Sodium dichloroisocyanurate toxicity in rats during a 90-day inhalation toxicity study

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Highlights

The total cell number in BALF increased in a concentration-dependent manner.

The histological changes showed in nasal cavity and bronchi of 10.0 mg/m3 group.

In the alveolar duct, the adverse effect of wall thickening by NaDCC was observed.

The adverse effect was observed to decrease during the recovery period.

The no-observed adverse effect concentration of NaDCC was 2.0 mg/m3 for both sexes.

Abstract

Sodium dichloroisocyanurate-96% (NaDCC) is commonly used to treat drinking water, industrial water, and wastewater. However, exposure to NaDCC by inhalation can have toxic pulmonary effects in humans. In the present study, we evaluated the potential toxicity of NaDCC following a 90-day inhalation toxicity study in Sprague-Dawley/Crl:CD (SD) rats. The animals were exposed to 0.4, 2.0, or 10.0 mg/m3 NaDCC for 90 days. In addition, male and female rats from the 10.0 mg/m3 group were set up as the recovery group for 14 days. The bronchoalveolar lavage fluid showed a concentration-dependent increase in the total cell count, with a significant increase in neutrophils in both the sexes in the 10.0 mg/m3 group compared to the negative control group. In the 10.0 mg/m3 group, lung organ weight was significantly increased among the female rats. Histopathological examination showed eosinophilic droplets in the olfactory/respiratory epithelium, mucous cell hyperplasia, atrophy/degeneration of the tracheal branches, and wall thickening of the alveolar ducts in the nasal cavity of both sexes in the 10.0 mg/m3 group. The adverse effects of NaDCC exposure were observed to decrease during the 14-day recovery period in both sexes. Based on pathological observations, the “no observed adverse effect concentration (NOAEC)” of inhaled NaDCC was 2.0 mg/m3 for both sexes. These results are expected to provide a scientific basis for inhalation toxicity data of NaDCC.

Graphical abstract

Unlabelled Image

Keywords

Sodium dichloroisocyanurate-96%

90-day inhalation toxicity study, Sprague-Dawley/Crl:CD rats

Biocide, Bronchoalveolar lavage fluid

Abbreviations

ALP

alkaline phosphatase

BALF

bronchoalveolar lavage fluid

Cl

chloride

BUN

blood urea nitrogen

GSD

geometric standard deviation

Na

sodium

NaDCC

sodium dichloroisocyanurate

NOAEC

no observed adverse effect concentration

LDH

lactate dehydrogenase

MMAD

mass median aerodynamic diameter

NC

negative control

OECD

Organization for Economic Co-operation and Development

PLT

platelets

RBC

red blood cells

SD

Sprague-Dawley/Crl:CD

1. Introduction

Sodium dichloroisocyanurate-96% (NaDCC) is the physicochemical properties, it is in the form of a white powder with a molecular weight of 219.9 g/mol (Sigma-Aldrich, 2021). In addition, sodium salt of 1,3-dichloro-1,3,5-triazine-2,4,6(1H,3H,5H)-trione, and is a synthetic organochlorine donor derived from isocyanurate (Seo and Jo, 2021). The range of use is known to be effective against a wide range of bacteria, fungi, algae and viruses (Clasen and Edmondson, 2006). NaDCC is a biocide generally used as a disinfectant and industrial deodorant in living environments, such as swimming pools and drinking water (Choi et al., 2020; Khazaei et al., 2020; Mao et al., 2018). However, in addition to its general known toxicity, it has potential toxic effects in humans if inhaled (Lantagen et al., 2010).

