Who TRS 530

This report contains the collective views of an international
group of experts and does not necessarily represent the deci-

sions or the stated policy of the World Health Organization.
WORLD HEALTH ORGANIZATION
TECHNICAL REPORT SERIES
No. 530
WHO EXPERT COMMITTEE ON
BIOLOGICAL STANDARDIZATION
Twenty-fifth Report
WORLD HEALTH ORGANIZATION
GENEVA
1973
World Health Organization 1973
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PRINTED IN SWITZERLAND
CONTENTS
PHARMACOLOGICAL SUBSTANCES
Antibiotics
1. Doxycycline
2. Minocycline
3. Neomycin .
4. Gramicidin S
Hormones, vitamins, enzymes, and miscellaneous substances
5. Glucagon
6. Heparin .
7. Vitamin D . . . . . . . . . . . . . . . . . .
8. Sulfarsphenamine, neoarsphenamine, oxophenarsine
9. Mel B (melarsoprol), dimercaprol, MSb
10. Thrombin . . . . . . . . .
11. Opacity Reference Preparation . .
IMMUNOLOGICAL SUBSTANCES
Antigens
12. Diphtheria toxoid, plain
13. Yellow fever vaccine.
14. Cholera vaccine
Antibodies
15. Human immunoglobulin IgE .....
16. Diphtheria antitoxin for flocculation test
17. Gas-gangrene antitoxins . . . . . . .
18. Gas-gangrene antitoxin (Clostridium histolyticumt
19. Anti-Salmonella pullorum sera. . . . . . . .
REQUIREMENTS FOR BIOLOGICAL SUBSTANCES
20. Requirements for inactivated influenza vaccine
21. Requirements for cholera vaccine . . . . . .
22. Requirements for rabies vaccine for human use
23. General requirements for the sterility of biological substances
ANNEXES
Page
5
6
6
6
6
7
7
8
8
8
9
9
10
10
10
II
II
12
12
12
13
13
13
Annex 1. Requirements for inactivated influenza vaccine (Addendum 1973) 15
Annex 2. Requirements for cholera vaccine (Revised 1968) (Addendum 1973) 18
Annex 3. Requirements for rabies vaccine for human use . . . . . . 22
Annex 4. General requirements for the sterility of biological substances
(Revised 1973) . . . . . . . . . . . . . . . . . . . . . . . .. 40
Annex 5. Requirements for biolo gical substances and other sets of recommendations 62
Annex 6. International standards and international reference preparations 64
INDEX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
3
WHO EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION
Geneva, 24-30 April 1973
Members:
Dr H. Cohen, Director, National Institute of Public Health, Bilthoven, Nether-
lands
Dr J. Desbordes, Director, Microbiology Section, National Public Health Laboratory,
Paris, France (Vice-Chairman)
Professor S. G. Dzagurov, Director, Tarasevic State Institute for the Standardization
and Control of Biological Preparations, Moscow, USSR
Professor D. G. Evans, Director, National Institute for Biological Standards and
Control, London, England (Chairman)
Dr Sutas Guptarak, Chief, Biological Division, The Government Pharmaceutical
Organization, Bangkok, Thailand
Dr P. Krag, Director, Department of Biological Standardization, Statens Serum-
institut, Copenhagen, Denmark
Dr H. Mirchamsy, Associate Director, Razi State Institute for Serum and Vaccine
Production, Teheran, Iran
Dr R. Murata, Director, The Second Department of Bacteriology, National Institute
of Health, Tokyo, Japan
Dr E. B. Seligmann, Jr, Director, Division of Control Activities, Bureau of Biologics,
Food and Drug Administration, Rockville, Md., USA
Dr M. Yu, Senior Pathologist, Department of Pathology, Singapore (Rapporteur)
Secretariat:
Dr A. S. Outschoorn, Chief Medical Officer, Biological Standardization, WHO,
Geneva, Switzerland (Secretary)
4
WHO EXPERT COMMITTEE ON
BIOLOGICAL STANDARDIZATION
Twenty-fifth Report
The WHO Expert Committee on Biological Standardization met in
Geneva from 24 to 30 April 1973. Dr T. A. Lambo, Assistant Director-
General, opened the Meeting on behalf of the Director-General. He
welcomed the Members of the Committee and thanked them for coming
to Geneva to participate in this Meeting. He drew attention to the long
history of biological standardization, an activity inherited by WHO from
the League of Nations Health Organization and the Interim Commission.
The WHO Expert Committees on Biological Standardization continue the
work carried out previously by the Permanent Commission on Biological
Standardization of the League and it was interesting to note that in the
year of WHO's 25th anniversary there was also convened the 25th Expert
Committee on Biological Standardization. He was sure that the discussions
of this Committee would be in keeping with the high standards and tradi-
tions maintained over the years.
PHARMACOLOGICAL SUBSTANCES
ANTIBIOTICS
1. Doxycycline
In regard to the authorization in the twenty-fourth report 1 for the
establishment of the preparation studied as the international reference
preparation of doxycycline the Committee was informed that it had not
yet been possible to establish this preparation. The Committee therefore
endorsed the previous authorization for the National Institute for Biolog-
ical Standards and Control, London, to establish the material as the Inter-
national Reference Preparation of Doxycycline on the basis of the results
of the collaborative assay and to define the international unit with the
agreement of the participants.
1 WId HItlz Org. techno Rep. Ser ., 1972, No. 486, p. 9.
5
2. Minocycline
The Committee was informed that the limited collaborative assay of the
preparation of minocycline referred to in the twenty-fourth report 1 with a
view to the establishment of an international reference preparation was in
progress.
3. Neomycin
The Committee noted 2 that, in accordance with the request in the
twenty-third report," the National Institute for Biological Standards and
Control, London, had obtained a preparation of neomycin that was more
representative of preparations of neomycin in use throughout the world
than the existing reference preparation. A collaborative assay had been
carried out and the results were being analysed.
The Committee authorized the National Institute for Biological Stand-
ards and Control to establish the material studied as the second Inter-
national Reference Preparation of Neomycin and to define the interna-
tional unit on the basis of the results of the collaborative assay with the
agreement of the participants.
4. Gramicidin S
Since replacement of the International Reference Preparation of Grami-
cidin S was no longer necessary because this antibiotic could be adequately
characterized by chemical and physical means, the Committee discontinued
this international reference preparation.
The Committee was informed that, in accordance with the request in
the twenty-fourth report," the WHO Secretariat was investigating the
possibility of providing this antibiotic in the form of a suitable chemical
reference substance.
HORMONES, VITAMINS, ENZYMES, AND MISCELLANEOUS
SUBSTANCES
5. Glucagon
The Committee noted 5 that the collaborative assay of the proposed
international standard for glucagon, referred to in the twenty-fourth
1 Wid Hlth Org. techno Rep. Ser., 1972, No. 486, P- 10.
2 Unpublished working document WHO/BS/73.1063.
3 Wid Hith Org. techno Rep. Ser ., 1971, No. 463, p. 11.
4 Wid Hltli Org. techno Rep. Ser., 1972, No. 486, p. 9.
6
report,' had been completed. The result had shown that the material was
suitable to serve as the international standard and the Committee therefore
established this material as the International Standard for Glucagon,
Porcine, for Bioassay. The Committee also noted 2 a proposal for the
definition of the international unit, which was equivalent to the only known
existing national unit and which was acceptable to the participants. The
Committee agreed with this proposal and defined the International Unit
for Glucagon, Porcine, for Bioassay as the activity contained in 4.5302 mg
of the International Standard for Glucagon, Porcine, for Bioassay.
6. Heparin
The Committee noted 3 that stocks of the second International Standard
for Heparin were nearly exhausted and agreed that it should be replaced.
A preparation of heparin of porcine mucosal origin had been examined in
a collaborative study of heparins from different sources 4 and found suit-
able to replace the current International Standard when this became nec-
essary. The Committee agreed that this preparation could serve as the
replacement without the necessity of a further collaborative assay. The
Committee therefore established the material as the third International
Standard for Heparin in replacement of the second international standard.
The Committee also noted 3 a proposal on which to base the unitage
assigned to the preparation from the results of the collaborative study.
The Committee was informed that this was acceptable to the participants
and therefore defined the International Unit for Heparin as the activity
contained in 0.005766 mg of the third International Standard for Heparin.
7. VitaminD
The Committee considered further the question of the discontinuation
of the International Standard for Vitamin D discussed in the twenty-fourth
report.' In response to the request in that report," the WHO Secretariat
had investigated the possibility of making vitamin D available as a chem-
ical reference substance and action was being taken with a view to providing
such a reference substance. As it would be advisable also to have available
for international use a well characterized, stable preparation of vitamin D
that could be used for biological assays, the Committee agreed that the
current International Standard should not be discontinued for the present.
1 Wid Hlth Org. techno Rep. Ser., 1972, No. 486, p. 11.
4 Bull. WId Hlth Org., 1970, 42, 129.
5 WId Hlth Org: techno Rep. Ser., 1972, No. 486, p. 12.
7
8. Sulfarsphenamine, Neoarsphenamlae, Oxophenarsine
The Committee was informed that, in response to the request in the
twenty-fourth report, 1 the WHO Secretariat had ascertained that there
was little or no use of the substances sulfarsphenamine, neoarsphenamine
and oxophenarsine in clinical medicine and hence no need for reference
materials. The Committee therefore discontinued the international ref-
erence preparations of these substances.
9. Mel B (Melarsoprol), Dimercaprol, MSb
The Committee noted 2 that, in response to the request in the twenty-
fourth report,' the WHO Secretariat had ascertained that there was a need
for reference material of Mel B (Melarsoprol) and of Dimercaprol for use
in toxicity tests. The Committee agreed therefore that it was advisable to
retain the International Reference Preparations of these two substances.
The WHO Secretariat had also ascertained that there was no interest
at present in the use of MSb.The Committee therefore discontinued this
international reference preparation.
In view, however, of current interest in research on trypanocidal drugs
as well as the possibility that there may be further work on the. use of anti-
monial compounds in filariasis, the Committee agreed that remaining
stocks of the discontinued preparation should be kept for possible use
in such studies.
10. Thrombin
The Committee noted 3 that studies of certain components of the blood
coagulation and fibrinolytic systems, referred to in the eighteenth4 and
nineteenth 5 reports, had been continued and that a collaborative study of
a preparation of human thrombin had been made with a view to assess-
ing its suitability for the assay of human and bovine thrombin prepara-
tions. In this study 5 preparations of thrombin, and a common substrate
of freeze-dried fibrinogen were provided. Various kinds of local substrates
were also included. The results showed that the preparation studied was
suitable for the assay of both human and bovine thrombin preparations.
There was, however, some indication that there were significant differences
in the potency estimates obtained with different species of the substrate
fibrinogen.
2 Unpublished working document WHOjBS/73.1068.
3 Unpublished working document WHOjBSj73.1069.
4. Wid Hlth Org; techno Rep. Ser., 1966, No. 329, p. 11.
5 Wid Hlth Org. techno Rep. Ser, 1967, No. 361, p. 13.
8
The Committee agreed that there was insufficient evidence at present for
the need for an international reference preparation of thrombin and
requested the WHO Secretariat to collect further information in order to
assess the need.
11. Opacity Reference Preparation
The Committee noted 1 the results of a study at the Statens Seruminsti-
tut, Copenhagen, in which certain comparisons had been made between
opacity determinations and estimation by weight of bacterial mass in
pertussis vaccine. These studies were made with the International Opacity
Reference Preparation and a number of samples of routine production
batches all made at the Statens Seruminstitut from the same strains of
Bordetella pertussis by the same procedure. The results showed that between
batches there was less variation in bacterial mass per millilitre of vaccine
than in opacity units as determined photoelectrically. The Committee
agreed that it would be of interest to undertake further investigations,
which might be carried out in individual laboratories, of the relationship
of various parameters of pertussis vaccine, including optical density and
weight of bacterial mass used, under differing conditions, with estimates of
immunizing potency and toxicity. Such determinations may also be useful
for calibrating photoelectric instruments for measuring optical density of
vaccines.
IMMUNOLOGICAL SUBST ANCES
ANTIGENS
12. Diphtheria Toxoid, Plain
In regard to the request in the twenty-fourth report 2 for information
on continued use of purified diphtheria toxoid as a non-adsorbed product,
the Committee noted 3 that the Statens Seruminstitut, Copenhagen, in
collaboration with the WHO Secretariat, had ascertained that non-adsorbed
diphtheria toxoid was in limited use. Some laboratories had also agreed
on the continuing need for the International Standard for control purposes,
particularly for combined vaccines. The Statens Seruminstitut would there-
fore obtain suitable material that could serve as a replacement for the
International Standard for Diphtheria Toxoid, Plain, and would arrange a
collaborative assay.
1 Unpublished working document WHO/BSj72.1061.
2 WId Hlth Org . techno Rep. Ser., 1972, No. 486, p. 14.
9
13. Yellow Fever Vaccine
The Committee noted 1 that certain collaborative research studies of
yellow fever vaccines were being arranged by the WHO Secretariat. They
were designed to develop a more practical method, using cell cultures
instead of mice, for titrating the virus content of such vaccines. The Com-
mittee also noted 1 that a further possible outcome of these studies might
be a suitable preparation of yellow fever vaccine to serve as a reference
preparation. Such material would be useful for the control of yellow fever
vaccines and should be considered in relation to the revision of the
Requirements for Yellow Fever Vaccine requested in the twenty-second
report."
14. Cholera Vaccine
The Committee considered certain problems in the use of the current
International Reference Preparations of Cholera Vaccine (Ogawa) and of
Cholera Vaccine (Inaba) in the evaluation of potency of cholera vaccine.
In view of the fact that there is recent evidence of a relationship between
the results of assays made in mice in one laboratory and protection of man
in field trials, the Committee agreed that it might be useful if international
units be assigned to these International Reference Preparations.
Since, however, in the international collaborative assay for the replace-
ment of the international reference preparations referred to in the twenty-
fourth report," a wide variation was obtained among laboratories in the
relative immunogenicities, the Committee asked the Statens Serumin-
stitut, Copenhagen, to collect further information to enable a decision to
be made.
ANTffiODIES
15. Human Immunoglobulin IgE
The Committee noted 4 the results of the collaborative studies of a
preparation of human immunoglobulin IgE referred to in the twenty-third
report." A further preparation of this immunoglobulin had also been made
and a collaborative assay of it had been completed. The results showed
that the latter preparation was suitable to serve as an international refer-
ence preparation for the assay of human immunoglobulin IgE prepara-
tions. The Committee therefore established the material as the Interna-
tional Reference Preparation of Human Immunoglobulin IgE.
1 Unpublished working document WHO/BS/73.IOn.
2 Wid Hltb Org. techno Rep. Ser., 1970, No. 444, p. 21.
4 Unpublished working document WHO/BS/73.I070.
5 Wid Hlth Org. techno Rep. Ser., 1971, No. 463, p, 21.
10
The Committee also noted 1 a proposal, which was agreeable to the
participants, for a. definition of the international unit, equivalent to the only
known national unit. The Committee therefore defined on this basis the
International Unit for Human Immunoglobulin IgE as the activity con-
tained in 0.006562 mg ofthe International Reference Preparation of Human
Immunoglobulin IgE.
16. Diphtheria Antitoxin for Flocculation Test
The Committee noted 2 that, in accordance with the authorization in the
twenty-fourth report," the material referred to had been established by the
Statens Seruminstitut, Copenhagen, as the fifth International Reference
Preparation of Diphtheria Antitoxin for Flocculation Test in replacement
of the fourth international reference preparation and that the Institute had
specified its flocculating activity as 1800 Lf equivalents per ampoule.
The Committee agreed that when this international reference preparation
is distributed a statement should also be issued to the effect that the rela-
tionship of antitoxic activity as determined by in vivo and in vitro methods
(in vivo[in vitro ratio) was not stated, since such a ratio depends on the
methods of determination and cannot be unequivocally defined.
