0.025 0.05 0.25 0.5 2.5 Medium only C Medium only 0 0 0.0005 0.0025 0.005 0.025 0.05 0.25 0.5 2.5 Medium only D Medium only 0 0 0.0005 0.0025 0.005 0.025 0.05 0.25 0.5 2.5 Medium only E Medium only 0 0 0.0005 0.0025 0.005 0.025 0.05 0.25 0.5 2.5 Medium only F Medium only 0 0 0.0005 0.0025 0.005 0.025 0.05 0.25 0.5 2.5 Medium only G Medium only 0 0 0.0005 0.0025 0.005 0.025 0.05 0.25 0.5 2.5 Medium only H Medium only 0 0 0.0005 0.0025 0.005 0.025 0.05 0.25 0.5 2.5 Medium only MAIN TEST/GENE TOXICITY TEST – SEEDING OF CELLS   CYTOTOXICITY & GENE TOXICITY Method: OECD 476 Application: Cytotoxicity & Gene toxicity Cell line: CHO Passage at experiment initiation: 14 Tested substance: Pigment Violet 27 Concentrations tested: 0 (vehicle control), 0.005, 0.01, 0.025 and 0.05 mM Day of seeding for HPRT mutation cleansing: October 26, 2015 Day of seeding prior chemical exposure: November 03, 2015 Day of chemical exposure: November 04, 2015     Flask 1 Flask 2 Number of cells in cell culture flask (cells/ml)   3.79 x 10^6   4.28 x 10^6 Total number of cells in cell culture flask   17.06 x 10^6   19.26 x 10^6 Number of cells to be seeded per well (for cytotoxicity)   2.0 x 10^5   2.0 x 10^5 Dilution(s) made to create a cell suspension with 2.0 x 10^5cells per 2 ml for cytotoxicity   792ml cell suspension + 14.21 ml cell medium   701ml cell suspension + 14.30 ml cell medium Number of cells to be seeded per well (for gene toxicity)   5.0 x 10^4   5.0 x 10^4 Dilution(s) made to create a cell suspension with 5.0 x 10^4cells per 2 ml for gene toxicity   198ml cell suspension + 14.80 ml cell medium     175ml cell suspension + 14.83 ml cell medium     MAIN TEST/GENE TOXICITY TEST – CYTOTOXICITY DATA   RE-SEEDING AFTER EXPOSURE Method: OECD 476 Application: Cytotoxicity Passage when re-seeding cells for cytotoxicity: 16 Concentrations tested: 0 (vehicle control), 0.005, 0.01, 0.025 and 0.05 mM Day of re-seeding cells for cytotoxicity testing: November 04, 2015 Number of cells to be re-seeded per well (24 h and 48 h): 5.0 x 10^4 Day for 24 h analysis of cytotoxicity: November 05, 2015 Day for 48 h analysis of cytotoxicity: November 06, 2015 Total number of cells (x 10^6) per well after 3 hrs of the test chemical exposure without or with metabolic activation (S9) and prior to re-seeding. A haemocytometer was used for identifying the total number of cells (x 10^6) per well:     Flask 1 Flask 2   Without S9 With S9 Without S9 With S9 Neg. control 0.440 0.270 0.313 0.243 Pos. control 0.680 0.223 0.207 0.290 0.005 mM 0.287 0.303 0.173^b 0.313 0.01 mM 0.283 0.143^a 0.240 0.297 0.025 mM 0.363 0.440 0.250 0.180c 0.05 mM 0.203 0.223 0.263 0.213 ^a)Since the number of cells was low in this sample, 750ml (corresponding to 35250 cells) was re-seeded to each well applicable for the upcoming 24 and 48 hour analysis. ^b)Since the number of cells was low in this sample, 750ml (corresponding to 43275 cells) was re-seeded to each well applicable for the upcoming 24 and 48 hour analysis. ^c)Since the number of cells was low in this sample, 750ml (corresponding to 45000 cells) was re-seeded to each well applicable for the upcoming 24 and 48 hour analysis.     CYTOTOXICTY AFTER 24 & 48 HOURS Method: OECD 476 Application: Cytotoxicity Concentrations tested: 0 (vehicle control), 0.005, 0.01, 0.025 and 0.05 mM Day for 24 h analysis of cytotoxicity: November 05, 2015 Day for 48 h analysis of cytotoxicity: November 06, 2015   Raw data of cytotoxicity at 24 hours after chemical exposure and re-seeding using haemocytometer for identifying the total number of cells (x 10^6) per well:   Without metabolic activation   Flask 1 Flask 2   Sample 1 Sample 2 Sample 1 Sample 2 Neg. control 0.133 0.097 0.150 0.143 Pos. control 0.097 0.067 0.123 0.137 0.005 mM 0.173 0.107 0.140 0.170 0.01 mM 0.143 0.140 0.107 0.090 0.025 mM 0.137 0.080 0.080 0.127 0.05 mM 0.097 0.063 0.053 0.053   With metabolic activation   Flask 1 Flask 2   Sample 1 Sample 2 Sample 1 Sample 2 Neg. control 0.206 0.097 0.097 0.113 Pos. control 0.183 0.163 0.160 0.070 0.005 mM 0.100 0.140 0.093 0.093 0.01 mM 0.107 0.150 0.130 0.093 0.025 mM 0.160 0.100 0.067 0.093 0.05 mM 0.117 0.083 0.057 0.067   Raw data of cytotoxicity at 48 hours after chemical exposure and re-seeding using haemocytometer for identifying the