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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:
02 - 23 Nov 2016
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:
adopted 21 Jul 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
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
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:
1, 3, 10, 33, 100, 333 and 1000 µg/plate with and without metabolic activation for TA 98 and TA 100
10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation for TA 1535, TA 1537 and WP2 uvr A
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)
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.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I: -S9: starting at 2500 µg/plate; +S9: starting at 1000 µg/plate Experiment II: -S9: -; +S9: at 5000 mg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I: -S9 and +S9: starting at 2500 µg/plate Experiment II: -S9: starting at 1000 µg/plate; +S9: starting at 2500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I: -S9 and +S9: starting at 333 µg/plate Experiment II: -S9 and +S9: at 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I: -S9 and +S9: starting at 333 µg/plate Experiment II: -S9: at 33µg/plate; +S9: at 333 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I: -S9: - ;+S9: starting at 2500 µg/plate Experiment II: -S9 and +S9: starting at 2500 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 item precipitated in the overlay agar in the test tubes from 1000 up to 5000 μg/plate. No precipitation of the test item was observed in the overlay agar on the incubated agar plates.


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


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): Please refer to table 1 in "Any other information on results incl. tables".

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: Historical control data

Strain

 

 

without S9 mix

 

with S9 mix

 

Mean

SD

Min

Max

Mean

SD

Min

Max

 

 

TA 1535

Solvent control

12

2.5

6

25

12

2.5

7

26

Untreated control

12

3.1

6

28

12

2.9

7

26

Positive control

1130

143.1

334

1816

388

58.2

176

668

 

 

TA1537

Solvent control

10

2.2

6

19

13

3.5

7

30

Untreated control

11

2.7

5

21

14

4

7

31

Positive control

82

12.7

43

157

191

60.8

83

434

 

 

TA 98

Solvent control

25

4.4

13

43

34

6.2

15

58

Untreated control

27

4.9

12

43

37

6.5

11

57

Positive control

378

73.7

211

627

3949

771.8

360

6586

 

 

TA 100

Solvent control

156

26

78

209

148

32.3

73

208

Untreated control

176

23.6

79

217

172

25.4

85

218

Positive control

1966

293.2

498

2767

3798

830.4

536

6076

 

 

WP2uvrA

Solvent control

41

5.6

27

63

50

6.8

28

72

Untreated control

42

5.8

30

63

52

6.8

36

88

Positive control

798

362.7

319

4732

378

112.6

167

1265

Mean = mean value of revertants/plate

SD = standard deviation

Min = minimal value/Max = maximal value                             

Data from November 2014 until November 2016 representing approx. 600 experiments (WP2 uvrA the historical data are based on approx. 350 experiments).

Table 2: Test results of Experiment I (plate incorporation).

 

Number of revertant colonies per plate

S9-Mix

Without

Test substance (µg/plate)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvr A

Solvent control (DMSO)

18 ± 3

13 ± 1

25 ± 4

134 ± 12

36 ± 8

Negative control (untreated)

15 ± 1

11 ± 6

26 ± 8

162 ± 15

38 ± 11

3

16 ± 5

9 ± 5

26 ± 7

125 ± 10

38 ± 4

10

17 ± 3

12 ± 2

24 ± 8

104 ± 6

41 ± 2

33

17 ± 2

12 ± 2

30 ± 6

83 ± 4

29 ± 7

100

22 ± 10

17 ± 3

29 ± 3

62 ± 6 r

40 ± 2

333

14 ± 3 rm

13 ± 2

7 ± 2 rm

2 ± 1 r

30 ± 2

1000

9 ± 1 rm

7 ± 2 rm

0 ± 1 rm

0 ± 0 r

30 ± 5

2500

8 ± 2 rm

2 ± 1 rm

0 ± 0 rm

1 ± 1 r

32 ±1 r

5000

4 ± 2 rm

0 ± 0 rm

0 ± 0 rm

0 ± 1 r

21 ± 6 r

Positive Control

NaN3

4-NOPD

4-NOPD

NaN3

MMS

Dose (µg/plate)

10

50

10

10

2

Number of revertant colonies/plate

1607 ± 89

100 ± 9

426 ± 35

2404 ± 194

1013 ± 144

S9-Mix

With

Test substance (µg/plate)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvr A

Solvent

control (DMSO)

16 ± 3

15 ± 6

35 ± 6

105 ± 8

53 ± 10

Negative

control (untreated)

