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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:
6 November 2002 - 21 January 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with GLP to appropriate OECD study guideline.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Principles of method if other than guideline:
Deviations from the protocol:
- The animals for the dose range finding toxicity test arrived on 6 November 2002, instead of 13 November 2002.
- The dose range finding toxicity test was started on 12 November 2002, instead of 19 November 2002.
- The termination date of the dose range finding toxicity test was 15 November 2002, instead of 22 November 2002.
- The animals for the main micronucleus test arrived on 4 December 2002, instead of 11 December 2002.
- The main micronucleus test was started on 10 December 2002, instead of 17 December 2002.
- The termination date of the main micronucleus test was 12 December 2002, instead of 20 December 2002.
- During a short period in the dose-range finding acute toxicity test, the relative humidity and pressure in animal room were slightly different from the normal values, due to cleaning activities.
- During a short period in the main micronucleus test, the relative humidity and pressure in animal room were slightly different from the normal values, due to cleaning activities.
- During a short period in the main micronucleus test, the relative humidity in animal room was slightly higher than 70 %, due to cleaning activities.
These deviations did not adversely influence the integrity and/or the validity of the study.
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetraoctyltin
EC Number:
222-733-7
EC Name:
Tetraoctyltin
Cas Number:
3590-84-9
Molecular formula:
C32H68Sn
IUPAC Name:
tetraoctylstannane
Details on test material:
- Name of test material (as cited in study report): TTOT
- Molecular formula (if other than submission substance): C32H68Sn
- Molecular weight (if other than submission substance): 571.6 g/mol
- Physical state: colourless to slightly yellow turbid liquid
- Analytical purity: 89.5 % (reported) 89.05 % (measured)
- Lot/batch No.: 346272
- Expiration date of the lot/batch: 31 July 2004
- Storage condition of test material: at < - 18 ° C, in the absence of light


Test animals

Species:
mouse
Strain:
Swiss
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: obtained from a colony maintained under SPF conditions at Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: adult
- Weight at study initiation: mean body weights were approx. 32-33 g
- Assigned to test groups randomly: Yes, prior to the start of the main micronucleus test, the animals were allocated by computer randomization to a vehicle control group A, three test groups treated with Tetraoctylstannane (B, C and D) and to a positive control group E.
- Fasting period before study: 2 hours 25 minutes
- Housing: The animals were housed in sterilised Macrolon cages (type I), fitted with a grid cover of stainless steel and with a bedding of sterilised softwood chips.
- Diet/water (e.g. ad libitum): With the exception of the fasting period prior to dosing, feed and drinking water were provided ad libitum from the arrival of the animals until the end of the study. Fresh pellet diet was provided once weekly.

The animals received a commercial rodent diet (Rat and Mouse No. 3 Breeding Diet, RM3). Batch 2305 was used for both the dose-range finding acute toxicity test and the main micronucleus test. Each batch of this diet is analysed by the supplier (SDS Special Diets Services, Witham, England) for nutrients and contaminants. The certificates of analysis pertaining to batch 2305 are stored in the archives of TNO Nutrition and Food Research.

The drinking water (tap-water) was given in polypropylene bottles, which were cleaned weekly and filled as needed. Tap-water for human consumption (quality guidelines according to Dutch legislation based on the EEC Council Directive 98/83/EEC), was supplied by N.V. Hydron Midden-Nederland. Results of the routine physical, chemical and microbial examination of the drinking water as conducted by the supplier are made available to TNO Nutrition and Food Research. In addition, the supplier periodically (twice per year) analyses water samples taken on the premises of TNO in Zeist for a limited number of variables. The results of the samples taken during or close to the conduct of the study are stored in the archives of TNO Nutrition and Food Research.

