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Description of key information

In a 104-week study, no evidence of carcinogenic effects were observed after oral administration of AHU 377 by gavage to rats at doses 50, 150 and 400 mg/kg/day. 

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 16 February 2010 to 28 March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 451 (Carcinogenicity Studies)
Version / remarks:
2018
Principles of method if other than guideline:
The purpose of this study was to investigate the carcinogenic potential of AHU377, when administered by daily oral gavage to the rat for a minimum of 104 weeks. In addition, the study confirmed exposure to AHU377 and its active metabolite LBQ657.

Wistar Hannover Crl:WI (Han) rats were supplied by Charles River St-Constant, Quebec, Canada. At initiation of dosing, rats were approximately 8 weeks of age, the males weighed 197 to 267 g and the females weighed 146 to 201 g. AHU377 (Batch no. 0824011, salt/base ratio of 1.046) was administered orally by gavage in 0.5% (w/v) hydroxypropylcellulose, to 4 groups of rats (50/sex/group) at doses of 0, 50, 150 or 400 mg/kg/day (base) for at least 104 weeks. In addition, four animals/sex/group served as toxicokinetic animals, while an additional 10 males and 10 females were selected from the total population and used as health screen animals (group 5).

Activities performed included clinical examinations (twice daily examinations for mortality and signs of ill health or reaction to dosing, daily post dosing cage side observations, weekly detailed examinations, palpable mass examinations on main study animals), body weights, food consumption, laboratory investigations (hematology) for health screen animals prior to dosing initiation and for main animals at study termination (blood smears). Toxicokinetic evaluations were performed during weeks 4 and 26 from 2 rats/sex/group at 0.5 and 1 hour post dose. Post-mortem evaluations for main study animals included macroscopic observations and microscopic observations.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Active ingredient(s): AHU377 (also identified as AHU377 CALCIUM MODB FINE, AHU377-BAA.002)
Lot no./Batch no.: 0824011
Formulation: Suspension in aqueous 0.5% (w/v) hydroxypropylcellulose
Salt/base ratio: 1.046
Content of active ingredient in % of declaration: 99.2%
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rattus norvegicus, Wistar Hannover Crl:WI (Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
3.2.1 Experimental animals:
Animal species and strain: Rattus norvegicus, Wistar Hannover Crl:WI (Han)
Breeder/Supplier: Charles River Canada, St-Constant, Quebec Canada
No. of animals ordered: 249 males and 275 females
No. of animals assigned to the dosing phase: 216 males and 216 females (200/sex for the main phase and 16/sex for toxicokinetics); 24 females were assigned as sentinel animals
Age: 8 weeks (at start of dosing)
Body weight range: 197 to 267 g for the males and 146 to 201 g for the females (at start of dosing)
Acceptability: Following arrival, each animal was given a general physical examination by a member of the veterinary staff to assess health status.In addition, red blood cell count and total and differential white blood cell counts (including blood cell morphology) were performed on health screen animals (10 per sex). Blood samples (using EDTA as anticoagulant) were collected from the abdominal aorta following isoflurane anesthesia.Following a gross pathological evaluation of the health screen animals, all animals were considered suitable for use on this study, with the exception of rejected animals following a pre-dosing ophthalmology exam or the general physical examination.

After the start of dosing, extra/unused animals were released from the study and their disposition documented in the raw data.

3.2.2 Animal quarters/Husbandry:
Building/Animal room: 1-167
Room temperature: 19 - 25°C (target range)
Room relative humidity: 30 - 70% (target range)
Lighting cycle: Fluorescent light for a 12-hour light/12-hour dark cycle(except during designated procedures).
Animal caging: Group housed (up to 3 animals of the same sex and same dosing group together) in polycarbonate bins containing appropriate bedding and equipped with an automatic watering
valve. On occasion, animals were separated for medical treatment as recommended by the veterinarian. The environmental enrichment program was in accordance with the appropriate
SOP. On occasion, animals were provided with nesting material as recommended by the veterinarian. Cages were arranged on the racks in group order and control group animals were housed on a separate rack from the test item-treated animals. To ensure that rats of each dose group were exposed for similar periods of time to different areas of the room, the position of the animal cage racks was rotated once per month. The order of dosing was rotated concurrently with the position of the cage racks within the animal room except on occasions stated in Section

