adequaciesandinadequaciesinassessingmurinetoxicitydatawith ... · drugroutemouse...

[CANCER RESEARCH 39, 2204-2210, June 1979]0008-5472/79/0039-0000$02.00

Adequacies and Inadequacies in Assessing Murine Toxicity Data withAntineoplastic Agents

Anthony M. Guarino,1Marcel Rozencweig,Ira Kline, John S. Penta, John M. Venditti, Harris H. Lloyd,Donald A. Holzworth, and Franco M. Muggia

Division of Cancer Treatment, National Cancer Institute, NIH, Bethesda, Maryland 20205 [A. M. G., M. R., I. K., J. S. P., J. M. V., F. M. M]; Southern ResearchInstitute, Birmingham, Alabama 35205 (H. H. L.]; Toxicology Program Office, Battelle Columbus Laboratories, Vienna, Virginia 22 180 (0. A. H.]

regardless of the number of treatments. Finally, implicit and/orexplicit assumptions were made regarding the reproducibilityof these experimental results.

This retrospective study takes advantage of the accumulationof additional data to further assess problems associated withtoxicological results in mice and their potential for use in doseprediction in humans.

MATERIALS AND METHODS

The cytotoxic antitumor agents selected for analysis wereconfined to those for which clinical toxicological data wereavailable for i.v. administration by at least one of the followingschedules: high intermittent doses, weekly doses, and dailyadministration for 5 or 7 days. These limitations were necessarybecause other routes of administration, particularly the p.o.route, may introduce further bias in correlation studies; theschedules chosen are those most frequently investigated inhumans.

Toxicological studies in mice were performed under contractto the Laboratory of Toxicology and Drug Evaluation Branch ofthe Division of Cancer Treatment in the National Cancer Institute, according to previously described methods (13, 25).Lethality was chosen as a nonspecific but easily measurabletoxicological end point. The lethal dose categories are LD10,LD50,and LDso;in this study, this period ranged from 14 to 60days after the last dose administration, but more than 90% ofall deaths actually occurred within 14 days.

Statistical analyses were performed to assess true differences between the related parameters of animal strain, injection site, and drug-dissolving or suspending vehicles. Probitanalyses and tests for parallelism of response were performedwherever possible (3, 10, 11, 16). Slopes of log probit linesshould be parallel if: (a) there is no statistically significantdifference between the data (11); and (b) the cause of death(i.e. , mechanism of toxicity) in the 2 treatment groups is thesame, that is, both groups show the same â€˜â€˜specificactionâ€•(8).

RESULTS

Clinical toxicity data were available for 58 drugs that havebeen administered i.v. in humans by at least one of the 3schedules of interest (Table 1). The daily for 5 or 7 daysschedule has been most widely used, for 49 (84%) of thedrugs, whereas high-dose intermittent administration has beenstudied for only 41 % of the drugs.

Of the 3 categories of lethal dose, the mouse LD50,estimatedby the probit model, is statistically more reliable than is theLD10or LDso (11, 15) and was most often available for our

2204 CANCER RESEARCH VOL. 39

ABSTRACT

Previous retrospective analyses have suggested a very positive correlation in toxic doses of antineoplastic agents betweenmice and humans. Additional toxicological information has nowbeen accumulated and reveals a noticeable variability in theexisting data base. Nevertheless, it is likely that mouse toxicological studies will become a principal determinant for estimating initial doses to be used in humans. Recognition of thefactors responsible for differences in determinations of toxicdose levels in mice will enhance the proper utilization of thisapproach.

INTRODUCTION

Antitumon agents are commonly administered in humans ator near their MTD.2 When a new agent is first introduced intoclinical trials, its MTD is reached by progressive increments ofan initial dose that has been derived from toxicological dataobtained in animals (4, 5, 9, 24). Significant interspecies differences in the rate of drug metabolism and excretion make itdifficult to accurately extrapolate to humans the iatrogeniceffects observed in animals (1, 6, 7). Nevertheless, based onthe clinical experience with 37 drugs, Homan (20) has estimated that an initial dose of one-third the MTD (in mg/sq m),as determined in the most sensitive large animal species (beagle dog or rhesus monkey), would be tolerated in humans inabout 94% of the cases. Whether such a determination constitutes the most efficient method for rapidly and safely reaching the effective dose in humans or whether rodent data maybe used to advantage has been the subject of recent analysis(14).

