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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Ames test (OECD 471): negative with S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA with and without metabolic activation.
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:
16 Feb - 15 Mar 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21 July 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
his operon (for S. typhimurium strains)
trp operon (for E. coli strain)
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone
Test concentrations with justification for top dose:
Pre-experiment: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation

The pre-experiment is reported as Experiment 1.

Experiment 2: 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation

Since the criteria of the acceptability of the assay (minimum of five analyzable doses) were not met in Experiment 2, the experiment was repeated and named as Experiment 2a.

Experiment 2a: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation
Vehicle:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to bacteria.
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA), methylmethanesulfonate (MMS)
Remarks:
+S9: 2-AA (2.5 µg/plate, TA1535, TA1537, TA98, TA100; 10 µg/plate, WP2 uvrA); -S9: sodium azide (10 µg/plate, TA1535, TA100); 4-NOPD (10 µg/plate, TA98; 50 µg/plate, TA1537); MMS (2 µL/plate, WP2 uvrA)
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (Experiment 1); preincubation (Experiment 2 and 2a)

DURATION
- Preincubation period: 1 h
- Exposure duration: at least 48 h

NUMBER OF REPLICATIONS: 3 replications each in 3 independent experiment

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn
Evaluation criteria:
A test substance is considered as mutagenic if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) 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.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second 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 second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
Mean values and standard deviations were calculated.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
exp. 2 and 2a: starting at 2500 µg/plate with and without S9 mix
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
exp. 2 and 2a: starting at 2500 µg/plate with and without S9 mix
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
exp. 2 and 2a: starting at 2500 µg/plate with and without S9 mix
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
exp. 2 and 2a: starting at 1000 µg/plate with and without S9 mix
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
(exp. 2: starting at 2500 µg/plate with and without S9 mix; exp. 2a: starting at 2500 µg/plate with S9 mix and at 5000 µg/plate without S9 mix)
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test substance occurred up to the highest investigated dose.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment is reported as Experiment I (all strains were tested in the pre-experiment).

ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation in Experiment 2 and 2a.

Table 1. Test results of Experiment 1 (plate incorporation).

With or without S9-Mix

Test substance concentration [μg/plate]

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA1535

TA100

WP2 uvrA

TA1537

TA98

-

0 (DMSO)

11 ± 4

152 ± 7

39 ± 9

10 ± 3

28 ± 5

-

0

10 ± 3

180 ± 4

46 ± 14

10 ± 3

32 ± 4

-

3

12 ± 6

147 ± 16

32 ± 7

8 ± 2

23 ± 3

-

10

12 ± 5

140 ± 7

48 ± 12

10 ± 6

23 ± 7

-

33

12 ± 2

169 ± 10

36 ± 1

10 ± 4

24 ± 6

-

100

13 ± 3

174 ± 26

41 ± 1

9 ± 3

28 ± 3

-

333

10 ± 2

190 ± 25

48 ± 8

10 ± 2

31 ± 10

-

1000

10 ± 4

147 ± 13

39 ± 7

8 ± 3

32 ± 10

-

2500

5 ± 0

94 ± 27

43 ± 4

6 ± 1

19 ± 3

-

5000

10 ± 5

178 ± 19

52 ± 8

4 ± 2

21 ± 4

Positive controls, –S9

Name

NaN3

NaN3

MMS

4-NOPD

4-NOPD

Concentration

[μg/plate]

10

10

2.0 µL

50

10

Mean No. of colonies/plate

(average of 3 ± SD)

1205 ± 64

2375 ± 120

1045 ± 48

91 ± 15

387 ± 7

+

0 (DMSO)

