Dibutyltin oxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

IN VITRO GENE MUTATION STUDY IN BACTERIA

Krul (2002): Bacterial reverse mutation test with DBTO (OECD 471, GLP), Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, E. coli strain WP2 uvrA, with and without metabolic activation (+/- S9): negative

 

IN VITRO CYTOGENICITY STUDY IN MAMMALIAN CELLS/MICRONUCLEUS STUDY

Morris (2022): in vitro Micronucleus test with DBTO (OECD 487, GLP), normal human primary lymphocytes, with and without metabolic activation (+/- S9); 4-h +S9: negative, 4-h -S9: equivocal, 24-h -S9: positive

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Dec 2020 - 10 Mar 2022 (date of report amendment)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 29 July 2016.

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: normal (primary) human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
For lymphocytes:
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore, using this average, the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
The details of the donors used are:
Preliminary Toxicity Test: male, aged 34 years
Preliminary Toxicity Test Repeat: female, aged 29 years
Main Experiment: female, aged 25 years
Main Experiment Repeat I: male, aged 34 years
- Whether whole blood or separated lymphocytes were used: The lymphocytes of fresh heparinised whole blood were stimulated
- Whether blood from different donors were pooled or not: blood was not pooled from different donors
- Mitogen used for lymphocytes: phytohaemagglutinin (PHA)

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
incubation at approximately 37 ºC, 5% CO2 in humidified air; culture medium:
9.3 - 9.4 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.40 – 0.50 mL heparinised whole blood

Cytokinesis block (if used):

Cultures were supplemented with Cytochalasin B, at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: S9 Microsomal Enzyme Fraction
- source of S9: purchased from Moltox and Lot no 4272 with the expiry date of 16 July 2022, and Lot no 4217 with the expiry date of 05 March 2022
- method of preparation of S9 mix: The protein content was adjusted to approximately 20 mg/mL prior to use.
The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM).
- concentration or volume of S9 mix and S9 in the final culture medium: The final concentration of S9, when dosed at a 10% volume (1 mL) of S9-mix into culture media, was 2%.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): S9 was pre-tested for acceptability by the supplier

Test concentrations with justification for top dose:

0, 0.125, 0.25, 0.5, 0.75, 1, 1.5, 2 µg/mL for 24-h without S9 and up to 4 μg/mL in the 4-h exposure experiments with and without S9.
The selection of the maximum concentration for the Main Experiment was based on test item-induced toxicity in all three of the exposure groups.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble in culture medium at 20, 10, and 5 mg/mL, in DMSO at 200, 100, and 50 mg/mL, in tetrahydrofuran at 200 mg/mL, and in acetone at 200, 100, and 50 mg/mL. A suspension suitable for dosing was formed at 25 mg/mL in DMSO and therefore the maximum practical concentration was 250 μg/mL. DMSO was selected as the solvent and prior to each experiment, the test item was accurately weighed, suspended in DMSO and serial dilutions prepared.

- Justification for percentage of solvent in the final culture medium: in line with the OECD TG, DMSO did not exceed 1% (v/v) in the final culture medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcine

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Duplicate lymphocyte cultures (A and B), (quadruplicate for the solvent) were established for each concentration
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in suspension (prior to each experiment, the test item was accurately weighed, suspended in DMSO and serial dilutions prepared)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 44 to 48 hours (lymphocyte cultures alone, without test material)
- Exposure duration/duration of treatment: 4 hours with or without S9 or 24 hours without S9
- Harvest time after the end of treatment (sampling/recovery times): After removal of the test material, cultures were incubated with CytoB for further 24 hours. At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: At the end of the incubation period, the cells were washed and Cytochalasin B was added at a final concentration of 4.5 μg/mL, and then the cells were incubated for a further 24 hours.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry with gentle warming. Each slide was permanently labeled with the appropriate identification data. When the slides were dry, they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a percentage of the solvent controls.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): The micronucleus frequency in 1000 binucleated cells was analysed per culture (2000 binucleated cells per concentration for the test item and positive control and 4000 binucleated cells for the solvent controls). Cells with 1, 2 or more micronuclei were recorded and included in the total. Following consultation with the Sponsor, an additional 1000 cells were scored for the vehicle control and test item dose levels in the 4 hour exposure groups in the absence and presence of metabolic activation due to the increases observed after the first 1000 binucleate cells were scored. Scoring additional cells increases the statistical power of the data and is considered to be acceptable under the OECD 487 Guideline. Experiments with human lymphocytes have established a range of micronucleus frequencies acceptable for control cultures in normal volunteer donors. The criteria for identifying micronuclei were that they were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): Although, fluorescence in situ hybridisation (FISH) with a centromeric DNA probe to determine the origin of the micronuclei in the 24-hour exposure group was an option it was not considered beneficial for this study due to the relatively low numbers of micronuclei available for assessment.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index (CBPI)
- Any supplementary information relevant to cytotoxicity: A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a percentage of the solvent controls. The CBPI indicates the number of cell cycles per cell during the period of exposure to Cytochalasin B. It was used to calculate cytostasis by the following formula: % Cytostasis = 100 - 100{(CBPIt – 1) / (CBPIc – 1)}
Where: CBPI: (No. mononucleate cells + (2x No. binucleate cells) + (3x No. multinucleate cells)) / Total number of cells
t = test chemical treatment culture c = solvent control culture

Rationale for test conditions:

Study design and concentrations were selected based on the preliminary toxicity test and in line with the OECD TG

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase when evaluated with an appropriate trend test.
3. The results in all evaluated dose groups are within the range of the laboratory historical control data.
The test item is then considered to be unable to induce chromosome breaks and/or gain or loss in this test system.
Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, the following are applicable:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. The increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test.
3. The results are substantially outside the range of the laboratory historical negative control data.
When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system. There is no requirement for verification of a clear positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. The Study Director may make a judgement based on experience and the biological relevance of the data and any justification for acceptance of the data will be included in the report.

Statistics:

The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent solvent control value using the Chi-squared Test on observed numbers of cells with micronuclei. A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei.
The dose-relationship (trend-test) was assessed using a linear regression model. An arcsin square-root transformation was applied to the percentage of binucleated cells containing micronuclei (excluding positive controls). A linear regression model was then applied to these transformed values with dose values fitted as the explanatory variable. The F-value from the model was assessed at the 5% statistical significance level.

Species / strain:

lymphocytes: normal human (primary) lymphocytes

Metabolic activation:

without

Genotoxicity:

ambiguous

Remarks:

after 4 hours

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: normal human (primary) lymphocytes

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

after 24 hours

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

lymphocytes: normal human (primary) lymphocytes

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH/osmolality: There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm. Please see any other information on results incl. tables below.
- Precipitation and time of the determination: No precipitate of test item was observed in any of the three exposure groups at the end of the exposure period.

RANGE-FINDING/SCREENING STUDIES (if applicable):
The Preliminary Toxicity test was initially performed with a dose range of 0.98 to 250 μg/mL where the maximum dose level was the maximum practical concentration. There were no surviving cells at any of the dose levels in this experiment due to excessive toxicity and it was therefore repeated with a lower dose range. The concentrations used for the repeat of the Preliminary Toxicity Test were 0, 0.016, 0.031, 0.063, 0.125, 0.25, 0.5, 1, 2, and 4 μg/mL. The maximum dose was limited by test item-induced toxicity.
No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure periods in any of the three exposure groups.
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 2 μg/mL in the 4-hour exposure groups, both in the absence and presence of metabolic activation. The maximum dose with binucleate cells present in the 24-hour continuous exposure was 1 μg/mL.
The test item induced evidence of marked toxicity in all three of the exposure groups.
The selection of the maximum concentration for the Main Experiment was based on test item-induced toxicity in all three of the exposure groups.

