Eye blinks as indicator for sensory irritation during constant and peak exposures to 2-ethylhexanol

Two experiments were performed to re-evaluate the sensory irritating properties of 2-ethylhexanol in relation to dose and time and to examine the usability of electromyographic eye blink recordings as indicator of sensory irritation. Mean exposure levels of 1.5, 10 and 20 ppm were realized in experimental models simulating either constant or variable 4 h exposure. Each study was carried out with two subject samples, healthy young men with self-reported multiple chemical sensitivity (sMCS) and age matched controls. Although 2-ethylhexanol exposure was below the occupational threshold limit value of 50 ppm, the study revealed strong dose–response relationships between airborne solvent concentrations and blink rates. During 40 ppm peak exposures the blink rate increased threefold. In the course of 4 h, exposure blink rates increased significantly showing no adaptation. Subjects with sMCS revealed, with one exception at start of exposure, no significantly higher blink rates than controls. The results indicate that the irritative potential of 2-ethylhexanol is higher than commonly expected. In both exposure scenarios with either constant or peak exposures, electromyographic eye blink recordings were an appropriate method for the examination of acute sensory irritations in time.     

Comparative assessment of the sensory irritation potency in mice and rats nose-only exposed to ammonia in dry and humidified atmospheres

Young adult male Wistar rats, a species commonly used in inhalation toxicity studies, and OF1 mice, a species often used in sensory irritation studies, were simultaneously nose-only exposed for 45-min to ammonia in concentrations from 92 to 1243 mg/m³. This study examined airway reflexes by the changes in respiratory patterns elicited by ammonia in either dry, steam-humidified (approximately 95% relative humidity) or aqueous aerosol containing atmospheres. This served the objective to explore whether high concentrations of anhydrous ammonia and/or high humidity and aqueous aerosol change the predominant nasal deposition site to more distal locations in the lung. Animals from all groups tolerated the exposure without evidence of respiratory tract irritation, changes in body and lung weights. The evoked changes on breathing patterns resembled those known to occur following exposure to ‘upper respiratory tract sensory irritants’, rapid in onset and reversibility. Reflex stimulation from the lower airways was not observed in any group. While mice showed some adaption during the 45-min exposure period, rats displayed more stable changes in respiratory patterns. In this species humidity- or aqueous aerosol-related changes in sensory irritation potency did not occur to any appreciable extent. The respiratory decrease 50%, RD₅₀, was 972 and 905 mg/m³ in dry and wet air, respectively. In contrast, mice appeared to more susceptible to ammonia in presence of dry air (the RD₅₀, was 582 and 732 mg/m³ in dry and wet air, respectively). In summary, it was shown that the sensory irritation potency of ammonia does not increase when inhaling wet atmospheres nor penetrates this gas into the lower airways up to 1243 mg/m³ × 45-min. The mild-to-moderate sensory irritation-related effects observed at ≈400 mg/m³ (571 ppm) × 45-min do not appear to be offensive enough to impair escape as a result of upper airway sensations. Interestingly, this rat-based estimate matches almost exactly the experienced-based RAM TRAC recommendations of 696 and 492 ppm for 30 and 60 min, respectively.     

Evaluation of airborne sensory irritants for setting exposure limits or guidelines: A systematic approach

Sensory irritation of eyes and upper airways is an important endpoint for setting occupational exposure limits (OELs) and indoor air guidelines. Sensory irritants cause a painful burning, stinging and itching sensation. Controlled chamber studies are the “golden standard” for evaluations. Well conducted workplace studies offer another possibility. For generalization, the number of participants and their age, smoking, gender, and prior exposure, experience and mood has to be considered. Exposure assessments have to be reliable and exposure duration sufficiently long to establish time-response relationships. A potential confounding by odour has to be assessed. For workplace exposures, mixed exposure and healthy worker effects have to be evaluated. The “Alarie test” is the only validated animal bioassay for prediction of sensory irritation in humans. The mouse bioassay uses the trigeminal reflex-induced decrease in the respiratory rate. The 50% decrease (RD₅₀) has been correlated with OELs set for sensory irritants; predicted OELs for sensory irritants are 0.03xRD₅₀. Evaluation of the bioassay comprises the number of mice and the strain, the reliability of the exposure concentrations and exposure-response relationships, and the similar mode-of-action in mice and humans. These approaches can be used for quality assurance of reported data to set air quality guidelines.     

