Biochemie der Ernhrung

1
Issue Semicarbazid

• A contaminant found in food jars

Update 14th April 2004
2
Semicarbazide (SEM)The Issue

• Semicarbazide is found in Baby Food
• SEM levels of up to 20 µg/kg were found in baby
  food
• Baby food was analysed for SEM as marker for
  illegal vetenary drug use
• SEM is a protein bound metabolite of the drug
  nitrofurazone

3
SemicarbazideChemical Data

• Chemical Names
• Semicarbazide hydrochloride1- Synonyms
  amidourea hydrochloride aminourea hydrochloride
  carbamylhydrazine
  hydrochloride hydrazinecarboxamide,
  monohydrochloride - CAS Nr. 563-41-7
  semicarbazide hydrochloride- formula CH6ClN3O
  MW 111.5 g/Mol

4
SemicarbazideToxicology

• Toxicity6,8 (available information 07/2003)

5
SemicarbazideToxicology

• Ames Test (Oct. 2003)
• No mutagenicity observed in Salmonella
  typhimurium strains TA1537, TA98, TA100, TA102
  /- S9 up to 5000 ug per plate
• Weak mutagenic response in strain TA1535 at 5000
  ug/plate
• NoteSalmonella typhimurium TA102 normally
  detects mutagenic hydrazine derivatives
• In-vivo UDS assay
• No increase in UDS, no genotoxic effect

6
SemicarbazideToxicology

• Exposition8
• infant, 4 months
• Not worst case 0,48 µg/kg body weight (daily
  intake of 190g jar food ( 1 glass jar),
  contaminated with 15 ppb SEM of a 6 kg weight
  baby)
• Worst case 1,25 µg/kg body weight(daily intake
  of 360g jar food ( 3 glass jar á 125g),
  contaminated with 20 ppb SEM of a 6 kg weight
  baby)
• Adults0,025 µg/kg body weight

Prof. Eisenbrand, Kaiserslautern, Germany
7
SemicarbazideToxicology

• Exposition (calculated by joint industry group,
  JIG)
• DONALD study Intakes of commercial baby food9

) 95th-percentile
An exposure of 1 µg/kg BW/day is unlikely to be
of concern for non-thresholded (cancer) effects.
The TD50 (dose producing tumor in 50 of the
animals) for SEM is 220 mg/kg BW/day.
8
SemicarbazideToxicology

• Exposition (calculated by joint industry group,
  JIG)
• Age-specific exposures to SEM (according to the
  intake of commercial baby food9 and assuming a
  contamination level of 20 ppb in all products)

) 95th-percentile
9
SemicarbazideToxicology

• Conclusions 6,8 (available information 07/2003)
• 2 studies indicated that SEM may cauce cancer in
  female mice ? data are insufficient for firm
  conclusions about mutagenicity
• Birth defects are only shown for exposure levels
  very high compared to SEM detected in foods ? no
  matter of concern for teratogenicity

10
SemicarbazideMetabolite of Illegal Veterinary
Drugs?

• Nitrofurans
• Derivatives of 2-Nitrofurane
• Nitrofurans are known mutagens and potentially
  cancerogenic
• Fast metabolisation in the farm animals
• Metabolites are used as indicators for the use of
  nitrofuranes
• Since 1995 the use of nitrofuranes in the EU is
  forbidden? zero-tolerance is 1 µg/kg food

11
SemicarbazideMetabolite of Illegal Veterinary
Drugs?
Semicarbazide (protein bound metabolite )
Nitrofurazone (antibiotic)
add 2-Nitrobenzaldehyd in 0.2 M HCl, 12h at
RTEtOAc extraction
derivatised metabolite, analysis by LC-MS/MS
12
SemicarbazideContaminant from the Cap Gasket?

• Industry labs showed that the food (used for
  filling the jars)was not the source of SEM
• All closures of baby food seem to be involved but
  only some glass containers filled with other
  foods
• Levels of 1 7 mg SEM/kg gasket material (baby
  food closures) have been determined(LUA,
  Dresden, Germany)
• SEM contamination in baby foods 95th percentile
  20µg/kg, in other products 3µg/kg
• High variation among different lotsof the same
  product mean 18ppb, range 5 50 ppb(migration
  by condensed water? Raeation with food matrix?)

