Joint FAO/WHO Technical Workshop on Working Paper

Residues of Substances without ADI/MRL in Food Bangkok, 24. – 26.8. 2004

Nitrofurans Case Study: Thailand’s experience*

Sujittra Phongvivat, Bangkok, Thailand

1 Introduction
Thailand exports agricultural commodities i.e. animal and aquaculture products to Europe,
Asia, Middle-east, Africa; she is world largest exporter of farmed shrimp and the fourth
largest exporter of poultry. Production and quality assurance system were mainly
implemented by the Department of Livestock Development (DLD) and the Department of
Fisheries (DoF), both under the Ministry of Agriculture and Cooperatives. Other Ministries
were involved partially (Ministry of Health, Ministry of Commerce).
Late in 2001 animal products from some countries (Vietnam, China and Brazil) were found to
contain residues related to nitrofurans and chloramphenicol, veterinary drugs which had been
banned in many countries due to their genotoxic and carcinogenic properties. In March 2002,
first cases of similar residues in Thai chicken and shrimp were reported, findings that affected
soon all consignments of poultry and shrimp imported from Thailand. Originally, the product
sampling and testing was done in order to demonstrate their wholesomeness. Value of exports
during 2002 dropped causing significant economic losses.
The Thai government addressed this problem immediately and seriously by revising the
existing legislation and regulations including import restrictions of 16 chemicals that were
prohibited according to Annex IV of EU Council regulation (EEC) No 2377/90 (MRL
regulation) and to the USFDA list of drugs prohibited from extra-label use in food animals by
Import and Export Act B.E. 2522 (1979). The objective was to ensure traceability of any
imports and restriction of uses for industrial and medical purpose only. Further steps included:
o Development of a regulation to control use of veterinary drugs and a proposal to
improve Drug Act B.E. 2510 (1967).
o Development of legislation and regulations to control importation and use of animal
feed and premix containing prohibited veterinary drugs including nitrofurans and
chloramphenicol under the Feed Quality Control Act B.E. 2522 (1979).
o Development of a regulation to control standard of broiler farm by promotion Good
Animal Health and Husbandry Practices (GAHP) concept at farm level.
o Implementation of the International Code of Practice for control the use of veterinary
drugs by veterinarian in standard farm accredited by DLD and DoF.
o Development of legislation to regulate veterinary profession and veterinarian practice
with morality including the possibility of suspension of veterinary license if prohibited
drugs are prescribed.
o Development of a regulation to facilitate safe production of food producing animals by
inspection of products during the slaughtering process allowing sampling for
laboratory test.
In addition an efficient nitrofurans testing laboratory for product exit control was intended.
An emergency budget of 288 million Baht (~ 7.2 million US.D.) by government allowed DLD
and DOF to purchase of six units of ultra-high sensitive and considerably expensive LC-MS-
MS system including toll-synthesis of some costly, but commercially unavailable deuterated

*
This case study was prepared during Dr Sujittra Phongvivat’s six-month visit to FAO (15.1.-15.7.2004) within
the framework of the FAO Partnership Program for Visiting Experts.

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internal standards, the recruitment of additional personnel and the training of technical staff.
The challenge was that this laboratory would be able to perform the most recent methodology
(as in the EU) with an expertise that would allow the operation of such a high technology
instrument LC-MS-MS and the interpretation of the results obtained from it. It should be
noted that expertise as described could not be created overnight. (Ref: 51).
The analysis of every lot before export was an extremely heavy and costly burden, but it is
recognized that it was necessary in order to guarantee the quality of and to protect the markets
for Thai goods. A significant decrease of the number of positive samples and a reduction of
trace levels of residues was observed after 21 September 2002. Thailand could prove that her
stringent measures and approach guarantee the soundness of animal products. EU
Commission issued accordingly two decisions of 2003/477/EC 24 June 2003 and
2003/895/EC of 19 December 2003 for amending Decision 2002/251/EC of 27 March 2002
which revoked the protective measures with regard to the fishery and aquaculture products
and certain consignments of poultry meat imported from Thailand.
Meanwhile the modern technology of nitrofurans metabolite residues analysis was made
available and training provided by RIKILT to involved regulatory laboratories’ personnel
from Europe in July 2002 and from third countries in November 2002. This new nitrofurans
metabolites analytical method with increased sensitivity was introduced in the residue
monitoring schemes of many countries during late year 2002 and 2003. As a consequence, a
metabolite (AMOZ) was found in Portuguese poultry in October 2002 and in March 2003.
(Ref: 45 http://www.foodlaw.rdg.ac.uk/news/eu-03029.htm, Ref: 46). The first round of
investigation uncovered of positive results of nitrofurans in 47 farms (36 chicken, 5 turkey, 4
quail, 1 rabbit and 1 swine farms) in Portugal. The second round investigation 12 farms were
confirmed positive, 9 were negative and 34 remain under restriction (Ref:
http://www.fsai.ie/alerts/archive/fa20030327.asp). Nitrofurans were identified in pork in 10
cases from Portugal and one case each from Italy and Greece, respectively. (Ref: 47) AMOZ
is the metabolite most frequently found in Brazilian chicken (Ref: 48). Another metabolite
(AOZ) was found in shrimps from India (Ref: 49) and in fish from Taiwan. (Ref: 50). SEM
and AOZ were found in egg powder from India. (Ref: 64) These initial observations showed
that nitrofurans residues resulting from possible illegal use were not limited to developing
countries.
Although raw poultry meat free of nitrofuran-related residues had been produced and
exported successfully from Thailand, such residues were still found in cooked or further
processed products. Levels of 1.1 µg/kg of semicarbazide (SEM) in cooked frozen chicken
breast meat onion stick, 13 µg/kg SEM in frozen fully cooked chicken oriental dim sum mix,
and AOZ and SEM in spring roll selection (frozen fully cooked chicken product) were
detected (Ref: 52). Additional investigation revealed the origin of the metabolites found in
such products: AOZ was found in egg powder, SEM in a broad range of ingredients derived
from animal and non-animal such as egg powder, milk, bread crumb, spice, flour and
carrageen have been analyzed. Recently a research project of EU concluded that SEM is
present as an impurity in the chemical azodicarbonamide (ADA), a substance used as a plastic
blowing in o-ring gasket producing of metal lid glass jar. (Ref: 38)
Using ultra-high sensitive machines and methods can increase the detection of undesirable
substances; not only nitrofurans at trace levels but sometimes also other banned substances
were found in European food e.g. chloramphenicol 2.7 µg/l in Spanish white wine and even in
environmental samples e.g. chloramphenicol at 0.56 µg/l in the effluent of a sewage treatment
plant in the south of Germany. (Ref: 53, 54)
These findings of trace levels of residues of banned veterinary drug in some food products
raise the more general question how foods may become contaminated? Besides the misuse of

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veterinary drugs other possible sources could be the contamination from historical use, from
human medical use, by a genuine environmental source, contamination in the laboratory
caused by inappropriate practice and, the worst case like in the case of SEM detected from the
use of ADA, a source regularly used in food manufacture. Any of these low levels of residues
will, if found in food, violate the EU regulation leading to the destruction of large amount of
food products worth billion of dollars.
Thailand and other export countries have confronted with a variety of issues during the
nitrofurans crisis. This paper is presented as a case study to learn from the Thai experience in
order to propose means to resolve or prevent similar problems which might arise in future
from the presence of residues of other substances for which the ADI and/or MRL are not
allocated. The existing different approaches to the theoretical zero tolerance limits, to any
other limits or parameters of analytical methods need to be harmonized in order to assure fair
trade.
2 Nitrofurans Residues in Food of Animal Products
2.1 Chemical Structures and Metabolites
The nitrofurans are synthetic antibiotics characterised by their basic chemical structure i.e. a
nitrofuran ring:
o Furazolidone or, 3-([(5-nitro-2-furanyl)methylene]amino)-2-oxazolidinone
o Furaltadone or 5-(morpholinomethyl)-3-[(5-nitrofurfurylidene)amilno]-2-
oxazolidinone,
o Nitrofurantoin or 1-[(5-nitrofurfurylidene) amino]hydantoin
o Nitrofurazone or 2-[(5-nitro-2-furanyl)methylene]-hydrazinecarboxamide.
They are cheap, effective and continued to be manufactured and used for companion animals,
aquarium fish and human medicine in many countries. In the past they were widely used in
feed and medication of food producing animal as well, however, due to evidence that they
have genotoxic and carcinogenic properties (ref WHO 832TRS) this use was terminated most
countries.