An attempt to define a just detectable effect for airborne chemicals on the respiratory tract in mice

 We have attempted to define a just detectable effect (JDE) for three different types of reactions along the respiratory tract: (a) sensory irritation of the upper airways (S), (b) airflow limitation along the conducting airways (A), and (c) pulmonary irritation at the alveolar level (P1 or P). Each type of reaction, S, A, P1 or P, was recognized by analyzing the breathing pattern of unanesthetized mice held in body plethysmographs. A rule-based computer program analyzed each breath during a period of 3.75 h and classified each breath as normal (N) or falling in any of the above categories (i.e., S, A, P1 or P). Eight groups of four mice were used for sham exposures: exposed to water vapor. These data sets were used, as sham exposure data, to define the variation which can occur with time in order to define an expected range of normal variation. Once this range was established, we defined JDE values for each type of effect and used such values to evaluate the results obtained in exposed animals. Eight groups of four mice were exposed to a mixture of airborne chemicals, machining fluid G (MFG), at concentrations from 0.17 to 55 mg/m³. Data sets for individual animals and for each group of animals exposed to MFG were analyzed to determine if and when a particular effect occurred. It was possible to recognize the effects of low exposure concentrations on groups of exposed animals or individual animals within each group. This procedure will be valuable when investigating the effect of airborne chemicals and when it is impossible to generate high exposure concentrations to define concentration-response relationships. Received: 4 October 1995/Accepted: 20 December 1995     

Characterization of the effects of an airborne mixture of chemicals on the respiratory tract and smoothing polynomial spline analysis of the data

 We expanded a previously published (Vijayaraghavan et al. 1994) computerized system to analyze the breathing pattern of unanesthetized mice in order better to recognize and quantify the effects of an airborne mixture of chemicals at three different levels of the respiratory tract. The airborne chemical mixture used was a machining fluid. Such fluids are widely used in industry and a large number of workers are exposed to these airborne mixtures. We found this mixture to be capable of inducing three types of effects on the respiratory tract: sensory irritation of the upper respiratory tract (S), airflow limitation along the conducting airways (A) and pulmonary irritation (P). Depending upon the exposure concentration, mainly S or P effects were obtained but an A effect was also identified. The three types of effects occurred at various times during the exposures and, furthermore, within a group of exposed animals some exhibited one type of effect while others exhibited another type. In order to analyze such complex data sets, two statistical methods for smoothing polynomial splines were utilized: the maximum likelihood (ML) method and generalized cross validation (GCV) method. The results indicated that previous methods used to characterize a single effect of airborne chemicals can now be extended to evaluate mixtures likely to induce multiple types of effects. However, statistical analysis methods, either the ML or GCV methods, or other appropriate methods are needed to evaluate the responses obtained due to the complex effects that a mixture can induce in comparison to single chemicals. Received: 3 November 1994/Accepted: 2 February 1995     

Characteristic modifications of the breathing pattern of mice to evaluate the effects of airborne chemicals on the respiratory tract

A system was developed for exposure of unanesthetized mice to airborne chemicals and for continuous measurement of their breathing pattern prior to, during and following exposure. By measuring inspiratory and expiratory airflows (VI and VE), and integration with time to yield tidal volume (VT), we obtained characteristic modifications to the normal breathing pattern. These permitted recognition that a specific portion of the respiratory tract was affected by the selected airborne chemicals. Following recognition, we also quantitated the degree of effect using one specific measurement in each case. An effect on the upper respiratory tract, induced by the sensory irritant, 2-chlorobenzylchloride, was quantitated by measuring a decrease in respiratory frequency. An effect on the conducting airways, induced by the airway constrictor, carbamylcholine, was quantitated by a decrease in VE at the mid-point of VT. An effect at the alveolar level, induced either by the vagal nerve ending stimulant, propranolol, or by the pulmonary irritant, machining fluid G, was quantitated by an increase in the length of a pause induced at the end of expiration. The system is easy to construct and operate and can be used to rapidly evaluate the effects of airborne chemicals on the respiratory tract.     

