Abstract
Background
Citric acid (CA) and sodium hypochlorite (NaOCl) have been used to disinfect animals to protect them against avian influenza and foot-and-mouth disease.
Objectives
We performed a good laboratory practice (GLP)-compliant animal toxicity study to assess the acute toxic effects of CA and NaOCl aerosol exposure in Sprague-Dawley rats.
Methods
Groups of five rats per sex were exposed for 4 h to four concentrations of the two chemicals, i.e., 0.00, 0.22, 0.67, and 2.00 mg/L, using a nose-only exposure. After a single exposure to the chemicals, clinical signs, body weight, and mortality was observed during the observation period. On day 15, an autopsy, and then gross findings, and histopathological analysis were performed.
Results
After exposure to CA and NaOCl, body weight loss was observed but recovered. Two males died in the CA 2.00 mg/L group and, two males and one female died in the 2.00 mg/L NaOCl group. In the gross findings and histopathological analysis, discoloration of the lungs was observed in the CA exposed group and inflammatory lesions with discoloration of the lungs were observed in the NaOCl exposed group. These results suggest that the lethal concentration 50 (LC50) of CA is 1.73390 mg/L for males and > 1.70 mg/L for females. For NaOCl, the LC50 was 2.22222 mg/L for males and 2.39456 mg/L for females.
Conclusions
The Globally Harmonized System is category 4 for both CA and NaOCl. In this study, the LC50 results were obtained through a GLP-based acute inhalation toxicity assessment. These results provide useful data to reset safety standards for CA and NaOCl use.
Keywords: Disinfectants, acute toxicity tests, lethal concentration 50, Sprague-Dawley rats
INTRODUCTION
During the past decade, Korea has been experiencing continuous outbreaks of lethal livestock infectious diseases such as avian influenza (AI) and foot-and-mouth disease (FMD). The constant outbreak of infections caused by such highly pathogenic viruses continuously harms workers [1,2]. Moreover, the use of animal disinfectants is increasing because of the constant need for an effective disinfectant to prevent the outbreak and spread of livestock infectious diseases. Compared to the increased amount of disinfectant usage, most users do not keep the proper amount and use it. It has been reported that citric acid (CA; CAS No. 77-92-9) and sodium hypochlorite (NaOCl; CAS No. 7682-52-9), which are widely used as animal disinfectants, have sufficient virucidal efficacy against AI and FMD viruses when used at 4%–5% dilution [3,4,5]. However, according to Kim et al. [6], when using disinfectants to combat the development of pathogenic livestock infectious diseases, approximately 47% of workers use concentrations greater than the effective amount and excessive use of high doses of animal disinfectants unintentionally exposes the respiratory tracts of the target animal and workers to the various chemicals in the disinfectants. Studies on the toxicity and effects of animal disinfectants on inhalation exposure remain limited.
CA is a weak, colorless organic acid compound, found in citrus fruits [7]. It has several applications, such as food production, chelating agent, cosmetics, pharmaceuticals, and dyeing [8,9], and is an important ingredient in animal disinfectants that inactivates viruses that cause AI and FMD [10]. CA, a relatively safe disinfectant chemical, is included in 65% of disinfectants used in livestock infection and disinfection facilities [6].
NaOCl is a powerful oxidizer and chlorinator. Recently, its use in combination with CA, in animal disinfectants, was brought into practice [11]. It is widely used in the food and medical industries as a bleaching and bactericidal agent and is more effective than 70% ethanol in inhibiting the biofilm formation of Staphylococcus aureus [12].
The outbreak of livestock diseases such as AI and FMD, and the COVID-19 pandemic have emphasized the importance of infection prevention, thus, disinfectants are currently more widely used. However, previous studies on the acute inhalation toxicity of CA and NaOCl have not clarified the exposure method, concentration, and aerosol type. Therefore, research to evaluate the toxic effects of inhalation exposure to disinfectant mist are similar to the actual exposure environment. In this study, our objective was to assess the effects and toxicity of acute inhalation exposure to two components of animal disinfectants–CA and NaOCl-based on the guidelines of the Organization for Economic Cooperation and Development (OECD), and to estimate the lethal concentration 50 (LC50) and determine the effects on the target organs. Furthermore, our findings intended to provide users with information on the toxic effects of acute inhalation exposure to disinfectant chemicals to establish the concentrations safe for use.
