Assessment of Respiratory Hypersensitivity in Guinea Pigs Sensitized to Toluene Diisocyanate: Improvements on Analysis of Respiratory Response

FUNDAMENTAL AND APPLIED TOXICOLOGY ARTICLE NO.

40, 211–219 (1997)

FA972393

Assessment of Respiratory Hypersensitivity in Guinea Pigs Sensitized to Toluene Diisocyanate: Improvements on Analysis of Respiratory Response Ju¨rgen Pauluhn Institute of Toxicology, BAYER AG, 42096 Wuppertal, Germany Received January 30, 1997; accepted October 15, 1997

Assessment of Respiratory Hypersensitivity in Guinea Pigs Sensitized to Toluene Diisocyanate: Improvements on Analysis of Respiratory Response. Pauluhn, J. (1997). Fundam. Appl. Toxicol. 40, 211–219. Groups of guinea pigs of the Hartley strain were sensitized to toluene diisocyanate (TDI) by combined single intradermal injection and repeated inhalation exposure (3 h/day for 5 consecutive days) to 0, 3.8, 11, 26, 46, and 51 mg TDI/m3 air. One group of animals was sensitized by intradermal injection only. Sham-exposed and TDI–polyisocyanate resin-sensitized guinea pigs served as controls. Three weeks after the first encounter with the inducing agent, animals were challenged with the free TDI (approximately 0.5 mg/m3) and 1 week later with TDI–guinea pig serum albumin conjugate. Breathing patterns were analyzed by objective mathematical procedures taking into account the intensity and duration of the respiratory rate exceeding {3 standard deviations of the individual prechallenge exposure period. In none of the animals challenged with TDI were conclusive immediate-onset respiratory responses identified. During the TDI conjugate challenge a characteristic increase in respiratory rate was observed in all groups sensitized with TDI. In each of the sham and TDI–resin control groups, 1 of 16 animals responded mildly to the conjugate challenge. With regard to analysis of the development of asthma-like dyspnea, the results obtained suggest that respiratory response can suitably be defined by objective mathematical analysis of breathing patterns. Moreover, the ‘‘duration’’ of response exceeding /3 1 standard deviation of prechallenge baseline data appears to show less variability when compared to the ‘‘intensity’’ of response (area). It can be concluded that this method of evaluation of respiratory response may be useful to compare more quantitatively this type of data and serves the objective of decreasing potential interlaboratory variability. q 1997 Society of Toxicology.

The guinea pig model for assessment of pulmonary allergenicity of low-molecular-weight chemicals has received rigorous review (Blaikie et al., 1995; Griffiths-Johnson and Karol, 1991; Hayes and Newman-Taylor, 1995; Karol and Thorne, 1988; Karol, 1988, 1994; Pauluhn and Eben, 1991; Sarlo and Clark, 1992; Sugawara et al., 1993). The model

utilizes various routes of exposure to the hapten for sensitization and inhalation to the hapten and/or hapten–protein conjugate for the elicitation phase of the response. This study summarizes attempts to further refine this animal model for a more objective assessment of respiratory response upon challenge exposures to free and conjugated toluene diisocyanate (TDI). The TDI guinea pig model has been used in numerous laboratories and the responses of animals have been reported to be quite similar. From the various reports, the robustness of the guinea pig respiratory hypersensitivity model is confirmed because both from repeated inhalation induction exposure to a range of concentrations and from (intra)dermal injection protocols, upon challenge with the TDI conjugate, TDI was identified as a respiratory sensitizer (Karol et al., 1981; Karol, 1983, 1988, 1994; Botham et al., 1988; Huang et al., 1993; Blaikie et al., 1995; Sarlo and Clark, 1992). Characteristic changes in breathing pattern were indicated by an increase in respiratory rate upon inhalation challenge with TDI conjugate. Challenge with the free TDI was reported to test negative (Botham et al., 1988; Blaikie et al., 1995) or positive (Aoyama et al., 1994). Previous investigations of the low-molecular-weight trimellitic anhydride (TMA) suggest that guinea pigs sensitized to TMA and challenged with the free or conjugated chemical experienced a high incidence of unequivocal, typical respiratory responses whereas it appears to be more difficult to duplicate this response with known human respiratory sensitizers of the (di)isocyanate class (Pauluhn and Mohr, 1994; Pauluhn, 1994a). It is therefore believed that for analysis of toluene diisocyanate-induced respiratory allergy in guinea pigs, a more refined and standardized analysis of respiratory function data may improve the validity of this animal model. Thus, the objective of this study is to analyze different mathematical procedures for quantifying their sensitivity to distinguish objectively respiratory responses occurring in animals sensitized by different routes in comparison to naive animals.

