Dicyclopentyldimethoxysilane

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Cyclohexyldimethoxymethylsilane

EC Number:

402-140-1

EC Name:

Cyclohexyldimethoxymethylsilane

Cas Number:

17865-32-6

Molecular formula:

C9H20O2Si

IUPAC Name:

Cyclohexyl-dimethoxy-methylsilane

Test material form:

liquid

Test animals

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

Administration / exposure

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

Doses / concentrations

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

Examinations

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 was not homogeneous, the variance ratio was calculated and the significance obtained from the tables of the variance ratio distribution.

Results and discussion

Test results

Key result

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

Conclusions:

In a well-conducted dominant lethal test, using a protocol similar to OECD guideline 478 and in compliance with GLP, there was no evidence of germ cell mutation in mice after oral dosing of cyclohexyl(dimethoxy)methylsilane 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 guideline 478 and to GLP.

Groups of 25 male mice were given oral gavage doses of 50, 250 or 1250 mg CHMS/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 CHMS at up to 1250 mg/kg bw/day, a toxic dose. A clear positive response was seen with the positive control.