Registration Dossier

Administrative data

Endpoint:
fertility, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Feb - Aug 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: ICH Harmonised Tripartite Guideline S5 (R2) Guideline for Industry: Detection of Toxicity to Reproduction for Medicinal Products & Toxicity to Male Fertility
GLP compliance:
yes
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).

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rattus norvegicus/Wistar Hannover Crl:WI (Han)
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

Sex:
male/female
Details on test animals 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

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.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% (w/v) hydroxypropylcellulose
Details on exposure:
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:
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.
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:
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:
60 days
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)
First day of dosing
18-Feb-2014 (females)
18-Feb-2014 (males)
04-Mar-2014 (females)
Last necropsy 25-Apr-2014
Experimental completion date 29-Aug-2014
Doses / concentrationsopen allclose all
Dose / conc.:
7.5 mg/kg bw/day
Dose / conc.:
25 mg/kg bw/day
Dose / conc.:
75 mg/kg bw/day
No. of animals per sex per dose:
24 males and 24 females per goup
Control animals:
yes, concurrent no treatment
Details on study design:
Group no./ Test material Dose level (mg/kg/day)* Dose volume (mL/kg) Dose concentration (mg/mL) Animal nos.
Males Females
1/ Vehicle control 0 10 0 1001-1024 1501-1524
2/ AHU377 75 10 7.5 2001-2024 2501-2524
3/ AHU377 250 10 25 3001-3024 3501-3524
4/ AHU377 750 10 75 4001-4024 4501-4524
* Dose levels and concentrations are expressed in base. Dose formulations were adjusted for the salt factor.
The salt/base ratio for AHU377 is 1.046.

Examinations

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.

Variables for inferential analysis Statistical method
ANOVA Non-parametric Incidence
Body weight X
Corpora lutea count X
Numbers of implants X
Live embryos X
Dead embryos X
Number of early resorptions X
Sum of resorptions and dead embryos X
Organ weights X
Body weight change* X
Pre implantation losses** X
Post implantation losses** X
Parental rates and indices X
Parental time to mate X
Organ weight relative to body weight X
Male reproductive assessments
(count and motility) X

* At each interval and overall values during the premating treatment period, post mating period (as applicable) and the gestation period (days 0 to 13 pc).
** Calculated based on litter means/values.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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
Other effects:
no effects observed
Description (incidence and severity):
Parental performance
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.
Male reproductive assessments
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.

Reproductive function / performance (P0)

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

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
paternal toxicity
Effect level:
250 mg/kg bw/day
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
Sex:
female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive function and early embryo-fetal development
Effect level:
750 mg/kg bw/day
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not specified

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
750 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
other: embryo-fetal development

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
750 mg/kg bw/day
Treatment related:
yes

Applicant's summary and conclusion

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.