Harmonization of guidelines for toxicity testing of pharmaceuticals by 1992

REGULATORY TOXICOLOGY AND PHARMACOLOGY 12, 179-211 (1990)

Harmonization of Guidelines for Toxicity Testing of Pharmaceuticals by 1992

LORNA H . SPEID, CYNTHIA E. LUMLEY, AND STUART R. WALKER

Centre for Medicines Research, Woodmansterne Road, Carshalton, Surrey, SM5 4DS, United Kingdom

Received March 17, 1990

In the past there has been considerable disagreement between various regulatory authorities regarding the type and design of animal tests that should be required before a new medicine can be used ethically and safely in the clinic. However, regulatory variations have largely been removed within politically and geographically similar regions (e.g., the U.S.A., the European Com- munity, the Nordic countries) and there now appears to be a consensus regarding the value of harmonizing international requirements. In order to assist the process of harmonization, a detailed table of preclinical toxicity requirements in the U.S.A., Canada, Japan, and the European Community for each test (acute, subacute, chronic, carcinogenicity, mutagenicity, reproduction) has been compiled. This has been circulated to the relevant regulatory authorities to ensure that it accurately reflects current requirements. The major differences between authorities were found to be the duration of chronic, repeated-dose tests and the design of reproduction studies. International pharmaceutical companies were asked to complete a questionnaire, indicating how they design their preclinical testing program to comply with varying regulatory requirements. Most of the respondent companies indicated that chronic tests of longer than 6 months were conducted solely to comply with some regulatory requirements. Many companies repeat reproduction studies in order to comply with Japanese requirements. This emphasizes the need to harmonize these guidelines and discussions are currently underway to attempt to develop protocols acceptable to the FDA, the EC, and the Japanese Ministry of Health and Welfare. �9 1990 Academic Press, Inc.

INTRODUCTION

The thalidomide tragedy in the 1960s catalyzed the introduction of medicine regulations, particularly those governing toxicity testing in animals. The original principle behind regulatory control was to state the nature of the information required rather than to describe the details of the tests necessary to achieve this. However, over the past 20 years there has been a tendency for national and international guidelines to become more specific. Some are now so detailed (for example, the reproduction tests), that they serve as testing protocols from which companies hesitate to deviate, because it is potentially too expensive to risk rejection of a product licence submission.

179

0273-2300/90 $3.00 Copyright �9 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

180 SPEID, LUMLEY, AND WALKER

TABLE 1

N U M B E R O F C O M P A N I E S S U R V E Y E D A N D R E S P O N D I N G

COUNTRY USA JAPAN GERMANY SWIT2ERLAND OK FRANCE OTHERS TOTAL

ND~ER OF COMPANIES SUR%'EYED 17 1O 7 3 6 3 5 51

NUMBER OF COMPLETED QUESTIONNAIRES 8 5 3 2 4 0 3 25

RETURNED

NUMBER OF NON-QUESTIONNAIRE RESPONSES 0 0 2 1 1 0 0 4

TOTAL RESPONSE (Z) 8 (47) 5 (50) 5 (71) 3 (100) 5 (83) 0 3 (60) 29 (57)

Various government agencies and health organizations issue guidelines indicating the manner in which studies should be conducted. In the past there has been considerable disagreement between agencies regarding the type and design of animal tests that should be required before a compound can be used ethically and safely in the clinic. As a result, in order to achieve international marketing of a new medicine, toxicology studies have had to be carried out according to experimental protocols recommended by the most exacting authority and even repeated with slight variations to meet the idiosyncrasies of some regulations in specific countries. Yet presumably, the rationale that leads to these differing requirements is based on the same data provided by pharmaceutical companies in their product licence submissions.

Recently, several agencies have evaluated and revised some of their guidelines, for example Japan (JMHW, 1990), and there appears to be a consensus regarding the value of harmonizing international requirements. However, some discrepancies still exist. In order to assist international harmonization ofpreclinical toxicity testing regulations, it is essential to be able to compare requirements across the major world markets for pharmaceuticals. Although the published regulatory requirements of different authorities have been documented (Alder and Zbinden, 1988), these are not in a format to allow the comparison of protocols for specific tests. Furthermore, the requirements of some regulatory authorities have changed since they were last published.

A comprehensive table has therefore been compiled and circulated to the relevant regulatory authorities to ensure that it accurately reflects current requirements. In order to ascertain how strictly pharmaceutical companies adhere to these guidelines, regulatory affairs managers and toxicologists within the pharmaceutical industry have been asked to comment on their companies' current practice. A similar attempt has been made to bring about the harmonization of international guidelines for toxicity studies involving chemicals (ECETOC, 1985).

METHOD

The regulatory bodies of seven countries (U.S.A., Canada, Japan, France, United Kingdom, West Germany, and Italy) were approached for information about their preclinical toxicity requirements for marketing authorization of new medicines. The European Community (EC) was included as a separate category to illustrate how the EC guidelines are derived from those of the constituent countries.

INTERNATIONAL TOXICITY TESTING GUIDELINES

TABLE 2

ACUTE-ToxICITY (SINGLE DOSE) STUDIES

1 8 1

c 0 ~ Y I u ~ SPECIES 13 tO 4 s p e c i e s , a t

and l e a s t one a n o n -

SEX r o d e n t .

A t l e a s t 2 s p e c i e s

s h o u l d be u s e d , one

s e l e c t e d f rom r o d e n t s

and t h e other from

non-rodents o t h e r

than rabbits. Both

sexes should be used

i n a t least 1 s p e c i e s

CANADA

N o r m a l l y , n o t more

two s p e c i e s

(one rodent, one

non-rodent) .

IEEC JAr l e a s t 2 ~-~alian

s p e c i e s of known str-

lain. Rodents such ms

[ ~ t L S e , rat and h a m s t -

l e t are suitable for

I t h e qualitative s t u d y

I o f t o x i c signs and

t h e quantitative det-

l e r ~ L u a t i o n of t h e a p -

J p r o x i m a t e lethal d o s e

I I f no d i f f e r e n c e i s

l o b s e r v e d b e t w e e n the

J s e x e s o f the first

rodent species, then

I o n l y 1 s e x n e e d be

u s e d i n other acute

studies.

( 1 , 2 , 6 , 9 , 1 1 , 2 0 ) ( 1 7 ) ] ( 1 9 ) (7)

........... , ..................... , ..................... , ..................... , .....................

The d r u g is admin-

istered by several

routes, including the

p o t e n t i a l route for

human exposure.

ROUTES O r a l & p a r e n t e r a l

r o u t e L n c l u d i n g t h e

p roposed clinical

r o u t e ( s ) i n r o d e n t s .

If t h e e x p e c t e d c l i n - !

i c a l r o u t e i s r e s -

t r i c t e d t o i n t r a v e n -

ous administration,

u s e o f t h i s r o u t e

a l o n e i n a n i m a l t e s t - I

is a c c e p t a b l e . In

non-rodents, u s e of

P a r e n t e r a l d rugs=

a l l p a r e n t e r a l r o u t e =

t o be u s e d c l i n i c a l l y

a s w e l l a s by t h e

i n t r a v e n o u s route

(even thoush the

i n t r a v e n o ~ r o u t e i s

n o t p r o p o s e d f o r

c l i n i c a l u s e ) .

O r a l d r u g s :

o r a l and p a r e n t e r a l

r o u t e s ( p r e f e r a b l y

I n t h e c a s e o f r oden~

t s i n g e n e r a I , 2

r o u t e s s h o u l d be u s e d

and when p o s s i b l e

s h o u l d i n c l u d e t h o s e

r o u t e s p r o p o s e d for

man- At l e a s t one

s h o u l d e n s u r e f u l l

access o f u n c h a n g e d

d r u g into t h e c i r c u -

l a t i o n . I f t h e p r o p -

o s e d r o u t e o f aclms

s t r a t i o n t o man i s

i n t r a v e n o u s , t h e n use

o f t h i s r o u t e a l o n e

i n a n i m a l s is a c c e p t ~

a b l e .

(7)

t h e e x p e c t e d c l i n i c a l ] i n t r a v e n o u s or

r o u t e a l o n e i s i n t r a p e r i t o n e a l ) .

a c c e p t a b l e . C u r r e n t 17 u n d e r

r e v i e w .

( 1 , 9 , 1 1 ) ( 1 7 ) ( 1 3 , 1 4 , 1 9 )

. . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . .

NUMBER NO SPECIFICATIONS F o r r o d e n t s a t l e a s t T h e r e i s no r e q u i r e - The maxL~um amount o f

OF GIVEN. 5 a n i m a l s p e r s e x merit f o r a s p e c i f i e d X n f o n e a t t o n s h o u l d be

ANIMALS s h o u l d be u s e d and number o f a n i m a l s , o b t a L n e d f rom t h e

f o r n o n - r o d e n t s a t [ a n i m a l s u s e d i n t h e

l e a s t 2 a n i m a l s per [ s t u d y . F o r e t h i c a l

s e x . ] r e a s o r ~ , i t i s n e c -

l e s s a x 7 t o r e p l a c e e x -

I s t L n g me thods a s

s o o n a s s c i e n t i f i c

t e c h n i c a l a d v a n c e s

So a l l o w , by methods

I i n v o l v i n & a s f ew i a b -

i o r a t o r y a n i m a l s a s

I p o s s l b l e .

( 1 7 ) ( 1 9 ) I (7 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


TABLE 2--Continued

COUMTRY USA JAPAR CARADA l ~ C

LDS0 DA does not require

the LDSO test.

Ch~racterisation of

dose response (toxic-

ity) c u r v e s and surv-

ival should be

monitored.

( 1 , 9 , 1 0 , 1 8 , 2 0 )

New guidelines indi-

cate that Jg~anno

longer requires am

accurate LDSO study,

as previously c a l c u -

l a t e d u s i n g a large

number o f animals.

Approximate lethal

dose in rodents

shauld be estimated

on the basis of mor-

tallty at different

dose levels. In non-

rodents the dose

causing apparent tox-

ic signs s h o u l d be

! d e t e r m i n e d .

( 5 , 1 7 )

The Drugs Directorate

does not require t h e

LDSO test. An a p p r o -

x i m a t e determination

o f acute toxielty, or

if necessary, the

mlpI~n lethal dose,

will mu~fiee. (These

requirmments will be

r e - e v a l u a t e d as new

walidatedmethods

become available). A

limit dose o f 20OOmg

l k g i s oon~s lde red

sufficient.

(19)

The single d o s e t o x l -

c i t y tests s h o u l d be

c o n d u c t e d i n s u c h a

way that signs o f

acute toxicity are

revealed and t h e mode

of death a s s e s s e d . A

g~la~tltative ewalua-

t t o n o f t h e approxi-

mate lethal dose and

information on the

dose-effect relation-

s h i p s h o u l d be

o b t a i n e d , b u t a h i g h

l e v e l o f p r e c i s i o n i s

n o t r e q u i r e d .

(7 )

OBSERVATION

PERIOD

A l l a n i m a l s a r e As a g e n e r a l r u l e ,

o b s e r v e d f o r 2 weeks a n i m a l s s h o u l d be

f o r s i g n s o f t o x i c g t y l o b s e r v e d f o r 14 d a y s .

and fatalities. The lToxic signs, their

time o f death and thelseverity, o n s e t , pro-

onset, severety, rew-lgression and re~ersi-

ersibility and durat-!bilicy s h o u l d be

i o n o f c l i n i c a l s i s n s o b s e r v e d . A u t o p s y i s

are noted as they required for all

occur, rodents, and for non-

rodents d y i n g d u r i n g

!the observation peel-

od. This makes the

re-use of surviving

non-rodent animals a

Ipossibllity.

J l

l l

(9,18,20) I (5,17)

14 d a y s for o r a l , and

7 d a y s for parenteral

E u t h a n a s i a s h o u l d be

i n v o k e d i f e x c e s s i v e

p a i n and p r o l o n g e d

sufferim~ is observed

(19)

The period during

w h i c h t h e test ani-

mals are observed

shall be fixed by the

investigator as being

adequate to reveal

tissue or organ dam-

age or recovery,

usually for a period

o f I ~ d a y s , but not

l e s s t i t a n 7 d a y s , b u t

without e~posin~ the

animals to prolonged

suffering. A n i m a l s

d y i n g d u r i n g t h e o b -

s e r v a t i o n period

sha tLld be subject to

autopsy as should all

animals surviving t o

the end of the obser-

vation period.

(7)

ADDITIONAL NO SPECIFICATIONS INO SPECIFICATIONS NO SPECIFICATIONS Exclpients used for

INFORMATION GIVEN IGIVEN GIWE~ the first time in the

I [ p h a r m a c e u t i c a l f i e l d

Ishall be treated as a

I I . . . . . t i r e ingredient

I [(7)

1. A l d e r & Z b i n d e n (1988)

2 . anon (1977 )

3. anon (1984)

4. anon (1988)

5 . anon (1989a)

6 . D ' A g u a n n o (1973) 11 . G o l d e n t h a l ( 1968 )

7. EC ~ t s s i o n (1989) 12 . G r i f f i n (1985)

8 . FDA (1985) 13 . HPB (1987a)

9 . FDA (1987) 14 . HPB (1987b)

10 . FDA (1988) 1 5 . J e f f e r y s (1989)

16 . ~ (1987)

17 . ~ (1990)

18. K e s t e ~ s o n (1982)

19 . Somers (1990 )

20 . W e t s s ~ n g e r (1990)

INTERNATIONAL TOXICITY TESTING GUIDELINES 183

TABLE 3

SUBACUTE (REPEAT DOSE UP TO 3 MONTHS) TOXICITY STUDIES

COUNTRY

SPECIES J U s u a l l y the rat t dog

J O u t b r e d rodents and

I n o n - r n d e n t s . Primate

Imay be c h o s e n a s n o n -

rodent if p h e r m a c o L o -

Jgy o r k i n e t i c s show

i t i s more s u i t a b l e .

JAPAN

At least 2 species,

lone selected f r m

rodents, the other

s174 n o n - r n d e n t s

o t h e r t h a n r a b b i t s .

