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Mixtures of chemicals with different target organs or the same target organ but different target sites or different modes of action did not appear to be distinctly more hazardous than the individual chemicals, provided the dose level of each chemical in the mixture did not exceed its own 'No-Observed-Adverse-Effect Level'. Clearly, for such mixtures and exposure conditions the additivity assumption did not hold. However, the additivity rule appeared to be applicable to mixtures of chemicals with the same target organ and the same mechanism of action or receptor. Fractional 2-factorial study designs were found to be promising tools for examining possible combined actions or interactions of chemicals in a mixture.

This article describes how scientists in the Netherlands set occupational exposure limits (OELs) for sensory irritants. When they tackle this issue, a number of key questions need to be answered. For example, did the studies indeed measure sensory irritation and not cytotoxicity? When the irritant is an odorant, can interference of olfactory stimulation be excluded? In the case of subjective measurements, can psychological irritation be excluded? When adaptation is an issue, did the studies indeed measure adaptation and not habituation? When OELs are established in the Netherlands, each of these issues is carefully addressed before a value is suggested. When setting an OEL in the Netherlands, human data carry more weight than animal data of comparable quality. As in the United States, documentation for the recommended OEL is written and a discussion of all available relevant and reliable data culminating in the selection of the key study for deriving the health-based recommended occupational exposure limit is provided. Special effort is dedicated to reconciling differences between the animal and human data. If the toxicological database is considered to be inadequate, the committee acknowledges this limitation and will not recommend a limit value due to insufficient data.Chemicals/CAS: Air Pollutants, Occupational; Irritants

Allergens are substances that may cause a hypersensitivity (allergy) of the immune system. After acquiring this hypersensitivity, further exposure to the same substance may result in allergic skin disease such as allergic contact dermatitis, or allergic airway disease such as allergic rhinitis or asthma. Occupational allergens are present in many sectors and occupations. Occupational allergens and the products containing them may be identified by means of their classification and labelling as well as by using various existing lists. Prevention should include combinations of measures at source, technical and organisational measures, personal protection and hygiene, and early warning.

Health risks resulting from occupational exposure to chemicals are controlled by the establishment of acceptable dermal and respiratory exposure levels. Due to a lack of route-specific toxicity data, acceptable levels are frequently established by means of route-to-route extrapolation. A pitfall in route-to-route extrapolation is the occurrence of local effects. Often, the results of acute irritation studies are used to assess the likelihood of the occurrence of local effects also following repeated exposure and thereby the validity of route-to-route extrapolation. We questioned this working practice and considered whether local effects observed in a given study are of any predictive value with respect to the occurrence of local effects after repeated exposure. Our database analysis indicates that substances inducing skin and/or eye irritation frequently induce local effects after repeated respiratory exposure. In contrast, observations made in any type of study show little or no positive predictive value for the occurrence of local effects after repeated dermal exposure. Notably, the absence of any indication of local effects in any type of study does not exclude the occurrence of local effects on repeated dermal or respiratory exposure. We conclude that the presumed reliability of route-to-route extrapolation in the absence of route-specific toxicity data can be questioned. © 2002 Elsevier Science (USA). Chemicals/CAS: Irritants

The enucleated eye test with chicken eyes (CEET) obtained from an abattoir proved to be a valuable and practical alternative for the 'traditional' enucleated eye test with eyes of laboratory rabbits. Since 1992, the CEET has been incorporated in standard contract toxicity testing at the Toxicology Division of the TNO Nutrition and Food Research Institute as a (pre)screen for the Draize eye test with rabbits. The results of the first 44 compounds tested showed excellent correlation with the in vivo results. The CEET identified non-irritating or severely irritating compounds, and predicted (slightly to moderately) irritating compounds. Statistical analysis of the CEET and the rabbit in vivo scores showed high linear correlations between the critical values of both tests and confirmed the relevance of this assay with respect to ocular effects. In general, tiered in vitro/in vivo testing was considered a meaningful approach for further validation of alternative methods and for reducing the use of suffering of laboratory animals to a minimum. Tiered testing of compounds in cases of eye irritation hazard assessment should be incorporated in the legislation of the European Community. Chemicals/CAS: Irritants

