Markus Roller

Results of current intraperitoneal carcinogenicity studies with mineral and vitreous fibres

The study includes some 50 groups of male or female Wistar rats tested in three series. Except for one untreated group and 3 vehicle control groups, the animals were injected intraperitoneally (i.p.) once or repeatedly with dust suspensions and then examined, after lifetime observation up to 30 months, for tumours in the abdominal cavity. 1 granular dust (silicon carbide), 2 asbestos dusts (crocidolite, tremolite) and 11 vitreous fibre dust samples were administered. 5 of the vitreous fibre types were fine fibre fractions from 4 commercial insulation wools and 1 experimental wool, the others were prepared by milling glass microfibres, which have, per se, a small diameter range. The dosage per rat differed over a wide range in accordance with experience from earlier studies. The lowest dose was 0.04 x 10(9) crocidolite fibres in 0.5 mg dust, and the highest amounted to 20 x 10(9) glass fibres in 1000 mg divided into 40 weekly injections. Two mesotheliomas were found in a total of 395 rats treated with saline or granular silicon carbide (250 or 1000 mg). Eleven fibre dusts produced dose-dependent mesotheliomas at rates of up to 97 %, but the calculated fibre number > 5 micrometers in length required for inducing a 25 % tumour risk differed between the fibre samples tested in the relation of 1 to about 1000. UICC-like crocidolite heads the ranking order; the glass fibre B-01, which possesses a low durability in the body, ends it together with a rather thin sample of glass fibre type B-09. The stone fibre MMVF-21 takes a high place in the ranking order, similar to the tremolite sample. The results correspond to those of earlier i.p. tests.

Tumours by the Intraperitoneal and Intrapleural Routes and their Significance for the Classification of Mineral Fibres

In 1967, the first author of this paper started intraperitoneal studies on the carcinogenicity of asbestos. Later Wagner and coworkers (1974) published their large inhalation experiment with five asbestos types and this provided the incentive to perform similar inhalation studies with man-made mineral fibres. This aim was achieved in 1983 after some years of planning and the construction of the facilities for inhalation toxicology at the Fraunhofer Institute in Hannover. It was a great disappointment that Muhle and coworkers (1987) at the new facilities could not even find a clear carcinogenic effect in the groups exposed to chrysotile or crocidolite. Even in a parallel injection study with about 109 chrysotile fibres there was no clear carcinogenic effect. In these experiments Californian chrysotile was used as a positive control following the recommendation of NIOSH. The crocidolite sample used gave an unequivocally positive result after intraperitoneal injection of a relatively low number of fibres but no significant carcinogenic effect could be detected in the inhalation study. This compares with the work of Wagner and others (1985, 1987) who also failed to reproduce the high tumour rates observed with crocidolite in their previous inhalation study.

Effect of subchronic 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure on immune system and target gene responses in mice: calculation of benchmark doses for CYP1A1 and CYP1A2 related enzyme activities

Abstract The dose-effect relationships were analysed for several noncarcinogenic endpoints, such as immunological and biochemical responses at subchronic, low dose exposure of female C57BL/6 mice to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The animals were treated i.p. with TCDD according to the initial- and maintenance-dose principle for a period of 135 days. The initial doses were 1, 10 and 100 ng TCDD/kg, the weekly maintenance doses were 0.2, 2 and 20 ng TCDD/kg, respectively. At days 23, 79 and 135 of TCDD treatment 10 animals of each dose group were killed. As immunological parameters the number of thymocytes and the pattern of thymocyte subpopulations were determined. In liver, lung and thymus, mRNA expression of TGF-α, TGF-β1, TGF-β2, TGF-β3, TNF-α, IL-1β and different CYP1 isoforms (CYP1A1, CYP1A2, CYP1B1) was analysed. In the livers, activities of 7-ethoxyresorufin-O-deethylase (EROD) and 7-methoxyresorufin-O-demethylase (MROD) were measured. TCDD content in the liver was determined. The main results are summarized as follows: (1) The TCDD doses were not sufficient to elicit dose-dependent changes of pattern of thymocyte subpopulation. (2) TCDD failed to change the mRNA expression of TGF-α, TGF-β and TNF-α, but led to an increase of IL-1β mRNA expression in liver, lung and thymus. The results show that the TCDD induced IL-1β mRNA increase is at least as sensitive a marker as the induction of CYP1A isoforms. (3) The expression of CYP1B1 mRNA remained unchanged at the doses tested, while CYP1A1 and CYP1A2 mRNA expression was dose-dependently enhanced. EROD and MROD activities in the liver paralleled the increases of CYP1A1 and CYP1A2 mRNA expression. (4) Regression analysis of the data showed that most of the parameters tested fit a linear model. (5) From the data, a benchmark dose for EROD/MROD activities in the livers of female C57BL/6 mice of about 0.03 ng TCDD/kg per day was calculated.

