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V a .•~)aKOiOG~( 0k1-01fXCt ;SS%079:-b:3] Cuo.,-gh: i;s'9 0. i;e So(,e:v oi Pnarmuo,og-cd, .r•.: en. ,u~ . enu, .d ^n ,. t,••: ln Vivo Carcinogenesis Testing F. H0k1BC•RGER, M.D. From the Bio•Research Institute. Cambridge, N1A 02141 and the Departmer,t of Patnology, Boston l.'ntverstty. School of Medic,ne• Bostcn. .`1A. A few years ago carcinogenests testing seemed to be a rather straightforward s2:en- tific matter and it was a simple legtslat,~a decision to extrapolate trom animals ';, ^::• mans. The Delaney clause, was quite ar:ept• able to regulators and scientists alike Th.s law mandated that "No additive s`;all -P deemed to be safe if it is found• af•er :• •'s which are'appropriate for the eva!uat:;.1 f the safety of food addittves, to induce _Jacer in man or animal." Since those days the of safety testing has become a confused ,t• tlefield of political optnton. with scientific ' formation being misused to justify dec:s:or,i which have little or no scientific basis. ':u- have to be made for practical and often com• pelling reasons (1, 2). There occurred, for example, the bann:ng of cyclamates, the saccharin embroglto. •'te banning of chloroform from such items as toothpastes. the more recent withdrawal oy the manufacturers of sleeping medicines con• tatntng methapyrilene. and the ongoing :on• troversy about the alleged carctnogcn:c::,r ~f such wtdel~' used medications as femair r. hormones and reserptne. The issue of carctnogentctty testing in . . , and its significance for man has :akrn n difficult and often awesome dimensions N'• cause of a strange and perhaps htsturicJttv unique combination of technologicJl ,a• vances and a pragmatic attitude throuynuut the world which has resulted n pressurs•s n the planning. execution end tnterprrtatcon nt carcinogenesis testing programs. Recent methodological refinernents ,uW permit the detection of trace amounts uf t•.• most complex chemical substances. :nclt. :inq naturally occurring carcincgens such as ,tia• toxins and nitrosamines. and tmpurtt:es :.,n• \:'cr a papcr prescn,cJ a!hc Ffih \nn.,• .i• .• \let..cal 7.T~OS..m •_n,.f(So.' Jf ,tJinc dl . .• . . ,.nc 15 :d'9 tamed in test substances pre%:ousi'. cins;c:- r.red acceptably pure. At the same ::me be• cause of budgetary pressures. I:t;le effort :s e:ng made :o improve !!:e !echnuiuv. , i jn• .mal testing by deselup;ttg ne%% ana -,ecer :pec;es and strains of test an;ma!s for such --sts. Increasingly. regulators and pol:;:c:a^s .:emand appltcable results from scient:sts %% ho nply do not know enoueh ,ts %er about t!-e _Juse of cancer to be of much heip in regula• •ory decision making. To br;dge thts gap :n uur icnowledge. stansttctans, are callea :n to apply :hetr methods to stretch the useab;i:n• of our data to the utmost. This leads :o debai• able conclusions such as eyuat;ng a rar s food :ntake with the number of bottles of some sof: artnk which a human may consume. , ie s:a:• ;sttcfans ignore the differences ;n body sur- face. rate of evaporatton. inaccuracy of water consumption aid food consumption cr,easure• ments in rodents. dtssfmtlarttv of inetaboltsm. gastrointestinal and renal phystolog} :n ra:s and humans. and many other parame!ers :r.a: render such compartsuns ;nappropr:ate .\ definttrr.e Jnswer :u n:s ~;._ .,, ...:. ,nly come when we underst,;nd :`:e pJ!:^.-.;¢e^ "sIs of eertain human c.:ncers 'Jr.:, :-e-: ...,: : ne pussibie to ue~,:se :es!s .n ier:a..^. :in.rr.J.s ..hich c.in pred;et the e,:rc:nuken:c Jc:;..t; af 'rst suhstances related to the ( onrrr•.nduc.r) k mer.h,inism knuwn to funLttun n ^umans. \une uf us is Iikrlv tu li%e to see :h:s ~;j-. .,f ,•nl;ghtenment unlrss thi!:;n%vrnrr.en, nit:a:es hea.•ier i,.upport of nun•mns&on•,,r:ented :e- search. ',.leanwhtlr. we c,in unly try :o Jppi~- 'he knowledge we p:esentlv possess :~, .^i• prove the methuds uf in vtvu testing Thts paper is concerned with :he c!to ce of ippropr:ate Jn;mals fur r~Jrc:nuu-e.n. 'ei:,^,q .:nd with some modtf;rrs "f ;..;:r .n.-Kenes:s t',e fulluwing six pu,tul,ites ...:i --nted. : It :s desiruhle 'o .i(1d thrr ~. .:~ 'u '.^,e CTR IlN 0427 ~0