Figure 1: Chemical structures of 4 nitrofurans and their correspond metabolites

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For furazolidone it was shown that the detection of the parent drug is not feasible (McCracken
et al. 1995 Nouws and Laurensen 1990) because residues of the parent drug are highly
unstable in vitro and exhibit a very short half-life in vivo and in post-mortem tissues. The
drug is therefore not found as a residue or only at very low concentrations at zero withdrawal
time. (WHO TRS 832). Despite the research has proved that tissue-bound metabolites are
formed, the free side chain of furazolidone, the molecule 3-amino-2-oxazolidinone (AOZ) is
more stable. It can be detected and measured in tissues of pigs for up to 7 weeks after
withdrawal of drug (McCracken et al. 1997) thus being a more suitable marker residue for
furazolidone. (Ref 7-9). By analogy, side-chain moieties of other metabolites of furaltadone,
nitrofurantoin and nitrofurazone are the molecules 5-methylmorpholino-3-amino-2-
oxazolidinone (AMOZ), 1-aminohydantoin (AHD) and semicarbazide (SEM) respectively
and serve as marker residues for these drugs.
2.2 Properties of nitrofurans metabolites (AOZ, AMOZ, AHD and SEM)
AOZ: Hoogenboom et al, (ref ) showed that free AOZ could be detected in the blood of pigs
with treated with furazolidone at levels up to 0.3 µg/ml. However, the liver of such treated pig
when given to rats, leads itself to the presence of free AOZ in the blood of rats. This was
evidence that the side-chain AOZ was bound to proteins and could be released not only from
the parent drug but also from tissue containing chemically bound AOZ. Further studies
showed that AOZ could be released from both the parent drug and protein-bound residues
under mild acidic condition (like in the stomach) by cleavage of the azomethine bond (15-
25% bound residues in pig liver could be released). AOZ’s possibly metabolises into the
mutagen and carcinogen 2-hydroxyethylhydrazine (HEH). In addition to the inhibition of the
enzyme monoamine oxidase and the formation of protein-adducts, AOZ gave a dose related
positive response in the Salmonella/microsome mutagenicity test in tester strains TA 1535
and TA 100, especially in the presence of rat liver homogenate. Furthermore, a positive
response was obtained in the chromosome aberration test with human lymphocytes and in
bone marrow micronucleus test with mice treated intraperitoneally with AOZ. Based on these
data it cannot be excluded that AOZ is also involved in the formation of tumours as observed
in rats and mice treated with furazolidone. These data lead to the conclusion that ingestion of
protein-bound residues of furazolidone may result in the release and absorption of AOZ, a
compound with potential genotoxic properties. (Ref: 33).
AMOZ: Pig hepatocytes, incubated with the AMOZ side-chain of furaltadone, showed a
decreased monoamine oxidase activity at high dose levels (IC50 3.7 mM), whereas exposure
to AOZ resulted in a clear inhibition at 10,000-fold lower concentrations (IC50 0.5 µM). This
AMOZ residue might therefore be of less toxicological concern than AOZ. (Ref: 34;
Xenobiotica. 1994 Aug; 24(8): 713-27.)
AHD: 1- Aminohydantoine, is the moiety of nitrofurantoin which has structural relationships
to the identified carcinogenic 5-nitrofuran compounds. There were no toxicological data
available for AHD but toxicological data from a study of nitrofurantoin led to the conclusion
that there was some evidence of carcinogenic activity of the parent drug for male F344/N rats
(increased incidences of uncommon kidney tubular cell neoplasms). Uncommon
osteosarcomas of the bone and neoplasms of the subcutaneous tissue were observed in dosed
male rats. There was clear evidence of carcinogenic activity of nitrofurantoin for female
B6C3F1 mice as shown by increased incidences of tubular adenomas, benigh mixed tumors,
and granulose cell tumors of the ovary. (Ref TR-341 No. 38)
SEM: Semicarbazide hydrochloride (or hydrazinecarboxamide monohydrochloride) belongs
to a family of hydrazines which are known to cause cancer in laboratory animals. SEM itself
demonstrated weak genotoxic activity in vitro and weak carcinogenic activity in female but
not male mice when given via diet or via drinking water. The types of tumours found with

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semicarbazide were lung and vascular tumours, which have also been found with other
hydrazines. However SEM was found to be one of the least potent carcinogens among several
hydrazines. (Ref: 58, 59, 60).
The European Food Safety Authority (EFSA) concluded that there is limited evidence that
SEM at high levels may be carcinogenic. There is no scientific evidence that SEM is
carcinogenic to humans, it is therefore not possible to conclude whether SEM may pose a
carcinogenic risk to humans. There is no risk of immediate illness to adults, children or
infants from consumption of foods containing semicarbazide. The concern relates to health in
the long term because of the possibility that semicarbazide may cause cancer.
Recent research showed that semicarbazide detected in foods is not an artefact of the
analytical method but originates from azodicarbonamide (ADA), a reagent used in the
production of o-rings/gaskets. (Ref 38) The levels of semicarbazide found in foods packed in
glass jars closed with metal lids that are sealed with such plastic gaskets are variable, in the
range from not detectable and up to 25 µg/kg and in the range 1-7 µg/kg in gaskets
themselves. For some baby food samples also higher concentrations have been reported. The
exposure estimate evaluated a scenario of a hypothetic worst case of a 6-month old baby with
7.5 kg body weight, eating 700 g of food that contains a level of 25µg/kg: the potential intake
of semicarbazide would be 2.3µg/kg body weight/day. Taking into consideration the types of
food packaged in glass jars and bottles in which semicarbazide had been found it could be
concluded that the estimated intake by adults and children on a body weight basis will be
many times lower than the preliminary estimate of the intake for babies. EFSA has concluded
and recommended that taking into account the information available to date, on the levels in
food, intake and toxicology, the risk, if any, to consumers eating products containing
semicarbazide is likely to be very small (Ref 8, 22).
2.3 Regulatory Status
The European Union banned the use of nitrofurans in food producing animals by classifying it
in ANNEX IV (list of pharmacologically active substances for which no maximum residue
limits can be fixed) of the Council Regulation 2377/90. The Food and Drug Administration
(FDA) of the United States of America prohibited furaltadone since February 1985 and
withdrew the approval for the other nitrofuran drugs (except some topical uses) in January
1992. The topical use of furazolidone and nitrofurazone was prohibited in 2002. Australia
prohibited the use of nitrofurans in food production in late 1992. Japan did not allocate MRLs
for nitrofurans leading to the implementation of a “zero tolerance or no residue standard”. In
Thailand, the Ministry of Health issued in 2001 Proclamation No. 231 MRL of veterinary
drug in food which did not allocate MRL for nitrofurans. The Ministry of Agriculture and
Cooperatives had already prohibited importation and use of furazolidone and nitrofurazone in
animal feed in 1999 which was extended to all nitrofurans in 2002. Furazolidone and
nitrofurazone were withdrawn from the list of veterinary drug formulations in 2002.
JECFA, on request from Codex, evaluated furazolidone and nitrofurazone at the 40th meeting
in 1992 (WHO TRS 832 and FAO 41/5). No ADI was established for furazolidone because of
its genotoxic and carcinogen, and the insufficiency of the residue data of both drugs
presented. The Committee could not identify marker residues and no information was
available on the quantity and nature of the total residues.
In most of the countries that recognize nitrofurans as toxic substances, the prohibition of their
use in food producing animal was implemented consequently. Food containing residues of
these drugs at any concentration is considered “not fit for human consumption”. Nitrofurans
continue to be manufactured as drugs of choice for treatment of urinary infections in humans.

