Dibutyltin oxide

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

Study period:

10 Jun - 17 Nov 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Dibutyltin oxide

EC Number:

212-449-1

EC Name:

Dibutyltin oxide

Cas Number:

818-08-6

Molecular formula:

C8H18OSn

IUPAC Name:

dibutylstannanone

Test material form:

solid: particulate/powder

Details on test material:

Batch number: 2004RDX103
Molecular weight: 248.94
Appearance: White powder
Storage conditions: Ambient temperature (15 to 25°C), in the dark
Purity: 97.91%

Test animals

Species:

rat

Strain:

other: Crl:CD (SD) rats

Details on species / strain selection:

In line with the OECD 474, mice, rats, or another appropriate mammalian species may be used. Young adult animals (rats) are chosen for use because of the high rate of cell division in the bone marrow, the wealth of background data on this species, and their general suitability for toxicological investigations.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England
- Age at study initiation: 49 - 55 days old
- Weight at study initiation: 222 g - 255 g
- Assigned to test groups randomly: yes
- Housing: All animals were given access to small soft white untreated wood (Aspen) chew blocks and a plastic shelter for environmental enrichment
- Diet (e.g. ad libitum): free access to pelleted Envigo Teklad 2014C diet
- Water: tap water ad libitum
- Acclimation period: a minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: peanut oil
- Concentration of test material in vehicle: Suspension of the test item were prepared in peanut oil at 100, 200 and 400 mg/mL
- Amount of vehicle (if gavage or dermal): dose volume of 5 mL/kg
- Lot/batch no. (if required): The peanut oil was obtained from Acros Organics (Batch number: A0400081, expiry: 23 June 2026).

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Suspension of the test item were prepared in peanut oil. Each formulation was prepared on the day of dosing by suspending an accurate weight of test item with a measured volume of vehicle and mixed by magnetic stirring until visually homogenous.

Duration of treatment / exposure:

The test item was administered on two occasions approximately 24 hours apart.

Frequency of treatment:

The test item was administered on two occasions approximately 24 hours apart.

Post exposure period:

Animals were killed 18 -24 hours after administration of the second dose.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 000 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

0 mg/kg bw/day

Remarks:

control (peanut oil)

No. of animals per sex per dose:

6 males per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

At least 3 bone marrow smears previously prepared from rats administered Cyclophosphamide in a separate study were stained and coded along with the bone marrow smears prepared in this study.

Examinations

Tissues and cell types examined:

One femur dissected out from each animal. The femurs were cleaned of all excess tissue and blood and the head of the femur removed from each bone. The bone marrow of one femur from each animal was flushed out and pooled in a total volume of 3 mL of filtered foetal bovine calf serum by aspiration.
The resulting cell suspensions were centrifuged at 1000 rpm for 5 minutes and the supernatant discarded. The final cell pellet was resuspended in a small volume of foetal bovine calf serum to facilitate smearing in the conventional manner on glass microscope slides.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Doses were selected on the basis of a preliminary toxicity study, testing increasing dose levels of DBTO (150 up to 2000 mg/kg/day) in male and female rats (3 animals per sex/dose).
No mortalities were observed throughout the duration of the preliminary test. No substantial differences in toxicity between sexes were observed in the preliminary toxicity test, in line with current guidelines the main test was performed using male animals only.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
The oral route was chosen for this particular study as directed by the Sponsor to maximise exposure. All animals in the vehicle control and test item dose groups were dosed orally by gavage using a dose volume of 5 mL/kg. The test item was administered on two occasions approximately 24 hours apart.
Animals were inspected at least twice daily for evidence of ill-health, mortality, or reaction to treatment. Any clinical signs were recorded at the time in respect of nature and severity, date, and time of onset. Following dosing, the animals were examined regularly during the working day and any mortalities or clinical signs of reaction during the experiment were recorded. Animals from the vehicle control and test item groups were killed 18 -24 hours after administration of the second dose.

DETAILS OF SLIDE PREPARATION:
The slides were fixed in methanol and allowed to air dry. They were then rinsed in purified water and stained using an acridine orange solution at 0.0125 mg/mL and stored at room temperature in the dark until required. Prior to scoring the slides were wet mounted with coverslips using purified water.