- Acclimation period: During the quarantine and acclimatization period the animals were observed daily for overt signs of ill health and anomalies.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): at least 30% and not exceeding 70% other than during room cleaning.
- Air changes (per hr): about 10 air changes per hour
- Photoperiod (hrs dark / hrs light): Lighting was artificial with a sequence of 12 hours light and 12 hours dark.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On day 0 (prior to dosing), the test substance was suspended in corn-oil, at concentrations of 100, 50 and 25 mg/ml. The orally (by gavage) given dosing volume was 20 ml/kg-bw. Prior to dosing, the animals were weighed. During dosing, the test substance was stirred on a magnetic stirrer.
Duration of treatment / exposure:
single oral dose
Frequency of treatment:
single oral dose
Post exposure period:
48 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000 & 2000 mg/kg-bw
Basis:
nominal conc.
No. of animals per sex per dose:
5 animals for doses 500, 1000 mg/kg-bw and the positive control group. 10 animals used for the negative control and dose group 2000 mg/kg-bw. Three reserve mice were treated with the highest dose-level of 2000 mg/kg-bw to replace any mortality in the highest dose-level group.
Control animals:
yes, concurrent vehicle
Positive control(s):
mitomycin C
- Route of administration: Sigma; intraperitoneal injection
- Vehicle: saline
- Doses / concentrations: stock-concentration: 0.075 mg/ml; dosing volume 10 ml/kg-bw; dose level: 0.75 mg/kg-bw
- Other: For safety reasons, the animals of the positive control group (main micronucleus test only) were housed in a laminar down-flow cabinet, just prior to administration and until sacrifice. The animal rooms were ventilated with about 10 air changes per hour and were maintained at a temperature of 22 +/- 3 ° C and a relative humidity of at least 30% and not exceeding 70% other than during room cleaning. Lighting was artificial with a sequence of 12 hours light and 12 hours dark.

Examinations

Tissues and cell types examined:
Bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
No acute toxicity (LD50) data for mice or rats, orally treated with Tetraoctylstannane, was available. Therefore, dose levels of 2000 (the limit dose level), 1000, 500 and 250 mg/kg-bw were selected for the dose-range finding acute toxicity test. The dose-range finding acute toxicity test was carried out with both male and female mice.

The dose levels and the choice of sex, selected for the main micronucleus test, were based on the clinical signs observed during the performance of the dose-range finding acute toxicity test. The results of the dose-range finding acute toxicity test (absence of toxicity and sex difference) were discussed with the sponsor. Thereafter, it was decided to perform the main micronucleus test with the three highest dose levels (2000, 1000 and 500 mg/kg-bw) administered in the dose-range finding acute toxicity test. Because no sex differences could be demonstrated in the dose-range finding acute toxicity test, the main micronucleus test was carried out with male mice only.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Signs of reactions to treatment were recorded from 1- 4 hours after treatment and daily thereafter. All clinical signs, observed during the performance of the study, were recorded.

At the sacrifice time of 24 hours after dosing, 5 mice treated with the vehicle control, 15 mice treated with the test substance Tetraoctylstannane (5 mice per dose-level) and 5 mice treated with the positive control substance mitomycin C, were killed by cervical dislocation. At the sacrifice time of 48 hours after dosing, 5 mice treated with the vehicle control, together with 5 mice treated with the highest dose-level of the test substance Tetraoctylstannane (2000 mg/kg-bw), were killed by cervical dislocation.


DETAILS OF SLIDE PREPARATION:
From each mouse, the bone marrow cells of both femur were immediately collected into foetal calf serum and processed into glassdrawn smears. Two bone marrow smears per animal were prepared, air-dried and fixed in methanol. One smear per animal was stained with a May-Grunwald Giemsa solution. The other smear was stored as reserve slide.

The slides were randomly coded by a person not involved in the scoring of slides. The slides (one slide per animal) were read by moving from the beginning of the smear (label end) to the leading edge in horizontal lines taking care that areas selected for evaluation were evenly distributed over the whole smear.

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.
Evaluation criteria:
The study is considered valid if the positive controls give a statistically significant increase in the mean number of MPE/2000 PE and if the negative controls are within the historical range.

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 substance 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 substance 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 substance or its metabolites are considered to have reached the general circulation and thereby the bone marrow, if the test substance statistically reduce the mean number of PE/E or causes systemic toxicity.
Statistics:
At time point 24 hours after administration, data on MPE and PE were subjected to a One Way Anova with factor group (A,B,C and D). 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 A versus treatment groups B, C and D. In addition, the positive control group E and the negative control group A were compared using pooled error variance t-tests or, if variances were not homogeneous, separate t-tests.

