2-Propenoic acid, 2-methyl-, tris(1-methylethyl)silyl ester

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 March 2010 to 09 May 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Recent study conducted by a GLP certified laboratory in accordance with an OECD guideline

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tripropan-2-ylsilyl 2-methylprop-2-enoate

EC Number:

700-182-8

Cas Number:

134652-60-1

Molecular formula:

C13 H26 O2 Si

IUPAC Name:

Tripropan-2-ylsilyl 2-methylprop-2-enoate

Details on test material:

Name: TIS-M
Lot No.: 9925
CAS Number: 134652-60-1
Appearance: Colorless to light yellow liquid
Storage condition: Room temperature (15-25oC), under nitrogen, in the dark
Manufacture date: 23 September 2009
Expiry Date: 02 December 2010

Test animals

Species:

rat

Strain:

other: CRL:(WI) BR Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and strain: CRL:(WI) BR Wistar rats
Source: TOXI COOP Ltd., 1103 Budapest, Cserkesz u. 90, Hungary
Hygienic level: SPF at arrival; standard laboratory conditions during the study
Justification of species/strain: The rat is regarded as suitable for reproduction studies and the test guideline is designed to use the rat. Wistar rat was selected due to experience with this strain in reproduction toxicity studies and known fertility.
Number of animals: 48 male and 48 female rats were used on the study, 12 male and 12 female rats/group, 4 groups; the spare animals were assigned to LAB Research Ltd. spare colony, as their use was not required (at least 8 pregnant females/group were achieved)
Age of animals: Young adult rats, 10-11 weeks at the start of treatment and 12-13 weeks at mating, as practical
Body weight: 368-429 g (males) and 228-280 g (females) at onset of treatment; did not exceed ± 20% of the mean weight for each sex per group at onset of treatment
Acclimation period: 20 days

Husbandry
Animal health: Only healthy animals were used, as certified by the veterinarian. Females were nulliparous and non-pregnant.
Room number: 523
Cage type: Type II and III polypropylene/polycarbonate
Bedding: Lignocel® Hygienic Animal Bedding produced by J.Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany).
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20 - 22.9°C
Relative humidity: 30 - 77 %
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Rodents were group-housed up to 5 animals of the same sex and dose group per cage, with the exception of the mating and gestation/delivery period, when they were paired or individually housed, respectively. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting).
The temperature and relative humidity values were checked and recorded twice daily during the study.

Food and water supply
Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum, and tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum. Water quality control analysis is performed once every three months and microbiological assessment is performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary).
The food and water were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Animal identification
Each parental animal (P Generation) was identified by a number unique within the study, written with indelible ink on the tail and cross-referenced to the Animal Master File at LAB Research Ltd. The animal number consisted of 4 digits, the first digit being the group number, the second, 0 for the males, and 5 for the females, and the last 2, the animal number within the group, as indicated in the Experimental design section. The boxes were marked by identity cards, with information about study code, sex, dose group, cage number and individual animal numbers, date of mating and delivery. Boxes were arranged in such a way that possible effects due to cage placement were minimized.

Randomization
All parental (P) male and female animals were sorted according to body weight by computer and divided to weight ranges. There were an equal number of animals from each weight group in each of the experimental groups assigned by randomisation to ensure that animals of all test groups were as nearly as practicable of a uniform weight. The grouping was controlled by SPSS/PC software, according to the actual body weight to ensure homogeneity/variability between/within the groups and cages.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Sunflower oil (dried)

Details on oral exposure:

The test item was formulated in the vehicle at 25, 125 and 500 mg/mL concentrations, in order to achieve the dose levels of 50, 250 and 1000 mg/kg bw/day, at a dose volume of 2 mL/kg, selected based on the preliminary dose range finding study LAB code 09/285-220PE and the appropriate dose volume for use in Wistar rats with this vehicle. Dose formulation preparation was conducted in the Central Dispensary of LAB Research Ltd. Formulations were prepared at the appropriate frequency to allow their use within 5 days when stored refrigerated (5±3ºC), or within 24 hours when stored at room temperature, according to stability assessment results.

Rationale for dose-selection and route of administration
The dose levels were selected by the Sponsor with the Study Director based on a preliminary dose range finding study performed at LAB Research Ltd. (09/285-220PE). The oral route is a possible route of exposure to the test item in humans.

Dosing procedure
The dosing formulations were administered daily by oral gavage on a 7 days/week basis, using a tipped gavage needle attached to a syringe. A constant volume of 2 mL/kg was administered to all animals. The actual volume administered was calculated and adjusted based on each animal’s most recent body weight. Animals were not treated on the day of scheduled necropsy. Dosing of both sexes began after 20 days of acclimatisation (A), after the animals have attained full sexual maturity and 2 weeks before mating, and continued during the mating period and up to and including the day before the necropsy.