17. Gas-Gangrene Antitoxins
The various International Standards for Gas-gangrene Antitoxins were
first established many years ago. The names of these standards were
originally assigned according to the practice of the time and are not in
conformity with the present principles of bacterial nomenclature; they are
also not named in a uniform manner.
The Committee therefore decided that the following International
Standards be renamed:
Old name
Gas-gangrene antitoxin (perfringens)
(Clostridium welchii type A
antitoxin)
Gas-gangrene antitoxin (vibrion septiquei
Gas-gangrene antitoxin (oedematiens)
Gas-gangrene antitoxin (histo1yticus)
Gas-gangrene antitoxin (Sorde1li)
New name
Gas-gangrene antitoxin (Clostridium
welchii type A)
septicum)
oedematiensi
histolyticum)
sordellii)
2 Unpublished working document WHO/BSj72.1056.
3 Wld Hlth Org. techno Rep. Ser ., 1972, No. 486, p. 17.
11
18. Gas-Gangrene Antitoxin (Clostridium histolyticum)
The Committee noted 1 that in accordance with the authorization in the
twenty-fourth report 2, the material referred to had been established by the
Statens Seruminstitut, Copenhagen, as the third International Standard for
Gas-Gangrene Antitoxin (Clostridium histolyticum) in replacement of the
second international standard and that the Institute had defined the Inter-
national Unit for Gas-Gangrene Antitoxin (Clostridium histolyticum) as
the activity contained in 0.2 mg of the third International Standard for Gas-
Gangrene Antitoxin (Clostridium histolyticum).
19. Anti-Salmonella pullorum Sera
The Committee noted 3 that the collaborative assay of the two prepara-
tions of anti-Salmonella pullorum serum referred to in the twenty-fourth
report 4 had been completed. The results showed that the preparations
were suitable to serve as international standards for assaying sera containing
Salmonella pullorum antibodies, one for antibodies of the standard form
S and the other for antibodies of the variant form V. The Committee
therefore established the material for the proposed standard S serum as the
International Standard for Anti-Salmonella pullorum Serum (Standard
Form S) and the material for the proposed standard V serum as the Inter-
national Standard for Anti-Salmonella pullorum Serum (Variant Form V).
The Committee also noted 3 a proposal, which was acceptable to the
participants, for the definition of the international unit for each of these
preparations. The Committee defined, on this basis, the International
Unit for Anti-Salmonella pullorum Serum (Standard Form S) as the activity
contained in 0.0838 mg of the International Standard for Anti-Salmonella
puflorum Serum (Standard Form S) and the International Unit for Anti-
Salmonella pullorum (Variant FormV) as the activity contained in 0.0814 mg
of the International Standard for Anti-Salmonella pullorum Serum (Variant
Form V).
REQUIREMENTS
FOR BIOLOGICAL SUBSTANCES
20. Requirements for Inactivated Influenza Vaccine
The Committee studied the proposed Addendum 5 to the Requirements
for Inactivated Influenza Vaccine (Requirements for Biological Substances
1 Unpublished working document WHOjBS/72.1055 Rev. 1.
3 Unpublished working document WHOjBSj73.1066.
4 Wid Hlth Org. techno Rep, Ser., 1972, No. 486, p. 18.
S Unpublished working document WHOjBSj72.1057 Rev. 1.
12
" -------- " ..-----_ .._-- ---
No. 17) 1 prepared by the WHO Secretariat in collaboration with various
experts. The Addendum was intended to enable specifications for haem-
agglutinin content in influenza virus vaccines to be made in terms of the
International Reference Preparation of Influenza Virus Haemagglutinin
(Type A).
The Committee, after making some modifications, adopted the Adden-
dum to these requirements and agreed that it should be annexed to the
present report (see Annex 1).
21. Requirements for Cholera Vaccine
The Committee studied the proposed Addendum 2 to the revised
Requirements for Cholera Vaccine (Requirements for Biological Sub-
stances No.4, Revised 1968) 3 prepared by the WHO Secretariat in collabo-
ration with a number of experts. These modifications were needed because
the international Reference Preparations of Cholera Vaccine (Ogawa) and
of Cholera Vaccine (Inaba) had been replaced since the revised require-
ments were formulated.
The Committee, after making some modifications, adopted the Adden-
dum to these requirements and agreed that it should be annexed to the
present report (see Annex 2).
22. Requirements for Rabies Vaccine for Human Use
The Committee studied 4 the proposed Requirements for Rabies Vaccine
for Human Use prepared by the WHO Secretariat in collaboration with a
number of experts. The Committee, after making some modifications,
agreed that the text of these requirements was satisfactory and that they
would be of value for the control of rabies vaccine for human use produced
in different countries.
The Committee adopted these requirements and agreed that they should
be annexed to the present report (see Annex 3).
23. General Requirements for the Sterility of Biological Substances
The Committee studied 5 the proposed General Requirements for the
Manufacture and Control of Sterile Pharmaceutical Preparations and
Biological Substances and for Sterility Testing which had been prepared by
the WHO Secretariat in collaboration with a number of experts. These
1 WId Hlth Org. techn, Rep. Ser, 1968, No. 384, Annex 2.
2 Unpublished working document WHO/BS/72.1059 Rev.I.
3 WId Hlth Org. techno Rep. Ser., 1969, No. 413, Annex 1.
4 Unpublished working document WHO/BS/72.1058 Rev.I.
5 Unpublished working document WHO/BSj73.1062; \VHOjPHARM/73.474.
13
proposed requirements had been prepared for consideration of the WHO
Expert Committee on Biological Standardization and the WHO Expert
Committee on Specifications for Pharmaceutical Preparations.
The Committee agreed that the formulation of such requirements by
these two Expert Committees, in liaison, would be useful for the control or'
sterility of relevant products in different countries, and made detailed
recommendations that could be considered at a later stage by the Expert
Committee on Specifications for Pharmaceutical Preparations.
In view, however, of the observation in the twenty-fourth report,' and
the need for an early revision of the Requirements for Biological Substances
No.6 (General Requirements for the Sterility of Biological Substances) 2,
the Committee agreed that the general requirements relevant to the sterility
of immunological biological substances should be considered for adoption
by the Committee at the present time.
The Committee therefore studied the material made available and,
after making certain modifications, prepared such requirements relating
to immunological biological substances. It was agreed that the require-
ments so prepared were satisfactory and that they would be useful for the
control of sterility of immunological biological substances produced in
different countries.
The Committee therefore adopted these revised general requirements and
agreed that they should be annexed to the present report (see Annex 4).
The Committee emphasized that when general requirements for sterility
were quoted in the requirements for individual biological products in the
series of Requirements for Biological Substances, the revised requirements
should apply.
ACKNOWLEDGEMENTS
The Committee wishes to record its thanks to the following members of the WHO
Secretariat for their special contributions to its deliberations:
Dr K. Bogel, Veterinary Public Health; Dr P. Bres, Virus Diseases; Dr W. C. Cockburn,
Chief, Virus Diseases; Dr M. Gonzalez-Pacheco, Biological Standardization; Mr K. O.
Wallen, Chief, Pharmaceuticals; and Dr Y. Watanabe, Bacterial Diseases.
2 Wid Hlth Org. techno Rep. Ser., 1960, No. 200, Annex.
14
Annex 1
REQUIREMENTS FOR INACTIVATED INFLUENZA VACCINE
(Requirements for Biological Substances No. 17)
Addendum 1973
The Requirements for Inactivated Influenza Vaccine were adopted by
the twentieth WHO Expert Committee on Biological Standardization
(Geneva, 25-30 September 1967).1 In these requirements it was specified
that tests for content of virus antigen in whole virus vaccines be made on
both monovalent bulk and final product, and that these tests be made in
comparison with the International Reference Preparation of Influenza
Haemagglutinin (Type A) (established in 1967) 2 or an equivalent reference
preparation approved by the national control authority. The purpose
was to enable the content of virus antigen to be expressed in terms of
international units.
International collaborative studies made after the establishment of the
international reference preparation were considered by the twenty-first and
twenty-second Expert Committees on Biological Standardization," These
showed that the international reference preparation, which was of Type A
virus haemagglutinin, was also suitable for tests of Type B virus haem-
agglutinin.s Further studies had also been made on the relation between
the immunizing effect, evaluated in various ways, of inactivated influenza
virus vaccines and their haemagglutinin content."
The twenty-first Expert Committee on Biological Standardization
requested that an addendum be prepared for the Requirements for Inac-
tivated Influenza Vaccine, taking into consideration the results of these
studies, since specification of the haemagglutinin content of influenza virus
vaccines in international units was now possible. In formulating this
addendum account has been taken of opinions and data received from the
experts listed below, whose assistance is gratefully acknowledged.
Professor A. de Barbieri, General Director, Istituto Sieroterapico Milanese" Serafino
Belfanti ", Milan, Italy
Dr W. C. Cockburn, Chief, Virus Diseases, WHO, Geneva, Switzerland
Dr H. Cohen, Director, National Institute of Public Health, Bilthoven, Netherlands
3 Wid Hlth Org. techno Rep. Ser., 1969, No. 413, p. 20; 1970, No. 444, p. 15.
4 Bull. Wid Hlth Org., 1971,45,473-486.
15
Mr V. F. Davey, Deputy Director (Technical), Commonwealth Serum Laboratories,
Parkville, Victoria, Australia
Paris, France
Professor D. G. Evans, Director, National Institute for Biological Standards and Control,
London, England
Dr Ch. B. Gerichter, Director, Division of Laboratories, Ministry of Health, Jerusalem,
Israel
Dr J. J. Graydon, Commonwealth Serum Laboratories, Parkville, Victoria, Australia
Professor G. Heymann, Federal Agency for Sera and Vaccines, Frankfurt-am-Main,
Federal Republic of Germany
Dr A. Krassnigg, Director-General of Public Health, Vienna, Austria
Dr W. G. Laver, Microbiology Department, The John Curtin School of Medical
Research, The Australian National University, Canberra, Australia
Dr R. Murray, Director, Division of Biologics Standards, National Institutes of Health,
Bethesda, Md., USA
Professor R. Negri, Chief, Laboratory of Microbiology, Istituto Superiore di Sanita,
Rome, Italy
Dr M. Pontecorvo, Director, Biological Division, Istituto Sieroterapico e Vaccinogeno
Toscano" Sclavo ", Siena, Italy
Dr G. C. Schild, Director, World Influenza Centre, National Institute for Medical
Research, Mill Hill, London, England
Dr J. R. Thayer, Chief Inspector, National Biological Standards Laboratory, Canberra,
Australia
The following amendments are made to the Requirements for Inactivated
Influenza Vaccine. It should be noted that, since these requirements apply
only to whole virus vaccines, the present addendum also applies only to
such vaccines.
Amendment 1
In Part A, section 3.5.3 Testfor content of virus antigen, insert after the
existing paragraph the following:
" The material passes the test if the content of haemagglutinin is not less
than 600 IV in the quantity corresponding to the largest recommended
single human dose, provided that not less than two-thirds of such virus
antigen is of Type A influenza virus, the remainder being of either Type A
or Type B.
There is now some indication that the protective activity of
influenza vaccines is related to their haemagglutinin content.
It is therefore advisable that vaccines should contain as much
haemagglutinin as has been found from experience to be pro-
tective and safe in man.
16
It is not possible to specify a maximum permissible content
of haemagglutinin for all kinds of vaccine. A content greater
than 600 IV may cause a high frequency of untoward reactions,
depending on the degree of purification of the vaccines. National
control authorities should decide on a maximum acceptable
content of antigen for each kind of vaccine...
Amendment 2
In Part A, section 8, Labelling, delete "the number of international
units or comparable units of haemagglutinin per dose for each strain" and
replace by " the number of international units of haemagglutinin for each
strain per largest recommended single human dose".
17
Annex 2
REQUIREMENTS FOR CHOLERA VACCINE
(Requirements for Biological Substances No.4)
Addendum1973
The requirements for cholera vaccine were adopted by a WHO Study
Group on Requirements for Biological Substances (1958).1 Arevised version
of the Requirements was adopted by the twenty-first Expert Committee
on Biological Standardization (1968).2 In the revised requirements it was
specified that tests of antigenicity be made on each vaccine lot, and that
these tests be made in comparison with the International Reference Prepara-
tions of Cholera Vaccine (Ogawa) and of Cholera Vaccine (Inaba) or
suitable national reference preparations. It was further specified that the
national control authority should provide or approve the reference prepara-
tion(s) to be used, the relationship of the preparation(s) to the corresponding
International Reference Preparations of Cholera Vaccines having been
previously established. In the active mouse protection test of antigenicity
in the revised requirements, an antigenicity ratio for each serotype, in terms
of the respective International Reference Preparation, was specified.
At the time the revised requirements for cholera vaccine were adopted,
the current International Reference Preparations of Cholera Vaccine
(Ogawa) and of Cholera Vaccine (Inaba) were those established in 1953.
The twentieth Expert Committee on Biological Standardization (1967),3
however, requested a collaborative assay of two monovalent cholera
vaccines whose antigenicity was greater than that of the relevant Inter-
national Reference Preparations and that resembled in antigenicity the
cholera vaccines then being produced. The twenty-fourth Expert Committee
on Biological Standardization (1971) 4 established the preparations studied
as the second International Reference Preparations of Cholera Vaccine
(Ogawa) and of Cholera Vaccine (Inaba) in replacement of the first inter-
national reference preparations.
The Expert Committee also pointed out that the provisions relating to
limits of antigenicity as determined in the active mouse protection test in
the revised requirements for cholera vaccine were no longer applicable and
requested the WHO Secretariat to arrange for suitable modifications to
1 Wid Hlth argo techno Rep. Ser., 1959, No. 179, Annex 2.
2 Wid Hlth argo techno Rep. Ser., 1969, No. 413, Annex 1.
3 Wid Hlth argo techno Rep. Ser., 1968, No. 384, p. 14.
4 Wid Hlth argo techno Rep. Ser., 1972, No. 486, p. 13.
18
these requirements. In formulating these modifications, account has been
taken of opinions and data received from the experts listed below, whose
assistance is gratefully acknowledged:
Dr D. R. Bangham, Division of Biological Standards, National Institute for Bio-
logical Standards and Control, London, England
Professor A. de Barbieri, General Director, Istituto Sieroterapico Milanese" Serafino
Belfanti ", Milan, Italy
Dr D. Barua, Medical Officer, Bacterial Diseases, WHO, Geneva, Switzerland
Dr H. Cohen, Director, National Institute of Public Health, Bilthoven, Netherlands
Dr B. Cvjetanovic, Chief Medical Officer, Bacterial Diseases, WHO, Geneva, Switzerland
Dr V. F. Davey, Deputy Director (Technical), Commonwealth Serum Laboratories,
Parkville, Australia
Mr I. Davidson, Central Veterinary Laboratory, Weybridge, Surrey, England
Paris, France
Professor D. G. Evans, Director, National Institute for Biological Standards and Control,
London, England
Dr J. C. Feeley, Chief, Bacterial Immunology Section, Bacteriology Branch, Center for
Disease Control, Atlanta, Ga., USA
Dr J. J. Graydon, Commonwealth Serum Laboratories, Parkville, Australia
Dr I. J60, " Human" Institute for Serobacteriological Production and Research, Buda-
pest, Hungary
Professor A. Lafontaine, Director, Institute of Hygiene and Epidemiology, Brussels,
Belgium
Mr J. W. Lightbown, Division of Biological Standards, National Institute for Biological
Standards and Control, London, England
Dr M. S. Nasution, Director, Perusahaan Negara "Bio Farma" (Pasteur Institute), Ban-
dung, Indonesia
Dr R. Netter, Director, Virology Section, National Public Health Laboratory, Paris,
France
Dr F. T. Perkins, Division of Immunological Products Control, National Institute for
Biological Standards and Control, London, England
Dr M. Pittman, 3133 Connecticut Avenue N.W., Washington, D.C., USA
Dr J. D. van Ramshorst, Chief, Department of Biological Standards, National Public
Health Laboratory, Bilthoven, Netherlands
Professor M. Saletti, Head, Microbiological Department, Istituto Sieroterapico e
Vaccinogeno Toscano" Sclavo ", Siena, Italy
Dr E. B. Seligmann, Jr, Chief, Division of Control Activities, Bureau of Biologics,
Dr J. Spaun, Deputy Director, Department of Biological Standardization, Statens
Seruminstitut, Copenhagen, Denmark
Dr A. F. B. Standfast, The Lister Institute of Preventive Medicine, Elstree, England
Dr J. S. Sumpaico, Director of Medical Bureau (Laboratories), Bureau of Research and
Laboratories, Department of Health, Manila, Philippines
19
/
Dr J. R. Thayer, Chief Inspector, National Biological Standards Laboratory, Canberra,
Australia
Dr W. Aeg, Timmerman, Blauwkapelseweg 29, De Bilt, Netherlands
Mr K. Uemura, Chief, Health Statistical Methodology, WHO, Geneva, Switzerland
Dr Y. Watanabe, Medical Officer, Bacterial Diseases, WHO, Geneva, Switzerland
Dr R. J. Wilson, Chairman and Director, Connaught Laboratories Limited, Ontario,
Canada
The requirements for antigenicity in this addendum have been formulated
on the basis that an individual lot of cholera vaccine is acceptable if it is
not significantly lower in antigenicity than the respective International
Reference Preparations of Cholera Vaccine. Related to the question of
significance is that of limits of error of the estimate of the antigenicity
ratio. In the existing requirements (1968) this question was not dealt with.
