Quantitative in Vitro to in Vivo Extrapolation (QIVIVE) for Predicting Reduced Anogenital Distance Produced by Anti-Androgenic Pesticides in a Rodent Model for Male Reproductive Disorders

Abstract

BACKGROUND: Many pesticides can antagonize the androgen receptor (AR) or inhibit androgen synthesis in vitro but their potential to cause reproductive toxicity related to disruption of androgen action during fetal life is difficult to predict. Currently no approaches for using in vitro data to anticipate such in vivo effects exist. Prioritization schemes that limit unnecessary in vivo testing are urgently needed. OBJECTIVES: The aim was to develop a quantitative in vitro to in vivo extrapolation (QIVIVE) approach for predicting in vivo anti-androgenicity arising from gestational exposures and manifesting as a shortened anogenital distance (AGD) in male rats. METHODS: We built a physiologically based pharmacokinetic (PBK) model to simulate concentrations of chemicals in the fetus resulting from maternal dosing. The predicted fetal levels were compared with analytically determined concentrations, and these were judged against in vitro active concentrations for AR antagonism and androgen synthesis suppression. RESULTS: We first evaluated our model by using in vitro and in vivo anti-androgenic data for procymidone, vinclozolin, and linuron. Our PBK model described the measured fetal concentrations of parent compounds and metabolites quite accurately (within a factor of five). We applied the model to nine current-use pesticides, all with in vitro evidence for anti-androgenicity but missing in vivo data. Seven pesticides (fludioxonil, cyprodinil, dimethomorph, imazalil, quinoxyfen, fenhexamid, o-phenylphenol) were predicted to produce a shortened AGD in male pups, whereas two (k-cyhalothrin, pyrimethanil) were anticipated to be inactive. We tested these expectations for fludioxonil, cyprodinil, and dimethomorph and observed shortened AGD in male pups after gestational exposure. The measured fetal concentrations agreed well with PBK-modeled predictions. DISCUSSION: Our QIVIVE model newly identified fludioxonil, cyprodinil, and dimethomorph as in vivo anti-androgens. With the examples investigated, our approach shows great promise for predicting in vivo anti-androgenicity (i.e., AGD shortening) for chemicals with in vitro activity and for minimizing unnecessary in vivo testing.

On the Use and Interpretation of Areola/Nipple Retention as a Biomarker for Anti-androgenic Effects in Rat Toxicity Studies

“…Exposure to the fungicides vinclozolin and procymidone also give rise to dose-dependent NR in male rat offspring ( see Table 1 ). Both chemicals display AR antagonism in vitro ( Kelce et al, 1994 ; Nellemann et al, 2003 ; Kleinstreuer et al, 2017 ; Scholze et al, 2020 ) and give strong effects on NR in vivo . Vinclozolin induces NR with a clear dose-response relationship and, like flutamide, can induce retention of all 12 nipples in prenatally exposed males.…”

“…Linuron is a well-characterized anti-androgenic herbicide that can antagonize the AR in vitro , but also inhibit the synthesis of androgens such as testosterone and androstenedione ( Scholze et al, 2020 ). The performed in vivo studies show decreased testosterone levels indicating that this compound could also affect steroidogenesis in vivo .…”

“…For instance, the three pesticides fludioxonil, cyprodinil and dimethomorph have all been shown to have AR antagonistic activity in vitro; cyprodinil also showed AR agonistic activity at low concentrations ( Orton et al, 2011 ). When tested in vivo , cyprodinil and fludioxonil had no effect on NR at doses between 20 and 180 mg/kg ( Scholze et al, 2020 ). Dimethomorph was tested twice in the same study at doses of 6.7, 20, 60 and 180 mg/kg, with data showing conclusive effects on NR at 180 mg/kg ( Scholze et al, 2020 ).…”

“…When tested in vivo , cyprodinil and fludioxonil had no effect on NR at doses between 20 and 180 mg/kg ( Scholze et al, 2020 ). Dimethomorph was tested twice in the same study at doses of 6.7, 20, 60 and 180 mg/kg, with data showing conclusive effects on NR at 180 mg/kg ( Scholze et al, 2020 ).…”

“…However, not all data supports this hypothesis, as some AR antagonists such as linuron and DDE induce a low number of nipples ( You et al, 1998 ; Loeffler and Peterson 1999 ; McIntyre et al, 2000 ; Hotchkiss et al, 2004 ). This is also the case for fludioxonil, cyprodinil and dimethomorph, albeit the cause-and-effect relationship is much less clear for these chemicals as they have only been tested in vivo in one study ( Scholze et al, 2020 ).…”