Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial gene mutation assay:

The study was performed to evaluate the potential of the test chemical to induce mutations in the histidine operon in fiveSalmonella typhimuriumstrains both in the presence and absence of exogenous metabolic activation system (S9) containing microsomal enzymes by Prival modification method. The Ames test was conducted by Prival modification method and 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 (30 % v/v cofactor supplemented post-mitochondrial S9 fraction, prepared from hamster liver) and absence of metabolic activation usingSalmonella 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 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 (30% v/v S9 mix) and absence of metabolic activation in triplicates. In TA98 and TA100 tester strains, no reduction in the revertant colony count and no diminution of the background lawn were observed at any concentrations tested, neither in the presence (30 % v/v S9 mix) nor in 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 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate concentrations. Trial I was performed according to the preincubation method using the strains TA98, TA100, TA102, TA1535, and TA1537, both in the presence (30 % 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 (30 % 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 with 0.0501,0.1585, 0.5007, 1.5823 and 5 mg/plate concentrations. Trial II was conducted according to the preincubation method using the tester strains TA98, TA100, TA102, TA1535, and TA1537, both in the presence (30 % 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. The Test Item doses were selected using concentration spacing factor of half log (√10). 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 (30 % 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 chemicalis 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 30% v/v S9 metabolic activation system, in any of the five tester strains ofSalmonella typhimurium (TA1537, TA1535, TA98, TA100 and TA102) as per the study performed by Ames assay (OECD guideline no. 471) with Prival modification and hence it is not likely to classify as a gene mutant in vitro

 

 

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:
19 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)
Version / remarks:
By Prival Modification Method
Principles of method if other than guideline:
The study was performed to investigate the potential of Test chemical to induce point mutations in the histidine operon in strains of Salmonella typhimurium TA98, TA100, TA1535, TA1537, and TA102 both in the presence (30 % v/v cofactor supplemented post-mitochondrial S9 fraction, prepared from hamster liver) and absence of exogenous metabolic activation system (S9) containing microsomal enzymes by Prival modification method.
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: Uninduced hamster liver microsomal enzymes (S9 homogenate).
- source of S9: Uninduced hamster liver microsomal enzymes (S9 homogenate) was procured from MASS Biotech.
- method of preparation of S9 mix: S9 mix [Cofactors (Cofactor ingredients: 20 mM D-Glucose-6-phosphate, 4 mM β-NADP, 8 mM magnesium chloride, 33 mM potassium chloride, 0.1 M sodium phosphate, 2 mM NADH, 2.8 U/ ml D-Glucose-6-phosphate dehydrogenase, 2 mM flavin mononucleotide (FMN) and liver homogenate] was prepared prior to use in the study.
- concentration or volume of S9 mix and S9 in the final culture medium: The post-mitochondrial fraction (hamster liver homogenate) was used at the concentration of 30 % (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 preliminary cytotoxicity test results, the main study was performed with the following concentrations:
For Trial I: 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate.
For Trial II: 0.0501,0.1585, 0.5007, 1.5823 and 5 mg/plate.
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 Control: Distilled Water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
congo red
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 and Trial II was performed according to the preincubation method.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): No data
- Test substance added in medium: After preincubation, 2 ml of overlay agar (45 ± 2 °C) was added to each tube. The mixture was poured on selective agar plates.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 30 minutes 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.: bacterial background growth inhibition
- Any supplementary information relevant to cytotoxicity: No data

METHODS FOR MEASUREMENTS OF GENOTOXICITY:
For each strain and dose level, including the controls, three plates (triplicate) were used.
In the preincubation method, the following materials were mixed in a test tube and incubated at 37 ± 2 °C for 30 minutes:
• 100 µl -Test solution at each dose level/vehicle control
• 500 µl - S9 mix (for tests with metabolic activation) or phosphate buffer (for tests without metabolic activation)
• 100 µl - Bacterial suspension
After preincubation, 2 ml of overlay agar (45 ± 2 °C) was added to each tube. The mixture was poured on selective agar plates.
After solidification, the plates were incubated upside down for 48 hours at 37 ± 2 °C in an incubator.

