ISO/FDIS 22467

Traditional Chinese medicine -- Determination of microorganism in natural products

Médecine traditionnelle chinoise -- Détermination des micro-organismes dans les produits naturels

General Information

• Status: Published
• ICS: 11.120.10 - Medicaments
• Technical Committee: ISO/TC 249 - Traditional Chinese medicine
• Drafting Committee: ISO/TC 249/WG 2 - Quality and safety of manufactured TCM products
• Current Stage: 5020 - FDIS ballot initiated: 2 months. Proof sent to secretariat
• Start Date: 21-Jul-2021
• Completion Date: 21-Jul-2021

Standards Content (sample)

FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 22467
ISO/TC 249
Traditional Chinese medicine —
Secretariat: SAC
Determination of microorganism in
Voting begins on:
2021­07­21 natural products
Voting terminates on:
2021­09­15
RECIPIENTS OF THIS DRAFT ARE INVITED TO
SUBMIT, WITH THEIR COMMENTS, NOTIFICATION
OF ANY RELEVANT PATENT RIGHTS OF WHICH
THEY ARE AWARE AND TO PROVIDE SUPPOR TING
DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
Reference number
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO­
ISO/FDIS 22467:2021(E)
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN­
DARDS TO WHICH REFERENCE MAY BE MADE IN
NATIONAL REGULATIONS. ISO 2021
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ISO/FDIS 22467:2021(E)
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ii © ISO 2021 – All rights reserved
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ISO/FDIS 22467:2021(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

Introduction ................................................................................................................................................................................................................................vi

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative reference ......................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Symbols and abbreviated terms ........................................................................................................................................................... 1

5 Test methods ............................................................................................................................................................................................................. 2

5.1 General ........................................................................................................................................................................................................... 2

5.2 Strains ............................................................................................................................................................................................................. 2

5.3 Test for sterility ...................................................................................................................................................................................... 2

5.3.1 General...................................................................................................................................................................................... 2

5.3.2 Culture media and incubation temperatures .......................................................................................... 2

5.3.3 Growth promotion test of aerobes, anaerobes and fungi ............................................................. 3

5.3.4 Method suitability test ................................................................................................................................................ 3

5.3.5 Test for sterility of the product to be examined .................................................................................... 4

5.4 Microbiological examination of non-sterile products: microbial enumeration tests................. 7

5.4.1 General...................................................................................................................................................................................... 7

5.4.2 Growth promotion test, suitability of the counting method and negative

controls .................................................................................................................................................................................... 7

5.4.3 Testing of products .....................................................................................................................................................13

5.4.4 Interpretation of the results ...............................................................................................................................14

5.5 Microbiological examination of non-sterile products: tests for specified

microorganisms ..................................................................................................................................................................................14

5.5.1 General...................................................................................................................................................................................14

5.5.2 Growth-promoting and inhibitory properties of the media, suitability of

the test and negative controls............................................................................................................................15

5.5.3 Testing of products .....................................................................................................................................................17

6 Acceptance criterion of test methods ..........................................................................................................................................20

6.1 Acceptance criterion of test for sterility ........................................................................................................................20

6.1.1 Acceptance criterion of sterility in test for culture medium ...................................................20

6.1.2 Acceptance criterion of growth promotion test of aerobes, anaerobes and

fungi .........................................................................................................................................................................................21

6.1.3 Acceptance criterion of method suitability test .................................................................................21

6.1.4 Acceptance criterion of test for sterility ...................................................................................................21

6.2 Acceptance criterion of microbial enumeration tests in microbiological

examination of non-sterile products .................................................................................................................................21

6.2.1 Acceptance criterion of preparation of test strains in microbiological

examination of non-sterile products: microbial enumeration tests .................................21

6.2.2 Acceptance criterion of negative control in microbiological examination of

non­sterile products ..................................................................................................................................................21

6.2.3 Acceptance criterion of media suitability in microbiological examination

of non­sterile products ............................................................................................................................................22

6.2.4 Acceptance criterion of the method suitability in microbiological

examination of non-sterile products ...........................................................................................................22

