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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18th December 2002 - 23rd June 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Principles of method if other than guideline:
- Responsible Personnel: D. Jonker M. Sc. and M.V.W. Wijnands DVM should read D. Jonker PhD and M.V.W. Wijnands PhD DVM , respectively; During the acclimatization period, the animals of the dose range finding study were not identified by a temporary tail mark, but were tattooed directly after allocation.
- The title of the FOB-manual should read: `Functional Observational Battery. Operational Definitions' (Lammers, 2000);
M.V.W. Wijnands was responsible for pathology, instead of R.A. Woutersen; Pups were identified by tattoos in paws and tail on PN 1; On each day of analysis of study samples, QC samples were freshly prepared, in order to check the derivatisation and extraction on that particular day.
- Validation of the analytical method used for determination of DOTC in diet at the 10 mg/kg level of the main study (10 mg/kg) was performed, because the 10 mg/kg level was outside the dose-range tested in the dose-range finding study . Validation of the analytical method at the dose levels of the dose-range finding study was performed in a separate study (TNO report V 4921). The validation criteria used for the validation of the 10 mg/kg level of the main study were identical to the validation criteria used for the other dose levels. These validation criteria are described in TNO report V4921; The 24 h-stability of the test substance in RM3 rat feed of the control group was erroneously not determined in the first batch of diets prepared for the 13-week study;
- In addition to the protocol, the animals of the satellite groups were weighed once before the start of the study; The definition used for runts was: pup weight less than mean pup weight of the control group minus 2 standard deviations; Calculation of the viability index was not mentioned in the protocol; In addition to the protocol, the intake of test substance per kg body weight per day was calculated;
- Food consumption and food efficiency were statistically analysed by anova followed by L.S.D. tests instead of anova followed by Dunnett's multiple comparison tests; Occasionally, a determination was or could not be conducted in an individual animal. The reasons are presented in the appendices (individual data).

These deviations were considered not to have influenced the validity of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Purity is 92.1%

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Deutschland, Sulzfeld, Germany.
- Age at study initiation: approx. 7 weeks old.
- Weight at study initiation: Body weights at the start of the treatment ranged from 130.2 g to 175.8 g (mean 154.3 g) in males and from 107.5 g to 133.4 g (mean 119.4 g) in females.
- Fasting period before study: NDA
- Housing: The animals were housed under conventional conditions in one room, in macrolon cages, with sterilized wood shavings (Woody Clean 3/4) as bedding material and environmental enrichment (shreds of paper), 2 (dose range finding study) or 5 rats per cage (13-week study), separated by sex.
- Diet (e.g. ad libitum): ad libitum until end of study, unless precluded by the performance of certain laboratory investigations (urine collection)
- Water (e.g. ad libitum): ad libitum until end of study, unless precluded by the performance of certain laboratory investigations (urine collection)
- Acclimation period: 13 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): of 22 ± 3 °C (except on 22 April 2003 (c. 20 min; min. 18.9°C))
- Humidity (%): 30 - 70 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours light / dark


IN-LIFE DATES: From: 18th December 2002 To: 23rd June 2003

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Prepared shortly before the start of the study and every 6 weeks thereafter.
- Mixing appropriate amounts with (Type of food): RM3 diet
- Storage temperature of food: < -18 ºC


VEHICLE
No vehicle used.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
From each diet sample, 2.0 g was transferred into a 50 ml Greiner tube. An aliquot of the internal standard solution (monoheptyltin trichloride, diheptyltin dichloride, tripropyltin chloride and tetrapropyltin in methanol) was added. Subsequently methanol, acetate buffer solution (pH 4.5), 20% aqueous NaBEt4 solution (STEB solution) and hexane (with naphthalene as internal standard) were added to each sample and this mixture will be shaken and heated to 60 °C. During this step, the organotin chlorides were converted into the corresponding ethylated tetraorganotin derivatives, which were extracted into the hexane layer. Prior to GC-MS analysis, the hexane layer was washed with 2 mol/L HCI in order to remove (most of) the ethylboron compounds that interfere with the GC-MS analysis. The concentration of each test substance in feed was determined by GC-MS analysis of the hexane extracts.

The homogenous distribution, stability and achieved concentration were determined
Duration of treatment / exposure:
13 consecutive weeks of treatment with test substance in diet.
Frequency of treatment:
ad libitum
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 10, 100, 300 mg/kg diet
Basis:
nominal in diet
No. of animals per sex per dose:
10 rats per sex per dose.
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: based on 14 day range finding study
- Rationale for animal assignment (if not random): Computer randomisation
- Rationale for selecting satellite groups: Computer randomisation
- Post-exposure recovery period in satellite groups: NDA
- Section schedule rationale (if not random): N/A
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily in the morning. On weekdays, also checked again in afternoon for dead or moribund animals.


