Ruud A. Woutersen

Inhalation toxicity of acetaldehyde in rats. III. Carcinogenicity study

Male and female Wistar rats were exposed to acetaldehyde vapour at nominal concentrations of 0, 750, 1500 or 3000/1000 ppm during 6 h/day, 5 days/week for up to 28 months. Major compound-related effects included increased mortality, growth retardation, nasal tumours, and non-neoplastic nasal changes in each of the test groups. The treatment-related nasal changes comprised: (1) degeneration, hyperplasia, metaplasia and adenocarcinomas of the olfactory epithelium at all exposure levels; (2) squamous metaplasia accompanied by slight to severe keratinisation and squamous cell carcinomas of the respiratory epithelium at the 2 highest exposure levels; and (3) slight to severe rhinitis and sinusitis in top-concentration rats. In the larynx hyperplasia and keratinized squamous metaplasia of the epithelium in the vocal cord region were seen in many rats of the mid- and top-concentration groups. One female rat of the 1500 ppm group had developed a laryngeal carcinoma in situ. It was concluded that under the conditions of the present study acetaldehyde is both cytotoxic and carcinogenic to the nasal mucosa of rats. The effects of acetaldehyde on the nose were compared to those of formaldehyde.

Dietary fat and carcinogenesis.

Epidemiologic investigations have suggested a relationship between dietary fat intake and various types of cancer incidences. Furthermore, epidemiologic studies as well as studies with animal models have demonstrated that not only the amount but also the type of fat consumed is important. At present, the mechanism by which dietary fat modulates carcinogenesis has not been elucidated. The effects of dietary fat on the development of tumours have been summarized in the present review with emphasis on colorectal, pancreas, breast and prostate cancer. It is concluded that influence on synthesis of prostaglandins and leukotrienes may be the universal mechanism by which dietary fats modulate carcinogenesis.

The use of in vitro toxicity data and physiologically based kinetic modeling to predict dose-response curves for in vivo developmental toxicity of glycol ethers in rat and man.

At present, regulatory assessment of systemic toxicity is almost solely carried out using animal models. The European Commissions REACH legislation stimulates the use of animal-free approaches to obtain information on the toxicity of chemicals. In vitro toxicity tests provide in vitro concentration-response curves for specific target cells, whereas in vivo dose-response curves are regularly used for human risk assessment. The present study shows an approach to predict in vivo dose-response curves for developmental toxicity by combining in vitro toxicity data and in silico kinetic modeling. A physiologically based kinetic (PBK) model was developed, describing the kinetics of four glycol ethers and their embryotoxic alkoxyacetic acid metabolites in rat and man. In vitro toxicity data of these metabolites derived in the embryonic stem cell test were used as input in the PBK model to extrapolate in vitro concentration-response curves to predicted in vivo dose-response curves for developmental toxicity of the parent glycol ethers in rat and man. The predicted dose-response curves for rat were found to be in concordance with the embryotoxic dose levels measured in reported in vivo rat studies. Therefore, predicted dose-response curves for rat could be used to set a point of departure for deriving safe exposure limits in human risk assessment. Combining the in vitro toxicity data with a human PBK model allows the prediction of dose-response curves for human developmental toxicity. This approach could therefore provide a means to reduce the need for animal testing in human risk assessment practices.

