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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 January 2004 to 8 March 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
OECD Guideline for Testing of Chemicals no. 422 (adopted 22 March 1996).
Deviations:
yes
Remarks:
See "Any other information" for details
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Dioctyltin oxide
EC Number:
212-791-1
EC Name:
Dioctyltin oxide
Cas Number:
870-08-6
Molecular formula:
C16H34OSn
IUPAC Name:
dioctylstannanone
Test material form:
solid
Details on test material:
Name: dioctyloxostannane
Common name: dioctyltin oxide
Abbreviation: DOTO
Physical appearance: colourless solid
CAS number: 870-08-6
Molecular formula: C16H34OSn
Molecular weight: 316.14g.mol-1
Solubility in water: poorly soluble
Melting point: 245-260°C
Density: 1.2 g.cm-3 at 20 °C

Purity: 94.64% dioctyltin oxide
1.48% monooctyltin oxide
3.33% dioctyltin dichloride
0.53% trioctyltin oxide

Storage temperature range: < -18°C
Protected from light: yes
ORTEP Lot no.: W01/61
Expiry date: 1 October 2003
Specific details on test material used for the study:
No further details specified in the study report.

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The study was conducted with rats. The rat was used because this species is considered one of the most suitable species for this type of study, and is usually required by regulatory agencies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Characterization of the test system
For the dose-range finding study 22 male and 22 female, and for the main study 52 male and 52 female, Wistar outbred (Crl:(WI)WU BR) rats of about 9-10 weeks of age were obtained from a colony maintained under SPF conditions at Charles River Deutschland, Sulzfeld, Germany.
At the start of the treatment period the rats were about 10-11 weeks old and the weight variation of the animals used did not exceed 20% of the mean weight for each sex.

Animal allocation
The animals of the dose-range finding study arrived on 5 November 2003 and the animals of the main study arrived on 7 January 2004. The rats from these studies were taken to room 5.1.13 and checked for overt signs of ill health and anomalies.
Upon arrival, serological controls of microbiological status were conducted in randomly chosen animals. After the results of serology indicated an acceptable microbiological status (on 7 November 2003 and 9 January 2004 for the dose-range finding study and main study, respectively), the animals were released for use in the studies. The animals were further acclimatised until the start of the study on 12 November 2003 and 14 January 2004, for the dose-range finding study and main study, respectively.
Shortly before the start of the studies, the animals (males and females separately) were allocated to the various treatment groups by computer randomization and proportionally to body weight. Surplus animals were kept in the animals rooms for monitoring during the study and discarded at the end of the study.

Identification of the test system
The studies were identified as study number 5374DRF (dose-range finding study) and 5374F0 (main study). During the acclimatization period, before allocation to the groups, the animals were identified by a temporary tailmark. After allocation to the different treatment groups, the rats were identified by a unique even (male) or odd (female) animal identification number. The identification numbers of the parental animals were clipped and tattooed in the ears. On PN 1, pups were identified by a tattoo in the paws and tail.
Each dosing group was coded by a letter and a colour.
Each cage was provided with a card showing the colour code, animal identification number(s), cage number, group letter and study number.

Animal maintenance
The animals were housed under conventional conditions in animal room 5 .1.13 for the dose-range finding study and main study. No other test system was housed in the same animal room during the study.
The room was ventilated with about 10 air changes per hour and was maintained at a temperature of 19-25°C. The relative humidity was between 30-70% except for some short periods during cleaning reaching a maximum of99.9 %. Furthermore, during the main study relative humidity was less than 30 % (minimum 26.6%) for less than 1 hour due to renovation activities.
Lighting was artificial with a sequence of 12 hours light and 12 hours dark.
During the dose-range finding study, the animals were housed in groups of 2/sex in type 3 (42.5x26.6x15 cm) macrolon cages with sterilized dust-free saw shavings (Woody Clean in the dose-range finding study and Espen E-001 in the main study) as bedding material and shreds of paper as environmental enrichment.
During the premating period of the main study the animals were housed in groups of 4/sex in macrolon cages type 4 (48x37.5x21 cm) with sterilized dust free saw shavings (Espen E-001) as bedding material and environmental enrichment (shreds of paper). For mating, one male and one female were housed together in type 3 (42.5x26.6x15.0 cm) macrolon cages. Mated females were housed individually in the same type of cages placed in another cage rack. The location of the mated females in the new cage rack was determined by the date of mating (females found sperm-positive on the same date were considered a "lot") and by animal number (within each lot the mated females were housed in the order of animal number).
After delivery, the cage containing the dam with litter was transferred to another cage rack, the location being determined by delivery date and animal number as described above.

