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

Carcinogenicity

Description of key information

In a two-year carcinogenicity study with TBBPA, groups of either male and female Wistar Han rats or male and female B6BC3F1 mice were administered TBBPA by gavage in corn oil at dose of 0, 250, 500 or 1000 mg/kg bw/day on five days/week for 104 to 105 weeks (NTP, 2014).
In rats, the MTD had been reached at the highest dose, while in mice, due to reduced survival of both sexes at 1000 mg/kg bw/day, meaningful analysis of tumour incidences was only possible at 250 and 500 mg/kg bw/day.  With regard to the different tumour types the NTP concluded for female mice there was no evidence of an increase, but for male mice there was some evidence (liver tumours), or equivocal evidence (tumours of the large intestine; haemangiomas and haemangiosarcomas). Also for male rats there was equivocal evidence (testicular interstitial cell adenomas), and for female rats, clear evidence (uterine tumours) of carcinogenic activity.
Considerations, however, of the mode of action (MoA) for carcinogenesis indicate that the tumours observed in rodents at elevated exposure levels are unlikely to be relevant to humans in particular when considering the possible human exposure situation.
Summaries of the 2-year Carcinogenesis studies of TBBPA and new data on the MoA (Borghoff et al. and Lai et al. 2015 studies) are attached.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliable source

Principles of method if other than guideline:

Principle of the method:
Groups of 60 male and 60 female rats were administered 0 or 1,000 mg tetrabromobisphenol A/kg body weight and 50 male and 50 female rats were
administered 250 or 500 mg/kg, in corn oil by gavage, 5 days per week for up to 104 (males) or 105 (females) weeks. Vehicle control animals receivedcorn oilonly. Rats were 6 to 7 weeks old at the beginning of the studies. The health of the animals was monitored during the studies according to the protocols
of the NTP Sentinel Animal Program. Rats were housed three (males) or five (females) per cage. Feed and water were available ad libitum. Cages and racks were rotated every 2 weeks.

Clinical Examinations and Pathology:
All animals were observed twice daily. Clinical findings were recorded every 4 weeks beginning week 5 and at the end of the studies.
Body weights were recorded on day 1, weekly for 13 weeks, monthly thereafter, and at the end of the studies.
Complete necropsies and microscopic examinations were performed on all animals.

At the 3-month interim evaluation in rats, the heart, right kidney, liver, lung, right testis, and thymus were weighed.
At necropsy, all organs and tissues were examined for grossly visible lesions, and all major tissues were fixed and preserved for microscopic examination.

After 2-years, samples of grossly observed tumors (uterine adeno-carcinomas) were collected at the time of necropsy, flash frozen in liquid nitrogen, and stored at −80° C for molecular analysis.

Species:

rat

Strain:

other: Wistar Han [Crl:WI(Han)]

Sex:

male/female

Details on test animals or test system and environmental conditions:

ANIMAL SOURCE:
Male and female Wistar Han [Crl:WI(Han)] rats were obtained from Charles River Laboratories (Raleigh, NC). The Wistar Han rat, an outbred rat stock, was selected because it was projected to have a long lifespan, resistance to disease, large litter size, and low neonatal mortality.

Animal strain:
The rationale for change of rat strain from F344/N to F344/NTac was a programmatic decision. For many years the NTP used the inbred F344/N rat for its toxicity and carcinogenicity studies. Over a period of time, the F344/N strain exhibited sporadic seizures and idiopathic chylothorax and
consistently high rates of mononuclear cell leukemia and testicular neoplasia. Because of these issues in the F344/N rat and the NTP’s desire to find a more fecund rat model that could be used in both reproductive and carcinogenesis studies for comparative purposes, a change in the rat model was explored. Following a workshop in 2005, the F344 rat from the Taconic commercial colony (F344/NTac) was used for a few NTP studies to allow the NTP time to evaluate different rat models between 2005 and 2006 (King-Herbert and Thayer, 2006). The Wistar Han rat, an outbred rat stock, was then selected because it was projected to have a long lifespan, resistance to disease, large litter size, and low neonatal mortality.

ANIMAL MAINTENANCE:
Animal care and use are in accordance with the Public Health Service Policy on Humane Care and Use of Animals.
All animal studies were conducted in an animal facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal CareInternational. Studies were approved by the Battelle Columbus Operations Animal Care and Use Committee and conducted in accordance with all relevant NIH and NTP animal care and use policies and applicable federal, state, and local regulations and guidelines.
The health of the animals was monitored during the studies according to the protocols of the NTP Sentinel Animal Program.

Rats were quarantined for 8 or 9 days and were housed three (males) or five (females) per cage. Feed and water were available ad libitum. Cages and racks were rotated every 2 weeks. Five male and five female rats were randomly selected for parasite evaluation and gross observation of disease.

Rats were 6 to 7 weeks old at the beginning of the studies.

Conditions:
Animals per Cage: 3 (males) or 5 (females.
Water- Tap water (Columbus municipal supply) via automatic rack watering system (Edstrom Industries, Waterford, WI), available ad libitum.
Cages- Polycarbonate (Lab Products, Inc., Seaford, DE), changed weekly (male mice) or twice weekly (rats and female mice) and rotated every 2 weeks.
Bedding- Irradiated Sani-Chips (P.J. Murphy Forest Products Corp., Montville, NJ), changed weekly (male mice) or twice weekly (rats and female mice).
Racks- Stainless steel (Lab Products, Inc., Seaford, DE), changed and rotated every 2 weeks.