34 HOMBC'RCER Tox:L:ot.oc:c P{ryo:.~c• I I / 1 animals presently sed for carctno¢en testing (3). 2. Because of a high :nc:dence of sponta• neous tumors. cer:atn rat and mouse strains are inappropriate for carcinogen !esting (4). 3. SuscNptihtltty to carcinogens varies %.,delv among inhrrd strains of mtce. rats .ind hamsters. Susceptible strains may and resistant strains ma_v not detect weak c;,ircinogens (3). 4. Dose per se may alter the incidence of carctnogen•tnducrd tumors of inbred mice 15). 5. Prenatal hormonal :reatment is a factor altering susceptibility 'o carctnogens'tn :.he progeny i6t 6 Carc:no¢ens such as df:ator:ns. nttros- amtnes. pesticides and c:hlortnated hy- drocarbons present in natural rodent d;ets may affect resul:s oi carc:nogenests •est:n¢ (7t. t. The use of multiple spec;es A s:Qntftcant r:sk to humans becomes more likely when se%eral species rather than only one are re• spondtng to an administered substance with !he development of similar neoplasms. Some- ttmes :t is possible to ascertain that a certain laboratory an:mal species and humans metab- olize a class of chemicals in a similar way. In such a case. testtnc in only one species may he aciequate. This is true• with lS•naphthyla- minr. for example. which is a potent bladder c-arr.:no¢en in humans and dogs. hut not in rats Ind mice. Since the latent time for blad• :t,r •,:mor formation in doqs approaches seven },-.irs !he newly discovered ~?•naphthvla- T:r.,,-uscrptthle hamster may oe a cetter :est ,r..:rI l Jt. Spontaneous Tumor Inc,dence :n Cerlcin Rats and Mtce Preclude Their Lse in Carcinogen Testing: Hamsters are better sub• lects for carcinogenicity testing because they have a much lower spontaneous tumor tnct- dence than rats or mice. especially of those neoplasms usually induced by administered carcinogens. There exists a most fortunate paradox. that an animal with hardly any spon- taneous tumors proves suscept:ble to admtn- tstered carcinogens (3. •31 3. Susceptibility to Corcinoeens Varies 'Xidely among Inbred Strains 'I nis :s beuer known in mice and hamsters •han in rats. Suscept:biltty appears to '-Io r...er.ted The select;on of the stra:n mav ae:er-:re ,he uut- come cf !he experiment. i-i% -•r ;: ;•r3:r.s ^ave been de%eloped which are .is ~..,.ep:.bie :o carcinogens tpol}•c}•citc hydrocaroons, as -~ parental strains. and these stra;ns may '_e- come the animals of ct:o:ce for czrc:ncg_er: testing (3). 4. The Importance of Dose• The dose le% et of administered carcinogen may alter the tr.• cidence of induced tumors in inbred mice and their hybrids, according to L. Prehn (5). Th:s observation deserves confirmation and exten- sion to the hamster. the only other species of which sufficient numbers of inbred strains are available. and their suscepttbilel%• to car• ctnogens is known. 5. The Importance of Sex Hormones in :he .1.tother of Test Animals It :s «•ell lcnos.•n tt:a: the two sexes often respond differently tc administered carcinogens. Rust:a and Shue:k t6) have found that the tngest:on of 10 mg ~¢ of diethylstilbestrol on !`te 14th day of pret2• nancy in hamsters alters :he suscept:btl::y : the progeny. The female of.'spr:ng %.•hen ;na• ture become more susceptible to genttal. mam- mary and forestomach tumors :nduced by !he intragastric administration of dtmeth}Iben• zanthracene. In males. no such effect ;s oo- served. This opens new perspectives ;n the controversial field of two-generat;on carc:no• genesis testing. where high .