[1,1'-Biphenyl]-4-pentanoic acid, .gamma.-[(3-carboxy-1-oxopropyl)amino]-.alpha.-methyl-, 4-ethyl ester, calcium salt (2:1), (.alpha.R,.gamma.S)-

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Administration of AHU377 by daily oral gavage at dosages of 75, 250 and 750 mg/kg/day to males and females resulted in adverse effects on body weights and food consumption in males, but not females, at 750 mg/kg/day. There was no evidence of effects on male or female reproductive function or embryo-lethality at any dose. The no-observed-adverse-effect level (NOAEL) for paternal toxicity was considered to be 250 mg/kg/day and for maternal toxicity, 750 mg/kg/day. The NOAEL for reproductive function and early embryo-fetal development was considered to be 750 mg/kg/day, the highest dose level administered.

Link to relevant study records

Reference

Endpoint:

fertility, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 Feb 2014 - 29 Aug 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline S5 (R2) Guideline for Industry: Detection of Toxicity to Reproduction for Medicinal Products & Toxicity to Male Fertility

Deviations:

yes

Remarks:

Minor deviations (see report)

GLP compliance:

yes (incl. QA statement)

Justification for study design:

The Wistar Hannover rat was selected as the test animal because 1) it is a standard species accepted for use in embryo-fetal development studies; 2) this species and strain has been demonstrated to be sensitive to developmental toxicants; and 3) historical data and experience
exist at the test facility.

The requirement for, and use of animals in this research was the responsibility of the Sponsor, in that the research did not unnecessarily duplicate previous animal experiments. The research was conducted in the absence of acceptable non-animal alternatives to provide meaningful data and was designed such that it did not require an unnecessary number of animals to accomplish its objectives.

The study plan, study plan amendments and procedures involving the care and use of animals in this study were reviewed and approved by PCS-MTL Institutional Animal Care and Use Committee (IACUC) before conduct. During the study, the care and use of animals was conducted in accordance with the guidelines of the USA National Research Council and the Canadian Council on Animal Care (CCAC). This non-clinical health and environmental safety study was reviewed and approved by the East Hanover Novartis Animal Care and Use Committee (EH-NACUC).

Specific details on test material used for the study:

Active ingredient: AHU377 (AHU377 BAA.002, calcium salt form)
Batch no.: 0723009
Retest date: 31-Jul-2014
Supplier: Novartis Technical Research and Development (TRD)
Purity (by HPLC): 100.2% (purity assumed to be 100% for dose calculation purposes)

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:

No. of animals assigned to the dosing phase: 96/sex
Age: Males 12 weeks and females 9 weeks at start of dosing
Body weight range: 298-355 g (males) and 167-215 g (females), at start of dosing
Acceptability: All assigned animals were considered suitable for use on study

Room temperature: 19°C to 25°C
Room relative humidity: 30-70%
Lighting cycle: Fluorescent light for a 12-hour light/12-hour dark cycle
Animal caging: Throughout the study, animals were housed in solid-bottomed cages equipped with an automatic watering valve on corn-cob bedding. On arrival, animals were individually housed until randomization; following randomization, animals were group housed (up to 3 animals of the same sex and same dosing group together), unless otherwise recommended by the clinical veterinarian (i.e., for animal nos. 3018 and 3017 that were single housed from 28-Feb-2014 to 20-Mar-2014 to allow for healing of a skin lesion at the dorsal cervical lesion of animal no. 3018). During cohabitation, each female was paired with a male and animals were returned to group housing following confirmation of mating or at the end of the cohabitation period, as applicable.
Acclimation period: An acclimation period of two weeks was allowed between animal arrival and the start of treatment in order to accustom the animals to the laboratory environment.
Diet: All animals had free access to standard certified pelleted commercial laboratory diet (PMI Certified Rodent 5002: PMI Nutrition International Inc.) throughout the study, except during designated procedures.
The feed is analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis are on file at the test facility.
Water: Municipal tap water softened, purified by reverse osmosis and exposed to ultraviolet light, was freely available (except during designated procedures).
Periodic analysis of the water is subcontracted to management authorized analytical laboratories. The analytical results are retained in the archives of PCS-MTL.
Animal enrichment: Animals were socially housed for psychological/environmental enrichment and were provided with items such as hiding tubes and a chewing object, except during designated activities.
There were no known contaminants in the food or water that would interfere with the conduct
of the study.

Route of administration:

oral: gavage

Vehicle:

other: 0.5% (w/v) hydroxypropylcellulose

Details on exposure:

Route and frequency: Oral (gavage), daily
Justification for route: Intended route in humans

Males were given the test or reference item formulations once daily beginning 28 days before cohabitation, during cohabitation and continuing through the day before euthanasia, for a total of at least 56 days. Females were given the test item and/or the reference item formulations once daily beginning 14 days before cohabitation, during cohabitation and continuing until day 6 postcoitum (i.e., of gestation). Females not mated after completion of the 14-day cohabitation period were given the test item and/or the reference item formulations until the day before necropsy.
Animals were given a daily oral dose (gavage) of the dose formulation using a plastic gavage tube. The dose volume was adjusted for each animal based on the most recently recorded body weight and administered at approximately the same time each day (i.e., ±1 hour for each group). The dosing formulations were placed on a stir plate for at least 30 minutes prior to and constantly during dosing and were administered within a 4-hour period following transfer to room temperature. Reflux was observed once between study days 1 and 63 for a few animals (male nos. 1014, 3007, 3010, 4007, 4009 and female nos. 1512, 2516 and 4512) in each group.

Details on mating procedure:

Within each dosage group, a consecutive order was used to assign rats to cohabitation (i.e., pairing), one male to one female. The cohabitation period consisted of a maximum of 14 days. Females with spermatozoa observed in a smear of the vaginal lavage and/or a copulatory plug observed in situ were considered to have mated (day designated as day 0 postcoitum [i.e., of gestation]) and were returned to group housing. Females not mated after completion of the 14-day cohabitation period were returned to group housing and were euthanized 8 days after completion of the mating period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Based on stability data provided by the Sponsor, AHU377 suspensions in 0.5% (w/v) hydroxypropylcellulose (grade HF), NF (Klucel), at 1.0 to 125.5 mg/mL (salt) are chemically
stable for 12 days at 6°C, 24 hours at room temperature and 4 hours stirring at room temperature.

Homogeneity of the dose formulations was assessed at the dose concentrations intended for use on the study. It was assessed from the preparation vessel and the dose aliquot jars
as follows:
Following the first preparation of the suspension (including stirring) but prior to aliquoting into daily containers, two sets of samples (target volume of 1 mL), one in duplicate for analysis and the other in triplicate as back-up samples, were sampled from approximately the top, middle, and bottom of the group 2 and 4 test item formulations and from the middle of the vehicle control and group 3 test item formulation. An additional sample (10 mL) was taken from each formulation for density measurement. As suspensions were prepared on a weekly basis and stored in daily dosing containers, the homogeneity was also assessed following resuspension of the
dosing container for groups 2 and 4 on the first day of use.
Two sets of samples (target volume of 1 mL), one in duplicate for analysis and the other in triplicate as back-up samples, were sampled from approximately the top, middle, and bottom of the group 2 and 4 test item formulations and from the middle of the vehicle control and group 3 test item formulation. All samples were transferred to HDPE
containers.
Concentration of the dose formulations was also assessed at the dose concentrations intended for use on the study. It was assessed from the preparation vessel as follows:
Following week 5 and the last preparation of the suspension (including stirring) but prior to aliquoting into daily containers, two sets of samples (target volume of 1 mL), one in duplicate for analysis and the other in triplicate as back-up samples, were sampled from approximately the
middle of the test item formulations and the vehicle control.
For concentration, the criterion for acceptability was mean sample concentration results within or equal to ± 15% of theoretical concentration (individual sample concentration result within or equal to ± 20%). For homogeneity, the criterion for acceptability was a relative standard deviation
(RSD) of concentrations of 5% for each group.

Duration of treatment / exposure:

Males: 28 days before cohabitation, during cohabitation and continuing through the day before euthanasia, for a total of at least 56 days.