METHOD OF ANALYSIS:
Coded slides were examined by fluorescence microscopy and 4000 polychromatic erythrocytes per animal were examined for the presence of micronuclei. One smear was examined per animal, remaining smears being held temporarily in reserve in case of technical problems with the first smear.
The proportion of polychromatic erythrocytes was assessed by examination of a total of at least 1000 erythrocytes per animal and the number of micronucleated normochromatic erythrocytes was recorded.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
a) None of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b) There is no dose-related increase at any sampling time when evaluated by an appropriate trend test.
c) All results are inside the distribution of the historical negative control data (95% confidence limits).
d) Bone marrow exposure to the test item(s) occurred.

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly positive if:
a) At least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent vehicle control.
b) This increase is dose-related at least at one sampling time when evaluated with an appropriate trend test.
c) Any of these results are outside the distribution of the historical negative control data (95% confidence limits).
There is no requirement for verification of a clear positive or clear negative response.

See further details under "any other information on materials and methods incl. tables" below.

Statistics:

For the proportion of polychromatic erythrocytes, an asymptotic one-tailed Jonckheere’s test for trend (Jonckheere 1954) with “step-down” was used on Groups 1 to 4 for a decrease from control. If significant, then the analysis was carried out on Groups 1 to 3. Exact one-tailed Wilcoxon pairwise tests (Wilcoxon 1945), for a decrease from control, were also carried out on Group 1 (control) versus Groups 2, 3, 4 and 5 (positive control).
For incidences of micronucleated polychromatic erythrocytes, an exact one-tailed Linear-by- Linear association test (Cytel 1995) with “step-down” was used on Groups 1 to 4 for an increase from control. If significant, then the analysis was carried out on Groups 1 to 3. Exact one-tailed pairwise Permutation tests (Cytel 1995), for an increase from control, were also carried out on Group 1 (control) versus Groups 2, 3, 4 and 5 (positive control).
If the exact version of a test could not be calculated (due to the amount of data), then the asymptotic version was used instead.
Statistical significance was declared at the 5% level for all tests.
The data were received in an Excel document and analyzed using SAS (SAS Institute Inc. 2002) (Jonckheere's and Wilcoxon tests) and StatXact (Cytel 1995) (Linear-by-Linear and Permutation tests).

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

clinical signs of toxicity were observed at all dose levels, overall group mean bw losses of 9.5%, 8.2% and 7.9% were observed in animals administered DBTO at 500, 1000 and 2000 mg/kg/day respectively (Day 1 to termination); no bw loss in control animals

