Migration Studies and Endocrine Disrupting Activities: Chemical Safety of Cosmetic Plastic Packaging.

The endocrine activity and endocrine disruptor (ED) chemical profiles of eleven plastic packaging materials covering five major polymer types (3PET, 1HDPE, 4LDPE, 2 PP, and 1SAN) were investigated using in vitro cell-based reporter-gene assays and a non-targeted chemical analysis using gas chromatography coupled to mass spectrometry (GC-MS). To mimic cosmetic contact, six simulants (acidic, alkaline, neutral water, ethanol 30%, glycerin, and paraffin) were used in migration assays performed by filling the packaging with simulant. After 1 month at 50 °C, simulants were concentrated by Solid Phase Extraction (SPE) or Liquid-Liquid Extraction (LLE). The migration profiles of seven major endocrine disrupting chemicals detected from GC-MS in the different materials and simulants were compared with Estrogen Receptor (ER) and Androgen Receptor (AR) activities. With low extraction of ED chemicals in aqueous simulants, no endocrine activities were recorded in the leachates. Paraffin was shown to be the most extracting simulant of antiandrogenic chemicals, while glycerin has estrogenic activities. Overall, ED chemical migration in paraffin was correlated with hormonal activity. The NIAS 2,4-di-tert-butyl phenol and 7,9-di-tert-butyl1-oxaspiro (4,5) deca-6,9-diene-2,8-dione were two major ED chemicals present in all polymers (principally in PP and PE) and in the highest quantity in paraffin simulant. The use of glycerin and liquid paraffin as cosmetic product simulants was demonstrated to be relevant and complementary for the safety assessment of released compounds with endocrine activities in this integrated strategy combining bioassays and analytical chemistry approaches.

In vitro and in silico approach to study the hormonal activities of the alternative plasticizer tri-(2-ethylhexyl) trimellitate TEHTM and its metabolites.

Tri-(2-ethylhexyl) trimellitate (TEHTM) is a plasticizer for polyvinyl chloride (PVC) material used in medical devices. It is an alternative to di-(2-ethylhexyl) phthalate (DEHP), a well-known reprotoxic and endocrine disruptor. As plasticizers are known to easily migrate when in contact with fatty biological fluids, patient exposure to TEHTM is highly probable. However, there is currently no data on the potential endocrine-disrupting effects of its human metabolites. To evaluate the effects of TEHTM metabolites on endocrine activity, they were first synthesized and their effects on estrogen, androgen and thyroid receptors, as well as steroid synthesis, were investigated by combining in vitro and in silico approaches. Among the primary metabolites, only 4-MEHTM (4-mono-(2-ethylhexyl) trimellitate) showed agonist activities on ERs and TRs, while three diesters were TR antagonists at non-cytotoxic concentrations. These results were completed by docking experiments which specified the ER and TR isoforms involved. A mixture of 2/1-MEHTM significantly increased the estradiol level and reduced the testosterone level in H295R cell culture supernatants. The oxidized secondary metabolites of TEHTM had no effect on ER, AR, TR receptors or on steroid hormone synthesis. Among the fourteen metabolites, these data showed that two of them (4-MEHTM and 2/1-MEHTM) induced effect on hormonal activities in vitro. However, by comparing the concentrations of the primary metabolites found in human urine with the active concentrations determined in bioassays, it can be suggested that the metabolites will not be active with regard to estrogen, androgen, thyroid receptors and steroidogenesis-mediated effects.

Comparative Effects of Di-(2-ethylhexyl)phthalate and Di-(2-ethylhexyl)terephthalate Metabolites on Thyroid Receptors: In Vitro and In Silico Studies.

