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Three Rs Approaches in the Production and
Quality Control of Avian Vaccines
The Report and Recommendations of ECVAM Workshop 41 1,2
Lukas Bruckner,3 Johan Bongers,4 Peter Castle, 5 Pieter H. Flore,6
Michèle Guittet,7 Marlies Halder,8 Carmen Jungbäck,9 François Xavier
Le Gros,10 Maria Tollis, 11 Venugopal K. Nair,12 Manfred Wilhelm,9 Joseph
Zeegers13 and Guy Zigterman14
3Institut
für Viruskrankheiten und Immunprophylaxe, 3147 Mittelhäusern, Switzerland;
P.O. Box 65, Edelhertweg 15, 8200 AB Lelystad, The Netherlands; 5European
Pharmacopoeia, Council of Europe, 226, avenue de Colmar, P.O. Box 907, 67029 Strasbourg
01, France; 6Lohmann Animal Health GmbH, Heinz-Lohmann-Strasse 4, 27472 Cuxhaven,
Germany; 7Laboratoire Central de Recherches Avicole et Porcine, Agence Francaise de
Securite Sanitaire des Aliment, BP 53, Zoopole, 22440 Ploufragan, France; 8ECVAM, JRC
Institute for Health & Consumer Protection, European Commission, 21020 Ispra (VA), Italy;
9Paul Ehrlich Institute, Paul-Ehrlich-Strasse 51–59, 63225 Langen, Germany; 10Merial
S.A.S., 29 Avenue Tony Garnier, P.O. Box 7123, 69348 Lyon 07, France; 11Laboratorio di
Medicina Veterinaria, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Roma,
Italy; 12Viral Oncogenesis Group, Institute for Animal Health, Compton, Berkshire RG20
7NN, UK; 13Fort Dodge Animal Health, C.J. van Houtenlaan 36, 1381 CP Weesp, The
Netherlands; 14Intervet International B.V., Wim de Korverstraat 35, 5831 AN Boxmeer, The
Netherlands
4ID-DLO,
Preface
This is the report of the forty-first of a series
of workshops organised by the European
Centre for the Validation of Alternative
Methods (ECVAM). ECVAM’s main goal, as
defined in 1993 by its Scientific Advisory
Committee, is to promote the scientific and
regulatory acceptance of alternative methods
which are of importance to the biosciences
and which reduce, refine or replace the use of
laboratory animals. One of the first priorities
set by ECVAM was the implementation of
procedures which would enable it to become
well informed about the state-of-the-art of
non-animal test development and validation,
and the potential for the possible incorporation of alternative tests into regulatory procedures. It was decided that this would be
best achieved by the organisation of ECVAM
workshops on specific topics, at which small
groups of invited experts would review the
current status of various types of in vitro
tests and their potential uses, and make recommendations about the best ways forward
(1).
The joint ECVAM/AGAATI (Advisory
Group on Alternatives to Animal Testing in
Address for correspondence: Dr Lukas Bruckner, Institut für Viruskrankheiten und Immunprophylaxe, 3147 Mittelhäusern, Switzerland.
Address for reprints: ECVAM, JRC Institute for Health & Consumer Protection, TP 580, 21020 Ispra (VA), Italy.
1ECVAM
— The European Centre for the Validation of Alternative Methods. 2This document represents the
agreed report of the participants as individual scientists.
242
Immunobiologicals) workshop on Three Rs
Approaches in the Production and Quality
Control of Avian Vaccines was held in Langen, Germany, on 11–13 June 1999, under
the co-chairmanship of Lukas Bruckner
(Institute of Virology and Immunoprophylaxis, Mittelhäusern, Switzerland) and Guy
Zigterman (Intervet, Boxmeer, The Nether lands). The participants, all experts in vaccine quality control, avian vaccines or avian
diseases, came from international regulatory
or government organisations, national control laboratories, vaccine manufacturers and
academia. The objectives of the workshop
were: a) to review the monographs of the
European Pharmacopoeia on avian live viral
vaccines in the light of animal welfare
aspects; b) to propose and recommend strategies to replace, reduce and/or refine the use
of animals; c) to review the state-of-the-art
and the suitability of the tests proposed for
their intended purposes; and d) to comment
on the draft monographs on avian live viral
vaccines which had been published in
Pharmeuropa (2–12).
The outcome of the discussions and the
recommendations agreed by the workshop
participants are summarised in this report.
Introduction
Vaccines are considered to be the most costeffective tools in the prevention of infectious
diseases; this applies, in particular, to avian
live viral vaccines. The minimal requirements for the production and quality control
of vaccines are given in the European Pharmacopoeia. Its actual Third Edition & Supplements include 13 monographs on avian
vaccines (live and inactivated) and the text
for extraneous agents testing (Table I).
Most of these monographs are more than
15 years old and have only occasionally been
revised since they were first published. It is
questionable whether all of the tests prescribed are still relevant. In particular, some
of the “safety tests” might be superfluous,
and it is debatable whether the high numbers of animals required can still be justified.
Tests in animals are prescribed for testing
for safety, extraneous agents and potency.
Since the quality of vaccines is controlled not
only during development, but also on a
batch-to-batch basis, large numbers of animals are used. The representatives of vac-
L. Bruckner et al.
cine manufacturers at the workshop estimated that about 51,000 chickens and other
birds are used each year for the quality control of 1800 batches of avian vaccines.
In recent years, the European Pharmacopoeia Commission and its Group of
Experts 15V, which is responsible for veterinary vaccines, have made great efforts to
revise these old monographs and to harmonise them. In March 1999, draft revisions
of the monographs on live avian vaccines
(2–9) and extraneous agents testing (10, 11),
and the draft monograph on Avian Viral
Tenosynovitis Vaccine (Live) (12), were published in Pharmeuropa. As soon as a draft
monograph is published in Pharmeuropa,
members of the public are invited to submit
comments on the text to the European Pharmacopoeia Commission. The comments
received are discussed in the relevant Group
of Experts and, if considered to be appropriate, the comments are incorporated into the
monographs. One of the aims of the workshop was to comment step-by-step on the
published texts in the light of the Three Rs.
Comments representing the conclusions of
the participants in the workshop were sent
to the European Pharmacopoeia Secretariat
in September 1999.
This workshop report summarises these
comments, and includes additional information on the current status of alternative
methods for the testing of inactivated poultry vaccines.