13
SemicarbazideDecomposition Product of
Azodicarbonamide?

• Closures for glass containers
• Different foods (jams, sauces, ketchups, honey,
  juice drinks babyfoods) are filled in glass jars
• Some closures are manufactured with a seal
  containing ADC

United States Department of Agriculture http//www
.agen.ufl.edu/foodsaf/he203.html
14
SemicarbazideDecomposition Product of
Azodicarbonamide?

• Basic parts of a glass jar

http//www.ehcan.com/JarsClosure.html
15
SemicarbazideDecomposition Product of
Azodicarbonamide?

• Closures for glass containers
• Twist-off cap
• Press-On Twist-Off (PT) closures

http//www.ehcan.com/JarsClosure.html
16
SemicarbazideDecomposition Product of
Azodicarbonamide?

• Closures for baby food in glass containers
• Nearly exclusively PT (Press-on Twist-off)
  closures Long shelf-life hermetic seal for
  Sterilised/Pasteurised foods in glass jars
• Gives total protection against microbiological
  re-contamination (top- and side seal)
• Gives consumer indication of leakage/spoilage
  (safety button)
• Sealing Gasket is an ADC-foamed PVC Plastisol

17
SemicarbazideDecomposition Product of
Azodicarbonamide?

• Foaming agents / Blowing agents5
• organic nitrogen compounds (carbonates do not
  produce the desired fine, closed-cell structure
  in rubbers and plastics)
• stable at normal storage and mixing temperatures
• undergo controllable gas evolution at reasonable
  well-defined decomposition temperatures
• are added to polymers in order to form minute gas
  cells throughout the plastic ? sealing gaskets
  for metal lids on glass bottles and jars
• gases should provide low permeability through the
  polymersfor rubber CO2 (260) gtgt H2 (100) gt
  O2 (46) gt N2 (17)) relative values based on
  hydrogen as 100, but most seals are based on PVC

18
Azodicarbonamide (ADC)

• Synonyms1 1,1'-azobiscarbamide
  1,1'-Azobisformamide Azodicarbonamide
  azobiscarboxamide azodicarboxylic acid diamide
  azoformamide Diazenedicarboxamide
  diazenedicarboxylic acid diamide
  C,C'-azodi(formamide) celosen az chkhz 21
  chkhz 21r delta(1,1')-biurea Genitron genitron
  ac genitron ac 2 genitron ac 4 kempore
  kempore 125 kempore r 125 lucel ada Nitropore
  pinhole ak 2 Porofor porofore 505 porofor
  adc/r porofor chkhz 21 porofor chkhz 21r
  Unifoam unifoam az uniform az yunihomu az
• CAS Nr. 123-77-3- formula C2H4N4O2 MW 116.1
  g/Mol
• Use2
• blowing agent in plastics
• Oxidative flour treatment (45 mg/kg flour, not
  EU)
• Decomposition temperature 195-202 CAmount of
  gas generated 220 245 mL/gSolubility
  0.2g/L water, 0.1g/L methanol
  2.6g/L DMF, 43.8g/L DMSO

19
Azodicarbonamide Decomposition

• decomposition speed and temperature of
  azodicarbonamide can be controlled by changing
  the type of resin and the use of different
  additives like metal salts4

20
Azodicarbonamide Synthesis

• Synthesis according to (5)(1) formation of the
  intermediate biurea from urea and hydrazine(2)
  oxidation yielding azodicarbonamide

21
Azodicarbonamide Decomposition Reactions I4
22
Azodicarbonamide Decomposition Reactions II5

• Dry decomposition (190 C)
• under liquid paraffin (190 C)

Gaseous products19.8 nitrogen, 11.2 CO,1
NH3, CO2
Decomposition depends on the specific curing
conditions (see part of biurea)
23
Azodicarbonamide Safety Evaluation

