Antimicrobial food additives characteristics, uses, effects

Preface to the second English Edition Since the publication of the first English translation of this book about 15 years ago, a great deal of new information on antimicrobial food additi

Antimicrobial Food Additives

Springer-Verlag Berlin Heidelberg GmbH

Erich L iick Martin J ager

Antimicrobial

Food Additives

Characteristics U ses Effects

2nd revised and enlarged edition

translated from the German by S F Laichena

Dr phil nat Erich Liick

Title of the German Edition:

E Ltick, M Jager: Chemische Lebensmittelkonservierung, 3 Auflage, 1995

ISBN 978-3-642-63896-1

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Liick, Erich:

Antimicrobial food additives : characteristics, uses, effects /

Erich Liick ; Martin Jager Trans! from the German by S F

Laichena - 2., rev and en! ed - Berlin; Heidelberg ; New

York; Barcelona ; Budapest ; Hong Kong ; London ; Milan ;

Paris; Santa Clara; Singapore; Tokyo: Springer, 1997

Einheitssacht.: Chemische Lebensmittelkonservierung <eng!.>

ISBN 978-3-642-63896-1 ISBN 978-3-642-59202-7 (eBook)

DOI 10.1007/978-3-642-59202-7

NE: Jager, Martin:

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Originally published by Springer·Veriag Berlin Heidelberg New York in 1997

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For Ulrike, Lena and Jonas

Preface to the second English Edition

Since the publication of the first English translation of this book about 15 years ago,

a great deal of new information on antimicrobial food additives has emerged in the fields of microbiology, toxicology and analysis These aspects have been given pro-minence in this edition

Despite a widespread and in most cases emotive aversion to food additives in general and preservatives in particular, the commercial use of preservatives is in-creasing rather than declining There are many reasons for this: the growing popularity of convenience foods is certainly as important as changes in consumer awareness, against a background of continuous rises in the incidence of food infection and food poisoning

The structure of the book remains unchanged Like the previous edition it sists of two sections The first section contains information applicable to preser-vatives in general The second section describes the properties and applications

con-of the various preservatives The chapters are grouped into those on inorganic and organic preservatives respectively

Each chapter has been thoroughly revised and updated Preservatives that have become less important and those of little commercial relevance have been sum-marized fairly briefly in a chapter on "Other preservatives" This edition still focuses primarily on the commercial use of preservatives in the food sector The book is aimed at those involved on the practical side in the food industry who have

an interest in the scientific aspects of their work Information on the regulatory status has deliberately been confined to the main points since a detailed and up-to-date description cannot be given against a background of constantly changing regulations

Foreword to the first English Edition

Although the preservation of food by chemical techniques is such an important field of research in food science and also a major branch of the food industry, no monograph on the subject has hitherto existed in modern literature Knowl-edge concerning this field is widely dispersed in a multiplicity of journals and handbooks The foremost works of reference on food microbiology and food technology treat the preservation of food by chemical means only as a peripheral aspect

This book first appeared as a German-language publication in the Federal

reviews all aspects of food preservation by chemical techniques, the majority

of which involve the use of chemical additives This, the first English-language edition, is more than a straight translation from its Geman predecessor since the text has been updated in the light of new knowlegde aquired in the interim Essentially, this book is a collection of facts augmented by information drawn

con-sists of two sections:

The sequence of chapters in the second edition of the book follows the usual system employed in inorganic and organic chemistry A separate chapter is devoted to each substance that is, or used to be, of major practical importance Preservatives which have attained a minor degree of importance at some time are then summarized in

a further single chapter This book also deals with those disinfectants and gases which may still be regarded as preservatives in the broader sense of the term be-

itself strictly to food preservation; only brief reference is made to food analysis since good books on the analysis of preservatives in foods already exist

