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Genetic toxicity: in vitro

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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:
11 August 2003 to 21 August 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Harmonised Tripartite Guidelines S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals
Qualifier:
according to guideline
Guideline:
other: ICH Harmonised Tripartite Guidelines S2B: Genotoxicity. A Standard Battery for Genotoxicity Testing of Pharmaceuticals.
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
452-330-3
EC Name:
-
Cas Number:
314020-40-1
Molecular formula:
C14H20N2O2
IUPAC Name:
2-(2,6-diethyl-4-methyl-phenyl)propanediamide
Test material form:
solid: particulate/powder
Details on test material:
- Physical state: Powder, yellowish
- Storage condition of test material: In the dark at ambient temperature

Method

Target gene:
S. typhimurium: Histidine synthesis

E. coli: Tryptophan synthesis
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: Top agar consisting of 0.6 % w/v agar and 0.5 % w/v sodium chloride in deionised water. Prior to testing, the molten top agar was prepared by adding sterile 0.5 mM histidine/0.5 mM biotin stock solution (10 mL solution: 100 mL) agar).
0.5 mL S9 mix (or S9 buffer) was then added to the number of aliquots of one strain required for one concentration, followed by 0.1 mL of the appropriate concentration of the test substance preparation. Finally 2.0 mL top agar was then added to each aliquot, and the resulting mixture poured onto the surface of a prepared pre-labelled Vogel Bonner plate (9 cm diameter vented Petri-dish prepared with 25 mL Vogel Bonner minimal medium and containing 1.5 % w/v agar and 2 % w/v glucose) and allowed to gel.
- Periodically checked for genotype stability: Yes. The overnight culture from each new frozen culture was screened for the deep-rough characters, DNA repair deficiency and Ampicillin resistance. The presence of the uvrB deletion was confirmed by testing the sensitivity of each culture to mitomycin C (10 µL of a 10 µg/mL solution) in the same manner as sensitivity to crystal violet was tested.
- Periodically "cleansed" against high spontaneous background: Yes. When fresh frozen stocks were prepared, the strains were tested for amino acid requirement.
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli, other: E. coli WP2P uvr A and E. coli WP2P
Details on mammalian cell type (if applicable):
- Type and identity of media: Top agar consisting of 0.6 % w/v agar and 0.5 % w/v sodium chloride in deionised water. Prior to testing, the molten top agar was prepared by adding sterile tryptophan solution (10 mL 0.5 mM stock: 100 mL agar).
0.5 mL S9 mix (or S9 buffer) was then added to the number of aliquots of one strain required for one concentration, followed by 0.1 mL of the appropriate concentration of the test substance preparation. Finally 2.0 mL top agar was then added to each aliquot, and the resulting mixture poured onto the surface of a prepared pre-labelled Vogel Bonner plate (9 cm diameter vented Petri-dish prepared with 25 mL Vogel Bonner minimal medium and containing 1.5 % w/v agar and 2 % w/v glucose) and allowed to gel.
- Periodically checked for genotype stability: Yes. The overnight culture from each new frozen culture was screened for the deep-rough characters, DNA repair deficiency and Ampicillin resistance. The presence of the uvrA mutation was confirmed by testing the sensitivity of each culture to mitomycin C (10 µL of a 10 µg/mL solution) in the same manner as sensitivity to crystal violet was tested.
- Periodically "cleansed" against high spontaneous background: Yes. When fresh frozen stocks were prepared, the strains were tested for amino acid requirement.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
5000, 2500, 1000, 500, 200 and 100 µg/plate both in the presence and absence of metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Details on positive control substance assignment is presented in table 1 in the field "Any other information on materials and methods incl. tables"
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: Plates were incubated inverted at 37 °C for three days in the dark.

NUMBER OF REPLICATIONS: Test concentrations were performed in triplicate

- EVALUATION PROCEDURE: Following the total incubation period the plates were examined for the lack of microbial contamination and evidence that the test was valid: i.e. there was a background lawn on the solvent control plates and on the plates for (at least) the lower concentrations of test substance, and that the positive controls had responded as expected. All plates were counted using an automated colony counter (Cardinal® automated counter linked to the Ames Study Manager system [Perceptive Instruments]) adjusted appropriately to permit the optimal counting of mutant colonies.
Evaluation criteria:
Test data from individual experiments are considered valid if:
a) the concurrent solvent control data are acceptable
b) the positive control data show acceptable increases

Failure of one or more tester strain/S9 combinations does not invalidate the data for the remainder of a concurrent experiment.

