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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): negative with and without 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 (OECD TG 471) (LPT, 2002).

In vitro mammalian chromosome aberration test: Negative with or without metabolic activation when tested up to cytotoxic concentrations in Chinese Hamster Ovary cells (OECD 473) (TNO, 2003).

Mutagenicity in mammalian cells: positive in mouse lymphoma L5178Y cells (similar to OECD TG 476) with metabolic activation, negative without metabolic activation (Dow Corning Corporation, 1997).

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

histidine operon

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

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.

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

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Qualifier:

according to guideline

Guideline:

other: EEC protocol B.10 (Mutagenicity: in vitro Mammalian Cytogenetic Test) of Council Directive 67/548/EEC (1992)

Qualifier:

according to guideline

Guideline:

other: FDA, 2000

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

Tested up to 5000
Concentrations evaluated:
Test 1: 20, 39, 78 µg/ml (+MA); 10, 20 39, 78 µg/ml (-MA)
Test 2: 10, 30, 50, 75 µg/ml (-MA); 50, 75, 100 µg/ml (+MA)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: none given in report

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without activation

Details on test system and experimental conditions:

ACTIVATION: Aroclor induced rat liver S9
Final concentrations:
- MgCl2 8 mM
- KCl 33 mM
- G-6-P 5 mM
- NADP 4 mM
- sodium phosphate 100 mM (pH 7.4)
- S9 40% (v/v)

METHOD OF APPLICATION: in medium

DURATION
-Incubated at 37°C
Test 1:
-Pulse treatment: 4 hours; Harvesting: 18 hours after onset of treatment (+/-MA)
-Continuous treatment: 18 hours; Harvesting: 18 hours after onset of treatment (-MA)
Test 2:
-Pulse treatment: 4 hr; Harvesting: 18 or 32 hours after onset of treatment (+MA)
-Continuous treatment: 18 and 32 hours (-MA)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.1 mM medium)
STAIN (for cytogenetic assays): Giesma

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED:
-Mitotic index: at least 1000 nuclei (500/slide)
-Chromosome analysis: 200 well-spread metaphases per concentration (100/culture); at least 3 concentrations of the test substance together with the negative and positive controls were selected for analysis

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: microscopic examination
- Determination of endoreduplication: microscopic examination
- Other: analysis of interstitial deletions and multiple aberrations: microscopic examination

Evaluation criteria:

A response is considered to be positive if a concentration-related increase or a reproducible increase in the number of cells with structural chromosomal aberrations is observed.
A response is considered to be equivocal if the percentage of cells with structural chromosomal aberrations is statistically marginal higher than that of the negative control (0.05A test substance is considered to be clastogenic if a concentration-related increase in the percentage of cells with structural chromosomal aberrations over the concurrent control frequencies is observed, or if a single positive test point is observed in both tests.
A test substance is considered to be negative in the chromosomal aberration test if it produces neither a dose-related increase in the number of structural chromosomal aberrations nor a reproducible positive response at any of the test points.
Both statistical significance and biological relevance are considered together in the evaluation of the results.

Statistics:

Fischer's exact probability test (two-sided)

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

75 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: No mutagenic potential

Chromosomal aberration test (200 cells observed):

Dose level of test substance (µg/ml)	Number of cells showing structural aberrations (%)	Relative mitotic index (%)	Number of cells showing numerical aberrations (%)
Test 1: 4 h treatment, 18 h harvest, with metabolic activation
Negative control	0.0	100	0.0
20	1.5	73	0.0
39	0.5	66	0.0
78	0.5	61	0.0
Positive control (3.0)*	22.5	29	0.0
Test 1: 4 h treatment, 18 h harvest, without metabolic activation
Negative control	1.0	100	0.0
20	0.0	107	0.0
39	0.5	89	0.0
78	0.0	78	0.0
Positive control (0.1)*	35.0	98	0.0
Test 1: 18 h treatment, 18 h harvest, without metabolic activation
Negative control	0.0	100	0.0
20	0.5	93	0.0
39	0.5	81	0.0
78	1.0	56	0.0
Positive control (0.05)*	43.5	60	0.0
Test 2: 18 h treatment, 18 h harvest, without metabolic activation
Negative control	1.0	100	0.0
30	0.5	97	0.0
50	0.5	72	0.0
75	1.0	48	0.0
Positive control (0.05)*	29.5	56	0.0
Test 2: 32 h treatment, 32 h harvest, without metabolic activation
Negative control	1.0	100	0.0
30	0.0	119	0.0
50	0.0	87	0.0
75	0.5	69	0.0
Positive control (0.025)*	36.0	53	0.0
Test 2: 4 h treatment, 18 h harvest, with metabolic activation
Negative control	0.0	100	0.0
30	0.5	72	0.0
50	1.5	62	0.0
75	0.0	41	0.0
Positive control (3.0)*	16.5	40	0.0
Test 2: 4 h treatment, 32 h harvest, with metabolic activation
Negative control	1.5	100	0.0
30	1.5	76	0.0
50	0.5	90	0.0
75	0.0	78	0.0
Positive control (3.0)**	9.0	79	0.0

