The results of our study demonstrate that there was no significant toxicity of low-level CD gas, not greater than 0.1 ppm, following 24 hours/day, 7 days/week and 6-months whole body exposure in rats. This exposure period was longer than that of other previously reported studies [17–20]. Additionally, we successfully regulated the CD gas concentration within ± 25% of the target concentration throughout the exposure period, in comparison with Dalhamn's study in which actual CD gas levels fluctuated widely between 0.05 ppm and 0.3 ppm against the target concentration of 0.1 ppm . According to the US OSHA, the long-time (8 hour) permissible exposure TWA level of CD gas in environmental air in a human workplace is 0.1 ppm. Our results support that this level is not toxic in rats, even if the exposure was continued for 24 hours. Also, in an experiment before the study, we confirmed that the existence of animals, chow, water and excreta did not affect the circulating CD gas concentration under our experimental condition, presumably because of the high cycle time of ventilation, i.e., 30 times per hour (data not shown). Moreover, we confirmed prior to the study that the CD gas level was equal in the chamber and in the cage, irrespective of where in the chamber it was measured.
Because of the inhalation exposure of the oxidant agent, it was expected that the primary target organs would be the respiratory organs. However, there were no significant changes of WBC in the blood and the BALF. Also, the cytokines (TNF-α and IL-6), which increase in the presence of inflammation [22, 23], were almost not detectable (Table 2), below the lower quantification limit (TNF-α: < 12.5 pg/ml; IL-6: < 62.5 pg/ml). In the high exposure group of male rats, IL-6 was detectable (63.8 pg/ml). However, this value was near the lower quantification limit level. Additionally, in the histopathological observation, no signs of toxicity in the nasal meatus, the nasal cavity, the pharynx, the trachea and the bronchus were observed. Thus, we concluded that the whole body exposure of CD gas, not greater than 0.1 ppm for 24 hours/day, 7 days/week for 6 months, did not cause the inflammatory sign in the respiratory organs in rats.
In biochemistry and hematology examinations, we observed that some parameters showed statistically significant changes. However, almost all of the changes in these parameters were not dose-related. EO%, which was reported to increase as a result of allergy, such as asthma or infection of parasitic arthropods [24–27], showed a statistically significant change between the exposed and the control groups. However, it was not a dose-dependent change, and decreased in the exposed group compared with the control group. To our speculation, since the presumable mechanism of action of CD is oxidation that is a physicochemical reaction, it's toxicity, if any, should appear in a dose-dependent manner. Thus, we concluded that the statistically significant change of EO% was not a sign of CD gas-related toxicity. There was a statistically significant change in BUN in the high exposure female rats. However, according to a report of Charles River Laboratories Japan for SD rats , mean ± standard deviation of BUN value in 30-week old SD rats were 16.9 ± 1.9 mg/dl. Also, the mean values in the control and low exposure groups were lower than the reference range. Thus, we concluded that the BUN value in the high exposure female rats was within the normal range and was not a toxic sign.
In previous studies, LOAEL of 2.5 ppm for 30 days' exposure , LOAEL of 1 ppm for two months , NOAEL of 5 ppm and LOAEL of 10 ppm for 4 weeks , and NOAEL of 0.1 ppm for 10 weeks and LOAEL of 10 ppm for 2 weeks , all studied in rats, were reported. However, since our primary objective in this study was to reveal that the CD gas up to 0.1 ppm (the concentration that exceeds the effective level against microbes) was safe, even after the longest period of exposure time. Therefore, although we should admit that we were unable to determine the ordinary NOAEL and/or LOAEL levels, our study was still valuable in terms of confirming that the CD gas concentration that was effective against microbes was not toxic.
It was reported that CD gas at a low concentration prevented an infectious respiratory disease in vivo, and also inactivated feline calicivirus, influenza A virus, Staphylococcus aureus and Escherichia coli in vitro [11–15]. According to these studies, effectiveness of CD gas was shown below 0.1 ppm. Hence, our study results suggest that the protective level of CD gas against infectious agents may not be a toxic level in rats, therefore the continuous exposure of a low level CD gas not greater than 0.1 ppm may have a possibility to be an effective measure to disinfect spaces where people live in or gather at, such as houses, offices, airport buildings and hospitals, although some uncertainty factors, such as inter-species differences and inter-individual variations, should be taken into account. Furthermore, a higher dose(s) may have to be employed so as to define the LOAEL as well as NOAEL. Also, additional toxicity studies, such as a developmental toxicity study and a reproduction toxicity study, will be necessary to warrant the safety of continuous exposure of low-level CD gas in public space for the purpose of controlling infection from various, airborne and surface-residing microorganisms.