Plasticizers added to polyvinylchloride (PVC) used in medical devices can be released into patients' biological fluids. Di-(2-ethylhexyl)phthalate (DEHP), a well-known reprotoxic and endocrine disruptor, must be replaced by alternative compounds. Di-(2-ethylhexyl) terephthalate (DEHT) is an interesting candidate due to its lower migration from PVC and its lack of reprotoxicity. However, there is still a lack of data to support the safety of its human metabolites with regard to their hormonal properties in the thyroid system. The effects of DEHT metabolites on thyroid/hormone receptors (TRs) were compared in vitro and in silico to those of DEHP. The oxidized metabolites of DEHT had no effect on T3 receptors whereas 5-hydroxy-mono-(ethylhexyl)phthalate (5-OH-MEHP) appeared to be primarily an agonist for TRs above 0.2 µg/mL with a synergistic effect on T3. Monoesters (MEHP and mono-(2-ethylhexyl)terephthalate, MEHT) were also active on T3 receptors. In vitro, MEHP was a partial agonist between 10 and 20 µg/mL. MEHT was an antagonist at non-cytotoxic concentrations (2-5 µg/mL) in a concentration-dependent manner. The results obtained with docking were consistent with those of the T-screen and provide additional information on the preferential affinity of monoesters and 5-OH-MEHP for TRs. This study highlights a lack of interactions between oxidized metabolites and TRs, confirming the interest of DEHT.

Lemon Juice, Sesame Paste, and Autoclaving Influence Iron Bioavailability of Hummus: Assessment by an In Vitro Digestion/Caco-2 Cell Model.

Hummus, an iron-containing plant-based dish mainly made from chickpea purée, tahini, lemon juice and garlic, could be a valuable source of iron when bioavailable. Since the processing and formulation of food influence iron bioavailability, the present study investigated for the first time, their effects on hummus. Firstly, iron bioaccessibility was assessed on eight samples (prepared according to the screening Hadamard matrix) by in vitro digestion preceding iron dialysis. Then, iron bioavailability of four selected samples was estimated by the in vitro digestion/Caco-2 cell model. Total and dialyzable iron were determined by the atomic absorption spectrometry and ferritin formation was determined using an ELISA kit. Only autoclaving, among other processes, had a significant effect on iron bioaccessibility (+9.5, p < 0.05). Lemon juice had the highest positive effect (+15.9, p < 0.05). Consequently, the effect of its acidic components were investigated based on a full factorial 23 experimental design; no significant difference was detected. Garlic's effect was not significant, but tahini's effect was negative (-8.9, p < 0.05). Despite the latter, hummus had a higher iron bioavailability than only cooked chickpeas (30.4 and 7.23 ng ferritin/mg protein, respectively). In conclusion, hummus may be a promising source of iron; further in vivo studies are needed for confirmation.

In vitro and in silico hormonal activity studies of di-(2-ethylhexyl)terephthalate, a di-(2-ethylhexyl)phthalate substitute used in medical devices, and its metabolites.

Plasticizers added to polyvinylchloride used in medical devices can be released into patients' biological fluids. The substitution of di-(2-ethylhexyl)phthalate (DEHP) by alternative plasticizers is essential but their safety must be demonstrated. DEHP, di-(2-ethylhexyl)terephthalate (DEHT) and their metabolites were investigated using level 2 Organization for Economic Co-operation and Development bioassays to screen for in vitro hormonal changes. Differences between the DEHP and DEHT metabolites were observed. Albeit weak, the hormonal activities of DEHT-derived metabolites, e.g., 5-OH metabolite of mono-(ethylhexyl)terephthalate (5-OH-MEHT), were detected and the results of docking experiments performed on estrogen receptor alpha and androgen receptor agreed with the biological results. A co-stimulation of human estrogen receptor alpha and human androgen receptor was also observed. With regard to steroidogenesis, a 16-fold increase in estrogen synthesis was measured with 5-OH-MEHT. Therefore, even if DEHT remains an interesting alternative to DEHP because of its low migration from medical devices, it seems important to verify that multi-exposed patients in neonatal intensive care units do not have urinary levels of oxidized metabolites, in particular 5-OH-MEHT, suggesting a potential endocrine-disrupting effect.

