Test chemical
NaDCC (both the dihydrate and anhydrous material), as well as cyanuric acid, are well-characterized substances. Physical and chemical properties are described in the Kirk-Othmer Encyclopedia of Chemical Technology [17], in a web-based document on chloroisocyanurates by Occidental Chemical Corporation [18, 19], in a monograph developed by OxyChem on the chemistry of the chloroisocyanurates [20], and in a Food Additive Petition (FAP) submitted by Occidental to the U.S. Food and Drug Administration [21]. NADCC was purchased from Sigma-Aldrich (Saint Louis, USA).
Experimental design
NaDCC used in humidifier disinfectants in the living environment has an exposure concentration of 0.1~0.2 mg/m3 (1~2 tablets a day, 4.53% per tablet, 320 mg per tablet, 24 h of use, average volume of use of 30.3 m3, and winter average ventilation of 0.2 times/h). In an acute inhalation toxicity test, five (5/5) males and two (2/5) females died at the exposure concentration of 250-mg/m3. At the exposure concentration of 40-mg/m3, five (5/5) males and one (1/5) females died. Therefore, it can be inferred that NaDCC contains 4~8 times the concentration ranging from 0.1 to 0.2-mg/m3. In order to confirm certain toxicity, we set 20-mg/m3 as the high concentration of exposure and applied a common ratio 5 to set 4- and 0.8-mg/m3 as the medium and low concentrations, respectively. In addition, since it was judged that the toxicity would be more strongly induced in male animals, an interim test group for bronchoalveolar fluid (BALF) examination 7 days after exposure and a recovery test group for the evaluation of the presence or absence of reversibility of toxicity were also assigned for males. The exposure period of the test substance was set at 6 h a day, 5 days a week for 14 days, and the recovery period was for 14 days after the end of exposure.
Exposure and analysis
For exposure to the test substance, NaDCC was dissolved in water, and an aerosol was generated using an atomizer-type mist generator (NB-2N, Sibata Co. Ltd., Japan). The aerosol generated to maintain the target concentration in the chamber was diluted with air from the Aerosol Dilution System and supplied to the whole body chamber (1.4 m3). The control group was supplied only clean air without test substances, but other environmental conditions in the chamber were the same for both control and test exposure groups.
Samples of the test substance in the chamber were collected three times using a 25-mm micro-glass filter and a personal sample collector (Model No. Airchek XR 5000, SKC Inc., USA) from the breathing area of the test animals during exposure to the test substance. The weight of the filter with the test substance collected was measured using an electronic balance (Model No. 770-60, KERN & SOHN GmbH Co. Ltd., German). The concentration of the test substance in the chamber was calculated by measuring the filter weight before and after collection. When measuring the weight of the filter, it was measured by excluding the influence of moisture. In addition, while the test substance aerosol was being generated, the number of aerosol particles was checked in real time using a Portable Aerosol Spectrometer (Model 1.109, GRIMM Aerosol Technik GmbH & Co.KG, Germany). The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) were determined for each exposure concentration using a Cascade impactor (Model 135, MiniMOUDI Impactor, MSP Co. LTD., USA) during exposure to the test substance to confirm the particle size distribution of the aerosols.
Test system
F344 rats (6 weeks of age) were purchased from Japan SLC, Inc. (Shizuoka, Japan); on the day of obtaining the animals, all animals were weighed using an electronic balance (QUINTIX3102-1SKR, Sartorius, Germany). The males weighed 83.94 to 118.22 g, and the females weighed 102.35 to 124.41 g. This species was selected for the study because of the availability of background information for this species as these animals are generally used in sub-chronic toxicity studies. Clinical signs were recorded on the day the animals were obtained. The rats were allowed to acclimate to their housing environment and quarantined for 7 days; no abnormality was observed in any animal.
Based on the weight of the animals, the test animals were allocated to 4 test groups-5 animals per group-such that the average weight of all the groups was the same. In addition, the interim and recovery groups were also formed in the same way as the test groups.
During the study period, ≤ 3 rats were housed in a polysulfone cage (W 310 × L 500 × H 200 mm), but the rats were housed individually in a 6-wire mesh cage (W 240 × L 1200 × H 200 mm) during the period of exposure. During the exposure time of the test substance, feed was not supplied, but water was supplied. The animal room conditions were as follows: temperature of 19.0~25 °C, humidity of 30~70%, light/dark cycle of 12 h/day, illuminance of 150~300 Lux, and ventilation frequency of 10~20 times/h.
