Comparison of Preservatives Used in Various Therapeutic Applications

Considerations for use of preservatives in topical, oral, ophthalmic, transmucosal, injectable, inhalation, and otic applications

A variety of preservatives can be used in therapeutic applications such as topical, oral, transmucosal, inhalation, and otic, as well as injectables. These excipients help to increase the shelf life of products and prevent microbial growth. When selecting a preservative for formulation, a number of factors should be considered as each has its own advantages and disadvantages depending on the application.

This article outlines considerations when using the following excipients as preservatives:

• Benzalkonium chloride
• Benzyl alcohol
• Parabens
• Benzoic acid and sodium benzoate

Benzalkonium Chloride

Considerations for Use of Benzalkonium Chloride as a Preservative

Benzalkonium chloride is widely used as a preservative due to its broad anti-microbial and pH range activity. Most frequent applications include ophthalmics and parenterals, but it is also very commonly applied in topical and transmucosal formulations.

Table 1. Applications of benzalkonium chloride based on FDA Inactive Ingredients Database information.

Benzalkonium Chloride Applications: General Overview
Inhalation	•
Nasal	•
Ophthalmic	•
Oral
Oromucosal
Otic	•
Parenteral	•
Rectal
Topical & transdermal	•
Vaginal

Benzalkonium chloride is not a single moiety; it is available as a combination of C₁₂ and C₁₄ carbon chains. For higher anti-microbial efficacy, the pharmacopeias recommend ≥ 40% w/w C₁₂ and ≥ 20% w/w C₁₄ homologues. High batch-to-batch consistency of benzalkonium chloride is essential for reproducible preservative efficacy activity and can vary among suppliers.

Summary of Benzalkonium Chloride Attributes

Table 2 summarizes several attributes of benzalkonium chloride.

Synergy with Other Preservatives

Benzalkonium chloride can be used in combination with EDTA and other preservatives including benzyl alcohol and boric acid. Benzalkonium chloride plus sodium dodecyl sulfate (SDS) has been shown to attenuate the irritation potential of SDS in transdermal patches.

Incompatibilities of Benzalkonium Chloride

Because benzalkonium chloride is cationic in nature, it is normally incompatible with other cationic surfactants and phospholipids. Benzalkonium chloride is adsorbed onto hydroxypropyl methylcellulose (HPMC) and thus presents challenges for preservative efficacy assays.

It is also incompatible with acidic active pharmaceutical ingredients (APIs) because it causes precipitation. Basic amino acids such as histidine, arginine, lysine, as well as tromethamine or meglumine, can be used with acidic APIs to form a complex with API and enable benzalkonium chloride to be used for preservative action.

Microbial Activity of Benzalkonium Chloride

Benzalkonium chloride offers microbial activity against yeast, bacteria, and mold. It is ineffective against some strains of pseudomonas aeruginosa but a combination of benzalkonium chloride with EDTA can increase efficacy.

Safety

Benzalkonium chloride should not be used in inhalation products such as those for asthma as it can induce bronchospasms.

Table 2. Synergies, incompatibilities, and microbial activity of benzalkonium chloride.

Synergy	EDTA, benzyl alcohol, 2-phenylethanol or 3-phenylpropanol, boric acid1
	Benzalkonium chloride plus sodium dodecyl sulfate (SDS) attenuates the irritation potential of SDS in transdermal patches2
Incompatibility	Phospholipids3
	Cationic and non-ionic surfactant1
	Adsorb on HPMC, kaolin, lanolin1
	Eye drops containing local anaesthetics4
	Soaps and other anionic surfactants, citrates, iodides, nitrates, permanganates, salicylates, silver salts, tartrates, zinc oxides and sulfates1
	Insoluble precipitate with acidic APIs. Recommended to use basic amino acids like histidine, arginine, lysine, tromethamine or meglumine in case of acidic APIs to form complex with API and allow benzalkonium chloride for preservative action5
Microbial activity	Higher biocide activity for C12 and C14 derivatives5
	Yeast, bacteria and molds5
	Ineffective against some strains of Pseudomonas aeruginosa5
Safety	Not for use in inhalation products targeting asthma patients due to the risk of bronchospasm7, 8

