Cyclohexyldimethoxymethylsilane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No mutagenicity was seen in the Ames test in the key study conducted according to Japanese guidelines and in compliance with GLP (Sogo Biomedical Laboratories., 1987) when cyclohexyl(dimethoxy)methylsilane was tested at up to 5000 μg/plate in a total of seven bacterial strains in the presence or absence of metabolic activation.

No increase in the frequency of chromosome aberrations was seen in the key study conducted according to OECD Test Guideline 473 and in compliance with GLP (SafePharm Laboratories Ltd., 1996) at concentrations of cyclohexyl(dimethoxy)methylsilane that caused a reduction in mitotic index, either with or without metabolic activation.

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:

3 June to 16 June 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(only 2 replicate plates/concentration)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine locus (S. typhimurium)
tryptophan locus (E. coli)

Species / strain / cell type:

bacteria, other: Salmonella typhimurium TA1535, TA1537, TA98, TA100, Escherichia coli WP2 uvrA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 microsomal fraction prepared from rat liver induced by phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

S. typhimurium TA100, TA98 and TA1537 were treated at 0, 10, 20, 39, 78 or 156 µg/plate without S9; TA1535 and E. coli WP2 uvrA were exposed at 20-313 µg/plate. With S9, all S. typhimurium strains were tested at 20, 39, 78, 156 or 313 µg/plate, apart from TA1537 which was tested at 10-156 µg/plate. E. coli WP2 uvrA was tested at 313, 625, 1250, 2500 or 5000 µg/plate with S9.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test material reacts with water, soluble in DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Migrated to IUCLID6: without S9 - 3 µg/plate TA100; 5 µg/plate TA1535; 2 µg/plate WP2 uvrA

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Migrated to IUCLID6: without S9 - 1 µg/plate TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Migrated to IUCLID6: without S9 - 80 µg/plate TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: with S9 - 5 µg/plate TA100, TA98, TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene; with S9 - 2 µg/plate TA1535; 20 µg/plate WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: plates incubated for 48 h

SELECTION AGENT (mutation assays): depleted histidine or tryptophan levels in medium for selection of heterotrophs

NUMBER OF REPLICATIONS: 2 plates/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER:
- type and composition of metabolic activation system:
- species and cell type: rat, S9 fraction
- quantity: 0.5 ml of S9 mix/culture
- induced or not induced: induced
- chemicals used for induction: phenobarbital and 5,6-benzoflavone
- co-factors used: MgCl2 8 µmol, KCl 33 µmol, G6P 5 µmol, NADPH 4 µmol, NADP 4 µmol, Na-phosphate buffer (pH 7.4) 100 µmol,

Evaluation criteria:

no data

Statistics:

not applicable, results negative

Species / strain:

other: Salmonella typhimurium TA1535, TA1537, TA98, TA100, Escherichia coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

156 µg/plate in TA98, TA100, TA1537 -S9, TA1537 +S9; 313 µg/plate in TA1535, WP2 uvrA -S9, TA98, TA100, TA1535 +S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no data
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: yes with concentrations of 10-5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA: no data

ADDITIONAL INFORMATION ON CYTOTOXICITY: Inhibition of the background lawn was seen without S9 at 156 µg/plate with TA100, TA98 and TA1537 and at 313 µg/plate with TA1535 and WP2 uvrA. Inhibition was seen with S9 at 156 µg/plate with TA1537 and at 313 µg/plate with TA100, TA1535 and TA98. No inhibition was observed in WP2 uvrA with S9 at levels of up to 5000 µg/plate.

Revertant numbers were reduced without S9 at 78 µg/plate for TA1535 and TA98 and at 156 µg/plate for TA100, TA1537 and WP2 uvrA. With S9, revertant frequency was reduced at 156 µg/plate with TA100, TA98, TA1535 and TA1537.

Remarks on result:

other: all strains/cell types tested

Table 1:Number of revertants per plate (1 plate) – Preliminary test

Bacterial strain	TA100			TA1535			WP2 uvrA
Concentration of test material, µg/plate	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	105	96	no	5	6	no	20	13	no
10	77	88	no	12	13	no	7	11	no
50	111	97	no	6	10	no	10	14	no
100	75	77	yes –MA no +MA	6	10	no	8	17	no
500	0	0	yes	0	5	yes	8	13	yes –MA no +MA
1000	0	0	yes	0	5	yes	1	13	yes –MA no +MA
5000	0	0	yes	0	3	yes	0	18	yes –MA no +MA
Positive control	ENNG 3.0 µg/plate	B[a]P 5.0 µg/plate		ENNG 5.0 µg/plate	2AA 2.0 µg/plate		ENNG 2.0 µg/plate	2AA 20.0 µg/plate
	328	823		130	87		361	419

 

Table 1. (cont’d)

	TA98			TA1537
	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	16	21	no	6	8	no
10	17	27	no	3	4	no
50	18	23	no	4	3	no
100	5	16	yes –MA no +MA	4	2	yes –MA no +MA
500	0	0	yes	0	0	yes
1000	0	0	yes	0	0	yes
5000	0	0	yes	0	0	yes
Positive control	4NQO 1.0 µg/plate	B[a]P 5.0 µg/plate		9AA 80.0 µg/plate	B[a]P 5.0 µg/plate
	458	343		1117	63

*solvent control (DMSO)

2AA, 2-aminoanthracene; 9AA, 9-aminoacridine; B[a]P,benzo(a)pyrene; ENNG, N-ethyl-N'-nitro-N-nitrosoguanidine; MA, metabolic activation; 4NQO, 4-nitroquinoline-N-oxide

 