Trichloroisocyanuric acid, known as a component with a structure similar to NaDCC,is confirmed by the 1979 research report, and toxic effects such as increased lung weight, nasal secretions and wet runny nose were observed by systemic exposure in the form of dust at concentrations of 3.2, 10.1 and 31 mg/m3 (ECHA, 1979). A study reported in 2011 reported mediastinal findings in two young patients due to acute exposure to chlorine gas produced from trichloroisocyanuric acid (Li et al., 2011). The significant damage caused by NaDCC exposure necessitates advisory to prevent recurrence, and toxicological evaluation of ingredients. In a general review of toxicity of NaDCC and recommendations for its use, the WHO reported it to have low acute oral toxicity and lack genotoxicity or carcinogenicity (Clasen and Edmondson, 2006). The International Committee of the Red Cross reported that exposure to NaDCC could cause pulmonary dysfunction and provides information on its toxicity (International Committee of the Red Cross, 2002). Furthermore, Seo et al. reported body weight loss and upper respiratory tract (nasal and larynx) toxicity in rats, exposed to inhalation of 4.0 mg/m3 NaDCC for 14 days (Seo and Jo, 2021). The Material Safety Data Sheet for NaDCC warns of potential irritation to the nose, mouth, trachea, and lungs if inhaled (Sigma-Aldrich, 2021). The U.S. Environmental Protection Agency reported the median lethal concentration (LC50) for Sprague-Dawley/Crl:CD (SD) rats exposed to NaDCC for 4 h by inhalation, as less than 1.17 mg/L but more than 0.27 mg/L (ECHA, 1985). According to the report on oral administration of NaDCC, rats were exposed to 400, 1200, 4000 and 8000 ppm of NaDCC. Body weight loss, reduced movement rate and death were observed in the exposure groups of 4000 and 8000 ppm. The NOAEL values of NaDCC in this study were 50 mg/kg/day (male) and 130 mg/kg/day (female) by oral administration (ECHA, 1980; Hammond et al., 1986). In the other study, food intake was performed for 13 weeks at concentrations of 2000, 6000 and 12,000 ppm in rats to evaluate the response of repeated exposure to NaDCC. Body weight and food consumption decreased, and relative liver and kidney weights increased in groups of 6000 and 12,000 ppm. The authors reported NOEALs of 100 mg/kg/day for dietary intake in both sexes (Hammond et al., 1986).

NaDCC may cause potential damage to the respiratory system in case of inhalation (Sigma-Aldrich, 2021); however, previous research on NaDCC focused on its toxic effects caused during sterilization and while using it as a disinfectant for drinking and domestic water (Clasen and Edmondson, 2006; Seo and Jo, 2021). In addition, most of the toxicity data for NaDCC are from oral toxicity evaluation (Clasen and Edmondson, 2006, ECHA, 1980, Hammond et al., 1986). Although pulmonary toxicity studies on animals have been reported using exposure to intratracheal instillation (ITI) and whole-body exposure by inhalation (Clasen and Edmondson, 2006, Seo and Jo, 2021), toxicity following long-term inhalation exposure to NaDCC has not been studied. In the present study, we evaluated the potential toxicity of NaDCC after subchronic inhalation exposure in SD rats in accordance with the Organization for Economic Cooperation and Development (OECD) test guideline 413 (OECD guidelines, 2018).

2. Material and methods

2.1. Study design and chemicals

SD/Crl:CD rats (6 weeks old; Orient Bio Inc., Seongnam, Korea), were reared at 22 ± 3 °C, 30–70% humidity, 12/12 h light/dark cycle, with access to food and water ad libitum (Lee et al., 2021, Li et al., 2021; OECD guidelines, 2018). After 6 days of acclimatization, the 7-week-old rats were separated into four groups (10 males and 10 females in each group). Each group was then exposed to different concentrations of NaDCC: 0 mg/m3 (negative control, NC), 0.4 mg/m3 (low group), 2.0 mg/m3 (middle group), and 10.0 mg/m3 (high group). Additionally, a 14-day recovery group was set up from the NC and high groups, including 5 males and 5 females from each group, to observe the effects of NaDCC exposure on assessed criteria. At the end of the experiment, all animals were euthanized by administering an overdose of isoflurane and necropsy was performed. NaDCC (product number: 218928, CAS number: 2893-78-9) were obtained from Sigma-Aldrich (St. Louis, MO, USA), and a 15% aqueous solution for inhalation was prepared by dissolving them in sterile distilled water.

The approval for the study was obtained from the Institutional Animal Care and Use Committee of the Korea Institute of Toxicology (IACUC #1905–0160). The procedure for inhalation exposure to NaDCC was in accordance with the OECD test guideline 413 (OECD guidelines, 2018).

