Lucie Dubugnon Dr ès sciences de la vie MAS Toxicologie
10th Biodetection conference Sorrento - Italia 6.4.17
Validation of the DR CALUX bioassay for the screening PCB and dioxins in food and feed in Switzerland
ICC Lausanne - Switzerland
¡ Clinical and toxicological laboratory
¡ Customers ¡ Medical doctors- Physicians / other
laboratories ¡ Police, prosecutors, forensic analyses ¡ Work place testing ¡ Customer goods ¡ Food/feed lab and industries
¡ Developed a CALUX lab since end of 2015
www.laboicc.ch
PCBs have been widely used as construction material (1955-1975)
3
Joints seals < 200’000 ppm (20%)
Industrial paintings < 20’000 mg/kg (ppm)
PCBs in Switzerland
2004
Profil GC-HRMS. © Restek
Gold-standard : GC-HRMS 5
Equivalent toxicity factor (TEF) 6
Indicator PCBs dioxin-like PCBs non planars planars
Toxic equivalent factor : relates to 2,3,7,8- TCDD toxicity
Toxicity is linked to the ability to bind to the AhR-receptor
Dioxin Receptor Chemically Activated LUciferase gene eXpression
CALUX assay : allows ligand –receptor activation measurement
7
Nucleus
Ligandbinding
Cytosol
Transcription
ProteinsEnzymesLuciferase
Add substrate (luciferine)
Light
ChemicalResponsiveElement(CRE)
Ligand
Transport protein
Hsp
Chemical receptor
CALUX-assay
« Gene reporter bioassay »
Dioxin/PCBs in men
¡ Lipophilic: Accumulate in body fat
¡ Is there any risk due to the exposure of worker ? Ø Sampling before and after a working period of 4 month
¡ Best sample : blood
¡ Plasma fat levels are proportional to the body levels
¡ Found no significant difference in the levels of PCBs/Dioxins in blood after the working time
¡ People protected with EPI
Prélèvement de surface (wipe-test) dans une usine d’incinération
10
TOXpro 2014, unpublished data
Valeurs seuils Allemagne 10 ng/m2
Biological monitoring in a incineration plant 2014 - now
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
100.00
TCDD TEQ / g fat Workplace testing
DR-CALUX serum levels > 200 workers - Clinical chemistry (liver enzymes,
thyroid hormones) - Heavy metals - PAH exposure (HAP and 1-OH-pyrene in
urine by GCMS)
- Sample once every second year - Budget dependant …
11
Gathering data to get more accurate trigger values for the working population Non exposed population …
Validation of the DR CALUX assay for feed / food
¡ Federal Food Safety and Veterinary Office (FSOV) asked for a validation of the method
12
Collaboration with Geneva consumption service
Set up
Comparison Screening GCMS vs Screening DR CALUX Comparison with CGHRMS data (to be obtained from Confederation)
22 23 10
(graisses)
European regulations 14
Teneur maximales et seuils d’intervention
Food Code Value Unit Reference