Animal Room Environment:
Temperature: 72° ± 3° F
Relative humidity: 50% ± 15%
Room fluorescent light: 12 hours/day
Room air changes: at least 10/hour

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

Corn Oil National Formulary-grade corn oil was obtained in multiple lots from Spectrum Chemicals and Laboratory Products (Gardena, CA) and from Sigma-Aldrich (St. Louis, MO) and was used as the vehicle in the 3-month and 2-year studies. Periodic analyses

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

PREPARATION AND ANALYSIS OF DOSE FORMULATIONS:

The dose formulations were prepared every 6 weeks for the 2-year studies by mixing tetrabromobisphenol A with corn oil.

Homogeneity studies of 0.5 and 600 mg/mL formulations and stability studies of a 0.5 mg/mL formulation were performed by the analytical chemistry labora-tory using HPLC/UV. Homogeneity was confirmed; stability was confirmed for at least 42 days for dose formulations stored in sealed glass vials, protected
from light, at temperatures up to 25° C, and for at least 3 hours under simulated animal room conditions.

The dose formulations were stored in sealed glass bottles protected from light for up to 42 days at room temperature. The study laboratory conducted homogeneity studies of 1, 2, 10, 25, 50, 100, and 200 mg/mL formulations using HPLC/UV; gavageability studies of 100 and 200 mg/mL formulations were also performed. Homogeneity was confirmed.

Periodic analyses of the dose formulations of tetrabromobisphenol A were conducted by the study laboratory using HPLC/UV. During the 2-year studies, the dose formulations were analyzed approximately every 3 months (Table J3); of the dose formulations analyzed and used during the studies, all 72 for rats were within 10% of the target concentrations. Animal room samples were also analyzed; 7 of 9 animal room samples for rats were within 10% of the target concentrations.

To ensure stability, the bulk chemical was stored in sealed glass bottles protected from light at room temperature. Periodic reanalyses of the bulk chemical were performed by the study laboratory during the 3-month and 2-year studies using HPLC/UV. No degra-dation of the test chemical was detected.

Duration of treatment / exposure:

3 months (interim evaluation rats), 104 weeks (male rats),or 105 weeks.

Frequency of treatment:

5 days per week for up to 104 (male rats) or 105 weeks.

Remarks:

Doses / Concentrations:
0, 250, 500, 1000 mg/kg body weight.
Basis:
actual ingested

No. of animals per sex per dose:

60 male and 60 female Wistar han rats were administered 0 or 1000mg/kg bw
50 male and 50 female rats were administered 250 or500 mg/kg bw

Control animals:

yes, concurrent vehicle

Details on study design:

Study Design:
General:
Groups of 60 male and 60 female Wistar Han rats were administered 0 or 1,000 mg tetrabromobisphenol A/kg body weight in corn oil by gavage.
50 male and 50 female rats were administered 250 or 500 mg/kg bw in corn oil by gavage, for 5 days per week for up to 104 (male rats) or 105 week.

Ten vehicle control and ten 1,000 mg/kg rats of each sex were evaluated at 3 months to allow comparison to 3-month endpoints in the F344/NTac rats.
Vehicle control animals received corn oil only. Dosing volumes was 5 mL/kg bw.

Rats were quarantined for 8 or 9 days before the beginning of the studies. Five male and five female rats were randomly selected for parasite evaluation and gross observation of disease.

Rats were 6-7 weeks old at the beginning of the studies. The health of the animals was monitored during the studies according to the protocols of the NTP
Sentinel Animal Program. Rats were housed three (males) or five (females) per cage. Feed and water were available ad libitum. Cages and racks were rotated every 2 weeks. Further details of animal maintenance are given in Table 1.

Clinical Examinations and Pathology:
All animals were observed twice daily. Clinical findings were recorded every 4 weeks beginning week 5 and at the end of the studies.

Body weights were recorded on day 1, weekly for 13 weeks, monthly thereafter, and at the end of the studies.

Complete necropsies and microscopic examinations were performed on all rats and mice. At the 3-month interim evaluation in rats, the heart, right kidney, liver, lung, right testis, and thymus were weighed. At necropsy, all organs and tissues were examined for grossly visible lesions, and all major tissues were fixed and preserved.

For all paired organs (e.g., adrenal gland, kidney, ovary), samples from each organ were examined. Original transverse and residual longitudinal reviews of uterine tissue from female Wistar Han rats, including the 3-month interim evaluation animals, were conducted as described for the 3-month study in F344/NTac rats. In addition, cytokeratin and vimentin immunohistochemical stains were used to better characterize specific lesions that occurred in the uterus. Tissues examined micro-scopically are listed in Table 1.

For the 2-year studies, samples of grossly observed tumors (uterine adeno-carcinomas) were collected at the time of necropsy, flash frozen in liquid nitrogen, and stored at −80° C for molecular analysis (Appendix M). Microscopic evaluations were completed by the study laboratory pathologist, and the pathology data were entered into the Toxicology Data Management System. The report, slides, paraffin blocks, residual wet tissues, and pathology data were sent to the NTP Archives for inventory, slide/block match, wet tissue audit, and storage. The slides, individual animal data records, and pathology tables were evaluated by an independent quality assessment laboratory. The individual animal records and tables were compared for accuracy, the slide and tissue counts were verified, and the histo-technique was evaluated. For the 2-year studies, a quality assessment pathologist evaluated slides from all tumors and all potential target organs, which included the liver and uterus of rats and mice; the nose of rats; and the forestomach, large intestine, and kidney of mice.