The book is aimed mainly at the practical man in the food industry with an interest in the scientific aspects of his work The scientific principles of food pre-servatives are all explained in sufficient detail for a clear understanding of the way

chap-ters dealing with the individual preservatives a deliberate attempt has been made

to provide systematic description enabling the student or other reader to obain a rapid overall picture Finally, in deciding on the book's layout and scope it has been born in mind that a book of this type can also serve as a source of information for government authorities, medical practitioners, nutritional scientists and, not least,

emotio-X Foreword to the first English Edition

nally charged discussion on the use of preservatives in foods and return such cussion to a scientific level

dis-The author would like to record his sincere gratitude to Grant F Edwards, Manager of the Translation Department of Hoechst UK, for his careful and con-scientious translation work Thanks are also due to the publishers, notably

Dr Boschke, for their critical comments, of which due account has been taken in both the German and English editions, as well as for their promptitude in com-pleting the task of publication

Table of Contents

Genera/Considerations

XII Table of Contents

Antimicrobial Action of Preservatives

General Mechanisms of Action

Inhibitory and Destructive Actions

Action on Microorganisms

Antimicrobial Spectrum of Preservatives

Acquired Resistance to Preservatives ,

Combinations of Preservatives with One Another

Broader Spectrum of Action

Changes in Antimicrobial Action

Combination of Preservatives with Physical Measures

Combination of Preservatives with the Use of Heat

Combination of Preservatives with Refrigeration Treatment

Combination of Preservatives with Irradiation

Preservation Against Toxin-Forming Microorganisms

Influences of Substrate Factors on the Action of Preservatives

Influence of the pH Value

Influence of the Partition Coefficient

Influence of the Water Activity

Influence of Other Physico-Chemical Substrate Factors

Influence of Food Ingredients

Decomposition of Preservatives

Test Methods for Preservatives

Nutrient Medium Test

Table of Contents XIII

XIV Table of Contents

XVI Table of Contents

Table of Contents XVII

XVIII Table of Contents

Table of Contents XIX

Table of Contents XXI

XXII Table of Contents

XXIV Table of Contents

31.2 Lime Water and Waterglass Solution

31.3 Mineral Oils and Fatty Oils

31.4 Waxes and Plastic Coatings

31.5 Antimicrobial Packagings and Coverings

Genera/Considerations

The Individual Preservatives

Aim and Development of Food Preservation

All foods are prone to spoilage and must therefore be consumed immediately or, where this is not possible or desirable, preserved so that they can be stored

1.1

Food Spoilage: Definition and Controlling Factors

The quality of food can be adversely affected by physical, chemical, biochemical and microbiological processes This review, however, will confine itself to food spoilage caused by microorganisms and to the substances that reduce the rate of spoilage or actually prevent it altogether

The biological processes involved in food spoilage due to microorganisms not take place unless certain extrinsic conditions are met:

can-1) The presence of spoilage microorganisms:

Spoilage cannot occur unless microorganisms are (still) present on or in the food

2) Nutrients for the microorganisms need to be bioavailable:

Unless the microorganisms have nutrients available which they can utilize, their life cycle cannot commence

3) Conditions favorable to life must exist in respect of:

4) Sufficiently long food storage time:

If the food is consumed before any undesired growth of microorganisms occurs, measures against intrinsically possible microbiological spoilage become super-fluous

5) The vital activity of the microorganisms must cause a deterioration in the lity of the food:

qua-Unless microorganisms on the food become recognizable and/or excrete ful or otherwise undesirable metabolic products, the term "spoilage" is inap-posite

harm-Not every microbiological change in a foodstuff is regarded as spoilage of the food The expression "food spoilage" is confined to what, by convention and in normal par-lance, would be termed undesirable Hence, the intentional fermentation of grape