3.3.5. Control animals were always dosed first.
Acclimation period: At least 14 days
Diet: Animals had free access to standard certified pelleted commercial laboratory diet (Certified Rodent Diet 18% no. 5LG3: PMI Feeds, Richmond, Indiana). On occasion, powdered diet (PMI Certified Rodent Diet 18% no. 5LG3- Meal), wet pellets and Nutragel, a non-medicated food supplement were provided as required.
Maximum allowable concentrations of contaminants in the diet (e.g., heavy metals, aflatoxin, organophosphate, chlorinated hydrocarbons, PCBs) were controlled and routinely analyzed by the manufacturers. The results of the analysis are retained at PCS-MTL.

Water: Municipal tap water which had been softened, purified by reverse osmosis and exposed to ultraviolet light was freely available (except during designated procedures). On occasion, water bottles were provided to the animals.
Periodic analysis of the water was subcontracted to management authorized analytical laboratories which were audited by the Quality Assurance department of PCS-SHB.
The analytical results are retained in the archives of PCS-MTL.
Environmental enrichment: Certified polycarbonate rat tunnels and/or 100% nylon bones (e.g., nylabones) and/or wood blocks were provided to the animals.
There were no known contaminants in the food or water that interfered with the conduct of the study.
Route of administration:
oral: gavage
Vehicle:
other: Klucel HF (Hydroxypropyl cellulose)
Details on exposure:
Route and frequency: Oral gavage, daily
Justification for route: Intended route in humans
Dose volume: 10 mL/kg
Administration method: Each rat was given a daily oral dose of vehicle (group 1) or test item (groups 2 to 4) using a plastic gavage tube. Occasionally, refluxes were observed during the course of the study. This was considered not to have affected the overall doses received by the animals or the integrity of the study due to the duration of the study. Each actual volume administered was based on the most recent practical body weight of each animal except on occasions mentioned in Section 3.3.5. All dosing formulations were stirred (via magnetic stir bar and plate) for at least 10 minutes prior to and during dosing except on occasions mentioned in
Section 3.3.5.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
AHU377 suspension of 5 mg/mL in 0.5% Klucel was found stable for 15 days when refrigerated.

AHU377 formulations, 0.96 mg/mL to 123 mg/mL as base (1 mg/mL to 128.7 mg/mL as salt),
were found to be stable for at least 4 hours stirring at room temperature, at least 12 days stored
at 6°C, and at least 1 day stored at room temperature (Long 22-Feb-2010). AHU377 formulations, 5 mg/mL to 120 mg/mL as base, were found to be stable for at least 8 hours stirring at room temperature (Long 12-Dec-2012). AHU377 5 mg/mL as base formulation was also found to be stable for at least 15 days stored at 6°C (Long 19-Apr-2012).
See Table 4-1 and Table 4-2. The control was found to be suitable for use for at least 15 days
stored at 6°C (Long 20-Apr-2012), at least 8 hours stirring at room temperature (Long 17-Dec-2010) and at least 24 hours stored at room temperature.
Duration of treatment / exposure:
at least 104 weeks
Frequency of treatment:
daily
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
base
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
base
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Remarks:
base
No. of animals per sex per dose:
50
Control animals:
yes, concurrent vehicle
Details on study design:
Pretest period: 14 days
Duration of dosing: At least 104 weeks


