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PLAINTIFF'S EXHIBIT IKITZ mtropm H.-I.'asmf ,,, iflkumuIatK.n ... < .-ll in . ,,(,IJPl* 43. ft | fi. VftON*, A. R., Oi.MKN, V, . '.`i3) Sequence ..f , i* and Vagina ..f i|,,. hTM n iif l'-i('M<l>.prfi;nKMc\ A two ..f Ooculuuniata. A,,, / -KRl.AAN. W. P. ,t MaVKII.S V livroxmo mid Ovclie AMP ,,Jj nntii'.K. Wn [`me . AO, .'..Ij .H'tion nf Mammary (VrniiK,mt iir.'d Rttt-s t mVu [1!( ;| 1 ' '"..'tradio!. 17ft. Pnig^ti.p,,,',,! ii.*. .Suture. Loan.. [[K), ;j-(i (oncer (1973) 28. 173 mesotheliomata in rats after inoculation with ASBESTOS AND OTHER MATERIALS .1. WARNER, R. JAERRV .\xo V. TIMBRELL Fmnt the Metliral- Resrarrh QnnnciVs Rne.'tmor.nnitisi.i ((nit. Uamlmtgh Hospital, Renarth. Glamorgan RreWvMl 21 Marrh 1973. Accepted 10 April 1973 Summary.--Four experiments in which SPF Wlstar rats were inoculated Intra pleurally with asbestos or other materials are described. Mesotheliomata were .observed in a considerable proportion of animals with all the samples of asbestos used and with a sample of brucite. A few were produced with synthetic aluminium silicate fibres and single ones with barium sulphate, glass powder and aluminium oxide. The risk of developing a mesothelioma at a given time after injection was approximately proportional to the dose. Of the UICC standard reference samples, crocidolite was the most carcinogenic and removal of the oils by benzene extraction . did not alter the carcinogenicity of these samples. Chemical properties also seem " unlikely to be the main factor producing mesotheliomata but the results support the hypothesis that the finer fibres are the m carcinogenic, and this is additional to (he known aerodynamic advantage which finer fibres have in penetrating to the .-periphery of the lung. A V-L. We report here the results of 4 experl ' ment-s in which asbestos and other test ^.materials were administered to rats by .`^intrapleural inoculation. These experiAments were planned to obtain more infor mation on the carcinogenic effect of .y'asbestos and other materials than could V be obtained from our original 2 experi` ments (Wagner and Berry, 1969). Pre liminary resuits of some of the present experiments were given by Wagner, Berry and Timbrel! (1970), Wagner (1970, 1972). In this paper the complete results are given, with emphasis on the light they throw on the aetiology of mesotheliomata, taking into account the oils and waxes present in asbestos, other chemical charac teristics and the physical characteristics. MATERIALS AND METHODS In all 4 experiments specific pathogen-free (SPF: rata, of the Wistar strain were used. These rats had been bred at the Unit from stocks given to us by imperial ('hemical Industries, Pharmaceutical Division at Aiderlev Edge, Cheshire in 1904 and 1968. The following; materials were used: !. itSFA chrysotile.--A super fine sample obtained from a Canadian mine, and pro duced by water sedimentation separation from grade 7. the most fully milled commer cial product. 2. Grar.idolite.--Prepared from virgin fibre from a mine in the North West- Cape. Both (1) and (2) were from the same samples as used in the earlier experiments (Wagner and Berry. 1969). 8. UICC Standard reference samples.-- .Samples of amosite. anthophvllite. Canadian ehrysotile, Rhodesian chrvsotiie and erocidolite (Timbrell, Gilson and Webster. 1968) prepared following recommendations of I'Union Internationale t'ontre U Cancer (UICC). 4. Benzene-extracted UWC Standard reference samples.--Samples of (3) which had been repeatedly extracted for H4 hours by hot benzene using a Soxhlet apparatus to remove oils and other benzene-soluble sub stances. After extraction the benzene was 174 J. C. WAGNER, U. BERRY AND V. TIMBRELL first allowed to evaporate naturally and finally the samples were warmed to 80'C for 24 hours to remove any remaining benzene. After this treatment the samples were tested for the presence of any residual benzene by extracting boat portions witli cyclohexane and examining the solutions by means of ultraviolet spectrophotometry; no benzene was detected in these solutions. 5. Canadinn chrysotile.it.--iSamplea from 8 mines (A. B, ... H) in Canada. These were the same samples used to prepare the UICC standard reference sample of Canadian ehrvsotile (Timbrell and Rendall, 1971) but were milled for our purpose, more finely than the reference sample. (5. Brucite..--A specimen of bracito, which however also contained ehrvsotile. This specimen was from Canadian mine H and consisted of long coarse brownish fibres above 50 cm in length. The sample was milled to respirable particle' size,' 7. Barium sulphate---Used as a control. This was prepared in the laboratory by the addition of sulphuric acid to barium chloride solution. x. ttaline.--Sterile physiological salute was also used as a control. 9. Ceramic .fibre---A synthetic aluminium silicate fibre. This fibre was prepared for experimental use by grinding in a ceramic ball mill and extracting the respirable traction by settlement in air. The fibre diameters were between 0-5 and 1 pm. 10. Fibreglass.