Tetrabutyltin

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18th December 2002 - 22nd April 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18th December 2002 - 22nd April 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age: aound 9-10 weeks of age on arrival (18 December 2002 and 19 February 2003 in the range finder and main study respectively)
- Diet: ad libitum from arrival until the end of the study
- Weight: the weight variation of the animals used did not exceed 20 % of the  mean weight for each sex
- Water: ad libitum from arrival until the end of the study
- Acclimation period: Acclimatised until study initiation on the 2 January 2003 (range finding study) and 27 February 2003

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 °C
- Humidity (%): 30-70 % with some short periods at 100 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): Artificial lighting on a 12 hours light and 12 hours dark sequence.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
The feed was provided as a powder, in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. The feed in the feeders was refreshed at least once per week and topped up where necessary. The test substance was incorportated int he basal diet by mixing in a mechanical blender. The experimental diets were prepared once shortly before the start of the studies and stored in a freezer (-18 °C) until use.

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 (PN4) or shortly after (PN 5). Sperm positive females that were not pregnant were killed 25 days after copulation. The non-mated female of the high-dose group D393 was sacrificed on day 54 of the study, 26 days after the last day of the mating period. The morning after birth was considered day 1 post partum.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before the start of the study, the analytical method for analysing test substance in the diet was validated in the matrix under examination (viz. diet). The validation was performed in a separate study. 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, tripopultin chloride and tetrapropyltin in methanol) was added. Subsequently methanol, acetate buffer solution (pH 4.5) 20 % aqueous sodium tetraethylborate (NaBEt4) 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 chlorides were converted into the corresponding ethylated tetraorganotin derivative, which were extracted into the hexane layer. Prior to GC-MS analysis, the hexane layer was washed with 2 mol/L HCl in order to remove most of the ethylboron compounds that interfere with the GC-MS analysis. The concentration of each test susbtance in feed was determined by GC-MS analysis of the hexane extracts. The homogenous distribution, stability and acheived concentration of the test substance in 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. Directly after mixiing 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 and right centre. Secondly, five samples (of about 50 g each) for examination of the stability were taken from the top part of the mixer. The samples taken for the homogeneity experiments were also used for determination of the content.
In addition, analyses to determine the content (acheived concentration) of the test substance in the batch of diet was used in the main study were conducted. Stability of the test susbtance in the diet was also tested after then end of the main study (after c. 55 days). Diet samples for the determination of content of the diets used were taken immediately after preparation of the diets and stored at ca. -18 °C.

ACTUAL DOSE RECEIVED BY DOSE LEVEL BY SEX
- Dose range finding study: The test substance intake of the male animals ranged from 6-7, 30-34, 102-131 and 206-636 mg/kg body weight/day in the 100, 500, 2000 and 10 000 mg/kg dose groups, respectively. The test substance intake of the female animals ranged from 6-7, 30-33, 111-129 and 253-555 mg/kg body weight/day in the 100, 500, 2000 and 10 000 mg/kg dose groups, respectively.
- Main study: The test substance intake of the male animals during the study ranged from 6-7, 17-20 and 109-130 mg/kg body weight/day the 100, 300 and 2000 mg/kg dose group, respectively. The test substance intake of the female animals during the premating, gestation and lactation period ranged from 5-8, 16-24 and 100-118 mg/kg body weight/day the 100, 300 and 2,000 mg/kg dose group, respectively.

TEST SUBSTANCE ANALYSIS IN THE DIET
The test substance was considered to be homogeneously distributed in all diets. Furthermore, the test substance was considered to be stable in the diets upon storage at room temperature for 7 days and upon storage at < -18 °C for 6 weeks. The test substance also met the criteria for stability upon storage at < -18 °C for 55 days in all diets from the main study, except the 300 mg/kg dose level, for which the relative difference was -22 %. This difference can at least be partially explained by analytical causes (i.e. varying extraction efficiency due to sticking of the test substance to diet). The content of the test substance was considered to be close to intended for all diets. Only the 2000 mg/kg dose level of the dose-range finding study exceeded the criteria (relative difference from intended concentration was -25 %), but when the low recovery of 82 % obtained during the validation of the test substance at this dose level is taken into account, the achieved concentration seems to be close to the intended concentration. In view of these conclusions regarding the analysis of the diets of both the dose-range finding and the main study, the nominal levels of the test substance were used to calculate the test substance intake.

Duration of treatment / exposure:

- Range finder: Animals were dosed for 14 days
- Main test: Males: for a period of 33 days; Females: during the period of 2 weeks premating, mating, gestation, and up to postnatal day 4 or 5

Frequency of treatment:

Feed containing the test substance was provided ad libitum during the whole study. Feed was refreshed daily.

Dose / conc.:

100 mg/kg diet

Dose / conc.:

300 mg/kg diet

Dose / conc.:

2 000 mg/kg diet

No. of animals per sex per dose:

- Dose-range finding study: The study comprised 5 groups of 4 male and 4  female rats each per dose group
- Main study: The main study comprised four groups of 12 male and 12 female ratseach per dose group.

Control animals:

yes, plain diet

Details on study design:

The oral route was used because this is a possible route of human exposure. The test substance was administered at constant concentrations in the diet which remained the same for each group during the dose-range finding study and during the main study.

- Dose-range finding study: The dose levels tested were: 0 (control), 100 (low dose), 500 (low-mid-dose), 2000 (high-mid-dose), 10000 (high-dose) mg  test substance/kg diet.
- Main study: The dose levels were: 0 (control), 100 (low-dose), 300  (mid-dose), 2000 (high-dose) mg test substance/kg diet.

Parental animals: Observations and examinations:

DOSE RANGE FINDING STUDY
- Clinical signs: Each animal observed daily in the morning hours by cage side observations (with handling where necessary) 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 Saturday, Sunday and public holidays only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded.
- Body weights: Recorded on day -3 (randomisation) and on days 0 (First day of dosing), 3, 7, 10 and 14 (day of sacrifice).
- Food consumption: Measured in periods, days 0-3, 3-7, 7-10 and 10-14.
- Gross necropsy: All animals were subjected to a complete gross necropsy. The animals were euthanised by exsanguination under CO2/O2-anaesthesia and then examined grossly for pathological changes. The following organs were weighed: kidney, liver, spleen, testes, thymus, brain.

MAIN STUDY
- Detailed clinical observations: Each animal observed daily in the morning hours by cage side observations (with handling where necessary) 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 Saturday, Sunday and public holidays only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded.
- Body weight: Recorded on day -2 (randomisation), and on days 0 (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 (postnatal days (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, litters were examined once daily for dead pups.
- Haematology: Prior to the end of the premating period, 5 rats/sex/group were fasted overnight and blood was taken whilst under CO2/O2 anaesthesia by orbital puncture: K2-EDTA was used as an anticoagulant. The following parameters were determined: 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 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 parameters were measured in the plasma: fasting glucose, alkaline phosphatase activity, aspartate aminotransferase activity, alanine aminotransferase activity, gamma glutamyl transferase activity, total protein, albumin, ration albumin to globulin, urea, creatinine, bilirubin (total), cholesterol (total), triglycerides, phospholipids, calcium, sodium, potassium, chloride and inorganic phosphate.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined:
- number of females with liveborn and (all) stillborn pups
- number of pups delivered (live and stillborn)
- number of live pups at day n
- number of pups lost
- number of litters lost entirely
- number of male pups at day n

GROSS EXAMINATION OF DEAD PUPS:
yes, for abnormalities

Postmortem examinations (parental animals):

