Document VYBdpX5mNdLazLzOjYxMDq54

Information obtained from animal experiments J. C. WAGNER1 & G. BERRY1 Evidence was presented at the New York meeting In a comparison of different doses, using crocido in 1964 that mesotheliomas could be produced in lite and chrysotile (not the UICC samples), the risk hamsters and rats by intrapleural inoculation of of developing a mesothelioma at a given age was asbestos dusts (Smith et a/., 1965; Wagner, 1965). found to be proportional to the dose (Wagner et/., This opened up the possibility of using animal 1970). The effect of dose was also demonstrated by experimentation as a means of investigating the Stanton & Wrench (1972) for UICC crocidolite, as etiology of mesotheliomas caused by asbestos. In well as for another sample of crocidolite. this paper, the results of experiments published or Mesotheliomas have also been produced by other made available to us since then are summarised. workers (Donna, 1970; Reeves et al., 1971; Smith et For convenience the experiments will be divided al., 1965) in rats, hamsters and rabbits, including into four categories according to the route by which 23 out of 50 rats inoculated with 25 mg of UICC the asbestos was administered; first, intrapleural; Rhodesian chrysotile (Wheldon, 1972 3). secondly, intraperitoneal; thirdly, intratracheal; and Thesuggestion that natural oils and waxes(Haring- fourthly, inhalation experiments. The different ton, 1962) and, subsequently, contaminating oils routes are considered by Wagner & Berry 2 at this both from the preparation of the fibre (Harington & meeting, and fuller details of the methodology are Roe, 1965; Roe et al., 1966) and from plastic storage given in the references below. bags (Commins & Gibbs, 1969) played a part in the development of the tumours, has been refuted. The sample of crocidolite from which the oils were re INTRAPLEURAL EXPERIMENTS moved by Harington gave very similar results to the untreated sample (Wagner & Berry, 1969). Also, all five UICC samples from which the oils were re Alltypes of asbestos have produced mesotheliomas moved by Commins have been compared with identi in rats. Wagner (1972) reported an experiment in cal untreated materials, and the oil-extracted samples which inoculations with all five UICC standard produced 58 mesotheliomas compared with 56 with reference samples (Timbrell et ctl., 1968) produced the untreated samples (Wagner, 1972). I mesotheliomas; with a dose of 20 mg these tumours occurred in 610 0 of animals for crocidolite, 36" ,, for amosite, 34,, for anthophvllite, 30"0 for Canadian chrysotile and 19" 0 for Rhodesian chrysotile. Stanton & Wrench (1972), with a dose of 40 mg of asbestos dust on gelatine-coated fibre-glass pledgets, found that three of the UICC samples, crocidolite, amosite and Rhodesian chrysotile, all produced mesotheliomas in about 60,, of rats. : MRC Pneumoconiosis Unit, Llandougli Hospital, Penarth, UK. Another suggested explanation for the carcino genicity of asbestos was that the presence of the trace metals might be relevant (Harington & Roe, 1965). This received support from Wagner et al., (1970). who reported that for samples of chrysotile from seven different Canadian mines there was a significant correlation between the production of mesotheliomas and their chromium content. How ever, Wagner (1972) discounted this finding, since the published chromium content of one of the . ________________ J Sec p. 85 of this publication. J Personal communication. - 285 - 286 BIOLOGICAL EFFECTS OF ASBESTOS samples had subsequently been amended and the correlation was no longer present. Wagner et al. (1970) suggested that the fineness of the grinding of the samples was of importance. Evi dence of this was found by Wagner (1972) from an experiment in which UICC Canadian chrysotile had been compared with each of the eight separate samples which had been combined to form the UICC sample (Timbrell & Rendall, 1971). All of the separate samples produced more mesotheliomas than did the UICC sample and had been ground more finely before experimental use. However, a different mill had been used, so that factors other than particle size could not be ruled out. Of the eight separate samples, one was from a mine in Western Canada whilst the others were from a rela tively small