13 ± 4

125 ± 16

50 ± 11

9 ± 1

32 ± 9

+

0

14 ± 4

175 ± 26

51 ± 10

9 ± 1

42 ± 8

+

3

14 ± 4

112 ± 14

43 ± 13

11 ± 1

40 ± 3

+

10

14 ± 4

109 ± 17

48 ± 9

11 ± 6

37 ± 6

+

33

15 ± 1

122 ± 15

44 ± 1

7 ± 3

31 ± 2

+

100

13 ± 6

132 ± 16

47 ± 5

12 ± 3

33 ± 9

+

333

13 ± 1

142 ± 14

49 ± 4

10 ± 3

34 ± 2

+

1000

14 ± 5

159 ± 17

60 ± 5

7 ± 3

30 ± 10

+

2500

14 ± 3

93 ± 25

69 ± 7

5 ± 1

25 ± 6

+

5000

15 ± 1

158 ± 11

49 ± 7

9 ± 5

37 ± 15

Positive controls, +S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentration

[μg/plate]

2.5

2.5

10

2.5

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

433 ± 44

3819 ± 36

359 ± 37

198 ± 26

5032 ± 617

DMSO: dimethyl sulfoxide

NaN3: sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

Table 2. Test results of Experiment 2 (preincubation).

With or without S9-Mix

Test substance concentration (μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA1535

TA100

WP2 uvrA

TA1537

TA98

-

0 (DMSO)

9 ± 1

128 ± 8

42 ± 4

9 ± 1

23 ± 4

-

0

7 ± 3

194 ± 15

37 ± 7

8 ± 3

24 ± 8

-

33

11 ± 3

143 ± 6

32 ± 4

8 ± 2

27 ± 4

-

100

8 ± 2

130 ± 17

39 ± 6

11 ± 5

18 ± 3

-

333

10 ± 3

117 ± 24

38 ± 12

11 ± 1

26 ± 10

-

1000

9 ± 1

74 ± 6

34 ± 4

10 ± 3

24 ± 6

-

2500

0 ± 0

1 ± 1

1 ± 1

2 ± 1

1 ± 1

-

5000

1 ± 2

0 ± 0

1 ± 1

1 ± 1

0 ± 1

Positive controls, –S9

Name

NaN3

NaN3

MMS

4-NOPD

4-NOPD

Concentration

[μg/plate]

10

10

2.0 µL

50

10

Mean No. of colonies/plate

(average of 3 ± SD)

1118 ± 46

1066 ± 73

639 ± 49

93 ± 14

424 ± 21

+

0 (DMSO)

12 ± 5

96 ± 7

45 ± 5

16 ± 4

35 ± 7

+

0

10 ± 3

189 ± 12

51 ± 3

11 ± 5

43 ± 9

+

33

12 ± 6

93 ± 13

53 ± 7

17 ± 4

33 ± 5

+

100

9 ± 3

103 ± 28

49 ± 9

12 ± 1

23 ± 4

+

333

13 ± 3

83 ± 22

46 ± 10

17 ± 5

29 ± 7

+

1000

7 ± 3

42 ± 14

35 ± 6

12 ± 4

34 ± 13

+

2500

1 ± 1

2 ± 2R

1 ± 1

0 ± 0R

4 ± 2R

+

5000

1 ± 1

1 ± 1R

0 ± 0

0 ± 0R

1 ± 1R

Positive controls, +S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentration

[μg/plate]

2.5

2.5

10

2.5

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

422 ± 6

1606 ± 371

501 ± 38

83 ± 11

4628 ± 345

DMSO: dimethyl sulfoxide

NaN3: sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

R: reduced background growth

Conclusions:
Under the conditions of the conducted Ames test the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) tested with and without metabolic activation.
Executive summary:
The mutagenicity of the test substance was studied with four mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and the Escherichia coli strain WP2 uvrA according to OECD 471. The investigations were carried out using the plate incorporation test and the pre-incubation test with and without liver homogenate (S9). In the concentration range investigated, the test substance did not induce a significant increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system. In conclusion, these results indicate that the test substance under the experimental conditions described, was not mutagenic to Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and the Escherichia coli strain WP2 uvrA in the presence and absence of a metabolizing system.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

A bacterial gene mutation assay with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (2016). In this study the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) tested with and without metabolic activation.

Justification for classification or non-classification

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighth time in Regulation (EU) No 2016/918.