the value of chronic animal toxicology studies of pharmaceutical compounds: a retrospective analysis
TRANSCRIPT
FUNDAMENTAL AND APPLIED TOXICOLOGY 5, 1007-1024 (1985)
ISSUES
The Value of Chronic Animal Toxicology Studies of Pharmaceutical Compounds: A Retrospective Analysis
CYNTHIAE.LUMLEYAND STUARTR.WALKER
Centre for Medicines Research, Carshalton, Surrey, England
The Value of Chronic Animal Toxicology Studies of Pharmaceutical Compounds: A Retro- spective Analysis. LUMLEY, C. E., AND WALKER, S. R. (1985). Fundam. Appl. Toxicol. 5, 1007- 1024. Extensive animal studies are carried out during the development of new medicines to assess toxicity and predict their safety for use in man. There are, however, differences of opinion con- cerning the nature of safety evaluation studies required prior to marketing. Any prospect of rationalizing the number and design of animal studies must stem from a reappraisal of conventional animal testing procedures and better use of available data on the toxicity of compounds previously investigated. The Centre for Medicines Research has therefore established a toxicology databank containing comprehensive data from repeated-dose animal safety evaluation studies provided by pharmaceutical companies. Thirteen companies within the United Kingdom have provided tox- icological data for 32 pharmaceutical compounds studied in the rat, dog, or primate, resulting in 45 case studies for which both short-term (~6 months) and long-term (>6 months) tests had been completed. A comprehensive analysis of these studies has been carried out to determine what new findings, if any, become apparent in studies after 6 months. The results do not support the need for animal toxicity studies of longer than 6 months duration, apart from those designed to investigate carcinogenicity. 8 1985 society of Totic&gy.
Toxicity studies in animals provide useful ev- The main objectives of repeated-dose tox- idence about potential hazards to man, and icity studies (with the exception of carcino- the scientific community, the research-based genicity investigations) are to reveal physio- drug industry, and drug regulatory authorities logical and/or pathological changes induced regard them as indispensable in the overall as- by repeated administration of the substance sessment of new medicines. However, there under consideration, to determine the dose- are major differences of opinion with regard response relationship of these changes, and to to the amount and nature of toxicity testing establish the maximum “no effect” dose for that is necessary during drug development. In each species (Poggiolini, 1983; Traina, 1983). particular the duration of chronic repeated- The ultimate goals are to collect data which dose animal studies is a subject of considerable will both aid in the decision as to whether the controversy. This is illustrated by the differ- drug should be tested in man (Dayan, 198 1; ences in international recommendations for Kesterson, 1982) and subsequently to indicate the minimum periods of repeated-dose ad- problems likely to be encountered during long- ministration to animals to support marketing term clinical administration. According to authorization (Table 1). It appears that the Traina (1983), the only difference between the duration of the animal studies is empirically conditions of subchronic (l-3 months) and related to the proposed duration of human ex- chronic (6-18 months) toxicity studies is the posure. dose. In a subchronic study doses are given
1007 0272-0590185 $3.00 Copyri&t 0 1985 by the Society of Toxicology. All rights of reproduction in any form reserved.
1008 LUMLEY AND WALKER
TABLE I
CURRENT RECOMMENDATIONS FORTHE MINIMUM DURATIONOFANIMAL TOXICITY STUDIES
Proposed duration of
human exposure
Duration of animal toxicity studies required to support marketing authorisation (studies required in a rodent (R) and a nonrodent (Non-R) species)
Canadaa EECb USA’ Japand
1 week 4-6 months 4 weeks Up to 3 months 3 months 1 month 18 months* 3 months Up to 6 months 6 months 3 months 18 months* 6 months Up to 6 months 12 months 6 months- 18 months* 6 months 12 months (Non-R) 12 months
unlimited 18 months (R)**
’ Health Protection Branch (198 I). b Alder ef al. (1980). ’ Kesterson (1982). d Koseisho (1984).
* Duration of studies should be extended according to existing toxicity data, nature of drug, proposed clinical use, and indications.
** Although 18-month studies in rodents are indicated in the guidelines, in practice I2-month studies appear to be sufficient.
that will establish the nature of the com- pound’s toxicity while in a chronic study doses are employed that will demonstrate if this or some other toxicity is manifested after pro- longed exposure at less stressful levels. It is common experience that doses used in sub- chronic studies may be higher than those in chronic investigations. Both Goldenthal (1968) and D’Aguanno (1972) consider that in certain instances longer studies may some- how compensate for unforeseen shortcomings in experimental design.
It has been suggested that the potential of any drug to produce organ damage in the test species (except carcinogenicity) can be esti- mated through well-designed studies of 3 to 6 months (Barnes and Denz, 1954; WHO, 1966; ABPI, 1968; Paget, 1968; Peck, 1968; D’Ag- uanno, 1972; Page, 1977; Gross, 1982; Hey- wood, 1983). In contrast, a WHO publication (WHO, 1978) declares that “Acute and sub- acute tests are of limited value in predicting chronic effects because:
(a) chemicals may produce different toxic responses when administered over a prolonged period; and
(b) during the aging process, factors such as altered tissue sensitivity, changing metabolic
and physiological capability and spontaneous disease may influence the degree and nature of toxic responses.”
The implication is therefore that longer- term studies are necessary, although an alter- native may be short-term studies in aging an- imals if there were adequate evidence to sup- port supposition (b). There have been claims that data in the files of the FDA (Goldenthal, 1968; D’Aguanno, 1972), the National Cancer Institute (Page, 1977), Huntingdon Research Centre (Worden, 1978), and the Canadian Health Protection Branch (HPB) (G. Freder- ick, 1984, personal communication) have demonstrated new toxic effects becoming ev- ident after dosing for 6 months or longer. Al- though no specific examples have been pub- lished, Goldenthal ( 1968), D’Aguanno ( 1972), and Worden (1978) cite general effects that were apparently not detected within 6 months with certain therapeutic classes of drug. How- ever, the design and nature of such studies must be very carefully considered before it can be concluded that they provide any justifica- tion for longer-term investigations.
Although the value of repeated-dose studies of longer than 6 months has not been conclu- sively established, there has been a growing
VALUE OF LONG-TERM ANIMAL TOXICOLOGY 1009
tendencv for rezulatorv agencies to reauire tablish a toxicology databank, for which the methodology
tests of increasing duration(Gross, 1979). On and data acquisition have been previously described in
the other hand, Gross (1982) stated that safety detail (Lumley and Walker, 1985a,b). To preserve the
cannot be achieved by simply prolonging confidentiality of the data, the company names and the compounds were given CMR codes before being entered
studies. into the Centre’s microcomputer. The databank is also One method of assessing the value of more password protected at several levels and all analyses were
carried out in-house. prolonged tests of medicines would be to make a retrospective analysis of the data available from both shorter-term (~6 months) and lon- ger-term (>6 months) studies. There appear to have been very few published attempts to do this, and these have been constrained by reliance on limited data from one laboratory (Peck, 1968) or by dependence solely on results from the literature (McNamara, 1976). Both these studies supported the conclusion that toxicological studies beyond 3-6 months are usually unnecessary.
Invaluable but dormant toxicological data are contained in the archives of the toxicology departments of pharmaceutical companies, chemical companies, and the regulatory agen- cies. Using data supplied by many companies, the Centre for Medicines Research (CMR) has established a unique computer-based toxicol- ogy databank to enable retrospective analyses to be carried out (Lumley and Walker, 1985a,b). This paper describes a retrospective analysis based on the comparison of shorter- term (~6 months) and longer-term (>6 months) repeated-dose animal toxicology in- vestigations to determine what new findings, if any, become apparent in studies lasting more than 6 months.
