Inhalation and injection experiments in rats to test the carcinogenicity of MMMF.
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Biomedical subjects
Publications and source records attributed to F Pott.
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Attapulgite (palygorskite) and sepiolite are fibrous clay minerals used commercially as components in a wide variety of products including oil and grease adsorbents, carriers for pharmaceuticals, cosmetics, and pesticides. They are also components of drilling muds and animal litter and they are used as paint thickeners. The current annual worldwide production of these minerals exceeds one million tons. Although fibrous in nature, the fibre length may vary greatly depending on the location of the geological deposits. American attapulgite is short (0.1-2.5 micron in length, median of 0.4 micron) but palygorskite from other parts of the world is much longer (30% longer than 5 micron). Several samples of these materials have been submitted to scanning transmission electron microscopy (STEM). This paper reports the results of microscopic evaluations and makes a comparison with the data from experimental carcinogenicity studies and it is concluded that fibre length is a most important carcinogenic property.
According to the findings that long, thin, and durable fibres have a high carcinogenic potency after intrapleural and intraperitoneal administration, the elongated shape of a particle represents a carcinogenic agent; this physical phenomenon is a special cause of cancer. It induces a biological process which can lead to cancer by several as yet unknown steps. However, the properties of the material the fibres are made of determine the carcinogenic potency of a fibre in a secondary way although they do not seem to be responsible for the true carcinogenic agent. For example, these properties determine the degree of solubility and flexibility. The persistence of fibres in the tissue is a very important property with regard to their carcinogenic effect because the formation of a tumour takes many years or some decades. It can be assumed that a fibre has to remain by the bronchial or serosa tissue until the induction of tumour cells occurs. If this hypothesis is correct, there could be a "durability threshold value" for fibres whose length and diameter would otherwise indicate a high carcinogenic potency. There are indications that other fibre properties apart from length, diameter and durability are important for tumour induction, however, at present, they cannot be included in a definition of carcinogenic fibres. It is proposed to classify all natural and man-made mineral fibres with an aspect ratio of greater than 5:1 as carcinogenic when they are longer than 3 microns, thinner than 1 micron (or can split into such fine fibres) and when they can persist in the tissue for more than 3 years.
A long-term exposure study with hamsters, mice and rats inhaling filtered and unfiltered diesel engine exhaust was carried out to investigate effects of chronic toxicity and, predominantly, carcinogenicity in the respiratory tract. The level of diesel exhaust in the exposure chambers corresponded to a concentration close to 4 mg m-3 in the unfiltered diesel exhaust. Satellite groups of animals were additionally treated with BaP, DBahA or nitrosamines in order to check for syncarcinogenic effects. In hamsters and rats, alveolar lung clearance and mechanical lung function tests as well as biochemical and cytological measurements in lung lavage fluids showed significant changes only after exposure to unfiltered diesel exhaust and, predominantly, in rats. No lung tumors were found in hamsters. Spontaneous tumor rates occurred in mice and both types of diesel exhaust increased the incidence of adenocarcinomas in the lungs. In rats, only the unfiltered diesel exhaust caused a lung tumor incidence. It amounted to 16% with no tumors in the controls. The heavy load of particulate matter in the lungs of rats was caused by an exposure-related impairment of the alveolar lung clearance and may have been instrumental in the induction of squamous cell tumors. However, an effect of particle-associated PAH cannot be excluded. Syncarcinogenic effects of diesel exhaust after initial carcinogen treatment were found only in the respiratory tract of rats.
Rats and mice were exposed to a PAH-rich emission to test the carcinogenic activity. The BaP content of the exposure atmosphere was only 2-3 times higher than the concentration of about 30 micrograms/m3 measured in older coke plants. Although only half of the rats exposed to the exhaust have been investigated histologically up to now, the lung cancer incidence already amounts to 11%, no lung tumours were found in the control animals. Also the mice exposed to the exhaust with and without additional treatment with BaP, DBahA or urethane to induce a "base tumour rate" in the lung, showed a clear exhaust exposure-related carcinogenic effect. The lung tumour multiplicity as well as the incidence was significantly higher. The epidemiological findings of an increased lung cancer risk in coke oven workers can now be supported by the results of this animal inhalation study.
