Estrogenic, androgenic, and genotoxic activities of zearalenone and deoxynivalenol in in vitro bioassays including exogenous metabolic activation.

Zearalenone (ZEN) and deoxynivalenol (DON) and their derivatives are well-known mycotoxins, which can occur not only in crops but also in water bodies, including drinking water sources. In vitro bioassays can be used to detect biological effects of hazardous compounds in water. To this, when studying biological effects and toxicity in vitro, metabolism is important to consider. In this study, ZEN, α-zearalenol (α-ZEL), DON, 3-acetyl DON, and 15-acetyl DON were evaluated in vitro for hormone receptor-mediated effects (estrogen receptor [ER] and androgen receptor [AR]) and genotoxicity (micronucleus assay) in the presence of an exogenous metabolic activation system (MAS). The ER bioassay proved to be a highly sensitive method to detect low concentrations of the ZEN compounds (EC10 values of 31.4 pM for ZEN, 3.59 pM for α-ZEL) in aqueous solutions. In the presence of the MAS, reduced estrogenic effects were observed for both ZEN compounds (EC10 values of 6.47 × 103 pM for ZEN, 1.55 × 102 pM for α-ZEL). Of the DON compounds, only 3-acetyl DON was estrogenic (EC10 of 0.31 µM), and the effect was removed in the presence of the MAS. Anti-androgenic effects of the ZEN compounds and androgenic effects of the DON compounds were detected in the micromolar range. No induction of genotoxicity was detected for ZEN or DON in the presence of the MAS. Our study highlighted that inclusion of exogenous MAS is a useful tool to detect biological effects of metabolites in in vitro bioassays.

Impact of sample acidification and extract storage on hormone receptor-mediated and oxidative stress activities in wastewater.

An underemphasized aspect of sampling strategies in effect-based in vitro testing is to determine suitable collection and preparation techniques. In the current study, the impact of sample acidification on bioactivities was assessed using in vitro bioassays for hormone receptor-mediated effects (estrogen receptor [ER] and androgen receptor [AR]) and the oxidative stress response (Nrf2 activity). Sampling was conducted at a recently upgraded Swedish wastewater treatment plant. Future plans for the treated wastewater include reuse for irrigation or as a potential drinking water source. In the AR and Nrf2 assays, acidification decreased bioactivities in the wastewater influent sample extracts, whereas acidification increased bioactivities following further treatment (disc filtration). In the ER assay, acidification had no impact on the observed bioactivities in the sample extracts. A secondary objective of the study was to assess the stability of the sample extracts over time. Lower activities were detected in the ER and AR assays in all extracts after storage for approximately 1 year. Nrf2 activities did not decrease over time, but rather increased in some of the acidified sample extracts. Overall, the findings suggest that sampling strategies involving acidification may need to be tailored depending on the selected bioassay(s) and the type of wastewater treatments being assessed.

Artificial infiltration in drinking water production: Addressing chemical hazards using effect-based methods.

Artificial infiltration is an established managed aquifer recharge method that is commonly incorporated into drinking water processes. However, groundwater sourced from this type of purification method is prone to contamination with chemical hazards. Such an instance was previously shown at a Swedish DWTP where the river water was contaminated by hazardous chemicals during artificial infiltration. Further, there remains a paucity of research studying the quality of drinking water following this type of treatment from an effect-based bioanalytical perspective. In the current study, an effect-based assessment for chemical hazards was conducted for a Swedish drinking water system comprised of two DWTPs fed artificially-infiltrated river water. In this system, artificial infiltration of the river water takes approximately six to eight months. A sampling event was conducted in the autumn season and the samples were enriched by solid phase extraction. A panel of cell-based reporter gene assays representing several toxicity pathways was selected: oxidative stress response (Nrf2 activity), aryl hydrocarbon receptor (AhR) activation, and hormone receptor-mediated effects (estrogen receptor [ER], androgen receptor [AR]). AhR and ER bioactivities were detected in samples collected from the river intake and in the open-air infiltration basins prior to artificial infiltration. However, the AhR activity decreased and ER activity was effectively removed following artificial infiltration. In the Nrf2 and AR assays, no bioactivities above cut-off levels were detected in any samples collected along the entire treatment process of the drinking water production from source to tap. Using a suite of bioassays, the current study highlighted the effectiveness of artificial infiltration in reducing bioactive compounds in this raw river water. Although artificial infiltration is a common purification method in drinking water production, the limited number of effect-based studies evaluating the effectiveness of this method emphasizes the need for further research to better understand the risks and benefits of this water treatment process.