--A borosilicate. The nom inal diameters of the fibres were between 1-5 and 2-5 pm but in fact only 30% were within this range, the range extending to 7 /tin. The sample was prepared by em bedding the fibres in water soluble wax. chopping in a microtome and washing awav the wax. Over 60% of the fibres were longer than 20 pm. 11. Glass powder.--A borosilicate all in the respirable range (less than 8 pm pro jected area diameter). 12. Aluminium oxide.--A non-fibrous mat erial all in the respirable range {less than Jo pm projected area diameter). 13. SFA rhrysotile (Second sample).--A sample from the same, mine and prepared similarly to (I), but taken several years later. Experiment 1--Varying dose There were 5 doses. 0-5, 1. 2. 4 and 8 mg per rat, of SFA chrysotile and crocidolite. There were about 12 rats per dose per (|Ust and inoculation was during March 15165, Experiment 2.---Canadian chrysolites The experimental materials were ", of the s (`anadian chrysotile samples. SFA ehrvsotile and saline control. The dose was 20 mg per rat. There were 16 rats for each Canadian sample, 32 for SFA chrysotile and 48 control* and inoculation was during December 1966. Experiment 3--UICC, samples and Canadian chrysolites The materials used were the 5 UICC reference samples both in the normal and nil-free forms, the X Canadian chrysotile samples, brucite and barium sulphate and saline controls. The dose was 20 rag per rat. There were 24 rats for each of the Canadian samples and 32 for each of the other treat ments. Inoculation took place between' November 1967'and Februar`y 1968. 7 Experiment 4.-- Various dusts : The materials injected were ceramic fibre* fibreglass, glass powder, aluminium oxide, .SFA chrysotile and also the second sample of' SFA chrysotile. The dose was 20 mg per rat and there were up to 36 rats per treatment (because of a shortage of animals it was not possible to allocate 36 to all treatments and in addition inoculation fatalities could not ' lie replaced). Inoculation took place in June and July 1969. For each experiment animals were allo cated at random to treatments. The age of the rats at inoculation was ahout 6 weeks for Experiments 1. and 3 and 13 weeks for Experiments 2 and 4. (n Experiment 1 and 3 there were equal numbers of male and female rats, whilst- in Experiment 2 there wert 3 times as many females as males, and in Experiment 4 there were twice as many males as females. Methods The experimental materials were made up in a suspension of physiological saline with * concentration of 56 rag/ml for Experiment* 2, 3 and 4 and for Experiment l the eon* centration was such that the required do** would be present in 0-4 mi of suspensionThe rats were anaesthetized with ether and * needle attached to a two-way rap "as then introduced into the right axilla at tin- love* ot the second nipple. One arm of the ' .io-"*y tap was attached to a capillary mi -'ineW. M Ext m .mull gave lie in i ' iciit-d the i'.. "id >il it U .i Mid Bern hun v rats were .jiiTi.ii niiir. Tlies liraml nf aiiloclai libitum. Each car liicil ur appeared t iii'iviipsy exammut. Ini- a lew a Inch lla flic results liav iiimiIcI given by Pi *. .-Ini for cxperimei Uaguer. (!I6!I|. F in the appendix, ar tins method of ana lie estimated for ea mid I hat this eonst as (!' carcinogei -me -lex sitmn c\|m nf each infoi.nation oil tl which developed times alter mociili tlicliimiata m-curre nt fsiiinari<m eiin Miller .-anses, ehai mortality her ween do not atfect the n do systematic ditto between different resulting from the and 4 being older I and 3. affect cot meats U'liere dignities me e iUv based n ,h'11' t likelih- i I'liere were I ''III V-' it lie % \ elirv-'e! ||(. % ' rbi v-lillli. W \ i-lu-v-i.tilr. '* ' `'bn -..tile 1 r,".|r|.,|,|e 1 fl-'lil..In,. [T dose , .March hin.< hri'rt c/in/snh'tex I materials were 7 of d,,. .-Slnplcs, SKA cln-ys, The dose was 20 inu jkt rats lor each ('auatliHii hrvsotile and 4-K omit mk during; December I'.lfiii. ' >vwnpiex mul ('anruliau f I i re-d were the ft t'Hc oth in the normal ,,m( S Canadian ehrysuiilc .(l liarium sulpha*. aiul dose was '2n me i it tor each ot' the < T h ui each of' the other ..c.it. M took place hetaccri Fehraary 1 !)l)8. ''nan tllists ected were ceramic films wder, aluminium oxide, also the second sample ot' e dose was 20 mg per rat to 36 rats per treatment oxe of animals it uas not 36 to all treatments and tion fataiities could not ifl^n took place in dune meat animals were alioeatmenfa. The out. the.. . was about 6 wet for l 3 and 13 week- tor f. In Experiments 1 and I numbers of male atul ; i Experiment 2 there, were 'males as males, ami in v were twice as many tl materials were made up hysioiogieal saline with a i mg/ml for Experiments Experiment I the eon- It that the required do-"' m 0-4 ml of suspension. tUtilized with ether mul a a two-way tap wa' Ic-n right- axilla at the h i >6 One. arm of the !"< i a capillary maiiuin.