The value of combined animal toxicological data, includingfindings in rodents, for predictive purposes in humans has beenrepeatedly advocated (12, 14, 26). This approach has beenhampered by the availability of only limited rodent data notexpressly obtained for toxicological purposes. In fact, previouscorrelations had to be based on various assumptions. Specifically, assumptions were made regarding the route of administration, considering i.p. and i.v. injections equivalent and neglecting the effect of schedule. The toxicity pen course wasconsidered to be a function of the total dose administered,

I To whom requests for reprints should be addressed, at Building 37, Room

5B22, Laboratory of Toxicology, National Cancer Institute, NIH, Bethesda, Md.20205.

2 The abbreviations used are: MTD, maximum tolerated dose; LD10, drug dose

that kills 10% of the non-tumor-bearing animals during the observation period;LD@,dose that kills 50% of the non-tumor-bearing animals during the observationperiod; LD@,dose that kills 90% of the non-tumor-bearing animals during theobservation period; 6-MP, 6-mercaptopurine; ara-C, 1-f@-n-arabinofuranosylcytosine; BCNU, 1,3-bis(2-chioroethyi)-1-nitrosourea; FU, 5-fluorouracil; TIC mustard, lmidazole-4-carboxamide, 5-(3,3-bis(2-chloroethyl)-1 -triazeno).

Received November 27, 1978; accepted March 2, 1979.

Research. on January 3, 2021. © 1979 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

â€” No. of drugs for which mouse Is

thality data are available forthecorrespondingschedule in humans

No. of drugsgiven iv. in hu- LD@+LD,0Schedule

mans LD@ and/orLD@Single

dose 24 20 (83)a13(54)Weekly34 11 (32) 2(6)Daily

for 5 (or 7) days 49 23 (47) 16(33)a

Numbers in parentheses, number of drugs for which mouse lethalitydataareavailable for the corresponding schedule in humans.

Drug Toxicity in Rodents and Humans

Table1Availability of mouse lethality data for 58 drugs used i. v. in humans

on the most common schedules

for FU. The slopes of the probit response lines were parallel forall of the drugs except daunomycin, where the LD50and LD10differed by 52 and 275%, respectively.

The variation in the total equitoxic dose per course could bestudied for 6 drugs, each given on 1 equitoxic dose on Day 1and 1 equitoxic dose on Days 1 to 5 schedules in a singlemouse strain using one vehicle and injection route. The L050orLD10is shown for each drug on both schedules in Table 5. Thedifferences in total equitoxic dose per course apparently varygreatly according to the drug in question. Thus, the differencewas modest for BCNU (7%), intermediate for ama-C(23%) andisophosphamide (28%), large for cis-diamminedichlonoplatinum (77%) and 6-MP (150%), and extreme for 2'-deoxythioguanosine (1440%).

Toxicity studies have been repeated, usually at the samelaboratory, with 16 drugs under the same conditions withregard to the animal species and strain, route of administration,vehicle, and schedule. The data for the vast majority of theseagents show striking discrepancies (Table 6). The averagedifference between each pair of lethality studies at LOsowas42%. The LD50was identical for 3 drugs (thiotepa, pseudourea,and BCNU) and differed by 20% or less for azasenine, actinomycin 0, cyclophosphamide, porfinomycin, and daunomycmn.Differences between 21 and 50% were noted in the LD50ofmethotrexate, mitomycin C, ama-C,and TIC mustard. The LD50differed by 71% with nitrogen mustard, by 82% with vincnistine,by 85% with cis-diamminedichloroplatinum, and by 245% withcamptothecin.

It is also noteworthy that there was a lack of parallelism inprobit slopes for more than one-half (9 of 16) of the companisons. Occasionally, the slopes diverged so dramatically withinthe LD10to LD@range that reversals of results were observedaccording to the lethal dose category considered. For example,the LD50of cyclophosphamide (58 mg/kg) in one study is 1@%smaller than that in the other (67 mg/kg). However, when theLD10 in the same pain of experiments is compared, the firststudy shows a 24% higher toxicity (26 mg/kg) than does thelatter one (21 mg/kg). In the case of pseudoumea,the L050 ofboth studies was equal, but reversal was evident when the LD10and LDso were considered. Similarly, examination of thelethal doses for ara-C and daunomycin showed reversals depending upon which lethal dose category was considered.