An in vitro-based hazard assessment of liquid smoke food flavourings.

Liquid smoke products are widely used as a food additive to create a desired smoke flavour. These products may contain hazardous chemicals generated during the wood-burning process. However, the toxic effects of these types of hazardous chemicals constituting in the commercially available products are largely unknown. Therefore, a test battery of cell-based in vitro methods, covering different modes of actions of high relevance to human health, was applied to study liquid smoke products. Ten liquid smoke flavourings were tested as non-extracted and extracted. To assess the potential drivers of toxicity, we used two different solvents. The battery of in vitro methods covered estrogenicity, androgenicity, oxidative stress, aryl hydrocarbon receptor activity and genotoxicity. The non-extracted samples were tested at concentrations 0.002 to 1 µL liquid smoke flavouring/mL culture medium, while extracted samples were tested from 0.003 to 200 µL/mL. Genotoxicity was observed for nearly all non-extracted and all hexane-extracted samples, in which the former had higher potency. No genotoxicity was observed for ethyl acetate-extracted samples. Oxidative stress was activated by almost all extracted and non-extracted samples, while approximately half of the samples had aryl hydrocarbon receptor and estrogen receptor activities. This study used effect-based methods to evaluate the complex mixtures of liquid smoke flavourings. The increased bioactivities seen upon extractions indicate that non-polar chemicals are driving the genotoxicity, while polar substances are increasing oxidative stress and cytotoxic responses. The differences in responses indicate that non-extracted products contain chemicals that are able to antagonize toxic effects, and upon extraction, the protective substances are lost.

Assessment of source and treated water quality in seven drinking water treatment plants by in vitro bioassays - Oxidative stress and antiandrogenic effects after artificial infiltration.

Drinking water quality and treatment efficacy was investigated in seven drinking water treatment plants (DWTPs), using water from the river Göta Älv, which also is a recipient of treated sewage water. A panel of cell-based bioassays was used, including measurements of receptor activity of aryl hydrocarbon (AhR), estrogen (ER), androgen (AR), peroxisome proliferator-activated receptor alpha (PPARα) as well as induction of oxidative stress (Nrf2) and micronuclei formation. Grab water samples were concentrated by solid phase extraction (SPE) and water samples were analyzed at a relative enrichment factor of 50. High activities of AhR, ER and AR antagonism were present in WWTP outlets along the river. Inlet water from the river exhibited AhR and AR antagonistic activities. AhR activity was removed by DWTPs using granulated activated carbon (GAC) and artificial infiltration. AR antagonistic activity was removed by the treatment plants, except the artificial infiltration plant, which actually increased the activity. Furthermore, treated drinking water from the DWTP using artificial infiltration exhibited high Nrf2 activity, which was not found in any of the other water samples. Nrf2 activity was found in water from eight of the 13 abstraction wells, collecting water from the artificial infiltration. No genotoxic activity was detected at non-cytotoxic concentrations. No Nrf2 or AR antagonistic activities were detected in the inlet or outlet water after the DWTP had been replaced by a new plant, using membrane ultrafiltration and GAC. Neither target chemical analysis, nor chemical analysis according to the drinking water regulation, detected any presence of chemicals, which could be responsible of the prominent effects on oxidative stress and AR antagonistic activity in the drinking water samples. Thus, bioanalysis is a useful tool for detection of unknown hazards in drinking water and for assessment of drinking water treatments.

Hormetic Dose Response of NaAsO2 on Cell Proliferation of Prostate Cells in Vitro: Implications for Prostate Cancer Initiation and Therapy.