MATERIALS AND METHODS
Animals
Seven-week-old male and female Sprague-Dawley rats were purchased (Orient Bio Inc., Korea) and acclimatized for six days in a stainless-steel cage with specific-pathogen-free conditions before the grouping. The animals were kept in a controlled environment (automated control based on standard working guidelines) with the following conditions: temperature, 22°C ± 3°C; relative humidity, 30%–70%; 12 h light/12 h dark cycle; illuminance, 150–300 Lux; and air ventilation, 10 to 20 times per h. Temperature and relative humidity were automatically monitored and recorded. Rats were provided ad libitum access to gamma-irradiated solid chow (Lab Diet 5053; PMI Nutrition International, USA) and UV sterilized water. All animal protocols described in this study were approved by the committee on Animal Research Committee of the Korea Institute of Toxicology. And the CA and NaOCl experimental protocol approval numbers are IAC-21-01-0413 and IAC-21-01-0414, respectively.
Experimental procedure
Twenty rats of each sex were randomly assigned to four groups of different exposure concentrations (n = 10; male: 5, female: 5); 0.00 (vehicle control [VC], filtered air), 0.22, 0.67, and 2.00 mg/L, and each group was exposed once to CA and NaOCl for 4 h. Because of the preliminary development test using the substances, the maximum analytical concentration was measured at 2.00 mg/L. Intermediate and low concentrations were fixed at 0.67 and 0.22 mg/L, respectively, by a scaling factor of 3. During observation periods, clinical observations were recorded more than once a day, including the day of exposure (Fig. 1). At the end of the experiment, all rats were anesthetized with isoflurane, then killed by exsanguination of the abdominal aorta. An autopsy, including gross findings, was performed on each rat.
Fig. 1. Schema of the experimental procedures. The rats were assigned to four treatment groups. They were exposed once to CA and NaOCl for 4 h. During the observation period (14 days), mortality, general symptoms, and weight change were observed after exposure to test the substances, and visual observation was performed during autopsy. Data are presented as mean (± SD) to VC rats (n = 5).
CA, citric acid; NaOCl, sodium hypochlorite; VC, vehicle control.
Chemicals
CA (CAS No: 77-92-9, Purity ≥ 99.5) and NaOCl (CAS No: 7681-52-9, Purity: 4.00%–4.99% chlorine basis) were purchased from Sigma-Aldrich (USA). Isoflurane was purchased from Hana Pharm. Co., Ltd (Korea).
Inhalation exposure
The environmental conditions of the chamber were monitored every 1 h using standard equipment (model No. PGM-6208; RAE Systems, Inc., USA). The concentration analysis of aerosolized CA and NaOCl (using a mist generator, model No. NB-2N; SIBATA, Japan) in the chambers was performed during 4 h of exposure using a nose-only exposure inhalation experiment system (model No. SIS-30BK; SIBATA). The particle size distribution was measured twice for each concentration group (except the VC group) on the day of exposure using a cascade impactor (Mini MOUDI 135-6S; MSP Corporation, USA). Based on the measured values, mass median aerodynamic diameter (MMAD) and geometric SD (GSD) were calculated using Microsoft Excel software (Microsoft Corporation, USA).
Histopathological assessment
The whole lung tissue, trachea, and nasal cavity were fixed in 10% neutral-buffered formalin and embedded in paraffin. Subsequently, the paraffin-embedded tissues were sliced into 4-μm-thick sections. The tissue sections were stained with hematoxylin and eosin (Sigma-Aldrich), followed by histological analysis. The sections were examined under a light microscope (BX51; Olympus, Japan).
Statistical analyses
Data are expressed as the mean ± SD. All statistical analyses were performed using the Pristima System or Statistical Analysis System (SAS/STAT; Xybion Digital, Canada), and multiple comparison analyses were performed to compare the groups. The data obtained were tested for equal variances using the Bartlett’s Test, followed by testing for equal variances using a one-way analysis of variance. The Dunnett’s Test was used to analyze the differences between the groups. Skewed data were analyzed using the Kruskal–Wallis test, and the differences between the exposure and VC groups were analyzed using the Dunn’s rank sum test. The LC50 was calculated at the test site using the SAS/STAT analysis system.
RESULTS
Inhalation chamber environment monitoring
During the exposure period, the oxygen concentration in the chamber was fixed at 20.9% ± 0.0% for rats in the CA and NaOCl exposure groups, as in well as the VC group, which was ≥ 19%. The carbon dioxide concentration in the chamber was maintained below 1% at 200 ± 0 and 100 ± 0 ppm in the CA and NaOCl groups, respectively, as well as in the VC group (Table 1).
