Benzalkonium chloride, a quaternary ammonium compound, is a mixture of several n-alkylbenzyldimethylammonium chlorides (n = 10–16) with different alkyl groups . It is a cationic surfactant used as a bactericide or preservative owing to its inhibitory action against bacteria and fungi . It is known that the long alkyl group of BAC interferes with the double-layered bacterial cell membrane, destroys it, and leaks the cell contents, and thus inhibits bacterial growth [14, 15]. Studies have reported the side effects of BAC, such as skin irritation and dermatitis due to exposure via inhalation [16,17,18].
Globally, BAC is commonly used as sterilizing and preserving agents in household chemical products such as spray-type antimicrobial agents, perfumes , and deodorants. Some studies have reported respiratory toxicity of BAC. Exposure to BAC induced cytotoxicity and DNA damage in human bronchial cell line (BEAS-2B) , and acute or repeated inhalation of BAC induced lung irritation, inflammation, and alveolar damage [3, 21, 22]. In addition, orally or intravenously administered BAC has been reported to accumulate predominantly in the lungs of rats, leading to pulmonary edema and pneumonia [23, 24]. Therefore, the lungs are considered to be the main target organ of BAC. Recently, BAC is suspected to be one of the causative substances of toxicity involving humidifier fungicides in Korea. In the present study, the inhalation toxicity test was performed to investigate the toxicity of BAC inhalation.
To evaluate the toxic effects of repeated exposure to BAC, F344 rats were exposed to 0.8, 4, and 20 mg/m3 BAC, 6 h/day for 14 days. We also set up a recovery period of two and 4 weeks to assess the reversibility of these effects. The mean concentration of BAC in the whole-body exposure chamber measured during the exposure period was in accordance with that used in the inhalation test for aerosols because the error from the target concentration was ≤20%. The MMAD and GSD of the aerosols measured during the exposure period also met the aerosol inhalation test requirements in the OECD TG412 .
During the exposure period, nasal discharge, rale, and deep respiration were observed in the T3 group, and nasal discharge observed in the T1 and T2 groups. These clinical signs are thought to be an emergency airway response caused by the irritation of BAC. The emergency airway defense response consists of glottal closure, airway constriction, pulmonary vessel dilation, cough, and copious gland secretion. The emergency airway defense response is centrally mediated and depends on intact vagal connections to the lungs . During the exposure period, a significant weight loss was observed in males in the T2 and T3 groups and females in the T3 test group, and more weight loss was observed in males than in females, but both sexes showed similar changes in weight. The body weight loss was accompanied by the decreased feed intake. These reductions were considered related to the exposure to the test substance.
The hematological test results showed changes in the RBC count, HCT level, HGB level, and MCHC in the male T3 group rats. The results also showed changes in the RBC count, and HCT and HGB levels in the male T2 group rats and the HGB level in the male T1 group rats. These changes are considered to be due to weight loss with dehydration .
The changes in the MCV, reticulocyte count and ratio, PLT count, APTT, and PT in the male T3 group rats; the reticulocyte count and ratio, PLT count, and PT in the male rats exposed to T2 group rats; the reticulocyte count and ratio, APTT, and PT in the female T3 group rats; and the PT in the female T2 group rats were observed. These changes can be attributed to the reduction in hematopoietic function due to the decrease in feed intake and the subsequent weight loss [28, 29]. The changes in the number of lymphocytes observed in the T3 group rats were inferred to be due to nutritional deficiency or stress due to weight loss . In addition, there was no toxicological significance in the basophil count of the male T2 and T3 group rats and in the monocyte count of the female T3 group rats.
In the blood biochemical test, the increase in the ALT activity in the male T3 group rats is considered to be related to liver atrophy observed in histopathological examination  (Additional file 1: Table S5). On the contrary, the changes in the ALP activity in the male T3 group rats and the TG level and CK activity in the male T3 group rats were considered to be due to the decrease in feed intake causing weight loss. In addition, the significant increase in the K and Na levels observed in female rats is thought to be due to dehydration by epithelial cell stimulation by BAC.
In the BALF analysis, the concentration of ROS/RNS, IL-1β, IL-6, and MIP-2 decreased dose dependently at the end of the exposure period, but did not show a concentration-dependent change at 4 weeks of recovery. In addition, the concentrations of TNF-α, IL-4, and TGF-β did not show changes associated with test substance exposure. Although IL-6 is traditionally considered as proinflammatory cytokine, it is highly polymorphic and its anti-inflammatory activity has been reported . In addition, a reduction in inflammatory cells, decrease in chemokine and inflammatory cytokine expression, and alteration in macrophage mobilization in JP-8-induced dermatitis strongly suggest the role of anti-inflammatory response, rather than inducing IL-6 inflammatory response. These changes in the expression or function of these cytokines might modulate stimulatory sensitivity in human . The concentration-dependent decrease in the concentrations of ROS/RNS, IL-1β, IL-6, and MIP-2 in this study was caused by the stimulation of the test substance, and cytokines such as IL-6, IL-1β, and MIP-2 are thought to function together.
