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The EFSA Journal (2005) 219, 1-36

Opinion of the Scientific Panel on Food Additives,

Flavourings, Processing Aids and Materials in Contact with Food

on a request from the Commission related to

Semicarbazide in food

Question number EFSA-2003-235

Adopted on 21 June 2005 by written procedure

SUMMARY

The Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact

with Food (AFC) has been asked to advise the European Commission on the occurrence of

semicarbazide (SEM) in food. The European Food Safety Authority (EFSA) issued

preliminary advice on SEM in 2003, when the occurrence of SEM in food, derived from food

packaging, was first discovered. The Panel was asked on this occasion to gather data on the

occurrence of SEM in all types of food, to explain the conditions under which SEM may be

formed in food and to evaluate the analytical methods used. In the light of this information, the

Panel was asked to assess the risks posed by semicarbazide in all types of food. The approach

taken by the Panel on this question was to search the scientific literature and to take account of

information from the Commission, national authorities and trade associations.

SEM has been found to occur in different types of foods and the source of SEM varies. SEM

is a metabolite of the veterinary medicine nitrofurazone, but since the use of nitrofurazone is

illegal in the EU, SEM from this source should not be detectable in foods. SEM can be present

in foods as a result of migration from sealing gaskets used in the metal lids of jars and bottles.

In this case, the origin of the SEM is thermal breakdown of azodicarbonamide, a blowing agent

used to foam the plastic gaskets. SEM has been found in food products made using flour in

which azodicarbonamide has been added as a dough-improver a practice that is not permitted

in the EU. Other sources have been suggested but are less well documented. SEM is

reportedly formed as a reaction product of the action of hypochlorite on food additives such ascarrageenan and on foods such as egg white powder. Finally, SEM may be present at

background levels naturally, may be formed at low levels when some foods are dried, and may

also derive from as yet unidentified sources.

The method of analysis used to test foods for SEM involves acid hydrolysis and a

derivatisation step with 2-nitrobenzaldehyde. The derivative is then determined using liquid

chromatography coupled to tandem mass spectrometry with a detection limit in the region of

0.2 g/kg. The acid hydrolysis step liberates bound residues for analysis and the analysis

method therefore measures total SEM (free and bound) in the sample. The acid hydrolysis

conditions used in the analytical method are not dissimilar to normal gastric conditions. Since

the current state of knowledge on the bioavailability of any bound residues is incomplete, inthis evaluation there is no distinction made between SEM that is present as such in a food


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Semicarbazide in food The EFSA Journal (2005) 219, p. 2 of 36

sample and any SEM that may have been formed from precursors in the food under acidic

conditions used in the analysis.

It is concluded that the method of testing for SEM provides concentration data that are suitable

for this risk evaluation.

On the basis of the information available, migration of SEM from the breakdown of

azodicarbonamide (ADC) in sealing gaskets is by far the largest source of exposure known.

The concentration data available from analyses of food undertaken by different countries were

similar. The highest potential intakes of SEM are in infants consuming ready-to-feed infant

milk and baby food, attributable to the larger gasket areas involved in the packaging and their

small body weight. Reasonable worst case estimates of intake for infants fed on products

packaged in glass jars and bottles range from 0.35 to 1.4 g/kg bw/day.

Adult exposures to SEM from this source are likely to be much lower than infant exposures,

due to the lower contribution of foods packaged in bottles and jars to the total diet of adults, the

lower contamination levels derived from the smaller gasket areas involved for that packaging,and the higher adult body weight. A reasonable worst case estimate of intake for an adult

would be 0.02 g/kg bw/day.

Commission Directive 2004/1/EC prohibits the use of azodicarbonamide in food contact

materials from 2ndAugust 2005. Once existing stocks of packaged foods are used up, exposure

of consumers by this route will have been eliminated.

Other possible sources of SEM in foods contribute far less to exposure than that estimated

above for packaging. Bread made using flour treated with ADC can contain SEM. In

laboratory tests the SEM concentration in bread was 28 g/kg. ADC is not permitted as a flour

treatment agent in the EU and the importation of bread and bakery ware is likely to be verylow. There is the potential for exposure from breaded animal products imported into the EU

(e.g. frozen breaded chicken or fish products). Taking an upper figure of 5 g/kg of product

this would give an intake of SEM of 1 g/person from a consumption of 200g of product.

For a high consumer of egg products that may be contaminated by 50 g/kg SEM as a result of

using hypochlorite as a sanitising solution on production equipment, a reasonable worst-case

estimate of exposure is 0.008 g/kg bw/day. For the food additive carrageenan, that may

become contaminated with SEM at a mean concentration of 65 g/kg from use of hypochlorite

in the production process, if consumption was up to the Acceptable Daily Intake (ADI) for

carrageenan then the intake of SEM from this source could be up to 0.005 g/kg bw/day.

SEM has been shown to be carcinogenic in mice, but not rats. Literature data on genotoxicity

together with the results of recent studies indicate that SEM is mutagenic but not clastogenic in

some test systems in vitro, notably in the absence of an exogenous metabolising system. In

vivo, negative results were reported in studies on DNA damage in liver and lung of mice, and

in the micronucleus assay in the mouse. Based on the overall weight of evidence provided by

the studies performed, which included a study using a highly sensitive methodology, the Panel

concluded that the weak genotoxicity exerted by SEM in vitrois not expressed in vivo.

The new data allaying the concern on genotoxicity in vivo, and the likely reductions in

exposure following replacement of the most significant, currently known source of SEM in the

diet (gaskets on glass jars and bottles), offer further support to the preliminary advice given byEFSA in 2003 that the risk, if any, from consumption of foods containing SEM is judged to be


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very small, not only for adult consumers but also for infants. In this respect the Panel noted

that SEM is a weak non-genotoxic carcinogen for which a threshold mechanism can be

assumed. A large margin of at least 5 orders of magnitude exists between the dose causing

tumours in experimental animals and human exposure, including that of infants.

The Panel therefore concluded that the issue of carcinogenicity is not of concern for humanhealth at the concentrations of SEM encountered in food.

KEYWORDS

Semicarbazide, CAS No 57-56-7, SEM, azodicarbonamide, CAS No 123-77-3 nitrofurazone,

CAS No 59-87-0, gaskets, hypochlorite.


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TABLE OF CONTENTS

SUMMARY........................................................................................................................1

KEYWORDS .....................................................................................................................3BACKGROUND................................................................................................................6

TERMS OF REFERENCE...............................................................................................7

INTRODUCTION .............................................................................................................7

ANALYTICAL ASPECTS ...............................................................................................7

SOURCES AND POSSIBLE MECHANISMS OF FORMATION OF SEM..............8

SEM from nitrofurazone use in veterinary medicine............................................8

SEM from azodicarbonamide (ADC) foamed plastic gaskets ..............................8

Regulation of ADC used in food contact plastics..............................................8

Detection of SEM in food originating from packaging materials .....................9SEM from hypochlorite-treated foods and food additives ...................................9

SEM in hypochlorite treated foods ....................................................................9

SEM in egg powder ...........................................................................................10

SEM in food additives........................................................................................11

SEM from azodicarbonamide-treated flour...........................................................11

SEM present at background levels naturally or from as yet unidentified

sources .......................................................................................................................11

OCCURRENCE AND EXPOSURE................................................................................12

SEM from nitrofurazone use in veterinary medicine............................................12

Migration of SEM from lid gaskets ........................................................................12Concentration data from different countries.....................................................12

Summary of levels reported ...............................................................................14

Food intake........................................................................................................14

Exposure estimates ............................................................................................15SEM from hypochlorite-treated foods and food additives ...................................16

SEM from the use of chlorinated wash water....................................................16

SEM from egg powder .......................................................................................16

SEM from carrageenan .....................................................................................17

SEM from azodicarbonamide-treated flour...........................................................18

TOXICITY OF SEM.........................................................................................................18

Metabolism and toxicokinetics. ...............................................................................18

Acute toxicity ............................................................................................................18

Developmental toxicity .............................................................................................18

Oral studies. ......................................................................................................18

Intraperitoneal studies.......................................................................................19

In vitro study......................................................................................................21

Carcinogenicity .........................................................................................................21

Genotoxicity ..............................................................................................................23

Bacterial mutagenicity assays ...........................................................................23

Mammalian cell gene mutation assay ...............................................................23

In vitro chromosomal aberration assays...........................................................24In vivo cytogenetics ...........................................................................................24

DNA damage in vitro/in vivo.............................................................................25


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Effects on DNA in acellular systems .................................................................25

Summary and discussion of the toxicity studies ....................................................25

CONCLUSIONS AND RECOMMENDATIONS ..........................................................27

The occurrence and the precursors of SEM in foods............................................27

The method of analysis used for SEM in foods......................................................27Estimates of exposure...............................................................................................28

Toxicity ......................................................................................................................29

INFORMATION PROVIDED TO EFSA .......................................................................30

REFERENCES ..................................................................................................................30

Annex 1. The EFSA advice on baby foods from October 2003.........................................35


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BACKGROUND

In early 2003 semicarbazide (SEM) was reported to have been found in a number of food

products from different manufacturers that are packaged in glass jars and bottles with metal

lids sealed with plastic PVC gaskets. Examples of the foods involved include some types ofbaby food, fruit juices, jams and conserves, honey, pickles and sterilized vegetables,

mayonnaise, mustard, sauces and ketchups. The presence of SEM was linked by industry to

the permitted use of azodicarbonamide (ADC) as a blowing agent to make a foamed plastic

which is suitable for gaskets. The amounts that were detected in foods were variable but low,

ranging from not detectable up to 20 microgrammes per kg of food (20 g/kg). Levels of 1 - 7

mg SEM per kg of gasket material have been detected in extracts of the gaskets themselves.