2.2. Whole-body inhalation exposure

The NaDCC aqueous solution was aerosolized using a mist generator (NB-2 N, Sibata, Japan), and the exposure concentration was adjusted by mixing the aerosol with filtered clean air. A whole-body exposure inhalation device (WITC-1.5, HCT, Korea) was used to deliver the aerosolized solution at specific concentrations (Fig. 1). Weight loss in rats was reported following exposure to 20.0 mg/m3 NaDCC by inhalation for 14 days (Seo and Jo, 2021). Subsequently, 10.0 mg/m3 was selected as the maximum concentration of NaDCC for this study at which observable prominent toxicity was expected following NaDCC inhalation exposure for 90 days. The other exposure concentrations were determined, by reducing 10.0 mg/m3 by a scaling factor of 5, as 2.0 and 0.4 mg/m3. The animals were exposed to 0.4, 2.0, or 10.0 mg/m3 NaDCC and fresh air for 6 h/day, 7 days per week, for 90 days (Kim et al., 2012). The number of exposures was slightly different depending on the sex: males were exposed for 90 days, while females were exposed for 91 days, more by one day. The trial design was based on a clinically planned route in humans. The inhalation chamber was exposed to NaDCC while maintaining at least 19% oxygen, 22 ± 3 °C temperature, and 30–70% relative humidity.

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Fig. 1. Schematic of whole-body inhalation chamber for sodium dichloroisocyanurate-96% (NaDCC).

2.3. Environmental monitoring analysis

Environmental factors, such as differential pressure, flow rate, temperature, and relative humidity, were measured for all groups during inhalation exposure. Oxygen and carbon dioxide concentration were measured at approximately 1 h intervals after initiating exposure, using a portable combined ISC M40 Multi-Gas monitor (Industrial Scientific Corp., Oakdale, PA) and MultiRAE Lite PGM-6208-CO2 monitor (RAE Systems, Sunnyvale, CA, USA), respectively. Aerosol particle size was calculated using the mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) values measured for each exposure group using a cascade impactor (Mini MOUDI 135-6S, MSP Corporation, Shoreview, MN, USA).

2.4. Clinical observations

The animals had access to food and water ad libitum except for the period when exposed to whole-body inhalation of NaDCC. During the NaDCC exposure period of 90 days, average daily food consumption (g/animal/day) was calculated for each group by calculating the difference between the added food amount/day and the amount of food remaining at a specific measurement time. The changes in the appearance and behavior of each animal, general symptoms, and number of dead or moribund animals were recorded at least once a day for the duration of NaDCC exposure.

2.5. Hematology and blood biochemistry analysis

The animals were fasted overnight, with free access to water, before blood collection. Blood samples were collected in a blood collection tube containing an anticoagulant (EDTA-2 K, BD, Bergen County, NJ, USA). For hematology analysis, approximately 1.5 mL of blood was drawn from all animals during autopsy, ∼0.5 mL of which was placed in a blood collection tube, containing anticoagulant, and analyzed using an ADVIA 2120i Hematology System (Siemens, München, Germany). For biochemical, approximately 1.5 mL of blood was drawn and placed in a blood collection tube without an anticoagulant, allowed to stand for at least 90 min at 25 °C, and then centrifuged (2095 g × 10 min; 25 °C) to separate the serum. The serum was analyzed using a single high-performance analyzer (TBATM-120FR, Toshiba, Tokyo, Japan). The remaining ∼1.0 mL blood was placed in a tube containing 3.2% sodium citrate and centrifuged to separate plasma (2095 g × 10 min; 25 °C), and blood clotting time was measured.