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The ability of particulate air pollutants (and possible constituents) to alter pulmonary host defenses was examined using an in vitro alveolar macrophage cytotoxicity assay and an in vivo bacterial infectivity screening test which employed intratracheal injection of the particles. A wide range of response between particles was seen at the 1.0-mg/ml level in vitro and the 0.1-mg/mouse level in vivo. A sample of fluidized-bed coal fly ash, bentonite, asbestos, some ambient air particles, and heavy metal oxides greatly increased susceptibility to pulmonary bacterial infection. Most coal fly ash samples and some air particles caused moderate increases in infectivity, while diesel particulates, volcanic ash, and crystalline silica caused only small increases. Cytotoxic effects on macrophages in vitro were observed with most of the particles. The in vivo and in vitro assays produced a similar ranking of toxicity; however, not all particles that were highly cytotoxic were potent in increasing bacterial infectivity. Increased toxicity measurable by either assay often appeared to be associated with small size or with the presence of metal in the particles.
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After intratracheal instillation of glass fibres and crocidolite in male Syrian golden hamsters, besides lung carcinomas a number of mesotheliomas of the epitheloid type occurred. These were examined with light microscopy (LM) and scanning electron microscopy (SEM); the characteristics of the tumours are described.
Most air pollution in West Germany is caused by combustion products. Particulate organic matter released by incomplete combustion is suspected to contribute to the "urban factor" of lung cancer frequency in urban-industrial centers. The carcinogenic potential of single components, groups of compounds and total source emissions of combustion processes was investigated in laboratory animals by subcutaneous injection, intratracheal instillation or inhalation. Tests by subcutaneous injection of condensates of automobile exhaust, extracts of coal furnace emissions and of airborne particles and different fractions of these extracts showed that the polycyclic aromatic hydrocarbons (PAH) with four to six benzene rings have the strongest experimental carcinogenicity. However, polar compounds (heterocyclic nitrogen-containing PAH, phenols, and others) also show remarkable carcinogenic potency. There were large differences between the dose-response relationships of several PAHs. In the subcutaneous tissue, benzo(a)pyrene and dibenz(a,h)anthracene are the most carcinogenic of the tested airborne PAHs. Furthermore, they can induce high tumor rates in the lung after subcutaneous injection in newborn mice and after intratracheal instillation of mice or hamsters. The tumor rate of benzo(a)pyrene did not further increase after simultaneous instillation of carbon black, but lead chloride may have a promoting effect. Far more than 100 PAHs are found in the urban atmosphere. However, because of the remarkable similarity of the PAH profiles in the examined samples, it may be sufficient to measure just a few stable PAHs in the urban air in order to facilitate an assessment of the carcinogenic potency of the PAH content in the atmosphere. To examine the carcinogenic or cocarcinogenic effects of gas and vapor emissions, studies with a two-phase model were carried out: phase 1 relates to the induction of a basic tumor rate in the lung by a well known carcinogen, while phase 2 is characterized by an inhalation of the substance under investigation. In an experiment with mice, the inhalation of a mixture of SO2 and NO2 seemed to increase the basic tumor rate induced by dibenz(a,h)anthracene. In a similar two-phase experiment conducted with hamsters, the inhalation of diesel exhaust (total exhaust as well as exhaust without particles) increased a basic tumor rate induced by diethyl nitrosamine. These experiments deserve confirmation before a detailed interpretation is attempted.
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The papers presented in this session are summarized. Although asbestos fibres produce tumours in a number of animal species tested, rats appear to be the most susceptible, in terms of latent period and numbers of tumours produced. The deposition, translocation and clearance of different types of fibres in the lung have been investigated in a number of experiments, and it has been shown that many of them migrate more readily than was previously thought; their penetration into the gut was the object of further investigation. The syncarcinogenicity with asbestos of various substances, such as benzo[a]pyrene, N-nitrosodiethylamine, cigarette smoke or radiation, is described. Experiments on the different carcinogenicities of different fibres are summarized; although it is pointed out that there is much controversy in this area. A hypothesis is presented whereby the carcinogenic potency of a fibre is dependent on various size parameters, based on length, diameter and length:diameter ratio. On the basis of this hypothesis, the carcinogenic potency of short fibres may be weak, but many short fibres may induce a tumour as easily as a few long fibres. Finally, a plea is made for a far greater number of well-defined standard samples of asbestos and man-made mineral fibres than exists at present, since there are currently great difficulties in comparing and interpreting results.