Chemical analysis and potential endocrine activities of aluminium coatings intended to be in contact with cosmetic water.

The objective of the work was to check the presence of Non-Intended Added Substances (NIAS) with hormonal activities in aluminium coatings extracts coded: AA, BBF, MC and RR, furnished by four different suppliers. Water samples were prepared at room temperature or at 40°C for three months to verify the storage effect on the coatings. Solid phase extraction was used to concentrate and to extract coating substances. Hormonal activities were checked in vitro using reporter gene bioassays. Except BBF, all extracts induced a weak but significant estrogenic agonist activity in the human cell line. Using an estrogenic antagonist (ICI-182, 780), the answer was demonstrated specific in the bioassay. RR was the only extract to induce a concentration dependent anti-androgenic response in the MDA-KB2 cell line. Analysis performed using GC-MS and HPLC-MS detected 12 substances in most of the extracts. 8 NIAS were present. Among them, 4 were identified with certainty: HMBT, BGA, DCU and BPA. Estrogenic potency was BPA>DCU>BGA>HMBT. HMBT was also anti-androgenic at high concentration. Combining chemical analysis and bioassays data, we demonstrated that in the RR and the RR40 extracts, the observed estrogenic response was mainly due to BPA, the anti-androgenic activity of RR could be due to a synergism between HMBT and BPA. For MC and AA, estrogenic responses appear to be due to the presence of DCU. Except BBF, storage conditions tended to increase estrogenic activities in all extracts. However, in term of risk assessment, activities observed were negligible. This work demonstrated that sensitive bioassays are pertinent tools in complement to chemical analysis to monitor and check the presence of NIAS with hormonal activity in coating extracts.

Use of bioassays to assess hazard of food contact material extracts: State of the art.

This review focuses on the use of in vitro bioassays for the hazard assessment of food contact materials (FCM) as a relevant strategy, in complement to analytical methods. FCM may transfer constituents to foods, not always detected by analytical chemistry, resulting in low but measurable human exposures. Testing FCM extracts with bioassays represents the biological response of a combination of substances, able to be released from the finished materials. Furthermore, this approach is particularly useful regarding the current risk assessment challenges with unpredicted/unidentified non-intentionally added substances (NIAS) that can be leached from the FCM in the food. Bioassays applied to assess hazard of different FCM types are described for, to date, the toxicological endpoints able to be expressed at low levels; cytotoxicity, genotoxicity and endocrine disruption potential. The bioassay strengths and relative key points needed to correctly use and improve the performance of bioassays for an additional FCM risk assessment is developed. This review compiles studies showing that combining both chemical and toxicological analyses presents a very promising and pragmatic tool for identifying new undesirable NIAS (not predicted) which can represent a great part of the migrating substances and/or "cocktail effect".

In vitro toxicity assessment of extracts derived from sol-gel coatings on polycarbonate intended to be used in food contact applications.

Polycarbonate is a widely used polymer in food contact applications all around the world. However, due to the potential release of Bisphenol A (BPA) during repeated washing cycles, its use becomes compromised as BPA is known for being an endocrine disruptor for rodents. In order to tackle this issue, sol-gel coatings based on organoalkoxysiloxane were developed on PC, to act as a physical barrier. To this end, two sol-gel systems based on tetraethylorthosilicate (TEOS), methyltriethoxysilane (MTES) and 3-glycidyloxypropyltriethoxysilane (GPTES), three common sol-gel precursors, were prepared. The coatings derived from the latter two systems were then studied with regards to their potential toxicity in vitro. Migration tests were performed in food simulants, and the maximal migration was obtained in ethanol 10% (v/v) for one system and in isooctane for the other one. In vitro genotoxicity was assessed with the Ames test (OECD 471) and the micronucleus assay (OECD 487), and no genotoxic effect was observed. Moreover, the estrogenic activity of the extracts was studied with a transcriptional activation assay using transient transfection in human cells; none of the extracts was found estrogenic. These negative in vitro results are highly promising for the future use of these new barrier coating formulations onto food contact materials.

