Tin

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay

Beevers 2008, a GLP compliant study performed to the standardised guideline OECD 471, tin powder (2-11 µm) was found not to induce mutations in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium.

In vitro mammalian cytogenicity

The key study for this endpoint, Lloyd 2009, was performed in compliance with GLP and to the OECD guideline 473. Under the conditions of the test, powdered tin with a particle size of 2-11 µm did not induce chromosome aberrations in cultured CHO cells when tested up to toxic concentrations in the absence of metabolic activation and up to the acceptable maximum concentration, 10 mM, in the presence of S-9.

In vitro gene mutation study in mammalian cells

Stankowski 2009, the key study for this endpoint was performed in compliance with GLP and to the current guideline OECD 476. Tin metal powder (2-11 µm) did not induce a positive response in a CHO/HPRT forward mutation assay performed with duplicate cultures and a confirmatory assay.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus

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.

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.

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.

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.

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 May 2008 to 17 July 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Evaluation of the clastogenic potential of powdered tin from its effects on the chromosomes of cultured Chinese Hamster Ovary cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: CHO cells, supplied by Dr S Galloway, West Point USA, maintained on McCoy's supplemented medium.
- Properly maintained: yes
- Periodically checked for karyotype stability: yes. Stocks of cells preserved in nitrogen were reconstituted for each assay to maintain karyotypic stability.
The cell lines were regularly subcultured. The cell cycle for cell line used at Covance was measured at circa 13 hours. Cell sheets removed from stock cultures were incubated for 24 hours and cultures with suitable confluence levels prepared for the study.

Metabolic activation:

with and without

Metabolic activation system:

Mammalian liver post-mitochondrial fraction (S-9) from Aroclor 1254 induced male Sprague Dawley rats

Test concentrations with justification for top dose:

Details on dosing are presented in the tables under section "Any other information on material and methods incl. tables"

For experiment 1 (20 hour treatment, 0 hour recovery, without S-9) aberrations were analysed in three concentrations - 600, 700 and 900 µg/mL
For experiment 2 (3 hour treatment 17 hour recovery without S-9), aberrations were analysed at four test concentrations - 600, 800, 1000 and 1187 µg/mL
For experiment 2 (3 hour treatment 17 hour recovery with S-9), aberrations were analysed at three test concentrations - 800, 1000 and 1187 µg/mL
For experiment 3 (3 hour treatment 17 hour recovery with S-9), aberrations were analysed at three test concentrations - 800, 1000 and 1187 µg/mL
Test concentrations were selected on the basis of results from the range-finding experiments, using population doubling or mitotic indices as the criteria for assessing cytotoxicity and suitable dosing concentrations.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Suspended in sterial water
- Justification for choice of solvent/vehicle: Powdered tin was found to be insoluble in the commonly used vehicles suitable for this assay but could be suspended in water for injection. Homogeneous suspensions were stable up to concentrations of 11.90 µg/mL. A ten-fold dilution of this stock added to culture medium showed precipitation and consequently the maximum treatment concentration used in the range finder was 1187 µg/mL and test concentrations were based on concentration ranges up to and including 1187 µg/mL.

Untreated negative controls:

yes

Remarks:

Sterile water for injection

Negative solvent / vehicle controls:

yes

Remarks:

Sterile water for injection

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

Remarks:

NQO used in assays without S-9 at 0.25 or 0.30 µg/mL and CPA used with S-9 at concentrations of 6.25 or 12.5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 or 20 hours
- Expression time (cells in growth medium): 17 or 0 hours for the 3 and 20 hour exposures respectively
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): Approximately 1.5 hours prior to harvest, colchicine was added to give a final concentration of approximately 1 μg/mL to arrest dividing cells in metaphase. At the defined sampling time, the monolayers of these cultures were then removed using trypsin/EDTA

STAIN (for cytogenetic assays): In all experiments, an aliquot of cell suspension from all cultures (with the exception of positive control treated cultures) was taken for determination of cell number by using a Coulter Counter. The remaining suspension from each flask was transferred to a plastic centrifuge tube and the cells pelleted by centrifuging at approximately 200 x 'g' for 5 minutes and cells resuspended in 4 mL pre-warmed (hypotonic) 0.075 M KCl and incubated at 37 ± 1 °C for 5 minutes to allow cell swelling to occur. Cells were fixed by dropping the KCl suspension into fresh, cold methanol/glacial acetic acid (3:1, v/v). The fixative was changed by centrifugation (approximately 200 x 'g' for 5 minutes) and resuspension. This procedure was repeated several times (centrifuging at approximately 1250 x 'g', 2-3 minutes) until the cell pellets were clean.
Cells were kept in fixative at 1-10 °C before slides were made. Cells were centrifuged and resuspended in a minimal amount of fresh fixative (if required) to give a milky suspension. Several drops of 45 % (v/v) aqueous acetic acid were added to each suspension to enhance chromosome spreading, and several drops of suspension were transferred to clean microscope slides labelled with the appropriate study details. Slides may have been flamed to improve quality. After the slides had dried, the cells were stained for 5 minutes in filtered 4 % (v/v) Giemsa in pH 6.8 buffer. The slides were rinsed, dried and mounted with coverslips.