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0272-0590/97 $25.00 Copyright q 1997 by the Society of Toxicology. All rights of reproduction in any form reserved.

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EXPERIMENTAL Test Materials Toluene diisocyanate (Desmodu T80), an 80:20 mixture of the 2,4- and 2,6-isomers (purity ú99.9%), and a solid, finely ground TDI polyisocyanate resin (reaction product of TDI with polyol, NCO content: approximately 16%; Desmodu L) were from Bayer AG (Leverkusen, Germany). Corn oil and dry acetone were supplied by Caesar and Loretz GmbH (Hilden, Germany) and by Merck (Darmstadt, Germany), respectively. Hapten–Protein Conjugate Conjugate batches of TDI with guinea pig serum albumin (GPSA) were prepared as follows: 2 g guinea pig serum albumin, supplied by Sigma Chemie (Deisenhofen, Germany), was dissolved in 200 ml ice-cold phosphate-buffered saline (PBS, pH 7.4). 522 mg of TDI was added dropwise over 15 min and the solution was stirred at 07C (ice bath) for an additional 30 min. After centrifugation (approximately 30 min at 1000g), the supernatant was dialyzed successively against PBS and distilled water, each for a period of 3 days at 47C. The conjugate was lyophilized and stored at 0207C. The same procedure was used to prepare the polyisocyanate resin conjugate, except that the resin (2.8 g) was dissolved in 20 ml dimethyl sulfoxide (Sigma Chemie) and that the supernatant was dialyzed successively against PBS and 50 mM ammonium hydrogen carbonate instead of distilled water. Animals Female Dunkin–Hartley guinea pigs (Crl:(HA)BR), with an initial weight range of 250–350 g, were obtained from Charles River (Sulzfeld, Germany). After an acclimatization period of 1–2 weeks the animals were randomly assigned to the groups. Animals were housed in type IV Makrolon cages, four animals per cage. Bedding material (low-dust wood shavings) was changed twice weekly. Altromin 3020 maintenance diet for guinea pigs and tap water were provided ad libitum, except during exposures. A 12-hr on/off light cycle was maintained in the animal housing room. Temperature was 23 { 27C and relative humidity was in the range 40–70%. Test Procedure Sensitization. Sensitization of the guinea pigs was induced by a single intradermal injection into the scapular region of the test or control material on day 0 (group 8, Table 1) by a combined repeated inhalation–single intradermal injection (groups 3–7, Table 1) or by brief, high-concentration inhalation exposures (groups 1–2, Table 1). In groups 3–8, TDI was injected (100 ml) as a 0.3% (w/v) solution in dried corn oil. In two groups of animals the polyisocyanate resin was injected (50 ml) as a 3% (w/v) solution in a 70:30 mixture of dried corn oil and acetone. The stability of test materials in each vehicle had been verified analytically. All inhalation exposures were made in a directed-flow nose-only inhalation chamber. Guinea pigs of groups 3–7 were exposed to TDI vapor for 3 h/day on 5 consecutive days, days 0–4. TDI vapor was generated by metering dry nitrogen through a water-jacketed glass bubbler (content approximately 25 ml, kept at 457C). Animals of the two polyisocyanate resin groups were subjected to the same exposure protocol. Concentrations were 50 and 250 mg dust/m3, respectively. The dust atmosphere was generated by a WrightDust-Feeder (BGI Inc., Waltham, MA) using micronized resin (micronization of frozen {liquid N2} material in a mortar mill). Before entrainment into the cyclone of the inhalation chamber this atmosphere was diluted further with conditioned air to achieve the desired concentration. The airflow rate through the chamber was at least 20 liters/min. Two groups of naive control animals were used; one was sham-exposed and the other received the vehicle (corn oil) alone. Each group consisted of at least eight guinea pigs. In the repeated inhalation induction groups 3–6, an additional

