Nicotine sulphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

Ames test of the test chemical was conducted by the plate incorporation and preincubation methods. The study was performed as per the OECD guideline No. 471 (Adopted: July 21, 1997, Corrected: June 26, 2020). Based on the solubility test, distilled water was selected as a vehicle for the Test Item in the study. The mutagenic potential of test chemical was tested in two independent experiments (Trial I and Trial II) and both in the presence (10 % v/v cofactor supplemented post-mitochondrial S9 fraction, prepared from rat liver) and absence of metabolic activation using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA102. The Test Item was tested along with solvent vehicle control (distilled water) and concurrent positive controls in triplicates. 

A preliminary cytotoxicity assay was performed using the strains TA98 and TA100 with the concentrations 0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate along with the vehicle and the positive controls both in the presence (10% v/v S9 mix) and absence of metabolic activation in triplicates. In tester strains, TA98 and TA100, no reduction in the revertant colony count and no diminution of the background lawn were observed at any of the concentrations, either in the presence (10 % v/v S9 mix) or the absence of metabolic activation, when compared to the vehicle control data. Based on the preliminary cytotoxicity test results, the main study was performed with the concentrations 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate for Trial I and II.

Trial I was performed according to the plate incorporation method, both in the presence (10 % v/v S9 mix) and absence of a metabolic activation system. The Test Item, together with the vehicle and the positive control were tested in triplicates. The Test Item doses were selected using concentration spacing factor of 2. No increase in the number of revertant colonies or diminution of the background lawn was observed up to the highest concentration of 5 mg/plate, either in the presence (10 % v/v S9 mix) or absence of metabolic activation when compared to the vehicle control data. Trial II was performed to confirm the negative results observed in Trial I. Trial II was conducted according to the preincubation method, both in the presence (10 % v/v S9 mix) and absence of a metabolic activation system. The Test Item, together with the vehicle, negative and positive controls, was tested in all the tester strains in triplicates. There was no increase in the number of revertant colonies or inhibition of the background lawn up to the highest concentration of 5 mg/plate, either in the presence (10 % v/v S9 mix) or absence of metabolic activation, when compared to the vehicle control. The numbers of revertant colonies of the vehicle (spontaneous revertant colonies) and positive controls (induced revertant colonies) of all the tester strains were within the range of historical data of the laboratory. The positive controls used in the study produced significant increases in the mean number of revertant colonies under both activated and non-activated conditions in all tester strains when compared to the control data confirming the validity of the mutagenicity test.

Based on the results of this study, it is concluded that the test chemical was found to be non-mutagenic as it does 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 in vitro as per the criteria mentioned in the CLP regulation.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 29, 2020 to October 27, 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 mutagenic potential of test item to induce point mutation in Histidine operon in two independent experiments (Trial I and Trial II) both in the presence (10 % v/v cofactor supplemented post-mitochondrial S9 fraction, prepared from rat liver) and absence of metabolic activation using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA102.

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 : In-house prepared Rat liver microsomal enzymes (S9 homogenate) ) were used for the assay.
- method of preparation of S9 mix : S9 mix [Cofactors (Cofactor ingredients: D-glucose-6-phosphate, β-NADP, magnesium chloride, potassium chloride, sodium phosphate and Liver homogenate] was prepared prior to use in the study.
- 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:

Based on the preliminary cytotoxicity test results, 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate concentration were selected for the main study.

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 (50 mg/ml). Hence, distilled water was selected as the vehicle for the study.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle: Distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

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: 20 min at 37 ± 2 °C
- Exposure duration/duration of treatment: 48 hours at 37 ± 2 °C
- Harvest time after the end of treatment (sampling/recovery times): No data

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Reduction of revertants per plate and/or clearing or diminution of the bacterial background lawn
- Any supplementary information relevant to cytotoxicity: No data

METHODS FOR MEASUREMENTS OF GENOTOXICIY

- OTHER: No data

Rationale for test conditions:

No data available

Evaluation criteria:

A Test Item is considered as a mutagen if a biologically relevant increase in the mean number of revertants exceeding the threshold of twice (strains TA98, TA100 and TA102) or thrice (strains TA1535 and TA1537) the colony count of the corresponding solvent control is observed.
A dose-dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
A dose-dependent increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent experiment.
A dose-dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent experiment.