NUMBER OF REPLICATIONS: 2 in the dosing groups and 4 in the vehicle controls

NUMBER OF CELLS EVALUATED: One hundred metaphases where possible (Only cells with 19 to 23 (modal number ± 2) chromosomes)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and population doubling
The concentrations for the main experiments and for chromosome aberration analysis were selected on the basis of toxicity. For this type of study, toxicity is usually measured by assessing (PD) in treated cultures, relative to controls, (11). For the 3+17 hour and 20+0 hour treatments in the absence of S-9, PD was calculated for each concentration as follows:
PD = [log (N ÷ Xo)] ÷ log 2

Where: N = mean final cell count/culture at each concentration
Xo = starting (baseline) count.

For the 3+17 hour treatments in the presence of S-9 in Range-Finders 1 and 2, PD was the only method used for toxicity measurement. PD was not considered as an appropriate method of toxicity measurement for the 3+17 hour treatments in the presence of S-9. For Range-Finder 3 and Experiments 2 and 3, for the 3+17 hour treatments in the presence of S-9 was assessed by two separate measures, PD and mitotic index. The Mitotic Index (MI) is a measure of the proliferative state of the culture at a particular moment in time and was calculated as follows:
MI = (number of cells in mitosis/total number of cells observed) x 100

MI = number of cells in mitosis x100
Total number of cells observed

Mitotic inhibition (MIH) was calculated as:

MIH (%)= [1 - (mean MIT/mean MIC)] x 100 %
(where T = treatment and C = negative control)

Slides from enough concentrations from each treatment group were scored to determine whether chemically induced mitotic inhibition had occurred. This is defined as a clear decrease in mitotic index compared with negative controls, (based on at least 1000 cells counted where possible) and is preferably concentration-related.

OTHER: Baseline cell counts were established to provide starting counts for toxicity calculations.
Various range-finding assays were completed to establish levels of cytotoxicity, with and without metabolic activation, covering both the 3 hour treatment (with 17 hours recovery) and the 20 hour treatment period with 0 hours recovery.
In the main study experiment 1, a 3 hour treatment was followed by 17 hours recovery in the absence and presence of S-9 (no chromosomal aberration data were obtained from the 3+17 -S-9 assay) and a 20 hour treatment with no recovery time, in the absence of S-9.
In experiment 2 the treatment regimen was 3 hour treatment + 17 hour recovery with and without S-9 .
In experiment 3, only one treatment occurred - a 3 hour + 17 hour recovery in the presence of S-9 metabolic activation.
The final culture volume in each case was 5 mL. Cultures were gassed with carbon dioxide and incubated at 37 ° C for 3 or 20 hours.
Cultures subject to continuous treatment retained media through to harvest. The 3-hour cultures were pulse treated.
Colchicine was added to the cultures approximately 1.5 hours prior to harvesting to arrest dividing cells in metaphase.
An aliquot of each cell suspension was taken for coulter counter determination of cell numbers. The remaining suspension was centrifuged to form a cell pellet which was resuspended, incubated and re-centrifuged and then fixed for evaluation after slides of the cell suspension were prepared.
Chromosome spreads were stained with Giemsa and slides analysed for aberrations.

Evaluation criteria:

ACCEPTANCE CRITERIA
The assay is considered valid if the following criteria are met:
1. the binomial dispersion test demonstrates acceptable heterogeneity between replicate cultures
2. the proportion of cells with structural aberrations (excluding gaps) in negative control cultures falls within the historical negative control (normal) range
3. at least 160 cells out of an intended 200 suitable for analysis at each concentration, unless 10 or more cells showing structural aberrations (per slide) other than gaps only were observed during analysis
4. the positive control chemicals induce statistically significant increases in the proportion of cells with structural aberrations.