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FIG. 1. Mathematical analysis of respiratory response. For assessment, the duration of response as well as the intensity of response (hatched area) was calculated exceeding the mean / 31 std of the 15-min prechallenge period (challenge, 30 min; postchallenge period, 60 min).

two guinea pigs per group were exposed as replacement animals for unscheduled events. The sham and vehicle controls as well as the 50 and 250 mg/m3 polyisocyanate groups were indistinguishable in all parameters measured and were therefore combined as groups 9 and 10, respectively (see Table 1). Two groups of guinea pigs (groups 1 and 2) were also sensitized with either 136 or 220 mg TDI/m3 by a single 15-min inhalation exposure (no intradermal injection). In the lower concentration, TDI was generated as vapor; in the higher it was aerosolized. The single, brief highconcentration induction protocol has previously been shown to be adequate to sensitize guinea pigs to the respiratory allergen TMA (Pauluhn and Eben, 1991) or diphenylmethane-4,4*-diisocyanate (Pauluhn and Mohr, 1994). Elicitation of respiratory sensitization. Starting on day 21 the guinea pigs were challenged with approximately 0.5 mg/m3 TDI (groups 1–10). About 1 week later, animals received an additional challenge with 30–50 mg/m3 TDI–GPSA conjugate (groups 1–9). The duration of each challenge was 30 min. Animals sensitized to polyisocyanate resin (group 10) were challenged subsequently with TDI–GPSA conjugate and resin–GPSA conjugate (É50 mg/m3), each for 15 min. For challenge exposures, the respective conjugate was dissolved in saline to obtain a concentration of 1.5% (w/v). This solution was nebulized into the baffle of the nose-only inhalation chamber at a rate of 200 ml/10 liters air/min. Inhalation chamber and analysis of atmospheres. The type of noseonly chamber used minimizes rebreathing of exhaled test atmosphere and prevents hydrolytic degradation of isocyanate. The temporal stability of the vapor atmospheres was monitored continuously using a total hydrocarbon analyzer equipped with a flame ionization detector (Compur, Munich, Germany), whereas the respective aerosol atmospheres were monitored using a RAM-1 or a RAS-2 real-time aerosol photometer (MIE, Bedford, MA). All air flows, including those for analytical sampling, were monitored and adjusted continuously by means of calibrated and computer-controlled mass-flow controllers. For all exposure groups, chamber humidity and temperature were approximately 20% and 237C, respectively. Previously pub-

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FIG. 2. Analysis of variability of selected breathing patterns of pooled prechallenge data. MV, respiratory minute volume; RR, respiratory rate; PEF(IT / ET)/TV (FDP), flow-derived, dimensionless breathing parameter.

lished validation data indicated that this inhalation system provides a high degree of spatial and temporal uniformity of test atmosphere (Pauluhn, 1994d). Chamber air from the vicinity of the breathing zone of the animals was sampled three times per exposure day during the induction phase and once during each challenge exposure. The TDI atmosphere was collected using glass powder (40/60 mesh, G. Karl, Part No. GK 26-48004)-loaded tubes containing nitro reagent (N-4-nitrobenzyl-N-(n-propyl)-amine, Riedel de Haen No. 33487) (Dunlap et al., 1976). This procedure involves derivatiza-

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tion of isocyanate in the air sample with nitro reagent, desorption of the TDI urea derivative from the glass powder with acetonitrile, and subsequent analysis by HPLC with ultraviolet (UV) detection. A calibration curve was constructed by plotting the detector response of the most prominent HPLC peak(s) from analysis of derivatized TDI versus the mass of TDI analyzed. Atmospheric concentrations of the protein conjugates or polyisocyanate resin were determined gravimetrically using filter sampling (glass fiber filters SM 13430, Sartorius, Go¨ttingen, Germany). Analyses of particle-size distributions of the conjugate and polyisocyanate resin atmospheres were

