Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2017-05-16 to 2017-11-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997-07-21
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008-05-30
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Target gene:
his/trp
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Remarks:
uvrA
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/beta-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 and IIa:
Strain TA 100 (Exp. II): 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
The remaining strains (Exp. II): 33; 100; 333; 1000; 2500; and 5000 µg/plate
Strain TA 98 (Exp. IIa): 3; 10; 33; 100; 333; 1000; and 2500 µg/plate
In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment is reported as experiment I.
Based on the toxic effects observed in experiment I, six, respectively seven concentrations were tested in experiment II or experiment IIa. The concentration range included two logarithmic decades. 5000 µg/plate were chosen as maximal concentration in experiment II. The additional experiment IIa was performed after discussion with the sponsor to confirm the data on bacteriotoxicity observed in experiment II.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 mix: 10 µg/plate in TA 1535 and TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
Without S9 mix: 10 µg/plate in strain TA 98, 50 µg/plate in strain TA 1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without S9 mix: 2.0 µL/plate in E.coli WP2 uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9 mix: 2.5 µg/plate in TA 1535, TA 1537, TA 98, TA 100; 10.0 µg/plate in E.coli WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

SELECTION AGENT (mutation assays): Plates with selective agar (without histidine/tryptophan) were used.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in the number of spontaneous revertants or a clearing 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:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9 mix: 1000 - 5000 µg/plate with S9 mix: 2500 - 5000 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:
1000 - 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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:
with and without S9 mix: 2500 - 5000 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:
with s9 mix: 5000 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 in experiment II at 5000 µg/plate with and without metabolic activation.

Any other information on results incl. tables

Table 1: Summary of Experiment I

Metabolic Activation

Test Group

Dose Level [µg/plate]

Revertant Colony Counts (Mean ± SD)

TA 1535

TA 1537

TA 98

TA 100

E. coli

Without

DMSO

 

12±4

11±4

25±5

159±24

41±7

Untreated

 

14±33

12±4

33±6

191±29

37±6

Test Item

3

9±2

11±5

24±5

186±17

37±1

10

12±5

10±2

19±2

169±12

39±12

33

9±1

9±2

24±6

192±6

41±6

100

10±3

10±1

18±3

190±10

39±4

333

13±6

9±3

31±6

188±10

37±4

1000

11±4

9±2

19±4

132±20

31±5

2500

11±2

8±2

18±5

77±7

25±3

5000

9±2

9±3

22±3

72±14

28±6

NaN3

10

1367±25

 

 

2411±77

 

4-NOPD

10

 

 

423±40

 

 

4-NOPD

50

 

86±13

 

 

 

MMS

2.0 µL

 

 

 

 

1085±44

With

DMSO

 

19±3

18±3

36±2

195±23

58±10

Untreated

 

19±2

19±7

45±11

178±16

73±1

Test Item

3

19±8

22±1

43±10

184±11

58±12

10

18±5

13±3

39±9

173±21

50±15

33

14±5

13±3

40±3

146±3

43±7

100

15±6

14±6

32±7

164±4

60±15

333

17±6

18±1

47±7

165±8

57±6

1000

21±7

13±4

44±12

193±21

52±6

2500

14±3 p

14±3 p

37±0 p

94±9 p

43±4

5000

6±1 pm

7±1 pm

20±5 pm

44±7 pm

28±3

2-AA

2.5

375±21

115±29

3439±12

3859±101

 

2-AA

10.0

 

 

 

 

331±23

p precipitate

m manual count

Table 2: Summary of Experiment II

Metabolic Activation

Test Group

Dose Level [µg/plate]

Revertant Colony Counts (Mean ± SD)

TA 1535

TA 1537

TA 98

TA 100

E. coli

Without

DMSO

 

9±3

11±5

28±11

131±27

39±3

Untreated

 

9±2

7±2

36±5

192±31

42±10

Test Item

10

 

 

 

130±3

 

33

9±5

10±4

25±2

130±11

40±3

100

10±1

11±5

28±6

134±17

47±10

333

9±3

7±3

30±3

98±7

42±13

100

7±3

7±4

11±1

44±9

28±6

2500

8±1

3±1

1±1

5±1

23±2

5000

5±2 pm

0±1 p

0±0 p

0±1 p

19±1 pm

NaN3

10

1187±26

 

 

1881±106

 

4-NOPD

10

 

 

323±7

 

 

4-NOPD

50

 

95±7

 

 

 

MMS

2.0 µL

 

 

 

 

668±63

With

DMSO

 

13±5

12±3

38±6

123±10

53±9

Untreated

 

18±6

13±2

39±10

176±15

67±8

Test Item

10

 

 

 

116±9

 

33

15±1

15±0

42±7

127±5

51±2

100

14±5

16±6

40±12

104±6

53±8

333

13±6

12±4

44±8

92±26

51±10

1000

15±7

10±2

31±5

26±1

39±3

2500

11±2

1±1

2±0

8±2

30±6

5000

6±2 pm

0±0 p

0±1 p

1±1 p

13±3 pm

2-AA

2.5

389±25

109±8

4249±284

2158±344

 

2-AA

10.0

 

 

 

 

498±31

p precipitate

m manual count

Applicant's summary and conclusion

Conclusions:
In a bacterial reverse mutation assay (Ames) according to OECD Guideline 471, the test item did not show mutagenic properties.
Executive summary:

In a bacterial reverse mutation assay (Ames) according to OECD Guideline 471, the potential of the test item to induce gene mutations was investigated in the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in three independent experiments with and without liver microsomal activation (S9 mix). 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 and IIa:

Strain TA 100 (Exp. II):                 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The remaining strains (Exp. II): 33, 100, 333, 1000, 2500, and 5000 µg/plate     

Strain TA 98 (Exp. IIa):                 3;10; 33; 100; 333; 1000; and 2500 µg/plate

The test item precipitated in the overlay agar in the test tubes in experiment I at 5000 µg/plate with metabolic activation and in experiment II at 5000 µg/plate with and without metabolic activation. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I from 2500 to 5000 µg/plate with metabolic activation and in experiment II at 5000 µg/plate with and without metabolic activation. In experiment IIa no precipitation was observed when tested up a top dose of 2500 µg/plate. The undissolved particles had no influence on the data recording of this study. 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. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in experiment I in strains TA 1535, TA 1537 and TA 100, in experiment II in all strains except strain TA 1535, and in experiment IIa in strain TA 98. 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.