Altern. Animal Test. EXperiment. 5, 162-172 (1998)

Original Article

Current Status of Conducting Alternative Testing toMammalian Toxicity Studies
in the Japan Pharmaceutical Manufacturers Association
Toshiaki Matsuzawa1, Akira Koda2, Masaharu Hashimato3 and Toshiji Igarashi4(General Toxicity Working Group, The Japan Pharmaceutical Manufacturers
Association)
1

Yamanouchi Pharmaceutical Co. Ltd., 2 Sumitomo Chemicals Co., Ltd.,
3
Fujisawa Pharmaceutical Co., Ltd. and 4 Eisai Co., Ltd.
Abstract
To measure the use of alternatives to in vivo mammalian toxicity studies during drug research and development, the Japan Pharmaceutical Manufacturers
Association (JPMA) distributed a questionnaire to its 99 member companies, ofwhich 85 responded and 50 (59%) of them had some experiences in using alternatives to in vivo mammalian toxicity studies.
The results show alternative methods have been used foremost in hepatic toxicity testing, followed by teratogenicity, renal toxicity, eye irritation, skin irritation, cardiac toxicity, bone marrow toxicity and testicular toxicity tests. The
alternative methods, regardless of category, provided screening and mechanistic
analyses of the results of toxicity. Alternative test systems included isolated cells,
primary culture cells, and established cell lines of mammalian species. For hepatic toxicity testing, primary culture cells were most commonly used. Other
materials, such as perfused or sliced organs, were also used. For cardiac toxicity
and neurotoxicity testing, primary culture cells were most popular; establishedcell lines were used in renal toxicity. Whole embryonic culture was mostly usedin the primary culture alternative method for teratogenicity studies. The results
also showed that animal welfare and cost savings were of minor importance inthe opinion of most companies that responded.

Most toxicological studies performed forpharmaceuticals before marketing are wholebody (in vivo) animal studies using laboratory
animals. These studies are required by the regu-

latory authorities in any developed countries inorder to assess the potential adverse effects inhumans. On the other hand, the concept of animal welfare and protection has recently been
accepted widely, up to regulatory authorities(e.g., USDA, 1989 and 1991). Substantial goalof “3 Rs” i.e., refinement, reduction and replace-* Correspondence : Toshiaki Matsuzawa, Ph.D., Safety
Research Laboratories, Yamanouchi PharmaceuticalCo., Ltd., 1-8 Azusawa 1-chome, Itabashi-ku, Tokyo
174-8511, Japan

Key words : status survey, alternative tests, mammalian
toxicity
Received : August 11, 1997.
Accepted : June 18, 1998.

Introduction– 162 –

Alternatives in J. Pharm. Man. Assoc.

