Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 May 2013 - 24 Jul 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Modern study carried out under GLP and in accordance with the appropriate guidelines.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
A small number of minor deviations from the protocol are reported; however none of the deviations were considered to have affected the outcome or integrity of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: solid
Details on test material:
- Name of test material (as cited in study report): BEPD
- Substance type:
- Physical state: White solid
- Analytical purity: 99.63%
- Lot/batch No.: B184/1339
- Expiration date of the lot/batch: 01 Dec 2013
- Storage condition of test material: Ambient, Dark

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Age at study initiation: 9 weeks
- Weight at study initiation: 197-273 g
- Fasting period before study: None
- Housing: 1 per cage. Suspended polycarbonate cages with stainless steel grid tops and solid bottoms, containing an integral food hopper and sterilised white wood shavings as bedding material
- Diet (e.g. ad libitum): SDS Rat and Mouse (modified) No. 3 Diet SQC Expanded was provided ad libitum
- Water (e.g. ad libitum): ad libitum from the public supply
- Acclimation period: 3-5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 44-55%
- Air changes (per hr): A minimum of 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

IN-LIFE DATES: From: 27 May 2013 To: 12 Jun 2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: hydroxypropyl methylcellulose (aqueous)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was weighed into a pre-labelled container. The appropriate amount of vehicle was then weighed into the pre-labelled container containing the test item to make up the required volume. Formulations were then magnetically stirred until a homogenous formulation was visually confirmed. The dosing formulations were prepared weekly, stored at 2-8°C and dispensed daily. Formulations was removed from the refrigerator and stirred for at least 30 minutes before dosing and continuously during dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): None given
- Concentration in vehicle: 1.5, 15, 100 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg
- Lot/batch no. (if required): XD05012N12 / DT357129
- Purity: 1.0% (w/v) Methocel E4M in Milli-Q Water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis from all groups at Week 1 and Week 2. They were tested for concentration and homogeneity. Analyses were performed by Gas Chromatography with Flame ionisation Detection using a validated analytical procedure
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy: day of detection of mating referred to as Day 0 of pregnancy
Duration of treatment / exposure:
14 days: from Days 6-19 of gestation
Frequency of treatment:
once daily
Duration of test:
15 days: Scheduled Euthanasia Day 20 of gestation
Doses / concentrations
Remarks:
Doses / Concentrations:
15, 150, 1000 mg/kg bw/d
Basis:
actual ingested
gavage
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were based on the results of a previous toxicity study (Huntingdon Life Sciences Study No. NTO 17/951955). Justification for dose level selection was provided by the Sponsor from evaluation of previous studies in which animals received 15, 150 or 1000 BEPD/kg/day for up to 28 days. In that study, a NOAEL was considered to be 1000 mg/kg/day with a NOEL considered to be 15 mg/kg/day based on liver and kidney findings noted in males.
- Rationale for animal assignment: random

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were checked early morning and as late as possible each day for viability
- Cage side observations: Mortality/Moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily, from the start of dosing

BODY WEIGHT: Yes
- Time schedule for examinations: Pretrial – Once (Day 4 of gestation), Dosing Period – Daily Day 6 – 20 of gestation

FOOD CONSUMPTION: Yes
- Food consumption was quantitatively measured daily from Day 4 of gestation (first measured quantity given on Day 3 of gestation).

WATER CONSUMPTION: Yes
- Time schedule for examinations: Regular basis throughout the study
- Water consumption was monitored by visual inspection of the water bottles.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: 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
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Placentae (size, color or shape)
- Number of live and dead foetuses
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
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. Unless otherwise stated, 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 appear 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 is significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remain heterogeneous, then a Kruskal-Wallis nonparametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test).
Foetal weight data was subjected to the Kruskal-Wallis non-parametric analysis.
For ANOVA summary tables, the results of analyses are reported indicating the level of significance (P<0.05, p<0.01 and p<0.001) for each pairwise comparison. Actual p-values will not be reported in the summary tables for these analyses.
Indices:
Not relevant
Historical control data:
Not required for assessment of the data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: at 1000 mg/kg bw/d

Details on maternal toxic effects:
Dosing of BEPD at 1000 mg/kg/day was associated transient clinical signs of: subdued behaviour, reduced activity, staggering locomotion or animals dragging limbs, slow, irregular or crackling/wheezing respiration and excess salivation; and there were isolated incidences of piloerection, body being held low and partially closed eyes.
At 1000 mg/kg/day, slight group mean body weights loss was noted on Days 7 and 8 of gestation, when compared to Day 6, with values being similar to pretrial and Controls from Day 9 of gestation onwards.
At 1000 mg/kg bw/d, group mean food consumption decreases of ~25% were noted on Days 7 and 8 of gestation when compared with pretrial, with individual decreases of up to ~55% being noted in some individuals. In general, recovery was first noted on Day 9 of gestation, with values being comparable with Controls from Day 12 of gestation onwards.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
Not relevant: no effects were observed

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

 

0

15

150

1000

Weight change (Day 6-7)

+4 g

+3 g

+2 g

-3 g

Weight change (Day 6-8)

+10 g

+8 g

+7 g

-1 g

Weight change (Day 6-9)

+16 g

+14 g

+13 g

+6 g

Weight change (Day 6-20)

+118 g

+117 g

+124 g

+109 g

Mated (#)

24

24

24

24

Pregnant (#)

24

24

24

23

Corpora lutea (#)

14.1

14.3

14.1

14.3

Implants (#)

13.8

13.8

13.8

13.9

Dead implants (#)

0.7

1.3

0.5

0.8

Foetal deaths (#)

-

-

-

0.04

Foetal weight (g)

3.749

3.75

3.85

3.73

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, it is concluded that the maternal NOAEL was 150 mg/kg bw/d. In the absence of any effects of treatment at the limit dose, a developmental NOAEL of 1000 mg/kg bw/d is determined.
Executive summary:
A prenatal development toxicity study was conducted to determine the effects of BEPD when the material was administered during the period of organogenesis to pregnant rats. Time-mated female Sprague-Dawley rats were randomised into 3 test groups, receiving 15, 150, 1000 mg/kg bw/d of BEPD and 1 control group receiving only the vehicle, 1.0% (w/v) aqueous Methocel. All animals were dosed via oral gavage over Days 6-19, inclusive of gestation, where the day of detection of mating was designated Day 0.

Animals were regularly monitored for clinical signs of toxicity, body weight and food consumption performance and were killed on Day 20 of gestation for examination of pregnancies and foetal development. Dosing of BEPD at 1000 mg/kg bw/d was associated with transient clinical signs which included subdued behaviour, reduced activity, staggering locomotion, animals dragging limbs, altered respiration and excess salivation. Additionally, on Days 7 and 8 of gestation body weight losses and food consumption decreases were noted when compared with pre-trial, with recovery in these parameters being noted from Day 9 of gestation.

Dosing at 15 and 150 mg/kg bw/d was generally well tolerated, with only a few isolated incidents of transient clinical signs being noted and no body weight or food consumption effects. There were also no treatment related gross necropsy findings at any dose level. Pregnancy performance and foetal weights were similar between treated groups and controls, and the type and distribution of foetal major abnormalities, minor visceral and skeletal abnormalities and skeletal ossification parameters did not indicate and association with treatment with BEPD.

Based on the results of this study, it was concluded that the maternal NOAEL was 150 mg/kg bw/d and the developmental NOAEL was 1000 mg/kg bw/d.