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Genetic toxicity in vitro

Description of key information

Negative in an in vitro Ames assay (OECD 471)

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Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Principles of method if other than guideline:
Method: other: comparable to guidelines established by OECD, USEPA, and USFDA
All tester strains were checked for the presence of the appropriate genetic markers. The S. typhimurium strains were obtained from Dr. Bruce Ames, University of California - Berkeley. The E. coli strain was obtained from Dr. M.H.L. Green, M.R.C. Cell Mutation Unit, University of Sussex, Falmer, England. For tests with metabolic activation, a reaction mixture was prepared with 8 mM of MgCl2, 33 mM KCl, 4 mM NADP, 5 mM glucose-6-phosphate, 100 mM Na2HPO4 (pH 7.4), and 6% (v/v) S9 Homogenate fraction. The S9 Homogenate was prepared at Pharmakon Research from Aroclor 1254-induced male Sprague-Dawley rat liver homogenate, according to the procedures of Maron and Ames (1983). The concentration of liver homogenate in the metabolic activation mixture was optimized for each lot of S9 prepared using 3 positive control articles (2-acetylaminofluorene, 2-anthramine, and benzo[a]pyrene). [Maron, D.M. and B.N. Ames. 1983. Revised methods for the Salmonella mutagenicity test. Mutation Res. 113:173-215.] All required dilutions of the test substance were made with dimethyl sulfoxide (DMSO). Dilutions were prepared the day of the test and used immediately after preparation. A preliminary toxicity screen was conducted, using both liquid pre-incubation and plate incorporation treatment conditions. Duplicate cultures of strains TA1537, TA100, and E. coli WP2 uvrA (treated with the test substance at doses of 50, 167, 500, 1670, and 5000 ug/plate), and a DMSO solvent control were prepared without metabolic activation. Following a 48-hour incubation at 37 deg. C, the background lawn and spontaneous revertants were scored for normal, inhibited, or no growth. Primary test cultures were prepared from working stock cultures in 25 mL of Oxoid nutrient broth #2, incubated for ~8 hours at 37 deg. C, and diluted 1:4 in de-ionized water. Optical densities were determined at 650 nm and cultures with optical densities of 0.4-0.6 (~1-2 x 10E9 cells/mL) were used for this study. The test substance was evaluated for mutagenicity using both liquid pre-incubation and plate incorporation treatment conditions. Triplicate cultures of all six test strains were prepared and evaluated at doses of 16.7, 50, 167, 500, 1670, and 5000 ug/plate, both with and without metabolic activation (S9). Positive and negative control articles (with and without activation) were prepared in triplicate for the primary mutagenicity testing:
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
selected histidine loci
tryptophan locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
exogenous metabolic activation system (S9)
Test concentrations with justification for top dose:
16.7, 50, 167, 500, 1670, and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the solvent giving the best solubility or suspension was selected.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
all tester strains plated with DMSO ±S9
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
N-ethyl-N-nitro-N-nitrosoguanidine
mitomycin C
other: 2-aminofluorene; 2-anthramine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); liquid preincubation;

DURATION
- Preincubation period: approximately 30 minutes for those undergoing liquid pre-incubation
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicate cultures


Evaluation criteria:
A positive result is defined as a statistically significant, dose-dependent increase in the number of histidine- or tryptophan- independent revertants, with at least one dose level inducing a revertant frequency that is two-fold the concurrent negative control value. Statistical analysis were performed using the program developed by Snee and Irr (1981), with significance established at the 95% confidence limit. If the tetst article does not induce a statistically significant, dose-dependent increase in revertant frequency, but does induce a revertant frequency at one dose level that is two-fold the spontaneous control value, the result is equivocal. A negative result is defined as the absence of a statistically significant or dose-dependent increase in the number of histidine or tryptophan-independent revertants. Inhibited growth was characterized by the absence of a confluent bacterial lawn nd /or the presence of pindot colonies.
Statistics:
Statistical analyses were conducted using the program developed by Snee and Irr (1981), with significance established at the 95% confidence limit. A positive result was defined as a statistically significant, dose-dependent increase in the number of histidine- or tryptophan-independent revertants, with at least one dose level inducing a revertant frequency that was two-fold the solvent control value. An equivocal result was declared if the test substance did not induce a statistically significant, dose-dependent increase in revertant frequency, but did induce a revertant frequency at one dose level that was two-fold the spontaneous control value. A negative result was defined as the absence of a statistically significant or dose-dependent increase in the number of histidine- or tryptophan-independent revertants. Statistical analyses were only conducted when a 50% increase in revertant frequency (relative to the concurrent negative controls) was observed.
Key result
Species / strain:
other: S. typhimurium TA1535, TA1537, TA98, TA100 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
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:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: none noted
- Water solubility: DMSO- solvent