EVALUATION CRITERIA
For valid data, the test article was considered to induce clastogenic events if:
1. A proportion of cells with structural aberrations at one or more concentrations that exceeded the historical negative control (normal) range was observed in both replicate cultures
2. A statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) was observed (p ≤ 0.05) 3. There was a concentration-related trend in the proportion of cells with structural aberrations (excluding gaps). The test article was considered as positive in this assay if all of the above criteria were met. The test article was considered as negative in this assay if none of the above criteria were met. Results which only partially satisfy the above criteria were dealt with on a case-by-case basis. Evidence of a concentration-related effect is considered useful but not essential in the evaluation of a positive result. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments, or effects occurring only at high or very toxic concentrations, and the types and distribution of aberrations.

Statistics:

After completion of scoring and decoding of slides, the numbers of aberrant cells in each culture were categorised as follows:
Category 1: cells with structural aberrations including gaps
Category 2: cells with structural aberrations excluding gaps
Category 3: polyploid, endoreduplicated or hyperdiploid cells.
The totals for category 2 in negative control cultures were compared with the current laboratory historical negative control (normal) ranges to determine whether the assay was acceptable or not (see Acceptance Criteria). The totals for category 2 in test article treated cultures were also compared with normal ranges. The statistical significance of increases in the percentage of cells with structural aberrations for any data set was only taken into consideration if the frequency of aberrant cells in both replicate cultures at one or more concentration exceeded the normal range. The statistical method used was Fisher's exact test. Probability values of p ≤ 0.05 were accepted as significant. The proportions of cells in categories 1 and 3 were examined in relation to normal ranges and may have been analysed by Fisher’s exact test, for example in the case of an equivocal result. The proportions of aberrant cells in each replicate were also used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test. Probability values of p ≤ 0.05 were to be accepted as significant.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at highest doses analysed

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed and is noted at the various concentrations in the tabulated data in the attached pdf.

RANGE-FINDING/SCREENING STUDIES: Appropriate levels of toxicity were observed following 3+17 hour and 20+0 hour treatments in the absence of S-9 in range-finder 1. However, for the 3+17 hour treatment in the presence of S-9, post-treatment cell counts indicated that there had been no change in the cell population since the time of treatment in all three range-finder experiments. These data appeared anomalous by comparison with the inspection of cultures prior to harvest, which indicated that the vehicle control cultures were >50 % confluent in and treated cultures showed evidence of a concentration-related decrease in toxicity. The reasons for this were unclear. Following range-finder 2, population doubling was deemed an unsuitable parameter for determining cytotoxicity for the 3+17 hour treatment in the presence of S-9, therefore in range-finder 3, toxicity was assessed by mitotic index.

The mitotic indices were low in the vehicle controls, but there was clearly no evidence of cytotoxicity.

Tabulated methods, results and appendices are presented in the attached pdf.

Conclusions:

Interpretation of results: negative with and without metabolic activation

With one exception, there were no increases in frequency of cells with numerical aberrations (that exceeded the controls or normal control ranges), for tin powder treated cultures either with or without S-9. Treatment of all cultures under all test conditions, with or without S-9, continuous or pulse treatment, resulted in no increase in the frequency of cells with structural aberrations, compared with negative controls.
Powdered tin with a particle size of 2-11 µm did not induce chromosome aberrations in cultured CHO cells when tested up to toxic concentrations in the absence of metabolic activation and up to the acceptable maximum concentration, 10 mM, in the presence of S-9.

Executive summary:

Powdered tin (particle size 2 -11 µm) was tested in an in vivo cytogenetic assay for clastogenicity using cultured Chinese Hamster ovary cells. Treatments, covering a broad range of test concentrations, were performed in the presence or absence of metabolic activation provided by S-9 fraction. The three experiments provided continuous or pulse treatment over 20 or 3 hours with 0 or 17 hour recovery phases respectively. Within the three experiment replicate treatments were investigated.

Experiment 1 - continuous treatment for 20 hours, no recovery time, without S-9; test concentrations selected on basis of population doubling figures in range-finding tests. The concentrations selected for analysis of chromosomal aberrations were 600, 700 and 900 µg/ml. 51 % cytotoxicity occurred at the highest concentration analysed.

Experiment 2 - three hour treatment, followed by a 17 hour recovery phase, was completed with and without metabolic activation. The test concentrations analysed for chromosomal aberrations were 600, 800, 1000 and 1187 µg/mL. At the highest concentration analysed, cytotoxicity (measured by population doubling) was 72 % without S-9 and, measured by the mitotic index, was 0 % with S-9.

Experiment 3 - Three hour treatment, followed by a 17 hour recovery phase, was completed with metabolic activation. Test concentrations analysed were 800, 1000 and 1187 µg/mL. At the highest concentration analysed 16 % cytotoxicity occurred, as measured by the mitotic index.

Powdered tin with a particle size of 2-11 microns did not induce chromosome aberrations in cultured CHO cells when tested up to toxic concentrations in the absence of metabolic activation and up to the acceptable maximum concentration, 10 mM, in the presence of S-9.