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FIG. 3. Results of delayed-onset measurements following TDI challenge. Left, controls; right, single intradermal induction of TDI.

determined using a critical orifice, low-pressure AERAS stainless steel cascade impactor (HAUKE, 4810 Gmunden, Austria). The individual impactor stages were covered with aluminum foil which had been weighed. For polyisocyanate resin and conjugate, the respective MMADs (GSDs) were approximately 2.6 (2.1) and 1.8 mm (1.9). Measurement of Immediate-Onset Pulmonary Response After acclimatization to the nose-only plethysmograph, baseline parameters were measured for approximately 15 min. Measurements were continued during the 30-min challenge period, followed by postchallenge measurements of approximately 60 min. Measurements were made with eight guinea pigs simultaneously. For evaluation of immediate-onset responses, the following respiratory parameters were evaluated: respiratory rate (RR), tidal volume (TV), respiratory minute volume (MV), peak expiratory flow rate (PEF), inspiratory (IT) and expiratory times (ET), the flow-derived dimensionless parameter PEF 1 (ET / IT)/TV (FDP), average duration of apneic period, and the number of apneic periods occurring during one averaging period exceeding 20% of the ET period (AC). Measurements were taken in a nose-only animal restrainer with a wire mesh pneumotachograph and a differential pressure transducer (MP 45 { 2 cm H2O, Validyne) fitted directly onto the exposure restrainer (plethysmograph). The head and body compartments were separated using a double-layer latex neck seal separated by a stabilizer. Precautions were taken to avoid artifacts due to restraint and tight-fitting seals around the neck. Data collection and evaluation were made using a commercially available system (PO-NE-MAH Digital Acquisition, Analysis & Archive System, Simsbury, CT) equipped with PLUGSYS amplifiers (H. Sachs, March, Germany). Signals were averaged during a logging period of 20 s. Potential artifacts due to the dependence on the

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calculated volume as a function of frequency were considered as described earlier (Pauluhn, 1994b). Measurement of Delayed-Onset Pulmonary Response Exploratory measurements for the detection of delayed-onset effects were made on animals sensitized by intradermal injection, including sham controls (four animals/group). The recording of data for delayed-onset effects commenced after cessation of measurements of the immediate-onset response following challenge with TDI. The delayed-onset respiratory hypersensitivity response was monitored on unrestrained guinea pigs using four water-jacketed, temperature-controlled (thermostat Julabo UC-5B/5) wholebody plethysmographs (average chamber temperature, 22.57C; duration of measurement, ca. 19 h; chamber volume, 2.4 liters, bias air-flow rate 2 litersrmin01). During the monitoring period, guinea pigs were kept on bedding material (low-dust wood shavings); diet and tap water were provided ad libitum. Signals (respiratory rate) were averaged during a logging period of 1 min and positive response was indicated by an increased respiratory rate (Karol et al., 1985; Karol and Thorne, 1988). Data Recording and Evaluation For each animal the individual baseline data collected during the 15-min prechallenge period were used to calculate the mean { 31 standard deviation (std). All data collected were normalized to the mean of the prechallenge period (Å 100%). Any response exceeding the mean { 31 std during the subsequent challenge and postchallenge periods was classified as a positive response. Breathing patterns were analyzed mathematically using the raw data whereas breathing patterns shown in the figures were smoothed

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TABLE 1 Evaluation of Immediate-Onset Respiratory Hypersensitivity Using Various Induction and Elicitation Protocols TDI challenge

N 1 2 3 4 5 6 7 8 9 10

Induction regimen 1 1 15 min 136 mg/m3 1 1 15 min 220 mg/m3 513h 3.8 mg/m3 513h 11 mg/m3 513h 26 mg/m3 513h 46 mg/m3 513h 51 mg/m3 id 1 1 0.3% Control 513h Resin