A positive response in a (valid) individual experiment is acheived when one or both of the following are met:
a) a significant, dose related increase in the mean number of revertants is observed; or
b) a two-fold or greater increase in the mean number of revertant colonies (over that observed for the concurrent solvent control plates) is observed at one or more concentrations.

A negative result in a (valid) individual experiment is achieved when:
a) there is no significant dose-related increase in the mean number of revertant colonies per plate observed for the test substance; and
b) in the absence of any such dose response, no increase in colony numbers is observed (at any test concentration) which exceeds 2x the concurrent solvent control.

For a positive response in an individual experiment to be considered indicative of an unequivocal positive, i.e. mutagenic, result for that strain/S9 combination, then the observed effect(s) must be consistently reproducible.
Statistics:
All derived calculations were carried out by the computerised data analysis system.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: S. typhimurium TA1535, TA1537, TA98 and TA100 and E. coli WP2P uvrA and WP2P
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
other: S. typhimurium TA1537 and TA98
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
other: S. typhimurium TA1535 and TA100 and E. coli WP2P uvrA and WP2P
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA:
The results of the study were found to be comparable with the historical control.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 2: Results with test material

Strain

Compound

Dose µg/plate

Mean revertants/plate

Treated/solvent

Individual revertant colony counts

Mean revertants/plate

Treated/solvent

Individual revertant colony counts

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Experiment 1

Experiment 2

TA 100

Test material

5000

95

194.7

1.1

1.5

97,97,91

219,223,142

94

259.7

1.0

1.3

88,93,101

276,278,225

2500

91.3

165

1.0

1.3

84,91,99

163,164,168

85.7

245

0.9

1.2

83,84,90

237,273,225

1000

78.7

138.3

0.9

1.0

77,51,108

152,135,128

95.3

214.3

1.0

1.0

72,106,108

219,224,200

500

82

124.7

0.9

0.9

86,77,83

138,112,124

101.3

219.7

1.1

1.1

101,114,89

201,257,201

200

103.7

125

1.2

0.9

101,110,100

110,125,140

96.3

212.3

1.0

1.0

100,101,88

226,219,192

100

101

112

1.2

0.8

95,117,91

130,106,100

94.3

220

1.0

1.1

96,105,82

212,235,213

DMSO

87.4

131.8

91,84,90,83,89

136,133,151,133,106

95.8

207.6

97,99,94,101,88

184,239,184,229,202

TA 1535

Test material

5000

4.0

8.7

0.8

1.5

4,4,M

10,6,10

3.3

8.7

0.6

1.0

4,2,4

9,11,6

2500

7.0

11.3

1.3

2.0

7,4,10

10,12,12

6.0

8.7

1.0

1.0

7,4,7

9,12,5

1000

4.0

9.7

0.8

1.7

6,1,5

4,23,2

5.7

7.0

1.0

0.8

7,6,4

4,11,6

500

4.7

7.0

0.9

1.2

1,10,3

9,5,7

5.3

5.0

0.9

0.6

2,7,7

6,5,4

200

7.0

10.7

1.3

1.8

10,7,4

15,11,6

6.0

8.3

1.0

1.0

10,7,1

10,9,6

100

5.7

13.7

1.1

2.4

5,6,6

9,5,27

4.3

9.3

0.7

1.1

5,4,4

7,10,11

DMSO

5.2

5.8

2,5,7,1,11

4,5,5,6,9

5.8

8.4

4,4,4,6,11

6,9,9,9,9

TA 1537

Test material

5000

8.3

27

0.8

3.5

6,12,7

21,32,28

4.0

31.3

0.4

4.2

2,6,4

27,29,38

2500

3.3

19.7

0.3

2.5

4,4,2

18,26,15

6.3

17.7

0.6

2.4

5,9,5

9,17,27

1000

8.0

11.7

0.7

1.5

6,6,12

18,13,4

8.7

9.7

0.9

1.3

9,11,6

13,4,12

500

6.0

15

0.5

1.9

7,6,5

11,15,19

6.3

8.0

0.6

1.1

6,6,7

7,6,11

200

8.7

9.3

0.8

1.2

13,6,7

5,6,17

8.7

6.7

0.9

0.9

10,1,15

11, 5, 4

100

10.3

11.3

0.9

1.5

9,12,10

15,10,9

8

7.0

0.8

0.9

10,9,5

10,5,6

DMSO

11

7.8

18,5,11,10,11

7,7,9,9,7

10

7.4

7,11,11,6,15

7,7,7,6,10

TA 98

Test material

5000

13.3

618.7

0.9

24.6

11,16,13

715,609,532

24.3

635.3

1.9

27.6

17,21,35

461,629,819

2500

17.7

252.7

1.2

10

19,18,16

267,252,239

14.7

206

1.1

9.0

17,11,16

223,134,261

1000

11

75

