o R G A N I Z A T ION SAN 0 I N 0 I V I 0 U ALS V I S I TED978-1-349-11762-8/1.pdf · Ministry of Health and Welfare: Mr Suichi Tani, Director, Health Science Division; Mr Takahisa

o R G A N I Z A T ION SAN 0 I N 0 I V I 0 U ALS V I S I TED

IN JAPAN

Govemment

Science and Technology Agency: Mr Koichi Soga, Director, Life Sciences Division, Research and

Development Bureau.

Dr Genya Chiba, Director, ERATO, STA; Hideo Ohara, Assistant.

Ministry of Agriculture, Forestry and Fisheries: Mr Keiji Kainuma, Director, Biotechnology

Division; Mr Naoya Nakamura, Assistant.

Ministry of Education, Science and Culture: Mr Akihiko Ueda, Research Aid Division; Dr

Chifumi Sato, Senior Specialist.

Ministry of Health and Welfare: Mr Suichi Tani, Director, Health Science Division; Mr Takahisa

Murakami, Deputy Director.

Ministry of International Trade and Industry: Mr Masaru Masuda, Director, Bioindustry Office.

197

Japanese Biotechnology

Universities & Research Institutes

Kinki University: Dr Hajime Kadota, Professor of Microbiology.

Kyoto University: Professor Mituru Takanami, Director, Institute for Chemical Research.

National Institute of Agrobiological Resources, Ministry of Agriculture, Forestry and Fisheries:

Dr Joh-E Ikeda, Lab Chief of Molecular Genetics.

Osaka Biosciences Institute: Dr Osamu Hayaishi, Director; Dr Frederick I. Tsuji, Head,

Department of Enzymes and Metabolism.

Osaka University, Institute for Molecular and Cell Biology: Professor Kenichi Matsubara,

Director; Professor Tadatsagu Taniguchi.

Protein Engineering Institute: Dr Morio Ikehara, Managing Director; Mr Yukio Takegawa,

Director Planning and Coordination Program.

RIKEN (Institute of Physical and Chemical Research), Science and Technology Agency: Mr

Yoshiki Chiba, Director, Life Science Promotion Division; Dr Yoji Ikawa, Vice-Director for

Scientific Research & Lab Chief of Molecular Oncology; Dr Shunsuke Ishii, Lab Chief. Dr H.

Amanuma.

Tokyo University: Professor Isao Karube, Research Center for Advanced Science and

Technology.

Tokyo University: Professor Teruhiko Beppu, Laboratory of Fermentation and Microbiology,

Department of Agricultural Chemistry.

198

Organizations and Individuals Vzsited

Tsukuba Research Consortium: Dr Genya Chiba; Noboru Kakita, Chief General Affairs

Department; and a few scientists from the ERA TO labs.

Industry

Ajinomoto: Dr Ryuichiro Tsugawa, Director, Ajinomoto Company, Inc., Deputy Laboratory

Manager, Central Research Laboratories.

Asahi Chemical Industry: Dr Daikichi Nishimura, Senior Scientist, Scientific Affairs and Licensing;

Dr Yu Saito, Manager; Dr Yukiji Shimojima, Deputy Plant Manager; Mr Jeffrey Encinas.

Daiichi Seiyaku Company Ltd.: Dr Mitsuru Furasawa, Director of Research Institute; Project

Director, MorphoGene Project, JRDC.

Dainippon Pharmaceuticals: Dr Susumu Yoshizawa, Director, Laboratory Products; Dr Hiroaki

Hattori, Manager, Product Planning Division; Dr Toshikazu Fukui, Chief Department of

Biotechnology.

Genzyme Japan: Ms Keiko Ohishi, Technical Coordinator.

Green Cross Corporation: Dr Minoru Hirama, Director, Research Division; Dr Haruhide

Kawabe, Director, Biotechnology Center; Dr Toshizumi Tanabe, Senior Principal Researcher.

Hayashibara: Mr Ken Hayashibara, President; Mr Masashi Kurimoto, Executive Director, Basic

Research Center, Fujisaki Institute; Dr Jun Minowada, Executive Director, Fujisaki Cell Center,

Mr Mohammad Raees, Deputy Director, Hayashibara Institute Corporation.

Kirin: Mr Noburu Miyadai, Adviser; Dr Takamoto Suzuku, Manager.

199


Kubota, Ltd: Dr Hidetaka Hori; Dr Tadaaki Kawasugi, Manager; Mr Hiroshi Noguchi,

Microbiology Research Team, R&D Headquarters.

Kyowa Hakko Kogyo: Dr Akira Furuya, Managing Director, New Products & Technology

Development Center and Technical Information Center, R&D Division.

Mitsubishi Kasei: Mr Tomoyuki Watanabe, Manager, Plantech Research Institute.

NEC: Dr Toshihide Kuriyama, Research Manager, Sensor Research Laboratory, Microelectronics

Research Laboratories; Mr Toru Murakami, Environment Protection Engineering Research

Laboratory.

Sankyo Company: Dr Sadamasa Minato, Director, Bioscience Research Laboratories; Dr Yo

Takiguchi, Vice-Director, Bioscience Research Labs.

Showa Yakuhin Kako Company: Dr Tomoyuki Ishikura, Executive Managing Director.

Takeda: Dr Yukio Sugino, Vice-President; General Manager of R&D Division.

Suntory: Dr Hiroshi Nakazato, Manager, Laboratory of Molecular Biology, Institute for

Biomedical Research.

Tanabe Seiyaku: Dr Tetsuya Tosa, General Manager, Research Lab of Applied Biochemistry.

200

Organizations anti Intiividuals Vzsited

U.S. Embassy

Economics Section: Mr Robert Ludan, First Secretary; Ms Fumiko Shioda, Economics Specialist.

Science Section: Dr Richard Getzinger, Science Counselor;

Other

BIDEC: Dr Yasuki Mori, General Manager.

Genetic and Engineering News: Dr Carol Cooper Martin, Freelance writer.

National Association for Food Industries, Japan: Mr Yohei Matsunobu, Executive Director.

Nikkei Biotechnology: Mr Mitsuru Miyata, Editor-in-Chief.

Pe at Marwick Minato: Mr Thomas W. Whitson, Partner.

SRI: Dr Akiko Fujiwara.

Techno-Venture: Mr Yu Makiuchi, Executive Managing Director.

201

R E F E REN CES

1. International Financial Statistics, 43(7), July 1990.

2. Oxender, D., C. Cooney, D. Jackson, G. Sato, R. Wiclmer, and J. Wilson. nECH Panel

Report on Biotechnology in Japan. (Science Applications International Corporation), U.S.

Department of Commerce, Washington, DC, June 1985.

3. Commercial Biotechnology: An International Analysis. U.S. Office of Technology

Assessment, Washington, DC, 1984.

4. Fujimura, R., Biotechnology in Japan. International Trade Administration, Washington, DC,

1988.

5. Uekusa, Masu and Ide, Hideki. Industrial Policy in Japan. In Industrial Poikies for Pacijic

Economic Growth. (Eds. Hiromichi Mutoh, Sueo Sekiguchi, Kotaro Suzumura, and Ippei

Yamazawa). Allen & Unwin, Sydney, London, and Boston. 1986. pp. 147-171.

6. The Report of the Advisory Group on Economic Structural Adjustment for International

Harmony submitted to the Prime Minister, Mr Yaushiro Nakasone, April 7, 1986.

7. Saxonhouse, Gary R. Industrial Policy and Factor Markets: Biotechnology in Japan and

the United States. In Japan's High Technology Industries. (Ed. Hugh Patrick). University

of Washington Press, Seattle and London. University of Tokyo Press. 1986.

8. Trends and Future Tasks in Industrial Technology - Developing Innovative Technologies to

Support the 21st Century and Contributing to the International Community, Ministry of

International Trade and Industry. September 1988.

9. Motoyuki, Fujii. Biotechnology: Government's Support for Pharmaceutical Industry.

Business Japan. July 1988.

10. Mr Suichi Tani, Director, Health Science Division, and Mr Takahisa Murakami, Deputy

Director MHW, personal communication, 1989.

11. Japan Health Sciences Foundation Report, April 1989.

12. STA, Its Roles and Activities 1987.

202

References

13. Mr Koichi Soga, Director, Life Sciences Division, Research and Development Bureau,

Science and Technology Agency, personal communication, 1989.

14. Research Development Corporation of Japan 1988.

15. ERATO, Research Development Corporation of Japan, 1990.

16. Mr Akihiko Ueda, Research Aid Division; Dr Chifumi Sato, Senior Specialist, personal

communication, 1989.

17. University-Industry Cooperation in Japan, Isao Karube, 1989.

18. Okimoto, Daniel I, Between MITI and the Market: Japanese Industrial Policy for High

Technology, Stanford University Press, Stanford, CA, 1989.

19. Mr Masaru Masuda, Director, Bioindustry Office, MITI, personal communication, 1989.

20. Agency of Industrial Science and Technology, Ministry of International Trade and Industry,

1988.

21. Biotechnology Research and Development in Agriculture, Forestry, Fisheries and the Food

Industry in Japan, 1989.

22. Drug Registration Requirements in Japan, 3rd Ed., Tokyo, Yakuji Nippo, Ltd., Tokyo, Japan,

1988; Standards and Certification Systems Conceming Drugs in Japan. Second Edition.

Tokyo, Yakugyo Jiho Co., Ltd., Tokyo, Japan, 1988 (in Japanese and English).

23. Yoshikawa, A, Japanese Biotechnology: New Drugs. BRIE working Paper No.33, Berkeley,

CA, 1988.

24. Dibner, MD, Drug Regulation in Japan: Can Foreign Companies Play on their Field,

Biopharm 2 (9): 34-42, October 1989.

25. The Institute for Chemical Research, Kyoto University, 1988.

26. The Institute for Molecular and Cellular Biology, Osaka University, 1987.

27. Research Center for Advanced Science and Technology, The University of Tokyo 1988.

28. Professor Isao Karube, personal communication, 1989.

29. Professor Teruhiko Beppu, personal communication, 1989.

30. Economist, May 6, 1989.

31. RIKEN 1987.

32. Professor Joh-E Ikeda, personal communication, 1989.

33. Tsukuba Research Consortium.

203


34. Protein Engineering Research Institute 1988.

35. Dr Morio Ikehara and Mr Yukio Takegawa, personal communication, 1989.

36. Osaka Bioscience Institute. Annual Report 1987-1988.

37. Mr Ken Hayashibara, personal communication, 1989.

38. The Scientist, September 4, 1989.

39. Science anti Technology in Japan, 9(33), 1990.

40. Biotechnology Newswatch, December 4, 1989, p. 9.

41. Business Week, Special Innovation Issue, June 1989, p. 17.

42. Japan Economic Journal, October 29, 1988, p. 23.

43. Science, December 8, 1989, pp. 1238-1239.

44. Wall Street Journal, February 21, 1989, P A12.

45. Nature, 342, December 21/28, 1989, p. 1850.

46. Wall Street Journal, November 10, 1988, p. 34.

47. Science anti Technology in Japan, 9(33) March 1990, p. 25.

48. Siddiqui, S., Nature, 340, 1989, p. 337-338.

49. Economist, May 20, 1989.

50. Economist, December 2, 1989.

51. Economist, July 8, 1989.

52. Economist, March 3, 1990.

53. James C. Abegglen & George Stalk, Jr., Kaisha. The Japanese Corporation, Basic Books,

New York, 1985.

54. IMR Survey: Instruments anti Supplies Jor Biotechnology Research anti Development in Japan,

prepared by Pacific Projects, Ltd., for the American Embassy, Japan, 1988.

55. Japan Economic Journal, March 11, 1989, p. 1, 6.

204

A B B R E V I A T ION S

AIDS . . . . . . . . . . . . . . . . . . . .. Acquired Immune Deficiency Syndrome

AIST . . . . . . . . . . . . . . . . . . . .. Agency of Industrial Science and Technology

ANF ..................... atrial natriuretic factor

BTI ...................... BioTechnology International

DNA . . . . . . . . . . . . . . . . . . . .. deoxyribonucleic acid

EEC ..................... European Economic Commission

ERATO . . . . . . . . . . . . . . . . . .. Exploratory Research for Advanced Technology

GILSP . . . . . . . . . . . . . . . . . . .. Good industriallarge-scale practice

GNP ..................... gross national product

HEP A . . . . . . . . . . . . . . . . . . .. high efficiency particulate air

JAFCO ................... Japan Associated Finance Company

JKTC . . . . . . . . . . . . . . . . . . . .. Japan Key Technology Center

JRDC .................... Research Development Corporation of Japan

JSPS ..................... Japan Society for the Promotion of Science

KAKEN . . . . . . . . . . . . . . . . . .. Scientific Research Institute, Ltd.

MAFF . . . . . . . . . . . . . . . . . . .. Ministry of Agriculture, Forestry and Fisheries

MESC

MHW

MIT ..................... .

MID .................... .

mRNA ................... .

NCBC ................... .

NHI ..................... .

NIe ..................... .

Ministry of Education, Science and Culture

Ministry of Health and Welfare

Massachusetts Institute of Technology

Ministry of International Trade and Industry

messenger ribonucleic acid

North Carolina Biotechnology Center

National Health Insurance

newly industrializing country

205


NIH ..................... .

OBI ..................... .

OECD ................... .

OEM .................... .

P AB .................... .

PERl .................... .

R&D .................... .

RCAST .................. .

rDNA ................... .

RlKEN .................. .

RNA .................... .

STA ..................... .

TAP

TNF

TRC

U.K.

VCR

VEC

206

National Institute of Health

Osaka Bioscience Institute

Organization for Economic Cooperation & Development

original equipment manufacturing

Pharmaceutical Affairs Bureau

Protein Engineering Research Institute

research and development

Research Center for Advanced Science and Technology

recombinant deoxyribonucleic acid

Institute of Physical and Chemical Research

ribonucleic acid

Science and Technology Agency

Takeda-Abbott Pharmaceuticals

tumor necrosis factor

Tsukuba Research Consortium

United Kingdom

video cassette recorder

Venture Enterprise Center

A P P END I X A

GUIDELINFS FOR REOOMBINANT DNA EXPERIMENTS IN

UNIVERSrrIES AND OTHER RESEARCH INSTIfUTIONS

Chapter 1 Introduction

#1 Purpose

These Guidelines for Recombinant DNA Experiments (hereafter referred to as 'Guidelines')

are intended as guidelines to be followed by investigators in Universities and other research

institutions to ensure safety in planning and conducting recombinant DNA experiments, and

to promote the use of safe and appropriate procedures for recombinant DNA experiments.

#2 Definitions

Definitions of major terms used in the Guidelines are as folIows.

1 'Recombinant DNA experiments' are aseries of experiments in which DNA

(deoxyribonucleic acid) moIecuIes capable of replicating in certain living cells are joined to

DNAs from different species by means of in vitro enzymatic reactions, introduced into living

ceIls, and propagated. (However, experiments involving cells carrying recombinant DNA

moIecuIes whose genetic composition if equivalent to those that can exist naturaIly, are

excluded from the defmition of recombinant DNA experiments.)

2 'Recombinant organisms' are living cells into which recombinant DNA moIecuIes have

been introduced by means of recombinant DNA experiments.

3 'Hosts' are living cells into which recombinant DNA moIecuIes are to be introduced by

recombinant DNA experiments.

207


4 IVectors' are DNAs to which foreign DNAs are joined to be introduced into hosts in

recombinant DNA experiments.

5 'Host-vector systems' are combinations of a host and a vector.

6 'DNA-donors' are microorganisms or cells which provide DNA to be joined to vectors.

When complementary DNA synthesized from RNA as template is inserted into a vector,

cells or microorganisms which are the source of the RNA are defined as the DNA-donors.

7 'Purified DNAs' are characterized DNAs which are prepared from DNA-donors. The

cloned or chemically synthesized DNAs are also categorized as purified DNAs.

8 'Unpurified DNAs' are DNA mixtures with unidentified genes prepared from DNA

donors.

9 'Laboratories' are rooms in which recombinant DNA experiments are conducted.

10 'Laboratory areas' are areas of laboratories and passage ways separated from the other

areas by specified anterooms which serve as access controlling areas.

11 'Special laboratory sections' in a laboratory area are sections of air-tight construction

where life support systems are provided.

12 'Safety cabinets' are box-type facilities so designed that contaminated aerosols produced

during experiments shall not leak outside. Their specifications are described in Supplement

one.

13 'Laboratory workers' are persons who actually conduct recombinant DNA experiments.

14 'Laboratory supervisors' are persons, among the laboratory workers, who have the

responsibility for conducting each experimental project.

15 'Universities and other research institutions' include universities, technical colleges, inter

university institutions, institutions controlled by the Minister of Education, Science and

Culture, and also corporations managed by the Minister of Education, Science and Culture

for scientific research under Article 34 of the Civil Law (Law No. 89, 1896).

#3 Scope

These Guidelines are to be applied to recombinant DNA experiments (which are hereafter

referred to as lexperiments') conducted in universities and other research institutions (which

are called 'universities etc.' hereafter). Theyare also to be applied to experiments in research

208

AppendixA

institutions other than universities etc. when theyare conducted with the support of a Grant-in

Aid from the Ministry of Education, Science and Culture.

#4 Methods for Containment Ensuring the Safety of Experiments

In order to ensure safety, experiments should be performed on the basis of the general

procedures employed in the pathogenic microbiallaboratories. For tbis purpose, experiments

shall be planned and conducted with a suitable combination of two containment methods,

namely physical containment and biological containment, according to the assessment of their

safety.

#5 Responsibility of Laboratory Workers

Laboratory workers must be weIl acquainted with and experienced in the handling of

pathogenic microorganisms and the techniques specifically required for the experiments to be

conducted, as weIl as associated procedures. When planning and conducting experiments,

laboratory workers must realize the importance of ensuring safety and shall take all necessary

precautions.

#6 Obligations of Presidents of Universities, etc.

The presidents of universities, etc., shall make every effort, for example by organizing a safety

advisory committee, to ensure the safety of the experiments conducted in their universities, etc.

#7 Procedures for Ensuring the Safety of Experiments

In order to ensure safety, all experiments should be first judged by the safety advisory

committees organized in universities etc., and them approved by the presidents of universities

etc. or by those whom are entrusted by the presidents (hereafter referred to 'administrators

of universities, etc!).

The experiments conducted with a Grant-in-Aid from the Ministry of Education, Science and

CuIture in institutes other than universities should be approved by directors of the institutes.

In this case, the directors have the responsibility to ensure the safety of experiments as do the

administrators of universities.

209


The experiments not described or specified in the guidelines should be first judged by The

Science Council and then approved by the Minister of Education, Science and culture

(hereafter this sort of arrangement will be referred to 'Approval of the Minister of Education,

Science and Culture').

Chapter 2 Methods for Containment

Section 1 Physical Containment

#1 Purpose and Outline of Physical Containment

1 The purpose of physical containment is to confine the recombinant organisms within the

equipment or the facilities, and thus to reduce the potentials for exposure to recombinant

organisms or laboratory personneI, other persons and the general environment.

2 Physical containment consists of three elements, namely 'containment equipment', 'special

laboratory design' and 'laboratory practices', and it is divided into four levels, namely, Pl,

P2, P3, or P4 depending on the level of containment.

#2 Levels of Physical Containment

1 Pllevel

210

(1) Containment equipment and laboratory design

Laboratories shall be equipped and designed at the same level as well designed ordinary

microbiological laboratories.

(2) Laboratory practices

1 Laboratory windows and doors shall be kept c10sed while experiments are in

progress.

2 Laboratory benches shall be decontaminated daily after the completion of the

experimental activity, and also immediately following spills of recombinant

organisms while experiments are in progress.

AppendixA

3 All biological wastes produced by the experiments shall be decontaminated before

disposal. Other contaminated materials shall be decontaminated before washing,

reuse, or disposal.

4 It is preferable to use mechanical pipetting devices. When pipetting by mouth,

cotton-plugged pipettes shall be employed.

5 Eating, drinking, smoking, and storage of food are not permitted in the laboratory.

6 Persons shall wash their hands after handling recombinant organisms, and when

they leave the laboratory.