A summary of the lethality data by factors of variabilityappears in Table 7. Of the 49 separate drug comparisonsincluded in the analyses, 38.8% showed a difference of @s20%in LD50.This 20% variability corresponds to the range traditionally accepted among toxicologists (19).

Differences exceeding 100% occurred in 7 (14.3%) of 49comparisons. The mouse strain used was associated withdifferences of 128% for camptothecin, and 125 and 145% in2 tests of vincnistine (Table 2). A change in the route of injection(Table 3) for vincnistine and daunomycin apparently modifiedthe results by 115 and 300%, respectively, between groups ofmice receiving the same treatment. Alternate vehicles seemedresponsible for a 250% difference in the LD50of FU (Table 4).Unknown variables led to a 245% difference in a pair ofexperiments where camptothecin toxicity was tested underapparently similar conditions (Table 6). Finally, the 3 knownvariables (strain, route, and vehicle) yielded a greater percentage of parallel probit response lines (84 to 100%) than the47% that was obtained when none of these factors could be

study. LD50's were known for 83, 32, and 47% of the drugstested at the single, weekly, and daily schedules, respectively(Table 1). Knowledge of other lethal dose levels does provideadditional information of obvious importance in the analysis oftoxicological data. However, fully usable lethality data (LD50plus LD10or LOso)for the 3 schedules were only available for54, 6, and 33% of the drugs.

Variations in the lethal dose levels according to the mousestrain relate to data obtained largely in Swiss and C57BL xDBA/2 F1 (hereafter called BD2F1) mice (Table 2). Occasionally, results were available for other strains, i.e. , AKA, BALB/c X DBA/2 F1(hereafter called CD2F1), and DBA2.

At least one comparison could be made for each of 16 drugsthat used identical routes, vehicles, and schedules. For anumber of these agents, noticeable variations existed in one onmore of the lethal dose levels (L010, LD50, and LDso). Theaverage difference between the LD50obtained in a given pairof mouse strains receiving the same drug was 45% and rangedbetween 0% (fluorouracil deoxyriboside) and 145% (vincnistine). For 5 drugs (6-MP, cyclophosphamide, pseudourea, yincnistine, and camptothecin), the LD50differed by more than50% between strains. In one case (fluonouracil deoxyniboside),the LD50's were exactly the same, but the LD10's differed by17%. In another case (6-MP), the LD10's were virtually thesame in both strains, but the LD50differed by 52%.

The last column of Table 2 shows the results of standardtests for parallelism of response to the drugs in each pair ofstrains tested. Of the 16 drugs in this table, 13 strain pairs hadparallel slopes. For the other 3 (6-MP, pseudourea, and amaC), either the 2 strains are not truly comparable from a statistical point of view or it may be assumed that there was a straindifference in the cause of death.

Because the primary screening protocols of the NationalCancer Institute (13) involve essentially i.p. administration, datacomparing the i.p. and i.v. routes were available for only 3drugs (Table 3). None of the lethal dose levels of Adniamycinvaried significantly with the route of injection. On the otherhand, both vincristine and daunomycin were severalfold moretoxic via the i.p. route. The well-known local toxicity (17) ofthese agents apparently shifted the dose-response curves infavor of greater i.p. toxicity, but the slopes of these curvesremained parallel. This observation contrasts with the usualexpectation that the route carrying the substance most rapidlyto the bloodstream is invariably the most toxic (27).

The average LD50difference was 72% between groups ofanimals receiving 10 separate drugs in 2 different drug vehicles(Table 4). This difference ranged from 9% for BCNU to 250%

2205JUNE 1979



time.Lethal

LD,0doses

(mg/kg/dose)

LD@ LD@Parallelism

ofprobit re

sponse linesc

A. M. Guarino et a!.

Table 2

Variation of toxicity according to mouse strainForeachdrug,thecomparedstudieswereperformedby the i.p. routeof administrationin thesamelaboratorybut notnecessarilyat thesame

0.0700.0680.432

0.2827.4

141.6

2.021

3012

1455

12714

60

0.0890.099

0.4850.563

1723

2.12.2

4256

1718

64146

3568

0.1140.144

0.546>0.680

3837

2.82.3

86101

2323

74169

8676

@!@:@!strainVehicleaSchedulebAlkylatingagents

Alanine mustardSwissBD2F,Dimethyl

sulfoxideDaily for 7 days5.25.96.6 8.98.4 13pd

@,CyciophosphamideSwiss

BD2F,0.9%NaCI solutionDaily on Days 1 and 850

147138266>200 479P.

