9) in the Wiley Library (Ver. 7) or comparison

with literature mass spectra; for 4-OPA (Fruekilde et al., 1998, Hutton et al., 2003 and Molander and Cameron, 1993) and IPOH (Calogirou et al., 1999a). The purity was based on GC peak area integration in full scan mode and averaged (n = 2). Inbred BALB/cA male mice were purchased from Taconic, Denmark. At the initiation of the study, the mean weight and SD of the mice was 25.8 ± 1.3 g. Mice were housed in polypropylene cages (380 mm × 220 mm × 150 mm) with pinewood sawdust bedding (Lignocel S8, Brogaarden, selleckchem Denmark). The photoperiod was from 6 a.m. to 6 p.m., and the temperature and relative humidity in the animal room were 22 ± 2 °C and 50 ± 5%, respectively. The cages were sanitized twice weekly. Food (Altromin no. 1324, Altromin, Lage, Germany) and tap water were available ad libitum. Treatment of the animals adhered to procedures approved by The Animal Experiment Inspectorate, Denmark with Permission numbers 2006/561-1123 and 2011/561-1990. The terpene reaction products were PS-341 research buy evaporated in Pitt No. 1 VOC generator (Wong and Alarie, 1982), diluted with medical dry air, and fed into a 24 L exposure chamber (Larsen and Nielsen, 2012). The airflow rates in

the chamber were set between 18.8 and 23.2 L/min. The chamber exposure concentrations were monitored every fourth minute by 15 sequential 1.0 mL air samples on Tenax TA steel tubes (PerkinElmer), taken by syringe (size: 2.0 mL) suction, followed by thermal desorption within 12 h, and GC/FID analysis, as described previously (Wolkoff, 1998). Six-point calibration of the weighed compound in methanol (0.08–2.5 μg/mL) was applied for determination of air concentrations (R2 ≥ 0.98), except for 4-OPA that was dissolved in pentane. Initially, a starting concentration was selected on the basis of the relation for non-reactive compounds according to Alarie et al. (1996). However, for reactive compounds, i.e. with an aldehyde group, a lower starting concentration was decided. Other exposure concentrations were decided upon the first observation of a bioresponse. The resulting exposure concentrations

are shown in Table 2. The respiratory effects were studied in a head out mouse bioassay (Alarie, 1998). The bioassay allows detection of respiratory effects on the upper airways (sensory irritation), effects on the conducting Metalloexopeptidase airways, and at the alveolar level by continuous computerized monitoring of the breathing pattern. The inhalation effects are investigated by analyses of the breathing patterns in mice (Alarie, 1973 and Nielsen et al., 1999). Briefly, the breathing pattern analysis recognizes and quantifies specific deviations from the normal breathing pattern (for terms and definitions, see Fig. 1 in Nielsen et al. (1999)). Thus, after end of inhalation, a short brake occurs before the exhalation is initiated, termed time of brake (TB, ms). An increase in TB leads to a decrease in the respiratory frequency (f, breaths/min).