7 Care shall be taken in the conduct of all procedures to minimize the creation of

aerosols.

8 Contaminated materials that are to be decontaminated at a site away from the

laboratory shall be placed in a durable leak-proof container which shall be tightly

closed before removal from the laboratory.

9 The laboratory shall be free of insects and rodents.

10 Use of hypodermic needles and syringes shall be avoided when alternative methods

are available.

11 Use of laboratory gowns or coats is at the discretion of the laboratory supervisor.

12 Other practices directed by the laboratory supervisor shall be used.

2 P2level

(1) Containment equipment

1 Biological safety cabinets shall be used to accommodate aerosol-producing

equipment such as blenders, lyophilizers, sonicators, and centrifuges when used to

process recombinant organisms, except where the equipment design provides for

containment of the potential aerosol.

2 Safety cabinets shall be set up in such a way that they can be inspected periodically,

have their HEP A filters (High Efficiency Particulate Air Filters) replaced, and be

fumigated with formaldehyde, without moving them. Immediately after setting up

and annually thereafter, the following inspections shall be performed. In cases

where air is exhausted from a safety cabinet into a laboratory, inspections are

required twice a year.

211


212

a Examination of air flow rate and volume or the supply air

b Examination of the degree of air-tightness

c Examination of the effectiveness of HEP A filters

(2) Laboratory design

A building containing a laboratory shall be equipped with an autoclave for the

sterilization of wastes and contaminated materials.


1 Laboratory windows and doors shall be kept closed while experiments are in

progress.

2 Laboratory benches and safety cabinets shall be decontaminated daily following the

completion of the experimental activity, and immediately following spills of

recombinant organisms while experiments are in progress.

3 All biological wastes produced by the experiments shall be decontaminated before

disposal. Other contaminated materials shall be decontaminated before washing,

reuse, or disposal.

4 Mechanical pipetting devices shall be used.

5 Eating, drinking, smoking, and storage of food are not permitted in the laboratory.

6 Persons shall wash their hands after handling recombinant organisms and when they

leave the laboratory.

7 Care shall be exercised to minimize the creation of aerosols in all manipulations.

For example, manipulations such as inserting an inoculation loop or ne edle so that

it splatters, and forceful ejection of fluids from pipettes or syringes shall be avoided.


laboratory shall be tightly placed in a durable leak-proof container which shall be

closed before removal from the laboratory.

9 The laboratory shall be free of insects and rodents.


are available.

11 The use of laboratory gowns or coats is required. Laboratory clothing shall not be

worn outside the laboratory.

AppendixA

12 Only persons who have been advised of the nature of the experiment being

conducted shall enter the laboratory.

13 A sign shall be posted on the entrance of the laboratory when experiments

requiring P2 containment are in progress. A sign shall also be posted on freezers

and refrigerators and other containers used to store recombinant organisms.

14 The laboratory shall be kept neat, clean, and free of materials not pertinent to the

experiments.

15 HEP A filters shall be decontaminated immediately before their exchange, at their

periodical inspections and when the experimental substances are changed by tightly

c10sing the safety cabinets, fumigation with formaldehyde at the concentration of

10 g/m3, and then leaving them for about 1 hour.

16 Experiments requiring PI containment can be carried out concurrently in carefully

demarcated areas of the same laboratory.


3 P3 level


1 Biological safety cabinets shaII be used for aII manipulations and equipment that

produce aerosols whenever recombinant organisms are processed, except where the

equipment design provides for containment of the potential aerosoI.


have their HEPA filters replaced, and be fumigated with formaldehyde, without

moving them. Immediately after setting up and annually thereafter, the following

inspections shall be performed. In cases where air is exhausted from a safety

cabinet into a laboratory, inspections are required twice a year.

a Examination of air flow rate and volume or the supply air


c Examination of the effectiveness of HEPA filters

213


214


1 A laboratory area shall be set up. The anteroom shall have two successive doors,

which shall be constructed so that both doors will not open at the same time, and

shall serve as achanging room.

2 A building containing a laboratory area shall be equipped with an autoclave for the

sterilization of wastes and contaminated materials.

3 The surfaces of walls, floors, and ceilings of the laboratory area shall be constructed

of materials and be of a design that are readily cleanable and capable of being

fumigated.

4 A foot- or elbow-operated, or automatically operated hand washing facility shall be

provided near the main exit of each laboratory and of each laboratory area.

5 Windows in the laboratory area shall be sealed.

6 Laboratory doors shall be self-closing.

7 Vacuum pumps for exclusive use shall be provided independently of ones located

outside the laboratory area. Vacuum outlets shall be protected by filter and liquid

disinfectant traps.

8 An exhaust air ventilation system shall be provided. This system shall be balanced

so that the direction of air flow is from the anteroom into the laboratory

environment. The exhaust air from the laboratory shall be discharged outdoors

after filtration or other treatment.


1 Laboratory doors shall be kept closed while experiments are in progress.

2 Work surfaces shall be decontaminated following the completion of the

experimental activity, and immediately following spills of organisms containing

recombinant DNA molecules while experiments are in progress.

3 All biological wastes shall be decontaminated before disposal. Other contaminated

materials such as glassware, animal cages, and laboratory equipment shall be

decontaminated before washing, reuse, or disposal.


AppendixA

5 Eating, drinking, smoking, and storage of food are not permitted in the laboratory

area.



7 Care shall be exercised to minimize the creation of aerosols in all manipulations.

For example, manipulations such as inserting an inoculation loop or needle so that

it splatters, and forceful ejection of fluids from pipettes or syringes shall be avoided.


laboratory shall be placed in a durable leak-proof container which shall be closed

before removal from the laboratory.

9 Tbe laboratory shall be free of insects and rodents.


are available.

11 Laboratory clothing, e.g., long-sleeved, solid-front gowns, button less slipover

jackets, etc., shall be worn in the laboratory. Laboratory clothing shall not be worn

outside the laboratory and shall be decontaminated before it is sent to the laundry.

12 Entry into the laboratory shall be through an anteroom. Only persons who have

been advised of the nature of the research being conducted shall enter the

controlled access area.

13 A sign shall be posted on the anteroom door and on all laboratory doors when

experiments requiring P3 level containment are in progress. A sign shall also be

pos ted on freezers and refrigerators used to store organisms containing

recombinant DNA molecules.

14 Tbe laboratory shall be kept neat, clean, and free of materials not pertinent to the

research.


periodical inspections and when the experimental substances are changed by tightly

closing the safety cabinets, fumigation with formaldehyde at the concentration of

10 g/m3, and then leaving them for about 1 hour.

215


16 Gloves shall be worn when handling materials. They shall be removed aseptically

immediately following the handling procedure and decontaminated.

17 Conducting experiments which require lower levels of containment is prohibited in

the same laboratory concurrently with experiments requiring P3 level containment.


4 P4level

216


1 A class 111 safety cabinet shall be set up for handling recombinant organisms.

However, persons wearing one-piece positive pressure isolation suits can conduct

experiments in Qass I or Class 11 safety cabinets that are located in a special

laboratory area.


have their HEPA filters (apparatus to trap fine grains through filtration) replaced,

and be fumigated with formaldehyde, without moving them. Immediately after

setting up and annually thereafter, the following inspections shall be performed.

In cases where air is exhausted from a safety cabinet into a laboratory, inspections

are required twice a year.

a Examination of air flow rate and volume


c Examination of the effectiveness of HEP A filters


1 The laboratory area shall be located in a restricted access facility which is either a

separate building or a clearly demarcated and isolated zone within a building, and

the approach of persons other than laboratory workers to the area shall be

prohibited.

2 An anteroom shall be provided equipped with achanging room, and a shower

room.

3 If materials, supplies, and equipment are not brought into the P4 facility through

the changing room, passage through an ultraviolet-irradiated anteroom having

AppendixA

successive doors shall be required. The anteroom doors shall be constructed so

that both doors will not open at the same time.

4 Walls, tloors, and ceilings of the laboratory area shall be constructed to allow vapor

phase decontamination and shall be animal- and insect-proof. The integrity of the

walls, tloors, and ceilings shall ensure adequate containment of a vapor phase

decontaminant under normal decontamination conditions. . However, this

requirement does not imply that these surfaces must be airtight.

5 A foot- or elbow-operated or automatically operated hand washing facility shall be

provided near each laboratory and primary laboratory exit area.

6 Laboratory doors shall be self-closing.

7 Where a central vacuum system is provided, it shall be exclusive to the facility to

which it belongs. The vacuum system shall include in-line HEP A filters as near as

practicable to each take-off point or service cock. The filters shall be installed so

as to permit in-place decontamination and replacement.

8 Water supply and liquid and gaseous services provided to the laboratory area shall

be protected by devices that prevent back tlow.

9 A pass-through autoclave equipped with successive doors (hereafter referred to as

an autoclave) shall be provided for the sterilization of material passing out of the

laboratory area. The autoclave doors shall be interlocked so that both doors will

not open at the same time.

10 A pass-through dunk tank or fumigation chamber shall be provided for the removal

of material and equipment from the laboratory area that cannot be heat sterilized.

11 An individual air supply and exhaust ventilation system shall be provided for the

laboratory area. The system shall maintain press ure differentials and directional

air tlow as required to assure intlow from areas outside towards areas of highest

potential risk. The system shall be designed to prevent the reversal of air tlow.

The system shall sound an alarm in the event of system malfunction.

12 Recirculation of air within individuallaboratories is permissible provided that this

air is filtered by a HEPA filter.

217


218

13 The exhaust air from the laboratory area shall be filtered by HEP A filters and

discharged outdoors so that it is dispersed clear of occupied buildings and air

intakes. The filter chambers shall be designed to allow in situ decontamination

before removal and to facilitate certification testing after replacement.

14 The treated exhaust air from Oass 111 cabinets shall be discharged outdoors. The

treated exhaust air from Oass land Oass 11 biological safety cabinets may be

discharged directly into the laboratory or outdoors. If the treated exhaust air from

these cabinets is to be discharged outdoors through the laboratory air exhaust

system, it shall be connected to this system in such a way as to avoid any

interference with the air balance of the cabinets or the laboratory air exhaust

system.

15 The speciallaboratory section in a laboratory area shall have the following facilities.

a A life support system shall be provided, with alarms and an emergency backup

air tank.

b Entry to the area shall be through an airlock fitted with airtight doors.

c A chemical shower area shall be provided to decontaminate the surfaces of the

suit before removal.

d The exhaust air from the speciallaboratory section shall be filtered by two sets

of HEP A filters installed in series.

e Two sets of exhaust fans shall be provided for safety.

f An emergency power source, emergency lighting and communication systems

shall be provided.

g The air pressure within the speciallaboratory section shall be less than that in

any adjacent area.

h An autoclave shall be provided for the sterilization of all waste materials to be

removed from the special laboratory section.


1 Laboratory doors shall be kept closed while experiments are in progress.

AppendixA

2 Work surfaces shall be decontaminated following the completion of the

experimental activity and immediately following spills of recombinant organisms

while experiments are in progress.

3 All biological wastes shall be decontaminated before disposal. Other contaminated

materials shall be decontaminated before washing, reuse, or disposal.


5 Eating, drinking, smoking, and storage of food are not permitted in the laboratory

area.



7 Care shall be exercised to minimize the production of aerosols. For example,

manipulations such as inserting a hot inoculation loop or needle into a culture,

flaming an inoculation loop or needle so that it splatters, and forceful ejection of

fluids from pipettes or syringes shall be avoided.

8 Biological materials to be removed from or brought into Oass III cabinets or

laboratory areas in a viable state shall be transferred to a non-breakable sealed

container which is then removed through a pass-through disinfectant dunk tank or

fumigation chamber.

9 All materials except for biological materials as described in 8 shall be sterilized or

decontaminated before removal from Class III cabinets or laboratory areas. All

wastes and other materials as well as equipment which will not be damaged by high

temperature or steam shall be sterilized in the autoclave. Other materials which

may be damaged by high temperature or steam shall be removed through a pass

through fumigation chamber.

10 Insects and rodents in the laboratory area shall be exterminated.

11 Use ofhypodermic needles and syringes shall be avoided when alternative methods

are available.

12 Only persons whose entry is required for experimental work or to confirm safety

shall be authorized to enter.

219


13 Personnel shall enter the experimental areas only through the anteroom. Personnel

shall shower at entry and egress.

14 After passing through a shower room in the laboratory area, personnel shall put on

full laboratory clothing including undergarments, pants and shirts or jumpsuits,

shoes, head cover, and gloves in the changing room of the experimental area.

Upon exit from the experimental area, this clothing shall be removed and stored

in collection hampers before personnel enter the shower room.

15 The universal biohazard sign is required on all doors, and freezers and refrigerators

used for storing recombinant organisms in the laboratory areas.

16 The laboratory shall be kept neat, clean, and free of materials not pertinent to the

research.


periodic inspections and when the experimental substances are changed by tightly

closing the safety cabinets, fumigating with formaldehyde at the concentration of

10 g/m3, then leaving them for about 1 hour.

18 Waste liquid to be removed from safety cabinets and laboratory sinks shall be

autoclaved. Waste water from shower rooms and hand washing facilities shall be

chemically decontaminated.

19 Conducting experiments which require lower levels of containment is prohibited in

the same laboratory concurrently with experiments requiring P4 level physical

containment.

20 Other practices directed by the laboratory supervisor shall be followed.

Section 2 Biological Containment

#1 Purpose and General Principles of Biological Containment

1 The purpose of biological containment is to prevent the propagation and dispersal of

recombinant organisms in the environment by using a host that can only survive under

220

AppendixA

restricted cultural conditions and a vector that can not be transmitted into living cells other

than those used for experiments.

Biological containment will also ensure biologically the safety of experiments involving

recombinant organisms by using a host-vector system that is ascertained to be safe.

2 For achievement of the purpose of biological containment, laboratory workers should

confirm, before and during the experiments, that both host and vector to be employed are

certified by the criteria for biological containment (defined in the guidelines).

3 When prokaryotes or lower eukaryotes are used as hosts, they are classified at either the

B1 or B2level of biological containment. The level of biological containment depends on

the degree of safety of the host-vector system. Based on consideration by the Science

Council Committee, the Minister of Education, Science and Culture will certify the level of

biological containment of individual host-vector systems (hereafter, this sort of arrangement

is referred to as 'Certification by the Minister of Education, Science and Culture').

4 When animal or plant-cultured cells are used as hosts, theyare classified at the B1level of

biological containment. However, if they are identified to be highly safe, they will be

classified at the B2 level after approval of the Minister of Education, Science and Culture.

#2 Levels of Biological Containment

1 B1level

The following cases will be categorized at the B1level.

Combination of a host with low potential of survival under natural conditions and a vector

with low potential for transmission to other cells because of the strong dependence on its

host propagation.

A host-vector system which provides biological safety to human beings on the basis of

evidence of characterization of its genetic, physiological, and ecological behavior under

natural conditions.

2 B2level

Among the host-vector systems categorized to the B1level, some (host-vector) systems that

are recognized to prevent the propagation and dispersal of recombinant organisms into the

221


environment can be categorized to the B2level. These host-vector systems can be provided

by the combination of a host with an extremely low potential for survival under the natural

conditions, and a vector with strong dependence on its host for propagation.

#3 Data to be Submitted for the Certification of Host-Vector Systems by the Minister of

Education, Science and Culture for Use in Biological Containment

In the certification of host-vector systems by the Minister of Education, Science and Culture,

the following factors shall be considered.

1 What is the reason to use the host-vector system for the experiment concemed?

2 Characterization of the host and vector to be used including the followings.

(1)

(2)

(3)

(4)

(5)

Data on ecological behavior in the natural environment

Data on physiological properties

Data on extent and mechanisms of genetic exchangeability

Data on pathogenicity and toxin production

Data on parasitism or saprophagy

(6) Data on history in terms of the relation of the strains to man

(7) Data on ease for sterilization in a laboratory

3 Properties of the host and the vector to be used, including the following:

(1) Source and background

(2) The range of organisms with which this organism normally exchanges genes

(3) Dependency of the vector on its host cells

4 For certification at the B2 level, data on procedures to construct the host and the vector,

with indication of the sources, properties, and methods of introduction of mutant genes, in

addition to the data itemized above.

5 Data that the host-vector systems achieve the B1 or B2 level of containment.

222

AppendixA

Chapter 3 Standards for Containment According to the Safety Assessment of

Experiments

Section 1 Safety Assessment of Experiments

#1 Principles of Safety Assessment in Experiments

In performing the experiments, laboratoryworkers should select suitable containment according

to the safety assessment for the components of the experiments. Safety in experiments shall

be ensured by means of a suitable combination of physical and biological containment along

with the general procedures usually employed in the pathogenic microbial laboratories.

In experiments using a host-vector which has been approved by the Minister of Education,

Science and Culture, the extent of safety shall be assessed, as described below, by consideration

of what new biological properties are donated to the host by inserting DNAs into the vector.

When unpurified DNAs are inserted iIito the vector, and when there is the possibility that all

genetic properties of the inserted DNAs are expressed in the recombinant organisms, safety in

experiments shall be assessed by the biological properties of the DNA-donor cells used. When

there is no possibility that the biological properties of DNA-donor cells itemized in #2 of this

chapter are expressed in the recombinant organisms, or when purified DNAs which do not

encode such biological properties are used, it can be assessed that the recombinant organisms

that are formed are safer then the DNA-donor cells.

When certain DNA donors in combination with a host-vector system which has been approved

by the Minister of Education, Science and Culture are indicated to be safe, safety in the

experiment with these DNAs should be assessed to be high.

#2 Estimation of the Degree of Safety based on the Biological Characteristics of DNA to be

inserted into vectors

223


Tbe degree of safety, taking into consideration the biological properties which are expressed

by DNAs to be inserted into the vector, is estimated by considering together the following

items.

(1) Is it pathogenic?

(2) Has it the ability to produce toxins?

(3) Is it parasitic or does it colonize?

(4) Has it carcinogenic genes or does it produce carcinogens?

(5) It is drug resistant?

(6) Does it produce substances, such as some kinds of hormones or metabolic

intermediates, which may disturb the metabolism in human beings and other

organisms?

(7) Does it cause ecological disturbances?

#3 Safety Assessment of DNA Donors in Experiments with Unpurified DNAs

1 Eukaryotes (except lower eukaryotes)

Laboratory workers should handle more carefully DNA from animals than that from plants,

because the former is phylogenically closer to human beings that the latter.

2 Lower Eukaryotes and Prokaryotes

DNAs from lower eukaryotes or prokaryotes, except the DNAs described in Supplement

2, will be assessed to be safe on the basis of accumulated evidence. Viruses of lower

eukaryotes or prokaryotes will be assessed as their host is.

3 Viruses, Rickettsials and Chlamydials of Eukaryotes (except those of lower eukaryotes)

Since the life cycle of viruses, rickettsials and chlamydials of eukaryotes is dependent on

their respective host cells, the experiments with whole DNA or DNA segment of these

parasites will be assessed to be safer then those with DNAs from their host cells.

#4 Safety Assessment in Experiments with Only Purified DNAs

When purified DNAs are characterized functionally and proven to carry only definitely non

hazardous genes, they may be considered to be safer than unpurified DNAs.

224

AppendixA

#5 Principle of Safety Assessment Based on the Number of Oones, and the Scale of Culture

1 Number of clones

When cloning unpurified DNAs, the probability of involvement of harmful genes decreases

with the decreasing number of clones involved, and the degree of safety thus increases.

2 Scale of culture

Even when potentially hazardous genes are involved, the degree of safety increases with a

decrease of their net amount as a result of decreasing the scale of experiments.

Section 2 Standards for Containment According to the Safety Assessment of Experiments

Based on the safety assessment of experiments, standards of containment except item (2) in

chapter 5 are established as folIows.

1 Standards for containment according to the safety assessment based on the biological

characteristics of DNA donors in experiments with unpurified DNAs are shown in the

following table. The experiments shall be conducted on a scale smaller that 20 liters except

those approved by the Minister of Education, Science and Culture.