METHODS
Eleven pharmaceutical companies with research and development activities dire&d from the United Kingdom have provided comprehensive repeated-dose toxicology data. Compounds that had been tested and evaluated be- tween 1965 and 1982 were included if information from both shorter-term (~6 months) and longer-term (>6 months) experiments which the companies considered to be comparable was available. Two additional companies provided information for such compounds where the data were generated by the European subsidiary. Some infor- mation for compounds which had been studied in two or more species was also provided. Carcinogenicity studies were excluded unless a combined carcinogenicity/toxicity study was carried out. These data have been used to es-
The UK database contains 74 compounds, 58 of which were tested in more than one species. This has resulted in 145 case studies; a case study being defined as the full information for one compound tested in one species for one or more time period(s). Ninety-three case studies in- clude tests carried out for a maximum period of 6 months or less, while in 52 case studies tests lasted longer than 6 months.
To determine whether any new information was ob- tamed from repeated-dose toxicity studies of longer than 6 months, it was necessary to have comparable studies of differing duration for each compound or complete interim and final analyses in a single study of appropriate total duration, Two or more studies were considered to be comparable if the same compound had been administered to the same species by the same route. Studies where a different strain or breed of the same species had been used were not excluded, and no differentiation was made be- tween oral administration by gavage, as tablets or capsules, or in the diet. Studies carried out in different institutions were also included when the company involved considered them to be comparable. The 52 long-term studies are summarized in Fig. I. For 5 out of the 52, data were avail- able only from long-term studies (two 1 I-month studies and three 12-month studies) and in 2 cases the routes of administration were different in the short- and long-term studies. There are 45 long-term studies (>6 months) for which comparable short-term (~6 months) data were available for analysis.
RESULTS
Analysis of the data shows that in 20 out of 45 case studies all toxicological effects were seen within 6 months (Fig. 1). The 25 case studies in which apparently new findings were seen after 6 months are summarized in Table 2. These 25 case studies were discussed in de- tail at a meeting attended by the toxicologists who had provided the data and representatives from academia. The comments and conclu- sions reached by this group with regard to each case are included in Table 2.
Figure 1 shows that in 9 of these 25 case studies no 6-month data were available and therefore definitive conclusions could not be
1010 LUMLEY AND WALKER
52 LONG-TERM CASE STUDIES
LONG-TERM +
-5 i!! 2
45
NO SHORT-TERM STUDIES
FOR COMPARISON
ROUTES OF ADMINISTRATION DIFFERENT
IN LONG- AND SHORT- TERM STUDIES
LONG-TERM STUDIES &6M) WITH
COMPARABLE SHORT-TERM STUDIES &6M)
NEW EFFECTS SEEN AFTER 6 MONTHS
3 CONSIDERED OF NO TOXICOLOGICAL SIGNIFICANCE (SHIVERING,
COLOURATION OF GUT CONTENTS) (TABLE 2: NOS. 7, 16, 20)
20 13 ALL EFFECTS SEEN SOME APPARENTLY NEW
WITHIN 6 MONTHS FINDINGS IN THE LONG-TERM STUDY
’
9 NO 6 MONTH STUbY 7 NEW EFFECTS: EXTENSION
(TABLE 2: Nos. 4. 5, 6, 9, 13.19, 22, 23, 24, ) TERM STUDY
(TABLE 2: NOS. 3, 8, 10, 12,
16, 17, 25,)
I: OF EFFECTS SEEN IN SHORT-
2 NEW EFFECT SEEN WITHIN 6
MONTHS IN ALTERNATIVE SPECIES
(TABLE 2: NOS. 14, 21)
4 NEW EFFECTS SEEN AFTER 6 MONTHS DID NOT FURTHER INFLUENCE ANY RISK/BENEFIT ASSESSMENTS (TABLE 2: Nos. 1. 2, 11, 15)
FIG. I. Summary of long-term case studies.
drawn. In 3 case studies the new effects seen after 6 months were considered by the group to be of no toxicological significance (shivering and coloration of gut contents). In 13 case studies some apparently new findings were observed in the long-term investigations. In 7 of these 13 the group considered the new ef- fects to be a progression of effects seen in the short-term studies and so they do not justify continuing the study beyond 6 months. In a further 2 case studies the new effects were seen within 6 months in the alternate species. In the remaining 4 case studies the toxicologists have indicated that the new effects seen after
6 months did not further influence the risk appraisal of the compound.
DISCUSSION
Analysis of the data provided by 13 phar- maceutical companies on 32 compounds (45 case studies) did not provide any evidence to justify prolonging chronic toxicity studies be- yond 6 months. In those cases in which some effects required longer than 6 months to be- come apparent or measurable this was a con- sequence of several factors including the
VALUE OF LONG-TERM ANIMAL TOXICOLOGY 1011
problems involved in carrying out a retro- spective analysis and in interpreting toxico- logical findings in aging animals, as well as inconsistencies in the design of studies of dif- ferent durations.
1. Dijiculties of Retrospective Analyses
One of the major problems encountered in this retrospective analysis was that the case studies were not originally designed with a view to determining the value of long- as com- pared to short-term studies, but rather merely to perform a safety study of an individual compound. Several points must therefore be considered when attempting to do so:
(a) Time. In many instances there were several years between the conduct of the short- and long-term studies. As toxicology is a rap- idly developing science, the “state of the art” at the time of the short-term study might not have permitted all the observations that were subsequently made in the long-term study to be undertaken.
(b) Location. In several cases the short- and long-term studies were carried out at different institutions, and even in different countries. Toxicological observations, for example, CNS effects, can be subjective, or require interpre- tation by experienced experts, and this can in- troduce variation in what is reported.
(c) Reexamination. When a new patholog- ical change is noted in a long-term study, the slides prepared during the short-term study may be reexamined in more detail. With the knowledge obtained from the long-term study, the change, or its onset, may subsequently be detected in the slides from the short-term study.
(d) Animals/vehicles. Different animal strains or different vehicles have occasionally been employed in the short- and long-term studies, the significance of which was not al- ways known.
(e) Personnel. In many cases the toxicolo- gist submitted data to this study which had been generated and reported by others, often before he had even joined the company. Dif-
ferences in terminology and reporting format can create difficulties in interpretation of data.
fl Salient eficts. Considerable time and effort was required to complete the CMR questionnaires and in some instances it was not always done by the most senior toxicolo- gist. Although the original protocol requested only salient drug-related effects to be noted, often all the effects seen were recorded. These then required subsequent interpretation by the toxicologist.
2. Study Design
Study design and the procedure followed when a toxicological investigation was under- taken differed between companies. In some cases evaluation of the short-term studies was completed before the long-term study was ini- tiated, whereas in others the short- and long- term studies submitted for comparison were carried out simultaneously. Three points arise from this.