Compatibility of Benzalkonium Chloride with Membrane Filters

Many preservatives bind or are adsorbed by membrane filters which causes a loss of the appropriate amount of preservatives in the final product. Benzalkonium chloride has this tendency which affects the preservative assay, yield of the process, and increases the flush volume. To avoid this problem, a polyethylenesulfone (PES) filter should be used. As shown in Figure 2, test results with a 0.01 % benzalkonium chloride solution showed the lowest binding and lowest preservative loss in the final product. For most competitive membranes, product losses were more than double that of the Millipore Express^® SHF membrane.

Figure 2. Benzalkonium chloride concentration in filtrate when using PES filters.

Pharmacopeia Compliance

Impurities in benzalkonium chloride such as benzyl chloride can react with the API. Use of a compendial grade of benzalkonium chloride where the limit of impurities is less than 0.05 % reduces the risk of interactions with the API. 

Benzyl Alcohol

Considerations for Use of Benzyl Alcohol as a Preservative 

Benzyl alcohol is a preservative used most frequently in injections, but also in topical, oral, transmucosal, and otic applications. It is also used as a solubilizer. Because of its anesthetic properties, it is sometimes used to reduce pain on injection or discomfort of the patient, e.g. when used in cough syrups or ophthalmics.⁸ While this preservative has numerous applications, it should not be used for neonates and used with caution in children older than four weeks.⁹

Table 3. Applications of benzyl alcohol based on FDA Inactive Ingredients Database information.

Benzyl Alcohol Applications: General Overview
Inhalation
Nasal	•
Ophthalmic
Oral	•
Oromucosal
Otic	•
Parenteral	•
Rectal	•
Topical & transdermal	•
Vaginal	•

Summary of Benzyl Alcohol Attributes

Table 4 summarizes several attributes of benzyl chloride.

Synergy with Other Preservatives

Benzyl alcohol can be used with parabens, potassium sorbate, BHT, EDTA, and benzyl benzoate. It can also be used as a solvent or cosolvent system for poorly soluble drugs.

Incompatibilities of Benzyl Alcohol

Benzyl alcohol is incompatible with methylcellulose and can be adsorbed by the rubber closures used in drug containers. Given this risk, rubber closures and stoppers should be coated with fluorinated polymers if benzyl alcohol is used in the formulation. Similarly, plastic containers are also not recommended for this preservative unless they are polypropylene containers or coated with fluorinated polymers.

Microbial Activity of Benzyl Alcohol

When used as a preservative, benzyl alcohol is more active against gram-positive bacteria compared to gram-negative. It is also active against yeast and molds.

Suitable API Chemistry

Benzyl alcohol is suitable for small and peptide-based products. It is incompatible with large molecules as it may induce aggregation; given this risk, it is instead used as a diluent so as there will be minimal duration of contact.

Safety

Benzyl alcohol should not be used in formulations intended for neonates and should be used with caution in children older than four weeks.

Table 4. Synergies, incompatibilities, and microbial activity of benzyl alcohol.

Synergy	EDTA,11chlorhexidine and benzalkonium chloride 11, 12
	Solvent-cosolvent for poorly soluble drugs and local anaesthetic activity; promoted for pain on injection type formulations8
Incompatibility	Oxidizing agents and strong acids, non-ionic surfactants, such as polysorbate 80, methylcellulose and plastic container (may be responsible for leachables)13
	Incompatible with methylcellulose and is only slowly absorbed by closures composed of natural rubber, neoprene, and butyl rubber; resistance can be enhanced by coating with fluorinated polymers13
Microbial activity	More active against Gram positive bacteria and less effective against Gram negative13
	Active against yeast and molds13
API chemistry	Suitable for small and peptide-based products; incompatible with large molecules, may induce aggregation and hence it is normally used as part of the reconstitution medium so that there is minimal duration of contact with the API (example Herceptin®)14
Therapeutic use	Pediculicide (it kills head lice but not the eggs and thus needs to be used after the eggs hatch as well)15
Safety	Should not be used in neonates and used with caution in children older than four weeks.9

Benzaldehyde Impurity in Benzyl Alcohol

Benzaldehyde is present as an impurity in benzyl alcohol (Figure 4). It interacts with benzyl alcohol as well as active pharmaceutical ingredients (APIs), resulting in accelerated oxidation of benzyl alcohol and degradation of the API^{16, 17}. For sensitive APIs and formulations, special grades of benzyl alcohol with reduced, controlled, and/or specified low benzaldehyde impurity levels are available.