Feed and drinking water
Feed and water were provided ad libitum from the arrival of the rats until the end of the study.
Upon arrival, the rats were fed a commercial rodent diet (Rat&Mouse No. 3, Breeding Diet, RM3) obtained from SDS Special Diets Services, Witham, England.
Each batch of this diet was analysed by the supplier for nutrients and contaminants.
The feed was provided as a powder, in stainless steel cans, covered by a perforated stainless steel plate that served to prevent spillage. The feed in the feeders was refreshed at least once per week and topped up when necessary.
The test substance was incorporated in the basal diet by mixing in a mechanical blender.
The experimental diets were prepared once shortly before the start of the studies; they were stored in a freezer (<-18 °C) until use.
The drinking water (tap-water) was supplied in polypropylene bottles with a rubber stopper and a stainless steel nipple that were cleaned approximately weekly and filled up when necessary. Tap water suitable for human consumption (quality guidelines according to Dutch legislation based on EEC Council Directive 98/83/EEC) was supplied by N.V. Hydron Midden-Nederland. Results of the routine physical, chemical and microbiological examination of drinking water as conducted by the supplier are made available to TNO Nutrition and Food Research.
In addition, the supplier periodically (twice per year) analyses water samples taken on the premises of TNO Nutrition and Food Research in Zeist for a limited number of physical, chemical and microbiological variables.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
The test substance was weighed in a tray of steel and moved to a small grinder. Thereafter the tray was rinsed with RM3 food which was also moved to the small grinder and subsequently mixed for 2 x 30 sec. This mixture was moved to the Stephan cutter and the small grinder was rinsed with RM3 food which was also moved to the Stephan cutter. The mixture was mixed with another approximately 3 kg weighed RM3 food in the Stephan cutter for 2 x 2 minutes and subsequently moved to the Lodige cutter. The Stephan cutter was rinsed with approximately 3 kg of RM3 food that was also moved to the Lodige cutter. Mixing was continued in the Lodige for 2 minutes with the total amount of RM3 food. The total amount of RM3 food used for all mixing and rinsing procedures was the same as mentioned under "RM3 food added".
Details on mating procedure:
After a premating period of 2 weeks each female was caged with a male from the same dosing group until pregnancy occurred. During the mating period, vaginal smears were made daily and examined for the presence of sperm cells. The day of observation of sperm in the vaginal smear was considered day 0 of pregnancy. Upon evidence of copulation, the females were caged individually for the birth and rearing of their pups until day 4 of lactation (PN 4) or shortly thereafter, when dams and pups were killed for necropsy. Sperm positive females that were not pregnant were killed 25 days after copulation. The morning after birth was considered day 1 post partum. Consequently, for litters that were born during the day, but after the morning observation, that day was considered day 0 post partum.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test substance analysis in the diet
Before the start of the study, the analytical method was validated in the matrix under examination (viz. RM3 diet).

Principle of the analytical method
From each diet sample, 2.0 g was transferred into a 50 ml Coming tube. An aliquot of the internal standard solution (monoheptyltin trichloride, diheptyltin dichloride, tripropyltin chloride and tetrapropyltin in methanol) was added. Acetic acid was added to each sample and the mixture was shaken for 1 hour. By addition of acetic acid, the test substance DOTO, as well as alkyltin chloride internal standards, were converted into the corresponding alkyltin acetates. Subsequently acetate buffer solution (pH 4.5), methanol, 20 % aqueous tetraethylborate (NaBE4) solution and hexane (with naphthalene as internal standard) were added to each sample and this mixture was shaken and heated to 60 °C. During this step, the organotin acetates were converted into the corresponding ethylated tetraorganotin derivatives, which were extracted into the hexane layer. Prior to GC-MS analysis, the hexane layer were washed with 2 mol/1 HCl 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. On each day of the study QC samples were freshly prepared, in order to check the derivatization and extraction on that particular day.

Analyses
The homogeneous distribution, stability and achieved concentration of the test substance in RM3 rat feed was analysed in the batch of diets prepared for the dose range finding study. The same diet preparation protocol was used in the main study. The homogenous distribution and achieved concentration in RM3 rat feed of the low-dose group (5 mg/kg) was determined in the batch of diets prepared for the main study.
Directly after mixing of each diet for the dose range finding study, samples for the homogeneity/stability experiments were taken from the mixer. Firstly, five homogeneity samples (about 50 g each) 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 were 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 (achieved concentration) of the test substance in the batch of diet used in the main study were conducted.
Diet samples for the determination of content of the diets used in this study were taken immediately after preparation of the diets and stored at ea. -18 °C pending analysis.

Criteria for homogeneity, stability and content of the test substance in the diet
Homogeneity:
For each group a one-way analysis of variance (Anova) was performed using the sample location (1-5) as grouping factor. An associated F-value with probability p <0.01 was considered to be significant (i.e. the mean concentrations differ significantly at the various locations in the sample). 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 grouping factor. An associated F-value with probability p < 0.01 was considered to be significant (i.e. the difference between the results of the first day and the last day is 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).
Achieved concentration:
For each concentration level, the mean of the concentrations as measured in the study samples used for the assessment of the homogeneity were considered to represent the achieved 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.
Duration of treatment / exposure:
Males 28 days
Females until PN 4 or 25 days after copulation if no live births.
Frequency of treatment:
The animals were fed diets containing test substance from the start of the treatment period until sacrifice.
Details on study schedule:
At the start of the treatment period the rats were about 10-11 weeks old
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg diet
Remarks:
Control group
Dose / conc.:
5 mg/kg diet
Remarks:
Low-dose group
Dose / conc.:
25 mg/kg diet
Remarks:
Mid-dose group
Dose / conc.:
250 mg/kg diet
Remarks:
High-dose group
No. of animals per sex per dose:
12 animals per sex/dose group
Control animals:
yes, concurrent no treatment
Details on study design:
The study comprised four groups of 12 male and 12 female rats each. The dose levels were selected in consultation with the sponsor and were based on the results of the dose-range finding study. The test substance concentrations in the diet remained the same for each group during the study. The animals were fed diets containing test substance from the start of the treatment period until sacrifice.
Detailed clinical observations were performed in all animals prior to the first day of dosing and once weekly thereafter. Spontaneous motor activity measurements and Functional Observational Battery (FOB) were performed in 5 males/group prior to the end of dosing and in 5 females/group prior to the end of lactation (PN 4) randomly selected from each group.
At the end of the premating period, blood was collected by orbital puncture for haematology and clinical chemistry.
Positive control:
Not specified in the study report.

Examinations

Parental animals: Observations and examinations:
Clinical signs
Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity starting from the beginning of the study.
On working days, all cages were checked again in the afternoon for dead or moribund animals to minimise loss of animals from the study. On Saturdays, Sundays and public holidays only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded.

