Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro Bacterial gene mutation assay:

Ames test of 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, 1997The study was performed to evaluate the mutagenic potential of test chemical using Salmonellatyphimurium strains TA98, TA100, TA1537, TA1535 and TA102 in Trial I (with 10% v/v S9 mix and without metabolic activation) and Trial II (with 10% v/v S9 mix and without metabolic activation) experiments including the vehicle (distilled water) and concurrent positive controls in triplicates. 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 positive controls both in the presence and absence of metabolic activation in triplicates Based on the solubility test, distilled water was selected as a vehicle for the test item in the study.

In tester strains TA98 and TA100, no reduction in revertant count and no inhibition in background lawn were observed at any of the concentrations, either in the presence or absence of metabolic activation, when compared to the vehicle control data.On the basis of preliminary cytotoxicity test results, main study was performed with the following concentrations: Trial I and II: 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate.

Trial I was performed both in the presence (10% v/v S9 mix) and absence of metabolic activation system along with the vehicle and the positive control with the test item concentration spacing factor of 2. There was no increase in the number of revertant colonies and no inhibition in background lawn up to the highest concentration of 5 mg/plate in the presence (10% v/v S9 mix) or absence of metabolic activation, when compared to the vehicle control data.

Trial II was conducted to confirm the negative results observed in Trial I. Trial II was conducted in the presence and in the absence of metabolic activation system with all the tester strains along with the vehicle and concurrent positive controls according to the preincubation method.

There was no increase in the number of revertant colonies and no background lawn inhibition up to the highest concentration of 5 mg/plate in the presence (10% v/v S9 mix) and absence of metabolic activation, when compared to the vehicle control.

The number of revertant colonies of the vehicle (spontaneous revertant colonies) and positive controls (induced revertant colonies) were in the range of historical data of the lab.The positive controls used in the study produced significant increase in the mean number of revertant colonies in all of the tester strains compared to the control data, thus confirming the validity of the test system to detect specific mutagens in the presence and in the absence of a metabolic activation system.

From the above data It can be concluded that the test chemical did not induce gene mutations 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 and hence it is not likely to classify as a gene mutant as per the criteria mentioned in 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:
06 February 2020 to 22 February 2020
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 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
The study was performed to evaluate the mutagenic potential of test chemical using Salmonella typhimurium strains TA98, TA100, TA1537, TA1535 and TA102 in Trial I (with 10% v/v S9 mix and without metabolic activation) and Trial II (with 10% v/v S9 mix and without metabolic activation) experiments including the vehicle (distilled water) and concurrent positive controls in triplicates.
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 : Rat liver microsomal enzymes (S9 homogenate) prepared in house 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) in the S9 mix.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No data
Test concentrations with justification for top dose:
0.3125, 0.625, 1.25, 2.5 and 5 mg/plate. Based on the result of cytotoxicity study, doses were selected.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Distilled water

- Justification for choice of solvent/vehicle: On the basis of solubility test, distilled water was selected as a vehicle for the study.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
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 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 (Table 4). A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An 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:
No data available
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No data
- Data on osmolality: No data
- Possibility of evaporation from medium: No data
- Water solubility: Soluble in water (upto 50 mg/ml)
- Precipitation and time of the determination: No precipitation was observed 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): On the basis of 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 (10% v/v S9 mix) and absence of a metabolic activation system. In this assay, the tester strains TA98 and TA100 were exposed to the test item, the vehicle and the positive controls according to the plate incorporation 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 and no inhibition in background lawn were observed at any of the concentrations, either in the presence or absence of metabolic activation, when compared to the vehicle control data (Table 1).

STUDY RESULTS
- Concurrent vehicle control data : vehicle control was within the acceptable range of historical data of lab.
- positive 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, thus confirming the validity of the test system to detect specific mutagens in the presence and absence of a metabolic activation system.

Ames test:
- Signs of toxicity : please reffer the table attached in result section (Appendix 4.)
- Individual plate counts : please reffer the table attached in remark section (Appendix 1, Appendix 2, Appendix 3)
- Mean number of revertant colonies per plate and standard deviation (Table 1, Table 2, Table 3)

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)

2.31

21.33

(NI)

1.53

101.67

(NI)

7.51

99.33

(NI)

9.07

T1

0.0390625

20.67

(NI)

3.06

19.33

(NI)

3.21

96.33

(NI)

3.06

94.00

(NI)

3.00

T2

0.078125

19.67

(NI)

3.51

20.33

(NI)

2.31

102.67

(NI)

4.51

97.33

(NI)

6.66

T3

0.15625

21.33

(NI)

2.31

21.67

(NI)

2.08

96.00

(NI)

6.00

105.33

(NI)

10.02

T4

0.3125

20.33

(NI)

1.15

21.33

(NI)

2.08

94.33

(NI)

4.16

92.67

(NI)

4.51

T5

0.625

22.00

(NI)

2.65

19.00

(NI)

2.00

100.33

(NI)

5.51

95.33

(NI)

4.04

T6

1.25

21.00

(NI)

5.00

20.67

(NI)

2.08

97.67

(NI)

5.03

92.00

(NI)

4.36

T7

2.5

20.33

(NI)

1.53

20.33

(NI)

1.15

94.33

(NI)

4.51

97.33

(NI)

6.03

T8

5.0

20.33

(NI)

2.31

18.33

(NI)

2.08

97.67

(NI)

4.73

95.00

(NI)

7.21

PC

392.33

20.21

407.33

13.20

728.67

24.99

725.00

23.07

Key: VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation, NI = No Inhibition.

