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A summary is given of the pathology results after long-term inhalation in rats of insulation wool representing the new biosoluble types. The pathology results are compared with previously conducted long-term inhalation study with MMVF21 (traditional stone wool). The biosoluble fiber MMVF34/HT (HT) is characterized by a relatively high content of aluminum and a relatively low content of silica compared to the older MMVF21. HT has a high in vitro dissolution rate at pH 4.5, and a relatively low dissolution rate at pH 7.5. Male Fischer 344 rats were exposed at one exposure level of 30 mg/m(3) by nose-only inhalation of a well-characterized fiber test atmosphere. The fibers had been size selected to be largely rat respirable. The negative control group was exposed to filtered air. The exposure duration was 6 h/day, 5 days/wk for 104 wk, with a subsequent nonexposure period lasting until approximately 20% survival in the air control group. Interim sacrifices were performed at wk 13, 26, 52, 78, and 104 to monitor the progression of pulmonary change and fiber numbers. Effectively the main protocol for the previously conducted chronic study with MMVF21 was the same, except that there were three concentration levels (3, 16, and 30 mg/m(3)). In addition to the endpoints measured in the previous study, slides from both studies were evaluated for collagen deposition using a quantitative morphometric method. The results of the comparative study clearly showed a marked difference in the pulmonary pathogenicity of the MMVF21 and HT in terms of their fibrogenic potential. MMVF21 caused pulmonary fibrosis, but the HT fiber did not. The incidence of tumors for both the HT and the MMVF21 fiber was comparable to the control groups.

A summary is given of the biopersistence and pathology after inhalation by rats of two different Man–made Vitreous Fibres, MMVF10 (traditional stone wool) and MMVF23 (HT stone wool), and the results are discussed in relation to biopersistence measured after intra–tracheal instillation. The results are given from a short–term inhalation biopersistence study, a completed chronic inhalation study, and interim results from an on–going chronic inhalation study. In both the short–term and chronic studies, laboratory rats were exposed by nose–only inhalation to well characterised fibre test atmospheres that had been selected to be largely rat respirable. The short–term inhalation study included groups exposed to aerosols targeted at 150 fibres longer than 20 mm per cm 2 . The exposure duration was 6 hours:day for 3 days, with subsequent post–exposure periods lasting up to 10 months. For lung burden analyses, interim sacrifices were performed at regular intervals. The ongoing chronic study comprises a group of rats exposed to the MMVF34 fibre at one exposure level of 30 mg:m 3 . The negative control group is filtered air. The exposure duration is 6 hours:day, 5 days:week for 2 years, with a subsequent post–exposure period lasting until approximately 20) survival in the test fibre group. Interim sacrifices are performed at months 3, 6, 10, 18 and 24 and biopersistence monitored for rats exposed for 3 and 12 months, with subsequent post–exposure periods lasting 6 months. Effectively the main protocol for the previously conducted chronic study was the same, except that there were 3 fibre exposure groups (3, 16 and 30 mg:m 3 ) and no specific biopersistence satellite groups were included. For MMVF34, the inhalation tests of different duration show a similar biopersistence pattern, while the intra–tracheal test gives longer elimination half–times especially for long fibres. The MMVF34 fibre is considerably less biopersistent than the traditional MMVF21 fibre when comparing the calculated elimination half times after short–term inhalation. When comparing the pathology after 3, 6, 10 and 18 months exposure, MMVF34 showed minor histopathological changes compared to MMVF21. The car–cinogenicity and toxicity results of the chronic study with MMVF21 suggest that this fibre does not pose a significant health risk to humans and the current results with MMVF34 indicate that this fibre consequently should pose an even smaller risk, if any. © 1998 British Occupational Hygiene Society. Published by Elsevier Science Ltd.

A wide range of fiber types was tested in two in vitro assays: toxicity to A549 epithelial cells, as detachment from substrate, and the production of the proinflammatory cytokine tumor necrosis factor (TNF) by rat alveolar macrophages. Three of the fibers were also studied in vivo, using short-term inhalation followed by a) bronchoalveolar lavage to assess the inflammatory response and b) measurement of cell proliferation in terminal bronchioles and alveolar ducts, using incorporation of bromodeoxyuridine (BrdU). The amount of TNF produced by macrophages in vitro depended on the fiber type, with the man-made vitreous fibers, and refractory ceramic fibers being least stimulatory and silicon carbide (SiC) whiskers providing the greatest stimulation. In the epithelial detachment assay there were dose-dependent differences in the toxicity of the various fibers, with long amosite being the most toxic. However, there was no clear relationship to known chronic pathogenicity. Fibers studied by short-term inhalation produced some inflammation, but there was no clear discrimination between the responses to code 100/475 glass fibers and the more pathogenic amosite and SiC. However, measurements of BrdU uptake into lung cells showed that amosite and SiC produced a greater reaction than code 100/475, which itself caused no more proliferation than that seen in untreated lungs. These results mirror the pathogenicity ranking of the fibers in long-term experiments. In conclusion, the only test to show potential as a predictive measure of pathogenicity was that of cell proliferation in lungs after brief inhalation exposure (BrdU assay). We believe that this assay should be validated with a wider range of fibers, doses, and time points.

