2,2',6,6'-tetrabromo-4,4'-isopropylidenediphenol

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9th August 2001 - 26th December 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study; conducted according to standardized guidelines; no analytical verification of test compound concentrations

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Reference 2

Reference Type:

publication

Title:

Unnamed

Year:

2006

Reference 3

Reference Type:

publication

Title:

Unnamed

Year:

2000

Reference 4

Reference Type:

review article or handbook

Title:

Unnamed

Year:

1996

Reference 5

Reference Type:

publication

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): Tetrabromobisphenol-A
- Molecular formula (if other than submission substance): N/A
- Molecular weight (if other than submission substance): N/A
- Smiles notation (if other than submission substance): N/A
- InChl (if other than submission substance): N/A
- Structural formula attached as image file (if other than submission substance): see Fig. N/A
- Substance type: Monoconstituent
- Physical state: Solid, white powder
- Analytical purity: 98.71 to 98.87%
- Impurities (identity and concentrations):
Tetrabromobisphenol-A: 98.71% (pre-study)
o,p-Tetrabromobisphenol-A: 0.04%
2,4,6-Tribromobisphenol: N.D.
Tribromobisphenol-A: 1.25%
- Composition of test material, percentage of components: See above
- Isomers composition: See above
- Purity test date: 15 November 2001 and 11 January 2002
- Lot/batch No.: 5381A
- Expiration date of the lot/batch: Not reported
- Radiochemical purity (if radiolabelling): N/A
- Specific activity (if radiolabelling): N/A
- Locations of the label (if radiolabelling): N/A
- Expiration date of radiochemical substance (if radiolabelling): N/A
- Stability under test conditions: Stable in corn oil for at least 14 days when refrigerated
- Storage condition of test material: At room temperature, in a dry, well-ventilated area

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, Mich. USA
- Age at study initiation: 8 weeks
- Weight at study initiation: Males- 128 to 156g; Females- 110 to 151g
- Fasting period before study:
- Housing: Individually in cages
- Diet (e.g. ad libitum): Diet Certified Rodent Diet #5002, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5 to 24C
- Humidity (%): 28 and 70%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hours light: 12 hours dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Corn oil, for gavage
- Storage temperature of food: Refrigerated

VEHICLE
- Justification for use and choice of vehicle (if other than water): None
- Concentration in vehicle: Sufficient to give dose levels of 100, 300, and 1000 mg/kg/day
- Amount of vehicle (if gavage): 5 mL/kg/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HPLC analysis with the following:
Column: Phenomex Luna C18 (250 mm x 4.6mm)
Mobile Phase: A. 10 mM Phosphate buffer; B. Acetonitrile
Flow rate: 1.25 mL per minute
Injection volume: 20uL
Expected retention time: ~12 minutes

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily gavage treatments

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 300, 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

15 males/15 females each in control and 1000 mg/kg/day exposure
10 males/10 females each in 100 and 300 mg/kg/day exposures

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on available data from previous studies
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: Randomized prior to study
- Post-exposure recovery period in satellite groups: 6-week recovery period

Positive control:

N/A

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice a day/ seven days a week
- Cage side observations checked: Morbidity, mortality, signs of injury, availability of food and water

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, calculated as g food/animal/day

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: At test initiation and termination
- Dose groups that were examined: All

HAEMATOLOGY: Yes / No / No data
- Time schedule for collection of blood: At test termination and at end of 6-week recovery period
- Anaesthetic used for blood collection: Yes, CO2
- Animals fasted: Yes, overnight
- How many animals: Only main study rats
- Parameters checked: TSH, T3, T4, serum

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Weekly
- Dose groups that were examined: all
- Battery of functions tested: activity, gait and posture, reactivity to handling or sensory stimuli, altered strength, and stereotypes or bizarre behavior

OTHER: Functional Observation Battery (FOB), at test initiation and at week 12

Sacrifice and pathology:

GROSS PATHOLOGY: Yes- cavity analysis, organ weights
HISTOPATHOLOGY: Yes-
- Adrenal (2)*#
- Aorta*
- Bone with marrow (femur)*
- Bone with marrow (stemum)*
- Bone marrow smear'*
- Brain (cerebrum, midbrain, cerebellum, medulla/pons) *#
- Eye including optic nerve and retina (2)*
- Gastrointestinal tract:
--esophagus*
--stomach (glandular and nonglandular)*
--duodenum*
--jejunum*
--ileum*
--cecum*
--colon*
--rectum *
- Gonads:
--ovary (2)*#
--testis (2)*# with epididymis (2)*#
- Gross Lesions*
- Harderian gland (2)
- Heart*#
- Kidney (2)*#
- Lacrimal gland, exorbital (2)
- Larynx*
- Liver (3 sections collected, 2 examined)*#
- Lung with mainstem bronchi (2 sections examined)*
- Lymph nodes: mandibular (2 collected, lexamined)*, mesenteric*, and regional lymph node*
- Mammary gland (females only)*
- Nose (4 sections examined)*
- Pancreas*
- Pharynx*
- Pituitary*
- Prostate* and seminal vesicle (2)*
- Salivary gland, mandibular (2 collected, i examined)*
- Sciatic nerve*
- Skeletal muscle, biceps femoris*
- Skin*
- Spinal cord (cervical, thoracic, and lumbar)*
- Spleen*#
- Thymus*#
- Thyroid/parathyroid (2)*#
- Tissue Masses*
- Tongue
- Trachea*
- Urinary bladder*
- Uterus (both)

Statistics:

See table below

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

All mortality due to dosing injuries

Mortality:

no mortality observed

Description (incidence):

All mortality due to dosing injuries

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See table below

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL CHEMISTRY: significant reduction in T4 levels (see table below)

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects obsreved even at 1000mg/kg test substance (highest dose tested).

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Mean TSH and T3 levels were statistically comparable between control and treated animals at all time points (Day 33, Day 90, and Recovery Sacrifice). (See table below). Mean T4 blood levels in male and female rats were statistically lower than their respective control means on Day 33 in the 100, 300 and 1000 mg/kg/d groups. Mean T4 levels were statistcally lower in all male groups on Day 90 compared to the control mean. Mean T4 levels were statistically comparable to the control mean on Day 90 in all female groups. At recovery euthanasia, mean T4 levels were comparable in the control and 1000 mg/kg/d male and female groups. The change in T4 levels was reversible on recovery.

Table 1: Male and female T4 levels (ng/dL)

Treatment	Sex	Mean T4 concentrations (Day 33)	Terminal T4 level
Control	Male	4.96±0.84	5.09± 0.80
100 mg/kg/day	Male	3.66± 0.88*	3.27± 0.67*
300 mg/kg/day	Male	3.42±0.71*	2.61± 0.87*
1000 mg/kg/day	Male	3.39± 0.55*	3.09± 0.91*
Control	Female	4.27± 0.96	5.41± 1.04
100 mg/kg/day	Female	3.31± 1.08*	5.22± 1.23
300 mg/kg/day	Female	3.24± 0.85*	4.95± 1.32
1000 mg/kg/day	Female	3.33±0.84*	4.95± 1.11

* Significantly different from control; (p < 0. 01)