number of cells per well:   Without metabolic activation   Flask 1 Flask 2   Sample 1 Sample 2 Sample 1 Sample 2 Neg. control 0.290 0.337 0.373 0.500 Pos. control 0.137 0.173 0.300 0.340 0.005 mM 0.160 0.523 0.463 0.397 0.01 mM 0.333 0.387 0.510 0.463 0.025 mM 0.187 0.223 0.153 0.233 0.05 mM 0.107 0.090 0.080 0.087   With metabolic activation   Flask 1 Flask 2   Sample 1 Sample 2 Sample 1 Sample 2 Neg. control 0.316 0.440 0.397 0.387 Pos. control 0.360 0.510 0.390 0.430 0.005 mM 0.383 0.380 0.273 0.317 0.01 mM 0.280 0.487 0.410 0.317 0.025 mM 0.367 0.453 0.217 0.223 0.05 mM 0.117 0.060 0.073 0.093   MAIN TEST/GENE TOXICITY TEST – GENE TOXICITY DATA   RE-SEEDING AFTER EXPRESSION TIME Method: OECD 476 Application: Gene toxicity Expression time: 7 days Selection time (if incubation with a selection agent): 14 days Selection agent used: 6-Thioguanine (10mM) Passage when re-seeding cells for gene toxicity: 16 Concentrations tested: 0 (vehicle control), 0.005, 0.01, 0.025 and 0.05 mM Day of re-seeding cells for gene toxicity testing: November 11, 2015 Number of cells to be re-seeded per well with selection agent: 5.0 x 10^4 Number of cells to be re-seeded per well without selection agent: 50   Total number of cells (x 10^6) per well after 3 hrs ofPigment Violet 27 exposure without or with metabolic activation (S9) and prior to re-seeding. A haemocytometer was used for identifying the total number of cells (x 10^6) per well:     Flask 1 Flask 2   Without S9 With S9 Without S9 With S9 Neg. control 1.755 1.450 1.240 1.633 Pos. control 1.427 1.320 1.250 1.920 0.005 mM 2.180 1.107 1.725 1.688 0.01 mM 2.068 1.695 2.510 1.950 0.025 mM 0.370 0.123^b 0.127^d 0.023^f 0.05 mM 0.000^a 0.047^c 0.033^e 0,037^g ^a)Since the amount of cells seemed to be low or absent, 740ml of the cell suspension was re-seeded to the applicable wells that should contain 50000 cells while 20ml of the cell suspension was re-seeded to the applicable wells that should contain 50 cells. ^b)Since the number of cells was low in this sample, 740ml (corresponding to 30825 cells) was re-seeded to each well applicable for the upcoming 24 and 48 hour analysis. ^c)Since the number of cells was low in this sample, 740ml (corresponding to 11625 cells) was re-seeded to each well applicable for the upcoming 24 and 48 hour analysis. ^d)Since the number of cells was low in this sample, 740ml (corresponding to 31650 cells) was re-seeded to each well applicable for the upcoming 24 and 48 hour analysis. ^e)Since the number of cells was low in this sample, 740ml (corresponding to 8325 cells) was re-seeded to each well applicable for the upcoming 24 and 48 hour analysis. ^f)Since the number of cells was low in this sample, 740ml (corresponding to 5775 cells) was re-seeded to each well applicable for the upcoming 24 and 48 hour analysis. ^g)Since the number of cells was low in this sample, 740ml (corresponding to 9165 cells) was re-seeded to each well applicable for the upcoming 24 and 48 hour analysis.     STAINING AND COUNTING OF COLONIES Concentrations tested: 0 (vehicle control), 0.005, 0.01, 0.025 and 0.05 mM Number of days for incubation with/without selection agent: 14 Selection agent used: 6-Thioguanine (10mM) Day for staining with crystal violet: November 25, 2015 Day for counting colonies: December 01, 2015 Number of colonies at 14 days of incubation without or with 6-Thioguanine (TG):   Without metabolic activation and without TG   Flask 1 Flask 2   Sample 1 Sample 2 Sample 1 Sample 2 Neg. control 142 144 181 188 Pos. control 121 105 187 191 0.005 mM 201 186 146 145 0.01 mM 155 167 180 200 0.025 mM 196 156 190 165 0.05 mM 0 0 2 3   Without metabolic activation and with TG   Flask 1 Flask 2   Sample 1 Sample 2 Sample 1 Sample 2 Neg. control 0 0 0 0 Pos. control 1 4 10 7 0.005 mM 0 0 0 0 0.01 mM 0 0 0 0 0.025 mM 0 0 0 0 0.05 mM 55 54 0 9   With metabolic activation and without TG   Flask 1 Flask 2   Sample 1 Sample 2 Sample 1 Sample 2 Neg. control 166 180 257 207 Pos. control 175 195 218 192 0.005 mM 126 152 172 182 0.01 mM 154 189 199 202 0.025 mM 145 166 4 1 0.05 mM 0 0 2 0   With metabolic activation and with TG   Flask 1 Flask 2   Sample 1 Sample 2 Sample 1 Sample 2 Neg. control 0 0 0 0 Pos. control 0 0 0 0 0.005 mM 0 0 0 0 0.01 mM 0 0 0 0 0.025 mM 0 0 0 0 0.05 mM 0 80 1 0

