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Toxicological information

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:
12 Oct - 25 Nov 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his operon (S. thyphimurium)
Species / strain
Species / strain / cell type:
other: TA 1535, TA97a, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix) (Trinova Biochem, Gießen, Germany), prepared from the livers of male Sprague Dawley rats intraperitoneally treated with 500 mg/kg bw Aroclor 1254
Test concentrations with justification for top dose:
First experiment: 50, 150, 500, 1500 and 5001 µg/plate with and without metabolic activation
Second experiment: 313, 626, 1251, 2501 and 5001 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle/solvent used: DMSO (for the test substance and the positive control substances 4-nitro-1,2-phenylene diamine, 2-amino-anthracene and benzo-a-pyrene) or water (for the positive control substance sodium azide); 0.1 mL for the plate-incorporation and the preincubation treatment
- Justification for choice of solvent/vehicle: DMSO was chosen as vehicle, since the test substance was completely soluble, and this solvent did not have any effect on the viability of bacteria or the number of spontaneous revertants.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylene diamine (20 µg/plate, -S9, TA 97a, TA98 and TA 102); sodium azide (1 µg/plate, -S9, TA100 and TA 1535); 2-amino-anthracene (1 µg/plate, +S9, TA 97a, TA100, TA 102 and TA 1535); benzo-a-pyrene (20 µg/plate, +S9, TA98)
Details on test system and experimental conditions:
METHOD OF APPLICATION 1: in agar (plate incorporation) for first experiment

DURATION
- Exposure duration: 48 h

METHOD OF APPLICATION 2: preincubation method for the second experiment

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: quadruplicate each in both experiments

DETERMINATION OF CYTOTOXICITY
- Method: determination of titre (cytotoxicity test) and observation of background lawn (plate incorporation and preincubation experiment)
Evaluation criteria:
The test substance was considered to have mutagenic potential, if a significant, reproducible increase in revertant colonies per plate (increase factor ≥ 2) in at least one of the test strains was observed. A concentration-dependent increase over a range of test concentrations was considered to be a sign of mutagenic activity.
Statistics:
Mean values and standard deviations of revertant colonies from each test group were calculated.

Results and discussion

Test results
Species / strain:
other: TA 1535, TA97a, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA:
The mean numbers of spontaneous revertants in the solvent/vehicle controls of the strains used were all within the normal historical ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In a preliminary study using the plate incorporation method, each strain was incubated with 1500 and 5001 µg/plate of the test substance. Two replicates for each concentration were performed both in the presence or absence of metabolic activation. For the determination of toxicity, the titre (colonies/plate) was determined in control and treated cultures. In control culture, the titre should give a number of at least 1E+9 cells/mL, correlating to 100 colonies/plate after dilution. A substance is considered non-toxic, if the quotient control titre/treatment titre is below 2. This criterion was fulfilled after treatment with 1500 and 5001 µg/plate of the test substance. In the main study, no signs of toxicity were observed in any strain and at any concentration tested in both experiments.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

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 4 plates ± standard deviation)

 

Base-pair substitution type

Frameshift type

 

TA 100

TA102

TA1535

TA 97a

TA 98

water

105 ± 5

151 ± 24

22 ± 4

97 ± 3

11 ± 3

DMSO

88 ± 5

171 ± 9

16 ± 1

108 ± 3

10 ± 2

50

79 ± 10

146 ± 9

15 ± 2

108 ± 3

13 ± 1

150

81 ± 9

147 ± 17

13 ± 3

110 ± 8

11 ± 1

500

96 ± 2

142 ± 9

14 ± 1

132 ± 9

17 ± 3

 

1500

90 ± 8

148 ± 9

14 ± 3

104 ± 14

10 ± 2

5001

90 ± 10

145 ± 6

14 ± 2

106 ± 9

14 ± 4

Positive controls, -S9

Name

NaN3

4NOPD

NaN3

4NOPD

4NOPD

Concentrations (μg/plate)

1

20

1

20

20

Mean No. of colonies/plate (average of 4 ± SD)

495 ± 60

604 ± 99

201 ± 18

456 ± 18

240 ± 19

+

water

128 ± 19

176 ± 17

15 ± 2

108 ± 8

13 ± 6

+

DMSO

112 ± 9

171 ± 4

14 ± 2

115 ± 2

14 ± 3

+

50

86 ± 11

154 ± 5

14 ± 3

102 ± 8

12 ± 2

+

150

80 ± 16

150 ± 8

14 ± 3

101 ± 3

13 ± 2

+

500

91 ± 12

146 ± 8

20 ± 2

105 ± 6

10 ± 3

+

1500

91 ± 15

154 ± 34

16 ± 3

108 ± 5

10 ± 2

+

5001

92 ± 5

149 ± 9

16 ± 4

92 ± 4

14 ± 1

Positive controls, +S9

Name

AAN

AAN

AAN

AAN

BP

Concentrations (μg/plate)

1

1

1

1

20

Mean No. of colonies/plate (average of 4 ± SD)

446 ± 63

594 ± 88

201 ± 11

499 ± 22

202 ± 13

Table 2. Test results of experiment 2 (preincubation)

With or without S9-Mix

Test substance concentration

Mean number of revertant colonies per plate

(μg/plate)

(Average of 4 plates ± standard deviation)

 

Base-pair substitution type

Frameshift type

 

TA 100

TA102

TA1535

TA 97a

TA 98

water

114 ± 23

162 ± 7

23 ± 6

115 ± 5

17 ± 4

DMSO

124 ± 4

153 ± 12

21 ± 3

113 ± 4

10 ± 3

313

115 ± 21

154 ± 4

22 ± 2

115 ± 1

11 ± 1

626

126 ± 16

148 ± 3

18 ± 3

113 ± 10

10 ± 3

1251

123 ± 11

141 ± 15

20 ± 3

109 ± 6

11 ± 3

2501

117 ± 8

160 ± 13

19 ± 4

114 ± 1

13 ± 2

5001

130 ± 21

150 ± 7

20 ± 1

118 ± 6

10 ± 3

Positive controls, –S9

Name

NaN3

4NOPD

NaN3

4NOPD

4NOPD

Concentrations (μg/plate)

1

20

1

20

20

Mean No. of colonies/plate (average of 4 ± SD)

487 ± 35

657 ± 40

122 ± 8

488 ± 56

192 ± 30

+

water

143 ± 11

141 ± 17

17 ± 4

117 ± 5

17 ± 3

+

DMSO

111 ± 15

165 ± 6

22 ± 2

115 ± 12

15 ± 3

+

313

127 ± 23

148 ± 9

19 ± 4

116 ± 4

14 ± 1

+

626

147 ± 8

144 ± 4

14 ± 3

106 ± 4

10 ± 2

+

1251

126 ± 9

143 ± 2

17 ± 2

116 ± 5

11 ± 1

+

2501

134 ± 25

154 ± 13

17 ± 3

118 ± 6

11 ± 3

+

5001

130 ± 7

152 ± 2

15 ± 2

116 ± 6

12 ± 2

Positive controls, +S9

Name

AAN

AAN

AAN

AAN

BP

Concentrations (μg/plate)

1

1

1

1

20

Mean No. of colonies/plate (average of 4 ± SD)

618 ± 164

516 ± 60

131 ± 15

460 ± 24

236 ± 26

NaN3 = sodium azide

4NOPD = 4-nitro-1,2-phenylene diamine

AAN = 2-aminoanthracene

BP = benzo-a-pyrene

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative