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Description of key information

Short-term toxicity oral (OECD 407, rats): LOAEL = 10 mg/kg bw/day; no NOAEL could be established

Repested dose toxicity-oral (Read Across to DOTO): NOAEL for general toxicity is established on the low-dose level (5 mg/kg diet which is equivalent to 0.3-0.4 mg/kg body weight/day for the male animals and to 0.3-0.5 mg/kg body weight/day for the female animals).

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Oct - 12 Dec 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted Oct 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
adopted Oct 2008
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3050
Version / remarks:
adopted 2000
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Remarks:
Food and Consumer Product Safety Authority (VWA), Utrecht, The Netherlands
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
Details on species / strain selection:
Outbred, SPF-Quality
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: 138 - 140 g (males); 121- 124 g (females)
- Housing: Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm) with
sterilized sawdust as bedding material and paper as cage-enrichment. During locomotor activity mo
nitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage-enrichment
or bedding material.
- Diet: Pelleted roden diet, SM R/M-Z (Ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4 - 21.9
- Humidity (%): 44 - 62
- Air changes (per hr): Approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

IN LIFE DATES: From: 13 Oct 2011 To: 12 Dec 2011
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing, and were homogenized to visually acceptable levels. In order to obtain homogeneity, the formulations were heated in a water bath with a maximum temperature of approximately 80 °C for a maximum of approximately 20 minutes. The formulations were allowed to cool down to a temperature below 33 °C prior to dosing. Adjustment was made for specific gravity of the test substance and vehicle.

VEHICLE
- Justification for use and choice of vehicle: Based on trial formulations performed at the testing facility and on information from the sponsor
- Amount of vehicle: 5 mL/kg bw
Analytical verification of doses or concentrations:
no
Remarks:
Analytical techniques such as ICP-MS, GC, HPLC and UV-Vis were not suitable for the determination of the test substance due to its hydrophobic molecular structure.
Duration of treatment / exposure:
at least 28 days
Frequency of treatment:
once daily, 7 days/week
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on the results of a foregoing dose range finding study. Three females per group were orally exposed to 200 mg/kg bw/day for 10 days, 500 and 1000 mg/kg bw/day for 5 days at the same testing facility (Project 498035), respectively. At every dose group hunched posture, piloerection and reduced food consumption were observed. A weight loss was determined at 200 mg/kg bw/d in 2/3 females (up to 5%), which was reversible by end of study. At 500 mg/kg bw/day a weight loss up to 6% in 2/3 females but a weight gain (4%) in 1/3 females was observed. At 1000 mg/kg bw/day the weight loss in all females amounted up to 15%. At every dose group no abnormalities were noted during the macroscopic examination and the liver and kidney weights were considered to be normal. One female at 200 mg/kg bw/day was sacrificed in extremis on Day 10 as it showed lethargy, hunched posture, uncoordinated movements, piloerection, chromodacryorrhoea, lean appearance, ptosis and hypothermia between Days 8 and 10. Based on the results of this range finding study, dose levels suggested for the main study (28 days toxicity study) were 10, 30 and 100 mg/kg bw/day.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, all animals were observed for mortality and morbidity.
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes, observations were made outside the cage in a standard arena.
- Time schedule: Immediately after dosing, once prior to start of treatment and at weekly intervals prior to dosing

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD COMSUMPTION: Yes, the food consumption was measured weekly and given as food consumption in g/kg bw/day.

HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: Yes (isoflurane)
- How many animals: All
- Parameters checked: white blood cells (WBC), differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), red blood cells, reticulocytes, red blood cell distribution width (RDW), haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelets, prothrombin time and activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Animals fasted: Yes, overnight for a maximum of 20 hours.
- How many animals: all
- Parameters checked: alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate (Inorg. Phos)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4 of treatment
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength, motor activity test
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Parameters checked: The weight of the following organs was recorded: liver, kidneys, adrenal glands, testes, epididymides, prostate, seminal vesicles including coagulating glands, thymus, spleen, brain, heart, ovaries, uterus including cervix and thyroid including parathyroid.

HISTOPATHOLOGY: Yes. All organ and tissue samples were processed, embedded in paraffin wax, sliced and stained with haematoxylin and eosin. The samples of all tissues collected at the scheduled sacrifice from animals in the control and high-dose group, all tissues from the high-dose female terminated in extremis, the thymus of all animals in the low-and mid-dose groups (based on possible treatment -related changes in this organ in the high-dose group), and all gross lesions were examined by a pathologist.
- The following organs and tissues were collected and fixed in a 10% buffered formalin: ovaries, adrenal glands, Peyer's patches (jejunum, ileum), brain (cerebellum, mid-brain, cortex), caecum, cervix, prostate gland, rectum, colon, duodenum, sciatic nerve, epididymides, seminal vesicles including coagulating gland, eyes including optic nerve and harderian gland, skeletal muscle, spinal cord (cervical, midthoracic, lumbar), femur including joint, spleen, heart, sternum with bone marrow, ileum, stomach, jejunum, testes, kidneys, thymus, thyroid including parathyroid, liver, trachea, lung (infused with formalin), urinary bladder, lymph nodes (mandibular, mesenteric), uterus, vagina and all gross lessions.
Following tissues/organs were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination:
pancreas, aorta, pituitary gland, preputial gland, clitoral gland, salivary glands (mandibular, sublingual), skin, female mammary gland area, larynx, oesophagus, lacrimal gland (exorbital), tongue, nasopharynx.
Statistics:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.

Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
100 mg/kg bw/day: Salivation was observed in 5/5 males and 2/5 females after dosing. This was considered to be a physiological response rather than a sign of systemic toxicity as it was observed after dosing. Alopecia and scabs were observed in 1/5 males, these were considered signs of no toxicological significance. Lethargy, a hunched posture, piloerection, dehydration and/or a lean appearance was observed from Day 13 in 1/5 high-dose females. This animal was sacrificed in extremis on Day 18.
30 mg/kg bw/day: Chromodacryhorrea was observed in 1/5 males on Day 2-4. This is considered to be an effect of the treatment, due to the limited duration of the effect.
10 mg/kg bw/day: Alopecia and scabs were observed in 1/5 males on Day 10-20, however, this observation is considered to be incidental.

The summary of the results of the observed clinical signs in males and females is shown in Table 1 under "Any other information on results incl. tables".
Mortality:
mortality observed, non-treatment-related
Description (incidence):
100 mg/kg bw/day: One female was sacrified in extremis on Day 18. No cause of death could be established based on histopathological assessment. As other animals of this dose group showed no toxicologically relevant clinical signs, this death was considered to be incidental in nature and unrelated to treatment with the test substance.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The percentages of the body weight and body weight changes were comparisons to a baseline day 0. There was no significant difference in body weight between the control group and the treatment groups.
100 mg/kg bw/day: In week 2, a statistically significant lower body weight gain of males (23%) compared with the control group (33%) was observed. This effect was also noted in week 3, when the animals of the high-dose group showed 50% body weight gain which was significantly lower than that of the control groups (67%). The body weight gain of the high-dose group (100%) in week 4 was not statistically significant compared with the control group (119%) due to the high standard deviation. However, this effect was considered to be of toxicological relevance.

The summary of the results of the body weight gain (%) in males and females is shown in Table 2 under "Any other information on results incl. tables".
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In high-dose males significantly lower values of haemoglobin, haematocrit, mean corpuscular volume and mean corpuscular haemoglobin were measured, compared with the control group. However, the values of each parameter were within the range of historical control data for rats of this strain according to data published by Charles River (reference: Charles River, Clinical Laboratory Parameters for Cri:WI(Han), March 2008) and were not dose-related. Therefore, the effects were considered to be non-treatment-related.
Significantly lower relative eosinophil counts in high-dose males and higher partial thromboplastin time in low-dose males, as well as the significantly lower white blood cell counts in low- and mid-dose females and lower haematocrit level in low-dose females were observed, compared with the control group. The changes fell within the range of historical control data for rats of this strain according to data published by Charles River (reference: Charles River, Clinical Laboratory Parameters for Cri:WI(Han), March 2008. These changes in haematological parameters were slight in nature and were not observed at higher dose levels. Therefore, these changes are considered to be incidental.

The summary of the results of the haematological findings in males and females are shown in Table 3 and 4 under "any other information on results incl. tables".

Reference: Charles River, Clinical Laboratory Parameters for Cri:WI(Han), March 2008; link: http://www.criver.com/files/pdfs/rms/wistarhan/rm_rm_r_wistar_han_clin_lab_parameters_08.aspx
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Significantly higher alkaline phosphatase activity was recorded in the high-dose females, compared with the control group. A dose-related increase was observed in the mid- and high-dose males, although this result was not statistically significant. This effect is considered to be an adaptive response, caused by the increased metabolic load on the liver due to the treatment. In high-dose females, a significant increase (> 100%) in the bile acids level was observed compared with the control group. The toxicological relevance of this observation is unclear, as no similar increase was observed in the males, but may be related to the increased hepatic load related to the treatment.

The statistically significant higher glucose level in low-dose females compared with the control group did not show a dose-related trend, and the lower creatinine level in high-dose females compared with the control group was minor in nature. Furthermore, no similar changes were observed in the males. These changes were therefore considered to have no toxicological relevance.

