Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 June 2015 to 24 June 2016
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: extremely viscous amber liquid
- Analytical purity:
- Lot/batch No.: E00031-68-1
- Expiration date of the lot/batch: 16 October 2010
- Storage condition of test material:room temperature in the dark, under nitrogen

Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/batch No.of test material: E00031-541
- Expiration date of the lot/batch: 01 November 2016
- Purity test date: > 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature, in the dark

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Han Wistar rat was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Kent, UK.
- Age at study initiation: 7-8 weeks old
- Weight at study initiation: Males: 190-233 g; females: 136-178 g
- Housing: Prior to stratification, animals were randomly housed up to 5 per cage per sex. After stratification, animals were housed randomly 2 per cage by sex with similar group mean body weights
- Diet (e.g. ad libitum): ad libitum, except during designated procedures
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23 °C (There were occasions where the target environmental conditions were not maintained. However these were considered to be transient, the animals didn't show any clinical signs and, therefore, did not have any effect on the outcome or integrity of the study).
- Humidity (%): 40-70%
- Air changes (per hr): 10 or more air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

IN-LIFE DATES: From: 07 July 2015 To: 06 October 2015

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test and control items were administered to the appropriate animals once daily by oral gavage from Day 1 to Day 90 and up to the day before scheduled euthanasia.
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations were prepared based on a method established at the Test Facility at appropriate concentrations to meet dosage level requirements. The dosing formulations were prepared at weekly intervals. The formulations were split into suitable volume aliquots for daily dispensing, and stored in a refrigerator set to maintain 4°C, and dispensed daily. The dosing formulations were removed from the refrigerator for at least 30 minutes before dosing.
The control formulation, Arachis Oil BP, was dispensed as received and was stored in a refrigerator set to maintain 4°C, and dispensed daily. The control formulation was removed from the refrigerator at least 30 minutes prior to dosing.

VEHICLE
- Concentration in vehicle: 0, 25, 75 and 250 mg/mL of EXP0700332 in vehicle
- Amount of vehicle (if gavage): 4 mL/kg
- Lot/batch no: A0344960
- Purity: 100%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test item dosing formulations were prepared based on a method established at the Test Facility at appropriate concentrations to meet dosage level requirements. The dosing formulations were prepared at weekly intervals. The dosing formulations were split into suitable volume aliquots for daily dispensing, and stored in a refrigerator set to maintain 4°C, and dispensed daily. The dosing formulations were removed from the refrigerator for at least 30 minutes before dosing.
Duplicate top, middle and bottom (duplicate middle only for control) samples (1 mL) for each sampling time point were sent to the analytical laboratory. In addition, triplicate top, middle, and bottom (triplicate middle only for control) samples (1 mL) were maintained at the Test Facility as backup samples. For concentration, the criterion for acceptability was mean sample concentration results within or equal to ± 10% of theoretical concentration. For homogeneity, the criterion for acceptability was a relative standard deviation (RSD) of concentrations of = 10% for each group.
Duration of treatment / exposure:
90 Days
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The Han Wistar rat was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies.
The total number of animals used in this study was considered the minimum required to properly characterise the effects of the test item. This study was designed such that it did not require an unnecessary number of animals to accomplish its objectives.
The animals were allowed to acclimate for 2 weeks before dosing. The test and control items were administered to the appropriate animals once daily by oral gavage from Day 1 to Day 90 and up to the day before scheduled euthanasia. The doses were given using a syringe with attached gavage cannula. The first day of dosing was designated as Day 1. The dosing formulations were stirred for at least 30 minutes before the start of administration and continuously during dose administration.
The oral route of administration was selected for this study as this route was defined by the Sponsor as a possible route of human exposure. The dose levels were agreed with the Sponsor following evaluation of a previous 28-day repeat dose oral (gavage) toxicity study in the rat (OECD Guideline 407) and a reproduction / oral gavage developmental toxicity screening study in the rat (OECD Guideline 421; Charles River Study No. 496381) performed on behalf of the Sponsor, where no significant signs of toxicity were noted at dosage levels of 30, 300 or 1000 mg/kg/day.
The animals were observed twice daily, once at the start and once towards the end of the working day, for general health/mortality and moribundity. Animals were not removed from their cage during observation, unless necessary for identification or confirmation of possible findings. The animals were removed from the cage and examined weekly commencing in Week -1 and continuing throughout the course of the study for a detailed clinical observation. Bosy weights, food consumption, water consumption, ophthalmic examinations, functional and cageside observations were performed throughout the study.
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
Posture/condition on first approach (animal undisturbed), checking for:
Prostration, Lethargy, Writhing, Circling, Breathing abnormalities, Gait abnormalities, Tremor, Fasciculation, Convulsions, Biting (of cage components or self mutilating), Vocalisations and Piloerection.
Ease of removal from the cage
Body temperature: A rectal temperature was taken.
Condition of the eyes, checking for:
Pupillary function, Miosis, Mydriasis, Exophthalmos, Encrustation and Lacrimation.
Condition of the coat.
Presence of salivation.
Overall ease of handling.