Vehicle
Name: Helianthi Annum Oleum Raffinatum, Ph. Hg. VIII (dried)
Alternative name: Sunflower oil (dried)
Lot No.: 19T/4/16.09.09
Manufacturer: Hungaropharma Zrt.
Expiry Date: 22 September 2010
Storage: Room temperature, protected from direct sunlight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of formulations (concentration and homogeneity) was performed in the Analytical Laboratory of LAB Research Ltd. Top, middle and bottom samples were taken in duplicate for analysis of concentration and homogeneity from each of the test-item dosing solutions (Groups 2, 3, and 4) on 3 occasions, during the first and last weeks of treatment for both males and females (17 March, 12 and 29 April 2010). Similarly, one sample was taken in duplicate on each occasion from the Group 1 (control) solution, for concentration measurements. The samples were diluted in two or three consecutive steps using tetrahydrofuran in the first and using acetonitrile in the next steps and then analysed by HPLC using UV detection.
Dose formulations were homogenous. No TIS-M was detected in the Control samples. In the test item dose formulations, the measured concentrations ranged from 95 to 108% of nominal concentrations (25, 125 and 500 mg/mL).

Duration of treatment / exposure:

Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period), then they were euthanized and subjected to necropsy examination. Females were dosed for 14 days pre-mating, for up to 14 days mating period, through gestation and up to and including the day before necropsy (at least 4 days post-partum dosing). The day of birth (viz. when parturition is complete) was defined as Day 0 postpartum. Females showing no-evidence of copulation were sacrificed as practical, up to 27 days after the last day of the mating period.

Frequency of treatment:

7 days/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
50 mg/kg bw/day
Basis:
other: nominal in sunflower oil (dried)

Doses / concentrations 2

Remarks:

Doses / Concentrations:
250 mg/kg bw/day
Basis:
other: nominal in sunflower oil (dried)

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
other: nominal in sunflower oil (dried)

No. of animals per sex per dose:

12 male + 12 female per dose

Control animals:

yes, concurrent vehicle

Details on study design:

See below under any other information for details of experimental design.

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS AND FUNCTIONAL OBSERVATION BATTERY (FOB)

All animals:
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day). Animals that showed severe clinical signs (female 2506 and male 4010) were isolated and pre-terminally sacrificed to prevent suffering, cannibalism or autolysis. All premature decedents were processed in the same way as the animals of the terminal necropsy. General clinical observations were made once a day, after treatment as practical. Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality were recorded including onset, degree and duration of signs as applicable. More detailed examinations were made once before the first exposure (to allow for within-subject comparisons), and at least once a week thereafter. These observations were made outside the home cage in a standard arena approximately at similar times. Signs evaluated included, but were not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also monitored. Special attention was directed to detect any tremors, convulsions, salivation, diarrhoea, lethargy, sleep or coma.

Five male and 5 female rats/group (“subgroup A”):

Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity was conducted on five randomly selected males and females. The assessment was performed during the last exposure week (males, on Day 27 am, females, on PND 4 am). General physical condition and behaviour of animals were tested. A modified Irwin test was performed. In order to avoid hyperthermia of pups, dams were removed from the pups for not more than approximately 30-40 minutes.

BODY WEIGHT MEASUREMENT

All adult animals were weighed with accuracy of 1 g for randomization purposes, then on Day 0, afterwards at least weekly and at termination. Parent females were weighed on gestation Days GD0, 7, 14 and 20 and on post-partal Days PPD0 (within 24 hours after parturition) and 4 (before termination). Body weights of the female animals were additionally measured on gestational Days GD10 and 17 in order to give accurate treatment volumes.

FOOD CONSUMPTION MEASUREMENT

Animal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Day 7 then at least weekly, as detailed in the Study Schedule.

OPHTHALMOLOGY

The fundus of eyes of all animals was examined before treatment, then in 5 male and 5 female Control and high-dose animals (“subgroup C”) randomly selected from groups 1 and 4, during the last week of treatment prior to necropsy (males, Day 27 pm, females, PND 4 pm). Mydriasis was produced after instillation of a mydriatic agent (eye drops "Mydrum") into the conjunctival sac. The examination was performed using a Gowlland ophthalmoscope. As no treatment-related alterations were found, no additional evaluation was carried out.

CLINICAL PATHOLOGY
Laboratory examinations for haematology and clinical chemistry evaluation were conducted at the end of pre-mating period, on blood samples collected from the sublingual vein, prior to the start of mating on Day 14 from 5 animals/sex/group randomly selected (“subgroup B”). Coagulation evaluation (APTT and PT) was performed at the completion of the treatment, on blood samples collected by cardiac puncture from subgroup B animals under pentobarbital anaesthesia, immediately prior to scheduled necropsy. After an overnight period of food deprivation of animals, 3 samples were taken from each animal: one for haematology (1.2 mL blood; tube contains K3-EDTA as anticoagulant, 1.6 mg/mL blood), one for blood clotting times (1.4 mL blood; APTT and PT; tube contains sodium citrate as anticoagulant) and one to obtain serum (approximately 1 mL blood as practical; tube contains no anticoagulant) for clinical chemistry. Urine sampling (approximately 16 hours sampling period) and urinalysis were performed prior to necropsy from the same animals in subgroup B (males, Day 28, females, PND 5).

Haematology and blood clotting times

The parameters evaluated are detailed below in a table under any other information on materials and methods.

