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Toxicological information

Genetic toxicity: in vivo

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Administrative data

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
Cross-reference
Reason / purpose for cross-reference:
other: read-across target
Reference
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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
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)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethylhexyl 10-ethyl-4-[[2-[(2-ethylhexyl)oxy]-2-oxoethyl]thio]-4-octyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecanoate
EC Number:
248-227-6
EC Name:
2-ethylhexyl 10-ethyl-4-[[2-[(2-ethylhexyl)oxy]-2-oxoethyl]thio]-4-octyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecanoate
Cas Number:
27107-89-7
Molecular formula:
C38H74O6S3Sn
IUPAC Name:
2-ethylhexyl 10-ethyl-4-({2-[(2-ethylhexyl)oxy]-2-oxoethyl}sulfanyl)-4-octyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecan-1-oate
Constituent 2
Reference substance name:
Octyltin tris(2-ethylhexylmercaptoacetate)
IUPAC Name:
Octyltin tris(2-ethylhexylmercaptoacetate)
Details on test material:
Chemical name: Octyltin tris(2-ethylhexylmercaptoacetate) (MOTE)
Purity: 97.7%
Lot No.: ESOC21.341
Storage conditions: 27107-89-7
Storage condition: 2-10°C in the dark. Air space flushed with nitrogen. The sample was kept away from air and water.
The sample was allowed to warm to room temperature before opening the bottle to prevent condensation of moisture into the sample and prevent any moisture condensed on the outside of the flask from dripping into the sample. Brief periods at room temperature up to 1-2 days did not harm the sample. For longer storage (weeks), refrigerated conditions were used to prevent long term thermal degradation.
Appearance: clear, colourless liquid
Date of receipt: 18 November 2011
Expiry date: 15 March 2012.
TNO dispense number: 11012B

Test animals

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.

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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

Results and discussion

Test results
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.


Applicant's summary and conclusion

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).