(a) Dose levels. Generally, if a subchronic study has been evaluated before commencing the long-term study, the dose levels in the latter would be based on the shorter-term results. Dose levels in chronic studies are usually cho- sen so that the top dose can be expected to produce some evidence of toxicity (D’Ag- uanno, 1972; WHO, 1978; Traina, 1983). Traina ( 1983) advises that the high dose should be selected near the midpoint of the inter- mediate and high doses of subchronic studies. However, from the data submitted it appears that the same or even higher dose levels were often used in the longer compared to the shorter-term studies (27 out of 45 case studies). In addition, as Zbinden ( 1980) and Heywood (1983) have pointed out, many target organ effects induced by artificially high doses are of no relevance to man. Moreover, higher dose levels do not necessarily result in a propor- tional increase in blood or tissue levels and one of two effects may result:
(i) A larger than expected increase in blood or tissue levels may occur due to accumulation
TABL
E 2
SUM
MAR
Y O
F ST
UD
IES
IN W
HIC
H A
PPAR
ENTL
Y N
EW F
IND
ING
S W
ERE
SEEN
AFIX
R 6
MO
NTHS
Gro
up
com
men
ts
and
conc
lusi
ons
(targ
et
Stud
y Sp
ecie
s rou
te
Tim
e“
orga
ns i
dent
ified
by
the
com
pany
U
O.
ther
cl
(mon
ths)
E
ffect
s obs
erve
d wi
&in
6 m
onth
s E
ffect
s ob
serv
ed a
fter 6
mon
ths
toxi
colo
gist
wh
o pr
ovid
ed
the
data
)
1 R
at
1 6
Ora
l 12
CVS
2 3
Rat
oral
CVS
Rat
oral
cvs
3 6 9 18
CLI
N:
Rat
e of
bod
y wt
gai
n, f
ood
inta
ke i
nc
LAB:
SA
P de
c; S
ALT,
SA
ST i
nc.
PATH
: Li
ver
and
mes
ente
ric l
ymph
no
des-
accu
mul
atio
n of
pi
gmen
t
CLI
N:
Dea
th
food
int
ake
dec;
sa
livat
ion;
hy
perre
spon
sivi
ty;
atax
ia;
resp
irato
ry
rate
dep
ress
ion
LAB
Ret
icul
ocyt
es i
nc;
sedi
men
tatio
n ra
te in
c;
chol
este
rol
inc;
glu
cose
dec
PATH
: Li
ver-w
eigh
t de
c G
I-gas
tric
ulce
rs
Thym
us-s
mal
l Ad
rena
ls-w
eigh
t in
c
CLI
N:
Brow
n co
lora
tion
of ti
ssue
s be
hind
Br
own
colo
ratio
n se
en a
t 12
mon
ths
is n
ot
the
eye
and
unde
r th
e sk
in
cons
ider
ed
a ne
w ef
fect
in v
iew
of
pigm
enta
tion
seen
with
in
6 m
onth
s;
rela
tive
hear
t we
ight
in
crea
se w
as
PATH
: So
ft tis
sues
-bro
wn
at p
ostm
orte
m
Hea
rt-w
eigh
t in
c.
expe
cted
fro
m t
he p
harm
acol
ogy
of th
e co
mpo
und,
sim
ilar
dose
leve
ls w
ere
used
in
all
stud
ies;
targ
et o
rgan
(so
ft tis
sues
) id
entif
ied
with
in
6 m
onth
s
The
appa
rent
ly
new
findi
ngs
may
be
rela
ted
to a
ge a
nd s
tress
as th
ey w
ere
seen
in
both
tre
ated
and
con
trol
grou
ps a
nd w
ere
not
dose
-rela
ted,
the
y wo
uld
not
have
fu
rther
in
fluen
ced
any
risk/
bene
fit
asse
ssm
ent o
f the
com
poun
d,
targ
et
orga
n (h
eart)
was
ide
ntifi
ed
with
in
9 m
onth
s.
An e
ffect
on h
eart
weig
ht (
Dee
) wa
s no
ted
in t
he d
og w
ithin
6
mon
ths
PATH
: H
eart-
rela
tive
weig
ht
inc
-scl
erot
ic
myo
card
ial
Pitu
itary
-hem
atic
su
lfusi
ons
Adre
nals-
hem
atic
sutfu
sion
s Se
min
ifero
us
tubu
les-
de
gene
ratio
n
6 C
LIN
: D
eath
, bo
dy w
t de
c/lo
ss;
food
C
LIN
: Fo
ot l
esio
ns
Foot
les
ions
(pr
essu
re s
ores
) are
ass
ocia
ted
24
inta
ke d
ec; b
ehav
iora
l Ab
norm
al
oest
rous
cyc
le
with
m
ale
rate
s on
wire
mes
h flo
ors;
the
y ch
ange
s; s
edat
ion;
act
ivity
ar
e no
t sp
ecifi
c to
thi
s co
mpo
und
dec,
poo
r gr
oom
ing;
al
thou
gh
incid
ence
an
d se
verit
y we
re
abno
rmal
op
thal
mol
ogy
grea
test
in t
he h
igh-
dose
gro
up;
this
co
mpo
und
is k
nown
to
affe
ct p
rola
ctin
4 5 6
Rat
Ora
l
cvs
Rat
oral
CVS
3 12
18 3 12
24
Rat
0.
5 3
Ora
l 18
24
cv
s
LAB:
C
hole
ster
ol,
sodi
um,
pota
ssiu
m d
ec; A
lb/G
lob,
pr
otei
n de
c; A
ST,
ALT
dec
PATH
: Lu
ng-p
hosp
holip
id
lade
n al
veol
ar
mac
roph
ages
CLI
N:
Body
wt
gain
, fo
od/w
ater
in
take
de
c; h
yper
sens
itivi
ty;
aggr
essi
vene
ss; re
duce
d gr
oom
ing
activ
ity
LAB:
G
luco
se d
ec; p
rote
in
dec
PATH
: lu
ng-in
c nu
mbe
rs
of
subp
leur
al
foci
of
alve
olar
m
acro
phag
es
CLI
N:
No
abno
rmal
ities
LAB:
R
ed c
ell p
aram
eter
s in
c
PATH
: H
eart-
rela
tive
weig
ht i
nc.