Clinical chemistry

The parameters evaluated are detailed below in a table under any other information materials and methods.

Urinalysis

Urine samples were collected over a period of approximately 16 hours from each subgroup B animal (males, start on Day 27, urine collection and analysis on Day 28; females, start on PND 4, urine collection and analysis on PND 5). The animals were food and water deprived during urine collection for approximately 16 hours, then water was provided at libitum for at least approximately 2 hours prior to necropsy and organ weight measurements.The parameters evaluated are detailed below in a table under any other information on materials and methods.

Sacrifice and pathology:

PATHOLOGY
Gross necropsy was performed on each animal irrespective of the date of death. Terminally (one day after the last treatment), animals were sacrificed under pentobarbital anaesthesia by exsanguination. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded. At the time of termination, body weight and weight of the following organs of all parental animals was determined:
- With a precision of 0.01 g: uterus (with and without cervix), vagina, testes, epididymides (total and cauda), prostate, seminal vesicles with coagulating glands, brain
- With a precision of 0.001 g: ovaries, pituitary

The weighed organs and all organs showing macroscopic lesions of all adult animals were preserved. Testes and epididymides were preserved in Bouin’s solution, all other organs in 10% buffered formalin solution. In addition, for 5 animals/sex/group (subgroup B), the following organs and tissues, or representative samples, were preserved:
Gross findings, Liver, Small intestine (8), Adrenals, Lungs with bronchi (5), Spinal cord (cervical, lumbar, and thoracic levels), Animal identification (1), Lymph nodes (6) , Aorta, Mammary gland (inguinal), Spleen, Brain (2), Ovaries with oviduct, Sternum with marrow, Epididymides, Pancreas, Stomach, Eyes with optic nerves (7), Pituitary Testes, Oesophagus, Prostate, Thymus, Femur with marrow, Salivary gland (mandibular), Thyroid with parathyroids (7), Heart (3) Sciatic nerve, Tongue, Kidneys, Seminal vesicles with coagulating glands, Trachea, Large intestine (4) , Urinary bladder, Lachrymal gland with Harderian glands, Skeletal muscle (quadriceps), Uterus (9), Skin and subcutis (inguinal), Vagina.

1. Fixation and preservation only.
2. Cerebral cortex, midbrain, cerebellum and medulla.
3. Section including both ventricles and atria, septum with papillary muscle.
4. Caecum, colon and rectum.
5. Lungs of euthanized animals were infused with formalin.
6. Mandibular and mesenteric.
7. Parathyroids and optic nerves, examined histologically only if present in routine sections.
8. Duodenum, ileum and jejunum with Peyer’s patches.
9. Horns, body and cervix.

From subgroup B animals, the following organs were weighed in addition to the ones
previously mentioned:
- With a precision of 0.01 g: heart, kidneys, liver, spleen and thymus
- With a precision of 0.001 g: adrenals.

Paired organs were weighed individually; absolute organ weights are reported. Relative organ weights (to body and brain weight) were calculated and are also reported.

For the parental animals, detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups, any animals found dead or euthanized pre-terminally during the study, in all groups, and all macroscopic findings (abnormalities) from all animals. Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Additional histopathology evaluation was performed on the liver and kidneys samples from the subgroup B Low and Mid dose groups 2 and 3 animals to provide additional information on possible test item effects under the conditions of this study. The retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6μ by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.

Other examinations:

Details of the examinations specific to the reproductive/developmental screening test are provided in Chapter 7.8.1.

Statistics:

Numerical data obtained during the conduct of the study were subjected as appropriate to calculation of group means and standard deviations and are reported in the final report.
The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to assess the significance of inter-group differences. Where significant result was obtained at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the
inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as required.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Not considered to be of toxicological importance

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Not considered to reflect a neurotoxicological effect

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

MORTALITY AND CLINICAL OBSERVATIONS

In the High dose 1000 mg/kg bw/day males group, rat 4010 (1/12) was euthanized preterminally in moribund condition after treatment on Day 8. Prior to its death, this animal was normal until Day 6, then displayed clinical signs such as, but not limited to, activity decreased, hunched back position, piloerection, uncoordination, emaciation, vocalization and/or difficult respiration, followed by death on Day 8. In addition, activity decreased and/or hunched back position were noted for up to 17 days in 2/12 High dose males (4003 and 4011).
In the High dose 1000 mg/kg bw/day females group, 1/12 female 4511 was found dead on GD22 (treatment Day 38). Moderately decreased activity, piloerection and hunched back position were observed one day before death in this animal. Similar clinical signs were noted in female 4506 from Day 39 to 41, but the animal recovered and underwent scheduled necropsy on Day 42 (PND5).
There were no clinical signs or mortality considered related to TIS-M administration at up to and including 250 mg/kg bw/day. One Low dose female 2506 was preterminally euthanized on Day 38 (GD24). No clinical signs were noted from the onset of treatment to Day 37. On Day 38, hunched back position, piloerection and clear vulvar discharge were noted, followed by moderate decrease in activity and poor clinical condition. At necropsy, placental haemorrhage and foetal deaths were observed, as well as spleen enlargement (which was considered agonal change unrelated to treatment). Based on the clinical signs and macroscopic findings recorded in this animal, and considering the isolated incidence within the group, the clinical condition of this female was regarded as individual variability associated with placental haemorrhage, followed by fetal death and retention, and not ascribed to systemic test item toxicity.
There were no toxicologically significant changes in behaviour, general physical condition, in the reactions to different type of stimuli, grip strength or motor activity in the Control or treated groups noted in the evaluation performed towards the end of the treatment period. Minor variations including piloerection, hunched back position, vocalisation or slightly increased positional struggle were observed, either with isolated incidence associated with the animal clinical status, or throughout all dose groups, including Controls when subjected to the modified Irwin test (functional observation battery), thus, they were not considered to reflect a neurotoxicological effect, but associated with individual variability or without toxicological significance in correlation with test item administration.