At time point 48 hours after administration, for treatment groups A and D, 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
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
No severe clinical signs and sex differences were observed in the dose-range finding acute toxicity test. The results of the dose-range finding acute toxicity test were reported to the sponsor. Thereafter, it was decided to perform the main micronucleus test with the three highest dose levels of the test substance Tetraoctylstannane (2000, 1000 and 500 mg/kg-bw), administered in the dose-range finding acute toxicity test and with male mice only.


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): No clinical signs were observed as a result of treatment with three dose levels (2000, 1000 and 500 mg/kg-bw) of the test substance Tetraoctylstannane.
- Induction of micronuclei (for Micronucleus assay):
- Ratio of PCE/NCE (for Micronucleus assay): The group mean numbers of MPE per 2000 PE for each group are presented in Table 1. The individual data of MPE per 2000 PE are presented in Appendix 1. The group mean numbers of PE per 200 E for each group are presented in Table 2. The individual data of PE per 200 E are presented in Appendix 1.
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 substance Tetraoctylstannane.
- Statistical evaluation:
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 with Tetraoctylstannane, up to 2000 mg/kg-bw (the limit dose level), 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 micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) was statistically significantly different (P<0.001) from the negative control A. This demonstrates the sensitivity of the test system.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
These data support the conclusion that, under the conditions used in this study, the test substance Tetraoctylstannane did not produce chromosomal damage or damage to the mitotic spindle apparatus in the bone marrow target cells of mice.
Executive summary:

The test substance Tetraoctylstannane (TTOT) [CAS# 3590-84-9] was examined for its mutagenic potential in a bone marrow micronucleus test in mice conducted in accordance with GLP to OECD Guideline 474.

The study consisted of a dose-range finding acute toxicity test carried out with male and female mice and a main micronucleus test with male mice only.

Results of the dose range finding acute toxicity test indicated that there were no sex differences in response and that a limit dose of 2000 mg/kg-bw could be tolerated. Male mice were chosen for the main study and doses of 2000, 1000 and 500 mg/kg-bw were adopted.

For the main micronucleus test, animals were treated once by gavage with three graded dose levels of the test substance Tetraoctylstannane. The high dose group (D), consisted of 10 males, and each animal received a dose of 2000 mg/kg-bw (the limit dose-level). The moderate dose group (C), consisted of 5 males, and each animal received a dose of 1000 mg/kg-bw. The low dose group (B), consisted of 5 males, and each animal received a dose of 500 mg/kg-bw. The vehicle control group (A), consisted of 10 males and each animal was dosed in a similar way with the corn-oil vehicle only. A positive control group, consisted of 5 males, and each animal was given a single intraperitoneal dose of mitomycin C at 0.75 mg/kg-bw. At 24 hours after treatment, 5 animals of each dose-level of the test substance, 5 negative control animals and 5 positive control animals, were euthanized. At 48 hours after treatment, the remaining 5 animals of group D (the high dose group) and the remaining 5 negative control animals, were euthanized. From both femurs of each animal, the bone marrow cells were collected in foetal calf serum and processed into smears for microscopic examination.

At both time points, 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 dose levels of 2000, 1000 and 500 mg/kg-bw of Tetraoctylstannane, were not statistically significantly different than the vehicle control mean. Therefore, the test substance Tetraoctylstannane, at dose-levels up to 2000 mg/kg-bw, was not genotoxic to bone marrow cells in mice.

For the mice of the positive control group, the mean number of MPE per 2000 PE was significantly (p<0.01) elevated compared to the mean of the vehicle control mice. This demonstrates the validity and sensitivity of the test system.

At 24 and 48 hours after treatment , the mean number of polychromatic erythrocytes (PE) per erythrocyte (E) in mice, at all treatment levels of Tetraoctylstannane, were not statistically significantly different from the mean of the vehicle control mice. Therefore, treatment with Tetraoctylstannane, at dose-levels up to 2000 mg/kg-bw, was not cytotoxic to the bone marrow of mice.

These data support the conclusion that, under the conditions used in this study, the test substance, Tetraoctylstannane did not produce chromosomal damage or damage to the mitotic spindle apparatus in the bone marrow target cells of mice.