Table 1. Environmental monitoring in the inhalation chamber.
| Variables | Concentrations (mg/L) | ||||
|---|---|---|---|---|---|
| 0.00 | 0.22 | 0.67 | 2.00 | ||
| Citric acid | |||||
| Chamber pressure (Pa) | −99.5 ± 3.1 | −95.0 ± 1.2 | −105.8 ± 5.1 | −102.4 ± 1.0 | |
| Main flow (L/min) | 32.5 ± 1.0 | 45.1 ± 0.5 | 36.2 ± 0.6 | 30.1 ± 0.0 | |
| Temperature (°C) | 23.4 ± 0.7 | 23.2 ± 0.8 | 22.9 ± 0.6 | 22.5 ± 0.7 | |
| Relative humidity (%) | 40.0 ± 2.3 | 62.6 ± 4.0 | 67.0 ± 2.9 | 60.0 ± 0.8 | |
| Oxygen concentration (%) | 20.9 ± 0.0 | 20.9 ± 0.0 | 20.9 ± 0.0 | 20.9 ± 0.0 | |
| Carbon dioxide concentrationa (ppm) | 200 ± 0 | 200 ± 0 | 200 ± 0 | 200 ± 0 | |
| Sodium hypochlorite | |||||
| Chamber pressure (Pa) | −97.4 ± 1.9 | −96.0 ± 2.9 | −95.5 ± 2.2 | −101.3 ± 1.5 | |
| Main flow (L/min) | 29.5 ± 0.0 | 39.8 ± 0.8 | 41.7 ± 0.0 | 31.0 ± 0.8 | |
| Temperature (°C) | 23.1 ± 0.6 | 22.7 ± 0.5 | 22.5 ± 0.2 | 22.4 ± 0.1 | |
| Relative humidity (%) | 38.0 ± 2.4 | 68.3 ± 2.8 | 61.5 ± 1.3 | 71.6 ± 3.5 | |
| Oxygen concentration (%) | 20.9 ± 0.0 | 20.9 ± 0.0 | 20.9 ± 0.0 | 20.9 ± 0.0 | |
| Carbon dioxide concentration (ppm) | 100 ± 0 | 100 ± 0 | 100 ± 0 | 100 ± 0 | |
Values are presented as mean ± SD.
a104 ppm = 1%.
Concentration and particle size distribution of CA and NaOCl in the nose-only exposure chamber
Data on actual CA concentrations and other conditions monitored in the exposure chambers are presented in Table 2. The mean (± SD) analyte concentrations for 0.22, 0.67, and 2.00 mg/L groups measured during the exposure were 0.20 ± 0.03 mg/L, 0.64 ± 0.05 mg/L, and 1.70 ± 0.08 mg/L for CA and 0.23 ± 0.03 mg/L, 0.69 ± 0.02 mg/L, and 2.16 ± 0.06 mg/L for NaOCl, respectively (Table 2).
Table 2. Analyte concentration.
| Variables | Concentrations (mg/L) | |||||
|---|---|---|---|---|---|---|
| Citric acid | Sodium hypochlorite | |||||
| 0.22 | 0.67 | 2.00 | 0.22 | 0.67 | 2.00 | |
| 1st | 0.23 | 0.70 | 1.63 | 0.20 | 0.70 | 2.17 |
| 2nd | 0.19 | 0.61 | 1.79 | 0.23 | 0.70 | 2.22 |
| 3rd | 0.18 | 0.63 | 1.69 | 0.26 | 0.66 | 2.10 |
| Mean ± SD | 0.20 ± 0.03 | 0.64 ± 0.05 | 1.70 ± 0.08 | 0.23 ± 0.03 | 0.69 ± 0.02 | 2.16 ± 0.06 |
The MMAD and GSD, based on the OECD test guideline 403 (TG 403) standard [13] which is the guideline for short-term exposure to test articles by inhalation, were calculated by measuring each concentration group twice to check if they were maintained within the range of 1–4 µm and 1.5–3.0, respectively. During exposure, the MMAD of CA was 1.32 ± 0.16, 1.20 ± 0.08, and 1.15 ± 0.06 µm in the 0.22, 0.67, and 2.00 mg/L groups, respectively. The GSD of CA was 2.13 ± 0.08, 2.12 ± 0.03, and 2.00 ± 0.02 in the 0.22, 0.67, and 2.00 mg/L groups, respectively. The MMAD of NaOCl was 1.55 ± 0.16, 1.48 ± 0.11, and 1.18 ± 0.03 µm in the 0.22, 0.67, and 2.00 mg/L groups, respectively, whereas the GSD of NaOCl was 1.97 ± 0.00, 1.91 ± 0.01, and 1.79 ± 0.00 in the 0.22, 0.67, and 2.00 mg/L groups, respectively (Table 3). All exposure groups met the OECD TG 403 standard and were exposed equally and stably.