Sodium meta-arsenite (NaAsO2) has been suggested to play a role both in initiation/progression of prostate cancer and as a future antiprostate cancer drug. We have studied the effects of NaAsO2 on cell proliferation of prostate cancer and noncancer cells, breast cancer cells, and adrenocortical carcinoma cells in vitro. Further, we have investigated the effect of NaAsO2 on the androgen receptor. We report that NaAsO2 alters the cell proliferation of prostate cells, in a hormetic manner, by increasing cell proliferation at low concentrations and decreasing the cell proliferation at high concentrations. No activation of the androgen receptor was detected. We conclude that NaAsO2 is able to increase cell proliferation of prostate cells in vitro at low concentrations, while it decreases cell viability at high concentrations. This novel finding has to be further addressed if NaAsO2 should be developed into an antiprostate cancer drug.

Re-evaluation of sulphuric acid and its sodium, potassium, calcium and ammonium salts (E 513, 514 (i), 514 (ii), 515 (i), 515 (ii), 516 and 517) as food additive.

The Panel on Food Additives and Flavourings (FAF) provided a scientific opinion re-evaluating the safety of sulphuric acid (E 513) and its sodium (E 514), potassium (E 515), calcium (E 516) and ammonium (E 517) salts when used as a food additive. The Panel considered that adequate exposure and toxicity data were available. Sulphuric acid and its sodium, potassium, calcium and ammonium salts (E 513, E 514, E 515, E 516, E 517) are authorised food additives in the EU, in accordance with Annex II and Annex III to Regulation (EC) No 1333/2008. In the refined estimated exposure non brand-loyal scenario, mean exposure ranged from 0.4 mg sulphate/kg body weight (bw) per day in infants to 35 mg sulphate/kg bw per day in toddlers. The high percentile of exposure ranged from 3 mg sulphate/kg bw per day in adolescents to 68 mg sulphate/kg bw per day in toddlers. The Panel considered sulphates of low acute toxicity and there is no concern with respect to genotoxicity and carcinogenicity. The Panel noted that the exposure to sulphates at mean and 95th percentile in the non brand-loyal scenario as well as in the other scenarios, is far below the 300 mg/kg a dose that induced laxative effect in humans. Based on the toxicological database available, the Panel concluded that the exposure to sulphuric acid (E 513), sodium sulphate (E 514), potassium sulphates (E 515), calcium sulphate (E 516) and ammonium sulphate (E 517) does not raise a safety concern at the reported uses and use levels and there is no need for a numerical acceptable daily intake (ADI).

Re-evaluation of benzyl alcohol (E 1519) as food additive.

The Panel on Food Additives and Flavourings (FAF) provided a scientific opinion re-evaluating the safety of benzyl alcohol (E 1519) when used as a food additive. The Panel considered that adequate exposure and toxicity data were available. Benzyl alcohol (E 1519) is authorised as a food additive in the EU in accordance with Annex III to Regulation (EC) No 1333/2008. The Panel considered benzyl alcohol of low acute toxicity with no concern with respect to genotoxicity and carcinogenicity and established an acceptable daily intake (ADI) of 4 mg/kg body weight (bw) per day based on a no observable adverse effect level (NOAEL) of 400 mg/kg bw per day from the carcinogenicity study in rats. The mean and high exposure estimates in the refined exposure scenarios were maximally 0.27 and 0.81 mg/kg bw per day in toddlers, respectively. The exposure estimates to benzyl alcohol (E 1519) were below the ADI of 4 mg/kg bw per day in all population groups. The Panel noted that also the exposure in the regulatory maximum level exposure assessment scenario is below the ADI in all population groups. The Panel concluded that the exposure to benzyl alcohol (E 1519) does not raise a safety concern at the reported uses and use levels.

Safety of use of Monk fruit extract as a food additive in different food categories.

