Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 06, 2015 - Oct 29, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
none
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Batch: E015012542
Appearance: White solid
Purity: 99.96 %
Released until: Mar 31, 2017

Test animals

Species:
rat
Strain:
other: Crl:CD (SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, Zona Industriale Azzida, 57, 33049 San Pietro al Natisone (UD), Italy on July 14th, 2015 (shipping slip No15003754).
- Age at study initiation: 9 weeks
- Weight at study initiation: (P) Males: 248.2 - 270.1 g; Females 174.0 - 193.4 g
- Fasting period before study: -
- Housing: single
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): 20 air changes per hour filtered on HEPA
- Photoperiod (hrs dark / hrs light): 12 / 12 hours

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Methocel KM4 Premium 0.25 % in Milli-Q
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0.25 % in pure water (Milli-Q)
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): 2K27012N01
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 7 evenings/week with a maximum of 14 evenings
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the formulated test item was checked twice during the dosing period (i.e., on formulations prepared on one day of the pre mating and post mating periods).The HPLC analytical method was validated. A stability and homogeneity study in 0.25% Methocel® K4M Premium in water was performed.
Duration of treatment / exposure:
Oral, by gavage
F0 Males: The test item or the vehicle was administered daily for 2 weeks before the mating
period and throughout the same. Males were further dosed after the mating period to achieve a
minimum total dosing period of 28 days.
F0 Females: The test item or the vehicle was administered daily for 2 weeks before the start of
the mating period and throughout the same. Treatment continued during pregnancy and until Day
3 of lactation.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
80 (40m / 40f)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on 28 day study with similar chemical
- Rationale for animal assignment (if not random): random
- Other:

Examinations

Parental animals: Observations and examinations:
Mating
The rats were mated, one male to one female. Each male was placed and left with the same
female for 7 days/week, for a maximum of 14 days, up to detection of pregnancy.
Every morning, a vaginal smear from each female was examined at the microscope. The day on
which the presence of spermatozoa was found (positive smear) was considered Day 0 of
pregnancy for that female.
The slides of vaginal smears were disposed of after evaluation.
Females that never showed a positive smear were separated from the male at the end of the
mating period.

Clinical Signs
Physical appearance, behavior and clinical signs were observed twice a day.
Any deviation from the norm was recorded.
The duration of gestation was recorded and calculated from Day 0 of pregnancy (day of positive
smear).
The day of parturition was defined as Day 0 of lactation.
Observations for birth were scheduled three times a day during working hours. If births occurred
after working hours, the following day was defined as Day 0 of lactation.
Each litter was examined as soon as possible after delivery to establish the number and sex of
pups, stillbirths, live births, runts (pups that are significantly smaller than corresponding control
pups) and the presence of gross abnormalities.

Body Weight and Food and Water Consumption
Males and females were weighed on the day before the start of dosing (Day -1 of the study), on
the first day of dosing (Day 1 of the study) and then weekly thereafter up to the end of treatment.
During pregnancy, females were weighed on days 0, 7, 14 and 20 and within 24 hours of
parturition (Day 0 of lactation) and on Day 4 of lactation.
Weights were reported individually for each adult animal.
Body weight of Female No. 32 of Group 2 was recorded on Days 0, 1, 2, 3 and 4 of lactation,
after dystocic parturition, to closely monitor the health of the animal.
During the pre-mating period and pregnancy, food consumption was measured weekly and
during lactation on Day 4.
Water consumption was measured twice a week on the following days:
- Pre-mating: Days -1, 4, 7, 11, 14 of the study;
- Pregnancy: Days 0, 4, 7, 11, 14, 17 and 20;
- Lactation: Days 0 and 4.

