Effects of methacrolein on the respiratory tract in mice

Toxicology Letters 114 (2000) 197 – 202 www.elsevier.com/locate/toxlet

Effects of methacrolein on the respiratory tract in mice Søren Thor Larsen *, Gunnar Damga˚rd Nielsen National Institute of Occupational Health, Lersø Parkalle´ 105, DK-2100 Copenhagen, Denmark Received 1 June 1999; received in revised form 24 November 1999; accepted 3 December 1999

Abstract The acute respiratory effects of airborne exposure to methacrolein were studied in a recent refinement of the standard test method with mice (ASTM, 1984. American Society for Testing and Materials, Philadelphia). Irritation of the upper respiratory tract caused a concentration-dependent decrease in the respiratory rate of 2 – 26 ppm methacrolein. In this range, only a minor airflow limitation occurred in the lower respiratory tract, suggesting that the main effect of methacrolein is sensory irritation. During exposure, the sensory irritation response maintained the same level, i.e. no desensitisation occurred. The concentration 10.4 ppm methacrolein reduced the respiratory rate by 50% (RD50). The extrapolated threshold for the respiratory depressing effect, RD0, was 1.3 ppm. The sensory irritation effect of methacrolein was compared with results from closely related compounds in order to elucidate the mechanism of the interaction between methacrolein and the sensory irritant receptor. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Methacrolein; Sensory irritation; Pulmonary irritation; Airflow limitation; BALB/c mice; Airborne exposure; Inhalation

1. Introduction Methacrolein (CH2C(CH3)CHO; CAS-number, 78-85-3) may occur as an intermediate in the production of methacrylonitrile or methacrylic acid (Hess et al., 1978). It also appears as a product of the reaction between ozone (O3) and isoprene (Sauer et al., 1999), which we have confirmed recently (Wolkoff et al., unpublished observation). This reaction may be of special interest for indoor air quality evaluations, as isoprene is a * Corresponding author. Tel.: +45-39-165248; fax: + 4539-165201. E-mail address: [email protected] (S.T. Larsen)

natural constituent of human breath (IARC, 1994). Methacrolein may thus be formed indoors or in the respiratory tract, especially if a local O3 source is present. Limited knowledge is available about the airway effects of methacrolein. It is suspected to be a potent airway irritant as it is structurally related to acrolein, which possesses this property (Nielsen, 1991). Therefore, the purpose of this study was to assess the acute effects of methacrolein on three different levels of the respiratory system by means of a recent refinement (Vijayaraghavan et al., 1993, 1994; Boylstein et al., 1996; Alarie, 1998) of the standard mouse bioassay (ASTM, 1984). The levels studied were

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the upper respiratory tract, i.e. sensory irritation, the conducting airways and the alveolar level. The closely related substance, acrolein, has been studied extensively (e.g. WHO, 1992). In contrast, little has been published about the toxicity of methacrolein.

2. Materials and methods

2.1. Animals Male BALB/cA mice were obtained from Bomholtgaard, Denmark. The animals were placed in polypropylene cages with sawdust bedding (Lignocel S8, Broga˚rden, Denmark), provided with tap water ad libitum, and given a standard diet (Altromin no. 1324, Broga˚rden, Denmark). The animal housing was maintained on a 12 h light/dark cycle. A group of four naive mice was used at each exposure concentration, i.e. n =24, as six concentrations were studied. At testing, their mean weight was 27.7 g with a S.D. of 1 g.

2.2. Chemical Methacrolein (about 95% pure) was purchased from Fluka Chemie AG, Switzerland. It was used without further purification.

2.3. Generation of test atmosphere Methacrolein was evaporated in a Pitt no. 1 aerosol generator (ASTM, 1984), diluted with room air and led to a 2.3-l exposure chamber. The airflow rate in the exposure chamber was set between 17.9 and 24.7 l min − 1. The nominal concentration was calculated from the delivery rate of evaporated chemical and the airflow in the exposure chamber. The six studied concentrations, ranging from 2.0 to 26.3 ppm, were monitored continuously by infrared spectroscopy (Miran 1A, Foxboro). The differences between the nominal and monitored exposure concentration were less than 10%.