-ier. l . I i MBSOTHELIOMATA IN HATS At'TKR INOCULATION WITH A.s K,ST< io 17.', whi<" aave a negative reading when the cached the pleural cavity. Details of {||t, a tho'd of inoculation were given by SVaguci' and Berry (11)611). Following injee- t,(1(r(hc rats wore caged in fours isolated in a spceial unit. They were fed on a proprietary brand of autoclaved cubes, and water ud libitum. Each rat was allowed to live until it died of appeared to he. distressed and a full necropsy examination was carried out. except for a few which had beeu cannibalized. The results have, been analysed using the model given by Bike (1966) and shown to he valid for experiments of this type fBerry and Wagner. 1969). Fuller details are given in in the appendix, audit need only be noted that this method of analysis allows a constant r. to be e-,1 .mated for each of the treatment groups and t; if this constant, which we will refer to as tin " carcinogenicity factor ", serves as a single index summarizing die mesothelioma experience of each group. It combines the information on the proportion of animals which developed a mesothelioma with the times after inoculation at- which the meso- t.heliomata occurred. Also, since the method of estimation eliminates mortality due to other causes, chance variations in natural mortality hetween different treatment groups do not affect the treatment comparisons, nor do systematic differences in natural mortality between different experiments, such as that resulting from the animals in Experiments 2 and 4 being older than those in Experiments 1 and 3, affect comparisons between experi- Tnent', ' Y\ nere significance levels are quoted they are. usually based on the eAt-squared approxi mation to a likelihood-ratio test. RESULTS There were 13 rata for which histo logical examination was nor possible, leaving 1112 rats included in (he results. The predominant rinding was that a high proportion of most- asbestos (reared groups developed mesotheliomata and the results will be given mainly in terms of die number of mesotheliomata and the time when they occurred. Sonic details nf the results are given in Tables (-IW A total of 3x6 mesotheliomata occurred hut the histological features of these tu muni's will not In* described here as there is nothing to add to the features descriheii for the, original experiments (Wagner and Berry, 1969). Also, in the presentation and analysis of the results, no account liasbeen taken of the sex of the rats. The original experiments show males and females equally likely to develop a meso thelioma, and the present experiments confirm this. Experiment I There is a relationship between the number of mesotheliomata and the dose ibr both SFA chrysotile and crocidolite. This implies that the carcinogenicity is related to dose (d) and we considered this relationship in the form of the carcino genicity factor being proportional to a power of dose, i.e. e --- hdv where h and p are constants. The power p was estimated separately for each dust, giving -73 for ohrvsotile and (Mi6 for crocidolite. Be cause of the small number of mesur.heliomata, however, these estimates are nor very precise and the approximate 96;, limits are n-3-1'3 and u-2-1-9 for ejhrvsotile and crocidolite respectively. There SKA chrysotile SKA chryuotiio SKA ohrydonfc SKA ohrysotilo SKA ohryaotilo Crno' inlite Crnr'-ioUto Croc -lolito CrmMtlolito u' ft mg 1 me me 4 msc S mu (i'A mp 1 mp 2 mu 1 me S mu Table .--Experiment 1 Result* Number m" rats Number with with histology a mtwjtht'lumiu Survival time* (day*; ot' drat uutrfut.hc'Uorrm 12 f .* 1 1 1 :i <Wft 12 A U!f 12 l 4-70 12 s mi 11 I 12 l - 12 13 i HIT II "HU Mwm 'iifviviil i<mvs] 7S4 72*J ti(i4 7 h2 <W2 M)<1 7<iO .<( NlH riSU I 7fi .1. 1`. WAONER. O. HERRV AND V. TIMBRWDL <'rmadiati ohrvKotiln A ('muuiian chryautil U < `tiriadiati uhryxntifo C fttmtdian clirysoulf* l> I'timuiian ctirybutdc E i 'tinutiuwi ehrvaotilo F t'Hmulmn hrvftOt.iln H SFA nhryantii Control Table i'i.- -Experiment 2 Results Number nf rats with histology III III (ti HI 16 III 16 32 48 Number with a mt'huthnlioma s Hi o in * HI 4 22 (l Survival time (duvsl of first mesothelioma 416 416 ( mi 1115 421 364 376 -- Mean survival (day 642 594 752 624 673 61!) 652 553 728 Table III.--Experiment 3 Results Number of rata with Alatorial histology / -fCC Humpies Amoftita 32 Amosite benzcnoxtr&cted 32 Anthophyllite Anthophyllite benzene-extracted 32 JV2 ' Chrysotile (Canadian) 32 Chrvsotilo (Canadian) henzeno-** xtracted 32 Chrysotile (Rhodefcian) 31 Chrysotile UihudoKian) benzono- extracted Orocidolite 32 32 Crocidolit benzeiuj**\t ractod 3(1 Canadian chrysotile A Canadian chrysotile B 2444 Canadian chrysofile (! 24 Canadian chrysotile U 23 Canadian chrysottlo E 21 Canadian chrysotile F 21 Canadian chry*wrile O 23 Canadian chrysotile F{ 23 Briteil* 32 Barium Miiphata 311 Saline control 32 Number with a mesothelioma 12 11 s. 14 1(1 '.) 7 5 19 HI 14 II `1 12 !l 13 16 14 IS 1 u Survival time (days) of first mesothelioma 377 51)0 . 498 533 f,41 632 .502 659 5X6 4HK 488 437 1611 534 489 484 576 129 55* 136 -- Mean survival (days) 716 718 761 728 747 753 693 686 682 657 712 636 717 669 660 075 659 663 685 783 sl8 Oramic fibre FihrcglAHH Olasx powder Aluminium oxide SFA chrysotile SKA chrysotile (2nd Mitnple) Table IV.