(2S)-[[(benzyloxy)carbonyl]amino](cyclohexyl)acetic acid

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-10-16 to 2007-10-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed according to OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents) without deviations.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

The testing facility indicated that the protocol was followed without deviation.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate from "The Department of Health of the Government of the United Kingdom"

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): VRT-126016 (Z-L-cyclohexylglycine)
- Molecular formula: no data
- Molecular weight: no data
- Smiles notation: no data
- InChl: no data
- Structural formula attached as image file: no data
- Substance type: no data
- Physical state: white powder
- Analytical purity: 99.8% (area - HPLC)
- Impurities: <0.1% chloride, <0.3% (area) Z-D-Cyclohexylglycine, <0.1% water
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: 2006-02-02
- Lot/batch No.: batch 25719, lot 1-4107-6-02-02
- Expiration date of the lot/batch: 2008-02
- Stability under test conditions: All formulations were shown to be homogenous in the vehicle and stable at ambient temperature for 2 days and for 8 days following refrigerated storage (approximately 4 deg C)
- Storage condition of test material: ambient temperature
- Other: received on 2006-09-15

Test animals

Species:

rat

Strain:

other: Crl:CD (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 40-46 days
- Weight at study initiation: 222-256 g (males) and 157-203 g (females)
- Fasting period before study: overnight before routine blood sampling
- Housing: Animals were housed inside a limited access rodent facility. Sensory reactivity and grip strength assessment were performed in separate rooms. The facility was designed and operated to minimize the entry of external biological and chemical agents and to minimize the transference of such agents between rooms. Animals were housed 5 of one sex per cage. The cages were made of polycarbonate with a stainless steel grid lid. Wood flakes were supplied as bedding. Cages, food hoppers and water bottle were changed at appropriate intervals. Each cage of animals was provided with an Aspen chew block for environmental enrichment. Chew blocks were provided throughout the study and were replaced when necessary. Prior to clinical pathology investigations, the blocks were removed for the same period as the food.
- Diet: The animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1 Maintenance Diet), except overnight before routine blood sampling.
- Water: Potable water taken from the public supply was freely available ad libitum, via polycarbonate bottles fitted with sipper tubes.
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18.5-22.2 deg C
- Humidity: 28-68 %
- Air changes: Each animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated. No further data was provided.
- Photoperiod: 12 hours light/12 hours dark

IN-LIFE DATES
- From: 2007-01-15 to 2007-02-12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1.0 % w/v Methylcellulose in water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
The required amount of the test substance was ground in a mortar using a pestle and mixed with some vehicle to form a paste. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogenizer. A series of suspension at the required concentrations were prepared by dilution of individual weighings of the test substance. All formulations were prepared freshly each week. Detailed records of test substance usage were maintained. The amount of test substance necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed. Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

DIET PREPARATION
- Rate of preparation of diet: not applicable
- Mixing appropriate amounts with: not applicable
- Storage temperature of food: not applicable

VEHICLE
- Justification for use and choice of vehicle: no data
- Concentration in vehicle: 0, 1.5, 15 and 100 mg of test substance/mL
- Amount of vehicle: 10 mL/kg
- Lot/batch no.: no data
- Purity: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before treatment commenced, the suitability of the proposed mixing procedure was determined and specimen formulations were analyzed at concentrations of 1 and 100 mg/mL to assess the homogeneity and stability of the test substance in the vehicle. Samples of each formulation prepared for administration in Week 1 of treatment were analyzed for achieved concentration of the test substance.

Duration of treatment / exposure:

4 consecutive weeks

Frequency of treatment:

Animals were dosed in sequence of cage-number within each group, once each day at approximately the same time each day, seven days per week.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 15, 150 and 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

5 per sex per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses used were selected after consultation with the Sponsor and were based on data from a 7 day preliminary oral toxicity study, where a dose level of 1000 mg/kg/day was well tolerated. The dose levels of 15 and 150 mg/kg/day were selected based on EC labelling points.
- Rationale for animal assignment: On arrival, the animals were removed from the transit boxes and allocated to study cages. Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately. The cages constituting each group were blocked together by sex and the groups were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable. Additionally, batteries of cages were rotated around the room at weekly intervals to further minimize possible spatial variations.
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rational: not applicable