E Lück et al., Antimicrobial Food Additives

© Springer-Verlag Berlin Heidelberg New York 1997

4 1 Aim and Development of Food Preservation

juice by yeast with a view to producing wine is not food spoilage, although the desired fermentation of the self-same grape juice would indeed class as spoilage if the intention were to retain the grape juice as such Vinegar results from the spoilage

un-of wine, hence the French word 'vinaigre' (vinegar); the intentional production un-of vinegar from wine by the same vinegar bacteria is not classed as spoilage, however Occasionally, the scale of the reaction and the course it takes is the determining fac-tor as to whether a microbiological change in the food is termed spoilage or not This principle is exemplified by the microbiological process occurring in the ripening of cheese Depending on its extent, the microbiological process is termed ripening (de-sired) or spoilage (undesired) The transition from one to the other is gradual, and sometimes the point at which spoilage begins is not clear-cut Since most countries operate a ban on the marketing of spoilt food, this uncertainty may also have legal consequences Without elaborating the point, it should nevertheless be mentioned

at this juncture that certain microorganism strains are an essential requirement for the production of certain foods, e g bread, yoghurt and wine

1.2

Definition of and Processes for Food Preservation

Food preservation in the broad sense of the term refers to all measures taken against any spoilage of food In its narrower sense, however, food preservation con-notes the processes directed against food spoilage due to microbial action Since ancient times two fundamentally different types of method have been used, na-mely physical and chemical methods In recent times biological methods of food preservation (see Sect 30.25 and 30.26) have become increasingly important The microorganisms need not necessarily be killed It is much more important

to produce conditions that merely prevent the microorganisms from causing food spoilage

Physical preservation methods, which will not be described in this book, are notable in that the food is subjected to a physical measure which counters micro-bial growth The best-known physical methods are those of sterilization and pasteurization (heat treatment), cooling and freezing (refrigeration), drying (de-hydration), and irradiation

The chemical methods are characterized by the addition of a chemically more

or less readily definable substance that inhibits the development of nisms or kills them These substances are known as preservatives A distinction is drawn between preservatives in the broader sense of the term and those in the narrower sense Examples ofthe former group are common salt and vinegar, whilst examples of the latter are sorbic acid and sulfur dioxide The essential difference between the groups is in their applied concentration The preservatives in the broader sense are used in concentrations upwards of 0.5 - 1 %, whereas those in the narrower sense can be employed in concentrations of 0.5 % or less because their antimicrobial action is more powerful

microorga-In the biological methods, high purity, harmless microorganism cultures, which have an inhibitory effect on undesirable spoilage microorganisms, are added to the foods These are known as "protective cultures"

1.4 History of Chemical Food Preservation 5

1.3

The Need for Food Preservation

Cereals, fruit and other foods of plant origin are available fresh only during a brief harvest period Consequently, since ancient times it has been necessary to keep supplies of harvested food edible over relatively long spells Rainy periods and droughts, which have occurred everywhere in the world and continue to do so, have likewise necessitated proper preservation of foods One of the earliest references

is in the Bible (Genesis, chapter 41, verses 34-36), where Pharaoh commanded Joseph to store one-fifth of the harvest in granaries during the seven fat years in order to provide supplies for the seven lean years This was the first recorded in-stance of food preservation by means of protective gas, since the cereal was pro-tected from spoilage as a result of the carbon dioxide it produced by respiration

In these instances the need for food preservation was engendered entirely by natural factors, but later the factors of civilization were also involved Man is living increasingly in towns and cities, where it is no longer possible to plant or harvest one's own food on any large scale In all the industrialized countries, a declining number of people are responsible for obtaining and producing food for a growing number of other people Such a development is possible only if the foods have adequate keeping power

Recently, moreover, there has been a change of living habits and requirements

in many countries, inasmuch as people now wish to enjoy foods and specialties from distant lands Many foods whose keeping properties are subject to highly exacting requirements have become branded articles These are products that can

be made available only by a suitable preservation technique specific to the product

Whereas food used to be preserved for commercial reasons alone, toxicological findings have recently become additional reason for preservation Since the nine-teen-sixties it has been known that some molds form afiatoxins and other myco-toxins, and that these molds can enter foods If the growth of molds is inhibited, for example by the use of preservatives, toxin formation can be reduced (Luck 1981)

As a form of preventive medicine therefore, the use of a toxicologically harmless preservative sometimes poses a smaller risk than failure to use one