BODY WEIGHT AND BODY WEIGHT GAIN

On several occasions, statistically lower than Control body weight and body weight gain mean values were noted in animals treated with TIS-M at 1000 and 250 mg/kg bw/day. In the males, the overall mean body weight gain from Day 0 to Day 28 was -31% lower than the Controls at 250 mg/kg bw/day (p<0.01) and -73% lower at 1000 mg/kgbw/day (p<0.01), with weekly body weight loss of up to -109% in the High dose group after the first week of treatment (-2.92 g versus 33 g body weight gain in the Control group, p<0.01).

In the Low dose 50 mg/kg bw/day males, the mean value was -17% lower than Control at the completion of the treatment between days 0 and 28, but without attaining statistical significance, showing a relatively large variability within the group and comparable with the physiological ranges; the only statistically lower than Control value noted in the group was recorded after the first week of treatment (-26%, p<0.05).

In the females, the differences to Controls were less significant compared to the males and statistically lower body weight and/or body weight gain were only noted at 1000 mg/kg bw/day during gestation period or shortly thereafter. Lower mean body weight was noted in the High dose group females on GD17, GD20 and on the day of delivery PPD0, with differences of up to -9% (p<0.01). In addition, the body weight gain was statistically lower up to -29% during the last week of gestation GD14-20 as well as when calculated overall, for the whole gestation period (GD0-20). No statistically significant variations were observed before the onset of gestation or during the post-partal period.

No effects on the animal body weight or body weight gain that could be considered adverse or associated with TIS-M administration were noted at 50 mg/kg bw/day dose level, as the variations observed were minor and/or within the contemporaneous Control data.

FOOD CONSUMPTION DATA

There were no effects considered related to test item administration, or toxicologically significant at 50 mg/kg bw/day.

At 1000 mg/kg bw/day, both male and female animals had statistically lower mean food consumption at most of the timepoints evaluated and correlated with the body weight values. Lower feed intake was also noted in the Mid dose 250 mg/kg bw/day males between Days 7 to 14, and in the females, between GD7 and GD14.

OPHTHALMOLOGY

No test item related changes compared to pre-treatment were noted during ophthalmoscopy examination of 5 male and 5 female Control and high-dose main animals during the last week of treatment prior to necropsy (males, Day 27 pm, females, PND 4 pm), thus no additional evaluation was required in other dose groups or recovery animals.

CLINICAL PATHOLOGY

HAEMATOLOGY

When compared to Control, statistically higher white blood cells count WBC were noted at 1000 mg/kg bw/day up to 63% in the males and 86% in the females (p<0.01) and were considered potentially related to TIS-M administration, although they were associated with a relatively low Control value in the females. In addition, a tendency for lower than Control RBC and APTT was observed in both sexes at 250 and/or 1000 mg/kg bw/day, although the dose response was not clear and the differences have not reached consistently statistical significance and thus they were considered equivocal in the conditions of this study in correlation with TIS-M administration.

CLINICAL CHEMISTRY

Lower total protein mean value was noted in both males and females at 1000 mg/kg bw/day compared to Controls, with differences of -25% in the males (p<0.05) and -17% in the females (p<0.05).

The mean albumin was also lower than the Controls at 1000 mg/kg bw/day, with differences of -25% in the males (p<0.05) and -20% in the females (p<0.01); however, no dose response was observed, the values were comparable with contemporaneous control data and these variations were considered equivocal in correlation with TIS-M administration under the conditions of this study.

Cholesterol mean value was 48% higher than Controls in the High dose males (p<0.01) and 15% higher in the High dose females, although did not attain statistical significance. Higher than Control urea and creatinine were noted at 250 and 1000 mg/kg bw/day, or in the 250 mg/kg bw/day group only, respectively, with no dose response or similar variations in the female animals, and not associated with correlated histopathological findings, thus considered unrelated to treatment.

At 1000 mg/kg bw/day, ALKP was statistically lower than Control in the males, with a mean value of 88.2 U/L vs. 171 U/L in the Control group (-48% lower, p<0.01). In the High dose females, the mean value remained -23% lower than Control (75 vs. 97.2 U/L) but without attaining statistical significance. These variations were not considered to reflect an adverse effect of the test item; in addition, although the response was similar in both genders, no dose response was observed.