Table 3. Particle size distribution in chambers during the exposure period.
| Parameter | Concentrations (mg/L) | |||||
|---|---|---|---|---|---|---|
| Citric acid | Sodium hypochlorite | |||||
| 0.22 | 0.67 | 2.00 | 0.22 | 0.67 | 2.00 | |
| MMDA (μm) | 1.32 ± 0.16 | 1.20 ± 0.08 | 1.15 ± 0.06 | 1.55 ± 0.16 | 1.48 ± 0.11 | 1.18 ± 0.03 |
| GSD ± SD | 2.13 ± 0.08 | 2.12 ± 0.03 | 2.00 ± 0.02 | 1.97 ± 0.00 | 1.91 ± 0.01 | 1.79 ± 0.00 |
MMDA, mass median aerodynamic diameter; GSD, geometric SD.
Measurement of mortality
After CA exposure, two male rats 2.00 mg/L group died on the day 1 of the exposure period, whereas no deaths occurred in females (Table 4). The LC50 because of CA exposure is 1.73390 mg/L for males and > 1.70 mg/L for females with no mortality. In the NaOCl group, two males and one female died on day 1 in the 2.00 mg/L group (Table 5). the LC50 of NaOCl was 2.22222 mg/L for males and 2.39456 mg/L for females.
Table 4. Summary of mortality with citric acid.
| Sex | Dose (mg/L) | Treatment day | Final mortalitya | |||
|---|---|---|---|---|---|---|
| Day 1 | ≤ Day 3 | ≤ Day 7 | ≤ Day 15 | |||
| Male | 0.00 | 0 | 0 | 0 | 0 | 0/5 |
| 0.22 | 0 | 0 | 0 | 0 | 0/5 | |
| 0.67 | 0 | 0 | 0 | 0 | 0/5 | |
| 2.00 | 2 | 0 | 0 | 0 | 2/5 | |
| Female | 0.00 | 0 | 0 | 0 | 0 | 0/5 |
| 0.22 | 0 | 0 | 0 | 0 | 0/5 | |
| 0.67 | 0 | 0 | 0 | 0 | 0/5 | |
| 2.00 | 0 | 0 | 0 | 0 | 0/5 | |
aNumber of rats with dead animals/total number of rats.
Table 5. Summary of mortality with sodium hypochlorite.
| Sex | Dose (mg/L) | Treatment day | Final mortalitya | |||
|---|---|---|---|---|---|---|
| Day 1 | ≤ Day 3 | ≤ Day 7 | ≤ Day 15 | |||
| Male | 0.00 | 0 | 0 | 0 | 0 | 0/5 |
| 0.22 | 0 | 0 | 0 | 0 | 0/5 | |
| 0.67 | 0 | 0 | 0 | 0 | 0/5 | |
| 2.00 | 2 | 0 | 0 | 0 | 2/5 | |
| Female | 0.00 | 0 | 0 | 0 | 0 | 0/5 |
| 0.22 | 0 | 0 | 0 | 0 | 0/5 | |
| 0.67 | 0 | 0 | 0 | 0 | 0/5 | |
| 2.00 | 1 | 0 | 0 | 0 | 1/5 | |
aNumber of rats with dead animals/total number of rats.
Body weight changes
A significant decrease in body weight was observed on day 2 of exposure, in the male CA 2.00 mg/L group (Fig. 2A) and the female 0.67 and 2.00 mg/L group (Fig. 2B), compared to that in the VC group. However, the body weights recovered over time (Fig. 2A and B). In the male NaOCl exposure group, there were significant changes in the post-exposure body weight of the 0.22, 0.67, and 2.00 mg/L groups as compared to that in the VC group. The 0.67 mg/L group showed a significant decrease in body weight gain on day 2 after exposure to autopsy (day 15) compared to the VC group. Likewise, weight gain was significantly less in the 0.67 (day 2) and 2.00 mg/L (days 2, 4, and 8) groups than in the VC group (Fig. 2C). In females, on days 2 and 4, we observed a significant decrease in weight gain in the 0.67 and 2.00 mg/L groups, respectively, compared to that in the VC group (Fig. 2D).
Fig. 2. Body weight changes in Sprague-Dawley rats after inhaling CA and NaOCl. Measured body weight after rats inhaled (A and B) CA and (C and D) NaOCl. Data are presented as mean (± SD) to VC rats (n = 5).
CA, citric acid; NaOCl, sodium hypochlorite; VC, vehicle control.
*p < 0.05, **p < 0.01 versus VC.