area of Eastern Canada. The sample from Western Canada was the least carcinogenic in each of two experiments, but it could not be separ ated from the other samples on the basis of its size distribution. However, the sample of chrysotile which unexpectedly produced mesotheliomas in as many as 66/ of rats (Wagner & Berry, 1969) was a super-fine sample, produced by sedimentation separ ation from grade 7 (the most fully milled of all commercial products). The effect of grinding was also investigated by Stanton & Wrench (1972), who found that UICC crocidolite when partially pulverised produced fewer mesotheliomas than did the standard sample. They postulated that submicroscopic fibres, i.e., smaller than about 1.25 x 3.75 g.m, could be discounted. The apparent disagreement on the significance of fibre size is discussed in the following two papers (Stan ton l; Timbrell 2). INTRA-PERITONEAL EXPERIMENTS Peritoneal mesotheliomas have been produced by Reeves et al. (1971) in Charles River caesareanderived rats after intraperitoneal inoculation with crocidolite and chrysotile, but not with amosite. They have also been produced by Davis (19723) in 17 out of 25 rats and 23 out of 50 mice injected with crocidolite. : See p. 289 of this publication. - See p. 295 of this publication. 3 Personal communication. INTRA-TRACHEAL INOCULATION This method has been used to study theco-carcinogenesis of chrysotile fibre with benzo(a)pyrene by Miller et al. (1965) and by Vosamae (1972). Miller and colleagues used hamsters, and Vosamae rats. The results of both experiments demonstrate that the presence of the chrysotile has a promoting action on the carcinogenicity of the benzo(a)pyrene. INHALATION EXPERIMENTS Gross et al. (1967) reported the production of carcinomas of the lungs of rats exposed to chrysotile dust. Of 72 rats which survived 16 months' expo sure with a mean concentration of 86 mg/m3 for 30 hours a week, 24 animals developed carcinomas and there was one mesothelioma. It was considered that trace metals from the worn hammer of the mill used to produce the respirable fibre could be a factor in the causation of these tumours. Reeves et al. (1971) found squamous carcinomas in 2 rats out of 31 which survived two years of croci dolite exposure with a concentration of 49 mg/m3 for 16 hours per week. Five of those exposed to chrysotile developed pulmonary adenomatosis, but there were no malignant tumours among those exposed to either chrysotile or amosite. Wagner (1972) reported an experiment in which rats were exposed to a concentration of 12 mg/m:l of respirable dust of each of the five UICC samples. There were two lengths of exposure, 1 day (7 hours) and 3 months (400 hours). The amount of chryso tile retained in the lungs at the end of the longer period of exposure was only one sixth that found with the three amphibole samples. Three meso theliomas were observed, and two of these occurred after the shorter exposure, one with amosite and one with crocidolite. The third was a peritoneal tumour occurring after the longer exposure to crocidolite; a squamous-celled carcinoma of the lung also occurred in this group. An excess of lung adenomas over non-exposed controls was observed after the longer exposure for all dusts except anthophyllite. No explanation could be offered for the surprising finding that two mesotheliomas occurred after the shorter exposure compared with only one after the fifty times more severe exposure. The probability of such an extreme result occurring by chance is only about 2 in 1000. The e' one with all types fibrosis \ Also, all an exces: from ade cancers , producei found oc Most i s cation ol 1 tion. TJ unrealist 'i % to play. 1 must be p- trate thr< xk differ wit u y ly, that ti f. exercises n inoculati 1 vity" nei ! y experime two type 1 intrapleu 1i for inve^ tion of ti In additi ! by the i j mental r From unlikely ! natural < ? developn trace ele: V cant rok v. support ? that the ent chen thelioma a chemi. Exper asbestos point. arcinoene by Miller ie rats, .hat the lion on tion of rysotile ' expo1 for 30 nas and red that lill used ictor in `inomas if croci- mg'nr* osed to sis, but > those INFORMATION OBTAINED FROM ANIMAL EXPERIMENTS 287 The experiment has now been supplemented by one with exposures of up to 2 years. In this study all