Cell lines were established in vitro from five peritoneal mesotheliomas produced in rats by the injection of crocidolite asbestos. These tumours exhibited the previously described diverse range of histological patterns normally associated with mesotheliomas. This diversity of appearance was also evident in culture but cell patterns in vitro did not necessarily correspond to the histology of the original tumour. With increased time in culture most cell lines tended towards a pattern of random orientation associated with the piling up of cells to form discrete masses. Despite this variation in cell pattern in culture, few ultrastructural differences were seen during electron microscope examination. Following varying periods in culture, some cell lines from these tumours were injected into either rats or athymic mice. Only one typical rat mesothelioma was produced but all cell lines produced tumours in athymic mice. The implications of these findings are discussed in relation to the aetiology of asbestos induced mesotheliomas.

It is generally accepted that to cause pulmonary disease, mineral fibers must be relatively long and thin but also able to remain in the lung for long periods. This "biopersistence" of fibers is limited by two main mechanisms of fiber clearance: removal by macrophages after phagocytosis and, for some fibers, by actual dissolution. The relative importance of these mechanisms has not been properly evaluated for any type of fiber and will certainly vary with mineral type. The efficiency of macrophage clearance is greatest with short fibers (< 5 microns long) and is reduced as fibers get longer. Fibers > 50 microns long cannot be cleared by macrophages and for some mineral types they may remain in the lung permanently. Others may fracture into shorter lengths, perhaps aided by chemical dissolution, and thus become susceptible to macrophage clearance. However, for a number of areas relating to fiber removal from the lung parenchyma detailed information is still needed: Do dusts differ in their ability to attract macrophages and stimulate these cells to phagocytosis? Following dust uptake what controls the movement of macrophages? Some may penetrate to the interstitium, some phagocytosing fibers in interstitial sites may migrate back to the alveolar space. Some move to the mucociliary escalator and some to the lymphatics. Some, most importantly, move to the pleura. Fibers are found and phagocytosed in the interstitium during the early stages of disease development, but with time many fibers appear isolated in areas of fibrous tissue. Are such fibers subsequently ignored or can they reenter the disease process after years of isolation? Finally, can phagocytosis by macrophages effect dissolution of fibers?(ABSTRACT TRUNCATED AT 250 WORDS)

The association between occupational asbestos exposure and the development of both pulmonary fibrosis or asbestosis and pulmonary carcinomas is well documented. It has been suggested that the two pathological conditions are associated with asbestos-related carcinomas developing from areas of asbestosis and not occurring when exposure has been too low to produce this type of pulmonary scarring. Experimental inhalation studies so far published have not been designed to examine this association specifically, but many publications have reported that asbestos samples producing high levels of fibrosis is experimental animals are also very carcinogenic. Samples of asbestos or man-made fibers that produce little fibrosis also produce few tumors. These works are reviewed. In order to examine the association between fibrosis and tumor production in more detail, groups of animals with and without pulmonary tumors and with individual fibrosis measurements were assembled from a number of inhalation studies undertaken over a period of years at this Institute. It was found that animals with pulmonary tumors had almost double the amount of pulmonary fibrosis as animals of similar age that did not. In a few of the animals where tumors were found at an early stage of development, their origin from fibrotic areas could be confirmed, although in most cases where tumor deposits were widespread this was not possible. Experimental confirmation of the site of origin of most pulmonary tumors in asbestos-treated rats would require new studies with rats examined specifically at an age when early tumors would be expected.

Rats were exposed to clouds of the following pneumoconiotic dusts: quartz, coal-mine dust, and chrysotile asbestos at 10 or 50 mg/m3 for 8, 32, and 75 days; for comparison, rats were also exposed to the non-pathogenic dust titanium dioxide (TiO2). The bronchoalveolar leukocytes (macrophages and neutrophils) from dust-exposed and control rats were obtained by lavage and tested for their ability to migrate toward zymosan-activated serum. Varying amounts of neutrophils were present depending on the ability of the dust to cause inflammation and the length of exposure. There was a marked loss of chemotactic ability in leukocytes from rats inhaling the pneumoconiotic dusts compared with controls; TiO2-exposed leukocytes had some impairment of chemotaxis, but this was substantially less than that found with the pneumoconiotic dusts. The loss of chemotactic activity did not correlate with the percentage of neutrophils in the lavage cells except when there were very high levels of neutrophils, and there was substantial impairment of chemotaxis with negligible numbers of neutrophils, showing that macrophage chemotaxis was impaired. A phagocytic burden within the leucocytes was not sufficient alone to inhibit chemotaxis, nor was the loss of chemotactic activity due to occupied receptors, since incubation failed to restore chemotaxis. Loss to chemotactic activity by leukocytes from pneumoconiotic dust-exposed lung could be an important factor in the development of pneumoconiosis.

This paper summarizes the biological effects of refractory ceramic fibres (RCFs). RCFs are aluminosilicate glass insulation wools with similar chemical properties to other synthetic vitreous fibres (SVFs) or 'man-made vitreous fibres' (MMVFs). There is concern that RCFs could be significantly more pathogenic than other SVFs. This paper critically reviews the data on which this perception is based. Morbidity studies on workers in RCF manufacturing indicated that, in the United states, RCF exposure was associated with an increased incidence of pleural plaques and in both the united states and Europe with statistically significant changes in some measures of lung function (though not at present exposure levels). No interstitial fibrosis was found. An ongoing mortality study of limited statistical power has failed to indicate any increased incidence of lung cancer or mesothelioma. Findings in several early animal studies led to a large series of inhalation studies where rats exposed to high levels of RCF developed fibrosis and tumours but not those exposed to other SVFs. Similarly hamsters exposed to one sample (RCF1) developed mesothelioma. Subsequent analyses of the data indicated that the RCF used in these experiments had a significantly greater proportion of non-fibrous particles than those present in the other types of SVFs tested or in workplace air. Short-term studies indicated that pulmonary overload occurred at the same as RCF tissue burdens as those in the long-term animal bioassay. When RCFs were prepared in the same way as the other SVFs, a sample resulted with a more representative ratio of particles to fibres; this sample did not produce overload in short-term tests. SVFs have various abilities to persist in the lung tissue and thus accumulate to varying degrees. It is suggested that biopersistence is a key property. While RCFs are among the more persistent they are similar to many other fibre types. The scientific and regulatory implications of these findings are examined.