Preliminary advice on the possible occurrence of SEM in certain packaged foods was issued by

EFSA in July 2003 and is available at

(http://www.efsa.eu.int/science/afc/afc_documents/365/p_afc_doc_0111.pdf).

In October 2003, an ad hoc group of experts was convened, including members of EFSAs

Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact

with Food (AFC Panel), its Panel on Contaminants in the Food Chain and its Panel on Dietetic

Products, Nutrition and Allergies. It heard a presentation from the food and packaging

industries updating their findings since July 2003. This new information covered industry

progress on development of alternative technologies and strategies to reduce or eliminate SEM

in foods, further information on the range of concentrations of SEM found so far in foods

packaged in glass jars and bottles, and exposure estimates for infants. The ad hoc expert group

was specifically asked by EFSA to advise further on possible risks to infants, given that this is

the consumer group for which potential exposure to SEM on a body weight basis is likely to be

the highest in Europe. The advice of the ad hoc group can be found at

http://www.efsa.eu.int/science/afc/afc_documents/364/p_afc_doc_03_en11.pdf and included

discussion of microbiological and nutritional aspects of baby foods (Annex 1), which need to

be considered alongside the toxicological aspects.

Member States informed the Commission through the Rapid Alert System that SEM had also

been found in a wide variety of food products which could not be attributed to food contact

materials. SEM was found in egg products and its presence was attributed to the production

process for these egg products rather than the illegal administration of nitrofurazone to laying

hens. Two further sources of possible SEM contamination of food were also recognised during

2003; breakdown of ADC used as a flour treatment agent and SEM formed by hypochloriteaction on food or food additives. ADC is not permitted as a flour treatment agent in the EU. In

addition SEM is a metabolite of nitrofurazone and had for a long time been used as a marker

residue to detect use of this banned veterinary medicine in animal products. The presence of

residues of nitrofurans and/or their breakdown products in foods is illegal in the EU.

In view of the potential impact of SEM on public health it is necessary for the Commission to

have a complete picture on occurrence in all types of food and feed; and especially whether the

presence of SEM is due to the intentional use of an avoidable component or processing step or

whether it results from contamination.


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TERMS OF REFERENCE

The Commission requests EFSA to advise on the scientific basis for the occurrence of SEM in

food. The EFSA is asked especially to:

Gather and collate data of occurrence of SEM in all types of food (e.g. food of animalorigin, animal feed, processed food, raw materials, etc.). The analytical method used and

the performance criteria would be needed in order to compare the results.

Explain the conditions under which SEM may be formed in food (e.g. during foodprocessing, during the analytical procedure, etc.)

Assess the risk of semicarbazide levels currently reported in food products, as well as toexamine new information which might become available, especially through the data

collection process.

Complete the full assessment of the risks posed by semicarbazide in all types of food.INTRODUCTION

The approach taken by the Panel on this question was to search the scientific literature and the

Panel also received information from the Commission, national authorities and trade

associations.

ANALYTICAL ASPECTS

Semicarbazide (H2N-NH-CO-NH2) is a small molecule that belongs to the hydrazine group of

chemicals. The current methods of analysis used to detect SEM in food involve acidhydrolysis and a derivatisation step with 2-nitrobenzaldehyde (2NBA). These steps are to help

extract and measure SEM bound to protein. The SEM-NBA derivative is then determined

using LC-MS/MS (liquid chromatography coupled to tandem mass spectrometry). The

methods were developed for the testing of meat for SEM as a marker for nitrofurazone, which

is rapidly metabolised to SEM. These methods may not be needed for the detection of SEM in

free solution and may in theory lead to artefacts, for example the production of SEM as a result

of hydrolysis of ADC or some of its impurities, during the analytical work-up.

The 2NBA method measures total SEM (free and bound) in the sample and cannot

distinguish between the two forms. The detection limit of the method is in the region of 0.2

g/kg. There are currently no methods for measuring free SEM with g/kg sensitivity infood matrices. For the purposes of this evaluation, no distinction is made between free and

bound SEM in estimating intakes - the prudent assumption is made that the all SEM measured

in foods is freely available. Tissue-bound residues of SEM are reported to be bioavailable in

the digestive systems of mammalian species (Hoogenboom et al. 1992; Gottschall & Wang

1995; McCracken et al. 1997).

A number of efforts are on-going to develop new analytical methods. For example, DG-JRC

(IRMM-Geel) has the following research objectives: a) identification of an alternative marker

residue for nitrofurazone use in animal derived food products; b) development and validation

of analytical methods for SEM in various food products; c) baby food analysis (method

validation, monitoring and investigation of source of contamination); d) organisation andcoordination of proficiency test exercises for the determination of SEM in food products.


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SOURCES AND POSSIBLE MECHANISMS OF FORMATION OF SEM

There are presently five chemical sources to which the presence of SEM detected in foods has

been attributed. These are, in approximate chronological order of discovery:

1. As a metabolite of the veterinary medicine nitrofurazone.2. As a thermal breakdown product arising from the use of azodicarbonamide to make

foamed plastic sealing gaskets for metal lids on glass food jars.

3. As a reaction product formed by the action of hypochlorite on food additives and/or

foods.

4. As a decomposition product of azodicarbonamide used as a flour treatment agent.

5. Present at background levels naturally or from as yet unidentified sources.

SEM from nitrofurazone veterinary medicine use

The foods in which SEM has now been found include foods not of animal origin and its

presence in such foods can not be explained by veterinary use of nitrofurazone. The limits of

detection for SEM are in the range of 0.2 to 1 g/kg. Minimum required performance limits

have been set up by Commission Decision 2002/657/EC for the metabolites of nitrofurans in

products of animal origin. Consequently, if the ban on nitrofurazone use is enforced

effectively then there should be no detectable levels of SEM from this source in the food of

European consumers.

SEM from azodicarbonamide (ADC) foamed plastic gaskets

ADC is structurally related to SEM as shown below.

ADC. Azodicarbonamide H2N-CO-N=N-CO-NH2

SEM. Semicarbazide H2N-NH-CO-NH2

ADC is authorized for use in the EU as a blowing agent for plastics in contact with food. These

plastics are used in sealing gaskets for metal lids on glass bottles and jars. Blowing agents are

added to polymers during processing to form minute gas cells throughout the plastic. During

high temperature processing, ADC decomposes to form gases, primarily nitrogen and carbon

monoxide together with some carbon dioxide and ammonia and non-volatile residues such as

biurea. Normally, the residues of non-volatiles are only small (biurea about 2% of addedADC). However under certain conditions the residues of biurea could be as high as 34%. Other

non-volatile products can be urazole, cyanuric acid, and cyamelide.

Regulation of ADC used in food contact plastics

ADC is listed in Commission Directive 2002/72/EC relating to plastics materials and articles

intended to come into contact with foodstuffs. The directive permits ADC as an additive with

the condition that it is used only as a blowing agent. Commission Directive 2004/1/EC of 6th

January 2004 has been issued and this amends Directive 2002/72/EC with the effect that, as

regards the additive ADC, its use in food contact materials is prohibited as from 2ndAugust2005. Foods packed before that date may still be sold after that date.


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ADC was re-evaluated by the EC Scientific Committee on Food in 2003 when it was

confirmed as acceptable for use in food contact materials, but only as a blowing agent and not

for materials in contact with alcoholic beverages. It was classified in List 3 (substances for

which an ADI or a TDI could not be established, but where the present use could be accepted).

SEM is not specifically regulated by EU food packaging directives but if it were present in

food packaging materials, for instance as an impurity or a reaction or degradation product, its

presence in food would be covered by the Framework Regulation EC 1935/2004.Under Article

3 of this Regulation it could be present in food contact

materials provided it did not transfer into foodstuffs in quantities that could endanger human

health.