Conclusions:

The test chemical does not give rise to gene mutations when exposed to the test chemical at ≤ 10.0 mM for 3 hrs or more, and the test chemical does not induce cytotoxic effects at concentrations ≤ 10.0 mM.Hence, the test chemical can be considered to be non mutagenic to CHO cells.

Executive summary:

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. The study was performed as per OECD 476 Guidelines. A preliminary dose-finding study was conducted prior to the main study. A range of different concentrations of the test chemical 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, 0.005, 0.01, 0.025 or 0.05 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 as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. The results showed indication of gene mutations occurring in the positive control ENU and when cells were exposed to 0.05 mM of the test chemical in the absence or presence of S9 liver microsomal fraction. No other treatment gave rise to gene toxicity. No cytotoxic effects was observed in CHO cells when exposed to the test chemical for 3 hrs. Based on the results of the current study, it was concluded that the test chemical give rise to gene mutations when exposed to the test chemical at concentrations of ≥ 0.05 mM for 3 hrs or more. Also, the test chemical does not give rise to cytotoxicity in CHO cells at concentrations of ≤ 0.05 mM. Hence, the test chemical can be considered to be non mutagenic to CHO cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Various studies have been reviewed to determine the in vitro mutagenic potential of the test chemical. The results are summarized below:

Ames Assay

Ames test as per the OECD guideline No. 471 (Adopted: July 21, 1997, Corrected: June 26, 2020) was performed to investigate the potential of test chemical to induce gene mutations in comparison to vehicle control according to the plate incorporation method (Trial I) and the pre-incubation method (Trial II) using the Salmonella 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, were tested in triplicate. The pre-experiment was performed with TA 100 and TA 98 strain ofSalmonella typhimurium and with eight concentrations spaced by (√10) half-log intervals in triplicates. Based on the solubility and precipitation test, 3.750 mg/plate was selected as the highest dose in the pre-experiment. The concentration 0.0 (NC), 0.0(VC), 0.001, 0.004, 0.012, 0.038, 0.119, 0.376, 1.187 and 3.750 mg/plate doses were selected for the pre-experiment. In TA 98 and TA 100 tester strains, the complete inhibition of background lawn and the absence of revertant colonies were observed at 3.750 and 1.187 mg/plate (T8-T7) both in the presence and absence of metabolic activation. Whereas, at 0.376 mg/plate (T6) the reduction in colony count and moderate inhibition of background lawn was observed both in the absence (-S9) and presence (+S9) of metabolic activation. No reduction in colony count and diminution of the background lawn were observed at 0.119 – 0.001 mg/plate (T5-T1) neither in the absence (-S9) nor the presence (+S9) of metabolic activation. Based on the results of the pre-experiment, 0.376 mg/plate was selected as the highest dose for the main study trials both in the absence and presence of metabolic activation.