ment, made clear what to be done. However,
the manufacturers tend to stick to the traditional
animal studies, as these toxicity tests have beenused and are presently required for the pharmaceuticals, in spite of some insufficiencies of theanimal toxicological testing such as the species
difference between animals and humans. In vitro
testing provide new aspects in the safety assessment, for screening, mechanistic investigationand economical impacts, and thus, the manufacturers especially the pharmaceutical companies
are becoming aware of the use of in vitro tests.
There are many reports on the status of useof alternatives to in vivo toxicological studies inindustries of Europe and US (Balls and Fentem,
1992, Rozenkranz et al., 1992, Zucco, 1992,
Fentem et al., 1992, Fry 1993). There has beenno such report so far in Japan.
The present paper is concerned with the current status of the use of alternative testing in pharmaceutical industries in Japan, reporting the survey conducted in 1995 by the General Toxicology Working Croup (GTW) of the Japan Pharmaceutical Manufacturers Association ( JPMA).
Methods
Alternative testing in this present questionnaire survey was defined as an in vitro study
designed as an alternative to an in vivo animal
study described in the Ministry of Health andWelfare (MHW,1989) and/or OECD (1981)
guidelines for toxicity study. These were classified into several categories such as hepatotoxicity, renal toxicity, cardiotoxicity, neurotoxicity,
sensitization and immunotoxicity, reproductive
organ toxicity, teratogenicity, carcinogenicity,
genotoxicity (excluding generally established invitro studies), local irritation test (eyes, skin andothers), drug-dependency and acute toxicity.
The questionnaire asked JPMA member companies to select one of the following statements
about their experience in conducting an alternative test : [A] usually conduct alternative tests;[B] sometimes conduct, but do not routinely conduct any alternative tests; and [C] no experience with alternative tests. In cases of [A] or[B], the questionnaire asked the member com-panies to state for conducting the validity of alternative testing.
The questionnaire also asked the member
companies to state their purpose for adopting thealternative test and which test systems they used,e.g., organ perfusion, sliced tissues, isolated celllines, subcellular fractions, non-biological materials or other systems.
Results
Overview of the survey
The questionnaire was sent to 99 member
companies, of which 85 companies responded.
Table 1 shows the current status of alternative
toxicology tests in JPMA. Forty (47%) out of
85 companies responded to the questionnairewith [A] for one or more categorized toxicity
studies, meaning they were running one or morekinds of alternative testing routinely. Ten companies answered with [B], which means they
have some experience but may not conduct suchtests routinely. The other respondents had noexperience with any alternative testing. Table 1
indicates the current status of alternative testingfor 23 categories of toxicity. Results analyzed
by the toxicity category revealed that hepatic
toxicity had the highest proportion of alternative testing followed by teratogenicity, renal toxicity, eye irritation, skin irritation, cardiac toxicity, bone marrow toxicity, neurotoxicity, vessel
irritation, testicular toxicity and carcinogenicity.
Current status of alternative testing by test
category
The reactions the companies gave for adopting alternative testing, and the test systems
adopted or tried are shown in Tables 2 and 3.
These data include those companies that haveany methods, are trying to establish new methods, or are planning new methods.
1. Acute Toxicity
Only one company was currently using somemethods. The alternative test under development
uses established cell lines to do some screening– 163 –

Conduct in vitro tests
Category of the test

Adrenal toxicity
Antigenicity/ Sensitization
Bone marrow toxicity
Carcinogenicity
Cardiovascular toxicity
Eye irritation
Gastro-intestinal toxicity
Hepatic toxicity
Immunotoxicity
Mucous membrane irritation
Muscle irritation
Neurotoxicity
Ocular toxicity
Pituitary toxicity
Prostate toxicity
Renal toxicity
Skin irritation
Teratogenicity
Testicular toxicity
Thyroid toxicity
Vessel irritation

OthersTotal*

5
82
7
78
80
69
2
82
81
3
3
81
3
1
1
82
68
84
12
3
6

Usually

Sometimes

0
0
1
1
0
2
0
6
1
0
1
0
1
1
0
3
1
3
0
0
1

2
3
4
2
7
8
1
19
0
1
1
4
2
0
1
7
8
12
3
2
3

(in vivo tests only)
3
79
2
75
73
59
1
57
80
2
1
77
0
0
1
72
59
69
9
1
2

Table 1. Prevalence of in vitro toxicity testing in the JPMA companies
Total : Number of companies that answered the questions in total
Usually : Number of companies conducting some tests usually as routine work
Sometimes : Number of companies conducting some tests sometimes on a case-by-case basis
Others : Number of experience with alternative tests (in vivo tests only)*The questionnaire was sent to 99 member companies of JPMA, and the maximum of 85 companies responded to the survey.