Table 2:Number of revertants per plate (mean of 2 or 3 plates) – Main test

Bacterial strain	TA100			TA1535			WP2 uvrA
Concentration of test material, µg/plate	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	127 108 99 (111)	116 114 126 (119)	no	18 16 6 (13)	14 18 13 (15)	no	16 17 26 (20)	25 19 14 (19)	no
10	78 118 (98)		no
20	112 92 (102)	122 118 (120)	no	12 7 (10)	8 14 (11)	no	22 24 (23)		no
39	103 104 (104)	127 120 (124)	no	10 7 (9)	10 11 (11)	no	17 20 (19)		no
78	108 95 (102)	89 110 (100)	no	10 3 (7)	10 11 (11)	no	16 17 (17)		no
156	56 66 (61)	83 81 (82)	yes –MA no +MA	7 4 (6)	6 7 (7)	no	11 17 (14)		no
313		0 0 (0)	yes	0 0 (0)	0 0 (0)	yes	0 8 (4)	19 18 (19)	yes –MA no +MA
625								11 13 (12)	no
1250								12 13 (13)	no
2500								9 16 (13)	no
5000								10 13 (12)	no
Positive control	ENNG 3.0 µg/plate	B[a]P 5.0 µg/plate		ENNG 5.0 µg/plate	2AA 2.0 µg/plate		ENNG 2.0 µg/plate	2AA 20.0 µg/plate
	230 239 (235)	913 1006 (960)		66 42 (54)	176 158 (167)		298 177 (238)	530 496 (513)

 

Table 2. (cont’d)

	TA98			TA1537
	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
0*	44 47 30 (40)	49 52 53 (51)	no	4 9 9 (7)	8 12 14 (11)	no
10	42 39 (41)		no	14 6 (10)	8 14 (11)	no
20	40 50 (45)	47 47 (47)	no	9 6 (8)	12 7 (10)	no
39	39 40 (40)	45 45 (45)	no	4 8 (6)	13 9 (11)	no
78	18 23 (21)	46 40 (43)	no	6 4 (5)	7 6 (7)	no
156	10 14 (12)	18 10 (14)	yes –MA no +MA	0 1 (1)	1 11 (6)	yes
313		0 0 (0)	yes
Positive control	4NQO 1.0 µg/plate	B[a]P 5.0 µg/plate		9AA 80.0 µg/plate	B[a]P 5.0 µg/plate
	595 601 (598)	416 458 (437)		712 545 (629)	88 99 (94)

*solvent control (DMSO)

2AA, 2-aminoanthracene; 9AA, 9-aminoacridine; B[a]P,benzo(a)pyrene; ENNG, N-ethyl-N'-nitro-N-nitrosoguanidine; MA, metabolic activation; 4NQO, 4-nitroquinoline-N-oxide

Conclusions:

In a key study conducted according to Japanese guidelines and in compliance with GLP, cyclohexyldimethoxymethylsilane showed no mutagenic potential in a bacterial reverse mutation (Ames) assay with four strains of Salmonella typhimurium and Escherichia coli WP2 uvrA, with or without S9.

Executive summary:

In a GLP study, conducted according to Japanese guidelines, CHMS was assessed for its ability to induce reverse mutation in bacteria in an Ames test.

A range-finding study was first conducted using the pre-incubation method in which the test material was tested at concentrations of up to 5000 µg/plate, with and without a rat metabolic activation fraction (S9), to determine the concentrations for the main study. In the main study, again using a pre-incubation method, Salmonella typhimurium strains TA100, TA98 and TA1537 were tested at up to 156 µg/plate and TA 1535 and Escherchia coli WP2 uvrA were tested at up to 313 µg/plate without S9. In the presence of S9, TA1537 was tested at up to 156 µg/plate, TA100, TA98 and TA1535 at up to 313 µg/plate and WP2 uvrA at up to 5000 µg/plate. The S9 was prepared from microsomes obtained from phenobarbital- and 5,6-benzoflavone-induced rat liver and the pre-incubation period was 20 min, after which top agar was added to the pre-incubation mix and poured onto the surface of agar plates. After incubation at 37 ^oC for 2 days the revertant colonies were counted. Vehicle controls were similarly prepared together with positive controls using known mutagens.

No increase in mutant frequency was observed with the test material when compared to the vehicle controls in either the range-finding or main study. The positive controls gave the expected increase in mutant frequency demonstrating the validity of the assay. With the test material, cytotoxicity was observed for each of the strains (apart from WP2 uvrA with S9) at the highest concentration tested as shown by inhibition of the background lawn and the number of mutants present.

Under the conditions of this assay, CHMS showed no mutagenic potential in a reverse bacterial mutation test with four strains of S. typhimurium and E. coli WP uvrA.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 August 1995 to 22 February 1996

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced S9, prepared in-house from the livers of male Sprague-Dawley rats.

Test concentrations with justification for top dose:

14.5 µg/ml
29 µg/ml
58.5 µg/ml
117 µg/ml
233.75 µg/ml
467.5 µg/ml
935 µg/ml
1870 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: well known solvent

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Migrated to IUCLID6: at 500 µg/ml for cultures without S9, dissolved in dimethyl sulphoxide.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: at 25 µg/ml for cultures treated with S9, dissolved in culture medium without serum.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 h
- Exposure duration: 4 h
- Expression time (cells in growth medium): 16 h (and 40 h - Experiment 2)
- Fixation time (start of exposure up to fixation or harvest of cells): > 4 h

SPINDLE INHIBITOR (cytogenetic assays): demecolcine (Colcemid 0.1 µg/ml)
STAIN (for cytogenetic assays): 5% Gurrs Giemsa R66

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per culture, 200 per concentration, where possible

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index, performed at time of experiment

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

OTHER:
- type and composition of metabolic activation system:
- species and cell type: rat, liver, S9 fraction
- quantity: 1ml of 10% S9 in standard co-factors
- induced or not induced: induced
- chemicals used for induction: Aroclor 1254
- co-factors used: 'standard' [no further information]

Evaluation criteria:

A positive response was recorded for a particular treatment if the percentage of cells with aberrations markedly exceeded that seen in the concurrent vehicle control, either with or without a clear concentration-relationship. For modest increases in aberration frequency, a concentration response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Metaphase spreads were searched for any changes in chromosome number, gaps, breaks or rearrangements.