In vitro genotoxicity assessment of MTES, GPTES and TEOS, three precursors intended for use in food contact coatings.

Organoalkoxysilanes are precursors that are used increasingly in the synthesis of food contact coatings. To comply with the EU regulation, their potential toxicity must be assessed, and very little information is known. The genotoxicity of three common precursors was studied, namely, tetraethylorthosilicate (TEOS), methyltriethoxysilane (MTES) and 3-glycidyloxypropyltriethoxysilane (GPTES). By the Ames test, MTES and TEOS were not mutagenic for bacteria. A significant positive response was observed with GPTES in the TA100 and TA1535 strains. The mutagenic effect was more pronounced in the presence of the exogenous metabolic activation system with an increase of the induction factor (ten-fold higher for the TA1535 strain). In the micronucleus assay performed with a human hepatoma cell line (HepG2 cells), GPTES gave negative results even in the presence of an exogenous activation system. To ascertain the possibility of using this precursor in food contact material, its migration must be monitored according to the coating formulation because migration might result in hazardous human exposure.

Evaluation of the sensitivity of three sublethal cytotoxicity assays in human HepG2 cell line using water contaminants.

The in vitro toxicological index IC50 (the millimolar concentration of compound which inhibits response assay by 50% compared to the solvent control) of 11 water contaminants (acrylamide, atrazine, B[a]P, BPA, 2,4-DAT, 17-alphaEE, H(2)O(2), 4-OP, sodium bromate, sodium chlorate, sodium nitrate) was evaluated on the human hepatoma (HepG2) cells using three short-term bioassays related to their morbidity status [radiometric RNA synthesis assay (RNA), luminometric ATP assay (ATP), fluorometric Alamar blue assay (AB)]. Among all substances, we were not able to determine atrazine IC50 value whatever the test used. Furthermore, B[a]P was not cytotoxic in the ATP and AB assays. Statistical analysis revealed a correlation between the IC50 values obtained in the three assays. Except with 4-OP, RNA assay was always inhibited at lower concentrations than those required in the other assays, suggesting that this assay is a very sensitive indicator of the presence of toxic compounds. ATP and AB assays responded to a similar pattern. Due to its higher sensitivity and its reliability, RNA synthesis assay using HepG2 cell line provides the most suitable tool for the screening of water contaminants.

2,4-Diaminotoluene (2,4-DAT)-induced DNA damage, DNA repair and micronucleus formation in the human hepatoma cell line HepG2.

2,4-Diaminotoluene (2,4-DAT) is a widely used industrial intermediate and human exposure is possible in the dye and plastics industries. We investigated the genotoxicity of the environmental pollutant, 2,4-DAT, in human HepG2 cells using the unscheduled DNA synthesis (UDS) test, the micronucleus (MN) assay and single-cell gel electrophoresis (SCGE). 2,4-DAT was first tested by the RNA synthesis inhibition test as a cytotoxicity assay: the IC(50) of 2,4-DAT was 5.2 mM after 20 h of exposure. The compound had a genotoxic effect at concentrations from 1.45 to 6.80 mM in both micronucleus and comet assays. In the micronucleus assay, the number of MN/1000 BNC was 3.5 times higher at a concentration of 6.80 mM 2,4-DAT than in the negative control. At the same concentration, DNA migration (SCGE) showed an Olive tail moment (OTM) of 3.56+/-0.45, as compared to 0.19+/-0.02 for the negative control. The UDS test detected genotoxic effects at lower concentrations than did the other assays (0.01-5 mM). The percentage of cells in repair increased in a concentration-dependent manner to a maximum of 57% at 1mM. At the highest concentration tested (5 mM), the NNG/cell score was 13.6+/-0.5 whereas it was -2.7+/-0.5 for the negative control. These data, based on various endpoints, show a midly genotoxic effect of 2,4-DAT in the HepG2 cells and confirm that this cell line is a suitable model to study the toxic effects of aromatic amines.