2.6. Bronchoalveolar lavage fluid (BALF) analysis

BALF was sampled from the right lung of each animal, and lactate dehydrogenase (LDH), total protein, as well as cell counts and differentials for alveolar macrophages, lymphocytes, neutrophils, and eosinophils were analyzed according to standard operating instructions (Song et al., 2010). The total number of cells was measured by adding and mixing solution-18 (AO/DAPI, Chemometec, Gydevang, Denmark) reagent with the cells and using the automated cell analyzer (NucleoCounter® NC-250™, Chemometec, Gydevang, Denmark). Differential cell counting was performed using the Diff-Quik staining kit (Sysmex Inc., Kobe, Janpan) and counting >400 cells per sample using a microscope. Total protein and LDH levels were estimated using a single high-performance analyzer (TBA™-120FR, Toshiba, Tokyo, Japan). After BALF sampling was complete, the right lung of each animal was placed in liquid nitrogen and stored at −80 °C until further analysis.

2.7. Measurement of organ weights

All animals were necropsied, and the absolute and relative (organ-to-body weight ratios) weights were measured for the designated organs (adrenal glands, brain, epididymides, heart, kidneys, liver, lung, ovaries, spleen, testes, thymus, thyroids, and uterus). If there were no gross abnormalities, the organ were weighed at once; however, if there was an abnormality, it was observed separately.

2.8. Histopathological analysis

All tissues were removed from each animal and fixed in 10% neutral-buffered formalin (product number: #GD4018, GD chemical, Korea) for analysis. The eyeball (including the optic nerve) was fixed using Davidson's solution (Formalin +95% alcohol + glacial acetic acid + distilled water), and the testes and epididymis were fixed in Bouin's solution (product number: #2012MIRA01, BBC biochemical, Washington, DC, USA) for approximately 24–72 h before being transferred to 70% ethanol. Additionally, formalin was injected and fixed in the left lung and bladder specimens. The organs with abnormal macroscopic findings in all groups were embedded in paraffin, sectioned tissue specimens were prepared, stained with hematoxylin and eosin, and histopathological examination was done under an optical microscope.

2.9. Statistical analysis

All results are expressed as mean ± standard deviation. Data were tested for equal variances using Bartlett's test, and further with one-way analysis of variance (ANOVA), and post hoc Dunnett's test. Data that were not evenly distributed were analyzed by the Kruskal-Wallis Test and the significance of inter-group differences between the negative control and treated groups will be assessed using Dunn's Rank Sum Test. All statistical analyses were performed using the Pristima System (Version 7.4, Xybion Medical Systems Co., USA), and differences with P-values <0.05 were considered to be statistically significant.

3. Results

3.1. Environmental monitoring data of exposure

Exposure conditions were set according to the guidelines of OECD TG 413 (OECD guidelines, 2018), and the environmental monitoring data during NaDCC exposure is shown in Table 1. The exposure concentration of NaDCC was within ±20% for each group, and this was considered appropriate for the targeted concentrations. During the exposure period, the environmental conditions, including the temperature and relative humidity in the chamber, were maintained within a set standard range (Table 2). The particle size distribution met the recommended MMAD standard (≤ 2.0 μm) presented in the aforementioned guideline. In the case of GSD, the average range of 1–3 was exceeded in the 0.4 mg/m3 exposure group, indicating a change in the range of distribution due to the characteristics and aerosol particles size of NaDCC (Table 3).

Table 1. Exposure conditions in the NaDCC exposure chamber.

Table 2. Stability of NaDCC exposure concentrations.

Values are represented as mean ± standard deviations.

a not determined.

Table 3. Particle size distribution of NaDCC aerosol.

Values are represented as mean ± standard deviations.

a not determined.

3.2. Clinical signs, body weight, and food consumption

No deaths occurred during the 90 days of study (data not shown). Hair loss and dirtiness were observed in both sexes. The number of animals with these signs increased in a concentration-dependent manner in each group. Most of the these signs stayed even during the recovery period (data not shown).Changes in body weight and food consumption over 90 days are presented in Fig. 2. No significant changes in body weight were observed in the both sexes of NaDCC-exposed groups compared to the NC group (Fig. 2A, B). During the recovery period also, no significant changes in body weight were observed in both sexes of the recovery group compared to the NC group (Fig. 2C, D). No significant changes in food consumption were observed in either sex of NaDCC-exposed groups except for a transiently significant decrease in food consumption in the males of 10.0 mg/m3 group compared to the NC group on exposure day 62 and 69 (Fig. 2E). No change in food consumption was observed in the both sexes compared to the NC group during recovery period (data not shown).