DETAILED CLINICAL OBSERVATIONS: Yes
All animals were examined for reaction to treatment regularly throughout the day. The onset, intensity and duration of these signs were recorded (if appropriate), particular attention being paid to the animals during and for the first hour after dosing.

BODY WEIGHT: Yes
All animals were weighed individually once during pretreatment and daily during the dosing period. Body weights were also recorded on the first day of scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption was quantitatively measured weekly, commencing at the start of Week -1

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Water consumption was visually assessed throughout the study. There were no differences seen that were considered to be related to treatment.

OPHTHALMOSCOPIC EXAMINATION: Yes
The eyes of all animals were examined during pretreatment and in Groups 1 and 4 during Week 13 using an indirect ophthalmoscope after the application of a mydriatic agent (1% Tropicamide, Mydriacyl®). The anterior, lenticular and fundic areas were examined.

HAEMATOLOGY: Yes
Red blood cell count, Haemoglobin, Haematocrit, Mean cell volume, Mean cell haemoglobin concentration, Mean cell haemoglobin, Reticulocytes, Reticulocyte count (absolute), Red blood cell distribution width, Platelets, White blood cell count, Neutrophils, Lymphocytes, Monocytes. Eosinophils, Basophils, Large unstained cells, Other cells (as appropriate)

CLINICAL CHEMISTRY: Yes
Blood samples (approximately 1.0 mL) were collected into lithium heparin, processed for
plasma, and analysed for the parameters:
Urea, Glucose, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Creatine phosphokinase, Lactate dehydrogenase, Sodium, Potassium, Chloride, Blood Urea Nitrogen, Total protein, Albumin, Globulin, Albumin/globulin ratio, Cholesterol, Creatinine, Total bilirubin, Calcium, Inorganic phosphate, Triglycerides

URINALYSIS: Yes
Urine was collected from animals individually housed in metabolism cages over approximately 18 h with absence of food/presence of water. After collection, samples were transferred to the appropriate laboratory for processing. Urine samples were processed and analysed for the parameters:
Microscopic evaluation of spun deposit, Colour, Turbidity, Specific gravity, Volume, pH, Protein, Glucose, Bilirubin, Ketones, Leukocytes, Blood Pigments, Urobilinogen

NEUROBEHAVIOURAL EXAMINATION: Yes
Battery of functions tested: sensory activity / grip strength / motor activity

