Livestock Promotional Subsidy fromthe Japan Racing Association
Livestock Promotional Subsidy fromthe Japan Racing Association

MINIMUM REQUIREMENTSMINIMUM REQUIREMENTSFOR
FOR
VETERINARY
BIOLOGICALVETERINARYPRODUCTSBIOLOGICALPRODUCTS
Volume 1
Volume 1

Japan Veterinary Products Association
Japan Veterinary Products
March Association
2024

March 2024

CONTENTS
General Rules・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・１
Monograph of Drugs・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・９
Vaccines ・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・11
Akabane Disease Vaccine, Live, Seed・・・・・・・・・・・・・・・・・・・・・・13
Bovine Respiratory Syncytial Virus Infection Vaccine, Live, Seed・・・・・・・・・・・21
Akabane Disease, Chuzan Disease, Ainovirus Infection , Peatonvirus Infection
Vaccine（adjuvant）, Inactivated, Seed・・・・・・・・・・・・・・・・・・・・・29
Infectious Bovine Rhinotracheitis, Bovine Viral Diarrhea (bivalent), Bovine
Parainfluenza, Bovine Respiratory Syncytial Virus Infection Vaccine(adjuvant),
Inactivated, Seed・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・44
Equine Influenza Vaccine, Inactivated・・・・・・・・・・・・・・・・・・・・・・65
Japanese Encephalitis Vaccine, Inactivated, Seed・・・・・・・・・・・・・・・・・70
Mycoplasma gallisepticum Vaccine, Live, Seed・・・・・・・・・・・・・・・・・・78
Rabies (tissue culture) Vaccine, Inactivated, Seed・・・・・・・・・・・・・・・・・84
General Tests・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・97
Test Methods・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・99
Properties Test ・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・100
Test for pH ・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・101
Vacuum Degree Test ・・・・・・・・・・・・・・・・・・・・・・・・・・・・102
Test for Moisture Content・・・・・・・・・・・・・・・・・・・・・・・・・・103
Sterility Test・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・104
Test for Freedom from Mycoplasma Contamination・・・・・・・・・・・・・・・・108
Test for Freedom from Salmonella Contamination・・・・・・・・・・・・・・・・111
Microbial Limit Test・・・・・・・・・・・・・・・・・・・・・・・・・・・・113
Test for Freedom from Extraneous Viruses for Live Vaccines and Sera・・・・・・・・115
Test for Thimerosal Content・・・・・・・・・・・・・・・・・・・・・・・・・124
Test for Phenol Content・・・・・・・・・・・・・・・・・・・・・・・・・・・126
Test for Formalin Content・・・・・・・・・・・・・・・・・・・・・・・・・・128
Test for Aluminum Content・・・・・・・・・・・・・・・・・・・・・・・・・129
Test for Macrogol Content・・・・・・・・・・・・・・・・・・・・・・・・・・131
Test for Protein Nitrogen Content・・・・・・・・・・・・・・・・・・・・・・・133

Test for Freedom from Abnormal Toxicity・・・・・・・・・・・・・・・・・・・135
Toxicity Limit Test・・・・・・・・・・・・・・・・・・・・・・・・・・・・・136
Test for Freedom from Extraneous Viruses ・・・・・・・・・・・・・・・・・・・137
Target Animal Immunogenicity Test・・・・・・・・・・・・・・・・・・・・・・145
Target Animal Safety Test・・・・・・・・・・・・・・・・・・・・・・・・・・146
Test for Absence of Reversion to Virulence・・・・・・・・・・・・・・・・・・・147

Test for Stability Confirmation of Recombinant Gene・・・・・・・・・・・・・・149
Reagents, Test Solutions, Etc. ・・・・・・・・・・・・・・・・・・・・・・・・・・・150
Specifications・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・163
Materials for Live Vaccine Production・・・・・・・・・・・・・・・・・・・・・165
Table 1 Inspection and Treatment of SPF Chicken Flocks・・・・・・・・・・・・168
Seed Lot Specifications・・・・・・・・・・・・・・・・・・・・・・・・・・・169

General Rules
1

These standards (”Minimum Requirement for Veterinary Biological Products”）specify the
manufacturing methods, properties, quality, storage, and other matters for veterinary biologicalproducts listed in the monograph of drugs (hereafter referred to as “monographs”). Thesestandards are abbreviated to “veterinary biological standards” in this document. Monographs
include bulk material or final bulk that serve as intermediate products.

2

The Japanese Pharmacopoeia is specified by the Act on Securing Quality, Efficacy and Safetyof Products Including Pharmaceuticals and Medical Devices (Law No. 145, 1960, hereafterreferred to as "Law"), and the Japanese Industrial Standards are specified by the Industrial
Standardization Law (Law No. 185, 1949).

3“Standard names” imply names listed in the monograph of drugs.
For bulk material or final bulk, however; standard names shall be specified by appending “bulkmaterial” or “final bulk” to the names listed in the respective monograph of drugs. Standard
names are considered nonproprietary names when applied to Article 50 of the Law.

4

Veterinary biological products are to be judged according to the provisions given in the General
Rules, pertinent monograph, and the provisions of the General Tests. For bulk material or final
bulk that served as intermediate products in the pertinent monograph, these products shall be
judged according to the rules specified in the sections related to bulk material or final bulk.

5

The details of the manufacturing methods and test items in the test methods may be changed
under the condition in which products manufactured after changes are made are equal to or better
than those manufactured in a specified manner only when such changes are approved as specified
in Article 14, Article 19 (2), Article 23 (2)-5, or Article 23 (2)-17 of the Law.

6

Standard names enclosed in single quotation marks (‘ ’) indicate that the properties and quality
conform to those specified in these standards; however, the standard names shown in the title ofpertinent monograph shall not be enclosed in these marks, and those enclosed in double quotation
marks (“ ”) indicate substances that exhibit specific biological activities.

7

The following abbreviations are used as main units of measurement:
Centimetercm

Kilogram
1kg

Millimeter

mm

Gram

g

Nanometernm

Milligram

mg

2

Square centimetercm

Microgram

μg

Liter

L

Mole per liter

mol/L

DeciliterdL

PascalPa

Milliliter

mL

GravityG

acceleration
Microliter
8μL

Mass percent, mass per volume percent, and volume percent shall be expressed as %, w/v% andvol%, respectively.

9

The Celsius scale is used for temperature measurement and “°C” is added after Arabic
numerals.

10

Solutions without a solvent name indicate water solutions.

11“Diluent” indicates an attached diluent or diluting solution approved as suitable for dissolvingof the relevant product.

12

For veterinary biological products freeze-dried or intended to be dissolved for use whose label
instructions state “Dissolve with diluent” only, these products shall be dissolved using diluent
according to the method stated on the container.

13

Unless otherwise specified, pharmaceuticals or chemical agents that are listed in the JapanesePharmacopoeia (JP) and that are used to manufacture drugs in the monographs shall comply with
the JP requirements, whereas those not listed in the JP but specified in the Japanese Industrial
Standards shall comply with their specifications depending on the purpose of use.

14

Inactivator and stabilizer specified by the term, for example, “use suitable agents” in themonograph of drugs shall be safe for use at the usual amount used and shall not inhibit thebeneficial effects of the drug or interfere with pharmaceutical tests.

15

The actual amount of drugs in the monographs and attached diluents shall be sufficient forcollecting the labeled amount and sufficient for the administration of the labeled dose.

216“Original strain” indicates a virus strain, strain, or coccidia strain specified as the strain used
for production in the monograph of drugs.
“Original virus,” “original bacteria,” or “original coccidia” indicates a passaged original strain
used for the production of the seed virus, seed bacteria, or seed coccidia for vaccines. It is
passaged according to the specified method in the part the vaccines in monograph of drugs or in
the specifications and has the same property as that of the original strain.
“Seed virus,” “seed bacteria,” or “seed coccidia” indicates a passaged original strain directly
used for production of vaccine. It is passaged according to the specified method in themonograph of drugs and has the same property as that of the original strain.

17“Primary cultured cells” indicate cultured cells obtained from appropriate tissues throughtrypsin digestion and etc. and basically having the same property as that of the original cells.
“Passaged cells” indicate cultured cells having the continuous propagation ability.

18“Seed lots” indicate homogeneous suspensions of specific viruses, bacteria, and cells etc.
obtained from single cultures and stored under proper conditions to maintain their genetic
properties.

19“Seed lot product” indicates a vaccine produced using seed lots.

20“Master seed” indicates a virus strain used for the production of seed lot products specified in
the monograph of drugs and passaged not more than approved passages and it is permanently
stored as a virus strain (hereafter referred to as “master seed virus”), as a bacterial strain(hereafter referred to as “master seed bacteria”), and as a coccidia strain (hereafter referred to as“master seed coccidia”).
“Working seed” indicates a virus strain derived from the master seed and not directly used for
manufacturing the products (hereafter referred to as “working seed virus”), a bacteria strain
derived from master seed and not directly used for manufacturing the products (hereafter referred
to as “working seed bacteria”), or a coccidia strain derived from master seed and not directly
used for manufacturing the products (hereafter referred to as “working seed coccidia”).
“Production seed” is a virus strain derived from the working seed and directly used for
manufacturing the products (hereafter referred to as “production seed virus”), a bacteria strain
derived from working seed and directly used for manufacturing the products (hereafter referred toas “production seed bacteria”), or a coccidia strain derived from working seed and directly used
for manufacturing the products (hereafter referred to as “production seed coccidia”).

321“Cell line” indicates cultured cells having the continuous propagation ability and used for
manufacturing seed lot products.
“Master cell seed” indicates cultured cells that have been passaged not more than approved
passages.
“Working cell seed” indicates a cell line derived from the master cell seed and not directly
used for manufacturing the products.
“Production cell seed” is a cell line derived from the working cell seed and directly used for
manufacturing the products.

22“Master primary cell seed” indicates primary cultured cells generated through not more than
five passages of cells that have been collected from animals.
“Working primary cell seed” indicates primary cultured cells derived from the master primarycell seed and not directly used for manufacturing the products.
“Production primary cell seed” is primary cultured cells derived from the working primary cell
seed and directly used for manufacturing the products.

23“Bulk material” indicates a veterinary biological product containing a single active ingredientthat is not dispensed as is into final containers.

24“Final bulk” indicates a bulk drug prepared in one vessel and ready for immediate filling into
final containers. The content shall be completely homogeneous in terms of pharmaceuticalproperties and qualities. Shaking is allowed to maintain homogeneity.

25“Final product” indicates the products filled into small containers from a final bulk, freezedried if necessary, and hermetically sealed.

26“Lot” usually indicates a group of final products derived from the final bulk. Also, the “lot” ofbulk material or final bulk in the monograph of drugs that serves as veterinary biologicalproducts indicates a group of products having a uniform character and quality that has been filled
in a series of manufacturing processes within a specified period.

27

In general, the same manufacturing number or manufacturing code is assigned to the productsof one lot. However, additional numbers or marks shall be assigned to the individual final lots or
freeze-dried lots, if dispensed and hermetically sealed under different operating conditions (theamount to be dispensed may vary) or if freeze-dried under different drying conditions,
respectively.
428

Generally, tests on specifications for bulk material or final bulk and final lot products requiredin the monographs are collectively performed with samples from the same lot. For final products
having individual final lots or individual freeze-dried lots, however, the following tests shall be
conducted on individual lots: properties test, sterility test, viable count test, spore count test, test
for freedom from contaminant microorganisms, test for virus content, vacuum degree test, test formoisture content, and additionally specified tests. Other tests shall be conducted by taking andmixing equal volumes of the test sample of individual lots.

29“Test for freedom from abnormal toxicity,” “Toxicity limit test,” “Safety test,” and “Potency
test” for final lot products specified in the monographs may be omitted when the requirements
otherwise specified by the Director of the National Veterinary Assay Laboratory are met.

30

“Test sample” indicates a final product used for tests or the final product dissolved withdiluent.
“Test article” indicates a product subject to tests on the final bulk or earlier stages that has notbeen manipulated in any way.
“Test material” indicates a test sample or article that has been diluted or otherwise
manipulated.

31

Generally, tests on veterinary biological products freeze-dried or intended to be dissolved drug
for use in the monographs, unless these tests are the “Vacuum degree test,” “Test for moisture
content,” “uniformity test per container of freeze-dried product before dissolution” in the“Properties test,” and otherwise specified, shall be conducted on solutions diluted or suspended
according to the method stated on the container using the diluents.

32

The “Inactivation test” evaluates the loss or reduction in biological activity of viablemicroorganisms used in the production of a veterinary biological product below the levelspecified in the requirements.
The “Detoxication test” evaluates the loss of toxicity of toxic substances present during the
production process of a veterinary biological product below the level specified in therequirements.
The “Test for freedom from (name of microorganism or substance)” determines the absence of
substances or microorganisms indicated in ( ) below the level specified in the requirements.

33

Unless otherwise specified, tests shall be conducted at room temperature.
5

Room temperature is defined as 15℃ to 25℃.
34

Unless otherwise specified, tests shall be conducted in accordance with the “Reagents, Test
Solutions, Etc.” of the General Tests.
The term “water” for use in the tests indicates purified water specified by the JP.

35

To “accurately weigh” means weighing to the specified digit.
To “exactly measure” means measuring the specified whole volume using pipettes, measuring
flasks, or burettes.
To “precisely weigh” means weighing to the lowest digit, for example, 0.1 mg, 0.01 mg, or
0.001 mg.

36

Numerical values shall indicate the specified value with a ± 5% variation.
“Approximately” in collecting the desired amount or quantity indicates a variation innumerical value within ±10%.

37

When a test result is judged by comparing a specified value (hereafter referred to as “specified
value”) with actually measured values obtained from a test (hereafter referred to as “experimental
values”) in the Physiochemical Test of the General Tests, experimental values shall be obtained to
one digit lower than that for the specified value and rounded off to the same digit with thespecified value.

38

Animals used in tests must be healthy. If animals exhibit incidental abnormalities during thetest, the veterinary biological product shall not meet the test requirements unless theabnormalities can be demonstrated to be unrelated to the product tested.

39

The test methods or the details of the test methods specified in the Minimum Requirement for
Veterinary Biological Products may be changed so far as it is equally or more precise andaccurate than the specified method. However, if the test results are different from the ones byusing the specified method of the Minimum Requirements for Veterinary Biological Products, thefinal judgement of the test shall be made in accordance with said specified method.

40

Unless otherwise specified, storage temperature shall be kept ≤ 10℃ for freeze-dried products
and 2℃ to 10℃ for liquid products in a light-shielded environment. However, this shall not
apply to bulk material or final bulk.

641“Delivery from the warehouse” means the shipping of the veterinary biological products fromthe storage of the manufacturing facility for sales or transport. Veterinary biological products
shall be stored at a constant temperature before shipping

42

Unless otherwise specified, the shelf life and expiry date shall be set based on the monthfollowing the month of the day when production is completed. However, for veterinary biologicalproducts to be subjected to national certification tests according to Article 43 (1) of the Law, thedate when the national certification test is completed may be substituted for the date when
production is completed.

43

In cases where veterinary biological products should be subject to the national certification
test but could not be submitted for application within the period specified in Article 152, Section
3, of the Regulatory Rules for Veterinary Medicinal Products (Ordinance of the Ministry ofAgriculture, Forestry and Fisheries No. 107, 2004) under unavoidable circumstance when
Director of National Veterinary Assay Laboratory determines that particularly there is any reason
for shortening the expiry date as the result of the national certification test, the shelf life of suchproducts shall be defined as the date specified by the director notwithstanding above provisions.

44

Unless specified otherwise, entries on the immediate container for monographs required by
Article 50 (9) of the Law shall be those listed below.
(1) Storage conditions(2) Expiry date(3) Live or inactivated for vaccines
However, these entries may be omitted when they are provided on the outer container orpackage if the following conditions apply:-The product is provided in an ampoule or direct container or package with a volume of 2 mLor less.
-The product is provided in an ampoule or container with a volume of more than 2 mL having
an area on which these entries are directly printed but less than 10 mL having an area insufficient
to clearly label the entries specified in the provisions of Article 50 of the Law.
-The product is designed for in vitro diagnostic purposes and provided in the outer container orpackage stating “For in vitro diagnostic purposes.”

45

When the monographs or pharmaceuticals used for the production of the monographs arederived from animals, these animals must be healthy, unless otherwise specified.

7

Monograph of Drugs

9Vaccines

11Akabane Disease Vaccine, Live, Seed
1 Definition
Akabane Disease Vaccine, Live, Seed is a freeze-dried vaccine of virus suspension obtained bypropagating attenuated Akabane virus that meets the seed lot specifications in cell lines that meetthese specifications.
2 Production methods
2.1 Virus strain used for production
2.1.1 Name
Attenuated Akabane virus TS-C2 strain or strain approved as equivalent thereof
2.1.2 Properties
Inoculation of this virus strain into pregnant cows causes neither abortion, stillbirth, orcongenital defects in newborns.
2.1.3 Master seed virus
2.1.3.1 Preparing, storing, and maximum passage number allowed for obtaining final products
The master seed virus shall be propagated in HmLu-1 cells or cells approved as suitable,
prepared in consecutive processes, and dispensed into storage containers.
Dispensed master seed virus shall be given a specific manufacturing number or manufacturingcode and stored frozen at -70℃ or lower or stored freeze-dried at 5℃ or lower.
The master seed virus shall be tested as specified in 3.1.1.
The master seed virus shall not be passaged for purposes other than production of vaccines. The
master seed virus shall not be passaged more than five times to obtain the final product.
2.1.4 Working seed virus
2.1.4.1 Propagation, passage and storage
The working seed virus shall be propagated and passaged in HmLu-1 cells or cells approved as
suitable.
The working seed virus shall be stored frozen at -70℃ or lower or be stored freeze-dried at 5℃
or lower.
The working seed virus shall be tested as specified in 3.1.2.
2.1.5 Production seed virus
2.1.5.1 Propagation and storage
The production seed virus shall be propagated in HmLu-1 cells or cells approved as suitable.
The production seed virus shall be stored frozen at -70℃ or lower or stored freeze-dried at 5℃
or lower, when it needs to be stored.
13

The production seed virus shall be tested as specified in 3.1.3 if it needs to be stored.
2.2 Materials for production
2.2.1 Cell line
HmLu-1 cells or cell lines approved as suitable for production shall be used.
2.2.2 Culture medium
The culture medium approved as suitable for production shall be used.
2.2.3 Master cell seeds
2.2.3.1 Preparing, storing, and maximum passage number allowed for obtaining production cellseeds
The master cell seeds shall be propagated in a culture medium in 2.2.2, prepared in consecutive
processes, and dispensed into storage containers.
The dispensed master cell seeds shall be given a specific manufacturing number or
manufacturing code and stored frozen at -70℃ or lower.
The master cell seeds shall be tested as specified in 3.2.1.
The master cell seeds shall not be passaged for purposes other than production or testing ofvaccines. The master cell seeds shall not be passaged more than 20 times to obtain the production
cell seeds.
2.2.4 Working cell seeds
2.2.4.1 Propagation, passage and storage
The working cell seeds shall be propagated and passaged using culture medium in 2.2.2.
The working cell seeds shall be stored frozen at -70℃ or lower. The working cell seeds shall betested as specified in 3.2.2.
2.2.5 Production cell seeds
2.2.5.1 Propagation and storage
The production cell seeds shall be propagated using culture medium in 2.2.2.
The production cell seeds shall be stored frozen at -70°C or lower when it needs to be stored.
The production cell seeds shall be tested as specified in 3.2.3 if it needs to be stored.
2.3 Bulk material
2.3.1 Culture of production cell seeds
The cultured cells obtained in one session shall be regarded as an individual cultured cells. No
abnormalities shall be detected prior to the inoculation of virus.
2.3.2 Virus cultivation
The production seed virus shall be cultivated in the cells in 2.3.1. The filtrate or supernatant
after centrifugation of culture medium collected from individual cultured cells at the peak of virus
propagation shall be mixed, and the mixture shall serve as the bulk material.
The bulk material shall be tested as specified in 3.3.
14

2.4 Final bulk
A stabilizer approved as appropriate shall be added to the bulk material to serve as the finalbulk.
2.5 Final product
The final bulk shall be dispensed into small containers and freeze-dried to serve as the final
product.
The final product shall be tested as specified in 3.4.
3 Test methods
3.1 Tests on virus strain used for production
3.1.1 Tests on master seed virus
3.1.1.1 Identification test
The tests given in 1.4.2.1.1.1 in the Seed Lot Specifications shall apply.
3.1.1.2 Sterility test
The test given in the Sterility Test given of the General Tests shall apply.
3.1.1.3Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.1.1.4 Test for freedom from extraneous viruses
3.1.1.4.1 Common test for freedom from extraneous viruses
The tests given in 1.1, 2.1 and 2.2 in the Test for Freedom from Extraneous Viruses of theGeneral Tests shall apply.
3.1.1.4.2 Test for freedom from specific viruses
3.1.1.4.2.1 General test for freedom from specific viruses
For Lymphocytic choriomeningitis virus, Bovine respiratory syncytial virus and Bluetongue
virus, the tests given in 1.1 and 3.1.1 in the Test for Freedom from Extraneous Viruses of theGeneral Tests shall apply.
3.1.1.4.2.2 Individual test for freedom from specific viruses
For Bovine viral diarrhea virus, Rotavirus, Bovine leukemia virus, Japanese encephalitis virus,
and Rabies virus, the tests given in 1.1, 3.2.5, 3.2.7, 3.2.8 and 3.2.9 in the Test for Freedom from
Extraneous Viruses of the General Tests shall apply.
3.1.1.5 Target animal immunogenicity test
The test given in the Target Animal Immunogenicity Test of the General Tests shall apply.
3.1.1.6 Target animal safety test
The test given in the Target Animal Safety Test of the General Tests shall apply.
3.1.1.7 Test for absence of reversion virulence
15

The test given in the Test for Absence of Reversion to Virulence of the General Tests shallapply.
3.1.2 Tests on working seed virus
3.1.2.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.1.2.2 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.1.3 Tests on production seed virus
3.1.3.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.1.3.2 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.2 Tests on cell line
3.2.1 Tests on master cell seeds
3.2.1.1 Test for confirmation of cell properties
The test given in 2.1.4.2.1.1 in the Seed Lot Specifications shall apply.
3.2.1.2 Test for identification of the animal species of the cell
The test given in 2.1.4.2.1.2 in the Seed Lot Specifications shall apply.
3.2.1.3 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.2.1.4 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.2.1.5 Test for freedom from extraneous viruses
3.2.1.5.1 Common test for freedom from extraneous viruses
The tests given in 1.2, 2.1 and 2.2 in the Test for Freedom from Extraneous Viruses of theGeneral Tests shall apply.
3.2.1.5.2 Test for freedom from specific viruses
3.2.1.5.2.1 General test for freedom from specific viruses
For Lymphocytic choriomeningitis virus, Bovine respiratory syncytial virus and Bluetongue
virus, the tests given in 1.2 and 3.1.1 in the Test for Freedom from Extraneous Viruses of theGeneral Tests shall apply.
3.2.1.5.2.2 Individual test for freedom from specific viruses
For Bovine viral diarrhea virus, Rotavirus, Bovine leukemia virus, Japanese encephalitis virus
16and Rabies virus, the tests given in 1.2, 3.2.5, 3.2.7, 3.2.8 and 3.2.9 in the Test for Freedom from
Extraneous Viruses of the General Tests shall apply.
3.2.1.6 Test for karyological (chromosomal) characterization
The test given in 2.1.4.2.1.6 in the Seed Lot Specifications shall apply.
3.2.1.7 Test for freedom from tumorigenicity
The test given in 2.1.4.2.1.7 in the Seed Lot Specifications shall apply.
3.2.2 Tests on working cell seeds
3.2.2.1 Test for confirmation of cell properties
The test given in 2.1.4.2.2.1 in the Seed Lot Specifications shall apply.
3.2.2.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.2.2.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.2.3 Tests on production cell seeds
3.2.3.1 Test for confirmation of cell properties
The test given in 2.1.4.2.3.1 in the Seed Lot Specifications shall apply.
3.2.3.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.2.3.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.3 Tests on bulk material
3.3.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.3.2 Test for virus content
3.3.2.1 Materials
3.3.2.1.1 Test materials
The test article shall be diluted ten-fold with virus growth medium (Note) and each level
dilution shall serve as a test material.
3.3.2.1.2 Cultured cells
HmLu-1 cells shall be cultured in small test tubes for one to three days, and monolayer cultures
shall be used.
3.3.2.2 Test procedures
A 0.1 mL portion of the test material shall be inoculated into at least four tubes of cultured cells
and allowed to stand for adsorption at 37℃ for 60 minutes. A 0.5 mL portion of virus growth17

medium shall be added, roller-cultured at 34℃ to 36℃ for seven days and observed.
3.3.2.3 Judgment
The cultured cells in which CPE is observed shall be regarded as infected to calculate TCID50.
The virus content in 1 mL shall be 106.5TCID50 or higher.
3.4 Tests on final product3.4.1 Properties test
When the test is performed as specified in the Properties Test of the General Tests, the final
product shall be dried substances with a specific color. The dissolved product shall be a liquidwith a specific color. No foreign matter and foreign odor shall be observed. The property of
product per small container shall be uniform.
3.4.2 Vacuum degree test
The test given in the Vacuum Degree Test of the General Tests shall apply.
3.4.3 Test for moisture content
The test given in the Test for Moisture Content of the General Tests shall apply.
3.4.4 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.4.5 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply. However, this test may be omitted if it is conducted during the intermediate processincluding the bulk material.
3.4.6 Test for virus content
When the test is performed as specified in 3.3.2, the virus content of the test sample shall be
5.0

10

TCID50 or higher per animal.

3.4.7 Test for freedom from abnormal toxicity
The test given in the Test for Freedom from Abnormal Toxicity of the General Tests shall apply.
3.4.8 Safety test
3.4.8.1 Materials
3.4.8.1.1 Injection materials
The test sample shall be used as an injection material.
3.4.8.1.2 Test animals
Cattle weighing 100–200 kg shall be used.
3.4.8.2 Test procedures
A dose of injection material for one animal shall be administered subcutaneously to one animaland observed for 14 days.
3.4.8.3 Judgment
During the observation period, a slight fever (40.5℃ or lower), if observed, shall not persist for18

more than three days, and no other abnormalities shall be identified.
3.4.9 Potency test
3.4.9.1Materials
3.4.9.1.1 Test animals
The animals used in the test specified in 3.4.8 shall be used.
3.4.9.1.2 Virus for neutralization test
Akabane virus JaGAr39 strains propagated in HmLu-1 cells shall be used.
3.4.9.1.3 Cultured cells
HmLu-1 cells shall be cultured in small test tubes for one to three days, and monolayer cultures
shall be used.
3.4.9.2 Test procedures
The serum obtained 14 days after the completion of test specified in 3.4.8 shall be subject to theneutralization test.
The serum shall be heat-inactivated, and then shall be diluted two-fold with virus growth
medium. With 0.5 mL of each diluted serum, 0.5 mL of the virus for the neutralization test withapproximately 200 TCID50 per 0.1 mL shall be mixed and reacted at 37℃ for 60 minutes. The 0.1
mL of the mixtures of each shall be inoculated into four tubes of cultured cells and allowed foradsorption at 37℃ for 60 minutes. To each tube, 0.5 mL of virus growth medium shall be added
and roller-cultured at 34℃ to 36℃ for seven days for observation.
3.4.9.3 Judgment
The maximum dilution factor of serum at which CPE is observed in at least two tubes ofcultured cells shall be defined as the neutralizing antibody titer.
The neutralizing antibody titer in test animals shall be at least two-fold or higher.
4 Storage and expiry date
The expiry date shall be two years and three months after the manufacturing unless otherwise
specified by the Minister of Agriculture, Forestry and Fisheries.
Note

Virus growth medium
1,000 mL consists of the following:
Tryptose phosphate broth

2.95 gGlucose1.0 g

Yeast extract

0.5 g

Bovine serum

5-20 mL

Eagle’s MEM

residual quantity

pH shall be adjusted to 7.2-7.6 with sodium hydrogen carbonate.
19

Bovine serum to be used shall be negative for neutralizing antibodies against the Akabane
virus.
Minimal amounts of antibiotics may be added.

20

Bovine Respiratory Syncytial Virus Infection Vaccine, Live,Seed
1 Definition
Bovine respiratory syncytial virus infection vaccine, live, seed is a freeze-dried vaccine of thevirus suspension obtained by propagating attenuated bovine respiratory syncytial virus that meetsthe seed lot specifications in cell lines that meet these specifications.
2 Production methods
2.1 Virus strain used for production
2.1.1 Name
Attenuated bovine respiratory syncytial virus rs-52 strain or strain approved as equivalent
thereof
2.1.2 Properties
This virus shows no pathogenic effects when inoculated in cows. The propagative ability of thisvirus at 30ºC in HAL cells established from cells derived from hamster lung is 100-fold higher
than that of the virulent virus.
2.1.3 Master seed virus
2.1.3.1 Preparing, storing, and maximum passage number allowed for obtaining final products
The master seed virus shall be propagated in HAL cells or cells approved as suitable, preparedin consecutive processes, and dispensed into storage containers.
The dispensed master seed virus shall be given a specific manufacturing number or
manufacturing code and stored frozen at -70ºC or lower or stored freeze-dried at 5ºC or lower.
The master seed virus shall be tested as specified in 3.1.1.
The master seed virus shall not be passaged for purposes other than the production of vaccines.
The master seed virus shall not be passaged more than five times to obtain the final product.
2.1.4 Working seed virus
2.1.4.1 Propagation, passage, and storage
The working seed virus shall be propagated and passaged in HAL cells or cells approved as
suitable. The working seed virus shall be stored frozen at -70ºC or lower or be stored freeze-driedat 5ºC or lower.
The working seed virus shall be tested as specified in 3.1.2.
2.1.5 Production seed virus
2.1.5.1 Propagation and storage
21

The production seed virus shall be propagated in HAL cells or cells approved as suitable.
The production seed virus shall be stored frozen at -70ºC or lower or stored freeze-dried at 5ºCor lower when it needs to be stored.
The production seed virus shall be tested as specified in 3.1.3 if it needs to be stored.
2.2 Materials for production
2.2.1 Cell line
HAL cells or cell lines approved as suitable for production shall be used.
2.2.2 Culture medium
The culture medium approved as suitable for production shall be used.
2.2.3 Master cell seeds
2.2.3.1 Preparing, storing, and maximum passage number allowed for obtaining production cellseeds
The master cell seeds shall be propagated in a culture medium in 2.2.2, prepared in consecutive
processes, and dispensed into storage containers.
The dispensed master cell seeds shall be given a specific manufacturing number or
manufacturing code and stored frozen at -70ºC or lower.
The master cell seeds shall be tested as specified in 3.2.1.
The master cell seeds shall not be passaged for purposes other than production or testing ofvaccines. The master cell seeds shall not be passaged more than 20 times to obtain the production
cell seeds.
2.2.4 Working cell seeds
2.2.4.1 Propagation, passage, and storage
The working cell seeds shall be propagated and passaged using culture medium in 2.2.2.
The working cell seeds shall be stored frozen at -70ºC or lower. The working cell seeds shall betested as specified in 3.2.2.
2.2.5 Production cell seeds
2.2.5.1 Propagation and storage
The production cell seeds shall be propagated using culture medium in 2.2.2.
The production cell seeds shall be stored frozen at -70°C or lower when it needs to be stored.
The production cell seeds shall be tested as specified in 3.2.3 if they need to be stored.
2.3 Bulk material
2.3.1 Culture of production cell seeds
The cultured cells obtained in one session shall be regarded as individual cultured cells. No
abnormalities shall be detected in production cell seeds prior to the inoculation of the virus.
2.3.2 Virus cultivation
The production seed virus shall be cultivated in the cells in 2.3.1. The filtrate or supernatant
22

after centrifugation of culture medium collected from individual cultured cells at the peak of thevirus propagation shall be mixed, and the mixture shall serve as the bulk material.
The bulk material shall be tested as specified in 3.3.
2.4 Final bulk
A stabilizer approved as appropriate shall be added to the bulk material to serve as the finalbulk.
2.5 Final product
The final bulk shall be dispensed into small containers and freeze-dried to serve as the final
product.
The final product shall be tested as specified in 3.4.
3 Test methods
3.1 Tests on virus strain used for production
3.1.1 Tests on master seed virus
3.1.1.1 Identification test
The tests given in 1.4.2.1.1.1.2 in the Seed Lot Specifications shall apply.
3.1.1.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.1.1.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.1.1.4 Test for freedom from extraneous viruses
3.1.1.4.1 Common test for freedom from extraneous viruses
The tests given in 1.1, 2.1 and 2.2 in the Test for Freedom from Extraneous Viruses of theGeneral Tests shall apply.
3.1.1.4.2 Test for freedom from specific viruses
3.1.1.4.2.1 General test for freedom from specific viruses
For the lymphocytic choriomeningitis virus and Bluetongue virus, the tests given in 1.1 and3.1.1 in the Test for Freedom from Extraneous Viruses of the General Tests shall apply.
3.1.1.4.2.2 Individual test for freedom from specific viruses
For the Bovine viral diarrhea virus, Rotavirus, Bovine leukemia virus, Japanese encephalitis
virus, and Rabies virus, the tests given in 1.1, 3.2.5, 3.2.7, 3.2.8 and 3.2.9 in the Test for Freedom
from Extraneous Viruses of the General Tests shall apply.
3.1.1.5 Target animal immunogenicity test
The test given in the Target Animal Immunogenicity Test of the General Tests shall apply.
3.1.1.6 Target animal safety test
23

The test given in the Target Animal Safety Test of the General Tests shall apply.
3.1.1.7 Test for absence of reversion virulence
The test given in the Test for Absence of Reversion to Virulence of the General Tests shallapply.
3.1.2 Tests on working seed virus
3.1.2.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.1.2.2 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.1.3 Tests on production seed virus
3.1.3.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.1.3.2 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.2 Tests on cell line
3.2.1 Tests on master cell seeds
3.2.1.1 Test for confirmation of cell properties
The tests given in 2.1.4.2.1.1 in the Seed Lot Specifications shall apply.
3.2.1.2 Test for identification of the animal species of the cell
The tests given in 2.1.4.2.1.2 in the Seed Lot Specifications shall apply.
3.2.1.3 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.2.1.4 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.2.1.5 Test for freedom from extraneous viruses
3.2.1.5.1 Common test for freedom from extraneous viruses
The tests given in 1.2, 2.1 and 2.2 in the Test for Freedom from Extraneous Viruses of theGeneral Tests shall apply.
3.2.1.5.2 Test for freedom from specific viruses
3.2.1.5.2.1 General test for freedom from specific viruses
For the Lymphocytic choriomeningitis virus, Bluetongue virus, and Bovine respiratorysyncytial virus, the tests given in 1.2 and 3.1.1 in the Test for Freedom from Extraneous Viruses of
the General Tests shall apply.
24

3.2.1.5.2.2 Individual test for freedom from specific viruses
For the Bovine viral diarrhea virus, Rotavirus, Bovine leukemia virus, Japanese encephalitis
virus, and Rabies virus, the tests given in 1.2, 3.2.5, 3.2.7, 3.2.8 and 3.2.9 in the Test for Freedom
from Extraneous Viruses of the General Tests shall apply.
3.2.1.6 Test for karyological (chromosomal) characterization
The tests given in 2.1.4.2.1.6 in the Seed Lot Specifications shall apply.
3.2.1.7 Test for freedom from tumorigenicity
The tests given in 2.1.4.2.1.7 in the Seed Lot Specifications shall apply.
3.2.2 Tests on working cell seeds
3.2.2.1 Test for confirmation of cell properties
The tests given in 2.1.4.2.2.1 in the Seed Lot Specifications shall apply.
3.2.2.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.2.2.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.2.3 Tests on production cell seeds
3.2.3.1 Test for confirmation of cell properties
The tests given in 2.1.4.2.3.1 in the Seed Lot Specifications shall apply.
3.2.3.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.2.3.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.3 Tests on bulk material
3.3.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.3.2 Test for virus content
3.3.2.1 Materials
3.3.2.1.1 Test materials
The test article shall be diluted ten-fold with the virus growth medium (Note 1) and each level
dilution shall serve as the test material.
3.3.2.1.2 Cultured cells
Vero cells shall be cultured in small test tubes for three to four days, and monolayer cultures
shall be used.
3.3.2.2 Test procedures
25

The 0.1 mL portions of the test material shall be inoculated into at least four portions ofcultured cells and allowed to stand for adsorption at 37ºC for 60 minutes. The 0.5 mL portions of
the virus growth medium shall be added, roller-cultured at 34ºC for 14 days, and observed.
3.3.2.3 Judgment
The cultured cells in which CPE is observed shall be regarded as infected to calculate TCID50.
The virus content in 1 mL shall be 106.3TCID50 or higher.
3.4 Tests on final product3.4.1 Properties test
When the test is performed as specified in the Properties Test of the General Tests, the final
product shall be dried substances with a specific color. The dissolved product shall be a liquidwith a specific color. No foreign matter and foreign odor shall be observed. The property of the
product per small container shall be uniform.
3.4.2 Vacuum degree test
The test given in the Vacuum Degree Test of the General Tests shall apply.
3.4.3 Test for moisture content
The test given in the Test for Moisture Content of the General Tests shall apply.
3.4.4 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.4.5 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
However, this test may be omitted if it is conducted during the intermediate process, including
the bulk materials.
3.4.6 Test for virus content
When the test is performed as specified in 3.3.2, the virus content of the test sample shall be
105.0 TCID50 or higher per animal.
3.4.7 Test for freedom from abnormal toxicity
The test given in the Test for Freedom from Abnormal Toxicity of the General Tests shall apply.
3.4.8 Safety test
3.4.8.1 Materials
3.4.8.1.1 Injection materials
The test samples shall be used as injection materials.
3.4.8.1.2 Test animals
Cattle weighing 100–200 kg shall be used.
3.4.8.2 Test procedures
A dose of injection material for one animal shall be administered intramuscularly to one animal
26

and observed for 14 days.
3.4.8.3 Judgment
During the observation period, a slight fever (40.5ºC or lower), if observed, shall not persist for
more than three days, and no other abnormalities shall be identified.
3.4.9 Potency test
3.4.9.1 Materials
3.4.9.1.1 Injection materials
The test samples shall be used as injection materials.
3.4.9.1.2 Test animals
Hamsters weighing approximately 100 g shall be used.
3.4.9.1.3 Virus for neutralization test
The Bovine respiratory syncytial virus NMK7 strain propagated in passage cells of bovine
kidneys.
3.4.9.1.4 Cultured cells
Vero cells shall be cultured in small test tubes for three to four days, and monolayer cultures
shall be used.
3.4.9.2 Test procedures
The 2 mL portions of the injection materials shall be injected intramuscularly twice at intervalsof 14 days in five test animals, and serum shall be obtained from individual animals 14 days after
the second injection to conduct a neutralization test.
The test serum shall be heat-inactivated and diluted with the dilution for neutralization test
(Note 2) in two-fold serial dilutions. A 0.5 mL portion of diluted serum and a 0.5 mL portion of
the virus suspension for neutralization test containing approximately 200TCID50 in 0.1 mL shallbe mixed and reacted at 22ºC for 24 hours. The 0.1 mL portion of each mixture shall be inoculated
into four tubes of cultured cells and allowed to stand for adsorption at 37ºC for 60 minutes. Then,the 0.5 mL portions of the virus growth medium shall be added, roller-cultured at 34ºC for 10days, and observed.
3.4.9.3 Judgment
The maximum dilution factor of serum at which inhibition of CPE is observed in at least two
tubes of cultured cells shall be defined as the neutralizing antibody titer. A two-fold or greater
increase in the neutralizing antibody titer shall be defined as positive.
The neutralizing antibody positive rate in test animals shall be 80% or higher.
4 Storage and expiry date
The expiry date shall be one year and three months after the manufacturing unless otherwise
specified by the Minister of Agriculture, Forestry and Fisheries.
27

Note 1 Virus growth medium
1,000 mL consists of the following:
Tryptose phosphate broth

2.95 gMonosodium glutamate

5g

Glucose1g

Fetal bovine serum

10–20 mL

Yeast extract

0.5 g

Eagle’s MEM

Residual quantity

The pH shall be adjusted to 7.2–7.6 with sodium hydrogen carbonate.
Fetal bovine serum to be used shall be negative for neutralizing antibodies against the bovine
respiratory syncytial virus.
Minimal amounts of antibiotics may be added.
Note 2 Dilution for neutralization
1,000 mL consists of the following:
Tryptose phosphate broth

2.95 gFetal bovine serum

100 mL

Kanamycin sulfate

100 mg (potency)

Eagle’s MEM

Residual quantity

The pH shall be adjusted to 7.2–7.4 with sodium hydrogen carbonate.
Fetal bovine serum to be used shall be negative for neutralizing antibodies against the bovine
respiratory syncytial virus.
Minimal amounts of antibiotics may be added.