tests. The majority of companies is not considering any alternative testing in this field. There
have been established and authorized alternative
methods as for the acute toxicity test applied tochemicals in general for the classification purpose. They may not be considered applicable tothe pharmaceuticals.
2. Adrenal toxicity
Two companies have established test systems,
and implemented them on a “case-by-case” basis. The purposes are for mechanistic analysesof toxicities and for screening. The main test
system was the primary cell culture of various
mammals (rat, hamster, monkey, cattle, rabbit,
dog, guinea pig). The effects were evaluated bymeasuring hormone secretion such as corticoid,the uptake abilities of neutral red (NR), releaseof the ADP or LDH content, or cell death(Higashijima et al., 1987; Hornsby et al., 1974;
Vernetti et al., 1993).
3. Antigenicity and sensitization
Only a few companies have actually done thistesting. The purposes of the testing were screening and mechanistic analyses of the toxicities.
Some companies did the tests to improve the predictability of toxicity for human extrapolation.
The test systems used were isolated cells(rat mast cells and human peripheral lymphocytes from blood). The amount of free histamine from rat mast cells was measured to check– 164 –

Alternatives in J. Pharm. Man. Assoc.

Category of tests

Adrenal toxicity
Bone marrow toxicity
Cardiovascular toxicity
Eye irritation
Hepatic toxicity
Immunotoxicity
Neurotoxicity
Renal toxicity
Skin irritation
Teratogenicity
Testicular toxicity

Screening

Mechanistic

Animal
WelfareCost
Reduction

Others

2
5
4
12
28
3
2
8
11
8
1

5
5
6
4
22
3
5
9
0
15
4

0
0
1
6
5
0
0
1
5
1
0

0
0
1
3
7
0
0
2
3
0
0

0
0
0
1
1
1
0
0
0
2
0

Table 2. Purpose of major alternative research to animal toxicity testing**Number reflects the total of those companies using, planning or developing an in vitro test.

the antigenicity. Also, the number of cells producing specific antibodies is measured to indicate an induction of an immune response after a
test material is added to human lymphocytes isolated from peripheral blood.

Most common test systems were establishedcell lines, examining the effects on gap binding,the phenotypic transformation test (Oyamada andYamasaki, 1989), and checking replicative DNA
synthesis (RDS) using perfused organs.

4. Bone Marrow Toxicity
One company was doing this test routinely.
A test method has been established by four companies. The purposes of the tests were mainly
mechanistic analyses of toxicities. The main test
system was primary culture cells, followed byisolated cells (Du et al., 1992; Letza et al., 1988).
The cells originated from bone marrow cells ofanimals and erythrocytes. The effects of hemopoietic cells are checked by the colony generation method. The cell division ability waschecked by adding 3H-thymidine to cultured cells
after the cells were treated with test materials.
Hemolysis of erythrocytes was also checked asa marker.

6. Cardiac toxicity
Ten companies had experienced alternative
testing for cardiac toxicity. The purpose in adopting the tests was mostly mechanistic analyses of
toxicities and screening. The main test systemused was primary cell culture. Evaluation was
done by measuring enzyme activities (e.g., GOT,
CPK) in cells, pulse changes, and checking morphological changes. In the perfused heart system, intra-cardiac pressure or ECG was measured.

5. Carcinogenicity
One company was doing in vitro carcinogenicity test routinely for the screening purpose.
Though a test method had been established, it
was carried out only on a case-by-case basis by
two companies. Two companies conducted thetests for screening and clarification of onset
mechanisms.

7. Drug dependency liability
There was no company doing any alternative
testing for dependency. A few alternative methods seem to have been developed in this field(Katsura et al., 1994).
8. Eye irritation
The tests were routinely done by two companies, and test methods had been established but
not routinely done by eight companies. The test
system used was isolated rabbit corneal epithelium (Minami et al., 1993 Sakemi et al., 1991;– 165 –

Category of tests
Adrenal toxicity
Bone marrow toxicity
Carcinogenicity
Cardiovascular toxicity
Eye irritation
Hepatic toxicity
Immunotoxicity
Neurotoxicity
Renal toxicity.
Skin irritation
Teratogenicity
Testicular toxicity

A

B

1
1
3

C

D

E

1
2

4
4

1

1

8
7
29
2
5
2
3
4
5

F

G

H

I

J

0
4

3

4
2

1
1

1

1

6
2

1

1
8
4
2
1

2

2

4
4
1

2

18

Table 3. Test systems* of major alternative tests to in vivo toxicity tests
A: perfused organ, B: slice organ, C: isolated cell, D: primary cell culture, E: cell line, F: sub-cellular fraction,
G: organ culture, H: non-mammalian, I: non-biological lines, J: whole embryo culture*Number of companies that answered the question include those which did the tests, are trying to establish a new
method, or are planning a new test.