Methylparaben and Propylparaben

Considerations for Use of Parabens as Preservatives

Methylparaben or methyl 4-hydroxybenzoate is commonly used as a preservative in combination with propyl-paraben or propyl 4-hydroxybenzoate due to their synergistic activity.^{18, 19}

The application range is very broad: Most frequently, methyl- and propylparaben are used in oral and topical applications, but their use includes also parenteral, transmucosal, and other formulations.

Table 5. Applications of methyl- and propylparaben based on FDA Inactive Ingredients Database information.

Methylparaben & Propylparaben Applications: General Overview
	Methylparaben	Propylparaben
Inhalation	•	•
Injection	•	•
Nasal	•	•
Ophthalmic	•	•
Oral	•	•
Oromucosal	•	•
Otic	•	•
Rectal	•	•
Topical & transdermal	•	•
Vaginal	•	•

Summary of Paraben Attributes

Table 6 summarizes several attributes of methylparaben and propylparaben.

Synergy with other Preservatives

Methylparaben and propylparaben are typically used in combination; both also show synergistic activity with EDTA.

Incompatibilities of Parabens

When parabens are in the presence of glycerin and boric acid, the pH shifts to the acidic range, and this promotes the transesterification reaction of parabens with glycerin. Formation of transesterification reaction products between methylparaben and selected sugars (glucose, fructose, sucrose, lactose, maltose, cellobiose) occurs only under mild reaction conditions (e.g., pH 7.4 at 50 ˚C).

Parabens can interact with cyclodextrins and polysorbate 80 resulting in a reduction in efficacy.  These preservatives are also incompatible with sorbitol, maltitol, and lactitol and may show discoloration in the presence of iron. Reaction with residual reducing sugars may lead to a Maillard reaction. At pH levels of 8.0 and above, parabens are unstable due to the formation of phenolate anion.

Microbial Activity of Parabens

When used as preservatives, methylparaben, and propylparaben are more active against gram-positive bacteria compared to gram-negative. They are also active against yeast and molds.

Safety

While parabens are among the most used preservatives, concerns exist regarding their safety and tolerability. Parabens can cause skin irritation and the European Medicines Agency (EMA) has published guidelines recommending caution when using them due to oestrogenic activity. In addition to the risk of oestrogenic activity in neonates, parabens have the potential for bilirubin binding activity which can lead to long-term accumulation and potentially hazardous effects for neonates.

Table 6. Synergies, incompatibilities, and microbial activity of methylparaben and propylparaben.

Synergy	Methylparaben and propylparaben are used in combination1, 18, 19
	EDTA1, 18, 19
Incompatibility	Potential transesterification reaction between methylparaben and selected sugars (glucose, fructose, sucrose, lactose, maltose, cellobiose)20
	In presence of glycerine and boric acid, pH gets shifted to acidic which can promote transesterification reaction of parabens with glycerine21
	Can interact with cyclodextrins and polysorbate 80 resulting in a reduction in effectiveness1, 18, 19, 21
	Incompatible with sorbitol, maltitol, lactitol, and may show discoloration with iron20
	Reacts with residual reducing sugars which may lead to a Maillard reaction20
	Above pH 8.0, unstable due to formation of phenolate anion (pKa 8.4)2
Microbial activity	More active against Gram-positive bacteria18, 19
	Molds and yeast18, 19
Safety	Parabens have oestrogenic activity and hence EMA has published guidelines for cautious use of parabens18, 19, 24 Potential for bilirubin binding activity which can results in long-term accumulation18, 25

Benzoic Acid and Sodium Benzonate

Considerations for Use of Benzoic Acid and Sodium Benzoate as Preservatives

Benzoic acid and sodium benzoates are typically used in moderate- to low-risk pharmaceutical and food applications. They are primarily utilized in oral formulations, but they also find applications in topical and transmucosal formulations, among others.