An unusual clinical chemistry profile possibly associated with a test item effect was noted in male 4002 (see table under any other information on results), correlated with increases in the absolute and relative liver weight within this group and compared to the Control animals, although no microscopic findings were recorded in this animal.

URINALYSIS

TIS-M administration by daily oral gavage at up to and including 1000 mg/kg bw/day did not result in any test item-related effect.

The specific gravity (SG) of urine was lower than Control in the low and High dose males and the volume, higher, with no similar changes in the other gender. In addition, higher pH was noted at 50 mg/kg bw/day in the males, however, with no dose response. These variations were minor, showed no clear dose or gender dependent tendency, and were thus not considered to be of toxicological importance in correlation with test item administration in the conditions of this study. The few other minor variations observed did not attain statistical significance and/or were regarded as normal background changes.

Minor variations were noted in other clinical pathology parameters (haematology, coagulation, clinical chemistry and/or urinalysis). Although occasionally these changes were statistically significant, no dose related response was observed, the results were within the historical range, and/or there was no consistent reaction in the male and female groups. These changes were neither considered toxicologically significant, nor indicated a test item related etiology.

PATHOLOGY EVALUATION AND ORGAN WEIGHT

Pre-terminal Euthanasia, Parental Generation

One High Dose parental male (4010) was euthanized pre-terminally on Day 8 and one Low Dose parental female (2506), on Day 38 (Gestation Day GD24, as no parturition was noted and the female clinical condition worsened).

Macroscopic Findings: In the High Dose parental male (4010) diffuse, dark/red discoloration in all lobes of the lungs and bilateral, multifocal, pale discoloration in the kidneys were observed. In the 2506 Low Dose parental female enlargement of the spleen and placental haemorrhage were found at necropsy. These findings were regarded as incidental, unrelated to treatment.

Microscopic Findings: In the High Dose parental male (4010) no significant findings were noted. In the 2506 Low Dose parental female minimal congestion was observed in the spleen; in addition, diffuse, severe hemorrhage and mild, diffuse, mixed cell infiltrate were observed in the placenta and correlated with necropsy. These findings were regarded unrelated to treatment.

Parental Generation, Found Dead

High Dose female 4511 was found dead on Day 38 (GD22) and 31 intact pups were found dead between Days 0-1 post partum.
Macroscopic Findings: In the 4511 High Dose female no macroscopic abnormalities were found.
Microscopic Findings: Minimal, perilobular, hepatocellular vacuolation in all lobes of the liver was seen in this female.

TERMINAL

Parental Generation
Macroscopic Findings: Liver enlargement and pale mottling of the liver, potentially associated with test item administration and correlated with the microscopic findings were noted in 3/11 High Dose surviving females.
Incidental findings such as, but not limited to, renal pelvic dilatation, dark, red discoloration in the lungs and/or thymus, white discoloration of the digestive mucosa and/or content, enlargement of the spleen or adrenals were noted throughout all groups, or with limited incidence and were not considered to reflect a toxicological effect, or to be associated with test item administration.

Microscopic Findings
Main animals except Subgroup B
There were no test item-related findings observed histologically in the High and Control animals in the reproductive organs examined. The follicular, luteal and interstitial compartments of the ovary as well as epithelial capsule and stroma were similar histological structure in both Control and High Dose females. Histopathological evaluation of the male gonads as well as testicular interstitial cell structure, the spermatogenic cells representing different phases of the development and differentiation of the spermatozoons were similar in Control and High Dose males. In one High dose male 4011, hypospermia and ductal atrophy occurred as incidental finding in the epididymis.

In summary, potentially TIS-M related, minimal/mild centrolobular hepatocellular vacuolation was noted in all lobes of the liver in five 5/12 High Dose female animals (4504, 4505, from Subgroup B, and 4506, 4509, 4512), with no similar findings noted in the male animals, or any findings in the organs examined of the Low and Mid dose subgroup B animals (liver, kidneys and examined tissues with macroscopic abnormalities). No test item-related pathological findings were noted in the reproductive organs in the parental animals. Low Dose female 2506 and High Dose male 4010 were pre-terminally euthanized on Days 38 and 8, respectively, based on the clinical signs noted. Findings such as minimal congestion in the spleen and diffuse,severe hemorrhage and mild, mixed, diffuse cell infiltrate in the placenta were recorded in the 2506 Low Dose female. One High Dose female (4511) died during the study, on Day 38. A specific cause of death was not determined; minimal, perilobular, hepatocellular vacuolation in all lobes of the liver were seen in this female, resulting in a total of 6/12 females with liver findings.

All other changes in the variety of organs including ovary, uterus, spleen, kidneys, thymus and lungs, epididymis and/or testes with low incidence and severity were considered to be incidental or procedure-related. No microscopic findings considered related to test item administration were noted in the organs examined of the Low and Mid dose subgroup B animals (liver, kidneys and examined tissues with macroscopic abnormalities).

