Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147, (24 November 2000)
Principles of method if other than guideline:
The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between Days 5 and 19 of gestation, inclusive, at dose levels 250, 500, or 1000 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (arachis oil) to serve as a control.
Clinical signs, body weight change, food and water consumptions were monitored during the study.
All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(2-ethylhexyl) tetrabromophthalate
EC Number:
247-426-5
EC Name:
Bis(2-ethylhexyl) tetrabromophthalate
Cas Number:
26040-51-7
Molecular formula:
C24H34Br4O4
IUPAC Name:
1,2-bis(2-ethylhexyl) 3,4,5,6-tetrabromobenzene-1,2-dicarboxylate
Test material form:
liquid
Details on test material:
Batch 12128E71
Specific details on test material used for the study:
Identification : Bis(2-ethylhexyl) tetrabromophthalate (CAS No. 26040-51-7)
Physical State/Appearance : Liquid, slight viscous, colorless
Purity : 90.6%
Storage Conditions : Room temperature in the dark; used/formulated in light

No correction for purity was made.

Test animals

Species:
rat
Strain:
other: Sprague-Dawley Crl:CD® (SD) IGS BR
Details on test animals or test system and environmental conditions:
Animal Information
A total of ninety-six time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in two batches, two days apart. The first delivery consisted of two batches of thirty-two animals each at Day 1 and Day 0 of gestation, respectively, whilst the second delivery consisted of one batch of thirty-two animals at Day 1 of Gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation (sperm in the vaginal smear). At the start of treatment (Day 5 of gestation), the females weighed 213 to 305g.

Animal Care and Husbandry
The animals were housed individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly mean temperatures and humidity are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3ºC and 50 ± 20% respectively; there were no deviations from these targets.

The animals were randomly allocated to treatment groups using a randomization 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 routinely used in these laboratories.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the purpose of the study, the test item was prepared at the appropriate concentrations as a solution in Arachis Oil. On each day of formulation preparation, for each concentration the required amount of Test Item was weighed out and added to a volume of vehicle. Additional vehicle was added to the resulting formulation to make it up to the required volume and shaken/mixed to give a homogeneous bulk formulation. These bulk formulations were subsequently divided into the required daily aliquots and stored at 4°C in the dark until the day of use.

The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK Analytical Services. The formulations were shown to be stable for at least sixteen days when stored at 4°C in the dark. Formulations for this study were made and used within the known stability period.

Representative samples of dosing formulations were analyzed for concentration of Bis(2-ethylhexyl) tetrabromophthalate (CAS No. 26040-51-7) at Envigo Research Limited Analytical Laboratory, Shardlow. The results indicate that the prepared formulations were within 90-96% of nominal concentration confirming the accuracy of the preparation procedure.
Details on mating procedure:
Time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were used.
Duration of treatment / exposure:
The test item was administered once daily, from Day 5 to Day 19 of gestation, by gavage. Control animals were treated in an identical manner with the vehicle alone.
Frequency of treatment:
Once daily
Duration of test:
All animals were killed on Day 20 of gestation.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
250 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
24 female animals per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The study was performed between 16 December 2015 and 15 September 2016. The in-life phase of the study was conducted between 20 December 2015 (first day of treatment) and 06 January 2016 (final day of necropsy).

Examinations

Maternal examinations:
Clinical Observations
Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral changes 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. All observations were recorded.

Body Weight
Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 5, 6, 7, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at terminal kill (Day 20).

