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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental phase of this study was performed between 12 May 2010 and 07 June 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted to GLP. Study refers to the tin compound of the reaction mass

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Dibutyltin di(acetate)
EC Number:
213-928-8
EC Name:
Dibutyltin di(acetate)
Cas Number:
1067-33-0
Molecular formula:
C12H24O4Sn
IUPAC Name:
dibutyltin di(acetate)
Details on test material:
Sponsor's identification: CAS No 1067-33-0
Description: Clear colourless liquid
Chemical name: Di-n-butyltindiacetate
Purity: >98%
Batch number: 156 004 10/1
Date received: 15 April 2010
Expiry date: 08 September 2010
Storage conditions: room temperature in the dark over silica gel

The integrity of supplied data relating to the identity, purity and stability of the test material is the responsibility of the Sponsor.

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Experiment 1: 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate.

Experiment 2: Salmonella strains: 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate.
E.coli strain WP2uvrA: 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test material was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/ml but was fully miscible in acetone at the same concentration in solubility checks performed in house. Acetone was therefore selected as the vehicle.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Without S9: N-ethyl-N-nitro-N-nitrosoguanidine, 9-Aminoacridine & 4-Nitroquinoline-1-oxide; With S9: 2-Aminoanthracene & Benzo(a)pyrene.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: In experiment 2, measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 ml of S9-mix or phosphate buffer and 0.05 ml of the vehicle or test material formulation and incubated for 20 minutes at 37°C with shaking at approximately 130 rpm prior to the addition of 2 ml of molten, trace histidine or tryptophan supplemented, top agar.
- Exposure duration: All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

NUMBER OF REPLICATIONS: triplicate

NUMBER OF CELLS EVALUATED: nda

DETERMINATION OF CYTOTOXICITY
- Method: In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material. The test material was, therefore, tested up to the toxic limit.
Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (6) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.

A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Acceptance Criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
- All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
- The appropriate characteristics for each tester strain have been confirmed, eg rfa cell wall mutation and pKM101 plasmid R-factor etc.
- All tester strain cultures should be in the range of 1 to 9.9 x 10^9 bacteria per ml.
- Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
- There should be a minimum of four non-toxic test material dose levels.
- There should be no evidence of excessive contamination.

Results and discussion

Test results
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
A precipitate (greasy in appearance) was noted in the preliminary toxicity test at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES:
The test material was found to be initially toxic at and above 50 and 500 µg/plate to the strains of bacteria used TA100 and WP2uvrA-, respectively. The test material formulation and S9 mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA: yes, see appendix 2

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary Toxicity Test

The test material was initially toxic at and above 50 and 500 µg/plate to the strains of bacteria used TA100 and WP2uvrA-, respectively. The test material formulation and S9-mix used in this experiment were both shown to be sterile.

The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-)

S9-mix

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

165

151

144

154

157

129

27S

0S

0T

0TP

0TP

+

TA100

169

150

159

148

155

133

12S

0S

0T

0TP

0TP

-

WP2uvrA-

23

19

18

22

20

21

16

16

0T

0TP

0TP

+

WP2uvrA-

33

24

34

21

24

14

11

11

0S

0TP

0TP

S = Sparse bacterial background lawn
T = Toxic, no bacterial background lawn
P = Precipitate

Mutation Test

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9-mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable. These data are not given in the report.

Results for the negative controls (spontaneous mutation rates) are presented in Table1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.

A history profile of vehicle and positive control values for 2008 and 2009 is presented in Appendix 2.

In the first experiment (plate incorporation method) the test material caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, in both the presence and absence of S9-mix, at and above 150 and 500 µg/plate for the Salmonella strains and Escherichia coli strain WP2uvrA-, respectively. A similar toxic response was noted in the second experiment (pre-incubation method) with weakened bacterial background lawns noted to all of the tester strains, initially from 50 µg/plate (TA100 and TA1535), 150 µg/plate (TA98 and TA1537) and 500 µg/plate for Escherichia coli strain WP2uvrA-. The test material was, therefore, tested up to the toxic limit. A precipitate (greasy in appearance) was noted in the preliminary toxicity test at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

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 Regulation (EC) Number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using both the Ames plate incorporation and pre-incubation methods at 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 was determined in a preliminary toxicity assay and was 0.5 to 500 µg/plate in the first experiment. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test material formulations. The test material dose range was amended slightly (ranging between 0.15 and 500 µg/plate) to allow for results from Experiment 1 and the change in test methodology.

Additional dose levels and an expanded dose range were selected in both experiments in order to achieve both four non-toxic dose levels and the toxic limit of the test material.

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.

In the first experiment (plate incorporation method) the test material caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, in both the presence and absence of S9-mix, at and above 150 and 500 µg/plate for the Salmonella strains and Escherichia coli strain WP2uvrA-, respectively. A similar toxic response was noted in the second experiment (pre-incubation method) with weakened bacterial background lawns noted to all of the tester strains, initially from 50 µg/plate (TA100 and TA1535), 150 µg/plate (TA98 and TA1537) and 500 µg/plate for Escherichia coli strain WP2uvrA-. The test material was, therefore, tested up to the toxic limit. A precipitate (greasy in appearance) was noted in the preliminary toxicity test at and above 1500 µg/plate, this observation 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 or exposure method.

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