Positive control:

No.

Observations and examinations performed and frequency:

Clinical Examinations and Pathology:
All animals were observed twice daily. Clinical findings were recorded every 4 weeks beginning week 5 and at the end of the studies.
Body weights were recorded on day 1, weekly for 13 weeks, monthly thereafter, and at the end of the studies.
Complete necropsies and microscopic examinations were performed on all animals.

Sacrifice and pathology:

Complete necropsies and microscopic examinations were performed on all animals.
At the 3-month interim evaluation in rats, the heart, right kidney, liver, lung, right testis, and thymus were weighed.

At necropsy, all organs and tissues were examined for grossly visible lesions.

Other examinations:

Complete histopathology was performed on all rats. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, cervix, clitoral gland, esophagus, eyes, gallbladder (mice only), Harderian gland, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, uterus, and vagina.

Statistics:

Statistical Analyses methods were used for calculating: the probability of survival, incidences of neoplasms or nonneoplastic lesions, analysis of Neoplasm and Nonneoplastic Lesion Incidences, and comparisons between dosed and control groups in the analysis of continuous variables data such as organ and body weight, hematology, clinical chemistry, thyroid hormone, cytochrome P450, UDP-GT, spermatid, and epididymal spermatozoal data.
Details on the methods are attached in the appropriate attachments section.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See details below

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

see details

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Females: Uterus and ovary Males: None. see details below.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Females: Uterus Males: testis. see details below

Details on results:

Details on findings;

Survival rates (per respective doses 0, 250, 500, or 1,000 mg/kg):
Survival of dosed groups was similar to that of the vehicle control groups.
Males: 33/50, 28/50, 38/50, 39/50
Females: 35/50, 34/50, 29/50, 33/50

Organ Weights
At the 3-month interim evaluation, the absolute and relative thymus weights of 1,000 mg/kg rats were
significantly less than those of the vehicle control groups and the relative liver weights of these dosed groups were significantly greater than those of the vehicle controls (Table H2), similar to those seen in the 3-month F344/NTac rats.

Body Weights:
The mean body weights of 500 and 1,000 mg/kg males were generally at least 10% less than those of the vehicle control group after week 25; body weights o f dosed groups of female rats were similar to those of the vehicle controls throughout the study (Tables 4 and 5; Figure 4).
Males: 500 and 1,000 mg/kg groups at least 10% less than the vehicle control group after week 25
Females: Dosed groups within 10% of the vehicle control group.

Clinical Findings
There were no clinical findings related to tetrabromobisphenol A administration.

Non-neoplasmic lesions:
Males: None
Females: Uterus: endometrium, hyperplasia, atypical (residual longitudinal review-2/50, 13/50, 11/50, 13/50)
Ovary: rete ovarii cyst (1/50, 0/49, 6/50, 6/49)

Neoplasmic lesions-
Males: Testis: interstitial cell, adenoma (0/50, 0/50, 1/50, 3/50) - Equivocal findings.
Females: Uterus: adenoma (original transverse review-0/50, 0/50, 3/50, 4/50); adenocarcinoma (original transverse review-3/50, 3/50, 8/50, 9/50;
original transverse and residual longitudinal reviews, combined-4/50, 10/50, 15/50, 16/50); malignant mixed Müllerian tumor (original transverse review- 0/50, 4/50, 0/50, 2/50); adenoma, adenocarcinoma, or malignant mixed Müllerian tumor (orignal transverse review-3/50, 7/50, 11/50, 13/50; original tran sverse and residual longitudinal reviews, combined-6/50, 11/50, 16/50, 19/50)- Clear evidence.


Mutation analyses
were performed comparing the mutation spectra of uterine adenocarcinomas from TBBPA-dosed Wistar Han rats and spontaneous
uterine adenocarcinomas from control Wistar Han rats from a variety of NTP studies. Results of these analyses indicated that the rate of Tp53 mutations was significantly increased in uterine adenocarcinomas from rats dosed with TBBPA (10/16, 63%) compared to spontaneous uterine adenocarcinomas from control Wistar Han rats (1/9, 11%).
The uterine adenocarcinomas from the TBBPA-dosed Wistar Han rats had not only higher incidences of Tp53 mutations, but they also harbored multiple mutations per tumor. Tp53 mutations in spontaneous uterine adenocarcinomas were observed in only one exon (exon 6), whereas two uterine adenocarcinomas from tetrabromobisphenol A-dosed animals harbored mutations in multiple exons, one animal with mutations in exons 6 and 7, and another had mutations in exons 6 and 8 (Table M3).

Relevance of carcinogenic effects / potential:

Findings and exposure relevancy:

In a two-year carcinogenicity study with TBBPA, groups of either male and female Wistar Han rats or male and female B6BC3F1 mice were administered TBBPA by gavage in corn oil at dose of 0, 250, 500 or 1,000 mg/kg bw on five days/week for 104 to 105 weeks (NTP, 2014).

