Registration Dossier

Administrative data

Description of key information

In a subacute repeated dose toxicity study according to OECD 407, three groups of ten male and ten female CD rats received FR-45B via the diet at concentrations of 200, 2 000 or 20 000 ppm (= ca. 21.97, 223.4 or 2331 mg/kg/day) for four weeks. A similarly constituted control group received untreated diet. There were no signs of reaction to treatment with FR-45B (bis(2-ethylhexyl) tetrabromophthalate). Slightly low overall bodyweight gain was recorded for females receiving the highest dietary concentration of FR-45B. Males treated with FR-45B were unaffected. Marginally low alanine amino-transferase activities were seen in females receiving the highest dietary concentration of FR-45B, and marginally low phosphorus concentrations were seen in all females and males receiving the highest dietary concentration of FR-45B. Urinary composition was unaffected by treatment with FR-45B. There were no organ weight changes in rats receiving FR-45B.There were no treatment-related histopathological changes in animals that received FR-45B. The NOAEL is 2000 ppm  = ca. 223.4 mg/kg bw (male + female  rats).

In a subchronic repeated dose toxicity study according to OECD 408, oral administration of bis(2-ethylhexyl) tetrabromophthalate (CAS No. 26040-51-7) to male and female Wistar Han™:RccHan™:WIST strain rats for ninety consecutive days at dose levels of 100, 300 and 1000 mg/kg bw/day resulted in no treatment related changes.  The No Observed Effect Level (NOEL) is considered to be 1000 mg/kg bw/day, the highest dose tested.

The effects observed in the 28 day feeding study were slight (marginally low alanine amino-transferase activities in the highest dietary concentration of FR-45B, and marginally low phosphorus concentrations were seen in all females and males receiving the highest dietary concentration) and are not confirmed in the 90 day gavage study.

Therefore the NOAEL of 1000 mg/kg bw/day determined in the 90 day study is used for the derivation of the DNELs because the 90 day study exposed animals longer, used more animals per group, investigated more parameter and is considered more relevant for human risk assessment.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 07 July 2017 Experimental Completion Date: 22 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
Deviations from the study plan were considered to have had no impact on the study
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is a preferred species of choice as historically it is used for safety evaluation studies and specified by appropriate regulatory authorities.
Sex:
male/female
Details on test animals or test system and environmental conditions:
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Oxon, UK. These animals were originally intended for use on another study which was cancelled, therefore this study was moved forward to utilise these animals to avoid animal wastage. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for nine days during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study. At the start of treatment the males weighed 193 to 239g, the females weighed 153 to 172g, and were approximately six to eight weeks old.

Animal Care and Husbandry
The animals were housed in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. During the acclimatization period a pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) was used (see Deviation from Study Plan). A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) was used throughout the treatment period. Certificates of analysis of the batches of diet used during the treatment period are given in Annex 5. 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 were 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 temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.
The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Route of administration:
oral: gavage
Details on route of administration:
The test item was administered daily, for ninety consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe.
Vehicle:
arachis oil
Details on oral exposure:
The dose levels were chosen in collaboration with the Sponsor and were based on the results of previous toxicity work, including a Fourteen Day Repeated Dose Oral (Gavage) Range-Finding Study in the Rat (Envigo study number NB70MD). The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.
The test item was administered daily, for ninety consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each test item formulation were taken on six occasions and analyzed for concentration of Bis(2-ethylhexyl) tetrabromophthalate (CAS No. 26040-51-7) at Envigo Research Limited, Shardlow, UK, Analytical Services.
Formulations for use on the first week of the study were analysed in advance of use and the measured concentration for the low dosage was found to be slightly lower than expected. This low dose formulation was discarded and a further low dose formulation was prepared. This second formulation was again found to be lower than expected but as there was insufficient time to produce and analyse another low dose formulation, this formulation was used for three days of the study (Days 1-3 for males and Days 1-2 for females). A third low dose formulation was prepared and found to be within specification and therefore replaced the previous lower formulation. Low dose formulations prepared for use on the study were also noted to be low on a further two occasions on the study but these were replaced prior to use. The results for the analysed formulations used to dose the low dosage animals were 86-103% of nominal concentration. The results for the analysed formulations used to dose intermediate and high dosage animals were 90-96% and 96-101% of nominal concentration respectively. Whilst the slightly lower measured concentration at the low dose level was not ideal, as the No Observed Effect Level (NOEL) for this study was the high dosage (1000 mg/kg bw/day), the observed discrepancies at the low dosage had no impact on the study.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control group
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low dose group
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Intermediate dose group
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose group
No. of animals per sex per dose:
10 males and 10 females at 0 mg/kg bw/day
10 males and 10 females at 100 mg/kg bw/day
10 males and 10 females at 300 mg/kg bw/day
10 males and 10 females at 1000 mg/kg bw/day
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health or behavioral change immediately before dosing, up to thirty minutes post dosing and one hour after dosing. All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and at weekly intervals thereafter. Body weights were also recorded at terminal kill.

