Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
The test item was considered to be negative in the Ames test.
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 2015-11-18 to 2015-11-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
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)
Remarks:
ENVIGO CRS GmbH, In den Leppsteinswiesen 19, 64380 Roßdorf
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:
Phenobarbital/β-naphthoflavone induced rat liver S9 mix
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(s)/solvent(s) used: DMSO
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9: Sodium azide (NaN3; TA 1535, TA 100), 4-nitro-o-phenylene-diamine (4-NOPD; TA 1537, TA 98), methyl methane sulfonate (MMS; WP2 uvrA); +S9: 2-aminoanthracene (2-AA; TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA)
Details on test system and 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

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

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.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
In experiment II toxic effects occured in strains TA 1537, TA 98 and TA 100.
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item occurred up to the highest investigated dose in the overlay agar in the test tubes. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 μg/plate in experiment II with and without S9 mix. The undissolved particles had no influence on the data recording.

COMPARISON WITH HISTORICAL CONTROL DATA: Done

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No 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 in experiment I. In experiment II toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in strains TA 1537, TA 98, and TA 100.

Table 1: Toxic effects, evidence 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

 

Without S9 mix

With S9 mix

Without S9 mix

With S9 mix

TA 1535

/

/

/

/

TA 1537

/

/

5000

5000

TA 98

/

/

5000

/

TA 100

/

/

2500-5000

/

WP2 uvrA

/

/

/

/

/ = no toxic effects, evident as a reduction in the number of revertants (below the induction factor 0.5)

Table 2: Summary of Experiment I (plate incorporation)

Test group

Dose level (µg/plate)

Revertant Colony Counts (Mean +/- SD)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

DMSO

 

11+/- 2

16 +/- 3

12 +/- 3

12 +/- 4

23 +/- 6

32 +/- 6

141 +/- 14

146 +/- 4 *

39 +/- 2

46 +/- 3

Untreated

 

8 +/- 3

12 +/- 4

10 +/- 4

17 +/- 3

23 +/- 2

34 +/- 9

154 +/- 9

182 +/- 11 *

42 +/- 9

49 +/- 8

Test item

3

9 +/- 3

16 +/- 4

10 +/- 3

12 +/- 3

25 +/- 5

38 +/- 7

133 +/- 29

134 +/- 16*

36 +/- 6

49 +/- 4

10

12 +/-3

11 +/- 4

12 +/- 5

16 +/- 6

21 +/- 1

52 +/- 5

122 +/- 8

123 +/- 13*

38 +/- 4

53 +/- 10

33

14 +/- 2

15 +/- 2

9 +/-3

11 +/- 3

28 +/- 1

36 +/- 4

124 +/- 9

148 +/- 26*

44 +/- 1

45 +/- 8

100

12 +/- 2

13 +/- 1

10 +/- 1

12 +/- 7

24 +/- 6

39 +/- 3

138 +/- 22

142 +/- 7*

42 +/- 5

50 +/- 5

333

11 +/- 5

11 +/- 2

8 +/- 2

12 +/- 2

21 +/- 1

45 +/- 8

132 +/- 14

143 +/- 15*

40 +/- 8

53 +/- 8

1000

11 +/- 3

11 +/- 3

11 +/- 3

13 +/- 4

22 +/- 2

32 +/- 5

135 +/- 25

140 +/- 20*

43 +/- 10

55 +/- 15

2500

9 +/- 1

10 +/- 2

8 +/- 2

13 +/- 4

19 +/- 5

35 +/- 9

84 +/- 17

121 +/- 17*

40 +/- 10

48 +/- 4

5000

8 +/- 1

12 +/- 6

6 +/- 3

16 +/- 3

24 +/- 3

32 +/- 10

70 +/- 7

78 +/- 6*

38 +/- 7

47 +/- 10

NaN3

10

1185+/- 65

 

 

 

 

 

2241 +/- 56

 

 

 

4-NOPD

10

 

 

 

 

318 +/- 27

 

 

 

 

 

4-NOPD

50

 

 

104 +/- 13

 

 

 

 

 

 

 

MMS

2.0

 

 

 

 

 

 

 

 

742 +/- 48

 

2-AA

2.5

 

359 +/- 23

 