The summary of the results of the clinical biochemistry in males and females is shown in Table 5 under "Any other information on results incl. tables".
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No toxicologically significant effects on motor activity were noted. The parameters hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all surviving animals. The statistically significant higher motor activity (total movements) of females at 30 mg/kg bw/day occurred in the absence of a dose-related trend, and was therefore considered to be of no toxicological relevance. All groups showed a similar motor activity habituation profile; with high activity in the first interval that decreased over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute and relative thymus weight was reduced at 10 mg/kg bw/day (35% and 40% reduction of the relative weight compared with the control group for males and females, respectively), at 30 mg/kg bw/day (59% and 65% reduction of the relative weight compared with the control group for males and females, respectively) and at 100 mg/kg bw/day (77% and 81% reduction of the relative weight compared with the control group for males and females, respectively) compared with the control group. These effects were also dose-related. The effects on the thymus were also observed during the gross pathology (reduced thymus size) and histopathological examination (loss of cortical and medullary differentiation, lymphoid atrophy and hyperplasia of undifferentiated cells). Therefore, the effect on thymus weight were considered to be toxicologically relevant.
High-dose females showed a significantly higher absolute liver weight and relative liver weight. An increase in the alkaline phosphatase activity was also observed in high-dose females. Therefore, the increase in the absolute liver weight is considered to be a treatment-related, adaptive effect, due to the increased metabolic load on the liver following the treatment.
The statistically significant lower prostate weight of the high-dose males and higher spleen weights of the high-dose females were non-adverse but not toxicologically relevant as the respective relative organ weight was not affected and no related histopathological results were observed. The slightly higher relative kidney weights of high-dose males may be related to a slightly lower terminal body weight. The absolute kidney weights were similar to control levels.
The higher heart weight at 30 and 100 mg/kg bw/day and the higher relative heart weight at 30 mg/kg bw/day of females showed no dose-related trend for relative weight, according to the study report the means were within the normal range and the control means were considered to be slightly low. In addition, no histopathological effects were present and no toxicological relevance was ascribed to these changes.

The summary of the results of the absolute (g) and relative (%) organ weights in males and females are shown in Table 6 and 7 under "Any other information on results incl. tables".
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
A reduced size of the thymus was noted in 1/5 males at 10 mg/kg, 2/5 males and 2/5 females at 30 mg/kg, and in 5/5 males and 4/5 females at 100 mg/kg bw/day with statistical significance compared with the control group. The reduction in thymus size was dose-related in both males and females. Based on the related effects observed on the organ weights and the histopathological findings these effects were considered to be toxicologically relevant.
Other findings among the control and treated animals were incidental and therefore considered to be of no toxicological relevance.

The summary of the results of the macroscopic findings in males and females are shown in Table 8 under "Any other information on results incl. tables".
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Substance-related findings were found in the thymus and consisted of:
− Lymphoid atrophy:
10 mg/kg bw/day: 1/5 males (grade 2) and 1/5 females (grade 1)
30 mg/kg bw/day: 4/5 males (2/5: grade 1, 1/5: grade 3 and 1/5: grade 4) and and 5/5 females (1/5: grade 2, 3/5: grade 3 and 1/5: grade 4)
100 mg/kg bw/day: 5/5 males (grade 4) and 4/4 females (1/4: grade 3, 3/4: grade 4).
− Increase in lymphocytolysis:
10 mg/kg bw/day: 4/5 males (2/5: grade 1, 1/5: grade 2, 1/5: grade 3)
30 mg/kg bw/day: 1/5 males (grade 2)
− Hyperplasia of undifferentiated cells (epithelial cells /immature lymphocytes):
30 mg/kg bw/day: 2/5 males (1/5: grade 2, 1/5: grade 3) and 5/5 females (2/5: grade 2, 3/5: grade 3)
100 mg/kg bw/day: 5/5 males (grade 4) and 3/4 females (3/4: grade 3, 1/4: grade 4).
− Loss of cortical and medullary differentiation:
10 mg/kg bw/day: in 1/5 males
30 mg/kg bw/day: in 2/5 males and 5/5 females
100 mg/kg bw/day: in 5/5 males and 4/4 females

The lymphoid atrophy, increase in lymphocytolysis, hyperplasia, loss of cortical and medullary differentiation all increased in incidence and severity with increasing dose level. This tendency was seen in both males and females. Related effects were observed on organ weight and thymus size. The effects on the thymus were considered to be toxicologically relevant at all dose levels.

The summary of the results of the macroscopic findings in males and females are shown in Table 9 under "Any other information on results incl. tables".
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Remarks on result:
other: Toxicologically relevant effects were observed at the lowest dose level and therefore a NOAEL could not be determined
Key result
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
10 mg/kg bw/day (nominal)
System:
immune system
Organ:
thymus
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Table 1: Clinical Signs Summary: Males and Females

Sign (max. grade)
(location)
  Treatment
Days
MALES   1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
Group 1 (control)   no clinical signs noted
                                                           
Group 2 (10 mg/kg bw/day)                                                          
Skin / fur                                                          
Alopecia (3) (Neck) G - - - - - - - - - - - - - - - - - - - - 1 1 1 1 - - - -
% - - - - - - - - - - - - - - - - - - - - 2 2 2 2 - - - -
Scabs (3) (Neck) G - - - - - - - - - - - - - 1 1 1 1 1 1 - - - - - - - - -
% - - - - - - - - - - - - - 2 2 2 2 2 2 - - - - - - - - -
                                                           
Group 3 (30 mg/kg bw/day)                                                          
Secretion / excretion                                                          
Chromodacryorrhoes (3) (Periorbital region left) G - 1 1 1 - - - - - - - - - - - - - - - - - - - - - - - -
% - 2 2 2 - - - - - - - - - - - - - - - - - - - - - - - -
                                                           
Group 4 (100 mg/kg bw/day)                                                          
Skin / fur                                                          
Alopecia (3) (Neck) G - - - - - - - - - - - - - - - - - - - - 1 1 1 1 1 1 1 1
% - - - - - - - - - - - - - - - - - - - - 2 2 2 2 2 2 2 2
Scabs (3) (Neck) G - - - - - - - - 1 1 1 1 1 1 1 1 1 1 1 1 - - - - - - - -
% - - - - - - - - 2 2 2 2 2 2 2 2 2 2 2 2 - - - - - - - -
Secretion / excretion                                                          
Salivation (3) G - - - - - - - - - - - - - - - - - - - - - - - - 1 1 1 1
% - - - - - - - - - - - - - - - - - - - - - - - - A A A A
FEMALES                                                          
Group 1 (control)   no clinical signs noted
                                                           
Group 2 (10 mg/kg bw/day)   no clinical signs noted
                                                           
Group 3 (30 mg/kg bw/day)   no clinical signs noted
                                                           
Group 4 (100 mg/kg bw/day)                                                          
Behaviour                                                          
Lethargy (3) G - - - - - - - - - - - - - - - 1 1 1 - - - - - - - - - -
% - - - - - - - - - - - - - - - 2 2 2 - - - - - - - - - -
Posture                                                           
Hunched Posture (1) G - - - - - - - - - - - - - 1 1 1 1 1 - - - - - - - - - -
% - - - - - - - - - - - - - 2 2 2 2 2 - - - - - - - - - -
Skin / fur                                                          
Pilorection (1) G - - - - - - - - - - - - - - 1 1 1 1 - - - - - - - - - -
% - - - - - - - - - - - - - - 2 2 2 2 - - - - - - - - - -
Secretion / excretion                                                          
Salivation (3) G - - - - - - - - - - - - - - - - - - - - - - - - 1 1 1 1
% - - - - - - - - - - - - - - - - - - - - - - - - 5 5 5 5
Various                                                          
Dehydrated (3) G - - - - - - - - - - - - - - - 1 1 1 - - - - - - - - - -
% - - - - - - - - - - - - - - - 2 2 2 - - - - - - - - - -
Lean (1) G - - - - - - - - - - - - - 1 1 1 1 1 - - - - - - - - - -
% - - - - - - - - - - - - - 2 2 2 2 2 - - - - - - - - - -

G: Median value of the highest individual daily grades

%: Percent of affected animals (0=less than 5%, 1=between 5% and 15%,..., A=more than 95%)

-: Observation performed, sign not present

Table 2: Body Weight Gain (%) Summary: Males and Females

MALES          
Treatment   Control 10 mg/kg bw 30 mg/kg bw 100 mg/kg bw
Day 1 Mean 0 0 0 0
Week 1 ST. DEV 0.0 0.0 0.0 0.0
  N 5 5 5 5
Day 8 Mean 33 30 28 23*
Week 2 ST. DEV 4.1 3.0 5.0 7.0
  N 5 5 5 5
Day 15 Mean 67 61 55 50*
Week 3 ST. DEV 7.9 5.6 8.2 13.4
  N 5 5 5 5
Day 22 Mean 99 90 86 83
Week 4 ST. DEV 8.9 5.9 9.8 15.5
  N 5 5 5 5
Day 28 Mean 119 105 105 100
Week 4 ST. DEV 11.4 5.7 11.3 18.0
  N 5 5 5 5
FEMALES          
Treatment   Control 10 mg/kg bw 30 mg/kg bw 100 mg/kg bw
Day 1 Mean 0 0 0 0
Week 1 ST. DEV 0.0 0.0 0.0 0.0
  N 5 5 5 5
Day 8 Mean 16 19 15 16
Week 2 ST. DEV 3.4 3.3 3.5 3.4
  N 5 5 5 5
Day 15 Mean 33 36 31 29
Week 3 ST. DEV 5.7 6.5 5.8 12.8
  N 5 5 5 5
Day 22 Mean 46 52 44 47
Week 4 ST. DEV 5.1 9.4 8.9 5.4
  N 5 5 5 4
Day 28 Mean 50 60 52 53
Week 4 ST. DEV 7.9 10.6 13.0 4.9
  N 5 5 5 4

*/** Dunnett−test based on pooled variance significant at 5% (*) or 1% (**) level

Table 3: Haemotology Summary: Males

End of Treatment   Control 10 mg/kg bw/day 30 mg/kg bw/day 100 mg/kg bw/day
Eosinophils (%) Mean 0.6 0.7 0.4 0.2+
  ST. DEV 0.1 0.2 0.3 0.1
  N 5 5 5 5
Haemoglobin Mean 10.0 9.9 9.7 9.2*
mmol/L ST. DEV 0.4 0.1 0.1 0.6
  N 5 5 5 5
Haematocrit Mean 0.459 0.456 0.442 0.429*
L/L ST. DEV 0.011 0.009 0.009 0.026
  N 5 5 5 5
MCV Mean 55.2 54.6 54.7 51.7**
fL ST. DEV 1.1 0.7 1.6 0.9
  N 5 5 5 5
MCH Mean 1.21 1.19 1.20 1.11
fmol ST. DEV 0.04 0.02 0.06 0.03
  N 5 5 5 5

+/++ Steel−test significant at 5% (+) or 1% (++) level

*/** Dunnett−test based on pooled variance significant at 5% (*) or 1% (**) level