VNitrogen

mustardSwissBD2F,0.9%

NaCI solutionDay 1 only3.13,53.5 4.84.0 6.5P.

VNitrominSwiss

BD2FOtherDailyfor 7 days22

1960 55>100 >120P.VAntimetabolites

CycloleucineSwissBD2F,OtherDaily

for 7 days74881

181401

88223P,

Vara-CBD2F

CD2F,0.9%NaCI solutionDay 1 only

Day 1 only2829 39623779 43365047 4745NP,C

NP,C5FUSwiss

BD2F,CarboxymethyicelluloseDailyfor 7 days39

3442 4446 56P.VFluorouracil

deoxyribosideSwiss BD2F,OtherDailyfor 7 days1 28

109178

178248 291P,C6-MPSwiss

BD2F,OtherDailyfor 7 days43

4691 60>146 77NP,VAntibiotics

Actinomycin DBD2F,AKR0.9%

NaCI solutionDaily for 7 daysP,VBD2F,

AKR0.9%NaCI solutionDaily on Days 1 and 4P,VAzaserineSwiss

BD2FOtherDailyfor 7 daysP.VMitomycin

CSwissBD2F,0.9%

NaCI solutionDaily for 7 daysP.VOthers

BCNUSwissBD2F0.9%

NaCI solutionDay 1 onlyP.VSwiss

BD2F0.9%NaCI solution

+ 2% ethanolDaily

for 7 daysP,CCamptothecinSwiss

BD2F,0.9%NaCi solutionDay 1 onlyP,CPseudoureaSwiss

BD2FCarboxymethylcelluloseDailyfor 5 daysNP,VVincristineSwiss

BD2FDBA20.9%

NaCI solutionDay 1 only0.8262.72.22.0

4.54.95.1

7.66.5P.

V (all 3)

2206 CANCERRESEARCHVOL. 39

aThevehiclewasthesameforeachcomparison.Other,actualvehiclenotrecorded(datafrom1961),butitwasidenticalforbothstrains.b Unless otherwise stated, doses were daily for Indicated number of consecutive days.C See@ â€˜Materials and Methods.â€•

d p probit lines parallel; NP, probit lines not parallel; C, computer-generated result; V, visual observation when data were insufficient for computer fit (1 1).

considered a possible contributor to variable results in toxicity and the route of administration. In addition, the cumulative totalstudies. dose per equitoxic course also appeared to vary greatly ac

cording to schedule.DISCUSSION This latter observation seems to contrast with findings re

ported by Griswold eta!. (18). Their study showed no consistentThree parameters were found to modify markedly the toxicity difference in the toxicity of a wide variety of agents in random

of anticancer agents in mice, i.e. , the strain, the drug vehicle, bred Swiss mice and inbred BO2F1 mice. Furthermore, when



DrugRouteMouse strainVehicle8ScheduleLethal

dose (mg/kg/dose)Parallelism of

probit response

llnesbLD,0LD@[email protected].

iv.Swiss0.9%NaCi solutionDay 1 only0.8

3.52.0 4.35.1 5.2p,CCDaunomycini.p.

iv.BD2F0.9%NaCI solutionDay 1 only5.4

298.0 3212

34P.CAdriamycini.p.

iv.BD2FBuffered 0.9% NaCI solutionDay1 only1 0

1218

1825 26P,V

DrugVehicleaMouse strainScheduleLethaI

dose (mg/kg/dose)Parallelismof probit re

sponsellnesbLD,0LD@LD@Alkylating

agentsMelphalan (L-

phenylalaninemustard)Carboxymethylcellulose

Hydroxypropylcellulose(klucel)BD2FDay

1 onlyI 11920 2936 46pC

VTIC

mustardCarboxymethylcellulose/ethanol

CarboxymethylcelluloseBD2F,Day

1 only520262871 4571458 796P.

CAntimetabolltes

FUCarboxymethylceiluloseOtherSwissDay

1 only622401

023571

67560P.

VMethotrexateWater/i

N NaOHNaHCO3BD2FDaily

on Days 1 and 872421

14631

81112P.

