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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 May 2017 - 10 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
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 forward mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I:
3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment II:
Strains TA 1535 and WP2 uvrA: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
The remaining strains: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Justification for top dose: according to OECD guideline 471
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Controls
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: TA 1537, TA 98 without metabolic activation, 2-aminoanthracene: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: experiment I: in agar (plate incorporation); experiment II: preincubation

DURATION
- Preincubation period: 60 minutes.
- Exposure duration: ≥ 48 hours

NUMBER OF REPLICATIONS: 3 (two independent experiments)

DETERMINATION OF CYTOTOXICITY
- Method: counting numbers of revertants and inspection of the bacterial background lawn
Evaluation criteria:
A test item is considered as a mutagen 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:
no statistical analysis

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations ≥ 333 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:
at concentrations ≥ 100 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:
at concentrations ≥ 333 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:
at concentrations ≥ 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:
at concentrations ≥ 333 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item occurred up to the highest investigated dose.

Any other information on results incl. tables

Table 1: Summary of Experiment I

Metabolic Activation

Test Group

Dose Level (per plate)

Revertant Colony Counts (Mean ± SD)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

Without Activation

DMSO

 

10 ± 4

12 ± 3

22 ± 5

162 ± 12

36 ± 6

Untreated

 

11 ± 4

9 ± 3

25 ± 2

175 ± 24

37 ± 3

Test item

3 µg

12 ± 2

12 ± 2

26 ± 9

151 ± 4

34 ± 7

10 µg

13 ± 3

13 ± 3

25 ± 5

142 ± 13

35 ± 10

33 µg

10 ± 1

11 ± 4

26 ± 5

137 ± 7

40 ± 2

100 µg

9 ± 5

7 ± 4

16 ± 5

110 ± 15

40 ± 9

333 µg

10 ± 4

8 ± 3R

20 ± 6R

54 ± 2R

41 ± 1

1000 µg

7 ± 3

10 ± 2R

17 ± 5R

51 ± 4R

37 ± 6

2500 µg

8 ± 3

8 ± 5R

16 ± 3R

57 ± 10R

40 ± 4

5000 µg

8 ± 2

10 ± 2R

13 ± 1R

35 ± 3M R

41 ± 8

NaN3

10 µg

1284 ± 13

 

 

2059 ± 79

 

4-NOPD

10 µg

 

 

346 ± 17

 

 

4-NOPD

50 µg

 

62 ± 5

 

 

 

MMS

2.0 µL

 

 

 

 

934 ± 30

With Activation

DMSO

 

14 ± 3

18 ± 6

26 ± 7

158 ± 7

47 ± 2

Untreated

 

14 ± 6

16 ± 5

33 ± 4

166 ± 8

54 ± 8

Test item

3 µg

12 ± 3

16 ± 3

32 ± 5

130 ± 26

47 ± 5

10 µg

13 ± 3

18 ± 8

31 ± 4

134 ± 8

39 ± 9

33 µg

13 ± 4

12 ± 1

28 ± 9

143 ± 16

51 ± 8

100 µg

12 ± 5

15 ± 2

32 ± 8

148 ± 9

53 ± 11

333 µg

9 ± 3

10 ± 4R

31 ± 4R

94 ± 24R

40 ± 11

1000 µg

11 ± 3R

4 ± 1M R

16 ± 2R M

25 ± 2R

29 ± 8

2500 µg

6 ± 2R M

3 ± 1M R

1 ± 1M R

1 ± 1R

21 ± 6

5000 µg

7 ± 3M R

3 ± 1M R

0 ± 0R

0 ± 0R

25 ± 2

2-AA

2.5 µg

533 ± 60

154 ± 14

3890 ± 433

4101 ± 294

 

2-AA

10.0 µg

 

 

 

 

444 ± 24

NaN3: sodium azide, 2-AA: 2-aminoanthracene, 4-NOPD: 4-nitro-o-phenylene-diamine, MMS:methyl methane sulfonate, R: Reduced background growth, M: Manual count

Table 2: Summary of Experiment II

Metabolic Activation

Test Group

Dose Level (per plate)

Revertant Colony Counts (Mean ± SD)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

Without Activation

DMSO

 

11 ± 4

8 ± 3

18 ± 4

182 ± 12

41 ± 9

Untreated

 

10 ± 2

10 ± 3

22 ± 1

213 ± 3

44 ± 7

Test item

3 µg

 

9 ± 3

21 ± 5

180 ± 3

 