ORGAN WEIGHT

Terminal mean body weights of the male animals were statistically lower at 250 and 1000 mg/kg bw/day, with differences to Control of -8% and -17% (p<0.05 and 0.01, respectively). In the females, the terminal body weights were only up to -10% lower than Control in the 1000 mg/kg bw/day High dose group and without attaining statistical significance.
Statistically higher absolute and/or relative liver and/or kidney weights were observed at 250 and/or 1000 mg/kg bw/day, with an apparent dose response and considered related to TIS-M administration.
The absolute liver weights in the High dose group were up to 32% higher than Control, p<0.01 in the males, and up to 48%, p<0.01, in the females. When adjusted for the terminal body weights, increases up to 55% and up to 68%, p<0.01 were noted in the males and females, respectively; when adjusted for the brain weight, 33% and 59% higher mean liver values were recorded at 1000 mg/kg bw/day compared to Control. In the 250 mg/kg bw/day group, statistically higher mean liver weights were noted in the females both as absolute value and when adjusted for the body and brain weight; in the males, only the mean liver weight relative to the body weight has attained statistical significance (29% higher than Control, p<0.01). These changes were considered to be correlated with the hepatic microscopic findings, noted however in 6/12 High dose females only. Increases in the kidneys weight considered potentially related to test item administration were observed at 1000 mg/kg bw/day in both sexes, with absolute values up to 25% and 17%, p<0.05 higher than Control in the High dose males and females, respectively. In addition, higher than Control relative kidneys weight when adjusted for the body weight occurred in the 250 mg/kg bw/day Mid dose males only (32%, p<0.01).

Lower absolute and/or relative mean seminal vesicle, testes and/or epididymides weights when adjusted to the brain weight were noted in the male animals at 1000 and/or 250 mg/kg bw/day. However, the relative organ weights were slightly higher, or comparable with the control values when adjusted for the animal terminal body weight; in the absence of any test item related associated microscopic findings in these organs, or of any effects on the reproductive parameters evaluated in the adult males, these variations were ascribed to the differences in the group mean terminal body weight and a correlation with test item administration was considered equivocal under the conditions of this study.

Other statistically significant variations were noted in the absolute or relative organ weights including brain, testes, uterus and/or thymus, however, these were either ascribed to biological variability after pregnancy, or there was no consistent dose response or correlated pathological and histopathological findings, thus, these variations were not considered toxicologically relevant or related to dose administration.

Results of the reproduction/development toxicology screening test are detailed in Chapter 7.8.1.

Effect levels

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the associated adverse effects including the body weight, food consumption, clinical pathology, organ weights and histopathology findings.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

An unusual clinical chemistry profile possibly associated with a test item effect was noted in male 4002:

Parameter	AST	ALT	ALKP	GGT	T-BIL	A/G	Bile acids
Unit	U/L	U/L	U/L	U/L	umol/L		umol/L
Male 4002	237	18	57	23	28.9	1.2	11.0
Mean Control value	113.80	47.00	171.00	b.q.l.	5.76	1.30	13.74

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, based on the associated adverse effects including the body weight, food consumption, clinical pathology, organ weights and histopathology findings, the no observed adverse effect level (NOAEL) for TIS-M in the parental generation for systemic toxicity was considered to be 50 mg/kg bw/day. Conclusion on the results of the reproduction/development toxicology screening test is detailed in Chapter 7.8.1.

Executive summary:

The purpose of this study was to obtain information on the possible toxic effects of the test item TIS-M following repeated, daily administration by oral gavage to CRL:(WI)BR rats at 3 dose levels, 50, 250 and 1000 mg/kg bw/day, at a dose volume of 2 mL/kg. A control group received the vehicle only, dried sunflower oil. The study also comprised a reproductive/developmental toxicity screening test, intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and also on the development of the F1 offspring from conception to Day 4 post-partum. The dose levels were based on findings obtained in previous studies conducted by the Sponsor in other laboratories, with the aim of inducing toxic effects but no death or suffering at the highest dose and a NOAEL at the lowest dose.

Twelve male and 12 female Wistar rats/group were treated according to the experimental design detailed under any other information on materials and methods.

Dose formulation preparation was conducted in the Central Dispensary and analysis in the Analytical Laboratory of LAB Research Ltd. Formulations of TIS-M were prepared in the vehicle (dried sunflower oil) at the appropriate frequency and stored refrigerated at 5±3ºC pending use within 5 days, or at room temperature pending use within 24 hours, according to stability assessment results conducted under study LAB code 09/285-316AN. Formulation samples were collected and analysed for concentration and/or homogeneity on 3 occasions, during the first and last weeks of treatment for both males and females (17 March, 12 April and 29 April 2010). Dose formulations were homogenous. No TIS-M was detected in the control samples. In the test item dose formulations, the measured concentrations ranged from 95 to 108% of nominal concentrations of 25, 125 and 500 mg/mL. These results were considered suitable for the study purposes.

Animals were inspected for signs of morbidity and mortality twice daily. Clinical observations were made daily, with detailed examination performed at least weekly. Body weight and food consumption were measured at least weekly.