Watanabe et al., 1989).
9. Gastrointestinal Toxicity
One company was doing a test for mechanistic analyses of toxicities, using perfused organs.
Gastric surface mucous cell lines from mice were
established by Sugiyama et al., 1993.
10. Hepatic toxicity
Alternative testing methods were most commonly adopted in the test for hepatic toxicity andsome correlation between in vivo and in vitro
test results have been shown.
The most common purpose for adopting thetest was for screening in exploratory research,followed in order by mechanistic analyses of
toxicities, cost saving and animal welfare (Table2). The test system used was most often basedon primary cell culture (Deboyser et al., 1989,
Viau et al., 1983, Ratanasavanh et al., 1988,
Nakamura et al., 1985, Jurima-Romet et al., 1991,
Boelsterli et al., 1987, Borenfreund and Puerner
1985), followed by fresh isolated cell suspension (e.g., Nakagawa et al., 1992), perfused organs (Shiota et al., 1985) or sliced organs (Smithet al., 1985, Barr et al., 1991, Wright and Paine
1992), and established cell lines (Viau et al.,1993). A primary cell cultures used were of rat

origin in most cases, but also came from dogs,
rabbits, and humans in some cases. Toxicity inthe primary culture system was determined byassessing various enzyme activities (e.g., LDH,
GOT, GPT, CPK) in more than half of the companies. Morphological observation of cells, thedamage to mitochondria examined by the MTT
assay, and quantitative analyses of lipid lines andlysosome damage by the neutral-red uptake assay were done in a small number of companies.
Other test items included albumin synthesis, phagocytosis of Kupper cells, cytochrome P-450
measurements, peroxisome proliferation of acompound, and interaction with other drugs.
Organ toxicity was assessed by measuring leaked
enzyme activities and morphological changes inother test systems. Bile excretion, oxygen consumption, and drug concentration were alsomeasured in perfused organ systems. Damage
to organelles was evaluated in established celllines. Some companies used liver slices of dogs
and monkeys in addition to those of rats
(Dogteron, 1993).
11. Immunotoxicity
No company had experienced doing in vitro
immunotoxicity test. Only one company was
doing ex vivo immunotoxicity tests routinely– 166 –

Alternatives in J. Pharm. Man. Assoc.