The EFSA Panel on Food Additives and Flavourings (FAF) provides a scientific opinion on the safety of Monk fruit extract proposed for use as a new food additive in different food categories. Monk fruit extracts are prepared by water extraction of the fruits of Siraitia grosvenorii. Cucurbitane glycosides, mogrosides, are the main components of the S. grosvenorii fruit and mogroside V is the main mogroside in the Monk fruit extract. Mogroside V is absorbed to some extent and is systemically bioavailable. Monk fruit extract containing 25% and 55% mogroside V were negative in the bacterial reverse mutation assay and did not induce structural and/or numerical chromosomal damage. However, the Panel noted that the in vitro toxicity studies including study with metabolic activation were not sufficiently informative to evaluate the genotoxic potential of the metabolites generated after microbial metabolism, including the aglycone. The effects on the testis observed in a 90-day study with monk fruit extract-52% mogroside V cannot be dismissed and the adversity of these effects cannot be ruled out. No effects on parental, reproductive or development toxicity were observed in a reproductive and developmental screening study in rats. For male animals, the time of exposure did not cover the full length of spermatogenesis and, therefore, a longer term study at higher doses would be needed to clarify the effects on testes observed in the 90-day study. No maternal and developmental toxicity was observed. Considering the systemic availability of mogroside V, the effects observed in the rat subchronic study and following the principles of EFSA Guidance on food additives evaluation, data from chronic/carcinogenicity toxicity testing would have been warranted. Exposure to mogroside V was calculated based on the proposed use levels. The Panel concluded that toxicity database on Monk fruit extract is insufficient to conclude on the safety of the use of Monk fruit extract as a food additive.

Re-evaluation of phosphoric acid-phosphates - di-, tri- and polyphosphates (E 338-341, E 343, E 450-452) as food additives and the safety of proposed extension of use.

The Panel on Food Additives and Flavourings added to Food (FAF) provided a scientific opinion re-evaluating the safety of phosphates (E 338-341, E 343, E 450-452) as food additives. The Panel considered that adequate exposure and toxicity data were available. Phosphates are authorised food additives in the EU in accordance with Annex II and III to Regulation (EC) No 1333/2008. Exposure to phosphates from the whole diet was estimated using mainly analytical data. The values ranged from 251 mg P/person per day in infants to 1,625 mg P/person per day for adults, and the high exposure (95th percentile) from 331 mg P/person per day in infants to 2,728 mg P/person per day for adults. Phosphate is essential for all living organisms, is absorbed at 80-90% as free orthophosphate excreted via the kidney. The Panel considered phosphates to be of low acute oral toxicity and there is no concern with respect to genotoxicity and carcinogenicity. No effects were reported in developmental toxicity studies. The Panel derived a group acceptable daily intake (ADI) for phosphates expressed as phosphorus of 40 mg/kg body weight (bw) per day and concluded that this ADI is protective for the human population. The Panel noted that in the estimated exposure scenario based on analytical data exposure estimates exceeded the proposed ADI for infants, toddlers and other children at the mean level, and for infants, toddlers, children and adolescents at the 95th percentile. The Panel also noted that phosphates exposure by food supplements exceeds the proposed ADI. The Panel concluded that the available data did not give rise to safety concerns in infants below 16 weeks of age consuming formula and food for medical purposes.

Re-evaluation of hydrochloric acid (E 507), potassium chloride (E 508), calcium chloride (E 509) and magnesium chloride (E 511) as food additives.

The Panel on Food Additives and Flavourings added to Food (FAF) provided a scientific opinion re-evaluating the safety of chlorides (E 507-509, E 511) as food additives. Chlorides are authorised food additives in the EU in accordance with Annex II and III to Regulation (EC) No 1333/2008. In the non- brand-loyal scenario, mean exposure to chlorides (E 507-509, E 511) as food additives ranged from 2 mg/kg body weight (bw) per day in the elderly to 42 mg/kg bw per day in toddlers. The 95th percentile of exposure ranged from 5 mg/kg bw per day in the elderly to 71 mg/kg bw per day in toddlers. Chloride is an essential nutrient and after absorption is distributed to organs and tissues. The Panel considered chlorides to be of low acute oral toxicity and there is no concern with respect to genotoxicity and carcinogenicity. No effects were reported in developmental toxicity studies in rats following administration of magnesium chloride hexahydrate at 800 mg/kg bw per day. Some animal studies suggested a role of chloride in increasing blood pressure but based on the toxicological database available the Panel considered human data more appropriate to identify a level of chloride intake which does not raise a safety concern. The Panel identified a human dose of 40 mg chloride/kg bw per day as a reference value for the assessment. Mean levels of exposure in all age groups were below or at this reference value, which indicates no safety concern. In some age groups (toddlers, children and adolescents), the 95th percentile exposure estimates were slightly above this reference value. The Panel concluded that the exposure to chloride from hydrochloric acid and its potassium, calcium and magnesium salts (E 507, E 508, E 509 and E 511) does not raise a safety concern at the reported use and use levels.