Testing for Extraneous Agents in
Avian Live Viral Vaccines
Testing for extraneous agents in avian live
viral vaccines is regulated by two monographs (Table I), which apply to all of the
individual monographs on avian live viral
vaccines. The methods proposed are animal
and non-animal tests that have to be performed on the seed lot and on each batch of
the finished product. Ten 2-week-old chicks
are twice inoculated in a given period with
multiple doses of the vaccine, by intramuscular injection and by eye drop. Serum is collected from each chick before the inoculation
and at the end of the test period. The animals are observed, and should not show
other than mild signs of infectious disease.
The serum samples are tested for antibodies
against a given list of infectious agents.
ECVAM Workshop 41: avian vaccines
243
Table I: European Pharmacopoeia texts on avian vaccines
Texts
Revision Reference
General texts
2.6.3 Avian Viral Vaccines: Tests for Extraneous Agents in
Seed Lots
2.6.4 Avian Live Virus Vaccines: Tests for Extraneous Agents
in Batches of Finished Product
Individual monographs
Avian Infectious Bronchitis Vaccine (Live)
Avian Infectious Bronchitis Vaccine (Inactivated)
Avian Infectious Bursal Disease Vaccine (Live)
Avian Infectious Bursal Disease Vaccine (Inactivated)
Avian Infectious Encephalomyelitis Vaccine (Live)
Avian Infectious Laryngotracheitis Vaccine (Live)
Avian Viral Tenosynovitis Vaccine (Live)
Duck Viral Hepatitis type 1 Vaccine (Live)
Egg Drop Syndrome ’76 Vaccine (Inactivated)
Fowl-pox Vaccine (Live)
Newcastle Disease Vaccine (Live)
Newcastle Disease Vaccine (Inactivated)
Marek’s Disease Vaccine (Live)
Comments on 2.6.3. Avian Viral Vaccines:
Tests for Extraneous Agents in Seed Lots
(10)
The current scheme requires the testing for
extraneous agents to be carried out with
embryonated hens’ eggs, in cell cultures and
in chicks. A polymerase chain reaction (PCR)
method is proposed for the detection of avian
leukosis virus (17). The workshop participants
agreed that the test in chicks is necessary with
regard to the safety of the vaccine; however, to
avoid unnecessary test repetitions, and to
enhance the safety of the vaccine, several
items should be stated more clearly.
1) It should be clearly indicated in the text
that the tests for extraneous agents in
chicks need to be performed only once
during development, preferably on the
master seed lot. If insufficient master
seed lot is available, testing should be
allowed on a working seed lot, preferably
as close as possible to the master seed lot.
yes
10
yes
11
yes
4
13
3
14
2
yes
yes
yes
new draft
yes
yes
yes
yes
9
12
5
15
6
7
16
8
2) The test for extraneous agents in chicks
must include clinical observation of the
animals and the testing of serum samples.
3) The list of agents against which the
serum samples should be tested, and the
list of test methods to be used, should
include the agents and methods that are
given in the general monograph on specific pathogen-free (SPF) eggs (Table II;
18). This would increase the certainty
that the product is free of these agents.
With regard to the testing of turkey vaccines, deletion of the extraneous agents
test for turkey lymphoproliferative disease virus is recommended, since there is
only a low incidence of the virus, and the
test currently performed has not been
validated.
4) It is recommended that the sera of the
test animals should be stored under suitable conditions as long as seed material
244
L. Bruckner et al.
Table II:
Agents against which serum samples should be tested in the extraneous
agents test using chicks (text 2.6.3)
Microorganism
Type of test
Avian adenoviruses
Avian encephalomyelitis virus*
Avian infectious bronchitis virus
Avian infectious laryngotracheitis virus
Avian leucosis viruses
Enzyme-linked immunosorbent assay
Enzyme-linked immunosorbent assay
Enzyme-linked immunosorbent assay
Serum neutralisation
Serum neutralisation
Avian nephritis virus*
Avian reoviruses
Avian reticuloendotheliosis virus*
Chicken anaemia virus
Haemagglutinating avian adenovirus
(egg drop syndrome EDS 76 virus)*
Fluorescent antibody
Enzyme-linked immunosorbent assay
Fluorescent antibody
Fluorescent antibody
Haemagglutination inhibition
Infectious bursal disease virus*
Influenza A virus
Marek’s disease virus*
Newcastle disease virus
Turkey rhinotracheitis virus*
Serum neutralisation
Enzyme-linked immunosorbent assay
Enzyme-linked immunosorbent assay
Haemagglutination inhibition
Enzyme-linked immunosorbent assay
Mycoplasma gallisepticum
Agglutination and, to confirm a positive test,
haemagglutination inhibition
Agglutination and, to confirm a positive test,
haemagglutination inhibition
Agglutination
Mycoplasma synoviae
Salmonella pullorum*
* = infectious agents included in the revised monograph.
obtained from the tested master seed lot
is being used for production of the vaccine. This will facilitiate the testing for
antibodies against agents which were not
known when the master seed was established, and will provide information on
the possible contamination of the product
with agents discovered subsequently.
Comments on 2.6.4. Avian Live Virus
Vaccines: Tests for Extraneous Agents in
Batches of Finished Product (11)
The proposed scheme requires tests in eggs,
cell cultures and chicks in order to cover
the range of possible contaminants. With
regard to the numbers of animals used, far
more animals are needed for the testing of
each batch than for testing seed lots.
The test in chicks is intended to cover 16
infectious agents. It is often not the most
sensitive method for the detection of contaminants, but its lack of sensitivity is partially
compensated for by inoculating the chicks
with multiple vaccine doses. Alternative
methods that are at least as sensitive as the
animal test (17, 19–24) are now available.
Some of the tests that have recently been
developed (for example, PCR methods for
detection of chick anaemia virus and avian
infectious haemorrhagic enteritis virus; 20,
ECVAM Workshop 41: avian vaccines
24) could be fully validated for use as routine
tests by the time the revised monographs are
expected to become official (January 2002).