1.4

History of Chemical Food Preservation

At the time when man was still a gatherer and hunter who lived, in the truest sense

of the term "from hand to mouth", he had no need of food preservation It was unnecessary for food to stay fresh for any lengthy period since nature constantly

6 1 Aim and Development of Food Preservation

provided fresh supplies in adequate quantities Not until the neolithic revolution some ten thousand years ago, in the New Stone Age, did man begin to adopt a settled life-style Then, instead of gathering and hunting, he took to tilling the soil and keeping animals This forced him increasingly to lay in stores Owing to his lack of specialist knowledge, man confined himself in those early days to simply stockpiling his food This he collected much as a squirrel collects its nuts; then pro-tected it against theft by his fellow-men and the vagaries of wind and weather In doing this he was forced to sacrifice much of the food's nutritive and organoleptic quality

Initially, the preservation technique involve drying and salting The diet was governed accordingly From careful reading of old accounts about the food of sea-farers or the winter diet of country-folk, who were partially or completely dependent

on preserved foods, at least in the temperate zones of the world, it will be found that the diet was dominated on the one hand by cereals and flour and on the other hand by dried, salted or pickled meat or salted/unsalted dry fish Thus a fourteenth-century Parisian merchant advised his customers to soak a twelve-year old stock-fish overnight and then beat it with a blacksmith's hammer until it was tender In

a number of countries it was normal practice to bake bread only two or three times

a year, after which it would be dried out and eaten in the ensuing months in the form of a softened mush Obviously, such a diet was monotonous and ailments due

to deficiency diseases were not uncommon, especially since little or nothing was known about the influences of the preservation methods on the constituents of the foods In time, the list of preservatives used grew to include alcohol, smoke, sulfur dioxide and a number of organic acids, such as acetic and lactic acid; then, for the next two thousand years these constituted the total range available

Food preservation changed with the commencement of industrialization The need for food preservation increased rapidly, and people became more fastidious

No longer were they satisfied with the preservatives mentioned above, since these produced a radical alteration in the structure and properties of the foods they preserved

The advances made in chemistry were also utilized in preservation techniques Thought began to be given to the principles underlying the preservatives employ-

ed up to that time In his investigations into smoke, K Reichenbach discovered, in pyroligneous acid and the tar of beechwood, an oil that he named creosote owing

to its property of preserving meat Reichenbach wrote in glowing terms of its serving action, although already at that time he stated that the substance involved health risks (Strahlmann 1974) Creosote was probably used on only a limited scale for sensory reasons; yet it is the only preservative to be described in any detail in

pre-a well-known book on food chemistry dpre-ating from 1848, apart from salt (whose use was correctly described as indirect drying), drying, heat treatment, lactic acid fermentation, sugars, alcohol, vinegar and smoke (Knapp 1848)

For rather more than a century now, increased efforts have been made to serve foods not simply by any feasible means but to do so in a way that leaves their frequently sensitive constituents unimpaired, as well as safeguarding their nutrient value and flavor against unfavourable influences In the first flush of excitement at the discovery of substances with an antiseptic action for medicinal purposes, "che-micals" such as hydrofluoric acid, fluorides, chlorates and the like were used in

pre-1.4 History of Chemical Food Preservation 7

Table 1 Historical development of chemical food preservation (Strahlmann 1974) Prehistoric times

Borax recommended by HOfer

Sulfur dioxide reconunended for preserving meat Creosote recommended for preserving meat by Reichenbach Antimicrobial action of boric acid discovered by Jaques Sorbic acid isolated from rowan berry oil by Hofmann Antimicrobial action of formic acid discovered by Jodin Antimicrobial action of salicylic acid discovered by Kolbe and Thiersch

Antimicrobial action of benzoic acid discovered by Fleck

Formaldehyde and hydrogen peroxide recommended for milk vation by von Behring

preser-Benzoic acid permitted for use in foods in the USA Antimicrobial action of p-chlorobenzoic acid discovered by Margolius Antimicrobial action of p-hydroxybenzoic acid esters discovered by Sabalitschka