In the international collaborative assay 1 for the replacement of the inter-
national reference preparations of cholera vaccines, the participants had
assayed the antigenicity of the proposed replacements in parallel with the
existing international reference preparations; a wide variation in the
relative antigenicities was found. The confidence limits (%), however,
varied little between laboratories arid were apparently independent of the
estimates of relative antigenicity. The 95% confidence interval for each
serotype, was between 45% and 227% of the relative antigenicity when 3
tests were performed and the results combined. In an informal survey of a
number of cholera vaccines produced in different countries, arranged by the
WHO Secretariat, it was also found that there was a wide variation in the
relative antigenicities.
The following amendments are made to the revised Requirements for
Cholera Vaccine:
Amendment 1
In Part A, section 1.3 : International standards or reference preparations and
international units
Replace the whole section by the following:
" The second International Reference Preparation of Cholera
Vaccine (Inaba) and the second International Reference Prepara-
tion of Cholera Vaccine (Ogawa) were established in 1971.
2
Each preparation is dispensed in ampoules and each ampoule
contains freeze-dried material from 5 ml of monovalent vaccine
of the particular serotype. These preparations are intended for
calibrating reference preparations that are used in antigenicity
testing (see Part A, section 5.4).
1 Unpublished working document WHO/BS/72.1032 Rev. 1.
2 WId Hlth Org, techno Rep. Ser., 1972, No. 486, p. 13.
20
The third International Opacity Reference Preparation
(established in 1965)1 is dispensed in ampoules containing 15ml
of a suspension of Pyrex-glass particles in water (10 IV of
opacity per ml).
These reference preparations are in the custody of the
International Laboratory for Biological Standards, Statens
Seruminstitut, Copenhagen. Samples are distributed free of
charge, on request, to national control laboratories."
Amendment 2
In Part A, section 5.4. 1 : Active mouse protection test
Replace the first paragraph by the following:
"The antigenicity of the two serotypes of the vaccine is
compared with that of the relevant monovalent or divalent
reference preparations by immunization of mice and subsequent
challenge with a virulent strain of the appropriate serotype of
V. cholerae.
" For each test the dilutions of the laboratory reference prep-
aration should be such that they correspond in antigenicity to
the dilutions made from the relevant International Reference
Preparation, reconstituted with 5 ml of fluid per ampoule.
Similar dilutions are made of the test vaccine.
" For the antigenicity test of each serotype the vaccine and
the relevant reference preparation should be tested in parallel.
" In some countries for each test a divalent reference prep-
aration is used in the form that corresponds in antigenicity
to a mixture of equal volumes of each International Reference
Preparation reconstituted with 5 ml of fluid per ampoule."
Replace (c) by the following:
"(c) Estimate of relative antigenicity. The median effective
immunization dose (ED.
o
) of each vaccine is calculated by a
conventional method. The antigenicity ratio for each of the
respective challenges (Ogawa and Inaba) is determined, and the
confidence limits of the ratio calculated.
" The vaccine passes the test if (i) the antigenicity of the
largest recommended human dose is not significantly less than
that of 1 ml of the reconstituted International Reference Prep-
aration of each serotype, as determined by the antigenicity
ratio, and (ii) the precision of the test, as determined by the
confidence limits of the ratio, is acceptable.
" If the vaccine does not meet these criteria, the test may
be repeated. The results of all the tests performed should be
used in calculating the final result. "
1 WId Hlth Org. techn, Rep. SeT., 1966, No. 329, p. 21.
21
/
/
Annex 3
REQUIREMENTS FOR RABIES VACCINE FOR HUMAN USE 1
Page
Introduction . 22
General considerations 24
Part A: Manufacturing requirements
1. Definitions 27
2. General manufacturing requirements 29
3. Production control 29
4. Filling and containers . 36
5. Control tests on final product 36
6. Records. 37
7. Samples. 37
8. Labelling 37
9. Distribution and shipping 38
10. Storage and expiry date 38
Part B: National control requirements
1. General 38
2. Release and certification . 39
Introduction
The WHO monograph Laboratory Techniques in RabiesF which was
first published in 1954 and has since been twice revised, includes material
that has been a useful guide for the manufacture and testing of rabies
vaccine. It was not, howerer, written to serve as international require-
ments for the manufacture and control of rabies vaccines. The fifth and
1 Prepared by the following members of the WHO Secretariat:
Dr M. Abdussalam, Chief, Veterinary Public Health, WHO, Geneva, Switzerland;
Dr K. Bogel, Veterinary Public Health, WHO, Geneva, Switzerland; Professor D. G.
Evans, Director, National Institute for Biological Standards and Control, London,
England (Consultant); Dr M. Kaplan, Director, Office of Science and Technology,
WHO, Geneva, Switzerland; Dr R. Netter, Director, Virology Section, National Public
Health Laboratory, Paris, France (Consultant); Dr A. S. Outschoorn, Chief, Biological
Standardization, WHO, Geneva, Switzerland; Dr F. T. Perkins, Division of Immu-
nological Products Control, National Institute for Biological Standards and Control,
London, England (Consultant); Dr E. B. Seligmann, Jr, Chief, Division of Control
Activities, Bureau of Biologics, Food and Drug Administration, Rockville, Md., USA.
2 Kaplan M. M. & Koprowski, H., ed. (1973) Laboratory techniques in rabies,
3rd ed., Geneva, World Health Organization (Monograph Series, No. 23).
22
sixth WHO Expert Committees on Rabies 1, 2 recommended that WHO
should take steps to develop requirements for rabies vaccine and expand
studies on tissue culture vaccines. The twenty-first WHO Expert Com-
mittee on Biological Standardization 3 agreed that international require-
ments for rabies vaccine are needed and that it would be feasible to
formulate them, especially as an International Reference Preparation of
Rabies Vaccine has been established.
The following international requirements for rabies vaccine (for human
use) have been fitted into the framework adopted in the Requirements for
Biological Substances Nos. 1 to 21, already published by WHO,4 and in
drafting them, account has been taken of the opinions of consultants, the
regulations and requirements for the manufacture and control of rabies
vaccine that have been formulated in a number of countries, as well as
information from both published and unpublished reports. In addition,
opinions and data have been received from a number of experts, whose
assistance is acknowledged below:
Dr P. Atanasiu, Pasteur Institute, Paris, France
Professor A. de Barbieri, General Director, Istituto Sieroterapico Milanese " Serafino
Belfanti ", }'Iilan, Italy
Professor Benhassine, Director General, Pasteur Institute, Algiers, Algeria
Dr K. Berger, Director, Federal Vaccine Production Institute, Vienna, Austria
Mr Ian Davidson, Central Veterinary Laboratory, Weybridge, England
Paris, France
Professor G. Edsall, Head, Department of Microbiology, London School of Hygiene and
Tropical Medicine, London, England
Dr P. Fenje, Connaught Laboratories Limited, Ontario, Canada
Professor G. Heymann, Federal Agency of Sera and Vaccines, Frankfurt-am-Main,
Federal Republic of Germany
Dr Hilary Koprowski, Director, The Wistar Institute, Philadelphia, Pa., USA
Dr M. Majer, Head, Virus Vaccine Production Department, Swiss Serum and Vaccine
Institute, Berne, Switzerland
Dr H. Mirchamsy, Razi State Institute for Serum and Vaccine Production, Teheran, Iran
Dr M. Pontecorvo, Director, Biological Division, Istituto Sieroterapico e Vaccinogeno
Dr J. B. Shrivastav, Director General of Health Services, New Delhi, India
Dr A. K. Thomas, Director, Central Research Institute, Kasauli, India
Dr W. Aeg, Timmerman, Blauwkapelseweg 29, De Bilt, Netherlands
Dr N. Veeraraghavan, Director, Pasteur Institute of Southern India, Coonoor, India
1 Wld HIth Org. techn . Rep. Ser., 1966, No. 321, p. 12.
2 Wld Hlth Org : techno Rep. Ser., 1973, No. 523, pp. 21 and 45.
3 Wld Hlth Org. techno Rep. Ser., 1969, No. 413, p. 25.
4 For a list of references, see WId Hlth Org. techno Rep. Ser ., 1973, No. 530, p. 62.
23
General Considerations
Various types of vaccine are described in the fifth report of the WHO
Expert Committee on Rabies." Vaccines presently in use in man in most
countries are derived from nerve tissue, either brain or brain with spinal
cord. Vaccines prepared in chick or duck embryos are in use in a few
countries and vaccine produced in primary hamster kidney-cell cultures has
also been used. A number of vaccines produced from cell culture are being
studied experimentally but only a few have been used in man. Because of
the widespread manufacture and use of rabies vaccine from nerve tissue
and embryos and the comparatively limited use of cell culture vaccines at
this time, these international requirements for rabies vaccine have been
restricted to those vaccines that are produced in nerve tissues or in embryos.
Furthermore, in view of numerous problems created by the use of rabies
vaccines containing living virus they have also been restricted to inactivated
vaccines.
A number of different manufacturing and testing procedures are in
use in various countries. The procedures differ in the rabies virus strain
used, the species of animal used for propagation of the virus, the method
of inactivation, the preservatives added, the form in which the vaccine is
issued, and the methods for testing potency.
The passage histories of the different strains of virus being used for
production are not well documented. Such a strain should be one known
to produce vaccine that is antigenically active against classical rabies virus
strains." While many strains have been derived from the original Pasteur
strain, others used for production have been isolated recently from man or
animals. Strains used for production should be limited to what is termed
a " fixed" strain of virus. It is desirable that studies be made in order to
define qualitatively and quantitatively the properties of a " fixed" strain.
This is one that has a short, stable, and reproducible incubation period
when injected intracerebrally into rabbits. However, it can be demon-
strated that strains in use at the present time differ considerably in their
ability to produce rabies in experimental animals when inoculated by routes
other than intracerebral. The present requirements recommend that the
Pasteur strain of rabies" fixed" virus maintained only in rabbits by intra-
cerebral inoculation be used for production. The use of the seed lot system
has also been specified. It is recommended, however, that a specific anti-
rabies serum be produced and be made available as an aid to establishing
the identity and purity of the seed virus, since the International Standard
for Anti-Rabies Serum is not made available - and may not be suitable-
1 Wid Hlth Org; techno Rep. Ser., 1966, No. 321, p. 10.
2 Classical rabies virus strains are those belonging to serotype 1 of the rabies sub-
group of rhabdoviruses.
24
---- " ------
for this purpose. It is also desirable that studies be made to establish
appropriate genetic markers to characterize the virus used for production.
It has been assumed that the ability of a rabies vaccine to produce high
neutralizing antibody titres in man is an indication of its effectiveness in
protection. It has also been assumed that good protection in experimental
animals is an indication that a vaccine will be effective in man. Neither
assumption may be entirely correct. In any event, it has not been possible
to specify the minimum level of activity that will afford protection for man.
Because of this difficulty, it is important that rabies vaccines be prepared
having maximum antigenicity in man and experimental animals, together
with an acceptable level of adverse reactions. For this reason rabies vac-
cine must not be diluted to the point where it just satisfies minimum potency
requirements.
Nerve tissue rabies vaccines have been in worldwide use for generations,
and experience has indicated that they are generally effective. It is univer-
sally recognized that such vaccines produce some incidence of post-vaccinal
complications of the central nervous system, but there is some evidence
that the risk is reduced when vaccines are produced in the brains of young
suckling animals. However, data are lacking on the degree of risk due to
the possible presence in the vaccine of adventitious agents (from mice and
rats) that may have survived the inactivation procedures. Duck-embryo
vaccine has a demonstrated low risk for central nervous system effects.
However, duck-embryo vaccine is somewhat less immunogenic in mouse
potency tests than is nerve tissue vaccine and it does not produce a good
level of neutralizing antibody in man. Nevertheless, it has been shown,
on a statistical basis, that the number of rabies cases following adminis-
tration of duck-embryo vaccine is not greater than the number occurring
when nerve tissue vaccine is used. Because of its greater safety, duck-embryo
vaccine is being increasingly used for pre-exposure immunization. Studies
have been made of the effectiveness of duck-embryo vaccine in combination
with minimal amounts of antirabies human immunoglobulin for post-
exposure treatment.
The development of new rabies vaccines is hindered by the difficulty
in evaluating the effectiveness in man. It has never been possible to deter-
mine accurately the degree of risk to an individual following exposure to a
rabid animal. Because of the nature of the disease, it is virtually impossible
with rabies vaccine to do controlled clinical studies involving an unvac-
cinated group to determine accurately the degree of effectiveness. Studies
are in progress to develop vaccines free from neurological and allergic
effects and to enhance the antigenicity of vaccines by adjuvants. When
improved rabies vaccines are put into general use, these requirements
will require revision.
The potency testing of rabies vaccine is of considerable concern. The
potency test must be capable of differentiating between vaccines of differing
25
activity. Preferably a single test should be applicable to all types of vaccine.
The rabies virus strain used for challenge should be one of reproducible
virulence for the test animal when given intracerebrally. In addition the
test should be reproducible and economical. A common reference pre-
paration of vaccine is of considerable aid in evaluating test results. Ideally,
such a reference preparation should be protective in animals and found to
produce antibodies in man. The second International Reference Preparation
of Rabies Vaccine (established in 1965) 1 is available but it has not been
tested in man nor examined for suitability for use in man. Classically
vaccine potency has been based on the wet weight (mg) of brain tissue
required for protection of 50% of the test animals. With the development
of purified vaccines that contain reduced amounts of host tissue, and cell
culture vaccines, the potency should be expressed on the basis of the dilution
of vaccine (injected in a defined volume) protecting 50% of the test animals
rather than on the basis of the tissue content. Although the minimum
potency ratio to a reference vaccine can be established with either procedure
it is not yet possible to determine what ratio represents protection in man.
