Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 March 2015 to 10 May 2016
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 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

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

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

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

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, UK
- Age at study initiation: 9-10 weeks old
- Weight at study initiation: 183 - 264 g
- Housing: 2 animals per cage in appropriately sized suspended polycarbonate/polypropylene cages.
- Diet (e.g. ad libitum): ad libitum throughout study, except during designated procedures
- Water (e.g. ad libitum): ad libitum
- Acclimation period: From arrival until Day 6 gestation.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-25 °C
- Humidity (%): 44-57%
- Air changes (per hr): at least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle

IN-LIFE DATES: From: 30th March 2015 To: 14th April 2015

Administration / exposure

Route of administration:
oral: gavage
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations were prepared based on a method established at the Test Facility at appropriate concentrations to meet dosage level requirements. The dosing formulations were prepared at appropriate intervals and were split into suitable volume aliquots for daily dispensing, stored in a refrigerator set to maintain 4°C, and dispensed daily. The dosing formulations were removed from the refrigerator for at least 30 minutes before dosing. The dosing formulations were stirred continuously from at least 30 minutes prior to dosing until dosing was completed each day. Any residual volumes were discarded with the exception of the residues from Day 17 of dosing; these residues were used for formulation analysis sampling; following completion of sampling the residues were discarded.

VEHICLE
Arachis Oil
- Amount of vehicle (if gavage): 4 mL/kg
The control formulation, Arachis Oil BP, was stored in a refrigerator set to maintain 4°C, and dispensed daily. The control formulation was removed from the refrigerator at least 30 minutes prior to dosing. The control formulation was stirred continuously from at least 30 minutes prior to dosing until dosing was completed each day. Any residual volumes were discarded with the exception of the residues from Day 17 of dosing; these residues were used for formulation analysis sampling; following completion of sampling the residues were discarded.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Test item and the control were administered from Days 6-20 of gestation.
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
24 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were agreed with the Sponsor following evaluation of a previous twenty-eight day repeated dose oral (gavage) toxicity study in the rat (OECD Guideline 407) and a reproduction/ developmental toxicity screening oral gavage study in the rat (OECD Guideline 421; Charles River Study No. 496381) performed on behalf of the Sponsor.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Animals were checked twice a day (once at the start and once at the end of the working day throughout the study) for general health, mortality or moribundity. The animals were not removed from the cage during these observations, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS: Yes
- The animals were removed frm the cage and examined daily from the first day of dosing (Day 6 of gestation)

BODY WEIGHT: Yes
- Body weights were recorded on Day and on Days 6 to 21 of gestation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal was quantitatively measured daily from Day 4 to Day 21 of gestation.

WATER CONSUMPTION
- Water consumption was monitored by visual inspection of the water bottles regularly throughout the study.

POST-MORTEM EXAMINATIONS: Yes / No / No data
- Animals were euthanised by carbon dioxide exposure on Day 21 of gestation. All adult 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 procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.
Ovaries and uterine content:
The reproductive tract was dissected from the abdominal cavity. The gravid uterus was weighed. The uterus was opened and the contents examined. The fetuses were then removed from the uterus.
The ovaries and uterus were examined for number and distribution of corpora lutea, implantation sites, placentae (size, color or shape) – any abnormalities were recorded as were the numbers of live and dead fetuses and early and late embryonic deaths.
Fetal examinations:
Fetuses were examined for external abnormalities. Late resorptions and dead fetuses were examined for external abnormalities to the extent possible.
Each implant was classified as being a live fetus, a dead fetus (dead full term fetus that showed no sign of maceration), a late embryonic death (macerated tissue identifiable as an embryo or fetus, with recognisable external features such as tail, limbs, mouth and nares present; attached to distinct identifiable placentae), or an early embryonic death (discrete, formless, discolored tissue mass attached to the internal uterine wall; may be of varying size).
Statistics:
Means and standard deviations were calculated for body weight, food consumption and pregnancy data.
Where required to assist interpretations, tests were applied to determine the statistical significance of observed differences between Control and groups receiving test item. All statistical tests were two-sided and performed at the 5% significance level using in house software. Pairwise comparisons were only performed against the control group. Body weight and food consumption data were analysed for homogeneity of variance using the ‘F-Max' test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test ie pairwise comparisons were made only if the overall F-test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal- Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test).
Following review of the other data obtained on the study, the Study Director considered that no further statistical analysis necessary.
Indices:
Post-implantation loss (%) per litter, group mean fetal weights for live fetuses and group mean litter weight were calculated.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Post dose ploughing in the cage shavings was noted in animals from all dose groups. As this observation was also recorded in control animals it is not considered related to the test item, but may be a result of the oil based vehicle.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
The total number of abortions was the same between the control and tested animals.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Pregnancy performance parameters were similar between the control animals and the animals that recieved EXP0700332.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Pregnancy performance parameters were similar between the control animals and the animals that recieved EXP0700332.
Early or late resorptions:
no effects observed
Description (incidence and severity):
Pregnancy performance parameters were similar between the control animals and the animals that recieved EXP0700332.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Pregnancy performance parameters were similar between the control animals and the animals that recieved EXP0700332.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOEL
Effect level:
ca. 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
ophthalmological examination
behaviour (functional findings)
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
maternal abnormalities
number of abortions
pre and post implantation loss
total litter losses by resorption
effects on pregnancy duration
early or late resorptions
dead fetuses
changes in pregnancy duration
changes in number of pregnant
necropsy findings

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There were no fetal abnormalities and variants, including those indicating the state of skeletal
ossification, that were considered to be related to EXP0700332 administration.
Skeletal malformations:
no effects observed
Description (incidence and severity):
There were no fetal abnormalities and variants, including those indicating the state of skeletal
ossification, that were considered to be related to EXP0700332 administration.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no fetal abnormalities and variants, including those indicating the state of skeletal
ossification, that were considered to be related to EXP0700332 administration.

Effect levels (fetuses)

Key result
Dose descriptor:
NOEL
Effect level:
ca. 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
external malformations
skeletal malformations
visceral malformations

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Administration of EXP0700332 at dose levels of up to 1000 mg/kg/day was not associated with any changes in clinical signs, body weights, food consumption, necropsy findings, pregnancy performance or fetal development. In conclusion, under the conditions of this study the maternal and fetal no observed effect level (NOEL) was considered to be 1000 mg/kg/day.
Executive summary:

The objective of this study was to determine the potential toxicity of EXP0700332 when administered by oral gavage to pregnant rats from implantation until the end of gestation. Dosage levels of 0, 100, 300 and 1000 mg/kg/day were administered to each animal once a day. The following parameters and end points were evaluated in this study: clinical signs, body weights, food consumption, gross necropsy findings, pregnancy performance and fetal examinations. Administration of EXP0700332 at dose levels of up to 1000 mg/kg/day was not associated with any changes in clinical signs, body weights, food consumption, necropsy findings, pregnancy performance or fetal development. In conclusion, under the conditions of this study the maternal and fetal no observed effect level (NOEL) was considered to be 1000 mg/kg/day.