Detection of SEM in food originating from packaging materials

In 2003, a laboratory conducting routine monitoring for the presence of residues of nitrofuranantimicrobials discovered traces of SEM in a purely vegetable-based product. Further

investigation revealed that SEM (tested as 2NBA-SEM) occurs in different food products that

are packaged in glass jars and bottles with metal lids and furnished with a foamed PVC seal.

The use of metal closures with a foamed gasket include baby food jars. These typically are

Press-on Twist-off closures which include a tamper-evident safety button. A feature of these

closures is that a greater surface area of the PVC seal is in contact with the food. The gaskets

are used in the packaging for a wide range of foods in jars and bottles including fruit juices,

jams and conserves, honey, baby food, pickles and sterilized vegetables, mayonnaise, mustard

and sauces.

SEM, ADC and biurea have similar chemical structures and it was necessary to prove thatSEM was not an artefact of the analytical method used. In November 2003 an independent test

method for free SEM was developed and validated in a two-laboratory exercise. This work

reported the finding of free SEM in the chemicals ADC, biurea and urazole. Subsequently, it

has been demonstrated that SEM is a minor thermal decomposition product of

azodicarbonamide used in the gaskets of certain food jar and bottles and that it migrates from

the gaskets to the packaged food.

SEM from hypochlorite-treated foods and food additives

SEM in hypochlorite treated foods

Gatermann et al. (2003) reported the results of laboratory experiments on the formation of

SEM in food subjected to hypochlorite treatment. Food and food products were treated with a

hypochlorite solution containing 12% active chlorine, levels far higher than would normally be

used, in order to investigate the source and mechanism of formation of SEM. They found that

SEM was formed at 10 65 g/kg in shrimps, venison, soybean flakes, carrots, and honey.

Levels of SEM were 350 450 g/kg in egg powder and gelatine after hypochlorite treatment.

These findings do not appear to have been confirmed yet by a second independent laboratory.

These same workers followed-up their findings by using lower hypochlorite levels (Hoenicke

et al. 2004). After incubation with 1% active chlorine at room temperature overnight, chicken,

milk, egg white powder, soybean flakes, red seaweed, carrageenan, locust bean gum, gelatine,


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and starch all showed a small formation of SEM. In all cases the formation was less than 1

g/kg except for carrageenan (7.7 g/kg), gelatine (4.5 g/kg) and starch (1 g/kg). When the

same foods/ingredients were treated with 0.05% active chlorine overnight, only egg white

powder, carrageenan and starch showed any SEM formation, in the range 0.1 to 0.3 g/kg. At

0.015% active chlorine any increase was barely detectable, with only carrageenan and starch

showing any formation, at 0.1 and 0.3 g/kg respectively, after treatment overnight.

Chlorinated water may be used as a processing aid to wash foods, for example fruits and

vegetables, provided that it meets the definition of a processing aid i.e. does not perform a

function such as preservation in the final product and leaves no harmful residues [Directive

89/107/EEC). It is a reasonably widespread practice to wash certain ready-to-eat foods using

water with a chlorine content up to 100 parts per million (0.0001%). Similarly, the Codex

committee on fish and fishery products stated that chlorinated water at a recommended level of

10 parts per million of free chlorine is used on fish and fishery products. Given that these

concentrations are 100-fold and 1000-fold lower respectively than the concentration of 0.015%

active chlorine that gave barely any detectable SEM formation in the tests described above,

given that the chlorine wash will be for a far shorter period that the overnight conditions used

in the laboratory tests, and given that the processes also generally incorporate a final rinse with

chilled water with just 2 to 4 parts per million free chlorine, it is considered that the use of

chlorinated water as a processing aid is highly unlikely to give any detectable residues of SEM

in the washed food.

Disinfection of equipment and surfaces with disinfecting agents such as sodium hypochlorite is

common practice in industrial food production. This should be rinsed off before the cleaned

surface is used again into contact with food. With effective rinsing, no subsequent formation

of SEM is to be expected.

SEM in egg powder

The presence of SEM in egg products was detected in Belgium in 2003 by the routine

monitoring of egg white powder for residues of the nitrofuran class of antibiotics. Low but

persistent contamination (1-150 g/kg) was found in many batches tested. The Belgian

authority took action since this finding was considered as a non conformity in regards to

Council regulation EEC/2377/90 which forbids the use of nitrofurazone (with SEM as marker)

in animal entering the food chain. The lots affected were sequestered, information was posted

using the Rapid Alert System for Food and Feed (RASFF), and action was started to trace the

origin of SEM. These investigations showed that contamination with SEM occurred during the

extraction of lysozyme from egg albumin because bleach solution was used to sanitize the

carrageenan column used for the extraction. Up to that time, in 2003, the possibility that

hypochlorite bleach could react with organic substances to form SEM (Gatermann et al., 2003)

was unknown and unexpected. Through the RASFF, the Belgian authorities announced that

batches under temporary seizure were released if they contained less than 50 g/kg of SEM

(RASFF, 2003).

Another possible cause of SEM formation in egg white powder could be any heat treatments

applied. It has been reported that heating at 70-80 C for 3 days yields up to 30 g/kg of SEM

if heated in an open system (Gatermann et al., 2004). In a closed system, in contrast, the

formation of SEM was very limited, no more than 1-2 g/kg. Other products were reported to

show similar results, although less pronounced, and the authors suggested a role for oxygen inthe heat-formation of SEM from compounds in foods.


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SEM in food additives

As described above, reports of SEM in processed egg products were attributed to the

carrageenan columns used during processing, which were sanitised using chlorine bleach.

Carrageenan is also used as a food additive (E 407) as a thickening, gelling and suspendingagent for example in ice cream, pudding, yoghurt, fruity jellies, chocolate milk, and different

meat products. It is a complex mixture of different polysaccharides obtained from red seaweed

(Rhodophyceae) by several processing steps including extraction, washing, precipitation,

drying and grinding. One type of carrageenan is bleached during production. This is semi-

refined carrageenan or Processed Euchema Seaweed (PES, E407a) which is bleached to

remove colour since the production process does not involve a precipitation step. Bleaching

with calcium hypochlorite is a standard practice in making PES from seaweed. The industry

association for producers of agar, alginates, carrageenan and processed eucheuma seaweed

(Marinalg) reported that they were initiating trials to replace calcium hypochlorite with an

alternative bleaching agent for the production of PES.

Information was received from the Norwegian Food Safety Authority on their analysis of 8

samples of food grade carrageenan from three different companies. Seven out of the eight

samples were below the detection limit of 5 g/kg and one sample contained SEM at 750

g/kg. The geographic location of the supplier of this sample was unknown.

SEM from azodicarbonamide-treated flour

ADC is not permitted as a flour treatment agent in the EU. Where permitted, for example in the

USA and Brazil, the use of ADC up to 45 mg/kg of flour is allowed. In a recently publishedpaper, the formation of SEM from ADC used to treat flour was studied and reported (Pereira et

al., 2004). The authors found that when ADC was added to flour it formed SEM at about 0.1%

yield. For example, they estimated that chicken products with added coatings made from flour

could contain 0.2-5 g/kg SEM in the whole product. They observed that this level would be

sufficient to be detected, with the result that the sample could fail EU import controls using

SEM as the metabolic marker for nitrofurazone use.

In a second publication, bread was made using commercial flour that had ADC listed as an

additive (Becalski et al., 2004). The level of treatment of the flour was considered by the

authors to have been about 20 mg/kg as is normal industry practice in Canada. The treated

flour contained SEM at 3 g/kg. Bread made using the flour contained 28 g/kg with most ofthe SEM concentrated in the crust.

SEM present at background levels naturally or from as yet unidentified sources

Other sources of SEM in foods that had been suggested, based on structural similarities,

included the herbicides triazophos, diflufenzopyr and roxarsone, but these possibilities have

since been discounted (van Rhijn et al., 2004).

Saari & Peltonen (2004) have reported finding SEM in all 18 samples of wild crayfish samplestested. The crayfish were trapped in various locations in Finland in Autumn 2003 and the


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authors ruled-out all possibility that the crayfish had been treated with nitrofurazone or that

they had become cross-contaminated during handling and transport. The crayfish were boiled

in tap water and the authors noted that Helsinki uses ozone and not chlorine for tap water

supplies. The meat of all 18 crayfish samples tested contained SEM, at 0.4 to 18 g/kg.

Chicken and shrimp samples were cooked and tested as blank controls and no SEM was

detected in them.

