validation of the dr calux bioassay for the screening pcb ...€¦ · dioxins in blood after the...
TRANSCRIPT
![Page 1: Validation of the DR CALUX bioassay for the screening PCB ...€¦ · Dioxins in blood after the working time ¡ People protected with EPI . Prélèvement de surface (wipe-test)](https://reader035.vdocuments.us/reader035/viewer/2022071117/60036e926ff41c4ccb385f32/html5/thumbnails/1.jpg)
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
![Page 2: Validation of the DR CALUX bioassay for the screening PCB ...€¦ · Dioxins in blood after the working time ¡ People protected with EPI . Prélèvement de surface (wipe-test)](https://reader035.vdocuments.us/reader035/viewer/2022071117/60036e926ff41c4ccb385f32/html5/thumbnails/2.jpg)
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
![Page 3: Validation of the DR CALUX bioassay for the screening PCB ...€¦ · Dioxins in blood after the working time ¡ People protected with EPI . Prélèvement de surface (wipe-test)](https://reader035.vdocuments.us/reader035/viewer/2022071117/60036e926ff41c4ccb385f32/html5/thumbnails/3.jpg)
PCBs have been widely used as construction material (1955-1975)
3
Joints seals < 200’000 ppm (20%)
Industrial paintings < 20’000 mg/kg (ppm)
![Page 4: Validation of the DR CALUX bioassay for the screening PCB ...€¦ · Dioxins in blood after the working time ¡ People protected with EPI . Prélèvement de surface (wipe-test)](https://reader035.vdocuments.us/reader035/viewer/2022071117/60036e926ff41c4ccb385f32/html5/thumbnails/4.jpg)
PCBs in Switzerland
2004
![Page 5: Validation of the DR CALUX bioassay for the screening PCB ...€¦ · Dioxins in blood after the working time ¡ People protected with EPI . Prélèvement de surface (wipe-test)](https://reader035.vdocuments.us/reader035/viewer/2022071117/60036e926ff41c4ccb385f32/html5/thumbnails/5.jpg)
Profil GC-HRMS. © Restek
Gold-standard : GC-HRMS 5
![Page 6: Validation of the DR CALUX bioassay for the screening PCB ...€¦ · Dioxins in blood after the working time ¡ People protected with EPI . Prélèvement de surface (wipe-test)](https://reader035.vdocuments.us/reader035/viewer/2022071117/60036e926ff41c4ccb385f32/html5/thumbnails/6.jpg)
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
![Page 7: Validation of the DR CALUX bioassay for the screening PCB ...€¦ · Dioxins in blood after the working time ¡ People protected with EPI . Prélèvement de surface (wipe-test)](https://reader035.vdocuments.us/reader035/viewer/2022071117/60036e926ff41c4ccb385f32/html5/thumbnails/7.jpg)
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
![Page 8: Validation of the DR CALUX bioassay for the screening PCB ...€¦ · Dioxins in blood after the working time ¡ People protected with EPI . Prélèvement de surface (wipe-test)](https://reader035.vdocuments.us/reader035/viewer/2022071117/60036e926ff41c4ccb385f32/html5/thumbnails/8.jpg)
CALUX-assay
« Gene reporter bioassay »
![Page 9: Validation of the DR CALUX bioassay for the screening PCB ...€¦ · Dioxins in blood after the working time ¡ People protected with EPI . Prélèvement de surface (wipe-test)](https://reader035.vdocuments.us/reader035/viewer/2022071117/60036e926ff41c4ccb385f32/html5/thumbnails/9.jpg)
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
![Page 10: Validation of the DR CALUX bioassay for the screening PCB ...€¦ · Dioxins in blood after the working time ¡ People protected with EPI . Prélèvement de surface (wipe-test)](https://reader035.vdocuments.us/reader035/viewer/2022071117/60036e926ff41c4ccb385f32/html5/thumbnails/10.jpg)
Prélèvement de surface (wipe-test) dans une usine d’incinération
10
TOXpro 2014, unpublished data
Valeurs seuils Allemagne 10 ng/m2
![Page 11: Validation of the DR CALUX bioassay for the screening PCB ...€¦ · Dioxins in blood after the working time ¡ People protected with EPI . Prélèvement de surface (wipe-test)](https://reader035.vdocuments.us/reader035/viewer/2022071117/60036e926ff41c4ccb385f32/html5/thumbnails/11.jpg)
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 …
![Page 12: Validation of the DR CALUX bioassay for the screening PCB ...€¦ · Dioxins in blood after the working time ¡ People protected with EPI . Prélèvement de surface (wipe-test)](https://reader035.vdocuments.us/reader035/viewer/2022071117/60036e926ff41c4ccb385f32/html5/thumbnails/12.jpg)
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
![Page 13: Validation of the DR CALUX bioassay for the screening PCB ...€¦ · Dioxins in blood after the working time ¡ People protected with EPI . Prélèvement de surface (wipe-test)](https://reader035.vdocuments.us/reader035/viewer/2022071117/60036e926ff41c4ccb385f32/html5/thumbnails/13.jpg)
Set up
Comparison Screening GCMS vs Screening DR CALUX Comparison with CGHRMS data (to be obtained from Confederation)
22 23 10
(graisses)
![Page 14: Validation of the DR CALUX bioassay for the screening PCB ...€¦ · Dioxins in blood after the working time ¡ People protected with EPI . Prélèvement de surface (wipe-test)](https://reader035.vdocuments.us/reader035/viewer/2022071117/60036e926ff41c4ccb385f32/html5/thumbnails/14.jpg)
