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

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8th January 2001 - 11th December 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study, conducted according to standardized guidelines, with analytical verification of test compound concentrations

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2003

Reference 2

Reference Type:

publication

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

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: 98.91%
- Impurities (identity and concentrations):
Tetrabromobisphenol-A: 98.91%
o,p- Tetrabromobisphenol-A: 0.05%
2,4,6-Tribromophenol: <0.01%
Tribromobisphenol-A: 1.04%
- Composition of test material, percentage of components: See above
- Isomers composition: See above
- Purity test date: 4 and 5 January 2001
- Lot/batch No.: Composite of 5381B, and 5381
- Expiration date of the lot/batch: Not reported
- Radiochemical purity (if radiolabelling):
- Specific activity (if radiolabelling):
- Locations of the label (if radiolabelling):
- Expiration date of radiochemical substance (if radiolabelling):
- Stability under test conditions: Not reported
- Storage condition of test material: Room temperature, Desiccated, 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 MI USA
- Age at study initiation: (P) 6 wks; (F1)3 wks
- Weight at study initiation: (P) Males: 258 to 338 g; Females: 190 to 248 g; (F1) 327.7 to g
- Fasting period before study: Not reported
- Housing: Individually in hanging wire mesh cages
- Diet (e.g. ad libitum): Lab Diet #5002, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 12 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 14 to 25C
- Humidity (%): 30 to 70%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hr light: 12hr dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: measured weight of TBBPA was mixed into known amount of vehicle

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, given (standard vehicle for gavage exposures)
- Concentration in vehicle: Not reported
- Amount of vehicle (if gavage): 5mL/kg/day
- Lot/batch no. (if required): 1940.01
- Purity: Not reported

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Maximum 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear
- Further matings after two unsuccessful attempts: None after the 14 day period
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: None reported

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At the 2.0 mg/mL concentration level (10 mg/kg/day) the mean analytical value for the 12 weeks of analysis was 2.05 mg/mL or 103% of nominal.
At the 20 mg/mL concentration level (100 mg/kg/day), the mean analytical value for the 12 weeks of analysis was 19.8 mg/mL or 99% of nominal.
At the 200 mg/mL concentration level (1000 mg/kg/day), the mean analytical value for the 12 weeks of analysis was 212 mg/mL or 106% of nominal.

Duration of treatment / exposure:

36 weeks

Frequency of treatment:

Daily

Details on study schedule:

- Selection of parents from F1 generation when pups were 35 days of age.
- Age at mating of the mated animals in the study:14 weeks

Doses / concentrations

Remarks:

Doses / Concentrations:
10, 100, 100 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

30 male and 30 female

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Results of previous studies
- Rationale for animal assignment (if not random): Random

Positive control:

N/A

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice per day, seven days per week
- Cage side observations included: morbidity, mortality, and signs of injury.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once per week

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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

Oestrous cyclicity (parental animals):

Vaginal smears (lavage) were performed daily beginning three weeks prior to pairing in all parental females (P and FJ to establish estrous cyclicity.

Sperm parameters (parental animals):

Parameters examined in all male parental generations: Daily sperm production, sperm motility, epididymal sperm count and morphology

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- If yes, maximum of 8 pups/litter ; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in all offspring: number and sex of pups, stillbirths, live births, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS: Yes

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals until completion of parturition
- Maternal animals: All surviving animals after the last litter of each generation was weaned

GROSS NECROPSY- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

HISTOPATHOLOGY / ORGAN WEIGHTS
See table 1.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at weaning
- These animals were subjected to postmortem examinations as follows: gross pathology, histopathology organ weights

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed

Statistics:

Group pair-wise comparisons, Fisher's extact test, Arcsin-Square-Root transformation

Reproductive indices:

Litter size, live pups, pup sex ratio, stillborn, mean pup weight

Offspring viability indices:

Not reported

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not specified

Description (incidence and severity):

Test substance intake: Assumed complete, as exposed via gavage

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

When compared to vehicle control means, treatment-related effects on total thyroxine (T 4) for TBBP A groups consisted of an increased incidence and magnitude of lower T 4 values for P and F1 parental generation individual male and female rats from the 100 mg/kg/day (~0.77 to 0.48x) and 1000 mg/kg/day (~0.67 to 0.30x) groups. Reductions (~0.84 to 0.63x) in tri-iodothyronine (T3) values were also observed for P generation male rats given 1000 mg/kg/day, and for
F1 generation male rats given 100 or 1000 mg/kg/day. Mild inconsistent alterations in T3 values were noted for some female rats that were considered of equivocal relationship to TBBP A.

