1987L0153 — EN — 31.05.1995 — 002.001 — 1
This document is meant purely as a documentation tool and the institutions do not assume any liability for its contents
►B COUNCILDIRECTIVE
of16February1987
fixingguidelinesfortheassessmentofadditivesinanimalnutrition
(87/153/EEC)
(OJ L 64, 7.3.1987, p. 19)
Amended by:
Official Journal
No page date
►M1Commission Directive 94/40/EC of 22 July 1994 L 208 15 11.8.1994
►M2Commission Directive 95/11/EC of 4 May 1995 L 106 23 11.5.1995
▼B
COUNCILDIRECTIVE
of16February1987
fixingguidelinesfortheassessmentofadditivesinanimalnutrition
(87/153/EEC)
THE COUNCIL OF THE EUROPEAN COMMUNITIES,
Having regard to the Treaty establishing the European Economic
Community,
Having regard to Council Directive 70/524/EEC of 23 November 1970
concerning additives in feedingstuffs(1), as last amended by Commis-
sion Directive 86/525/EEC(2), and in particular Article 9 thereof,
Having regard to the proposal from the Commission,
Whereas Directive 70/524/EEC provides that the examination of addi-
tives must be performed on the basis of a dossier forwarded officially
to the Member States and to the Commission;
Whereas such dossiers must make it possible to verify that additives
comply, in respect of their proposed use, with the general principles
laid down in the Directive for their inclusion in the Annexes thereto;
Whereas it has been found necessary to provide for the dossiers to be
compiled in accordance withcommon guidelines defining th e scientificdata which make it possible to identify and characterize the productsconcerned and the studies necessary in order to evaluate, in particular,their efficacy and their safety for man, animals and the environment;
Whereas the guidelines are intended primarily as a general guide;
whereas, depending on the nature of the additive or its conditions ofuse, the extent of the studies necessary in order to evaluate its proper-ties or its effects may vary;
Whereas it is indispensable to apply the principles of good laboratory
practice when developing additives intended for use in feedingstuffs to
ensure that the results of laboratory tests are not disputed; whereas
recourse to procedures involving the use of laboratory animals forexperimental or other scientific purposes should be kept to a minimum;
Whereas the guidelines have been drawn up on the basis of present
scientific and technical knowledge and they may be adapted if neces-sary to any developments in this sphere,
HAS ADOPTED THIS DIRECTIVE:
Article 1
Member States shall prescribe that the dossiers which must accompany
every request for the inclusion of an additive or a new use of an addi-tive in the Annexes to Directive 70/524/EEC are to be compiled in
accordance with the guidelines set out in the Annex to this Directive.
Article 2
This Directive shall apply without prejudice to provisions on:
(a) good laboratory practice for the purposes of mutual acceptance of
data for the evaluation of chemical products; and
(b) the protection of animals used for experimental or other scientific
purposes.1987L0153 — EN — 31.05.1995 — 002.001 — 2
(1) OJ No L 270, 14. 12. 1970, p. 1.
(2) OJ No L 310, 5. 11. 1986, p. 19.
▼B
Article 3
Member States shall bring into force the laws, regulations or adminis-
trative provisions necessary in order to comply withth is Directive by
31 December 1987 at the latest. They shall forthwith inform the
Commission thereof.
Article 4
This Directive is addressed to the Member States.1987L0153 — EN — 31.05.1995 — 002.001 — 3
▼M1
ANNEX
GUIDELINES FOR THE ASSESSMENT OF ADDITIVES IN FEEDING-
STUFFS
GENERAL ASPECTS
These guidelines are intended as a guide for establishing dossiers on substances
and preparations being submitted for authorization as additives in feedingstuffs.
These dossiers must enable an assessment to be made of the additives based on
the present state of knowledge and make it possible to ensure their compliance
with the fundamental principles laid down for their admission, which are the
subject of the provisions of Article 7 (2) of Directive 70/524/EEC.
All the studies outlined in these guidelines may be required and, if necessary,
additional information will be requested. As a general rule, studies to establish
the identity, conditions of use, physico-chemical properties, methods of determi-
nation and efficacy of the additive, and also its metabolism, biological and
toxicological effects on target species must be provided. When the additive is
intended for a category of animals belonging to a defined species, these studies
must be performed on this target category. The studies necessary for the evalua-
tion of risks to human health or the environment will depend essentially on the
nature of the additive and the circumstances of its use. In this respect, no strict
rule is applicable.
It may not always be necessary to subject additives intended exclusively for pet
food to as exhaustive a program of chronic toxicity, mutagenicity and carcino-
genicity testing as that required for additives intended for feeding to livestock
from which products for human consumption are derived. To determine chronic
toxicity, studies on two target species or on one target species and rats for a
period of one year will generally suffice. Mutagenesis and carcinogenesis
studies can generally be dispensed with if the chemical composition, practical
experience, or other considerations do not indicate the likelihood of changes. It
is possible to dispense withth e analysis of residues in pet animals.
