Dichlorocyclohexylmethylsilane

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 TA1535, 1537, 98, 100 and 102 (OECD TG 471) (LPT Laboratory of Pharmacology and Toxicology, 2002)

Cytogenicity in mammalian cells: read-across from analogous substance cyclohexyldimethoxymethylsilane: negative in cultured human lymphocytes (OECD TG 473, 1983) (Huntingdon Research Centre, 1989)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-05-13 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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

3.16, 10, 31.6, 100 and 316 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethylene glycol dimethylether

- Justification for choice of solvent/vehicle: Solubility properties and relative non-toxicity to bacteria

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)

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)

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)

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)

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)

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)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION

- Preincubation period: 60 minutes

- Expression time (cells in growth medium): 48 hours

NUMBER OF REPLICATIONS: 3 plates for each test concentration

DETERMINATION OF CYTOTOXICITY
- Method: Background lawn assessment, revertant colony counts

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.

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 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(316 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:

cytotoxicity

Remarks:

(316 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

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

	TA100
Conc. (µg/plate)	Plate 1	Plate 2	Cytotoxic (yes/no)
0*	137	124	No
0.316	154	166	No
1	133	147	No
3.16	157	162	No
10	154	147	No
31.6	139	172	No
100	135	147	No
316	143	151	Yes
1000	154	165	Yes
3160	0	0	Yes
5000	0	0	Yes

*solvent control withEthylene glycoldimethylether

Table 3: Experiment 1 Plate incorporationNumber of revertants per plate (mean of 3 plates)

	TA98			TA100			TA102
Conc. (µg/plate)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
0*	36.7	35	No	147.7	141.3	No	281.7	285	No
3.16	23.7	29	No	150	148.7	No	271.7	260	No
10	23.3	34	No	143.7	151.3	No	300	252.7	No
31.6	27.7	27.3	No	143.7	152	No	287.7	269.3	No
100	35	34.7	No	133.3	173	No	276.3	272.7	No
316	29	29.7	Yes	143	159	Yes	279	268.7	Yes
Positive control	871	867	No	1027.7	1035.7	No	1025.7	285	No

*solvent control withEthylene glycoldimethylether

Table 3: Experiment 1 Plate incorporationNumber of revertants per plate (mean of 3 plates)

	TA1535			TA1537
Conc. (µg/plate)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
0*	15.3	14	No	8.7	7	No
3.16	15.7	16.7	No	6.3	5.7	No
10	15	14.7	No	6.3	7.3	No
31.6	15.7	17.3	No	7	6	No
100	11.3	16	No	6	6.3	No
316	13.3	11.7	Yes	5	7	Yes
Positive control	420.7	422	No	424.7	424	No

*solvent control withEthylene glycoldimethylether

Table 4: Experiment 2 PreincubationNumber of revertants per plate (mean of 3 plates)

	TA98			TA100			TA102
Conc. (µg/plate)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+  MA	Cytotoxic (yes/no)	— MA	+  MA	Cytotoxic (yes/no)
0*	35.3	31.7	No	159.3	128	No	271.7	267.3	No
3.16	27	25.7	No	160.7	142	No	271	270.3	No
10	25.7	25	No	137.3	130	No	266.3	263.3	No
31.6	24.7	25.3	No	140	137	No	270	273	No
100	29.3	36.7	No	129.3	125.7	No	264	263.3	No
316	29.7	28.7	Yes	134.7	129	Yes	256.3	271.3	Yes
Positive control	526	535	No	1059	1051.7	No	1032.3	1066	No

*solvent control withEthylene glycoldimethylether

Table 4: Experiment 2 PreincubationNumber of revertants per plate (mean of 3 plates)

	TA1535			TA1537
Conc. (µg/plate)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
0*	12.3	15	No	4	6	No
3.16	13.7	13.3	No	4	3.3	No
10	12.7	14.3	No	3.7	5	No
31.6	14.3	13.7	No	4.7	4.3	No
100	13.3	12	No	2.7	4	No
316	11.7	13.3	Yes	4	4	Yes
Positive control	527.7	521.3	No	517	522	No

