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Fishing for an answer: How did melamine a non-toxic compound cause so much toxicity in pets and kids? Renate Reimschuessel VMD PhD Center for Veterinary Medicine Gieseker, C.

1 Fishing for an answer: How did melamine a non-toxic compound cause so much toxicity in pets and kids? Renate Reimschuessel VMD PhD Center for Veterinary Medicine Gieseker, C., Eric Evans, Nick Hasbrouck, Tamara Mayer Miller, R.A., Rummel, N., Heller, D., Nochetto, C. de Alwis, H.A., Andersen, W.C., Turnipseed, S.B., Karbiwnyk, C.M., Satzger, R.D., Crowe, J., Wilbur, N., Roberts, J., Reinhard, M.K., Witkowski, M.

2 Center for Veterinary Medicine s Aquaculture Research Infectious Studies Radioactive Drug Studies

3 Why is a fish vet talking to you about the Pet food recall?

4 2007 FDA s Largest Recall 350 people at CVM CVM s Web site attracted 1.3 million hits FDA s Pet Food recall page attracted 6.1 million hits 18,700 calls in an 11 week period (5-6000/ year)

5 ? R. Reimschuessel Practicing Veterinarian U. of Maryland School of Medicine Aquatic Diagnostic Path Kidney Research Teaching Vet & Medical Students

6 Pet food company came forward alerting FDA 2007 Pet Food Recall: What Happened? Mid February MENU FOODS received consumer complaints Early March Cats died during a feeding (taste test) trial Mid March MENU FOODS issues pet food recall March 15

7 What was in the food? March 30, 2007 H 2 N N NH 2 N N Melamine NH 2 Found in pet food Found in cat urine Toxicologists say NOT really toxic??? Is it the cause???

8 Melamine Target.com Plastics Fertilizer Flame-retardant with Cyanuric acid Drug delivery Neerman-et al-2004 Sigma-Aldritch

Menu Foods traced the wheat gluten to China http://standeyo.

9 Menu Foods traced the wheat gluten to China BAGS got mixed up somehow?

10 April 19, 2007 RICE Protein Concentrate contains Melamine And it was really wheat flour

11 If not an accident then Why? PROTEIN = $

12 How was melamine causing kidney failure in the pets?

13 Conference call on Unusual Crystals Vet Pathologists saw crystals in kidney but not enough to cause clinical signs not enough to cause pathology Pathology photos sent to FDA

14 Crystal Morphology Michigan State University- Apr 3, 2007 Needle like crystals arranged in a radial sphere These crystals looked familiar to me

15 Human Gout Uric acid crystals URIC ACID Needle like crystals, dissolve in formalin

Uric Acid Spherulites Human Gout Ascidian

16 Uric Acid Spherulites Human Gout Ascidian Renal Sac Spherulites Urate Spherulites in Gouty Synovia Fiechtner et al. JAMA : Uric Acid Accumulation in the Solitary Ascidian Corella inflata Lambert et al. J. Exp Zool. 282: (1998)

17 Massive literature search on melamine

18 2 year melamine - cancer study Ogasawara, et al (1995) Urinary bladder carcinogenesis induced by melamine in F344 male rats: Correlation between carcinogenicity and urolith formation. Carcinogenesis, 16, Rats fed melamine for 2 years developed Bladder stones: Melamine:Uric acid 1:1 Microcrystals in renal pelvis Ogasawara et al. 1995

19 Uric Acid Crystals Dissolve in Formaldehyde Formaldehyde is routinely used to preserve tissue for pathology Could crystals be dissolving?

20 Hypothesis Could a combination of melamine with uric acid or cyanuric acid be toxic? Is the pathogenesis similar to Acute uric acid nephropathy? Submitted April 6, 2007

21 April 20, 2007 CYANURIC ACID Another high nitrogen compound had was found in pet food HO N N OH N OH

April 27, 2007 85,000 HOGS May 1, 2007 http://earthfood.wordpress.

22 April 27, ,000 HOGS May 1, Million CHICKENS

23 May 9, 2007 NOW FISH

24 HOW? Contaminated ingredients Pet food manufacture Unused Scraps Pet food Processed into other feeds Fed to livestock Pet food recall Disposed

25 Filler in Animal Feed Is Open Secret in China Using scrap melamine Nationwide recall of pet foods Melamine and Cyanuric Acid

26 May 9, 2007

com/product/50326245/51452118/feeds/chinese_shrimp_feed.

