Use of the TIPS Technique as a Possible Method of Acute Inhalation Toxicity Assessment

ABSTRACT

Acute inhalation toxicity studies provide crucial information regarding short‐term exposure to volatile chemicals and help to ensure the safe handling and use of these chemicals. However, acute inhalation toxicity assessment requires specialized equipment, and most animal facilities are not able to carry out such studies. Consequently, an extremely large number of chemicals remain unassessed. Therefore, we investigated intra‐Tracheal Intra‐Pulmonary Spraying (TIPS) as a method for testing the acute inhalation toxicity of volatile chemicals. TIPS does not require specialized equipment and consequently is an approach that can be used by essentially all animal facilities. We diluted 12 chemicals in physiological saline and administered the chemicals to test animals using TIPS. When the acute LD50 values obtained by TIPS were converted to LC50 values, there was good agreement between the TIPS LC50 values and the reported inhalation LC50 values of nine of 11 water‐soluble chemicals. Two water‐soluble chemicals had TIPS LC50 values that were more than 2.5‐fold higher than the published inhalation LC50 values, and water‐insoluble xylene had an LC50 value that was more than 2.5‐fold lower than the published inhalation LC50 value. This strong agreement between TIPS LC50 values and published LC50 values suggests that TIPS has the potential to be used for acute inhalation toxicity testing. However, further studies are needed to establish testing systems that will identify solutes that can and cannot be used for TIPS testing of different water‐soluble chemicals and for TIPS testing of water‐insoluble chemicals.

Short abstract

Acute inhalation toxicity assessment requires specialized equipment, and most animal facilities are not able to carry out such studies. Consequently, an extremely large number of chemicals remain unassessed. In this study, we investigated intra‐Tracheal Intra‐Pulmonary Spraying (TIPS) as a method for testing the acute inhalation toxicity of volatile chemicals. There was good agreement between TIPS‐derived LC50 values and published inhalation LC50 values for nine of the 12 tested chemicals.

1. Introduction

According to the Globally Harmonized System (GHS), acute inhalation toxicity assessment should be used for the safety classification of substances that present inhalation exposure hazards. Currently, in vivo studies using whole‐body or nose‐only inhalation chambers have been conducted in accordance with OECD TG 436 (OECD ²⁰⁰⁹). However, because of the vast number of unassessed chemicals, the high cost of experiments, the difficulty of quality control, and the lack of facilities that are equipped to carry out in vivo inhalation exposure studies, less expensive and easier approaches to ensure the safe use of chemicals are urgently needed: The large/expensive facilities required when whole‐body exposure is used are well known; however, even nose‐only inhalation studies require specialized equipment (see Annexes 1, 2, and 3 in OECD ²⁰⁰⁹). We have been evaluating the inhalation toxicity of several kinds of carbon nanotubes and particles using the intra‐Tracheal Intra‐Pulmonary Spraying (TIPS) method (Abdelgied et al. 2019; Numano et al. ²⁰¹⁴; Numano et al. ²⁰¹⁹; Saleh et al. ²⁰²⁰; Saleh et al. ²⁰²²; Sheema et al. ²⁰²⁴; Suzui et al. ²⁰¹⁶). This method does not require specific equipment or large facilities, and its use is thus applicable for general animal facilities. The purpose of the present study was to investigate the use of TIPS for testing acute inhalation toxicity of air‐borne chemicals. The in vitro cytotoxicity of the test chemicals against A549 cells was also evaluated to assess whether a preliminary in vitro analysis would provide useful data for setting doses for in vivo studies.

2. Materials and Methods

2.1. Animals

Twelve‐week‐old male F344 rats were purchased from Charles River Japan Inc. (Yokohama, Japan). The animals were housed in the Center for Experimental Animal Science of Nagoya City University Medical School, maintained on a 12‐h light–dark cycle, and received Oriental MF basal diet (Oriental Yeast Co., Tokyo, Japan) and tap water ad libitum. The experimental protocol was approved by the Animal Care and Use Committee of Nagoya City University Medical School (approval number 24‐005), and the research was conducted according to the Guidelines for the Care and Use of Laboratory Animals of Nagoya City University. The experiment was started after a 2‐week acclimation and quarantine period.

2.2. Chemicals

Twelve chemicals were tested: 1,4‐dioxane, 2‐ethoxyethyl acetate, acetylacetone, 1,2‐dichloroethane, glycidyl methacrylate, xylene, glycidol, N,N‐dimethylformamide, N,N‐dimethylacetamide, acrolein, polyacrylic acid 5000, and tert‐butyl alcohol. All test chemicals were purchased from FUJIFILM Wako Chemicals: 1,4‐dioxane (purity > 99.7%; Cat. # 045‐24491); 2‐ethoxyethyl acetate (purity > 99.0%; Cat. # 052‐03186); acetylacetone (2,4‐pentanedione; purity > 99.0%; Cat. # 013‐00493); 1,2‐dichloroethane (purity > 99.5%; Cat. # 058‐00866); glycidyl methacrylate (purity > 95.0%; Cat. # 079‐03632); xylene (purity > 80.0%; Cat. # 241‐00096); glycidol (2,3‐epoxy‐1‐propanol; purity > 90.0%; Cat. # 056‐04941); N,N‐dimethylformamide (purity > 99.5%; Cat. # 041‐02913); N,N‐dimethylacetamide (purity > 98.0%; Cat. # 048‐02543); acrolein (10 mg/mL in water; Cat. # 513‐66081); polyacrylic acid 5000 (Wako 1st grade powder; MW approximately 5000; Cat. # 165‐18571); and t‐butyl alcohol (purity > 99.0%; Cat. # 028‐03386). The test chemicals and their acute toxicity classifications by GHS of classification and labelling of chemicals are listed in Table 1: See GHS ²⁰²³ (Chapter 3.1) for a description of acute toxicity classification by GHS.

TABLE 1.

Globally harmonized system (GHS) of classification and labelling of chemicals. Acute inhalation toxicity category.