28

Akabane Disease, Chuzan Disease, Aino Virus Infection,
Peaton Virus Infection Vaccine (adjuvant), Inactivated, Seed
1 Definition
This Vaccine, Inactivated, Seed is made by mixing the inactivated virus suspensions obtainedby propagating the Akabane virus, Kasba virus, Aino virus and Peaton virus that meet the seed lot
specifications in cell lines that meet these specifications, respectively, and by adding an adjuvant.
2 Production methods
2.1 Virus strain used for production
2.1.1 Akabane virus
2.1.1.1 Name
Akabane virus E-24 strain or strain approved as equivalent thereof
2.1.1.2 Properties
This virus shall be propagated in the following cells in which CPE is confirmed: primarycultured cells of bovine kidney, primary cultured cells of swine kidney, HmLu-1 cells, HmLu-SC
cells, and Vero cells.
2.1.1.3 Master seed virus
2.1.1.3.1 Preparing, storing, and maximum passage number allowed for obtaining final products
The master seed virus shall be propagated in HmLu cells or cells approved as suitable, preparedin consecutive processes, and dispensed into storage containers.
The dispensed master seed virus shall be given a specific manufacturing number or
manufacturing code and stored frozen at -70ºC or lower or stored freeze-dried at 5ºC or lower.
The master seed virus shall be tested as specified in 3.1.1.
The master seed virus shall not be passaged for purposes other than the production of vaccines.
The master seed virus shall not be passaged more than five times to obtain the final product.
2.1.1.4 Working seed virus
2.1.1.4.1 Propagation, passage, and storage
The working seed virus shall be propagated and passaged in HmLu cells or cells approved as
suitable.
The working seed virus shall be stored frozen at -70ºC or lower or be stored freeze-dried at 5ºCor lower.
The working seed virus shall be tested as specified in 3.1.2.
2.1.1.5 Production seed virus292.1.1.5.1 Propagation and storage
The production seed virus shall be propagated in HmLu cells or cells approved as suitable.
The production seed virus shall be stored frozen at -70ºC or lower or stored freeze-dried at 5ºCor lower when it needs to be stored.
The production seed virus shall be tested as specified in 3.1.3 if it needs to be stored.
2.1.2 Kasba virus
2.1.2.1 Name
Kasba virus K-47 strain or strain approved as equivalent thereof
2.1.2.2 Properties
Inoculation of this virus strain into the brain of calves causes fever, anorexia, and leukopenia,
followed by neurological symptoms.
This virus shall be propagated in the following cells in which CPE is confirmed: BHK-21 cells,
BHK-SC cells, HmLu-1 cells, HmLu-SC cells, and Vero-T cells.
2.1.2.3 Master seed virus
2.1.2.3.1 Preparing, storing, and maximum passage number allowed for obtaining final products
The master seed virus shall be propagated in HmLu cells or cells approved as suitable, preparedin consecutive processes, and dispensed into storage containers.
The dispensed master seed virus shall be given a specific manufacturing number or
manufacturing code and stored frozen at -70ºC or lower or stored freeze-dried at 5ºC or lower.
The master seed virus shall be tested as specified in 3.1.1.
The master seed virus shall not be passaged for purposes other than the production of vaccines.
The master seed virus shall not be passaged more than five times to obtain the final product.
2.1.2.4 Working seed virus
2.1.2.4.1 Propagation, passage, and storage
The working seed virus shall be propagated and passaged in HmLu cells or cells approved as
suitable.
The working seed virus shall be stored frozen at -70ºC or lower or be stored freeze-dried at 5ºCor lower.
The working seed virus shall be tested as specified in 3.1.2.
2.1.2.5 Production seed virus
2.1.2.5.1 Propagation and storage
The production seed virus shall be propagated in HmLu cells or cells approved as suitable.
The production seed virus shall be stored frozen at -70ºC or lower or stored freeze-dried at 5ºCor lower when it needs to be stored.
The production seed virus shall be tested as specified in 3.1.3 if it needs to be stored.
2.1.3 Aino virus302.1.3.1 Name
Aino virus JaNAr28 strain or strain approved as equivalent thereof
2.1.3.2 Properties
When this virus strain is inoculated intravenously in cows, viremia is present, but no clinicalsymptoms such as fever are observed.
This virus shall be propagated in the following cells in which CPE is confirmed: BHK-21 cells,
HmLu-1 cells, HmLu-SC cells, and Vero cells.
2.1.3.3 Master seed virus
2.1.3.3.1 Preparing, storing, and maximum passage number allowed for obtaining final products
The master seed virus shall be propagated in HmLu cells or cells approved as suitable, preparedin consecutive processes, and dispensed into storage containers.
The dispensed master seed virus shall be given a specific manufacturing number or
manufacturing code and stored frozen at -70ºC or lower or stored freeze-dried at 5ºC or lower.
The master seed virus shall be tested as specified in 3.1.1.
The master seed virus shall not be passaged for purposes other than the production of vaccines.
The master seed virus shall not be passaged more than five times to obtain the final product.
2.1.3.4 Working seed virus
2.1.3.4.1 Propagation, passage, and storage
The working seed virus shall be propagated and passaged in HmLu cells or cells approved as
suitable.
The working seed virus shall be stored frozen at -70ºC or lower or be stored freeze-dried at 5ºCor lower.
The working seed virus shall be tested as specified in 3.1.2.
2.1.3.5 Production seed virus
2.1.3.5.1 Propagation and storage
The production seed virus shall be propagated in HmLu cells or cells approved as suitable.
The production seed virus shall be stored frozen at -70ºC or lower or stored freeze-dried at 5ºCor lower when it needs to be stored.
The production seed virus shall be tested as specified in 3.1.3 if it needs to be stored.
2.1.4 Peaton virus
2.1.4.1 Name
Peaton virus NS/3 strain or strain approved as equivalent thereof
2.1.4.2 Properties
When this virus strain is inoculated intravenously in cows, viremia is present, but no clinicalsymptoms such as fever are observed.
This virus shall be propagated in the following cells in which CPE is confirmed: BHK-21 cells,
31

HmLu-1 cells, HmLu-SC cells, and Vero cells.
2.1.4.3 Master seed virus
2.1.4.3.1 Preparing, storing, and maximum passage number allowed for obtaining final products
The master seed virus shall be propagated in HmLu cells or cells approved as suitable, preparedin consecutive processes, and dispensed into storage containers.
The dispensed master seed virus shall be given a specific manufacturing number or
manufacturing code and stored frozen at -70ºC or lower or stored freeze-dried at 5ºC or lower.
The master seed virus shall be tested as specified in 3.1.1.
The master seed virus shall not be passaged for purposes other than the production of vaccines.
The master seed virus shall not be passaged more than five times to obtain the final product.
2.1.4.4 Working seed virus
2.1.4.4.1 Propagation, passage, and storage
The working seed virus shall be propagated and passaged in HmLu cells or cells approved as
suitable.
The working seed virus shall be stored frozen at -70ºC or lower or be stored freeze-dried at 5ºCor lower.
The working seed virus shall be tested as specified in 3.1.2.
2.1.4.5 Production seed virus
2.1.4.5.1 Propagation and storage
The production seed virus shall be propagated in HmLu cells or cells approved as suitable.
The production seed virus shall be stored frozen at -70ºC or lower or stored freeze-dried at 5ºCor lower when it needs to be stored.
The production seed virus shall be tested as specified in 3.1.3 if it needs to be stored.
2.2 Materials for production
2.2.1 Cell line
HmLu cells or cells approved as suitable shall be used.
2.2.2 Culture medium
The culture medium approved as suitable for production shall be used.
2.2.3 Master cell seeds
2.2.3.1 Preparing, storing, and maximum passage number allowed for obtaining production cellseeds
The master cell seeds shall be propagated in a culture medium in 2.2.2, prepared in consecutive
processes, and dispensed into storage containers.
The dispensed master cell seeds shall be given a specific manufacturing number or
manufacturing code and stored frozen at -70ºC or lower.
The master cell seeds shall be tested as specified in 3.2.1.
32

The master cell seeds shall not be passaged for purposes other than production or testing ofvaccines. The master cell seeds shall not be passaged more than 20 times to obtain the production
cell seeds.
2.2.4 Working cell seeds
2.2.4.1 Propagation, passage, and storage
The working cell seeds shall be propagated and passaged using culture medium in 2.2.2.
The working cell seeds shall be stored frozen at -70ºC or lower.
The working cell seeds shall be tested as specified in 3.2.2.
2.2.5 Production cell seeds
2.2.5.1 Propagation and storage
The production cell seeds shall be propagated using culture medium in 2.2.2.
The production cell seeds shall not be stored.
2.3 Bulk material
2.3.1 Akabane virus bulk material
2.3.1.1 Culture of production cell seeds
The cultured cells obtained in one session shall be regarded as individual cultured cells. No
abnormalities shall be detected prior to the inoculation of the virus.
2.3.1.2 Virus cultivation
The production seed virus shall be cultivated in the cells in 2.3.1.1. The filtrate, supernatant
after centrifugation, or concentrate of the culture medium collected from individual cultured cells
at a time deemed appropriate shall serve as the virus suspension.
The virus suspension shall be tested as specified in 3.3.1 and 3.3.2.1.
2.3.1.3 Inactivation
Virus suspension shall be inactivated by the addition of an inactivator approved as appropriate
and shall serve as the inactivated virus suspension.
The inactivated virus suspension shall be tested as specified in 3.4.1.1.
2.3.1.4 Bulk material
The inactivated virus suspension shall be concentrated by a method approved as suitable andshall serve as the bulk material.
The bulk material shall be tested as specified in 3.5.
2.3.2 Kasba virus bulk material
2.3.2.1 Culture of production cell seeds
The cultured cells obtained in one session shall be regarded as individual cultured cells. No
abnormalities shall be detected prior to the inoculation of the virus.
2.3.2.2 Virus cultivation
The production seed virus shall be cultivated in the cells in 2.3.2.1. The filtrate, supernatant
33

after centrifugation, or concentrate of the culture medium collected from individual cultured cells
at a time deemed appropriate shall serve as the virus suspension.
The virus suspension shall be tested as specified in 3.3.1 and 3.3.2.2.
2.3.2.3 Inactivation
Virus suspension shall be inactivated by the addition of an inactivator approved as appropriate
and shall serve as the inactivated virus suspension.
The inactivated virus suspension shall be tested as specified in 3.4.1.2.
2.3.2.4 Bulk material
The inactivated virus suspension shall be concentrated by a method approved as suitable andshall serve as the bulk material.
The bulk material shall be tested as specified in 3.5.
2.3.3 Aino virus bulk material
2.3.3.1 Culture of production cell seeds
The cultured cells obtained in one session shall be regarded as individual cultured cells. No
abnormalities shall be detected prior to the inoculation of the virus.
2.3.3.2 Virus cultivation
The production seed virus shall be cultivated in the cells in 2.3.3.1. The filtrate, supernatant
after centrifugation, or concentrate of the culture medium collected from individual cultured cells
at a time deemed appropriate shall serve as the virus suspension.
The virus suspension shall be tested as specified in 3.3.1 and 3.3.2.3.
2.3.3.3 Inactivation
Virus suspension shall be inactivated by the addition of an inactivator approved as appropriate
and shall serve as the inactivated virus suspension.
The inactivated virus suspension shall be tested as specified in 3.4.1.3.
2.3.3.4 Bulk material
The inactivated virus suspension shall be concentrated by a method approved as suitable andshall serve as the bulk material.
The bulk material shall be tested as specified in 3.5.
2.3.4 Peaton virus bulk material
2.3.4.1 Culture of production cell seeds
The cultured cells obtained in one session shall be regarded as individual cultured cells. No
abnormalities shall be detected prior to the inoculation of the virus.
2.3.4.2 Virus cultivation
The production seed virus shall be cultivated in the cells in 2.3.4.1. The filtrate, supernatant
after centrifugation, or concentrate of the culture medium collected from individual cultured cells
at a time deemed appropriate shall serve as the virus suspension.
34

The virus suspension shall be tested as specified in 3.3.1 and 3.3.2.4.
2.3.4.3 Inactivation
Virus suspension shall be inactivated by the addition of an inactivator approved as appropriate
and shall serve as the inactivated virus suspension.
The inactivated virus suspension shall be tested as specified in 3.4.1.4.
2.3.4.4 Bulk material
The inactivated virus suspension shall be concentrated by a method approved as suitable andshall serve as the bulk material.
The bulk material shall be tested as specified in 3.5.
2.4 Final bulk
The Akabane virus bulk material, Kasba virus bulk material, Aino virus bulk material and
Peaton virus bulk material shall be adjusted for their antigen levels and mixed, and then mixed
with an aluminum gel adjuvant or adjuvant approved as suitable and shall serve as the final bulk.
2.5 Final product
The final bulk shall be dispensed into small containers to serve as the final product.
The final product shall be tested as specified in 3.6.
3 Test methods
3.1 Tests on virus strain used for production
3.1.1 Tests on master seed virus
3.1.1.1 Identification test
The tests given in 1.4.2.3.1.1 in the Seed Lot Specifications shall apply.
3.1.1.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.1.1.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.1.1.4 Test for freedom from extraneous viruses
3.1.1.4.1 Common test for freedom from extraneous viruses
The tests given in 1.1, 2.1 and 2.2 in the Test for Freedom from Extraneous Viruses of theGeneral Tests shall apply.
3.1.1.4.2 Test for freedom from specific viruses
3.1.1.4.2.1 General test for freedom from specific viruses
The tests given in 1.1 and 3.1 in the Test for Freedom from Extraneous Viruses of the General
Tests shall apply.
3.1.1.4.2.2 Individual test for freedom from specific viruses
35For the Bovine viral diarrhea virus, Rotavirus, Bovine leukemia virus, Japanese encephalitis
virus, and Rabies virus, the tests given in 1.1, 3.2.5, 3.2.7, 3.2.8 and 3.2.9 in the Test for Freedom
from Extraneous Viruses of the General Tests shall apply.
3.1.2 Tests on working seed virus
3.1.2.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.1.2.2 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.1.3 Tests on production seed virus
3.1.3.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.1.3.2 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.2 Tests on cell line
3.2.1 Tests on master cell seeds
3.2.1.1 Test for confirmation of cell properties
The tests given in 2.1.4.2.1.1 in the Seed Lot Specifications shall apply.
3.2.1.2 Test for identification of the animal species of the cell
The tests given in 2.1.4.2.1.2 in the Seed Lot Specifications shall apply.
3.2.1.3 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.2.1.4 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.2.1.5 Test for freedom from extraneous viruses
3.2.1.5.1 Common test for freedom from extraneous viruses
The tests given in 1.2, 2.1 and 2.2 in the Test for Freedom from Extraneous Viruses of theGeneral Tests shall apply.
3.2.1.5.2 Test for freedom from specific viruses
3.2.1.5.2.1 General test for freedom from specific viruses
The tests given in 1.1 and 3.1 in the Test for Freedom from Extraneous Viruses of the General
Tests shall apply.
3.2.1.5.2.2 Individual test for freedom from specific viruses
For the Bovine viral diarrhea virus, Rotavirus, Bovine leukemia virus, Japanese encephalitis
36

virus, and Rabies virus, the tests given in 1.2, 3.2.5, 3.2.7, 3.2.8 and 3.2.9 in the Test for Freedom
from Extraneous Viruses of the General Tests shall apply.
3.2.1.6 Test for karyological (chromosomal) characterization
The tests given in 2.1.4.2.1.6 in the Seed Lot Specifications shall apply.
3.2.1.7 Test for freedom from tumorigenicity
The tests given in 2.1.4.2.1.7 in the Seed Lot Specifications shall apply.
3.2.2 Tests on working cell seeds
3.2.2.1 Test for confirmation of cell properties
The tests given in 2.1.4.2.2.1 in the Seed Lot Specifications shall apply.
3.2.2.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.2.2.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.3 Tests on virus suspension
3.3.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.3.2 Test for virus content
3.3.2.1 Akabane virus
3.3.2.1.1 Materials
3.3.2.1.1.1 Test materials
The test article shall be diluted ten-fold with the virus growth medium approved as suitable, andeach level dilution shall serve as the test material.
3.3.2.1.1.2 Cultured cells
HmLu-1 cells shall be used.
3.3.2.1.2 Test procedures
The 0.1 mL portions of the test material shall be inoculated into at least four portions (wells) ofcultured cells and allowed to stand for adsorption at 37ºC for 60 minutes. The 1.0 mL portions of
the virus growth medium approved as suitable shall be added, cultured at 37ºC for seven days, andobserved.
3.3.2.1.3 Judgment
The cultured cells in which CPE is observed shall be regarded as infected to calculate TCID50.
The virus content in 1 mL shall be 106.5TCID50 or higher unless otherwise specified by the
Minister of Agriculture, Forestry and Fisheries.
3.3.2.2 Kasba virus
3.3.2.2.1 Materials
37

3.3.2.2.1.1 Test materials
The test article shall be diluted ten-fold with the virus growth medium approved as suitable, andeach level dilution shall serve as the test material.
3.3.2.2.1.2 Cultured cells
Vero-T cells shall be used.
3.3.2.2.2 Test procedures
The 0.1 mL portions of the test material shall be inoculated into at least four portions (wells) ofcultured cells and allowed to stand for adsorption at 36ºC for 60 minutes. The 1.0 mL portions of
the virus growth medium approved as suitable shall be added, cultured at 37ºC for seven days, andobserved.
3.3.2.2.3 Judgment
The cultured cells in which CPE is observed shall be regarded as infected to calculate TCID50.
The virus content in 1 mL shall be 107.5TCID50 or higher unless otherwise specified by the
Minister of Agriculture, Forestry and Fisheries.
3.3.2.3 Aino virus
3.3.2.3.1 Materials
3.3.2.3.1.1 Test materials
The test article shall be diluted ten-fold with the virus growth medium approved as suitable, andeach level dilution shall serve as the test material.
3.3.2.3.1.2 Cultured cells
HmLu-1 cells shall be used.
3.3.2.3.2 Test procedures
The 0.1 mL portions of the test material shall be inoculated into at least four portions (wells) ofcultured cells and allowed for adsorption at 36ºC for 60 minutes. The 1.0 mL portions of the virus
growth medium approved as suitable shall be added, cultured at 37ºC for seven days, andobserved.
3.3.2.3.3 Judgment
The cultured cells in which CPE is observed shall be regarded as infected to calculate TCID50.
The virus content in 1 mL shall be 106.5TCID50 or higher unless otherwise specified by the
Minister of Agriculture, Forestry and Fisheries.
3.3.2.4 Peaton virus
3.3.2.4.1 Materials
3.3.2.4.1.1 Test materials
The test article shall be diluted ten-fold with the virus growth medium approved as suitable, andeach level dilution shall serve as the test material.
3.3.2.4.1.2 Cultured cells
38

HmLu-1 cells shall be used.
3.3.2.4.2 Test procedures
The 0.1 mL portions of the test material shall be inoculated into at least four portions (wells) ofcultured cells and allowed to stand for adsorption at 37ºC for 60 minutes. The 1.0 mL portions of
the virus growth medium approved as suitable shall be added, cultured at 37ºC for seven days, andobserved.
3.3.2.4.3 Judgment
The cultured cells in which CPE is observed shall be regarded as infected to calculate TCID50.
The virus content in 1 mL shall be 107.5TCID50 or higher unless otherwise specified by the
Minister of Agriculture, Forestry and Fisheries.
3.4 Tests on inactivated virus suspension
3.4.1 Inactivation test
3.4.1.1 Akabane virus
3.4.1.1.1 Materials
3.4.1.1.1.1 Test materials
A 5 mL portion of the test article shall be dialyzed overnight at 4ºC using at least a 100-foldvolume of phosphate-buffered saline to remove the inactivator, and it shall be used as the test
material.
3.4.1.1.1.2 Cultured cells
HmLu-1 cells shall be cultured, and monolayer cultures shall be used.
3.4.1.1.2 Test procedures
Whole test material shall be inoculated to the cultured cell sheet of at least 3 cm2 per 1 mL
portion, allowed to stand for adsorption at 34ºC for 60 minutes, cultured with culture medium
approved as suitable at 37ºC for seven days, and observed.
3.4.1.1.3 Judgment
The cultured cells in which no CPE is observed shall be judged to be negative for active virus.
No active virus shall be observed in the test material.
3.4.1.2 Kasba virus
3.4.1.2.1 Materials
3.4.1.2.1.1 Test materials
A 5 mL portion of the test article shall be dialyzed overnight at 4ºC using at least a 100-foldvolume of phosphate-buffered saline to remove the inactivator, and it shall be used as the test
material.
3.4.1.2.1.2 Cultured cells
Vero-T cells shall be cultured, and monolayer cultures shall be used.
3.4.1.2.2 Test procedures
39

Whole test material shall be inoculated to the cultured cell sheet of at least 3 cm2 per 1 mL
portion, allowed to stand for adsorption at 34ºC for 60 minutes, cultured with culture medium
approved as suitable at 34 – 36ºC for five days, and then the cells shall be passaged to the next
generation. After the cell layers are formed, culture media shall be sampled. The culture medium
approved as suitable shall be added, cultured at 34ºC to 36ºC for five days, passaged tosubsequent generations, and then cultured in the same manner as the second generation andobserved.
3.4.1.2.3 Judgment
The cultured cells in which no CPE is observed shall be judged to be negative for active virus.
No active virus shall be observed in the test material.
3.4.1.3 Aino virus
3.4.1.3.1 Materials
3.4.1.3.1.1 Test materials
A 5 mL portion of the test article shall be dialyzed overnight at 4ºC using at least a 100-foldvolume of phosphate-buffered saline to remove the inactivator, and it shall be used as the test
material.
3.4.1.3.1.2 Cultured cells
HmLu-1 cells shall be cultured, and monolayer cultures shall be used.
3.4.1.3.2 Test procedures
Whole test material shall be inoculated to the cultured cell sheet of at least 3 cm2 per 1 mL
portion, allowed to stand for adsorption at 34ºC for 60 minutes, cultured with culture medium
approved as suitable at 34ºC to 36ºC for seven days, and observed.
3.4.1.3.3 Judgment
The cultured cells in which no CPE is observed shall be judged to be negative for active virus.
No active virus shall be observed in the test material.
3.4.1.4 Peaton virus
3.4.1.4.1 Materials
3.4.1.4.1.1 Test materials
A 5 mL portion of the test article shall be dialyzed overnight at 4ºC using at least a 100-foldvolume of phosphate-buffered saline to remove the inactivator, and it shall be used as the test
material.
3.4.1.4.1.2 Cultured cells
HmLu-1 cells shall be cultured, and monolayer cultures shall be used.
3.4.1.4.2 Test procedures
Whole test sample shall be inoculated to the cultured cell sheet of at least 3 cm2 per 1 mL
portion, allowed to stand for adsorption at 34ºC for 60 minutes, cultured with culture medium
40approved as suitable at 34ºC to 36ºC for seven days, and observed.
3.4.1.4.3 Judgment
The cultured cells in which no CPE is observed shall be judged to be negative for active virus.
No active virus shall be observed in the test article.
3.5 Tests on bulk material
3.5.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.6 Tests on final product3.6.1 Properties test
When the test is performed as specified in the Properties Test of the General Tests, the final
product shall be a homogeneous suspension with a specific color, and no foreign matter andforeign odor shall be observed. The property of the product per small container shall be uniform.
3.6.2 Test for pH
When the test is performed as specified in the Test for pH of the General Tests, the pH shallhave an intrinsic value.
3.6.3 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.6.4 Test for formalin content
For products inactivated with formalin when the test given in the Test for Formalin Content of
the General Tests is applied, the formalin content shall be no higher than 0.05 vol%.
3.6.5 Test for aluminum content
When the test is performed as specified in the Test for Aluminum Content of the General Tests,
the content of aluminum shall have a specific value. When the Minister of Agriculture, Forestry
and Fisheries specifies the test procedure, follow it.
3.6.6 Test for freedom from abnormal toxicity
The test given in the Test for Freedom from Abnormal Toxicity of the General Tests shall apply.
3.6.7 Potency test
3.6.7.1 Materials
3.6.7.1.1 Injection materials
The test samples shall be used as injection materials.
3.6.7.1.2 Test animals
Guinea pigs weighing approximately 350 g shall be used.
3.6.7.1.3 Virus for neutralization test
3.6.7.1.3.1 Akabane virus
Akabane virus JaGAr 39 strain propagated in HmLu cells or strain approved as equivalent
thereof shall be used.
41

3.6.7.1.3.2 Kasba virus
Kasba virus K-47 strain propagated in BHK-21 cells or strain approved as equivalent thereof
shall be used.
3.6.7.1.3.3 Aino virus
Aino virus JaNAr 28 strain propagated in HmLu cells or strain approved as equivalent thereof
shall be used.
3.6.7.1.3.4 Peaton virus
Peaton virus NS/3 strain propagated in HmLu cells or strain approved as equivalent thereof
shall be used.
3.6.7.1.4 Cultured cells
HmLu-1 cells and Vero-T cells shall be used.
3.6.7.2 Test procedures
A 0.5 mL each of the injection materials shall be injected intramuscularly twice at intervals of
three weeks in five test animals, and serum shall be obtained from individual animals 10 days
after the second injection to conduct a neutralization test.
The test serum shall be heat-inactivated, and then diluted with culture medium approved as
suitable in two-fold serial dilutions. Mix equal quantities of each diluted serum and virus
suspension for neutralization test containing approximately 200 TCID50 in 0.1 mL and neutralize
at 37ºC for 60 minutes. For the Kasba virus, the mixture shall be neutralized for 90 minutes. The
mixtures obtained shall be inoculated at a dose of 0.1 mL into four portions (wells) of cultured
HmLu-1 cells for the Akabane virus, Aino virus, and Peaton virus, respectively, and into four
portions (wells) of cultured Vero-T cells for the Kasba virus, respectively, and allowed to stand foradsorption at 37ºC for 60 minutes. Then, culture media approved as suitable shall be added at adose of 1.0 mL and cultured at 37ºC for seven days for the Akabane virus, Kasba virus, andPeaton virus and at 34ºC to 36ºC for seven days for the Aino virus.
3.6.7.3 Judgment
The maximum dilution factor of serum at which CPE is observed in at least two portions ofcultured cells shall be defined as the neutralizing antibody titer.
A 16-fold or greater increase in the neutralizing antibody titer against the Akabane virus andPeaton virus, a 32-fold or greater increase in the neutralizing antibody titer against the Kasba virus
and an 8-fold or greater increase in the neutralizing antibody titer against the Aino virus shall be
regarded as positive for neutralizing antibodies.
The neutralizing antibody positive rates in test animals shall be 80% or higher against eachvirus.

42

4 Storage and expiry date
The expiry date shall be two years and three months after the manufacturing unless otherwise
specified by the Minister of Agriculture, Forestry and Fisheries.

43

Infection Bovine Rhinotracheitis, Bovine Viral Diarrhea(bivalent), Bovine Parainfluenza, Bovine Respiratory
Syncytial Virus
Infection Vaccine (adjuvant), Inactivated, Seed
1 Definition
This Vaccine, Inactivated, Seed is made by mixing the inactivated virus suspensions obtainedby propagating Infections bovine rhinotracheitis virus, Bovine viral diarrhea virus type 1, Bovine
viral diarrhea virus type 2, Bovine parainfluenza type 3 virus, and Bovine respiratory syncytial
virus that meet the seed lot specifications in cell lines that meet these specifications, respectively,
and by adding an adjuvant.
2 Production methods
2.1 Virus strain used for production
2.1.1 Infectious bovine rhinotracheitis virus
2.1.1.1 Name
Infectious bovine rhinotracheitis virus No. 758-KB strain or strain approved as equivalent
thereof
2.1.1.2 Properties
This virus shows the pathogenic effects on cows, such as fever, respiratory symptoms, andleukopenia.
This virus shall be propagated in cultured cells derived from bovine and cultured cells derived
from swine in which CPE is confirmed.
2.1.1.3 Master seed virus
2.1.1.3.1 Preparing, storing, and maximum passage number allowed for obtaining final products
The master seed virus shall be propagated in MDBK-NSC cells or cells approved as suitable,
prepared in consecutive processes, and dispensed into storage containers.
The dispensed master seed virus shall be given a specific manufacturing number or
manufacturing code and stored frozen at -70ºC or lower or stored freeze-dried at 5ºC or lower.
The master seed virus shall be tested as specified in 3.1.1.1.
The master seed virus shall not be passaged for purposes other than the production of vaccines.
The master seed virus shall not be passaged more than five times to obtain the final product.
44