Knowledge of the metabolism of the additive in productive livestock and of the
residues and their bio-availability is essential. In particular it must enable the
extent of the toxicological studies to be performed on laboratory animals in
order to assess the risks, if any, to the consumer to be determined. This evalua-
tion cannot be based solely on data confined to determining the direct effects of
the additives on laboratory animals. The latter does not provide specific infor-
mation on the actual effects of residues resulting from the metabolism in the
species for which the additive is intended.
Any application for authorization of an additive or a new usage for an additive
shall be supported by a dossier which should include detailed reports presented
in the order and with the numbering proposed in these guidelines. Reasons must
be given for the omission from the dossier of any data prescribed in these guide-
lines. Publications to which reference is made must be attached to it the reports
of experiments must include the plan, the reference number, the date of the
beginning and the end of the experiment, detailed description of the tests, results
and their analysis and also the name, address and signature of the person respon-
sible for the study. Each batch of the feedingstuffs used in the experimental
studies on animals must be analysed for the concentration of the relevant active
substances using an appropriate method and a report of the analytical results
must be provided. The report must also mention the individual doses determined
in each experimental study, the corresponding dates, the name, address and
signature of the person responsible for the controls. Furthermore, a certificate
drawn up by the laboratory or laboratories where the experiments were
conducted to the effect that the tests were carried out in accordance with the
principles of good laboratory practices pursuant to Council Directive 87/18/
EEC, on the harmonization of laws, regulations and administrative provisions
relating to the application of the principles of good laboratory practice and the
verification of their applications for tests on chemical substances(1), must be
attached to the report.
The determination of physico-chemical, toxicological and ecotoxicological prop-
erties shall be performed in accordance with the methods established by Council
Directive 67/548/EEC on the approximation of laws, regulations and administra-
tive provisions relating to the classification, packaging and labelling of
dangerous substances(2), as last amended by Commission Directive 93/105/1987L0153 — EN — 31.05.1995 — 002.001 — 4
(1) OJ No L 15, 17. 1. 1987, p. 29.
(2) OJ No L 196, 16. 8. 1967, p. 1.
▼M1
EC(1), or with methods internationally recognized by scientific bodies. The use
of other methods should be justified.
Each dossier shall contain an adequate summary. The dossiers relating to anti-
biotics, coccidiostats and other medicinal substances, growth promoters,
microorganisms and/or enzyme preparations, must be accompanied by a mono-
graph, conforming with the model provided in Section V, enabling the additive
concerned to be identified and characterized in accordance with Article 8 (1) of
Directive 70/524/EEC.
The term ‘additive’, as used in these guidelines, refers to the active substances
or the preparations containing active substances in the state in which they will
be incorporated in premixtures and feedingstuffs. An active substance may be a
chemically specified substance, a microorganism or an enzyme preparation.
In these guidelines the expression ‘Chemically specified substances’, means
chemical substances for which a chemical name is agreed according to IUPAC
nomenclature.
The Commission must be notified within a reasonable time by the Member State
which forwarded the dossier to it of any modification to the manufacturing
process or the composition of an additive, its field of application or its condi-
tions of use. This could necessitate the submission of documentation suitable
for a new assessment. These requirements will be especially necessary for
products derived from microorganisms, the genetic characteristics of which
have been modified or which arise as natural mutants.
CONTENTS
SECTION I: Summary of the data in the dossier
SECTION II: Identity, characterization and conditions of use of the
additive
Methods of control
SECTION III: Studies concerning the efficacy of the additive
1. Studies concerning improvements in the characteris-
tics of feedingstuffs
2. Studies concerning the effects of additives on animal
production
3. Studies concerning the quality of animal produce
SECTION IV: Studies concerning the safety of use of the additive
1. Studies on target species
1.1. Toxicological studies of the additive
1.2. Microbiological studies of the additive1.3. Study of the metabolism and residues
2. Studies on excreted residues
3. Studies on laboratory animals
SECTION V: Form of monograph1987L0153 — EN — 31.05.1995 — 002.001 — 5
(1) OJ No L 294, 30. 11. 1993, p. 21.
▼M1
SECTION I
SUMMARYOFTHEDATAINTHEDOSSIER
SECTION II
IDENTITY, CHARACTERIZATION AND CONDITIONS OF USE OF
THEADDITIVE
METHODSOFCONTROL
1.Identityoftheadditive
1.1. Proposed proprietary name(s)1.2. Type of additive according to its main function1.3. Qualitative and quantitative composition (active substance, other compo-
nents, impurities)
1.4. Physical state, particle size1.5. Manufacturing process including any specific processing procedures
N.B.:If the active substance is a mixture of active components, each of
which is clearly definable, the main components must be described
separately and the proportions in the mixture given.