VAntibiotics

Actinomycin DOther0.9% NaCI solutionBD2FDaily

for 7 days0.0980.0700.1

180.0890.142 0.114P,

VAdriamycinBuffered

0.9% NaCI solution0.9% NaCI solutionBD2FDay

1 only1 0141

82336 37P.

VDaunomycin0.9%

NaCI solutionWaterBD2F,Daily

on Days 1 and 43.00.83.5 2.34.1 6.5NP,

COthers

BCNU0.9% NaCI solution/ethanol0.9% NaCI solutionBD2F,Day

1 only303061 561

24101P.

VDecarbazineCarboxymethylcellulose

Hydroxypropylcellulose(kiucel)BD2F,Day

1 only626856859 14001

1772020P,

V5-Hydroxypicoilnaide

hydethiosemicarbazone0.9%

NaCI solution/IN NaOH0.9% NaCI solution/Tween

80BD2F1Daily

for 5 days91289283 467884 756P.

C


Table 3

Variation of toxicity with route of administration

Foreachdrug, the comparedstudieswereperformedin the samelaboratorybut notnecessanilyat the sametime.

a The vehicle was the same for each comparison. Buffered 0.9% NaCI solution, 6.5 g Na2HPO4-4 g NaH2PO4 in 1 lIter 0.9% NaCI solutionat final pH 6.9.

b See Table 2.C p probit lines parallel; C, computer-generated result.

Table 4

Variation of toxicity with drug vehicle

For each pair of vehicles,studywas performedby the i.p. route in the samemousestrainat the samelaboratorybut not necessarilyat thesame time.

a@ additional definitions in previous tables.b@ Table 2, Footnote c.C See Tabe 2, Footnote d.

the LD10in BD2F1mice for 70 anticancer agents was comparedfor 2 schedules (daily for 7 and 11 days), there appeared to beevidence of cumulative drug toxicity. These findings with 2similar subacute dose schedules led to the assumption that allanimal dose schedules could be converted by considering that

the total dose per equitoxic course was independent of theschedule. Griswold et a!. (18) also reported that in more than200 lethal dose determinations (daily for 7 days in Swiss mice;daily for 7 and I 1 days in BD2F1) the median range betweenthe lower and upper confidence limits at the 0.05 probability

JUNE 1979 2207



Variation of total equitoxic doseper course accordingto scheduleDose/course

(mgikg)DrugMouse

strainRouteVehicleToxic dose levelDay1

onlyDailyfor

5 daysVariation(%)6-MP

ara-C2'-Deoxythioguanosinelsophosphamidecis-Diamminedichloroplatinum

BCNUSwiss

SwissBALB/cSwissBD2F

BD2Fi.p.

iv.i.p.p.p.

i.p.0.9%

NaCI solution0.9% NaCI solutionWater0.9% NaCi solution0.9% NaCI solution

Tween 800.9 NaCI solutionLD@

LD@LD@LD@LD0

LD@200

20506i 6565

13

56500

166040

72523

60150

231440

2877

7

A. M. Guarino et a!.

Table 5

Table 6

Variability of toxicity data in repeated studies

Fora givendrug, the comparedstudieswereperformed,usuallyin the samelaboratory,usingthe samemousestrain,vehicle,and routeofadministration but at time intervals ranging from 1 month to several years apart.

Lethal dose (mg/kg/dose) Parallelismofprobitre

sponseDrug Mouse strain Vehicle8 Schedule LD,0 L0@ LD@ linesb

Alkylating agentsCyclophosphamide BD2F 0.9% NaCI solution Daily for 5 days 26 58 131 NP,CV

21 67 >200

Nitrogen mustard Swiss Other Daily 7 days 0.7 1.2 2.2 P, C0.4 0.7 1.2

Thiotepa BD2F 0.9% NaCI solution Day 1 only 24 27 31 P. C16 27 46

TIC mustard Swiss Carboxymethylcelluiose/ Daily for 5 days 49 88 158 P. Vethanol 31 63 131

Antimetabolitesara-C BD2F, 0.9% NaCi solution Day 1 only 1.872 4,689 >10,000 NP, V