Detailed clinical observations and neurobehavioural observations and motor activity assessment (arena testing, FOB and motor activity)
Detailed clinical examinations were conducted outside the home cage prior to the first exposure and then once weekly in all animals.
Spontaneous motor activity measurements and Functional Observational Battery (FOB) were performed in 5 males/group prior to the end of dosing and in 5 females/ group prior the end oflactation (PN 4) randomly selected from each group.

Body weights
Body weights of male and female rats were recorded on day -2 (randomization) and on days O (first day of dosing), 7 and 13 of the premating period.
Males were weighed weekly during the mating period until sacrifice. Females were weighed during mating (day 0, 7 and 13) and mated females were weighed on days 0, 7, 14 and 21 during presumed gestation and on day 1 and 4 of lactation.
All animals were weighed on the day of sacrifice.

Food consumption
Food consumption of male rats was measured weekly (days 0-7, 7-13 and 21-28), except during the mating period. Food consumption of female rats was measured weekly during the premating period ( days 0-7, 7-13). Food consumption of mated females was recorded weekly during gestation (gestation days (GD) 0-7, 7-14 and 14-21) and once during lactation (PN 1-4).

Parturition and litter evaluation
At the end of the gestation period, females were examined twice daily for signs of parturition. Any difficulties that occurred during parturition were recorded. To keep nest disturbance to a minimum, the litters were examined only once daily for dead pups.

Haematology
Prior to the end of the premating period, 5 rats/sex/group (the 2 animals with the lowest identification numbers of the first and second cages of each group and the animal with the lowest identification number of the third cage of each group) were fasted overnight and blood was taken, whilst under CO2/O2 anaesthesia by means of orbital puncture.
K2-EDTA was used as anticoagulant. In each sample the following determinations were carried out:
haemoglobin; packed cell volume; red blood cell count; reticulocytes; total white blood cell count; differential white blood cell count; prothrombin time; thrombocyte count; mean corpuscular volume (MCV); mean corpuscular haemoglobin (MCH); mean corpuscular haemoglobin concentration (MCHC).

Clinical chemistry
Prior to the end of the premating period, 5 rats/sex/group (the 2 animals with the lowest identification numbers of the first and second cages of each group and the animal with the lowest identification number of the third cage of each group) were fasted overnight and blood was taken, whilst under CO2/O2 anaesthesia by orbital puncture.
Blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation.
The following measurements were made in the plasma collected:
fasting glucose; 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; bilirubin (total); cholesterol (total); triglycerides; phospholipids; calcium (Ca); sodium (Na); potassium (K); chloride (Cl); inorganic phosphate.
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Not examined
Litter observations:
Litter size, sexes and pup weight
The total litter size and numbers of each sex as well as the number of stillbirths, livebirths and grossly malformed pups and pups showing abnormalities were recorded on PN 1 and 4. The pups were weighed individually on PN 1 and 4. Mean pup weights were calculated as litter weight/number pups. The number of runts (pup weight less than mean pup weight of the control group minus 2 standard deviations) was calculated.
Postmortem examinations (parental animals):
Female animal D181 of the high-dose group was found dead on GD 24; a gross necropsy was performed on this animals. All remaining male and female parent rats were euthanized by exsanguination from the abdominal aorta after CO2/O2 anaesthesia and examined grossly for pathological changes.
Samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes which were preserved in Bouin's fixative:
- ovanes
- uterus (after counting the implantation sites1)
- testes
- epididymides,
- seminal vesicles
- prostate
- organs and tissues showing macroscopic abnormalities

In addition for 5 animals/sex/group, randomly selected from each group, the following organs were preserved:
adrenals; axillary lymph node; bone marrow (femur); brain; caecum; coagulation glands*; colon; duodenum; eyes; heart; ileum; jejunum; lungs; kidneys; liver; mammary gland (females only)*; mesenteric lymph node; parathyroids; Peyer' s patches; pituitary; rectum; sciatic nerve; spinal cord; spleen; stomach; thymus; thyroids; trachea; urinary bladder
* Tissues marked with an asterisk were preserved but not processed for histopathological examination.
The underlined organs were weighed (paired organs together) as soon as possible after dissection to avoid drying.
Tissues for microscopic examination were embedded in paraffin wax, sectioned at 5 μm, and stained with haematoxylin and eosin, except for sections of the testes which were stained with PAS haematoxylin. Microscopic examination was performed on the collected organs of all rats of the control and high-dose groups. After consultation with the sponsor, examination was extended to the liver and ovaria of the female rats and the thymus of the male and female rats of the low and mid-dose groups; because of the effect observed in the high-dose group.
In addition, reproductive organs of males that failed to sire (mated female which was not pregnant) and females that were non-mated or non-pregnant, of the low- and mid-dose groups, were microscopically examined.
Postmortem examinations (offspring):
Pathology of pups
A necropsy was performed on stillborn pups and pups that died during the study; macroscopic abnormalities were recorded. Pups were examined externally for gross abnormalities and killed by hypothermia at < -18°C on PN 4 or shortly thereafter.
Statistics:
Statistical procedures used in the evaluation of the data were as follows:
- Fisher's exact probability test was used to evaluate the number of mated and pregnant females and females with live pups.
- Number of implantation sites, live and dead pups were evaluated by Kruskal- Wallis nonparametric analysis of variance followed by the Mann Whitney U-test
All tests were two-sided. A level of probability of <0.05 (p< 0.05) was considered significant. Effects of treatment on habituation were analysed using repeated measures analysis of variance on time blocks. Each session consisted of 5 time blocks of 6 minutes each.
Statistical evaluations on variables associated with pups were considered on a litter basis. Additional evaluations on a pup basis were performed to attempt to identify any specific dose-related effect that may have occurred.
Reproductive indices:
The following parameters are 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
Offspring viability indices:
The following parameters are calculated:
- 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
- pre-implantation loss = [ (number of corpora lutea- number of implantation sites)/number of corpora lutea] x 100
- post-implantation loss= [(number of implantation sites - number of pups born alive)/number of implantation sites] x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Male animal D176 of the high-dose group showed exophthalmus from week 2 and complete degeneration of the eye from week 3 onwards. This observation was first made after orbital punction on day 13 and was most probably caused by this action. No other clinical signs were observed in the males from the start of the study until sacrifice.
The only finding during the gestation period on GD 21, was a sparsely haired animal in the 250 mg/kg group (Table 8). During the lactation period, sparsely haired animals were found in the control (n=l), 5 mg (n=l) and in the 250 mg (n=l) groups.
No other findings were observed in the female animals during the premating, gestation and lactation periods.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
Female animal D181 of the high-dose (500 mg) group was found dead on GD 24; this animal was pregnant and at necropsy 11 dead fetuses were found in the uterus. No other mortalities were observed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight of the male animals of the dioctyloxostannane-treated groups was comparable to the control group (Table 10). Body weight change of the male animals was statistically significantly decreased in the 250 mg (high-dose) group from days 13-21.
Mean body weight of the dams of the high-dose group was statistically significantly decreased on GD 21 and PN 1 (Tables 12 and 14). Mean body weight change of the dams of the high-dose group was statistically significantly decreased from GD 14-21. Body weight and body weight change of the dams of all dioctyloxostannane-treated groups was comparable to the control group during all other periods of the premating, gestation and lactation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption
Mean food consumption (g/kg/day) of the male animals of the 250 mg (high-dose) was statistically significantly decreased from day 7-13. No other treatment related effects were observed in the male animals.
During the gestation period (GD 7-14 and GD 14-21) and lactation period (PN 1-4) food consumption (expressed as g/animal/day and as g/kg body weight/day) of the dams of the 250 mg (high-dose) group was statistically significantly decreased. No other treatment related effects were observed during the premating, gestation and lactation periods.