185 +/- 13

 

4104 +/- 292

 

4899 +/- 171*

 

 

2-AA

10.0

 

 

 

 

 

 

 

 

 

331 +/- 12

Table 3: Summary of Experiment II (preincubation)

Test group

Dose level (µg/plate)

Revertant Colony Counts (Mean +/- SD)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

DMSO

 

14 +/- 1

16 +/- 5

8 +/- 2

11 +/- 3

26 +/- 4

27 +/- 5

130 +/- 9

111 +/- 8

36 +/- 8

49 +/- 2

Untreated

 

22 +/- 2

15 +/- 4

10 +/- 3

10 +/- 2

30 +/- 9

28 +/- 3

180 +/- 17

180 +/- 34

55 +/- 4

57 +/- 4

Test item

33

11 +/- 4

15 +/- 7

9 +/- 5

9 +/- 4

21 +/- 5

31 +/- 4

122 +/- 8

119 +/- 10

36 +/- 6

53 +/- 3

 

100

10 +/- 3

14 +/- 2

9 +/- 0

7 +/- 3

23 +/- 8

28 +/- 5

130 +/- 14

123 +/- 12

42 +/- 1

56 +/- 8

 

333

14 +/- 2

15 +/- 6

9 +/- 3

9 +/- 1

28 +/- 9

31 +/- 3

141 +/- 4

128 +/- 17

30 +/- 3

58 +/- 11

 

1000

15 +/- 2

14 +/- 5

8 +/- 2

10 +/- 3

29 +/- 4

34 +/- 2

100 +/- 12

125 +/- 8

25 +/- 2

52 +/- 4

 

2500

10 +/- 2

13 +/- 6

8 +/- 3

8 +/- 1

23 +/- 7

27 +/- 3

34 +/- 7

98 +/- 18

38 +/- 3

53 +/- 3

 

5000

14 +/- 1 (P)

8 +/- 2 (P,M)

0 +/- 1 (P)

3 +/- 1 (P,M)

11 +/- 3 (P)

22 +/- 3 (P)

37 +/- 6 (P, M)

69 +/- 22 (P)

27 +/- 1 (P)

52 +/- 8 (P)

NaN3

10

1195 +/- 37

 

 

 

 

 

1787 +/- 163

 

 

 

4-NOPD

10

 

 

 

 

319 +/- 5

 

 

 

 

 

4-NOPD

50

 

 

81 +/- 12

 

 

 

 

 

 

 

MMS

2.0

 

 

 

 

 

 

 

 

509 +/- 50

 

2-AA

2.5

 

352 +/- 31

 

153 +/- 12

 

4087 +/- 458

 

4012 +/- 671

 

 

2-AA

10.0

 

 

 

 

 

 

 

 

 

310 +/- 25

NaN3: sodium azide

2-AA: 2-aminoanthracene

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

Conclusions:
Under the experimental conditions reported, the test item is considered negative in the Ames test.
Executive summary:

The genetic toxicity of the test item was tested according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the Escherichia coli strain WP2 uvrA (Ames test). The assay was performed with and without liver microsomal activation. 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
No substantial increase in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Therefore, the test substance is considered to be non-mutagenic in this 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

Key study
The genetic toxicity of the test item was tested according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the Escherichia coli strain WP2 uvrA (Ames test). The assay was performed with and without liver microsomal activation. 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
No substantial increase in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Therefore, the test substance is considered to be non-mutagenic in this reverse mutation assay.


Supporting studies:
Yoo, 1986 (J. Osaka City Med. Centre, 34: 267-288)

In a reverse gene mutation assay in bacteria (Ames test), strain WP2 uvrA of E. coli was exposed to the concentrations of 0.5 - 4.0 mg/plate. As a result, the test substance was considered to be non mutagenic under the test conditions chosen.

Ono et al., 2012 (Food and Chemistry Toxicology 50(5): 1538 -1546)
In this study the information on the results of 367 flavor items in the Ames test were summarised. The test substance was one of the substances studied and was considered negative in the Ames test. However, no information about test system and no information about strains are given.

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 genetic toxicity under Regulation (EC) No 1272/2008, as amended for the ninth time in Regulation (EC) No 2016/1179.