Females: 14 days before cohabitation, during cohabitation and continuing until day 6 postcoitum (i.e., of gestation)

Frequency of treatment:

once daily

Details on study schedule:

Major activities Date or study day no.
Study plan signed 03-Feb-2014
Experimental start date 04-Feb-2014
Animal arrival/Start of acclimation 04-Feb-2014 (males) 18-Feb-2014 (females)
First day of dosing 18-Feb-2014 (males) 04-Mar-2014 (females)
Last necropsy 25-Apr-2014
Experimental completion date 29-Aug-2014

Dose / conc.:

75 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

24 males and 24 females per goup

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses for this study were based on preliminary findings in the AHU377 26-week oral (gavage) toxicity study in rats conducted at doses of 50, 150 and 600 mg/kg/day [Novartis reference no. 1370484]; on the results of the AHU377 oral (gavage) embryo-fetal development study in rats conducted at doses of 75, 250 and 750 mg/kg/day [Novartis reference no. 0570301] and on the AHU377 oral (gavage) pre and postnatal study in rats conducted at doses of 50, 250 and 750 mg/kg/day [Novartis reference no. 1070349]. Based on results from those studies, doses of 75, 250 and 750 mg/kg/day were selected for the current study.

- Rationale for animal assignment (if not random): One week before initiation of treatment, animals were assigned to groups using a
computer-based randomization procedure. Males and females were randomized separately. No animals at the extremes of the weight range or in poor health were assigned to the study. Animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. The disposition of all animals was documented in the study records. All animals remaining unassigned to groups were released from the study on study day 3 and their disposition documented.

- Fasting period before blood sampling for clinical biochemistry: not relevant.

Parental animals: Observations and examinations:

3.5.1 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.

3.5.2 Clinical signs
Cageside observations: On non-dosing days, once daily starting on the day of randomization when no detailed examinations were scheduled; on days of dosing, predose (when no detailed
examinations were scheduled) and within 1 to 3 hours post dose.
Detailed examinations: On the days of body weight assessment

3.5.3 Body weight
Individual body weights were measured twice weekly commencing on the day of randomization and extending through the treatment period, including the day animals were placed for mating and, for males, the day of scheduled necropsy. Mated females were weighed on days 0, 3, 7, 10 and 13 postcoitum.

3.5.4 Food consumption
Food consumption (cage measurements) was quantitatively measured twice weekly commencing the day of randomization and until initiation of the mating period.
Food consumption for mated females was measured on days 0 to 3, 3 to 7, 7 to 10 and 10 to 13 post coitum.

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage for 14 consecutive days beginning with the first day of dosage administration and then until spermatozoa were observed in a smear of the vaginal contents and/or a copulatory plug was observed in situ during the cohabitation period.

Postmortem examinations (parental animals):

3.6.3 Ovarian and uterine examinations
The reproductive tract was dissected from the abdominal cavity. The uterus was opened and the contents examined. The embryos were removed from the uterus.
The ovaries and uterus were examined for number and distribution of:
¿ Corpora lutea
¿ Implantation sites
¿ Placentae (size, color or shape)
¿ Early resorptions
¿ Live and dead embryos

Uteri of apparently nonpregnant females were stained with 10% aqueous (v/v) ammonium sulfide solution and examined for implantation sites (Salewski, 1964).

3.6.5 Necropsy
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.
Necropsy examinations were conducted under the supervision of a board-certified veterinary pathologist or other qualified personnel with appropriate training and experience in animal anatomy and gross pathology.

3.6.6 Organ weights
The organs identified for weighing in the Tissues collection and preservation table were weighed at necropsy for all animals. Paired organs were weighed individually, unless otherwise indicated.

3.6.7 Tissue collection and preservation
Representative samples of the tissues identified in the Tissue collection and preservation table were collected at scheduled euthanasia and were preserved in 10% neutral buffered formalin. Unless specifically cited below, all other tissues were discarded. A standard list of tissues were collected at scheduled necropsy from the first two gravid control females and the first two control males examined, as per PCS-MTL SOP, and were retained for possible future comparison. No histopathological evaluation was performed and all tissues have been discarded.

Statistics:

Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation were reported whenever possible. Inferential statistics were performed according to
the matrix below when possible, but excluded semi-quantitative data, and any group with less
than 3 observations. Litter values were used where appropriate.

Reproductive indices:

3.5.6 Cohabitation/mating.
Within each dosage group, a consecutive order was used to assign rats to cohabitation (i.e., pairing), one male to one female. The cohabitation period consisted of a maximum of 14 days. Females with spermatozoa observed in a smear of the vaginal lavage and/or a copulatory plug observed in situ were considered to have mated (day designated as day 0 postcoitum [i.e., of gestation]) and were returned to group housing. Females not mated after completion of the 14-day cohabitation period were returned to group housing and were euthanized 8 days after completion of the mating period.

3.6.4 Male reproductive assessments
To assess the potential toxicity of the test item on the male reproductive system, the endpoints
listed below were evaluated for each main study male at scheduled euthanasia.

3.6.4.1 Sperm motility
Sperm motility was evaluated using computer-assisted sperm analysis (CASA). Motility was evaluated following dispersion, into an appropriate medium, of sperm from the left vas deferens.

3.6.4.2 Sperm concentration
A homogenate was prepared from the left cauda epididymis for evaluation to determine sperm concentration (sperm per gram of tissue weight; manual count using a hematocytometer). Sperm concentration (millions/gram of epididymis) was assessed from two counts of sperm obtained from the cauda epididymis.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation and/or wet fur on the lower jaw and/or muzzle were observed in a few males at
75 mg/kg/day and most males and females at 250 and 750 mg/kg/day during the treatment
period, including the gestation period. A few males at 250 and 750 mg/kg/day were noted to
be hypersensitive sporadically during the treatment period. These observations were
considered to be non-adverse.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There was no unscheduled mortality.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

For males at 250 and 750 mg/kg/day, significantly lower body weight gains, compared to
controls, were noted during the first week of the premating treatment period. At
750 mg/kg/day, this continued throughout the remaining 3 weeks of this period of the study.
As a result, the overall weight gains (study days 1 to 28) were significantly decreased at
250 and 750 mg/kg/day, being 12 and 35% lower than controls. The body weights at
750 mg/kg/day were significantly lower from study day 15 until the end of the study (-4 to
-6%). These lower body weights for males at 750 mg/kg/day continued during the mating and
post mating treatment periods. Body weight decreases in males at 750 mg/kg were of
sufficient magnitude to be considered adverse. An a few occasions, statistically significant
differences were noted for body weight gain values for males at 75 mg/kg/day but these were
considered to be the result of random variation and not test item related.
There were no AHU377-related effects upon body weights or body weight gains at any dose
level for the females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There were slightly lower food intakes for males at 250 and 750 mg/kg/day during the
premating treatment period, particularly for the first week of treatment. Overall, all values
(study days 1 to 28) were reduced by 5% and 8% at 250 and 750 mg/kg/day, respectively,
compared to controls.
The food consumption of the males at 75 mg/kg/day and females at <= 750 mg/kg/day was
unaffected.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The estrous cycles of the AHU377-treated females were not affected.

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

There was no effect of AHU377 upon the day to mating, mating or fertility indices, or the conception rates.

Ovarian and uterine findings:
For AHU377-treated females, there were no effects upon the numbers of corpora lutea, implantations, live embryos, dead embryos, resorptions or the pre or post-implantation losses. The increased pre or post-implantation loss values noted for the treated groups were not considered related to the test item since there was no dose response noted and because values were within the historical control values for these parameters.

There were no toxicologically significant differences in mean for sperm parameters, including
sperm motility and concentration, between AHU377-treated animals and vehicle-treated control animals.