Effect levels (P0)

1

Dose descriptor:

NOEL

Remarks:

thyroid hormone levels

Effect level:

10 mg/kg bw/day

Sex:

male

Basis for effect level:

other: NOEL for effects of TBBPA on thyroid hormone levels was 10 mg/kg/day based on lower T3 and T4 levels at the 100 and 1000 mg/kg/day dose levels

Remarks on result:

other: Generation: P and F1 parental (migrated information)

2

Dose descriptor:

NOEL

Remarks:

reproductive performance and pup toxicity

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: The NOEL for reproductive performance and pup toxicity was 1000 mg/kg/day, the highest dose level evaluated.

Remarks on result:

other: Generation: all generations tested (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

Although no effects on thyroid hormones were observed for rats given 10 mg/kg/day of TBBPA, daily oral dosing with 100 or 1000 mg/kg/day resulted in treatment-related effects on some thyroid parameters for P and F1 generation male and female rats, when compared to vehicle control rats.

Effect levels (F1)

Dose descriptor:

NOEL

Remarks:

parental toxicity

Generation:

F1

Effect level:

100 mg/kg bw/day

Sex:

male

Basis for effect level:

other: Lower body weights and body weight gain in males at the 1000 mg/kg/day dose level

Results: F2 generation

Effect levels (F2)

Dose descriptor:

NOEL

Remarks:

developmental neurotoxicity/neuropathology

Generation:

F2

Effect level:

100 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: In the DNT/NP component, the NOEL was 100 mg/kg/day based on subtle morphometric changes in the parietal cortex in the brains of the Day 11 F2 pups in the 1000 mg/kg/day group.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1: Summary of P Male Mean Thyroid Hormone Values

Endpoint	0 mg/kg/day	10 mg/kg/day	100 mg/kg/day	1000 mg/kg/day
TSH ng/mL	15.10	11.47	14.4	14.93
T4 ng/dL	4.70	5.08	3.9*	3.38*
T3 ng/dL	102.7	92.8	97.5	83.2*

* significantly different from controls p<0.05

Table 2: Summary of P Female Mean Thyroid Hormone Values

Endpoint	0 mg/kg/day	10 mg/kg/day	100 mg/kg/day	1000 mg/kg/day
TSH ng/mL	10.80	9.77	10.32	9.70
T4 ng/dL	4.23	3.45	3.5	2.39*
T3 ng/dL	94.8	96.0	87.5	90.8

* significantly differrent from controls p<0.05

Table 3: Summary of F1 Male Mean Thyroid Hormone Values

Endpoint	0 mg/kg/day	10 mg/kg/day	100 mg/kg/day	1000 mg/kg/day
TSH ng/mL	11.92	10.67	13.34	9.12
T4 ng/dL	6.29	5.98	3.91*	3.33*
T3 ng/dL	116.8	112.6	105.9	108.2

* significantly different from controls, p<0.05

Table 4: Summary of F1 Female Mean Thyroid Hormon Values

Endpoint	0 mg/kg/day	10 mg/kg/day	100 mg/kg/day	1000 mg/kg/day
TSH ng/mL	10.23	8.90	11.74	7.40
T4 ng/dL	6.00	4.42	3.40*	3.41*
T3 ng/dL	112.7	102.2	101.3	140.7

* significantly different from controls, p<0.05

Applicant's summary and conclusion

Conclusions:

In this 2-generation reproduction study with TBBPA the No Observed Effect Level (NOEL) for parental toxicity was 100 mg/kg/day based on lower body weights and body weight gain in males at the 1000 mg/kg/day dose level. The NOEL for effects of TBBPA on thyroid hormone levels was 10 mg/kg/ day based on lower T3 and T 4 levels at the 100 and 1000 mg/kg/day dose levels. TSH levels, however, were not affected at any of the dose levels in either generation. The NOEL for reproductive performance and pup toxicity was 1000 mg/kg/day, the highest dose level evaluated. In the DNT/NP component, the NOEL was 100 mg/kg/day based on subtle morphometric changes in the parietal cortex in the brains of the Day 11 F2 pups in the 1000 mg/kg/day group.