OCCURRENCE AND EXPOSURE

SEM from nitrofurazone veterinary medicine use

If the ban on nitrofurazone use is enforced effectively then there should be no detectable levels

of SEM from this source in the food of European consumers. The detection limits of the

surveillance methods used are in the range 0.2 to 1 g/kg. Taking the UK as an illustrativeexample, the average consumption of all animal products (meat, meat products, fish, poultry)

excluding milk, is 103 gram per person per day. For high adult consumers of animal products

(average x 3), consumption of 300g of food and making the unlikely assumption that it was all

contaminated with SEM at the highest detection limit of 1 g/kg, the intake would be 0.005

g/kg bw/day for a 60 kg bw person.

Migration of SEM from lid gaskets

Concentration data from different countries

Analytical surveys of the concentration of SEM in foods packaged in glass jars and bottles with

metal lids have been reported recently for several European countries (Information provided to

EFSA see list page 30). The levels reported are reasonably consistent between different

surveys. The individual surveys are described below. For the calculation of the average

concentrations, the upper-bound assumption was made, that samples reported as not detected

contained SEM at the detection limit stated in the survey.

The Netherlands. In a survey of 40 samples of baby foods, covering 7 brands, purchased in

The Netherlands in October 2003, all samples contained detectable SEM. The samples of baby

food were fruit, vegetables, meat, pasta, and combinations thereof. The range ofconcentrations found was 3 to 26 g/kg and the average was 13 g/kg. The authors of the

report noted that there appeared to be no relation between the level of SEM, type of food,

content (size of jar), or brand. They also measured high levels (ppm) of SEM in gaskets and so

concluded that migration from lids was the most probable origin of the SEM in foods. The

same workers also conducted a smaller survey of general foods (i.e. not specifically baby

foods) including fruit and vegetables packaged in glass jars and bottles. The 22 samples were

purchased in October 2003. SEM was found in a range up to 2 g/kg. 10 of the 22 samples

had no detectable SEM (


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Industry data. The European industry has supplied results of analyses to EFSA. Levels of

SEM in 63 samples of baby foods, of all types and covering several brands and countries of

production, were in the range 0.1 to 27 g/kg. The average for all samples was 11 g/kg.

Results for 7 samples of ready-to-feed infant milk were also provided. The range of results

was 5 to 14 g/kg and the average SEM concentration in the 7 samples was 9 g/kg. Results

for 45 samples of general foods packed in glass jars and bottles with metal lids were alsoprovided. The foods were sauces, jams, pickles etc. The range of concentrations of SEM was

0.3 to 3.3 g/kg and the average of all 45 results was 1.2 g/kg. 24 of the 45 samples

contained no detectable SEM, with the limit of detection generally being 1 g/kg or lower.

Spain. From surveys covering several regions of Spain, results for 88 samples of baby foods in

glass jars and bottles have been reported. A small number of other results were reported too,

but it was noted by the researchers that these samples may not have been made adequately

homogeneous before analysis and so these results have not been included here. The baby food

samples included fish, fruit, vegetables, meat, etc. The range of SEM concentrations was 1 to

87 g/kg and the average for the 88 samples was 16 g/kg. The same laboratories also

conducted smaller surveys of general foods (i.e. not specifically baby foods) including

vegetables, jams, sauces and fish packaged in glass jars and bottles. The samples were 30 in

number. SEM was in the range 0.5 to 6 g/kg. The average of the 30 samples was 0.9 g/kg.

France. In a small laboratory study, 4 different types of fruit and vegetable baby foods were

studied for SEM concentration. More than 10 samples were tested and the range of SEM

concentrations was 6 to 15 g/kg. The method of showed good repeatability when different

specimens for the same jar were analysed (c.v. ca. 10%) and the variability when different jars

of the same product were analysed was not great (c.v. ca. 15%). The authors noted that for

some samples there was a concentration gradient down the jar consistent with the proposal that

migration from the gasket was the source of SEM. The authors also noted that further warmingof the foods in the jars, to 50C as if in preparation for feeding, did not increase SEM levels

further.

Germany. Results have been reported for 133 samples of all types of baby foods purchased in

Germany. The range of concentrations of SEM was


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average was 7 g/kg. Seven other miscellaneous food products were tested and none contained

detectable SEM,


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Table. Intakes (average and 95th

percentile, consumers only) of commercial baby foods and

drinks known to be potential sources of SEM exposure in infants (Kersting et al., 1998)

Age in

months

Average

bodyweight in kg

Average intake

in gram per

infant per day

P95 intake in

gram per infant

per day

3 5.8 67 +/- 65 192

6 7.8 195 +/- 114 407

9 8.8 234 +/- 127 464

12 9.8 208 +/- 128 424

Beyond the first year, studies in France and UK have indicated that the contribution of baby

foods in jars to the total diet decreases significantly. Regarding adults, the quantity of available

data on target foods is very limited, but it is expected that the contribution of food in jars to the

total diet of adults is likely to be much smaller than in the diet of infants.

Exposure estimates

Exposure assessment was targeted to highly exposed subjects i.e. to subjects who

systematically consume foods which are likely to contain semicarbazide. Available analytical

data suggest that variability in semicarbazide concentration in products packaged in jars and

bottles with metal lids and PVC gaskets is generally low and is of the same order of magnitude

between and within the different brands of processed foods. Therefore, it was considered

appropriate to estimate chronic exposure to semicarbazide on the basis of the average observed

semicarbazide concentration and that such exposure assessment would be valid even for

subjects who are loyal to specific brands of processed food.

To estimate the exposure of infants to SEM from commercial baby foods and juices, an

average contamination value of 13 g/kg was combined with the average and the 95 th

percentile of food consumption. The average contamination value of 13 g/kg was derived

from the survey data summarised above. The survey data are dominated by baby foods in jars

with relatively few samples of juices for drinking tested. However, the UK (Mills & Tyler,

1992) and the more recent French studies (Even et al., 2001) indicated that a significant

proportion (probably up to 40%) of the baby foods consumed are vegetable or fruit products

and/or fruit juices. Since lower levels of SEM are generally found in fruit juices compared to

baby foods, using an average concentration level of 13 g/kg is likely to be conservative.

With regards to taking the average and the 95thpercentile of food consumption, a conservative

assumption is that all baby foods consumed are packaged in glass jars and bottles with metal

seals, rather than in other packaging materials such as metal cans or plastic pouches. Such an

assumption is not unrealistic.


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Table. Exposure (average and 95th

percentile, consumers only) to SEM taking an average

concentration of 13 g/kg in all commercial baby foods and drinks known to be potential

sources of SEM exposure in infants

Age in months Average

bodyweight inkg

Average

exposure ing/kg bw/day

P95 exposure in

g/kg bw/day

3 5.8 0.15 0.43

6 7.8 0.33 0.68

9 8.8 0.35 0.69

12 9.8 0.28 0.56

To estimate the exposure of infants to SEM from ready-to-feed infant milk pre-packaged in

glass bottles with metal lids, the average SEM concentration of 9 g/kg (from 7 samples) can

be combined with a consumption of 700 mL each day by a 4.5 kg bodyweight infant. (SCF,

2002) The resulting exposure would be 1.4 g/kg bw/day.

Adult exposures to SEM are likely to be much lower than infant exposures, due to the lower

contribution of foods in bottles and jars to the total diet of adults, the lower contamination

levels derived from the smaller gasket areas involved for that packaging, and the higher adult

body weight. Taking an assumption that 1 kg of food contaminated with SEM at an average

concentration of 1.0 g/kg is consumed each day, the exposure for a 60 kg bodyweight adult

would be 0.02 g/kg bw/day.

SEM from hypochlorite-treated foods and food additives

SEM from the use of chlorinated wash water

Based on the foregoing discussions, it is considered that the use of chlorinated water as a

processing aid is highly unlikely to give any detectable residues of SEM in washed food.

SEM from egg powder

In the analysis of production from one manufacturing facility in Belgium that sourced eggs

from a number of countries, 78% (478 of 613 samples) contained no detectable SEM,


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Using this figure of 10% dilution, taking 50 g/kg as a high but not unrealistic level, the final

food product could contain 5 g/kg of SEM. At a 1% usage dilution the concentration in the

final foods could be 0.5 g/kg. The average UK consumption of eggs and egg products is 14

gram per person per day. For a high consumer of egg products, consuming 1 kg/day of food

made with 1% dry egg powder or 100g/day of food made using the maximum likely usage of

10% egg powder, the intake of SEM would 0.008 g/kg bw/day for a 60 kg bw person.