Table 4: Haemotology Summary: Females

End of Treatment   Control 10 mg/kg bw/day 30 mg/kg bw/day 100 mg/kg bw/day
WBC Mean 7.8 5.7* 5.3** 6.4
10E9/L ST. DEV 1.5 1.1 0.6 1.0
  N 5 5 5 4
Haematocrit Mean 0.423 0.398* 0.415 0.414
L/L ST. DEV 0.009 0.015 0.010 0.011
  N 5 5 5 4

+/++ Steel−test significant at 5% (+) or 1% (++) level

*/** Dunnett−test based on pooled variance significant at 5% (*) or 1% (**) level

Table 5: Clinical Biochemistry Summary: Females and Males

End of Treatment   Control 10 mg/kg bw 30 mg/kg bw 100 mg/kg bw
FEMALES          
ALP Mean 116 146 143 213*
  ST. DEV 42 68 24 29
  N 5 5 5 4
Creatinine Mean 42.4 45.0 43.1 37.8*
umol/L ST. DEV 2.2 3.2 2.1 1.9
  N 5 5 5 4
Bile acids Mean 21.4 19.1 15.4 51.9**
umol/L ST. DEV 11.4 10.2 5.3 9.8
  N 5 5 5 4
MALES          
ALP Mean 283 278 414 504
  ST. DEV 66 122 65 226
  N 5 5 5 5
Creatinine Mean 36.5 38.1 35.9 35.8
umol/L ST. DEV 1.4 1.9 2.0 1.3
  N 5 5 5 5
Bile acids Mean 45.5 74.5 47.9 55.1
umol/L ST. DEV 15.2 39.6 20.6 14.1
  N 5 5 5 5

*/** Dunnett−test based on pooled variance significant at 5% (*) or 1% (**) level

Table 6: Organ Weights (g): Males and Females

End of Treatment   Control 10 mg/kg bw 30 mg/kg bw 100 mg/kg bw
MALES          
Thymus (g) Mean 0.550 0.332** 0.209** 0.116**
  ST. DEV 0.106 0.070 0.084 0.020
  N 5 5 5 5
FEMALES          
Thymus (g) Mean 0.406 0.255** 0.144** 0.082**
  ST. DEV 0.061 0.062 0.027 0.030
  N 5 5 5 4
Liver (g) Mean 5.01 5.40 5.28 6.08**
  ST. DEV 0.51 0.15 0.37 0.43
  N 5 5 5 4

*/** Dunnett−test based on pooled variance significant at 5% (*) or 1% (**) level

Table 7: Relative Organ Weight (%) Summary: Males and Females

End of Treatment   Control 10 mg/kg bw 30 mg/kg bw 100 mg/kg bw
MALES          
Thymus (%) Mean 0.192 0.124** 0.078** 0.045**
  ST. DEV 0.036 0.027 0.028 0.009
  N 5 5 5 5
FEMALES          
Thymus (%) Mean 0.239 0.144** 0.083** 0.046**
  ST. DEV 0.030 0.033 0.015 0.016
  N 5 5 5 4
Liver (%) Mean 2.95 3.05 3.07 3.43**
  ST. DEV 0.26 0.04 0.14 0.11
  N 5 5 5 4

*/** Dunnett−test based on pooled variance significant at 5% (*) or 1% (**) level

Table 8: Macroscopic Findings Summary: Males and Females

End of Treatment Control 10 mg/kg bw/day 30 mg/kg bw/day 100 mg/kg bw/day
MALES        
Thymus        
reduced size 0 1 2 5 ##
FEMALES        
Thymus        
reduced size 0 0 2 4 ##

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

Table 9: Summary of histopathological findings: Males and Females

Dose
[mg/kg bw/day]
Lymphoid atrophy Increase in lymphocytolysis Hyperplasia of undifferentiated cells (epithelial cells /immature lymphocytes) Loss of cortical and medullary differentiation
MALES
10 1/5: grade 2 2/5: grade 1
1/5: grade 2
1/5: grade 3
- 1/5
30 2/5: grade 1
1/5: grade 3
1/5: grade 4
1/5 males: grade 2 1/5: grade 2
1/5: grade 3
2/5
100 5/5: grade 4 - 5/5 males: grade 4 5/5
FEMALES
10 1/5: grade 1 -
30 1/5: grade 2
3/5: grade 3
1/5: grade 4
- 2/5: grade 2
3/5: grade 3
5/5
100 1/4: grade 3
3/4: grade 4
- 3/4: grade 3
1/4: grade 4
4/4
Conclusions:
Based on the effects on the reduced thymus weight, the reduced thymus size and histopathological findings in the thymus at 10 mg/kg bw/day, the substance is classified as STOT-RE 1, H372, oral, thymus.
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
14 January 2004 to 8 March 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Please refer to analogue justification provided in IUCLID section 13
Reason / purpose for cross-reference:
read-across source
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
No further details specified in the study report.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The study was conducted with rats. The rat was used because this species is considered one of the most suitable species for this type of study, and is usually required by regulatory agencies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Characterization of the test system
For the dose-range finding study 22 male and 22 female, and for the main study 52 male and 52 female, Wistar outbred (Crl:(WI)WU BR) rats of about 9-10 weeks of age were obtained from a colony maintained under SPF conditions at Charles River Deutschland, Sulzfeld, Germany.
At the start of the treatment period the rats were about 10-11 weeks old and the weight variation of the animals used did not exceed 20% of the mean weight for each sex.

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

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

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

Feed and drinking water
Feed and water were provided ad libitum from the arrival of the rats until the end of the study.
Upon arrival, the rats were fed a commercial rodent diet (Rat&Mouse No. 3, Breeding Diet, RM3) obtained from SDS Special Diets Services, Witham, England.
Each batch of this diet was analysed by the supplier for nutrients and contaminants.
The feed was provided as a powder, in stainless steel cans, covered by a perforated stainless steel plate that served to prevent spillage. The feed in the feeders was refreshed at least once per week and topped up when necessary.
The test substance was incorporated in the basal diet by mixing in a mechanical blender.
The experimental diets were prepared once shortly before the start of the studies; they were stored in a freezer (<-18 °C) until use.
The drinking water (tap-water) was supplied in polypropylene bottles with a rubber stopper and a stainless steel nipple that were cleaned approximately weekly and filled up when necessary. Tap water suitable for human consumption (quality guidelines according to Dutch legislation based on EEC Council Directive 98/83/EEC) was supplied by N.V. Hydron Midden-Nederland. Results of the routine physical, chemical and microbiological examination of drinking water as conducted by the supplier are made available to TNO Nutrition and Food Research.
In addition, the supplier periodically (twice per year) analyses water samples taken on the premises of TNO Nutrition and Food Research in Zeist for a limited number of physical, chemical and microbiological variables.
Route of administration:
oral: feed
Details on route of administration:
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 study.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
The test substance was weighed in a tray of steel and moved to a small grinder. Thereafter the tray was rinsed with RM3 food which was also moved to the small grinder and subsequently mixed for 2 x 30 sec. This mixture was moved to the Stephan cutter and the small grinder was rinsed with RM3 food which was also moved to the Stephan cutter. The mixture was mixed with another approximately 3 kg weighed RM3 food in the Stephan cutter for 2 x 2 minutes and subsequently moved to the Lodige cutter. The Stephan cutter was rinsed with approximately 3 kg of RM3 food that was also moved to the Lodige cutter. Mixing was continued in the Lodige for 2 minutes with the total amount of RM3 food. The total amount of RM3 food used for all mixing and rinsing procedures was the same as mentioned under "RM3 food added".
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test substance analysis in the diet
Before the start of the study, the analytical method was validated in the matrix under examination (viz. RM3 diet).

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

Analyses
The homogeneous distribution, stability and achieved concentration of the test substance in RM3 rat feed was analysed in the batch of diets prepared for the dose range finding study. The same diet preparation protocol was used in the main study. The homogenous distribution and achieved concentration in RM3 rat feed of the low-dose group (5 mg/kg) was determined in the batch of diets prepared for the main study.
Directly after mixing of each diet for the dose range finding study, samples for the homogeneity/stability experiments were taken from the mixer. Firstly, five homogeneity samples (about 50 g each) were taken in the order: top centre, middle centre, bottom centre, left centre, right centre and labelled as such. Secondly, five samples (of about 50 g each) for examination of the stability were taken from the top centre part of the mixer. All samples were labelled with the diet-codes (TNO study number), the colour-codes, the nominal concentrations of the test substance and the date of preparation.
The samples taken for the homogeneity experiments were also used for dose confirmation.
In addition, analyses to determine the content (achieved concentration) of the test substance in the batch of diet used in the main study were conducted.
Diet samples for the determination of content of the diets used in this study were taken immediately after preparation of the diets and stored at ea. -18 °C pending analysis.

Criteria for homogeneity, stability and content of the test substance in the diet
Homogeneity:
For each group a one-way analysis of variance (Anova) was performed using the sample location (1-5) as grouping factor. An associated F-value with probability p <0.01 was considered to be significant (i.e. the mean concentrations differ significantly at the various locations in the sample). The test substance was considered to be homogeneously distributed in the diets if p ≥ 0.01 and/or if the relative standard deviation (RSD) between the sample means was less than or equal to 15 %.
Stability:
For each group a one-way analysis of variance (Anova) was performed using time as grouping factor. An associated F-value with probability p < 0.01 was considered to be significant (i.e. the difference between the results of the first day and the last day is significant). The test substance was considered to be stable in the diets if p ≥ 0.01 and/or if the mean concentration on the last day was between 80 % and 120 % of the mean concentration on the first day (t = 0).
Achieved concentration:
For each concentration level, the mean of the concentrations as measured in the study samples used for the assessment of the homogeneity were considered to represent the achieved concentration. The content of the test substance in diet was considered to be 'close to intended' if the mean measured concentration was between 80 % and 120 % of the intended concentration.
Duration of treatment / exposure:
Males 28 days
Females until PN 4 or 25 days after copulation if not pregnant
Frequency of treatment:
The animals were fed diets containing test substance from the start of the treatment period until sacrifice.
Dose / conc.:
0 mg/kg diet
Remarks:
Control group 0 Dose-range finding study & Main study
Dose / conc.:
5 mg/kg diet
Remarks:
Low-dose group - Main study
Dose / conc.:
25 mg/kg diet
Remarks:
Mid dose group - Main study
Low-dose group - Dose-range finding study
Dose / conc.:
250 mg/kg diet
Remarks:
High dose group - Main study
Dose / conc.:
75 mg/kg diet
Remarks:
Low-mid-dose group - Dose-range finding study
Dose / conc.:
200 mg/kg diet
Remarks:
High-mid-dose group - Dose-range finding study
Dose / conc.:
500 mg/kg diet
Remarks:
High-dose group - Dose-range findign study
No. of animals per sex per dose:
Dose-range finding study 4 animals per sex/dose
Main study - 12 anmals per sex/dose
Control animals:
yes, concurrent no treatment
Details on study design:
Dose-range finding study
The study comprised 5 groups of 4 male and 4 female rats each. The dose levels were selected by the sponsor and the test substance concentrations in the diet remained the same for each group during the study.
Main study:
The study comprised four groups of 12 male and 12 female rats each. The dose levels were selected in consultation with the sponsor and were based on the results of the dose-range finding study. The test substance concentrations in the diet remained the same for each group during the study.