2.Specificationsconcerningtheactivesubstance
2.1. For chemically specified substances: generic name, chemical name
according to Iupac nomenclature, other generic international names and
abbreviations. Chemical Abstracts Service Number (CAS).
For microorganisms: name and taxonomic description according to the
internatinal Codes of Nomenclature. Other internationally recognized
manuals of classification can also be used(
1).
For enzyme preparations: name according to main enzymatic activities as
described by IUB/Iupac. Einecs and CAS Number.
2.2 Formula, empirical and structural, and molecular weight. Qualitative and
quantitative composition of the main components, if the active substance
is a fermentation product.
For micro-organisms: name and place of the culture collection recognized
as an International Depositary Authority(2), if possible in the European
Union, where the strain is deposited and accession number, genetic modi-
fication and all relevant properties for its identification. In addition,
origin, appropriate morphological and physiological characteristics, devel-
opmental stages, relevant factors that may be involved in its biological
activity (as an additive) and other genetic data for identification. Number
of colony forming units (CFU) per gram.
For enzyme preparations: biological origin (in case of microbial origin:
name and place of the culture collection recognized as an International
Depositary Authority if possible in the European Union, where the strain
is deposited and accession number, genetic modification and all relevant
properties for its identification including genetic data), the activities
towards relevant chemically pure model substrates and other physico-
chemical characteristics.
A copy of the receipt of deposit of the micro-organism with an Interna-
tional Depositary Authority, precising the name and the taxonomic
description of the micro-organism according to the international Codes
of Nomenclature must, in all cases, be provided.
2.3.Degree of purity
Qualitative and quantitative composition of the impurities.For microorganisms: genetic stability and purity of strains cultivated.1987L0153 — EN — 31.05.1995 — 002.001 — 6
(1) Suchas Bergey's Manual of Systematic Bacteriology, The Yeasts, a taxonomic study by
Lodder and Kreger van Rij, Ainsworth and Bisby's Dictionary of the Fungi by Hawks-
worth, Sutton and Ainsworth or The Genus Aspergillus by Raper and Fennell.
(2) International Depositary Authority according to Article 7 of the Budapest Treaty on the
International Recognition of Micro-organisms for the Purposes of Patent Procedure▼M2
▼M1
▼M1
For enzyme preparations:
— purity, by checking the level of contaminating microorganisms, heavy
metals, absence of toxins derived from the source organism (e.g.
mycotoxins), as shown by a suitable method;
— absence of antimicrobial activity at feed concentration level as deter-
mined by a suitable method;
— composition of the non-enzymatic components (particularly Total
Organic Solids TOS(1)).
2.4.Relevant properties
For chemically specified substances: Electrostatic properties, melting
point, boiling point, decomposition temperature, density, vapour pressure,
solubility in water and organic solvents, mass and absorption spectra and
any other appropriate physical properties.
For microorganisms: properties relevant to identification and proposed
use (e.g. vegetative or sporulated form, CFU per g)
For enzyme preparations: optimal PH (value(s), optimal temperature(s)
and other appropriate properties).
2.5.Manufacturing, purification processes and media used
Variation in the composition of the batches in the course of production.
3.Physico-chemical, technological andbiological propertiesoftheaddi-
tive
3.1. Stability (for microorganisms: loss of biological activity, e.g. viability) on
exposure to environmental conditions suchas ligh t, temperature, pH,
moisture and oxygen. Shelf life.
3.2. Stability (for microorganisms: loss of biological activity, e.g. viability)
during the preparation of premixtures and feedingstuffs, in particular
stability to heat, pressure and moisture. Possible decomposition products.
3.3. Stability (for microorganisms: loss of biological activity, e.g. viability)
during the storage of premixtures and feedingstuffs under defined condi-
tions. Shelf life.
For enzyme preparations: Details of the presence of unexpected reaction
products formed by either enzymatic or chemical reactions of the enzyme
preparation withfeed constituents or by degradation of th e enzyme
preparation during storage of the feedingstuff.
3.4. Other appropriate physico-chemical, technological or biological proper-
ties suchas ability to obtain h omogeneous mixtures in premixtures and
feedingstuffs, dust-forming properties, and for microorganisms and/or
enzyme preparations, assessment of resistance to degradation or loss of
biological activity in the digestive tract or by systems of simulation in
vitro.
3.5. Physico-chemical or biological incompatibilities or interactions (e.g. with
feedingstuffs, other approved additives or with medicinal products).
4.Conditionsofuseoftheadditive
4.1. Proposed use in animal nutrition (e.g. species and category of animal,
type of feedingstuff, period of administration and withdrawal period).
4.2.Contra-indications
4.3. Proposed dosing in premixtures and feedingstuffs expressed as:
— a percentage of the active substance by weight for premixtures and as
mg/kg for feedingstuffs, in case of chemically specified substances,
— appropriate units of biological activity suchas CFU per gram of
product for microorganisms or relevant activity units for enzyme
preparations.
