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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a bacterial reverse mutation test (plate incorporation test) according to OECD TG 471 the test item was not mutagenic in the absence and presence of a rat liver metabolizing system (S9 mix) (reference 7.6.1-1).

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:
April 24, 2018- May 04, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
1993
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
All Salmonella typhimurium strains contain mutations in the histidine operon, thus imposing a requirement for histidine in the growth medium. The Escherichia coli strain carries a defect in one of the genes for tryptophan biosynthesis, imposing a requirement for tryptophan in the growth medium.
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:
Due to migration, the value was transferred to one of the current document's attachments
Test concentrations with justification for top dose:
5; 15.8; 50; 158; 500; 1580; 5000 µg/plate (with and without S9 mix)
The test material concentrations used were selected according to the EEC, OECD and Japanese guidelines for this test system.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle for test item: DMSO showed best performance and was thus used for this experiment at a maximum concentration of 17.8 µL/plate.

Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ultrapure water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Daunomycin
Remarks:
without metabolic activation, 2 µg/plate, TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without metabolic activation, 2 µg/plate, E.coli WP2 uvrA
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ultrapure water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation, 2 µg/plate, TA100, TA 1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation, 50 µg/plate, TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
with metabolic activation, all strains
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 3 parallel plates were used for each concentration step of the test material and the positive controls. Twice as many solvent control plates were used for each bacterial strain.

METHOD OF TREATMENT/ EXPOSURE:
- Test substance in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: The incubation of plates was performed at 36 - 38 °C for 2 days.



Evaluation criteria:
A test material was to be defined as positive or mutagenic in this assay if
- the assay is considered valid and
- a biologically relevant increase in the mean number of revertants above a threshold of 2-fold (TA 98, TA 100, WP2 uvrA) or 3-fold (TA 1535, TA 1537) as compared to the concurrent negative controls is observed
- an increase exceeding the threshold at only one concentration is considered as biologically meaningful if reproduced in a second independent experiment
- a concentration-dependent increase is considered biologically meaningful if the threshold is exceeded at more than one concentration
A test material is defined as negative or non-mutagenic in this assay if
- the assay is considered valid and
- none of the above-mentioned criteria are met
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Following treatment with the test item, precipitation of the test material on the agar plates occurred at concentrations > 5000 µg/plate.

Ames test:
- Signs of toxicity : No toxicity to the bacteria was observed.
- Mean number of revertant colonies per plate and standard deviation : see Tables 1 and 2

Table 1: Summary 1st Series

Metabolic Activation

Test Material

Concentr.

[µg/plate]

Revertants per plate (Mean ± SD)

TA 98

TA 100

TA1535

TA 1537

WP2 uvrA

Without Activation

DMSO

 

23±7

121±8

17±6

12±4

37±6

Test item

5.00

21± 7

126 ±6

16 ±5

9±5

33 ±7

15.8

15 ± 2

128 ±9

21 ±8

13±4

43 ±1

50.0

20 ±1

125 ±11

18±2

12±2

42 ±6

158

16 ±7

112 ±23

16±4

13±4

43 ±7

500

20 ±2

121 ±5

19 ±3

9±7

38 ±3

1580

21±7

126 ±15

23±3

10±2

39 ±9

5000

21 ±4E

122 ±16E

16±4E

17±2E

31 ±4E

DAUN

2.00

67 ±1

 

 

 

 

NaN3

2.00

 

1478 ±27

747 ±34

 

 

9-AA

50.0

 

 

 

938±130

 

NQO

2.00

 

 

 

 

1857±103

With Activation

DMSO

 

21 ±8

122 ±13

13±4

12±4

32 ±4

Test item

5.00

19 ±5

119±9

16±3

12±3

31 ± 11

15.8

19 ±6

136 ±2

16±1

11±4

28 ± 12

50.0

13±3

113±10

13±5

11±7

31±3

158

22±10

137±5

12±2

11±3

43±14

500

23±2

142±3

9±1

12±2

36±4

1580

17±2

143±6

12±6

11±2

31±12

5000

26±2E

128±6E

20±7E

9±5E

34±6E

2-AA

2.00

195 ±38

1310±17

 

 

 

2-AA

5.00

 

 

173±17

413±6

 

2-AA

10.0

 

 

 

 

319±18

 Key to Positive Controls                                                        Key to Plate Postfix Codes

NaN3 Sodium azide                                                   E        Precipitation until end of experiment

2-AA 2-Aminoanthracene

9-AA 9-Aminoacridine

DAUN Daunomycin

NQO 4-Nitroquinoline-N-oxide

Table 2 Summary 2nd Series

Metabolic Activation

Test Material

Concentr.

[µg/plate]

Revertants per plate (Mean ± SD)

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Without Activation

DMSO

 

24±12

115±15

23±4

9±5

32±5

Test tem

50.0

26 ±6

106 ±10

17±5

11±6

33±2

158

35 ±9

121±7

23±1

13±7

40±5

500

27±4

126 ±4

20 ±5

8±5

44 ±8

1580

28±1

115 ±9

20±3

7±1

42 ±6

5000

28 ±4E

136 ±10E

24±5E

12±5E

36 ±6E

DAUN

2.00

243±64

 

 

 

 

NaN3

2.00

 

1728±58

957±15

 

 

9-AA

50.0

 

 

 

2031±574

 

NQO

2.00

 

 

 

 

1785±210

With Activation

DMSO

 

31±11

120 ±12

15±7

10±4

40±6

Test item

50.0

28±3

130±15

19±4

15±3

43±5

158

25±3

132±13

18±4

10±2

43±2

500

29±8

148±12

16±5

7±2

42±3

1580

41±6

143±12

18±7

12±3

41±8

5000

55±3

125±14

19±5

9±3

33±5

2-AA

2.00

485±55

788±72

 

 

 

2-AA

5.00

 

 

247±47

219±24

 

2-AA

10.0

 

 

 

 

330±28

Key to Positive Controls                                                        Key to Plate Postfix Codes

NaN3 Sodium azide                                                   E        Precipitation until end of experiment

2-AA 2-Aminoanthracene

9-AA 9-Aminoacridine

DAUN Daunomycin

NQO 4-Nitroquinoline-N-oxide

 

Table 3: Historical Data

The historical data have been obtained in experiments between 01/2017 and 12/2017.

