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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:
19 May - 6 Jun 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21 Jul 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon (for S. typhimurium strains)
trp operon (for E. coli strain)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
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 for TA1535 and WP2 uvrA
10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation for TA1537 and TA98
0.3, 1, 3, 10, 33, 100, 333 and 1000 µg/plate with and without metabolic activation for TA100
Vehicle / solvent:
- 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.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA)
Remarks:
2-AA (+S9, 2.5 µg/plate for TA1535, TA1537, TA98, TA100; 10 µg/plate for WP2 uvrA); 4-NOPD (-S9, 10 µg/plate for TA98; 50 µg/plate for TA1537)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (Experiment 1); preincubation (Experiment 2)

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

NUMBER OF REPLICATIONS: 3 replications each in 2 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 a 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 / choice of top concentrations:
cytotoxicity
Remarks:
Exp. 1: +S9: starting at 1000 µg/plate; Exp. 2: -S9: 5000 µg/plate, + S9: starting at 2500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp. 1: -S9: starting at 2500 µg/plate, +S9: starting at 1000 µg/plate; Exp. 2: -S9 and +S9: starting at 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp. 1 and 2: -S9 and +S9: starting at 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp. 1: -S9 and +S9: starting at 100 µg/plate; Exp. 2: -S9: starting at 33 µg/plate, +S9 : starting at 100 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp. 2: +S9: starting at 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate with metabolic activation in both experiments. Precipitation of the test substance in the overlay agar on the incubated agar plates was observed at 5000 µg/plate with metabolic activation in both experiments.

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

ADDITIONAL INFORMATION ON CYTOTOXICITY: The plates incubated with the test substance showed reduced background growth in all tester strains used. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all tester strains used.

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 ± SD)

Base-pair substitution type

Frameshift type

TA1535

TA100

WP2 uvrA

TA1537

TA98

-

0 (DMSO)

8 ± 3

183 ± 7

40 ± 6

7 ± 2

32 ± 8

-

0

13 ± 1

186 ± 20

37 ± 6

9 ± 2

32 ± 4

-

3

11 ± 2

168 ± 15

29 ± 6

10 ± 3

26 ± 8

-

10

10 ± 4

185 ± 15

42 ± 8

11 ± 4

34 ± 5

-

33

9 ± 2

115 ± 17

42 ± 11

11 ± 5

28 ± 3

-

100

9 ± 5

60 ± 10R

42 ± 1

7 ± 3

24 ± 2

-

333

10 ± 5

50 ± 13R

38 ± 1

13 ± 2

22 ± 6

-

1000

7 ± 3

37 ± 1R

33 ± 8

8 ± 3

23 ± 9R

-

2500

7 ± 3

19 ± 5M, R

31 ± 5

9 ± 6R

19 ± 6R

-

5000

10 ± 3

13 ± 3M, R

32 ± 9

3 ± 1R

15 ± 5R

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 plates ± SD)

952 ± 61

2025 ± 39

729 ± 15

79 ± 1

435 ± 12

+

0 (DMSO)

9 ± 6

161 ± 13

47 ± 6

12 ± 6

31 ± 1

+

0

14 ± 3

180 ± 17

49 ± 8

15 ± 2

45 ± 10

+

3

13 ± 5

164 ± 8

48 ± 13

12 ± 4

38 ± 9

+

10

9 ± 4

163 ± 13

48 ± 8

15 ± 5

33 ± 8

+

33

9 ± 2

181 ± 25

46 ± 6

15 ± 3

44 ± 1

+

100

9 ± 5

44 ± 7R

38 ± 5

15 ± 5

31 ± 5

+

333

12 ± 5

34 ± 12R

44 ± 3

13 ± 3

33 ± 11

+

1000

10 ± 5R

30 ± 3R

32 ± 5

14 ± 3R

43 ± 13R

+

2500

6 ± 1R

27 ± 9R

25 ± 9

17 ± 3R

30 ± 2R

+

5000

5 ± 2P, R

13 ± 3P, M, R

29 ± 3P

12 ± 2P, R

4 ± 1P, R, M

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 plates ± SD)

430 ± 17

3889 ± 175

369 ± 2

186 ± 3

4431 ± 443

NaN3: sodium azide

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

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

M: manual count

P: precipitate

R: reduced background growth

 

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 ± SD)

Base-pair substitution type

Frameshift type

TA1535

TA100

WP2 uvrA

TA1537

TA98

-

0 (DMSO)

15 ± 4

168 ± 11

43 ± 3

9 ± 3

31 ± 6

-

0

12 ± 3

189 ± 36

32 ± 6

7 ± 3

36 ± 8

-

0.3

-

178 ± 31

-

-

-

-

1

-

156 ± 14

-

-

-

-

3

-

174 ± 9

-

-

-

-

10

-

144 ± 19

-

10 ± 4

23 ± 7

-

33

10 ± 1

45 ± 18

40 ± 12

11 ± 2

20 ± 3

-

100

11 ± 2

60 ± 4R

39 ± 4

10 ± 1

21 ± 3

-

333

12 ± 4

26 ± 1R

33 ± 6

11 ± 2

29 ± 7

-

1000

12 ± 3

16 ± 3M, R

33 ± 14

13 ± 1R

27 ± 6R

-

2500

10 ± 1

-

27 ± 3

9 ± 2R

18 ± 2R

-

5000

5 ± 1

-

24 ± 5

3 ± 1M, R

1 ± 1M, R

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 plates ± SD)

1175 ± 69

1824 ± 144

676 ± 35

71 ± 12

334 ± 12

+

0 (DMSO)

14± 6

185 ± 9

49 ± 2

13 ± 4

37 ± 8

+

0

8± 3

208 ± 8

48 ± 12

13 ± 3

44 ± 3

+

0.3

-

183 ± 18

-

-

-

+

1

-

183 ± 18

-

-

-

+

3

-

186 ± 28

-

-

-

+

10

-

184 ± 23

-

11 ± 4

42 ± 6

+

33

17 ± 5

152 ± 12

51 ± 6

10 ± 1

45 ± 6

+

100

17 ± 7

62 ± 9

59 ± 7

19 ± 3

33 ± 8

+

333

9 ± 3

30 ± 4R

43 ± 3

18 ± 2

29 ± 9

+

1000

7 ± 2

5 ± 1P, M, R

45 ± 2R

17 ± 3R

21 ± 1R

+

2500

4 ± 2

-

29 ± 6R

15 ± 3M, R

1 ± 1M, R

+

5000

3 ± 1P, M

-

18 ± 3P, M

9 ± 2P, M, R

1 ± 1P, M, R

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 plates ± SD)

365 ± 21

5342 ± 111

436 ± 26

191 ± 16

191 ± 16

NaN3: sodium azide

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

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

M: manual count

P: precipitate

R: reduced background growth

Conclusions:
Under the conditions of the conducted 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.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

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 up to 5000 µg/plate or cytotoxic concentrations.

Justification for classification or non-classification

The available data on genetic toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008.