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Nortestosterone and Casualty Animals

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Nortestosterone and Casualty Animals Nortestosterone and Hormone testing in Northern Ireland casualty animals National Surveillance Scheme “Random” No detention of carcases Non-compliant: Investigate Meat Inspection All animals inspected D. Glenn Kennedy Ante & post mortem D. Glenn Kennedy Suspects detained UK-NRL, AFBI, Belfast Non-compliant: Destroy meat Northern Ireland, UKUK Investigate Hormones and ß-agonists in NI 500 ImplantImplant PelletsPellets HormonesHormones 400 ß-Agonistsß-Agonists 300 LowLow levellevel ofof abuseabuse or 200 Smarter abusers ? Positives 100 0 0 1 4 97 98 99 00 01 02 03 9 9 9 0 0 0 0 00 199 199 1992 1993 1994 1995 1996 1 1 1 2 2 2 2 2 Year The far west of 17-β-19-Nortestosterone the European Union OH H H H H The Wild West of O the European Union ?? 1 17-α-19-Nortestosterone 19-Nortestosterone OH Occurs naturally in pregnant cows (α) boars (β) stallions (β) sheep (α) H H Another “difficult” compound boldenone H H zeranol testosterone oestradiol O progesterone Does not occur naturally in male cattle No stolen animals : but very wellwell-finished-finished 2004 2 XEL-S 2005 1919-Nortestosterone-Nortestosterone decanoate Trace 17ß17ß-oestradiol-oestradiol Major 17ß17ß-oestradiol-oestradiol benzoate Major Testosterone propionate Major Trenbolone acetate Trace Laboratory findings Quattro Premier XE & Acuity UPLC Anabolic preparations: none found Injection sites none found Syringes β-Nortestosterone decanoate Urine from steers No nortestosterone High progesterone 3 Hormones covered by NI testing schemes Proficiency Testing Schemes ADD Methyl Boldenone α-Boldenone-Boldenone Methyltestosterone α-Nortestosterone β-Boldenone-Boldenone Norethandrolone CLAD D(-) Norgestrel Last 6 years Dienestrol α-Nortestosterone-Nortestosterone Diethylstilbestrol ß-Nortestosterone 1 RIVM – CRL Ring Test Epitestosterone Progesterone 8 Progetto Trieste Ring Tests α-Estradiol-Estradiol Stanozolol β-Estradiol-Estradiol Taleranol No false positives Ethisterone Testosterone No false negatives Fluoxymesterone α-Trenbolone-Trenbolone Hexestrol α-Zearalenone-Zearalenone All z-scores between –2 and +2 16ß-Hydroxy stanozolol β-Zearalenol-Zearalenol Medroxyprogesterone Zearalenone Analytical method under control Methenolone Zeranol Run time As 2006 crisis developed…. 50 X 2.1 mm UPLC Column Urine Biosensor Screen Compliant Non-Compliant LC (± MS/MS) 31 analytes eluted in 5 minutes GC-MS/MS (heptafluorobutyric AA) System re-equilibrated and ready to injectinject againagain afterafter 66 minutesminutes (heptafluorobutyric AA) d3-ß-Nortestosterone d3-ß-Nortestosterone α-NT β-NT α-NT β-NT 4 OFES positive sample d3-ß-Nortestosterone d3-ß-Nortestosterone α-NT β-NT α-NT β-NT α-NT β-NT α-NT β-NT GC-MS/MS 2006 Emergency 17α-19-Nortestosterone in bovine urine Meat Inspection Scheme Fortification Mean % Within day Between day Intermediate SD n thth Level recovery Recovery CV CV Precision CV 10 March 2006 0.50 0.56 0.06 19 111.6 9.5 6.0 11.2 1.00 1.08 0.09 19 107.9 6.9 5.6 8.9 α-Nortestosterone - 1.03 ppb 1.50 1.69 0.13 21 112.4 6.6 5.0 8.3 2.00 2.26 0.16 21 113.1 5.3 5.5 7.7 β-Nortestosterone – Compliant Carcass condemned Subsequently found out: “casualty” animal CCα 0.30 µg/kg Subsequently found out: “casualty” animal CCβ 0.59 µg/kg Casualty animal types Test all casualties !! FCI Food Chain Information Farmer declares animal injured Instruction went out from Ministry vets AMF Ante-Mortem Finding All Meat Plants in Northern Ireland “walking wounded” Injury discovered at Meat Plant “Sample all casualty animals” OFES On-Farm Emergency Slaughter More non-compliant animals found Killed on farm, certified by Vet Sent to Plant dead (< 3 hours) 5 Sample splitting Sample splitting Intense media/political interest Allegations !!! 