CANADA [EEC

At least 2, including[At least 2 species of

anon-rodent o t h e r [~nimal (normally both

t h = n t h e r a b b i t . [ s e x e s ) , one o f w h i c h

] s b o u J . d b e a n o n -

] r o d e n t . The c h o i c e o f

I s p e c l e s s h o u l d be

JJustlfied. As far a s

p o s s i b l e , the s p e c i e s

J s h o u l d be c h o s e n on

I t he b a s i s o f their

simllarlty to man

with regard to the

] p h e r m a c o k ~ e t i c s ,

i ~ e l u d l n g the b i o -

t r a n s f o r m a t i o n o f the

active ingredient.

( 9 , 1 8 ) ( 1 7 ) ( 1 3 , 1 4 ) ( 7 )

. . . . . . . . . . . i . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROUTE As c l o s e to intended

human route as possi-

b l e . D~s i n t e n d e d

f o r o r a l & p a r e n t e r a l

u s e a r e a d m i n i s t e r e d

o r a l l y ( g a v a E e o r

c a p s u l e ) and by i n -

t e n d e d p a r e n t e r a l

r o u t e r e s p e c t i v e l y . I f !

more ~ one r o u t e

of adm~istration is

l i k e l y s t u d i e s may be

c o n d u c t e d by both t o

define differences in

toxicity. If oral

route is used, ensure

d r u g is a b s o r b e d .

( 9 , 1 8 )

N o r m a l l y , t h e

e x p e c t e d c l i n i c a l

r o u t e s h o u l d be u s e d .

(17 )

A d m i n i s t r a t i o n s h o u l d

i n c l u d e t h e r o u t e s to

be u s e d i n ~he c l i n -

i c a L s t u d i e s . When

d r u g iS a d m i n i s t e r e d

o r a l X y , e v i d e n c e f o r

a b s o r p t l o n ( b l o o d l e -

v e l s , p h a r m a c o l o g i c

e f f e c t s ) s h o u l d be

I p r o v i d e d . O r a l a d m i n -

Istration m y be s u b -

I s t l t u t e d f o r p a r e n -

J t e r a l i f s t u d i e s show

[ r h a t t h e m e t a b o l i c

t p a t t e r n s o b t a i n e d by

e a c h r o u t e a r e t om-

[ p a r a b l e . C u r r e n t l y

u n d e r r e v i e v .

( 1 3 , 1 4 , 1 9 )

Whenever t h i s i s

t e c h n i c a l l y f e a s i b l e ,

t h e a c t i v e i n g r e d i e n t

s h o u l d be a d m i n i s t e r -

ed by t h e r o u t e

l n t e n d e d f o r u s e i n

man a n d i t i s d e s i r -

a b l e t h a t t h e phel"m~-

c o l o g i e a I e f f e c t i s

d e | by t h i s

r o u t e . When t h i s c a n -

t o t be shown, t h e use

o f o t h e r routes

should be considered.

A t t e n t i o n s h o u l d be

p a l d t o t h e p o s s l b i l -

I t y o f L o c a l t o x i c i t y

a t t h e s i t e o f a p p l i -

c a t i o n .

1(7) . . . . . . . . . . . | . . . . . . . . . . . . . . . . . . . . . ' . . . . . . . . . . . . . . . . . . . . . " . . . . . . . . . . . . . . . . . . . . . [ . . . . . . . . . . . . . . . . . . . . .

DURATION F o r up t o 2 w e e k n u s e Depends on d u r a t i o n V a r i e s f r o m 4 t o 6 J ~ n AnlmaL

i n , a n , t e s t f o r 2 o s I n man. weeks ; i n s e n e r a l , I one o r s e v e r a l

weeks t o 3 monrJ~s i n Man An ima l does n o t e x c e e d 3m. [ d o s e s im I d a y 2wks

a n i m a l s . A d m i n i s t e r Jl wk o r l e s s lm Depends on d u r a t i o n [ t o 7 d a y s 4vks

d r u g f o r 7 d a y s a

week.

I I I I I I J ( 1 , 6 , 9 , 1 8 ) I

> I v k t o 4 wks 3m

A d m s s h o u l d Dosage r e g i m e n s h o u l d

be p e r f o r m e d 7 d a y s a [ b e c o m p a r a b l e w i t h

week . I t h a t i n t e n d e d i n man.

F o r s t u d i e s o f 3m, I [ A d r u g t o be a d m i ~ l -

month p r e l i m i n a r y [ s t e r e d d a l l y t o man

t e s t s a r e r e q u i r e d . I s h o u l d be g i v e n 7

These c o n t r i b u t e t o I day8 a w e e k .

t h e s e l e c t i o n o f doseJ

l e v e l s and h e l p t o J

c l a r i f y t o x i c i t y . I

( 4 , 1 7 ) [ (13 ,L4 ,19) t

o f USe i n man. lup t o 30 d a y s 3m

I f t h e i n v e s t i g a t o r

s e e s f l t t o c a r r y o u t

e x p e r i m e n t s o f g r e a t -

e r o r l e s s e r d u r a t i o n

t l u m t ~ d L c a t e d a b o v e ,

h e m u s t g i v e a d e q u a t e

r e a s o n f o r d o i n g s o .

1(7) I

184 SPE1D, LUMLEY, AND WALKER

TABLE 3--Continued

COtP~TRY

DOSE LEVELS

USA

3 dose levels. High

dose i n d u c e s severe

t o x i c i t y . Interme-

diate dose chosen to

characterise toxic

dose-response curve.

Low dose to produce

Ipharmacologlcal

effects but without

t o x i c e f f e c t s .

I JAPAN

JAr least 3 dose

l e v e l s e a c h s e x . f o r

lOne dose l e v e l should

n o t c a u s e t o x l c dam-

l a t e (no e f f e c t dose)

I& 1 dose l e v e l should

lproduce overt toxici-

ty. I t is desirable

to set the dose le-

vels s o that a dose-

response relationship

i s achieved. A

vehicle control group

should be included

and if necessary an

untreated and/or a

positive control

g r o u p may b e added.

CANADA

At least 3 different

dose l e v e l s . The

highest dose should

cause t o x i c e f f e c t s

( f u n c t i o n a l ,

h a e m a t o l o g l c altera-

tions) hut it should

also allow survival

o f t h e majority of

a n i m a l s . The l o w e s t

dose should exert

appropriate pharmaco-

logic o r therapeutic

effects in the

species concerned.

EEC

3 dose levels.

High dose - to cause

target organ toxicity

or non-specific toxi-

city. The low dose

s h o u l d p r o d u c e a

p h a r m a c o d y n a m i c o r

d e s i r a b l e t h e r a p e u t i c

effect. The inter-

mediate d o s e can be

the geometric mean of

the high and low

doses.

(2,9,20) ( 4 , 1 7 ) ( 1 3 , 1 4 ) (7 )

When decidln 8 on the Treatment groups

number of animals per should be large

group, consideration enough to:

should be given to Is) reveal all toxi-

the fact that at [cologically important

times, at least some leffects due to treat-

animals at the high- Iment;

est dose level and i n J b ) allow sacrifice of

the control group

should be retained

for observation after

the end of the admin-

istration period.

Non-rodent studies

generally involve a

smaller number of

animals.

(13,14,19)

animals at intervals

before the end of the

study without inter-

ferln 8 with the final

statistical a n a l y s e s ;

c)allow an assessment

of reversibility or

irreversibility.

These conslderats

lalso apply to chronic

Istudies (see Table 4)

1(71

The final group size

will depend, to a

Large e x t e n t , upon

s t u d y d e s i g n , s u c h a s

inclusio~ of interim

sacrifices, evaluati-

on of reversibility

of toxic effects,

flndlngs from range

finding studies and

other preliminary

data.

NUMBER OF

ANIMALS

( 1 , 2 , 9 , 1 8 , 2 0 )

At l e a s t I 0 o f e a c h

s e x p e r g r o u p f o r r o -

d e n t s t u d i e s . A t

least 3 of each sex

per group for non-

rodent studies. Where

i n t e r ~ e x a m i n a t i o n s

o r r e c o v e r y t e s t s a r e

s c h e d u l e d , t h e n u m b e r

of animals necessary

for these tests

should be added. Con-

slderation should be

g i v e n to i n c o r p o r a -

t i n g a r e c o v e r y g r o u p

f o r 1 a n d 3 m o n t h

studies to evaluate

reverslbillty or

d e l a y e d t o x c i t y .

(17)

To facilitate comparisons, the table was divided into sections by toxicity test (acute toxicity studies, subacute toxicity studies, chronic toxicity studies, carcinogenicity tests, mutagenicity tests, and reproduction studies). The resulting table was circulated to the regulatory authorities (8) and major international pharmaceutical companies (51) in the U.S.A., UK, Switzerland, Germany, Japan, France, Belgium, Italy, Den- mark, Sweden, and the Netherlands. The regulators were asked to make corrections to the table so that it accurately reflected their current guidelines, while the companies were asked, by means of a questionnaire survey, to indicate to what extent they adhered to the written regulations.

I N T E R N A T I O N A L T O X I C I T Y T E S T I N G G U I D E L I N E S

T A B L E 3--Continued

185

c0~Y l.sA IJAP~ ICA.~A I~.C

l l c a l l y . A d e t a i l e d

I g r o s s n e c r o p s y l s

l p e r f o r m e d o n a l l

animals that die, or

are killed,

AUTOPSY �9 I A co~p=ehe~Ive l l s t l ~ l ~ I s ~ .~ lud~os l~ l =~a . . . . S~"~ o f ITermioal o b . . . . t ions 81SX0L~Y Io f ORBS- ~ t l . . . . . I o o . t r o l ~ l . . . . I*11 r~nts ~d ~ 0 - I s h ~ l d b . . . . ~ l e t e

[ i S e x a m i n e d h i s t o l o g - l t o b e a u t o p s l e d . H l s - l r o d e n t s t h a t d i e o r [ a s p o s s i b l e . A u t o p s y

l t o p a t h o l o g l c a l e x a m - l a t e k i l l e d s h o u l d h e l=~t he c o n d u c t e d o n

l~ t l o , shonld he le~-~d for 8 . . . . l - l l ~ . l s .isto- i p e r f o r m e d o n o r g a n ~ & i p a t h o l o 8 1 c c h a n g e s . [ p a t h o l o g y s h o u l d be

( 1 , 2 , 6 , 9 , 1 8 )

tissues of all non-

rodents & O f at least

t h e h i g h e s t d o s e

g r o u p a n d t h e c o n t r o l

StOUp of rodents. If

m a c r o s c o p i c e x a m i n a -

t i o n o f o r g a n s a n d

tissues of rodents in

other dose groups

reveals a n y changes,

o r i f c o n s i d e r e d a p -

lpropriate b a s e d o n

l i n f o r m a t i o n o b t a i n e d

I f r o m t h e h i g h e s t d o s e

I g r o u p , hlstopathelo-

8ical examination

Ishould be performed

I o n t h o s e organs ~ d

tissues from all anl-

mls in those groups.

[(17)

H l s t o p a t J ~ o l o g y s h o u l d l p e r f o z m e d o n a l l o r -

t e p e r f o z m e d o n a l l [ 8 a n s a n d t i s s u e s o f

n o n - r o d e n t s a n d a l l t h e h i g h d o s e a n d

r o d e n t s i n t h e h i g h c o n t r o l g r o u p s . I n

dose and c o n t r o l r o d e n t s , t h e e x a m l n a -

8 r o u p s . t l o n o f t h e l o w e r

d o s e 8 r o u p s may be

r e s t r i c t e d t o t h o s e

o r g a n s a n d t i s s u e s

s h o w i n g p a t h o l o g i c a l

changes a t a u t o p s y . I n

o t h e r s p e c i e s w h e r e

small numbers o f ani-

mals a r e u s e d , d o

h i s t o p a t h o l o g y o n all

listed tissues.

Attention should be

paid to possible

I n t e r f e r e n c e w i t h t h e

i m m u n e s y s t e m .

(14) ( 7 )

1. A l d e r & Z b l n d e n ( 1 9 8 8 )

2 . a n o n ( 1 9 7 7 )

3 . a n o n ( 1 9 8 4 )

4 . a n o n ( 1 9 8 8 )

5 . a n o n ( 1 9 8 9 a )

6 . D ' A g u a r m o ( 1 9 7 3 ) 1 1 . G o l d e n t h a l ( 1 9 6 8 )

7 . EC C o m m i s s i o n ( 1 9 8 9 ) 12 , G r i f f i n ( 1 9 8 5 )

8 . FDA ( 1 9 8 5 ) 1 3 . HPB ( 1 9 8 7 a )

9 . FD& ( 1 9 8 7 ) 14 . HPB ( 1 9 8 7 b )

10. FDA (1988) 15. J e f f e r y s (1989)

16 . JTHHW (1987 )

17 . J ~ (1990)

18 . K e s t e r s o n (1982)

19 . Somers (1990)

20 . W e i s s l n g e r (1990)

R E S U L T S

Replies were received from regulatory authorities in Japan, the U.S.A., Canada, the United Kingdom, Germany, and Italy and from over 50% of the companies surveyed (Table 1).

Following the survey, the table was altered to reflect the comments of the regulators and the companies. The separate EC countries (UK, France, Germany, and Italy) were deleted, as companies and regulators indicated that the EC regulations are accepted by the constituent countries. The final version of the table, which accurately reflects the current position of these regulatory authorities, is shown in Tables 2 to 10.

D I S C U S S I O N

Differences in regulations between countries may have arisen for cultural, histori- cal, and economic reasons and not because of critical needs in patient protection

1 8 6 SPEID, LUMLEY, AND WALKER

TABLE 4

CHRONIC (REPEAT DOSE LONGER THAN 3 MONTHS) TOXICITY STUDIES

COUNTRY USA

SPECIES 2 m~lian s p e c i e s ,

lone a non-rodent.