11 ± 5

15 ± 3

43 ± 6

136 ± 11

48 ± 1

3

17 ± 5

18 ± 8

43 ± 1

94 ± 13

50 ± 8

10

16 ± 2

21 ± 4

30 ± 3

107 ± 8

51 ± 3

33

16 ± 6

12 ± 3

29 ± 2

83 ± 18

44 ± 16

100

14 ± 2

16 ± 6

32 ± 7

60 ± 7

41 ± 14

333

11 ± 5

17 ± 2

15 ± 5 rm

15 ± 3 rm

37 ± 8

1000

7 ± 2 rm

9 ± 2 rm

1 ± 1 rm

1 ± 1 r

25 ± 5

2500

7 ± 2 rm

5 ± 1 rm

0 ± 0 rm

0 ± 0 r

18 ± 4 r

5000

4 ± 2 rm

4 ± 1 rm

0 ± 0 rm

0 ± 0 r

6 ± 1r

Positive

Control

2-AA

2-AA

2-AA

2-AA

2-AA

Dose (µg/plate)

2.5

2.5

2.5

2.5

10

 Number of revertant colonies/plate

396 ± 51

121 ± 5

4563 ± 298

2777 ± 315

439 ± 51

NaN3: sodium acide; 2-AA: 2-Aminoanthracene; 4-NOPD: 4-nitro-o-phenylene-diamine; MMS: methyl methane sulfonate

r: reduced background growth

m: manual count

 

 

 Table 2: Results of Experiment II (pre-incubation)

 

Number of revertant colonies per plate

S9-Mix

Without

Test substance (µg/plate)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvr A

Solvent control (DMSO)

10 ± 2

11 ± 5

31 ± 5

169 ± 12

41 ± 2

Negative control (untreated)

11 ± 1

16 ± 5

29 ± 3

203 ± 11

42 ± 6

1

 

 

27 ± 7

157± 20

 

3

 

 

24 ± 8

168 ± 6

 

10

9 ± 2

11 ± 4

36 ± 5

169 ± 15

37 ± 6

33

10 ± 1

8 ± 2

19 ±2

70 ± 10

38 ± 10

100

11 ± 4

8 ± 2

25 ± 3

69 ± 13

35 ± 3

333

7 ± 3

11 ± 2

16 ± 2

43 ± 6 r

22 ± 3

1000

5 ± 1 rm

3 ± 1 rm

2 ± 1

1 ± 1 rm

26 ± 5

2500

6 ± 2 rm

1 ± 1 rm

 

 

16 ± 4 rm

5000

5 ± 1 rm

0 ± 1 rm

 

 

10 ± 2 rm

Positive Control

NaN3

4-NOPD

4-NOPD

NaN3

MMS

Dose (µg/plate)

10

50

10

10

2

Number of revertant colonies/plate

1761 ± 16

93 ± 6

548 ± 32

2534 ± 43

1052 ± 96

S9-Mix

With

Test substance (µg/plate)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvr A

Solvent control (DMSO)

13 ± 6

13 ± 3

33 ± 6

156 ± 11

52 ± 8

Negative control (untreated)

13 ± 1

16 ± 4

47 ± 8

206 ± 6

54 ± 11

1

 

 

33 ± 13

167± 1

 

3

 

 

38 ± 6

149 ± 10

 

10

10 ± 4

14 ± 4

39 ± 10

161 ± 4

49 ± 10

33

14 ± 3

15 ± 4

44 ± 8

137 ± 18

48 ± 6

100

10 ± 4

17 ± 2

39 ± 9

85 ± 4

52 ± 10

333

9 ± 2

18 ± 2

25 ± 7 r

28 ± 7 rm

47 ± 9

1000

11 ± 1

18 ± 4 r

1 ± 1 rm

0 ± 1 rm

28 ± 3

2500

7 ± 1 rm

4 ± 1 rm

 

 

17 ± 5 rm

5000

6 ± 1 rm

2 ± 1 rm

 

 

11 ± 2 rm

Positive Control

2-AA

2-AA

2-AA

2-AA

2-AA

Dose (µg/plate)

2.5

2.5

2.5

2.5

10

Number of revertant colonies/plate

453 ± 31

109 ± 2

5475 ± 288

4801 ± 140

385 ± 10

NaN3: sodium acide; 2AA: 2-Aminoanthracene; 4-NOPD: 4-nitro-o-phenylene-diamine; MMS: methyl methane sulfonate

r: reduced background growth

m: manual count

 

 

 

 

 

Conclusions:
Under the conditions of the conducted test the substance was not mutagenic in any of the five bacterial strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) tested with and without metabolic activation.
Executive summary:

This study was performed to investigate the potential of test substance to induce gene mutations according to the plate incorporation test (experiment I) and the preincubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:

Strains TA 98 and TA 100: 1; 3; 10; 33; 100; 333; and 1000 µg/plate

Strains TA 1535, TA 1537, and WP2 uvrA: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

No Precipitation of the test item was observed in the overlay agar on the incubated agar plates. 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.

No substantial increase in revertant colony numbers of any of the five tester strains was observed at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used and is considered to be non-mutagenic.

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 (2017). In this study the substance was not mutagenic in any of the five bacterial strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvr A) 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.