In vitro cytotoxic effects of secondary metabolites of DEHP and its alternative plasticizers DINCH and DINP on a L929 cell line

https://doi.org/10.1016/j.ijheh.2019.03.005Get rights and content

Highlights

  • 7-oxo-MMeOCH and 5-oxo-MEHP induce no action on cell proliferation at 0.1 mg/mL.

  • Among secondary metabolites of DEHP, 5-oxo-MEHP induce an action on L929.

  • Among secondary metabolites of MMeOCH, 7-oxo-MMeOCH induce an action on L929.

  • Secondary metabolites of MMeOCH are less toxic than MMeOCH.

  • Secondary metabolites of MMeOP are no toxic like MMeOCH.

Abstract

Background

Phthalic acid esters are widely used to improve the plasticity of PVC in medical devices (MD). The most famous plasticizer is DEHP, whose use in medical devices has been contested by the European authorities since 2008. Several alternative plasticizers are being considered to replace DEHP, such as DEHT, TOTM, DINP or DINCH, but they are also released from the PVC throughout their life cycle and are metabolized in the same way as DEHP.

Objectives

Our study focuses on the in vitro cytotoxicity of two alternative plasticizers (DINCH and DINP) contained in certain medical devices. They are likely to migrate and be transformed in vivo into the primary and secondary metabolites by a metabolism similar to that of DEHP. This preliminary study is the first to assess the in vitro cytotoxicity of oxidized metabolites of DINCH and DINP based on the EN ISO 10-993-5 standards documents.

Methods

We have studied the complete multi-step organic synthesis of secondary metabolites of DINP and DINCH and have performed cytotoxicity tests on L929 murine cells according to the EN ISO 10993-5 standard design for the biocompatibility of a MD. The tested concentrations of obtained metabolites (0.01, 0.05 and 0.1 mg/mL) covered the range likely to be found for DEHP (total metabolism) in biological fluids coming into direct contact with the MD. The concentrations tested in our study were chosen based on a complete transformation of the plasticizers released after direct contact between a MD and the patient's blood.

Results

Only 7-oxo-MMeOCH is cytotoxic at the highest concentration (0.1 mg/mL) after 7 days of exposure, just like 5-oxo-MEHP for the same concentration. By contrast, 7-OH-MMeOP, 7-cx-MMeOP, 7-oxo-MMeOP, 7-OH-MMeOCH and 7-cx-MMeOCH were not found to be cytotoxic.

Conclusion

The known concentrations of these secondary metabolites in urinary samples are in the μg/L range, i.e. about 100–1000 times lower than the concentrations tested in this study. Cytotoxicity is known to be dose-dependent but it is not always the case for endocrine perturbations and the secondary metabolites could induce endocrine perturbations at very low doses.

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.

References (38)

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Both authors contributed equally.

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