10 µg

11 ± 5

8 ± 4

18 ± 8

172 ± 4

44 ± 5

33 µg

9 ± 2

10 ± 3

18 ± 3

153 ± 18

46 ± 9

100 µg

10 ± 2

6 ± 1R

19 ± 6

71 ± 25R

35 ± 3

333 µg

9 ± 3

9 ± 3R

18 ± 5R

47 ± 9R

36 ± 8

1000 µg

8 ± 2

8 ± 1R

19 ± 3R

46 ± 6R

31 ± 7

2500 µg

11 ± 3R

10 ± 4R

7 ± 3R M

26 ± 5M R

41 ± 9

5000 µg

10 ± 5R

2 ± 1M R

6 ± 1M R

8 ± 2M R

40 ± 7

NaN3

10 µg

1218 ± 30

 

 

1468 ± 71

 

4-NOPD

10 µg

 

 

290 ± 7

 

 

4-NOPD

50 µg

 

81 ± 6

 

 

 

MMS

2.0 µL

 

 

 

 

537 ± 47

With Activation

DMSO

 

11 ± 2

10 ± 1

34 ± 6

181 ± 13

52 ± 9

Untreated

 

12 ± 1

12 ± 4

38 ± 3

201 ± 3

71 ± 14

Test item

3 µg

 

13 ± 4

30 ± 8

166 ± 14

 

10 µg

14 ± 2

12 ± 4

38 ± 7

171 ± 21

58 ± 11

33 µg

10 ± 2

10 ± 4

38 ± 3

158 ± 5

57 ± 8

100 µg

13 ± 2

11 ± 3

36 ± 9

133 ± 18

62 ± 9

333 µg

7 ± 2M R

3 ± 1R M

6 ± 1R M

71 ± 7M R

36 ± 8R

1000 µg

5 ± 1M R

2 ± 1M R

1 ± 1R

28 ± 9M R

27 ± 4R

2500 µg

3 ± 1M R

0 ± 0R

0 ± 0R

7 ± 2R M

9 ± 2M R

5000 µg

2 ± 1M R

0 ± 0R

0 ± 0R

0 ± 0R

11 ± 1M R

2-AA

2.5 µg

376 ± 1

119 ± 20

3852 ± 420

4094 ± 290

 

2-AA

10.0 µg

 

 

 

 

492 ± 60

NaN3: sodium azide, 2-AA: 2-aminoanthracene, 4-NOPD: 4-nitro-o-phenylene-diamine, MMS:methyl methane sulfonate, R: Reduced background growth, M: Manual count

Table 2: Historicla data

Strain

 

without S9 mix

with S9 mix

Mean

SD

Min

Max

Mean

SD

Min

Max

TA 1535

Solvent control

Untreated control

Positive control

12

12

1130

2.5

3.1

143.1

6

6

334

25

28

1816

12

12

388

2.5

2.9

58.2

7

7

176

26

26

668

TA 1537

Solvent control

Untreated control

Positive control

10

11

82

2.2

2.7

12.7

6

5

43

19

21

157

13

14

191

3.5

4.0

60.8

7

7

83

30

31

434

TA 98

Solvent control

Untreated control

Positive control

25

27

378

4.4

4.9

73.7

13

12

211

43

43

627

34

37

3949

6.2

6.5

771.8

15

11

360

58

57

6586

TA 100

Solvent control

Untreated control

Positive control

156

176

1966

26.0

23.6

293.2

78

79

498

209

217

2767

148

172

3798

32.3

25.4

830.4

73

85

536

208

218

6076

WP2uvrA

Solvent control

Untreated control

Positive control

41

42

798

5.6

5.8

362.7

27

30

319

63

63

4732

50

52

378

6.8

6.8

112.6

28

36

167

72

88

1265

Applicant's summary and conclusion

Conclusions:
Based on the results of this study it is concluded that the test item is non-mutagenic in the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2uvrA in the absence and presence of S9-mix.
Executive summary:

The genetic toxicity of the test item was assessed using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2uvrA, in accordance with OECD 471 guideline and GLP principles in two independent experiments (direct plate assay and a preincubation assay). No precipitation of the test item occurred up to the highest investigated dose. The negative and strain-specific positive control values were within the laboratory background historical control data ranges.

The plates incubated with the test item showed reduced background growth up in all strains with metabolic activation and in all strains, except of strain WP2uvrA without metabolic activation.

Toxic effects, evident as a reduction in the number of revertants, were observed in all strains with metabolic activation and in strains TA 1537, TA 98, and TA 100 without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item 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.

Based on the results of this study it is concluded that the test item is non-mutagenic in the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2uvrA in the absence and presence of S9-mix.