Ruminants meat** fo-rum 2,5 pg WHO-PCDD/F-TEQ/g fat Note 1
4,0 pg WHO-PCDD/F-PCB-TEQ/g fat Note 1
1,7 (2,7) 2/3 of limit Note 3
Poultry / farmed meat** fo-pou 1,75 pg WHO-PCDD/F-TEQ/g fat Note 1
3,0 pg WHO-PCDD/F-PCB-TEQ/g fat Note 1
1,2 (2,0) 2/3 of limit Note 3
Pigs meat** fo-pig 1,0 pg WHO-PCDD/F-TEQ/g fat Note 1
1,25 pg WHO-PCDD/F-PCB-TEQ/g fat Note 1
0,70 (0,80) 2/3 of limit Note 3
Liver fo-liv 0,30 pg WHO-PCDD/F-TEQ/g fresh weight Note 4
0,50 pg WHO-PCDD/F-PCB-TEQ/g fresh weight Note 4
0,20 (0,33) 2/3 of limit Note 3
Liver Sheep fo-livs 1,25 pg WHO-PCDD/F-TEQ/g fresh weight Note 4
(products) 2,00 pg WHO-PCDD/F-PCB-TEQ/g fresh weight Note 4
0,80 (1,3) 2/3 of limit Note 3
Fish liver* fo-fis --- pg WHO-PCDD/F-TEQ/g fresh weight Note 1
20 pg WHO-PCDD/F-PCB-TEQ/g fresh weight Note 1
---, (13) 2/3 of limit Note 3
Fish* fo-fis 3,5 pg WHO-PCDD/F-TEQ/g fresh weight Note 1
6,5 pg WHO-PCDD/F-PCB-TEQ/g fresh weight Note 1
2,3 (4,3) 2/3 of limit Note 3
Eel* fo-eel 3,5 pg WHO-PCDD/F-TEQ/g fresh weight Note 1
10 pg WHO-PCDD/F-PCB-TEQ/g fresh weight Note 1
2,3 (6,7) 2/3 of limit Note 3
Milk** fo-mil 2,5 pg WHO-PCDD/F-TEQ/g fat Note 1
5,5 pg WHO-PCDD/F-PCB-TEQ/g fat Note 1
1,7 (3,7) 2/3 of limit Note 3
Egg** fo-egg 2,5 pg WHO-PCDD/F-TEQ/ g fat Note 1
5,0 pg WHO-PCDD/F-PCB-TEQ/g fat Note 1
1,9 (3,3) 2/3 of limit Note 3
Ruminants fat fo-ruf 2,5 pg WHO-PCDD/F-TEQ/g fat Note 1
4,0 pg WHO-PCDD/F-PCB-TEQ/g fat Note 1
1,7 (2,7) 2/3 of limit Note 3
Poultry/farmed fat fo-pof 1,75 pg WHO-PCDD/F-TEQ/g fat Note 1
3,0 pg WHO-PCDD/F-PCB-TEQ/g fat Note 1
1,2 (2,0) 2/3 of limit Note 3
Pigs fat fo-pif 1,0 pg WHO-PCDD/F-TEQ/g fat Note 1
1,25 pg WHO-PCDD/F-PCB-TEQ/g fat Note 1
0,70 (0,80) 2/3 of limit Note 3
Mixed fat fo-mif 1,5 pg WHO-PCDD/F-TEQ/g fat Note 1
2,5 pg WHO-PCDD/F-PCB-TEQ/g fat Note 1
1,0 (1,7) 2/3 of limit Note 3
Vegetable oil fo-voi 0,75 pg WHO-PCDD/F-TEQ/g fat Note 1
1,25 pg WHO-PCDD/F-PCB-TEQ/g fat Note 1
0,50 (0,83) 2/3 of limit Note 3
Fish oil fo-foi 1,75 pg WHO-PCDD/F-TEQ/g fat Note 1
6,0 pg WHO-PCDD/F-PCB-TEQ/g fat Note 1
1,2 (4,0) 2/3 of limit Note 3
Fruits, vegetables and cereals
fo-fvc 0,6 pg WHO-PCDD/F-PCB-TEQ/g product Note 2
Food for infants fo-inf 0,1 pg WHO-PCDD/F-TEQ/g wet weight Note 1
0,2 pg WHO-PCDD/F-PCB-TEQ/g wet weight Note 1
0,070 (0,13) 2/3 of limit Note 3
Analytical steps
1. Sampling 2. Standard fat extraction 3. Acid silica clean up / fractionation
4. Cell culture (maintenance) 5. Cell culture (experiment) 6. Exposure in 96-well plates / bio
degradation 7. Quantification light emission
8. Data handling
ICC
Ref BioDetection Systems, DR CALUX® analysis guide, P-BDS-051, Version R, 2015
Extraction out of the lab: difficulty to sort out problems
¡ Fish : Dried extracts.