The colonies were counted manually. The mean number of revertant colonies and the standard deviation within the plates for each concentration were compared to the spontaneous reversion rates of the control.

- 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:
Remarks:
TA1535, TA1537, TA98, TA100 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No data
- Data on osmolality: No data
- Possibility of evaporation from medium: No data
- Water solubility: The test Item was found to be soluble in distilled water (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 a 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):
Based on the solubility and precipitation test, the preliminary cytotoxicity test was performed at the concentrations of 0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate, both in the presence (30 % v/v S9 mix) and absence of a metabolic activation system. In the cytotoxicity test, the tester strains TA98 and TA100 were exposed to the Test Item, vehicle, and positive controls according to the preincubation method. Each concentration of the Test Item, including the controls, was tested in triplicates.
In tester strains TA98 and TA100, no reduction in revertant count or inhibition of the background lawn were observed at any concentrations tested either in the presence (30% v/v S9 mix) or absence of metabolic activation when compared to the vehicle control data.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: For each tester strain, the frequency of the spontaneous revertant colonies in the vehicle control was within the acceptable range of historical data of the lab. 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.

Ames test:
- Signs of toxicity: Increase in Revertant colony count and inhibition of the 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 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: No data
- Negative (solvent/vehicle) historical control data: Please refer the table attached in remark section
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

20.33

(NI)

3.21

21.00

(NI)

2.00

94.33

(NI)

3.06

99.33

(NI)

4.73

T1

(0.0390625)

21.33

(NI)

2.52

20.67

(NI)

1.15

96.00

(NI)

5.29

101.00

(NI)

8.19

T2

(0.078125)

18.00

(NI)

2.00

22.00

(NI)

1.73

90.33

(NI)

0.58

95.67

(NI)

4.16

T3

(0.15625)

20.33

(NI)

2.52

20.67

(NI)

2.08

95.67

(NI)

2.08

97.00

(NI)

4.36

T4

(0.3125)

18.33

(NI)

4.04

20.00

(NI)

2.00

95.67

(NI)

4.16

94.67

(NI)

3.21

T5

(0.625)

19.00

(NI)

1.73

19.33

(NI)

0.58

91.67

(NI)

3.79

97.00

(NI)

2.00

T6

(1.25)

19.67

(NI)

1.15

17.00

(NI)

2.65

95.67

(NI)

2.89

98.00

(NI)

4.00

T7

(2.5)

18.67

(NI)

1.53

18.67

(NI)

1.53

92.00

(NI)

4.58

93.33

(NI)

4.93

T8

(5.0)

17.67

(NI)

1.53

19.33

(NI)

1.53

91.67

(NI)

3.06

92.00

(NI)

2.65

PC

375.67

12.66

319.00

11.36

742.00

15.10

618.33

4.04

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, NI = No Inhibition.

Note:  No reduction in the revertant colony count and/or diminution of the background lawn was noted at 5 mg/plate; therefore Trial I (preincubation 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)

Congo red

:

TA98 and TA100 (presence of metabolic activation)

Table 2 :  Mean Revertant Colony Count-Trial I(Preincubation Method)

Absence of
metabolic activation (-S9)

Presence of
metabolic activation (+S9 30 % 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

11.33

1.53

6.00

0.00

232.33

3.06

12.33

1.15

6.67

1.15

234.67

12.34

T1 (0.3125)

12.33

1.15

6.33

2.31

228.67

7.09

12.33

2.08

5.67

1.15

235.00

6.00

T2 (0.625)

13.33

2.08

7.33

1.15

245.00

5.20

12.33

2.31

5.33

0.58

237.33

4.04

T3 (1.25)

13.33

0.58

4.67

2.31

234.00

9.64

14.00

1.00

4.67

1.15

233.00

10.15

T4 (2.5)

13.33

2.08

6.00

1.00

243.00

10.54

13.67

0.58

5.00

1.00

246.00

11.79

T5 (5.0)

13.67

1.15

4.67

1.15

229.67

8.50

13.33

2.52

5.67

0.58

234.00

6.56

PC

358.00

14.93

223.00

10.82

1607.67

15.18

361.00

13.00

215.33

16.01

1629.00

30.20

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)

Congo red

:

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 30% v/v S9 Mix)

Test Item

Concentration

(mg/plate)

TA 98

TA 100

TA 1535

TA 1537

TA 102

TA 98

TA 100

TA 1535

TA 1537

TA 102

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

VC

19.67

2.31

104.33

7.02

12.33

1.15

5.67

0.58

237.33

4.04

20.67

2.31

104.33

7.02

14.00

2.65

6.33

0.58

236.67

4.04

T1(0.0501)

17.67

1.73

98.67

3.51

12.33

2.08

6.67

0.58

233.67

7.37

18.00

1.73

98.67

4.16

12.67

1.53

5.67

0.58

237.33

10.97

T2(0.1585)

21.00

0.58

99.33

7.37

11.67

1.53

7.33

1.15

233.33

6.81

19.67

0.58

101.33

5.86

13.33

3.06

6.00

1.73

246.00

4.36

T3(0.5007)

19.00

1.53

99.00

4.00

13.00

1.00

8.00

2.65

233.33

3.06

21.33

1.53

94.00

5.00

13.33

0.58

6.67

1.15

235.33

9.29

T4(1.5823)

20.33

0.58

98.00

3.61

13.33

1.53

6.67

2.08

231.00

7.00

19.33

0.58

98.33

5.13

13.00

2.00

6.33

2.31

236.33

11.59

T5(5.0)

19.00

1.73

96.00

5.57

13.33

2.08

6.00

2.00

231.67

4.51

22.00

1.73

96.67

7.37

13.67

0.58

6.33

0.58

239.00

7.55

PC

383.67

10.58

746.00

14.18

355.67

13.05

212.33

10.07

1616.67

22.05

360.00

10.58

761.33

11.24

359.33

8.50

219.00

3.61

1647.33

36.50

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)

Congo red

:

TA98, TA100, TA1535, TA1537 and TA102 (presence of metabolic activation)

Table 4 : Fold Increase

Trial I - Preincubation Method

 

Trial II – Preincubation Method

Test Item

Concentration

(mg/plate)

TA 98

TA 100

TA 1535

TA 1537

TA 102

Test Item

Concentration

(mg/plate)

TA 98

TA 100

TA 1535

TA 1537

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

VC

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

T1

(0.3125)

0.90

0.95

1.01

0.95

1.09

1.00

1.06

0.85

0.98

1.00

T1

(0.0501)

0.90

0.87

0.95

0.95

1.00

0.90

1.18

0.89

0.98

1.00

T2

(0.625)

0.93

0.92

0.97

0.98

1.18

1.00

1.22

0.80

1.05

1.01

T2

(0.1585)

1.07

0.95

0.95

0.97

0.95

0.95

1.29

0.95

0.98

1.04

T3

(1.25)

0.97

0.81

1.01

0.99

1.18

1.14

0.78

0.70

1.01

0.99

T3

(0.5007)

0.97

1.03

0.95

0.90

1.05

0.95

1.41

1.05

0.98

0.99

T4

(2.5)

0.92

0.89

0.98

0.94

1.18

1.11

1.00

0.75

1.05

1.05

T4

(1.5823)

1.03

0.94

0.94

0.94

1.08

0.93

1.18

1.00

0.97

1.00

T5

(5.0)

0.87

0.92

0.97

0.93

1.21

1.08

0.78

0.85

0.99

1.00

T5

(5.0)

0.97

1.06

0.92

0.93

1.08

0.98

1.06

1.00

0.98

1.01

PC

18.48

15.19

7.87

6.22

31.59

29.27

37.17

32.30

6.92

6.94

PC

19.51

17.42

7.15

7.30

28.84

25.67

37.47

34.58

6.81

6.96

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)