Usually conducted in

rat ~d dog (Choice

o f s p e c i e s will be

i n f l u e n c e d by t h e

!~esults of the subac-

ute toxicity tests -

table 2).

JAPAN

;At least two species,

one selected from

! r oden ts and the o t h e r

from non-rndents,

other rh~n rabbits.

ezcretion are con-

cerned. Use species

with similar parent

e u m p o u n ~ m e r a h o l i c

pattern toman.

CANADA [EEC

Use species which ]At least 2 species of

:sost closely resem- [m~Is (normally

bles man as far as ] b o t h sexes), one of

a b s o r p t i o n , distribu-lwhichmust be a non-

riot, metaholismand [rodent. The choice of

[species should be

ljustified. As far as

[possible, t h e species

] s h o u l d be chosen on

the basis of their

similarity to man

with regard to the

I pharmacokinet ic s, i n -

c lndJng the biotrans-

f o r m a t i o n of the

active ingredient.

ROUTE

( 1 , 6 , 9 , 1 8 , 2 0 ) (17) ( 1 3 , 1 4 ) ( 7 , 1 2 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

[Administration of Normally the expected Route to he used in

drug to rodents & clinical route should clinical studies. If

Don-rodents duriJx E be used. !oral, evidence for

chronic tests is absorption should be

usually by the [ p r o v i d e d . When d r u ~

[intended route. [in diet, stability &

little to be monitor-

led. Oral Lnstead of

parenteral route is

I p o s s i b l e i f m e t a b o l i c

p a t t e r n s o f b o t h a r e

shown as comparable.

(1,9,18) (17) (13,14) (7)

........... .......................................................................................

DOSE LEVELS[These are determined At least 3 dose At least 3 different 3 dose levels. The

[by the results of the levels. One dose levels. HiF~hest maximu~ dose should

[90 day t o x i c i t y l e v e l s h o u l d n o t ! s h o u l d i n d u c e t o x i c be c h o s e n so a s t o

Istudies. There are 3 cause toxic damaEe effects,but it should bring harmful effects

llevels. The highest (no effect dose) and also allow the sur- to llght. The lower

[dose should induce one dose level s h o u l d viral of the majority doses will enable the

ls~ toxicity, lprnduce overt toxiei- of animals, animals' tolerance of

[ [ty. It is desirable t he product to be

to set the dose le- determined. The in-

v e l s so that a d o s e - t e r m e d i a t e d o s e may

] response relatlonshipl he the geometric mean

I is a c h i e v e d . [ b e t w e e n t h e hish and

] I l ow d o s e s , r e a s o n s

I I should he g i v e n f o r

[ ] choice o f doses.

[ ( 1 , 6 , 9 ) (17) 1 (13 ,14 ) (7 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C h o i c e o f r o u t e s

w i l l d e p e n d on t h e

i n t e n d e d t h e r a p e u t i c

u s e ~ d t h e p o s s i b i l -

i t i e s of systemic

a b s o r p t i o n . A t t e n t i o n :

s h o u l d be p a i d t o t h e

p o s s i b i l i t y o f l o c a l

t O x i c i t y a t t he s i t e

o f 8 p p l l c a t i o n .


T A B L E 4-Continued

187

DURATION lDepends on d u r a t i o n lDepends on d u r a t i o n

[o f u s e i n man. [o f u s e i n man.

[1 mon th o r 14 v k - Gm 6m

l o n g e r 12m L o n g e r t h a n ~ 12m

( 1 , 2 , 6 , 9 , 1 8 . 2 0 )

Administer tO animals

for 7 days per week.

12m test required f o r

lom~-te~m repetitive

administration o f >6

months or studies

where this i s j u d g e d

to be appropriate.

Preliminary studies

of 1 month for 6

month tests, and 3

~onths for 12 month

tests, s h o u l d be

[ p e r f o ~ d . ( s e e s u b -

l a c u t e , T a b l e 3)

I(4,5,17)

Depends on duration

o f use i n man.

Man A n i m a l

Beyond Im 12m

(Current ly under

review).

( 1 3 , 1 ~ , 1 9 )

Depends o n duration

o f u s e i n man.

Man A n i m a l

Beyond 30 d a y s 6~*

Whemhtmmn e x p o s u r e

i s l i k e l y t o be l o * ~ -

t e r m , e g w h e n f r e -

I cJ~ent discontinuous

administration re-

sults in a total per-

i n d o f e x p o s u r e o f l m

or more in a period

of 1 year, or when

retention in body of

a single d o s e of d r u g

is prolonged, dura-

tion will be 6 m.

*Do after 2-4 wk dose

range finding study.

( 7 )

................................. i .................................................................

NUMBER OF i0 - 25 rodents and 21Rndent: at least I0 Rodents - sufficient

ANIMALS -3 ~n-rndents/sexl Imales and I0 f~males animals to ])emit

group. The selection [per group. ]perindlc laboratory

o f group size for [Non-rodent: at least

chronic studies will 13 males and 3 females

depend on the toxlei- Where interim exa~In-

ty/~ortality in the ations or recovery

preceding subacute tests are scheduled,

studies, the number o f animals

necessary f o r these

tests should be

added.

( 1 , 2 . 9 ) ( 5 , 1 7 )

i~vestlgatlons, his-

t o p a d l o l o g y and i n -

f o r m a t i o n i n reversi-

bility study to be

o b t a i n e d .

N o n - r n d e n t s : smaller

number o f animals.

(13,14)

L a r g e e n o u g h to :

a ) a l l o w a l l t o x i c o -

l o g i c a l l y important

effects due to treat-

ment t o be r e v e a l e d ;

b ) p e r m i t s a c r i f i c e o f

a n i m a l s b e f o r e t h e

end o f t h e s t u d y

w i t h o u t i n t e r f e r i n g

with the final stat-

i s t l e a l a n a l y s e s ;

c ) a l l o w some animals

t o be r e t a i n e d f o r

r e v e r s i b i l i t y s t u d y .

S i z e o f g r o u p s w i l l

be limited f o r p r a c -

tical, financial and

h~ane reasons,

These co~s Iderations

a l s o a p p l y t o s u b -

a c u t e s t u d i e s ( s e e

T a b l e 3 ) .

(7)

(Lasagna, 1977). These differences can force companies to repeat studies, or to perform additional studies, to meet the requirements of certain regulatory authorities in order to obtain marketing authorization. Such repetition is likely to produce little information of value, and its acquisition implies an irrational allocation of available resources (Binns et al., 1976). Consequently, it is necessary to distinguish regulations


TABLE 4--Continued

COUNTRY

CONTROL

GROUPS

USA

Appropriate c o n t r o l

groups.

! J~AS

A v e h i c l e c o n t r o l

group s h o u l d b e in-

e l u d e d a n d i f n e c e s -

s a r y , an untreated

control StOUp and/or

a positive (reference

d r u ~ ) c o n t r o l group

may be added.

CANADA J EEC

C o n t r o l groups m i g h t [Wherever possible,and

i n c l ude u n t r e a t e d , [always in experiments

sham, o r vehicle [on small rodents, the

treated and p o s i t i v e [ d e s l E n o f t h e experi-

controls as required Iment and the control

for varioUS co~parat-lprocedures Exist be

!i~e purposes, suited to the sca le

o f t he p r o b l e ~ b e i n E

tackled, and enable

flduelal l~mits to be

determined.

(20) (4 ,17) (13 ,14) (7) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Eye examinations Exciplents used for

should be carried out the first time in the

on all non-rodents [pharmaceutical field

Iprlor to and at the [shall be treated like

end o f the study, fan active ingredient.

Recovery 8roup of fin the case of new

anlmals from the hi~hlcomb~nations of known

dose group will pro- ~substances that have

v i d e ~nfor~tion on [been investigated in

t h e reversibility of [ a c c o r d a n c e with the

any bioche~ical al- Iprovision~ of Direc-

!teratlons or histo- tlve 75/318/EEC, the

patholoEical lesions, lons-term tests may,

except where acute

a n d s u b a c u t e t o x i c i t y

t e s t s h a v e d e m o n s t r a -

t e d potentiation or

n o v e l t o x i c e f f e c t s ,

be s u i t a b l y mod i f i ed

[ by t h e i n v e s t i g a t o r .

A t t e n t i o n s h o u l d be

p a i d t o p o s s i b l e i n -

t e r f e r e n c e w i t h t h e

l lmmune s y s t e m .

; (13 ,14) ] ( 7 )

Autopsy all animals

a t the end of the

study, as for sub-

acute tests. (Table

Coc~arative d r u ~

metabollsm~d pharm-

acoklnetic studies in

animals and~ are

u s e f u l i n t o x i c o l o - 13)

l g l c a l evaluation,

extrapolatlonand

species s e l e c t i o n .

Enzyme induction

should be considered

when plannillg and

evaluatln 8 chronic

t o x i c i t y studies.

(11,18,20) (17)

ADDITIONAL

INFORMATION

1. Alder & Z b i n d e n (1988)

2. anon (1977)

3. anon (1984)

4. anon (1988)

5. anon (1989a)

6. D'Asuanno (1973) 11. Go lden tha l (1968)

7. EC Co~mission (1989) 12. G r i f f i n (1985)

8. FDA (1985) 13. HPB (1987a)

9. FDA (1987) 14. HPB (1987b)

10. FDA (1988) 15. Jefferys (1989)

16 . ~ ( 1 9 8 7 )

17 . JMHW ( 1 9 9 0 )

18 . K e s t e r s o n ( 1 9 8 2 )

19 . S o m e r s ( 1 9 9 0 )

20 . Weissin~er ( 1 9 9 0 )

which are obsolete and scientifically irrelevant from those which must be adhered to by the industry because of scientific rationale. This survey was unique in that it en- abled both the regulated (the companies) and the regulators to comment on the current situation, and it highlighted certain key issues which can serve as a focal point for discussion between these two parties. The following discussion of results reflects the questionnaire responses which were obtained from 25 companies. (Although 29 companies participated in the study, 4 companies sent back detailed comments, without completing the questionnaire.)


T A B L E 5

C A R C I N O G E N I C I T Y T E S T S

189

COUNTRY

REQUIREMENT

USA

' [ R e q u i r e d for:

]i. Drugs w h i c h a r e

Icheml~ally related t o

Iuaown carci~1ogens.

2 . DruBs that producel

]metabolltes similar

[ t o t l ~ s e of known

carcinogens.

] 3 . D r u g s k n o w n to

I d a m a s e o r affect

]rapidly divldLn 8

tissues.

] 4 . Drugs for chronic

use.

New d r u E s must be Unnecessary for anti- 1.Substances having a

tested if o n e o f t h e

f o l l o w i n g a p p l i e s :

1 . Carcinogenieity

is suspected fr=:

a . c h e m i c a l s t r u c t u r e

o r p h a r m a c o l o g i c a l

a c t i o n ;

b. the results of re-

Ipeated d o s e tozlcity

studies or other tox-

icity tests;

c . other information.

2. Clinical use is

expected to extend

over a long period of

Itlme.

T h e s e r u l e s d o n o t

a p p l y t o s u b s t a n c e s

u s e d o n l y for v e r y

l i m i t e d t a r g e t

diseases o r patient

groups, and where the

substance is highly

beneficial for such

patients.

( 9 , 1 8 ) (17)

n e o p l a s t i c d r u g s t g

be u s e d i n temiDally[

Ill patients. Carcln-

o g e n l c l t y t e s t i ~

s h o u l d b e c a r r i e d g u t

d u r i n g c l i n i c a l s t n d -

i e s a n d p r i Q r t o m ~ r -

k e t i n 8 , o n d r u g s t h a t

are intended f o r l~n8

- t e r m u s e , a n d a r e

usually essential

1.chemical and biolo-

gical p r o p e r t i e s r~-

s e ~ b l e those o f chem-

ical c a r c i n o g e n s .

2 . s i m i l a r r e a c t i v e

m e t a b o l i t e s a r e f o ~ m -

e d f r o m t h e d r u g a~_d

& k n o w n c a r c i n o g e n .

3 . d r u g damages / ~ f f e c t

mitosis.

( 1 3 , 1 4 , 1 9 )

c l o s e c h e m i c a l a n a l -

o g y w i t h k n o v n c a r -

c i n o g e n t c campom~ds;

2 . S ~ s t a n c e s w h i c h

b a r e g i v e n r i s e t o

s u s p i c i o u s c h a n g e s

d u r i n g t h e long-term

t o x i c o l o g i c a l t e s t s ;

3 . S u b s t a n c e s w h i c h

hAVe g i v e n rise to

suspicious results in

the mutagenic poten-

:tial tests or i~i

other short-ter~

carc~ogenicity tests

4. Tests may b e r e q u -

i r e d f o r products t o

he used long-term (6

m o n t h ~ e i t h e r c o n t i n -

u o u s l y or intermit-

tently totaling a

similar period).

5 . S u b s t ~ c e s w h i c h

c a u s e c o n c e r n d u e to

some s p e c i f i c a s p e c t

o f their biological

a c t i v i t y ,

( 7 )

PRELIMINARY

STUDIES

90 d a y d o s e - r a n g e

f i n d i n E s t u d i e s a r e

r e c o m m e n d e d t o

a p p r o p r i a t e l y s e l e c t

t h e p r o p e r d o s e s .

(20)

P r e l i m ~ r y s t u d i e s

a r e p e r f o r m e d t o s e t

t h e d o s e l e v e l s f o r

full-scale careino-

g e n i c i t y studiesj

Unless t h r o u g h l y

reliable data a r e

available.

(17)

Number of Animals Used in Toxicity Tests

The number of animals required by the authorities can vary for the same toxicity test. Respondent pharmaceutical companies indicated the numbers of animals generally used for each test, and these are summarized in Table 11. It must be born in mind, however, that the determination of the number of animals to be used can depend on the compound under investigation, together with the purpose and nature of the studies.