Note: Since there was no reduction in revertant count and no background lawn inhibition at 5 mg/plate, Trial I (plate incorporation method) was performed with 5 mg/plate as a highest concentration.  

Positive control:

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)]

 

Test Item Concentration

(mg/plate)

TA 1537

TA 1535

TA 102

Mean

SD

Mean

SD

Mean

SD

VC

5.33

1.15

13.33

2.52

234.33

8.08

T1(0.3125)

6.33

0.58

12.67

2.89

219.33

3.51

T2(0.625)

4.00

1.00

13.67

1.15

219.67

12.22

T3(1.25)

5.67

2.31

14.33

0.58

234.67

7.09

T4(2.5)

5.33

1.15

13.00

2.00

223.00

4.00

T5(5.0)

5.67

1.15

14.33

1.15

233.00

12.29

PC

209.33

20.79

349.67

12.22

1431.67

88.01

Plate Incorporation Method [Presence of metabolic activation (+S9 10 % v/v S9 Mix)]

Test Item

Concentration

(mg/plate)

TA 1537

TA 1535

TA 102

Mean

SD

Mean

SD

Mean

SD

VC

6.33

1.53

13.33

2.52

222.67

9.07

T1(0.3125)

6.00

0.00

14.33

1.15

229.33

8.74

T2(0.625)

7.33

1.53

13.67

1.15

224.00

5.57

T3(1.25)

7.00

1.00

13.67

0.58

232.00

6.24

T4(2.5)

6.00

0.00

14.67

2.31

223.00

14.73

T5(5.0)

6.33

0.58

13.67

1.15

212.00

6.24

PC

206.33

15.53

362.67

9.29

1474.00

38.74

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

Positive Control:

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]

Test Item

Concentration

(mg/plate)

TA 1537

TA 1535

TA 102

TA 98

TA 100

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

VC

6.67

1.15

13.33

2.08

219.67

22.90

21.67

3.06

101.33

6.66

T1(0.3125)

5.67

1.15

13.33

0.58

220.33

25.40

21.33

2.08

99.00

9.17

T2(0.625)

6.67

1.15

11.67

1.15

217.67

17.90

20.67

3.21

104.00

8.19

T3(1.25)

6.33

1.53

13.33

1.53

225.67

15.95

21.33

2.08

94.00

3.61

T4(2.5)

6.33

0.58

13.67

1.15

225.33

10.02

20.33

2.00

97.33

8.50

T5(5.0)

7.00

1.73

12.33

0.58

222.67

9.07

19.67

3.79

99.67

4.93

PC

233.00

15.39

360.67

15.14

1586.00

9.02

266.33

9.17

732.00

14.00

Preincubation Method [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

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

VC

6.00

2.00

13.00

1.00

222.67

9.07

19.67

3.06

94.67

2.08

T1(0.3125)

7.33

1.15

12.33

1.53

222.67

13.87

22.33

2.08

97.33

8.50

T2(0.625)

5.33

0.58

13.67

1.15

223.00

4.00

20.67

3.21

99.67

4.93

T3(1.25)

7.33

0.58

16.33

0.58

225.33

8.62

20.67

2.08

97.00

8.72

T4(2.5)

6.67

1.15

13.67

1.15

222.00

9.85

22.00

2.00

105.00

9.85

T5(5.0)

6.67

0.58

12.00

1.73

219.67

3.06

21.33

3.79

95.33

9.45

PC

245.00

8.19

372.67

10.02

1593.33

71.67

426.00

9.17

794.67

18.93

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

Positive Control:

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

Test Item

Concentration

(mg/plate)

TA 1537

TA 1535

TA 102

TA 98

TA 100

-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

T1

(0.3125)

1.19

0.95

0.95

1.08

0.94

1.03

0.94

1.00

0.93

0.93

T2

(0.625)

0.75

1.16

1.03

1.03

0.94

1.01

1.02

0.89

0.99

0.96

T3

(1.25)

1.06

1.11

1.08

1.03

1.00

1.04

0.97

0.97

0.96

0.93

T4

(2.5)

1.00

0.95

0.98

1.10

0.95

1.00

0.94

0.95

0.93

0.98

T5

(5.0)

1.06

1.00

1.08

1.03

0.99

0.95

0.94

0.86

0.96

0.96

PC

39.25

32.58

26.23

27.20

6.11

6.62

18.11

19.09

7.17

7.30

Trial II – Preincubation Method

Test Item

Concentration

(mg/plate)

TA 1537

TA 1535

TA 102

TA 98

TA 100

-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

T1

(0.3125)

0.85

1.22

1.00

0.95

1.00

1.00

0.98

1.14

0.98

1.03

T2

(0.625)

1.00

0.89

0.88

1.05

0.99

1.00

0.95

1.05

1.03

1.05

T3

(1.25)

0.95

1.22

1.00

1.26

1.03

1.01

0.98

1.05

0.93

1.02

T4

(2.5)

0.95

1.11

1.03

1.05

1.03

1.00

0.94

1.12

0.96

1.11

T5

(5.0)

1.05

1.11

0.93

0.92

1.01

0.99

0.91

1.08

0.98

1.01

PC

34.95

40.83

27.05

28.67

7.22

7.16

12.29

21.66

7.22

8.39

Key: NC = Negative control, VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation.