-- Experiment 4 Results Number of rats with histology 31 35 35 35 3fi 32 Number with ft mesothelioma 3 (i 1 t 23 21 Survival rime idaysi of first mesothelioma 743 - 516 646 325 382 Mean survival (day. 736 774 751 710 568 639 arc. some theoretical grounds for choosing p to ho. an integer and therefore p was raken as unity. The values of (ho carcinogenicity factor adjusted to a dose of -i.i mg were then 4-10 >; in-8 for chrvsotile and 1-71) 10-9 for orocidolite. Experiments 2. 3 and 4 Comparing (irst the effects of the separate Canadian samples (Table V), (here is considerable variation between Experiments 2 and 3 and this is m: mly because, of the small number of anim: - in MEritr; Tmu.k V.--l'Jx (> UP) ft .M.mltuu rhrv^milo. `.inmhn-n chrv.sutilc ``utuithitn ohrv^otilo 'jirmdiau chrysotile `"diuulian ohrvsotilo Omudinn chrvsonli* i'imuth&n (ihry&trtle ('mmdian ohrysouie SKA thrvsofiio M*'A chrysotile (2nd Hrurtr.e Human sulphate < 'name fibre powder Mumimum oxide ciich group. T 'l1-'",, more card] - ' n in Experi i- >t signifiean li.is the lowest experiment and < least carcinogen from this no i samples were d have been anal (Holmes, Morga Morgan and Cra \'l the results of together with fl obtained by ooml I'be correlation carcinogenicity f iiw <.iU are -- <! ; ium, (M)4 . --0-3H to i"1 manganese, iicanf and it is "\ainination of tl ''mpie C that th this sample is not "f any of these in Turning now 'Hmples (Table \ leant differences i'cir/.one-oxrraoteo ,fi mesothelioma i'ciizene-extracterl t,u' untreated sa H" most earcino M KSOTHK LiO M ATA IN RATS AKTKR I NO(*l NATION WITH ASUKSTOS 177 |' .|,e V.--RdiwrtPS nf (.'arcinutiaiin'hf others in order atnosile. atitliophyllite. y,, ,- (/, Ml9) for Kxpmtnvrihi 2. 3 and 4 Canadian chrysotile and Rhodesian chry iir*r Moan -nrvivnl f(In\ >.j Experiment 1 3 4 sotile. The difference between the two samples of chrysotile is not significant. Holmes / <//. (19711 also carried out 042 594 7irj 024 573 HIM 6U2 553 72* \al time i . if tirsr. 'Iw-lioma Mortt. 'UrviN (`iuukIisk chrysotile A I -25 1 211 -- chemical analyses mi the UK'C reference Caiieultiiu ehrysutilo H Cmirtdian chrysotile (.! Crtiiaiiiaa chrysotile D 2-23 1-1(1 0-08 n-117 2-31) 1-23 -- - -hiu))les. There were very large differ ences between the samples in the amount Ciiimilian ehrysotile K 1 -89 (|.`(7 - of the different, metals present and it is i ('muulian chrysotile F Crtiijulmii chrysotile 4 1 -!m 1 -4(1 2-ll!l - clear that these hear no relation to the Ciiiiadiaii ehrvsotile H 1 111 I-S4 - carcinogenicity. SKA chrysotile SKA chrysotile (2nd sample) Urueitc 4-72 - 2 xS -- - 2 2x ... 1-21 -- The sample of brucite proved .is carcinogenic as the Canadian .-.ampies of Marinin nulphuti- - O'04 -- chrysotile (Table V). Non-asbestos (Vt-Hinic tibre tiln>- powder { .Uiimniiiim oxide .- -- it -1 ii -- ii 14 materials which produced the occasional _ (MIS mesothelioma were ceramic fibre. Imrium sulphate, giass powder and aluminium eacr. group. There was overall about oxide. 477 710 V.lil 7tK Hix 701 .133 728 .741 747 753 .7112 693 US!) 080 7KU 082 WS 657 (XX 712 more carcinogenicity in Experiment 2 than in Experiment 3 but the difference is not significant (P > <> 1). Sample has the lowest carcinogenicity in each experiment and overall is significantly the least carcinogenic (P < iM)5) but apart from this no differences between the samples were detected. These samples The second sample of SFA chrysotile proved similar in carcinogenic effect in (he original sample. ntscusstox The application of the test materials by intrapleural inoculation may he. criti cized as unrealistic in comparison with 137 tint 734 429 .702 436 . 036 717 009 OHO 075 059 063 0k 783 818 have been analysed for certain metals (Holmes, Morgan and Sandalis, 1971: Morgan and Oralley. 1973) and in Table VI the results of these analyses are shown, together with the carcinogenicity factor obtained by combining the 3 experiments. The correlation coefficients between the carcinogenicity factor and the different- human exposure, about which the animal experiments are intended to provide relevant information. Nevertheless, this type of experiment lias an important part to play. With an inhalation experi ment. which provides a realistic route of entry of the test material, there net* 2 factors involved. First, the penetration metals are --0-13 for iron. for of dust through the airways and alveoli ( urrm 4` first Immu Moan survival > chromium, 0-04 for cobalt, -0-02 for will differ with different samples of dust I nickel. --0-39 for scandium and --u-()4 (Timbrell. 19H5). The second factor is the / for manganese. None of these is signi effect of the dust, given that it- has reached ficant and it is reasonably clear from the pleura. In inoculation experiments l 736 774 77.1 7UJ .768 o2o examination of the chemical properties of sample C- that the low carcinogenicity of this sample is not because of a low content of any of these metals. Turning now to the UTCO reference only the second factor is relevant anti hence these experiments arc simpler to interpret. This makes intrapleural inocu lation a more suitable method for the investigation of questions such as whether samples (Table VT.I), there are no signi extraction of the oils alters the carcino / 4 I the effects ot' tile i samples (/Table ' i. Iile variation bet > I 3 and this is nv II (lumber nf animat-- <<i ficant differences between the normal and henzene-extracted samples, and overall S3 nesotheiiomata occurred with the bercene-extracted samples and oH with the untreated samples. Crocidolite was the most carcinogenic sample with the genicity of an asbestos sample. The two types of experiment supplement one another and we will he reporting separ ately on 2 experiments in which rats were exposed to dust clouds of the I'FCr reference samples. ITS {. WAGNER. 