A functional observation battery, ophthalmology evaluation and/or clinical pathology assessment were performed on subgroups of 5 animals/sex/group, randomly selected. Gross necropsy was conducted at the end of the treatment period. During necropsy, a macroscopic examination was performed in all adult animals. The absolute and relative organ weights of selected organs and tissues were determined in the adult animals. For the parental animals, detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups, any animals found dead or euthanized preterminally during the study, in all groups, and all macroscopic findings (abnormalities) from all animals. Additional histopathology evaluation was performed of liver and kidneys samples from the subgroup B Low and Mid dose groups 2 and 3 animals to provide additional information on possible test item effects under the conditions of this study.

One (1/12) High dose male was euthanized pre-terminally in moribund condition after treatment on Day 8. Prior to its death, this animal was normal until Day 6, then displayed clinical signs such as, but not limited to, activity decreased, hunched back position,piloerection, uncoordination, emaciation, vocalization and/or difficult respiration, followed by death on Day 8. In addition, activity decreased and/or hunched back position were noted for up to 17 days in 2/12 High dose males (4003 and 4011).

One 1/12 High dose female was found dead on GD22 (treatment Day 38). Activity moderately decreased, piloerection and hunched back position were observed one day before death in this animal. Similar clinical signs were noted in female 4506 from Day 39 to 41, but the animal recovered and underwent scheduled necropsy on Day 42 (PND5).

There were no clinical signs or mortality considered related to TIS-M administration at up to and including 250 mg/kg bw/day. One 1/12 Low dose female 2506 was preterminally euthanized on Day 38 (GD24) due to placental haemorrhage, followed by fetal death and retention, observations considered incidental or individual finding, not ascribed to test item systemic toxicity.

The behaviour and the general condition of the adult animals were normal during the study. There was no treatment related effect on motor activity or in the functional observation battery parameters across groups of treated male or female animals as compared to Controls and no findings indicative for neurotoxicity were observed. No changes were noted at ophthalmology examination of Control and High dose animals.

On several occasions, statistically lower than Control body weight and body weight gain mean values were noted in the 1000 and 250 mg/kg bw/day animals. In the males, the overall mean body weight gain from Day 0 to Day 28 was -31% lower than the Controls at 250 mg/kg bw/day and -73% lower at 1000 mg/kg bw/day, with weekly body weight loss of up to -109% in the High dose group after the first week of treatment (-2.92 g versus 33 g body weight gain in the Control group). The differences were less significant in the female animals compared to the males and statistically lower body weight and/or body weight gain were only noted at 1000 mg/kg bw/day during gestation period or shortly thereafter. Lower mean body weight was noted in the High dose group females on GD17, GD20 and on the day of delivery PPD0, with differences of up to -9%. In addition, the body weight gain was statistically lower up to -29% during the last week of gestation GD14-20 as well as when calculated overall, for the whole gestation period (GD0-20). No statistically significant variations were observed before the onset of gestation or during the postpartal period.

At 1000 mg/kg bw/day, both male and female animals had statistically lower mean food consumption at most of the timepoints evaluated and correlated with the body weight values. Lower feed intake was also noted in the Mid dose 250 mg/kg bw/day males between Days 7 to 14, and in the females, between GD7 and GD14. No adverse effects on the animal body weight, body weight gain, or food consumption were noted at 50 mg/kg bw/day dose level.

When compared to Control, statistically higher white blood cells count WBC were noted at 1000 mg/kg bw/day up to 63% in the males and 86% in the females and were considered potentially related to TIS-M administration, although they were associated with a relatively low Control value in the females. In addition, a tendency for lower than Control RBC and APTT was observed in both sexes at 250 and/or 1000 mg/kgbw/day, although the dose response was not clear and the differences have not reached consistently statistical significance and thus they were considered equivocal in the conditions of this study in correlation with TIS-M administration.

Statistically lower total protein mean value was noted at 1000 mg/kg bw/day compared to Controls, with differences of -25% in the males and -17% in the females. The mean albumin was also statistically lower than the Controls at 1000 mg/kgbw/day, with differences of -25% in the males and -20% in the females; however, no dose response was observed, the values were comparable with contemporaneous control data and these variations were considered equivocal in correlation with TIS-M administration under the conditions of this study. Cholesterol mean value was 48% statistically higher than Controls in the High dose males and 15% higher in the High dose females, although did not attain statistical significance. Higher than Control urea and creatinine were noted at 250 and 1000 mg/kg bw/day, or in the 250 mg/kg bw/day group only, respectively, with no dose response or similar variations in the female animals, and not associated with correlated histopathological findings, thus considered unrelated to treatment. At 1000 mg/kg bw/day, ALKP was statistically lower than Control in the males, with a mean value of 88.2 U/L vs. 171 U/L in the Control group (-48% lower, p<0.01). In the High dose females, the mean value remained -23% lower than Control (75 vs. 97.2 U/L) but without attaining statistical significance. These variations were not considered to reflect an adverse effect of the test item; in addition, although the response was similar in both genders, no dose response was observed.

An unusual clinical chemistry profile possibly associated with a test item effect was noted in male 4002, correlated with increases in the absolute and relative liver weight within this group and compared to the Control animals, although no microscopic findings were recorded in this animal.