2.1.1.4 Working seed virus
2.1.1.4.1 Propagation, passage, and storage
The working seed virus shall be propagated and passaged in MDBK-NSC cells or cells
approved as suitable.
The working seed virus shall be stored frozen at -70ºC or lower or be stored freeze-dried at 5ºCor lower.
The working seed virus shall be tested as specified in 3.1.2.
2.1.1.5 Production seed virus
2.1.1.5.1 Propagation and storage
The production seed virus shall be propagated in MDBK-NSC cells or cells approved as
suitable.
The production seed virus shall be stored frozen at -70ºC or lower or stored freeze-dried at 5ºCor lower when it needs to be stored.
The production seed virus shall be tested as specified in 3.1.3 if it needs to be stored.
2.1.2 Bovine viral diarrhea virus type 1
2.1.2.1 Name
Bovine viral diarrhea virus type 1 Nose-KB strain or strain approved as equivalent thereof
2.1.2.2 Properties
Inoculation of this virus into the nasal cavity of cows causes respiratory symptoms, fever
viremia, and leukopenia.
This virus shall be propagated in cultured cells derived from bovine in which CPE is confirmed,
and if co-infected with cells infected with the non-cytopathogenic strain, CPE is inhibited.
2.1.2.3 Master seed virus
2.1.2.3.1 Preparing, storing, and maximum passage number allowed for obtaining final products
The master seed virus shall be propagated in MDBK-NSC cells or cells approved as suitable,
prepared in consecutive processes, and dispensed into storage containers.
The dispensed master seed virus shall be given a specific manufacturing number or
manufacturing code and stored frozen at -70ºC or lower or stored freeze-dried at 5ºC or lower.
The master seed virus shall be tested as specified in 3.1.1.2.
The master seed virus shall not be passaged for purposes other than the production of vaccines.
The master seed virus shall not be passaged more than five times to obtain the final product.
2.1.2.4 Working seed virus
2.1.2.4.1 Propagation, passage, and storage
The working seed virus shall be propagated and passaged in MDBK-NSC cells or cells
approved as suitable.
The working seed virus shall be stored frozen at -70ºC or lower or be stored freeze-dried at 5ºC45

or lower.
The working seed virus shall be tested as specified in 3.1.2.
2.1.2.5 Production seed virus
2.1.2.5.1 Propagation and storage
The production seed virus shall be propagated in MDBK-NSC cells or cells approved as
suitable.
The production seed virus shall be stored frozen at -70ºC or lower or stored freeze-dried at 5ºCor lower when it needs to be stored.
The production seed virus shall be tested as specified in 3.1.3 if it needs to be stored.
2.1.3 Bovine viral diarrhea virus type 2
2.1.3.1 Name
Bovine viral diarrhea virus type 2 KZ-91-KB strain or strain approved as equivalent thereof
2.1.3.2 Properties
Inoculation of this virus into the nasal cavity of calves causes respiratory symptoms, fever,viremia, and leukopenia.
This virus shall be propagated in cultured cells derived from bovine in which CPE is confirmed,
and if co-infected with cells infected with the non-cytopathogenic strain, CPE is inhibited.
2.1.3.3 Master seed virus
2.1.3.3.1 Preparing, storing, and maximum passage number allowed for obtaining final products
The master seed virus shall be propagated in MDBK-NSC cells or cells approved as suitable,
prepared in consecutive processes, and dispensed into storage containers.
The dispensed master seed virus shall be given a specific manufacturing number or
manufacturing code and stored frozen at -70ºC or lower or stored freeze-dried at 5ºC or lower.
The master seed virus shall be tested as specified in 3.1.1.3.
The master seed virus shall not be passaged for purposes other than the production of vaccines.
The master seed virus shall not be passaged more than five times to obtain the final product.
2.1.3.4 Working seed virus
2.1.3.4.1 Propagation, passage, and storage
The working seed virus shall be propagated and passaged in MDBK-NSC cells or cells
approved as suitable.
The working seed virus shall be stored frozen at -70ºC or lower or be stored freeze-dried at 5ºCor lower.
The working seed virus shall be tested as specified in 3.1.2.
2.1.3.5 Production seed virus
2.1.3.5.1 Propagation and storage
The production seed virus shall be propagated in MDBK-NSC cells or cells approved as46

suitable.
The production seed virus shall be stored frozen at -70ºC or lower or stored freeze-dried at 5ºCor lower when it needs to be stored.
The production seed virus shall be tested as specified in 3.1.3 if it needs to be stored.
2.1.4 Bovine parainfluenza type 3 virus
2.1.4.1 Name
Bovine parainfluenza type 3 virus BN1-1-KB strain or strain approved as equivalent thereof
2.1.4.2 Properties
This virus shows the pathogenic effects on cows, such as fever and respiratory symptoms.
This virus shall be propagated in cultured cells derived from bovine and cell lines derived fromAfrican green monkey kidneys (hereinafter referred to as “Vero cells” in this paragraph) in which
CPE is confirmed.
2.1.4.3 Master seed virus
2.1.4.3.1 Preparing, storing, and maximum passage number allowed for obtaining final products
The master seed virus shall be propagated in MDBK-NSC cells or cells approved as suitable,
prepared in consecutive processes, and dispensed into storage containers.
The dispensed master seed virus shall be given a specific manufacturing number or
manufacturing code and stored frozen at -70ºC or lower or stored freeze-dried at 5ºC or lower.
The master seed virus shall be tested as specified in 3.1.1.4.
The master seed virus shall not be passaged for purposes other than the production of vaccines.
The master seed virus shall not be passaged more than five times to obtain the final product.
2.1.4.4 Working seed virus
2.1.4.4.1 Propagation, passage, and storage
The working seed virus shall be propagated and passaged in MDBK-NSC cells or cells
approved as suitable.
The working seed virus shall be stored frozen at -70ºC or lower or be stored freeze-dried at 5ºCor lower.
The working seed virus shall be tested as specified in 3.1.2.
2.1.4.5 Production seed virus
2.1.4.5.1 Propagation and storage
The production seed virus shall be propagated in MDBK-NSC cells or cells approved as
suitable.
The production seed virus shall be stored frozen at -70ºC or lower or stored freeze-dried at 5ºCor lower when it needs to be stored.
The production seed virus shall be tested as specified in 3.1.3 if it needs to be stored.
2.1.5 Bovine respiratory syncytial virus
472.1.5.1 Name
Bovine respiratory syncytial virus rs-52-KB strain or strain approved as equivalent thereof
2.1.5.2 Properties
Inoculation of this virus on cows cause no pathogenic effects.
This virus shall be propagated in the following cells in which CPE is confirmed: cells derived
from bovine, cells derived from hamster lung and Vero cells.
2.1.5.3 Master seed virus
2.1.5.3.1 Preparing, storing, and maximum passage number allowed for obtaining final products
The master seed virus shall be propagated in HmLu-SC cells or cells approved as suitable,
prepared in consecutive processes, and dispensed into storage containers.
The dispensed master seed virus shall be given a specific manufacturing number or
manufacturing code and stored frozen at -70ºC or lower or stored freeze-dried at 5ºC or lower.
The master seed virus shall be tested as specified in 3.1.1.5.
The master seed virus shall not be passaged for purposes other than the production of vaccines.
The master seed virus shall not be passaged more than five times to obtain the final product.
2.1.5.4 Working seed virus
2.1.5.4.1 Propagation, passage, and storage
The working seed virus shall be propagated and passaged in HmLu-SC cells or cells approved
as suitable.
The working seed virus shall be stored frozen at -70ºC or lower or be stored freeze-dried at 5ºCor lower.
The working seed virus shall be tested as specified in 3.1.2.
2.1.5.5 Production seed virus
2.1.5.5.1 Propagation and storage
Production seed virus shall be propagated in HmLu-SC cells or cells approved as suitable.
The production seed virus shall be stored frozen at -70ºC or lower or stored freeze-dried at 5ºCor lower when it needs to be stored.
The production seed virus shall be tested as specified in 3.1.3 if it needs to be stored.
2.2 Materials for production
2.2.1 Infectious bovine rhinotracheitis virus, Bovine viral diarrhea virus type 1, Bovine viral diarrhea
virus type 2, and Bovine parainfluenza type 3 virus
2.2.1.1 Cultured cells
MDBK-NSC cells or cell lines approved as suitable shall be used.
2.2.1.2 Culture medium
The culture medium approved as suitable for production shall be used.
2.2.1.3 Master cell seeds48

2.2.1.3.1 Preparing, storing, and maximum passage number allowed for obtaining production cellseeds
The master cell seeds shall be propagated in a culture medium in 2.2.1.2, prepared inconsecutive processes, and dispensed into storage containers.
The dispensed master cell seeds shall be given a specific manufacturing number or
manufacturing code and stored frozen at -70ºC or lower.
The master cell seeds shall be tested as specified in 3.2.1.1.
The master cell seeds shall not be passaged for purposes other than production or testing ofvaccines.
The master cell seeds shall not be passaged more than 20 times to obtain the production cell
seeds.
2.2.1.4 Working cell seeds
2.2.1.4.1 Propagation, passage, and storage
The working cell seeds shall be propagated and passaged using culture medium in 2.2.1.2.
The working cell seeds shall be stored frozen at -70ºC or lower.
The working cell seeds shall be tested as specified in 3.2.2.1.
2.2.1.5 Production cell seeds
2.2.1.5.1 Propagation and storage
The production cell seeds shall be propagated using culture medium in 2.2.1.2.
The production cell seeds shall be stored frozen at -70°C or lower when it needs to be stored.
The production cell seeds shall be tested as specified in 3.2.3.1 if they need to be stored.
2.2.2 Bovine respiratory syncytial virus
2.2.2.1 Cultured cells
HmLu-SC cells or cell lines approved as suitable shall be used.
2.2.2.2 Culture medium
The culture medium approved as suitable for production shall be used.
2.2.2.3 Master cell seeds
2.2.2.3.1 Preparing, storing, and maximum passage number allowed for obtaining production cellseeds
The master cell seeds shall be propagated in a culture medium in 2.2.2.2, prepared inconsecutive processes, and dispensed into storage containers.
The dispensed master cell seeds shall be given a specific manufacturing number or
manufacturing code and stored frozen at -70ºC or lower.
The master cell seeds shall be tested as specified in 3.2.1.2.
The master cell seeds shall not be passaged for purposes other than production or testing ofvaccines. The master cell seeds shall not be passaged more than 20 times to obtain the production
49

cell seeds.
2.2.2.4 Working cell seeds
2.2.2.4.1 Propagation, passage, and storage
The working cell seeds shall be propagated and passaged using culture medium in 2.2.2.2.
The working cell seeds shall be stored frozen at -70ºC or lower.
The working cell seeds shall be tested as specified in 3.2.2.2.
2.2.2.5 Production cell seeds
2.2.2.5.1 Propagation and storage
The production cell seeds shall be propagated using culture medium in 2.2.2.2.
The production cell seeds shall be stored frozen at -70°C or lower when it needs to be stored.
The production cell seeds shall be tested as specified in 3.2.3.2 if they need to be stored.
2.3 Bulk material
2.3.1 Infectious bovine rhinotracheitis virus
2.3.1.1 Culture of production cell seeds
The cultured cells obtained in one session shall be regarded as individual cultured cells. No
abnormalities shall be detected prior to the inoculation of the virus.
2.3.1.2 Virus cultivation
The production seed virus shall be cultivated in the cells in 2.3.1.1. The filtrate, supernatant
after centrifugation, or concentrate of the culture medium collected from individual cultured cells
at a time deemed appropriate shall serve as the virus suspension.
The virus suspension shall be tested as specified in 3.3.1 and 3.3.2.1.
2.3.1.3 Inactivation
The virus suspension inactivated by treating it with a method approved as suitable shall be usedas the bulk material.
The bulk material shall be tested as specified in 3.4.1 and 3.4.2.1.
2.3.2 Bovine viral diarrhea virus type 1
2.3.2.1 Culture of production cell seeds
The cultured cells obtained in one session shall be regarded as individual cultured cells. No
abnormalities shall be detected in production cell seeds prior to the inoculation of the virus.
2.3.2.2 Virus cultivation
The production seed virus shall be cultivated in the cells in 2.3.2.1. The filtrate, supernatant
after centrifugation, or concentrate of the culture medium collected from individual cultured cells
at a time deemed appropriate shall serve as the virus suspension.
The virus suspension shall be tested as specified in 3.3.1 and 3.3.2.2.
2.3.2.3 Inactivation
The virus suspension inactivated by treating it with a method approved as suitable shall be used50

as the bulk material.
The bulk material shall be tested as specified in 3.4.1 and 3.4.2.1.
2.3.3 Bovine viral diarrhea virus type 2
2.3.3.1 Culture of production cell seeds
The cultured cells obtained in one session shall be regarded as individual cultured cells. No
abnormalities shall be detected prior to the inoculation of the virus.
2.3.3.2 Virus cultivation
The production seed virus shall be cultivated in the cells in 2.3.3.1. The filtrate, supernatant
after centrifugation, or concentrate of the culture medium collected from individual cultured cells
at a time deemed appropriate shall serve as the virus suspension.
The virus suspension shall be tested as specified in 3.3.1 and 3.3.2.3.
2.3.3.3 Inactivation
The virus suspension inactivated by treating it with a method approved as suitable shall be usedas the bulk material.
The bulk material shall be tested as specified in 3.4.1 and 3.4.2.1.
2.3.4 Bovine parainfluenza type 3 virus
2.3.4.1 Culture of production cell seeds
The cultured cells obtained in one session shall be regarded as individual cultured cells. No
abnormalities shall be detected prior to the inoculation of the virus.
2.3.4.2 Virus cultivation
The production seed virus shall be cultivated in the cells in 2.3.4.1. The filtrate, supernatant
after centrifugation, or concentrate of the culture medium collected from individual cultured cells
at a time deemed appropriate shall serve as the virus suspension.
The virus suspension shall be tested as specified in 3.3.1 and 3.3.2.4.
2.3.4.3 Inactivation
The virus suspension inactivated by treating it with a method approved as suitable shall be usedas the bulk material.
The bulk material shall be tested as specified in 3.4.1 and 3.4.2.1.
2.3.5 Bovine respiratory syncytial virus
2.3.5.1 Culture of production cell seeds
The cultured cells obtained in one session shall be regarded as individual cultured cells. No
abnormalities shall be detected in production cell seeds prior to the inoculation of the virus.
2.3.5.2 Virus cultivation
The production seed virus shall be cultivated in the cells in 2.3.5.1. The filtrate, supernatant
after centrifugation, or concentrate of the culture medium collected from individual cultured cells
at a time deemed appropriate shall serve as the virus suspension.
51

The virus suspension shall be tested as specified in 3.3.1 and 3.3.2.5.
2.3.5.3 Inactivation
The virus suspension inactivated by treating it with a method approved as suitable shall serveas the bulk material.
The bulk material shall be tested as specified in 3.4.1 and 3.4.2.2.
2.4 Final bulk
Mix Infectious bovine rhinotracheitis virus bulk material, Bovine viral diarrhea virus type 1bulk material, Bovine viral diarrhea virus type 2 bulk material, Bovine parainfluenza type 3 virus
bulk material, and Bovine respiratory syncytial virus bulk, add adjuvants and preservatives
approved as suitable, and then adjust the concentration of the mixture to serve as the final bulk.
2.5 Final product
The final bulk shall be dispensed into small containers to serve as the final product.
The final product shall be tested as specified in 3.5.
3 Test methods
3.1 Tests on virus strain used for production
3.1.1 Tests on master seed virus
3.1.1.1 Infectious bovine rhinotracheitis virus
3.1.1.1.1 Identification test
The tests given in 1.4.2.3.1.1.2 in the Seed Lot Specifications shall apply.
3.1.1.1.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.1.1.1.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.1.1.1.4 Test for freedom from extraneous viruses
3.1.1.1.4.1 Common test for freedom from extraneous viruses
The tests given in 1.1, 2.1 and 2.2 in the Test for Freedom from Extraneous Viruses of theGeneral Tests shall apply.
3.1.1.1.4.2 Test for freedom from specific viruses
3.1.1.1.4.2.1 General test for freedom from specific viruses
For the Bovine respiratory syncytial virus and Bluetongue virus, the tests given in 1.1 and 3.1 in
the Test for Freedom from Extraneous Viruses of the General Tests shall apply.
3.1.1.1.4.2.2 Individual test for freedom from specific viruses
For the Bovine viral diarrhea virus, rotavirus, Bovine leukemia virus, Japanese encephalitis
virus, and Rabies virus, the tests given in 1.1, 3.2.5, 3.2.7, 3.2.8 and 3.2.9 in the Test for Freedom
52

from Extraneous Viruses of the General Tests shall apply.
3.1.1.2 Bovine viral diarrhea virus type 1
3.1.1.2.1 Identification test
The tests given in 1.4.2.3.1.1.2 in the Seed Lot Specifications shall apply.
3.1.1.2.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.1.1.2.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.1.1.2.4 Test for freedom from extraneous viruses
3.1.1.2.4.1 Common test for freedom from extraneous viruses
The tests given in 1.1, 2.1 and 2.2 in the Test for Freedom from Extraneous Viruses of theGeneral Tests shall apply.
3.1.1.2.4.2 Test for freedom from specific viruses
3.1.1.2.4.2.1 General test for freedom from specific viruses
For the Bovine respiratory syncytial virus and Bluetongue virus, the tests given in 3.1 in the
Test for Freedom from Extraneous Viruses of the General Tests shall apply.
3.1.1.2.4.2.2 Individual test for freedom from specific viruses
For the Bovine viral diarrhea virus, Rotavirus, Bovine leukemia virus, Japanese encephalitis
virus, and Rabies virus, the tests given in 1.1, 3.2.5, 3.2.7, 3.2.8 and 3.2.9 in the Test for Freedom
from Extraneous Viruses of the General Tests shall apply.
3.1.1.3 Bovine viral diarrhea virus type 2
3.1.1.3.1 Identification test
The tests given in 1.4.2.3.1.1.2 in the Seed Lot Specifications shall apply.
3.1.1.3.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.1.1.3.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.1.1.3.4 Test for freedom from extraneous viruses
3.1.1.3.4.1 Common test for freedom from extraneous viruses
The tests given in 1.1, 2.1 and 2.2 in the Test for Freedom from Extraneous Viruses of theGeneral Tests shall apply.
3.1.1.3.4.2 Test for freedom from specific viruses
3.1.1.3.4.2.1 General test for freedom from specific viruses
For the Bovine respiratory syncytial virus and Bluetongue virus, the tests given in 3.1 in the53

Test for Freedom from Extraneous Viruses of the General Tests shall apply.
3.1.1.3.4.2.2 Individual test for freedom from specific viruses
For the Bovine viral diarrhea virus, Rotavirus, Bovine leukemia virus, Japanese encephalitis
virus, and Rabies virus, the tests given in 1.1, 3.2.5, 3.2.7, 3.2.8 and 3.2.9 in the Test for Freedom
from Extraneous Viruses of the General Tests shall apply.
3.1.1.4 Bovine parainfluenza type 3 virus
3.1.1.4.1 Identification test
The tests given in 1.4.2.3.1.1.2 in the Seed Lot Specifications shall apply.
3.1.1.4.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.1.1.4.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.1.1.4.4 Test for freedom from extraneous viruses
3.1.1.4.4.1 Common test for freedom from extraneous viruses
The tests given in 1.1, 2.1 and 2.2 in the Test for Freedom from Extraneous Viruses of theGeneral Tests shall apply.
3.1.1.4.4.2 Test for freedom from specific viruses
3.1.1.4.4.2.1 General test for freedom from specific viruses
For the Bovine respiratory syncytial virus and Bluetongue virus, the tests given in 1.1 and 3.1 in
the Test for Freedom from Extraneous Viruses of the General Tests shall apply.
3.1.1.4.4.2.2 Individual test for freedom from specific viruses
For the Bovine viral diarrhea virus, Rotavirus, Bovine leukemia virus, Japanese encephalitis
virus, and Rabies virus, the tests given in 1.1, 3.2.5, 3.2.7, 3.2.8 and 3.2.9 in the Test for Freedom
from Extraneous Viruses of the General Tests shall apply.
3.1.1.5 Bovine respiratory syncytial virus
3.1.1.5.1 Identification test
The tests given in 1.4.2.3.1.1.2 in the Seed Lot Specifications shall apply.
3.1.1.5.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.1.1.5.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.1.1.5.4 Test for freedom from extraneous viruses
3.1.1.5.4.1 Common test for freedom from extraneous viruses
The tests given in 1.1, 2.1 and 2.2 in the Test for Freedom from Extraneous Viruses of the54General Tests shall apply.
3.1.1.5.4.2 Test for freedom from specific viruses
3.1.1.5.4.2.1 General test for freedom from specific viruses
For the Lymphocytic choriomeningitis virus and Bluetongue virus, the tests given in 1.1 and 3.1
in the Test for Freedom from Extraneous Viruses of the General Tests shall apply.
3.1.1.5.4.2.2 Individual test for freedom from specific viruses
For the Bovine viral diarrhea virus, Rotavirus, Bovine leukemia virus, Japanese encephalitis
virus, and Rabies virus, the tests given in 1.1, 3.2.5, 3.2.7, 3.2.8 and 2.9 in the Test for Freedom
from Extraneous Viruses of the General Tests shall apply.
3.1.2 Tests on working seed virus
3.1.2.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.1.2.2 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.1.3 Tests on production seed virus
3.1.3.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.1.3.2 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.2 Tests on cultured cells
3.2.1 Tests on master cell seeds
3.2.1.1 MDBK-NSC cells
3.2.1.1.1 Test for confirmation of cell properties
The tests given in 2.1.4.2.1.1 in the Seed Lot Specifications shall apply.
3.2.1.1.2 Test for identification of the animal species of the cell
The tests given in 2.1.4.2.1.2 in the Seed Lot Specifications shall apply.
3.2.1.1.3 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.2.1.1.4 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.2.1.1.5 Test for freedom from extraneous viruses
3.2.1.1.5.1 Common test for freedom from extraneous viruses
The tests given in 1.2, 2.1 and 2.2 in the Test for Freedom from Extraneous Viruses of the55

General Tests shall apply.
3.2.1.1.5.2 Test for freedom from specific viruses
3.2.1.1.5.2.1 General test for freedom from specific viruses
For the Bovine respiratory syncytial virus and Bluetongue virus, the tests given in 1.2 and 3.1 in
the Test for Freedom from Extraneous Viruses of the General Tests shall apply.
3.2.1.1.5.2.2 Individual test for freedom from specific viruses
For the Bovine viral diarrhea virus, Rotavirus, Bovine leukemia virus, Japanese encephalitis
virus, and Rabies virus, the tests given in 1.2, 3.2.5, 3.2.7, 3.2.8 and 3.2.9 in the Test for Freedom
from Extraneous Viruses of the General Tests shall apply.
3.2.1.1.6 Test for karyological (chromosomal) characterization
The tests given in 2.1.4.2.1.6 in the Seed Lot Specifications shall apply.
3.2.1.1.7 Test for freedom from tumorigenicity
The tests given in 2.1.4.2.1.7 in the Seed Lot Specifications shall apply.
3.2.1.2 HmLu-SC cells
3.2.1.2.1 Test for confirmation of cell properties
The tests given in 2.1.4.2.1.1 in the Seed Lot Specifications shall apply.
3.2.1.2.2 Test for identification of the animal species of the cell
The tests given in 2.1.4.2.1.2 in the Seed Lot Specifications shall apply.
3.2.1.2.3 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.2.1.2.4 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.2.1.2.5 Test for freedom from extraneous viruses
3.2.1.2.5.1 Common test for freedom from extraneous viruses
The tests given in 1.2, 2.1 and 2.2 in the Test for Freedom from Extraneous Viruses of theGeneral Tests shall apply.
3.2.1.2.5.2 Test for freedom from specific viruses
3.2.1.2.5.2.1 General test for freedom from specific viruses
For the Bovine respiratory syncytial virus, Bluetongue virus, and Lymphocytic
choriomeningitis virus, the tests given in 1.2 and 3.1 in the Test for Freedom from Extraneous
Viruses of the General Tests shall apply.
3.2.1.2.5.2.2 Individual test for freedom from specific viruses
For the Bovine viral diarrhea virus, Rotavirus, Bovine leukemia virus, Japanese encephalitis
virus, and Rabies virus, the tests given in 1.2, 3.2.5, 3.2.7, 3.2.8 and 3.2.9 in the Test for Freedom
from Extraneous Viruses of the General Tests shall apply.
56

3.2.1.2.6 Test for karyological (chromosomal) characterization
The tests given in 2.1.4.2.1.6 in the Seed Lot Specifications shall apply.
3.2.1.2.7 Test for freedom from tumorigenicity
The tests given in 2.1.4.2.1.7 in the Seed Lot Specifications shall apply.
3.2.2 Tests on working cell seeds
3.2.2.1 MDBK-NSC cells
3.2.2.1.1 Test for confirmation of cell properties
The tests given in 2.1.4.2.2.1 in the Seed Lot Specifications shall apply.
3.2.2.1.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.2.2.1.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.2.2.2 HmLu-SC cells
3.2.2.2.1 Test for confirmation of cell properties
The tests given in 2.1.4.2.2.1 in the Seed Lot Specifications shall apply.
3.2.2.2.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.2.2.2.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.2.3 Tests on production cell seeds
3.2.3.1 MDBK-NSC cells
3.2.3.1.1 Test for confirmation of cell properties
The tests given in 2.1.4.2.3.1 in the Seed Lot Specifications shall apply.
3.2.3.1.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.2.3.1.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.2.3.2 HmLu-SC cells
3.2.3.2.1 Test for confirmation of cell properties
The tests given in 2.1.4.2.3.1 in the Seed Lot Specifications shall apply.
3.2.3.2.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.2.3.2.3 Test for freedom from Mycoplasma contamination
57

The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.3 Tests on virus suspension
3.3.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.3.2 Test for virus content
3.3.2.1 Infectious bovine rhinotracheitis virus
3.3.2.1.1 Materials
3.3.2.1.1.1 Test materials
The test article shall be diluted ten-fold with cell growth medium (Note 1) and each level
dilution shall serve as the test material.
3.3.2.1.1.2 Cultured cells
Cell line suspension derived from bovine kidneys (hereinafter referred to as “MDBK cells” inthis paragraph) shall be used.
3.3.2.1.2 Test procedures
The 100 μL portions of the test material shall be inoculated into 100 μL portions of cultured
cells dispensed into at least 4 wells in a 96-well microplate, cultured in an air containing 5 vol%carbon dioxide at 37ºC for seven days, and observed.
3.3.2.1.3 Judgment
The cultured cells in which CPE is observed shall be regarded as infected to calculate TCID50.
The virus content in 1 mL shall be 108.0TCID50 or higher.
3.3.2.2 Bovine viral diarrhea virus type 1
3.3.2.2.1 Materials
3.3.2.2.1.1 Test materials
The test article shall be diluted ten-fold with cell growth medium and each level dilution shallserve as the test material.
3.3.2.2.1.2 Cultured cells
Bovine testis cell suspension shall be used.
3.3.2.2.2 Test procedures
The 100 μL portions of the test sample shall be inoculated into 100 μL portions of cultured cells
dispensed into at least 4 wells in a 96-well microplate, cultured in an air containing 5 vol% carbon
dioxide at 37ºC for seven days, and observed.
3.3.2.2.3 Judgment
The cultured cells in which CPE is observed shall be regarded as infected to calculate TCID50.
The virus content in 1 mL shall be 107.6TCID50 or higher.
3.3.2.3 Bovine viral diarrhea virus type 2
58

3.3.2.3.1 Materials
3.3.2.3.1.1 Test materials
The test article shall be diluted ten-fold with cell growth medium and each level dilution shallserve as the test material.
3.3.2.3.1.2 Cultured cells
Bovine testis cell suspension shall be used.
3.3.2.3.2 Test procedures
The 100 μL portions of the test sample shall be inoculated into 100 μL portions of cultured cells
dispensed into at least 4 wells in a 96-well microplate, cultured in an air containing 5 vol% carbon
dioxide at 37ºC for seven days, and observed.
3.3.2.3.3 Judgment
The cultured cells in which CPE is observed shall be regarded as infected to calculate TCID50.
The virus content in 1 mL shall be 106.4TCID50 or higher.
3.3.2.4 Bovine parainfluenza type 3 virus
3.3.2.4.1 Materials
3.3.2.4.1.1 Test materials
The test article shall be diluted ten-fold with cell growth medium and each level dilution shallserve as the test material.
3.3.2.4.1.2 Cultured cells
MDBK cell suspension shall be used.
3.3.2.4.2 Test procedures
The 100 μL portions of the test sample shall be inoculated into 100 μL portions of cultured cells
dispensed into at least 4 wells in a 96-well microplate, cultured in an air containing 5 vol% carbon
dioxide at 37ºC for seven days, and observed.
3.3.2.4.3 Judgment
The cultured cells in which CPE is observed shall be regarded as infected to calculate TCID50.
The virus content in 1 mL shall be 107.9TCID50 or higher.
3.3.2.5 Bovine respiratory syncytial virus
3.3.2.5.1 Materials
3.3.2.5.1.1 Test materials
The test article shall be diluted ten-fold with cell growth medium and each level dilution shallserve as the test material.
3.3.2.5.1.2 Cultured cells
Vero cell suspension shall be used.
3.3.2.5.2 Test procedures
The 100 μL portions of the test sample shall be inoculated into 100 μL portions of cultured cells
59dispensed into at least 4 wells in a 96-well microplate, cultured in an air containing 5 vol% carbon
dioxide at 34ºC for seven days, and observed.
3.3.2.5.3 Judgment
The cultured cells in which CPE is observed shall be regarded as infected to calculate TCID50.
The virus content in 1 mL shall be 106.6TCID50 or higher.
3.4 Tests on bulk material
3.4.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.4.2 Inactivation test
3.4.2.1 Infectious bovine rhinotracheitis virus, Bovine viral diarrhea virus type 1, Bovine viraldiarrhea virus type 2 and Bovine parainfluenza type 3 virus
3.4.2.1.1 Materials
3.4.2.1.1.1 Test materials
The test article shall be dialyzed overnight at 4ºC using at least a 100-fold volume ofphosphate-buffered saline to remove the inactivator, and it shall be used as the test material.
3.4.2.1.1.2 Cultured cells
MDBK cells shall be cultured for one to three days, and monolayer cultures shall be used.
3.4.2.1.2 Test procedures
A 1 mL portion of the test material shall be inoculated to the cultured cell sheet of at least 3
cm2, allowed to adsorb for 90 minutes at 37ºC, cultured with the virus growth medium (Note 2) at
37ºC for seven days, and observed.
3.4.2.1.3 Judgment
The cultured cells in which no CPE is observed shall be judged to be negative for active virus.
No active virus shall be observed in the test materials.
3.4.2.2 Bovine respiratory syncytial virus
3.4.2.2.1 Materials
3.4.2.2.1.1 Test materials
The test article shall be dialyzed overnight at 4ºC using at least a 100-fold volume ofphosphate-buffered saline to remove the inactivator, and it shall be used as the test material.
3.4.2.2.1.2 Cultured cells
Vero cells shall be cultured for one to three days, and monolayer cultures shall be used.
3.4.2.2.2 Test procedures
A 1 mL portion of the test sample shall be inoculated to the cultured cell sheet of at least 3 cm2
or more, allowed to adsorb for 90 minutes at 37ºC, cultured with the virus growth medium at 34ºC
for seven days, and observed.
3.4.2.2.3 Judgment
60

The cultured cells in which no CPE is observed shall be judged to be negative for active virus.
No active virus shall be observed in the test materials.
3.5 Tests on final product3.5.1 Properties test
When the test is performed as specified in the Properties Test of the General Tests, the final
product shall be a homogeneous suspension with a specific color, and no foreign matter andforeign odor shall be observed. The property of the product per small container shall be uniform.
3.5.2 Test for pH
When the test is performed as specified in the Test for pH of the General Tests, the pH shallhave an intrinsic value.
3.5.3 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.5.4 Test for formalin content
When the test given in the Test for Formalin Content of the General Tests is applied, theformalin content shall be no higher than 0.08 vol%.
3.5.5 Test for aluminum content
When the test is performed as specified in the Test for Aluminum Content of the General Tests,
the content of aluminum shall be 1.35–1.65 mg in 1 mL.
3.5.6 Toxicity limit test
The test given in the Toxicity Limit Test 2 of the General Tests shall apply. However, verify by
a t-test of population mean differences assuming equal variances (unpaired, two-tailed, 5% levelof significance) that the body weight on the third day after injected at a dose of 1 mL is equal to or
greater than that before injected.
3.5.7 Potency test
3.5.7.1 Potency on infectious bovine rhinotracheitis
3.5.7.1.1 Materials
3.5.7.1.1.1 Injection materials
The test samples shall be used as injection materials.
3.5.7.1.1.2 Test animals
Guinea pigs weighing approximately 300 g shall be used.
3.5.7.1.1.3 Virus for neutralization test
Infectious bovine rhinotracheitis virus No. 758 strain or strain approved as suitable shall be
used.
3.5.7.1.1.4 Cultured cells
MDBK cell suspension shall be used.
3.5.7.1.2 Test procedures
61

Three mL each of the injection materials shall be injected twice at intervals of two weeks in fivetest animals. Each test material shall be injected intramuscularly at a volume of 1 mL into twosites and subcutaneously at a volume of 1 mL into one site. The serum shall be obtained fromindividual animals 10 days after the second injection to conduct a neutralization test.
The serum shall be heat-inactivated, and then shall be diluted two-fold with the virus growth
medium. Mix equal quantities of each diluted serum and virus for neutralization test containing
approximately 200 TCID50 in 25 μL and neutralize at 37ºC for 18 hours. The 25 μL portions of themixed solution shall be inoculated into 4 wells in a 96-well microplate, the 0.1 mL portions of thecultured cells shall be added, and cultured in an air containing 5 vol% carbon dioxide at 37ºC forseven days, and observed.
3.5.7.1.3 Judgment
The maximum dilution factor of serum at which inhibition of CPE is observed in at least two
wells of cultured cells shall be defined as the neutralizing antibody titer.
When a 128-fold or greater increase in the neutralizing antibody titer is regarded as positive, theneutralizing antibody positive rate in test animals shall be 80% or higher.
3.5.7.2 Potency on bovine viral diarrhea
3.5.7.2.1 Materials
3.5.7.2.1.1 Injection materials
The test samples shall be used as injection materials.
3.5.7.2.1.2 Test animals
Rats weighing approximately 100 g shall be used.
3.5.7.2.1.3 Virus for neutralization test
3.5.7.2.1.3.1 Bovine viral diarrhea virus type 1
Bovine viral diarrhea virus type 1 Nose strain or strain approved as suitable shall be used.
3.5.7.2.1.3.2 Bovine viral diarrhea virus type 2
Bovine viral diarrhea virus type 2 KZ-91-cp strain or strain approved as suitable shall be used.
3.5.7.2.1.4 Cultured cells
MDBK cell suspension shall be used.
3.5.7.2.2 Test procedures
Two mL each of the injection materials shall be injected intramuscularly at a volume of 1 mLeach in thighs of both hindlimbs in five test animals. Serum shall be obtained from individual
animals 21 days after the injection to conduct a neutralization test.
The serum shall be heat-inactivated, and then shall be diluted two-fold with the virus growth
medium. Mix equal quantities of each diluted serum and virus for neutralization test containing
approximately 200 TCID50 in 25 μL and neutralize at 37ºC for 60 minutes. The 25 μL portions of
the mixed solution shall be inoculated into 4 wells in a 96-well microplate, the 0.1 mL portions of62

the cultured cells shall be added, and cultured in an air containing 5 vol% carbon dioxide at 37ºC
for seven days, and observed.
3.5.7.2.3 Judgment
The maximum dilution factor of serum at which inhibition of CPE is observed in at least two
wells of cultured cells shall be defined as the neutralizing antibody titer.
When a two-fold or greater increase in the neutralizing antibody titer is regarded as positive, theneutralizing antibody positive rates in test animals shall be 80% or higher against bovine viraldiarrhea virus type 1 and bovine viral diarrhea virus type 2, respectively.
3.5.7.3 Potency test on bovine parainfluenza
3.5.7.3.1 Materials
3.5.7.3.1.1 Injection materials
The injection materials specified in 3.5.7.1.1.1 shall be used.
3.5.7.3.1.2 Test animals
The test animals specified in 3.5.7.1.1.2 shall be used.
3.5.7.3.1.3 Virus for neutralization test
Bovine parainfluenza type 3 virus BN1-1 strain or strain approved as suitable shall be used.
3.5.7.3.1.4 Cultured cells
Vero cell suspension shall be used.
3.5.7.3.2 Test procedures
The test method given in 3.5.7.1.2 shall apply.
However, the serum to be tested shall be neutralized at 37ºC for 60 minutes.
3.5.7.3.3 Judgment
The maximum dilution factor of serum at which inhibition of CPE is observed in at least two
wells of cultured cells shall be defined as the neutralizing antibody titer.
When an at least four-fold increase in the neutralizing antibody titer is regarded as positive, theneutralizing antibody positive rate in test animals shall be 80% or higher.
3.5.7.4 Potency on bovine respiratory syncytial virus infection
3.5.7.4.1 Materials
3.5.7.4.1.1 Injection materials
The injection materials specified in 3.5.7.1.1.1 shall be used.
3.5.7.4.1.2 Test animals
The test animals specified in 3.5.7.1.1.2 shall be used.
3.5.7.4.1.3 Virus for neutralization test
Bovine respiratory syncytial virus NMK7 strain or strain approved as suitable shall be used.
3.5.7.4.1.4 Cultured cells
Vero cells which are grown as monolayers in a 96-well microplate shall be used.
63

3.5.7.4.2 Test procedures
A neutralization test shall be performed on the serum obtained in 3.5.7.1.2.
The serum shall be heat-inactivated, and then shall be diluted two-fold with the virus growth
medium. Equal quantities of each diluted serum and virus for neutralization test containing
approximately 200 TCID50 in 25 μL shall be mixed and neutralized at 37ºC for 60 minutes.
Removing the culture medium of Vero cells, the 25 μL portions of the mixture shall be added into
4 wells, respectively, and adsorbed in an air containing 5 vol% carbon dioxides at 37ºC for 60
minutes. After the adsorption, the 0.1 mL portions of the virus growth medium shall be added,
cultured in an air containing 5 vol% carbon dioxides at 34ºC for seven days, and observed.
3.5.7.4.3 Judgment
The maximum dilution factor of serum at which inhibition of CPE is observed in at least two
wells of cultured cells shall be defined as the neutralizing antibody titer.
When a two-fold or greater increase in the neutralizing antibody titer is regarded as positive, theneutralizing antibody positive rate in test animals shall be 80% or higher.
4 Storage and expiry date
The expiry date shall be one year and six months after the manufacturing unless otherwise
specified by the Minister of Agriculture, Forestry and Fisheries.
Note 1 Cell growth medium
1,000 mL consists of the following:
Tryptose phosphate broth

2.95 gFetal bovine serum

50–100 mL

Eagle’s MEM

Residual quantity

The pH shall be adjusted to 7.2–7.6 with sodium hydrogen carbonate.
Fetal bovine serum used shall be suitable for cell maintenance and virus growth.
Minimal amounts of antibiotics may be added.
Note 2 Virus growth medium
1,000 mL consists of the following:
Tryptose phosphate broth