Test substance intake
The test substance intake of the male animals of the low-, mid- and high-dose groups was respectively:
Premating period
days 0-7 0.4, I. 7 and 17.4 mg/kg body weight/day, days 7-13 0.3, 1.6 and 15.4 mg/kg body weight/day,
After mating
days 21-28 0.3, 1.5 and 14.5 mg/kg body weight/day.
The test substance intake of the female animals of the low-, mid-and high-dose groups was respectively:
Premating period
days 0-7: 0.3, I. 7 and 16.4 mg/kg body weight/day, days 7-13: 0.3, 1.6 and 16.0 mg/kg body weight/day,
Gestation period
GD 0-7: 0.4, 2.0 and 17.4 mg/kg.body weight/day, GD 7-14: 0.4, 1.9 and 16.7 mg/kg body weight/day, GD 14-21: 0.3, 1.4 and 11.2 mg/kg body weight/day,
Lactation period
PN 1-4: 0.5, 2.4 and 17.4 mg/kg body weight/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Red blood cell- and coagulation variables and mean total and differential white blood cell counts were comparable in all groups for male and female animals.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In the 250 mg group, the alkaline phosphatase (U/1) was statistically significantly increased in the male animals and bilirubin (μmol/1) in the female animals; these findings were considered to be a treatment related finding. The statistically significant increase in chloride in the male rats of the 25 mg (mid-dose) group and statistically significant decrease in calcium in the female rats of the 5 mg (low-dose) group were not considered to be related to dioctyloxostannane administration as these effects were not observed in the high-dose group and mid-and high-dose groups, respectively. All other parameters were comparable to the control group.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Treatment-related changes were not observed during the neurobehavioural testing of males and females at arena testing during the study and FOB and motor activity assessment at the end of the study. Therefore, no evidence was obtained for a neurotoxic potential of the test substance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Examination of the thymus revealed moderate to very severe lymphoid depletion in all animals (both sexes) of the 250 mg/kg (high-dose) group and in all females of the 25 mg/kg (mid-dose) group. Lymphoid depletion was characterized by a decrease in the size of the thymic lobules because of an extensive loss of cortical and medullary small lymphocytes. Consequently the distinction between the cortical and medullary areas was blurred. In the (very) severe cases the cortex was very small, or absent. The remaining lymphoid cells visible in the cortical areas were mainly lymphoblasts.
Lymphoblastic cells and reticuloepithelial cells had increased, and/or higher numbers of these cells were visible because of the disappearance of small lymphocytes and collapse of thymic stroma. In 3 high-dose females thymic lymphoid depletion was accompanied by lymphoid depletion in the PALS areas in the spleen. Lymphoid depletion of the PALS, i.e. the periarteriolar lymphocyte sheath, is a common feature associated with thymic atrophy because of its T-cell dependence. The macroscopically observed small thymi in 5 control and 7 low-dose (5 mg/kg) females revealed no abnormalities in 3 control and 5 low-dose females. In the thymi of 2 control and 2 low-dose females pregnancy/lactation involution was observed. The thymic lobules were decreased in size but exhibited normal architecture with the histological appearance of age-involution.
Increased glycogenic vacuolation, viz moderate versus (very) slight, was seen in the liver of 4 high-dose females and probably accounts for the statistically significant increase in relative liver weight.
Examination of the reproductive organs revealed a statistically significant increase in the incidence of cysts in the ovaries of 8 high-dose females.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not specified
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
In each group 12 females were placed with males and all females were mated. Pre-coital time was comparable for the control, low-, mid-dose, and high-dose groups. The number of pregnant females and the number of males that became sires amounted to 11, 12, 11 and 10 for the control, low-, mid- and high-dose groups, respectively.
The number of females with liveborn pups was 11, 12, 11 and 8 for the control, low-,mid- and high-dose groups, respectively.
The mating index was 100% in all groups. The female fecundity index, female fertility index and male fertility index were comparable among the control, low-, mid- and high-dose groups and ranged from 83-100%. The gestation index was comparable 100% in the control, low- and mid-dose groups and 80% in the high-dose group. The duration of gestation was statistically significantly increased in the 250 mg (high-dose) group.
In addition 1 female (D181) of the high-dose group was found dead on GD 24. This animal appeared to be pregnant and 11 dead fetuses were found in the uterus. No difference in the duration of gestation was observed amongst the control and the low and mid-dose groups. Stillborn pups were observed in 0, 0, 0, and 3 litters of the control, low-, mid- and high-dose group, respectively. In the high-dose group 1 female with all stillborn pups was observed.
Pre-implantation loss was 8.4, 2.2, 12.3 and 6.2% for the control, low-, mid- and high-dose groups, respectively and was comparable in all groups.
The statistically significantly increased number of implantation sites in the 5 mg (low-dose) group was not considered as a treatment-related effect.
Post-implantation loss was 9.8, 11.6, 7.5 and 38.7% for the control, low-, mid- and high-dose groups, respectively; the post-implantation loss in the 250 mg group was statistically significantly increased when compared to the control group and this effect was considered to be related to treatment.