Subchronic inhalation toxicity of amorphous silicas and quartz dust in rats

The inhalation toxicity of three amorphous silicas (Aerosil 200, Aerosil R 974 and Sipernat 22S) was compared with that of quartz dust. Rats were exposed to 1, 6 or 30 mg Aerosil 200/m3, 30 mg Aerosil R 974/m3, 30 mg Sipernat 22S/m3 or 60 mg quartz/m3 for 6 hr/day, 5 days/wk for 13 wk. Some rats were killed at the end of the exposure period and some were killed 13, 26, 39 or 52 wk after the end of exposure. Clinical signs, body weight, haematology, biochemistry, urinalyses, organ weights, retention of test material in the lungs and regional lymph nodes, collagen content of the lungs, and gross and microscopic pathology were determined in order to disclose possible adverse effects and to study the reversibility, stability or progression of the effects. All test materials induced increases in lung weight, and pulmonary lesions such as accumulation of alveolar macrophages, inflammation, alveolar bronchiolization and fibrosis. In addition, rats exposed to Aerosil 200, Aerosil R 974 or quartz developed granulomatous lesions. Silicosis was observed only in quartz-exposed animals. At the end of the exposure period, Aerosil 200 and quartz had induced the most severe changes. Quartz dust was hardly cleared from the lungs and the changes in the lungs progressed during the post-treatment period, and eventually resulted in lesions resembling silicotic nodules and in one squamous cell carcinoma. Although Aerosil 200 was very quickly cleared from the lungs and regional lymph nodes, the changes in these organs were only partly reversed during the post-exposure period in rats exposed to 30 mg/m3. Aerosil R 974 and the lower levels of Aerosil 200 resulted in less severe, and mostly reversible, changes. The slightest changes were found after exposure to Sipernat 22S, notwithstanding the persistence of this silica in the lungs during the major part of the post-treatment period. The results of this study revealed that only quartz induced progressive lesions in the lungs resembling silicotic nodules. Of the amorphous silicas examined Aerosil 200 induced the most severe changes in the lungs, which only partly recovered, whereas Sipernat 22S induced the least severe, completely reversible lung changes.

Relative Developmental Toxicity of Glycol Ether Alkoxy Acid Metabolites in the Embryonic Stem Cell Test as compared with the In Vivo Potency of their Parent Compounds

The embryonic stem cell test (EST) has been proposed as an in vitro assay that might reduce animal experimentation in regulatory developmental toxicology. So far, evaluation of the EST was not performed using compounds within distinct chemical classes. Evaluation within a distinct class of chemically related compounds can define the usefulness of the assay for the chemical class tested. The aim of the present study was to evaluate the relative sensitivity of the EST for a selected series of homologous compounds and to compare the data to the relative developmental toxicity of the compounds in vivo. To this end a series of proximate developmentally toxic glycol ether alkoxy acid metabolites was tested in the EST. All glycol ether alkoxy acid metabolites tested showed a concentration-dependent inhibition of cardiomyocyte differentiation at noncytotoxic concentrations, with methoxyacetic acid as the most potent compound followed by ethoxyacetic acid, butoxyacetic acid, and phenoxyacetic acid, respectively. The potency ranking of the compounds in the EST corresponds with the available in vivo data. The relative differences between the potencies of the compounds appeared more pronounced in the in vivo studies than in the EST. A possible explanation for this discrepancy could be the difference in the kinetics of the compounds in vivo as compared with their in vitro kinetics. This study illustrates that the EST can be used to set priorities for developmental toxicity testing within classes of related compounds.

Nasal tumours in rats after short-term exposure to a cytotoxic concentration of formaldehyde

Male Wistar rats were exposed to 0, 10 or 20 ppm formaldehyde vapour for 4, 8 or 13 weeks (6 h/day; 5 days/week), and were then observed for periods up to 126 weeks. Transient growth retardation occurred in both test groups. Death rate was not noticeably affected by formaldehyde. Despite recovery periods of at most 126 weeks, the nasal respiratory and olfactory epithelium of many rats of the 20 ppm group exhibited non-neoplastic histopathological changes. Similar but much less severe changes of the respiratory epithelium were seen in a small number of rats of the 10 ppm group; the olfactory epithelium was not visibly affected in rats of this group. Nasal tumours considered to be induced by formaldehyde were seen only in the 20 ppm group and mainly in rats that had been exposed for 13 weeks, the incidence being 4.5% (6/132). These tumours comprised 3 squamous cell carcinomas, 1 carcinoma in situ and 2 polypoid adenomas, all originating from respiratory epithelium. It was concluded that rat nasal respiratory epithelium severely damaged by formaldehyde vapour often does not regenerate and in some cases develops tumours.