225


Standards for Containment

DNAdonor

1. Animals (except those belonging to eukaryotes)

2. Plants (except those belonging to lower eukaryotes)

3. Lower eukaryotes and prokaryotes in Supplement 2-(1) and their viruses

4. Lower eukaryotes and prokaryotes characterized to be pathogenic, and their viruses

5. Lower eukaryotes and prokaryotes mentioned in Supplement 2-(2) and their viruses

6. Lower eukaryotes and prokaryotes mentioned in Supplement 2-(3) and their viruses

7. Lower eukaryotes and prokaryotes not included in item (3) or item (6), and their viruses

8. Viruses, rickettsials and chlamydials of eukaryotes (except those of lower eukaryotes) mentioned in Supplement 3-(1)

9. Viruses, rickettsials and chlamydials of eukaryotes mentioned in Supplement 3-(2)

10. Viruses, rickettsials and chlamydials of eukaryotes mentioned in Supplement 3-(3)

II.Viruses, rickettsials and chlamydials of eukaryotes mentioned in Supplement 3-(4)

226

Containment of BiologicIll cl: Physical Containment to be Adopted Bl B2

P2 PI

PI PI

Each experiment should be mentioned approved by the Minister of Education, Science and Culture

P3 P2

P2 PI

PI PI

Each experiment should be approved by the Minister of Education, Science and Culture

P3 P2

P2 PI

PI PI

12. Viruses, rickettsials and chlamydials of eukaryotes not included in item (9) to (11)

AppendixA

Each experiment should be approved by the Minister of Education, Science and Culture

2 In the following cases, (1) and (2), a single step reduction in physical or biological

containment described in the above table may be permitted after approval of the Minister

of Education, Science and Culture. The reduction does not apply to the case of PI and BI

levels:

(1) When the number of clones is very small (less than 100 clones in the case of

mammalian chromosomal segments with a mean molecular weight of 1()6).

(2) When purified DNAs are used.

3 Experiments with a host-vector system which has been certified by the Minister of

Education, Science and Culture, may be performed, after being reviewed by the Minister,

with a single step reduction in physical containment described in the table, if the DNA

donor used is confirmed to be not harmful by the Science Council. However, this reduction

does not apply to experiments in which certain specified DNA donor is used.

Chapter 4 Handling Recombinant Organisms

Section 1 Principle of Handling Recombinant Organisms

The physical containment level required for construction of recombinant organisms will also be

adapted for the handling of recombinant organisms.

Section 2 Propagation of Recombinant Organisms

#1 For propagation of recombinant organisms which have been constructed by use of a host

vector system certified by the Minister of Education Science and Culture, the administrators

227


of Universities etc. may give approval for a single step reduction in physical containment

corresponding to move then the P2 level mentioned in the previous chapter, after the

institutional safety advisory committee determines that the specified cloned DNAs are

probably free from harmful genes.

#2 For propagation of recombinant organisms which have been constructed by use of a host

vector system described in item (1) or chapter 5, the Minister of Education, Science and

Culture may give approval for a single step reduction in physical containment on receipt of

evidence indicating that the recombinant organisms are free from harmful genes.

#3 When an experiment described in item (2) of this chapter, which has been approved by the

Minister of Education, Science and Culture, is confirmed to be safe by the Science Council,

it can be conducted under the same experimental conditions (in terms of the same cloned

DNA, host-vector system, and physical containment) with the approval ofthe administrators

of universities etc. on behalf of the Minister of Education, Science and Culture.

Section 3 Preservation of Recombinant Organisms

#1 SampIes containing recombinant organisms should be marked "Recombinant Organisms"

and preserved in a laboratory or specified area where the conditions of physical

containment required for their preparation are certified.

#2 Safety officers must prepare and retain the list of sampies containing recombinant

organisms to be preserved.

Section 4 Distnbution and Shipment of Recombinant Organisms

#1 When laboratory workers distribute a sampie containing recombinant organisms form a

specified area to outside, they must put it in a bottle or can sealed tightly to avoid leakage

228

AppendixA

and pack it in a tight box with cotton or its equivalent resistant to damage by external

pressure.

#2 Mark 'Handle Carefully' with red marker on the surface of the container containing the

sampIe of recombinant organisms to be distributed.

#3 Whenever recombinant organisms are distributed from a specified area to outside, safety

officers must record the name and quantity of sampIes and the address (including names

of investigator and institute) to which the sampIe is sent.

#4 SampIes containing recombinant organisms requiring physical containment of more that the

P3level shall be shipped in compliance with the requirement issued by the Japanese postal

law, chapter 8, section 3, The Foreign Postallaw, chapters 68 and 69 and the International

Postal Treatment, chapters 119 and 120 (In this case, safety officers have to make records

for shipping the sampIe as is done in #3).

Chapter 5 Special Case Experiments

Experiments corresponding to any of the following items require approval of the Minister of

Education, Science and Culture, whenever planned. However, once the experiments cited in

item (1), which have been approved by the Minister of Education, Science and Culture, are

proven to be safe by the Science Council Committees, they can be {continued, performed,

conducted} only under the same experimental conditions (in terms of same DNA-donor, host

vector system and physical containment) with the approval of the presidents (administrators)

of universities on behalf of the Minister.

(1) Development of a new host-vector system that is not approved yet by the Minister of

Education, Science and Culture (Experiments concerning the development of a new

host-vector system).

(2) Experiments concerning the cloning of genes encoding proteineous toxins to vertebrates.

229


(3) Experiments concerning the inoculation of recombinant organisms into plants and

animals.

(4) Experiments involving the spreading of recombinant organisms in nature.

Chapter 6 Education, Training and Medical Precautions

#1 Education and Training

Laboratory supervisors and the administrators of universities etc. should ensure that laboratory

workers are familiar with the Guidelines and the internal rules, set up according to the

Guidelines to ensure safety (which are hereafter referred to as 'internal rules'). Moreover,

they should provide an opportunity for laboratory workers to obtain instruction and training on

the following subjects.

(1) Techniques for safe handling of microorganisms corresponding to the level of their

possible hazard

(2) Knowledge and techniques for physical containment

(3) Knowledge and techniques for biological containment

(4) Knowledge of the degree of hazard involved in experimental systems to be dealt with

(5) Knowledge of the procedures for dealing with accidents

#2 Medical Precautions

1 The administrators of universities etc. shall ensure that laboratory workers take medical

examinations before starting experiments and at intervals of less than one year after starting

the experiments.

2 When pathogenic microorganisms are handled by laboratory workers, the administrators of

institutions shall survey appropriate medical precautions and treatments and, if necessary,

make preparations before starting experiments, for example by obtaining antibiotics,

vaccinating agents, sera, etc. They shall ensure that laboratory workers take medical

examinations at intervals of less than 6 months.

230

AppendixA

3 When experiments are performed in experimental areas over the P3 level, the

administrators of universities etc. shall obtain sera for the laboratory workers before the

start of the experiments, and the sera should be stored for two years after the experiments

are finished.

4 When there is a possibility that a laboratory worker may have been infected in a laboratory,

the administrator of his university etc. shall request him to take a medical examination

immediately, and appropriate treatment should be carried out.

S The administrators of universities etc. shall record and file the results of such medical

examinations.

6 When one of the following accidents occurs or when areport prescribed by 7 is received,

the administrator of the university etc. shall investigate immediately, and shall arrange for

appropriate medical precautions.

(1) Accidental ingestion or inhalation of recombinant organisms.

(2) Contamination of the skin by recombinant organisms.

(3) Serious contamination of experimental areas by recombinant organisms when a

laboratory worker is present.

7 Bach laboratory worker shall pay attention to his health, and when his health becomes

irregular or when he has a serious long term illness, he shall report his condition to the

administrator of his university etc ..

Chapter 7 Organization for Ensuring the Safety of Experiments

#1 Laboratory Supervisors

Laboratory supervisors shall be weil acquainted with the Guidelines and the internat rules and

shall have knowledge of and training in the necessary techniques for preventing biohazards and

other relevant matters. They shall be responsible for the following measures.

231


1 To follow the Guidelines and the internal roles in planning and conducting experiments, and

to manage and supervise all experiments properly in close association with the Safety

Officer.

2 To perform necessary educational instroction for laboratory workers as described in #1 of

Chapter 4.

3 To submit experimental plans to the administrator of the university etc. and to obtain bis

approval. This also applies when an experimental plan is changed.

4 To take necessary steps to ensure the safety of experiments.

#2 Administrators of universities etc.

Administrators of universities etc. bear the responsibility for the safety of experiments

performed by researchers belonging to their institutions. They shall take the following

measures.

1 To set up a Safety Committee and to appoint a Safety Officer.

2 To approve or to disapprove individual experimental plans following examination by the

Safety Committee, and to obtain prior judgement by the Minister of Education, Science and

Culture for work requiring certification or approval by the Minister of Education, Science

and Culture according to the Guidelines.

3 To establish internal roles on matters necessary for ensuring experimental safety.

4 To take medical precautions on behalf of laboratory workers following the advice of the

Safety Committee as described in #2 of Chapter 4.

5 To carry out other tasks necessary to ensure experimental safety.

#3 Safety Committee

1 A Safety Committee shall be established in an university etc. conducting experiments.

2 The Safety Committee shall consist of members representing the relevant fields, and having

high standards of both professional and technical knowledge and judgement.

232

AppendixA

3 The Safety Committee shall investigate and deliberate the following matters upon

eonsultation by the administrator of the university ete., and advise hirn on the necessary

proeedures.

(1) Establishment and revision of internat rules

(2) Acceptability of experimental plans in terms of the Guidelines and the internal

rules

(3) Planning of education and training programs

(4) Neeessary aetions and proeedures to improve seeurity based on the results of

investigation of the cireumstanees in the event of an aeeident in a laboratory

(5) Other matters neeessary to ensure experimental safety

4 The Safety Committee may request the Safety Offieer to present neeessary reports, and to

give adviee on relevant matters.

#4 Safety Offieer

1 To assist administrators of universities ete. a Safety Offieer shall be appointed in eaeh

institution.

2 The Safety Offieer shall be a person having knowledge of the Guidelines and the internal

rules, and shall be bighly experienced in the teehniques of biohazard prevention. His role

is as folIows.

(1) To confirm that experiments are being performed properly according to the

Guidelines and the internat rules.

(2) To give guidance to laboratory supervisors, and to give them necessary advice.

(3) To carry out other tasks necessary to ensure experimental safety.

3 The Safety Offieer shall maintain dose eontaet with the Safety Committee in bis task of

ensuring safety and shall present reports to the eommittee as neeessary.

233


Notes:

1 These Guidelines are effective August 31, 1982.

2 In this guideline, EK1 defined in Chapter 2, Section 2, #2-1-(1) of the Guidelines for

Recombinant DNA Experiments in Universities and Other Research Institutes (Notice #42

of The Ministry of Education, Science and Culture, 1979) (hereafter referred to 'Old

Guideline'), SC1 (Notice #55 and 171, 1980), and BS1 (Notice #46, 1981) are approved

to be a host-vector system of B1level, respectively, by the Minister of Education, Science

and Culture.

3 In this guideline, EK2 defined in Chapter 2, Section 2, #1-3 of the old guideline is approved

to be a host-vector system of B2 level.

234

AppendixA

Supplement 1 Standards of Safety Cabinets

#1 Oass land Oass 11

1 General remarlcs

A Oass I cabinet is a protective unit for laboratory workers. It is a ventilated cabinet

having a fixed open front (hereafter called an 'open front') for carrying out experiments.

The cabinet is designed so that air flows inwards away from the operator and is exhausted

from the cabinet through a HEP A filter, thus protecting the operator from possible leakages

of contaminated aerosols produced during experiments.

A Class 11 cabinet is equipped for the protection of laboratory workers and experimental

materials. In addition to the common features with the Cl ass I cabinet, it is designed so

that clean air always flows from the top to the bottom in the cabinet, and air is exhausted

from the cabinet through a HEPA filter.

2 Standard for average face velocity of air flow through the open front

The face velocity of the inward flow of air through the open front shall be 0.4 rn/sec or

greater. This applies not only to a simple cabinet but also to a cabinet connecting to a duct.

The face velocity of air through the open front shall be estimated by measuring the air

velocity in the duct and dividing it by the area of the open front.

3 Standard of downward air flow (Oass 11)

Oean air passed through a HEP A filter shall be recirculated as downward air flow in a

Class 11 safety cabinet. The velocity of air flow of the recirculated air shall be 0.2 rn/sec or

greater, and the coefficient of variation shall be within +/- 20% when the velocity is

measured at 15 - 30 different points above the open front in the cabinet.

4 Standard of gas-tight construction

The standard of gas-tight construction when the open front and the exhaust duct are closed

shall be as follows.

(1) When the difference between the air pressure in the cabinet and atmospheric

pressure is 50 mm H20, soapsuds rubbed on junctures do not foam.

235


(2) When the cabinet is under a positive pressure of 50 mm H20 of Freon gas, the

amount of the gas escaping through the junctures is 10-4 cc/sec or less at points 5

mm distant from the surface.

5 Test of air flow using spores (Class 11)

The procedures used to test the air flow through the open front in Oass 11 cabinets, are as

folIows.

(1) Spraying spores of B. subtüis var. niger in the safety cabinet, and measuring the

fraction that escapes from the cabinet.

(2) Spraying the spores outside the cabinet, and measuring the fraction that enters the

cabinet.

(3) Spraying the spores at a point in the cabinet, and measuring the spreading of the

spores within it.

6 Specification of the HEP A filter

The HEP A filter shall have the ability to remove particles of 0.3" or greater diameter

contained in air passed through it at an efficiency of 99.99% or greater. HEP A filters are

tested for leakage using DOP (dioctyl phthalate) particles (0.3" in diameter) and a partic1e

counter.

7 Volume of liquid receptacles

A liquid receptac1e in a safety cabinet shall have a capacity of more than 4 liters and be

soundly constructed.

#2 Oass 111

A Oass 111 cabinet is a closed front, ventilated cabinet of gas-tight construction, which is fitted

with arm length rubber gloves through which experiments are performed by outside operators.

An autoclave and a pass-through dunk tank or a facility for sterilization by fumigation shall be

provided. The air supply is filtered through HEP A filters. Exhaust air is filtered through

HEP A filters or incinerated before being discharged to the outside environment. The cabinet

is opera ted under a negative pressure of 1.5 cm H20.

236

AppendixA

Supplement 2

Oassification of the degree of safety of lower eukaryotes and prokaryotes used as the DNAdonors.

(1) Bartonella bacilliformis Oostridium botulinum Oostridium tetani Corynebacterium diphtheriae Mycoplasma mycoides Pasteurella multocida (B:6, E:6, A:5, A:8, A:9) Pseudomonas (Actinovacillus) mallei Pseudomonas pseudomallei Shigella dysenteriae Yersinia pestis (Yersinia pseudotuberculosis sub sp. pestis)

(2) Bacillus anthracis Brucella abortus Brucella melitensis Brucella suis Coccidioides immitis Cryptococus neoformans Francisella tularensis Histoplasma capsulatum Histoplasma duboisii Mycobacterium africanum Mycobacterium bovis Mycobacterium tuberculosis Salmonella paratyphi-A Salmonella typhi

(3) Actinobacillus

Actinomyces

Aeromonas

Arizona

All species except for A. mallei (Pseudomonas mallei)

A. bovis A israelii A naeslundii

A hydrophila (Toxin producing strain) A punctata (Toxin producing strain )

A hinshawee (all antigenic type)

237


Bacillus

Blastomyces

Bordete11a

Borrelia

Brucella

Calymmatobacterium

Campylovacter

Clostridium

Corynebacterium

Entamoeba

Erysipelothrix

Escherichia

Haemophilus

Hartmane11a

Here11ea

238

B. cereus (Toxin producing strain B. thuringiensis

B. dermatitidis

All species

All species

B. canis

C. granulamatis

All species

C. chauvoei C. difficile C. equi C. haemolyticum C. histolyticum C. novyi C. perfringens (Toxin producing strain) C. septicum

C. equi C. haemolyticum C. pseudotuberculosis C. pyogenes C. renale

E. histolytica

E. rhusiopathiae E. insidiosa

E. coli (all enteropathogenic, antigenic type)

H. ducreyi H. influenzae

All species

H. vaginicola

Klebsiella

Legionella

Leishmania

Leptospira

Listeria

Mima

Moraxella

Mycobacterium

Mycoplasma

Naegleria

Neisseria

Nocardia

Paracoccidioides

Pasteurella

Plasmodium

Plesiomonas

All species

L pneumophila

All species

L interrogans (all antigenie type)

L monocytogenes

M. polymorpha

All species

M. avium - M. intracellulare complex M. kansasii M. marinum M. paratuberculosis M. scrofulaceum M. ulcerans

M. pneumoniae

All species

N. gonorrhoeae N. meningitidis

N. asteroides N. brasiliensis N. caviae N. farcinica

P. brasiliensis

All species except P. multocida

P. falciparum P. malariae P. ovale P. vivax Simian malarial parasites

P. shigelloides

AppendixA

239


Salmonella

Shigella

Sphaerophorus

Staphylococcus

Streptobacillus

Streptococcus

Treponema

Trichinella

Toxocara

Toxoplasma

Trypanosoma

Vibrio

Yersinia

240

All serotypes except S. paratyphi-A and S. typhi

All species except S. Dysenteriae

S. necrophorus

S. aureus

S. moniliformis

S. pneumoniae S. phygenes

T. carateum T. pallidum T. pertenue

T. spiralis

T. canis

T. gondii

T. cruzi T. gambiense T. rhodesiense

V. cholerae (including Biotype EI Tor) V. parahaemolyticus

Except Y. enterocolitica and Y. pseudotuberculosis (yercinia pestis (Yercinia pseudotuberculosis subsp. pestis))

AppendixA

Supplement 3

Classification of the degree of safety of viruses, rickettsials and chlamydials of eukaryotes (except those parasites of lower eukaryotes) used as DNA donors

(1) African horse sickness virus African swine fever virus Colorado tick fever virus Congo hemorrhagic fever virus Coxiella burnetii Creutzfeldt-Jakob disease agent Ebola virus Foot-and-mouth disease virus Hemorrhagic fever with renal syndrome virus Herpes B virus Junin virus Kyasanur forest disease virus Lassa fever virus Machupo virus Marburg disease virus Rickettsia prowazekii Rickettsia rickettsii Rickettsia tsutsugamushe Rickettsia typhi Rift Valley Fever virus Rinderpest virus Russian spring-summer encephalitis virus Scrapie agent Tick-borne encephalitis virus Variola major virus Variola minor virus Venezuelan encephalitis virus Whitepox virus Yellow fever virus

(2) Adult T -cell leukemia virus California encephalitis virus Chikungunya virus Chlamydia psittaci Herpes ateles virus Herpes saimiri virus Hog cholera virus

241


Human T -celllymphoma virus Japanese encephalitis virus La Crosse virus LCM virus Monkeypox virus Murray Valley encephalitis virus O'nyong-nyong virus Powassan virus Rabis (street) virus St. Louis encephalitis virus Tacaribe virus Vesicular stomatitis virus West Nile virus

(3) Adenovirus (human) A vian reticuloendotheliosis virus Batai virus BK virus Bovine papilloma virus Chlamydia trachomatics Cowpox virus Coxsackie virus (A, B) Cytomegalovirus (human, animal) Dengue virus (1-4) Bastern equine encephalitis virus EB virus Echovirus (1-34) Ectromelia virus Enterovirus (68-71) Equine infectious anemia virus Equine rhinopneumonitis virus Hepatitis A virus Hepatitis B virus Hepatitis (non-A, non-B) virus Herpes simplex virus (1, 2) Human wart virus (Human papilloma virus) HVJ Influenza virus (human) JC virus Mammalian retrovirus (except Adult T-cellleukemia virus and Human T-celllymphoma vin.6 Measles virus Molluscum contagiosum virus

242

Mouse hepatitis virus Mumps virus NDV Parainfluenza virus (1-4) Pichinde virus Poliovirus (1-3) Polyoma virus Rabis (fixed, attenuated) virus Rhinovirus Rinderpest virus (vaccine strain) Rotavirus Rubella virus Semliki Forest virus SSPE agent SV 40 Tanapox virus Vaccinia virus Varicella virus Western equine encephalitis virus Yaba virus

(4) Adenovirus (avian, bovine, porcine) Aino virus Akavane virus Avian encephalomyelitis virus Avian enterovirus Avian poxvirus Avian retrovirus (except Avian reticuloendotheliosis virus) Bluetongue virus Bovine enterovirus Bunyamwera virus Canine distemper virus Coronavirus Duck hepatitis virus Fish viruses (IPN, IHN, EVA, EVE, LV) Getah virus Influenza virus (avian, equine, swine) Insect viruses (except the viruses pathogenic to vertebrates, such as Arbovirus) Langat virus Uve virus vaccine strains (except Rindepest vaccine strain) Lucke virus Mareck's disease virus

AppendixA

243


Parvovirus Plant viruses Poikilothermal vertebrate retrovirus Reovirus (1-3) Ross River virus Shope fibroma virus Simbu virus Sindbis virus Swinepox virus Viroids

244

A P P END I X B

MITI'S REGUlATIONS FOR

LARGE-SCALE INDUSTRIAL PROCESSES

Chapter 1. General Provisions

Section 1. Purpose

It is the purpose of the present guidelines to provide the basic conditions for securing adequate

safety in the application of DNA recombinant technology to various industrial processes, including

manufacturing and mining, thus providing complete safety and promoting appropriate use when

applying recombinant DNA technology.