Trial-I was performed with five concentrations of the test item along with the negative, vehicle, and concurrent positive controls with the remaining three strains, i.e., TA 1537, TA1535, and TA 102 by the plate incorporation method. For TA 98 and TA 100 tester strains, the revertant colony counts were directly incorporated in the Trial-I from the pre-experiment up to the five concentrations [T2 (0.004 mg/plate) to T6 (0.376 mg/plate)]. The 0.0 (NC), 0.0 (VC), 0.004, 0.012, 0.038, 0.119 and 0.376 mg/plate concentrations of test item were prepared using (√10) half-log intervals, both in the absence (-S9) and presence of metabolic activation (+S9). The plates were treated and incubated at 37°C for 48 hours (approximately). No biologically relevant increase in the revertant count in any of the five strains was observed at any concentrations tested.

Trial-II was performed independently with all the five tester strains along with the negative, vehicle, and positive controls by the pre-incubation method for the confirmation of the Trial-I results. The 0.0 (NC), 0.0 (VC), 0.004, 0.012, 0.038, 0.119 and 0.376 mg/plate concentrations of test item were prepared using (√10) half-log intervals,both in the absence (-S9) and the presence of metabolic activation (+S9) for Experiment -I. The concentration of positive controls used was the same as used in the plate incorporation assay. The test item, negative, vehicle and positive controls were pre-incubated along with 500 µL of metabolic activation mix (+S9) and Buffer (-S9) and 100µL of bacterial culture for 60 minutes at 37 °C 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 treated plates were incubated for 48 hours (approximately) in an incubator. No biologically relevant increase in the revertant count was observed in any of the five tester strains pre-incubated with the test item.The positive controls showed distinct increases in the number of revertant colonies in all the tester strains both in the presence and absence of metabolic activation, thus confirming the validity of the assay.The positive controls showed distinct increases in the number of revertant colonies in all the tester strains both in the presence and absence of metabolic activation, thus confirming the validity of the assay.

From the above data it can be concluded that, the test chemical did not induce gene mutations in the histidine operon by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system as per the study performed by Ames assay (OECD guideline no. 471) and hence it is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.

 

In vitro chromosomal aberration study

 

This study was conducted to determine the chromosomal aberration induction potential of the test chemical in human peripheral blood lymphocyte cultures. The methods followed were as per OECD guideline No. 473, adopted on 29th July 2016 “ In Vitro Mammalian Chromosome Aberration Test. The experiment was conducted using human peripheral blood lymphocytes. Blood was drawn from a healthy volunteer, by venous puncture using heparinised syringe. The experiment was performed both in the presence and in the absence of metabolic activation system after 48 h mitogenic stimulation. The test chemical was dissolved in DMSO and used at dose level of 0.0 (NC), 0.0 (VC) 0.016 (T1), 0.031 (T2) and 0.062 (T3) mg/mL in the presence and absence of S9 metabolic activation system in phase 1 and phase 2. Phase I of experiment was performed by short term treatment method both in the presence and absence of metabolic activation system (1%). Phase II of experiment was performed by short term treatment as well as long term treatment method. Long term treatment was performed in absence of metabolic activation to confirm the negative results obtained in the absence of metabolic activation in Phase I. Short term treatment method was performed with increased metabolic activation (2%) condition to confirm the negative results obtained in the presence of metabolic activation in Phase I. The doses for the main study were based on the cytotoxicity study conducted both in the presence and absence of metabolic activation system. 3 test concentrations (0.062, 0.125 and 0.250 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 negative control. The medium of the proliferating blood culture was removed by centrifugation at 1500 rpm for 10 minutes. The cells were suspended in plain medium (medium without serum) mixed with S9 mix (Phase I - 1 % and Phase II - 2 % v/v) and in complete media mixed with phosphate buffer for the treatment in presence and in absence of metabolic activation system respectively. A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks. Each tube/flask according to treatment groups was identified. Negative control tubes were treated with 80 µL of RPMI media and treatment group were treated with 80 µL of respective test item stock solution. The cultures were incubated at 37 ± 2 °C for duration (exposure period). For Phase I, after incubation cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in Phosphate Buffer Saline (PBS). The washing procedure was repeated once again. After washing the cells were re-suspended in complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37 ± 2 °C for 1.5 normal cell cycle lengths (22 - 25 hours). The cultures were harvested at the end of incubation of 24 hours after treatment. Before 3 hours of harvesting, 240 µL of colcemid (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each of the culture tube, and kept under incubation at 37 ± 2 °C. The cultures were harvested 24 hours after beginning of treatment by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded and the cells were re-suspended in 7 mL of freshly prepared, pre-warmed (37 ± 2 °C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37 ± 2 °C for 30 minutes in water bath. After hypotonic treatment, the culture was centrifuged and supernatant was removed. After that 5 mL of freshly prepared, chilled Carnoy’s fixative (3:1 methanol: acetic acid solution) was added and left for 5 min. The cells were collected by centrifugation and washed twice with Carnoy’s fixative. After the final centrifugation, the supernatant was removed completely, and the cell pellet resuspended in 0.5 mL of Carnoy’s fixative. The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The slides were dried over a slide warmer and labelled. At least two slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation. 300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pulverization, polyploidy (including endo-reduplication) and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. Only metaphases with 46± 2 centromere regions were included in the analysis. The test chemical is non-clastogenic at the highest tested concentration of 0.062 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 vitro mammalian cell gene mutation assay