Key result

Dose descriptor:

NOAEL

Remarks:

paternal toxicity

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

750 mg/kg bw/day

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Food efficiency:

not specified

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

750 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

In conclusion, administration of AHU377 by daily oral gavage at dosages of 75, 250 and
750 mg/kg/day to males and females resulted in adverse effects on body weights and food
consumption in males, but not females, at 750 mg/kg/day. There was no evidence of effects
on male or female reproductive function or embryolethality at any dose. The
no-observed-adverse-effect level (NOAEL) for paternal toxicity was considered to be
250 mg/kg/day and for maternal toxicity, 750 mg/kg/day. The NOAEL for reproductive
function and early embryo-fetal development was considered to be 750 mg/kg/day, the highest
dose level administered.

Executive summary:

This study was conducted at Charles River Laboratories Preclinical Services Montreal, 22022 Transcanadienne, Senneville, Quebec, Canada, H9X 3R3 and was based on requirements of the OECD Principles of Good Laboratory Practice (GLP) and as accepted by Regulatory Authorities throughout the European Union, United States of America (FDA), Japan (MHLW), and other countries that are signatories to the OECD Mutual Acceptance of Data Agreement. Exceptions included: the test item characterization and stability testing performed by the Sponsor followed FDA Good Manufacturing Practice (GMP) regulations.

 

The design of this study was based on the study objective and the following study design guidelines: ICH Harmonised Tripartite Guideline S5 (R2) Guideline for Industry: Detection of Toxicity to Reproduction for Medicinal Products & Toxicity to Male Fertility.
The purpose of this study was to determine the potential adverse effects of AHU377, a neutral endopeptidase inhibitor, on Wistar Hannover Crl:WI (Han) rats before cohabitation, through mating and implantation. This study evaluated ICH Harmonised Tripartite Guideline stages A and B of the reproductive process and was designed to detect effects on the estrous cycle, tubal transport, implantation, and development of preimplantation stages of the embryos of females and permit detection of functional effects (e.g., effects on libido or epididymal sperm maturation) that may not be detected by histological examinations of male reproductive organs.

 

AHU377 (batch no. 0723009) was administered by oral gavage at doses of 75, 250 and 750 mg/kg/day (base) to three groups (n = 24/sex) of male and female rats. An additional group of rats (n = 24/sex) received the vehicle, 0.5% (w/v) hydroxypropylcellulose (grade HF), NF (Klucel), aqueous solution, at an equivalent dose volume (10 mL/kg), and served as controls. Males were given the test or reference item (vehicle) formulations once daily beginning 28 days before cohabitation, during cohabitation and continuing through the day before euthanasia, for a total of at least 56 days. Females were given the test item and/or the reference item formulations once daily beginning 14 days before cohabitation, during cohabitation and continuing until day 6 postcoitum (i.e., of gestation).

 

Rats Wistar Han (Crl:WI[Han]) were obtained from Charles River Laboratories, Kingston, New York, USA. At the start of dosing, males were 12 weeks and weighed 298 to 355 g; females were 9 weeks of age and weighed 167 to 215 g at the onset of treatment. Cesarean sections were performed on day 13 post coitum (pc) for main study animals.

 

The following parameters and endpoints were evaluated: mortality and moribundity, clinical signs, body weights, food consumption, estrous cycles, reproductive performance, organ weights, ovarian and uterine examinations, sperm evaluations, and gross pathology.

 

There was no mortality. Signs of salivation/wet fur were observed primarily for males and females at 250 and 750 mg/kg/day during the study although salivation was also noted for few males at 75 mg/kg/day.

For males, overall weight gains (study day 1 to 28) were significantly decreased at 250 and 750 mg/kg/day. The male body weights at 750 mg/kg/day were significantly lower from study day 15 until the end of the study. There were lower food intakes for males at 250 and 750 mg/kg/day during the premating treatment period, with differences from controls ranging from 5% to 8%. There were no AHU377-related effects upon body weights or food intake at any dose level for the females.

 

The estrous cycles of females and the parental performance parameters (mean day to mating, mating and fertility indices and the conception rate) of the males and females were not affected by treatment with AHU377. There were no compound-related effects upon the ovarian and uterine parameters (numbers of corpora lutea, implantations, live embryos, dead embryos, resorptions or the pre or post-implantation losses).

 

No macroscopic observations attributed to the effect of treatment with AHU377 were observed. There were no differences in mean absolute or relative organ weights between

 

AHU377-treated animals and vehicle-treated control animals. Sperm parameters, including sperm motility and concentration, were unaffected by treatment with AHU377.

 

In conclusion, administration of AHU377 by daily oral gavage at dosages of 75, 250 and 750 mg/kg/day to males and females resulted in adverse effects on body weights and food consumption in males, but not females, at 750 mg/kg/day. There was no evidence of effects on male or female reproductive function or embryolethality at any dose. The NOAEL for paternal toxicity was considered to be 250 mg/kg/day and for maternal toxicity, 750 mg/kg/day. The no-observed-adverse-effect level (NOAEL) for reproductive function and early embryo-fetal development was considered to be 750 mg/kg/day, the highest dose
level administered. 

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

750 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

All available studies related to the developmental toxicity of AHU377 have been included in the dossier, to cover the key developmental milestones in foetal and pup development in a weight of evidence approach. In a study performed in rats treated with AHU377 by gavage, a NOAEL of 100 mg/kg/day has been reported based on clinical chemistry, gastrointestinal tract (stomach) and bone development (femur and tibia) changes. 

In an embryo-fetal development study performed on rabbits (2008), after administration of AHU377 by gavage at dose of 500 mg/kg/day, a decrease of fetal body weight and increase in late resorptions have been reported but considered as a consequence of maternal toxicity (decreased food consumption and body weight loss).  Consequently, the NOAEL of 100 mg/kg/day observed in the Juvenile rat study for bone development is taken as the point of departure for developmental toxicity. 

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 29 June 2009 to 12 November 2009

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

other: ICH guidelines on detection of toxicity to reproduction for medicinal products

Version / remarks:

Fed. Reg., Vol. 59, No. 183, 22-Sep-1994

Deviations:

yes

Remarks:

See report

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Active ingredient: AHU377
Batch number: 0722008
Drug content: 98.9%
Salt/Base ratio: 1.046

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Rattus norvegicus, Wistar Hannover Crl:WI (Han)

3.2.1 Experimental animals
Animal species and strain: Rattus norvegicus, Wistar Hannover Crl:WI (Han)
Breeder/Supplier: Charles River Canada Inc., St. Constant, Quebec
Number of animals ordered: 78 dams with Day 3 post partum litters
Number of animals assigned to the dosing phase: A minimum of 278 male pups and 278 female pups.
Age: Pups were on day 7 post partum at the start of treatment.
Body weight range: 10.5 to 18.9 g at the start of treatment.
Acceptability: Following arrival, all animals were given a general physical examination by a member of the veterinary staff to ensure normal health status.

3.2.2 Animal quarters/husbandry
Room temperature: 19 - 25°C
Room relative humidity: 30 - 70%
Lighting cycle: 12 hours light and 12 hours dark (except during designated procedures).
Animal caging: F0 generation dams and their litters were housed in solid-bottomed cages equipped with an automatic watering valve on corn-cob bedding. Following weaning on day 21
post partum, the F1 animals were housed 2 or 3 per cage according to sex and group, in stainless steel wire mesh bottomed cages equipped with an automatic watering valve.
Water bottles were also placed on cages until day 28 post partum. On day 28 post partum, F1 animals were housed individually, in similar cages. In addition, a certified chewing object was placed in each cage.
Unmated females were placed in solid bottomed plastic cages at the end of the mating period.
Diet: All animals had free access to a pelleted commercial laboratory diet (Certified Rodent 5002: PMI Nutrition International Inc.), except during designated procedures.
Powdered food (PMI Rodent Laboratory Meal 5002) was provided on occasion to a few animals.
Analysis of food: 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 were retained at PCS-MTL.
Water: Municipal tap water, which was softened, purified by reverse osmosis and exposed to ultraviolet light, was freely available. Water bottles were provided when required.
Analysis of water: Periodic analysis of the water was subcontracted to management authorized analytical laboratories, which were audited by the quality assurance department of PCS-MTL.
The analytical results were retained in the archives of PCS-MTL.
It was considered that there were no known contaminants in the dietary materials that interfered with the objectives of the study.

