Bioassays in food safety context: the IZSTO CALUX experience

Transcription

1 Bioassays in food safety context: the IZSTO CALUX experience Daniela Meloni

2 The network 10 Institutes in Italy

3 The Piemonte Liguria and Valle d'aosta Institute Three Regions Headquarter in Torino 10 peripheral laboratories

4 General aims of Istituto To ensure public health To support competent authority Through activities in: animal health and wellbeing food hygiene and food safety

5 Istituti Zooprofilattici Sperimentali The network: 10 Institutes in Italy

6 IZSTO activities: Control of food & feedstuffs and live animals. Detection of contaminants, drug residues, agents of zoonoses, animal diseases.

7 National Reference Centers Feed control Beached cetacean Anabolic Treatments Animal encephalopathies Wild animal diseases Veterinary oncology

8 CALUX BIOASSAY Chemical Activated Luciferase gene expression cells produce light when exposed to compounds or groups of compounds with the same mode of action. Selectivity and biological relevance of CALUX assays are not achievable by instrumental techniques.

9 CALUX BIOASSAY Relatively cheap (screening) Biological response of total mixtures Allow broad monitoring High throughput

10 IZSTO CALUX experience DR in milk, eggs and fish GR in bovine urine ER, AR, GR in food contact materials ER in water D E P O S I T I O N ARI A SUO LO FIUMI LAGHI MARI D E P O S I T I O N 95% FOOD

11 CALUX experience IZSTO Laboratory

12 CALUX experience The Equipment

13 DR CALUX - DIOXINS From July 2002 maximum limit values in the EU for dioxins and dioxin-like compounds in food and feed Dioxins are toxic man-made compounds and can be measured with the DR CALUX. Suitable for rapid screening of large numbers of samples. Positive samples need to be confirmed by HRGC-MS.

14 DR CALUX - raw milk Scatter plot of logarithm CALUX values versus HRGC-HRMS results ICC= 0,83

15 DR CALUX - eggs Scatter plot of logarithm CALUX values versus HRGC-HRMS results Log GC-HRMS ICC= 0, logcalux Fitted values loghrgchrms

16 DR CALUX - raw milk and eggs Good infraclass correlation (ICC) between HRGC- HRMS methods and DR CALUX DR CALUX gives a biological response of total mixture Suspect samples need to be confirmed by HRGC- HRMS to look for the concentration of specific chemical compounds S. Sciuto, F. Martucci et al. Large Animal Review 2013; 19:

17 MAGGIORE lake DR CALUX - fish COMO lake (LECCO saide) Acquacolture Perca fluviatilis Rutilus rutilus Oncorhyncus mykiss

18 DR CALUX - fish Results Lago Mean BEQ TCDD-pg/g Specie Mean BEQ TCDD-pg/g Como Maggiore 0.13 Allevamento Persico Rutilo Trota S. Sciuto et al Fondazione CRT (project 13D02)

19 S.C. Istopatologia e Test Rapidi Glucocorticoids SYNTHETIC molecules (dexamethasone - betamethasone - flumethasone ) Therapeutic agents in veterinary practice: metabolic diseases, shock, inflammatory disorders (respect of withdrawal time for meat-producing animals). Illegally (EU) used as growth promoters alone or in combination with other drugs (low-dose cocktails). 8% of slaughtered cattle in Italy.

20 S.C. Istopatologia e Test Rapidi Glucocorticoids NATURAL molecules SPECIFICITY!! Endogenous hormones (cortisol - cortisone) secreted by the adrenal cortex for physiological activities: stress response inflammation immune function hydroelectrolyte balance

21 S.C. Istopatologia e Test Rapidi GR CALUX in bovine urine Glucocorticoids HISTOLOGICAL METHOD GR CALUX ELISA

22 S.C. Istopatologia e Test Rapidi GR CALUX validation study Applicability as a screening method of synthetic glucocorticoids in bovine urine (according to the EC Decision 657/2002): 1. Activity of glucocorticoids as measured in solvent (DMSO). 2. Activity of glucocorticoids as measured in matrix (urine). 3. Assessment of CCα and CCβ: - Ruggedness - Stability - Specificity

23 S.C. Istopatologia e Test Rapidi GR CALUX validation study GR CALUX fully passed the validation study fulfilling all the requirements. Prednisolone selected as reference molecule as showing the lower hormonal activity. Suspect samples need to be confirmed by HRGC- HRMS to look for the concentration of specific chemical compounds. Pitardi D., Meloni D., et al. 2015

24 IZSTO CALUX experience Food Contact Materials Food contact materials should not release harmful substances. Challenge to identify potential migrating substances from FCM. Several compounds have hormonal activity and migration to food simulators was shown (Bisphenol A, phthalates, fluorinated). A strategy based on effect directed analysis combined with advanced analytical chemistry is recommended.

25 S.C. Istopatologia e Test Rapidi FCM CALUX study design 1. Selection of macro-compounds. 2. Activity as measured in solvent (DMSO) by ER, AR, GR CALUX. 3. Selection of micro-compounds and? CALUX. 4. Set up of extraction methods for food simulants. 5. Activity of selected molecules as measured in simulators. 6. Test of field samples.

26 Endocrine Disruptors in Food Contact Materials First steps Almost 32 molecules reported to have hormonal activity are used as additives and/or monomers in plastic FCM across the EU. 20 of them showed estrogenic properties.

27 ER CALUX activity - some molecules

28 THANKS