Pathology results after subchronic inhalation in rats of three separate fibres representing the new biosoluble high-aluminium low-silica HT type stone wool are given, and the results were compared with the results from a similar study done with the traditional stone wool MMVF21. Male Wistar rats were exposed at one exposure level by nose-only inhalation to well-characterized fibre test atmospheres. The fibres had been size selected to be largely rat respirable. The target dose was an exposure to 150 long fibres/ml (length>20 microm) in each group, and this dose was achieved for all the fibres. The negative control groups were exposed to filtered air. The exposure duration was 6 h/day, 5 days/week for 3 months, with a subsequent non-exposure period lasting 3 months. The rats were killed 1 week after the last exposure and additional post-exposure kills were performed at 1.5 and 3 months to monitor the progression of pulmonary change and fibre numbers in the lung. The assessments included bronchoalveolar lavage fluid (BALF) for evaluation of inflammatory response (e.g. protein content, enzymes, increase in polymorphonuclear leucocytes) and measurement of cell proliferation, assessment of early fibrosis through histological examination and comparison of body weight and lung lobe weights. After exposure of rats to the new biosoluble fibres no biologically significant effects were observed except that a statistically significant increase in lung weight was observed up to 1.5 months post-exposure in all three treatment groups. At 3 months post-exposure, the small increase was no longer significant. The increase in lung weight was still present in the MMVF21 group at the 3 months post-exposure kill. After 3 months exposure, lung retention of long fibres (length>20 microm) varied from 0.4 to 5.2 x 10(6) per lung for the biosoluble fibres. At 3 months post-exposure, the long fibre concentration in the lungs had decreased to 1-7% of this figure. The fibre with the relatively highest biopersistence (RIF41001) showed the highest fibre retention. The retention of the more biopersistent traditional stone wool MMVF21 was 5.7 x 10(6) per rat lung after 3 months exposure and had decreased to 64% of this figure at 3 months post-exposure. There was no clear difference in the bronchoalveolar lavage fluid cell concentration and percentage of cells between MMVF21 and the HT groups. Fibre inhalation caused a significant increase after 3 months in all the biochemical parameters measured in the BALF. Cell proliferation was enhanced at the end of exposure for MMVF21 for all three labelling indices, but only for the bronchiolar epithelium in the RIF41001 group and for alveolar parenchymal cells in the RIF43006-1 group. At the termination of the 3 month exposure period, as well as after 1.5 and 3 month recovery periods, minimal morphological changes were diagnosed in the biosoluble fibre groups. These changes included alveolar macrophage aggregation and/or microgranulomas at the bronchiolar-alveolar junction in the few rats affected. No fibrogenic potential was noted for any of the three fibres. No clear-cut difference between the different biosoluble fibre types was noted. In the MMVF21 group, minimal interstitial fibrosis was observed that gradually decreased after the 1.5 and 3 month non-exposure periods. In this study, the pathological changes found in the lungs of exposed rats were in accordance with the pathology previously reported from full lifespan inhalation studies. This may indicate that for fibres belonging to the man-made vitreous fibres group a well conducted biopersistence study is sufficient to predict possible pathogenic effects for new fibre types. The biological parameters examined in a 90 day study may indicate little additional information to contribute to the prediction of the outcome of carcinogenicity studies.

Cellulose fibres are used to manufacture productssuch as textiles, paper and cardboard, to give struc-tural strength to cement and other construction prod-ucts and to insulate buildings. This manufacture anduse can produce respirable airborne cellulose fibres.Cellulose fibres have not been subjected to thesame rigorous toxicity testing that has been applied toasbestos and man-made mineral fibres (Davis, 1993).Previously, there have been only limited numbers ofshort-term studies in animals using lung instillationand inhalation, all with relatively high doses. Thoseexperiments produced some pathological changes,such as granuloma, alveolitis, epithelial hyperplasiaand fibrosis (see references in Davis, 1993; Cullen

Previous results from the Colt Fibre Research Programme supported the idea that the number of long thin fibres and their ability to persist in the lung are predictors for the risk of cancer. The results were based on nine fibres, including one glass microfibre (100/475) which had low biological activity. New results with a second glass microfibre (104E) show greater cell proliferation than with 100/475 following short-term inhalation and much greater carcinogenic potential in both chronic inhalation and i.p. experiments, equalling or exceeding that of amosite asbestos. New measurements are used to characterize the durability of the fibres. The extended data still support a dependence of pathogenicity on dose and durability, but with some other factor, perhaps differential leaching affecting surface properties, modifying the relative potency of the two microfibres.