Details on results (P0)

One female animal of the 250 mg/kg group (high-dose) was found dead on GD 24.
Clinical signs related to the treatment with dioctyloxostannae were not observed.
In male animals tested after 4 weeks of treatment and in females animals tested on PN 4, no changes indicative of neurotoxic potential of the test substance were observed in the neurobehavioural observations and motor activity assessment.
Mean body weight of the male animals of the dioctyloxostannane-treated groups was comparable to the control group. Body weight change of the male animals was statistically significantly decreased in the 250 mg (high-dose) group.
Mean body weight of the dams of the high-dose group was statistically significantly decreased on GD 21 and PN 1. Mean body weight change of the dams of the high-dose group was statistically significantly decreased from GD 14-21. Body weight and body weight change of the dams of all dioctyloxostannane-treated groups was comparable to the control group during all other periods of the premating, gestation and lactation.
Administration of 250 mg dioctyloxostannane per the diet resulted in a decreased food consumption in male animals from day 7-13 and in female animals during the gestation period (GD 7-14 and GD 14-21) and lactation period (PN 1-4). No other treatment related effects were observed during the premating, gestation and lactation periods.
The test substance intake of the male animals during the study ranged from 0.3-0.4, 1.5-1.7 and 14.5-17.4 mg/kg body weight/day for the low-, mid- and high-dose group, respectively.
The test substance intake of the female animals during the premating, gestation and lactation period ranged from 0.3-0.5, 1.4-2.4 and 11.2-17.4 mg/kg body weight/day for the low-, mid- and high-dose group, respectively.
The statistically significant increase in alkaline phosphatase in the male animals and bilirubine in the female animals of the 250 mg group were the only treatment-related findings in clinical chemistry and haematology.
Male animals were sacrificed on day 28. In the 25 mg (mid-dose) and 250 mg (high-dose) groups, the absolute thymus weight of the male animals was statistically significantly decreased. Relative thymus weight of the male animals was statistically significantly decreased in the high-dose group.
Female animals with a litter were sacrificed on PN 4 or 5. The absolute and relative thymus weight of the female animals of the high-dose group was statistically significantly decreased. In the mid-dose group the relative thymus weight was also statistically significantly decreased.
In the female animals of the high-dose group, the relative kidney and liver weights were statistically significantly increased.
Decreased thymus weights and macroscopically observed small thymi were accompanied by microscopic changes in the thymus of all animals (both sexes) of the 250 mg/kg (high-dose) group and in 11 females of the 25 mg/kg (mid-dose) group. The thymus showed overt lymphoid depletion, accounting for the decreased thymus weights. The microscopic appearance of the affected thymi resembled thymus atrophy described in the literature for organotin compounds in that there was no small lymphocyte necrosis, whereas lymphoblasts, reticuloepithelial cells and macrophages were more accentuated. Examination of the ovaries revealed a statistically significant increased incidence of ovarian cysts in 8 female animals of the high-dose group. At microscopic examination, glycogenic vacuolation in the liver of 4 female animals of the high-dose group was consistent with the increased relative liver weights.
Based on the observed effects on reproductive parameters in the 250 mg/kg (high-dose) group, mean duration of gestation, post-implantation loss, total number of stillborn pups, pup mortality PN 4, pup weight PN 1 and number of runts, the NOAEL for reproductive toxicity was established on the mid-dose (25 mg/kg diet which is equivalent to 1.5-1.7 mg/kg body weight in the male animals and 1.4-2.4 mg/kg body weight in the female animals).

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
5 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Target system / organ toxicity (P0)

Key result
Critical effects observed:
not specified
Lowest effective dose / conc.:
5 mg/kg diet
System:
immune system
Organ:
thymus