Within the framework of risk assessment of existing substances in the EC the irritating properties on the respiratory tract should be considered. Since no standardized test is available it was studied whether the Alarie test could be used for this purpose, as proposed by the Technical Guidance Document for new and existing substances. The available literature on respiratory tract irritation, seen as a local inflammatory response and/or tissue damage, after single and repeated (few-day) exposure was evaluated and compared with data on sensory irritation. No relation was found between the sensory irritation potential (as measured by the Alarie test) and local tissue damage (histopathological changes) in the respiratory tract after single or repeated exposure. It was concluded that the Alarie test is inappropriate to evaluate respiratory tract irritation. In addition, the available data do not support a quantitative potency ranking for man based on the RD50 obtained with experimental animals. Chemicals/CAS: Air Pollutants; Irritants

In vitro techniques make a major contribution to the development of alternatives to the in vivo 'Draize' skin irritation test, and the development of sensitive and generally applicable in vitro endpoints of cutaneous toxicity is an area of intensive research. To investigate in vitro characteristics of cutaneous irritation, skin explants of rabbit and human origin were topically exposed to chemical irritants, after which the culture medium was analyzed for the presence of metabolites of both arachidonic and linoleic acid. In rabbits exposed to the potent irritant benzalkonium chloride, a direct relation was established between clinical signs of irritation and in vitro release of the proinflammatory mediator 12-hydroxyeicosatetraenoic acid (12-HETE) by the exposed skin. Histological examination revealed varying degrees of epidermal damage. 12-HETE was also the predominant hydroxy fatty acid released in a dose-dependent way by rabbit skin cultures after in vitro exposure to sodium dodecyl sulfate (SDS), benzalkonium chloride (BC), and formaldehyde (FA). Human skin cultures released, in addition to 12-HETE, predominantly 15-HETE and 13-hydroxyoctadecadienoic acid (13-HODE), ω-6 oxygenase products of arachidonic acid and linoleic acid, respectively. The irritant-induced release of hydroxy fatty acids was strongly inhibited by the lipoxygenase inhibitor eicosatetraynoic acid, indicating enzyme-mediated generation of these bioactive lipids. Comparison of hydroxy fatty acid release to more established markers of cytotoxicity (leakage of the cellular enzymes, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH)) revealed that increased levels of 13-HODE, 9-HODE, 12-HETE, and ALT were specific markers of cutaneous irritancy in rabbit skin cultures. AST and LDH were more sensitive endpoints compared to ALT or hydroxy fatty acids, but failed to identify the difference in irritation capacity between SDS and BC reported in vivo. In human skin cultures, release of 12-HETE or 15-HETE occurred at irritant concentrations that did not result in enzyme leakage into the culture medium. In vitro effect levels of irritants were found comparable to the in vivo situation. We conclude that release of hydroxy fatty acids, in particular of 12-HETE, offers a mechanism-based characteristic of skin irritation which may be applicable to in vitro toxicity testing.

Objectives: Chemosensory effects of stimulation by a chemical can either be irritating (trigeminal stimulation) or odorous (olfactory stimulation) or both. For odorous irritants, a clear-cut distinction between odour and irritation is difficult to make. The differences in the lowest concentration found to be irritating to the respiratory tract in humans when compared to experimental animals has brought much debate in the process of setting occupational exposure limits (OELs) for such chemicals. In this paper it will be discussed as to how far subjectively measured sensory irritation threshold levels can be used to establish OELs. Methods: Data on respiratory irritation of four odorous irritants were retrieved from public literature and discussed, viz. acetone, formaldehyde, furfural and sulphur dioxide. Results: Objective measures of irritation yielded results that differed from subjective evaluations. Important factors modulating the reported levels of irritation and health symptoms include the perception of odour intensity, exposure history and the individual's bias to report irritation on the basis of his/her prejudice or knowledge of the compound. Conclusions: Subjective measures alone are less appropriate for establishing sensory irritation thresholds of odorous irritants and are, therefore, less suitable to establish OELs without supporting evidence. Objectively measured irritation in humans, the Alarie assay (an experimental animal test assessing the concentration that results in a 50% reduction of the breathing frequency) and repeated exposure studies in animals may be of help to study objective irritation. If subjective measurements are used to study sensory irritation, the study design should at least include: Measurement of both incidence and severity determined at several concentrations, an appropriate (0 ppm) control condition, preferably a non-irritant odorant control exposure, validated questionnaires and correct concentration measurements. © Springer-Verlag 2005.