*solvent control withEthylene glycoldimethylether

Conclusions:

In a highly reliable test, conducted under OECD 471 with GLP, no mutagenic effect was observed for the test substance tested up to cytotoxic concentration in any of the test strains in two independent experiments without and with metabolic activation. The test substance is non-mutagenic in test strains used.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The restrictions were that the study lacks details regarding source of cells and definition of aberrations recorded, which was not in accordance with the current guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(1983)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: cultivated human

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

1, 2, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: The test compound was miscible with the solvent at a concentration of 125 mg/ml. However, all final concentrations greater than 500 µg/ml were immiscible with aqueous culture medium. 500 µg/ml is regarded and referred to as the maximum achievable concentration. Dilutions were made immediately before use.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation Migrated to IUCLID6: 20 µg/ml

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation Migrated to IUCLID6: 1000 µg/ml

Details on test system and experimental conditions:

Human blood cells were collected aseptically and prepared for cell culture by gradient centrifugation, suspended in RPMI medium and cultured in a multiwell tissue culture plate at 37 ⁰C for 48 hours. After 48 hours, 50 µl aliquots of the test material were added to one set of duplicate cultures in the range of final concentrations. Where appropriate, metabolic activation was pursued by the addition of S9 mix which was left in place for 2 hours before removal after which the cells were suspended in fresh medium and incubated for further 22 hours.

22 hours after treatment with test compound, mitotic activity was arrested by addition of colchine to each culture at final concentration of 0.25 µg/ml, and left for 2 hours. The cells were then centrifuged and made hypotonic, and thereafter fixed by addition of freshly prepared fixative. They were then used for preparation of slides and stained using Giemsa solution, and finally mounted in DPX.

The prepared slides were examined by light microscopy at a magnification of x160. The proportion of metaphase figures in each culture was recorded. From these results the dose level causing a decrease in mitotic index of approximately 40-80% of the solvent control value or, if there was no decrease, maximum achievable concentration was used as the highest dose level for the metaphase analysis. The intermediate and low doses were 50 and 12.5% of the highest concentration.

The slides were then coded. Metaphase figures were identified, using a magnification of x160 and examined at a magnification of x1000 using an oil immersion objective. Approximately 100 metaphase figures were examined, where possible, from each culture, with normally a maximum of 25 from each slide.

Evaluation criteria:

None given in report

Key result

Species / strain:

lymphocytes: cultivated human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

MI 22% of solvent control at 500 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Both positive control compounds caused large, statistically highly significant, increases in the proportion of metaphase figures containing chromosomal aberrations.

Conclusions:

Cyclohexyldimethoxymethylsilane has been tested in accordance with OECD guideline 473 and under conditions of GLP. No substance related statistically significant increase in chromosomal damage was observed in either the presence or absence of metabolic activation in human lymphocytes cultured in vitro at any concentration analysed . The test was conducted in accordance with OECD guideline 473 and compliant with GLP, but was lacking in detail regarding the source of cells and the definition of aberrations recorded. The experiment was not repeated. It is concluded that the test substance is negative for the induction of chromosome aberrations in vitro under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Micronucleus assay oral gavage study in mouse: read-across from analogous substance cyclohexyldimethoxymethylsilane, positive in male mice at 48 hours; negative at other sampling times and in female mice. Overall conclusion for genetic toxicity was negative (OECD TG 474) (Life Science Research 1987; Rel 2)
Rat dominant lethal Assay, oral administration: read-across from analogous substance cyclohexyldimethoxymethylsilane negative (similar to EU method B22) (Life Science 1989b; Rel 2)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9-Apr-1987 to 21-Aug-1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The restrictions were that the number of erythrocytes scored for the detection of micronucleus was not in accordance with the current guideline and the administered dose exceeded the recommended limit dose for this assay.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(old guideline)

Deviations:

yes

Remarks:

: only 1000 PCE's per animal examined instead of the 2000 required by current regulation.