Detailed clinical observations were performed in all animals prior to the first day of dosing and once weekly thereafter. Spontaneous motor activity measurements and Functional Observational Battery (FOB) were performed in 5 males/group prior to the end of dosing and in 5 females/group prior to the end of lactation (PN 4) randomly selected from each group.
At the end of the premating period, blood was collected by orbital puncture for haematology and clinical chemistry.
Positive control:
Not specified in the study report.
Observations and examinations performed and frequency:
Dose-range finding study
Clinical signs
Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity starting from the beginning of the study. On working days, all cages were checked again in the afternoon for dead or moribund animals to minimise loss of animals from the study. On Saturdays, Sundays and public holidays only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded.

Body weights
Body weights of male and female rats were recorded on day -2 (randomisation), 0 (first day of dosing), 3, 7, 10 and 14 (day of sacrifice).

Food consumption
Food consumption was measured over the following periods: days 0-3, 3-7, 7-10 and 10-14.

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

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

Body weights
Body weights of male and female rats were recorded on day -2 (randomization) and on days 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 (PN 1-4).

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

Clinical chemistry
Prior to the end of the premating period, 5 rats/sex/group (the 2 animals with the lowest identification numbers of the first and second cages of each group and the animal with the lowest identification number of the third cage of each group) were fasted overnight and blood was taken, whilst under CO2/O2 anaesthesia by orbital puncture.
Blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation.
The following measurements were made in the plasma collected:
fasting glucose; alkaline phosphatase activity (ALP); aspartate aminotransferase activity (ASAT); alanine aminotransferase activity (ALAT); gamma glutamyl transferase activity (GGT); total protein; albuminl ratio albumin to globulin; urea; creatinine; bilirubin (total); cholesterol (total); triglycerides; phospholipids; calcium (Ca); sodium (Na); potassium (K); chloride (Cl); inorganic phosphate.
Sacrifice and pathology:
Dose-range finding study
Gross necropsy of animals
All animals were subjected to a complete gross necropsy. The animals were euthanized by exsanguination under CO2/Oz-anaesthesia and then examined grossly for pathological changes.
At necropsy the following organs were weighed:
kidney; liver; spleen; testes; thymus; brain

Main Study
Gross necropsy and histology of parental animals
Female animal D181 of the high-dose group was found dead on GD 24; a gross necropsy was performed on this animals. All remaining male and female parent rats were euthanized by exsanguination from the abdominal aorta after CO2/O2 anaesthesia and examined grossly for pathological changes.
Samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes which were preserved in Bouin's fixative:
- ovanes
- uterus (after counting the implantation 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*; colon; duodenum; eyes; heart; ileum; jejunum; lungs; kidneys; liver; mammary gland (females only)*; mesenteric lymph node; parathyroids; Peyer' s patches; pituitary; rectum; sciatic nerve; spinal cord; spleen; stomach; thymus; thyroids; trachea; urinary bladder
* Tissues marked with an asterisk were preserved but not processed for histopathological examination.
The underlined organs were weighed (paired organs together) as soon as possible after dissection to avoid drying.
Tissues for microscopic examination were embedded in paraffin wax, sectioned at 5 μm, and stained with haematoxylin and eosin, except for sections of the testes which were stained with PAS haematoxylin. Microscopic examination was performed on the collected organs of all rats of the control and high-dose groups. After consultation with the sponsor, examination was extended to the liver and ovaria of the female rats and the thymus of the male and female rats of the low and mid-dose groups; because of the effect observed in the high-dose group.
Statistics:
Statistical procedures used in the evaluation of the data were as follows:
- 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.
- Red blood cell and coagulation variables, total white blood cell counts, absolute differential white blood cell counts, clinical chemistry values and organ weights: oneway 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 measurement scales (e.g., continuous, rank, categorical). Continuous measures were analyzed by one-way analysis of variance at each test time point, followed by posthoe group comparisons in case of a significant result. Rank order data were analyzed 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 analyzed by Pearson chi-square analysis.
- Motor activity data were analyzed using one-way analysis of variance at each test time point, followed by post-hoe 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. Effects of treatment on habituation were analysed using repeated measures analysis of variance on time blocks. Each session consisted of 5 time blocks of 6 minutes each.
Clinical signs:
no effects observed
Description (incidence and severity):
Dose-range finding study
No clinical signs were observed.

Main study
Male animal DI 76 of the high-dose group showed exophthalmus from week 2 and complete degeneration of the eye from week 3 onwards. This observation was first made after orbital punction on day 13 and was most probably caused by this action. No other clinical signs were observed in the males from the start of the study until sacrifice.
The only finding during the gestation period on GD 21, was a sparsely haired animal in the 250 mg/kg group. During the lactation period, sparsely haired animals were found in the control (n=l), 5 mg (n=l) and in the 250 mg (n=l) groups.
No other findings were observed in the female animals during the premating, gestation and lactation periods.
Mortality:
no mortality observed
Description (incidence):
Dose-range finding study
No mortalities were observed.

Main study
Female animal D181 of the high-dose (500 mg) group was found dead on GD 24; this animal was pregnant and at necropsy 11 dead fetuses were found in the uterus. No other mortalities were observed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Dose-range finding study
No treatment-related changes were observed in the mean body weight and body weight change of the male animals. Mean body weight of the female animals of the high-dose group (500 mg/kg diet) was statistically significantly decreased on days 3, 7 and 10 of the study. Mean body weight change of the female animals of the high-dose group was statistically significantly decreased from day 0-3. No other relevant differences in mean body weight or body weight change were observed between the control and the dioctyloxostannane-treated groups.

Main study
Mean body weight of the male animals of the dioctyloxostannane-treated groups was comparable to the control group. Body weight change of the male animals was statistically significantly decreased in the 250 mg (high-dose) group from days 13-21.
Mean body weight of the dams of the high-dose group was statistically significantly decreased on GD 21 and PN 1. Mean body weight change of the dams of the high-dose group was statistically significantly decreased from GD 14-21. Body weight and body weight change of the dams of all dioctyloxostannane-treated groups was comparable to the control group during all other periods of the premating, gestation and lactation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Dose-range finding study
Food consumption
Mean food consumption (expressed as g/kg body weight/day) was statistically significantly decreased in the males of the 200 and the 500 mg groups from days 0-7.
In the female animals, mean food consumption (g/animal/day and or g/kg body weight/day) was statistically significantly decreased in the 200 mg group from days 0-7 and 10-14 and in the 500 mg group during the entire administration period (expressed as g/animal/day) and from day 0-10 (expressed as g/kg body weight).
In all other cases food consumption was similar among the groups or the differences were considered to be not related to treatment.

Test substance intake
The test substance intake of the male animals of the low-, low-mid-, high-mid- and high-dose groups was respectively:
days 0-3: 1.7, 5.2, 11.9 and 28.1 mg/kg body weight/day, days 3-7: 1.8, 5.6, 13.3 and 30.7 mg/kg body weight/day, days 7-10: 1.7, 5.1, 12.5 and 31.5 mg/kg body weight/day, days 10-14: 1.6, 5.1, 12.8 and 31.5 mg/kg body weight/day.
The test substance intake of the female animals of the low-, low-mid-, high-mid and high-dose groups was respectively:
days 0-3: 1.7, 4.9, 11.1 and 21.8 mg/kg body weight/day, days 3-7: 1. 7, 5.0, 12.8 and 23. 7 mg/kg body weight/day, days 7-10: 1.7, 4.8, 12.5 and 22.9 mg/kg body weight/day, days 10-14: 1.7, 5.1, 12.4 and 32.8 mg/kg body weight/day.

Main study
Food consumption
Mean food consumption (g/kg/day) of the male animals of the 250 mg (high-dose) was statistically significantly decreased from day 7-13. No other treatment related effects were observed in the male animals.
During the gestation period (GD 7-14 and GD 14-21) and lactation period (PN 1-4) food consumption (expressed as g/animal/day and as g/kg body weight/day) of the dams of the 250 mg (high-dose) group was statistically significantly decreased. No other treatment related effects were observed during the premating, gestation and lactation periods.

