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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was considered to be non-mutagenic with and without metabolic activation in the bacterial reverse mutation assay, according to OECD 471.

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:
24 February 2017 - 19 June 2017
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:
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 reverse mutation assay
Test material information:
Composition 1
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9, induced by phenobarbitone/p-naphthoflavone
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate
Vehicle:
- Vehicle/solvent used: DMSO
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine / 2-aminoanthracene
Details on test system and conditions:
METHOD OF APPLICATION: plate incorporation test (experiment I), pre-incubation test (experiment II)

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

NUMBER OF REPLICATIONS: 3 (two independent experiments)

DETERMINATION OF CYTOTOXICITY
- Method: counting numbers of revertants
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
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
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:
at a concentration of 5000 µg/plate, induction factor < 0.5
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:
no
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:
at a concentration of 5000 µg/plate, induction factor < 0.5
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:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

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

 

13 ± 1

12 ± 3

28 ± 7

201 ± 37

41 ± 5

Untreated

 

8±2

9±3

34±7

211±6

32±10

Test Item

3 µg

12± 2

12±3

28±4

198±17

41±10

10 µg

12±4

11±1

35±3

203±16

43±7

33 µg

10±3

11±2

29±8

196±18

39±7

100 µg

13±3

12±1

30±4

211±11

40±3

333 µg

15±3

11±3

26±5

217±18

39±6

1000 µg

11±1

10±1

26±1

214±20

33±9

2500 µg

9±4

8±1

26±6

200±22

31±5

5000 µg

12±2

5±1

24±3

151±18

24±6

NaN3

10 µg

1467±38

 

 

2217±69

 

4-NOPD

10 µg

 

 

564±50

 

 

4-NOPD

50 µg

 

118±21

 

 

 

MMS

2.0 µL

 

 

 

 

932± 18

With Activation

DMSO

 

14±5

13±4

34±3

162±22

50±9

Untreated

 

12±3

26±7

43±6

211±24

51±2

Test Item

3 µg

13±3

16±5

41±3

161±13

57±9

10 µg

14±3

13±3

39±2

151±5

49±4

33 µg

14±3

16±3

39±8

166±8

42±4

100 µg

13±2

17±2

37±6

185±23

55±1

333 µg

13±4

15±5

46±8

152±9

46±8

1000 µg

9±2

18±4

36±4

162±10

46±10

2500 µg

13±5

11±2

42±1

196±19

35±7

5000 µg

13±3

13±2

33±4

189±7

33±10

2-AA

2.5 µg

396±6

205±4

4701±749

3889±254

 

2-AA

10.0 µg

 

 

 

 

463±19

NaN3: sodium azide, 2-AA: 2-aminoanthracene, 4 -NOPD: 4-nitro-o-phenylene-diamine, MMS: methyl methane sulfonate

Table 2: Summary of Experiment II

MetabolicActivation

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 ± 3

9 ± 2

26 ± 7

148 ± 19

34 ± 5

Untreated

 

10± 4

10± 5

31±7

206± 7

40± 8

Test Item

33 µg

10± 4

9± 2

25± 8

134± 4

39± 6

100 µg

9± 3

7± 2

26± 6

140± 28

33± 5

333 µg

10± 4

7± 0

22± 3

115±18

39± 5

1000 µg

9± 2

8± 1

22± 3

120± 9

35± 3

2500 µg

9±2

5±1

22± 7

90± 5

24± 4

5000 µg

8± 3

7± 3

21± 5

63± 4

26± 8

NaN3

10 µg

1121± 154

 

 

1888± 171

 

4-NOPD

10 µg

 

 

284± 17

 

 

4-NOPD

50 µg

 

74± 5

 

 

 

MMS

2.0 µL

 

 

 

 

547± 13

With Activation

DMSO

 

12± 2

14± 5

35± 2

120± 9

45± 8

Untreated

 

14± 3

12± 4

40± 9

143± 36

51±6

Test Item

33 µg

13±3

20± 3

39± 6

104± 10

43± 9

100 µg

15±3

12± 4

35± 10

121± 26

42± 5

333 µg

12± 2

15± 6

25± 6

118± 9

44± 10

1000 µg

14± 2

15± 4

42± 9

92±23

41± 7

2500 µg

11± 1

17± 3

34± 8

119±17

27± 9

5000 µg

12± 3

12± 4

37± 1

79± 4

29± 7

2-AA

2.5 µg

343± 12

155± 7

4001± 547

2322± 303

 

2-AA

10.0 µg

 

 

 

 

392±38

NaN3:sodium azide,2-AA: 2-aminoanthracene,4 -NOPD: 4-nitro-o-phenylene-diamine,MMS: methyl methane sulfonate

Table 3: Historical 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

TA 1537

Solvent control

10

2.2

6

19

13

3.5

7

30

Untreated control

11

2.7

5

21

14

4.0

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

26.0

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

Conclusions:
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.
Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

A bacterial reverse mutation assay (OECD 471) was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate) and the pre-incubation test (experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate) using the Salmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia colistrain WP2uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. No precipitation of the test item occurred up to the highest investigated dose. The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.

Only slight toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in strain TA1537 and TA100.

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.

The controls confirmed the validity of the study.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial Reverse Mutation Test

A bacterial reverse mutation assay (OECD 471) was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate) and the pre-incubation test (experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. No precipitation of the test item occurred up to the highest investigated dose. The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.

Only slight toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in strain TA1537 and TA100.

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.

The controls confirmed the validity of the study.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are 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 tenth time in Regulation (EU) No 2017/776.