types of fibre produced asbestosis. and the fibrosis was progressive after exposure was ended. Also, all samples, including anthophyllite. produced an excess of lung tumours. These tumours varied from adenomas to adeno-carcinomas; and squamous cancers and the occasional mesothelioma were also produced. Surprisingly, a few of the mesotheliomas found occurred with Canadian chrysotile. DISCUSSION Most of the experimental work has involved appli cation of asbestos either intrapleurally or by inhala tion. The former may be rightly criticised as being unrealistic, but it has nevertheless an important part to play. In an inhalation experiment two factors must be borne in mind: firstly, the dust must pene trate through the airways and alveoli, and this may differ with different samples of asbestos; and second ly, that the dust, given that it has reached the pleura, exercises a "biological activity". In contrast, in inoculation experiments only the "biological acti vity" needs to be considered, and this makes these experiments the more readily interpretable. The two types of experiment supplement one another, intrapleural application being much more suitable for investigating questions such as whether extrac tion of the oils alters the carcinogenicity of a sample. In addition, the ease of production of mesotheliomas by the intrapleural route provides a useful experi mental model. From the results of intrapleural studies it seems unlikely that the presence of oils and waxes, whether natural or acquired, plays a significant part in the development of mesotheliomas. The possibility that trace elements contaminating the fibre play a signifi cant role as carcinogenic factors also receives no support from the experimental evidence. The fact that the different types of asbestos, with very differ ent chemical compositions, can all produce meso theliomas, also makes it unlikely that there would be a chemical explanation for the carcinogenicity of asbestos. The contamination of the fibre may have had some significance in the inhalation experiments of Gross and his co-workers 0967), but not in the larger study with standard reference samples. The most interesting pointer obtained from these experimental results is that development of meso theliomas is associated with the presence of fine fibrous material within the pleural cavity. This theme is elaborated in the following two papers (Stanton *; Timbrell 12). If physical characteristics are the important factors as far as carcinogenesis is concerned, then one might expect that other fibrous material would produce mesotheliomas. In fact, other materials have pro duced mesotheliomas after intrapleural application. It should be noted, however, that the tumours pro duced by silica and reported as being similar to meso theliomas by Wagner (1965) have now been shown to be lymphomas and not mesotheliomas (Wagner & Wagner, 1972). Stanton & Wrench (1972) re ported the occurrence of mesotheliomas with silicon dioxide, glass-wool and fibre-glass, the finer samples of fibre-glass producing the highest incidence (18%). Occasional tumours were also produced by Webster (1965) with carbon black, and in our own experi ments by synthetic aluminium silicate fibre, barium sulphate, glass-powder and aluminium oxide. Experiments designed to investigate in more detail the role of the size and shape of asbestos and other fibres would seem to be a profitable way of advancing current knowledge. Additionally, intrapleural methods have provided a source of tumours, histologically indistinguishable from those seen in man, which can be used for im munological investigations and to study the response to therapy. Inhalation experiments have provided evidence for estimating the dose required to produce a low inci dence of tumours. This opens up the possibility of using inhalation to investigate the effects of combined exposure to asbestos with other agents, such as smoking. 1 See p. 289 of this publication. 