Fig. 2. Effects of a 90 day inhalation toxicity study to NaDCC on body weight and food consumption. Body weight males (A and C, 90-day exposure period and recovery period); body weight females (B and D, 90-day exposure period and recovery period) and food consumption (E; males and F; females). Recovery period; 14 days.

⁎ P < 0.05 vs. compared with the negative control (NC) group.

3.3. Hematology and blood biochemistry parameters

The findings of hematological and biochemical analysis are shown in Table 4, Table 5. In hematological analysis, there was a statistically significant increase in the number of platelet (PLT) in the males of 10.0 mg/m3 group and the number of red blood cells (RBC) in the females of 2.0 mg/m3 group (Table 4). In blood biochemical analysis, the levels of blood urea nitrogen (BUN) in the males and alkaline phosphatase (ALP) in the females of 10.0 mg/m3 group, sodium (Na) and chloride (Cl) in both males and females of 10.0 mg/m3 group, and the level of Cl in the males of 2.0 mg/m3 and females of 0.4 mg/m3 group significantly increased compared to the NC group (Table 5). During the recovery period, no hematological and blood biochemical changes were observed in either sexes of recovery group compared to the NC group.

Table 4. Hematological parameters after a 90 day inhalation NaDCC exposure.

Abbreviations: NaDCC, sodium dichloroisocyanurate-96%; NC, negative control; RBC, total red blood cell count; HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; PLT, platelet count; RET, relative reticulocyte count; RETA, absolute reticulocyte count; WBC, total leukocyte count; NEU, relative neutrophils count; NEUA, absolute neutrophils count; LYM, relative lymphocytes count; LYMA, absolute lymphocytes count; EOS, relative eosinophils count; EOSA, absolute eosinophils count; MON, relative monocytes count; MONA, absolute monocytes count; BAS, relative basophils count; BASA, absolute basophils count; LUC, relative large unstained cells count; LUCA, absolute large unstained cells count; PT, prothrombin time; APTT, activated partial thromboplastin time.

Values are means ± standard deviation.

• p < 0.05 and **p < 0.01, compared with the NC group.

Table 5. Blood chemical parameters after a 90 day inhalation NaDCC exposure.

Abbreviations: NaDCC, sodium dichloroisocyanurate-96%; NC, negative control; GLU, glucose; BUN, blood urea nitrogen; CREA, creatinine; TP, total protein; ALB, albumin; A/G, albumin/globulin ratio; AST, aspartate aminotransferase; ALT, alanine aminotransferase; TBIL, total bilirubin; GGT, gamma glutamyl transpeptidase; ALP, alkaline phosphatase; TCHO, total cholesterol; TG, triglyceride; Ca, calcium; IP, inorganic phosphorus; K, potassium; CK, creatine phosphokinase; PL, phospholipid; Na, sodium; Cl, chloride.

Values are means ± standard deviation.

• p < 0.05 and **p < 0.01, compared with the NC group.

3.4. Gross finding and organ weight changes

No NaDCC-related gross findings were observed in any exposure and recovery groups (data not shown). The weights of designated organs and body weights were measured for all animals, and the organ to body weight ratios are shown in Fig. 3. No changes in organ weight were observed except that the relative weight of the testes of the males of 0.4 and 10.0 mg/m3 groups (Fig. 3A), and lungs of the females in 10.0 mg/m3 group, (Fig. 3B), increased significantly compared to the NC group. However no histological changes were observed. No increase in lung weight was observed in the recovery group (data not shown).

Fig. 3. Effect of a 90 day inhalation toxicity study to NaDCC on organ weight. A, male; B, female.

⁎ P < 0.05 and ⁎⁎ P < 0.01 vs. compared with the NC group.