CLI
N:
Dea
th;
dose
-rela
ted
redu
ctio
n in
bod
y wt
pai
n; f
ood
inta
ke
RdUC
4Xl
LAB:
SA
P an
d/or
tra
nsam
inas
es i
nc;
chol
este
rol
inc;
urin
e-
keto
ne h
odie
s
PATH
: N
o ab
norm
alitie
s
PATH
: G
I-enl
arge
d ca
ecum
ov
ary-
folli
cuIa
r cy
sts
Sem
inal
ve
sicl
es d
isten
ded
PATH
: R
elat
ive
uter
ine
weig
ht
dec
LAB:
U
rine-
volu
me
inc
5% c
f. co
ntro
ls
PATH
: Li
ver
and
kidn
ey-re
lativ
e we
ight
in
c Lu
ngs-
intra
alve
olar
ag
greg
ates
of
mac
roph
ages
-Y
ello
w
foci
leve
ls a
nd a
bnor
mal
es
trous
cyc
le w
ould
have
bee
n pr
edict
ed
from
oth
er t
ests
; 6-
mon
th
dose
leve
ls a
re o
nly
slig
htly
hig
her
than
24m
onth
do
ses;
no
targ
et o
rgan
s we
re i
dent
ified
in
thi
s st
udy
12-m
onth
st
udy
carri
ed
out o
n a
diffe
rent
st
rain
at a
diff
eren
t in
stitu
tion
and
ther
efor
e is
not
com
para
ble;
th
ere
are
no
6-m
onth
da
ta a
nd i
t is
not
pos
sible
to
conc
lude
wh
ethe
r th
e ne
w fin
ding
s (a
ll ex
pect
ed f
rom
the
acu
te t
oxic
ity o
f th
is
com
poun
d)
would
ha
ve b
een
appa
rent
w
ithin
6
mon
ths;
no
tar
get o
rgan
s we
re
iden
tifie
d in
thi
s st
udy
Endo
crin
e we
ight
ch
ange
s ha
ve b
een
obse
rved
with
re
late
d co
mpo
unds
w
ithin
6
mon
ths,
bu
t da
ta a
t 6 m
onth
s ar
e no
t av
aila
ble
in t
his
stud
y; th
e nu
mbe
r of
an
imal
s ex
amin
ed
in t
he 3
-mon
th
stud
y ( 1
0 fe
mal
es)
was
lowe
r th
an i
n th
e 12
- m
onth
st
udy
(25
fem
ales
); ta
rget
org
an
(hea
rt)
was
iden
tifie
d w
ithin
3
mon
ths
In t
his
stud
y a
3- a
nd a
n 1 I
-mon
th
stud
y ar
e co
mpa
red,
an
d it
is th
eref
ore
not
poss
ible
to
conc
lude
wh
ethe
r th
e ef
fect
s se
en a
t 18
mon
ths
would
ha
ve b
een
appa
rent
w
ithin
6
mon
ths;
in
add
ition,
do
se le
vels
use
d in
the
long
-term
st
udy
are
3X
thos
e us
ed in
the
sho
rt te
rm;
targ
et o
rgan
(liv
er)
was
iden
tifie
d w
ithin
18
mon
ths
TABL
E 2-
Con
tinue
d z
Gro
up
com
men
ts
and
conc
lusi
ons
(targ
et
;;
Stud
y Sp
ecie
s rou
te
Tim
e”
orga
ns i
dent
ified
by
the
com
pany
no
. th
er c
l (m
onth
s)
Effe
cts o
bser
ved
with
in
6 m
onth
s E
ffect
s obs
erve
d uf
te 6
mon
ths
toxi
colo
gist
wh
o pr
ovid
ed
the
data
)
7 D
og
6 12
oral
cvs
8 9
Dog
0.
5 3
Ora
l 12
cvs
Dog
Ora
l
1 C
LIN
: D
eath
, re
duce
d gr
owth
ra
te;
3 sa
livat
ion;
po
stm
edic
atio
n 12
he
art
rate
inc
; EC
G c
hang
es
cvs
CLI
N:
Body
wt
gain
ret
ard&
, fo
od
inta
ke d
ec; s
aliv
atio
n;
conv
ulsi
ons;
st
iff un
stea
dy
gait;
abn
orm
al
ECG
, va
sodi
lata
tion;
vo
miti
ng
LAB:
R
BC,
Hb,
PC
V de
c;
pota
ssiu
m,
ALT
inc
PATH
: N
o ab
norm
alitie
s
CLI
N:
Wei
ght
loss
; ECG
cha
nges
; ec
zem
atou
s le
sion
s
LAB:
Po
tass
ium
dec
PATH
: H
eart-
wei
ght
inc
-end
ocar
dial
he
mor
rhag
e/
necr
osis
.
LAB:
Se
rum
cho
lest
erol
de
c
PATH
: H
eart-
rela
tive
weig
ht
inc
-ven
tricu
lar
fibro
sis
--my-
dial
fib
rosi
s/
necr
osis
Li
ver-w
eigh
t in
c; h
epat
ocyt
e hy
pertr
ophy
PATH
: Li
ver-s
light
we
ight
in
c
PATH
: H
eart-
myo
card
ial
necr
osis
/ fib
rosi
s
LAB:
Er
ythr
ocyt
e co
unt
and
Hb
dec
PATH
: Ki
dney
-sm
all
area
s of
tubu
lar
dege
nera
tion
Sple
en-re
lativ
e we
ight
in
c
Live
r we
ight
in
crea
se w
as m
argi
nal
and
does
not
just
ify a
12-
mon
th
stud
y; o
nly
6 an
imal
s we
re e
xam
ined
at
6 m
onth
s an
d th
e do
se le
vels
wer
e ha
lf th
ose
used
in
the
12-m
onth
st
udy
wher
e 10
ani
mal
s we
re e
xam
ined
. Li
ver
weig
ht
incr
ease
wa
s se
en in
the
rat
with
in
1 m
onth
; th
e si
gnifi
canc
e of
live
r we
ight
ch
ange
s wi
th
no a
ccom
pany
ing
lesi
ons
is d
ebat
able
; no
ta
rget
org
ans
were
ide
ntifi
ed
in t
his
stud
y E $
Endo
card
ial
hem
orrh
age/
necr
osis
was
seen
at
2 w
eeks
; the
myo
card
ial
lesio
n 5
obse
rved
at
12 m
onth
s wa
s ju
dged
to
be
a co
nseq
uenc
e of
the
phar
mac
olog
ical
z pr
oper
ties
of th
e co
mpo
und;
al
thou
gh
< th
e 12
-mon
th
dose
s ar
e lo
wer
than
tho
se
K us
ed in
the
2-w
eek
stud
y th
ey a
re 1
.5 X
hi
gher
tha
n th
e 3-
mon
th
dose
s; ta
rget
or
gan
(hea
rt) w
as id
entif
ied
with
in
2 w
eeks
The
sple
en w
as n
ot w
eigh
ted
in t
he s
hort-
te
rm s
tudi
es;
rena
l tu
bula
r de
gene
ratio
n wa
s se
en in
onl
y l/8
hi
gh-d
ose
anim
al;
thes
e ne
w fin
ding
s do
not
just
ify a
12-
m
onth
st
udy;
targ
et o
rgan
(he
art)
was
iden
tifie
d w
ithin
3
mon
ths
10
Prim
ate
I C
LIN
: D
eath
; bo
dy w
t lo
ss; f
ood
6 in
take
dec
; con
stip
atio
n;
Ora
l 12
di
arrh
ea;
vom
iting
cvs
LAB:
An
emia
; ch
oles
tero
l de
c; S
AST
inc;
blo
od
suga
r low
11
Prim
ate
Ora
l
6 12
cvs
12
Rat
Old
CN
S
6 12
PATH
: Li
ver-d
egen
erat
ive
chan
ges
Kidn
ey-n
ecro
tic
epith
elia
l ce
lls
Gl-u
lcera
tion,
in
flam
mat
ion,
in
flam
mat
ory
infil
trate
s Ad
reno
corti
cal
hype
rpla
sia
CLI
N:
Atax
ia,
redu
ced
sens
ory
refle
xes;
su
bdue
d; h
eart
rate
dec
; eye
- or
ange
tin
ge
in
optic
di
sc;
vom
iting
; ex
cess
saliv
atio
n
LAB:
N
o ab
norm
alitie