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 150, 300, 500, 800, 1600, 2000 mg/kg bw/day
- Solubility: Suspension of the test item were prepared in peanut oil
- Clinical signs of toxicity in test animals:
To determine suitable dose levels for use in the main test, one group consisting of three male and three female animals were scheduled to be administered Dibutyltin Oxide (DBTO) at 150 mg/kg/day on two consecutive days approximately 24 hours apart.
At 150 mg/kg/day, clinical signs of toxicity observed in male animals included elevated gait, pilo erection, hunched posture and wet fur. All animals survived until scheduled termination; a group mean body weight loss of 7.9% was observed (Day 1 to scheduled termination).
At 150 mg/kg/day, clinical signs of toxicity observed in female animals included elevated gait and pilo erection. All animals survived until scheduled termination; a group mean body weight loss of 7.1% was observed (Day 1 to scheduled termination).
On the basis of this result, 150 mg/kg/day had clearly not exceeded the MTD in either male or female animals. A subsequent group of three male and three female animals were therefore administered Dibutyltin Oxide (DBTO) at 300 mg/kg/day on two consecutive days approximately 24 hours apart.
At 300 mg/kg/day, clinical signs of toxicity observed in male animals included pilo erection, loose faeces and wet fur. All animals survived until scheduled termination; a group mean body weight loss of 6.2% was observed (Day 1 to scheduled termination).
At 300 mg/kg/day, clinical signs of toxicity observed in female animals included pilo erection, elevated gait and loose faeces. All animals survived until scheduled termination; a group mean body weight loss of 3.0% was observed (Day 1 to scheduled termination).
On the basis of this result, 300 mg/kg/day had clearly not exceeded the MTD in either male or female animals. A subsequent group of three male and three female animals were therefore administered Dibutyltin Oxide (DBTO) at 500 mg/kg/day on two consecutive days approximately 24 hours apart.
At 500 mg/kg/day, clinical signs of toxicity observed in male animals included pilo erection, hunched posture, elevated gait, loose faeces and wet fur. All animals survived until scheduled termination; a group mean body weight loss of 5.5% was observed (Day 1 to scheduled termination).
At 500 mg/kg/day, clinical signs of toxicity observed in female animals included pilo erection, wet fur, hunched posture, elevated gait and loose faeces. All animals survived until scheduled termination; a group mean body weight loss of 5.0% was observed (Day 1 to scheduled termination).
On the basis of this result, 500 mg/kg/day had clearly not exceeded the MTD in either male or female animals. A subsequent group of three male and three female animals were therefore administered Dibutyltin Oxide (DBTO) at 800 mg/kg/day on two consecutive days approximately 24 hours apart.
At 800 mg/kg/day, clinical signs of toxicity observed in male and female animals included pilo erection and loose faeces. All animals survived until scheduled termination; group mean body weight losses of 4.1 and 5.0% were observed in male and female animals respectively (Day 1 to scheduled termination). It was noted during euthanisation of the animals at scheduled termination that all animals had a firm area in the mid-ventral region which was believed to be a firm stomach.
On the basis of this result, 800 mg/kg/day had clearly not exceeded the MTD in either male or female animals. A subsequent group of three male and three female animals were therefore administered Dibutyltin Oxide (DBTO) at 1600 mg/kg/day on two consecutive days approximately 24 hours apart.
At 1600 mg/kg/day, clinical signs of toxicity observed in male animals included pilo erection, elevated gait, decreased activity, loose faeces and hunched posture. All animals survived until scheduled termination; a group mean body weight loss of 6.9% was observed (Day 1 to scheduled termination).
At 1600 mg/kg/day, clinical signs of toxicity observed in female animals included pilo erection, elevated gait and loose faeces. All animals survived until scheduled termination; a group mean body weight loss of 6.6% was observed (Day 1 to scheduled termination).
On the basis of this result, 1600 mg/kg/day had clearly not exceeded the MTD in either male or female animals. A subsequent group of three male and three female animals were therefore administered Dibutyltin Oxide (DBTO) at 2000 mg/kg/day (the standard test limit) on two consecutive days approximately 24 hours apart.
At 2000 mg/kg/day, clinical signs of toxicity observed in male animals included pilo erection, loose faeces, wet fur, elevated gait and salivation. All animals survived until scheduled termination; a group mean body weight loss of 6.8% was observed (Day 1 to scheduled termination).
At 2000 mg/kg/day, clinical signs of toxicity observed in female animals included fast breathing, pilo erection, wet fur, elevated gait and loose faeces. All animals survived until scheduled termination; a group mean body weight loss of 5.1% was observed (Day 1 to scheduled termination).
On the basis of this result, the standard test limit of 2000 mg/kg/day, was considered to be the maximum dose in both male and female animals.
No substantial differences in toxicity were observed between the sexes, therefore, in line with current guidelines the main test was performed using male animals only, dose levels of 500, 1000 and 2000 mg/kg/day were selected.
- Rationale for exposure:
The preliminary toxicity test was performed to identify the maximum tolerated dose (MTD), defined as the highest dose that will be tolerated without evidence of study-limiting toxicity, relative to the duration of the study period (for example, by inducing body weight depression or hematopoietic system cytotoxicity, but not death or evidence of pain, suffering or distress necessitating humane euthanasia) up to a standard limit of 2000 mg/kg/day.


