Bioanalytical screening of low levels of dioxins and dioxin-like PCBs in pig meat (pork) for checking compliance with EU maximum and action levels using highly sensitive “third generation” recombinant H4L7.5c2 rat hepatoma cells

Abstract

Background Low maximum and action levels set by the European Union for polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (DL-PCBs) in pig meat (pork) have led to a demand for reliable and cost-effective bioanalytical screening methods implemented upstream of gas chromatography/high-resolution mass spectrometry confirmatory technology, that can detect low levels of contamination in EU-regulated foods with quick turn-around times. Results Based on the Chemically Activated LUciferase gene eXpression (CALUX) bioassay, extraction and clean-up steps were optimized for recovery and reproducibility within working ranges significantly lower than in current bioassays. A highly sensitive “3rd generation” recombinant rat hepatoma cell line (H4L7.5c2) containing 20 dioxin responsive elements was exposed to pork sample extracts, and their PCDD/Fs and DL-PCBs levels were evaluated by measuring luciferase activity. The method was validated according to the provisions of Commission Regulation (EU) 2017/644 of 5 April 2017 with spiking experiments performed selectively for PCDD/Fs and DL-PCBs and individual calibration for PCDD/Fs, DL-PCBs and the calculated sum of PCDD/Fs and DL-PCBs. The resulting performance parameters met all legal specifications as confirmed by re-calibration using authentic samples. Cut-off concentrations for assessing compliance with low maximum levels and action levels set for PCDD/Fs and DL-PCBs within a range of 0.50–1.25 pg WHO-TEQ/g fat were derived, ensuring low rates of false-compliant results (ß-error < 1%) and keeping the rate of false-noncompliant results well under control (α-error < 12%). Conclusions We present a fast and efficient bioanalytical routine method validated according to the European Union’s legal requirements on the basis of authentic samples, allowing the analyst to reliably identify pork samples and any other EU-regulated foods of animal origin suspected to be noncompliant with a high level of performance and turn-around times of 52 h. This was facilitated in particular by a quick and efficient extraction step followed by selective clean-up, use of a highly sensitive “3rd generation” H4L7.5c2 recombinant rat hepatoma cell CALUX bioassay, and optimized assay performance with improved calibrator precision and reduced lack-of-fit errors. New restrictions are proposed for the calibrator bias and the unspecific background contribution to reportable results. The procedure can utilize comparably small sample amounts and allows an annual throughput of 840–1000 samples per lab technician. The described bioanalytical method contributes to the European Commission's objective of generating accurate and reproducible analytical results according to Commission Regulation (EU) 2017/644 across the European Union.

Mixture effect assessment applying in vitro bioassays to in-tissue silicone extracts of traditional foods prepared from beluga whale blubber

“…The TCDD-EQ chem were shown to agree reasonably well with the TCDD-EQ bio of POPs extracted from pork with exhaustive extraction/cleanup using different AhR-CALUX assays, among them also the cell line we used. 6 Pork meat contained 0.30 to 5.29 pg WHO-PCDD/F-TEQ g lipid −1 , which would be 49 to 860 timed lower than the TCDD-EQ bio found in the baseline beluga whale samples. This comparison is an indication that a lot of BEQ bio detected in beluga stemmed from nonpersistent organics but could partially be contributed by a higher POP level in beluga than in pork.…”

“…The TCDD-EQ chem were shown to agree reasonably well with the TCDD-EQ bio of POPs extracted from pork with exhaustive extraction/cleanup using different AhR-CALUX assays, among them also the cell line we used. 6 Pork meat contained 0.30 to 5.29 pg WHO-PCDD/F-TEQ g lipid −1 , which would be 49 to 860 timed lower than the TCDD-EQ bio found in the baseline beluga whale samples.…”

“…Specically, the AhR-CALUX assay has been widely used to assess dioxin-like residues in food and feed for 20 years, [36][37][38] and recently detailed recommendations have been given for the use of AhR-CALUX assays to quantify dioxins and PCBs in EUregulated foods. 4,39,40 These bioassay methods are typically only applied to exhaustive lipid extracts, from which lipids were removed through a clean-up process that is targeted to remove not only lipids but also nonpersistent organics. This is appropriate if one is only interested in dioxin-like chemicals, but bioactivity can also be introduced by nonpersistent chemicals.…”

“…The maximum level of mixtures of PCDD/Fs and dioxin-like PCBs accepted in pork for consumption is defined as 1.25 pg WHO-PCDD/F-PCB-TEQ g lipid −1 and the action levels is 0.75 pg WHO-PCDD/F-TEQ g lipid −1 . 6 These values were derived for mixtures of 7 PCDDs, 10 PCDF and 12 planar PCBs from chemical analysis and would be equivalent to TCDD-EQ chem accounting only for these 29 POPs. The TCDD-EQ chem were shown to agree reasonably well with the TCDD-EQ bio of POPs extracted from pork with exhaustive extraction/cleanup using different AhR-CALUX assays, among them also the cell line we used.…”

“…Such assays based on the aryl hydrocarbon receptor (AhR) have been applied for decades in the monitoring of food and feed for dioxin-like chemicals. 4–6…”