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Charles River Italia S.p.A., Calco, Italy
- Weight at study initiation: 28-37g (males), 24-33g (females)
- Assigned to test groups randomly: no data
- Fasting period before study: overnight
- Acclimation period: 5 days

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil

Duration of treatment / exposure:

Single exposure.

Frequency of treatment:

Single administration.

Post exposure period:

Animals were sacrificed at 24, 48 and 72 hours after treatment.

Dose / conc.:

10 other: mL/kg (nominal)

Remarks:

vehicle

Dose / conc.:

2 500 mg/kg bw/day (nominal)

Remarks:

test material

Dose / conc.:

5 000 mg/kg bw/day (nominal)

Remarks:

test material

Dose / conc.:

5 mg/kg bw/day (nominal)

Remarks:

Mitomycin C

No. of animals per sex per dose:

5 male, 5 female

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C in sterile distilled water was prepared immediately prior to dosing.

Tissues and cell types examined:

After sacrifice, the femurs were removed and marrow cells obtained by flushing with foetal calf serum.

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 24, 48, 72 hours

DETAILS OF SLIDE PREPARATION: The removed marrow cells obtained by flushing with foetal calf serum were centrifuged and a concentrated suspension prepared to make smears on slides. These slides were air - dried overnight and then stained with May-Gruenwald and Giemsa, and mounted with Eukitt. Three slides were made from each animal.

METHOD OF ANALYSIS: The slides were examined under medium magnification and one slide from each animal was selected according to staining and quality of smears. Where the toxicity of the test substance was not so great as to inhibit cell proliferation, at least 1000 PCEs were examined at high magnification (x1000) for the presence or absence of micronuclei. At the same time the number of normochromatic erythrocytes was also recorded.

Statistics:

The incidence of micronucleated cells per 1000 erythrocytes (mature or polychromatic) are given as group means by sex, and for the sexes combined. The standard error of the means and rages are also shown. The animals presenting less than 200 PCEs per 1000 NCEs scored were excluded from the calculations and subsequent statistical analyses.

Key result

Sex:

male

Genotoxicity:

positive

Remarks:

at 48 hours increase in micronucleated PCEs at high dose level.

Toxicity:

yes

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Key result

Sex:

female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
In preliminary toxicity trials it was established that treatment with Cyclo-hexyl-dimethoxy-silane at 5000 mg/kg by oral gavage did not result in excessive lethality, and accordingly dose-levels of 5000 and 2500 mg/kg were selected for this study. The oral route of administration was used in this study on the instruction of the Sponsor.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):
- Ratio of PCE/NCE (for Micronucleus assay): A dose related increase in the ration of mature to polychromatic erythrocytes was observed at the 48 hour sampling time in the test substance treatment groups. Similar increases were observed at the 24 hour sampling time. This indicates that the treatment with the test substance exerted an inhibitory effect on cell division of bone marrow erythropoietic cells. The ratio of mature to polychromatic erythrocytes increased at all sampling times following treatment with the positive control compound., indicating that this treatment resulted in inhibition of the cell division of the bone marrow erythropoietic cells.

Table 1:Results of in vivo micronucleustest at 24 hours (both sexes combined)

		Positive control 5 mg/kg	0 mg/kg	2500 mg/kg	5000 mg/kg
Number of cells evaluated		1000	1000	1000	1000
Number of erythro-cytes	normo­chromatic	12675	11579	13891	12003
	poly­chromatic	9449	10070	9464	8948
	polychromatic with micronuclei	23.1 ± 2.9	1.4±0.5	1.6± 0.5	1.9±0.6
Ratio of erythro­cytes	polychromatic / normochromatic	1.39	1.15	1.52	1.45

 Table 2:Results of in vivo micronucleus test at 48 hours (females)