Table III includes the agents to be tested,
and possible alternative methods and their
stages of development. Efforts should be
made to develop and validate the PCR
method for the detection of avian infectious
haemorrhagic enteritis virus, and to validate
the PCR method for the detection of chick
anaemia virus. Some of the alternative methods are already included in the draft text of
2.6.3. Avian Viral Vaccines: Tests for Extraneous Agents in Seed Lots (10).
In the light of the availability of alternatives methods that are at least as relevant
and reliable as the animal test, it is recommended, in accordance with obligations
under the European Convention for laboratory animal protection (26) and Council
Directive 86/609/EEC (27), that the test for
extraneous agents on batches of the finished
product using chicks should be deleted.
A Good Manufacturing Practice (GMP)
environment minimises the risk of contamination of the product. Extraneous agents
testing should be performed on the agents
which are handled in the individual production facility. This would significantly reduce
the list of agents to be tested, and the
reduced number of tests to be carried out
might increase willingness to apply the nonanimal methods. In situations where such
viruses as Newcastle disease virus (NDV),
infectious bursal disease virus (IBDV), infectious laryngotracheitis virus (ILTV), and
infectious bronchitis virus (IBV), which are
not included in the table given in the revised
text (11), are handled in the production facility, they should be considered for extraneous
agents testing. PCR methods are available
for NDV (21), IBV (22) and ILTV (23), and
are under development for IBDV (Table III).
Avian Live Viral Vaccines
The revised monographs on avian live viral
vaccines are divided into three sections: Definition, Production and Batch Tests. The Definition section briefly describes the vaccine
and its scope. The Production section covers
the requirements on the preparation of the
vaccine, the substrate for virus propagation,
seed lots, and choice of vaccine virus, which
includes tests for safety, increase in viru-
245
lence and immunogenicity. These tests have
to be fulfilled for the licensing of a vaccine.
Most of them involve the use of animals. The
Batch Tests section includes the requirements that have to be met by each batch to
be released. Animals are used for the tests
for extraneous agents and for safety testing.
General comments
A number of animal tests are stipulated for
each monograph, and have been harmonised
during revision (for example, the number of
animals used for the safety testing).
Production
Safety testing.
As the text reads in the revised monographs,
at least 20 animals are inoculated with a 10fold dose of the test vaccine for each route
and method of administration and category of
animal. The animals are observed for 21
days, and should not show noticeable clinical
signs of disease or die from causes attributable to the vaccine virus. As the text reads
now, far more animals would be used than
necessary, since the safety test should be performed in different categories of chicken. The
general opinion was that differences in the
susceptibility between the various categories
versus the various viruses are not evident,
except in the case of IBDV. SPF chickens are
regarded as a fully susceptible category of
animals. In addition, the monograph does not
clearly define the categories, and the current
vague requirement is therefore subject to
individual interpretation (for example, broilers, layers, different breeds, and white or
brown lineage).
It is recommended that SPF chickens
should be the only category of animals to be
used.
During revision of the monographs, the
observation period has been prolonged from 14
days to 21 days. If this prolongation is adopted,
all the vaccines on the market will have to be
retested, which would require a large number
of chickens. Retesting of these vaccines is not
considered to be appropriate, since they have
already been tested with a 14-day observation
period during their development and, in addition, batch safety tests have been carried out
with a 21-day observation period.
It is recommended that only newly
licensed vaccines need be tested with a 21day observation period.
246
L. Bruckner et al.
Table III: Agents against which serum samples should be tested and available
non-animal methods for extraneous agents testing of batches of the
finished product (monograph 2.6.4)
Agent
Alternative method
Status and Reference
Infectious avian encephalomyelitis Eggs (intravitelline injection)
Covered by paragraph 2.6.3.1 in (10)
Avian leucosis viruses
Cell culture
PCR
Covered by paragraph 2.6.3.3 in (10)
Available (17)
Avian nephritis virus
Kidney cell culture
Covered by paragraph 2.6.3.2 of (10)
Avian reticuloendotheliosis virus
Chick (or duck) embryo fibroblasts Covered by paragraph 2.6.3.4 in (10)
+ immunofluorescence;
PCR
Available (19)
Egg drop syndrome virus
Production substrate
(fibroblasts, duck eggs)
or other sensitive cells
Available for substrates
given in 2.6.3.1 and
2.6.3.2 in (10)
Chicken anaemia virus
Cells;
PCR
Covered by paragraph 2.6.3.5 in (10)
Available (20)
Marek’s disease virus
Chick embryo fibroblasts
Covered by paragraph 2.6.3.2 in (10)
Newcastle disease virus
Eggs (intra-allantoic inoculation)
PCR
Covered by paragraph 2.6.3.1 in (10)
Available (21)
Infectious bursal disease virus
Eggs (chorio-allantoic membrane
and intra-allantoic inoculation)
PCR
Covered by paragraph
2.6.3.1 in (10)
Under development
Infectious bronchitis virus
Eggs (intra-allantoic inoculation)
PCR
Covered by paragraph 2.6.3.1 in (10)
Available (22)
Infectious laryngotracheitis virus
Eggs (chorio-allantoic membrane
inoculation)
PCR
Covered by paragraph 2.6.3.1 in (10)
Salmonella pullorum
Culture
Covered by the sterility
test stipulated in (25)
Turkey rhinotracheitis virus
Vero cells, chick embryo
fibroblasts
Available for substrates
given in 2.6.3.2 in (10)
Chlamydia spp.
Eggs (intravitelline injection)
Covered by paragraph 2.6.3.1 in (10)
Avian infectious hemorrhagic
enteritis virus
PCR
Available (24)
Avian paramyxovirus-3
Eggs
Covered by paragraph in 2.6.3.1
in (10)
Duck/goose parvoviruses
Duck eggs/duck embryo
fibroblasts
Available for substrates given in
2.6.3.1 and 2.6.3.2 in (10)
Duck enteritis virus
Duck embryo fibroblasts
Available for substrates given in
2.6.3.1 and 2.6.3.2 in (10)
Duck hepatitis viruses I and II
Eggs
Covered by paragraph 2.6.3.1 in (10)
Available (23)
ECVAM Workshop 41: avian vaccines
Increase in virulence
Avian live viral vaccines contain attenuated
virus, which means that their virulence is
reduced. During its spread (passaging), the
virus can increase in virulence and can eventually induce an outbreak of the disease.
Only sufficiently attenuated strains, which
show no increase of virulence, should be used
for the production of a vaccine.
