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Genetic toxicity in vitro

Description of key information

- Ames: Arni, 1981 (similar to POECD 471, GLP not specified) - K2 (test material 67%:33% MOTE: DOTE mixture)

In the experiments performed without and with microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of the test material revealed no marked deviations.

No evidence of the induction of point mutations by the test material or by the metabolites of the substance' formed as a result of microsomal activation was detectable in the strains of S. typhimurium and E. coli used in these experiments.

- Chromosome Aberration: De Vogel, 2010 (OECD 473, GLP) - K1 (test material 97.9% MOTE)

From the results obtained in two chromosomal aberration tests it is concluded that, under the conditions used in this study, the test substance Octyltin tris (2-ethylhexylmercaptoacetate) (MOTE) was not clastogenic to cultured human lymphocytes as it did not induce structural chromosomal aberrations.

- Gene mutation in mammalian cells: Steenwinkel, 2010 (OECD 476, GLP) - K1 (test material 97.9% MOTE)

It is concluded that under the conditions used in this study, the test substance Octyltin tris (2-ethylhexylmercaptoacetate) (MOTE) is mutagenic at the TK-locus of mouse lymphoma L5178Y cells, although clastogenicity can not be excluded.

More specifically, the findings are positive without metabolic activation and ambiguous with metabolic activation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8th August - 11th September 1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
not specified
Remarks:
pre-existing study
Type of assay:
bacterial reverse mutation assay
Target gene:
S. typhimurium: histidine locus
E. coli: tryptophan locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
N/A
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
N/A
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
N/A
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of rat livers
Test concentrations with justification for top dose:
5, 10, 50, 100, 500, 1000, 5000 µg/0.1 ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone for test substance, DMSO for positve controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: See 'Any other information on materials and methods incl. tables' for information
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Preincubation period: NDA
- Exposure duration: 48 hours
- Expression time (cells in growth medium): NDA
- Selection time (if incubation with a selection agent): NDA
- Fixation time (start of exposure up to fixation or harvest of cells): NDA

SELECTION AGENT (mutation assays): NDA
SPINDLE INHIBITOR (cytogenetic assays): NDA
STAIN (for cytogenetic assays): NDA

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: NDA

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the number of bacteria in the treated and control cultures that have undergone back-mutation to histidine- or tryptophan-prototrophism. To ensure that mutagenic effects of metabolites of the test substance formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture (rat liver microsomes and co-factors).
Statistics:
no
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
NDA

Table 1. Results of tests - mean colony counts.

Material Concentration of testing sample (µg/plate) Presence of S9 mix Back mutation - Number of colonies/plate (mean)
Base conversion type Frame shift type
TA100 TA1535 WP2UVrA TA98 TA1537 TA1538
Vehicle control N/A - 148 15 22 22 6 12
TK 12152 5 - 133 13 25 25 5 6
10 - 136 12 26 17 5 12
50 - 134 17 21 23 7 17
100 - 139 15 18 25 6 12
500 - 122 11 17 20 5 15
1000 - 132 13 23 16 5 11
5000 - 117 15 23 16 8 8
Vehicle control N/A + 132 14 23 34 5 54
TK 12152 5 + 116 8 22 45 11 53
10 + 135 7 22 40 7 50
50 + 128 7 28 36 8 55
100 + 122 11 29 41 8 43
500 + 138 9 21 39 6 40
1000 + 107 10 23 33 5 38
5000 + 110 8 26 27 4 24
Conclusions:
In the experiments performed without and with microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of the test material revealed no marked deviations.
No evidence of the induction of point mutations by the test material or by the metabolites of the substance' formed as a result of microsomal activation was detectable in the strains of S. typhimurium and E. coli used in these experiments.
Executive summary:

Octyltin tris(2-ethylhexylmercaptoacetate was tested for mutagenic effects on histidine-auxotrophic strains of Salmonella typhimurium and on a tryptophan auxotrophic strain of E. coll. The investigations were performed with the following concentrations of the trial substance without and with microsomal activation: 5, 10, 50, 100, 500, 1000 and 5000 µg/0.1 ml.

In the experiments performed without and with microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of the test material revealed no marked deviations.

No evidence of the induction of point mutations by the test material or by the metabolites of the substance' formed as a result of microsomal activation was detectable in the strains of S. typhimurium and E. coli used in these experiments.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 December 2009 to 4 February 2010
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:
yes
Remarks:
see below
Principles of method if other than guideline:
After arrival at the testing facility, the test substance was stored for two days at ambient temperature. Thereafter, the test substance was stored at 2-10ºC, in the dark. Based upon the physico-chemical information (see section 3) this deviation was considered to have no impact upon the reliability of the study.
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable to test type.
Species / strain / cell type:
lymphocytes: Human
Details on mammalian cell type (if applicable):
- Type and identity of media:
The medium for culturing the human peripheral blood lymphocytes consisted of RPMI-1640 medium (with Glutamax-I), supplemented with heat-inactivated (30 min, 56ºC) foetal calf serum (20%), penicillin (100 IU/ml medium), streptomycin (100 μg/ml medium) and phytohaemagglutinin (2.4 μg/ml).

- Properly maintained:
yes

- Periodically checked for Mycoplasma contamination:
not reported

- Periodically checked for karyotype stability:
not reported.

- Periodically "cleansed" against high spontaneous background:
note reported.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Test 1: 2000, 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.8 & 3.9 µg/ml
Test 2: 500, 300, 200, 150, 100, 50 & 25 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:
DMSO

- Justification for choice of solvent/vehicle:
The test substance could be suspended in DMSO up to 500 mg/ml, the stock formulation.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Cyclophosphamide (indirect acting clastogen) & Mitomycin C (direct acting clastogen).
Details on test system and experimental conditions:
METHOD OF APPLICATION:
in medium

DURATION
- Preincubation period:
48 hours at 37ºC in humidified air containing 5% CO2, conducted in sterile loose tubes.

- Exposure duration:
4 hours

- Expression time (cells in growth medium):
22 hours after onset of treatment

- Incubation time with spindle inhibitor:
2 hours

- Fixation time (start of exposure up to fixation or harvest of cells):
24 hours

SPINDLE INHIBITOR (cytogenetic assays):
colcemid

STAIN (for cytogenetic assays):
Giemsa

NUMBER OF REPLICATIONS:
2 slides were prepared for each selected culture

NUMBER OF CELLS EVALUATED:
50 well spread metaphase cells per slide (100 per culture, 200 per concentration).

DETERMINATION OF CYTOTOXICITY
- Method:
The number of metaphases containing one or more aberrations was compared with those of the concurrent negative controls using Fisher's exact test (one-sided). The results are considered statistically significant when the p-value of the Fisher’s exact test is less than 0.05.
The study was considered valid because the positive controls gave a statistically significant increase in the number of aberrant cells and the negative controls were within the historical range.
There are several criteria for determining a positive response, such as a statistically significant concentration-related increase or a reproducible statistically significant increase in the number of metaphases containing one or more aberrations at one or more test concentrations.
A response was considered to be equivocal if the percentage of aberrant cells was statistically marginal higher than that of the negative control (0.05A test substance was considered to be negative if it produces neither a statistically significant concentration-related increase nor reproducible statistically significant increase in the number of metaphases containing one or more aberrations, at any of the test concentrations analysed. A dose related but not significant increase in the number of aberrant cells (i.e., a dose related response in the range of negative historical control values) was judged as negative.
The total number of metaphases containing one or more aberrations (excluding cells with only gaps) of the negative control was compared to the total number of metaphases containing one or more aberrations (excluding cells with only gaps) of the test substance treated groups.
Statistical methods were used as an aid in evaluating the test results but were not the only determining factor for a positive response. Both statistical methods and biological relevance of the results were considered together in the evaluation.