Statistics:

The frequency of cells with aberrations (including and excluding gaps), and the frequency of polyploid cells, were compared to the vehicle control using Fisher's Exact test or a Chi-squared test.

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

mild toxicity (mitotic index at 69 % of control) observed at the top concentration tested (1870 µg/ml)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

steep toxicity concentration-response relationship with no scorable metaphases at 233.75 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

no scorable metaphases at doses above 175.3 µg/ml (for which the mitotic index was 24% of the control)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

only slight effects at 1870 µg/ml (mitotic index of 82% compared to control)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

no scorable metaphases observed at 175.32 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no data
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: no data

COMPARISON WITH HISTORICAL CONTROL DATA: Based on historical aberration ranges for vehicle and untreated control cultures, a frequency of 0 to 4% of cells is judged to be acceptable for structural aberrations, including gaps, in lymphocyte control cultures. Excluding gaps, it is acceptable for 0 to 2% of cells to have structural aberrations, and 0 to 1% of cells to exhibit polyploidy.

ADDITIONAL INFORMATION ON CYTOTOXICITY: no data

Remarks on result:

other: all strains/cell types tested

The treatment concentrations were chosen for metaphase spread evaluation following the mitotic index determination for cytotoxicity.

Experiment 1:

For cultures harvested at 20 hours, the following concentrations were analysed:

With S9 treatment: 0, 467.5, 935, 1870 µg/ml

Without S9 treatment: 0, 29, 58.5, 117 µg/ml

Experiment 2:

For cultures harvested at 20 hours, the following concentrations were analysed:

With S9 treatment: 0, 467.5, 935, 1870 µg/ml

Without S9 treatment: 0, 29, 58.5, 117, 175.3 µg/ml

For cultures harvested at 44 hours, the following concentrations were analysed:

With S9 treatment: 0, 1870 µg/ml

Without S9 treatment: 0, 117 µg/ml

 

 

Table 1: Results of chromosome analysis – Experiment 1 – 20-hour harvest without metabolic activation

Concentration of test material		Control*	Low dose 29 µg/ml	Mid dose 58.5 µg/ml	High dose 117 µg/ml	Positive control EMS 500 µg/ml
Cytotoxicity		no	no	no	no	yes
		Mean (% of control)
Mitotic index		3.65 (100)	2.93 (81)	3.05 (84)	2.58 (71)	1.53 (42)
Chromosome aberrations		Total (per 100 cells)
Gaps		0 (0.0)	2 (1.0)	0 (0.0)	2 (1.0)	33 (22.0)
Chromatid aberrations	breaks	1 (0.5)	0 (0.0)	0 (0.0)	2 (1.0)	17 (11.3)
	interchanges	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	4 (2.7)
Isochromatidaberrations	breaks	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	2 (1.3)
	interchanges	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Total number of aberrant cells	including gaps	1 (0.5)	2 (1.0)	0 (0.0)	4 (2.0)	47 (31.3)
	excluding gaps	1 (0.5)	0 (0.0)	0 (0.0)	2 (1.0)	22 (14.7)
		Mean frequency (%)
Polyploidy		0.0	0.5	0.0	0.0	0.0

* solvent control (acetone)

EMS, ethylmethanesulphonate

 

Table 2: Results of chromosome analysis – Experiment 1 – 20-hour harvest with metabolic activation

Concentration of test material		Control*	Low dose 467.5 µg/ml	Mid dose 935 µg/ml	High dose 1870 µg/ml	Positive control CP 25 µg/ml
Cytotoxicity		no	no	no	no	no
		Mean (% of control)
Mitotic index		4.55 (100)	4.18 (92)	4.43 (97)	3.15 (69)	2.43 (53)
Chromosome aberrations		Total (per 100 cells)
Gaps		2 (1.0)	2 (1.0)	3 (1.5)	7 (3.5)	12 (6.0)
Chromatid aberrations	breaks	1 (0.5)	0 (0.0)	2 (1.0)	5 (2.5)	19 (9.5)
	interchanges	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	4 (2.0)
Isochromatidaberrations	breaks	1 (0.5)	0 (0.0)	2 (1.0)	0 (0.0)	5 (2.5)
	interchanges	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Total number of aberrant cells	including gaps	4 (2.0)	2 (1.0)	6 (3.0)	11 (5.5)	31 (15.5)
	excluding gaps	2 (1.0)	0 (0.0)	3 (1.5)	4 (2.0)	23 (11.5)
		Mean frequency (%)
Polyploidy		0.5	0.0	1.0	0.0	0.0

* solvent control (acetone)

CP, cyclophosphamide

 

Table 3: Results of chromosome analysis – Experiment 2 – 20-hour harvest without metabolic activation

Concentration of test material		Control*	Low dose 29 µg/ml	Low-mid dose 58.5 µg/ml	Mid-high dose 117 µg/ml	High dose 175.3 µg/ml	Positive control EMS 500 µg/ml
Cytotoxicity		no	no	no	no	yes	no
		Mean (% of control)
Mitotic index		6.48 (100)	5.53 (85)	5.40 (83)	4.95 (76)	1.55 (24)	3.63 (56)
Chromosome aberrations		Total (per 100 cells)
Gaps		2 (1.0)	1 (0.5)	4 (2.0)	3 (1.5)	6 (3.0)	27 (13.5)
Chromatid aberrations	breaks	0 (0.0)	2 (1.0)	0 (0.0)	1 (0.5)	3 (1.5)	29 (14.5)
	interchanges	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.5)
Isochromatidaberrations	breaks	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.5)	0 (0.0)	5 (2.5)
	interchanges	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Total number of aberrant cells	including gaps	2 (1.0)	3 (1.5)	4 (2.0)	5 (2.5)	6 (3.0)	45 (22.5)
	excluding gaps	0 (0.0)	2 (1.0)	0 (0.0)	2 (1.0)	2 (1.0)	27 (13.5)
		Mean frequency (%)
Polyploidy		0.0	0.0	0.5	1.0	0.0	0.0