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2.4 Method of Analysis
2.4.1 Previous methods
During the last two decades furazolidone drug use was monitored in most countries by
detecting the residues of the parent drug. Analytical methods for nitrofurans up to 1984 were
reviewed by Kalim (1985). The methods were all based on chromatography coupled with
various detectors i.e. HPLC with UV, diode array or electrochemical detection or GC with
electron capture detection system. The methods detect residues in parent form and have limits
of detection (LOD) around 0.2 – 2.0 µg/kg. In Thailand, the Veterinary Public Health
Laboratory of the Department of Livestock Development used for the monitoring of residues
of the four parent forms (furazolidone, furaltadone, nitrofurazone and nitrofurantoin) the
HPLC-UV method described by Winterlin et al, 1981 (limit of quantification, LOQ, of 10
µg/kg) in the annual residue monitoring plan. Up to year 2001, the results were reported to the
EU Commission.
Since the parent drugs are metabolized in the body and disappear rapidly, these methods are
not used anymore.
2.4.2 Current methods
Due to the instability of the parent drugs and the difficulties of their detection, researchers had
to study and look for other suitable markers that can replace the parent drugs.
The development of a method to use AOZ as a marker residue started at RIKILT
(Netherlands) in the late 1980’s. It involves the following steps: homogenization of the liver,
incubation with weak hydrochloric acid to hydrolyse or cut the side-chain (AOZ) from the
metabolite and subsequent derivatisation with 2-nitrobenzaldehyde, extraction of the
derivative NPAOZ with ethyl acetate and determination with HPLC-UV at 275 nm detection.
The derivatisation was necessary since the metabolites of nitrofurans are very small molecules
which do not absorb at UV wave length. Using nitrobenzaldehyde as a derivatising agent
yields a nitrophenyl chromophore derivative with intense UV absorption in the molecule and
hence being detectable by an LC-UV detector.
Furthermore the FoodBRAND (Bound Residues and Nitrofurans Detection) project funded by
European Commision was launched in January 2000. The project consortium includes four
National Reference Laboratories (NRLs), one research laboratory, one SME and consumer
representatives from UK, Ireland, the Netherlands, Belgium, Hungary and the Czech
Republic. Objectives were (1) to control abuse of the nitrofurans antibiotics by improving the
methods; (2) to detect 4 marker residues (side chain) of AOZ, AMOZ, AHD and SEM the
metabolite forms of furazolidone, furaltadone, nitrofurantoin and nitrofurazone respectively
which are more stable; (3) to develop two different types of screening test and a confirmatory
method and to improve the ability of control laboratories across the EU to be able to detect
residues of these drugs.
2.4.2.1 Screening tests
ELISA immunological screening test kits were developed for nitrofurans metabolites under
the FoodBRAND project. Late in 2003 there were only two kits for AOZ and AMOZ
commercially available (by FoodBRAND partner r-biopharm AG: RIDASCREEN). The
detection limits of the ELISA test kits claimed for AOZ are approximately 0.1 µg/kg and 0.2
µg/kg in meat, fish, whole egg, milk and liver, shrimp respectively, for AMOZ is
approximately 0.2 µg/kg in shrimp, meat, fish, liver and whole egg. At present no other
ELISA screening test kits or other immunochemical methods have been successfully
developed for the detection of all four metabolites.

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2.4.2.2 Confirmatory Method
The FoodBRAND project developed a confirmatory method that detects the four main
metabolites (AOZ, AMOZ, AHD and SEM) of nitrofurans residues using very sophisticated
and high technology equipment of HPLC coupled with tandem mass spectrometry (LC-MS-
MS) because the previously used HPLC-UV method gave high interference with matrix
compounds and had a low sensitivity.
Although such an alternative LC-MS has proven to be a valuable tool, this technique, even
with careful optimization of separating conditions may be suitable for screening purpose at
Minimum Required Performance Limit of 1 µg/kg, but is insufficient for identification
according to EU requirements (Decision 2002/657/EC).
The extraction and derivatisation steps are the same as described above (2.4.2.) and yield the
nitrophenyl derivative forms (NPAOZ, NPAMOZ, NPAHD and NPSEM) which are more
stable than the parent drugs. Due to the increased molecular mass, the mass spectra contain
more characteristic ions that are better suitable for the identification of the analytes. The
derivatives are extracted with ethyl acetate; the extracts are dried and reconstituted in the
solution of methanol/water which is analysed using HPLC, the substances are identified and
quantified by tandem mass spectrometer (LC-MS-MS). At present this LC-MS-MS is the only
technique accepted for nitrofurans metabolite residues regulatory testing and in general is
considered to be acceptable in legal procedures as reliable and unequivocal.
2.4.3 Extraction procedures of metabolites
Before derivatisation and extraction, two different approaches, one without washing the other
involving additional washing steps are applied (Diagram 1). The first technique aims to
quantify total metabolite residue which comprises protein/tissue-bound and free/unbound
residue. The second one, with washing steps, removes unbound metabolites and yields only
tissue-bound residues. The question to be answered now is whether both extraction
procedures lead to the same results with respect to the proof of illegal use of nitrofurans.
The Department of Agriculture and Rural Development in Northern Ireland (DARDNI),
United Kingdom, used the technique including washing steps before extraction and has
validated the method for testing tissue-bound metabolites in animal tissues. This tissue-bound
metabolite method is confirmed to test appropriately for the use of nitrofurans in food
producing animal; it is also recommended to test only the meat part not the whole processed
product.
Whereas RIKILT, Netherlands, when conducting training courses for analysts from EU
member states laboratories in July 2002 and from third countries in November 2002,
introduced a standard operating procedure without washing steps to determine and confirm
the analysis of the sum of residues of free and tissue-bound metabolites of nitrofurans drugs
in fresh muscle of poultry, rabbit and aquaculture (like shrimp, prawn and fish) products
(cooked, battered, ready-to-eat). (Ref 57)
Analysts from the Veterinary Public Health Laboratory (VPHL) of the Department of
Livestock Development (DLD) in Thailand were trained at DARDNI in April 2002 and
adopted therefore the same technique and validated it for tissue-bound residue for the control
of raw poultry meat and liver. Products for export are tested with this method since May
2002. Analysts from the DOF were advised and trained to test shrimp and shrimp products
using the total residue method from the beginning (2002) to February 2003 when further
advice was made by DARDNI to continue with the total residue method for screening and to
re-test using the tissue-bound method for confirmation. (Diagram 1)

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VPHL decided to test each lot of poultry meat for tissue-bound residues, and to allow only
meat with negative results to enter the food chain and to be used as raw material for further
processing. Surprisingly, Thai animal products continued to be alerted for finding dangerous
substances, in most cases SEM was detected in processed poultry and shrimp products as
well.
Diagram 1: Nitrofurans Metabolite Residues Extraction Flow Chart

First Technique Second Technique

Total metabolite (with no washing steps) Tissue-bound metabolite extraction.

Tissue Tissue
add Internal standard homogenise with water & methanol
hydrolysed with HCl centrifuge,
Metabolite (tissue-bound) collect pellet, discard supernatant
derivatise with 2-NBA, Pellet
incubate 37 ˚C, 16 Hr. wash with 3x MeOH, 2x EtOH, 2x
diethylether
adjust pH to 7.4
(to remove free form of metabolites)
Metabolite derivatives form
discard washings
extracted with 2x EtOAc
Pellet
Total metabolite residue
add Internal standard
(Unbound + bound residue)
hydrolysed with HCl
Metabolite (tissue-bound)
derivatised with 2-NBA,
incubate 37 ˚C, 16 Hr.
adjust pH to 7.4
Metabolites as derivatives
extracted with 2x EtOAc
Tissue-bound metabolite residue

From mid of 2002 to 2003, EU member states’ laboratories continued to detect nitrofuran
related residues in samples from consignments of imported poultry and aquaculture products
that ranged from frozen raw meat to various already cooked products (e.g. battered chicken,
fried products, chicken essence, spring roll, dim sum, etc). Such processed products contain
not only meat but also various ingredients that originate from animal and non- animal sources
e.g. vegetables, flour, bread crumb, spices like pepper, salt, sugar, soy sauce, oil and
carrageenan. The method employed analysed samples as a whole which were homogenized
and extracted without washing technique yielding total residues (sum of free and tissue
bound).

