Time-to-tumor dose threshold analysis for intratracheal particle instillation-induced lung tumors in a large carcinogenicity study.

BACKGROUND AND OBJECTIVES: A comprehensive time-to-tumor analysis of the 16 dose groups which received intratracheal instillations of "respirable granular bio-durable particles without known significant specific toxicity" (GBP) in a large carcinogenicity study with rats should be conducted. METHODS: The primary lung tumors were mathematically treated as observed in an incidental context (non-fatal occult tumors), based on biological observations and on the fact that lifetime was not considerably reduced even in groups with high tumor frequency. Maximum likelihood estimates of the parameters of time-to-tumor multistage Weibull models were calculated. RESULTS: Retained dust volume is a highly significantly better dose measure than instilled dust mass, where particle size is taken into account; there is no empirical support for a dose threshold from this study. CONCLUSIONS: Carcinogenicity studies with intratracheal instillation can lead to results that are relevant for the assessment of relative carcinogenic potencies of particles. A dose threshold for GBP is not supported.

In vitro genotoxicity data of nanomaterials compared to carcinogenic potency of inorganic substances after inhalational exposure.

Literature data of epidemiological studies, carcinogenicity studies and in vitro studies on inorganic substances were surveyed with the aim to determine sensitivity and specificity of in vitro tests of nanomaterials. Asbestos, quartz and chromium and cadmium compounds were assigned to classes of highest carcinogenic potency. After 20 years of occupational exposure to long-term average concentrations of 0.5mg/m(3) of these dusts - or to even lower concentrations - an epidemiologically detectable increased lung cancer risk has to be expected. In contrast, diesel engine emissions, some nickel species and "ultrafine" versions (nanomaterials) of titanium dioxide and carbon black were also carcinogenic in inhalation studies, but show varied epidemiological results. The high frequency of lung cancer in the male general population due to cigarette smoking hampers unequivocal detection of occupationally caused lung cancer risks. Based on the experience from the inhalation studies, workers had to be exposed to long-term concentrations of 1mg/m(3) or more to identify epidemiologically a clear cause-and-effect relationship for a specific substance of intermediate potency. Respirable granular biodurable particles without known significant specific toxicity with primary particle sizes of more than 1µm have also shown carcinogenicity in rats. Their potency was even lower; and partially results after instillation rather than inhalation are available. Nearly all types of nanomaterials and control dusts used in the in vitro assays showed genotoxic effects in cell cultures (e.g., CoCr particles, diesel soot, SiO(2) crystalline and amorphous, TiO(2), carbon black), but not consistently in all studies; overall, the proportion of positive results was about 50%. No clear correlation of the probability of a positive in vitro test with particle properties was seen. I recommend trying and calibrating a sensitive in vitro model (e.g., micronucleus assay) against the described rank order of carcinogenic potency by testing a series of inorganic substances.

Carcinogenicity of inhaled nanoparticles.

Large epidemiological studies in the United States have shown a statistical association between air concentration of the fine dust fraction PM(2.5) in the general environment and increased risk of lung cancer. A quantitative risk assessment for lung cancer based on these studies corresponds to risk estimates based on studies at workplaces with exposure to diesel engine emissions; its magnitude cannot be explained by the known carcinogenicity of organic substances or metals adsorbed to the insoluble particle core. Carcinogenic effects of diesel particles were observed after inhalation in rats independently in several studies. The surprisingly strong effect of diesel particles was partially attributed to their small size. This hypothesis was corroborated by inhalation studies with synthetic nanoparticles virtually free of organic compounds. IARC found sufficient evidence for the carcinogenicity of carbon black and of titanium dioxide in experimental animals. Long-term studies by the method of intratracheal instillation confirmed the carcinogenic effects in rats for an even broader spectrum of synthetic nanoparticles. Non-positive studies with hamsters are not valid because hamsters did not develop lung tumors after inhalation of some known human carcinogens. In recent years, the number of publications reporting in vitro genotoxicity of TiO(2) and of carbon black nanomaterials has increased. Overall, there is clear positive evidence for carcinogenicity in rats, together with supporting evidence from human data of structurally related substances. Therefore, the European Union (EU) criteria for category 2 of carcinogenic substances appear to be fulfilled for bio-durable nanoparticles consisting of matter without known significant specific toxicity.

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.

Differences between the data bases, statistical analyses, and interpretations of lung tumors of the 19-dust study: two controversial views.

