Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23.05.2016 - 21.06.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
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 preparation
Test concentrations with justification for top dose:
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 / 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.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 4-NOPD; 2-aminoanthracene, 2-AA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

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

NUMBER OF REPLICATIONS: 3

ACCEPTANCE CRITERIA
- Regular background growth in the negative and solvent control
- The spontaneous reversion rates in the negative and solvent control must be in the range of the historical data
- The positive control substances should produce an increase above 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
- A minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.
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
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
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
with S9 at 2500 - 5000 µg/plate in Experiment I and with andwithout S9 at 5000µg/plate in Experiment II
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
with andwithout S9 at 5000µg/plate in Experiment II
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
with S9 at 2500 - 5000 µg/plate in Experiment I
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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
Positive controls validity:
valid
Additional information on results:
Only one single toxic event (reduction in the number of revertants below the indication factor of 0.5), occurred in the strain TA 100 at the highest tested dose (5000 μg/plate) in the second experiment without metabolic activation.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any concentration 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. They showed a distinct increase in induced revertant colonies.

Any other information on results incl. tables

Summary results:

Experiment I

 

Without Metabolic Activation

Test Group

Dose level (µg/plate)

Revertant Colony Counts (mean±SD)

TA1535

TA1537

TA98

TA100

WP2uvrA

DMSO

n.a.

11±4

10±1

21±5

113±22

42±5

Untreated

n.a.

12±4

9±2

26±8

113±3

41±3

Test item

3

11±4

8±1

25±7

117±14

50±11

 

10

9±3

11±1

20±3

113±17

45±2

 

33

13±4

9±3

23±4

100±6

42±12

 

100

10±6

9±3

20±0

106±6

36±2

 

333

10±1

7±3

17±3

114±18

37±9

 

1000

11±4

11±1

25±9

105±8

40±8

 

2500

12±1

11±1

24±3

89±13

41±9

 

5000

14±4

7±3

21±8

78±12

38±9

NaN3

10

1188 ± 58

 

 

2360 ± 203

 

4-NOPD

10

 

 

315 ± 32

 

 

4-NOPD

50

 

79±18

 

 

 

MMS

2.0 µL

 

 

 

 

979±24

 

With Metabolic Activation

Test Group

Dose level (µg/plate)

Revertant Colony Counts (mean +/- SD)

TA1535

TA1537

TA98

TA100

WP2uvrA

DMSO

n.a.

14±2

 

13±7

26±6

 

101±3

46±7

 

Untreated

n.a.

15±4

18±8

33±9

105±8

54±7

Test item

3

14±1

13±3

36±8

102±2

59±8

 

10

11±5

8±2

34±3

119±16

57±5

 

33

12±3

11±3

32±8

106±24

52±9

 

100

15±2

14±4

29±2

117±10

48±10

 

333

10±4

14±3

37±6

113±6

59±9

 

1000

12±4

7±2

37±6

102±18

51±6

 

2500

14±3

10±5R

30±1

93±3R

48±8

 

5000

13±3

12±2R

24 ± 7

93±13R

46 ± 3

2-AA

2.5

348±42

208 ± 27

3155 ± 391

4755 ± 207

 

2-AA

10

 

 

 

 

371 ± 21

 

 

NaN3: sodium azide

2-AA: 2-aminoanthracene

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

MMS: methyl methane sulfonate

R: Reduced background growth


 

Experiment II

 

Without Metabolic Activation

Test Group

Dose level (µg/plate)

Revertant Colony Counts (mean±SD)

TA1535

TA1537

TA98

TA100

WP2uvrA

DMSO

n.a.

18±4

10±4

29±9

144±4

34 ± 6

Untreated

n.a.

11±2

8±1

26±6

171±16

34 ± 5

Test item

33

16±7

8±2

27±8

152±5

34 ± 10

 

100

15±5

10±1

31±8

145±8

31±5

 

333

18±1

11±5

37±6

138±6

35±2

 

1000

12±3

8±3

27±6

120±8

39±6

 

2500

15±2

12±4

24±6

95±15

34±10

 

5000

9±6

10±1R

23±3R

58 ± 10

29 ± 2

NaN3

10

1089 ± 43

 

 

1987 ± 45

 

4-NOPD

10

 

 

431±13

 

 

4-NOPD

50

 

75±6

 

 

 

MMS

2.0

 

 

 

 

759 ± 42

 

With Metabolic Activation

Test Group

Dose level (µg/plate)

Revertant Colony Counts (mean +/- SD)

TA1535

TA1537

TA98

TA100

WP2uvrA

DMSO

n.a.

18±3

15±1

43±7

131±7

50 ± 6

Untreated

n.a.

16±6

15±6

36±9

184±28

48 ± 6

Test item

33

15±4

12±4

43±9

124±6

45 ± 3

 

100

14±6

15±4

39±1

120 ± 8

49 ± 8

 

333

17±4

17±2

41±12

132 ± 13

49 ± 3

 

1000

14±6

13±3

39±3

117 ± 7

45 ± 12

 

2500

13±4

11±4

35 ± 5

127 ± 8

47 ± 2

 

5000

13±2

11±1R

42 ± 9R

118 ± 17

42 ± 3

2-AA

2.5

394±3

197 ± 19

5018 ± 368

3837 ± 314

 

2-AA

10

 

 

 

 

413 ± 14

 

NaN3: sodium azide

2-AA: 2-aminoanthracene

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

MMS: methyl methane sulfonate

R: reduced background growth

Applicant's summary and conclusion

Conclusions:
The test item is considered to be non-mutagenic.
Executive summary:

This 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) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and Escherichia coli strain WP2uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation (S9). Each concentration, including the controls, was tested in triplicate. The concentrations in the pre-experiment (experiment I) were: 3; 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate. The concentrations in experiment II were 33; 100; 333; 1000; 2500 and 5000 μg/plate

No precipitation of the test item occurred up to the highest tested dose.

In the strains TA 1537, TA 98 and, TA 100 the plates incubated with the test item showed reduced background growth.

Only slight toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in strain TA 100 without metabolic activation.

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. The acceptance criteria were met.

In the current study 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.