DETAILED CLINICAL OBSERVATIONS: Yes, handled if necessary to detect signs of toxicity
- Time schedule: daily, if necessary.


BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded once during the acclimatization period (data not reported but kept as raw data), at initiation of the study prior to introduction of feed, and twice (dose range finding study) or once weekly (13-week study) thereafter


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food /kg bw / day and g food / animal / day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


FOOD EFFICIENCY:
- Body weight gain in g/food consumption in g per animal per day. The efficiency of food utilization was calculated and expressed in g weight gain per g food consumed. Food consumption of male rats of the 13 week study was not measured in weeks 10 (all animals) and 11 (some animals), because this measurement was hampered by the mating procedure.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): N/A
- Time schedule for examinations: N/A


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmoscopic observations were made prior to the start of treatment in all animals and towards the end of the treatment period.
- Dose groups that were examined: In all surviving animals of the control group (group A) and the 300 mg/kg group (group D)


HAEMATOLOGY: Yes
- Time schedule for collection of blood: At necropsy at the end of treatment, blood samples were taken from the abdominal aorta of all surviving rats.
- Anaesthetic used for blood collection: Yes (identity) C02/02-anaesthesia
- Animals fasted: Yes, overnight
- How many animals: all surviving animals.

Parameters checked below were examined:
- haemoglobin
- packed cell volume
- red blood cell count
- reticulocytes
- total white blood cell count
- differential white blood cell count
- prothrombin time
- thrombocyte count

The following parameters were calculated:
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin (MCH)
- mean corpuscular haemoglobin concentration (MCHC)



CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At necropsy at the end of treatment.
- Animals fasted: Yes, overnight
- How many animals: all surviving rats

Parameters checked below were examined:
- alkaline phosphatase activity (ALP) aspartate
- aminotransferase activity (ASAT) alanine
- aminotransferase activity (ALAT)
- gamma glutamyl transferase activity (GGT) total protein
- albumin
- ratio albumin to globulin
- urea
- creatinine
- total bile acid
- bilirubin (total and direct)
- cholesterol (total)
- triglycerides phospholipids
- calcium (Ca)
- sodium (Na)
- potassium (K)
- chloride (Cl)
- inorganic phosphate
- fasting glucose



URINALYSIS: Yes
- Time schedule for collection of urine: shortly before the end of treatment (days 86/87).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Deprived of water for 24 hours and food for 16 hours.

Parameters checked below were examined:
- appearance
- protein
- glucose
- bilirubin
- pH
- urobilinogen
- occult blood
- microscopy of the sediment
- ketones



NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Weekly
- Dose groups that were examined: all rats.

Battery functions tested:
- Posture
- Convultions, tremors
- Palpebral closure
- Lacrimation
- Piloerection
- Salivation
- Vocalisation
- Rearing
- Urination
- Defecation
- Gait
- Arousal
- Mobility
- Ease of removal
- Handling reactivity
- Palpebral closure
- Approach response
- Click response
- Tail pinch response
- Touch response
- Righting response
- Landing foot splay
- Forelimb grip strength
- Handlimb grip strength
- Pupil response
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, see below

All animals were subjected to a complete gross necropsy. After 14 days of treatment, the animals of the dose range finding study were killed. In week 14, the animals of the 13-week study were killed on a number of successive working days, in such a sequence that the average time of killing was approximately the same for each group. The animals were killed by exsanguination from the abdominal aorta under C02/02-anaesthesia and then examined grossly for pathological changes.
In the dose range finding study the adrenals, kidneys, liver, spleen, testes and ovaries of all survivors were weighed and preserved in formalin, and were discarded at the end of the main study.
At final necropsy in the 13-week study, the following organs were weighed (paired organs together) as soon as possible after dissection to avoid drying:

- adrenals
- ovaries
- brain
- spleen
- epididymides
- testes
- heart
- thymus
- kidneys
- thyroid (with parathyroids)
- liver

To preserve any potential evidence of effects of tin compounds on the hippocampus or other regions of the brain, the brain was removed as soon as possible at the final necropsy and placed in fixative for histopathological evaluation.
uterus

HISTOPATHOLOGY: Yes, see below

The tissues to be examined microscopically were embedded in paraffin wax, sectioned at 5µm and stained with haematoxylin and eosin. Histopathological examination was performed on all tissues and organs listed above - except those marked with an asterisk - of all animals of the control group (group A) and of the 300 mg/kg group (group D). In addition, lungs, liver, kidneys and gross lesions were examined microscopically in all rats of the intermediate dose¬groups.
Since treatment-related changes were found in the thymus of males and females of the 300 mg/kg group, histopathology on this organ was extended to males and females of the intermediate-dose groups.