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Confronted with this confusing situation the question arose one could be sure that the residues
found in finished products did originate from the meat part only and not from other
ingredients? Could the detected residues be the result of some unidentified contamination?
Was there the possibility of false positive results which were not related to illegal use of
nitrofurans? In order to clarify this confusion, the Thai government decided to find out how
laboratories in UK, the Netherlands, Germany and France actually perform their analysis.
Three examples illustrate that there is no harmonisation of sample preparation between the
official laboratories of one single EU member state:
Example 1: Reply from the BVL (Mr. Bernhard Kuhnle, 21 November 2003) which
referred to an investigation of the 16 German state control laboratories.
Answer to the question “Do all laboratories examine only for bound residues?”
Determination can either be done for total residues (sum of the bound and the free
residues) or for the so-called bound residues of a sample only. This does not depend
exclusively on the related reconditioning method or the nature of the sample (exclusively
animal matrix or animal matrix plus processing contingent, suspending other materials as
well as breadcrumbs or sheets of dough, sauce etc.).
Answer to the question “Do the laboratories examine exclusively the animal matrix or
finished product?” A survey of the official laboratories of the states by the national
reference laboratory was carried out in the past month. 16 answers out of 12 states were
included into the analysis. 14 out of 15 laboratories indicated that they undertake a
separation of the animal matrix from other components. This separation is achieved using
different methods (washing, mechanical separation).
Evidence is available that sample preparation without washing steps continued to be
performed for residue testing of imported products:
Example 2: RASFF Notification No. 2003/BBG, dated 15.05.2003: 0.7 µg/kg of SEM was
found in a sample of pooled Frozen Soft Shell Crab. The analytical method used was SOP
BIO 220 V 2 “Determination of Total Nitrofuran Residues in Tissue using LC-MS-MS”
and was carried out by Institut fur Hygiene und Umwelt , Marckmannstr, 129a/b, 20539
Hamburg. (Ref: 32)
Example 3: The report on results of Nitrofurans in Shrimps and Chicken Meat,
Interlaboratory Study NIFU_06/03 proficiency testing scheme provided by European
Reference Laboratory for Residues of Veterinary Drugs CRL/NRL Berlin, 25 November
2003 has shown that the laboratory participants perform the routine test which mostly
used no-washing (total residue) method. (Ref: 40)
The different approach in sample preparation prior to the extraction can distinguishably result
in amounts of residues. Especially SEM could originate from the substance called ADA
which is used in some countries to improve the characteristic of dough and in gasket
production. The fact that cooked products’ ingredients cannot be removed completely may
lead to the wrong assumption that the contamination resulted from the illegal use of
nitrofurans.
In 2003, VPHL carried out the 2nd tissue-bound analysis of 82 processed food and various
ingredient samples were they found SEM (range 0.16 – 2.56 µg/kg) at first time in total
residue analysis. The results are shown in Table 1. Sixty three out of 82 samples were found
to contain no tissue-bound SEM and 19 of 82 samples were found having tissue-bound SEM
at concentration range 0.22 – 1.09 µg/kg. The ratio of bound SEM and total SEM, the
frequency and concentration of bound-SEM and total SEM are displayed in Figure 2 to 5. As
the experimental results of 2nd tissue bound analysis found SEM at maximum of 1.09 µg/kg to

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could be discussed whether of SEM originated from contamination of the irremovable
ingredient/substances during heat treatment of food or from Nitrofurazone abuse.
Table1. Experimental analysis of total and tissue bound SEM in processed chicken products
and ingredients. Decision limit (CCα) of SEM for chicken tissue is 0.15 µg/kg
No-washing Washing Washing
Sample ID technique technique Sample ID No-washing technique technique
Total SEM Tissue bound Tissue bound
DRP (µg/kg) SEM (µg/kg) DRP Total SEM (µg/kg) SEM (µg/kg)
1 31366 0.42 ND 42 45265 0.66 ND
2 37930 0.21 ND 43 45266 0.76 ND
3 38209 0.28 ND 44 45283 0.6 ND
4 38270 0.23 ND 45 45286 0.24 ND
5 38274 0.36 ND 46 45287 0.36 ND
6 40530 0.54 ND 47 45288 0.33 ND
7 41469 0.24 ND 48 45290 0.99 ND
8 41470 0.16 ND 49 45291 0.92 ND
9 41570 0.38 ND 50 45292 0.27 ND
10 41571 0.6 ND 51 45293 0.61 ND
11 41574 0.36 ND 52 45294 0.86 ND
12 41575 0.3 ND 53 45295 0.89 ND
13 42273 0.87 ND 54 45296 1.68 ND
14 42380 0.25 ND 55 45297 0.52 ND
15 42462 0.23 ND 56 45298 1.32 ND
16 42525 1.09 ND 57 45314 0.28 ND
17 43039 0.7 ND 58 45315 0.32 ND
18 43041 0.26 ND 59 45486 0.28 ND
19 43071 0.23 ND 60 45487 0.87 ND
20 43099 1.05 ND 61 45488 0.36 ND
21 43525 0.53 ND 62 45489 0.21 ND
22 43585 0.36 ND 63 45495 0.17 ND
23 43633 0.34 ND 64 27662 0.38 0.33
24 43634 0.34 ND 65 38271 0.42 0.22
25 43645 0.56 ND 66 40531 1.28 0.28
26 43802 0.27 ND 67 43047 1.31 0.54
27 43803 0.34 ND 68 43048 1.12 0.62
28 44109 0.24 ND 69 43415 0.5 0.25
29 44130 0.66 ND 70 43416 0.4 0.24
30 44468 0.28 ND 71 43417 1.14 0.74
31 44474 0.34 ND 72 43632 0.34 0.22
32 44599 0.39 ND 73 43879 0.77 0.3
33 44627 0.19 ND 74 44131 2.56 0.64
34 44668 0.28 ND 75 44161 1.11 0.37
35 44748 0.34 ND 76 44383 0.72 0.55
36 44848 0.26 ND 77 44503 0.54 0.51
37 44882 0.22 ND 78 44504 1.28 1.09
38 44888 0.79 ND 79 44944 1.26 0.3
39 45058 0.66 ND 80 44945 1.04 0.22
40 45263 0.61 ND 81 45648 1.38 0.67
41 45264 0.3 ND 82 45651 1.81 0.78
Total n = 82 samples

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Figure 2: Bound-SEM and total SEM concentration found in processed food and ingredients.

Tissue-bound and Total residue of SEM in

processed food
SEM concentration

3
(ug/kg)

0 276 382 405 430 430 4341 4341 4341 436 438 4413 4416 443 445 445 449 449 456 4565
62 71 31 47 48 5 6 7 32 79 1 1 83 03 04 44 45 48 1

SEM Total (µg/kg) 0.38 0.42 1.28 1.31 1.12 0.5 0.4 1.14 0.34 0.77 2.56 1.11 0.72 0.54 1.28 1.26 1.04 1.38 1.81
SEM Tissue-bound(µg/kg) 0.33 0.22 0.28 0.54 0.62 0.25 0.24 0.74 0.22 0.3 0.64 0.37 0.55 0.51 1.09 0.3 0.22 0.67 0.78

Sample ID (DRP)

Figure 3: Bound-SEM in processed food sample is calculated in percentage.