Propionic acid recommended for the preservation of baked goods by

Hoffman, Dalby and Schweitzer

Antimicrobial action of sorbic acid discovered by Miiller and, pendently of this, by Gooding in 1940

inde-Antimicrobial action of dehydroacetic acid discovered by Coleman and

Wolf Worldwide revision of approvals for new preservatives Industrial-scale production of sorbic acid conunences

Antimicrobial action of diethyl pyrocarbonate discovered by

Bern-hard, Thoma and Genth Increasing use of protective gases

food preservation In no sense was this done unscrupulously as a means of

profi-teering or with intent to deceive; it was merely due to ignorance of the products' potential harm, since toxicological investigations were unknown People simply believed that the small quantities of a substance just sufficient to act as a preser-vative could scarcely be harmful; so at first they were undiscriminating in their choice of substances Hence, the introduction of salicylic acid and boric acid to food preservation about a hundred years ago should be regarded as an advance, although nowadays both these preservatives are considered outmoded At the end

of the second half of the nineteenth century, formic acid joined the preservatives The beginning of the twentieth century saw the first use of benzoic acid, which is still widely used in food preservation As an aromatic compound benzoic acid, like salicylic acid, was regarded unfavorably from the outset Effective derivatives were therefore sought and, as a result, p-chlorobenzoic acid and the esters of p-hy-

droxybenzoic acid were discovered This was followed in the late nineteen-thirties

by the salts of propionic acid, and in the post-war years by sorbic acid and its salts The introduction of sorbic acid is largely the result of the worldwide toxicological re-evaluation, which commenced around 1950, of food ingredients in general and

8 1 Aim and Development of Food Preservation

preservatives in particular As an unsaturated fatty acid, sorbic acid is the most thoroughly researched and harmless of all preservatives in widespread use Over the last 15 - 20 years the trend towards consuming fresh foods has grown substantially Logistical measures implemented by the food industry have pro-gressively shortened the transportation times between production sites and the consumer Refrigeration techniques, also employed during the transport of food, have shown vast improvements in the industrialized nations

Another current trend is the increasing discussion concerning "harmless" additives for food preservation purposes, i e the use of antimicrobial substances produced from plants and microorganisms Many laymen regard such products as less suspicious than others merely because of their natural origin

It is worth noting that in the many articles attacking the use of additives it is the preservatives that receive the least criticism, on the whole This is because even cri-tics realize that in certain cases preservatives do indeed protect consumers from harmful effects on their health In future, therefore, antimicrobial food additives will retain their importance in certain areas

1.S

Literature

Knapp Fe (1848) Die Nahrungsmittel in ihren chemischen und technischen Beziehungen weg, Braunschweig, p 101-109

Vie-Luck E (1981) SchutzmaBnahmen gegen Lebensmittelverderb durch Schimmelpilze In ReiB J:

Mykotoxine in Lebensmitteln Gustav Fischer, Stuttgart - New York, p 437 - 457

Strahlmann B (1974) Entdeckungsgeschichte antimikrobieller Konservierungsstoffe fUr bensmittel Mitt Geb Lebensmittelunters Hyg 65, 96 -130

sub-or not Fsub-or this purpose the foodstuff to be investigated, possibly after ate dilution, is inoculated with defined strains of microorganism, on which the preservative is known to have an antimicrobial action A study is then made over

appropri-a pappropri-articulappropri-ar period of time to appropri-ascertappropri-ain whether the microorgappropri-anisms multiply Many microorganisms, especially yeasts, release carbon dioxide, the evolution of which can be measured Known as the "fermentation test", this method used to be

of some importance, but is rarely used today because it is not sufficiently specific Defined strains of microorganism can be obtained from the "DSM" (German microorganism collection) in Braunschweig, Germany or the ''American Type Culture Collection" (ATCC, 12301 Parklawn Drive, Rockville, MD 20852, USA) Wet assay and instrumental methods of determination for virtually all food preservatives have been described in the literature The principles of each of these methods are described in the chapters on the individual substances themselves The purpose of these descriptions is to present the range of methods available for detecting each preservative, examining the advantages and drawbacks of each method This should enable the analytical chemist to select the appropriate method However, full experimental details are not given; these can be found in the literature references