Several acid anhydrides are known for their sensitizing and irritative properties. Since both irritation and respiratory allergy can cause changes of lung function, proper testing of allergen-dependent effects on the respiratory tract requires knowledge of the respiratory irritant effects. To study the latter effects, groups of female Brown Norway (BN) and Wistar rats were exposed for 30 min to a range of concentrations (10 to 300 mg/m3) of the well-known respiratory allergen trimellitic anhydride (TMA). Breathing pattern and frequency were monitored before, during, and after exposure. Animals were necropsied and lung weights were determined 1 day after exposure. In BN rats, changes in breathing pattern were seen at levels of 29 mg/m3 and higher and decreases in frequency at 60 mg/m3 and higher, whereas in Wistar rats changes in both pattern and frequency (increases followed by decreases) were seen at levels of 34 mg/m3 and higher. Changes in breathing pattern consisted of a spiked form instead of a wave form of the respiratory cycle, with a pause between breaths at the end of expiration. The length of the pause increased with increasing concentrations of TMA while the duration of the respiratory cycle decreased slightly, implying that breathing frequency was mainly determined by the magnitude of the increase in pause. These reversible changes in breathing pattern and frequency were considered to be suggestive of lower airway irritation, rather than upper airway irritation. No concentration-related changes in lung weights were observed. The highest level at which no acute airway irritation as based on both breathing pattern and frequency was observed in both rat strains was 14 mg/m3.

At present, there are no widely applied or fully validated test methods to identify respiratory LMW allergens, i.e. compounds that are considered capable of inducing allergic asthma. Most tests have been investigated using strong respiratory allergens. Moreover, they are meant to detect the potential of a chemical to induce respiratory sensitisation at relatively high doses. Consequently, the sensitivity of the tests is not well-known, and they do not provide information on low doses such as generally found in occupational situations, and on threshold levels to be used in risk assessment. In addition, the various test methods use different application routes, i.e. intradermal, topical or inhalation exposure, and different parameters. Therefore standardised and validated dose-response test methods are urgently required in order to be able to identify respiratory allergens and to recommend safe exposure levels for consumers and workers. In the present paper, methods or testing strategies are described to detect respiratory sensitisation and/or allergy. Overall, assays that utilize only an induction phase may serve as indicators of respiratory sensitisation potential whereas assays that use both an induction and an elicitation or challenge phase may provide information on potency and presence of thresholds. The dermal route as sensitisation route has the advantage of the respiratory tract not being exposed to the allergen prior to challenge which facilitates the distinction between irritant and allergic effects. © 2006 Elsevier Inc. All rights reserved.

Studies on the irritative effects of acetone vapor in humans and experimental animals have revealed large differences in the lowest acetone concentration found to be irritative to the respiratory tract and eyes. This has brought on much confusion in the process of setting occupational exposure limits for acetone. A literature survey was carried out focusing on the differences in results between studies using subjective (neuro)behavioral methods (questionnaires) and studies using objective measurements to detect odor and irritation thresholds. A critical review of published studies revealed that the odor detection threshold of acetone ranges from about 20 to about 400 ppm. Loss of sensitivity due to adaptation and/or habituation to acetone odor may occur, as was shown in studies comparing workers previously exposed to acetone with previously unexposed subjects. It further appeared that the sensory irritation threshold of acetone lies between 10,000 and 40,000 ppm. Thus, the threshold for sensory irritation is much higher than the odor detection limit, a conclusion that is supported by observations in anosmics, showing a ten times higher irritation threshold level than the odor threshold found in normosmics. The two-times higher sensory irritation threshold observed in acetone-exposed workers compared with previously nonexposed controls can apart from adaptation be ascribed to habituation. An evaluation of studies on subjectively reported irritation at acetone concentrations < 1000 ppm shows that perception of odor intensity, information bias, and exposure history (i.e., habituation) are confounding factors in the reporting of irritation thresholds and health symptoms. In conclusion, subjective measures alone are inappropriate for establishing sensory irritation effects and sensory irritation threshold levels of odorants such as acetone. Clearly, the sensory irritation threshold of acetone should be based on objective measurements.