I.p. injection of glass fibres to rats of different strains resulted partly in different tumour rates. Young rats showed more marked coalescence and earlier growth of tumours but also earlier mortality without tumours, as compared with older rats. Haemorrhagic ascites from tumour-bearing rats induced tumours after i.p. injection. Tumours were also obtained after i.p. injection of fibres in European hamsters and rabbits, but these species are less susceptible than rats. Histological studies in rats show that fibrosis is not an absolute requirement for the development of a fibre-induced mesothelioma. After intratracheal instillation of either crocidolite or glass fibres in European hamsters, the respective fibres showed up in the diaphragm and in the spleen.
Airborne particulate matter was collected on filters at various times in Duisburg, Duesseldorf, Muenster and in 2 rural areas. After extraction with organic solvents the extract was partly fractionated and analyzed for BaP and 7 other polycyclic aromatic hydrocarbons (PAH). The carcinogenicity was examined with the subcutaneous test using 7000 mice in total. The amount of extract required was relatively small. Extracts from suspended matter with a BaP content of 0.37 to 1.1 microgram BaP brought about tumour rates in the range of 30%. On account of the results the following can be concluded: 1. The total extract of atmospheric particulates results in definite dose-response relationship, when the part of the compounds not belonging to the PAH is rather low. 2. The PAH fractions derived from the suspended matter collected in different cities show the same carcinogenic potency, when they are adjusted to the same BaP level by means of concentration or dilution. 3. The PAH bear the most important share with respect to the carcinogenic potency of an extract from suspended matter. 4. Numerous compounds (polar substances) which are not classed with the PAH are carcinogenic to some extent, partly they are able to inhibit the carcinogenic effect. 5. The dose-response relationship of BaP is strongly leveled off in general when all the substances of an extract are present. That means when the dose of an extract is low, the importance of BaP becomes less. Therefore a fixed ratio concerning the effect of BaP within an extract cannot be given. It has to be examined in particular, which PAH and non-PAH are necessary to analyse in order to describe the carcinogenic effect due to an extract from airborne particulates. Much more attention is to be payed to the polar substances.
The relative proportion of eight polycyclic aromatic hydrocarbons (PAH) in suspended particles from several sampling stations with different level of air pollution (cities and rural districts) was investigated. The PAH-profiles of most samples collected in three winters (1974/75, 1975/76, 1976/77) and in one summer (1975) were very similar. On the other hand the PAH-profiles differ distinctly from those of several exhausts, especially regarding automobiles, coke and oil stoves. It can be presumed, that the mixture of the emissions in the atmosphere leeds to a rather uniform relative proportion of the eight PAH in the immission as much as the difference in stability of the PAH.
Automobile exhaust condensate (AEC), either mixed with benzo[a]pyrene (BaP) or suspended or dissolved in tricaprylin, was injected subcutaneously into NMRI mice in a series of experiments. The addition of AEC decreased the incidence of tumours which developed with 30, 90 and 270 microgram BaP. Reduction of tumour incidence was proportional to the amount of AEC added. With an injection of 10 microgram BaP, the latent period was greatly increased when AEC was added, but the occurrence of tumours was the same. Components of AEC appear to inactivate BaP, at least temporarily. In further experiments AEC and nine fractions thereof were injected subcutaneously into mice. The fraction comprising only polycyclic aromatic hydrocarbons (PAH) induced the highest incidence of tumours. In contrast, when it was administered in combination with other fractions the PAH fraction was less active. Application of the products of further fractionation of PAH showed that polycyclic compounds with seven or more rings can also induce tumours in this model.