Chemical	Acute inhalation GHS classification
1,4‐Dioxane (inhalation of vapors) a	Category 4
Glycidol (2,3‐epoxy‐1‐propanol) (inhalation of vapors) a	Category 3
N,N‐Dimethylformamide (inhalation of vapors) a	Category 3
N,N‐Dimethylacetamide (inhalation of vapors) a	Category 3
t‐Butyl alcohol 1	Category 3
Acrolein 2	Categories 1 and 2
Polyacrylic acid 3	Category 3
2‐Ethoxyethyl acetate (inhalation of vapors) a	Category 4
Acetylacetone (inhalation of vapors) a	Category 3
1,2‐Dichloroethane (inhalation of vapors) a	Category 3
Glycidyl methacrylate 4	Category 3
Xylene (inhalation of vapors) a	Category 4

^a The GHS classifications were obtained from FUJIFILM Wako Chemicals Safety Data Sheet.

¹ The GHS hazard statement in PubChem for t‐butyl alcohol is H332: Category 3. The state of the inhaled substance is not stated.

² The GHS hazard statement in PubChem for acrolein is H330: Categories 1 and 2. The state of the inhaled substance is not stated.

³ The GHS hazard statement in PubChem for polyacrylic acid is H335: Category 3. The state of the inhaled substance is not stated.

⁴ The GHS hazard statement in PubChem for glycidyl methacrylate is H335: Category 3. The state of the inhaled substance is not stated.

2.3. In Vitro Assay

The in vitro cytotoxicity assay was conducted using Neutral Red (MERCK/Sigma‐Aldrich, N4638) according to Repetto et al. ²⁰⁰⁸. The A549 human lung adenocarcinoma cell line, derived from human lung cancer, was obtained from American Type Culture Collection (ATCC, Virginia, USA) and seeded into 35‐mm dishes (growth surface, 9.2 cm²) at a saturation density of 900,000/dish in RPMI (FUJIFILM Wako, 189‐02025) + 10% FBS (FUJIFILM Wako, FB‐1001/500). The next day, the chemicals being tested were diluted in 4 mL RPMI and in 4 mL RPMI + 10% FBS at 0, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6, and 51.2 μL/mL. The media was removed from the tissue culture dishes, and 2 mL of the media containing each dilution of the test chemical was added to the dishes: two dishes each for the test chemical diluted in RPMI and two dishes each for the test chemical diluted in RPMI + 10% FBS. Cells in the remaining two dishes were exposed to a lethal dose of 2‐ethoxyethyl acetate: In preliminary experiments, exposure to 250 μL/mL 2‐ethoxyethyl acetate in RPMI or RPMI + 10% FBS for 15 min had been shown to induce 100% cell death (data not shown). Polyacrylic acid polymer powder was solubilized in physiological saline (Otsuka Normal Saline [injection 20 mL]) at 1 g of powder in a 10‐mL final total volume. Cells were incubated in the test chemical for 15 min. The media containing the test chemical was then aspirated from the plate, and the cells were washed twice with PBS. Two milliliters of neutral red solution (33‐μg/mL neutral red in RPMI or RPMI + 10% FBS) was added to each of the plates, and the cells were incubated for 3 h at 37°C, then washed twice with PBS, and solubilized with 1‐mL acid‐ETOH (50% EtOH in water with 1% glacial acetic acid), and the absorbance was read at 540 nm. 100% viability was the reading in the 0‐μL chemical dishes, and 0% viability was the reading in the 250‐μL/mL 2‐ethoxyethyl acetate dishes. If the series of dilutions of a chemical did not include a dose that caused 100% cell death, a second series of dishes was exposed to higher levels of the chemical. Once an LD50 was determined, triplicates of dishes were exposed to a final series of dilutions centered around the LD50 determined in the initial experiments to determine the in vitro LD50s (Table 2). These LD50s were used to calculate doses for a 300‐g rat as described below.

TABLE 2.

Conversion of in vitro LD50 values to single TIPS doses expected to result in 100% survival and 100% death of the treated rats.

	LD50 (μL/mL) (RPMI)	LD50 (μL/mL) (RPMI + 10% FBS)	Lower LD50 ÷ 2 (μL/mL)	Higher LD50 × 4 (μL/mL)	Density (g/mL = mg/μL)	Single TIPS doses (mg/kg) corresponding to the lower LD50/2 and the higher LD50 × 4
1,4‐Dioxane	148	150	73.8	598	1.0325	152	1235
Miscible with water
Glycidol (2,3‐epoxy‐1‐propanol)	82.5	75.0	37.5	330	1.1175	83.8	738
Miscible with water
N,N‐Dimethylformamide	198	190	95.0	790	0.9495	180	1500
Miscible with water
N,N‐Dimethylacetamide	198	190	95.0	792	0.9405	179	1490
Miscible with water
T‐Butanol	93.7	93.8	46.9	375	0.7800	73.1	585
Miscible with water
Acrolein	6.37	6.36	3.2	25.5	0.0100	0.06	0.51
Dissolved in water. Solution is miscible with water.
Polyacrylic acid (10%)	117	118	58.5	472	0.1000	11.7	94.4
Dissolved in saline. Saline solution is miscible with water.
2‐Ethoxyethyl acetate	73.2	74.0	36.6	296	0.9735	71.3	576
Solubility in water: 187 g/L
Acetylacetone	36.6	33.3	16.7	147	0.9735	32.4	285
Solubility in water: 166 g/L
1,2‐Dichloroethane	9.38	11.7	4.7	46.7	1.2560	11.8	117
Solubility in water: 8.6 g/L Density: 1.256 g/cm3 Will sink in aqueous solutions
Glycidyl methacrylate	6.7	7.0	3.3	27.9	1.0740	7.19	59.9
Solubility in water: 5–10 g/L Density: 1.074 g/cm3 Will sink in aqueous solutions
Xylene	43.0	46.0	21.5	184	0.8650	37.2	318
Solubility in water: 106 mg/L Density: 0.864 g/cm3 a Will not sink in aqueous solutions

Note: TIPS doses were calculated for a 300‐g rat administered 600‐μL TIPS solution. In vitro μL chemical/mL values were converted to mg/kg values as follows: μL/mL × 0.6 mL × chemical density (mg/μL)/0.3 kg = mg/kg. Doses in mg/kg could then be applied to all rats.