4.4. Other known uses of the active substance or the preparation (e.g. in food-
stuffs, human or veterinary medicine, agriculture and industry). For each
use give the proprietary names, indications and contraindications.
4.5. If necessary, measures for the prevention of risks and means of protection
during manufacture and handling.1987L0153 — EN — 31.05.1995 — 002.001 — 7
(1) TOS (%) = 100 − (% ash+ water + % diluents and/or additives and ingredients).
▼M1
5.Controlmethods
5.1. Description of the methods used for the determination of the criteria
listed under items 1.3., 2.3., 2.4., 2.5., 3.1., 3.2., 3.3., 3.4. and 4.3.
5.2. Description of the qualitative and quantitative analytical methods for
routine control of the additive in premixtures and feedingstuffs.
5.3. Description of the qualitative and quantitative analytical methods for
determining residues of additives in animal produce.
N.B.The methods specified and the results should be accompanied by
information as to percentage recovery, specificity, sensitivity, limits
of detection, possible interferences, reproducibility and to the
sampling method used. Reference standards of the preparation and
of the active substance must be available.
In the case of microorganisms state methods of detection, enumeration,
identification and relevant markets.
SECTION III
STUDIESCONCERNINGTHEEFFICACYOFTHEADDITIVE
1.Studies concerning improvements in the characteristics of feeding-
stuffs
These studies concern technological additives such as antioxidants,
preservatives, binders, emulsifiers, stabilizers, gelling agents etc., which
are intended to improve or stabilize the characteristics of premixtures
and feedingstuffs. Some microorganisms and/or enzyme preparations
could also be considered as technological additives, if they improve rele-
vant feed characterictics.
Evidence of the efficacy of the additive should be provided by means of
appropriate criteria under the intended conditions of use in comparison
withnegative control feedingstuffs and, possibly, feedingstuffs containing
technological additives of known effectiveness.
The precise nature of the active substances, preparations, premixes and
feedingstuffs examined, the reference number of the batches, the concen-
tration of the active substances in premixtures and feedingstuffs, the
testing conditions (e.g. temperature and humidity) and also the dates and
duration of testing, the adverse and negative effects which occurred
during testing shall be specified for each experiment
2.Studiesconcerningtheeffectsofadditivesonanimalproduction
These studies concern zootechnical additives which have effects on
animal production. The following studies, including dose/response rela-
tionship, should be performed on each target species in comparison with
negative control groups and, possibly, groups receiving feedingstuffs
containing additives of known effectiveness.
If the active substance is a mixture of active components, the presence of
eachcomponent must be justified.
2.1. For coccidiostats and other medicinal substances, importance should
primarily be attached to evidence of the specific effects and particularly
prophylactic properties (e.g. morbidity, oocyst count and lesion score).
Information on the effect on feed effciency, animal growth and market-
able quantity and quality of the animal produce may be added.
2.2. For other zootechnical additives (including if appropriate microorgasn-
isms and/or enzyme preparations) information should be provided on the
effects on: nutritional efficiency, animal growth, animal-product charac-
teristics and yield, animal welfare and other parameters having a
positive influence on animal production.
2.3.Experimental conditions
The test performed must be described and the results presented individu-
ally in detail. The statistical evaluation and the methods employed should
be reported. The following data must be provided:
2.3.1. Species, breed, age and sex of the animals, identification procedure.2.3.2. Number of test and control groups, number of animals in eachgroup. Th e
number of animals of bothsexes must be sufficient for statistical
purposes.1987L0153 — EN — 31.05.1995 — 002.001 — 8
▼M1
2.3.3. Concentration of the active substance (and, where it is the case,
substances used for comparative purposes) in the feedingstuffs established
by a control analysis, using the appropriate recognized method. Reference
number of the batches. Nutritional composition of the diet in terms of
quality and quantity.
2.3.4. Location of each experiment. Animal health, physiological, feeding and
rearing conditions as usually practised in the Community. Feed control
and measures taken to avoid contamination of control groups during the
experiment (particularly for microorganisms through cross-contamination
of feed by the microorganism).
2.3.5. Date and exact duration of testing. Date and nature of examinations
performed.
2.3.6. Unfavourable effects and other incidents which occurred during the
experiment and time of their appearance.
3.Studiesconcerningthequalityofanimalproduce
Studies on the organoleptic, nutritional, hygienic and technological quali-
ties of produce from animals fed withfeedingstuffs containing th e
additive.
SECTION IV
STUDIESCONCERNINGTHESAFETYOFUSEOFTHEADDITIVE
The studies outlined in this section are intended to permit assessment of:
— the safety of use of the additive in the target species,
— the from risks inhalation, other mucosal, eye or cutaneous contact for
persons likely to handle the additive as such or as incorporated into premix-
tures or feedingstuffs,
— the risks to the consumer which could result from the consumption of food
containing residues of the additive, or its metabolites,
— the risks of pollution or persistence in the enviroment from the additive itself
or by products derived from the additive and excreted by animals,
— possible risks to non-target species.