It is concluded that powdered tin (particle size 2-11 µm) did not induce chromosome aberrations in cultured Chinese hamster ovary (CHO) cells when tested up to toxic concentrations in the absence of rat liver metabolic activation system (S-9) and up to a concentration of 1187 µg/mL (equivalent to 10 mM), an acceptable maximum for in vitro chromosome aberration studies according to current regulatory guidelines, in the presence of S-9.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 April 2009 to 17 June 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT is a purine salvage enzyme that allows cells to utilize hypoxanthine and guanine for use in DNA synthesis. It catalyzes the reaction between 5-phosphoribosyl-1-pyrophosphate and hypoxanthine or guanine to form inosine or guanosine monophosphate, respectively. If the purine analog TG is included in the growth medium, it will be phosphorylated by HPRT and incorporated into nucleic acids, resulting in cell death.
The hprt locus is located on the X chromosome. Since only one of the two X chromosomes is functional in these female-derived cells, a single-step forward mutation from hprt+ to hprt- in the functional X chromosome results in loss of HPRT activity and renders the cell unable to utilize purine analogs. Such mutant cells are TGr and are assumed to have mutated, either spontaneously or as the result of treatment, to the hprt- genotype. However, these mutants still are viable, since DNA synthesis can proceed by the de novo purine biosynthetic pathway that does not involve hypoxanthine or guanine.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The cell line used in this assay, clone CHO-K1-BH4, was isolated by Hsie et al. (1975), and has been demonstrated to be sensitive to many chemical mutagens. Cells to be used in this study were obtained from A.W. Hsie, Oak Ridge National Laboratories (Oak Ridge, TN) and are stored frozen in liquid nitrogen.
- Type and identity of media: Cells used in this study were maintained in Ham's F12 medium supplemented with 5 % heat-inactivated and dialyzed fetal bovine serum (F12FCM5). Cleansing medium (THGM) was F12FCM5 supplemented with 5 μM thymidine, 10 μM hypoxanthine, 100 μM glycine, and 3.2 μM methotrexate. Recovery medium (THG) was THMG without methotrexate. Treatment medium was serum-free Ham’s F12. Cloning medium (for determination of cloning efficiency) was hypoxanthine-free F12FCM5 (HX-F12FCM5). Selection medium (for selection of TGr mutants) was HX-F12FCM5 supplemented with 10 μM TG. All media contained antibiotics (gentamicin and/or Fungizone).
- Properly maintained: yes. Laboratory cultures for the mutation assays were maintained in logarithmic growth by serial subculture for up to 4 months and then were replaced by cells from the frozen stock. Working stock cultures were maintained as monolayer cultures under standard conditions (35 to 38 °C in a humidified atmosphere containing 4 to 6 % CO2). To reduce the frequency of spontaneous HPRT- mutants prior to use in a mutation assay, the stock cultures were exposed to cleansing medium for 3 days, followed by growth in recovery medium for 1 day.
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Liver homogenate was purchased from Molecular Toxicology, Inc. (Lot 2278). The homogenate was prepared from male Sprague Dawley rats injected i.p. with Aroclor 1254 (200 mg/mL in corn oil), at a dose of 500 mg/kg given 5 days before sacrifice

Test concentrations with justification for top dose:

Preliminary test concentrations: with and without S9, 2.35, 4.70, 9.40, 18.8, 37.5, 75.0, 150, 300, 600, and 1200 µg/mL
Initial mutagenicity assay: 37.5, 75.0, 150, 300, 600, and 1200 µg/mL with and without S9.
Confirmatory mutagenicity assay: 37.5, 75.0, 150, 300, 600, and 1200 µg/mL with and without S9.

Dose Analyses: Quadruplicate 1.0-mL samples from the top, middle, and bottom of the low, mid, and high dose formulations used in the definitive mutation assays were collected into amber glass vials and stored at room temperature. Duplicate 2.5-mL samples also were collected from the concurrent vehicle control and stored under the same conditions. Samples were not collected from the dose range-finding assay.

Vehicle / solvent:

- Vehicle used: de-ionised water

Tin metal powder was found to be incompletely soluble in di-H2O at all concentrations prepared for treatment (0.0235 to 120 mg/mL) and was administered as a suspension. The test article was used as received (no correction factor was used for dose formulation calculations). All dose formulations were prepared on the day of use.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

de-ionised water 10 % w/v

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;
Cleansed cells were seeded (on Day -1) in 75-cm^2 flasks at a density of approximately 4 x 10^6 in 10 mL F12FCM5. Following an overnight incubation (on Day 0), the cultures were washed twice with 10 mL PBS and re-fed with 10 mL treatment medium (adjusted for the test article dose volume and S9, as appropriate). Following addition of the test or control articles, the cultures were incubated for 5 hours. After the treatment media was removed, the cultures were washed twice with 10 mL PBS, re-fed with 10 mL culture media, and incubated overnight.