Food Consumption
Food consumption was recorded for each cage group at weekly intervals throughout the study. Food conversion efficiency was calculated retrospectively.

Water Consumption
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

Specialist Evaluations
Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. During Week 12 functional performances tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.

Behavioral Assessment
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait Hyper/Hypothermia
Tremors Skin color
Twitches Respiration
Convulsions Palpebral closure
Bizarre/Abnormal/Stereotypic behavior Urination
Salivation Defecation
Pilo-erection Transfer arousal
Exophthalmia Tail elevation
Lachrymation
This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.

Functional Performance Tests
Motor Activity. Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail 1979).

Forelimb/Hindlimb Grip Strength. An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.
The following parameters were observed:
Grasp response Touch escape
Vocalization Pupil reflex
Toe pinch Blink reflex
Tail pinch Startle reflex
Finger approach

Ophthalmoscopic Examination
The eyes of all control and treated animals were examined pre-treatment and all control and high dose animals were examined before termination of treatment (during Week 12). Examinations included observation of the anterior structures of the eye and following pupil dilation with 0.5% Tropicamide solution (Mydriacyl® 0.5%, Alcon Laboratories (UK) Ltd., Pentagon Park, Boundary Way, Hemel Hampstead, Hertfordshire), detailed examination of the internal structure of the eye using an ophthalmoscope was performed.

In-Life Sampling and Analysis
Hematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 90). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91. Animals were not fasted prior to sampling.

Hematology
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices - mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea Inorganic phosphorus (P)
Glucose Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.) Alanine aminotransferase (ALAT)
Albumin Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation) Creatinine (Creat)
Sodium (Na+) Total cholesterol (Chol)
Potassium (K+) Total bilirubin (Bili)
Chloride (Cl-) Bile acids
Calcium (Ca++)
Sacrifice and pathology:
Necropsy
On completion of the dosing period all animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals Ovaries
Brain Spleen
Epididymides Testes
Heart Thymus
Kidneys Uterus (with cervix and oviducts)
Liver

Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:
Adrenals Ovaries
Aorta (thoracic) Pancreas
Bone & bone marrow (femur including stifle joint)• Pituitary
Bone & bone marrow (sternum) Prostate
Brain (including cerebrum, cerebellum and pons) Rectum
Cecum Salivary glands (submaxillary)
Colon Sciatic nerve
Duodenum Seminal vesicles
Epididymides ♦ Skin
Esophagus Spinal cord (cervical, mid-thoracic
Eyes* and lumbar)
Gross lesions Spleen
Heart Stomach
Ileum (including Peyer’s patches) Testes ♦
Jejunum Thymus
Kidneys Thyroid/Parathyroid
Liver Tongue•
Lungs (with bronchi) # Trachea
Lymph nodes (mandibular and mesenteric) Urinary bladder
Mammary glands Uterus (with cervix and oviducts)
Muscle (skeletal)• Vagina
All tissues were dispatched to the Test Site (Propath UK Ltd., Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU, UK) for processing. All tissues from control and 1000 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed.