^a The solubility and density of mixed xylenes are from Toxicological Profile for Xylene Table 4‐2: available at <https://www.ncbi.nlm.nih.gov/books/NBK600783/table/ch4.tab2/>.

2.4. Calculation of Doses

TIPS doses were calculated based on the LD50 of the in vitro analysis as follows. The in vitro LD50s (in μL/mL) using RPMI and RPMI + 10% FBS, determined as described above, are shown in Table 2. The lower LD50 was divided by two and used to approximate a dose for a 300‐g rat that would result in 0% death after four administrations of the chemical. The higher LD50 was multiplied by four and used to approximate the dose for a 300‐g rat that would result in 100% death after four administrations of the chemical. These doses were converted to mg/kg for a 300‐g rat instilled with a 600‐μL solution (2‐μL/g body weight) as follows: μL chemical/mL × 0.6 mL × chemical density (mg/μL)/0.3 kg. The chemical densities provided by FUJIFILM Wako Chemicals were used in this calculation. Once the mg/kg doses were determined, the mg/kg doses could be applied to all rats. The single TIPS doses are shown in Table 2.

2.5. TIPS

Rats were divided into groups of six to eight rats for each chemical dose (Table 3). Rats were administered 600 μL of the TIPS solution containing the test chemical by TIPS as previously described (Abdelgied et al. 2019; Numano et al. ²⁰¹⁹). Briefly, for each group of rats, the test chemical was suspended in physiological saline (Otsuka Normal Saline) such that administration of 2 μL of the diluted chemical per gram body weight resulted in administration of the target dose (see Table 3). Rats were anesthetized with 3% isoflurane and weighed. 2.0 μL of the test solution per gram body weight of the rat was drawn into a disposable 1‐mL syringe. The syringe was connected to an aerosol sprayer (Series IA–1B Intratracheal Aerosolizer Penn‐century, Philadelphia, PA, USA), and the nozzle of the sprayer was inserted into the trachea to the level of the carina, and the solution was sprayed into the lungs of the rat. To mimic a 4‐h inhalation exposure, administration was carried out by TIPS four times over the course of 3 h (0, 1, 2, and 3 h). The rats were then observed for 14 days. (The sprayer we used in the present study is no longer available. Penn‐Century Inc. is closed and no longer sells these microsprayers.) Aerosol sprayers that can be used for TIPS administration of liquid materials are available from Natsume Seisakusho, Tokyo, Japan, and Braintree Scientific Inc. Lab Research Products, and these sprayers are currently being used by our collaborators.

TABLE 3.

Range of doses administered to the rats (four administrations) and the LD50 (mg/kg) of the TIPS administered chemicals.

	Range of doses (mg/kg) hypothesized to include the in vivo LD50		Doses (mg/kg) administered in vivo (2‐week survival)						LD50 (mg/kg)
1,4‐Dioxane	609	4939	0	960	1280	1600	1920	2240	1720
Miscible with water			7/7	7/7	6/7	3/7	3/7	2/7
Glycidol (2,3‐epoxy‐1‐propanol)	335	2950	0	160	320	640			209
Miscible with water			8/8	8/8	0/8	0/8
N,N‐Dimethylformamide	722	6001	0	640	1280	2560			1321
Miscible with water			8/8	8/8	6/8	0/8
N,N‐Dimethylacetamide	715	5959	0	640	1280	2560			1321
Miscible with water			8/8	8/8	6/8	0/8
T‐Butanol	292	2341	0	200	400	800			817
Miscible with water			6/6	6/6	6/6	4/6
Acrolein	0.25	2.04	0	0.5	1.0	2.0			1.06
Dissolved in water. Solution is miscible with water.			8/8	8/8	7/8	0/8
Polyacrylic acid (10%)	46.8	378	0	60	120	240			170
Dissolved in saline. Saline solution is miscible with water.			8/8	8/8	7/8	1/8
2‐Ethoxyethyl acetate	285	2305	0	500	650	800	1000		893
Solubility in water: 187 g/L			8/8	8/8	7/8	7/8	1/8
Acetylacetone	130	1141	0	100	200	400	800		521
Solubility in water: 166 g/L			8/8	8/8	8/8	8/8	0/8
1,2‐Dichloroethane	47.1	470	0	60	120	240	480		270
Solubility in water: 8.6 g/L Density: 1.2454 g/cm3 Will sink in aqueous solutions			6/6	6/6	5/6	4/6	0/6
Glycidyl methacrylate	28.8	240	0	120	240	480	960		480
Solubility in water: 5–10 g/L Density: 1.08 g/cm3 Will sink in aqueous solutions			8/8	8/8	8/8	4/8	0/8
Xylene	149	1273	0	25	100	400	1600		315
Solubility in water: 106 mg/L Density: 0.864 g/cm3 a Will not sink in aqueous solutions			8/8	8/8	6/8	3/8	2/8

^a The solubility and density of mixed xylenes are from Toxicological Profile for Xylene Table 4‐2: available at <https://www.ncbi.nlm.nih.gov/books/NBK600783/table/ch4.tab2/>.

The in vivo TIPS LD50s (Table 3) were calculated using nonlinear regression analysis performed with GraphPad Prism version 8 (GraphPad Software, San Diego, CA).

2.6. Comparison of In Vivo Inhalation LC50 (mg/L) Values to Converted In Vivo TIPS LC50 (mg/L) Values

Table 4 lists inhalation LC50 values and the sources from which these values were obtained.

TABLE 4.

Inhalation LC50 values.