Route of administration:

oral: gavage

Vehicle:

other: 0.5% (w/v) hydroxypropylcellulose (grade HF), NF (Klucel), aqueous solution.

Details on exposure:

Route and frequency: Once daily by oral gavage
Justification for route: Intended route in humans
Dose volume: 6.5 (groups 1 and 4) or 5 (groups 2 and 3) mL/kg/day
Administration method: The test/control article suspensions were administered to pups assigned to the study from day 7 to day 70 post partum, inclusive. Single dose toxicokinetic animals were dosed on day 7 post partum only. Animals were given a daily oral dose (gavage) of dose formulation at a dose volume of 6.5 or 5 mL/kg/day using a plastic gavage tube at approximately the same time each day. Control animals received the vehicle alone at the same dosage volume as the 800 mg/kg/day treated animals. Each dose was based upon each animal’s most recent body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity of the low and mid dose formulations in the preparation vessels was confirmed. Although the high dose formulation in the preparation vessel did not meet the homogeneity specification in the primary analysis (82% of target), the concentration of the
retained high dose top preparation vessel sample was 103% of target. The homogeneity of the
low and high dose formulations in the dosing containers was confirmed. The mid dose formulation in the dosing container did not meet the homogeneity specification in the primary analysis (70% of target), but the concentration of the retained mid dose top dosing container sample was 101% of target, meeting acceptance criteria. Samples of AHU377 in 0.5% Klucel from weeks 1, 3, 5, 7 and 10 were analyzed and found to be within acceptance criteria except for the mean concentration of the week 5 high dose initial sample, which was outside of specification (78% of target). However, the mean concentration of the week 5 high dose retained sample was 91% of target. As all retention samples were found to be within specification for samples found to be out of acceptable range in the primary analysis, dose formulations were considered to be within acceptance criteria, and these deviations were considered to have had no impact on the study results. All other mean results were within the concentration specifications. Five control samples were analyzed, and no AHU377 was detected.

Details on mating procedure:

At approximately 105 days of age, half of the main study animals were subjected to mating procedures whereby 1 female was placed with 1 male (sibling matings were avoided) in the same dosage group for up to 14 days. The females were examined for mating by examination of the vaginal lavage for spermatozoa and/or presence of vaginal plug. The day of positive identification of spermatozoa and/or presence of vaginal plug was termed day 0 of gestation. All unmated females were placed in solid-bottomed plastic cages at the end of the mating period.

Duration of treatment / exposure:

Day 7 to 70 post partum

Frequency of treatment:

Daily

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

400 mg/kg bw/day (actual dose received)

Dose / conc.:

800 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

40 for main study
12 for pathology subset

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses for this study were selected based on preliminary results of a dose range-finding study in juvenile rats [study no. 901864, Novartis ref. no. 0870735] in which dose levels of 0, 100, 400 and 800 mg/kg/day were administered. In that study, one control pup, 4 pups at 400 mg/kg/day and 8 pups at 800 mg/kg/day were found dead or euthanized in poor condition. Gross pathological examinations revealed findings suggestive of gavage accidents in the majority of these animals. Therefore, the toxicological significance of the increased mortality rate at 400 and 800 mg/kg/day was uncertain. There were transient decreases in body weight gains, following initiation of treatment at doses = 400 mg/kg/day. Subsequently, decreased mean body weights were noted in males at doses = 400 mg/kg/day and transiently in females at a dose of 800 mg/kg/day. On day 34 post partum, male body weights were 6.4 and 7.1% less than controls at doses of 400 and 800 mg/kg/day, respectively. Female body weights rebounded and were comparable to, or in excess of, concurrent controls on post partum day 34. There were no treatment-related gross macroscopic changes noted. The dose of 100 mg/kg/day was considered the no observable effect level. Based on these results, dose levels of 100, 400 and 800 mg/kg/day were selected for this study.

- Rationale for animal assignment (if not random): The main study (including pathology) pups were assigned to cross-fostered litters such that no more than one male and one female sibling were assigned to a cross-fostered litter, and no pup was assigned to its biological mother. The cross-fostered litters were randomly assigned to each of the treatment groups 1 to 4 using a computer-based randomization. Animals were allocated to dose groups as per Table 3-1.

Maternal examinations:

Signed and dated records of activities relating to the day-to-day running and maintenance of the study within the animal room as well as the activities relating to the observations and examinations outlined in the protocol were recorded. The data collected from F0 generation dams were retained with the raw data but were not reported

Mortality:
Observations for mortality or signs of ill health were conducted twice daily (am and pm)
following arrival.

Clinical signs:
Detailed examinations were performed on the days of body weight assessment

Body weight:
Individual body weights were measured on days 7, 14 and 21 post partum.

Fetal examinations:

Litter observations (pretreatment):
Clinical examination: A litter check was conducted daily until weaning.

Body weight
Individual body weight was measured on day 4 post partum for randomization.

Observations (pups - F1 generation - treatment);:
Clinical examination: After weaning, animals were observed twice daily for mortality and signs of ill health or reaction to treatment (SIRTs). Detailed examinations (DEs) were performed once daily
during the pre-weaning period (as part of the litter check) and on days of body weight determination thereafter. More frequent observations were undertaken when considered appropriate. Death and observed clinical signs were individually recorded.

Body weight
In addition to the assessment for randomization, the pups were weighed individually, each day
from days 7 to 21 post partum and then twice weekly thereafter until termination, and on the
day of scheduled euthanasia. Mated females assigned to the fertility phase were weighed on
gestation days 0, 3, 6, 9 and 13. Toxicokinetic pups dosed once on day 7 post partum were
weighed on days 4 and 7 post partum.

Weaning
On day 21 post partum, the F1 generation pups were separated from their dams and were
housed 2 or 3 per sex/cage, according to dose level, until day 28 post partum and individually
thereafter. Before or at weaning, each animal was uniquely identified using the AIMS® tail
tattoo system. Any treated spares not assigned to the study were euthanized at weaning and
discarded without examination.

Food consumption
Food consumption was measured on main study animals twice weekly from day 28
post partum onwards on days of body weight assessment until termination or, if selected for
fertility assessment, until cohabitation. Mated females assigned to the reproductive subset
had food consumption measured from days 0 to 3, 3 to 6, 6 to 9 and 9 to 13 of gestation.

Visual function
On day 21 post partum, the pupillary closure and visual placing responses were assessed on
all main study animals.

Physical development
On all main study animals, eye opening was assessed from days 14 to 17 post partum. Vaginal opening was assessed from day 26 post partum until development for females and preputial separation was assessed from day 35 post partum until development for males.

Behavioral performance (recovery subset)
Auditory startle: At day 28 (±1) post partum, the startle habituation (San Diego Instruments) was measured.
The animals were given a 4-minute acclimation period and then the startle response was
measured in 50 identical trials at a sound level of 120dBA with an 8-second inter-trial
interval.

Motor activity
Locomotor activity was assessed for 1 hour in a figure 8 enclosure on day 91 (±5) post partum. Animals from the control and treated groups were balanced across chambers using a randomization procedure. The sessions were of 1 hour’s duration and were of six 10-minute intervals. The sound level was kept constant at approximately 70dBA in the test room using exterior white noise generation. Room illumination was approximately 600 to 800 Lux.

‘E’ water maze
The ‘E’ water maze assessments were conducted commencing on day 98 (±5) post partum. The learning and memory tests were conducted using an ‘E’ water maze. The time to exit the maze and the number of errors (incorrect turns) were recorded for 5 tests, each of a maximum time of 1 minute on the first day of testing. Each test was performed at least 15 minutes apart. On the following day, two tests were performed (at least 25 hours after the end of the first days’ trials). Any abnormality in swimming was recorded.