All male and female parent rats were sacrificed by decapitation after CO2/O2 anaesthesia, except the 5 animals/sex/group which were sacrificed by exsanguination from the abdominal aorta under CO2/O2 anaesthesia. Males were killed after 33 days of exposure for necropsy. All sacrificed animals were 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 was preserved in Bouin’s fixative:
- Ovaries
- Uterus (after counting of the implantation sites)
- 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 (preserved but not processed)
- Colon
- Duodenum
- Eyes
- Heart
- Ileum
- Jejunum
- Lungs
- Kidneys
- Liver
- Mammary gland (females only) (preserved but not processed)
- Mesenteric lymph node
- Parathyroids
- Peyer’s patches
- Pituitary
- Rectum
- Sciatic nerve
- Spinal cord
- Spleen
- Stomach
- Thymus
- Thyroids
- Trachea
- Urinary bladder
In addition, reproductive organs of males that failed to sire (did not mate or mated female was not pregnant) and females that were non-mated or non-pregnant, of the low- and mid-dose groups, were microscopically examined. In females, the number of implantation sites and number of lost implantations were examined.
Tissues for microscopic examination were embedded in paraffin wax, sectioned at 5 µm, and stained with haemotoxylin and eosin, except for sections of the testes which were stained with PAS haemotoxylin. Microscopic examination was performed on the collected organs of all rats of the control and high-dose groups. Examination was extended to the mesenteric lymph nodes of the male and female rats of the low- and mid-dose groups and to the thymus of the female rats of the low and mid-dose groups.

Postmortem examinations (offspring):

SACRIFICE
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 after.

Statistics:

- Clinical findings were evaluated by Fisher's exact probability test.
- Body weight, body weight gain, organ weights and food consumption data were subjected to one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests.
- 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.
- Red blood cell and coagulation variables, total white blood cell counts, absolute differential white blood cell counts, clinical chemistry values and organ weights: one-way ANOVA followed by Dunnett's multiple comparison tests (treatment period).
- Reticulocytes and relative differential white blood cell counts: Kruskal-Wallis non-parametric ANOVA followed by Mann-Whitney U-tests.
- Histopathological changes: Fisher's exact probability test.
- Parameters assessed during functional observations were measured on different measurements scales (e.g., continuous, rank, categorical). Continuous measures were analysed by one-way analysis of variance at each test time point, followed by post-hoc group comparisons in case of a significant result. Rank order data were analysed by Kruskal-Wallis analysis of variance at each test time point, followed by planned multiple comparisons between dose groups in case of a significant result. Categorical data were analysed by Pearson chi-square analysis.
- Motor activity data were analysed using one-way analysis of variance at each test time point, followed by post-hoc group comparisons in case of a significant result.
All tests were two-sided. A level of probability of <0.05 (p<0.05) was considered significant. Statistical evaluations on variables associated with pups were considered on a litter basis. Additional evaluations on a pup basis were performed to identify any specific dose-related effect.

Reproductive indices:

FERTILITY AND REPRODUCTIVE PERFORMANCE
For each mating the following were recorded for each group:
- number of females placed with males
- number of males mated with females
- number of successful copulations (= number of females mated)
- number of males that became sires
- number of pregnant females as demonstrated by the presence of implantation sites observed at autopsy
- number of females surviving delivery
- number of females with liveborn and (all) stillborn pups
- number of pups delivered (live and stillborn)
- number of live pups at day n
- number of pups lost
- number of litters lost entirely
- number of male pups at day n
- number of implantation sites
- number of lost implantations

From which 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

Offspring viability indices:

> 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 implantation sites – number of pups born alive
> Post implantation loss = [(number of implantation sites - number of pups born alive)/number of implantation sites] x 100

Clinical signs:

no effects observed

Description (incidence and severity):

- Dose-range finding study: Apart from 1 male animal of the 10 000 mg/kg dose group which showed emaciation and weakness from day 8 until sacrifice, no clinical signs were observed.

- Main study: Two female animals of the 2000 mg/kg dose group showed treatment-related findings; from gestation days 21-22 one animal showed piloerection and the other animal was emaciated from postnatal days 1-5.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Dose-range finding study: Mean body weights of the male animals of the 10 000 mg/kg dose group were statistically significantly decreased from days 3-14. Body weight change of the male animals of the 10 000 mg/kg dose group was statistically significantly decreased between days 0-3 and 3-7. Mean body weight change of the females of the 10 000 mg/kg dose group was statistically significantly decreased between days 0-3.

- Main study: On gestation day 14, mean body weight of the pregnant females of the 2000 mg/kg dose group was statistically significantly decreased. Mean body weight change of the pregnant females of the 2000 mg/kg dose group was statistically significantly decreased from gestation days 0-7 and gestation days 7-14. Body weight and body weight change of the lactating females was comparable amongst the groups. Body weight change of the male animals of the 2000 mg/kg dose group was statistically significantly decreased from days 21-28.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Dose-range finding study: In the 10 000 mg/kg dose group, food consumption (expressed as g/animal/day and as g/kg body weight/day) was decreased from days 3-7.

- Main study: In the female animals of the 2000 mg/kg dose group a decreased food consumption (expressed as g/animal/day and g/kg body weight/day) was observed during the premating, gestation and lactation period; this effect was statistically significant except for gestation days 14-21.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- Main study: At the end of the premating period, statistically significant differences in haematology parameters between animals given the test substance and control animals were limited to a higher number of thrombocytes and a shorter prothrombin time in the females of the 2000 mg/kg dose group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- Main study: At the end of the premating period, statistically significant differences in clinical chemistry parameters of female animals of the 2000 mg/kg dose group were limited to an increase in gamma glutamyl transferase, cholesterol, triglycerides and phospholipids; these findings suggest an effect on the liver. However, these findings were not accompanied by changes in liver weight or histopathology.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- Main study: Neurobehavioural testing of female animals at the end of the study on postnatal day 4 revealed minor differences in arousal and rearing in 2000 mg/kg dose females and an impaired righting reflex in two of these 2000 mg/kg dose females. As the differences were only minor and were not supported by behavioural changes in measures of the same functional domain, they were not considered as clear evidence of neurotoxicity induced by 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):

Treatment-related effects on thymus weights were accompanied by lymphoid depletion in the thymus of 5/5 2000 mg/kg dose and 2/5 300 mg/kg dose females. In addition, the paracortex of the mesenteric lymph nodes, which is a thymus dependent area, showed slight lymphoid depletion in male and female animals of the 2000 mg/kg dose group. In two 2000 mg/kg dose male animals the mesenteric lymph nodes were red discoloured at macroscopic examination. In these and other 2000 mg/kg dose male and female animals the mesenteric lymph nodes showed microscopically a treatment-related increased severity of red blood cells in the sinusoids, with conspicuous erythrophagocytosis and brown pigment accumulation (haemosiderin deposits), pointing to removal of red blood cells. Although spleen weight was decreased in 2000 mg/kg dose male animals, the spleen did not show periarteriolar lymphocyte sheath (PALS, a thymus dependent area) lymphoid depletion upon microscopic examination. The toxicological significance of this finding is unknown.