Sensory irritation of formaldehyde (FRM), acrolein (ACR) and acetaldehyde (ACE) as measured by the decrease in breathing frequency (DBF) was studied in male Wistar rats using nose-only exposure. Groups of four rats were exposed to each of the single compounds separately or to mixtures of FRM, ACR and/or ACE. Exposure concentrations of the mixtures were chosen in such a way that summation of the effects of each chemical would be expected not to exceed 80% reduction of the breathing frequency. FRM, ACR and ACE appeared to act as sensory irritants as defined by Alarie (1966, 1973). With FRM and ACR desensitization occurred, whereas with ACE the breathing frequency gradually decreased with increasing exposure time (up to 30 min). For mixtures, the observed DBF was more pronounced than the DBF for each compound separately, but was less than the sum of the DBFs for the single compounds. A model for three compounds competing for the same receptor was applied to predict the DBF of mixtures of FRM, ACE and ACR. The results also showed that with mixtures no desensitization occurred; in fact, the breathing frequency further decreased in the last 15 min of exposure. These observations were similar to those found for ACE alone, and might have been caused by effects on the upper respiratory tract. The results of the present study allow the conclusion that sensory irritation in rats exposed to mixtures of irritant aldehydes is more pronounced than that caused by each of the aldehydes separately, and that the DBF as a result of exposure to a mixture could well be predicted using a model for competitive agonism, thus providing evidence that the combined effect of these aldehydes is basically a result of competition for a common receptor (trigeminal nerve).

Objectives: The critical health effects of formaldehyde exposure include sensory irritation and the potential to induce tumours in the upper respiratory tract. In literature, a concentration as low as 0.24 ppm has been reported to be irritating to the respiratory tract in humans. Nasal tumour-inducing levels in experimental animals seem to be 1-2 orders of magnitude larger. In this paper, the subjectively measured sensory irritation threshold levels in humans are discussed in line with findings obtained in animal experiments. In addition, a Benchmark dose (BMD) analysis of sensory irritation was used to estimate response incidences at different formaldehyde concentrations. Methods: Data on respiratory irritation and carcinogenicity of formaldehyde were retrieved from public literature and discussed. BMD analysis was carried out on human volunteer studies using the US-EPA BMD software. Results: Subjective measures of irritation were the major data found in humans to examine sensory (eye and nasal) irritation; only one study reported objectively measured eye irritation. On a normalized scale, mild/slight eye irritation was observed at levels ≥1 ppm, and mild/slight respiratory tract irritation at levels ≥2 ppm. With the BMD software, it was estimated that at a level of 1 ppm, only 9.5% of healthy volunteers experience 'moderate' (i.e., annoying) eye irritation (95% upper confidence limit). An important factor modulating the reported levels of irritation and health symptoms most probably includes the perception of odour intensity. In several studies, the 0-ppm control condition was missing. From the results of the long-term inhalation toxicity studies in experimental animals, a level of 1 ppm formaldehyde has been considered a NOAEL for nasal injury. Conclusions: Sensory irritation is first observed at levels of 1 ppm and higher. From both human and animal studies, it was concluded that at airborne levels for which the prevalence of sensory irritation is minimal both in incidence and degree (i.e., <1 ppm), risks of respiratory tract cancer are considered to be negligibly low. © 2005 Elsevier Inc. All rights reserved.