These studies will be required in their entirety or in part depending on the
nature of the additive and the conditions proposed for its use.
As a general principle microorganisms and/or enzyme preparations must be, or
be derived from microorganisms that are non-pathogenic and non-toxicogenic
for target species and humans under the expected conditons of use.
In the case of microorganisms and/or enzyme preparations appropriate safety
tests must be performed, unless other satisfactory documentation for safety of
use is provided. For microorganisms at least a tolerance test on target species
must be performed.
In principle toxicological tests for enzymes derived from edible parts of animals
or plants are not required; where such products are not generally considered a
normal part of the diet used, some toxicological testing may be required.
If the active substance is chemically specified the knowledge of their metabo-
lism in the various target species and also of the composition and the bio-
availability of the tissue residues will be essential for determining the extent of
studies on laboratory animals to assess the risks for the consumer. Furthermore,
knowledge of the composition and of the physico-chemical and biological prop-
erties of the excreted residues deriving from the additive will be indispensable
to define the extent of the studies necessary for assessment of the risk of pollu-
tion or persistence in the enviroment.
1.Studiesontargetspecies
1.1.Toxicological studies of the additive
Tolerance tests
Study of the biological, toxicological, macroscopic and histological
effects. Determination of the safety margin between the maximum
proposed dose-level and the level resulting in unfavourable effects. It
may be sufficient to indicate a minimum or approximate value for this
margin if it can be shown that the level resulting in unfavourable effects
greatly exceeds the maximum proposed dose-level.1987L0153 — EN — 31.05.1995 — 002.001 — 9
▼M1
1.2.Microbiological studies of the additive
1.2.1. If the active substance is chemically specified and possesses antimicrobial
activity at feed concentration level, studies of the antibacterial spectrum
of action of the additive by determination of the minimum inhibition
concentration (MIC) in various pathogenic and non-pathogenic Gram-
negative and Gram-positive species of bacteria must be provided.
1.2.2. Studies on the cross-resistance to therapeutic antibiotics by determination
of the MIL, in mutants produced in vitro which exhibit chromosomal
resistance to the additive. In the case of microorganisms which are resis-
tant to therapeutic antibiotics, the genetic basis of this resistance should
be shown.
1.2.3. Tests to find out whether the additive is capable of selecting resistance
factors. These tests are to be performed under field conditions in the
animal species for which the additive is primarily intended. Subsequently,
it should be determined whether R factors which may have been found
carry multiple resistance and are transmissible.
1.2.4. Tests to determine the effect of the additive,
— on the microflora of the digestive tract,
— on the colonization of the digestive tract, if it is a microorganism or a
mixture of several strains of microorganisms,
— on the shedding or excretion of pathogenic microorganisms, if the
active substance is chemically specified and has antimicrobial
activity.
1.2.5. In cases where the active substance shows an antimicrobial action, field
studies to monitor the percentage of bacteria resistant to the additive
should be provided. These are to be carried out at major intervals before,
during and after (one month) the use of the additive.
1.2.6. If the active substance is a microorganism, it should be determined if it is
resistant to antibiotics.
1.2.7. If the active substance (e.g. enzyme preparations) is produced by a micro-
organism the level of the respective viable organism should be
determined.
1.2.8. If the additive contains or consists of a genetically modified organism
within the meaning of Article 2 (1) and (2) of Council Directive 90/220/
EEC the following information must be provided:
— A copy of any written consent or consents of the competent authori-
ties to the deliberate release of the genetically modified organisms for
researchand development purposes pursuant to Article 6 (4) of Direc-
tive 90/220/EEC and the summary of the notification as referred in
Article 9 of Directive 90/220/EEC according to the model set up in
Council Decision 91/596/EEC(1);
— the complete technical file with the information required in Annex II
of Directive 90/220/EEC, extended as necessary to take into account
the diversity of sites of use of the additive, including information on
data and results obtained from researchand developmental releasesconcerning the ecosystems which could be affected by the use of the
additive and an assessment of any risks for human health, animal
health and the environment related to the GMO(s) contained in the
product including information obtained from the research and devel-
opment stage on the impact of the release on human health and the
environment;
— the conditions for the placing on the market of the additive, including
specific conditions of use and handling and a proposal for labelling
and packaging which should comprise at least the requirements laid
down in Annex III of Directive 90/220/EEC.
If on the basis of any release notified under Part B of Directive 90/220/
EEC, or on substantive, reasoned scientific grounds, the person respon-
sible for the dossier considers that the placing on the market or the use
of the additive does not pose a risk to human health, animal health and
the environment, he may propose not to comply with one or more of the
requirements of Annex III B.