Effect-based assessment of recipient waters impacted by on-site, small scale, and large scale waste water treatment facilities - combining passive sampling with in vitro bioassays and chemical analysis.

Waste water treatment facilities are a major sources of organic micropollutants (MPs) in surface water. In this study, surface water samples were collected from seven sites along a river system in Uppsala, Sweden, during four seasons and evaluated based on the occurrence of MPs in the samples and bioactivity using in vitro bioassays. The sampling sites were differentially impacted by on-site sewage treatment facilities (OSSFs), small scale, and large scale waste water treatment plants (WWTPs). The bioassays used included activation of aryl hydrocarbon receptor (AhR), estrogen receptor (ER), nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB), nuclear factor erythroid 2-related factor 2 (Nrf2), and androgen receptor (AR). Occurrence of 80 MPs, were analyzed using liquid chromatography coupled to tandem mass spectrometry. Most water samples induced AhR activity, and all sampling sites showed a similar profile regarding this activity. With the exception of one water sample, we did not detect any NFkB, Nrf2 or AR activity of the water samples. The exception was a sample impacted by OSSFs, which showed an activity in multiple bioassays, but the activity could not be explained by the occurrence of target MPs. The occurrence of MPs showed a spatial trend, with the highest number and amount of MPs detected in the samples collected downstream of the WWTPs, where up to 47 MPs were detected in one single sample. A seasonal variation was observed with highest levels of MPs and highest AhR activities in samples collected in June and September 2015. However, neither the seasonal activity nor the on-site activity could be explained by the measured MPs, suggesting unknown contributory agents in the water.

Identification of a xenobiotic as a potential environmental trigger in primary biliary cholangitis.

BACKGROUND & AIMS: Primary biliary cholangitis (PBC) is an autoimmune-associated chronic liver disease triggered by environmental factors, such as exposure to xenobiotics, which leads to a loss of tolerance to the lipoic acid-conjugated regions of the mitochondrial pyruvate dehydrogenase complex, typically to the E2 component. We aimed to identify xenobiotics that might be involved in the environmental triggering of PBC. METHODS: Urban landfill and control soil samples from a region with high PBC incidence were screened for xenobiotic activities using analytical, cell-based xenobiotic receptor activation assays and toxicity screens. RESULTS: A variety of potential xenobiotic classes were ubiquitously present, as identified by their interaction with xenobiotic receptors - aryl hydrocarbon receptor, androgen receptor and peroxisome proliferator activated receptor alpha - in cell-based screens. In contrast, xenoestrogens were present at higher levels in soil extracts from around an urban landfill. Furthermore, two landfill sampling sites contained a chemical(s) that inhibited mitochondrial oxidative phosphorylation and induced the apoptosis of a hepatic progenitor cell. The mitochondrial effect was also demonstrated in human liver cholangiocytes from three separate donors. The chemical was identified as the ionic liquid [3-methyl-1-octyl-1H-imidazol-3-ium]+ (M8OI) and the toxic effects were recapitulated using authentic pure chemical. A carboxylate-containing human hepatocyte metabolite of M8OI, bearing structural similarity to lipoic acid, was also enzymatically incorporated into the E2 component of the pyruvate dehydrogenase complex via the exogenous lipoylation pathway in vitro. CONCLUSIONS: These results identify, for the first time, a xenobiotic in the environment that may be related to and/or be a component of an environmental trigger for PBC. Therefore, further study in experimental animal models is warranted, to determine the risk of exposure to these ionic liquids. LAY SUMMARY: Primary biliary cholangitis is a liver disease in which most patients have antibodies to mitochondrial proteins containing lipoic acid binding site(s). This paper identified a man-made chemical present in soils around a waste site. It was then shown that this chemical was metabolized into a product with structural similarity to lipoic acid, which was capable of replacing lipoic acid in mitochondrial proteins.