At the terminal evaluation of the 2-year studies, the incidences of interstitial cell adenomaof the testis were slightly increased in male rats at 500 (1/50) and 1,000 mg/kg-bw/day (3/50) as compared to controls (0/50). In the female rats, there were significant positive trends in the incidences of adenoma and adenocarcinoma of the uterus, and the incidences of adenocarcinoma of uterus in the 500 and 1,000 mg/kg/day females were greater than that in the vehicle control group. When combined, the incidences of adenoma, adenocarcinoma, or malignant mixed Müllerian tumor of the uterus were significantly increased in the 500 and 1,000 mg/kg-bw/day groups;the incidences in control, low-, mid-, and high-dose groups were: 3/50, 7/50, 11/50, 13/50, respectively.
In addition to the traditional NTP histopathology review of transverse sections through the cervix of the uterus, an extended review of longitudinal sections of residual tissues was conducted to detect additional tumors/lesions. When the two evaluations were combined, there were significant positive trends in the incidences of adenocarcinoma and of adenoma, adenocarcinoma, or malignant mixed Müllerian tumor (combined), and the incidences were significantly increased in the 500 and 1,000 mg/kg-bw/day groups; the incidences in control, low-, mid-, and high-dose groups were: 6/50, 11/50, 16/50, 19/50,respectively (Table 1).
 
Uterine adenocarcinomas from Wistar Han rats chronically exposed to TBBPA had a significantly higher incidence of Tp53 mutations compared to those arising spontaneously in controls. The Tp53 tumor suppressor gene is responsible for cell cycle checkpoint maintenance, regulation of apoptosis, and genomic stability (Blagosklonny, 2000), and loss of this tumor suppressor function via mutation or dysregulation of the Tp53 signaling pathway is an important event in the pathogenesis of many different types of cancer in rodents and humans. In this study, the high rate of Tp53 mutations in uterine adenocarcinomas from TBBPA-dosed Wistar Han rats compared to spontaneous uterine adenocarcinomas suggests that the increased incidence of uterine adenocarcinomas in TBBPA -dosed animals may be driven at least in part through a Tp53-mediated mechanism.

The NTP concluded that under the conditions of these 2-year gavage studies, there was equivocal evidence of carcinogenic activityof TBBPA in male Wistar Han rats based on the slight increased incidence of testicular adenoma and a clear evidence of carcinogenicactivityof TBBPA in female Wistar Han rats based on increased incidences of uterine epithelial tumors (predominantly uterine adenocarcinoma).
However, studies on the mode of action have shown that high concentrations of TBBPA seem to compete for glucuronosyltransferases and/or sulfotransferases, therefore indirectly resulting in higher serum levels of oestrogen. This leads to subsequent promotion of pre-existing Tp53 mutations in the uterus through increased DNA synthesis and cell proliferation, which leads to uterine tumours and is consistent with the findings of the NTP study.
Therefore since TBBPA becomes an inhibitor of sulfotransferase activity in high and prolonged (chronic) doses only, it is considered that the NOAEL can be set at 250 mg/kg bw/day.


Exposure to TBBPA

Industry estimates indicate that the majority of TBBPA is used in reactive applications (i.e., TBBPA is reacted with other products to form new materials) where no human exposure is likely to occur. The primary reactive applications for TBBPA are in the production of epoxy resins for printed circuit boards and as a reactant to make other higher molecular weight flame retardants. TBBPA is also used as an additive in certain polymers commonly used for electrical and electronic products to ensure that they meet fire safety standards.

In the event that exposure does occur, TBBPA is rapidly metabolized and eliminated from the body. Peak levels of TBBPA and/or its metabolites are reached within 4 to 6 hours of administration and subsequently diminish rapidly. Studies have shown that TBBPA and its metabolites are eliminated within 72 hours of oral or intravenous administration.

Biomonitoring surveys conducted in Europe and North America have consistently found little if any detectable amounts of TBBPA. In fact, a recent survey of over 50,000 Canadians reported no measurable amounts of TBBPA in the blood samples of people tested

Based on their survey, Health Canada estimated that total exposure to TBBPA ranged from 10 to 195 nanograms per kg body weight per day (ng/kg/day). The lowest dose to which the animals were exposed in the NTP study was 250 milligrams/kg/day or about 1.25 million times higher than Health Canada’s highest estimate of human exposures.

Summary of carcinogenicity data of TBBPA (NTP 2014):

Species	Tumor site	Dose (mg/kgbw/day for 5 days/week for 103-104 weeks)
		0	250	500	1000
Male rat	Testis: interstitial cell adenoma	0/50	0/50	1/50	3/50
Female rat	Uterus: adenoma, adenocarcinoma, or malignant mixed Mullerian tumor (original transverse	6/50	11/50	16/50	19/50
Male mice	Liver: hepatoblastoma	2/50	11/50	8/50	n.a
Female mice	None	-	-	-	-

 

Conclusions:

The NTP concluded that under the conditions of these 2-year gavage studies, there was equivocal evidence of carcinogenic activityof TBBPA in male Wistar Han rats based on the slight increased incidence of testicular adenoma and a clear evidence of carcinogenicactivityof TBBPA in female Wistar Han rats based on increased incidences of uterine epithelial tumors (predominantly uterine adenocarcinoma).
However, studies on the mode of action have shown that high concentrations of TBBPA seem to compete for glucuronosyltransferases and/or sulfotransferases, therefore indirectly resulting in higher serum levels of oestrogen. This leads to subsequent promotion of pre-existing Tp53 mutations in the uterus through increased DNA synthesis and cell proliferation, which leads to uterine tumours and is consistent with the findings of the NTP study.
Therefore since TBBPA becomes an inhibitor of sulfotransferase activity in high and prolonged (chronic) doses only, it is considered that the NOAEL can be set at 250 mg/kg bw/day.