Observation of clinical signs
One female rat in the CA 2.00 mg/L group showed mild dyspnea, irregular breathing, and weakness after 2 and 3 days of exposure, followed by remission (data not shown). No specific abnormal clinical signs could be observed in other CA exposure groups (data not shown). Contamination of the hair on the nose, mouth, and chin was observed in all male exposure groups, except the VC group and deceased animals, and in the female NaOCl exposure groups of 0.67 and 2.00 mg/L groups. Furthermore, hair loss was observed in two males and one female in the NaOCl 2.00 mg/L group (data not shown). Three males and one female in the NaOCl 2.00 mg/L group showed irregular breathing (Table 6).
Table 6. Summary of clinical signs in the NaOCl inhalation group.
| Sex | Clinical signs | Concentrations (mg/L) | |||||||
|---|---|---|---|---|---|---|---|---|---|
| 0.00 | 0.22 | 0.67 | 2.00 | ||||||
| aa | bb | aa | bb | aa | bb | aa | bb | ||
| Male | No abnormalities detected | 5 | 15.00 | 5 | 14.40 | 5 | 12.20 | 5 | 5.00 |
| Irregular respiration | 0 | 0.00 | 0 | 0.00 | 0 | 0.00 | 3 | 2.67 | |
| Female | No abnormalities detected | 5 | 15.00 | 5 | 15.00 | 5 | 13.20 | 5 | 9.80 |
| Irregular respiration | 0 | 0.00 | 0 | 0.00 | 0 | 0.00 | 2 | 3.00 | |
NaOCl, sodium hypochlorite.
aNumber of rats affected; bMean number of days with observed clinical signs.
Gross findings and histopathological analysis
In the CA 2.00 mg/L group, discoloration of the lungs was observed in the two deceased male rats. A discharge around the nose and mouth was observed on one of the deceased rats (data not shown). Whereas no gross findings were observed in all the rats subjected to autopsy. Histopathological changes were observed under a light microscope to confirm the effects of inhalation exposure to CA on the target organs (lung, trachea, and nasal cavity). In the CA exposure groups, no abnormal histopathological changes were observed in the lung, trachea, and nasal cavity compared to those in the VC group, in both the deceased and euthanized rats (Fig. 3). Discoloration of the lungs and discharge around the nose and mouth was observed in the two deceased males and one deceased female rat in the NaOCl 2.00 mg/L group (data not shown). Additional histopathological change observations were made to evaluate the effect of NaOCl inhalation exposure. Perivascular/inflammatory cell infiltration, alveolar macrophage aggregation, and congestion in the lungs of the two deceased males, and one female rat exposed to 2.00 mg/L NaOCl were observed (Fig. 4A). Mild perivascular/inflammatory cell infiltration, alveolar macrophage aggregation, and congestion were observed in the lungs of one female in the 0.67 mg/L NaOCl euthanized group (Fig. 4B), but no pathological changes were observed in any of the other euthanized rats (Fig. 4C). Similarly, no abnormal histopathological changes were observed in the trachea and nasal cavity compared to those in the VC group in all exposure groups (Fig. 4D and E).
Fig. 3. Histological features of the target organs in the CA exposure group. Representative photomicrograph at 20× magnification demonstrating H&E staining of the (A and B) lung, (C) trachea, and (D) nasal cavity in CA exposure and VC group rats (scale bar = 100 μm).
CA, citric acid; H&E, hematoxylin and eosin; VC, vehicle control.
Fig. 4. Histological features of the target organs in the NaOCl exposure groups. Representative photomicrograph at 20× magnification demonstrating H&E staining of the (A, B, and C) lung, (D) trachea, and (E) nasal cavity NaOCl exposure and VC group rats. The red arrows indicate alveolar macrophage aggregation, the blue arrows indicate inflammatory cell infiltration in the perivascular/alveolar, and the green arrows indicate congestion/hemorrhage (scale bar = 100 μm).
NaOCl, sodium hypochlorite; H&E, hematoxylin and eosin; VC, vehicle control.
