Purpose
Inhalation exposure is essential for toxicological and pharmacological studies of aerosol such as dusts, gaseous substances, complex mixtures. Introduction of inhalation exposure system typically requires a large scale facility and investment. There are two types of inhalation exposure methods; whole-body and nose-only inhalation exposure methods. The negative effect of nose-only inhalation exposure method caused by restraint holder is under debate. We studied a performance test with Multipurpose Inhalation Cage Unit (MIC Unit) (Shibata Biotechnology Inc., Tokyo, Japan) which is newly developed small scale inhalation exposure system. We also studied effect of restriction stress of rats between whole-body and nose-only exposure methods
Method
Multipurpose Inhalation Cage Unit (Shibata Biotechnology Inc.) (Pic.1) was used for whole-body and nose-only inhalation exposure experiment.
Picture 1: Multipurpose Inhalation Cage Unit, Model SBT-MIC-A06
Animals
Male Slc:SD (Japan SLC, Inc., Shizuoka, Japan) rats were obtained at 6 wk. After 1wk of quarantine, the animals were allocated by stratified randomization method into 3 groups (10 each rats); cage-control, whole-body exposure (WBE), and nose-only exposure (NSE) groups. (Pic.2)
Picture 2:Rats are devided into 3 groups (2 exposure, 1 control). Whole-body exposure (WBE) group (top-left), and Nose-only exposure (NSE) group (top-right). WBE and NSE groups were divided into 5 rats/chamber sub- groups. Each chambers were set to one Multipurpose Inhalation Cage Unit(bottom).
Experimental design
Laboratory room air was controlled to 23 °C, 55 % relative humidity (RH), and 6 rph ventilation, at all times. For the 4 wk study, animals were exposed to air for 6 h/day, 5 days/wk, for 4 wk. Each animal of NSE was placed into plastic restraint holder (Shibata Biotechnology Inc.) during the period of inhalation exposure experiment. Each group was exposed to laboratory room air which was suctioned to each chamber through supply air system of MIC Unit. Air flow rate for each chamber was set to 15 l/min. Ventilation rate of each chamber was set to 25 rph. Target temperature inside each chamber was between 20 and 26 degrees Celsius. Target relative humidity inside each chamber was between 40 and 70 %RH. (Fig.1)
Observations and pathological examinations
Temperature and relative humidity inside chambers were measured every day. Food consumption was observed once a week. Body weight was measured everyday for first 2 wk period of experiment, then measured once a 2 days. After experimental period, body weight, organ weight (thymus, heart, lung, liver, kidney, adrenal gland, testis) were measured.
Table 1: Body weight change
Results
Temperature inside chamber
The mean temperature inside chamber was about 24 degree Celsius during the period of inhalation exposure experiment. The mean RH inside chamber during the period was 66.6±7.3 %RH, and was lower down to 63.7±7.3 %RH at the end of the period each day.
Survival, body weights, and food consumption
There was suppression of body weight in NSE group compared with WBE and cage control group, beginning the 6th day. Beginning the 15th day, There was statistically significant(p<0.01) low value of body weight in NSE groups through the end of experiment. There were no significant
difference of food consumption amount between WBE, NSE, and cage control groups. (Tab.1, Fig.2)
Organ weights
There were statistically significant lower value of heart and thymus weights (absolute) in NSE group compared with WBE and cage control groups. There were statistically significant higher value of kidney weights (absolute) in both exposure groups compared with cage control group. There were statistically significant lower value of thymus weight (relative) in NSE group compared with WBE group. There were statistically significant higher value of adrenal gland weight (relative) in NSE group compared with cage control group. There were statistically significant higher value of testes weight (relative) in NSE group compared with WBE and cage control groups. There were no abnormity in any rats by visual examination at autopsy.
Discussion
It is an ongoing discussion about suppression of body weight caused by restriction stress using restrainer holder in nose-only inhalation exposure method. There are reports of more than 10% suppression of body weight in nose-only group compared with cage control group especially for long term period of experiments elsewhere. Also, our study showed that restriction stress by nose-only inhalation method influences body weight.
There are also reports of effects on thymus and adrenal gland caused by restriction stress by nose-only inhalation method elsewhere. Our study showed statistically significant lower value of thymus weight in nose-only inhalation exposure group compared with whole-body inhalation exposure and cage control groups.
OECD test guideline suggests to use nose-only inhalation method, but it also refers to the need to whole-body inhalation exposure method where appropriate. The newly-developed Multipurpose Inhalation Cage (MIC) Unit is practical for not only whole-body method but also nose-only method using restrainer holder. The mean temperature inside chamber during experiment is within OECD test guideline(22±3 °C). Average relative humidity is under 70 %RH.
It is possible to operate MIC Unit with smaller generation amount of test substance compared with traditional inhalation exposure system. Combination of dosage and numbers of chambers can be easily modified to meet experimental demands. Therefore, MIC Unit would be especially suitable for short and medium term repeated dose inhalation exposure.
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