Executive summary:

2-YEAR STUDY IN WISTAR HAN RATS:

Groups of 60 male and 60 female rats were administered 0 or 1,000 mg tetrabromobisphenol A/kg body weight and 50 male and 50 female rats were administered 250 or 500 mg/kg, in corn oil by gavage, 5 days per week for up to 104 (males) or 105 (females) weeks. Mean body weights of 500 and 1,000 mg/kg males were at least 10% less than those of the vehicle control group after week 25. Ten vehicle control and ten 1,000 mg/kg rats of each sex were evaluated at 3 months to allow comparison to 3-month endpoints in the F344/NTac rats. Survival of dosed groups was similar to that of the vehicle control groups. At the 3-month interim evaluation, there were no treatment-related lesions in males or females, but thymus weights of 1,000 mg/kg rats were significantly less than those of the vehicle control groups, and there were increased liver weights in the 1,000 mg/kg groups similar to those seen in the 3-month F344/NTac rats.

In the original evaluation of the uterus, there were significant positive trends in the incidences of adenoma and adenocarcinoma, and the incidences of adenocarcinoma in the 500 and 1,000 mg/kg groups were greater than that in the vehicle control group. Malignant mixed Müllerian tumors were also found in treated rats. When combined, the incidences of adenoma, adenocarcinoma, or malignant mixed Müllerian tumor were significantly increased in the 500 and 1,000 mg/kg groups. Additional evaluations of residual uterine tissue were conducted and more neoplasms were identified. When the two evaluations were combined, there were significant positive trends in the incidences of adenocarcinoma and of adenoma, adenocarcinoma, or malignant mixed Müllerian tumor (combined), and the incidences were significantly increased in the 500 and 1,000 mg/kg groups. In the residual tissue evaluation, a new and potentially preneoplastic lesion of endometrial atypical hyperplasia was identified as statistically significant in all dosed groups.

Mutation analyses were performed comparing mutation spectra between uterine adenocarcinomas from tetrabromobisphenol A-dosed Wistar Han rats and spontaneous uterine adenocarcinomas from control Wistar Han rats from a variety of NTP studies.

The Uterine adenocarcinomas from Wistar Han rats chronically exposed to TBBPA had a significantly higher incidence ofTp53mutations compared to those arising spontaneously in controls. The Tp53 tumor suppressor gene is responsible for cell cycle checkpoint maintenance, regulation of apoptosis, and genomic stability (Blagosklonny, 2000), and loss of this tumor suppressor function via mutation or dysregulation of the Tp53 signaling pathway is an important event in the pathogenesis of many different types of cancer in rodents and humans. In this study, the high rate of Tp53 mutations in uterine adenocarcinomas from TBBPA-dosed Wistar Han rats compared to spontaneous uterine adenocarcinomas suggests that the increased incidence of uterine adenocarcinomas in TBBPA -dosed animals may be driven at least in part through a Tp53-mediated mechanism.

In the testis, incidences of interstitial cell adenoma were slightly increased in 500 and 1,000 mg/kg males. In the ovary, the incidences of rete ovarii cyst in 500 and 1,000 mg/kg females were significantly greater than that in the controls.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

chronic

Species:

other: rat and mouse

Quality of whole database:

Reliable. NTP study.

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the MoA studies submitted, high concentrations of TBBPA seem to compete for glucuronosyltransferases and/or sulfotransferases therefore indirectly resulting in higher serum levels of oestrogen. This leads to subsequent promotion of pre-existing Tp53 mutations in the uterus through increased DNA synthesis and cell proliferation, which leads to uterine tumours.

Uterine tumours induced by TBBPA in rats may be qualitatively applicable to humans via this MoA, despite the fact that Wistar Han strain rats resemble the Sprague-Dawley strain, which are known to exhibit elevated levels of oestrogens and therefore the observed uterine tumours in the carcinogenicity study may be solely due to a strain-related effect. 

In addition, it is unlikely that this MoA is quantitatively plausible for humans, especially taking into account absorption, distribution, metabolism, and elimination, as well as kinetics factors. In all in vivo studies, TBBPA was administered in corn oil at high doses by gavage to maximize GI tract absorption, whereas in humans, the major route of exposure to TBBPA particles/powders is inhalation – little will be absorbed by the lungs or reach the GI tract (through the respiratory tract) for absorption. The absence of a genotoxic MoA for uterine tumour induction by TBBPA and the low levels of human exposure clearly indicate a tumour risk in humans after environmental exposures to TBBPA is very low.

Since further data are required to determine whether this MoA is relevant for humans, in accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, it is proposed to classify TBBPA for carcinogenicity as Category 2 (H351; Suspected of causing cancer).

Additional information

The NTP studies on TBBPA in rodents were performed to evaluate the toxic and carcinogenic potential of this substance.

The 3-month dose range finding studies were conducted in F344/NTac rats and B6C3F1/N mice, and 2-year studies were conducted in Wistar Han rats and B6C3F1/N mice. A special 3-month interim evaluation using Wistar Han rats was conducted as part of the 2-year study in order to compare the results in this rat strain with those from the 3-month F344/NTac rat study. 

The results of the 3-month interim evaluation in the 2-year Wistar Han rat study (vehicle control and 1000 mg/kg bw/day groups) were similar to those in the 3-month F344/NTac rat study, where there was no treatment-related mortality, mean body weights in the treated groups were similar to those of the vehicle control groups, and there were no treatment related lesions. Liver weights at 1000 mg/kg bw/day were 4 to 7% greater than those of the vehicle controls. The high dose selected for the 2-year rat and mouse studies was 1000 mg/kg bw/day because, although there were some increases in liver weights and alterations in clinical pathology endpoints in rats and/or mice in the 3-month studies, these effects were not considered to be severe enough to compromise the conduct of a 2-year study.

