Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Aug - 2 Sep 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 Jul 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:
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)
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 experiment

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 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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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. 2: +S9: starting at 1000 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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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 from 2500 to 5000 µg/plate. Precipitation of the test substance in the overlay agar on the incubated agar plates was observed at 5000 µg/plate. 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 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 indication factor of 0.5), occurred in strain TA 100 in the presence of metabolic activation in Experiment II at concentrations ranging from 1000 to 5000 µg/plate.

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)

11 ± 5

181 ± 23

37 ± 5

10 ± 1

30 ± 3

-

0

9 ± 2

191 ± 6

45 ± 2

9 ± 2

28 ± 10

-

3

14 ± 7

184 ± 5

38 ± 8

10 ± 1

30 ± 12

-

10

11 ± 1

176 ± 17

33 ± 5

8 ± 3

29 ± 10

-

33

12 ± 4

171 ± 5

35 ± 6

12 ± 3

26 ± 8

-

100

12 ± 4

165 ± 27

41 ± 5

10 ± 2

30 ± 4

-

333

10 ± 1

172 ± 12

36 ± 8

10 ± 1

31 ± 6

-

1000

15 ± 6

166 ± 15

41 ± 13

12 ± 6

29 ± 3

-

2500

12 ± 3

175 ± 13

42 ± 4

11 ± 5

28 ± 7

-

5000

14 ± 3P

172 ± 17P

45 ± 2P

12 ± 3P

39 ± 3P

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)

1318 ± 26

2258 ± 81

985 ± 16

79 ± 10

461 ± 21

+

0 (DMSO)

12 ± 3

171 ± 6

39 ± 9

11 ± 4

34 ± 5

+

0

16 ± 6

198 ± 7

57 ± 12

16 ± 4

37 ± 7

+

3

15 ± 6

138 ± 18

46 ± 11

14 ± 4

42 ± 11

+

10

14 ± 2

149 ± 18

47 ± 7

12 ± 1

39 ± 4

+

33

14 ± 4

134 ± 10

53 ± 9

13 ± 3

32 ± 10

+

100

11 ± 4

165 ± 14

44 ± 8

11 ± 2

39 ± 8

+

333

11 ± 3

156 ± 32

48 ± 9

13 ± 3

34 ± 8

+

1000

13 ± 3

138 ± 17

44 ± 12

13 ± 6

36 ± 12

+

2500

10 ± 4

128 ± 6

47 ± 8

12 ± 6

32 ± 8

+

5000

14 ± 3P

128 ± 10P

38 ± 9P

9 ± 3P

33 ±7P

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)

383 ± 20

5126 ± 220

488 ± 17

311 ± 25

5169 ± 523

NaN3: sodium azide

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

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

P: precipitate

 

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)

9 ± 2

125 ± 1

39 ± 12

9 ± 4

30 ± 8

-

0

12 ± 2

195 ± 5

37 ± 9

13 ± 4

26 ± 4

-

33

8 ± 3

113 ± 17

45 ± 9

8 ± 3

25 ± 3

-

100

7 ± 1

129 ± 7

38 ± 4

10 ± 4

24 ± 11

-

333

7 ± 2

126 ± 9

45 ± 8

8 ± 2

21 ± 7

-

1000

9 ± 1

111 ± 16

32 ± 7

11 ± 2

26 ± 5

-

2500

11 ± 2

105 ± 5

41 ± 5

8 ± 2

24 ± 4

-

5000

12 ± 2P

120 ± 20P

51 ± 3P

14 ± 2P

31 ± 6P

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)

1214 ± 64

1739 ± 67

758 ± 31

75 ± 12

489 ± 14

+

0 (DMSO)

10 ± 3

132 ± 35

49 ± 3

18 ± 2

45 ± 8

+

0

8 ± 3

174 ± 12

54 ± 12

20 ± 5

44 ± 2

+

33

10 ± 5

93 ± 16

53 ± 8

19 ± 5

32 ± 5

+

100

11 ± 4

118 ± 10

50 ± 12

20 ± 2

37 ± 8

+

333

8 ± 3

76 ± 11

51 ± 5

12 ± 3

30 ± 2

+

1000

10 ± 3

44 ± 5

54 ± 7

19 ± 7

33 ± 7

+

2500

11 ± 3

53 ± 12

39 ± 11

18 ± 3

34 ± 13

+

5000

9 ± 3P

49 ± 3P

38 ± 16P

13 ± 4P

35 ± 6P

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)

346 ± 28

4134 ± 107

446 ± 27

266 ± 8

2980 ± 776

NaN3: sodium azide

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

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

P: precipitate

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 strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) tested with and without metabolic activation up to 5000 µg/plate.