Trichlorooctylstannane

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26th February - 10th June 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Guideline study conducted under GLP. The method of analysis involved derivatization. This method only measures the amount of the alkyltin moiety, MOT, present and does not identify the other ligands attached to the tin. All measured MOT was attributed to the parent substance. Currently there is no analytical method available that can quantify the actual named substance, i.e., the entire organotin compound with its associated chloride ligand.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD 408

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD 421

Deviations:

not specified

Principles of method if other than guideline:

Type: other: reproduction/developmental screening study
Method: other: OECD Guide-line 421

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Trichlorooctylstannane

EC Number:

221-435-4

EC Name:

Trichlorooctylstannane

Cas Number:

3091-25-6

Molecular formula:

C8H17Cl3Sn

IUPAC Name:

trichlorooctylstannane

Details on test material:

SOURCE: ORTEP Association Stabilizer Task Force
PURITY: 89.2% Trichlorooctylstannane (monooctyltin trichloride)
ANY OTHER INFORMATION:
Alkyl group distribution % (mass/mass)
Monooctyltin trichloride 85.50
Dioctyltin dichloride 11.07
Trioctyltin chloride 0.50
Tin tetrachloride 0.03
Mono-i-butyltin trichloride 0.42
Monobutyltin trichloride 0.03
Monooctyltin trichloride isomers 0.91
Octyl-i-butyltin dichloride 0.12
Octylbutyltin dichloride 0.05
Monododecyltin trichloride 0.03
Dioctyltin dichloride isomer 0.06
Monohexadecyltin trichloride 1.10
Octylhexadecyltin dichloride 0.17

The alkyl group distribution of trichlorooctylstannane was determined using a Grignard ethylation (ethylmagnesium bromide) followed by GC-analysis with FID detection. The concentration of trichlorooctylstannane in feed was determined by GC-MS analysis of the hexane extracts. The homogeneity and stability of the test substance were determined for each group by a one way ANOVA (sample location and time as grouping factor, respectively).

Test animals

Species:

rat

Strain:

other: Wistar outbred (Crl:(WI)WU BR)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Obtained from a colony maintained under SPF conditions at Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: In the 13 weeks study, the animals were 7 weeks old at study initiation, in the satelite group, the animals were 13-14 weeks of age.
- Weight at study initiation: 137.7 - 168.0 g (mean 154.4 g) for males; 110.4 - 135.1 g (mean 122.4 g) for females and 187.1 g to 214..2 g (mean 200.4 g) in the satelite group.
- Housing: Animals were housed under conventional conditions in one room, in macrolon cages, with sterilised 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. During the premating period females of the satelite groups were housed 3 or 4 per group per cage. During the gestation and lactation period the females were housed individually.
- Diet : RM3 (commercial Rat & Mouse No. 3 Breeding Diet) diet was provided ad libitum as a powder, refreshed once a week, provided in stainless steel cans covered by a perforated steel plate to prevent spillage.
- Drinking water: Tap water was provided ad-libitum in polypropylene bottles.
- Acclimation period: The quarantine and acclimatisation periods between arrival and experimental start date were 5, 13 and 13 days (range finder, definitive test and satelite groups respectively).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): at least 30% not exceeding 70 % other than during room cleaning (max 99.9 %)
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light): Artificial light with a sequence of 12 hours light and 12 hours dark.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): The experimental diets were prepared shortly prior to study initiation, and roughly every six weeks thereafter.
- Mixing appropriate amounts with (Type of food): The test material was mixed with RM3 diet with a mechanical mixer.
- Storage temperature of food: <-18 °C freezer until use

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

GC-MS was used to determine the achieved concentration, homogeneous distribution and stability of the test substance in diet samples. The method of analysis involved derivatization. This method only measures the amount of the alkyltin moiety, MOT, present and does not identify the other ligands attached to the tin. All measured MOT was attributed to the parent substance. Currently there is no analytical method available that can quantify the actual named substance, i.e., the entire organotin compound with its associated chloride ligand.