 

2-YEAR STUDY IN RATS AND MICE

In two long-term carcinogenicity studies (2 years) with TBBPA (NTP 2014), Wistar Han rats (60 rats/sex/dose in the control and top-dose group; 50 rats/sex/dose for the other groups) and B6C3F1 mice (50 mice/sex/dose) were exposed over 2 years to TBBPA by gavage in corn oil (5 days/week) at dose levels of 0, 250, 500, 1000 mg/kg bw/day.

Dose selection was based on 3-month studies in F344/NT rats and B6C3F1 mice indicating that the MTD for the long term study was expected at the highest subchronic dose of 1000 mg/kg bw/day. In male rats, mean body weights at 500 and 1000 mg/kg bw/day were at least 10% less than those of the controls after week 25, such that the MTD had been reached at the highest dose group. A 3-month bridging study with Wistar Han rats at 0 and 1000 mg/kg bw/day (10/sex/dose) indicated that both rat strains have about the same sensitivity to TBBPA. In mice, survival of the 1000 mg/kg bw/day males and females was significantly less than that of controls and a meaningful analysis of tumour incidences was only possible for males dosed with 250 and 500 mg/kg bw/day.

No treatment related tumours were induced in female mice.

Regarding liver tumours in male mice, hepatocellular adenomas, hepatocellular carcinomas and hepatoblastomas form a biological and morphological continuum of increasing malignancy and hepatoblastomas are uncommon spontaneous neoplasms. Hepatoblastomas at 250 and 500 mg/kg bw/day were significantly increased as compared to controls and exceeded the historical control range. But for both dose groups combined incidence of hepatocellular carcinomas and hepatoblastomas was within the historical control range, a significant increase compared to concurrent controls only occurred for the 250 mg/kg bw/day group and the trend test was not positive. Similarly, for the combined incidences of adenomas, carcinomas and blastomas there was no indication for a substance induced increase and the trend test or the group wise comparisons were not significant. Therefore the conclusion for liver tumours in male mice was some evidence of carcinogenic activity. There was supportive evidence for liver tumour formation by increased incidences of liver foci and multiple hepatocellular adenomas. As judged by the day when the first incidence was noted, progression to malignancy or reduced tumour latency were not affected.

Regarding tumours of the large intestine (cecum or colon) in male mice, the combined incidences of carcinomas or adenomas showed a significant trend test and at 500 mg/kg bw/day exceeded the historical control ranges for corn oil gavage studies and all routes of administration (each one carcinoma in cecum and colon, one adenoma in colon). This finding was considered as equivocal evidence as the tumour incidence was very low, similar effects were not seen in females and the pairwise comparison was not significant. As judged by the day when the first incidence was noted, progression to malignancy or reduced tumour latency were not affected.

Haemangiosarcomas occurred with a positive trend and the incidence was significantly increased at 500 mg/kg bw/day by pair wise comparison. Haemangiomas or haemangiosarcomas also showed a positive trend. These neoplasms were found in a wide variety of different organs. The incidences were within the historical control range for corn oil gavage studies and for all routes of administration, while the control incidence was at the low end of historical controls. This finding was considered as equivocal evidence. As judged by the day when the first incidence was noted, tumour latency was not reduced.

The incidences of testicular interstitial cell adenomas in male rats were slightly, but not significantly increased at 500 and 1000 mg/kg bw/day and exceeded at 1000 mg/kg bw/day the historical control incidence for all routes that was obtained only from 150 animals (3 studies with an incidence of 4/150 = 2.7%). The trend test was positive. But as the incidence in the concurrent control group was at the low end of the (very limited) historical control data and the incidence of the high dose group was only slightly in excess, this tumour type was considered to be of equivocal evidence. There was no indication for progression to malignancy (no interstitial cell carcinomas observed) and the benign tumours were first observed at termination of study.

In the original transverse sections of the uterus neoplasms were observed in all groups including controls. Therefore additional longitudinal sections were evaluated and only the combined incidences of both histopathological evaluations will be discussed here. Adenomas, adenocarcinomas and malignant mixed Müllerian tumours (MMMT) were observed. For adenocarcinomas as well as for the combined incidences of adenomas, adenocarcinomas and MMMT the trend tests were positive and by pairwise comparisons the incidences at 500 and 1000 mg/kg bw/day were significantly increased. Some adenocarcinomas invaded distant organs. MMMTs are uncommon tumours and are composed of epithelial and mesenchymal cells; all tumours were very large and infiltrative and the 4 tumours at 250 mg/kg bw/day had extensive metastases. MMMTs were combined with adenocarcinomas for statistical analysis because based on human literature the epithelial component of MMMTs is the “driving force” for this tumour type. Endometrial atypical hyperplasia as a potential preneoplastic lesion was significantly increased in all dose groups. As judged by the day when the first incidence was noted, progression to malignancy or reduced tumour latency may have been affected by TBBPA treatment for adenocarcinomas. The occurrence of uterine tumours (predominately adenocarcinomas) in treated rats was considered as clear evidence for carcinogenicity basically because the tests for trend and pairwise comparisons were significant. In addition, according to NTP the incidences exceeded the (limited) historical control ranges. But this comparison must be viewed very critically because of the limited database for this strain of rats, the different modes of application and as it only refers to the standard transverse sections.