Executive summary:

The objective of this reproduction study was to provide general information concerning the effects of Tetrabromobisphenol A (TBBPA) over the course of two generations (P and F1) and the growth and development of the offspring (F1 and F2). The study

consisted of three treatment groups (10, 100 and 1000 mg/kg/day) and a vehicle (corn oil)- treated control group (30 CD Sprague-Dawley rats/sex/group/generation). TBBPA was administered orally via gastric intubation. Animals were treated seven days a week throughout the study. Dosing suspensions were prepared fresh weekly. For breeding of the P and F1 generations, one male was paired with one female from the same treatment group continuously until mating occurred or for 14 consecutive days. At weaning of each F1 litter, at least one pup/sex/litter was selected to continue on study as the F1 parental generation (30 pups/sex/group). These pups started treatment on PND 22.

Detailed clinical examinations, body weights, and food consumption were recorded periodically throughout the study for the P and F1 parental animals. All pups were monitored for appearance, growth, and survival throughout the lactation period

Several days before terminal euthanasia of the P and F1 animals, blood was collected from 10 randomly selected animals/sex/group and analyzed for thyroid hormone levels (TSH, T3 and T4) At necropsy, P and F1 animals received a macroscopic examination and reproductive tissues and other designated tissues were taken, weighed, and preserved. Reproductive tissues were evaluated microscopically for all P and F1 animals in the control and 1000 mg/kg/day groups. Gross lesions were also examined microscopically for all parental animals. Sperm evaluations (motility, caudal epididymal sperm counts, homogenization-resistant testicular sperm head counts, and morphology) for P and F1 males and a count of primordial follicles were conducted for P and F1 females. At weaning, the unselected F1 pups and one F1 pup/sex/litter were euthanized, necropsied, specific organs weighed (brain, spleen, and thymus), and gross lesions preserved.

Dosing formulations were homogeneous at the batch size prepared and stable when refrgerated to 14 days. Mean recoveries from the periodic analyses of dosing suspensions used on study were 101 %, 99%, and 105% of nominal for the 10, 100, and 1000 mg/kg/day groups, respectively.

No effect of treatment was seen for mortality in the P and F1 generations. The low incidence of mortality seen in these animals was considered incidental and unrelated to treatment with TBBPA.

In the parental generations, the only effect of treatment with TBBPA was seen in the F1 males at 1000 mg/kg/day and involved lower body weights for several weekly intervals during the study and lower weight gain over the entire Week 1-11 premating period. At 100 and 1000 mg/kg/day, some treatment-related effects on some thyroid hormone parameters (T3 and T4) were seen. No effect of treatment in either generation was evident from other endpoints. No effect on body weights or body weight gain was seen in the P animals or F1 parental females. Likewise, no adverse effect on food consumption was seen in the treated groups for either generation.

Thus, in this 2-generation reproduction study with TBBPA the No Observed Effect Level (NOEL) for parental toxicity was 100 mg/kg/day based on lower body weights and body weight gain in males at the 1000 mg/kg/day dose level. The NOEL for effects of TBBPA on thyroid hormone levels was 10 mg/kg/ day based on lower T3 and T4 levels at the 100 and 1000 mg/kg/day dose levels. TSH levels, however, were not affected at any of the dose levels in either generation. The NOEL for reproductive performance and pup toxicity was 1000 mg/kg/day, the highest dose level evaluated. In the DNT/NP component, the NOEL was 100 mg/kg/day based on subtle morphometric changes in the parietal cortex in the brains of the Day 11 F2 pups in the 1000 mg/kg/day group.