A quantitative structure-activity relationship for the eye irritation potential of neutral organic chemicals

Quantitative structure-activity relationships (QSARs) have been derived relating eye irritation data of a set of neutral organic chemicals to log(octanol-water partition coefficient), the minor principal inertial axes (R_y and R_z) and dipole moment. Datasets were analysed using principal components analysis; plots of the first 2 principal components of the above parameters showed that the analysis was able to discriminate well between the irritant and non-irritant chemicals in the dataset. The derived QSAR could be useful for the prediction of the eye irritation potential of new or untested chemicals within this category.     

Sensory irritation to mixtures of formaldehyde,acrolein, and acetaldehyde in rats

 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). Received: 24 April 1995/Accepted: 26 September 1995     

Formaldehyde and chemosensory irritation in humans: a controlled human exposure study

OBJECTIVES: The objective of this study was to examine the possible occurrence of sensory irritation and subjective symptoms in human volunteers exposed to formaldehyde concentrations relevant to the workplace. The set up of the study included formaldehyde exposures with and without peaks, the presence and absence of a masking agent, and evaluation of the influence of personality factors. METHODS: Testing was conducted in 21 healthy volunteers (11 males and 10 females) over a 10-week period using a repeated measures design. Each subject was exposed for 4h to each of the 10 exposure conditions on 10 consecutive working days. The 2-week exposure sequences were randomized, and the exposure to formaldehyde and the effect measurements were conducted in a double-blind fashion. During 4 of the 10 exposure sessions, 12-16 ppm ethyl acetate (EA) was used as a 'masking agent' for formaldehyde exposure. Measurements consisted of conjunctival redness, blinking frequency, nasal flow and resistance, pulmonary function, and reaction times. Also subjective ratings of discomfort as well as the influence of personality factors on the subjective scoring were examined. These were carried out pre-, during and/or post-exposure, and were used to evaluate the possible irritating effects of formaldehyde at these concentrations. RESULTS: The results indicated no significant treatment effects on nasal flow and resistance, pulmonary function, and reaction times. Blinking frequency and conjunctival redness, ranging from slight to moderate, were significantly increased by short-term peak exposures of 1.0 ppm that occurred at a baseline exposure of 0.5 ppm formaldehyde. Results of the subjective ratings indicated eye and olfactory symptoms at concentrations as low as 0.3 ppm. Nasal irritation was reported at concentration levels of 0.5 ppm plus peaks of 1.0 ppm as well as at levels of 0.3 and 0.5 ppm with co-exposure to EA. However, exposure to EA only was also perceived as irritating. In addition, volunteers who rated their personality as 'anxious' tended to report complaints at a higher intensity. When 'negative affectivity' was used as covariate, the level of 0.3 ppm was no longer an effect level but 0.5 ppm with peaks of 1.0 ppm was. Increased symptom scores were reversed 16 h after the end of the exposures. CONCLUSIONS: The results of the present study indicated eye irritation as the most sensitive parameter. Minimal objective eye irritation was observed at a level of 0.5 ppm with peaks of 1 ppm. The subjective complaints of ocular and nasal irritation noted at lower levels were not paralleled by objective measurements of eye and nasal irritation and were strongly influenced by personality factors and smell. It was concluded that the no-observed-effect level for subjective and objective eye irritation due to formaldehyde exposure was 0.5 ppm in case of a constant exposure level and 0.3 ppm with peaks of 0.6 ppm in case of short-term peak exposures.     

Setting an indoor air exposure limit for formaldehyde: factors of concern

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.     

In vitro human skin irritation test for evaluation of medical device extracts

The aim of this study was to determine if the EpiDerm? reconstructed human skin model (MatTek Corp.) could be an acceptable alternative to the ISO 10993-required rabbit skin irritation test for assessing medical device biocompatibility. Eleven medical device polymers were tested. Four extracts were prepared per polymer, two each with saline and sesame oil; half were spiked with two R-38 irritants, lactic acid for saline extracts and heptanoic acid for the sesame oil extracts. Tissue viability was assessed by MTT reduction and the proinflammatory response was assessed by IL-1α release. LOAELs of 2% for lactic acid in saline and 0.7% for heptanoic acid in sesame oil were determined. A cell viability reduction of >50% was indicative of skin irritation. Cells exposed to saline extracts spiked with 3.25% lactic acid had significantly reduced mean cell viabilities (12.6–17.2%). Cells exposed to sesame oil extracts spiked with 1.25% heptanoic acid also exhibited reduced mean cell viabilities (25.5%–41.7%). All spiked cells released substantial amounts of IL-1α (253.5–387.4 pg/ml) signifying a proinflammatory response. These results indicate that the EpiDerm? model may be a suitable in vitro replacement for the assessment of the irritation potential of medical device extracts.     