Pathology
Microscopic examination was conducted by the Study Pathologist (W Henderson). A peer review of the findings observed was conducted by V Mowat at Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS.

* Eyes fixed in Davidson’s fluid
♦ Preserved in Modified Davidson’s fluid
# Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative
• Retained only and not processed
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.
Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs observed that indicated any systemic effect of treatment at dosages of 100, 300 and 1000 mg/kg bw/day.
Incidental results included one control male which was observed to have a scab between Days 77 and 80 which was later followed by generalized fur loss from Day 81 to termination, and one female dosed at 100 mg/kg bw/day which had a mass approximately 1mm x 1mm under the right eye from Day 88 to termination. One male dosed with 300 mg/kg bw/day had noisy respiration on five occasions (Days 51, 56, 63, 77 and 78), which is likely to be associated with difficulty during the dosing procedure for this particular animal.
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):
There was no effect of treatment on bodyweight or body weight gains for either sex at 100, 300 or 1000 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect of treatment on food consumption for either sex at 100, 300 or 1000 mg/kg bw/day.
Food efficiency:
no effects observed
Description (incidence and severity):
There was no effect of treatment on food conversion efficiency for either sex at 100, 300 or 1000 mg/kg bw/day.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There was no effect of treatment on water consumption for either sex at 100, 300 or 1000 mg/kg bw/day; daily visual inspection of water bottles revealed no overt intergroup differences.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmoscopic examination of animals of both sexes from the control and 1000 mg/kg bw/day dose groups during Week 12 of the treatment period did not indicate any treatment-related differences.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Assessment of hematology parameters did not indicate any obvious effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day.
Platelet values for males dosed with 300 and 1000 mg/kg bw/day were statistically significantly higher than controls and mean corpuscular volume values for all treated females were statistically significantly higher than controls. For male platelet values, 2/10 control values were lower than the historical control range, compared to 0/10 and 1/10 for males treated with 300 and 1000 mg/kg bw/day respectively. For female mean corpuscular volume values, 7/10 control values were lower than the historical control range, compared to 2/10, 5/10 and 1/10 for females treated with 100, 300 and 1000 mg/kg bw/day respectively. The majority of the values for treated animals were within normal ranges, and there was no true dosage relationship for mean female mean corpuscular volume values. Furthermore, no dose response is seen and the observations are reported only in one gender and in the absence of any supporting histopathological changes lead to the conclusion that these findings are considered to be incidental and unrelated to treatment.
A statistically significant increase in circulating neutrophils was identified in either sex treated at 1000 mg/kg bw/day, in comparison with controls, although no dosage relationship was present. For animals dosed at 1000 mg/kg bw/day all individual values exceeded the historical control ranges, but so did 8/10 and 9/10 values for control males and females respectively. Therefore the finding can be considered most likely to be incidental and unrelated to treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Assessment of blood chemistry parameters did not indicate any obvious effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day.
Mean calcium values for both sexes dosed with 300 and 1000 mg/kg bw/day were statistically significantly higher than controls; however, all individual values for both sexes treated with 300 and 1000 mg/kg bw/day were within historical control ranges. Females treated at 1000 mg/kg bw/day also showed a statistically significant increase in creatine level. However, the majority (8/10) of individual values for these treated animals were within the historical control, and no similar effect was apparent for males.