Japan is the only country that specifies a minimum number of animals to be used for acute tests (5 rodents and 2 nonrodents per group, with both sexes to be studied


TABLE 5--Continued

COUNTRy

SPECIES

NUMBER OF

ANIMALS

IJ~AN [2 species,usually ratlAt least 2 species [At least 2 - the

~d mouse. Strain of [for both preliminary Imost useful species

a n l ~ a l u s e d s h o u l d b e l a n d f u l l s e a l . . . . . I . . . . . t . . . . . . .

sensitive to known dies. Rats, mice o r [hamster. Sometimes

carcinogens, hamsters are co~u~Dly[~e d o g , primate, O~

u s e d . Other are u s e d .

D e l ~ S o n t h e o b j e c -

t i v e o f ~ study.

(2,9,18,20) (17) (13,14)

The number of animals

is a statistical

issue. A m i n i m u m o f

50 animals of each

rodent species & sex

in each d~ug and

conErol group should

be u s e d .

EEC

The studies should

usually be conducted

on 2 species. Those

u n d e r t a k i n g t h e s t u d y

s h o u l d s e l e c t s p e c i e s

a n d s t r a i n s known t o

be sen~igive to one

or more carcinogens.

The metahoLic h a n d -

l i n g o f t h e d r u g

s h o u l d he ~ o w n i n

the species used and

should preferably

show similarities to

t h e metabolism in

man.

(7)

(9,18)

At least 50 animals

of each sex/gp. A

negative control

group should be inc-

luded. If varlous ve-

hicle5 or emulsifiers

are required tO ad-

minister t h e test

substance, the nega-

tive control group

should receive such

vehicles or e~mulsl-

fiefs alone, It is

desirable to e m p l o y

another untreated

control group. (pre-

liminary studies

should use groups

consistin~ of about

10 of each sex.)

(4,5,17)

At least 50 animals/

sex and per treatment

group. Two control

groups each with 50

anlmals/sex are re-

cammended. Sometime5

a d d i t i o n a l ~ntrol

groups are necessary

For routine tests

with mice, rats and

h~m~ters - suggest

50[sex/gp & 2 control

groups of 50/sex/gp

w i t h v e h i c l e , by t h e

s~ route.

IcE v e h i c l e c o n t r o l o r ]

e x p o s u r e ~o n i t r o g e n

i n s t u d i e s wLch l n l m -I

lation anaesthetics.

(13,14) ( 7 )

for at least one species), and most companies use this number or less. However, five companies use 10 rodents per sex per group and three use more than 2 nonrodents per sex per group (one of these conducts acute tests in only one sex in nonrodents).

No company responding to this survey uses more than 25 rodents/sex/group for subacute studies, and four of the seven employing this number indicated that these included animals to enable an assessment of recovery/reversibility. Similarly, for chronic studies only six companies use more than the maximum recommended number (U.S.A., 10-25); these include one company that carries out a combined chronic toxicity/carcinogenicity test, and one each that includes extra animals for interim sacrifice, kinetic studies, and a recovery group. The situation is very similar for nonrodents; 15 companies use only 3/sex/group for subacute tests as required by Japan while six use 3 or 4 and only four on occasion use up to 6 (one includes recovery


TABLE 5--Continued

191

COUNTRy USA [ JAPAN CANADA EEC

DURATION Rat: 24 ~onths IAd~inlstration period Rat: 24 ~onths Rat: 24 months (max

Mouse: 24 months is Ishould last from 24 ~)use & hamster: l~m is 30 months).

preferable. Ito 30 m (max) for Mouse or hamster:18

]rats & 18 to 24 m months. (Maximum is 24

[(max) for mice and months), or for the

hamsters. The study lifespan o f the a n i -

[should be terminated Dis ie to 20Z surv-

at, o r 1 - 3 m after, ival in the controls.

end o f a~minlstration

Iperiod. When cumula-

tire mortality reach-

t e s 75Z i n low dose or

control 8roup of ei-

ther sex, the survi-

vors Of that sez

sho u ld be sacrificed

& study terminated.

Admilxistrat ion is

n o r m a l l y 7 days per

week, but i f f o r c e d

administration is

employed , 5 days a

week is acceptable

from a practical

[ p o i n t o f v i e w . (Pre-

liminary studies are

I for 90 days, or lonE-

Jet if the substance

lhas delayed toxic ef-

f e c t s or cumulative

I effects.

(2,9,20) I (5,17) (13,14) (7,12)

....................................................................................................

animals). Slightly larger numbers of nonrodents than required by the authorities appear to be used in chronic tests, but for several companies these include recovery groups or extra animals for interim sacrifice. This also reflects the fact that with small numbers of animals per group, early deaths can complicate the interpretation of the results and extra animals are often included to cover this eventuality.

The situation is less clear-cut for reproduction studies, as is apparent from Table l 1. Several companies appear to u s e more animals, both rodents and nonrodents, than recommended for these tests. This probably reflects the fact that extra animals are included to ensure a sufficient number of pregnancies as well as to take into ac- count variations in the protocol between the FDA, EC, and Japan.

With the natural desire on ethical and humane grounds to explore alternatives to animals for toxicity testing and research, it is important to examine the use of animal resources. Some regulatory authorities avoid specifying exact numbers, and expect the investigator to decide and justify the number of animals needed. One of the reasons given for the revision of the Japanese guidelines is the growing concern about the numbers ofanimals used for toxicity testing (Anonymous, 1989a). However, their


TABLE 5--Continued

COUNTRy IUSA JAPAN CANADA I Izl~C

DOSE LEVELS[3 d o s e l e v e l s . Top

d o s e s h o u l d be max-

t o l e r a t e d d o s e .

A t l e a s t 3 d o s e

l e v e l s . P r e l i m i n a ~ y

studies: It is desir-

able to s e t t h e c o -

mon r a t i o o f d o s e l e -

v e l s at 2 or 3. The

highest dose s h o u l d

cause some toxicity.

If no t o x i c c h a n g e s

occu~ at the maximum

dose technically p o s -

s i b l e to administer,

this should be taken

as the maximum dose.

Full-scale studies:

The t o p d o s e s h o u l d

be determined frc~

the results of the

)reliminary s t u d y .

The low d o s e s h o u l d

n o r m a l l y be s e t so

that some p h a r m a c o l o -

lgical effect occurs,

or by considerin~ the

expected clinical

dose. It is desirable

that the intermediate

dose is the geometric

mean of the high and

low d o s e s . In e x c e p -

t i o n ~ l e a s e s w h e r e

the test substance

has a v e r y low toxi-

city coa~)ared to the

therapeutic dose in

humans, the highest

dose should be a b o u t

I00 X the c l i n i c a l

dose. In this case it

is necessary to

indicate the justif-

ication for such a

decision . It is gen-

erally desirable that

that low d o s e iS >10%

of the highest dose,

u n l e s s t h e l o w d o s e

i s far different from

t h e clinical d o s e .

At least 3. Highest [No~lZy 3. The top

dose is the maximum Idose should p~oduce a

t o l e r a t e d d o s e which [mLnimum t o x i c e f f e c t ,

d i d not p r o d u c e m o r t - l e g , IOZ ~ l o s s o r

allty, clinical signs failure of growth, or

o f toxicity o r patho- minimal target organ

logical lesions, toxicity. Target or-

w h i c h wou ld be e x - g a n t o x i c i t y w i l l be

) e c t e d t o s h o r t e n t h e d e m o n s t r a t e d by f a i l -

an~mal's lifespan, ure of physiological

functions and ultima-

tely by p a t h o l o g i c a l

ch=n~es. The lowest

d o s e s h o u l d be o f the

order of 2 to 3 times

the max human thera-

Ipeutic d o s e or the

d o s e that gives a

I p h a r m a c o l o g i c a l e f f -

e c t i n a n i m a l s . I n -

ter1~dlate d o s e is

the geometric mean.

For d r u g s with v e r y

low toxicity, the

maximum dose may be

a b o u t l O O x the clin-

ical d o s e .

( 9 , 1 8 , 2 0 ) ( 1 7 ) ( 1 3 , 1 4 ) ; ( 7 )

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

requirement for separate dose-range finding studies to be conducted prior to carcinogenicity tests using at least 10 animals per dose, appears to be excessive, as it is possible to perform meaningful dose-finding studies with fewer animals.


TABLE 5--Continued

193

COUNTRy

ROUTE

[USA IJAPAB

IBy t h e r o u t e t h a t [The e x p e c t e d c l i n i c a l

[ d u p l i c a t e s o r mos t [ r o u t e should be used

[ c l o s e l y a p p r o x l m a t e s ] i f p o s s i b l e . O r a l a d -

tO t h e l ~ t e n d e d shou ld route m l n i s t r a t I o n

lof human exposure, lelther involve forced

Incorporation into a~inlstratlon or ad

drlnklng water or libltum If m ~ e d with

feed may also be food or water. If the

appropriate for oral test substance is

drugs. Palatability mixed with food, the

should be evaluated. ~Imu~ concentration

in the fond should be

5Z. (Route used in

preliminary studies

should be the same as

that 111 c a r c i n o g e n i c -

i t y s t u d i e s ) . When

s u b s t a n c e i s admEnis -

t e r e d i n f o o d o r

water, measure f o o d

or water IJltake to

calculate the amount

of test s u b s t a n c e

I n g e s t e d , f o r b o t h

[ p r e l i m i n a r y and f u l l

scale s t u d i e s .

( 9 , 1 8 , 2 0 ) ( 4 , 1 7 )

CANADA I EEC

I n 8 e n e r a l a d m i n l s - l ~ l e r e p o s s i b l e , u s e

t r a t i o n o f t h e t e s t [ p r o p o s e d c l i n i c a l

agent should be by Iroute of ad~inlstra-

same route as the Ilion. Wherever rele-

p r o p o s e d exposure ] v a n t , e v i d e n c e o f

route in man.When rhe[absorptlon should be

proposed c [ s [provided.

route is by oral ad - [

m i n i s t r a t i o n a d e c i - [

s i o n w h e t h e r t o ad- I

minister the d r u g by [

garage o r incorporate)

it into the diet or [

d r i ~ k l n g w a t e r must I

be made.When t h e druE[

i s applied t o p i c a l l y

t o man, i t s h o u l d be

a d m i n l s t e r e d by t h e

same r o u t e i n an ima ls

Perti~lent stability

data are necessary

when d r u g i s admin-

i s t e r e d i n f o o d o r

water.

( 1 3 , 1 4 ) (7 )

ANIMAL

HUSBANDRY

S p e c i f i e d under GLP L o s s o f a n i m a l s due [ C o n t r o l t h e f o l l o v i n g ] C u ~ n e r c i a l diets a r e

R e s u l a t i o n s . t o a u t o l y s i s , c a n n i - 1. I m p u r i t i e s I n a i r [ v a r i a b l e and s t e p s

balism or problems in or water. 2. Housing [should be t~en to

husbandry should not conditions. 3. Crowd-tprovide as uniform

exceed 10X. [ l n g & stress. 4. Dis- adiet as posslble

(20) (17)

eases . 5. See GLP

[ 8 u l d e l i n e s . Cons ide r

I t he use o f s e ~ l -

I s y n t h e t i c d i e t s o f

Iknownand constant

[ c u ~ p o s i t i o n .

[ ( 1 3 , 1 4 )

t h r o u s h o u t t h e

duration of the

icarcinoEenlclty study

Full specification of

the diet shou td be

given.

(7)

1. Alder & Zbinden (1988)

2. anon (1977)

3. anon (1984)

4. anon (1988)

5. anon (1989a)

6. D'Asuanno (1973) 11. Go ldenthaL (1968)

7. EC Commiss ion (1989) 12. G r i f f i n (1985)

8. FDA (1985) 13. HPB (1987a)

9. FDA (1987) 14. BPB (1987b)

1 0 . FDA (1988) 15. J e f f e r y s (1989)

*Currentl 7 under review

16, ~ (1987)

17, Jt~d (1990)

18. Kesterson (1982)

19, Somers (1990)

20. @eisslnger (1990)

The Number of Species Used in Toxicity Tests

The number of species used and required for most tests was fairly consistent, with the exception of acute toxicity tests, for which the requirements vary from two species for the EC to three to four species for the U.S.A. (Table 2). Until recently, both Japan


TABLE 6

M U T A G E N I C I T Y S T U D I E S

REQUIRJ~4F~T[At the present time, [As a r u l e , every new

the FDA does not re- [pharma~eutlcal drug

.quire mutagenicity [should be subjected

!testing for human [ t o at least three

d r u g s or f o o d addi- mutagenicity tests.

tives, although they Detailed procedures

are generally sub- are given.

mitted.

CANADA l EEC

Currently u n d e r I D i r e c t i v e 7513181EEC

review. H i g h prloritylstates that any new

l.drugs which are re-]substance for use in

fated to known muta- Jmediclnal products

g e n s / c a r c L n o g e n s , lprior to b e i n E mar-

2.drugs[~tabolites keted has to be in-

that produce certain

biological effects

!Such as depression of

gametogenesis, fert-

ility, i~uns or

haemat opeie s is.

3 . d r t ~ s which a r e

~ o f t e n u s e d o v e r a

period o f years, par-

ticularly in children

& young adults, and

!d rugs w h i c h come in

contact with sperma-

tozoa.

vesti~ated f o r mta-

genie p r o p e r t i e s . The

combination o f tests

a p p l i e d should in

each case depend on

t h e specific charac-

teristics of the sub-

stances t o be tested.

The procedure should

be capable of detect-

ing t h e main classes

of genetic damage,

notably gene muta-

tion, chro~oso|

mutation, and when

possible genome

mutation.

