Hershberger Assays for Di-2-ethylhexyl Phthalate and Its Substitute Candidates

INTRODUCTION

Phthalates or phthalate esters, are esters of phthalic acid, and they are mainly used as plasticizers, i.e., substances added to plastics to increase their flexibility, transparency, durability, and longevity (Cao, 2009). Since the plasticized materials are indispensable in everyday lives, the majority of people in modernized nations are exposed to some level of phthalates (Meeker et al., 2008). For example, multiple phthalates metabolites were detected in human urine from several Asian countries including South Korea (~Guo et al., 2011).

Di-2-ethylhexyl phthalate (DEHP) is the most common member of the phthalates family, and along with other phthalates, DEHP is known to cause endocrine disruption. Indeed, numerous studies have demonstrated a potential for DEHP to adversely impact the male and female reproductive systems such are abnormal reproductive tract formation, lower fertility, pregnancy loss, abnormal puberty onset, endometriosis, and abnormal breast development (Gray et al., 2000; Lyche et al., 2009; Martino-Andrade & Chahoud, 2010; Huang et al., 2012; Kay et al., 2013).

Due to many detrimental effects of DEHP, particularly on the fetus and newborn, the task making plastic products with DEHP alternatives becomes much more urgent. Although there are some biological alternatives on the market, the common problem is that they are typically expensive and not compatible as a primary plasticizer. Concurrently, the potential negative effects of the candidates should be accurately and efficiently screened. Previously we evaluated the two bisphenol-A (BPA) substitute candidates whether they have androgenic or antiandrogenic activity using Hershberger assay, and found isosorbide could be a safer candidate while cyclohexanedimethanol exerted more detrimental effects (Kim et al., 2017). In the present study, we employed the identical in vivo animal model system to determine possible androgenic or antiandrogenic activities of three DEHP substitute candidates.

MATERIALS AND METHODS

1. Chemicals

Testosterone propionate (TP), bis(2-ethylhexyl) phthalate (DEHP, CAS number 117-81-7), triethyl 2-acetylcitrate (ATEC, CAS number 77-89-4) and corn oil were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2-ethylhexyl oleate (IOO, CAS number 26399-02-0) and 2-ethylhexyl stearate (IOS, CAS number 22047-49-0) were donated from SFC Co. (Seoul, Korea). All substances were stored in glass containers at room temperature (Table 1).

Table 1. Chemical structure of DEHP and its substitute candidates

RESULTS

No abnormal clinical wounds or body weight changes were observed in the rats administered with the test chemicals.

DISCUSSION

DEHP is well known to have potential hazards on animal and human health, such as reproductive and developmental toxicity via disruption of the endocrine system (Zarean et al., 2016). As a typical example, DEHP and some other phthalates act as anti-androgens and can cause cryptorchidism in rodents and probably in human. One plausible hypothesis for occurrence of this symptom is an exposure to a chemical (or a mixture of chemicals) with anti-androgenic or estrogenic properties before and during the critical timing of external genital differentiation (Toppari et al., 2006). Indeed, maternal DEHP exposure could disrupt fetal testicular development (Shirota et al., 2005) and testosterone production (Parks et al., 2000; Howdeshell et al., 2008), and these findings support the hypothesis.

Since DEHP is the most common phthalate plasticizer in medical devices such as intravenous tubing and bags, special concern has been expressed about leachates of DEHP transported into the patient, especially for those requiring extensive infusions or those who are at the highest risk of developmental abnormalities, e.g. neonates, premature babies, lactating and pregnant women (Tickner et al., 2001; Latini et al., 2010). Therefore, development of plastic products with DEHP alternatives which can ensure safety becomes an urgent issue. Several bio-based plasticizers have been and are being developed as alternatives to phthalates. Among them, a plasticizer based on vegetable oil which is compatible as a primary plasticizer has been developed and is a ready substitute for dioctyl phthalate (Privas et al., 2013). Isosorbide, a BPA replacement, is a non-toxic biodegradable diol derived from bio-based feedstock, and it can be used for preparing thermoplastic starch through a semi-industrial process of extrusion, allowing some technological advantages with respect to classical plasticizers (Battegazzore et al., 2015).

Although there are some biological alternatives on the market, the problem is that they are typically expensive and not coISmpatible as a primary plasticizer. Furthermore, the potential negative effects of the candidates could not be screened accurately and efficiently. For example, there is ample of evidence on the adverse effects of bisphenol-S (BPS), an alternative of BPA (Usman & Ahmad, 2016; Rosenfeld, 2017). The Hershberger Bioassay is a short-term in vivo screening test using accessory tissues of the male reproductive tract to detect compounds with potential to exert androgenic or antiandrogenic activities (EPA, 2011; OECD, 2008). This assay is relatively convenient and reliable because five androgen-dependent organs, in castrated rats, sensitively respond to chemical with androgenic or antiandrogenic activities as an increase or decrease in absolute weights (Marty & O’Connor, 2014). We previously evaluated the two BPA substitute candidates whether they have androgenic or antiandrogenic activity using Hershberger assay, and found isosorbide could be a safer candidate while cyclohexanedimethanol exerted more detrimental effects (Kim et al., 2017).

In the present study, we found that all test materials including DEPC did not exhibit any androgenic activity in the Hershberger assay. Rather, antiandrogen-like activities were found in all test materials, and the order of the intensity was ATEC < DEHP < ISO < IOO in 5 androgen-sensitive organs. There is no available information on the action mechanism of the test materials (i.e. their receptors), so we tentatively concluded the weight reduction effects of these materials are derived from antiandrogen-like activities. Meanwhile, no androgenic or antiandrogenic activities of the test materials were found in the androgen-insensitive organs used in this study. Generally, the adverse effects of ATEC treatment on the accessory sex organs were much less or not present at all when compared to those of DEHP. So we concluded that ATEC could be a DEHP substitute. IOO treatment brought most severe weight reduction in all of androgen-sensitive organs, so this material should be excluded for further screening of DEHP substitute selection.