RANGE-FINDING/SCREENING STUDIES:
Toxicity of the test article first was evaluated in a preliminary toxicity screen using both liquid pre-incubation and plate incorporation treatment conditions. Duplicate cultures of strains TA1537, TA100 and WP2 urvA were treated with the test substance at doses of 50.0, 167, 500, 1670 and 5000 µg/plate, and the DMSO solvent control in the absence of S9 activation. Results of the pre-screen indicated that the test substance produced inhibited growth (characterised by the absence of a confluent bacterial lawn and /or the presence of pindot colonies) in both Salmonella tester strains at doses ≥500 µg/plate under liquid pre-incubation conditions. In addition, the test substance was found to be incompletely soluble at doses ≥500 µg/plate.
Preliminary test results showed that the test substance produced inhibited growth in both Salmonella tester strains at doses ≥500 µg/plate, under liquid pre-incubation conditions. Primary test results showed that, except for strain WP2 uvrA, inhibited growth was again observed for all test strain/S9/treatment combinations at the 3 highest dose levels (i.e., 500, 1670 and 5000 µg/plate). In addition, the test substance again was seen to be incompletely soluble at levels ≥500 µg/plate. Statistically significant increases in revertant frequencies (to ~1.6 -2.0 fold control values) were observed in strains TA1537 and TA1535 at a dose level of 16.7 µg/plate, without S9, under liquid pre-incubation and plate incorporation conditions, respectively. However, these increases were not determined to be dose dependent and the observed revertant frequencies approximated historical negative control values. Revertant frequencies for all other dose/test strain/S9/treatment condition combinations approximated or were less than control values. All positive and negative control values (in both assays) were within acceptable ranges. Therefore, the slight increases observed in strains TA1535 and TA1537 were considered statistical aberrations.

COMPARISON WITH HISTORICAL CONTROL DATA: Historical data were provided for positive and negative controls. Six dose levels were evaluated in case of unacceptable toxicity and/or insolubility at the highest dose levels evaluated in the mutation assay. After a 48-hour incubation at 37 °C, all plates were scored for bacterial growth. Solvent and positive controls were scored first, and test substance-treated cultures were scored only if the average negative control values were within historical ranges.
Conclusions:
Negative in the Ames/Salmonella-E.coli Reverse Mutation Assay, using liquid pre-incubation and plate incorporation treatments, under the conditions of this study.
Executive summary:

The test article, methyltin tris(2­ethylhexylthioglycolate), was evaluated in the Ames/Salmonella­E. coli Reverse Mutation Assay to determine its ability to induce reverse mutations at selected histidine loci in five tester strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100 and TA102), and at the tryptophan locus in one Escherichia coli tester strain (WP2 uvrA), in the presence and absence of an exogenous metabolic activation system (S9).

Toxicity of the test article first was evaluated in a preliminary toxicity screen using both liquid pre­incubation and plate incorporation treatment conditions. Duplicate cultures of strains TA1537, TA100 and WP2 uvrA were treated at doses of 50.0, 167, 500, 1670 and 5000 µg/plate, and the DMSO solvent control, in the absence of S9. Results of the pre-screen indicated the test material produced inhibited growth (characterised by the absence of a confluent bacterial lawn and/or the presence of pindot colonies) in both Salmonella tester strains at doses ≥500 µg/plate under liquid pre­incubation conditions. In addition, the test article was found to be incompletely soluble at doses ≥500 µg/plate. 