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: see below
- Cage side observations included: Animals were inspected visually at least twice daily, for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of ill-health amongst the occupants. Any deviation from normal was recorded at the time, in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate. Daily throughout the treatment period, detailed observations were performed immediately before dosing and between one and two hours after completion of dosing of all groups. A further observation was performed as late as possible in the working day during Week 1 of treatment. During the acclimation period, observations of the animals and their cages were recorded once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment, detailed physical examinations and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group to which the animal belonged. After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs for neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior. Findings were either reported as "present" or assigned a severity group of slight, moderate or marked.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded one week before treatment commenced (Day -7), on the day the treatment commenced (Day 1), weekly throughout the treatment period (last scheduled bodyweight was recorded on Day 28) and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal determined and mean weekly diet consumption calculated as g food/animal/week: Yes; The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started (Week -1) and each week throughout the treatment period. From these records, the mean weekly consumption per animal (g/rat/week) was calculated for each cage.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: not applicable

FOOD EFFICIENCY
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: no

WATER CONSUMPTION AND COMPOUND INTAKE: Not applicable
- Time schedule for examinations: Water consumption was assessed by daily visual observation. No treatment related changes were suspected therefore precise measurements were not undertaken.

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: not applicable
- Dose groups that were examined: not applicable

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period (Day 29), blood samples (nominally 0.5 mL) were obtained from the sublingual vein of animals and collected into tubes containing EDTA as anticoagulant for the majority of parameters listed below, except prothrombin time and activated partial thromboplastin time in which additional samples were taken and added to tubes containing citrate anticoagulant.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: yes
- How many animals: all animals
- Parameters examined: The following were measured using a Bayer Advia 120 haematology analyzer: haematocrit, haemoglobin, erythrocyte count, mean cell haemoglobin, mean cell haemoglobin concentration, mean cell volume, total white cell count, differential WBC count (neutrophils, lymphocytes, eosinophils, basophils, monocytes, large unstained cells), and platelet count. Morphology flags were generated by the Advia 120 analyzer. The most common morphological changes, anisocytosis, micro/macrocytosis and hypo/hyperchromasia were recorded as follows: (-) = no abnormalities detected, (+) = slight, and (++) = moderate. Blood film (prepared for all samples)-Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate.
Prothrombin time was measured using an ACL 3000 Plus analyzer and IL PT-Fibrinogen reagent. Activated partial thromboplastin time was measured using an ACL 3000 Plus Analyzer and IL APTT reagent.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period (Day 29), further blood samples (nominally 0.7 mL) were collected at the same time and using the same animals as for peripheral haematology, into tubes containing lithium heparin as anticoagulant. All tubes were mechanically agitated for at least two minutes and the sample subsequently centrifuged at 2000 G for 10 minutes in order to separate the plasma. After separation, the plasma was examined using a Hitachi 917 Clinical Chemistry Analyzer.
- Animals fasted: yes
- How many animals: all animals
- Parameters examined: alanine aminotransferase, asparate amino-transferase, urea, creatinine, glucose, total cholesterol, sodium, potassium, total protein, albumin, albumin/globulin ratio (calculated from total protein concentration and analyzed albumin concentration)


URINALYSIS: No
- Time schedule for collection of urine: not applicable
- Metabolism cages used for collection of urine: not applicable
- Animals fasted: not applicable
- Parameters examined: not applicble


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: test were performed (before dosing) during Week 4 of treatment. Animals were not necessarily all tested on the same day, but the number of animals and the times of testing were balanced across the groups on each day of testing. These observations were performed before any laboratory investigations.
- Dose groups that were examined: all animals in all groups
- Battery of functions tested: approach response, touch response, auditory startle response, tail pinch response, grip strength and motor activity

OTHER: Not applicable

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Animals were killed by carbon dioxide asphyxiation. The sequence in which the animals were killed after completion of treatment, was selected to allow satisfactory inter-group comparison. All animals were subjected to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external futures and orifices were examined visually. The cranial roof was removed, to allow observation of the brain, pituitary gland and cranial nerves. After ventral mid-line incision, the neck and associate tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded.
- The requisite organs (adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus) were weighed and external and cut surfaces of the organs and tissues were examined as appropriate. The organs were dissected free of adjacent fat and other contiguous tissue, and the weights were recorded. Bilateral organs were weighed together. Organ weights were also adjusted for terminal bodyweight, using the weight recorded immediately before necropsy. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative. The retained tissues were check before disposal of the carcass.