Histopathological findings: neoplastic:

not specified

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

- Fertility and reproductive performance: One female of the high-dose group was not mated. No statistically significant difference was observed in the pre-coital time. The number of pregnant females and the number of males that became sires were 10, 9, 10 and 9 for the control, low-, mid- and high dose groups, respectively. One animal of the high dose group (D377) delivered no pups, but at necropsy 11 implantations were observed in the uterus. The number of females with live-born pups was comparable in all groups and was 10, 9, 10 and 8 for the control, low-, mid- and high-dose groups, respectively. The mating index, female fecundity index, female and male fertility index and gestation index were comparable among the groups and ranged from 75 - 100 %. No difference was observed in the duration of gestation. Stillborn pups were observed in 1, 0, 1 and 1 litter of the control, low-, mid- and high-dose group, respectively. No females with all stillborn pups were observed. Post-implantation loss was 10.1, 11.5, 6.9 and 21.5 % for the control, low-, mid- and high-dose groups, respectively. In summary, all reproductive parameters were comparable amongst the groups except for the post-implantation loss, which was not statistically significantly increased in the high-dose group.

Key result

Dose descriptor:

NOAEL

Remarks:

General toxicity

Effect level:

100 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Remarks on result:

other: Equivalent to 6-7 mg/kg body weight/day for the male animals and 5-8 mg/kg body weight/day for the female animals.

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive effects

Effect level:

300 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: Equivalent to 17-20 mg/kg body weight/day for the males and 16-24 mg/kg body weight/day in the females.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg diet

System:

haematopoietic

Organ:

spleen

thymus

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Pup observations: On PN 1 the number of cold pups was highest in the control group; 10 of the 11 pups of litter A319 were cold. No cold pups were observed in the tetrabutylstannane-treated groups. The number of pale pups was statistically significantly increased in the high-dose group on PN1. The number of runts (pup weight less than mean pup weight of the control minus 2 standard deviations) was increased in the high-dose group on PN1 and 4; only on PN4 this finding was statistically significant. The other findings were incidental and normal for pups of that age and were considered not to be related to treatment.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Decreased number of live pups, increased pup mortality on PN 4 and increased number of dead pups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Litter weight: Mean pup weights on PN1 and 4 and the pup weight change from PN 1-4 of the high-dose group were decreased when compared to the control group; a level of significance was not reached for these findings.

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

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

- Pathology: No abnormalities were observed in still born or dead pups.

Histopathological findings:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

- Litter size and sex: The number of pups delivered per litter in the high-dose group was statistically decreased when compared to the control group; the number of pups delivered per litter amounted 10.4, 10.6, 9.7 and 8.0 for the control, low-, mid-, and high-dose groups, respectively. The number of liveborn pups amounted 103, 95, 96 and 63 for the control, low-, mid- and high-dose groups, respectively and the number of stillborn pups 1, 0, 1 and 1 for the control, low-, mid and high-dose groups, respectively. Pup mortality of PN 4 was statistically significantly increased in the high-dose groups, respectively. Pup mortality on PN 4 was statistically significantly increased in the high-dose groups and amounted to 0, 1, 4, 26 (incidences 0, 1.1, 4.2 and 41 %) in the control, low-, mid- and high-dose groups, respectively. In the control, low-, mid-, and high-dose groups 0, 0, 0 and 3 litters were lost entirely between PN 0-4. The number of live pups per litter on PN 1 and 4 was statistically significantly lower in the high-dose group than in the control group. The sex ratio was comparable in all groups.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive effects

Generation:

F1

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Remarks on result:

other: Equivalent to 17-20 mg/kg body weight/day for the males and 16-24 mg/kg body weight/day in the females.

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg diet

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Litter observations

Effect	2000 mg/kg group	Statistically Singnificant	Control group
post-implantation loss (%)	21.5	NS	10.1
number of pups delivered/litter	8.0	S	10.4
number of live pups/litter postnatal day 1	7.9	S	10.3
number of live pups/litter postnatal day 4	7.4	S	10.3
pup mortality postnatal day 4 (%)	41	S	0
pup weight postnatal day 1 (g)	4.6	NS	5.0
pup weight postnatal day 4 (g)	6.1	NS	7.0
pup weight change (g)	1.4	NS	2.0
number of runts postnatal day 1 (%)	15.9	NS	5.8
number of runts postnatal day 4 (%)	18.9	S	3.9

Conclusions:

Under the conditions of this study, based on the reproductive effects in the high-dose group (increased implantation loss, decreased number of live pups, increased pup mortality on PN 4 and increased number of dead pups), the NOAEL for reproductive toxicity is established on the mid-dose level (300 mg/kg diet which is equivalent to 16-24 mg/kg body weight/day for the female animals).

Executive summary:

The objective of this study was to provide data on the possible reproductive and developmental effects of the test material after oral administration via the diet to Wistar rats of both sexes. The study was conducted in accordance with the standardised guideline OECD 422 under GLP conditions.

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, 100, 500, 2000 and 10 000 mg/kg diet for 14 days. In the main study, rats were fed diets containing 0, 100, 300 and 2000 mg/kg diet for up to 33 days (males) or during 2 weeks of pre-mating, mating, gestation and up to postnatal day 4 or 5 (females).

In each group 12 females were placed with males. One female of the high-dose was not-mated. When comparing the high dose group to the control, effects were seen regarding post-implantation loss (%), number of pups delivered/litter, number of live pups/litter PN 1, number of live pups/litter PN 4, pup mortality PN 4 (%), pup weight PN 1 (g), pup weight PN 4 (g), pup weight change (g), number of runts PN 1 (%) and number of runts PN 4 (%). The effects were statistically significant for the number of pups delivered/litter, number of live pups/litter PN 1, number of live pups/litter PN 4, pup mortality PN 4 (%) and number of runts PN 4 (%).

Pre-coital time, female fecundity, male and female fertility, gestation index, number of females with liveborn pups, number of females with stillborn pups, number of females with only stillborn pups, number of liveborn pups and number of stillborn pups were comparable to the control group.

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

In conclusion, based on the observed effects in the mid-dose group (decrease of spleen weight (males only), thymus weight, microscopic findings in the thymus) the NOAEL for general toxicity is established on the low-dose level (100 mg/kg diet which is equivalent to 5-8 mg/kg body weight/day for the female animals).

Under the conditions of this study, based on the reproductive effects in the high-dose group (increased implantation loss, decreased number of live pups, increased pup mortality on PN 4 and increased number of dead pups), the NOAEL for reproductive toxicity is established on the mid-dose level (300 mg/kg diet which is equivalent to 16-24 mg/kg body weight/day for the female animals).

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18th December 2002 - 22nd April 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

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

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age: aound 9-10 weeks of age on arrival (18 December 2002 and 19 February 2003 in the range finder and main study respectively)
- Diet: ad libitum from arrival until the end of the study
- Weight: the weight variation of the animals used did not exceed 20 % of the  mean weight for each sex
- Water: ad libitum from arrival until the end of the study
- Acclimation period: Acclimatised until study initiation on the 2 January 2003 (range finding study) and 27 February 2003

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 °C
- Humidity (%): 30-70 % with some short periods at 100 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): Artificial lighting on a 12 hours light and 12 hours dark sequence.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
The feed was provided as a powder, in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. The feed in the feeders was refreshed at least once per week and topped up where necessary. The test substance was incorportated int he basal diet by mixing in a mechanical blender. The experimental diets were prepared once shortly before the start of the studies and stored in a freezer (-18 °C) until use.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before the start of the study, the analytical method for analysing test substance in the diet was validated in the matrix under examination (viz. diet). The validation was performed in a separate study. 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, tripopultin chloride and tetrapropyltin in methanol) was added. Subsequently methanol, acetate buffer solution (pH 4.5) 20 % aqueous sodium tetraethylborate (NaBEt4) 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 chlorides were converted into the corresponding ethylated tetraorganotin derivative, which were extracted into the hexane layer. Prior to GC-MS analysis, the hexane layer was washed with 2 mol/L HCl in order to remove most of the ethylboron compounds that interfere with the GC-MS analysis. The concentration of each test susbtance in feed was determined by GC-MS analysis of the hexane extracts. The homogenous distribution, stability and acheived concentration of the test substance in 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. Directly after mixiing 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 and right centre. Secondly, five samples (of about 50 g each) for examination of the stability were taken from the top part of the mixer. The samples taken for the homogeneity experiments were also used for determination of the content.
In addition, analyses to determine the content (acheived concentration) of the test substance in the batch of diet was used in the main study were conducted. Stability of the test susbtance in the diet was also tested after then end of the main study (after c. 55 days). Diet samples for the determination of content of the diets used were taken immediately after preparation of the diets and stored at ca. -18 °C.