The paper aims to evaluate the indoor air limit of 1 μg/m3 (0.8 ppb) formaldehyde as advised by the European Commission [the INDEX project; Kotzias, D., Koistinen, K., Kephalopoulos, S., Schlitt, C., Carrer, P., Maroni, M., Jantunen, M., Cochet, C., Kirchner, S., Lindvall, T., McLaughlin, J., Mølhave, L., de Oliveira Fernandes, E., Seifert, B., 2005. Critical appraisal of the setting and implementation of indoor exposure limits in the EU. European Commission, Institute for Health and Consumer Protection, Physical and Chemical Exposure Unit, Ispra, Italy, pp. 1-50]. The limit has been based on a nose and throat irritation threshold of 0.1 mg/m3 (0.08 ppm; LOAEL), a NOAEL of 0.03 mg/m3 (0.025 ppm) and an assessment factor of 30, including a factor of 3 for the higher sensitivity of children. Nose and throat irritation, at concentrations below which hyperplasia/metaplasia occurs, are most likely the manifestation of trigeminal nerve stimulation (sensory irritation). The threshold for sensory irritation in human volunteers is 1 ppm, much higher than the 0.1 mg/m3 indicated above. Eye irritation is the most sensitive effect reported in human volunteers but has been mentioned only occasionally in the studies used by the European Commission. Moreover, sensory irritation is a local reaction that requires a low assessment factor, if any. It is difficult to judge the sensitivity for sensory irritation in children because of the potential confounding factors in the evaluated studies. It is concluded that an indoor air level of 0.1 ppm (0.12 mg/m3) formaldehyde, as indicated by Appel et al. (2006) [Appel, K.E., Bernauer, U., Herbst, U., Madle, S., Schulte, A., Richter-Reichhelm, H.B., Gundert-Remy, U. 2006. Kann für Formaldehyd eine "sichere" Konzentration abgeleitet werden?-Analyse der Daten zur krebserzeugenden Wirkung (Can a "safe" concentration be established for formaldehyde?-Analysis of carcinogenicity data)? Umweltmed. Forsch. Prax. 11, 347-361], can be considered a safe and appropriate level. © 2008 Elsevier Inc. All rights reserved. Chemicals / CAS: formaldehyde, 50-00-0; Air Pollutants; Carcinogens; Formaldehyde, 50-00-0; Irritants

Five rat strains were compared for their performance in the local lymph node assay (LLNA) a promising test system for the identification of the skin-sensitizing potential of chemicals in the mouse. The contact sensitizer 2,4-dinitrochlorobenzene (DNCB) and the contact and respiratory sensitizer trimellitic anhydride (TMA) were used as model chemicals and responses in rats were compared with those in BALB/c mice. The chemicals were applied to the dorsum of both ears, once daily for three consecutive days; 2 days (mice) or 3 days (rats) thereafter, proliferating cells were labelled by ip injection of BrdU 2 hr before the animals were killed. Systemic effects were subsequently assessed by determination of spleen liver and kidney weights, skin effects by determination of swelling and inflammatory cell infiltration of the ears, and immune effects by determination of weight and proliferative activity of the local lymph nodes (LLN). Following application ( x 3) of DNCB or TMA, minor systemic effects were observed, as indicated by slightly elevated spleen and liver weights in a few rat strains and the mice. Skin effects, consisting of increased ear thickness and presence of mononuclear inflammatory cell infiltrates, were observed in all rat strains treated with DNCB or TMA. LLN weights had increased, as had the proliferative activity in these nodes. It was concluded that effects induced by DNCB and TMA in all five rat strains were comparable with those in mice. Chemicals/CAS: Allergens; Antimetabolites; Bromodeoxyuridine, 59-14-3; Dinitrochlorobenzene, 97-00-7; Irritants; Phthalic Anhydrides; trimellitic anhydride, 552-30-7

