International Journal of Hygiene and Environmental Health
In vitro cytotoxic effects of secondary metabolites of DEHP and its alternative plasticizers DINCH and DINP on a L929 cell line
Graphical abstract
Introduction
Some phthalate esters present a general concern for public health due to their wide use in the manufacturing of polymeric materials and various consumer products. Some phthalate esters can be classified into two categories according to the number of carbons in the ester chain. On the one hand, there are those with a low molecular weight (number of carbon inferior to 6) which are used as industrial solvents, lubricants, and as components in personal-care products. On the other hand, those with a high molecular weight are commonly used as plasticizers, conferring better flexibility and durability to everyday polyvinyl chloride (PVC) consumer products, such as flooring, food packaging, clothing, children's toys and medical devices (Hauser and Calafat, 2005). These PVC plasticizers can be released from the polymers by volatilization into the air, abrasion of the polymer, leaching into liquids and direct diffusion from the polymer into dust on the polymer surface, resulting in subsequent human exposure which might cause adverse health effects (Giovanoulis G et al., 2018; Zhang, S. et al., 2016; Engel, A. et al., 2017, 2018). With regard to their presence in PVC medical devices, the most famous plasticizers is DEHP, whose use has been contested by the European authorities (SCENHIR, 2008, 2016). The toxic potential of DEHP is largely due to the metabolic transformation into more toxic metabolites by hydrolysis (MEHP) and subsequent oxidation reactions (5-OH-MEHP, 5-oxo-MEHP, 5-cx-MEHP) (Fig. 1A). Numerous studies have been carried out on DEHP, leading to its classification as carcinogenic, mutagenic or toxic for reproduction (CMR1B) because of its toxic effects on reproduction and fertility in rodents. Its use is now highly controlled in medical devices. The European regulation 2017/745 recommends not to exceed a rate of 0.1% in mass fraction in each medical device (Regulation (EU) 2017/745). Two alternative plasticizers (diisononyl phthalate (DINP) and 1,2-cyclohexane dicarboxylic acid (DINCH)) have been considered by manufacturers as a replacement for DEHP in many PVC products, such as medical devices and food packaging, but are limited in toys with concentrations inferior to 0.1% by weight of the plasticized material (European Chemical Agency, 2013; SCENHIR, 2016), due to their physicochemical properties suggesting a lower migration and more favorable toxicological profile (Fromme et al., 2016; Bhat et al., 2014) than DEHP. DINP and DINCH respectively constitute a complex commercial mixture composed mainly of isomers of phthalate or dialkyl cyclohexanoate with 9 carbon chains. DINCH (Hexamoll®) is manufactured by catalytic hydrogenation of the aromatic ring of diisononyl phthalate (Palatinol® N) into the cyclohexane ring existing predominantly in the most stable chair configuration, allowing cis- and trans-configuration. The typical commercial products consist of 90% cis- and 10% trans-isomers. In both molecules, the 4-methyl isomer forms a fraction that represents less than 50% of the total C9 alcohols (Schütze et al., 2017). Like DEHP, they are not covalently bonded to the PVC to which they are mixed. As a result, they are released from the products throughout their life cycle (Bernard et al., 2015; Jeon et al., 2016). Because they are a mixture of isomers, their metabolites are also isomeric mixtures (Silva et al., 2013). Both are initially hydrolyzed in rodents and humans into their monoester (MINP or MINCH), followed by oxidative processes into several metabolites including mono-hydroxy, mono-oxo and mono-carboxymetabolites (Silva et al., 2007a, 2007b; Koch et al., 2007, Koch and Angerer, 2007, 2013) (Fig. 1B).
Some biomonitoring studies carried out in humans have made it possible to demonstrate the presence of DINCH and DINP metabolites in biological media such as urine, blood and nails (Giovanoulis et al., 2016, 2018, Correia-Sa et al., 2017, Schütze et al., 2012, 2017, Zeman et al., 2013; Koch et al., 2017, Johns et al., 2015). However, despite the animal toxicology data and the in vitro toxicity of the plasticizers, there is almost no information on the in-vitro toxicity of the oxidized metabolites of DINP and DINCH from medical devices except the urinary metabolites of DINCH, which seem to have an impact on the activities of the human nuclear receptors ERα, ERβ, AR, PPARα and PPARγ. However, the in vitro data does not support the notion that DINCH or any of the investigated metabolites may exert considerable endocrine effects in vivo at relevant human exposure levels. (Engel et al., 2017, 2018). In a previous study we investigated the cytotoxicity of DEHP, DINCH, DINP and their corresponding primary unconjugated metabolites (MEHP, MINCH, MMeOCH, MINP, respectively), and showed that the primary metabolites are more cytotoxic than their corresponding plasticizer at concentrations between 0.01 and 0.1 mg/mL. Only MMeOP, the primary metabolite of DINP, is not cytotoxic at these concentrations (Eljezi et al., 2017). Taking into account that DINP and DINCH are metabolized in the same way as DEHP, secondary metabolites are also found in the urine (7-oxo-MMeOCH, 7-OH-MMeOCH, 7-cx-MMeOCH, 7-oxo-MMeOP, 7-OH-MMeOP and 7-cx-MmeOP) in a glucuroconjugated form or not. According to the data in the literature, it appears that 26% of cx-MINCH are excreted in its unconjugated form in urine (Koch et al., 2013). These results are similar to the previous work concerning DEHP and DINP (Silva et al., 2013). In continuation of our work on alternative plasticizers to DEHP, we therefore undertook the total synthesis of the secondary metabolites of DINP and DINCH in order to be able to carry out a first cytotoxicity study on the NCTC clone 929 in accordance with the EN ISO 10-993-5 standards documents.
Section snippets
Chemicals, biochemicals and reagents
Unless otherwise mentioned, all manipulations were performed under an argon atmosphere. All reagents were purchased from the following commercial suppliers: Sigma-Aldrich, Acros Organics, Carlo Erba, TCI Europa, and Alpha Aesar. Anhydrous DMF and anhydrous triethylamine were purchased from Acros Organics. THF was distilled over benzophenone and sodium. Dichloromethane was distilled over calcium hydride. Nuclear magnetic resonance (NMR) spectra were acquired on a Bruker AC-200 or AC-400
Synthesis of MeDINCH and MeDINP secondary metabolites
The three metabolites of the MeDINCH plasticizer, i.e. 7-oxo-MMeOCH, 7-OH-MMeOCH and 7-cx-MMeOCH, were synthesized from monoprotected alcohol 1 (Eljezi et al., 2017) and hexahydroisobenzofuran-1,3-dione (Scheme 1). Acid compound 2 was esterified, followed by a catalytic hydrogenation to produce compound 4. The hydroxy group at ω-position of the alkyl chain was oxidized into an aldehyde. Compound 5 reacted with isopropyltriphenylphosphonium iodide (Wittig reaction) to obtain vinyl derivative 6.
Discussion
For several years, our research program has focused on DEHP and some of the corresponding alternatives in medical devices. DINCH and DINP are two plasticizers commonly used as alternatives to DEHP in PVC medical devices. DINCH is used in parenteral and enteral nutrition tubings as well as in hemodialysis lines (Strømmen et al., 2016). DINP is added to some PVC infusions tubings or blood transfusion sets (Frederiksen et al., 2014; Bernard et al., 2015; Bourdeaux et al., 2016). Like DEHP, these
Declaration of interest
This study is a part of the ARMED® project and received financial support from the French National Agency for the Safety of Medicines and Health Products (ANSM) Grant/award number : AAPR-2012-009.
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Both authors contributed equally.