2,828 3,779 5,049CD2F 0.9% NaCI solution Day I only 3,961 4,336 4,747 NP, V

1,925 6.275 >6,700

Methotrexate BD2F Water/i N NaOH Daily on Days 1 and 8 72 114 118 P. V54 86 139

AntibioticsActinomycin D Swiss Other Daily 7 days 0.046 0.095 0. 195 NP, V

0.053 0.079 0.116

Azaserine BD2F Other Daily 7 days 11 25 55 NP, C21 29 40

Daunomycin BD2F1 0.9% NaCI solution Day 1 only 3.0 9.2 14 NP, V5.4 8.0 12

Mitomycin C BD2F 0.9% NaCI solution Day 1 only 9 14 24 P. C6 11 19

Porfiromycin Swiss Other Day 1 only 26 49 89 P, V25 50 100

OthersBCNU Swiss 0.9% NaC1solution/ Day 1 only 21 42 86 P. V

alcohol 23 42 78

Camptothecin BD2F 0.9% NaCi solution Day 1 only 58 73 91 NP, V141 252 449

cis-diamminodichloro- BD2F 0.9% NaCI solution/ Daily for 5 days 4.6 8.9 16 NP, Vplatinum Tween 80 4.5 4.8 5.2

Pseudourea Swiss Carboxymethyicellulose Daily for 5 days 14 35 86 NP, V9 35 135

Vincristine BD2F 0.9% NaCi solution Day 1 only 2.7 4.5 7.6 P, V4.2 8.2 15.4

a See definitions in previous tables.b See Table 2, Footnote c.

C See Table 2, Footnote d.




Effect of variables 0fl L050 levels inmiceNo.

of drugDifference

in LD@sNo. of comparisons yielding

parallel probit reVariable factoracomparisons<20%21 -50% 51-1 00%>1 00%sponselinesStrain1

994 331 6(84)bRoute310023(100)Vehicle1013

519(90)Noneofabove1785318(47)Totals491912

ii7C%10038.824.522.414.3


Table 7

a@ Tables2to4and6.b Numbers in parentheses, number of comparisons yielding parallel probit response lines.C Includes 3 of >200%: daunomycin (route), 300%: FU (vehicle), 250%; camptothecin (unknown

variable), â€”245%.

level corresponds to about 22% variability.Further analysis of our data showed wide variation in lethal

dose levels even when using the same mouse strain, drugvehicle, route of administration, and schedule, which indicatesthat, in fact, the actual impact ot these parameters is difficult toassess accurately in retrospective analyses.

The variability of the data does not necessarily reflect a lackof reproducibility of such determinations. Some of the discrepancies in this paper could be accounted for by the heterogeneity of study circumstances and sources. It should be emphasized that part of the available toxicity data in nontumor-beaningrodents was extrapolated from experiments treating groups ofmice in parallel to tumor-bearing mice or in pilot studies todetermine the potential therapeutic range for the screening ofnew drugs. Thus, the toxicity results often were not obtained inexperiments primarily designed (e.g. , with respect to numberof animals per test group on selection of dosage levels fordetermining dose-response curves) to define LD10,LD50or LDsoper se by the usual mathematical methods, such as that ofLitchfield and Wilcoxon (23).

Moreover, it is likely that different laboratories and expenimentalists might have used different standards for animal supplies, number of animals involved per experiment, type of diet,degree of fasting, concentration of drug solution, size of needleused for injection, and the like. However, even when suchfactors are rigidly controlled, as in collaborative studies, a highdegree of variability is not uncommon in the results of toxicitytests. For example, Swoap (28) has reported that there was adifference of as much as 71% in the LD50 obtained by 6different laboratories giving injections of the same drug. Evenreplicates of one drug in a single laboratory differed by up to63%. Similarly, in a study involving 8 laboratories testing 4drugs (2), themewas a range of 12 to 58% difference betweenLDso's for the same drug.

Perhaps toxicologists, in their haste to elevate their field toa quantitative science, have created what Ipsen (21) refers toas an â€˜@ LD50-fixation.â€˜â€˜From this analysis, we must caution theclinician, who is anxiously awaiting completion of animal toxicological studies, not to accept LDso'5 as readily as one accepts melting points on pure organic compounds. More than60% of the drug comparisons (Table 7) varied by >20%, andthere was >1 00% variability in 14%, including 3 examples(daunomycin, FU, and camptothecin) where the LD50values inmice receiving the same drug differed by >200%.