In vitro bioanalysis of drinking water from source to tap.

The presence of chemical pollutants in sources of drinking water is a key environmental problem threatening public health. Efficient removal of pollutants in drinking water treatment plants (DWTPs) is needed as well as methods for assessment of the total impact of all present chemicals on water quality. In the present study we have analyzed the bioactivity of water samples from source to tap, including effects of various water treatments in a DWTP, using a battery of cell-based bioassays, covering health-relevant endpoints. Reporter gene assays were used to analyze receptor activity of the aryl hydrocarbon receptor (AhR), estrogen receptor (ER), androgen receptor (AR), peroxisome proliferator-activated receptor alpha (PPARα) and induction of oxidative stress by the nuclear factor erythroid 2-related factor 2 (Nrf2). DNA damage was determined by Comet assay. Grab water samples were concentrated by HLB or ENV solid phase extraction and the water samples assayed at a relative enrichment factor of 50. The enrichment procedure did not induce any bioactivity. No bioactivity was detected in Milli-Q water or drinking water control samples. Induction of AhR, ER and Nrf2 activities was revealed in source to tap water samples. No cytotoxicity, PPARα or AR antagonist activity, or DNA damage were observed in any of the water samples. A low AR agonist activity was detected in a few samples of surface water, but not in the samples from the DWTP. The treatment steps at the DWTP, coagulation, granulated activated carbon filtration, UV disinfection and NH2Cl dosing had little or no effect on the AhR, Nrf2 and ER bioactivity. However, nanofiltration and passage through the distribution network drastically decreased AhR activity, while the effect on Nrf2 activity was more modest and no apparent effect was observed on ER activity. The present results suggest that bioassays are useful tools for evaluation of the efficiency of different treatment steps in DWTPs in reducing toxic activities. Bioassays of AhR and Nrf2 are useful for screening of effects of a broad range of chemicals in drinking water and ER activity can be monitored with a high sensitivity.

Toxicity bioassays with concentrated cell culture media-a methodology to overcome the chemical loss by conventional preparation of water samples.

The use of in vitro bioassays for studies of toxic activity in environmental water samples is a rapidly expanding field of research. Cell-based bioassays can assess the total toxicity exerted by a water sample, regardless whether the toxicity is caused by a known or unknown agent or by a complex mixture of different agents. When using bioassays for environmental water samples, it is often necessary to concentrate the water samples before applying the sample. Commonly, water samples are concentrated 10-50 times. However, there is always a risk of losing compounds in the sample in such sample preparation. We have developed an alternative experimental design by preparing a concentrated cell culture medium which was then diluted in the environmental water sample to compose the final cell culture media for the in vitro assays. Water samples from five Swedish waste water treatment plants were analyzed for oxidative stress response, estrogen receptor (ER), and aryl hydrocarbon receptor (AhR) activity using this experimental design. We were able to detect responses equivalent to 8.8-11.3 ng/L TCCD for AhR activity and 0.4-0.9 ng/L 17ß-estradiol for ER activity. We were unable to detect oxidative stress response in any of the studied water samples. In conclusion, we have developed an experimental design allowing us to examine environmental water samples in toxicity in vitro assays at a concentration factor close to 1, without the risk of losing known or unknown compounds during an extraction procedure.

Impact of natural organic matter in water on in vitro bioactivity assays.

Surface water can be contaminated with pollutants from multiple sources and contain a vast number of various chemicals. In vitro bioassays are valuable tools to assess the total bioactivity of micropollutants in water samples. Besides anthropogenic chemicals, natural organic matter (NOM) is ubiquitous in water, which also may have an impact on the bioactivity in water samples. In the present study we investigated concentration-dependent effects of Nordic Aquatic fulvic acid (NA-FA) and Nordic reservoir NOM (NR-NOM) on bioactivity measured by a panel of luciferase reporter gene assays. The assays included measurements of both induction of activities and inhibition of induced activation on aryl hydrocarbon receptor (AhR), androgen receptor (AR), estrogen receptor (ER), peroxisome proliferator-activated receptor alpha, and on the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) activity as a marker of oxidative stress. At non-cytotoxic concentrations both NA-FA and NR-NOM induced AhR activity, inhibited AR activity with and without the known inducer dihydrotestosterone, inhibited Nrf2 activity, and NR-NOM induced ER activity. The results indicate that the presence of NOM in water samples may lead to false positive results for AhR activity and false positive results for AR and Nrf2 activity, when assessing inhibition of induced bioactivities from anthropogenic substances. We have demonstrated that NA-FA and NR-NOM have an impact on in vitro bioactivities and conclude that the impact of NOM in water should be considered in the evaluation of results from bioactivity assays.