3.4.3 Peripheral quantitative computed tomography (pQCT)
In vivo
Peripheral QCT was performed on the right proximal tibia on day 28 and 105 (±1 day) post partum on the first ten surviving animals/sex/group assigned to the recovery subset. Peripheral QCT was used to measure bone mineral content, bone mineral density, and geometric parameters of the right proximal tibia. Animals were anesthetized using isoflurane before and during the scans. An ophthalmic lubricating ointment was administered to each eye following anesthesia induction and reapplied as necessary.
Peripheral QCT scans were performed using a XCT Research SA or SA + bone scanner with software version 5.50D. A single scan was obtained in the proximal tibia metaphysis with an additional site in the diaphysis. Additional slices were obtained as considered appropriate to ensure the optimal scans and data acquisition. The exact position of the scan slices were documented in the raw data. For follow-up scans, positioning and placement of CT scan lines were verified using the scout scan and were compared with the scout scan obtained during the initial scanning occasion. Reanalysis using different modes was performed when the data was considered to better represent the scans. The diaphysis site was analyzed using Cortmode 2 for cortical bone measurements only. All scan analysis data was retained.
The metaphysis scans were evaluated for area, bone mineral content and bone mineral density of the total slice and the trabecular and cortical/subcortical regions. The scans obtained at the diaphysis site were evaluated for cortical bone mineral content, cortical bone mineral density, total area, cortical area, cortical thickness, periosteal circumference and endosteal circumference.

Ex vivo
Peripheral QCT scans were performed ex vivo using an XCT Research SA or SA + bone scanner with software version 5.50D. Scans were obtained for the excised left tibia for all animals assigned to the pathology subset surviving to scheduled termination.
A single scan was obtained of the proximal tibia metaphysis with an additional site in the diaphysis. The diaphysis site was analyzed using Cortmode 2 for cortical bone measurements. The exact position of the scan slices were documented in the raw data.
All analyses was performed using the LOOP option of the analysis software. All other data generated as a result of the LOOP option was retained with the raw data but not reported. The most appropriate analysis mode for the metaphysis was used and documented in the raw data and the analysis details were included in the final report. All scan analysis data was retained.
Scanning parameters for excised left tibia were the same as the in vivo scanning parameters.

3.4.4 Radiographs
In vivo
Radiographs were performed on day 28 and day 105 (±1 day) post partum on the first ten surviving animals/sex/group assigned to the recovery subset, and measurements of the femur, tibia and axial skeleton were derived from the 2 radiographs taken from each animal. Radiographs were obtained while animals were under isoflurane anesthesia. An ophthalmic lubricating ointment was administered to each eye following anesthesia induction and reapplied as necessary.

Ex vivo
Radiographs were performed ex vivo from the femur (right) and tibia (left) collected at scheduled necropsy from the pathology subset, and measurements of the femur and tibia were derived from 2 radiographs taken for each specimen.

Reproductive phase (reproductive subset)
Mating procedures (F1 adult generation):At approximately 105 days of age, half of the main study animals were subjected to mating procedures whereby 1 female was placed with 1 male (sibling matings were avoided) in the same dosage group for up to 14 days. The females were examined for mating by examination of the vaginal lavage for spermatozoa and/or presence of vaginal plug. The day of positive identification of spermatozoa and/or presence of vaginal plug was termed day 0 of gestation. All unmated females were placed in solid-bottomed plastic cages at the end of the mating period.

3.5.3 Organ weight assessment (pathology and recovery subsets)
For each animal from the pathology subset and from the first 10 surviving animals/sex/group in the recovery subset euthanized at completion of the observation period, the following organs were dissected free of fat and weighed: adrenals ovaries testes
brain pituitary* thymus heart prostate thyroid and parathyroid* kidneys spleen uterus liver
* weighed post fixation
Paired organs were weighed together and organ weight ratios relative to body weights and to
brain weight were calculated.

On completion of the necropsy of each animal in the pathology subset and the first ten surviving animals/sex/group in the recovery subset, and preterminally euthanized and found dead from all subsets (except the single-dose toxicokinetic subset), the following tissues and organs were retained. No tissues were retained for the remaining animals in the recovery subset or for the animals in the reproductive subset. Neutral buffered 10% formalin was used for fixation and preservation unless otherwise indicated:
abnormalities sciatic nerve animal identification a kidneys seminal vesicles adrenals lacrimal glands skeletal muscle aorta (thoracic) larynx (1 level) skin (inguinal) bone (femurs and tibiae) b,g liver (sample of 2 lobes) spinal cord (cervical, thoracic and lumbar) bone and marrow (sternum) b
brain (forebrain, midbrain, cerebellum and medulla oblongata) lungs (sample of 2 lobes) spleen
cecum lymph nodes (mandibular, unilateral; mesenteric) stomach colon mammary gland(thoracic
and inguinal) testes duodenum nasal cavities (1 level) thymus epididymides optic nerves thyroid lobes (and parathyroids) esophagus ovaries, oviducts tongue eyes pancreas trachea harderian glands pituitary ureters heart (including section of aorta) prostate urinary bladder ileum rectum a uterus (horns, body and cervix) jejunum salivary gland h (mandibular, parotid, sublingual)
vagina.
For all euthanized animals, 3 sternal bone marrow smears were prepared and stained but not
examined.

3.6 Bone measurements
Femur dimensions (length and width bilaterally) were measured at scheduled necropsy from the pathology subset and the first 10 surviving animals/sex/group in the recovery subset.

3.6.1 Histopathology
Tissues from all animals sampled were prepared for histopathological examination by embedding in paraffin wax, sectioning and staining with hematoxylin and eosin and examined as follows: A complete histopathological evaluation was performed for animals assigned to the pathology subset whereas the histopathological evaluation was limited to the tissues showing gross abnormalities and potential target organ (stomach) for animals assigned to the recovery subset.
Remaining tissues were retained in fixative.

3.7 Histomorphometry
At scheduled termination, the right tibia of the first ten surviving animals/sex/group in each of the pathology and recovery subsets were embedded in plastic, stained with Goldner’s trichrome and a qualitative evaluation performed. In addition, growth plate thickness was measured.

Statistics:

For each pairwise group comparison of interest, significance was reported at the 0.05, 0.01 or
0.001 levels. The data collected from F0 generation dams and treated spare animals, and body weights and clinical signs from day 7 post partum toxicokinetic animals were not reported but retained with the raw data.
Data for parameters not required by protocol but automatically generated by analytical
devices were retained on file, but were not reported.

Remarks on result:

not measured/tested

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Pre-weaning period:
Daily body weight gains were significantly decreased in a dose dependent manner between
days 7 and 11 post partum in both sexes in animals given 400 and 800 mg/kg/day and
continuing until day 14 post partum at a dose of 800 mg/kg/day. These decreased body
weight gains resulted in statistically significantly lower body weights for males and females
from day 8 post partum, until day 19 post partum for animals given 400 mg/kg/day and
lasting until weaning for animals given 800 mg/kg/day. The sporadic affects in the body
weight gains in animals given 100 mg/kg/day did not result in significant changes in body
weight.
Post weaning period:
Overall, in all subsets, body weight gains were generally decreased in animals given 100,
400 and 800 mg/kg/day up to day 35 post partum for females and day 42 post partum for
males, subsequently attaining levels comparable to controls. This generally resulted, in lower
body weights up to day 49 post partum in males and day 45 post partum in females, although
values did not reach statistical significance in all groups of each subset for these periods.
After day 49 post partum for males and 38 post partum for females, the 800 mg/kg/day group
tended to have similar or higher body weight gains than controls. During gestation, body
weight gains were significantly increased in animals given 800 mg/kg/day between days 9 and
13 of gestation, resulting in slightly increased body weights for those animals.
In the post dosing period (reproductive and recovery subsets) there were sporadic significant
increases in body weight gains in all treated groups with a tendency to recover to levels
comparable to controls

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

Pre-weaning period:
Several pups from the 100 and 800 mg/kg/day dose groups were found dead or euthanized between days 8 and 13 post partum due to gavage accident (nos. 251-2, 254-1, 255-3 (100 mg/kg/day) and 459-8 (800 mg/kg/day)). The causes of deterioration for pup nos. 256-8, 451-6 and 459-2 euthanized between days 8 and 11 post partum were undetermined. Body weight gains were decreased in some of these animals. Clinical signs noted prior to death/euthanasia included: activity decreased, empty stomach suspected, cold to touch, skin, pallor, thin, breathing labored and abnormal breathing sounds. There was no clear pattern of treatment-related mortality and considering the low incidence, these deaths were considered unrelated to the administration of AHU377.

