Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 May - 05 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
/ During the study, 2-Aminoanthracene was used as sole indicator of the efficacy of the S9-mix

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:
21 July 1997
Deviations:
yes
Remarks:
/ During the study, 2-Aminoanthracene was used as sole indicator of the efficacy of the S9-mix
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 July 1992/ 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, tryp operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Remarks:
and 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 and β-naphthoflavone
Test concentrations with justification for top dose:
First experiment (dose-finding study):
all strains: 3 - 5000 μg/plate with and without metabolic activation (tested up to the limit concentration)

Second experiment:
TA 1535 and TA 1537: 10 - 5000 μg/plate with and without metabolic activation (tested up to the limit concentration)
TA 98, TA 100, WP 2 uvrA: 3 - 5000 μg/plate with and without metabolic activation (tested up to the limit concentration)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen based on its solubility properties and its relative nontoxicity for 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: plate incorporation test (experiment I = pre-experiment) and preincubation (experiment II = main assay)

DURATION
- Preincubation period: 1 h (experiment I)
- Exposure duration: 48 h (exp. I and II)

NUMBER OF REPLICATIONS: The dose-finding assay and main assay were carried out in triplicate plating for each dose level.

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn
Evaluation criteria:
Acceptance criteria
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of historical data
- the positive control substances induce a duplication (TA 98, TA 100, and WP2 uvrA) or triplication (TA 1535 and TA 1537) of the colony count compared to 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
When the test substance shows a biologically relevant increase in the number of revertant colonies to at least twice (TA 98, TA 100, and WP2 uvrA) or three times (TA 1535 and TA 1537) as many as that of the solvent control, the response is judged to be positive. TA 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, if 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:
- S9: reduced background growth at ≥ 2500 µg/ plate; + S9: reduced background growth at ≥ 2500 µg/ plate and reduced number of revertant colonies at ≥ 5000 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:
- S9: reduced background growth at ≥ 2500 µg/ plate and reduced number of revertant colonies at ≥ 5000 µg/ plate; + S9: reduced background growth at ≥ 1000 µg/ plate and reduced number of revertant colonies at ≥ 2500 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:
- S9: reduced background growth at ≥ 1000 µg/ plate and reduced number of revertant colonies at ≥ 5000 µg/ plate; + S9: reduced background growth at ≥ 1000 µg/ plate and reduced number of revertant colonies at ≥ 2500 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:
- S9: reduced background growth at ≥ 1000 µg/ plate and reduced number of revertant colonies at ≥ 2500 µg/ plate; + S9: reduced background growth and reduced number of revertant colonies 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:
cytotoxicity
Remarks:
- S9: reduced background growth at ≥ 2500 µg/ plate and reduced number of revertant colonies at ≥ 5000 µg/ plate; + S9: reduced background growth and reduced number of revertant colonies at ≥ 2500 µg/ plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
yes (for further details please refer to "Any other information on results incl. tables", Table 1)

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
yes (for further details please refer to "Any other information on results incl. tables", Table 3)

Any other information on results incl. tables

Table 2: Summary of test results (experiment 1; dose-finding assay)

With or without S9-Mix

Test substance concentration (μg/plate)

Mean number of revertant colonies per plate (average of 3 plates)

Frameshift type

Base-pair substitution type

TA1537

TA98

TA100

TA1535

WP2 uvrA

Solvent control (DMSO)

7 ± 2

33 ± 6

169 ± 16

13 ± 3

44 ± 7

Untreated control

12 ± 3

28 ± 2

173 ± 10

8 ± 5

42 ± 1

3

8 ± 2

31 ± 8

149 ± 7

10 ± 5

36 ± 1

10

9 ± 3

29 ± 4

167 ± 5

13 ± 3

38 ± 2

33

7 ± 2

30 ± 5

185 ± 5

13 ± 2

39 ± 15

100

9 ± 1

34 ± 3

160 ± 24

13 ± 3

43 ± 9

333

10 ± 6

31 ± 5

159 ± 21

12 ± 2

38 ± 10

1000

8 ± 2

39 ± 7

127 ± 32

15 ± 3

42 ± 3

2500

4 ± 1

25 ± 9

65 ± 5

13 ± 6

41 ± 1

5000

3 ± 1

11 ± 2

51 ± 4

11 ± 3

45 ± 6

Positive controls (unit/plate)

4-NOPD
(50 µg)

4-NOPD
(10 µg)

SA
(10 µg)

SA
(10 µg)

MMS
(2 µl)

Mean No. of colonies/plate (average of 3 plates)

