October 17, 2008 Seminar Melamine-Contaminated Chinese Milk: Public health Perspective and Management in Thailand Plernpit Suwan-ampai, Ph.D. (Environmental Epidemiologist) Bureau of Occupational and Environmental Diseases Ministry of Public Health

Melamine: Outbreak Chronology What happened? March 2007 Kidney failure-related death of cats and dogs in the US, Europe, South Africa Pet food incorporated Chinese imported “wheat gluten” and “rice protein” adulterated with melamine Sept 18, 08 WHO* reported >6240 cases of kidney stones in infants with 3 death in China – related to Melamine-contaminated powdered infant formula – global concern Exact onset date of illness is unknown but customer complained since March’08 Melamine has been available commercially since the late 1930s. *Resource: WHO-International Food Safety Authorities Network (INFOSAN)

Melamine: Outbreak Chronology (cont.) What happened? Sept 18, 08 cont. >22 dairy manufacturers found to have melamine (range 0.09-2560 mg/kg), recall Sept 22, 08 >53,000 sick and 12,900 hospitalized with additional 1 death (82% affected children <2 yrs of age) >25 countries banned dairy products from China Sept 25, 08 3 manufacturers involved with the outbreak: Sanlu, Guandong Yashili*, Qingdao Suokang* (*Big exporters)

Melamine: Outbreak Chronology (cont.) What happened? Early Oct Thai FDA-an ongoing test >96 products for sale and imports from China Found 0.38-0.55 mg/kg melamine in powdered milk from Shuangwa dairy Co. (Action level* = 1 mg/kg) Found traces in some product i.e., M&Ms, Snickers bars, Oreo wafer sticks, and Dove milk chocolate bars. Oct 13, 08 Switzerland found high melamine concentration in Thai S&P milk cookies Oct 15, 08 Found 92.82 mg/kg melamine in “Mali” unsweetened condensed milk Action level เป็นค่าที่กำหนดขึ้นเพื่อใช้สำหรับตรวจสอบเฝ้าระวัง/การปล่อยสินค้าเพื่อใช้ดำเนินการตาม พรบ อาหาร พ.ศ. 2522 ที่ใช้อยู่ในขณะนี้

พบเมลามีนปนเปื้อนในนมข้นแปลงไขมันไม่หวาน สูตรน้ำมันปาล์ม ตรามะลิ (15 ตุลาคม 2551) สธ. เผย ผลตรวจวิเคราะห์ นมข้นแปลงไขมันไม่หวาน สูตรน้ำมันปาล์ม ตรามะลิ ชนิดกระป๋อง เลขสารบบ อย. 14-1-02323-1-0037 วันหมดอายุ 160109 พบสารเมลามีน 92.82 มิลลิกรัม/กิโลกรัม ซึ่งเป็นปริมาณที่สูงมาก จึงขอให้ผู้บริโภค ผู้ประกอบการ งดใช้ผลิตภัณฑ์รุ่นดังกล่าว เนื่องจากอาจเป็นอันตรายต่อร่างกายได้ อย. ทำหนังสือแจ้งบริษัท ผู้ค้าปลีก และ สสจ. ให้เรียกคืนสินค้าออกจากท้องตลาดแล้ว

What is Melamine? Melamine (C3H6N6) 1,3,5-triazine-2,4,6-triamine Organic base, white solid powder, less water soluble, contained 66% nitrogen by mass Uses: melamine resin (houseware items), glue, countertops, laminates, flame retardants Melamine itself is very low toxic, not a genotoxic or mutagenic agent Melamine is not metabolized and rapidly excrete in urine (T1/2 ~ 3hrs) Melamine use as non-protein nitrogen (NPN) for cattle was described in a 1958 patent. In 1978, however, a study concluded that melamine "may not be an acceptable non-protein N source for ruminants" because its hydrolysis in cattle is slower and less complete than other nitrogen sources such as cottonseed meal and urea. *Source: http://en.wikipedia.org/wiki/Melamine