I(7) ( 1 , 2 0 ) ( 1 7 ) ( 1 , 1 3 , 1 4 )

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I . . . . . . . . . . . . . . . . . . . . . TESTS TO BE T h e r e a r e no FDA]CDER At l e a s t 3 t e s t s a r e Recommeoded t e s t s ] ~ e p u r p o s e o f t h e

pERFORMED [guidelines on muta- required: ]are Salmonella/ [study o f mutagenie

Igenicity test re- I. Reversion test m-~lian microsome ]potential is to re-

[quirements, but data with bacteria; assay, m ~ l i a n veal the changes

on m u t a g e n i c p o t e n - 2 . Chromosomal a ~ e r r - i n v i t r o and i n v i v o w h i c h a s u b s t a n c e m~y

tial are normally ation test with [chromosome assays, cause in the genetic

included in IND ~lian cells in mterial o f individu-

and NDA submissiozls, culture; als or cells and

3. Micronucleus test which has o r may have

with rodents, the effect of making

s u c c e s s o r s p e r m a n e n t -

As these tests o f f e r [ly & hereditarily

o n l y minimum i n f o r m - [different from their

ation on mutagenielty Ipredecessors. Types

o f drugs, other mtt t a - ~of test categories:

genicity tests s h o u l d [ a ) gene mutations in

be added and perform- [bacteria. b) chromo-

ed ~s t h e occasion [somal a b e r r a t i o n s i n

demands, lady, allan cells in

(Several possible Ivitro. c) test for

tests are listed) [gene ~tatlons in

eukaryotic systems

[In Vitro. d) genetlc

damage test in v i v o .

( 1 , 2 0 ) ( 1 7 ) ( 1 9 ) [ ( 7 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


TABLE 6--Continued

195

COUNTRY

INTERPRETA-[

TION OF

RESULTS

USA

NO SPECIFICATIONS

GIVEN.

JAPAN

NO SPECIFICATI~$

GIVEN.

I CANADA I EEC tAt the present time, lif atl res.lts ~dr

Jit Is not p o s s i b l e onleate that s u b s t a n c e

Ithe basis of mutagen-lhas no effect in any

l ielty tests to afflrmlof the tests then the

Jwlth absolute cer- Ipossibllity o f muta-

Itainty that a d r u g Igenln hazard is low.

lw111 be a ~tasen in

lh,~.i. However it is

] p o s s l b l e t o tdentlfy

Idrlags w h t ~ are pot-

l e n r i a / l y c a p a b l e o f

I producing heritable

effects Ln ~ l s a~i humans.

( 1 3 , 1 4 )

If a l l resus b o t h

I n v l t r o a n d in v l v o

i . n d i ~ a t e that the

compound h a s

mutaEenEc p r o p e r t i e s ,

then existence of a

risk for humans is

high. If the results

are non-uniform, the

significance of the

[results is to be

ljudged not by their

Inumber b u t by their

I i~u~rure.

1(1,7)

I. Aider & Zbinden (1988)

2. anon (1977)

3. anon (1984)

4. anon (1988)

5. anon (1989a)

6, D'Aguanno (1973) 11. C~ldenthal (1968)

7. EC Commission (1989) 12. Griffin (1985)

8. FDA (1985) 13. RI~B (1987a)

9, FDA (1987) 14. HPB (1987b)

10. FDA (1988) 15. Jefferys (1989)

16. ~ (1987)

17. ~ (1990)


19. Somers (1990)

20. Weissi~er (1990)

and Canada required three species; however, this has now been dropped to two (Anon, 1989a; JMHW, 1990; Somers, 1990). Eleven companies responding to the survey indicated that they conduct acute tests in only two species, while an additional eight use two or three and five companies routinely use three or four species (one company did not answer this question). Generally, it is reasonable to expect the larg- est number of species to be used at an early stage of toxicity testing, particularly when the type of compound is new to the company. Although the U.S.A., Japan, and Can- ada still request that acute toxicity studies be conducted in nonrodents, seven companies do not carry out these studies, suggesting that these authorities might be per- suaded to bring this requirement into line with that of the other countries. The new Japanese guidelines do not require the nonrodents used in acute tests to be sacrificed and autopsied, which makes their reuse possible (Anonymous, 1989a).

The German regulatory authority permits the use of only one rodent species for carcinogenicity studies where there is sufficient scientific reason (Anonymous, 1989b), while all other authorities require two species. Most companies would use the rat and the mouse. The need for two species has been questioned by Schach yon Wittenau et al. (1983), who showed that rats and mice responded similarly to the majority of the 273 chemicals they reviewed. They argue that the justification for the two-rodent concept is ill-defined and in fact constitutes a paradox; if "carcinogens" possess the inherent property of causing cancer, one species should suffice for detect- ing this quality, but if "carcinogenic" activity is species-dependent, then the validity


TABLE 7

REPRODUCTION STUDIES

COMMENTS

COUNTRY [ USA

iGuideiines were

]issued 1966 and are

icurrently ~der re-

Iview. The 1966

]guidelines are

Isu=~arised in Tables

[ 8 - 1 0 .

I ~ guidelines a r e

[designed t o 8 i ~ r e c o n -

[sideration to a c c u r -

]ately~derstanding

ithe adverse effects ion reproduction and

(3 ,11 ,20 )

CANADA

NO SPECIFIC~S

[develolment b y dlvld-

[ L u g the tL~e f r c ~

] p r e g z m n e y to weanln8

[ i n t o t h r e e s e g m e n t s .

I H o ~ v e r , o t h e r ~ p p r o -

~priate methods will

also be accepted as

long as the obtained

findings satisfy the

p u r p o s e of the s t u d y

and l e a d to a logical

relatio~hlp helpful

for the clinical eva-

luation.

(17)

[The notes f o r quid-

l a n c e a r e not rigid

[requirements a n d m y

~noc b e u n i ~ e r s a l i y

a p p l i c a b l e . I n t e r p r e -

t a t i o n s h o u l d t h e r e -

f o r e be f l e x i b l e

a n d r e l a t e d to t h e

p r o p o s e d u s e o f t h e

d r u g ; j u s t i f i c a t i o n

f o r choice o f s t u d i e s

m u s t b e g i v e n . A c c o u n t

s h o u l d be take~ o f

] t h e p h a r m a c o k i n e t i c s

l o f the d r u g in the

Ipre~n~nt a~li~L~l. The

]level o f exposure of

]the foetus to the

[ d r u g s h o u l d h ~ v e b e e n

[determined a s f a r a s

]technically possible.

[ ( 7 )

GENERAL R e q u i r e d f o r a l l As a g e n e r a l r u l e , Some d r u g s , such a s IAny s t u d y o f d r u g

[ a l l new drugs s h o u l d t h o s e t o be u s e d i n ] e f f e c t s o n r e p r o d u c -

[be s u b j e c t e d to s t u d - g e r i a t r i c p a t i e n t s , J t i o n c o n d u c t e d o n n e w

lies in which the d~l~y ~ot requis re- Ides should be done

REQUIREMENT drugs intended for

Juse in males or r e -

[males of child bear-

[ l n g p o t e n t i a l .

]Not r e q u i r e d for

[ d r u g s w h i c h a r e n o t

{absorbed syst~ical-

[ly.

(1 ,20)

[is administered

[I. prior to ~d in

l t he early stages o f

I p r e g n a n c y ; If. d u r l x ~

Jthe p e r i o d o f f o e t a l

org~nogenesis ; I I I . l ~ l

the p e r i n a t a l & lact-

ation periods. In the

case o f drugs wlth

suspected e f f e c t s on

reproduction & devel-

opment from findings

elsewhere, w i t h poss-

Ible admLnlstration

t o many p r e g n a n t wo-

men or when consider-

ed necessary from re-

sults of above stu-

dies, It i s desirable

to conduct more de-

tailed studies such

as determination of

the c o n c e n t r a t i o n o f

t e s t substance in

b l o o d & other o r g a n s

& tissues o f dams a n d

ifoetuses, or in milk,

[ e x a m i n a t i o n o f d r u g

[ m e t a b o l i s m a n d direct

[ a d m i n i s t r a t i o n o f

I t b e dr~ to neonates.

[ ( 1 7 )

I ~ r o d u c t i v e s t u d i e s a t l L u s u c h a m a n n e r a s

all. J w o u l d r e v e a l t h e

[ p r e s e n c e o f a n y

e f f e c t on mar inE be-

h a v i o u r and o f any

effect t h a t might re-

sult in fetal loss,

fetal abnormality and

damage t o t h e o f f -

s p r i n g i n L a t e r l i f e .

(13,14) [ (7 )

1. A l d e r t 2 b i n d e n ( 1 9 8 8 )

2 . a n o n ( 1 9 7 7 )

3 . a n o n ( 1 9 8 4 )

4. a n o n ( 1 9 8 8 )

5 . a n o n ( 1 9 8 9 a )

6 . D ' A g u a r m o ( 1 9 7 3 ) 11 . G o l d e n t h a l ( 1 9 6 8 )

7 . EC C o m m i s s i o n ( 1 9 8 9 ) 1 2 . G r i f f i n ( 1 9 8 5 )

8 . FDA ( 1 9 8 5 ) 13 . HPg ( 1 9 8 7 a )

9. FDA (1987) 14. HPB (1987b)

10 . FDA ( 1 9 8 8 ) 1 5 . J e f f e r y s ( 1 9 8 9 )

16 . JE~W ( 1 9 8 7 )

17 . ~ ( 1 9 9 0 )

18 . K e s t e r s o n ( 1 9 8 2 )

19 . S o m e r s ( 1 9 9 0 )

20. W e i s s i n g e r (1990)


TABLE 8

REPRODUCTION SEGMENT I: FERTILITY STUDIES

1 9 7

COUNTRY

SPECIES

USA

One s p e c i e s .

R a t mOSt f r e q u e n t l y

u s e d (Mouse c a n b e

u s e d i n s t e a d ) .

JAPAN [CANADA

At l e a s t 1 s p e c i e s o f JOne s p e c i e s .

a n l m a l s u c h a s r a t o r J T h e rat is u s e d most

~ u s e , selected from [ o f t e n .

among t h o s e u s e d f o r

t e r a t o l o K y s t u d i e s .

[ EEC

JAr least one species.

JWhere metabolism of a

ldrug in a particular

[species is known to

I be s $ ~ i l a r t o t~t o f

]man~ i t is desirable

t o I J n e l u d e t h i s s p e -

c l e s . It i s desirable

[~t 0~ o f the

I s~cies is the same

las in Io~-tem toxi ~

city studies.

' (2,~,9,18) (17) I(13,14) I(7) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I . . . . . . . . . . . . . . . . . . . . .

AGE ~[ale: at least 40 IMale: at least 40d ofMale rats: minLmum [Animals should be

d a y s ( p e a k f e c u n d i t y ) [ a B e , a g e o f 40 d a y s b e f o r e a b o u t 40 d a y s o f a g e

l d r u g a d m L ~ i s t r a t i o n a t t h e Commencement

] b e g t J a s , o f d o s i ~ E .

( 2 , 4 ) I ( 1 , 1 6 , 1 7 ) ( 1 3 , 1 ~ ) ( 7 )

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i . . . . . . . . . . . . . . . . . . . . . ' . . . . . . . . . . . . . . . . . . . . ' . . . . . . . . . . . . . . . . . . . . . ROUTE A n i m a l s a r e d o s e d b y lThe a d m l a a i s t r a t i o n As c l o s e t o t h e human D o s i ~ E s h o u l d be c o n -

t h e a p p r o p r i a t e r o u t e r o u t e s h o u l d u s u a l l y r o u t e a s p o s s i b l e , d u e l e d by t h e p r o p o s -

b e t h e e x p e c t e d o l i n - e d r o u t e ( s ) o f c l i n i -

l c a l r o u t e . I f t h i s c a t a d m i n i s t r a t i o n .

i s n o t a p p l i c a b l e ,

a ~ o t h e r a p p r o p r i a t e

r o u t e e a n b e e m p l o y e d

F o r oral d o s i n g ,

I f o r c e d a d m i n l s t r a t l o n

l l s s u p e r i o r t o o t h e r

I ~ t h o d s b e c a u s e i t

p e r m i t s a s d o s e

I v i thout f a i l . (18) I(17) (i~) (7)

...................................................................................................

of the models as surrogates for man is questionable. Furthermore, the validity of the mouse as a test system has been questioned (Grasso and Crampton, 1972; Grasso et al., 1977; Butler, 1981). In view of the need to minimize animal use, consideration of only one species would be beneficial. The rat was favored by Schach von Wittenau as the species of choice. He further suggested that some of the cost savings achieved by using only one species could be used to expand the design of carcinogenicity bioassays to yield toxicological data more comprehensive than mere tumor counts, so that the proper perspective could be obtained on the results. (The latter point could be reiterated in respect of other long-term toxicity tests.)

The Duration of Long- Term Toxicity Tests

T h e m o s t e x p e n s i v e , t i m e - c o n s u m i n g , a n d c o n t r o v e r s i a l a n i m a l t o x i c o l o g y t e s t i s

the long-term chronic study (Heywood, 1983). Some regulatory authorities still re-


TABLE 8--Continued COUNTRY [USA JAPAN CANADA [FI~C

DOSE

LEVELS

[Animals are normally

[dosed with 3 dose

[levels, including a

[high dose t h a t is

[ p r e d i c t e d from a dose

-range finding study.

( 2 , 3 , 9 , 1 8 , 2 0 )

At least 3 dose lev-

els ~ a control group

should be used. The

h i g h e s t dose l e v e l

should cause some

toxic si~ such as

d e c r e a s e d f o o d intake

or inhibition of body

w e i g h t g a i n . When n o

tOXiC signs appear

with the maximomap-

[plicahle dose, such a

I d o s e level should

serve as the highest

d o s e . T h e lowest dose

~should not cause an y

adverse effect in

[parent animals, foe-

tuses or offspring.

The intermediate dose

(or d o s e s ) s h o u l d

n o r m a l l y b e t h e g e o -

m e t r i c mea~(s) o f the

highest and lowest

dose level. It is

desirable t h a t a dose

is i n c l u d e d w h i c h e x -

h l b i t s t h e p h a r m a c o l -

o g i c a l effects i n t h e

species used o r that

is c l o s e to t h e e x -

p e t t e d clinical dose.