Section 2. Definition of terms

In these guidelines, terms are defined as follows:

1. 'Recombinant DNA technology' refers to techniques involving preparation of a recombinant

deoxyribonucleic acid (DNA) molecule - a gene - replicable in a vital cell, and a heterologous

DNA with the use of, for example, an enzyme in vitro, and introducing the obtained

recombinant molecule into said vital cell to thereby replicate the heterologous DNA. It

further includes techniques wherein the vital cell to which the recombinant DNA molecule

is introduced thereby, which will be called a 'recombinant' hereinafter, is used, except in

such a case where a vital cell having the same genetic construction as that of the vital cell to

which the recombinant DNA is introduced occurs in nature.

2. A 'recombinant DNA molecule' refers to a recombinant molecule of DNA replicable in a

vital cell with a heterologous DNA.

245


3. A 'recipient organism' refers to avital ce11 to which a recombinant DNA molecule is

introduced.

4. A 'vector' refers to DNA wbich carries a heterologous DNA to a recipient organism in the

recombinant DNA technology.

5. A 'DNA donor' refers to a ceIl, a microorganism, or the like which provides the DNA to

be introduced into a vector. When DNA synthesized with the use of RNA as a template is

to be introduced in a vector, a ceIl, a microorganism or the like which provides the RNA is

caIled the 'DNA donor'.

6. 'Purified DNA' refers to identified DNA synthesized from a DNA donor and includes

cloned DNA as weIl as chemicaIly synthesized DNA

7. 'Work area' refers to an area where operations required for manufacturing, mining or the

like are performed.

8. 'Work site' refers to each site in a work area where a recombinant is directly handled using

equipment and apparatus for operations such as culture, fermentation, isolation and

purification.

Chapter 2 Evaluation of Recombinant's Safety

Section 1. Rules

Tbe person in charge of a working organization shaIl evaluate thoroughly the safety of a

recombinant through evaluation of the safety of the employed recipient organism and recombinant

DNA molecule and comparison of the properties of the recombinant with those of the recipient

organism. Since it is difficult to uniformly evaluate every recombinant, he (she) shaIl evaluate the

necessary items for the safety of recombinants selected from among those cited in Section 2, using

bis (her) particular business as a basis in classifying the recombinant's safety

Section 2 Items for evaluation

1. Recipient organism

(1) Taxonomy

246

AppendixB

a. Names and designation

b. Characteristics of the organism which permit identification: Traits in common with

and departing from a species having an authorized scientific name; source of strain;

the organization with which the derived-type culture is deposited and the

deposition number etc.

c. Reproductive cycle (sexuaVasexual)

(2) Genetic characteristics

a. History of prior genetic manipulation

b. Techniques for detecting the organism

c. Factors which might limit the reproduction, growth and survival of the recipient

organism; stability of genetic traits

(3) Pathogenic and physiological traits of recipient organism

a. Capacity for colonization

b. Nature of pathogenicity

c. Details of the pathogenic effects, if any;

Diseases caused in humans; nature ofvirulence (Le., invasiveness, communicability,

infective dose and toxigenicity); recipient organism range and possibility of

alteration; possibility of survival outside of human recipient; presence of vectors or

means of dissemination; biological stability; antibiotic resistance patterns;

allergenicity; availability of appropriate prophylaxis and therapies; availability of

medical surveillance, etc.

d. Other physiologically important properties, e.g., possibility of acquiring

pathogenicity through infection by a particular phage.

e. Stabilities of these properties

(4) Prior reports of an extended history of safe industrial use, if any

2. Recombinant DNA molecule

(1) Construction of recombinant DNA molecule

a. Identification of a vector and its properties

247


b. Nature, functions and method of preparation of the DNA to be inserted into the

vector

c. Method for the introduction of the DNA to be inserted into the vector

(2) Description of the method by which the recombinant has been constructed

a. Description of methods for introducing the recombinant DNA molecule into the

recipient organism and procedure for selection of the modified organism

b. Stability of the inserted DNA

c. Frequency of mobilization of inserted vector and/or genetic transfer capability

In addition to the items cited in (1) and (2) above, the following properties shall be evaluated

in cases where the evaluation of a recombinant DNA molecule is insufficient

248

(3) Properties of DNA donor and vector donor

1. Taxonomy

a. Name and designation

b. Characteristic of the organism which permits identification: Traits in common with

and departing from a species having an authorized repository with which the

derived-type culture is deposited and the deposition number etc.

2. Pathogenic and physiological traits of recipient organism

a. Capacity for colonization

b. Nature of pathogenicity

c. Details of the pathogenic effects, if any: Diseases caused in humans; nature of

virulence (Le., invasiveness, communicability, infective dose and toxigenicity);

recipient organism range and possibility of alteration; possibility of survival outside

of human recipient; presence of vectors or means of dissemination; biological

stability; antibiotic resistance patterns; allergenicity; availability of appropriate

prophylaxis and therapies; availability of medical surveillance, etc.

d. Other physiological important properties, e.g., possibility of acquiring pathogenicity

through infection by a particular phage.

e. Stabilities of these properties

AppendixB

3. Recombinant

(1) Gene expression

a. Method for expression

b. Product, rate and level (when the product is a protein or peptide) of the

introduced genetic material and method and sensitivity of the measurement

(2) Comparison with recipient organism

a. Conditions restricting survival and growth

b. Capacity for colonization

c. Nature of pathogenicity

d. Details of the pathogenic effects, if any: Diseases caused in humans; nature of

virulence (i.e., invasiveness, communicability, infective dose and toxigenicity);

recipient organism range and possibility of alteration; possibility of survival outside

of human recipient; presence of vectors or means of dissemination; biological

stability; antibiotic resistance patterns; allergenicity; availability of appropriate

prophylaxis and therapies; availability of medical surveillance, etc.

e. Other physiological important properties, e.g., possibility of acquiring pathogenicity

through infection by a particular phage.

f. Stabilities of these properties

Section 3. Safety evaluation and cIassification

1. Safety evaluation and cIassification of recipient organism. The person in charge of working

organization shall evaluate the safety of a recipient organism and assign it to one of the

following cIassifications.

(1) Good Industrial Large-Scale Practice (GILSP)

A recipient organism should be non-pathogenic; should not contain adventitious agents

such as pathogenic viruses, phages and plasmids; and should have an extended history

of safe industrial use, or have built-in environmental limitations that permit optimum

249


growth in an industrial setting but limited survival without adverse consequences in the

environment.

(2) Category 1

A non-pathogenic recipient organism which is not included in the above GILSP.

(3) Category 2

A recipient organism having undeniable pathogenicity for humans that might cause

infection when directly handled. However, the infection will probably not result in a

serious outbreak in cases where effective preventive and therapeutic methods are

known.

(4) Category 3

A recipient organism capable of resulting in disease and not included in Category 2

above. It shall be carefully handled but there are known effective preventive and

therapeutic methods for said disease.

A recipient organism which, whether directly handled or not, might be significantly

harmful to human health and result in a disease for which no effective preventive nor

therapeutic method is known, shall be assigned a classification separate from Category

3 and treated in a special manner.

2. Safety evaluation and classification of recombinant

250

The person in charge of a working organization shall completely evaluate the safety of a

recombinant based on evaluation of the safety of the recipient organism and a comparison

of the properties of the recombinant DNA molecule and those of the recipient organism, and

shall classify the same into GILSP, Categories 1, 2, 3, or any other established categories.

When the recombinant DNA molecule has safe properties and a comparison of the

recombinant with the recipient organism that the safety level of the former seems to be the

same or higher than that of the latter, the safety or the recombinant shall be regarded as the

same as that of the recipient organism. In order to be classified into GILSP, recombinant

shall meet the following conditions.

(1) Recipient organism

It shall be a recipient organism to be classified into GILSP.

Appendix B

(2) Recombinant DNA molecule

The DNA to be inserted shall be well-characterized and free from known harmful

sequences, limited in size as much as possible to the DNA required to perform the

intended function, poorly mobilizable, not capable of transferring any resistance marker

to microorganisms not known to acquire them naturally (if such acquisition could

compromise use of drugs to control disease agents).

(3) Recombinant

It shall be non-pathogenic, not increase the stability of the construct in the environment,

as safe in the industrial setting as the recipient organism, and without any adverse

consequence in the environment unless it is a requirement of the intended function.

Chapter 3. Equipment, Apparatus, Operations and Management for Recombinants

Section 1. Rules

The person in charge of a working organization shall evaluate the safety of equipment and

apparatus by which a recombinant is treated. Further he (she) shall keep equipment and

apparatus required for preventing or minimizing leakage of the recombinant at the work site or

work area in perfect operating condition depending on the safety level of the recombinant and

obey the operation and control rules so as to secure the greatest amount of safety in the treatment

of the recombinant.

Section 2 Equipment and Apparatus

1. Rules for safety evaluation

The person in charge of a working organization shall evaluate the safety of equipment and

apparatus based on the specifications set forth in Chapter 2, Section 3 and shall make a

classification based on an evaluation of the safety of the recombinant's properties as set forth

in Chapter 2, Section 2.

251


Since industrial equipment and apparatus are employed in various ways depending on, for

example, the scale of production and application of the recombinant, he (she) shall evaluate

the safety of equipment and apparatus used in each step such as culture and fermentation,

isolation, and purification.

2. Items for evaluation

252

(1) Extent to which equipment and apparatus can be sealed effectively

a. Viable organisms should be handled in a system that physically separates the

process from the external environment (a closed system)

b. Treatments of exhaust gases from the closed system

c. Performance of seals

(2) The closed system should be located within a work site

a. Designation of a work site

b. If a work site has been designated:

(a) Biohazard signs should be posted

(b) An air lock controlling ingress and egress should be established

(c) Decontamination and washing facilities should be provided for personnel

(d) Personnel should shower before leaving the work site

( e ) Effluent from sinks and showers should be collected and inactivated before

release

(f) Work site should be adequately ventilated to minimize contamination

(g) Work site should be maintained at less than atmospheric air pressure

(h) Input air and extract air to the work site should be HEPA filtered

(i) Work si te should be designed to contain spillage of the entire contents of the

closed system

0) Work site should be sealable to permit fumigation

3. Speeification for evaluating equipment and apparatus:

Classification

0/ recombinant

Evaluation item 1

(1) a

b

e

(2) a

b (a)

(b)

(e)

(d)

(e)

(f)

(g)

(h)

(i)

0) Notes:

GILSP

semi-closed2

minimize

release3

minimize

release

optional

no

no

optional

no

no

optional

no

no

no

no

Category

1

yes

minimize

release

minimize

release

optional

optional

no

yes

no

no

option al

no

no

no

no

Category

2

yes

prevent

release

prevent

release

yes

yes

no

yes

optional

optional

optional

optional

optional

optional

optional

Corresponding to the items as set forth in Chapter 2.

AppendixB

Category

3

yes

prevent

release

prevent

release

yes and

purposebuilt

yes

yes

yes

yes

yes

yes

yes

yes

yes

yes

2 A system wherein well-repaired equipment and apparatus are used and follows a closed

system. 3 Release shall be deereased to an appropriate level depending on the safety level of

reeombinant.

253


Section 3. Operation and Management

The person in charge of a working organization shall obey the following operation and control

roles.

1. Inoculation, transfer and sampling of recombinant

(1) Recombinants classified into GILSP or Category 1, which will be hereinafter referred

to as GILSP or Category 1 respectively, shall be inoculated into a culture and

fermentation apparatus in such a manner as to minimize its release. This will be

achieved by, for example, preventing the recombinant from adhering to the external wall

of the culture and fermentation apparatus. Recombinants classified into Category 2 or

3 shall not be released. If release occurs, disinfection shall be performed by validated

means.

(2) A GILSP or Category 1 recombinant shall be transferred from one vessel to another as

to minimize its release. Category 2 or 3 recombinants shall be transferred in such a

manner as to prevent their release with the use of, for example, pipettes. If release

occurs, disinfection shall be performed by validated means.

(3) A GILSP or Category 1 recombinants shall be sampled from a culture and fermentation

apparatus or other equipment or apparatus in such a manner as to minimize its release.

This will be achieved by, for example, preventing the recombinant from adhering to the

external wall of said apparatus. Category 2 and 3 recombinants shall be sampled in

such a manner as to prevent their release. If release occurs, disinfection shall be

performed by validated means.

2. Washing, disinfection or sterilization of equipment and apparatuses

Equipment and apparatuses where a GILSP or Category 1 recombinant is treated shall be

disinfected and washed following use. Those where a Category 2 or 3 recombinant is treated shall

be inactivated by validated means.

254

AppendixB

3. Treatment of waste, including liquid waste

In order to prevent release of a recombinant outside a work area, bulk quantities of culture fluids

obtained from the treatment of Category 1 recombinant should not be removed from the system

unless the viable organisms have been inactivated by validated means. Those obtained from

treatment of a Category 2 or 3 recombinant should not be removed from the system unless the

viable organisms have been inactivated by validated chemical or physical means.

4. General attention to operational performance

(1) Personnel should wear clothing in a work site. In work sites where a Category 3

recombinant is treated, personnel shall completely change his (her) cloth es and shower

before leaving work site.

(2) A controlled area shall be indicated 'Treating GILSP', 'Treating Category 1',

'Treating Category 2' or 'Treating Category 3' during the operation depending on the

evaluation of the safety of the recombinant to be treated.

(3) In a work site where a GILSP or Category 1 recombinant is treated, exhaust gases

should be treated to minimize the release of viable organisms. Care shall be taken to

avoid the recombinant being inhaled or swallowed inadvertently. In a work site where

a Category 2 or 3 recombinant is treated, the release of viable organisms shall be

prevented.

(4) A work area shall be kept clean. Rodents such as mice and insects such as mosquitoes

shall be exterminated at a work site where a GILSP or Category 1 recombinant is

treated. A work site where a Category 2 or 3 recombinant is treated shall be free from

all rodents and insects.

5. Storage of recombinants

(1) A material containing a recombinant shall be so labelIed. Those containing a Category

2 or 3 recombinant shall be stored containing a recombinant shall, at a prominent place,

display signs reading 'Treating GILSP', 'Treating Category 1', 'Treating Category 2'

or 'Treating Category 3' depending on the evaluation of the safety of the stored

recombinant.

255


(2) A detailed catalog shall be made of all stored materials containing recombinants.

6. Transportation of recombinants

(1) A material containing a recombinant shall be transported outside a work site in a sealed

bottle or can to prevent the contents from being released. A Category 2 or 3

recombinant shall be transported in a particularly rigid sealed container.

(2) A box in which a material containing a recombinant is placed shall be labelIed 'Handle

with Care' in prominent red lettering.

7. Maintenance of equipment and apparatus

(1) The extent to which equipment or apparatus can be sealed shall be regularly and

adequately examined. An apparatus for removing bacteria from a work site where a

Category 2 or 3 recombinant is treated shall be examined twice a year.

(2) The extent to which modified or exchanged parts of sealing equipment or apparatuses

can be sealed shall be examined with each change.

(3) In the treatment of Category 2 or 3 recombinant, the sealing capacity of a culture and

fermentation apparatus, and equipment or apparatuses directly connected thereto, shall

be examined in an appropriate manner during the operation.

(4) All equipment and apparatuses used in the treatment of a Category 2 or 3 recombinant

shall be given an identifying number and strictly controlled.

(5) An apparatus for removing bacteria shall be sterilized by validated me ans at each

exchange and routine examination as weIl as whenever the operation is varied.

Chapter 4. Management and Responsibility System

Section 1. Organizer of working organizations

The organizer of working organizations shall perform the following duties.

256

Appendix B

1. He (She) shall continuously collect information regarding recombinant DNA technology.

When he (she) notices something affecting the evaluation of the corresponding recombinant,

he (she) shall immediately report this to the Minister of International Trade and Industry.

2. In order to secure the maximum amount of safety, he (she) shall make sure the working

organization carries out the prescribed duties as set forth in Section 2.

Section 2. The head of working organizations shall perform the following duties.

1. (1) He (She) shall appoint directors(s) of operation and manager(s) for safe operation to

assist the former.

(2) He (She) shall appoint in advance substitutes for the director(s) of operation and the

manager(s) for safe operation who are capable of executing during such times as when

travel or disease prevents the director( s) or manager( s) from performing their

prescnbed duties.

2. He (She) shall set up a committee for safe operation and appoint members whose role will

include questioning all aspects affecting the safety of the operation.

3. He (She) shall make sure the director( s) of operation carry out their duties as set forth in

Section 3.

4. He (She) shall make sure the manager(s) of safety operations carry out their duties as set

forth in Section 6.

5. He (She) shall pay special attention to the health of the personnel.

Section 3. Director of operations

A director of operations shall fully understand the present guidelines and perform the following

duties.

1. He (She) shall fully obey the present guidelines in the planning and carrying out of an

industrial process and shall adequately manage the whole operation under close contact with

the manager(s) to provide maximum safety.

2. He (She) shall train personnel for operation prior to the initiation of the operation.

257


3. He (She) shall indicate prominently information indicating the category of recombinants

under treatment and in storage.

4. He (She) shall restrict entrance of persons other than regular personnel into a work site and

shall forbid those who have no knowledge about the operation to enter the same. When

persons other than the regular personnel enter the work site, they shall follow personnel's

instructions.

5. He (She) shall prepare books and note the following items therein. There books shall be

stored for five years after the completion of the corresponding industrial application.

(1) Name of recombinant and identifying number on its container

(2) Storage and passage of recombinant

(3) Biological properties of recombinant and date of the examination of the same

(4) Name, address, purpose and system of organization with which the recombinant is

deposited

(5) Result of medical examination

(6) Record of review by committee for safe operation including references used for judging

the suitability of the treatment of the recombinant

(7) Record of regular examination and operation of equipment and apparatuses

Section 4. Operations personnel

Personnel for operation shall perform the following duties.

1. He (She) shall fully und erstand how to exercise sufficient care to ensure safety.

2. He (She) shall give appropriate instructions to those not concerned with operation who must

enter the work site.

Section 5. Committee for safe operations

1. A committee for safe operations shall be established for every working organization.

258

AppendixB

2. Tbe committee for safe operations shall consist of members in the appropriate fields since

appropriate judgement requires highly expert knowledge of techniques as well as an overall

view.

3. In response to questions brought up by the person in charge of the working organization, the

committee for safe operations shall discuss and provide advice on the following items.

(1) Suitability of recombinant treatment

(2) Safe operations training and adequate health measures for workers

(3) Necessary treatment and improvements designed to minimize accidents

(4) Other items required for securing the safe operations

4. Tbe committee for safe operations can request areport from director( s) of operations or

manager(s) for safe operations if necessary.