Increase in virulence is tested by artificially
passaging the virus at least five times in five
1-day-old chicks. The individual monographs
state that a quantity of the vaccine virus that
will allow maximum recovery of virus has to
be used for the passaging. The determination
of this dose (maximum recovery) would
require many animals. However, the exact
determination of this dose is not relevant for
the outcome of the test, and the use of additional animals is therefore not justified.
The word “maximum” should be deleted
from avian live vaccine monographs.
With regard to the state-of-the-art of the
test, two comments were made: animals of
the minimum age recommended for vaccination should be used for the test, and passaging under natural conditions should be
allowed for viruses showing natural spread,
since artificial passaging is very often not as
effective as natural passaging from a group
of animals to another group of animals.
Immunogenicity
Since the immunogenicity tests vary in the
individual monographs, depending on the
scope of the vaccine, a general description of
the test is not possible. However, some of the
individual monographs require that the test
should be performed in “each category of
chickens for which the vaccine is intended”.
As explained under Safety testing, the general opinion was that differences in the susceptibility between the various categories
versus the various viruses are not evident,
except for IBDV. SPF chickens are regarded
as a fully susceptible category of animals. It
is therefore not necessary to perform the test
with different categories.
Batch tests
Two of the tests prescribed for the batch
quality control of avian live virus vaccines,
which have to be performed on each batch of
the finished product before its release,
involve the use of animals: the test for extraneous agents and the batch safety test.
247
Extraneous agents test
The monographs stipulate that the vaccines
have to comply with the tests for extraneous
agents in batches of finished products (2.6.4).
As explained earlier under Testing for Extraneous Agents in Avian Live Viral Vaccines,
the workshop participants recommend the
replacement of the extraneous agent test in
chicks by alternative test methods. On the
basis of the figures given above, the release
of 1800 batches/year by four manufacturers,
and the use of ten chicks in the test, a total
reduction of 18,000 animals could be
achieved by deleting this test.
Batch safety test
At least ten chickens are inoculated by a given
route with ten doses of the vaccine to be tested.
The animals are observed for 21 days and, for
compliance with the requirements, should not
show “abnormal clinical signs or die from
causes not attributable to the vaccine”.
This safety test in the target animal is
required by most of the Europoean Pharmacopoeia monographs and various EU guidelines on veterinary vaccines. Its initial
purpose was the detection of non-specific contamination. During recent years, the relevance of the safety test has increasingly been
questioned (28–30), because the introduction
of Good Laboratory Practice and Good Manufacturing Practice into the manufacture of
vaccines has significantly increased the safety
and quality of the products, and contamination with non-specific agents should not
occur. Two studies are currently being performed by a working group at the Paul
Ehrlich Institute (Langen, Germany) and by
AGAATI (with the financial support of the
European Commission through ECVAM).
Data on the target animal safety test are
being collected from vaccine manufacturers
and control authorities in Europe, then
analysed retrospectively. The results concerning poultry vaccines show that all batches
tested in the last few years passed the test.
Passing the test had no influence on the number of the reports on vaccinovigilance collected from the field. Therefore, the workshop
participants questioned the purpose and relevance of this test, and recent experience has
shown that the test is not capable of detecting
major problems with vaccine batches.
Comparison of the number of animals stipulated for the target animals safety test in veterinary monographs reveals that two animals
are required for mammalian vaccines, but at
248
least ten are necessary for poultry vaccines.
There is no scientific or statistical rationale for
this difference. The data given in Appendix 1
show that, in order to obtain statistically reliable results, it would be necessary to increase
the numbers of animals markedly. For the
given requirement (ten out of ten animals do
not show abnormal clinical signs or die), it can
be concluded with 95% confidence that 74.1%
of the true population would not show abnormal clinical signs or die. A sample size of at
least 14 animals would be required to conclude
with 95% confidence that 80% of the true population met the requirement.
However, in the interests of animal welfare and good science, and for economic rea sons, such an increase cannot be justified for
a test which might no longer be relevant.
In the opinion of the workshop participants, the batch safety test should be
restricted to two situations: a) for demonstration of consistency of production, when
the batch safety test should be carried out on
a limited number of batches; and b) after relevant changes in the production process (for
example, new batches of critical components
of the final product), when the batch safety
test should be carried out on a limited number of batches.
In all other cases, the need for batch safety
testing is not justified. Therefore, it is recommended that the batch safety test should
be deleted from the individual monographs
on avian vaccines.
On the basis of the figures given above, the
release of 1800 batches/year by four manufacturers, and the use of ten chicks in the
test, a total reduction of 18,000 animals used
could be achieved by deleting this test.
Humane endpoints
Whenever applicable to a test stipulated in
these monographs, animals showing typical
signs of disease should be humanely killed to
avoid unnecessary suffering.
Specific comments on individual
monographs
Most of the specific comments on avian live
vaccines apply to the tests stipulated for the
choice of the vaccine strain in the Production
section. In general, animal tests are required
for assessing safety, increase in virulence, and
immunogenicity. Depending on the vaccine
virus, additional animal tests are included in
L. Bruckner et al.
the monographs, which should cover certain
aspects of safety and immunogenicity.
Avian Infectious Bronchitis Vaccine (Live)
A safety test for the reproductive tract
should be conducted during development, if
the vaccine is intended to be used for hens;
however, a detailed design of the test is not
given. The participants of the workshop
underlined the need for a defined model, and
forwarded a proposal to the European Pharmacopeia Commission.
Two animal tests are stipulated for the
immunogenicity testing of this vaccine.
Equivalent information on the vaccine strain
can be achieved with either of the tests. It is
therefore recommended that the monograph
should clearly state that performance of one
of the tests is sufficient.
Avian Infectious Bursal Disease Vaccine
(Live).
As already stated in the general comments,
SPF chickens might be too sensitive for the
safety testing of this vaccine. The monograph should state that “commercial animals
with representative levels of maternally
derived antibodies should be used”. This recommendation applies also to the test for
damage to the bursa of Fabricius and the test
for immunosuppression, which are two additional tests in animals to be performed during development.
The present test for damage to the bursa
of Fabricius involves 20 chickens, which are
inoculated with the vaccine virus; after given
observation periods, the animals are killed
and the bursa of Fabricius is examined histologically. Clear criteria for estimation of the
degree of the bursal damage are not given,
which might lead to subjective interpretation
of the test. It is recommended that clear criteria should be established and incorporated
into the test protocol.