[(&,5,16,17)

A t l e a s t 3 d o s e l D o s i n g s h o u l d n o r m a l -

l e v e l s s h o u l d b e f l y be c o n d u c t e d a t 3

used. The highest ldose levels. The high

dose s h o u l d cause [dose should usually

only minimal toxlcity[produce evidence of

such as relatively lso~e maternal toxici-

decreased body weight[ty eg decreased body

g a i n , b u t s h o u l d n o t

c a u s e s i g n i f i c a n t

a n o r e l l a , s e d a t i o n

or o t h e r O~vert effec-

ts in the dams. The

lowest dose should be

close to the pharmac-

ologically active or

therapeuti~ effective

dose in the selected

species.

weight g a i n . The low

dose should be suffi-

cient t o produce a

phazmacodynamic eff-

ect similar to the

desired therapeutic

effect, or to produce

blood levels compar-

able to those requir-

ed to p r o d u c e t h e

e f f e c t . The i a t e r m e -

d i a t e dose should be

the geommEtric ~a~ of

t h e s e .

(19) [ (7 )

quire that long-term testing be extended to 12 months, although Lumley and Walker (1985, 1986) have shown that extending tests beyond 6 months does not appear to contribute to the safety assessment of pharmaceutical compounds. All companies still conduct 12-month studies, probably a reflection that the U.S. and Japan are seen as important markets. However, several companies expressed the opinion that long- term studies beyond 6 months are unnecessary, indicating that they are only conducted to satisfy the requirements of the regulators, rather than because of scientific rationale. This is currently under investigation by the American FDA, who have es- tablished a committee to assess the value of 12-month repeat-dose toxicity tests.

Reproduction Studies

In the fertility study, dosing is usually commenced well before mating, but for Ja- pan it is then stopped at about the time of blastocyst implantation, while for Europe and the U.S.A., dosing is continued throughout pregnancy. Again, in the teratology study, the European and American guidelines require all dams and fetuses to be examined at the end of pregnancy, while those for Japan require an additional 10 rodents per group to litter and rear their young as a test for late toxicity (JMHW, 1990).

I N T E R N A T I O N A L T O X I C I T Y T E S T I N G G U I D E L I N E S 1 9 9


COUNTRY

NID4BEROF

ANIMALS/

CROUP

USA

Minimum: 20 males

and 20 females.

[ A t l e a s t 20 m l e s and

~20 femalesJgp shou ld

Jhe mated. F o r s p e c i e s

lother than rats, mice

Jot r a b b i t s , a suffl-

~cient number o f a n i -

Im~is for evaluation

Io f the results should

[be used.

As a minlmum, 15 males

shou ld he mated w i t h

30 fema le a n i m a l s .

EEC

Minimum of 24 females

aud24males per dose

group,except primates

Dose males for 60

' t o 80 days & sexu -

a l l y matu re fema les

for 14 days before

mating and through

lactation. Mate

treated males with

created or untreat-

ed females. Sacri-

fice half t he fe-

males on day 13 of

pregnancy; allow

remaining dams to

litter normal ly .

Record d u r a t i o n o f

gestation, litter

size, nL~ber of

stillborn, live

weight, gross ano-

malies, growth up

to 21 days.

(18)

................................. + ..................................................................

METHOD IM~les: dose f o r 60

[days or more be fore

mat ing , and con t inue

until s u c c e s s f u l

c o p u l a t i o n .

Females: dose f o r a t

l e a s t 14 days before

[matl~ng & eonttnui/1~

l u n t i l the hegLmatng

o f f e t a l orga-nogene-

i s i s ; 7 ( r a t s ) or 6

[(mice) days a f t e r

c o p u l a t i o n confirmed.

JCopula t ion and ferti-

llty s should

Ibe calculated.

IDurs the e x p e r i -

Imental p e r i o d all

[animals should be

l examlned for mortal-

ity g general signs.

[Body weights & food

intake should be

measured.

I (zT)

Trea t f o r 80 days DoslIX~ should e ~ -

p r i o r t o mmting.A~ter mence i n males & l e -

t14 days o f drug od- [ r o l e s a t s u f f i c i e n t

Im/~xls t ra t ion , female [t ime before the p ro-

J ~ l s are exposed [posed mting so thet

[to males. Half of the[any drug effects on

[females should be Igametogenesis can be

[sacrificed on day 13 Jrevealed (Rodents -

~of preRJ~m~cies. Exa - IMates : > 60 days ;

m ~ f o r numbers of J Y m l e s : >_ 14 days)

embryos i n t h e u t e r - [salf of the females

[he horn, presence of Jshould be killed

[empty i m p l a n t a t i o n Jduri-n8 g e s t a t i o n ,

s i t e s and embryos [p r e f e r ab ly some days

undergoLng r e s o r p t i o n [ b e f o r e the expected

In a d d i t i o n , any ab- [date o f p a r t u r i t i o n ,

Inormai c o n d i t i o n i n [and the foe tuses r e -

[the u t e r u s which m y [moved by caesarian

have contributed to Isectlon and examined.

embryonic dea th JThe remainder o f the

should be noted . [females should be

a l lowed t o l a t t e r ~-~d

[ rear progeny.

[

(14) [(7)

[female & non-treated [ [duct[re defect, the

Ira ale, and vi ........ [ [study should b ....

Ishouid be mated. ~peated uss dosed

I animals mated with

tmdosed @ r t n e r s .

[ After rating, dosed

[ females should con-

J tlnue to be dosed

t h ~ o ~ o u t p r e ~ e y .

( 1 , 2 ) [ ( 1 , 5 , 1 6 , 1 7 ) ( 1 3 , 1 4 ) 1(7)

(3,9) [ (i,3,5,i6,17) [(i~.14) (7)

................................. I ..................... l ...........................................

MATING Mate t r e a t e d males [Treated males with [Male an imals from Dosed an imals may be

w i t h e i t h e r t r e a t e d [ t r e a t e d females . The Jsubacute or ch ron ic mated w i th dosed

. . . . . . . . . d f emales . [mating p e r i o d should [ s t u d i . . . . be mated [pa . . . . . . bu t i n the

]be about 2 weeks. I f ]wi th e i t h e r t r e a t e d ]event o f p o s i t i v e

l . . . . . . . r y , a t r e a t e d [ o r ~ t r e a t e d f ~ l e ~ . [ f ' - d i ~ s o f a r e p r o -


TABLE 8--Continued

SACRIFICES ISacrifiee half of the

]females o n d a y 13 of

lpregnaney. Sacrifice

re~ainlng animals at

weaning.

TERMINAL

STUDIES

(3,9)

Observe the number &

distribution of

embryos, empty

implantation sites,

resorptions, abnon~l

conditions of uterus.

a n d f e m a l e s wi~out

s u e o e s s f u l c o p u l a t i o n

s h o u l d b e n e c r o p s l e d

at an appropriate

time.

( 2 , 9 , 1 8 )

When copulation is IOne half of females

successful, all fe- [under test should be

males should be Isacrlfleed on day 13

necropsied at tem. ]of their pre~ies.

Males used for matlng[The remainlng

should continue to

receive drug and

be a l l o w e d t o litter

normally.

(17) (19)

H a l f t h e f e m a l e s

s h o u l d be k i l l e d

d u r f n 8 g e s t a t i o n ,

p r e f e r a b l y some d a y s

b e f o r e t h e e x p e c t e d

dare o f parturition,

the fetuses re-

moved b y caesarian

section a n d examined.

The remainder of the

females should be al-

lowed to litter nor-

mally and rear their

progeny.

(12)

Examine for number of It my be advisable

corpora lurea, suet- to allow a large

essful pregnancy, enough number of

mortality of foetuseslprogeny to ]lye to

e t c . Findings for

estimating the time

of death in utero

should be noted as

much as possible.

Gross observations on

o r g a n s a n d tissues

should be ~de for

d a m s , males used f o r

mating and females

without successful

c o p u l a t i o n .

(17)

maturity. Late eff-

ects of the d r u g o n

the p r o g e n y should

be assessed, as

well as effects on

tholr reproductive

capacity.

(13 ,14)

Late effects of the

progeny should be ob-

Iserved (auditory,vis-

] u a l & behav iou r ) a n d

reproductive function

]assessed by allowing

Ii male & 1 female

from each litter of

dosed animals to

b r e e d & produce lEt-

ters. The no. of cor-

pora lutea, implanta-

tion sites, r e s o r p -

tion~, w e i g h t & sex

etc of individual fe-

tuses should be de-

termined. Examine fe-

tuses for external &

internal defects.

(7)

I. Alder & Zbinden (1988)

2. anon (1977)

3. anon (1984)

4. anon (1988)

5. anon (1989a)

6. D'Aguanno (1973) 11. Goldenthal (1968)

7. EC Commission (1989) 12. Griffin (1985)

8. FDA (1985) 13. HPB (1987a)

9. FDA (1987) 14. HPB (1987b)

10. FDA (1988) 15. Jefferys (1989)

16. ~ (1987)

17. ~ (1990)


19. Somers (1990)

20. We lss inger (1990)

This survey revealed reproduction study design to be a major area of concern to the companies. The majority of participants emphasised the need to harmonize these requirements as until recently they have had to repeat reproduction studies to comply with the Japanese guidelines. However, because of the recognized need for harmonized guidelines to facilitate the mutual adoption of data between nations, (Anony- mous, 1989a) the new Japanese Guidelines state that appropriate methods other than those they specify will also be accepted, as long as they "satisfy the purpose of the study and lead to a logical relation helpful for clinical evaluation" (JMHW, 1990). In addition, discussions are currently underway in Europe to attempt to develop protocols which will be acceptable to the FDA, the EC, and the Japanese Ministry of Health and Welfare (Bass, 1990).


TABLE 9

R E P R O D U C T I O N S E G M E N T II : T E R A T O L O G Y S T U D I E S

COUNTRY

SPECIES

IUSA [ JAPAM

[At l e a s t 2 species, [At least two s p e c i e s :

lfrom rat,mouse and lone rodent, s u c h as

lrabbit. Other species[rats or mice and one

l i~cluding dogs, cats, [ ~ n rodent e g rabbit.

lpigs, mice , guinea- lit is preferable t o

Ip igs �9 hamsters b.ave, luse ~ t s ,*hich I h o w e v e r , been used in[have similar metabol-

[the past. If the dis-[ism of the drug to

lposition and/or meta-[man. At least two

bolism of a d r u g in alspecies are mentioned

species other than

rat is more similar

t o t h a t o f m a n , t h a t

s p e c i e s c o u l d be

cons idered. The same

con~Iderat ion sheuld

apply w i t h regard to

transplaeental pass-

o f a d ~ .

for teratology and

only one for fertil-

Ity because more

i m p o r t a n c e is a t t a c h -

e d t o t e r a t o l o g y

studies.

CANADA EEC

I ( 2 , 9 , 1 8 , 2 0 ) ( 2 7 ) ( 1 9 ) ( 7 )

. . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . .

SEX F e m a l e Fema le U n t r e a t e d m a l e s F e m a l e .

s h o u l d be u s e d t o

p r o d u c e p r e ~ j ~ n c i e s

i n t r e a t e d f e m a l e s .

( 2 , 9 , 1 8 ) ( 1 7 ) ( 1 3 , 1 4 ) [ ( 7 )

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i . . . . . . . . . . . . . . . . . . . . . DOSE L E V E L S I T h r e e . T h e h i g h d o s e A t l e a s t 3 d o s e A t l e a s t 3 d o s e l e v e - J D o s t ~ 8 s h o u l d n o r m a l -

should be sub-toxic, levels and a control Is should be used. fly be conducted at 3

s the maximum toler- group. The highest The hlshest dose [dose levels. The high

a t e d d o s a g e , whilst d o s e l e v e l s h o u l d s h o u l d c a u s e o n l y l d o s e s h o u l d be s u c h

t h e low d o s a g e s h o u l d c a u s e some t o x i c m i n i m a l t o x i c i t y s u c h [ t h a t e v i d e n c e o f some

take i n t o a c c o u z l t t h e s i g r ~ , s u c h a s d e - a s r e l a t i v e l y d e c r e a - [ m t e r v ~ l t o x i c i t y i s

p r o p o s e d t h e r a p e u t i c c r e a s e d f o o d i n t a k e Ised body w e i g h t g a i n , I p r o d u c e d , e g d e c r e a s e

d o s a g e , a s p r e d i c t e d o r i n h i b i t i o n o f b u t s h o u l d n o t c a u s e f i n body w e i g h t g a i n .

from the dose r~n~e body weight gain. significant anorexia, IThe low dose should fLRdi-ng s t u d y . When no t o x i c s i g n s s e d a t i o n o r o t h e r lhe s u f f I c L e n t t o p r o -

a p p e a r w l t h t h e o v e r t e f f e c t s in Iduce a p h ~ r m a c o d y r ~ -

~axlmum applicable dams. The lowest }mic effect similar to

dose, this ~y s e r v e dose should be close Ithe d e s i r e d therapeu-

a s t h e h l g h d o s e . T h e l t o t h e p h a r m a c o l o g i - ~ t l c e f f e c t , o r t o

low d o s e s h o u l d n o t l c a l l y a c t i v e o r t h e r - l p r o d u c e b l o o d l e v e l s

c a u s e a n y adverse l a p e u t i c e f f e c t i v e c o m p a r a b l e t o t h o s e

effect i n parent, l d o s e i n the selected required to produce

f o e t u s e s o r young . I s p e c l e s - t h e effect. I n t e r m e -

I n t e r m e d i a t e d o s e ( s ) ] C o n s i d e r t h e k i n e t i c s d i a t e d o s e s h o u l d be

s h o u l d be t h e g e e - [ o f t h e d r u g and b l o o d t h e g e o m e t r i c mean o f

m e t r i c man(s) of the 'levels in order to high & low doses.

high and low dose. It ensure adequate

is desirable t o dosin 8.

i n c l u d e a d o s e t h a t

exhibits the p h a r m a -

c o l o g i c a l e f f e c t s , o r

that is close t o the

expected o l i n d o s e .