-loses of carc:no- gens are fed to pregnant Tothers and :^e:r progeny for the duration of :aetr I:fe. 6. The Role of Dietary Contaminants: A beginning is being made :n ~!:e use of ser^,- synthetic diets which may :ecuce !he r:sk of carcinogenic contaminant n :he feed 9',. However. our own experience with I:fel:rr;e feeding of one such diet :o a smail numoer : animals has been dtscour.tQ:ng :n that :':e antmals ate less than was -•,• essar•: :o ass::e 2rowth similar to that of -`:s :eze..:cz ~ natural atet. Co%ccs,sir~. A., Ex,.,P:: . -, .. - : ~:- _•, . NEw CARCINOCE•t,•• '•eu~Ee Unttl recently the carc:nuelen,c:ty of c:¢a- rette smoke has never been -7nc:us;•~ ei~ dem- onstrated in a rodent. Hams•ers -,ierate m,ore nicotine by tntraperttone3i ^ ec-:on ! ;ar. either rats or mice. This perr^::s exposure of hamsters to far greater amoe:^:s af ;:Qare:-e smoke than is possible in :^e ither spec:es. Two inbred strains of !tar^s•ers •-%h;ch have been shown to be suscept:-.r •;,..oc.:taneous and intragastric pol%•c•.•...-: `:,:crocaroons have been exposed to sm~ft~ '-I referer,ce ctctarette. A 20 o tnctdence ..;r-.ncma of e larynx was induced ;n i :.n i:~J _nly a'> of the same cancer :n :`e - A :°re::- " ~ um MN 042'e=`'r

/i r Vol. 7. No. 1, 1979 a IN VIVO CARCINOCENESIS TESTING tion of this experiment. with other cigarettes and a more massive exposure. vielded a40 -. incidence of carcinoma of the larynx. histo- logically identical with a similar disease in man, known to be cigarette smoke-related (10). Dose-response experiments with smoke dtlu- tion and with dilution of the tobacco in the cigarettes shows linear dose-effect relation- ships. These types of experiments may be used as a quantitative assay method to ascer- tain the relative carcinogenicity of various cigarette smokes (11). This kind of systamatic approach must be made for any given class of carcinogens to be tested. It is only by such procedures that the field of in vivo carcinogenesis testing can be made truly sc:enttfic and to yield factual in- formation reliable enough to permit a certain degree of cauttous extrapolation to human problems. RErE2E%CE:, .i. Kr:ybtll HF Proper Perspectr.es in cc:rapo, a•.on f Expertmenul Carc:nogenesis Data to riur.:ans F:,oC Technology August 1978. .. Kraybtll HF Proceedings of the 10th Inter•Amer:can Conference on Toxicology and Occupauonai Medt• ctne. Miami. FL. October 1978 7 Homburger F. Adams RA. Soto E.'.'an Dongen CC Susceptibility and resistance to chemical carc:no• gens in inbred Syrian hamsters. In The Syrion Ham• ster n Tcrtcoloay :n t \•nl .2i,if PrnKrraa in F.p, rim- r, .. ". n F Homburqer Ed . S Kdr¢cr :\i; 3,s Nrl. :9'9 pp 215-:-t 4 Homourger F. Russf r _' \° 'Ae.snu:,- .. Chak SP \\cishur¢er F.K Al:nst hanr•: n Cf) i`: Ha~l%ICR mu,e reared under condiUons I tiutl Cuncer Inst 55,1!"-i5 :9'; S. Prehn LM. Lawler EM Rank order of s,rcoma sus ceptibility among mouse strains re%erses ..i:h ::%,, concentrations of carcinogen Science 2W(i390; ix- 110. 1979. 6. Rustia M. Shubik P Effect of transplacental expo sure to DES on carc:noqenic suscepeib ,.••. _.. - postnatal life tn hamster prn¢enp C:rrer ?es press Edwards CS. Fox IC. Pnl c3scro P:•„) if '. MH. Fine DH \:niatile airrosam,r.e ..... _... ..., . . of laboratory animal d:e+s C.:ncer Res :Y :-=- 19'9 8 \'ewberne 10 \1cC,nnrii RG golden hamsttr In Thr 1).nun Hu^+arer -' .qy onu Carc7nogenes.s 3rsrur:^ . .. -s . - , ress in Experimental Turr.or Res•3r:- F.._...... ger. Ed. S Karger AC dasr: %e.+ 'r'... .• ., -1?8 9 3ernfeia P Hcn:bureer F R_ss `erences in the :espor.se f -.,. . . . -• • . 10 c earece smoke nna,a:.on : . - 3](i11141-1157 :974 t0 Homburger F. Soto H-X:thoff I Da.y.:en P-? : P Animal model carc,no^ta oi :he .artir.. - 1-s-< exposed to c garette smoke im I?:r" r-^< I1 8ernfetd P. HomburFer F Soto E. P3. smoke inhaiauon stud es •n inor_a Sy c a- .~-.. I.votl Cancer Inst in press iSeptemoer CTR NN 042~0-~~t-2