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the test chemical when administered to Chinese Hamster Ovary (CHO) cells. The study was performed as per OECD 476 Guidelines. A preliminary dose-finding study was conducted prior to the main study. A range of different concentrations of the test chemical 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, 0.005, 0.01, 0.025 or 0.05 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 as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. The results showed indication of gene mutations occurring in the positive control ENU and when cells were exposed to 0.05 mM of the test chemical in the absence or presence of S9 liver microsomal fraction. No other treatment gave rise to gene toxicity. No cytotoxic effects was observed in CHO cells when exposed to the test chemical for 3 hrs. Based on the results of the current study, it was concluded that the test chemical give rise to gene mutations when exposed to the test chemical at concentrations of ≥ 0.05 mM for 3 hrs or more. Also, the test chemical does not give rise to cytotoxicity in CHO cells at concentrations of ≤ 0.05 mM. Hence, the test chemical can be considered to be non mutagenic to CHO cells.

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

The above results are supported by a study conducted according to OECD 476 Guidelines where the test chemical was assayed for gene mutations at the tk locus of mouse lymphoma cells both in the absence and presence of S9 metabolic activation.Liver S9 fraction from phenobarbital/β-naphthoflavone-induced rats was used as exogenous metabolic activation system. The test chemical's test concentrations were based on the results of a cytotoxicity assay with a wide range of concentration levels both in the presence and absence of S9-mix measuring cell survival relative to the concurrent vehicle control cell cultures. In the main test cells were treated for 3 h or 24 h (experiment 2 with S9-mix only) followed by an expression period of 7-9 days to fix the DNA damage into a stable tk mutation. Toxicity was measured as percentage relative total growth of the treated cultures relative to the survival of the solvent control cultures. Negative and positive controls were in accordance with the OECD 476 Guidelines. In the cytotoxicity test both in the absence and presence of S9-mix and at 3 and 24 h treatment, severe toxicity was observed at the highest dose levels tested, reducing survival below 5% of the negative control level. On the basis of these findings 1.5 and 2.75 μg/ml were selected as maximum dose level used in the first experiment with and without S9-mix respectively and 0.4 μg/ml in experiment 2 for the 24 h treatment group. In the second experiment, in the absence of S9-mix the appropriate level of toxicity (reduction of the relative total growth after the highest dose to 10-20%) was reached; in the experiments in the presence of S9-mix the appropriate level of reduction in relative total growth was not reached. No biologically relevant and dose dependent increases in the mutant frequency following treatment with the test chemical were found at any dose level, in the absence or presence of S9-mix at any treatment time. Based on all the available data, it was concluded that the test chemical was not mutagenic in the mouse lymphoma assay at the tk locus.

Based on the available data, the test chemical was not mutagenic in the mouse lymphoma assay at the tk locus. Hence, it can be classified under the category “Not Classified” as per CLP Regulation.

Justification for classification or non-classification

Based on the available data, the test chemical was not mutagenic when tested in-vitro in bacterial and mammalian cell lines. Hence, it can be classified under the category “Not Classified” as per CLP Regulation.