6.2.5 Acceptance criterion of the validity of the results in microbiological

examination of non-sterile products ...........................................................................................................22

6.3 Acceptance criterion of tests for specified microorganisms in microbiological

examination of non-sterile products .................................................................................................................................22

6.3.1 Acceptance criterion of preparation of test strains in microbiological

examination of non-sterile products: tests for specified microorganisms .................22

6.3.2 Acceptance criterion of negative control in microbiological examination of

non-sterile products: tests for specified microorganisms ........................................................22

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ISO/FDIS 22467:2021(E)
6.3.3 Acceptance criterion of media suitability in microbiological examination

of non-sterile products: tests for specified microorganisms ..................................................22

6.3.4 Acceptance criterion of the method suitability in microbiological

examination of non-sterile products: tests for specified microorganism ....................23

6.3.5 Acceptance criterion of the validity of the results in microbiological

examination of non-sterile products ...........................................................................................................23

Annex A (Normative) Microbiological quality of natural products ...................................................................................24

Annex B (Informative) Recommended solutions and culture media ...............................................................................35

Bibliography .............................................................................................................................................................................................................................40

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ISO/FDIS 22467:2021(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non­governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/ patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www .iso .org/

iso/ foreword .html.

This document was prepared by Technical Committee ISO/TC 249, Traditional Chinese medicine.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/ members .html.
© ISO 2021 – All rights reserved v
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ISO/FDIS 22467:2021(E)
Introduction

Natural products used in traditional Chinese medicine are widely used around the world due to their

high medicinal values and mild side effects. It is a common phenomenon that natural products are

contaminated by microorganisms which not only impact their quality and efficacy, but also restrict the

international trade in them and related products. Although the Pharmacopeia of the People’s Republic

of China, the British Pharmacopoeia, the Japanese Pharmacopoeia, the European Pharmacopoeia and

the United States Pharmacopeia have stipulated the microbial limits of natural products, there is no

International Standard for microorganism detection methods, which adversely affects communication

and trade between researchers and factories in different countries. Furthermore, microorganism

levels on or in natural products usually exceed the maximum limit levels set by many international

organizations and countries due to the lack of an International Standard.
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FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 22467:2021(E)
Traditional Chinese medicine — Determination of
microorganism in natural products
1 Scope

This document specifies test methods to determine microorganisms in natural products. It is applicable

only to natural products used in traditional Chinese medicine, including raw materials, herbal pieces

and preparations.
2 Normative reference
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
sterility
state of being free from viable microorganisms

Note 1 to entry: In practice, no such absolute statement regarding the absence of microorganisms can be proven.

[SOURCE: ISO 11139:2018, 3.274]
3.2
microbial enumeration test

quantitative counting of mesophilic bacteria and fungi which may grow under aerobic conditions

4 Symbols and abbreviated terms
ATCC American Type Culture Collection
CMCC National Center for Medical Culture Collections
CIP Collection de Bactéries de I'Institut Pasteur
IMI International Mycological Institute
IP Institut Pasteur
MPN most­probable­number
NBRC Biological Resource Center, National Institute of Technology and Evaluation
NCIMB National Collection of Industrial and Marine Bacteria Ltd
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ISO/FDIS 22467:2021(E)
NCPF National Collection of Pathogenic Fungi
NCTC National Collection of Type Cultures
TAMC total aerobic microbial count
TYMC total combined yeast and mould count
5 Test methods
5.1 General

The test shall be carried out under aseptic conditions. In order to achieve such conditions, the test

environment shall be adapted to the way in which the sterility test is performed. The precautions

taken to avoid contamination shall be such that they do not affect any microorganisms which are to

be revealed in the test. The working conditions in which the tests are performed shall be monitored

regularly by appropriate sampling of the working area and by carrying out appropriate controls.

5.2 Strains

The standardized stable suspensions of test strains as stated in Table 1 shall be used. Seed­lot culture

maintenance techniques (seed-lot systems) shall be used so that the viable microorganisms used for

inoculation are not more than five passages removed from the original master seed-lot.