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
On PN 1 the number of runts (pup weight less than mean pup weight of the control group minus 2 standard deviations) was statistically significantly increased in the 250 mg (high-dose group). The other findings were normal for pups of this age and are considered not to be related to treatment.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The number of pups delivered per litter was comparable in all groups and amounted to 9.8, 10.8, 9.6 and 8.0 for the control, low-, mid- and high-dose groups, respectively.
The number of live born pups amounted to 108, 129, 105 and 64 for the control, low-, mid- and high-dose groups, respectively and was statistically significantly decreased in 250 mg (high-dose) group. The number of stillborn pups was 0, 0, 0 and 8 for the control, low-, mid- and high-dose groups, respectively; in the high-dose group the number was statistically significantly increased. Pup mortality on PN 4 was comparable in all groups except for the high-dose group and amounted to 1, 2, 2, 22 (incidences 0.9, 1.6, 1.9 and 34.0 %) in the control, low-, mid- and high-dose groups, respectively; in the high-dose group the number was statistically significantly increased. In the control, low-, mid- and high-dose groups 0, 0, 0 and 3 litters, respectively were lost entirely between PN 0-4. The number of live pups per litter on PN 1 (9.8, 10.8, 9.6, 8.0 for the control, low-, mid- and high-dose groups, respectively) and PN 4 (9.7, 10.6, 9.4, 7.0 for the control, low-, mid- and high-dose groups, respectively). Although the number of live pups of the high-dose group was decreased on PN 1 and 4, no statistical significant difference was observed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
On PN 1 and 4, a statistical significant effect was observed in the pup weight of the 250 mg (high-dose) group. The pup weight change (PN 1-4) was statistically significantly decreased in male pups of the high-dose group. No effect on pup weight and pup weight change was observed in the 5 mg (low-) and 25 mg (mid-dose) groups when compared to the control group.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observation of the stillborn pups revealed 3 partly cannibalized pups and 3 autolytic pups in the high-dose group; in the latter pups no abnormalities were observed. In addition 2 pups with no abnormalities were examined in the high-dose group.
Histopathological findings:
not examined
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

In each group 12 females were placed with males and all females were mated. The number of pregnant females and the number of males that became sires amounted to 11, 12, 11 and 10 for the control, low-, mid- and high-dose groups, respectively.
The following effects on reproduction and litter data were observed in the 250 mg (high-dose) group:
high-dose vs. control
- mean duration of gestation (days) 21.7 (S) 21.0
- number of females with live born pups 8 11
- gestation index (%) 80 100
- post-implantation loss(%) 38.7(S) 9.8
- mean number of pups delivered 8.0 9.8
- mean number of live pups/litter PN 1 8.0 9.8
- mean number of live pups/litter PN 4 7.0 9.7
- total number of stillborn pups 8 (S) 0
- pup mortality PN 4 (%) 34 (S) 0.9
- pup weight PN 1 (g) 4.5 (S) 5.1
- pup weight PN 4 (g) 6.5 7.6
- percentage of runts PN 1 (%) 34 (S) 2
(S) = Statistically significant difference relative to the control group

No effect on reproduction and litter data was observed in the low- and mid-dose group.
Based on the observed effects on reproductive parameters in the 250 mg/kg (high-dose) group, mean duration of gestation, post-implantation loss, total number of stillborn pups, pup mortality PN 4, pup weight PN 1 and number of runts, the NOAEL for reproductive toxicity was established on the mid-dose (25 mg/kg diet which is equivalent to 1.5-1.7 mg/kg body weight in the male animals and 1.4-2.4 mg/kg body weight in the female animals).

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
25 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
mortality

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

A summary of relevant treatment-related changes is given in the table below:

Parameter

Dose levels

5 mg/kg

25 mg/kg

250 mg/kg

Reproductive and/or developmental changes

 

 

 

Duration of gestation

 

 

Post-implantation loss

 

 

Number of stillborn pups

 

 

Pup mortality PN 4

 

 

Pup weight PN 1

 

 

Number of runts PN 1

 

 

↓ decrease; ↑ increase

 

NATURAL DELIVERY DATA AND LITTER DATA-SUMMARY

 

 

A

Control

B

5 mg/kg

C

25 mg/kg

D

250 mg/kg

Females place with male

Females mated

N

N

12

12

12

12

12

12

12

12

Number of males mated with females

N

12

12

12

12

Number of males that became sire

N

11

12

11

10

Pre-coital time (days)

MEAN

S.E.

2.50 u

0.337

3.08

0.941

2.33

0.284

2.83

0.297

Day 1 to 4

N

%

11 f

92

11

92

12

100

12

100

Day 5 to 7

N

%

1 f

8.3

0

0.0

0

0.0

0

0.0

Day 8 to 14

N

%

0 f

0.0

1

8.3

0

0.0

0

0.0

Day 1 to 14

N

%

12 f

100

12

100

12

100

12

100

Females pregnant

Females with liveborn

N

N

11 f

11 f

12

12

11

11

10

8

Mating index

Female fecundity index

Female fertility index

Male fertility index

Gestation index

%

%

%

%

%

100

92

92

92

100

100

100

100

100

100

100

92

92

92

100

100

83

83

83

80

Duration of gestation

MEAN

S.E.

21.00 u

0.135

21.17

0.112

21.36

0.152

21.67*

0.167

Females which have birth

as % of pregnant females

N

%

11 f

100

12

100

11

100

9

90

Females with stillborn pups

as % of pregnant females

N

%

0 f

0.0

0

0.0

0

0.0

3

30

Females with all stillborn pups

as % of pregnant females

N

%

0 f

0.0

0

0.0

0

0.0

1

10

Pups delivered (total)

 

Liveborn

Live Birth Index

 

Stillborn

Pup mortality day 1

N

MEAN

S.E.

N

%

 

N

%

108

9.82 u

0.483

108 f

100

 

0 f

0.0

129

10.75

0.641

129

100

 

0

0.0

105

9.55

0.366

105

100

 

0

0.0

72

8.00

0.726

64***

89

 

8**

11

Number of pups lost (dying, missing, and/or cannibalized) on the following days:

 

 

 

 

Day 1-4

Pup mortality day 4

N

%

1 f

0.9

2

1.6

2

1.9

22#

34

Pups alive day 4

Viability index day 1-4

N

%

107 f

99

127

98

103

98

42

66

Number of litters lost entirely (stillborn, dying, missing, cannibalized, and/or culled) in the period between:

 

 

 

 

Day 1-4

N

%

0 f

0.0

0

0.0

0

0.0

3

38

Live pups/litter

 

 

 

 

 

Day 1

MEAN

S.E.

9.82 u

0.483

10.75

0.641

9.55

0.366

8.00

0.756

Day 4

MEAN

S.E.