		Positive control 5 mg/kg	0 mg/kg	2500 mg/kg	5000 mg/kg
Number of cells evaluated		1000	1000	1000	1000
Number of erythro-cytes	normo­chromatic	5219	3467	7110	7206
	poly­chromatic	3080	5062	4426	3770
	polychromatic with micronuclei	11.3±3.5	1.4±0.7	2.3±0.8	1.8±1
Ratio of erythro­cytes	polychromatic / normochromatic	2.12	0.69	1.69	2.2

Table 3:Results of in vivo micronucleus test at 48 hours (males)

		Positive control 5 mg/kg	0 mg/kg	2500 mg/kg	5000 mg/kg
Number of cells evaluated		1000	1000	1000	1000
Number of erythro-cytes	normo­chromatic	3632	5609	4450	5387
	poly­chromatic	1610	5000	4013	3600
	polychromatic with micronuclei	16.1±1.3	1.6±0.6	1.5±0.6	5.6±2.2
Ratio of erythro­cytes	polychromatic / normochromatic	2.84	1.12	1.11	2.02

 

Table 4:Results of in vivo micronucleustest at 72 hours (both sexes combined)

		Positive control 5 mg/kg	0 mg/kg	2500 mg/kg	5000 mg/kg
Number of cells evaluated		1000	1000	1000	1000
Number of erythro-cytes	normo­chromatic	8856	9861	8041	8617
	poly­chromatic	5567	9016	9579	7000
	polychromatic with micronuclei	4±0.9	2±0.2	1.7±0.5	1.4±0.4
Ratio of erythro­cytes	polychromatic / normochromatic	1.91	1.09	1.23	1.91

 

Conclusions:

There was an increase in micronuclei 48 hours after dose administration, but only at a dose which exceeds the limit dose recommended for this assay, and only in male mice, not in female. No statistically significant increase was found when the results for male and female mice were combined. Considerable toxicity to bone marrow was observed and the number of polychromatic erythrocytes (PCEs) scored was only 3600 for the group, whereas it should have been 4000 per animal according to current guideline. The increase in micronuclei was observed in just one male animal (1000 cells scored for this animal). In view of the low number of cells scored, the negative result when results for male and female animals were combined, and the negative result from the in vivo rodent dominant lethal assay, indicating lack of cytogenicity in germ cells, the conclusion of negative for the in vivo micronucleus study is reasonable. The study was conducted in accordance to OECD 474 with deviations, and compliant with GLP..

Reference 2

Endpoint:

in vivo mammalian somatic and germ cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09.08.1988-30.11.1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.22 (Rodent Dominant Lethal Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

rodent dominant lethal assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (UK), Margte, Kent, England
- Weight at study initiation: 24-31g (males), 23-31g (females)
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Housing: Male mice were housed in groups of 4 (Preliminary test) or five (main test) during acclimatisation and dosing. Females were caged in groups of 4 prior to and after mating. High density polypropylene cages with stainless steel tops were used, in a limited access animal holding room. Cages housing animals of different treatment groups were distributed so that the effects of any spatially -variable components were equalised between groups as far as possible.
- Diet: LAD 1 laboratory animal diet was fed ad libitum through out the study
- Water: ad libitum
- Acclimation period: minimum five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): target 21⁰C (acceptable range 19-25⁰C)
- Humidity (%): 55 +/- 15%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil

Details on exposure:

Five groups, each comprising 25 male mice, were dosed orally by gavage using a volume-dosage of 10 ml/kg. The treatment schedule is shown in Table 1. Treatment of group 4 was changed during the dosing period, as indicated, due to observed toxicity of the initial dosage.

Duration of treatment / exposure:

6 weeks.

Frequency of treatment:

Dosing regime A: dosed once daily, Monday-Friday in weeks 1-5, then once daily, Monday-Sunday in week 6.
Dosing regime B: as A, but initial dosage of 1250 mg/kg/day reduced to 750 mg/kg/day from Monday of week 3 onwards.
Dosing regime C: dosed once daily on the Friday, Saturday and Sunday of week 6 only.
(See Table 1 )

Post exposure period:

The first mating period commenced on the monday of week 7, and three successive matings were established.