2.2

Quantitative Determination

Foodstuffs have an extremely complex composition and consist of many dual constituents which may interfere with chemical detection of preservatives For this reason and sometimes also in order to increase their concentration, the pre-servatives to be determined nearly always have to be isolated from the food as a first step To do this it is necessary to use solid-liquid or liquid-liquid extraction,

indivi-E Lück et al., Antimicrobial Food Additives

© Springer-Verlag Berlin Heidelberg New York 1997

10 2 Analysis of Preservatives

steam distillation or special methods specific to individual cases Possibly after ther purification, the extract can then be made available for actual analysis This method is generally known as sample preparation

fur-Quantitative determination of the preservatives can be successfully carried out with gas, thin-layer or liquid chromatography, wet chemical processes, colorimetry, photometry and other methods, depending on the substances to be tested In view

of the increasingly widespread use of high-performance liquid chromatography (HPLC), the focus will be on this method in the analytical section of each chapter describing an individual substance and on the relevant detection techniques

2.3

Purity Requirements

Only preservatives of special and standardized purity can be used as food tives Hence, in most modern regulations on the acceptance of preservatives, there are increasing numbers of special purity requirements for the individual substances (see 2.4 General literature) In general they are concerned chiefly with the content of toxicologically relevant heavy metals and specific impurities deriv-ing from the process of synthesis

Horowitz E (1984) Official methods of analysis of the Association of Official Analytical mists 14th edition Association of Official Analytical Chemists, Washington

Che-King RD (1978) Developments in food analysis techniques Applied Science Publishers, don

Lon-Kommission des Bundesgesundheitsamtes zur Durchfiihrung des § 35 LMBG (Publisher) Amtliche Sammlung von Untersuchungsverfahren nach § 35 LMBG, Beuth, Berlin, loose- leaf collection (1980 onwards)

Glandorf KK, Kuhnert P, Luck E (1991) Handbuch Lebensmittelzusatzstoffe, Behr, Hamburg, chapter CV, loose-leaf collection (1990 onwards)

National Research Council (US) Food and Nutrition Board (1981), Food Chemicals Codex: (FCC), 3rd edition National Academy Press inc supplements

Verordnung uber das Inverkehrbringen von Zusatzstoffen und einzelnen wie Zusatzstoffe wendeten Stoffe (Zusatzstoff-Verkehrsverordnung) of July 10, 1984

of vegetables or cyanogenic glycosides in cassava Alternatively such substances may either form in plants themselves as a reaction to exogenous noxae (so-called phytoalexins such as solanine or chaconin in potatoes) or, after microbial attack, may help contaminate food with mycotoxins or other toxins In view of the high toxicological potential of most mycotoxins, this particular type of contamination

is a problem that cannot be ignored (Goto 1990, Jelinek et al.19B9) Preservatives, when used appropriately and correctly, can prevent the formation of mycotoxins

in many foods and, as a result, greatly improve food safety

Auxiliaries and additives, including preservatives, used to be employed in foods without prior testing The absence of adverse effects on the health after the con-sumption of food treated with preservatives was accepted as adequate proof that they were harmless This meant that people were "testing" the substances added

to the food as well as the food itself

Deriving from that time, when "chemicals" were used in food preservation out safeguards, there is still a discernible but diminishing aversion in some circles

with-to food preservation generally, even with-today Since the beginning of this century and especially since the nineteen-fifties, the situation has changed radically with the development of toxicology as a specialist discipline Auxiliaries and additives are now permitted and employed for food purposes only if the toxicological tests con-ducted upon them in accordance with the level of scientific knowledge at the time give no reason for believing they are in any way harmful, or even that they might

be so In this connection it should be pointed out that in the case of additives the legislators refer to the "principle that they are prohibited unless expressly per-mitted" This means that all additives are prohibited and only those expressly ap-proved (in terms of permissible maximum quantity and field of use) may actually

be used Today the harmlessness to health of auxiliaries and additives for foods is better researched than that of some foods and food ingredients It is interesting to note that the natural occurrence of toxic ingredients in foods arouses much less public attention than the presence of food additives