% of Tissue-bound SEM in processed food

100.00
% of Tissue bound SE

80.00

60.00

40.00
20.00

0.00 27662 38271 40531 43047 43048 43415 43416 43417 43632 43879 44131 44161 44383 44503 44504 44944 44945 45648 45651 aver age

%of Ti ssue-bound SEM 86.84 52.38 21.88 41.22 55.36 50.00 60.00 64.91 64.71 38.96 25.00 33.33 76.39 94.44 85.16 23.81 21.15 48.55 43.09 51.96

Sample ID (DRP)

Figure 4: Figure 5:

Frequency and concentration of Total SEM Frequency and concentration of Tissue-bound

(ug/kg) found in processed food samples SEM found in processed food samples
(Non-washing method) (Washing method)

80 52.63
57.89 60.00 42.11
60
sample (n) 40.00 %
40 21.05 21.05
%

11 % 20.00 10 8 5.26 1 sample (n)

20 4 4
0 0.00
(min) 0.34- 0.5-<1.0 1.0-2.56 (min) 0.22- 0.5-<1.0 1.0-1.09
<0.5 (max) <0.5 (max)
Concentration range (ug/kg) of Concentratin Range (ug/kg) of
Total SEM Tissue-bound SEM

The FAPAS Proficiency Testing of Series 2 Round 48, March – May 2004, report No. 0248
can give information of concentration of total and tissue-bound of 2 metabolites, AHD and

11 (26)
AMOZ analyzed by different techniques. The consensus assigned values were statistically
calculated from valid participants’ data. The values for total AHD, total AMOZ, bound AHD,
and bound AMOZ were 1.51, 3.51, 0.58 and 0.68 µg/kg, respectively. (Ref: 65, Table 3)
Analytical method without washing steps measures total residues and does not discriminate
between free and tissue-bound residues; total method certainly gives higher amount of
residue, such method should not be used for regulatory purposes to determine illegal presence
of nitrofuran related residues resulting from drug use. They may be useful for screening
purposes, but for confirmation samples need to be washed and any non-meat part should be
mechanically removed as much as possible before extraction. This recommendation was made
and communicated by the French Community Reference Laboratory (AFSSA) only in
December 2003; the implementation would harmonize analytical procedures and reduce
unfair difficulties. (Ref: 61)
2.4.4 Analytical method performance and validation
Method Performance: The LC-MS-MS analytical method for nitrofurans metabolites were
developed only recently and are performed only by a limited number of laboratories. The
detection limits (LOD) or decision limits (CCα) of this method are at very low levels, less
than one part per billion. The performance of historic and current analytical methods for the
determination of nitrofurans is summarized in Table 2.
Table2. Performance of nitrofurans analytical method
Year Limit of Detection (µg/kg) Techniques Limitations References
1979 FZD, 2 HPLC-UV 1 parent drug J. AOAC 62, 257 – 261
1980 FZD, 2 TLC 1 parent drug J. AOAC 63, 720 – 726
1981 FZD, 0.5 HPLC-UV 1 parent drug J.AOAC 64, 1055 -1059
1989 FZD & NFZ, 2.5 HPLC-ECD 2 parent drugs J.AOAC 72, 567 – 569
(screening)
2003 AOZ, 0.1 – 0.2 ELISA 1 metabolite RIDASCREEN
AMOZ, 0.2 ELISA 1 metabolite RIDASCREEN
2002 AHD, 0.1 LC-MS-MS 4 metabolites Validation data of VPHL,
AOZ, 0.04 Bureau of QC of
SEM, 0.15 Livestock Products,
AMOZ, 0.02 DLD, Thailand
The validated values shown are
calculated as CCα (Decision Limit)
2003 Chicken/shrimp LC-MS-MS 4 metabolites The lab No. 10 data from
AHD, 0.09/0.11 Proficiency Test Report,
AOZ, 0.06/0.06 Nitrofuran
SEM, 0.06/0.10 Interlaboratory Study
AMOZ, 0.06/0.05 NIFU_06/03, European
The validated values shown are Reference Laboratory for
calculated as CCα (Decision Limit) Residues of Veterinary
Drugs (Ref: 40)
2003 AHD, 0.217 LC-MS-MS 4 metabolites Unpublished data,
AOZ, 0.118 personal contact
SEM, 0.179 (Dra. Adriana Fernandez
AMOZ, 0.175 Suarez)
L.O.D.s were calculated as the
background + 3 S.D.
2003 SEM in liquid milk/ powder LC-MS-MS AgriQuality Ultra-Trace
L.O.D. 0.15/ 0.75 Laboratory, New Zealand
L.O.Q. 1.0 / 2.5 (Ref 44 )

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Table 3: Description of confirmatory methods for the determination of nitrofuran metabolites
Lab Analyte CCα CCβ Recovery Internal Technique
Code Chicken/shrimp Chicken/shrimp corrrection standards
(µg/kg) (µg/kg)
0006 AOZ 0.12±0.06a 0.52/0.29b Yes (matrix D4-AOZ, LC-MS-MS
AMOZ 0.12±0.06a 0.43/0.32b calibration) d5-AMOZ,
SEM 0.23±0.22a 0.53/0.47b C13N15N15-
AHD 0.49±0.20a 2.6/1.13b SEM
0010 AOZ 0.06/0.06 0.10/0.11 Yes (stable D4-AOZ, LC-MS-MS
AMOZ 0.06/0.05 0.12/0.08 isotope labelled d5-AMOZ
SEM 0.06/0.10 0.10/0.19 internal
AHD 0.09/0.11 0.21/0.24 standard)
0012 AOZ 0.04/___ 0.06/___ Yes (stable D4-AOZ, LC-MS-MS
(DLD) AMOZ 0.06/___ 0.03/___ isotope labelled d5-AMOZ
SEM 0.15/___ 0.25/___ internal
AHD 0.1/___ 0.15/___ standard)
0019 AOZ 0.5/0.5c ___/___d Yes (stable D4-AOZ, LC-MS-MS
AMOZ 0.5/0.5c ___/___d isotope labelled d5-AMOZ,
SEM 0.5/0.5c ___/___d internal standard, C13N15N15-
AHD __/___d ___/___d matrix SEM
calibration)
0020 AOZ __e/__ __e/0.25b Yes (in D4-AOZ, LC-MS-MS
AMOZ __e/__ __e/0.25b correspondence
SEM __e/__ __e/0.50b with validation)
AHD __e/__ __e/0.50b
0021 AOZ 0.2/0.2a 0.5/0.5b Yes (stable D4-AOZ, LC-MS
AMOZ 0.1/0.1a 0.4/0.4b isotope labelled d5-AMOZ Ion Trap
SEM 0.4/0.4a 1.0/1.0b internal standard,
AHD 0.5/0.5a 1.0/1.0b SEM external
recovery)
0025 AOZ 0.25/0.25f 0.5/0.5g Yes (stable D4-AOZ, LC-MS-MS
AMOZ 0.25/0.25f 0.5/0.5g isotope labelled d5-AMOZ
SEM 0.25/0.25f 0.5/0.5g internal
AHD 0.25/0.25f 0.5/0.5g standard)
0028 AOZ 0.14/___i 0.16/__i Yes (stable D4-AOZ, LC-MS-MS
AMOZ 0.24/___i 0.27/__i isotope labelled d5-AMOZ
SEM 0.24/___i 0.28/__i internal
AHD 2.58/___i 3.16/__i standard)
0031 AOZ 0.50/0.50h 0.50/0.50h Yes (stable D4-AOZ, LC-MS-MS
AMOZ 0.50/0.50h 0.50/0.50h isotope labelled d5-AMOZ,
SEM 0.50/0.50h 0.50/0.50h internal C13N15N15-
AHD 0.50/0.50h 0.50/0.50h standard) SEM
a =limit of detection, b = limit of quantification, c = preliminary figures, validation not yet completed
d = validation not yet completed, e = only shrimps were examined, f = limit of detection derived from additional
examinations concerning the blank matrix, g = limit of quantification derived from additional examinations concerning the
blank matrix, h = lowest point of calibration curve was fixed as limit of detection and limit of quantification,
i = only chicken meat was examined

Method validation: In May 2002 the Veterinary Public Health Laboratory of Department of
Livestock Development started the nitrofurans residue analysis of export poultry products
with the first LC-MS-MS unit. In accordance with Council Directive 96/23/EC (Ref 41) and
Commission Decision 2002/657/EC (Ref 42) VPHL, a competent authorized laboratory
accredited according to international standards had completed the validation for the
determination of tissue-bound nitrofurans metabolite residues in chicken tissues by December
2002. In June 2003 VPHL participated in proficiency testing scheme NIFU_06/03
“Nitrofurans in Shrimps and Chicken Meat” which was organized by the German Bundesamt
für Verbraucherschutz und Lebensmittelsicherheit (BVL), one of the European Reference
Laboratory for Residues of Veterinary Drugs. The aim of NIFU_06/03 was to promote the