However, for the determination of such a ratio the following considerations
apply. For nerve tissue vaccines the equivalent of at least 2 ml of a 5%
tissue suspension has been recommended as the single dose for man." The
International Reference Preparation of Rabies Vaccine when reconstituted
from the dry form as instructed is equivalent to a 10% brain tissue sus-
pension. Hence 1 ml is equivalent to one dose for man. For determining
the relative potency of any vaccine in animals, the dilution is made, starting
from the concentration at which the vaccine is administered to man or the
equivalent concentration of the reference vaccine as the case may be. By
this procedure a single reference vaccine may be used for routine testing
of potency of all types of rabies vaccine. This has the additional advantage
of providing a common basis for comparing the potency of the classical
nerve tissue vaccines, with which there have been many years of experience,
and the potency of the newer types of vaccine.
Tests for factors in the vaccine that may induce allergic encephalo-
myelitis have not been included in these requirements because no techniques
have been described that can be relied upon. The degree of reproducibility
of existing procedures has not been evaluated and there is evidence that
considerable variation in results can be expected. Existing tests, however,
can be used to assess the period during which the factor causing allergic
encephalomyelitis develops in the brain of young animals. Studies should
be encouraged for improving such tests.
Because of the need for large quantities of vaccine where rabies is
prevalent, in some countries vaccine is used without fulfilling all of the
26
requirements for the potency test in order to avoid serious curtailment of
vaccine production. This, however, is not a satisfactory procedure and
every effort should be made to achieve compliance with all the present
requirements. In cases, however, where the risks of using vaccine that
may not itself have been tested for potency have to be weighed against the
need for protecting the population at risk against rabies, the decision rests
with the national control authority of the country in which the vaccine is
to be used.
The use of healthy animals has been specified in these requirements,
bUL no provisions are incorporated for tests for extraneous viruses.
National control authorities should, however, pay attention to the prob-
lems of ensuring that the animals used are free from infectious agents that
might contaminate rabies vaccine.
Each of the following sections constitutes a recommendation. The parts
of each section that are printed in large type have been written in the form
of requirements so that, if a health administration so desires, these parts
as they appear may be included in definitive national requirements. The
parts of each section that are printed in small type are comments and
recommendations for guidance.
Should individual countries wish to adopt these requirements as the
basis of their national regulations concerning rabies vaccine, it is recom-
mended that a clause be included permitting modifications of manufac-
turing requirements on the condition that it be demonstrated, to the satis-
faction of the national control authority, that such modified requirements
ensure a degree of safety and potency of the vaccine at least equal to those
provided by the requirements formulated below. It is desirable that the
World Health Organization should then be informed of the action taken.
The terms " national control authority" and " national control labor-
atory " as used in these requirements, always refer to the country in which
the vaccine is manufactured.
Part A : Manufacturing Requirements
1. Definitions
1. 1 International name and proper name
The international name shall be " Vaccinum rabiei (for Human Use) ".
The proper name shall be the equivalent of the international name in the
language of the country of origin.
The use of the international name should he limited to
vaccines that satisfy the requirements formulated below.
27
1.2 Descriptive definition
Vaccinum rabiei (for Human Use) is a fluid or dried preparation of
rabies" fixed" virus grown in the brains or spinal cords of rabbits, sheep,
goats, mice, or rats, or in embryonated eggs, and inactivated by a suitable
method. The preparations for human use shall satisfy all the requirements
formulated below.
1.3 International reference preparation and international standard
The International Reference Preparation of Rabies Vaccine,
established in 1965,1 is stored and distributed in ampoules con-
taining 121 mg of freeze-dried material. The vaccine in each
ampoule, when reconstituted with 8 ml of sterile distilled water,
is equivalent to a 10% rabbit brain tissue suspension. This
reference preparation is intended for the calibration of national
reference preparations for use in tests of potency of rabies
vaccine (see Part B, section 1).2 After reconstitution the Inter-
national Reference Preparation may be stored for subsequent
animal immunizations provided that the storage temperature
is below -60C and that the period of storage is not longer
than one month.
The International Standard for Anti-Rabies Serum estab-
lished in 1955,3 is stored and distributed in ampoules containing
86.6 mg of dried hyperimmune horse serum. The International
Unit is defined as the activity contained in 1 mg of the Inter-
national Standard. Each ampoule therefore contains 86.6 IU.
This standard is intended for use in the laboratory assay of
potency of antirabies immunoglobulin preparations used in man.
It can also be used for the assay of rabies antibodies in man
and animals.
The reference preparation and the standard are in the custody
of the International Laboratory for Biological Standards,
Statens Seruminstitut, Copenhagen. Samples are distributed free
of charge to national control laboratories upon request.
1.4 Terminology
Seed lot. A quantity of virus that has been processed together and
has a uniform composition. A seed lot is used for vaccine preparation or
for the preparation of further seed lots.
Viral harvest. Tissue harvested from a single animal or from a group of
suckling animals or a group of embryonated eggs inoculated at the same
2 Use of the International Reference Preparation for administration to man is not
authorized. A national reference preparation should not be considered as suitable for use
in man unless it has been approved by the national control authority.
28
time and harvested together. The virus in the harvest is without intervening
passage from the seed lot.
Bulk material. A pool of viral harvests before preparation of the final
bulk. Bulk material may be prepared from one or a number of viral harvests
and may yield one or more final bulks.
Final bulk. A quantity of vaccine present in a single container from
which the final containers are filled.
Filling lot (final lot). A collection of sealed final containers that are
homogeneous with respect to the risk of contamination during filling or
drying. A filling lot must, therefore, have been filled in one working session
and (if applicable) have been dried together.
2. General manufacturing requirements
The general requirements for manufacturing establishments contained
in the revised Requirements for Biological Substances No. 1 (General
Requirements for Manufacturing Establishments and Control Labor-
atories) 1 shall apply to establishments manufacturing rabies vaccine (for
human use).
3. Production control
3. 1 Control of source materials
3.1.1 Strain of virus
The strain of virus used in the production of all seed lots shall be a
cc fixed" strain and shall be identified by historical records. It shall have
been shown to the satisfaction of the national control authority to yield
immunogenic vaccines 'when the virus has been inactivated. In addition
the vaccine strain shall be characterized by serological tests and animal
inoculation.
Records shall be maintained of all tests made periodically for verifi-
cation of strain characters. Such tests shall include the titration in animals
of various species and age and by various routes of inoculation as well as
serum neutralization tests.
Preferably, the production strain should be the Pasteur
strain of rabies" fixed" virus maintained only in rabbit pass-
age.
2
It should be capable of producing characteristic paralysis
within 5 to 7 days in rabbits inoculated intracerebrally.
1 Wid Hlth Org. techn, Rep. Ser., 1965, No. 323, p. 11.
2 This strain is available to laboratories on request from the World Health Organi-
zation, 1211 Geneva 27, with the approval of the national authorities.
29
3. I .2 Animals or embryonated eggs for the production of seed virus and
vaccine
For vaccine production only healthy animals, or embryos obtained from
healthy flocks, shall be used. They shall conform to all the requirements
given in Part A, section 3.2. I and section 3.2.2 respectively.
Different species of animals may be used for vaccine pro-
duction or for preparing seed virus. Rabbits (adult or suckling),
sheep, goats, suckling mice, suckling rats, and chicken or duck
embryonated eggs are used successfully in different countries.
3. I .3 Seed lot system
Preparation of rabies vaccine (for human use) shall be based on the use
of the seed lot system. A seed lot shall not be more than 10 passages
removed from the characterized strain. Vaccines shall be made from a seed
lot without further intervening passage. If a seed lot is used for the
preparation of a further seed lot such further seed lot shall be made
without intervening passage. Seed lots shall be maintained either in dried
or frozen form. If frozen, the seed shall be kept continuously at a temper-
ature below -60C.
Seed lots should have been shown, to the satisfaction of the
national control authority, to be capable of yielding vaccine
that meets all of these requirements
3. I .4 Tests on seed lots
The seed lot, in the dilution used as inoculum for the production of
vaccine shall be tested for bacterial and fungal contamination by appropriate
tests according to the requirements of Part A, section 5.2 of the revised
Requirements for Biological Substances No.6 (General Requirements for
the Sterility of Biological Substances}!
Each seed lot shall be identified as rabies virus by methods approved by
the national control authority.
A titration of virus content of the seed lot shall be made.
Such titrations may be done by the intracerebral inoculation
of suitable dilutions in mice. The mice are observed for 14 days.
The virus activity of the seed lot should be such that all mice
are killed when so inoculated with 0.03-m1 quantities of a dilution
of not less than 10-
6
3.2 Production precautions

The general precautions as formulated in the requirements of Part A,
section 3, of the revised Requirements for Biological Substances No. 1
1 Wid Hlth Org. techno Rep. Sen., 1973, No. 530, p. 49.
30
(General Requirements for Manufacturing Establishments and Control
Laboratories) 1 shall apply to the manufacture of rabies vaccine (for human
use) with the addition of the following:
Penicillin and streptomycin preparations shall not be used at any stage
of manufacture of the vaccine.
3.2.1 Vaccines produced in animal brain and spinal cord
The animals intended for production shall be kept in quarantine under
veterinary supervision for at least 2 weeks prior to inoculation of the seed
virus, except in the case of suckling animals when this requirement shall
apply to the mothers. Only animals free from all signs of disease shall be
used. Seed virus shall be inoculated intracerebrally. Methods for inocula-
tion and harvesting approved by the national control authority shall be used.
While inoculation of virus is always made intracerebrally, the
technique used varies with the species of animal. A satisfactory
technique is one that consistently produces paralysis in the
animals inoculated but does not introduce other infection.
In order to obtain the maximum virus titre, nerve tissues
from inoculated animals, apart from suckling animals, should be
harvested on an individual basis when the animal is moribund
and estimated to be close to death from rabies.
If suckling animals are used, the dose and date of inoculation
should allow for harvesting of brain tissue before neuroaller-
genic activity becomes demonstrable. This can be done for the
animal species and particular strain used for vaccine produc-
tion by immunizing guineapigs with brain material suspended
in complete Freund adjuvant. It is essential that positive and
negative controls should be included in the test. On the basis
of the results of the test, the period can be assessed during
which acceptable material can be harvested. The time of harvest
used by some production laboratories is 8 days for mice, 6
days for rabbits, and 7 to 11 days for rats.
Nerve tissue shall not be taken from dead animals, whether death is
due to rabies or other causes.
All animals used in the production of vaccine should be
examined by autopsy after removal of nerve tissue. If evidence
of tuberculosis or any nerve disease other than rabies is found,
the nerve tissue from the animal should be discarded, or if
nerve tissues have been pooled, the pool containing nerve tissue
from such an animal should be discarded. If there is evidence
of a communicable disease among the animals, the viral harvest
from that group should be discarded.
1 Wid Hlth Org: tec1I11. Rep. Ser., 1965, No. 323, p. 11.
31
When other than suckling animals are used, the tissue harvested from
each animal shall be kept separate until completion of the sterility test
(Part A, section 3.2.3). When suckling animals are used, .the harvest
composed of a pool of tissue from a group of animals inoculated at the
same time and harvested together shall similarly be kept separate until
completion of the sterility test.
3.2.2 Vaccines produced in embryonated eggs
The eggs shall be derived from healthy flocks free from micro-organisms
known to be pathogenic for man.
Such agents include Salmonella pullorum, Mycobacterium
tuberculosis, mycoplasma and avian leucosis viruses. If eggs are
used from flocks that have not been shown to be free from avian
leucosis viruses and mycoplasma, the method of inactivation
used should have been shown to the satisfaction of the national
control authority to be capable of killing these organisms.
After the eggs have been incubated for a suitable period they shall be
inoculated with seed virus. After further incubation for a suitable period,
the living embryos shall be harvested with aseptic precautions. Embryos
inoculated at the same time and harvested together may be pooled and the
viral harvest kept separate until completion of the sterility test (Part A,
section 3.2.3).
3.2.3 Sterility tests of the viral harvest
A sample removed from each viral harvest shall be tested for bacterial
and fungal contamination by appropriate tests according to the require-
ments of Part A, section 5.2 of the revised Requirements for Biological
Substances No. 6 (General Requirements for the Sterility of Biological
Substances);' Any viral harvest in which contamination is detected shall
be discarded.
3. 3 Control of bulk material
3.3. 1 Pooling of viral harvests
Only viral harvests satisfying the requirements for sterility given in
Part A, section 3.2. 3 of these requirements shall be pooled for bulk
material.
32
3.3.2 Homogenization and virus titration
The apparatus used for homogenizing the tissues shall be of such a
design as to prevent any escape of aerosols.
Grinding and blending of tissues should be done at as low
a temperature as possible to avoid destruction of virus.
Nerve tissue vaccines should be prepared such that a single
dose for man in the immunization course is contained in not
more than 2 ml of a 5% nerve tissue suspension or its equivalent,
e.g., 1 ml of a 10% suspension
Bulk material shall not be frozen.
A sample should be taken from the homogenized bulk
material prior to inactivation for determination of virus titre
in mice. The titre of virus in nerve tissue harvests should be
not less than 10
5
LD
50
1 per 0.03-ml dose; however, titres of the
order of 10
6
are achievable.
3.3.3 Time of inactivation
Inactivation shall be initiated immediately after homogenization and
removal of the sample for determination of virus titre.
3.3.4 Inactivation procedure
Methods and agents approved by the national control authority shall
be used for inactivation. The method shall be demonstrated to be consist-
ently effective in the hands of the manufacturer. The inactivation process
shall also have been shown, to the satisfaction of the national control
authority, to be capable of inactivating mycoplasma as shown by in vitro
tests and also, in the case of vaccine produced in embryonated eggs, avian
leucosis viruses as shown by tests in tissue culture, or, in the case of vaccine
produced in the brain of suckling animals, any adventitious agent that may
be present as shown by tests using tissue culture or by animal inoculation.
Various methods for inactivating rabies virus have been
used with success. The concentration of the inactivating chem-
ical, temperature, and length of time necessary for inactivation
must be developed for the particular type of vaccine being
manufactured. The most widely used agent is phenol, generally
at a concentration between 0.5% and 1% and at a temperature of
20'-30'Cfor several days until complete inactivation has occurred
as shown by the results of the test specified in section 3.3.5
below. Etherized vaccines are produced in some countries by
combining the action of ether and phenol in the inactivation
procedure. Beta-propiolactcne (BPL) has also been used. Satis-
factory vaccines may be prepared by treating 10% nerve tissue
1 LD
50
is the quantity of virus that kills 50% of mice when injected intracerebrally.
33
homogenates at 20C with a concentration of 1: 3500 to
I : 5000 BPL for 24 hours or until complete inactivation has
occurred as shown by the results of the .test specified in sec-
tion 3.3.5 below.
Ultraviolet light irradiation has also been used but the
equipment required and the procedures involved make it diffi-
cult to prepare vaccine in large volumes. The dosage range and
time of application needed to accomplish complete inactivation
of the virus without reducing antigenicity are critical, but when
the radiation dose is regulated properly, highly antigenic vaccines
may be prepared. The time required for inactivation is short
compared with chemical methods, and hence the vaccine may
be kept at a low temperature throughout; this also aids in con-
serving antigenicity. Vaccines produced by this procedure may
be freeze-dried. For best results the time from inactivation to ini-
tiation of the freeze-drying cycle should be kept to a minimum.
3. 3. 5 Test for effective inactivation
A test approved by the national control authority shall be used to test
each bulk material for inactivation of virus prior to the addition of preserv-
atives and other substances.
The test should be performed with undiluted bulk material
injected intracerebrally into not less than 10 mice.
In some cases the concentration of inactivating agent or
tissue in undiluted bulk material may be toxic to the test animals.
In this case the test should be performed on final bulk material,
which may be diluted, if necessary, but not more than 1 : 2.
In some countries 2 species of animal are used, e.g., rabbits
and mice, for testing effective inactivation. In such cases at least
3 rabbits should be used. If the virus was propagated in an
animal other than the rabbit, consideration should be given to
using the production species rather than the rabbit.