1.1. BURRY AND V. TIMURfOU. 'fhe varying dose experiment pave results which indicated that the risk of developing a mesothelioma at a given time after injection was proportional to the dose. This form of dose relationship was also found, by Pike and Doll (1965) for lung cancer and smoking in man whereas Lee and O'Neill (1971) showed that after repeated applications of benzopyrene to the hacks of mice the incidence rate of tumours was proportional to the square of the <lose. The carcinogenicity of the iSFA chry- sotile sample was similar in Experiments 1 and 2, after adjusting the former to a dose of -J() mg, and in Experiment 4 was lower but not significantly so. fn all these 3 experiments the carcinogenicity of the SFA chrysotile was significantly greater than in the earlier experiment (Wagner and Berry, 1999) when the estimate of the carcinogenicity factor was L-68 x 10~. The erocidolite was also more carcinogenic in Experiment I than iu the earlier experi- tiient (c -- M6 L<>--") hut not signi ficantly so. These differences could he the result of a change In susceptibility of the rats or of a change in the dust during storage. " The suggestion that natural oils and waxes (Harington. 1962), or contaminating oils from the preparation of the fibre (Harington and Roe, 1995; Roe, Walters and Harington. 1996) or from plastic storage bags (Commins and (fibbs, 1969) might contribute to the carcinogenicity of asbestos receives no support from our present experiments, which is in agree ment with our original experiments (Wagner and Berry. 1969) when removal of the oils from the erocidolite sample resulted iti no detectable change in carcinogenicity. Harington and Roe (1965) also ad vanced the possibility that the presence of trace metals might be relevant to the carcinogenicity of a.-. -os. [n our experi ments with the Canadian samples the carcinogenicity was not related to the content of iron, chromium, cobalt, nickel, scandium or manganese. Also, the fact that all the types of asbestos, having very different chemical compositions, produce mesotheliomata makes it unlikely thar die carcinogenicity of asbestos could be me to chemical properties. Our experiments offer soma evidence that the development of mesotheliomata is associated witli (he presence of fine fibrous material within the pleural cavity. First. UICC Canadian chrysotile is a mixture of batches of material from s Canadian mines, and the separate samples used were taken from the same hatches (Timbrell and Rendall. 1971). The main difference iu the subsequent preparation of the material was that the separate Canadian samples were ground more finely than the composite UICC sample. Comparing Tables V and VII, the carcuio- . . genicities of all the separate Canadian 1 | samples were greater than that of the ' UICC Canadian chrysotile. Also, the samples of SFA chrysotile were from mine T). These were superfine samples and resulted in a very high carcinogenicity. It should be noted that of the Canadian samples the one with the lowest carcino genicity (C) in both experiments was from a mine in British Columbia whilst the others were from 7 mines in the Quebec area. However, sample C could not be distinguished from the other samples by its size distribution. A full quantitative analysis of our experimental results will only be possible when techniques are available for -omplete size characterization of t he ex peri- j mental materials, both before injection and present in the lungs at postmortem. iSueh techniques to determine the muss and the diameter and length distributions of the particles are being developed. However, even with the characterization methods at present- available, a relation ship emerges between the observed in cidence of mesotheliomata and the physical factors. A further factor that must be faken into account is the tendency of chrysotile ( fibres to fragment longitudinally int tine fibrils in lung fluids, the degree of ag- j M KSU'I Taki.e VI ' urn <i O B H K 'I'aisle vn.- /<;* Fm'tnr < 11 Sitw [j(px in Fix \l* > , 'hvllitc > "--rlr (t'anniliai ` ' . it* iRhoflt'sui 1 i.lift' mentation and fibres and fibril* tile precise ph; conditions. Amj on t he. other ii fibre-diameter d appear to retain I'.tlll ey and W'ag 'I'o illustrate ron mierog 111- * ,:als are | I f their 1*'* a.mi us give -if..II consider . f Ilf IX that are les, and also greate! The non-ciirysot' M* if`l*ed first. Iu for Urn* epoc' umosite (Fig. - (r itr. u. ceramic fibre (Fig. S) it is of significant ' tlccre using carein. 