Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
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
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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
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
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
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
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
Gross pathological findings:
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: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
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.
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)
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)
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
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.
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
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.
Reproductive effects observed:
not specified

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

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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Both the key and supporting two generation reproductive toxicity studies were conducted in accordance with standardised guidelines under GLP conditions. The quality of the database is considered to be high.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The key study, an oral 2-generation reproduction study in the rat with a developmental neurotoxicity component was conducted using a composite of three commercial TBBPA products. The study was performed according to Good Laboratory Practices and US EPA OPPTS guidelines.

The objective of study was to provide information concerning the effects of TBBPA over the course of two generations (P and F1) on the growth and development of the offspring (F1 and F2). A developmental neurotoxicity/neuropathology assessment was also conducted on the F2 offspring. The study consisted of three treatment groups (10, 100 and 1000 mg/kg/day) and a vehicle (corn oil)-treated control group (30 CD®[Crl: CD® (SD) IGS BR]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. Parental animals were treated for at least 10 weeks prior to mating (premating treatment period) to produce the F1 and F2 litters. In the developmental neurotoxicity/neuropathology (DNT/NP) component, F2 pups were randomly selected to continue on study for the following evaluations (unique sets of animals [10 pups/sex/group] were randomly selected for each assessment): PND 60 brain weights, PND 60 perfusion and neuropathology, special detailed clinical examinations (PND 4, 11, 21, 35, 45, and 60), motor activity [MA] (PND 13, 17, 21, and 60), auditory startle habituation [ASH] (PND 22 and 60), and learning and memory [L&M] (PND 22, 60 and 110). Additionally, 10 F2 pups/sex/group were selected randomly on PND 11 for collecting, weighing, and preserving of the brains.

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. The day of mating evidence was considered Day 0 of gestation. During mating of the F1 generation, cohabitation of littermates was avoided. Females delivered and nursed litters over a 21-day lactation period. On Day 4 of lactation all litters were culled if necessary to 8 pups (F1) or 10 pups (F2) with sex distribution equalized, when possible. Litters with fewer pups than required at culling were not adjusted.

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. The premating period formally initiated after the last litter weaned. Thus, there was a maximum of two weeks difference in age for the F1 animals within each treatment group at initiation of the premating growth period.

Detailed clinical examinations, body weights, and food consumption were recorded periodically throughout the study for the P and F1 parental animals. Oestrous cyclicity was evaluated in the P and F1 females the last three weeks of the premating period, and these evaluations continued until the female was confirmed mated or to the end of the mating period. Females were allowed to deliver and nurse the litter to weaning. Litters were evaluated at birth and throughout the lactation period. Each pup was individually identified at birth (paw tattoo), sexed, examined externally for defects, and weighed. All pups were monitored for appearance, growth, and survival throughout the lactation period. Clinical examinations, body weights, food consumption, and occurrence of maturation landmarks (vaginal opening [VO] and preputial separation [PS]) were recorded for F) parental animals. 

Several days before terminal euthanasia of the P and F1 animals, blood was collected from 10 randomly selected animals/sex/group and analysed 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. Microscopic examinations were also performed for reproductive tissues of the few low- and mid-dose P and F1 animals that failed to mate, conceive or sire. Gross lesions were also examined microscopically for all parental animals. Sperm evaluations (motility, caudal epididymal sperm counts, homogenisation-resistant testicular sperm head counts, and morphology) for P and F, males and a count of primordial follicles were conducted for P and F1 females. The latter evaluations were conducted only in the control and 1000 mg/kg/day groups. At weaning, the unselected F1 pups and one F2 pup/sex/litter were euthanised, necropsied, specific organs weighed (brain, spleen, and thymus), and gross lesions preserved.

In the DNT/NP component, brains from F2 pups euthanized on PND 11 (10/sex/group) were weighed, and preserved in fixative for neuropathological evaluation and morphometric measurements. These examinations were initially conducted in the control and high-dose animals and were expanded to include the lower dose groups. F2 pups retained post-weaning were observed twice daily cage side for mortality and were weighed and given detailed clinical examinations periodically during the study. Sexual maturation (VO and PS) was evaluated for the 40 animals/sex/group retained for the neurobehavioral assessments (i.e., special clinical examinations, MA, L&M, and ASH). These animals were euthanized after all the behavioural tests had been completed. At termination, all F2 animals were weighed, given a detailed clinical examination and necropsied. The F2 animals euthanised on PND 60 for neuropathological evaluation were anesthetised with sodium pentobarbital and perfused with 3 % paraformaldehyde and 3 % glutaraldehyde. The whole brain, sections of the spinal cord, and selected peripheral nerves were collected and processed for neuropathological examination in the control and 1000 mg/kg/day groups.