• Scientific Committee on Food (SCF) opinion (2003)
  on Azodicarbonamide (ADC)
• Azodicarbonamide per se seems of minor
  toxicological significance except ist ability to
  induce asthmatic conditions in exposed workers
• main problem are the formation of undesirable
  by-products
• Formation of ethyl carbamate from
  azodicarbonamide containing materials in contact
  with alcoholic beverages3,4
• Ethyl carbamate is a well known animal cancerogen
• Conclusion Restriction for use only as a blowing
  reagent and not for
  materials intendet to come into contact with
  alcoholic beverages

24
Azodicarbonamide Safety Evaluation

• SEM is present as an impurity in ADC, but at
  insignificant amounts compared to the formation
  of SEM after thermal treatment of ADC

25
Azodicarbonamide Substitution Products (Joint
Industry Group)

• Non-PVC technology
• Under development by cap suppliers
• Major capital investment for babyfoods
• long term option for industry
• Sodium bicarbonate
• First trial too high leakage rate
• Last trials encouraging
• Reduced bicarbonate particle size
• Water scavenger to prevent reagglomeration

26
Azodicarbonamide Substitution Products (Joint
Industry Group)

• First Trial

Sodium bicarbonate
Azodicarbonamide
TEMPERATURE
27
Azodicarbonamide Substitution Products (Joint
Industry Group)

• First Trial
• Trial volume 1,8 Mio glass jars (Aug. Dec.
  2003)
• Dud rates (leakage) for pasteurized products are
  accepatable
• Dud rates for sterilized products (80 on the
  market) about 50 times higher than minimum
  required
• Major improvement required

28
Azodicarbonamide Substitution Products (Joint
Industry Group)
Second/Third TrialCompound with reduced
bicarbonate particle size
Improved cell structure
Compound 284 Ver. 1
Compound 071 (ADC)
Compound 284 Ver. 3
Sodium bicarbonate (1st trial) Sodium
bicarbonate (2nd trial) Azodicarbonamide
Conclusion Optimized / reduced sodium bicarbonate
particle size further improves compound cell
structure and surface.
29
Food Sources of SEMHypochlorite treated
Carrageenan10,11

• SEM was detected in Carrageenan
• Polysaccharide extracted from red seaweeds
• Food additive (E 407) thickening agent
• Applications
• Ham, bacon, processed poultry, sausages
• chocolate milk, dressings
• Ice cream, yoghurt, pudding
• Dessert jellies, fruit jam
• Petfood
• Toothpaste, airfreshner gels

30
Food Sources of SEMHypochlorite treated
Carrageenan10,11
drying
alkaline extraction
Red seaweed
drying
filtration, concentration, alcohol or salt
precipitation, drying
bleaching
grinding, blending
Semirefined Carrageenan (PES)
Carrageenan
31
Food Sources of SEMHypochlorite treated
Carrageenan10,11

• SEM analyses (WEJ, Hamburg, Germany)
• raw red seaweed 0.2 ppb
• red seaweed, dried 2 ppb
• Semirefined carrageenan (PES) before bleaching 7
  - 9 ppb
• Semirefined carrageenan (PES) 10 - 100 ppb
• Carrageenan lt 0.2 - 9 ppb

E 407a
E 407
32
Food Sources of SEMHypochlorite treated
Carrageenan10,11

• Precursors11

33
Food Sources of SEMHypochlorite treated
Carrageenan10,11
NH3 NaOCl
NH2Cl
SEM
chloramine hypochlorite
creatine
arginine
urea
creatinine
citrulline
34
Semicarbazide Conclusion

• semicarbazide is formed from azodicarbonamide
  which is used as blowing agent in sealing gaskets
  
• Additional toxicological data have to be provided
• Ames test (OECD Guideline 471)
• In-vitro mammalian cell gene mutation test (OECD
  Guideline 476)
• In-vitro mammalian chromosome aberration test
  (OECD Guideline 473)
• Formation mechanism and conditions have to be
  elucidated
• technical feasability of reduction or avoidance
  of SEM migration has to be checked
• But using substitution products, the integrity
  of the seal must be garanteed and safely
  evaluated (microbiological contamination may be
  fatal for babies and infants)!
• Flour treatment with azodicarbonamide has to be
  re-evaluated (risk benefit estimation)

