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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test

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.

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:
from 2019-04-12 to 2019-06-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
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:
- Type and composition of metabolic activation system:
An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 10 % (v/v) in the S9 mix. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
- method of preparation of S9 mix : according to Ames et al. (1977)
- concentration or volume of S9 mix and S9 in the final culture medium: 33.3 mg/mL
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
Since the acceptance criterion of at least three concentrations showing no signs of toxic effects was not fulfilled in experiment II in strain TA 100 without S9 mix, this part of experiment II was repeated at the following concentrations (reported as experiment IIa):
Experiment IIa: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Vehicle / solvent:
- Solvent used: DMSO (purity > 99 %)
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the 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 - without S9 mix: TA 1537, TA 98; 2-aminoanthracene, 2-AA - with S9 mix: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 3

METHOD OF TREATMENT/ EXPOSURE:
A pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. Toxicity of the test item results in a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn. The pre-experiment is reported as main experiment I since the acceptance criteria are met.

Experiment I (Plate Incorporation)
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 μL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 μL Bacteria suspension,
2000 μL Overlay agar

Experiment II and IIa (Pre-Incubation)
The following materials were mixed in a test tube and incubated at 37 °C for 60 minutes.
100 μL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 μL Bacteria suspension.
After pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube.

The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.
In parallel to each test a sterile control of the test item was performed. Therefore, 100 μL of the stock solution, 500 μL S9 mix / S9 mix substitution buffer were mixed with 2.0 mL overlay agar and poured on minimal agar plates.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
An increase of revertant colonies equal or above 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.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True 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:
cytotoxicity
Remarks:
at 2500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True 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:
cytotoxicity
Remarks:
at 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True 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:
cytotoxicity
Remarks:
at 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True 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:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No precipitation of the test item occurred in the overlay agar in the test tubes. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I and II at 5000 μg/plate in the absence of S9 mix and from 2500 to 5000 μg/plate in the presence of S9 mix and in experiment IIa at 5000 μg/plate in the absence of S9 mix.
The plates incubated with the test item showed reduced background growth in strains TA 1537, TA 98, and TA 100 in experiments I, II, and IIa, and additionally in strain TA 1535 with S9 mix in experiment II.
Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in strain TA 100 in experiments I and IIa and in strains TA 98 and TA 100 in experiment II.
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 plates incubated with the test item showed reduced background growth at the following concentrations (μg/plate):

Strain

Experiment I

Experiment II

Experiment IIa

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

without S9 mix

TA 1535

/

/

/

5000

n.p.

TA 1537

5000

2500 – 5000

2500 – 5000

2500 – 5000

n.p.

TA 98

2500 – 5000

1000 – 5000

2500 – 5000

1000 – 5000

n.p.

TA 100

2500 – 5000

1000 – 5000

1000 – 5000

1000 – 5000

1000 – 5000

WP2 uvrA

/

/

/

/

n.p.

/ = normal background growth
n.p. = not performed

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (μg/plate):

Strain

Experiment I

Experiment II

Experiment IIa

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

without S9 mix

TA 1535

/

/

/

/

n.p.

TA 1537

/

/

/

/

n.p.

TA 98

/

/

/

2500 – 5000

n.p.

TA 100

/

5000

333 – 5000

1000 – 5000

1000 – 5000

WP2 uvrA

/

/

/

/

n.p.

/ = no toxic effects (induction factor ≥ 0.5)
n.p. = not performed

Summary results from 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 (Solvent control)

 

18 ± 3

14 ± 4

24 ± 6

115 ± 10

51± 5

Untreated control

 