0.8

3.0

10,11,12

84,55,86

11.3

64.3

0.9

2.8

12,13,9

60,65,68

500

10.7

51.7

0.7

2.1

12,9,11

44,60,51

18.7

48.3

1.4

2.1

12,27,17

49,47,49

200

19.7

31.7

1.3

1.3

23,17,19

38,29,28

14.3

31.3

1.1

1.4

11,16,16

35,21,38

100

15

25.3

1.0

1.0

16,13,16

24,26,26

17.7

28.7

1.4

1.2

26,9,18

39,28,19

DMSO

14.6

25.2

16,17,10,19,11

23,17,23,28,35

13

23

10,13,13,12,17

28,27,17,15,28

WP2 (pKM101)

Test material

5000

53.7

55.7

0.8

0.6

39,55,67

49,56,62

39.7

56

1.0

0.9

47,46,26

56,57,55

2500

63.3

78.7

0.9

0.9

66,73,51

66,95,75

37.7

57.3

0.9

0.9

30,49,34

39,86,47

1000

64.3

75

0.9

0.8

72,47,74

65,82,78

46

71.3

1.1

1.1

43,50,45

73,79,62

500

80.7

106

1.2

1.2

83,97,62

105,116,97

37

65.3

0.9

1.0

39,29,43

65,57,74

200

104

92.3

1.5

1.0

101,116,95

89,105,83

39.7

64.3

1.0

1.0

41,35,43

73,62,58

100

118.3

94.3

1.7

1.0

83,154,118

99,83,101

35.7

57

0.9

0.9

39,45,23

58,47,66

DMSO

69.6

90

51,66,82,69,80

61,75,114,106,94

40.6

63.6

39,44,41,32,47

58,56,82,61,61

WP2 uvrA (pKM101)

Test material

5000

158.3

160.3

1.0

0.6

141,149,185

118,158,205

154.7

258

1.1

1.4

129,160,175

235,263,276

2500

186

294

1.2

1.1

169,188,201

274,296,312

139.7

199.3

1.0

1.1

130,145,144

202,213,183

1000

198.7

227

1.3

0.9

185,214,197

206,223,252

147.7

202.7

1.1

1.1

139,166,138

186,225,197

500

217

270

1.4

1.0

256,218,177

242,283,285

137.7

174.3

1.0

0.9

140,117,156

166,183,174

200

232

269

1.5

1.0

233,246,217

265,284,258

130

177

0.9

1.0

101,119,170

144,206,181

100

238.3

248.3

1.5

0.9

272,237,206

261,216,268

138.7

185.7

1.0

1.0

140,146,130

198,173,186

DMSO

156.8

263.4

173,149,147,135,180

259,219,304,267,268

137.8

184.2

128,125,151,161,124

168,157,190,209,197

M = Plate missing

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative without metabolic activation All strains tested
negative with metabolic activation All strains tested except S. typhimurium TA1537 and TA98
positive with metabolic activation S. typhimurium TA1537 and TA98 strains

Under the specific conditions of this assay, the test material gave a negative (i.e. non-mutagenic), response in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strain WP2P and WP2P uvrA in the absence of metabolic activation and in all strains, except for TA1537 and TA98, in the presence of metabolic activation. The test material gave a positive (i.e. mutagenic) response in S. typhimurium strains TA1537 and TA98 in the presence of metabolic activation. The study is considered to be reliable, relevant and adequate for risk assessment and classification and labelling purposes.
Executive summary:

The potential of the test material to cause gene mutation in bacterial strains was determined in accordance with standardised guidelines OECD 471, EU Method B.13/14 and EPA OPPTS 870.1500. Four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and two Escherichia coli strains (WP2P and WP2PuvrA) were treated in the presence and absence of at rat liver derived metabolic activation system (S9 mix). In two separate assays, the test material did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the tester strains used in the absence of metabolic activation, nor in strains TA1535, TA100, WP2P and WP2PuvrA in the presence of metabolic activation. In both assays, the test material induced reproducible, dose related increases in revertant colony numbers in strains TA1537 and TA98 in the presence of metabolic activation. The test material gave a negative (i.e. non-mutagenic), response in S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strain WP2P and WP2P uvrA in the absence of metabolic activation and in all strains, except for TA1537 and TA98, in the presence of metabolic activation. The test material gave a positive (i.e. mutagenic) response in S. typhimurium strains TA1537 and TA98 in the presence of metabolic activation.