Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From September 2012 to July 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD, EPA, etc)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No. 8147, (24 November 2000)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): XP-7866
- Substance type: white powder
- Physical state: Solid
- Analytical purity: 99.5% (certificate of analysis provided by the sponsor included in the original report)
- Purity test date: 99.5%
- Lot/batch No.: 10188-1 JM
- Expiration date of the lot/batch: 28 February 2015
- Stability under test conditions: responsability of the Sponsor
- Storage condition of test material: Ambient in the dark

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Sprague-Dawley Crl:CD(R) (SD) IGS BR strain rats, obtained from Charles River (UK) Limited, Margate, Kent.
- Age at study initiation: time-mated female
- Weight at study initiation: 216-299 g (day 5 of gestation)
- Housing: housed individually in solid-floor polypropylene cages with stainless steel lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK).
- Diet (e.g. ad libitum): free access
- Water (e.g. ad libitum): free access (polycarbonate bottles)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3ºC
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): 12hrs dark /12hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
distilled water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
the test item was prepared as a solution in Distilled Water. Thet formulations were homogeneous and stable for at least thirteen days at approximately 4°C in the dark. Bulk formulations were therefore prepared on two occasions and each bulk formulation subdivided into daily aliquots sufficient for nine days of dosing. Formulations were made ahead of the first day of use to allow results of analyses to become available before use. Results from these analyses showed that formulations were 83-102% of nominal concentration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
* ANALTYCAL PROCEDURE:
- HPLC : Agilent Technologies 1200, incorporating autosampler and workstation
- Column : luna phenyl 3μm (50 x 2.0 mm id)
- Column temperature : 40 ºC
- Mobile phase : acetonitrile:0.1% phosphoric acid in water 50:50 v/v
- Flow-rate : 1.0 ml/min
- UV detector wavelength : 210 nm
- Injection volume : 10 μl
- Retention time : ~ 0.5 mins
Details on mating procedure:
- The female rats were delivered as time-mated in two batches. Each batch containing two sets of animals mated over two consecutive days and contained females at Day 0 or Day 1 of gestation. The day that positive evidence of mating was observed at the animal supplier was designated Day 0 of gestation.
Duration of treatment / exposure:
14 days
Frequency of treatment:
The test item was administered daily, from Day 5 to 19 of gestation, by gavage.
Duration of test:
20 days
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw/day .
Basis:
actual ingested
Remarks:
Doses / Concentrations:
250 mg/kg bw/day
Basis:
actual ingested
concentration 50 mg/ml
Remarks:
Doses / Concentrations:
500 mg/kg bw
Basis:
actual ingested
concentration 100 mg/ml
Remarks:
Doses / Concentrations:
1000 mg/kg bw
Basis:
nominal in water
concentration 200 mg/ml
No. of animals per sex per dose:
24 females per dose
Control animals:
yes
Details on study design:
- Dose selection rationale: Dosages were selected, based on available toxicological data including a preliminary oral (gavage) pre-natal development toxicity
study in the rat conducted at these laboratories (Harlan Laboratories Ltd. Project No.:41104828). In this preliminary study, a dosage of 1000 mg/kg bw/day was well tolerated with no adverse effect on maternal body weight or food consumption being apparent.
- Rationale for animal assignment (if not random): The animals were randomly allocated to treatment groups using a randomisation procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system.

Examinations

Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily during the gestation period. Additionally, during the dosing period observations were recorded immediately before and soon after dosing and one hour post dosing. An additional observation was also performed five hours after dosing during the normal working week. All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 3 and on Days 5 (prior to treatment), 6, 7, 8, 11, 14 and 17 of gestation. Body weights were also recorded for surviving animals at terminal kill (Day 20).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption was recorded for each surviving individual animal for the periods, Days 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20.


POST-MORTEM EXAMINATIONS: Yes
All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded.
Ovaries and uterine content:
The ovaries and uteri of pregnant females were removed, examined and the following data recorded:
i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Foetal sex
iv) External foetal appearance
v) Foetal weight
vi) Placental weight
vii) Gravid uterus weight

The ovaries and uterine content was examined after termination: Yes
The uteri of any apparently non-pregnant females were immersed in 10% ammonium sulphide to reveal evidence of implantation.