¡ Results were good
¡ Extraction of meats gave variable fat percentages
¡ No good homogenisation of the samples, difficult to compare to HRMS values
¡ Not the right control used for the calculation of extraction ratios
¡ Blanks often too high for the TCDD/F fraction …
Matières grasses Viandes OSAV CALUX Annoncé OSAV Résultats AG Ecart% Matières grasses Matières grasses
N° d'analyse g/100g g/100g
3269 A 46.80 45.75 -2%
3271 A 48.10 40.68 -15%
3279 A 16.70 48.74 192%
3281 A 41.00 26.50 -35%
3283 A 25.00 47.05 88%
3285 A 24.00 28.58 19%
3287 A 30.30 30.31 0%
3289 A 29.40 43.85 49%
3291 A 30.60 25.98 -15%
3332 A 30.70 35.69 16%
3334 A 28.90 40.41 40%
3336 A 39.20 28.62 -27%
3338 A 42.60 49.48 16%
3340 A 45.40 51.65 14%
3349 A 36.90 33.38 -10%
3353 A 45.40 54.85 21%
3358 A 42.10 35.47 -16%
3368 A 59.50 40.40 -32%
3370 A 28.50 30.00 5%
3372 A 25.60 25.24 -1%
3374 A 37.30 40.91 10%
22
23
¡ Started new extractions with other hexane and DEE
Solvent testing
Type d'échantillon Volume initial (ml)
ΣPCDD/F-PCB CALUX
Hexane 50 3.3
DEE 25 1.9 DCM 25 0.9
Toluène 25 0.1 2-Propanol 50 0.1
DMSO -0.5
10 new meat extracts
(graisses)
10 ¡ Fat from the meat
¡ Homogenized
¡ New HRMS measurements
Interpretation and maximum limits
Maxium limit Total TEQ
(PCDD/F-dlPCB)
6.5 pg TEQ/g
3.5 pg TEQ/g
2.3 pg BEQ/g
Maxium limit Dioxins TEQ
(PCDD/F)
3.5 pg BEQ/g Action limit Total BEQ
(PCDD/F-dlPCB)
Action limit Dioxins BEQ
(PCDD/F)
19
4.0 pg TEQ/g
2.5 pg TEQ/g
1.7 pg BEQ/g
2.7 pg BEQ/g
Somme : PCBs + Dioxins – furanes
Dioxins – furanes
GCHRMS
CALUX
Results interpretation
IN GENERAL: a suspect analysis results always need to be Confirmed by re-analysis of the sample (possible 48 hrs)
Total BEQ analysis
PCDD/F- and dlPCB-specific BEQ analysis
• Total BEQ response (PCDD/F-dlPCBs) sample < than PCDD/F EU action limits: COMPLIANT
• Total BEQ response (PCDD/F-dlPCBs) sample > than PCDD/F EU action limits: POSSIBLE SUSPECT re-analysis including separation by florisil
• PCDD/F-specific BEQ response sample < than PCDD/F EU action limits: COMPLIANT
• PCDD/F-specific BEQ response sample > than PCDD/F EU action limits: SUSPECT
• PCDD/F-specific + dlPCB-specific BEQ response < than PCDD/F-dlPCB EU action limit: COMPLIANT
• PCDD/F-specific + dlPCB-specific BEQ response > than PCDD/F-dlPCB EU action limit: SUSPECT
Fish samples 22
Meat samples 23
Method comparision
Fish DR - CALUX
Positifs Négatifs
GCHRMS Positifs 22 0
Négatifs 6 16
Meat DR - CALUX
Positifs Négatifs
GCHRMS Positifs 22 1
Négatifs 4 18
23
sensibility 100% specificity 73%
sensibility 96% specificity 81%
22
23
DR - CALUX
Fat from meat
Due to technical problems: No HR-MS data for the DD/DF fraction
10
No conclusion possible on these samples
- Validation is successful
- False negatif: as less as possible þ
- False positive: always possible to repeat the measurement with a longer exposure time
- Always confirm any positive results with CG/HRMS
Conclusions 25
False POS False NEG Below 5% by EU law accepted
Fish 27% 0% þ
Meat 22% 4% þ
2017 projects on-going in ICC
¡ DR-CALUX ¡ Wipe tests ¡ House dust testing and ambient air testing ¡ Human biomonitoring
¡ Hormone CALUX (ER, AR; PR–CALUX) ¡ Work place testing national funded study: exposure
of volunteers to chemical compounds and analysis of the hormonal perturbation in women
¡ Water analysis (effluent, rivers)
Why does it takes time to develop the market
• Many screening possibilities • Each type of sample and field has his own
regulations and maximal limits or threshold • These values are sometimes not yet clearly
defined • Using CALUX in routine requires that the assay is
already recognized in the targeted domain • CALUX has many positive aspects: allows to
screen for effects, not only for “known substances”, some regulators might be afraid
• Many different Canton in Switzerland J
Merci pour votre attention!
www.laboicc.ch [email protected]
SCAV Vaud
SCAV Genève: Dr Patrick Edder, Dr Didier Ortelli Dr Aurélie Bugey Ngoc Huy Ho Joëlle Nançoz Vincent Perret
ICC Olga Martin Sylvie Perriraz Emmanuel Strahm phD Christelle Vogne phD Jean-Luc Dubugnon phD
