Odontology (2001) 89:12–20
© The Society of The Nippon Dental University 2001
ORIGINAL ARTICLE
James Huff
Carcinogenicity of bisphenol-A in Fischer rats and B6C3F1 mice
Received: January 23, 2001 / Accepted: June 1, 2001
Abstract Bisphenol-A (BP-A; 4,4¢-isopropylidenediphenol) is a monomer of plastics commonly used in various consumer products, and is used as an intermediate in the manufacture of epoxy, polycarbonate, and polyester-styrene resins. A National Toxicology Program carcinogenesis bioassay of BP-A (>98% pure) was conducted by feeding diets containing 0, 1000, or 2000 ppm BP-A to groups of 50 male and 50 female Fischer (F)344 rats; 0, 1000, or 5000 ppm to groups of 50 male B6C3F1 mice; and 0, 5000, or 10 000 ppm to groups of 50 female B6C3F1 mice for 103 weeks. The mean body weights of the low- and high-dose rats and of female mice and high-dose male mice were lower than those of the controls throughout much of the study. Lower body weight gains in rats were likely caused by reduced food consumption. Survivals were comparable among groups. Regarding neoplasia, leukemias occurred at increased incidences in BP-A-dosed rats of both sexes: male, 13/50 controls vs 12/50 low-dose and 23/50 high-dose (P < 0.03); in females, the respective findings were 7/50, 13/50, and 12/50. Interstitial-cell tumors of the testes were increased in BP-A-dosed male rats: 35/49 controls vs 48/50 (P < 0.01) and 46/49 (P < 0.01); and an increasing trend was observed for mammary gland fibroadenomas in male rats (P < 0.05, 0/50 controls vs 0/50 and 4/50). In male mice, lymphomas/leukemias were increased: 2/49 controls vs 9/50 (P < 0.05) and 5/50. Multinucleated giant hepatocytes were observed in male mice (1/49 controls vs 41/49 and 41/50), whereas there was no increase of liver tumors. In their BPA bioassay report, the National Toxicology Program concluded that there was no convincing evidence that BP-A was carcinogenic for rats or mice. However, the marginal increases in leukemias in male and female rats, along with increases in the combined incidence of lymphomas and leukemias in male mice, suggest that BP-A may be associated with increased cancers of the hematopoietic system. J. Huff (*) National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA Tel. +1-919-541-3780; Fax +1-919-541-5002 e-mail:
[email protected]
Increases in interstitial-cell tumors of the testes in rats were also evidence of carcinogenesis, as was the unusual occurrence of mammary gland fibroadenomas in male rats. Key words Bisphenol-A · BP-A · Carcinogenesis bioassay · Estrogen disrupter · Hormonomimetic · Leukemia · Mammary gland fibroadenoma · National Toxicology Program · Testes tumor · 2-Year study · Xenoestrogen
Preamble Chemical carcinogenesis bioassays are carried out to identify potential carcinogenic effects for humans.1,2 Carcinogenesis results in rodents (mainly in rats and mice) have been shown to be consistent and reliable indicators and predictors of human cancer risks.3–7 Hence, for chemicals discovered to be carcinogenic to laboratory animals, prudent public health policy suggests that reductions in human exposures to identified carcinogens would prevent or reduce cancers in humans exposed to these chemical carcinogens.8–10 This was the rationale when the National Cancer Institute (NCI) in the 1970s initiated their bioassay program11 and selected bisphenol-A [BP-A] as one of many chemicals for studying the potential carcinogenicity of these relatively common chemicals. In 1978, the National Toxicology Program (NTP) was formed, and continued the NCI bioassay activities.12 In October 1980, the NTP presented their evaluation of the bioassay findings in a public meeting, and the NTP Technical Report was issued in 1982.13 Because these findings have never been published in the journal literature, because no other long-term studies on this chemical have been reported, and because of the heightened interest in endocrine disrupters such as BP-A, the bioassay results form the basis of this paper.