OTHER EXAMINATIONS:
If heavily damage sells, endoreplicated cells or polyploid cells were observed these cells were recorded but the cells were not counted and included in the 200 analysed cells.
Evaluation criteria:
There are several criteria for determining a positive response, such as a statistically significant concentration-related increase or a reproducible statistically significant increase in the number of metaphases containing one or more aberrations at one or more test concentrations.

A response was considered to be equivocal if the percentage of aberrant cells was statistically marginal higher than that of the negative control (0.05
A test substance was considered to be negative if it produces neither a statistically significant concentration-related increase nor reproducible statistically significant increase in the number of metaphases containing one or more aberrations, at any of the test concentrations analysed. A dose related but not significant increase in the number of aberrant cells (ie, a dose related response in the range of negative historical control values) was judged as negative.

Statistical methods were used as an aid in evaluating the test results but were not the only determining factor for a positive response. Both statistical methods and biological relevance of the results were considered together in the evaluation.
Statistics:
The number of metaphases containing one or more aberrations was compared with those of the concurrent negative controls using Fisher's exact test (one-sided). The results are considered statistically significant when the p-value of the Fisher’s exact test is less than 0.05.
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Precipitation of the test substance was noted in the preliminary solubility test between 5000 and 156µg/ml, therefore test concentrations in excess of 250 µg/ml were not scored for clastogenic effects.

RANGE-FINDING/SCREENING STUDIES:
A preliminary solubility observed precipitation of the test material shortly after preparation with and without 20% serum (metabolic activation) at concentrations between 5000 µg/ml (the highest concentration tested) and 625 µg/ml, slight precipitation was also observed after preparation at concentrations of 313 µg/ml. After 24 hours precipitation was observed in concentrations between 156 and 5000 µg/ml.

COMPARISON WITH HISTORICAL CONTROL DATA:
not applicable

ADDITIONAL INFORMATION ON CYTOTOXICITY:
none reported.

See Tables 1 -8 (attached) for individual and mean per concentration results.

Conclusions:
From the results obtained in two chromosomal aberration tests it is concluded that, under the conditions used in this study, the test substance Octyltin tris (2-ethylhexylmercaptoacetate) (MOTE) was not clastogenic to cultured human lymphocytes as it did not induce structural chromosomal aberrations.
Executive summary:

The test substance Octyltin tris (2-ethylhexylmercaptoacetate) (MOTE) was examined for its potential to induce structural chromosomal aberrations in cultured human lymphocytes, in both the absence and presence of a metabolic activation system (S9-mix). Dimethylsulfoxide (DMSO) was used as solvent for the test substance. Two separate tests were conducted for which blood was obtained from two different donors. Dose levels, ranging from 0.1 to 2000 μg/ml (final concentrations in the culture medium), were tested. The purity of the test substance (97.9%) was taken into account while preparing the dosing solutions. In all instances, duplicate cultures were used and both solubility and cytotoxicity were used for dose level selection. Reduction of the mitotic index was used as indication for cytotoxicity. Cyclophosphamide, an indirect acting clastogenic compound which requires metabolic activation, was used as positive control in the presence of S9-mix. Mitomycin C, a direct acting clastogenic compound was used as positive control in the absence of S9-mix.

In the first test, in the presence and absence of S9-mix, the treatment/harvesting times were 4/24 hours (pulse treatment). In the presence of S9-mix, the cultures of three dose levels of the test substance (7.8, 62.5 and 125 μg/ml), the cultures of the solvent control (DMSO) and the cultures of the positive control Cyclophosphamide were analysed for the induction of chromosomal aberrations. At higher dose levels the test substance was too toxic for the cells (mitotic index reduction >70% at the dose level of 250 μg/ml). In the absence of S9-mix, the cultures of three dose levels of the test substance (7.8, 125 and 250 μg/ml), the cultures of the solvent control (DMSO) and the cultures of the positive control Mitomycin C were analysed for the induction of chromosomal aberrations. At higher dose levels (500, 1000 and 2000 μg/ml) the test substance was too toxic for the cells, demonstrated by a lack of stimulated lymphocytes and metaphases.

In the second test, in the presence and absence of S9-mix, the treatment/harvesting times were 4/24 hours (pulse treatment) and 24/24 hours (continuous treatment), respectively. In the presence of S9-mix, the cultures of three dose levels of the test substance (150, 300 and 500 μg/ml), the cultures of the solvent control (DMSO) and the cultures of the positive control Cyclophosphamide were analysed for the induction of chromosomal aberrations. Dose-related toxicity could be demonstrated in this treatment group. In the absence of S9-mix, the cultures of three dose levels of the test substance (50, 100 and 150 μg/ml), the cultures of the solvent control (DMSO) and the cultures of the positive control Mitomycin C were analysed for the induction of chromosomal aberrations. At higher dose levels the test substance was too toxic for the cells, demonstrated by a mitotic index reduction of >70% and a lack of stimulated lymphocytes and metaphases at the dose level of 300 μg/ml and a poor quality of the metaphases at the dose level of 200 μg/ml.

Treatment with the positive controls Cyclophosphamide and Mitomycin C resulted in statistically significant increases in the numbers of metaphases containing one or more chromosomal aberrations, when compared to the numbers observed in the cultures treated with the solvent control. This demonstrates the validity of the study.

In both chromosomal aberration tests, the test substance did not induce a statistically significant increase in the number of metaphases containing one or more chromosomal aberrations at any of the dose levels and time points analysed.