15 ± 1

9 ± 2

29 ± 2

121 ± 15

52 ± 6

Test Item

3 µg

15 ± 1

13 ± 4

25 ± 7

105 ± 2

47 ± 5

10 µg

16 ± 5

15 ± 1

27 ± 6

112 ± 9

58 ± 5

33 µg

17 ± 3

11 ± 7

33 ± 4

120 ± 11

57 ± 5

100 µg

16 ± 1

12 ± 3

30 ± 9

113 ± 16

54 ± 6

333 µg

13 ± 3

14 ± 2

22 ± 1

110 ± 11

51 ± 3

1000 µg

15 ± 1

11 ± 5

19 ± 5

89 ± 3

57 ± 10

2500 µg

14 ± 5

13 ± 2

18 ± 4R

102 ± 7R

50 ± 5

5000 µg

11 ± 1P

8 ± 3R,P

17 ± 5P,R

78 ± 4P,M,R

58 ± 14P

NaN3

10 µg

1308 ± 71

 

 

2083 ± 13

 

4-NOPD

10 µg

 

 

368 ± 9

 

 

4-NOPD

50 µg

 

94 ± 7

 

 

 

MMS

2.0 µL

 

 

 

 

1137± 33

With activation

DMSO (Solvent control)

 

13 ± 4

10 ± 2

32 ± 5

124 ± 15

62± 11

Untreated control

 

17 ± 4

11 ± 5

32 ± 4

137 ± 10

57 ± 11

Test Item

3 µg

12 ± 3

11 ± 0

34 ± 6

134 ± 16

52 ± 9

10 µg

14 ± 4

10 ± 3

31 ± 6

151 ± 9

66 ± 12

33 µg

13 ± 2

15 ± 1

35 ± 6

145 ± 25

55 ± 8

100 µg

14 ± 3

16 ± 2

30 ± 9

141 ± 5

63 ± 12

333 µg

12 ± 3

10 ± 3

26 ± 1

115 ± 9

60 ± 8

1000 µg

11 ± 3

9 ± 3

27 ± 6R

91 ± 6R

55 ± 7

2500 µg

8 ± 2P

7 ± 2R,P

21 ± 7P,R

73 ± 6P,M,R

53 ± 6P

5000 µg

8 ± 1P

7 ± 3R,P

16 ± 2P,R,M

42 ± 7P,M,R

41 ± 6P

2-AA

2.5 µg

440 ± 27

298 ± 36

3576 ± 236

4288 ± 385

 

2-AA

10.0 µg

 

 

 

 

282 ± 13

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

Plate Postfix Codes: R – Reduced background growth, P – Precipitate, M – Manual count.

Summary results from 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 (Solvent control)

 

11 ± 2

13 ± 3

33 ± 8

214 ± 7

47± 8

Untreated control

 

11 ± 1

10 ± 4

40 ± 8

217 ± 19

40 ± 3

Test Item

33 µg

11 ± 4

15 ± 3

28 ± 9

197 ± 26

41 ± 13

100 µg

12 ± 6

11 ± 3

23 ± 3

104 ± 13

38 ± 4

333 µg

9 ± 3

9 ± 5

24 ± 3

82 ± 7

36 ± 6

1000 µg

12 ± 2

11 ± 4

23 ± 2

22 ± 4M,R

32 ± 2

2500 µg

7 ± 3

8 ± 3R

24 ± 5R

18 ± 2M,R

44 ± 7

5000 µg

7 ± 2P

8 ± 3P,R

26 ± 1P,R

18 ± 2P,M,R

34 ± 8P

NaN3

10 µg

1211 ± 65

 

 

1257 ± 44

 

4-NOPD

10 µg

 

 

401 ± 21

 

 

4-NOPD

50 µg

 

98 ± 11

 

 

 

MMS

2.0 µL

 

 

 

 

573± 21

With activation

DMSO (Solvent control)

 

13 ± 5

19 ± 2

45 ± 8

207 ± 12

46± 15

Untreated control

 

14 ± 4

15 ± 1

37 ± 4

209 ± 11

56 ± 5

Test Item

33 µg

13 ± 1

21 ± 5

49 ± 9

214 ± 22

53 ± 10

 

100 µg

12 ± 4

14 ± 3

40 ± 9

141 ± 12

48 ± 6

 