Relationship between peroxisome proliferator-activated receptor alpha activity and cellular concentration of 14 perfluoroalkyl substances in HepG2 cells.

Peroxisome proliferator-activated receptor alpha (PPARα) is a molecular target for perfluoroalkyl substances (PFASs). Little is known about the cellular uptake of PFASs and how it affects the PPARα activity. We investigated the relationship between PPARα activity and cellular concentration in HepG2 cells of 14 PFASs, including perfluoroalkyl carboxylates (PFCAs), perfluoroalkyl sulfonates and perfluorooctane sulfonamide (FOSA). Cellular concentrations were determined by high-performance liquid chromatography-tandem mass spectrometry and PPARα activity was determined in transiently transfected cells by reporter gene assay. Cellular uptake of the PFASs was low (0.04-4.1%) with absolute cellular concentrations in the range 4-2500 ng mg-1 protein. Cellular concentration of PFCAs increased with perfluorocarbon chain length up to perfluorododecanoate. PPARα activity of PFCAs increased with chain length up to perfluorooctanoate. The maximum induction of PPARα activity was similar for short-chain (perfluorobutanoate and perfluoropentanoate) and long-chain PFCAs (perfluorododecanoate and perfluorotetradecanoate) (approximately twofold). However, PPARα activities were induced at lower cellular concentrations for the short-chain homologs compared to the long-chain homologs. Perfluorohexanoate, perfluoroheptanoate, perfluorooctanoate, perfluorononanoate (PFNA) and perfluorodecanoate induced PPARα activities >2.5-fold compared to controls. The concentration-response relationships were positive for all the tested compounds, except perfluorooctane sulfonate PFOS and FOSA, and were compound-specific, as demonstrated by differences in the estimated slopes. The relationships were steeper for PFCAs with chain lengths up to and including PFNA than for the other studied PFASs. To our knowledge, this is the first report establishing relationships between PPARα activity and cellular concentration of a broad range of PFASs.

Re-evaluation of celluloses E 460(i), E 460(ii), E 461, E 462, E 463, E 464, E 465, E 466, E 468 and E 469 as food additives.

Following a request from the European Commission, the EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS) was asked to deliver a scientific opinion re-evaluating the safety of microcrystalline cellulose (E 460(i)), powdered cellulose (E 460(ii)), methyl cellulose (E 461), ethyl cellulose (E 462), hydroxypropyl cellulose (E 463), hydroxypropyl methyl cellulose (E 464), ethyl methyl cellulose (E 465), sodium carboxy methyl cellulose (E 466), enzymatically hydrolysed carboxy methyl cellulose (E 469) and cross-linked carboxy methyl cellulose (E 468) as food additives. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) and the Scientific Committee on Food (SCF) established an acceptable daily intake (ADI) 'not specified' for unmodified and modified celluloses. Celluloses are not absorbed and are excreted intact in the faeces; in addition, microcrystalline cellulose, powdered and modified celluloses could be fermented by the intestinal flora in animals and humans. Specific toxicity data were not always available for all the celluloses evaluated in the present opinion and for all endpoints. Given their structural, physicochemical and biological similarities, the Panel considered it possible to read-across between all the celluloses. The acute toxicity of celluloses was low and there was no genotoxic concern. Short-term and subchronic dietary toxicity studies performed with E 460(i), E 461, E 462, E 463, E 464, E 466 and E 469 at levels up to 10% did not indicate specific treatment related adverse effects. In chronic toxicity studies performed with E 460(i), E 461, E 463, E 464, E 465 and E 466, the no observed adverse effect level (NOAEL) values reported ranged up to 9,000 mg/kg body weight (bw) per day. No carcinogenic properties were detected for microcrystalline cellulose and modified celluloses. Adverse effects on reproductive performance or developmental effects were not observed with celluloses at doses greater than 1,000 mg/kg bw by gavage (often the highest dose tested). The combined exposure to celluloses (E 460-466, E 468 and E 469) at 95th percentile of the refined (brand-loyal) exposure assessment for the general population was up to 506 mg/kg bw per day. The Panel concluded that there was no need for a numerical ADI and that there would be no safety concern at the reported uses and use levels for the unmodified and modified celluloses (E 460(i); E 460(ii); E 461-466; E 468 and E 469). The Panel considered an indicative total exposure of around 660-900 mg/kg bw per day for microcrystalline, powdered and modified celluloses.

