Better GRAS than sorry: Excipient toxicity in pulmonary formulations

Abstract

Similar to other routes of administration, pulmonary formulations typically contain excipients. The panel of substances that can be used in pulmonary formulations is limited compared to other routes of administration, and most of them have the generally regarded as safe (GRAS) status. New excipients must undergo in vitro and complete in vivo testing to be approved by regulatory authorities. The toxic effects induced by excipients contained in the most common pulmonary formulations (dry powder inhalers, pressurized metered dose inhalers, and nebulizers) were summarized to compare the cytotoxic effects to the approved concentrations. The hypothesis was that a concentration much higher than the approved one could serve as an indicator of the safety of the compound. While the cytotoxic concentration of many excipients was higher than the approved concentration, the opposite was found for some of them. For most of these compounds, adverse lung effects were reported with long-term administration or in specific populations. This suggests that concentrations lower than the cytotoxic threshold are unlikely to cause adverse respiratory effects. Combining excipients can increase toxic effects, but few studies address this. Since neither cytotoxicity testing nor animal testing can reliably identify toxic concentrations of excipients, additional in vitro tests have been proposed.

1. Introduction

Pharmaceutical formulations contain not only the active pharmaceutical ingredient (API), but usually also excipients, which are all ingredients other than the API [1]. They can be fillers, extenders, absorption enhancers, diluents, wetting agents, solvents, emulsifiers, preservatives, flavors, sustained release matrices and colorants. In general, the proportion of excipients (70–75 %) is higher than that of the API (25–30 %). For the treatment of respiratory diseases, oral inhalation is the preferred route of administration because high local concentrations can be achieved with low systemic effects [2]. In pulmonary formulations, excipients typically represent 70–90 % in dry powder inhalers (DPIs) and 90 % in pressurized metered dose inhalers (pMDIs) and nebulizers. Excipient levels are usually indicated as v/v or w/v in pMPIs, w/w in DPIs and as w/v in nebulizers. Excipients are not only important for formulation but also determine toxicity. The respiratory barrier is highly permeable to small hydrophobic molecules, making the pulmonary route a promising administration method [3]. To minimize the toxicity risk, the Food and Drug Administration (FDA) recommends using only molecules that are generally recognized as safe (GRAS) for pulmonary formulations. This definition is based on use in food and may not apply to inhaled products. For example, vitamin E, which has GRAS status, was reported to have toxic effects on the lungs in vaporized e-liquids [4]. Existing formulations and FDA-approved concentrations provide insight into the toxic potential of excipients. To decide if increasing the concentration of a given excipient in a pulmonary formulation will result in adverse effects, it is helpful to have cytotoxicity data. It is hypothesized that a large difference between the cytotoxic concentration and the concentration in the formulation indicates that the formulation will not cause cell damage.

To illustrate the different toxicological potential of excipients, the text lists adverse effects on (preferentially airway) cells and in vivo inhalation toxicity data of the excipients contained in the three most commonly used inhalers, pMDIs, DPIs and nebulizers. The less commonly used soft-mist inhalers contain water, citric acid, ethylenediaminetetraacetic acid (EDTA), sodium chloride (NaCl) and benzalkonium chloride (BAC), which are also present in the other inhalers [5].

2. Function and use of excipients in different pulmonary formulations

Excipients should ensure the safety and efficacy of the drug during formulation, storage, and during and after administration [6]. Databases for excipients are provided by the FDA and the Japanese Pharmaceutical Excipients Dictionary. Countries in Europe do not have common lists, but country-specific lists such as “Die Rote Liste” in Germany, “Dictionnaire Vidal” in France, and “Electronic Medicines Compendium” in the United Kingdom. The Excipient Biological Evaluation Guidelines provide information on what testing is required or conditionally recommended for new excipients. Although not a rare adverse effect of excipients, pulmonary sensitization is only conditionally recommended for inhaled formulations.

Predicting the toxicity of excipients in humans is difficult for several reasons. Impurities may be present, excipients may degrade or interact with other molecules in the formulation, and toxicity may be route-specific. Cumulative exposure is difficult to assess, and limited animal data are available. Additionally, animal data may be misleading due to specific differences, and vulnerable groups exist in the human population.