The monograph includes two tests for
immunosuppression. The first test is a
potency test for Newcastle disease (ND) vaccines carried out in groups of chickens inoculated with: a) the vaccine virus and a given
ND vaccine; b) the ND vaccine only; or c) no
vaccine. The three groups of animals are
challenged with NDV and the protection
rates are compared. This test differs significantly from the potency test given in the
monograph on Newcastle Disease Vaccine
(Live) with regard to the challenge strain
used and the observation period. Since there
ECVAM Workshop 41: avian vaccines
is no scientific rationale for this difference,
this test should be carried out as the
immunogenicity test stipulated for ND live
vaccines.
The second test for immunosuppression
stipulated by the monograph is carried out
with 20 chickens, of which 10 are immunised
with the vaccine virus. The animals are inoculated with Brucella abortus and the
immune responses of the immunised and
non-immunised groups to B. abortus are
compared. The workshop participants criticised the lack of evaluation criteria for this
test, and the fact that vaccination against B.
abortus is not performed in practice. In addition, the test cannot provide additional information on any immunosuppressive effects of
the vaccine strain.
There was agreement that the first test is
better for this purpose, and it is recommended
that the second test should be deleted.
Increase in virulence should also be evaluated with two animal tests after passaging of
the vaccine virus, the safety test and the test
for damage to the bursa of Fabricius. However, performance of the latter test appears
to be sufficient for this purpose, since it is
more sensitive than the safety test.
Avian Infectious Encephalomyelitis Vaccine
(Live)
The test for intracerebral virulence should
be deleted, since it cannot discriminate
between attenuated and virulent virus
strains, due to the inappropriate 8-day observation period. Prolongation of the observation period might improve the test, but
vaccines that are currently in use and have
proved to be efficacious would be rejected. In
addition, the vaccine is mainly intended for
use in animals above the age of 10 weeks, at
which age, the animals are no longer susceptible to the agent.
Two tests are required for the testing of
immunogenicity, the test for active immunity in the chicken and the test for passive
immunity in chicks. There is no need to
demonstrate active immunity in adult chickens, since it is not relevant to the purpose of
this vaccine. Deletion of the test for active
immunity in adult chickens is therefore recommended.
The purpose of the vaccine is protection
against vertical transmission and the conferment of passive immunity. Conferment of passive immunity could be tested by
demonstrating the neutralisation of mater-
249
nally derived or egg yolk-derived antibodies.
An embryonated-egg challenge test (31)
should be incorporated into the monograph
for this purpose.
In the Batch tests section, the virus titre of
the vaccine can be estimated by titration in
embryonated hens’ eggs or in cell cultures.
However,
titration
of
the
avian
encephalomyelitis virus in tissue culture is not
yet feasible. In order to permit titration in
eggs, the monograph should state “suitable in
vitro methods” instead of “cell cultures”.
Avian Infectious Laryngotracheitis Vaccine
(Live).
See General comments.
Avian Viral Tenosynovitis Vaccine (Live)
It is questionable whether the proposed test
for immunogenicity reflects the use of the
vaccine in the field. Live tenosynovitis vaccine is usually only applied for the priming of
chicks. Boosting is performed with an inactivated vaccine. It cannot be expected that vaccines, which are recommended for use with a
booster injection, will confer protection if
used for priming alone.
In the Batch tests section, immunofluorescence methods should be allowed for the
identification of the vaccine, since neutralisation of tenosynovitis virus with antiserum
might be difficult or even impossible.
Duck Viral Hepatitis type 1 Vaccine (Live)
It is recommended that the tests stipulated in
the monograph are carried out with only one
species of duck, namely, the domestic duck
(Anas platyrhynchus), since differences in susceptibility between the various species of
ducks versus duck hepatitis virus 1 are not
evident. In the immunogenicity test, which is
performed with vaccines for active immunisation of ducklings, virus shedding in the faeces
should be used for test evaluation, in addition
to observation for clinical signs and mortality.
Fowl-pox Vaccine (Live)
In the test for increase of virulence, the passage material is administered by cutaneous
scarification of the chicken comb. Since this
could lead to cannibalism, it is recommended
that scarification of other unfeathered parts
of the body should be permitted.
Newcastle Disease Vaccine (Live)
This monograph stipulates that nine passages
should be performed in the test for increase in
virulence; however, the NDV is no more likely
250
to revert to virulence than other avian viruses
are. Therefore, only five passages should be
required, as in the other monographs. For
detection of increased virulence, three tests
should be carried out with the unpassaged vaccine virus and the maximally passaged virus.
In addition to the two specific tests — the
intracerebral pathogenicity index and the test
for amino-acid sequence — the more-general
safety test is stipulated. The workshop participants agreed that the safety test provides neither additional information nor additional
assurance that reversion to virulence has not
occurred, and therefore should be deleted.
Two tests for immunogenicity are given in
the monograph, and should be applied
according to the use of the vaccine. The
design of the immunogenicity test for vaccines for use in chickens deviates from the
test protocol in the monograph currently
used. For vaccines on the market, the introduction of a new test protocol would require
retesting in animals, which would not
increase the quality of the vaccines, but
would unnecessarily increase the numbers of
animals used. Therefore, maintenance of the
current protocol is recommended.
With regard to vaccines for use in avian
species other than chickens, prolongation of
the observation period from 14 days to 21
days is recommended, since the development
of the disease could be different in birds
other than SPF chickens. In addition to
death and clinical signs, virus shedding in
faeces should be monitored, and the criteria
for a valid test should be modified accordingly.
Marek’s Disease Vaccine (Live)
The monograph stipulates that two safety
tests should be carried out during development. The first test can be regarded as a
safety test comparable with the ones
required by the other monographs. In addition to the modifications given above in the
general comments, the observation period
should be extended to 120 days. The purpose
of the second test is to demonstrate that SPF
chicks are susceptible to Marek’s disease
virus, and that lesions similar to those of
Marek’s disease virus do not occur naturally
in SPF chicks. Both criteria are fulfilled by
the available SPF chicks, and that these criteria have to be demonstrated for each product is not justified. Since the second test does
not provide any additional information to the
first test, its deletion is recommended.
L. Bruckner et al.
Inactivated Avian Vaccines
Four monographs on inactivated avian vaccines are included in the European Pharmacopoeia (Table I). Since the workshop was
focused on the live avian vaccines currently
under revision, only the current status of
alternative methods for the potency testing
of inactivated avian vaccines was discussed.