1 m* glass fibre, fi Ii 1 >rf. bur t|ie ina III:. Or Jos. havilii: ly mf^siliuns. pitiiiucc s it unlikely that lu. hestos could lie <lu,. offer sonic evidence it. of mesotheliomatu i lie presence of tine in the pleural cavity, liaii chrysotile is of material from s \ the separate samples on the same hatches ill. 1971). The main bsef|uent prepari-'oti is that, the se( mwere ground niposite r.TTCC sample, and VII. the earcinoe separate Canadian er than that of the hrvsotile. Also, the rsotile were from mine aperfine samples anrl gh carcLnogenicity. It hat of the Canadian th. the lowest carcino^Hjeriments was from ^^imbui whilst the ^Bies in the Quebec imple C could not he the oflier- sample- Cm.. itive analysis of mr s will only he possible ire available for coni' rizat-ion of the experihoth before injection lungs at postmortem, o determine the mass <nd length distributions are being developed, th the characterization it available, a relation- veen the observed iniiomata and the physical | I t f ir that must be f :cu tendency of chry i1, longitudinally intc w ds. "the degree of twin- MESOTHELIOMATA IN RATS AFTER INOCULATION WITH ASBESTOS 171/ Table VI.--< 'arcinoijenicitij and Chemical Analysis of Canadian dam pm* Experiments - and .1 ('ombined i'mnuKi-itii`irv trim t'hruimum I'uLoU Nickel snuii/mm Maimanc-i .Slllll|)ll- factor i m;,) part*/1 [iarti( tun purrs/10 part-/ (0 puff ^ i; D K B H E A t' 2-37 L - 70 1 -!l :jsn r.:t (40(f 7 .1 420 Ml *u?o 7X fii.W <Mi mm to|u a-H 730 7.H 1`juu i - Mu l :.<( 11-4 :xn III! *2100 4-1 .ixu 1 .74 :t-2 4XO 12 .7.10 .1 fi .7:10 l -:i;t .7 -i 43-1 .77 Sl)f> .7-4 hill 1 - C2 ti-ii7 4-S .11.1 HU 1 1.70 7-0 \40 oil (2oo HU [SUU 12-0 420 Table Vfl.--Estimates of CarcitUHjeuicil!/ Factor ( - (rtyi for vice Reference Samples in Experiment 3 Normal form Benzeneextracted form Amo-'ic Aiitliopliyllite Chrysotiln (Batiaciiaui ChrvsoWe (Rhodoamn) Crociilolito n-iih lied mSO <1-44 1 *4-fi n*mi M-77 n-4u-3! l-s7 mentation and lienee the number of fibres and fibrils produced depending on the precise physical and physiological conditions. Amphibole types of asbestos, on the other hand, have characteristic fibre-diameter distributions which they appear to retain in lung tissue (Timbrell, .Poohv and Wagner. 1970). To illustrate this relationship, the electron micrographs of some of the materials are presented in decreasing order of their carcinogenicity (Fig. L-R). For reasons given by Timbrell (L973), we shall consider as " significant " fibres those that are less than 0*5 ,m in diameter and also greater than 10 //m in length. The non-ehrvsotile materials will be con sidered first, in the electron micrographs for UICC! croc-idolite (Fig. 3), UICC amosite (Fig. 4-), ITTCO anthophyllite (Fig. 5), ceramic fibre (Fig. 7) and glass fibre (Fig. S) it is evident that the number of significant" fibres decreases with deer rasing carcinogenicity of the materials. The glass fibre, for instance, contains long fibres but the majority of these are thicker than u-5 /im. Un the other hand, whereas a high proportion by weight of the brueitc consists of large fibres, rbere are also present a number of very fine long fibrils.. It is difficult to compare chrysorile samples (Fig. I and 0) with other types of material on this basis. But even so. rlie relative positions of the chrysotiles in the classifi cation hy carcinogenicity appears in correspond with the number of " signifi cant " fibres present. For example, al though the SFA ohrysotile <Fig. 1) contains a high proportion by weight of non-fibrous particles, even before injection the fibres were in a highly dispersed state. The enormous number of fibres that complete fragmentation of chrvsotile can produce will be clear from the illustration that a single fibre may fragment into 1000 fibrils. The fact (hat this SFA sample was the most carcinogenic of all the materials used corresponds to its highly dispersed state- and its high content of " significant " fibres. The above theory has been examined further using the results of Stanton anti Wrench (1972b Their experiments were similar to ours and they used 17 samples, including several materials (UICC samples and glass fibres) after partial pulveriza tion. They analysed their results by iliscounting submieroseopic fibrils and converting all longer fibres into micro fibres of standard size (I-25 3-7n ;nni on the assumption that fragmentation of both glass and asbestos oei-urred in eim. The numbers of mierofibres were then compared with the carcinogenicity of the materials. At first sight their results seem to conflict with our findings. But, | fU'l C. WAGNER. (.!. BERRY AND V. TrMBRELL MESOT / .jll.r u v u .