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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:
2002-04-15 to 2002-09-12
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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EC 92/69/EEC L383 A: part B Determination of Toxicity-Mutagenicity
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH S2A Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH S2B: A Standard Battery for Genotoxicity Testing of Pharmaceuticals
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4,7,7-tetraethoxy-3,8-dioxa-4,7-disiladecane
EC Number:
240-212-2
EC Name:
4,4,7,7-tetraethoxy-3,8-dioxa-4,7-disiladecane
Cas Number:
16068-37-4
Molecular formula:
C14H34O6Si2
IUPAC Name:
4,4,7,7-tetraethoxy-3,8-dioxa-4,7-disiladecane

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:
100, 316, 1000, 3160, 5000 µg/plate: all strains, with and without metabolic activation, plate incorporation test and preincubation test
10, 31.5 µg/plate: preincubation test, strains TA1535, TA1537, without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Abs. ethanol

- Justification for choice of solvent/vehicle: Solubility properties and relative non-toxicity to bacteria
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Abs. ethanol
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 1254-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 aqua ad injectabilia ad 100 mL

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

SELECTION AGENT: histidine deficient agar

NUMBER OF REPLICATIONS: 3 plates for each test concentration

DETERMINATION OF CYTOTOXICITY
A reduction in the number of colonies by more than 50% compared with the solvent control and/or a scarce background lawn
Evaluation criteria:
A result is positive 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 experiments. In addition, a significant concentration related effect in observed.

Positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking on histidine-free agar plates.

Cytotoxicity is defined as a reduction in the number of colonies by >50% compared with the solvent control and/or a sparse background lawn.
Statistics:
MANN and WHITNEY and Spearman’s rank.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
1000-5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
3160 and 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
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:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
316-5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
316-5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA: Results were within range of historical control data

Remarks on result:
other: No mutagenic potential

Any other information on results incl. tables

Table 2: Dose range-finding study Number of revertants per plate (TA 100)

Conc.
(µg/plate)

Plate 1

Plate 2

Cytotoxic (Y/N) 

solvent control

124

164

 N

0.316

149

155

 N

1

145

173

N

3.16

171

189

 N

10

158

164

 N

31.6

161

166

 N

100

148

134

 N

316

155

159

 N

1000

185

154

 N

3160

179

159

 N

5000

185

165

 N

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

36.7

47.0

123.0

140.3

264.7

296.0

19.0

18.7

7.0

9.3

100

40.7

42.7

122.3

130.3

260.7

244.0

15.3

14.0

7.3

11.3

316

36.3

43.0

123.3

134.3

256.7

261.0

19.3

17.3

11.0

10.7

1000

33.0

37.3

124.0

142.3

249.7

271.7

16.7

13.0

5.3

8.7

3160

39.0

36.0

125.3

131.3

253.3

261.0

16.3

15.7

6.3

7.3

5000

40.7

44.0*

127.7

136.0*

265.0

244.0

17.3

18.3*

9.3

6.0*

Positive control

271.0

258.0

1177.0

1198.0

121.0

1209.3

420.0

1001.7

1123.3

1135.0

*cytotoxicity seen

Preincubation test: 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

29.0

27.3

130.7

147.7

287.3

287.7

11.0

15.0

4.0

4.0

10

-

-

-

-

-

-

11.0

-

2.3

-

31.5

-

-

-

-

-

-

10.0

-

4.0

-

100

27.0

28.0

139.0

154.7

386.0

316.0

14.0

13.0

6.7

5.7

316

24.7

25.7

142.0

165.0

300.7

287.3

0.0*

14.7

0.0*

7.0

1000

0.0*

24.3

158.3

141.0

291.0

284.3

0.0*

13.7

0.0*

4.0

3160

0.0*

23.0

0.0*

148.0

280.7

281.7

0.0*

14.3

0.0*

6.7

5000

0.0*

29.0

0.0*

154.3

289.7

289.0

0.0*

16.7

0.0*

6.0

Positive control

591.0

311.3

1233.7

1237.0

988.3

1305.7

525.0

936.7

422.0

594.3

*cytotoxicity seen

Applicant's summary and conclusion

Conclusions:
4,4,7,7-Tetraethoxy-3,8-dioxa-4,7-disiladecane has been tested for mutagenicity to bacteria, in a study which was conducted according to the OECD TG 471, EC 92/69/EEC L383 A, ICH S2A and ICH S2B, compliant 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 or the repeat experiments 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.