Sperm parameters (parental animals):
A detailed qualitative examination of one testis and the equilateral epididymis was done in 10male rats of the control (vehicle) group, as well as in 10 males of the high dose group, taking intoaccount the tubular stages of the spermatogenic cycle. The examination was conducted in orderto identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and disorganization of the germinal epithelium.
Litter observations:
On Day 0 of lactation, the pups of each litter will be identified with a mark made by appropriately
coloring different sides of the body; this mark will be renewed when necessary.
Live pups will be counted and sexed and litters weighed within 24 hours of parturition (Day 0 of
lactation) and on Day 4 post partum.
In addition to the observations on parent animals, any abnormal behavior of the offspring will be
recorded.
At birth and during the lactation period, all the young will be individually observed for:
- Live and stillbirths;
- Mortality ensuing after live birth, ascertained daily. Whenever possible, any pup found dead
will be examined externally and internally in an attempt to determine the cause of death;
- Sex (on Day 0 of lactation by an external examination and measurement of the ano-genital
distance and by internal examination, at sacrifice, on Day 4 of lactation);
- External abnormalities at birth;
- Pup weight (on Days 0 and 4 of lactation).
Postmortem examinations (parental animals):
Gross pathologyHistopathology
Postmortem examinations (offspring):
Gross pathologyHistopathology
Statistics:
Body weight, food and water consumption, mortality, clinical signs, dosing, reproduction data,and fetal weight data were recorded and stored in the PROVANTIS™ system.
Reproductive indices:
-Mating index: percent ratio between the animals with positive smear + females found to bepregnant but without positive smear and the animals mated.
- Fertility index: percent ratio of females having evident signs of pregnancy with respect to thefemales that had positive vaginal smear and females found to be pregnant but without positivesmear.
- Pregnancy index: the percent ratio of females with live births with respect to the pregnantfemales.
- Pregnancy period: the duration of pregnancy was determined for all those dams that reachedpregnancy term as being the time that elapsed between the positive vaginal smear and the start ofparturition.
- Mean mating time: mean mating time will be calculated on the dams proved pregnant and will be expressed for each group as the mean time lapse (in days) between the beginning of the mating period and the ascertainment that copulation occurred. The females found to be pregnant but without positive smear will be excluded from this calculation.
Offspring viability indices:
- The mean F1 body weight was obtained by averaging the mean weight of each litter at thevarious times.- The F1 probability of survival was analyzed per group. Animals that died owing to accidentalcauses or were sacrificed at the end of lactation were statistically censored.- The mean value per litter of live pups (number of males and females and total) was calculatedat different times (Days 0, 4 of lactation).- The following indices of each litter were calculated:Birth index:No. of live newborns at birth----------------------------------- x 100No. of implantationsViability index on Day 4:No. live pups on Day 4 after birth----------------------------------------- x 100No. of live birthsGroup mean values were calculated from individual data in two ways:Mean A: calculated on all the surviving females having evident signs of pregnancy, includingthose that presented 100% post-implantation losses.Mean B: calculated only on those females with viable fetuses at term.The external, visceral and skeletal malformations and variations found were described for eachlitter.All data were recorded and stored in PROVANTIS TM System.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One Female of the 1000 mg/kg/day treated group showed piloerection and hunched posture on
Day 10-12 of the study. On Day 11 of the study, due to the severe clinical signs, this female did
not receive treatment. This female was found dead on day 13 of the study; at histopathology
evaluation this animal showed severe inflammatory reactions around the esophagus and lungs
(thoracic cavity), typical of a gavage error.

One Female of the 100 mg/kg/day had a dystocic parturition after showing pale ear and tail
with piloerection and slight blood discharge from the vagina on Day 21-22 of pregnancy up to
Day 2 of lactation.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One Female of the 1000 mg/kg/day treated group showed piloerection and hunched posture on
Day 10-12 of the study. On Day 11 of the study, due to the severe clinical signs, this female did
not receive treatment. This female was found dead on day 13 of the study; at histopathology
evaluation this animal showed severe inflammatory reactions around the esophagus and lungs
(thoracic cavity), typical of a gavage error.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Males did not show any compound-related effects on body weight at any time point during the
study at 100, 300 and 1000 mg/kg in comparison to controls.
During the pre-mating period, pregnancy and lactation no compound-related effects were
observed on body weight and body weight gain in females at 100, 300 and 1000 mg/kg in
comparison to controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No changes were observed in the mean food consumption of the 100, 300 and 1000 mg/kg/day
treated males, in comparison to controls, except for a statistically significantly lower mean food
consumption observed at 300 and 1000 mg/kg during the Day 1-7of the pre-mating period.
No effects were seen on food consumption of females at 100, 300 and 1000 mg/kg in comparison
to the control group during the pre-mating period, gestation and lactation.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Higher mean water consumption was observed in males treated at 100, 300 and 1000 mg/kg/day
during the study, reaching statistically significantly higher mean values in the Day 7-11 and 11-
14 periods.
During the 2-week pre-mating period higher mean water consumption was observed at 100, 300
and 1000 mg/kg in the Day 7-11 period. This observation was not confirmed thereafter since
during pregnancy and lactation no differences were observed in the mean water consumption of
females treated at 100, 300 and 1000 mg/kg/day in comparison to control females.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: higher mean water consumption was observed in males of the treated groups in the Day 7-11 and 7-14 periods in comparison to controls

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

Offspring development was not affected by the test-item.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
On the basis of the above findings 1000 mg/kg was considered the parental NOAEL and 1000 mg/kg was considered the NOEL for reproductive / developmental toxicity.
Executive summary:

The informtion for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD TG 421.

On the basis of this study 1000 mg/kg was considered the parental NOAEL and 1000 mg/kg was considered the NOEL for reproductive / developmental toxicity.