Section 6. Manager for safe operations

1. A manager for safe operations shall be selected from among those having sufficient technical

knowledge pertaining to the prevention of biohazards and related issues.

2. He (She) shall fully understand the present guidelines and perform the following duties.

(1) He (She) shall confirm whether the operation is properly performed according to the

present guidelines.

(2) He (She) shall advise director( s) of operations.

3. Tbe manager for safe operations shall perform his (her) duties under close contact with the

committee for safe operations. Further, he (she) shall report necessary items in response to

requests from the committee for safe operations.

Section 7. Training of personnel

A director of operations shall ensure that personnel fully understand the present guidelines prior

to the initiation of operations and shall train them on the following items.

(1) Knowledge of the safety of recombinants

(2) Treatment of different recombinants depending on their level of safety

259


(3) Knowledge and techniques regarding equipment and apparatuses

(4) Knowledge of the safety of the operations to be performed

(5) Knowledge on the occurrence of accidents

Section 8. Hea1th care

1. The person in charge of a working organization shall perform a medical examination of

personnel prior to the initiation of the operation and at intervals of not longer than one year

thereafter.

2. He (She) shall discuss in advance preventive and therapeutic methods for workers who treat

Category 2 or 3 recombinants

3. He (She) shall ensure that personnel at work sites, who are in the danger of being

contaminated by Category 2 or 3 recombinants, are medica1ly examined immediately and he

(she) take proper action.

Serum of personnel treating a Category 3 recombinant shall be collected before the

beginning, and stored for two years after the completion of the operation.

Chapter 5. Others

1. In order to secure their safety, the organizer of a working organization can request the

Minister of International Trade and Industry to authorize that the equipment, apparatuses,

operations and management of the industrial application of recombinant DNA technology

conforms with the present guidelines.

2. The present guidelines shall be applied tentatively to cases where the organism used as a

DNA donor belongs to the same species as that of the organism used as a recipient organism

from a taxonomical viewpoint, and where avital cell having the same genetic construction

as that of the vital cell to which a recombinant DNA molecule is introduced occurs in nature.

260

A P P END I X C

FUIL TEXT OF NOTIFICATION OF APPLICATION DATA

FOR RECOMBINANT DNA DRUGS

Notification No. 243 ofthe Pharmaceutical Affairs Bureau, Ministry ofHealth and Welfare, March

30, 1984

To: The Prefectural Governments

Director of the Evaluation and Registration Division

Director of the Biologics and Antibiotics Division

Pharmaceutical Affairs Bureau

Ministry of Health and Welf are

Preparation of Data Required for Approval Applications for Drugs Manufactured by the

Application of Recombinant DNA Technology

These matters will be handled as follows and your cooperation in informing the related persons

in your jurisdiction of the following specifications would be appreciated.

This notification shall apply to drugs with peptides or proteins manufactured by the application

of recombinant DNA technology as the main ingredients (hereinafter referred to as 'recombinant

drugs').

The terminology in this notification shall be defined as folIows:

261


1 Reeombinant DNA teehnology shall refer to teehnology by wbieh reeombinant moleeules

of DNA whieh ean be propagated in living cells and heterogenie DNA and produeed in

vitro by enzymes, ete., are transferred into the appropriate living cells and produee

heterogenous DNA [teehniques whieh use living eells into wbieh DNA reeombinant

moleeules have been transferred as a result of the applieation of these teehniques

(hereinafter referred to as 'reeombinants') shall be included. However, eases in whieh

living eells with the same genetie strueture as the living eells into whieh the DNA

reeombinant moleeules were transferred and present in nature shall be excluded.]

2 Host shall refer to the living eells into which the DNA reeombinant moleeules are

transferred, veetor shall mean the DNA earrying the heterologous DNA transferred into

the host by reeombinant DNA teehnology, and the host-veetor system shall mean the

eombination of the two.

Contents

I. Qassifications eoneeming the ranges of attaehed data

Sinee reeombinant DNA teehnology is a eompletely new manufaeturing method, drugs

manufaetured by tbis method shall be handled as follows for the time being until data are

aeeumulated eoneeming safety and effieacy.

262

1. Drugs in a to e shall, in principle, be handled as drugs with new aetive ingredients in 1-

(1) of table 2-(1) in Notifieation No. 698 of the Pharmaeeutieal Affairs Bureau, dated

May 30, 1980.

a. Reeombinant drugs with aetive ingredients which have not previously been

approved for reeombinant drugs.

b. Reeombinant drugs for whieh the method of obtaining the struetural genes of the

target peptides, ete. differ from those approved reeombinant drugs.

Appendix C

c. Recombinant drugs for which the host-vector system used in the manufacture

differs from that of approved recombinant drugs.

2. Drugs in d to f shall, in principle, be handled as 1-(8) other drugs in the table of the

above Notifications.

d. Recombinant drug for which the composition of the medium used in the culture

of the recombinants differs from that of the approved recombinant drugs.

e. Recombinant drugs for which the purification method differs from that of

approved recombinant drugs.

f. Other recombinant drugs.

However, in the case of the drugs in d and e, the following data shall be submitted in

addition to those specified in 1-(8) in the table.

1) Manufacturing method, determination of the structure, physicochemical

properties, etc.

2) Toxicity tests, antigenicity and pyrogenicity tests related to impurities.

3) Oinical trials with detailed investigations to confirm safety (at least 2 institutions,

at least 20 cases per institution).

11. Method of preparation of attached data

The data which must be attached to applications for approval of manufacture or import of

recombinant drugs shall be prepared with consideration given to a) to g) for each classification.

(a) Data conceming the origin and background of the discovery, use in foreign countries, etc.

When there are recombinant drugs of the same type developed or approved in foreign

countries, detailed explanations shall be given conceming conditions of use, incidence of side

effects, etc.

263


(b) Physicochemical properties and standards and test methods

264

1. Manufacturing method

Detailed data shall be collected conceming the following points.

(1) Structural genes of the peptide or protein concemed

1) The method of obtaining the structural genes concemed or their

corresponding mRNA fragments shall be clear.

2) The total base sequence shall be clear.

3) The function of the cloned genes and the stability of the base sequence shall

be confirmed.

4) When mRNA corresponding to the structural genes concemed is extracted

from tumor tissue, it is recommended that equivalence with normal tissue be

confmned.

(2) Host-vector relation

The properties of the host-vector relation shall be clear.

(3) Cultures

1) The stability of the recombinant (such as the stability during preservation

and passaging of the recombinant) shall be clear. Consideration shall also

be given to mass cultures.

2) The identification method of the recombinant shall be clear.

3) The composition of the culture medium shall be clear. It is recommended

that synthetic media with as simple a composition as possible be used.

(4) Purification

1) The purification process shall be explained as part of the flowsheet of the

manufacturing method.

2) The method of separating the target peptide, etc. and the heterogenous

protein or polysaccharide, etc. of microbial origin shall be clear.

3) In cases where there is chemical separation of excess peptide, etc. added to

the N-terminal by means of bromocyano decomposition, etc. to stabilize

proteins which are apt to decompose in the cells, the reagents used and the

method used to eliminate the separated peptide, etc. shall be clear.

Appendix C

4) When the target substance is separated from a high molecular protein, etc.

as aprecursor such as proinsulin, the enzymes used in the separation and the

method of elimination of the separated peptide, etc. shall be c1ear.

2. Determination of the structure and physicochemical properties

In principle, data concerning the following properties shall be collected.

(1) Structure and composition

The following items shall be c1ear.

1) Amino acid structure

2) Terminal amino acids

3) When there are disulfide bonds, their position

4) Peptide analysis

5) Amino acid sequence (in cases of high molecular substances, the terminal

amino acid sequence in the possible range)

(2) Physicochemical properties

The following items shall be investigated.

1) Spectral properties (ultraviolet absorption spectrum, etc.)

2) Electrophoretic properties (polyacrylamide gel electrophoresis, etc.)

3) Isoelectric point (sucrose density gradient, isoelectric point electrophoresis,

gel isoelectric point electrophoresis, etc.)

4) Molecular weight (SDS gel electrophoresis, gel filtration chromatography,

ultracentrifugation, etc.)

5) Liquid chromatograph pattern

6) Higher order structure (optical rotatory dispersion, circular dichroism, etc.)

(3) Immunochemical properties

These properties shall be investigated using such methods as immunoassay

and immunoelectrophoresis.

(4) Biological properties

The following items shall be c1ear.

265


266

1) Biological aetivity, content and purity (specifie aetivity, ete.)

2) In the ease of enzymes, enzymoehemical properties, ete.

3. Standards and test methods

The standards and test methods used to clarify the eharaeteristics of the reeombinant

drug shall be speeified for the following items.

(1) Origin and nature

It shall be clearly indicated that the substance eoneemed is a reeombinant

drug.

(2) Appearance

1) Color, taste and odor

2) Solubility, erystallinity and stability (hygroseopicity, photolysis, ete.)

(3) Identification

In addition to tests utilizing ehemieal reaetions, bioassays and immunoassays

shall be used.

(4) Component amino acids

(5) Peptide analysis

It shall be specified in prineiple.

(6) Purity

In addition to the same items for ordinary drugs sueh as solubility, items

eoneeming separation or deteetion of polypeptides, proteins, deeomposition

produets, ete. origination from baeteria or the eulture medium shall be

speeified using liquid ehromatography, radioimmunoassay, enzyme

immunoassay, ete. Tests for heavy metal and arsenie shall be specified in

eonsideration of the manufaeturing method, direetions and dosage, ete.

(7) Loss on drying or water eontent

(8) Residue on ignition

(9) Biological aetivity

Reeombinant drugs are generally 'biologieal preparations' and in many

eases it is diffieult to prove the equivalenee, purity, potency or safety of

Appendix C

complex substances of biological origin only by physicochemical methods.

Therefore, the use of special property tests shall be investigated.

(10) Antigenicity

The test shall be specified in principle.

(11) Pyrogenicity

(12) Assay

( c) Stability

The assay shall be specified using either physicochemical or biological activity

tests. When physicochemical tests are specified, the correlation with the

activity shall be confirmed.

Stability shall be investigated in the same way as for ordinary drugs.

( d) Toxicity

The amino acid sequence which is the active ingredient of recombinant drugs is completely

the same as ingredients originating from the living body, and if the toxicity of such ingredients

has already been studied, the toxicity tests within the range of toxicity originating from

impurities which has been sufficiently confirmed may be omitted.

The standards for performing toxicity tests shall be as follows in principle. When toxicity

tests are performed, they shall be based on the tests given in the Toxicity Test Guideline.

1. Acute toxicity, subacute toxicity and mutagenicity

The tests shall be performed in accordance with the methods used for

ordinary drugs.

2. Chronic toxicity, effects on reproduction, dependence, local irritation and

carcinogenicity

The tests shall be performed in accordance with the methods used for

ordinary drugs. However, such tests can be omitted if there is a valid reason.

267


3. Antigenicity

(1) The antigenicity of the active ingredients shall be investigated. However, this can

be omitted if there is a valid reason.

(2) Since there is a possibility of an immune response to minute amounts of

contaminants originating from the manufacturing method, this shall be

investigated using such methods as radioimmunoassay or enzyme immunoassay.

In such cases, the detection sensitivities of the test methods shall be clear.

4. Pyrogenicity

Pyrogenicity shall be investigated by the pyrogen test using rabbits or by the

limulus test. It is also recommended to investigate other test methods to

detect other pyrogens.

( e ) Pharmacological action

The investigation shall be the same as in the case of ordinary drugs. However, if the amino

acid sequence which is the active ingredient of the recombinant drug is exactly the same as

an ingredient originating from the living body, and the pharmacology of that ingredient has

already been studied, the investigation can be omitted with the exception of the following

tests.

1. Basic pharmacological tests on efficacy including comparisons with similar drugs

originating from the living body.

2. When required, tests confirming the following properties including proof of the

equivalence of the higher order structure with that of similar drugs originating from the

living body.

(1) Binding to receptors, bin ding conditions and binding affinity

(2) When there are various types of target cells, biological effects on such cells

(f) Absorption, distribution, metabolism and extraction

These shall be investigated in the same way as for ordinary drugs.

268

Appendix C

(g) Oinical trials

The trials shall be performed with care phase I, 11 and 111, and precise and objective

observations shall be made.

The following items shall be investigated in detail for recombinant drugs.

1. Local and systemic allergies

2. Antibody production (antibodies to the active ingredients and antibodies responding to

host antigens)

3. Changes at administration sites

4. Changes in pharmacokinetics due to interaction with circulating antibodies

5. Pyrogenicity

In cases where the substance originating from the living body is already applied clinically,

alterations in antibodies, variations in action, etc. shall be observed and compared in patients using

the substance originating from the living body and in those using the recombinant drug. If

required in consideration of the predicted treatment period, number of patients, etc., precise and

objective comparative trials shall be performed.

269

A P P END I X D

GUIDELINES FOR MANUFAcruRING DRUGS EIe.

BY APPUCATION OF RECOMBINANT DNA TECHNOLOGY

Chapter 1. General Provisions

1. Purpose

The purpose of the Guidelines is to establish basic requirements for the application of

recombinant DNA technology in the manufacture of drugs, quasi-drugs, cosmetics, and medical

devices (hereinafter referred to as 'drugs etc.'), so that the quality and manufacturing safety of

drugs etc. is assured.

2. Definitions

(1) 'Recombinant DNA technologyl means a technology by which recombinant molecules

are produced in vitro by joining in vivo replicable DNA (deoxyribonucleic acid, basic

principle of gene, and hereinafter referred to as such) molecules to heterologous DNA

segments (including DNA synthesized by the use of an RNA template, and hereinafter

referred to as such) under the effect of enzyme( s) or the like, and then making the

recombinant molecules migrate into living cells to propagate the heterologous DNA

molecules. This technology also includes the use of living cells into which recombinant

DNA molecules have migrated by application of the above method (hereinafter referred

to as 'recombinantsl).

(2) 'Hostl means a living cell into which the recombinant DNA molecule migrates.

(3) 'Vectorl means a DNA molecule which carries the heterologous DNA to the host by

recombinant technology.

270

Appendix D

(4) 'GILSP' (Good Industrial Large-Scale Praetiee, hereinafter referred to as sueh),

'Category 1,' 'Category 2,' and 'Category 3' mean, respeetively, the biosafety levels

of manufaeturing operations aeeording to the safety evaluation of a reeombinant.

(5) 'Manufaeturing area' means an area where the reeombinants are handled in the

manufaeturing operation.

(6) 'Manufaeturing plant' means a loeation where a manufaeturing operation is performed,

including, in addition to the manufaeturing area, an area where the reeombinants are

not direetly handled.

(7) 'Master eell bank' means an original seed lot of a reeombinant from which all the

manufaeturing seed lots are made. Generally, a master eell bank eonsists of aliquots of

a single eulture of the reeombinants, which have been prepared and cloned in the

experimental stage and stored in a manner whieh gives reasonable assuranee of genetie

stability, after the genetie eharaeter of obtained reeombinants was eonfirmed to be

sufficiently stable within a eertain range of serial subeultures.

3. Manufaeturing Conditions

The manufaeturing of drugs ete. by the applieation of reeombinant DNA teehnology must be

earried out under eonditions deseribed in Chapter 2 and subsequent ehapters.

Seleetion ofthe level ofthe manufaeturing operation from among GILSP, and Categories 1, 2 and

3, for the reeombinant to be employed in the manufaeture of a drug ete. shall be based on

information (viz., eharaeteristies of the host, veetor, genetie information inserted into the veetor,

eharaeteristies of the reeombinant, ete.) obtained during the experimental stage. (N otes)

The essential characteristics of living cells employed in manufacturing in accordance with GILSP

are described in Table D-l, and the eharaeteristies of recombinants which can be employed for

manufaeturing in accordance with Category 1, 2 or 3 in Table D-2.

Notes:

(1) The safety evaluation of a recombinant should be made, in principle, on the nature of

the recombinant itself. In addition to the safety evaluation of the host, review of

271


characteristics of the vector/inserted DNA and comparison of the properties of the

recombinant and the host cells should be made.

(2) A recombinant to be used for the manufacture of a drug etc. has been cloned in the

experimental stage. Characteristics of the vector and of the inserted genetic

information, physicochemical and biological properties of the product obtained, and the

nature of the recombinant itself have also been thoroughly studied in that stage. These

studies in the experimental stage provide useful data for the safety evaluation of the

recombinant to be used in the manufacture of a drug etc.

Conceming the safety evaluation of recombinants, the following can be stated on the basis

of research carried out so far.

272

(i) The majority of microorganisms now used in traditional manufacturing can be

regarded as safe on the ground that they have rarely given rise to safety problems

in the course of long periods of industrial use. In addition, microorganisms which

exhibit optimum proliferation und er conditions of industrial application but only

limited proliferation in the natural environment, and free of any adverse effects

on the environment, are also considered to be safe.

(ii) In the same way, modified microorganisms obtained by introducing segments of

DNA that are well-characterized, and free from known harmful base sequences

into microorganisms, are also unlikely to pose any incremental risk compared to

the unmodified host microorganisms.

(iii) In cases where microorganisms, which have been known to be safe, are modified

by introducing segments of DNA to facilitate the manufacture of a new product

which does not present any safety problems beyond those that might be posed by

the products themselves. In extremely rare cases where a safety problem is posed,

manufacturing should be carried out under appropriate containment.

(3) Accordingly, the manufacture of a drug etc. using a recombinant obtained by introducing

highly safe genetic information into a host-vector system which is well-recognized as safe,

can be carried out by employing the facilities which have been used in conventional

methods of manufacturing drugs etc. after safety verification of the recombinant. Such

AppendixD

manufacturing method is called GILSP-conforming manufacture. The characteristics

required for hosts, vector/inserted gene (DNA), and recombinants which are acceptable

for GILSP are set forth in Table D-1.

In the case of living cells which do not meet the criteria stipulated in Table D-1,

manufacture should be carrled out at the level of Category 1, 2 or 3 on the basis

of a safety evaluation made on the organism in question.

The classification into GILSP and a manufacturing level of Category 1, 2 or 3

should be performed on the basis of safety evaluations of the recombinant in

accordance with the above section (1).

Chapter 2 Premises and FaciIities

1. Buildings and Facilities for GILSP

(1) There should be a manufacturing area.

(2) The manufacturing area should maintain the following standards:

(i) The manufacturing area should be separated from other areas.

(ü) The manufacturing area should be provided with a well-arranged culture

apparatus.

(3) Facilities for analysis and testing of the biological properties of the recombinant should

be provided.

(4) The following facilities should be provided:

(i) Facilities for storage of recombinants.

(ü) Facilities for the preparation of culture media.

(iii) Facilities for washing and sterilization of equipment, utensils, containers, and other

items which are used in the manufacturing operations or in analysis and testing.

(iv) Dressing facilities for manufacturing personnel.

273


(5) Any other necessary facilities and equipment should be provided.

2. The buildings and facilities for Categories 1, 2 and 3 should satisfy the conditions of

containment criteria described in Table D-3, in addition to the conditions described in 1

above.

Chapter 3. Personnel and Organization

1. Manufacturer

A person who employs recombinant DNA technology in the manufacturing process of drugs etc.

(hereinafter referred to as 'manufacturer') should perform the following duties:

(i) To designate at each manufacturing plant the manufacturing director (identical

with 'responsible technician' or 'responsible engineering manager' in the case

of quasi-drugs, cosmetics and medical devices, and hereinafter referred to as such),

and a manufacturing safety manager.

(ii) To establish a manufacturing safety committee, and to designate the members

thereof in order to assure manufacturing safety. To re quest the manufacturing

safety committee to investigate and discuss manufacturing safety assurance.

(iii) To ensure that the manufacturing director fulfills his duties without impediments.

2. Manufacturing Director

The manufacturing director should have a full understanding of the Guidelines and perform the

following duties:

274

(i) To comply with the provisions of the Guidelines in the formation and execution

of the manufacturing plan, and to maintain appropriate control and supervision

of all manufacturing operations in close communication with the manufacturing

safety manager.

(ii) To give education and training to manufacturing personnel.

(iii) To keep the manufacturing plant clean and to make efforts to eliminate rodents

such as rats, and insects such as flies and mosquitoes.