Sodium benzoate offers better aqueous solubility than benzoic acid, making it a preferred choice in certain applications and formulation types.²⁶

Table 7. Applications of benzoic acid and sodium benzoate based on FDA Inactive Ingredients Database information.

Benzoic Acid & Sodium Benzoate Applications: General Overview
	Benzoic acid	Sodium benzoate
Inhalation
Nasal
Ophthalmic
Oral	•	•
Oromucosal	•	•
Otic
Parenteral	•	•
Rectal	•	•
Topical & transdermal	•	•
Vaginal	•

Summary of Benzoic Acid and Sodium Benzoate Attributes

Table 8 summarizes several attributes of benzoic acid and sodium benzoate.

Synergy with other Preservatives

Sodium benzoate and benzoic acids have synergy with other excipients such as propylene glycol and sorbic acid. These preservatives are active at an acidic pH; for oral liquid preparations for which the pH should be acidic, benzoic acid and sodium benzoate are good choices.

Incompatibilities of Benzoic Acid and Sodium Benzoate

The preservative activity of benzoic acid and sodium benzoate may be reduced with interaction with kaolin due to alkalis or heavy metals.

Microbial Activity of Sodium Benzoate and Benzoic Acid

When used as preservatives, sodium benzoate and benzoic acid more active against gram positive bacteria compared to gram negative. They are also active against yeast and molds.

Formulation Challenge of Benzoic Acid and Sodium Benzoate

Benzoic acid is less soluble in water compared to sodium benzoate and therefore used in non-aqueous or oily formulations.

Therapeutic Use of Sodium Benzoate

Sodium benzoate is used for the treatment of patients with urea cycle enzymopathies (i.e., hyper-ammonaemia due to inborn errors of urea synthesis) to facilitate an alternative pathway of nitrogen excretion.

Safety

These preservatives may also cause skin or eye irritation and are therefore not used in ophthalmic applications. For topical applications, a minimal concentration should be used. Benzoic acid and sodium benzoates are toxic to neonates; when used in parenteral dosage forms, these preservatives may lead to jaundice, metabolic acidosis, and neurotoxicity in neonates.

Table 8. Synergies, incompatibilities, and microbial activity of benzoic acid and sodium benzoate.

Synergy	Propylene glycol27, sodium benzoate, sorbic acid28
	Acidic preservatives and active at acidic pH29
Incompatibility	With alkalis or heavy metals, preservative activity may be reduced by interaction with kaolin26, 27
Microbial activity	More active against Gram-positive bacteria & less effective against Gram negative27
	More effective against mold and yeast growth27
Formulation challenge	Benzoic acid has lower water solubility than sodium benzoate, making it suitable for non-aqueous or oily formulations. Its antimicrobial properties require low pH values, so it may be necessary to add acids like citric acid.27
Therapeutic use	Used for treatment of patients with urea cycle enzymopathies (i.e., hyper-ammonaemia due to inborn errors of urea synthesis) to facilitate an alternative pathway of nitrogen excretion28
Safety
	Irritant to skin and eye, hence normally not used in ophthalmics. For topical application, minimal concentration is used.27
	A minimal concentration should be used for topical applications
	When used in parenteral dosage forms, benzoic acid and sodium benzoate may increase the risk of jaundice in neonates30
	A build-up of benzoic acid in neonates can cause metabolic acidosis and neurotoxicity25

References

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29. European Medicines Agency (EMA), Committee for Human Medicinal Products (CHMP). 2017: Questions and answers on benzoic acid and benzoates used as excipients in medicinal products for human use (EMA/CHMP/508189/2013). Available from: https://www.ema.europa.eu/en/documents/scientific-guideline/questions-answers-benzoic-acid-benzoates-used-excipients-medicinal-products-human-use_en.pdf

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