Adenosine sensory transduction pathways contribute to activation of the sensory irritation response to inspired irritant vapors.

The molecular mechanisms through which sensory irritants stimulate nasal trigeminal nerves are poorly understood. The current study was aimed at evaluating the potential contribution of purinergic sensory transduction pathways in this process. Aerosols of 4-36 mM adenosine 5'-triphosphate (ATP) and adenosine both acted as sensory irritants. Large dose capsaicin pretreatment to induce degeneration of transient receptor potential vanilloid type-1 (TRPV1)-expressing C fibers greatly reduced, but did not abolish, the sensory irritation response to ATP aerosol and was without effect on the response to adenosine aerosol, indicating that ATP acts largely on capsaicin-sensitive (primarily C fibers) and adenosine acts on capsaicin-insensitive (primarily Adelta fibers) nerves. The response to adenosine was diminished by pretreatment with the broad-based adenosine receptor antagonist theophylline (20 mg/kg) and A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (0.1 mg/kg), providing evidence that adenosine stimulates capsaicin-insensitive nerves via the A1 receptor. The sensory irritation responses to 275 ppm styrene and 110 ppm acetic acid vapors were significantly reduced by theophylline pretreatment suggesting a role for adenosine signaling pathways in activation of the sensory irritant response by these vapors. If sensory nerves are activated by mediators that are released from injured airway mucosal cells, then nasal sensory nerve activation may be a reflection of irritant-induced alterations in airway cell integrity.     

Second-phase validation study of short time exposure test for assessment of eye irritation potency of chemicals

A Short Time Exposure (STE) test is a cytotoxicity test that uses SIRC cells (rabbit corneal cell line) to assess eye irritation potency following a 5-min chemical exposure. This second-phase validation study assessed the predictive capacity of the STE test using 40 coded test substances at three laboratories. A Validation Management Team (VMT) then evaluated the predictivity of the STE test for United Nation (UN) Globally Harmonized System (GHS) categories using 63 test substances including the results of the first-phase validation study.The STE test can assess not only the severe or corrosive ocular irritants (corresponding to the UN GHS Category 1) but also non-irritant (corresponding to UN GHS Non Category) from other toxicity classes, especially for limited types of test substances. The predictivity by STE test, however, was insufficient for identification of UN GHS categories (Category 1, Category 2, or Non Category).These results suggest that the STE test can be recommended as an initial step in a top-down approach to identification of severe irritants and test substances that require classification for eye irritation (UN GHS Category 1) as well as an initial step in a bottom-up approach to identification of test substances that do not require classification for eye irritation (UN GHS Non Category) from other toxicity classes, especially for limited types of test substances. On the other hand, the STE test is not considered adequate for the identification of mild or moderate irritants (i.e., UN GHS Categories 2A and 2B) and severe irritants (UN GHS Category 1).     

Identifying an indoor air exposure limit for formaldehyde considering both irritation and cancer hazards

