| Concentration and dosing |
In vitro and in vivo studies should employ at least three concentrations/doses or five if assessing non-monotonicity, including equivalent in vitro low doses below 50 ng/ml or in vivo doses below the NOAEL and/or TDI of BPA; the same concentrations/doses should be maintained for BPA alternatives. Animal dosing of bisphenols via oral delivery is preferable if assessing them within a dietary contaminant context, in line with US FDA requirements.
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| Employment of multiple bisphenols |
In vitro and in vivo: Any BPA alternative such as BPS, BPF, BPB, BPAF etc. being assessed either in isolation or in combination with other bisphenols should be examined alongside BPA as an additional treatment group to characterize and compare the mechanisms of action. BPA can be used to provide a point of reference to build the profile of BPA analogues that are not yet well established within the literature; also generating additional evidence for BPA. Human studies in the fertility clinic setting should aim to incorporate the relevant alternative bisphenols (BPS, BPF, BPB, BPAF, etc.) in their detection protocols alongside BPA.
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| Endpoint measures |
In vitro and in vivo: Studies prioritizing molecular and mechanistic endpoints should pair these with one or more comparable endpoints associated with oocyte or ovarian adverse effects (follicle counts, oocyte yield, oocyte meiotic capacity, oocyte and/or cumulus cell morphology, and oocyte meiotic spindle integrity). Human studies: Spindle assessment or morphological assessment of discarded oocytes from participants should be included where possible alongside current endpoints of oocyte yield and antral follicle count to link to animal study outcomes. ART outcomes including measures of embryo quality, embryo development, and implantation rates are also ideal to report in order to assess the developmental potential of oocytes selected for use in IVF/ICSI.
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| Methodological reporting |
In vitro and in vivo: For study results to be considered by governing bodies, studies must include detailed reporting of methodologies including all aspects of animal use, experimental replicates, cell numbers, and dosing protocols as per US FDA guidelines. These guidelines such as the Redbook 2000 should be clearly defined, updated, and made accessible by the US FDA. Peer review processes for publication of these studies should hold authors to the correct reporting standards of methodologies in this toxicological context. Human studies should report the full range of statistical population parameters for BPA concentration in samples (means, standard deviation, quartiles/percentiles, maximums, the limit of detection, and percent of participants samples that were below the limit of detection, unadjusted and adjusted urinary parameters) for ease of interpretation and comparison between study population.
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| Sample collection and detection |
Human observational studies should aim to collect multiple urine samples across different time points using a prospective cohort approach, consider these as separate data points, and where possible, measure unconjugated BPA. To obtain accurate results, it is critical that detection and quantification of BPA and BPA alternatives is conducted using the direct method (not indirect) as detailed in Gerona et al. 2020).
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