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 Sep - 14 Oct 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
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 2:
10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative low toxicity 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 (4-NOPD), 2-aminoanthracene (2-AA)
Remarks:
+S9: 2-AA (2.5 µg/plate, TA1535, TA1537, TA98, TA100; 10 µg/plate, WP2 uvrA); -S9: NaN3 (10 µg/plate, TA1535, TA100); 4-NOPD (10 µg/plate, TA98; 50 µg/plate, TA1537); MMS (2 µL/plate, WP2 uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation, Experiment 1); preincubation (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
Rationale for test conditions:
The test conditions were chosen according to OECD 471.
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
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 and +S9: starting at 2500 µg/plate; Exp. 2: -S9 and +S9: starting at 1000 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:
cytotoxicity
Remarks:
Exp. 1: -S9 and +S9: starting at 2500 µg/plate; Exp. 2: -S9 and +S9: starting at 1000 µg/plate
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 and +S9: starting at 2500 µg/plate; Exp. 2: -S9 and +S9: starting at 1000 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 and +S9: starting at 2500 µg/plate; Exp. 2: -S9: starting at 1000 µg/plate, +S9: starting at 333 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exp. 1: -S9 and +S9: starting at 2500 µg/plate; Exp. 2: -S9 and +S9: starting at 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 substance precipitated in the overlay agar in the test tubes at 5000 µg/plate in the presence of metabolic activation. No precipitation of the test substance was observed in the overlay agar on the incubated agar plates.

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 normal background growth up to 5000 µg/plate with and without metabolic activation in all strains used. Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed in all strains with and without metabolic activation.

Any other information on results incl. tables

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

With or without S9-Mix

Test substance concentration [μg/plate]

Mean number of revertant colonies per plate

(average of 3 plates ± SD)

Base-pair substitution type

Frameshift type

TA1535

TA100

WP2 uvrA

TA1537

TA98

-

0 (DMSO)

12 ± 6

195 ± 6

35 ± 6

18 ± 2

34 ± 3

-

0

14 ± 6

202 ± 6

45 ± 9

20 ± 7

38 ± 4

-

3

10 ± 1

185 ± 25

40 ± 5

13 ± 4

33 ± 4

-

10

17 ± 3

180 ± 13

42 ± 3

18 ± 5

41 ± 6

-

33

14 ± 7

200 ± 14

41 ± 14

17 ± 1

44 ± 4

-

100

17 ± 3

198 ± 7

36 ± 5

13 ± 3

46 ± 3

-

333

11 ± 5

181 ± 25

35 ± 13

11 ± 5

44 ± 3

-

1000

11 ± 1

107 ± 20

32 ± 3

13 ± 2

31 ± 3

-

2500

15 ± 6R

50 ± 5M,R

20 ± 4R

5 ± 1M,R

22 ± 5R

-

5000

7 ± 2R

25 ± 3M,R

18 ± 1R

2 ±1M,R

5 ± 2M,R

Positive controls, –S9

Name

NaN3

NaN3

MMS

4-NOPD

4-NOPD

Concentration

[μg/plate]

10

10

2.0 µL

50

10

Mean No. of colonies/plate

(average of 3 plates ± SD)

1531 ± 96

2556 ± 91

1006 ± 112

102 ± 7

547 ± 25

+

0 (DMSO)

14 ± 6

136 ± 21

49 ± 6

20 ± 2

48 ± 1

+

0

13 ± 2

147 ± 19

46 ± 12

23 ± 6

52 ± 8

+

3

14 ± 4

136 ± 15

49 ± 10

21 ± 9

57 ± 10

+

10

12 ± 2

118 ± 8

64 ± 7

19 ± 3

57 ± 10

+

33

13 ± 3

125 ± 9

51 ± 12

16 ± 6

52 ± 9

+

100

12 ± 3

152 ± 16

48 ± 6

20 ± 5

41 ± 3

+

333

14 ± 2

177 ± 18

46 ± 7

20 ± 2

50 ± 10

+

1000

13 ± 6

157 ± 5

46 ± 9

21 ± 1

56 ± 6

+

2500

11 ± 3M,R

44 ± 11R

20 ± 0R

12 ± 2R

43 ± 2R

+

5000

6 ± 3M,R

0 ± 1R

8 ± 2M,R

0 ± 1M,R

21 ± 6M,R

Positive controls, +S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentration

[μg/plate]

2.5

2.5

10

2.5

2.5

Mean No. of colonies/plate

(average of 3 plates ± SD)

422 ± 41

4195 ± 108

459 ± 12

231 ± 22

5194 ± 506

NaN3: sodium azide

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

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

M: manual count

R: reduced background growth

 

Table 2. Test results of Experiment 2 (preincubation).

With or without S9-Mix

Test substance concentration [μg/plate]

Mean number of revertant colonies per plate

(average of 3 plates ± SD)

Base-pair substitution type

Frameshift type

TA1535

TA100

WP2 uvrA

TA1537

TA98

-

0 (DMSO)

14 ± 4

162 ± 23

43 ± 8

10 ± 1

25 ± 5

-

0

12 ± 4

182 ± 9

48 ± 8

11 ± 3

32 ± 7

-

10

10 ± 1

137 ± 14

33 ± 3

10 ± 2

32 ± 7

-

33

15 ± 5

144 ± 16

37 ± 5

8 ± 3

27 ± 3

-

100

13 ± 2

145 ± 7

36 ± 5

12 ± 6

27 ± 7

-

333

19 ± 4

125 ± 13

37 ± 11

7 ± 0

16 ± 3

-

1000

19 ± 3R

9 ± 4M,R

26 ± 2R

9 ± 2R

23 ± 3R

-

2500

4 ± 3M,R

0 ± 0R

0 ± 0R

2 ± 1R

1 ± 0R

-

5000

0 ± 0R

0 ± 0R

0 ± 0R

0 ± 0R

0 ± 0R

Positive controls, –S9

Name

NaN3

NaN3

MMS

4-NOPD

4-NOPD

Concentration

[μg/plate]

10

10

2.0 µL

50

10

Mean No. of colonies/plate

(average of 3 plates ± SD)

1320 ± 33

2360 ± 59

910 ± 21

93 ± 22

541 ± 56

+

0 (DMSO)

12 ± 5

121 ± 14

45 ± 4

14 ± 2

38 ± 5

+

0

13 ± 4

180 ± 14

59 ± 8

11 ± 5

38 ± 3

+

10

16 ± 5

125 ± 11

51 ± 6

17 ± 6

35 ± 3

+

33

11 ± 3

126 ± 6

39 ± 9

10 ± 5

40 ± 4

+

100

14 ± 3

146 ± 17

44 ± 5

13 ± 3

35 ± 14

+

333

10 ± 4

74 ± 4R

47 ± 11

13 ± 4

50 ± 4

+

1000

4 ± 2M,R

2 ± 1R

34 ± 3R

10 ± 2R

6 ± 1R

+

2500

6 ± 1R

0 ± 0R

2 ± 1R

1 ± 0R

0 ± 0R

+

5000

0 ± 0R

0 ± 0R

0 ± 0R

0 ± 0R

0 ± 0R

Positive controls, +S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentration

[μg/plate]

2.5

2.5

10

2.5

2.5

Mean No. of colonies/plate

(average of 3 plates ± SD)

355 ± 31

4197 ± 260

384 ± 11

161 ± 20

3778 ± 306

NaN3: sodium azide

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

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

M: manual count

R: reduced background growth

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.
Executive summary:

A bacterial gene mutation assay with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (2016). In this study 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 up to 5000 µg/plate or cytotoxic concentrations