(Luster et al., 1988, 1992). The principal usesof the tests were mechanistic analyses of toxicities and screening. The test systems used wereprimary cell culture and isolated cells, which
originated from the spleen of mice and rats andlymphocytes of peripheral blood in rats or humans (Wood et al., 1992).
12. Mucous membrane irritation
One company was doing alternative testing
using urinary bladder membrane as a screening
prior to in vivo irritation studies.
13. Muscle irritation
Alternative testing in this field had been conducted by two companies. A primary cell culture was used in mechanistic analyses of toxicities, and CPK or histopathologic examinations
were used by one company to evaluate the damage to the muscle (Kato et al., 1992). At anothercompany, primary cell culture or established celllines had been established for the purposes ofscreening, mechanistic analyses of toxicities, andanimal welfare. The tests were not done routinely by then.
14. Neurotoxicity
Four companies had experienced alternative
testing for neurotoxicity. The main uses of thetests were mechanistic analyses of toxicities andscreening. The test system used was mainly primary cell culture. Various indicators such as
GABA receptor binding, cell death rate, and enzyme activity were measured (Ogura and Kudo,
1988; Sher, 1991). Other systems included cultured organs with nerve projections as an indicator and a nerve-muscle combination specimen
to observe muscle constriction in response tonerve stimulation.
15. Ocular toxicity
Three companies which have established test
systems for screening and mechanistic analysesof toxicities responded. The test systems usedwere included to measure GSH levels and ATP
ase activity in the lens, to examine direct effects
of test materials, and cultured rat lens to measure the proliferation rate of epithelial cells (Xuet al., 1992).
16. Pancreatic Toxicity
One company was currently trying to establish a test method to check the secretion of insulin from the islet of Langerhans using the primary cell culture of rats. This was being doneto evaluate the side effects in a similar type ofcompound to those with pancreatic toxicity(Sako et al., 1986).
17. Phototoxicity
Two companies which have established test
systems for screening responded. Establishedcell lines were used. There are some in vitro
phototoxicity methods such as neutral-red uptake using BALB/3T3 and NB1RGB, red blood
cell photohemolysis, hemoglobin photooxidationand measurement of singlet oxygen (Arakane etal., 1996, Tabuchi et al., 1995).
18. Pituitary Toxicity
One company was doing tests routinely formechanistic analyses of toxicities. Membranecurrent was measured by a patch clamp method,and hormones were measured using establishedcell lines (Oxford and Tes, 1993).
19. Prostate Toxicity
A test method has been established by justone company for mechanistic analyses of toxicities. The binding of test material to testosterone receptors was measured by using isolated
cell fraction (Hosokawa et al., 1993).
20. Renal toxicity
Ten companies had experience in alternative
testing for renal toxicity. The primary purposeof adopting alternative testing for renal toxicity
was the greater part of screening and mechanistic analyses of toxicities. The test systems usedwere mostly established cell lines. Various kindsof enzyme activity (e.g. LDH, NAG) were measured in rat-derived NRK52E cells or swine-derived LLCPK1 cells in testing with establishedcell lines. The indicators were mostly cell morphology and enzyme activity. Cell death, cellproliferation and lipid content were also mea-

– 167 –

sured by some workers (Fukunishi et al., 1989,
Williams et al., 1988, Boogaard et al., 1989,
Bruggeman et al., 1992).
21. Skin irritation
The test was routinely done or a method had
been established for one company each. Eightcompanies were experimenting with alternative
methods.
The main purpose of testing was screening,
followed by mechanistic analyses of toxicities.
Companies routinely using in vitro tests mentioned animal welfare and cost reduction as compelling reasons for implementing the methods.
The test systems for both eye and skin irritation
were primary culture cells and established celllines. Some nonbiological test kits were also
used (Sasaki et al., 1992). Cultured organ systems were also used for eye irritation.
22. Teratogenicity
This was the second most popular field forwhich the in vitro testing was done. Fifteencompanies had experience with in vitro teratogenicity testing. The main uses of the tests weremechanistic analyses of teratogenicity andscreening. Cost saving was not a factor for any
company responding. The most common test
system was a whole embryo culture, obtained
mostly from rats. The micro-mass culture
method is used to check the effects on differentiation in limb buds of central nervous tissue
removed from rat embryos. Systems using mammalian cells included primary cell culture andestablished cell lines. Those used were strains
for measuring proteoglycan synthesis ability inlimb buds, checking the effects of lectin adherence ability in Ehrlich cells, and evaluating proliferation of palate cells of human embryos (Peters and Piersma, 1990; Gray and Kimber, 1989).
Chick embryo was used by 7% of the companies instead of the mammalian species, but thisis still on a trial basis.
In the whole embryo culture systems(Ninomiya et al., 1993, Brown and Fabro, 1985,
Klug et al., 1992, Uphill et al., 1990, Peters andPiersma, 1990), fetuses of mice, rats, or rabbits
are removed between Gestation Days 8 and 12