In isolation, and in absence of any supporting histopathological changes, these findings are likely to represent normal biological variation. They are considered to be incidental and of no toxicological significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Behavioral Assessments did not indicate any effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Assessment of organ weights did not indicate any adverse effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day.
At the end of the treatment period, statistically significant decreases in absolute and body weight relative heart weights were identified in all treated males in comparison with controls. However, for absolute values, 6/10 control values exceeded the historical control range, compared to only 2/10, 2/10 and 1/10 values for animals dosed with 100, 300 and 1000 mg/kg bw/day respectively. For body weight relative heart values, 6/10 control values exceeded the historical control range, compared to only 2/10, 3/10 and 1/10 values for animals dosed with 100, 300 and 1000 mg/kg bw/day respectively. Therefore this statistical significance is likely to be due to unusually high control values. Furthermore there was no consistent dosage relationship for either absolute or relative heart weights, and in the absence of any supporting histopathological change, this finding was considered to be incidental and of no toxicological significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The incidence, type and distribution of macroscopic findings observed at terminal necropsy did not indicate any effect of treatment at dosages of 100, 300 or 1000 mg/kg bw/day.
Red coloration of the lungs was observed for 1/10 males and 3/10 females treated with 300 mg/kg bw/day, and 5/10 females treated with 1000 mg/kg bw/day. However, this finding is often observed for animals (including controls) within this laboratory when using the exsanguination procedure employed on this study, and in the absence of any histopathological change is considered to be incidental and unrelated to treatment.
An isolated incidence of increased pelvic space (hydronephrosis) in the right kidney was observed in a control male. Findings of this nature are consistent with normally expected low incidence findings in laboratory maintained rats within this laboratory.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment at levels of 100, 300 and 1000 mg/kg bw/day did not cause any treatment related findings; all findings noted were considered to be incidental and not related to the administration of the test item.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Functional Performance Tests
Functional performance tests did not indicate any obvious effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day.
Motor activity assessment during Week 12 revealed activity in animals treated with 100, 300 and 1000 mg/kg bw/day was comparable to controls.
Forelimb/Hindlimb Grip Strength Tests during Week 12 revealed a statistically significant reduction in hindlimb grip strength in the third trial in all treated females when compared to controls. These differences were evident in only one of three tests, with hindlimb grip strengths for treated females not statistically different from controls in the first and second trials. The observations were not dose dependant (454.3, 331.4, 279.4 and 327.2 g for the for the female control, 100, 300 and 1000 mg/kg bw/day dose groups respectively), and no effect was seen on forelimb grip strength at any dose or trial. There were no statistically significant differences from control apparent for forelimb or hindlimb grip strength of males at any dose tested. Overall, the isolated finding of hindlimb strength reduction in females at one trial was considered to be incidental and unrelated to treatment.
Sensory Reactivity Assessments
Intergroup differences observed in the scores for sensory reactivity did not indicate any effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day.
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
food efficiency
gross pathology
haematology
histopathology: non-neoplastic
mortality
ophthalmological examination
organ weights and organ / body weight ratios
water consumption and compound intake
Key result
Critical effects observed:
no
Conclusions:
Oral administration of Bis(2-ethylhexyl) tetrabromophthalate (CAS No. 26040-51-7) to male and female Wistar Han™:RccHan™:WIST strain rats for ninety consecutive days at dose levels of 100, 300 and 1000 mg/kg bw/day resulted in no treatment related changes. The No Observed Effect Level (NOEL) is considered to be 1000 mg/kg bw/day, the highest dose tested.
Executive summary:

Introduction

The study was designed to investigate the systemic toxicity of Bis(2-ethylhexyl) tetrabromophthalate (CAS No. 26040-51-7) and was conducted according to the following regulatory guideline:

·        The OECD Guidelines for Testing of Chemicals No. 408 "Subchronic Oral Toxicity - Rodent: 90 Day Study” (Adopted 21 September 1998).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for ninety consecutive days, at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopic examination was also performed on all animals prior to the start of treatment and on all control and high dose animals during Week 12 of the study.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed.

Mortality

There were no unscheduled deaths during the study.

Clinical Observations

There were no clinical signs observed that indicated any systemic effect of treatment at dosage of 100, 300 and 1000 mg/kg bw/day.

Behavioral Assessment

Behavioral assessments did not indicate any effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day.

Functional Performance Tests

Functional performance tests did not indicate a treatment related effect for either sex at 100, 300 or 1000 mg/kg bw/day.

Sensory Reactivity Assessments

Sensory reactivity assessments did not indicate any effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day.

Body Weight

There was no effect of treatment on body weight or body weight gains for either sex at 100, 300 or 1000 mg/kg bw/day.

Food Consumption

There was no effect of treatment on food consumption or food conversion efficiency for either sex at 100, 300 or 1000 mg/kg bw/day.

Water Consumption

There was no effect of treatment on water consumption for either sex at 100, 300 or 1000 mg/kg bw/day.

Ophthalmoscopy

Ophthalmoscopic examination of animals receiving 1000 mg/kg bw/day did not indicate any effect of treatment for either sex.

Hematology

Assessment of hematology parameters did not indicate treatment related effects for either sex at 100, 300 or 1000 mg/kg bw/day.

Blood Chemistry

Assessment of blood chemistry parameters did not indicate treatmemt related effects for either sex at 100, 300 or 1000 mg/kg bw/day.

Necropsy

The incidence, type and distribution of macroscopic findings observed at terminal necropsy animals did not indicate any effect of treatment at dosages of 100, 300 or 1000 mg/kg bw/day.

Organ Weights

Assessment of organ weights did not indicate treatment related effects for either sex at 100, 300 or 1000 mg/kg bw/day.

Histopathology

Treatment at levels of 100, 300 and 1000 mg/kg bw/day did not cause any treatment related findings.

Conclusion

Oral administration of Bis(2-ethylhexyl) tetrabromophthalate (CAS No. 26040-51-7) to male and female Wistar Han™:RccHan™:WIST strain rats for ninety consecutive days at dose levels of 100, 300 and 1000 mg/kg bw/day resulted in no treatment related changes. The No Observed Effect Level (NOEL) is considered to be 1000 mg/kg bw/day, the highest dose tested.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Principles of method if other than guideline:
Three groups of ten male and ten female CD rats received FR-45B via the diet at concentrations of 200, 2 000 or 20 000 ppm (= ca. 21.97, 223.4 or 2331 mg/kg/day) for four weeks. A similarly constituted control group received untreated diet. A positive control group of five male and five female CD rats received di-2-ethyl hexyl phthalate (DEHP) via the diet at a concentration of 15 000 ppm (ca. 1507 mg/kg bw) for four weeks.
GLP compliance:
yes
Specific details on test material used for the study:
FR-45B, Code No. 6458-68-1, a clear pale yellow liquid received from Pennwalt Corporation on 20 July 1987.
Purity >95%
Species:
rat
Strain:
other: CD rats
Sex:
male/female
Route of administration:
oral: feed
Vehicle:
other: diet
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0 (control), 200, 2000, or 20000 ppm = ca. 21.97, 223.4 or 2331 mg/kg/day
Basis:
nominal in diet
No. of animals per sex per dose:
Three groups of ten male and ten female CD rats per dose
Control animals:
other: A positive control group of five male and five female CD rats received di-2-ethyl hexyl phthalate (DEHP) via the diet at a concentration of 15 000 ppm for four weeks.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no signs of reaction to treatment in animals receiving FR-456.
Mortality:
no mortality observed
Description (incidence):
Male No. 31, receiving 2 000 ppm FR- 5B, died following routine blood sampling in Week 4. Necropsy and histopathology examination did not indicate an significant contributory findings.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Slightly low overall bodyweight gain was recorded for females receiving the highest dietary concentration of FR-45B. Males treated with FR-45B were unaffected.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The amounts of food consumption by rats receiving FR-45B was essentially similar to the amounts consumed by negative controls.
Food efficiency:
no effects observed
Description (incidence and severity):
Food conversion ratios of rats receiving FR-45B were similar to those of negative controls.
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Marginally low alanine amino-transferase activities were seen in females receiving the highest dietary concentration of FR-45B, and marginally low phosphorus concentrations were seen in all females and males receiving the highest dietary concentration of FR-45B.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinary composition was unaffected by treatment with FR-458.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no organ weight changes in rats receiving FR-45B.
Gross pathological findings:
no effects observed
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related histopathological changes in animals that received FR-45B.
Key result
Dose descriptor:
NOAEL
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: 2000 ppm = ca. 223.4 mg/kg bw (male + female rats)
Critical effects observed:
yes
Lowest effective dose / conc.:
20 000 ppm
System:
other: blood
Organ:
blood
Treatment related:
yes
Dose response relationship:
yes