3.5. Alteration of cell distribution and biomarker in BALF

The results of the total and differential cell counts in BALF after NaDCC exposure are shown in Fig. 4. In both sexes of NaDCC-exposed groups, the total cell count in BALF, and the macrophages (Fig. 4A, E and Fig. 4B, F, respectively) increased in a concentration-dependent manner compared to the NC group, but no statistically significant difference were observed. No increase was observed in the recovery group (data not shown). Neutrophils significantly increased in both the sexes of 10.0 mg/m3 group compared to the NC group (Fig. 4C, G), and no change was observed in the recovery group (data not shown). Lymphocyte levels of both sexes of NaDCC-exposed groups were similar to those of the NC group (Fig. 4D, H, L, P). No eosinophils were observed in any group (data not shown).

⁎

P

< 0.05 and

⁎⁎

P

< 0.01 vs. compared with the NC group.

P, recovery period).

LDH and total protein levels were measured as sub-chronic toxicity biomarkers of the exposure to NaDCC; the findings are presented in Fig. 5. LDH levels were increased in males of all the NaDCC-exposed groups (Fig. 5A) and females of the 0.4 and 10.0 mg/m3 exposure groups compared to those in the NC group (Fig. 5C). During the recovery period, LDH levels were increased in all sexes of the 10.0 mg/m3 exposure group (Fig. 5E, G). There was a change in total protein levels at 2.0 and 10.0 mg/m3 in males (Fig. 5B), but no such change was observed in the recovery group (Fig. 5F). No changes in total protein levels were observed in all females of the exposure and recovery groups (Fig. 5D, H).

Fig. 5. Effects of a 90 day inhalation toxicity study to NaDCC on lactate dehydrogenase (LDH) and total protein levels. LDH (A and C, NaDCC exposure groups; E and G, recovery group), total protein (B and D, NaDCC exposure groups; F and H, recovery group).

3.6. Histopathological changes following NaDCC exposure

After 90 days of NaDCC inhalation exposure, histopathological changes were observed in all organs (Fig. 6 and Table 6). In the microscopic findings, abnormal changes were observed in respiratory tissues upon NaDCC exposure. Eosinophilic globules (minimal to moderate) and mucous cell hyperplasia (minimal to slight) were observed in the both sexes of 10.0 mg/m3 group (Fig. 6A, B and Table 6). These changes were mostly reverted during the recovery period (Fig. 6C, D). Epithelial atrophy of tracheal carina (minimal) was observed in the females of 10.0 mg/m3 group (Fig. 6E and Table 6), and this finding was not observed during the recovery period (Fig. 6F). In addition, minimal or slight thickened alveolar duct walls in lung tissue was observed in the both sexes of 10.0 mg/m3 group (Fig. 6G and Table 6). The incidence and severity of this finding were decreased during the recovery period (Fig. 6H).

Fig. 6. Effects of a 90 day inhalation toxicity study to NaDCC on the nasal cavity, trachea, and lung with bronchi tissues of female rats. Black arrow, eosinophilic globules; blue arrow, epithelial and carinal atrophy; red arrow, thickened wall and alveolar duct; scale bars, 200 μm (original magnification: 100×). Nasal cavity (A and B, 90-day exposure period; C and D, recovery period), trachea (E, 90-day exposure period; F, recovery period), and lung with bronchi (G, 90-day exposure period; H, recovery period). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 6. Histopathological analysis after a 90-day inhalation NaDCC exposure.

a): no findings.

Abbreviations: NaDCC, Sodium dichloroisocyanurate-96%; NC, negative control.

4. Discussion

A biocide is a substance used to remove harmful organisms other than humans and animals (European Union Regulation, 2012; Hwang et al., 2021). It is used as a general living environment disinfectant, preservative, disinfectant, and pesticide, but even exposure to a small amount of biocide it can cause serious damage (Mahmood et al., 2020). It has been reported that quaternary ammonium contained in products used for disinfection of COVID-19 also has a toxic effect on the respiratory tract in case of inhalation (Luz et al., 2020). NaDCC has been used to disinfect swimming pools or drinking water, but it also exhibits toxic effects when inhaled or exposed to skin. Previous studies have evaluated the toxicity of NaDCC mostly following oral administration or after acute inhalation (ECHA, 1985; Seo and Jo, 2021; Yoo et al., 2022).