2.4. Bioassay A computerised system (Vijayaraghavan et al., 1993, 1994; Boylstein et al., 1995, 1996; Alarie, 1998) was used to determine the effects on the respiratory system at three levels.

2.4.1. Sensory irritation If a substance stimulates the trigeminal nerve endings in the upper respiratory tract, it may cause a burning and painful sensation in humans (Alarie, 1973). In mice, it causes a reflexively induced decrease in the respiratory rate ( f ). The decrease is caused by an elongation of the period from the end of the inspiration until the start of the expiration, termed ‘time of brake’ (TB). 2.4.2. Airflow limitation This effect may be caused by bronchoconstriction, oedema, and accumulation of mucus in the conducting airways. In particular, this causes an elongation of the time of expiration (TE) and a decrease in expiratory flow rate. The parameter used to quantify airflow limitation is the expiratory flow rate at half of the tidal volume (VT), which is abbreviated VD. If VT changes, it is expected that VD changes as well. We adjusted for differences in VT by plotting the VD/VT ratio versus the exposure concentration. 2.4.3. Pulmonary irritation Stimulation of vagal nerves at the alveolar level may result in two types of respiratory effects. One effect is rapid, shallow breathing, which increases f and reduces VT (Nielsen et al., 1999). Another type of vagal stimulation causes an increase in the time from the end of the expiration to the initiation of the following inspiration, termed ‘time of pause’ (TP). This results in a decrease in respiratory rate, and thus pulmonary irritation can in this case be recognised and quantified either from the effect on TP or from the decrease in f. TP is, however, the more specific of the two parameters (Vijayaraghavan et al., 1994). The concentration which causes a 50% decrease in respiratory rate is termed RD50. The extrapolated threshold concentration, obtained from the log concentration–effect relationship, is termed RD0.

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2.5. Exposure conditions For each experiment, four naive animals were placed in separate body plethysmographs and installed head-only into the exposure chamber. Details are given by Vijayaraghavan et al. (1993) and Nielsen et al. (1999). The animals were allowed to acclimatise 10–15 min before initiating a 15 min control period (baseline period) followed by a 30 min exposure period and a 15 min recovery period. The exposure concentrations were 2.0, 4.4, 6.6, 10.2, 13.1 and 26.3 ppm.

2.6. Data analysis Fig. 1. Decrease in respiratory rate in BALB/c mice due to airborne exposure to methacrolein. The decrease is expressed as percent decrease from base line (Df ) in different parts of the exposure period. In the period 0–10 min, the Df values were selected as the maximum decrease during a 1 min period. In the other periods, the Df values were average values from the indicated periods. The regression line was obtained from the mean values using the entire exposure period (0–30 min). Each point represents the mean value of four naive animals. The methacrolein concentrations studied were 2.0, 4.4, 6.6, 10.2, 13.1 and 26.3 ppm.

To facilitate comparisons, the effects ( f, TB, TP, VD and VT) were expressed either as percentages of baseline (defined as 100%) or as relative decrease from baseline, e.g. Df = (( fcontrol − fexposure)/fcontrol)100%. The trend over time, i.e. time-response relationship, was studied by twoway analysis of variance and regression analysis. Calculations were performed by use of the Minitab Statistical Software, Release 10 Xtra (Minitab Inc.). P values B 0.05 were considered statistically significant.

3. Results

Fig. 2. Effect of exposure to airborne methacrolein on the TB and TP in BALB/c mice. TB is a specific marker of sensory irritation and TP is a specific marker of pulmonary irritation. In the period 0 – 10 min, the TB and TP values were selected as the maximum increase during a 1 min period. In the other periods, the values were the mean values from the indicated periods. The TP symbols are strongly overlapping, showing that no trend over time occurred. The methacrolein concentrations studied were 2.0, 4.4, 6.6, 10.2, 13.1 and 26.3 ppm.