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residue analysis of nitrofurans in shrimp and chicken and to enable the participants to check
their routine methods which allowed a comparison between 2 national reference labs
(RIKILT, the Netherlands and BVL, Germany, 5 German routine laboratories and two Thai
labs (DLD and DOF). The results of analysis of each lab participant were sent to BVL for
evaluation. The report on result of NIFU_06/03 “Nitrofurans in Shrimps and Chicken Meat”
had been made and submitted back to participants in November 2003. A summary of the
confirmatory methods used is in table 3 (ref: 40).
All participants received a questionnaire concerning the quality assurance measures
performed during the confirmatory analysis. What had to be indicated were the performance
parameters in accordance with point 3 of the Annex of Commission Decision 2002/657/EC.
Only three (laboratory 0010, 0012 and 0028) of the nine participating laboratories had already
finished the method validation and thus had given full details as regards the performance
parameters. The other laboratories answered the questions only partially. One laboratory did
not submit data for this result form.
Only three laboratories (laboratory 0010, 0012 and 0028) indicated the decision limit (CCα)
and the detection capability (CCβ) in accordance with Commission Decision 2002/657/EC.
Laboratory 0019 stated preliminary data. The remaining laboratories made indications
concerning “limits of detection and limit of quantification” which were still used but base on
different calculations. The values indicated for AOZ, AMOZ, and SEM generally lie
significantly below the Minimum Required Performance Limit (MRPL) value (1.0 µg/kg) for
these substances. The AHD values handed in by laboratory 0006, 0021 and 0028 did not lie
below MRPL.
The additional SOP for aquaculture products – on the detection, identification and
quantitation of residues of metabolites of furazolidone, furaltadone, nitrofurantoin and
nitrofurazone in muscle of poultry, rabbit and aquaculture products by LC-MS/MS
confirmatory analysis, draft 2002-11-01, provided by RIKILT for the training course in
November 2002 stated in chapter 2 that the described method is experimental and not yet
validated. (Ref: 57)
VPHL has received only recently the report (No. 0248) of the latest FAPAS Proficiency
Testing Scheme for Veterinary Drug Residue (Nitrofurans metabolite) Series 2, Round 48,
March – May 2004, on date 11August 2004 which gives the following information.
Instruction was given to participants to treat the test material as if it was a sample for routine
analysis. 1st un-cleared proforma result was provided to participant. The 2nd proforma result
was sent again asking participants to confirm what kind of residue (total or bound) they
analysed. The participants were asked to specify the form of residue and return the answer of
the 2nd proforma before 28 May 2004. There were 39 participants’ results submitted before
closing date (7 May 2004) for this round. Two participants submitted their 2 sets of results
for total (bound plus free) and tissue-bound. Thirty one participants submitted results for only
total residue. Seven participants (including VPHL) submitted results for only tissue-bound
but results of VPHL were mistakably assessed as total residue and unsatisfactorily results (Z-
score >-3) were given to VPHL. These figures could give an idea that around 3/4 of the
participants prefer to test total residue for nitrofurans metabolites as their routine methods.
The test sample was incurred pig kidney which AHD and AMOZ should be detected. The
L.O.Q.s for total AHD, total AMOZ, bound AHD and bound AMOZ stated by (not all)
participants are all below 1 µg/kg. (Ref: 65)
This chapter demonstrates that a number of laboratories have not yet finished the validation of
nitrofurans analytical methods. With their workload of routine testing most of the laboratories
seem to apply rather a policy of “test first, validate later” which raises the question whether

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the test results from non-validated methods may be used when controlling for implementation
of legal analytical methods?
2.4.5 Reporting limits and Minimum Required Performance Limits (MRPL)
For the European Union, the Commission Decision 2002/657/EC established so-called
Minimum Required Performance Limits (MRPL) for analytical methods to be used for
substances for which no permitted limit has been established. A later decision (March 2003)
set the MRPL for nitrofuran related residues detected in Poultry meat or aquaculture products
at 1.0 µg/kg for each metabolite. Table 4 illustrates the differences between European
countries in setting value of reporting limits which are equal to or less than MRPL. These
reporting limits are used to judge/report the positive or negative finding status of products. In
other words the products are judged to be condemned or to be released by the reporting limits.
Table 4: Reporting limits of Nitrofurans metabolites expressed in µg/kg
AOZ AMOZ AHD SEM Remarks
RIKILT 1.0 1.0 1.0 1.0 updated in Jan 2003
Netherlands 0.5 0.5 for decreasing R.L. of
AOZ and AMOZ to
0.5 ppb
DARDNI, 0.3 0.3 0.3 1.0 updated on 29 Jun
UK 1.0 1.0 1.0 2003 R.L. for 4
metabolites are 1 ppb
(Ref: 37)
BgVV, (BVL) 0.5 ? 0.5 ? 0.5 ? 0.5 ?
Germany > CCα > CCα > CCα > CCα
AFSSA any limits any limits any limits any limits R.L. can be daily
France 0.1-0.5 0.1-0.5 0.1-0.5 0.1-0.5 changed depend on
CCα
Belgium 1.0 1.0 1.0 1.0
Italy 1.0 1.0 1.0 1.0
Notes: Bold numbers are current values of Reporting Limits.
These informations were compiled from the replies to questions of Thai government and from internet.

One of the reasons for the UK to change the reporting limit, were clear indications that
importers had started to divert consignments for import to ports in other Member States
subsequently transporting them after import to the UK (without any additional testing
requirements). Since nitrofurans are genotoxic carcinogens, the Agency consulted the
Chairman of the Committee on Carcinogenicity of Chemical in Food, Consumer Products and
the Environment (COC). His judgement was that changing the UK reporting level from 0.3
ppb to 1 ppb for nitrofurans would not result in a meaningful change in risk to consumers.
When deciding to harmonise the reporting limit for nitrofurans it was acknowledged that it
would not possible to quantify the risks associated with low level exposure to nitrofuran
residues and that levels should be kept as low as reasonably practicable. The agency accepted
the opinion of the COC chairman that changing the UK reporting level (from the present level
of 0.3 ppb to 1.0 ppb) for nitrofurans would not result in meaningful change in risk to
consumers.(Ref: 37)
The member states of EU have implemented their own zero tolerance policy with different
criteria for establishing reporting and decision limits, thereby rather creating technical barrier
to trade.
2.5 Sources of residues in foods and food ingredients
The presence of residues of veterinary drugs in food products may not be caused by the
veterinary use as drugs. Sources of residues of prohibited substances could be:

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 o Historical veterinary use before a ban.
o Illegal veterinary use after ban or drug abuse or extra label use.
o Cross contamination from feed
o Environmental contamination from human or pet/aquarium fish medical use
o Contamination in the laboratory
o From other chemicals i.e. SEM originated from ADA, hypochlorite etc.
2.5.1 Investigation of possible source of nitrofurans metabolites found in foods
After Thailand had submitted her action plan to the EU Commission and had implemented
stringent measures in order to solve the nitrofuran residues crises it was expected that
significant improvement should be observed after 21September 2002. However, exported
products from Thailand continued to be notified by the weekly Rapid Alert System for Food
and Feed (RASFF) due to presence of SEM and AOZ (Ref 63). These astonishing findings
prompted a vast investigation in order to identify the sources of these residues which were
mostly SEM but to a lesser extent also AOZ and which were found mainly in
cooked/processed food. The investigation tested therefore beyond raw poultry meat/shrimp a
rather bizarre range of ingredients from both animal and non-animal sources (egg/milk
powder, starch, wheat flour, pre-dust, pepper, salt, soy sauce, sea weed derived carrageen
etc.). SEM was found from traces to high concentration of hundreds ppb, AOZ was also
detected in egg and milk products. It should be noted that the mentioned ingredients were
from local or foreign production. (Table 5)
Being informed by the European food industry (CIAA) that an independent laboratory during
routine analysis of nitrofuran antibiotic had identified SEM in food from non-animal sources,
the European Food Standard Agency (EFSA) started in July 2003 her own investigations.
Almost in parallel in April 2003, an increase in samples of Brazilian chicken containing
semicarbazide was reported. Further investigation showed that most of the chicken products
containing semicarbazide had been coated with flour, salt and spices. (Ref: 20 &
http://www.foodproductiondaily.com/news/printnews-NG.asp?id=49643). These events in
different countries confirmed our finding that SEM originates from food other than animal
products.
Table 5: Nitrofurans metabolites residues in food ingredient from local and imported product
Type of ingredient and its composition NFS metabolites conc. (µg/kg)
A. Local product
white pepper SEM = 47.5
liquid egg yolk AOZ = 2.1
wheat flour SEM = 0.47
pre-dust mix SEM = 0.77
(wheat flour, food starch)
bread crumb SEM = 0.27
Batter mix SEM = 0.23
B. Imported product
carrageenan SEM = 255
(carrageenan,vegetable gum, Sodium carbonate)
hen egg albumin powder AMOZ = 5.89, SEM = 3.31
egg yolk powder AOZ = 34
bread crumb (wheat flour, salt, yeast emulsifier, AMOZ = 1.47
fluor treatment agent) SEM = 3.31
pre-dust (specially baked, dense, white breader, SEM = 0.17
wheat flour, salt, alumen)
protein powder SEM = 0.57
(isolated soy protein)

