Polyhexamethylene guanidine hydrochloride (PHMG-HCl), an antimicrobial additive in humidifier disinfectants, was associated with the pulmonary disease outbreak in South Korea. However, PHMG-mediated oxidative stress has only been studied in vitro. Here, we evaluated PHMG-induced oxidative stress in the lungs of rats exposed to PHMG-HCl. Male F344 rats were exposed to different concentrations of PHMG-HCl for 13-weeks via whole-body inhalation. Histopathological examination of the exposed rats showed the presence of lung lesions, including alveolar/interstitial fibrosis with inflammatory cell infiltration, bronchioalveolar hyperplasia, bronchiolar/alveolar squamous metaplasia, bronchial/bronchiolar epithelial detachment, and alveolar hemorrhage. Immunohistochemical analysis showed that 4-hydroxynonenal (4-HNE) was expressed in the bronchiolar epithelium, mainly in Clara cells and macrophages of the fibrotic tissue. The number of 4-HNE-positive cells increased significantly in a dose-dependent manner. This is the first in vivo study to report PHMG-induced oxidative stress. Our study provides clues to elucidate the mechanisms underlying PHMG-induced damage in patients affected by humidifier disinfectants.

Recently, there has been an increase in the use of several nephrotoxicity biomarkers in preclinical experiments. In addition, it has been indicated that the result may have been influenced by secondary factors, such as sample storage condition or storage period. In this study, we have assessed the variation in urinary nephrotoxicity biomarkers as a result of urine storage conditions and storage period of the urine. Urine was sampled from specific pathogen-free Sprague-Dawley rats (19 weeks old), which were housed individually in hanged stainless steel wire mesh cages. Urine was stored at 20℃, at 4℃, or at −70℃ after sampling. The levels of the biomarkers such as beta-2 microglobulin (B2M), cystatin-C (Cys-C), N-acetyl-β- D-glucosaminidase (NAG), micro albumin (MA), micro protein (MP) were measured at 6, 24, 48 and 144 hr after sampling. The B2M level was significantly decreased at 6, 24, 48, and 144 hr compared to 0 hr at −70℃ (p < 0.05, p < 0.01, p < 0.05, and p < 0.05, respectively) and 24 and 144 hr at 20℃ (p < 0.01, p < 0.01, respectively). The Cys-C level was significantly decreased at 144 hr compared to 0 hr at 4℃ (p < 0.01), at 20℃ (p < 0.05) and at 70℃ (p < 0.01). MP and MA levels were not different for 144 hr in all storage conditions. Taken together, B2M and Cys-C levels were modulated by storage temperature and period. For the enhancement of test accuracy, it is suggested that strict protocols be established for samples to minimize the effects of the storage conditions on the detected levels of biomarkers.

Ethyl formate, a volatile solvent, has insecticidal and fungicidal properties and is suggested as a potential fumigant for stored crop and fruit. Its primary contact route is through the respiratory tract; however, reliable repeated toxicological studies focusing on the inhalation route have not been published to date. Therefore, the present study was conducted to investigate the safety of a 90-day repeated inhalation exposure in rats. Forty male and 40 female rats were exposed to ethyl formate vapor via inhalation at concentrations of 0, 66, 330, and 1,320 ppm for 6 hr/day, 5 days a week for 13 weeks. Clinical signs, body weights, food consumption, urinalysis, hematologic parameters, serum chemistry measurements, organ weights, necropsy, and histopathological findings were compared between the control and ethyl formate-exposed groups. Locomotor activity decreased during exposure and recovered afterward in male and female rats exposed to 1,320 ppm ethyl formate. Body weight and food consumption continuously decreased in both sexes exposed to 1,320 ppm ethyl formate from week 1 or 3 compared with the control values. The increases in adrenal weight and decreases in thymus weight were noted in both sexes exposed to ethyl formate at 1,320 ppm. Degeneration, squamous metaplasia of olfactory epithelium in the nasopharyngeal tissue, or both were noted in the male and female rats at 1,320 ppm and female rats at 330 ppm ethyl formate. Taken together, our results indicate that ethyl formate-induced changes were not observed in male and female rats at 330 and 66 ppm, respectively. This indicates that exposure to ethyl formate at concentrations below 66 ppm for 90 days is relatively safe in rats. This is the first report of a full-scale repeated inhalation toxicity assessment in rats and could contribute to controlling occupational environmental hazards related to ethyl formate.

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