Food Consumption
Food consumption was recorded for each individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

Water Consumption
Water intake was observed daily by visual inspection of the water bottles for any overt changes.
Ovaries and uterine content:
Necropsy
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. 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) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight
vii) Gravid uterus weight
The uteri of any apparently non-pregnant females were immersed in 0.5% ammonium polysulphide solution 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/fetal and placental tissue visible
Dead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths for reporting purposes
All implantations and viable fetuses were numbered according to their intrauterine position as follows (as an example):

Left Horn Cervix Right Horn

L1 L2 L3 L4 L5 L6 L7 L8 R1 R2 R3 R4 R5 R6 R7 R8
V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13 V14 V15 V16

V = viable fetus

The fetuses were killed by subcutaneous injection of sodium pentobarbitone. Fetuses from each litter were divided into two groups and examined for either skeletal alterations (skeletal examinations) or soft tissue alterations (visceral examinations).
Fetal examinations:
Visceral Examinations
At necropsy, alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. These fetuses were subsequently transferred to distilled water prior to examination for visceral anomalies under a low power binocular microscope and following examination transferred to 10% Buffered Formalin for storage.
Visceral examinations commenced with an external assessment of general appearance, including the limbs, digits, genitals, anus, tail and umbilicus. Once completed the head, and subsequently the tongue was removed, for examination of the tongue, palate and surrounding tissue including the teeth, genioglossus and nasopharynx. Serial sections were made of the head and lower jaw to enable a detail examination of brain morphology and the lower jaw was also sectioned to allow examination of the incisors, multicuspid teeth and genioglossus muscle.
The skin was opened up to allow further visceral examination and the internal sex of the fetuses was confirmed. The position of the umbilical artery and the liver, stomach spleen, pancreas and intestines assessed. These tissues were then removed to enable examination of the under lying abdominal tissues including the kidneys which were transversely cut to enable assessment of the cortex, medulla and papilla.
Following these examinations the thoracic tissues were examined, commencing first with the diaphragm lungs, azygo us vein and thymus. The lungs and thymus were subsequently removed to allow assessment of the heart and cervicothoracic blood vessels. Examination of the heart included the external size and shape of the ventricles and atria, the entry and exit of the blood vessels around the heart and assessment of the foramen ovale, atrio-ventricular valves, semi-lunar valves, ventricular septum and general proportions of ventricular walls and cavities.

Skeletal Examinations
At necropsy, fetuses not allocated to visceral examinations were identified using cardboard tags marked with chinagraph pencil and placed in 70% IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and alterations and storage.
For assessment, fetuses were placed in a petri dish containing glycerol and examined using a microscope. Fetuses were examined whole but for evaluation and reporting purposes the skeleton was divided into the following regions: skull, vertebral column, ribs, sternebrae, pectoral girdle, pelvic girdle, fore limbs and hind limbs. A peer review (approximately 10% of the total number of litters examined) was performed as part of the overall skeletal examination procedures for the study.
Statistics:
The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:
Female body weight change, food consumption and gravid uterus weight: Shapiro Wilk normality test and 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, on alternative multiple comparison test.
All caesarean necropsy parameters and fetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.
Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis non-parametric analysis of variance and Mann-Whitney ‘U’ test.
Probability values (p) are presented as follows:
p<0.05 *
p≥0.05 (not significant)
Indices:
Pre and Post Implantation Loss
Percentage pre-implantation loss was calculated as:
[(number of corpora lutea - number of implantations) : (number of corpora lutea)] x 100

Percentage post-implantation loss was calculated as:
[(number of implantations - number of live fetuses) : (number of implantations)] x 100

Sex Ratio
Sex ratio was calculated as:
% male fetuses (sex ratio) = [(Total number of fetuses : Number of male fetuses)] x 100
Historical control data:
Historical control data are available for:
Normal Range Data for Pre-Natal Study Gestation Body Weights in the Sprague-Dawley Crl:CD® (SD) IGS BR Rat
Normal Range Data for Pre-Natal Study Gestation Food Consumption in the Sprague-Dawley Crl:CD® (SD) IGS BR Rat
Normal Range Data for Pre-Natal Litter Data in the Sprague-Dawley Crl:CD® (SD) IGS BR Rat
Normal Range Data for Pre-Natal Study External Fetal Observations in the Sprague-Dawley Crl:CD® (SD) IGS BR Rat
Normal Ranges for Pre-Natal Study Visceral Fetal Findings in the Sprague-Dawley Crl:CD® (SD) IGS BR Rat
Normal Ranges for Pre-Natal Study Skeletal Fetal Findings in the Sprague-Dawley Crl:CD® (SD) IGS BR Rat

