Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
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

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
PRE-EXPERIMENT / EXPERIMENT I:
3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate

EXPERIMENT II:
- Without S9 mix: 33, 100, 333, 1000, 2500 and 5000 µg/plate
- With S9 mix: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: better solubility properties, relative nontoxic to bacteria
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine; 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION:
- Preincubation period: 1 hour
- Exposure duration: 72 hours

NUMBER OF REPLICATIONS: 3 plates

DETERMINATION OF CYTOTOXICITY:
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) 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 TA 102) 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 results
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100, TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
with S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: none
- Precipitation: Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate in the presence of metabolic activation in both experiments. The undissolved particles had no influence on the data recording.

COMPARISON WITH HISTORICAL CONTROL DATA: performed

Any other information on results incl. tables

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. Toxic effects, evident 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

With S9

Without S9

With S9

TA 1535

no

5000

no

no

TA 1537

no

5000

no

5000

TA 98

no

5000

no

no

TA 100

no

5000

no

no

TA 102

no

5000

no

2500-5000


RESULTS

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with 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.

SUMMARY EXPERIMENT I

 

Metabolic

Activation

Test

Group

Dose

per plate

Revertant Colony Counts (Mean ±SD)

TA 1535

TA 1537

TA 98

TA 100

TA 102

Without

Acetone

 

15 ± 7

11 ± 3

31 ± 10

126 ± 7

458 ± 29

Untreated

 

12 ± 3

10 ± 3

34 ± 1

128 ± 13

416 ± 19

test item

3 µg

15 ± 6

12 ± 3

27 ± 4

130 ± 12

417 ± 30

 test item

10 µg

15 ± 6

10 ± 2

29 ± 5

127 ± 10

443 ± 5

 test item

33 µg

15 ± 7

11 ± 2

34 ± 11

131 ± 6

461 ± 15

test item

100 µg

14 ± 5

13 ± 4

26 ± 9

137 ± 12

476 ± 26

test item

333 µg

15 ± 4

10 ± 4

31 ± 10

130 ± 9

411 ± 6

test item

1000 µg

15 ± 8

11 ± 2

26 ± 5

130 ± 20

421 ± 32

test item

2500 µg

16 ± 5

10 ± 2

27 ± 5

142 ± 11

430 ± 49

test item

5000 µg

13 ± 5

8 ± 4

26 ± 1

141 ± 12

389 ± 15

NaN3

10 µg

1972 ± 38

 

 

2206 ± 29

 

4-NOPD

10 µg

 

 

292 ± 25

 

 

4-NOPD

50 µg

 

74 ± 2

 

 

 

MMS

2.0 µL

 

 

 

 

4996 ± 449

With

Acetone

 

19 ± 7

17 ± 4

43 ± 2

162 ± 24

583 ± 13

Untreated

 

18 ± 3

17 ± 5

36 ± 6

171 ± 12

564 ± 7

test item

3 µg

15 ± 4

20 ± 3

38 ± 4

152 ± 14

551 ± 55

test item

10 µg

17 ± 2

21 ± 4

43 ± 6

165 ± 20

610 ± 35

test item

33 µg

16 ± 5

17 ± 1

38 ± 6

175 ± 5

592 ± 39

test item

100 µg

16 ± 5

18 ± 3

40 ± 3

182 ± 15

626 ± 24

test item

333 µg

13 ± 1

18 ± 4

46 ± 8

155 ± 12

576 ± 27

test item

1000 µg

19 ± 2

22 ± 1

39 ± 9

165 ± 21

528 ± 28

test item

2500 µg

23 ± 3P

8 ± 3P M

38 ± 5P

165 ± 5P

177 ± 1P

test item

5000 µg

4 ± 2P M

3 ± 1P M

2 ± 1P M

52 ± 6P M

28 ± 9P M

2-AA

2.5 µg

285 ± 21

190 ± 28

1384 ± 35

1850 ± 50

 

2-AA

10.0 µg

 

 

 

 

2101 ± 78

  

SUMMARY EXPERIMENT II

 

Metabolic

Activation

Test

Group

Dose per plate

Revertant Colony Counts (Mean ±SD)

TA 1535

TA 1537

TA 98

TA 100

TA 102

Without

Acetone

 

12 ± 3

9 ± 1

32 ± 7

127 ± 17

389 ± 6

Untreated

 

16 ± 4

9 ± 1

33 ± 3

131 ± 8

384 ± 14

test item

33 µg

14 ± 1

10 ± 1

33 ± 3

121 ± 6

412 ± 14

test item

100 µg

12 ± 5

9 ± 2

28 ± 4

120 ± 21

435 ± 19

test item

333 µg

15 ± 1

10 ± 3

29 ± 4

128 ± 9

412 ± 18

test item

1000 µg

12 ± 4

12 ± 3

26 ± 2

129 ± 8

364 ± 36

test item

2500 µg

12 ± 5

8 ± 2

33 ± 6

117 ± 12

372 ± 31

test item

5000 µg

15 ± 2

7 ± 2

28 ± 5

129 ± 14

388 ± 20

NaN3

10 µg

2113 ± 40

 

 

2345 ± 48

 

4-NOPD

10 µg

 

 

343 ± 17

 

 

4-NOPD

50 µg

 

75 ± 1

 

 

 

MMS

2.0 µL

 

 

 

 

4245 ± 164

With

Acetone

 

18 ± 5

15 ± 1

44 ± 2

149 ± 17

512 ± 10

Untreated

 

16 ± 3

20 ± 7

46 ± 7

159 ± 7

509 ± 29

test item

3 µg

15 ± 3

17 ± 3

43 ± 7

134 ± 7

450 ± 11

test item

10 µg

15 ± 2

17 ± 2

46 ± 4

144 ± 22

446 ± 48

test item

33 µg

17 ± 4

16 ± 1

45 ± 4

153 ± 3

506 ± 55

test item

100 µg

17 ± 6

18 ± 5

41 ± 3

157 ± 22

498 ± 93

test item

333 µg

16 ± 3

18 ± 7

51 ± 5

125 ± 12

434 ± 49

test item

1000 µg

16 ± 5

16 ± 6

43 ± 3

157 ± 14

435 ± 50

test item

2500 µg

20 ± 7P

16 ± 5P

37 ± 4P

139 ± 8P

160 ± 24P

test item

5000 µg

13 ± 4P M

7 ± 3P M

25 ± 4P M

115 ± 11P M

116 ± 10P M

2-AA

2.5 µg

251 ± 12

102 ± 9

1069 ± 17

1045 ± 150

 

2-AA

10.0 µg

 

 

 

 

2439 ± 35

 

Key to Positive Controls

NaN3          sodium azide

2-AA           2-aminoanthracene

MMS          methyl methane sulfonate

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

 

Key to Plate Postfix Codes

P                Precipitate

M               Manual Count

Applicant's summary and conclusion

Conclusions:
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.
Executive summary:

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 the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

The assay was performed in two independent experiments both 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:

- without S9 -mix: 33, 100, 333, 1000, 2500 and 5000 µg/plate

- with S9 -mix: 3, 10, 33, 100, 333, 1000, 2500 and 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 in the presence of metabolic activation in both experiments. The undissolved particles had no influence on the data recording.

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.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups without metabolic activation. With metabolic activation toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred.

No substantial increase in revertant colony numbers of any of the fivetester 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.