9.73 u

0.488

10.58

0.609

9.36

0.279

7.00

1.155

Number of male pups at day 1

Sex ratio at day 1

N

%

50 f

46

59

46

53

50

26

41

Number of male pups at day 21

Sex ratio at day 21

N

%

50 f

47

59

46

53

51

17

40

No. of corpora lutea

MEAN

S.E.

12.0 u

0.447

12.1

0.229

11.9

0.392

11.2

0.249

No. of lost corpora lutea

Pre-implantation loss

N

%

S.E.

12

8.37 u

2.758

3

2.15

1.122

17

12.30

3.375

7

6.24

2.380

No of implantation sites

MEAN

S.E.

10.9 u

0.315

11.8*

0.297

10.4

0.364

10.5

0.342

No of lost implantations

Post implantations loss

N

%

S.E.

12

9.78 u

3.937

17

11.57

4.608

9

7.53

2.866

41

38.70*

11.168

Statistical key: f = Fishers exact test      u = Kruskal-Wallis & Mann-Whitney U    * = p<0.05        ** = p<0.01      # = p<0.001

 

SUMMARY OF PUP WEIGHTS (g)

 

 

A

Control

B

5 mg/kg

C

25 mg/kg

D

250 mg/kg

Day 1 males

MEAN

S.E.

N

5.28 d

0.177

1

4.98

0.107

12

5.44

0.104

11

4.48**

0.266

7

Day 1 females

MEAN

S.E.

N

4.95 d

0.117

11

4.65

0.097

12

5.12

0.100

11

4.49

0.234

8

Day 1 males+females

MEAN

S.E.

N

5.08 d

0.130

11

4.82

0.110

12

5.27

0.097

11

4.54*

0.242

8

Day 4 males

MEAN

S.E.

N

7.84 d

0.353

11

7.56

0.231

12

8.30

0.221

11

6.12**

0.278

5

Day 4 females

MEAN

S.E.

N

7.35 d

0.320

11

7.14

0.193

12

7.93

0.204

11

6.38

0.589

6

Day 4 males+females

MEAN

S.E.

N

7.55 d

0.328

11

7.37

0.230

12

8.10

0.198

11

6.45

0.538

6

Statistical key: d = ANOVA & Dunnett test         * = p<0.05        ** = p<0.01

 

SUMMARY OF PUP BODY WEIGHT CHANGES (g)

 

 

A

Control

B

5 mg/kg

C

25 mg/kg

D

250 mg/kg

Day 1 – 4 males

Pre-culling

MEAN

S.E.

N

2.56 d

0.202

11

2.57

0.138

12

2.86

0.155

11

1.38**

0.284

5

Day 1-4 females

Pre-culling

MEAN

S.E.

N

2.40 d

0.226

11

2.49

0.111

12

2.81

0.149

11

1.64

0.511

6

Day 1-4 males+females

Pre-culling

MEAN

S.E.

N

2.47 d

0.218

11

2.55

0.133

12

2.83

0.146

11

1.66

0.456

6

Statistical key: d = ANOVA & Dunnett test         * = p<0.05        ** = p<0.01

 

 

SUMMARY OF CLINICAL OBSERVATIONS IN PUPS RECORDED ON DAYS 1-4 OF LACTATION

 

A

Control

B

5 mg/kg

C

25 mg/kg

D

250 mg/kg

 

Number of pups showing the observations

DAY 1

Runt2

Pale

Cold

Subcutaneous haemorrhage: head

No milk in stomach

Missing/dead tailtip

 

2(2)1

0

10(1)

1

10(1)

1

 

10(4)

0

0

0

0

0

 

1

0

0

0

0

0

 

21#(3)**

1

8(1)

0

0

0

DAY 4

Runt

Missing/dead tailtip

 

6(2)

1

 

2(2)

0

 

0

0

 

6(2)

0

Statistical key: Fisher’s exact test          * p<0.05           ** p<0.01         # p<0.001

1Figures in brackets represent the number of litters with pups showing the observation

2Criterion for runt: pup weight less than mean pup weight of the control group minus 2 standard deviations

 

SUMMARY OF MACROSCOPIC OBSERVATIONS IN STILL BORN PUPS AND PUPS THAT DIED DURING LACTATION

OBSERVATIONS

A

Control

B

5 mg/kg

C

25 mg/kg

D

250 mg/kg

Autolytic

Partly cannibalized

No abnormalities

0

0

0

0

0

0

0

0

0

3(1)

3(1)

2(2)

Figures in brackets represent the number of litters with pups showing the observation

Applicant's summary and conclusion

Conclusions:
In conclusion, based on the observed effects in the animals of the 25 mg/kg (mid-dose) group, decrease in thymus weight (male and female animals) and macroscopic and microscopic findings in the thymus (female animals), the NOAEL for general toxicity is established on the low-dose level (5 mg/kg diet which is equivalent to 0.3-0.4 mg/kg body weight/day for the male animals and to 0.3-0.5 mg/kg body weight/day for the female animals).
Based on the observed effects on reproductive parameters in the 250 mg/kg (high-dose) group, mean duration of gestation, post-implantation loss, total number of stillborn pups, pup mortality PN 4, pup weight PN 1 and number of runts, the NOAEL for reproductive toxicity was established on the mid-dose (25 mg/kg diet which is equivalent to 1.5-1.7 mg/kg body weight in the male animals and 1.4-2.4 mg/kg body weight in the female animals).
Executive summary:

The objective of this study was to provide data on the possible reproductive and developmental effects of dioctyloxostannane [Dioctyltin oxide, CAS # 870-08-6] after oral administration via the diet to Wistar rats of both sexes. The study was combined with a repeated dose toxicity study and preceded by a dose-range finding study. In the dose-range finding study, rats were fed diets containing 0, 25, 75, 200 and 500 mg dioctyloxostannane/kg diet for 14 days. In the main study, rats were fed diets containing 0, 5, 25 and 250 mg dioctyloxostannane/kg diet for up to 28 days (males) or during 2 weeks premating, mating, gestation and up to day 4 or 5 of lactation (females).