Estimation of a lifetime unit lung cancer risk for benzo(a)pyrene based on tumour rates in rats exposed to coal tar/pitch condensation aerosol

Female Wistar rats were exposed to coal tar/pitch condensation (CTP) aerosol containing either 20 or 46 micrograms/m3 benzo(a)pyrene (BaP) among other polycyclic aromatic hydrocarbons (PAH) 17 h/day and 5 days/week for 10 or 20 months followed by a clean air period of up to 20 or 10 months, respectively. Based on the inhaled BaP, given as BaP exposure concentration multiplied by the total exposure time, the cumulative dose of inhaled BaP of the 4 exposure groups was 71, 142, 158 and 321 mg BaP/m3 x h and the corresponding lung tumour rates were 4.2, 33.3, 38.9 and 97.2%. There was no lung tumour in the control group. Using the US Environmental Protection Agency (EPA) linearized multistage model, the lifetime lung tumour risk for rats exposed to 1 microgram/m3 BaP as a constituent of a complex PAH mixture may be 2% or correspondingly 2 per 100,000 with a BaP concentration of 1 ng/m3. The estimation of the unit lung cancer risk for BaP based on epidemiological data from coking plants was 7-9%.

Lung Tumor Risk Estimates from Rat Studies with Not Specifically Toxic Granular Dusts

Abstract:  Since 1985 several carcinogenicity studies have been published about lung tumors in rats after exposure to respirable granular biodurable particles without known significant specific toxicity (abbreviation of this complex definition by the three letters GBP to substitute the former term inert dusts). During this time, the relevance of the carcinogenicity of GBP in rats was questioned, for example, because no lung tumors from GBP were found in hamsters and carcinogenicity in mice was questionable. However, the carcinogenesis and the tumor risk from quartz appear similar in men and rats, and the effects of GBP in rats appear not to differ, on principle, from that of quartz, but at a much higher dose level. We calculated the excess risk (ER) of GBP in rats from the final results of an instillation study with 16 GBP types in connection with results of inhalation experiments with carbon black, titanium dioxide, and diesel particles. Retained particle volume together with some indicator of particle size was identified as the best suitable dose metric and the dose‐response relationships were analyzed on the basis of the multistage model. By relating the results to the available dose–response slopes after inhalation, ER for workplace‐like exposure were calculated for three particle size classes and an exposure to 0.3 mg/m3 (density 2–2.5 g/mL); mean diameter 1.8–4 μm (GBP‐fine‐large): ER 0.1%; 0.09–0.2 μm (GBP‐fine‐small): ER 0.2%; 0.01–0.03 μm (GBP‐ultra‐fine): ER 0.5%.

Intraperitoneal Injection Studies for the Evaluation of the Carcinogenicity of Fibrous Phyllosilicates

A review of the inhalation studies performed with crocidolite shows that, in the case of mineral fibres, realistic exposure can lead to irrelevant (false negative) results. The intraperitoneal test with mineral dusts in rats is much more sensitive and the qualitative analogy to the carcinogenicity found in the lung is good. Evaluation of the carcinogenicity of different fibre types according to their activity after i.p. injection may be justified. New measurements of fibres tested in earlier experiments indicate that at least 0.5 to 1 × 109 fibres longer than 5 pm should be injected for a negative result to become valid. About 109 fibres of chrysotile Calidria did not show a clear carcinogenic effect in contrast to 4 × 107 fibres of UICC chrysotile from Canada which induced tumours in about 30% of the rats.