SEM from carrageenan

The industry association for producers of agar, alginates, carrageenan and processed eucheuma

seaweed (Marinalg) provided a description of two industrial process to obtain carrageenan and

one process to obtain PES. They also provided analytical results from testing production

batches from across the industry for SEM. For 34 batches of carrageenan made by Process A

the range of results was 0 to 4 g/kg SEM in the food additive with a mean of 1 g/kg. For

carrageenan prepared using Process B the average for 13 batches was 6 g/kg and the range

was 0 to 46 g/kg. (Hoenicke et al. (2004)) have reported that the finding of SEM as a natural

constituent in red seaweed which can be detected after drying, suggests that there may be other

natural sources of SEM. If seaweed or seaweed products that contained such background levels

of SEM were to be used in animal feed, it is not expected that any detectable SEM would occur

in products for human consumption that were derived from those production animals. For

example, when broiler chickens were fed a diet containing 200 mg/kg SEM the level of tissue-

bound SEM in the muscle meat was 60 g/kg a transmission factor of just 0.03% (Kennedy

& van Rhijn, 2003).

For PES prepared using a bleaching step, for the 25 batches reported by Marinalg the range for

SEM was 9 to 380 g/kg with a mean of 65 g/kg. Industry stated that carrageenan and PES

are used interchangeably in many food additive applications, depending on customer

requirements. Using the average reported value for SEM in PES and using the available

information on maximum usage levels of this food additive, the SEM levels which could in

principle be found in various foods have been calculated and are shown in the table.

Table. Potential concentrations of SEM that may result from the use of additive E407a at the

maximum usage levels and assuming an average contamination of 65 g/kg of the food

additive

Food Additive usage levels SEM concentrationBeverages e.g. milk-based

drinks

0.02 to 0.04% additive used up to 0.026 g/kg

Foods In the range 0.02 (ice cream) to

1% (jams, marmalade)

up to 0.65 g/kg

Confectionery Up to 2% (e.g. gums) up to 1.3 g/kg

The ADI for carrageenan is 75 mg/kg bw/day (SCF, 2003b). If consumption was up to the full

ADI and if all of this food additive contained SEM at 65 g/kg then the intake of SEM from

this source could be up to 0.005 g/kg bw/day.

JECFA (2002) summarised data on intake from Europe, Canada and the USA, noting that the

resulting mean intake estimates were consistent, falling within a range of 30-50


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mg/person/day from the use of carrageenan and PES as food additives. If all of this food

additive was PES and contained SEM at 65 g/kg, then the intake of SEM from this source

could be up to 0.003 g/person/day.

SEM from azodicarbonamide-treated flour

In Canadian tests, bread made using the ADC-treated flour contained 28 g/kg SEM. ADC is

not permitted as a flour treatment agent in the EU and the importation of bread and bakery

ware is likely to be very low. There is the potential for exposure from breaded animal products

imported into the EU (e.g. frozen breaded chicken or fish products). Taking the upper figure of

5 g/kg of product from the study by Pereira et al (2004) this would give an intake of SEM of

1 g/person from a consumption of 200g of product.

TOXICITY OF SEM

Metabolism and toxicokinetics

Most biochemical data on SEM concern its uses as an enzymatic (amine oxidase) inhibitor and

as an aldehyde trapping agent. No information on its toxicokinetics or in vivometabolism is

available.In vitro, SEM was shown to be metabolised by rat liver homogenate to formaldehyde

and N2at a rate of 0.028 and 2.5 mol/g of liver per hr, respectively (Kroeger-Koepke et al.,

1981).

Acute toxicity

Doses causing acute toxicity in mice (LD50s by oral, intraperitoneal (i.p.), subcutaneous and

intravenous routes) ranged from 123-176 mg/kg bw (IARC, 1974). At high doses it caused

convulsions (Jenney and Pfeiffer, 1958).

A dose of 10 mg/kg bw was reported not to cause death in the rat (NTP Chemical Repository,

1991). The i.p. LD50for rats was reported to be 212 mg/kg bw (de la Fuente del Rey 1986).

Developmental toxicity

Some studies have been reported on the effects of SEM hydrochloride on development in chick

embryos (Shephard, 1989), however these are not considered relevant for human risk

assessment.

Oral studies.

SEM was included in a study on lathyrogenic agents (Steffek et al., 1972). Lathyrism is a

disease of collagen cross-linking caused by copper sequestration by nitriles. Groups of 3-11

female Sprague-Dawley rats were given SEM hydrochloride by aqueous, oral gavage at doses

of 5, 10, 25, 50 or 100 mg/day on days 10-16 or 12-15 of gestation. The rats were killed one

day before term and were examined for the presence of cleft palate. At the highest dose, 3 out


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of 9 pregnant rats died. The incidence of resorptions (embryo-foetal deaths) was 3%, 0%, 3%,

38% and 56% and the incidence of cleft palate in surviving foetuses was 0%, 0%, 43%, 95%

and 100% in 5, 10, 25, 50 or 100 mg/day dose groups respectively. Other lathyrogenic agents

also caused cleft palate. From this study a no-effect level of 10 mg/day (equivalent to around

30-40 mg/kg bw/day) for cleft palate and intrauterine death was apparent. It should be noted

that this was not a comprehensive study of developmental toxicity; dosing did not cover thewhole of embryogenesis and the foetuses were not examined for internal or skeletal

abnormalities.

In a study on hamsters (Wiley and Joncja, 1978), groups of 5 or 6 pregnant animals were given

100, 150 or 200 mg/kg bw of SEM hydrochloride orally by gavage on gestation day 7. The

animals were killed on gestation day 14 and the contents of the uterus examined. The two

higher doses caused death of all the mothers, usually within 48h of dosing and all implants

showed signs of resorptions. At 100 mg/kg bw, 1.3% of implants were dead or resorbed. Of the

live fetuses, mean fetal weight was not significantly reduced, but 16.5% of fetuses showed

growth retardation (defined as weighing


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rear their litters to 21 days of age. The numbers of live fetuses and fetal weights were

significantly reduced in a dose-related manner compared to saline controls following all

treatments except 50 mg/kg on day 13. Increased incidences of gross, skeletal and soft tissue

abnormalities were found in all treated groups. These included anophthalmia, haemorrhages in

the brain, liver and intestine, hydronephrosis, absent testes, incomplete ossification, absent

sternum and rib abnormalities. Postnatally, there were significant, increases in offspringmortality in all treated groups, ranging from 25-63% compared with


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In vitro study

In a study reported in abstract only (Simpson and Freeman, 1986), 9.5 day rat embryos in

culture were exposed to SEM at concentrations of 10, 50, 100 or 250 g/ml for 48 h. There

were no effects on growth or development at 10 g/ml, but at higher exposures there wereconcentration-related deficits in growth and differentiation, with haemorrhage in or absence of

yolk sac development. These studies suggest a direct toxic and teratogenic effect of SEM on

the embryo perhaps mediated via a disturbed yolk sac circulation.

Carcinogenicity

In a study by Mori et al. (1960) primarily aimed at evaluating the induction of pulmonary

tumours in female dd mice with isonicotinic acid hydrazide a group of mice treated with SEM

hydrochloride was included for comparison. Thirteen 6-8-week old female dd mice were fed a

basal diet containing 0.1% SEM hydrochloride for 7 months (equivalent to approximately 150mg/k bw.) It is stated that the animals gained weight on the diet. After the 7 months on the diet

6 of 8 survivors (75%) had developed 8 lung tumours compared to 1 tumour in 1 of 20 (5%)

control animals.

Groups of 50 male and 50 female Swiss albino mice, 6 weeks old (47 days) at the beginning of

the experiment, were administered SEM hydrochloride at a concentration of 0.0625% (625

mg/L) in the drinking water for the rest of their life span. Based on the recordings of the

average daily intake of drinking water the authors estimated the average daily intake of SEM

hydrochloride to be 3.3 mg for females and 4.8 mg for males (130 mg/kg bw/day for females

weighing 25 gram and 160 mg/kg bw/day for males weighing 30 gram). Groups of 100 male

and 100 female mice, observed from weaning (5 weeks of age), served as controls. Thelifespan was reduced in the treated animals, especially in the males. Complete necropsies were

performed on all animals. All organs were examined macroscopically and histological

examination was carried out on the liver, spleen, kidney, bladder, thyroid, hearth, pancreas,

testis, brain, nasal turbinale, and lungs as well as on organs showing gross pathological

changes. The occurrence of lung tumours and tumours of vascular origin were increased in

treated females compared to controls. A total of 37 lung tumours occurred in 25 treated female

mice (50%) compared to 31 lung tumours in 21 of the 100 control animals (21%). In the

treated females 20 had 31 adenomas and 5 had 6 adenocarcinomas. In the treated males, 15

(30%) developed 21 lung tumours whereas 23 (23%) of control males developed 35 lung

tumours. In the treated males 11 had 12 adenomas, 3 had 5 adenocarcinomas, and 1 had an

adenoma and three adenocarcinomas. Tumours of vascular origin were observed in 9 treatedfemales (18%) compared to 5 (5%) in the controls. The 9 vascular tumours in the treated

females were 3 angiosarcomas and 2 angiomas in the liver, 2 angiosarcomas and 1 angioma in

the ovaries, and 1 angioma in the lymph nodes. In the treated males, 3 animals (6%) developed

vascular tumours compared to 6 (6%) of the control males. The incidences of a number of

other tumours did not differ between treated and control mice. The authors also reported that

induction of lung and blood vessel tumours in mice was a common finding during the testing

of a number of substituted hydrazines in their laboratory (Toth et al.1975). Toth and Shimizu

(1974) has given a detailed description of the occurrence and morphology of the lung tumours

in the control animals.