There were no signs of reaction to treatment with FR-45B (bis(2-ethylhexyl) tetrabromophthalate). Hairloss from the dorsal surface of rats receiving DEHP was recorded from the second week of treatment.

One male receiving FR-45B at 2 000 ppm died at routine blood sampling in Week 4.

Markedly low food consumption and bodyweight gain, and high food conversion ratio, were recorded for rats receiving DEHP.

Slightly low overall bodyweight gain was recorded for females receiving the highest dietary concentration of FR-45B. Males treated with FR-45B were unaffected.

Rats receiving DEHP had higher platelet numbers than controls.

Marginally low alanine amino-transferase activities were seen in females receiving the highest dietary concentration of FR-45B, and marginally low phosphorus concentrations were seen in all females and males receiving the highest dietary concentration of FR-45B.

Changes in rats receiving DEHP were minor and comprised high alkaline phosphatase activities, urea and albumin concentrations

and albumin to globulin ratios in males, and low alanine and aspartate amino-transferase activities in females.

Urinary composition was unaffected by treatment with FR-458.

Organ weight analysis indicated markedly high liver and markedly low testes weights in rats receiving DEHP. There were no organ

weight changes in rats receiving FR-45B.

At necropsy males receiving DEHP were found to have small, flaccid testes.

There were no treatment-related histopathological changes in animals that received FR-45B.

Histopathological changes in animals that received DEHP comprised panacinar hepatocytic granular eosinophilic extensive cytoplasm in males and females, a lack of centriacinar hepatocytic glycogen in males, and a lack of germinal epithelium in the testes.

Conclusions:
Dietary administration of FR-45B to CD rats at 20 000 ppm (ca. 2331mg/kg bw) produced only minor changes and that there was no evidence of toxicity at 200 ppm (ca.21.97 mg/kg bw) or 2000 ppm (ca. 223.4 mg/kg bw).
Executive summary:

Three groups of ten male and ten female CD rats received FR-45B (bis(2-ethylhexyl) tetrabromophthalate)via the diet at concentrations of 200, 2 000 or 20 000 ppm (= ca. 21.97, 223.4 or 2331 mg/kg/day) for four weeks. A similarly constituted control group received untreated diet. A positive control group of five male and five female CD rats received di-2-ethyl hexyl phthalate (DEHP) via the diet at a concentration of 15 000 ppm for four weeks.

Dietary administration of FR-45B to CD rats at 20 000 ppm (ca. 2331mg/kg bw) produced only minor changes and that there was no evidence of toxicity at 200 ppm (ca.21.97 mg/kg bw) or 2000 ppm (ca. 223.4 mg/kg bw). FR-45B did not cause the liver and testes changes seen in rats receiving the positive control (DEHP), particularly in respect of peroxisome proliferation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP guideline study.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The effects observed in the 28 day feeding study were slight (marginally low alanine amino-transferase activities in the highest dietary concentration of FR-45B, and marginally low phosphorus concentrations were seen in all females and males receiving the highest dietary concentration) and are not confirmed in the 90 day gavage study.