ACTUAL DOSE RECEIVED BY DOSE LEVEL BY SEX
- Dose range finding study: The test substance intake of the male animals ranged from 6-7, 30-34, 102-131 and 206-636 mg/kg body weight/day in the 100, 500, 2000 and 10 000 mg/kg dose groups, respectively. The test substance intake of the female animals ranged from 6-7, 30-33, 111-129 and 253-555 mg/kg body weight/day in the 100, 500, 2000 and 10 000 mg/kg dose groups, respectively.
- Main study: The test substance intake of the male animals during the study ranged from 6-7, 17-20 and 109-130 mg/kg body weight/day the 100, 300 and 2000 mg/kg dose group, respectively. The test substance intake of the female animals during the premating, gestation and lactation period ranged from 5-8, 16-24 and 100-118 mg/kg body weight/day the 100, 300 and 2,000 mg/kg dose group, respectively.

TEST SUBSTANCE ANALYSIS IN THE DIET
The test substance was considered to be homogeneously distributed in all diets. Furthermore, the test substance was considered to be stable in the diets upon storage at room temperature for 7 days and upon storage at < -18 °C for 6 weeks. The test substance also met the criteria for stability upon storage at < -18 °C for 55 days in all diets from the main study, except the 300 mg/kg dose level, for which the relative difference was -22 %. This difference can at least be partially explained by analytical causes (i.e. varying extraction efficiency due to sticking of the test substance to diet). The content of the test substance was considered to be close to intended for all diets. Only the 2000 mg/kg dose level of the dose-range finding study exceeded the criteria (relative difference from intended concentration was -25 %), but when the low recovery of 82 % obtained during the validation of the test substance at this dose level is taken into account, the achieved concentration seems to be close to the intended concentration. In view of these conclusions regarding the analysis of the diets of both the dose-range finding and the main study, the nominal levels of the test substance were used to calculate the test substance intake.

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 (PN4) or shortly after (PN 5). Sperm positive females that were not pregnant were killed 25 days after copulation. The non-mated female of the high-dose group D393 was sacrificed on day 54 of the study, 26 days after the last day of the mating period. The morning after birth was considered day 1 post partum.

Duration of treatment / exposure:

- Range finder: Animals were dosed for 14 days
- Main test: Males: for a period of 33 days; Females: during the period of 2 weeks premating, mating, gestation, and up to postnatal day 4 or 5

Frequency of treatment:

Feed containing the test substance was provided ad libitum during the whole study. Feed was refreshed daily.

Dose / conc.:

100 mg/kg diet

Dose / conc.:

300 mg/kg diet

Dose / conc.:

2 000 mg/kg diet

No. of animals per sex per dose:

- Dose-range finding study: The study comprised 5 groups of 4 male and 4  female rats each per dose group
- Main study: The main study comprised four groups of 12 male and 12 female ratseach per dose group.

Control animals:

yes, plain diet

Details on study design:

The oral route was used because this is a possible route of human exposure. The test substance was administered at constant concentrations in the diet which remained the same for each group during the dose-range finding study and during the main study.

Maternal examinations:

DOSE RANGE FINDING STUDY
- Clinical signs: Each animal observed daily in the morning hours by cage side observations (with handling where necessary) 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 Saturday, Sunday and public holidays only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded.
- Body weights: Recorded on day -3 (randomisation) and on days 0 (First day of dosing), 3, 7, 10 and 14 (day of sacrifice).
- Food consumption: Measured in periods, days 0-3, 3-7, 7-10 and 10-14.
- Gross necropsy: All animals were subjected to a complete gross necropsy. The animals were euthanised by exsanguination under CO2/O2-anaesthesia and then examined grossly for pathological changes. The following organs were weighed: kidney, liver, spleen, thymus, brain.

MAIN STUDY
- Detailed clinical observations: Each animal observed daily in the morning hours by cage side observations (with handling where necessary) 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 Saturday, Sunday and public holidays only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded.
- Body weight: Recorded on day -2 (randomisation), and on days 0 (first day of dosing), 7 and 13 of the premating period. 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 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 (postnatal days (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, litters were examined once daily for dead pups.
- Haematology: Prior to the end of the premating period, 5 rats/sex/group were fasted overnight and blood was taken whilst under CO2/O2 anaesthesia by orbital puncture: K2-EDTA was used as an anticoagulant. The following parameters were determined: 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 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 parameters were measured in the plasma: fasting glucose, alkaline phosphatase activity, aspartate aminotransferase activity, alanine aminotransferase activity, gamma glutamyl transferase activity, total protein, albumin, ration albumin to globulin, urea, creatinine, bilirubin (total), cholesterol (total), triglycerides, phospholipids, calcium, sodium, potassium, chloride and inorganic phosphate.

SACRIFICE
All parent rats were sacrificed by decapitation after CO2/O2 anaesthesia, except the 5 animals/sex/group which were sacrificed by exsanguination from the abdominal aorta under CO2/O2 anaesthesia. All sacrificed animals were 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:
- Ovaries
- Uterus (after counting of the implantation sites)
- 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 (preserved but not processed)
- Colon
- Duodenum
- Eyes
- Heart
- Ileum
- Jejunum
- Lungs
- Kidneys
- Liver
- Mammary gland (preserved but not processed)
- Mesenteric lymph node
- Parathyroids
- Peyer’s patches
- Pituitary
- Rectum
- Sciatic nerve
- Spinal cord
- Spleen
- Stomach
- Thymus
- Thyroids
- Trachea
- Urinary bladder
In addition, reproductive organs of females that were non-mated or non-pregnant, of the low- and mid-dose groups, were microscopically examined. In females, the number of implantation sites and number of lost implantations were examined.
Tissues for microscopic examination were embedded in paraffin wax, sectioned at 5 µm, and stained with haemotoxylin and eosin, except for sections of the testes which were stained with PAS haemotoxylin. Microscopic examination was performed on the collected organs of all rats of the control and high-dose groups. Examination was extended to the mesenteric lymph nodes of the rats of the low- and mid-dose groups and to the thymus of the female rats of the low and mid-dose groups.

Ovaries and uterine content:

In females, the number of implantation sites and number of lost implantations were examined. If necessary the implantation sites were made visible following Salewski E (1964), Arch. Exp. Path. Pharmacol. 247, 367.

Fetal examinations:

PARAMETERS EXAMINED
The following parameters were examined:
- number of females with liveborn and (all) stillborn pups
- number of pups delivered (live and stillborn)
- number of live pups at day n
- number of pups lost
- number of litters lost entirely
- number of male pups at day n

GROSS EXAMINATION OF DEAD PUPS:
yes, for abnormalities

SACRIFICE
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 after.