Dosing formulations were homogeneous at the batch size prepared and stable when refrigerated 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. No effect of treatment in either generation was evident from the clinical examinations, oestrous cyclicity, reproductive performance, gestation/lactation body weights or food consumption, gestation length, litter data, or from the macroscopic and microscopic evaluations, organ weights, sperm evaluations, and primordial follicle counts. 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.

No effect of treatment with TBBPA was evident in the F1 and F2 pups in regard to body weights, clinical findings, sex ratios, survival to weaning, macroscopic findings, or organ weight data (Day 21).  No effects on thyroid hormone levels (TSH, T3 and T4) were observed at the 10 mg/kg/day dose level in either generation. At 100 and 1000 mg/kg/day, some treatment-related effects on some thyroid hormone parameters (T3 and T4) were seen. TSH levels were unaffected, however, in either generation. Treatment with TBBPA demonstrated an increased incidence and magnitude of lower T4 values in the 100 and 1000 mg/kg/day groups. P males given 1000 mg/kg/day, and F, males given 100 or 1000 mg/kg/day also had mild reductions in T3 values. In the absence of increases in TSH hormone levels, moderate reductions in circulating serum T4 levels, with only mild decreases in T3 for a few 1000 mg/kg/day P males, and 100 and 1000 mg/kg/day F, males, are suggestive of induction of hepatic T4-uridine diphosphate glucuronyl transferase (UDP-GT) enzymes that increase the removal of thyroxine. TBBPA has been shown in vitro to competitively displace T4 from human transthyretin, a serum carrier protein. The decreases in T4 and T3 observed in this study did not exceed the threshold for stimulation of TSH production. Thus, repeat daily dosing with TBBPA at doses of 100 or 1000 mg/kg/day to P and F1 generation rats resulted in effects on thyroid function, probably secondary to enzyme induction, without alteration in TSH activity. The 10 mg/kg/day dose was determined to be a no observed effect level (NOEL) for TBBPA and its response on thyroid function.

In the DNT/NP component, no effects of treatment were seen in F2 pups with respect to: PND 60 brain weights, PND 60 perfusion and neuropathology, special detailed clinical examinations (PND 4, 11, 21, 35, 45, and 60), motor activity (PND 13, 17, 21, and 60), auditory startle habituation (PND 22 and 60), and learning and memory (PND 22, 60 and 110).  The only suggestion of a treatment-related effect was a reduction in the thickness of the parietal cortex of Day 11 pups at the 1000 mg/kg/day dose level, but not in pups at this dose level on Day 60. This change on Day 11 was not accompanied by any histologic changes in the parietal cortex, such as degeneration, necrosis, cell loss, demyelination, proliferative changes, or changes in neuronal cell density. A likely explanation for the decreased thickness of parietal cortex would be a decreased number of cells without changes in cell density. The brain weights of the 11-day-old rats were virtually equal across groups. However, it is possible that other regions of the brain were enlarged and compensated for the decrease in the thickness of parietal cortex in the affected groups. The thickness of the parietal cortex for the animals at the 10 and 100 mg/kg/day dose levels was comparable to the control. No microscopic alterations were observed in brain, spinal cord, nerves, and ganglia in the 60-day-old rats. Therefore, this apparent test-article related effect in the Day 11 F2 pup brains must be interpreted with caution, given the limitations of morphometric analysis. No effect of treatment was evident from the other parameters evaluated in the DNT/NP component. This would include the special detailed clinical observations, developmental maturation landmarks (vaginal opening and preputial separation), neurobehavioral evaluations (motor activity, learning and memory, auditory startle habituation), or Day 60 brain weights or parietal cortex thickness.

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 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, but not Day 60 F2 pups, in the 1000 mg/kg/day group. In this component no changes at any dose level were seen in the pups at any time point from clinical findings, sexual maturation landmarks, growth, or from the various behavioural assessments. 

In the supporting study, the potential of the test material to cause toxicity was investigated in a reproductive study conducted in accordance with the standardised guidelines OECD 416 and US EPA OPPTS 870.3800 under GLP conditions.

The effects of the test material at oral doses of 10, 100 or 1000 mg/kg bw/day over the course of 2 generations (30 rats/sex/dose) on growth as well as behavioural, neurological and neuropathologic functions in offspring were investigated.

