Silicic acid (H4SiO4), tetraethyl ester, reaction products with bis(acetyloxy)dioctylstannane

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Oct - 12 Dec 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted Oct 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

adopted Oct 2008

Deviations:

not specified

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

Silicic acid (H4SiO4), tetraethyl ester, reaction products with bis(acetyloxy)dioctylstannane

EC Number:

300-346-5

EC Name:

Silicic acid (H4SiO4), tetraethyl ester, reaction products with bis(acetyloxy)dioctylstannane

Cas Number:

93925-43-0

Molecular formula:

C4H8O2, C20H44O4SiSn, C24H52O6SiSn, C40H84O8SiSn2, C60H128O12Si2Sn3, C80H172O16Si3Sn4, C100H216O20Si4Sn5

IUPAC Name:

Silicic acid (H4SiO4), tetraethyl ester, reaction products with bis(acetyloxy)dioctylstannane

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day (nominal)

Doses / concentrations 3

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.

Examinations

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.

Results and discussion

Results of examinations

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

Effect levels

1

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

2

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

Target system / organ toxicity

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.