- Toxicokinetic data: yes
- Dose selection rationale: Dose levels for this study were based on results of the 13-week segment of a 4-week (interim necropsy) and 13-week oral dose range-finding toxicity study in rats with a 4-week recovery period (Novartis study no. 0770711). AHU377 was administered orally by gavage (10 mL/kg) in an aqueous solution of 0.5% Klucel to 4 groups of Wistar Hannover rats at doses of 0, 400, 800 or 1200 mg/kg/day (base). Four early deaths/sacrifices occurred (one male at 800 mg/kg/day and 3 females at 1200 mg/kg/day). The cause of moribundity or death was not identified upon histopathologic assessment of these animals, as there were no microscopic changes observed that were considered directly related to the test item. There was an increase in the incidence and/or frequency of the following clinical signs at 800 and 1200 mg/kg/day: hunched posture and piloerection of the fur in males and females, unkempt coat and slightly decreased locomotor activity in males and abdominal distension in females. Additional observations at 1200 mg/kg/day included red oral stains around the mouth in males and decreased locomotor activity and perineal stains in females. Test item-related decreases in mean body weight parameters were noted at all dose levels in the males, associated with decreases in food consumption at = 800 mg/kg/day. In females, no test item-related effects on mean body weight were noted; however, transient decreases in body weight gain were noted on days 8 and 15 at 800 mg/kg/day and at 1200 mg/kg/day on days 8-22. In the females at 1200 mg/kg/day, these decreases were accompanied by concurrent decreases in mean food consumption values.
There were no test item-related changes in hematology. In clinical chemistry, minimal (less than 2 fold), dose dependent increases in serum alanine aminotransferase activity were apparent in females at all dose groups (= 400 mg/kg/day) and males at = 800 mg/kg/day. As there was no histological correlate and the increase was of minimal magnitude, the change was not considered adverse.
No test item-related macroscopic or microscopic findings were present after dosing for 13 weeks. Test item-related organ weight changes included minimally decreased absolute and relative (to brain) heart weights in males at doses = 800 mg/kg/day. The decreased heart weights could be related to the expected hypotensive effects of AHU377 in lowering stroke volume and decreasing the force of ventricular contraction resulting in decreased cardiac
workload.
Toxicokinetics analysis revealed systemic concentrations of AHU377 and its active metabolite LBQ657 at all doses. No consistent differences in exposure to AHU377 and LBQ657 were observed between males and females following single and multiple oral doses of AHU377. Exposure to AHU377 and LBQ657 increased proportionally with increasing dose. After multiple dosing, the exposures to AHU377 and LBQ657 were higher on day 72 compared to day 1 for both genders. However, there was no evidence of AHU377 or LBQ657 retention.
With the exception of one possible test item-related death at 1200 mg/kg/day, AHU377 was tolerated for 13 weeks. However, based on the increased incidence and/or frequency of dosing-related clinical signs, notably at 1200 mg/kg/day, and the severity of the decreases in mean body weight and food consumption parameters at 1200 mg/kg/day, the maximun tolerated dose (MTD) was considered to be 800 mg/kg/day following 13 weeks of dosing. Based on the results of the 13-week dose range-finding study, the dose levels selected for this 104-week carcinogenicity study were 0, 50, 150 and 400 mg/kg/day (base).

- Rationale for animal assignment (if not random): Prior to the start of dosing, 10 male and 10 female rats were selected from the total population using random numbers for the provision of blood samples and gross pathology examination for health screen purposes. Prior to dosing initiation, all animals were weighed and assigned to dosing groups using a randomization procedure. Randomization was by stratification using body weight as the parameter. Males and females were randomized separately. Animals at extremes of body weight range, in poor health or recommended for rejection by the ophthalmologist based on ocular findings were not assigned to groups.

- Rationale for selecting satellite groups: not relevant
- Post-exposure recovery period in satellite groups: not relevant
Observations and examinations performed and frequency:
3.6 In-life examinations:
Data were collected on an individual animal basis with the exception of food consumption
(when group housed). Veterinary treatments and other appropriate actions (including early euthanasia) were authorized by the Study Director/designee in consultation with a staff Veterinarian and the study Sponsor monitor (if possible), as necessary during the study to minimize discomfort and pain to the animals. All treatments/actions were documented in the raw data file and are included in the report (see Section 3.4). Signed and dated records of activities relating the day-to-day running and maintenance of the study within the animal room, as well as the activity relating to the observations and examinations outlined in the study plan for this study were recorded.

