Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 Jun - 26 Jul 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:
adopted: 21 Jul 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
adopted: 30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his operon (for S. typhimurium strains)
trp operon (for E. coli strain)
Species / strainopen allclose all
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:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone
Test concentrations with justification for top dose:
Pre-experiment: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation

The pre-experiment is reported as Experiment 1.

Experiment 2a:
33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation for TA1537
10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation for TA98, TA100, TA1535 and WP2 uvrA

Experiment 2b:
10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation for TA98
100, 333, 1000, 2000, 3000, 4000 and 5000 µg/plate without metabolic activation for TA1535
Vehicle / solvent:
- Vehicle(s/solvent used: ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Controls
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA)
Remarks:
Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation (Experiment 1); pre-incubation (Experiment 2a and b)

DURATION
- Preincubation period: 1 h
- Exposure duration: at least 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

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:
Mean values and standard deviations were calculated.

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:
Exp 1: +S9: at 5000 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:
no cytotoxicity, but tested up to precipitating concentrations
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:
Exp 1: +S9: at 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:
Exp 1: +S9: at 2500 µg/plate and above
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
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 from 333 to 5000 μg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 μg/plate in all experiments and at 2500 μg/plate in Experiment 1 with S9 mix. The undissolved particles had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment is reported as Experiment 1 (all strains were tested in the pre-experiment).

ADDITIONAL INFORMATION ON CYTOTOXICITY: 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. Slight toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in strain TA 1535, TA 98 and TA 100.

Any other information on results incl. tables

A slight dose dependent increase in the number of revertant colonies was observed in Experiment 2a without S9 mix. The threshold of thrice the number of the corresponding solvent control was neither reached nor exceeded and the result was not reproducible in Exp 2b and can therefore be judged as biologically irrelevant.

Table 1. Test results of Experiment 1 (plate incorporation method)

 

Number of revertant colonies (mean of 3 plates±SD)

Without S9

Test substance (µg/plate)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

Ethanol

12±2

8±2 b,m

29±7

114±26

40±8

Untreated

10±5

7±1 b,m

29±9

140±27

45±7

3

10±1

7±1 b,m

27±7

117±21

45±4

10

13±3

8±3 b,m

29±8

95±5

40±4

33

11±1

7±2 b,m

34±8

70±17

46±9

100

9±1

8±2 b,m

31±6

75±6

46±1

333

10±1

8±2 b,m

33±10

63±16

36±8

1000

10±1

8±3 b,m

28±6

66±4

38±7

2500

11±1

8±2 b,m

34±4

68±6

36±1

5000

11±4 p

6±2 p,b,m

36±3 p

66±8 p

45±1 p

Positive Control

NaN3

4-NOPD

4-NOPD

NaN3

MMS

Dose (µg/plate)

10

50

10

10

2

Number of revertant colonies/plate

1166± 111

81±13 b,m

392±4

1963±80

833±56

With S9

Test substance (µg/plate)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvr A

DMSO

18±4

9±2 b,m

56±4

135±5

49±7

Untreated

15±1

9±1 b,m

45±5

149±17

61±8

3

15±3

9±2 b,m

48±13

124±9

53±5

10

19±2

9±1 b,m

51±6

129±11

51±6

33

20±4

9±2 b,m

56±13

118±6

48±12

100

16±6

9±2 b,m

43±5

112±8

51±4

333

10±4

8±1 b,m

39±8

79±8

44±3

1000

12±4

9±1 b,m

44±3

78±10

51±15

2500

11±3 p,m

8±1 p,b,m

26±3 p,m

59±5 p,m

27±2 p,m

5000

7±2 p,m

9±2 p,b,m

24±5 p,m

46±4 p,m

26±2 p,m

Positive Control

2-AA

2-AA

2-AA

2-AA

2-AA

Dose (µg/plate)

2.5

2.5

2.5

2.5

10

Number of revertant colonies/plate

297±52

117±9 b,m

3492±860

3213±171

358±48

NaN3: sodium azide

2AA: 2-Aminoanthracene

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

MMS: methyl methane sulfonate

p: precipitate

m: manual count

b: extensive bacterial growth

Table 2: Test results of Experiment 2a (pre-incubation method)

 

Number of revertant colonies (mean of 3 plates±SD)

Without S9

Test substance (µg/plate)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

Ethanol

14±3

18±6

 

155±11

42±8

Untreated

8±3

15±0

 

165±8

48±12

10

8±2

 

 

144±26

38±6

33

9±5

21±5

 

123±25

40±3

100

13±2

19±7

 

98±15

38±11

333

8±2

25±2

 

90±12

45±3

1000

6±1

27±5

 

89±11

40±4

2500

9±2

33±5

 

93±11

39±5

5000

10±3 p

51±6 p

 

96±14 p

43±3 p

Positive Control

NaN3

4-NOPD

4-NOPD

NaN3

MMS

Dose (µg/plate)

10

50

10

10

2

Number of revertant colonies/plate

1078±22

83±11

 

1701±99

776±5

With S9

Test substance (µg/plate)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvr A

Ethanol

11±1

27±3

 

161±30

41±5

Untreated

7±1

20±4

 

177±1

59±14

10

13±2

 

 

154±25

50±7

33

13±1

28±9

 

157±14

54±4

100

16±1

25±2

 

147±5

41±5

333

10±2

24±3

 

107±12

52±9

1000

12±6

21±5

 

93±13

46±5

2500

13±2

26±6

 

94±14

50±6

5000

13±4 p

28±2

 

97±12 p

46±16 p

Positive Control

2-AA

2-AA

2-AA

2-AA

2-AA

Dose (µg/plate)

2.5

2.5

2.5

2.5

10

Number of revertant colonies/plate

384±9

144±30

 

3366±216

373±21

NaN3: sodium azide

2AA: 2-Aminoanthracene

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

MMS: methyl methane sulfonate

p: precipitate

 

Table 3. Results of Experiment 2b (pre-incubation method)

 

Number of revertant colonies (mean of 3 plates±SD)

Without S9

Test substance (µg/plate)

TA 1537

TA 98

Ethanol

11±2

38±4

Untreated

13±3

32±6

10

 

39±3

33

 

34±7

100

11±6

40±5

333

9±5

38±12

1000

9±3

27±1

2000

12±2

 

2500

 

30±6

3000

7±3

 

4000

10±3

 

5000

13±4 p

33±7 p

Positive Control

4-NOPD

4-NOPD

Dose (µg/plate)

50

10

Number of revertant colonies/plate

119±12

374±26

With S9

Test substance (µg/plate)

TA 1537

TA 98

Ethanol

 

46±2

Untreated

 

54±9

10

 

49±7

33

 

43±12

100

 

48±4

333

 

47±7

1000

 

55±7

2500

 

42±10

5000

 

42±1 p

Positive Control

2-AA

2-AA

Dose (µg/plate)

2.5

2.5

Number of revertant colonies/plate

 

4016±153

NaN3: sodium azide

2AA: 2-Aminoanthracene

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

MMS: methyl methane sulfonate

p: precipitate

Applicant's summary and conclusion

Conclusions:
Under the conditions of the conducted test the substance was not mutagenic in any of the five bacterial strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) tested with and without metabolic activation.
Executive summary:

Under the conditions of the conducted test the substance was not mutagenic in any of the five bacterial strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) tested with and without metabolic activation.