TIS-M administration by daily oral gavage at up to and including 1000 mg/kg bw/day did not result in any effects considered test item-related on the analyzed urinalysis parameters.

Minor variations were noted in other clinical pathology parameters (haematology, coagulation, clinical chemistry and/or urinalysis). Although occasionally these changes were statistically significant, no dose related response was observed, the results were within the historical range, and/or there was no consistent response between genders. These changes were neither considered toxicologically significant, nor indicated a test item related etiology.

Potentially TIS-M related, minimal/mild centrolobular hepatocellular vacuolation was noted in all lobes of the liver in five 5/12 High Dose female animals (4504, 4505, from Subgroup B, and 4506, 4509, 4512), with no similar findings noted in the male animals, or any findings in the organs examined of the Low and Mid dose subgroup B animals (liver, kidneys and examined tissues with macroscopic abnormalities).

Low Dose female 2506 and High Dose male 4010 were pre-terminally euthanized on Days 38 and 8, respectively, based on the

clinical signs noted. Findings such as minimal congestion in the spleen and diffuse, severe haemorrhage and mild, mixed, diffuse cell infiltrate in the placenta were recorded in the 2506 Low Dose female. One High Dose female (4511) died during the study, on Day 38. A specific cause of death was not determined; minimal, perilobular, hepatocellular vacuolation in all lobes of the liver were seen in this female, resulting in a total of 6/12 females with liver findings.

Terminal mean body weights of the male animals were statistically lower at 250 and 1000 mg/kg bw/day, with differences to Control of -8% and -17%. In the females, the terminal body weights were only up to -10% lower than Control in the 1000 mg/kgbw/day High dose group and without attaining statistical significance.

Potentially TIS-M related, minimal/mild centrolobular hepatocellular vacuolation was noted in all lobes of the liver in five 5/12 High Dose female animals (4504, 4505, from Subgroup B, and 4506, 4509, 4512), with no similar findings noted in the male animals, or any findings in the organs examined of the Low and Mid dose subgroup B animals (liver, kidneys and examined tissues with macroscopic abnormalities). No test item-related pathological findings were noted in the reproductive organs in the parental animals, or in F1 Generation. Low Dose female 2506 and High Dose male 4010 were pre-terminally euthanized on Days 38 and 8, respectively, based on the clinical signs noted. Findings such as minimal congestion in the spleen and diffuse, severe haemorrhage and mild, mixed, diffuse cell infiltrate in the placenta were recorded in the 2506 Low Dose female. One High Dose female (4511) died during the study, on Day 38. A specific cause of death was not determined; minimal, perilobular, hepatocellular vacuolation in all lobes of the liver were seen in this female, resulting in a total of 6/12 females with liver findings.

Necropsy was performed on 31 pups found dead and intact between PND 0-1 (9, 9, 8 and 5, from Control, Low, Mid and High Dose groups, respectively). A negative flotation test of the lungs was observed in 5, 9, 1 and 1 offspring from Control, Low, Mid and High Dose groups, respectively. Dark/red discoloration and/or noncollapsing of the lungs were seen in 3 Control (1508/18, 1509/7 and 1509/9) and 2 High Dose pups (4503/4 and 4505/8). In addition, 3/9 Control and 4/5 High Dose pups had no digestive content in the stomach. A specific cause of death of these pups could not be determined. Terminal mean body weights of the male animals were statistically lower at 250 and 1000 mg/kg bw/day, with differences to Control of -8% and -17%. In the females, the terminal body weights were only up to -10% lower than Control in the 1000 mg/kg bw/day High dose group and without attaining statistical significance.

Statistically higher absolute and/or relative liver and/or kidney weights were observed at 250 and/or 1000 mg/kg bw/day, with an apparent dose response and considered related to TIS-M administration. The absolute liver weights in the High dose group were up to 32% higher than Control in the males, and up to 48% in the females When adjusted for the terminal body weights, increases up to 55% and up to 68% were noted in the males and females, respectively; when adjusted for the brain weight, 33% and 59% higher mean liver values were recorded at 1000 mg/kg bw/day compared to Control. In the 250 mg/kg bw/day group, statistically higher mean liver weights were noted in the females both as absolute value and when adjusted for the body and brain weight; in the males, only the mean liver weight relative to the body weight has attained statistical significance (29% higher than Control). These changes were considered to be correlated with the hepatic microscopic findings, noted however in 6/12 High dose females only.

Increases in the kidneys weight considered potentially related to test item administration were observed at 1000 mg/kg bw/day in both sexes, with absolute values up to 25% and 17% higher than Control in the High dose males and females,

respectively. In addition, higher than Control relative kidneys weight when adjusted for the body weight occurred in the 250 mg/kg bw/day Mid dose males only (32%).

In conclusion, based on the associated adverse effects including the body weight, food consumption, clinical pathology, organ weights and histopathology findings, the no observed adverse effect level (NOAEL) for TIS-M in the parental generation for systemic toxicity was considered to be 50 mg/kg bw/day. See Chapter 7.8.1 for conclusion on the results of the reproduction/developmental toxicity screening test.