Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006-10-16 to 2007-10-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study performed according to OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents) with the following deviation from the protocol reported by the testing facility: During Week 3 of treatment, an error was made in the recording of the food data at the mid-week top up feed. The weight of the food residue and food given was not correctly recorded. This operator error resulted in the exclusion of the food data for Week 3. This deviation was considered to have not affected the integrity or validity of the study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
During Week 3 of treatment, the weight of the food residue and food given was not correctly recorded. This resulted in the exclusion of the food data for Week 3. This deviation was considered to have not affected the integrity or validity of the study.
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. certificate)
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: VRT-753136 HCL [(3s,2s)-N-cyclopropyl-3-amino-2-hydroxyhexanamide hydrochloride]
- 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: 100 area % (HPLC)
- Impurities: 0.03 area % of individual unknowns
- Composition of test material, percentage of components: 99.4 % w/w test substance
- Isomers composition: no data
- Purity test date: 2006-05-25
- Lot/batch No.: batch 25515, lot AF6-001
- Expiration date of the lot/batch: 2008-05
- 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: at ambient temperature
- Other: received on 2006-09-15

Test animals

Species:
rat
Strain:
other: Crl:CD (SD)IGS BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 36-42 days
- Weight at study initiation: 181-232 g (males) and 142-174 g (females)
- Fasting period before study: no data
- Housing: Animals were housed inside a barriered rodent facility. 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. Before the study, the room was cleaned and disinfected with a bactericide. The animals were housed five of one sex per cage. The cages were made of polypropylene with a solid bottom, with Lignocel 3/4 wood flakes bedding. 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. Each cage was provided with an Aspen chew block for environmental enrichment. Chew blocks were provided throughout the study and were replaced when necessary, but at a minimum frequency of every two weeks.
- 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.
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (deg C): 19-23
- Humidity (%): The relative humidity was maintained within the range of 40 to 70%. However, there were isolated fluctuations which resulted in an overall range of 23-59 %. This minor deviation was not considered to have affected the integrity of the study.
- 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-recirculated. Periodic checks were made on the number of air changes in the animal rooms. No further data were provided.
- Photoperiod: 12 hours light/12 hours dark


IN-LIFE DATES: From: 2007-01-18 To: 2007-02-15

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was prepared for administration as a series of graded concentrations in the vehicle. The required amount of the test substance was accurately weighed, and appropriate amount of vehicle was added and the formulation was mixed by using a high shear homogenizer and then magnetic stirring until dissolution was achieved. The solution was then diluted to volume with the vehicle and finally mixed by magnetic stirring. A series of solutions of the required concentrations were prepared by serial dilution of the primary solution with the vehicle. The test substance was used as supplied. All formulations were prepared freshly each week and stored at 4 deg C. 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.

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/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 formations 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:
All 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 males and 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses used in the study were selected in conjunction with the Sponsor with reference to a 7 day preliminary toxicity study with this test substance. In the preliminary study, a dose level of 1000 mg/kg/day was well tolerated. The dose levels were selected based on EC labeling 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.
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale: not applicable
Positive control:
none

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. 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 the first week of treatment. During the acclimatisation 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 examination 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 behaviour 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 of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behaviour. Findings were either reported as "present" or assigned a severity grade - 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 that treatment commenced (Day 1), weekly throughout the treatment (last scheduled bodyweight was recorded on Day 28), and before necropsy.


FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean weekly consumption calculated as g food/kg body weight/day: 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: No
- Other: During Week 3 of treatment, an error was made in the recording of the food data at the mid-week top up feed. The weight of the food residue and food given was not correctly recorded. This operator error resulted in the exclusion of the food data for Week 3. This deviation was considered to have not affected the integrity or validity of the study.


FOOD EFFICIENCY: No
- 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: Not applicable


WATER CONSUMPTION AND COMPOUND INTAKE: No
- 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)
- Anaesthetic used for blood collection: Yes; isoflurane
- Animals fasted: Yes; overnight
- How many animals: all animals
- Parameters examined: Blood samples withdrawn from the sublingual vein (nominally 0.5 mL) were collected into tubes containing EDTA as an anticoagulant and examined for the following characteristics 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 white blood cell 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 change, anisocytosis, micro/macrocytosis and hypo/hyperchromasia were recorded as follows: (-) = no abnormalities detected, (+) = slight, (++) = moderate, and (+++) = marked.
- Blood film (prepared for all samples)-Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer.
- Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrated anticoagulant and examined in respect of: prothrombin time using an ACL 3000 Plus Analyzer and IL PT-Fibrinogen reagent and activated partial thromboplastin time 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)
- Animals fasted: Yes; overnight
- How many animals: all animals
- Parameters examined: At the same time and using the same animals as for haematology, further blood samples (nominally 0.7 mL) were collected into tubes containing lithium heparin as an anticoagulant. All tubes were mechanically agitated for at least two minutes and the samples subsequently centrifuged at 3000 rpm for 10 minutes in order to separate the plasma. After separation, the plasma was examined using a Hitachi 917 Clinical Chemistry Analyzer in respect of: alanine aminotransferase, aspartate aminotransferase, urea, creatinine, glucose, total cholesterol, sodium, potassium, total protein and albumin.
- Albumin/globulin ratio was 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 applicable


