Chris Winder

The sensitivity and specificity of the MTS tetrazolium assay for detecting the in vitro cytotoxicity of 20 chemicals using human cell lines.

A number of studies reported that the MTS in vitro cytotoxicity assay is a convenient method for assessing cell viability. The main features found with this assay are its ease of use, accuracy and rapid indication of toxicity. It might well be a useful tool in human health risk assessment if it can be shown that this assay also has an acceptable sensitivity and specificity. This is of interest particularly when exposure to unknown chemical substances requires the rapid detection and evaluation of toxic effects. In this study, the cytotoxicity of 20 chemicals selected from the MEIC priority list was determined with the MTS assay. Since it could be shown that interactions between detection reagents and test chemicals might influence the results of this assay, preliminary experiments were carried out such that artifactual results due to test chemical interference could be excluded from this study. IC50 (50% inhibitory concentration) were established for each test chemical in two human cell lines (F1-73 and HeLa) and later compared with published toxicity data of the same chemicals established with in vitro and in vivo toxicological test systems. Direct comparisons of the data showed a generally lower sensitivity of the MTS assay, which is influenced by biological test organisms, cell type and exposure time. In terms of the specificity of the MTS assay, the results showed a good correlation between data obtained with the MTS assay and published data. The lowest correlation was found when the MTS assay was compared with in vivo studies, however, this finding corresponds well with other published in vitro-in vivo correlations. The highest correlation was found when the MTS assay was compared with test systems using human cell lines or exposure times of 3-24 h. Since the sensitivity of the MTS assay might be increased using different cell types or by extended incubation, this assay is found to provide ideal features of a good measurement system that might also be used for on site toxicological assessments.

Toxicity assessment of industrial chemicals and airborne contaminants: Transition from in vivo to in vitro test methods : A review

Exposure to occupational and environmental contaminants is a major contributor to human health problems. Inhalation of gases, vapors, aerosols, and mixtures of these can cause a wide range of adverse health effects, ranging from simple irritation to systemic diseases. Despite significant achievements in the risk assessment of chemicals, the toxicological database, particularly for industrial chemicals, remains limited. Considering there are approximately 80,000 chemicals in commerce, and an extremely large number of chemical mixtures, in vivo testing of this large number is unachievable from both economical and practical perspectives. While in vitro methods are capable of rapidly providing toxicity information, regulatory agencies in general are still cautious about the replacement of whole-animal methods with new in vitro techniques. Although studying the toxic effects of inhaled chemicals is a complex subject, recent studies demonstrate that in vitro methods may have significant potential for assessing the toxicity of airborne contaminants. In this review, current toxicity test methods for risk evaluation of industrial chemicals and airborne contaminants are presented. To evaluate the potential applications of in vitro methods for studying respiratory toxicity, more recent models developed for toxicity testing of airborne contaminants are discussed.

Mechanisms of multiple chemical sensitivity.

Sensitivity to chemicals is a toxicological concept, contained in the dose-response relationship. Sensitivity also includes the concept of hypersensitivity, although controversy surrounds the nature of effects from very low exposures. The term multiple chemical sensitivity has been used to describe individuals with a debilitating, multi-organ sensitivity following chemical exposures. Many aspects of this condition extend the nature of sensitivity to low levels of exposure to chemicals, and is a designation with medical, immunological, neuropsychological and toxicological perspectives. The basis of MCS is still to be identified, although a large number of hypersensitivity, immunological, psychological, neurological and toxicological mechanisms have been suggested, including: allergy; autosuggestion; cacosomia; conditioned response; immunological; impairment of biochemical pathways involved in energy production; impairment of neurochemical pathways; illness belief system; limbic kindling; olfactory threshold sensitivity; panic disorder; psychosomatic condition; malingering; neurogenic inflammation; overload of biotransformation pathways (also linked with free radical production); psychological or psychiatric illness; airway reactivity; sensitisation of the neurological system; time dependent sensitisation, toxicant induced loss of tolerance. Most of these theories tend to break down into concepts involving: (1) disruption in immunological/allergy processes; (2) alteration in nervous system function; (3) changes in biochemical or biotransformation capacity; (4) changes in psychological/neurobehavioural function. Research into the possible mechanisms of MCS is far from complete. However, a number of promising avenues of investigation indicate that the possibility of alteration of the sensitivity of nervous system cells (neurogenic inflammation, limbic kindling, cacosomia, neurogenic switching) are a possible mechanism for MCS.