 

Analyses of the diets

Dioctyloxostannane was considered to be homogeneously distributed in all diets and was considered to be stable in the diets upon storage at room temperature for 7 days, and upon storage at< -18 °C for 5 weeks.

The content of the test substance was considered to be close to intended for all diets.

 

Dose-range finding study

It was concluded that treatment with dioctyloxostannane via the diet up to a level of 75 mg/kg diet (low-mid-dose group) for a period of 14 days had no effect on body weight and food intake in male and female rats. In male animals, thymus weight (absolute and relative) was affected from 200 mg/kg diet (high-mid-dose group) onwards and in female animals, thymus weight (absolute and relative) was affected in all dioctyloxostannane-treated groups.

 

Main study

One female animal of the 250 mg (high-dose) group was found dead on GD 24.

No other mortalities or treatment related clinical signs were observed.

 

In male animals tested after 4 weeks of treatment and in female animals tested on postnatal day (PN) 4, no changes indicative of neurotoxic potential of the test substance were observed in the neurobehavioural observations and motor activity assessment.

 

Mean body weight of the male animals of the dioctyloxostannane-treated groups was comparable to the control group. Body weight change of the male animals was statistically significantly decreased in the 250 mg (high-dose) group from days 13-21.

Mean body weight of the dams of the high-dose group was statistically significantly decreased on GD 21 and PN 1. Mean body weight change of the dams of the high-dose group was statistically significantly decreased from GD14-21. Body weight and body weight change of the dams of all dioctyloxostannane treated groups was comparable to the control group during all other periods of the premating, gestation and lactation.

 

Administration of 250 mg dioctyloxostannane per the diet resulted in a decreased food consumption in male animals from day 7 -13 and in female animals during the gestation period (GD 7-14 and GD 14-21) and lactation period (PN 1-4). No other treatment related effects were observed during the premating, gestation and lactation periods.

 

The test substance intake of the male animals during the study ranged from 0.3-0.4, 1.5-1.7 and 14.5-17.4 mg/kg body weight/day for the low-, mid- and high-dose group, respectively.

The test substance intake of the female animals during the premating, gestation and lactation period ranged from 0.3-0.5, 1.4-2.4 and 11.2-17.4 mg/kg body weight/day for the low-, mid- and high-dose group, respectively.

 

In each group 12 females were placed with males and all females were mated. The number of pregnant females and the number of males that became sires amounted to 11, 12, 11 and 10 for the control, low-, mid- and high-dose groups, respectively.

The following effects on reproduction and litter data were observed in the 250 mg (high-dose) group:

high-dose vs.control

- mean duration of gestation (days)            21.7(S)          21.0

- number of females with liveborn pups       8                    11

- gestation index (%)                                    80                  100

- post-implantation loss(%)                          38.7 (S)         9.8

- mean number of pups delivered               8.0                 9.8

- mean number of live pups/litter PN 1         8.0                 9.8

- mean number of live pups/litter PN 4         7.0                 9.7

- total number of stillborn pups                     8 (S)              0

- pup mortality PN 4 (%)                              34 (S)            0.9

- pup weight PN 1 (g)                                   4.5 (S)           5.1

- pup weight PN 4 (g)                                   6.5                 7.6

- percentage of runts PN 1 (%)                    34 (S)            2

(S) = statistically significant

 

No effect on reproduction and litter data was observed in the low- and mid-dose group.

 

The statistically significant increases in alkaline phosphatase in the male animals and bilirubin in the female animals of the 250 mg group were the only treatment related findings in clinical chemistry and haematology.

 

Male animals were sacrificed on day 28. In the 25 (mid-dose) and 250 mg (high-dose) groups, the absolute thymus weight of the male animals was statistically significantly decreased. Relative thymus weight of the male animals was statistically significantly decreased in the high-dose group.

Female animals with a litter were sacrificed on PN 4 or 5; non-pregnant females on GD 25. The absolute and relative thymus weight of the female animals of the high-dose group was statistically significantly decreased. In the mid-dose group the relative thymus weight was statistically significantly decreased.

In the female animals of the 250 mg/kg group, the relative kidney and liver weights were statistically significantly increased.

 

Decreased thymus weights and macroscopically observed small thymi were accompanied by microscopic changes in the thymus of all animals (both sexes) of the 250 mg/kg (high-dose) group and in 11 females of the 25 mg/kg (mid-dose) group. The thymus showed overt lymphoid depletion, accounting for the decreased thymus weights. Examination of the ovaries revealed a statistically significant increased incidence of ovarian cysts in 8 female animals of the high-dose group.

At microscopic examination, glycogenic vacuolation in the liver of 4 female animals of the high-dose group was consistent with the increased relative liver weights.

 

In conclusion, based on the observed effects in the animals of the 25 mg/kg (mid-dose) group, decrease in thymus weight (male and female animals) and macroscopic and microscopic findings in the thymus (female animals), the NOAEL for general toxicity is established on the low-dose level (5 mg/kg diet which is equivalent to 0.3-0.4 mg/kg body weight/day for the male animals and to 0.3-0.5 mg/kg body weight/day for the female animals).

Based on the observed effects on reproductive parameters in the 250 mg/kg (high-dose) group, mean duration of gestation, post-implantation loss, total number of stillborn pups, pup mortality PN 4, pup weight PN 1 and number of runts, the NOAEL for reproductive toxicity was established on the mid-dose (25 mg/kg diet which is equivalent to 1.5-1. 7 mg/kg body weight in the male animals and 1.4-2.4 mg/kg body weight in the female animals).

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