The decrease in serum T4 levels was considered a possible effect of test article administration. TBBPA has been shown to competitively displace T4 from transthyretin (TTR), a major serum T4 -binding protein in the rat, in vitro (Meerts et al. 2000). That portion of serum T4 displaced from its serum binding site would be available for metabolism and elimiantion, thereby leading to a decrease in serum levels. The half-life of T4 in the rat is short due to its transport by TBBP, and thus this species is sensitive to perturbations in T4 levels. For example, the plasma T4 half-life in rats is 12 -24 hours while the human half-life is 5 -9 days (Capen C 1996). In humans, circulating T4 is bound primarily to thyroxin binding globulin, but htis high affinity binding protein is not present in

rodents. This mechanism, displacement of T4 from TTR by TBBPA, may have accounted for the decreased mean T4 levels in treated animals. However, as shown by Schauer et al. (2006), TBBPA has a low systemic bioavailability in rats (and humans) and undergoes extensive first-pass hepatic metabolism to sulfate and glucuronide conjugates. Hepatic induction of glucuronidation could result in a decreased half-life of T4 in the rat. Because the derease in T4 levels was not of sufficient magnitude to alter mean serum TSH or T3 levels, thyroid histopathology, thyroid weight, or other parameters indicative of thyroid pathology (e.g. body weigth, tec.), the decrease in serum T4 levels was not considered adverse. The reduction in serum T4 levels was not accompanied by evidence of toxicity or adverse effects, and the animals were clinically normal.

Applicant's summary and conclusion

Conclusions:

The 90 D No Observed Adverse Effect Level (NOAEL) in the rat was 1000 mg/kg/day, the highest dose tested.

Executive summary:

This study was conducted using a composite of the commercial products produced by Albemarle Corporation, Dead Sea Bromine Group, and Great Lakes Chemical Corporation. It was performed according to Good Laboratory Practices and according to US EPA OPPTS and OECD guidelines.

This study was conducted to evaluate the subchronic toxicity of TBBPA in CD® [Crl: CD® (SD) IGS BR] rats. The study consisted of three treatment groups and one vehicle (corn oil) control group (ten rats/sex/group). Recovery animals (five rats/sex) were

included in the control and high-dose group and evaluated over a 6-week post-treatment period. TBBPA was administered orally by gavage daily for 13 weeks at dose levels of 0, 100,300, and 1000 mg/kg/day at a constant volume of 5 mL/kg/day. The control animals received the vehicle at the same volume and dosing regimen as the treated groups. Animals were observed daily cage side for survivability, injury, and availability of feed and water. Other observations conducted weekly during the study included detailed physical and neurobehavioral evaluations, and measurements of body weights and food consumption. A Functional Observational Battery (FOB) was conducted pretest and at Week 12. Motor activity (MA) was also evaluated during Week 12. Ophthalmoscopic examinations were conducted pretest, study termination, and following recovery .Other evaluations conducted at termination and following recovery included: hematology, clinical chemistry , urinalysis, organ weights, and pathological examinations (macroscopic and microscopic). Thyroid hormone levels [Thyroid Stimulating Hormone (TSH), T3 (3,5,3' -triiodothyronine), and T4 (thyroxine

or 3,5,3'5'-tetraiodothyronine)] were evaluated of animals at 33 days and at termination. These same hormone levels were evaluated following recovery. Homogeneity of the dosing suspensions at the low and high concentration levels was determined on mixes used the first week of study. Mean concentration recoveries from the periodic analyses of dosing suspensions used on study were 102.5%, 110.2%, and 106.8% of nominal for the l00, 300, and 1000 mg/kg/day groups, respectively.

A total of six females (two control and four in the 1000 mg/kg/day group) died or were euthanized in extremis.The mortality/moribundity seen in these groups was considered related to dosing injury and not treatment related.

No effect of treatment was seen in clinical or neurobehavioral evaluations, body weights, food consumption, ophthalmological examinations, MA, FOB evaluations, hematology or urinalysis evaluations. Likewise, no effect of treatment was evident from organ weights, or from the macroscopic or microscopic examinations. After 90 days of dosing, total bilirubin values were statistically higher than the control means (males: 0.14 ± 0.05; females: 0.13 ± 0.05) in males in the 1000 mg/kg/day dose (0.34 ± 0.024) (p<0.01) group and in females in the 300 (0.19 ± 0.03) (p<0.05) and 1000 mg/kg/day (0.2 ± 0.06) groups (p<0.01 ). Mean serum alkaline

