A Practical Approach, Second Edition=Ronald D. Ho.pdf

THE U.S. EPA ENDOCRINE DISRUPTOR SCREENING PROGRAM 505following the last of three daily s.c. injections of bisphenol A. However, in the later study theincreased uterine weight at 24 h was still significantly different from the control. The higher dosesof bisphenol A used in the OECD validation studies (e.g., 600 mg/kg by oral gavage and 300 mg/kgfor s.c. injection) did result in significant increases in uterine weight 24 h following the last dose. 91Thus, it is imperative that the final protocol for the uterotrophic assay include dose selection criteriato allow a sufficient dose of the test chemical for each treatment route that will enable the detectionof estrogenic activity at the time of necropsy.B. Hershberger AssayThe Hershberger assay is an in vivo short-term test similar in concept to the rodent uterotrophicassay discussed above. Both assays measure changes in specific tissues that normally respond toendogenous steroid hormones. The assay conditions are designed to achieve low endogenoushormone levels and examine target tissues that are highly responsive to administration of exogenoushormones. The Hershberger assay provides a method to determine whether a compound possessesandrogenic or antiandrogenic activity. It involves weighing tissues in castrated, sexually immaturemale rats following exposure to a test chemical with or without androgen supplementation. 99 Thebiological relevance of the assay is based upon the fact that accessory sex tissues and glands dependon androgen stimulation to gain and maintain weight during and after puberty. 100The Hershberger assay may also present the opportunity to detect inhibitors of 5-α-reductaseby taking advantage of a differential response among the accessory sex glands and tissues. Testosteroneis converted by 5-α-reductase to DHT, which has significantly higher binding affinity thantestosterone for the androgen receptor. The levator ani and bulbocavernosus (LABC) muscles lackthe enzyme 5-α-reductase, while in other tissues the enzyme is present (e.g., ventral prostate,seminal vesicles, and coagulating glands). It is plausible then that tissues with 5-α-reductase activityare more responsive to compounds that are metabolized to DHT than tissues without the enzyme.Acknowledging the substantial amount of historical data and the long standing use of theHershberger assay for the detection of androgens and antiandrogens, 99–104 the EDSTAC recommendedthat the Hershberger be a component of the TIS battery. As with the uterotrophic assay,the OECD agreed to assist the OSCP, U.S. EPA, by directing the validation process for theHershberger assay. Using an approach similar to that used for the validation of the uterotrophicassay, the OECD designed a series of studies that will be conducted in multiple laboratoriesthroughout the world. Phase I was designed to evaluate the standardized protocol, using a referenceandrogen and an antiandrogen. Phase II of the process will evaluate the ability of the assay to detectother androgens, several weak antiandrogens, and a 5-α-reductase inhibitor. Estimates of intra- andinterlaboratory variance will be determined throughout the validation process.The protocol being evaluated currently by the OECD uses castrate, sexually immature malerats. The animals are castrated after peripuberty (generally occurs after postnatal day [PND] 42)by removing both the testes and epididymides. In most rat strains, such as the Sprague-Dawley,Long-Evans, or Wistar, peripuberty is expected to take place at approximately 6 weeks of age,within an average age range of 5 to 7 weeks. Peripuberty is marked by prepuce separation fromthe glans penis (GP). Experimentally, preputial separation is necessary so that the GP can beproperly dissected and accurately weighed. At the peripubertal stage of sexual development, theGP and other androgen-dependent sex accessory tissues are sensitive to androgens, having bothandrogen receptors and the appropriate steroidogenic enzymes. The advantage of using a rodentof this age is that the sex accessory tissues have a high sensitivity and small relative weight, whichminimizes variation in responses between individual animals. In addition, castration removes theinhibitory feedback of testosterone on the hypothalamic-pituitary axis, resulting in a characteristicincrease in the concentration of LH. Depending upon the action of the test substance in thehypothalamus, the pattern of LH secretion with increasing doses of the test substance may be altered.© 2006 by Taylor & Francis Group, LLC