27 Shrimp and Fish feeds sold in same area that produced the wheat gluten and rice protein concentrate What about imported fish eating fish food from Asia?

28 Concerns for human food safety We had Hogs Chickens Fish that were exposed to contaminated feed WE NEEDED METHODS NOW!

29 AQUACULTURE METHODS GROUP Dose Animals Develop Methods Method Trials Official Regulatory Method FDA COMPLIANCE PROGRAM Harmful Residues 1914 NOW

30 We Needed Methods! Current program for providing dosed animal tissues already in place at CVM (esp for fish) Collaborations between FDA Centers already in place Within 2 weeks we had begun dosing animals to provide tissues to chemists

31 Dosing 400 mg/kg BW for 3 days Melamine (MEL) Cyanuric acid (CYA) Both (400 mg/kg of each drug) Species Fish Hogs Chicken

32 CVM dosing fish

33 Christine Karbiwnyk Mark Madson Collaborating FDA Labs Developed Methods for Edible Fish Tissues Sam Gratz Martha Schwartz Sherri Turnipseed Wendy Andersen Animal Drugs Research Center, Denver Bryan Gamble FDA Forensic Chemistry Center Cincinnati Alex Krynitsky CVM (kidneys) Dave Heller

34 Results: THE METHODS WORK Detected Melamine in Muscle and Kidneys 1, 3, 6, 14 days post-dose in PPM (mg/kg) Now we could screen for Melamine and Cyanuric acid

35 What about the pets and those unusual crystals? Were they the cause of the renal failure? What was their chemical composition?

36 Cat Case Frozen tissue available U. Florida Crystals present in tubules Yellow Crystals with spherical radial pattern like urate crystals and cellular debris

37 Crystal Properties? Crystals resembled uric acid crystals Uric acid crystals are soluble in formalin Examine tissues as a wet mount to evaluate crystals without fixative

38 Wet mounts Debris caught in tubule upstream

39 Many crystals in the medulla

slide Same field photo taken unfixed, then flooded with formalin,

40 Crystals Response to Formalin 60 hours later no crystals Kidney unfixed Friday PM Dirt on slide Formalin fixed Monday AM Dirt on slide Same field photo taken unfixed, then flooded with formalin, left on microscope over weekend. Crystals not visible on Monday (same field)

41 0 hr 2 hr 5 hr Response to Formalin or HCL 10% Neutral Buffered Formalin 0 min 4 min 6 min 8 min 0.2 N HCL 6 um frozen sections

42 Shared this information AVMA AAVLD

43 Cat Crystal Analysis: Optical microscopic and micro Raman chemical analysis FDA Forensic Chemistry Center Raman Polarizing Light Microscope (PLM) FT-IR Raman / FT-IR Spectrometer System Microscope with a laser to get fingerprint of compounds John Crowe Mark Witkowski

44 Cat crystals consistent with Melamine:Cyanurate spectrum Fourier Transform Infrared Spectroscopy FDA Forensic Chemistry Center

45 June 2007 What about the fish kidneys? Dosing showed residues in the muscle How were the kidneys?

46 Glomerulus Renal Lesions? Neck Fish kidneys are similar to Mammalian (minus Loop of Henle) Proximal Distal Catfish Tilapia Warmwater Collecting Duct Trout Salmon Coldwater Freshwater Fish

47 Glomeruli Catfish Kidney CONTROL Melamine Only Cyanuric acid Only (Normal appearance) Glomeruli Crystals MELAMINE+CYANURIC ACID DOSED

48 Catfish Melamine + Cyanuric acid Crystals

49 Catfish day 1 after dose Melamine:Cyanurate Combination

50 Crystal Histopathology Color: gold-brown, bluish, pink, pink ring Size: µm Location in kidney: cat & pig Medulla/Cortex fish no regions Proximal, distal and CD Clusters or single Location in tubule: lumen or wall