2 See p. 295 of this publication. SUMMARY Experiments in which animals hate been exposed lo asbestos are reviewed, mainly from the carcinogenic view point. Most work has involved applying the do'C either intrapleurally or by inhalation; and the animal used most has been the rat. After intrapleural administration a high proportion of animals usually developed nieso- 288 BIOLOGICAL EFFECTS OF ASBESTOS theliomas, and result'; show that the carcinogenesis of asbestos is unlikely to be due to the oils and waxes present in asbestos or to trace elements. There are indi cations that fibre size and shape are important, and it is suggested that further work on this aspect might be profitable. Mesotheliomas have been produced with fibre-glass and other non-asbestos materials. Meso theliomas have occurred only occasionally after experi mental inhalation, but adenomas and adeno- and squa mous carcinomas occurred more frequently. REFERENCES Commins, B. T. & Gibbs, G. W. (1969) Contaminating organic material in asbestos. British Journal ofCancer, 23, 358-362 Donna. A. (1970) Tumori sperimentali da amianto di crisotilo. crocidolite e amosite in ratio Sprague-Dawley. La Medicina del Lavoro, 61, 1-17 Gross, P.. dc Treville, R. T. P,, Tolker. E. B., Kaschak, M. & Babyak, M. A. (1967) Experimental asbestosis. The development of lung cancer in rats with pulmonary deposits of chrysotile asbestos dust. Archives of Environmental Health (Chicago), 15, 343-355 Harington, J. S. (1962) Occurrence of oils containing 3:4benzpyrene and related substances in asbestos. Mature (London), 193, 43-45 Harington, J. S. & Roe, F. J. C. (1965) Studies of carcino genesis of asbestos fibres and their natural oils. Annals of the New York Academy of Sciences, 132, 439-450 Miller, L-, Smith, W. E. & Berliner, S. W. (1965) Tests for effect of asbestos on benzo(a)pyrene carcinogenesis in the respiratory tract. Annals of the New York Academy of Sciences, 132, 489-500 Reeves, A. L,, Puro, H. E., Smith, R. G. & Vorwald, A. J. (1971) Experimental asbestos carcinogenesis. Environmental Research, 4, 496-511 Roc, F. J. C., Walters, M. A. & Harington, J. S. (1966) Tumour initiation by natural and contaminating asbes tos oils. International Journal of Cancer. 1, 491-495 Smith, W. E., Miller, L., Elsasser. R. E. & Hubert, D. D. (1965) Tests for carcinogenicity of asbestos. Annals of the New York Academy of Sciences, 132. 456-488 Stanton, M. F. & Wrench, C. (1972) Mechanisms of mesothelioma induction with asbestos and fibrous glass. Journal of the National Cancer Institute, 48, 797-821 Timbrell, V., Gilson, J. C. & Webster, I. (1968) UICC standard reference samples of asbestos. International Journal of Cancer, 3, 406-408 Timbrell, V. & Rendall, R. E. G. (1971) Preparation of the UICC standard reference samples of asbestos. Powder Technology, 5, 279-287 Vosamae, A. (1972) In: International Agency for Research on Cancer, Annual Report, 1971. Lyon, p. 46 Wagner, J. C. (1965) Contribution to discussion. Annals of the New York Academy of Sciences, 132, 505-506 Wagner, J. C. (1972) The significance of asbestos in tissue. In: Grundmann, E. & Tulinius. H., eds.. Recent Re sults in Cancer Research, 39, 37-46 Wagner, J. C. & Berry, G. (1969) Mesotheliomas in rats following inoculation with asbestos. The British Jour nal of Cancer, 23, 567-581 Wagner, J. C., Berry, G. & Timbrell, V. (1970) Meso theliomas in rats following the intra-pleural inocula tion of asbestos. In: Shapiro, H. A., ed., Pneumoconio sis. Proceedings of the International Conference, Johannesburg, 1969, Cape Town, Oxford University Press, pp. 216-219 Wagner, M. M. F. & Wagner, J. C. (1972) Lymphomas in the Wistar rat after intrapleural inoculation of silica. Journal of the National Cancer Institute, 49, 81-91. Webster, I. (1965) Mesotheliomalous tumours in South Africa: pathology and experimental pathology. An nals of the New York Academy of Sciences, 132, 623 646 t Sorr The cause o groups. ix There i- ?! bers of 6 mechar ft Asbesti l are cat\ it is of has atti the prehydroc; through for the hand, p % of virus it is cot could whateu On ti dence tl carcinc conclus (1) T toses a carcino1 carcinohydroc; carcino: at al, I (2) V i various types ot cinogen Wagner 1 The Bethesd. WORLD HEALTH ORGANIZATION lill INTERNATIONAL AGENCY FOR RESEARCH ON CANCER BIOLOGICAL EFFECTS OF ASBESTOS Proceedings of a Working Conference held at the International Agency for Research on Cancer, Lyon, France, 2-6 October 1972 P. BOGOVSKI V. TIMBRELL EDITORS J. C. GILSON J. C. WAGNER TECHNICAL EDITOR FOR IARC W. DAVIS IARC Scientific Publications No. 8 INTERNATIONAL AGENCY FOR RESEARCH ON CANCER LYON 1973