s
PATH
: Li
ver-w
eigh
t in
c; d
epos
ition
of
yello
w p
igm
ent
Lym
ph
node
s-de
posit
ion
of y
ello
w
pigm
ent
CLI
N:
Dea
th,
body
w-t g
ain
inc/
dec;
fo
od in
take
dec
; sal
ivat
ion
inc;
con
vuls
ions
, ag
gres
sion,
hy
pers
ensi
tivity
; al
opec
ia;
absc
esse
s; re
d te
ars
LAB:
a-
glob
de
c; &
glob
ln
c; u
rea
inc;
SAS
T,
SALT
, SA
P in
c
PATH
: Li
ver-d
egen
erat
ive
chan
ges,
PA
TH:
Live
r-wei
ght
inc;
vac
uola
tion
of
fat c
onte
nt
inc
hepa
tccy
tes
CLI
N:
Leth
argy
, se
vere
hem
orrh
agic
diar
rhea
; sl
ight
ata
xia;
ski
n pa
le,
yello
w;
poor
bod
y co
nditio
n;
sign
s of
deh
ydra
tion
LAB:
U
rea
inc
PATH
: Li
ver--
occa
sion
al
hepa
tocy
te
hype
~oph
y
PATH
: H
eart-
wei
ght
dec
Trea
tmen
t-rel
ated
co
lora
tion
of ti
ssue
s
All
appa
rent
ly
addi
tiona
l fin
ding
s at
12
mon
ths
retle
ct 6
-mon
th
chan
ges
and
do
not
just
ify c
ontin
uing
th
e st
udy
beyo
nd
this
per
iod,
th
e cl
inic
al
sign
s ar
e al
l re
late
d to
Gl
chan
ges
and
this
sys
tem
is
clea
rly i
dent
ified
as
a ta
rget
by
6 m
onth
s;
alth
ough
he
patc
qte
hype
rtrop
hy
was
not
seen
unt
il 12
mon
ths,
deg
ener
ativ
e ch
ange
s we
re s
een
in t
he l
iver
with
in
6 m
onth
s an
d th
ese
were
con
sider
ed
to b
e m
ore
serio
us t
han
hype
rtrop
hy;
the
sum
e do
se le
vels
wer
e us
ed a
t 6 a
nd
12
5
mon
ths;
ta
rget
org
an (
Gl
tract
) id
entif
ied
$ w
ithin
1
mon
th
a
Col
orat
ion
of t
issu
es o
bser
ved
at 1
2 m
onth
s is
not
con
sider
ed
an a
dditio
nal
findi
ng
in
8 vi
ew o
f pi
gmen
tatio
n of
live
r an
d ly
mph
$
node
s se
en w
ithin
6
mon
ths;
onl
y 6
anim
als
were
exa
min
ed
in t
he 6
-mon
th
stud
y, t
oo
E
few
to d
etec
t org
an w
eigh
t ch
ange
; an
effe
ct
on
hear
t we
ight
wa
s ex
pect
ed
from
th
e g
phar
mac
olog
y of
this
com
poun
d an
d th
e 2
Obs
eNd
decr
ease
doe
s no
t ju
stify
a
12-
$
mon
th
stud
y; ta
rget
org
an (
Gl
tract
) wa
s 2
iden
tifie
d w
ithin
6
mon
ths
x 3 $ Th
e ne
w fin
ding
s (li
ver
weig
ht
incr
ease
and
he
pato
cyte
vac
uola
tion)
ar
e an
ext
ensi
on
8 4 of
effe
cts s
een
in t
he s
hort-
term
as
he
patic
tox
icity
was
not
ed w
ithin
6
mon
ths
(live
r fa
t con
tent
in
crea
sed
and
dege
nera
tive
chan
ges)
; tar
get
orga
n (C
NS-
phar
mac
olog
ical
targ
et)
iden
tifie
d w
ithin
1
mon
th
TABL
E 2-
Con
tinue
d
Gro
up
com
men
ts
and
conc
lusi
ons
(targ
et
Stud
y Sp
ecie
s rou
te
Tim
d or
gans
ide
ntifi
ed
by th
e co
mpa
ny
UO.
ther
cl
(mon
ths)
E
ffect
s obs
erve
d w
ithin
6
mon
ths
Effe
cts o
bser
ved
afte
r 6 m
onth
s to
xico
logi
st
who
prov
ided
th
e da
ta)
Kidn
ey-w
eigh
t de
c;
dege
nera
tive
chan
ges
13
14
Rat
oral
CN
S
1 C
LIN
: D
eath
: bo
dv w
t. fo
od i
ntak
e C
LIN
: C
age
sore
s le
adin
g to
acu
te
The
mea
ning
of
thes
e la
bora
tory
ch
ange
s in
24
de
cf s
eda>
on’a
fter
dosin
g;
exci
tem
ent;
abno
rmal
be
havi
or;
aggr
essio
n
necr
otizi
ng
derm
atiti
s
LAB:
C
hole
ster
ol
dec;
glu
cose
dec
PATH
: Li
ver-w
eigh
t de
c, g
lyco
gen
dec
Kidn
ey-w
eigh
t de
c H
eart-
wei
ght
dec
Sple
en-w
eigh
t de
c
LAB:
R
etic
uloc
ytes
, er
ythr
ocyt
es,
hem
atoc
rit,
Hb
dec;
MC
HC
, M
CH
inc;
AST
, AL
T in
c; K
and
cr
eatin
ine
inc;
sod
ium
in
c/de
c ur
ine-
vol
inc,
dilu
te,
Na
and
K in
c
old
rats
is u
ncle
ar;
in p
artic
ular
th
e si
gnifi
canc
e of
effe
cts s
een
only
at t
he
inte
rmed
iate
do
se (
dec
in r
etic
uloc
ytes
, er
ythr
ocyt
es,
hem
atoc
rit,
Hb,
and
Inc
in
MC
HC
an
d M
CH
), an
d th
ose
para
met
ers
that
sho
w bo
th a
n in
crea
se
and
a de
crea
se a
re q
uest
iona
ble;
th
ere
are
no d
ata
betw
een
1 m
onth
an
d 24
m
onth
s ap
art
from
a 6
mon
th
im s
tudy
wh
ich
is n
ot c
ompa
rabl
e;
thes
e da
ta d
o no
t pr
ovid
e an
y ev
iden
ce i
n su
ppor
t of
lo
ng-te
rm
stud
ies;
targ
et o
rgan
(h
emop
oiet
ic sy
stem
) was
iden
tifie
d w
ithin
3
mon
ths
Dog
Ora
l
NSA
I
0.5
CLI
N:
Dea
th,
body
wt
dec;
ano
rexia
; C
LIN
: Sh
iver
ing
follo
win
g do
sing
All
maj
or
effe
cts w
ere
seen
with
in
6 1
subd
ued
afte
r do
sing;
m
onth
s; a
ll ta
rget
org
ans
(GI
tract
, liv
er,
1.5
diar
rhea
; vo
miti
ng;
kidn
eys,
eye
s) w
ere
iden
tifie
d w
ithin
1
2 sa
livat
ion;
eye
-lent
icul
ar
mon
th
6 ch
ange
s, c
omea
l ed
ema,
10
in
traoc
ular
te
nsio
n de
c
LAB:
R
ed c
ell p
aram
eter
s de
c;
LAB:
Bl
ood
in u
rine
lym
phoc
ytes
inc
/dec
; al
bum
in,
gluc
ose,
chl
orid
e de
c; c
reat
inin
e,
SAP,
BU
N
inc;
cho
lest
erol
in
c/de
c; (
u-2
glob
inc
/dec
; ur
ine-
pr
otei
n,
vol
inc;
SG
, O
P de
c; p
H i
nc/d
ec
PATH
: Li
ver-w
eigh
t in
c, f
atty
ch
ange
s, c
loud
y sw
ellin
g,
glyc
ogen
de
c Ki
dney
-wei
ght
inc,
tub
ular
di
latio
n/fa
tty
chan
ges,
gl
omer
ular
sc
hler
osis
, clo
udy
swel
ling,
co
nges
tion/
IW
XOSiS
Eye-
cata
ract
s,
dege
nera
tion,
va
cuol
atio
n H
eart-
necr
osis
/cal
cific
atio
n,
myo
card
ial
fatty
ch
ange
s/cl
oudy
sw
ellin
g Lu
ng-c
onge
stio
n,
pneu
mon
itis,
emph
ysem
a, e
dem