DURATION
- Exposure duration: 5 hours
- Expression time (cells in growth medium): The 60-mm dishes were incubated for 7 days for colony development and subsequent determination of adjusted initial relative survival as described previously).
The large dishes were subcultured for 8 days, at 2- to 3-day intervals, to maintain logarithmic growth and permit expression of induced mutants. At each subculture, the duplicate dishes were trypsinized, combined, counted, and reseeded at 1.5 x 10^6 cells/150 mm dish (in duplicate; duplicate dishes were used for each culture to increase the population size as well as to serve as a back-up should one dish be lost due to technical issues).
- Selection time (if incubation with a selection agent): plates were incubated for 7 days in selection medium
- Fixation time (start of exposure up to fixation or harvest of cells): Cells were fixed with methanol after 7 days incubation with the selection agent

SELECTION AGENT (mutation assays): HX-F12FCM5 selection medium supplemented with 10 μM TG.

NUMBER OF REPLICATIONS: Duplicate cultures prepared in each of two replicate assays

DETERMINATION OF CYTOTOXICITY
- Method: A dose range finding study was performed, the highest dose tested approximated the limit dose for the assay. On the day after treatment (Day 1), the cultures were washed twice with PBS and then trypsinized and counted. An aliquot of cells from each culture was plated at a density of 200 cells/60-mm dish in triplicate. These cloning efficiency dishes were incubated for 9 days, and the resulting colonies were fixed in methanol, stained with Giemsa and counted manually. The cloning efficiency was calculated for each culture, and cytotoxicity was expressed as the adjusted relative initial survival (%; Day 1 cell density x cloning efficiency, as compared to the concurrent vehicle control).

Evaluation criteria:

Criteria for a Positive Response
The test article will be considered to have produced a positive response if it induces a statistically significant increase in mutant frequency (p≤ 0.05) that represents an increase of ≥15 TGr mutants/10^6 clonable cells over the concurrent vehicle controls (this threshold is based upon historical data from this laboratory as well as published results). In addition, any response should be dose dependent (p≤ 0.05) and reproducible.

Criteria for a Negative Response
A test article will be considered to have produced a negative response if no significant, dose-dependent, or ≥15 TGr mutants/10^6 clonable cells are observed.

Criteria for an Equivocal Response
Even after repeated trials, a test article may produce results that are neither clearly positive nor clearly negative (e.g., responses that do not meet the dose-dependency or increase requirements but are reproducible). In those rare instances, the test article may be considered to have produced an equivocal response.

Other Considerations
Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological significance, etc.). If such a case arised, the Study Director used sound scientific judgment and clearly reported and described any such considerations.

Statistics:

Statistical analysis was performed using the method of Snee and Irr (1981), with significance established at the 0.05 level.

The adjusted relative survival was calculated by:
cell density x cloning efficiency

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

clone CHO-K1-BH4

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

limited evidence of cytotoxicity (decreases in adjusted relative survival) was observed in the absence of S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Cytogenicity:
Tin metal powder was evaluated in a preliminary dose range-finding assay, in single cultures with and without S9, at concentrations of 2.35, 4.70, 9.40, 18.8, 37.5, 75.0, 150, 300, 600, and 1200 μg/mL. The highest concentration evaluated approximated the limit dose for this assay (10 mM). di-H2O was evaluated concurrently as the vehicle control. Little or no cytotoxicity was observed in the presence of S9, while limited evidence of cytotoxicity (decreases in adjusted relative survival) was observed in its absence. Adjusted relative survival values at concentrations of 600 and 1200 μg/mL without S9 were 58.85 and 47.76 %, respectively. In addition, the test article was incompletely soluble at concentrations ≥18.8 μg/mL with and without S9.

Initial Mutagenicity Assay
Based upon the results of the dose range finding assay, tin metal powder was evaluated in the initial mutagenicity assay at concentrations of 37.5, 75.0, 150, 300, 600, and 1200 µg/mL with and without S9. The vehicle control was evaluated concurrently. All test and control article concentrations were evaluated in duplicate cultures. Little or no cytotoxicity was observed for tin metal powder: average adjusted relative survivals at a concentration of 1200 µg/mL were 85.64 and 62.38 %, with and without S9, respectively. Those cultures treated at a concentration of 37.5 µg/mL with and without S9 were discarded because a sufficient number of higher concentrations were available. The average mutant frequencies of the vehicle control cultures were 1.7 and 5.2 TGr mutants/10^6 clonable cells with and without S9, respectively, while those of the remaining cultures treated with tin metal powder ranged from 2.6 to 5.6 TGr mutants/10^6 clonable cells with S9, and 2.7 to 7.7 TGr mutants/106 clonable cells without S9. There were no statistically significant or dose-dependent increases in average mutant frequency observed with or without S9 (p >0.05).

