Results of long-term carcinogenicity bioassays of rockwool on Sprague-Dawley rats

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1 Eur. J. Oncol., vol. 3, n. 3, pp , 1998 Tematiche specifiche/specific topics Fibre minerali/mineral fibres Results of long-term carcinogenicity bioassays of rockwool on Sprague-Dawley rats F. Minardi and C. Maltoni Cancer Research Centre, European Foundation for Oncology and Environmental Sciences B. Ramazzini, Bologna, Italy Summary Rockwool was tested for carcinogenicity by a single intraperitoneal and intrapleural injection, at the dose of 25 mg in water, on groups of 40 Sprague-Dawley rats (20 males and 20 females). The fibres caused mesotheliomas: 10% following intraperitoneal injection, 2.5% following intrapleural injection. The importance and possible implications of these findings are discussed. Key words: rockwool, experimental study, rat, mesothelioma Riassunto La lana di roccia è stata sottoposta a saggi di cancerogenicità mediante iniezione intraperitoneale ed intrapleurica, alla dose di 25 mg in acqua, una tantum, su gruppi di 40 ratti Sprague-Dawley (20 maschi e 20 femmine). Le fibre hanno causato mesoteliomi: 10% dopo iniezione intraperitoneale, 2,5% dopo iniezione intrapleurica. L importanza e le possibili implicazioni di questi risultati vengono discusse. Parole chiave: lana di roccia, studio sperimentale, ratto, mesotelioma Introduction The chemical and physical characteristics, production and uses of man-made mineral fibres (MMMF) and specifically of rockwool, were summarised by the IARC (1988). The term MMMF includes inorganic fibres produced by glass, clay, rock and slag. These fibres may be classified in 4 groups: glass fibres (glass filaments and glass wool), ceramic fibres, rockwool and slagwool. The production of MMMF consists in 3 steps: 1) the fusion of the basic material, 2) the formation of fibres, and 3) the conversion of fibres into the final product. In the last few decades, the production and uses of MMMF have expanded considerably, in large part owing to the gradual reduction in production and use of asbestos, given the harmful effects of this material. In those exposed to it, asbestos causes mesotheliomas at different sites, lung carcinomas, a variety of other tumours, and lung fibrosis (IARC, 1977; Selikoff and Lee, 1978). Indirizzo/Address: Franco Minardi, Centro di Ricerca sul Cancro, Castello di Bentivoglio, Bentivoglio (BO), Italia Already in 1993 the production of MMMF was estimated at over 4,585,000 tons. The expansion, production and use of MMMF, particularly as asbestos substitutes, has not be paralleled by adequate biomedical research (experimental, epidemiological and pathological) aimed at identifying and quantifying the carcinogenic and otherwise pathological effects of these materials. The existing studies are scanty in number, fragmentary and often incomplete (IARC, 1988). In the Cancer Research Centre (CRC) of the European Foundation of Oncology and Environmental Sciences B. Ramazzini (FR), a widescale integrated project of experimental research to study the carcinogenic potential of fibrous and nonfibrous, natural and man-made materials of industrial impact was started at the beginning of the 1980s. These materials have been tested mainly by intraperitoneal injection and in some cases by intrapleural injection and intratracheal instillation. The project includes longterm carcinogenicity bioassays on various types of rockwool, by intraperitoneal and intrapleural injection. Rockwool and slagwool have many chemical and physical characteristics in common, as well as alternative and also similar uses. Mixed forms also exist: rockwool-slagwool. This is the reason why some data presented here deal with both of them. 251