The test article next was evaluated for mutagenicity using both treatment conditions. Based upon the results of the pre-screen, the test material was evaluated in triplicate cultures in all six tester strains at doses of 16.7, 50.0, 167, 500, 1670 and 5000 µg/plate with and without S9. Six doses were evaluated in the event of unacceptable toxicity and/or insolubility at the highest dose levels in the mutation assay. The S9 mixture included 6 % (v/v) Aroclor 1254­induced male Sprague­Dawley rat liver homogenate with the appropriate buffer and cofactors.

Except for strain WP2 uvrA, inhibited growth again was observed for all tester strain/S9/treatment combinations at the highest 1­3 doses evaluated. In addition, the test article again was found to be incompletely soluble at doses ≥500 µg/plate. Statistically significant increases in revertant frequencies, to approximately 1.6­ to 2.0­fold control values, were observed in strains TA1537 and TA1535 at a dose of 16.7 µg/plate without S9 under liquid pre­incubation and plate incorporation conditions, respectively. However, these increases were not dose dependent, and the observed revertant frequencies approximated historical negative control values. In addition, revertant frequencies for all other dose/tester strain/S9/treatment condition combinations approximated or were less than control values. All positive and negative control values in both assays were within acceptable ranges. Thus, the slight increases observed in strains TA1535 and TA1537 are considered to be statistical aberrations due to random fluctuation of the spontaneous revert frequencies.

Therefore, the results for the test article, methyltin tris(2­ethylhexylthio­glycolate), were negative in the Ames/Salmonella­E. coli Reverse Mutation Assay, using liquid pre­incubation and plate incorporation treatments, under the conditions of the study according to the criteria of the test protocol.


Endpoint:
in vitro gene mutation study in mammalian cells
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint:
in vitro cytogenicity / micronucleus study
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
In accordance with Column 2 of REACH Annex VIII, information requirement 8.4.2, an in vitro cytogenicity study in mammalian cells study does not need to be conducted if adequate data are available from an in vivo cytogenicity study. Data from an in vivo cytogenicity study are available and testing is therefore omitted.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Positive in an in vivo micronucleus assay (OECD 474) by read across based on category approach

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Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
other: read-across based on grouping of substances (category approach)
Adequacy of study:
key 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 test result from source substance MMTC to target substance MMT(EHTG). This data requirement is fulfilled by read-across. Justification for the read-across of this test result is provided in Section 7.1 Toxicokinetics. The simulated gastric hydrolysis study shows rapid and complete conversion of MMT(EHTG) to MMTC. Based on this result MMTC fulfils the requirements of being a source compound for studies required for MMT(EHTG) where the endpoint is based on oral exposure. The study on MMTC can be applied both quantitatively and qualitatively for the end point for MMT(EHTG).
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
The results of the total tin analysis indicated that the amount of total tin in the bone marrow samples was less than the detection limit of 250 ng. The main test was performed without total tin analysis. Deviation did not adversely influence the integ
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
rat
Strain:
other: Wistar outbred CrI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: young adult
- Weight at study initiation: 162.6 to 203.4
- Assigned to test groups randomly: yes, by computer randomization
- Fasting period before study: yes, 19 hours prior to dosing
- Housing: sterilized Macrolon cages (type IV for the main test and type III for the dose-range finding test) fitted with a grid cover of stainless steel
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): at least 30 % not exceeding 70 % except during room cleaning
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark


IN-LIFE DATES: From: October 1, 2002 To: October 3, 2002
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 0.9 % sodium chloride
- Justification for choice of solvent/vehicle:
- Concentration of test material in vehicle: 50, 16.67, 5.56, and 1.85 mg/mL
- Amount of vehicle (if gavage or dermal): 20 mL/kg-bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: On the day before dosing the test substance was weighed and stored. On day 0, the test substance was dissolved and diluted in 0.9 % sodium chloride at concentrations of 50, 16.67, 5.56 and 1.85 mg/mL. The orally (by gavage) given dosing volume was 20 mL/kg-bw.

