Commonly used preservative systems reviewed

Microbial growth depends on many environmental factors, such as temperature, air, PH, osmotic pressure, nutrition, etc.

Temperature: Different microbes have their own optimal growth temperatures. The optimal growth temperature for bacteria is 30-37°C, and for mould and yeast is 20-25°C. When temperature is lower than 0°C, water will freeze and the metabolism of microbes will stop; and when it is higher than 70°C, microbes die due to condensation and denaturing of cellular proteins.

Humidity and water content: Water is an essential factor for microbial growth. Higher relative humidity in the air leads to greater microbial growth. On the other hand, higher water content in a cosmetic product is more prone to microbial growth, and thus more demanding for the preservative system.

Oxygen content: Many microbes are aerobic that can only survive in oxygen while anaerobes can grow well without oxygen. Facultative anaerobes can live with or without oxygen.

PH: Different microbes have different optimal pH for growth. Bacteria can survive at a pH range of 3.5-9.5, with the optimal pH of around 7 while mould can survive at a pH range of 2-11 with an optimal pH of 6, whereas yeast can survive at a pH range of 1.5-8.5 with preference to the acidic side.

Osmotic pressure: Osmotic pressure can inhibit microbial growth because it can cause the cell membranes breakage and make the cell dehydrated.

Nutrition: Microbial growth is also dependent on nutrients that are available to it. The usual forms of nutrients are those substances that are good sources of carbon, nitrogen, phosphor and sulfur; minerals such as sodium, potassium, magnesium, iron; organic growth factors (most of which are water-soluble vitamins in the “B” group including VB1, VB2, VB3, VB5) and water. There is about 90% water in a living cell, so water is an essential element for microbial growth.

Some ingredients in cosmetics, such as esters, hydrocarbons and soluble polymers tend to provide not only abundant nutrition with carbon and nitrogen sources that are necessary for microbial growth but also chemical energy with its adduction. The microorganisms in cosmetics grow, reproduce, and even cause the products to be spoiled once they live under appropriate conditions. Microorganisms can cause cosmetic deteriorations, which are usually demonstrated by product turning opaque, occurrence of precipitation and/or colour change; change in pH value, gas generation, etc. Microorganisms may even secrete toxins, which can cause harm and skin anaphylaxis to humans. It is essential for a cosmetic product to have an effective preservative system that keeps the product safe and stable.

Mechanisms of action and factors influencing preservatives efficacy

Mechanisms of action
Many preservatives function through inhibiting microbial growth and reproduction by acting on the cell membranes, cellular wall and cellular enzymes of the microbes. Preservatives do not have strong instant killing effect until they become in direct contact with the cells of the microbes at sufficient concentration. Preservatives with different molecular structures have different mechanisms of action. There are mainly three types of mechanisms of action:

Preservatives acting on cellular walls of microbes – by blocking the syntheses of the materials that form the cellular walls and thus breaking the cells and making them lose their protective effects. Examples are penicillin and cationic surfactants.

Preservatives acting on cell membrane – by destroying cell membranes to induce cellular apnea or metabolism disorder, which in turn causes the leakage of cellular materials and thus death of microbes. Examples are benzyl alcohol, benzoic acid, salicylic acid, etc.

Preservatives acting on protein and/or enzymes – by interfering with the synthesis of cellular protein and/or enzymes, which leads to denature of the protein/enzyme and microbial death. Some preservatives can oxidise the thiohydroxy group in protein into bisulfide, which causes microbial death. Examples are boric acid, benzoic acid, salicylic acid, sorbic acid etc. Other preservatives can make protein denature which leads to condensation and precipitation. Examples are heavy metals, formaldehyde, ethanol, etc.

In short, preservatives interfere with cellular growth through inhibiting synthesis of enzymes, proteins and nucleic acids.

Frequently used preservatives
This section presents the individual classes of preservatives while section 3 will discuss the preservative systems that are combinations of these individual classes of preservatives and often used in real cosmetic products. There are eight major classes of preservatives, which are detailed below.

Parabens
Parabens refer to the group of chemicals that consist of methylparaben, ethylparaben, propylparaben and butylparaben. Their target of action on microbes is cell membrane and they inhibit microbial growth by destroying the difference of cell membrane potential. The longer the carbochain, the better the effect of preservation. More favourable effects are obtained when these chemicals are used in combination. The most popular combination is the blend of methylparaben and propylparaben. Further more, combinations of parabens with other preservatives may also achieve synergistic effects, which will be discussed in more detail in later section of this article.

Phenol derivatives
Phenol derivatives are often used as antiseptics, although phenol and its sodium salt, and p-chloro-meta-xylenol (PCMX) are used as preservatives in cosmetics. Their target of action is cell membrane like parabens.

Alcohols
Some alcohols are very effective preservatives in cosmetics. The  frequently-used alcohol preservatives include ethanol, isopropanol, propanetriol, 2, 4-dichlorobenzyl alcohol (DCBA) and phenoxyethanol, etc.

