Natural variability and the influence of concurrent control values on the detection and interpretation of low-dose or weak endocrine toxicities.

Abstract

While defining the no effect level for the 5α-reductase inhibitor finasteride in the Hershberger assay, we encountered an inverted-U low-dose trophic effect on the prostate gland of the rat. Two attempts to confirm this observation were unsuccessful, and we concluded that the positive effect initially observed was associated with normal biologic variability. During the same period we attempted, unsuccessfully, to repeat our own observation of weak uterotrophic activity in the rat for the sunscreen 3-(4-methylbenzylidene)camphor (4MBC). Further evaluation led us to conclude that 4MBC is uterotrophic only when the control uterine weights are at the low end of their normally encountered range. This led us to reevaluate our earlier mouse uterotrophic assay data for bisphenol A (BPA). Originally we had concluded that BPA gave irreproducible evidence of weak uterotrophic activity, but upon ordering the eight experiments we had conducted, according to decreasing control uterine weight, we confirmed reproducible weak uterotrophic activity for BPA when the control uteri were at the low end of their normal range. In this article, we describe these observations, together with a reanalysis of the data associated with several reported instances of weak or low-dose endocrine effects that have proven difficult to confirm in independent laboratories. These include the activity of BPA on the CF1 mouse prostate; the activities of BPA, octylphenol, and nonylphenol on the rat testis; and the effect of polycarbonate caging on control mouse uterine weight. In all of these cases, variability among controls provides a major obstacle to data interpretation and confirmation. Our recommendations on experimental design are also presented, with a view to ending the current impasse on the reality, or otherwise, of low-dose or weak endocrine toxicities. Key words: 3-(4-methylbenzylidine)camphor, bisphenol A, endocrine disruption, finasteride, low dose, nonylphenol.

Effect of Gestational Exposure to Nonylphenol on the Development and Fertility of Mouse Offspring

“…Many other groups have also shown a marked dose-dependent increase in uterus weight, diameter of the uterine ducts, and luminal epithelial height of the vagina and uterus after exposure to NP [12,13,24,25]. However, we found that females administered NP during the gestational stage had slightly (not significantly) increased uterine weights.…”

“…There is controversy concerning the "inverted U" responses of EDs at dose levels lower than the no effect level defined by classical toxicity [13]. Interestingly, in our results, the NP1/100 treatment increased ovary weight by about 180% compared with the control group, whereas the NP1/1000 and NP1/10 treatments had no effect on ovary weight.…”

“…The effects of NP are consistent with ER-mediated actions. However, a direct link between exposure to NP and endocrine-related effects has only been established in some in vivo tests, such as uterotrophic assays and mammary gland developmental studies [12][13][14]. Although the uterotrophic assay is regarded as sensitive model for the detection of chemicals with e s t r o g e n i c / a n t i e s t r o g e n i c a c t i v i t y , d i r e c t extrapolation to humans is difficult because of the complexity of the effects of endogenous hormones, such as gonadotrophins and ovarian steroid h o r mo n e s d u r i n g t h e d e v e l o p m e n t o f t h e reproductive organs and sexual maturation.…”

Influence of dietary soy isoflavones on immature hamster uterotrophic and Hershberger assays

“…However, this is contraindicated by current literature, which specifically states that diets containing less than this value still have the potential to alter the results of vaginal opening and uterotrophic assay (29). This remains to be an unresolved issue, and is especially critical for the detection of weak acting oestrogenic substances (2).…”

“…Therefore, besides the appropriate animal model and control groups (negative and positive), feed is of significant importance in interpreting results of the studies on endocrine disruptors (EDs) (1,2,31). Selection of the most appropriate diet for ED research conducted on rodent experimental models, is not only a current subject providing the current proposals to mass screen a wide range of endocrine-disrupting compounds (EDCs), but it is also a decision that should be put under a serious consideration before standardising or initiating such studies (29).…”