TDI–GPSA conjugate challenge

Duration

Area

Duration

Area

Duration

Area

Duration

Area

RR

RR

FDP

FDP

RR

RR

FDP

FDP

5/8

5/8

2/8

3/8

2/8

3/8

4/8

4/8

4/8

3/8

5/8

4/8

3/8

3/8

6/8

5/8

3/8

6/8

2/8

1/8

4/8

4/8

0/8

2/8

0/8

2/8

2/8

2/8

5/8

6/8

5/8

4/8

1/8

1/8

3/8

0/8

5/8

5/8

4/8

3/8

2/8

2/8

3/8

2/8

7/8

5/8

6/8

5/8

1/8

2/8

2/8

0/8

3/8

1/8

1/8

6/8

0/8 3/16 0/16

0/8 4/16 0/16

5/8 3/16 1/16

0/8 4/16 3/16

3/8 1/16 1/16

3/8 1/16 1/16

3/8 1/16 1/16

3/8 1/16 1/16

Note. Area, intensity of response (area under the curve exceeding 31 std of the individual prechallenge period); duration, number of events exceeding 31 std of the individual prechallenge period); RR, respiratory rate; FDP, dimensionless flow-derived parameter; #/#, number of animals showing positive response/number of challenged animals; GPSA, guinea pig serum albumin.

using a low-pass filter to eliminate breathing patterns of high frequency (except those depicted in Fig. 1). Two modes of mathematical analysis of response were used. First, the area under the curve was calculated to quantify the ‘‘intensity’’ of response (hatched area in Fig. 1). Second, the number of events above the individual mean / 31 std was counted for the same time interval to quantify the ‘‘duration’’ of response. To accommodate for responses observed in naive animals, an individual response was rated positive when values of RR and FDP exceeded 500 and 150 (intensity) and 25 and 15 (duration), respectively. This approach was assumed to provide an objective means for the quantitative assessment of the intensity of response (area under the curve) as well as the duration of response. The analysis of regression curves and their 95% prediction intervals as well as the iterative calculations were made by Sigma Plot for Windows (Jandel Scientific, Erkrath, Germany). The metaanalysis of respiratory patterns was made using Microsoft FORTRAN code.

RESULTS

Delayed-Onset Response Delayed-onset respiratory response was monitored on four guinea pigs per group after the measurements of immediateonset reactions had been completed (measurements were only made in four of eight animals in group 8 and sham controls). Figure 3 illustrates delayed-onset changes in respiratory rate occurring in TDI-sensitized guinea pigs and sham controls after TDI challenge (see below). Despite placement of guinea pigs in the bias-flow whole-body plethysmographs (no restraint), increases in respiratory rate of variable intensity occurred approximately 5 h after the start of data collection. Due to the difficulty in distinguishing spontaneous and specific responses of delayed onset, this study had been focused on the analysis of data of immediate-onset measurements.

Analysis of Prechallenge Data The usefulness of pooled data of the respective preexposure control periods for comparison and assessment was analyzed and summarized in Fig. 2. From the statistical evaluation of the prediction intervals of the means or means / 11 std it appears that pooled data are less suitable than individual baseline data to set the criteria for positive response. Therefore, the comparison based on individual animal prechallenge baseline data was given preference to pooled data.

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TDI Challenge For elicitation of specific respiratory hypersensitivity, guinea pigs of all groups were exposed for 30 min to an analytically determined concentration of 0.5 mg TDI/m3 air (std { 12%) on about day 21 (range, days 20–23). A summary of results is given in Table 1. Individual response data with regard to the total intensity and duration of response exceeding the /31 std of baseline prechallenge values are

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FIG. 4. Analysis of (a) intensity (area under the curve exceeding 31 std of the individual prechallenge period) or (b) duration (number of events exceeding 31 std of the individual prechallenge period) of respiratory rate (RR) and dimensionless flow-derived parameter (FDP) of guinea pigs sensitized to and challenged with TDI (for allocation of groups see Table 1). Values in parentheses indicate the incidence of responding animals per number of challenged animals.