Introduction Bisphenol-A (BP-A; Chemical Abstracts Service [CAS] Registry No: 80-05-7; 4,4¢-isopropylidenediphenol; 2,2-bis[4-
13
hydroxyphenyl] propane; C15H16O2; C 79%, H 7%, O 14%) is a monomer of plastics commonly used in various consumer products, and is used as an intermediate in the manufacture of epoxy, polycarbonate, and corrosionresistant unsaturated polyester-styrene resins.13 These resins may be found in reinforced pipe, adhesives, flooring, watermain filters, artificial teeth, nail polish, food-packing materials, and interior coatings for cans and drums.14 BP-A has also been used as a fungicide. As BP-A is used to produce polymers for food-contact applications (inner coatings for food cans by BP-A diglycidyl ether, and baby bottles), there is potential for oral exposure of humans to trace amounts via the diet. As biomaterials in health applications, such as BP-A-based filling materials in preventive and restorative dentistry, there is added opportunity for exposure.15 Additionally, BP-A has been found in recycled and virgin paper for household use16 and in hazardous waste landfill leachates.17 Thus, BP-A finds common and widespread consumer uses in various plastics and paper, and leads to occupational, environmental, and consumer exposures. A major concern is what biological effects this xenoestrogen (endocrine disrupter) might exert on wildlife and human populations exposed to this hormonomimetic chemical.18,19 Manufactured from phenol and acetone, BP-A was patented in 1949. As background information, and for completeness, a few words follow on each of the BP-A starting chemicals. Whether and at what percentages either of these chemicals is found in BP-A products is not clear.
Purpose In this article, results of 2-year NTP carcinogenicity studies of dietary BP-A in Fischer rats and B6C3F1 mice are presented.13,14 No other carcinogenicity bioassays or epidemiological studies on BP-A were located in the literature.
Materials and methods Two-year carcinogenesis bioassays were designed such that groups of 50 male and 50 female Fischer rats received diets containing 0, 1000, or 2000 ppm BP-A; groups of 50 male B6C3F1 mice were given diets having 0, 1000, or 5000 ppm BP-A, and groups of 50 female B6C3F1 mice received feed holding 0, 5000, or 10 000 ppm BP-A. These exposure concentrations were chosen after shorter-term (14- and 90-day) toxicology studies to establish appropriate and optimal dietary levels of BP-A for the longer-term studies.23,24 At the end of the 2-year studies, all animals were killed and necropsied. Histopathology was done on 35–40 tissues/organs per animal. These are relatively standard designs for long-term chemical carcinogenesis bioassays.2,12,23,25–27
Results Survival and body weight
Phenol (CAS no: 108-95-2; hydroxybenzene; carbolic acid). Phenol is a basic feedstock for the production of phenolic resins, BP-A, caprolactam, chlorophenols, and several alkylphenols and xylenols.20 Phenol is also used in disinfectants and antiseptics.The carcinogenicity of phenol has been studied in rats and mice.14,20,21 Interestingly, increases in leukemia, pheochromocytomas of the adrenal glands, and C-cell carcinomas of the thyroid glands were observed in male rats treated with the lower dose of phenol but not in higher-dose male rats, or in female rats, or in mice. Because these effects were not dose-related, the National Cancer Institute opined that these increases did not constitute real carcinogenic effects.21 The International Agency for Research on Cancer considered the available studies inadequate for evaluation.20 However, phenol has been shown to be a promoter of skin carcinogenesis.22 Also, BP-A has a phenolic odor. Acetone (2-propanone; CAS no: 67-64-1). Acetone, used to make plastic, fibers, drugs, and other chemicals, is a highly flammable and volatile general solvent with many and extensive uses. Acetone is a manufactured chemical that is also found in the environment. Acetone occurs naturally in plants, trees, volcanic gases, forest fires, and as a product of body fat catabolism, and is present in vehicle exhaust, tobacco smoke, and landfill sites. Industrial processes contribute more acetone to the environment than natural processes. Toxicologically, other than for skin carcinogenesis, for which acetone is uniformly negative, acetone has not been studied adequately for potential carcinogenicity.
Survivals at 2 years were generally high, and were comparable among groups (male rats: 46% controls vs 60% lowdose and 54% high-dose; female rats: 70% vs 70% and 74%; male mice: 86% vs 74% and 76%; female mice: 78% vs 77% and 85%) (Figs. 1 and 2). Mean body weights of dosed rats of either sex and of low- and top-dose female mice and topdose male mice were lower than those of the controls throughout much of the study (Figs. 3 and 4). Because the food consumption of BP-A female rats was only 70% to 80% that of the controls throughout most of this study, reduced body weight gain was likely caused by reduced food consumption. However, food consumption by dosed male rats was 90% that of controls. Food consumption among all groups of mice was similar. Why only one group out of four had decreased food consumption is not clear.