* solvent control (acetone)

EMS, ethylmethanesulphonate

 

Table 4: Results of chromosome analysis – Experiment 2 – 20-hour harvest with metabolic activation

Concentration of test material		Control*	Low dose 467.5 µg/ml	Mid dose 935 µg/ml	High dose 1870 µg/ml	Positive control CP 25 µg/ml
Cytotoxicity		no	no	no	no	yes
		Mean (% of control)
Mitotic index		3.85 (100)	4.58 (119)	2.90 (75)	4.53 (118)	1.55 (40)
Chromosome aberrations		Total (per 100 cells)
Gaps		0 (0.0)	3 (1.5)	0 (0.0)	3 (1.5)	6 (3.0)
Chromatid aberrations	breaks	0 (0.0)	3 (1.5)	0 (0.0)	1 (0.5)	10 (5.0)
	interchanges	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.5)
Isochromatidaberrations	breaks	0 (0.0)	2 (1.0)	0 (0.0)	0 (0.0)	2 (1.0)
	interchanges	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Total number of aberrant cells	including gaps	0 (0.0)	8 (4.0)	0 (0.0)	4 (2.0)	15 (7.5)
	excluding gaps	0 (0.0)	5 (2.5)	0 (0.0)	1 (0.5)	13 (6.5)
		Mean frequency (%)
Polyploidy		1.0	2.0	0.0	1.5	0.0

* solvent control (acetone)

CP, cyclophosphamide

 

Table 5: Results of chromosome analysis – Experiment 2 – 44-hour harvest without metabolic activation

Concentration of test material		Control*	116.9 µg/ml
Cytotoxicity		no	no
		Mean (% of control)
Mitotic index		3.38 (100)	2.35 (70)
Chromosome aberrations		Total (per 100 cells)
Gaps		1 (0.5)	1 (0.5)
Chromatid aberrations	breaks	0 (0.0)	0 (0.0)
	interchanges	0 (0.0)	0 (0.0)
Isochromatidaberrations	breaks	1 (0.5)	0 (0.0)
	interchanges	0 (0.0)	0 (0.0)
Total number of aberrant cells	including gaps	2 (1.0)	1 (0.5)
	excluding gaps	1 (0.5)	0 (0.0)_
		Mean frequency (%)
Polyploidy		0.0	1.0

* solvent control (acetone)

 

Table 6: Results of chromosome analysis – Experiment 2 – 44-hour harvest with metabolic activation

Concentration of test material		Control*	1870 µg/ml
Cytotoxicity		no	no
		Mean (% of control)
Mitotic index		4.88 (100)	4.00 (82)
Chromosome aberrations		Total (per 100 cells)
Gaps		1 (0.5)	3 (1.5)
Chromatid aberrations	breaks	0 (0.0)	0 (0.0)
	interchanges	0 (0.0)	0 (0.0)
Isochromatidaberrations	breaks	2 (1.0)	1 (0.5)
	interchanges	0 (0.0)	0 (0.0)
Total number of aberrant cells	including gaps	3 (1.5)	4 (2.0)
	excluding gaps	2 (1.0)	1 (0.5)
		Mean frequency (%)
Polyploidy		0.0	0.5

* solvent control (acetone)

Conclusions:

In an in vitro cytogeniticity study conducted according to OECD Test Guideline 473 and in compliance with GLP, cyclohexyldimethoxymethylsilane was not clastogenic to human lymphocytes in vitro at up to 1870 µg/ml (limited by cytotoxicity), either in the presence or in the absence of metabolic activation by S9.

Executive summary:

In this GLP study, carried out according to OECD Test Guideline 473 (and EU method B.10), cyclohexyldimethoxymethylsilane was examined for clastogenic activity in two separate experiments on primary cultures of lymphocytes, taken from the peripheral blood of a human volunteer. Half of these lymphocyte cultures were incubated with the liver enzyme metabolising system S9 prior to treatment with cyclohexyldimethoxymethylsilane.

In Experiment 1, cell cultures were treated with cyclohexyldimethoxymethylsilane at 0, 14.5, 29, 58.5, 117, 233.75, 467.5, 935 or 1870 µg/ml for four hours, then incubated for a further 16 hours, before harvesting and fixation. Experiment 2 was carried out in the same way, but in addition to the 16 hour incubation period, 40 hour incubations were carried out for cells treated with cyclohexyldimethoxymethylsilane at 0, 467.5, 935 and 1870 µg/ml (with S9), or 0, 58.5, 117, 175.3 and 233.75 µg/ml (without S9).

Following fixation, cell cultures were checked for the effects of cytotoxicity, which was observed to be significantly higher in cultures which had not been pre-treated with S9. Using this information, appropriate concentrations were selected for metaphase spread analysis. In Experiment 1, concentrations chosen were 0, 467.5, 935 and 1870 µg/ml for cells treated with S9 and 0, 29, 58.5 and 117 µg/ml for cells without S9. In Experiment 2, the selected concentrations for the cultures incubated for 16 hours following treatment were 0, 467.5, 935 and 1870 µg/ml for cells treated with S9, and 0, 29, 58.5, 117 and 175.3 µg/ml for cells without S9. For the 40 hour cultures of Experiment 2, only metaphase spreads from the highest concentration that did not cause excessive toxicity (1870 µg/ml with and 117 µg/ml without S9) and the vehicle-only control were evaluated.