The Wagner grading system is a qualitative histopathologic method designed to describe the severity of nonmalignant respiratory disease (NMRD) as it pertains to the pathology induced by fibrous particulates in humans and later in rats. However, once the method had been used in several rodent fiber studies it was found that it did not adequately differentiate the magnitude of early fibrosis. This article describes a modification of the Wagner scoring system that incorporates a semiquantitative yet simple approach to assuage this problem.

A summary is given of the pathology results after intraperitoneal (i.p.) injection in rats of insulation wool HT, representing the new biosoluble types. The pathology results are compared with a previously conducted i.p. study with traditional stone wool D6 (with similar chemical composition to MMVF21). The HT fibre is characterized by a relatively high content of aluminium and a relatively low content of silica compared to MMVF21. HT has a high in vitro dissolution rate at pH 4.5, a relatively low dissolution rate at pH 7.5 and is less biopersistent than the MMVF21 fibre. Female Wistar rats received a dose of 2 x 10(9) WHO HT fibres by i.p. injection. The fibres had been size-selected to be largely rat respirable. The negative control group was exposed to saline. Following exposure, the animals were maintained until survival in one group fell below 20%. At this time, all animals were killed. All animals were subjected to a necropsy examination; any gross abnormalities observed at necropsy were subjected to histopathological examination. In addition, histopathology was carried out on a predefined list of tissues. The incidences of lesions and survival in the control and fibre dosed animals were compared using appropriate statistical methods to determine whether the dosed animals showed adverse effects on survival or a positive carcinogenic response. The main protocol for the previously conducted study with D6 (MMVF21) was similar, but the animals were maintained as long as they survived, and the WHO fibre dose was lower. The results of the comparative study showed a marked difference in the i.p. pathogenicity of D6 (MMVF21) and HT in terms of their carcinogenic potential. D6 (MMVF21) caused a statistically significant increase of mesotheliomas in the peritoneal cavity compared to the negative control, but the HT fibre did not cause any mesotheliomas or any increase in other tumour types.

Experimental inhalation in a number of studies has demonstrated that chrysotile asbestos can cause pulmonary fibrosis and both benign and malignant pulmonary tumours, two lesions which are associated in that the studies reporting high tumour rates also found high levels of asbestosis. One comparison reported that animals with malignant tumours had approximately twice the amount of fibrosis in the lung parenchyma as those of similar age without tumours. Many studies have examined the pathogenicity of asbestos administered by ingestion and most of these included chrysotile asbestos: the results have been universally negative apart from one study with amosite that contained no control animals and is best discarded. Only one inhalation study has reported an examination of the larynxes of animals: this found no pathological changes. In many studies, tumours other than the lung had been listed, but significant numbers of kidney tumours have never been recorded. Injection studies inducing mesothelioma have indicated that fibre geometry is important with long thin fibres (> 8 microns in length and < 0.25 microns in diameter) being the most carcinogenic. This has been difficult to confirm for inhaled fibres although fibres less than 5 microns in length appear to cause neither fibrosis nor pulmonary tumours. Similar results have been found with amosite for fibres up to 10-15 microns although longer fibres do produce these conditions. It is suggested that to produce pulmonary fibrosis and neoplasia fibres may need to be longer than 20 microns. Chrysotile has been shown in many studies to be removed from lung tissue much more rapidly than amphibole fibres.(ABSTRACT TRUNCATED AT 250 WORDS)

We used a special-purpose glass microfiber sample, Johns-Manville Code 100/475, to study the effects of various acid and alkali treatments on biological activity as assessed by inflammation in the mouse peritoneal cavity, the leaching of Si, and the phase contrast optical microscopy (PCOM) fiber number. We used mild and medium treatments with oxalic acid and Tris buffer and harsh treatment with concentrated HCl and NaOH. Mild oxalic acid and Tris treatment for 2 weeks had no effect on any of the end-points, but prolonging the mild oxalic acid treatment time to 2 months reduced the biological activity and the fiber number. Medium oxalic acid treatment reduced the biological activity and the fiber number and caused a loss of Si. Medium Tris alkali treatment reduced the PCOM-countable fibers and the biological activity but did not cause a substantial loss of Si. Harsh treatment with strong HCl did not affect the fiber number or cause leaching but the biological activity was reduced; strong NaOH reduced the fiber number and biological activity, and caused marked leaching of Si. The medium oxalic acid conditions (pH 1.4) were more acid than those found in lung cells but produced the same effects (reduction in fiber number and biological activity) as the more physiological mild treatment (pH 4.0), when prolonged. This study suggests that medium oxalic acid treatment can be used as a short-term assay to compare loss of Si, reduction in fiber number, and change in biological activity of vitreous fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

The in vitro effects due to phagocytosis of asbestos by mouse peritoneal macrophages in various stages of activation have been compared. The amphiboles proved relatively inert; chrysotile, however, expressed a greater degree of cytotoxicity toward those populations of macrophages induced in vivo with asbestos, than toward any of the other populations of cells. These results are compared with data concerning the enzyme release from the different populations of macrophages following phagocytosis of asbestos. The results indicate that those macrophages that have been exposed to a prior stimulation of either amphibole or serpentine asbestos in vivo are particularly sensitive to exposure to a second dose of a toxic fiber.