Confirmatory Mutagenicity Assay
Tin metal powder was re-evaluated in the confirmatory mutagenicity assay under identical conditions, and similar results were observed. Little or no cytotoxicity was observed for tin metal powder: average adjusted relative survivals at a concentration of 1200 µg/mL were 71.50 and 51.40 %, with and without S9, respectively. Those cultures treated at a concentration of 37.5 µg/mL with and without S9 were discarded because a sufficient number of higher concentrations were available. The average mutant frequencies of the vehicle control cultures were 3.7 and 4.2 TGr mutants/10^6 clonable cells with and without S9, respectively, while those of the remaining cultures treated with tin metal powder ranged from 2.2 to 4.6 TGr mutants/10^6 clonable cells with S9, and 1.9 to 3.0 TGr mutants/10^6 clonable cells without S9. There were no statistically significant or dose-dependent increases in average mutant frequency observed with or without S9 (p >0.05).
Thus, none of the treatments, in either trial with or without S9, induced a statistically significant, dose-dependent increase in mutant frequency that was greater than 15 TFTr mutants/106 clonable cells above the average concurrent vehicle control mutant frequency.

Dose Analysis: Results of the dose formulation analyses indicated that the dose formulations were not homogeneous. In addition, all dose formulations were lower than the concentration stated, and the vehicle controls did not contain detectable test article. The variation between samples, and from stated concentrations, is attributed to difficulties in handling and sub-sampling tin suspensions rather than the analytical procedure. However, the test article was evaluated at concentrations far in excess of the limit of solubility. Therefore, the variation from stated concentrations is not considered to have had an adverse impact upon the integrity of the study or the conclusions derived from it.

Dose range finding assay
Treatment		+ S9			-S9
		Cloning Efficiency (%)¹	Relative Cloning Efficiency (%)²	Adjusted Relative Survival (%)²	Cloning Efficiency (%)¹	Relative Cloning Efficiency (%)²	Adjusted Relative Survival (%)³
Water (% v/v)	10.0	77.50	100.00	100.00	91.50	100.00	100.00
Tin metal powder (µg/mL)	2.35	83.83	108.17	108.17	88.00	96.17	101.07
	4.70	77.67	100.22	102.41	90.33	98.72	106.25
	9.40	101.17	130.54	121.97	87.33	95.44	101.10
	18.8	81.67	105.38	99.23⁴	92.50	101.09	95.10⁴
	37.5	81.33	104.94	98.81⁴	94.83	103.64	105.40⁴
	75.0	94.00	121.29	105.35⁴	98.33	107.46	102.00⁴
	150	97.17	125.38	111.65⁴	111.50	121.86	111.53⁴
	300	82.50	106.45	97.90⁴	90.50	98.91	81.30⁴
	600	81.83	105.59	90.94⁴	69.83	76.32	58.85⁴
	1200	86.50	111.61	94.50⁴	73.67	80.51	47.76⁴
¹Number of colonies / number of cells plated x 100 % (average of three plates) ²Relative cloning efficiency as compared to concurrent vehicle control ³Day 1 cell count x cloning efficiency, as compared to the average of the concurrent vehicle control ⁴Test article incompletely soluble at end of treatment

Initial Mutation Assay with S9
Treatment (+S9)	Adjusted Relative Survival (%)¹	Total Mutant Colonies	Cloning Efficiency (%)²	TGr Mutants/ 10^6 Clonable Cells³
Water (%, v/v)
10.0	91.15	4	94.00	1.8
10.0	108.85	5	126.50	1.6
Tin metal powder (µg/mL)
37.5⁴	125.62	-	-	-
37.5⁴	94.54	-	-	-
75.0⁴	104.86	2	112.83	0.7
75.0⁴	78.06	11	104.83	4.4
150⁴	99.70	12	116.17	4.3
150⁴	97.45	6	107.50	2.3
300⁴	91.39	6	98.00	2.6
300⁴	97.29	7	92.67	3.1
600⁴	87.74	17	96.50	7.3
600⁴	91.16	9	99.50	3.8
1200⁴	93.37	10	88.50	4.7
1200⁴	77.91	13	108.83	5.0
MCA (µg/mL)
5.00	33.53	175	82.83	88.0**
5.00	43.68	237	94.83	104.1**
¹Day 1 cell count x cloning efficiency, as compared to the average of the concurrent vehicle controls (average cloning efficiency = 70.00 %) ²Number of colonies / number of cells plated x 100 % (average of three plates; average cloning efficiency = 110.25 %) ³Total mutant colonies / number of cells selected, corrected for cloning efficiency (at time of selection) ⁴Test article incompletely soluble at end of treatment. - = Not determined **Significant increase (p<0.01); Snee and Irr, 1981