Statistics:

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

Species / strain:

S. typhimurium, other: TA98, TA100, TA102, TA1535, and TA1537

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: The test item was found to be soluble in distilled water (upto 50 mg/ml)
- Precipitation and time of the determination: The precipitation test of Test Item was performed by adding 100 µl of the highest test Item concentration (50 mg/ml) to 2 ml of top agar and plated on to minimal glucose agar plate. No precipitation was observed on minimal glucose agar plate at the tested concentration of 5 mg/plate.
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES (if applicable): To evaluate the toxicity of the test item, a preliminary cytotoxicity assay was performed using the strains TA98 and TA100 with the 0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate concentrations along with the vehicle and the positive controls both in the presence (10% v/v S9 mix) and absence of metabolic activation in triplicates. In tester strains, TA98 and TA100, no reduction in the revertant colony count and no diminution of the background lawn were observed at any of the concentrations, either in the presence (10 % v/v S9 mix) or the absence of metabolic activation, when compared to the vehicle control data.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The numbers of revertant colonies of the vehicle (spontaneous revertant colonies) and positive controls (induced revertant colonies) of all the tester strains were within the range of historical data of the laboratory. The positive controls used in the study produced significant increases in the mean number of revertant colonies under both activated and non-activated conditions in all tester strains when compared to the control data confirming the validity of the mutagenicity test.

Ames test:
- Signs of toxicity : Revertant count and inhibition in background lawn
- Individual plate counts : Please refer 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)
Please refer the table attached in remark section.
- Positive historical control data: The positive controls used in the study produced significant increases in the mean number of revertant colonies in all of the tester strains when compared to the control data.
- Negative (solvent/vehicle) historical control data: The frequency of the spontaneous revertant colonies in the vehicle control were within the acceptable range of historical data of the lab.

Remarks on result:

other: No mutagenic potential

Table 1  :        Mean Revertant Colony Count – Preliminary Cytotoxicity Assay

Test Item Concentration (mg/plate)	TA 98				TA 100
	- S9		+ S9		- S9		+ S9
	Mean	SD	Mean	SD	Mean	SD	Mean	SD
VC	21.67 (NI)	3.21	22.00 (NI)	2.00	102.67 (NI)	4.51	98.00 (NI)	4.58
T1 (0.0390625)	19.33 (NI)	0.58	20.00 (NI)	3.00	96.00 (NI)	6.24	97.00 (NI)	2.65
T2 (0.078125)	19.67 (NI)	3.06	20.00 (NI)	1.00	103.33 (NI)	11.72	96.33 (NI)	6.51
T3 (0.15625)	18.67 (NI)	1.53	19.67 (NI)	3.51	97.33 (NI)	6.03	96.67 (NI)	5.51
T4 (0.3125)	17.67 (NI)	1.15	22.33 (NI)	2.08	101.00 (NI)	3.61	99.00 (NI)	7.00
T5 (0.625)	20.33 (NI)	2.52	19.67 (NI)	0.58	102.67 (NI)	4.73	96.67 (NI)	2.52
T6 (1.25)	22.00 (NI)	2.65	19.00 (NI)	3.46	98.00 (NI)	4.58	98.67 (NI)	5.13
T7 (2.5)	19.67 (NI)	0.58	19.67 (NI)	3.06	98.67 (NI)	3.06	99.33 (NI)	6.81
T8 (5.0)	19.00 (NI)	3.46	20.67 (NI)	2.08	97.67 (NI)	4.04	95.33 (NI)	5.86
PC	375.67	12.58	368.33	11.68	754.00	11.79	730.33	30.66

Key:   VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation, T1-T8 = Test Item concentration from lower to higher.

Note:  No reduction in the revertant colony count and/or diminution of the background lawn was noted at 5 mg/plate; therefore Trial I (plate incorporation method) was performed with 5 mg/plate as the highest concentration.

Positive Controls:

2-Nitrofluorene	:	TA98 (absence of metabolic activation)
Sodium azide	:	TA100 (absence of metabolic activation)
Benzo[a]pyrene	:	TA98 and TA100 (presence of metabolic activation)

Table 2  :        Mean Revertant Colony Count Trial I(Plate Incorporation Method)