Chemical	LC50 a	Reference
1,4‐Dioxane	14,261 ppm (4 h)	ATSDR Chapter 3.2.1.1 (Wilbur et al. 2012)
	46 gm/m3 (2 h)	PubChem Section on Acute Effects (National Center for Biotechnology Information 2025a) PubChem Section on Toxicity Data (National Center for Biotechnology Information 2025a) NIOSH 2018: Section on Acute Toxicity Data (NIOSH 2018a)
Glycidol (2,3‐epoxy‐1‐propanol)	580 ppm (8 h)	PubChem Section on Acute Effects (National Center for Biotechnology Information 2025b) PubChem Section on Toxicity Data (National Center for Biotechnology Information 2025b) NIOSH 2019: Section on Acute Toxicity Data (NIOSH 2019a)
	1260 mg/m3 (4 h)	NIOSH 2019: Section on Acute Toxicity Data (NIOSH 2019a)
N,N‐Dimethylformamide	3421 ppm (1 h)	PubChem Table in Section on NIOSH Toxicity Data (National Center for Biotechnology Information 2025c) NIOSH 2018: Section on Acute Toxicity Data (NIOSH 2018b)
	3421 ppm (3 h)	PubChem Table in Section on NIOSH Toxicity Data (National Center for Biotechnology Information 2025c)
	1948 ppm (4 h)	PubChem Table in Section on NIOSH Toxicity Data (National Center for Biotechnology Information 2025c)
	> 5900 mg/m3 (4 h)	OECD Chapter 3.2.2 (OECD SIDS 2001b)
N,N‐Dimethylacetamide	2475 ppm (1 h)	PubChem Section on Acute Effects (National Center for Biotechnology Information 2025d) PubChem Section on Toxicity Data (National Center for Biotechnology Information 2025d) NIOSH 2019: Section on Acute Toxicity Data (NIOSH 2019b)
	8.81–10.1 mg/L (1 h)	OECD Chapter 4.2 (OECD SIDS 2001c)
	1.47 mg/L (7 h)	OECD Chapter 4.2 (OECD SIDS 2001c)
t‐Butyl alcohol	14,100 ppm (4 h)	PubChem Table in Section on NIOSH Toxicity Data (National Center for Biotechnology Information 2025e) NIOSH 2016: Section on Acute Toxicity Data (NIOSH 2016)
	> 10,000 ppm (4 h)	PubChem Section on Acute Effects (National Center for Biotechnology Information 2025e) NIOSH 2016: Section on Acute Toxicity Data (NIOSH 2016)
	10,000 ppm (4 h)	PubChem Section on Toxicity Data (National Center for Biotechnology Information 2025e)
Acrolein	18 mg/m3 (4 h)	PubChem Section on Acute Effects (National Center for Biotechnology Information 2025f) PubChem Section on Toxicity Data (National Center for Biotechnology Information 2025f) NIOSH 2019: Section on Acute Toxicity Data (NIOSH 2019c)
	8 ppm (4 h)	NIOSH 2019: Section on Acute Toxicity Data (NIOSH 2019c)
	131 ppm (30 min)	NIOSH 2019: Section on Acute Toxicity Data (NIOSH 2019c)
Acrylic acid polymers	The report “Acrylic acid polymer, neutralized, cross‐linked (Chapter 5.1.1)” cites two acute inhalation toxicity studies, both unpublished reports, indicating that the LC50 (4 h) of acrylic acid polymers was 2.17–57.1 mg/L (The MAK Collection for Occupational Health and Safety 2001) (The 4‐h exposure is stated in the references to the unpublished reports.)
2‐Ethoxyethyl acetate	> 1500 ppm (8 h)	PubChem Table in Section on NIOSH Toxicity Data (National Center for Biotechnology Information 2025g) NIOSH 2013: Section on Acute Toxicity Data (NIOSH 2013)
	12,100 mg/m3 (8 h)	PubChem Section on Acute Effects (National Center for Biotechnology Information 2025g) PubChem Section on Toxicity Data (National Center for Biotechnology Information 2025g) NIOSH 2013: Section on Acute Toxicity Data (NIOSH 2013)
Acetylacetone (2,4‐pentanedione)	1224 ppm (4 h)	PubChem Section Non‐Human Toxicity Excerpts (National Center for Biotechnology Information 2025h) OECD Chapter 3.1.2 (OECD SIDS 2001a)
	5.1 mg/L (4 h)	PubChem Section Non‐Human Toxicity Excerpts (National Center for Biotechnology Information 2025h) OECD Chapter 3.1.2 (OECD SIDS 2001a)
1,2‐Dichloroethane	1000 ppm (8 h)	ATSDR Chapter 2.2 (Toxicological Profile for 1,2‐Dichloroethane. Agency for Toxic Substances and Disease Registry (ATSDR) Toxicological Profiles; Atlanta (GA) 2024)
	1000 ppm (7 h)	PubChem Section on Acute Effects (National Center for Biotechnology Information 2025i) PubChem Section on Toxicity Data (National Center for Biotechnology Information 2025i) NIOSH 2018: Section on Acute Toxicity Data (NIOSH 2018c)
	5100 mg/m3 (6 h)	PubChem Table in Section on NIOSH Toxicity Data (National Center for Biotechnology Information 2025i) NIOSH 2018: Section on Acute Toxicity Data (NIOSH 2018c)
	4100 mg/m3 (7.2 h)	OECD Chapter 3.1.2 (OECD SIDS 2002)
	49,400 mg/m3 (0.5 h)	OECD Chapter 3.1.2 (OECD SIDS 2002)
	1650 ppm (6 h)	OECD Chapter 3.1.2 (OECD SIDS 2002)
	6670 mg/m3 (6 h)	OECD Chapter 3.1.2 (OECD SIDS 2002)
	8000 mg/m3 (4 h)	OECD Chapter 3.1.2 (OECD SIDS 2002)
	1900 ppm (4 h)	OECD Chapter 3.1.2 (OECD SIDS 2002)
Glycidyl methacrylate	45 ppm (4 h)	PubChem Section on Acute Effects (National Center for Biotechnology Information 2025j)
	> 2394 mg/m3 (LC0, 4 h)	OECD Chapter 4.2 Section (b) Acute toxicity (OECD SIDS 2000)
Mixed xylenes	6350–6700 ppm (4 h)	ATSDR Chapter 3.2.1.1 (Toxicological Profile for Xylene 2007)
p‐Xylene	4740 ppm (4 h)	ATSDR Chapter 3.2.1.1 (Toxicological Profile for Xylene 2007)
	4550 ppm (4 h)	PubChem Section on Acute Effects (National Center for Biotechnology Information 2025k) PubChem Section on Toxicity Data (National Center for Biotechnology Information 2025k) NIOSH 2019: Section on Acute Toxicity Data (NIOSH 2019d)
o‐Xylene	4330 ppm (6 h)	PubChem Section Non‐Human Toxicity Excerpts (National Center for Biotechnology Information 2025l)
	> 6125 ppm (LC0, 12 h)	NIOSH 2019: Section on Acute Toxicity Data (NIOSH 2019e)
m‐Xylene	5984 ppm (6 h)	PubChem Section Non‐Human Toxicity Excerpts (National Center for Biotechnology Information 2025m)
	> 8000 ppm (LC0, 4 h)	NIOSH 2019: Section on Acute Toxicity Data (NIOSH 2019f)