* P≤0.001

** P≤0.01

Conclusions:

4,4,7,7-Tetraethoxy-3,8-dioxa-4,7-disiladecane has been tested in a valid study conducted according to OECD 473 and in compliance with GLP. No statistically and biologically significant increase in the number of cells with chromosomal aberrations was observed with or without metabolic activation when tested up to cytotoxic concentrations in Chinese Hamster Ovary cells. It is concluded that the test substance is negative for cytogenicity to mammalian cells under the conditions of the test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

Preliminary toxicity assay:
Initial:
Independent repeat:
Expt 1: 20-150 (-S9), 40-500 µg/ml (+S9)

Vehicle / solvent:

ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

-MA

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 7,12-dimethyl-benz(a)anthracene

Remarks:

+MA

Details on test system and experimental conditions:

ACTIVATION:
S9 mix:
- 250 µl S9
- 6.0 mg NADP
- 11.25 mg DL-isocitric acid
- 750 µl F0P/ml mix (pH 7)

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period:
- Exposure duration:
- Expression time (cells in growth medium): plates incubated at 37±1°C in a humidified 5±1% CO2 atmosphere for 10-14 days
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF REPLICATIONS: 3 plates, initial and independent repeat assays

DETERMINATION OF CYTOTOXICITY
- Method:

Evaluation criteria:

The result was considered to induce a positive response if a concentration-related increase in mutant frequency was observed and one or more dose levels with 10% or greater total growth exhibited mutant frequencies of ≥100 mutants. per 1x10^6 clonable cells over the background level.
A result was considered equivocal if the mutant frequency in treated cultures was between 55 and 99 mutants per 1x10^6 clonable cells over the background level.
Test articles producing fewer than 55 mutants per 1x10^6 clonable cells over the background level were concluded to be negative.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

250 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

100 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Mutagenic potential

Initial assay (mean of 3 plates)

- MA							+ MA
Concentration (µg/ml)	Mutant colonies	SD (±)	Viable count	SD (±)	Mutant frequency	% total growth	Concentration (µg/ml)	Mutant colonies	SD (±)	Viable count	SD (±)	Mutant frequency	% total growth
Vehicle (1)	33	1	199	18	33	-	Vehicle (1)	42	3	188	2	45	-
Vehicle (2)	38	6	222	22	34	-	Vehicle (2)	41	3	161	13	51	-
30	30	6	184	2	33	101	40	60	7	179	18	68	106
40	35	1	223	10	31	124	60	51	11	174	8	58	109
50	27	8	203	12	27	107	80	59	6	193	28	61	115
60	37	8	201	14	37	108	100	42	3	161	18	52	95
70	-	-	211	6	-	111	125	57	12	181	6	63	109
80	28	2	157	15	36	80	150	35	7	114	9	61	70
90	35	2	226	9	31	102	200	58	8	167	6	69	85
100	30	2	163	6	37	18	250	53	10	88	4	120	12
Positive control (10)	207	9	148	19	280	58	Positive control (2.5)	160	18	186	12	172	111
Positive control (20)	162	3	54	6	598	14	Positive control (4.0)	10	10	157	19	269	81

Independent repeat assay (mean of 3 plates):

- MA							+ MA
Concentration (µg/ml)	Mutant colonies	SD (±)	Viable count	SD (±)	Mutant frequency	% total growth	Concentration (µg/ml)	Mutant colonies	SD (±)	Viable count	SD (±)	Mutant frequency	% total growth
Vehicle (1)	57	9	210	11	55	-	Vehicle (1)	64	3	146	11	87	-
Vehicle (2)	53	3	201	12	53	-	Vehicle (2)	68	5	194	13	70	-
75	63	3	204	12	62	8	150	72	14	167	4	86	7
80	59	7	189	11	63	9	175	93	7	187	25	99	21
85	58	6	187	17	62	8	200	80	7	172	13	93	14
90	83	9	205	9	81	27	210	140	10	149	10	189	110
95	125	8	159	10	158	104	220	399	11	106	2	750	672
97.5	64	12	177	13	72	18	230	516	12	119	19	870	791
100	74	7	167	6	88	34	240	473	14	98	4	965	886
105	237	9	153	15	309	255	250	++	-	72	7	-	-
Positive control (10)	228	9	145	14	314	260	Positive control (2.5)	181	20	159	11	228	149
Positive control (20)	218	8	61	4	714	560	Positive control (4.0)	263	13	156	13	336	257

