validation of the dr calux bioassay for the screening pcb

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)

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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

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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

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TOXpro 2014, unpublished data

Valeurs seuils Allemagne 10 ng/m2

Biological monitoring in a incineration plant 2014 - now

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10.00

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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 …

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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

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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


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


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


1,7 (3,7) 2/3 of limit Note 3

Egg** fo-egg 2,5 pg WHO-PCDD/F-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


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


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


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


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


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


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%

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¡  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)

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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

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sensibility 100% specificity 73%

sensibility 96% specificity 81%

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DR - CALUX

Fat from meat

Due to technical problems: No HR-MS data for the DD/DF fraction

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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

validation of the dr calux bioassay for the screening pcb

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