Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-05-22 to 2008-06-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material : Tin metal powder (2 – 11 µm)
- Physical state: Solid, grey powder
- Analytical purity: 99.99 %
- Impurities (identity and concentrations): Pb 0.0035 %, Sb 0.0010 %, Cu 0.0003 %, Au 0.0001 %, Al < 0.0001 %, Cd < 0.0001 %, Zn < 0.0002 %, Ag 0.0005 %, Bi 0.0018 %, As 0.0014 %, Fe < 0.0001 %, In 0.0002 %, Ni 0.0002 %, P 0.0016 % and S 0.0028 %
- Purity test date: 05/07/2008
- Lot/batch No.: 061184
- Expiration date of the lot/batch: August 2008
- Storage condition of test material: When not in use the test article was stored in a sealed container, at room temperature in the dark.

Method

Target gene:
Histidine locus
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
mammalian liver post-mitochondrial fraction (S-9) from Aroclor induced male rats
Test concentrations with justification for top dose:
Range finder and mutation experiment 1 concentrations of powdered tin were:
0.0032; 0.016; 0.08; 0.4; 2.00; 10.0 mg/mL equivalent to 1.6, 8, 40, 200, 1000, 5000 µg/plate
For mutation experiment 2 concentrations were:
0.3125; 0.625; 1.25; 2.50; 5.00; 10.00 mg/mL equivalent to 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate

2-nitrofluorene (2-NF), 50 µg/mL, 5.0 µg/plate formulated in DMSO was the positive control for TA98 strain without S-9
Sodium azide (NaN3), 20 µg/mL, 2.0 µg/plate, formulated in water was the positve control for TA100 and TA 1535 without S-9
9-aminoacridine (AAC), 500 µg/mL, 50.0 µg/plate, formulatd in DMSO was the positive control for TA 1537 without S-9
Mitomycin C (MMC), 2 µg/mL, 0.2 µg/plate, formulated in water was the positive control for TA102 without S-9
Benzo[a]pyrene (B[a]P), 100 µg/mL, 10.0 µg/plate, formulated in DMSO was the positive control for TA98 with S-9
2-aminoanthracene (AAN), 50 µg/mL, 5.0 µg/plate, formulated in DMSO was the positive control for TA 100, TA1535 and TA 1537 with S-9
2-aminoanthracene (AAN), 200 µg/mL, 20.0 µg/plate, formulated in DMSO was the positive control for TA 102 with S-9.
For mutagenicity experiment 2 with pre-incubation, the concentrations of BaP and AAN were doubled to allow the volume additions to be reduced to 0.05 mL and thereby avoid vehicle-induced toxicity
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Purified water
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that powdered tin (particle size 2-11 µm) was insoluble in water for irrigation (purified water). The test article was therefore treated as a homogenous (doseable) suspension.
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: As the results of experiment 1 proved negative, experiment 2 included a pre-incubation step of 1 hour
- Exposure duration: 3 days

SELECTION AGENT (mutation assays): Histidine

NUMBER OF REPLICATIONS: Mutagenicity plates were performed in triplicate. Negative (vehicle) controls were included in quintuplicate, and positive controls were included in triplicate in both assays without and with S-9.

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (colony counting)
Evaluation criteria:
Electronic colony counts were recorded for revertant colonies and the background lawn was checked for indications of toxicity.
Individual plate counts were recorded for each treatment. Background or hisorical control data were checked for acceptability against other spontaneous ranges and positive and negative control revertant numbers were within normal ranges.