CD® [Crl: CD® (Sprague-Dawley) IGS BR] rats were exposed to the test material in corn oil by gavage once daily. Parental males and females were treated prior to and during mating; females were treated through gestation and lactation. The P generation was dosed for 10 weeks prior to pairing for mating, and the mating period lasted a maximum of 14 days. 

After the weaning of F1 pups, on Lactation Day (LD) 21 F1 animals to continue the study were selected and dosed for at least 10 weeks prior to mating. All F1 females were treated until the F2 pups were weaned.

In the P and F1 generations, there were no significant effects on mortality, body and tissue weights, clinical observations and tissue histopathology. There were no marked effects on various reproductive parameters including time to vaginal opening and anogenital distance.

Exposure to ≥100 mg/kg bw/day resulted in a decrease in circulating, peripheral thyroxine (T4) levels that were not accompanied by any marked alterations in triiodothyronine (T3) and thyroid stimulating hormone (TSH). These findings are explainable on the basis of induction of rat liver catabolism, a phenomenon that may be species-specific and not relevant for humans. Doses up to 1000 mg/kg bw/day were not associated with any significant non-neurological effects on reproduction, growth and development. A subtle reduction, of unknown biological relevance, in the thickness of the parietal cortices of 11-day-old F2 pups in the 1000 mg/kg bw/day group was noted. This change was not accompanied by evidence of micro-anatomic changes. No oestrogenic effects sufficient to affect macro and micro anatomy, fertility, reproduction, development, survival or behaviour were detected. No other test material-related effects on developmental neurotoxicity/neuropathology were detected.

The NOAEL in P and F1 generation animals was ≥1000 mg/kg bw/day when considering effects on mortality, body and tissue weights, clinical observations and tissue histopathology and reproductive parameters. The NOAEL in P and F1 generation animals was 10 mg/kg bw/day for effects of the test material on serum thyroid hormone levels. Thinning of the brain parietal cortex in F2 generation animals was observed at day 11 of life at the maternal oral dose of 1000 mg/kg bw/day. The NOEL for this effect was thus the maternal oral dose of 100 mg/kg bw/day.

Under the conditions of this study, the no observed effect level (NOEL) for maternal and developmental toxicity was considered to be 1000 mg/kg bw/day, the highest dose evaluated.


Short description of key information:
Guideline- and GLP-compliant oral two generation reproduction study with a developmental neurotoxicity component in the rat.

Justification for selection of Effect on fertility via oral route:
Three studies are available to address this endpoint; one key, one supporting and one disregarded. The key study was selected on the basis that, although both the key and supporting studies were conducted in accordance with standardised guidelines under GLP conditions, the key study was a full study report and therefore deemed to contain more detail than the supporting study which was published in the literature.

Effects on developmental toxicity

Description of key information
Guideline- and GLP-compliant prenatal developmental in the rat.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26th January 2001 - 20th September 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to standardized OECD/EPA guidelines, with analytical verification of test compound concentrations administered
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portgage MI USA
- Age at study initiation: 10 weeks old at study initiation
- Weight at study initiation: 203 to 274 g
- Fasting period before study: Not reported
- Housing: Individually in hanging wire cages
- Diet (e.g. ad libitum): Rodent Chow #5002, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks prior to mating

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 26C
- Humidity (%): 38 to 63%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hours light: 12 hours dark

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Measured weights of TBBPA added pre-determined quantities of corn oil

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): Not given, standard vehicle
- Concentration in vehicle: Sufficient to give 100, 300, and 1000 mg/kg/day
- Amount of vehicle (if gavage): 5mL per kg
- Lot/batch no. (if required): PU0041, PO0173
- Purity: Not reported
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to initiation oftest article administration, test batches ofthe test article suspensions at the low and high concentrations used in the study (20 and 200 mg/mL, respectively) were prepared to assess the homogeneity of the test preparations employing the same method and batch size to be used during the study.
Validation was performed by HPLC with UV detection, with an internal standard operating procedure. LLOQ = 0.0100 mg/mL
Column: Phenomenex Luna C i 8, (250 mm x 4.6 mm), 5 urn
Guard Column: 0.45 urn ¡nline filter canridge
Column Temp: Ambient
Mobile Phase: A: 10 mM Phosphate buffer (pH = 4) B: Acetonitrle

Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: Not reported
- Further matings after two unsuccessful attempts: Not reported
- Verification of same strain and source of both sexes: Yes
- Proof of pregnancy Not reported, referred to as day 0 of pregnancy
- Any other deviations from standard protocol: None reported
Duration of treatment / exposure:
Exposed to 100, 300, and 1000 mg/kg/day from Day 0 of gestation to Day 19
Frequency of treatment:
Daily
Duration of test:
20 days
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
25 females per dose plus control
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
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, seven days a week for morbidity, mortality, and signs of injury