3.6.1 Mortality:
Twice daily (AM and PM) on weekdays and weekends. Once daily on the day of animal arrival and on the last day of necropsy.

3.6.2 Clinical signs
Pretest and dosing periods:
Complete detailed examinations were performed weekly starting the week prior to the dosing period on main study animals, except on occasions stated in Section 3.3.5. In addition, a detailed examination was conducted on all main study animals prior to necropsy. Daily cage side observations were performed at least 2 hours post dose, during the dosing period except on occasions stated in Section 3.3.5. In addition, main study animals were examined for the presence of palpable masses during the detailed examination, except on occasions stated in Section 3.3.5. More observations were undertaken when considered appropriate. The site, size and appearance of these masses were recorded when first detected and, following this initial
description, the presence or disappearance of these masses was monitored. Any mass borne by an animal was given a numerical designation (e.g. M1, M2, etc.) according to order of appearance. Death and observed clinical signs were individually recorded.

3.6.3 Body weight:
Pretest: Once prior to randomization and once on day -1.
Dosing period: Main and TK animals: Once weekly during weeks 1 to 14, every four weeks from weeks 18 to 78 and every 2 weeks thereafter for the remainder of the dosing period. Additional body weights were obtained as part of a health check and were documented in the raw data. Terminal body weights were collected from scheduled necropsy animals.

3.6.4 Food consumption:
Pretest: Once
Dosing period: Cage measurements, measured weekly during weeks 1 to 14, every fourth week from weeks 18 to 78 and every 2 weeks thereafter up to and including week 104. In the event that
food consumption could not be determined, the reason was documented in the raw data file.

3.6.5 Ophthalmology
Pretest: Once on all animals for selection purposes only. All animals (excluding health screen animals) were subjected to funduscopic (indirect ophthalmoscopy) and biomicroscopic
(slit lamp) examinations. The mydriatic used was 1% mydriacil. Examinations were performed by a board certified veterinary ophthalmologist. Pre-dosing examinations revealed no significant ocular defects. Pre-dosing examinations were done only to eliminate rats with major ocular findings from assignment to study. Data for the rejected animals were retained in the raw
data file and not reported.
Sacrifice and pathology:
3.7 Clinical pathology:
Red blood cell count and total and differential white blood cell counts (including blood cell
morphology) were performed on health screen animals (10 per sex). Blood samples (using EDTA as anticoagulant) were collected from the abdominal aorta following isoflurane
anesthesia. In addition, two peripheral blood smears (abdominal aorta) were performed for all euthanized animals (both preterminal and terminal), when possible (according to the condition of the animals). The smears were stained and retained but evaluation was not deemed necessary by the Study Director and the study pathologist. The blood smears were collected in EDTA tubes.

3.8.1 Necropsy procedures
Prior to dose initiation, 10 male and 10 female health screen animals were euthanized by
exsanguination from the abdominal aorta following isoflurane anesthesia and blood sample
collection. These animals were subjected to external and internal gross examinations.
Tissues were not retained.
All animals found dead during the study were subject to necropsy and tissue samples were
preserved. Prior to necropsy, the carcass was stored refrigerated (set to maintain 4°C).

3.8.2 Euthanasia:
Main study animals euthanized on completion of the dosing period, as well as those
euthanized for humane reasons, underwent exsanguination from the abdominal aorta
following isoflurane anesthesia and blood sample collection (from the abdominal aorta).
A similar proportion of animals from each group and sex, as appropriate, were euthanized on
any one day. In order to avoid autolytic change, a complete gross pathology examination of
the carcass was conducted immediately on all animals which were euthanized. Where
possible, the order of necropsy for each prosection group was vehicle control (group 1),
high dose (group 4), mid dose (group 3) and low dose (group 2). All animals were not fasted
overnight prior to scheduled necropsy. All necropsies were conducted under the supervision
of a pathologist and necropsy consisted of an external examination, including identification of
all clinically recorded lesions, as well as a detailed internal examination.After completion of their blood collection, the toxicokinetic animals were euthanized by exsanguination from the abdominal aorta following carbon dioxide asphyxiation and the carcasses were discarded without examination. No necropsy was performed and no tissues were collected.