No significant, concentration-related increases in the frequency of cells displaying gaps, breaks, rearrangements or abnormalities in chromosome number were observed. Cyclohexyldimethoxymethylsilane was not clastogenic in human lymphocytes in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Data from two in vivo studies are also available. No evidence of germ cell mutation was seen in a dominant lethal test conducted according to OECD Test Guideline 478 and in compliance with GLP in mice treated for two weeks with up 1250 mg/kg bw/day, a toxic dose (Life Science Research, 1989).

There was an increased frequency of micronuclei in the bone marrow of male mice dosed at 5000 mg/kg bw, a highly toxic dose, but not in females and not in either sex at 2500 mg/kg bw, which was also toxic, in a mammalian erythrocyte micronucleus test conducted according to OECD Test Guideline 474 and in compliance with GLP (Life Science Research, 1987).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 August 1988 to 30 November 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)

Deviations:

no

Principles of method if other than guideline:

A 6-week dominant lethal study in mice

GLP compliance:

yes

Type of assay:

rodent dominant lethal assay

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (UK), Margate, Kent, England
- Age at study initiation: no data
- Weight at study initiation: 24-31 g
- Assigned to test groups randomly: no data
- Fasting period before study: no data
- Housing:group-housed (4 or 5/cage) during acclimation and dosing, high density polypropylene cages with stainless steel tops
- Diet: ad libitum, laboratory animal diet LAD 1
- Water: ad libitum
- Acclimation period: at least 5 days prior to treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): target 21, acceptable range 19-25
- Humidity (%): 55+/- 15
- Air changes (per hr): 15/hr
- Photoperiod (hrs dark / hrs light): 12 hr light/12 hr dark

IN-LIFE DATES: From: 19-Aug-1988 To:30-Nov-1988

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: test material freely miscible with corn oil and adequately stable in corn oil over a 72-hr period
- Concentration of test material in vehicle: dosed at 10 ml/kg bw, so presumably 5-125 mg/ml
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw
- Type and concentration of dispersant aid (if powder): not applicable
- Lot/batch no. (if required): no data
- Purity: no data

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Required volumes of CHMMS violently mixed with corn oil initially during dose preparation. Inversion of the dose container employed prior to dosing to ensure maintenance of an adequate mixture and to minimise introduction of air bubbles.

DIET PREPARATION
not applicable

Duration of treatment / exposure:

6 weeks

Frequency of treatment:

Vehicle control and treatment groups treated 5 days/week for 5 weeks and daily for week 6; positive control group treated daily for 3 days during week 6.

Post exposure period:

Mating to untreated females (2 females/male) on day following final treatment, then repeated with fresh females seven days later and repeated until 3 separate weekly matings were completed

Remarks:

Doses / Concentrations:
50, 250 or 1250 mg/kg bw/day (1250 mg/kg bw/day dose reduced to 750 mg/kg bw/day from Monday of week 3 onwards)
Basis:
actual ingested

No. of animals per sex per dose:

25 males

Control animals:

yes, concurrent vehicle

Positive control(s):

ethylmethanesulphonate
- Justification for choice of positive control(s): guideline recommendation
- Route of administration: oral gavage
- Doses / concentrations: 200 mg/kg bw/day for 3 consecutive days during week 6

Tissues and cell types examined:

Treated males were mated with untreated females and the pregnancy rate, pre-implantation loss and post-implantation loss assessed

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: based on a preliminary toxicity test

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): On the day following the final treatment, each male mouse was housed for mating with two virgin females. The individual males were re-caged with fresh females seven days later, and this was repeated until 3 separate weekly matings were completed. Females were inspected daily, in the mornings, during the mating period for the presence of vaginal plugs. Sixteen days after introduction to the males, the pregnant females were killed and examined for total implant number and early and late deaths.

Evaluation criteria:

Comparisons were made between treated and vehicle control groups each week for pregnancy rate, average number of implants (live and dead) and proportions of total implants found dead. Vaginal plugs were recorded to identify mated females and comparisons were made between treated and control groups for the proportion of females found to be pregnant. The average number of implants, live or dead, was calculated for each individual male and for each individual female of the groups, non-pregnant females being excluded, and treated and control groups were compared. Counts of early and late deaths were combined and the proportion of total implants found dead was obtained for each male. Individual male values were then used to test the homogeneity of each group.

Statistics:

Proportion of pregnant females: The variation between each treated group and the vehicle control group was assessed by calculation of the term X2B and the significance of the difference between the groups determined from chi-squared distribution tables.
Average number of implants per male and per female: The values for the treated and control groups were compared using a computer-based calculation of the non-parametic Mann-Whitney "U" test.
Proportion of implants found dead per male: A modified chi-squared calculation was performed giving X2W values and subsequent pairwise comparisons of test and control groups were undertaken, with calculation of X2B values. Where X2W values for both groups were non-significant, the significance of any difference between the groups was assessed by consideration of X2B alone. Where one or both groups were not homogeneous, the variance ratio was calculated and the significance obtained from the tables of the variance ratio distribution.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Overt signs of toxicity and death in a single male at the top dose level

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 500, 750, 1250 or 2000 mg/kg bw/day
- Clinical signs of toxicity in test animals: A single male in the top dosed group was killed in extremis, showing general lethargy and hind-limb paralysis, after receiving its sixth treatment. At this time and on later days of treatment, the remaining 3 animals of this group showed hyperactivity and/or partial hind-limb paralysis (transient, with full recovery by the morning after each dose). A single male dosed at 1250 mg/kg bw/day, also showed partial hind-limb paralysis on the last day of dosing. No other significant treatment-related effects were observed.
- Rationale for exposure: With the exception of a single male dosed at 2000 mg/kg bw/day, surviving males impregnated both of the two females with which they were caged during the subsequent mating period. Based on these findings and the overt toxicity seen, a maximum dosage of 1250 mg/kg bw/day was selected for the main study.

