Bisphenol A: findings of a multigenerational rat study.

I read with great interest John Heinze's letter (2003) about the article by Schonfelder et. al. (2002) titled "Parent Bisphenol A Accumulation in the Human Maternal--Fetal-Placental Unit" and the response to Heinz by Ibrahim Chahoud (2003). I am the study director and first author of the muhigenerational study of dietary bisphenol A (BPA) in CD (Sprague-Dawley) rats (Tyl et al. 2002) Chahoud referred to when he mentioned "the problem of the interpretation of the so-called negative studies." Specifically, he referred to our interpretation of our finding of reduced absolute and relative ovarian weights observed in [F.sub.0], [F.sub.1], and [F.sub.2] (but not [F.sub.3]) adult females at 500 mg/kg/day BPA (7,500 ppm) and the reduced ovarian weights observed only in the [F.sub.2] females at 1 (0.0015 ppm) and 300 [micro]g/kg/day (4.5 ppm), but not at 20 [micro]g/kg/day (0.3 ppm), 5 mg/kg/day (75 ppm), or 50 mg/kg/day (750 ppm). In our multigenerational study, we used six BPA dose groups (0.0015-7,500 ppm) and a concurrent vehicle control group, 30 animals per sex per group per generation, with the [F.sub.3] animals raised to adulthood. In such a study, the [F.sub.1], [F.sub.2], and [F.sub.3] generations are essentially replicates (although the [F.sub.3] animals were not bred), so the findings observed in the multiple generations (e.g., ovarian effects at 500 mg/kg/day) can be compared in context with the findings observed only in one generation (e.g., ovarian effects at 1 and 300 [micro]g/kg/day). In the latter example, an effect in only one generation, with no dose-response pattern, is most likely not treatment or dose related but is most likely due to biologic variation. In the former example, the reduced [F.sub.0], [F.sub.1], and [F.sub.2] maternal ovarian weights and the reduced [F.sub.1], [F.sub.2], and [F.sub.3] litter sizes at birth at 500 mg/kg/day BPA were clearly related to treatment and dose. However, there was a clear indication at this high dietary dose (7,500 ppm) of profound systemic toxicity in both sexes in all four generations; therefore, the effects on the ovaries and litters are confounded by the toxicity at this dose, which exceeded the maximum tolerated dose.

I agree with Chahoud that "investigators are in the position to interpret the adversity of their own data ..." We are not only in position to, but are scientifically and morally responsible to interpret our data. In our paper (Tyl et al. 2002), we concluded that there was systemic toxicity in the form of decreases in body weight gain at the 50- and 500-mg/kg/day doses (750 and 7,500 ppm); therefore, the NOAEL (no-observed-adverse-effect level) for adult systemic toxicity of BPA was 5 mg/kg/day (75 mg/kg/day). Because reproductive toxicity occurred only at 500 mg/kg/day, the NOAEL for reproductive toxicity of BPA was 50 mg/kg/day. These conclusions are fully supported by our data (Tyl et al. 2002); this opinion was also held by the statistics subcommittee of the National Toxicology Program (NTP)/U.S. Environmental Protection Agency Endocrine Disruptors Low-Dose Workshop (NTP 2001), who reviewed all of our data on this study and concurred with all of our results and interpretations.

The fact that our statistically powerful study, compliant with good laboratory practices (and the study by Ema et al. 2001 and others), could not confirm the low-dose effects of BPA was also acknowledged by the Statistics Subpanel of the Endocrine Disruptors Low-Dose Workshop as follows:

   There is credible evidence that low doses of BPA
   can cause effects on specific endpoints. However,
   due to the inability of other credible studies in
   several different laboratories to observe low dose
   effects of BPA, and the consistency, of these negative
   studies, the Subpanel is not persuaded that a
   low dose effect of BPA has been conclusively
   established as a general or reproducible finding.
   In addition, for those studies in which low dose
   effects have been observed, the mechanism(s) is
   uncerrain (i.e., hormone related or otherwise) and
   the biological relevance is unclear.

The absence of effects of BPA at low dietary doses in rats (Ema et al. 2001; Tyl et al 2002) and mice (Ashby et al. 1999; Tyl et al. Unpublished data) and the absence of any consistent or persistent adverse effects exhibiting a "nonmonotonic inverted-U dose-response curve" mitigate against any "need for mechanistic experimental studies as well as follow-up studies in humans regarding low-dose effects" (Chahoud 2003). One cannot and should not perform mechanistic studies on nonreproducible findings. My laboratory has shown (Tyl et al. Unpublished data) that CD-1 mice can tolerate 5,000 (0.5%; BPA intake ~850 mg/kg/day) and 10,000 (1.0%; BPA intake ~1,700 mg/kg/day) ppm BPA in the diet, with dose-related systemic toxicity in parental livers and kidneys and reduced litter size at birth found only at 10,000 ppm. Our data confirm and extend those of Reel et al. (1985). The conclusion by the NTP Low-Dose Review and the detailed analyses published in individual articles provide strong support for the absence of reproducible, convincing effects after exposure to low doses of BPA. Also, my laboratory (Tyl et al. 2002) showed that a) dietary BPA < 500 mg/kg/day does nor produce reproductive, fertility, or developmental effects, and b) BPA is not a selective reproductive toxicant in any generation in either sex at doses < 500 mg/kg/day, it is my understanding that workplace and consumer exposures are orders of magnitude below these doses.

The author declares she has no conflict of interest.

Rochelle W. Tyl

Center for Life Sciences and Toxicology Research Triangle Institute

Research Triangle Park, North Carolina

E-mail: [e-mail]

REFERENCES

Ashby J, Tinwell H, Haseman J. 1999. Lack of effects for low dose levels of bisphenol A and diethylstilbestrol on the prostate gland of CF1 mice exposed in utero. Regul Toxicol Pharmacol 30:156-166.

Chahoud I. 2003. Adverse health effects of bisphenol A: Chahoud's response. Environ Health Perspect 111:A383.

Ema M, Fujii S, Furukawa M, Kiguchi M, Ikka T, Harazono A. 2001. Rat two-generation reproduction study of bisphenol A. Reprod Toxicol 15:505-523.

Heinze JE. 2003, Adverse health effects of bisphenol A in early life. Environ Health Perspect 111:A382-A383.

NTP. 2001. National Toxicology Program's Endocrine Disruptors Low-Dose Peer Review. Research Triangle Park, NC:National Toxicology Program. Available: http://ntp-server.niehs.nih.gov/htdocs/liason/ LowDosePeerFinalRpt.pdf [accessed 23 April 2002].

Reel JR, George JD, Myers CB, Lawton AD, Lamb JC. 1985. Reproduction and Fertility Assessment in CD-1 Mice when Administered in the Feed. Final Study Report. Research Triangle Park, NC:National Toxicology Program.

Schonfelder G, Wittfoht W, Hopp H, Talsness CE, Paul M, Chahoud I, 2002. Parent bisphenol A accumulation in the human maternal fetal-placental unit. Environ Health Perspect 110:A703 A707.

Tyl RW, Myers CB, Marr MC, Thomas BF, Keimowitz AR, Brine DR, et al. 2002. Three-generation reproductive toxicity study of dietary bisphenol A (BPA) in CD (Sprague-Dawley) rats. Toxicol Sci 68:121 146,