3.8.3 Sampling and histological processing of tissues:
The bone was decalcified prior to sectioning. The animal identification and nasal cavities
were retained but not processed. The drainage lymph node of clinically observed external
masses was retained only if a gross lesion was present. The epididymides and testes were
fixed in modified Davidson’s fluid (euthanized animals only). The eyes and optic nerves
were fixed in Davidson’s fluid (euthanized animals only). The lungs and nasal cavities were
infused with neutral buffered 10% formalin (all animals). In addition, each clinically observed external mass, or sample of the mass if it was large, (with the nearest identifiable drainage lymph node, when applicable) was preserved and labeled according to the numerical designation on the animal’s clinical observation sheet to facilitate future identification. For all euthanized animals (both preterminal and terminal), three femoral bone marrow smears were prepared. The smears were stained and retained but not evaluated.

3.8.4 Microscopic examination and peer review:
Tissues were prepared for histopathological examination by embedding in paraffin wax,
sectioning and staining with hematoxylin and eosin and examined from all animals.
The optic nerves, mammary glands (males) and thymic lymphoid tissue were examined
histopathologically only if present in routine sections of eyes, skin or thymus, respectively.
Oviducts, salivary glands and ureters were submitted to a unilateral histopathological
examination (although collected bilaterally). All tissue sections (except blood and bone marrow smears) were assessed with all observations being recorded in the raw data. Incidences of diagnosed lesions were tabulated. All suspected tumors were diagnosed, and the incidences of benign and malignant tumors of different cell types in the various treatment groups were tabulated. Histopathological evaluation was performed by a board-certified veterinary pathologist.
Statistics:
The statistical comparisons were performed on main study animals (separated by sex)
excluding health screen animals. All statistical analyses were performed using release 8.2 of
SAS System.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinically observed palpable masses, which were present during the dosing phase and at
necropsy, were generally noted in a similar proportion of animals from control and
AHU377-treated groups. The onset, distribution and incidence of the palpable masses
showed no indication of any difference that could be attributable to AHU377. There were no AHU377-related clinical signs observed during the study.
Clinical signs associated with generally poor or deteriorating condition were observed in
animals from the control and AHU377-treated groups found dead, euthanized prior to the
scheduled necropsies or at terminal necropsies.
These clinical signs included, but were not limited to decreased activity, dehydration, reduced
appetite, prominent backbone, decreased feces output, decreased muscle tone, lying on
side/prostrate, hunched posture, thin body condition, weak condition, generalized skin pallor,
partly closed eyes and breathing difficulties.
Other clinical signs were observed across all groups including controls and were considered
to be incidental (unrelated to test item) in view of their minimal incidence and/or lack of
dose-relationship
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no AHU377-related effects on survival.
During the course of the study, between 12 and 20 rats per group/sex died or were euthanized
prior to the end of the study.