STUDY RESULTS
- Concurrent vehicle negative and positive control data:
The solvent control cultures had frequencies of cells with micronuclei within the expected range and were considered acceptable for addition to the laboratory historical negative control data range.
The positive control items induced statistically significant increases in the frequency of cells with micronuclei with responses that were compatible with those in the laboratory historical positive control data range. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

HISTORICAL CONTROL DATA: Please see any other information on results incl. tables below

The pH and osmolality readings are presented in the following table:

Concentration (μg/mL)	0	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250
pH	7.39	7.37	7.36	7.38	7.39	7.40	7.41	7.41	7.40	7.41
Osmolality mOsm	483	491	-	493	-	490	490	491	488	489

- = Not performed for this concentration

The test item was formulated within two hours of it being applied to the test system; it is assumed that the test item formulation was stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines.

Conclusions:

The test item, Dibutyltin Oxide (DBTO), did not demonstrate any statistically significant increases in the frequency of binucleate cells with micronuclei in the 4-hour exposure group in the absence of a metabolising system. The result in the 4-hour exposure group in the presence of a metabolising system was considered to be equivocal due to small but statistically significant increases in the frequency of micronuclei. In the 24-hour exposure group in the absence of a metabolising system statistically significant increases in the frequency of binucleate cells with micronuclei were demonstrated. The test item was therefore considered to be clastogenic and/ or aneugenic to human lymphocytes in vitro.

Executive summary:

This study was conducted according to OECD 487 and in compliance with GLP and includes the results of the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes.

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at up to four concentrations, together with solvent (quadruplicate cultures) and positive controls (duplicate cultures). Three exposure conditions were used for the study using a 4-hour exposure in the presence and absence of a standard metabolising system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.
The concentrations used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited by test item induced-toxicity. The concentrations selected for the Main Experiment and subsequent repeat of the 24-hour exposure group were as follows:

 

Main Experiment

4-hour without S9: 0, 0.125, 0.25, 0.5, 0.75, 1, 1.5, 2, 4 µg/mL DBTO

4-hour with S9 (2%): 0, 0.125, 0.25, 0.5, 0.75, 1, 1.5, 2, 4 µg/mL DBTO

24-hour without S9: 0, 0.125, 0.25, 0.5, 0.75, 1, 1.5, 2 µg/mL DBTO

 

Main Experiment Repeat I

24-hour without S9: 0, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.75, 1, 1.5, 2 µg/mL DBTO

 

Results

All solvent (DMSO) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes and were considered acceptable for addition to the laboratory historical negative control data range.
The positive control items induced statistically significant increases in the frequency of cells with micronuclei with responses that are compatible with those in the laboratory historical positive control data range. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei in the 4-hour exposure group in the absence of metabolic activation. The results were within the distribution of the historical solvent data (within 95% control limits), and there was no statistically significant concentration related increase when evaluated with a trend test.
The test item induced a statistically small but significant increase in the frequency of binucleate cells with micronuclei in the 4-hour exposure group in the presence of metabolic activation, at the maximum dose level scored (0.75 μg/mL) and there was also a statistically significant concentration related increase when evaluated with a trend test. However, the results were within the distribution of the historical solvent data (within 95% control limits). The response was therefore considered to be equivocal.
The test item induced some small but statistically significant increases in the frequency of binucleate cells with micronuclei in the 24-hour exposure group in the absence of metabolic activation in the initial experiment. However, although the responses observed were within the historical control range for a vehicle (within 95% control limits), the 0.5 μg/mL concentration exceeded the upper limit of acceptable toxicity and therefore the toxicological significance of this equivocal finding was unclear. Therefore, the 24-hour exposure group was repeated using a refined range of treatment concentrations. In the repeat experiment, statistically significant increases in the frequency of binucleate cells were observed at 0.4 μg/mL and 0.5 μg/mL. There was also a statistically significant concentration related increase when evaluated with a trend test and the results were also outside the distribution of the historical solvent data (outside 95% control limits). Therefore, the criteria for a positive result have been met and the test item is considered to be weakly clastogenic and/or aneugenic.