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical observations for any of the animals throughout the study.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Throughout the dosing period, there was no effect of treatment with the test item at any dose level on body weight development. Overall body weight gains were comparable across all dose groups including controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Throughout the dosing period, there was no effect of treatment with the test item at any dose level on food consumption.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles did not reveal any obvious intergroup differences.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination of adult females on Day 20 of gestation did not indicate any effect of treatment with the test item at 250, 500 or 1000 mg/kg bw/day.
One female from the control group showed a uterus filled with colorless fluid. The female was not pregnant and, in the absence of any treatment with the test item, this observation was deemed to be incidental.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
The oral administration of Bis(2-ethylhexyl) tetrabromophthalate (CAS No. 26040-51-7) to pregnant rats by oral gavage during gestation at dose levels of 250, 500 or 1000 mg/kg bw/day was well tolerated. There was no effect of treatment with the test item on body weight development or associated dietary intake and the ‘No Observed Effect Level’ (NOEL) for maternal toxicity was considered to be 1000 mg/kg bw/day within the confines of this type of study.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
With the exception of one female each from the control, 250 and 1000 mg/kg bw/day dose groups, all the remaining females on the study were found to be pregnant at necropsy. There were no incidences of total litter loss by resorption and the following assessment is based on the 23, 23, 24 and 23 females with live fetuses at Day 20 of gestation at 0 (control), 250, 500 and 1000 mg/kg bw/day, respectively.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No treatment-related effects were detected on litter data as assessed by the mean number of implantations, in utero offspring survival (as assessed by the mean number of early or late resorptions), live litter size, post-implantation loss or sex ratio at 250, 500 or 1000 mg/kg bw/day.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
With the exception of one female each from the control, 250 and 1000 mg/kg bw/day dose groups, all the remaining females on the study were found to be pregnant at necropsy. There were no incidences of total litter loss by resorption and the following assessment is based on the 23, 23, 24 and 23 females with live fetuses at Day 20 of gestation at 0 (control), 250, 500 and 1000 mg/kg bw/day, respectively.
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
With the exception of one female each from the control, 250 and 1000 mg/kg bw/day dose groups, all the remaining females on the study were found to be pregnant at necropsy.
Other effects:
no effects observed

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Remarks on result:
other: The ‘No Observed Effect Level’ (NOEL) for maternal toxicity was 1000 mg/kg bw/day (highest dose tested).

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
No treatment-related effects were detected in fetal growth and development.
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):
For all dose groups, there were no treatment-related trends in the proportion of fetuses (or litters) with evidence of external or visceral abnormalities.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Skeletal examination of fetuses/litters revealed some statistically significant variations of ossification in relation to controls. At 1000 mg/kg bw/day, these included a slight increase in unossified area(s) in frontal (controls and high dose: 0.0 and 5.0*, respectively) or occipital (supra-occipital) (controls and high dose: 0.7 and 4.7*, respectively) regions. Although there was no dose-relationship, group mean values for these alterations were above the historical control data ranges and, as such their relation to treatment cannot be ignored. Minor variations of this type, however, are frequently seen in control populations of this strain of rat, are normally temporary alterations with postnatal skeletal remodeling and, are not mechanistically linked to any malformation (Carney and Kimel, 2007). In the absence of any treatment-related effects on any associated parameters, these observations were considered not to be adverse in nature.

