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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 Oct - 10 Nov 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted Jul 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Principles of method if other than guideline:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches 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; trp operon
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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
Species / strain:
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:
First experiment (plate incorporation test): 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate with and without metabolic activation
Second experiment (pre-incubation test): 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate with and without metabolic activation
Vehicle:
- Solvent: DMSO
- Justification for choice of vehicle: Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle
Controls
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9: sodium azide (10 µg/plate) for TA100 and TA1535; 4-nitro-o-phenylene-diamine (10 µg/plate and 50 µg/plate) for TA1537 and TA98; methyl methane sulfonate (2 µl/plate) for WP2 uvrA; +S9: 2-aminoanthracene (2.5 µg/plate and 10 µg/plate) for all strains
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation; Experiment I); pre-incubation (Experiment II)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each of two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of the number of spontaneous revertants and 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 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 deviation 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:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
reduction on the number of revertants at 5000 µg/plate (Experiment I with and without S9 mix; Experiment II with S9 mix)
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
reduction on the number of revertants at 5000 µg/plate (Experiment I with and without S9 mix; Experiment II with S9 mix)
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
reduction on the number of revertants at 2500 - 5000 µg/plate (Experiment I with and without S9 mix; Experiment II with S9 mix)
Vehicle controls valid:
yes
Negative controls valid:
no
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item precipitated in the overlay agar in the test tubes from 333 to 5000 μg/plate in experiment I and from 1000 to 5000 μg/plate in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 1000 to 5000 μg/plate in both experiments. The undissolved particles had no influence on the data recording and the tested number of concentration levels that showed no precipitation was sufficient according to the guideline.

Any other information on results incl. tables

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

With or without S9-Mix

Test substance concentration

Mean number of revertant colonies per plate

(μg/plate)

(average of 3 plates ± Standard deviation)

 

Base-pair substitution type

Frameshift type

Base-pair substitutions and others

 

TA 100

TA1535

TA98

TA1537

WP2 uvrA

DMSO

112 ± 9

9 ± 4

19 ± 2

7 ± 1

37 ± 3

Untreated

131 ± 8

9 ± 2

22 ± 6

8 ± 1

43 ± 1

3

111 ± 8

9 ± 3

20 ± 1

6 ± 3

32 ± 5

10

103 ± 8

8 ± 2

25 ± 8

8 ± 4

37± 7

33

113 ± 24

12 ± 3

23 ± 8

7 ± 0

46 ± 9

100

85 ± 8

9 ± 1

20 ± 1

8 ± 2

46 ± 6

333

76 ± 11

10 ± 2

27 ± 10

10 ± 4

42 ± 13

1000

91 ± 12 P

11 ± 1 P

17 ± 2 P

8 ± 2 P

57 ± 7 P

2500

49 ± 3 P

9 ± 2 P

19 ± 4 P

6 ± 1 P

39 ± 8 P

5000

46 ± 7 P

7 ± 2 P

6 ± 2 P

3 ± 1 P

30 ± 4 P

Positive controls, –S9

Name

NaN3

NaN3

4-NOPD

4-NOPD

MMS

Concentrations (μg/plate)

10

10

10

50

2 [µL]

Mean No. of colonies/plate (average of 3 ± SD)

2261 ± 165

1077 ± 131

382 ± 22

77 ± 8

988 ± 24

+

DMSO

87 ± 4

12 ± 4

24 ± 6

10 ± 2

47 ± 5

+

Untreated

127 ± 12

10 ± 2

34 ± 7

11 ± 3

49 ± 5

+

3

83 ± 4

13 ± 1

28 ± 7

9 ± 5

50 ± 5

+

10

86 ± 8

15 ± 2

24 ± 2

10 ± 2

40± 10

+

33

83 ± 4

12 ± 2

27 ± 6

8 ± 1

49 ± 8

+

100

65 ± 13

12 ± 4

29 ± 2

11 ± 4

37 ± 9

+

333

65 ± 13

12 ± 3

32 ± 10

9 ± 5

53 ± 11

+

1000

67 ± 12 P

15 ± 2 P

25 ± 3 P

10 ± 2 P

56 ± 8 P

+

2500

53 ± 3 P

9 ± 2 P

14 ± 3 P

6 ± 1 P

30 ± 3 P

+

5000

46 ± 2 P

9 ± 2 P

9 ± 1 P

2 ± 1 P

25 ± 4 P

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations (μg/plate)

2.5

2.5

2.5

2.5

10

Mean No. of colonies/plate (average of 3 ± SD)