A novel in vitro exposure technique for toxicity testing of selected volatile organic compounds

Exposure to vapours of volatile chemicals is a major occupational and environmental health concern. Toxicity testing of volatile organic compounds (VOCs) has always faced significant technological problems due to their high volatility and/or low solubility. The aim of this study was to develop a practical and reproducible in vitro exposure technique for toxicity testing of VOCs. Standard test atmospheres of xylene and toluene were generated in glass chambers using a static method. Human cells including: A549-lung derived cell lines, HepG2-liver derived cell lines and skin fibroblasts, were grown in porous membranes and exposed to various airborne concentrations of selected VOCs directly at the air/liquid interface for 1 h at 37 degrees C. Cytotoxicity of test chemicals was investigated using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) and NRU (neutral red uptake) assays following 24 h incubation. Airborne IC(50) (50% inhibitory concentration) values were determined using dose response curves for xylene (IC(50)=5350+/- 328 ppm, NRU; IC(50)=5750+/- 433 ppm, MTS in skin fibroblast) and toluene (IC(50)=0 500+/- 527 ppm, NRU; IC(50)=11,200 +/- 1,044 ppm, MTS in skin fibroblast). Our findings suggest that static direct exposure at the air/liquid interface is a practical and reproducible technique for toxicity testing of VOCs. Further, this technique can be used for inhalational and dermal toxicity studies of volatile chemicals in vitro as the exposure pattern in vivo is closely simulated by this method.

The dermal toxicity of cement.

Cement and concrete are products used widely in the construction sector, with a traditional perception that any hazards that they have are limited to dermatitis in a small number of workers. In some cases, employers and builders do not think that concrete is a chemical. However, contact dermatitis is one of the most frequently reported health problems among construction workers. A review of the available literature suggests that cement has constituents that produce both irritant contact dermatitis and corrosive effects (from alkaline ingredients such as lime) and sensitization, leading to allergic contact dermatitis (from ingredients such as chromium). These findings indicate that cement and concrete should be treated as hazardous materials, and that workers handling such products should reduce exposure wherever possible. Initiatives to reduce the chromium content of cement have been shown to be successful in reducing the incidence of allergic dermatitis, although the irritant form remains.

In vitro cytotoxicity testing of airborne formaldehyde collected in serum-free culture media

The purpose of this study was to identify a suitable sampling model for on-site toxicity assessment of soluble air contaminants such as formaldehyde, a well known industrial and indoor air contaminant. The in vitro cytotoxicity of formaldehyde, the selected model for soluble air contaminants, was studied using the MTS (tetrazolium salt) assay in two carcinoma cell lines, A549 epithelial lung and HepG2 hepatocarcinoma, and in skin fibroblasts. The cytotoxic effects of airborne formaldehyde were evaluated using test atmospheres in concentrations below 10 ppm (12.3 mg/m3), generated by a dynamic diffusion method and bubbled (0.3 L/min) through serum-free culture media for one or four hours. Human cells were treated with formaldehyde air samples, and cell viability was determined after four hours incubation. In parallel, the concentration of airborne formaldehyde was monitored, using the 3500 NIOSH method. Cell viability of the HepG2 cells exposed to formaldehyde air samples (8.75 ppm-4 h) was reduced to less than 50% (31.69/1.24%). The HepG2 cell lines were found to be more sensitive (IC50=103.799/23.55 mg/L) to formaldehyde than both A549 cell lines (IC50=198.369/9.54 mg/L) and skin fibroblasts (IC50=196.689/36.73 mg/L) (PB/0.01). An average of 96.8% was determined for collection efficiency of formaldehyde in serum-free culture media. The results of this study suggest that absorption of soluble air contaminants, such as formaldehyde, in serum-free culture media can be used as a suitable sampling model for on-site toxicity assessments.

