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Toxicological information

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:
22 Feb - 04 Apr 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
2-aminoanthracene was used as the only positive control in the presence of S9-mix, at least a second positive control substance must be included for the efficacy testing of S9-mix.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his operon (for S. typhimurium strains) and trp operon (for E. coli strain)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
Preliminary toxicity test:
- 6.7, 10, 33, 67, 100, 333, 667, 1000, 333 and 5000 µg/plate (with and without metabolic activation, TA 100 and E. coli WP2 uvrA)
Experiment I+II:
- 100, 333, 1000, 3333 and 5000 µg/plate (with and without metabolic activation)
Experiment III:
- 100, 333, 1000, 3333 and 5000 µg/plate (TA 100 without metabolic activation)
5000 µg/plate is the highest recommended dose level based on the current OECD guideline
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Solubility properties and relative non-toxicity to bacteria
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: +S9-mix: 2-aminoanthracene (2-AA); -S9-mix: 2-nitrofluorene (2-NF), sodium azide (NaN), 9-aminoacridine (9-AA), methyl methanesulfonate (MMS)
Remarks:
2-AA: 1 µg/plate (all TA strains), 10 µg/plate (E. coli WP2 uvrA), 2-NF: 1 µg/plate (TA 98), NaN: 1 µg/plate (TA 100, TA 1535), 9-AA: 75 µg/plate (TA 1537), MMS: 1000 µg/plate (E. coli Wp2 uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar ; preincubation

DURATION
- Preincubation period: 60 min
- Expression time (cells in growth medium): 48 h

NUMBER OF REPLICATIONS: 3 plates for each test concentration in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: A dose level is considered toxic if one or both of the following criteria are met: a >50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control - this reduction must be accompanied by an abrupt dose-dependent drop in the revertant count and/or a reduction in the background lawn.
Evaluation criteria:
The mean of each positive control must exhibit at least a three-fold increase in the number of revertants over the mean value of the respective vehicle control. A minimum of three non-toxic dose levels are required to evaluate assay data. For the test material to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain with a minimum of two increasing concentrations of test material. Data sets for strains TA 1535 and TA 1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than three times the mean vehicle control value. Data sets for strains TA 98, TA 100 and E. coli WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose is equal to or greater than two times the mean vehicle control value.
Statistics:
Mean values and standard deviation were calculated.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA: All results of the vehicle and positive controls as well as the test groups were within range of historical control data.

Any other information on results incl. tables

Table 2: Dose range-finding study Number of revertants per plate (2 plates per strain)

TA 100

WP2 uvrA

Concentration (µg/Plate)

Plate 1

+ MA

Plate 2

- MA

Cytotoxic (Yes/No)

Plate 1

+ MA

Plate 2

- MA

Cytotoxic (Yes/No)

0

142

112

No

13

16

No

6.7

138

119

No

16

20

No

10

126

101

No

9

18

No

33

146

128

No

15

16

No

67

129

105

No

12

16

No

100

140

109

No

8

12

No

333

127

112

No

12

16

No

667

111

119

No

5

13

No

1000

125

120

No

12

12

No

3333

133

129

No

10

12

No

5000

144

121

No

13

14

No

*solvent control with Acetone

MA: metabolic activation (S9 -mix)

Table 3: Experiment 1 Mutagenicity Assay Number of revertants per plate (mean of 3 plates)

 

TA98

TA100

TA1535

Conc.µg/plate

— MA

+

MA

Cytotoxic
(yes/no)

MA

+

MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

0*

14

24

No

125

156

No

12

9

No

100

18

23

No

125

143

No

9

15

No

333

26

30

No

133

159

No

12

11

No

1000

21

37

No

139

169

No

11

12

No

3333

23

31

No

133

162

No

9

11

No

5000

24

31

No

129

169

No

8

14

No

Positive control

523

2672

No

910

2698

No

673

285

No

*solvent control with Acetone

MA: metabolic activation (S9 -mix)

Table 4: Experiment 1 Mutagenicity Assay Number of revertants per plate (mean of 3 plates)

 

TA1537

WP2uvrA

Conc.
µg/plate

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

0*

7

9

No

14

17

No

100

8

7

No

19

15

No

333

7

10

No

18

20

No

1000

6

10

No

20

18

No

3333

6

10

No

17

19

No

5000

6

10

No

20

15

No

Positive control

736

424

No

610

204

No

*solvent control withAcetone

MA: metabolic activation (S9 -mix)

Table 5: Experiment 2 Mutagenicity Assay Number of revertants per plate (mean of 3 plates)

 

TA98

TA100

TA1535

Conc.
µg/plate

— MA

+ MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

0*

13

15

No

116

98

No

8

10

No

100

13

15

No

114

84

No

7

9

No

333

12

16

No

116

97

No

9

8

No

1000

16

16

No

232

82

No

7

9

No

3333

17

19

No

125

95

No

5

9

No

5000

14

18

No

137

92

No

5

11

No

Positive control

248

876

No

638

891

No

227

134

No

*solvent control withAcetone

MA: metabolic activation (S9 -mix)

Table 6: Experiment 2 Mutagenicity Assay Number of revertants per plate (mean of 3 plates)

 

TA1537

WP2 uvrA

Conc.
µg/plate

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

0*

6

4

No

12

11

No

100

2

3

No

8

9

No

333

2

4

No

10

10

No

1000

4

4

No

9

13

No

3333

5

4

No

10

11

No

5000

5

5

No

11

10

No

Positive control

226

147

No

369

45

No

*solvent control withAcetone

MA: metabolic activation (S9 -mix)

Applicant's summary and conclusion

Conclusions:
Under test conditions with GLP, no mutagenic effect and no cytotoxicity was observed for the test substance tested up to limit concentration in any of the test strains without and with metabolic activation in the Ames test. The test substance is non mutagenic in the strains used.