Conclusions:
The test chemical did not induce gene mutations 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 and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.
Executive summary:

Ames test of 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 The study was performed to evaluate the mutagenic potential of test chemical using Salmonella typhimurium strains TA98, TA100, TA1537, TA1535 and TA102 in Trial I (with 10% v/v S9 mix and without metabolic activation) and Trial II (with 10% v/v S9 mix and without metabolic activation) experiments including the vehicle (distilled water) and concurrent positive controls in triplicates. 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 positive controls both in the presence and absence of metabolic activation in triplicates Based on the solubility test, distilled water was selected as a vehicle for the test item in the study.

In tester strains TA98 and TA100, no reduction in revertant count and no inhibition in background lawn were observed at any of the concentrations, either in the presence or absence of metabolic activation, when compared to the vehicle control data.On the basis of preliminary cytotoxicity test results, main study was performed with the following concentrations: Trial I and II: 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate.

Trial I was performed both in the presence (10% v/v S9 mix) and absence of metabolic activation system along with the vehicle and the positive control with the test item concentration spacing factor of 2. There was no increase in the number of revertant colonies and no inhibition in background lawn up to the highest concentration of 5 mg/plate in the presence (10% v/v S9 mix) or absence of metabolic activation, when compared to the vehicle control data.

Trial II was conducted to confirm the negative results observed in Trial I. Trial II was conducted in the presence and in the absence of metabolic activation system with all the tester strains along with the vehicle and concurrent positive controls according to the preincubation method.

There was no increase in the number of revertant colonies and no background lawn inhibition up to the highest concentration of 5 mg/plate in the presence (10% v/v S9 mix) and absence of metabolic activation, when compared to the vehicle control.

The number of revertant colonies of the vehicle (spontaneous revertant colonies) and positive controls (induced revertant colonies) were in the range of historical data of the lab.The positive controls used in the study produced significant increase in the mean number of revertant colonies in all of the tester strains compared to the control data, thus confirming the validity of the test system to detect specific mutagens in the presence and in the absence of a metabolic activation system.

From the above data It can be concluded that the test chemical did not induce gene mutations 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 and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro study:

In vitro Bacterial gene mutation assay:

Ames test of 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). The study was performed to evaluate the mutagenic potential of test chemical using Salmonella typhimurium strains TA98, TA100, TA1537, TA1535 and TA102 in Trial I (with 10% v/v S9 mix and without metabolic activation) and Trial II (with 10% v/v S9 mix and without metabolic activation) experiments including the vehicle (distilled water) and concurrent positive controls in triplicates. 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 positive controls both in the presence and absence of metabolic activation in triplicates Based on the solubility test, distilled water was selected as a vehicle for the test item in the study.

In tester strains TA98 and TA100, no reduction in revertant count and no inhibition in background lawn were observed at any of the concentrations, either in the presence or absence of metabolic activation, when compared to the vehicle control data.On the basis of preliminary cytotoxicity test results, main study was performed with the following concentrations: Trial I and II: 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate.

Trial I was performed both in the presence (10% v/v S9 mix) and absence of metabolic activation system along with the vehicle and the positive control with the test item concentration spacing factor of 2. There was no increase in the number of revertant colonies and no inhibition in background lawn up to the highest concentration of 5 mg/plate in the presence (10% v/v S9 mix) or absence of metabolic activation, when compared to the vehicle control data.

Trial II was conducted to confirm the negative results observed in Trial I. Trial II was conducted in the presence and in the absence of metabolic activation system with all the tester strains along with the vehicle and concurrent positive controls according to the preincubation method.

There was no increase in the number of revertant colonies and no background lawn inhibition up to the highest concentration of 5 mg/plate in the presence (10% v/v S9 mix) and absence of metabolic activation, when compared to the vehicle control.

The number of revertant colonies of the vehicle (spontaneous revertant colonies) and positive controls (induced revertant colonies) were in the range of historical data of the lab.The positive controls used in the study produced significant increase in the mean number of revertant colonies in all of the tester strains compared to the control data, thus confirming the validity of the test system to detect specific mutagens in the presence and in the absence of a metabolic activation system.

From the above data It can be concluded that the test chemical did not induce gene mutations 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 and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Based on the experimental data available for the target chemical, it does not induce gene mutation in vitro. Hence the target chemical is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the experimental data available for the target chemical, it does not induce gene mutation in vitro. Hence the target chemical is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.