Statistics:

- Clinical findings were evaluated by Fisher's exact probability test.
- Body weight, body weight gain, organ weights and food consumption data were subjected to one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests.
- 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.
- Red blood cell and coagulation variables, total white blood cell counts, absolute differential white blood cell counts, clinical chemistry values and organ weights: one-way ANOVA followed by Dunnett's multiple comparison tests (treatment period).
- Reticulocytes and relative differential white blood cell counts: Kruskal-Wallis non-parametric ANOVA followed by Mann-Whitney U-tests.
- Histopathological changes: Fisher's exact probability test.
- Parameters assessed during functional observations were measured on different measurements scales (e.g., continuous, rank, categorical). Continuous measures were analysed by one-way analysis of variance at each test time point, followed by post-hoc group comparisons in case of a significant result. Rank order data were analysed by Kruskal-Wallis analysis of variance at each test time point, followed by planned multiple comparisons between dose groups in case of a significant result. Categorical data were analysed by Pearson chi-square analysis.
- Motor activity data were analysed using one-way analysis of variance at each test time point, followed by post-hoc group comparisons in case of a significant result.
All tests were two-sided. A level of probability of <0.05 (p<0.05) was considered significant. Statistical evaluations on variables associated with pups were considered on a litter basis. Additional evaluations on a pup basis were performed to identify any specific dose-related effect.

Indices:

FERTILITY AND REPRODUCTIVE PERFORMANCE
For each mating the following were recorded for each group:
- number of females placed with males
- number of males mated with females
- number of successful copulations (= number of females mated)
- number of males that became sires
- number of pregnant females as demonstrated by the presence of implantation sites observed at autopsy
- number of females surviving delivery
- number of females with liveborn and (all) stillborn pups
- number of pups delivered (live and stillborn)
- number of live pups at day n
- number of pups lost
- number of litters lost entirely
- number of male pups at day n
- number of implantation sites
- number of lost implantations

From which 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 implantation sites – number of pups born alive
> Post implantation loss = [(number of implantation sites - number of pups born alive)/number of implantation sites] x 100

Clinical signs:

no effects observed

Description (incidence and severity):

- Dose-range finding study: Apart from 1 male animal of the 10 000 mg/kg dose group which showed emaciation and weakness from day 8 until sacrifice, no clinical signs were observed.

- Main study: Two female animals of the 2000 mg/kg dose group showed treatment-related findings; from gestation days 21-22 one animal showed piloerection and the other animal was emaciated from postnatal days 1-5.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Dose-range finding study: Mean body weight change of the females of the 10 000 mg/kg dose group was statistically significantly decreased between days 0-3.

- Main study: On gestation day 14, mean body weight of the pregnant females of the 2000 mg/kg dose group was statistically significantly decreased. Mean body weight change of the pregnant females of the 2000 mg/kg dose group was statistically significantly decreased from gestation days 0-7 and gestation days 7-14. Body weight and body weight change of the lactating females was comparable amongst the groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Dose-range finding study: In the 10 000 mg/kg dose group, food consumption (expressed as g/animal/day and as g/kg body weight/day) was decreased from days 3-7.

- Main study: In the female animals of the 2000 mg/kg dose group a decreased food consumption (expressed as g/animal/day and g/kg body weight/day) was observed during the premating, gestation and lactation period; this effect was statistically significant except for gestation days 14-21.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- Main study: At the end of the premating period, statistically significant differences in haematology parameters between animals given the test substance and control animals were limited to a higher number of thrombocytes and a shorter prothrombin time in the females of the 2000 mg/kg dose group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- Main study: At the end of the premating period, statistically significant differences in clinical chemistry parameters of female animals of the 2000 mg/kg dose group were limited to an increase in gamma glutamyl transferase, cholesterol, triglycerides and phospholipids; these findings suggest an effect on the liver. However, these findings were not accompanied by changes in liver weight or histopathology.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- Main study: Neurobehavioural testing of female animals at the end of the study on postnatal day 4 revealed minor differences in arousal and rearing in 2000 mg/kg dose females and an impaired righting reflex in two of these 2000 mg/kg dose females. As the differences were only minor and were not supported by behavioural changes in measures of the same functional domain, they were not considered as clear evidence of neurotoxicity induced by the test substance.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Dose-range finding study: Absolute and relative thymus weights of the females of the 2000 and 10 000 mg/kg dose groups were statistically significantly decreased; the decrease is considered to be treatment-related.

- Main study: In the females of the 300 and 2000 mg/kg dose group, the absolute thymus weight was 0.121 g (control) versus 0.097 g (300 mg/kg) and 0.097 g (2000 mg/kg) and relative thymus weight was 0.580 g/kg body weight (control) versus 0.446 g/kg body weight (300 mg/kg) and 0.459 g/kg body weight (2000 mg/kg); the decrease in absolute and relative thymus weights of the female animals was not statistically significant.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Dose-range finding study: In the female animals a small thymus was observed in 3 and 2 animals of the 2000 and 10 000 mg/kg dose groups, respectively.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related effects on thymus weights were accompanied by lymphoid depletion in the thymus of 5/5 2000 mg/kg dose and 2/5 300 mg/kg dose females. In addition, the paracortex of the mesenteric lymph nodes, which is a thymus dependent area, showed slight lymphoid depletion in female animals of the 2000 mg/kg dose group. In 2000 mg/kg dose female animals the mesenteric lymph nodes showed microscopically a treatment-related increased severity of red blood cells in the sinusoids, with conspicuous erythrophagocytosis and brown pigment accumulation (haemosiderin deposits), pointing to removal of red blood cells. Although, the spleen did not show periarteriolar lymphocyte sheath (PALS, a thymus dependent area) lymphoid depletion upon microscopic examination. The toxicological significance of this finding is unknown.

Histopathological findings: neoplastic:

not specified

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Post-implantation loss was 10.1, 11.5, 6.9 and 21.5 % for the control, low-, mid- and high-dose groups, respectively. The post-implantation loss was not statistically significantly increased in the high-dose group.

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

no effects observed

Description (incidence and severity):

The number of females with liveborn pups was comparable in all groups and was 10, 9, 10 and 8 for the control, low-, mid- and high-dose groups, respectively.
Stillborn pups were observed in 1, 0, 1, and 1 litter of the control, low-, mid- and high-dose group, respectively. No females with all stillborn pups were observed.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No difference was observed in the duration of gestation.
The gestation index was comparable among the groups.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

In each group 12 females were placed with males and all females of the control and low- and mid-dose group were mated. One female of the high-dose group was not mated. No statistically significant difference was observed in the pre-coital time.
The number of pregnant females and the number of males that became sires amounted 10, 9, 10 and 9 for the control, low-, mid- and high-dose groups, respectively. One animal of the high-dose group (D377) delivered no pups but at necropsy 11 implantations were observed in the uterus.
The mating index, female fecundity index, female and male fertility index were comparable among the groups and ranged from 75-100 %.

Other effects:

no effects observed

Description (incidence and severity):

In summary, all reproductive parameters were comparable amongst the groups except for the post-implantation loss which was, not statistically significantly, increased in the high-dose group.

Key result

Dose descriptor:

NOAEL

Remarks:

General toxicity

Effect level:

100 mg/kg diet

Based on:

test mat.

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: Equivalent to 5-8 mg/kg body weight/day for the female animals.

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive effects

Effect level:

300 mg/kg diet

Based on:

test mat.

Basis for effect level:

dead fetuses

pre and post implantation loss

Remarks on result:

other: Equivalent to 16-24 mg/kg body weight/day in the females.

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean pup weights on PN 1 and 4 and the pup weight change from PN 1-4 of the high­ dose group were decreased when compared to the control group; a level of significance was not reached for these findings.
The number of runts (pup weight less than mean pup weight of the control group minus 2 standard deviations) was increased in the high-dose group on PN 1 and 4; only on PN 4 this finding was statistically significant.