From each diet sample, 2.0 g was transferred into a 50 mL Greiner tube. An aliquot of the internal standard solution (monoheptylin trichloride, diheptylin dichloride, tripopyltin chloride and tetrapropyltin in methanol) was added. Subsequently methanol, acetate buffer solution (pH 4.5), 20 % aqueous NaBEt4 solution (STEB solution) and hexane (with naphtalene as internal standard) were added to each sample, this mixture was agitated 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 HCL to remove the majority of the ethylboron compounds that interfere with the GC-MS analysis of the hexane extracts.
The homogenous distribution, stability and acheived concentration of the test substance in RM3 rat feed was analysed in the batch of diets prepared for the dose range finding study. The homogenous distribution and acheived concentration of the test substance in RM3 rat feed of the high dose-group of the 13-week study (500 mg/kg) was determined in the first batch of diets prepared for the 13 wek study. Validation of the high dose level was performed simultaneously with the homogeneity and content analyses in the first batch of diets prepared for the 13-week study.#
The same diet preparation protocol was used in the dose range finding study and the 13-week studies.
The criteria for homogeneity, stability and content of the test substance in the diet were:
Homogeneity:- For each group a one way analysis of variance was performed using the sample location (1-5) as a grouping factor. An associated F-value with probability p < 0.01 were considered to be significant. The test substance was considered to be homogeneously distributed in the diets if p = 0.01 and/or if the relative standard deviation (RSD) between the sample means was less than or equal to 15%.
Stability:- For each group a one way analysis of variance (Anova) was performed using time as a grouping factor. An associated F-value with probability p < 0.01 was considered to be significant. The test substance was considered to be stable in the diets if p = 0.01 and/or if the mean concentration on the last day was between 80 and 120 % of the mean concentration on the first day (t = 0).
Acheived concentration: For each concentration level, the mean of the concentrations as measured in the study samples used for the assessment of the homogeneity was considered to represent the acheived concentration. The content of the test substance in diet was considered to be "close to intended" if the mean measured concentration was between 80 % and 120 % of the intended concentration. Directly after mixing of each diet for the dose range finding study, samples for the homogeneity/stability experiments were taken in the order; top centre, middle centre, bottom centre, left centre, right centre and labelled as such. Secondly, five samples (of about 50 g each) for examination of the stability were taken from the top centre part of the mixer. All samples wer labelled with the diet codes (TNO study number), the colour-codes, the nominal concentrations of the test substance and the date of preparation. The samples taken for the homogeneity experiments were also used for dose confirmation. In addition, analyses to determine the content (acheived concentration) of the test substance in three batches of diets used in the 13 week study were conducted.
Diet samples for the determination of the content of the diets used in this study were taken immediately after preparation of the diets and stored at ca. -18 °C.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal smear, considered as gestation day 0

If a male died before or during the mating period, before the female is found positive, then the female was mated with a proven male of the same group.

Duration of treatment / exposure:

The test substance was administered at constant concentrations in the diet for 14 consecutive days in the dose range finding study or 13 consecutive weeks in the reproduction/developmental screening test. In the reproduction/developmental screening study, the test material was administered via the diet for two weeks premating, during gestation and up to euthanasia at or shortly after postnatal day (PN) 4.

Frequency of treatment:

Daily

Duration of test:

13 weeks

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 10, 30, 100 and 300 mg MOTC/kg diet in the range finding study
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
10, 100 and 500 mg trichlorooctylstannane/kg diet in the main test
Basis:
nominal in diet

No. of animals per sex per dose:

Range finder study: five groups of 4 rats/sex/group
Main study: four groups of 10 rats/sex/group
Satelite group: four groups of 10 female rats

Control animals:

yes, plain diet

Details on study design:

On nominal day 0 the animals were allocated (males and females separately) to the various groups by computer randomization. Female rats of the satellite groups were allocated to the various groups by computer randomization on nominal day -4.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily (in the morning) and on working days also once in the afternoon

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: once during the acclimatization period, once at initiation of the study prior to introduction of feed. Thereafter, body weights were recorded once weekly. Furthermore, all animals were weighed on the day of necropsy in order to determine their correct organ to body weight ratios.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
The intake of substance per kg/bw/day was calculated from the nominal dietary concentration of the substance, the food consumption and the mean body weight in the period for which the intake of the substance is calculated.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes
Measured per cage over weekly periods by weighing the feeders (in g/animal/day). The efficiency of food utilization was calculated and expressed in g weight gain/g food consumed. Food consumption of male rats of the main study was not measured in weeks 10 (all animals) and 11 (some animals), because this measurement was hampered by the mating procedure (male rats of the 13-week study were used to mate with female rats from the satellite group).