Table 1 — Standard strains
Escherichia coli ATCC 8739, NCIMB 8545, CIP 53.126 NBRC 3972 or CMCC (B) 44102

Pseudomonas aeruginosa ATCC 9027, NCIMB 8626, CIP 82.118, NBRC 13275 or CMCC (B) 10104

Clostridium sporogenes CMCC (B) 64941 or ATCC 11437 (NBRC 14293, NCIMB 12343, CIP 100651) or

ATCC 19404 (NCTC 532 or CIP 79.03) or NBRC 14293
Staphylococcus Aureus ATCC6538, NCIMB9518, CIP 4.83, NBRC 13276 or CMCC (B)26003
Bacillus subtilis ATCC 6633, NCIMB 8054, CIP 52.62, NBRC 3134 or CMCC (B) 63501
Candida albicans ATCC 10231, NCPF 3179, IP 48.72 or NBRC 1594 or CMCC (F) 98001

Aspergillus brasiliensis ATCC 16404, IMI 149007, IP 1431.83 NBRC 9455 or CMCC (F) 98003

(Aspergillus niger)

Salmonella paratyphi B CMCC(B)50094, ATCC 14028 or, as an alternative, Salmonella enteric subsp. enteric

serovar Abony such as NBRC 100797, NCTC 6017 or CIP 80.39
Shigella dysenteriae type 1 ATCC 11835, ATCC 9361, CMCC 51252
5.3 Test for sterility
5.3.1 General

The test is applied to raw materials, herbal pieces and preparations which are required to be sterile.

However, a satisfactory result only indicates that no contaminating microorganism has been found

in the sample examined under the conditions of the test. The acceptance criteria for microbiological

quality of products to be tested shall be done according to A.1 in Annex A.
5.3.2 Culture media and incubation temperatures

Recommended media for the test may be prepared as shown in Annex B, or equivalent commercial

media may be used provided that they conform to the growth promotion test.
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ISO/FDIS 22467:2021(E)

The culture media have been found to be suitable for the test for sterility. Fluid thioglycollate medium

is primarily intended for the culture of anaerobic bacteria. However, it will also detect aerobic bacteria.

Soya-bean casein digest medium is suitable for the culture of both fungi and aerobic bacteria.

Fluid thioglycollate medium shall be incubated at 30 °C to 35 °C. For products containing a mercurial

preservative that cannot be tested by the membrane-filtration method, fluid thioglycollate medium

incubated at 20 °C to 25 °C may be used instead of soya-bean casein digest medium provided that it has

been validated as described in the growth promotion test.
5.3.3 Growth promotion test of aerobes, anaerobes and fungi

Test each lot of ready-prepared medium and each batch of medium prepared either from dehydrated

medium or from ingredients. Suitable strains of microorganisms are indicated in Table 2.

Inoculate portions of fluid thioglycollate medium with a small number (not more than 100 cfu) of

microorganisms, using a separate portion of medium for each of the following species of microorganism:

Clostridium sporogenes, Pseudomonas aeruginosa and Staphylococcus aureus.

Inoculate portions of alternative thioglycollate medium with a small number (not more than 100 cfu) of

Clostridium sporogenes. Inoculate portions of soya-bean casein digest medium with a small number (not

more than 100 cfu) of microorganisms, using a separate portion of medium for each of the following

species of microorganism: Aspergillus brasiliensis, Bacillus subtilis, and Candida albicans. Incubate for

not more than 3 days in the case of bacteria and not more than 5 days in the case of fungi. Seed lot

culture maintenance techniques (seed-lot systems) are used so that the viable microorganisms used for

inoculation are not more than five passages removed from the original master seed-lot.

Table 2 — Strains of the test microorganisms suitable for use in the growth promotion test and

the method validation
Staphylococcus aureus
ATCC 6538, CIP 4.83, NCTC 10788, NCIMB 9518, NBRC 13276 or CMCC (B)26003
Bacillus subtilis
Aerobic bacteria
ATCC 6633, CIP 52.62, NCIMB 8054, NBRC 3134 or CMCC (B) 63501
Pseudomonas aeruginosa
ATCC 9027, NCIMB 8626, CIP 82.118, NBRC 13275 or CMCC (B) 10104
Clostridium sporogenes
Anaerobic bacterium
ATCC 19404, CIP 79.3, NCTC 532, ATCC 11437, NBRC 14293 or CMCC (B) 64941
Candida albicans
ATCC 10231, IP 48.72, NCPF 3179, NBRC 1594 or CMCC (F) 98001
Fungi
Aspergillus brasiliensis
ATCC 16404, IP 1431.83, IMI 149007, NBRC 9455 or CMCC (F) 98003

An alternative microorganism in Aerobic bacteria is Micrococcus luteus (ATCC 9341).