In a meeting abstract, Ulland et al.(1973) reported that SEM was non-carcinogenic in Charles

River CD rats administered the maximal tolerated dose and half that level for 18 months


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followed by an observation period of 6 months. However, no further information were given

except that SEM at the highest dose level had a lathyrogenic effect and that the animals

suffered severe skeletal deformities.

Weisburger et al.(1981) reported more details on the study of SEM hydrochloride in Charles

River CD rats apparently mentioned by Ulland et al. (1973) in an abstract. SEM hydrochloridewas among 14 different environmental and industrial chemicals that were tested for

carcinogenicity in Charles River CD rats. Groups of 26 male and 26 female Charles River CD

rats, 5-6 weeks old, were given diets containing SEM hydrochloride at concentrations of 500 or

1000 mg/kg (equivalent to 25 or 50 mg/kg bw/day, respectively). The authors considered the

highest dose to be the maximal tolerated dose (MTD). A total of 184 animals per sex served as

controls. The animals receiving the diet containing 500 mg SEM hydrochloride/kg were treated

for 78 weeks. They thereafter received control diet and were observed for a further 26 weeks (a

total of 104 weeks on test). In each of the groups receiving the diet containing 1000 mg SEM

hydrochloride/kg the number of early deaths was so large that the treatment was discontinued

at week 32. They therefore received control diet for the remainder of the study period of 104

weeks. The authors state that the survival was adequate in all the groups (at least 20 animals) at

78 weeks except for the high-dose males of which only 13 survived to 52 weeks. The mean

weights of the treated animals were less than those of the matched controls. All animals were

necropsied and an extensive tissue histopathology examination was carried out. The authors

reported that SEM hydrochloride produced negative results as regards potential carcinogenic

activity. However, no details were provided on the tumour incidences after treatment with

SEM hydrochloride as well as the other compounds reported to be negative in the study,

although detailed information was provided in this respect for the control animals and the

chemicals reported to be carcinogenic. Osteolathyrism was diagnosed grossly commencing

with the 10thweek in all treated groups. Signs of the disorder were rough coat, protrusion of

the sternum and bowing of the legs, and stiffness of the joints with bony growths.Histologically, osteoporosis was noted in the long bones. In conclusion, the authors stated that

SEM hydrochloride had been tested at sufficiently high dose levels to cause toxic effects in

both sexes and except for the high-dose males, survival was adequate in the treated groups.

However, there was no indication of tumour induction from the treatment.

A number of hydrazine and related substances were evaluated by IARC in 1974 whereas

carbazides, including SEM was evaluated by IARC in 1976 (IARC 1974; 1976). In its update

of IARC Monographs Volumes 1 to 42 IARC evaluated that the evidence for carcinogenicity is

inadequate in humans and inadequate or limited in experimental animals and placed SEM

hydrochloride in Group 3 (IARC 1987).

Gold et al.(2004) in their Carcinogenic Potency Database have calculated TD50values for the

three statistically significant carcinogenic effects of SEM hydrochloride observed in the male

and female mice in the study by Toth et al.(1975). The TD50is defined as follows: For any

particular sex, strain, species and set of experimental conditions, the TD50 is the dose rate (in

mg/kg bw/day) that, if administered chronically for a standard period the standard lifespan

of the species will halve the mortality-corrected estimate of the probability of remaining

tumourless throughout that period. A TD50 of 223 mg/kg bw/day was calculated for lung

tumours combined in females and of 833 mg/kg bw/day for males, whereas a TD50 of 395

mg/kg bw/day was estimated for blood vessel tumours combined in females. Cheeseman and

co-workers (1999) have grouped 709 positive animal carcinogens in the Carcinogenic Potency

Database according to structural class and used the TD50s to compare their potencies. In thestructural group of hydrazines/triazenes/azides/azoxy compounds more than 30 substituted


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hydrazines are included with TD50s ranging from 0.102 mg/kg bw/day for 1,2-

dimethylhydrazine hydrochloride to 561 mg/kg bw/day for 1,2-diformylhydrazine. Among the

hydrazines tested, SEM hydrochloride was the fourth-least potent (TD50of 223 mg/kg bw/day).

Genotoxicity

Bacterial mutagenicity assays

A sample of commercial semicarbazide (carbamyl hydrazine, ICN-K&K Lab., purity and

solvent unspecified) was tested in the Salmonella typhimurium reversion test using the plate

incorporation method, with and without S9 from Aroclor induced rat liver and from Aroclor

induced mouse liver and lung. In experiments without S9, SEM was weakly positive toward

strain TA1535 (0.0009 induced revertants/nmol); the mutagenic response was partially or

totally depressed in the presence of liver and lung S9, respectively. Negative results were

obtained with strains TA1537, TA1538, TA98 and TA100, either with or without S9 (De Flora.(1981), De Flora et al. (1984).

Negative results were obtained in a study using a modified test protocol with S.typhimurium

strains G46, C3076, D3052, TA1535, TA1537, TA1538, TA98, TA100 and the E.coli strains

WP2 and WP2 uvrA, with and without metabolic activation by Aroclor induced rat liver S9 (no

experimental details given) (McMahon et al. 1979).

A sample of pure SEM hydrochloride (99.7%, Sigma-Aldrich) was dissolved in water and

tested in the plate incorporation test with S.typhimurium strains TA1535, TA1537, TA98,

TA100 and E.coli WP2 uvrA at doses ranging from 62 to 5000 ug/plate, with and without

exogenous metabolic activation by Aroclor induced rat liver S9. In the absence of S9, aclearcut mutagenic response was observed in strain TA1535, with a 16-fold increase in

revertant colonies over control value at the highest dose applied. A two-fold increase in

revertants colonies was also observed in strain TA1535 in the presence of S9. Borderline

mutagenicity was also observed also in strain TA100, only in the absence of exogenous

metabolism. No mutagenicity was observed in the other bacterial strains (TNO 2004a).

A sample of pure (99.7%, Fluka) SEM hydrochloride and tested in the plate incorporation test

with S.typhimuriumstrains TA1535, TA1537, TA98, TA100 and TA102 at doses ranging from

50 to 5000 ug/plate, with and without exogenous metabolic activation by Aroclor induced rat

liver S9. A weak mutagenic response was observed toward the strain TA1535, only in the

absence of S9, with two-fold increases in revertant colonies at 3000 ug/plate and above. Nomutagenicity was observed in the other bacterial strains (Herbold, 2003).

Mammalian cell gene mutation assay

A sample of pure SEM hydrochloride (99.7%, Sigma-Aldrich) was dissolved in culture

medium and tested in the forward mutation assay at the tk locus in mouse lymphoma cells

L5178Y. The substance was applied at nine dose levels, from 0.21 to 10.0 mM (maximum

recommended dose level), with and without metabolic activation. Treatments in the absence of

S9 produced mild toxicity, with 50 % inhibition of growth at the highest dose. No toxicity wasobserved without S9. A dose related increase in mutant colonies, which exceeded twice the


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spontaneous frequency at 10 mM, was observed in the experiment without S9. A borderline

increase in mutant colonies was also observed at the highest dose in the presence of S9 (TNO

2004b).

In vitro chromosomal aberration assays

SEM hydrochloride (99.7 %, Bayer) was tested in a chromosomal aberration assay in Chinese

hamster V79 cells. SEM was applied for 4 hours at 1120 ug/ml (10 mM, maximum

recommended dose level) and at two lower doses (500 and 250 ug/ml), with and without S9.

Cells were harvested after 18 hours and 30 hours (top dose only). In addition, a continuous

treatment of 18 hours was applied without S9. Chromosomal aberrations were scored in 200

metaphases per dose (100 for each duplicated culture). Mild toxicity (about 20 % decrease of

mitotic index and survival) was observed at the highest dose, only in the absence of S9. No

increase in the frequency of aberrant metaphases and polyploidy was observed in any of the

cultures treated with SEM compared with those treated with the vehicle only (deionized water)(Herbold, 2004).