Airborne exposure to methacrolein resulted in a concentration-dependent decrease in respiratory rate (Fig. 1). Also, TB increased in a concentration-dependent manner (Fig. 2). The regression line describing the relationship between decrease in f and exposure concentration showed no statistical difference in slope and intercept for the intervals 0–10, 11–20 and 21–30 min and, thus, a common regression line was obtained from the mean values of the entire exposure period (0–30 min). The relationship between relative decrease in respiratory rate (Df ) and the exposure concentration, c was Df(% decrease) = − 5.66+54.8× log c (ppm), r 2 = 0.94. The RD50 value was 10.4 (6.6–16.4) ppm and the RD0 was 1.3 (0.8–2.1) ppm, with the 95% confidence intervals given in brackets.

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Fig. 3. Effect of methacrolein on the VD at half of the VT, the VT and the ratio, VD/VT are shown. The parameters are given as mean values from the entire exposure period 0–30 min. Each point represents the mean value of four naive BALB/c mice. The methacrolein concentrations studied were 2.0, 4.4, 6.6, 10.2, 13.1 and 26.3 ppm.

Both VD and VT decreased due to exposure (Fig. 3). Overall, no time-dependent effect was seen on VD, VT and the ratio VD/VT (data not shown). Thus, further analyses were performed from the mean values from the entire exposure periods (Fig. 3). The effect on VT did not occur in a consistent concentration-dependent manner. The decrease in VD was analysed from the VD/ VT ratio (Fig. 3). This ratio decreased very slightly with increasing exposure concentration. Thus, only a slight exposure related airflow limitation was apparent. The other lung effects studied, rapid shallow breathing and effects causing elongation of TP, did not occur (Figs. 1 and 2).

4. Discussion

4.1. Upper respiratory tract Methacrolein caused a conspicuous increase in TB as well as a decrease in f, which indicates that it is a potent sensory irritant. The level of sensory irritation was constant during exposure, indicating the absence of desensitisation. Activation of the sensory irritant receptor can occur by two mechanisms (Alarie et al., 1998a,b), adsorption to

the receptor (nonreactive irritants) or by reaction with the receptor (chemically reactive irritants). In general, the chemically reactive irritants are more potent than the nonreactive ones (Alarie et al., 1998b). The two mechanisms can be distinguished by means of the ratio of the equipotent concentration (e.g. RD50) and the saturation vapour concentration (P 0) of the irritant. The ratio adjusts in a simple manner for differences in lipophilicity of the substances and it can thereby provide information on affinity to receptors that are in a lipophilic receptor compartment (Alarie et al., 1998b). For nonreactive irritants, the RD50/P 0 ratios are generally above 0.01, whereas for chemically reactive irritants, the ratios are below 0.01. As the ratio for methacrolein (Table 1) is below 0.01, it suggests that a chemical reaction is involved in the activation of the sensory irritant receptor. The importance of the CC double bond for the activation of the sensory irritant receptor can be deduced by comparing the effect of methacrolein with the effect of 2-methyl propanal (Table 1), which contains the aldehyde group and a closely related hydrocarbon structure. The high potency of methacrolein is seen both from the RD50 ratio of these two chemicals (4167/10.4 = 401) and the RD50/P 0 ratio (1.1× 10 − 2/3.2×

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10 − 5 = 338), suggesting that the CC double bond is important for the activation of the receptor. This is in agreement with the generally proposed theory that the oxygen atom of the oxyallylic compounds (CCCO) allows the molecule to be adsorbed to the receptor with the CC double bond in close proximity to a nucleophilic group of the receptor (Nielsen et al., 1984; Nielsen and Bakbo, 1985; Nielsen, 1991; Alarie et al., 1998b), possibly a thiol group (Nielsen, 1991). The type of the functional group carrying the oxygen atom is less important (Nielsen, 1991). This is also seen from the potency of allyl ether and allyl alcohol (Table 1). Comparing the sensory irritating effect of acrolein and methacrolein may add further information on the reaction with the CC double bond. Accepting that the receptor activation occurs in a lipophilic compartment (Alarie et al., 1998b), it suggests that acrolein should, due to the shorter hydrocarbon chain, distribute less efficiently than methacrolein to this compartment. Nevertheless, methacrolein is less potent as seen both from the RD50 values and the RD50/P 0 values. Indeed, the last mentioned ratio, (3.2×10 − 5/5.1 × 10 − 6), suggests that methacrolein should be about six times less potent than acrolein. However, the same trend is seen for the corresponding substances without the CC double bond, i.e. 2-methyl propanal Table 1 Concentrations depressing the respiratory rate by 50% (RD50) due to sensory irritation and the saturation vapour concentration (P 0) at 37°C of allylic compounds related to methacrolein Substance