51 Variable color Melano-macrophages Hematopoietic tissue Day 1 Tilapia Day 3 Trout Day 14 Salmon

52 Variable size Large accumulations Small crystals o14 Day 1 Tilapia

53 Location- clusters

54 Day 1 catfish Location - Scattered

55 Location in tubule: lumen

56 Location in tubule: wall Necrotic cells and blebs Pink ring Embedded tubule wall Day 6 Salmon

57 Day 6 Salmon Location in tubule: wall

58 Cat histopathology similar

59 Renal Response Inflammation Fibrosis Epithelial Hyperplasia Regeneration along basement membrane Nephron neogenesis (warm water fish)

60 Day 6 Tilapia Inflammatory response

61 Inflammation and fibrosis around crystals 1 week after dosing Day 6 Tilapia

62 Fibrosis 2 weeks after dosing Day 14 Catfish

63 Day 14 Tilapia Hyper/dysplastic epithelium

64 Regenerative epithelium Polarized light helps locate crystals Day 3 catfish

65 Gastrointestinal tract White feces from tank White ppt in intestine Day 1 Trout Hemorrhagic mucosa

66 Crystals in lumen, on mucosa, in lamina propria, in submucosa, occasionally in muscularis Stomach Intestine S32 Ulceration Muscularis Day 3 Salmon

67 Similar morphology to those seen in pets Thus by mid June 2007, the agency had it s own experimental evidence that the crystals form when animals are given a combination of melamine and cyanuric acid

68 Peri-renal edema Pig Kidney M:C Crystals Small hemorrhages This pig had elevated BUN and Creatinine

69 H 2 N Low Toxicity N NH 2 Low Toxicity HO N OH N N N N NH 2 OH Melamine Cyanuric Acid Bonding Interactions Renal crystals Crystal Lattice Structure

Creatinine Dobson et al Rat Diuresis then anuria BUN, Creatinine, uric acid

70 Clinical findings in experimental animals given both MEL & CYA Reimschuessel et al. Pig Bloody diarrhea BUN, Creatinine Ensley et al Pig Bloody diarrhea BUN, Creatinine Dobson et al Rat Diuresis then anuria BUN, Creatinine, uric acid Urine ph Puschner et al Cat Anorexia BUN, Creatinine ALL STUDIES CLINICAL RENAL FAILURE

71 wikipedia Why does the combination cause crystals?

72 Crystals form in tubules Plugging the Plumbing Like Acute Uric Acid Nephropathy in humans Locations vary depending on species

73 September 11, 2008 Melamine in infant formula in China 6 deaths Over 300,000 sickened

74 China Pediatricians noted unusual number of babies with kidney stones (usually very rare) Consumers reported dark urine, sandy urine, painful urination, no urine (stones) Melamine found in infant formula (esp Sanlu)

75 News Reports Children on formula a long time Bloody urine Sandy urine Stones urinary obstruction Renal Failure (?-Obstruction? Crystals?)

melamine in possession) Melamine was being added to watered down milk by

Added to powered products???(powdered milk-infant formula-others)?? http://www.

76 What is happening? Melamine was being added to watered down milk by dairy farmers (arrests with melamine in possession) Melamine was being added to watered down milk by producers/distributers (arrests with melamine in possession) Melamine? Added to powered products???(powdered milk-infant formula-others)??

77 What is causing the toxicity Melamine alone in rats causes stones within 13 weeks (melamine:uric acid) Our data shows crystal nephropathy if given melamine+cyanuric acid Could both of these things be happening in the affected children chronic melamine effects acute combined triazine effects LUCKILY NOT??

78 Crystal interaction wikipedia Melamine-Cyanuric acid interaction to form crystals Uric acid Melamine Melamine Uric acid May form a complex, perhaps less stable

79 2008 melamine in formula as high as 5000 ppm Primary contaminant MELAMINE pure (very very little cya) Stones: melamine uric acid