Another source of variation arises when we consider that thei.p. route of injection, which is used by the National Cancer

Institute for most drug tests in rodents, has involved an approximate 20% chance that part of the drug â€˜â€˜wasnot injectedinto the peritoneal cavity' â€˜(22).

The findings in the present analysis point out variables toconsider while establishing retrospective correlations betweenanimal and human quantitative toxicity. The wide mangeofexperimental values available precludes firm conclusions fromretrospective toxicological studies for prospectively choosingstarting doses for clinical trials.

In the earlier report of Goldsmith et a!. (14), one-third LD10,expressed in mg/sq m, was greater than the human MTD for2 of 28 drug schedules (bleomycin and camptothecin). Thisupdated survey of rodent toxicology would caution that use ofone-third LD10might, according to the data base used (high orlow LD10),potentially lead to exceeding the human MTD for 6additional drugs: dacanbazine, vincristine, methotmexate, thiotepa, L-phenylalanine mustard, and nitmomin.Six drug schedules in our study were also reported by Goldsmith et a!. (14) sothat the combined studies represent 51 distinct drug scheduleswith available LD10's.For at least 43 (84%) of these, one-thirdLD10in mice would have been a tolerated dose in humans. This84% mateof safe estimation for clinical trials is very similar tothat found by using one-third toxic dose low in larger animals(14) as a starting dose in Phase 1 trials in humans. Thus, fromthis point of view, neither lange animal or rodent quantitativetoxicometric results present overWhelming differences.

The application of dose levels toxic to animals in determiningtolerable starting doses for clinical trials in humans should beevaluated further in the light of the dose escalation schemechosen to reach the human MTD, as well as the number ofsuch escalations required. Decreasing the fraction of a specifictoxic dose level obtained in a particular animal species todetermine the initial dose in humans will always improve thetolerability of the so-defined clinical starting dose, but at theexpense of a greaten number of dose escalations necessary toreach a therapeutic dose.

The potential of mouse data for selecting starting doses inPhase 1 clinical trials has held considerable appeal eventhough the strain was not stipulated (14, 26). The selection ofa fraction of a mouse toxic dose level must allow a margin ofsafety for the range of data which may be observed with anumber of drugs. However, studies designed primarily fortoxicology with the specific purpose of determining LD10's,LDso'S,and LD@'smay in the future prove to be more accurate.This represents an added factor favoring the use of mousetoxicology data as a principal determinant of the starting dose

JUNE 1979 2209



Abbot, B. J. Protocols of screening chemical agents and natural productsagainst animal tumors and other biological systems. Cancer Chemother.Rep., 3 (Part 3): 1-103, 1972,

14. Goldsmith, M. A., Slavik, M., and Carter, S. K. Quantitative prediction ofdrug toxicity in humans from toxicology in small and large animals. CancerRes., 35: 1354-1364, 1975.

15. Goldstein, A., Aronow, L., and Kalman, S. M. Principles of drug action. Ed.2, p. 382. New York: John Wiley & Sons, Inc., 1974.

16. Gray, A. R. Probit regression analysis. A computer program, statisticalsoftware section. Bethesda, Md.: Laboratory of Statistical and MathematicalMethodology, Division of Computer Research and Technology, NIH, 1977.

17. Greenwaid, E. S. Cancer Chemotherapy, Ed. 2, pp. 259â€”270.Flushing, N.Y. Medical Examination Publishing Co., Inc., 1973.

18. Griswold, D. P., Laster, W. R., Snow, M. Y., Schabel, F. M., and Skipper, H.E. Experimental evaluation of potential anticancer agents. xll. Quantitativedrug response of SA 180, CA 755, and leukemia Li2iO systems to a. standard listâ€• of â€â€˜active' â€ and â€œinactiveâ€• agents. Cancer Res., 23: 271 â€”

520, 1963.19. Hagan, E. C. Acute toxicology. In: appraisal of the safety of chemicals in

foods, drugs and cosmetics, pp. 17â€”25.Austin, Texas; Association of Foodand Drug Officials of the United States, 1959.

20. Homan, E. R. Quantitative relationships between toxic doses of antitumorchemotherapeutic agents in animals and man. Cancer Chemother, Rep., 3(Part 3): 13-19, 1972.