An alternative to Clostridium sporogenes, when a nonspore­forming microorganism is desired, is Bacteroides vulgatus

(ATCC 8482).
5.3.4 Method suitability test
5.3.4.1 Membrane filtration

After transferring the contents of the container or containers to be tested to the membrane, add an

inoculum of a small number of viable microorganisms (not more than 100 cfu) to the final portion of

sterile diluent used to rinse the filter.
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ISO/FDIS 22467:2021(E)
5.3.4.2 Direct inoculation

After transferring the contents of the container or containers to be tested to the culture medium, add

an inoculum of a small number of viable microorganisms (not more than 100 cfu) to the medium.

In both cases, use the same microorganisms as those described in 5.3.3. Perform a growth promotion

test as a positive control. Incubate all the containers containing medium for not more than 5 days.

5.3.5 Test for sterility of the product to be examined
5.3.5.1 General

Unless otherwise specified elsewhere in this document or in the individual monograph, test the number

of articles specified in Table 3. If the contents of each article are of sufficient quantity (see Table 3), they

may be divided so that equal appropriate portions are added to each of the specified media. (Perform

sterility testing employing two or more of the specified media.) If neither article contains sufficient

quantities for each medium, use twice the number of articles indicated in Table 4.

The test may be carried out using the technique of membrane filtration or by direct inoculation of

the culture medium with the product to be examined. Appropriate negative controls are included.

The technique of membrane filtration is used whenever the nature of the product permits; that is, for

filterable aqueous preparations, for alcoholic or oily preparations, and for preparations miscible with,

or soluble in, aqueous or oily solvents, provided these solvents do not have an antimicrobial effect in the

conditions of the test.
5.3.5.2 Membrane filtration
5.3.5.2.1 General

Use membrane filters having a nominal pore size not greater than 0,45 μm, in which the effectiveness to

retain microorganisms has been established. Cellulose nitrate filters, for example, are used for aqueous,

oily and weakly alcoholic solutions. Cellulose acetate filters, for example, are used for strongly alcoholic

solutions. Specially adapted filters may be needed for certain products.

Sterility test assumes that membranes about 50 mm in diameter will be used. If filters of a different

diameter are used, the volumes of the dilutions and the washings should be adjusted accordingly. The

filtration apparatus and membrane shall be sterilized by appropriate means. The apparatus is designed

so that the solution to be examined can be introduced and filtered under aseptic conditions. It permits

the aseptic removal of the membrane for transfer to the medium or it is suitable for carrying out the

incubation after adding the medium to the apparatus itself.
5.3.5.2.2 Aqueous solutions

If appropriate, transfer a small quantity of a suitable, sterile diluent such as a 1 g/l neutral solution of

meat or casein peptone pH 7,1 ± 0,2 onto the membrane in the apparatus and filter. The diluents may

contain suitable neutralizing substances, appropriate inactivating substances or both, for example in

the case of antibiotics.

Transfer the contents of the container or containers to be tested to the membrane or membranes, if

necessary, after diluting to the volume used in 5.3.4 with the chosen sterile diluent; in any case, using

not less than the quantities of the product to be examined prescribed in Table 3. Filter immediately. If

the product has antimicrobial properties, wash the membrane not less than three times by filtering

through it each time the volume of the chosen sterile diluent used in 5.3.4. Do not exceed a washing

cycle of five times 100 ml per filter, even if during the method suitability test it has been demonstrated

that such a cycle does not fully eliminate the antimicrobial activity. Transfer the whole membrane to the

culture medium or cut it aseptically into two equal parts and transfer one half to each of two suitable

media. Use the same volume of each medium as in the method suitability test. Alternatively, transfer

the medium onto the membrane in the apparatus. Incubate the media for not less than 14 days.