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 4 of treatment before dosing. (Animals were not necessarily all tested on the same day, but the numbers 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
- Battery of functions tested: Sensory reactivity and grip strength assessments were performed. The following measurements, reflexes and responses were recorded: approach response, touch response, auditory startle response, tail pinch response, and grip strength. In addition, motor activity was measured.


OTHER:
not applicable









Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- All 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 subject to a detailed necropsy.
- After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features 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 associated 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 were weighed and external and cut surfaces of the organs and tissues were examined as appropriate. 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 checked before disposal of the carcass.
- The following organs, taken from each animal killed after 4 weeks of treatment, were dissected free of adjacent fat and other contiguous tissue and the weights recorded: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes and thymus.
- Bilateral organs were weighed together. Organ weights were also adjusted for terminal bodyweight, using the weight recorded immediately before necropsy.

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 other following tissues from all animals were preserved in 10% neutral buffer 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 (including bronchi), lymph nodes (mandibular and mesenteric), oesophagus (not processed for examination), ovaries, pancreas (not processed for examination), prostate, rectum, sciatic nerves (only one processed for examination), seminal vesicles, spinal cord, spleen, sternum (not processed for examination), stomach, testes, thymus, thyroid with parathyroids, trachea, urinary bladder, uterus and cervix.
- Samples of any abnormal tissues were also retained and processed for examination. 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 samples (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 subject 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 (section from all main lobes), lungs (section from two major lobes, to include bronchi), spinal cord (transverse and longitudinal section at the cervical, lumber 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:
1. All tissues preserved for examination (as specified above) were examined for all animals of the control and high dose group sacrificed on completion of the scheduled treatment period.
2. Tissues reported at macroscopic examination as being grossly abnormal were examined for Main animals in line with current practice.
Findings were either reported as "preset" or assigned a severity grade. In the latter case, one of the following grades was used - minimal, slight and moderate.
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:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths.
Bright yellow urine was noted occurring among animals treated at 1000 mg/kg/day from Day 4 and to a lesser extent among animals treated at 150 mg/kg/day from Day 13. This was associated with the bright yellow staining of the bedding noted from Day 4 among animals treated at 1000 mg/kg/day and transiently from Days 20 to 23 among animals treated at 150 mg/kg/day. In addition, an incidental finding of chin rubbing was observed occurring in one or more animals treated at 1000 mg/kg/day from Day 4. Chin rubbing was noted immediately after dosing, but was not generally seen by 1 to 2 hours after dosing in the majority of animals, although on occasions it did persist until the end of the day observation. Chin rubbing was also noted on odd occasions from Day 9 among some animals treated at 150 mg/kg/day.


BODY WEIGHT AND WEIGHT GAIN
Bodyweight gain during the period Weeks 0 to 4 of treatment was slightly higher than control for both sexes treated at 1000 mg/kg/day and for males treated at 150 mg/kg/day, although the individual gains were generally within the concurrent control range. Mean bodyweight gain for both sexes treated at 15 mg/kg/day and for females treated at 15 or 150 mg/kg/day, was essentially similar to that of their respective controls.

FOOD CONSUMPTION AND COMPOUND INTAKE
Food intake during the treatment period was considered to have been unaffected by treatment. The slightly higher food intake recorded for males treated at 150 mg/kg/day, in comparison with controls was not seen in animals treated at 1000 mg/kg/day, thus it was considered not to be attributable to treatment.

FOOD EFFICIENCY
not applicable

WATER CONSUMPTION AND COMPOUND INTAKE
not applicable

OPHTHALMOSCOPIC EXAMINATION
not applicable

HAEMATOLOGY
There were no findings considered to be related to treatment. After 4 weeks of treatment higher than control monocyte counts were noted for all males treated at 1000 mg/kg/day, with the mean value attaining statistical significance. The individual values, however, were within the background control range for this parameter (5% = 0.09, 50% = 0.24, 95% = 0.56). In the absence of a similar change among females treated at 1000 mg/kg/day or other treatment-related changes in the haematology parameters, this increase in monocyte values was considered not to be associated with treatment.
There were no other differences from control in the haematology parameters measured at termination (including the minor differences for female mean MCH, MCV and eosinophil values, which attained statistical significance) that were considered to be attributable to treatment.