2 F. Minardi and C. Maltoni Table 1 - Composition (%/weight) of rockwool and slagwood used for insulation (1) Components Rockwool (samples) Slagwool (samples) SiO Al 2O CaO FeO TiO MnO Na 2O K 2O P 2O Fe 2O CaS 1.04 S F (2) 0.4 (1) From IARC, 1988 (2) Fluorine present in glass possibly in the form of fluorides Rockwool and slagwool are produced by blowing, centrifuging or drawing molten rock or slag. Rockwool is typically made from igneous rocks such as diabase, basalt and olivine, and calcium and magnesium carbonate rocks. It dissolves in diluted hydrochloric acid. Slagwool is made from the fused agglomerate by-products of certain metal smelting processes. The properties of these fibres vary, not only according to the sources of raw material, but also from batch to batch. Slagwool is usually slightly soluble in hydrochloric acid. The composition of some rockwool and slagwool insulation materials is given in Table 1. Most rockwool and slagwool is used for thermal and acoustic insulation, in constructions and shipbuilding. The population categories potentially exposed to these fibres include: workers who produce and use them, workers engaged in the maintenance, repair and destruction of products containing them, workers operating in buildings insulated with materials containing them, consumers of fibre containing products, and the population at large, living in an environment exposed to fibre pollution. The results of long-term experimental studies on rockwool, reported in the scientific literature, are reported in Table 2. In all there are 3 experimental studies. Two of them gave positive results. When rockwool was injected into the serosal cavities (pleura and peritoneum), it produced mesotheliomas at the site of injection. The peritoneum seems to be more responsive than the pleura. Table 2 - Long-term experimental carcinogenicity bioassays on rockwool, reported in the literature Characteristics Experimental animals Route of Concentration Neoplastic response References of fibres (1) (species, strain, sex) (2) administration in air/dose Tumours No. % Rockwool Wistar rats 10 PS Inhalation 5 mg/m 3 (3) 0 Le Bouffant et al., (L40% <10 µ; Af/Han, M and F 1984 D 23% <1 µ) Rockwool SPF Fischer rats Inhalation 10 mg/m 3 (4) Pulmonary Wagner et al., without resin (sex unspecified) adenomas 1984 (L71% 5-20 µ; D 58% 1 µ) Rockwool SPF Sprague-Dawley Intrapleural 20 mg x 1 Mesotheliomas Wagner et al., with resin rat (sex unspecified) injection 1984 (L70% <5 µ; D 52% <0.6 µ) Rockwool SPF Sprague-Dawley Intrapleural 20 mg x 1 Mesotheliomas Wagner et al., without resin rat (sex unspecified) injection 1984 (L70% 5 µ; D 52% <0.6 µ) Rockwool Wistar rats, F Intraperitoneal 15 mg x 5 NOS abdominal Pott et al., 1987 (Germany) injection tumours (macro- (L50% <20 µ; scopic diagnosis D 50% <1.8 µ) only) Rockwool Sprague-Dawley Intraperitoneal 25 mg x 5 NOS abdominal Pott et al., 1987 (Sweden) rats, F injection tumours (macro- (L50% <23 µ; scopic diagnosis D 50% <1.9 µ) only) Rockwool Sprague-Dawley Intraperitoneal 10 mg x 1 NOS abdominal Pott et al., 1987 (Sweden) rats, F injection tumours (macro- (L50% <4.1 µ; scopic diagnosis D 50% <0.64 µ) only) (1) L = length; D= diameter (2) M = males; F = females (3) 5 hours/daily, 5 days/weekly, for 12 or 24 months (4) 7 hours/daily, 5 days/weekly, for 12 months 252