Frequency and Occurrence of Ld50 Values for Materials in the Workplace

The main routes of occupational exposure for chemicals encountered in occupational environments are by skin contact and by inhalation. Acute toxicity data were collected for all chemicals with Australian Exposure Standards. For the 582 chemical entities, data were available as follows: oral toxicity (364, 63%), dermal toxicity (154, 26%) and inhalational toxicity (171, 29%). No acute toxicity data were available for 177 of these chemicals (177, 30%). Chemicals with data for more than one acute toxicity test were limited to: oral/dermal (151, 26%), oral inhalation (131, 23%), dermal/inhalation (73, 13%) and oral/dermal/inhalation (70, 12%). Analysis of subsets of groups of chemicals indicated that toxicity data were available for many pesticides and organic chemicals, but not for inorganic chemicals. The lack of acute inhalational and dermal toxicity data for chemicals used extensively enough to warrant exposure standards is at variance with the amount of toxicity information for other categories of chemicals (e.g. agricultural chemicals, therapeutic substances and food additives). This may also indicate scant regard given to toxicological information in the exposure standard setting process.

An Integrated in Vitro Approach for Toxicity Testing of Airborne Contaminants

While it is possible to establish the chemical composition of air pollutants through conventional air sampling and analytical techniques, such data do not provide direct measures of toxicity and the potential mechanisms that induce adverse effects. The aim of this study was to optimize in vitro methods for toxicity testing of airborne contaminants. An integrated approach was designed in which appropriate exposure techniques were developed. A diversified range of in vitro assays using multiple human cell systems were implemented. Direct exposure of cells to airborne contaminants was developed by culturing cells on porous membranes in conjunction with a horizontal diffusion chamber system. Concentration-response curves were generated allowing the measurement of toxicity endpoints. Regression analysis indicated a significant correlation between in vitro and published in vivo toxicity data for the majority of selected chemical contaminants. Airborne IC50 values were calculated for selected volatile organic compounds (xylene, 5350 ± 328 ppm > toluene, 10500 ± 527 ppm) and gaseous contaminants (NO2, 11 ± 3.54 ppm > SO2, 48 ± 2.83 ppm and > NH3, 199 ± 1.41 ppm). Results of this study indicate the significant potential of in vitro methods as an advanced technology for toxicity assessment of airborne contaminants.

Toxicity of ricin

Ricin is a potent protein toxin derived from the beans of the castor plant (Ricinus communis). Castor beans are found in many parts of the world, and the toxin can be produced simply. Ricins significance as a potential terrorist weapon relates in part to its wide availability and ease of extraction. Ricin comprises a number of polypeptide chains that act to assist cellular uptake of the ricin molecule, cause membrane damage, and once in the cell, inhibit protein synthesis. The toxic dose in animals is low, although there is little toxicity data in humans. Most probable exposure in humans would be from inhalation of aerosols or mists containing ricin, or by ingestion. There is one established case of assassination by injection. Signs and symptoms of toxicity in humans would probably include weakness, fever, cough and pulmonary edema occuring within 18–24 hours of inhalation exposure, followed by severe respiratory distress and death from hypoxemia in 36–72 hours.

Assessment of fogging resistance of anti-fog personal eye protection

The propensity for occupational eye protectors to fog in warm and moist conditions is often offered as a reason by workers not to wear occupational eye protection even where mandatory eye protection areas have been specified. A study of eye protection practices in the New South Wales coal mining industry identified the number one issue in underground coal mine conditions as being fogging of eye safety wear. Conventional anti‐fog treatments and cleaners were considered by the miners as completely inadequate in these conditions. At the time of the study claims were being made for a new generation of lens treatments. These merited evaluation. Spectacles and goggles claimed to be fog resistant were obtained from manufacturers and suppliers and subjected to the test set out in BS EN 168:1995 and the compliance criterion of BS EN 166:1996. Some lenses claimed to be fog resistant failed the requirement, some new technology lenses showed arguably superior performance but failed the criterion of the standard. Modifications to the test procedure of BS EN 168 and acceptance criteria of BS EN 166 are proposed.