The mortality rate was comparable between all groups for both genders. For each preterminal
decedent rat, the most probable cause of death was determined. The cause of death could not
be determined for a small number of animals per group.
No AHU377-related effect was seen in the distribution of neoplastic or non-neoplastic lesions
contributory to preterminal death or euthanasia of animals in this study.
The most frequent neoplastic causes of early death/euthanasia recorded in control animals and
animals given AHU377 were pituitary adenoma/carcinoma, mammary gland tumor
(fibroadenoma/adenocarcinoma) and sarcomas of various origin whereas the most frequent
non-neoplastic cause of death/early euthanasia was obstructive uropathy
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no AHU377-related effects on mean body weight or mean absolute body weight
gain observed during the study.
All other alterations in mean body weight and/or absolute mean body weight gain that
occurred during the study, were considered incidental and unrelated to dosing based on the
lack of a dose response, the transient nature of the effect or individual variability
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no AHU377-related effects on mean food consumption observed during the study.
Any differences were considered related to individual variation.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The spectrum and distribution of macroscopic findings were not meaningfully different
between the preterminal and terminal animals and between animals from the different dose
groups (including control). These findings were typical of the changes commonly
encountered at necropsy in rats of this strain and age range. It was considered that these
changes were spontaneous in origin and were not associated with the administration of
AHU377.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No AHU377-related effects were seen in the incidence of any non-neoplastic lesion in male
and female rats in this study.
All non-neoplastic changes described in this study were lesions commonly encountered in rats
of this strain and age range, and were considered to be spontaneous, procedural or agonal in
origin, therefore, they were not considered to be of any toxicological importance.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No AHU377-related effects were seen in the incidence of any neoplastic lesion in male and
female rats in this study. The various neoplastic lesions observed were generally typical of
those commonly encountered in rats of this strain and age range.
A statistically significant (trend test analysis p-value of 0.0046) increased incidence of
pituitary adenoma of the pars distalis was recorded in males given 400 mg/kg/day AHU377
when compared to the control group. However, the pairwise comparison (group 1 vs group 4)
did not result in statistical significance for a common tumor (p-value of 0.0153). Although
the overall incidence was slightly above the historical range for this laboratory(incidence
between 20% to 37% for pituitary adenoma in males), this change was considered equivocal
and not likely to be an indication of carcinogenic potential of AHU377.
A few other tumors or tumor combinations reached statistical significance including follicular
adenoma of the thyroid with a p-value of 0.0248 (overall trend test) in females only,
combined hemangioma/hemangiosarcoma with a p-value of 0.0085 in males only (pairwise
comparison test: group 1 vs group 3) and uterus endometrial stroma polyp in females
(pairwise comparison test was significant (group 1 vs group 2) with a p-value of 0.0077) were
observed. Follicular adenoma in the thyroid is a common tumor, the p-value did not reach
statistical significance for common tumors and this increased incidence in females was
therefore considered of no toxicological significance. Since the overall trend test was not
significant and the pairwise comparison test with the high dose group did not reach statistical
significance for the hemangioma/hemangiosarcoma combination in males and for endometrial
polyp in the uterus of females, and the incidence of the uterine endometrial polyp was within
range of the historical control data at this laboratory (incidence between 6% to 22% for
uterine endometrial polyp, however no historical control data are available for combined
hemangioma/hemangiosarcoma), the higher incidence of these tumor/tumor combinations in
the intermediate or low dose group were interpreted to be incidental and of no toxicological
significance.
All other tumor incidences recorded were within the range of spontaneous occurrence
reported for aged Wistar Hannover rats, were generally randomly distributed in control and
treated groups and/or lacked a dose-related pattern. In addition, no increase in the incidence
of any hyperplastic lesions was noted.
Conclusions:
In conclusion, AHU377 given orally to Wistar Han rats at 50, 150 and 400 mg/kg/day (base)
for a minimum of 104 consecutive weeks was well tolerated. There were no AHU377-related
effects on survival rate, clinical signs, body weights and food consumption. No evidence of
any carcinogenic effect was seen in male or female rats given up to 400 mg/kg/day of
AHU377. No gross or microscopic changes were seen in any organs or tissues examined that
were attributed to the oral gavage administration of AHU377 up to 400 mg/kg/day. Under the
conditions of this study, the Maximum Tolerated Dose (MTD) was considered to be
400 mg/kg/day for males and females.
Executive summary:

This study was conducted at Charles River Laboratories Preclinical Services Montreal, Sherbrooke site, 1580 Ida-Métivier, Sherbrooke, Quebec, Canada, J1E 0B5 in accordance with the OECD Principles of Good Laboratory Practice and as accepted by Regulatory Authorities throughout the European Union, United States of America (FDA) and Japan(MHLW). Dosing formulation and toxicokinetic analyses were performed in accordance with the U.S. Department of Health and Human Services, Food and Drug Administration. United States Code of Federal Regulations, Title 21, Part 58: Good Laboratory Practice for Nonclinical Laboratory Studies and as accepted by Regulatory Authorities throughout the European Union (OECD Principles of Good Laboratory Practice) and Japan (MHLW).