Test substance intake
The test substance intake of the male animals of the low-, mid- and high-dose groups was respectively:
Premating period
days 0-7 0.4, 1.7 and 17.4 mg/kg body weight/day, days 7-13 0.3, 1.6 and 15.4 mg/kg body weight/day,
After mating
days 21-28 0.3, 1.5 and 14.5 mg/kg body weight/day.
The test substance intake of the female animals of the low-, mid-and high-dose groups was respectively:
Premating period
days 0-7: 0.3, I. 7 and 16.4 mg/kg body weight/day, days 7-13: 0.3, 1.6 and 16.0 mg/kg body weight/day,
Gestation period
GD 0-7: 0.4, 2.0 and 17.4 mg/kg.body weight/day, GD 7-14: 0.4, 1.9 and 16.7 mg/kg body weight/day, GD 14-21: 0.3, 1.4 and 11.2 mg/kg body weight/day,
Lactation period
PN 1-4: 0.5, 2.4 and 17.4 mg/kg body weight/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Main study
Red blood cell- and coagulation variables and mean total and differential white blood cell counts were comparable in all groups for male and female animals.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Main study
In the 250 mg group, the alkaline phosphatase (U/1) was statistically significantly increased in the male animals and bilirubin (μmol/1) in the female animals; these findings were considered to be a treatment related finding. The statistically significant increase in chloride in the male rats of the 25 mg (mid-dose) group and statistically significant decrease in calcium in the female rats of the 5 mg (low-dose) group were not considered to be related to dioctyloxostannane administration as these effects were not observed in the high-dose group and mid-and high-dose groups, respectively. All other parameters were comparable to the control group.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Main study
Treatment-related changes were not observed during the neurobehavioural testing of males and females at arena testing during the study and FOB and motor activity assessment at the end of the study. Therefore, no evidence was obtained for a neurotoxic potential of the test substance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Dose-range finding study
Terminal body weights of the male and female animals were comparable in all groups.
Absolute and relative thymus weight was statistically significantly decreased in the males of the 200 mg and 500 mg groups. Absolute and relative thymus weight was statistically significantly and dose-related decreased in the females of all dioctyloxostannane-treated groups. Relative liver weight of the female animals of the 500 mg group was statistically significantly increased; absolute weight of the liver of the females of this group was increased but the difference did not reach a level of statistical significance. No other statistically significant differences were observed in the organ weights (kidneys, liver, spleen, testes, thymus and brain) among the groups.

Main study
Male animals were sacrificed on day 28. In the 25 mg (mid-dose) and 250 mg (high-dose) groups, the absolute thymus weight of the male animals was statistically significantly decreased. Relative thymus weight of the male animals was statistically significantly decreased in the high-dose group.
Female animals with a litter were sacrificed on PN 4 or 5; non-pregnant animals were sacrificed on GD 25. The absolute and relative thymus weight of the female animals of the high-dose group was statistically significantly decreased. In the mid-dose group the relative thymus weight was also statistically significantly decreased.
In the female animals of the high-dose group, the relative kidney and liver weights were statistically significantly increased.
No other effects on organ weights (absolute and relative) were observed in the male and female animals.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Dose-range finding study
At necropsy, a small thymus was observed in all animals of the 200 mg and 500 mg groups. Two males of the 75 mg group showed unilateral hydronephrosis and one female of this group a flabby kidney. These latter observations are normal findings for this strain of animals.

Main study
Male animals were sacrificed on day 28. Female animals with a litter were sacrificed on PN 4 or 5; non-pregnant animals were sacrificed on GD 25.
At necropsy a decrease in thymic size was seen in all animals (both sexes) of the 250 mg/kg (high-dose) group, in 11 females of the 25 mg (mid-dose) group (the female which failed to get pregnant exhibited normal thymic size), in 7 females of the 5 mg/g (low-dose) group and in 5 females of the control group.
Examination of the pregnant female (D181 of the high-dose group) which was found dead on GD 24, revealed hydrothorax, haemorrhagic lungs, dilatation of the vena cava and haemorrhagic discharge in the vagina. These observations in late gestation are indicative of problems during parturition (dystocia) with congestion of the lungs as the ultimate cause of death.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Main study
Examination of the thymus revealed moderate to very severe lymphoid depletion in all animals (both sexes) of the 250 mg/kg (high-dose) group and in all females of the 25 mg/kg (mid-dose) group. Lymphoid depletion was characterized by a decrease in the size of the thymic lobules because of an extensive loss of cortical and medullary small lymphocytes. Consequently the distinction between the cortical and medullary areas was blurred. In the (very) severe cases the cortex was very small, or absent. The remaining lymphoid cells visible in the cortical areas were mainly lymphoblasts.
Lymphoblastic cells and reticuloepithelial cells had increased, and/or higher numbers of these cells were visible because of the disappearance of small lymphocytes and collapse of thymic stroma. In 3 high-dose females thymic lymphoid depletion was accompanied by lymphoid depletion in the PALS areas in the spleen. Lymphoid depletion of the PALS, i.e. the periarteriolar lymphocyte sheath, is a common feature associated with thymic atrophy because of its T-cell dependence. The macroscopically observed small thymi in 5 control and 7 low-dose (5 mg/kg) females revealed no abnormalities in 3 control and 5 low-dose females. In the thymi of 2 control and 2 low-dose females pregnancy/lactation involution was observed. The thymic lobules were decreased in size but exhibited normal architecture with the histological appearance of age-involution.
Increased glycogenic vacuolation, viz moderate versus (very) slight, was seen in the liver of 4 high-dose females and probably accounts for the statistically significant increase in relative liver weight.
Examination of the reproductive organs revealed a statistically significant increase in the incidence of cysts in the ovaries of 8 high-dose females.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Details on results:
Dose-range finding study
No clinical signs or mortalities were observed.
No treatment-related changes were observed in the mean body weight and body weight change of the male animals. Mean body weight of the female animals of the high-dose group (500 mg/kg diet) was statistically significantly decreased on days 3, 7 and 10 of the study. Mean body weight change of the female animals of the high-dose group was statistically significantly decreased from day 0-3. No other relevant differences in mean body weight or body weight change were observed between the control and the dioctyloxostannane-treated groups.
Mean food consumption (expressed as g/kg body weight/day) was statistically significantly decreased in the males of the 200 and the 500 mg groups from days 0-7.
In the female animals, mean food consumption (g/animal/day and or g/kg body weight/day) was statistically significantly decreased in the 200 mg group from days 0-7 and 10-14 and in the 500 mg group during the entire administration period (expressed as g/animal/day) and from day 0-10 (expressed as g/kg body weight).
In all other cases food consumption was similar among the groups or the differences were considered to be not related to treatment.
The test substance intake of the male animals ranged from 1.6-1.8, 5.1-5.6, 11.9-13.3 and 28.1-31.5 mg/kg body weight/day for the low-, low-mid-, high-mid and high-dose group, respectively.
The test substance intake of the female animals in the low-dose group was 1. 7 mg/kgbody weight/day and ranged from 4.8-5.1, 11.1-12.8 and 21.8-32.8 mg/kg body weight/day for the low-mid-, high-mid and high-dose group, respectively.
Terminal body weights of the male and female animals were comparable in all groups.
Absolute and relative thymus weight was statistically significantly decreased in the males of the 200 mg and 500 mg groups. Absolute and relative thymus weight was statistically significantly and dose-related decreased in the females of all dioctyloxostannane-treated groups. Relative liver weight of the female animals of the 500 mg group was statistically significantly increased; absolute weight of the liver of the females of this group was increased but the difference did not reach a level of statistical significance. No other statistically significant differences were observed in the organ weights (kidneys, liver, spleen, testes, thymus and brain) among the groups.
At necropsy, a small thymus was observed in all animals of the 200 mg and 500 mg groups. Two males of the 75 mg group showed unilateral hydronephrosis and one female of this group a flabby kidney. These latter observations are normal findings for this strain of animals.
It was concluded that treatment with dioctyloxostannane via the diet up to a level of 75 mg/kg diet (low-mid-dose group) for a period of 14 days had no effect on body weight and food intake in male and female rats. In male animals, thymus weight (absolute and relative) was affected from 200 mg/kg diet (high-mid-dose group) onwards and in female animals, thymus weight (absolute and relative) was affected in all dioctyloxostannane-treated groups.
In consultation with the sponsor the following dose levels were chosen for the main study: 0, 5, 25 and 250 mg/kg diet.

Main study
One female animal of the 250 mg/kg group (high-dose) was found dead on GD 24.
Clinical signs related to the treatment with dioctyloxostannae were not observed.
In male animals tested after 4 weeks of treatment and in females animals tested on PN 4, no changes indicative of neurotoxic potential of the test substance were observed in the neurobehavioural observations and motor activity assessment.
Mean body weight of the male animals of the dioctyloxostannane-treated groups was comparable to the control group. Body weight change of the male animals was statistically significantly decreased in the 250 mg (high-dose) group
Mean body weight of the dams of the high-dose group was statistically significantly decreased on GD 21 and PN 1. Mean body weight change of the dams of the high-dose group was statistically significantly decreased from GD 14-21. Body weight and body weight change of the dams of all dioctyloxostannane-treated groups was comparable to the control group during all other periods of the premating, gestation and lactation.
Administration of 250 mg dioctyloxostannane per the diet resulted in a decreased food consumption in male animals from day 7-13 and in female animals during the gestation period (GD 7-14 and GD 14-21) and lactation period (PN 1-4). No other treatment related effects were observed during the premating, gestation and lactation periods.
The test substance intake of the male animals during the study ranged from 0.3-0.4, 1.5-1.7 and 14.5-17.4 mg/kg body weight/day for the low-, mid- and high-dose group, respectively.
The test substance intake of the female animals during the premating, gestation and lactation period ranged from 0.3-0.5, 1.4-2.4 and 11.2-17.4 mg/kg body weight/day for the low-, mid- and high-dose group, respectively.
The statistically significant increase in alkaline phosphatase in the male animals and bilirubine in the female animals of the 250 mg group were the only treatment-related findings in clinical chemistry and haematology.
Male animals were sacrificed on day 28. In the 25 mg (mid-dose) and 250 mg (highdose) groups, the absolute thymus weight of the male animals was statistically significantly decreased. Relative thymus weight of the male animals was statistically significantly decreased in the high-dose group.
Female animals with a litter were sacrificed on PN 4 or 5. The absolute and relative thymus weight of the female animals of the high-dose group was statistically significantly decreased. In the mid-dose group the relative thymus weight was also statistically significantly decreased.
In the female animals of the high-dose group, the relative kidney and liver weights were statistically significantly increased.
Decreased thymus weights and macroscopically observed small thymi were accompanied by microscopic changes in the thymus of all animals (both sexes) of the 250 mg/kg (high-dose) group and in 11 females of the 25 mg/kg (mid-dose) group. The thymus showed overt lymphoid depletion, accounting for the decreased thymus weights. The microscopic appearance of the affected thymi resembled thymus atrophy described in the literature for organotin compounds in that there was no small lymphocyte necrosis, whereas lymphoblasts, reticuloepithelial cells and macrophages were more accentuated. Examination of the ovaries revealed a statistically significant increased incidence of ovarian cysts in 8 female animals of the high-dose group. At microscopic examination, glycogenic vacuolation in the liver of 4 female animals of the high-dose group was consistent with the increased relative liver weights.
In conclusion, based on the observed effects in the animals of the 25 mg/kg (mid-dose) group, decrease in thymus weight (male and female animals) and macroscopic and microscopic findings in the thymus (female animals), the NOAEL for general toxicity is established on the low-dose level (5 mg/kg diet which is equivalent to 0.3-0.4 mg/kg body weight/day for the male animals and to 0.3-0.5 mg/kg body weight/day for the female animals).
Key result
Dose descriptor:
NOAEL
Effect level:
> 0.3 - < 0.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
5 mg/kg diet
System:
immune system
Organ:
thymus
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified

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

Parameter*

Dose levels

5 mg/kg

25 mg/kg

250 mg/kg

Main study

 

 

 

Body weight: GD 21, PN 1 (females)

 

 

Body weight change: GD 14-21 (females)

 

 

Food consumption:

PM 7-13 (males)

GD 7-14, 14-21 and PN 1-4 (females)

 

 

Bilirubin (females)

 

 

Alkaline phosphatase (males)

 

 

Relative liver weight (females)

 

 

Relative kidney weight (females)

 

 

Absolute and/or relative thymus weight

 

Thymus: lymphoid depletion (males)

 

 

Thymus: lymphoid depletion (females)

 

Ovary: cysts (females)

 

 

Liver: glycogenic vacuolation (females)

 

 

*in case finding is only observed in one sex, sex is mentioned

↓ decreased; ↑ increased

 

SUMMARY OF CLINICAL OBSERVATIONS DURING PREMATING (Main Study)          MALES

 

Group#

Week of Study

0

1

2

3

4

TOTAL

# of rats with clin. Obs.

 

 

 

 

 

 

 

 

Control

5 mg/kg

25 mg/kg

250 mg/kg

A

B

C

D

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

1

0

0

0

1

 

EYES

 

 

 

 

 

 

 

EYES: exophthalums

 

 

 

 

 

 

 

 

Control

5 mg/kg

25 mg/kg

250 mg/kg

A

B

C

D

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

1

0

0

0

0

0

0

0

1

EYES: complete degeneration

 

 

 

 

 

 

 

 

Control

5 mg/kg

25 mg/kg

250 mg/kg

A

B

C

D

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

1

0

0

0

1

SUMMARY OF CLINICAL OBSERVATIONS DURING PREMATING (Main Study)          FEMALES

 

Group#

Week of Study

0

1

2

# of rats with clin. Obs.

 

 

 

 

 

Control

5 mg/kg

25 mg/kg

250 mg/kg

A

B

C

D

0

0

0

0

0

0

0

0

0

0

0

0

Statistical key: Fishers exact test * p<0.05 ** p<0.01 # p<0.001

 

SUMMARY OF MATERNAL CLINICAL OBSERVATIONS DURING GESTATION (Main Study)                        FEMALES

 

 

Group#

Day of gestation

 

 

 

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

TOTAL

# of rats with clin. Obs.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Control

5 mg/kg

25 mg/kg

250 mg/kg

A

B

C

D

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

 

SKIN/FUR

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Skin: sparsely haired

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Control

5 mg/kg

25 mg/kg

250 mg/kg

A

B

C

D

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

1

Statistical key: Fishersexact test * p<0.05 ** p<0.01 # p<0.001

 

SUMMARY OF MATERNAL CLINICAL OBSERVATIONS DURING LACTATION(Main Study)            FEMALES

 

Group#

Day of Lactation

0

1

2

3

4

TOTAL

# of rats with clin. Obs.

 

 

 

 

 

 

 

 

Control

5 mg/kg

25 mg/kg

250 mg/kg

A

B

C

D

1

0

0

1

1

1

0

1

1

1

0

1

1

1

0

1

1

1

0

1

 

SKIN/FUR

 

 

 

 

 

 

 

SKIN: sparsely haired

 

 

 

 

 

 

 

 

Control

5 mg/kg

25 mg/kg

250 mg/kg

A

B

C

D

1

0

0

1

1

1

0

1

1

1

0

1

1

1

0

1

1

1

0

1

1

1

0

1

Statistical key: Fishers exact test * p<0.05 ** p<0.01 # p<0.001

 

MEAN BODY WEIGHTS DURING PREMATING (g) (Main Study)             MALES

 

 

A

Control

B

5 mg/kg

C

25 mg/kg

D

250 mg/kg

DAY 0

Mean

S.E.

N

262.67 d

3.136

12

264.68

2.201

12

266.57

1.666

12

266.75

3.154

12

DAY 7

MEAN

S.E.

N

295.05 d

4.505

12

298.16

3.319

12

295.83

3.046

12

296.58

4.659

12

DAY 13

MEAN

S.E.

N

319.67 d

4.956

12

320.05

4.274

12

321.77

3.842

12

317.45

5.587

12

DAY 21

MEAN

S.E.

N

333.83 d

4.473

12

336.23

4.662

12

329.81

5.067

12

321.86

7.309

12

DAY 28

MEAN

S.E.

N

354.93 d

5.537

12

354.14

4.805

12

352.91

5.772

12

341.32

7.498

12

MEAN BODY WEIGHTS DURING PREMATING (g) (Main Study)             FEMALES

 

 

A

Control

B

5 mg/kg

C

25 mg/kg

D

250 mg/kg

DAY 0

Mean

S.E.

N

179.32 d

1.675

12

177.97

1.316

12

179.14

1.367

12

182.68

2.449

12

DAY 7

MEAN

S.E.

N

186.80 d

2.246

12

187.76

1.146

12

187.72

1.873

12

191.07

2.353

12

DAY 13

MEAN

S.E.

N

193.04 d

2.453

12

193.66

1.256

12

192.42

2.169

12

196.67

2.309

12

Statistical key: d = ANOVA & Dunnett test

 

MEAN BODY WEIGHTS CHANGE DURING PREMATING (g) (Main Study)                    MALES

 

 

A

Control

B

5 mg/kg

C

25 mg/kg

D

250 mg/kg

DAY 0 TO 7

Mean

S.E.

N

32.35 d

2.981

12

33.47

1.919

12

29.26

1.701

12

29.82

2.895

12

DAY 7 TO 13

MEAN

S.E.

N

24.64 d

1.434

12

21.89

1.874

12

25.94

1.389

12

20.88

1.310

12

DAY 13 TO 21

MEAN

S.E.

N

14.17 d

1.662

12

16.18

1.473

12

8.04

2.256

12

4.41*

3.685

12

DAY 21 TO 28

MEAN

S.E.

N

21.09 d

1.353

12

17.92

0.943

12

23.10

2.113

12

19.46

1.829

12

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

MEAN BODY WEIGHTS CHANGE DURING PREMATING (g) (Main Study)                    FEMALES

 

 

A

Control

B

5 mg/kg

C

25 mg/kg

D

250 mg/kg

DAY 0 TO 7

Mean

S.E.

N

7.48 d

1.347

12

9.79

1.307

12

8.58

1.207

12

8.38

1.236

12

DAY 7 TO 13

MEAN

S.E.

N

6.24 d

0.901

12

5.90

0.941

12

4.69

1.249

12

5.60

0.953

12

Statistical key: d = ANOVA & Dunnett test

 

MEAN MATERNAL BODY WEIGHTS DURING GESTATION (g) (Main Study)                 FEMALES

 

 

A

Control

B

5 mg/kg

C

25 mg/kg

D

250 mg/kg

DAY 0

Mean

S.E.

N

188.71 d

1.918

11

193.21

1.759

12

193.75

2.888

11

189.60

2.899

9

DAY 7

MEAN

S.E.

N

210.08 d

2.532

11

214.25

1.755

12

212.87

2.386

11

207.14

2.223

9

DAY 14

MEAN

S.E.

N

230.88 d

2.955

11

238.98

2.001

12

234.72

2.298

11

225.63

3.165

9

DAY 21

MEAN

S.E.

N

263.50 d

4.160

10

264.63

3.041

12

263.77

4.101

11

241.03**

5.610

9

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

 

MEAN MATERNAL BODY WEIGHT CHANGE DURING GESTATION (g) (Main Study)               FEMALES

 

 

A

Control

B

5 mg/kg

C

25 mg/kg

D

250 mg/kg

DAY 0 TO 7

Mean

S.E.

N

21.37 d

1.354

11

21.04

0.920

12

19.12

0.993

11

17.54

1.613

9

DAY 7 TO 14

MEAN

S.E.

N

20.80 d

1.253

11

24.73

1.375

12

21.85

0.871

11

18.49

1.237

9

DAY 14 TO 21

MEAN

S.E.

N

32.14 d

2.608

10

25.64

2.040

12

29.05

2.558

11

15.40**

4.382

9

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

 

MEAN MATERNAL BODY WEIGHTS DURING LACTATION (g) (Main Study)                 FEMALES

 

 

A

Control

B

5 mg/kg

C

25 mg/kg

D

250 mg/kg

DAY 1

Mean

S.E.

N

195.55 d

3.409

11

198.92

3.470

12

192.22

4.109

11

177.27**

3.773

9

DAY 4

MEAN

S.E.

N

209.97 d

3.623

11

214.49

3.965

12

212.79

3.925

11

199.37

4.465

9

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

 

MEAN MATERNAL BODY WEIGHT CHANGE DURING LACTATION (g) (Main Study)               FEMALES

 

 

A

Control

B

5 mg/kg

C

25 mg/kg

D

250 mg/kg

DAYS 1 TO 4

Mean

S.E.

N

14.43 d

3.870

11

15.57

2.043

12

20.57

2.020

11

22.10

2.998

9

Statistical key: d = ANOVA & Dunnett test

 

MEAN ORGAN WEIGHTS ABSOLUTE (g) (Main Study)                MALES

 

 

A

Control

B

5 mg/kg

C

25 mg/kg

D

250 mg/kg

TERMINAL BODY WEIGHT (g)

MEAN

S.E.