European regulations 14
![Page 15: Validation of the DR CALUX bioassay for the screening PCB ...€¦ · Dioxins in blood after the working time ¡ People protected with EPI . Prélèvement de surface (wipe-test)](https://reader035.vdocuments.us/reader035/viewer/2022071117/60036e926ff41c4ccb385f32/html5/thumbnails/15.jpg)
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
![Page 16: Validation of the DR CALUX bioassay for the screening PCB ...€¦ · Dioxins in blood after the working time ¡ People protected with EPI . Prélèvement de surface (wipe-test)](https://reader035.vdocuments.us/reader035/viewer/2022071117/60036e926ff41c4ccb385f32/html5/thumbnails/16.jpg)
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
![Page 17: Validation of the DR CALUX bioassay for the screening PCB ...€¦ · Dioxins in blood after the working time ¡ People protected with EPI . Prélèvement de surface (wipe-test)](https://reader035.vdocuments.us/reader035/viewer/2022071117/60036e926ff41c4ccb385f32/html5/thumbnails/17.jpg)
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
![Page 18: Validation of the DR CALUX bioassay for the screening PCB ...€¦ · Dioxins in blood after the working time ¡ People protected with EPI . Prélèvement de surface (wipe-test)](https://reader035.vdocuments.us/reader035/viewer/2022071117/60036e926ff41c4ccb385f32/html5/thumbnails/18.jpg)
¡ 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
![Page 19: Validation of the DR CALUX bioassay for the screening PCB ...€¦ · Dioxins in blood after the working time ¡ People protected with EPI . Prélèvement de surface (wipe-test)](https://reader035.vdocuments.us/reader035/viewer/2022071117/60036e926ff41c4ccb385f32/html5/thumbnails/19.jpg)
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
![Page 20: Validation of the DR CALUX bioassay for the screening PCB ...€¦ · Dioxins in blood after the working time ¡ People protected with EPI . Prélèvement de surface (wipe-test)](https://reader035.vdocuments.us/reader035/viewer/2022071117/60036e926ff41c4ccb385f32/html5/thumbnails/20.jpg)
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
![Page 21: Validation of the DR CALUX bioassay for the screening PCB ...€¦ · Dioxins in blood after the working time ¡ People protected with EPI . Prélèvement de surface (wipe-test)](https://reader035.vdocuments.us/reader035/viewer/2022071117/60036e926ff41c4ccb385f32/html5/thumbnails/21.jpg)
Fish samples 22
![Page 22: Validation of the DR CALUX bioassay for the screening PCB ...€¦ · Dioxins in blood after the working time ¡ People protected with EPI . Prélèvement de surface (wipe-test)](https://reader035.vdocuments.us/reader035/viewer/2022071117/60036e926ff41c4ccb385f32/html5/thumbnails/22.jpg)
Meat samples 23
![Page 23: Validation of the DR CALUX bioassay for the screening PCB ...€¦ · Dioxins in blood after the working time ¡ People protected with EPI . Prélèvement de surface (wipe-test)](https://reader035.vdocuments.us/reader035/viewer/2022071117/60036e926ff41c4ccb385f32/html5/thumbnails/23.jpg)
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
![Page 24: Validation of the DR CALUX bioassay for the screening PCB ...€¦ · Dioxins in blood after the working time ¡ People protected with EPI . Prélèvement de surface (wipe-test)](https://reader035.vdocuments.us/reader035/viewer/2022071117/60036e926ff41c4ccb385f32/html5/thumbnails/24.jpg)
Fat from meat
Due to technical problems: No HR-MS data for the DD/DF fraction
10
No conclusion possible on these samples
![Page 25: Validation of the DR CALUX bioassay for the screening PCB ...€¦ · Dioxins in blood after the working time ¡ People protected with EPI . Prélèvement de surface (wipe-test)](https://reader035.vdocuments.us/reader035/viewer/2022071117/60036e926ff41c4ccb385f32/html5/thumbnails/25.jpg)
- 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
![Page 26: Validation of the DR CALUX bioassay for the screening PCB ...€¦ · Dioxins in blood after the working time ¡ People protected with EPI . Prélèvement de surface (wipe-test)](https://reader035.vdocuments.us/reader035/viewer/2022071117/60036e926ff41c4ccb385f32/html5/thumbnails/26.jpg)
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)
![Page 27: Validation of the DR CALUX bioassay for the screening PCB ...€¦ · Dioxins in blood after the working time ¡ People protected with EPI . Prélèvement de surface (wipe-test)](https://reader035.vdocuments.us/reader035/viewer/2022071117/60036e926ff41c4ccb385f32/html5/thumbnails/27.jpg)
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
![Page 28: Validation of the DR CALUX bioassay for the screening PCB ...€¦ · Dioxins in blood after the working time ¡ People protected with EPI . Prélèvement de surface (wipe-test)](https://reader035.vdocuments.us/reader035/viewer/2022071117/60036e926ff41c4ccb385f32/html5/thumbnails/28.jpg)
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