a G
I-hem
orrh
age,
ul
cera
tion,
in
flam
mat
ion
Thyr
oid-
colu
mna
r ep
ithel
ial
CdIS
Sple
en-e
nlar
ged,
co
nges
tion,
at
roph
y Ly
mph
no
de-s
inus
ca
tarrh
Bo
ne m
arro
w-ly
mph
o-
cyte
s dec
, m
ono-
cy
tes
inc
Pitu
itary
an
d ad
rena
ls-w
eigh
t inc
15
Prim
ate
Ora
l
NSA
I
1 C
LIN
: de
ath;
bod
y wt
and
/or
gain
6
dec;
food
inta
ke
dec;
loos
e 12
fe
ces w
ith
pass
age
of b
lood
/ m
ucus
/tiss
ue d
ebris
, ed
ema
of fa
ce, c
hest
, abd
omen
; de
terio
ratio
n in
con
dition
Inte
rstit
ial
neph
ritis
was
first
obs
erve
d at
8
mon
ths
and
is p
roba
bly
not
treat
men
t re
late
d; t
hym
us w
eigh
t de
crea
se is
pr
obab
ly
a no
nspe
cific
ef
fect
due
to
stre
ss; th
ese
findi
ngs
do n
ot ju
stify
a
12-
;5
mon
th
stud
y; d
ose
leve
ls u
sed
in t
he
12-
:
TABL
E 2-
Con
tinue
d
;;,
Gro
up
com
men
ts
and
conc
lusi
ons
(targ
et
Stud
y Sp
ecie
s rou
te
Tim
e”
orga
ns i
dent
ified
by
the
com
pany
no
. th
er c
l (m
onth
s)
Effe
cts o
bser
ved
with
in
6 m
onth
s E
ffect
s obs
erve
d af
ter
6 m
onth
s to
xico
logi
st
who
urov
ided
th
e da
ta)
16
17
Prim
ate
oral
CN
S
I 6 12
Prim
ate
Ora
l
NSA
I
1 C
LIN
: Bo
dy w
t ga
in,
food
int
ake
dec;
6
saliv
atio
n; r
ash;
poo
r wo
und
12
heal
ing;
los
s of
con
ditio
n;
18
feca
l OB+
; vo
miti
ng
LAB:
R
ed c
ell p
aram
eter
s de
c;
prot
ein,
al
bum
in
dec;
LAP
, SA
LT
dec;
urin
e-br
own,
os
mol
ality
de
c
PATH
: G
I-hem
orrh
age/
eros
ion
-sub
muc
osal
ed
ema
in
colo
n
CLI
N:
deat
h; b
ody
wt l
oss;
food
in
take
dec
; dia
rrhea
, bl
ood
in f
eces
; abd
omin
al
dist
ensi
on;
skin
ulce
ratio
n
LAB:
An
emia
; er
ythr
ocyt
e hy
poch
rom
asia
; ES
R i
nc;
retic
uloc
ytos
is;
leuc
ocyt
osis
; ur
ea i
nc;
SAP
dec;
urin
e-
bloo
d,
cast
s
mon
th
stud
y ar
e hi
gher
th
an t
hose
use
d in
the
6-m
onth
st
udy;
targ
et o
rgan
(G
I tra
ct)
was
iden
tifie
d w
ithin
3
mon
ths
PATH
: Ki
dney
-inte
rstit
ial
neph
ritis
Thym
us-w
eigh
t de
c
PATH
: Ki
dney
-epi
thel
ial
necr
obio
tic
PATH
: Ki
dney
-pap
illary
tip
nec
rosi
s/
chan
ges
in
hem
orrh
age/
edem
a co
llect
ing
tubu
les
GI-u
lcera
tion/
eros
ion
LAB:
R
BC,
Hb,
PC
V de
c; c
lotti
ng
LAB:
LD
H
inc;
SAP
inc
; po
tasi
um
dec/
te
sts
abno
rmal
; AL
T,
AST
inc
inc;
circ
ulat
ing
thyr
oid
horm
one
leve
ls d
ec; u
rine-
Epith
elia
l ne
crob
iotic
ch
ange
s we
re
obse
rved
in
the
rena
l co
llect
ing
tubu
les
with
in
6 m
onth
s; t
he k
idne
y wa
s th
eref
ore
iden
tifie
d as
a ta
rget
org
an,
and
5
the
12 m
onth
s st
udy
did
not
elic
it an
y co
mpl
etel
y ne
w to
xico
logi
cal
findi
ngs:
5
papi
llary
tip
cha
nges
wer
e se
en fi
rst a
t 12
&
mon
ths
in o
nly
l/10
high
-dos
e an
imal
G
an
d th
is d
ose
had
been
inc
reas
ed a
t 6
mon
ths;
th
ese
data
do
not
prov
ide
any
evid
ence
in
supp
ort
of lo
ng-te
rm
stud
ies.
Ta
rget
or
gans
(G
I tra
ct a
nd k
idne
ys)
were
ide
ntifi
ed
with
in
3 m
onth
s
Alth
ough
liv
er w
eigh
t in
crea
se a
nd e
nzym
e ch
ange
s we
re n
ot o
bser
ved
until
12
mon
ths,
tox
ic l
iver
inj
ury
had
been
no
ted
with
in
6 m
onth
s; e
ffect
s on
the
liver
wer
e ex
pect
ed b
ased
on
the
acut
e to
xici
ty o
f thi
s co
mpo
und,
th
e ef
fect
on
pota
ssiu
m s
een
at 1
2 m
onth
s is
pro
babl
y no
t si
gnifi
cant
in
view
of t
he v
aria
ble
chan
ge; t
he f
indi
ngs
afte
r 6
mon
ths
do
bile
pig
men
ts,
lack
of
conc
entra
ting
abilit
y
PATH
: Li
ver-t
oxic
in
jury
, ce
ntril
obul
ar
PATH
: Li
ver-w
eigh
t in
c
swel
ling
swol
len
hepa
tocy
tes
Kidn
ey-w
t in
c, d
isten
ded
tubu
les,
ne
cros
is
G&c
onge
stio
n,
hem
orrh
age,
in
flam
m.,
dege
nera
tion,
ne
cros
is,
ulce
ratio
n
18
Prim
ate
Ora
l
NSA
I
6 C
LIN
: D
eath
/kille
d du
e to
poo
r C
LIN
: Sh
iver
ing
12
cond
ition;
bo
dy w
t de
c;
leth
argy
; po
or b
ody
cond
ition;
vo
miti
ng;
bloo
d/
muc
us i
n fe
ces;
abdo
min
al
pain
; sk
in u
lcera
tion
and
pale
are
as
LAB:
N
eutro
philia
an
d ly
mph
open
ia;
WBC
in
c;
plat
elet
s in
c; a
nem
ia;
hypo
chro
mas
ia
and
anis
ocyt
osis
; ES
R in
c;
albu
min
de
c; S
AP d
ec; u
rea
dec
PATH
: N
ervo
us s
yste
m-C
SF
inc
GI
tract
-ulc
erat
ion,
ed
ema,
in
flam
mat
ory
cell
infil
trate
s Ad
rena
ls-en
larg
ed,
corti
cal
hype
rpla
sia
Thym
us-a
troph
y Su
bcut
aneo
us
edem
a; e
xces
s pe
riton
ea&e
ricar
dial
flu
id;
perit
oniti
s;
mes
ente
ric
lym
ph
node
hyp
erpl
asia
not
ther
efor
e ju
stify
a 1
Zmon
th
stud
y;
targ
et o
rgan
s (k
idne
y, l
iver
, an
d G
I tra
ct)
were
ide
ntifi
ed
with
in
6 m
onth
s
Shiv
erin
g fir
st s
een
at 1
2 m
onth
s in
2/2
5 an
imal
s is
not
a m
ajor
ef
fect
and
not
4)
suffi
cien
t re
ason
for
con
tinui
ng
the
stud
y be
yond
6 m
onth
s; t
arge
t or
gan
(GI
tract
) E;
was
iden
tifie
d w
ithin
3
mon
ths
6 i4
E
TABL
E 2-
Con
tinue
d E;
Gro
up
com
men
ts
and
conc
lusi
ons
(targ
et
ki
Stud
y Sp
ecie
s rou
te
Tim
@
orga
ns i
dent
ified
by
the
com
pany
IlO
. th
er c
l (m
onth
s)
Effe
cts o
bser
ved
with
in
6 m
onth
s E
