Chlorotrioctylstannane

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 October 2002 to 8 January 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese MHLW (1994): "Guidelines for Toxicity Studies of Drugs", New Drugs Division, Pharmaceutical Affairs Bureau and MHLW/MITI (1997): "Guidelines for Screening Toxicity Testings of Chemicals. Testing Methods for New Chemical Substances".

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Chlorotrioctylstannane

EC Number:

219-969-8

EC Name:

Chlorotrioctylstannane

Cas Number:

2587-76-0

Molecular formula:

C24H51ClSn

IUPAC Name:

chlorotrioctylstannane

Test material form:

liquid

Specific details on test material used for the study:

- Physical appearance: Transparent colourless liquid
- Storage conditions of the test material: <- 18 ° C, in the absence of light
- Expiry date: 31 July 2004

Test animals

Species:

mouse

Strain:

Swiss

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Young adult
- Average weight at study initiation: 33.1 g
- Assigned to test groups randomly: Yes. The animals were allocated by computer randomisation.
- Fasting period before study: 3 hours 20 minutes
- Housing: Sterilised cages, fitted with a grid cover of stainless steel and with a bedding of sterilised softwood chips. The animals of the positive control group were housed in a laminar down-flow cabinet, just prior to administration and until sacrifice.
- Diet: ad libitum; fresh pellet diet was provided once weekly
- Water: ad libitum tap water provided in polypropylene bottles
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 to 70 % (relative)
- Air changes: about 10 per hour
- Photoperiod: Lighting was artificial with a sequence of 12 hours light and 12 hours dark

IN-LIFE DATES
- From: 20 November 2002
- To: 28 November 2002

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Corn oil
- Concentration of test material in vehicle: 100, 50 and 25 mg/mL
- Amount of vehicle (if gavage or dermal): 20 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test material was suspended in the vehicle.

Duration of treatment / exposure:

24 hours (all dose groups) and 48 hours (control and high dose only)

Frequency of treatment:

Single dose

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 male animals per dose for the control and 2000 mg/kg bw dose group (3 reserve mice were additionally treated with 2000 mg/kg bw in order to replace any mortality); 5 male animals per dose for the 1000 and 500 mg/kg bw dose groups.

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C
- Route of administration: Intraperitoneal
- Doses / concentrations: 0.75 mg/kg kw

Examinations

Tissues and cell types examined:

- Clinical signs: Signs of reaction to treatment were recorded from 1 to 4 hours after treatment and daily thereafter.
- Bone marrow: the number of micronucleated polychromatic erythrocyte (MPE) per 2000 polychromatic erythrocytes (PE) were counted for each mouse.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION
Based on the results of a dose-finding acute toxicity study; the acute toxicity (LD50) for rats, orally treated with the test material was known to be >4000 mg/kg bw. Dose levels of 2000, 1000 and 500 mg/kg bw were selected for the dose-range finding acute toxicity test in male and female mice (per sex per dose). No severe clinical signs were observed. As no sex differences could be demonstrated in the dose-range finding acute toxicity test), the main micronucleus test was performed with male mice only.

DETAILS OF SLIDE PREPARATION
From each mouse, the bone marrow cells of both femurs were immediately collected into foetal calf serum and processed into glassdrawn smears according to the method described by Schmid (1976). Two bone marrow smears per animal were prepared, air-dried and fixed in methanol. One smear per animal was stained with a May-Grünwald Giemsa solution. The other smear was stored as a reserve slide.

METHOD OF ANALYSIS
The numbers of polychromatic and normochromatic erythrocytes (PE and NE, respectively) were recorded in a total of 200 erythrocytes (E) per animal; if micronuclei were observed, these were recorded as micronucleated polychromatic erythrocytes (MPE) or micronucleated normochromatic erythrocytes (MNE). Once a total number of 200 E (PE + NE) had been scored, an additional number of PE was scored for the presence of micronuclei until a total number of 2000 PE had been scored. Thus the incidence of MPE was recorded in a total of 2000 PE per animal and the number of MNE was recorded in the number of NE.

OTHER: Animals were sacrificed by cervical dislocation

Evaluation criteria:

A response is considered to be positive if the mean number of MPE/2000 PE is statistically significantly higher, when compared to the mean number of the vehicle controls.
A test material is considered to cause chromosomal damage and/or damage to the mitotic apparatus, if a clear dose-related increase in the mean numbers of MPE/2000 PE is observed, when compared to the mean number of the vehicle controls.
A test material is considered to be negative in the micronucleus test if it produces no positive response at any of the dose-levels and time points analysed.
The test material or its metabolites are considered to have reached the general circulation and thereby the bone marrow, if the test material statistically reduce the mean number of PE/E or causes systemic toxicity.
Both statistical significance and biological relevance are considered together in the evaluation.