16 (26)
 egg white powder AOZ = 0.07
Remarks: (unpublished data of analysis carried out by VPH lab, DLD, Thailand)

2.5.2 SEM resulting from other sources

Azodicarbonamide (ADA), 1,1’-Azobisformamide; is a yellow to orange red crystalline solid,
odourless substance with a molecular weight of 116.08.

The chemical is used as

o Aging and bleaching ingredient (FDA §172.806)
o Dough conditioner in bread baking (FDA §172.806)
o Exothermic foaming agent for the plastic gaskets sealed of metal lids of glass
containers.
ADA has been extensively studied and the possible presence of unconverted ADA was
studied in experiments using flour treated above technological levels and bread made from
such flour. The evidence strongly supports the view that ADA is rapidly and completely
converted to biurea on wetting and that this substance is stable in bread. Biurea itself is
metabolically inert, has low toxicity and does not present any carcinogenic hazard. (Ref: 19,
31) Considering the resemblance of the molecular structure of biurea with semicarbazide, it
has been suggested that upon acid treatment, semicarbazide residues could be formed. A
mechanism of two hydrolysis reactions has been proposed for conversion of biurea to SEM.
(Figure 6).
By products of ADA released at decomposed temperature (195 to 202˚C) are Urazol (UR),
Biurea (BU), Isocyanuric acid and Cyamelide approximately 36%, 24%, 15% and 7%
respectively (Figure 7).
Figure 6: A proposed mechanism for conversion of biurea in SEM by two hydrolysis reactions
(Ref: 20)

17 (26)
Figure 7: ADA and its by-products (solids and sublime substances)

ADA has been adequately studied in several species and is similarly free from carcinogenic
hazard. Long-term studies in mice are in progress (Frazer, 1966).
Semicarbazide (SEM) is a known metabolite of the drug “nitrofurazone” and is monitored as
a marker for nitrofurazone abuse in food animal origin, but SEM has also been reported in
seaweed derived products, which are widely used as food additives (Ref: 62). SEM was found
in food products packaged in glass jar with metal lids that have foamed plastic seals.
An investigation conducted by the National Food Agency Finland found SEM in 15 baby
foods and five other food stuffs at similar levels than European average level.(SEM ranged
from 1 µg/kg to 28.4 µg/kg, average was 11.3 µg/kg).
It was also discussed whether SEM could possibly originate at high concentrations from
reactions between hypochlorite and some amino acids. Hypochlorite is used for the bleaching
of ingredients such as carrageenan, egg powder. (Ref: 53 Mad Science Discussion Board
http://www.sciencemadness.org/talk/viewthread.php?tid=148&page=2#pid.
An investigation of SEM found in chicken essence soup packed in glass jar and comparison
results of bleached and un-bleached pepper analysed for nitrofurans metabolite were carried
out by VPHL, DLD in 2003 has shown in table below.
Table 6: SEM found in glass jar product and bleaching pepper
Sample description Sample ID Reference SEM µg/kg or µg/L Remarks
Chicken essence soup in DRP 12642/0346 9.01 SEM was analysed as
glass jar with plastic DRP12643/0346 11.60 total residue.
seal under twist-top cap DRP12644/0346 20.25 Unpublished data of
DRP12645/0346 5.18 VPHL, BQCLP, DLD.
DRP14703/0346 8.22
DRP14704/0346 5.41
Bleaching pepper DRP26331/0546 19.20 Survey of different
(involved with Calcium DRP26333/0546 374 processing of peppers,
hypochlorite CaCl2O2) DRP26336/0546 50.20 unpublished analytical
DRP26338/0546 15.50 result of VPHL in 2003
DRP26339/0546 77.10
Pepper DRP26330/0546 (black pepper) Not detected
(no bleaching) DRP26332/0546 (white pepper) Not detected
DRP26334/0546 (white pepper) Not detected
DRP26335/0546 (white pepper) Not detected
DRP26337/0546 (black pepper) Not detected

The EU research project A03037 concluded that SEM is present as impurity in all three
chemicals ADA, Biurea and Urazol. (Ref: 38)
Based on current information, the only likely source of positive results involving SEM
originates from its occasional presence in ingredients of finished products other than meat.
This is of concern if total residues are determined for the whole product. Experts from the EU

18 (26)
therefore agreed and advised that the most appropriate methods try to determine tissue-bound
residues in animal tissue which minimize the risks of false positive results and will remove
any criticism that the methods are being applied to sample matrices for which they have not
been appropriately validated. (Ref: 61)
3 Socio-economical impact
Agricultural products and processed foods are Thailand’s second highest export income next
to industrial goods. Thailand is the 7th largest poultry producer and ranks at No. 4 among
poultry exporters following U.S.A., Brazil and the EU. Most important Thai export markets
are Japan and the EU. Livestock products constitute 10% of agro-food products for export.
The figures in Table 5 reflect the decrease of amount and value of poultry exports from
Thailand to EU in year 2002 and to a lesser extent in year 2003. If we assume an annual
increase of poultry exports from Thailand to Europe of 10 %, the actual value dropped by
approximately 20% in 2002 and still by 7% in year 2003 (Figures 10 and 11).
Table 7: Demonstration figure in metric tons (MT) and price of chicken raw meat and
products export to Europe of year 2001, 2002 and 2003
Year 2001 2002 2003
Product
Raw MT 99,119 79,777 103,747
Mil. Baht 7,414 5,823.7 7,573.5
Mil. US $ 166.61 135.21 189.34
Processed MT 49,854 50,720 61,650
Mil. Baht 6,481 6,466.8 7,860
Mil. US $ 145.64 150.14 196.5
Total MT 148,973 130,497 165,397
Mil. Baht 13,895 12,290.5 15,433.9
Mil. US $ 312.25 285.36 385.85
Source: Thai Broiler Processing Exporters Association
Remarks: Year 2001 Price of Raw Meat is 74.8 Baht/Kg and Cooked Product is 130 Baht/Kg
Year 2002 Price of Raw Meat is 73 Baht/Kg and Cooked Product is 127.5 Baht/Kg
Year 2003 Price of Raw Meat is 73 Baht/Kg and Cooked Product is 127.5 Baht/Kg
Currency Exchange rate: Year 2001, 44.50 Baht = 1 US $, Year 2002, 43.07 Baht = 1 US $,
Year 2003, 40.00 Baht = 1 US $

In addition to the government’s emergency budget of approximately 7.2 Million US Dollars

that were provided to address the residue problem, the decrease of country’s income as shown
in table 7 and Figure 8 to 11, and the value of products destroyed due to nitrofurans residues
found in them, add up to significant losses. Costs results also from the expenses paid for
retesting products and the renting of cold storage for longer periods to keep the products fresh
while waiting for the analytical results. However, all such amounts of money do not include
the social impact and chain reaction to the grass root farmers and their household when
chicken is tested to contain residues, product is not exported, and the company would not pay
or pay less to farmers. They are directly or indirectly punished if the residue found in their
chicken even in the case of false positives (see SEM!). Some of them had to face hard
circumstances and tragedies in life e.g. bankruptcy, no sufficient income for daily life
expense.