333 µg

13 ± 3

14 ± 3

39 ± 7

105 ± 13

54 ± 3

 

1000 µg

12 ± 4

15 ± 5

42 ± 3R

22 ± 4M,R

41 ± 6

 

2500 µg

11 ± 1P

16 ± 7P,M,R

14 ± 4P,M,R

9 ± 2P,M,R

37 ± 6P

 

5000 µg

10 ± 5P,R

9 ± 2P,M,R

9 ± 1P,R,M

5 ± 2P,M,R

34 ± 6P

2-AA

2.5 µg

417 ± 2

136 ± 22

3787 ± 652

2401 ± 153

 

2-AA

10.0 µg

 

 

 

 

489 ± 3

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

Plate Postfix Codes: R – Reduced background growth, P – Precipitate, M – Manual count.

 

Summary results from Experiment IIa:

Metabolic Activation

Test Group

Dose Level (per plate)

Revertant Colony Counts (Mean ± SD)

TA 100

Without activation

DMSO (Solvent control)

 

102 ± 8

Untreated control

 

120 ± 10

Test Item

3 µg

103 ± 13

10 µg

99 ± 12

33 µg

91 ± 10

100 µg

90 ± 8

333 µg

62 ± 8

1000 µg

39 ± 2M,R

2500 µg

16 ± 4M,R

5000 µg

1 ± 1P,M,R

NaN3

10 µg

1995 ± 86

Positive Controls: NaN3 – sodium azide.

Plate Postfix Codes: R – Reduced background growth, P – Precipitate, M – Manual count.

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

A study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II and IIa) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The test was conducted according to OECD 471 (1997) and EU Method B.13/14 (30 May 2008). The assay was performed in three independent experiments. Experiment I and II were performed with and without liver microsomal activation, experiment II without liver microsomal activation, only. Each concentration, including the controls, was tested in triplicate. 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: 33; 100; 333; 1000; 2500; and 5000 μg/plate

Since the acceptance criterion of at least three concentrations showing no signs of toxic effects was not fulfilled in experiment II in strain TA 100 without S9 mix, this part of experiment II was repeated at the following concentrations (reported as experiment IIa):

Experiment IIa: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

No precipitation of the test item occurred in the overlay agar in the test tubes. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I and II at 5000 μg/plate in the absence of S9 mix and from 2500 to 5000 μg/plate in the presence of S9 mix and in experiment IIa at 5000 μg/plate in the absence of S9 mix. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed reduced background growth in strains TA 1537, TA 98, and TA 100 in experiments I, II, and IIa, and additionally in strain TA 1535 with S9 mix in experiment II. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in strain TA 100 in experiments I and IIa and in strains TA 98 and TA 100 in experiment II.

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

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

OECD 471 - Ames test

A study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II and IIa) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The test was conducted according to OECD 471 (1997) and EU Method B.13/14 (30 May 2008). The assay was performed in three independent experiments. Experiment I and II were performed with and without liver microsomal activation, experiment II without liver microsomal activation, only. Each concentration, including the controls, was tested in triplicate. 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: 33; 100; 333; 1000; 2500; and 5000 μg/plate

Since the acceptance criterion of at least three concentrations showing no signs of toxic effects was not fulfilled in experiment II in strain TA 100 without S9 mix, this part of experiment II was repeated at the following concentrations (reported as experiment IIa):

Experiment IIa: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

No precipitation of the test item occurred in the overlay agar in the test tubes. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I and II at 5000 μg/plate in the absence of S9 mix and from 2500 to 5000 μg/plate in the presence of S9 mix and in experiment IIa at 5000 μg/plate in the absence of S9 mix. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed reduced background growth in strains TA 1537, TA 98, and TA 100 in experiments I, II, and IIa, and additionally in strain TA 1535 with S9 mix in experiment II. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in strain TA 100 in experiments I and IIa and in strains TA 98 and TA 100 in experiment II.

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

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. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) 2019/521.