The potency of inactivated avian vaccines
has to be tested on each vaccine batch. This
requires a large number of animals and
involves suffering to the animals if, under
certain circumstances, challenge with the
infectious agent is required. In most cases,
serological methods are used for antibody
estimation after immunisation.
In recent years, efforts have been made to
develop antigen-estimation-based in vitro
methods which do not require animals. Most
progress has been achieved with models for
the potency testing of inactivated ND vaccines
(32–34). The approach used is based on the
quantification of the NDV antigens, the HNprotein and F-protein, which induce the production of neutralising antibodies after
vaccination. Two ELISA procedures have been
developed for this purpose. Preliminary
results show that the method is sensitive, specific and reproducible. There is a good correlation between the amount of virus estimated in
the vaccine and the antibodies detected in the
serum after immunisation (34).
Whether antigen quantification can be
used for the potency testing of inactivated
bursal disease vaccine is also now under
investigation.
The participants of the workshop recommended that further research activities for
the development of alternative methods for
the potency testing of inactivated avian vaccines should be encouraged and undertaken;
and that the in vitro method developed for
the potency testing of inactivated ND vaccines should be prevalidated/validated under
the auspices of independent institutions such
as ECVAM and the European Directorate for
the Quality of Medicines (EDQM).
Summary of Recommendations
Extraneous agents testing
1.
The tests for extraneous agents of seed lots
in chicks should be performed only once
during development, preferably on the
ECVAM Workshop 41: avian vaccines
251
master seed lot. If sufficient material of the
master seed lot is not available, testing
should be on a working seed lot, preferably
as close as possible to the master seed lot.
2.
The test for extraneous agents of seed
lots in chicks must include clinical observation of the animals and the testing of
the serum samples. It is recommended
that the sera of the test animals should
be stored under suitable conditions, as
long as seed material obtained from the
tested master seed lot is being used for
production of the vaccine. This will
facilitiate the testing for antibodies
against agents which were not known
when the master seed was established,
and will provide information on the possible contamination of the product with
agents discovered subsequently.
stipulated during production, since they
are regarded as a fully susceptible category of animal and differences between
the categories versus the various viruses
are not evident.
9.
In order to avoid unnecessary retesting
of already licensed vaccines, a 21-day
observation period for safety testing during production should be required only
for newly licensed vaccines.
10. For the outcome of increase in virulence
testing, it is not necessary that passaging of the virus is performed with a dose
which allows maximum recovery of the
virus. Therefore, the word “maximum”
should be deleted from avian live vaccine
monographs, since the use of additional
animals for the determination of the necessary virus dose is not justified.
3.
The list of agents against which the
serum samples should be tested, and the
list of test methods to be used, should
also include the agents and methods that
are given in the general monograph on
specific pathogen-free (SPF) eggs.
11. Animals of the minimum age recommended for vaccination should be used
for the test, and passaging under natural
conditions should be allowed for viruses
showing natural spread.
4.
With regard to turkey vaccines, the test
for extraneous agents of seed lots
against turkey lymphoproliferative disease virus should be deleted. There is
only a low incidence for the virus, and
the test currently performed has not
been validated.
12. The test for extraneous agents on
batches of the finished product using
chicks should be replaced with the available alternative test methods.
5.
The test for extraneous agents on
batches of the finished product in chicks
should be replaced with the available
non-animal methods.
6.
Efforts should be undertaken to further
develop and validate the PCR method for
the detection of avian infectious haemorrhagic enteritis virus.
7.
Efforts should be made to validate scientifically the available alternative methods
which have not yet been incorporated into
Monograph 2.6.3. Avian Viral Vaccines:
Tests for Extraneous Agents in Seed Lots ,
and to promote their acceptance.
Avian live viral vaccines
General recommendations with regard to the
Production section
8.
Only SPF chickens should to be used for
the safety and immunogenicity tests
General recommendations to the Batch test
section
13. The batch safety test should be deleted
from the individual monographs on
avian vaccines. It should only be allowed
for two situations: a) for demonstration
of consistency of production, when the
batch safety test should be carried out on
a limited number of batches; and b) after
relevant changes in the production
process (for example, new batches of
critical components of the final product),
when the batch safety test should be carried out on a limited number of batches.
14. Whenever applicable to a test stipulated
in these monographs, animals showing
typical signs of disease should be
humanely killed to avoid unnecessary
suffering.
Specific recommendations to individual
monographs
15. The monograph, Avian Infectious Bronchitis Vaccine (Live), should include a
detailed design for the safety test for the
252
L. Bruckner et al.
reproductive tract stipulated for vaccines intended to be used in hens. The
proposal forwarded by the participants
in the workshop should be considered by
the Group of Experts 15V of the European Pharmacopoeia Commission.
16. It should be clearly stated in the monograph, Avian Infectious Bronchitis Vaccine (Live), that only one of the two tests
for immunogenicity needs to be performed.
17. Clear criteria should be stated in the
monograph, Avian Infectious Bursal
Disease Vaccine (Live), for the histological examination of the bursa of Fabricius
in the test for damage to the bursa of
Fabricius.
18. In the monograph, Avian Infectious Bursal Disease Vaccine (Live), of the tests
for immunosuppression, only the
potency test for ND vaccines should be
stipulated. The second test for immunosuppression, involving challenge with B.
abortus, should be deleted.
19. Performance of the test for damage to
the bursa of Fabricius appears to be sufficient for increase in virulence testing of
avian infectious bursal disease live vaccine. The safety test stipulated for this
purpose should be deleted from the
monograph, Avian Infectious Bursal
Disease Vaccine (Live).
20. The test for intracerebral virulence in
the monograph, Avian Infectious
Encephalomyelitis Vaccine (Live), should
be deleted, since it cannot discriminate
between attenuated and virulent virus
strains, due to the inappropriate 8-day
observation period. Prolongation of the
observation period might improve the
test, but would lead to the rejection of
vaccines that are currently in use and
have proven to be efficacious.
21. Deletion of the test for active immunity
in the chicken from the monograph,
Avian Infectious Encephalomyelitis Vaccine (Live), is recommended. Since one of
the purposes of this vaccine is protection
against vertical transmission, there is no
need to demonstrate active immunity in
adult chickens.
