Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Read-across to structurally similar substance (mixture MOTE: DOTE. 70:30) (CAS No 27107-89-7 and CAS 15571-58-1)

In vitro Ames

Under the conditions of this study, the test material was determined to be non-mutagenic in both the presence and absence of metabolic activation.

Read-across to structurally similar substance Octyltin tris(2-ethylhexylmercaptoacetate) (MOTE) (CAS No 27107 -89 -7)

In vitro Mouse Lymphoma assay

It is concluded that under the conditions used in this study, the test material is mutagenic at the TK-locus of mouse lymphoma L5178Y cells, although clastogenicity can not be excluded.

In vitro Chromosome aberration assay

Under the conditions of this study, the test material was not clastogenic to cultured human lymphocytes as it did not induce structural chromosomal aberrations.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
8th August 1981 to 11th September 1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
other: read-across target
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
not specified
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 remarks section.
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
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and 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
Key result
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

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
Test material 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
Test material 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:
Under the conditions of this study, the test material was determined to be non-mutagenic in both the presence and absence of metabolic activation.
Executive summary:

The test material was evaluated 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 test material without and with microsomal activation: 5, 10, 50, 100, 500, 1000 and 5000 µg/0.l 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.

Under the conditions of this study, the test material was determined to be non-mutagenic in both the presence and absence of metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Justification for type of information:
Read Across to Octyltin tris(2-ethylhexylmercaptoacetate) (MOTE) (EC Number 248-227-6 and CAS No 27107-89-7) based on structural similarity and hydrolytical behaviour, see attached justification.
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and 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
Key result
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
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
Reason / purpose for cross-reference:
other: read-across target
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 2-10°C, in the dark. Based upon the physico-chemical information 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.
Key result
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
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 250µg/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 313µg/ml. After 24 hours precipitation was observed in concentrations between 156 and 5000µg/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:
Under the conditions of this study, the test material was not clastogenic to cultured human lymphocytes as it did not induce structural chromosomal aberrations.
Executive summary:

The test material 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). The study was performed in accordance with the standardised guideline OECD 476.

Dimethylsulfoxide (DMSO) was used as solvent for the test material. 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 material (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 material (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 material 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 material (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 material 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 material (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 material (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 material 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 material 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.

Under the conditions of this study, the test material was not clastogenic to cultured human lymphocytes as it did not induce structural chromosomal aberrations.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Justification for type of information:
Read Across to Octyltin tris(2-ethylhexylmercaptoacetate) (MOTE) (EC Number 248-227-6 and CAS No 27107-89-7) based on structural similarity and hydrolytical behaviour, see attached justification.
Reason / purpose for cross-reference:
read-across source
Key result
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
Positive controls validity:
valid
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2010 to May 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
other: read-across target
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:
mammalian cell gene mutation assay
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:
3-methylcholanthrene
methylmethanesulfonate
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
Key result
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
Positive controls validity:
valid
Key result
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
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 µg/ml and equivocally increased at 9.8 µg/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

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

2 large 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

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

2 large 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 material 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 absence 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 µg/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.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Justification for type of information:
Read Across to Octyltin tris(2-ethylhexylmercaptoacetate) (MOTE) (EC Number 248-227-6 and CAS No 27107-89-7) based on structural similarity and hydrolytical behaviour, see attached justification.
Reason / purpose for cross-reference:
read-across source
Key result
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
Positive controls validity:
valid
Key result
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
Positive controls validity:
valid

Genetic toxicity in vivo

Description of key information

Read-across to structurally similar substance Octyltin tris(2-ethylhexylmercaptoacetate) (MOTE) (CAS No 27107-89-7)

In vivo Mouse Micronucleus Assay

Under the conditions used in this study, it is concluded that the test material, 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).

Link to relevant study records

Referenceopen allclose all

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
Reason / purpose for cross-reference:
other: read-across target
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
other: mammalian erythrocyte micronucleus test (migrated information)
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
Dose / conc.:
250 mg/kg bw (total dose)
Dose / conc.:
500 mg/kg bw (total dose)
Dose / conc.:
1 000 mg/kg bw (total dose)
No. of animals per sex per dose:
5 males per 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
Key result
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.


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:
Under the conditions of this study, the test material 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:

The study was performed in accordance with the standardised guideline OECD 474, under GLP conditions. Five male Wistar rats were treated at each dose (25 0, 500 and 1000 mg/kg/bw).

Under the conditions used in this study, it is concluded that the test material, 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:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Justification for type of information:
Read Across to Octyltin tris(2-ethylhexylmercaptoacetate) (MOTE) (EC Number 248-227-6 and CAS No 27107-89-7) based on structural similarity and hydrolytical behaviour, see attached justification.
Reason / purpose for cross-reference:
read-across source
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Read-across to structurally similar substance (mixture MOTE: DOTE. 70:30) (CAS No 27107-89-7 and CAS 15571-58-1)

In vitro Ames Test

The test material was evaluated 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 test material without and with microsomal activation: 5, 10, 50, 100, 500, 1000 and 5000 µg/0.l mL. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

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.

Under the conditions of this study, the test material was determined to be non-mutagenic in both the presence and absence of metabolic activation.

Read-across to structurally similar substance Octyltin tris(2-ethylhexylmercaptoacetate) (MOTE)

In vitro Mouse Lymphoma assay

The test material was tested and evaluated for mutagenicity in both the absence and presence of metabolic activation (S9-mix), in accordance with OECD 476. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

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 µg/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 material is mutagenic at the TK-locus of mouse lymphoma L5178Y cells, although clastogenicity can not be excluded.

In vitro Chromosome aberration assay

The test material 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). The study was performed in accordance with the standardised guideline OECD 476, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Dimethylsulfoxide (DMSO) was used as solvent for the test material. 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 material (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 material (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 material 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 material (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 material 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 material (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 material (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 material 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 material 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.

Under the conditions of this study, the test material was not clastogenic to cultured human lymphocytes as it did not induce structural chromosomal aberrations.

In vivo Mouse Micronucleus Assay

The study was performed in accordance with the standardised guideline OECD 474, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Five male Wistar rats were treated at each dose (25 0, 500 and 1000 mg/kg/bw).

Under the conditions used in this study, it is concluded that the test material, 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).

Justification for classification or non-classification

Based upon the information presented in the in vitro tests and the in vivo test the registered substances does not meet the criteria for classification according to Regulation EC (No) 1272/2008.