From the results obtained in two chromosomal aberration tests it is concluded that, under the conditions used in this study, the test substance Octyltin tris (2-ethylhexylmercaptoacetate) (MOTE) was not clastogenic to cultured human lymphocytes as it did not induce structural chromosomal aberrations.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April-May 2010
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 (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
TK gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The L5178Y tk +/- 3.7.2C cells to be used in the TK-assay were obtained from Dr. J. Cole, MRC Cell Mutation Unit, University of Sussex, United Kingdom. The cells are stored as frozen stock cultures in liquid nitrogen. Subcultures are prepared from these stocks for experimental use. At 3-5 days prior to freezing the cells are cleansed with methotrexate-containing growth medium in order to reduce the background level of spontaneous TK-deficient cells. The cells are checked at regular intervals for the absence of mycoplasma contamination.
L5178Y cells will be grown in growth medium consisting of RPMI 1640 medium (with HEPES and L-Glutamine) supplemented with heat-inactivated horse serum (10% v/v for growing in flasks, and 20% for growing in microtiter plates), sodium pyruvate and penicillin/streptomycin.
Metabolic activation:
with and without
Metabolic activation system:
a liver homogenate fraction (S9)
Test concentrations with justification for top dose:
-Without S9 / 4h of exposure : 0, 4.8, 6.9, 9.8, 14, 20, 29 and 32 µg/ml
-Without S9 / 24h of exposure : 0, 2.4, 3.4, 4.8, 6.9, 9.8, 14, 16, 18 and 20 µg/ml
-With S9 / 4h of exposure : 0, 4.6, 6.5, 9.3, 13, 19, 27, 39 and 55 µg/ml
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Methyl methanesulphonate (MMS) without S9 and 3-methylcholanthrene (MCA) with S9
Details on test system and experimental conditions:
Cell culturing
The cells will be cultured in a humidified incubator at ca 37 °C in air containing ca 5% CO2. Five to seven days prior to treatment, the cells will be generated from a frozen stock culture by seeding them in sterile, screw-capped tissue culture flasks (about 10,000,000 cells per flask: growth area ± 75 cm²) containing 50 ml growth medium (with 10% horse serum). Near-confluent cells will be harvested from a number of culture flasks and suspended in growth medium (with 10% horse serum), and the number of cells will be counted. For the cytotoxicity and gene mutation assays portions of ca. 3,000,000 and ca. 5,000,000 cells will be used per culture in the absence and presence of metabolic activation, respectively.
Each flask, microtiter plate or tube, used for the conduct of the test, will be identified by a code representing the test substance and the test substance concentration, and identifying the presence or absence of a metabolic activation system (S9-mix).

Preparation of the test substance solutions and dose levels
The test substance will be dissolved or suspended in an appropriate vehicle and added to single cell cultures at 13 serial dilutions if toxicity is expected, and to duplicate cell cultures at 6 serial dilutions if no or hardly no toxicity is expected. The intervals between the concentrations will be 0.7 at the higher concentrations and 0.5 at the lower concentrations. RPMI medium will preferably be used as solvent for the test substance. Test substance solutions in RPMI medium will be sterilized by passage through a micropore filter. If RPMI medium is not appropriate, dimethyl sulfoxide (DMSO), ethanol, methanol, and THF will be successively evaluated as vehicle, in which case the final concentration of solvent in the cultures will be 1% (v/v). The maximum concentration of the test substance in the final culture medium will be 5 mg/ml based on a purity of 97.9%. For relatively insoluble substances the highest dose tested will be the lowest insoluble concentration in the final culture medium.
If toxicity is the limiting factor the highest dose used should cause at least 80% toxicity (reduction in initial cell yield or relative total growth, see §4.5). If no data on toxicity are available, preceding the main study, a dose range finding study will be performed.

Study
Prior to treatment, the growth rate (doubling time of 9-14 h) and viability (> 90%) of the cells will be checked. To 3,000,000 or 5,000,000 cells in 5 ml growth medium (with 10% horse serum), the test substance, negative control or positive control and, if required, growth medium (with 0% horse serum) will be added for treatment without the S9-mix to a final volume of 10 ml. For treatment with S9-mix, 1 ml S9-mix will also be added. The cells will be exposed to the test substance, the vehicle control or the positive control for 4 and/or 24 h in the absence of S9-mix and for 4 h in the presence of S9-mix, at ca 37 °C and ca 5% CO2 in a humidified incubator. At the start of the treatment all cell cultures will be checked visually for precipitation of the test substance, discolouring of the medium due to pH change and/or any other aberrancy. If appropriate, the pH and osmolality will be checked.

Assessment of cytotoxicity
The cytotoxicity of the test substance will be determined by counting the cells after exposure and by measuring the relative suspension growth (RSG) and the relative total growth (RTG) of the cells. The RSG is a measure for the cumulative growth rate of the cells 24 h and 48 h after treatment compared with untreated control cultures; the RTG is the product of the relative initial cell yield after treatment, the RSG and the relative colony-forming ability (‘cloning efficiency’) of the cells 48 h after treatment compared with untreated control cultures.
Evaluation criteria:
The following criteria will be used to validate the data obtained:
a) the average cloning efficiency of the negative controls should not be less than 60% or more than 140%,
b) the average suspension growth of the negative controls should be between 8 and 32.
c) the average mutant frequency of the negative controls should fall within the range of 40-300 TFT-resistant mutants per 1,000,000 clonable cells,
d) the mutant frequency of the positive controls should be higher than 400 TFT-resistant mutants per 1,000,000 clonable cells, and should at least be 2-fold higher than the corresponding negative control,
e) unless the test substance shows no cytotoxicity at the highest possible concentration (determined by its solubility, pH and osmolar effects), the highest test substance concentration should result in a clear cytotoxic response. The RTG value of one of the data points should be between 10 and 20%, or one data point should be between 1 and 10% and another between 20 and 30%.
Statistics:
no
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Dose levels and visual observations before and after treatment
-First test
In the absence and presence of S9-mix, the dose levels of the test substance used ranged from 0.3 to 120 μg/ml and 2.4 to 500 μg/ml, respectively.
At the start of the 24 hours treatment in the absence of S9-mix, no abnormalities were observed; at the end of the treatment, the viability of the cells at and above 59 μg/ml was decreased.
At the start of the 4 hours treatment in the presence of S9-mix, dose related turbidity was observed at and above 120 μg/ml. At the end of the treatment period no abnormalities were observed. The viability of the cells at and above 172 μg/ml was decreased.
-Second test
In the absence of S9-mix, the dose levels of the test substance used ranged from 1.7 to 30 μg/ml and 4.8 to 60 μg/ml, for 24 or 4 hours treatment, respectively. In the presence of S9-mix, the dose levels of the test substance used ranged from 4.6 to 120 μg/ml.
At the start and end of the 4 hours treatment in the absence of S9-mix, no abnormalities were observed. At the end of the treatment period the viability of the cells at and above 54 μg/ml was decreased.
At the start and end of the 24 hours treatment in the absence of S9-mix, no abnormalities were observed. At the end of the treatment, the viability of the cells at and above 24 μg/ml was decreased.
At the start of the 4 hours treatment in the presence of S9-mix, dose related turbidity was observed at and above 87 μg/ml. At the end of the treatment period no abnormalities were observed and the viability of the cells at 120 μg/ml was above 90%.

Cytotoxicity / Cytotoxicity is defined as a reduction by more than 10% initial cell yield and/or suspension growth.
-First test
In the absence of S9-mix during 24 hours exposure the test substance was toxic to the cells. Cytotoxicity, was observed at and above at 1.2 μg/ml. The highest dose level of the test substance that could be evaluated for mutagenicity was 29 μg/ml; the RTG at this dose was 7%.
In the presence of S9-mix, after 4 hours treatment the test substance was toxic to the cells. Cytotoxicity was observed at and above 59 μg/ml. The highest dose level of the test substance that could be evaluated for mutagenicity was 84 μg/ml; the RTG at this dose was 48%.
-Second test
In the absence of S9-mix after 4 hours exposure cytotoxicity was observed at and above 9.8 μg/ml. The highest dose level of the test substance that could be evaluated for mutagenicity was 32 μg/ml; the RTG at this dose was 5%.
In the absence of S9-mix after 24 hours exposure cytotoxicity was observed at and above 2.4 μg/ml. The highest dose level of the test substance that could be evaluated for mutagenicity was 20 μg/ml; the RTG at this dose was 6%.
In the presence of S9-mix cytotoxicity was observed at and above 13 μg/ml. The highest dose that could be evaluated for mutagenicity was 55 μg/ml; the RTG at this dose was 8%.