DISCUSSION
Various forms of disinfectants for animals exist such as spray, fumigation, and heated smoke screen. Among them, the spray form is commonly used, and CA and NaOCl are the main active ingredients in these products. CA is an essential chemical produced and consumed worldwide. It is used mainly as an acidulant in the food and beverage industry. NaOCl is commonly used as both a bleaching agent and topical antiseptic agent. Owing to the widespread use of CA and NaOCl, previous studies have evaluated the toxic effects of different exposure concentrations, frequencies, and various types of aerosol [14,15,16,17]. However, published studies on acute inhalation toxicity of CA and NaOCl do not provide clarity on the aerosol types and methods of these chemicals. Alternatively, most of the data in acute inhalation toxicity studies conducted in mist or aerosol form are outdated. To confirm the acute toxic effect of CA, a study in 1986, conducted a systemic exposure of CA to guinea pigs for 3 min, but the clear aerosol type was unknown [18]. An NaOCl (in vapor form) exposure study evaluated acute inhalation toxicity in male albino rats exposed for 1 h; however, the exact exposure method was not described and the test was conducted in 1962 [16]. In addition, toxicity is evaluated by the LC50 value, which is a common dose-response descriptor for acute inhalation toxicity. However, there are previous studies showing that oral administration of CA has an LD50 of 300 to 1,200 mg/kg body weight (bw), and the LD50 by skin exposure is greater than 2,000 mg/kg bw [19]. Clinical studies also reported only vomiting and near death in women who took 25 g of CA once. In the case of NaOCl, the LC50 in rats is > 10.5 mg/L, but the test was carried out in rats using an unspecified commercial solution of NaOCl [16,20].
Our study was conducted in accordance with OECD TG 403, applying good laboratory practice (GLP) to acute inhalation exposure toxicity studies for CA and NaOCl. Additionally, we evaluated the effects of CA and NaOCl exposure in target organs such as the lungs, trachea, and nasal cavity. In the male CA 2.00 mg/L group, two rats were found dead on day 1 of exposure. These results suggested that the LC50 of CA is 1.73390 mg/L for males and > 1.70 mg/L for females, with no mortality. Discoloration of the lungs and discharge around the nasal and perioral area were observed in the deceased rats. CA exposure group did not cause significant histopathological changes in deceased and euthanized rats. Cui et al. [17], demonstrated that guinea pigs continually exposed to 0.5 M CA by inhalation for 43 days may exhibit coughing, increased tracheal basement membrane thickness, and airway constriction. Nakaji et al. [21], also demonstrated that repeated exposure to 0.5 M CA induces coughing. Furthermore, liver damage caused by increased inflammation, decreased blood cell count, and induced apoptosis in mice administered CA for 30 days; furthermore, cytotoxicity was shown in macrophage cells exposed to CA for 7 days [22,23]. Based on these previous reports, repeated inhalation exposure studies are required to elucidate the toxic effects and mechanisms of CA.
In the NaOCl 2.0 mg/L exposure group, two males and one female died on day 1. These results calculated that the LC50 of NaOCl was 2.22222 mg/L for males and 2.39456 mg/L for females. And the deceased rats showed lung discoloration and discharge around the nasal and perioral area. In the euthanized rats, lung discoloration was observed in one female in the 0.67 mg/L exposure group. Abnormal symptoms of irregular respiration, ranging from weak to severe, were observed in males and females in the 2.00 mg/L group, but the symptoms subsided during the observation period. The deceased rats in the NaOCl exposure groups demonstrated infiltration of inflammatory cells and/or alveolar macrophage aggregation, and the euthanized rats exhibited congestion in the lungs. Similar symptoms were observed in previous clinical toxicity studies on NaOCl exposure. Exposure to high concentrations of NaOCl in the skin causes blemishes because of exothermic reactions such as a burning sensation, pain, blistering, and edema [24,25,26]. Inhaled respiratory irritation has been reported to be induced when inhaled, and symptoms such as nausea, vomiting, dizziness, dyspnea, and headache are exhibited [27,28,29]. There are also clinical reports that some people demonstrated asthma-like symptoms after long-term use of bleach and disinfectant, including NaOCl [28,30,31]. These findings demonstrate that long-term inhalation exposure of NaOCl can cause abnormalities due to respiratory irritation. Furthermore, in this study, similar histopathological lesions were observed in the lungs in both NaOCl-exposed deceased and euthanized rats. Likewise, additional research such as repeated exposure is necessary to identify the toxic effects and mechanisms of NaOCl exposure. Together, these results suggest that the lung is a target organ for inhalation exposure to CA and NaOCl, since no chemical exposure-related changes were observed in other organs, including the nasal cavity and trachea.
In conclusion, according to the LC50, the Globally Harmonized System is category 4 for both CA and NaOCl. In this study, the LC50 results were obtained through a GLP-based acute inhalation toxicity assessment. We propose a helpful template to reset safety standards for CA and NaOCl use.
ACKNOWLEDGEMENTS
The authors thank the Korea Institute of Toxicology (KIT) technical staff of the Inhalation Toxicology Research Group for their technical support.
Footnotes
Funding: This research was supported by the Animal and Plant Quarantine Agency (20201201131-00) and Korea Institute of Toxicology (KK-2207), Republic of Korea.
Conflict of Interest: The authors declare no conflicts of interest.
- Investigation: Park CM.
- Resources: Choi SH, Lee JY, Jeon BS, Ku HO.