OTHER:
Morality/Moribundity Checks:
Animals were observed twice daily, once at the start and once towards the end of the working day, for general health/mortality and moribundity. Animals were not removed from their cage during observation, unless necessary for identification or confirmation of possible findings.
Sacrifice and pathology:
All animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. Scheduled necropsy examinations were conducted by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.
All animals were weighed, and euthanised by exposure to a rising concentration of carbon dioxide, followed by exsanguination. When possible, the animals were euthanised rotating across dose groups such that similar numbers of animals from each group, including controls were necropsied at similar times throughout the day. Animals were not fasted before their scheduled necropsy.
Histopathological evaluation was performed by the Principal Investigator at Charles River Pathology Associates, who is a board-certified veterinary pathologist.
Statistics:
Unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in-house software. Males and females were analysed separately.
Body weight, food consumption, selected functional observational battery and motor activity data, haematology, coagulation, clinical chemistry and selected urinalysis data were analysed for homogeneity of variance using the ‘F-Max' test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test i.e. pairwise comparisons were made only if the overall F-test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test).
In circumstances where it was not possible to perform the F Max test due to zero standard deviation in at least one group, the non-parametric ANOVA resultswere reported.
Organ weights were analysed using ANOVA as above and by analysis of covariance (ANCOVA) using terminal kill body weight as covariate. In addition, organ weights as a percentage of terminal body weight were analysed using ANOVA as above as an exploratory analysis.
In circumstances where the variances in the ANCOVA remained heterogeneous following log or square root transformations, the data was subjected to a rank transformation prior to analysis.
In the ANOVA and ANCOVA summary tables, the results of the analysis were reported indicating the level of statistical significance (p<0.05, p<0.01 and p<0.001) of each pairwise comparison. Actual p-values were not reported in the summary tables for these analyses.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical Observations:
Excess salivation was noted immediately post dose on Days 59, 70-73 in up to 3 animals given 100 mg/kg/day or males given 300 or 1000 mg/kg/day. Due to the isolated incidences, it was considered that the clinical observations were due to the dosing technique rather than the test item.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weight gain at the end of the treatment period was statistically significantly lower in females given 100 mg/kg/day when compared with controls. However, due to the relatively small magnitude of change and the lack of a dose relationship this was considered to be incidental.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption was slightly higher throughout the study in males given 1000 mg/kg/day when compared with controls, with statistical significance being achieved on Day 42 only. However, due to all values, with the exception of Day 42, being within 10% variation of the control values this was considered not to be toxicologically significant.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
Coagulation - There were no differences in any coagulation parameter that were considered to be treatment related.
Prothrombin time was longer in animals given 1000 mg/kg/day and males given 100 mg/kg/day when compared with controls. In addition, activated partial thromboplastintime was longer in males given 1000 mg/kg/day when compared with controls. However, due to the small magnitude of change and the lack of a correlating change in fibrinogen this was considered not to be treatment related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Creatine phosphokinase was lower females given 1000 mg/kg/day and animals given 100 mg/kg/day when compared with controls. However, due to the small magnitude of difference and the direction of the change this was considered not to be treatment related.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional Observations:
Hind grip values were statistically significantly higher and tail flick values were statistically significantly lower at Week 12 in males given 1000 mg/kg/day when compared with controls. However, due to the small magnitude of difference from pre-treatment values, the lack of any other neurotoxicity signs and larger differences in control animals these differences were considered to be incidental.

Motor Activity:
There were isolated incidences of statistical significance noted in the X and Y-ambulation when compared to controls. However, due to the small magnitude of difference, the lack of consistency and this was not seen in any other parameters this was considered to be incidental.
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Absolute and covariate spleen weight were statistically significantly lower in females given 100 mg/kg/day when compared with controls. However due to the small magnitude of change and the lack of any dose relationship this was considered incidental and unrelated to EXP0700332 administration.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test item-related gross findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of EXP0700332.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No test item-related microscopic findings were noted. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of EXP0700332.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Urinalysis - no differences in any urinalysis parameter that were observed were considered treatment related

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
ophthalmological examination
haematology
clinical biochemistry
urinalysis
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, administration of EXP0700332 by once daily oral gavage was well tolerated in rats at levels of 100, 300 and 1000 mg/kg/day. There were no treatment-related clinical observations or target organ effects. Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg/day.
Executive summary:

The objective of this study was to determine the potential toxicity of EXP0700332 when given orally for 91 consecutive days to rats.

40 male and 40 female rats were randomly organized into 4 (1-4) dose groups of 10 animals per sex per dose with dosage levels of 0, 100, 300 and 1000 mg/kg/day.

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight changes, food consumption, ophthalmology, detailed functional observations, clinical pathology parameters (haematology, coagulation, clinical chemistry, and urinalysis), gross necropsy findings, organ weights, and histopathologic examinations.

All analysed formulation concentrations were found to be within the acceptable range and homogeneous.

There were no unscheduled deaths during the study.

There were no clinical observations noted that were considered to be treatment related. Excess salivation was noted in 3 animals, however, these were isolated incidences and were considered to be related to the dosing technique.

Any differences seen were considered unrelated to the administration of EXP0700332.

In conclusion, administration of EXP0700332 by once daily oral gavage was well tolerated in rats at levels of 100, 300 and 1000 mg/kg/day. There were no clinical observations or target organ effects. Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg/day.