- ( l.V77. --. x v . i. im . - i. - f is -t I 'l i V ( 1S7.7). MESOTHELIOMATA IN RATS AFTER INOCULATION WITH ASBESTOS i:t cirt'amiv.' ti* " I K4 J. C. WAGNER. G. BERRY AND V. TIMBRELL ME.SOTHE when. their materials were assessed in the manner used for our own samples, the order obtained by classification of the materials according to the number of " significant " fibres was in good agree ment with the reported order of carcino genicity. The smaller number of mesotheliomata observer! from pulverized materials did not correlate with the estimated total number of particles that this pulverization would produce, but did correlate with the estimated number of significant '' fibres. dtanton and Wrench i, 1972) also pro ducer! mesotheliomata with very line fibreglass. Mesotheliomata were not pro duced by our sample of fibreglass but they were by synthetic aluminium silicate fibre, which was finer than our glass fibre (Fig. 7 and 8). Again, the apparent con tradiction is explicable in terms of phy sical characterizations. The 2 samples of fibregiass can only be compared using Light microscope size data; in our sample u/5% of the fibres had diameters exceeding 2-5 /an compared with less than 10% of .Stanton and Wrench's sample. Although a direct association between physical factors and the development of mesotheliomata has not been demon strated, these characteristics appear to be the relevant properties. If the finer fibres arc the more carcinogenic when applied to the pleura then, since the finer fibres are also able to penetrate to the pleura more easily after inhalation, these 2 factors would combine together to give the finer fibres more relative importance than, even the aerodynamic differences would suggest. Clearly, the experiments described in this paper have involved a large amount of daily effort over a number of years and we are grateful to all our colleagues who have contributed to (his. We are also grateful to Dr B. T. Commins of the MB.C Air Pollution Unit who prepared the benzene-extracted samples and the barium sulphate sample. REFERENCES Hurry. G. & Waunbk. J. {iimsi) The Application of a Mathematical Model Describing the Tirm-i ,,f Oreurrence of Mesothelioma* ui Rats foil- wine Inoculation with Asbestos. Hr../. Cancer. 21 . :,s`2. Oomkin's, IS. T. it Gibbs. G. IV. (1009) Contt.-nina- twsr Organic Material in Asbestos., fir. ./, r ,,,,etr 23.358. ' Harington. .1. 8. (1962) Occurrence of Oils Con i . t. tint* following I ' ' -r. 23, r,ii7 J 1'. AGKlty. iiolionm, ,,i Kilt, ; oiiim ..i A.-t*. ' lutemat. iwoni\ II 1 Shapiro. <V[;t i'l`" p. 2Ki, ` taining 3 : J-Benzpyrono anil Related Sub. stances in Asbestos. Xature, Land., 193, 43. Harington. J. 8. Si Roe. F. .1. C. liUtSS) Studies of CarciimgeneHm of Asbestos Fibres and their APP Natural Oils. .inn. A'. Y. Acad. Set., 132. 4!IH. Holmes, A.. Morgan. A. & Sanualls, F. J. (1071) Determination of Iron. Chromium. Cobalt, Nickoi and Scandium in Asbestos by Neutron Activation The t'isults havi * the model given by ui In* valid for exp Analysis. Am. imluxlr. Hyg. Ass../., 32, 281. Lee. P. N. & O'Neill. J. A. (1971) The Effect of Time and Dose Applied mi Tumour Incidenco Kate in Benzopyrene Skin Painting Experiments. Hr. J. Caitrrr, 25, 759. Morgan, A, & Cralley, L. .1. 11973) Chemical Characteristics of Asbestos and Associated Trace Elements. In fine. Conf. Biological Effect* of Aubcutoe, Lyon. 2-5 October, 1972. In print. Pike. At. 0. (196(1) A Method of Analysis of a Vi+ain, , Class of Experiments m Carcinogenesis., Bio-.. i , ; f , (Kerry and Wagm .jweiflc deatii rate < n mc.suilieliorna t <: taken us mnslants. These ostium red separately eaci neriment bu the uiiates make metric*. 22. 1+2. Pike, M. 0. <fe Doll. R. (L965) Age at Onset of Lung Cancer: Significance in Relation to Effect of Smoking. Lancet, i, 665. the " i miual uxperin 417 mesotheliomata vinilil lie taken as 3 Roe. F. J. Walters, At. A. A Harinoton. J. S. (1966) Tumour Initiation by Natural and Conlaminating Asbestos Oils. /id. J. Cancer, 1, 491. Stanton, M. F. & Wrench. 0. (1972) Mechanisms (hut i/\ the lapse pe tlieliomata '?<itrrec menr.; in particular of Mesothelioma Induction with Asbestos and Fibrous Glass. ./. natn. Cancer [nut.. 48. 797. TtMBRELL, V. (1965) The Inhalation of Fibrous Dusts. Ann. .V. Y. Acad. Bd.. 132. 255. umositp was found lunger than rhat iToeiilolite but tiff Timbrell. V, (1973) Physical Factors as Aetiological mesotheiioma occurr Mechanisms, In Proc. Conf. Biological Effect* of AsbcHtae. Lyon. 2-5 October, 1972. fn print. only :i!ih days. For Timbrell. V., Gilson, J. 0. Sc Webster. I. (1968) UTCC Standard Reference Samples of Asbestos. Int../. Cancer, 3. 406. TtMBRELL, V.. POOLEV. F. & WAGNER. J. ' (197) Characteristics of Respirable Ashesti.. Fibre*. In Pneumoconiosis. Proc. Internal. Com. .fohan- lie fin theimprec iimi i is no stro nesburg, 1969. Ed. H. A. Shapiro. Cape Towns on all i use a c>. Oxford University Press, p. 120. Timbbell. V. & Kenuall. R. E. G. (1971) l'repsra. tion of the UICC .Standard Reference Sample* "f : I here no evidcnct iieiiiu dependent on Asbestos. Powder Technol.. 5, 279. lhi' analysis the bus Wagner. 9. C. (19701 The Pathogenesis ofTuumum following iho Intrapleural Injection of A-busKi* and Silica. In Morphology of Erpertaiental Imsec 1 nit all our those are ir -- 270 .* Hespiratorv Carcinogenexix. Proc. Conf. Gatlin1 burg 13-16 .Mav 1970. Ed. P. Nettesheim, M. G. Hanna Jr. and 4. W. Deathenige Jr. with all the data th ten- precise; for ex; U.S. Atomic Energy Commission Symposium Soriea No. 21. p. 347. Wagner, J. C. (1972) The Significance.* if m Tissue. In Recent Result* i/t Cancer Reseafcth Vol, 39. Current Problems In the of Cancer und Lymphoma*. Bd. B. < nmdnuin and H. TuUniue. Berlin: Springer-WrW Waunbr, J. 0. & Berry, G. (1969) -Me- <""nU MESOTHELIOMATA IN RATS AFTER INOCULATION WITH ASBESTOS I xr> fi'ftl Tim A(I,,,,, "i^Pwjribuiit (hi* "I'm.......... liumurf in Kate t < u,u i,,, Br../. Camtr, 23. . tVV. t f liOy t O'UtHHuiui II Aabc^fllH. Br. .1, t r i rJramronnv -a- Oil, f,,,, mi ttiift Ri-lrtti`<l Sijh Mature. Lomt.. 193. +3. . F. )>'.