HISTOPATHOLOGY: Yes
- Testes and epididymides were fixed in Bouin's solution prior to transfer to 70% industrial methylated spirit. Samples (or the whole ) of the following tissues from all animals were preserved in 10% neural buffered formalin: adrenals, brain, caecum, colon, duodenum, epididymides, femurs (only one processed for examination), head (not processed for examination), heart, ileum with Peyer's patch, jejunum, kidneys, liver, lungs, lymph nodes (mandibular and mesenteric), oesophagus, ovaries, pancreas, prostate, rectum, sciatic nerves, seminal vesicles, spinal cord, spleen, sternum, stomach, testes, thymus, thyroid with parathyroids, trachea, urinary bladder, uterus and cervix. Samples of abnormal tissues were also retained and processed for examination if appropriate. In those cases where a lesion was not clearly delineated, contiguous tissue was fixed with the grossly affected region and sectioned as appropriate. Samples of the head (including nasal cavity, paranasal sinuses and nasopharynx), oesophagus, pancreas, sternum and the remaining femur and sciatic nerve were not examined histologically, but were retained against any future requirement for microscopic examination.
- Tissues used for histopathology were dehydrated, embedded in paraffin wax, sectioned at approximately four to five micron thickness and stained with haematoxylin and eosin, except the testes which were stained using a standard periodic acid/Schiff (PAS) method. Those tissues subjected to histological processing included the following regions: Adrenals (cortex and medulla), brain (cerebellum, cerebrum and midbrain), femur with joint (longitudinal section including articular surface, epiphysial plate and bone marrow), heart (included auricular and ventricular regions), kidneys (included cortex, medulla and papilla regions), liver (sectioned from all main lobes), lungs (section from two major lobes, to include bronchi), spinal cord (transverse and longitudinal section at the cervical, lumbar and thoracic levels), sternum (included bone marrow), stomach (included keratinised, glandular and antrum in sections), thyroid (included parathyroids in section where possible) and uterus (uterus section separate from cervix section). For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required for microscopic pathology.
- Microscopic examination was performed as follows: tissues preserved for examination (as specified above) were examined for all animals in the control and high dose group sacrificed on completion of the scheduled treatment period. The following tissues, which were considered to exhibit a reaction to the treatment at the high dose were examined for all animals: Liver, thyroid (all animals) and spleen (all females). Findings were either reported as "present" or assigned a severity grade. In the latter case, one of the following five grades was used: minimal, slight, moderate, marked or severe.

Other examinations:

none

Statistics:

see below

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

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:

CLINICAL SIGNS AND MORTALITY
- There were no unscheduled deaths. No treatment related clinical signs were observed for any rats in any of the dose groups. Salivation and rales were recorded for one female receiving 1000 mg/kg/day, one to two hours after dosing on Day 21 only. As only one rat was affected, on one day at one time point, these signs were not considered to be treatment related.

BODY WEIGHT AND WEIGHT GAIN
- Group mean bodyweight gains for males receiving 15 or 1000 mg/kg/day were slightly lower than control between Days 1 and 28. As no similar difference was recorded for males receiving 150 mg/kg/day and overall individual gains for males at 15 or 1000 mg/kg/day were within or very close to the concurrent control range, these differences from control were considered not to be treatment related. Body weight gain for all treated groups of females was similar to controls.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
- Overall food consumption values were similar to control for all treated groups.