DNA Fingerprint Analysis Reveals Differences in Mutational Patterns in Experimentally Induced Rat Peritoneal Tumors, Depending on the Type of Environmental Mutagen

We performed tumor DNA fingerprint analysis using the synthetic minisatellite probe S3315x2 based on the 33.15-repeat unit. The aim of the study was to investigate fingerprinting patterns of peritoneal tumors induced experimentally in Wistar rats by two carcinogens with unknown mechanism of action (crocidolite asbestos and nickel powder) and, as a positive control, benzo[a]pyrene. The carcinogens were administered intraperitoneally into rats. The banding patterns obtained with DNA from 71 peritoneal tumors were compared to the corresponding normal tissues. DNA derived from peritoneal tumors induced by the three carcinogens differed with respect to mutation frequencies and mutation patterns. The mutation frequencies in these tumors, revealed by DNA fingerprinting, were 18.2% for benzo[a]pyrene, 14.8% for crocidolite asbestos, and 40.9% for nickel powder. The alterations detected in the banding pattern of benzo[a]pyrene-induced peritoneal tumors were exclusively additional bands. On the contrary, in the DNA from asbestos-induced peritoneal tumors, only deletions of bands were observed on the autoradiographs. In the DNA from nickel-induced peritoneal tumors, both types of mutations occurred. The different mutation frequencies and mutation patterns appear to discriminate between benzo[a]pyrene, crocidolite asbestos, and nickel powder, and may be related to the mechanisms of action of these compounds.

P53 mutations in tumours induced by intraperitoneal injection of crocidolite asbestos and benzo[a]pyrene in rats

Mutation analysis of the tumour suppressor gene p53 in tumours induced in the peritoneal cavity of rats revealed differences in the mutational pattern with regard to the carcinogenic substances applied. In tumours induced by benzo[a]pyrene a considerable amount of p53 mutations resulting in an altered protein structure could be detected. For the development of these tumours an escape from the p53 mediated cell cycle control can be assumed. However, in tumours of the same tumour type induced by crocidolite asbestos no mutations could be observed. Since there were even no spontaneous p53 mutations detectable in this tumour group, it is obvious that in these tumours the escape from cell cycle control does not take place via inactivation of p53. Therefore, it is concluded that the molecular mechanisms of carcinogenesis and tumour development in this tumour type depend on the type of carcinogen applied.

Statistical analysis of in vitro data for risk assessment – Exemplified for a case of Ames test data☆

Ames test data of experiments with smoke of six cigarette types were used for dose-response analysis and for derivation of a measure of mutagenic potency. Each cigarette type had been tested using a smoking machine and four dilutions of the smoke of each of seven cycles (one to seven cigarettes). Three plates had been exposed per cigarette number/smoke dilution combination and three control plates had been simultaneously exposed to clean air with each set of smoke-exposed plates. It was the aim of the statistical analysis to determine the slopes of dose-response relationships of various cigarette types and to compare them using statistical tests. Basically, the following procedure is recommended: (1) calculate a dose measure on the basis of the number of smoked cigarettes per cycle and dilution air flow. (2) Use the absolute count values of the individual plates as effect variable. (3) Describe the dose-response relations of the individual cigarette types on the basis of all available data with a polynomial model by means of Poisson regression analysis accounting for overdispersion. (4) Identify the linear dose-response region using the likelihood ratio test and restrict the data set to this region. (5) Use the slope of the linear model in the restricted data set as the basis of the mutagenicity measure. (6) Compare the slope for the individual cigarette type with the slope for a reference cigarette by means of multivariate Poisson regression using the likelihood ratio test and accounting for overdispersion. It is finally recommended to express the mutagenic potency as percentages related to the reference cigarette K2R4F. This type of cigarette was set here equal to 100%; the following values are then obtained for some commercially available cigarette types: type A 25%, type B 90%, type C 119%, type D 13%, type E 59%. The differences are statistically significant.

Carcinogenicity Testing of Bladder Carcinogens and Other Organic Compounds by the Intraperitoneal and Intravesical Route

Up to now no simple and sensitive experimental test system is available to assess the carcinogenic properties of substances which are suspected to cause bladder cancer in humans. The usual animal model in this case is the oral application (IARC, 1987). The data show, however, that it is greatly a matter of chance to select a species which is sufficiently sensitive to a certain tested substance. Bladder carcinogens were detected in the past especially with dogs. High doses and long observation periods have to be used with this species. It can be deduced from some studies, that a total of more than one kilogram of benzidine or s-naphthylamine had to be applied (Bonser et al., 1956; Walpole et al., 1954). Naturally this leads to many technical problems. Therefore, we started to investigate the applicability of two other test models which we expected to allow successful carcinogenicity testing with much smaller amounts of test substances.