Mutagenicity
-First test
In the absence of S9-mix dose related significant increases in mutant frequency (MF) were observed at concentrations of 20 and 29 μg/ml (more than 126 mutants per 1,000,000 clonable cells compared to the negative control). The RTG at these concentrations were 14% and 7%, respectively.
In the presence of S9-mix the mutant frequency was equivocally increased (more than 88 and less than 126 mutants per 1,000,000 clonable cells compared to the negative control) at a concentration of 84 μg/ml. The RTG at this concentration was 48%.
-Second test
Since in the first test, in the absence of S9-mix after 24 hours exposure, at a single concentration (20 μg/ml), causing less than 90% cytotoxicity, a significant positive response was observed, and in the presence of S9-mix (at 84 μg/ml) an equivocal response was observed at a concentration causing about 50% cytotoxicity, it was decided to repeat the assay with more data points at concentrations causing 50 to 90% cytotoxicity.
Additionally, in the absence of S9-mix cells were exposed for 4 hours since in the first test, due to cytotoxicity, the highest dose level based on physiological parameters (5 mg/ml, 5 Xl/ml, 10 mmol/l or solubility) could not be evaluated for mutagenicity.
In this second test, in the absence of S9-mix after 4 hours treatment the mutant frequency was significantly increased at concentrations ranging from 14 to 32 μg/ml. The RTG at these concentrations ranged from 39 to 5%, respectively.
In the absence of S9-mix after 24 hours treatment the mutant frequency was significantly increased at concentrations ranging from 14 to 20 Xg/ml and equivocally increased at 9.8 Xg/ml. The RTG at these concentrations ranged from 41 to 6% respectively.
In the presence of S9-mix the mutant frequency was significantly increased at concentrations ranging from 39 to 55 μg/ml and equivocally increased at 27 µg/ml. The RTG at these concentrations ranged from 60 to 8%, respectively.
In general, increases in mutant frequency at concentrations causing more than 90% cytotoxicity (RTG <10%) were considered to be artificial and not indicative of genotoxicity. However, in all three assays of the second test in at least 2 concentrations per assay a significant or equivocal increase in mutant frequency was observed at concentrations causing less than 90% cytotoxicity (RTG >10%).

Colony sizing
At concentrations causing an increase in mutant frequency and resulting in less than 90% cytotoxicity (RTG >10%) the mutant colonies were scored using the criteria of small and large colonies.
In the absence of S9-mix generally slightly more small than large colonies were formed. In the first test at 20 μg/ml 61% small colonies compared to 39% large colonies were formed. In the second test the mean percentage of small colonies after 4 hours at 14 and 20 μg/ml was 60% compared to 40% of large colonies, and after 24 hours the mean percentage of small colonies at 9.8, 14 and 16 μg/ml was 65% compared to 35% of large colonies. Overall, the mean percentage of small colonies was 63% (mean of 61, 65, 56, 68. 68 and 60%).
In the presence of S9-mix also more small than large colonies were formed. In the first test at 84 μg/ml the percentage small colonies was 67% compared to 33% of large colonies and in the second test the mean percentage of small colonies at 27 and 39 μg/ml was also 67% compared to 33% of large colonies.
Although in general more small than large colonies were formed, overall the number of both small and large colonies was increased, and therefore, neither mechanism (clastogenicity and mutagenicity) can be excluded.

Positive and negative controls
Methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA) were used as positive control substances in the absence and in the presence of S9-mix, respectively; Dimethyl sulphoxide (DMSO) served as negative control. The negative controls were within historical background ranges and treatment with the positive controls yielded the expected significant increase in mutant frequency compared to the negative controls.

Table 1 : Results of the gene mutation assay with MOTE,

Treatment in the absence of S9-mix (24h treatment) ; first assay

Dose (mmol/l)

Initial cell yield (x10^6)

Relative1initial cell yield (%)

Suspension growth

Relative1suspension growth (%)

Cloning efficiency

Relative1cloning efficiency (%)

Relative total growth (%)

Mutant cloning efficiency (x10^6)

Mutant frequency (x10^6)

Mutant2colonies

large (%)

Small (%)

MMS 0.1mM

7.33

69

8.96

47

0.33

34

11

384

1161

37

63

120

1.60

15

1.28

7

*

 

 

 

 

 

 

84

1.74

16

1.20

6

*

 

 

 

 

 

 

59

2.01

19

1.30

7

*

 

 

 

 

 

 

41

3.32

31

3.58

19

*

 

 

 

 

 

 

29

4.56

43

6.38

33

0.51

52

7

186

366

42

58

20

4.91

46

7.91

41

0.71

72

14

213

300

39

61

14

6.66

63

13.75

72

1.01

102

46

138

137

52

48

10

7.61

72

15.22

80

0.87

88

51

104

120

58

42

6.9

8.19

77

#

 

 

 

 

 

 

 

 

4.8

8.26

78

17.22

90

0.80

81

57

61

76

 

 

2.4

9.35

88

17.22

90

0.85

86

69

61

71

 

 

1.2

9.14

86

16.79

88

0.82

83

63

91

111

 

 

0.6

10.10

95

17.67

92

0.91

92

81

49

54

 

 

0.3

10.12

95

18.22

95

0.88

90

82

55

62

 

 

0

10.42

98

18.57

97

0.94

96

91

58

61

76

24

0

10.81

102

19.68

103

1.03

104

109

78

76

68

32

1values are given relative to the mean of that of the vehicle negative control

2large and small mutant colonies are given as percentage of all mutant colonies

# cultures discarded because they were superfluous

* cultures discarded due to toxicity

 

 

Table 2 : Results of the gene mutation assay with MOTE,

Treatment in the presence of S9-mix (4h treatment) ; first assay

Dose (mmol/l)

Initial cell yield (x10^6)

Relative1initial cell yield (%)

Suspension growth

Relative1suspension growth (%)

Cloning efficiency

Relative1cloning efficiency (%)

Relative total growth (%)

Mutant cloning efficiency (x10^6)

Mutant frequency (x10^6)

Mutant2colonies

large (%)

Small (%)

MCA 10

4.88

95

19.74

90

0.74

71

60

485

656

58

42

500

2.52

49

*

 

 

 

 

 

 

 

 

350

2.24

43

*

 

 

 

 

 

 

 

 

245

1.53

30

*

 

 

 

 

 

 

 

 

172

1.56

30

*

 

 

 

 

 

 

 

 

120

2.84

55

1.35

6

<0.1

<10

<1

 

 

 

 

84

4.05

79

15.09

69

0.92

88

48

178

194

33

67

59

4.61

89

20.08

92

1.04

99

81

123

119

 

 

41

4.75

92

19.94

91

1.01

97

81

106

105

 

 

29

5.06

98

20.07

92

0.85

81

73

92

109

 

 

20

4.96

96

21.18

97

0.89

85

79

92

104

 

 

14

5.02

97

21.87

100

1.08

103

100

38

35

 

 

10

5.16

100

20.71

95

1.23

118

111

66

54

 

 

6.9

5.20

101

#

 

 

 

 

 

 

 

 

4.8

5.27

102

22.33

102

1.25

119

124

64

51

 

 