19 (26)
Figure 8: The Export Quantity of Poultry Figure 10: Forecast and Actual Value in
Products from Thailand to EU display in Thousand Metric Tonnes of Poultry Export
graph to EU
Export Quantity of Poultry Products Forecast and Actual Value in Thousand Metric
from Thailand to EU tonnes of Poultry Exportation to EU
thousand metric tonnes

200
200
150
150
100
100
50
50 0
0 -50
2001 2002 2003 2001 2002 2003

Raw 99.1 79.8 103.7 Forecasted 149 164 179

(Thousand MT)
Processed 49.9 50.7 61.7
actual 149 130 165
Total 149 130.5 165.4 (Thousand MT)
YEAR Change rate % 0 -20 -7
YEAR

Figure 9: The Export Value (in million US Figure 11: The Forecast and Actual Value
$) of Poultry Products from Thailand to in Million US $ of Poultry Export to EU.
EU.
Export Value (in million US $) of Poultry Products
from Thailand to the EU Forecast and Actual Value in Million US $ of
Poultry Export to EU
600
500
Million US $

400 400
Million US $

300
200
200
100
0
2001 2002 2003 0
-100
Raw 166.61 135.211 189.34 2001 2002 2003
Processed 145.64 150.14 196.5 Forcasted M. 323 355 390
Total 312.25 285.36 385.85 US $
actual M.US $ 312 285 385
YEAR
% change rate 0 -19.7 -1.28
YEAR

Examples of trade barriers by different reporting limits, un-unified analytical method and
SEM at trace level below 25 µg/kg found in glass jar packaged product, resulting in product
destroyed could be given below.
o RASFF Notification No. 2003/BBG, dated 15.05.2003: SEM 0.7 µg/kg was found in
sample of pooled Frozen Soft Shell Crab taken from three consignments of 800 kg, 200 kg
and 1500 kg Products were rejected and destroyed. With the reporting limit of 0.5 µg/kg
and SOP BIO 220 V 2, “Determination of Total Nitrofuran Residues in Tissue using LC-
MSMS”, the analysis was carried out by Institut fur Hygiene und Umwelt ,
Marckmannstr. 129a/b, 20539 Hamburg.
o RASFF 2002/AXS, 30 July 2002, Sweden, Metabolites of Nitrofurazone (SEM) 2.4
µg/kg, Crab Paste with Soya Bean Oil (CPB: ACDCF 200 g x 24 Jars/Carton, 50 Cartons,
Net weight 240 Kg). (Place of test: RIKILT)

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o RASFF 2002/AXS, 30 July 2002, Sweden, Metabolites of Nitrofurazone (SEM) 2.8
µg/kg, Shrimp Paste with Soya Bean Oil (SPB: ACDCF 200 g x 24 Jars/Carton, 50
Cartons, Net weight 240 Kg). (Place of test: RIKILT)
o RASFF 2002/274, 1 August 2002, Germany, Nitrofuran metabolite AOZ 0.5 ug/kg,
Thailand Black Tiger Shrimps 26/30 Raw, Minus Shells, Deep-frozen (Penaeus Spp.).
o RASFF 2002/BLI, 12 November 2002, Germany, Nitrofurans metabolite SEM 0.8 ug/kg,
Bottled Shrimp in Brine, Pan Asia (1981) Ref Nr./BEZUGS-NR. :P>A>/050702/595
DATE: 20.09.2002
o RASFF 2002/BPF, 16 December 2002, Germany, AOZ 0.9 ug/kg, Frozen Shrimp Ball
Breaded Black Tiger Shrimp
Example of discrimination of same residues of SEM found in carrageen (2003/086 add 43)
and AOZ found in egg powder (2003/086 add 57) products which were placed under
temporary seizure and were later released.
o RASFF 2003/086, Brussels,12 December 2003, Subject: Nitrofuran (metabolite)-
Furazolidone (AOZ) in egg products from India, Measures taken in Belgium, Release of
batches of carrageenan and egg powder. (Ref: 65)
4 Conclusion
Thailand’s stringent measures have shown her sincerity to solve the drug residue problem
because it is most important to produce safe and sound food for the consumers of the world
not only for export markets but for local consumer as well.
The “nitrofurans crisis” was a severe problem not only in developing countries but impacted
on worldwide trade especially developing countries. The nitrofurans case is an example from
which many lessons can be learnt and can prevent occurrence of similar problems in the
future:
o Analytical method for substances without ADI/MRL substances should be discussed,
harmonized internationally and approved as a regulatory method.
o Criteria or policies of setting critical parameters which would affect trade such as
“reporting limit” should be discussed and be based on risk assessments rather than be
based on the sensitivity of technology or instruments.
o Substances should be banned on the basis of their toxicity risk to human not because
of insufficient data or unfinished evaluation.
o Marker residues should be appropriate and valid and not create false positive.
o Residue of veterinary drug should be test from target tissues or organs of animal e.g.
liver, kidney, muscle, fat, skin, egg, milk, etc, not from processed or finished product.
o Terms or parameters involved in regulatory aspects and analytical aspects i.e. MRPL,
Zero Tolerance, L.O.D., L.O.Q., CCα, CCβ etc. lead to confusion and need to be
clarified, defined and harmonized.
o A consultation of experts would be necessary to solve scientifically the problems and
unify/harmonize different approaching/implementation of countries which should be
transparency and stop or minimize conflict and unfair trade.

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62. RASFF week 2003/31, Ref: 2003.201, date 31.07.2003, SEM found in carrageen, derived
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report – March 2003, dinsdag 1 april 2003, p 33 to p 36 of 43.
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Relevant web-pages and other documents available on-line
http://www.afsni.ac.uk/foodbrand/
http://www.efsa.eu.int/pdf/p_afc_doc_01.pdf ( Advice on the possible occurrence of
Semicarbazide in packaged foods, 28 July 2003 AFC/adhoc SEM/1
http://www.efsa.eu.int/pdf/p_afc_doc_02_en.pdf ( Statement of the Scientific Panel on Food
Additives, Flavourings, Processing Aids and Materials in Contact with Food. Updating the
advice available on Semicarbazide in Packaged foods, Adopted on 1 October 2003.
http://www.europa.eu.int/comm/food/fs/sc/scf/out181_en.pdf Opinion of the SCF on the 21st
additional list of monomers and additives for food contact materials
http://www.naturalovens.com/lib/pdf/betterhealth/Toxin_Additives_in_Food_and_Drink.PDF
ADDITIVES PERMITTED IN U.S. FOR BAKED FOODS (F.D.A.)
http://www.crl.fougeres.afssa.fr/publicdoc/Microsoft%20Word%20-%2003-09PS-SEM-
L7349-NoteCRL211103.pdf
http://www.foodstandards.gov.uk/multimedia/pdfs/semicarbazide.pdf
http://www.rikilt.dlo.nl/Services/Info%20Nitrofuran.htm (screening test)
http://www.efsa.eu.int/pdf/pressrel20031015_en.pdf (SEM advice by EFSA)
http://www.efsa.eu.int/pdf/p_afc_doc_01.pdf
http://www.efsa.eu.int/p_foodadd_en.html
http://www.nzfsa.govt.nz/dairy
http://www.rikilt.wur.nl/Services/Asorption_of_a_genotoxic_metabo.htm
http://www.efsa.eu.int/pdf/p_afc_doc_01.pdf
http://www.efsa.eu.int/p_foodadd_en.html
http://www.efsa.eu.int/pdf/pressrel20031015_en.pdf)
(http://www.rikilt.dlo.nl?Services/Info%20Nitrofuran.htm)
[http://www.afsni.ac.uk/foodbrand/]
(Ref: r-biopharm AG: leaflet, http://www.rikilt.dlo.nl/Services/Info%20Nitrofuran.htm)
http://www.rikilt.dlo.nl/Services/Info%20Nitrofuran.htm

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