WATER CONSUMPTION : No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Opthalmoscopic observations were made prior to the start of treatment in all animals and towrds the end of the treatment period. Examinations were carried out using an ophthalmoscope after induction of mydriasis by a solution of atropine sulphate.
- Dose groups that were examined: All surviving animals of the control group (Group A) and the high-dose 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. K2-EDTA was used as an anticoagylant.
- Anaesthetic used for blood collection: Yes, CO2/O2-anaesthesia
- Animals fasted: Yes, overnight fasting at necropsy.
- How many animals: All surviving rats
- Parameters checked: haemoglobin, packed cell volume, red blood cell count, reticulocytes, total white blood cell count, prothrombin time and thrombocyte count. The following parameters were calculated; mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpsucular haemoglobin concentration (MCHC).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At necropsy at the end of the treatment. Blood was collected from the abdominal aorta in heparinised plastic tubes and plasma was prepared by centrifugation
- Animals fasted: Yes
- How many animals: All survivng animals.
- Parameters checked: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGCT), 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 and 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, during the last 16 hours, the rats were kept in metabolism cages (one rat per cage)
- Animals fasted: Yes, all rats were deprived of water for 24 hours and of food during the last 16 hours of this period.
- Parameters checked: appearance, glucose, pH, occult blood, ketones, protein, bilirubin, urobilinogen and microscopy of the sediment.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Detailed neurobehavioural examinations were conducted in the experimental room outside the home cage prior to the first exposure and then once weekly up to and including week 12.
- Dose groups that were examined: Each animal of the main group.
- Battery of functions tested: The Functional Observational Battery used in the study included:
Autonomic - lacrimation, salivation, pupil response to light, palpebral closure, piloerection, defecation, urination
Neuromuscular: gait, mobility, forelimb and hindlimb gripstrength, landing foot splay, righting reflex
Sensorimotor: response to tail pinch, click, touch and approach of a visual object
Convulsive: clonic and tonic movements
Excitability: ease of removal, handling reactivity, arousal, vocalisations
Activity: rearing, motor activity
Physiological: body temperature

ORGANS EXAMINED AT NECROPSY:
After 14 days treatment, the animals of the dose range finding study were killed. In week 13, the animals of the 13 week study were killed on a number of successive working days, in such a sequence that the average killing was approximately the same for each group. The animals were killed by exsanguinations from the abdominal aorta under CO2/O2 anaesthesia and then examined for grossly pathological changes.

TOXIC EFFECTS
- Macroscopic: organs weighed included adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, thyroid (with parathyroids), uterus.
-Histopathological examination was performed on tissues and organs of all animals of the control and 500 mg/kg group and included adrenals, aorta, brain (brain stem, cerebrum, cerebellum), caecum, colon, epididymides, eyes, GALT (gut associated lymphoid tissue, including Peyer's patches), heart, kidneys, liver, lungs, mammary gland (females), mesenteric lymph nodes, nerve-peripheral (sciatic), oesophagus, ovaries, pancreas, parathyroid, pituitary, prostate, rectum, skin (flank), small intestine (duodenum, ileum, jejunum), spinal cord (at 3 levels), spleen, sternum with bone marrow, stomach (glandular and non-glandular), sublingual salivary glands, submaxillary salivary glands, testes, thymus, thyroid, trachea/bronchi, urinary bladder, uterus (with cervix) and all gross lesions
- Microscopic: lungs, liver, kidneys and gross lesions were examined microscopically in all intermediate dose groups.