What else is related to Melamine incidents? Cyanuric acid (CNOH)3 1,3,5-triazine-2,4,6-triol Melamine analogue, white & odorless solid Uses: stabilizer & disinfectant in swimming pool, herbicides, dyes, antimicrobial agents Melamine + Cyanuric acid (CA) – leads to crystals formation and highly nephrotoxicity Unclear whether CA was added intentionally or a by-product of melamine preparation added (melamine  ammeline  ammelide  CA) *Source: http://en.wikipedia.org/wiki/Melamine

Other potential sources of human exposure Consumption meat from animal fed melamine-contaminated pet foods - unlikely to pose a human health risk Melamine-based kitchenware used with hot acidic foods - leached Melamine ~0.007 mg/day which probably no health impact CA is an FDA-accepted of component of feed-grade biuret, a ruminant food additive CA in swimming pool Recommended 100 ppm (may be higher) Estimated ingestion of H2O up to 154 mL (children) ~15.4 mg CA

Acute toxicity of Melamine & Cyanuric acid LD50oral,rat = 3,161 mg/kg LD50oral,mice = 3,296 mg/kg Cyanuric acid LD50oral,rat >10,000 mg/kg LD50dermal,rabbit >7,940 mg/kg Toxicity levels comparable to NaCl

Chronic Toxicity of Melamine No human data on oral toxicity of melamine Toxic effects (from animal feeding studies): Calculi formation, inflammatory reaction & hyperplasia in the urinary bladder  renal failure May leads to reproductive damage, bladder/kidney stones  bladder cancer Dogs fed 3% melamine 1 yr: 1) reduced specific gravity, 2) increased output, 3) melamine crystalluria, and 4) protein & occult blood Carcinogenicity Class 2B (IARC 1999) Calculi are a mixture of melamine protein, uric acid, and phosphate and are distinct from other kidney stone. Class 2B = Sufficient evidence in animal models but inadequate evidence for carcinogenicity in humans.

Chronic Toxicity of Cyanuric acid (CA) No human data on oral toxicity of CA Toxic effects (from animal feeding studies): Sub-chronic oral tox. studies in animal - caused renal tissue damage incld. 1) dilatation of renal tubules, 2) necrosis and hyperplasia of tubular epithelium, 3) increased basophilic tubules, 4) neutrophilic tubules, 5) mineralization, and 6) fibrosis In human, 98% of an oral dose is excreted unchanged in urine within 24 hrs. Not include genotoxic, carcinogenic, and mutagenic effects Calculi are a mixture of melamine protein, uric acid, and phosphate and are distinct from other kidney stone.

Melamine + CA toxicity – Why? absorption in GI tract Systemic distribution Water solubility: Melamine = 3,240 mg/L Cyanuric acid = 2,000 mg/L Melamine cyanurate = 2.2 mg/L Melamine cyanurate precipitation in kidneys Melamine and cyanuric acid are of low acute toxicity. However, malamine cyanurate has very low solubility and it is hypothesized that this leads to the formation of malamine cyanurate crystals in kidney. Synergistic Toxicity *Source: National Toxicology Program Research Concept: Melamine/Cyanuric Acid, USA

Melamine Cyanurate Crystal University of California, Davis, veterinary toxicologist Birgit Puschner and colleagues reported on the results of feeding melamine and cyanuric acid individually and in combination to a few cats (J. Vet. Diagn. Invest. 2007, 19, 616). The researchers found that a single 32 mg/kg dose of the combination can cause acute renal failure in cats. Puschner suggested that the compounds cause renal damage at least in part through precipitation of melamine cyanurate crystals out of acidic urine in the kidneys. As Puschner suggested, the behavior of melamine and cyanuric acid depends on pH. Tolleson determined that different pH values in different zones in the body influence the solubility of melamine and cyanuric acid Fig: PERILOUS CRYSTALS-Pets that ate the tainted food developed kidney stones. Polarized light microscopy (right) highlights the greenish brown kidney stones shown in kidney tissue (left) *Source: http://pubs.acs.org/cen/science/86/8619sci3.html

