Downloaded from orbit.dtu.dk on: Oct 07, 2021 Grouping of endocrine disrupting chemicals for mixture risk assessment - Evidence from a rat study Christiansen, Sofie; Axelstad, Marta Axelstad; Scholze, Martin; Johansson, Hanna Katarina Lilith; Hass, Ulla; Mandrup, Karen Mandrup; Frandsen, Henrik Lauritz; Frederiksen, Hanne; Isling, Louise Krag; Boberg, Julie Published in: Environment International Link to article, DOI: 10.1016/j.envint.2020.105870 Publication date: 2020 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Christiansen, S., Axelstad, M. A., Scholze, M., Johansson, H. K. L., Hass, U., Mandrup, K. M., Frandsen, H. L., Frederiksen, H., Isling, L. K., & Boberg, J. (2020). Grouping of endocrine disrupting chemicals for mixture risk assessment - Evidence from a rat study. 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Environment International 142 (2020) 105870 Contents lists available at ScienceDirect Environment International journal homepage: www.elsevier.com/locate/envint Grouping of endocrine disrupting chemicals for mixture risk assessment – Evidence from a rat study T ⁎ Sofie Christiansena, , Marta Axelstada, Martin Scholzeb, Hanna K.L. Johanssona, Ulla Hassa, Karen Mandrupa, Henrik Lauritz Frandsenc, Hanne Frederiksend, Louise Krag Islinga, Julie Boberga a Division of Diet, Disease Prevention and Toxicology, National Food Institute, Technical University of Denmark, Kemitorvet Building 202, Kgs. Lyngby DK-2800, Denmark b Institute of Environment, Health and Societies, Brunel University London, Quad North, Kingston Lane, Uxbridge UB8 3PH, UK c Research Group for Analytical Food Chemistry, National Food Institute, Technical University of Denmark, Kemitorvet Building 202, DK-2800 Kgs. Lyngby, Denmark d Department of Growth and Reproduction, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark ARTICLE INFO ABSTRACT Handling Editor: Olga Kalantzi Exposure to mixtures of endocrine disrupting chemicals may contribute to the rising incidence of hormone- Keywords: related diseases in humans. Real-life mixtures are complex, comprised of chemicals with mixed modes of action, Mixture risk assessment and essential knowledge is often lacking on how to group such chemicals into cumulative assessment groups, Endocrine disruptive chemicals which is an essential prerequisite to conduct a chemical mixture risk assessment. EDC We investigated if mixtures of chemicals with diverse endocrine modes of action can cause mixture effects on Adverse outcome pathway hormone sensitive endpoints in developing and adult rat offspring after perinatal exposure. Wistar rats were exposed during pregnancy and lactation simultaneously to either bisphenol A and butylparaben (Emix), die- thylhexyl phthalate and procymidone (Amix), or a mixture of all four substances (Totalmix). In male offspring, the anogenital distance was significantly reduced and nipple retention increased in animals exposed to Amix and Totalmix, and the mixture effects were well approximated by the dose addition model. The combination of Amix and Emix responded with more marked changes on these and other endocrine-sensitive endpoints than each binary mixture on its own. Sperm counts were reduced by all exposures. These experimental outcomes suggest that the grouping of chemicals for mixture risk assessment should be based on common health outcomes rather than only similar modes or mechanisms of action. Mechanistic-based approaches such as the concept of Adverse Outcome Pathway (AOP) can provide important guidance if both the information on shared target tissues and the information on shared mode/mechanism of action are taken into account. 1. Introduction performing Mixture Risk Assessment (MRA), where the risk of com- bined exposures to multiple chemicals is assessed (Boberg et al., 2019; Chemical exposure is suspected to contribute to a high prevalence of Bopp et al., 2018; EC European Commision, 2012; EFSA, 2019; endocrine disorders in the Western world (WHO/UNEP, 2013). This Howdeshell et al., 2017). calls for an improved understanding of how exposure to complex che- There is clear experimental evidence that mixture effects can be mical mixtures in our everyday life may lead to adverse health effects anticipated for chemicals with similar mechanisms of action even at such as hormone-dependent