REPRODUCTIVE EFFECTS
- Macroscopic: all adult animals were subjected to a complete gross necropsy. Organs weighed included: ovaries, uterus (after counting of the implantation sites), thymus and all gross lesions. Samples of latter organs were preserved for microscopic examination.
- Microscopic: microscopic examination of the ovaries, uterus and thymus of the control and high dose group (500 mg/kg) was performed. Microscopic examination was extended to the thymus of the females of the 10 and 100 mg/kg groups because of the effects observed in the 500 mg/kg group for this tissue/organ. Furthermore, the reproductive organs of the males of the 10 and 100 mg/kg groups that failed to sire (did not mate or female was not pregnant), and the reproductive organs of females of the 10 and 100 mg/kg groups that were non-mated or non-pregnant, were microscopically examined.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:

The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Uterus weight: Yes
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of implantation loss: Yes

Fetal examinations:

- Parturition and litter evaluation: at the end of the gestation period, females were examined twice daily for signs of parturition. Any difficulties occurring during parturition were recorded. To keep nest disturbance to a minimum, the litters were examined only once daily for dead pups.
- Litter size, sexes and weights: the total litter size and numbers of each sex as well as the number of stillbirths, live and dead pups and grossly malformed pups were evaluated on days 1 and 4 of lactation. The pups were weighed individually and litter weight was calculated for days 1 and 4 of lactation. Mean pup weights were calculated as litter weight/number pups. The number of runts (< 2 sd from the litter mean) were noted and reported.

Statistics:

Body weight data were evaluated by one way analysis of covariance (covariate: body weight on day 0) followed by Dunnett's multiple comparison tests.
For 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 were analysed by 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 and organ weights were all analysed by 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 were assessed by Kruskal-Wallis non-parametric anova followed by Mann-Whitney U-test.
Histopathological changes were assessed with Fisher’s exact probability test.
All tests were two-sided. Probability values of p < 0.05 were considered significant.

Indices:

With regard to fertility and reproductive performance, the following parameters were calculated:
-pre-coital time = time between the start of mating and successful copulation
-duration of gestation = time between gestation day 0 and day of delivery
-mating index = (number of females mated/number of females placed with males) x 100
-male fertility index = (number of males that became sires/number of males placed with females) x 100
-female fertility index = (number of pregnant females/number of females placed with males) x 100
-female fecundity index = (number of pregnant females/number of females mated) x 100
-gestation index = (number of females with live pups/number of females pregnant) x 100
-live birth index = (number of pups born alive/number of pups born) x 100
-pup mortality day n = (number of dead pups on day n/total number of pups on day n) x 100
-sex ratio day n = (number of live male pups on day n/ number of live pups on day n) x 100
-number of lost implantations = number of implantations sites - number of pups born alive
-post-implantation loss = [(number of implantation sites - number of pups born alive)/number of implantation sites] x 100
-viability index day 1-4 = (number of pups surviving 4 days/total number of live pups on day 1) x 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
In the main groups, no treatment-related clinical signs were observed. In the satelite groups, one animal (D179) was found dead on post natal day 2. During the premating, mating, gestation and lactation period no treatment-related clinical signs were observed.

BODY WEIGHT AND WEIGHT GAIN
In the main groups, body weights were similar among the groups in males and females throughout the study. An occasional statistically significant difference was seen. In the satelite groups, during the premating, gestation and lactation periods, mean body weight and body weight change of the female animals were similar among groups.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption was similar among the groups in males and females throughout the study. An occasional statistically significant difference was seen. The overall intake of the test substance for the 10, 100 and 500 mg/kg groups respectively was approximately 0.6, 6.4 and 31.5 mg/kg body weight/day in males and 0.7, 6.8 and 32.9 mg/kg body weight/day in females.

FOOD EFFICIENCY
Food conversion efficiency was similar among the groups in males and females throughout the study. An occasional statistically significant difference was seen in the main groups. In the satellite groups, the test substance intake of the female animals of the 10, 100 and 500 mg/kg groups was:
Premating period during days 0-7 the intake was 0.6, 6.1 and 29.1 mg/kg body weight/day, and on days 7-14, the intake was 0.6, 6.1 and 28.3 mg/kg body weight/day. During gestation, during days 0-7 the intake was 0.7, 6.9 and 31.7 mg/kg body weight/day, gestation days 7-14 0.7, 6.8 and 32.6 mg/kg body weight/day, and on gestation days 14-21 intake was 0.5, 4.8 and 22.3 mg/kg bodyweight/day. During lactation, PN 1-4, the consumption of the test material was 0.7, 7.7 and 33.0 mg/kg body weight/day.