21. Ipsen, J. Appropriate scores in bio-assays using death-times and survivorsymptoms. Biometrics, 11: 465â€”480,1955,

22. Lewis, R. E., Kunz, A. L., and Bell, R. E. Error of Intraperitoneal injections inrats. Lab. Anim. Care, 16: 505-509, 1966.

23. Litchfieid, J. T., Jr., and Wilcoxon, F. A simplified method of evaluatingdose-effect experiments. J. Pharmacol. Exp. Ther., 95: 99-i 13, 1949.

24. Owens, A. H. Predicting anticancer drug effects in man from laboratoryanimal studies. J. Chronic Dis., 15: 223â€”228,1963.

25. Prieur, D. J., Young, D. M., Davis, R. D., Cooney, D. A., Homan, E. R., Dixon,A. L., and Guarino, A. M. Procedures for preclinical toxicologic evaluationof cancer chemotherapeutic agents. Protocols of the Laboratory of Toxicology. Cancer Chemother. Rep., 4 (Part 3): 1â€”30,1973.

26. Schein, P. S. Preclinical toxicology of anticancer agents. Cancer Res., 37:1934-1937, 1977,

27. Spector, W, S. (Ed.), Handbook of Toxicology, Vol 2, p. 1. Philadelphia: W.B. Saunders Co., 1957.

28. Swoap, 0. F. A collaborative study on the use of mice in acute toxicitytesting. J. Pharm. Sci., 44: 11â€”16,1955.


A. M, Guarino et a!.

for Phase 1 clinical trial of anticancer drugs in humans. Attention to experimental design and appreciation of variable factorsinvolved will enhance the proper utilization of this approach.

ACKNOWLEDGMENTS

The authors acknowledge the editorial work of William Soper and the secretarlal assistance of Karen Brownell and Judy Williams in the preparation of thispaper.

REFERENCES

1. Adamson, R. H. Metabolism of anticancer agents in man. Ann. N. Y. Acad.Sd., 179:432â€”441,1971.

2. Alimark, M. G. A collaborative study on the acute toxicity testing of severaldrugs. J. Am. Pharm. Assoc., 40: 27-31, 1951.

3. Barr, A. J., Goodnight, J. H., Sail, J. P., Helwig, J. T. A user's guide toSAS76. pp. 206â€”211. Raleigh, N. C.: SAS Institute, Inc., 1976.

4. Blackwell, B. For the first time In man. Clin. Pharmacol. Ther., 13:812-820,1972.

5. Brlndley, C. 0. Methodology of preliminary chemotherapeutic trials in patients with solid tumors. Cancer Chemother. Rep., 32: 27â€”33,1963.

6. Brodie, B. B. Difficulties in extrapolating data on metabolism of drugs fromanimal to man. Clin. Pharmacol. Ther., 3: 374â€”380,1962.

7. Brodle, B. B., Cosmides,G. S., and Rail, D. P. Toxicology and the biomedicalsciences. Science, 148: 1547â€”1554,1965.

8. Bum, J. H., Finney, D. J., and Goodwin, L G. Biological standardization,Ed. 2, p. 3. London: Oxford Univ. Press, 1950.

9. Carter, D. K., Selawry, 0., and Slavik, M. Phase I clinical trials. NatI. CancerInst. Monogr., 45: 75â€”80,1977.

10. Daum, R. J. A revision of two computer programs for probit analysis. Bull.Entomol. Soc. Am., 16: 10-15, 1970.

11. Finney, D. J. Probit analysis. Ed. 3, pp. 100â€”109.Cambridge, England:Cambridge University Press, 1971.

12. Freirelch, E. J, Gehan, E. A., Rail, D. P. Schmidt, L. H., and Skipper, H. E,Quantitative comparison of toxicity of anticancer agents In mouse, rat,hamster, dog, monkey, and man. Cancer Chemother. Rep., 50: 219-244,1966.

13. Geran, R. I., Greenberg, N. H., Macdonald, M. M., Schumacher, A. M., and



1979;39:2204-2210. Cancer Res Anthony M. Guarino, Marcel Rozencweig, Ira Kline, et al. Data with Antineoplastic AgentsAdequacies and Inadequacies in Assessing Murine Toxicity

Updated version

http://cancerres.aacrjournals.org/content/39/6_Part_1/2204

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/39/6_Part_1/2204To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



adequaciesandinadequaciesinassessingmurinetoxicitydatawith ... · drugroutemouse...

Documents