Absence of metabolic activation (-S9)							Presence of metabolic activation (+S9 10 % v/v S9 Mix)
Test Item Concentration (mg/plate)	TA 1535		TA 1537		TA 102		TA 1535		TA 1537		TA 102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
VC	12.67	1.53	6.33	1.53	234.33	6.51	12.00	2.65	6.67	1.15	237.67	8.96
T1 (0.3125)	11.67	2.08	6.67	1.15	238.00	3.00	12.00	1.73	7.67	1.15	235.00	14.18
T2 (0.625)	12.33	0.58	9.00	1.00	235.00	6.56	13.33	2.08	6.67	2.31	227.33	10.41
T3 (1.25)	12.33	2.08	7.00	0.00	234.67	6.51	11.00	1.73	7.00	1.73	232.33	8.50
T4 (2.5)	13.00	2.65	6.67	2.08	235.67	11.02	13.33	2.08	9.67	1.53	240.00	6.56
T5 (5.0)	11.33	1.53	7.33	1.53	230.33	13.65	12.33	2.08	6.33	0.58	236.33	11.24
PC	364.33	6.51	205.33	8.74	1582.33	30.37	366.00	6.08	207.00	16.52	1684.33	34.65

Key:   VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation, T1-T5 = Test Item concentration from lower to higher.

Positive Controls:

2-Nitrofluorene	:	TA98 (absence of metabolic activation)
Sodium azide	:	TA100 and TA1535 (absence of metabolic activation)
9-Aminoacridine	:	TA1537 (absence of metabolic activation)
Mitomycin-C	:	TA102(absence of metabolic activation)
Benzo[a]pyrene	:	TA98, TA100, TA1535, TA1537 and TA102 (presence of metabolic activation)

Table 3 :         Mean Revertant Colony Count Trial II (Preincubation Method) 

	Absence of metabolic activation										Presence of metabolic activation (+S9 10% v/v S9 Mix)
Test Item Concentration (mg/plate)	TA 1537		TA 1535		TA 102		TA 98		TA 100		TA 1537		TA 1535		TA 102		TA 98		TA 100
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
VC	6.33	1.53	12.33	2.08	244.33	8.50	20.33	3.61	104.67	8.33	6.67	1.15	13.33	2.08	236.33	5.69	22.00	3.61	97.67	6.35
T1(0.3125)	6.00	2.00	11.33	0.58	244.33	10.60	21.67	1.15	105.33	5.77	6.00	1.00	12.67	1.53	237.33	4.04	19.33	1.15	104.00	6.24
T2(0.625)	7.67	1.15	11.00	2.65	238.00	11.27	21.33	2.52	98.67	5.03	6.33	2.08	12.33	2.08	247.00	7.55	20.67	2.52	104.33	2.08
T3(1.25)	7.00	1.73	12.33	1.15	243.67	12.22	20.00	2.08	97.33	6.11	6.67	1.15	11.33	1.53	244.00	7.55	22.33	2.08	102.33	8.50
T4(2.5)	5.00	1.73	12.33	2.08	248.00	13.53	20.33	1.53	105.33	4.93	6.00	2.65	12.67	1.53	239.00	5.00	22.33	1.53	96.00	2.65
T5(5.0)	6.33	2.08	12.33	1.53	247.00	12.12	22.67	3.06	93.33	4.04	6.67	1.15	12.67	1.53	234.00	6.56	20.67	3.06	98.00	3.61
PC	226.33	14.74	364.33	9.50	1662.67	66.53	397.33	13.75	736.33	47.50	213.00	10.54	365.67	18.50	1667.67	47.08	376.00	13.75	856.33	10.07

Key:     VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation, T1-T5 = Test Item concentration from lower to higher.

Positive Controls:

2-Nitrofluorene	:	TA98 (absence of metabolic activation)
Sodium azide	:	TA100 and TA1535 (absence of metabolic activation)
9-Aminoacridine	:	TA1537 (absence of metabolic activation)
Mitomycin-C	:	TA102 (absence of metabolic activation)
Benzo[a]pyrene	:	TA98, TA100, TA1535, TA1537 and TA102 (presence of metabolic activation)

Table 4  :        Fold Increase

	Trial I - Plate Incorporation Method										Trial II – Preincubation Method
Test Item Concentration (mg/plate)	TA 98		TA 100		TA 1537		TA 1535		TA 102		TA 98		TA 100		TA 1537		TA 1535		TA 102
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
VC	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
T1 (0.3125)	0.82	1.02	0.98	1.01	1.05	1.15	0.92	1.00	1.02	0.99	1.07	0.88	1.01	1.06	0.95	0.90	0.92	0.95	1.00	1.00
T2 (0.625)	0.94	0.89	1.00	0.99	1.42	1.00	0.97	1.11	1.00	0.96	1.05	0.94	0.94	1.07	1.21	0.95	0.89	0.93	0.97	1.05
T3 (1.25)	1.02	0.86	0.95	1.01	1.11	1.05	0.97	0.92	1.00	0.98	0.98	1.02	0.93	1.05	1.11	1.00	1.00	0.85	1.00	1.03
T4 (2.5)	0.91	0.89	0.96	1.01	1.05	1.45	1.03	1.11	1.01	1.01	1.00	1.02	1.01	0.98	0.79	0.90	1.00	0.95	1.02	1.01
T5 (5.0)	0.88	0.94	0.95	0.97	1.16	0.95	0.89	1.03	0.98	0.99	1.11	0.94	0.89	1.00	1.00	1.00	1.00	0.95	1.01	0.99
PC	17.34	16.74	7.34	7.45	32.42	31.05	28.76	30.50	6.75	7.09	19.54	17.09	7.04	8.77	35.74	31.95	29.54	27.43	6.80	7.06