2.95 gFetal bovine serum

20–100 mL

Eagle’s MEM

Residual quantity

The pH shall be adjusted to 7.2–7.6 with sodium hydrogen carbonate.
Fetal bovine serum used shall be suitable for cell maintenance and virus growth.
Minimal amounts of antibiotics may be added.
64Equine Influenza Vaccine, Inactivated
1 Definition
Equine Influenza Vaccine, Inactivated is an inactivated vaccine produced by the purificationand concentration of the virus suspension obtained by propagating Equine influenza viruses inembryonated chicken eggs.
2 Production methods
2.1 Virus strain used for production
2.1.1 Name
Strains of Equine influenza A virus specified separately
2.1.2 Properties
The virus strains shall be propagated in the allantoic cavities of embryonated chicken eggs, andit shall agglutinate chicken red blood cells.
2.1.3 Passage and storage
The original strain and seed virus shall be propagated in the embryonated chicken eggs.
The original strain and seed virus shall not be passaged more than five times and three times,
respectively.
This does not apply if especially specified.
The original strain and seed virus shall be stored frozen at -70ºC or lower or be stored freezedried at 5ºC or lower.
2.2 Materials for production
2.2.1 Embryonated chicken eggs
Embryonated chicken eggs aged 10–12 days shall be used.
2.3 Bulk material
2.3.1 Virus cultivation
Each strain of virus shall be separately inoculated into the allantoic cavities of embryonatedchicken eggs, and the inoculated eggs shall be incubated. The allantoic fluid containing the
propagated virus shall be harvested to serve as the virus suspension of each strain.
The virus suspension shall be tested as specified in 3.1.
2.3.2 Virus purification and concentration
Each virus suspension shall be purified and concentrated by the ultracentrifugal method,
alcohol precipitation method, or other methods approved as suitable. In this case, a stabilizer
approved as suitable may be added.
2.3.3 Inactivation
65Each purified and concentrated virus suspension shall be inactivated by the addition of formalin
to serve as the virus suspension of each strain.
The inactivated virus suspensions shall be tested as specified in 3.2.
2.3.4 Bulk material
Virus suspension shall be mixed and diluted with a dilution approved as suitable to adjust theconcentration to serve as the bulk material.
The bulk material shall be tested as specified in 3.3.
2.4 Final bulk
The bulk material shall be mixed to serve as the final bulk. In this case, a stabilizer approved as
suitable may be added.
2.5 Final product
The final bulk shall be dispensed into small containers to serve as the final products.
The final products shall be tested as specified in 3.4.
3 Test methods
3.1 Test on virus suspension
3.1.1 Hemagglutination titer test
3.1.1.1 Materials
3.1.1.1.1 Test materials
A series of two-fold dilutions of the test article shall be prepared using physiological saline.
Each dilution shall be used as the test material.
3.1.1.2 Test procedures
To each 0.4 mL portion of the test material, add 0.4 mL portion of chicken red blood cells
adjusted to 0.5 vol% and allow to react for 60 minutes.
3.1.1.3 Judgment
The highest dilution of test article that causes the hemagglutination is considered thehemagglutination titer.
The hemagglutination titer of the test article shall be within its specific range.
3.2 Test on inactivated virus suspension
3.2.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.2.2 Inactivation test
3.2.2.1 Materials
3.2.2.1.1 Injection materials
The test article shall be used as the injection material.
3.2.2.1.2 Embryonated chicken eggs
66

Eggs aged 9–10 days shall be used as specified in 1.1 in the Materials for Live Vaccine
Production.
3.2.2.2 Test procedures
The injection material shall be inoculated at a dose of 0.2 mL into the allantoic cavities of atleast six embryonated chicken eggs. The inoculated eggs shall be incubated at 32℃ to 36℃. The
allantoic fluid shall be passaged every three days to the second generation. To the secondpassaged allantoic fluid, red blood cells given in 3.1.1.2 shall be added and observed.
If the allantoic fluid of each egg shows hemagglutination, equal volumes of these allantoic
fluids shall be mixed and passaged to the third generation. The third-passaged allantoic fluid shallbe retested in a similar manner and then judged.
3.2.2.3 Judgment
If no hemagglutination is observed, the test article shall be judged to be negative for active
virus.
No active virus shall be observed in the test article.
3.3 Tests on bulk material
3.3.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.3.2 CCA test
3.3.2.1 Materials
The test article and standard influenza vaccine (for CCA) (Note 1) shall be used as thematerials.
3.3.2.2 Test procedures
The CCA value of the test article shall be determined by the modified Miller-Stanley's method.
3.3.2.3 Judgment
The CCA value in 1 mL of the test article shall be within its specific range.
3.4 Tests on final product3.4.1 Properties test
When the test is performed as specified in the Properties Test of the General Tests, the final
product shall be a liquid or a homogeneous suspension with a specific color, and no foreign
matter and foreign odor shall be observed. The property of product per small container shall be
uniform.
3.4.2 Test for pH
When the test is performed as specified in the Test for pH of the General Tests, the pH shallhave an intrinsic value.
3.4.3 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
67

3.4.4 Test for formalin content
For products added with formalin, when the test given in the Test for Formalin Content of theGeneral Tests is applied, the formalin content shall be no higher than 0.1 vol%.
3.4.5 Test for thimerosal content
For products added with thimerosal, the test given in the Test for Thimerosal Content of theGeneral Tests shall apply.
3.4.6 Test for protein nitrogen content
When the test is performed as specified in the Test for Protein Nitrogen Content of the General
Tests, the protein nitrogen content shall be 50 μg or lower in 1 mL.
3.4.7 Test for freedom from abnormal toxicity
The test given in the Test for Freedom from Abnormal Toxicity of the General Tests shall apply.
3.4.8 Potency test
3.4.8.1 Materials
3.4.8.1.1 Injection materials
The test sample shall be diluted ten-fold with phosphate-buffered saline to serve as the injection
material.
3.4.8.1.2 Test animals
Mice aged 4 weeks shall be used.
3.4.8.1.3 Hemagglutination antigens
Hemagglutination antigens of each virus strain contained in the test sample shall be used.
3.4.8.2 Test procedures
Twenty test animals inoculated with 0.5 mL portions of injection materials into the peritoneal
cavities, and then divided into four groups. Sera obtained after 14 days shall be pooled for eachgroup to perform a hemagglutination inhibition test. The sera to be tested shall be prepared by
RDE, trypsin, potassium periodate, or a method approved as suitable and make two-folddilutions. To 0.2 mL of each dilution, a 0.2 mL portion of hemagglutination antigens containing 8
units per 0.4 mL shall be added and treated at 37℃ for 60 minutes. To the treated mixtures, 0.4
mL portions of red blood cells given in 3.1.1.2 shall be added, allowed to stand for 60 minutes,
and observed for the presence of hemagglutination.
3.4.8.3 Judgment
The highest dilution of the pooled sera that causes the inhibition of hemagglutination isconsidered the hemagglutination inhibition titer. In at least two of four groups, thehemagglutination inhibition titer shall be no less than 8.
4 Storage and expiry date
The expiry date shall be one year and a half after the manufacturing unless otherwise specified68

by the Minister of Agriculture, Forestry and Fisheries.
Note 1 Standard influenza vaccine (for CCA)
A standard provided by the National Veterinary Assay Laboratory that contains 1,000 CCA ofvirus in 1 mL

69

Japanese Encephalitis Vaccine, Inactivated, Seed
1 Definition
Japanese encephalitis, vaccine, inactivated, seed is a vaccine of inactivated virus suspension
obtained by propagating the Japanese encephalitis virus that meets the seed lot specifications incell lines that meets the specifications.
2 Production methods
2.1 Virus strain used for production
2.1.1 NameJapanese encephalitis virus Nakayama-Yakken strain or strain approved as equivalent thereof
2.1.2 Properties
This virus strain shall be propagated in primary cells of swine kidneys, and goose, day-old
chick and pigeon red blood cells shall be hemagglutinated.
2.1.3 Master seed virus
2.1.3.1 Preparing, storing, and maximum passage number allowed for obtaining final products
The master seed virus shall be propagated in cells approved as suitable, prepared in consecutive
processes, and dispensed into storage containers.
The dispensed master seed virus shall be given a specific manufacturing number or
manufacturing code and stored frozen at -60ºC or lower or stored freeze-dried at 5ºC or lower.
The master seed virus shall be tested as specified in 3.1.1.
The master seed virus shall not be passaged for purposes other than the production of vaccines.
The master seed virus shall not be passaged more than five times to obtain the final product.
2.1.4 Working seed virus
2.1.4.1 Propagation, passage, and storage
The working seed virus shall be propagated and passaged in cells approved as suitable.
The working seed virus shall be stored frozen at -60ºC or lower or be stored freeze-dried at 5ºCor lower.
The working seed virus shall be tested as specified in 3.1.2.
2.1.5 Production seed virus
2.1.5.1 Propagation and storage
The production seed virus shall be propagated in cells approved as suitable.
The production seed virus shall be stored frozen at -60ºC or lower or stored freeze-dried at 5ºCor lower when it needs to be stored.
The production seed virus shall be tested as specified in 3.1.3 if it needs to be stored.
70

2.2 Materials for production
2.2.1 Cell line
Vero cells or cell lines approved as suitable for production shall be used.
2.2.2 Culture medium
The culture medium approved as suitable for production shall be used.
2.2.3 Master cell seeds
2.2.3.1 Preparing, storing, and maximum passage number allowed for obtaining production cellseeds
The master cell seeds shall be propagated in a culture medium in 2.2.2, prepared in consecutive
processes, and dispensed into storage containers.
The dispensed master cell seeds shall be given a specific manufacturing number or
manufacturing code and stored frozen at -70ºC or lower.
The master cell seeds shall be tested as specified in 3.2.1.
The master cell seeds shall not be passaged for purposes other than production or testing ofvaccines.
The master cell seeds shall not be passaged more than 20 times to obtain the production cell
seeds.
2.2.4 Working cell seeds
2.2.4.1 Propagation, passage, and storage
The working cell seeds shall be propagated and passaged using culture medium in 2.2.2.
The working cell seeds shall be stored frozen at -70ºC or lower.
The working cell seeds shall be tested as specified in 3.2.2.
2.2.5 Production cell seeds
2.2.5.1 Propagation and storage
The production cell seeds shall be propagated using culture medium in 2.2.2.
The production cell seeds shall be stored frozen at -70°C or lower when it needs to be stored.
The production cell seeds shall be tested as specified in 3.2.3 if they need to be stored.
2.3 Bulk material
2.3.1 Culture of production cell seeds
The cultured cells obtained in one session shall be regarded as individual cultured cells. No
abnormalities shall be detected in the cultured cells prior to the inoculation of the virus.
2.3.2 Virus cultivation
The production seed virus shall be cultivated in the cells in 2.3.1. The filtrate, supernatant after
centrifugation, or concentrate of the culture medium collected from individual cultured cells at thepeak of virus propagation shall serve as the virus suspension.
The virus suspension shall be tested as specified in 3.3.
71

2.3.3 Inactivation
The virus suspension shall be inactivated by the addition of formalin or inactivator approved as
suitable and shall serve as the inactivated virus suspension.
The inactivated virus suspension shall be tested as specified in 3.4.
2.3.4 Bulk material
The inactivated virus suspension shall be mixed to make the final bulk. The bulk material shallbe tested as specified in 3.5.
2.4 Final bulk
The bulk material shall be mixed, diluted with a dilution approved as suitable to adjust its
concentration and shall serve as the final bulk.
In this case, preservatives approved as suitable may be added.
2.5 Final product
The final bulk shall be dispensed into small containers to serve as the final product.
The final product shall be tested as specified in 3.6.
3 Test methods
3.1 Tests on virus strain used for production
3.1.1 Tests on master seed virus
3.1.1.1 Identification test
The tests given in 1.4.2.3.1.1 in the Seed Lot Specifications shall apply.
3.1.1.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.1.1.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.1.1.4 Test for freedom from extraneous viruses
3.1.1.4.1 Common test for freedom from extraneous viruses
The tests given in 1.1, 2.1 and 2.2 in the Test for Freedom from Extraneous Viruses of theGeneral Tests shall apply.
3.1.1.4.2 Test for freedom from specific viruses
3.1.1.4.2.1 General test for freedom from specific viruses
For the Equine infectious anemia virus, Porcine reproductive and respiratory syndrome virus,
and Endogenous retrovirus (Types C and D particles) in a test using cells derived from monkeys,
the test given in 1.1 and 3.1.1 of the Test for Freedom from Extraneous Viruses of the General
Tests shall apply.
3.1.1.4.2.2 Individual test for freedom from specific viruses
72For the Classical swine fever virus, Porcine circovirus, Bovine viral diarrhea virus, Rotavirus,
and Rabies virus, the test given in 1.1, 3.2.3, 3.2.4, 3.2.5, 3.2.7 and 3.2.9 in the Test for Freedom
from Extraneous Viruses of the General Tests shall apply.
3.1.2 Tests on working seed virus
3.1.2.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.1.2.2 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.1.3 Tests on production seed virus
3.1.3.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.1.3.2 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.2 Tests on cell line
3.2.1 Tests on master cell seeds
3.2.1.1 Test for confirmation of cell properties
The tests given in 2.1.4.2.1.1 in the Seed Lot Specifications shall apply.
3.2.1.2 Test for identification of the animal species of the cell
The tests given in 2.1.4.2.1.2 in the Seed Lot Specifications shall apply.
3.2.1.3 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.2.1.4 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.2.1.5 Test for freedom from extraneous viruses
3.2.1.5.1 Common test for freedom from extraneous viruses
The tests given in 1.2, 2.1 and 2.2 in the Test for Freedom from Extraneous Viruses of theGeneral Tests shall apply.
3.2.1.5.2 Test for freedom from specific viruses
3.2.1.5.2.1 General test for freedom from specific viruses
For the Equine infectious anemia virus, Porcine reproductive and respiratory syndrome virus,
and Endogenous retrovirus (Types C and D particles), the test given in 1.2 and 3.1.1 of the Test forFreedom from Extraneous Viruses of the General Tests shall apply.
3.2.1.5.2.2 Individual test for freedom from specific viruses
73For the Classical swine fever virus, Porcine circovirus, Bovine viral diarrhea virus, Rotavirus,
Japanese encephalitis virus, and Rabies virus, the test given in 1.2, 3.2.3, 3.2.4, 3.2.5, 3.2.7 and3.2.9 of the Test for Freedom from Extraneous Viruses of the General Tests shall apply.
3.2.1.6 Test for karyological (chromosomal) characterization
The tests given in 2.1.4.2.1.6.1 in the Seed Lot Specifications shall apply.
3.2.1.7 Test for freedom from tumorigenicity
The tests given in 2.1.4.2.1.7 in the Seed Lot Specifications shall apply.
3.2.2 Tests on working cell seeds
3.2.2.1 Test for confirmation of cell properties
The tests given in 2.1.4.2.2.1 in the Seed Lot Specifications shall apply.
3.2.2.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.2.2.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.2.3 Tests on production cell seeds
3.2.3.1 Test for confirmation of cell properties
The tests given in 2.1.4.2.3.1 in the Seed Lot Specifications shall apply.
3.2.3.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.2.3.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.3 Tests on virus suspension
3.3.1 Test for virus content
The test shall be performed through the inoculation test in mice or inoculation test in cultured
cells.
3.3.1.1 Inoculation test in mice3.3.1.1.1 Materials
3.3.1.1.1.1 Test materials
The test article shall be diluted ten-fold with phosphate-buffered saline or diluent solution
approved as suitable, and each level dilution shall serve as the test material.
3.3.1.1.1.2 Test animals
Mice aged approximately 3 weeks shall be used.
3.3.1.1.2 Test procedures
A 0.03 mL each of the test materials shall be injected into the brain of at least four test animals
74

and observed for 14 days.
3.3.1.1.3 Judgment
Mice that showed encephalitis symptoms and died shall be judged to be infected to calculate
LD50.
The virus content in 1 mL shall be 107.5LD50 or higher.
3.3.1.2 Inoculation test in cultured cells
3.3.1.2.1 Materials
3.3.1.2.1.1 Test materials
The test article shall be diluted ten-fold with the virus growth medium (Note 1), and each level
dilution shall serve as the test material.
3.3.1.2.1.2 Cultured cells
Primary cultured cells of chicken embryo in accordance with 2.1.1 of the Materials for Live
Vaccine Production or cultured cells approved as suitable shall be cultured in small test portions,and cells that are grown as monolayers shall be used.
3.3.1.2.2 Test procedures
The 0.1 mL portions of the test material shall be inoculated into at least four portions ofcultured cells and allowed to stand for adsorption at 37ºC for 60 minutes. The 0.5 mL portions of
the virus growth medium shall be added, cultured at 37ºC for seven days, and observed.
3.3.1.2.3 Judgment
The cultured cells in which CPE is observed shall be regarded as infected to calculate TCID50.
The virus content in 1 mL shall be 107.5TCID50 or higher.
3.4 Tests on inactivated virus suspension
3.4.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.4.2 Inactivation test
3.4.2.1 Materials
3.4.2.1.1 Injection materials
The test article shall be used as the injection materials.
3.4.2.1.2 Test animals
Mice aged 3 weeks shall be used.
3.4.2.2 Test procedures
A 0.03 mL each of the injection materials shall be injected into the brain of 10 test animals, andobserved for 14 days.
3.4.2.3 Judgment
If there are no test animals that showed encephalitis symptoms and died, the test article shall be
judged to be negative for active virus.
75No active virus shall be observed in the test article.
3.5 Tests on bulk material
3.5.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.6 Tests on final product3.6.1 Properties test
When the test is performed as specified in the Properties Test of the General Tests, the final
product shall be a liquid with a specific color or a homogeneous suspension, and no foreign matterand foreign odor shall be observed. The property of the product per small container shall be
uniform.
3.6.2 Test for pH
When the test is performed as specified in the Test for pH of the General Tests, the pH shallhave an intrinsic value.
3.6.3 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.6.4 Test for thimerosal content
For products added with thimerosal, the test given in the Test for Thimerosal Content of theGeneral Tests shall apply.
3.6.5 Test for formalin content
For products added with formalin when the test given in the Test for Formalin Content of theGeneral Tests is applied, the formalin content shall be no higher than 0.2 vol%.
3.6.6 Test for freedom from abnormal toxicity
The test given in the Test for Freedom from Abnormal Toxicity of the General Tests shall apply.
3.6.7 Potency test
3.6.7.1 Materials
3.6.7.1.1 Injection materials
The test sample diluted ten-fold with phosphate-buffered saline shall be used as the injection
materials.
3.6.7.1.2 Test animals
Mice aged 2–3 weeks shall be used.
3.6.7.2 Challenge virus
The Japanese encephalitis virus Nakayama-Yakken strain or strain approved as suitable (Note2) shall be used.
3.6.7.3 Test procedures
Thirty and sixty animals shall be assigned to the test group and control group, respectively.
On Day 1 and Day 4 of the test, 0.1 mL each of the injection materials shall be injected76

intraperitoneally into the animals in the test group.
On Day 8 of the test, a 0.2 mL portion of challenge virus shall be injected intraperitoneally into
30 animals each of the test group and control group. Then, other 30 animals in the control groupshall be divided into three groups (10 animals per group), and these groups shall be injected
intraperitoneally with a 0.2 mL portion of the challenge virus diluted 10-fold, 100-fold, and 1,000fold, respectively. The test group and control group shall be observed for 14 days.
3.6.7.4 Judgment
Animals that died showing encephalitis symptoms and animals that survived but still showing
encephalitis symptoms shall be considered dead, and the mortality rate of each group and LD50 ofchallenge virus shall be calculated.
The tolerability of the test group must be more than 40%. In this case, the mortality rate of thecontrol group injected with challenge virus must be more than 90%, and the challenge virus
content must be less than 103LD50 in 0.2 mL.
4 Storage and expiry date
The expiry date shall be one year after the manufacturing unless otherwise specified by the
Minister of Agriculture, Forestry and Fisheries.
Note 1 Virus growth medium
1,000 mL consists of the following:
Tryptose phosphate broth

2.95 gBovine serum or goat serum

0–50 mL

Eagle’s MEM

Residual quantity

The pH shall be adjusted to 7.0–7.6 with sodium hydrogen carbonate.
Minimal amounts of antibiotics may be added.
Note 2 Challenge virus
The Japanese encephalitis virus Nakayama-Yakken strain or strain approved as suitable shallbe inoculated into the brain of mice aged 3-4 weeks, sampled at the peak of disease onset, anddiluted with phosphate-buffered saline or dilutions approved as suitable to serve as the 10-foldemulsion.
The supernatant obtained by centrifuging this solution shall be used as the challenge virus.

77

Mycoplasma gallisepticum Vaccine, Live, Seed
1 Definition
Mycoplasma gallisepticum vaccine, live, seed is a freeze-dried vaccine of culture bacteria
medium of attenuated Mycoplasma gallisepticum that meets the seed lot specifications.
2 Production methods
2.1 Virus strain used for production
2.1.1 Name
Mycoplasma gallisepticum G210 strain or strain approved as equivalent thereof
2.1.2 Properties
This virus strain shall agglutinate chicken red blood cells and have the immunogenicity toprevent Mycoplasma gallisepticum.
2.1.3 Master seed bacteria
2.1.3.1 Preparing, storing, and maximum passage number allowed for obtaining final products
Master seed bacteria shall be propagated in a medium approved as suitable and dispensed intostorage containers.
The dispensed master seed bacteria shall be given a specific manufacturing number or
manufacturing code and stored frozen at -70ºC or lower or stored freeze-dried at 5ºC or lower.
The master seed bacteria shall be tested as specified in 3.1.1.
The master seed bacteria shall not be passaged for purposes other than the production of
vaccines. The master seed bacteria shall not be passaged more than 10 times to obtain the final
product. However, the number of passages shall apply if approved by the Minister of Agriculture,
Forestry and Fisheries.
2.1.4 Working seed bacteria
2.1.4.1 Propagation, passage, and storage
The working seed bacteria shall be propagated and passaged in medium approved as suitablefor production.
The working seed bacteria shall be frozen at -70ºC or lower or be stored freeze-dried at 5ºC or
lower.
The working seed bacteria shall be tested as specified in 3.1.2.
2.1.5 Production seed bacteria
2.1.5.1 Propagation and storage
The production seed bacteria shall be propagated in a medium approved as suitable for
production.
78

The production seed bacteria shall be prepared before use for each vaccine production.
2.2 Materials for production
2.2.1 Medium
A medium approved as suitable for production shall be used.
2.3 Bulk material
2.3.1 Cultivation
The production seed bacteria inoculated into a medium and cultured shall serve as the cultured
bacterial medium. This may be concentrated by methods approved as suitable.
The cultured bacterial medium shall be tested as specified in 3.2.
2.3.2 Bulk material
Bulk material shall be obtained by adjusting, if necessary, the concentration of bacteria in thecultured bacterial medium. A stabilizer approved as suitable may be added.
The bulk material shall be tested as specified in 3.3.
2.4 Final bulk
A stabilizer approved as appropriate shall be added to the bulk material to serve as the finalbulk.
2.5 Final product
The final bulk shall be dispensed into small containers and freeze-dried to serve as the final
product.
The final product shall be tested as specified in 3.4.
3 Test methods
3.1 Tests on bacterial strain used for production
3.1.1 Tests on master seed bacteria
3.1.1.1 Identification test
The tests given in 1.4.2.2.1.1 in the Seed Lot Specifications shall apply.
3.1.1.2 Test for freedom from contaminant microorganisms
The test given in the Sterility Test of the General Tests shall apply.
3.1.1.3 Target animal immunogenicity test
The test given in the Target Animal Immunogenicity Test of the General Tests shall apply.
3.1.1.4 Target animal safety test
The test given in the Target Animal Safety Test of the General Tests shall apply.
3.1.1.5 Test for absence of reversion virulence
The test given in the Test for Absence of Reversion to Virulence of the General Tests shallapply.
3.1.2 Tests on working seed bacteria
793.1.2.1 Test for freedom from contaminant microorganisms
The test given in the Sterility Test of the General Tests shall apply.
3.2 Test on cultured bacterial medium
3.2.1 Test for freedom from contaminant microorganisms
The test given in the Sterility Test of the General Tests shall apply.
3.3 Tests on bulk material
3.3.1 Test for freedom from contaminant microorganisms
The test given in the Sterility Test of the General Tests shall apply.
3.3.2 Viable count test
3.3.2.1 Materials
3.3.2.1.1 Test materials
The test article shall be diluted ten-fold with the dilution medium (Note 1) or medium approved
as suitable, and each level dilution shall serve as the test material.
3.3.2.1.2 Medium
Dilution medium and agar medium (Note 2) or medium approved as suitable shall be used.
3.3.2.2 Test procedures
A 25 μL portion of the test material shall be inoculated into two agar plates or cultured by thepour plate culture method in an air containing 5 vol% carbon dioxide at 37ºC for 14 days.
3.3.2.3 Judgment
Calculate the viable count from the number of colonies formed. The viable count of the test
material per 1 mL shall be 109.2 or greater. This number shall not apply if otherwise specified.
3.4 Tests on final product3.4.1 Properties test
When the test is performed as specified in the Properties Test of the General Tests, the final
product shall be dried substances with a specific color. The dissolved product shall be a liquidwith a specific color. No foreign matter and foreign odor shall be observed. The property of the
product per small container shall be uniform.
3.4.2 Vacuum degree test
The test given in the Vacuum Degree Test of the General Tests shall apply.
3.4.3 Test for moisture content
The test given in the Test for Moisture Content of the General Tests shall apply.
3.4.4 Test for freedom from contaminant microorganisms
The test given in the Sterility Test of the General Tests shall apply.
3.4.5 Viable count test
When the test is performed as specified in 3.3.2, the viable count of the test sample per bird
shall be 106.9 or greater. This number shall not apply if otherwise specified.
80

3.4.6 Identification test
This test may be omitted on products for which it is not specified.
3.4.6.1 Materials
3.4.6.1.1 Test materials
Dissolve the quantity of test samples for 1,000 birds in 30 mL of phosphate-buffered saline ordiluent solution.
3.4.6.1.2 Mycoplasma gallisepticum positive serum
Mycoplasma gallisepticum positive serum (Note 3) shall be used.
3.4.6.2 Test proceduresMix 30 μL of the test material and 30 μL of Mycoplasma gallisepticum positive serum on areaction glass plate and observe the reaction while tilting the plate back and forth, left and right.
3.4.6.3 Judgment
Aggregated granules must be observed in one minute.
3.4.7 Marker test
This test may be omitted on products for which it is not specified.
3.4.7.1 Materials
3.4.7.1.1 Inoculation materials
The test materials given in 3.4.5 shall be used.
3.4.7.1.2 Embryonated chicken eggs
The embryonated chicken eggs aged six days in 1.1 in the Materials for Live Vaccine
Production shall be used.
3.4.7.2 Test procedures
A 0.1 mL each of the test materials shall be injected into the yolk sac of 10 embryonatedchicken eggs, cultured at 37ºC for 14 days, and observed.
3.4.7.3 Judgment
Chicken embryos in which the specific lesions are observed shall be regarded as infected, andEID50 shall be calculated. Of them, the embryos that died within 24 hours of inoculation shall be
excluded from the calculation. The viable count per EID50 must be 105 or greater when calculated
from the viable count obtained by the test given in 3.4.5.
3.4.8 Safety test
3.4.8.1 Materials
3.4.8.1.1 Inoculation materials
Dilute the test sample with phosphate-buffered saline or diluent so that a 0.1 mL portion of thedilution contains the quantity of test sample for 10 birds to serve as the inoculation materials.
3.4.8.1.2 Test animals
The chickens aged 2–4 weeks derived from 1.1 in the Materials for Live Vaccine Production.
81

3.4.8.2 Test procedures
Ten and five test animals shall be assigned to the test group and control group, respectively.
Inoculate 0.1 mL of inoculation materials ophthalmically into the test group and observe bothgroups for three weeks. On the final day of the test, the animals shall be autopsied and observed
for the presence of lesions in the nasal cavity, suborbital sinus, trachea, and air sacs.
3.4.8.3 Judgment
During the period of observation, no clinical abnormalities shall be observed in the test andcontrol groups. No abnormalities shall be observed in the autopsy.
3.4.9 Potency test
3.4.9.1 Materials
3.4.9.1.1 Inoculation material
Dilute the test sample with phosphate-buffered saline or diluent so that a 0.03 mL of thedilution contains the quantity of test sample for one bird to serve as the inoculation material.
3.4.9.1.2 Test animals
The chickens aged 2–4 weeks derived from 1.1 in the Materials for Live Vaccine Production
shall be used.
3.4.9.1.3 Agglutination antigen
Inactivated antigen against Mycoplasma gallisepticum (Note 4) shall be used.
3.4.9.2 Test procedures
Ten and three animals shall be assigned to the test group and control group, respectively.
Inoculate 0.03 mL portions of inoculation material ophthalmically into the test group. After 4
weeks, the sera of individual animals obtained from the test and control groups are subjected to an
agglutination reaction test using the agglutination antigen.
3.4.9.3 Judgment
At least 70% of the test group must be positive for agglutination antibodies in three minutes. In
this case, all of the control group must be negative for agglutination antibodies.
4 Storage and expiry date
The expiry date shall be three years and three months after the manufacturing unless otherwise
specified by the Minister of Agriculture, Forestry and Fisheries.
Note 1 Dilution medium
1,000 mL consists of the following:
Proteose peptone

7.4 g

Yeast extract

2.5 g

Dextrose

4.0 g82

Sodium chloride

5.0 g

Sodium dihydrogen phosphate dihydrate

1.8 g

Phenol red

15 mg

Water

Residual quantity

After adjusting the pH to 8.0, sterilize by filtration.
Note 2 Agar medium
The agar medium used is obtained by adding 1.0 g of bactoagar to 90.0 mL of dilution
medium from which phenol red is removed, sterilizing by autoclaving at 121ºC for 15 minutes,
cooling to approximately 50ºC, and adding 10 mL of swine serum.
Note 3 Mycoplasma gallisepticum positive serum
A serum obtained by immunizing the chickens in 1.1 in the Materials for Live Vaccine
Production with the R strain of Mycoplasma gallisepticum or strain approved as suitable andshowing positive serum plate agglutination reactions. It shall have the potency to show
agglutination in a minute.
Note 4 Inactivated antigen of Mycoplasma gallisepticum
Inactivated antigen of Mycoplasma gallisepticum used is obtained by harvesting bacteria
from the liquid culture medium of Mycoplasma gallisepticum S6 strain and diluting withphosphate-buffered saline so that the viable count is 1010 per mL. Then, they shall beinactivated with thimerosal and stained with crystal violet.
Agglutination is observed in three minutes in a serum plate agglutination reaction test using
sera immunized with Mycoplasma gallisepticum S6 strain; however, no agglutination isobserved in three minutes in the test using serum obtained from non-immunized control
chickens or chickens immunized with Mycoplasma synoviae.

83

Rabies (tissue culture) Vaccine, Inactivated, Seed
1 Definition
Rabies (tissue culture) vaccine, inactivated, seed is a vaccine of purified and inactivated virus
suspension obtained by propagating rabies virus adapted to tissue culture that meets the seed lot
specifications in cell lines that meet the specifications.
2 Production methods
2.1 Virus strain used for production
2.1.1 Name
Cell culture adapted RC･HL strain of Rabies virus
2.1.2 Properties
Injection of this virus into the brain of suckling mice aged 3 days or less causes the onset of thedisease and death of the infected animals; however, the injection of this virus into the brain ofmice aged 3 weeks or older, guinea pigs weighing approximately 300 g, rabbits weighing
approximately 1.5 kg and dogs aged 1.5 months have almost no pathogenic effects.
This virus propagates in HmLu cells with CPE.
2.1.3 Master seed virus
2.1.3.1 Preparing, storing, and maximum passage number allowed for obtaining final products
The master seed virus shall be propagated in HmLu cells, prepared in consecutive processes,
and dispensed into storage containers.
The dispensed master seed virus shall be given a specific manufacturing number or
manufacturing code and stored freeze-dried at 5ºC or lower.
The master seed virus shall be tested as specified in 3.1.1.
The master seed virus shall not be passaged for purposes other than the production of vaccines.
The master seed virus shall not be passaged more than five times to obtain the final product.
2.1.4 Working seed virus
2.1.4.1 Propagation, passage, and storage
The working seed virus shall be propagated and passaged in HmLu cells.
The working seed virus shall be stored freeze-dried at 5ºC or lower.
The working seed virus shall be tested as specified in 3.1.2.
2.1.5 Production seed virus
2.1.5.1 Propagation, passage, and storage
The production seed virus shall be propagated in HmLu cells.
The production seed virus shall be stored frozen at -70ºC or lower when it needs to be stored.
84

The production seed virus shall be tested as specified in 3.1.3 if it needs to be stored.
2.2 Materials for production
2.2.1 Cultured cells
HmLu cells shall be used.
2.2.2 Culture medium
The culture medium approved as suitable for production shall be used.
2.2.3 Master cell seeds
2.2.3.1 Preparing, storing, and maximum passage number allowed for obtaining production cellseeds
The master cell seeds shall be propagated in a culture medium in 2.2.2, prepared in consecutive
processes, and dispensed into storage containers.
The dispensed master cell seeds shall be given a specific manufacturing number or
manufacturing code and stored frozen at -100ºC or lower.
The master cell seeds shall be tested as specified in 3.2.1.
The master cell seeds shall not be passaged for purposes other than production or testing ofvaccines. The master cell seeds shall not be passaged more than 20 times to obtain the production
cell seeds. When using the suspension culture method, approximately 3-fold increase in cell countthan increase in the population doubling time shall be regarded as passage of one generation.
2.2.4 Working cell seeds
2.2.4.1 Propagation, passage, and storage
The working cell seeds shall be propagated and passaged using culture medium in 2.2.2.
The working cell seeds shall be stored frozen at -100ºC or lower.
The working cell seeds shall be tested as specified in 3.2.2.
2.2.5 Production cell seeds
2.2.5.1 Propagation, passage, and storage
The production cell seeds shall be propagated using culture medium in 2.2.2.
The production cell seeds shall be stored frozen at -100°C or lower when it needs to be stored.
The production cell seeds shall be tested as specified in 3.2.3 if they need to be stored.
2.3 Bulk material
2.3.1 Culture of production cell seeds
In the monolayer culture method, cells obtained and cultured in one session shall be regarded as
individually cultured cells, and in the suspension culture method, cells cultured in the final
fermenter shall be regarded as individually cultured cells, respectively. No abnormalities shall be
detected in the cultured cells prior to inoculation of the virus.
2.3.2 Virus cultivation
The production seed virus shall be inoculated into the cultured cells, cultured at 32ºC to 34ºC,
85

and harvested at the peak of CPE or G protein production. Centrifugal supernatant or filtrate of theculture medium obtained shall serve as the virus suspension.
The virus suspension shall be tested as specified in 3.3.
2.3.3 Purification
The virus suspension shall be purified and concentrated with macrogol or in a manner approved
as suitable to serve as the purified virus suspension. In the suspension culture method, purification
may be followed by the inactivation process given in 2.3.4.
2.3.4 Inactivation
The purified virus suspension shall be inactivated with β-propiolactone or in a manner approved
as suitable to serve as the inactivated virus suspension.
The inactivated virus suspension shall be tested as specified in 3.4.
2.3.5 Adjustment of bulk material
Inactivated virus suspension shall be diluted with phosphate-buffered saline to adjust its
concentration and serve as the bulk material. To inactivated virus suspension or inactivated andpurified virus suspension, in the suspension culture method, add preservatives approved as
suitable to serve as the bulk material.
The bulk material shall be tested as specified in 3.5.
2.4 Final bulk
The bulk material shall be mixed, diluted with phosphate-buffered saline to adjust its
concentration in the suspension culture method, and serve as the final bulk. In this case,preservatives approved as suitable may be added.
2.5 Final product
The final bulk shall be dispensed into small containers to serve as the final product.
The final product shall be tested as specified in 3.6.
3 Test methods
3.1 Tests on virus strain used for production
3.1.1 Tests on master seed virus
3.1.1.1 Identification test
The tests given in 1.4.2.1.1.1 in the Seed Lot Specifications shall apply.
3.1.1.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.1.1.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.1.1.4 Test for freedom from extraneous viruses
863.1.1.4.1 Common test for freedom from extraneous viruses
The tests given in 1.1, 2.1 and 2.2 in the Test for Freedom from Extraneous Viruses of theGeneral Tests shall apply.
3.1.1.4.2 Test for freedom from specific viruses
3.1.1.4.2.1 General test for freedom from specific viruses
For the Feline leukemia virus/Feline sarcoma virus and Lymphocytic choriomeningitis virus,
the test given in 1.1 and 3.1.1 of the Test for Freedom from Extraneous Viruses of the General
Tests shall apply.
3.1.1.4.2.2 Individual test for freedom from specific viruses
For the Bovine viral diarrhea virus, Canine parvovirus, Feline panleukopenia virus, andJapanese encephalitis virus, the test given in 1.1, 3.2.5, 3.2.6 and 3.2.9 in the Test for Freedom
from Extraneous Viruses of the General Tests shall apply.
3.1.2 Tests on working seed virus
3.1.2.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.1.2.2 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.1.2.3 Efficiency and immunogenicity test
3.1.2.3.1 Tests using guinea pigs
3.1.2.3.1.1 Materials
3.1.2.3.1.1.1 Test materials
Dissolve the new and old working seed viruses in phosphate-buffered saline and adjust theconcentrations so that the virus content in 1 mL is approximately 107.0TCID50. To the virus
solutions, add β-Propiolactone so that the concentration is 0.0125 vol%, sensitize at 4ºC for 48
hours and inactivate the virus to serve as the test material.
3.1.2.3.1.1.2 Test animals
Guinea pigs weighing approximately 400 g shall be used.
3.1.2.3.1.1.3 Challenge virus
Rabies virus CVS strain (Note 1) shall be used. Adjust the concentration with physiologicalsaline containing 2 vol% horse serum to be 10 LD50 of the virus in 0.2 mL when injecting into themasseter muscle of guinea pigs and serve as the challenge virus.
3.1.2.3.1.2 Test procedures
Inject the old and new test materials at a dose of 0.5 mL subcutaneously into the inner thigh of
10 test animals, respectively. These treated animals shall be regarded as the test groups. Another10 animals shall be regarded as the untreated control group. On the 21st day after injection, inject87

