Toxicology 311 (2013) 13–26 Contents lists available at SciVerse ScienceDirect Toxicology jo u r n al homep age: www.elsevier.com/locate/toxicol The influence of study design and sex-differences on results from developmental neurotoxicity studies of bisphenol A, implications for toxicity testing Anna Beronius a,∗ , Niklas Johansson a , Christina Rudén b , Annika Hanberg a a Institute of Environmental Medicine, Karolinska Institutet, PO Box 210, 171 77 Stockholm, Sweden b Department of Applied Environmental Science, Stockholm University, 106 91 Stockholm, Sweden a r t i c l e i n f o Article history: Received 6 December 2012 Received in revised form 4 February 2013 Accepted 13 February 2013 Available online 26 February 2013 Keywords: Bisphenol A Developmental neurotoxicity Sex-differences Regulatory testing Risk assessment a b s t r a c t Developmental neurotoxicity (DNT) of bisphenol A (BPA) has been investigated in a large number of studies. However, there are discrepancies in the results reported between the studies. The aim of this study was to identify and analyze factors that may contribute to these differences and to assess whether there are sex-differences in the sensitivity of certain endpoints or tests used in DNT-studies. Forty-four DNT studies of BPA were identified from the open literature. Details about study design and results from each study, as well as the criteria for DNT testing according to the standardized OECD test guideline (TG) 426, were collected in a database. This enabled systematic and detailed comparisons between studies as well as to the criteria and recommendations stated in TG 426. Multivariate analyses were also used to investigate how different factors of the study design contributed to differences in study results. The analyses showed behavioral effects were often observed for endpoints that are not required accord- ing to OECD TG 426, such as anxiety-related, social and sexual behaviors, especially at very low doses and in female offspring. On the other hand relatively few studies observed any effects on motor activity, which is commonly used in screening for neurotoxic effects in regulatory testing. However, varied and to some extent seemingly contradictory results have been reported in these studies, especially for endpoints related to motor activity and anxiety and exploration. Many studies were also poorly reported, limiting these analyses. No strong conclusions could be drawn from the multivariate analyses. A few factors of study design, such as the size of the dose and number of dose levels used and the use of litter or individual pup as statistical unit seemed to have some influence on study results. In conclusion, this analysis suggests that DNT-studies conducted according to the standardized OECD TG 426 may overlook sensitive effects of BPA, and possibly other potential endocrine disruptors, especially in female offspring. © 2013 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Bisphenol A (BPA) is an endocrine disrupting compound (EDC), with mainly estrogenic properties, for which health risk assess- ment has proven particularly complicated. Compared to most other chemicals the toxicity of BPA is very well studied. Still, there is disagreement among scientists as well as regulators as to Abbreviations: BPA, bisphenol A; DNT, developmental neurotoxicity; EDC, endocrine disrupting compound; EPM, elevated plus maze; LOAEL, lowest observed adverse effect level; NMDR, non-monotonic dose–response; NOAEL, no observed adverse effect level; OF, open field; PCA, principal components analysis; PLS, par- tial least squares projection to latent structures modeling; RfD, reference dose; TDI, tolerable daily intake; TG, test guideline. ∗ Corresponding author. Tel.: +46 08 524 87528. E-mail addresses: anna.beronius@ki.se (A. Beronius), niklas.johansson@ki.se (N. Johansson), christina.ruden@itm.su.se (C. Rudén), annika.hanberg@ki.se (A. Hanberg). the nature and size of the health risks posed by this compound (Beronius et al., 2010). The estimated exposure to BPA, a high production volume chemical mainly used in the manufacture of polycarbonate plastics and epoxy materials, is in the range of 0.01–4.5 g/kg body weight (bw) and day (FAO/WHO, 2011) and is widespread in the general population (Calafat et al., 2008; Vandenberg et al., 2007). Currently, the tolerably daily intake (TDI) and reference dose (RfD) established in Europe and the United States, respectively, are both set to 50 g BPA/kg body weight (bw) and day (EFSA, 2006, 2008; US FDA, 2008). These guidance values were derived from a no observed adverse effect level (NOAEL) of 5 mg/kg bw/day, identified from two multi-generation reproduction studies in rats and mice conducted according to standardized and internationally accepted toxicity test guidelines and where BPA was administered via the feed to dams and off- spring (Tyl et al., 2002, 2008). However, over the last decade a large number of research studies, i.e. studies that were not con- ducted according to standardized test guidelines, have reported 0300-483X/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tox.2013.02.012