22. The second purpose of avian infectious
encephalomyelitis live vaccines, which is
conferment of passive immunity, could be
tested by demonstrating the neutralisation
of maternally derived or egg yolk-derived
antibodies. An embryonated-egg challenge
test should be incorporated into the monograph, Avian Infectious Encephalomyelitis
Vaccine, for this purpose.
23. It is recommended that the tests stipulated
in the monograph, Duck Viral Hepatitis
type 1 Vaccine (Live), should be carried out
with only one species of duck, namely, the
domestic duck (A. platyrhynchus).
24. In the test for increase of virulence in the
monograph, Fowl-pox Vaccine (Live), the
passage material is administered by cutaneous scarification of the chicken comb.
Since this could lead to cannibalism, it is
recommended that scarification of other
parts of the unfeathered body be permitted.
25. In the monograph, Newcastle Disease Vaccine (Live), only five passages should be
required for the test for increase in virulence, as in the other monographs for
avian live vaccines.
26. For detection of an increase in virulence,
it is sufficient to estimate the intracerebral pathogenicity index and to conduct
the amino-acid sequence test. The third
test stipulated for this purpose, the safety
test, should be deleted from the monograph, Newcastle Disease Vaccine (Live).
27. The currently used immunogenicity test
for vaccines for use in chickens should not
be modified as proposed in the revised
monograph, Newcastle Disease Vaccine
(Live), since the modification would
require retesting of the vaccines on the
market, and, although this would not
increase the quality of the vaccines, it
would unnecessarily increase the numbers of animals used.
28. The second safety test included in the
revised monograph, Marek’s Disease Vaccine (Live), does not provide additional
information and should be deleted. One
safety test in an SPF chicken, with an
observation period of 120 days, would be
appropriate.
Inactivated Avian Vaccines
29. Further research activities for the development of alternative methods for the
ECVAM Workshop 41: avian vaccines
253
potency testing of inactivated avian vaccines should be undertaken and encouraged.
30. The in vitro method developed for the
potency testing of inactivated ND vaccines should be prevalidated/validated
under the auspices of independent institutions such as ECVAM and EDQM.
References
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Council of Europe (1999). Avian Infectious Bursal
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Council of Europe (1999). Avian Infectious Bron chitis Vaccine (Live). Pharmeuropa 11, 163–166.
Council of Europe (1999). Duck Viral Hepatitis
Type 1 Vaccine (Live). Pharmeuropa 11, 166–168.
Council of Europe (1999). Fowl-pox Vaccine
(Live). Pharmeuropa 11, 168–170.
Council of Europe (1999). Newcastle Disease Vaccine (Live). Pharmeuropa 11, 170–172.
Council of Europe (1999). Marek’s Disease Vaccine (Live). Pharmeuropa 11, 172–175.
Council of Europe (1999). Avian Infectious Laryn gotracheitis Vaccine (Live). Pharmeuropa 11,
175–177.
Council of Europe (1999). 2.6.3. Avian Viral Vaccines: Tests for Extraneous Agents in Seed Lots.
Pharmeuropa 11, 188–191.
Council of Europe (1999). 2.6.4. Avian Live Virus
Vaccines: Tests for Extraneous Agents in Batches
of Finished Product. Pharmeuropa 11, 191–193.
Council of Europe (1999). Avian Viral Tenosynovitis Vaccine (Live). Pharmeuropa 11, 156–158.
Council of Europe (1997). Avian Infectious Bron chitis Vaccine (Inactivated). In European Pharmacopoeia, 3rd edn. Strasbourg, France: Council
of Europe.
Council of Europe (1997). Avian Infectious Bursal
Vaccine (Inactivated). In European Pharmacopoeia, 3rd edn. Strasbourg, France: Council of
Europe.
Council of Europe (1998). Egg Drop Syndrome ’76
Vaccine (Inactivated). European Pharmacopoeia,
3rd edn, Suppl. Strasbourg, France: Council of
Europe.
Council of Europe (1997). Newcastle Disease Vaccine (Inactivated). European Pharmacopoeia, 3rd
edn. Strasbourg, France: Council of Europe.
Häuptli, D., Bruckner, L. & Ottiger, H.P. (1997).
Use of reverse transcriptase polymerase chain
reaction for detection of vaccine contamination by
avian leukosis virus. Journal of Virological Methods 66, 77–81.
Council of Europe (1997). 5.2.2. Chicken flocks
free from specified pathogens for the production
and quality control of vaccines. European Pharmacopoeia, 3rd edn. Strasbourg, France: Council
of Europe.
Tagaki, M., Ishikawa, K., Nagai, H., Sasaki, T.,
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Gotoh, K. & Koyama, H. (1996). Detection of contamination of vaccines with the reticuloendotheliosis virus by reverse transcriptase polymerase
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Falcone, E., Tarantino, M., Massi, P., Di
Pasquale, I., Vignolo, E. & Tollis, M. (2000).
Detection of vaccine contamination by chicken
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Stäuber, N., Brechtbühl, K. Bruckner, L. & Hofmann, M.A. (1995). Detection of Newcastle disease virus in poultry vaccines using polymerase
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Falcone, E., D’Amore, E., Di Trani, L., Puzelli, S.
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Hess, M., Raue, R., & Fafez, H.M. (1999). PCR for
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Roberts, B. & Lucken, R.N. (1996). Reducing the
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Zeegers, J.J.W., de Vries, W.F. & Remie, R.
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32. Maas R.A., Oei, H.L., Kemper, S., Koch, G. &
Visser, L. (1998). The use of homologous virus in
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Halder), in press.
ECVAM Workshop 41: avian vaccines
255
Appendix 1
Sample Size Determination with Dichotomous Responses
Manfred Wilhelm
Biostatistics Section, Paul Ehrlich Institute, 63207 Langen, Germany
Dichotomous Response
A variable of interest in an animal trial that
is designed to result in two categories (for
example, survival/death, presence/absence of
disease, positive/negative outcome) is called
a dichotomous response; it can be quantified
by rates and proportions. Thresholds of test
procedures, as specified in monographs on
avian live virus vaccines, are usually given as
rates that allow the decision to be made: is
the test valid and/or does the vaccine virus
comply with the test?
