Bis(tributyltin) oxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Bis(tributyltin) oxide

EC Number:

200-268-0

EC Name:

Bis(tributyltin) oxide

Cas Number:

56-35-9

Molecular formula:

C24H54OSn2

IUPAC Name:

tributyl[(tributylstannyl)oxy]stannane

Details on test material:

Chem. Bez. / Chemical name (lupac)
Bis (tri-n-butyltin) oxide
Synonyms: Hexa-n-butyldistannoxane, Tri-n-butyltin oxide (TBTO)
Manufacturer: Schering Bergkamen
Product No. 2288
U. No: 86/2245
Stability: at least 1.5 yr in tightly closed light proof bottle, stabilized against destabilizer (0.1% stabilize, identity disclosed upon request)
Product report dated 21May1986
Solutions prepared immeidately before start of test
Sovent: DMSO
Structure: (n-C4H9)3Sn-O-Sn(n-C4H9)3

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Whole blood cultures were prepared by adding 0.4 mL heparinzed blood, obtained by venipuncture from healthy adult male to 4.67 mL of cluture medium containing McCoy's 5a medium, 15% fetal calf serum, antibiotics, and PHA; incubated with loose caps at 37°C in humidified incubator (c. 5% CO2).

Metabolic activation:

with and without

Metabolic activation system:

S9 from sprague dawley rats treated with Aroclor (Litton Bionetics)

Test concentrations with justification for top dose:

Without metabolic activation: 0.0025. 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5 and 1 µg/mL
Positive control: triaziquone 5x10-8M
With metabolic activation: 0.01, 0.025, 0.05. 0.1, 0.25, 0.5, 1.0, 2.5, 5 µg/mL
Positive control: cyclophosphamide, 2x10-5 M

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO 50 µL

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: cyclophosphamide (with activation); triaziquone (without activation)

Details on test system and experimental conditions:

Without activation: Test material (50 µL), positive control (triziqone, 5x10-8 M), or solvent control (DMSO, 50 µL) was added to respective whole blood cultures approximately 22 h after culture initiation/stimulation for a further incubation period of 31 h. Duplicate cultures prepared for each concentration and controls.
With activation: Test material (60 µL), positive control (cyclophosphamide, 2x10-5 M), or solvent control (60 µL) were added to whole blood cultures approximately 26 h after culture initiation. Immediately after, 1 mL of freshly prepared S9 mix was added to a 5 mL blood culture to give a final concentration of 2%.
After 3 h incubation at 37°C, the cells were washed 3 times with McCoy’s 5a culture medium and re-incubated for approximately 22 h. For evaluation of cell cycle kinetics, paralleled blood samples were treated the same with except that BrdU was added after treatment. Duplicate cultures performed. To arrest cells in the metaphase, colcemide (0.08 µg/mL) was added 3.5 h before harvest. After cells were collected by centrifuge they were exposed to hypotonic solution and fixed in glacial acetic acid/methanol. Drops of concentrated cell suspension placed on slides, air dried then stained with Giemsa (10%) and mounted with Eukitt the following day.
For assessment of cell cycle kinetics, the slides were stained by a modified fluorescence-plus-Giemsa technique (FPG-technique). The slides were stained for 15 minutes with Hoechst 33258 (final concentration 4.5 µg/mL) in phosphate buffer (pH 6.9), mounted in the same buffer and exposed at 60°C to "black-light-blue" UV from 2x20 Watt tubes (Philips, Type TL 20W/08,F20T12BLB) for the time required for differentiation between chromatids (15 - 30 min). Finally, slides were stained with 5 % Giemsa for 4-8 min and air dried. After drying the slides were mounted with Eukitt the following day.

Evaluation criteria:

100 cells per replicate were scored if possible (200 cells per concentration level, 100 cells for the positive control). Sides were scored "blind" using a photomicroscope with attached TV with 1000X magnification. Only metaphases with 2n = 46 chromosomes were included in the analysis. All metaphase spreads were examined for both chromatid and chromosome aberrations, notably achromatic lesions (gaps), breaks, acentric fragments, deletions and exchange figures, according to UKEMS (1983). Endoreduploidy was classified as quasi-tetraploid metaphases exhibiting chromosome pairing; polyploidy cell is any metaphase containing multiple copies of the haploid number (n) of the chromosome complement, e.g. 3n, 4n etc. All structural chromosome aberrations were assigned artificial lesion (or break) scores as follows:
- chromatid and chromosome (isochromatid) breaks, acentric fragments and deletions were scored as one lesion each
- exchange, rings and dicentrics were designated 2 lesions each.
Breakage rate and the percentage of aberrant cells excluding ALs was calculated and compared to solvent; a break incidence up to 3% was classified as a negative response, especially if a dose dependent increase did not exist. The highest dose chosen for evaluation should be one which causes a reduction in the mitotic index of approx. 50% or corresponds to the substance's solubility limit. Based on these considerations at least 3 concentrations were selected for the evaluation of chromosomal aberrations. Mitotic indices were compiled from counts of 1000 stimulated cells per parallel culture.
For determination of the cell cycle progression, 100 metaphase spreads per culture, treated with BUdR and stained with the FPG technique, were analyzed for the occurrence of 1st, 2nd and 3rd mitosis. The differentiation was necessary in the assay with metabolic activation because of the limited ZK 21.955 incubation period.