In the measurement of organ weights, changes in the weights of the brain, heart, lungs, liver, spleen, and kidneys of the male and female T2 and T3 group rats, size reduction and atrophy of the liver and spleen in the male and female T3 group rats (Additional file 1: Table S5) were considered to be due to weight loss [33, 34]. In the recovery group, the body and organ weights of rats gradually improved.
The autopsy results showed that the black lesions in the lungs of male T3 group rats were consistent with the hemoglobin crystallographic findings of the alveolar muscle in the histopathological examination. The size reduction observed in the thymus, testis, epididymis, seminal vesicles, prostate, uterus, and vagina was consistent with the atrophic findings in the macroscopic examination. Furthermore, the squamous metaplasia of the respiratory epithelium and transitional epithelium, mucinous cell hypertrophy and proliferation of the respiratory epithelium, mucinous cell metaplasia of the transitional epithelium in the nasal cavities, and mucinous cell hypertrophy and proliferation of terminal bronchiole are considered adaptive changes after tissue injury . The atrophy of the thymus was considered to be due to stress from weight loss .
In histopathological examination, changes in the respiratory epithelium and transition epithelium of the nasal cavity, atrophy of the erosive and olfactory epithelium with necrosis, denaturation and regeneration of the respiratory bronchial epithelium, hypertrophy of the smooth muscle in the bronchial alveolar junction, and cell debris indicated damage due to stimulation by the test substance. Peripheral eosinophil infiltration in the lung is thought to be an allergic reaction because it is associated with increased eosinophil ratio and eosinophil count.
Histopathological findings in the nasal cavity and lungs verified that BAC induced irritation in the nasal cavity and the lungs, which were the main organs affected in the respiratory system. ROS/RNS, IL-1β, IL-6, and MIP-2 levels decreased in a BAC concentration-dependent manner, indicating that BAC exposure caused oxidative damage. The decrease in the level of IL-6, an anti-inflammatory agent, led to a decrease in ROS/RNS, an indicator of oxidative damage. At this time, IL-1β and MIP-2, together with IL-6, are considered to be acting as cytokines. Therefore, additional research is needed to clarify this hypothesis.
Inhaled substances may affect the respiratory system at various levels according to various factors, such as the characteristics of substances, environment, and host factors. The nasal cavity is important in inhalation toxicology because nose is the first part of the respiratory tract that contacts and filters airborne particles . The mucosa in the nasal cavity is the first tissue of defense against inhaled particles in upper airway [38, 39]. Inhaled particles are trapped in mucus and removed to be swallowed by the coordination with the movement of ciliary epithelium [38, 39]. BAC has been shown to inhibit the nasal mucociliary activities via damaging the ciliated nasal epithelial cells [40, 41, 42]. BAC is a human skin and severe eye irritant . It is a suspected respiratory toxicant, immunotoxicant, gastrointestinal toxicant, and neurotoxicant [44,45,46]. BAC for consumer use are dilute solutions. Concentrated solutions are toxic to humans, causing corrosion/irritation to the skin and mucosa, and death if taken internally in sufficient volumes. Several studies have shown the nasal toxicity of BAC in animals and humans. The administration of 0.05 and 0.10% (w/v) BAC solutions to the nasal cavity of rats caused epithelial inflammation, desquamation, and edema in the dorsal meatus and adjacent nasal septum . BAC-containing nasal decongestant sprays induced or aggravated nasal swelling and stuffiness in healthy volunteers and patients with rhinitis compared with those of sprays without BAC [48,49,50]. Recently, the adverse effects of BAC through inhalation exposure and the target organ of BAC toxicity can be shifted to the upper respiratory organs rather than the deeper lower airway . In this study, more exposure-related effects were observed in the upper airway. As mentioned above, BAC is thought to be more exposed to the upper respiratory tract due to mucociliary clearance and emergency airway response caused by the irritation of BAC. In addition, NOAEL is considered to be less than 0.8 mg/m3 because the effects associated with BAC exposure were also observed in the nasal cavity of rats exposed to a concentration of mg/m3.
Thus, from these results, we calculated the BMD value because the exposure criteria are required to protect the health of workers handling BAC. The BMD recommended by the Environmental Protection Agency should be calculated to overcome the drawbacks of NOAEL, which is dependent on exposure concentration. The BMD calculation yielded the BMDL, the 95% lower confidence of the dose corresponding to 10% reaction incidence, and the lowest value was selected as the BMD value. The toxicity data for dose response were body weight, lung weight, RBC count, HCT level, Hb level, MCHC, MCV, RETA, RET%, PLT count, LYMA, APTT, PT, TG level, and ALT, ALP, and CK activities, and the models were Exponential and Hill. The BMDL values obtained were 0.10, 0.0031, 0.63, 1.53, 1, 37.5, 33.5, 0.55, 0.39, 0.89, 0.29, 0.12, 0.26, 0.86, 0.085, 0.082, and 0.208 mg/m3, respectively. Therefore, we chose 0.0031 mg/m3 as the BMD value, the dose corresponding to lung weight-related dose-response, and the DNEL was 0.000062 mg/m3, determined by applying an interspecies factor of 2.5, intraspecies factor of 5, and exposure duration factor of 4 as default assessment factors .