Post weaning period:
There was no mortality attributed to the administration of AHU377 post weaning.

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: hindlimb

visceral/soft tissue: gastrointestinal tract

Description (incidence and severity):

minimal/slight hyperplasia of the squamous mucosa minimally increased incidence >= 400 mg/kg/day.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

400 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

presumably yes

4.2.2 Clinical signs
Clinical signs noted that were considered to be AHU377-related were a firm internal abdominal structure, wet fur and salivation. A firm internal abdominal structure was seen for both males and females primarily in animals at a dose of 800 mg/kg/day, commencing during the first week of treatment and generally abating by the end of the second week of dosing. Wet fur and salivation commenced post dose in animals given 400 and 800 mg/kg/day on day 21 post partum and continued through the treatment period.

All other clinical signs noted were considered incidental and unrelated to treatment.

 

4.2.4 Food consumption
Food intake in the treated groups, particularly in animals given 800 mg/kg/day, tended to be higher initially for the females and generally comparable to or higher than controls for the males commencing around day 52 during the post-weaning treatment period. Overall values tended to be increased at a dose of 800 mg/kg/day in both sexes compared to control values for the post dosing period.
The food consumption in the reproductive subset females during gestation was unaffected.

 

4.2.5 Visual function
There was no AHU377-related effect on visual placing or pupillary closure.

 

4.2.6 Physical development
There was no AHU377-related effect on eye opening, vaginal opening or preputial separation.

The mean day of development of preputial separation was 41.7, 42.9, 42.8 and 43.0 days for the 0, 100, 400 and 800 mg/kg/day groups, respectively. The values for all AHU377-treated groups showed statistical significance. As the historical control range for this parameter in this facility is 43.4 to 43.7 days, it was considered that the low value in the control group was the reason for the statistical significance and, therefore, this finding was not considered to be of any toxicological significance.

 

4.2.7 Motor activity
There was no AHU377-related effect noted on motor activity.

 

4.2.8 Auditory startle
There was no AHU377-related effect on auditory startle habituation.
Results that showed statistical significance were considered to be due to biological variation.

 

4.2.9 ‘E’ water maze
There were no AHU377-related differences noted for performance in the ‘E’ water maze.
Any values that showed statistical significance were considered to be due to biological variation.

 

4.2.10 Peripheral quantitative computed tomography (pQCT)
4.2.10.1 Proximal tibia metaphysis
No test article related effect was noted in animals given 100 mg/kg/day. On day 28 post partum, males and females at doses of 400 and 800 mg/kg/day had slightly lower (-7 to -20% and -6 to -19%, respectively, for total, trabecular and cortical/subcortical) bone mineral content (BMC) and bone mineral density (BMD) when compared to controls. The differences attained statistical significance for all parameters at 800 mg/kg/day and for total and cortical/subcortical BMD at a dose of 400 mg/kg/day. Slightly lower values were noted for the total slice area at a dose of 800 mg/kg/day.

In males at the end of the treatment period, no meaningful effect was noted at doses of 100 and 400 mg/kg/day, suggesting reversibility of effects at a dose of 400 mg/kg/day. However the mean BMC and BMD values remained lower (up to 13%) for AHU377-treated males compared to vehicle controls at a dose of 800 mg/kg/day, attaining statistical significance for cortical/subcortical and total BMC and BMD.

In females at the end of the treatment period, the mean BMC and BMD values for AHU377-treated animals were generally lower compared to vehicle controls at all dose levels, attaining statistical significance for total and trabecular BMC and BMD at a dose of 800 mg/kg/day, total and trabecular BMC at a dose of 400 mg/kg/day and total BMD and trabecular BMC and BMD at a dose of 100 mg/kg/day.
At the end of the recovery period, effects on pQCT parameters were generally normalized for bone densitometry values for all dose levels, however, trabecular BMC and BMD remained slightly lower compared to vehicle controls at doses of 400 and 800 mg/kg/day (6 - 16%-attaining statistical significance for BMC at 800 mg/kg/day). All other differences including slightly lower total and cortical/subcortical BMC at doses of 100 and
400 mg/kg/day were attributed to slightly lower total slice area.

 

4.2.10.2 Tibia diaphysis
On day 28 post partum, statistically lower periosteal circumference and total slice area (5 and 10% respectively) were noted for males at a dose of 800 mg/kg/day compared to vehicle controls. Endosteal circumference was also proportionally lower (6%) for treated males compared to vehicle control males, resulting in comparable cortical thickness. The mean cortical area and cortical BMC were also slightly lower, attaining statistical significance for cortical BMC, which was consistent with lower values for periosteal circumference and total nslice area. A greater decrease in BMC, relative to cortical area, was associated with significantly lower (4%) cortical BMD. For males at a dose of 400 mg/kg/day, slightly (but statistically significant) lower values were noted for cortical BMD, consistent with the statistically lower cortical BMC value. Cortical thickness was slightly lower (5%) and associated with marginally higher endosteal circumference (2%) and marginally lower (1%) periosteal circumference. No meaningful effect was noted at a dose of 100 mg/kg/day. The results noted for females on day 28 post partum at a dose of 800 mg/kg/day were similar to
males with lower (4 - 13%) values for periosteal circumference, endosteal circumference, cortical BMC, cortical BMD, total slice area and cortical area compared to vehicle controls.
The differences attained statistical significance for bone length, cortical area, cortical BMD, and cortical BMC.
At the end of the treatment period in males, the effects on bone size and bone mass were generally reversed at a dose of 400 mg/kg/day. Evidence for normalization on bone mass and bone size (length and diameter) were also noted at a dose of 800 mg/kg/day. However the mean cortical BMC and BMD remained statistically significantly lower at a dose of 800 mg/kg/day when compared to vehicle controls. The mean cortical thickness and cortical area were also slightly lower and were attributed to the marginally lower periosteal circumference and higher endosteal circumference. No meaningful effect was noted at a dose of 100 mg/kg/day, except slightly lower cortical thickness attributed to slightly higher endosteal circumferences. In females at a dose of 800 mg/kg/day at the end of the treatment period, the mean BMC and cortical area remained slightly lower (but still statistically significant) compared to vehicle controls. The mean cortical thickness was slightly lower (4%) and was attributed to marginally lower periosteal circumference and higher endosteal circumference. At the end of the treatment period, similar responses were also noted females at doses of 100 and 400 mg/kg/day. The mean cortical area, cortical BMC and cortical thickness were lower for treated females compared to vehicle controls, generally attaining statistical significance (except cortical area at a dose of 100 mg/kg/day). Similar to males, these effects seemed transient and evidence of recovery were noted for both bone mass and bone geometry parameters at the end of the treatment period.
No meaningful effect was noted at the end of the recovery period in males. At the end of the recovery period in females, the mean bone densitometry and bone geometry parameters were comparable to vehicle controls.

 

4.2.11 Radiographs
On day 28 post partum, mean bone length values for femur and tibia (left and right) were slightly lower (maximum of -4%) in treated males and females, attaining statistical significance at a dose of 800 mg/kg/day. Slightly lower values were also noted in the width of the long bones in animals (females up to -5%) at a dose of 800 mg/kg/day when compared to controls. Effects on the axial skeleton were limited to a slight reduction in length in males and females and in width only in females at a dose of 800 mg/kg/day.
At the end of the treatment period, a trend towards normalization was evident in both males and females when compared to controls although mean bone length was still minimally lower in males (-2%) and females at a dose of 800 mg/kg/day.
At the end of recovery, males at a dose of 800 mg/kg/day were completely recovered when compared to controls, although some differences persisted in the females.
The statistically significantly lower value for the right tibia width at the end of the recovery period in females was not consistent with the observations at doses of 100 or 800 mg/kg/day and, therefore, was likely incidental in nature.