Formaldehyde and its donors
Formaldehyde solutions are seldom used directly in cosmetics, although formaldehyde donors are commonly used in cosmetic and personal care products. Formaldehyde donors can slowly release free formaldehyde molecule in hydrous products, which is safer than direct use of formaldehyde solution. The formaldehyde donors include 2-bromo-2-nitropropane-1, 3-diol (Bronopol), monomethylol dimethylhydantion (MDMH), DMDM Hydantoin, Diazolidinyl urea, Imidazolidinyl urea, and so on. These formaldehyde donors are often used in combination with other anti-fungal agents to achieve broad spectrum of antimicrobial effects.

Organic acids and their salts
The most widely used organic acids and their salts as preservatives include benzoic acid and sodium benzoate, sorbic acid and its sodium salts, and so on. This kind of preservative can only function effectively in the formulation with pH lower 5. Only under acidic conditions can these compounds penetrate the cell membrane and kill microbes.      

Isothiazolinone compounds
These kinds of compounds exhibit a broad spectrum of antimicrobial effect at very low concentrations. It has been shown by researchers that they are the kind of compounds that work at multi-targets of the microbes. They do not only destroy the base enzymes of cells but also interfere with the permeability of cell membrane and the composition of nucleic acid. The mixture of 5-chloro-2-methyl-4- isothiazoline-3-one and 2-methyl-4- isothiazoline-3-one are commonly used in cosmetics. These compounds are highly effective at low usage levels, and they only cost about 1/3 of parabens.

Bromonitro compounds
Bromonitro compounds have effective anti-fungal and antibacterial activity at low concentrations. These compounds inhibit cell growth by reacting with the enzymes inside the cells.

Chelating agents
Chelating agents are mild antimicrobial agents, which can strongly inhibit gramnegative bacteria. More importantly, they have excellent synergistic effect with other preservatives to improve the antimicrobial activity. In cosmetics, EDTA and its salts are the most commonly used chelating agents, which are usually utilised in addition to other preservatives.

Factors influencing preservative efficacy
There are many factors that can influence the efficacy of a preservative system. The same preservative system can have different efficacy in different cosmetic formulations. The main factors that influence the efficacy of a preservative system are discussed below.

Influence of PH in formulations
Most of the preservatives can provide adequate antimicrobial effect under acidic and neutral conditions, whereas under alkaline conditions their antimicrobial effects reduce sharply and even become ineffective entirely. One exception is the quaternaries, which are effective when PH is greater than 7. For organic acid type of preservatives, the PH of a cosmetic formulation can affect its efficacy through influencing the dissociation of organic acid. For example, Bronopol (2- Bromo-2-nitropropane-1, 3-diol) is very stable when its PH is 4; it remains effective for a year when its PH is 6; but its activity only lasts several months when the PH is 7.

Solubility
The ideal preservative should be soluble in water as well as in oil at the same time, so that it could cross the cellular membrane of microorganisms which are made of protein and phospholipids, and thus penetrate into the cell and destroy the microbes. Short chain alcohols, such as ethanol, isopropanol and propanetriol themselves have preservative activity. Their addition enables an increase in preservative solubility and a reduction of preservatives in micelles in the water phase and thus they enhance the efficacy of the preservative system. In general, the O/W type of emulsion is more prone to microbial contamination than the W/O type. Some nonionic surfactants can interfere with the activity of certain preservatives by changing their distribution coefficients in water and oil phases, which could be detrimental to the efficacy of the preservatives.

Chemical compatibility
The efficacy of preservatives can be influenced by the chemical compatibility between the preservatives and other ingredients in cosmetics.

Adsorption: Certain carbohydrates, talc, Kaolin or magnesium aluminum silicate can reduce preservative efficacy because of its adsorption of preservatives. On the other hand, the particles of these materials could sometimes strengthen the bacteriostatic effects by adsorbing the microbes as well.

Water soluble polymers: The preservatives’ performance may be affected by combination with water-soluble high molecular weight polymer, which can bring down the concentration of free preservatives in the formulation.

Metal ions: Metal ions, such as Mg2+, Ca2+, Zn2+, can influence preservative efficacy significantly.

Surfactants: a small amount of surfactants can enhance the performance of preservatives by increasing the permeation of preservatives into the cell membrane; whereas a large amount of surfactants may reduce the preservatives’ performance because they can form micelles which attract preservative molecules and reduce the concentration of preservatives available in the water phase of an emulsion.

Initial amount of microbes in cosmetic products
The effective concentration of preservatives in cosmetic products can also be impacted by the initial amount of microbes that are present in finished product on completion of the manufacturing process. In general, the effective concentration of preservatives decreases somewhat on completion of the production process because killing the initial microbes consumes certain amount of preservatives.