++ = too toxic to count, total growth <10%

Conclusions:

4,4,7,7-Tetraethoxy-3,8-dioxa-4,7-disiladecane has been tested in a valid study conducted according to a protocol that is similar to OECD 476. In the initial assay, an increase in the mutant frequency was observed in the presence of metabolic activation in mouse lymphoma L5178Y cells. The maximum dose tested produced a reduction in total growth to 12%, therefore the increase was not likely to be caused by excessive toxicity. It is considered by the reviewer that only the highest dose would be considered positive by currently accepted evaluation criteria. No increase in mutant frequency was observed in the absence of metabolic activation in the initial assay. In the repeat assay, a dose-dependent increase in mutant frequency was observed in the presence of metabolic activation, with suspension growth of 13% at the maximum dose. There was an increase in mutant frequency which was not dose dependent in two concentrations in the absence of metabolic activation, and the study author's conclusion was that the results were ambiguous without metabolic activation. These increases are considered by the reviewer to be above the Global Evaluation Frequency, but probably not biologically significant because of the absence of a dose response. In the opinion of the reviewer, the result of the repeat assay would be considered to be positive in the presence of metabolic activation when assessed by currently accepted criteria.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo mammalian erythrocyte micronucleus test: Negative with no significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female ICR mice (OECD 474) (Dow Corning Corporation, 1998).

The potential for mutagenicity to mammalian cells which was observed in vitro, needs to be followed up in vivo and therefore a Comet Assay is proposed.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6-8 weeks
- Weight at study initiation: males 25.3-33.0 g, females 21.6-28.8 g
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study:
- Housing: mice of the same sex were house 1 per cage in polycarbonate solid bottom cages which were maintained on stainless steel racks equipped with automatic watering manifolds and were covered with filter material. Heat-treated hardwood chips were used for bedding
- Diet (e.g. ad libitum): ad libitum - certified laboratory rodent chow (certified Rodent 7012C)
- Water (e.g. ad libitum): ad libitum - tap water
- Acclimation period: no less than 7 days after receipt

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72±3
- Humidity (%): 50±20
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

sesame oil

Details on exposure:

Male and female mice were dose by a single oral gavage of 75, 150 or 300 mg/kg bw which was administered in a total volume of 10 ml test article-vehicle mixture/kg bw.

Duration of treatment / exposure:

24 or 48 hours

Remarks:

Doses / Concentrations:
75, 150, 300 mg/kg bw
Basis:

No. of animals per sex per dose:

10 - vehicle control, low test dose (75 mg/kg), mid test dose (150 mg/kg)
15 - high test dose (300 mg/kg)
5 - CP (60 mg/kg), MMC (2 mg/kg)

Control animals:

no

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): none given
- Route of administration: oral (gavage)
- Doses / concentrations: 10 ml/kg bw

mitomycin
- Justification for choice of positive control(s): none given
- Route of administration: intraperitoneal injection
- Doses / concentrations: 10 ml/kg

Tissues and cell types examined:

At 24 and 48 hours post-exposure, bone marrow was collected from the femur of 5 animals in all groups for micronucleus assay.

Details of tissue and slide preparation:

The bone marrow cells were pelleted by centrifugation at approximately 100 x g for 5 minutes and the supernatant was drawn off.
The cells were resuspended by aspiration with a capillary pipette and a small drop of bone marrow suspension was spread onto a clean glass slide.
2-4 slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and mounted.

Evaluation criteria:

The test article was considered to induce a positive response if a dose-response increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control.
If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose-response, the assay was considered equivocal and a repeat assay recommended.
The test article was considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.