OPHTHALMOSCOPIC EXAMINATION
No treatment related ocular changes were observed.

HAEMATOLOGY
The haematological parameters assessed were found to be similar across all groups.

CLINICAL CHEMISTRY
In males and females of the 500 mg/kg group ALP was statistically significantly increased. In males of the 10 and 100 mg/kg groups the A/G ratio was statistically lower than controls. No change was observed in the measured parameters (total protein and albumin) and no clear dose-response relation was present, these findings were considered coincidental. The statistically significant increase in the level of calcium in 10 mg/kg females was considered also coincidental. No other statistically significant differences were noted.

URINALYSIS
Urinary volume and density were similar among the groups. Semi-quantitative and microscopic urinary observations were similar among the groups.

NEUROBEHAVIOUR
Results fromf the neurobehavioural observations and motor activity assessment did not indicate any neurotoxic potential of the test substance.

ORGAN WEIGHTS
In the main groups, the absolute liver weights were statistically significantly decreased in males and females of the 500 mg/kg group by 25-28 % and 47-50 % respectively. No other statistically significant differences were observed among the groups. In the satelite groups, mean absolute uterus weight was statistically increase in the 10 mg/kg group when compared to the control group. This difference was considered to be a chance finding. No other differences were observed in absolute and relative uterus and ovary weights. Mean absolute and relative thymus weights of the females of the 100 and 500 mg/kg groups were decreased by 26-24 % and 31-35 % respectively. However, these were not found to be statistically significant.

GROSS PATHOLOGY
In the main groups, gross examination of male and female rats at necropsy did not reveal any treatment-related changes with the exception of the decreased weight of the thymus. The decreased thymic size was not reported as a gross lesion since decreases in thymus weights were considered to reflect the diminished size of the thymus in individual animals more accurately. In the satelite groups, gross examination of all female rats including the animal that was found dead on PN 2 revealed no treatment related changes. In both the satelite and main groups, the other observations at necropsy represented common background pathological findings in rats of that age and strain.

HISTOPATHOLOGY: NON-NEOPLASTIC
In the main groups, examination of the thymus revealed slight to severe lymphoid depletion in 9/10 females of the 500 mg/kg group. Lymphoid depletion was characterised by a decrease in the size of the thymic lobules due to an extensive loss of cortical en medullary small lymphocytes. The distinction between the cortical and medullary areas became blurred. In the more severe cases, the cortex was very small or partially absent. The remaining lymphoblastic cells and reticuloepithelial cells had increased or were more pronounced because of the disappearance of small lymphocytes and collapse of thymic stroma. In the satellite group, only the reproductive organs and in females, thymi were examined microscopically. All histopathological changes in the testes, epididymides, prostate and in the ovaries and uterus were considered common findings in rats of that age and strain. These only appeared to occur only incidentally or at similar levels amongst the treatment groups, including controls. Therefore, they were not considered to be related to treatment. No cause of death could be established for animal D179 which died on post-natal day 2. Examination of the thymus revealed moderate to severe lymphoid depletion in 8/9 females of the 500 mg/kg group (autolytic changes hindered the evaluation of the thymus of animal D179). Lymphoid depletion was characterised by a decrease in the size of the thymic lobules which were attributed to extensive loss of cortical en medullary small lymphocytes. The distinction between the cortical and medullary areas became blurred. In severe cases the cortex was very small, or even partially absent. The remaining lymphoid cells visible in the cortical areas were predominantly lymphoblasts. Lymphoblastic cells and reticuloepithelial cells had increased in number or these cells were more pronounced fue to the disappearance of small lymphocytes and collapse of thymic stroma. In three females of the 100 mg/kg group and in one female of the 500 mg/kg group only remnants of thymic tissue were seen, describes as "very severe atrophy". This was characterised by very small lobules, with complete loss of the normal architecture, consisting predominantly of reticulo-epithelial cells/stroma and only a few lymphoblasts and lymphocytes. The statistically significant decrease in incidence of pregnancy/lactation involution seen in the thymus of animals of the 500 mg/kg group, was ascribed to the presence of overt treatment-related lymphoid depletion, which obscured the lactation-related involution. Pregnancy/lactation involution, characterised by a decreased size of thymic lobules exhibiting normal architecture, is a common observation in pregnant or lactating animals. All other observations made in both the main and satelite groups represented common background pathological findings in rats of that strain and age and occurred only incidentally or at a similar level amongst the groups, including the control animals. These were considered not to be the result of treatment.