3. Potential local adverse effects of excipients (cytotoxicity and irritation)

The FDA guidance on Nonclinical Studies for the Safety Evaluation of Pharmaceutical Excipients [7] recommends that International Council of Harmonization (ICH) guidelines, specifically ICH Guideline for Industry S7A Safety Pharmacology Studies for Human Pharmaceuticals, be used to evaluate acute, repeated dose, and chronic toxicity. The studies should demonstrate local and systemic effects in animals. In addition, genotoxicity, reproductive and developmental toxicity, carcinogenicity, and immunotoxicity should be evaluated. Studies should be conducted in two different species.

Excipients in pulmonary formulations may cause effects on the lining epithelium of the respiratory tract. These effects include induction of apoptosis, plasma membrane damage (necrosis), and inhibition of proliferation. These processes are visible as a decrease in cell viability. These changes can, in principle, be evaluated in any cells. However, usually cell lines representing the respiratory tract, e.g. BEAS-2B, Calu-3 and A549 cells, are used [8]. The predictive value of toxicity recorded in these cells for pulmonary toxicity in humans is reported differently. The predictive value of human pulmonary toxicity was high when cell viability, A549 cell transcriptomics, and BEAS-2B phenotypic markers were used together (Huang, 2023, #7243; Lee, 2018, #7245). However, respiratory toxicity in rats performed no better than A549 cells and reconstructed human tissues for prediction (Sauer, 2013, #6000). However, rat data may not be the most relevant because the predictive value of rodent toxicity studies for human toxicity is moderate (43 %) (Olson, 2000, #7246). Another limitation is interlaboratory variation. For viability studies, differences exist in cell batches and passages. For animal studies, genetic differences and environmental factors are present (Hughes, 2007; Witjes, 2020). In both types of experiments, exposure duration greatly impacts the toxic effect.

Irritation refers to effects that do not necessarily lead to cell damage, such as release of inflammatory markers, changes in permeability and functional changes (e.g. changes in ciliary beating frequency (CBF) and airway hyperreactivity). These changes, as well as genotoxicity, can be assessed by in vitro and in vivo exposures. Animal studies are required to assess complex effects such as sensitization and pulmonary changes beyond the epithelial layer (fibrosis, vascular dysfunction, and systemic toxicity) [9].

Download the full article as PDF here Better GRAS than sorry

or read it here

Table 1. Overview of excipient discussed in this review with indication of main function, preferential use in which type of formulation and at which concentration, and concentrations of reported cytotoxic effects and toxic effects in vivo.

