4,4,7,7-tetraethoxy-3,8-dioxa-4,7-disiladecane

Genetic toxicity: in vitro

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EC 92/69/EEC L383 A: part B Determination of Toxicity-Mutagenicity

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: ICH S2A Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals

Deviations:

no

Test guideline 4

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

Test material information

Constituent 1

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

Controls

Controls 1

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

Controls 2

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

Controls 3

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

Controls 4

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

Controls 5

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

Controls 6

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 results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Test results 6

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.