0.2 mL of challenge virus into masseter muscle of the three groups each, and observe the animals
clinically for 14 days.
3.1.2.3.1.3 Judgment
The animals that did not develop the disease shall be regarded as tolerant, and the tolerability
shall be calculated.
The tolerability of both groups shall be 70% or higher. In this case, the tolerability of thecontrol group shall be 20% or lower.
3.1.2.3.2 Tests using dogs
3.1.2.3.2.1 Materials
3.1.2.3.2.1.1 Test materials
The test materials specified in 3.1.2.3.1.1.1 shall be used.
3.1.2.3.2.1.2 Test animals
Dogs aged approximately 4 months and negative for antibody against rabies virus shall be used.
3.1.2.3.2.2 Test procedures
Inject 1 mL portions of the old and new test material subcutaneously into three test animals
each, and one month later, collect the blood from each animal, and measure the neutralizing
antibody titer using the cell culture adapted RC-HL strain of rabies virus.
3.1.2.3.2.3 Judgment
The neutralizing antibody titers in both study groups must be greater than ten-fold geometricmean, respectively.
3.1.3 Tests on production seed virus
3.1.3.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.1.3.2 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.2 Tests on cell line
3.2.1 Tests on master cell seeds
3.2.1.1 Test for confirmation of cell properties
The tests given in 2.1.4.2.1.1 in the Seed Lot Specifications shall apply.
3.2.1.2 Test for identification of the animal species of the cell
The tests given in 2.1.4.2.1.2 in the Seed Lot Specifications shall apply.
3.2.1.3 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.2.1.4 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
88

shall apply.
3.2.1.5 Test for freedom from extraneous viruses
3.2.1.5.1 Common test for freedom from extraneous viruses
The tests given in 1.2, 2.1 and 2.2 in the Test for Freedom from Extraneous Viruses of theGeneral Tests shall apply.
3.2.1.5.2 Test for freedom from specific viruses
3.2.1.5.2.1 General test for freedom from specific viruses
For the Feline leukemia virus/Feline sarcoma virus and Lymphocytic choriomeningitis virus,
the test given in 1.2 and 3.1.1 of the Test for Freedom from Extraneous Viruses of the General
Tests shall apply.
3.2.1.5.2.2 Individual test for freedom from specific viruses
For the Bovine viral diarrhea virus, Canine parvovirus, Feline panleukopenia virus, Japanese
encephalitis virus, and Rabies virus, the test given in 1.2, 3.2.5, 3.2.6 and 3.2.9 in the Test forFreedom from Extraneous Viruses of the General Tests shall apply.
3.2.1.6 Test for karyological (chromosomal) characterization
The tests given in 2.1.4.2.1.6 in the Seed Lot Specifications shall apply.
3.2.1.7 Test for freedom from tumorigenicity
The tests given in 2.1.4.2.1.7 in the Seed Lot Specifications shall apply.
3.2.2 Tests on working cell seeds
3.2.2.1 Test for confirmation of cell properties
The tests given in 2.1.4.2.2.1 in the Seed Lot Specifications shall apply.
3.2.2.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.2.2.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.2.3 Tests on production cell seeds
3.2.3.1 Test for confirmation of cell properties
The tests given in 2.1.4.2.3.1 in the Seed Lot Specifications shall apply.
3.2.3.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.2.3.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3.3 Tests on virus suspension
3.3.1 Test for virus content
893.3.1.1 Materials
3.3.1.1.1 Test materials
The test article shall be diluted ten-fold with cell growth medium (Note 2) and each level
dilution shall serve as the test material.
3.3.1.1.2 Cultured cells
Primary cultured cells of chicken embryo shall be used.
3.3.1.2 Test procedures
The 0.1 mL portions of the test material shall be inoculated into at least four portions (wells) ofcultured cells and allowed to stand to culture at 37ºC for two days. On the second day, the culture
medium shall be replaced by virus growth medium 1 (Note 3), allowed to stand to culture at 32ºC
for eight days, and observed.
Note that if a 96-well microplate is used, each 25 μL portion of the test material shall be
inoculated into 10 wells of cultured cells, then 0.2 mL portions of the virus growth medium 2(Note 4) shall be added, cultured at 37ºC for 10 days and observed.
3.3.1.3 Judgment
The cultured cells in which CPE is observed shall be regarded as infected to calculate TCID50.
The virus content in the test article shall be 107.5 TCID50 or greater in 1 mL in the monolayer
culture method, and 107.0 TCID50 or greater in 1 mL in the suspension culture method.
3.4 Tests on inactivated virus suspension
3.4.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.4.2 Inactivation test
3.4.2.1 Materials
3.4.2.1.1 Injection materials and inoculation materials
The test article shall be used as the injection material and inoculation material.
3.4.2.1.2 Test animals and cultured cells
Suckling mice aged three days or less and primary cultured cells of chicken embryo shall be
used.
Note that if the primary cultured cells of chicken embryo are used, the 10 mL portions of thecells shall be dispensed into a culture bottle, and cells that are grown as monolayers ofapproximately 36 cm2 shall be used.
3.4.2.2 Test procedures
The 0.02 mL portions of the injection materials shall be injected into the brain of 10 test
animals, and observed for 14 days.
The 2 mL portions of the inoculation material shall be inoculated into at least four portions ofcultured cells and adsorbed at 37ºC for 60 minutes. The inoculation material shall be removed, the90

10 mL portions of the virus growth medium 1 shall be added, allowed to stand to culture at 37ºC
for 10 days, and observed.
3.4.2.3 Judgment
If the test animals show no symptoms of rabies virus, and the cultured cells in which no CPE isobserved, the material shall be judged to be negative for active virus.
No active virus shall be observed in the test article.
3.5 Tests on bulk material
3.5.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.5.2 Potency test
3.5.2.1 Materials
The test article, reference vaccine (Note 5), antibody adsorption plate (Note 6), and enzyme
labeled antibody (Note 7) shall be used.
3.5.2.2 Test procedures
To 6 mL of the test article, add 327.6 mg of disodium hydrogen phosphate dodecahydrate and38.6 mg of potassium dihydrogen phosphate, and mix thoroughly by shaking. Dissolve thereference vaccine in the method specified by the National Veterinary Assay Laboratory. These test
articles and reference vaccine shall be sonicated for 30–60 seconds each, and then centrifuged at
approximately 200 G for 5 minutes. Previously filter each centrifuged supernatant through amembrane filter with a pore size of 450 nm pre-treated with phosphate-buffered saline containing
1 w/v% bovine serum albumin, and 4 mL of the filtrate obtained shall be gel-filtered (Note 8).
Collect 8 mL of the filtrate containing the peak of the first fraction and prepare original (highdose), two-fold dilution (medium dose) and four-fold dilution (low dose) using the antigen
diluting solution (Note 9). To two wells in an antibody adsorption plate, add 100 μL portions ofantigen solutions to serve as the negative control wells. Seal the plate and allow to react at 37ºC
for 60 minutes. After washing five times with washing solution (Note 10), add 100 μL portions ofenzyme-labeled antibody to the wells each. The plate shall be sealed, and shielded from light, andallowed to react at 37ºC for 90 minutes. After washing five times with washing solution, add 100
μL of substrate chromogenic solution (Note 11) to the wells each. The plate shall be allowed toreact at room temperature for 30 minutes avoiding light. Immediately after the reaction iscompleted, add the 50 μL portions of the stop solution (Note 12) to the wells each, and measure
the absorbance at the main wavelength of 492 nm and at the reference-wavelength of 630 nm,
respectively. The value obtained by subtracting the absorbance value of each negative control wellfrom that of each well to which the test article and reference vaccine are added shall be defined as
the absorbance value of each antigen. Measure the absorbance twice for each antigen solution
after gel filtration and then calculate the relative titer of the test article against the reference91vaccine from the absorbance values of the test article and the reference vaccine according to therelative titer calculation method (Note 13).
3.5.2.3 Judgment
The relative titer of the test article against the reference vaccine shall be 0.683 or higher.
3.6 Tests on final product3.6.1 Properties test
When the test is performed as specified in the Properties Test of the General Tests, the final
product shall be a liquid with a specific color. No foreign matter and foreign odor shall beobserved. The property of the product per small container shall be uniform.
3.6.2 Test for pH
When the test is performed as specified in the Test for pH of the General Tests, the pH shallhave an intrinsic value.
3.6.3 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
3.6.4 Test for thimerosal content
The test given in the Test for Thimerosal Content of the General Tests shall apply.
3.6.5 Test for macrogol content
When the test is performed as specified in the Test for Macrogol Content of the General Tests,
the macrogol content shall be 0.5 mg or lower in 1 mL.
3.6.6 Test for protein nitrogen content
When the test is performed as specified in the Test for Protein Nitrogen Content of the General
Tests, the protein nitrogen content shall be 100 μg or lower in 1 mL.
3.6.7 Safety test
3.6.7.1 Materials
3.6.7.1.1 Injection materials
The test samples shall be used as injection materials.
3.6.7.1.2 Test animals
Dogs weighing approximately 5–10 kg and cats weighing approximately 1 kg shall be used.
3.6.7.2 Test procedures
A 5 mL each of the injection materials shall be injected subcutaneously into two dogs and 2 mLeach into two cats and observed clinically for 10 days.
3.6.7.3 Judgment
During the period of observation, no abnormalities shall be observed in the test group.
3.6.8 Potency test
The test given in 3.5.2 shall apply.

92

4 Storage and expiry date
The expiry date shall be two years after the manufacturing unless otherwise specified by the
Minister of Agriculture, Forestry and Fisheries.
Note 1 Rabies virus CVS strain
A sample of brain inoculated with rabies virus CVS strain in 3-week-old mice exhibiting
symptoms. This sample is emulsified with physiological saline containing 2 vol% horse serum
to make homogenates.
When this homogenate is administered into the masseter muscle of guinea pigs weighing
approximately 400 g, the virus content shall be 102.5LD50 or higher in 0.2 mL.
Note 2 Cell growth medium
1,000 mL consists of the following:
Tryptose phosphate broth

2.95 gFetal bovine serum or calf bovine serum

50 mL

Eagle’s MEM

Residual quantity

The pH shall be adjusted to 7.1–7.3.
Minimal amounts of antibiotics may be added.
Note 3 Virus growth medium 1
1,000 mL consists of the following:
Tryptose phosphate broth

2.95 gEagle’s MEM

Residual quantity

The pH shall be adjusted to 7.4–7.6.
Minimal amounts of antibiotics may be added.
Note 4 Virus growth medium 2
1,000 mL consists of the following:
Glucose5g

L-glutamine

0.4 g

Fetal bovine serum or Calf serum

5–25 mL

Eagle’s MEM

Residual quantity

The pH shall be adjusted to 7.1–7.3.
Minimal amounts of antibiotics may be added.

93Note 5 Reference vaccine
The reference vaccine used is obtained by adjusting the concentration of rabies (tissue
culture) vaccine, inactivated, for reference provided by the National Veterinary Assay
Laboratory as specified by the National Veterinary Assay Laboratory.
Note 6 Antibody adsorption plate
The antibody adsorption plate used is obtained by adding 100 μL portions of the dilution of
purified monoclonal antibody against Rabies virus G protein of the concentration specified by
the National Veterinary Assay Laboratory to each wells of a 96-well plate, sealing the plate, andallowing to adsorb at 37ºC for 18 hours.
The antibody adsorption plate shall be used after washing four times with phosphate-bufferedsaline.
Note 7 Enzyme labeled antibody
The enzyme labeled antibody used is obtained by adjusting the concentration of peroxidaselabeled monoclonal antibody against Rabies virus G protein with the following dilution solution
to the concentration specified by the National Veterinary Assay Laboratory.
In 100 mL:
Bovine serum albumin

0.3 g

Polysorbate 20

0.05 mL

Phosphate-buffered saline

Residual quantity

Sterilize by filtration through a membrane filter with a pore size of 450 nm.
Note 8 Gel filtration
Using a column specified by the National Veterinary Assay Laboratory, separate the fractions
by liquid chromatography at a flow rate of 1 mL per minute with antigen diluting solution as themobile phase.
The detection wavelength shall be 280 nm.
Note 9 Antigen diluting solution
In 1,000 mL:
Disodium hydrogen phosphate dodecahydrate

54.66 gPotassium dihydrogen phosphate

6.44 gWater

Residual quantity

After adjusting the pH to 7.2, filter through a membrane filter with a pore size of 450 nm.

94Note 10 Washing solution
The washing solution used is obtained by adding polysorbate 20 to phosphate-buffered saline
so that the concentration is 0.05 vol%.
Note 11 Substrate chromogenic solution
In 1,000 mL:
Citric acid21.0 gAnhydrous monobasic sodium phosphate

28.4 gWater

Residual quantity

The dissolved solution shall be filtered through a membrane filter with a pore size of 450 nm.
To 20 mL of the solution, add 10 mg of o-phenylenediamine dihydrochloride to dissolve.
Immediately before use, add 5 μL of hydrogen peroxide (30).
Note 12 Stop solution
The stop solution used is obtained by adding 150 mL of sulfuric acid to 1,000 mL of water.
Note 13 Relative tier calculation method
1 Validity test
Multiply each absorbance value obtained by 1,000 and convert it to the ordinary logarithm.
Evaluate the total values of the doses for the reference vaccine and the test article andcalculate the following formula. The low, medium, and high doses of the reference vaccine
shall be represented by S1, S2, and S3, respectively, and those of the test article shall be
represented by T1, T2, and T3, respectively.
Difference between the test article and reference vaccine SPa ＝(S1+S2+S3)-(T1+T2+T3)
Linearity SPb ＝(S3-S1)+(T3-T1)
Curvilinearity SPc ＝[(S1+S3)-2S2]+[(T1+T3)-2T2]
Line non-parallelism SPb´＝(S3 -S1)-(T3-T1)
Curve non-parallelism SPc´＝[(S1+S3)-2S2]-[(T1+T3)-2T2]
If SPb obtained by the above formula is greater than or equal to 0.50, the absolute value of
SPc is less than or equal to 0.86, that of SPb´ is less than or equal to 0.49, and that of SPc´ isless than or equal to 0.86: the validity test is met.
2 Calculation of relative tierIf the validity test given in 1 is judged as compliant, calculate the relative titer by thefollowing formula:M = - (4 × SPa × log 2) / (3 × SPb)
Furthermore, calculate the relative titer of the test article to the reference vaccine by95finding the true number of M (inverse logarithm of M, 10M).
P= 10M96

General Tests

97

Test Methods
In this Test Methods, "the test article etc." refers to the test article and the test sample.
Freeze-dried products not supplied with a diluent shall be tested by dissolving in physiologicalsaline to make the volume indicated on the direct container unless otherwise specified.

99

Properties Test
The Properties Test is a method to examine the properties of the test article etc. by visual andolfactory senses.
In the tests given in 1.1 and 1.2, when the volume of small container used exceeds 20 mL or it is
not made of clear and colorless glass, the test article shall be transferred to a clear and colorless glass
container with a diameter of 18–20 mm before the tests.
The criterion for judgment shall be given in the monographs.
1 Test procedure
1.1 Color test
Inspect the color tone of the test article etc. against a white background under natural light or at
1,000 lux or higher under a white light source.
1.2 Transparency test
Inspect the transparency under natural light or at 1,000 lux or higher under a white light source.
In this test, transparent means no scattering of light rays by particles in the liquid, opaquemeans that the background is invisible, and translucent means none of the above.
1.3 Foreign matter test
Inspect foreign matters with the unaided eye against a white or black background under naturallight or at 1,000 lux or higher under a white light source.
1.4 Odor test
Bring your nose to the mouth of the container and smell it.
1.5 Uniformity test for each container
For at least seven portions of final products, examine each container for its properties.
For freeze-dried products examine the properties before and after dissolution.

100

Test for pH
The Test for pH is a method to determine the hydrogen ion concentration of the test article etc.
using a glass electrode system. The hydrogen ion concentration is usually represented by pH.
The criterion for judgment shall be given in the monographs.
1 Test procedure
The pH determination method specified in General Tests of the Japanese Pharmacopoeia shallapply.

101

Vacuum Degree Test
The Vacuum Degree Test is a method to determine the vacuum degree of dried final products
sealed under reduced pressure.
1 Test procedure
All the dried final products and test samples shall be tested by non-polar discharge using a tesla
coil at a distance of 5 to 10 mm in a dark place.
2 Judgment
The test results show discharge: the final products and test samples shall comply with the test.

102Test for Moisture Content
The Test for Moisture Content is a method to measure the weight loss of the dried product of test
article etc. by drying under heating and reduced pressure to determine the moisture content of thetest article etc.
1 Test procedure
The test shall be performed as follows or the method specified in the guideline of theInternational Cooperation on Harmonisation of Technical Requirements for Registration of
Veterinary Medicinal Products (GL26: Testing of residual moisture).
The test shall be performed at a relative humidity of not higher than 45%. Dry a weighing bottle
and measure the weight of the bottle precisely. Crush the test article etc., precisely weighapproximately 100 mg of it in the weighing bottle, and use as the test material.
Place the weighing bottle in a vacuum dryer with its surface of mouth slightly opened and dry
the bottle at a pressure of not more than 0.65 kPa and at 60°C for 3 hours, if necessary, overphosphorus oxide (V) or silica gel.
When drying is completed, place dried air in a vacuum dryer to return the pressure to normal
level. Take out the weighing bottle and place in a desiccator containing phosphorus oxide (V) or
silica gel, allow to cool to room temperature and weigh precisely.
Calculation of the moisture content
The moisture content is calculated by the following formula:
Moisture content (%) =weight loss of the test material by drying
weight of the test material before drying

× 100

2 Judgment
The test results show that the moisture content is not more than 3% unless otherwise specified:
the test article etc. shall comply with the test.

103

Sterility Test
The Sterility Test is a method to determine the absence of detectable microorganisms (bacteria or
fungi) in the test article etc. by the following tests or the Sterility Test under the General Tests of theJapanese Pharmacopoeia unless otherwise specified.
1 Test for freedom from bacteria
1.1 Medium
Unless otherwise specified, the fluid thioglycolate medium consisting of the following
components shall be used. The volume of medium used shall be not less than 15 mL per test tube .
1.1.1 Fluid thioglycolate medium
1.1.1.1 Composition
L-Cystine
0.5 g
Agar
0.75 gSodium chloride
2.5 g
Glucose5.0 g
Yeast extract
5.0 g
Casein peptone
15.0 gSodium thioglycolate
0.5 g
0.1 w/v% resazurin sodium solution
1.0 mL
Water
1,000 mL
Adjust the pH so that after sterilization it is 6.9–7.3, sterilize by autoclaving at 121ºC for 15
minutes, and store in a dark place at 2ºC to 25ºC. Do not use the medium, if more than theupper one-third of the medium has acquired a pink color. A dried product of suitable quality
may be used.
1.1.1.2 Suitability of the medium
The media used shall comply with the following tests:
1.1.1.2.1 Growth promotion test
When inoculated with not more than 100 CFU of Clostridium sporogenes, Pseudomonas
aeruginosa and Staphylococcus aureus, respectively, or with not more than 100 CFU of
Streptococcus equi subspecies zooepidemicus and Clostridium sporogenes, respectively, andcultured at 30ºC to 35ºC for 72 hours, clearly visible growth must be observed.
1.1.1.2.2 Sterility test
Culture portions of the media at 30–35ºC for 14 days. No growth of microorganisms shall beobserved.
1.2 Culture materials
The test article and the test sample shall be used. Freeze-dried products not supplied withdiluents shall be dissolved in suitable solubilizing solvent, such as phosphate-buffered saline, to104

make the volume specified in the Dosage and Administration.
1.3 Quantity of test article etc.
When testing the test article, unless otherwise specified, take a sufficient quantity of it from thecontainer to perform the test. When testing the test sample, unless otherwise specified, at least 7
containers shall be tested.
1.4 Quantity of inoculum for each medium
When testing the test article, use 4 tubes of medium for each material. Inoculate 1 mL ofmaterial into two tubes, respectively, while inoculate 0.5 mL of material to other two tubes,
respectively.
The test sample shall be tested according to the reference volume of each small container andquantity of inoculum for each medium, as shown in Table 1.
Table 1 Reference Volume of Small Container and Quantity of Inoculum and Number of Tubes ofMedium for Each Container (Test for Freedom from Bacteria)
Reference volume
Quantity of
Number of tubes of
Quantity of inoculum per
inoculum per
medium per
mediumcontainer
container< 3 mL
1/4 quantity
1 tube
1/4 quantity × 1 tube
3 mL≤ R < 5 mL
1 mL
2 tubes
0.5 mL × 2 tubes
5 mL≤ R <10 mL
1.5 mL
2 tubes
1 mL × 1 tube
0.5 mL × 1 tube
≥ 10 mL
3 mL
2 tubes
2 mL × 1 tube
1 mL × 1 tube
1.5 Culture and observation
The test article etc. shall be inoculated to a medium, mixed thoroughly, cultured at 30ºC to 35ºC
for at least 14 days, and observed for bacterial growth between the 3rd day and 5th day of culture
and between the 7th day and 9th day, and on the 14th day.
If the preparation causes turbidity in the medium or if otherwise necessary, subculture in a new
medium on the 7th day, culture at the same temperature for at least 8 days, and observed.
1.6 Judgment
The test results show no bacterial growth: the test article shall comply with the test.
2 Test for freedom from fungi
2.1 Medium
Unless otherwise specified, fluid thioglycolate medium shall be used. For the test article etc. notincluding thimerosal, unless otherwise specified, soybean-casein digest broth shall be used.
The volume of medium used shall be not less than 15 mL per test tube.
2.1.1 Fluid thioglycolate medium
2.1.1.1 Composition
The composition shall be applied to 1.1.1.1.
105

2.1.1.2 Suitability of the medium
The medium shall comply with the following tests.
2.1.1.2.1 Growth promotion test
When inoculated with not more than 100 CFU of Aspergillus brasiliensis, Bacillus subtilis andCandida albicans, respectively, or with not more than 100 CFU of Streptococcus equi subspecies
zooepidemicus, Aspergillus brasiliensis and Candida albicans, respectively, and cultured at 20ºC
to 25ºC for five days, clearly visible growth must be observed.
2.1.1.2.2 Sterility test
The test shall be performed as specified in 1.1.1.2.2. The culture temperature shall be 20ºC to
25ºC.
2.1.2 Soybean-casein digest broth
2.1.2.1 Composition
Casein peptone
17.0 gSoybean peptone
3.0 g
Sodium chloride
5.0 g
Dipotassium hydrogen phosphate
2.5 g
Glucose
2.3 g
Water
1,000 mL
Adjust the pH so that after sterilization it is 7.1–7.5, sterilize by autoclaving at 121ºC for 15
minutes, and store in a dark place at 2ºC to 25ºC. A dried product of suitable quality may be
used.
2.1.2.2 Suitability of the medium
The suitability shall be applied to 2.1.1.2.
2.2 Culture materials
The culture materials shall be applied to 1.2.
2.3 Quantity of the test article etc.
The quantity of the test article etc. shall be applied to 1.3.
2.4 Quantity of inoculum for each medium
When testing the test article, use 4 tubes of medium for each material, and inoculate 1 mL ofeach material.
The test sample shall be tested according to the reference volume of each small container andquantity of inoculum for each medium, as shown in Table 2.

106Table 2 Reference Volume of Small Container and Quantity of Inoculum and Number of Portions ofMedium for Each Container (Test for Freedom from Fungi)
Reference volume
Quantity of
Number of tubes
Quantity of inoculum per
inoculum per eachof medium per
mediumcontainer
container< 3 mL
1/2 volume
2 tubes
1/4 volume × 2 tubes
3 mL≤ R < 5 mL
1 mL
2 tubes
0.5 mL × 2 tubes
5 mL≤
2 mL
2 tubes
1 mL × 2 tubes
2.5 Culture and observation
The test article etc. shall be inoculated to a medium, mixed thoroughly, cultured at 20ºC to 25ºC
for at least 14 days, and observed for bacterial growth between the 3rd day and 5th day of culture
and between the 7th day and 9th day, and on the 14th day.
If the product causes turbidity in the medium or if otherwise necessary, subculture in a new
medium on the 7th day, culture at the same temperature for at least 8 days, and observed.
2.6 Judgment
The criterion for judgment shall be applied to 1.6.
3 Retest
When doubt exists about the result for Tests 1 and 2, the test shall be repeated using at least
twice the amount of the test article etc.

107

Test for Freedom from Mycoplasma Contamination
The Test for Freedom from Mycoplasma Contamination is conducted by the following method toverify the absence of Mycoplasma in the test article etc. or the method specified in the guideline of
the International Cooperation on Harmonisation of Technical Requirements for Registration of
Veterinary Medicinal Products (GL34: Testing for the detection of Mycoplasma contamination).
1 Medium
Unless otherwise specified, use broth medium for Mycoplasma with the following composition.
The volume of broth medium shall be 100 mL per test tube.
1.1 Broth medium for Mycoplasma
1.1.1 Composition
1.1.1.1 Basal medium
Each 1,000 mL consists of:
50 w/v% beef heart infusion broth
100 mL
Proteose peptone
10 g
Sodium chloride
5g
Glucose1g
L-monosodium glutamate
0.1 g
L-arginine hydrochloride
1g
Water
Residual quantity
Sterilize by filtration through a membrane filter with a pore size of 220 nm or sterilize by
autoclaving at 121ºC for 15 minutes. Adjust the pH of the solution so that it is 7.2–7.4 aftersterilization.
A dried product of suitable quality may be used.
1.1.1.2 Medium additives
To 77 mL of basal medium in 1.1.1.1, add the following additives:
Horse serum
10 mL
5 mL
Heat-inactivated porcine serum
25 w/v% fresh yeast extract
5 mL
1 w/v% β-nicotinamide adenine dinucleotide oxidized form
1 mL
1 w/v% L-cysteine hydrochloride test solution
1 mL
0.2 w/v% phenol red solution
1 mL
Previously filter sterilize the additives and aseptically add to the sterilized basal medium.
Note that the additives that can be sterilized by high pressure may be autoclaved.
Benzylpenicillin potassium, 500 units/mL of the medium, and/or Thallium acetate, 0.02
w/v%, may be added.
1.1.2 Performance
When inoculated with not more than 100 CFU of Mycoplasma gallisepticum, Mycoplasma108synoviae, Mycoplasma hyopneumoniae and Mycoplasma orale, respectively, and cultured at 35ºC
to 37ºC for seven days, clearly visible growth must be observed.
1.2 Agar medium for Mycoplasma
1.2.1 Composition
1.2.1.1 Basal medium
Add 1 g of agar to 78 mL of basal medium given in 1.1.1.1.
1.2.1.2 Medium additives
Phenol red solution shall be removed from the media additives specified in 1.1.1.2.
After sterilization, 5 mL portions of media dissolved with the aid of heat shall be dispensed into
sterilized Petri dishes with a diameter of 45–55 mm, cooled, solidified, and used as agar plates for
Mycoplasma.
1.2.2 Performance
As specified in 1.1.2, when inoculated with not more than 100 CFU of Mycoplasma strains,respectively, and cultured in an air containing 5 vol% carbon dioxides at 35ºC to 37ºC for 10 days,
these strains must form their own specific colonies.
2 Culture materials
The test article or the test sample shall be used. Freeze-dried productions not supplied withdiluents shall be dissolved in suitable solubilizing solvent, such as phosphate-buffered saline, tomake the volume specified in the Dosage and Administration. For preparations for oral
administration (administration by drinking water) and preparations for administration by puncture,these preparations shall be diluted with suitable solubilizing solvent, such as phosphate-bufferedsaline, so that the quantity of inoculum is equal to that of a single dose.
3 Quantity of the test article etc.
For the test article, a test shall be performed per container. For the test sample, mix equal
quantities of it taken from at least 2 small containers to perform the test.
4 Quantity of inoculum for each medium
A 1 mL portion of the test article shall be inoculated into 100 mL of broth medium. Then, a 0.1
mL portion of each broth medium shall be inoculated into agar plates.
5 Culture and observation
The test article etc. shall be inoculated into the broth medium, mixed thoroughly, and thencultured at 35ºC to 37ºC for 14 days. If the test article etc. includes live cells, the pH of cultured
medium shall be adjusted timely.
Inoculate the agar plate for Mycoplasma with the culture medium on the 3rd, 7th, 10th, and14th days of culture, cultured in an air containing 5 vol% carbon dioxides at 35ºC to 37ºC for 10
days to observe the plates for Mycoplasma colonies.
In this case, the plates inoculated as the control with the medium and Mycoplasma synoviae
109shall be observed in a similar manner.
6 Judgment
The test results show no Mycoplasma colonies in the plates inoculated with the test article etc.
and medium and show colonies in the plate inoculated with Mycoplasma synoviae: the test article
shall comply with the test.
If the test results show no colonies in plate inoculated with Mycoplasma synoviae, or show
Mycoplasma colonies in the plate inoculated with the medium: the test shall be repeated.
7 Retest
When doubt exists about the result for tests, the test shall be repeated using at least twice thequantity of the test article etc.

110

Test for Freedom from Salmonella Contamination
Unless otherwise specified, the Test for Freedom from Salmonella Contamination is a method to
examine the absence of Salmonella detectable in the test sample by the following test:
1 Medium
Unless otherwise specified, soybean-casein digest broth (SCD broth medium), selenite medium,
BTB lactose agar medium (Drigalski improved medium), and DHL agar medium shall be used.
The volume of broth medium used in this test shall be 100 mL per test tube.
1.1 Soybean-casein digest broth
1.1.1 Composition
Dissolve a dried product of appropriate quality as specified and sterilize by autoclaving at
121ºC for 15 minutes. The pH after the sterilization shall be 7.1–7.3.
1.1.2 Performance
When inoculated with not more than 100 CFU of Escherichia coli and Salmonella typhimurium,
respectively, and cultured at 35ºC to 37ºC for 18–24 hours, clearly visible growth must be
observed.
1.2 Selenite medium
1.2.1 Composition
Dissolve a dried product of appropriate quality as specified. The pH shall be 7.1–7.3.
1.2.2 Performance
When inoculated with not more than 100 CFU of Salmonella typhimurium and cultured at 35ºC
to 37ºC for 18–24 hours, clearly visible growth must be observed.
1.3 BTB lactose agar medium
1.3.1 Composition
Dissolve a dried product of appropriate quality as specified and sterilize by autoclaving at
121ºC for 15 minutes. The pH after the sterilization shall be 7.3–7.5.
1.3.2 Performance
When inoculated with not more than 100 CFU of Escherichia coli, Salmonella pullorum andSalmonella typhimurium, respectively, and cultured at 35ºC to 37ºC for 18–24 hours, thesemicroorganisms shall form their own specific colonies.
1.4 DHL agar medium
1.4.1 Composition
Dissolve a dried product of appropriate quality as specified. The pH shall be 6.9–7.1.

111

1.4.2 Performance
When inoculated with not more than 100 CFU of Escherichia coli, Salmonella pullorum, andSalmonella typhimurium, respectively, and cultured at 35ºC to 37ºC for 18–24 hours, thesemicroorganisms shall form their own specific colonies.
2 Culture material
The test sample shall be used. Freeze-dried products not supplied with diluents shall bedissolved in suitable solubilizing solvent, such as phosphate-buffered saline, to make the volume
specified in the Dose and Administration. For products for oral administration (administration bydrinking water) and products for administration by puncture, these products shall be diluted withsuitable solubilizing solvent, such as phosphate-buffered saline, so that the quantity of inoculum isequal to that of a single dose.
3 Quantity of the test article etc.
Mix equal quantities of the test article taken from at least two small containers to perform thetest.
4 Culture and observation
The 5 mL portions of the test article etc. shall be inoculated into SCD broth medium andselenite medium, respectively, mixed thoroughly, and cultured for enrichment at 35ºC to 37ºC for
18–24 hours. A 0.1 mL portion of each culture medium shall be inoculated into BTB lactose agar
medium and DHL agar medium, respectively, cultured at 35ºC to 37ºC for 18–24 hours, andobserved for the presence of Salmonella colonies.
5 Judgment
The test results show that no colonies of Salmonella are observed: the test article shall comply
with the test.
6 Retest
When doubt exists about the result for Tests, the test shall be repeated using at least twice theamount of the test article etc.

112

Microbial Limit Test
The Microbial Limit Test is a method to verify that the viable count of bacteria in the test article
etc. is below a certain level.
1 Medium
Unless otherwise specified, heart infusion agar medium shall be used.
1.1 Heart infusion agar medium
1.1.1 Composition
Dissolve a dried product of appropriate quality as specified and sterilize in an autoclave at
121ºC for 15 minutes. The pH after the sterilization shall be 7.3–7.5.
1.1.2 Performance
When inoculated with not more than 100 CFU of Escherichia coli and cultured at 35ºC to 37ºC
for 18–24 hours, the microorganism shall form their own specific colonies.
2 Culture materials
The test article or the test sample shall be used. For the test sample, the following preparations
shall be used:
2.1 Freeze-dried productPrepared by diluting with the supplied diluent or suitable solubilizing solvent, such as
phosphate-buffered saline, so that the quantity of inoculum is equal to that of a single dose
2.2 Liquid productPrepared by diluting with suitable solubilizing solvent, such as phosphate-buffered saline, incases where the quantity of inoculum is not equal to that of a single dose.
3 Quantity of the test article etc.
For the test article a test shall be performed per container. For the test sample, mix equal
quantities of it taken from at least two small containers to perform the test.
4 Quantity of inoculum for each medium
Dispense 1 mL portions of the test article or single-dose portions of the test sample to four Petridishes with a diameter of 90 mm, add 15 mL portions of agar medium dissolved below 50ºC, mix
thoroughly, and allow to solidify.
5 Culture and observation
Culture two of four plates at 35ºC to 37ºC for 48 hours and other two plates at 22ºC to 24ºC for
four days and count the number of colonies grown.
6 Judgment
The test results indicate that the mean number of colonies at any culture temperatures is not113more than 10 per 1 mL portion of the test article, while that is not more than one per single-dose
portion of the test sample: the test article etc. shall comply with the test.
7 Retest
When doubt exists about the test results, the test shall be repeated using at least twice thenumber of Petri dishes for the test article etc. or using twice the number of containers for the test
sample.