shown in Figs. 4a and 4b, respectively. Animals sensitized according to the single, brief high-level exposure regimen (136 or 220 mg TDI/m3 air for a duration of 15 min, groups 1 and 2) appeared to be more responsive to the TDI challenge when compared to naive animals (group 9) and the FDP indicates greater response than the analysis of RR. Based on the evaluation of RR and FDP, animals that received one intradermal injection only (group 8) showed no response whereas those of groups 3 to 7, which where subjected to the additional 5 1 3 h/day inhalation induction exposures of 3.8, 11, 26, 46, and 51 mg TDI/m3 air, respectively, experienced mild or inconclusive changes in breathing patterns. Respiratory responses observed in the polyisocyanate resin and control groups (group 10 versus group 9) were virtually indistinguishable. TDI–GPSA Challenge Bronchial provocation challenges with the TDI–GPSA conjugate were performed on day 28 ({1). During challenge,

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positive response was indicated by increases in PEF, MV, RR, and AC as well as by a decrease in ET. In relation to the fluctuations in breathing patterns, evaluation of RR was given preference. Characteristic changes in breathing pattern observed in the more severely responding animals are depicted in Figs. 5a and 5b. Results of challenge with the conjugate are summarized in Table 1. As shown in Figs. 6a and 6b, none of the control animals displayed marked changes in respiratory rate (group 9). By contrast, several animals of the brief high-level (groups 1 and 2) and of the repeated inhalation exposure regimen displayed pulmonary sensitivity. Animals sensitized to the polyisocyanate resin (group 10) responded neither to the TDI conjugate nor to the resin conjugate. Comparison of animals of group 8 (single intradermal injection) with groups 3 to 7 (additional repeated inhalation exposures) demonstrates that additional inhalation exposures appear to enhance both the duration and the intensity of pulmonary response. From the data summarized it appears that the analysis of the duration of response provides

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FIG. 5. Respiratory parameters measured during challenge with TDI–GPSA conjugate for 30 min. Respiratory response data were normalized to the mean of the prechallenge exposure period (Å100%). Breathing parameters shown: PEF, peak expiratory flow rate during tidal breathing; TV, tidal volume; MV, respiratory minute volume; RR, respiratory rate; IT, ET, inspiratory and exspiratory times; AT, apnea time; AC, apneic periods per logging period.

more consistent results than the analysis of intensity of response. Additional analysis of the composite dimensionless FDP did not improve the sensitivity of evaluation of response. DISCUSSION

This investigation utilized the experience gained with the low-molecular-weight chemical TMA in previous studies (Pauluhn and Eben, 1991; Pauluhn, 1994a–c, 1995). Those studies showed severe and unequivocal respiratory responses upon TMA and TMA–protein conjugate challenges and the intensity of response appeared to be most pronounced when a low-dose single intradermal injection regimen rather than

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a repeated inhalation sensitization regimen was used. However, when the same protocol was used to explore whether the intradermal injection of TDI into guinea pigs, followed by the analysis of pulmonary responses to an inhalation challenge with either the free or conjugated chemical, no conclusive respiratory responses occurred during the TDI challenge. During the TDI–protein conjugate challenge, in turn, characteristic changes in respiratory patterns of various intensities were observed. One of the most pronounced effects recorded is illustrated in Figs. 5a and 5b. However, in none of the various sensitization regimens summarized in Table 1 was the typical ‘‘waveform’’ breathing pattern that occurred upon challenge with the free or conjugated TMA (Pauluhn and Eben, 1991; Pauluhn, 1994a,b) observed.

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FIG. 6. Analysis of (a) intensity (area under the curve exceeding 31 std of the individual prechallenge period) or (b) duration (number of events exceeding 31 std of the individual prechallenge period) of respiratory rate (RR) and dimensionless flow-derived parameter (FDP) of guinea pigs sensitized to TDI and challenged with TDI–GPSA conjugate (for allocation of groups see Table 1). Values in parentheses indicate the incidence of responding animals per number of challenged animals.

