Practical experience with the use of the DR CALUX® bioassay for ...

Practical experience with the use of theDR CALUX ® bioassay for feed and foodRon Hoogenboom

January 2006: Dioxins in recycled fat from gelatin productionSamplenumberLevelResponse (RLUs)Decisionpg TEQ/gmeanSDBlank fat6713Ref 10.4752Ref 21.01026Ref 31.516813Ref 43.124637200163193?Hoogenboom et al. 2007

January 2006: Dioxins in recycled fat from gelatin productionSamplenumberLevelResponse (RLUs)Decisionpg TEQ/gmeanSDBlank fat6713Ref 10.4752Ref 21.01026Ref 31.516813Ref 43.124637200163193?94177suspectedHoogenboom et al. 2007

Application of DR CALUX assay at RIKILT• Gelatin first incident discovered by DR CALUX ® ,• but DR CALUX widely used for monitoring and,• in other incidents, like ….

Food and feed incidentsBrazilian citrus pulp 1998Belgian PCB fat 1999Wild eelGerman kaolinic clay 1999Belgian cholin chloride 2002German bakery waste 2003Free range eggsPotato peels/kaolinic clay 2004Gelatin fat/Hydrochloric acid 2006

Screening method for dioxins• Requirements in 1990• quick and relatively cheap• high sample throughput• rapid expansion of capacity (crisis)• no false-negatives, few false-positives• sensitive at low levels• obeying the TEQ-principle• Still valid in 2007?• Clean-up GC/HRMS improved• And thus throughput, expansion, and costs?

Current use of DR CALUX ®• Test especially useful in large monitoring programs• Few samples with high response• DR CALUX will detect other (persistent) AhR-agonists• Brominated dioxines• Mixed halogenated dioxins• Others?

Application for dioxins and dl-PCBs in food and feed• Clean-up procedure• Sensitivity• Validation• Test strategy

DR CALUX bioassay: screening assayClOClClOClClClOOClClLucLuciferin+ATPAarts et al. 1993

Clean-up of samples (increased selectivity)• Fat extraction• acid silica clean-up (6-8 h)• CALUX-assay (24 h incubation)

Quantitative approach: calibrationSeveral options:• TCDD calibration curve (most common)• Dioxin/PCB mixture (not applied)• “Reference” samples (RIKILT)

Quantitative approach35003000Response (RLUs)2500200015001000Level in testsample50000.1 1 10 100 1000Concentration TCDD (pM)

JRC: Results chicken feed (n=10)FeedGC/MSMeanUnc. Int.srRSD rsRRSD R10.0420.7631.8543.95Gizzi et al. 2005

Chicken feed: individual results3CALUX (ng TCDD eq/kg)21y=x0762985 with Corr.Coeff. > 0.9700.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50GC/MS (ng TEQ/kg)

JRC: Results chicken feed (n=10)FeedGC/MSMeanUnc. Int.srRSD rsRRSD R10.040.250.14-0.360.09370.187120.760.690.46-0.930.25370.375331.851.030.87-1.180.14140.252443.951.631.15-2.100.85520.7546Background highRecovery lowGizzi et al. 2005

Use of reference samples• Matrix/pattern specific reference materials:• to correct for possible contaminants from solvents• Including interaction fat and acid silica• to correct for possible “recovery losses”

“Recovery”Determined by different factors:• Loss of dioxins and PCBs during clean-up• Evaporation is critical step• Loss of DMSO (keeper) during evaporation• DMSO increases response in the test• Quality of curve-fit, especially at lower concentrations• Often poor fit at low levels: underestimation of levelSee Hoogenboom et al. TRAC 2006

Curve-fit and DMSO-effectB0.5%DMSO

“Recovery”Determined by different factors:• Loss of dioxins and PCBs during clean-up• Evaporation is critical step• Loss of DMSO (keeper) during evaporation• DMSO increases response in the test• Quality of curve-fit, especially at lower concentrations• Often poor fit at low levels: underestimation of level• Difference between REPs and WHO-TEF values• REPs in general lower than WHO-TEQ: underestimationSee Hoogenboom et al. TRAC 2006

• Relationship CALUX-REPs and WHO-TEFs

Dose-response curves for dioxins and PCBsBovee et al. 199

CALUX REPs versus WHO-TEFsCongenerWHO-TEFBoveeLaierBenischScippoNew TEFs2,3,4,7,8-PeCDF0.50.30.60.50.40.31,2,3,6,7,8-HxCDF0.10.070.040.090.12,3,7,8-TCDD1111111,2,3,7,8-PeCDD10.510.50.511,2,3,6,7,8-HxCDD0.10.30.10.060.1PCB 1260.10.070.20.070.040.1PCB 1690.010.0020.0030.00080.03PCB 1181.10 -45.10 -63.10 -6ndrcNr3.10 -5PCB 1051.10 -42.10 -61.10 -5Nr3.10 -5PCB 1565.10 -44.10 -52.10 -42.10 -42.10 -53.10 -5

Role of CALUX-REPs in “recovery”Lower REPs lead tounderestimation of content1010.10.01HxCDDTCDDPeCDDPeCDFPCB 126CALUX-REPs10TCDD10.1HxCDDPeCDDPeCDF0.01PCB 1260.001PCB 1690.0001PCB 1560.00001PCB 105PCB 1181e-061e-06 0.00001 0.0001 0.001 0.01 0.1 1 10WHO-TEF0.001PCB 1690.0001PCB 1560.00001PCB 1051e-06PCB 1181e-06 0.00001 0.0001 0.001 0.01 0.1 1 10WHO-TEFImportant to introduce newTEF values into legislation