 

Conclusion

The test item, Dibutyltin Oxide (DBTO), did not demonstrate any statistically significant increases in the frequency of binucleate cells with micronuclei in the 4-hour exposure group in the absence of a metabolising system. The result in the 4-hour exposure group in the presence of a metabolising system was considered to be equivocal due to small but statistically significant increases in the frequency of micronuclei. In the 24-hour exposure group in the absence of a metabolising system statistically significant increases in the frequency of binucleate cells with micronuclei were demonstrated. The test item was therefore considered to be clastogenic and/ or aneugenic to human lymphocytes in vitro.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 5-18, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

The test substance was stored at ,-20 degrees Celsius, instead of between -16 and -20 degrees celsius. However, it was stated in the test substance information sheet that the storage conditions should be <-18 degrees Celsius, therefore the deviation is c

Principles of method if other than guideline:

No further information required.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

rfa, uvrB/A,and R-factor

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate (S9-mix)

Test concentrations with justification for top dose:

Assay 1: 7, 21, 62, 185, 556, 1667, 5000 µg/plate
Assay 2: 1.25, 2.5, 5, 10, 20 µg/plate

Vehicle / solvent:

Methanol

Untreated negative controls:

yes

Remarks:

solvent

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

For strain TA1535 and strain TA100 in the absence of the S9-mix

Untreated negative controls:

yes

Remarks:

solvent

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

For strain TA1537 in the absence of the S9-mix

Untreated negative controls:

yes

Remarks:

solvent

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

For strain TA1535, TA98, TA100, and WP 2 uvrA in the presence of the S9-mix

Untreated negative controls:

yes

Remarks:

solvent

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

For strain TA98 in the absence of the S9-mix

Untreated negative controls:

yes

Remarks:

solvent

Positive controls:

yes

Positive control substance:

other: N-ethyl-N-nitrosourea

Remarks:

For strain WP 2 uvrA in the absence of the S9-mix

Untreated negative controls:

yes

Remarks:

solvent

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

For strain TA1537 in the presence of the S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 37 °C for 48-72 hours

Evaluation criteria:

The mutagenicity study is considered valid if the mean colony counts of the control values of the strains are within acceptable ranges, if the results of the positive controls meet the criteria for a positive response, and if no more than 5% of the plates are lost through contamination of other unforeseen events.

A test substance is considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plates is concentration-related increased or if a reproducible two-fold or more increase is observed compared to that on the negative control plates.

A test substance is considered to be negative in the bacterial gene mutation test if it produces neither a dose-related increase in the mean number of revertant colonies nor a reproducivle positive response at any of the test points.

Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base substitutions or frameshifts in the genome of either Salmonella typhimurium and/or E. coli. Negative results indicate that under the test conditions, the test substance is not mutagenic in the tested strains.

In case of an inconclusive first assay, a second independant assay was conducted. The first mutagenicity assay is regarded inconclusive if a positive or equivocal response at only one concentrations is observed or if a positive or equivocal responses at several concentrations without a concentration-related increase are observed.

Statistics:

No statistical analysis was performed. Both numerical significance and biological relevance are considered together in the evaluation.

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Genotoxic effects: negative both with and without metabolic action.
In both the absence and presence of S9-mix and in all strains, dibutyloxostannane did not cause a more than two-fold or a dose-related increase in the mean number of revertant colonies appearing in the test plates compared to the background spontaneous reversion rate observed with the negative control.

The mean number of his+ and trp+ revertant colonies of the negative controls were within the acceptable range, and the positive controls gave the expected increase in the mean number of revertant colonies.