DIET PREPARATION N/A- gavage administration
Duration of treatment / exposure:
single dose
Frequency of treatment:
single dose
Post exposure period:
24 hours for all doses including controls and 48 hours for the negative control and two highest dose levels
Dose / conc.:
37 mg/kg bw/day (nominal)
Dose / conc.:
111 mg/kg bw/day (nominal)
Dose / conc.:
333 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Group A, D, E: 10 males per group. Group B, C,  F: 5 males per group.
Control animals:
yes, concurrent vehicle
other: positive control- Mitomycin C
Positive control(s):
mitomycin C
- Justification for choice of positive control(s): no data
- Route of administration: intraperitoneal injection; vehicle: saline
- Doses / concentrations: 10 mL/kg-bw
Tissues and cell types examined:
- Clinical observations: Clinical signs were recorded from 1-4 hours and  at 24 and 48 hrs after treatment. - Organs examined at necropsy: From each rat, the bone marrow cells of  one femur were immediately collected into foetal calf serum and processed  into glassdrawn smears according to the method described by Schmid  (1976).  No  additional organs were examined.
Details of tissue and slide preparation:
OTHER: From each rat, the bone marrow cells of one femur were immediately 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 prepared, air-dried and  fixed in methanol. One smear per animal was stained with a May-Grünwald  Giemsa solution. The second fixed smear was stored as a reserve slide.  Slides were independently coded.
The slides (one slide 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 were evenly distributed over the whole smear.
Evaluation criteria:
The numbers of polychromatic and  normochromatic erythrocytes (PE and NE, respectively) were recorded in a  total of 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 number of  200 E (PE + NE) had been scored, an additional number of PE was scored  for the presence of micronuclei until a total number of 2000 PE had been  scored. Thus the incidence of MPE/2000 PE was registered, and the number  of MNE/NE. The study is considered valid if the positive controls give a  statistically significant increase in the mean number of MPE/2000 PE and  if the negative controls are within the historical range. A response is considered to be positive if the mean number of MPE/2000 PE  is statistically significantly higher, when compared to the mean number  of the vehicle controls. A test substance is considered to cause chromosomal damage and/or damage  to the mitotic apparatus, if a clear dose-related increase in the mean  numbers of MPE/2000 PE is observed, when compared to the mean number of  the vehicle controls. A test substance is considered to be negative in the micronucleus test if  it produces no positive response at any of the dose-levels and time  points analysed. The test substance or its metabolites are considered to have reached the  general circulation and thereby the bone marrow, if the test substance  statistically reduce the mean number of PE/E or causes systemic toxicity. Both statistical significance and biological relevance are considered  together in the evaluation.
Statistics:
STATISTICAL METHODS: 1) At time point 24 hours after administration, data  on micronucleated polychromatic erythrocytes (MPE) and polychromatic  erythrocytes (PE) were subjected to a One Way Anova with factor group (A,  B, C, D and E). If the Anova yielded a significant effect (p<0.05), it  was followed by pooled error variance t-tests or, if variances were  inhomogenous, separate variance t-tests. These t-tests were applied to  the negative control group A versus treatment groups B (37 mg/kg bw), C  (111 mg/kg bw), D (333 mg/kg bw) and E (1000 mg/kg bw). In addition, the  positive control group F and the negative control group A, at time point  24 hours after administration, were compared using t-tests and a linear  trend test across groups A, B, C, D and E (orthogonal contrasts). 2) At time point 48 hours after administration, data on MPE and PE in  group A, D and E were subjected to a One Way Anova. If the Anova yielded  a significant effect (p<0.05), it was followed by pooled error variance  t-tests or, if variances were inhomogenous, separate variance t-tests. In  addition a linear trend test across groups A, D and E were applied  (orthogonal contrasts).
Key result
Sex:
male
Genotoxicity:
positive
Remarks:
weakly genotoxic because of a potential to produce micronuclei and induce damage to the mitotic spindle apparatus in the bone marrow target cells.
Toxicity:
no effects
Remarks:
No treatment related cytotoxicity could be demonstrated in the target cells of the rat bone marrow.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 25, 50, 100 and 200 mg/kg bw
- Clinical signs of toxicity in test animals: No clinical signs were observed at any of the dose levels.
- Rationale for exposure: Performed in order to determine the dose level(s) to be used in the main micronucleus test.
- Harvest times: On day 2 (48 hours after administration), the negative control rats (2 males and 2 females) and the rats (2 males and 2 females) of the highest dose-level (200 mg/kg bw) were killed and the bone marrow cells of two femurs of each rat were sampled in 0.9 % sodium chloride for total tin analysis with the atomic absorption spectrometry with graphite furnace technique (GF-AAS) at a wavelength of 224.6 nm
- High dose with and without activation: The limit dose of 2000 mg/kg bw could not be administered because the test substance is known to be harmful to the rats and the acute toxicity (LD50) is between 200 and 2000 mg/kg bw (data provided by the sponsor). Therefore, 200 mg/kg bw was selected as highest dose level for the dose-range finding acute toxicity test.
- Other: The results of these analyses indicated that the amount of total tin in the bone marrow samples was less than the detection limit of 250 ng. In agreement with the sponsor, it was decided to continue the study with a main micronucleus test excluding total tin analysis in bone marrow samples. No sex differences were observed in the dose range-finding study; therefore, the main study was performed with male rats only.
RESULTS OF DEFINITIVE STUDY EFFECT ON PCE/NCE RATIO: 24 hours after treatment, measured as the mean number of MPE, the negative vehicle control group A (0.9 % sodium chloride) was statistically significantly (weak effect) different from treatment group B (test substance; 37 mg/kg bw; *p<0.05), treatment group D (test substance; 333 mg/kg bw; *p<0.05) and treatment group E (test substance; 1000 mg/kg bw; *p<0.05). In group C (test substance; 111 mg/kg bw), the mean number of MPE was not statistically significantly higher than the mean number of MPE found in the vehicle control group A. The linear trend test showed a weak significant effect (*p<0.05). 48 hours after treatment no statistical differences between the negative control group and the treated groups could be demonstrated. At both time points of 24 and 48 hours after treatment, the numbers of polychromatic erythrocytes (PE) per number of (E) erythrocytes in the rats treated with four different dose levels (37, 111, 333 and 1000 mg/kg bw) of the test substance Trichloromethylstannane, were not statistically significantly different from the numbers of polychromatic erythrocytes per number of erythrocytes found in the vehicle controls.
- INDUCTION OF MICRONUCLEI (FOR MICRONUCLEUS ASSAY): Weakly genotoxic because of a potential to produce micronuclei and induce damage to the mitotic spindle apparatus in the bone marrow target cells. LOAEL (C): The lowest concentration at which a weak genotoxic effect was observed, was 37 mg/kg bw.
- MPE/2000 PE FREQUENCY: After 24 hrs the observed MPE/2000 PE frequencies were: group A (control), 1.2 ± 0.4; group B (37 mg/kg bw), 3.0 ± 1.2; group C (111 mg/kg bw), 1.8 ± 0.4; group D (333 mg/kg bw), 3.0 ± 1.4; group E (1000 mg/kg bw), 3.4 ± 1.7; group F (positive control), 26.8 ± 3.3. After 48 hrs the observed frequencies were: group A (negative control), 2.4 ± 1.8; group D (333 mg/kg bw), 1.8 ± 1.1; group E (1000 mg/kg bw), 1.6 ± 0.9.
- STATISTICAL EVALUATION: At the 24-hour time point, the PCE/NCE ratio in group B, D and E was significantly higher than for the negative control group, p<0.05. The difference in PCE/NCE ratio was not statistically significant between the negative control group and group C at the 24-hour and 48-hour time points, and the 48-hour time points for group B, D and E. The numbers of polychromatic erythrocytes (PE) per number of (E) erythrocytes were not statistically significantly different from the numbers of polychromatic erythrocytes per number of erythrocytes found in the vehicle controls for any of the dose groups or time points.
- MORTALITY: None. No treatment related cytotoxicity could be demonstrated in the target cells of the rat bone marrow.
Conclusions:
Interpretation of results: positive
From the results obtained, under the conditions used, it was demonstrated that the test substance was weakly genotoxic because of a potential to produce micronuclei and induce damage to the mitotic spindle apparatus in the bone marrow target cells.
Executive summary:

The test substance was examined in a bone marrow micronucleus test in rats. The study consisted of a dose-range finding acute toxicity test and a main micronucleus test.

In the dose range finding acute toxicity test, bone marrow cells of 2 males and 2 females, treated with the highest dose level of the test substance (200 mg/kg-bw), was sampled for total tin analysis. The results of this analysis were not satisfactory because the amount of total tin in the bone marrow samples was less than the detection limit. Therefore, the main micronucleus test was performed without total tin analysis in bone marrow samples.

For the main micronucleus test, animals were treated once by gavage with four graded dose levels of the test substance. One group, consisting of 10 males, was treated with a dose level of 1000 mg/kg. One group, consisting of 10 males, was treated with a dose level of 333 mg/kg. One group, consisting of 5 males, was treated with a dose level of 111 mg/kg. One group, consisting of 5 males, was treated with a dose level of 37 mg/kg. The rats of the vehicle control group were treated in a similar way with the vehicle only (saline; 0.9 % sodium chloride). A positive control group (5 males) was given a single intraperitoneal dose of the mutagen mitomycin C. At time point 24 hours after treatment, 5 animals of each dose level of the test substance, 5 negative control animals and 5 positive control animals, were sacrificed. At time point 48 hours after treatment, 5 animals of the two highest dose levels of the test substance together with 5 negative control animals were sacrificed. From one femur of each animal, the bone marrow cells were collected in foetal calf serum and processed into smears for microscopic examination.

At time point 24 hours after treatment, the mean numbers of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) in rats, treated with three different dose levels (37, 333 and 1000 mg/kg) of the test substance, were weakly statistically significantly higher than the mean number found in the vehicle control rats. The linear trend test also yielded a weak significant effect. Therefore, the study indicates that treatment with the test substance at dose levels up to 1000 mg/kg, resulted in a weak genotoxic effect to bone marrow cells in rats. The mean number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes in rats, treated with a dose level of 111 mg/kg, was not statistically significantly higher than the mean number found in the vehicle control rats. For the rats of the positive control group, the mean number of MPE per 2000 PE was statistically significantly higher than the mean number found in the vehicle control rats. This demonstrates the validity and sensitivity of the test system.

At time point 48 hours after treatment, the mean numbers of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes in rats, treated with the two highest dose levels (333 and 1000 mg/kg) of the tet substance, were not statistically significantly higher than the mean number found in the vehicle control rats.

At both time points of 24 hours and 48 hours after treatment, the numbers of polychromatic erythrocytes per number of erythrocytes in rats, treated with four different dose-levels (1000, 333, 111 and 37 mg/kg) of the test substance, were not statistically significantly different from the polychromatic erythrocytes per number of erythrocytes found in the vehicle control rats. Therefore, no treatment related cytotoxicity to bone marrow cells of male rats could be demonstrated in the target cells.

From the results obtained in this micronucleus test with the test substance, it was demonstrated that the test substance was weakly genotoxic to bone marrow cells of rats and that the test substance has the potential to induce damage to the mitotic spindle apparatus of the bone marrow target cells.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

The test substance was negative in an Ames assay with Salmonella typhimurium (OECD 471).

A rat micronucleus assay, conducted according to OECD Test Guideline 474 on a surrogate compound, demonstrated that methyltin trichloride produced a statistically significant increase in the number of micronucleated polychromatic erythrocytes (MPE) at dose levels of 37 mg/kg bw and above. The MPE response did not increase with increasing dose and was transient, appearing only 24 hours after treatment, but not at 48 hours after treatment. These results could be judged equivocal or characterised as weakly positive for induction of MPE from bone marrow cells in rats. Methyltin trichloride did not increase the number of polychromatic erythrocytes (PE) in the dosed animals.

A paper is included as supporting information for completeness and in order to provide further detail on how organotin compounds influence cyto- and genotoxic effects. 

In this study, the alkylated tin derivatives monomethyltin trichloride (MMT), dimethyltin dichloride (DMT), trimethyltin chloride (TMT) and tetramethyltin (TetraMT) were investigated for cyto- and genotoxic effects in CHO-9 cells in relation to the cellular uptake. After forced uptake by electroporation MMT, DMT and TMT induced significant DNA damage at non-cytotoxic concentrations. The results indicate a considerable toxicological potential of some organotin species but demonstrate clearly that the toxicity is modulated by the cellular uptake capability.

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 requires classification with respect to mutagenicity as Muta Category 2 (H341: Suspected of causing genetic defects).