To evaluate the potential toxicity of NaDCC after long-term inhalation exposure, we investigated the effects of inhalation of NaDCC at various concentrations for 90 days in rats based on OECD TG 413 guidelines (OECD guidelines, 2018).

During the NaDCC 90-day inhalation exposure period, no changes in mortality, body weight, and food consumption resulting from NaDCC exposure were observed. Body weight loss in males and decreased food consumption in females were reported following 2 weeks of inhalation exposure to NaDCC at 20.0 mg/m3, but there were no significant changes at 0.8 and 4.0 mg/m3 (Seo and Jo, 2021). Based on previously reported studies, we hypothesized that there is no effect on mortality, body weight, and food consumption after inhalation exposure to NaDCC up to a concentration of 10.0 mg/m3, which we set up as a limit to observe discernible toxicity. In addition, during the NaDCC exposure period, hair-related symptoms such as hair loss and dirtiness were observed in both sexes, and were maintained during the recovery period. It is necessary to further confirm whether these hair-related symptoms are symptoms of toxic effects from NaDCC inhalation exposure.

Hematology and blood biochemical analysis showed significant increases in several parameters as follows: hematology analysis (male; 10.0 mg/m3- PLT, female; 2.0 mg/m3- RBC), blood biochemical analysis (male; 2.0 mg/m3- Cl, 10.0 mg/m3- BUN, Na, Cl, female; 0.4 mg/m3-Cl, 10.0 mg/m3-ALP, Na Cl). However, hematological changes were considered not to be an effect of NaDCC exposure as they were not concentration-dependent. In addition, biochemical changes did not appear to be NaDCC-related and were insignificant. Previous studies reported that in the case of inhalation exposure to polyhexamethylene guanidine phosphate (PHMG-p) and xylitol, several parameters (MPV, MCHC, RBC, HCT, HGB, etc.) increased in hematology and blood biochemical analysis, but it was difficult to suggest the association with PHMG-p and xylitol (Lee et al., 2021; Tian et al., 2022). Likewise, the increase in several parameters (PLT, RBC, Cl, BUN, Na, and ALP) was observed with inhalation exposure to NaDCC for 90 days, but no toxic effects were considered. Therefore, hematology and blood biochemical changes following NaDCC inhalation exposure were not considered toxicologically relevant.

Analysis of total cell and differentiated cell of BALF recovered from the lung is useful for the diagnosis and treatment of interstitial lung diseases including idiopathic pulmonary fibrosis (Ronan et al., 2018). Alterations of immune cells count and lung injury biomarkers were observed in BALF, after 90 days of inhalation exposure to NaDCC. Thus, the total cell count and macrophages increased in a concentration-dependent manner in both sexes compared to the NC group, and the number of neutrophils significantly increased in 10.0 mg/m3 group. However, lymphocyte levels were not different than those in the NC group, and eosinophils were not observed. Neutrophils are important components of the immune defense mechanism that link innate and adaptive immunity (Liew and Kubes, 2019). Neutrophils are known to be involved in the clearance of exogenous pathogens and endogenous cellular debris as well as the pathogenesis of respiratory diseases (Mokra and Kosutova, 2015). The studies of didecyldimethylammonium chloride and benzalkonium chloride showed an increase in neutrophils, and these changes were found to be associated with lung injury (Choi et al., 2020; Kim et al., 2017). These data suggested that a 90-day inhalation exposure on NaDCC has the potential to cause progressive lung injury by increasing neutrophils.

A significant increase in LDH level was observed in female rats in the 10.0 mg/m3 recovery group, and the total protein content did not change in the NaDCC exposure and recovery groups. In toxicological evaluation, measurement of LDH generally can confirm tissue damage caused by foreign chemicals, and changes in total protein content are used as data to predict diseases related to liver, kidney, and lung dysfunction (Fahmi et al., 2018; Kim et al., 2017 Mahmutovic Persson et al., 2020). The results of these changes are insignificant and it is difficult to confirm they are related to NaDCC, because they are within the toxicity standard (Moudgal et al., 2008).

