Tetrabutyltin

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Tetrabutyltin

EC Number:

215-960-8

EC Name:

Tetrabutyltin

Cas Number:

1461-25-2

Molecular formula:

C16H36Sn

IUPAC Name:

tetrabutylstannane

Test material form:

liquid

Details on test material:

SOURCE: ORTEP Association Stabilizer Task Force.

Test animals

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Stated as young
- Fasting period before study: Animals were fasted prior to dosing
- Housing: All animals, except the positive controls group, were housed in sterilised cages (type I + II) fitted with a grid cover over stainless steel with a bedding of sterilised softwood chips. The positive control were housed in a laminar down-flow cabinet prior to sacrifice.
- Diet: ad libitum
- Water: Tap water ad libitum in polypropylene bottles
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hour cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle: corn oil.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing volume was 20 mL/kg bw
The dose levels 500 (group B), 1000 (group C) and 2000 (group D)  mg/kg bw were administered orally, by gavage. Just before dosing, the  animals were weighed and the test substance was dissolved and diluted in corn oil at concentrations of 25, 50 and 100 mg/mL. The dosing volume was 20 mL/kg bw.

Duration of treatment / exposure:

Animals were dosed once by gavage, after 2 hrs and 20 minutes fasting.

Frequency of treatment:

Single dose

Post exposure period:

The animals were observed for 24 or 48 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

6 males and 6 females for the dose-range finding toxicity test and 38 males in the micronucleus test.
10 males were given 20 mL/kg bw corn oil by gavage as a negative control (group A). 5 males were treated as a positive control (group E).

Control animals:

yes, concurrent vehicle

Positive control(s):

mitomycin C
- Route of administration: intra-peritoneal
- Doses / concentrations: 0.75 mg/kg bw

Examinations

Tissues and cell types examined:

Sampling times and number of samples: At the first sacrifice (24 hours after dosing) 5 vehicle control mice, 15 mice treated with the test substance (5 per dose level) and 5 mice treated with the positive control were killed by cervical  dislocation. 48 hours after dosing, 5 vehicle control mice and 5 mice treated with 2000 mg/kg bw Tetrabutylstannane were killed by cervical  dislocation.

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). 

EXAMINATIONS
- Clinical observations: Signs of reactions to treatment were recorded from 1-4 hours and at 24 and 48 h after treatment. A dose-range finding acute toxicity test was performed in order to determine the dose level(s) to be used in the main test. The limit dose of 2000 mg/kg bw was administered because the acute oral toxicity data (LD50) for mice is known to be >6000 mg/kg bw. After a fasting period of ca. 2 hours and 30 minutes, two males and two females per dose level were treated once (by gavage, dosing volume 20 mL/kg bw) with one of the three selected dose levels (500, 1000 and 2000 mg/kg bw) of the test substance. Observations with respect to all signs of reaction to the treatment were recorded 1, 4, 24 and 48 h after administration. Body weights were recorded prior to dosing (day 0) and on day 2. No clinical signs were observed during the first 24 h after exposure to the test substance. 48 hrs after administration, piloerection was observed in 1 male administered 500 mg/kg bw, both females administered 1000 mg/kg bw, and 1 female and 1 male administered 2000 mg/kg bw Tetrabutylstannane. In addition, 1 female administered 500 mg/kg bw and 1 female in each of the two higher dose groups (with piloerection) displayed a hunched back. The main study was performed with male mice only, as no significant sex differences were observed in the dose range-finding study.

Details of tissue and slide preparation:

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 second fixed smear was stored as a reserve slide. No additional organs were examined. Slides were  independently coded.

Evaluation criteria:

- Criteria for evaluating results: 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.
The study is 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 mitotic apparatus, if a clear dose related increase in the mean 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 deemed to have reached the general circulation and thereby the bone marrow, if the test substance statistically reduce the number of PE/E or causes systemic toxicity. Both statistical significance and biological relevance were considered together in the evaluation.

