Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02.09.2016 - 14.09.2016
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:
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 for TA 100: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate and for all others: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate.
Vehicle:
- 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
Negative controls:
yes
Solvent 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 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 our 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:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
at 333 µg/plate in Experiment I, not in Experiment II
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
In Experiment I, without S9 at 100-5000 µg/plate and with S9 at 333-5000µg/plate. In Experiment II, without S9 at 333-5000 µg/plate and with S9 at 333-5000µg/plate.
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
No relevant toxic effects, evident as a dose dependent reduction of the number of revertants below 0.5 times the corresponding solvent control, occurred in the test groups with and without metabolic activation in strain TA 1537. The moderate reduction in the number of revertants in strain in experiment I without S9 mix was not dose dependent and thus judged to be based upon statistical fluctuations at such low numbers of colonies and does not represent a true toxic effect.

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.

8±2

17±2

21±5

176±30

35±3

Untreated

n.a.

10 ± 5

19 ± 2

23 ± 12

184 ± 10

47 ± 1

Test item

3

7 ± 3

27 ± 10

25 ± 3

177 ± 7

35 ± 7

 

10

11 ± 1

24 ± 9

21 ± 7

186 ± 10

34 ± 2

 

33

12 ± 1

23 ± 5

19 ± 4

157 ± 6

35 ± 2

 

100

12 ± 5

9 ± 1

20 ± 4

60 ± 4

34 ± 6

 

333

9 ± 2

7 ± 1

26 ± 10

45 ± 15

40 ± 6

 

1000

7 ± 3

8 ± 3

30 ± 5

55 ± 6

40 ± 6

 

2500

14 ± 6P

14 ± 2P

28 ± 4P

47 ± 2P

35 ± 3P

 

5000

24 ± 1P

10 ± 3P

27 ± 8P

55 ± 1P

36 ± 11P

NaN3

10

1236 ± 41

 

 

2100 ± 87

 

4-NOPD

10

 

 

475 ± 65

 

 

4-NOPD

50

 

86 ± 10

 

 

 

MMS

2.0

 

 

 

 

954 ± 46

 

With Metabolic Activation

Test Group

Dose level (µg/plate)

Revertant Colony Counts (mean +/- SD)

TA1535

TA1537

TA98

TA100

WP2uvrA

DMSO

n.a.

11 ± 3

27 ± 5

30 ± 7

177 ± 13

54 ± 11

Untreated

n.a.

10 ± 2

25 ± 1

42 ± 7

175 ± 10

54 ± 11

Test item

3

10 ± 5

23 ± 2

40 ± 10

139 ± 13

48 ± 8

 

10

15 ± 5

31 ± 4

30 ± 8

157 ± 10

48 ± 8

 

33

11 ± 6

24 ± 4

34 ± 5

163 ± 8

53 ± 6

 

100

9 ± 3

30 ± 2

31 ± 3

105 ± 24

54 ± 3

 

333

9 ± 4

20 ± 3

29 ± 8

51 ± 8

47 ± 5

 

1000

12 ± 5

20 ± 3

27 ± 5

24 ± 7

41 ± 10

 

2500

6 ± 1

25 ± 2

21 ± 1

10 ± 2

32 ± 8

 

5000

10 ± 4P

23 ± 7P

25 ± 5P

11 ± 2P

34 ± 8P

2-AA

2.5

394 ± 25

421 ± 28

4235 ± 1180

5176 ± 85

 

2-AA

10

 

 

 

 

438 ± 12

 

NaN3: sodium azide

2-AA: 2-aminoanthracene

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

MMS: methyl methane sulfonate

P: Precipitate

Experiment II

 

Without Metabolic Activation

Test Group

Dose level (µg/plate)

Revertant Colony Counts (mean±SD)

TA1535

TA1537

TA98

TA100

WP2uvrA

DMSO

n.a.

12±3

12±2

22±3

145±16

37±6

Untreated

n.a.

9 ± 5

12 ± 3

38 ± 8

181 ± 21

40 ± 11

Test item

3

 

 

 

128 ± 3

 

 

10

13 ± 2

13 ± 4

28 ± 6

140 ± 10

43 ± 5

 

33

13 ± 3

12 ± 5

28 ± 3

94 ± 9

39 ± 8

 

100

10±2

14±2

27±7

81±18

34±5

 

333

10 ± 3

10 ± 5

28 ± 4

62 ± 7

35 ± 7

 

1000

10 ± 4

12 ± 3

28 ± 3

46 ± 7

31 ± 8

 

2500

11 ± 2

15 ± 5

26 ± 4

48 ± 5

32 ± 4

 

5000

13 ± 2P

15 ± 1P

27 ± 5P

25 ± 6P M

38 ± 4P

NaN3

10

1215 ± 100

 

 

2004 ± 55

 

4-NOPD

10

 

 

505 ± 28

 

 

4-NOPD

50

 

91 ± 2

 

 

 

MMS

2.0

 

 

 

 

569 ± 47

 

With Metabolic Activation

Test Group

Dose level (µg/plate)

Revertant Colony Counts (mean +/- SD)

TA1535

TA1537

TA98

TA100

WP2uvrA

DMSO

n.a.

11±1

18±5

37±12

131±9

56±9

Untreated

n.a.

15 ± 5

16 ± 6

49 ± 10

189 ± 3

65 ± 6

Test item

3

 

 

 

110 ± 12

 

 

10

14 ± 4

19 ± 3

41 ± 6

134 ± 21

63 ± 10

 

33

14 ± 4

20 ± 4

41 ± 10

128 ± 7

50 ± 5

 

100

15±1

18±5

36±3

112±15

53±3

 

333

11 ± 4

25 ± 3

37 ± 5

39 ± 2

49 ± 6

 

1000

15 ± 1

26 ± 1

42 ± 1

35 ± 2

44 ± 4

 

2500

13 ± 4

21 ± 0

31 ± 5

21 ± 2

32 ± 1

 

5000

13 ± 1

22 ± 4

30 ± 7

13 ± 2

39 ± 4

2-AA

2.5

326 ± 38

242 ± 4

3304 ± 213

4011 ± 287

 

2-AA

10

 

 

 

 

439 ± 23

 

NaN3: sodium azide

2-AA: 2-aminoanthracene

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

MMS: methyl methane sulfonate

P: Precipitate

M Manual count

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 for TA 100: 3; 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate and for all others: 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate.

The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 μg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 μg/plate without metabolic activation and at 5000 μg/plate with metabolic activation in the first experiment and at 5000 μg/plate without metabolic activation in the second experiment.

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), occurred in strain TA 100 in both experiments.

There was no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance with the exception of strain TA 1535 in the absence of metabolic activation in experiment I. This strain showed an increase in revertant colony numbers at the highest applied concentration, which showed precipitation too. However, the absolute numbers of colonies did not reach the threshold of 3.0.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. The acceptance criteria were met.

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 the
reverse mutation assay.