77 ± 3

427 ± 30

1999 ± 68

1253 ± 30

908 ± 42

+

Solvent control (DMSO)

14 ± 0

40 ± 6

178 ± 15

10 ± 4

52 ± 5

Untreated control

19 ± 5

45 ± 2

195 ± 20

11 ± 4

49 ± 2

3

15 ± 3

40 ± 7

169 ± 21

13 ± 3

53 ± 7

10

16 ± 3

30 ± 5

186 ± 14

11 ± 5

52 ± 11

33

13 ± 3

37 ± 5

164 ± 11

12 ± 5

49 ± 3

100

16 ± 5

47 ± 7

171 ± 2

12 ± 4

53 ± 1

333

15 ± 6

40 ± 7

164 ± 9

9 ± 3

48 ± 11

1000

11 ± 4

37 ± 10

144 ± 9

12 ± 4

36 ± 12

2500

14 ± 4

17 ± 1

58 ± 13

13 ± 1

23 ± 4

5000

5 ± 2

6 ± 2

10 ± 3

6 ± 2

19 ± 3

Positive controls (µg/plate)

2AA
(2.5)

2AA
(2.5)

2AA
(2.5)

2AA
(2.5)

2AA
(10)

Mean No. of colonies/plate (average of 3 plates)

224 ± 27

4313 ± 457

5077 ± 151

396 ± 13

478 ± 13

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

SA = sodium azide

MMS = methyl methane sulfonate

2AA = 2-aminoanthracene

SD = standard deviation

 

Table 3:Summary of test results (experiment 2; main assay) 

With or without S9-Mix

Test substance concentration (μg/plate)

Mean number of revertant colonies per plate (average of 3 plates)

Frameshift type

Base-pair substitution type

TA1537

TA98

TA100

TA1535

WP2 uvrA

Solvent control (DMSO)

11 ± 1

28 ± 6

147 ± 6

8 ± 2

32 ± 6

Untreated control

11 ± 1

30 ± 6

188 ± 12

10 ± 3

34 ± 2

3

29 ± 7

138 ± 4

29 ± 5

10

8 ± 1

26 ± 10

137 ± 11

13 ± 3

35 ± 2

33

10 ± 1

31 ± 5

138 ± 19

10 ± 1

33 ± 0

100

8 ± 3

30 ± 3

132 ± 23

11 ± 1

31 ± 3

333

13 ± 3

38 ± 4

105 ± 6

9 ± 2

37 ± 5

1000

13 ± 1

37 ± 9

81 ± 4

16 ± 4

36 ± 7

2500

12 ± 3

29 ± 3

16 ± 1

13 ± 3

26 ± 6

5000

4 ± 4

7 ± 4

0 ± 1

12 ± 4

12 ± 4

Positive controls (unit/plate)

4-NOPD
(50 µg)

4-NOPD
(10 µg)

SA
(10 µg)

SA
(10 µg)

MMS
(2 µl)

Mean No. of colonies/plate (average of 3 plates)

87 ± 12

578 ± 35

1967 ± 33

1199 ± 32

443 ± 23

+

Solvent control (DMSO)

16 ± 0

35 ± 6

123 ± 19

12 ± 4

43 ± 6

Untreated control

11 ± 3

34 ± 3

179 ± 14

13 ± 3

41 ± 5

3

 

33 ± 3

120 ± 13

 

50 ± 7

10

12 ± 4

44 ± 8

127 ± 16

11 ± 3

41 ± 1

33

16 ± 3

38 ± 13

121 ± 9

10 ± 1

41 ± 6

100

14 ± 2

39 ± 6

117 ± 13

8 ± 3

39 ± 3

333

14 ± 3

42 ± 6

98 ± 3

9 ± 2

34 ± 6

1000

15 ± 4

24 ± 2

46 ± 5

10 ± 3

40 ± 2

2500

0 ± 0

15 ± 4

2 ± 2

7 ± 1

8 ± 3

5000

0 ± 0

0 ± 0

0 ± 0

3 ± 3

0 ± 1

Positive controls (µg/plate)

2AA
(2.5)

2AA
(2.5)

2AA
(2.5)

2AA
(2.5)

2AA
(10)

Mean No. of colonies/plate (average of 3 plates)

179 ± 19

4913 ± 498

3863 ± 364

378 ± 43

355 ± 22

 4-NOPD = 4-nitro-o-phenylene-diamine

SA = sodium azide

MMS = methyl methane sulfonate

2AA = 2-aminoanthracene

SD = standard deviation 

Applicant's summary and conclusion

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