30 samples treated this way Interference with samples in transit 27 in-lab split samples Interference with samples in laboratory 3 Meat Plant split samples Sample splitting either in the lab (QA Unit) No contradictory results found or in Meat Plant (Lab vet) Renumber (blind) Analyse - original sample screened positive Retesting in other labs RIVM The Netherlands (n=11) LABERCA France (n= 6) Mixture of compliant and non-compliant No contradictory results obtained “Where is it coming Finally “ Lab testing is reliable ! ” from?” 6 Positives by casualty type Nortestosterone concentrations α--NortestosteroneNortestosterone NT result Gender present in all 72 nonnon-compliant-compliant samples Count of NT result Negative Positive Grand range CCα ––17.217.2 ppb Negative Positive α Total Total Total levellevel similarsimilar toto treatedtreated animalsanimals Casualty type Bull Male Bull Male AMF 7 1522191032 β--NortestosteroneNortestosterone FCI 224 4 present in only 5 nonnon-compliant-compliant samples OFES 20 46 66 8 54 62 128 range CCα – 1.4 ppb Grand Total 29 63 92 9 63 72 164 1.60 1.40 R2 = 0.8708 1.20 1.00 All 72 positive animals removed from food chain 0.80 0.60 -Nortestosterone OFES animals predominate (86%) β 0.40 0.20 0.00 Male (castrates) predominate (88 %) 0.00 5.00 10.00 15.00 20.00 α-Nortestosterone Clinical History – OFES only Can NT use be out of control ? Number of animals Nortestosterone Do the farmers have a “history” ? History Negative Positive Grand Total Did the prevalence change ? Ataxia/Down 1 1 What was found on farms ? Back Injury 8 13 21 Dislocation 1 1 2 Is ““DeadDead on ArrivalArrival”” relevant ? Fracture 33 25 58 Hip Injury 1 1 2 What about normal animals? Lameness 14 13 27 Nerve Paralysis 3 2 5 Does it happen elsewhere ? Unknown 1 2 3 Down 3 3 Have any follow-up animals been injured ? Other 5 1 6 What other explanation is possible ? Grand Total 66 62 128 Do the farmers have a “history” ? Did the prevalence change ? 100 10 80 60 22 16 29 54 40 29 20 % positive Samples testing 0 10-14 15-18 19-22 23-26 27-30 31-34 Weeks 7 In previous cases: In previous cases: Urine clenbuterol in 1990s Liver clenbuterol in 1990s 70 70 60 60 50 50 40 40 30 30 20 20 Number of positives Number of positives 10 10 0 0 1990 1991 1992 1993 1990 1991 1992 1993 On-farm investigations On-farm investigations First cases All cases Police and Ministry vets Batch of animals defined Raid farm and house No movement off farm Urine samples taken Later cases Tested in laboratory Ministry vets Release only if compliant Raid farm 700+ urine samples tested No nortestosterone detected All negative Is “Dead on Arrival” relevant ? What is found in normal animals ? Normal animals killed at Meat Plant OFES dead for up to 3 hours Other testing schemes 5 untreated steers killed and bled in lab 422 samples tested for nortestosterone Urine from bladder at 0, 3, 6 and 24 hr Three positives Frozen or stored 4°C for 72 hr then –20°C 1) condemned emaciated 2) condemned TB reactor (transportation(transportation time)time) 3) under investigation Analysed by GC-MS/MS 1) & 2) not “normal” No production of α-NT-NT oror β-NT-NT 3) at follow up, all urine negative 8 Does it happen anywhere else ? Have any follow-up animals been injured ? Case 1 Casualty (AMF 06/04/06) Irish Republic (OFES) Hormone problems in the past α-Nortestosterone 1.5 ppb 16 samples On farm visit 24/05/06 10 non-compliant for α-NT 18 animals sampled All negative Great Britain (OFES & AMF) All negative No known problem with hormones 1/18 fracture 28/06/06 8 samples Retested 1 non-compliant for α-NT α-Nortestosterone 1.88 ppb β-Nortestosterone compliant Have any follow-up animals been injured ? Can NT use be out of control ? Case 2 Casualty (OFES 30/6/06) Farmer “history” ? α-Nortestosterone 1.5 ppb Problem goes away ? No On farm visit 24/07/06 On farm findings ? Nothing 10 animals sampled 10 animals sampled Is “DoA” relevant ? No All negative Normal animals? Nothing obvious 1/10 fracture 07/08/06 Just North Ireland ? No Retested Retested animals ? Two positives α-Nortestosterone 3.41 ppb Other explanations ? Stress of the injury β-Nortestosterone compliant Stress What do I need now ? Biochemical link between stress & NT EU awareness of problem Difficult to model – can’t break a leg Simulated in laboratory Debate on natural occurrence Young bulls Sampling in other Member States De-horn & castrate Collect urine & analyse Implementation of hair test No NT produced Investigations continue Proof that it’s natural 9 Thanks to: Armstrong Hewitt Cheryl Rainey Paul Young Robert McCracken Debbie Catherwood Sandy Millar Dermot Faulkner Des Shortt Henrietta Price John Cooper Steven Crooks Imelda Traynor Jean Bowers Pauline Ross Rosie Martin Staff at RIVM ((NL),NL), Laberca ((F),F), State Lab ((IR).IR). Question time Thank you ! 10.
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