and are cultured in 100% plasma. Lethality,
growth suppression, and teratogenicity of thecultured fetuses exposed to test materials were
measured. It was not completely alternative testing, because the duration of the culture was limited, and the viable period was short. However,it was often used to clarify mechanisms of teratogenicity in relation to “in vivo” studies. The
survey results also indicated the main reason for
the test was mechanistic analyses. The disadvantages of this test method include financial
impact and ethical issues. In addition to culture
media, 100% plasma requires many animals
from which to harvest plasma, which also affects study cost. If an artificial culture medium
is developed in the future, this issue might be
resolved.
In the micro-mass culture method (Friedman,
1987, Shiota et al., 1990ab, Uphill, 1990), using mouse palate or rat embryos, the primordial
four limbs and tissue of mesencephalon from theembryos are removed and cultured to evaluate
the effects of exposure to test materials on development and differentiation. Generally speaking, there are some problems in the correlationof these results with “ in vivo” study results.
Evaluation by using multiple strains may be necessary. The uses of this test were mechanistic
analyses and screening. The method used in
mammalian cell culture (Pratt and Wills, 1985)
is the same as in the micro-mass culture method;that is, tissues are removed, cells are isolated andcultured, and biochemical parameters in cell toxicity and cell differentiation are measured. The
main use of this test was for screening rather thanmechanistic analyses, according to the survey.
Some responses raised questions on the correlation to in vivo study results.
23. Testicular toxicity
Three companies thought some alternative
testing in this field was necessary. The purposesof testing were mainly mechanistic analyses of
toxicities and screening. The test systems usedwere mainly primary cell culture, followed byisolated cells, organ slices, and cultured organs(Foster et al., 1987, Kazawa et al., 1990, Noguchi
et al., 1987). Evaluation indicators were test-– 168 –

Alternatives in J. Pharm. Man. Assoc.

osterone measurements, the measurement offunctional markers on Sertoli cells, and cell
death.
24. Thyroid Toxicity
Two companies formerly did the tests. They
no longer do the tests for the following reasons.
In the established cell line systems, FRTL-5
cells were used to measured the inhibition rateof test material on Na125I uptake into cells. The
data were highly variable, and the correlation
with in vivo results was questionable. The system requires specially designated facilities forradiolabeled material, which adds to the problem. In primary cell culture systems (AmbesiImpiobato et al.,1980), the method was examined to assess whether isolated and incubatedthyroid follicles or follicular cells could be used
to observe the effects of a test material (Tamaki
et al., 1984).
25. Vascular irritation
Four companies were doing alternative testing in this field for mechanistic analyses of toxicity, screening and cost savings. The test systems used endoepithelial cells or smooth muscle
cells of blood vessels. Quantitation of FITCmarked albumin or enzyme activities or a morphological technique was used for evaluation(Hoorn et al., 1993).
Consideration on validity of alternative testing
Reasoning for adopting alternative tests wereasked. Forty-two out of 49 companies responded
to the questionnaire with usually conduct validation or sometimes conduct. Five had usedalternative tests with showed no reasoning validations. The responses have been classified
roughly as follows of the companies : 32% used
a comparison with a positive control substanceas the reasoning 22% a correlation with in vivo
study results; 15% a comparison with background data (including comparison with a negative control substance); 13% comparative studies among facilities (interlaboratory validation
studies); 13% a comparison with literature data