The bulk material passes the test if the product has been shown, to the
satisfaction of the national control authority, to be free from residual live
virus.
3.4 Preparation and control offinal bulk
3.4. I Preservatives and other substances added
In preparing the final bulk, only those preservatives or other substances
approved by the national control authority shall be added. Such substances
shall have been shown by appropriate tests not to impair the safety or
effectiveness of the product in the amounts used.
If phenol has been used for inactivation, its concentration in the final
bulk shall be such that it will not exceed 0.25% in the final product.
34
Phenolized vaccines cannot be frozen without destroying
antigenicity and hence should not be freeze- dried unless a pro-
tective stabilizer has been added.
If beta-propiolactone has been used for inactivation, the procedure shall
be such that there is no detectable amount ofthe chemical in the final bulk.
No antibiotics shall be added to rabies vaccine (for human use).
3.4.2 Potency tests on the final bulk
In the case of liquid vaccine a test for potency shall be made on each
final bulk unless such a test is made on each filling lot. The test shall be
one in which mice are immunized and subsequently challenged with rabies
virus and shall be made in parallel with a reference vaccine. The challenge
strain 1 and reference vaccine as well as the test procedure used shall be
those approved by the national control authority (see Part B, section 1).
Reproducibility of the test depends upon the strain of rabies
virus used for challenge and its maintenance in a large homo-
geneous working pool kept below -60C. The strain of mice
may also affect reproducibility.
When the NIH test is used," the potency relative to a refer-
ence preparation is determined. An acceptable potency ratio for
a vaccine under test should be not less than 0.3 of the inter-
national reference preparation or its equivalent. The relative
potency is obtained by dividing the mg ED
50
of the reference
vaccine by the mg ED
50
of the vaccine under test. When testing
vaccines of low tissue content, the NIH procedure may be modi-
fied to employ dilution endpoints rather than endpoints expressed
as mg of brain tissue. A single dose for man is equivalent to
1 ml of reconstituted International Reference Preparation of
Rabies Vaccine (see Part A, section 1.3) or its equivalent of
a national reference preparation, serial dilutions being made
on this basis. In this case the relative potency is determined
by dividing the reciprocal of the ED
50
dilution of the vaccine
under test by the reciprocal of the ED
50
dilution of the refer-
ence vaccine and multiplying, if necessary, by a factor that
corrects for the dose (human) difference between the test and
reference vaccines.
When the Habel test is used,2 inclusion of the reference
vaccine (see Part A, section 1.3) would enable the sensitivity
of the test system to be monitored in the testing of successive
batchesP
1 A suitable challenge strain, CVS, is available to laboratories on request from the
World Health Organization, 1211 Geneva 27, with the approval of the national authorities.
2 See Kaplan, M. M. & Koprowski, H., ed. (1973) Laboratory techniques in rabies,
3rd edition, Geneva, World Health Organization (Monograph Series, No. 23), Part V.
3 The reconstituted International Reference Preparation of Rabies Vaccine when
injected into mice in volumes of 0.25 ml of a strength corresponding to a 0.5% suspension
has been shown to protect against more than 10000 LD
50
of CVS rabies virus under
the conditions of the test.
35
3 .4. 3 Sterility tests
Each final bulk shall be tested for sterility according to the requirements
given in Part A, section 5 of the revised Requirements for Biological
Substances No. 6 (General Requirements for the Sterility of Biological
Substances)."
4. Filling and containers
The requirements concerning filling and containers given in Part A,
section 4 of the revised Requirements for Biological Substances No. 1
Laboratories) 2 shall apply with the addition of the following:
Containers of dried vaccine shall be hermetically sealed under vacuum
or after filling with pure, dry, oxygen-free nitrogen or any other gas not
deleterious to the vaccine. All containers shall be tested for leaks and all
defective containers shall be discarded.
Generally only single-dose containers are used.
5. Control tests on final product
5. 1 Identity test
An identity test shall be performed on at least one labelled container
from each filling lot by an appropriate method.
The test for potency as described in Part A, section 3.4.2
may serve as an identity test.
5.2 Sterility tests
Each fillinglot shall be tested for bacterial and mycotic sterility according
to the requirements given in Part A, section 5, of the revised Require-
ments for Biological Substances {General Requirements for the Sterility
of Biological Substancesj.!
5. 3 Innocuity tests
Each filling lot shall be tested for abnormal toxicity by appropriate
tests in mice and guineapigs using parenteral injections. The test procedures
shall be those approved by the national control authority.
5.4 Potency test of vaccine infinal containers
A test for potency as described in Part A, section 3.4.2, shall be made
on each filling lot in the case of dried vaccine and also in the case of liquid
1 Wid Hlth Org; techno Rep. Ser., 1973, No. 530, p. 48.
36
vaccine if such a test was not made on the final bulk. Before the test is
made, dried vaccine shall be reconstituted to the form in which it is to be
used in man.
5.5 Stability test for freeze-dried vaccine
A test for potency as described in Part A, section 3.4.2, shall be made
on each filling lot after storage of samples for 4 weeks at 37C and to be
satisfactory the lot shall show no loss in potency.
5.6 Residual moisture tests on freeze-dried vaccine
In the case of dried vaccine it is advisable to test for residual
moisture as a guide to the optimum content for the stability of
the product.
With some vaccines it is possible to dry the product to less
than 1% residual moisture without impairing its stability and
potency. However, depending on the type of stabilizer pres-
ent, higher values may be accepted by the national control
authority.
5.7 Inspection offinal containers
Each container in each filling lot shall be inspected and those showing
abnormalities shall be discarded.
6. Records
The requirements given in Part A, section 6, of the revised Require-
ments for Biological Substances No.1 (General Requirements for Manu-
facturing Establishments and Control Laboratories) 1 shall apply.
7. Samples
ments for Biological Substances No. 1 (General Requirements for Manu-
8. Labelling
ments for Biological Substances No.1 (General Requirements for 'Manu-
facturing Establishments and Control Laboratories) 2. shall apply with the
addition of the following:
1 WId Hlth Org. techn, Rep. Ser ., 1966, No. 323, p. 17.
2 WId Hlth Org . techn. Rep. Ser., 1966, xo. 323, p. 18.
37
The leaflet accompanying the package shall include the following
information:
the tissue and animal species in which the vaccine was prepared;
the method used for inactivating the virus;
if the vaccine is in the dried form, a statement that, after its recon-
stitution it shall be used as soon as possible or stored at 50 3C
and discarded at the end of the day.
9. Distribution and shipping
10. Storage and expiry date
10. 1 Storage conditions
Rabies vaccine (for human use) shall be stored at a temperature of
53C.
10.2 Expiry date
The date after which liquid vaccine may not be used shall be not more
than 6 months after passing the last test for potency. The date after which
dried vaccine may not be used shall be not more than 18 months after
passing the last test for potency.
Part B : National Control Requirements
1. General
The general requirements for control laboratories contained in Part B of
the revised Requirements for Biological Substances No.1 (General Require-
ments for Manufacturing Establishments and Control Laboratories) 2 shall
apply.
1 WId Hlth Org; techno Rep. Ser., 1966, No. 323, p, 18.
2 WId Hlth Org; techno Rep. Ser., 1966, No. 323, p. 19.
38
The national control authority shall give directions to manufacturers
concerning the strain of rabies virus 3 to use for production of vaccine.
The national control authority shall provide or approve the strain for
challenge 1 and the reference vaccine for use in the potency test (Part A,
section 3.4.2).
2. Release and certification
A vaccine lot shall be released only if it fulfils Part A of these require-
ments.
A statement signed by the appropriate official of the national control
laboratory shall be provided at the request of the manufacturing estab-
lishment and shall certify whether or not the lot of vaccine in question
meets all national requirements as well as Part A of the present requirements.
The certificate shall further state the date of the last satisfactory test for
potency, the lot number, the number under which the lot was released, and
the number appeasing on the labels of the containers. In addition, a copy
of the official national release document shall be attached.
The purpose of the certificate is to facilitate the exchange of
rabies vaccine (for human use) between countries.
1 The Pasteur strain for vaccine production and the CVS virus are available to labor-
atories on request from the World Health Organization, 1211 Geneva 27, with the approval
of the national authorities.
39
Annex 4
GENERAL REQUIREMENTS FOR THE
STERILITY OF BIOLOGICAL SUBSTANCES
(Revised 1973) 1
Page
Introduction. . . . . 41
General considerations 41
Part A. Manufacturing requirements
1. Terminology . . . . . . . . . . . 43
2. General precautions against microbial
contamination in manufacture . . 44
contamination from materials used for
manufacture. . . . . . . . . . 47
contamination in sterility testing 47
5. Sterility tests . . . . . . . . . . . 48
5.1 Sampling . . . . . . . . . . 48
5.2 Sterility tests for bacteria and fungi 49
5.3 Sterility test for mycoplasmas. . . 52
5.4 Sterility test for viruses . . . . . 52
5.5 Tests for specific micro-organisms 52
6. Records . . . . . . . . 53
7. Additional samples. . . . . . . . . . 53
Part B. National control requirements
1. General. . . . . . . . . . . . . . . 53
2. Release and certification . . . . . . . 53
Appendix 1. Media for the detection of aerobic and
anaerobic bacteria and fungi 54
Appendix 2. Procedure for sterility test using
membrane filtration 55
Appendix 3. Tests for mycoplasma. 56
Appendix 4. Acknowledgements . . 57
1 These revised requirements have been derived from the document entitled" Sterility
and Sterility Testing of Pharmaceutical Preparations and Biological Substances" (unpub-
lished workingdocumentWHOjBSf73.1062; WHOjPHARMj73.474) which was prepared
for consideration of the WHO Expert Committee on Biological Standardization and of the
WHO Expert Committee on Specifications for Pharmaceutical Preparations; additional
information was obtained from a number of other sources. The names of those who
prepared the original unpublished working document and the names of those who have
submitted suggestions and comments to date on the document are given in Ap-
pendix 4 on p. 57 et seq.
40
Introduction
General requirements for the sterility of biological substances (Require-
ments for Biological Substances No.6) were formulated by a WHO Study
Group in 1959.
1
These general requirements were applicable to any biolog-
ical product from which the exclusion of microbial contamination is
imperative, and they have been quoted in all the sets of requirements for
individual biological substances that have since been formulated. The
twenty-fourth Expert Committee on Biological Standardization 2 agreed
that in view of recent developments III methods of sterility testing of biolog-
ical products and the improvements in control measures that were now
feasible, the general requirements for sterility should be revised. This task
was therefore undertaken. Since the preparation of a set of amendments or
alterations would be an unsatisfactory way of revising the requirements, it
was decided to provide a complete document embodying all the require-
ments concerned. Where provisions formulated by the earlier Study
Group were retained unchanged they have been embodied as such in the
present revised requirements.
In preparing these revised requirements account has been taken of the
opinions of consultants, the relevant regulations and requirements that
have been formulated in a number of countries, as well as information
from both published and unpublished reports. In addition, opinions and
data relevant to these requirements have been received from a number of
experts to whom grateful acknowledgement is made (see Appendix 4,
page 57).
General Considerations
The requirements in this document apply to all immunological biolog-
ical substances, i.e., vaccines and sera, that must be sterile and are used for
administration to man. Many of the provisions, however, may be used for
the control of sterility of other biological preparations, or of blood and
blood products, with suitable modifications, where necessary.
1 WId Hlth Org. technoRep. Ser., 1960, No. 200. The members of the Study Group were:
Dr M. Weis Bentzon, Statens Seruminstitut, Copenhagen, Denmark; Dr P. H. Bonnel,
Centre de Transfusion - Reanimation de l'Armee, Seine, France (Rapporteur) ; Dr P. de
Goes, Institute of Microbiology, Rio de Janeiro, Brazil; Dr J\L Pittman, Division of
Biologics Standards, National Institutes of Health, Bethesda, Md., USA (Chairman);
Dr G. Penso, Laboratory of Microbiology, Istituto Superiore di Sanita, Rome, Italy;
Dr R. H. Regamey, Institut d'Hygienc de l'Universite, Geneva, Switzerland; Dr Sumiatno,
Pasteur Institute, Bandung, Indonesia (Vice-Chairman); Dr J. O'H. Tobin, Biological
Standards Control Laboratory, Medical Research Council, Hampstead, London, England
(Rapporteur); Dr G. V. Vygodchikov, N. F. Gamaleja Institute of Epidemiology and
Microbiology, Moscow, USSR, Secretariat: Dr B. K. Bhattacharya, Medical Officer,
Biological Standardization, WHO; Dr N. K. Jerne, Chief Medical Officer, Biological
Standardization, \VHO, acted as Secretary.
2 WId Hith Org. techno Rep. Ser., 1972, No. 486, p. 19.
41
The Study Group on General Requirements for the Sterility of Biolog-
ical Substances 1 (in 1959) pointed out that confidence in the sterility of
biological preparations depended on two important considerations; first,
adequate control tests for the sterility of biological preparations in their
final containers, using a sufficient number of random samples, and secondly
the use of suitable precautions with respect to source materials and
manufacturing procedures.' The Study Group considered available infor-
mation on sampling procedures adopted in several countries and also
discussed a suggested schedule for sampling of final containers to ensure an
acceptably low probability of the release of contaminated final containers.
Theoretically, sterility may be defined as the absence of all micro-
organisms capable of multiplying.s Sterility tests employing a reasonable
number of samples are capable of detecting contamination only in a lot
with a high percentage of contaminated units. Accordingly, it is not
possible to detect, on the basis of acceptable confidence limits, a low per-
centage of contaminated units in a homogeneous lot of final product. If
the finished product is a single final container of bulk material, then the
control measures are applicable on the basis of an assumed homogeneity
of material in such a single final container. Sterility, therefore, cannot be
assured in the control laboratory, but must be built into the product during
processing. Experience in many countries over the years has confirmed
that greater reliance must be placed on appropriate techniques and proce-
dures throughout the manufacture of the product (including in-process
sterility testing at various stages) rather than simply depending on sterility
tests made on a number of samples of the final batch as the sole basis for
criteria of sterility. Notwithstanding the limitations of sterility tests, every
effort should be made to use the most effective procedure and to strengthen
and validate the tests in the light of the most recent knowledge and ex-
perience.
The tests for sterility mentioned in the previous general requirements
have been revised in the present document, both in nature and in scope.
They now include recommendations for test procedures for mycoplasmas
and for tests using membrane filtration. Tests for viral contaminants are
not dealt with in detail because requirements concerning such contaminants
of biological products and appropriate tests for viral sterility have been
included in the sets of Requirements for Biological Substances
3
that have
been formulated for individual products. Similarly, information on when
1 WId Hlth Org. techno Rep. Ser., 1960, No. 200, p. 4.
2 In the case of a product consisting of living micro-organisms, e.g., certain vaccines,
sterility consists of the absence of contamination by other micro-organisms. Practically,
howerer, assertions regarding sterility relate to the probability that all units in the lot are
sterile. For example, in some countries, a lot of a product is accepted as sterile if there
be no more than one living micro-organism in one million units.
3 See WId Hlth Org; techno Rep. Ser ., 1973, No. 530, p. 62, for a complete list.
42
and where sterility tests are to be made will be found in the requirements
for individual products and this question is not discussed in detail in the
present document.
Each of the following sections constitutes a recommendation. The parts
of each section that are printed in large type have been written in the form
of requirements so that, if a health administration so desires, these parts
as they appear may be included in definitive national requirements. The
parts of each section that are printed in small type are comments and
recommendations for guidance.
Should individual countries wish to adopt these requirements as the
basis for their national regulations concerning sterility of biological prod-
ucts, it is recommended that a clause be included that would permit modifi-
cations of manufacturing requirements on the condition that it be demon-
strated, to the satisfaction of the national control authority, that such
modified requirements ensure a degree of sterility of the product at least
equal to that provided by the requirements formulated below. It is
desirable that the World Health Organization should then be informed of
the action taken.