3 Carcinogenicity bioassays of rockwool The results of the 2 major epidemiological studies available on workers engaged in the production of rockwool and slagwool, one performed in the USA and one in Europe, are reported in Table 3. These studies show an upward trend in the incidence of lung cancer. The risk seems to increase with the duration of the period from the start of exposure. The present report gives the results of one the two long-term experiments, performed at the CRC/FR, studying the carcinogenic potential of rockwool fibres on rats, following intraperitoneal and intrapleural injection. Materials, methods and experimental plan The rockwool fibres tested were supplied by CERAM-SNA (Canada). Male and female Sprague-Dawley rats from the colony of the CRC/FR were used. This rat colony has been used for various experiments in the BT laboratory for nearly 25 years. Data are available on more than 10,000 historical controls, kept under observation for life-span and submitted to systematic necropsy and standardized histopathological examination. Data are therefore available on the expected incidence of the different types of tumours Table 3 - Mortality from lung cancer and mesothelioma in the major USA and European epidemiological studies, on workers engaged in the production of rockwool and slagwool Parameter USA Europe Combined data study (1) study (2) from the two studies (3) No. of workers 1,846 10,115 11,961 Person-years of follow-up 48, , ,254 Lung cancer deaths Lung cancer SMRs compared to local reference rates Lung cancer mortality 134 (p<0.05) (p<0.01) Time since first exposure <10 years years years years Duration of exposure <20 years years Technological phase Early 257 (p<0.05) Intermediate 141 Late 141 By time from first exposure (early phase only) <10 years years years 317 (p<0.05) 30+ years 295 (p<0.05) Mesothelioma (1) From Enterline et al., 1987 (2) From Simonato et al., 1987 (3) From IARC, 1988 No excess in control animals and on its fluctuations: the incidence of spontaneous mesotheliomas in these animals is extremely low. After weaning, at 4-5 weeks of age, the experimental animals were identified by ear punch, randomized in order to have no more than one male and one female of each litter in the same group, and housed 5 per cage. The animals were 8 weeks old at the start of the experiment. The plan of the experiment is shown in Table 4. The test compound was administered by a single intraperitoneal or intrapleural injection in 1 cc of water, at the dose of 25 mg. The animals were kept under observation up to 112 weeks of age, under highly standardized housing and diet conditions, the same as used in the CRC/FR laboratories over the last 25 years. Individual animal weight was measured every 2 weeks for the first 14 weeks from the start of the experiment, and then every 4 weeks. In order to detect and register all gross lesions, the animals were examined every 2 weeks from the start until the end of the experiment. The biophase of the experiment terminated after 104 weeks, when surviving animals were sacrificed. Upon death, the animals were submitted to systematic necropsy. Histopathology was routinely performed on the site of injection (peritoneum or pleura), the brain, thymus and mediastinal lymph nodes, lung and mainstem bronchi, heart, liver, spleen, pancreas, kidneys and adrenal glands, stomach (fore and glandular), uterus, gonads, subcutaneous and mesenteric lymph nodes, and any other organ or tissue with pathological lesions. All organs and tissues were preserved in 70% ethyl alcohol. The normal specimens were trimmed, following the standard procedures of the BT laboratory: i.e. parenchymal organs were dissected through the hilus to expose the widest surface, and hollow organs were sectioned across the greatest diameter(s). The pathological tissue was trimmed through the largest surface, including normal adjacent tissues. The trimmed specimens were processed as paraffin blocks, and 3-5 micron sections of every specimen were obtained. Sections were routinely stained with haematoxylineosin. Specific stainings were performed when needed. All slides were examined microscopically by the same group of pathologists; a senior pathologist reviewed all the tumours and any other lesion of oncological interest. All pathologists followed the same criteria of histopathological evaluation and classification. Table 4 - Long-term carcinogenicity bioassays on rockwool: : plan of the experiment Group Route of Dose Animals administration (mg) Sex No. I Intraperitoneal 25 M 20 injection F 20 M+F 40 II Intrapleural 25 M 20 injection F 20 M+F 40 III Intraperitoneal 0 M 20 injection F 20 M+F 40 IV Intrapleural 0 M 20 injection F 20 M+F

4 F. Minardi and C. Maltoni Results The results of the experiment, i.e. the incidence of peritoneal and pleural mesotheliomas, are reported in Table 5. Since spontaneous mesotheliomas of the peritoneum and pleura are very rare in our colony of Sprague-Dawley rats, the results presented indicate that rockwool fibres cause mesotheliomas at both sites of injection and therefore must be considered carcinogenic. All 5 mesotheliomas were observed in the cavity in which rockwool had been injected. Four of them arose in the peritoneal cavity: in the same experimental conditions, the peritoneum seems to be more sensitive than the pleura. Four of the 5 mesotheliomas were observed at necroscopy. On histopathological examination of the 4 peritoneal mesotheliomas, 2 showed a predominat fusocellular pattern (cases 1 and 2) (figs. 1-4), 1 a mixed solid and fusocellular pattern (case 3) (figs. 5, 6), and 1 various patterns typical of this neoplasia (case 4) (figs. 7, 8). Pleural mesothelioma showed a mixed pattern: solid and fusocellular (predominant) (figs. 9, 10). In the peritoneum of some animals treated by intraperitoneal injection of rockwool, fibrous plaques and omental nodules containing several fibres of tested material were observed (figs. 11, 12). Conclusions The results presented show that rockwool is carcinogenic and, in the experimental conditions tested, specifically causes mesothelioma of the peritoneum and pleura at the site of injection. The mesotheliomatogenic potency of rockwool seems to be lower than that of asbestos of various kinds, or ceramic refractory fibres, studied at our Institute in the same experimental conditions (Maltoni and Minardi, 1989; Minardi and Maltoni, in this volume). Table 5 - Long-term carcinogenicity bioassays on rockwool: incidence and latency time of mesotheliomas Group Route of Dose Animals Mesotheliomas administration (mg) Sex No. Incidence Latency time No. % (weeks) I Intraperitoneal 25 M injection F M+F II Intrapleural 25 M 20 0 injection F M+F III Intraperitoneal 0 M 20 0 injection F 20 0 M+F 40 0 IV Intrapleural 0 M 20 0 injection F 20 0 M+F 40 0 Our data confirm those available in the scientific literature, indicating the mesotheliomatogenic effect of rockwool. By and large, the data presented here are a further proof of the potential carcinogenic risk of MMMF. These fibres, and particularly glass fibres, ceramic fibres, and rockwool and slagwool, are asbestos substitutes. On the basis of the data from the literature (IARC, 1988) and from the results of our experimental project on the carcinogenicity of ceramic fibres (Minardi and Maltoni, in this volume), and rockwool (presented in this report), we must emphatically point out that, in spite of many historical lessons, the old policies still remain: 1) to substitute dangerous materials with materials of unproven safety where the risk is either unknown or little known, and 2) when positive results become available, to ignore them. Fig. 1 - Epitheliomorphic peritoneal mesothelioma of fusocellular pattern (liver) (case 1). H. E. x

