Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 Feb - 14 Mar 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:
Commission Regulation (EC) No. 440/2008
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 / 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:
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 2:
33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation for TA 100 and TA 1535
10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation for remaining strains
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: 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:
DMSO
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)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (Experiment 1); pre-incubation (Experiment 2)

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 substance is considered as a mutagenic 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
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp 2: -S9: at 5000 µg/plate; +S9: at and above 2500 µg/plate
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:
Exp 1: -S9: at 5000 µg/plate; Exp 2: +/-S9: at and above 2500 µg/plate
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:
Exp 1: +S9: at 5000 µg/plate; Exp 2: +S9: at and above 1000 µg/plate
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:
Exp 1: +S9: at 5000 µg/plate; Exp 2: -S9: at 5000 µg/plate; +S9: at and above 1000 µg/plate
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:
cytotoxicity
Remarks:
Exp 1 and 2: +/- S9: at and above 1000 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 at 5000 μg/plate with and without S9 mix in both experiments. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in Experiment 1 at 5000 μg/plate with S9 mix. In Experiment 2 no precipitation of the test susbtance was observed on the incubated agar plates. The undissolved particles had no influence on the data recording.

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

ADDITIONAL INFORMATION ON CYTOTOXICITY:The plates incubated with the test substance showed reduced background growth in Experiment 2 in all strains used, except of strain WP2 uvrA. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in all strains used.

Any other information on results incl. tables

Table 1: Results of Experiment 1 (plate incorporation).

 

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

Without S9

Test substance (µg/plate)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvr A

DMSO

13 ± 2

8 ± 2

27 ± 2

194 ± 10

33 ± 6

Untreated

12 ± 2

7 ± 3

29 ± 6

208 ± 16

38 ± 5

3

14 ± 2

9 ± 4

26 ± 4

184 ± 22

30 ± 6

10

10 ± 2

8 ± 1

27 ± 5

200 ± 13

33 ± 3

33

9 ± 3

6 ± 1

23 ± 4

181 ± 19

28 ± 4

100

11 ± 4

8 ± 3

28 ± 4

181 ± 18

33 ± 7

333

12 ± 4

10 ± 1

28 ± 7

193 ± 14

30 ± 3

1000

13 ± 3

4 ± 1

26 ± 4

179 ± 8

15 ± 5

2500

12 ± 0

4 ± 2

25 ± 5

166 ± 21

13 ± 3

5000

11 ± 4

3 ± 1

23 ± 4

169 ± 21

14 ± 2

Positive Control

NaN3

4-NOPD

4-NOPD

NaN3

MMS

Dose (µg/plate)

10

50

10

10

2

Number of revertant colonies/plate

930 ± 93

100 ± 18

478 ± 53

2300 ± 55

923 ± 20

With S9

Test substance (µg/plate)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvr A

DMSO

13 ± 3

9 ± 4

37 ± 4

177 ± 8

43 ± 6

Untreated

19 ± 3

9 ± 3

42 ± 8

186 ± 17

46 ± 7

3

9 ± 2

8 ± 4

38 ± 3

183 ± 13

44 ± 11

10

11 ± 2

8 ± 3

40 ± 2

179 ± 6

42 ± 7

33

10 ± 3

7 ± 2

31 ± 2

180 ± 22

44 ± 11

100

12 ± 4

11 ± 3

31 ± 4

188 ± 21

48 ± 7

333

15 ± 1

11 ± 6

41 ± 5

157 ± 5

34 ± 7

1000

9 ± 2

11 ± 2

27 ± 5

164 ± 14

18 ± 4

2500

12 ± 5

12 ± 5

18 ± 1

99 ± 7

8 ± 2

5000

8 ± 1 p

5 ± 1 p

11 ± 2 p

36 ± 10 p

2 ± 1 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

219 ± 34

173 ± 24

4806 ± 849

4719 ± 196

346 ± 48

NaN3: sodium azide

2AA: 2-Aminoanthracene

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

MMS: methyl methane silfonate

p: Precipitate

Table 2: Results of Experiment 2 (pre-incubation).

 

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

Without S9

Test substance (µg/plate)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvr A

DMSO

9 ± 2

8 ± 3

26 ± 8

149 ± 9

44 ± 8

Untreated

10 ± 3

13 ± 3

22 ± 9

189 ± 5

42 ± 10

10

 

9 ± 3

29 ± 8

 

44 ± 8

33

11 ± 2

9 ± 4

25 ± 3

143 ± 12

40 ± 3

100

9 ± 3

10 ± 2

25 ± 4

151 ± 6

42 ± 1

333

8 ± 2

9 ± 3

26 ± 5

126 ± 17

25 ± 4

1000

13 ± 3

8 ± 3

21 ± 6

114 ± 26

14 ± 5

2500

10 ± 2

11 ± 3 r

20 ± 3

75 ± 17

16 ± 1

5000

10 ± 1 r

9 ± 6 r

18 ± 3

48 ± 23 r

17 ± 8

Positive Control

NaN3

4-NOPD

4-NOPD

NaN3

MMS

Dose (µg/plate)

10

50

10

10

2

Number of revertant colonies/plate

1225 ± 59

89 ± 10

462 ± 22

1884 ± 169

732 ± 13

With S9

Test substance (µg/plate)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvr A

DMSO

13 ± 2

11 ± 5

32 ± 4

136 ± 7

43 ± 10

Untreated

10 ± 6

8 ± 3

44 ± 9

206 ± 10

59 ± 5

10

 

13 ± 2

39 ± 7

 

52 ± 7

33

9 ± 2

11 ± 5

35 ± 7

130 ± 6

49 ± 8

100

13 ± 3

8 ± 2

30 ± 6

153 ± 21

42 ± 12

333

10 ± 3

11 ± 3

33 ± 6

145 ± 14

36 ± 4

1000

10 ± 4

10 ± 4

39 ± 10 r

85 ± 1 r

18 ± 4

2500

5 ± 2 r, m

5 ± 2 r, m

9 ± 1 r, m

5 ± 1 r, m

18 ± 4

5000

2 ± 1 r, m

7 ± 4 r, m

6 ± 2 r, m

5 ± 1 r, m

8 ± 2

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

369 ± 24

160 ± 7

4201 ± 214

2905 ± 543

377 ± 14

NaN3: sodium azide

2AA: 2-Aminoanthracene

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

MMS: methyl methane silfonate

r: reduced background growth

m: manual count

Applicant's summary and conclusion

Conclusions:
Under the conditions of the conducted test the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) tested with and without metabolic activation up to 5000 µg/plate.
Executive summary:

Ames test according to OECD 471 and GLP was performed. 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: Strains TA 1535 and TA 100: 33; 100; 333; 1000; 2500; and 5000 µg/plate; the remaining strains: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate.

No substantial increase in revertant colony numbers of any of the five tester strains was observed any dose level, neither in the presence nor absence of metabolic activation (S9 mix). In conclusion, the test item did not induce gene mutations by base pair

changes or frameshifts in the genome of the strains used.