adrenals
parathyroid
aorta
* parotid salivary glands
*axillary lymph nodes pituitary
brain (brain stem, cerebrum, cerebellum)
prostate
caecum
rectum
colon
* seminal vesicles with coagulating glands
epididymides
* skeletal muscle (thigh)
* exorbital lachrymal glands
skin (flank)
eyes
small intestine (duodenum, ileum, jejunum)
*femur with joint spinal cord (at three levels)
GALT (gut associated lymphoid tissue, including Peyer's patches)
spleen
sternum with bone marrow
* Harderian gland stomach (glandular and non-glandular)
heart
sublingual salivary glands
kidneys
submaxillary salivary glands
liver
testes
lungs
thymus
mammary gland (females)
thyroid
* mandibular (cervical) lymph nodes
* tongue
mesenteric lymph nodes
trachea/bronchi
* nasal cavity
urinary bladder
nerve-peripheral (sciatic)
uterus (with cervix)
oesophagus
* vagina
ovaries
* Zymbals gland
pancreas
all gross lesions.

* The tissues marked with an asterisk were preserved but not processed for histopathological examination, since histopathological examination was not considered necessary on the basis of the results of gross observations.
Other examinations:
N/A
Statistics:
The statistical procedures used in the evaluation of data were as follows:
• Body weight: one way analysis of covariance (covariate: body weight on day 0) followed by Dunnett's multiple comparison tests;
• Neurobehavioural observations: parameters assessed during functional observations were measured on different measurement scales (e.g. continuous, rank, categorical). Continuous measures were analysed by analysis of variance techniques and other parametric tests where appropriate. Rank and categorical data were analysed non-parametrically. Motor activity data were analysed using analysis of variance techniques;
• Food consumption and food efficiency: one way analysis of variance (anova) followed by L.S.D. tests;
• Red blood cell and clotting potential variables, total white blood cell counts, absolute differential white blood cell counts, clinical chemistry values, volume and density of the urine, organ weights: one way analysis of variance (anova) followed by Dunnett's multiple comparison tests;
• Relative differential white blood cell counts, urinary parameters except volume and density: Kruskal-Wallis non-parametric anova followed by Mann-Whitney U-tests;
• Histopathological changes: Fisher's exact probability test.

All tests were two-sided. Probability values of p<0.05 were considered significant.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No treatment-related clinical signs were observed.

BODY WEIGHT AND WEIGHT GAIN
Body weights were statistically significantly decreased by about 9% in males and females of the 300 mg/kg group and females throughout the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption was slightly decreased in males and females of the 300 mg/kg group (by about 8 and 11%, respectively). On a number of days the difference reached the level of statistical significance. Food consumption was generally similar among the control, 10 and 100 mg/kg groups in males and females. An occasional statistically significant difference was seen among these groups.

FOOD EFFICIENCY
Food conversion efficiency was similar among the groups in males and females throughout the study. An occasional statistically significant difference was seen.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study) N/A


OPHTHALMOSCOPIC EXAMINATION
No treatment-related ocular changes were observed.

HAEMATOLOGY
The following statistically significant changes in haematology parameters were observed:
- Hb was decreased in females of the 300 mg/kg group;
- PCV was decreased in females of the 300 mg/kg group;
- MCV was decreased in males of the 100 mg/kg group;
- MCH was decreased in the 100 (males) and 300 mg/kg groups (males and females);
- Reticulocytes were decreased in males of the 100 mg/kg group;
- Prothrombin time was increased in females of the 300 mg/kg group;
- Total WBC was decreased in males of the 300 mg/kg group. Atlthough not statistically significant, a similar decrease was also seen in emales of the 100 and 300 mg/kg groups;
- The absolute number of lymphocytes was decreased in males of the 300 mg/kg group;
- The absolute numbers of monocytes were decreased in females of all treated groups.


CLINICAL CHEMISTRY
The following statistically significant changes in clinical chemistry parameters were observed:
- ALP was increased in males and females of the 100 and 300 mg/kg groups;
- TP was decreased in females of the 300 mg/kg group;
- The A/G ratio was increased in the 10 (females) and 300 mg/kg groups (males and females);
- Total bilirubin was increased in females of the 100 and 300 mg/kg groups;
- Direct bilirubin was increased in females of the 300 mg/kg group;
- Cholesterol was decreased in females of the 300 mg/kg group;
- Bile acids were increased in males of the 300 mg/kg group. Although not statistically
significant, a similar increase was also seen in females of the 300 mg/kg group;
- Phospholipids was increased in males of the 10 mg/kg group and decreased in females of the 300 mg/kg group;
- Calcium was decreased in females of the 300 mg/kg group;
- Sodium was decreased in males of the 100 and 300 mg/kg groups.


URINALYSIS
Urinary volume and density were similar among the groups. Urinary crystals were statistically significantly increased in females of the 300 mg/kg group. Further semi-quantitative and microscopic urinary observations were similar among the groups.