80 Melamine-Uric Acid STONE Melamine-Uric Acid Grases et al 2009 Spain- adopted 11 mo girl

81 Zoo animals with kidney stones and 1500 Raccoon dogs died ight-to-survive-revisited-part-2-one-year-ago/

82 What has CVM been doing? Reimschuessel et al. Evaluation of the renal effects of experimental feeding of melamine and cyanuric acid to fish and pigs. Am. J. Vet. Res.69: Andersen et al. Determination and confirmation of melamine residues in catfish, trout, tilapia, salmon, and shrimp by LC-MS-MS. J Agri Food Chemistry. 56: Karbiwnyk et al. Determination of cyanuric acid residues in catfish, trout, tilapia, salmon and shrimp by liquid chromatography-tandem mass spectrometry. Analytica Chimica Acta IN PRESS Dobson et al. Identification and characterization of toxicity of contaminants in pet food leading to an outbreak of renal toxicity in cats and dogs. Toxicological Sciences 106: Heller et al. Simultaneous determination and confirmation of melamine and cyanuric acid in animal feed. Rapid Communications in Mass Spectrometry, 2008 in press. Publishing our findings from May and June 2007

83 CVM adapted feed method to use for infant formula New methods posted Oct 8, 2008 Melamine and Cyanuric Acid

84 What else has CVM been doing? Depletion study in fish Threshold dose study in fish Sequential dose study in fish Starting a Threshold study in pigs

85 Melamine in feed still a concern Nov 1,

86 Risk Assessments Level unlikely to pose health risks 1 ppm infant formula 2.5 ppm other products

87 Depletion Single dose 20 mg/kg BW oral Catfish 24 o C Trout 12 o C

88 Crystals for in fish given melamine first then cyanuric acid later

89 Sequential Dosing of Melamine and Cyanuric Acid: MEL CYA CYA CYA CYA CYA Days Crystals trout catfish /3 2/3 1/3 0/3 2/3 1/3 1/3 0/3 0/3 CYA MEL MEL MEL Days Crystals trout 2/3 0/3 0/3 catfish 1/3 0/3 0/3

21 3/3 2/3 1/3 0/3 2/3 1/3 1/3 0/3 0/3 CYA MEL MEL MEL

90 Sequential Dosing of Melamine and Cyanuric Acid: MEL CYA CYA CYA CYA CYA Days Crystals trout catfish /3 2/3 1/3 0/3 2/3 1/3 1/3 0/3 0/3 CYA MEL MEL MEL Days Crystals trout 2/3 0/3 0/3 catfish 1/3 0/3 0/3

91 NO OBSERVABLE EFFECT LEVEL (NOEL) Single dose? Several doses? Many doses?

92 Threshold Dose- in catfish not found yet Dose mg/kg bw Dose ppm in diet # Days dosed 10 NOEL NOEL Crystals 0.1?

93 Threshold Dose- Preliminary Data in pigs Dose mg/kg bw Dose ppm in diet # Days dosed 10 crystals 3.3 NOEL? 1.0 NOEL?

94 MELAMINE EVENTS Dogs and Cats Kidney Failure Death MEL +CYA 100s 1000s Babies Kidney Stones Death (6) MEL only 300,000

95 White rabbit candy

97 Questions? Malaysian authorities supervising the dumping and burying of contaminated biscuits and ammonium bicarbonate into a landfill Taiwanese authorities detected melamine in 469 tons of baking ammonia imported from China. Samples tested showed up to 2,470ppm of melamine

98 RISK ASSESSMENT STAND UP!

99 Risk assessment Chapter 4: Risk analysis of food additives D R Tennant, Consultant, UK

100 What is in there? How toxic is it? How much is in there? Gluten Risk assessment Animal feed Scraps to livestock feed

101 How much is in there? How much did you eat?

102 Questions How much accumulates in muscle? How long does it stay there? What are the risks to humans? OOPS We DON T HAVE METHODS to look for these compounds in muscles

103 Chicken study (excrete uric acid normally) 0 sec 5 sec 10 um 40 sec 60 sec Crystals from chicken treated with Melamine 400 mg/kg Frozen 6 um sections treated with formalin. Crystals URIC ACID+melamine

104 0 sec 3 hours 10 um Crystals from chicken treated with MEL + CYA 400 mg/kg Frozen 6 um sections treated with formalin. Dissolved after 8 hours

105 Previous melamine studies: Histopathology Fixed in Formalin If melamine uric acid spherulites formed they would have dissolved in MINUTES 600 sec

Melamine and Analogues in Food

Melamine and Analogues in Food Yan Gu Office of Food Additive Safety Center of Food Safety and Applied Nutrition Food and Drug Administration April 23, 2009 1 Toxicology of Melamine and Analogues Sources