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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:
2002-03-04 to 2002-09-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
other: EC directive 92/69/EEC (Methods for the Determination of Toxicity-Mutagenicity.
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Guideline S2A (Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Guideline S2B (A Standard Battery for Genotoxicity Testing of Pharmaceuticals)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Target gene:
histidine operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
10, 31.6, 100, 316, 1000 µg/plate: all strains, with and without metabolic activation, plate incorporation test and preincubation test
1, 3.16 µg/plate: preincubation test, strain TA1537, with metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Abs. ethylene glycol dimethylether

- Justification for choice of solvent/vehicle: Solubility properties and relative nontoxicity to bacteria
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 1535, TA 100 without activation, 10 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA 98 without activation, 10 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA 1537 without activation, 100 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
TA 102 without activation, 1300 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-anthracene amide
Remarks:
TA 98, TA 102, TA 1537 with activation, 2 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
TA 100, TA 1535 with activation, 1500 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

ACTIVATION: Aroclor induced rat liver S9
S9 mix components (per 100 mL):
- 5.0 mL rat liver s9
- 2.0 mL 0.4 M MgCl2 + 1.65 M KCl-salt solution (sterile stock solution)
- 141.0 mg glucose-6-phosphate
- 306.5 mg NADP
- 50.0 mL 0.2 M phosphate buffer, pH 7.4 (sterile stock solution)
- sterile water for injection to 100 mL

DURATION:
-Plate incorporation: 48 hours at 37°C
-Preincubation 60 minutes at 37°C
- Preincubation period: 60minutes
- Exposure duration: 24hours
- Expression time (cells in growth medium): 24hours

NUMBER OF REPLICATIONS: 3 plates per concentration and experiment

SELECTION AGENT: histidine-deficient agar

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:
A test chemical is considered to be show a positive response if the number of revertants is significantly increased compared with the solvent control to at least 2-fold of the solvent control for TA 98, TA 100 and TA 102 and 3-fold of the solvent control for TA 1535 and TA 1537 in both independent experiments.
Positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples in histidine free agar plates
Statistics:
Mann Whitney U test and Spearman's rank correlation.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
reduction in background lawn was observed at 1000 µg/plate with strain TA 100 in the plate incorporation assay, and from 100 µg/plate in the pre-incubation experiment.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA: Controls were within range of historical data.

Any other information on results incl. tables

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

 

TA100

Conc.
(µg/plate)

Plate 1

Plate 2

Cytotoxic
(yes/no)

solvent control

155

136

no

0.316

151

157

no

1

145

128

no

3.16

144

159

no

10

155

146

no

31.6

127

157

no

100

149

151

no

316

155

182

no

1000

154

144

yes

3160

0

0

yes

5000

0

0

yes

Preincubation: number of revertants per plate (mean of 3 plates)

Conc(µg/plate)

TA 98

TA 100

TA 102

TA 1535

TA 1537

- MA

+ MA

- MA

+ MA

- MA

+ MA

- MA

+ MA

- MA

+ MA

Solvent control

30.7

42.3

137.0

136.0

285.3

281.0

15.0

16.0

5.3

3.3

1

-

-

-

-

-

-

-

-

-

3.0

3.16

-

-

-

-

-

-

-

-

-

3.0

10

38.7

42.7

119.7

144.3

282.3

263.3

14.7

15.0

5.3

5.0

31.6

32.7

43.0

127.7

121.7

285.7

270.3

13.0

17.7

4.0

2.3

100

35.0*

45.3*

142.7*

135.0*

274.0*

301.7

11.0*

13.3

3.7*

4.3*

316

37.3*

41.7*

138.3*

124.7*

260.3*

261.0*

14.7*

15.0*

4.7*

5.0*

1000

0.0*

0.0*

129.3*

136.0*

255.3*

277.0*

16.3*

16.7*

4.7*

3.7*

Positive control

1270.3

1266.0

1375.7

1379.7

1277.0

1373.3

1384.0

1376.7

844.3

420.3

 *cytotoxic

Plate incorporation:number of revertants per plate (mean of 3 plates)

Conc(µg/plate)

TA 98

TA 100

TA 102

TA 1535

TA 1537

- MA

+ MA

- MA

+ MA

- MA

+ MA

- MA

+ MA

- MA

+ MA

Solvent control

38.3

45.3

116.7

134.0

289.3

272.3

22.3

17.3

5.3

5.7

10

35.7

43.0

129.7

131.0

303.0

276.0

14.0

21.3

4.3

6.0

31.6

37.7

47.7

129.0

139.3

276.3

269.0

14.3

18.7

3.0

3.3

100

38.7

38.7

132.0

118.3

279.3

290.3

16.7

20.3

4.3

3.7

316

41.3

41.0

121.0

140.3

261.0

271.7

19.3

20.3

2.7

5.0

1000

37.0

45.3

117.3*

126.3*

272.0

284.3

17.3

20.7

4.7

6.3

Positive control

1173.7

1163.3

1186.7

1196.0

1329.7

1300.7

657.7

667.7

623.7

718.3

*cytotoxic

Applicant's summary and conclusion

Conclusions:
Triethylsilane has been tested for mutagenicity to bacteria, in a study which was conducted according to the OECD TG 471 and in compliance with GLP. No evidence of a test-substance related increase in the number of revertants was observed with or without metabolic activation in Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 or TA 1537 in the initial plate incorporation assay or the repeat experiment using the pre-incubation method up to cytotoxic concentrations. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.