Excipient	Main function in the formulation	Preferential use	Regulatory acceptance [1]	Decrease of viability in vitro / adverse effects in vivo effects	Reference
Citric acid	Stabilizer, pH adjustment, flavoring agent	pMDI nebulizer	0.13 %	0.23–6.5 %/-	[2,3]
Ethanol (alcohol)	Co-solvent	pMDI	5 %	5 %/-	[4]
Glycerin	Co-solvent, tonicity agent, preservative, humectant	Nebulizer	5 %	2–4 %/irritation	[5]/[5]
Propylene glycol	Co-solvent, preservative, humectant	Nebulizer	25 %	2 %/cellular changes	[6]/[7]
Hydrochloric acid	pH adjustment	Nebulizer	0.7 %	0.03 %/-	[61]
Sodium hydroxide	pH adjustment	Nebulizer	8 %	−/epithelial necrosis	[8]
Glycine	pH adjustment, tonicity agent	Nebulizer	2 mg MDE	none	[9]
Benzalconium chloride	Preservative	Nebulizer	0.02 %	0.004 %/-	[10]
Methyl- and propylparaben	Preservative	Nebulizer	0.03 % and 0.01 %	−/disruption of ciliary function	[11]
Thymol	Preservative	Nebulizer	0.01 %	0.02 %/none	[12]/ [13]
Benzyl alcohol	Preservative, co-solvent	Nebulizer	Nasal: 0.5 %	−/irritation	[14]
EDTA	Chelating agent, preservative	Nebulizer	no	0.07 %/-	[15]
Ascorbic acid	Antioxidant, pH adjustment	Nebulizer	0.04–0.11 %	0.088 %/-	[80]
Sodium bisulfite	Antioxidant, preservative	Nebulizer	0.3 %	−/pulmonary edema	[16]
Sodium metabisulfite	Antioxidant, preservative	Nebulizer	0.3 %	0.047 %/cell damage	[17]/ [18]
Soy lecithin	Surfactant	pMDI	0.0002 %	>100 µg/ml/-	[19]
Poloxamer 188	Surfactant	Nebulizer	Oral: 3 mg	>10 %/-	[20]
Polyvinyl alcohol	Surfactant, stabilizer	DPI nebulizer	Oral: 1.67 mg	1 %/-	[21]
Phosphatidylcholine	Surfactant	DPI	51 mg MDE	−/-
Polysorbate 20, polysorbate 80	Surfactant	Nebulizer	Polysorbate 80 MDE 1 mg	0.03 %/none	[22]/ [23]
Polyvinylpyrrolidone	Surfactant	DPI	Oral: 5–6374 mg MDE	8–20 %/-	[19]/ [24]
Polyethylene glycol	Surfactant, co-solvent, humectant	DPI nebulizer	Oral: 1 %	2.25 %/-	[25]
Oleic acid	Surfactant	pMDI	0.01 %	>0.00047 %/-	[26]
Menthol	Flavoring agent	pMDI DPI nebulizer	0.02	0.02–1.5 %/irritation	[27,28]/[29]
Saccharin	Flavoring agent	pMDI nebulizer	0.05 %	1.3 %/-	[30]
Hydroxypropyl β-cyclodextrin	Absorption enhancer	DPI nebulizer	no	6.6 %/-	[31]
Sodium taurocholate Sodium deoxycholate	Absorption enhancer	DPI	no Injection: 3 mg no	−/- 0.0012 %/inflammation	[32]/ [33]
Leucine	Surface modifier, surfactant	DPI	Injection: 1.1 %	−/-
Trileucine	Surface modifier, surfactant	DPI	no	−/-
Magnesium stearate	Surface modifier, surfactant	DPI	0.13 mg	0.001 %/irritation	[34]/[35]
Sucrose tristearate	Surface modifier, surfactant	DPI nebulizer	no	−/-
Fumaryldiketopiperazine	Surface modifier	DPI	no	−/-
Poly(lactic glycolic acid)	Polymer	DPI	no	>0.03 %/irritation	[36]/ [37]
Polycaprolactone	Polymer	DPI	no	0.1 %/-	[38]
Hydroxypropylmethylcellulose	Polymer	DPI nebulizer	no	−/-
Dipalmitoylphosphatidylcholine	Lipid	DPI nebulizer	Epidural: 2 mg MDE	−/impaired gas exchange	[39]
Distearoylphosphatidylcholine	Lipid	DPI	51 mg, MDE	−/impaired gas exchange	[39]
Dimyristoylphosphatidylcholine	Lipid	DPI nebulizer	no	−/impaired gas exchange	[39]
Cholesterol	Lipid	DPI nebulizer	i.v.: 0.32 %	−/impaired gas exchange	[40]
Lactose	Carrier	DPI	90 mg	17.6 %/-	[41]
Mannitol	Carrier, tonicity agent	DPI nebulizer	6 mg, MDE	−/bronchial hyperreactivity	[42]
Trehalose	Carrier, stabilizer	DPI	s.c.: 22 mg MDE	10.2 %	[43]
Sorbitol	Carrier, stabilizer, humectant, tonicity agent	Nebulizer	Oral: 60 %	−/irritation	[44]
Xylitol	Carrier, stabilizer, humectant, tonicity agent	Nebulizer	Oral: 21.6 g MDE	−/none	[45]
Bovine serum albumin	Carrier, stabilizer	DPI nebulizer	no	−/asthma	[46]
Carboxylmethylcellulose	Suspending agent	Nebulizer	Oral: 1 %	−/irritation	[47]
NaCl	Tonicity agent	Nebulizer	3 mg	2.8 %/-	[19]/[48]
Sodium sulfate	Tonicity agent	Nebulizer	0.03 %	−/>0.5 %	[48]

Eleonore Fröhlich, Better GRAS than sorry: Excipient toxicity in pulmonary formulations, European Journal of Pharmaceutics and Biopharmaceutics, 2025, 114814, ISSN 0939-6411, https://doi.org/10.1016/j.ejpb.2025.114814.