Formaldehyde is a well-studied chemical and effects from inhalation exposures have been extensively characterized in numerous controlled studies with human volunteers, including asthmatics and other sensitive individuals, which provide a rich database on exposure concentrations that can reliably produce the symptoms of sensory irritation. Although individuals can differ in their sensitivity to odor and eye irritation, the majority of authoritative reviews of the formaldehyde literature have concluded that an air concentration of 0.3?ppm will provide protection from eye irritation for virtually everyone. A weight of evidence–based formaldehyde exposure limit of 0.1?ppm (100 ppb) is recommended as an indoor air level for all individuals for odor detection and sensory irritation. It has recently been suggested by the International Agency for Research on Cancer (IARC), the National Toxicology Program (NTP), and the US Environmental Protection Agency (US EPA) that formaldehyde is causally associated with nasopharyngeal cancer (NPC) and leukemia. This has led US EPA to conclude that irritation is not the most sensitive toxic endpoint and that carcinogenicity should dictate how to establish exposure limits for formaldehyde. In this review, a number of lines of reasoning and substantial scientific evidence are described and discussed, which leads to a conclusion that neither point of contact nor systemic effects of any type, including NPC or leukemia, are causally associated with exposure to formaldehyde. This conclusion supports the view that the equivocal epidemiology studies that suggest otherwise are almost certainly flawed by identified or yet to be unidentified confounding variables. Thus, this assessment concludes that a formaldehyde indoor air limit of 0.1?ppm should protect even particularly susceptible individuals from both irritation effects and any potential cancer hazard.     

A new cell-based method for assessing the eye irritation potential of chemicals: an alternative to the Draize test

Using a human corneal cell line (HCE-T cells) and 2 evaluation criteria, we developed a new alternative method to assess the eye irritation potential of chemicals. We exposed HCE-T cells to different concentrations of 38 chemicals for 1h and measured relative cell viability (RCV) as an endpoint at each concentration. Using the RCV values, we calculated the RCV50. We also exposed HCE-T cells to 3 fixed concentrations of the 38 chemicals (5%, 0.5%, and 0.05%) for 1h and measured the RCV at each concentration. Using the RCV values at 5%, 0.5%, and 0.05%, we developed a new criterion for eye irritation potential (total eye irritation score, TEIS) and estimated the ocular irritancy. We then assessed the correlation of the results of RCV50 and TEIS with those of the Draize rabbit eye irritation. Both the RCV50 and TEIS results exhibited good positive correlations (sensitivity: 80.77%, specificity: 83.33%, and accuracy: 81.58% for TEIS; sensitivity: 73.08-76.92%, specificity: 75.00%, and accuracy: 73.68-76.32% for RCV50). We conclude that the new in vitro model using HCE-T cells is a good alternative evaluation model for the prediction of the eye irritation potential of chemicals.     

眼刺激性评价体外替代方法研究进展

眼用药物及可能接触眼睛的受试物均应考虑进行眼刺激试验。Draize试验一直是公认的测定急性眼刺激性的国际标准,但随着"3R"原则的实施,体外替代方法逐渐成为眼刺激性评价研究的发展方向。目前已有多种体外替代方法得到了欧洲化学品局(European Chemical Agency, ECHA)、美国国家环境保护局(US Environmental Protection Agency, US EPA)及经济合作与发展组织(The Organization for Economic Co-operation and Development, OECD)的认可,其中有5种替代试验得到了OECD的认可,分别是牛角膜不透明度和渗透性试验,离体鸡眼试验、短时暴露试验、重组人角膜上皮模型试验试验以及荧光素渗漏试验。在使用体外替代方法时,由于单一的方法不能蕴含所有的观察终点,所以应根据每种替代方法的特点及应用范围进行组合以覆盖整个分层测试策略。相信随着体外替代试验研究的不断深入,有希望在未来能够完全替代体内试验。本文就眼刺激评价的体外替代方法和评价标准进行了综述。     

Refinement of the Episkin® protocol for the assessment of acute skin irritation of chemicals: follow-up to the ECVAM prevalidation study

The Episkin® model took part in the prevalidation study on in vitro tests for acute skin irritation of chemicals, which was carried out during 1999 and 2000. This prevalidation study was co-ordinated and supported by the European Centre for the Validation of Alternative Methods (ECVAM). During Phase 1 and Phase 2 of this study, reproducibility and transferability of the method were verified. Unfortunately, the performance of the method in terms of predictive ability was considered insufficient, due to a low specificity. In order to improve the performance of the Episkin® method, the existing protocol was refined. This refinement consisted in reducing the exposure time of epidermis with chemicals. Sensitivity, specificity and accuracy of the new method were 70, 80 and 75%, respectively, thus meeting the acceptance criteria as defined by the Management Team. The Episkin® method is now ready to enter a validation study of in vitro tests for acute skin irritation.