^a Grams/m³ = mg/L.

When the values were reported as ppm, ppm was converted to mg/L using 24.45 L as the molar volume of an ideal gas:

$$\mathrm{ppm}=\text{moles}\left(\text{chemical}\right)\text{in}{10}^6\text{moles}\left(\mathrm{gas}\right)$$

$$\mathrm{ppm}\times \mathrm{MW}\left(\text{grams chemical}/\text{mole}\right)=\text{grams chemical in}{10}^6\text{moles}\left(\mathrm{gas}\right)$$

$$\mathrm{ppm}\times \mathrm{MW}\left(\text{grams chemical}/\text{mole}\right)=\text{grams chemical in}{10}^6\times 24.45\mathrm{L}\left(\mathrm{gas}\right)$$

$$\left(\mathrm{ppm}\times \mathrm{MW}\right)/\left(24.45\times {10}^6 \mathrm{L}\right)=\text{grams chemical in}1\mathrm{L}\left(\mathrm{gas}\right)$$

$$\left(\left(\mathrm{ppm}\times \mathrm{MW}\right)/\left(24.45\times {10}^6 \mathrm{L}\right)\right)\times 1000=\mathrm{mg}\text{chemical in}1\mathrm{L}\left(\mathrm{gas}\right)$$

The reported inhalation ppm LC50 values, the inhalation ppm LC50 values converted to mg/L, and the inhalation LC50 values reported as mg/L are shown in Table 5, along with the duration of exposure. The values were then converted to LC50 values in mg/L for a 4‐h exposure period. For this conversion, it was assumed that the LC50 values were proportional to the exposure period.

TABLE 5.

Comparison of inhalation LD50 values (mg/L) of the tested chemicals with the TIPS values converted to mg/L.

Compound name Solubility in water	LC50 (ppm) inhalation a	MW	LC50 (mg/L) inhalation a , 1	Duration (h) a	LC50 (mg/L) inhalation 4 h	Convert TIPS LD50 (from Table 3) to LC50 (mg/L) 2
1,4‐Dioxane	14,261	88.11	51.4	4	51.4	39.8
Miscible with water	—	—	46.0	2	23.0
Glycidol (2,3‐epoxy‐1‐propanol)	580	74.08	1.8	8	3.5	4.84
Miscible with water	—	—	1.26	4	1.26
N,N‐Dimethylformamide	3421	73.09	10.2	1	2.6	30.6
Miscible with water	3421	73.09	10.2	3	7.7
	1948	73.09	5.8	4	5.8
	—	—	> 5.9	4	> 5.9
N,N‐Dimethylacetamide	2475	87.12	8.8	1	2.2	30.6
Miscible with water	—	—	8.8	1	2.2
	—	—	10.1	1	2.5
	—	—	1.47	7	2.6
T‐Butanol	14,100	74.12	42.7	4	42.7	18.9
Miscible with water	> 10,000	74.12	> 30.3	4	> 30.3
	10,000	74.12	30.3	4	30.3
Acrolein	8	56.06	0.018	4	0.018	0.025
Dissolved in water	131	56.06	0.300	0.5	0.038
Solution is miscible with water.	—	—	0.018	4	0.018
Polyacrylic acid polymer			2.17	4	2.17	3.94
Dissolved in saline. Saline solution is miscible with water.			57.1	4	57.1
2‐Ethoxyethyl acetate	> 1500	132.16	> 8.1	8	> 16.2	20.7
Solubility in water: 187 g/L	—	—	12.1	8	24.2
Acetylacetone (2,4‐pentanedione)	1224	100.12	5.0	4	5.0	12.1
Solubility in water: 166 g/L	—	—	5.1	4	5.1
1,2‐Dichloroethane	1000	98.96	4.0	8	8.1	6.25
Solubility in water: 8.6 g/L	1000	98.96	4.0	7	7.1
	—	—	5.1	6	7.7
	—	—	4.1	7.2	7.4
	—	—	49.4	0.5	6.2
	1650	98.96	6.67	6	10.0
	—	—	6.67	6	10.0
	—	—	8.0	4	8.0
	1900	98.96	7.7	4	7.7
Glycidyl methacrylate	45	142.15	0.26	4	0.26	11.1
Solubility in water: 5–10 g/L	—	—	> 2.394	4	> 2.394
Mixed xylenes b	6350	106.16	27.6	4	27.6	7.29
Solubility in water: 106 mg/L	6700	106.16	29.1	4	29.1
p‐Xylene b	4740	106.16	20.6	4	20.6
Solubility in water: 162 mg/L	4550	106.16	19.8	4	19.8
o‐Xylene b	4330	106.16	18.8	6	28.2
Solubility in water: 178 mg/L	> 6125	106.16	> 26.6	12	> 79.8
m‐Xylene b	5984	106.16	26.0	6	39.0
Solubility in water: 161 mg/L	> 8000	106.16	34.7	4	> 34.7

^a The reported LC50 values in ppm and mg/L, the duration of exposure, and the sources from which these values were obtained are shown in Table 4.