In order to allow an objective mathematical analysis of respiratory data, pooled data of the respective preexposure control periods were considered for comparison. However, as shown in Fig. 2, the use of prediction intervals of the means or means / 11 std would have resulted in a high number of false-positive responses. This may possibly be due to stress-related artifacts of some individual animals placed in the nose-only restraining plethysmographs, though periods of increased respiratory rate were also observed in unrestrained guinea pigs in bias-flow whole-body plethysmographs. Due to these as yet ill-defined factors, the comparison of the various induction regimens has been focused on the analysis of immediate-onset responses based on individual animals’ prechallenge baseline data rather than pooled data. As can be seen from Fig. 2, the analysis of MV appears to be less suitable for evaluation due to its higher variability. Taking into account the variability of data obtained during the prechallenge period, analysis of data was focused on the respiratory rate (mean, 96 breaths/min; std, {13%) and the FDP (mean, 2.5; std, {8%). Most interestingly, other authors

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have also found the mean respiratory rate in control groups to be 98 breaths/min (std, {10%) (Aoyama et al., 1994) and 99 breaths/min (std, {9%) (Huang et al., 1993). Karol (1983) considered an increase in respiratory rate greater than 31 std above the mean response to inhalation challenge in nonsensitized guinea pigs to be indicative of respiratory allergy, i.e., 47% increase in respiratory rate. As can be seen from Fig. 2, this value is almost identical to the upper prediction interval of respiratory rate data determined during prechallenge in nose-only plethysmographs. Indeed, delayed-onset respiratory responses were reported to occur in guinea pigs following TDI challenge (Karol et al., 1985). However, as reported previously (Pauluhn and Eben, 1991), an adventitious increase of the respiratory rate appear also to occur in sham controls under the conditions of this study. Based on findings depicted in Fig. 3, no further emphasis was made to address measurements of delayedonset respiratory response. With regard to analysis of the development of asthmalike dyspnea, the comparison of the results summarized in

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Figs. 6a and 6b suggests that the respiratory responses can be defined by objective mathematical analysis of breathing patterns. Moreover, the duration of response exceeding /31 std of prechallenge baseline data appears to show less variability when compared to the intensity of response (area). This increased variability appears to be associated with periods of high respiratory rate transiently occurring in either type of plethysmographs. For the TDI or TDI–GPSA challenge the analysis of other respiratory parameters did not show any advantage over measurements of respiratory rate alone. The analysis of the duration of increased respiratory rate appears to be the most straightforward evaluation of response and may be productive in reducing the interlaboratory variability of this animal model. REFERENCES Aoyama, K., Huang, J., Ueda, A., and Matsushita, T. (1994). Provocation of respiratory allergy in guinea pigs following inhalation of free toluene diisocyanate. Arch. Environ. Contam. Toxicol. 26, 403–407. Blaikie, L., Morrow, T., Wilson, A. P., Hext, P., Hartop, P. J., Rattray, N. J., Woodcock, D., and Botham, P. A. (1995). A two-centre study for the evaluation and validation of an animal model for the assessment of the potential of small molecular weight chemicals to cause respiratory allergy. Toxicology 96, 37–50. Botham, P. A., Hext, P., Rattray, N. J., Walsh, S. T., and Woodcock, D. R. (1988). Sensitization of guinea pigs by inhalation exposure to low-molecular-weight chemicals. Toxicol. Lett. 41, 159–173. Briatico-Vangosa, C., Braun, C. J. L., Cookman, G., Hofmann, T., Kimber, I., Loveless, S. E., Morrow, T., Pauluhn, J., Sørensen, T., and Niessen, H. J. (1994). Review. Respiratory allergy: Hazard identification and risk assessment. Fundam. Appl. Toxicol. 23, 145–158. Dunlap, K. L., Sandridge, R. L., and Keller, J. (1976). Determination of isocyanates in working atmosphere by high performance liquid chromatography. Anal. Chem. 48, 497–499. Griffiths-Johnson, D. A., and Karol, M. H. (1991). Validation of a noninvasive technique to assess development of airway hyperreactivity in an animal model of immunologic respiratory hypersensitivity. Toxicology 65, 283–294. Hayes, J. P., and Newman-Taylor, A. J. (1995). In vivo models of occupational asthma due to low molecular weight chemicals. Occup. Environ. Med. 52, 539–543. Huang, J., Aoyama, K., and Ueda, A. (1993). Experimental study on respiratory sensitivity to inhaled toluene diisocyanate. Arch. Toxicol. 67, 373– 378. Karol, M. H. (1983). Concentration-dependent immunologic response to toluene diisocyanate (TDI) following inhalation exposure. Toxicol. Appl. Pharmacol. 68, 229–241.

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