The article presents a comparison of two opposing evaluations and interpretations of the carcinogenicity study with 19 granular dusts. It is one of the two responses to a Letter to the Editor of Exp Toxicol Pathol by Morfeld and Borm(1), who had analysed selected data from the primary source of our study [Borm et al., Inhal Toxicol 2000;12(Suppl 3):225-31; Borm et al., Int J Cancer 2004;110:3-14; Morfeld et al., Inhal Toxicol 2006:18:215-25]. In contrast, our statistical analyses are based on the authentic and complete results of the study published with a detailed description of materials, methods and results [Pott and Roller, Eur J Oncol 2005,10:249-81; Mohr et al. Exp Toxicol Pathol 2006;58:13-20; Roller and Pott, Ann NY Acad Sci 2006,1076:266-80]. Analysis of the tumor incidences from non-specifically toxic dusts in terms of a usual multistage model leads to reasonable dose-response curves and to the finding that tumor risk depends on the retained dust volume and on mean particle size. The model is adequate because survival of the groups included in the analysis was not reduced. In contrast to this analysis, Morfeld et al. (2006) calculated an implausible threshold of 10mg instilled total dust per rat and a saturation dose of 20mg. These values are not compatible with the data because three dose groups with highly significantly increased tumor incidences (up to 67%) lie within the 95% confidence interval of the threshold dose of Morfeld et al. (2006). The claim of Morfeld [Zbl Arbeitsmed 2004;54(7):246-58] to consider as "of interest" only carcinogenicity tests with doses similar to current occupational exposure limit values would make it impossible to identify the carcinogenicity of non-specifically toxic dusts even if the excess risk is higher than 1 in 100.

Lung tumor risk estimates from rat studies with not specifically toxic granular dusts.

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/m(3) (density 2-2.5 g/mL); mean diameter 1.8-4 microm (GBP-fine-large): ER 0.1%; 0.09-0.2 microm (GBP-fine-small): ER 0.2%; 0.01-0.03 microm (GBP-ultra-fine): ER 0.5%.

Pulmonary tumor types induced in Wistar rats of the so-called "19-dust study".

The incidences of primary lung tumor types histologically diagnosed in 28 groups of Wistar rats of the so-called "19-dust study" are described, the total study having been already presented by Pott and Roller (Carcinogenicity study with nineteen granular dusts in rats. Eur J Oncol, 2005; 10: 249-81). Each exposed group was repeatedly instilled intratracheally with a suspension of one type and dose of 13 non-mining dusts differing in at least one of the following properties: chemical composition, density, specific surface area, and mean particle size. Eleven of the 13 dusts were classified as respirable granular bio-durable particles without known significant specific toxicity (abbreviation of the nine-word definition: GBP). In 579 (58%) lungs of 1002 rats which survived more than 26 weeks after the first instillation of GBP, at least one primary lung tumor type was observed, and in 306 (31%) at least two types. Three benign tumor types were diagnosed in the 579 tumor-bearing rats: bronchiolo-alveolar adenoma in 46%, cystic keratinizing epithelioma in 53%, and non-keratinizing epithelioma in 2.6% of the rats. Two of three malignant tumor types (bronchiolo-alveolar carcinoma and squamous cell carcinoma) occurred in 46% and 31% of the tumor-bearing rats, respectively, and adenosquamous carcinoma was diagnosed in 0.9%. Numerous lungs with a malignant tumor also showed one or more benign tumor types. In addition, single or multiple metastases from primary tumors of other sites (mainly carcinoma of the uterus) were diagnosed in 14% of the 1002 lungs. The proportionate incidences of the four predominantly diagnosed tumor types were compared with three summarized experimental groups which were exposed either to carbon black (two size classes), to titanium dioxide (two size classes), or to the total of the other nine GBP. A significant difference was not detected. The combination of dust volume with particle size correlated best with the carcinogenic effect, in contrast to dust mass and surface area.

Nephrotoxicity of organic solvents: biomarkers for early detection.

OBJECTIVES: Evidence for a relationship between chronic kidney diseases or progression of already existing diseases (glomerulonephritides) and occupational solvent exposure has been found in case reports, in case-control studies and also in cross-sectional studies. An analysis of the available literature was performed with respect to markers measured in cross-sectional studies that might be useful for an early detection of solvent-induced effects on the kidney. METHODS: The relevant cross-sectional studies were evaluated and the following markers were analyzed with respect to their suitability as biomarker for renal damage: total protein, albumin, transferrin, IgG, beta(2)-microglobulin, retinol-binding protein, N-acetyl-beta-D: -glucosaminidase, alanine aminopeptidase, beta-galactosidase, beta-glucuronidase, leucin aminopeptidase, alkaline phosphatase, lysozyme, Tamm-Horsfall protein and laminin fragments in urine as well as E-selectin, laminin and anti-laminin antibodies and anti-glomerular basement membrane antibodies in serum. RESULTS: An increased albumin excretion was observed more frequently in groups of workers exposed to various solvents (like toluene, styrene, aliphatic/aromatic hydrocarbon mixtures, tetrachloroethene, mixtures of chlorinated hydrocarbons) than in controls. No clear pattern emerged for the other markers. CONCLUSIONS: The determination of albumin excretion in the urine appears to be a useful parameter for monitoring solvent-exposed workers.