2.4

5.05

98

#

 

 

 

 

 

 

 

 

0

5.14

100

21.97

100

0.95

91

91

81

85

52

48

0

5.18

100

21.86

100

1.14

109

109

67

59

58

42

1values are given relative to the mean of that of the vehicle negative control

2large and small mutant colonies are given as percentage of all mutant colonies

# cultures discarded because they were superfluous

* cultures discarded due to toxicity

Conclusions:
It is concluded that under the conditions used in this study, the test substance Octyltin tris (2-ethylhexylmercaptoacetate) (MOTE) is mutagenic at the TK-locus of mouse lymphoma L5178Y cells, although clastogenicity can not be excluded.
Executive summary:

The test substance Octylyin tris (2-ethylhexylmercaptoacetate) (MOTE) was tested and evaluated for mutagenicity in both the absence and presence of metabolic activation (S9-mix). In addition to treatment for 4 hours in the absences and presence of a metabolic activation system, an extended treatment for 24 hours in the absence of S9- mix was used. In the absence of S9-mix significant, reproducible and dose related increases in mutant frequency were observed at concentrations causing less than 90% cytotoxicity (RTG>10%). In the presence of S9-mix in the first test the mutant frequency was equivocally increased at a single dose (84 Xg/ml) causing about 50% cytotoxicity. In the second test a dose related and significant increase was observed at dose levels causing less than 90% cytotoxicity (RTG>10%). In addition, at concentrations causing an increase in mutant frequency, relatively more small than large colonies were formed. However, since the number of both small and large colonies was increased, neither mechanism (clastogenicity and mutagenicity) can be excluded. It is concluded that under the conditions used in this study, the test substance Octylyin tris (2-ethylhexylmercaptoacetate) (MOTE) is mutagenic at the TK-locus of mouse lymphoma L5178Y cells, although clastogenicity can not be excluded.

Genetic toxicity in vivo

Description of key information

- Micronucleus: Reus, 2012 (OECD 474, GLP) - K1 (test material 97.7% MOTE)

Octyltin tris (2-ethylhexylmercaptoacetate) MOTE did not show any indication of chromosomal damage and/or damage to the mitotic spindle apparatus of the bone marrow target cells of male rats.

- COMET Assay: Rashmi, 2019 (OECD 489, GLP) - K1 (test material 96.1% MOTE)

The data obtained under the conditions employed during this experiment support the conclusion that the test item, MOTE, did not induce any increase in DNA damage in cells from the liver, glandular stomach or duodenum of Wistar rats at any oral dose up to and including a maximum tolerated dose of 300 mg/kg.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
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 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
29. July 2016
tissues: liver, glandular stomach and duodenum
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian comet assay
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rat is one of the recommended species by regulatory agencies for conducting in vivo Comet assay among rodents.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Source of supply : In-house bred animals
No. of groups : 5 groups (1 Vehicle Control,1 Positive Control and 3 Treatment groups)
No. of animals /group and sex: 10 (5 Males + 5 Females)
Females used were nulliparous and non pregnant
Age at treatment : 7 weeks
Body weight range at receipt (g): Male - 194.01 to 220.12 g, Female - 190.11 to 217.01 g
Animal identification : Acclimatization Period - Cage cards with minimum details of study no., animal no., sex and tail marking with marker pen.
Treatment Period - All the animals were identified by body marking using turmeric solution, potassium permanganate (f roup five) and additionally, a cage card was displayed which includes study no., cage no., sex, animal no. (permanen reatment date and date of necropsy.

Environmental Conditions: Animals were housed under standard laboratory conditions, in an environmentally monitored air-conditioned room with adequate
fresh air supply (12 to 15 air changes per hour), room temperature 19.8 to 22.9°C and relative humidity 48 to 67% with 12 hours light and 12 hours dark cycle. The temperature and relative humidity were recorded once daily.

Housing Maximum of three animals of same sex and group were housed together in a standard polypropylene cage
(L 430 × B 285 × H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water in water bottle fitted with stainless steel sipper tube. Sterilized paddy husk was used as a bedding material.

Feed: Altromin Maintenance Diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to
the animals throughout the experimental period. The contaminant analysis test reports of feed are included as Annexure 4.

Water: Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through rever se osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.

Route of administration:
oral: gavage
Vehicle:
Peanut Oil
Lot No.: AG07110
Expiry Date: 17/02/2022
Make: MP Biomedicals
Details on exposure:
The test item was administered by the oral route once a day for 2 consecutive days using gavage cannula. The test item was administered in a dose volume of 5 mL/kg with the doses of 100, 200 and 300 mg/kg designated as low (G2), mid (G3) and high dose (G4), respectively. The vehicle control group (G1) animals were administered the peanut oil (vehicle) only at a dose volume of 5 mL/kg body weight.

The positive control group (G5) animals were administered with Ethyl methanesulfonate dissolved in distilled water at the volume of 10 mL/kg body weight. Ethyl methanesulfonate was administered at a dose of 250 mg/kg.

The dose volume to be administered was calculated for individual animals based on treatment day body weight of the animal.
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes
Positive control(s):
EMS 250 mg/kg
Tissues and cell types examined:
Animals were euthanized by cervical dislocation post 2 to 6 hours after the second treatment. Selected tissue (liver, glandular stomach and duodenum) were removed, dissected and a portion was collected for the comet assay. The tissues for the comet assay were placed into ice-cold mincing and stored on ice. Tissues were rinsed sufficiently with cold mincing buffer to remove residual blood and were stored in ice- cold mincing buffer until processed. All the animals were subjected to gross pathological examination

Single cell suspensions were prepared by chopping the tissue with scalpels in a small amount of ice-cold mincing solution. The scraped tissue solution was transferred into a homogenization tube and then gently homogenized using a homogenizer. Homogenate was filtered through a 30 micron pore nylon mesh and was then centrifuged at 800xg for 10 minutes. Single cells were suspended in DPBS. Cell suspensions were maintained at 2 to 8°C
Details of tissue and slide preparation:
Three slides were prepared for each tissue from each animal and labelled with the study number, animal no., tissue, slide number (e.g. 1/3 to 3/3) and sex of each animal using pencils. To reduce the possibility of detachment of the agarose during the procedure, slides were pre coated with 100 µL of liquid agarose and the agarose was allowed to dry to a thin film. Approximately 75 µL of cell suspension with 75 µL of
1.0% low-melting agarose gel was mixed and rapidly pipette onto the surface of the pre coated slides and a coverslip was placed on it. Slides were placed on ice packs until the Agarose layer hardened (20 minutes). Slides were immersed in chilled lysing solution in the dark for overnight. After completion of lysing, the slides were rinsed in distilled water to remove residual detergent and salts prior to alkali unwinding step.