Key:   VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, T1-T5 = Test Item concentration from lower to higher.

Table 5 :         S9 Efficiency Check- Summary

Summary of S9 efficiency check
	TA100		TA1535
	Mean	SD	Mean	SD
VC Distilled water (-S9)	102.67	4.51	12.67	1.53
VC Distilled water (+S9)	98.00	4.58	12	2.65
PC Benzo[a]pyrene (-S9)	105.00	8.19	13	2.65
PC Benzo[a]pyrene (+S9)	730.333	30.66	366	6.08

Key:   VC = Vehicle control, PC = Positive control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation.

Conclusions:

Bacterial Reverse Mutation Assay of the test chemical was carried out in compliance with the OECD Guideline No. 471 (Adopted: July 21st 1997, Corrected: June 26th 2020). Under the conditions described in this study, it is concluded that the test chemical is non-mutagenic when tested in five tester strains of Salmonella typhimurium (TA1537, TA1535, TA98, TA100 and TA102) in the presence and absence of S9 metabolic activation system and hence is likely to be non-mutagenic.

Executive summary:

Ames test of the test chemical was conducted by the plate incorporation and preincubation methods. The study was performed as per the OECD guideline No. 471 (Adopted: July 21, 1997, Corrected: June 26, 2020). Based on the solubility test, distilled water was selected as a vehicle for the Test Item in the study. The mutagenic potential of test chemical was tested in two independent experiments (Trial I and Trial II) and both in the presence (10 % v/v cofactor supplemented post-mitochondrial S9 fraction, prepared from rat liver) and absence of metabolic activation using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA102. The Test Item was tested along with solvent vehicle control (distilled water) and concurrent positive controls in triplicates. 

A preliminary cytotoxicity assay was performed using the strains TA98 and TA100 with the concentrations 0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate along with the vehicle and the positive controls both in the presence (10% v/v S9 mix) and absence of metabolic activation in triplicates. In tester strains, TA98 and TA100, no reduction in the revertant colony count and no diminution of the background lawn were observed at any of the concentrations, either in the presence (10 % v/v S9 mix) or the absence of metabolic activation, when compared to the vehicle control data. Based on the preliminary cytotoxicity test results, the main study was performed with the concentrations 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate for Trial I and II.

Trial I was performed according to the plate incorporation method, both in the presence (10 % v/v S9 mix) and absence of a metabolic activation system. The Test Item, together with the vehicle and the positive control were tested in triplicates. The Test Item doses were selected using concentration spacing factor of 2. No increase in the number of revertant colonies or diminution of the background lawn was observed up to the highest concentration of 5 mg/plate, either in the presence (10 % v/v S9 mix) or absence of metabolic activation when compared to the vehicle control data. Trial II was performed to confirm the negative results observed in Trial I. Trial II was conducted according to the preincubation method, both in the presence (10 % v/v S9 mix) and absence of a metabolic activation system. The Test Item, together with the vehicle, negative and positive controls, was tested in all the tester strains in triplicates. There was no increase in the number of revertant colonies or inhibition of the background lawn up to the highest concentration of 5 mg/plate, either in the presence (10 % v/v S9 mix) or absence of metabolic activation, when compared to the vehicle control. The numbers of revertant colonies of the vehicle (spontaneous revertant colonies) and positive controls (induced revertant colonies) of all the tester strains were within the range of historical data of the laboratory. The positive controls used in the study produced significant increases in the mean number of revertant colonies under both activated and non-activated conditions in all tester strains when compared to the control data confirming the validity of the mutagenicity test.