NTP (2014) concluded that under the conditions of these 2-year studies, there was equivocal evidence of carcinogenic activity of TBBPA in male Wistar Han rats based on the occurrence of testicular adenoma. There was clear evidence of carcinogenic activity TBBPA in female Wistar Han rats based on increased incidences of uterine epithelial tumours (pre-dominantly uterine adenocarcinoma). There was some evidence of carcinogenic activity of TBBPA in male B6C3F1/N mice based on increased incidences of hepatoblastoma. The increased incidences of large intestine neoplasms and haemangioma-sarcoma (all organs) may have been related to chemical administration. There was no evidence of carcinogenic activity of TBBPA in female B6C3F1/N mice administered 250 or 500 mg/kg bw/day. TBBPA administration resulted in increased incidences of non-neoplastic lesions of the uterus and ovary in female rats, the liver and kidney in male mice, and the forestomach in male and female mice.

The results are also presented in Table 1.

 Table 1: Summary of carcinogenicity data of TBBPA (NTP 2014)

Species	Tumour site	Dose (mg/kg bw/day for 5 days/week for 103-104 weeks)
		0	250	500	1000
Male rat	Testis: interstitial cell adenoma	0/50	0/50	1/50	3/50
Female rat	Uterus: adenoma, adenocarcinoma, or malignant mixed Mullerian tumor (original transverse	6/50	11/50	16/50	19/50
Male mice	Liver: hepatoblastoma	2/50	11/50	8/50	n.a
Female mice	None	-	-	-	-

 

Mode of Action (MoA) possibilities to explain the carcinogenicity findings on TBBPA were discussed in the NTP study as follows: Uterine tumours have been attributed to both oestrogenic and non-oestrogenic effects. Several hypotheses with regard to the origin of uterine tumours in the current study have been discussed: it may be related a possible ability of TBBPA-derived metabolites to disrupt hormone signalling or the possible potential of TBBPA to cause oxidative damage. Conjugation is the major biotransformation pathway for TBBPA in rodents and this pathway is shared by oestrogen and its potentially genotoxic catechol metabolite. Competition for glucuronosyltransferases and/or sulfotransferases by TBBPA could result in higher circulating levels of oestrogen and increased formation of oestrogen-derived reactive species, especially following exposure to high concentrations of the chemical. Glucuronidases in the uterus or other organs may release free TBBPA from its conjugated form, thus increasing the possible potential for free radical formation at target sites. Either process may contribute to tumorigenesis in the uterus. It has been postulated that TBBPA might disrupt endocrine signalling through direct interaction with endocrine receptors or indirectly, through binding to oestradiol-sulfotransferase (ES), thereby preventing sulfation of oestradiol and its subsequent elimination. TBBPA has a low IC50 (12 to 33 nM) sulfotransferase enzyme (SULT1E1) inhibition level. Crystallography studies show that TBBPA can bind to SULT1E1 and that the phenolic ring is critical for stable binding.

 

Proposed MoA for the occurrence of uterine tumours after TBBPA administration in rats

A number of MoA can be delineated from the MoA of chemicals which increase uterine tumour incidences in rats (i.e., genotoxic, direct oestrogen receptor binding; induction of cytochrome P450 1A enzymes). However, available data do not support these MoA for uterine tumorigenesis by TBBPA and there are no data on the dopamine receptor agonist MoA.

There is evidence on the two key events of the hypothesized MoA. Metabolism studies have shown that conjugations (glucuronidation and sulfation) are the major biotransformation pathways for excretion of TBBPA in rats. These pathways are shared by oestrogen and its catechol metabolite. A significant increase in the incidence of mutations of the Tp53 tumour suppression gene was noted in uterine adenocarcinomas of TBBPA treated rats (NTP, 2014). Competition for glucuronosyl-transferases and/or sulfotransferases by high concentrations of TBBPA indirectly resulting in higher serum levels of oestrogen which leads to subsequent promotion of pre-existing Tp53 mutations in the uterus through increased DNA synthesis and cell proliferation is the most likely MoA for the induction of uterine tumours by TBBPA in rats (a diagram of the proposed mechanism/ MOA of uterus carcinogenesis in rats is attached).

Data on associated events such as DNA synthesis and cell proliferation in the uterus of TBBPA-treated animals are unavailable and there are no data on dose-response and temporal relationship between the serum levels of oestrogen and uterine tumour formation. 

A summary document is attached to this endpoint that was previously prepared to assess the potential requirement for classification of TBBPA as a carcinogen. This is entitled 'TBBA classification GE aug 19 final.doc'. The report includes a thorough summary of the different types of tumours observed and the potential modes of action that may cause them. On the basis of the available data, the author concluded that overall, Category 2 classification for carcinogenicity (CLP) was appropriate to reflect the findings of the NTP (2014) bioassay. It was also considered that the mechanisms responsible for the tumours leading to classification may not be relevant to humans; it was therefore determined that further studies are necessary in support of any of the potential modes of action.

There is now new evidence available (see attached draft of the Borghoff et al. 2015 study) to address the mode of action (MoA). In order to further clarify the MoA, Borghoff et al. evaluated the effect of dose and repeated administration of TBBPA on the level of TBBPA, TBBPA-glucuronide (GA) and TBBPA-sulfate (S) conjugates in plasma, liver and uterus of female Wistar Han rats (6 rats/group). The animals were administered TBBPA at dose levels of 50, 100, 250, 500 and 1000 mg/kg bw/day for 28 consecutive days, using TBBPA sulfation as a surrogate for evaluating the potential for oestradiol sulfation to be limited at high dose levels of TBBPA. Blood samples were collected at 4 and 8 hours post dosing on study day 7, 14, and 28 while liver and uterus were collected at the same time points following 28 days of dosing. Tissue samples were analysed for TBBPA, TBBPA-GA and TBBPA-S by LC-MS/MS.