¹ ppm values were converted to mg/L as described in Section 2.

² TIPS mg/kg values were converted to mg/L as described in Section 2.

^b The solubility of mixed xylenes and the xylene isomers is from Toxicological Profile for Xylene Table 4‐2: available at <https://www.ncbi.nlm.nih.gov/books/NBK600783/table/ch4.tab2/>.

The TIPS LD50s in mg/kg (Table 3) were converted to corresponding LC50s in mg/L using a minute ventilation rate of 54 mL/min for 12‐week‐old male Fisher 344 rats (Derelanko and Auletta 2014: Table 6.36). Therefore, the volume of air in liters inhaled by a 12‐week‐old rat (average body weight 300 g) in 4 h is 0.054 L/min × 240 min:

$$\mathrm{LC}50\text{in}\mathrm{mg}/\mathrm{L}=\left(\mathrm{LD}50\text{in}\mathrm{mg}/\mathrm{kg}\times 0.3\mathrm{kg}\right)/\left(0.054\mathrm{L}/\min \times 240\min \right)$$

Table 5 shows the inhalation LC50 values in mg/L alongside the converted TIPS LC50 values.

3. Results

3.1. In Vitro Neutral Red Toxicity Test

A549 cells were exposed to the test chemicals in both RPMI and RPMI + 10% FBS. The in vitro LD50s (μL/mL) of the 12 test chemicals are shown in Table 2. The presence of FBS had little or no effect on the LD50s. The in vitro LD50s were used to identify a range of doses that would likely include the LD50s of the in vivo TIPS administration experiments. The amount of test chemical that was projected to result in rat survival rates of 100% was one‐half of the lower in vitro LD50, and the amount of test chemical that was projected to result in rat survival rates of 0% was the higher in vitro LD50 ×4 (Table 2). These values were converted to mg/kg TIPS doses (single administration) for a 300‐g rat as described in Section 2.4. These TIPS doses (single administrations) are shown in Table 2.

3.2. In Vivo TIPS Toxicity Test

In vitro LD50s were converted to TIPS doses in mg/kg (single administration) that were predicted to result in 0% rat death and 100% rat death as described above (Table 2). The test chemicals were administered in vivo using TIPS. Rats were administered the vehicle control and test chemical four times over the course of 3 h (0, 1, 2, and 3 h). After administration, the rats were observed for 2 weeks to detect the acute toxic effects of the instilled chemicals and to observe survival rates among the experimental animals. Table 3 shows the total TIPS doses derived from the in vitro LD50s projected to result in rat survival rates of 0% and 100%: This range of doses, which is hypothesized to include the in vivo LD50, is the total dose administered by four TIPS administrations. Table 3 also shows the doses of test chemicals that were administered to the rats, the survival rate of the rats, and the TIPS LD50 (mg/kg) values obtained from the survival curves.

1,4‐Dioxane was the initial chemical tested. Rats were administered five doses of 1,4‐dioxane. In the next set of experiments, rats were administered four doses of 2‐ethoxyethyl acetate, acetylacetone, 1,2‐dichloroethane, glycidyl methacrylate, and xylene. For glycidyl methacrylate, the initial range of doses did not include the in vivo LD50. Therefore, a second series of rats was administered 0‐, 120‐, 240‐, 480‐, and 960‐mg/kg glycidyl methacrylate (shown in Table 3). In the final set of experiments, rats were administered three doses of glycidol, N,N‐dimethylformamide, N,N‐dimethylacetamide, polyacrylic acid, acrolein, and T‐butanol in multiples of 2 or 4. The initial range of doses administered did not include the LD50s of glycidol or T‐butanol. Therefore, a second series of rats was administered 0‐, 160‐, 320‐, and 640‐mg/kg glycidol (shown in Table 3). For T‐butanol, the dose responses of the other chemicals strongly suggested that the next higher dose of T‐butanol would result in 100% lethality. Therefore, for the purposes of the present study, a second set of rats was not used, and the LD50 was derived from the initial set of rats administered T‐butanol.

3.3. Comparison of Reported 4‐h Inhalation LC50 Values vs. TIPS‐Derived LC50 Values

The acute inhalation LC50 values of the test chemicals are shown in Table 4. The ppm values were converted to mg/L as described in Section 2.6, and LC50s derived from exposures other than 4 h were converted to 4‐h LC50s (mg/L) (Table 5). The TIPS LD50 (mg/kg) values shown in Table 3 were converted to LC50 (mg/L) values as described in Section 2.6 and are also shown in Table 5. Comparison of the inhalation LC50 values and the converted TIPS LC50 values shown in Table 5 indicates that the inhalation LC50 mg/L values and the converted TIPS LC50 mg/L values are similar in nine of the 12 chemicals tested. Two water‐soluble chemicals, N,N‐dimethylformamide and N,N‐dimethylacetamide, had TIPS LD50 values that were significantly greater than the inhalation LC50 values, and the single water‐insoluble chemical tested, xylene, had a TIPS LD50 value that was lower than the inhalation LC50 value.

3.4. Comparison of Reported 4‐h Inhalation LC50 Values vs. In Vitro Derived LC50 Values

The in vitro LD50s were converted to mg/kg for a 300‐g rat as described in Section 2.4. These values are shown in Table 2. These mg/kg values (single doses) were multiplied by four to account for the four doses administered and converted to mg/L for a 300‐g rat using the same procedure for converting in vivo TIPS LD50s to mg/L (Section 2.6). The four dose mg/L values for the in vitro LD50s are shown in Table 6, alongside the inhalation 4‐h LC50 mg/L values for ease of comparison. Eight chemicals, 1,4‐dioxame, 2‐ethoxyethyl acetate, acetylacetone, 1,2‐dichloroethane, glycidyl methacrylate, acrolein, polyacrylic acid polymers, and tert‐butanol, had LD50 concentrations that, when converted to TIPS LC50 mg/L values, were within 2.5‐fold of reported inhalation LC50 mg/L values.

TABLE 6.