ffect
s obs
erve
d af
rer 6
mon
ths
toxi
colo
gist
wh
o pr
ovid
ed
the
data
)
19
Prim
ate
Ora
l
NSA
I
20
Rat
oral
GI
21
Rat
oral
1 C
LIN
: Bo
dy w
t de
c; s
ubdu
ed
12
beha
vior
LAB
No
abno
rmal
ities
PATH
: N
o ab
norm
alitie
s
1 C
LIN
: Bo
dy w
t ga
in d
ec; e
xces
s 6
saliv
atio
n 12
LA
B R
BC,
PCV
dec;
chl
orid
e in
c;
CPR
, LD
H
inc
PATH
: Ki
dney
-cor
tical
tu
bula
r ne
cros
is
1 C
LIN
: Pe
rinea
l w
etne
ss; e
xces
s 3
saliv
atio
n
LAB:
H
b de
c; c
hole
ster
ol
inc
GI
PATH
: Li
ver-w
eigh
t in
c Ki
dney
-wei
ght
inc
Thyr
oid-
hist
olog
ical
chan
ges
Ther
e is
no
stud
y wi
th
a du
ratio
n be
twee
n 1
mon
th
and
12 m
onth
s an
d th
e sa
me
dose
leve
ls w
ere
used
in t
hese
two
stud
ies;
it i
s th
eref
ore
not
know
n wh
ethe
r th
e ki
dney
effe
cts w
ould
have
PA
TH:
Kidn
ey-w
eigh
t in
c; m
icro
scop
ic
been
app
aren
t w
ithin
6
mon
ths
in t
he
corti
cal
scar
ring,
foc
al
prim
ate;
in
bot
h th
e ra
t an
d th
e do
g pa
pilla
ry
chan
ges
kidn
ey c
hang
es w
ere
obse
rved
with
in
6 m
onth
s wi
th t
his
com
poun
d,
targ
et
orga
n (k
idne
y)
was
not
iden
tifie
d un
til 12
mon
ths
beca
use
ther
e wa
s no
pa
thol
ogy
exam
inat
ion
betw
een
1 an
d 12
&
mon
ths
<
PATH
: G
I-yel
low
co
lora
tion
of g
ut
cont
ents
Col
orat
ion
of g
ut c
onte
nts
seen
afte
r 6
mon
ths
does
not
jus
tify
a 12
-mon
th
%
stud
y, a
s no
thin
g of
toxi
colo
gica
l E
sign
ifica
nce
was
lear
ned
from
thi
s F
findi
ng;
targ
et o
rgan
(ef
fect
s on
body
wt
gain
) id
entif
ied
with
in
6 m
onth
s
LAB:
U
rinar
y ca
sts,
giyc
osur
ia
A ki
dney
effe
ct (w
eigh
t in
crea
se)
had
been
se
en b
y 3
mon
ths;
ur
inar
y ca
sts
and
glyc
osur
ia w
ere
seen
in t
he d
og w
ithin
3
mon
ths
so a
lthou
gh
this
is a
new
effe
ct
for
the
rat i
t is
not
a n
ew e
ffect
for
the
com
poun
d;
the
sam
e do
se le
vels
wer
e us
ed in
all
stud
ies;
one
tar
get
orga
n (li
ver)
was
iden
tifie
d w
ithin
3
mon
ths;
th
e ki
dney
was
con
clus
ivel
y id
entif
ied
as
a ta
rget
in
this
spe
cies
with
in
12 m
onth
s,
but
had
been
ide
ntifi
ed
as a
targ
et w
ithin
3
mon
ths
in t
he d
og
22
Rat
Oral
GI
1 9 18
23
Dog
1.5
12
oral
GI
24
Rat
3 18
oral
ANTI
INF
25
Rat
6
Ora
l 18
ANTI
-NE0
1 IM
MUN
OSU
P
CLI
N:
Body
wt
gain
, fo
od i
ntak
e de
c;
lack
of g
room
ing
LAB:
C
opio
us,
dilu
te
urin
e
PATH
: N
o ab
norm
alitie
s
CLI
N:
Occ
asio
nal
saliv
atio
n;
loos
enes
s of
fece
s and
vo
miti
ng;
abno
rmal
ga
it;
hype
rexc
itabi
lity;
oc
casio
nal
rela
xatio
n of
nic
tatin
g m
embr
ane
DAB
: N
o ab
norm
alitie
s
PATH
: N
o ab
norm
alitie
s
CLI
N:
Body
wt
gain
dec
LAB:
N
o ab
norm
alitie
s
PATH
: Sa
livar
y gl
and
duct
hyp
erpl
asia
CLI
N:
Dea
th;
body
wt
dec
LAB:
R
BC,
Hb
dec;
pan
cyto
peni
a;
ALT
inc
LAB:
So
dium
in
c
PATH
: Li
ver-n
ecro
sis,
m
itosi
s PA
TH:
Bone
mar
row
-hyp
opla
stic
PATH
: Li
ver-w
eigh
t in
c R
espi
rato
ry
syst
em-h
pido
sis
CLI
N:
Dea
th
Eye-
-tran
sien
t ef
fect
on t
apet
um
lucid
um
PATH
: Li
ver
and
pros
tate
-wei
ght
dec
Ther
e ar
e no
3 o
r 6
mon
th
data
and
the
ne
w ef
fect
s wer
e se
en a
t dos
es u
p to
20X
hi
gher
tha
n th
e do
ses
used
in t
he l
- m
onth
st
udy;
no
conc
lusi
ons
caa
be
draw
n as
to w
heth
er
the
new
effe
cts
would
ha
ve b
een
seen
with
in
6 m
onth
s wi
th
a di
ffere
nt s
tudy
des
ign;
targ
et o
rgan
(lu
ng)
was
iden
tifie
d w
ithin
18
mon
ths
Dos
e le
vels
in t
he 1
2-m
onth
st
udy
were
m
ore
than
tw
ice t
hose
in t
he 6
-wee
k st
udy
and
were
dou
bled
at
Wee
k 47
; ne
w 2
findi
ngs
at 1
2 m
onth
s we
re s
een
5 w
eeks
i
a&r
this
dos
e in
crea
se,
aad
inclu
ded
1 de
ath,
a d
efin
ite
eye
effe
ct in
1 a
nim
al,
8
and
an o
bscu
re e
ye e
ffect
in 2
ani
mal
s;
g no
thin
g fro
m v
isio
n te
sts s
ugge
sted
any
to
xic
effe
ct; it
is
likel
y th
at t
hese
effe
cts
6
would
ha
ve b
een
obse
rved
with
in
6 4
mon
ths
if th
e hi
gher
dos
e le
vels
had
bee
n 2
used
thro
ugho
ut
the
stud
y; n
o ta
rget
or
gans
wer
e id
entif
ied
5 2 Th
is ca
se st
udy
com
pare
s a
3- a
ad a
n 18
- $:
m
onth
st
udy;
as
ther
e ar
e no
6-m
onth
da
ta i
t is
not
pos
sible
to
conc
lude
wh
ethe
r th
ese
orga
n we
ight
cha
nges
i2
would
ha
ve b
een
seen
with
in
6 m
onth
s;
?i
dose
le
vels
are
the
sam
e in
bot
h st
udie
s P
and
only
5 a
nim
als
were
exa
min
ed
at 3
3
mon
ths
(15
were
exa
min
ed
at I
8 m
onth
s);
no t
arge
t or
gans
wer
e id
entif
ied
in t
his
stud
y
Chan
ge i
n th
e bo
ne m
arro
w (e
xpec
ted
base
d on
the
pha
rmac
olog
y of
the
com
poun
d)
doea
not
just
ify a
long
-term
st
udy
as p
ancy
tope
aia
indi
catin
g m
arro
w da
mag
e wa
s se
en w
ithin
1
mon
th;
sodi
um
incr
ease
was
not
con
lirm
ed
in
K
LUMLEY AND WALKER
of the compound or an active metabolite. This could be brought about by high dose levels exceeding the body’s ability to detoxify or eliminate the compound or by increasing the conversion of an inactive chemical to an active metabolite.