Table 1. Bacterial reverse mutation test with dibutyloxostannane (first assay)
	Average number of revertants per plate
Dose ug/plate	TA 1535		TA1537		TA98		TA 100		E-coli
	without S9	with S9	without S9	with S9	without S9	with S9	without S9	with S9	without S9	with S9
0	25	24	17	12	35	51	168	171	37	40
7	17	17	20	12	34	49	155	169	34	39
21	11	15	5	5	23	29	109	105	26	25
62	0	0	0	0	0	0	0	0	24	13
185	0	0	0	0	0	0	0	0	11	5
556	0	0	0	0	0	0	0	0	0	0
1667	0	0	0	0	0	0	0	0	0	0
5000	0	0	0	0	0	0	0	0	0	0
Positive control	713	525	1220	348	2078	810	886	2013	228	1720

Table 2. Bacterial reverse mutation test with dibutyloxostannane (second assay)
	Average number of revertants per plate
Dose ug/plate	TA 1535		TA1537		TA98		TA 100		E-coli
	without S9	with S9	without S9	with S9	without S9	with S9	without S9	with S9	without S9	with S9
0	26	24	12	15	25	35	153	161	21	27
1.25	19	23	13	14	25	38	174	169	25	26
2.5	11	17	5	9	20	31	142	180	25	26
5	16	21	9	8	23	35	153	174	25	21
10	13	17	6	6	20	35	132	138	21	32
20	10	12	3	4	16	18	95	95	23	22
Positive control	489	428	811	295	1170	965	509	1635	195	1045

Conclusions:

It is concluded that the results obtained with the test substance in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and in the E. coli strain WP2 uvrA, in both the absence and presence of the S9-mix, indicate that dibutyloxostannane was not mutagenic under the conditions employed in this study.

Executive summary:

Dibutyloxostannane was examined for mutagenic activity in the bacterial reverse mutation test using the histidine-requiring Salmonella typhimurium strains TA 1535, 1537, 98 and 100 and the tryptophan-requiring E. coli strain WP2 uvrA, and a liver fraction of Aroclor 254 -induced rats from metabolic activation (S9 -mix). In both the absence and the presence of S9 -mix and in all strains, dibutyloxostanane did not cause a more than two-fold or a dose-related increase in the mean number of revertant colonies appearing in the test plates compared to the background spontaneous reversion rate observed with the negative control. The mean number of his+ and trp+ revertant colonies of the negative controls were within the acceptable range, and the positive controls gave the expected increase in the mean number of revertant colonies. It is concluded that dibutyloxostannane was not mutagenic under the conditions employed in this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

IN VIVO MAMMALIAN SOMATIC CELL STUDY - CYTOGENICITY/ERYTHROCYTE MICRONUCLEUS STUDY

Best (2021): in vivo Micronucleus test with DBTO (OECD 474, GLP), Crl:CD (SD) rats, 500, 1000, 2000 mg/kg bw in peanut oil, administered twice via oral gavage approx. 24 h apart: negative

Link to relevant study records

Reference

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

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

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

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.

Dose / conc.:

2 000 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

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.

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).

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

IN VITRO GENE MUTATION STUDY IN BACTERIA

This study was performed according to the OECD 471 and under GLP compliance. Dibutyloxostannane was examined for mutagenic activity in the bacterial reverse mutation test using the histidine-requiring Salmonella typhimurium strains TA 1535, 1537, 98 and 100 and the tryptophan-requiring E. coli strain WP2 uvrA, and a liver fraction of Aroclor 254 -induced rats from metabolic activation (S9 -mix). In both the absence and the presence of S9 -mix and in all strains, dibutyloxostanane did not cause a more than two-fold or a dose-related increase in the mean number of revertant colonies appearing in the test plates compared to the background spontaneous reversion rate observed with the negative control. The mean number of his+ and trp+ revertant colonies of the negative controls were within the acceptable range, and the positive controls gave the expected increase in the mean number of revertant colonies. It is concluded that dibutyloxostannane was not mutagenic under the conditions employed in this study.

