Tetrabutyltin

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation in bacteria

Using Salmonella typhimurium strains TA 1535, TA 1573, TA 98 and TA 100, and Escherichia coli strain WP2 uvrA, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9 in standard co-factors) the test material was considered to be non-mutagenic under the conditions of this test.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 9 June 2004 to 1 August 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

other: Guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW, and MAFF

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Mutant strains of Salmonella and E. coli that are unable to synthesize either histidine or tryptophan were exposed to test material to determine if the material is a mutagenic agent that can cause the bacteria to undergo a reverse mutation to histidine or tryptophan independence.

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: Salmonella was incapable of synthesizing histidine.

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: E. coli was incapable of synthesizing tryptophan

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate metabolizing system (10 % liver S9 in standard co-factors)

Test concentrations with justification for top dose:

PRELIMINARY TEST:
0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.

EXPERIMENT 1
Salmonella strains: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
E. coli strain WP2uvrA-: 50, 150, 500, 1500 and 5000 µg/plate
Additional dose levels were included where applicable to allow for test material induced toxicity, ensuring that a minimum of four non-toxic dose levels were achieved.

EXPERIMENT 2
Salmonella strains: (except TA98) with S9: 15, 50, 150, 500, 1500 and 5000 µg/plate
E. coli strain WP2uvrA- and TA 98 with S9: 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test material was insoluble in distilled water and only partially soluble in dimethyl sulphoxide at 50 mg/mL in solubility checks performed in-house. The test material was fully soluble in acetone at the same concentration.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION

- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: in triplicate

Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of the test material formulation, vehicle or positive control, and either 0.5 mL of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate for each bacterial strain and for each concentration of test material both with and without S9 mix. All plates were incubated at 37 degrees C for 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

Evaluation criteria:

Reverse mutation to histidine independent forms which are detected by their ability to grow on a histidine deficient medium. Test material should have induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.

Statistics:

Dunnett's method of linear regression

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused a visible reduction in the growth of the bacterial background lawn and/or a significant reduction in revertant colony frequency to all of the Salmonella strains, initially at 500 and 1500 µg/plate in the absence and presence of S9, respectively. No toxicity was noted to E.coli strain WP2uvrA- at any test material dose level either with or without S9. The sensitivity of the tester strains to the toxicity of the test material varied slightly between strain type, exposures with or without S9-mix and experiment number. These findings were not indicative of toxicity sufficiently severe to prevent the test material being tested up to the maximum recommended dose level of 5000 µg/plate. An oily precipitate was observed at and above 1500 µg/plate; this did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Conclusions:

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

The method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines. The study was carried out under GLP conditions.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9 in standard co-factors). The dose range for the first experiment was determined in a preliminary toxicity assay and was 5 to 5000 µg/plate for the Salmonella strains and 50 to 5000 µg/plate for E. coli strain WP2uvrA. The experiment was repeated on a separate day using an amended dose range based on results from Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

Additional dose levels were included (where applicable) to allow for test material induced toxicity, ensuring that a minimum of four non-toxic dose levels were achieved.

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused a visible reduction in the growth of the bacterial background lawn and/or a significant reduction in revertant colony frequency to all of the Salmonella strains, initially at 500 and 1500 µg/plate in the absence and presence of S9 respectively. No toxicity was noted to E. coli strain WP2uvrA at any test material dose level either with or without S9. The sensitivity of the tester strains to the toxicity of the test material varied slightly between strain type, exposures with or without S9-mix and experiment number. These findings were not indicative of toxicity sufficiently severe to prevent the test material being tested up to the maximum recommended dose level of 5000 µg/plate. An oily precipitate was observed at and above 1500 µg/plate; this did not prevent the scoring of revertant colonies.

 

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

The test material was considered to be non-mutagenic under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Mouse micronucleus test

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

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

Route of administration:

oral: gavage

Vehicle:

- Vehicle: corn oil.

Details on exposure:

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

Duration of treatment / exposure:

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

Frequency of treatment:

Single dose

Post exposure period:

The animals were observed for 24 or 48 hours

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

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

Control animals:

yes, concurrent vehicle

Positive control(s):

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

Tissues and cell types examined:

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

From each mouse, the bone marrow cells of  both femurs were immediately collected into foetal calf serum and  processed into glassdrawn smears according to the method described by Schmid (1976). 

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

Details of tissue and slide preparation:

Two bone marrow smears per animal were prepared, air-dried  and fixed in methanol. One smear per animal was stained with a May-Grünwald Giemsa solution. The second fixed smear was stored as a reserve slide. No additional organs were examined. Slides were  independently coded.