NEUROBEHAVIOUR
No neurotoxic effects of treatment were observed from neurobehavioural measures and motor activity assessment in any of the groups at any time point during the 13-week treatment period. Some abnormalities were observed in individual animals that were not considered to be related to treatment.
On one occasion during arena testing, a tilted head was observed in one female. This single observation was not considered to be related to treatment.
Tiptoe walking was observed in some females of different groups in various weeks of the study. Tiptoe walking was not considered to be related to treatment, for it was observed in females only, but in all groups, including the control group. Further, it was not consistently observed in the concerned animals from first occurrence towards the end of the test period and the severity of this gait abnormality did not increase over time.
The results of the neurobehavioural observations and motor activity assessment did not indicate any neurotoxic potential of the test substance.


ORGAN WEIGHTS
The following statistically significant changes in organ weights were observed:
Absolute adrenal weights were decreased in females of the 300 mg/kg group and relative adrenal weights were decreased in females of the 100 mg/kg group;
Absolute and relative thymus weights were decreased in the 10 (females; c.14%), 100 (males and females; c. 48% and 69%, respectively) and 300 mg/kg groups (males and females; c. 74% and 72%, respectively);
Absolute spleen weights were decreased in females of the 300 mg/kg group;
Relative kidney weights were increased in females of the 300 mg/kg group;
Relative liver weights were increased in males and females of the 300 mg/kg group; Relative testes weights were increased in animals of the 300 mg/kg group.


GROSS PATHOLOGY
At necropsy, treatment-related gross changes were not observed.


HISTOPATHOLOGY: NON-NEOPLASTIC
At microscopical examination, treatment-related histopathological changes were observed in the thymus. The histopathological changes comprised lymphoid depletion, characterised by a decrease in the size of the thymic lobules which can be ascribed to extensive loss of cortical en medullary small lymphocytes. Consequently, the distinction between the cortical and medullary areas was blurred.
The thymic change was observed in 5/10 males of the 100 mg/kg group and in 9/10 males of the 300 mg/kg group and in 10/10 females of the 100 mg/kg group and in 9/10 females of the 300 mg/kg group. Lymphoid depletion was not observed in any of the controls or 10 mg/kg animals.
All other histopathological changes were common findings in rats of this strain and age. They were about equally distributed amongst the different treatment groups or occurred in one animal only. Therefore, they were not considered to be related to treatment.


HISTOPATHOLOGY: NEOPLASTIC (if applicable) N/A


HISTORICAL CONTROL DATA (if applicable) N/A

Effect levels

Dose descriptor:
LOAEL
Effect level:
0.7 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Absolute and relative thymus weight.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the decreased thymic weights and associated histopathological findings in animals of the 100 and 300 mg/kg groups, these dose-levels were considered overt adverse effect levels. The decreased absolute and relative thymus weight in females of the 10 mg/kg group was considered to be the result of the treatment with Dichlorodioctylstannane. Although this change was not accompanied by histopathological changes, it was considered to reflect a toxicologically relevant change in the thymus, which was in accordance with the shown toxicity profile of the test substance (i.e. thymotoxicity). For this reason, a No Observed Adverse Effect Level (NOAEL) for sub-chronic toxicity cannot be established. The Lowest Observed Effect Level (LOAEL) in the present sub-chronic toxicity study was placed at the low dose of 10 mg Dichlorodioctylstannane/kg diet. This level was equivalent to 0.7 mg/ kg body weight/day in males and females.
Executive summary:

The toxicity of Dichlorodioctylstannane [CAS # 3542-36-7] in Wistar rats was examined using continuous administration via the diet for 13 consecutive weeks (OECD Test Guideline 408).The main study used four groups of 10 rats/sex (13-week study) and the satellite study used four groups of 10 female rats (reproduction/developmental screening study). The control group was kept on untreated diet and the three test groups received experimental diets containing 10, 100 and 300 mg/kg (ppm) of the test substance. The dose levels used in both studies were selected based on the results of a preceding dose range finding study.

Based on the decreased thymic weights and associated histopathological findings in animals of the 100 and 300 mg/kg groups, these dose-levels were considered overt adverse effect levels. The decreased absolute and relative thymus weight in females of the 10 mg/kg group was considered to be the result of the treatment with Dichlorodioctylstannane. Although this change was not accompanied by histopathological changes, it was considered to reflect a toxicologically relevant change in the thymus, which was in accordance with the shown toxicity profile of the test substance (i.e. thymotoxicity). For this reason, a No Observed Adverse Effect Level (NOAEL) for sub-chronic toxicity cannot be established. The Lowest Observed Effect Level (LOAEL) in the present sub-chronic toxicity study was placed at the low dose of 10 mg Dichlorodioctylstannane/kg diet. This level was equivalent to 0.7 mg/ kg body weight/day in males and females.