Dibutyltin oxide

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

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

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%

Method

Species / strain

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

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.