Neoplasia Leukemias occurred at increased incidences in rats of both sexes. In male rats, the dose-related trend (13/50 controls vs 12/50 and 23/50) and the high-dose effect were both statistically significant (P < 0.05) (Table 1). Leukemias were, likewise, increased in both dosed groups of female rats, but were not statistically significant (7/50 controls vs 13/50 and 12/50). Interstitial-cell tumors of the testes occurred at statistically significant (P < 0.01), dose-related incidences in lowand top-dose male rats (35/49 controls vs 48/50 and 46/49) (Table 1). Conversely, diffuse testicular atrophy was lower
14 Fig. 1a,b. Survival curves for rats fed diets containing bisphenol A. a Male rats; b female rats. Squares, Controls; circles, low dose; triangles, high dose
a
b
in dosed rats than in controls (controls 25/49 vs 5/50 lowdose, 12/49 high-dose). Also, for male rats, the trend statistic was positive for fibroadenomas of the mammary glands (P < 0.05: 0/50 controls vs 0/50 and 4/50), an unusual observation, as these are rarely occurring tumors in male rats. No increase in these fibroadenomas was seen for female rats: 8/50 vs 8/50 and 5/50; there was one adenoma of the mammary gland found in a low-dose female. In rats, moreover, there were decreases in tumors observed, or negative trends in tumor incidence, for adrenal
glands at both doses in male (medulla: 15/48 controls vs 7/50 low-dose and 7/47 high-dose) and female (cortical: 12/50 vs 5/50 and 4/50) rats. As is obvious and interesting, the decrease in males was for pheochromocytomas, whereas in females the decrease was for adenomas. Further, males rats showed a numerical increase in cortical adenomas/ carcinomas: 1/48 controls vs 3/50 and 5/47. In female rats, lower incidences of endometrial stromal polyps were seen in the BP-A-exposed groups: controls 15/48 vs 10/50 and 6/50.
15 Fig. 2a,b. Survival curves for mice fed diets containing bisphenol A. a Male mice; b female mice. Symbols, as in Fig. 1
a
b
In male mice, there was a marginal increase of leukemias or lymphomas combined (2/49 controls vs 9/50 and 5/50), and the increase in the low-dose group was statistically significant (P < 0.05) (Table 2). In male mice chromophobe carcinomas of the pituitary gland were observed only in the top-dose group (0/37 controls vs 0/36 low dose and 3/42 top dose). A compound-related increased incidence of multinucleated giant hepatocytes was observed in male mice (1/49 controls vs 41/49 and 41/50), but there was no increase of liver tumors.
Discussion BP-A was selected and tested for potential carcinogenicity, both because of consumer and occupational exposures, and because no other studies had been done. BP-A has varied uses, including use in dentistry, especially as the diepoxy resin BP-A diglycidyl ether (BADGE) and BP-A diglycidylether methacrylate (Bis-GMA). The BP-A-based filling materials have gained use in preventive and restora-
16 Fig. 3a,b. Growth curves for rats fed diets containing bisphenol A. a Male rats; b female rats. Symbols, as in Fig. 1
a
b
tive dentistry, which includes extensive manipulation and in-situ polymerization.15 These various polymers of BP-A have been used since the 1960s in restorative materials, including tooth-fissure sealants. Typically, resin-based composites consist of an organic resin matrix and an inorganic filler, whereas sealants – used to protect children’s teeth – are composites without a filler. Importantly, BP-A has been shown to leach from a variety of resin-based and plastic products, including dental sealants and food and beverage containers, in concentrations that are sufficient to induce
cell proliferation in vitro and biologic effects in vivo.15,28 Regarding estrogenic activity, BP-A induced estrogenic changes in the uterus of the CD-1 mouse28 and uterotrophy in the immature uterotrophic assay in Crj:CD (SD) rats.29 Further, BP-A inhibited testicular function30 and induced testicular toxicity in rats at dietary concentrations as low as 0.25%.31 To estimate carcinogenic potential, BP-A was evaluated for long-term carcinogenicity in one study in rats and in one study in mice.13,14 No other carcinogenicity bioassays were
17 Fig. 4a,b. Growth curves for mice fed diets containing bisphenol A. a Male mice; b female mice. Symbols, as in Fig. 1
a
b
located in the literature. In my opinion, long-term carcinogenesis bioassays are the most universal, reliable, valued, and predictive means of identifying potential carcinogenic hazards of various agents to humans.1–10,26,32–35 While not perfect, bioassays are the scientific cornerstone of carcinogen identification and the eventual foundation underlying primary prevention of cancer risks to humans.9,10,35 In the NTP BP-A studies, slight increases in hematopoietic system cancers – leukemias/lymphomas – were observed for both rats and mice. These marginally signifi-