In another study, a sample of pure SEM hydrochloride (> 99.0 %, Sigma-Aldrich), dissolved in

culture medium, was tested in a chromosomal aberration assay in CHO cells. A range finding

experiment showed that the maximum recommended dose of 10 mM (1115 ug/ml) was

exceedingly toxic. On this basis, in the first experiment the substance was applied at the doses

of 150, 300 and 600 ug/ml for 4 hours, with and without S9, and cells were harvested 14 hours

later; in the absence of S9 a continuous treatment of 18 hours was also applied. In the second

test, 700, 800 or 900 ug SEM/ml were applied with continuous treatments of 18 or 32 hours

without S9; with S9 cells were treated for 4 hours and harvested 14 or 28 hours later.

Treatments produced mild toxicity, with 20-50 % reductions of mitotic index, and no

significant increase in the incidence of cells with structural chromosomal aberrations. A

significant increase of endoreduplicated cells was observed only after treatments in the

presence of S9, in cells harvested at the early sampling time (TNO 2004c). The relevance of

endoreduplication with respect to genotoxicity hazard identification is questionable, as this

end-point is mainly consequence of alterations in cell cycle control rather that DNA damage.

In vivo cytogenetics

Semicarbazide hydrochloride (Sigma-Aldrich, Sweden) was tested in a flow cytometry-basedmicronucleus assay in vivo. Male CBA and Balb/C mice were injected intraperitoneally with 0,

40, 80 and 120 mg/kg bw SEM dissolved in phosphate buffer solution (maximum tolerated

dose, determined in a toxicity pretest). Three or four animal per dose were used in the first

experiment, with Balb/C mice, and five animal per dose (only 80 and 120 mg/kg bw) in the

second experiment, performed with CBA mice. Blood samples were collected from each

animal 42 hours after treatment. The incidence of micronuclei was determined in about

200,000 polychromatic erythrocytes per animal using a FACSVantage SE flow cytometer.

Neither in CBA or Balb/C mice the treatment with SEM resulted in a frequency of

micronucleated polychromatic erythrocytes (MnPCE) different from control values, nor did

cell proliferation (% of PCE) show any supression. A distinct increase in MnPCE was inducedby the positive control substance colchicine (Abramsson-Zetterberg and Svensson, 2005).


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In a study in grasshopper germ cells, SEM hydrochloride was given by injection (5 ul/animal

of 1 M solution in water). Increased incidences of chromatid and chromosome aberrations

(breaks, dicentrics, etc) were observed in spermatocytes (overall incidence 0.3% in treated vs

< 1 % in controls) (Bhattacharya 1976). The relevance of this study, based on an unvalidated

test method in insects, for the assessment of the clastogenic/aneugenic potential of SEM in

mammalian cells is limited.

DNA damage in vitro/in vivo

Negative results in a DNA repair test with SEM, using rat primary hepatocytes, are reported

with no details in an abstract (Sugie et al. 1987).

The DNA damaging activity of a series of hydrazine derivatives, including semicarbazide, was

evaluated in vivo in mouse liver and lung using the alkaline elution technique. SEM (95-99%

pure) was given according to the following treatment schedules to groups of 6 11 male Swissmice: i) single i.p. injection of 3.28 nmol/kg b.w. (LD50) 6 h before sacrifice; ii) five daily i.p.

injections of 0.55 nmol/kg b.w. Treatments did not induce any significant increase of DNA

elution rate over controls, and SEM was evaluated as inactive (Parodi et al. 1981).

Pure (>99 %, Fluka) semicarbazide hydrochloride was also tested in the in vivo/in vitro liver

UDS assay. SEM was given to female CD-1 mice as a single oral dose of 100 or 200 mg/kg

b.w. (MTD, based on clinical signs). Animals were sacrificed after 2 and 16 hours from

treatment, and UDS was evaluated by autoradiography. No significant increase in net nuclear

grains or in cells in repair was observed in hepatocytes isolated from animals treated with SEM

(Central Toxicology Laboratory 2004).

Effects on DNA in acellular systems

SEM has been shown to specifically modify cytosine, replacing the C4 amino group with a

semicarbazide residue. The reaction occurs at low pH (4.2), and with lower efficiency in

double stranded DNA (Hayatsu et al. 1966; Hayatsu and Ukita 1966).

Treatment of naked DNA with SEM (10- 100 M) and Cu (II) (20 M CuCl2), but not SEM

alone, led to DNA strand breakage and base oxidation (8-oxodG) due to the formation by

autooxidation of the carbamoyl radical CONH2and ROS production (Hirakawa et al. 2003).Whether these reactions also can occur at physiologically free copper levels (< 10-6uM), is not

known.

Summary and discussion of the toxicity studies

None of the developmental toxicity studies available so far are comprehensive or conducted to

current guidelines. In the hamster, a single oral dose of SEM hydrochloride of 100 mg/kg bw

on gestation day 7 was not teratogenic but did cause minor growth retardation. Studies in rats

showed that high doses of around 50-300 mg/kg bw/day in the rat, given either orally overseveral days or as single i.p. doses during gestation, cause maternal deaths, embryo-fetal deaths


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and congenital abnormalities. Lower doses of 50 and 75 mg/kg bw given i.p. on single days of

gestation also increased congenital abnormalities as did 17 mg/kg bw/day given i.p. throughout

gestation. The relationship of these lower doses, below 100 mg/kg bw, to any maternal toxicity

was not reported. Doses of 50 mg/kg bw and above given i.p. on single days during gestation

also caused high postnatal mortality in the rat; a NOAEL for this effect has not been

established. The no-effect level observed for induction of cleft palate in the rat (30-40 mg/kgbw/day orally) may not be applicable for other abnormalities. It can be noted however that the

doses causing adverse effects are around three or more orders of magnitude above likely

exposures of humans.

None of the carcinogenicity studies available were conducted and reported to current

guidelines. They show that SEM hydrochloride at a dose of approximately 130 160 mg/kg

bw/day in the drinking water increased the incidence of lung tumours (adenomas +

adenocarcinomas) in female and male mice and of blood vessel tumours in females. Induction

of lung and blood vessel tumours is a common finding in mice following oral administration of

substituted hydrazines. Comparison of the TD50

s for a number of substituted hydrazines to be

found in the Carcinogenic Potency Database reveals that SEM hydrochloride is among the least

potent hydrazines tested for carcinogenicity so far.

In rats, no carcinogenic effects were observed in either sex fed a diet containing 500 mg SEM

hydrochloride/kg for 72 weeks (corresponding to 25 mg/kg bw/day; considered to be half the

maximal tolerated dose) and observed for an additional 26 weeks. In the study, 14 different

environmental and industrial chemicals were tested for carcinogenicity in rats and no detailed

findings were reported for SEM hydrochloride and the other chemicals showing negative

effects, whereas adequate information is given for the compounds found to be carcinogenic. At

a higher concentration of 1000 mg/kg diet SEM hydrochloride was toxic to the rats and the

treatment was discontinued after 32 weeks. Also in this group no carcinogenic effect wasobserved after 104 weeks.

Literature data, and the results of recent studies performed to current standards with pure SEM

hydrochloride, indicate that SEM is mutagenic in some test systems in vitro. Direct

mutagenicity was observed, especially in the absence of an exogenous metabolising system, in

bacterial strains detecting base pair substitutions and in a forward mutation test in mouse

lymphoma cells. No clastogenic activity was observed in cytogenetic tests in vitro. A treatment

related increase of endoreduplicated cells was observed in one study, but not confirmed in

another investigation using a different cell line. The relevance of endoreduplication for

genotoxic hazard identification is questionable.

In vivo, the intraperitoneal administration of SEM to mice did not induce micronuclei in

peripheral blood in a sensitive flow-cytometer-based micronucleus assay, nor DNA damage, as

measured by alkaline elution, in liver and lung, whereas positive results were obtained with

known genotoxic hydrazine derivatives. Negative results were also obtained in the liver UDS

assay after oral administration of SEM to in female CD-1 mice, the mouse strain where long

term administration of SEM resulted in increased incidence of lung and vascular tumours

(IARC, 1976).


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CONCLUSIONS

The occurrence and the precursors of SEM in foods

SEM has been found to occur in different types of foods depending on the source of this

substance. SEM is a metabolite of the veterinary medicine nitrofurazone but since the use of

nitrofurazone is illegal in the EU, SEM from this source should not be detectable in foods.

SEM arises in foods, and especially baby foods, as a result of migration from sealing gaskets

used in the metal lids of jars and bottles. In this case, the origin of the SEM is thermal

breakdown of azodicarbonamide, the blowing agent used to foam the plastic gaskets. These

first 2 potential sources, nitrofurazone use and azodicarbonamide-blown gaskets, are the best

documented with independent verification. Other sources have been suggested too. SEM has

been found in food products made using flour in which azodicarbonamide has been added as a

dough-improver; a practice that is not permitted in the EU. SEM is also reportedly formed as a

reaction product of the action of hypochlorite on food additives such as carrageenan and onfoods such as egg white powder. Finally, SEM may be present at background levels naturally,

made be formed at low levels when some foods are dried, and may also derive from as yet

unidentified sources.