RESULTS OF DEFINITIVE STUDY
- Appropriateness of dose levels and route: The maximum level of CHMMS administration employed was sufficient to produce overt toxicity, demonstrating that the test material was absorbed.
- Statistical evaluation: There were no statistically significant effects on pregnancy rate, pre-implantation loss or post-implantation loss in any of the CHMMS treated groups.

During week 2 of dosing, males given 1250 mg/kg bw/day showed transient ataxia, loss of gripping reflex and other behavioural abnormalities. The effects included a brief period of hyperactivity post-dose, followed by ataxia and, in some cases, apparent deep sedation. Recovery was complete within 3 -4.5 hours of dosing. A single male of this group was found dead at the end of week 2. After reducing the dose to 750 mg/kg bw/day, some animals showed post-dose hyperactivity during weeks 3 and 4, and ataxia and prominent testes were also observed. No obvious signs of adverse reactions were seen at the mid- and low-dose levels.

Table: Summary of group data  

Treatment group	Mating week	Pregnant females		Total implants		Dead implants		Mutagenic index^b
		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 0.5	5.7 4.2 4.6
CHMMS, 50 mg/kg bw/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 bw/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 bw/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 bw/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 1.0	50.3** 40.7** 8.1*

 

a - Percentage of all females caged with surviving, treated males and later confirmed as pregnant.

b - Mutagenic Index = (Total dead implants/total implants) x 100

* - Significantly different from Group 1 value, p<0.05 (based on "per male" analysis)

** - Significantly different from Group 1 value, p<0.001

Conclusions:

In a well-conducted dominant lethal test, using a protocol similar to OECD Test Guideline 478 and in compliance with GLP, there was no evidence of germ cell mutation in mice after oral dosing of cyclohexyldimethylmethylsilane at up to 1250 mg/kg bw/day, a toxic dose.

Executive summary:

A well-conducted dominant lethal test was performed using a protocol similar to OECD Test Guideline 478 and in compliance with GLP.

Groups of 25 male mice were given oral gavage doses of 50, 250 or 1250 mg cyclohexyldimethylmethylsilane/kg bw/day, 5 days/week for 5 weeks and 7 days/week during the 6th week. The highest tested dose level was reduced to 750 mg/kg bw/day from the beginning of the 3rd week of dosing following marked adverse reactions to treatment (hyperactivity, ataxia and sedation). A control group received vehicle only (corn oil) and a positive control group received ethylmethanesulphonate at 200 mg/kg bw/day on the last 3 days of week 6.

On the day following the last day of dosing, each male was individually caged with 2 virgin females and allowed to mate; evidence of mating was inspected daily. After 7 days, the females were removed and replaced with fresh virgin females. This process was repeated once more to give a total of 3, 1-week mating periods. Sixteen days after introduction to the males, each group of females was killed and the numbers of live and dead (early or late death) implants were recorded. Three separate parameters were investigated: pregnancy rate, pre-implantation loss and post-implantation loss.

There was no evidence of germ cell mutation in mice after oral dosing with cyclohexyldimethylmethylsilane at up to 1250 mg/kg bw/day, a toxic dose. A clear positive response was seen with the positive control.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 April 1987 to 21 August 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The study was conducted according to an appropriate OECD test guideline with acceptable restrictions. The restrictions were: only 2 dose levels (guideline recommends 3)

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

(only 2 dose levels, guideline recommends 3)

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

(only 2 dose levels, guideline recommends 3)

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)

Deviations:

not specified

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
- Age at study initiation: 5-6 weeks
- Weight at study initiation: males 28-37 g, females 24-33 g
- Assigned to test groups randomly: yes
- Fasting period before study: overnight
- Housing: 5 animals of one sex/cage, clear polycarbonate cages measuring 35.5 x 23.5 x 19 cm with stainless steel mesh lid and floor
- Diet: ad libitum, Altromin MT diet
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): monitored daily
- Humidity (%): monitored daily
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hr light/12 hr dark

IN-LIFE DATES: From: 9-Apr-1987 To:21-Aug-1987

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: no data, but well-known vehicle
- Concentration of test material in vehicle: no data, but presumably 250 or 500 mg/ml
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw
- Type and concentration of dispersant aid (if powder): not applicable
- Lot/batch no. (if required): no data
- Purity: no data

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Fresh solutions of the test material were prepared for each day's work; solutions being prepared on a weight/volume basis without correction for the displacement due to the volume occupied by the test material.

Duration of treatment / exposure:

Single exposure, with harvest times of 24, 48 or 72 hours

Frequency of treatment:

Once

Post exposure period:

24, 48 and 72 hours

Remarks:

Doses / Concentrations:
2500 or 5000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5/sex per dose for each exposure period

Control animals:

yes, concurrent vehicle

Positive control(s):

mitomycin C
- Justification for choice of positive control(s): a well-known clastogen
- Route of administration: oral, gavage
- Doses / concentrations: 5.00 mg/kg bw

Tissues and cell types examined:

Femoral bone marrow cells obtained by flushing with foetal calf serum

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on a preliminary toxicity test

DETAILS OF SLIDE PREPARATION: Cells were centrifuged at 1000 rpm for 5 min and the supernatant completely removed. A concentrated suspension of the cells of the sediment was then prepared to make smears on slides. The slides were air-dried overnight and then stained with May-Gruenwald and Giemsa, and mounted with Eukitt. Three slides were made for each animal.

METHOD OF ANALYSIS: The slides were randomly coded by a person not involved in the subsequent microscope scoring. They were then 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 polychromatic erythrocytes (PCEs) were examined per animal at high magnification (100x) for the presence or absence of micronuclei. At the same time, the number of normochromatic erythrocytes (NCEs) and micronucleated NCEs was also recorded.