cant increases in leukemias in male rats, along with borderline increases in leukemias in female rats and in the combined incidence of lymphomas and leukemias in male mice (Tables 1 and 2), suggest that BP-A may be associated with increased cancers of the hematopoietic system. This finding may be somewhat supported by a marginal increase in leukemias observed in male rats exposed to phenol,14,21 a starting material for making BP-A. However, this is likely moot, because there is little evidence that phenol or acetone are found in BP-A products or metabolism. Less than 1%
18 Table 1. Neoplastic findings in Fischer 344 rats given bisphenol-A in the diet for 2 yars Organ: Neoplasm
Male rats Controls
Female rats 1000 ppm
2000 ppm
Controls
1000 ppm
2000 ppm
12/50
Incidence rates (animals with tumors/total animals in group) Leukemia Adrenal glands Cortical adenoma or carcinoma Pheochromocytoma Mammary glands: fibroadenoma Testes: interstitial cell Uterus: endometrial stromal polyps
13/50*
12/50
23/50**
7/50
13/50
1/48 15/48# 0/50* 35/49+
3/50 7/50# 0/50 48/50++
5/47 7/47## 4/50 46/49++
12/50# 2/50 8/50
5/50 4/50 8/50
4/50## 2/50 5/50
15/48#
10/50
6/50##
* Increasing trend, P < 0.05; ** increase compared with controls, P < 0.05; # decreasing trend, P < 0.05; ## decrease compared with controls; + increasing trend, P < 0.01; ++ increase compared with controls, P < 0.01
Table 2. Neoplastic findings in B6C3F1 mice given bisphenol-A in the diet for 2 years Organ: Neoplasm
Male mice Controls
Female mice 1000 ppm
5000 ppm
Controls
5000 ppm
10 000 ppm
Incidence rates (animals with tumors/total animals in group) Lymphoma Pituitary gland: chromophobe adenoma or carcinoma
2/49 0/37*
8/50*# 0/36
3/50 3/42
13/50 2/44
10/48 0/40
8/48 1/40
* Increasing trend, P < 0.05; ** increase compared with controls, P < 0.05; # another animal had leukemia (9/50)
phenol was found in a commercial product used for metabolism studies.36 BP-A was also associated with a dose-related increase in interstitial-cell tumors of the testes in male rats (Table 1). These testicular tumors are considered comparatively common in control Fischer rats, and often may not be considered to be clearly related to chemical exposures. However, at both BP-A exposure levels, the increases were indeed statistically increased (71% controls vs 96%; P < 0.01 and 94%; P < 0.01). Also, male rats exposed to low and high doses of phenol showed marginal increases in this tumor (42/48 controls vs 49/50; P = 0.05 and 47/50). Interestingly, Haseman and Elwell37 identified a number of chemicals tested by the NTP, including BP-A and phenol, that showed significant (P < 0.05) increases in interstitial-cell tumors of the testes in male rats (35/194; 18%). These increases are typically discounted because of the high background rates of these tumors, and because, in most cases, the control incidence rate was thought to be lower than typical. These authors concluded that certain of these increases may be false-negatives, and thus related to chemical exposure. Perhaps this BP-A-tumor effect could easily be interpreted as a more likely positive response. Overall, only two chemicals have been considered carcinogenic for the testis in the near 500 reported by the NTP: ethylbenzene and isoprene, both in inhalation studies. Also likely hormonally related, slight increases in fibroadenomas of the mammary gland were observed in male rats in the top exposure group: 0/50 controls vs 0/50 low dose and 4/50 top dose; importantly, male rats are considered particularly nonresponsive to chemicals that cause mammary tumors. Only six chemicals out of 500 tested by
the NTP have caused tumors of the mammary gland in male rats, whereas 43 of these 500 have caused tumors of the mammary glands overall, mainly in female rats and less so in female mice.38,39 Further, given that BP-A is a hormonomimetic, or endocrine mimic/disrupter, one would certainly expect to see carcinogenic effects in organs under hormone control. As with other estrogens, BP-A has the capacity to induce proliferative and stimulatory changes in estrogen target tissues.28 Thus, perhaps what is most surprising in these carcinogenesis studies is that the testis and mammary gland in male rats were the only hormonally “controlled” organs that showed any significant carcinogenic effect in rats and mice exposed for 2 years to BP-A. Interestingly, there were decreases in tumors observed, or negative