RD50 (ppm)

P 0 (ppm)

RD50/P 0

Methacrolein 2-Methyl propanal Acroleind Propanal Allyl alcohold Allyl etherd

10.4 4167b

320 022a 373 678c

3.2×10−5 1.1×10−2

2.9 2052b 3.9 5.0

565 000 673 000d 66 000 111 000

5.1×10−6 3.0×10−3 5.9×10−5 4.5×10−5

a

Calculated from Richardson and Gangolli (1994). Data from Steinhagen and Barrow (1984). c Calculated from Seprakova et al. (1959). d Data from Alarie et al. (1998b). b

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and propanal with the RD50/P 0 ratio (1.1 × 10 − 2/3.0× 10 − 3) giving a factor of three. As aldehyde groups have the same resonance and field-induction effects (Hansch et al., 1991), the aldehyde groups of propanal and 2-methyl propanal should possess the same chemical reactivity. In consequence, the spatial configuration, i.e. steric effect, should be responsible for the difference in reactivity of propanal and 2-methyl propanal. When comparing acrolein and methacrolein, the same spatial difference as that of propanal and 2-methyl propanal can be expected due to the methyl group in the two position. Thus, it is reasonable to suggest that the spatial configuration in the case of methacrolein and acrolein also contributes to a factor of three in potency. A factor of two is then left to be explained, as the difference in potency of acrolein and methacrolein, is a factor of six. Thus, the factor of two has to be explained by differences in the chemical reactivity. A straightforward explanation to the chemical reactivity of methacrolein; as the methyl group donates electrons (hyper conjugation) to the CC double bond, making the end carbon atom of the CC double bond less positive and, thus, less willing to participate in an attack from a nucleophilic group of the receptor. In addition, both effects may act in concert. All together, this study lends support to the hypothesis that the CC double bond is heavily involved in the sensory irritation mechanism of methacrolein.

4.2. Effect on the lungs The concentration-dependent effect of methacrolein on the lungs was limited to a very slight airflow limitation. Neither rapid shallow breathing (increase in f ) nor pulmonary irritation (increase in TP) were observed. This may be explained by the absorption of methacrolein in the upper airways as the closely related substances acrolein and propanal were extensively absorbed (\95%) in this part of the airways (Egle, 1972). Thus, only low concentrations may reach the lungs, thereby explaining the limited effect on this part of the respiratory system.

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4.3. Practical applications As sensory irritation is a prominent biological property of methacrolein exposure, this endpoint should be considered when setting its occupational exposure limit, e.g. the threshold limit value (TLV). It has previously been shown that TLV values and RD50 values for sensory irritants are highly correlated, TLV:0.03 ×RD50 (Schaper, 1993). Based on this relationship, it seems justifiable to suggest that the TLV for methacrolein should not exceed 0.3 ppm.

Acknowledgements This project has been supported by the Centre for Indoor Air Research, 1099 Winterson Road Suit 280, Linthicum, USA (Award No. 98-11) and by the Danish Working Environment Fund (Award No. 1997-11), to whom we are indebted. We also thank Anders Weng for technical assistance.