AppendixD

To effect complete extermination of these animals and insects in the

manufacturing area for Categories 2 and 3 operations.

(iv) To post a list of necessary information conceming the manufacturing level of the

recombinant in conspicuous places in the manufacturing area and the recombinant

storage facility.

(v) To limit the entry of persons other than manufacturing personnel into the

manufacturing area, and to direct such persons to follow the instructions of the

manufacturing personnel when they enter the area.

(vi) To maintain good communications with the manufacturing safety committee, and

to report any necessary information to the committee.

(vii) To take, in addition to the above, measures necessary for the assurance of the

quality and the manufacturing safety of drugs etc.

3. Manufacturing Safety Manager

(1) The manufacturing safety manager should assist the manufacturing director in the

operation conceming recombinant DNA technology, and possess advanced knowledge

to assure safety in manufacturing operations.

(2) The manufacturing safety manager should have a full understanding of the Guidelines

and perform the following duties:

(i) To confirm that the manufacturing operations are carried out in conformity with

the Guidelines.

(ii) To give advice and submit reports to the manufacturing director.

(iii) To handle, in addition to the above, any necessary items with regard to

manufacturing safety assurance.

4. Manufacturing Personnel

(1) Manufacturing personnel should be persons who have received education and training

from the manufacturing director.

(2) Manufacturing personnel should observe the following rules:

275


(i) To be fully aware of, and to pay much attention to, the maintenance of quality of

the product and safety during the manufacturing operation.

(ii) To wear in the manufacturing area special working clothes according to the

manufacturing level.

(üi) To displaya sign in the respective manufacturing area describing the operation

being performed during the manufacturing operation which is in accordance with

the GILSP or Category 1, 2 or 3.

5. Manufacturing Safety Committee

(1) The manufacturing safety committee, which is in a position to make judgments on the

basis of highly specialized knowledge, technology, and a broad understanding, should be

composed of members from appropriate fields.

(2) The manufacturing safety committee should investigate the following matters at the

request of the manufacturer, and report the results to the manufacturer:

(i) Conformity of manufacturing standards to the Guidelines.

(ii) Status of safety education and training, and health care of manufacturing

personnel.

(üi) Countermeasures and methods of improvement in the event of an accident.

(iv) In addition to the above, any necessary matters related to the safety assurance in

the manufacturing operations.

(3) The manufacturing safety committee may, as occasion demands, request reports from

the manufacturing director and the manufacturing safety manager.

Chapter 4. Operating Control

1. Control of Facilities and Equipment

276

(1) After completion of manufacturing operations, used facilities and equipment should be

thoroughly disinfected or sterilized.

Appendix D

(2) Culture equipment, sterile filtration facilities, and the Iike should be inspected for

airtightness and other functions immediately after their installation and periodically

thereafter.

(3) Whenever parts which may influence the function of the facilities and equipment are

modified or replaced, the airtightness and other functions of such facilities and

equipment should be tested.

(4) In the manufacturing areas of Category 2 or 3, the functions of culture equipment and

machinery, and facilities attached directly thereto should be verified by appropriate

measures during the manufacturing operations.

(5) Sterile filtration facilities should be sterilized by a method previously determined to be

effective at the time of replacement, periodic inspections and a change in products to

be manufactured.

(6) In the case of manufacturing under Categories 2 and 3, a11 facilities and equipment

employed in the manufacturing operations should bear serial numbers and be kept

under strict control.

2. Prevention of Contamination

(1) In a Category 1 manufacturing area, care should be taken to minimize the leakage of

aerosols from the facilities and equipment. In Category 2 and 3 manufacturing areas,

leakage of aerosols should be prevented.

(2) When seeding the culture equipment with a recombinant, or when collecting sampIes

from the culture equipment in a Category 1 manufacturing area, care should be taken

to minimize contamination of the outer wall or other parts of the culture equipment.

In Category 2 and 3 manufacturing areas, such contamination should be prevented. In

the event of contamination, such areas should be disinfected immediately by a method

previously recognized as effective.

(3) When transferring a recombinant from one culture to another or to other facilities or

equipment in a Category 1 manufacturing area, care should be taken to minimize

contamination by leakage of the recombinant. In Category 2 and 3 manufacturing areas,

277


such contamination should be prevented. In the event of contamination, such areas

should be disinfected immediately by a method previously recognized as effective.

(4) When the isolation process of an intended product, which is easily inactivated like a

protein, is carried out in Category 1, 2 or 3 manufacturing areas, treatment of the

culture fluid may be carried out in a Category 1 area under conditions that minimize a

leakage of recombinants, and in Category 2 and 3 areas und er the condition that a

leakage of recombinant is prevented.

(5) Attention should also be paid in a GIlSP manufacturing area to contamination with

recombinants during the operations described above.

(6) Countermeasures for massive leakage of culture fluid containing recombinants and

specific emergency procedures should be established.

3. Handling of Recombinants

(1) Storage

278

(i) Materials which include recombinants should be clearly labeled Recombinants.

Category 2 and 3 recombinants should be safely stored in a storage facility within

the manufacturing area.

(ii) In storage facilities for recombinants, a sign corresponding to the level of

manufacturing operations, i.e., tGILSP recombinants in storage,' tCategory 1

recombinants in storage,' tCategory 2 recombinants in storage,' or tCategory 3

recombinants in storage,' should be pos ted in a conspicuous place.

(iii) The manufacturing director should prepare and retain a list of any materials in

storage which contain recombinants.

(2) Transport

(i) In the case of transporting materials containing recombinants outside the

manufacturing area, such material should be placed in a bottle or can, and the

container should be sealed hermetically to prevent leakage of the contents.

In the case of transporting materials containing Category 2 or 3

recombinants outside the manufacturing area, care should be taken to

AppendixD

prevent the contents of the container from leaking in the event of damage

to the container.

(ü) The box used as a container for a material containing recombinants should be

clearly labeled on a conspicuous part of its surface with a waming in red 'Handle

with care.'

(3) Tests of Biological Properties

(i) At the time of preparation and during storage of the master cell bank, the stability

of the recombinant should be verified by conducting tests on the following items:

a. Actual production of the intended substance.

b. Maintenance of the basic structure of the vector/inserted genes

preserved in the recombinant.

c. Other items conceming the identification and homogeneity of the

recombinant.

(ii) In cases where the tests performed during storage of the master cell bank indicate

occurrence of a mutation affecting the quality or safety of the product,

manufacture should be stopped immediately, and appropriate countermeasures

should be taken.

(üi) Any other tests, deemed to be necessary, should be conducted.

4. Education and Training

The manufacturing director, prior to initiating the manufacturing operations, should familiarize

the manufacturing personnel with the provisions of the Guidelines, and should give them

education and training on the following matters:

(i) Information on safety of the recombinant.

(ii) Techniques related to the safe handling of recombinants for manufacture.

(üi) Information and techniques conceming the facilities and equipment.

(iv) Information on safety in the manufacturing processes.

(v) Information conceming countermeasures to be taken in the event of an accident.

279


5. Health Care

(1) The manufacturer shall arrange for periodic health examinations of manufacturing

personnel. In addition, the manufacturer should not allow any person unsuitable for

handling drugs to be engaged in manufacturing operations.

(2) The manufacturer should investigate measures for prevention and treatment of infection

with the recombinant prior to allowing manufacturing personnel to engage in Category

2 or 3 manufacturing operations.

(3) In cases where there is a possibility of infection with the recombinant occurring in the

manufacturing area for Category 2 or 3 manufacturing operations, the manufacturer

should immediately arrange for health examinations of the manufacturing personnel, and

should take appropriate countermeasures.

The manufacturer should collect aserum sampIe from manufacturing personnel

in Category 3 manufacturing operations prior to the initiation of the manufacturing

operations, and should preserve this sampIe for two years after the day the

personnel stops working in the manufacturing operations.

6. Records and Their Retention

280

(1) The manufacturing director should maintain arecord book, and should record the

following items:

(i) The name of the recombinant and the number attached to the container for the

recombinant.

(ü) The status of storage and passage of the recombinant.

(iii) The biological properties of the recombinant, and the date tests were performed.

(iv) The name and address of the parties from which the recombinant was obtained.

(v) Results of physical examinations.

(vi) Records of discussions by the manufacturing safety committee (including materials

used as a basis for confirming the manufacturing standards conform to the

Guidelines ).

(vü) Records of periodic inspections of facilities, equipment, and manufacturing

records.

AppendixD

(2) The record book should be retained for five years from the day manufacture of the drug

was completed.

7. Reports

The manufacturer should collect information on recombinant DNA technology. In cases where

there is some information which might influence the evaluation of the recombinant, the

manufacturer should immediately submit areport on that information to the Minister for Health

and Welfare.

Chapter 5. Miscellaneous Provisions

(1) In order to assure the quality of drug etc. and the safety in the manufacturing processes

for the use of recombinant DNA technology, the manufacturer may ask the Minister for

Health and Welf are whether the facilities and equipment, and the method of operation

conform to the Guidelines.

(2) The Guidelines should also apply, with necessary modifications, to the manufacture of

investigational new drugs and investigational medical devices involving recombinant

DNA technology.

281


282

Table D-l.

Essential Cbaracteristics of living Ceßs To Be Used in GILSP Manufacturing

Host Vector/lnserted Gene Recombinant

1. Nonpathogenic. 1. Well-characterized and 1. Nonpathogenic. free from known harmful base sequences.

2. Not contaminated by 2. To provide the intended 2. In the case of industrial exogenous factors (viruses function, minimize the size use, the safety should be etc.) related to of the inserted gene. equal to that of the host. pathogenicity. Except in cases where Should have a limited

required in view of the proliferation ability in the intended function, do not environment and should use a vector/inserted gene not exert any adverse capable to increase effect on the environment. stability of the recombinant in the environment.

3. A long record of safety 3. Poor ability to be in industrial use, or transmitted. capable of optimal proliferation in an industrial setting, while showing only a limited proliferation ability in the environment without adverse effects on the environment.

4. Do not transmit a resistance marker to a living cell which has never been known to acquire the resistance in question.

Table D-2.

Characteristics of Recombinants for Category 1, 2, and 3 Manufacturing

AppendixD

Category Characteristics 0/ Recombinant

1 Nonpathogenic. Exclude recombinants corresponding to GILSP.

2 Rarely develops a disease, though infectious, in humans, and has preventive measures and effective therapy.

3 Is pathogenic in humans, and requires very careful handling. Infections and diseases, even if caused, are comparatively less dangerous, and have preventive measures and effective therapy.

Note: Recombinants for manufacture of drugs etc. possessing pathogenicity in excess of Category 3 require special handling.

283

~

1. P

roce

ss f

or h

andl

ing

a re

cod)

inan

t

2. E

xhau

st g

as f

rom

a c

lose

d sy

stem

3.

Sam

plin

g,

addi

tion

of

mat

eria

ls t

o a

clos

ed s

yste

m,

and

tran

sfer

of

reco

mbi

nant

to

oth

er c

lose

d sy

stem

s

4. T

rans

fer

of c

ult

ure

flu

id f

rom

the

cl

osed

sys

tem

to

an

open

sys

tem

5.

Des

ign

for

seal

ing

a cl

osed

sys

tem

6. C

ondi

tion

s of

a m

anuf

actu

ring

are

a ha

ving

a c

lose

d sy

stem

6A.

Bio

haza

rd s

igns

68.

Ent

ry o

f pe

rson

s ot

her

than

ass

igne

d m

anuf

actu

ring

per

sonn

el

6C.

Clo

thin

g of

man

ufac

turi

ng p

erso

mel

6D.

Dec

onta

min

atio

n fa

ci l

itie

s an

d w

ashi

ng

faci

l iti

es

for

man

ufac

turi

ng p

erso

mel

6E.

Show

er f

acil

itie

s fo

r m

anuf

actu

ring

pe

rson

nel

whe

n le

avin

g a

man

ufac

turi

ng a

rea

6F.D

ispo

sal

faci

liti

es f

or d

rain

age

wat

er

from

was

hing

fac

ilit

ies

and

show

er

room

6G.

Ven

tila

tion

fac

ilit

ies

to m

inim

ize

air

poll

utio

n

6H.

Mai

nten

ance

of

nega

tive

pre

ssur

e in

a

man

ufac

turi

ng a

rea

61.

Pass

age

of i

nput

air

and

out

put

air

in

a m

anuf

actu

ring

are

a th

roug

h ef

fici

ent

dust

fi

lters

lab

le D

-3.

can:

titi

OM

of

can

tair

.en

t

Cat

egor

y 1

Cat

egor

y 2

Clo

sed

syst

em

Clo

sed

syst

em

Min

imiz

e le

akag

e of

rec

od)i

nant

Pr

even

t le

akag

e of

rec

ombi

nant

Min

imiz

e le

akag

e of

rec

od)i

nant

Pr

even

t le

akag

e of

rec

ombi

nant

Car

ry o

ut t

he t

ran

sfer

aft

er

Car

ry o

ut t

he t

ran

sfer

aft

er

inac

tiva

tion

of

the

reco

mbi

nant

in

acti

vati

on o

f th

e re

com

bina

nt

by

val

idat

ed m

eans

b

y v

al h

fate

d ch

emic

al

or

phys

i ca

l m

eans

Min

imiz

e le

akag

e of

rec

ombi

nant

Pr

even

t le

akag

e of

rec

ombi

nant

Opt

iona

l N

eces

sary

Min

imiz

ed,

if p

ossi

ble

Res

tric

ted

Spe

cial

wor

king

clo

thes

S

peci

al w

orki

ng c

loth

es

Nec

essa

ry

Nec

essa

ry

Not

nec

essa

ry

Opt

iona

l

Not

nec

essa

ry

Opt

iona

l

Opt

i ona

l O

ptio

nal

Not

nec

essa

ry

Opt

iona

l

Not

nec

essa

ry

Opt

i ona

l

Cat

egor

y 3

Clo

sed

syst

em

Prev

ent

leak

age

of r

ecom

bina

nt

Prev

ent

leak

age

of r

ecom

bina

nt

Car

ry o

ut t

he t

ran

sfer

aft

er

inac

tiva

tion

of

the

reco

mbi

nant

by

val f

date

d ch

emic

al

or p

hysi

cal

mea

ns

Prev

ent

leak

age

of r

ecom

bina

nt

Nec

essa

ry

Res

tric

ted.

M

anuf

actu

ring

pe

rsom

el s

houl

d en

ter

thro

ugh

an a

irlo

ck

Cha

nge

com

plet

ely

into

spe

cial

w

orki

ng c

loth

es

Nec

essa

ry

Nec

essa

ry

Nec

essa

ry

Nec

essa

ry

Nec

essa

ry

Nec

essa

ry

~

~

;: ~ b:l 5'

~ s.. 5 C"

~

----

-

Cat

egor

y 1

6J.

Des

ign

of a

man

ufac

turi

ng a

rea

prev

enti

ng l

eaka

ge t

o th

e en

viro

nmen

t ev

en

Not

nec

essa

ry

in t

he e

vent

of

tota

l le

akag

e of

the

co

nten

ts o

f th

e cl

osed

sys

tem

6K.

Des

ign

of a

man

ufac

turi

ng a

rea

allo

win

g N

ot n

eces

sary

d

isin

fect

ion

by

funi

gati

on

7. D

ispD

Sal

of w

aste

sol

utio

ns a

nd w

aste

C

arry

out

dis

posa

l af

ter

mat

eria

ls

inac

tiva

tion

by

val i

date

d m

eans

~

Cat

egor

y 2

Opt

iona

l

Opt

iona

l

Car

ry o

ut d

ispo

sal

afte

r in

acti

vati

on b

y va

l ida

ted

chem

i calo

r ph

ys i c

al m

eans

----

----

---

Cat

egor

y 3

Nec

essa

ry

Nec

essa

ry

Car

ry o

ut d

ispD

Sal

afte

r in

acti

vati

on b

y va

lida

ted

chem

i calo

r ph

ys i c

al m

eens

-

~

~ ~ ~

A P P END I X E

GUIDELINES FOR THE APPUCATION OF RECOMBINANT DNA

ORGANISMS IN AGRICULTURE, FORESTRY, FISHERIES, THE FOOD

INDUSTRY AND OTHER REIATED INDUSTRIES IN JAPAN

Chapter 1 General Provisions

Section 1 Purpose

The purpose of these guidelines in agriculture, forestry, fisheries, the food industry and other

industries controlled by the Ministry of Agriculture, Forestry and Fisheries Cagro-industries,'

hereinafter), is to promote the progress of agro-industries by defining general principles for the

appropriate application of recombinant DNA CrDNA,' herein after) organisms transduced by

rDNA techniques and ensuring safety in the use of rDNA organisms.

Section 2 Definition of Terms

1. 'rDNA' is defined as deoxyribonucleic acid (DNA) molecules constructed in vitro by joining

heterologous DNA segments (where 'heterologous DNA' refers to DNA derived from organisms

taxonomically different from the recipient cell mentioned below) to DNA molecules that can

replicate in a living cello This construction uses enzymes or other methods to propagate the

heterologous DNA molecules CrDNA techniques,' hereinafter).

'rDNA organisms' are defined as either 1) living cells into which rDNA molecules have

been introduced, except living cells possessing the same genetic structure as that of naturally

existing cells, or 2) cells or organisms derived from the living cells described in (1) above.

286

Appendix E

2. 'Host ceHs' refers to living ceHs into which rDNA molecules are introduced.

3. 'Vectors' refers to DNA molecules to which heterologous DNA segments are joined for

transferring the segments to bosts using rDNA technique.

4. 'Donor DNA' refers to heterologous DNA segments inserted into vectors.

5. 'rDNA plants' refers to rDNA organisms (excluding rDNA organisms in an undifferentiated

condition) whose host cells are derived from plants (including fungi which form sporophores

and excluding microalgae).

6. 'rDNA microorganisms' refers to rDNA organisms whose host cells are microorganisms

(including microalgae and excluding fungi which form sporophores). However, rDNA

organisms in an undifferentiated condition, whose host ceHs are animal or plant cells are

regarded as rDNA microorganisms in these guidelines.

7. 'Work areal is defined as an area where rDNA organisms are directly handled.

8. 'Work site' is defined as a site where the production or evaluation of characteristics of

rDNA organisms is performed, including sites where rDNA organisms are not necessarily

handled directly.

Chapter 2 Safety Evaluation of Recombinant DNA Organisms

Section 1 Fundamental Principles

Any person or organization (the organization shall designate an individual to be the 'responsible

person' mentioned below) who intends to produce or seIl rDNA organisms, or to produce

materials made using rDNA organisms in agro-industries ('responsible person,' hereinafter)

except in cases involving the application of rDNA organisms in an environment without specific

measures for containment (noncontainment system, hereinafter), where the safety of the organisms

287


has been previously confirmed in the noncontainment system, shall evaluate the characteristics of

the rDNA organisms on the basis of the characteristics of the hosts, rDNA moleeules and vectors

involved, and evaluate the safety of the rDNA organisms through comprehensive comparisons

between the rDNA organisms and their hosts according to the evaluation items described in

Section 2, and then apply the rDNA organisms in eomplianee with following, regarding their kinds,

situations or application and appropriate degree of safety.

1. rDNA plants

(1) In the case of propagating rDNA plants to develop breeding materials, prior to

applieation in the noneontainment system, they shall be applied in the simulated model

environment defined in Seetion 3-1-(1) and their safety must be eonfirmed after the

safety evaluation has been eompleted.

(2) rDNA plants whose safety has been eonfirmed as in Seetion 1-1-(1) above can be

applied in the noneontainment system defined in Seetion 3-1-(2).

2. rDNA mieroorganisms

288

(1) Applieation of rDNA mieroorganisms to produetion proeesses

rDNA microorganisms shall be c1assified into divisions of application defined in Section

3-2-(1) according to the appropriate degree of safety required.

(2) Application of rDNA microorganisms intended to be applied in the noneontainment

system

(a) rDNA mieroorganisms shall be eultivated aeeording to Seetion 1-2-(1) above.