 

Initial Mutation Assay without S9
	Treatment (-S9)	Adjusted Relative Survival (%)¹	Total Mutant Colonies	Cloning Efficiency (%)²	TGr Mutants/ 10^6 Clonable Cells³
	Water (%, v/v)
	10.0	103.74	13	74.00	7.3
	10.0	96.26	7	96.33	3.0
	Tin metal powder (µg/mL)
	37.5⁴	79.10	-	-	-
	37.5⁴	94.18	-	-	-
	75.0⁴	76.71	6	84.17	3.0
	75.0⁴	89.64	26	87.83	12.3
	150⁴	77.80	13	89.50	6.1
	150⁴	63.75	11	75.67	6.1
	300⁴	91.02	6	82.17	3.0
	300⁴	71.74	6	90.83	2.8
	600⁴	84.51	14	80.17	7.3
	600⁴	77.01	14	93.33	6.3
	1200⁴	68.96	4	94.50	1.8
	1200⁴	55.80	8	96.33	3.5
	EMS (µg/mL)
	200	46.74	299	73.67	169.1**
	200	65.31	374	94.33	165.2**
¹Day 1 cell count x cloning efficiency, as compared to the average of the concurrent vehicle controls (average cloning efficiency = 77.92 %)  ²Number of colonies / number of cells plated x 100 % (average of three plates; average cloning efficiency = 85.17 %) ³Total mutant colonies / number of cells selected, corrected for cloning efficiency (at time of selection) ⁴Test article incompletely soluble at end of treatment. - = Not determined **Significant increase (p<0.01); Snee and Irr, 1981.

 

Confirmatory Mutation Assay with S9
Treatment (+S9)	Adjusted Relative Survival (%)¹	Total Mutant Colonies	Cloning Efficiency (%)²	TGr Mutants/ 10^6 Clonable Cells³
Water (%, v/v)
10.0	85.78	10	95.17	4.4
10.0	114.22	8	112.83	3.0
Tin metal powder (µg/mL)
37.5⁴	113.12	-	-	-
37.5⁴	100.81	-	-	-
75.0⁴	107.82	8	100.00	3.3
75.0⁴	108.99	9	114.17	3.3
150⁴	93.86	6	112.00	2.2
150⁴	100.16	12	101.00	5.0
300⁴	77.54	1	91.83	0.5
300⁴	83.97	9	97.17	3.9
600⁴	81.76	12	86.67	5.8
600⁴	77.36	8	97.67	3.4
1200⁴	65.83	9	78.00	4.8
1200⁴	77.17	6	101.67	2.5
MCA (µg/mL)
5.00	47.28	251	91.17	114.7**
5.00	39.42	220	100.00	91.7**
¹Day 1 cell count x cloning efficiency, as compared to the average of the concurrent vehicle controls (average cloning efficiency = 81.33 %)  ²Number of colonies / number of cells plated x 100 % (average of three plates; average cloning efficiency = 104.00 %) ³Total mutant colonies / number of cells selected, corrected for cloning efficiency (at time of selection) ⁴Test article incompletely soluble at end of treatment. - = Not determined **Significant increase (p<0.01); Snee and Irr, 1981.

 

Confirmatory Mutation Assay without S9
	Treatment (-S9)	Adjusted Relative Survival (%)¹	Total Mutant Colonies	Cloning Efficiency (%)²	TGr Mutants/ 10^6 Clonable Cells³
	Water (%, v/v)
	10.0	107.57	8	88.00	3.8
	10.0	92.43	9	81.33	4.6
	Tin metal powder (µg/mL)
	37.5⁴	98.02	-	-	-
	37.5⁴	119.12	-	-	-
	75.0⁴	104.59	5	93.00	2.2
	75.0⁴	120.83	9	115.83	3.2
	150⁴	111.42	10	103.50	4.0
	150⁴	114.74	2	116.33	0.7
	300⁴	90.67	10	95.67	4.4
	300⁴	84.22	4	111.00	1.5
	600⁴	79.84	5	101.67	2.0
	600⁴	94.51	5	113.00	1.8
	1200⁴	56.03	9	104.50	3.6
	1200⁴	46.76	6	103.17	2.4
	EMS (µg/mL)
	200	67.42	291	92.83	130.6**
	200	77.17	315	94.83	138.4**
¹Day 1 cell count x cloning efficiency, as compared to the average of the concurrent vehicle controls (average cloning efficiency = 82.33 %)  ²Number of colonies / number of cells plated x 100 % (average of three plates; average cloning efficiency = 84.67 %) ³Total mutant colonies / number of cells selected, corrected for cloning efficiency (at time of selection) ⁴Test article incompletely soluble at end of treatment. - = Not determined **Significant increase (p<0.01); Snee and Irr, 1981.