Impact of new TEF values on levels in eel (2006)50TEQ new TEFs (pg TEQ/g eel)40302010y = 0.61x - 0.80R 2 = 0.986Primarily due to much lowerTEFs for mo-PCBsLevels 40%lower00 10 20 30 40 50 60 70 80TEQ old TEFs (pg TEQ/g eel)

Determination of CALUX-REPs• Difficult, due to:• Impurities in standards• Concentration of standards in DMSO• Currently repeated at RIKILT• Important for detection of novel risks (other AhRagonists):• When is there a discrepancy between CALUX and GC/MS,pointing at considerable levels of other agonists?

Test strategy- link to GC/MS- accreditation

When to declare a sample suspected ?

Test strategy: decision limit• Approach 1: calculate level and compare to limit• In TEQs, or better BEQs• Approach 2: compare with reference samples• Which level?

Target limits, action limits and residu limitsResidu limit1 pg TEQ/gAction limit0.6 pg TEQ/gTarget limit? pg TEQ/gAction limits should be target DR CALUX (during monitoring) !

New EU-limits (4 th November 2006)• Inclusion dioxin-like PCBs• Based on 95% interval of current levels (database)• Tolerance limits for dioxins and total TEQ• Action limits for dioxins and dl-PCBs• In the future only limits for total TEQ?• End of 2008?

Current limitsProductTolerancelimitTolerancelimitAction limitAction limitdioxinsdioxins anddl-PCBsdioxinsdl-PCBsMilk fat3622Poultry fat241.51.5Beef fat34.51.51Pig fat11.50.50.5Eggs3322Fish4833Vegetable oil0.751.50.50.5Feed0.751.250.50.35

New EU-limits (4 th November 2006)• Separation of dioxins and dl-PCBs ?• Use of action limits dl-PCBs as decision limit CALUX• What about poor response mo-PCBs ?• Use of new WHO-TEF values ?

Separation of dioxins and dl-PCBs?• Thus far not applied at RIKILT• More work• High risk of introducing extra problems• Difference between background and limits high• But, false-positive rate increased

Quantitative or screening approach• Qualitative statement often sufficient:• A quantitative statement implies that the response iscaused by dioxins or planar PCBs• Elevated samples always require GC/MS confirmation• Approach more acceptable for GC/MS labs

Principle of the screening approach• Comparison test sample with reference sample:• response test sample < response reference:sample declared negative• response test sample > response reference:sample declared suspected and GC/MS analysis required

Example: screening of milk from cows fed citrus pulplimitsuspectedreferencenegativeblanc

Adaptation of the screening approach• Disadvantage is the number of different action limits• Not always the same as levels in reference samples• Use of calibration curve with reference samples• Qualitative result: negative or suspected

Validation• Show repeatability/reproducibility (CV

Feed 5 gram (n=4)2000response (RLUs)1500100050000.03 0.34 0.58 0.76 1.84 3.95concentration (ng TEQ/kg)

Milk fat 0.5 g fat500response (RLUs)40030020010000.4 0.5 0.9 1.9 2.7 5.7concentration (pg TEQ/g)

Improved clean-up of samples• Fat extraction• acid pretreatment• acid silica clean-up• CALUX-assay

Milk fat (2 gram fat)400response (RLUs)30020010000.4 0.5 0.9 1.9 2.7 5.7concentration (pg TEQ/g)

Conclusions based on incidents and monitoring• Incidents best way to validate assay• many positive samples, required to show “

Case: dioxins in Brazilian citrus pulp (1998)

Validation for citrus pulp (repeatability)Concentration(pg i-TEQ/kg)undilutedResponse(RLUs)1 2 3 4 5 6 mean ± SD CV (%)0 653 708 742 639 788 935 695 ± 63 9400 940 1018 892 882 1105 1078 913 ± 39 41000 1557 1625 1225 1620 1638 1480 1424 ± 91 62000 1689 1760 1402 1943 1807 1596 1643 ± 138 85x diluted0 47 44 53 47 55 49 49 ± 4 9400 63 49 67 57 64 55 59 ± 7 111000 104 106 133 111 138 124 120 ± 15 122000 175 196 170 162 187 183 179 ± 12 7

Validation CALUX based on field samplesGC/MSnegativepositiveCALUXnegativesuspected

Screening of citrus pulpGC/MSContent(pg i-TEQ/kg)Number ofSamplesnegativesuspected500 or lower 4138 3500-6000 300 30Reference of 400 pg TEQ/kgHoogenboom 2001

Performance CALUX: animal fat (mostly pigs)CALUXGC/MSnegativepositive> actionlimitnegative601suspected212Hoogenboom et al. 2004

Future needs• Further international validation• Development of reference materials• Further improvement of sensitivity of the cells• Response at lower dioxin concentrations• Development of rapid high-troughput clean-upprocedures• Development of strategies and methods foridentification of novel agonists• Adaptation of EU-guidelines ?

And most of all …

AcknowledgementsMy collegues at RIKILTToine Bovee, Liza Portier, Karin van Ede, Astrid Hamers,Gerrit Bor, Theo Polman, Gerard van Bruchem, RichardHelsdingen, Wim Traag

Thank you for your attention