35
Semicarbazide Conclusion

• Commission Directive 2004/1/ECSuspension of the
  use of ADC as blowing agent from 2nd August 2005
  (filling date)
• SEM was detected in different types of animal and
  vegetable tissues after a drying process
• Thickening agents like carrageenan may be
  bleached by hypochlorite treatment, thereby
  reasonable amounts of SEM (10 100 ppb) may be
  formed from natural precursors
• Urea, arginine, creatine, creatinine
• Again, SEM cannot be taken to detect the use of
  the banned antibiotic nitrofurazone

36
SemicarbazideThe Issue - Chronology

• prior to 1950 azodicarbonamide was suggested as
  potential foaming agent for plastics in Germany
  
• End of 2002 routine analysis on nitrofuranes
  (antibiotics) and their metabolites by
  LC-MS/MS
• Beginning 2003 unusual findings of semicarbazide
  (a nitrofurane metabolite) in food samples
  Semicarbazide levels of up to 15 µg/kg were
  found in baby food
• 28.July 2003 The European Food Safety Authority
  (EFSA) gives a preliminary advice
  semicarbazide levels (up to 10 ppm) were
  detected in the cap gaskets of food jars
• 31. July 2003 Comment of the German BfR
• Oct. 2003 Ames test results (of ADC producer) are
  submitted to European and German authorities

37
SemicarbazideThe Issue - Chronology

• Nov. 2003 2 matagenicity tests (chromosomal
  aberration in mammalian cells, HPRT test in
  mammalian cells) and mouse liver UDS in-vivo
  study shall be submitted to European and German
  authorities
• Jan. 2004 Commission Directive 2004/1/EC Suspens
  ion of the use of ADC as blowing agent - Use
  of ADC prohibited from 2nd August 2005 (filling
  date)
• mid-end 2004 industry tries to introduce SEM
  reduced or SEM free closure systems,
  respectively

38
References

• http//www.chemfinder.com
• Opinion of the Scientific Committee on Food on
  the 23rd additional list of monomers and
  additives for food contact materials, April
  2003http//europa.eu.int/comm/food/fs/sc/scf/out
  181_en.pdf
• MJ. Dennis, RC. Massey, R. Ginn, P. Willetts, C.
  Crews, I. Parker The contribution of
  azodicarbonamide to ethyl carbamate formation in
  bread and beer. Food Addit. Contam. 14101-108
  (1997).
• http//www.otsukany.com/decompos.htm
• BA. Hunter Chemical Foaming AgentsChemistry and
  Decomposition Mechanismshttp//www.cromptoncorp.c
  om/ck_prd/gnsg_ax_ex_x_cfachemistrydecompositionme
  chanisms.pdf
• Advice of the ad hoc expert group set up to
  advice the European Food Safety Authority (EFSA)
  on the possible occurence of semicarbazide in
  packaged foods 28.07.2003http//www.efsa.eu.int/p
  df/p_afc_doc_01.pdf
• FAO Nutrition Meetings, Report Series No.
  40A,B,C WHO/Food Add./67.29Toxicological
  Evaluation of some antimicrobials, antooxidants,
  emulsifiers, stabilizers, flour-treatment agents,
  acids and baseshttp//www.inchem.org/documents/je
  cfa/jecmono/40abcj28.htm
• BfR Semicarbazid als Kontaminante in
  glasverpacktn Lebensmitteln, vorläufige Bewertung
  vom 31. Juli 2003http//www.bgvv.de/cms/detail.ph
  p?templateinternet_de_index_js
• Kersting M., Alexy U., Sichert-Hellert W., Manz
  F. and Schöch G. Measured consumption of
  commercial infant food products in German
  infants Results from the DONALD study. J.
  Pediatr. Gastroenterology Nutr. 27 547-552
  (1998).
• Gatermann, R., Hoenicke, K., Mandix, M.
  (Wierts-Eggert-Jörissen, Hamburg, Germany)
  Formation of semicarbazide (SEM) from natural
  compounds in different kind of food products,
  Lecture, Prague Nov. 2003.

39
References

• Hoenicke, K., Gatermann, R., Hartig, L., Mandix,
  M. and Otte, S. Formation of Semicarbazide (SEM)
  in Food by hypochlorite treatment Is SEM a
  Specific Marker for Nitrofurazone Abuse? Food
  additives and Contaminants (in press)