At 250 or 500 mg/kgbw/day, the incidence of incomplete ossification of sacral (neural) arch was statistically significantly lower than controls (controls, low and intermediate dose: 17.6, 5.5* and 7.1*, respectively) but without a dose-relationship. Group mean values for these test item-treated dose groups were within the historical control data ranges and in the absence of a similar effect for the high dose group (1000 mg/kg bw/day), this finding was considered likely to be incidental.
Visceral malformations:
no effects observed
Description (incidence and severity):
For all dose groups, there were no treatment-related trends in the proportion of fetuses (or litters) with evidence of external or visceral abnormalities.
Other effects:
no effects observed

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: The ‘No Observed Adverse Effect Level’ (NOAEL) for developmental toxicity, was 1000 mg/kg bw/day (highest dose tested).

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

The oral administration of Bis(2-ethylhexyl) tetrabromophthalate (CAS No. 26040-51-7) to pregnant rats by oral gavage during gestation at dose levels of 250, 500 or 1000 mg/kg bw/day was well tolerated. There was no effect of treatment with the test item on body weight development or associated dietary intake and the ‘No Observed Effect Level’ (NOEL) for maternal toxicity was 1000 mg/kg bw/day (highest dose tested).

No adverse changes were detected in the offspring parameters measured. The observed statistically significant variations of skeletal ossification in the high dose fetuses are considered to be not adverse and the ‘No Observed Adverse Effect Level’ (NOAEL) for developmental toxicity, was 1000 mg/kg bw/day (highest dose tested).

Applicant's summary and conclusion

Executive summary:

Methods

The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between Days 5 and 19 of gestation, inclusive, at dose levels 250, 500, or 1000 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Arachis oil) to serve as a control.

Clinical signs, body weight change, food and water consumptions were monitored during the study.

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

Results

Mortality

There were no unscheduled deaths during the study.

Clinical Observations

No clinical signs were detected for any of the animals throughout the study.

Body Weight

There was no effect of treatment with the test item at any dose level on body weight development.

Food Consumption Dietary intake across all groups of females receiving the test item remained similar to controls throughout the treatment period.

Water Consumption

Daily visual inspection of water bottles did not reveal any obvious intergroup differences.

Post Mortem Studies

There were no macroscopic findings for any of the females receiving the test item at dose levels of 250, 500 or 1000 mg/kg bw/day.

Litter Data and Litter Placental and Fetal Weights

No treatment-related effects were detected in the uterine parameters examined, in fetal viability or in fetal growth and development.

Fetal Examination

No adverse effects were detected on external fetal development or in the type and incidence of skeletal or visceral findings. Skeletal examination of fetuses/litters revealed some statistically significant variations of ossification in relation to controls. At 1000 mg/kg bw/day, these included a slight increase in unossified area(s) in frontal (controls and high dose: 0.0 and 5.0*, respectively) or occipital (supra-occipital) (controls and high dose: 0.7 and 4.7*, respectively) regions. Although there was no dose-relationship, group mean values for these alterations were above the historical control data ranges and, as such their relation to treatment cannot be ignored. Minor variations of this type, however, are frequently seen in control populations of this strain of rat, are normally temporary alterations with postnatal skeletal remodeling and, are not mechanistically linked to any malformation (Carney and Kimel, 2007). In the absence of any treatment-related effects on any associated parameters, these observations were considered not to be adverse in nature.

Conclusion

The oral administration of Bis(2-ethylhexyl) tetrabromophthalate (CAS No. 26040-51-7) to pregnant rats by oral gavage during gestation at dose levels of 250, 500 or 1000 mg/kg bw/day was well tolerated. There was no effect of treatment with the test item on body weight development or associated dietary intake and the ‘No Observed Effect Level’ (NOEL) for maternal toxicity was 1000 mg/kg bw/day (highest dose tested).

No adverse changes were detected in the offspring parameters measured. The observed statistically significant variations of skeletal ossification in the high dose fetuses are considered to be not adverse and the ‘No Observed Adverse Effect Level’ (NOAEL) for developmental toxicity, was 1000 mg/kg bw/day (highest dose tested).