N

354.4 d

5.75

12

354.2

4.63

12

353.4

5.89

12

341.1

7.39

12

TESTES

MEAN

S.E.

N

3.093 d

0.0303

12

3.222

0.0728

12

3.038

0.1067

12

3.240

0.1694

12

EPIDIDYMIDES

MEAN

S.E.

N

1.167 d

0.0184

12

1.167

0.0208

12

1.160

0.0132

12

1.168

0.0402

12

ADRENALS

MEAN

S.E.

N

0.055 d

0.0023

5

0.058

0.0036

5

0.051

0.0029

5

0.055

0.0085

5

BRAIN

MEAN

S.E.

N

1.920 d

0.0255

5

1.909

0.0176

5

1.864

0.0345

5

1.859

0.0242

5

HEART

MEAN

S.E.

N

1.176 d

0.0452

5

1.207

0.0433

5

1.022

0.0405

5

1.064

0.0626

5

KIDNEYS

MEAN

S.E.

N

2.267 d

0.0553

5

2.130

0.0410

4

2.193

0.0511

5

2.125

0.0772

5

LIVER

MEAN

S.E.

N

14.363 d

0.4265

5

13.811

0.1806

5

12.761

0.8012

5

12.292

0.5417

5

SPLEEN

MEAN

S.E.

N

0.684 d

0.0341

5

0.712

0.0448

5

0.659

0.0291

5

0.629

0.0667

5

THYMUS

MEAN

S.E.

N

0.498 d

0.0411

5

0.545

0.0294

5

0.385*

0.0313

5

0.102#

0.0110

5

Statistical key: d – ANOVA & Dunnett test         * = p<0.05        # = p<0.001

 

MEAN ORGAN WEIGHTS ABSOLUTE (g) (Main Study)                FEMALES

 

 

A

Control

B

5 mg/kg

C

25 mg/kg

D

250 mg/kg

TERMINAL BODY WEIGHT (g)

MEAN

S.E.

N

209.7 d

3.63

11

214.7

3.78

12

212.3

3.81

11

199.1

4.54

9

ADRENALS

MEAN

S.E.

N

0.059 d

0.0025

5

0.060

0.0036

5

0.060

0.009

5

0.062

0.0032

5

BRAIN

MEAN

S.E.

N

1.724 d

0.0384

5

1.739

0.0266

5

1.735

0.0327

5

1.687

0.0490

5

HEART

MEAN

S.E.

N

0.701 d

0.0295

5

0.752

0.0421

5

0.687

0.0269

5

0.658

0.0259

5

KIDNEYS

MEAN

S.E.

N

1.354 d

0.0156

5

1.424

0.0649

5

1.342

0.0451

5

1.430

0.0497

5

LIVER

MEAN

S.E.

N

8.090 d

0.3437

5

7.789

0.4470

5

8.203

0.4267

5

9.124

0.4285

5

SPLEEN

MEAN

S.E.

N

0.394 d

0.0140

5

0.424

0.0078

5

0.384

0.0262

5

0.403

0.0194

5

THYMUS

MEAN

S.E.

N

0.124 d

0.0107

5

0.146

0.0237

5

0.082

0.0084

5

0.039**

0.0045

5

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

 

MEAN ORGAN WEIGHTS RELATIVE TO TERMINAL BODY WEIGHT (g.kg b.w.) (Main Study)           MALES

 

 

A

Control

B

5 mg/kg

C

25 mg/kg

D

250 mg/kg

TERMINAL BODY WEIGHT (g)

MEAN

S.E.

N

354.4 d

5.75

12

354.2

4.63

12

353.4

5.89

12

341.1

7.39

12

TESTES

MEAN

S.E.

N

8.747 d

0.1459

12

9.112

0.2264

12

8.606

0.2985

12

9.478

0.4438

12

EPIDIDYMIDES

MEAN

S.E.

N

3.299 d

0.0586

12

3.299

0.0639

12

3.289

0.0577

12

3.429

0.1130

12

ADRENALS

MEAN

S.E.

N

0.151 d

0.0048

5

0.158

0.0075

5

0.152

0.0092

5

0.165

0.0211

5

BRAIN

MEAN

S.E.

N

5.275 d

0.0887

5

5.241

0.1520

5

5.514

0.0948

5

5.706

0.2128

5

HEART

MEAN

S.E.

N

3.222 d

0.0532

5

3.302

0.0643

5

3.021

0.0901

5

3.242

0.0932

5

KIDNEYS

MEAN

S.E.

N

6.227 d

0.1646

5

5.883

0.0718

4

6.502

0.2528

5

6.491

0.0832

5

LIVER

MEAN

S.E.

N

39.446 d

0.3918

5

37.859

0.5298

5

37.547

1.0986

5

37.531

0.7574

5

SPLEEN

MEAN

S.E.

N

1.878 d

0.0853

5

1.950

0.1098

5

1.948

0.0748

5

1.906

0.1518

5

THYMUS

MEAN

S.E.

N

1.365 d

0.1068

5

1.491

0.0601

5

1.134

0.0779

5

0.313#

0.0357

5

Statistical key: d – ANOVA & Dunnett test         # = p<0.001

 

MEAN ORGAN WEIGHTS RELATIVE TO TERMINAL BODY WEIGHT (g. kg b.w.) (Main Study)           FEMALES

 

 

A

Control

B

5 mg/kg

C

25 mg/kg

D

250 mg/kg

TERMINAL BODY WEIGHT (g)

MEAN

S.E.

N

209.7 d

3.63

11

214.7

3.78

12

212.3

3.81

11

199.1

4.54

9

ADRENALS

MEAN

S.E.

N

0.275 d

0.0148

5

0.274

0.01705

0.280

0.0093

5

0.311

0.0258

5

BRAIN

MEAN

S.E.

N

8.009 d

0.2285

5

7.984

0.2088

5

8.025

0.1377

5

8.470

0.2967

5

HEART

MEAN

S.E.

N

3.255 d

0.1493

5

3.438

0.1271

5

3.174

0.0783

5

3.301

0.1219

5

KIDNEYS

MEAN

S.E.

N

6.295 d

0.1746

5

6.512

0.1497

5

6.203

0.1762

5

7.168**

0.1542

5

LIVER

MEAN

S.E.

N

37.537 d

1.4765

5

35.642

1.5792

5

37.903

1.7806

5

45.701**

1.6180

5

SPLEEN

MEAN

S.E.

N

1.838 d

0.1040

5

1.952

0.0844

5

1.768

0.0794

5

2.025

0.1033

5

THYMUS

MEAN

S.E.

N

0.585 d

0.0387

5

0.659

0.0905

5

0.376*

0.0323

5

0.200#

0.0279

5

Statistical key: d – ANOVA & Dunnett test         * = p<0.05        ** = p<0.01      # = p<0.001

 

SUMMARY OF MACROSCOPIC OBSERVATIONS

CHANGES

INCIDENCE OF LESIONS (NUMERIC)

Males

Females

TREATMENT

Contr.

5

mg/kg

25

mg/kg

250 mg/kg

Contr.

5

mg/kg

25

mg/kg

250 mg/kg

ADRENALS

Dark appearance and enlarged

 

 

 

 

 

 

 

 

1

AXILLARY LYMPH NODES

Uni-lateral red appearance

 

1

 

 

 

 

 

 

 

EPIDIDYMIDES

Hyalin tissue/small

 

 

 

 

1

 

 

 

 

KIDNEYS

Pale appearance

Uni-lateral hydronephrosis

 

 

 

1

 

 

 

 

 

 

1

LIVER

Dark margins(s)

 

 

 

 

 

 

 

 

1

LUNGS

Haemorrhagic content

 

 

 

 

 

 

 

 

1

OVARIES

Red nodule

Uni-lateral cyst(s)

 

 

 

 

 

 

1

 

 

 

2

SEMINAL VESICLES

Small and flabby

 

 

1

 

 

 

 

 

 

SKIN

Sparsely haired

 

 

 

 

 

 

1

 

 

STOMACH

Several petechia(e)

No folds

 

 

 

 

 

 

 

 

1

1

TESTES

Small and flabby

 

 

 

 

1

 

 

 

 

THORACIC CAVITY

Hydrothotax

Vena cava dilated

 

 

 

 

 

 

 

 

1

1

THYMUS

(Uni-lateral) red spot(s)/patches

Small

 

1

 

1

 

 

 

12

 

 

5

 

 

7

 

 

11

 

 

11

URETER

Uni-lateral dilated

 

 

1

 

 

 

 

 

 

UTERUS

Hydrometra

 

 

 

 

 

 

 

1

 

VAGINA

Haemorrhagic discharge

 

 

 

 

 

 

 

 

1

 

SUMMARY OF MICROSCOPIC OBSERVATIONS

CHANGES

INCIDENCE OF LESIONS (NUMERIC)

Males

Females

TREATMENT

Contr.

5

mg/kg

25

mg/kg

250 mg/kg

Contr.

