Testing of endocrine active substances using an enhanced OECD test
guideline 407: Experiences from studies on flutamide and ethinylestradiol
A. Freyberger, P. Andrews, E. Hartmann, R. Eiben, I. Loof, U. Schmidt,
M. Temerowski, A. Folkerts, M. Becka, B. Stahl, and M. Kayser
Alexius Freyberger , Peter Andrews, Elke Hartmann,
Rolf Eiben, Ingo Loof, Ulrich Schmidt, Michael Temerowski, Andree Folkerts,
Michael Becka, Bernhard Stahl, and Martin Kayser
Abstract: Groups of five male and five female Wistar rats were
treated by gavage with 0, 1, 10, and 100 mg/kg body weight (b.w.) flutamide
(FLU) or 0.01, 0.05, and 0.2 mg/kg b.w. of ethinylestradiol (EE2) for
at least 28 days according to an enhanced Organization for Economic
Cooperation and Development (OECD) test guideline (TG) 407 to investigate
which of the current and/or additional parameters would detect effects
on the endocrine system and to provide data on intralaboratory variability.
Two identical studies were performed in parallel on each compound. Common
enhancements were determination of thyroid hormones (T3, T4) and thyroid
stimulating hormone (TSH), of the stage of the estric cycle to ensure
necropsy of females in diestrus, of the number and morphology of epididymal
sperm, and of additional organ weights (e.g., male accessory sex organs,
MASO) and histopathology of additional organs (e.g., pituitary, vagina).
Endocrine-mediated findings consistently observed in these studies were
decreased relative weights of MASO at 100 mg/kg FLU and at 0.2 mg/kg
EE2, histological changes in pituitary and testes at > or = 10 mg/kg
and in MASO, epididymis and adrenals at 100 mg/kg in FLU-treated males,
histological changes in the mammary gland at > or = 0.05 mg/kg and
in testes, MASO and adrenals at 0.2 mg/kg in EE2-treated males, estrogenization
of uterus and vagina (despite necropsy in diestrus) at > or = 0.01
mg/kg EE2, and changes in the ovary at 0.2 mg/kg EE2. Spermatology was
insensitive (EE2) or revealed changes only at the maximum tolerated
dose (MTD). Determination of T3, T4, and TSH did not contribute to the
detection of the endocrine effects (FLU) or provided equivocal results.
Doubling the group size to 10 animals by combining the studies run in
parallel did not increase the sensitivity of detection of endocrine-mediated
effects above the level obtained by histopathological examination of
groups of five animals. Only some of the proposed enhancements evaluated
were helpful in detecting the endocrine-mediated effects of FLU and
EE2. Evaluation of studies according to an enhanced TG 407 on 10 compounds
with different endocrine activities will identify the most appropriate
enhancements.
*Report from a SCOPE/IUPAC project: Implication of
Endocrine Active Substances for Human and Wildlife (J. Miyamoto and
J.Burger, editors). Other reports are published in this issue,
pp. 1617-2615.