In tests to determine the toxicological properties of auxiliaries and additives, the assumption is made that a dose-effect relationship exists (a mathematically quantifiable relationship between dosage, duration of effect and the extent of the effect) as well as the fact that there is a limit below which the substance has no ef-

E Lück et al., Antimicrobial Food Additives

© Springer-Verlag Berlin Heidelberg New York 1997

un-As long ago as 1538, in the third of his seven "Carinthian Defences" (Epistola dicorata St Veit), Paracelsus formulated the principle that substances must be able

de-to exist in doses de-too small de-to produce an acute effect To quote Paracelsus himself (in translation):

'Wl)at tJ:iftttl), but tl)at it bt poifonfomt1 1Ul tl)ingf bt poifonfomt

anb nont tl)trt bt witl)out poifon

Uougl)t but tl)t boft mafttl)

tl)at poifonf alfo bt poifonfomt

Paracelsus did not imply by this that a substance becomes a poison only above a certain dosage but that a poison ceases to have an acutely toxic effect below a cer-tain dosage

The scope of the required tests and the way in which they are conducted depends

on the use the substance being tested is specified for or expected to perform Nowadays animal experiments are preceded by a number of tests known as in-vitro short-time assays These are used to assess parameters such as genotoxicity and mutagenicity (e.g micronucleus assay, HGPRT test, Ames mutagenicity test, see Sects 3.4 and 3.5) Aspects such as "irritant effect" or "penetration of the skin" can also be assessed by in-vitro test systems that are already available (e g HET -CAM

"chorionallantois" test), as can acute cytotoxicity (e g neutral red uptake bition test) Even if every known in-vitro test is used in assessing a substance, this cannot entirely obviate the need for animal experiments Preliminary in-vitro testing may simply reduce the number of animal experiments (Spielmann 1989) It

inhi-is also necessary in each individual case to clarify whether such in-vitro results can

be applied firstly to animals and secondly to man and whether they can be

validat-ed ("What is the significance of a positive result in an in-vitro assay for man and how far are the results comparable?")

For obvious reasons, the basic knowledge required for a toxicological ment of a substance is derived from the results of animal experiments Most of the animals involved are small, short -lived creatures such as mice and rats, and, de-pending on the parameters being studied, other rodents, dogs, monkeys and, in special instances, other animals, too The animals used are always bred specially

assess-3.1 General Considerations 13

for the purpose and kept under defined conditions prior to the experiment For studies of certain aspects that are more pharmacological in nature, genetic engineering is used to breed experimental animal species which serve as excellent animal models

In advanced stages of trials, as in the case of pharmaceuticals, the biochemical behavior and metabolic reactions in man are investigated in human volunteers un-der medical supervision This greatly reduces the risk involved in transferring ex-perience gained in animal experiments to man

The following criteria are nowadays regarded as especially important for sing the harmlessness of an auxiliary or additive:

asses-1) Acute toxicity,

2) Metabolic investigations and toxicokinetics,

3) Genotoxicity/mutagenicity,

4) Reproductive toxicity including fertility toxicity and teratogenicity,

5) Sub chronic toxicity,

6) Chronic toxicity,

7) Carcinogenicity

This list of criteria forms the basis for the flow chart in Fig 1 ments are combined, e g chronic toxicity experiments and carcinogenicity tests During metabolic experiments, tests are carried out to discover whether the sub-stance being studied accumulates in the organism It is also necessary to check whether the biotransformation (absorption, distribution, metabolism and elimin-ation) of a test substance proceeds along similar lines