Evaluation criteria:

The ratio of PCEs to NCEs gives an indication of the toxicity of treatment; an increase in the ratio indicates inhibition of cell division. The incidence of micronucleated NCEs gives an indication of the pre-treatment status of the animals. The incidence of micronucleated PCEs provides an index of induced genetic damage. The test material will be considered to have induced micronuclei if a statistically significant and biologically meaningful increase in micronucleus incidence (at p<0.05) is observed in any treatment group, in the pooled data for both sexes, or in the data for male or female groups alone.

Statistics:

Only counts from polychromatic cells were subjected to statistical analysis. Using the original observations (and not the micronucleus frequencies per 1000 cells) a modified chi-squared calculation was employed to compare treated and control groups. The degree of heterogeneity within each group was first calculated, and where it was significant it was taken into account in the comparison between groups. If there was no significant within-group heterogeneity, the chi-squared test was used to compare treated groups with the controls. If there was significant within-groups heterogeneity, then that group was compared with the controls using a variance ratio (F) value calculated from the between-group and within-group chi-squared values.

Sex:

male

Genotoxicity:

positive

Remarks:

5000 mg/kg bw

Toxicity:

yes

Remarks:

See "any other information" section below

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Sex:

female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

See "any other information" section below

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

See "any other information" section below

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 5000 mg/kg bw
- Solubility: no data
- Clinical signs of toxicity in test animals: There was no excessive lethality
- Evidence of cytotoxicity in tissue analyzed: not applicable
- Rationale for exposure: There was no excessive lethality in the preliminary study.
- Harvest times: not applicable
- High dose with and without activation: not applicable
- Other: no data

RESULTS OF DEFINITIVE STUDY
There was no marked increase in the number of micronucleated PCEs in any of the treatment groups at the 24- and 72-hour sampling times. At the 48-hour sampling time, a pronounced increase was observed at the high-dose level. A dose-related increase in the ratio of mature to polychromatic erythrocytes (NCE:PCE ratio) was seen at the 4- hour sampling time in the test material treatment groups. Similar increases were also seen at 24 hours, indicating that the test material exerted an inhibitory effect on the cell division of bone marrow erythropoietic cells.

When analysed by treatment group and the male and female data were combined, no statistically significant increases in the incidence of micronucleated PCEs were observed when compared with the vehicle control group at the 24-, 48- or 72-hour sampling times. When analysed by sex, no statistically significant increases in the incidences of micronucleated PCEs were seen for the female animals when compared with the vehicle controls at any of the sampling times. In the males, a single statistically significant increase was observed at the high-dose level, at the 48-hour sampling time (p<0.01), with increased micronucleus incidence being present in 3 of the 5 animals. This observed micronucleated PCE incidence lay outside the historical negative control values for the laboratory.

A range of toxic signs was observed including loss of consciousness, hyperactivity and hypoactivity, loss of equilibrium and unusual gait, fibrillations and tremors, blanching, piloerection and ungroomed appearance. At the high-dose level, seven of the 30 animals died following treatment and were substituted by reserve animals.

Table 1: Summary of Incidence of Micronucleated Cells

 

Sampling time: 48 hours

 

MALES
				Incidence of Micronuclei per 1000 cells
Dose-level	Scored Cells		NCE/PCE	Polychromatic				Normochromatic
mg/kg	PCE	NCE	Ratio	Mean	SE	Min	Max	Mean	SE	Min	Max
0.00	5000	5609	1.12	1.6	0.6	0.0	3.0	1.1	0.6	0.0	2.8
2500	4013	4450	1.11	1.5	0.6	0.0	3.0	1.1	0.7	0.0	2.5
5000	3600	5387	2.02	5.6	2.2	0.0	11.0	1.9	0.7	0.9	4.5
Mitomycin C
5.00	1610	3632	2.84	16.1	1.3	13.5	18.0	2.5	1.0	0.6	4.0
FEMALES
				Incidence of Micronuclei per 1000 cells
Dose-level	Scored Cells		NCE/PCE	Polychromatic				Normochromatic
mg/kg	PCE	NCE	Ratio	Mean	SE	Min	Max	Mean	SE	Min	Max
0.00	5062	3467	0.69	1.4	0.7	0.0	3.0	0.8	0.5	0.0	2.3
2500	4426	7110	1.69	2.3	0.8	0.0	5.0	0.9	0.2	0.0	1.3
5000	3770	7206	2.20	1.8	1.0	0.0	5.0	0.4	0.2	0.0	1.0
Mitomycin C
5.00	3080	5219	2.12	11.3	3.5	4.0	23.3	5.1	1.7	1.1	10.0
BOTH SEXES
				Incidence of Micronuclei per 1000 cells
Dose-level	Scored Cells		NCE/PCE	Polychromatic				Normochromatic
mg/kg	PCE	NCE	Ratio	Mean	SE	Min	Max	Mean	SE	Min	Max
0.00 /
0.00	10062	9076	0.9	1.5	0.4	0.0	3.0	0.9	0.4	0.0	2.8
2500 /
2500	8439	11560	1.43	1.9	0.5	0.0	5.0	1.0	0.3	0.0	2.5
5000 /
5000	7370	12593	2.11	3.7	1.3	0.0	11.0	1.1	0.4	0.0	4.5
Mitomycin C
5.00	4690	8851	2.39	13.1	2.3	4.0	23.3	4.2	1.2	0.6	10.0

MEAN = group mean incidence of micronucleated PCEs/NCEs

SE = standard error of the mean incidence

MIN = Minimum value observed in an individual animal

MAX = maximum value observed in an individual animal

Table 2: Statistical analysis

 

Sampling time: 48 hours

 

STATISTICAL ANALYSIS – BOTH SEXES
Dose level mg/kg			Within animals of one group		Between each group and control group
Males		Females	X2	Sign.	X2	Sign.	F	Sign.
0.00		0.00	10.82	N.S.
2500		2500	10.05	N.S.	0.45	N.S.
5000		5000	25.25	**			4.17	N.S.
Mitomycin C		5 mg/kg	17.61	*				37.75***
MALES ONLY
0.00		0.00	4.51	N.S.
2500		2500	3.37	N.S.	0.02	N.S.
5000		5000	9.23	N.S.	10.09	**
Mitomycin C		5 mg/kg		N.C.		N.C.		N.C.
FEMALES ONLY
0.00	0.00		6.37	N.S.
2500	2500		5.66	N.S.	1.01	N.S.
5000	5000		8.63	N.S.	0.31	N.S.
Mitomycin C	5 mg/kg		14.09	**			9.24	*
DIFFERENCES BETWEEN SEXES
					Between male and female groups
0.00	0.00				0.08	N.S.
2500	2500				0.65	N.S.
5000	5000				6.90	**
Mitomycin C	5 mg/kg						2.73	N.S.