Based on the results of this study, it is concluded that the test chemical was found to be non-mutagenic as it does 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 in vitro as per the criteria mentioned in the CLP regulation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic toxicity: invitro

Various studies have been reviewed to evaluate the mutagenic potential of the test chemical in bacterial strains when tested in vitro. These include experimental study for the target chemical along with details from reliable publication. The results are mentioned below:

Ames test of the test chemical was conducted by the plate incorporation and preincubation methods. The study was performed as per the OECD guideline No. 471 (Adopted: July 21, 1997, Corrected: June 26, 2020). Based on the solubility test, distilled water was selected as a vehicle for the Test Item in the study. The mutagenic potential of test chemical was tested in two independent experiments (Trial I and Trial II) and both in the presence (10 % v/v cofactor supplemented post-mitochondrial S9 fraction, prepared from rat liver) and absence of metabolic activation using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA102. The Test Item was tested along with solvent vehicle control (distilled water) and concurrent positive controls in triplicates. A preliminary cytotoxicity assay was performed using the strains TA98 and TA100 with the concentrations 0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate along with the vehicle and the positive controls both in the presence (10% v/v S9 mix) and absence of metabolic activation in triplicates. In tester strains, TA98 and TA100, no reduction in the revertant colony count and no diminution of the background lawn were observed at any of the concentrations, either in the presence (10 % v/v S9 mix) or the absence of metabolic activation, when compared to the vehicle control data. Based on the preliminary cytotoxicity test results, the main study was performed with the concentrations 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate for Trial I and II. Trial I was performed according to the plate incorporation method, both in the presence (10 % v/v S9 mix) and absence of a metabolic activation system. The Test Item, together with the vehicle and the positive control were tested in triplicates. The Test Item doses were selected using concentration spacing factor of 2. No increase in the number of revertant colonies or diminution of the background lawn was observed up to the highest concentration of 5 mg/plate, either in the presence (10 % v/v S9 mix) or absence of metabolic activation when compared to the vehicle control data. Trial II was performed to confirm the negative results observed in Trial I. Trial II was conducted according to the preincubation method, both in the presence (10 % v/v S9 mix) and absence of a metabolic activation system. The Test Item, together with the vehicle, negative and positive controls, was tested in all the tester strains in triplicates. There was no increase in the number of revertant colonies or inhibition of the background lawn up to the highest concentration of 5 mg/plate, either in the presence (10 % v/v S9 mix) or absence of metabolic activation, when compared to the vehicle control. The numbers of revertant colonies of the vehicle (spontaneous revertant colonies) and positive controls (induced revertant colonies) of all the tester strains were within the range of historical data of the laboratory. The positive controls used in the study produced significant increases in the mean number of revertant colonies under both activated and non-activated conditions in all tester strains when compared to the control data confirming the validity of the mutagenicity test. Based on the results of this study, it is concluded that the test chemical was found to be non-mutagenic as it does 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 in vitro as per the criteria mentioned in the CLP regulation.

The test chemical was also tested for mutagenicity in bacterial reversion-assay systems with 2 strains (TA100, TA98) of Salmonella typhimurium. Most procedures were performed according to the methods described by Ames et al. These strains were cultured overnight in liquid nutrient broth and stored at -80°C. Their genetic markers and other characteristics, such as response to positive controls and the number of spontaneous revertants, were checked .To 2 ml of the top agar were added 0.1 ml of one of bacterial suspensions in phosphate buffer (1/15 M), 0.1 ml of a solution of the test chemical in suitable solvent (water or dissolved in dimethyl sulfoxide (DMSO) based on solubility) and, when required, 0.5 ml of the S9 mix (0.3 ml of S9 fraction/ml S9 mix). The contents were poured onto the surface of a minimal agar plate with modified Vogel-Bonnet E medium (Moriya et al., 1978). Plates were incubated at 37°C for 2 days, after which the number of revertant colonies on each plate was counted. The test chemical was tested up to a dose of 5000 microgram/plate, unless it showed toxicity to bacteria at the dose.The test chemical failed to show mutagenic response in the presence and absence of S9 mix when tested on 2 strains (TA100, TA98) of Salmonella typhimurium. Hence, it can be considered to be non-mutagenic in nature.

Based on the available results, the test chemical can be considered to be non- mutagenic to Salmonella typhimurium strains when tested in vitro either in the presence or absence of metabolic activation system. Hence, the test chemical can be classified under the category “Not Classified’ as per CLP Regulation.

Justification for classification or non-classification

Based on the available results, the test chemical can be considered to be non- mutagenic toSalmonella typhimuriumstrains when tested in vitro either in the presence or absence of metabolic activation system. Hence, the test chemical can be classified under the category “Not Classified’ as per CLP Regulation.