cancers, fertility problems and congenital doses where each chemical is ineffective (Christiansen et al., 2012, malformations. Risk assessment of chemicals with mixed modes and 2009; Conley et al., 2018, 2016; Hass et al., 2012, 2007; Metzdorff mechanisms of action presents an unsolved challenge. et al., 2007; Silva et al., 2002). Experiments show that cumulative ef- Over the last decades, our understanding of combined exposures to fects of chemicals with the same molecular initiating events (e.g. AR environmental chemicals and how to assess the humans and the en- antagonism) follow the dose-addition principles (Christiansen et al., vironment has improved substantially (Kortenkamp and Faust, 2018). 2008; Hannas et al., 2011; Hass et al., 2007; Howdeshell et al., 2015, Consequently, scientific and regulatory focus has shifted from only 2008b, 2007; Metzdorff et al., 2007; Rider et al., 2009). Additionally, performing risk assessments for an individual substance towards dose-additive effects on androgen sensitive endpoints have been ⁎ Corresponding author. E-mail address: [email protected] (S. Christiansen). https://doi.org/10.1016/j.envint.2020.105870 Received 14 February 2020; Received in revised form 26 May 2020; Accepted 26 May 2020 Available online 24 June 2020 0160-4120/ © 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). S. Christiansen, et al. Environment International 142 (2020) 105870 demonstrated after exposure to anti-androgenic compounds with dif- variation in quality and outcomes (EFSA, 2015). Moreover, numerous in ferent mechanisms of action (Christiansen et al., 2009; Conley et al., vitro studies have demonstrated a weak anti-androgenic activity for BPA 2018) and even for multi-causal effects such as reduced birth weight and butylparaben (Chen et al., 2007; Kjærstad et al., 2010; Reif et al., (Hass et al., 2017). This evidence for mixture effects of chemicals with 2010; Rosenmai et al., 2014; Satoh et al., 2005). different mechanisms of action implies that mixture risk assessment In the current study we exposed pregnant and lactating rats to a should go beyond grouping based on shared mechanisms of action. binary mixture of DEHP and Procymidone (Amix), a binary mixture of In the present paper, we propose to group chemicals based on a BPA and butylparaben (Emix), and a combination of all four com- common target tissue rather than common mechanisms of action. In pounds (Totalmix) and we evaluated both early androgen-sensitive line with this, EFSA and ECHA propose tiered strategies for component- endpoints (AGD, nipple retention, reproductive organ weights) and based mixture risk assessment, where at the first tiers chemicals are late-life effects such as sperm count. Doses were selected based on our grouped on basis of shared effects on target organs, and at more refined previous rat studies showing effects on a common adverse endpoint, tiers chemicals are grouped based on mode or mechanism of action male anogenital distance (AGD) (Boberg et al., 2016; Christiansen et al., (ECHA, 2017; EFSA, 2019, 2013). 2010; Christiansen et al., 2014; Hass et al., 2012, 2007), see Fig. 1. The In developmental and reproductive toxicity studies, commonly used lowest dose of each compound corresponded to around 5% reduction in predictive endpoints for male reproductive disorders are the anogenital AGD in previous studies (Emix-low, Amix-low, Totalmix-low). The distance (AGD) and nipple retention (NR). Both are considered an- highest doses were twice the lowest dose. drogen-sensitive and are expected to be primarily affected by anti-an- Acknowledging that androgens play a key role not only in early life drogens (Christiansen et al., 2008; Schwartz et al., 2019). Other end- sexual development but also during puberty (Monosson et al., 1999), points such as sperm count and development of testis, mammary gland we wanted to investigate if exposure to the same mixtures of EDs both and prostate can be influenced via both androgen and estrogen sensitive perinatally and during puberty would lead to an increased sensitivity pathways. for adverse effects later in life. This part of the study design was in- Importantly, numerous EDCs act via more than one endocrine me- spired by a study showing how adverse effects of perinatal BPA ex- chanism of action, thus