Evaluation criteria:

- Criteria for evaluating results: The numbers of polychromatic and normochromatic erythrocytes (PE and NE, respectively) were recorded in a total of 200 erythrocytes (E) per animal; if micronuclei were observed, these were recorded as micronucleated polychromatic erythrocytes (MPE) or micronucleated normochromatic erythrocytes (MNE). Once a total number of 200 E (PE + NE) had been scored, an additional number of PE was scored for the presence of micronuclei until a total number of 2000 PE had been scored. Thus the incidence of MPE was recorded in a total of 2000 PE per animal and the number of MNE was recorded in the number of NE.
The study is valid if the positive controls give a statistically significant increase in the mean number of MPE/2000 PE and if the negative controls are within the historical range. A response is considered to be positive if the mean number of MPE/2000 PE is statistically significantly higher, when compared to the mean number of the vehicle controls. A test substance is considered to cause chromosomal damage and/or damage to mitotic apparatus, if a clear dose related increase in the mean MPE/2000 PE is observed, when compared to the mean number of the vehicle controls. A test substance is considered to be negative in the micronucleus test if it produces no positive response at any of the dose-levels and time points analysed. The test substance or its metabolites are deemed to have reached the general circulation and thereby the bone marrow, if the test substance statistically reduce the number of PE/E or causes systemic toxicity. Both statistical significance and biological relevance were considered together in the evaluation.

Statistics:

1. At time point 24 hours after administration, data on MPE and PE were subjected to a One Way Anova with factor group (A, B, C, D). If the Anova yielded a significant effect (p < 0.05), it was followed by pooled error variance t-tests or, if variances were not homogenous, separate variance t-tests. These t-tests were applied to the negative control group A versus treatment groups B, C and D. In addition, the positive control group E and the negative control group A were compared using pooled error variance t-tests or, if variances were not homogenous, separate t-tests.
2. At time point 48 hours after administration, for treatment groups A and D, data on MPE and PE were subjected to pooled error variance t-tests or, if variances were not homogenous, separate variance t-tests.
All statistical tests were performed using BMDP statistical software (W.J. Dixon, BMDP Statistical Software Manual, University of California Press, Berkeley, 1992).

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

MORTALITY: None.

CLINICAL SIGNS: None.

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

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

NOAEL (C): 2000 mg/kg bw.

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

Conclusions:

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

Executive summary:

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

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

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro

Gene mutation in bacteria: The key study was conducted according to the OECD 471 guideline. The study was awarded a reliability score of 1 according to the criteria set forth by Klimisch et al. (1997).

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9 in standard co-factors). The dose range for the first experiment was determined in a preliminary toxicity assay and was 5 to 5000 µg/plate for the Salmonella strains and 50 to 5000 µg/plate for E. coli strain WP2uvrA. The experiment was repeated on a separate day using an amended dose range based on results from Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

Additional dose levels were included (where applicable) to allow for test material induced toxicity, ensuring that a minimum of four non-toxic dose levels were achieved.

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused a visible reduction in the growth of the bacterial background lawn and/or a significant reduction in revertant colony frequency to all of the Salmonella strains, initially at 500 and 1500 µg/plate in the absence and presence of S9 respectively. No toxicity was noted to E. coli strain WP2uvrA at any test material dose level either with or without S9. The sensitivity of the tester strains to the toxicity of the test material varied slightly between strain type, exposures with or without S9-mix and experiment number. These findings were not indicative of toxicity sufficiently severe to prevent the test material being tested up to the maximum recommended dose level of 5000 µg/plate. An oily precipitate was observed at and above 1500 µg/plate; this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

The test material was considered to be non-mutagenic under the conditions of this test.

Supporting studies are also available, the results are as follows:

- In a bacterial reverse mutation assay (Hamasaki, 1993) Tetra-butyltin was not found to be mutagenic.

- In a bacterial reverse mutation assay (Land & Ritthaler, 1989) the data does not indicate that ZK 24.942 is a mutagen in the Ames Salmonella/microsome test.

- In a bacterial reverse mutation assay (Land & Ritthaler, 1989, the data does not indicate that ZK 24.942 is a mutagen in the Ames Salmonella/microsome test using the preincubation modification.

- In a SOS chromotest (Hamasaki, 1992) Tetrabutyltin was not shown to to produce DNA damage by the SOS chromotest.

- In a Bacillus subtilis recombination assay test (Hamasaki, 1992) Tetrabutyltin was not shown to be genotoxic under the conditions of the test.

In vivo

A micronucleus test was carried out in Swiss mice in accordance with the standardised guideline OECD 474 under GLP conditions. The study was awarded a reliability score of 1 according to the criteria set forth by Klimisch et al. (1997).

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

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

Justification for classification or non-classification

In accordance with the criteria for classification as set forth in Regulation (EC) No 1272/2008, the substance does not meet the criteria for classification.