The chi-squared statistic (X2) and significance level (Sign.) are presented for within-group heterogeneity.

The Chi-squared (X2) or F-statistic (F), and significance level (Sign.) are shown for the comparison between the control and treatment group (or between males and females in the same treatment groups, as appropriate)

N.C. = not calculated

N.S. = not significant

* = statistically significant at p<0.05

** = statistically significant at p<0.01

*** = statistically significant at p<0.001

Conclusions:

In a reliable study, conducted according to OECD Test Guideline 474 and in compliance with GLP, cyclohexyldimethylmethylsilane induced micronuclei in the polychromatic erythrocytes of male mice given 5000 mg/kg bw by oral gavage, a toxic dose. No such effect was seen in females or when the 2 groups were combined.

Executive summary:

In a reliable study, conducted according to OECD Test Guideline 474 and in compliance with GLP, five male and five female mice were treated with a single oral, gavage, dose of cyclohexyldimethylmethylsilane at 2500 or 5000 mg/kg bw. Negative control animals received the vehicle only (corn oil) and positive control animals were treated with mitomycin C. Animals were sacrificed 24, 48 or 72 hours after treatment and bone marrow smear slides were prepared, stained and assessed for micronucleus induction in the polychromatic erythrocytes. The results obtained at each sampling time were subjected to statistical analysis using a modified chi-squared test.

A dose-related increase in the ratio of mature to polychromatic erythrocytes was observed at the 48-hour sampling time in the treatment groups, indicating that the test material exerted an inhibitory effect on the cell division of bone marrow erythropoietic cells. Similar increases were observed at the 24 -hour sampling time. There were no statistically significant increases in the incidences of micronucleated PCEs at any dose-level or sampling time when the results for the male and female animals were combined or those of the females were considered separately and compared with the vehicle control values. For the male animals, a single statistically significant increase was observed at 5000 mg/kg bw at the 48 -hour sampling time, with increases in the micronucleus incidence being seen in three of the five animals and the observed micronucleated PCE incidence lying outside the historical negative control values for the laboratory.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro genetic toxicity data on cyclohexyl(dimethoxy)methylsilane (CAS 17865-32-6) is available from five reliable bacterial mutagenicity tests.

The key bacterial mutagenicity study was conducted according to Japanese test guidelines and in compliance with GLP. Cyclohexyl(dimethoxy)methylsilane showed no mutagenic potential in this reverse mutagenicity (Ames) assay in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 at up to 313 µg/plate and Escherichia coli WP2 uvrA at up to 5000 µg/plate, with or without metabolic activation (Sogo Biomedical Laboratories., 1987).

The four supporting studies were conducted according to, or using protocols similar to, OECD Test Guideline 471 and in compliance with GLP. Cyclohexyl(dimethoxy)methylsilane showed no mutagenic potential with or without metabolic activation in these Ames assays in S. typhimurium strains TA97, TA98, TA100, TA1535, TA1537 and TA1538 at concentrations up to 5000 µg/plate (Life Science Research., 1986; Huntingdon Research Centre Ltd., 1989 and 1988; SafePharm Laboratories Ltd., 1995).

 

Further in vitro information is available from two reliable cytogenicity tests, both conducted according to OECD Test Guideline 473 and in compliance with GLP. In the key study, cyclohexyl(dimethoxy)methylsilane was not clastogenic to human lymphocytes in vitro at up to 1870 µg/ml (limited by cytotoxicity), either in the presence or in the absence of metabolic activation (SafePharm Laboratories Ltd., 1996). In the supporting study, no clastogenicity was seen in human lymphocytes at up to 250 µg/ml (limited by cytotoxicity), either in the presence or absence of metabolic activation (Huntingdon Research Centre Ltd., 1989).

 

There are also two key in vivo genotoxicity studies available for cyclohexyl(dimethoxy)methylsilane.

In a well-conducted dominant lethal test, using a protocol similar to OECD Test Guideline 478 and in compliance with GLP, there was no evidence of germ cell mutation in mice after oral dosing of cyclohexyl(dimethoxy)methylsilane for 2 weeks at up to 1250 mg/kg bw/day, a toxic dose (Life Science Research, 1989). In a reliable study, conducted to OECD Test Guideline 474 and in compliance with GLP, cyclohexyl(dimethoxy)methylsilane induced micronuclei in the polychromatic erythrocytes of male mice given 5000 mg/kg bw by oral gavage, a dose that was lethal to some of the animals. No statistically significant effect was seen in females, or when data for males and females were combined, and there was no increase in the frequency of micronuclei in either sex at 2500 mg/kg bw, a toxic dose (Life Science Research, 1987). The occurrence of a positive genotoxic response only at a dose high enough to induce severe toxicity is of questionable relevance to humans and OECD Test Guideline 474 requires testing only up to "the dose producing signs of toxicity such that higher dose levels... would be expected to produce lethality."

The most reliable studies were chosen as key. Where there was more than one reliable study, the most recent was selected as the key study. The results of all the studies are in agreement. 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

Based on the available in vitro and in vivo genotoxicity data, cyclohexyl(dimethoxy)methylsilance is not classified for mutagenicity according to Regulation (EC)1272/2008