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

The number of pups delivered per litter in the high-dose group was statistically decreased when compared to the control group; the number of liveborn pups amounted 103, 95, 96 and 63 for the control, low-, mid- and high-dose groups, respectively and the number of stillborn pups 1, 0, 1 and 1 for the control, low-, mid- and high-dose groups, respectively.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex ratio was comparable in all groups.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

The number of pups delivered per litter in the high-dose group was statistically decreased when compared to the control group; the number of pups delivered per litter amounted 10.4, 10.6, 9.7 and 8.0 for the control, low-, mid- and high-dose groups, respectively.

Changes in postnatal survival:

effects observed, treatment-related

Description (incidence and severity):

Pup mortality on PN 4 was statistically significantly increased in the high-dose group and amounted 0, 1, 4, 26 (incidences 0, 1.1, 4.2 and 41 %) in the control, low-, mid- and high-dose groups, respectively. In the control, low-, mid- and high-dose groups 0, 0, 0 and 3 litters were lost entirely between PN 0-4. The number of live pups per litter on PN 1 and 4 was statistically significantly lower in the high-dose group than in the control group.

External malformations:

no effects observed

Description (incidence and severity):

No abnormalities were observed in stillborn or dead pups.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

On PN 1 the number of cold pups was highest in the control group; 10 of the 11 pups of litter A 319 were cold. No cold pups were observed in the treated groups. The number of pale pups was statistically significantly increased in the high­ dose group on PN 1.
The other findings were incidental and normal for pups of this age and are considered not to be related to treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in postnatal survival

Remarks on result:

other: Equivalent to 16-24 mg/kg body weight/day in the females.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg diet

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Litter observations

Effect	2000 mg/kg group	Statistically Singnificant	Control group
post-implantation loss (%)	21.5	NS	10.1
number of pups delivered/litter	8.0	S	10.4
number of live pups/litter postnatal day 1	7.9	S	10.3
number of live pups/litter postnatal day 4	7.4	S	10.3
pup mortality postnatal day 4 (%)	41	S	0
pup weight postnatal day 1 (g)	4.6	NS	5.0
pup weight postnatal day 4 (g)	6.1	NS	7.0
pup weight change (g)	1.4	NS	2.0
number of runts postnatal day 1 (%)	15.9	NS	5.8
number of runts postnatal day 4 (%)	18.9	S	3.9

Conclusions:

Under the conditions of this study, based on the reproductive effects in the high-dose group (increased implantation loss, decreased number of live pups, increased pup mortality on PN 4 and increased number of dead pups), the NOAEL for reproductive toxicity is established on the mid-dose level (300 mg/kg diet which is equivalent to 16-24 mg/kg body weight/day for the female animals).

Executive summary:

The objective of this study was to provide data on the possible reproductive and developmental effects of the test material after oral administration via the diet to Wistar rats of both sexes. The study was conducted in accordance with the standardised guideline OECD 422 under GLP conditions.

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, 100, 500, 2000 and 10 000 mg/kg diet for 14 days. In the main study, rats were fed diets containing 0, 100, 300 and 2000 mg/kg diet for up to 33 days (males) or during 2 weeks of pre-mating, mating, gestation and up to postnatal day 4 or 5 (females).

In each group 12 females were placed with males. One female of the high-dose was not-mated. When comparing the high dose group to the control, effects were seen regarding post-implantation loss (%), number of pups delivered/litter, number of live pups/litter PN 1, number of live pups/litter PN 4, pup mortality PN 4 (%), pup weight PN 1 (g), pup weight PN 4 (g), pup weight change (g), number of runts PN 1 (%) and number of runts PN 4 (%). The effects were statistically significant for the number of pups delivered/litter, number of live pups/litter PN 1, number of live pups/litter PN 4, pup mortality PN 4 (%) and number of runts PN 4 (%).

Pre-coital time, female fecundity, male and female fertility, gestation index, number of females with liveborn pups, number of females with stillborn pups, number of females with only stillborn pups, number of liveborn pups and number of stillborn pups were comparable to the control group.

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

In conclusion, based on the observed effects in the mid-dose group (decrease of spleen weight (males only), thymus weight, microscopic findings in the thymus) the NOAEL for general toxicity is established on the low-dose level (100 mg/kg diet which is equivalent to 5-8 mg/kg body weight/day for the female animals).

Under the conditions of this study, based on the reproductive effects in the high-dose group (increased implantation loss, decreased number of live pups, increased pup mortality on PN 4 and increased number of dead pups), the NOAEL for reproductive toxicity is established on the mid-dose level (300 mg/kg diet which is equivalent to 16-24 mg/kg body weight/day for the female animals).

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:

adopted 22 March 1996

Deviations:

no

GLP compliance:

yes

Remarks:

The study was carried out in accordance with the OECD Principles of Good Laboratory Practice (as revised in 1997), OECD, Paris. ENV/MC/CHEM(98)17.

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tetrabutyltin

EC Number:

215-960-8

EC Name:

Tetrabutyltin

Cas Number:

1461-25-2

Molecular formula:

C16H36Sn

IUPAC Name:

tetrabutylstannane

Test material form:

liquid

Details on test material:

SOURCE: ORTEP Association Stabilizer Task Force.
[CAS no. 1461-25-2], colourless liquid.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age : aound 9-10 weeks of age on arrival (18 December 2002 and 19 February 2003 in the range finder and main study respectively)
- Diet : ad libitum from arrival until the end of the study
- Wieght: the weight variation of the animals used did not exceed 20 % of the  mean weight for each sex
- Water: ad libitum from arrival until the end of the study
- Acclimation period: Acclimatised until study initiation on the 2 January 2003 (range finding study) and 27 February 2003

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 °C
- Humidity (%): 30-30 % with some short periods at 100 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): Artificial lighting on a 12 hours light and 12 hours dark sequence.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
The feed was provided as a powder, in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. The feed in the feeders was refreshed at least once per week and topped up where necessary. The test substance was incorportated int he basal diet by mixing in a mechanical blender. The experimental diets were prepared once shortly before the start of the studies and stored in a freezer (-18 °C) until use.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before the start of the study, the analytical method for analysing test substance in the diet was validated in the matrix under examination (viz. diet). The validation was performed in a separate study. 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, tripopultin chloride and tetrapropyltin in methanol) was added. Subsequently methanol, acetate buffer solution (pH 4.5) 20 % aqueous sodium tetraethylborate (NaBEt4) 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 chlorides were converted into the corresponding ethylated tetraorganotin derivative, which were extracted into the hexane layer. Prior to GC-MS analysis, the hexane layer was washed with 2 mol/L HCl in order to remove most of the ethylboron compounds that interfere with the GC-MS analysis. The concentration of each test susbtance in feed was determined by GC-MS analysis of the hexane extracts. The homogenous distribution, stability and acheived concentration of the test substance in 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. Directly after mixiing 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 and right centre. Secondly, five samples (of about 50 g each) for examination of the stability were taken from the top part of the mixer. The samples taken for the homogeneity experiments were also used for determination of the content.
In addition, analyses to determine the content (acheived concentration) of the test substance in the batch of diet was used in the main study were conducted. Stability of the test susbtance in the diet was also tested after then end of the main study (after c. 55 days). Diet samples for the determination of content of the diets used were taken immediately after preparation of the diets and stored at ca. -18 °C.