 

4.2.12 Bone measurements
At the end of treatment, femur length was significantly lower for males (-2%) and slightly lower for females at a dose of 800 mg/kg/day attaining statistical significance only for the left femur of the males. Slightly lower femur width (-4%) was noted in males at a dose of 800 mg/kg/day.
At the end of recovery, there was no apparent effect noted in males, although in females the decreases in bone length persisted slightly (femur and tibia).

 

4.2.13 Mating procedures (F1 adult generation)
No significant differences were noted between groups in terms of days to mating, the conception rate or mating and fertility indices.

 

4.2.14 Uterine findings
There were no AHU377-related effects on total numbers of corpora lutea, numbers of implantation sites, live/dead embryos, resorptions and the pre and post implantation loss.

 

4.3 Clinical pathology
4.3.1 Hematology
There were no test article-related changes in hematology parameters associated with administration of AHU377 at doses of 100, 400 or 800 mg/kg/day in males and females.
Noteworthy, but not considered test-article related, due to low incidence of changes within a group: one male given 800 mg/kg/day (no. 4047) had moderate to marked increases in neutrophil count, mild decreases in lymphocyte counts, minimal decreases in red blood cell parameters (red blood cell count, hemoglobin and hematocrit), a marked decrease in reticulocyte count and a moderate increase in platelet count. These changes correlated with microscopic bone marrow hematopoietic hypercellularity and splenic/thymus lymphoid atrophy, and were considered secondary to gastrointestinal inflammatory changes observed in this animal.

At the end of the recovery period, there were no AHU377-related changes in hematology.
All differences in hematology parameters, including those determined to be statistically significant, were judged to be due to biological variation or considered unrelated to test-article treatment based on the inconsistency of the changes and/or lack of dose-response over the course of the study.

 

4.3.2 Clinical biochemistry
AHU377-related changes in clinical biochemistry were observed at doses ≥ 100 mg/kg/day compared to controls. There were minimal increases in urea (UREA, 14% to 17%) in males and females at a dose of 800 mg/kg/day. There were mild non dose-related increases in phosphorus (PHOS, 13% to 20%) in males and females at all dose levels.
These changes had no observed direct microscopic correlate, although increases in UREA were supportive of subclinical dehydration, which may have resulted from observed gastric lesions.
At the end of recovery there were no important changes in PHOS, indicating reversibility of the changes previously described for this parameter. There was a persistent mild increase in urea (16%) in males at a dose of 800 mg/kg/day. There were mild increases in glucose (40%) and triglycerides (55%) in males at a dose of 800 mg/kg/day. These changes were not observed during the treatment period, but were dose-related and considered AHU377-related.
Moderate to marked increases in aspartate aminotransferase and mild increases in alanine aminotransferase were observed in one male at a dose of 100 mg/kg/day (no. 2022) and 4 females at a dose of 800 mg/kg/day (animal nos. 4922, 4924, 4927 and 4928), with no concurrent increase in creatine kinase. A mild increase in alkaline phosphatase was also noted in no. 2022. These changes were not observed during the treatment period and had no microscopic correlate, except for animal no. 4927 who presented a mild hepatic chronic inflammation. Despite their low incidence, these changes were considered AHU377-related at a dose of 800 mg/kg/day, however they were considered of uncertain relation at at a dose of 100 mg/kg/day.
Other differences in clinical biochemistry parameters, including those that reached statistical significance, were judged to be due to biological variation or considered unrelated to test article treatment based on the inconsistency of the changes and/or lack of dose-response over the course of the study.

 

4.3.3 Urinalysis
There were no test article-related changes in urinalysis parameters associated with administration of AHU377 at all doses tested in males and females.

At the end of the recovery period, there were no AHU377-related changes in urinalysis parameters.
Other differences in urinalysis parameters were considered to be due to normal biological variation and not due to the administration of the test-article.

 

4.4 Pathology
Summary data are presented in the Summary tables section, and individual data are included in Appendix 4.

 

4.4.1 Histomorphometry
There were no microscopic abnormalities detected in the proximal tibia of the pathology and recovery subsets.
The administration of AHU377 had no effects on the growth plate thickness in two subset groups (pathology and recovery). All variations noted were regarded as normal biological variations.

 

4.4.2 Macroscopic observations
One pathology subset rat from group 4 (animal no. 4047) showed a thickening of the wall in the jejunum and ileum that correlated microscopically with villous/mucosal hyperplasia observed in the respective intestinal sections. These changes were considered of uncertain toxicological significance.
No macroscopic findings were considered to be related to treatment with AHU377 following the recovery period. A few macroscopic findings observed in various organs and tissues appeared to be agonal or incidental, and of no toxicological significance.
All other macroscopic findings observed in various organs and tissues from the pathology subset appeared to be agonal or incidental and were of no toxicological significance.

4.4.3 Organ weights (pathology and recovery subsets)
No organ weight changes were considered to be related to treatment with AHU377 in either examined subset. All organ weight changes noted, including those that reached statistical significance, were regarded as normal biological variations.

 

4.4.4 Microscopic observations
Histopathological examination revealed a possible exacerbation of microscopic changes in the stomach of rats of both sexes at ≥ 100 mg/kg/day, as summarized in the table below.
In the stomach, there was a slight increase in incidence of hyperplasia of the squamous mucosa that was localized at the limiting ridge in all treated groups, with no dose-response compared to the control rats. In a few animals, one additional change at the same location was recorded as vacuolation and was usually seen in combination with submucosal inflammation. Because these changes (hyperplasia and vacuolation, squamous mucosa) were also observed in one control rat (no. 1951), the slightly increased, although comparable incidence observed in all treated groups was suggestive of an exacerbation by the test-article of a change seen associated with gavage administration. This interpretation was further corroborated by identical changes seen in control animals following the recovery period, as described below.

One high dose animal (no. 4047) showed a few uncommon gastro-intestinal changes such as minimal increased single cell necrosis of parietal cells (stomach), slight villous/mucosal hyperplasia (jejunum and ileum), and moderate transmural inflammation (jejunum) extending into the mesentery. Because of the rare spontaneous occurrence of such gastrointestinal changes, a test-article related effect cannot be excluded. However, this interpretation remained equivocal due to the very low incidence noted in 1 of 24 high dose rats. Additional changes in the same animal included minimal hematopoietic hypercellularity in the bone marrow, minimal atrophy in the genital tract (prostate and seminal vesicles) and slight to moderate lymphoid atrophy in various lymphoid organs (thymus and spleen). They were all regarded as indirect, secondary to the gastrointestinal changes and/or stress-related.
Other microscopic findings seen in various organs and tissues at the end of treatment were considered to be agonal or incidental, and of no toxicological significance.
Microscopic changes were still observed in the stomach of rats euthanized at completion of the recovery period, as summarized in the table below.

Minimal to slight hyperplasia of the squamous mucosa was observed in both controls and/or treated male and female rats with a low incidence. The toxicological significance of the minimal increased incidence noted at ≥ 400 mg/kg/day remained equivocal and could be interpreted as to be in the possible range of variability in rats of this age given compounds by
oral gavage.
Other microscopic findings seen in various organs and tissues were considered to be agonal or incidental, and of no toxicological significance.

Conclusions:

In conclusion, doses of 100, 400 and/or 800 mg/kg/day, resulted in transient clinical signs,
effects on body weight, food consumption and phosphorous levels. At the end of recovery,
mild to marked increases in glucose and triglycerides for males and alanine aminotransferase
and/or aspartate aminotransferase were observed in a few females at a dose of 800 mg/kg/day.
Equivocal microscopic changes in the stomach of rats (hyperplasia and vacuolation of the
squamous mucosa) in both sexes at doses >= 100 mg/kg/day were noted at the end of treatment,
and at completion of the recovery period, hyperplasia of the squamous mucosa was observed
in the stomach of rats with a minimally increased incidence noted at doses >= 400 mg/kg/day.
AHU377 administration resulted in generally dose dependent and slight decreases in bone
length (femur and tibia) in animals at a dose of 800 mg/kg/day. Treatment related effects
were noted on the bone mass (BMD and BMC) at the metaphysis (at >= 400 mg/kg/day for
both males and females) and diaphysis (at >= 400 mg/kg/day for males and at 800 mg/kg/day
for females). At the end of the recovery period, the bone length, diameter and mass were
generally comparable to vehicle controls. Based on the findings in this study, the
No Observed Adverse Effect Level (NOAEL) was considered to be 100 mg/kg/day.

