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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 15 June 2012 to 30 August 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD 471 guideline sudy in compliance with the GLP.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
No 2011/40
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid at ambient temperature
Details on test material:
- Name of test material (as cited in study report): Dermalcare MAP L-213/S (or Dermalcare MAP L213S)
- Physical state: yellowish liquid
- Stability under test conditions: the test item is assumed to be stable by the sponsor
- Storage condition of test material: at room temperature

Method

Target gene:
Each strain contains one mutation in the histidine operon, resulting in a requirement for histidine.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
other: additional mutations in rfa and uvrB genes. For the strains TA98, TA100 and TA102 presence of an additional plasmid pKM101 in order to enhance their sensitivity of detection of some mutagens. See Table 7.6.1/1
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction obtained by Moltox (USA) and obtained from the liver of the rats treated with Aroclor 1254 (500 mg/kg bw) by the intraperitoneal route. Each batch is tested and validated by Moltox for its ability to activate B[a]P and 2- amino anthracene.
Test concentrations with justification for top dose:
Preliminary assay: 10; 100; 500; 1000; 2500 and 5000 µg/plate
First experiment -S9 mix (TA1535; TA 1537; TA98): 0.82; 2.47; 7.41; 22.2; 66.7 and 200 µg/plate
First experiment - S9 mix (TA 100 and TA102) and +S9 mix (TA1535; TA1537; TA98 and TA102): 2.47; 7.41; 22.2; 66.7; 200 and 600 µg/plate
First experiment + S9 mix (TA100): 7.41; 22.2; 66.7; 200; 600 and 1800 µg/plate
Second experiment -S9 mix (TA1535; TA1537; TA98) and +S9 mix (TA98): 0.82; 2.47; 7.41; 22.2; 66.7 and 200 µg/plate
Second experiment - S9 mix (TA 100 and TA102) and +S9 mix (TA1535; TA1537 and TA102): 2.47; 7.41; 22.2; 66.7; 200 and 600 µg/plate
Second experiment + S9 mix (TA100): 7.41; 22.2; 66.7; 200; 600 and 1800 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water for injections
- Justification for choice of solvent/vehicle: the test item is soluble in water for injections at 50 mg/mL.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water for injections (batch No. 2F0703 (CDM Lavoisier, France)).
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-anthramine
Remarks:
see details in table 7.6.1/2
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) for the first experiment and the second experiment without metabolic activation; preincubation for the second experiment with metabolic activation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 or 72 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: in preliminary assay: one plate/concentration, in the main studies: 3 plates/concentration; 2 independent experiments

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants and/or a thinning of the bacterial lawn

OTHER EXAMINATIONS:
not applicable
Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship would have been considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.
Statistics:
no statistics were performed.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
see tables 7.6.1/3 to 7.6.1/6
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in all strains, in both experiments, with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no precipitate was observed up to 5000 µg/plate
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: In the absence of S9 mix, a moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose-levels ≥ 500 µg/plate towards the TA 98 and TA 102 strains and at dose-levels ≥ 1000 µg/plate towards the TA 100 strain.
In the presence of S9 mix, a moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose-levels ≥ 1000 µg/plate towards the TA 98 and TA 102 strains and at dose-levels ≥ 2500 µg/plate towards the TA 100 strain.

COMPARISON WITH HISTORICAL CONTROL DATA: The number of revertants for the vehicle and positive controls met the acceptance criteria. The study was therefore considered to be valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Without S9 mix,a strong toxicity was noted at dose-levels ≥ 200 µg/plate towards the TA 1535, TA 98 and TA 102 strains and at 600 µg/plate towards the TA 100 strain in both experiments. For the TA 1537 strain, a moderate to strong toxicity was noted at dose-levels ≥ 66.7 µg/plate and at dose-levels ≥ 22.2 µg/plate in the first and second experiments, respectively.
With S9 mix, using the direct plate incorporation method, a strong toxicity was noted at 600 µg/plate towards the TA 1535, TA 1537, TA 98 and TA 102 strains, and a moderate toxicity was observed at 1800 µg/plate towards the TA 100 strain. Using the pre-incubation method, a moderate to strong toxicity was noted at dose-levels ≥ 200 µg/plate towards the TA 1535, TA 1537, TA 98 and TA 102 strains, and at 1800 µg/plate towards the TA 100 strain.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/3:Number of revertants per plate in the absence of metabolic activation in the first test (direct plate incorporation method)