Conclusions:

Interpretation of results: negative both with and without metabolic activation

Under the conditions of the test, tin metal powder did not induce a positive mutagenic response.

Executive summary:

Tin metal powder (ground to produce a particle diameter range of 2 -11 µm ) was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) in Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system (S9). 

In the preliminary assay single cultures, with and without S9, were prepared at concentrations of 2.35, 4.70, 9.40, 18.8, 37.5, 75.0, 150, 300, 600, and 1200 mg/mL. The highest concentration evaluated approximated the limit dose for this assay (10 mM). 

Deionised water (di-H2O) was evaluated concurrently as the vehicle control. Little or no cytotoxicity was observed in the presence of S9, while limited evidence of cytotoxicity (decreases in adjusted relative survival) was observed in its absence. Adjusted relative survival values at concentrations of 600 and 1200 mg/mL without S9 were 58.85 and 47.76 %, respectively.  In addition, the test article was incompletely soluble at concentrations ≥18.8 mg/mL with and without S9.

In the main study, tin metal powder was evaluated in the initial mutagenicity assay at concentrations of 37.5, 75.0, 150, 300, 600, and 1200 mg/mL, with and without S9.  The vehicle control was evaluated concurrently.  All test and control concentrations were evaluated in duplicate cultures. Little or no cytotoxicity was observed for tin metal powder: the average adjusted relative survivals at 1200 mg/mL were 85.64 and 62.38 %, with and without S9, respectively.  Cultures treated at 37.5 mg/mL, with and without S9, were discarded because a sufficient number of higher concentrations were available. The average mutant frequencies of the vehicle control cultures were 1.7 and 5.2 TGr mutants/10^6 clonable cells with and without S9, respectively.

The average mutant frequencies of the colonies treated with tin metal powder ranged from 2.6 to 5.6 TGr mutants/10^6 clonable cells with S9, and 2.7 to 7.7 TGr mutants/10^6 clonable cells without S9. There were no statistically significant or dose-dependent increases in average mutant frequency observed with or without S9 (p >0.05).

Tin metal powder was re-evaluated in the confirmatory mutagenicity assay under identical conditions, and similar results were observed.  Little or no cytotoxicity was observed for tin metal powder.  Average adjusted relative survival at the high concentration of 1200 mg/mL were 71.50 and 51.40 %, with and without S9, respectively.  Those cultures treated at a concentration of 37.5mg/mL with and without S9 were discarded because a sufficient number of higher concentrations were available.  The average mutant frequencies of the vehicle control cultures were 3.7 and 4.2 TGr mutants/10^6 clonable cells, with and without S9, respectively, while the average mutant frequencies for the cultures treated with tin metal powder ranged from 2.2 to 4.6 TGr mutants/10^6 clonable cells with S9, and 1.9 to 3.0 TGr mutants/10^6 clonable cells without S9.  There were no statistically significant or dose-dependent increases in average mutant frequency observed with or without S9 (p >0.05).

Treatment with metallic tin powder, with or without S9, did not induce a statistically significant, dose-dependent increase in mutant frequency in either assay, that was greater than or equal to 15 TGr mutants/10^6 clonable cells above the average concurrent vehicle control mutant frequency. All control values were within acceptable ranges, and all criteria for a valid assay were met.

Tin metal powder was negative in the CHO/HPRT Forward Mutation Assay according to the criteria of the protocol/ test guidelines.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

All three submitted key studies (Beevers 2008, Lloyd 2009 and Stankowski 2009) were all performed in compliance with GLP and to the respective current standardised guideline. As such all studies were considered adequate for classification and labelling purposes, all studies were assigned a reliability score of 1 in line with the criteria of Klimisch et al. (1997).

Tin metal powder failed to induce a positive response in any of the genetic toxicity studies performed. As the results of all three studies are conclusive and concur, further in vivo studies are not required in accordance with point 8.4 under column 2 (specific rules for adaptation from column 1), Annex IX of the regulation EC No. 1907/2006, a study need only be proposed if a positive result is reported in the in vitro studies.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.