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

1 250 mg/kg bw/day (actual dose received)

Dose / conc.:

750 mg/kg bw/day (actual dose received)

Remarks:

The highest dose of 1250 mg/kg bw/day was reduced to 750 mg/kg bw/day from the beginning of the third dosing week due to toxicity.

No. of animals per sex per dose:

Three test groups of 25 males mice were dosed. A concurrent control group and a positive control group of the same size were included.

Control animals:

yes, concurrent vehicle

other: positive control group, ethylmethanesulphonate, 2000 mg/kg/day on the last three days only of the dosing period.

Positive control(s):

ethylmethanesulphonate

- Route of administration: oral
- Doses / concentrations: 200 mg/kg/day on the last three days of the dosing period.

Tissues and cell types examined:

sperm cells

Evaluation criteria:

The following observations were made and recorded for each animal: total implant number, early deaths (dead implants not showing embryonic development), late deaths (dead implants showing both placental and embryonic tissues). Corpora lutea counts were not made or recorded. Male animals were killed after completion of a total of 3 matings without necropsy.

Statistics:

The basic (treatment) unit of the assay is the treated male. For this reason, the critical parameterfor detection of any induced dominant lethal mutations, post-implantation losses, were evaluated on a 'per male' basis. Although factors related to individual female might affect the survival of the implanted embryos, the low incidence of multiple implant deaths in test and vehicle control groups, together with the cler absence of treatment related effects seen after comparison of test and control groups on the 'per male' basis, suggested that there would be little value in repeating the analysis on a 'per female' basis. However, pre-implantation losses were only assessed indirectly, since corpora lutea counts were not taken: accordingly, these losses were assessed by comparison of total implant numbers in test and control groups, and to ensure sensitivity of this indirect assessment the values were calculated and compared on both 'per male' and 'per female' bases. Comparisons were made between treated and vehicle control groups each week for three parameters: pregnancy rate, average number of implants (live and dead), proportions of total implants found dead.

Pregnancy rate was assessed by the chi-squared test, average number of implants per male was compared using computer based calculation of the non-parametric Mann-Whitney 'U' test.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

at 1250 mg/kg

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

None of the parameters investigated were affected by treatment with the test material. Statistical analysis confirmed that pregnancy rates, implant numbers and proportions of implants found dead in test groups were similar to vehicle control values. In EMS - treated animals, all three parameters were markedly affected at the first and/or second matings.

Table 2:Dominant lethal test: Summary of group data

 

Treatment group	Mating week	Pregnant females		Total implants		Dead implants		Mutagenic index
		No.	%^a	No.	Mean/female	No.	Mean/female
Vehicle controls (corn oil)	1 2 3	48 46 46	96 92 92	559 577 568	11.6 12.5 12.3	32 24 26	0.7 0.5 0.6	5.7 4.2 4.6
CHMMS, 50 mg/kg./day	1 2 3	42 47 48	91 94 96	529 573 566	12.6 12.2 11.8	29 30 41	0.7 0.6 0.9	5.5 5.2 7.2
CHMMS, 250 mg/kg./day	1 2 3	43 46 46	86 92 92	524 555 546	12.2 12.1 11.9	23 30 26	0.5 0.7 0.6	4.4 5.4 4.8
CHMMS, 1250-750 mg/kg./day	1 2 3	39 44 39	89 92 81	461 555 456	11.8 12.6 11.7	29 18 25	0.7 0.4 0.6	6.3 3.2 5.5
EMS, 200 mg/kg./day	1 2 3	43 26 43	86 52*** 86	398 246 507	9.3 9.5* 11.8	200 100 41	4.7 3.8 1	50.3*** 40.7*** 8.1*

 ^aPercentage of all females caged with surviving, treated males and later confirmed as pregnant.