Evaluation criteria:
All the slides were coded before evaluation to avoid group bias during evaluation. Before scoring, slides were rehydrated with chilled distilled water for 30 minutes and stain with Ethidium bromide, covered with a fresh coverslip and cells were scored under 200 X magnification. At least 150 cells were analyzed per sample. The comet endpoints collected was % tail DNA, tail length in microns measured from the estimated edge of the head region closest to the anode. The frequency of hedgehogs were determined of at least 150 cells per sample.
Statistics:
Body weight were analyzed by SPSS at a 95% level of confidence (p<0.05) of significance. Inter group comparison of Body weight (day 1 and day 2) and percent tail DNA. The dose correlation was done using ‘t’ test.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
The positive control [G5], Ethyl methanesulfonate at a dose of 250 mg/kg produced a statistically significant increase in % tailing of DNA in cells from all the organs which were assessed (Liver, glandular stomach and duodenum)
Conclusions:
The data obtained under the conditions employed during this experiment support the conclusion that the test item, MOTE, did not induce any increase in DNA damage in cells from the liver, glandular stomach or duodenum of Wistar rats at any oral dose up to and including a maximum tolerated dose of 300 mg/kg.
Executive summary:

The results for the assessment of the test item, MOTE, to cause DNA strand breaks are provided for the doses of 100 [G2], 200 [G3] and 300 [G4] mg/kg, respectively, in male and female Wistar rats. The average % tail DNA observed in liver is 2.43, 2.40, 2.53 and 2.83 in males and 2.54, 2.46, 2.51 and 2.90 in females dosed at 0, 100, 200 and 300mg/kg, respectively. In glandular stomach, the observed average % tail DNA is 3.21, 3.35, 3.75 and3.85 in males and 3.34, 3.41, 3.88 and 3.94 in females dosed at 0, 100, 200 and 300 mg/kg, respectively. The average % tail DNA observed in duodenum was 2.85, 2.88, 2.89 and 3.26 in males and 2.96, 3.07, 3.00 and 3.19 in females dosed at 0, 100, 200 and 300 mg/kg, respectively. There was no dose-related or statistically significant increase in the % tailing of DNA from cells of any organ for any of the MOTE groups when compared to the vehicle control group.

The positive control [G5], Ethyl methane sulfonate at a dose of 250 mg/kg produced a statistically significant increase in % tailing of DNA in cells from all the organs which were assessed (Liver, glandular stomach and duodenum) when compared to the equivalent cells from organs of vehicle control animals [G1]. These data support the conclusion that the test conditions and sensitivity of the COMET assay for this test of MOTE were fully adequate.

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)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
Source:
- Age at study initiation: 6 weeks old
- Assigned to test groups randomly: yes
- Fasting period before study: yes
- Housing:
During the DRF study and the bone marrow micronucleus test, the animal room
was ventilated with about 10 air changes per hour and maintained at a temperature of 20-24°C.
The relative humidity in the animal room was between 45-65%, except during short periods on 27 January and 6, 7 and 10 February 2012, when the relative humidity in the animal room exceeded the lower and/or upper limit (minimum recorded value,
34.3%; maximum recorded value, 67.1%).

Caging
The animals were housed in groups of five, in macrolon cages (type IV) with wood shavings (Lignocel, type ¾) as bedding material and a wooden block and strips of paper as environmental enrichment (Enviro-dri).

- Diet (e.g. ad libitum) and water :
Feed (with the exception of the fasting period prior to dosing) and drinking water were provided ad libitum from the arrival of the rats until the end of the study. The rats received a cereal-based (closed formula) rodent diet (Rat & Mouse No. 3
Breeding Diet, RM3; pelleted) from a commercial supplier (SDS Special Diets Services, Witham, England). Each batch of RM3 diet is analysed by the supplier for nutrients and contaminants. Each cage was supplied with domestic mains tap water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC). The water was given in polypropylene bottles, which were cleaned weekly and filled as needed. Results of the routine physical, chemical and microbiological examination of drinking water as conducted by the supplier were made available to the test facility. In addition, the supplier periodically (twice per year) analyses water samples taken at the premises for a limited number of physical, chemical and microbiological variables. Copies of certificates of analysis for the diet and water are available on request.

Identification
The study was identified as study 20204/01. On 7 February 2012 (one day prior to the first oral dosing) animals were identified by a transient tail mark. Thereafter, the animals were allocated to the different dose groups by computer randomization and proportionally to body weight. After allocation, each rat was identified by a tail mark followed by an animal identification number (V-shaped ear cut). Each cage was provided with a coloured card showing the study number, group number, colour code, cage number and animal identification number.

Route of administration:
oral: gavage
Vehicle:


Positive control

Name : Mitomycin C
Lot No: 089k0731
: 50-07-7
CAS Reg No: C15H18N4O5
Molecular weight : 334.3 g/mol
Date of receipt : 19 July 2011
Expiry date : 1 March 2014
Storage conditions : 2-10ºC
Supplier : Sigma-Aldrich
Dispense number : 1100C1



Negative control
Name : Physiological saline
Lot. No. : B1810-2
Date of receipt : 13 September 2010
Expiry date : 1 May 2013
Storage conditions : ambient temperature
Supplier : Eurovet
Dispense number : 10018B

Details on exposure:
The test article preparations were stored at 2-10 °C in the dark between dosing occasions. Rats were dosed orally by gavage twice, on two consecutive days with approximately 24 h interval, with 250, 500 or 1000 mg/kg/bw of initial formulation as follows:

Experiment Concentration of dosing preparation Dose administered¶
(mg/mL) (mg/kg)¶
Range-finder 100.0 1000
Main study 25.0 250
500.0 500
1000.0 1000
Duration of treatment / exposure:
2 administrations at 24 hour interval.
Frequency of treatment:
daily
Post exposure period:
24 hours then sacrifice
Remarks:
Doses / Concentrations:
250, 500 and 1000 mg/kg/bw
Basis:

No. of animals per sex per dose:
5 MALES/DOSE
Control animals:
yes
Positive control(s):
Mitomycin C

- Route of administration: Intraperitoneally
- Doses / concentrations:1.5 mg/kg/bw
Tissues and cell types examined:
Signs of reactions to treatment were recorded at least once during the first 4 h post- treatment and at least once daily thereafter until necropsy. All abnormalities, signs
of ill health or reactions to treatment (clinical signs) were recorded.
Details of tissue and slide preparation:
Bone marrow collection and processing
Immediately following sacrifice, the bone marrow cells of one of the femurs was collected into foetal calf serum and processed into glassdrawn smears according to the method described by Schmid (1976). Two bone marrow smears per animal
were made, air-dried and fixed in methanol. One fixed smear was stained with a May-Grünwald Giemsa solution. The other fixed smear was kept in reserve and discarded after analysis of the stained smear.
Microscopic examination of the bone marrow smears
The slides were randomly coded by a person not involved in the scoring of the slides. Slides (one per animal) were read by moving from the beginning of the smear (label end) to the leading edge in horizontal lines taking care that areas selected for evaluation are evenly distributed over the whole smear.

The following criteria were used for the scoring of cells:
− A polychromatic erythrocyte (PE) is an immature erythrocyte that still contains ribosomes and can be distinguished from mature, normochromatic erythrocytes by a faint blue stain.
− A normochromatic erythrocyte (NE) is a mature erythrocyte that lacks
ribosomes and can be distinguished from immature, polychromatic erythrocytes by a yellow stain.
− A micronucleus is a small, normally round, nucleus with a diameter of circa 1/20 to 1/5 of an erythrocyte, distinguished from the cytoplasm by a dark blue stain.

The numbers of PE and NE were recorded in a total of at least 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 of 200 E (PE + NE) were scored, an additional number of PE were scored for the presence of micronuclei until a total of 2000 PE were scored.
Evaluation criteria:
Statistical evaluation of the data and evaluation criteria
The statistical procedures and evaluation criteria used to evaluate the data (ratio
PE/E and MPE/2000 PE) are described below.