OTHER: RANGE FINDING STUDY Dietary doses of 10, 30, 100, and 300 mg/kg as MOTC in feed (ppm) were administered for 14 days. The test substance was not homogeneously distributed in the two low dose levels (10 mg/kg and 30 mg/kg). No clinical signs were observed. Body weights were significantly lower in the 100 mg/kg group (females: days 4 and 14). Food consumption was significantly lower in the 30 and 100 mg/kg groups (females: day 14) and in the 300 mg/kg group (females: days 7 and 14). Food conversion efficiency was similar among the groups. Absolute and relative organ weights (ovaries, testes, adrenals, kidneys, spleen and liver) were not significantly different among the groups, except for a lower absolute liver weight in the 30 mg/kg group (females). Macroscopic examination at necropsy did not reveal any treatment-related changes. Dietary exposure to trichlorooctylstannane up to 300 mg/kg for 14 days was well tolerated; however, the body weight and food consumption decreases were deemed palatability effects. The significantly lower liver weights of females of the 30 mg/kg group was considered a chance finding. Doses for the main and satellite studies were chosen as 10, 100 and 500 mg/kg in the diet.

MATERNAL TOXIC EFFECTS:
- Mortality and day of death: One animal was found dead on PN 2 (i.e. 39 days after the start of exposure).
- Pre-coital time: comparable among the control and treated groups
- Mating index: 100% for all groups
- Number pregnant per dose level: 9, 9, 7 and 7 for the control, 10, 100 and 500 mg/kg groups, respectively.
- Female fecundity index: comparable among the control and treated groups
- Female fertility index: comparable among the control and the treated groups
- Male fertility index: comparable among the control and the treated groups.
- Number aborting: No data available.
- Number of resorptions: No data available.
- Number of implantations: 11-15 (control group), 10-14 (10 mg/kg), 9-13 (100 mg/kg), 10-14 (500 mg/kg).
- Gestation index: 100, 100, 100 and 71% in the control, 10, 100 and 500 mg/kg groups, respectively.
- Live birth index: 100, 99, 97 and 74% in the control, 10, 100 and 500 mg/kg groups, respectively.
- Number of females with live born pups: 9, 9, 7 and 5 in the control, 10, 100 and 500 mg/kg groups, respectively.
- Number of females with stillborn pups: 0, 1, 1 and 3 for the control, 10, 100 and 500 mg/kg groups, respectively.
- Number of females with all stillborn pups: Only in the 500 mg/kg group; 2 females with all still born pups were observed.
- Post implantation loss: 6.4, 6.3, 14.5 and 41.3% for the control, 10, 100 and 500 mg/kg groups, respectively.
- Number of corpora lutea: No data available.
- Duration of Pregnancy: 21-23 days

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Remarks:

Dose-Range Findings test (Toxicity)

Effect level:

6.8 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

4.8 - 7.7 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: other:

3

Dose descriptor:

NOAEL

Effect level:

6.4 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: other:

4

Dose descriptor:

NOAEL

Effect level:

0.5 - 0.7 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
- Litter size: The mean number of pups delivered per litter amounted to 11.6, 11.4, 10.3 and 8.9 for the control, 10, 100 and 500 mg/kg groups, respectively. The number of pups delivered was statistically significantly decreased in the 500 mg/kg group.
- Litter weight: Mean pup weights and pup weight changes were comparable among the control and treated groups.
- Pup mortality: 0, 1.0, 2.8 and 26% for the control, 10, 100 and 500 mg/kg groups, respectively (PN 1); 1, 7.8, 0 and 20% for the control, 10, 100 and 500 mg/kg groups, respectively (PN 4).
- Number viable: The viability index (PN 1-4) was 99, 92, 100 and 80% in the control, 10, 100 and 500 mg/kg groups, respectively.
- Number live pups per litter: 11.6, 11.3, 10.0 and 9.2 for the control, 10, 100 and 500 mg/kg groups, respectively (PN 1); 11.4, 10.4, 10.0 and 9.3 mg/kg for the control, 10, 100 and 500 mg/kg groups, respectively (PN 4)
- Sex ratio: No difference was observed in the sex ratio between the groups.
- Grossly visible abnormalities: The incidence of runts in the 10 mg/kg group was statistically significantly increased on PN 4; this observation was only significant on pup basis. No statistically significant difference in the number of runts was observed between the treated groups and the control group. For these reasons, the effect in the 10 mg/kg group is considered to be a chance finding. Macroscopic observation of the stillborn pups revealed no abnormalities of the pups. Four stillborn pups of the 500 mg/kg group were (partly) cannibalized; as far as possible no abnormalities were observed in these pups.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the incidences in several parameters which together are considered post-implantation losses at the 500 mg/kg dose, the mid-dose level of 100 mg Trichlorooctylstannane/kg (equivalent to 6.4 mg/ kg body weight/day in males and 4.8-7.7 mg/kg body weight for females) is judged to be the NOAEL for reproductive effects.
Based on the observed histopathological changes in the thymus (severe lymphoid depletion and very severe atrophy) of the 100 and 500 mg/kg female animals of the satellite groups, 10 mg Trichlorooctylstannane/ kg diet (equivalent to 0.5-0.7 mg/kg body weight/day) can be considered as a NOAEL for maternal toxicity.
The NOAEL for general sub-chronic toxicity, fertility and developmental effects was placed at 100 mg/kg diet. The NOAEL for maternal toxicity was placed at 10 mg trichlorooctylstannane/kg diet.

Executive summary:

In satellite groups of female rats a reproduction/developmental screening test (OECD Test Guideline 421) was performed to provide initial data on possible reproductive and developmental effects of trichlorooctylstannane. In the satellite study female rats were fed their respective test diets beginning 2 weeks prior to the mating period, and continued on test diets through mating, gestation and up to PN 4 or shortly thereafter. Male rats from the main study were mated after a premating period of 10 weeks with female rats of the satellite groups which were fed the same dose of test diets.

The calculated doses for the females receiving 10, 100, or 500 mg/kg trichlorooctylstannane in the diet were comparable to those in the main study during pre-mating, but varied with gestation stage and in the post-partum period. The ranges observed for the 10, 100, or 500 mg/kg groups were 0.5-0.7, 4.8-7.7, and 22.3-33.0 mg/kg body weight/day.

Mean body weight, body weight change and food consumption were similar in the control and the Trichlorooctylstannane treated groups.

No treatment-related effects on reproduction and development of the pups were observed in the 10 and 100 mg/kg groups.

In the 500 mg/kg group the following effects were indicative of a reproductive or developmental effect of Trichlorooctylstannane in the diet:

Two animals of the 500 mg/kg group delivered only dead pups and in 1 dam all pups died between PN 1-4. In the 500 mg/kg group the gestation index was 0.71 versus 1.00 in the control group, the live birth index was 0.74 versus 0.99 in the control group, and the viability index PN1-4 was 0.80 versus 0.99 in the control group. Post-implantation losses in the 500 mg/kg group were 41.3% versus 6.4% in the control group.

The majority of females of the 500 mg/kg group showed overt lymphoid depletion in the thymus. This microscopic change was considered treatment-related.

Very severe atrophy of the thymus was seen in 1/6 lactating female of the 500 mg/kg group and 3/7 lactating females of the 100 mg/kg group. This is an uncommon and toxicologically relevant observation in pregnant or lactating animals and is considered treatment-related.

The microscopic appearance of the affected thymi in the main study resembled the thymic atrophy described in the literature for some, but not all, organotin compounds. These gross and histopathological changes in the thymus suggest that some alkyl-organotins may have an immunotoxic effect, but no functional assessment of immune competency has been made in alkyltin-exposed animals so no definitive conclusion can be drawn.

Macroscopic observation of the stillborn pups revealed no abnormalities of the pups. Four stillborn pups of the 500 mg/kg group were (partly) cannibalized; as far as possible no abnormalities were observed in these pups.