5

mg/kg

25

mg/kg

250 mg/kg

ADRENALS

Multifocal haemorrhages(s)

Focal necrosis

Uni-lateral mononuclear cell infiltrate

(5)

0

0

0

 

 

(5)

0

0

1

(5)

0

0

0

 

 

(6)

1

1

0

AXILLARY LYMPH NODES

Microhaemorrhage(s)

(5)

1

 

 

(5)

0

(5)

0

 

 

(5)

0

BONE

No abnormality detected

(5)

5

 

 

(5)

5

(5)

5

 

 

(5)

5

BRAIN

No abnormality detected

(5)

5

 

 

(5)

5

(5)

5

 

 

(5)

5

CAECUM

No abnormality detected

(5)

5

 

 

(5)

5

(5)

5

 

 

(5)

5

COLON

No abnormality detected

(5)

5

 

 

(5)

5

(5)

5

 

 

(5)

5

EPIDIDYMIDES

Devoid of spermatozoa

Focal mononuclear cell infiltrate

Uni-lateral focal spermatocoele(s)

(12)

0

0

0

 

(1)

0

0

0

(12)

1

1

1

 

 

 

 

EYES

No abnormality detected

(5)

5

 

 

(5)

5

(5)

5

 

 

(5)

5

GALT (PEYER’S PATCHES)

No abnormality detected

(5)

5

 

 

(5)

5

(5)

5

 

 

(5)

5

HEART

Focal myocardial vaculation

Focal myocardial mononuclear cell infiltrate

(5)

0

3

 

 

(5)

0

5

(5)

1

0

 

 

(6)

0

0

KIDNEYS

(Uni-lateral) basophilic tubules

(Uni-lateral) focal mononuclear cell infiltrate

(Uni-lateral) mineralization

Uni-lateral focal transitional cell hyperplasia

Uni-lateral hydronephrosis

Uni-lateral focal tubular dilation

(5)

3

0

1

0

0

1

(1)

1

1

0

1

1

0

 

(5)

3

1

0

0

0

0

(5)

1

0

2

0

0

0

 

 

(5)

0

0

4

0

0

0

LIVER

Mononuclear cell aggregates/necrotic hepatocytes

Glycogenic vacuolation

           Very slight

           Slight

           Moderate

           Score Expanded Totals

Focal fibrosis

Focal polymorphonuclear leukocytic infiltration

(5)

2

 

0

0

0

0

0

0

 

 

(5)

0

 

0

0

0

0

0

0

(5)

1

 

2

1

0

3

1

0

(5)

0

 

2

1

0

3

0

1

(5)

0

 

1

1

0

2

0

0

(6)

0

 

0

1

4

5

0

0

LUNGS

Focal perivascular polymorphonuclear leukocyctic infiltration

Congestion

(5)

1

 

0

 

 

(5)

1

 

0

(5)

0

 

0

 

 

(6)

0

 

1

MESENTERIC LYMPH NODES

Erythrophagocytosis

(5)

0

 

 

(5)

0

(4)

0

 

 

(5)

1

NERVE-PERIPHERAL

No abnormality detected

(5)

5

 

 

(5)

5

(5)

5

 

 

(5)

5

OVARIES

Reduced number of corpora lutea

(Uni-lateral) cyst(s)

Interstitial-cell atrophy

 

 

 

 

(12)

1

2

1

(12)

0

6

0

(12)

0

5

0

(12)

0

8*

0

PARATHYROIDS

No abnormality detected

(3)

3

 

 

(5)

5

(5)

5

 

 

(5)

5

PITUITARY

Remnant(s) rathkes pouch

Pars distalis cyst(s)

(5)

0

1

 

 

(5)

0

0

(5)

0

0

 

 

(5)

1

0

PROSTATE

Focal prostatitis

Focal mononuclear cell infiltrate

(12)

2

0

 

(1)

0

0

(12)

0

1

 

 

 

 

RECTUM

No abnormality detected

(5)

5

 

 

(5)

5

(5)

5

 

 

(5)

5

SEMINAL VESICLES

No abnormality detected

(12)

12

(1)

1

(1)

1

(12)

12

 

 

 

 

SMALL INTESTINES

No abnormality detected

(5)

5

 

 

(5)

5

(5)

5

 

 

(5)

5

SPINAL CORD

No abnormality detected

(5)

5

 

 

(5)

5

(5)

5

 

 

(5)

5

SPLEEN

PALS lymphoid depletion

Increased extramedullary haematopoiesis

(5)

0

0

 

 

(5)

0

0

(5)

0

0

 

 

(5)

3

3

STOMACH

Focal glandular stomach mucosal pigment deposit(s)

Focal hyperkeratosis

Erosion(s)/ulceration

(5)

0

0

0

 

 

(5)

1

0

0

(5)

0

1

0

 

 

(6)

0

0

1

TESTES

(Uni-lateral) seminiferous tubular atrophy

(12)

0

 

(1)

0

(12)

2

 

 

 

 

THYMUS

Microhaemorrhage(s)

Ductular structure

Lymphoid depletion

           Moderate

           Severe

           Very severe

           Score Expanded Totals

Involution

(5)

4

2

 

0

0

0

0

0

(5)

1

0

 

0

0

0

0

0

(5)

2

0

 

0

0

0

0

0

(12)

4

0

 

1

5

6

12***

0

(8)

4

6

 

0

0

0

0

2

(10)

4

4

 

0

0

0

0

2

(11)

4

7

 

0

9

2

11***

0

(11)

0

1

 

0

2

9

11***

0

THYROID

No abnormality detected

(5)

5

 

 

(5)

5

(5)

5

 

 

(5)

5

TRACHEA/BRONCHI

No abnormality detected

(5)

5

 

 

(5)

5

(5)

5

 

 

(5)

5

URETER

No abnormality detected

 

(1)

1

 

 

 

 

 

 

URINARY BLADDER

No abnormality detected

(5)

5

 

 

(5)

5

(5)

5

 

 

(5)

5

UTERUS

Squamous metaplasia

Endometrial glandular dilation

(Uni-lateral) luminal dilation

 

 

 

 

(12)

1

0

0

 

(1)

0

0

1

(12)

0

1

2

VAGINA

Cystic dilation

Luminal debris

 

 

 

 

 

 

 

(2)

1

1

Statistics: 2-sided Fisher’s exact test between the controls & each of the treatment groups. * p<0.05, ** p<0.01, *** p<0.001

Figures in brackets represent the number of animals from which this tissue was examined microscopically.

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

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

 

Analyses of the diets

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

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

 

Dose-range finding study

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

 

Main study

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

No other mortalities or treatment related clinical signs were observed.

 

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

 

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

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

 

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

 

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

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

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

 

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

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

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

 

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

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

 

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

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
0.3 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study from a reference substance which is a hydrolysis product of the test substance. Read-across is justified based on rapid hydrolysation of the test substance in the stomach under acidic conditions (refer to analogue justification for further details). The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII/IX, 8.6, in accordance with Annex XI, 1.5, of Regulation (EC) No. 1907/2006.
System:
immune system
Organ:
thymus

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Subacute toxicity

The test substance was tested in a repeated dose oral toxicity study according to OECD guideline 407 and in compliance with GLP (Key, 2012). Five Wistar rats per sex and dose were treated via gavage with test substance at concentrations of 10, 30 and 100 mg/kg bw/day, respectively. The selected dose levels were based on the results of a range-finding study, in which adverse effects were observed in rats administered 200, 500 and 1,000 mg/kg bw/day for up to 10 days. The control group received the vehicle corn oil.

One female in the high-dose group was sacrificed in extremis on Day 18. No cause of death could be established based on histopathological assessment. As other animals of this dose group showed no toxicologically relevant clinical signs, this death was considered to be incidental in nature and unrelated to treatment with the test substance. In the high dose group salivation was observed in 5/5 males and 2/5 females after dosing. This was considered to be a physiological response rather than a sign of systemic toxicity as it was observed after dosing. Further clinical signs as alopecia and scabs and chromodacryhorrea were considered to incidental findings. In the high dose group in week 2, a statistically significant lower body weight gain of males (23%) compared with the control group (33%) was observed. This effect was also noted in week 3, when the animals of the high-dose group showed 50% body weight gain which was significantly lower than that of the control groups (67%). The body weight gain of the high-dose group (100%) in week 4 was not statistically significant compared with the control group (119%) due to the high standard deviation. However, this effect was considered to be of toxicological relevance. No treatment-related findings were noted in functional observations and food consumption. The gross necropsy revealed a significant reduction in absolute and relative thymus weight at all dose levels (along with reduced size of the thymus in several animals at 10 and 30 mg/kg bw/day and all surviving animals at 100 mg/kg bw/day). Mean relative thymus weight was reduced up to 40%, 65% and 81% at 10, 30 and 100 mg/kg bw/day, respectively. At 30 and 100 mg/kg bw/day, these findings were supported histopathologically by lymphoid atrophy (and lymphocytolysis at 30 mg/kg bw/day) and hyperplasia of undifferentiated cells (both moderate to marked), and loss of cortical and medullary differentiation in the thymus of all surviving animals. At 10 mg/kg bw/day, histopathological support in the thymus was limited to lymphoid atrophy in one male and female (minimal-slight degree) with loss of cortical and medullary differentiation in the same male, and lymphocytolysis in four males (minimal to moderate). Females at 100 mg/kg bw/day also showed a significant higher liver weight (16.3%) and liver to body weight ratio (3.43% vs. 2.95% in controls) and significant higher bile acid level (142%) and alkaline phosphatase activity (83.6%). Since histopathologically there were no treatment-related liver lesions, these changes were considered not to be adverse in nature. In conclusion, at the lowest dose of 10 mg/kg bw/day, the magnitude of thymus weight reduction of 40% along with loss of cortical and medullary differentiation in one male were considered to be adverse in nature. Furthermore, a dose-related effect on the thymus weight and severity of histopathological findings was noted at all dose levels. Therefore, a No Observed Adverse Effect Level (NOAEL) for the test substance could not be established. The LOAEL systemic is 10 mg/kg bw/day for male and female rats.

Subchronic toxicity:

No data is available on the subchronic toxicity of the test substance. Reliable data is available for the hydrolysis product dioctyltin oxide (CAS 870 -08 -6). Dioctyltin oxide (DOTO) was tested in a repeated dose oral toxicity study according to OECD Guideline 422 and in compliance with GLP (key, 2004).

The study was combined with a repeated dose toxicity study and preceded by a dose-range finding study. In the dose-range finding study, rats were fed diets containing 0, 25, 75, 200 and 500 mg dioctyloxostannane/kg diet for 14 days. In the main study, ratswere fed diets containing 0, 5, 25 and 250 mg dioctyloxostannane/kg diet for up to 28 days (males) or during 2 weeks premating, mating, gestation and up to day 4 or 5 of lactation (females).

 

Analyses of the diets

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

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

 

Dose-range finding study

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

 

Main study

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

No other mortalities or treatment related clinical signs were observed.

 

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

 

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

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

 

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

 

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

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

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

 

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

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

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

 

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

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

 

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

Justification for classification or non-classification

The available data on oral repeated dose toxicity with the test substance and the hydrolysis product meet the criteria for classification according to Regulation (EC) No 1272/2008 and therefore the test substance will be classified as STOT RE Cat. 1 (H372) with the thymus as the target organ.