Information on data or results from releases of the same GMO or the
same combination of GMOs previously or currently notified and/or
carried out by the person responsible for the dossier either inside or
outside of Community, shall be included.1987L0153 — EN — 31.05.1995 — 002.001 — 10
(1) OJ No L 322, 23. 11. 1991, p. 1.
▼M1
Other data or results from notifications previously submitted by other
persons may also be referred, provided that the latter have given their
agreement in writing.
1.3.Studies of the metabolism and residues (1)(2)(If the active substance is
chemically specified).
1.3.1. Study of metabolism,
— metabolic balance: rate and extent of absorption and elimination of
the active substance,
— identification of the metabolic pathways and main metabolites,
— distribution and excretion (biliary, urinary, faecal) of the metabolites,
— if appropriate, influence of the intestinal or ruminal microflora, of
enterohepatic cycle, of caecotrophy, on the metabolism.
1.3.2. Analytical studies of the residues: qualitative and quantitative composi-
tion of the residues (active substance, metabolites) in the various animal
food products at metabolic equilibrium and under practical conditions of
use of the additive.
1.3.3. Kinetic study of the residues (following repeated administration of the
additive according to the purposal use): persistence of the active
substance and the main metabolites in the various organs and tissues after
withdrawal of the supplemented feedingstuff.
1.3.4. Study of the bio-availability of the residues in animal food products. (see
3.7.).
1.3.5. Methods of monitoring: qualitative and quantitative method of determina-
tion used in the studies mentioned under items 1.3.1. to 1.3.4. with
information as to percentage recovery, specificity and limits of detection.
The methods of determination of the residues must be sufficiently sensi-
tive to permit detection of residues at levels which are toxicologically
negligible.
2.Study on excreted residues (where the active substance is chemically
specified)
2.1. Nature and concentration of the residues derived from the additive (active
substance, metabolites) in the excreta.
2.2. Persistence (half-life value) and kinetics of elimination of these residues
in slurries, farm yard manure and litter.
2.3. Effects on methanogenesis.2.4. Degradation, persistence (half-life value) and kinetics of elimination in
soils (contrasting soil types).
2.5. Effects on soil fauna and microbial processes of transformation (e.g.
decomposition of plant and animal residues).
2.6. Effects on terrestrial plants (e.g. seed germination, plant growthand plant
uptake). These studies should be carried out under controlled conditions
and field conditions, using different plant species.
2.7. Solubility and stability in water of the products derived from the additive
(active substance, metabolites).
2.8.Effects onaquatic life
2.8.1. Effects on flora (e.g. Chlorella).2.8.2. Toxicity in non-vertebrates (e.g. Daphnia magna).2.8.3. Toxicity in fish(at least two wild species found in th e Community terri-
tory).
3.Studiesonlaboratoryanimals
These studies must be carried out with the active substance and its major
metabolites or products, if the latter are also present in edible animal
produce and are bioavailable. As far as possible attempts should be1987L0153 — EN — 31.05.1995 — 002.001 — 11
(1) The studies mentioned under 1.3.1., 1.3.3. and 1.3.4. should be carried out with labelled
molecules or other appropriate methods, in each case the choice of the method utilizedshould be justified. The labelling should be suitable for the purpose intended.
(
2) If the active substance is produced by fermentation, these studies should be extended to
related substances derived from the production process.
▼M1
made to select laboratory animals which may be expected to digest and
metabolize the additive in a similar way to man or the target species.
Full detailed descriptions must be provided of the tests performed. These
should cover the animal species and strains employed, the size and
number of test and control groups, the dose levels administered, the
composition of the diet and the results of feed analyses, the rearing
conditions, the exact duration of the tests, the dates of the various exam-
inations performed and mortality. Full details must be given of the
macroscopic pathological and histopathological findings in all animals
tested with an indication of the time of appearance of all pathological
lesions. All results, including statistical assessment, must be presented in
detail.
3.1.Acute toxicity (not relevant for microorganisms)
3.1.1. Acute oral toxicity studies must be carried out on two animal species
(preferably the rat should be one). The maximum dosage should not be
higher than 2,000 mg/kg body weight. Detailed observations should be
reported of the biological effects observed during a period of at least
two weeks after ingestion. These studies are not relevant for enzyme
preparations.
3.1.2. Studies on acute inhalational toxicity, skin and, where necessary, mucous
membranes irritancy and also allergenic potential must be performed by
appropriate tests for the assessment of possible risks associated with the
handling of the additive.
3.2.Mutagenicity
3.2.1. If the active substance is chemically specified
In order to identify active substances or their metabolites or products that
possess mutagenic properties a selected combination of mutagenicity
tests, based on different genetic endpoints, must be carried out. Tests
must be performed, in the presence and absence of a microsomal
mammalian preparation, for metabolic activation.
The following package of tests is recommended:
(a) a test for gene mutations in a prokaryotic system,
(b) a test for gene mutations in an in-vitroeukaryotic system or a sex-
linked recessive lethal test in Drosophila melanogaster,
(c) a test for chromosomal aberrations in-vitroandin-vivo.