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): There were no statistically significant increases or trend observed in the group mean MPCE of male Crl:CD (SD) rats administered Dibutyltin Oxide (DBTO) at 500, 1000 and 2000 mg/kg/day, compared to the concurrent control. The individual and group mean %MPCE values from all groups were within the current vehicle historical control range (95% confidence limits).
There were no substantial increases in micronucleated normochromatic erythrocytes observed over the duration of the study.
- Ratio of PCE/NCE (for Micronucleus assay):
There were no statistically significant decreases in %PCE observed in male Crl:CD (SD) rats administered Dibutyltin Oxide (DBTO) at 500 mg/kg/day, compared to the concurrent vehicle control. The group mean %PCE value was within the current vehicle historical control range (95% confidence limits).
Statistically significant decreases in %PCE were observed in male Crl: CD(SD) rats administered Dibutyltin Oxide (DBTO) at 1000 and 2000 mg/kg/day, compared to the concurrent control (p<0.01). There was also a statistically significant trend observed from Groups 1 to 4 (p<0.001), this continued to be statistically significant following the exclusion of Group 4 (p<0.01). The group mean %PCE values from both groups fell outside of the current vehicle historical control range (95% confidence limits). As a depression in the immature erythrocyte ratio was observed, there is considered to be evidence that exposure to the bone marrow had occurred.

Applicant's summary and conclusion

Conclusions:

It is concluded that Dibutyltin Oxide (DBTO) did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes in male Crl:CD (SD) rats when administered orally by gavage in this in vivo test procedure.

Executive summary:

This study was conducted according OECD 474 and GLP and designed to assess the potential induction of micronuclei by Dibutyltin Oxide (DBTO) in the bone marrow cells of Crl:CD (SD) rats.

Animals were treated with Dibutyltin Oxide (DBTO) orally by gavage on two occasions approximately 24 hours apart using a dose volume of 5 mL/kg. The vehicle control group received peanut oil.

On the basis of results from a preliminary toxicity test, dose levels of 500, 1000, 2000 mg/kg/day were selected for the micronucleus test. No substantial differences in toxicity between sexes were observed in the preliminary toxicity test, in line with current guidelines the main test was performed using male animals only.

Bone marrow smears were obtained from animals in the vehicle control and in each of the test item groups 18 - 24 hours after administration of the second dose.

In addition, slides prepared from a separate study (Study Number: MV12VK) from animals treated with Cyclophosphamide (CPA), a well characterised clastogen, were stained and coded along with the bone marrow smears prepared from this study.

One smear from each animal was examined for the presence of micronuclei in 4000 polychromatic erythrocytes. The proportion of polychromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated normochromatic erythrocytes was also kept.

Results

The data for the concurrent vehicle control (group mean % polychromatic erythrocytes [%PCE] and % micronucleated polychromatic erythrocytes [%MPCE]) were within the ranges determined by the laboratory historical data (95% confidence limits). The coded positive control slides prepared from Covance study MV12VK demonstrated the ability of the analyst to detect increases in micronucleated polychromatic erythrocytes and there was a statistically significant increase compared to the vehicle control (p<0.05).

There were no statistically significant increases or trend observed in the group mean MPCE of male Crl:CD (SD) rats administered Dibutyltin Oxide (DBTO) at 500, 1000 and 2000 mg/kg/day, compared to the concurrent control. The individual and group mean %MPCE values from all groups were within the current vehicle historical control range (95% confidence limits). Therefore, there was considered to be no increase in the induction of micronucleated polychromatic erythrocytes following exposure to Dibutyltin Oxide (DBTO) up to the maximum tolerated dose.

There were no statistically significant decreases in %PCE observed in male Crl:CD (SD) rats administered Dibutyltin Oxide (DBTO) at 500 mg/kg/day, compared to the concurrent vehicle control. The group mean %PCE value was within the current vehicle historical control range (95% confidence limits).

Statistically significant decreases in %PCE were observed in male Crl: CD(SD) rats administered Dibutyltin Oxide (DBTO) at 1000 and 2000 mg/kg/day, compared to the concurrent control (p<0.01). There was also a statistically significant trend observed from Groups 1 to 4 (p<0.001), this continued to be statistically significant following the exclusion of Group 4 (p<0.01). The group mean %PCE values from both groups fell outside of the current vehicle historical control range (95% confidence limits). As a depression in the polychromatic erythrocyte ratio was observed, there is considered to be evidence that exposure to the bone marrow had occurred.

Conclusion

It is concluded that Dibutyltin Oxide (DBTO) did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes in male Crl:CD (SD) rats when administered orally by gavage in this in vivo test procedure.