Statistics:

Statistical analysis is only performed, if the number of aberrant cells exceeds 3 % in one of the treated groups. For statistical analysis of the percentage of aberrant cells (excl. gaps) two Chi-squared tests will be performed (a = 0.05): (1) positive control vs. solvent control; (2) test compound concentrations vs. solvent control (in case of significance the global test will be followed by comparisons of the individual groups with the solvent control; least significant difference test).

Results and discussion

Test results

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Cell cycle kinetics
In the test with metabolic activation parallel blood cultures were treated with BUdR for assessment of the cell cycle kinetics, because in this case the cultures were incubated with the test compound only for a restricted interval of 3 h. In the control and two middle ZK 21.955 concentrations (0.05 and 0.5 µg/mL) between 99 and 100 % M1 metaphases were observed, indicating that almost all cells scored for chromosomal aberrations (0,01, 0.05, 0.5 and 1.0 µg/mL) were in the first mitosis.

Chromosome aberration test without metabolic activation
The negative and the positive control values fell within the expected range for the testing laboratory.
The ZK 21.955 concentrations employed were 0.0025, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5 and 1.0 µg/mL. The corresponding mitotic indices showed a dose dependent reduction, with a suppression of 57 % at 0.1 µg/mL, the highest concentration scored for chromosomal aberrations.
There was no increase in the percentage of aberrant cells in the blood cultures treated with 0.005, 0.01, 0.05 or 0.1 µg ZK 21.955/mL as compared with the solvent control. Because the results were obviously negative and the frequencies of aberrant cells were too small for a valid test Performance, in this case the Chi-squared test was not done. No polyploid cells could be observed.
Triaziquone, the positive control, proved to be clearly clastogenic (P < 0.05).

Chromosome aberration test with metabolic activation
The solvent and the positive control values fell within the expected range reported for the testing laboratory. Due to the lower cytotoxic effect of ZK 21.955 in the test with S9 mix, it was possible to evaluate 1.0 µg/mL as the highest concentration, whereby the mitotic index showed a suppression of 58 %. There was no increase in the percentage of aberrant cells in the cultures treated with ZK 21.955 at final concentrations of 0.01, 0.05, 0.5 or 1.0 µg/mL as compared with the solvent control. Because the results were obviously negative and the frequencies of aberrant cells were too small for a valid test Performance, in this case the Chi-squared test was not done. No polyploid cells could be observed.
Cyclophosphamide, the positive control, proved to be clearly clastogenic (P < 0.05).

Applicant's summary and conclusion

Conclusions:

Evaluation of the data does not indicate any clastogenic potential of ZK 21.955 in the human lymphocyte test in vitro with or without an added metabolic activation system. In both assays ZK 21.955 was clearly cytotoxic at the highest concentration as proven by the reduction of the mitotic index.

Executive summary:

ZK 21.955 was examined for clastogenic activity in human lymphocytes from the peripheral blood (whole blood culture). The studies, which were conducted in the absence and presence of an extrinsic activating system derived from Aroclor 1254 induced rat liver (S9 mix), employed a range of ZK 21.955 concentrations from 0.0025-5.0 µg/ml. Because the highest concentration tested should either reduce the mitotic Index by approx. 50 % or correspond to the substance's solubility limit, the following concentrations were selected for the analysis of chromosomal aberrations (200 metaphases each): 0.005, 0.01, 0.05 and 0.1 µg/ml in the assay without metabolic activation and 0.01, 0.05, 0.5 and 1.0 µg/ml in the assay with metabolic activation, whereby the mitotic index was reduced by 57 % and 58 % at the highest concentration level, respectively. Negative controls and positive controls with known mutagens (-S9 mix: triaziquone, +S9 mix: cyclophosphamide) produced the expected range of chromosomal aberrations. Neither assay showed an increase in structural chromosomal aberrations in the cultures treated with ZK 21.955 as corapared with the negative controls. Evaluation of the data does not indicate any clastogenic potential of ZK 21.955 in the human lymphocyte test in vitro with or without an added metabolic activation system. In both assays ZK 21.955 was clearly cytotoxic at the highest concentration as proven by the reduction of the mitotic index.