The method of analysis used for SEM in foods

The method of analysis used to test foods for SEM involves acid hydrolysis and a

derivatisation step with 2-nitrobenzaldehyde (2-NBA). The SEM-NBA derivative is then

determined using LC-MS/MS (liquid chromatography coupled to tandem mass spectrometry).The detection limit of the method is in the region of 0.2 g/kg. This approach is used

worldwide and it was developed for the testing of animal products using SEM as a marker for

illegal nitrofurazone use. The performance of the method meets the requirements of

Commission Decision 2002/657/EC, Article 4, on minimum performance criteria for

quantitative analytical methods for toxicants in foods. In animals treated with nitrofurazone,

the veterinary drug is metabolised rapidly to SEM which becomes covalently tissue-bound.

The acid hydrolysis step is used to liberate the bound residue for analysis. The analysis

method therefore measures total SEM (free and bound) in the sample. There is evidence

that when SEM originates from the other sources described above, a fraction may also become

bound to the matrix. However, the acid hydrolysis conditions used in the analytical method are

not dissimilar to normal gastric conditions. Tissue-bound residues of SEM are reported to bebioavailable in the digestive systems of mammalian species (Hoogenboom et al. 1992;

Gottschall & Wang 1995; McCracken et al. 1997). Therefore, in this evaluation there is no

distinction made between free or bound SEM and the total result from the 2-NBA-LC-MS/MS

method is used. Similarly, given that a number of chemical source of SEM have been

identified and that there may still be as-yet unidentified sources and mechanisms of formation,

it is possible that the acid hydrolysis step used in the test method may form low levels of SEM

from labile precursors. Again, while the current state of knowledge is incomplete, in this

evaluation there is no distinction made between SEM that is present as such in a food sample

and any SEM that may have been formed from precursors in the food under acidic conditions

used in the analysis. It is concluded that the method of testing for SEM provides concentration

data that are suitable for this risk evaluation.


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Estimates of exposure

On the basis of the information available, migration of SEM from gaskets is by far the largest

source of exposure known. The concentration data supplied by for different countries weresimilar. Taking a conservative scenario for a 9-month old infant with a body weight of 8.8 kg

and eating exclusively food and drink from glass jars and bottles contaminated with SEM at an

average concentration of 13 g/kg, the intake of SEM would be 0.35 g/kg bw/day for an

average (consumers only) consumption of 234 gram per day and the intake would be 0.69

g/kg bw/day for consumption at the 95thpercentile of 464 gram per day. For an infant with a

body weight of 4.5 kg and fed exclusively each day with 700 mL of infant milk pre-packaged

in glass bottles with metal lids, containing SEM at an average concentration of 9 g/kg, the

intake would be 1.4 g/kg bw/day.

Adult exposures to SEM are likely to be much lower than infant exposures, due to the lower

contribution of foods packaged in bottles and jars to the total diet of adults, the lower

contamination levels derived from the smaller gasket areas involved for that packaging, and the

higher adult body weight. Taking an assumption that 1 kg of food contaminated with SEM at

an average concentration of 1.0 g/kg is consumed each day, the exposure for a 60 kg

bodyweight adult would be 0.02 g/kg bw/day. Commission Directive 2004/1/EC prohibits

the use of ADC in food contact materials from 2ndAugust 2005 and so, once existing stocks of

packaged foods are used up, exposure of consumers by this route will have been eliminated.

The Panel was informed that industry is making significant progress on the development of

new seal technology and expects to be able to meet the date of August 2005 for the ban on the

use of azodicarbonamide in food contact materials.

Other possible sources of SEM in foods contribute far less to exposure than estimated above

for packaging. Bread made using flour treated with azodicarbonamide can contain SEM. In

laboratory tests the SEM concentration in bread was 28 g/kg. ADC is not permitted as a flour

treatment agent in the EU and the importation of bread and bakery ware is likely to be very

low. There is the potential for exposure from breaded animal products imported into the EU

(e.g. frozen breaded chicken or fish products). Taking the upper figure of 5 g/kg of product

from the study by Pereira et al (2004) this would give an intake of SEM of 1 g/person from a

consumption of 200g of product.

For a high consumer of egg products that may be contaminated by 50 g/kg SEM as a result ofusing hypochlorite as a sanitising solution on production equipment, a reasonable worst-case

estimate of exposure is 0.008 g/kg bw/day. For carrageenan food additive that may likewise

become contaminated with SEM, if consumption was up to the full ADI and if all of this food

additive contained SEM at 65 g/kg then the intake of SEM from this source could be up to

0.005 g/kg bw/day. The Panel notes that SEM may be formed at low levels when some foods

are dried. SEM, may also derive from as yet unidentified sources, and SEM may be present at

very low background levels naturally.


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Toxicity

The only new toxicological information that has become available since EFSA issued its

preliminary advice on SEM in July and October 2003 concerns genotoxicity. There are no new

data on carcinogenicity, reproductive and developmental toxicity, or other aspects of repeat-dose toxicity and the available data on all these aspects are not as comprehensive as the Panel

would wish. While these data deficiencies add some uncertainties to the risk assessment, given

the generally low exposures to SEM via food, it is the aspects of genotoxicity and

carcinogenicity which remain the focus of this assessment.

SEM has been shown to be carcinogenic in mice, but not rats. Literature data on genotoxicity

together with the results of recent studies with pure SEM hydrochloride, indicate that SEM is

mutagenic but not clastogenic in some test systems in vitro, notably in the absence of an

exogenous metabolising system. In vivo, negative results were reported in adequately

performed studies on DNA damage in liver and lung of mice, and in the micronucleus assay in

the mouse. Based on the overall weight of evidence provided by the studies performed, whichincluded a study using a highly sensitive methodology, it is concluded that the weak

genotoxicity exerted by SEM in vitrois not expressed in vivo.

The new data allaying the concern on genotoxicity in vivo, and the likely reductions in

exposure following replacement of the most significant, currently known source of SEM in the

diet (gaskets on glass jars and bottles), offer further support to the preliminary advice given by

EFSA in 2003 that the risk, if any, from consumption of foods containing SEM is judged to be

very small, not only for adult consumers but also for infants. In this respect the Panel noted

that SEM is a weak non-genotoxic carcinogen for which a threshold mechanism can be

assumed. A large margin of at least 5 orders of magnitude exists between the dose causing

tumours in experimental animals and human exposure, including that of infants.

The Panel therefore concludes that the issue of carcinogenicity is not of concern for human

health at the concentrations of SEM encountered in food.


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INFORMATION PROVIDED TO EFSA

From Industry

Communication Possible contaminant in gaskets of metal lids from industry to the European

Food Safety Authority, dated 25 June 2003 and. Communicated to EFSA on 8 July 2003.Semicarbazide in food, submitted November 2003.

In vivo mouse liver unscheduled DNA synthesis assay, Danone

In vitro chromosome aberration test with Chinese Hamster V79 cell, submitted by Industry.

Formation of semicarbazide in seaweeds used as food additives. Submitted by Marinalg.

From Member States:

Belgium: Semicarbazide in powdered eggs

Finland: Semicarbazide in baby food and some other items

France: Semicarbazide in baby food

Germany: Semicarbazide in baby food

Ireland: Semicarbazide in baby foodNetherlands Semicarbazide in baby food

Norway: Semicarbazide in carrageenan

Spain: Semicarbazide in baby food

REFERENCES

Abramsson-Zetterberg,L., and Svensson, K. (2005) Semicarbazide is not genotoxic in the flow

cytometry-based micronucleus assay in vivo. Toxicol. Lett. 155: 211-217

Bhattacharya, A.K. (1976) Chromosome damage induced by semicarbazide in spermatocytes

of a grasshopper. Mutat. Res. 40: 237-242

Becalski, A., Lau, B. P-Y., Lewis, D. & Seaman, S. W. (2004) Semicarbazide formation in

azodicarbonamide-treated flour: a model study. J. Ag. Fd. Chem., 52, 6730-5734.

Bernard, A. (2003). Nitrofurazone residues in Belova egg products. Evaluation of the health

risks. Statement of April 5 2003.

Central Toxicology Laboratory (2004) Semicarbazide: in vivo mouse liver unscheduled DNA

synthesis assay. CTL study n. SM1207

Cheeseman, M.A., Machuga, E.J. and Bailey, A.B. (1999). A tiered approach to threshold of

Regulation. Fd Chem. Toxicol., 37, 387-412.

De Flora S. (1981) Study of 106 o

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EFSA journal 219