Comparison of 4‐h inhalation LD50 values (mg/L) of the tested chemicals with the in vitro LD50 values converted to mg/L.

Compound name Solubility in water	LC50 (mg/L) inhalation 4 h	LD50 (mg/L) (RPMI) 2	LD50 (mg/L) (RPMI + 10% FBS) 2
1,4‐Dioxane	51.4	28.20	28.58
Miscible with water	23.0
Glycidol (2,3‐epoxy‐1‐propanol)	3.5	17.07	15.52
Miscible with water	1.26
N,N‐Dimethylformamide	2.6	34.73	33.41
Miscible with water	7.7
	5.8
	> 5.9
N,N‐Dimethylacetamide	2.2	34.49	33.09
Miscible with water	2.2
	2.5
	2.6
T‐Butanol	42.7	13.53	13.55
Miscible with water	> 30.3
	30.3
Acrolein	0.018	0.01	0.01
Dissolved in water	0.038
Solution is miscible with water.	0.018
Polyacrylic acid polymer	2.17	2.17	2.19
Dissolved in saline. Saline solution is miscible with water.	57.1
2‐Ethoxyethyl acetate	> 16.2	13.20	13.34
Solubility in water: 187 g/L	24.2
Acetylacetone (2,4‐pentanedione)	5.0	6.61	6.01
Solubility in water: 166 g/L	5.1
1,2‐Dichloroethane	8.1	2.18	2.72
Solubility in water: 8.6 g/L	7.1
	7.7
	7.4
	6.2
	10.0
	10.0
	8.0
	7.7
Glycidyl methacrylate	0.26	1.33	1.39
Solubility in water: 5–10 g/L	> 2.394
Mixed xylenes a	27.6	6.89	7.37
Solubility in water: 106 mg/L	29.1
p‐Xylene a	20.6
Solubility in water: 162 mg/L	19.8
o‐Xylene a	28.2
Solubility in water: 178 mg/L	> 79.8
m‐Xylene a	39.0
Solubility in water: 161 mg/L	> 34.7

¹ The LC50 (mg/L) inhalation 4‐h values are from Table 5.

² In vitro μL/mL LD50 values in RPMI and RPMI + 10% FBS shown in Table 2 were converted to mg/kg as described in Section 2. These mg/kg values were converted to mg/L as described in Section 2 for the conversion of TIPS mg/kg values to mg/L.

^a The solubility of mixed xylenes and the xylene isomers is from Toxicological Profile for Xylene Table 4‐2: available at <https://www.ncbi.nlm.nih.gov/books/NBK600783/table/ch4.tab2/>.

4. Discussion

The present study is an initial proof‐of‐concept study. Twelve chemicals were tested to investigate the possibility of using intratracheal instillation to determine the acute inhalation toxicity of volatile chemicals. Since the TIPS vehicle in this study was saline, we concentrated on chemicals and chemical solutions provided by FUJIFILM Wako Chemicals that are miscible in water. As shown in Table 2 (and also in Tables 3, 5, and 6), seven of the 12 test chemicals or their solutions were miscible in water. Two chemicals had water solubilities of 187 and 166 g/L, two chemicals had water solubilities of 5–10 g/L, and one chemical was not soluble in water.

In vitro exposure of A549 lung carcinoma cells to increasing amounts of the test chemical was used to determine a dosage range that would likely include the LD50 in mg/kg of the chemical administered using TIPS. As shown in Table 3, for 10 of the 12 chemicals, the dose range included the LD50 in mg/kg of the test chemical administered using TIPS. The two chemicals for which the preliminary in vitro exposure of A549 cells did not include the LD50 in mg/kg of the test chemicals were glycidyl methacrylate and glycidol (Table 3).

For glycidyl methacrylate, the dose determined by the A549 in vitro assay predicted to result in 100% lethality of the rats administered the chemical by TIPS was 240 mg/kg (Table 3). However, administration of this dose by TIPS did not result in the death of any of the rats. This indicates that the in vivo toxicity of glycidyl methacrylate is much lower than predicted by the in vitro assay described in this study. Notably, as discussed below, the inhalation LC50 (mg/L) of glycidyl methacrylate and the LC50 (mg/L) of glycidyl methacrylate determined by TIPS were comparable.

The other test chemical for which the A549 in vitro assay did not predict the range of doses that included the LD50 of the chemical administered using TIPS was glycidol. For glycidol, the dose determined by the A549 in vitro assay predicted to result in 0% lethality of the rats administered the chemical by TIPS was 335 mg/kg (Table 3). However, administration of this dose by TIPS resulted in 100% lethality of the rats. This indicates that the in vivo toxicity of glycidol is much greater than predicted by the in vitro assay described in this study. Notably, the inhalation LC50 (mg/L) of glycidol and the LC50 (mg/L) of glycidol determined by TIPS were comparable (Table 5).

For chemicals such as glycidol, if the lowest amount of the chemical identified by the in vitro assay was more lethal than expected in vivo, a small second set of animals could be administered a few lower doses of the test chemical. Similarly, for chemicals such as glycidyl methacrylate, if the highest amount of the chemical identified by the in vitro assay was less lethal than expected in vivo, a small second set of animals could be administered a few higher doses of the test chemical. In addition, in order to lessen animal testing, we propose that animals surviving acute inhalation studies could continue to be monitored for up to 2 years for use in chronic toxicity/carcinogenicity evaluation.

One possible reason for the lower toxicity of glycidyl methacrylate in vivo compared with in vitro is that the solubility of glycidyl methacrylate in physiological saline (the TIPS administration vehicle) affects its lethality in vivo. However, the solubility of 1,2‐dichloroethane in water is approximately the same as that of glycidyl methacrylate (see Table 3), suggesting that a factor other than solubility in water affected the lethal dose of glycidyl methacrylate in vivo. It is possible that modifications of the in vitro assay may lead to assays that would allow better determination of the lowest and highest doses to be used in vivo. Currently, Vachiraarunwong et al. are developing an in vitro system designed to help assess acute inhalation toxicity (Vachiraarunwong et al. 2025).