Acceptance criteria were as follows:
1. negative control data fell wihin normal ranges
2. positive controls induced clear increases in revertant colony numbers
3. Not more than 5 % of the plates were lost to contamination or other experimental circumstances

Evaluation criteria:
The test material was considered to be mutagenic if
1 Dunnett's test gave a significant concentration related response (p<0.01) and
2. the positive results/trends were reproducible
Statistics:
Individual plate counts from all experiments were recorded separately and the mean and standard deviation of the plate counts for each treatment were determined. Control counts were compared with the accepted normal ranges for our laboratory for numbers of spontaneous revertants on vehicle control plates and numbers of induced revertants on positive control plates. The ranges that are quoted are based on a large volume of historical control data accumulated from experiments where the correct strain and assay functioning are considered to have been confirmed. Data for our laboratory are consistent with ranges of spontaneous revertants per plate considered acceptable elsewhere. For evaluation of test article and positive control data there are many statistical methods in use, and several are acceptable. Dunnett's test was used to compare the counts at each concentration with the control. The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example toxicity, precipitation or 5000 µg/plate).
Statistical analysis of test and control data used Dunnett's where appropriate.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
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:
RANGE-FINDING/SCREENING STUDIES:
An initial toxicity Range-Finder Experiment was carried out in the absence and in the presence of S-9 in strain TA100 only, using final concentrations of powdered tin (particle size 2-11 microns) at 1.6, 8, 40, 200, 1000 and 5000 μg/plate, plus negative (vehicle) and positive controls. Following these treatments, no evidence of toxicity was observed. These data were considered to be acceptable for mutation assessment and are presented in this report as the TA100 mutagenicity data for Experiment 1.

MAIN STUDY
In experiment 1, all tester strains were treated at same concentrations as used in the range-finder. No evidence of toxicity was observed. Precipitation occurred at 5000 µg/plate in all strains in the presence or absence of S-9. No evidence of mutagenicity was seen in the first experiment.

In experiment 2, all strains were tested with or without S-9 at concentrations from 156.3 up to 5000 µg/plate, following a pre-incubation phase in order to investigate powdered tin at the concentration limit, where any potential mutagenicity might be expected to be exhibited. No evidence of toxicity or mutagenicity was apparent. In this assay precipitation was apparent for all strains at 2500 µg/plate and above without S-9 and at 1250 µg/plate and above without S-9.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test article was observed following Experiment 1 treatments at 5000 µg/plate in all strains in the absence and presence of S-9. Following Experiment 2 treatments, evidence of precipitation was observed in all strains at 2500 µg/plate and above in the absence of S-9 and 1250 µg/plate and above in the presence of S-9.

COMPARISON WITH HISTORICAL CONTROL DATA:
Results were comparable.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1: treatments of the remaining test strains were performed in the absence and in the presence of S-9 and retained the same test concentrations as employed for the Range-Finder Experiment treatments. Following these treatments no evidence of toxicity was observed.
Experiment 2: treatments of all the tester strains were performed in the absence and in the presence of S-9 with the maximum test concentration of 5000 µg/plate. Narrowed concentration ranges were employed (156.3 – 5000 µg/plate), in order to examine more closely those concentrations of powdered tin (particle size 2-11 microns) approaching the maximum test concentration and therefore considered most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments no evidence of toxicity was observed.

Any other information on results incl. tables

All tables are presented in the attached pdf document.

Less than 5 % of plates were lost, leaving adequate numbers of plates at all treatments. The study therefore demonstrated correct strain and assay functioning and was accepted as valid.

No statistically significant increases in revertant numbers were observed following any strain treatments in the absence or presence of metabolic activation, and therefore this study was considered to have provided no evidence of any powdered tin (particle size 2-11 µm) mutagenic activity.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative with and without metabolic activation

In conclusion, powdered tin (particle size 2-11 µm) did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/plate, in the absence and in the presence of a rat liver metabolic activation system (S-9).
Executive summary:

Powdered tin (particle size 2 -11 µm) was tested for mutagenic potential in five strains of histidine dependent S.typhimurium, in the presence or absence of metabolic activation according to the standard Ames test methods (OECD 471). Since the first assay gave no indication of an increase in the revertant colony numbers the second assay used a closer range of concentrations and incorporated to a pre-incubation phase to increase the assay sensitivity.

Neither experiment gave any indication of mutgaenic potential for powdered tin.

Powdered tin metal prepared as a fine (2 -11 µm particle size) powder was found to be negative for mutagenic potential in the bacterial reverse mutation test.