The terms "national control authority" and " national control labor-
atory" as used in these requirements, always refer to the country in which
the product is manufactured.
Part A. Manufacturing Requirements
1. Terminology
. Contamination: The presence of live extraneous micro-organisms. The
term extraneous applies not only to micro-organisms that may enter the
product during processing, but also to those that may be present in the
materials used for preparing the product, such as SV40 virus in tissues used
to propagate certain viruses for vaccine production. Micro-organisms, as
referred to in this document, include bacteria, fungi, viruses, rickettsias,
mycoplasmas, and chlamydia.
Bulk material is a quantity of partly or wholly processed material,
present in a single container at any stage prior to distribution into final
containers.
Final bulk material is the homogeneous finished preparation present in
a single container from which the final containers are filled, either directly
or indirectly, through one or more intermediate containers.
In some cases a final bulk may constitute the manufacturer's
final product.
Product lot: All finished material, in sealed final containers, that has
been derived from the same final bulk, all of which, at the last stage of
43
processing capable of altering its composition, has been processed together
and therefore has a uniform composition.
In the case, for example, of a vaccine such a lot may be
termed a vaccine lot. The terms product batch and vaccine
batch may be used as appropriate.
Final lot: A collection of sealed final containers derived from a single
final bulk, which are homogeneous with respect to the risk of contamination
during filling, sealing and, if applicable, during drying. A final lot must
therefore have been filled from a single container, and, if applicable, dried
in one continuous operation, e.g., in one working session, the processing
being so arranged that each such operation yields a single homogeneous
batch of the product.
A final lot may consist of the whole or part of a product lot.
In appropriate cases the terms final batch, filling lot, filling
batch or drying lot may be used.
2. General precautions against microbial contamination in manufacture
Every manufacturing establishment should in addition have
a separate and independent quality audit to which every batch
of product would be subjected at the end of processing; the
purpose of such an audit would be to ensure that the measures
adopted for control of microbial contamination of the product
have been carried out satisfactorily.
The general manufacturing requirements given in Part A of the revised
Requirements for Biological Substances No.1 (General Requirements for
Manufacturing Establishments and Control Laboratories) 1 shall apply
which the addition of the following:
The requirements concerning buildings and equipment given
in Part A, section 2, of the above requirements are applicable.
The most desirable situation might be the provision of separate
buildings for each product. This, however, is feasible or neces-
sary only in special circumstances or for very large-scale manu-
facture. The situation usually encountered is one in which
more than one product is manufactured in one building or
laboratory.
If the manufacturing process requires that micro-organisms
be cultured and processed to yield concentrated suspensions
these may become the sources of aerosols or may contaminate
equipment, clothing, and personnel. The potential for cross-
contamination is high and barriers should be established to
prevent contamination of the environment and cross-conta-
mination.
1 Wid Hlth Org: techno Rep. Ser., 1966, No. 323, p. 13.
44
The various measures to avoid contamination are essentially
barriers that may be spatial, temporal, or operational. The
nature of these barriers depends on the risk of contamination
and on the mechanisms by which contamination and cross-
contamination may occur, and it will vary with the nature of the
potential hazards involved. The barriers may include (a) meas-
ures to restrict certain organisms and/or operations to separate
buildings or separate areas within the one building, (b) restric-
tion of the movement of personnel and materials, (c) the treat-
ment of air to remove or destroy aerosols containing micro-
organisms and to control relative humidity, (d) the establish-
ment of pressure gradients to control the direction of air move-
ment, (e) the provision of special work stations such as laminar
flow stations or biological safety cabinets, and (I) wearing of
suitable clothing, including head-covering and footwear, by
personnel.
If the same area is used for the successive manufacture of different
products, the area as well as all equipment it contains shall be adequately
cleaned and disinfected after the processing of one product has been com-
pleted and before the processing of another product is commenced.
Manufacturing procedures involving the handling of microbial spores
shall be confined to a separate building or to a separate area within a
building. Those involving live viruses shall be arranged so that at any
time each distinct virus is confined to an area that is physically isolated by
appropriate barriers. Virus product testing that requires the exclusion of
adventitious agents shall also be subjected to this requirement.
The precautions against contamination given in Part A, section 3.4, of
the revised Requirements for Biological Substances No. I (General Require-
ments for Manufacturing Establishments and Control Laboratories) 1 shall
apply.
There should be careful control of the air filtration and
routine microbial counts of the air in the manufacturing areas
should be carried out during manufacturing operations or period-
ically using an appropriate method of air sampling. The results
of such counts should be checked against criteria, established
for the particular manufacturing area, and adequate records of
the counts should be maintained. Where air filters are used in
manufacturing areas, filters that remove 99.98% :i:: 0.01 % of
particles greater than O.3 urn in diameter are suitable. The effi-
ciency of the filters should be checked routinely.
The recommendations concerning filling and containers given in Part A,
section 4, of the revised Requirements for Biological Substances No. I
Laboratories) 2 shall apply.
45
Filling operations shall be conducted in such a way as to avoid any
contamination or alteration of the product.
These operations should take place in controlled areas
specially designed for the purpose, in which steps are taken to
reduce to a minimum the number of micro-organisms to which
the product being filled is exposed. The use of laminar flow
work stations, disinfecting agents, air filters, and sterilized
clothing, including head-covering and footwear for personnel
conducting filling operations, is recommended. Germicidal
lamps and disinfecting aerosols should not be used during
filling operations.
The operations where liquid preparations are filled should be
checked. This may be done, e.g., at least twice each year at
the close of a working day, by filling not less than 1000 con-
tainers with a nutrient medium containing no antibiotics or
bactericidal substances, and incubating the complete batch of
filled containers at 30 - 36C for at least 14 days. If containers
filled and incubated in this manner show a contamination rate
above 0.3%, some countries do not consider the procedure
acceptable for the filling of sterile preparations with or without
an antimicrobial agent.
The presence of micro-organisms in the air at the filling
point should be monitored, for example, by means of settle
plates or slit-sampling devices.
All material from an area in which virus is being processed shall be
autoc1aved before being sent to the washing and sterilizing departments,
irrespective of whether each area has its own facilities or common facilities
are employed.
A common washing and sterilization area for glassware and
culture media may be used where two or more virus vaccines are
being produced, provided that only heat-sterilized materials are
issued for use in the production areas and that each production
area has its own equipment suitably marked. All media that
are sterilized. by filtration alone should be prepared in a separ-
ate area with its own equipment for the preparation of such
media. '
In controlled areas, apart from specific organisms or cells
being processed, all material introduced from the main
washing and sterilization area should preferably be sterilized by
heat. It is preferable that autoclaves or chambers used for the
sterilization of materials passing in or out of an area be of the
" double-ended" type, built into the wall in a suitable manner
and subjected to control procedures such that the only passage
way for materials between areas containing unsterilized and
sterilized materials is through the autoclave or sterilization
chamber itself.
Cell cultures for vaccine production shall be prepared in a special area
separated from any area in which virus is being handled. Where different
46
cell lines are handled in an area measures shall be taken to prevent cross-
contamination of one cell line with another.
The room and equipment used for filling a live virus vaccine into con-
tainers shall be used solely for this particular vaccine, unless it is adequately
disinfected when it may be used later for the filling of other products.
When animals are used for obtaining tissues for cell culture,
appropriate recommendations should apply."
3. General precautions against microbial contamination from materials used
for manufacture
Precautions shall be taken to avoid contamination from source or other
materials used for manufacture that may affect the sterility of the final
product.
All strains of micro-organisms used as seed materials for biological
preparations shall be maintained in a manner that will assure freedom from
contaminating micro-organisms.
As contamination of a seed lot introduces the possibility of
subsequent contamination of a number of production lots,
special precautions are necessary when preparing or manipu-
lating seed lots.
All blood and blood-derived source materials shall be obtained under
conditions that ensure a safe and effective final product. In the case of
human blood material the requirements for source material given in Part A,
section 3.1, of the Requirements for Human Immunoglobulin (Require-
ments for Biological Substances No. 14) 2 shall apply.
Particular attention should be paid to any regulations that
are applicable to the manufacturer and/or organization that
collects such human material.
4. General precautions against microbial contamination in sterility testing
The appropriate requirements for manufacture given in Part A, section
2, shall apply to those areas in which, and those procedures by which,
sterility testing is done.
The risk of accidental contamination from the environment
should be kept to a minimum by the use of disinfecting agents,
germicidal lamps, and air filters. Germicidal lamps and disin-
fecting aerosols should not be used during actual testing opera-
1 See, for example, Health aspects of the supply and use of non-human primates for
biomedical purposes, report of a WHO Scientific Group (WId Hlth Org . techn, Rep. Ser .,
1971, No. 470).
2 WId Hlth Org . techn, Rep. Ser ., 1967, No , 361, p. 49.
47
tions. The test manipulations should be carried out in a
filtered air environment or under a laminar flow hood, and the
operators should be dressed in sterilized, electrically neutral
clothing, including head-covering and footwear. The air pres-
sure in the testing room should be greater than the exterior
area. The performance of the laminar flow hood should be
monitored by settle plates or slit-sampling devices and the
efficiency of the filters and germicidal lamps checked routinely.
Sterility testing shall not be carried out in production areas.
5. Sterility tests
Sterility tests shall be performed whenever specified in the requirements
for individual products.
Tests for sterility are often performed advantageously at
various stages of manufacture, in addition to those given in
requirements. The tests described in these requirements may
be used for this purpose.
Detailed rules and precautions for the sterility test pro-
cedures should be written and carefully followed.
The staff performing the sterility tests should have experi-
ence in the duties assigned. Supervision of the work and inter-
pretation of sterility tests should be undertaken by an individual
with training in scientific subjects, who should also have been
trained in microbiology or be given training in that subject.
Where sterility tests for bacteria and fungi are required, such tests shall
be made as described in PartA, section 5.2. Appropriate tests for viral and
rickettsial sterility, when required, shall be made as specified in the require-
ments for the individual products. Tests for mycoplasmas, when required,
shall be made as described in Part A, section 5.3.
5 . I Sampling
The specifiednumber of suitable samples shall be taken from the product.
Such samples shall be taken at least from each final bulk, as well as from
each final lot.
5. 1. 1 Sampling from bulk
A sample shall be taken from each bulk to be tested in such a manner as
to be representative of the material to be tested. The amount taken shall
be sufficient to perform the tests and any repeat tests that may be required.
Such samples shall be taken so as to maintain Intact the level of sterility
of the material, or, if this is not possible, the samples shall be taken at the
stage of further processing.
48
Since any microbial contaminants in a liquid may settle out,
thorough mixing is required before the sample is taken.
5.1 .2 Sampling from final lot
Samples of final containers from each final lot to be tested shall be
taken in such a manner as to be representative of the lot to be tested.
Appropriate periodic samples shall be taken, includingsamples at the begin-
ning and the end of the filling operation.
If a product lot is filled through several outlets from a
single bulk, samples should be taken from each outlet (filling
lot) so. as to be representative of the filling assembly.
The number of samples taken shall be at least that approved by the
national control authority, provided that for final lots containing 500 or
more containers, at least 20 samples shall be taken, including samples at
the beginning and the end of the filling operation.
For final lots containing fewer than 500 containers, not less
than 10 containers from the lot should be taken for a sample,
except that for lots of less that 100 containers, only 10% of the
lot need be tested.
A suggested rule 1 for calculating the number of samples
is to take 0.4 \IN samples, where N is the number of final
containers in the lot.
5.2 Sterility tests for bacteria andfungi
5.2. 1 Culture media
The culture media used for sterility tests for bacteria and fungi shall be
those approved by the national control authority. Such media shall have
been shown to be capable of supporting the growth of a wide variety of
micro-organisms, with both aerobic and anaerobic growth characteristics,
including the types found in the environment of the manufacturing oper-
ations.
More than one culture medium, generally, will be needed
to fulfil these criteria. The media used in many countries for
sterility tests are fluid thioglycolate medium and soybean-casein
digest medium.s Any other media that are used, however,
should have been demonstrated to have at least equivalent growth
supporting properties.
For testing the growth supporting qualities of each culture
medium, strains of micro-organisms should be used with exact-
ing nutritive and aerobic-anaerobic requirements, in an inoc-
ulum of only a small number of the organisms (less than 100).
1 Wid Hlth Org. techn, Rep. Ser., 1960, No. 200, p. 3l.
2 The formulae of these culture media are given in Appendix 1.
49
The media should be incubated at the temperatures at which
they will be used in the sterility test.
Each lot of dehydrated medium or each lot of medium
prepared from basic ingredients should be tested for its growth
supporting properties, since every lot may not support the
growth of micro-organisms as well as desirable. It is desirable
that lots so tested should be kept separate and reserved for use
in sterility testing. This is a safeguard against occasional
unsatisfactory components in a particular lot, or destruction of
certain components by over-heating or over-sterilization, which
may cause differences in growth response.
In some countries an additional test is made to verify the
growth supporting properties of the medium, after the completion
of a sterility test in which no growth has occurred in the medium,
by inoculation with a small number of suitable organisms (less
than 100).
5.2.2 Performance of the test
Prior to conducting a sterility test on any product, it shall have been
determined wheter or not the material to be tested itself has the property
of killing or inhibiting the growth of micro-organisms, or contains preserv-
atives or other substances that have this effect. If such an effect is shown,
the sterility test shall be made using a suitable procedure to counteract
the effect.
The inhibitory effect in a preparation to be tested for steril-
ity may be overcome by increasing the volume of culture medium
used, so that the inhibitors are rendered ineffective, or if the
preparation can be filtered, the inhibitors removed by membrane
filtration." The volume of medium required to overcome the
inhibitory effect should be determined. Once established, these
quantities may be used for subsequent sterility tests unless a
change is made in the composition of the product.
5.2.2. 1 Inoculum
For sterility testing of bulk material, at least 5 ml must be used for
inoculation into each culture mediumand for each temperature of incubation.
For sterility testing of a final lot from each of the final containers that
contain not more than 20 ml, an amount of at least 1.0 ml shall be inoculated
into each culture medium and for each temperature of incubation. If the
volume in each final container is less than 1.0 ml, the amount to be inocu-
lated shall be the entire content of the container for each culture medium
and for each temperature of incubation. If the volume in each final con-
tainer is greater than 20 ml, but not more than 100 ml, the amount to be
inoculated shall be at least 5 ml for each culture medium and for each
temperature of incubation.
1 See Appendix 2 for a description. of the procedure using membrane filtration.
50
5.2.2.2 Medium
The quantity of medium put into each vessel for conducting the sterility
test shall be sufficiently large to ensure that the volume of material inoc-
ulated does not impair the growth supporting properties of the medium
by dilution.
All vessels of inoculated media shall be clearly identified by labelling
that is adequate to identify the product being tested, each medium used,
and each temperature of incubation.
5.2.2.3 Incubation
All vessels shall be incubated at the appropriate temperatures for the
media. The temperatures selected shall be those approved by the national
control authority and shall include 20-25C and 30=-36C.
If only one temperature is selected for a particular medium,
generally fluid thioglycolate medium is incubated at 30-36C
and casein digest medium at 20'-25'C.
In some countries additional temperatures are used for
psychrophilic and thermophilic organisms.
It is desirable that the incubation apparatus should have a
continuous temperature recording device.
All vessels shall be incubated for a period of at least 14 days and shall
be examined at regular intervals and on the last day of incubation for
evidence of microbial growth.
If the preparation inoculated into the test vessels has clouded
the medium to such an extent that it is difficult to recognize
whether or not growth has taken place, subcultures of not less
than 1.0 ml should be made from the cloudy medium between
the third and seventh days after the start of the test. The orig-
inal and transfer vessels should each be incubated and observed
for a total of not less than 14 days.