114

Test for Freedom from Extraneous Viruses for Live Vaccines
and Sera
The Test for Freedom from Extraneous Viruses for Live Vaccines and Sera is a method to verify
that no extraneous viruses in the test material are detected in live vaccines and sera.
1 Test material
1.1 Live vaccine
Unless specified in the monographs, the test article and liquid product shall be used as the test
materials as they are. If the test sample is freeze-dried, dissolve in the supplied diluent orphosphate-buffered saline. To the dissolved test sample, add each immune serum specified inmonographs, allow to neutralize at 37ºC for one hour or at 4ºC overnight, neutralize completely,
and use as the test material.
Unless specified in the individual monographs, adjust the quantity of test material to be
inoculated once so that it contains viral amount equivalent to the amount for one-dose before
neutralization.
1.2 Sera, etc.
The test article etc. shall be dialyzed at 2ºC to 5ºC overnight using at least a 100-fold volume ofphosphate-buffered saline to remove the preservative, and it shall be used as the test material.
2 Test procedure
2.1 Inoculation test using embryonated chicken egg
2.1.1 Inoculation into allantoic cavity
2.1.1.1 Observation of embryo
2.1.1.1.1 Test procedure
As specified in 1.1 in the Materials for Live Vaccine Production, embryonated chicken eggs
aged 9–11 days shall be used. The 0.1 mL portions of the test material shall be injected into theallantoic cavity of 10 embryonated chicken eggs and cultured at 37ºC for seven days. On the final
day of culture, the eggs shall be opened to examine abnormalities of the chicken embryos.
2.1.1.1.2 Judgment
The chicken embryos are properly developed, and no abnormalities are observed: the test
material shall comply with the test.
2.1.1.2 Hemagglutination test
2.1.1.2.1 Test procedure
To allantoic fluid collected on the final day of culture given in 2.1.1.1, an equal quantity of 0.5
115vol% chicken red blood cell suspension shall be added, allowed to stand at 4ºC for 60 minutes,
and observed for the presence of hemagglutination.
2.1.1.2.2 Judgment
No hemagglutination is observed in the allantoic fluid: the test material shall comply with thetest.
2.1.2 Inoculation onto chorioallantoic membrane
2.1.2.1 Test procedure
As specified in 1.1 in the Materials for Live Vaccine Production, embryonated chicken eggs
aged 10–12 days shall be used. The 0.1 mL portions of the test material shall be inoculated onto
the chorioallantoic membrane of 10 embryonated chicken eggs and cultured at 37ºC for five days.
On the final day of culture, these eggs shall be opened to examine abnormalities of chicken
embryos and chorioallantoic membranes.
2.1.2.2 Judgment
The chicken embryos are properly developed, and no abnormalities are observed in thechorioallantoic membranes: the test material shall comply with the test.
2.2 Inoculation test using cells derived from chicken
2.2.1 Inoculation test using chicken kidney cells
2.2.1.1 Observation of cultured cells
2.2.1.1.1 Test procedure
As specified in 2.2.1 in the Materials for Live Vaccine Production, primary cells of chicken
kidneys shall be used. These cells shall be cultured in at least four Petri dishes with a size of notless than 20 cm2. The 0.2 mL portions of the test material shall be inoculated and cultured at 37ºC
for seven days. The culture suspension and cells shall be harvested, and 0.2 mL portions shall be
inoculated and passage into the cultured cells in at least four Petri dishes, cultured at 37ºC forseven days, and observed for the presence of CPE.
2.2.1.1.2 Judgment
No CPE is confirmed in the cultured cells during the observation period: the test material shallcomply with the test.
2.2.1.2 Hemadsorption test
2.2.1.2.1 Test procedure
On the final day of observation given in 2.2.1.1, the culture suspension shall be removed fromthe cultured cells in the Petri dishes, overlaid with 0.1 vol% chicken red blood cell suspension,
allowed to stand at 4ºC for 60 minutes, and observed microscopically for the presence ofhemadsorption.
2.2.1.2.2 Judgment
No hemadsorption is observed in the cultured cells: the test material shall comply with the test.
1162.2.2 Inoculation test using chicken embryonic cells
2.2.2.1 Observation of cultured cells
2.2.2.1.1 Test procedure
As specified in 2.1.1 in the Materials for Live Vaccine Production, primary cells of chicken
embryo susceptible to avian leukosis virus shall be used. These cells shall be cultured into at least
eight Petri dishes with a size of not less than 20 cm2, and the 0.2 mL portions of the test material
shall be inoculated within 24 hours and cultured at 37ºC. These cells shall be passaged every three
to five days to the third passage, and the cells of each passage shall be observed for the presenceof CPE.
2.2.2.1.2 Judgment
No CPE is confirmed in the cultured cells during the observation period: the test material shallcomply with the test.
2.2.2.2 Hemadsorption test
2.2.2.2.1 Test procedure
For the third-passaged cells in at least four Petri dishes given in 2.2.2.1, culture suspension shallbe removed and overlaid with 0.1 vol% chicken red blood cell suspension, allowed to stand at 4ºC
for 60 minutes, and observed for the presence of hemadsorption.
2.2.2.2.2 Judgment
No hemadsorption is observed in the cultured cells: the test material shall comply with the test.
2.2.2.3 COFAL test
2.2.2.3.1 Test procedure
After washing the third-cultured cells in four Petri dishes given in 2.2.2.1, add one-twentiethvolume of culture medium of Veronal-buffered saline containing 1 w/v % gelatin. Freeze-thaw the
harvested cell suspension three times and collect centrifugal supernatant to prepare the cell
extract. Using this as an antigen, perform a COFAL test by the Kolmer’s method in pigeon, rabbit,
or hamster serum specific to avian leukosis virus, three units of hemolytic elements, four units ofguineapig complements and 2 vol% sheep erythrocytes.
2.2.2.3.2 Judgment
The COFAL test indicates that the cell extract is negative for the virus: the test material shallcomply with the test.
2.2.2.4 Test for freedom from Reticuloendotheliosis virus
2.2.2.4.1 Test procedure
The test material given in 2.2.2.1.1 shall be inoculated. The cultured primary cell suspension
shall be inoculated into a newly cultured primary cells of chicken embryos and cultured at 37ºC
for four days. When the cell suspension is passaged to the third generation, the cell suspension
shall be inoculated into the cultured cells in at least four Petri dishes with cover glasses and117cultured at 37ºC for four days, tested by fluorescence antibody assay with antireticuloendotheliosis virus serum, and then observed.
2.2.2.4.2 Judgment
No specific fluorescent antigens are observed in the cultured cell: the test material shall comply
with the test.
2.3 Inoculation test using cells derived from swine
2.3.1 Inoculation test using swine kidney cells
2.3.1.1 Observation of cultured cells
2.3.1.1.1 Test procedure
Primary or passaged swine kidney cells shall be used.
A 4 mL portion of the test material shall be inoculated into the cultured cell sheet of at least 20
cm2 per 1 mL of the inoculum, cultured at 37ºC for five days, and observed for the presence ofCPE. Then, the cells shall be passaged to the next generation, cultured at 37ºC for seven days, andobserved for the presence of CPE.
2.3.1.1.2 Judgment
No CPE is observed in the cultured cells during the observation period: the test material shallcomply with the test.
2.3.1.2 Hemadsorption test
2.3.1.2.1 Test procedure
On the final day of observation in 2.3.1.1, the cell surfaces shall be washed twice withphosphate-buffered saline. The cultured cells shall be divided into three groups, overlaid with 0.1
vol% red blood cell suspensions of guinea pig, goose, and chicken aged 7 days or younger,
respectively, allowed to stand at 4ºC for 60 minutes and at 37ºC for 30 minutes, and observed
microscopically for the presence of hemadsorption.
2.3.1.2.2 Judgment
No hemadsorption is observed in the cultured cells: the test material shall comply with the test.
2.3.1.3 Test for freedom from Classical swine fever virus
The test shall be performed as specified in 2.3.1.3.1 or 2.3.1.3.2 as follows:
2.3.1.3.1 Fluorescent antibody assay
2.3.1.3.1.1 Test procedure
Culture supernatant shall be collected on the 5th day of inoculation of the test material given in
2.3.1.1.1, a 1 mL portion shall be inoculated into the cells in 2.3.1.1 cultured on a cover glass witha size of 3 cm2 or larger, cultured at 37ºC for 24–48 hours, tested with anti-classical swine fever
virus serum by fluorescent antibody assay, and then observed.
2.3.1.3.1.2 Judgment
No specific fluorescent antigens are observed in the cultured cells: the test material shall118

comply with the test.
2.3.1.3.2 END method and interference method
2.3.1.3.2.1 Test procedure
On the 5th day of inoculation of the test material given in 2.3.1.1.1, culture supernatant shall becollected, the 0.1 mL portions of it shall be dispensed into at least 20 small test tubes (wells), andthe 0.5 mL portions of primary cells of swine testis suspended in the cell growth medium shall be
added. After allowing to culture at 37ºC for four days, the cultured cells shall be divided into twogroups and tested by the END method and interference method using the WEE virus.
2.3.1.3.2.2 Judgment
No Classical swine fever virus is observed in the cultured cells: the test material shall comply
with the test.
2.3.1.4 Test for freedom from Porcine circovirus
2.3.1.4.1 Test procedure
PPK-3F cells shall be used.
A 2 mL portion of the test material shall be inoculated into the cultured cell sheet of at least 20
cm2 per 1 mL of the inoculum, treated with glucosamine, cultured at 37ºC for five days, andpassaged to the next generation. The passaged cells shall be cultured at 37ºC for four days,
passaged to the next generation, and the passaged cells shall be cultured at 37ºC for three days,
and tested with anti-porcine circovirus serum by fluorescent antibody assay.
2.3.1.4.2 Judgment
No specific fluorescent antigens are observed in the cultured cells: the test material shallcomply with the test.
2.3.2 Inoculation test using swine testis cells
2.3.2.1 Observation of cultured cells
2.3.2.1.1 Test procedure
Primary or passaged cells of swine testis shall be used.
A 2 mL portion of the test material shall be inoculated into the cultured cell sheet of at least 20
cm2 per 1 mL of the inoculum, cultured at 37ºC, and observed for 10 days.
2.3.2.1.2 Judgment
No CPE is confirmed in the cultured cells during the observation period: the test material shallcomply with the test.
2.3.2.2 Hemadsorption test
2.3.2.2.1 Test procedure
On the final day of observation in 2.3.2.1, the cell surfaces shall be washed twice withphosphate-buffered saline. The cultured cells shall be divided into three groups, overlaid with 0.1
vol% red blood cell suspensions of guinea pig, goose, and chicken aged 7 days or younger,119respectively, allowed to stand at 4ºC for 60 minutes and at 37ºC for 30 minutes, and observed
microscopically for the presence of hemadsorption.
2.3.2.2.2 Judgment
No hemadsorption is observed in the cultured cells: the test material shall comply with the test.
2.4 Inoculation using cells derived from bovine
2.4.1 Inoculation test using bovine kidney cells
2.4.1.1 Observation of cultured cells
2.4.1.1.1 Test procedure
Passaged cells of bovine kidney shall be used.
A 2 mL portion of the test material shall be inoculated into the cultured cell sheet of at least 20
cm2 per 1 mL of the inoculum, cultured at 34ºC to 36ºC for five days, observed for the presence ofCPE, and passaged to the next generation. When the cells are grown as monolayers, the cellsurfaces shall be washed with phosphate-buffered saline. To the cells, culture medium for
maintenance shall be added, cultured at 37ºC for five days, and observed for the presence of CPE.
2.4.1.1.2 Judgment
No CPE is confirmed in the cultured cells during the observation period: the test material shallcomply with the test.
2.4.1.2 Hemadsorption test
2.4.1.2.1 Test procedure
On the final day of observation in 2.4.1.1, the cultured cells shall be divided into two groups,
overlaid with 0.1 vol% guinea pig and goose red blood cell suspensions, respectively, allowed tostand for 60 minutes, and observed microscopically for the presence of hemadsorption.
2.4.1.2.2 Judgment
No hemadsorption is observed in the cultured cells: the test material shall comply with the test.
2.4.2 Inoculation test using bovine testis cells
2.4.2.1 Test procedure
Passaged bovine testis cells shall be used.
A 2 mL portion of the test material shall be inoculated into the cultured cell sheet of at least 20
cm2 per 1 mL of the inoculum, cultured at 34ºC to 36ºC for five days, and observed for thepresence of CPE. Then, these cells shall be passaged in 10 small test tubes, cultured for five days,
and observed for the presence of CPE. After removing the culture suspension, a 0.5 mL portion ofculture medium for maintenance containing approximately 105TCID50/mL of Bovine viraldiarrhea virus Nose strain shall be added to each test tube, roller-cultured at 34ºC to 36ºC forseven days, and observed for the presence of CPE.
2.4.2.2 Judgment
During the observation period, CPE is not observed in the cultured cells before inoculation with120

Bovine viral diarrhea virus, but CPE is observed in the cultured cells after inoculation with thevirus: the test material shall comply with the test.
2.5 Inoculation test using cells derived from canine
2.5.1 Inoculation test using canine kidney cells
2.5.1.1 Observation of cultured cells
2.5.1.1.1 Test procedure
Primary or passaged canine kidney cells shall be used.
A 2 mL portion of the test material shall be inoculated into the cultured cell sheet of at least 20
cm2 per 1 mL of the inoculum, cultured at 36ºC for five days, and observed for the presence ofCPE. Then, these cells shall be passaged to the next generation, cultured at 36ºC for 10 days, andobserved for the presence of CPE.
2.5.1.1.2 Judgment
No CPE is confirmed in the cultured cells during the observation period: the test material shallcomply with the test.
2.5.1.2 Hemadsorption test
2.5.1.2.1 Test procedure
On the final day of observation in 2.5.1.1, after removing the culture suspension, the cells shallbe overlaid with 0.1 vol% guinea pig red blood cell suspensions, allowed to stand at 4ºC for 60
minutes, and observed microscopically for the presence of hemadsorption.
2.5.1.2.2 Judgment
No hemadsorption is observed in the cultured cells: the test material shall comply with the test.
2.6 Inoculation test using cells derived from feline
2.6.1 Inoculation test using feline kidney cells
2.6.1.1 Observation of cultured cells
2.6.1.1.1 Test procedure
Primary or passaged cells of feline kidney shall be used.
A 2 mL portion of the test material shall be inoculated into the cultured cell sheet of at least 20
cm2 per 1 mL of the inoculum, cultured at 36ºC for five days, and observed for the presence ofCPE. Then, these cells shall be passaged to the next generation, cultured at 36ºC for 10 days, andobserved for the presence of CPE.
2.6.1.1.2 Judgment
No CPE is confirmed in the cultured cells during the observation period: the test material shallcomply with the test.
2.6.1.2 Test for freedom from Canine parvovirus and Feline panleukopenia virus
2.6.1.2.1 Test procedure
To the culture medium obtained on the final day of observation given in 2.6.1.1, add an equal
121quantity of borate buffered saline containing 0.2 w/v% bovine serum albumin.
Additionally, add 0.5 vol% swine red blood cells prepared with a quantity of VAD 6.0 solution
equal to that of the mixture, allow to stand at 4ºC for 18 hours, and observe for the presence ofhemagglutination.
2.6.1.2.2 Judgment
No hemagglutination is observed: the test material shall comply with the test.
2.7 Inoculation test using cells derived from other animals
2.7.1 Inoculation test using guinea pig kidney cells
2.7.1.1 Observation of cultured cells
2.7.1.1.1 Test procedure
Primary cells of guinea pig kidney shall be used.
A 2 mL portion of the test material shall be inoculated into the cultured cell sheet of at least 20
cm2 per 1 mL of the inoculum, cultured at 37ºC for 10 days, and observed for the presence ofCPE.
2.7.1.1.2 Judgment
No CPE is confirmed in the cultured cells during the observation period: the test material shallcomply with the test.
2.7.1.2 Hemadsorption test
2.7.1.2.1 Test procedure
On the final day of observation in 2.7.1.1, the cell surfaces shall be washed twice withphosphate-buffered saline. The cultured cells shall be divided into three groups, overlaid with 0.1
vol% red blood cell suspensions of guinea pig, goose, and chicken aged 7 days or younger,
respectively, allowed to stand at 4ºC for 60 minutes and at 37ºC for 30 minutes, and observed
microscopically for the presence of hemadsorption.
2.7.1.2.2 Judgment
No hemadsorption is observed in the cultured cells: the test material shall comply with the test.
2.7.2 Inoculation test using MA-104 cells
2.7.2.1 Test for freedom from Rotavirus
The test shall be performed as specified in 2.7.2.1.1 or 2.7.2.1.2 as follows:
2.7.2.1.1 Observation of cultured cells
2.7.2.1.1.1 Test procedure
A 0.1 mL portion of the test material shall be inoculated in at least 10 test tubes of cultured cells
and allowed to adsorb at 37ºC for 60 minutes. The cell surfaces shall be washed with phosphatebuffered saline. Culture medium containing an appropriate amount of trypsin shall be added,
roller-cultured at 37ºC for seven days, and observed for the presence of CPE.

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2.7.2.1.1.2 Judgment
No CPE is confirmed in the cultured cells during the observation period: the test material shallcomply with the test.
2.7.2.1.2 Fluorescent antibody assay
2.7.2.1.2.1 Test procedure
A 0.1 mL portion of the test material shall be inoculated into the cells cultured on a cover glasswith a size of 3 cm2 or larger, cultured at 37ºC for 48 hours, tested with anti-rotavirus serum byfluorescent antibody assay, and then observed.
2.7.2.2.2 Judgment
No specific fluorescent antigens are observed in the cultured cells: the test material shallcomply with the test.
2.8 Animal inoculation test
2.8.1 Cattle or sheep inoculation test
2.8.1.1 Test for freedom from Bovine leukemia virus
2.8.1.1.1 Test procedure
Healthy cattle weighing 100–200 kg or healthy sheep weighing 30–50 kg shall be used.
A 10 mL portion of the test material shall be intramuscularly injected into one cattle or sheep,and serum shall be obtained through blood sampling in the 2nd and 3rd months. The obtained
serum shall be tested by passive hemagglutination assay, enzyme immunoassay, or agar gel
immunoprecipitation assay to detect the bovine leukemia virus antibody.
2.8.1.1.2 Judgment
No antibody against bovine leukemia virus is detected: the test material shall comply with thetest.
2.8.2 Suckling mice inoculation test
2.8.2.1 Test procedure
Mice aged 3 days or younger shall be used.
The 0.02 mL portions of the test material shall be injected into the brain of 10 mice.
The centrifugal supernatant of pooled brain mixed emulsion collected on the 5th day shallsubsequently be injected into the brain of 10 mice, and observed for 10 days.
2.8.2.2 Judgment
No mice show death or neurological symptoms: the test material shall comply with the test.

123Test for Thimerosal Content
The Test for Thimerosal Content is a method to determine the thimerosal content of the test article
etc. from the absorbance of the chelate compound with maximum specific absorption at awavelength of 480 nm formed by the reaction of thimerosal with dithizone in a sulfuric acidsolution.
1 Standard solution and test solutions
1.1 200 μg/mL thimerosal standard solution
Weigh precisely 20 mg of thimerosal and add water to make exactly 100 mL.
1.2 1 mol/L sulfuric acid test solution
Add 20 mL of sulfuric acid in 340 mL of water.
1.3 Dithizone test solution
Dissolve 2 mg of dithizone in carbon tetrachloride to make 100 mL.
If necessary, add 100 mL of 100-fold diluted ammonia solution (28), transfer dithizone to thewater layer, wash several times with 20–30 mL of carbon tetrachloride, neutralize withhydrochloric acid, and transfer dithizone to carbon tetrachloride layer to adjust the concentration
is 0.002 w/v%.
1.4 Ammonia test solution
Add water to 60 mL of ammonia solution (28) to make 100 mL.
2 Test procedure
Pipet 2.5, 5 and 7.5 mL of thimerosal standard solution, add water to make exactly 10 mL, anduse as the standard dilutions of 50, 100 and 150 µg in 1 mL, respectively.
Pipet 0.5 mL of the test article etc., add water to make exactly 5 mL, and use as the test
material.
Proceed with the standard solutions as directed above.
To these solutions, add 5 mL portions of 1 mol/L sulfuric acid test solution and 10 mL portionsof dithizone test solution, and shake for 5 minutes vigorously. Allow to stand, and then collect theresulting carbon tetrachloride layer. Shake with 10 mL of water and allow to stand. Discard theresulting water layer, shake the rest with 10 mL of the ammonia test solution, and allow to standand discard the water layer. Repeat this procedure of washing three times; then add 10 mL ofwater, shake, and mix.
Discard the resulting water layer and filter the remaining carbon tetrachloride layer through afilter paper. Measure the absorbance of the filtrate at a wavelength of 480 nm.
Since mercury dithizonate is unstable in direct sunlight and heat, extract at a liquid temperature
124

below 20ºC, kept in a dark place, and measure the absorbance as quickly as possible.
Prepare a calibration curve from the absorbance of the standard dilutions and insert theabsorbance of the test material in the calibration curve to calculate the thimerosal content of thetest article etc. Correct by measuring the absorbance of water treated in the same manner.
3 Judgment
Unless otherwise specified, the test results indicate that the thimerosal content is not more than
0.01 w/v%: the test article etc. shall comply with the test.

125Test for Phenol Content
The Test for Phenol Content is a method to determine the phenol content of the test article etc.
from its absorbance at a wavelength of 550 nm by utilizing the color development by the reaction
with 4-nitroaniline and nitrous acid.
The criterion for judgment shall be given in the monographs.
1 Standard solution and test solutions
1.1 5 mg/mL phenol standard solution
Weigh precisely 0.5 g of phenol and add water to make exactly 100 mL.
1.2 4-nitroaniline-sodium nitrite test solution
Before use, add 0.75 mL of sodium nitrite test solution to 25 mL of 4-nitroaniline test solution
as below and mix thoroughly to prepare the solution.
1.2.1 4-nitroaniline test solution
Dissolve 1.5 g of hydrochloric acid in 40 mL of 4-nitroaniline and add water to make 500 mL.
If necessary, heat in a water bath.
1.2.2 sodium nitrite test solution
Dissolve 10 g of sodium nitrite in water to make 100 mL.
1.3 50 w/v% sodium acetate test solution
Dissolve 50 g of sodium acetate trihydrate in water to make 100 mL.
1.4 sodium carbonate test solution
Dissolve 10.5 g of anhydrous sodium carbonate in water to make 100 mL.
2 Test procedure
Pipet 1, 2, 3, 4, and 5 mL of phenol standard solutions, add water to make exactly 5 mL, anduse as standard dilutions at concentrations of 1, 2, 3, 4, and 5 mg/mL.
Pipet 1 mL each of the test article etc. and standard solutions, add water to make exactly 50 mL,
and use as the test materials.
For the test article etc., if necessary, pipet a 1 mL portion and add water to make approximately
10 mL. To this solution, add approximately 10 mL of 5 w/v% trichloroacetic acid solution, andadd water to make exactly 50 mL. Allow to stand at room temperature for 30 minutes, filter thesolution, and use the filtrate obtained as the test materials.
Pipet 1 mL each of the test materials and add water to make 30 mL. To these solutions, add 1
mL of a 50 w/v% sodium acetate solution and then 1 mL of 4-nitroaniline-sodium nitrite test
solution and shake thoroughly. Add 2 mL of the sodium carbonate test solution and water to make
50 mL and then shake thoroughly. Allow to stand at room temperature for 10 minutes. Take
126

portions of the mixtures to measure absorbance at a wavelength of 550 nm immediately.
Prepare a calibration curve from the absorbance of the standard dilutions and insert theabsorbance of the test material in the calibration curve to calculate the phenol content of the test
article etc. Compensate by separately measuring the absorbance of water treated in the samemanner for reference.

127

Test for Formalin Content
The Test for Formalin Content is a method to determine the formalin content of the test article etc.
from its absorbance at a wavelength of 410 nm by means of the property that the formaldehyde
contained in the formalin gives the yellowish orange color of 3.5-diacetyl-1.4-dihydrolutidine
resulting from the reaction with acetylacetone in the presence of excess ammonia under slightly
acidic conditions. The formalin content shall be determined by the following method or the method
specified in the guideline of International Cooperation on Harmonisation of Technical Requirements
for Registration of Veterinary Medicinal Products (GL25: Testing of residual formaldehyde).
The criterion for judgment shall be given in the monographs.
1 Standard solution and test solutions
1.1 Formalin standard solution
Precisely dilute formalin 500-fold with water.
1.2 Acetic acid-ammonium acetate buffer solution (pH 6.25)
Mix 40 mL of the following acetic acid solution and 1,000 mL of ammonium acetate solution
and store in a dark place.
1.2.1 Acetic acid solution
Add water to 12.9 mL of acetic acid (100) to make 100 mL.
1.2.2 Ammonium acetate solution
Dissolve 173.4 g of ammonium acetate in water to make 1,000 mL.
1.3 Acetylacetone test solution
Mix 7 mL of acetylacetone and 14 mL of ethanol (99.5) and add water to make 1,000 mL.
2 Test procedure
Dilute the test article etc. accurately with water so that it contains 0.01–0.05 % formalin and use
as the test materials. Pipet 0.5, 1, 1.5, 2, and 2.5 mL of formalin standard solutions accurately, andadd water to make exactly 10 mL, respectively. Use these dilutions as 0.01, 0.02, 0.03, 0.04, and0.05 vol% standard dilutions.
Pipet 0.1 mL each of the test material and standard dilutions and add 2 mL of acetic acidammonium acetate buffer solution to these dilutions, respectively. Add 2 mL of acetylacetone test
solution and mix, heat at 60ºC for 15 minutes, and then cool for 5 minutes in cold water. Allow tostand for 20 minutes and measure the absorbance at a wavelength of 410 nm.
Prepare a calibration curve from the absorbance of the standard dilutions, and insert theabsorbance of the test material in the calibration curve to calculate the formalin content of the test
article etc. Compensate by separately measuring the absorbance of water treated in the samemanner for reference.
128

Test for Aluminum Content
The Test for Aluminum Content is a method to determine the aluminum content of the test article
etc. from the absorbance at a wavelength of 510 nm by utilizing the color development of chelatecompound formed by the reaction of stilbazo with the insoluble salt of aluminum contained in thetest article etc. which is dissolved in nitric acid.
The criterion for judgment shall be given in the monographs.
1 Standard solution and test solutions
1.1 Aluminum standard solution (4 μg/mL)
Weigh precisely 895 mg of aluminum (III) chloride hexahydrate, add water to make exactly 100
mL. Pipet 2 mL of the solution and add water to make exactly 500 mL.
1.2 1 mol/L acetate buffer (pH 5.55–5.75)
Add nine parts of 1 mol/L sodium acetate solution to one part of 1 mol/L acetic acid solution as
below and mix.
1.2.1 1 mol/L acetic acid solution
Add water to 6.0 mL of acetic acid (100) to make 100 mL.
1.2.2 1 mol/L sodium acetate solution
Dissolve 13.6 g of sodium acetate trihydrate in water to make 100 mL.
1.3 Stilbazo test solution
Weigh approximately 60 mg of stilbazo and grind it in a mortar. Add water to make 100 mL andfilter and use the filtrate. However, when pipet 1 mL of the filtrate, add 10 mL of 1 mol/L acetate
buffer and 14 mL of water to the filtrate and allow to stand at approximately 25°C for 20 minutes,
the absorbance of the mixture at 420 nm shall be 0.85 or more. Store the solution in a dark place,protected from sunlight, and use within 2 weeks after preparation.
2 Test procedure
Shake the test article etc. to make a homogeneous suspension and pipet 1 mL of the suspension.
To this suspension, add 0.2 mL of nitric acid solution and boil to dissolve. Dilute the test article
etc. with water so that the aluminum content is not more than 4 μg in 1 mL and use as the test
material.
Dilute the aluminum standard solution with water to make 2 μg/mL and 4 μg/mL standard
dilutions.
Pipet 1 mL each of the test dilutions and the standard dilutions. To the test solutions, add 2.5
mL of water, 1 mL of 1 mol/L acetic acid buffer, and 0.5 mL of the stilbazo test solution
accurately. Allow the mixtures to stand at room temperature for 20 minutes and then determine the129absorbance at a wavelength of 510 nm with a spectrophotometer immediately.
Prepare a calibration curve from the absorbance of the standard solution, insert themeasurement of the test material in the calibration curve to calculate the aluminum content in thetest material and calculate the aluminum content in each mL of the test article.
Compensate by separately measuring the absorbance of water treated in the same manner for
reference.

130Test for Macrogol Content
The Test for Macrogol Content is a method to determine the macrogol content of the test article
etc. from its absorbance at 470 nm of the yellowish orange color of precipitated complex salt formedby the addition of phosphomolybdic acid to a polyoxyethylene nonionic surfactant and treatment
with ammonium thiocyanate and tin (II) chloride.
The criterion for judgment shall be given in the monographs.
1 Standard solution and test solutions
1.1 Macrogol standard solution (10 μg/mL)
Weigh precisely 200 mg of Macrogol 4,000 or Macrogol 6,000 and dissolve in water to makeexactly 1,000 mL. Pipet exactly 5 mL of this solution and add water to make exactly 100 mL.
1.2 Barium chloride test solution
Dissolve 10 g of barium chloride in water to make exactly 100 mL.
1.3 Phosphomolybdic acid test solution
Dissolve 10 g of phosphomolybdic acid in water to make 100 mL.
1.4 Ammonium thiocyanate test solution
Dissolve 10 g of ammonium thiocyanate in water to make 100 mL.
1.5 Tin (II) chloride test solution
Dissolve 2 g of tin (II) chloride dihydrate in 10 mL of hydrochloric acid and add water to make100 mL. Filter if necessary.
1.6 Dilute hydrochloric acid test solution
Add water to 23.6 mL of hydrochloric acid to make 100 mL.
2 Test procedure
Take exactly an appropriate volume of the test article etc., add the equal volume of acetone, andallow to stand at room temperature for 10–15 minutes. For the period of time, agitate occasionally.
Centrifuge it at 3,000 rpm for 10 minutes, dilute the supernatant with water so that it contains 10–
50 μg of Macrogol in 1 mL, and use as the test material.
Pipet exactly 1, 2, 3, 4, and 5 mL of macrogol standard solution, add water to each portion of it
to make exactly 5 mL, and use as the standard dilutions at concentrations of 2, 4, 6, 8 and 10
μg/mL. Pipet exactly 1 mL of the test material and add water to make exactly 5 mL. To thisportion of it and each standard dilution, add three drops of dilute hydrochloric acid test solution,
two drops of barium chloride test solution, and two drops of phosphomolybdic acid test solution,
mix thoroughly, and allow to react at 40℃ for 10 minutes. Centrifuge for 10 minutes, discard thesupernatant, and wash by centrifuge several times. If necessary, invert the centrifuge tube for 1–2131minutes to remove water. Add 1.2 mL of sulfuric acid to each solution, and heat to dissolve theprecipitate. Add water to make 6 mL. Add 1 mL of an ammonium thiocyanate test solution and 0.5
mL of a tin (II) chloride test solution, add water to make exactly 10 mL, and mix thoroughly. After
20 minutes, measure the absorbance at 470 nm.
Prepare a calibration curve from the absorbance of the standard dilutions, and insert theabsorbance of the test material in the calibration curve to calculate the macrogol content of the test
article etc. Correct by measuring the absorbance of water treated in the same manner.

132

Test for Protein Nitrogen Content
The Test for Protein Nitrogen Content is a method to determine the protein nitrogen content of thetest article etc. by measuring nitrogen in heated trichloroacetic acid-precipitable protein in the test
article by the micro-Kjeldahl method.
The criterion for judgment shall be given in the monographs.
1 Test solutions and reagents
1.1 50 w/v% trichloroacetic acid test solution
Dissolve 500 g of trichloroacetic acid in water to make 1,000 mL.
1.2 5 w/v% trichloroacetic acid test solution
Dilute the test solution in 1.1 tenfold with water.
1.3 1 mol/L sodium hydroxide test solution
Dissolve 4.0 g of sodium hydroxide in water to make 100 mL.
1.4 Decomposition accelerator
Use a powdered mixture of 100 g of potassium sulfate and 10 g of copper (II) sulfate
pentahydrate.
1.5 30 w/v% sodium hydroxide test solution
Dissolve 300 g of sodium hydroxide in water to make 1,000 mL.
1.6 Indicator
Dissolve 0.15 g of bromocresol green and 0.1 g of methyl red in 90 vol% ethanol to make 200
mL.
1.7 Boric acid test solution
Dissolve 40 g of boric acid in water to make 1,000 mL.
1.8 0.005 mol/L sulfuric acid (standard solution for volumetric analysis)
Contain 0.4904 g of sulfuric acid in 1,000 mL.
Preparation of 0.05 mol/L sulfuric acid
After adding 3 mL of sulfuric acid to an appropriate volume of water, make it 1,000 mL withwater and standardize the solution as follows:Standardization of 0.05 mol/L sulfuric acidHeat sodium carbonate at 500℃ to 650℃ for 40–50 minutes, allow to cool in a desiccator(silica gel), weigh precisely approximately 0.08 g of it, dissolve in 30 mL of water, add three
drops of methyl red standard solution, titrate with the prepared sulfuric acid, and calculate thenormality factor. The endpoint shall set boil the content carefully, stopper the flask loosely,
allow to cool, and continue the titration until the color of the solution changes to persistent133

orange to orange-red.
0.05 mol/L sulfuric acid 1mL = 5.299 mg of Na2CO3
Preparation of 0.005 mol/L sulfuric acid
Before use, dilute 0.05 mol/L sulfuric acid with water to make exactly 10 times the initial
volume.
2 Test procedure
Pipet exactly a quantity of the test article etc. corresponding to 10–200 μg of protein nitrogen,add one-tenth of its volume of a 50 w/v% trichloroacetic acid test solution, heat in a boiling water
bath for 15 minutes, and allow to cool to room temperature. With regard to antitoxins and sera
listed in the monograph of drugs, this heat treatment is replaced by warming at an appropriate
temperature for 15 minutes. Centrifuge the mixture, add an appropriate amount of a 5 w/v%
trichloroacetic acid solution to the precipitate, and wash by centrifuge. Then, dissolve theprecipitate in a small volume of 1 mol/L sodium hydroxide test solution to serve as the test
material.
Place approximately 70 mg of decomposition accelerator and test material in a decomposition
flask and pour 1 mL of sulfuric acid along the inner wall of the flask. Heat for approximately 5
hours to decompose until the solution changes to a clear blue, and the inside wall of the flask isfree from a carbonaceous material. During the decomposition process, if necessary, add hydrogen
peroxide (30) in the decomposition flask. After allowing to cool, add approximately 5 mL of water
to cool the solution and rinse in the distillation flask washed beforehand by passing steam throughit. Place 6 mL of a 30 w/v% sodium hydroxide test solution in the distillation flask.
To the absorption flask, add 5 mL of a boric acid test solution and some drops of the indicator
to immerse the lower end part of the condenser tube.
Pass stream through the distillation flask and continue until the distillate measures 70 to 80 mL.
Remove the absorption flask from the lower end of the condenser tube, rinse the lower end partwith a small quantity of water, and titrate the distillate with 0.005 mol/L sulfuric acid until thecolor of the solution changes from green through pale grayish blue to pale grayish red-purple.
Perform a blank determination in the same manner with water and make any necessary correction.
1 mL of 0.005 mol/L sulfuric acid = 140.07 μg of N

134

Test for Freedom from Abnormal Toxicity
The Test for Freedom from Abnormal Toxicity is a method by conducting the following tests 1
and 2 to confirm that the injection of the test article etc. into the experimental animals causes noabnormalities:
1 Test on guinea pigs
1.1 Test materials
1.1.1 Injection materials
The test article etc. shall be used.
1.1.2 Test animals
Guinea pigs weighing approximately 350 g that show no abnormal signs and a normal increasein body weight during an observation period of at least four days prior to use shall be used.
1.2 Test procedure
To two guinea pigs, the 5 mL portions of the injection material shall be given by intraperitoneal
injection, and the animals shall be observed for seven days after the injection. Regarding those
approved in particular, however, follow the approved observation period.
1.3 Judgment
None of the animals shall show any abnormal signs during the observation period: the test
article etc. shall comply with the test.
2 Test on mice
2.1 Test materials
2.1.1 Injection materials
The test article etc. shall be used.
2.1.2 Test animals
Mice aged approximately 5 weeks that show no abnormal signs and a normal increase in bodyweight during an observation period of at least four days prior to use shall be used.
2.2 Test procedure
To 10 mice, the 0.5 mL portions of the injection material shall be given by intraperitoneal
injection, and the animals shall be observed for seven days after the injection. Regarding those
approved in particular, however, follow the approved observation period.
2.3 Judgment
None of the animals shall show any abnormal signs during the observation period: the test
article etc. shall comply with the test.