94.33

3.06

11.33

1.53

VC (Distilled water) (+S9)

99.33

4.73

12.33

1.15

PC (Congo red) (-S9)

92.67

4.04

14.33

1.15

PC (Congo red) (+S9)

618.33

4.04

361.00

13.00

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

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 30 % v/v S9 metabolic activation system as per the study performed by Ames assay (OECD guideline no. 471) with Prival modification and hence it is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.
Executive summary:

The study was performed to evaluate the potential of the test chemical to induce mutations in the histidine operon in five Salmonella typhimurium strains both in the presence and absence of exogenous metabolic activation system (S9) containing microsomal enzymes by Prival modification method. The Ames test was conducted by Prival modification method and 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 (30 % v/v cofactor supplemented post-mitochondrial S9 fraction, prepared from hamster 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 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 (30% v/v S9 mix) and absence of metabolic activation in triplicates. In TA98 and TA100 tester strains, no reduction in the revertant colony count and no diminution of the background lawn were observed at any concentrations tested, neither in the presence (30 % v/v S9 mix) nor in 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 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate concentrations. Trial I was performed according to the preincubation method using the strains TA98, TA100, TA102, TA1535, and TA1537, both in the presence (30 % 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 (30 % 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 with 0.0501,0.1585, 0.5007, 1.5823 and 5 mg/plate concentrations. Trial II was conducted according to the preincubation method using the tester strains TA98, TA100, TA102, TA1535, and TA1537, both in the presence (30 % 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. The Test Item doses were selected using concentration spacing factor of half log (√10). 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 (30 % 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 chemicalis 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 30% v/v S9 metabolic activation system, in any of the five tester strains of Salmonella typhimurium (TA1537, TA1535, TA98, TA100 and TA102) as per the study performed by Ames assay (OECD guideline no. 471) with Prival modification and hence it is not likely to classify as a gene mutant in vitro

 

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

Additional information

Experimental data available for the target chemical was reviewed to determine the mutagenic nature of the test chemical. The details are as mentioned below:

Bacterial gene mutation assay:

The study was performed to evaluate the potential of the test chemical to induce mutations in the histidine operon in fiveSalmonella typhimuriumstrains both in the presence and absence of exogenous metabolic activation system (S9) containing microsomal enzymes by Prival modification method. The Ames test was conducted by Prival modification method and 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 (30 % v/v cofactor supplemented post-mitochondrial S9 fraction, prepared from hamster 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 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 (30% v/v S9 mix) and absence of metabolic activation in triplicates. In TA98 and TA100 tester strains, no reduction in the revertant colony count and no diminution of the background lawn were observed at any concentrations tested, neither in the presence (30 % v/v S9 mix) nor in 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 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate concentrations. Trial I was performed according to the preincubation method using the strains TA98, TA100, TA102, TA1535, and TA1537, both in the presence (30 % 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 (30 % 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 with 0.0501,0.1585, 0.5007, 1.5823 and 5 mg/plate concentrations. Trial II was conducted according to the preincubation method using the tester strains TA98, TA100, TA102, TA1535, and TA1537, both in the presence (30 % 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. The Test Item doses were selected using concentration spacing factor of half log (√10). 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 (30 % 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 chemicalis 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 30% v/v S9 metabolic activation system, in any of the five tester strains of Salmonella typhimurium (TA1537, TA1535, TA98, TA100 and TA102) as per the study performed by Ames assay (OECD guideline no. 471) with Prival modification and hence it is not likely to classify as a gene mutant in vitro

 

 

Justification for classification or non-classification

From the experimental data available, it can be concluded that the target chemical does not induce gene mutation in the histidine operon by base-pair changes or frameshifts, in the presence or the absence of metabolic activation system as per the study performed by Ames assay (OECD guideline no. 471) with Prival modification. Hence the target chemical is likely to be non-mutagenic in vitro as per the criteria mentioned in CLP regulation.