Alpha 1 antitrypsin (alpha 1 AT) serum concentration was determined for a group of coalminers with particularly low levels of lung function, not thought to be explicable in terms of age and dust exposure, and compared with two other groups of coalminers who had average or above average lung function. There was no evidence for an alpha 1 AT deficiency in coalminers with poor lung function. On the contrary, a reactionary increase was evident, even in non-smokers, which may have resulted from the high frequency of chest disease in these men. Within the non-smoking group men with poor lung function had been exposed to higher mean levels of dust, and a greater proportion had early signs of bronchitis. There was no indication that the presence or degree of pneumoconiosis was affecting the results.

The pathology and dust content of lungs from 261 coalminers in relation to the appearances of their chest radiographs taken within four years of death were examined. Radiological opacities of coalworkers' pneumoconiosis were more profuse the more dust was retained in lungs. Among the men who had mined low rank coal--that is, with a relatively high proportion of ash--the increase in profusion was most closely related to the ash component of the dust, whereas in men who had mined high rank coal both coal and ash increased in the lungs in relation to radiological profusion. The fine p type of opacity was found to be associated with more dust and a higher proportion of coal and less ash than the nodular r opacity, and was also more likely to be associated with emphysema. The pathological basis of the different types of opacity found on the radiographs of coalminers related to the number, size, and nodularity of the dust lesions. Larger fibrotic lesions were likely to appear as r opacities, whereas fine reticular dust deposition was most likely to present as p opacities, q opacities showing a mixture of appearances. The study has shown that the composition of dust retained in the lung, as well as its amount, makes an important contribution to the radiographic appearances of pneumoconiosis. In particular, the r type of lesion on the radiograph of a low rank coalminer indicates the possibility of a silicotic like lesion.

Samples of the chrysotile taken during and after treatment by the wet dispersion process have been tested for their cytotoxic effect in vitro and the results compared with both a UICC chrysotile A sample and a dust prepared from a standard chrysotile textile yarn. Results were obtained from three different in vitro assay systems utilizing P388D1, V79-4 and A549 cells. A sample which still contained the wetting agent used in the wet dispersion process failed to show activity in any of these assays. The other samples, however, were all active with those dusts obtained by milling the final product and by sampling the air of the factory consistently proving significantly more cytotoxic than the standard chrysotile controls. Preliminary results from a parallel in vivo study suggest that these samples are also more active in producing mesotheliomas in rats.

Three different laboratories independently investigated the ability of ingested mineral fibers to penetrate tissues in rats. All concluded that there was no evidence of tissue penetration by ingested mineral fibers. These experimental observations are supported by the findings in coal and hard-rock miners who swallow, during their lifetime, nearly 100 times the amount of dust that is stored in their lungs. The intestinal wall or mesenteric lymph nodes of these people show no evidence of storage of the ingested dust particles. Animals fed asbestos over much of their lifetime and allowed to live to the age of cancer production, failed to provide evidence of a cancerogenic effect.

In order to compare methods of counting asbestos fibers in lung tissue, seven laboratories participated in an interlaboratory trial in which tissue samples from five human lungs were analyzed. In some laboratories, fiber concentrations were assessed with the light microscope and, in others, with either scanning or transmission electron microscopes. Within each laboratory the ranking of the results was similar, but there were marked differences in the absolute values obtained by the different laboratories. It is concluded that the laboratories participating in this trial appear to produce internally consistent results, but there is difficulty in directly comparing results from one laboratory to the next.

In this study, the cytotoxicity of 13 fibrous samples of known fiber number and dimensions has been established in P388D1 cells. The cells were exposed in vitro to dust concentrations of 10 or 50 micrograms/ml and, after incubation for 24 or 48 hours, any changes in cellular viability, lactate dehydrogenase, and glucosaminidase levels were determined. In general, there was a close association between the reduction in cellular viability and the loss of intracellular enzymes induced by each dust, the chrysotile asbestos samples proving more cytotoxic than the amphiboles. The cytotoxicity of the fibrous dusts was shown to be related to the number of fibers greater than 8 micron in length in the samples.

Previous studies have shown that long thin asbestos fibres are more pathogenic in in vivo and more active in in vitro assays than short fibre samples. In the present study a long fibre amosite asbestos sample and a short fibre sample prepared from it were tested for ability to cause inflammation in the peritoneal cavity of the mouse; a UICC sample intermediate in fibre size and an inert compact dust, TiO2, were also tested. The ability of the dust samples to cause inflammation, as judged by macrophage and neutrophil recruitment, was ranked in the order long fibre greater than UICC greater than short fibre greater than TiO2. Ability of amosite samples to cause inflammation was therefore related to the proportion of long fibres. The enhanced ability of long fibres to cause inflammation and cause macrophage activation is probably a key factor in the ability of long fibres to cause pulmonary fibrosis and may also be important in fibre carcinogenesis.

This work formed part of a series of studies devised to investigate immunological markers which it was considered might reflect individual susceptibility to the development of coalworkers' pneumoconiosis and especially progressive massive fibrosis. Estimations of T and B lymphocytes and white blood cell counts (WBCC) were performed on blood samples from 324 coalworkers grouped according to radiographic category of pneumoconiosis, age, and smoking habits. A subgroup of 43 men was further screened for both humoral anti-lung antibodies (ALA) and direct lymphocyte-mediated cellular cytotoxicity (LMCC) towards human embryonic lung fibroblasts (HEL). In addition 673 serum samples obtained during a previous study of autoimmune factors in coalworkers were screened for humoral antibodies to HEL cells. The only correlation found between category of pneumoconiosis and alterations in WBCC, percentage or absolute numbers of lymphocytes, or the presence of anti-lung antibodies was a decrease in a subgroup of T lymphocytes with increasing severity of pneumoconiosis. The major factor influencing these parameters in this study was the smoking status of the man. Smokers showed increased WBCC and lymphocyte numbers and a higher frequency of ALA and LMCC when compared with nonsmokers.