135

Toxicity Limit Test
The Toxicity Limit Test is a method by conducting either of the following tests 1 or 2 to confirm
that the injection of the test article etc. into the experimental animals causes no safety concerns.:
1 Test on mice
1.1 Test materials
1.1.1 Injection materials
The test article etc. shall be used.
1.1.2 Test animals
Mice aged approximately 5 weeks that show no abnormal signs and a normal increase in bodyweight during an observation period of at least four days prior to use shall be used.
1.2 Test procedure
To 10 mice, the 0.5 mL portions of the injection material shall be given by intraperitoneal
injection, the body weight shall be measured before the injection and on Day 3 of injection, andthe animals shall be observed for seven days after the injection.
1.3 Judgment
If the body weight on Day 3 of injection shall be no less than that measured before the time of
injection, and none of the animals shall show any abnormal signs during the observation period:
the test article etc. shall comply with the test.
2 Test on guinea pigs
2.1 Test materials
2.1.1 Injection materials
The test article etc. shall be used.
2.1.2 Test animals
Guinea pigs weighing approximately 350 g that show no abnormal signs and a normal increasein body weight during an observation period of at least four days prior to use shall be used.
2.2 Test procedure
To 5 guinea pigs, the 5 mL portions of the injection material shall be given by intraperitoneal
injection, the body weight shall be measured before the injection and on Day 3 of injection, andthe animals shall be observed for seven days after the injection.
2.3 Judgment
If the body weight on Day 3 of injection shall be no less than that measured before the time of
injection, and none of the animals shall show any abnormal signs during the observation period:
the test article etc. shall comply with the test.
136

Test for Freedom from Extraneous Viruses
The Test for Freedom from Extraneous Viruses is a method to verify that no detectable extraneous
viruses are observed in the master seed virus, master cell seed and master seed coccidia.
1 The test material
1.1 Master seed virus
To a 1 mL portion of the test article, add immune serum, neutralize at 37ºC for 1 hour or at 4ºCovernight, neutralize completely, and use as the test material.
1.2 Master cell seed
Divide the cultured cells grown as monolayers into two groups for culture. Freeze-thaw thecells in one group three times. With the freeze-thawed mixture, mix the culture supernatant fromthe other group and use the obtained mixture as the test material.
1.3 Master seed coccidia
The test article processed in a method approved as suitable, such as centrifugation, shall be usedas the test material.
2 Common test for freedom from extraneous viruses
Master seed virus, master cell seed and master seed coccidia shall be tested as specified in 2.1
and 2.2 or either thereof in consideration of the appropriate range of viruses that may infect the
animal species of origin and viruses that are pathogenic to the animals targeted for vaccination.
2.1 Inoculation test using susceptible cell
2.1.1 Test materials
2.1.1.1 Cultured cells
Use cells susceptible to an appropriate range of viruses that may infect the animal species fromwhich the cultured cells are derived and cells susceptible to an appropriate range of viruses that
may infect the animal species targeted for vaccination.
2.1.2 Test procedure
2.1.2.1 Culture
The test material shall be inoculated into the cultured cell sheet of at least 70 cm2 per 1 mL of
the inoculum, cultured at 37ºC for seven days, and the cells shall be passaged to the next
generation.
The passaged cells shall be cultured at 37ºC for seven days, and then shall be passaged tosubsequent generations at 37ºC for seven days. If using cells derived from fish, these cells shall be
cultured in a manner specified in the respective sections of the part of vaccine (seed lot product).
2.1.2.2 Observation of cultured cells
137The cells shall be observed for the presence of CPE at least every two days during all theculture periods.
2.1.2.3 Cell staining
On the final day of culture, cultured cell sheets of at least 12 cm2 shall be subjected to
Hematoxylin-Eosin stain or May-Grünwald-Giemsa stain, and observed microscopically for CPE,
inclusion bodies and any cell abnormalities due to other extraneous factors.
2.1.2.4 hemadsorption
On the final day of culture, the cultured cell sheet shall be divided in three groups so that thesize of each sheet is at least 18 cm2.The cell surfaces shall be washed twice with phosphatebuffered saline, and then overlaid with 0.1 vol% red blood cell suspensions of guinea pig, goose,and chicken aged 7 days or younger, respectively, allowed to stand at 4ºC for 30 minutes followed
at 20ºC to 25ºC for 30 minutes. Then washed with phosphate-buffered saline, and observed
microscopically for the presence of hemadsorption.
2.1.3 Judgment
During the period of observation, no CPE and inclusion bodies and other cell abnormalities areobserved in the cultured cells, and no hemadsorption is observed in the cultured cells: the test
material shall comply with the test.
2.2 Inoculation test using embryonated chicken egg
2.2.1 Inoculation into allantoic cavity
2.2.1.1 Observation of embryo
2.2.1.1.1 Test procedure
As specified in 1.1 in the Materials for Live Vaccine Production, embryonated chicken eggs
aged 9–11 days shall be used. The 0.1 mL portions of the test material shall be injected into theallantoic cavity of 10 embryonated chicken eggs and cultured at 37ºC for seven days. On the final
day of culture, these eggs shall be opened to examine abnormalities of chicken embryos.
2.2.1.1.2 Judgment
If chicken embryos are properly developed and no abnormalities are observed: the test material
shall comply with the test.
2.2.1.2 Hemagglutination test
2.2.1.2.1 Test procedure
To the allantoic fluid collected on the final day of culture in 2.2.1.1.1, an equal volume of 0.5
vol% chicken red blood cell suspension shall be added, allowed to stand at 4ºC for 60 minutes,
and observed for the presence of hemagglutination.
2.2.1.2.2 Judgment
No hemagglutination is observed in the allantoic fluid: the test material shall comply with the
test.
138

2.2.2 Inoculation onto chorioallantoic membrane
2.2.2.1 Test procedure
As specified in 1.1 in the Materials for Live Vaccine Production, embryonated chicken eggs
aged 10–12 days shall be used. The 0.1 mL portions of the test material shall be inoculated onto
the chorioallantoic membrane of 10 embryonated chicken eggs and cultured at 37ºC for five days.
On the final day of culture, these eggs shall be opened and observed to examine abnormalities ofchicken embryos and chorioallantoic membranes.
2.2.2.2 Judgment
If chicken embryos are properly developed and no abnormalities are observed in thechorioallantoic membranes: the test material shall comply with the test.
3 Test for freedom from specific viruses
In principle, for the master seed virus, master cell seed, and master seed coccidia, the Test forFreedom from Specific Viruses shall be performed in addition to the Common Test for Freedom
from Extraneous Viruses in consideration of the appropriate range of viruses that may infect the
animal species of origin and viruses that are pathogenic to the target animals. Viruses specified in
3.2 Individual Test for Freedom from Specific Viruses shall be tested as directed in the Individual
Test for Freedom from Specific Viruses.
3.1 General test for freedom from specific viruses
3.1.1 Fluorescent antibody assay
3.1.1.1 Test materials
3.1.1.1.1 Cultured cells
Use cells susceptible to an appropriate range of viruses that may infect the animal species fromwhich the cultured cells are derived and cells susceptible to an appropriate range of viruses that
may infect the animal species targeted for the vaccination. If using cells derived from fish, thesecells shall be cultured in a manner specified in the respective sections of the part of vaccine (seedlot product).
3.1.1.2 Test procedure
3.1.1.2.1 Culture
The test material shall be inoculated into the cultured cell sheet of at least 70 cm2 per 1 mL of
the inoculum, cultured at 37ºC for seven days, and the cells shall be passaged to the next
generation. The passaged cells shall be cultured at 37ºC for seven days, and then the cells shall bepassaged to the following generation and cultured at 37ºC for seven days. If using cells derived
from fish, these cells shall be cultured in a manner specified in the respective sections of the partof the vaccine (seed lot product).
3.1.1.2.2 Fluorescent antibody assay
A fluorescent antibody assay shall be performed with antisera against the viruses specified in139the respective sections of the part of vaccine (seed lot product) fluorescent antibody assay.
3.1.1.3 Judgment
No specific fluorescent antigens are observed in the cultured cells: the test material shallcomply with the test.
3.1.2 Inoculation to chicken
3.1.2.1 Test animals
Chickens aged 2 weeks or older derived from 1.1 in the Materials for Live Vaccine Production
shall be used.
3.1.2.2 Test procedure
The test material shall be inoculated at a dose of 1 mL intramuscularly and at a dose of 0.03 mL
ophthalmically or in other manners approved as suitable and shall be re-inoculated two weeks
later. Obtain the sera at the time of initial inoculation and five weeks after initial inoculation tomeasure the antibodies against the viruses specified in the respective sections of the part ofvaccine (seed lot product)
3.1.2.3 Judgment
No specific antibodies are detected in the sera obtained at the time of initial inoculation and atfive weeks after initial inoculation: the test material shall comply with the test.
3.2 Individual test for freedom from specific viruses
3.2.1 Test for freedom from Avian leukosis virus
3.2.1.1 Test procedure
As specified in 2.1.1 in the Materials for Live Vaccine Production, primary cells of chicken
embryo susceptible to avian leukosis virus shall be used.
A 1 mL portion of the test material shall be inoculated into the cell sheet of at least 70 cm2
cultured within 24 hours. These cells shall be cultured at 37ºC for three to five days and passaged
to the next generation. The passaged cells shall be cultured at 37ºC three to five days, passaged tosubsequent generations, and cultured at 37ºC for three to five days.
After washing the third cultured cell sheet of at least 70 cm2, add one-twentieth volume ofculture medium of Veronal-buffered saline containing 1 w/v% gelatin. Freeze-thaw the harvested
cell suspension three times and collect centrifugal supernatant to prepare cell extract. Using this as
an antigen, perform a COFAL test by the Kolmer’s method in pigeon, rabbit, or hamster serum
specific to avian leukosis virus, three units of hemolytic elements, four units of guinea-pig
complements, and 2 vol% sheep red blood cells suspension.
3.2.1.2 Judgment
The COFAL test indicates that the cell extract is negative for the virus: the test material shallcomply with the test.
3.2.2 Test for freedom from Reticuloendotheliosis virus
1403.2.2.1 Test procedure
As specified in 2.1.1 in the Materials for Live Vaccine Production, primary cells of chicken
embryo susceptible to avian leukosis virus shall be used.
A 1 mL portion of the test material shall be inoculated into the cell sheet of at least 70 cm2
cultured within 24 hours, the primary cell suspension cultured at 37ºC for three to five days shallbe inoculated into a newly cultured primary cells of chicken embryo, and cultured at 37ºC for fourdays. The cell suspension shall be passaged in the same manner to the third generation, cultured at
37ºC for four days, tested with anti-reticuloendotheliosis virus by fluorescent antibody assay, andthen observed.
3.2.2.2 Judgment
No specific fluorescent antigens are observed in the cultured cells: the test material shallcomply with the test.
3.2.3 Test for freedom from Classical swine fever virus
The test shall be performed as specified in 3.2.3.1 or 3.2.3.2 as follows:
3.2.3.1 Fluorescent antibody assay
3.2.3.1.1 Test procedure
Primary or passaged cultured cells of swine kidney shall be used.
A 4 mL portion of the test material shall be inoculated into the cultured cell sheet of at least 20
cm2 per 1 mL of the inoculum and cultured at 37ºC for five days.
On the 5th day of inoculation of the test material, culture supernatant shall be collected, a 1 mL
portion of it shall be inoculated into other cultured cell sheet of at least 3 cm2, cultured at 37ºC for
24–48 hours, tested with anti-classical swine fever virus serum by fluorescent antibody assay, andthen observed.
3.2.3.1.2 Judgment
No specific fluorescent antigens are observed in the cultured cells: the test material shallcomply with the test.
3.2.3.2 END method and interference method
3.2.3.2.1 Test procedure
Primary or passaged swine kidney cells shall be used.
A 4 mL portion of the test material shall be inoculated into the cultured cell sheet of at least 20
cm2 per 1 mL of the inoculum and cultured at 37ºC for five days.
On the 5th day of inoculation of the test material, culture supernatant shall be collected, 0.1 mL
portions of it shall be dispensed into at least 20 small test tubes (wells), and 0.5 mL portions ofprimary cells of swine testis suspended in the cell growth medium shall be added. After allowing
to stand to culture at 37ºC for four days, the cultured cells shall be divided into two groups andtested by the END method and interference method using WEE virus.
1413.2.3.2.2 Judgment
No Classical swine fever virus is detected in the cultured cells: the test material shall comply
with the test.
3.2.4 Test for freedom from Porcine circovirus
3.2.4.1 Test procedure
PPK-3F cells shall be used.
A 2 mL portion of the test material shall be inoculated into the cultured cell sheet of at least 20
cm2 per 1 mL of the inoculum, treated with glucosamine, cultured at 37ºC for five days, and thecells shall be passaged to the next generation. The passaged cells shall be cultured at 37ºC for fourdays, and these cells shall be passaged to the next generation. The passaged cells shall be cultured
at 37ºC for three days, tested with anti-porcine circovirus serum by fluorescent antibody assay,
and then observed.
3.2.4.2 Judgment
No specific fluorescent antigens are observed in the cultured cells: the test material shallcomply with the test.
3.2.5 Test for freedom from Bovine viral diarrhea virus
3.2.5.1 Test procedure
Passaged bovine testis cells shall be used.
A 2 mL portion of the test material shall be inoculated into the cultured cell sheet of at least 20
cm2 per 1 mL of the inoculum, cultured at 34ºC to 36ºC for five days, and observed for thepresence of CPE. Then, these cells shall be dispensed and passaged in 10 small test tubes, cultured
for five days, and observed for the presence of CPE. After removing the culture suspension, a 0.5
mL portion of culture medium for maintenance containing approximately 105TCID50/mL of
Bovine viral diarrhea virus Nose strain shall be added to each test tube, roller-cultured at 34ºC to
36ºC for seven days, and observed for the presence of CPE.
3.2.5.2 Judgment
During the observation period, CPE is not observed in the cultured cells before inoculation with
the Bovine viral diarrhea virus, but CPE is observed in the cultured cells after inoculation with thevirus: the test material shall comply with the test.
3.2.6 Test for freedom from Canine parvovirus and Feline panleukopenia virus
3.2.6.1 Test procedure
Primary or passaged cells of feline kidney shall be used.
A 2 mL portion of the test material shall be inoculated into the cultured cell sheet of at least 20
cm2 per 1 mL of the inoculum, cultured at 36ºC for five days, passaged to the next generation, andcultured at 36ºC for 10 days.
To the culture medium obtained on the final day of culture, add an equal quantity of borate142buffered saline containing 0.2 w/v% bovine serum albumin. Additionally, add 0.5 vol% swine red
blood cells prepared with a quantity of VAD 6.0 solution equal to that of the mixture, allow tostand at 4ºC for 18 hours, and observe the presence of hemagglutination.
3.2.6.2 Judgment
No hemagglutination is observed: the test material shall comply with the test.
3.2.7 Test for freedom from Rotavirus
The test shall be performed as specified in 3.2.7.1 or 3.2.7.2 as follows:
3.2.7.1 Observation of cultured cells
3.2.7.1.1 Test procedure
MA104 cells shall be used.
A 0.1 mL portion of the test material shall be inoculated in at least 10 portions of cultured cells
and allowed to adsorb at 37ºC for 60 minutes. The cell surfaces shall be washed with phosphatebuffered saline. Culture suspension containing an appropriate amount of trypsin shall be added,
roller-cultured at 37ºC for seven days, and observed for the presence of CPE.
3.2.7.1.2 Judgment
No CPE is confirmed in the cultured cells during the observation period: the test material shallcomply with the test.
3.2.7.2 Fluorescent antibody assay
3.2.7.2.1 Test procedure
A 0.1 mL portion of the test material shall be inoculated into the cultured cell sheet of at least 3
cm2, cultured at 37ºC for 48 hours, tested with anti-rotavirus serum by fluorescent antibody assay,
and then observed.
3.2.7.2.2 Judgment
No specific fluorescent antigens are observed in the cultured cells: the test material shallcomply with the test.
3.2.8 Test for freedom from bovine leukemia virus
3.2.8.1 Test procedure
Healthy cattle weighing 100–200 kg or healthy sheep weighing 30–50 kg shall be used.
A 10 mL portion of the test material shall be intramuscularly injected into one cattle or sheep,and serum shall be obtained through blood sampling in the 2nd and 3rd months. The obtained
serum shall be tested by passive hemagglutination assay, enzyme immunoassay, or agar gel
immunoprecipitation assay to detect the bovine leukemia virus antibody.
3.2.8.2 Judgment
No antibody against bovine leukemia virus is observed: the test material shall comply with the test.
3.2.9 Test for freedom from Japanese encephalitis virus or Rabies virus
3.2.9.1 Test procedure143Mice aged 3 days or younger shall be used.
The 0.02 mL portions of the test material shall be injected into the brain of 10 mice. The
centrifugal supernatant of pooled brain mixed emulsion collected on the 5th day shallsubsequently be injected into the brain of 10 mice, and observed for 10 days.
3.2.9.2 Judgment
No mice show death or neurological symptoms: the test material shall comply with the test.
3.2.10 Test for freedom from Avian encephalomyelitis virus
3.2.10.1 Test procedure
As specified in 1.1 in the Materials for Live Vaccine Production, embryonated chicken eggs
aged 6 days shall be used.
The 0.1 mL portions of the test material shall be inoculated into the yolk sac of 10 embryonatedchicken eggs and cultured at 37ºC for 12 days. On the final day of culture, 5 eggs shall be opened,
and observed to examine abnormalities of chicken embryos. The remaining eggs shall be hatchedand observed for 10 days.
3.2.10.2 Judgment
No deaths or neurological symptoms are observed in the chicken embryos and chickens: the test
material shall comply with the test.
3.2.11 Test for freedom from Koi herpesvirus
The test shall be performed as specified in 3.2.11.1 or 3.2.11.2 as follows:
3.2.11.1 Observational test of cultured cells
3.2.11.1.1 Test procedure
KF-1 or CCB cells shall be used.
A 0.1 mL portion of the test material shall be inoculated in at least 10 portions of cultured cells
and allowed to adsorb at 25ºC for 60 minutes. The cell surfaces shall be washed with phosphatebuffered saline, cultured at 25ºC for 21 days, and observed for the presence of CPE.
3.2.11.1.2 Judgment
No CPE is confirmed in the cultured cells during the observation period: the test material shallcomply with the test.
3.2.11.2 Fluorescent antibody assay
3.2.11.2.1 Test procedure
A 0.1 mL portion of the test material shall be inoculated into KF-1 cell or CCB cell sheet of atleast 3 cm2, cultured at 25ºC for seven days, tested with anti-Koi herpesvirus serum by fluorescentantibody assay, and then observed.
3.2.11.2.2 Judgment
No specific fluorescent antigens are observed in the cultured cells: the test material shallcomply with the test.
144

Target Animal Immunogenicity Test
The Target Animal Immunogenicity Test is a method to verify that master seeds of live vaccinesretain the immunogenicity to the animals targeted for vaccination even through passages.
1 Test materials
1.1 Test article
For live virus vaccines, use master seed virus obtained by 5 passages as the test article. For live
bacterial vaccines, use master seed bacteria obtained by 10 passages as the test article. For live
coccidia vaccines, use master seed coccidia obtained by 10 passages as the test article. However,this number of passages does not apply if approved by the Minister of Agriculture, Forestry and
Fisheries.
1.2 Test animals
Animals used for the test shall be known for their breeds and strains (SPF, etc.), healthy andeligible for vaccination, and suitable for immunogenicity evaluation.
2 Test procedure
2.1 Setting of test groups (including the control group)
The animals shall be assigned to two test groups (at least three animals per group for mammals
and at least 10 birds per group for chickens) and one control group.
2.2 Administration method
Administration shall be given in the same manner as applied for clinical use. In principle,
administration shall be given up to once, but if unable to evaluate the immunogenicity by a single
administration, administration can be given at appropriate intervals as frequent as it should be for
evaluation.
2.3 Dose
Administration shall be given at a dose estimated to be a single dose applied for clinical use.
The level of antigen administered shall be the same between individual animals in the test groups.
2.4 Evaluation method
Using approved methods (methods that can verify protection against infection by antibody
response, cellular immunity, or challenge test etc.), evaluate the immunogenicity of passaged
master seed in an objective manner.
3 Judgment
The immunogenicity of passaged master seed shall be tested in an appropriate manner.

145

Target Animal Safety Test
The Target Animal Safety Test is a method to examine the absence of pathogenic factors against
the animals targeted for vaccination in the master seeds of live vaccines.
1 Test materials
1.1 Test article
Use master seed as the test article. If a sufficient quantity of master seeds for the test is notavailable, use the working seeds whose number of passages is the lowest as the test article.
1.2 Test animals
1.2.1 Master seeds of live vaccines for mammals
Use at least four healthy target animals aged days at which the clinical symptoms related to thesafety evaluation are most likely to occur within the scheduled vaccination days old.
1.2.2 Master seeds of live vaccines for birds
Use at least 20 healthy chickens aged days at which the clinical symptoms related to the safety
evaluation are most likely to occur within the scheduled vaccination days old.
2 Test procedure
2.1 Administration
2.1.1 Test on master seeds of live vaccine targeted mammals
Test animals shall be divided into two groups, each with at least two animals. One group shallbe given the test article prepared from the master seeds via the planned route of administration.
The other group shall not be given the test article as the control group.
2.1.2 Test on master seeds of live vaccine targeted birds
Test animals shall be divided into two groups, each with at least ten animals. One group shall begiven the test article prepared from the master seeds via the planned route of administration. The
other group shall not be given the test article as the control group.
2.2 Matters to be observed
Animals shall be observed for clinical symptoms for 21 days after administration.
3 Judgment
No clinical abnormalities shall be observed during the observation period.

146

Test for Absence of Reversion to Virulence
The Test for Absence of Reversion to Virulence is a method to ensure that the virulence of thestrain used for live vaccine production is not reversed or enhanced in the animals targeted for
inoculation.
1 Test materials
1.1 Test article
The master seeds for live vaccine production shall be used as the test article. The test article
shall be adjusted to include the maximum amount of the strain in the shipment. However, if a
sufficient quantity of master seeds is not available for the test, use the working seeds whose
number of passages is the lowest as the test article. In this case, the test article shall also be
adjusted to include the maximum amount of the strain in the shipment.
1.2 Passage material
From the test animals receiving the test article, sample the parts inbody and etc. at whichmicroorganisms derived from the strain used for production are frequently isolated, and prepare
the passage materials. For each passage, microorganisms derived from the strain used for
production shall be quantified using a suitable method. Passage materials shall not be grown orcultured in the laboratory.
1.3 Test animals
Target healthy animals suitable for the test shall be used as the test animals.
2 Test procedure
2.1 Administration route
Administer the test article to at least two test animals(birds) via the route planned in theapplication or natural infection route as appropriate to investigate reversion or enhancement ofvirulence. The route of administration planned in the application shall be appropriate forinvestigating the reversion to or enhancement of virulence.
2.2 Material sampling
Based on the properties of the test article, sample the passage materials from the test animals at
an appropriate time.
2.3 Passage
From the initial inoculation with the test materials up to the 4th-passaged materials, at least two
test animals(birds) each shall be used. For the final 5th-passaged material, at least eight test
animals(birds) shall be used.
If the microorganisms derived from the strain used for production are not recovered during the147

course of passage, use the last passage material recovered to repeat passage with 10 animals(birds). During the repeated passage, if microorganisms derived from the virus strain used for
production are recovered from at least one animal(bird), the passage shall be continued using thematerials recovered during the repeated passage as the materials for the next passage. Even if thepassage is repeated, the number of passages in the test shall be regarded as once.
2.4 Test period
Test animals used from initial inoculation up to the 4th-passaged materials shall be observed
within 21 days, and those inoculated with up to the 5th-passaged materials shall be observed for
21 days, respectively.
2.5 Observation
During the test period, the test animals shall be clinically observed for the occurrence of clinicalsymptoms of the target diseases associated with the reversion to or enhancement of virulence.
2.6 Comparative test
During the observation period, if clinical symptoms associated with the reversion to or
enhancement of virulence are identified in the test animals used for inoculated with the 5thpassaged materials during their observation period, the test article and passage materials used forthe final passage shall be adjusted to the maximum amount of the strain in the shipment andinoculated to at least eight test animals (birds) per group to compare the clinical symptoms
associated with the reversion to or enhancement of virulence.
3 Judgment
In all the test animals inoculated with up to the 4th-passaged material, no clinical symptoms
associated with the reversion to or enhancement of virulence shall be observed during theobservation period. Also, in the comparative test, no differences in clinical symptoms associatedwith the reversion to or enhancement of virulence shall be identified between the animals
inoculated with the test article and those inoculated with the final passage material.

148

Test for Stability Confirmation of Recombinant Gene
The Test for Stability Confirmation of Recombinant Gene is a method to confirm that the masterseeds produced by genetic recombination technology have the stability of the modified nucleic acids
and properties during the passages.
1 Test materials
1.1 Test article
For genetically modified viruses, master seed viruses and their 5th-passaged viruses shall be
used as the test article. For genetically modified bacteria, master seed bacteria and their 10th passaged bacteria shall be used as the test article. However, if a sufficient quantity of master seeds
is not available for the test, use the working seeds whose number of passages is the lowest as thetest article unless otherwise approved by the Minister of Agriculture, Forestry and Fisheries.
1.2 Cultured cells or medium
Cultured cells or medium used as materials for production of vaccine seeds shall be used.
2 Test procedure
2.1 Test for gene stability confirmation
The nucleic acids at the modified sites in the master seeds and their passaged ones shall becompared for their states and sequences using an approved test method.
2.2 Test for property stability confirmation
The properties obtained by modification of nucleic acids in the master seeds and their passaged
ones shall be compared by an approved test method and evaluated objectively.
3 Judgment
3.1 Test for gene stability confirmation
Compare the master seeds and their passaged ones and there are no differences in the states ofnucleic acids at the modified sites and sequences: the test article shall comply with the test.
3.2 Test for property stability confirmation
Compare the master seeds and their passaged ones and there are no changes in their properties:
the test article shall comply with the test.

149

Reagents, Test Solutions, Etc.
Reagents, test solutions, etc. are used for the tests in this standard. In addition to those specified
herein, reagents and test solutions shall proceed as directed in Reagents, Test Solutions, etc.
specified in the General Tests, Processes and Apparatus in the Japanese Pharmacopoeia. In thisstandard, the reagents noted with [JP], [JP monograph], [Special grade], or [1st grade] represent thatthey conform to the Reagents, Test solutions specified in the General Tests, Processes and Apparatus
in the Japanese Pharmacopoeia, to the pharmaceuticals specified in the Official Monographs in thesame, and to the reagents noted with the Special grade or 1st grade in the Japanese Industrial
Standards. The reagent is marked with an asterisk (*) in the name of the reagent shall meet thesuitable quality for their intended uses.

150

A
Acetic acid (100) [Glacial acetic acid, Special grade]
Acetone [Special grade]
Acetylacetone [Special grade]
Acriflavine*
0.2 w/v% acriflavine
Dissolve 2.0 g of acriflavine in water to make 1,000 mL and sterilize at 121 ℃ for 15 minutes.
Agar [Agar, JP monograph]
Agar (for sterility test)
An agar that conforms to the following specifications shall be used:(1) Nitrogen content: not more than 0.5%.
(2) When conducting a test by the method specified in the Japanese Industrial Standards, thejelly strength at a concentration of 1.5% is 300 to 500 g/cm2.
Agar medium containing 0.02 w/v% acriflavine
Add acriflavine to nutrient agar medium so that it is contained at a rate of 0.02 w/v%.
Agar medium containing 10 vol% horse serum
Add horse serum in nutrient agar medium so that it is contained at a concentration of 10 vol%.
Aluminum (III) chloride hexahydrate [Aluminum chloride, Special grade]
4-Aminobenzoic acid [p-Aminobenzoic acid, Special grade]
2-Amino-2-hydroxymethyl-1,3-propanediol [tris (hydroxymethyl) aminomethane, Special grade]Ammonia water (28) [Ammonia water, Special grade]Ammonium acetate [Special grade]Ammonium thiocyanate [Special grade]
Anhydrous calcium chloride

See calcium chloride, anhydrous.

Anthrone [Special grade]
L-arginine monohydrochloride [Special grade]
B
Bacto peptone*
Barbital [JP monograph]
Barbital sodium salt [JP]Barium chloride [Special grade]
Benzylpenicillin potassium [JP monograph]
N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid*
Boric acid [JP monograph]
Bovine serum albumin
151

Bovine serum albumin is a pale-yellow or pale-brown powder purified from bovine serum by amethod that does not affect albumin and other plasma proteins, and it conforms to the following
specifications:(1) The 10 w/v% solution is clear, and its pH ranges from 5.0 to 5.5.
(2) When conducting a test using electrophoresis, albumin must be not less than 97% of total
protein.
5.5 w/v% bovine serum albumin solution
Dissolve 5.5 g of bovine serum albumin in water to make 100 mL.
Bromocresol green [Special grade]C
Calcium chloride, anhydrous [Special grade]
Calcium chloride dihydrate [Calcium chloride, Special grade]
3 w/v% calcium chloride solution
Dissolve 3.0 g of calcium chloride dihydrate in water to make 100 mL and sterilize at 121℃
for 15 minutes.
Carbon tetrachloride [Special grade]
Casein peptone
Description:

Casein peptone is a grayish yellow powder. It has a characteristic odor but has
no putrefactive odor. It is soluble in water but insoluble in ethanol (95) or
diethyl ether.

Digestibility:

Dissolve 1 g of casein peptone in 10 mL of water and use as the sample
solution for conducting the following tests:(1) On 1 mL of sample solution, superimpose 0.5 mL of solution prepared by adding 1 mL ofacetic acid (100) to 10 mL of dilute ethanol: no ring or precipitate is produced at the zone ofcontact. When mixing this solution, no turbidity is produced.
(2) To 1 mL of sample solution add 4 mL of saturated zinc sulfate solution: a small amount ofprecipitate is produced.
(3) Filter the mixture prepared in (2). To 1 mL of the filtrate add 3 mL of water and 0.2 mL ofbromine test solution: a red-violet color develops.
Casein peptone (for sterility test)
In addition to its specifications, casein peptone used conform to the following specifications:(1) The 2 w/v% solution is pale yellow clear, and its pH ranges from 6.5 to 7.0.
(2) When measured by the Van Slyke method and Kjeldahl method, amino nitrogen is 25% to
50% of total nitrogen.
(3) When measured by the microbioassay or other assays, the content of tryptophan is not less152

than 1.5%.
(4) Prepare the following five types of media A–E, adjust the pH to 7.2 to 7.4, and conduct thefollowing tests a–e: the media conform to the specifications:
MediumSampleSodium chloride

Others

Water

A

2.0 g

0.5 g

–

100 mL

B

1.0 g

0.5 g

–

100 mL

C

0.1 g

0.5 g

–

100 mL

D

1.0 g

0.5 g

Glucose 0.5 g

100 mL

E

2.0 g

0.5 g

Agar 1.5 g

100 mLaFermentable carbohydrates test
To 5 mL of Medium A, add an appropriate amount of phenol red test solution, put aDurham tube, inoculate a loopful of Escherichia coli incubated at approximately 36 ºC for
24 hours, and incubate it at 35ºC to 37°C for 48 hours: the bacteria grow well, and no acidor gas is produced.

b

Hydrogen sulfide production test
To 5 mL of Medium B, inoculate Salmonella typhi, place a piece of lead acetate paper
between the cotton stopper and the mouth of the test tube at a height of about 5 cm above
the medium surface, and incubate at 35ºC to 37°C for 24 hours: the bacteria grow well,and when incubated for 48 hours, the piece of paper shows a marked dark brown color.

cIndole production test
Inoculate Escherichia coli in 5 mL of Medium C at 35ºC to 37°C for 24 hours:
Escherichia coli grows well; layer 0.5 mL of Indole test solution 0.5 mL: a red-violet color
develops clearly at the zone of contact.

dAcetyl methyl carbinol production test
Inoculate Klebsiella pneumoniae in 5 mL of Medium D at 35ºC to 37 ºC for 24 hours:
Klebsiella pneumoniae grows well; add 5 mL of 10 w/v% sodium hydroxide solution,
shake, and allow to stand at approximately 25 ºC for 5 hours: a pale-red color develops.

eBacterial growth-promoting ability test(a) Puncture Brucella melitensis in a stab medium of Medium E at 35ºC to 37 ºC for 48
hours: bacteria grow along the puncture line.
(b) Inoculate Escherichia coli, Klebsiella pneumoniae, Salmonella Typhi, Pseudomonas
aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis in a slant mediumof Medium E at 35ºC to 37 ºC for 24 hours: all bacteria grow well.
(c) To Medium E, add defibrinated blood from a rabbit so that it is contained at aconcentration of approximately 5 vol%, inoculate Streptococcus pneumoniae or βhemolytic Streptococcus pneumoniae, mix well, fix on a plate, and incubate at 35ºC to153

37 ºC for 48 hours: both bacteria grow well, showing a characteristic α or β- hemolyticzone.
(d) To Medium E, add defibrinated bovine blood so that it contained at a concentration of
approximately 10 vol%, fix on a plate, heat at 80ºC to 90 ºC until the medium turns tochocolate brown in color, grow Neisseria gonorrhoeae, and incubate gas at 35ºC to 37ºC for 48 hours: the bacteria grow well when incubated in an incubator maintaining
10% carbon dioxide.
Chicken water*
Chloroform [Special grade]
Copper (II) sulfate pentahydrate [Copper sulfate, Special grade]
L-cystine [Special grade]
L-cysteine hydrochloride monohydrate [Special grade]
1 w/v% L-cysteine monohydrochloride test solution
Dissolve 1.0 g of L-cysteine hydrochloride monohydrate in water to make 100 mL.
D
Dibasic sodium phosphate anhydrous

See sodium dihydrogen phosphate, anhydrous.

Dipotassium hydrogen phosphate [Dipotassium hydrogen phosphate, Special grade]
Disodium hydrogen phosphate dodecahydrate [Disodium hydrogen phosphate 12-water, Special
grade]
Dithizone [Special grade]E
Eagle’s MEM
Dissolve a dried product of suitable quality according to the direction and sterilize.
Earle's solution
Calcium chloride dihydrate

0.2 g

Potassium chloride

0.4 g

Magnesium sulfate heptahydrate

0.2 g

Sodium chloride

6.8 g

Sodium hydrogen carbonate

2.2 g

Sodium dihydrogen phosphate dihydrate

0.163 gGlucose

1.0 g

Phenol red

0.05 g

Dissolve all the above-listed ingredients in water to make 1,000 mL, and filter sterilize.
A dried product of suitable quality may be used if it is prepared by dissolving according to the154direction and sterilized.
Ethanol (95) [ethyl alcohol, Special grade]
Ethanol (99.5) [Anhydrous ethanol, Special grade]
90 vol% ethanol
Add water to 180 mL of ethanol (99.5) to make 200 mL.
F
Formalin [JP monograph]
F10 medium
Dissolve the dried F10 medium of suitable quality according to the direction and sterilize.
G
Gelatin [JP monograph]
Glacial acetic acidSee acetic acid (100).

Glucosamine*
Glucose [JP monograph]
Glycerin [Special grade]H
Hank’s solution
Sodium chloride

8.0 g

Potassium chloride

0.4 g

Disodium hydrogen phosphate dodecahydrate

0.06 gGlucose

1.0 g

Magnesium chloride hexahydrate

0.2 g

Calcium chloride dihydrate

0.14 gSodium hydrogen carbonate

0.35 gPhenol red

0.02 g

Dissolve all the above-listed ingredients in water to make 1,000 mL, filter sterilize.
A dried product of suitable quality may be used if it is prepared by dissolving according to
the direction and sterilized.
Heart infusion brothHydrochloric acid [Special grade]
Hydrogen peroxide (30) [Hydrogen peroxide solution, Special grade]

155

I
Indole test solution*
K
Kaolin [JP monograph]
25 w/v% kaolin solution
Suspend 25 g of kaolin in water to make 100 mL.
L
Lactalbumin hydrolysate*
Lactose*Lactose agar*
Liquid paraffin [Light liquid paraffin, JP monograph]
M
Macrogol 4,000 [JP monograph]
Macrogol 6,000 [JP monograph]
Magnesium chloride hexahydrate [Magnesium chloride, Special grade]
Magnesium sulfate heptahydrate [Magnesium sulfate, Special grade]
Malachite green oxalate [Malachite green (oxalate), Special grade]
2 w/v% malachite green solution
Dissolve 2.0 g of malachite green oxalate in water to make 100 mL.
Meat extract broth*
Meat peptone*
Meat water*
Methyl red [Special grade]
Methyl red test solution
Dissolve 0.1 g of methyl red in ethanol (95), and filter if necessary.
Monosodium glutamate*
Mucin*N
Neutral red [Special grade]
1 w/v% β-Nicotinamide-adenine dinucleotide [oxidized form] solution*
Nitric acid [Special grade]
4-Nitroaniline [p-nitroaniline, 1st grade]156

Nutrient agar medium
Dissolve 15 g of agar in 1,000 mL of nutrient broth with the aid of heat, add water to make up
for the loss, adjust the pH to between 6.4 and 7.0, and filter. Dispense the filtrate and sterilize
by autoclaving at 121℃ for 15 minutes.
Nutrient broth
Dissolve 5 g of meat extract and 10 g of peptone in 1,000 mL of water by gentle heating. Adjust
the pH of the mixture between 6.4 and 7.0 after sterilization, cool, add water to make up for theloss, and filter. Sterilize the filtrate by autoclaving for 30 minutes at 121℃.
Nutrient broth containing 10 vol% horse serum
Add horse serum in nutrient broth so that it is contained at a concentration of 10 vol%.
P
Peptone*
Peptone for toxins*
Peroxidase-conjugated anti-mouse immunoglobulin*
Phenol [Special grade]
Phenol red [Special grade]
0.2 w/v% phenol red solution
Dissolve 2.0 g of phenol red in water to make 1,000 mL.
Phosphate-buffered saline (pH 7.4–7.45)
Dissolve 8.0 g of sodium chloride, 0.2 g of potassium chloride, 1.15 g of dibasic sodium
phosphate anhydrous, and 0.2 g of potassium dihydrogen phosphate in water to make 1,000 mL
and sterilize.
1/60 mol/L phosphate-buffered saline (pH 7.0)
Dissolve 14.45 g of dibasic sodium phosphate anhydrous, 8.83 g of potassium dihydrogen
phosphate, and 85.0 g of sodium chloride in water to make 10,000 mL and sterilize.
Phosphate-buffered saline containing 2 vol% horse serum
Add horse serum in phosphate-buffered saline so that it is contained at a concentration of 2
vol%.
1/60 mol/L phosphate-buffered saline containing 0.2 w/v% gelatin (pH 7.0)
Add gelatin in 1/60 mol/L phosphate-buffered saline so that it is contained at a concentration of
0.2 w/v%, heat until dissolved and sterilize.
Phosphate-buffered saline containing 0.5 w/v% phenol (pH 7.0)
1.45 g of potassium dihydrogen phosphate, 15.28 g of disodium hydrogen phosphate
dodecahydrate, 4.8 g of sodium chloride, and 5.0 g of phenol in water to make 1,000 mL, andfilter sterilize.
157

Phosphomolybdic acid n-hydrate [Phosphomolybdic acid, Special grade]
Phosphorus (V) oxide [Phosphorus (V) oxide, Special grade]
Physiological saline [JP monograph]
Physiological saline containing 0.3 vol% formalin
Add 3.0 mL of formalin to physiological saline to make 1,000 mL.
Physiological saline containing 50 vol% glycerin
Add 50 mL of physiological saline in 50 mL of glycerin.
Physiological saline containing 2 vol% horse serum
Add horse serum in physiological saline so that it is contained at a concentration of 2 vol%.
Physiological saline containing 0.1 w/v% magnesium sulfate
Dissolve 1.0 g of magnesium sulfate heptahydrate in physiological saline to make 1,000 mL
and sterilize.
Physiological saline containing 0.5 w/v% phenol
Dissolve 5.0 g of phenol in physiological saline to make 1,000 mL.
Polysorbate 80 [JP monograph]
Potassium chloride [Special grade]
Potassium dihydrogen phosphate [Potassium dihydrogen phosphate, Special grade]
Potassium periodate [Potassium periodate, Special grade]
Potassium sulfate [JP monograph]
Potato extract*
R
RDE*
Resazurin [JP]0.1 w/v% resazurin solution
Dissolve 1.0 g of resazurin in water to make 1,000 mL.
S
Silica gel [JP]Skimmed milk powder*
Sodium acetate trihydrate [Sodium acetate (trihydrate), Special grade]
Sodium azide
Description:

Sodium azide is white or practically white crystals. It is easily soluble inwater, and practically insoluble in diethyl ether. The solution in water isalkaline.