Therefore the NOAEL of 2000 ppm  = ca. 223.4 mg/kg bw (male + female  rats) determined in the 28 day study is regarded as obsolete and the NOAEL of 1000 mg/kg bw/day determined in the 90 day study is used for the derivation of the DNELs.

Assessment of potential endocrine activity (toxicity/human health) - summary and conclusion

A comprehensive literature search was conducted to identify data on potential endocrine modulating properties of TBPH. The available data was assigned to the OECD Conceptual Framework for Testing and Assessment of Endocrine Disrupters levels 1 to 5.

TBPH can be described as tetrabrominated DEHP. Whereas DEHP is a known endocrine active substance TBPH falls into disrepute due to its molecular similarity with DEHP. This assessment shows that the bromination in TBPH results in a substantial different behaviour compared to DEHP with respect to metabolism, toxicity, reproduction toxicity and binding affinities to endocrine receptors.

It is postulated that DEHP and its brominated counterpart TBPH are metabolized in a similar manner to the monoester metabolites MEHP and TBMEHP, respectively. This metabolic pathway is proven for DEHP, and MEHP is identified as the responsible metabolite for the toxicity of DEHP. For TBPH this metabolism is also proven, however, only in vitro in the presence of porcine esterases. No formation of TBMEHP was identified for TBPH under realistic conditions, neither with human hepatocellular fractions nor in vivo in rats or in mice under realistic exposure conditions.

TBPH is determined as ‘non-binder to the estrogen receptor’ by the OECD Toolbox Version 3.3.5 based on a MW of > 500 g/mol.

TBPH showed mainly negative and few positive results in some of the in vitro screening assays for anti/androgenic and anti/estrogenic activity, but no conclusive picture is given. The activities were seen in general only at concentrations in the micromolar range and at magnitudes higher concentrations than TBPH detected in human body fluids, and are thus not relevant for the in vivo situation. Based on the available information there is no indication of a specific endocrine activity in vitro.

This conclusion is in line with the available animal studies.

In animal studies TBPH is well tolerated. The overall NOEL for repeated dose toxicity is determined with 1000 mg/kg bw/day.

There were no gross or microscopic findings to the reproductive system and to reproductive parameters in repeated oral dose toxicity studies (28- and 90-day studies) and a developmental toxicity study in rats that would point to an endocrine activity of TBPH. This is in contrast to the effects seen for the unbrominated DEHP, used as positive control in the 28-day oral toxicity study with TBPH. For DEHP strong general toxicity and severe testes effects in rats at daily doses lower than that of TBPH were recorded.

In conclusion, there is no reliable evidence of a specific and biologically relevant endocrine activity of TBPH throughout all OECD CF level studies, i.e. in vitro assays and experimental animal studies.

An endocrine disruptor is defined by WHO as an ‘exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse effects in an intact organism, or its progeny, or (sub) populations.’

Based on the information available it can be concluded that TBPH does not meet the WHO definition of an endocrine disruptor with respect to human health.

The complete assessment report is attached as pdf to the endpoint summary of IUCLID chapter 7.9.4 and to IUCLID chapter 13.2.


Justification for classification or non-classification

No relevant changes were seen in rats treated for 28 days with FR 45B (bis(2-ethylhexyl) tetrabromophthalate). Only a slightly low body weight gain in females and marginally low alanine amino-transferase activities in females receiving the highest dietary concentration of FR-45B, and a marginally low phosphorus concentrations in all females and males receiving the highest dietary concentration of FR-45B were reported.

In a subchronic repeated dose toxicity study according to OECD 408, oral administration of bis(2-ethylhexyl) tetrabromophthalate (CAS No. 26040-51-7) to male and female Wistar Han™:RccHan™:WIST strain rats for ninety consecutive days at dose levels of 100, 300 and 1000 mg/kg bw/day resulted in no treatment related changes.  The No Observed Effect Level (NOEL) is considered to be 1000 mg/kg bw/day, the highest dose tested.

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.