Dermalcare MAP L-213/S Concentration
(µg/plate)

TA 1535

TA1537

TA 98

TA 100

TA 102

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

0*

12

5

4

2

34

8

156

38

397

36

0.82

13

3

7

3

23

4

-

-

-

-

2.47

14

3

6

3

36

18

240

84

377

16

7.41

13

0

4

2

43

18

206

33

414

46

22.2

11

3

6

2

30

9

184

9

299

9

66.7

11

5

3

2

15

3

264

69

277

48

200

3

2

9

4

0

1

302

11

38

45

600

-

-

-

-

-

-

93

25

8

2

1800

-

-

-

-

-

-

-

-

-

-

Positive control**

512

35

471

108

107

14

471

76

1771

47

$: Mean of triplicate

*Solvent control = negative control: 100 µL water

**Mutagens positive controls:

- NaN3(1 µg/plate) in TA1535 and TA100 strains

- 9AA (50 µg/plate) in TA1537 strain

- 2NF (0.5 µg/plate) in TA 98 strain

- MMC ( 0.5 µg/plate) in TA 102 strain

 

 

Table 7.6.1/4: Number of revertants per plate in the presence of metabolic activation (S9 mix) in the first test (direct plate incorporation method) 

Dermalcare MAP L-213/S Concentration
(µg/plate)

TA 1535

TA1537

TA 98

TA 100

TA 102

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

0*

17

3

11

2

36

7

149

3

552

101

0.82

-

-

-

-

-

-

-

-

-

-

2.47

29

8

10

4

33

2

-

-

786

125

7.41

21

5

10

2

29

6

203

56

735

35

22.2

15

9

14

4

35

8

144

10

609

75

66.7

19

2

7

4

28

6

171

17

422

61

200

11

3

8

3

17

3

223

27

294

73

600

3

3

3

3

9

7

165

24

91

16

1800

-

-

-

-

-

-

139

11

-

-

Positive control**

159

25

105

15

788

39

535

66

3673

1457

$: Mean of triplicate

*Solvent control = negative control: 100 µL water

**Mutagens positive controls:

-2AM(2µg/plate) in TA1535, TA1537, TA98 strains

- BAP (5µg/plate) in TA100 strain

-2AM(10µg/plate) in TA102 strain

 

Table 7.6.1/5: Number of revertants per plate in the absence of metabolic activation in the second test (direct plate incorporation method)

Dermalcare MAP L-213/S Concentration
(µg/plate)

TA 1535

TA1537

TA 98

TA 100

TA 102

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

0*

13

4

11

5

30

6

145

29

367

12

0.82

18

2

7

3

23

2

-

-

-

-

2.47

17

4

10

8

40

9

122

7

421

33

7.41

20

3

11

6

38

8

180

49

540

112

22.2

8

7

4

3

30

7

172

38

367

31

66.7

14

11

1

1

16

4

133

19

282

18

200

8

1

6

6

2

2

116

6

23

7

600

-

-

-

-

-

-

143

37

27

23

1800

-

-

-

-

-

-

-

-

-

-

Positive control**

450

10

981

507

132

39

472

81

1825

219

$: Mean of triplicate

*Solvent control = negative control: 100 µL water

**Mutagens positive controls:

- NaN3(1 µg/plate) in TA1535 and TA100 strains

- 9AA (50 µg/plate) in TA1537 strain

- 2NF (0.5 µg/plate) in TA 98 strain

- MMC ( 0.5 µg/plate) in TA 102 strain

 

Table 7.6.1/6: Number of revertants per plate in the presence of metabolic activation (S9 mix) in the second test (pre-incubation method)

 

Dermalcare MAP L-213/S Concentration
(µg/plate)