Statistics:

24 hours after administration, data on MPE and PE were subjected to a One Way Anova with factor group. If the Anova yielded a significant effect (p<0.05), it was followed by pooled error variance t-tests or, if variances were not homogeneous, separate variance t-tests. These t-tests were applied to the negative control group versus treatment groups. In addition, the positive control group and the negative control group were compared using pooled error variance t-tests or, if variances were not homogeneous, separate t-tests.
48 hours after administration, for the control and high dose groups, data on MPE and PE were subjected to pooled error variance t-tests or, if variances were not homogeneous, separate variance t-tests.
All statistical tests were performed using BMDP statistical software (W.J. Dixon, BMDP Statistical Software Manual, University of California Press, Berkeley, 1992).

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No clinical signs were observed as a result of treatment with three dose levels (2000, 1000 and 500 mg/kgbw) of the test material.

At both sacrifice times of 24 hours and 48 hours after treatment, the two-way ANOVA did not yield a statistically significant effect for MPE and PE. This indicates that treatment up to 2000 mg/kg bw (the limit dose) did not result in genotoxicity or clastogenicity to the bone marrow target cells. At the sacrifice time of 24 hours, in the positive control group, the incidence of MPE per 2000 PE was statistically significantly different (P<0.001) from the negative control. This demonstrates the sensitivity of the test system.
The results of this micronucleus test did not show any indication of chromosomal damage and/or damage to the mitotic apparatus of the bone marrow target cells in male mice, treated orally with the test material.
The group mean numbers of MPE per 2000 PE for each group are presented in Table 1. The group mean numbers of PE per 200 E for each group are presented in Table 2.

Any other information on results incl. tables

Table 1: Micronucleated Polychromatic Erythrocytes (MPE) 

The group mean numbers of MPE per 2000 PE
Group		Negative Control (corn-oil)	Test Material (mg/kg bw)			Positive Control (MMC 0.75 mg/kg bw)
			500	1000	2000
Sex	Time (hrs)
Male	24	2.6 ± 0.5	2.8 ± 1.3	3.2 ± 2.4	1.7 ± 0.8	32.6 ± 4.2***
Male	48	3.4 ± 1.5	-	-	2.8 ± 1.5	-

MMC = Mitomycin C

***p<0.001 (t-tests)

 

Table 2: Polychromatic Erythrocytes (PE)

The group mean numbers of PE per 200 E
Group		Negative Control (corn-oil)	Test Material (mg/kg bw)			Positive Control (MMC 0.75 mg/kg bw)
			500	1000	2000
Sex	Time (hrs)
Male	24	92.2 ± 13.4	89.0 ± 7.5	92.8 ± 18.1	82.7 ± 18.4	91.6 ± 10.4
Male	48	90.4 ± 8.1	-	-	80.8 ± 5.3	-

 

Applicant's summary and conclusion

Conclusions:

The test material did not produce chromosomal damage or damage to the mitotic spindle apparatus in the bone marrow target cells of mice.

Executive summary:

The test material was examined for its mutagenic potential in a bone marrow micronucleus test in mice conducted in accordance with the standardised guidelines OECD 474, EU Method B.12, US EPA OTS 798.5395 and Japanese guidelines under GLP conditions.

Following a dose range-finding test, in which no sex differences were observed and it was determined that the limit dose could be tolerated, male mice were treated in the main study at dose levels of 500, 100 and 2000 mg/kg bw.

Animals were treated once by gavage with three graded dose levels of the test material in corn oil. Ten animals were treated in the control and high dose groups, with 5 being treated at the intermediate dose levels. A concurrent positive control group consisting of 5 males was treated with a single intraperitoneal dose of Mitomycin C.

24 hours after treatment, 5 animals of each dose-level of the test material, 5 negative control animals and 5 positive control animals, were euthanised. 48 hours after treatment, the remaining 5 animals of the control and high dose-group were euthanised. From both femurs of each animal, the bone marrow cells were collected in foetal calf serum and processed into smears for microscopic examination.

24 and 48 hours after treatment, the number of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) were counted for each mouse. The mean number of MPE per 2000 PE at all dose-levels was not statistically significantly different than the vehicle control mean.

24 and 48 hours after treatment, the mean numbers of polychromatic erythrocytes (PE) per erythrocytes (E) in mice, at all treatment levels, were not statistically significantly different from the mean of the vehicle control mice.

Therefore, the test material, at dose-levels up to 2000 mg/kg-bw, was not genotoxic or cytotoxic to bone marrow cells in mice. For the mice of the positive control group, the mean number of MPE per 2000 PE differed significantly (p<0.001) from the mean number found in the vehicle control mice. This demonstrates the validity and sensitivity of the test system.

Under the conditions of this study, the test material was not genotoxic; it did not produce chromosomal damage or damage to the mitotic spindle apparatus in the bone marrow target cells of mice.