- Implantation types were divided into:
* Early Death: No visible distinction between placental/decidual tissue and embryonic tissue
* Late Death: Separate embryonic/foetal and placental tissue visible
* Dead Foetus: A foetus that had died shortly before necropsy. These were included as late deaths for reporting purposes

All implantations and viable foetuses were numbered according to their intrauterine position as follows:
* Left Horn
L1 L2 L3 L4 L5 L6 L7 L8
V1 V2 V3 V4 V5 V6 V7 V8
* Cervix
* Right Horn
R1 R2 R3 R4 R5 R6 R7 R8
V9 V10 V11 V12 V13 V14 V15 V16
V = viable foetus
Fetal examinations:
The foetuses were killed by subcutaneous injection of sodium pentobarbitone. Foetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate foetuses were identified using an indelible marker and placed in Bouin’s fixative. Foetuses were transferred to 70% industrial methylated spirits (IMS) in distilled water and examined for visceral anomalies under a low power binocular microscope. The remaining foetuses were identified using colour coded wires and placed in 70% IMS in distilled water. The foetuses were eviscerated, processed and the skeletons stained with alizarin red. The foetuses were examined for skeletal development and anomalies. Foetuses that were examined for skeletal development were placed in 100% glycerol.
Statistics:
* Female body weight change, food consumption and gravid uterus weight: Bartlett’s test for homogeneity of variance and one way analysis of variance, followed by Dunnett’s multiple comparison test or, if unequal variances were observed, an alternative multiple comparison test was performed.

* All caesarean necropsy parameters and foetal parameters: Kruskal-Wallis nonparametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.

*Foetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis nonparametric analysis of variance and Mann-Whitney ‘U’ test.

NOTE:
* Percentage pre-implantation loss was calculated as:
((number of corpora lutea - number of implantations)/ number of copora lutea) x 100

* Percentage post-implantation loss was calculated as:
((number of implantations - number of foetuses )/ number of implantations) x 100

Sex ratio was calculated for each litter value on Day 1 and 4 post partum, using the following formula:
% male foetuses (sex ratio) = (Total number of foetuses/Number of male foetuses) x 100
Historical control data:
Not followed

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Effect levels (maternal animals)

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: fetotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

* Mortality

All animals survived to scheduled termination on Day 20 of gestation.

* Clinical Observations

There were no clinical signs observed that were considered to be associated with treatment at 100, 300 or 1000 mg/kg bw/day.

* Body weight

Body weights and body weight gain, including values adjusted for the contribution of the gravid uterus, were unaffected by treatment at 100, 300 and 1000 mg/kg bw/day.

* Food Consumption

Food consumption was unaffected by treatment at 100, 300 and 1000 mg/kg bw/day.

* Post Mortem Studies

No macroscopic abnormalities were detected for adult animals at Day 20 of gestation.

*Litter Responses

- Litter Data and Litter Placental and Foetal Weights

There was no effect of treatment on in-utero offspring survival, as assessed by the mean numbers of early or late resorptions, live litter size and pre and post-implantation losses at 100, 300 and 1000 mg/kg bw/day.

At 1000 mg/kg bw/day, one female showed total litter resorption but, in the absence of any increased post-implantation loss for remaining litters at this dosage, this finding was considered to be incidental and unrelated to treatment.

There was no effect of treatment on mean foetal or litter weight at 100, 300 or 1000 mg/kg bw/day.

* Foetal Examination

Neither the type, incidence or distribution of findings observed externally at necropsy examination and subsequently during detailed visceral and skeletal assessment indicated any effect of treatment on foetal development.

Applicant's summary and conclusion

Conclusions:
The oral administration of XP-7866 to pregnant rats did not result in any treatment related effects for the parental females or their developing offspring at dose levels up to 1000 mg/kg bw/day and therefore the No Observed Effect Level (NOEL) was considered to be 1000 mg/kg bw/day.