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: On Days 0, 3,6, 9, 12, 15, 18, and 20 of gestation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: uterus, placenta, standard gross necropsy on maternal rats, fetal examinations

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data
Statistics:
Group Pair-Wise Comparisons; Fisher's Exact Test; Arcsin-Square-Root transformation; Covariate analysis; Descriptive
Indices:
Fertility indices
Historical control data:
See table below
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
One mortality determined to be result of dosing injury; salivation effects determined to be sporadic and not related to dose
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The few external findings (malformations and variations) seen in the treated groups occurred with low incidence on both a per fetus and per litter basis and were considered unrelated to treatment. The litter incidences for these findings for the treated groups did not differ statistically from controls.
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
In this oral developmental toxicity study in rats with Tetrabromobisphenol A, the NOAEL (No Observable Adverse Effect Level) for maternal and developmental toxicity was 1000 mg/kg/day, the highest dose level evaluated.
Executive summary:

The objective of this study was to provide general information concerning the effects of oral treatment of the pregnant rat with Tetrabromobisphenol A (TBBP A) on the developing organism. This included death, structural abnormalities or altered growth, and assessment of maternal effects. Female CD rats were mated in-house and received the TBBPA at dose levels of 0, 100, 300, and 1000 mg/kg/day at a constant volume of 5 mL/kg. The test article was administered orally by gavage as a single daily dose.

Observations of the dams included clinical signs, gestational body weights, and food consumption. Females were euthanized on Day 20 of gestation and given a postmortem macroscopic examination. All fetuses were given a gross external examination for malformations and variations. Approximately one-half of the fetuses in each litter were fixed in Bouin's solution, and the remaining fetuses were skinned and preserved in alcohol.

Pretest analyses confirmed that the suspensions as prepared were homogeneous and stable for at least 14 days when stored refrgerated. Periodic analysis of dosing suspensions used on study ranged from 88 to 113% of nominal and confirmed that animals were receiving the appropriate dose levels.

No treatment-related mortality was seen. The death of 1 animal in the 300 mg/kg/day group on Gestation Day 5 was attributed to an intubation injury. All other animals survived to scheduled euthanasia.

Salivation was seen among the TBBPA-treated animals, occurring most frequently at the 300 and 1000 mg/kg/day dose levels, Because of its sporadic occurrence, this was not considered to represent a direct effect of treatment with TBBPA, but more likely was in response to the taste of residual amounts oftest article on the dosing catheter. No other effects of treatment were seen from the clinical examinations, and no effect of treatment was evident from gestational parameters (body weight, body weight gain, or food consumption), uterine implantation data, liver weights, or necropsy findings, Likewise, no effect of treatment was evident from fetal body weights, fetal sex distribution, or from fetal external, visceral, or skeletal examinations.

Thus, in this oral developmental toxicity study in rats with TBBPA, the NOAEL (No Observable Adverse Effect Level) for maternal and developmental toxicity was 1000 mg/kg/day, the highest dose level evaluated.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Both the key and supporting studies were conducted in accordance with standardised guidelines under GLP conditions. The quality of the database is considered to be high.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In the key study, an oral prenatal developmental toxicity study was performed using a composite of 3 manufacturers' commercial products as test article. The study was performed according to Good Laboratory Practices and US EPA OPPTS/OECD guidelines.

The objective of the study was to provide information concerning the effects of oral treatment of the pregnant rat with TBBPA on the gestating dam and developing organism. This included death, structural abnormalities or altered growth, and assessment of maternal effects. Female CD rats were mated in-house and received the TBBPA at dose levels of 0, 100, 300, and 1000 mg/kg/day at a constant volume of 5 mL/kg. The test article was administered orally by gavage (corn oil) as a single daily dose.

Observations of the dams included clinical signs, gestational body weights, and food consumption. Females were euthanized on Day 20 of gestation and given a post-mortem macroscopic examination. All foetuses were given a gross external examination for malformations and variations. Approximately one-half of the foetuses in each litter were fixed in Bouin's solution, and the remaining foetuses were skinned and preserved in alcohol.

Pre-test analyses confirmed that the suspensions as prepared were homogeneous and stable for at least 14 days when stored refrigerated. Periodic analysis of dosing suspensions used on study ranged from 88 to 113 % of nominal and confirmed that animals were receiving the appropriate dose levels.

No treatment-related mortality was seen. The death of 1 animal in the 300 mg/kg/day group on Gestation Day 5 was attributed to an intubation injury. All other animals survived to scheduled euthanasia.