Two ANOVA models were applied for both PE and MPE. In the first ANOVA model it was tested if the positive control differed from the negative control. If there was a significant difference for MPE, the animal model was considered as valid. In a second ANOVA model it was tested if the negative control differed from the test substance (different dose). For both ANOVA models it was checked if the ANOVA assumptions were valid. If this was not the case non-parametric testing was performed and Kruskal-Wallis p-values were reported. In all statistical tests a significance level of 5% was used (α = 0.05). All statistical tests were performed using SAS V9.1 statistical software Copyright © 2002-2003 by SAS Institute Inc. Cary. NC, USA.

The study was considered valid if the positive controls showed a statistically significant increase in the mean number of MPE/2000 PE and the negative controls where within the historical range.

A response was considered to be positive if the mean number of MPE/2000 PE was statistically significantly higher compared to the negative control group (group 1).

A test substance was considered to cause chromosomal damage and/or damage to the mitotic apparatus if it showed a positive response at one or more dose levels, namely: if the mean number of MPE/2000 PE was statistically significant higher compared to the negative control group (group 1).

A test substance was considered to be negative in the micronucleus test if it did not produce a positive response at any of the dose levels analysed.




The test substance or its metabolites were considered to be cytotoxic to the bone marrow via general circulation, if the test substance statistically significantly reduced the mean number of number of PE.
Statistics:
SEE SECTION ABOVE
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
Dose range finding study

Based on the preliminary results of the acute oral toxicity study with the test substance in female Sprague Dawley rats (TNO Triskelion study number 9910/06), a dose level of 1000 mg/kg-bw Octyltin tris(2-ethylhexylmercaptoacetate) (MOTE) was selected as starting dose level to be used in the DRF study that was performed prior to the bone marrow micronucleus test. The test substance was formulated in
corn oil instead of dosing the undiluted test substance, because lower dose levels in addition to the MTD (e.g. ½ MTD and ¼ MTD) should be applied in the bone marrow micronucleus test than in the acute oral toxicity study. With the test substance formulated in corn oil the dosing volume should be sufficiently high in the bone marrow micronucleus test for administration of the lower dose levels. Approximately 4 h after dosing of the test substance formulated in corn oil at a dose level of 1000 mg/kg-bw Octyltin tris(2-ethylhexylmercaptoacetate) (MOTE) the two rats were lethargic, and the day after dosing they were found dead. These results were not in line with the results observed in the acute oral toxicity study.

In mutual agreement with the sponsor it was decided to dose one rat with the undiluted test substance. No abnormalities were observed at approximately 4, 6 and 24 h after the first dose of the undiluted test substance at 1117 mg/kg-bw. Approximately 4 h after the second dosing of 1000 mg/kg-bw piloerection was observed and approximately 6 h after the second dosing piloerection, nasal encrustations and an increased breathing pattern were observed. No abnormalities were observed at approximately 24 h after the second dosing. Based on these observations and those observed in the acute oral toxicity study the dose level of
1000 mg/kg-bw of Octyltin tris(2-ethylhexylmercaptoacetate) (MOTE) was considered the MTD and for the bone marrow micronucleus test dose levels of
1000, 500 and 250 mg/kg-bw of the undiluted test substance were selected as high, mid and low dose, respectively.

Main study

Body weights
The individual body weights prior to the first and second administration and prior to sacrifice are presented Appendix 2. Animals of groups 2, 3 and 4 showed a reduced body weight gain compared to animals of group 1.

Clinical signs
Approximately 4 h and 24 h after the first dose, no abnormalities were observed in any of the groups. Approximately 4 h after the second dose, all animals of group 4 (highest dose group, 1000 mg/kg-bw Octyltin tris (2-ethylhexylmercaptoacetate) MOTE) showed nasal encrustations and a hunched back. The other groups did not show any abnormalities. Approximately 24 h after the second dose (just before sacrifice), no abnormalities were observed in any of the groups. No mortality occurred during the study; therefore the three reserve rats were not dosed and the bone marrow cells were not collected.

Analysis of the results
The group mean numbers of micronucleated polychromatic erythrocytes (MPE) per
2000 polychromatic erythrocytes (PE) and the group mean numbers of PE per 200 erythrocytes (E) are presented in Table 1. The individual data of MPE per 2000 PE and PE per 200 E are presented in Appendix 1. The historical negative and positive control data for MPE per 2000 PE are presented in Appendix 3.

Positive control group 5: ►Mitomycin C (1.5 mg/kg-bw) for MPE:
Statistical analysis of the test results indicated there was a statistically significant increase (p value: 0.0086) in the mean number of MPE in the positive control mitomycin C (group 5), when compared to the negative control (group 1). The mean number of MPE found in the positive control mitomycin C was within the range of means of the historical data. This indicates that the positive control substance mitomycin C reached the bone marrow and induced damage to the chromosomes and/or to the spindle apparatus of the bone marrow cells of male rats. These
results, together with the MPE/PE ratio in the negative control group, demonstrate
the validity of the test system.

Treatment groups 2, 3 and 4: ► Octyltin tris (2-ethylhexylmercaptoacetate) MOTE (250, 500 and 1000 mg/kg-bw/day, respectively) for MPE:
Statistical analysis of the test results indicated there was no statistically significant
increase in the group mean of MPE/2000 PE, when compared to the negative control (group 1). This indicates that treatment with the test substance, up to 1000 mg/kg-bw/day, did not result in damage to the chromosomes and/or to the spindle apparatus of the bone marrow cells of male rats.

Positive control group 5: ►Mitomycin C (1.5 mg/kg-bw) for PE:
There were no statistically significant differences in the mean number of PE between the males of the positive control group and the males of the negative control group (group 1).

Treatment groups 2, 3 and 4: ► Octyltin tris (2-ethylhexylmercaptoacetate) MOTE (250, 500 and 1000 mg/kg-bw/day, respectively) for PE:
Statistical analysis of the test results indicated there was no statistically significant difference in the group mean of PE/200 PE, when compared to the negative control (group 1, which reflects a lack of toxic effects of the test substance on erythropoiesis.


Conclusions:
Octyltin tris (2-ethylhexylmercaptoacetate) MOTE did not show any indication of chromosomal damage and/or damage to the mitotic spindle apparatus of the bone marrow target cells of male rats.
Executive summary:

Under the conditions used in this study, it is concluded that the test item octyltin tris (2 -ethylexylmercaptoacetate) MOTE, did not show any indication of chromosomal damage and/or damage to the mitotic spindle apparatus of the bone marrow target cells of male rats, orally dosed once daily on two consecutive days with the test substance, when tested up to the MTD (1000 mg/kg-bw).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

> In vitro

- Ames: Arni, 1981 (similar to POECD 471, GLP not specified) - K2 (test material 67%:33% MOTE: DOTE mixture)

Arni (1981) (67%:33% MOTE: DOTE mixture), Similar to OECD 471, GLP not specified, K2

Octyltin tris(2-ethylhexylmercaptoacetate was tested for mutagenic effects on histidine-auxotrophic strains of Salmonella typhimurium and on a tryptophan auxotrophic strain of E. coll. The investigations were performed with the following concentrations of the trial substance without and with microsomal activation: 5, 10, 50, 100, 500, 1000 and 5000 µg/0.1 ml.