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ISO/FDIS 22467:2021(E)
5.3.5.2.3 Soluble solids

Use for each medium not less than the quantity prescribed in Table 3 of the product dissolved in a

suitable solvent, such as the solvent provided with the preparation, water for injections, saline or a 1 g/l

neutral solution of meat or casein peptone. Proceed with the test as described in 5.3.5.2.2 or aqueous

solutions using a membrane appropriate to the chosen solvent.
5.3.5.2.4 Oils and oily solutions

Use for each medium not less than the quantity of the product prescribed in Table 3. Oils and oily

solutions of sufficiently low viscosity may be filtered without dilution through a dry membrane. Viscous

oils may be diluted as necessary with a suitable sterile diluent such as isopropyl myristate shown not

to have antimicrobial activity in the conditions of the test. Allow the oil to penetrate the membrane

by its own weight then filter, applying the pressure or suction gradually. Wash the membrane at least

three times by filtering through it each time about 100 ml of a suitable sterile solution, such as 1 g/l

neutral meat or casein peptone containing a suitable emulsifying agent at a concentration shown to be

appropriate in 5.3.4, for example polysorbate 80 at a concentration of 10 g/l. Transfer the membrane or

membranes to the culture medium or media or vice versa as described in 5.3.5.2.2, and incubate at the

same temperature for the same time.
5.3.5.2.5 Ointments and creams

Use for each medium not less than the quantities of the product prescribed in Table 3. Ointments in

a fatty base and emulsions of the water-in-oil type may be diluted to 1 % in isopropyl myristate as

described in 5.3.5.2.4 by heating, if necessary, to not more than 40 °C. In exceptional cases it may be

necessary to heat to not more than 44 °C. Filter as rapidly as possible and proceed as described in

5.3.5.2.4.
5.3.5.2.6 Sterile aerosol products

For fluid products in pressurized aerosol form, freeze the containers in an alcohol-dry ice mixture of at

least –20 °C for about 1 hour. If feasible, allow the propellant to escape before aseptically opening the

container and transfer the contents to a sterile pooling vessel. Add 100 ml of fluid D (see Table B.2) to

the pooling vessel and mix gently. Proceed as described in 5.3.5.2.2 or 5.3.5.2.4, whichever applies.

Table 3 — Minimum quantity to be used for each medium
Minimum quantity to be used for each medium unless
Quantity per container
otherwise justified and authorized
Liquids
— less than 1 ml The entire contents of each container
— 1 ml to 40 ml Half the contents of each container but not less than 1 ml
— greater than 40 ml and less than 100 ml 20 ml
10 % of the contents of the container but not less than
— greater than 100 ml
20 ml
Antibiotic liquids 1 ml

Insoluble preparations, creams and ointments to be Use the contents of each container to provide not less

suspended or emulsified than 200 mg
Solids
Less than 50 mg The entire contents of each container
Half the contents of each container but not less than
50 mg or more but less than 300 mg
50 mg
300 mg to 5 g 150 mg
Greater than 5 g 500 mg
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ISO/FDIS 22467:2021(E)
Table 4 — Minimum number of items to be tested
Number of items in the batch Minimum number of items to be tested for each
medium, unless otherwise justified and author­
ized
Parenteral preparations
Not more than 100 containers 10 % or four containers, whichever is greater
More than 100 but not more than 500 containers 10 containers
More than 500 containers 2 % or 20 containers (10 containers for
large­volume parenterals), whichever is less
Ophthalmic and other non-injectable
Not more than 200 containers 5 % or two containers, whichever is greater
More than 200 containers 10 containers
If the product is presented in the form of single­dose contain­
ers, apply the scheme shown for preparations for parenteral
use
Bulk solid products
Up to four containers Each container

More than four containers but not more than 50 containers 20 % or four containers, whichever is greater

More than 50 containers 2 % or 10 containers, whichever is greater
5.3.5.3 Direct inoculation of the culture medium
5.3.5.3.1 Gene
...