5 Carcinogenicity bioassays of rockwool Fig. 2 - Epitheliomorphic peritoneal mesothelioma of fusocellular pattern (omentum) (case 2). H. E. x 200. Fig. 3 - Same case of fig. 2, with involvement of pancreas. H. E. x

6 F. Minardi and C. Maltoni Fig. 4 - Same case of fig. 2: fusocellular pattern and limited area of solid pattern. H. E. x 320. Fig. 5 - Epitheliomorphic peritoneal mesothelioma of mixed pattern (solid and fusocellular) (case 3). H. E. x

7 Carcinogenicity bioassays of rockwool Fig. 6 - Same case of fig. 5, with prevailing solid pattern. H.E. x 320. Fig. 7 - Epitheliomorphic peritoneal mesothelioma of mixed pattern (tubular and solid) (case 4). H. E. x

8 F. Minardi and C. Maltoni Fig. 8 - Same case of fig. 7, with mixed pattern (solid and fusocellular). H.E. x 320. Fig. 9 - Epitheliomorphic pleural mesothelioma: area with solid pattern. H. E. x

9 Carcinogenicity bioassays of rockwool Fig Same case of fig. 9: area with fusocellular pattern. H.E. x 200. Fig Fibrous nodule, in the omentum, containing several rockwool fibres. H. E. x

10 F. Minardi and C. Maltoni Fig Fibrous nodule, in the omentum, containing several rockwool fibres. H. E. x 320. In the case of rockwool, we think that the time has come to perform further and more adequate epidemiological studies and clinical surveillance on working groups, and at the same time to perform preventive measures. The urgency of these measures is heightened by the fact that, since rockwool is used in building industry, it also widely pollutes living environments. References Enterline P.E., Marsh G.M., Henderson V., et al.: Mortality update of a cohort of US man-made mineral fibres workers. Ann. Occup. Hyg., 31, , IARC: Monographs on the evalution of carcinogenic risk of chemicals to man, vol. 14, Asbestos. IARC, Lyon, IARC: Monographs on the evaluation of carcinogenic risks to humans, vol. 43, Man-made mineral fibres and radon. IARC, Lyon, Le Bouffant L., Henin J.P., Martin J.C., et al.: Distribution of inhaled MMMF in the rat lung - long term effects. In: Biological effects of man-made mineral fibres, Proceedings of a WHO/IARC Conference, vol. 2, Copenhagen, Maltoni C., and Minardi F.: Recent results of carcinogenicity bioassays of fibres and other particulate materials. In: J. Bignon, J. Peto and R. Saracci, Non-occupational exposure to mineral fibres, IARC, Lyon, Minardi F., and Maltoni C.: Results of long-term carcinogenicity bioassays of ceramic fibres ( Fiberfrax ) on Sprague-Dawley rats. Eur. J. Oncol., in this volume. Pott F., Ziem U., Reiffer F.-J., et al.: Carcinogenicity studies on fibres, metal compounds, and some other dusts in rats. Exp. Pathol., 32, , Selikoff I.J., and Lee D.H.K.: Asbestos and disease. Academic Press, New York, Simonato L., Fletcher A.C., Cherrie J., et al.: The International Agency for Research on Cancer historical cohort study of MMMF production workers in seven European countries: extension of the follow-up. Ann. Occup. Hyg., 31, , Wagner J.C., Berry F., Hill R.J., et al.: Animal experiments with MMM(V)F - effects of inhalation and intrapleural inoculation in rats. In: Biological effects of man-made mineral fibres, Proceeding of a WHO/IARC Conference, vol. 2, Copenhagen,