The effects of respirable dust from three commercially produced calcium silicate insulation materials were examined in laboratory rats by long-term inhalation and injection techniques. These calcium silicate products have been used as replacements for asbestos in the insulation of the engine rooms of ships, and the particle size distribution of the dust clouds generated for the experimental study closely matched those found in ships during the installation of this type of material. One year of exposure to a dust cloud of 10 mg/m3 of respirable dust had no discernible effect on the length of survival of treated animals compared to controls. No pulmonary lesions were found that appeared associated with the inhalation of calcium silicate per se, but one sample did contain significant amounts of quartz and this did produce a few small pulmonary nodules. While two small pulmonary neoplasms, one malignant and one benign, were found in dusted animals, neither was the cause of death, and the incidence was not significantly different from the control group where no tumors were found. One peritoneal mesothelioma was found in an animal from one of the inhalation groups, but this was considered to be a spontaneous tumor as none of over 100 animals injected intraperitoneally with 25 mg of calcium silicate developed these tumors. While the white blood cell count of dusted animals, compared to controls, was significantly raised in all treated groups at the end of the dusting period, these figures were within the published normal ranges for laboratory rats. It was concluded that the three tested calcium silicate products were harmless to the rats of this species at the doses tested.

The effect of a dose of 450 roentgen on regenerating liver cells is to cause the disappearance of all alpha cytomembranes and glycogen. This loss is accompanied by complete cytoplasmic vacuolation. The cytoplasm remains vacuolated for about 9 to 10 hours after irradiation but after this new alpha cytomembranes and glycogen are formed. (auth)

A method of tissue preparation for electron microscopy is described which allows the observation of more nuclear structure than has previously been reported. Regenerating rat-liver cells were used for this study. During the early stages of regeneration the nucleolar size was found to be much greater than normal, although nucleolonema fibres and pars amorpha were still present. At 16 to 18 h after operation, however, when there is considerable production of new $\alpha$ -cytomembranes in the tissue, much of the material of the pars amorpha was lost from many cells, and the nucleoli consisted solely of nucleolonema fibres. Later the pars amorpha was re-formed, but just before the start of mitosis at 25 to 26 h after operation, many of the nucleoli became very irregularly shaped. The various mitotic stages seen in liver cells with the electron microscope were recognizably similar to those previously seen in living mammalian cells with the light microscope. In the earliest stages of prophase the chromosomes appeared as strands approximately 1000 $\overset{\circ} {\mathrm A}$ in diameter, which were made up of thin fibres approximately 100 $\overset{\circ}{\mathrm A}$ in diameter. By mid-prophase these strands could be seen running diagonally across the chromosomes in a manner suggestive of coiling, but by late prophase this coiling was no longer visible and the chromosomes appeared as homogeneous masses of fibres 100 $\overset{\circ}{\mathrm A}$ in diameter. The double nature of the chromosomes was, however, evident during metaphase. Telophase nuclei were seen to contain fibrous inclusions approximately 1000 $\overset{\circ}{\mathrm A}$ in diameter which it is suggested may represent stages in the re-formation of the nucleolus after mitosis. The effect of X radiation on the ultrastructure of the nuclear components of regenerating liver cells was also studied in these experiments. With small doses (450 r) no ultrastructural changes were found in the chromosomes during cell division, but some changes were visible in the nucleoli just prior to mitosis. Larger doses (2000 r) caused chromosome fragmentation and a loosening of the fibrous chromosome structure. In telophase it was found that when the separation of the two daughter nuclei is inhibited or delayed, the nuclear membrane re-forms around the two partially separated chromosome masses.

After the intrapleural injection of asbestos dust in experimental animals, granulomas were produced and large areas of the pleural surface became thickened with fibrous tissue. In guinea-pigs injected with chrysotile dust, alveolar epithelial cells often migrated through the lung surface and were found lining spaces in the areas of pleural fibrosis. Initially these cells exhibited the exact structure of the surfactin-secreting type of alveolar cell, but later many of the cells lost the surfactin granules from their cytoplasm and became ciliated. In some animals cystic formation occurred in the lung tissue and groups of cysts grew out from the lung to invade the areas of pleural fibrosis. These cysts were also lined with surfactin-secreting alveolar cells. The possible association of these epithelial outgrowths and the well-known difficulties of diagnosing pleural mesotheliomas are discussed. Excroissances Epitheliales dans le Tissu Pulmonaire Aprks Injection Intrapleurale de Poussikre D'Amiante A des Animaux D'Experience Apres l'injection intrapleurale de poussiere d'amiante a des animaux d'experience, des granulomes sont apparus et de vastes zones de la surface pleurale ont ete epaissies par des tissus fibreux. Chez les cobayes ayant recu une injection de poussiere de chrysotile, les cellules epitheliales alveolaires emigrent souvent a la surface du poumon et vont se grouper en certains points des zones de fibrose pleurale. Ces cellules presentent d'abord la structure exacte du type de cellule alveolaire qui secrete la surfactine, mais par la suite un grand nombre d'entre elles perdent les granules de surfactine de leur cytoplasme et deviennent ciliees. Chez certains animaux, des formations kystiques sont apparues dans le tissu pulmonaire et des groupes de kystes se sont formes sur le poumon et ont envahi les zones de fibrose pleurale. Ces kystes etaient egalement recouverts de cellules alveolaires secretant de la surfactine. Les auteurs etudient la possibilite d'une association entre ces excroissances epitheliales et les mesotheliomes pleuraux, dont les difficultes du diagnostic sont bien connues.