TA 1535

TA1537

TA 98

TA 100

TA 102

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

0*

15

6

10

3

20

4

108

3

468

40

0.82

-

-

-

-

20

3

-

-

-

-

2.47

15

2

8

0

29

9

-

-

523

33

7.41

12

1

9

1

17

8

130

10

505

72

22.2

18

2

10

2

19

3

96

17

478

59

66.7

14

3

8

5

21

12

119

7

561

37

200

11

7

5

2

21

7

121

7

349

73

600

5

2

0

0

-

-

68

14

91

16

1800

-

-

-

-

-

-

44

8

-

-

Positive control**

123

9

152

15

959

43

486

387

1639

124

$: Mean of triplicate

*Solvent control = negative control: 100 µL water

**Mutagens positive controls:

-2AM(2µg/plate) in TA1535, TA1537, TA98 strains

- BAP (5µg/plate) in TA100 strain

-2AM(10µg/plate) in TA102 strain

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Under the test conditions, Dermalcare MAP L-213/S did not show any mutagenic activity in the bacterial reverse mutation test using Salmonella typhimurium.
Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD No.471 guideline, Dermalcare MAP L-213/S diluted in water was tested inS. typhimuriumTA1535, TA1537, TA100, TA98 and TA102 in the presence and the absence of mammalian metabolic activation (S9) using the direct incorporation or the preincubation method. Dermalcare MAP L-213/S as an aqueous solution contains 25.6% of solid content. Therefore a correction factor of 3.91 was applied to calculate the tested concentrations in order to test the solid content of Dermalcare MAP L-213/S at 100%. Six known mutagens (Sodium azide; 9-Aminoacridine; 2-Nitrofluorene; Mitomycin C; 2-Anthramine and Benzoapyrene), dissolved in dimethylsulfoxide (except for Mitomycin C which was dissolved in distilled water), were used to check the sensitivity of the test system.

A preliminary study (one plate/concentration) was performed in order to determine the appropriate concentrations for the two independent main studies (3 plates/concentration). In the preliminary study, the bacterial strains TA98; TA100 and TA102 were exposed to the test substance at the following concentrations: 0; 10; 100; 500; 1000; 2500 and 5000 µg/plate. The test item was freely soluble up to the highest tested concentration. Cytotoxicity, assessed by the decrease in the number of revertants and/or the thinning of the bacterial lawn, was observed in all strains. In the absence of S9 mix, a moderate to strong toxicity was noted at dose-levels ≥ 500 µg/plate towards the TA 98 and TA 102 strains and at dose-levels ≥ 1000 µg/plate towards the TA 100 strain. In the presence of S9 mix, a moderate to strong toxicity was noted at dose-levels ≥ 1000 µg/plate towards the TA 98 and TA 102 strains and at dose-levels ≥ 2500 µg/plate towards the TA 100 strain.

In the main studies Dermalcare MAP L-213/S was tested without S9 mix at2.47,7.41, 22.2, 66.7, 200 and 600 µg/plate for the TA 100 and TA 102 strains in both experiments and at 0.82,2.47,7.41, 22.2, 66.7 and 200 µg/plate for the TA 1535, TA 1537 and TA 98 strains in both experiments. In the presence of metabolic activation, Dermalcare MAP L-213/S was tested at7.41,22.2, 66.7, 200, 600 and 1800 µg/plate in TA 100 strain in both experiments,2.47,7.41,22.2, 66.7, 200 and 600 µg/plate in TA 1535, TA 1537 and TA 102 strains in both experiments and in the TA98 inthe first experiment, 0.82,2.47,7.41, 22.2, 66.7 and 200 µg/plate in TA 98 strain in the second experiment.

Cytotoxicity was observed in all strains in both experiments and whatever the metabolic condition used (i.e. with or without S9 mix). The number of revertants for the vehicle and positive controls met the acceptance criteria. The study was therefore considered to be valid. The test item did not induce any noteworthy or biologically relevant increase in the number of revertants, in any of the other tested strains.

 

Under the test conditions, Dermalcare MAP L-213/S did not show any mutagenic activity in the bacterial reverse mutation test using Salmonella typhimurium. This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 471.