trends in tumor incidence, for two other hormonal organs: for adrenal glands at both doses in male (medulla: 31% controls vs 14% low-dose and 15% highdose) and female (cortical: 24% vs 10% and 8%) rats, and for uterus in female rats (31% vs 20% and 12%). Decreases in tumor incidence in hormonally sensitive organs are often affected by decreases in body weights,40,41 which were somewhat common in these studies. However, there is no direct evidence that reduced body weights affect these two organs. Reductions in body weights were largely ascribed to decreased food consumption, perhaps because of unpalatability or disagreeable taste. BP-A comes in crystals or flakes and has a mild phenolic odor, which would, likely, influence taste. Regarding distribution and metabolism, BP-A was detected in the blood, brain, and preferentially in brown adipose tissues of BP-A-treated rats.42 BP-A glucuronide
19
appears to be the major metabolite in rats, and as Elsby and colleagues43 state: because human liver microsomes apparently do not glucuronidate BP-A as extensively as rat liver microsomes, estrogen target tissues in humans may be subject to greater exposure to BP-A. Further, BP-A glucuronide does not appear to have estrogenic activity, and likely represents a detoxication metabolite of BP-A.44 In male rats, BP-A is excreted via urine mainly as glucuronide (about 27%; 1% as free BP-A), whereas 56% of 14C was found in feces (35% unchanged BP-A, 35% as hydroxylated BP-A, and 30% as a conjugate).36 Mechanistically, using the Syrian hamster embryo (SHE) cell model to simultaneously assess in-vitro carcinogenicity and related activities, Tsutsui et al.45 examined the ability of BP-A to induce cellular transformation and genetic effects. Morphological transformation of SHE cells was induced by treatment with BP-A at 50 to 200 mM. Over this same dose range, BP-A failed to induce gene mutations at the Na+/K+ ATPase locus or the hprt locus. Whereas no chromosomal aberrations were detected, BP-A induced numerical chromosomal changes in the near diploid range, and DNA adduct formation in a dose-dependent fashion, as measured using 32P-postlabeling. These results indicate that BP-A has cell-transforming and genotoxic activities in cultured mammalian cells, and potential carcinogenic activity. Further, Tsutsui et al.46 posit that BP-A induction of aneuploidy may be a causal mechanism of the cellular transforming and carcinogenic activity. With this genotoxic activity one would have anticipated a clearer carcinogenic response in the long-term studies. Regarding a possible phenol contribution, benzene and its principal metabolites – catechol, hydroquinone, and phenol – were also examined for their ability to induce SHE cell transformation and genotoxic effects.47 Each of the four compounds induced morphological transformation. All four compounds induced gene mutations at two loci in SHE cells, with both ouabain-resistant and 6-thioguanineresistant mutant frequencies being increased. The three metabolites, but not benzene, induced chromosomal aberrations, sister chromatid exchanges, and unscheduled DNA synthesis. Benzene and catechol significantly induced aneuploidy. Whether these findings on phenol are relevant to the marginal carcinogenic effects of BP-A need further thought, especially in regard to whether any residual phenol is found in current commercial BP-A and products. In summary, marginal increases in leukemias in male and female rats, along with an increase in the combined incidence of lymphomas and leukemias in male mice, suggests that exposure to BP-A may be associated with cancers of the hematopoietic system. Increases in interstitial-cell tumors of the testes in rats were also evidence of carcinogenesis, as was the unusual occurrence of mammary gland fibroadenomas in male rats. Conversely, in their BP-A bioassay report, the NTP concluded that there was no convincing evidence that BP-A was carcinogenic for rats or mice.13 Given the small amounts of BP-A released from dental appliances, and the weak carcinogenicity displayed by BP-A in rodents, one would not expect any significant increased risk of cancer. However, one should not ignore
these bioassay observations or the cell-transforming and genetic activity of BP-A, especially in occupations dealing with making or using BP-A. Acknowledgments I thank John Bucher and Joseph Haseman for reviewing this paper and for offering useful comments. Douglas Bristol led me to several sources of valuable information on bisphenol-A. I am grateful to Takeki Tsutsui for inviting me to prepare this paper.
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