Statistics:

Kastenbaum-Bowman Tables

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

on bone marrow cells

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: No mutagenic potential

Additional information on results:

Lethargy observed at 300 mg/kg

Treatment (mg/kg)	Sex	Time	Number of mice	PCE/total erythrocytes (mean)	Change from control (%)	Micronucleated polychromatic erythrocytes
						Number per 1000 PCEs (mean)	Number per PCEs scored (/10 000)
Sesame oil	M	24	5	0.46	-	0.6	6
	F	24	5	0.46	-	0.5	5
75	M	24	5	0.37	-20	0.3	3
	F	24	5	0.41	-11	0.5	5
150	M	24	5	0.42	-9	0.3	3
	F	24	5	0.40	-13	0.4	4
300	M	24	5	0.35	-24	0.6	6
	F	24	5	0.31	-33	0.4	4
CP (60)	M	24	5	0.38	-17	31.2	312*
	F	24	5	0.27	-41	24.99	249*
MMC (2)	M	24	5	0.51	11	18.3	183*
	F	24	5	0.49	7	18.4	184*
Sesame oil	M	48	5	0.48	-	0.5	5
	F	48	5	0.44	-	0.5	5
75	M	48	5	0.59	23	0.5	5
	F	48	5	0.46	5	0.4	4
150	M	48	5	0.46	-4	0..2	2
	F	48	5	0.56	27	0.6	6
300	M	48	5	0.49	2	0.6	6
	F	48	5	0.55	25	0.4	4

*p ≤0.05

Conclusions:

Interpretation of results (migrated information): negative
4,4,7,7-Tetraethoxy-3,8-dioxa-4,7-disiladecane did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female ICR mice. It is concluded that the test substance is negative in the in vivo micronucleus test under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Additional information

Information is available from reliable studies for all the required in vitro endpoints. Where there was more than one result for an endpoint the most reliable study available was chosen as key study. Where there was more than one reliable study, the most recent study was selected. The results of all of the studies were not in agreement. There was no evidence for mutagenicity in bacteria (LPT, 2002), or clastogenicity (causing chromosomal aberrations) in mammalian cells (TNO, 2003) in the presence and absence of metabolic activation in vitro. However, a positive result was evident in mouse lymphoma L5178Y cells with metabolic activation, but not without metabolic activation (Dow Corning Corporation, 1997). An in vivo mammalian erythrocyte test was also available, with a negative result (Dow Corning Corporation, 1998). The potential for mutagenicity to mammalian cells which was observed in vitro, therefore needs to be followed up in vivo and a Comet Assay is proposed.

 

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, compliant with GLP (LPT, 2002). 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.

A supporting study for bacterial mutagenicity is also available, conducted according to OECD guideline, and in compliance with GLP (Bioreliance, 1999). 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 1535, TA 1537 or E. coli WP2 uvrA in the initial or the repeat experiments up to cytotoxic/limit 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, and the findings support those of the key study.

4,4,7,7 -Tetraethoxy-3,8-dioxa-4,7-disiladecane has been tested in a valid study conducted according to OECD 473 and in compliance with GLP (TNO, 2003). No statistically and biologically significant increase in the number of cells with chromosomal aberrations was observed with or without metabolic activation when tested up to cytotoxic concentrations in Chinese Hamster Ovary cells. It is concluded that the test substance is negative for cytogenicity to mammalian cells under the conditions of the test.

4,4,7,7 -Tetraethoxy-3,8-dioxa-4,7-disiladecane has been tested in a valid study conducted according to a protocol that is similar to OECD 476 (Dow Corning Corporation, 1997). In the initial assay, an increase in the mutant frequency was observed in the presence of metabolic activation in mouse lymphoma L5178Y cells. The maximum dose tested produced a reduction in total growth to 12%, therefore the increase was not likely to be caused by excessive toxicity. No increase in mutant frequency was observed in the absence of metabolic activation in the initial assay. In the repeat assay, a dose-dependent increase in mutant frequency was observed in the presence of metabolic activation, with suspension growth of 13% at the maximum dose. There was an increase in mutant frequency which was not dose dependent in two concentrations in the absence of metabolic activation, and the study author's conclusion was that the results were ambiguous without metabolic activation. These increases are considered by the reviewer to be above the Global Evaluation Frequency, but probably not biologically significant because of the absence of a dose response. In the opinion of the reviewer, the result of the repeat assay would be considered to be positive in the presence of metabolic activation when assessed by currently accepted criteria. 

4,4,7,7 -Tetraethoxy-3,8-dioxa-4,7-disiladecane has been tested in a valid study conducted according to a protocol that is similar to OECD 474 (Dow Corning Corporation, 1997). The test material did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female ICR mice. It is concluded that the test substance is negative in the in vivo micronucleus test under the conditions of the test.

The potential for mutagenicity to mammalian cells which was observed in vitro, needs to be followed up in vivo and therefore a comet assay is proposed.

Justification for classification or non-classification

Based on the available data for 4,4,7,7 -tetraethoxy-3,8-dioxa-4,7-disiladecane, no classification is proposed for genetic toxicity according to Regulation (EC) No 1272/2008.