Intraperitoneal asbestos injection in mice has previously been reported to elicit an activated macrophage population. In the present study supernatants from such macrophages were tested for their effect on thymocyte mitogenesis in response to concanavalin A; control supernatants were obtained from saline- and latex-elicited macrophages. Supernatants from asbestos-elicited macrophages were significantly inhibitory to thymocyte mitogenesis while saline- and latex-elicited macrophages did not release significant amounts of such activity. Asbestos-activated macrophage supernatants were inhibitory in a dose-dependent way and the activity was not secreted by macrophages from mice which had received asbestos in the long term. The inhibitory activity was partially dialysable. Supernatants prepared by treating macrophages in vitro with a lethal dose of asbestos were not inhibitory suggesting that the inhibitory activity in the supernatants of asbestos-activated macrophages did not leak from dead or dying cells. The asbestos macrophage supernatant was also significantly inhibitory to mature T-cell-enriched spleen cells but had no effect on fibroblasts, suggesting that the inhibitory effect could be lymphoid cell specific.

Samples of commercially used asbestos, especially chrysotile, are frequently contaminated by small amounts of other fibrous minerals. Among these are tremolite and brucite although pure tremolite is also produced commercially in relatively small quantities. In order to determine how harmful commercially exploited tremolite might be in comparison with other asbestos types and to explore the possibility that small amounts of tremolite and brucite as contaminants could significantly affect the pathogenicity of industrially used chrysotile, long-term animal inhalation and injection studies using rats were undertaken with what were considered to be mineralogically pure samples of these minerals. Rats treated with tremolite developed very high levels of pulmonary fibrosis as well as 16 carcinomas and two mesotheliomas in a group of 39 animals. Tremolite thus proved to be the most dangerous mineral that we have studied. Animals treated with 'brucite' developed moderate levels of pulmonary fibrosis and two carcinomas. Both tremolite and brucite produced mesotheliomas in greater than 90% of animals following i.p. injection. However, it was found that the supposedly pure brucite in fact contained 10% chrysotile, a level of contamination that could well have been responsible for the pathological changes found in both inhalation and intraperitoneal injection studies. The greatest care should be exercised by industry in handling tremolite or materials contaminated with it.

The kinetics of the bronchoalveolar response was assessed in rats exposed, at equal airborne mass concentration (10 mg/m3), to titanium dioxide--a non-pathogenic dust--and the two pathogenic mineral dusts quartz and chrysotile asbestos. Rats were killed at intervals over a 75 day exposure period and groups of rats exposed for 32 and 75 days after recovery for two months. Bronchoalveolar lavage was carried out and the lavage fluid characterised for cellular content, macrophage activation, and concentrations of free total protein, lactate dehydrogenase, and N-acetyl-beta-D-glucosaminidase. Inhalation exposure to the two pathogenic dusts resulted in an increased number of leucocytes, macrophage activation, and increased levels of free enzymes and total protein. The pattern and magnitude of the responses to quartz and chrysotile differed. Chrysotile caused less inflammation than quartz, and the main cellular response peaked around the middle of the period of dust exposure whereas the highest levels of enzymes occurred towards the end. The difference in timing suggests that macrophages were not available for lavage towards the end of the exposure, owing to their playing a part possibly in deposition of granulation tissue. Quartz caused a greater cellular and enzyme response than chrysotile, particularly towards the end of the dust exposure phase. There was a noticeable progression of inflammation in the quartz exposed groups left to recover for two months, but not in the chrysotile recovery groups.

Two groups of 48 rats of the AF/HAN strain were exposed for one year to respirable dust clouds of UICC chrysotile asbestos at a dose level of 10 mg/m3. One group was treated with dust carrying the normal electrostatic charge produced during dust generation, whereas the other was exposed to dust discharged by exposure to ionising radiation from a thallium-204 source. After dusting most animals were retained for their full life span. At the end of the dusting period those animals treated with normally charged dust had significantly more chrysotile retained in their lungs than animals exposed to discharged dust. Subsequently, animals treated with normally charged dust developed more pulmonary fibrosis and more pulmonary tumours. These findings suggest that the charge carried by airborne fibres should be taken into account when considering the health risks from exposure to chrysotile. Highly charged fibres are more likely to be deposited in lung tissue and thus constitute a greater hazard.

Much has been learned concerning the harmful effects of mineral dusts and other toxic materials by both in vivo and in vitro techniques. Both approaches have disadvantages, however. With in vivo studies exposure can be conducted under physiologically normal conditions but it is more difficult to unravel the complex sub-cellular and molecular events. In vitro, target cells can be examined in much greater detail but dose levels may have to be excessive to produce effects in an acceptable time scale and the absence of the whole of the body’s defensive systems and factors such as recruitment of new populations limit this approach. An approach now being adopted more frequently where target cells are readily available by simple techniques such as pulmonary lavage is to undertake exposure to harmful substances in vivo followed by examination of target cells and their products in vitro. This paper reports studies in which rats were exposed to three coalmine dusts, two varieties of asbestos, quartz and titanium dioxide administered by inhalation. Subsequently, their lungs were lavaged and a series of studies undertaken with the cell populations obtained.