Skin irritants and contact allergens reduce the number of Langerhans cells (LCs). It has been assumed that this reduction is due their migration to the draining lymph node (LN) for initiating immune sensitization in a host. Skin irritation, however, as opposed to contact allergy is not considered to be an immunological disease. Nevertheless, skin irritants are also known for their adjuvant-like effects on contact allergy, resulting in skin hypersensitivity reactions like toxic dermatitis. The human organotypic skin explant culture (hOSEC) model is used to study the characteristics of chemical-induced migration of CD1a+ LCs out of the epidermis in relation to irritancy or toxicity. We analysed cells emigrating out of hOSEC for CD1a+ LCs, CD83+ mature dendritic cells (DCs) and CCR7+ LN homing cells. After exposure to a toxic concentration of a non-immunogenic irritant, an increase of CD1a+CD83+ LCs was found in the culture medium. A non-toxic concentration of an sensitizer induced an increase of CD1a+ cells. About 50% of skin emigrating CD1a+ LCs were CD83- (immature) but all were CCR7+. Skin irritation by both non-allergenic and allergenic compounds induces LC migration and maturation. In contrast, only allergenic compounds induced LC migration with partial maturation at subtoxic concentration. This effectively demonstrates that irritation is physiologically needed stimuli for inducing LC maturation. © Blackwell Munksgaard, 2006.

The objective of this study was to establish the possible occurrence of eye irritation and subjective symptoms in human volunteers exposed to propylene glycol monomethyl ether (PGME) vapour at concentrations of 0, 100 and 150 ppm. Testing was conducted in 12 healthy male volunteers using a repeated measures design. Each subject was exposed for 2.5 h to each of the three exposure conditions that were spaced 7 days apart. The exposure sequences were counterbalanced and the exposure to the test substance and the effect measurements were conducted in a double-blind fashion. During all exposure sessions, 20 ppm diethyl ether was used as a 'masking agent' for vapour exposure. Measurements of pre- and post exposure eye redness, corneal thickness, tear film break-up time, conjunctival epithelial damage, blinking frequency, and subjective ratings on discomfort were used to evaluate the possible irritating effects of PGME. The results indicated no significant treatment effects for any of the objective parameters. Results of the subjective ratings indicated very slight effects on the eyes in the 150 ppm PGME condition only. No significant effects of treatment were found for the remaining questions concerning the perceived intensity of the smell in the room, the (un)pleasantness of the smell, the perceived effects on the skin, effects on the throat, shivering, muscle aching, and intestinal cramps. In conclusion, the results of the present study indicated minimal subjective eye effects at 150 ppm only, and no impact on the objective measures of eye irritation at either of the two exposure levels. It was concluded that the no adverse effect concentration for eye irritation due to PGME vapour was at least 150 ppm. © 2003 Elsevier Science Ireland Ltd. All rights reserved.

The objectives of this study were to examine the respiratory irritancy of boron trifluoride (BF3) and fluorosulfonic acid (FSA) following acute inhalation exposure. Testing was conducted using groups of 10 male and 10 female rats (BF3) or groups of 6 male rats (FSA). Rats were exposed for a single 4-h period (BF3) or a single 1-h period (FSA) and necropsied 1 or 14 days after exposure (BF3) or 14 days after exposure (FSA). Measurements consisted of clinical signs, body weight, kidney and lung weight, histopathology (BF3), and breathing parameters (FSA) and were used to evaluate the possible irritating effects of these compounds. The results indicated treatment-related findings in the larynx and trachea in the rats exposed to 74.4 mg/m3 BF3, consisting of ventral cartilage necrosis, hemorrhage, and an increase in ventral epithelial hyperplasia and ventral inflammatory cell inflammation 24 h postexposure. In the animals sacrificed 14 days postexposure, the only notable observation was ventral cartilage necrosis, present in 2 animals. The next lower level tested, 24.6 mg/m3 BF, was considered a no-observed-adverse-effects level (NOAEL). A concentration of 4125 mg/m3 FSA resulted in a clearly decreased breathing rate during and shortly after exposure with 67% (4/6) mortality on days 5-9 after exposure. A concentration of 845 mg/m3 FSA resulted in only minor signs of irritation, consisting of slight changes in breathing pattern shorlty after exposure. The results of the present 4-h inhalation study with BF3 indicated that respiratory irritation was present at a level of 74.4 mg/m3 whereas 24.6 mg/m3 was a NOAEL. A single 1-h exposure to 845 mg/m3 FSA resulted in only minor signs of respiratory irritation, indicating that on a mass basis FSA is no more toxic or irritating than hydrogen fluoride (HF) or sulfuric acid. Copyright © Informa Healthcare USA, Inc.