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William Owens,¹ L. Earl Gray Jr.,² Errol Zeiger,³ Michael Walker,⁴ Kanji Yamasaki,⁵ John Ashby,⁶ and Elard Jacob⁷

¹Procter & Gamble, Cincinnati, Ohio, USA; ²U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, USA; ³Errol Zeiger Consulting, Chapel Hill, North Carolina, USA; ⁴Health Canada, Ottawa, Ontario, Canada; ⁵Chemicals Evaluation and Research Institute, Oita, Japan; ⁶Syngenta Central Toxicology Laboratory, Macclesfield, Cheshire, UK; ⁷BASF, Ludwigshafen, Germany

Abstract
Objective: The Organisation for Economic Co-operation and Development (OECD) has completed phase 2 of an international program to validate the rodent Hershberger bioassay.

Design: The Hershberger bioassay is designed to identify suspected androgens and antiandrogens based on changes in the weights of five androgen-responsive tissues (ventral prostate, paired seminal vesicles and coagulating glands, the levator ani and bulbocavernosus muscles, the glans penis, and paired Cowper's or bulbourethral glands) . Protocol sensitivity and reproducibility were tested using two androgen agonists (17α-methyl testosterone and 17β-trenbolone) , four antagonists [procymidone, vinclozolin, linuron, and 1,1-dichoro-2,2-bis-(p-chlorophenyl) ethylene (p,p´-DDE) ], and a 5α-reductase inhibitor (finasteride) . Sixteen laboratories from seven countries participated in phase 2.

Results: In 40 of 41 studies, the laboratories successfully detected substance-related weight changes in one or more tissues. The one exception was with the weakest antiandrogen, linuron, in a laboratory with reduced sensitivity because of high coefficients of variation in all tissue weights. The protocols performed well under different experimental conditions (e.g., strain, diet, housing protocol, bedding, vehicle) . There was good agreement and reproducibility among laboratories with regard to the lowest dose inducing significant effects on tissue weights.

Conclusions: The results show that the OECD Hershberger bioassay protocol is reproducible and transferable across laboratories with androgen agonists, weak androgen antagonists, and a 5α-reductase inhibitor. The next validation phase will employ coded test substances, including positive substances and negative substances having no androgenic or antiandrogenic activity.

Key words: androgen, antiandrogen, bulbocavernosus, Cowper's glands, DDE, endocrine disruption, Finasteride, glans penis, Hershberger, levator ani, seminal vesicles, linuron, methyl testosterone, procymidone, trenbolone, validation, ventral prostate, vinclozolin. Environ Health Perspect 115:671–678 (2007) . doi:10.1289/ehp.9666 available via http://dx.doi.org/ [Online 17 January 2007]

Address correspondence to J.W. Owens, Central Product Safety, The Procter & Gamble Company, P.O. Box 538707, Cincinnati, OH 45253-8707 USA. Telephone: (513) 627-1385. Fax: (513) 627-1208. E-mail: owens.jw@pg.com

Supplemental material is available online (http://www.ehponline.org/docs/2007/9666/suppl.pdf) .

We appreciate the participating laboratories' efforts and work in generating these data: BASF, Germany ; BAYER-AG, Germany ; Bayer CropScience, France ; CIT, France ; Chemicals Evaluation and Research Institute, Japan ; Exxon Biomedical Sciences Inc., United States ; the Food Drug Safety Centre, Japan ; Huntingdon Life Sciences, United Kingdom ; the Institute of Environmental Toxicology, Japan ; the Institute for Food Safety, Denmark ; Japan Bioassay Research Centre, Japan ; Mitsubishi Chemical Safety Institute, Japan ; the National Institute of Toxicological Research, Korea ; Panapharm Laboratories, Ltd., Japan ; Sumitomo Chemical Company, Japan ; and Syngenta Toxicology Laboratory, United Kingdom. We thank Sigma-Aldrich for reserving a single lot of trenbolone, a controlled substance, for the validation study and ensuring its distribution to the individual laboratories. We acknowledge European and Japanese chemical industries for providing the reference chemicals and for supporting the chemical repository. We thank H.B.W.M. Koëter (OECD Secretariat and manager of the Test Guidelines Programme) for his constant support and efforts in initiating and conducting this and other endocrine-validation programs.

This article represents only the authors' opinions and may not represent official U.S. EPA policy or those of the OECD and its member countries.

W. O. participated in this program under a secondment to the OECD Secretariat. E.Z. participated in this program as a consultant to the OECD Secretariat.

W.O. is employed by Procter & Gamble, J.A. is employed by Syngenta, and E.J. is employed by BASF. The other authors declare they have no competing financial interests.

Received 31 August 2006 ; accepted 17 January 2007.