Potential Biomarkers of Melamine – Nephrotoxicity relationship Exposure Internal dose Biologically effective dose Early biological response Altered structure/ function Disease Susceptibility Melamine content analysis in Milk & dairy products Pet foods Vegetable proteins 4-Hydroxyproline Homovanillic acid (HVA) sulfate NAG* Kim-1* (used by US FDA) Oliguria/anuria Kidney stones Renal failure Age Race Sex SES Others Macroscopic/microscopic hematuria Altered renal fnc. Urine melamine (HPLC/MS) but not validated Melamine can be detected in urine and directly from tissue by liquid chromatography " tandem mass spectroscopy. Rapid testing procedures have not been validated. Fourier-transform infra-red microscopic analysis of crystals and ELISA tests have been described. 4-Hydroxyproline is a hydroxylated form of the imino acid proline. A deficiency in ASCORBIC ACID can result in impaired hydroxyproline formation. NAG = N-acetyl-{beta}-D-glucosaminidase - a marker of tubular damage Homovanillic acid sulfate is one of urine cathecolamines Kidney Injury Molecule-1 (Kim-1) is a type-1 membrane protein with extracellular immunoglobulin and mucin domains *NAG = N-acetyl-{beta}-D-glucosaminidase; Kim-1 = Kidney Injury Molecule 1

Melamine Analysis: Laboratories available in Thailand บริษัท ห้องปฏิบัติกลางการตรวจสอบผลิตภัณฑ์เกษตรและอาหาร จำกัด (LCFA) บริษัท เอส จี เอส (ประเทศไทย) จำกัด (SGS) 2.1. Melamine (LOD = 0.05 ppm) 2.2. Cyanuric acid (LOD = 1.0 ppm) 2.3. Ammeline (LOD = 0.1 ppm) 2.4. Ammelide (LOD = 0.5 ppm) OMIC Bangkok Laboratory Thai-FDA Hotline: 0 2590 7212 & 0 2590 7175

Strategic plans & actions on Health impact of Melamine Activities: 1st Prevention – Remove Exposure (Thai-FDA) - Ban Chinese milk/dairy products immediately if imported from involved manufacturers* - Survey and monitor suspected products esp. quarantine & restricted test for products imported from China - Educate consumers (risk communication to public) - Encourage relevant manufacturers to remove suspected product from shelves for investigation - Law enforcement – พรบ อาหาร พ.ศ. 2522 มาตรา 26(1) จำคุกไม่เกิน 2 ปี/ปรับไม่เกิน 20,000 บาท/ทั้งจำทั้งปรับ พรบ อาหาร พ.ศ. 2522 มาตรา 26(1) จำคุกไม่เกิน 2 ปี/ปรับไม่เกิน 20,000 บาท/ทั้งจำทั้งปรับ ว่าด้วยอาหารที่มีสิ่งที่น่าเป็นอันตรายต่อสุขภาพเจือปนอยู่ *Note: Banning products currently is not an action plan in Thailand

Strategic plans & actions on Health impact (cont.) Activities: 2nd Prevention – Early detection (DDC) - Screening biomarker of exposure i.e., internal dose – not yet available - Coordinate with provincial PH offices and pediatricians in relevant agencies to set a surveillance program – to early detection cases 3rd Prevention – Reduce severity (Tertiary service providers) - Appropriate treatment, referral system, and compensation - Review hospital discharge data for ICD-10 codes related to renal failure and urolithiasis for infant aged <10 yrs* *Note: recommended strategic plan

Possible strategies to be considered for Thai government ออกข้อกำหนดทางกฎหมายเพิ่มเติม เช่น กำหนดปริมาณการปนเปื้อนเมลามีนและสารที่เกี่ยวข้องสูงสุดที่ยอมให้พบในอาหาร (TDI) พิจารณา ห้าม ผลิต นำเข้า และจำหน่ายผลิตภัณฑ์ที่ปนเปื้อนเมลามีน ศึกษาวิธีการตรวจวิเคราะห์สารเมลามีนและสารที่เกี่ยวข้องที่เหมาะสม การประสานความร่วมมือ: 1) การร่วมมือระหว่างหน่วยงาน, 2) ระบบการสื่อสารความเสี่ยง, 3) มาตรการป้องกันเมลามีนปนเปื้อนเข้าสู่วงจรอาหาร (Food Chain) *TDI = Tolerable daily intake