FOOD EFFICIENCY
- not applicable

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
- not applicable

OPHTHALMOSCOPIC EXAMINATION
-not applicable

HAEMATOLOGY
- After 4 weeks of treatment lower than control group mean haematocrit and haemoglobin values were recorded for both sexes receiving 1000 mg/kg/day. Slightly lower than control mean cell haemoglobin values were recorded for both sexes receiving 1000 mg/kg/day, lower than control red blood cell and mean cell haemoglobin concentration values were recorded for females receiving 1000 mg/kg/day and a lower than control group mean cell volume was recorded for males receiving 1000 mg/kg/day. Females receiving 1000 mg/kg/day did tend to show higher than control lymphocyte values, which impacted on the mean total white blood cell value. A similar difference was not clearly evident in treated males. Group mean platelet counts were higher than control for females receiving 1000 mg/kg/day. Group mean prothrombin times were longer than control for females receiving 150 or 1000 mg/kg/day, although there was no dose relationship and individual values for females at 1000 mg/k/day were within the concurrent control range. Therefore the differences from controls were considered to represent normal biological variation rather than treatment. Slight to moderate hyper-chromasia was recorded for all females and 2/5 males receiving 1000 mg/kg/day. These changes do not clearly correlate with the effects on red blood cells and thus the toxicological importance of the finding was doubtful. No abnormalities of the blood morphology were recorded for the control or any other treated groups.


CLINICAL CHEMISTRY
- After 4 weeks of treatment higher than control group mean blood urea values were recorded for both sexes receiving 1000 mg/kg/day, with statistical significance attained for the females. Higher than control statistically significant asparate amino-transferase and glucose values were recorded for males receiving 1000 mg/kg/day and higher than control cholesterol values were recorded for females receiving 1000 mg/kg/day, however, statistical significance was not attained. In addition a higher than control albumin mean, resulting in higher mean total protein and albumin/globumin ratio was recorded for males receiving 1000 mg/kg/day.

URINALYSIS
- not applicable

NEUROBEHAVIOUR
- Sensory reactivity observations and grip strength values during Week 4 of treatment were unaffected by treatment. Motor activity scores were considered to be unaffected by treatment with the test substance. High beam scores (rearing activity) and low beam scores (cage floor activity) for males in all treated groups, tended to be slightly high when compared with controls but there was no dose relationship and the group mean total scores were all within historical control data range and showed no statistical significance. High beam and low beam scores for females receiving 1000 mg/kg/day were low when compared with control, but the differences failed to achieve statistical significance and comparisons with historical control data showed that the low beam scores for the concurrent control group were atypically high. These inter-group differences were, therefore, not attributed to treatment.

ORGAN WEIGHTS
- Higher than control group mean bodyweight adjusted liver weights were recorded for both sexes receiving 1000 mg/kg/day and females receiving 150 mg/kg/day with a dose relationship observed for the females. The bodyweight adjusted group mean spleen weight for females receiving 1000 mg/kg/day was elevated above control and statistical significance was attained. All treated female groups showed higher than control bodyweight adjusted mean adrenal weights. There was however no dose relationship and the absolute values showed good concordance with controls. Therefore an effect of treatment was doubtful and in the absence of any corresponding histopathological finding on the adrenals these differences from controls, if treatment related, were considered not to be of toxicological importance. Slightly lower than control absolute ovary weights and higher body weight adjusted kidney weights were recorded for females receiving 1000 mg/kg/day. The majority of individual absolute ovary and kidney values were however within the concurrent control range and in the absence of any corresponding adverse microscopic findings in either organ, the differences from control were considered not be of toxicological relevance. Organ weights for treated groups were similar to control for all other organs examined.

GROSS PATHOLOGY
- An increased incidence of liver enlargement was noted in male (3/5) and female (4/5) rats treated with 1000 mg/kg/day compared with none in the control rats. The incidence and distribution of all the other findings were considered to fall within the background range of macroscopic changes.

HISTOPATHOLOGY: NON-NEOPLASTIC
- Generalized hepatocyte hypertrophy/eosinophilia was seen in both sexes receiving 1000 mg/kg/day (2/5 minimal and 3/5 slight in males and 4/5 minimal and 1/5 slight in females). Follicular cell hypertrophy was seen in females receiving 1000 mg/kg/day (3/5 minimal and 1/5 slight). A slight increase in the severity of extramedullary haemopoiesis in the spleen was seen in females receiving 1000 mg/kg/day (2/5 minimal and 3/5 slight). Seminiferous tubules were evaluated with respect to their stage in the spermatatogenic cycle and the integrity of various cell types present within the different stages. No cell or stage abnormities were noted. All other findings were considered to be incidental and unrelated to the test substance.