Table 4 shows the reported LC50 ppm and mg/L values for acute inhalation toxicity studies with rats: ppm values were converted to mg/L as described in Section 2.6, and LC50s derived from exposures other than 4 h were converted to 4‐h LC50s (shown in Table 5). Table 3 shows the LD50 (mg/kg) values of each compound administered by the TIPS technique calculated using nonlinear regression analysis. These values were converted to LC50 mg/L values as described in Section 2.6, and the TIPS LC50 mg/L values are shown in Table 5. Eight compounds have converted TIPS LC50 mg/L values that are very similar to inhalation LC50 (4 h) values, that is, different from the inhalation LC50 values by 2.5‐fold or less: Notably, the inhalation LC50 (4 h) values of four compounds (glycidyl methacrylate, glycidol, N,N‐dimethylformamide, and polyacrylic acid polymer) also differed by more than 2.5‐fold between inhalation studies. As shown in Table 5, the TIPS LD50 of glycidyl methacrylate (11.1 mg/L) was more than 40‐fold higher than the LC50 reported in one study (45 ppm = 0.26 mg/L). However, another study reported that no mortality was observed in rats exposed to 2.394‐mg/L glycidyl methacrylate for 4 h (Table 4), indicating that the LC50 was greater, possibly much greater, than 2.394 mg/L. This indicates that for glycidyl methacrylate, the converted TIPS LC50 (4 h) and the inhalation LC50 (4 h) values will very likely be comparable once the acute inhalation toxicity LC50 of glycidyl methacrylate has been determined.

Of the three chemicals with TIPS LC50 mg/L values that differed by more than 2.5‐fold from the 4‐h inhalation LC50 mg/L values, two were higher than the inhalation values. The TIPS LC50 value of N,N‐dimethylformamide was approximately sixfold higher than the inhalation LC50 (4 h) values, and the TIPS LC50 (4 h) value of N,N‐dimethylacetamide was more than 10‐fold higher than the inhalation values (Table 5). This indicates that the toxicity of these chemicals when administered by TIPS was much lower than the toxicity of these chemicals administered by inhalation. Both of these chemicals are miscible with water; therefore, the reason for the low toxicity of these chemicals does not appear to be related to their solubility in saline (the TIPS administration vehicle). It is notable that the TIPS LD50 mg/L of 1,4‐dioxane, glycidol, acrolein, and T‐butanol, which are also miscible with water, agree well with the inhalation LD50 (4 h) values. Currently, it can be surmised that the toxicity of aqueous 1,2‐dimethylformamide and 1,2‐dimethylacetamide is lower than that of their gaseous vapors. This possibility needs to be investigated, and if found to be correct, a TIPS vehicle that does not affect the toxicity of the test chemical needs to be determined.

The other chemical that differed from the inhalation LC50 (4 h) value is xylene (Table 5). Xylene had a lower TIPS LC50 (4 h) value compared with the inhalation LC50 (4 h) values. This indicates that xylene administered by TIPS was more toxic than xylene administered by inhalation exposure. One factor is that the actual absorption rate of chemical substances into the fluid lining the lung epithelia is expected to vary depending on environmental factors and individual differences. For the purposes of the present study, when comparing inhalation LC50 values with converted TIPS LC50 values, a 100% absorption rate was assumed, and this assumption may need to be modified. Thus, when administered by TIPS, a higher level of xylene may have been present in the TIPS solution compared with the level of xylene that was absorbed in the fluid lining the lung epithelia during inhalation exposure. This would have resulted in increased interaction of xylene with lung epithelial cells in rats administered xylene by TIPS, resulting in increased toxicity.

Although the primary focus of the present study was to investigate the possibility of using TIPS to determine the acute inhalation toxicity of volatile chemicals, it is notable that the conversion of the in vitro LD50s to LC50 mg/L values identified eight chemicals with converted LC50s that differed from reported acute inhalation LC50s by 2.5‐fold or less (Table 6). This suggests that further studies on the use of in vitro experiments in the assessment of acute inhalation toxicity are warranted. As noted above, Vachiraarunwong et al. are developing an in vitro system designed to help assess acute inhalation toxicity (Vachiraarunwong et al. 2025).

5. Summary and Future Directions

The present study is an initial proof‐of‐concept study. Consequently, only 12 volatile chemicals were assessed. The strong agreement between TIPS LC50 values and the published inhalation LC50 values of nine of the 12 tested chemicals suggests that TIPS has the potential to be used to determine the acute inhalation toxicity of volatile chemicals.

Acute inhalation toxicity testing of volatile chemicals requires specialized equipment (see Annexes 1, 2, and 3 in OECD ²⁰⁰⁹). Consequently, only a limited number of facilities are able to carry out such studies. In contrast, the instillation of test materials into the lung can be carried out by essentially all institutions with toxicology research facilities. Thus, the ease of administration using intratracheal instillation and the small amount of material that needs to be used would significantly increase the number of institutions able to carry out acute inhalation toxicity testing. This is a major incentive for developing intratracheal instillation as a means of determining the acute inhalation toxicity of volatile chemicals.

Further studies using a wide range of volatile chemicals are being planned. In addition, studies to identify suitable solvents and solvents that are not suitable for different types of chemicals need to be undertaken. Also, future studies will include the possibility that the gender and age of the rats used to test the acute inhalation toxicity of chemicals may affect the results.

Finally, further studies on the use of in vitro experiments in the assessment of acute inhalation toxicity are warranted. Importantly, in vitro experiments have the potential to identify solvents suitable for TIPS and identify chemicals that are suitable and chemicals that are not suitable for the determination of acute inhalation toxicity using TIPS.

Conflicts of Interest

The authors declare no conflicts of interest.

Saleh, D. , Ahmed O., Alexander D., et al. 2026. “Use of the TIPS Technique as a Possible Method of Acute Inhalation Toxicity Assessment.” Journal of Applied Toxicology 46, no. 4: 1232–1245. 10.1002/jat.4940.

Data Availability Statement

The data that support the findings of this study are available from the corresponding authors upon reasonable request.