Modulation of pancreatic carcinogenesis by antioxidants

Previously performed short-term (4-month) studies demonstrated that vitamins C and E, beta-carotene and selenium modulate growth of early putative preneoplastic acinar lesions induced in rat pancreas by azaserine. The present paper summarizes the results of long-term studies performed with azaserine-treated rats maintained on diets high in either beta-carotene, vitamins C and E or selenium. It appeared that rats given a diet high in beta-carotene, vitamin C or selenium, but not vitamin E, developed fewer pancreatic tumours than controls. The chemopreventive effects of these micronutrients were most pronounced when beta-carotene and/or selenium were given during the promotion phase of the carcinogenic process. Surprisingly, cell proliferation in azaserine-induced preneoplastic acinar lesions was higher in rats given beta-carotene and/or selenium via the diet in comparison to controls. It is considered unlikely that any antioxidant alone can be associated with protection against cancer. It is concluded that dietary supplementation of combinations of antioxidants may have practical application in chemoprevention of cancer.

Safety Evaluation of Synthetic β-Carotene

The safety of β-carotene was reassessed by evaluating the relevant literature on the beneficial and adverse effects of β-carotene on cancer and, in particular, by evaluating the results of toxicity studies. β- Carotene appeared neither genotoxic nor reprotoxic or teratogenic, and no signs of organ toxicity have been found in subacute, subchronic, or chronic oral toxicity studies in experimental animals receiving doses of up to 1000 mg/day β-carotene per kg body weight via the diet. Synthetic β-carotene did not exert any carcinogenic effect in Sprague-Dawley rats or in CD1 mice. An enhanced risk of lung cancer was found in two human intervention studies. Although dose and (timing of) exposure, smoking status, and imbalance of antioxidant defense have been recognized as potential factors accounting for the outcome of these studies, a conclusive explanation has not yet been found. It is concluded that exposure to β-carotene resulting in mean plasma concentrations of no more than 2.2 μmol/l (1.2 μg/ml) is safe to the general population. By contrast, in heavy smokers higher plasma concentrations may be associated with a higher lung cancer risk.

The determination of exogenous formaldehyde in blood of rats during and after inhalation exposure

Formaldehyde (FA) is suspected of being associated with the development of leukemia. An inhalation experiment with FA was performed in rats to study whether FA can enter the blood and could thus cause systemic toxicity in remote tissues such as the bone marrow. Therefore, a sophisticated analytical method was developed to detect blood concentrations of FA during and after single 6-h exposure by inhalation. In order to differentiate between exogenous and endogenous FA the rats were exposed to stable isotope ((13)C) labeled FA by inhalation. During and after exposure of the rats to (13)C-FA their blood was analyzed to determine the ratio between labeled and natural FA in blood and the total blood concentration of FA. With respect to sensitivity, with the applied method exogenous (13)C-FA could have been detected in blood at a concentration approximately 1.5% of the endogenous FA blood concentration. Exogenous (13)C-FA was not detectable in the blood of rats either during or up to 30 min after the exposure. It was concluded that the inhalation of (13)C-FA at 10 ppm for 6h did not result in an increase of the total FA concentration in blood.

Toward in vitro biomarkers for developmental toxicity and their extrapolation to the in vivo situation

Introduction: Reliable in vitro and in silico assays as alternatives for in vivo developmental toxicity studies are urgently needed, for the replacement, reduction and refinement (3Rs) of animal use in toxicological research. Therefore, relevant biomarkers for in vivo developmental toxicity in in vitro assays are needed. Areas covered: The present review gives an overview of alternative assays, as described in literature, for in vivo developmental toxicity, including the effects (readouts) assessed in these assays. The authors discuss how these data may be used to obtain relevant biomarkers for in vivo developmental toxicity, and how in vitro effect data can be translated to the in vivo situation using physiologically based kinetic (PBK) modeling. Expert opinion: Relevance of readouts in in vitro developmental toxicity assays as predictive biomarkers for in vivo developmental toxicity should be evaluated by comparing the obtained in vitro effect concentrations with in vivo internal concentrations at dose levels causing developmental toxicity. Extrapolation of the in vitro effect concentrations to in vivo dose levels using PBK modeling (i.e., reverse dosimetry) is promising in its use to derive points of departure for risk assessment, enabling the use of in vitro toxicity data in the safety assessment of compounds.