Observations, analysis, and pathological examination
On the day of necropsy, all surviving animals were anesthetized using isoflurane. Blood was sampled, the abdominal artery or vein was exsanguinated, and a gross examination was performed. The sampled blood was subjected to hematological and blood biochemical analyses. The following hematological parameters were evaluated with an automatic blood cell automatic analyzer (ADVIA 2120i, SIEMENS, Germany) and an automatic coagulation time meter (Coapresta 2000, SEKISUI, Japan): leucocyte (WBC), platelet (PLT) count, erythrocyte (RBC) count, hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), reticulocyte (RET) count, prothrombin time (PT), and activated partial thromboplastin time (APTT). Blood biochemical parameters that were measured with an automatic analyzer (TBA-120FR NEO, Toshiba Co., Japan) were glucose (GLU), total bilirubin (TBIL), blood urea nitrogen (BUN), potassium (K), total protein (TP), calcium (Ca), albumin (ALB), chloride (Cl), creatinine (CREA), inorganic phosphorus (IP), total cholesterol (TCHO), sodium (Na), triglyceride (TG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and γ-glutamyl transpeptidase (γ-GTP), lactate dehydrogenase (LDH), creatinine phosphokinase (CPK), and albumin/ globulin (A/G) ratio.
BALF of only male rats was analyzed 1 and 2 weeks after exposure. To obtain BALF, the upper end of the trachea was cut, and a polypropylene tube attached to a syringe was inserted; the trachea was then washed three times with 4 mL of phosphate-buffered saline (PBS). The collected BALF was centrifuged at 450 g for 10 min, and the supernatant was stored at -80 °C. The cell pellet was re-suspended in fresh PBS, and the total immune cell count was determined by using a Hematology Analyzer (ADVIA 2120i). The re-suspended cell pellet was centrifuged at 270 g for 10 min using a Cytospin centrifuge (Cellspin; Hanil, Gimpo, Korea) and stained using Diff-Quick staining solution. Differential cell counts were determined using a light microscope at 100× magnification.
The supernatant separated from the BALF was thawed at ~20 °C just before cytokine analysis. A commercially available cytokine multi-magnetic bead array kit (R&D Systems, Minneapolis, MN 55413) was used to determine the concentrations of interleukin (IL)-1β, IL-6, IL-4, tumor necrosis factor alpha (TNF-α), and macrophage inflammatory protein 2-alpha (MIP-2) in the BALF. The Magnetic Bead Single Plex Kit (MILLIPLEX MAP; Merck Millipore, Darmstadt, Germany) was used to measure the concentration of transforming growth factor β (TGF-β). Reactive oxygen species (ROS)/reactive nitrogen species (RNS) was analyzed using an OxiSelect™ In Vitro ROS/RNS Assay Kit (Catalog No. STA-347; Cell Biolab, Inc., USA) and Varioskan Flash Reader (Thermo Fisher Scientific, Finland). The assays were performed per the manufacturers' instructions. The median fluorescence intensity of the samples was measured using a Luminex 100 instrument (Luminex, Austin, TX, USA), and standard curves were obtained using MasterPlex software (MasterPlex QT 2010; Miraibio, Hitachi, CA, USA). Cytokine concentrations were calculated using the standard curves.
The following organs of all animals were harvested and the absolute and relative (organ-to-body weight ratios) weights were measured: brain, liver, heart, spleen, lung, and kidneys. Bilateral organs were weighed together. The following tissues obtained from all animals in the control and high-concentration groups were stained with hematoxylin and eosin for histopathological examination: brain, heart, lung, nasal cavity, larynx, trachea, liver, spleen, and kidney. The nasal, larynx, tracheal, liver, kidney, spleen, thymus, seminal vesicles, prostate, epididymis, testis, uterus, and vaginal organs of the high-concentration group, for which effects of the test substance were predictable, were also examined for the low- and medium-concentration groups.
Statistical analysis
The data obtained during the study period are presented as means and standard deviation values. The data were statistically analyzed using PRISTIMA version 7.1.0 (Xybion Medical Systems Corporation, Morris Plains, NJ, USA). Levene’s test was performed to determine the homogeneity of the variances. When variances were homogeneous, one-way analysis of variance (ANOVA) was performed, and statistical differences between the control and exposed groups were analyzed by Dunnett’s test. When variances were not homogeneous, Kruskal-Wallis test was performed, and statistical differences between the control and exposure groups were analyzed by Dunn’s rank sum test.