Frequentlyexperi-The present -day practice whereby all toxicological investigations are conducted

at institutes specializing in the relevant sectors ensures that suitable types of animal are used, that the animals are properly kept and fed, and that the substance under test is administered properly and in the correct dosage Appropriate test guidelines can be obtained from the OBCD, for example Finally it is vital that results should

be properly interpreted

Before the toxicological investigation of an auxiliary or additive can begin, its tity must be determined The substance being tested must be chemically and physic-ally identical to the substance actually used subsequently in practice Therefore the first step is to draw up precise specifications of the test substance Besides the usual chemical data these must state the quantity of pure substance and any impurities the test substance may contain It is generally easier to draw up specifications for syn-thetic or natural substances with a precisely defined chemical specification than to

iden-do so for natural substances with a complex composition In the case of the

precise-ly defined substances the possibility of by-products occurring can be deduced from the method of synthesis, where this is known These by-products are, however, usually removed from the substances to be tested by the purification operations normallyem-ployed in chemical technology today, such as recrystallization and distillation Al-though a residual quantity of impurities may in some cases be very important, the toxicological evaluation should not overlook the question of dosage in this context either Auxiliaries and additives are used in foods in small to minute quantities Im-purities in auxiliaries and additives in the ppm to ppb range must therefore be view-

ed differently from equivalent amounts of contaminants in foods themselves

+ unfavorable results obtained which could be relevant to the use

of the substance tested in the food sector

Fig 1 Stepwise assessment of the effect of auxiliaries and additives on health

It is regarded as fundamentally desirable for food additives not to display any pharmacological effects in the concentrations used In principle, this requirement also includes preservatives It has to be borne in mind, however, that preservatives also have an effect on pathogenic microorganisms, mainly owing to their anti-microbial action, and could thus theoretically also be used to combat them, e g fungal infections of the skin The effect of the substances used as food preservatives

in controlling pathogenic microorganisms is, however, much too weak for them to

be realistically considered for such an application To prevent the development of resistance, substances used in medical practice are not permitted for use as food additives This applies in particular to a number of antibiotics

3.2 Acute Toxicity 15

3.2

Acute Toxicity

Acute toxicity, expressed in the form of the LD50 , is merely an approximate means

of measuring the toxicological properties of a substance after single-dose administration (Zbinden and Flury-Roversi 1981) The LD50 is the dose at which

50 % of the animals in an experimental group are expected to die It is governed

by a number of external factors, e g the species of animal, the age, the weight, the sex and the conditions under which the animal is kept All these factors must

be standardized

The LD50 is stated in milligrams of test substance per kg body weight of the experimental animal The higher the value, the lower the acute toxicity of the substance

For gaseous substances an LC50 (= median lethal concentration) is given in mgtl air The LD50 and LC50 are different from the ED50 (effective dose 50%), i.e the quantity of a substance at which a particular effect is produced in 50 % of the ani-mals

To determine the LD50 , the test substance is administered in various doses to groups of animals, each comprising five males and five females For auxiliaries and additives used in foods, only peroral administration is important The amount being investigated is administered as a single dose through the esophagus If the quantity is too great because the substances are of very low toxicity, it can also be administered in several smaller portions, though on the same day The animals are observed for at least one to two weeks

First, the LD50 forms the basis for classifying a substance in comparison with similar substances of known acute toxicity This it does by indicating the dose at which effects on the animal can be expected It also indicates the profile of toxi-cological effects and thus acts as a basis for determining the range of doses used

in sub chronic and chronic feeding tests It is also possible in some cases, after autopsies on the animals, to discover which organs were particularly affected by the test substance These observations also provide guidance on planning and eva-luating subsequent toxicity investigations Finally, the acute toxicity determined yields information on expected risks to man in the event of accidents or misuse,

or after handling of the relevant substance at work

Accordingly, the LD 50 can be used to place test substances in the following poison classes:

Poison class 1 = LD50 < 5 mgtkg body weight

Poison class 2 = LD50 5 -49 mglkg body weight

Poison class 3 = LD50 50 - 499 mgtkg body weight

Poison class 4 = LD50 500-4999 mg/kgbodyweight

Poison class 5 = LD50 > 5000 mgtkg body weight

The following table shows that all substances with a preservative action (other than nitrites = poison class 3) belong in poison classes 4 and 5