- Supervision: Kim MS.
- Visualization: Yang MJ.
- Writing - original draft: Kim J.
References
- 1.Mo IP, Bae YJ, Lee SB, Mo JS, Oh KH, Shin JH, et al. Review of avian influenza outbreaks in South Korea from 1996 to 2014. Avian Dis. 2016;60(1) Suppl:172–177. doi: 10.1637/11095-041715-Review. [DOI] [PubMed] [Google Scholar]
- 2.Joo YS, An SH, Kim OK, Lubroth J, Sur JH. Foot-and-mouth disease eradication efforts in the Republic of Korea. Can J Vet Res. 2002;66(2):122–124. [PMC free article] [PubMed] [Google Scholar]
- 3.Zou S, Guo J, Gao R, Dong L, Zhou J, Zhang Y, et al. Inactivation of the novel avian influenza A (H7N9) virus under physical conditions or chemical agents treatment. Virol J. 2013;10(1):289. doi: 10.1186/1743-422X-10-289. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Chae WS, Cha CN, Yoo CY, Kim S, Lee HJ. Virucidal efficacy of a disinfectant solution composed of citric acid, malic acid and phosphoric acid against avian influenza virus. J Prev Vet Med. 2018;42(1):16–21. [Google Scholar]
- 5.Gamal AM, Samaha HA, Elagrab HM, Ismael IE, Nasr AE, Moubarak ST, et al. Inactivation of avian influenza virus using commercial chemical disinfectants in small scale poultry production. Alex J Vet Sci. 2014;41(1):102–108. [Google Scholar]
- 6.Kim S, Chung H, Lee H, Myung D, Choi K, Kim S, et al. Evaluation of the disinfectant concentration used on livestock facilities in Korea during dual outbreak of foot and mouth disease and high pathogenic avian influenza. J Vet Sci. 2020;21(3):e34. doi: 10.4142/jvs.2020.21.e34. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Torrado AM, Cortés S, Manuel Salgado J, Max B, Rodríguez N, Bibbins BP, et al. Citric acid production from orange peel wastes by solid-state fermentation. Braz J Microbiol. 2011;42(1):394–409. doi: 10.1590/S1517-83822011000100049. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Singh Dhillon G, Kaur Brar S, Verma M, Tyagi RD. Recent advances in citric acid bio-production and recovery. Food Bioprocess Technol. 2011;4(4):505–529. [Google Scholar]
- 9.Berovic M, Legisa M. Citric acid production. Biotechnol Annu Rev (Amst) 2007;13:303–343. doi: 10.1016/S1387-2656(07)13011-8. [DOI] [PubMed] [Google Scholar]
- 10.Hong JK, Lee KN, You SH, Kim SM, Tark D, Lee HS, et al. Inactivation of foot-and-mouth disease virus by citric acid and sodium carbonate with deicers. Appl Environ Microbiol. 2015;81(21):7610–7614. doi: 10.1128/AEM.01673-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Mahdizadeh S, Sawford K, van Andel M, Browning GF. Efficacy of citric acid and sodium hypochlorite as disinfectants against Mycoplasma bovis . Vet Microbiol. 2020;243:108630. doi: 10.1016/j.vetmic.2020.108630. [DOI] [PubMed] [Google Scholar]
- 12.Tiwari S, Rajak S, Mondal DP, Biswas D. Sodium hypochlorite is more effective than 70% ethanol against biofilms of clinical isolates of Staphylococcus aureus . Am J Infect Control. 2018;46(6):e37–e42. doi: 10.1016/j.ajic.2017.12.015. [DOI] [PubMed] [Google Scholar]
- 13.Organization for Economic Cooperation and Development (OECD) Test No. 403: Acute Inhalation Toxicity. Paris: OECD; 2009. [Google Scholar]
- 14.Mei B, Cui F, Wu C, Wen Z, Wang W, Shen M. Roles of citric acid in conjunction with saline nebulization in experimental tracheostomy in guinea pigs. Exp Lung Res. 2018;44(10):433–442. doi: 10.1080/01902148.2018.1516832. [DOI] [PubMed] [Google Scholar]
- 15.Martyny JW, Harbeck RJ, Pacheco K, Barker EA, Sills M, Silveira L, et al. Aerosolized sodium hypochlorite inhibits viability and allergenicity of mold on building materials. J Allergy Clin Immunol. 2005;116(3):630–635. doi: 10.1016/j.jaci.2005.05.008. [DOI] [PubMed] [Google Scholar]