5.2. 3 Membrane filtration of test samples
The performance of sterility tests with the aid of membrane
filtration of the test samples is a valuable method of improving
results of such testing in certain situations."
The extra manipulations involved in these procedures addi-
tional to those for the performance of the test described in
section 5. .2.2 above may, however, be a source of extraneous
contamination. Hence the routine use of positive and negative
controls is advisable for validating the results obtained. A
suitable positive control is the occasional use of known con-
1 See Appendix 2 for a description of a procedure for sterility tests using membrane
filtration.
51
taminated solutions containing a few micro-organisms of dif-
fering types; if the product does not contain a preservative,
approximately 10 viable microbial cells may be used in the
total volume employed. If the product contains a preservative,
the positive control may be added to the wash fluid.
5.2.4 Interpretation of test results
If no evidence of growth is found in any of the vessels inoculated for the
test for sterility in Part A, section 5.2, the bulk material or final lot, which-
ever is applicable, meets the requirements for this test. If evidence of
growth is found, the preparation tested fails to meet the requirements for
the test for sterility, unless it can be demonstrated to the satisfaction of the
national control authority, by retests or by other means, that the test was
invalid.
The decision between failure of the product to pass the test
and invalidity of the test procedure requires competent judge-
ment of a trained individual.
5.3 Sterility test for mycoplasmas
For viral vaccines made by growing the virus in animal tissues or cell
cultures, sterility tests for mycoplasmas of virus culture fluid and control
fluid specifiedfor the particular product shall be made by a method approved
by the national control authority.'
The samples taken may include material prior to clarifi-
cation or filtration in the case of live vaccines produced from
in vitro living cell cultures or animal tissues. In the case of
inactivated virus vaccines produced from such cell cultures or
tissues, the samples may be taken prior to inactivation, from
each virus harvest pool and from control pooled fluid.
Control cultures should be included in each test.
5.4 Sterility test for viruses
The sterility tests for the detection of extraneous viruses in products
where their presence is unacceptable shall be performed as specified in the
requirements for the individual products.
5. 5 Tests for specific micro-organisms
The tests to ensure the non-survival of micro-organisms in inactivated
vaccines and additional tests for the absence of specific extraneous micro-
organisms in such products shall be performed as specified in the require-
ments for the individual products.
1 A description of tests for mycoplasmas is given in Appendix 3.
52
6. Records
Records shall be permanent and clearly indicate all steps in the sterility
testing of the product, including temperatures and incubation times, as
well as the relationship of the samples under test to the manufacturing
operations followed in the production of the lot under test. The records
shall be of a type approved by the national control authority. Written
records shall be kept of all tests performed, irrespective of their results.
They shall be retained throughout the dating period of the product, and
be available for inspection by the national control authority. Records
shall be maintained of all cultures kept in the establishment. Such records
shall include clear labelling for the identification of the cultures and the
purposes for which the cultures are used.
For each sterility test performed a record shall be kept of the name and
identifying numbers of the product under test, the quantities inoculated,
the batch, type and tests of culture media used, the temperatures of incuba-
tion, the dates of inoculation and the results.
7. Additional samples
For each final lot for which sterility tests are made, additional samples
shall be retained as reference material throughout the dating period of the
product, in a manner that ensures the identity of the lot or batch of the
product.
It is desirable that, where possible, manufacturers should
retain sufficient additional samples to permit repetition of the
sterility tests.
Part B. National Control Requirements
1. General
The general requirements for control laboratories given in Part B of
the revised General Requirements for Manufacturing Establishments and
Control Laboratories (Requirements for Biological Substances No.1) 1
shall apply.
2. Release and certification
The requirements given in Part B, section 3.3, of the revised General
Requirements for Manufacturing Establishments and Control Laboratories
(Requirements for Biological Substances No.1) 2 as well as the relevant
provisions in the requirements for the individual products shall apply.
53
Appendix 1
MEDIA FOR THE D E T E C T I O ~ OF AEROBIC AND ANAEROBIC
BACTERIA AND FlJNGI *
1. Fluid thiogIycoIate medium
r.-cystine . . . .
sodium chloride .
glucose (C6H1206.H20)
agar .
yeast extract, water soluble
pancreatic digest of casein.
distilled water . . . . .
thioglycolic acid . . . . .
(or sodium thioglycolate 1 .
resazurin sodium (0.10% 'fresh solution)
Final pH=7 .0-'-7.2
0.5 g
2.5 g
5.5 g
0.75 g
5.0 g
15.0 g
1000 ml
0.3 ml
0.5 g)
1.0 ml
Preparation:
Thoroughly grind the first six ingredients, in the order given above, in a mortar.
Stir in some heated water, transfer to a suitable container, add the remainder of the
water and complete the solution by heating in a boiling water bath, taking special
care to ensure complete solution of the L-cystine. Add the thioglycolic compound,
then I N sodium hydroxide so that the pH of the completed and sterilized medium will
be 7.0-7.2. Reheat the solution, but do not boil, filter (if necessary) through a moistened
filter paper and add the resazurin solution. Distribute into suitable vessels and sterilize
by autoclaving for 18-20 minutes at 121C. Cool promptly to 25C and store at
20_30C, avoiding excessive light. -If the uppermost portion of the medium has chan-
ged to a pink colour and this, exceeds one-third of the depth of the medium, it is
unsuitable for use, but may be restored once by heating in steam. Medium more than
3 weeks old should not be used.
2. Soybean-casein digest medium
pancreatic digest of casein.
papaic digest of soybean meal
sodium chloride .. . . . .
dipotassium hydrogen phosphate .
glucose (C6H1206.H20) . . .
distilled water'. . . . . . .
Final pH=7 .1-7.5
17.0 g
3.0 g
5.0 g
2.5 g
2.5 g
1000 ml
Preparation
Dissolve the solids in the water, warming slightly, then cool the solution to room
temperature. Adjust the reaction .with 1 N sodium hydroxide if necessary, so that the
pH of the completed and sterilized mediumwill be 7. 1-7.5. Filter, if necessary, to clarify,
distribute into suitable vessels and sterilize in an autoclave for 18-20 minutes at 121C.
* The quality of the components should be in accordance with Specifications for
Reagents mentioned in the International Pharmacopoeia, Geneva, World Health Organi-
zation, 1963, unless otherwise stated.
1 Reagent quality sodium thioglycolate is more stable than thioglycolic acid.
54
Appendix 2
PROCEDURE FOR STERILITY TEST USING MEMBRANE FILTRATION
Apparatus
A suitable unit consists of a closed reservoir and a receptacle, separated from one
another by a properly supported membrane of appropriate porosity. Membranes
generally suitable for sterility testing have a nominal porosity of 0.22 urn or 0.45 urn, a
diameter of approximately 47 mm, and a flow rate of 55.-75 ml of water per minute at a
pressure of 70 ern of mercury. Preferably assemble and sterilize the entire unit with
the membrane in place, prior to use. Where the sample to be tested is an oil, it may
be necessary to sterilize the membrane separately and, after thorough drying, assemble
the unit, using aseptic precautions. If each entire membrane is to be cultured (see below).
at least two filter units are set up.
Diluting fluids
Fluid A : Dissolve 1 g of peptic digest of animal tissue 1 or the equivalent in distilled
water to make one litre, filter or centrifuge to clarify, adjust to pH 7.1 0.2. dispense
into flasks in 100-ml quantities. and sterilize at 121C for 18-20 minutes.
Fluid B : If the test sample contains lecithin or oil, use Fluid A to each litre of which
has been added 1 ml of (p-tert-octylphenoxy)polyethoxyethanol, adjust to pH 7.1 0.2.
dispense into flasks, and sterilize at 121C for 18-20 minutes.
Note: Any sterile diluent that does not manifest antimicrobial activity and does not
affect the porosity of the membrane may be suitable for dissolving a preparation under
test for sterility.
Test procedure
Liquids
The number of sample containers or the volume of bulk sample that is specified for
conducting the sterility test is taken. Aseptically transfer the required volumes from each
container directly to a membrane filter previously moistened with sterile .water or the
diluting fluid used or to two sterile vessels for pooling prior to transfer to a moist mem-
brane. Immediately draw each sample through the filter with the aid of vacuum.
If the substance is a viscous liquid or suspension not adaptable to rapid filtration,
aseptically add a sufficient quantity of Fluid A to the pooled sample to increase the flow
rate. Sterile enzyme preparations such as penicillinase or cellulase may be added to the
diluting fluid to aid in dissolving insoluble substances. If the substance under test has
inherent antimicrobial properties or contains a preservative, wash the filter with sufficient
100-ml portions of Fluid A. If the substance under test contains lecithin or oil, replace
Fluid A by Fluid B. The number of portions of fluid used should be sufficient to allow
growth of a small inoculum of organisms (approximately 50) sensitive to the antimicrobial
substance in the presence of the residual inhibitory material on the membrane.
Upon completion of filtration, transfer each entire membrane to 100 ml of the culture
medium (or approximately one-half of each membrane to 100 ml of each of two culture
media) selected for the sterility test. The samples are then incubated at the selected
1 Example: Peptone, dried, R, as described in Specifications for Reagents mentioned
in the International Pharmacopoeia, Geneva, World Health Organization, 1963, p. 137.
An autoclaved solution (2 in 100) is clear and is neutral or nearly so in its reaction.
55
temperatures and for the appropriate periods and examined as described in Part A, section
5.2 of these requirements. The period of incubation of membrane-filtered test samples
used by some workers is shorter than the l4-day period prescribed in this section, since
growth occurs more rapidly when micro-organisms concentrated on filters are inoculated.
Appendix 3
TESTS FOR MYCOPLASMAS
All varieties of mycoplasma are not capable of growth in the same medium. Some
strains require special culture media, Suitable media should therefore be selected having
regard to particular conditions, e.g., the tissue in which a virus is grown or source of
animal serum, and the growth requirements of the potential contaminants, e.g., the
need for yeast extract. Solid and semi-solid or liquid media containing serum fractions
rich in certain lipids (sterols 1 and phospholipids) are used to provide the nutritive
requirements. Control cultures on solid and semi-solid or liquid media of known
fastidious strains of mycoplasma, both sterol-requiring and non-sterol-requiring, are
included in each test to show that the media are capable of supporting the growth of
mycoplasmas.
Samples of the material of not less than 6 ml for a single test (i.e., using one solid
and one semi-solid or liquid medium) are stored either between 2 and SoCfor no longer
than 24 hours or at -20
DC
or lower if stored for longer than 24 hours.
Not less than 2.0 ml of each sample are inoculated and evenly distributed over the
surface of at least 10 plates of each solid medium used, and not less than 1.0 ml of each
sample is inoculated into each of at least 4 tubes of each semi-solid or liquid medium
used. Each tube should contain 10 ml of medium. Larger volumes of inoculum, e.g.,
making a total of 50 ml of material in 400 ml of medium, have been used advantageously.
Ultracentrifugation before testing such samples has also been used.
The plates and tubes are incubated at 36
DC
l.OC for not less than 14 days. One
half of each set is incubated aerobically in an environment of adequate humidity, and
the other half is incubated anaerobically in an environment of 5-10% carbon dioxide
in nitrogen, also of adequate humidity. At the end of 3 days and again at 14 days,
0.5 ml from each of 2 tubes being incubated aerobically are combined and inoculated
on to not less than 4 plates. The 2 tubes being incubated anaerobically are treated likewise.
Since some mycoplasmas may not grow on media in Petri dishes, such subcultures on
solid media may also be made in tubes."
All material seeded originally on solid media is subcultured on the day after the
primary inoculation, on the third day and on the sixth day, on other suitable solid and
liquid media, and these subcultures are incubated similarly, half aerobically and the
other half anaerobically as described, and examined after 10 days, while the original
cultures are examined at the end of the 14-day period. All plates (and tubes) after
incubation for not less than 14 days are observed for the growth of mycoplasmas by
appropriate procedures.
If no evidence is found of the presence of mycoplasmas the material, e.g., virus pool,
meets the requirements of these tests. If evidence of growth of mycoplasmas is found,
the mycoplasmas should be identified and the source traced.
1 Non-sterol-requiring mycoplasmas will grow on media containing sterols.
2 In order to induce mycoplasmas to grow in liquid media, similar subculturing could
be done, but three or four passages may be needed.
56
For detection and identification of mycoplasmas the following procedures are suggested
but other methods are available.
1. For detection of mycoplasmas
(a) typical colonies or specific coloration (blue) by Dienes's stain of colonies spread
on blocks of agar in Petri dishes, and viewed under the microscope;
(b) phase contrast microscopy of colonies (strips or plaques) for globular bodies,
filaments and granules;
(c) examination under the electron microscope of the pellet centrifuged from a semisolid
culture;
(d) examination under the light microscope (x 1000) of a film stained by Giemsa.
2. For separation of mycoplasmas into c1asess
(a) enzymatic effects; fermentation of glucose, breakdown of arginine and reduction
of triphenyltetrazole, using decoloration of phenol red as indicator for the first two effects
and red colour of the tetrazole derivative for the last;
(b) haemolysis in a 4% suspension of washed guineapig cells, added and incubated for
18--48 hours aerobically at 37C;
(c) haemadsorption of a 4% suspension of guineapig cells poured over the growth
surface or fixation of red blood corpuscles by the colonies after incubation at 37C for
30 minutes.
3. For specific identification
Growth inhibition by specific anti-mycoplasma serum (prepared in the rabbit) ; such
inhibition might be demonstrated by depositing paper discs impregnated with the serum
on 2--4hour growth cultures and observing them from the third day.
Immunofluorescence techniques for this purpose are being developed.
Appendix 4

The unpublished working document WHO/BS/73.1062; WHO/
PHARM/73.474 on which these requirements were based was prepared by
the following experts and members of the WHO Secretariat:
Dr E. A. Christensen, Control Department, Statens Seruminstitut, Copenhagen,
Denmark
Dr H. Cohen, Director, National Institute of Public Health, Bilthoven, Nether-
lands (Consultant)
Paris, France (Consultant)
Dr L. F. Dodson, Director, National Biological Standards Laboratory, Common-
wealth Department of Health, Canberra, Australia (CoJ1sultallt)
57
Professor G. Edsall, Head, Department of Microbiology, London School of Hygiene
and Tropical Medicine, London, England (Consultant)
Professor D. G. Evans, Director, National Institute for Biological Standards and
Control, London, England (Consultant)
Mr J. W. Lightbown, Division of Biological Standards, National Institute for
Biological Standards and Control, London, England (Consultant)
Dr T. J. Macek, 16541 S. Westland Drive, Gaithersburg, Md., USA
Mr A. P. Mechkovski, Pharmaceutical Officer, Pharmaceuticals, WHO, Geneva,
Switzerland
Dr A. S. Outschoorn, Chief Medical Officer, Biological Standardization, WHO,
Geneva, Switzerland
Dr M. Gonzalez-Pacheco, Scientist, Biological Standardization, WHO, Geneva,
Switzerland
Dr F. T. Perkins, Division of Immunological Products Control, National Institute
for Biological Standards and Control, London, England (Consultant)
Dr H. D. Piersrna, 2512 Patterson Ave. S.E., Grand Rapids, Michigan, USA
(Consultant)
Dr M. Pittman, 3133 Connecticut Avenue N.W., Washington, D.C., USA (Con-
sultant)
Mr K. O. Wallen, Chief Pharmaceutical Officer, Pharmaceuticals, WHO, Geneva,
Switzerland
Miss A. Wehrli, Pharmaceutical Officer, Pharmaceuticals, WHO, Geneva, Switzerland
Grateful acknowledgement is made to the experts and institutions listed
below for their comments and advice and for supplying additional data:
Dr F. E. Andre, Research Laboratories, Philips-Duphar B.V., Amsterdam,
Netherlands
Dr W. R. Ashford, Assistant Director, Connaught Laboratories Limited, Willowdale,
Ontario, Canada
Professor M. A. Attisso, Faculty of Pharmacy, Montpellier, France
Dr Sami Baglum, P. Director, Refik Saydam, Central Institute of Hygiene, Ankara,
Turkey
Dr M. Baharsefat, Razi State Institute for Serum and Vaccine Production, Teheran,
Iran
Mr A. J. M. Bailey, Executive Officer, The Association of the British Pharmaceutical
Industry, London, England
Professor A. J. Bandoni, Lima 1425, Buenos Aires, Argentina
Dr T. Bican, Head, Chemistry Department, Institute for the Control of Drugs,
Zagreb, Yugoslavia
Dr Carrol Brock, Food and Drug Administration, Washington, D.C., USA.