HISTOPATHOLOGY: NEOPLASTIC
not applicable

HISTORICAL CONTROL DATA
see above results

OTHER FINDINGS
not applicable

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Sex:

not specified

Basis for effect level:

other: The effects on red blood cells in females at 1000 mg/kg/day were considered to represent toxicity. There were no findings of toxicological importance at 150 mg/kg/day.

2

Dose descriptor:

NOEL

Effect level:

15 mg/kg bw/day (nominal)

Sex:

not specified

Basis for effect level:

other: overall effects

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Formulation Chemistry:

All formulations were shown to be homogenous in the vehicle and stable at ambient temperature for 2 days and for 8 days following refrigerated storage (approximately 4 deg C). The mean concentrations of the test substance in Weeks 1 and 4 were between 0.9 and 3.7% of intended and were therefore considered satisfactory.

Discussion

This study was performed to assess the systemic toxic potential of the test substance to rats following daily oral gavage administration for 4 weeks at dose levels of 15, 150 or 1000 mg/kg/day. Treatment was generally well tolerated, however, treatment related changes were recorded for haematology, blood chemistry and pathology parameters in both sexes receiving 1000 mg/kg/day and to a lesser extent in rats receiving 150 mg/kg/day.

A high spleen weight and high incidence of extramedullary haemopoiesis in the spleen was observed for females receiving 1000 mg/kg/day when compared with control. This change is likely to be an adaptive response to the effects seen on the red blood cells in these animals. The adaptive response itself is not considered to be adverse but considering that the magnitude of effect on the red blood cells in females at 1000 mg/kg/day (0.89X control) was sufficient to drive an adaptive response, the red blood cell effects in these females are considered to be indicative of toxicity. Low red blood cell parameters were also recorded, for males receiving 1000 mg/kg/day, however, the lack of microscopic abnormalities in the spleen was considered to be at least partly due to the severity of the red blood cell effect in these males being less than in similarly treated females.

The heavy, enlarged livers seen in both sexes receiving 1000 mg/kg/day and females receiving 150 mg/kg/day are considered to be a consequence of the hepatocyte hypertrophy. This is a common finding on rat toxicity studies and in the absence of any adverse effects these findings are taken as indicative of an adaptive response, not toxicity. These differences from control may be linked to the high blood glucose, albumin and A/G ratio recorded for males at 1000 mg/kg/day and the high cholesterol level recorded for females receiving 1000 mg/kg/day; resulting from a general increase in metabolic activity in the liver. These disturbances in blood chemistry are considered to be treatment related but not adverse within the context of this study. The aetiology of the elevated aspartate aminotransferase is unknown, but in the absence of any adverse microscopic changes in the liver and the small magnitude of difference from controls, within the context of this study, this finding is considered not to be of toxicological importance.

The follicular cell hypertrophy observed in the thyroid is a common finding on rat toxicity studies such as this where hepatocyte hypertrophy and hence increased metabolic activity of the liver is evident. This thyroid change occurs due to increased metabolism of thyroid hormone in the liver. This in turn decreases the levels of circulating thyroid hormones, which then feedback onto the thyroid via the pituitary resulting in the thyroid increasing thyroid hormone production to try and maintain homeostasis. This mechanism is not relevant to humans due to species differences in thyroid hormone metabolism and thus the thyroid finding as evidenced on this study is not of toxicological importance when predicting possible adverse human events.

The aetiology of the elevated lymphocyte values in some females receiving 1000 mg/kg/day was not determined from this study. There were however no indications that these slightly elevated values were the cause/linked with any adverse events on this study and thus they are considered not of biological/toxicological relevance.

Higher than control group mean blood urea values were also recorded for both sexes receiving 1000 mg/kg/day, however, in the absence of any corresponding pathological findings, this difference from control is not considered to be of toxicological importance.

Applicant's summary and conclusion

Conclusions:

The systemic toxic potential of the test substance to Crl:CD (SD) rats by oral administration was assessed over a period of 4 weeks. The NOAEL was determined to be 150 mg/kg/day and the NOEL was determined to be 15 mg/kg/day.

Executive summary:

Not applicable