Mineral fibers in the lungs of 13 Pittsburghers and 10 Cbarlestonians as well as of 7 workers exposed to asbestos dust have been quantitated, and the methodology has been described in some detail. Although wide variations in fiber concentration were found in people of the same sex and of the same community, the average concentration of optically visible pulmonary mineral fibers of Pittsburghers was about seven times as high as that of Charlestonians. The average pulmonary concentration of optically visible fibers in people not occupationally exposed to asbestos was about 0.7% of the average concentration of optically visible fibers in asbestotic lungs. From 54 to 71% of the EM-size fibers were less than 5 μm long. Of the EM-size mineral fibers in the lungs of people not occupationally exposed to asbestos, an average of 6.8% were identifiable as chrysotile.

Preliminary results are reported from an International study on the mineral content of respirable coalmine dusts, the toxicity of these dusts as recorded by short-term in vitro and in vivo tests and their relationship to epidemiological data on levels of pneumoconiosis in the collieries concerned. The results from the mineralogical analyses show extremely good correlation between the different laboratories involved. In general, the correlations between the in vitro biological results are poor, although in most cases there was agreement on which were the most toxic dusts. At present, no information is available from the in vivo studies or from epidemiological comparisons.

An inhalation study has been undertaken to examine the relative pathogenicities of coalmine dusts containing different proportions of quartz. To minimise confounding effects the airborne dusts were collected from within a single seam at one colliery. Animals were exposed either to a high (25%) quartz dust, a low (7%) quartz dust, a medium (13%) quartz dust or an alternating regimen of 2 weeks high quartz and 6 weeks low quartz dusts. The respirable dust concentration was 20 mg/m 3 in all chambers and exposure lasted 12 or 18 months. Groups of animals were killed immediately after the end of the dusting phase and, thereafter, at 4 monthly intervals. Detailed pathological examinations were undertaken on each animal and lung and lymph node dust burdens and compositions were determined. The most important differences in response between treatments were in the development of discrete cellular pigmented pulmonary nodules which were most profuse in animals exposed to high quartz dust. Details of these differences and other pathological responses are described in relation to challenge dust composition and the lung and lymph node dust burdens and composition. In addition, some evidence for the inhibition of pulmonary response from continuing dusting was found.

The activities of 13 carcinogenic fibrous dust samples were compared in two in vitro test systems using V79/4 and A549 cells. Eleven of the dusts had similar ranks in both assays but two samples had disparate results. The relationship between the fibre length and diameter distributions of 12 of the samples and their activity in each assay was examined. A significant association between fibre length and biological activity occurred in both assays. This relationship showed increasing strength of association with increasing fibre length. The only significant association between fibre diameter and activity in vitro was demonstrated with fibres greater than 0.2 micron in the A549 assay.

Sera from 2421 coalminers, representing all the radiological categories of pneumoconiosis, and from 260 healthy blood donors, as controls, were examined for antinuclear factor and rheumatoid factor. Antinuclear factors were present in 21.5% of sera from the controls and in 23.1% from the coalminers' group. Rheumatoid factor was present in 5.3% of coalminers and as expected occurred particularly in the few men with progressive massive fibrosis who also had rheumatoid disease. The combined prevalence of both factors showed an increase with age at all disease levels and a significant association with pneumoconiosis category only in men older than 60 years. This study provides no evidence that autoantibodies are likely to be of value in detecting men predisposed to the development of massive fibrosis other than those with rheumatoid disease.

In order to explain why differences in frequency of pneumoconiosis are observed between European coalmines, 23 samples of respirable dust were collected from selected collieries in Belgium, West Germany, France and the United Kingdom. Their physical and mineralogical characteristics were determined and their cell and tissue toxicity measured (the work being funded in part by the European Coal and Steel Community). The relationship between these two sets of data was researched and the results compared with the available epidemiological data. Samples of a high mineral content show the highest concentration of fine particles. The main mineral components are, in order of decreasing concentration, mica, kaolinite and quartz, the relative proportion of the three being variable. The dusts were not particularly toxic, regardless of whether testing was in vitro or in vivo. The most representative tests were selected and the 23 samples distributed among three classes: marked, moderate and low toxicity. Toxicity is well correlated with mineralogical composition. In vivo tests show that toxicity increases not only with mineralogical content but also with quartz content. The protective effect of mica is confirmed. The epidemiological data correlate poorly with in vitro tests, but better with in vivo tests.

At the Institute of Occupational Medicine in Edinburgh, we have studied the in vitro effects of a series of mineral fibre samples using three different permanent cell lines. The results of this work have been summarised in the companion paper by Gormley et al.. Relatively consistent grading of the dusts was obtained by the different test systems with preparations of chrysotile asbestos consistently being found to be more active than varieties of amphibole or other mineral fibres. In addition there was an overall relationship between toxicity and fibre length. Most of the dust preparations used in these in vitro studies have also been tested in rats using either long term dust inhalation or intraperitoneal injection (Davis et al. 1978, 1980, 1984; Bolton et al. 1982). This paper reports how the results of the animal studies compare with the in vitro work.

The ability of mineral fibers to cause disease in man is well established and the work of Stanton et al. (1977), Stanton and Layard (1978) and Pott (1978) has indicated that the ability of a dust to induce intrapleural tumors depends on the number of fibers in the sample that are greater than 8 pm in length and less than 1.5 μm in diameter.