Safety of hydroxyanthracene derivatives for use in food.

The Panel on Food Additives and Nutrient Sources added to Food (ANS) was asked to deliver a scientific opinion on the safety of hydroxyanthracene derivatives and to provide advice on a daily intake that does not give rise to concerns about harmful effects to health. Hydroxyanthracene derivatives are a class of chemical substances naturally occurring in different botanical species and used in food to improve bowel function. The ANS Panel reviewed the available scientific data on a possible relationship between hydroxyanthracene derivatives exposure and genotoxic and carcinogenic effects. On the basis of the data currently available, the Panel noted that emodin, aloe-emodin and the structurally related substance danthron have shown evidence of in vitro genotoxicity. Aloe extracts have also been shown to be genotoxic in vitro possibly due to the presence of hydroxyanthracene derivatives in the extract. Furthermore, aloe-emodin was shown to be genotoxic in vivo and the whole-leaf aloe extract and the structural analogue danthron were shown to be carcinogenic. Epidemiological data suggested an increased risk for colorectal cancer associated with the general use of laxatives, several of which contain hydroxyanthracene derivatives. Considering the possible presence of aloe-emodin and emodin in extracts, the Panel concluded that hydroxyanthracene derivatives should be considered as genotoxic and carcinogenic unless there are specific data to the contrary, such as for rhein, and that there is a safety concern for extracts containing hydroxyanthracene derivatives although uncertainty persists. The Panel was unable to provide advice on a daily intake of hydroxyanthracene derivatives that does not give rise to concerns about harmful effects to health.

Re-evaluation of oxidised soya bean oil interacted with mono- and diglycerides of fatty acids (E 479b) as a food additive.

The EFSA Panel on Food Additives and Flavourings (FAF) provides a scientific opinion re-evaluating the safety of thermally oxidised soya bean oil interacted with mono- and diglycerides of fatty acids (TOSOM) (E 479b) when used as a food additive. The Scientific Committee on Food (SCF) and the Joint FAO/WHO Expert Committee on Food Additives (JECFA) derived an acceptable daily intake (ADI) of 25 and 30 mg/kg body weight (bw) per day, respectively. There was no reliable information regarding the absorption, distribution, metabolism, excretion (ADME) for TOSOM. No adverse effects have been detected in a limited subchronic toxicity study in pigs. The Panel identified a no observed adverse effect level (NOAEL) of 5,400, the highest dose tested, from a chronic and carcinogenicity study in rats. No genotoxicity data were available. No reliable studies for reproductive or developmental toxicity were available. From the chronic and carcinogenicity study, no lesions in reproductive organs were described and the lack of carcinogenic effect alleviated the concern for genotoxicity at the first site of contact. The Panel concluded that the available toxicological data were insufficient to support the current ADI, in particular, due to the lack of ADME data and absence of developmental toxicity studies TOSOM (E 479b) is only authorised in one food category and only one reported use level that equals the maximum permitted level was submitted. The estimated high (P95) exposure reached an upper value of 10.1 mg/kg bw per day for toddlers. When comparing the highest estimated exposure of 10 mg/kg bw per day in toddlers with the NOAEL of 5,400 mg/kg bw per day (the highest dose tested), the margin of safety (MoS) would be 540. Therefore, the Panel considered the use of TOSOM (E 479b) to be of no safety concern, in particular when considering the limited current use of this food additive. The Panel also recommended some modifications of the EU specifications for E 479b.