(b) Prior to application in the noneontainment system, rDNA microorganisms shall be

applied in the simulated model environment defined in Section 3-2-(2)-a and their

safety must be confirmed after the safety evaluation has been eompleted.

(e) rDNA mieroorganisms whose safety has been confirmed as in Section 1-2-(2)-b

above can be applied in the noncontainment system defined in Section 3-2-(2)-b.

Appendix E

Section 2 Evaluation iteIDS

The following factors shall be evaluated.

1. rDNA plants

(1) Purposes of the application of rDNA plants

(2) Rosts or biological species to which the hosts belong

(a) Taxonomic position

(b) Previous applications and distribution in the natural world

( c) Reproduction and propagation styles and genetic characteristics

(d) Weediness

( e ) Production of toxic substances

(f) Other principal physiologieal eharaeteristies

(3) Donor DNA

(a) Identified or not identified

(b) Strueture and origin

( e) Funetion of target genes

(4) Veetors

(a) Names and origin

(b) Charaeteristies

(5) rDNA plants

(a) Preparatory methods of rDNA plants

(i) Strueture and eonstruetion methods of rDNA moleeules

(ii) Methods of the introduetion of target genes into hosts

(iii) Development proeess of rDNA plants

(b) Loeation of target genes in the host and stability of their expression

(e) Differenees between rDNA plants and hosts or biologieal species to whieh the hosts

belong

(i) Reproduction and propagation styles and genetie eharaeteristies

(ii) Weediness

(iii) Produetion of toxie substanees

289


(iv) Other principal physiologieal eharaeteristics

(6) Other items (knowledge acquired in the course of rDNA experiments or by the

development of other rDNA plants, ete.)

2. rDNA mieroorganisms

290

(1) Purposes of the applieation of rDNA mieroorganisms

(2) Hosts or biologieal species to whieh the hosts belong

(a) Taxonomie position

(b) State of applieation and distribution in the natural world

(c) Propagation style and genetic charaeteristies

(d) Pathogenicity

( e ) Production of toxic substances

(f) Other principal physiologieal eharaeteristics

(3) Donor DNA

(a) Identified or not identified

(b) Structure and origin

( e) Funetions of target genes

(4) Vectors

(a) Names and origin

(b) Characteristics

(5) rDNA microorganisms

(a) Methods of preparation of rDNA microorganisms

(i) Structure and construction methods of rDNA molecules

(ii) Methods of the introduction of target genes into hosts

(iii) Development proeess of rDNA microorganisms

(b) State of existence and stability of expression of target genes

( e) Differenees of r D NA microorganisms from hosts or biological species to which the

hosts belong

(i) Propagation style and genetic characteristics

(ii) Pathogenicity

Appendix E

(iii) Production of toxic substances

(iv) Other principal physiological characteristics

(d) Survivability and monitoring methods in the natural world

(6) Other items (knowledge acquired through the process of rDNA experiments or the

development of rDNA microorganisms, etc.)

Section 3 Classification of application

1. rDNA plants

(1) Application in the simulated model environment

This refers to the experimental application of rDNA plants in a specifically restricted

area which is designed to simulate the environment of actual cultivation under such

conditions as to prevent the rDNA plants from either naturally propagating or from

influencing plants outside the area (e.g., via pollen).

(2) Application in the noncontainment system

This refers to the application of rDNA plants in the noncontainment system, whose

safety has been confirmed in the simulated model environment.

2. rDNA microorganisms

(1) Application of rDNA microorganisms to production process

(a) Good Industrial Large-Scale Practice (GILSP)

This refers to the application of rDNA microorganisms which satisfy the following

criteria under minimum containment.

(i) Hosts must be

1. non-pathogenic to humans

2. uncontaminated by exogenous factors (viruses, etc.) which are

pathogenic to humans

3. possessing either a long record of safe industrial use or an inability to

propagate except under specific cultivation condition not found in

nature

291


292

(ü) Donor DNA and vectors must be

1. well-characterized and free from known harmful base sequences

2. limited in size as much as possible to the DNA required to perform

the intended function

3. unable to transmit resistant markers to organisms which have never

been known to acquire such resistance naturally

(iü) rDNA microorganisms must be

1. non-pathogenic to humans

2. less capable of propagation that their hosts

(b) Category 1

This refers to the application of nonpathogenic rDNA microorganisms which do not

satisfy the criteria of GILSP

(c) Category 2

This refers to the application of rDNA microorganisms which have the possibility of

infection in spite of minimal likelihood of pathogenicity when handled directly, little

possibility of causing disease in case of infection. Effective preventive measures and

therapeutic methods must exist for infection by rDNA microorganisms under Category

2 application.

(d) Category 3

This refers to the application of rDNA microorganisms which have significant likelihood

of pathogenicity to humans than do rDNA microorganisms classified un~er Category 2

application. Effective preventive measures and therapeutic methods must exist for

infection by rDNA microorganisms under Category 3 application.

( e) Special class

rDNA microorganisms possessing pathogenicity in excess of that of rDNA

microorganisms classified under Category 3 application shall be classified under a special

division of application.

(2) Application of rDNA microorganisms intended to be applied in the noncontainment

system

(a) Application in the simulated model environment

Appendix E

This refers to the experimental application of rDNA microorganisms classified under

GILSP or Category 1 application in Section 3-2-(1) above in a specifically restricted area

under such conditions as to minimize both the spread of rDNA microorganisms outside

the area and the transmission of the genetic characteristics of rDNA microorganisms to

organisms outside this area.

(b) Application in the noncontainment system

This refers to the application of rDNA microorganisms in the noncontainment system,

whose safety has been confirmed in the simulated model environment.

Chapter 3 FaciIities, Apparatus and Operations for Handling Recombinant DNA

Organisms

Section 1 Facilities and apparatus for handling rDNA organisms

1. Facilities and apparatus for handling rDNA plants

In the case of the application of rDNA plants in the simulated model environment, facilities

and apparatus for handling rDNA plants shall be installed to satisfy the following criteria.

(1) A work area definitely distinguished from other areas shall be marked off, and

biohazard signs shall be posted if necessary.

(2) An isolated field to prevent spread of rDNA plants shall be marked off in the work area

taking into consideration the reproduction and propagation styles, the castration

treatment, the physiological characteristics and the situation of application in the

noncontainment system of rDNA plants as weil as the surrounding biota.

2. Facilities and apparatus for handling rDNA microorganisms

In the case of the application of rDNA microorganisms under GILSP, Category 1, Category

2 and Category 3, facilities and apparatus for handling rDNA microorganisms shall be

instalIed to satisfy the criteria listed in Table E-l.

293


In the case of the application of rDNA microorganisms in the simulated model

environment, facilities and apparatus for handling rDNA microorganisms shall be instalIed

to satisfy the following criteria.

(1) A work area definitely distinguished from other areas shall be marked off, and

biohazard signs shall be posted if necessary. See Table E-1.

(2) An isolated field shall be marked off or a management facility to prevent spread of

rDNA microorganisms shall be set up in the work area taking into consideration the

propagation style, the restriction treatment for propagation ability, the physiological

characteristics and the situation of application in the noncontainment system of rDNA

microorganisms as well as the surrounding biota.

Section 2 Operations for handling rDNA organisms

1. Operations for handling rDNA plants

294

In the case of the application of rDNA plants in the simulated model environment,

operations for handling rDNA plants shall be in compliance with the following:

(1) Cultivation management of rDNA plants

(a) The seeding and planting of rDNA plants shall be done in the work area so as to

prevent their seedlings and other parts from spreading outside the area.

(b) The propagation of plants unrelated to the application of rDNA plants shall be

minimized in the cultivation area and its vicinity.

(c) In the case of cuItivating rDNA plants whose pollen, seeds or other parts can easily

disperse, the dispersion shall be minimized by castration, bagging or other methods.

(d) In the case of cultivating rDNA plants whose sterns, leaves, tubers, rhizomes, roots

and other parts can easily regenerate, they shall be removed from the facility of

cultivation, and treatment to prevent their regeneration shall be performed after

the operation of handling rDNA plants ends.

(2) Disposal of wastes related to rDNA plants

Wastes related to rDNA plants shall be disposed of after inactivation appropriate to the

degree of safety required.

Appendix E

(3) Storage of rDNA plants

(a) rDNA plants or rDNA plant materials shall be clearly labeled as rDNA plants on

the container, and safety stored in a storage facility set up in advance. A sign,

'rDNA Plants in Storage (Application in the simulated model environment),' shall

be posted in clearly visible places in the storage facility.

(b) A catalogue of the stored materials including rDNA plants shall be prepared and

maintained.

(4) Transportation of rDNA plants

(a) In the case of transporting rDNA plants or materials outside the work area, they

shall be sealed in a container to prevent them from spreading.

(b) A sign, 'Handle with Care,' in red lette ring shall be clearly displayed on any

container which contains rDNA plants or materials.

(5) Maintenance and management of facilities and apparatus

The performance of facilities and apparatus for handling rDNA plants shall be tested,

their performance at the time of installation and regularly thereafter, and their original

performance shall be maintained.

(6) Others

(a) A sign, 'Application in the Simulated Model Environment (rDNA Plants),' shall

be posted at the work area for handling rDNA plants.

(b) The work area shall be kept clean.

(c) Working clothes shall be worn in the work area.

(d) Workers shall take special care not to spread the pollen, seeds or other parts of

rDNA plants outside the work area by bringing them out on their bodies.

2. Operations for handling rDNA microorganisms

In the case of the application of rDNA microorganisms in GILSP, Category 1, Category 2,

Category 3 applications and the simulated model environment, operations for handling rDNA

microorganisms shall be in compliance with the following:

(1) Cultivation management of rDNA microorganisms

295


296

(a) In the case of seeding rDNA microorganisms in cultivation or fermentation

apparatus, harvesting rDNA microorganisms from cultivation or fermentation

apparatus, or transplanting rDNA microorganisms from one apparatus to another,

the escape of rDNA microorganisms shall be minimized by preventing rDNA

microorganisms from sticking on the outside wall of the cultivation or fermentation

apparatus and other methods in GILSP and Category 1 applications. In Category

2 and Category 3 applications, the escape of rDNA microorganisms shall similarly

be prevented, and if such escape occurs, the rDNA microorganisms shall be

immediately disinfected by methods whose effectiveness has been previously

confirmed. In the case of application in the simulated model environment, the

escape of rDNA microorganisms outside the work area shall be minimized

according to the situation of application.

(b) The leakage of aerosols from cultivation or fermentation apparatus shall be

minimized in GILSP and Category 1 applications, and prevented in Category 2 and

Category 3 applications.

(c) After the conc1usion of the operation for handling rDNA microorganisms under

GILSP or Category 1 application and in the simulated model environment, the

facilities and apparatus related to the application of rDNA microorganisms shall

be washed and disinfected, and the facilities related to the application of rDNA

microorganisms und er Category 2 and Category 3 applications shall be sterilized

by methods whose effectiveness has been previously confirmed.

(2) Disposal of wastes related to rDNA microorganisms

Wastes related to rDNA microorganisms shall be disposed of after inactivation

appropriate to the degree of safety required. They shall be inactivated by methods

whose effectiveness has been confirmed in Category 1 application (inc1uding the

application of rDNA microorganisms in the simulated model environment classified

under Category 1 application), and sterilized in Category 2 and Category 3 applications.

(3) Storage of rDNA microorganisms

(a) Materials inc1uding rDNA microorganisms shall be c1early labeled as rDNA

microorganisms on the container, and safely stored in a storage facility set up in

Appendix E

advance. Especially in the case of Category 2 and Category 3 applications, rDNA

microorganisms shall be safely stored in a storage facility in the work area. A sign

'rDNA Microorganisms in Storage (GILSP application),' 'rDNA Microorganisms

in Storage (Category 1 application),' 'rDNA Microorganisms in Storage (Category

2 application),' 'rDNA Microorganisms in Storage (Category 3 application)' or

'rDNA Microorganisms in Storage (Application in the simulated model

environment)' shall be posted in clearly visible pI aces in each storage facility

according to each division of application.

(b) A catalogue of the stored materials including rDNA microorganisms shall be

prepared and maintained.

(4) Transportation of rDNA microorganisms

(a) In the case of transporting materials including rDNA microorganisms outside the

work area, they shall be sealed in a fairly strong container to prevent leaking.

Precaution shall be taken so that the contents of the sealed container will not leak

even if the container breaks, especially in Category 2 and Category 3 applications.

(b) A sign 'Handle with Care' in red lette ring shall be clearly displayed on any

container which contains materials including rDNA microorganisms.

(5) Maintenance and management of facilities and apparatus

(a) The performance of facilities and apparatus for handling rDNA microorganisms

shall be tested at the time of their installation and regularly thereafter, and special

note shall be taken of the degree of sealing. The apparatus for destroying rDNA

microorganisms used in Category 2 and Category 3 applications shall be tested

every half year.

(b) Their performance and the degree of sealing of the facilities and apparatus shall

be tested each time the sealing parts of the facilities and apparatus are modified

or replaced.

(c) The degree of sealing of cultivation or fermentation apparatus and the apparatus

connected thereto shall be confirmed by appropriate methods under operation in

Category 2 and Category 3 applications.

297


(d) The facilities and apparatus shall be strictly controlled with serial numbers in

Category 2 and Category 3 applications.

(e) The apparatus for destroying rDNA microorganisms shall be sterilized by methods

whose effectiveness has been previously confirmed prior to the replacement,

periodic inspection or operational alteration of the apparatus.

(6) Others

(a) Sign saying 'GILSP application,' 'Category 1 application,' 'Category 2

application,' 'Category 3 application' or 'Application in the simulated model

environment' shall be posted at each work area according to each division of

application under operation for handling rDNA microorganisms.

(b) The work area shall be kept clean, and especially in the case of application to

production process, insects and rodents shall be exterminated.

(c) Working clothes shall be worn in the work area. Exclusive working clothes shall

be worn in operations of Category 2 and Category 3 applications. These clothes

shall be completely changed at the time of entering and leaving the work area and

a shower shall be taken at the time of leaving the work area in operations of


Chapter 4 Management Systems

Section 1 Management by the responsible person

The responsible person (This term refers a trustee in such cases where the responsible person

entrusts the operation of application in the simulated model environment, hereinafter in tbis

chapter ) shall establish management systems in compliance with the following to ensure the safety

of the application of rDNA organisms.

1. He( she) shall appoint an administrator of operation and a manager for safe operation to

assist the administrator for each work site or each factory or institute where the work site

298

Appendix E

belongs, and also appoint substitutes for the administrator and manager, in advance, to

execute their duties in case of their absence due to disease or other reasons.

2. He( she) shall establish a committee for safe operation, appoint the members of the

committee and ask them to investigate and consider the safety of the application of rDNA

organisms.

3. He(she) shall see that the administrator executes the duties prescribed in Section 2 below.

4. He(she) shall see that the manager executes the duties prescribed in Section 3-2 below.

Section 2 Administrator of operation

An administrator of operation shall fully understand these guidelines and execute the following

duties.

1. He(she) shall observe these guidelines on the occasion of planning and executing operation,

and appropriately manage and supervise the whole operation under cIose cooperation with

the manager.

2. He(she) shall be responsible for the training of the personnel of operation prior to the

initiation of the operation.

3. He(she) shall post necessary information related to the handling of rDNA organisms in

cIearly visible places in the work area and the storage facility of rDNA organisms.

4. He( she) shall restriet the entrance of unauthorized persons into the work area according to

the situation of operation, and ensure that visitors obey the directions of the authorized

personnel when such persons enter the work area.

5. He(she) shall prepare books recording the following items and maintain the books for five

years after the termination of operation.

(1) Name of each rDNA organism and number written on its container

(2) Storage and culturing of rDNA organisms

(3) Biological properties of rDNA organisms and date of their examination

(4) Name, address, purpose of application and organization of acceptors to whom rDNA

organisms are transferred

(5) Results of medical examinations of personnel

299


(6) Record of the review of the committee for safe operation (including basic documents

to confirm whether the methods for handling rDNA organisms are proper or not)

(7) Record of the periodic inspection and operation of facilities and apparatus

Section 3 Manager for safe operation

1. A manager for safe operation who is to assist the administrator of operation shall be

appointed from personnel who are knowledgeable and acquainted with rDNA technology to

ensure the safety of the application of rDNA organisms.

2. He (she) shall fully understand these guidelines and execute the following duties.

(1) He (she) shall confirm that the operation is carried out in accordance with these

guidelines.

(2) He (she) shall advise or report to the administrator.

(3) He (she) shall conduct necessary operations for safety securement.

Section 4 Penronnelofoperation

Personnel of operation shall execute the following duties.

1. They shall fully understand and maintain the safety securement of operation.

2. They shall give instructions to persons other than the personnel of operation for safety

securement when such persons enter the work area.

Section 5 Committee for safe operation

1. A committee for safe operation shall consist of members from appropriate fields, since the

committee requires highly specialized knowledge of rDNA organisms and technology as weIl

as judgement from an overall stand point.

2. The committee shall investigate and consider the following items and give necessary advice

to the responsible person at his (her) request.

(1) Suitability of the methods for handling rDNA organisms

300

Appendix E

(2) Suitability of personnel training and health care with regard to the safety of the

personnel of operation

(3) Necessary treatment and improvement of operational procedure in the case of accident

(4) Other necessary items with regard to the safety securement of operation

3. Tbe committee shall be able to ask the administrator of operation and the manager of safe

operation to submit reports as the occasion demands.

Section 6 Training of personnel

Tbe administrator shall make certain that the personnel of operation fully understand these

guidelines and be responsible for their training with regard to the following items on them prior

to initiating the operation of the application of rDNA organisms.

1. Knowledge of the safety of rDNA organisms

2. Knowledge of proper handling of rDNA organisms in accordance with safety evaluation

3. Knowledge of proper use of facilities and apparatus required for handling rDNA organisms

4. Knowledge of the safety of operation to be carried out

5. Knowledge of measures to be taken in case of accident

Section 7 HeaIth care

1. Tbe responsible person shall ensure that medical examinations of the personnel of operation

are conducted prior to the initiation of operation and at intervals of no longer than one year.

2. He( she) shall examine the measures of prevention and treatment, in advance, when he( she)

intends to have the personnel of operation engage in the operation of Category 2 or


3. He(she) shall immediately require medical examination and take appropriate measures if

there is a possibility that the personnel of operation have been infected in the work area of

Category 2 or Category 3 applications. Serum of the personnel of operation who will be

301


engaged in the operations of Category 3 applications shall be collected prior to the initiation

of operation and stored for two years after the personnel cease working for the operation.

Chapter 5 Approval and Reports

1. At the time of the application of rDNA organisms, in order to ensure the safety of the

application, the responsible person can ask the Minister of Agriculture, Forestry and

Fisheries to approve that the facilities, apparatus and procedures utilized for the safe

application of rDNA organisms conform with these guidelines.

2. He(she) shall collect information relating to rDNA organisms and their application, and

report immediately to the Minister of Agriculture, Forestry and Fisheries if he( she) finds new

knowledge which may influence the safety evaluation of rDNA organisms.

Chapter 6 Others

1. The responsible person shall make efforts to accumulate enough knowledge to ensure the

safety of rDNA microorganisms intended to be applied in the noncontainment system.

2. rDNA orgamsms whose hosts are animal cells (excluding rDNA organisms In an

undifferentiated condition) shall be kept in a specifically controlled environment for the

present. If the responsible person asks the Minister of Agriculture, Forestry and Fisheries

to approve the safety evaluation of rDNA organisms whose hosts are animal cells, the

approval shall be made on a case-by-case basis.

3. Provisions relating to rDNA microorganisms in these guidelines shall be applied to non

cellular organisms (containing rDNA molecules) which are directly injected into plants or

animals. In such cases, the plants or animals into which non-cellular organisms are injected

shall not be classified as rDNA organisms.

302

Appendix E

4. For the time being, living cells into which rDNA molecules, constructed by joining DNA

segments derived from organisms taxonomically identical to the living cell to DNA moleeules

which can replicate in the living cell, have been introduced, or the application of organisms

derived from these living cells, shall be dealt with as rDNA organisms in spite of the

provisions in Section 2-1 in Chapter 1.