3458 ± 146

488 ± 30

301 ± 59

159 ± 11

370 ± 26

NaN3 = Sodium azide

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

MMS = methyl methane sulfonate

2AA = 2-Aminoanthracene

P = Precipitate

Table 2. Test results of experiment 2 (pre-incubation test)

With or without S9-Mix

Test substance concentration

Mean number of revertant colonies per plate

(μg/plate)

(average of 3 plates ± Standard deviation)

 

Base-pair substitution type

Frameshift type

Base-pair substitutions and others

 

TA 100

TA1535

TA98

TA1537

WP2 uvrA

DMSO

118 ± 3

10 ± 2

22 ± 5

11 ± 2

30 ± 10

Untreated

184 ± 21

9 ± 5

29 ± 5

8 ± 4

43 ± 6

3

111 ± 3

9 ± 3

23 ± 3

11 ± 2

35 ± 3

10

112 ± 22

10 ± 5

25 ± 5

10 ± 2

33 ± 9

33

112 ± 18

8 ± 3

23 ± 7

8 ± 3

44 ± 5

100

95 ± 16

10 ± 4

31 ± 2

9 ± 1

36 ± 7

333

60 ± 4

11 ± 3

21 ± 1

8 ± 2

45 ± 2

1000

60 ± 15 P

11 ± 3 P

24 ± 6 P

12 ± 0 P

35 ± 11 P

2500

45 ± 3 P

8 ± 1 P

19 ± 3 P

9 ± 2 P

34 ± 3 P

5000

44 ± 6 P

5 ± 2 P

22 ± 5 P

5 ± 1 P

33 ± 2 P

Positive controls, –S9

Name

NaN3

NaN3

4-NOPD

4-NOPD

MMS

Concentrations (μg/plate)

10

10

10

50

2 [µL]

Mean No. of colonies/plate (average of 3 ± SD)

1787 ± 97

938 ± 55

261 ± 39

90 ± 9

665 ± 51

+

DMSO

105 ± 11

10 ± 3

34 ± 1

13 ± 3

53 ± 12

+

Untreated

145 ± 22

10 ± 2

39 ± 9

15 ± 4

57 ± 8

+

3

116 ± 8

9 ± 3

41 ± 7

15 ± 2

44 ± 10

+

10

133 ± 16

10 ± 5

40 ± 2

17 ± 3

46 ± 8

+

33

112 ± 10

10 ± 5

40 ± 6

12 ± 3

47 ± 8

+

100

89 ± 8

11 ± 2

27 ± 9

12 ± 3

48 ± 15

+

333

85 ± 3

10 ± 3

23 ± 3

11 ± 3

52 ± 12

+

1000

93 ± 2 P

13 ± 3 P

27 ± 3 P

13 ± 2 P

55 ± 5 P

+

2500

92 ± 3 P

11 ± 2 P

20 ± 7 P

10 ± 1 P

40 ± 8 P

+

5000

85 ± 3 P

8 ± 1 P

13 ± 4 P

4 ± 1 P

27 ± 5 P

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations (μg/plate)

2.5

2.5

2.5

2.5

10

Mean No. of colonies/plate (average of 3 ± SD)

2907 ± 251

350 ± 42

3610 ± 212

175 ± 21

270 ± 26

NaN3 = Sodium azide

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

MMS = methyl methane sulfonate

2AA = 2-Aminoanthracene

P = Precipitate

Table 3: Historical control data

Strain   - S9 + S9
Mean SD Min Max Mean SD Min Max
TA1535 Solvent control 16 2.84 7 27 18 4.50 7 36
Untreated control 15 3.35 7 27 19 4.99 9 34
Positive control 2248 468.15 803 3722 418 119.81 182 683
TA1537 Solvent control 10 2.19 6 23 18 4.37 8 32
Untreated control 10 2.52 5 22 19 4.64 8 30
Positive control 74 10.30 50 159 300 126.74 77 651
TA98 Solvent control 26 4.27 16 46 38 6.29 21 59
Untreated control 28 5.15 16 54 41 6.40 21 55
Positive control 326 56.74 211 560 2306 944.29 330 4691
TA100 Solvent control 107 20.07 74 184 132 22.97 83 205
Untreated control 113 21.29 82 204 142 21.11 82 210
Positive control 1987 376.71 478 2594 2864 946.03 530 4983
WP2uvrA Solvent control 52 7.73 34 70 58 7.87 41 77
Untreated control 52 7.91 32 81 57 7.71 39 74
Positive control 716 259.60 226 1296 354 112.61 180 642

Mean = mean value if revertants/plate

SD = Standard Deviation

Min = minimal value

Max = maximal value

Applicant's summary and conclusion