Statistics:

1. 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, 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 homogenous, 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 homogenous, separate t-tests.
2. 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 homogenous, 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 specified

Positive controls validity:

valid

Additional information on results:

MORTALITY: None.

CLINICAL SIGNS: None.

EFFECT ON MITOTIC INDEX OR PCE/NCE RATIO: Group means of MPE/2000 PE ± standard deviation at 24 and 48 hrs, by dose level:
Negative control: 3.0 ± 1.6 (24 h), 3.6 ± 1.5 (48 h)
500 mg/kg bw: 2.2 ± 1.1 (24 h), not determined at 48 h 1000 mg/kg bw: 3.0 ± 1.4 (24 h), not determined at 48 h
2000 mg/kg bw: 1.8 ± 1.1 (24 h), 4.4 ± 1.8 (48 h) Positive control: 55.2 ± 8.8*** (24 h), not determined at 48 h
*** P<0.001 (t-tests), group size 5.
The positive control group E was statistically significantly different from the negative control group A, at 24 h after exposure (p<0.001). Incidences of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) in mice treated with 500, 1000 or 2000 mg/kg bw Tetrabutylstannane were not statistically significantly different than the incidences found in the vehicle controls, measured 24 or 48 h after treatment. The data indicate that oral treatment with the test substance did not result in genotoxicity (determined as chromosomal damage and/or damage to the mitotic apparatus) to bone marrow cells of mice. Group means of PE/200 erythrocytes (E) ± standard deviation at 24 and 48 h, by dose level: Negative control: 88.8 ± 19.8 (24 h), 90.2 ± 6.5 (48 h) 500 mg/kg bw: 92.0 ± 17.7 (24 h), not determined at 48 h 1000 mg/kg bw: 89.6 ± 19.8 (24 h), not determined at 48 h 2000 mg/kg bw: 87.8 ± 10.0 (24 h), 82.6 ± 21.0 (48 h) Positive control: 72.8 ± 3.0 (24 h), not determined at 48 h The number of polychromatic erythrocytes (PE) per number of erythrocytes in mice treated with 500, 1000 or 2000 mg/kg bw Tetrabutylstannane was not statistically significantly different than in the vehicle control mice, measured 24 or 48 h after treatment. This indicates that the test substance was not cytotoxic to the bone marrow.

GENOTOXIC EFFECTS: No cytotoxicity was observed. The results did not indicate any chromosomal damage and or damage to the mitotic apparatus of the target cells in the bone marrow.

NOAEL (C): 2000 mg/kg bw.

STATISTICAL RESULTS: The difference in PCE/NCE ratio was not statistically significant between the negative control group and groups B, C and D at any time points. The numbers of polychromatic erythrocytes (PE) per number of (E) erythrocytes were not statistically significantly different from the numbers of polychromatic erythrocytes per number of erythrocytes found in the vehicle controls for any of the dose groups or time points.

Applicant's summary and conclusion

Conclusions:

The data indicate that oral treatment with the test substance Tetrabutylstannane did not result in genotoxicity (determined as chromosomal damage and/or damage to the mitotic apparatus) to bone marrow cells of mice.

Executive summary:

A micronucleus test was carried out in Swiss mice in accordance with the standardised guideline OECD 474 under GLP conditions.

Incidences of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) in mice treated with 500, 1000 or 2000 mg/kg bw Tetrabutylstannane were not statistically significantly different than the incidences found in the vehicle controls, measured 24 or 48 h after treatment. The data indicate that oral treatment with the test substance Tetrabutylstannane did not result in genotoxicity (determined as chromosomal damage and/or damage to the mitotic apparatus) to bone marrow cells of mice.

The number of polychromatic erythrocytes (PE) per number of erythrocytes in mice treated with 500, 1000 or 2000 mg/kg bw Tetrabutylstannane was not statistically significantly different than in the vehicle control mice, measured 24 or 48 h after treatment. This indicates that the test substance was not cytotoxic to the bone marrow.