References Alarie, Y., 1973. Sensory irritation by airborne chemicals. CRC Crit. Rev. Toxicol. 2, 299–363. Alarie, Y., 1998. Computer-based bioassay for evaluation of sensory irritation of airborne chemicals and its limit of detection. Arch. Toxicol. 72, 277–282. Alarie, Y., Schaper, M., Nielsen, G.D., Abraham, M.H., 1998a. Structure-activity relationship of volatile organic chemicals as sensory irritants. Arch. Toxicol. 72, 125–140. Alarie, Y., Nielsen, G.D., Abraham, M.H., 1998b. A theoretical approach to the Ferguson principle and its use with non-reactive and reactive airborne chemicals. Pharmacol. Toxicol. 83, 270 – 279. ASTM, 1984. Standard Test Method for Estimating Sensory Irritancy of Airborne Chemicals, E981-84. American Society for Testing and Materials, Philadelphia. Boylstein, L.A., Anderson, S.J., Thompson, R.D., Alarie, Y., 1995. Characterisation of the effect of an airborne mixture of chemicals on the respiratory tract and smoothing polynomial splice analysis of the data. Arch. Toxicol. 69, 579 – 589. Boylstein, L.A., Luo, J., Stock, M.F., Alarie, Y., 1996. An attempt to define a just detectable effect for airborne chemicals on the respiratory tract in mice. Arch. Toxicol. 70, 567 – 578. .

Egle, J.L., 1972. Retention of inhaled formaldehyde, propionaldehyde, and acrolein in the dog. Arch. Environ. Health 25, 119 – 124. Hansch, C., Leo, A., Taft, R.W., 1991. A survey of Hammett substituent constants and resonance and field parameters. Chem. Rev. 91, 165 – 195. Hess, L.G., Kurtz, A.N., Stanton, D.B., 1978. Acrolein and derivatives. In: Kirk-Othmer (Ed.), Encyclopedia of Chemical Technology, vol. 1, third ed. Wiley, New York, pp. 277 – 297. IARC, 1994. Isoprene. In: IARC Monographs on the Evaluation of the Carcinogenic Risks to Humans, 60, 215 – 228. Nielsen, G.D., 1991. Mechanisms of activation of the sensory irritant receptor by airborne chemicals. CRC Crit. Rev. Toxicol. 21, 183 – 208. Nielsen, G.D., Bakbo, J.C., 1985. Sensory irritating effects of allyl halides and a role for hydrogen bonding as a likely feature at the receptor site. Acta Pharmacol. Toxicol. 57, 106 – 116. Nielsen, G.D., Bakbo, J.C., Holst, E., 1984. Sensory irritation and pulmonal irritation by airborne allyl acetate, allyl alcohol, and allyl ether compared to acrolein. Acta Pharmacol. Toxicol. 54, 292 – 298. Nielsen, G.D., Hougaard, K.S., Larsen, S.T., Hammer, M., Wolkoff, P., Clausen, P.A., Wilkins, C.K., Alarie, Y., 1999. Acute airway effects of formaldehyde and ozone in BALB/c mice. Hum. Exp. Toxicol. 18, 400 – 409. Richardson, M.L., Gangolli, S., 1994. The Dictionary of Substances and Their Effects, vol. 4. The Royal Society of Chemistry, UK, pp. 451 – 452. Sauer, F., Scha¨fer, C., Neeb, P., Horie, O., Moortgat, G.K., 1999. Formation of hydrogen peroxide in the ozonolysis of isoprene and simple alkenes under humid conditions. Atmos. Environ. 33, 229 – 241. Schaper, M., 1993. Development of a database for sensory irritants and its use in establishing occupational exposure limits. Am. Ind. Hyg. Assoc. J. 54, 488 – 544. Seprakova, M., Paulech, J., Dykyj, J., 1959. Tlak pa´r butyraldehydov. Chem. Zvesti 13, 313 – 316. Steinhagen, W.H., Barrow, C.S., 1984. Sensory irritation structure-activity study of inhaled aldehydes in B6C3F1 and Swiss-Webster mice. Toxicol. Appl. Pharmacol. 72, 495 – 503. Vijayaraghavan, R., Schaper, M., Thompson, R., Stock, M.F., Alarie, Y., 1993. Characteristic modifications of the breathing pattern of mice to evaluate the effects of airborne chemicals on the respiratory tract. Arch. Toxicol. 67, 478 – 490. Vijayaraghavan, R., Schaper, M., Thompson, R., Stock, M.F., Boylstein, L.A., Luo, J.E., Alarie, Y., 1994. Computer assisted recognition and quantitation of the effects of airborne chemicals acting at different areas of the respiratory tract in mice. Arch. Toxicol. 68, 490 – 499. WHO, 1992. Environmental Health Criteria 127. Acrolein, World Health Organisation, Geneva.