Statistical Inference
In general, statistical inference is the procedure by which we reach a conclusion about a
population on the basis of the information
contained in a sample that has been randomly drawn from that population. Inference from a sample to a population relies on
the assumption that the animals in the random sample are representative of all animals
in the population. The empirical rate ∧
p∧ (0% ≤
∧
∧
p ≤ 100%) of positive (negative) test results
in a sample of animals due to monograph
procedures is an estimate of the true rate of
positive (negative) test results in the population. Since ∧
p∧ is only a point estimate, we have
to take into consideration its precision by
calculating its confidence limits, which
depend heavily on sample size. With regard
to these principles of statistics, it remains
unclear whether the thresholds specified in
the monographs under investigation are
intended to be controlled in the sample or in
the population.
Confidence limit and sample size
Assuming a Binomial distribution, the exact
two-sided lower (1 – α)% confidence limit for
an observed proportion of p ≡ x/n < 100%, is
given (1) by:
x
πlower, 1–α = x + (n – x + 1) F
[1]
df1 ,df2, 1 –α/2
where x = number of positive (negative)
events (x < n), n = sample size, Fdf1,df2, 1–α/2
= 1 – α/2)% quantile of F distribution with
df1 = 2(n – x + 1) and df2 = 2x degrees of
freedom.
In the case of x = n, formula [1] reduces to:
n
πlower, 1–α = n + F
[2]
2,2n,1–α
which is now the exact one-sided lower
(1 – α)% confidence limit for an observed pro∧
∧
portion of p = 100%.
Based on formulae [1] and [2], Figures 1–3
illustrate the relation between the sample
size and the exact two-sided and one-sided
∧
∧
lower 95% confidence limits for p
= 80%, p∧∧ =
90% and ∧
p∧ = 100% (2). In practice, determination of sample size has to be based on
visual inspection of graphs such as those in
Figures 1–3 or on statistical tables, as in (1).
Nevertheless, after having chosen an adequate sample size n visually, we can check it
by applying formulae [1] and [2].
Example of Newcastle disease B1 strain AHI
(PEI project # 92.2063.3-01.100, p. 13–29)
The efficacy test revealed that all of n = 22
vaccinated and challenged chicks survived
∧
∧
and did not show any signs of disease, i.e. p
= 22/22 ≅ 100%. Apparently, there is stated
∧
∧
an empirical test threshold of p = 90%, i.e. at
least x = 20 (> 0.9·22) animals have to survive without showing any signs of disease in
the sample. Given the least acceptable sam∧
∧
ple of size n = 22, i.e. p = 20/22 > 90%, from
[1] and F6,40,0.975 = 2.74, , it follows that:
256
L. Bruckner et al.
Figure 1: Lower 95% confidence limit for observed proportion of 80%
100
90
80
70
60
50
40
30
20
10
0
0
5
10
15
20
25
30
35 40
sample size
45
50
55
60
65
Figure 2: Lower 95% confidence limit for observed proportion of 90%
100
90
80
70
60
50
40
30
20
10
0
0
5
10
15
20
25
30
35 40
sample size
45
50
55
60
65
ECVAM Workshop 41: avian vaccines
257
Figure 3: Lower 95% confidence limit for observed proportion of 100%
100
90
80
70
60
50
40
30
20
10
0
0
5
10
15
20
25
30
35 40
sample size
45
50
55
60
65
20
πlower,0.95 = 20 + 3·2.74 = 0.708 ≅ 70.8% [3]
10
πlower,0.95 = 10 + 3.49 = 0.741 ≅ 74.1% [4]
Thus, if a proportion of at least 20 out of 22
chicks (> 90%) are observed to be healthy,
we can be 95% confident that the lowest possible true proportion of surviving chicks that
do not show any signs of disease in the population of all vaccinated chicks is 70.8%. By
using only a sample of n = 22 chicks, we cannot be 95% confident that a true 90% threshold of surviving birds which show no signs of
disease does hold in the population of all vaccinated chicks! Even in the actual situation
that all of n = 22 chicks were observed to be
healthy, a lower 95% confidence threshold of
90% would require at least n = 29 animals in
vaccine testing for efficacy, as can be seen
from Figure 3.
i.e. we can be 95% confident that the true proportion of surviving chicks that do not show any
serious clinical respiratory signs in the population of all chicks to be vaccinated is ≥ 74.1%, or
reversibly ≤ 25.9% of all chicks to be vaccinated
are expected to show signs of disease and/or die.
By using a sample of only n = 10 chicks, we cannot be 95% confident that a true threshold of,
say, 80% of surviving chicks that show no serious clinical respiratory signs can be expected in
the population of all vaccinated chicks! Again, as
can be seen from Figure 3, a lower 95% confidence limit of 80% (i.e. the desired true threshold in the population) requires at least n = 14
animals in vaccine testing for safety.
The safety test revealed that all of n = 10 vaccinated and challenged chicks survived and
did not show any serious clinical respiratory
signs, i.e. ∧
p∧ = 10/10 ≅ 100%. Due to [2] and
since F2,20,0.95 = 3.49, we get:
The sample size n and minimum number of
required positive (negative) events x out of n to
hold an exact one-sided or two-sided lower
confidence limit of at least π% in the population are:
Summary
258
L. Bruckner et al.
π = 80
n
n–x
14*–25
0
26–33
1
29*–53
0
54–69
1
34–40
2
41–47
3
48–54
4
55–61
5
π = 90
n
n–x
* smallest possible sample size n.
Discussion
The main objective in biostatistics is to make
statistical inferences from a random sample
(of animals) of a certain size to the underlying
population (of all such animals); the statistical validity of such conclusions is guaranteed
by choosing the right sample size.
Since it is apparently not the primary
objective of almost all monographs under
investigation to do significance testing, it is
not feasible to perform a sample size calculation, which is usually based on type I and II
errors, the underlying variability, the relevant effect size, the kind of testing hypotheses and methods, and so forth (3, 4). The
method for sample-size determination in this
case, where almost no information exists, is
based on confidence limits for estimated
rates of interest.
It is accepted that the monograph is not
intended to relate statistical conclusions from
safety and efficacy testing under experimental
conditions (for example, artificially high dose,
kind of application) to the animal populations
in the field, the statistical sample size considerations presented above are not applicable in
this context. A reasonable sample size based
on general requirements such as relevance,
cost and animal welfare should be used
instead. Without any power calculations, sample sizes of n ≥ 10 are recommended as a common guideline in such situations (4).
References
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MathSoft, 425pp.
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