< lyrt'i) Stmlii-H t.|' .bcsiiw Filin'!' tiiul t tu-ir . Y. Acad. Bci.. 132. . A S\NUAtxa. F. .1. i In; . Chromium. Cobalt. \'te|;,.| ins l>y Neutron Acti\-nti..i, Huy. Aw. !.. 32. 2a I. .1. A. 11971) The Eti',.,.1 twd. on Tumour l i..||(... skin l'aintim< Ex|- ui. v. I.. 3. (1973) ... ..t irntoa and Atwnetttteii tun-,- Conf. Bialtiyio.il Kill <t* .if October. 1972. In print, t hod ot" Analysts ui a l '--n .wit , in Carcmoe;em>ei!<. Bn>. 4 (t < i \ i f 11003) Age at. t Inaet. nf l.tiiii: in Relation to KfTcct ..I rtrtfl. ,, M. A. & Harington, ). S lUtrn by Natural and ('mi. Oils. lilt. ). Cancer, l. 491. (inch, 0. (1972) Mechamimt" (action with Anhosto* ami it^^Aancrr Last.. 4S, 797. J^^Knhalation of l-'iliron-. 132, 25.-,. ts^mal Factors as Aetiolnctritl it. Conf. Biological Kfi'-rt* at . October, 1972. In' at. ,1. C. & Webktek. 1 ..rence Samples of .. I, . F. & Wagner. .1. C. , 197.11 Respirable Asbesto-. Film-.. Proc. Internal. Conf. .Inhtitt. K. A. Sha[nro. t'a|ic Timn: 'rcss. p. 129. ini.. 14. E. U. t La"l) I'rcimiii audard Refe.ruuce Sampl." "I r.i-Mnoi., 5, 279. The Pathoaenesm ul'Tuuimn- pleurul Injection of Anbe-t'iMorphology of Experimental .seiion*. Croc. Conf. Oatlm ' 1970. Ed. P. Netti'-lu-mi. and .f. IV'- Oeatheraac -Irv^jy Commission Symti'i'Ui"1 .47. . i The Siaindcance of .\-li--i-" .. ReauCta in Cancer -aril.. Problem* in the Kpt ' phomae. Ed. E. ierlui: Springer-V'erlu. A"t ' tuty, 0. (1909) ...................... . t ii i t< following Inoculation with A-la-stos. iir < 'aimer. 23, 007. iVn.Ni C-. Berry, O. & Ti.hbrell. V. (1970) jli'.jittn-ltomitH m Rats Fullowtna the Intra.plcural illoctilHtlon of Asbestos. In Pneuwnronioria. Prar. Intermit. Conf. .T.ihanne'bunf. 1909. Kd. H. A. Shapiro. Cape T.nvns Oxford University Press, p. 219. APPENDIX The results have boon analysed using the model given by Pike (19(50) and shown to he valid for experiments of this type (Berry and Wagner 19H9). The agespecific death rate of animals dying witli mesothelioma f. days after injection is taken cifcff-MJ)*-1 where c. k and w are consta ita. Those 3 constants could be estimated separately for each treatment of each experiment but correlations between the estimates make them imprecise. In the original experiments, with a total of 417 mesotheliomata, it was shown that k could be taken as 3 for all treatments but that ui, the lapse period before any meso theliomata occurred, varied with treat ment; in particular the lapse period for amosite was found to be about 200 days longer than that for chrysotile and erocidolite but there was an isolated mesothelioma occurring with amosite after only 398 days. For the experiments being reported here, although the estimates of the lapse period vary widely over the different treatments, this wide range could be due to the imprecision of the estimates, and there is no strong evidence that it is invalid to use a common value. Also, there is no evidence of the lapse period being dependent on dose. Therefore, in the analysis the best estimates of k and w based on all our evidence were used; these are w ~ 270 and k -- 3-25. Even with all the data these estimates are notvery precise; for example the pairs (300, 2'9) and (2'2o, 3*8) would be acceptable, as also would the estimates (250. 3-0) which we used ill our preliminary reports. However, these alternatives all lead to similar conclusions. The constant c was estimated for each of t.he..treatment groups and this constant, which we will refer to as the " carcino genicity factor ", serves as a single index summarizing the mesothelioma experience of each treatment group. It combines the information on the proportion of animals which developed a mesothelioma with the rimes after inoculation at which the meso theliomata occurred. Also, the method of estimation eliminates mortality due to other causes, so that neither chance nor systematic variations in natural mortality between different groups will affect- com parisons between these groups. As an example of the elimination of natural mortality, the proportion of rats developing mesotheliomata after injection with SFA chrysotile in Experiments 2 and 4 (69;, and 54%) were similar to the pro portion (05%) in SPF rats in our original experiment (Wagner and Kerry, 1969). However, the rats in Experiments- 2 and 4 were injected at 13 weeks of age opposed to 6 weeks in our original experiment, and also the natural mortality was less in our original experiment, oven after allowing for this age difference. Hence the carci nogenicity of t-he chrysotile was least in our original experiment and this is re flected in the values of the carcinogenicity factor which were 1-7 >, 10-" in (he original experiment-. 4-7 \ lo-9 in Experi ment 2 and 2-9 X 10-9 in Experiment 4 (Table V). The non-significant difference between the values for Experiments 2 and 4 was revealed after eliminating the chance lower natural mortality in the group used in the latter experiment.