In the experiments performed without and with microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of the test material revealed no marked deviations.

No evidence of the induction of point mutations by the test material or by the metabolites of the substance' formed as a result of microsomal activation was detectable in the strains of S. typhimurium and E. coli used in these experiments.

- Chromosome Aberration: De Vogel, 2010 (OECD 473, GLP) - K1 (test material 97.9% MOTE)

The test substance Octyltin tris (2-ethylhexylmercaptoacetate) (MOTE) was examined for its potential to induce structural chromosomal aberrations in cultured human lymphocytes, in both the absence and presence of a metabolic activation system (S9-mix). Dimethylsulfoxide (DMSO) was used as solvent for the test substance. Two separate tests were conducted for which blood was obtained from two different donors. Dose levels, ranging from 0.1 to 2000 μg/ml (final concentrations in the culture medium), were tested. The purity of the test substance (97.9%) was taken into account while preparing the dosing solutions. In all instances, duplicate cultures were used and both solubility and cytotoxicity were used for dose level selection. Reduction of the mitotic index was used as indication for cytotoxicity. Cyclophosphamide, an indirect acting clastogenic compound which requires metabolic activation, was used as positive control in the presence of S9-mix. Mitomycin C, a direct acting clastogenic compound was used as positive control in the absence of S9-mix.

In the first test, in the presence and absence of S9-mix, the treatment/harvesting times were 4/24 hours (pulse treatment). In the presence of S9-mix, the cultures of three dose levels of the test substance (7.8, 62.5 and 125 μg/ml), the cultures of the solvent control (DMSO) and the cultures of the positive control Cyclophosphamide were analysed for the induction of chromosomal aberrations. At higher dose levels the test substance was too toxic for the cells (mitotic index reduction >70% at the dose level of 250 μg/ml). In the absence of S9-mix, the cultures of three dose levels of the test substance (7.8, 125 and 250 μg/ml), the cultures of the solvent control (DMSO) and the cultures of the positive control Mitomycin C were analysed for the induction of chromosomal aberrations. At higher dose levels (500, 1000 and 2000 μg/ml) the test substance was too toxic for the cells, demonstrated by a lack of stimulated lymphocytes and metaphases.

In the second test, in the presence and absence of S9-mix, the treatment/harvesting times were 4/24 hours (pulse treatment) and 24/24 hours (continuous treatment), respectively. In the presence of S9-mix, the cultures of three dose levels of the test substance (150, 300 and 500 μg/ml), the cultures of the solvent control (DMSO) and the cultures of the positive control Cyclophosphamide were analysed for the induction of chromosomal aberrations. Dose-related toxicity could be demonstrated in this treatment group. In the absence of S9-mix, the cultures of three dose levels of the test substance (50, 100 and 150 μg/ml), the cultures of the solvent control (DMSO) and the cultures of the positive control Mitomycin C were analysed for the induction of chromosomal aberrations. At higher dose levels the test substance was too toxic for the cells, demonstrated by a mitotic index reduction of >70% and a lack of stimulated lymphocytes and metaphases at the dose level of 300 μg/ml and a poor quality of the metaphases at the dose level of 200 μg/ml.

Treatment with the positive controls Cyclophosphamide and Mitomycin C resulted in statistically significant increases in the numbers of metaphases containing one or more chromosomal aberrations, when compared to the numbers observed in the cultures treated with the solvent control. This demonstrates the validity of the study.

In both chromosomal aberration tests, the test substance did not induce a statistically significant increase in the number of metaphases containing one or more chromosomal aberrations at any of the dose levels and time points analysed.

From the results obtained in two chromosomal aberration tests it is concluded that, under the conditions used in this study, the test substance Octyltin tris (2-ethylhexylmercaptoacetate) (MOTE) was not clastogenic to cultured human lymphocytes as it did not induce structural chromosomal aberrations.

- Gene mutation in mammalian cells: Steenwinkel, 2010 (OECD 476, GLP) - K1 (test material 97.9% MOTE)

The test substance Octylyin tris (2-ethylhexylmercaptoacetate) (MOTE) was tested and evaluated for mutagenicity in both the absence and presence of metabolic activation (S9-mix). In addition to treatment for 4 hours in the absences and presence of a metabolic activation system, an extended treatment for 24 hours in the absence of S9- mix was used. In the absence of S9-mix significant, reproducible and dose related increases in mutant frequency were observed at concentrations causing less than 90% cytotoxicity (RTG>10%). In the presence of S9-mix in the first test the mutant frequency was equivocally increased at a single dose (84 Xg/ml) causing about 50% cytotoxicity. In the second test a dose related and significant increase was observed at dose levels causing less than 90% cytotoxicity (RTG>10%). In addition, at concentrations causing an increase in mutant frequency, relatively more small than large colonies were formed. However, since the number of both small and large colonies was increased, neither mechanism (clastogenicity and mutagenicity) can be excluded. It is concluded that under the conditions used in this study, the test substance Octylyin tris (2-ethylhexylmercaptoacetate) (MOTE) is mutagenic at the TK-locus of mouse lymphoma L5178Y cells, although clastogenicity can not be excluded.

> In vivo

- Micronucleus: Reus, 2012 (OECD 474, GLP) - K1 (test material 97.7% MOTE)

Under the conditions used in this study, it is concluded that the test item octyltin tris (2 -ethylexylmercaptoacetate) MOTE, did not show any indication of chromosomal damage and/or damage to the mitotic spindle apparatus of the bone marrow target cells of male rats, orally dosed once daily on two consecutive days with the test substance, when tested up to the MTD (1000 mg/kg-bw).

- COMET Assay: Rashmi, 2019 (OECD 489, GLP) - K1 (test material 96.1% MOTE)

The results for the assessment of the test item, MOTE, to cause DNA strand breaks are provided for the doses of 100 [G2], 200 [G3] and 300 [G4] mg/kg, respectively, in male and female Wistar rats. The average % tail DNA observed in liver is 2.43, 2.40, 2.53 and 2.83 in males and 2.54, 2.46, 2.51 and 2.90 in females dosed at 0, 100, 200 and 300mg/kg, respectively. In glandular stomach, the observed average % tail DNA is 3.21, 3.35, 3.75 and3.85 in males and 3.34, 3.41, 3.88 and 3.94 in females dosed at 0, 100, 200 and 300 mg/kg, respectively. The average % tail DNA observed in duodenum was 2.85, 2.88, 2.89 and 3.26 in males and 2.96, 3.07, 3.00 and 3.19 in females dosed at 0, 100, 200 and 300 mg/kg, respectively. There was no dose-related or statistically significant increase in the % tailing of DNA from cells of any organ for any of the MOTE groups when compared to the vehicle control group.

The positive control [G5], Ethyl methane sulfonate at a dose of 250 mg/kg produced a statistically significant increase in % tailing of DNA in cells from all the organs which were assessed (Liver, glandular stomach and duodenum) when compared to the equivalent cells from organs of vehicle control animals [G1]. These data support the conclusion that the test conditions and sensitivity of the COMET assay for this test of MOTE were fully adequate.

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.