(ii) A smaller than expected increase in blood or tissue levels may occur due to the absorption and retention mechanisms be- coming saturated or due to the chemical stim- ulating its own metabolism.
Of the 25 case studies in which apparently new findings were seen after 6 months, similar doses were used in the long- and short-term studies in 8 (Studies 2.1, 2.4, 2.14,2.19, 2.20, 2.21, 2.24, 2.25). Higher doses were used in the long-term as compared to the short-term studies in a further 6 (Studies 2.6, 2.7, 2.15, 2.17,2.22,2.23). Thus in 14/25 cases in which apparently new effects were seen after 6 months, the maximum dose was either the same or higher in the long-term as compared to the short-term study, and this may well have had a significant influence on whether or not all toxicological effects were elicited within 3- 6 months.
(b) Number of animals. In some cases the number of animals examined in the short term was too small to allow the demonstration of the pathological changes that were observed in the long-term study. This occurred in two case studies (2.5 and 2.11).
(c) Time of pathological examination. In several cases there was a large gap between the times of pathological examinations. In one case, while there were new pathological changes at 12 months, no animals were sac- rificed for examination between 1 and 12 months (2.25). No animals were sacrificed be- tween 3 and 12 months in five case studies (2.4, 2.5, 2.8, 2.9, 2.21) and in four of them new pathological changes were observed at 12 months. Again new pathological changes were seen at 18 months in the two studies in which there was no sacrifice between 3 and 18 months (2.6, 2.24). In all, these seven cases (with no 6 months’ data) where new patho-
VALUE OF LONG-TERM ANIMAL TOXICOLOGY 1023
logical changes were seen in the long-term study, there is no way of knowing when after 1 or 3 months these would have first occurred.
No 18-month dog studies were available for this analysis and therefore no insight could be gained as to changes that might occur in this species between 12 and 18 months. Toxicol- ogists of the Canadian Health Protection Branch have attempted a retrospective analysis of data contained in their files (G. Frederick, 1984, personal communication). Although they are in a similar situation of having no 18- month dog data for comparison, their current guidelines suggest 18-month toxicological data in a nonrodent species (HPB, 198 1). This is in contrast to the American FDA and the EEC which suggest that data be provided from 12- and 6-month tests in nonrodents, respectively, in support of product licence applications.
3. Dificulties in Interpreting Toxicology Studies in Aging Animals
Comments made by the company toxicol- ogists such as “terminal effects were those of senility” and “the significance of laboratory findings in old animals is not clear” emphasize the problems associated with interpreting tox- icological effects in studies of 18 and 24 months duration in rats. A differentiation must be made between stress effects resulting from the fact that the animals are generally unwell or are having to adapt to the stress of active treatment, and direct toxic effects. This may become more difficult as the age of the animal increases and according to Paget ( 1968) changes due to age are not only difficult to distinguish from those due to the administra- tion of a drug, but they may even obscure such changes. Boorman (198 1) has emphasized the importance of taking into account the back- ground of spontaneous lesions in long-term and lifetime studies. He lists several familial and aging lesions common to the rat, including nephrosis, non-neoplastic hepatic lesions such as bile duct hyperplasia, focal chronic hepa- titis, large hepatic nuclei and cysts, myocardial fibrosis, and retinal degeneration. The ques-
tion that must always be considered is whether or not observed lesions are related to, or ex- acerbated by, the compound being tested.
CONCLUSIONS
Unfortunate experiences with marketed drugs causing severe adverse reactions, despite extensive premarketing toxicological and clinical investigations, have created an at- mosphere of public apprehension and over- caution. This has resulted in a trend toward a proliferation of preclinical requirements to be completed before obtaining marketing au- thorization, including increased durations of chronic repeated-dose toxicity tests (Gross, 1979; Aldridge, 1981; Dayan, 1984). This CMR study is the first comprehensive attempt to determine whether any new findings be- came apparent during toxicity studies of longer than 6 months duration by analyzing data ob- tained from pharmaceutical companies on in- vestigational medicines. A limited number of compounds have been assessed to date and further data are currently being collected from other European countries and the United States. The information presented in this paper does not support the need for animal toxicity studies of longer than 6 months duration (with the exception of those to assess carcinogenic- ity) as these do not add materially to the overall safety evaluation of a given compound. The scientific rationale for conventional long-term studies, and in particular the premise that they are necessary in order to compensate for pos- sible shortcomings in the design of subchronic studies, must therefore be questioned.
The ultimate test of animal safety evalua- tion studies is how well they predict adverse reactions for man and clinical information for compounds contained in the CMR database is therefore being collected. Comparisons be- tween salient findings in animals and the toxic effects observed in man will be the subject of a future paper.
ACKNOWLEDGMENTS
We are grateful to the following companies for sup porting this study and particularly for providing data: The
1024 LUMLEY AND WALKER
Boots Company plc, Beecham Pharmaceuticals, Ciba- Geigy Pharmaceuticals, Fisons plc, Glaxo Group Research Limited, May & Baker Limited, Plizer Limited, Reckitt & Colman plc, Roche Products Limited, Smith, Kline and French Research Limited, Sterling-Winthrop Group Lim- ited, Wellcome Research Limited, and Wyeth Research (UK) Limited. The group which met to discuss the data was composed of representatives from these companies and three independent toxicologists from academia. The help of our colleagues at the CMR is gratefully acknowl- edged and in particular the assistance of Mrs. Elaine Smith and Mrs. Sheila Wright with the preparation of this manu- script.
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