Registration Dossier

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:
11 October 2002 to 1 November 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to OCED guidelines
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Principles of method if other than guideline:
No deviations from guidelines.
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetraoctyltin
EC Number:
222-733-7
EC Name:
Tetraoctyltin
Cas Number:
3590-84-9
Molecular formula:
C32H68Sn
IUPAC Name:
tetraoctylstannane
Details on test material:
- Name of test material (as cited in study report): tetraoctyltin
- Molecular formula (if other than submission substance): Not stated
- Molecular weight (if other than submission substance): 571.6
- Smiles notation (if other than submission substance): Not stated
- InChl (if other than submission substance): Not stated
- Structural formula attached as image file (if other than submission substance): Not stated
- Substance type: Monoconstituent
- Physical state: Slightly yellow turbid liquid, density = 0.96 g/cm3
- Analytical purity: 89.5% (reported); 90.79% (measured)
- Impurities (identity and concentrations):
Trioctyltin chloride = 8.44%
Dioctyltin dichloride = 0.74%
Monooctyltin trichloride = 0.03%
- Composition of test material, percentage of components: See above
- Isomers composition: N/A
- Purity test date: Not stated in this report ( 11-31 December 2002 given in 7.5.1, repeated dose toxicity report, for same compound lot)
- Lot/batch No.: 346272
- Expiration date of the lot/batch: 31 July 2004
- Radiochemical purity (if radiolabelling): N/A
- Specific activity (if radiolabelling): N/A
- Locations of the label (if radiolabelling): N/A
- Expiration date of radiochemical substance (if radiolabelling): N/A
- Stability under test conditions: Not stated
- Storage condition of test material: At less than -18C in the absence of light
- Other:

Method

Target gene:
Salmonella typhimurium: four strains with the following mutation (one per strain)- His D3052, His G46 (+R), His G46 (-R), His C3076
Escherichia coli: Trp
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: Molten top agar (0.6% agar + 0.05mM L-histidine.HCl/0.05 biotin) and minimal glucose agar plates (1.5% agar in Vogel and Bonner medium E with 2% glucose)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: no
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Type and identity of media: Molten top agar (0.6% agar + 0.05mM L-histidine.HCl/0.05 biotin supplemented with 0.05 mM tryptophane) and minimal glucose agar plates (1.5% agar in Vogel and Bonner medium E with 2% glucose)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: no
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenate
Test concentrations with justification for top dose:
0, 62, 185, 556, 1667, 5000 µg tetraoctyltin/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: No justification given
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: See table 2
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)

ORIGIN OF STRAINS:
S. typhimurium strains - Dr. B.N. Ames (University of California, Berkeley, USA)
E. coli strain- Dr. C. Voogd National Institute of Public Health, Bilthoven, the Netherlands)

DURATION
- Preincubation period: Not stated
- Exposure duration: 48 to 72 hours
- Expression time (cells in growth medium): Not stated
- Selection time (if incubation with a selection agent): Not stated
- Fixation time (start of exposure up to fixation or harvest of cells): Not stated

SELECTION AGENT (mutation assays): L-histidine for S. typhimurium assays; tryptophan for E. coli assay

SPINDLE INHIBITOR (cytogenetic assays): N/A

STAIN (for cytogenetic assays): N/A

NUMBER OF REPLICATIONS: Three plates per treatment level in both dose-range finding test and main study

NUMBER OF CELLS EVALUATED: Not stated

DETERMINATION OF MUTAGENICITY
- Method: 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 niether a dose-related increase in the mean number of revertant colonies nor a reproducible positive at any of the test points.

OTHER EXAMINATIONS:
- Determination of polyploidy: N/A
- Determination of endoreplication: N/A
Evaluation criteria:
The mutagenicity study is considered valid if the colony counts of the control values of the strains are within acceptable ranges, it 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 or other unforseen events.

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 S. typhimurium and/or E. coli. Negative results indicate that under test conditions, the test substance is not mutagenic in the tested strains.

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

Results and discussion

Test resultsopen allclose all
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 determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not stated/measured
- Effects of osmolality: Not stated/measured
- Evaporation from medium: Not stated/measured
- Water solubility: Not stated/measured
- Precipitation: Not stated/measured
- Other confounding effects: Not stated/measured

RANGE-FINDING/SCREENING STUDIES: Yes. A dose range finding test to assess the toxicity of the test substance to the bacteria was performed with TA98 both in the absence and presence of S9-mix. DMSO was chosen as vehicle. Just before use, the test substance was dissolved in the vehicle at 50 mg/mL, assumed that the purity ws 89.5%. A colourless slightly viscous solution was obtained. Serial 3-fold dilutions in DMSO were prepared and in total ten dilutions were tested, ranging from 0.3 to 5000 µg/plate.

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

Any other information on results incl. tables

Table 1: Average number of revertants per plate- main study

 

TA 1535

TA 1537

TA 98

TA 100

E. coli

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

0µg/ plate

28

15

17

19

35

60

181

202

36

44

62µg/ plate

20

15

25

22

47

68

184

192

41

41

185µg/ plate

22

16

19

23

36

64

176

187

25

40

556µg/ plate

22

19

15

26

29

59

177

176

33

35

1667µg/ plate

20

17

19

21

43

54

177

174

42

49

5000µg/ plate

23

22

12

20

43

58

183

171

40

34

Positive control

659

544

2050

336

1818

1930

751

3169

160

1482

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that the results obtained with the test substance in S. typhimurium strains TA 1535, TA 1537, TA98, and TA 100, and in the E. coli strain WP2 uvrA, in both the absence and presence of the S9-mix indicate that Tetraoctyltin was not mutagenic under the conditions employed in this study.
Executive summary:

1) The test substance Tetraoctyltin [CAS # 3590 -84 -9] was examined for mutgenic activity in the bacterial reverse mutation test using the histidine-requiring Salmonella typhimurium strains TA 1535, TA 1573, TA 98, and TA 100, the tryptophan-requiring Escherichia coli strain WP2 uvrA, and a liver fraction of Arochlor 1254 -induced rats for metabolic activation (S9 -mix).

2) The test substance was dissolved in DMSO. A dose range finding test was performed with TA 98 both in the absence and presence of S9 -mix with ten different concentrations of the test substance, ranging from 0.3 -5000 µg/plate. Tetraoctyltin was not toxic at any concentration.

3) In the main bacterial reverse mutation test, five different concentrations were tested ranging from 62 -5000 µg/plate. Negative controls (solvent) and positive controls were run simultaneously with the test substance.

4) Tetraoctyltin was not toxic to any strain at any concentration, as was evidenced by the absence of a decrease in the mean number of revertant colonies.

5) In both the absence and the presence of S9 -mix and in all strains, Tetraoctyltin did not cause a more than two-fold 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.

6) 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.

7) It is concluded that Tetraoctyltin was not mutagenic under the conditions employed in this study.