Executive summary:

This study was conducted in accordance with the United States Food and Drug Administration Good Laboratory Practice Regulations Fed. Reg., Vol. 43, 21 CFR Part 58, 22-Dec-1978, and all the subsequent amendments to these regulations. This study was performed according to the recommendations of the International Conference on Harmonization (ICH) guidelines on detection of toxicity to reproduction for medicinal products; availability; notice. Fed. Reg., Vol. 59, No. 183, 22-Sep-1994.

The objective of this study was to determine the potential adverse effects of AHU377 on the postnatal development of the rat. The juvenile animal was administered AHU377 from day 7 post partum through to day 70 post partum (young adult). Animal exposure to AHU377 was determined by toxicokinetic analysis.

 

AHU377 (batch no. 0722008) solutions in 0.5% (w/v) hydroxypropylcellulose (grade HF), NF (Klucel), aqueous solution, were administered orally, via gavage, at doses of 100, 400 and 800 mg/kg/day (base) to three groups (n = 52/sex) of male and female Wistar Hanover Crl:WI(Han) rats from days 7 through 70 post partum. An additional group (n = 52/sex) of animals received the vehicle at dose volumes equivalent to the high dose (6.5 mL/kg) and served as controls. The 100 and 400 mg/kg/day dose groups received dose volumes of 5 mL/kg. The animals were obtained from Charles River Canada Inc., St. Constant, Quebec and weighed between 10.5 and 18.9 grams at the initiation of dosing. Satellite animals (n = 20/sex for the AHU377-treated groups and n = 10/sex for controls) received single doses of AHU377 on post partum day 7 and were utilized for toxicokinetic evaluations. The following parameters were evaluated: mortality, clinical signs, body weights, food intake, physical development (eye opening, vaginal opening, preputial separation), sensory development (auditory startle habituation, visual function), behavioral performance (motor activity, water maze), reproductive phase (mating, gestation day 13 uterine examination), hematology, biochemistry, urinalysis, toxicokinetics, peripheral quantitative computed tomography, radiographs, histomorphometry, organ weights, gross and histopathological evaluations.

 

There was no treatment related mortality during the course of this study.
There were no test article related effects noted on eye opening, visual placing, pupillary closure, vaginal opening, preputial separation, motor activity, auditory startle habituation, swimming ability or performance, hematology, urinalysis, days to mating, conception rate, mating and fertility indices, histomorphometry, organ weights, macroscopic findings, number of corpora lutea, number of implantation sites, number of live/dead embryos, resorptions, or pre and post implantation loss.

 

Clinical signs typically noted that were considered to be AHU377-related were firm internal ,abdominal structure (transient and primarily seen at a dose of 800 mg/kg/day), fur wet and salivation.

 

Overall, in all subsets, body weight gains were generally decreased at all dose levels until just past weaning. This generally resulted in transient, lower body weights up to day 49 post-partum in males and day 45 post partum in females. After day 49 post partum for males and 38 post partum for females, the 800 mg/kg/day group tended to have higher body weight gains than controls. During gestation, body weight gains were significantly increased in animals at a dose of 800 mg/kg/day between days 9 and 13 of gestation, resulting in slightly increased body weights for these animals. In the post dosing period (reproductive and recovery subsets) there were sporadic, significant increases in body weight gains in all treated groups with a tendency to recover to levels comparable to controls.

 

Food intake in the treated groups tended to be higher when compared to controls during the post-weaning treatment period. Overall values were increased at a dose of 800 mg/kg/day compared to control values for the post dosing period. There was no effect on food consumption noted in the reproductive females during gestation. Treatment with AHU377 resulted in generally dose dependent and slight decreases in the length and width of appendicular (at doses ≥ 100) and axial skeleton (at a dose of 800 mg/kg/day) at day 28 pp, consistent with slightly reduced body weight gains. Slight decreases were noted in pQCT bone geometry parameters (periostal circumference and total slice area) for both males and females at a dose of 800 mg/kg/day, consistent with the effect on bone length. In addition to the bone length, treatment related effects were noted on the bone mass (BMD and BMC) at the metaphysis (at doses ≥ 400 mg/kg/day for both males and females) and diaphysis (at doses ≥ 400 mg/kg/day for males and at a dose of 800 mg/kg/day for females). The effects on bone dimension (X-ray measurements and pQCT) were transient and were generally normalized at the end of the treatment period. In general, the effect on bone mass persisted throughout the treatment period at the metaphysis however evidences of normalization were noted at the diaphysis. At the end of the recovery period, the bone length, diameter and mass were generally comparable to vehicle controls.

 

Mild AHU377-related changes in clinical biochemistry were observed in urea and/or phosphorus at all doses tested during the treatment period. Phosphorus changes did not persist at the end of the recovery period. Mild dose-related increases in glucose and  triglycerides were observed in males at 800 mg/kg/day at the end of the recovery period, and were considered to be AHU377-related. Mild to marked increases in alanine aminotransferase and/or aspartate aminotransferase were observed in a few individual females at a dose of 800 mg/kg/day. Despite their low incidence, these changes were considered
AHU377-related. Similar changes observed in 1 male at a dose of 100 mg/kg/day were considered of uncertain significance to test-article. Microscopic changes thought to be related to AHU377 were noted in the stomach of rats (hyperplasia and vacuolation of the squamous mucosa) in both sexes at doses ≥ 100 mg/kg/day at the end of the treatment period. At completion of the recovery period, hyperplasia of the squamous mucosa was observed in the stomach of rats with a minimally increased incidence noted in animals at doses ≥ 400 mg/kg/day. However, the toxicological significance of this finding remained equivocal and could be interpreted as to be in the possible range of variability in the rats of this age administered compounds by oral gavage.

 

Toxicokinetic evaluations indicated that all treated animals were exposed to the test article.

 

On day 64, there was a trend towards an over-proportional increase in exposure with increasing dose for AHU377 in both sexes. For the metabolite LBQ657, an over-proportional increase was only noted in females for the high dose group. Mean AUC values for AHU377 on day 64 were 874/402, 4590/5360 and 16300/12100 ng*h/mL for males/females, respectively. Mean AUC values for LBQ657 on day 64 were 46600/37300, 157000/207000  and 465000/802000 ng*h/mL for males/females, respectively. The total exposure to AHU377 plus LBQ657 on day 7 was proportional with increasing dose.  

 

In conclusion, doses of 100, 400 and/or 800 mg/kg/day, resulted in transient clinical signs, effects on body weight, food consumption and phosphorous levels. At the end of recovery, mild to marked increases in glucose and triglycerides for males and alanine aminotransferase and/or aspartate aminotransferase were observed in a few females at a dose of 800 mg/kg/day. Equivocal microscopic changes in the stomach of rats (hyperplasia and vacuolation of the squamous mucosa) in both sexes at doses ≥ 100 mg/kg/day were noted at the end of treatment, and at completion of the recovery period, hyperplasia of the squamous mucosa was observed in the stomach of rats with a minimally increased incidence noted at doses ≥ 400 mg/kg/day. AHU377 administration resulted in generally dose dependent and slight decreases in bone length (femur and tibia) in animals at a dose of 800 mg/kg/day. Treatment related effects were noted on the bone mass (BMD and BMC) at the metaphysis (at ≥ 400 mg/kg/day for both males and females) and diaphysis (at ≥ 400 mg/kg/day for males and at 800 mg/kg/day for females). At the end of the recovery period, the bone length, diameter and mass were generally comparable to vehicle controls. Based on the findings in this study, the No Observed Adverse Effect Level (NOAEL) was considered to be 100 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the lack of significant developmental or fertility adverse effect observed in the available studies, according to the Regulation (EC) No 1272/2008, AHU 377 does not have to be classified and has no obligatory labelling requirement for reproductive toxicity.