( 2 , 9 , 2 0 ) ( 1 7 ) ( 1 3 , 1 4 , 1 9 ) ( 7 )

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Two s p e c i e s , I o f At l e a s t 2 s p e c i e s ; a

w h i c h s h o u l d be a b r e e d o f r a b b i t s e n s -

n o n - r o d e n t . Mos t I t i v e to known t e r a -

c o l o r . y , mouse and t o g e n i c s u b s t a n c e s

r a b b i t a r e u s e d . A and r a t s o r m i c e

t h i r d s p e c i e s i s ( s p e c i f y i n g t h e

r e q u i r e d i f e q u i v o c a l s t r a i n ) o r i f a p p r o -

a r e o b t a s [ p r l a t e , some o t h e r r e s u l t s

m ~ l i a ~ s p e c i e s .

2 0 2 SPEID, LUMLEY, AND WALKER

TABLE 9--Continued c0~Y 0 ~ J~AN I ~ I~c ROUTE The route of admtn- The administration JRoute of a~minlstra- [DosEnS should be con-

istratlon should route should norm~lly]tlon should be slmi- [ducted by the pro-

approximate that of be the expected far to the proposed [posed route(s) of

cls use. cl~Ical route. If clinical route, clinical adminlstra-

this is not application.

ble, another appropr-

opriate route can be

e ~ p l o y e d . Fox oral

dosLng , f o r c e d a d m i n -

i s t r a t i o n l s s u p e r i o r

t o o t h e r m e t h o d s

because it permits a

fixed dose without

fail.

I(2) (17) (14) (7)

................................................................................................

NUMBER OF Rodents: At least 20 Rodents:At least 30 IRodents: at least 20 Rodents: 20 pregnant

ANIMALS/ ]pregnant females. [pregnant females/gp. [ p r e g n a n t females, females per group.

GROUP [Non-rodents: At least Rabbits: At least 12 ]Non rodents: at least Non-rodents: 12 preg-

[12 pregnant females, pregnant females/gp. [15 pregnant females, nant females/group

I [ (except primates)

](3,9,20) (17) [(13,14) (7)

........... I ........................................... l ...........................................

METHOD Dose pregnant animals

I dur~ng the entire ] period of organogene- Isis. This period ex-

fiends from gestation

Iday 7-17 i n rodents

land from day 6-20 in

Dose pregnant females[Treat animals during

from days 7 - 17 of Ithe period of embryo-

preEjla~cy in rats, [genesis~ Mouse & rat:

days 6-15 in mice, & [day 6 - 15 of preg-

days 6-18 in rabbits.lnancy. Day O is the

In the case of rats I daY that sperm were

or mice, approxlmate-lfound. Rabbit cortes-

Fuk~mination should be

made of the foetuses

from animals dosed

during the period of

e~bryogenesis.Ani~als

s h o u l d be killed and

the foetuses removed

]rabbits. 24 hours be-[ly 2 thirds of dams ]pondi~g period should[by caesarian section.

]fore r~tural parturi- s group and in ]be day 6 - 18. Deli-

[tion (rodents day 20 the case of non-rod- [ver foetuses by r

lot 21 of gestation: ents such as rabblts,[sarfan section, 1 or

rabbits day 29 of !all dams in each [2 days prior t o the

gestation), dams are group should be he- l anticlpated date of

killed and the pups cropsied at term. Forlparturition.

removed by caesarian rodents the remaLn- Rabbits: it might be

section. Eng 1 third of dams desirable to incubate

should be allowed to half the foetuses for

deliver and nurse for 24 hours.

their young. The ges-

:tation index should

be calculated.

(9,18) (1,4,17) (13,14) (7)

....................................................................................................

The European Community and the Approach of 1992

By 1992, it is envisaged that the trade barriers which separate Europe will be removed. This goal presupposes that member states will agree on the abolition of barriers of all kinds, including differences in regulatory guidelines, approximation of legis- lation, and tax strictures. The objectives identified by the Commission of the Euro- pean Communities for authorization of the marketing of medicinal products within the EC include a single scientific evaluation valid for the whole community and the

I N T E R N A T I O N A L T O X I C I T Y T E S T I N G G U I D E L I N E S 203


COUNTRY

PARAMETERS

1. Corpora lutea

2. Implantat ions

3. Resorp~ion.s

Foetal examlnatio~ :

1.Number and sex

12. Welght

3. External examlna-

tlon~

4. Internal (visceral

d e f e c t s should be

sought even if no

eASe rub I ano~al ie s

are s e e n .

M a t e r n a l examinatlon: I I )u r l~g the experim-

Number of: Jental period, all

l~ams in each Eroups

l s h o - l d be examined for mortality and

lseneral slsns~ body lweights & food intake Ishould be measured.

JAr autopsy, gross

lobservations of org-

t a n s & t i s s u e s shou ld

lbe made o n ~ . T h o s e

I s a c r i f i c e d a t t e r m

shou ld be examined

for s u c c e s s f u l pregn-

ancy and mortality of

foetuses. Body weight

measurement & morpho-

l o g i c a l examination

s h o u l d be made on

llve foetuses. Those

f allowed to deliver shou ld be e x a m i n e d

for a b n o r m a l i t y on

delivery. Litter size

mortality, sex, ex-

ternal changes & body

weight of newborns

should be noted. They

s h o u l d be e x a m i n e d

for growth & d e v e l o -

Ipu~nt through morpho-

I l o g l c a l , f u n c t l o . a l t I I b e h a v l o u r a l exam~na-

I t l o n s : and reproduc-

Itive performance on

b a s i s of successful

Ipregnaney. Birth, vl-

lability and weanin8

l i n d e s e s s h o u l d be

[ c a l c u l a t e d . When an

l a b n o r ~ a l finding i s

I d e t e c t e d s t h e o f f -

I s p r i n s , a n additional

llactation s t u d y with

f o s t e r dams may be

l p e r f o r m e d if n e c e s -

Isary to determine at

w h a t stage o f the

mpre- and post-natal

period the animals

lwere affected.

Numbers o f f o e t u s e s , IIr e x a m i n a t i o n :

thelr p l a c e m e n t i n l..~er of= uterine horn,cor-ml, corpora lutea.

relatlo~ with ~he Iz implantations.

numbers of corpora ]3. resorpts

lutea, llve and dead lFoetal examination:

foetuses, and early &ll. number and sex.

late r e s o r p r l o n ~ 12. w e l E h t .

s h o u l d a l l be deter- [3. examination for mined .Foe tuses should I external abnormal-

!be weighed ~ i v i d u - J l t i e s .

a l l y and be c a r e f u l l y m 4 , adequate examina -

e x a m i n e d ~ d l a t e l y

f o r e x t e r n a l ~ c ~ a l -

ies. ExamLne f o r i n -

t e r n a l d e f e c t s .

R a b b i t s : e x a m i n e all

foetuses for

a n o m a l i e s .

lion of the skele-

ton or v i s c e r a o r

b o t h on a l l f o e

ruses.

S p e c i a l attention

shous be paid to ab-

normaly high numbers

o f resorptlon~ as

t h i s m iE~t need f u r -

t h e r s t u d y .

(3,~) I(i;) I(13,14) (3,7) .................................................................................................


TABLE 9--Continued

COUNTRY

TERMINAL

STUDIES

[USA, [ J / ~ / ~

[A u t e r i n e map d e p l c t - l T h e l i v e f o e t u s e s I n

ing the distribution ~the late stages of

of live & dead f o e - Ipregn~ncy are Eerier-

ruses, resorption and[ally examined for set

degeneratin 8 f o e t u s e s l m o r p h o l o g i c a l changes

is drawn. Examine for[in t h e external and

skeletal and visceral[internal organs and

abnormalities, etc. [tissues, and for ~r- Iphology in the skele-

[ t o n and o s s i f i c a t i o n

with cleared and

Istained speelmens. I f l

necessary, further

[detailed histological

[or histochemieal exa-

minations may be

[made.

CANADA

Rat o c m o u s e f o e t u s e s

c a n b e r a n d o m i z e d

I n t o 2 g r o u p s , i e one

t h i r d e i t h e r f o r d i s -

s e c t i o n o r t h e s y s -

t e m a t i c s l l c i ~ s m e -

t h o d o f W i l s o n t o d e -

t e c t v i s c e r a l anomal-

ies & two-thirds for

c l e a r l y 8 & bone s t a i n

with alfzarln.

(9.18) [(17) [(13.14,19)

]F~C

A c c o r d i n g t o t h e

s t a t e o f s c i e n t i f i c

k n o w l e d g e .

(7)

1. Alder L Zbs (1988) 6. D'Aguanno (1973) 11. Goldenthal (1968)

2 . a n o n ( 1 9 7 7 ) 7 , EC C o m m i s s i o n ( 1 9 8 9 ) 1 2 . G r i f f E n ( 1 9 8 5 )

3 . a n o n ( 1 9 8 4 ) 8 , FDA ( 1 9 8 5 ) 1 3 . HPB ( 1 9 8 7 a )

4 . a n o n ( 1 9 8 8 ) 9 , FDA ( 1 9 8 7 ) 1 4 . IIPB ( 1 9 8 7 b )

5 . a n o n ( 1 9 8 9 a ) 1 0 , FDA ( 1 9 8 8 ) 1 5 . J e f f e r y s ( 1 9 8 9 )

16 . JMHW ( 1 9 8 7 )

1 7 . ~ ( 1 9 9 0 )

18 . K e s t e r s o n ( 1 9 8 2 )

19. Somers (1990)

2 0 . W e i s s l n g e r ( 1 9 9 0 )

provision of a credible European authorization (EC Commission, 1990). This means that harmonized, clear, flexible data requirements, applied consistently, are needed. Despite the fact that some differences still exist between the EC countries in their national guidelines, the companies made it very clear that they follow the European Community guidelines, paying little if any attention to the peculiarities of any individual country within the EC. For example, companies generally use two species for teratology studies, as specified in the EC guidelines. Even EC countries with different individual requirements, such as France, which requires three species (Ministere de la Sante, 1985), seem in practice disposed to accept studies carried out to EC guidelines. This emphasizes that the EC guidelines are now the accepted standard for all its constituent members. In fact, the United Kingdom, German, and Italian regulatory authorities have already indicated that they accept the guidelines of the European Community.

The Regulators versus the Regulated

A number of companies indicated that, in their opinion, certain requirements such as chronic tests longer than 6 months, acute testing in more than two species, and specific behavioral tests in reproduction and acute toxicity studies, are unnecessary. These need to be reassessed as to their value for patient safety. To utilize resources efficiently, most companies put together a core package to suit the majority of major markets, mainly based upon the U.S., EEC, and Japanese guidelines (seven companies), the U.S. and EEC guidelines (five companies), the EC guidelines (six compa-


TABLE 10

REPRODUCTION SEGMENT III: PER1- AND POSTNATAL STUDIES

COUNTRy USA JAPAN CANADA

SPECIES One s p e c i e s , usually

rat

At least 1 s p e c i e s :

rat or mouse. Select

from amon~ those used

in teratogenicity

studies.

A t least one rodent

species ie t h e rat o r

the mouse.

EEC

At least o n e species.

Where metabolism of a

d r u g i n a particular

s p e c i e s is knowt~t tO

be s i m i l a r t o man , it

Is desirable to

include this species.

(9,18) (1,12,16,17) (13,14) (7)

...................................................................................................

ROUTE I n t e n d e d c l i n i c a l I n a c c o r d a n c e w i t h S i m i l a r t o p r o p o s e d D o s i n g s h o u l d be c o n -

r o u t e , e~qpec ted c l i n i c a l c l i n i c a l r o u t e , d u c t e d b y t h e p r o p o s -

route as a rule. If ed clinical route or

this is not appllc- routes.

a b l e , a n o t h e r a p p r o -

lprlate route can be

employed. For oral

dosing, f o r c e d admin-

istratlon is superior 1

to other ~ethods b e -

cause it permits a

fixed dose without

fail.

( 3 , 9 ) ( 1 , 1 6 , 1 7 ) ( 1 , 1 3 ) ( 1 , 7 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

DOSE LEVELS Three dose levels

plus one control

group.

At l e a s t t h r e e d o s e

l e v e l s a n d a c o n t r o l

s h o u l d b e u s e d . The

h i g h e s t d o s e s h o u l d

cause so~e t o x i c

s i gns such as decrea-

sed food i n t a k e o r

i n h i b i t i o n o f b o d y

w e i g h t g a i n . When n o

T h r e e d o s e l e v e l s .

C o n s l d e r k i n e t i c s o f

[ t h e d r u g a n d b l o o d

l e v e l s , i n o r d e r t o

[ e r ~ u r e a d e q u a t e

d o s J ~ 8 . A c o n t r o l

[ g r o u p s h o u l d be i n c l -

u d e d i n e v e r y s t u d y .

I f t h e e f f e c t o f t h e

t o x i c s i g n s appear [ v e h i c l e on r e p r o d u c -

w i t h t h e maxLmum [ t i o n i s unknown an

a p p l i c a b l e dose, s u c h l e x t r a c o n t r o l group

la dose l e v e l shou ld I w h i c h r e c e i v e s app ro -

J s e ~ e a s t h e h i g h e s t J p r l a t e h a n d l i n g b u t

I d o s e . The l o w e s t d o s e

J s h o u l d n o t c a u s e a n y

l a ~ i v e r s e e f f e c t i n

[ p ~ r e n t a n i m a l s , f o e -

t U s , o r y o u n g . The

[ i ~ t e ~ e d l a t e d o s e ( s )

J s h o u t d u s u a l l y b e t h e

[geometric ~an(s) of [the hish and lov d o s -

es. I t i s d e s i r a b l e

I t o i n c l u d e a dose l e -

I V e l t h a t e x h i b i t s ch e

[pha~cologic. i ef- [ f e c t s i n t he spec ies

f u s e d o r i s c l o s e t o

t h e e x p e c t e d c l i n i c a l !

J d o s e .

T h r e e d o s e l e v e l s a n d

one c o n t r o l g r o u p .

The h i g h e s t d o s e u s u -

a l l y s h o u l d be s u c h

t h a t e v i d e n c e o f m a t -

e r n a l t o x i c i t y iS

Iproduced, eg decrease

i n b o d y w e i g h t g a i n .