ACTUAL DOSE RECEIVED BY DOSE LEVEL BY SEX
- Dose range finding study: The test substance intake of the male animals ranged from 6-7, 30-34, 102-131 and 206-636 mg/kg body weight/day in the 100, 500, 2000 and 10 000 mg/kg dose groups, respectively. The test substance intake of the female animals ranged from 6-7, 30-33, 111-129 and 253-555 mg/kg body weight/day in the 100, 500, 2000 and 10 000 mg/kg dose groups, respectively.
- Main study: The test substance intake of the male animals during the study ranged from 6-7, 17-20 and 109-130 mg/kg body weight/day the 100, 300 and 2000 mg/kg dose group, respectively. The test substance intake of the female animals during the premating, gestation and lactation period ranged from 5-8, 16-24 and 100-118 mg/kg body weight/day the 100, 300 and 2,000 mg/kg dose group, respectively.

TEST SUBSTANCE ANALYSIS IN THE DIET
The test substance was considered to be homogeneously distributed in all diets. Furthermore, the test substance was considered to be stable in the diets upon storage at room temperature for 7 days and upon storage at < -18 °C for 6 weeks. The test substance also met the criteria for stability upon storage at < -18 °C for 55 days in all diets from the main study, except the 300 mg/kg dose level, for which the relative difference was -22 %. This difference can at least be partially explained by analytical causes (i.e. varying extraction efficiency due to sticking of the test substance to diet). The content of the test substance was considered to be close to intended for all diets. Only the 2000 mg/kg dose level of the dose-range finding study exceeded the criteria (relative difference from intended concentration was -25 %), but when the low recovery of 82 % obtained during the validation of the test substance at this dose level is taken into account, the achieved concentration seems to be close to the intended concentration. In view of these conclusions regarding the analysis of the diets of both the dose-range finding and the main study, the nominal levels of the test substance were used to calculate the test substance intake.

Duration of treatment / exposure:

Males: for a period of 33 days
Females: during the period of 2 weeks premating, mating, gestation, and up to postnatal day 4 or 5

Frequency of treatment:

Feed containing the test substance was provided ad libitum during the whole study. Feed was refreshed daily.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg diet

Doses / concentrations 2

Dose / conc.:

300 mg/kg diet

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg diet

No. of animals per sex per dose:

- Dose-range finding study: The study comprised 5 groups of 4 male and 4  female rats each per dose group
- Main study: The main study comprised four groups of 12 male and 12 female ratseach per dose group.

Control animals:

yes, plain diet

Details on study design:

The oral route was used because this is a possible route of human exposure. The test substance was administered at constant concentrations in the diet which remained the same for each group during the dose-range finding study and during the main study.

- Dose-range finding study: The dose levels tested were: 0 (control), 100 (low dose), 500 (low-mid-dose), 2000 (high-mid-dose), 10000 (high-dose) mg  test substance/kg diet.
- Main study: The dose levels were: 0 (control), 100 (low-dose), 300  (mid-dose), 2000 (high-dose) mg test substance/kg diet.

Examinations

Observations and examinations performed and frequency:

DOSE RANGE FINDING STUDY
- Clinical signs: Each animal observed daily in the morning hours by cage side observations (with handling where necessary) 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 Saturday, Sunday and public holidays only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded.
- Body weights: Recorded on day -3 (randomisation) and on days 0 (First day of dosing), 3, 7, 10 and 14 (day of sacrifice).
- Food consumption: Measured in periods, days 0-3, 3-7, 7-10 and 10-14.
- Gross necropsy: All animals were subjected to a complete gross necropsy. The animals were euthanised by exsanguination under CO2/O2-anaesthesia and then examined grossly for pathological changes. The following organs were weighed: kidney, liver, spleen, testes, thymus, brain.

MAIN STUDY
- Detailed clinical observations: Each animal observed daily in the morning hours by cage side observations (with handling where necessary) 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 Saturday, Sunday and public holidays only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded.
- Body weight: Recorded on day -2 (randomisation), and on days 0 (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 (postnatal days (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, litters were examined once daily for dead pups.
- Haematology: Prior to the end of the premating period, 5 rats/sex/group were fasted overnight and blood was taken whilst under CO2/O2 anaesthesia by orbital puncture: K2-EDTA was used as an anticoagulant. The following parameters were determined: 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 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 parameters were measured in the plasma: fasting glucose, alkaline phosphatase activity, aspartate aminotransferase activity, alanine aminotransferase activity, gamma glutamyl transferase activity, total protein, albumin, ration albumin to globulin, urea, creatinine, bilirubin (total), cholesterol (total), triglycerides, phospholipids, calcium, sodium, potassium, chloride and inorganic phosphate.

Sacrifice and pathology:

All male and female parent rats were sacrificed by decapitation after CO2/O2 anaesthesia, except the 5 animals/sex/group which were sacrificed by exsanguination from the abdominal aorta under CO2/O2 anaesthesia. Males were killed after 33 days of exposure for necropsy. All sacrificed animals were 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 was preserved in Bouin’s fixative:
- Ovaries
- Uterus (after counting of the implantation sites)
- 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 (preserved but not processed)
- Colon
- Duodenum
- Eyes
- Heart
- Ileum
- Jejunum
- Lungs
- Kidneys
- Liver
- Mammary gland (females only) (preserved but not processed)
- Mesenteric lymph node
- Parathyroids
- Peyer’s patches
- Pituitary
- Rectum
- Sciatic nerve
- Spinal cord
- Spleen
- Stomach
- Thymus
- Thyroids
- Trachea
- Urinary bladder
In addition, reproductive organs of males that failed to sire (did not mate or mated female was not pregnant) and females that were non-mated or non-pregnant, of the low- and mid-dose groups, were microscopically examined.
Tissues for microscopic examination were embedded in paraffin wax, sectioned at 5 µm, and stained with haemotoxylin and eosin, except for sections of the testes which were stained with PAS haemotoxylin. Microscopic examination was performed on the collected organs of all rats of the control and high-dose groups. Examination was extended to the mesenteric lymph nodes of the male and female rats of the low- and mid-dose groups and to the thymus of the female rats of the low and mid-dose groups.

Statistics:

- Clinical findings were evaluated by Fisher's exact probability test.
- Body weight, body weight gain, organ weights and food consumption data were subjected to one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests.
- 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.
- Red blood cell and coagulation variables, total white blood cell counts, absolute differential white blood cell counts, clinical chemistry values and organ weights: one-way ANOVA followed by Dunnett's multiple comparison tests (treatment period).
- Reticulocytes and relative differential white blood cell counts: Kruskal-Wallis non-parametric ANOVA followed by Mann-Whitney U-tests.
- Histopathological changes: Fisher's exact probability test.
- Parameters assessed during functional observations were measured on different measurements scales (e.g., continuous, rank, categorical). Continuous measures were analysed by one-way analysis of variance at each test time point, followed by post-hoc group comparisons in case of a significant result. Rank order data were analysed by Kruskal-Wallis analysis of variance at each test time point, followed by planned multiple comparisons between dose groups in case of a significant result. Categorical data were analysed by Pearson chi-square analysis.
- Motor activity data were analysed using one-way analysis of variance at each test time point, followed by post-hoc group comparisons in case of a significant result.
All tests were two-sided. A level of probability of <0.05 (p<0.05) was considered significant. Statistical evaluations on variables associated with pups were considered on a litter basis. Additional evaluations on a pup basis were performed to identify any specific dose-related effect.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

- Dose-range finding study: Apart from 1 male animal of the 10 000 mg/kg dose group which showed emaciation and weakness from day 8 until sacrifice, no clinical signs were observed.