Qualitative test:

When treated with iron (III) nitrate test solution, sodium azide solution turns
158to reddish brown in color. When sodium azide is heated in a colorless flame,
the flame turns yellow in color.
Content:

Not less than 97.0%

10 w/v% sodium azide solution
Dissolve 10 g of sodium azide in water to make 100 mL.
Sodium carbonate, anhydrous [Anhydrous sodium carbonate, Special grade]
Sodium carbonate decahydrate [Sodium carbonate, Special grade]
Sodium carboxymethyl cellulose [JP monograph]
Sodium chloride [Special grade]
Sodium dihydrogen phosphate, anhydrous [Disodium hydrogen phosphate anhydrous, Special grade]
Sodium dihydrogen phosphate dihydrate [Sodium dihydrogen phosphate, Special grade]
Sodium hydrogen carbonate [Special grade]
Sodium hydroxide [Special grade]
Sodium nitrite [Special grade]
Sodium thioglycolate [Special grade]
Soybean-casein digest agar
Dissolve a dried soybean-casein digest agar of suitable quality according to the direction andsterilize.
Soy peptone*
Stilbazo(1) A black-brown powder.
(2) Shows yellow at pH 3–7, orange at pH 9 and red at pH 11 when dissolved in water. No
insoluble matter is produced when 50 mg of stilbazo is dissolved in 100 mL of water.
(3) When igniting 0.5 g of sodium hydroxide with 1 mL of stilbazo and weighing, the weight of
the residue shall not exceed 10 mg.
(4) The absorbance of 0.002 w/v% solution of stilbazo at 410 nm must be not less than 0.7.
(5) Add water to 5 mL of 0.05 w/v% solution of stilbazo, 10 mL of 0.0001 mol/L aluminum
chloride and 10 mL of 1 mol/L of acetate buffer solution to make 100 mL. The absorbanceof the diluted solution at wavelength 505 nm and optical path length 10 mm is higher by
0.42 or more than that of stilbazo alone.
Sulfuric acid [Special grade]T
Thallium acetate*
Thiamine chloride [JP monograph]
Thimerosal C9H9HgNaO2S
159

Description:

Thimerosal is a white or pale-yellow crystalline powder, having a slightcharacteristic odor. The pH of the solution at a concentration of 1.0 g/100 mL
ranges from 6.0 to 7.0.

Purity(1) Clarity and color of solution: Colorless and clear (solution at a concentration of 1.0 g/10
mL)(2) Diethyl ether-soluble substances: Weigh precisely about 0.5 g of powdered thimerosal, put
in a 50-mL glass-stoppered conical flask, add 20 mL of anhydrous diethyl ether, stopper,
shake for 10 minutes, filter in a weight-known beaker through filter paper previously
washed with diethyl ether, wash the residues with 5 mL of anhydrous diethyl ether, combine
the filtrate and washings, evaporate the combined solution on a water bath, and dry in a
desiccator (in vacuum, silica gel) for 24 hours: the mass of the residue is not more than
0.60%.
(3) Other soluble mercury salt: Dissolve 0.10 g of thimerosal in 10 mL of water, add 3 drops ofacetic acid (31) in 5 mL of this solution: a white precipitate is produced. To this solution,
add 1 drop of sodium sulfide, and allow to stand for 10 minutes: no dark color develops inthe solution.
(4) Readily carbonizable substances: Take 0.20 g of thiomersal and conduct the test as specified
the General Tests, Processes and Apparatus in the Japanese Pharmacopoeia. The solution
has no more color than that of J. the matching fluid for color. The test must be conducted ata standard temperature.
Loss on drying: Not more than 0.5% (1 g, in vacuum, silica gel, 5 hours)Content:

Not less than 98.0%

Assay:

Weigh precisely about 0.3 g of previously dried thimerosal for the assay, put ina 300 mL Kjeldahl flask, add 10 mL of sulfuric acid and 4 mL of fuming nitricacid, heat gently on a sand bath, and gradually increase the heat until thecontents in the flask become almost colorless and white smoke is produced.
After cooling, transfer the contents with 100 mL of water to a beaker and heaton a water bath for 15 minutes while occasionally shaking. Then add 0.5 g ofurea, shake, and add potassium permanganate test solution dropwise until aslight pale red color develops. After cooling, add hydrogen peroxide test
solution dropwise until the red color of the solution disappears, and titrate with
0.1 mol/L ammonium thiocyanate solution (indicator: 2 mL of ammonium iron(III) sulfate test solution).
1 mL of 0.1 mol/L ammonium thiocyanate solution = 20.240 mg
C9H9HgNaO2S
160

Storage:

Preserve in a light-resistant tight container.

Thiourea [Special grade]Tin (II) chloride dihydrate [Stannous chloride, Special grade]
Trichloroacetic acid [trichloroacetic acid, Special grade]
Tris-HCl buffer solution
Dissolve 20.5 g of 2-Amino-2-hydroxymethyl-1,3-propanediol, 13.6 g of sodium acetate
trihydrate, 2.2 g of calcium chloride dihydrate and 0.68 g of zinc chloride in 700 mL of water,
add hydrochloric acid, and adjust the pH to 7.4 to make 1,000 mL in total.
Tris(hydroxymethyl)aminomethaneSee 2-Amino-2-hydroxymethyl-1, 3-propanediol.

Trypsin*
Tryptose phosphate broth*V
Veronal-buffered saline (pH 7.2)
Add 85.0 g of sodium chloride, 5.75 g of barbital and 3.75 g of barbital sodium salt to water tomake 1,000 mL, and filter sterilize.
Veronal-buffered saline containing 1 w/v% gelatin (pH 7.5)
Add gelatin in veronal-buffered saline so that it is contained at a concentration of 1 w/v%, heat
until dissolved, and sterilize.
Y
Yeast extract
A peptone-like substance which represents all the soluble product of yeast cells(Saccharomyces) prepared under optimum conditions, clarified, and dried by evaporating to a
powder. Yeast extract (1 g) represents not less than 7.5 g of yeast. A reddish yellow to brown
powder with a characteristic odor. Soluble in water and forms a yellow to brown solution.
(1) The nitrogen content of yeast extract is 7.2% to 9.0%.
(2) The loss on drying at 105℃ at constant mass of yeast extract is not more than 5%.
(3) The residue of yeast extract when ignited is not more than 15%.
(4) Heat 5 w/v% yeast extract solution until boiling: no precipitate is produced.
(5) Chloride (as sodium chloride): not more than 5%.
Z
Zinc chloride [Special grade]161

Specifications

163

Materials for Live Vaccine Production
Embryonated eggs, cultured cells, and bovine serum used as materials for live vaccine production
shall comply with the following specifications. However, embryonated eggs and cultured cells used
for seed lot products shall comply with the Seed Lot Specifications.
1 Embryonated eggs
1.1 Embryonated chicken eggs
Embryonated chicken eggs used for live vaccine production must be derived from SPF chicken
flocks that have been confirmed to be free from the pathogens listed in Table 1 by the inspection
and treatment listed in the same table, or by the inspections and treatment approved as equivalent
thereof.
1.2 Embryonated quail eggs
Embryonated quail eggs used for live vaccine production must be derived from SPF quail
flocks that have been confirmed to be free from the pathogens listed in Table 2 by the inspection
and treatment listed in the same table, or by the inspections and treatment approved as equivalent
thereof.
1.3 Embryonated duck eggs
Embryonated duck eggs used for live vaccine production must be derived from SPF duck flocks
that have been confirmed to be free from the pathogens listed in Table 3 by the inspection andtreatment listed in the same table, or by the inspections and treatment approved as equivalent
thereof.
2 Cultured cells
2.1 Cells derived from chicken embryos
2.1.1 Primary cultured cells of chicken embryos
Primary cultured cells of chicken embryos (chicken embryo fibroblasts) used for live vaccine
production must be prepared from a chicken embryo derived from embryonated chicken eggs
compliant with the specification given in 1.1.
2.1.2 Primary cultured cells of chicken embryo kidneys
Primary cultured cells of chicken embryo kidneys used for live vaccine production must be
prepared from a chicken embryo kidney derived from embryonated chicken eggs compliant withthe specification given in 1.1.
2.1.3 Primary cultured cells of chicken embryo livers
Primary cultured cells of chicken embryo livers used for live vaccine production must be
prepared from a chicken embryo liver derived from embryonated chicken eggs compliant with the165specification given in 1.1.
2.2 Cells derived from chickens
2.2.1 Primary cultured cells of chicken kidneys
Primary cultured cells of chicken kidneys used for live vaccine production must be preparedfrom a chicken kidney derived from embryonated chicken eggs compliant with the specification
given in 1.1.
2.3 Cells derived from quail embryos
2.3.1 Primary cultured cells of quail embryos
Primary cultured cells of quail embryos (quail embryo fibroblasts) used for live vaccine
production must be prepared from a quail embryo derived from embryonated quail eggs compliant
with the specification given in 1.2.
2.4 Cells derived from duck embryos
2.4.1 Primary cultured cells of duck embryos
Primary cultured cells of duck embryos (duck embryo fibroblasts) used for live vaccine
production must be prepared from a duck embryo derived from embryonated duck eggs compliant
with the specification given in 1.3.
2.5 Cells derived from ducks
2.5.1 Primary cultured cells of duck kidneys
Primary cultured cells of duck kidneys used for live vaccine production must be prepared froma duck kidney derived from embryonated duck eggs compliant with the specification given in 1.3.
2.6 Cells derived from swine
2.6.1 Primary cultured cells of swine kidneys
Primary cultured cells of swine kidneys used for live vaccine production must be prepared froma lesion-free kidney removed from swine kept under health care for more than seven days prior toslaughter and have no abnormal findings including fever.
2.6.2 Primary cultured cells of swine testis
Primary cultured cells of swine testis used for live vaccine production must be prepared from alesion-free testis removed from swine kept under health care for more than seven days prior to
removal and have no abnormal findings including fever.
2.7 Cells derived from bovine
2.7.1 Primary cultured cells of bovine kidneys
Primary cultured cells of bovine kidneys used for live vaccine production must be preparedfrom a lesion-free kidney removed from bovine kept under health care for more than seven days
prior to slaughter and have no abnormal findings including fever.
2.7.2 Primary cultured cells of bovine testis
Primary cultured cells of bovine testis used for live vaccine production must be prepared from a166lesion-free testis removed from bovine kept under health care for more than seven days prior toslaughter and have no abnormal findings including fever.
3 Bovine serum
Bovine serum used for live vaccine production shall be separated from fresh blood of healthy
bovine or bovine fetus, sterilized by filtration, dispensed, and heat-inactivated. When this is usedas the test material and tested as specified in the Sterility Test and Test for Freedom from
Mycoplasma Contamination of the General Tests and in 2.4.1 and 2.4.2 in the Test for Freedom
from Extraneous Viruses for Live Vaccines and Sera of the General Tests, this test material shallcomply with these tests. Bovine serum used for materials for production of live vaccines for cattle
also shall comply with 2.8.1 in the Test for Freedom from Extraneous Viruses for Live Vaccines
and Sera of the General Tests when tested as specified.

167

Table 1 Inspection and Treatment of SPF Chicken Flocks

Antigen underinspection1

Inspection timing and number ofchickens inspected
1st
2nd and subsequent
Inspection
NumberNumbermethod2ofof
TimingTimingchickens
chickens(%)(%)

Newcastle disease virus

Ishii strain

8–12
weeks
old

20

Every 3months

10

HI

Avian infectious bronchitis virus
Avian leukosis virus

M-41 strain
Sub-A, B
Van Roekel
strainG-4260 strain
NS-175 strain
T strain
JM strain
J1 strain
Uchida strain
Ote strain
JPA-1 strain
5331 strain
Gifu-1 strain
MM-1 strain
Yucaipa strain

〃
〃

〃
〃

〃
〃

〃
〃

ELISA
SN

All the chickens positive
to the inspection andcohabitant chickens3
sacrificed
〃
〃

〃

〃

〃

〃

ELISA

〃

〃
〃
〃
〃
〃
〃
〃
〃
〃
〃
〃
〃

〃
〃
〃
〃
〃
〃
〃
〃
〃
〃
〃
〃

〃
〃
〃
〃
〃
〃
〃
〃
〃
〃
〃
〃

〃
〃
〃
〃
〃
〃
〃
〃
〃
〃
〃
〃

FA
ELISA
FA
FA
ELISA
DID
DID
HI
DID
FA
FA
HI

〃
〃
〃
〃
〃
〃
〃
〃
〃
〃
〃
〃

〃

〃

〃

〃

HI

〃

〃

〃

〃

〃

HI

〃

〃
〃

〃
〃

〃
〃

〃
〃

AGG
AGG

〃
〃

〃

〃

〃

〃

AGG

〃

〃

〃

〃

〃

Everyday

100

Everyday

100

Pathogen

Avian encephalomyelitis virus

Avian nephritis virus
Infectious laryngotracheitis virus
Reticuloendotheliosis virus
Marek's disease virus
Infectious bursal disease virus
Avian reovirus
Avian adenovirus
EDS-76 virus
Avian influenza virus
Chicken anemia virus
Turkey rhinotracheitis virus
Avian paramyxovirus
Haemophilus paragallinarum type221 strain
A
Haemophilus paragallinarum typeS1 strain
C
Salmonella pullorum
9-25 strain
Mycoplasma gallisepticumS6 strain
WVU-1853
Mycoplasma synoviae
strainSalmonella (except for Salmonella
pullorum)
Chickenpox virus

Note

Treatment

Bacterial〃
isolation
Clinical
Chickens positive to theobservation inspection sacrificed

Health conditions and abnormalities of the chickens shall be completely recorded. For deadchickens, histopathological examinations shall be performed.

1) For antigens under inspection, other appropriate strains may be used.
2) Other similar inspection methods may be used, if any. The inspections shall be performed by amethod whose validity has been verified and guaranteed. HI: Hemagglutination test
Enzyme-linked immunosorbent assay
antibody test

SN: Serum neutralization test

DID: Double immunodiffusion test

ELISA:

FA: Fluorescence

AGG: Agglutination test3) Cohabitant chickens refer to the chickens not completely isolated from those positive to theinspection.

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Seed Lot Specifications
1 Vaccine seeds
1.1 Range of passage numbers
To obtain the final product, the master seed virus shall not be passaged more than 5 times, themaster seed bacteria shall not be passaged more than 10 times, the master seed coccidia shall not
be passaged more than 10 times, unless otherwise approved by the Minister of Agriculture,
Forestry and Fisheries.
1.2 Preparation method
Master seeds shall be prepared in accordance with the approved method in consecutive process
to ensure their homogeneity and stability and prevent contamination.
Master seed virus shall be dispensed at a virus concentration at which it can be adequately
neutralized by antiserum in the Test for Freedom from Extraneous Viruses.
1.3 Storage
Master seeds shall be stored under the approved conditions.
1.4 Origin of seeds and specifications and test methods
1.4.1 Records on origin
1.4.1.1 Origin
The following information on the origin of seeds shall be recorded: Method of isolation, placeof isolation, timing of isolation, animal species of origin, and characteristics of isolates of viruses,bacteria, etc. isolated from animal species of origin.
If the master seed was distributed (or purchased), from whom it was distributed (or from whom
it was purchased) and the time of distribution (or purchase) shall also be recorded.
If the seed was produced by the genetic recombination technology, the characteristics of thegenetically modified microorganism (description of the host or the taxonomic species to which thehost belongs, description of the donor nucleic acid, description of the vector, method of
conditioning genetically modified microorganisms, method of identifying the genetically modified
microorganism, differences from the host or the taxonomic species to which the host belongs, etc.)
shall be recorded.
1.4.1.2 Passage history
With regard to passages after the isolation, the animals used, cultured cells, and media, cloning
and attenuation methods shall be recorded. If the master seed virus has been distributed, its
passage history before and after the distribution shall also be recorded.
1.4.2 Specifications and test methods
1.4.2.1 Live virus vaccines1691.4.2.1.1 Master seed virus
1.4.2.1.1.1 Identification test
The test shall be performed as specified in 1.4.2.1.1.1.1 or 1.4.2.1.1.1.2.
1.4.2.1.1.1.1 Fluorescent antibody assay
When the cultured cells inoculated with and without the test article are stained with afluorescence-labeled antibody, respectively, fluorescence characteristic of the virus shall be
detected in the cells inoculated with the test article but shall not be detected in those inoculated
without it.
1.4.2.1.1.1.2 Serum neutralization test
During the growth of the test article using appropriate cultured cells, virus-specificcytopathogenic changes shall be detected, and the growth shall be neutralized by the specificantiserum.
1.4.2.1.1.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
1.4.2.1.1.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
1.4.2.1.1.4 Test for freedom from extraneous viruses
The tests given in the Test for Freedom from Extraneous Viruses of the General Tests shallapply.
1.4.2.1.1.5 Target animal immunogenicity test
The test given in the Target Animal Immunogenicity Test of the General Tests shall apply.
1.4.2.1.1.6 Target animal safety test
The test given in the Target Animal Safety Test of the General Tests shall apply.
1.4.2.1.1.7 Test for absence of reversion to virulence
The test given in the Test for Absence of Reversion to Virulence of the General Tests shallapply.
1.4.2.1.1.8 Test for stability confirmation of recombinant geneIf gene recombination technology is used for preparation of the master seed virus, the test givenin the Test for Stability Confirmation of Recombinant Gene of the General Tests shall apply.
1.4.2.1.2 Working seed virus
1.4.2.1.2.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
1.4.2.1.2.2 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
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1.4.2.1.3 Production seed virus
For storage, the following tests shall be performed:
1.4.2.1.3.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
1.4.2.1.3.2 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
1.4.2.2 Live bacteria vaccine
1.4.2.2.1 Master seed bacteria
1.4.2.2.1.1 Identification test
The test for identification of bacteria species shall be performed in compliance with anappropriate morphological characterization test method, biochemical characterization test method
or other approved test methods.
1.4.2.2.1.2 Test for freedom from contaminant microorganisms
The test given in the respective sections of the parts of vaccine (seed lot product) shall apply. If
no test method is given in the respective sections of the parts of vaccine (seed lot product), the test
shall be performed in the approved manner.
1.4.2.2.1.3 Target animal immunogenicity test
The test given in the Target Animal Immunogenicity Test of the General Tests shall apply.
1.4.2.2.1.4 Target animal safety test
The test given in the Target Animal Safety Test of the General Tests shall apply.
1.4.2.2.1.5 Test for absence of reversion to virulence
The test given in the Test for Absence of Reversion to Virulence of the General Tests shallapply.
1.4.2.2.1.6 Test for stability confirmation of recombinant geneIf gene recombination technology is used for preparation of the master seed bacteria, the test
given in the Test for Stability Confirmation of Recombinant Gene of the General Tests shall apply.
1.4.2.2.2 Working seed bacteria
1.4.2.2.2.1 Test for freedom from contaminant microorganisms
The test given in the respective sections of the parts of vaccine (seed lot product) shall apply. If
no test method is given in the respective sections of the parts of vaccine (seed lot product), the test
shall be performed in the approved manner.
1.4.2.2.3 Production seed bacteria
For storage, the following tests shall be performed:
1.4.2.2.3.1 Test for freedom from contaminant microorganisms
The test given in the respective sections of the parts of vaccine (seed lot product) shall apply.
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If no test method is given in the respective sections of the parts of vaccine (seed lot product),
the test shall be performed in the approved manner.
1.4.2.3 Inactivated virus vaccine
1.4.2.3.1 Master seed virus
1.4.2.3.1.1 Identification test
The test shall be performed as specified in 1.4.2.3.1.1.1 or 1.4.2.3.1.1.2.
1.4.2.3.1.1.1 Fluorescent antibody assay
Stain the cultured cells inoculated with and without the test article with a fluorescence-labeled
antibody, respectively: the cells inoculated with the test article show fluorescence characteristic of
the virus and those not inoculated do not show it.
1.4.2.3.1.1.2 Serum neutralization test
Propagate the test article with appropriate cultured cells: virus-specific cytopathic changes shallbe identified, and the propagation shall be neutralized by a specific antiserum.
1.4.2.3.1.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
1.4.2.3.1.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
1.4.2.3.1.4 Test for freedom from extraneous viruses
The tests given in the Test for Freedom from Extraneous Viruses of the General Tests shallapply.
1.4.2.3.1.5 Test for stability confirmation of recombinant geneIf gene recombination technology is used for preparation of the master seed virus, the test givenin the Test for Stability Confirmation of Recombinant Gene of the General Tests shall apply.
1.4.2.3.2 Working seed virus
1.4.2.3.2.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
1.4.2.3.2.2 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
1.4.2.3.3 Production seed virus
For storage, the following tests shall be performed:
1.4.2.3.3.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
1.4.2.3.3.2 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
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shall apply.
1.4.2.4 Inactivated bacteria vaccine
1.4.2.4.1 Master seed bacteria
1.4.2.4.1.1 Identification test
The test for identification of bacteria species shall be performed in compliance with anappropriate morphological characterization test method, biochemical characterization test method,or other approved test methods.
1.4.2.4.1.2 Test for freedom from contaminant microorganisms
The test given in the respective sections of the parts of vaccine (seed lot product) shall apply. If
no test method is given in the respective sections of the parts of vaccine (seed lot product), the test
shall be performed in the approved manner.
1.4.2.4.1.3 Test for stability confirmation of recombinant geneIf gene recombination technology is used for preparation of the master seed bacteria, the test
given in the Test for Stability Confirmation of Recombinant Gene of the General Tests shall apply.
1.4.2.4.2 Working seed bacteria
1.4.2.4.2.1 Test for freedom from contaminant microorganisms
The test given in the respective sections of the parts of vaccine (seed lot product) shall apply. If
no test method is given in the respective sections of the parts of vaccine (seed lot product), the test
shall be performed in the approved manner.
1.4.2.4.3 Production seed bacteria
For storage, the following tests shall be performed:
1.4.2.4.3.1 Test for freedom from contaminant microorganisms
The test given in the respective sections of the parts of vaccine (seed lot product) shall apply. If
no test method is given in the respective sections of the parts of vaccine (seed lot product), the test
shall be performed in the approved manner.
1.4.2.5 Recombinant protein vaccine
1.4.2.5.1 Expression system in recombinant virus
1.4.2.5.1.1 Master seed virus
1.4.2.5.1.1.1 Identification test
The test given in 1.4.2.1.1.1 shall apply.
1.4.2.5.1.1.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
1.4.2.5.1.1.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
1.4.2.5.1.1.4 Test for freedom from extraneous viruses
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The tests given in the Test for Freedom from Extraneous Viruses of the General Tests shallapply.
1.4.2.5.1.1.5 Test for stability confirmation of recombinant gene
The test given in the Test for Stability Confirmation of Recombinant Gene of the General Tests
shall apply.
1.4.2.5.1.2 Working seed virus
1.4.2.5.1.2.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
1.4.2.5.1.2.2 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
1.4.2.5.1.3 Production seed virus
For storage, the following tests shall be performed:
1.4.2.5.1.3.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
1.4.2.5.1.3.2 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
1.4.2.5.2 Expression system in recombinant bacteria
1.4.2.5.2.1 Master seed bacteria
1.4.2.5.2.1.1 Identification test
The test for identification of bacteria species shall be performed in compliance with anappropriate morphological characterization test method, biochemical characterization test method,or other approved test methods.
1.4.2.5.2.1.2 Test for freedom from contaminant microorganisms
The test given in the respective sections of the parts of vaccine (seed lot product) shall apply. If
no test method is given in the respective sections of the parts of vaccine (seed lot product), the test
shall be performed in the approved manner.
1.4.2.5.2.1.3 Test for stability confirmation of recombinant gene
The test given in the Test for Stability Confirmation of Recombinant Gene of the General Tests
shall apply.
1.4.2.5.2.2 Working seed bacteria
1.4.2.5.2.2.1 Test for freedom from contaminant microorganisms
The test given in the respective sections of the parts of vaccine (seed lot product) shall apply. If
no test method is given in the respective sections of the parts of vaccine (seed lot product), the test
shall be performed in the approved manner.
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1.4.2.5.2.3 Production seed bacteria
For storage, the following tests shall be performed:
1.4.2.5.2.3.1 Test for freedom from contaminant microorganisms
The test given in the respective sections of the parts of vaccine (seed lot product) shall apply. If
no test method is given in the respective sections of the parts of vaccine (seed lot product), the test
shall be performed in the approved manner.
1.4.2.6 Live coccidia vaccine
1.4.2.6.1 Master seed coccidia
1.4.2.6.1.1 Identification test
The test for identification of coccidia shall be performed in compliance with an appropriate
morphological characterization test method, PCR, enzyme electrophoresis, or other approved test
methods.
1.4.2.6.1.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
1.4.2.6.1.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
1.4.2.6.1.4 Test for freedom from extraneous viruses
The tests given in the Test for Freedom from Extraneous Viruses of the General Tests shallapply.
1.4.2.6.1.5 Target animal immunogenicity test
The test given in the Target Animal Immunogenicity Test of the General Tests shall apply.
1.4.2.6.1.6 Target animal safety test
The test given in the Target Animal Safety Test of the General Tests shall apply.
1.4.2.6.1.7 Test for absence of reversion to virulence
The test given in the Test for Absence of Reversion to Virulence of the General Tests shallapply.
1.4.2.6.2 Working seed coccidia
1.4.2.6.2.1 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
1.4.2.6.2.2 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
1.4.2.6.3 Production seed coccidia
For storage, the following tests shall be performed:
1.4.2.6.3.1 Sterility test
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The test given in the Sterility Test of the General Tests shall apply.
1.4.2.6.3.2 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
2 Cell seeds
2.1 Cell line
2.1.1 Range of passage number
The master cell seeds shall not be passaged more than 20 times to obtain the production cell
seeds.
When using the suspension culture method, approximately a three-fold increase in cell counts
than an increase in the population doubling time shall be regarded as the passage of one
generation. Note that this does not apply if otherwise approved by the Minister of Agriculture,
Forestry and Fisheries based on the test results that assure their suitability as cells for production.
2.1.2 Preparation method
Cell suspension shall be dispensed in one continuous working session with a series of processes
in accordance with the approved method to ensure their homogeneity and stability and prevent
contamination.
2.1.3 Storage
Store under the approved conditions.
2.1.4 Origin, specifications and test methods of cell seeds
2.1.4.1 Records on origin
2.1.4.1.1 Origin
Record the name of animal and organ from which the cells are derived and backgrounds ofestablishment (passages, cloning, establisher, timing) to the extent possible.
2.1.4.1.2 Passage history
Record the passage history and cloning, etc. after established and after distributed (orpurchased).
2.1.4.1.3 Culture medium
Record the culture media used for passage, propagation, and preservation.
2.1.4.2 Specifications and test methods
2.1.4.2.1 Master cell seeds
2.1.4.2.1.1 Test for confirmation of cell properties
When observed for microscopic findings, cell growth rate, acid production, morphologicalcharacteristics, and other characteristics from which the cells are considered normal as the celllines, the cells shall comply with the test.
2.1.4.2.1.2 Test for identification of the animal species of the cell176

When the test is performed by fluorescent antibody assay, the animal species shall be identicalto “animal and organ from which the cells are derived” in 2.1.4.1.1 Origin.
2.1.4.2.1.3 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
2.1.4.2.1.4 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
2.1.4.2.1.5 Test for freedom from extraneous viruses
The tests given in the Test for Freedom from Extraneous Viruses of the General Tests shallapply.
2.1.4.2.1.6 Test for karyological (chromosomal) characterization
The test given in either of the following sections shall apply:
2.1.4.2.1.6.1 Test on master cell seeds and their passaged cells
The following tests shall be performed on each of the master cell seeds and the maximum
passaged cells.
In a test on chromosomes in at least 50 dividing cells, the modal number (modal chromosome
number) in the maximum passaged cells shall be within ±15% of that in the master cell seeds. All
indicator chromosomes present in the master cell seeds shall also be identified in the maximum
passaged cells.
2.1.4.2.1.6.2 Test on passaged cells
When the cultured cells are tested as specified in 2.1.4.2.1.6.2.1, 2.1.4.2.1.6.2.2, 2.1.4.2.1.6.2.3,
2.1.4.2.1.6.2.4, and 2.1.4.2.1.6.2.5, there are no differences between all the four cultures of thecells that passaged at not less than the number of passages used for production and master cell
seeds: the cultured cells shall comply with the test.
2.1.4.2.1.6.2.1 Polyploidy test
A total of more than 300 cells from four cultures of the cells shall be examined for polyploidy.
2.1.4.2.1.6.2.2 Aneuploidy test
A total of more than 100 cells from four cultures of the cells shall be examined for aneuploidy.
2.1.4.2.1.6.2.3 Morphology abnormalities test
A total of more than 100 cells from four cultures of the cells shall be examined for morphology
abnormalities of chromosomes.
2.1.4.2.1.6.2.4 Chromosome cleavage test
A total of more than 100 cells from four cultures of the cells shall be examined for chromosome
cleavage.
2.1.4.2.1.6.2.5 Karyotype analysis test
A karyotype analysis test shall be performed on one cell from one of four cultures of the cells.
1772.1.4.2.1.7 Test for freedom from tumorigenicity
If findings show that malignant tumors are suspected to be induced in the animals targeted for
inoculation of seed lot products using the cell lines, culture by passage the master cell seeds andpassage at least four cultures of the cells used for production of seed lot products up to or morethan the maximum number: these cultured cells shall comply with the following test.
Athymic mice (nu/nu), or immunosuppressed mice or hamsters shall be used for the test. At
least five animals shall be given by subcutaneous injection with more than 2×106 cells,
respectively, and observed for 28 days.
During the period, no animal shall show evidence of tumor formation. As control, at least fiveanimals shall be similarly injected with more than 2×106 HeLa cells, which are known to form
tumors. At the end of observation for 28 days, at least 80% of the animals shall show tumor
formation.
2.1.4.2.2 Working cell seeds
2.1.4.2.2.1 Test for confirmation of cell properties
When observed for microscopic findings, cell growth rate, acid production, morphologicalcharacteristics, and other characteristics from which the cells are considered normal as the celllines, the cells shall comply with the test.
2.1.4.2.2.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
2.1.4.2.2.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
2.1.4.2.3 Production cell seeds
For storage, the following tests shall be performed:
2.1.4.2.3.1 Test for confirmation of cell properties
When observed for microscopic findings, cell growth rate, acid production, morphologicalcharacteristics, and other characteristics from which the cells are considered normal as the celllines, the cells shall comply with the test.
2.1.4.2.3.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
2.1.4.2.3.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
2.2 Primary cells
2.2.1 Use conditions
For production of seed lot products, cell lines shall be used. Note that this does not apply if178primary cells compliant with 2.2.4 Specifications and test methods are available.
2.2.2 Range of passage number
To obtain the production primary cell seed, the cells prepared from the animals shall not bepassaged more than 10 times. However, this does not apply if otherwise approved by the Ministerof Agriculture, Forestry and Fisheries based on the test results that assure their suitability as cells
for production.
2.2.3 Storage
Store under the approved conditions.
2.2.4 Specifications and test methods
2.2.4.1 Animals from which primary cells are collected
The animals shall comply with the SPF Animal Specifications.
2.2.4.2 Specifications and test methods for primary cells
2.2.4.2.1 Master primary cell seed
2.2.4.2.1.1 Test for confirmation of cell properties
When observed for microscopic findings, cell growth rate, acid production, morphologicalcharacteristics, and other characteristics from which the cells are considered normal as the primarycells, the cells shall comply with the test.
2.2.4.2.1.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
2.2.4.2.1.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
2.2.4.2.2 Working primary cell seed
2.2.4.2.2.1 Test for confirmation of cell properties
When observed for microscopic findings, cell growth rate, acid production, morphologicalcharacteristics, and other characteristics from which the cells are considered normal as the primarycells, the cells shall comply with the test.
2.2.4.2.2.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
2.2.4.2.2.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
2.2.4.2.3 Production primary cell seed
For storage, the following tests shall be performed:
2.2.4.2.3.1 Test for confirmation of cell properties
The test given in the culture observation of cells for production specified in the parts of vaccine
179

(seed lot product) shall apply. If no test method is given in the respective sections of the parts ofvaccine (seed lot product), the test shall be performed in the approved manner.
2.2.4.2.3.2 Sterility test
The test given in the Sterility Test of the General Tests shall apply.
2.2.4.2.3.3 Test for freedom from Mycoplasma contamination
The test given in the Test for Freedom from Mycoplasma Contamination of the General Tests
shall apply.
3 Embryonated eggs
3.1 Animals from which embryonated eggs are collected
The animals shall comply with the SPF Animal Specifications.
3.2 Specifications and test methods for embryonated eggs
3.2.1 Observation of embryonic eggs
The control embryonated eggs shall be incubated and observed without being inoculated withvaccine seeds under the same conditions as the culture of vaccine seeds. No abnormalities shall be
detected in the embryos.
4 Chickens
4.1 Chickens
Chickens used for the production of seed lot products shall be derived from SPF chicken flocks
as specified in 1.1 in the Materials for Live Vaccine Production. However, this does not apply to
Chicken anemia virus among the pathogens listed in Table 1 in the Materials for Live Vaccine
Production, if the absence of the virus is confirmed in the intermediate manufacturing process of
productions, including the bulk material.
4.2 Specifications and test methods for chickens
4.2.1 Growth test
The control chickens shall be bred and observed without being inoculated with vaccine seeds
under the same conditions as the culture of vaccine seeds. No abnormalities shall be detected.
5 Other materials
The materials for use necessary to manufacture or maintain the seeds such as the media,
digestive fluid and other materials shall be free from microorganisms and foreign matter.

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公益社団法人日本動物用医薬品協会
Japan Veterinary Products Association〒103-0023
東京都中央区日本橋本町 4−−6−−10 サトービル６階
6F Sato Building, 4-6-10 Nihonbasshi- Honcyo, Chuo-ku, Tokyo, Japan

TEL:03-5204-0440

FAX:03-5204-0442