CLINICAL CHEMISTRY
There were no findings considered to be related to treatment. The low mean urea value for females treated at 1000 mg/kg/day in comparison with controls, that attained statistical significance, was mainly due to the low value of one female, which was also below the lower end of the control background range for this parameter (5% = 3.93, 50% = 5.41, 95% = 7.67). The individual values for the remaining females in this group were within the background control range for this parameter. The other differences in the blood chemistry parameters from control or between the treated groups were considered to be a reflection of normal biological variation.


URINALYSIS
not applicable

NEUROBEHAVIOUR
Assessment of the detailed physical examination and arena observations carried out during the treatment period did not reveal any clinical signs or changes in behaviour that were considered indicative of neurotoxicity. Sensory reactivity observations and grip strength values were unaffected by treatment. Motor activity scores showed no effect of treatment.

ORGAN WEIGHTS
Group mean liver weights (adjusted for bodyweight) were higher than control among females treated at 150 and 1000 mg/kg/day, with the values attaining statistical significance. Other minor variations in organ weight when compared with controls, including the slightly higher brain weight for males treated at 1000 mg/kg/day that attained statistical significance, were considered not to be related to treatment, but to be a reflection of normal biological variation.

GROSS PATHOLOGY
The macroscopic examination performed at termination revealed no lesions attributable to treatment with the test substance. The incidence and distribution of all the findings were considered to fall within the background range of macroscopic changes.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no findings considered to be related to treatment with the test substance. Findings of uncertain relationship to treatment included the following:
- Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage-specific abnormalities were noted.
- In the testes, slightly increased incidences of seminiferous tubular vacuolation at a minimal level, seminiferous tubular epithelial degeneration at a mainly minimal level and a case of unilateral seminiferous tubular epithelial degeneration at a slight level were seen in males treated at 1000 mg/kg/day. As single incidences of each of these findings were also seen in controls, they are considered to be a chance event and not related to the treatment of
the test substance.

All other findings were considered to be incidental and unrelated to the test substance.

HISTOPATHOLOGY: NEOPLASTIC
not applicable


HISTORICAL CONTROL DATA
see above sections

OTHER FINDINGS
none

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: In the study, minor treatment-related changes were observed among both sexes treated at 1000 mg/kg/day. However, there was an absence of any clear evidence of toxicity.
Dose descriptor:
NOEL
Effect level:
15 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No observed 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 Week 1 were between 3.1 and 5.7% of nominal and were therefore considered satisfactory.

Discussion

This study was performed to assess the systemic toxic potential of VRT-753136.HCL to rats following daily oral gavage administration for four weeks at dose levels of 15, 150 or 1000 mg/kg/day. A further group received the vehicle alone and acted as controls.

Treatment at these dose levels was well tolerated. The principal effect of treatment was the bright yellow urine and staining of the bedding material, which may be a result of excretion of a metabolite. There was no indication from this study that this finding was of toxicological importance. Although chin rubbing was observed among animals treated at 1000 mg/kg/day and to a lesser extent among animals treated at 150 mg/kg/day, the finding is often associated with the taste of the test material. Thus in the absence of any other clinical finding or behavioural change this finding is considered not to be of toxicological importance. The liver weights among females treated at 150 or 1000 mg/kg/day were slightly higher than control, however, there were no associated microscopic changes observed in the liver, thus, this weight change is considered to be a sub-clinical adaptive response by the liver to cope with the elimination of the test material. Bodyweight gain was also affected, being slightly increased mainly among both sexes treated at 1000 mg/kg/day. However, these changes were considered to be minor and in the absence of any clear finding that might be indicative of systemic toxicity, they are considered not to be of toxicological importance.

There were no treatment-related findings observed among both sexes treated at 15 mg/kg/day.

Applicant's summary and conclusion

Conclusions:
The systemic toxic potential of the test substance to male and female rats by oral administration was assessed over a period of 4 weeks. In this study, minor treatment-related changes were observed among both sexes treated at 1000 mg/kg/day. In the absence of any clear evidence of toxicity, it was concluded that a dose level of 1000 mg/kg/day was assigned the highest No Observed Adverse Effect Level (NOAEL). A dose level of 15 mg/kg/day was assigned as the No Observed Effect Level (NOEL).
Executive summary:

not applicable