^bMutagenic index = Total dead implants/Total implants x 100

*significantly different from group1 value p<0.05 (based on ‘per male’ analysis)

***significantly different from group1 value p<0.001

Conclusions:

In this study, subacute oral administration of the test material at dosages up to a level showing marked toxicity induced no detectable dominant lethal mutations in the sperm of treated male mice. The sensitivity of the test system was demonstrated by the marked responses observed following male treatment with the alkylating agent, ethylmethansulphonate. The study was carried out according to an appropriate protocol, similar to guideline and compliant with GLP.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In a reliable bacterial mutagenicity study conducted according to OECD TG 471 and under GLP, no evidence of mutagenicity was observed with or without metabolic activation when dichloro(cyclohexyl)methylsilane was tested in Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA1537 (LPT Laboratory of Pharmacology and Toxicology, 2002).

In a reliable in vitro chromosome aberration assay conducted according to OECD TG 473 (1983) and under GLP, no evidence of cytogenicity was observed when cyclohexyldimethoxymethylsilane (CAS 17865-32-6) was tested in peripheral human lymphocytes (Huntingdon Research Centre, 1989).

In a reliable in vivo micronucleus study in mice, the read-across substance, cyclohexyldimethoxymethylsilane (CAS 17865-32-6) was given by oral gavage at a dose level of 5000 mg/kg bw. There was an increase in micronuclei 48 hours after dose administration, but only at a dose which exceeds the limit dose recommended for this assay, and only in male mice, not in female. No statistically significant increase was found when the results for male and female mice were combined. Considerable toxicity to bone marrow was observed and the number of polychromatic erythrocytes (PCEs) scored was only 3600 for the group, whereas it should have been 4000 per animal according to current guideline. The increase in micronuclei was observed in just one male animal (1000 cells scored for this animal). In view of the low number of cells scored, the negative result when results for male and female animals were combined the conclusion of negative for the in vivo micronucleus study is reasonable and no further follow up of the positive result is needed. The study was conducted in accordance to OECD 474 with deviations, and compliant with GLP (Life Science Research 1987).

In addition to that, in a reliable in vivo study conducted according to a protocol similar to EU Method B.22 (Rodent Dominant Lethal Test), no evidence of the induction of dominant lethal mutation was observed when cyclohexyldimethoxymethylsilane (CAS 17865-32-6) was tested in a mouse model (Life Science 1989b). The guideline followed by this assay is comparable to OECD TG 478 which states that “The purpose of the Dominant lethal (DL) test is to investigate whether chemicals produce mutations resulting from chromosomal aberrations in germ cells”. Therefore, the negative result from the in vivo rodent dominant lethal assay indicates lack of cytogenicity in germ cells.

Although the source substance, cyclohexyldimethoxymethylsilane (CAS 17865-32-6), induced an increase in micronuclei somatic cells in male mice only at a dose higher than the recommended limit dose by the OECD TG 471 in the in vivo micronucleus assay, no evidence for germ cell mutagenicity was observed in the in vivo dominant lethal mutation assay in male and female mice.

In view of the availability of in vivo data from studies in somatic cells and in germ cells, and the overall weight of evidence of the information available, it is considered that additional testing for mutagenicity to mammalian cells in vitro is not required, as the overall conclusion for genetic toxicity in the studies was negative.

Justification for classification or non-classification

There is no evidence of genetic toxicity from the one available bacterial mutagenicity study on dichloro(cyclohexyl)methylsilane (CAS number 5578 -42 -7). The closely related read-across substance, cyclohexyldimethoxymethylsilane, CAS number 17865-32-6, did not induce chromosome aberrations when tested in peripheral human lymphocytes, but did induce micronuclei when tested in mice in vivo, but only in male animals at 48 hours. Although the test substance acted as a somatic cell mutagen in the in vivo micronucleus assay, no evidence for germ cell mutagenicity was observed in the in vivo dominant lethal mutation assay.

As the only evidence of genetic toxicity in the closely related substance was obtained from one of 6 groups of treated mice in a micronucleus assay, and there is no evidence of genetic toxicity to germ cells for the read-across substance from a rodent dominant lethal assay, it is concluded that classification according to Regulation (EC) No 1272/2008 is not required.