 

IN VITRO CYTOGENICITY STUDY IN MAMMALIAN CELLS/MICRONUCLEUS STUDY

This study was conducted according to OECD 487 and in compliance with GLP and includes the results of the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes.

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at up to four concentrations, together with solvent (quadruplicate cultures) and positive controls (duplicate cultures). Three exposure conditions were used for the study using a 4-hour exposure in the presence and absence of a standard metabolising system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.
The concentrations used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited by test item induced-toxicity. The concentrations selected for the Main Experiment and subsequent repeat of the 24-hour exposure group were as follows:

Main Experiment

4-hour without S9: 0, 0.125, 0.25, 0.5, 0.75, 1, 1.5, 2, 4 µg/mL DBTO

4-hour with S9 (2%): 0, 0.125, 0.25, 0.5, 0.75, 1, 1.5, 2, 4 µg/mL DBTO

24-hour without S9: 0, 0.125, 0.25, 0.5, 0.75, 1, 1.5, 2 µg/mL DBTO

Main Experiment Repeat I

24-hour without S9: 0, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.75, 1, 1.5, 2 µg/mL DBTO

Results

All solvent (DMSO) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes and were considered acceptable for addition to the laboratory historical negative control data range.
The positive control items induced statistically significant increases in the frequency of cells with micronuclei with responses that are compatible with those in the laboratory historical positive control data range. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei in the 4-hour exposure group in the absence of metabolic activation. The results were within the distribution of the historical solvent data (within 95% control limits), and there was no statistically significant concentration related increase when evaluated with a trend test.
The test item induced a statistically small but significant increase in the frequency of binucleate cells with micronuclei in the 4-hour exposure group in the presence of metabolic activation, at the maximum dose level scored (0.75 μg/mL) and there was also a statistically significant concentration related increase when evaluated with a trend test. However, the results were within the distribution of the historical solvent data (within 95% control limits). The response was therefore considered to be equivocal.
The test item induced some small but statistically significant increases in the frequency of binucleate cells with micronuclei in the 24-hour exposure group in the absence of metabolic activation in the initial experiment. However, although the responses observed were within the historical control range for a vehicle (within 95% control limits), the 0.5 μg/mL concentration exceeded the upper limit of acceptable toxicity and therefore the toxicological significance of this equivocal finding was unclear. Therefore, the 24-hour exposure group was repeated using a refined range of treatment concentrations. In the repeat experiment, statistically significant increases in the frequency of binucleate cells were observed at 0.4 μg/mL and 0.5 μg/mL. There was also a statistically significant concentration related increase when evaluated with a trend test and the results were also outside the distribution of the historical solvent data (outside 95% control limits). Therefore, the criteria for a positive result have been met and the test item is considered to be weakly clastogenic and/or aneugenic.

Conclusion

The test item, Dibutyltin Oxide (DBTO), did not demonstrate any statistically significant increases in the frequency of binucleate cells with micronuclei in the 4-hour exposure group in the absence of a metabolising system. The result in the 4-hour exposure group in the presence of a metabolising system was considered to be equivocal due to small but statistically significant increases in the frequency of micronuclei. In the 24-hour exposure group in the absence of a metabolising system statistically significant increases in the frequency of binucleate cells with micronuclei were demonstrated. The test item was therefore considered to be clastogenic and/ or aneugenic to human lymphocytes in vitro.

 

IN VIVO MAMMALIAN CELL STUDY - CYTOGENICITY/ERYTHROCYTEMICRONUCLEUS STUDY

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.

 

Justification for classification or non-classification

Based on the above mentioned study results, classification of the registered substance DBTO for germ cell mutagenicity is not warranted according to Regulation (EC) No 1272/2008.