The battery of tests suggested above does not imply, however, that other
tests are inappropriate or that other tests, in particular in-vivotests, would
not be acceptable as alternatives.
In all cases reasons fo the choice of tests should be given. Tests must be
carried out according to established validated procedures. Depending on
the outcome of the tests and taking into consideration the whole toxicity
profile of the substance as well as the intended use, additional investiga-
tions may be indicated.
3.2.2. In the case of enzyme preparations derived from microorganisms the
following tests are normally required:
(a) a test for gene mutations in bacteria,
(b) a test for chromosomal aberrations (preferably invitro).
The toxicological tests shall, where possible, be performed on a batch
from the final purified fermentation product, before addition of carriers,
diluents or other substances. They should, as a general rule, be performed
in accordance withestablish ed guidelines(1) from recognized interna-
tional Institutions although, because of the effects exerted at the cellular
level by the proteinaceous nature and/or enzymatic activities of certain
enzyme preparations, some modifications of the standard test protocols,
particularly in the case of in vitro tests, may be necessary. Such devia-
tions will be acceptable if accompanied by adequate supporting
arguments.
The test system is designed to uncover unspecified toxic reactions and to
reveal genotoxic effects. The combined information from the general1987L0153 — EN — 31.05.1995 — 002.001 — 12
(1) Suchas:
— Presentation of an application for assessment of a food additive prior to its author-
ization. 1989. (ISBN 92-826-0135-B)
— Report of the Scientific Committee for Food on guidelines for the safety assessment
of food additives. 1980. 10threport series, (EUR 6892).
▼M1
specifications and this test battery make it possible to evaluate the
product for the presence of both specific, well known toxins and
unknown toxic compounds.
The toxicological report shall contain satisfactory documentation that the
tests have been performed on the material which forms the basis of the
commercial product as described in the technological dossier.
3.3.Pharmacokinetics aspects
If the active substance is chemically specified balance studies and identi-
fication of metabolites must be performed using suitable labelled
molecules or other appropriate techniques and should cover both single
and multiple dose administration of the active substance over appropriate
periods. Metabolism studies must also include investigation of the phar-
macokinetics of the active substance and of the major metabolites.
Consideration must be given to the differences in the way that various
species metabolize the active substance when selecting the most relevant
species for subsequent toxicological investigations.
3.4.Subchronic toxicity
These studies must be carried out in general on two animal species
(preferably the rat should be one). The second species may in some
instances be a target species. The test substance may be administered
orally and a dose-response relationship must be established. The duration
in rodents must be at least 90 days.
In certain cases investigations extending over six months to two years in
non-rodents may be desirable to establishth e variation in sensitivity of
different animal species to the test substance.
These studies are not relevant for microorganisms. In the case of enzyme
preparations derived from microorganisms a 90-day oral toxicity test in a
rodent species may be sufficient.
3.5.Chronic toxicity/carcinogenicity
Chronic toxicity studies must be carried out on one species (preferably
the rat), carcinogenicity studies preferably on two species of rodent. The
substance must be administered orally at several dose levels. A combined
chronic toxicity/carcinogenicity study with in-utero exposure is also
acceptable. Experiments must extend for a minimum of 24 months in
rats and 18 months in mice. If continued beyond the minimum period,
the test must be terminated when survival in any but the highest dose
level groups has fallen to 20 %.
Full clinical chemistry, haematological and urine examinations must be
carried out at appropriate intervals throughout the experiment. Full
macroscopic and histological examinations must be carried out on all
animals dying during the test and on all survivors at the termination of
the study.
These studies are not relevant for microorganisms and enzyme prepara-
tions.
3.6.Reproductive toxicity (where the active substance is chemically specified)
Studies on reproduction must be carried out preferably on the rat. They
must extend over at least two filial generations and may be combined
withembryotoxicity including teratogenic studies. All relevant fertility,
gestation, parturition, peri- and postnatal parameters must be carefully
observed and reported. Specific teratogenic studies must be carried out
in at least two suitable species.
3.7.Toxicology of metabolites (where the active substance is chemically
specified)
Information for the calculation of residue concentration is required as a
basis for assessing the risk for man.
The basis for calculation of the proposed withdrawal period must be
made available. The studies mentioned in 1.3.4. must be carried out on
laboratory animals.
3.8.Other relevant studies
Any further special study providing additional information useful for the
assessment of test substance may be made available (e.g. bio-availability,
neurotoxicity or immunotoxicity).1987L0153 — EN — 31.05.1995 — 002.001 — 13
▼M1
SECTION V
FORMOFMONOGRAPH
1.Identityoftheadditive
1.1.Proposed proprietary name(s)
1.2.Type of additive according to its main function
1.3.Qualitative and quantitative composition (active substance, other compo-
nents, impurities)
1.4.Physical state, particle size
1.5.Possible specific processing
N.B.If the active substance is a mixture of active components, each of
which is to be clearly definable, the main components must be
described separately and the proportions in the mixture given.