A dose-related increase in the concentration of all three analytes occurred in all tissues at both 4 and 8 hours post dose. The plasma concentration of TBBPA-GA and TBBPA-S was higher in animals dosed for 28 days compared to those dosed for 7 or 14 days showing an increase in systemic circulation of these conjugates with repeated administration. The balance of these conjugates was also different in tissues with TBBPA-S > TBBPA-GA at high doses in the liver and TBBPA-GA > TBBPA-S in both plasma and uterus. In all three tissues the ratio of TBBPA-S/TBBPA-GA showed a decreasing trend with dose, suggesting that at high TBBPA dose levels sulfation of TBBPA becomes limited. This effect was most apparent in the liver and plasma at 28 days of administration. 

Together these data show that administration of high doses of TBBPA associated with the induction of uterine tumours results in a disruption in the balance of conjugates reflected by a decrease in the TBBPA-S/TBBPA-GA ratio. A decrease in TBBPA sulfation in vivo supports in vitro data that TBBPA is an inhibitor of ES activity, thus further supporting that the proposed MoA occurs under conditions of high dose, chronic exposure in Wistar Han rats. Furthermore, these findings are consistent with the data reported in the NTP study, supporting the role of kinetics in the underlying MoA. Data from the MoA study demonstrate that alterations in conjugation pathways occur at repeated dose levels of 250 mg/kg bw/day and greater. This observation is consistent with alterations in the pre-neoplastic lesion identified as atypical endometrial hyperplasia that was observed in all dose groups at a similar incidence (e.g., 4, 26, 22, and 26% at 0, 250, 500, and 1000 mg/kg bw/day, respectively). A statistically significant increase in uterine tumours (combined) were only observed in the 500 and 1000 mg/kg bw/day groups, highlighting the combined influence of both high dose and prolonged (chronic) exposure associated with the development of this carcinogenic response. In conclusion, the study by Borghoff et al. demonstrates that metabolic capacities are altered in female Wistar Han rats following repeated, high dose administration of TBBPA – in pathways in which key enzyme pathways, common to both TBBPA and oestradiol, become saturated at high doses (i.e., perturbation of homeostatic conditions). 

 

Uterine tumours induced by TBBPA in rats may be qualitatively applicable to humans via this MoA. However, Wistar Han strain rats resemble the Sprague-Dawley strain, which are known to contain elevated levels of oestrogens (Lai et al., 2015) and the uterine tumours detected in aged Wistar Han rats treated with high-doses of TBBPA may be solely due to a strain-related effect. Uterine tumours can also be induced by dopamine receptor agonists in the rat through another indirect oestrogen pathway. This MoA is irrelevant to humans since prolactin is the luteotrophic hormone in rodents but not primates.

Even if uterine tumours induced by TBBPA in rats is qualitatively applicable to humans via this MoA, it is unlikely that this MoA is quantitatively plausible for humans, especially taking into account absorption, distribution, metabolism, and elimination, as well as kinetics factors. In the two year NTP bioassays, as well as in the Borghoff et al. study, TBBPA was administered in corn oil at high-doses by gavage to maximize GI tract absorption, whereas in humans, the major route of exposure to TBBPA particles/powders is inhalation – little will be absorbed by the lungs or reach the GI tract (through the respiratory tract) for absorption. The absence of a genotoxic MoA for uterine tumour induction by TBBPA and the low levels of human exposure clearly indicate a tumour risk in humans after environmental exposures to TBBPA is very low.

 

Exposure to TBBPA

Industry estimates indicate that the majority of TBBPA is used in reactive applications (i.e., TBBPA is reacted with other products to form new materials) where no human exposure is likely to occur. The primary reactive applications for TBBPA are in the production of epoxy resins for printed circuit boards and as a reactant to make other higher molecular weight flame retardants. TBBPA is also used as an additive in certain polymers commonly used for electrical and electronic products to ensure that they meet fire safety standards.

In the event that exposure does occur, TBBPA is rapidly metabolized and eliminated from the body. Peak levels of TBBPA and/or its metabolites are reached within 4 to 6 hours of administration and subsequently diminish rapidly. Studies have shown that TBBPA and its metabolites are eliminated within 72 hours of oral or intravenous administration.

Justification for selection of carcinogenicity via oral route endpoint:
There was equivocal evidence of carcinogenic activity of TBBPA in male Wistar Han rats based on the occurrence of testicular adenoma. The increased incidences of large intestine neoplasms and haemangio-sarcoma (all organs) may have been related to chemical administration. For female rats, there was clear evidence (uterine tumours) of carcinogenic activity.

There was no evidence of carcinogenic activity of TBBPA in female B6C3F1/N mice administered 250 or 500 mg/kg, but for male mice there was some evidence (liver tumours), or equivocal evidence (tumours of the large intestine; haemangiomas and haemangiosarcomas).

Administration TBBPA resulted in increased incidences of nonneoplastic lesions of the uterus and ovary in female rats, the liver and kidney in male mice, and the forestomach in male and female mice.    

Carcinogenicity: via oral route (target organ): urogenital: uterus