- 16.The European Chemicals Agency (ECHA) Sodium hypochlorite [Internet] Helsinki: The European Chemicals Agency; [Updated 2022]. [Accessed 2022 Sep 8]. https://echa.europa.eu/substance-information/-/substanceinfo/100.028.790 . [Google Scholar]
- 17.Cui S, Ito I, Nakaji H, Iwata T, Matsumoto H, Oguma T, et al. Induction of airway remodeling and persistent cough by repeated citric acid exposure in a guinea pig cough model. Respir Physiol Neurobiol. 2019;263:1–8. doi: 10.1016/j.resp.2019.02.002. [DOI] [PubMed] [Google Scholar]
- 18.The European Chemicals Agency (ECHA) Citric acid acute toxicity: inhalation [Internet] Helsinki: The European Chemicals Agency; [Updated 2022]. [Accessed 2022 Sep 8]. https://echa.europa.eu/registration-dossier/-/registered-dossier/15451/7/3/3 . [Google Scholar]
- 19.Australian Industrial Chemicals Introduction Scheme. IMAP Group Assessment Report. Citric Acid: Human Health Tier II Assessment. Sydney: Australian Industrial Chemicals Introduction Scheme; 2013. [Google Scholar]
- 20.Australian Industrial Chemicals Introduction Scheme. IMAP Single Assessment Report. Hypochlorous Acid, Sodium Salt: Human Health Tier II Assessment. Sydney: Australian Industrial Chemicals Introduction Scheme; 2014. [Google Scholar]
- 21.Nakaji H, Niimi A, Matsuoka H, Iwata T, Cui S, Matsumoto H, et al. Airway remodeling associated with cough hypersensitivity as a consequence of persistent cough: An experimental study. Respir Investig. 2016;54(6):419–427. doi: 10.1016/j.resinv.2016.06.005. [DOI] [PubMed] [Google Scholar]
- 22.Atia MM, Mahmoud FA. Soya milk alleviates toxicity caused by citric acid in male mice: histopathological and hematological studies. J Food Biochem. 2021;45(6):e13773. doi: 10.1111/jfbc.13773. [DOI] [PubMed] [Google Scholar]
- 23.Amaral KF, Rogero MM, Fock RA, Borelli P, Gavini G. Cytotoxicity analysis of EDTA and citric acid applied on murine resident macrophages culture. Int Endod J. 2007;40(5):338–343. doi: 10.1111/j.1365-2591.2007.01220.x. [DOI] [PubMed] [Google Scholar]
- 24.Slaughter RJ, Watts M, Vale JA, Grieve JR, Schep LJ. The clinical toxicology of sodium hypochlorite. Clin Toxicol (Phila) 2019;57(5):303–311. doi: 10.1080/15563650.2018.1543889. [DOI] [PubMed] [Google Scholar]
- 25.Harley EH, Collins MD. Liquid household bleach ingestion in children: a retrospective review. Laryngoscope. 1997;107(1):122–125. doi: 10.1097/00005537-199701000-00023. [DOI] [PubMed] [Google Scholar]
- 26.Telmon N, Allery JP, Dorandeu A, Rougé D. Concentrated bleach burns in a child. J Forensic Sci. 2002;47(5):1060–1061. [PubMed] [Google Scholar]
- 27.Deschamps D, Soler P, Rosenberg N, Baud F, Gervais P. Persistent asthma after inhalation of a mixture of sodium hypochlorite and hydrochloric acid. Chest. 1994;105(6):1895–1896. doi: 10.1378/chest.105.6.1895. [DOI] [PubMed] [Google Scholar]
- 28.Matulonga B, Rava M, Siroux V, Bernard A, Dumas O, Pin I, et al. Women using bleach for home cleaning are at increased risk of non-allergic asthma. Respir Med. 2016;117:264–271. doi: 10.1016/j.rmed.2016.06.019. [DOI] [PubMed] [Google Scholar]
- 29.Goswami M, Chhabra N, Kumar G, Verma M, Chhabra A. Sodium hypochlorite dental accidents. Paediatr Int Child Health. 2014;34(1):66–69. doi: 10.1179/2046905512Y.0000000042. [DOI] [PubMed] [Google Scholar]
- 30.Dumas O, Wiley AS, Henneberger PK, Speizer FE, Zock JP, Varraso R, et al. Determinants of disinfectant use among nurses in U.S. healthcare facilities. Am J Ind Med. 2017;60(1):131–140. doi: 10.1002/ajim.22671. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Peck B, Workeneh B, Kadikoy H, Patel SJ, Abdellatif A. Spectrum of sodium hypochlorite toxicity in man-also a concern for nephrologists. NDT Plus. 2011;4(4):231–235. doi: 10.1093/ndtplus/sfr053. [DOI] [PMC free article] [PubMed] [Google Scholar]