2.Specificationsconcerningtheactivesubstance
2.1. For chemically specified substances: generic name, chemical name
according to IUPAC nomenclature, other generic international names
and abbreviations. Chemical Abstracts Service Number (CAS).
For microorganisms: name and taxonomic description according to the
international Codes of Nomenclature. Other internationally recognized
manuals of classification can also be used(1).
For enzyme preparations: name according to main enzymatic activities as
described by IUB/Iupac, Einecs and CAS Number.
2.2 Formula, empirical and structural, and molecular weight. Qualitative and
quantitative composition of the main components, if the active substance
is a fermentation product.
For micro-organisms: name and place of the culture collection recognized
as an International Depositary Authority, if possible in the European
Union, where the strain is deposited and accession number, genetic modi-
fication and all relevant properties for its identification.
For enyzme preparations: biological origin (in case of microbial origin:
name and place of the culture collection recognized as an International
Depositary Authority if possible in the European Union, where the strain
is deposited and accession number, genetic modification and all relevant
properties for its identification including genetic data), the activities
towards relevant chemically pure model substrates and other physico-
chemical characteristics.
2.3.Degree of purity
Qualitative and quantitative composition of the impuritiesFor microorganisms: genetic stability and purity of strains cultivated.For enzyme preparations:
— purity [by checking the level of contaminating microorganisms, heavy
metals, absence of toxins (e.g. mycotoxins) derived from the source
organism as shown by a suitable method];
— absence of antimicrobial activity at feed concentration level as deter-
mined by a suitable method;
— composition of the non-enzymatic components (particularly Total
Organic Solids T.O.S.)
2.4.Relevant properties
For chemically specified substances: Electrostatic properties, melting
point, boiling point, decomposition temperature, density, vapour pressure,
solubility in water and organic solvents, mass and absorption spectra and
any other appropriate physical properties.
For microorganisms: properties relevant to identification and proposed
use (e.g. vegetative or sporulated form, CFU per g).1987L0153 — EN — 31.05.1995 — 002.001 — 14
(1) Suchas Bergey's Manual of Systematic Bacteriology, The Yeasts, a taxonomic study by
Lodder and Kreger van Rij, Ainsworth and Bisby's Dictionary of the Fungi by Hawks-
worth, Sutton and Ainsworth or The Genus Aspergillus by Raper and Fennell.▼M2
▼M1
▼M1
For enzyme preparations: optimal pH value(s), optimal temperature(s)
and other appropriate properties.
3.Physico-chemical, technological andbiological propertiesoftheaddi-
tive
3.1. Stability (for microorganisms: loss of biological activity, e.g. viability) on
exposure to environmental conditions suchas ligh t, temperature, pH,
moisture and oxygen. Shelf life.
3.2. Stability (for microorganisms: loss of biological activity, e.g. viability)
during the preparation of premixtures and feedingstuffs, in parlticular
stability to heat, pressure and moisture. Possible decomposition products.
3.3. Stability (for microorganisms: loss of biological activity, e.g. viability)
during the storage of premixtures and feedingstuffs under defined condi-
tions. Shelf life.
3.4. Other appropriate physico-chemical, technological or biological proper-
ties suchas ability to obtain h omogeneous mixtures in premixtures and
feedingstuffs, dust-forming properties, and for microorganisms and/or
enzyme preparations, assessment of resistance to degradation or loss of
biological activity in the digestive tract or by systems of simulation in
vitro.
3.5. Physico-chemical or biological incompatibilities or interactions (e.g. with
feedingstuffs, other approved additives or with medicinal products).
4.Controlmethods
4.1. Description of the methods used for the determination of the criteria
listed under items 1.3., 2.3., 2.4., 3.1., 3.2., 3.3. and 3.4. of this Section.
4.2. Description of the qualitative and quantitative analytical methods for
determining residues of additives in animal produce.
4.3. If the said methods have been published the literature references may
suffice and the corresponding reprints should be given.
5.Biological propertiesoftheadditive
5.1. Particulars of the prophylactic effects for coccidiostats and other medic-
inal substances (e.g. morbidity, oocyst count and lesion score).
5.2. For zootechnical additives other than those listed in 5.1. including if
appropriate microorganisms and/or enzyme preparations: particulars of
the effects on nutritional efficiency, animal growth, animal product char-
acteristics and yield, animal welfare and other parameters having a
positive influence on animal production.
5.3. Any contra-indications or warnings, including biological incompatibil-
ities, withparticulars of th eir justification.
6.Details of the quantitative and qualitative residues, if any, found in
animalproducefollowingenvisageduseoftheadditive.
7.Othercharacteristics suitableforidentificationoftheadditive.1987L0153 — EN — 31.05.1995 — 002.001 — 15