Guidance on levels of health concern in food TDI (Tolerable daily intake) for Melamine & its analogues – from risk assessments in 2007 incident TDI: US FDA = 0.63 mg/kg BW/day TDI: EFSA* = 0.50 mg/kg BW/day แนวทางการกำหนดค่าสูงสุดที่ยอมให้มีในอาหาร WHO guideline: assuming TDI = 0.50 mg/kg, if an infant wt 4 kg  then TDI = 2 mg/day assuming infant has 750 mL-milk/day  TDI = (2 x 1000)/750 == 2.7 mg/L Risk assessment: โดยใช้ข้อมูลการบริโภคของประเทศไทย และข้อมูลการปนเปื้อนในจีน จากค่าเฉลี่ย = 29 mg/kg & highest level = 2500 mg/kg Risk = (ปริมาณการบริโภคอาหาร * ปริมาณการปนเปื้อนที่พบอาหาร * 100)/ค่าความปลอดภัย (TDI) WHO guideline ’08 - น้ำนม < 2.7 mg/L - นมผง < 27 mg/kg Risk assessment - น้ำนม < 1.3 mg/L - นมผง < 13 mg/kg *EFSA = European Food Safety Agency

Case Scenarios – Approaches? Hx of 1 time/occasionally consuming Malamine-contamination milk product with no clinical complaint? Hx of regularly consuming Malamine-contamination product with no clinical complaint? Case 2 years old girl presented with clinical symptoms of nephrotoxicity? How to r/o Melamine-related nephrotoxicity?

WHO-Guideline: Criteria for Diagnosis History of consumption contaminated products Having 1 or more following symptoms: 2.1. Unexplained fever arising from UTI/bacteraemia 2nd to urine stasis from obst’n 2.2. Macro-/microscopic hematuria 2.3. Acute obstructive renal failure: oligouria/anuria 2.4. Stone discharged while passing urine 2.5. HT, edema, painful when knocked on kidney area Lab test results: UA with macro-/microscopic haematuria; blood biochemistry; LFT & RFT; Urine Ca/Cr ratio (~normal); normal morphology of rbc Imaging examination: prefer U/S B exam

WHO Recommended Clinical treatment guideline Stop using melamine-contaminated food products Medical treatments of kidney stones: 2.1. Infusion & Urine alkalinization – dispel stones 2.2. Correct water, electrolytes, & acid-base imbalance, & symptomatic treatments Treatment of complicated acute renal failure Clinical treatment: Immediately stop using melamine-contaminated food products. Medical treatment: use infusion and urine alkalinization to dispel the stones. Correct the water, electrolyte and acid-base imbalance. Closely monitor routine urine tests, blood biochemistry, renal functions, ultrasound findings (with particular attention to the renal pelvis, ureter expansion, and the change of the stones in shape and location). If the stones are loose and sand-like, they are very likely to be passed out with urine. Treatment of complicated acute renal failure: priority should be given to the treatment of life-threatening complications such as hyperkalemia. Measures include the administration of sodium bicarbonate and insulin. If possible, blood dialysis and peritoneal dialysis can be used early. Surgical measures can be taken to remove the obstruction if necessary.

References: http://www.fda.moph.go.th/www_fda/fda_melamine/index-melamine.php http://en.wikipedia.org/wiki/Melamine http://www.medicalnewstoday.com/articles/124130.php http://www.who.int/foodsafety/fs_management/infosan_events/en/index3.html รายงานการประชุมคณะกรรมการอาหาร เรื่อง การปนเปื้อนสารเมลามีนในผลิตภัณฑ์อาหาร, 29 กันยายน 2551