Histopathological data showed a 90-day inhalation exposure of NaDCC did not result in serious histopathological symptoms in other organs (thymus, heart, kidney, spleen, liver, brain etc.), but toxicity-related symptoms were observed in the respiratory system (nasal, tracheal, and lung). Hyperplasia of mucous cells and eosinophilic globules were observed in the nasal cavity and pharyngeal duct, and epithelial atrophy was observed in carina of the trachea. In addition, the alveolar duct thickened wall owing to proliferation of pulmonary interstitial fibroblasts and collagen deposition were observed in lung. In general, nasal and tracheal histopathological changes are an adaptive response to a 90-day inhalation exposure of NaDCC, and are not considered to be toxic changes because they are not accompanied by inflammation and cell damage (Baldrick et al., 2020; Huang et al., 2021; Matute-Bello et al., 2008). However, the thickened alveolar duct walls observed in the both sexes of 10.0 mg/m3 group was considered as an adverse effect of NaDCC inhalation exposure because it affects the function of gas exchange and can contribute to hypoxia and hypercapnia in severe lesions (Hedenstierna and Edmark, 2015). Previous papers were reported that inhalation of chlorine supplements can induce changes in respiratory mechanics and an influx of neutrophils, eosinophils and lymphocytes, causing enhanced pulmonary inflammation (Bernard, 2007; Deschamps et al., 1994; Woolcock and Peat, 1997). Instillation of NaDCC was shown to increase necrotizing bronchiolitis and bronchioloalveolar inflammation around the airways (Yoo et al., 2022). Our results demonstrated a higher influx of neutrophils to NaDCC at both the sexes of 10.0 mg/m3 exposure. This inflammation may be related to the thickened alveolar duct walls in the lung and the enhancement of the lung mechanical response in our experiment. Our data indicate that a 90-day inhalation exposure on NaDCC affects respiratory tissues, and most of the induced lesions are non-serious and reversible.

In conclusion, several abnormal changes were observed in respiratory tissues upon NaDCC exposure. Eosinophilic globules, mucous cell hyperplasia, and epithelial atrophy were observed in the nasal cavity and trachea of the some exposure group. These changes were mostly reverted during the recovery period. In addition, the thickened alveolar duct walls of lung was observed in the both sexes of 10.0 mg/m3 group. Although this finding decreased during the recovery period, it is considered an adverse effect, because it can impair respiratory mechanics. Therefore, A 90-day inhalation exposure on NaDCC is considered to have progressive toxic effects on the respiratory system, and based on all results, the “no observed adverse effect concentrations (NOAECs)” of inhaled NaDCC is 2.0 mg/m3 for both sexes. The conversion of the NOAECs for NaDCC to the NOAELs value in this study was 0.34 mg/kg/day in both sexes, which was lower than oral administration (50 mg/kg/day; male and 130 mg/kg/day; female) (ECHA, 1980; Hammond et al., 1986). These results suggest the possibility that toxicity by human exposure to NaDCC may occur more easily by the inhalation route than by the oral route. The results of present study are expected to be used as inhalation toxicity data useful for the risk assessment of human exposure to NaDCC. Further study is necessary to clarify more details for toxicity of NaDCC.

CRediT authorship contribution statement

Chul-Min Park: Data curation, Writing – original draft, Writing – review & editing. Seulgi Jeon: Writing – original draft, Writing – review & editing. Yong-Hyun Kim: Conceptualization, Methodology, Investigation, Data curation, Project administration. Jinhee Kim: Writing – review & editing. Seong-Jin Choi: Project administration, Methodology, Writing – review & editing. Ilseob Shim: Project administration, Methodology, Writing – review & editing. Ig-Chun Eom: Project administration, Methodology, Writing – review & editing. Su-cheol Han: Writing – review & editing. Min-Seok Kim: Conceptualization, Methodology, Investigation, Data curation, Writing – review & editing, Supervision, Project administration.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.

Acknowledgements

This research was supported by grants from the National Institute of Environmental Research, Republic of Korea [NIER-2018-04-02-004] and the Korea Institute of Toxicology, Republic of Korea [grant number KK-2207].

Data availability

No data was used for the research described in the article.

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