; and 5% reproducibility of the test results.
Most companies with alternative testing experience responded that some kind of validation had been achieved (Zucco 1992).
Future Plans for Alternative Testing
Fifty-nine companies among JPMA members
gave their opinions in response to the question
about the future of adopting alternative testing.
Comments were as follows : 73% plan to accept
them; 22% have no plan to accept them; and 5%
will use them on a case-by-case basis.
The purposes of the testing, as conveyed bythose who would accept the testing with certain
conditions, were shared equally between mechanistic analyses and screening.
The actual toxicity fields for which alternative testing may be used in the future were local
irritation, hepatic toxicity, testicular toxicity, teratogenicity, renal toxicity, cardiac toxicity, bonemarrow toxicity, and carcinogenicity.
Comments from the JPMA member companies on Alternative Testing
Fifty-two companies responded. Most of thecomments were divided between acceptance andrejection of alternative testing for animal studies. The opinions were sometimes mixed, evenin the same company, which made a clear understanding of opinion difficult. But most of thetime, progressive and conservative opinions weresplit evenly down the center. The opinion of the
progressive group was that alternative testing
would be useful in screening and mechanistic
analyses of toxicity. Those of the conservative
opinion pointed out that the new methods had
not yet been organized enough by the regulatory
authorities, that they had not been established orvalidated as alternatives to current toxicology
studies, and that there was insufficient correlation between in vivo and in vitro study results.
Animal welfare was mentioned by bothgroups. The conservative group thought the
problem could be handled by reducing the number of animals used in current toxicology studies. Other comments indicated that alternative– 169 –

testing methods should be developed for extrapolation of results to humans rather than to laboratory animals, and that relevant information about
the testing should be available so that alternative testing methods could be used more frequently.
Discussion
The survey results indicated that about 60%of the companies had done some alternative testing one way or another. When correlation ofalternative testing results to study results in humans, study costs, and animal welfare issues
were considered by the pharmaceutical industry, it was recognized that the subject had been
actively discussed. The main purposes of thetests were mechanistic analyses of toxicity andscreening, but some of the test methods had not
yet been established, and it was expected to takesome time before they were validated. Cooperation and discussion by many companies areneeded to establish alternative test methods.
Many companies recognized the future needfor alternative testing for screening and mechanistic analyses of toxicity, but only a few companies have established these test systems andhave done the tests routinely. At present, alternative testing is not better than in vivo toxicity
testing; however, when alternative testing methods are established and serve to clarify mechanisms of toxicities, they could aid in the extrapolation of results to humans. Much research isnecessary to establish these alternative methods.
About half the companies are doing some research and development as an aid in establishing alternative testing. It is very important topursue this research in consideration of futureneeds. Further developments in the already advanced fields of hepatic toxicity, renal toxicity,
teratogenicity, and local irritation are expected
due to social trends (animal welfare and environmental issues).
The present purposes for testing are mechanistic analyses of toxicities and screening, butof equal importance is establishing an accurate
correlation with in vivo studies and convince theregulatory authorities to accept data from alter-

native testing as submittal information for new
drugs.
There is a fourth R concerning the alternative testing concept. Gad (1990) reviewed theprevious first three R’s of replacement, reduction and refinement. A fourth R, responsibility,
was not in the first proposal. Research toxicologists must stand by their responsibility to be conservative in ensuring safety. At the same time,
the responsibility of regulatory toxicologists andregulatory agents must be recognized to accept
IND and NDA. Also, the correlation between invivo toxicity and in vitro toxicity must be monitored closely and constantly.
During the past decade, issues of animals use
and care in toxicological research and testing
have become one of the fundamental concernsof both scientists and the general public.
To evaluate alternative testing in mammalian
toxicity studies, regulatory toxicologists andregulatory agents should provide a current overview of the general concepts, status and progressof in vitro alternatives to research toxicology.
The general public clearly supports animal use
in research when the need and benefit are perceived.
Acknowledgment
We wish to thank the experts who responded
to the survey from the JPMA member companies and the members (Mr. A. Asari of Seikagaku
Corp., Dr. T. Ochiai of Mochida Pharma., Dr.
M. Omosu of Hoechst Japan, Mr. Y. Kaneshiroof Nihon Pharma. Co.,Ltd., Mr. S. Tagishi ofTokyo Tanabe Co., Ltd., Dr. H. Tanaka of Rohto
Pharma. Co., Ltd., Mr. S. Nakamura of Kyoto
Pharma. Indust. Ltd. , Dr. T. Harada of Lederle
Japan Ltd., and Mr. M. Maeda of Ciba-Geigy
Japan Ltd.) of the non-clinical subcommittee of
JPMA who helped summarize the data surveyed.
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