- Main study: Two female animals of the 2000 mg/kg dose group showed treatment-related findings; from gestation days 21-22 one animal showed piloerection and the other animal was emaciated from postnatal days 1-5.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Dose-range finding study: Mean body weights of the male animals of the 10 000 mg/kg dose group were statistically significantly decreased from days 3-14. Body weight change of the male animals of the 10 000 mg/kg dose group was statistically significantly decreased between days 0-3 and 3-7. Mean body weight change of the females of the 10 000 mg/kg dose group was statistically significantly decreased between days 0-3.

- Main study: On gestation day 14, mean body weight of the pregnant females of the 2000 mg/kg dose group was statistically significantly decreased. Mean body weight change of the pregnant females of the 2000 mg/kg dose group was statistically significantly decreased from gestation days 0-7 and gestation days 7-14. Body weight and body weight change of the lactating females was comparable amongst the groups. Body weight change of the male animals of the 2000 mg/kg dose group was statistically significantly decreased from days 21-28.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Dose-range finding study: In the 10 000 mg/kg dose group, food consumption (expressed as g/animal/day and as g/kg body weight/day) was decreased from days 3-7.

- Main study: In the female animals of the 2000 mg/kg dose group a decreased food consumption (expressed as g/animal/day and g/kg body weight/day) was observed during the premating, gestation and lactation period; this effect was statistically significant except for gestation days 14-21.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- Main study: At the end of the premating period, statistically significant differences in haematology parameters between animals given the test substance and control animals were limited to a higher number of thrombocytes and a shorter prothrombin time in the females of the 2000 mg/kg dose group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- Main study: At the end of the premating period, statistically significant differences in clinical chemistry parameters of female animals of the 2000 mg/kg dose group were limited to an increase in gamma glutamyl transferase, cholesterol, triglycerides and phospholipids; these findings suggest an effect on the liver. However, these findings were not accompanied by changes in liver weight or histopathology.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- Main study: Neurobehavioural testing of female animals at the end of the study on postnatal day 4 revealed minor differences in arousal and rearing in 2000 mg/kg dose females and an impaired righting reflex in two of these 2000 mg/kg dose females. As the differences were only minor and were not supported by behavioural changes in measures of the same functional domain, they were not considered as clear evidence of neurotoxicity induced by the test substance.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Dose-range finding study: Terminal body weight of the male animals of the 10 000 mg/kg dose group was statistically significantly decreased. Absolute and relative thymus weights of the females of the 2000 and 10 000 mg/kg dose groups were statistically significantly decreased. Although the decrease in absolute and relative weights of the thymus of the male animals of the 2000 and 10 000 mg/kg dose groups did not reach a level of statistical significance, the decrease is considered to be treatment-related.

- Main study: In the male animals of the 2000 mg/kg dose group, the absolute and relative thymus weights were statistically significantly decreased. In addition, thymus weights (absolute and relative) of the male animals in the 300 mg/kg dose group were decreased (but not significantly) relative to the control: absolute 0.354 g (control) versus 0.308 g (300 mg/kg) and relative 1.066 g/kg body weight (control) versus 0.882 g/kg body weight (300 mg/kg). Relative spleen weights of the male animals of the 300 and 2000 mg/kg dose groups were statistically significantly decreased when compared to the weight of the control group. In the females of the 300 and 2000 mg/kg dose group, the absolute thymus weight was 0.121 g (control) versus 0.097 g (300 mg/kg) and 0.097 g (2000 mg/kg) and relative thymus weight was 0.580 g/kg body weight (control) versus 0.446 g/kg body weight (300 mg/kg) and 0.459 g/kg body weight (2000 mg/kg); the decrease in absolute and relative thymus weights of the female animals was not statistically significant.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Dose-range finding study: In 1 male animal of the control group, 2 male animals of the 2000 mg/kg dose group and 4 male animals of the 10 000 mg/kg dose group a small thymus was observed. In the female animals a small thymus was observed in 3 and 2 animals of the 2000 and 10 000 mg/kg dose group, respectively.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related effects on thymus weights were accompanied by lymphoid depletion in the thymus of 5/5 2000 mg/kg dose and 2/5 300 mg/kg dose females. In addition, the paracortex of the mesenteric lymph nodes, which is a thymus dependent area, showed slight lymphoid depletion in male and female animals of the 2000 mg/kg dose group. In two 2000 mg/kg dose male animals the mesenteric lymph nodes were red discoloured at macroscopic examination. In these and other 2000 mg/kg dose male and female animals the mesenteric lymph nodes showed microscopically a treatment-related increased severity of red blood cells in the sinusoids, with conspicuous erythrophagocytosis and brown pigment accumulation (haemosiderin deposits), pointing to removal of red blood cells. Although spleen weight was decreased in 2000 mg/kg dose male animals, the spleen did not show periarteriolar lymphocyte sheath (PALS, a thymus dependent area) lymphoid depletion upon microscopic examination. The toxicological significance of this finding is unknown.

Histopathological findings: neoplastic:

not specified

Details on results:

NOAEL: Based on the observed effects in the 300 mg/kg dose group, i.e., decrease of spleen weight (males only), thymus weight, microscopic findings in the thymus, the NOAEL for general toxicity is established at the 100 mg/kg dose level (which is equivalent to 6-7 mg TTBT/kg body weight/day for the male animals and 5-8 mg TTBT/kg body weight/day for the female animals).

Treatment with tetrabutylstannane caused treatment-related lymphoid depletion in the thymus in female animals of the mid- and high-dose groups. Paracortical lymphoid depletion in the mesenteric lymph nodes was observed in male and female animals of the high-dose group. An increased severity of blood in the sinusoids, accompanied by erythrophagocytosis and, occasional haemosiderin deposits were seen in the mesenteric lymph nodes of males and females of the high-dose group. The toxicological significance of this is unknown.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

>= 6 - <= 7 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

2

Key result

Dose descriptor:

NOAEL

Effect level:

>= 5 - <= 8 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

haematopoietic

Organ:

spleen

thymus

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the observed effects in the mid-dose group (decrease of spleen weight (males only), thymus weight and microscopic findings in the thymus), the low-dose level (100 mg/kg diet which is equivalent to 6 - 7 mg/kg weight/day for the male animals and 5 - 8 mg/kg body weight/day for the female animals).

Executive summary:

10 to 11-week-old male and female Wistar rats were dosed with tetrabutyltin in a repeat dose oral study carried out in accordance with OECD test guideline 422 and conducted under GLP conditions.

The oral route was used because this is a possible route of human exposure. The test substance was administered at constant concentrations in the diet which remained the same for each group during the dose-range finding study and during the main study.

- Dose-range finding study: The dose levels tested were: 0 (control), 100 (low dose), 500 (low-mid-dose), 2000 (high-mid-dose), 10000 (high-dose) mg test substance/kg diet.

- Main study: The dose levels were: 0 (control), 100 (low-dose), 300 (mid-dose), 2000 (high-dose) mg test substance/kg diet.

Treatment with tetrabutylstannane caused treatment-related lymphoid depletion in the thymus in female animals of the mid- and high-dose groups. Paracortical lymphoid depletion in the mesenteric lymph nodes was observed in male and female animals of the high-dose group. An increased severity of blood in the sinusoids, accompanied by erythrophagocytosis and, occasional haemosiderin deposits were seen in the mesenteric lymph nodes of males and females of the high-dose group. The toxicological significance of this is unknown.

Based on the observed effects in the mid-dose group (decrease of spleen weight (males only), thymus weight and microscopic findings in the thymus), the NOAEL was determined as 100 mg/kg diet which is equivalent to 6 - 7 mg/kg weight/day for the male animals and 5 - 8 mg/kg body weight/day for the female animals.