phosphatase (ALP) levels after 90 days of dosing in the female 1000 mg/kg/day (98.9 ± 49.47) group was statistically higher than that of the control mean (58.4 ± 28.46) (p<0.05). A slight increase, but non-statistically different, was also observed in males. Although these differences were considered possibly due to test article administration, neither of these changes was of sufficient magnitude as to be biologically or toxicologically meaningful or adverse. Serum bilirubin and ALP levels in control and treated groups of both sexes were comparable after the end of the recovery period. With respect to serum hormone levels, mean TSH and T3 levels were statistically comparable between control and treated animals at all time points (Day 33, terminal and recovery euthanasia). Mean T4 levels were statistically lower than the control mean (Day 33: 4.96 ± 0.84; terminal: 5.09 ± 0.80) in the 100 (Day 33: 3.66 ± 0.88; terminal: 3.27 ± 0.67), 300 (Day 33: 3.42 ± 0.71; terminal: 2.61 ± 0.87) and 1000 (Day 33: 3.39 ± 0.55; terminal: 3.09 ± 0.91) mg/kg/day male dose groups at days 33 and 90 (p<0.01). Mean T4 1evels were also statistically lower than the control mean (4.27 ± 0.96) in females in the l00 (3.31 ± 1.08), 300 (3.24 ± 0.85) and 1000 (3.33 ± 0.84) mg/kg/day dose groups at Day 33 (p<0.05). Mean T4 1evels in all female dose groups were statistically comparable to the control mean at Day 90. At the recovery euthanasia, mean T4 1evels were comparable in the control and 1000 mg/kg/day male and female groups. The change in T41evels seen in the 1000 mg/kg/day group was reversible and levels comparable to control were seen following recovery. The decrease in serum T 4 levels was considered a possible effect of test article administration. TBBPA has been shown to competitively displace T4 from transthyretin (TTR), a major serum T4-binding protein in the rat,in vitro(Meerts et al. 2000. Toxicological Sciences, 56,95-104). That portion of serum T4 displaced from its binding site would be available for metabolism and elimination, thereby leading to a decrease in serum levels. The half-life of T4 in the rat is short due to its transport by TTR, and thus this species is sensitive to perturbations in T4 levels. For example, the plasma T4 half-life in rats is 12-24 hours while T4's half-life in humans is 5-9 days (Capen, C. 1996. Chapter 21. Toxic Responses of the Endocrine System. In: Casarett & Doull's Toxicology, The Basic Science of Poisons. Fifth Edition. Ed. Curtis Klaassen. McGraw-Hill, New York. 474-006). In humans circulating T4 is bound primarily to thyroxin binding globulin, but this high affinity binding protein is not present in rodents.

This mechanism, displacement of T4 from TTR-binding by TBBPA with subsequent metabolism and elimination in the liver, may account for the decreased mean serum T4 levels in treated animals. Because the decrease in T4 1evels was not of sufficient magnitude to alter mean serum TSH or T3 levels, thyroid histopathology, thyroid weight, or other parameters indicative of thyroid pathology (e.g. body weight, etc.), the decrease in serum T4 1evels was not considered adverse. The reduction in serum T4 1evels was not accompanied by evidence of toxicity or adverse effects, and the animals were clinically normal.

Thus, in this rat 90-day oral toxicity study with TBBP A, the No Observed Adverse Effect Level (NOAEL) was 1000 mg/kg/day, the highest dose tested. No effect on mortality, clinical signs, body or organ weights, histopathology, urinalysis, ophthalmology, FOB, MA, serum TSH, serum T3 or serum chemistries was observed. Differences were observed for bilirubin and ALP, but neither of these changes were found to be biologically or toxicologically meaningful or adverse. Serum T4 levels were decreased in treated animals, but the decrease was not of sufficient magnitude to induce adverse effects.