Salivation was seen among the TBBPA-treated animals, occurring most frequently at the 300 and 1000 mg/kg/day dose levels, Because of its sporadic occurrence, this was not considered to represent a direct effect of treatment with TBBPA, but more likely was in response to the taste of residual amounts of test article on the dosing catheter. No other effects of treatment were seen from the clinical examinations, and no effect of treatment was evident from gestational parameters (body weight, body weight gain, or food consumption), uterine implantation data, liver weights, or necropsy findings. Likewise, no effect of treatment was evident from foetal body weights, foetal sex distribution, or from foetal external, visceral, or skeletal examinations.

Thus, in this oral developmental toxicity study in rats with TBBPA, the NOAEL (No Observable Adverse Effect Level) for maternal and developmental toxicity was 1000 mg/kg/day, the highest dose level evaluated.

In the first supporting study, a pilot study with mated Charles River CD female rats was performed to determine dosage levels for a teratology study.

Tetrabromobisphenol A was administered by gavage at dosage levels of 30, 100, 300, 1000, 3000 and 10 000 mg/kg/day from gestation day 6 through day 15. A control group received the vehicle, corn oil, at 10 mL/kg/day.

During gestation the females were observed for clinical signs of effect, mortality and body weight changes. The rats were sacrificed on gestation day 20 by carbon dioxide inhalation, and the uterine contents examined for viable and nonviable foetuses, early and late resorptions, and total implantations.

There were no changes in appearance, behaviour, or body weights which were attributed to Tetrabromobisphenol A, for those females receiving 3000 mg/kg/day or less. There were no differences in the mean number of viable or nonviable foetuses, resorptions, implantations, or corpora lutea when compared to the controls which were attributable to treatment at 10 000 mg/kg/day or less. There

were slight decreases in body weight gains between gestation days 6 and 15 in the 10 000 mg/kg/day dose group which may be attributed to treatment.

Three of the five rats in the 10 000 mg/kg/day dosage group died. All rats at this dosage level showed signs of toxicity. A dosage level of 10 000 mg/kg/day is considered too high for meaningful evaluation of the potential teratogenicity of the test material.

In the second supporting study, the potential of the test material to cause toxicity was investigated in a developmental toxicity study conducted in accordance with the standardised guidelines OECD 414 and US EPA OPPTS 870.3700 under GLP conditions.

The effects of the test material at oral doses of 100, 300 or 1000 mg/kg bw/day over the period of organogenesis (Gestation days 0 to 19) were investigated. CD® [Crl: CD® (Sprague-Dawley) IGS BR] rats were exposed to the test material in corn oil by gavage once daily (25 females per dose).

Mated females were given a detailed clinical/physical examination on a daily basis from gestation day (GD) 0 to 20. The clinical/physical investigation included, but was not limited to, examination of skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs, feet, respiration and palpation of soft tissues. Individual bodyweights were measured on GD0 and every 3 days during the gestational phase. Food consumption was also measured.

Ovarian and uterine examinations were performed following post-euthanasia laparohysterectomy. A necropsy was conducted on each dam. Special emphasis was placed on structural abnormalities or pathologic changes that may have influenced pregnancy.

Approximately half of the foetuses per litter were designated for soft tissue examination. The remaining foetuses in each litter were fixed and processed for staining of mineralised and cartilaginous tissues. Foetal findings were classified as malformations or developmental variations.

It was determined that the test material is not an adult maternal toxicant at oral doses of up to 1000 mg/kg bw/day.

It was also determined that the test material is not an overt teratogen at oral doses of up to 1000 mg/kg bw/day. It is thus not considered a developmental toxicant in rats. There was no evidence of prenatal or postnatal developmental delay, reduced body mass or reduced weight gain.

This study demonstrated that the test material exerted no marked developmental effects at oral gavage doses of up to and including 1000 mg/kg BW/day, the highest dose tested.

Therefore under the conditions of this study the NOAEL for developmental toxicity was 1000 mg/kg bw/day.


Justification for selection of Effect on developmental toxicity: via oral route:
The key study was selected on the basis that it was conducted in accordance with standardised guidelines under GLP conditions and was awarded the highest reliability score. Although one of the supporting studies was also conducted in accordance with standardised guidelines under GLP conditions, it was published in the literature and the information was not as detailed as that presented in the full study report.

Justification for classification or non-classification

Tetrabromobisphenol A does not require classification as a reproductive toxicant according to the criteria described in Directive 67/548/EEC and the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No.1272/2008.