5. Other than the provisions contained in these guidelines, the Director General of the relevant

bureau shall establish the necessary regulations with regard to the operation of these

guidelines.

303

w ~

lab

le E

-1.

Cri

teri

a fo

r H

andl

ing

rDNA

Mic

roo

rgan

i_

GIL

SP

Cat

egor

y 1

Cat

egor

y 2

Cat

egor

y 3

1. E

xten

t of

sea

ling

of

faci

liti

es

and

appa

ratu

s

1A.

Han

dlin

g of

rDN

A M

inim

izat

ion

of

leak

agea

M

inim

izat

ion

of

leak

age

Pre

vent

ion

of

leak

age

Pre

vent

ion

of

leak

age

mic

roor

gani

sms

in e

xhau

st g

as se

s

1B.

Perf

orm

ance

of

adju

stin

g M

inim

izat

ion

of

leak

age

Min

imiz

atio

n of

le

akag

e P

reve

ntio

n of

le

akag

e P

reve

ntio

n of

le

akag

e va

lves

2. C

ondi

tion

s of

wor

k ar

ea

2A.

Bio

haza

rd s

ign

Not

nec

essa

ry

Opt

iona

l N

eces

sary

N

eces

sary

2B.

Air

lo

ck o

n do

orw

ay

Not

nec

essa

ry

Not

nec

essa

ry

Not

nec

essa

ry

Nec

essa

ry

2C.

Dec

onta

min

atio

n an

d w

ashi

ng

Opt

iona

l N

eces

sary

N

eces

sary

N

eces

sary

fa

cil

itie

s fo

r op

erat

ion

pers

onne

l

20.

Show

er f

acil

itie

s N

ot n

eces

sary

N

ot n

eces

sary

O

ptio

nal

Nec

essa

ry

2E.

Dis

posa

l fa

cil

itie

s fo

r w

aste

w

ater

fro

m d

econ

tam

inat

ion

was

hing

N

ot

nece

ssar

y N

ot n

eces

sary

O

pt i o

nal

Nec

essa

ry

and

show

er f

acil

itie

s

2F.

Ven

tila

tion

O

ptio

nal

Opt

iona

l O

ptio

nal

Nec

essa

ry

2G.

Mai

nten

ance

of

air

pre

ssur

e N

ot

nece

ssar

y N

ot n

eces

sary

O

ptio

nal

Nec

essa

ry

nega

tive

to

atm

osph

ere

in w

ork

area

2H.

App

lica

tion

of

HEPA

fil

ters

to

Not

nec

essa

ry

Not

ne

cess

ary

Opt

iona

l N

eces

sary

v

enti

lati

on

facil

itie

s

21.

Des

ign

of w

ork

area

to

prev

ent

cont

ents

fro

m s

prea

ding

out

side

N

ot n

eces

sary

N

ot

nece

ssar

y O

ptio

nal

Nec

essa

ry

the

area

in

case

sp

illa

ge

occu

rs

2J.

Des

ign

for

seal

ing

of w

ork

area

to

ena

ble

ster

iliz

atio

n b

y N

ot

nece

ssar

y N

ot n

eces

sary

O

ptio

nal

Nec

essa

ry

fum

igat

ion

a:

the

exte

nt o

f le

akag

e w

ill

be

redu

ced

to a

per

mis

sibl

e le

vel,

acc

ordi

ng t

o th

e sa

fety

of

the

spec

ific

rDN

A m

icro

orga

nism

s in

volv

ed

~ ] ~ ~ b::I 5·

~ ~ c 5"

~

Amino acids

Antibiotic

Antibody

Antigen

Apo-

Applied research

Apyrene

Aspartame

Basic research

Bioconversion

GLOSSARY

The building blocks of proteins. There are 20 common amino acids.

A specific type of chemical substance that is administered to fight infections, usually bacterial infections, in humans or animals. Many antibiotics are produced by using microorganisms; others are produced synthetically.

A protein (immunoglobulin) produced by humans or higher animals in response to exposure to a specific antigen and characterized by specific reactivity with its complementary antigen. (See also monoclonal antibodies.)

A substance, usually a protein or carbohydrate which, when introduced in the body of a human or higher animal, stimulates the production of an antibody that will react specifically with it.

Formed from; related to.

Research to gain knowledge or understanding necessary for determining the means by which a recognized and specific need may be met (National Science Foundation definition).

Lacking a nucleus.

A low calorie sweetener (NutraSweet).

Research to gain fuller knowledge or understanding of the fundamental aspects of phenomena and of observable facts without specific applications toward processes or products in mind (National Science Foundation definition).

A chemical conversion using a biocatalyst.

305


Bioengineering

Bioprocess

Biotechnology

Botany

Cardiovascular

Cell culture

Cellulase

Cephalosporin

Characterization

Clinical trial

306

Engineering relating to the biosynthesis or processing of animal or plant products; specifically engineering relating to fermentation processes.

Any process that uses complete living cells or their components (e.g., enzymes, chloroplasts) to effect desired physical or chemical changes.

Commercial techniques that use living organisms, or substances from those organisms, to make or modify a product, and including techniques used for the improvement of the characteristics of economically important plants and animals and for the development of micro-organisms to act on the environment. In this report, biotechnology is used to mean 'new' biotechnology, which only includes the use of novel biological techniques -- specifically, recombinant DNA techniques, cell fusion techniques, especially for the production of monoclonal antibodies, and new bioprocesses for commercial production.

The science of plants.

Relating to, or involving the heart and blood vessels.

The in vitro growth of cells isolated from multicellular organisms. These cells are usually of one type.

Any of a group of enzymes that are found in various fungi, bacteria, insects, and lower animals and that hydrolyze cellulose.

A form genus of imperfect fungi with conidia (any asexual spore not borne within an enclosing structure) held together by a slimy secretion in more or less spherical heads at the ends of the fertile branches.

Portrayal of any trait, function, structure, or substance of an organism resulting from the effect of one or more genes as modified by the environment.

One of the final stages in the collection of data for drug approval where the drug is tested in humans.

Qone

Qoning

Collagen

CSF

Dextrin

Diagnostic products

DNA

DNAprobe

Downstream processing

Drug

E. coli

Endotoxin

Glossary

A group of genetically identical cells or organisms produced asexually from a common ancestor.

The amplification of segments of DNA, usually genes.

The protein that yields gelatin on boiling, contained in connective tissue and banes.

Colony stimulating factor.

Any of various water-soluble dextrorotatory gummy polysaccharides obtained from starch by the action of heat, acids or enzymes as a yellow or white powder or granule, capable of yielding maltose or glucose by further hydrolysis, and used as adhesives, as sizes for paper and textiles, as gum substitutes, and in making syrups and beer --- also called British gum.

Products that recognize molecules associated with disease or other biologic conditions and are used to diagnose these conditions.

Deoxyribonucleic acid. The genetic material of all living organisms. Every inherited characteristic has its origin somewhere in the code of each individual's complement of DNA

A sequence of DNA that is used to detect the presence of a particular nucleotide sequence.

After bioconversion, the purification and separation of the product.

Any chemical compound that may be administered to humans or animals as an aid in the treatment of disease.

Escherichia coli. A species of bacteria that inhabits the intestinal tract of most vertebrates. Some strains are pathogenic to humans and animals. Many nonpathogenic strains are used experimentally as hosts for rDNA

Atoxin of internalorigin; specifically: any of a class of poisonous substances present in bacteria but separable from the cell body only on its disintegration.

307


Entomology

Enzyme

Enzymology

Ethical drugs

Expression

Factor VIII

Fermentation

Fine chemical

Fractionation

Gene expression

Genetic engineering

308

The branch of zoology dealing with insects.

Any of a group of catalytic proteins that are produced by living cells and that mediate and promote the chemical processes of life without themselves being altered or destroyed.

The science that deals with the chemistry, biochemistry and biology of enzymes.

Drugs sold only upon medical prescription.

Something that manifests, represents, reflects, embodies, or symbolizes something else (i.e., the first clinical expression of the disease).

Used in the treatment of hemophiliacs; essential for blood clotting.

An anaerobic bioprocess. Fermentation is used in various industrial processes for the manufacture of products such as a1cohols, acids, and cheese by the action of yeasts, molds, and bacteria.

A chemical (as a photographic chemical, a perfume, or a pharmaceutical) produced and handled in relatively small amounts and usually in a more or less pure state.

To separate (a mixture ) into its ingredients or into portions having different properties, as by distillation or crystallization.

The mechanism whereby the genetic directions in any particular cell are decoded and processed into the final functioning product, usually a protein.

A technology used at the laboratory level to alter the hereditary apparatus of a living cell so that the cell can produce more or different chemicals, or perform completely new functions. These altered cells are then used in industrial production. Genetic engineering transfer techniques include cell fusion (protoplast fusion, hybridoma ), incubation of cells with a calcium phosphate precipitate of DNA fragments,

Genetics

Globin

Glutathione

Hepatoma

Hormone

Human Growth Hormone

Hyaluronic acid

Hybridoma

Immobilized cell

Immobilized enzyme

Immuno-

Immunoassay

Glossary

direct intracellular nuclear injection of DNA fragments, or transformation of cells with modified vectors (recombinant DNA).

The science of heredity, dealing with resemblances and differences of related organisms resulting from the interaction of their genes and the environment.

The protein component of hemoglobin.

A peptide of glutamic acid, cysteine, and glycine found in blood and in animal and plant tissues, and important in tissue oxidations and in the activation of some enzymes.

A tumor of the liver that is usually malignant.

A chemical messenger found in the circulation of higher organisms that transmits regulatory messages to cells.

Used in the treatment of dwarfism. Potentially (hGH) could improve wound healing and treat osteoporosis.

A viscous mucopolysaccharide acid that occurs chiefly in connective tissues or their derivatives.

Product of fusion between myeloma cell (which divides continuously in culture and is 'immortal') and lymphocyte antibody-producing cell); the resulting cell grows in culture and produces monoclonal antibodies.

Techniques used for the fixation of cells or enzymes onto solid supports. Immobilized cells and enzymes are used in continuous bioprocessing.

Techniques used for the fIXation of enzymes or cells onto solid supports. Immobilized cells and enzymes are used in continuous bioprocessing.

immune

The use of antibodies to identify and quantify substances. The binding of antibodies to antigen, the substance being measured, is often followed by tracers such as radioisotopes.

309


Immunology

Infrastructure

Interferons

Interleukin-2 (IL-2)

In vivo

Joint venture

Kinetics

Lipase

Lipoprotein

Methylation

Microbiology

Molecular biology

Monoclonal antibodies

310

The branch of medicine dealing with immunity from disease and the production of such immunity.

Foundation, groundwork.

A class of glycoproteins (proteins with sugar groups attached at specific locations) important in immune function and thought to inhibit viral infections.

Used in treatment of immune patients: AIDS, cancer, pre-operative, surgical patients, etc.

Within a living organism.

Form of association of separate business entities which falls short of a formal merger but uni ted certain agreed on resources of each entity for a limited purpose; in practice most joint ventures are partnerships.

The branch of mechanics that deals with the actions of forces in producing or changing the motion of masses.

Any of a class of enzymes that break down fats, produced by the liver, pancreas, and other digestive organs or by certain plants.

Any of the class of proteins that contain a lipid combined with a simple protein.

To replace one or more hydrogen atoms with the methyl group.

The science dealing with the structure, function, uses, etc., of microscopic organisms.

A branch of biology dealing with the ultimate physicochemical organization of living matter.

(MAbs) Homogeneous antibodies derived from a single clone of cells; MAbs recognize only one chemical structure. MAbs are useful in a variety of industrial and medical capacities since they are easily produced in large quantities and have remarkable specificity.

Mutagenesis

Mycology

Oncogenic

Papilloma

Pathology

Pharmaceuticals

Phenylalanine

Polysaccharide

Propionic acid

Protein

Protoplast

Reagent

Glossary

The occurrence or introduction of mutation in the genetic material of an organism; researchers may use physical or chemical means to cause mutations that improve the production of capabilities of organisms.

The branch of botany dealing with fungi.

Relating to tumor formation: tending to cause tumors.

A benign tumor (as a wart or condyloma) resulting from an overgrowth of epithelial tissue on papillae of vascularized connective tissue of skin and other organs that forms projections or ridges. An epithelial tumor caused by a virus.

The study of abnormality, especially the study of diseases, their essential nature, causes, and development, and the structural and functional changes produced by them.

Products intended for use in humans, as weIl as in vitro applications to humans, including drugs, vaccines, diagnostics, and biological response modifiers.

An amino acid essential to the nutrition of man and most animals, obtained chiefly from egg white or skim milk.

A carbohydrate, as starch, insulin, cellulose, etc., containing more than three monosaccharide units per molecule and capable of hydro lysis by acids or enzymes to monosaccharides.

A colorless, oily, water-soluble liquid having a pungent odor: used in making bread-mold-inhibiting propionates, in perfumery, etc., and in medicine as a topical fungicide.

A polypeptide consisting of amino acids. In their biologically active states, proteins function as catalysts in metabolism and, to some extent, as structural elements of cells and tissues.

The living content of a cell: the nucleus, cytoplasm, and plasma membrane constituting a living unit distinct from ergastic substances and inert walls.

A substance that takes part in a chemical reaction.

311


rDNA

Reeombination

RIA

Seale-up

Strain

Streptomyees

Synthetase

Teehnology transfer

Tetracycline

Thalassemia

Thermophilie

Ti plasmid

312

Recombinant DNA The hybrid DNA produced by joining pieces of DNA from different organisms together in vitro.

Formation of a new association of genes or DNA sequenees from different parentalorigins.

Radio Immunoassay.

The transition of a process from an experimental seale to an industrial seale.

A group of organisms of the same speeies having distinctive eharaeteristics but not usually eonsidered aseparate breed or variety. A genetically homogenous population of organisms at a subspecies level that can be differentiated by a bioehemical, pathogenie, or other taxonomie feature.

The type genus of Streptomyeetaeeae comprising numerous baeteria that produee chains of conidia from arial hyphae and including some that form antibioties as by-produets of their metabolism.

An enzyme that catalyzes the union of two moleeules with eoneurrent breakdown of a pyrophosphate bond in a triphosphate.

The movement of technieal information and/or materials, used for producing a product or process, from one sector to another; most often refers to flow of information between publie and private seetors or between countries.

An antibiotic used to treat a broad variety of infections.

A familial hypochromie anemia charaeterized by mierocytie anemia, splenomegaly, and ehanges in the bones and skin and oeeurring especially in ehildren of Mediterranean parents.

An organism growing at a high temperature (as various baeteria that thrive at 122-131 degrees Fahrenheit).

Plasmid from Agrobacterium tumerfaciens used as a plant vector.

Tissue culture

TNF

TPA

Transduction

Trypanosome

Urea

Urease

Vaccine

Glossary

Refers to processes and apparatus which permit the growth and maintenance of cell lines. Used in the production of vaccines, steroids, antibodies and skin and organ transplant materials.

Tissue necrosis factor. Used in treatment of tumors.

Tissue plasminogen activator. Blood dot dissolving agent for use in heart attacks and deep vein thrombosis.

The act or process of leading or conveying over; specifically the transfer of genetic determinants from one microorganism to another or from one strain of microorganism to another by a viral agent (as a bacteriophage).

A protozoa of the genus Trypanosoma, a genus comprising flagellates that as adults are elongated and somewhat spindle-shaped, have a posteriorly arising flagellum which passes forward at the margin of an undulating membrane and emerges near the anterior end of the body as a short free flagellum, and are parasitic in the blood or rarely the tissues of vertebrates, that in the development phase which occurs in the digestive tract of a blood-sucking invertebrate and usually an insect pass through aseries of changes comparable to the typical forms of members of the genera Leishmania, Leptomonas, and Crithidia, multiply freely, and pass ultimately to the mouthparts or salivary structures whence they may be inoculated into a new vertebrate host bitten by the invertebrate host, and that are responsible for various serious diseases of men and domestic animals.

A compound occurring in urine and other body fluids as a product of protein metabolism. Commercial form used as a fertilizer, animal feed, and in organic synthesis.

An enzyme that changes urea into ammonium carbonate, found in bacteria, fungi, etc.

A suspension of attenuated or killed bacteria or virus es, or portions thereof, injected to pro du ce active immunity.

313


Vector

Venture capital

Virus

Yeast

Zoology

314

DNA moleeule used to introduce foreign DNA into host cells. Vectors include plasmids, bacteriophages (virus), and other forms of DNA. A vector must be capable of replicating autonomously and must have cloning sites for the introduction of foreign DNA

Venture capital funds. Money that is invested in companies with which a high level of risk is associated.

An infectious agent, especially any of a group of ultramicroscopic, infectious agents that reproduce only in living cells.

A fungus of the family Saccharomycetacea that is used especially in the making of alcoholic liquors and as leavening in baking. Yeast are also commonly used in bioprocesses.

The science or branch of biology dealing with animals.

I N 0 E X

Agency of Industrial Science and Technology (AIST),

48-50 Asahi Chemical Industry Company, Ltd., 123-126 Bio-Oriented Technology Research Advancement

Institution (BRAIN), 59

Bioindustry Development Center (BIDEC), 168-170

Corporate research institutes, 97-99 Daiichi Pharmaceutical Company, Ltd., 127-129

Doing business in Japan, 181-188

Exchange rates, xv Exploratory Research for Advanced Technology

(ERATO), 38-41 Frontier Research Program, 86, 88

Fujisaki Cell Center, 97-98 Fujisaki Institute, 97-98

Government incentives, 64-65 Government policy, 2-5, 18-69

Green Cross Corporation, 130-133

Hayashibara Biochemical Laboratories, Inc., 97-98, 134-137

Human Frontier Science Program, 51-52

Industry, 9-11, 107-165 Institute of Physical and Chemical Research

(RIKEN), 35, 81-88

International cooperation, 59-60

Investment capital, 176-178 Japan Society for the Promotion

of Science Program (JSPS), 45-46

Japan Key Technology Center (JKTC), 49, 51

Japan Health Sciences Foundation, 27-29 Kirin Brewery Company, Ltd., 138-142 Kubota, Ltd., 143-146 Kyoto University, 71-73

Kyowa Hakka Kogyo Company, Ltd., 147-150 Ministry of Agriculture, Forestry

and Fisheries (MAFF), 54-59 Ministry of Education, Science and

Culture (MESC), 41-46

Ministry of Health and Welfare (MHW), 23-29

Ministry of International Trade and

Industry (MITI), 46-52

315

Mitsubishi Kasei Corporation, 151-155 National Laboratory of AgrObiological

Resources, 81, 86, 89 National research institutes, 80-89

Osaka University, 72, 74-76

Osaka Bioscience institute (OBI), 95-97

Plantech Research Institute, 152-155 Protein Engineering Research

Institute (PERl), 92-95

Regulations, 60-64: MAFF, 286-304

MESC, 207-244 MHW, 261-269, 270-285

MITI, 245-260 Research Promotion Fund for Pharmaceuticals,

Medical Devices and Cosmetics, 29 Research Development Corporation of Japan

(JRDC), 35-38

Science and Technology Agency (STA), 30-41 Strategie alliances (also see individual

companies), 172-174

Suntory, Ltd., 156-158 Suntory Research Center, 98-99

Suntory Institute for Biomedical Research, 98-99

Takeda Chemical Industries, Ltd., 159-163 Technology transfer, 12, 166-174 Technopolis Program, 52-53

Tsukuba Science City, 80-82 Tsukuba Research consortium, 89-92

Universities and research institutes, 6-8, 70-106 University of Tokyo, 72, 77-80:

Advanced Devices Department, 77-78, 79 Department of Agricultural Chemistry, 78, 80 Laboratory of Fermentation and Microbiology, 78, 80 Research Center for Advanced Science and

Technology (RCASn, 77-78, 79 Venture capital, 179-180

Documents

o R G A N I Z A T ION SAN 0 I N 0 I V I 0 U ALS V I S I TED978-1-349-11762-8/1.pdf · Ministry of Health and Welfare: Mr Suichi Tani, Director, Health Science Division; Mr Takahisa