 

Negative Controls

Strain

TA 98

TA 100

TA 1537

WP2 uvrA

TA 1538

S9 Mix

Without

With

Without

With

Without

With

Without

With

Without

With

Compound

Solvent

Solvent

Solvent

Solvent

Solvent

Solvent

Solvent

Solvent

Solvent

Solvent

Total Plates

486

486

482

488

394

394

434

433

44

44

Number of Values

90

90

89

90

68

68

77

77

11

11

Minimum

19

20

94

90

4

6

19

28

6

11

Maximum

52

58

159

172

11

16

44

47

15

21

Mean

36

42

118

123

8

10

30

3

12

15

Standard Deviation

7.1

7.3

12.1

13.4

1.5

2.3

4.9

4.3

2.9

2.7

 

Positive Controls

Strain

TA 98

TA 100

TA 1537

WP2 uvrA

TA 1538

S9 Mix

Without

With

Without

With

Without

With

Without

With

Without

With

Compound

DAUN

2-AA

NaN3t

2-AA

9-AA

2-AA

NQO

2-AA

2-NF

2-AA

Total Plates

243

243

242

244

203

203

217

217

22

22

Number of Values

90

90

89

90

70

70

77

77

11

11

Minimum

68

112

821

437

247

72

317

154

1087

461

Maximum

779

3015

2376

3429

1485

705

2275

696

2511

1323

Mean

243

738

1550

1386

736

293

1677

353

1909

1038

Standard Deviation

134.2

508.4

213.6

724.3

284.9

161.7

381.9

129.2

470.3

285.3

 

Table 4: Optical Density

 Salmonella typhimurium

Optical Density at 650 nm

(Range 0.75- 1.45)

 

TA 98

TA 100

TA 1535

TA 1537

1stseries

1.093

0.979

1.033

1.032

2ndseries

1.123

1.006

1.075

1.037

  

Escherichia coli

Optical Density at 650 nm

(Range 0.8-1.2)

 

WP2 uvrA

1stseries

0.981

2ndseries

1.074

 

Conclusions:
The test item did not induce gene mutations by base-pair or frameshift changes in the genome of the strains used. Therefore, it was concluded that with and without addition of S9 mix as the exogenous metabolizing system, the test item was not mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation test.
Executive summary:

The test item was examined for its mutagenic activity in an in vitro bacterial reverse mutation test according OECD TG 471 employing Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA as indicator organisms. The plate incorporation test with and without addition of liver S9 mix from Phenobarbital/p-Naphthoflavone-pretreated rats was used. In this study, two experimental series were performed. In the experiments with S9 mix, 10% and 20% S9 in the S9 mix were used. Treatments of all tester strains were performed using formulations prepared in DMSO in the absence and in the presence of S9 mix, using final concentrations between 5 and 5000 µg/plate, plus vehicle and positive controls.

The mean numbers of revertant colonies of the current negative controls were within the range of historical negative control values.

The strain-specific positive controls, namely daunomycin, sodium azide, 4-nitroquinolin-N-oxide, and 9-aminoacridine in the absence of S9 mix, yielded the expected mutant frequencies that were greatly exceeding the negative controls. The genotype of the tester strains used was thus confirmed. 2-Aminoanthracene which requires metabolic activation, was strongly mutagenic. This indicates that the exogenous metabolizing system used in the present investigation (S9 mix) was active. Thus, the requirements predetermined have been met in total and the study is considered valid. Following treatment with the test item, precipitation of the test material on the agar plates occurred at concentrations > 5000 µg/plate. No toxicity to the bacteria was observed.

Under the conditions described, there were no relevant increases in revertant numbers observed after exposure to the test item in the absence and presence of S9 mix.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The test item was examined for its mutagenic activity in an in vitro bacterial reverse mutation test according OECD TG 471 employing Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA as indicator organisms.The plate incorporation test with and without addition of liver S9 mix from Phenobarbital/p-Naphthoflavone-pretreated rats was used. In this study, two experimental series were performed. In the experiments with S9 mix, 10% and 20% S9 in the S9 mix were used.Treatments of all tester strains were performed using formulations prepared in DMSO in the absence and in the presence of S9 mix, using final concentrations between 5 and 5000 µg/plate, plus vehicle and positive controls.

The mean numbers of revertant colonies of the current negative controls were within the range of historical negative control values.

The strain-specific positive controls, namely daunomycin, sodium azide, 4-nitroquinolin-N-oxide, and 9-aminoacridine in the absence of S9 mix, yielded the expected mutant frequencies that were greatly exceeding the negative controls. The genotype of the tester strains used was thus confirmed. 2-Aminoanthracene which requires metabolic activation, was strongly mutagenic. This indicates that the exogenous metabolizing system used in the present investigation (S9 mix) was active. Thus, the requirements predetermined have been met in total and the study is considered valid.Following treatment with the test item, precipitation of the test material on the agar plates occurred at concentrations > 5000 µg/plate. No toxicity to the bacteria was observed.

Under the conditions described, there were no relevant increases in revertant numbers observed after exposure tothe test item in the absence and presence of S9 mix (reference 7.6.1-1).

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.