 


The purpose of this study was to investigate the carcinogenic potential of AHU377, when administered by daily oral gavage to the rat for a minimum of 104 weeks. In addition, the study confirmed exposure to AHU377 and its active metabolite LBQ657. Wistar Hannover Crl:WI (Han) rats were supplied by Charles River St-Constant, Quebec, Canada. At initiation of dosing, rats were approximately 8 weeks of age, the males weighed 197 to 267 g and the females weighed 146 to 201 g. AHU377 (Batch no. 0824011, salt/base ratio of 1.046) was administered orally by gavage in 0.5% (w/v) hydroxypropylcellulose, to 4 groups of rats (50/sex/group) at doses of 0, 50, 150 or 400 mg/kg/day (base) for at least 104 weeks. In addition, four animals/sex/group served as toxicokinetic animals, while an additional 10 males and 10 females were selected from the total population and used as health screen animals (group 5). Activities performed included clinical examinations (twice daily examinations for mortality and signs of ill health or reaction to dosing, daily post dosing cage side observations, weekly detailed examinations, palpable mass examinations on main study animals), body weights, food consumption, laboratory investigations (hematology) for health screen animals prior to dosing initiation and for main animals at study termination (blood smears). Toxicokinetic evaluations were performed during weeks 4 and 26 from 2 rats/sex/group at 0.5 and 1 hour post dose. Post-mortem evaluations for main study animals included macroscopic observations and microscopic observations.


 


There was no AHU377-related effect on survival. At the end of the dosing period, the survival rates varied from 62 to 76% for males and 60 to 68% for females, throughout all groups, including control.


 


There were no AHU377-related effects on clinical signs, palpable masses, body weights or food consumption observed during the study.
Clinically observed palpable masses, which were present during the dosing phase and at necropsy, were generally noted in a similar proportion of animals from control and AHU377-treated groups.


There was no evidence of carcinogenic effect of AHU377 in male or female rats at histopathology examination. In addition, no gross changes were seen in any organs or tissues examined that were attributed to the administration of AHU377 at doses up to 400 mg/kg/day.


All AHU377-treated animals displayed systemic exposure to the test item and to the active metabolite LBQ657 on study days 24 and 178 following multiple daily oral doses of AHU377. In general, mean plasma concentrations of AHU377 and LBQ657 increased approximately proportionally with increasing dose for the 0.5 h and 1 h samples within the dose range tested for both males and females. The average plasma concentration of AHU377 was 2.1% of the LBQ657 plasma concentration (range 0.7% to 6.0%) at 0.5 h and 1 h post dose, suggesting rapid conversion of AHU377 to the metabolite LBQ657. There was no consistent difference in exposure to AHU377 and LBQ657 between males and females. With few exceptions, the plasma concentrations for AHU377 and LBQ657 were generally higher on day 178 compared to day 24 for both males and females. A definitive statement on the time dependency of exposure cannot be made due to the sparse nature of the data, as only two sampling time points were taken.


 


In conclusion, AHU377 given orally to Wistar Han rats at 50, 150 and 400 mg/kg/day (base) for a minimum of 104 consecutive weeks was well tolerated. There were no AHU377-related effects on survival rate, clinical signs, body weights and food consumption. No evidence of any carcinogenic effect was seen in male or female rats given up to 400 mg/kg/day of AHU377. No gross or microscopic changes were seen in any organs or tissues examined that were attributed to the oral gavage administration of AHU377 up to 400 mg/kg/day. Under the conditions of this study, the Maximum Tolerated Dose (MTD) was considered to be 400 mg/kg/day for males and females.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
chronic
Species:
rat

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

No carcinogenic effect was observed in a 104-week study performed on rats after oral administration by gavage. Based on this result and according to the Regulation (EC) No 1272/2008, AHU 377 does not have to be classified and has no obligatory labelling requirement for carcinogenicity.

Additional information