The l ow dose shou ld

be s u f f i c i e n t t o p r o -

duce a p h a r m a c o d y r ~ -

m lc e f f e c t s i m i l a r t o

t h e d e s i r e d t h e r a p e u -

t i c e f f e c t , o r b lood

n e i t h e r t h e v e h i c l e l e v e l s c o m p a r a b l e t o

o r t h e d r u 8 s h o u l d be t h o s e r e q u i r e d t o

i n c l u d e d . J p r o d u c e t h i s e f f e c t .

I n t e r m e d i a t e d o s e i s

t h e g e o m e t r i c m e a n o f

t h e h i g h a n d l o w d o s e

(3,9) 1(1,17) (1,13,14) 1(7) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


TABLE lO--Continued

COUNTRY USA

Nt~4BERS OF At least 20 pregnant

ANIMALS females per test

Broug.

IJ~AN Each Stoup s h o u l d

Consist of at least

20 pre~j~nt females.

lCA~D* IEEC IRodent: zarse ~aot~a Ix2 pr.--~t fe~z . s / I t * satisfy statisti- [81t~cbup ( e x c e p t pri-

leal requis (min]mates).

120 preKna.nt f e m a l e s ) . J

Ill non-rodents are [

[u~ed, numbers s h o u l d [

Iho enough as p z a c t i - ]

IcabZe t o produce re- I

I p r o d u c i b t e r e s u l t s eg I

115 p regnan t females. J

(1,3,9) (6,17) 1(13,14) 1(7) . . . . . . . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I . . . . . . . . . . . . . . . . . . . . . I . . . . . . . . . . . . . . . . . . . . .

DURATION Dose for final third After the estimated [AdmLnlster dx~ 8 [Treat during period

of gestation (day 15)[period of foetal J d u r i n g fillal one [of gestation from the

t h r o u g h parturition oEganogenesis ends, I t h i r d of gestation ]end of organogenesis,

~d lactation until administration should[throughout lactation [parturition and

weanin 8 on day 21. be performed daily Ito weanIJn~, throushout the perLod

u~til the ti~e o f o f lactation up to

weaning. (Rats: day we~ning.

17 of pregnancy - 21

~ys after delivery=

Mice: day 15 of preg-

nancy - 21 days after

delivery).

(2,9,18) (1,16,17) (1,13) (7)

.......... l .....................................................................................

METHOD At ~eani t~g, all dams

and thei~ pu~s a r e

killed and n e e r o p -

sied. Dead yoor~

should be preserved

!for a study of abnor-

m~lities.S~ litters

could be exchanged

!be tween control and

h i g h d o s e dams t o

elucidate causes o f

Ip~r survival if s u c h

lis observed. I~ s~ue

leases, the study need

I o n l y look a t r e p r o -

]duetion in the off-

spring.

[(9,18)

All dams should be Some litters can be

allowed to deliver exceed between

and nurse t h e i r young l c o n t r o l arui h i g h dose

!and should be examLn-ld~ms to elucidate

ed for abnormality onlcauses of poor su~vi-

! d e l i v e r y . At ~ . lval, i f such a r e o b s -

a p p r o p r i a t e t~me, [ e r e e d .

a u t o p s y & g r o s s o b s - ]

e r v a t l o n s on orgarLs &l

t i s s u e s s h o u l d be

m~ie on t r e a t e d d~ms.

I I f necessary, exams

a t i o ~ of the second

litters should be

done.

( 1 ,12 ,17 ) (1,13,14)

The f e m a l e s s h o u l d be

a l lowed t o l i t t e r

spontaneously and t h e

p r o g e n y examined a t

w e a n i n g . A l l a n i m a l s

k i l l e d a t t h e end o f

l a c t a t i o n s h o u l d be

sub jec ted to a

t h o r o u g h a u t o p s y .

UDder certain circum-

stances, some of the

l p r o g e n y s h o u l d be

~a l l owed t o l i v e and

I r e a c h m a t u c i t y so

lthat their reproduc-

I t i v e c a p a c i t y can be

lassessed.

I ( 7 , 1 5 )

nies), or the U.S. and Japanese guidelines (two companies). This inevitably means some studies are repeated. For example, some companies indicated that they have had to repeat reproduction studies in order to comply with Japanese requirements.

From an economic point of view, repetition of studies causes a considerable waste of limited resources, especially when the studies are complex and costly. Data on new medicines which fulfill the necessary scientific requirements, including state of the art, should, in principle, be considered an adequate basis for approval by national health authorities, irrespective of national origin. Any request for repetition of preclinical or clinical studies should be justified on scientific and ethical grounds, and


TABLE lO--Continued

207

COUNTRy

PARAMETERS

USA ]JAPAN

O b s e r v a t i o n s r e c o r d e d l D u r i n g t h e e x p e r l m e n -

during t h e s t u d y i n - I t a l p e r i o d , a l l dams

e l u d e d u r a t i o n o f [ s h o u l d be e x a m l n e d

gestation, labour ~dlfor mortality and

d e l i v e r y , litter size[general signs, body

!lactation, pup viabi- weights and f o o d In-

lity at b i r t h , 4 L 21 t a k e s h o u l d be meas-

days o f a g e , as w e l l u r e d . The g e s t a t i o n

as b o d y w e i g h t s d u r - i n d e x s h o u l d be c a l e -

I L t ~ the nursing per- ulated. Litter size,

iod. Continuous a d - mortality, sex and

m i n i s t r a t i o n t h r o u g h - e x t e r n a l changes o f

o u t t h e n u r s i n g p e r - new h e r n J & body

lod will allow for weights should be n o -

detection of adverse ted. Offspring should

effects on lactation be examined for

and nursiaxE instinct, growth & d e v e l o p m e n t

a s w e l l a s t o x i c a e - ( i n c l u d e m o r p h o l o g i -

tion of the drug or c a l f u n c t i o n a l and

i t s metabolites, b e h a v i o u r a l examina-

tions), & for reprod-

uctive performance on

the basis of s u c c e s s -

f u l pregnancy. Birth,

viability and w e a n i n g

i n d e s e s s h o u l d be d e -

t e r m i n e d . When an

abnormal finding is

d e t e c t e d i n the o f f -

spring, all additional

lactation study may

be performed if n e c e -

s s a r y , t o d e t e r m i n e

at what stage of peri

and post-natal period

t h e animals were

affected.

(2,9) (1,12,17)

CANADA

L a b o u r , d e l i v e r y ,

duration o f 8 e s t a t i o n

s i z e o f litter, pup

weight e r e s h o u l d be

r e c o r d e d .

C o n t i n u o u s a d m i n i s -

t r a t i o n o f d r u g

throushout the ~ u r s -

in period will allow

l f o r d e t e c t i o n o f ~ l -

v e r s e e f f e c t s on l a e -

ratio, nursin 8 i n -

s t i n c t s a s well a s

a l ly t o x i c a c t i o n s o f

t h e d r u g o r i t s m e t a -

b o l i t e s on the new-

b o r n by i~agestion o f

milk.

( 1 , 1 3 , 1 4 )

~ C

L a t e e f f e c t s o f t h e

d ~ t 8 on t h e p r o g e n y

i n t e r m s o f b e h a v -

i o u r a l , v i s u a l and

auditozy i m p a i r m e n t

s h o u l d be d e t e r m i n e d .

1(7)

1. A l d e r & Z b l n d e n ( 1 9 8 8 )

2 . anon ( 1 9 7 7 )

3 . anon ( 1 9 8 4 )

4. anon ( 1 9 8 8 )

5 . anon ( 1 9 8 9 a )

6. D'Agua~o (1973) 11. Goldenthal (1968)

7. EC C~isslon (1989) 12. Griffi~ (1985)

8 . FDA ( 1 9 8 5 ) 13 . BPB ( 1 9 8 7 a )

9. FDA (1987) 14 . EIP8 (1987b)

10 . FDA (1988) 15 . J e f f e r y s (1989)

16 . JE~W ( 1 9 8 7 )

17 . ~ ( 1 9 9 0 )

18 . K e s t e r s o n ( 1 9 8 2 )

19 . Somers ( 1 9 9 0 )

20. Welssinger (1990)

wherever possible should also aim at the acquisition of new information rather than merely at the validation of that already existing and accepted by others. In 1976, Binns stated that international cooperation needs to be encouraged at both the preclinical and the clinical level, as an efficient means of enlarging the basis of inference and of providing a more complete and integrated knowledge of drugs. Fourteen years later, this must still be an objective for all those concerned with evaluating the safety and efficacy of new medicines. Certainly, the introduction of Good Laboratory Prac- tice has increased the confidence with which results can be accepted across regulatory authorities (Purchase, 1984).


TABLE 11

NUMBERS OF ANIMALS ROUTINELY USED BY INTERNATIONAL PHARMACEUTICAL COMPANIES

A. RODENTS

Test Requirement: No. Companies: No. Number of

Animals/Sex/Group Animals/Sex/Group Co~npanies

Acute Japan: at least 5/~roup <5 6

Others: not specified 5 14

i0 5

Subacute I0 7

Japan: at least I0 I0 - 15 11

Others: not specified 15 - 25 7*

Chronic USA: i0 - Z5 i0 - 20 14

Japan: at least i0 15 - 25 5

Others: not specified ~30 6**

Teratology USA: at least 20 pregnant f

Japan: at least 30 pregnant f

Canada: at least 20 pregnant f

EC: 20 pregnant f

<20f 2

20 - 28f g

30 - 40f 13

No comment 1

Peri- & Post-natal USA: at least 20 pregnant f

Japan: at least 20 pregnant f

Canada: at least 20 presnant f

EC: 12 pregnant f

<20f 1

20f i0

22 - 25f ii

30f 2

No comment I

Fertility USA: minimum 20m + 20f

Japan: aL least 20m + 20f

Canada: minimum 15m + 30f

EC: minimum 24m + 24f

20 - 24f

I~+ 30f

20-25~n+ 20-25f

20m + 40f

2.~m+ 50f

30-36m+ 30-36f

2***

3

8

2

1

9

* four include extra recovery animals

4. one cou~ined chronic/carcinogenicity

one includes interim sacrifice

one includes recovery animals

one includes Kinetic study animals

*** number of males not specified


TABLE 1 l--Continued

B. NON-RODENTS

209

Test Requirement: H o . Co~panies: No.

Animals~Sex~Group Animals/Sex/Group

Number of

Companies

Acute Japan: at least 2 1 - 2 15

Others: Not Specified >2 3*

Not Conducted 7

Subacute 3

Japan: at least 3 3 - 4

Others: not specified Up to 6

15

6

4**

Chronic USA: 2 - 3 3 - 5

Japan: at least 3 6 - i0

Others: not specified No comment

15"**

9****

1

Teratology USA: at least 12 pregnant f Up to 12f 5

Japan: at least 12 pregnant f 15 - 20f 17

Canada: at least 15 pregnant f 24f 1

EC: 12 pregnant f No comment 2

Peri- & Post-natal n/a

Fertility n/a

* o n e c o m p a n y c o n d u c t s s t u d i e s i n o n l y 1 s e x ( 3 a n i m a l s )

* * o n e i n c l u d e s r e c o v e r y a n i m a l s

*** Lwo include recovery animals

**** two include recovery animals; one includes extra animals for interim sacrifice

one company uses 6 monkeys/sex/sroup but fewer doss

one company u~es 4-5 animals~sex~stoup

Finally, both regulators and regulated need to discuss ways in which differences can be eliminated. In areas in which they will still exist, greater emphasis should be placed upon the state of the art defence of new drug submissions if they deviate from written guidelines. Furthermore, toxicologists need encouragement to develop sim- pler and more effective toxicity tests which have a good scientific basis. This should allow the regulatory authorities to modify their demands on the industrial community, in line with present-day needs (Purchase, 1984).

CONCLUSION

This study sustains ideas and views which were previously suspected to be true, but for which there was little corroborative evidence; it would appear that regulatory variations which exist are to the greater extent unnecessary. This is further borne out by the fact that they have largely been removed within politically and geographically similar regions (for example, U.S.A., EC, and Nordic regions). It is important for the issues of regulatory differences raised in this paper to be discussed between the regulatory authorities and pharmaceutical companies.


The impor tance of harmoniza t ion of international regulatory requirements for toxicity testing of pharmaceuticals has been recognized by regulatory agencies, pharmaceutical manufacturers , health-related and scientific societies, politicians, and animal protection organizations. Therefore, there has been an initiative, at the sugges- t ion of the EC Commiss ion and the Japanese Ministry of Heal th and Welfare, to set up talks between these two regulatory authorities. This has subsequently been supported by the U.S. FDA under the auspices of international organizations like I U T O X and W H O , together with regulatory authorities and pharmaceut ical manufacturers' associations. This initiative is to discuss the state of the art concerning single-dose toxicity (acute toxicity) testing and reproductive studies and how these should be laid down and defined in such a way that the resulting guidelines can be applied to any pharmaceut ical manufacturer and the results generated in accordance with these guidelines can be acceptable to all drug regulatory authorities (Bass, 1990). The Canadian Drugs Directorate has also indicated their c o m m i t m e n t to harmonization, as far as possible, with the U.S. and EC testing requirements (Somers, 1990).

A C K N O W L E D G M E N T S

The help of Dr. J. Weissinger (Food and Drug Administration, U.S.A.), Dr. E. Somers (Health Protection Branch, Canada), Professor R. Bass (Bundesverband der Pharma Industrie, Germany), and Dr. D. Jefferys (Medicines Control Agency, UK) in making detailed comments on the tables to ensure that they accurately reflect current guidelines is gratefully acknowledged, as is the assistance of the pharmaceutical companies responding to the questionnaire. Thanks are also expressed to Miss J. Winch for her patient assistance in preparing the manuscript and tables.

REFERENCES

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Anonymous ( 1977). Pharmaceutical Manufacturer's Association Guideline for the Assessment of Drug and Medical Device Safety in Animals. PMA, Washington, DC.

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Harmonization of guidelines for toxicity testing of pharmaceuticals by 1992