Dr A. Brunzell, Associate Professor, The Galenical Section, Division of Pharmacy,
Department of Drugs, National Board of Health and Welfare, Stockholm, Sweden
Dr J. R. Burianek, State Institute for the Control of Drugs, Prague, Czechoslovakia
Professor A. Calo, Chief, Laboratory of Chemistry, Istituto Superiore di Sanita,
Rome, Italy
58
Mr K. C. Chatterjee, Bombay College of Pharmacy, Kalina, Santa Cruz, Bombay,
India I
Dr E. A. Christensen, Control Department, Statens Seruminstitut, Copenhagen,
Denmark
Dr V. F. Davey, Deputy Director (Technical), Commonwealth Serum Laboratories,
Parkville, Victoria, Australia
Mr I. Davidson, Central Veterinary Laboratory, Weybridge, Surrey, England
Dr Nils Diding, Apotekens Centrallaboratorium, Solna, Sweden
Professor 1. Dony, Scientific Director, Belgian Pharmaceutical Association, Drug
Control Service, Brussels, Belgium
Mr J. Evans, Acting Chief, Drug Microbiology Branch, Division of Drug Biology,
Office of Pharmaceutical Research and Testing, Washington, D.C., USA
Mr J. T. Faragher, Central Veterinary Laboratory, Weybridge, Surrey, England
Dr M. Fors, Secretary General, Nordic Pharmacopoeia Council, Stockholm, Sweden
Dr Ch. B. Gerichter, Director, Division of Laboratories, Ministry of Health, Jeru-
salem, Israel
Dr D. Ghosh, Director, Central Drugs Laboratory, Calcutta, India
Mr N. Goddard, Sartorius-Membranfilter GmbH, Gottingen, Federal Republic of
Germany
Dr J. 1. Graydon, Commonwealth Serum Laboratories, Parkville, Victoria, Australia
Dr F. Hartley, Dean, The School of Pharmacy, University of London, and Chairman,
British Pharmacopoeia Commission, London, England
Dr L. Hayflick, Department of Medical Microbiology, Stanford University School
of Medicine, Stanford, California, USA
Dr John Hampton, National Biological Standards Laboratory, Canberra, Australia
Professor R. Hazard, Faculty of Medicine, Institute of Pharmacology, Paris, France
Professor G. Heymann, Director, Paul Ehrlich Institute, Federal Agency for Sera
and Vaccines, Frankfurt-am-Main, Federal Republic of Germany
Dr L. Higy-Mandic, Final Control and Biological Standardization Section, Institute
of Immunology, Zagreb, Yugoslavia
Dr N. \V. Holm, Head, Accelerator Department, Danish Atomic Energy Commission,
Research Establishment Rise, Roskilde, Denmark
Dr D. W. Howes, Assistant Director, National Biological Standards Laboratory
(Viral Products Section), Parkville, Victoria, Australia
Mr E. C. Hulse, Deputy Director, Central Veterinary Laboratory, Weybridge,
Surrey, England
Dr T. Inoue, Chief, Department of Drugs, National Institute of Hygienic Sciences,
Tokyo, Japan
Dr S. Iwahara, Chief, Department of Microbiology, National Institute of Hygienic
Sciences, Tokyo, Japan
Professor L. Jannes, State Serum Institute, Helsinki, Finland
Mr C. A. Johnson, Scientific Director, British Pharmacopoeia Commission, London,
England
Mr S. C. Jolly, Director, Department of Pharmaceutical Sciences, The Pharmaceu-
tical Society of Great Britain, London, England
Dr J. Kaneko, Medical Officer, The Vaccination Research Center, Tokyo, Japan
59
60
Dr J. C. Kelsey, Deputy Director, Public Health Laboratory Service, Central Public
Health Laboratory, London, England
Dr K. Kihara, National Institute of Health, Tokyo, Japan
Mr E. Knoll, Chief, Sterility Testing Branch, National Center for Antibiotic Analysis,
Office of Pharmaceutical Research and Testing, Washington, D.C., USA
Mr J. Kramer, Acting Director, Division of Drug Biology, Office of Pharmaceutical
Research and Testing, Washington, D.C., USA
Professor K. G. Krebs, Director, E. Merck AG, Darmstadt, Federal Republic of
Germany
Dr Hanne Kristensen, Statens Seruminstitut, Copenhagen, Denmark
Dr M. Kurokawa.. Chief, Department of General Biologics Control, National
Institute of Health, Tokyo, Japan
Dr A. Lafontaine, Director, Institute of Hygiene and Epidemiology, Brussels,
Belgium
Dr E. Lang, Assistant Manager, CIBA-GEIGY Limited, Basle, Switzerland
Dr H. Lansberg, National Institute of Public Health, Bilthoven, Netherlands
Dr P. Laroux, Scientific Director, SPECIA, Paris, France
Dr F. J. Ley, Irradiated Products Ltd., Elgin Estate, Swindon, Wilts, England
Dr J. P. Lowenthal, Chief, Department of Biologics Research, Division of Com-
municable Disease & Immunology, Walter Reed Army Institute of Research,
Walter Reed Medical Center; Washington, D.C., USA
Dr J. Lyng, Statens Seruminstitut,' Copenhagen, Denmark
Mrs de Maeyer-Cleempoel, Chief, Bacteriology Section, Institute of Hygiene and
Epidemiology, Brussels, Belgium
Mr A. G. Mathews, Chief of Quality Control, Commonwealth Serum Laboratories,
Parkville, Victoria, Australia '
Mr F. A. Maurina, 3485 Bedford, Detroit, Mich., USA
Dr J. A. McKiel, Director General, Laboratory Centre for Disease Control, Health
Protection Branch, Ottawa, Ontario, Canada
Mr H. F. Meldahl, Kabi Ltd, Technical Department, Stockholm, Sweden
Dr H. Mirchamsy, Associate Director, Razi State Institute for Serum and Vaccine
Production, Teheran, Iran
Dr R. Murray, Special Assistant to the Director, National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Bethesda, Md., USA
Dr R. Netter, Director, Virology Section, National Public Health Laboratory,
Paris, France
Dr E. Nieminen, Director, Laboratory for the Examination of Pharmaceutical
Products, Helsinki, Finland
Miss M. Ozsandik, Bacteriologist, Central Institute of Hygiene, Ankara, Turkey
Mr B. V. Patel, Dr Vikram Sarabhai Marg, Ahmedabad, India
Professor G. Penso, Istituto Superiore di Sanita, Rome, Italy
Professor M. Pernarowski, Faculty of Sciences, The University of
British Columbia, Vancouver, Canada
Professor A. Puech, Director, Montpellier Section, National Laboratory for the
Control of Drugs, Montpellier, France
Dr Chaloem Puranananda, Director, National Blood Centre, Thai Red Cross Society,
Bangkok, Thailand
Dr J. D. van Ramshorst, Chief, Department of Biological Standards, National
Institute of Public Health, Bilthoven, Netherlands
Mr M. K. Rangnekar, Commissioner, Food and Drug Administration, Maharashtra
State, Bombay, India
Dr O. Ringertz, Associate Professor, Swedish National Bacteriological Laboratory,
Sweden
Dr E. B. Seligmann, Jr, Director, Division of Control Activities, Bureau of Biologics,
Professor P. L. Senov, Flat 17, 16 Teplyi pereulok, Moscow, USSR
Dr J. Spaander, Director General, National Institute of Public Health, Bilthoven,
Netherlands
Dr 1. Suzuki, Chief, Department of Synthetic Chemistry, National Institute of
Hygienic Sciences, Tokyo, Japan
Dr 1. Di Tommaso, Head, Quality Control, Istituto Sieroterapico e Vaccinogeno
Dr F. Tomicek, Pharmaceutical and Biochemical Research Institute, Prague,
Czechoslovakia
Mr K. Topsy, Senior Government Chemist, Government Chemist Division, Candos,
Mauritius
Professor A. Vegh, Institute for Pharmaceutical Chemistry of Semmelweis University,
Budapest, Hungary
Mr Voggel, Sartorius-Membranfilter GmbH, Gottingen, Federal Republic of Germany
Dr J. Volckringer, Honorary Inspector-General of Health, 3 rue Louis Rolland,
Montrouge, France
Professor M. Welsch, Director, Faculty of Medicine, Chair of Microbiology, Uni-
versity of Liege, Belgium
Dr W. VI'. Wright, Acting Director, Office of Pharmaceutical Research and Testing,
Bureau of Drugs, Food and Drug Administration, Washington, D.C., USA
Valuable suggestions were also obtained from the report of a discussion
on recommendations for WHO Requirements that took place at the Sym-
posium on Sterility and Sterility Testing of Biological Substances held
from 9 to 12 April 1973 in Madrid, Spain, by the International Association
of Biological Standardization.
61
Annex 5
REQUIREMENTS FOR BIOLOGICAL SUBSTANCES
AND OTHER SETS OF RECOMMENDATIONS
The specification of requirements to be fulfilled by preparations of bio-
logical substances is necessary in order to ensure that these products are
safe, reliable, and potent prophylactic or therapeutic agents. International
recommendations on requirements are intended to facilitate the exchange
of biological substances between different countries and to provide guidance
to workers responsible for the production of these substances as well as to
others who may have to decide upon appropriate methods of assay and
control.
Recommended requirements and sets of recommendations concerned
with biological substances formulated by international groups of experts
and published in the Technical Report Series of the World Health Organi-
zation are listed hereunder:
No. Year
178 1959 Requirements for Biological Substances:
1. General Requirements for Manufacturing Establishments and Control
Laboratories
2. Requirements for Poliomyelitis Vaccine (Inactivated)
3. Requirements for Yellow Fever Vaccine
4. Requirements for Cholera Vaccine
5. Requirements for Smallpox Vaccine
7. Requirements for Poliomyelitis Vaccine (Oral)
274 1964 WHO Expert Committee on Biological Standardization:
8. Requirements for Pertussis Vaccine
9. Requirements for Procaine Benzylpenicillin in Oil with Aluminium
Monostearate
10. Requirements for Diphtheria Toxoid and Tetanus Toxoid
62
- - ~ ~ - - - - ~ ~ ~ - - - -
No. Year
323 1966 WHO Expert Group:
Requirements for Biological Substances (Revised 1965)
1. General Requirements for Manufacturing Establishments and Control
Laboratories
2. Requirements for Poliomyelitis Vaccine (Inactivated)
7. Requirements for Poliomyelitis Vaccine (Oral)
5. Requirements for Smallpox Vaccine
11. Requirements for Dried BCG Vaccine
12. Requirements for Measles Vaccine (Live) and Measles Vaccine (Inac-
tivated)
13. Requirements for Anthrax Spore Vaccine (Live - for Veterinary Use)
14. Requirements for Human Immunoglobulin
15. Requirements for Typhoid Vaccine
9. Requirements for Procaine Benzylpenicillin in Oil with Aluminium
Monostearate (Revisions adopted 1966)
16. Requirements for Tuberculins
17. Requirements for Inactivated Influenza Vaccine
4. Requirements for Cholera Vaccine (Revised 1968)
18. Requirements for Immune Sera of Animal Origin
19. Requirements for Rinderpest Cell Culture Vaccine (Live) and Rinder-
pest Vaccine (Live)
20. Requirements for Brucella abortus Strain 19 Vaccine (Live - for
Veterinary Use)
Development of a National Control Laboratory for Biological Substances
(A guide to the provision of technical facilities)
21. Requirements for Snake Antivenins
7. Requirements for Poliomyelitis Vaccine (Oral) (Revised 1971)
4. Requirements for Cholera Vaccine (Revised 1968) (Addendum 1973)
(Revised 1973)
17. Requirements for Inactivated Influenza Vaccine (Addendum 1973)
22. Requirements for Rabies Vaccine for Human Use
63
Annex 6
INTERNATIONAL STANDARDS AND INTERNATIONAL
REFERENCE PREPARATIONS
NOTE: The lists of international biological standards, international
biological reference preparations, and international biological reference
reagents previoulsy included as annexes to the reports of the WHO Expert
Committee on Biological Standardization are now issued as a separate
publication.' Copies may be obtained direct, or through booksellers,
from the agents listed on the back cover of this report, or they may be
ordered from: World Health Organization, Distribution and Sales Service,
1211 Geneva 27, Switzerland:
The twenty-fifth Expert Committee made the following changes to the
lists already published.
Established :
Human Immunoglobulin IgE
Anti-Salmonella pullorum Serum
(Standard Form S)
Anti-Salmonella pullorum Serum
(Variant Form V)
Glucagon, Porcine, for Bioassay
Heparin
1
st
Reference Preparation 1973
2
1st Standard 1973
3
1st Standard 1973
3
1st Standard 1973
4
3
r d
Standard 1973
4
Discontinued :
International reference preparations of the following substances:
Gramicidin S
Sulfarsphenamine
Neoarsphenamine
Oxophenarsine
MSb
1 World Health Organization (1977) Biological substances - International standards,
reference preparations, and reference reagents, 1972, Geneva. Revised editions, incor-
porating the latest additions and amendments, will be published every few years. The
changes made between editioris will be listed in the reports of the Expert Committee.
2 Held and distributed by the International Laboratory for Biological Standards,
Statens Seruminstitut, 80 Amager Boulevard, Copenhagen, Denmark.
Central Veterinary Laboratory, Weybridge, Surrey, England.
National Institute for Biological Standards and Control, Holly Hill, Hampstead, London
England.
64
The following International Standards were renamed:
Old name New name
Gas-gangrene antitoxin (perfringens) 5
t h
Standard 1963
(Clostridium welchii type A
antitoxin)
Gas-gangrene antitoxin (vibrion septique) 3
r d
Standard 1957
Gas-gangrene antitoxin (oedematiens) 3
r d
Standard 1966
Gas-gangrene antitoxin (histolyticus) JTd Standard 1971
Gas-gangrene antitoxin (Sordelli) 1
8t
Standard 1938
Gas-gangrene antitoxin
(Clostridium
welchii type A)
(Clostridium
septicum)
(Clostridium
oedematiens)
(Clostridium
histolyticumt
(Clostridium
sordelliii
65
INDEX
Anti-Salmonella pullorum sera 12 Mel B (melarsoprol) . 8
Cholera vaccine 10
Minocycline 6
MSb.
Cholera vaccine, requirements for. 13,18
8
Dimercaprol 8
Neoarsphenamine. 8
Diphtheria antitoxin for flocculation
Neomycin 6
test 11
Diphtheria toxoid, plain 9
Opacity Reference Preparation 9
Doxycycline 5
Oxophenarsine 8
Gas-gangrene antitoxins 11
Rabies vaccine for human use,
requirements for. 13, 22
histolyticum) . 12
Glucagon 6
Sterility, requirements for 13, 40
Gramicidin S . 6
Sulfarsphenamine . 8
Heparin 7
Thrombin 8
Human Immunoglobulin 19B . 10
Vitamin D 7
Influenza vaccine, inactivated,
requirements for 12, 15 Yellow fever vaccine 10
66

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