Other factors
Other factors including sunlight, heat and chemical reaction, can reduce the efficacy of preservatives. Sunlight or heat can cause preservative decomposition while chemical reaction changes the structure of the preservatives which inactivates them. There are many other factors, including distribution coefficient of preservatives between oil/water phase, packaging, fragrance, chelating agents, which may all affect the performance of a preservative system.

Preservative systems for cosmetic products
In order to achieve optimal performance, a preservative system in real cosmetic and personal care products is often composed of two or more different preservative entities that are mixed in a certain ratio. The advantages for these blends are:

A broader spectrum of antimicrobial activity: the components in a preservative system of blends can complement each other and thus achieve a broader spectrum of antimicrobial activity, which is not possible with a single preservative entity.

Increases in antimicrobial efficacy and safety: when two or more preservative entities with different mechanisms of action are mixed together, a resultant synergistic effect occurs which usually lowers the use level of each individual preservative component and thus improves the safety of the preservative system.

Protection against microbial recontamination: some preservative entities are powerful bactericides with strong short-term killing effect, but with weak long-term inhibitory effects, while others are excellent bacteriostatic agents with strong long-term inhibitory effects, but with weak short-term killing effects. When preservative components from these two categories are combined together, they can provide adequate product preservation so as to maintain product quality intact during product distribution and shelf life as well as to protect against microbial re-contamination during user consumption.

Avoidance of drug resistance: drug resistance of microbes can occur with one individual preservative component, but drug resistance of microbes against two or more preservative components at the same time is very rare. Therefore, it is highly effective to avoid drug resistance of microbes against a preservative system by using combinations of preservative components.

In the following paragraphs, the most commonly used preservative systems in recent years will be presented and discussed.

Blends of Parabens and other preservatives
Blends of Parabens and Phenoxy Ethanol. A number of commercial products for these blends are available. These blends demonstrate excellent synergistic effects without the usual solubility problem of parabens.

Blends of Parabens, Phenoxy Ethanol and Bromopol (2-Bromo-2-nitropropane-1, 3-diol ). Commercial products for these blends are also readily available. These blends exhibit stronger antimicrobial efficacy than that when used alone individually at a similar concentration.

Blends of DMDMH and IPBC
DMDMH provides excellent antibacterial activity while IPBC (3-Iodo-2-Propynyl butyl carbamate ) is an excellent anti-fungal agent. By combining these two components into one preservative system, the blend results in excellent, broad spectrum antimicrobial activity against bacteria, mould and yeast.

Blends of Diazolidinyl urea and other preservatives
Diazolidinyl urea provides excellent broad spectrum antibacterial effect, but poor anti-fungal effect. Due to its antifungal weakness, it is commonly used in combination with other preservative entities to achieve broad spectrum antimicrobial activity. Popular commercial combinations are :

Blends of Diazolidinyl urea, 3-Iodo-2- Propynyl butyl carbamate (IPBC) and Methylparaben.

Blends of Diazolidinyl urea, IPBC, Methylparaben and Propylene glycol.

Blends of Diazolidinyl urea and IPBC.

Blends of Diazolidinyl urea, IPBC and Propylene glycol.

Blends of Diazolidinyl urea, Methylparaben, Propylparben and Propylene glycol.

Blends of Isothiazolinone compound
This type of preservative system has the advantage of very low use level with excellent efficacy. The disadvantage is that it is only recommended for rinse-off products. Commercial products of these blends are readily available, and the popular combinations are:

Blends of 5-chloro-2-methyl-4- isothiazoline-3-one (CIT) and 2-methyl- 4-isothiazoline-3-one (CIT).

Blends of 2-methyl-4-isothiazoline-3- one and 2-methyl-3-isothiazolinone.

Efficacy evaluation for a preservative system
Two test methods are often used to evaluate the efficacy of a preservative system; namely Minimum Inhibitory Concentration (MIC) test and the Challenge Test. The minimum inhibitory concentration or MIC is defined as the minimum concentration of an antimicrobial agent which will inhibit the growth of the isolated microorganism. This MIC test method is mainly used as a research tool to determine the in vitro activity of new antimicrobials, whereas the challenge test is commonly used to evaluate the preservative system in a real cosmetic product.

There are a number of published challenge test methods, which include the various pharmacopeia methods – US Pharmacopeia (USP) method, British Pharmacopeia (BP) method and European Pharmacopeia (EP) method, American Society for Testing and Materials (ASTM) method and the Cosmetic, Toiletry and Fragrance Association (CTFA) method. In 1995, the BP method was revised in accordance with EP method. In the US, however, the personal care industry most commonly uses the CTFA challenge test method. This method recommends at least 99.9% reduction in bacterial counts and at least 90% reduction in fungal counts within seven days, with later counts remaining at or below that level.

There are also a number of more rapid test methods, which include linear regression method, presumptive challenge test and accelerated preservation test methods. These test methods are largely similar, although there are differences in test time and performance criteria. A study by Farrington et al1 on the ability of six commonly used laboratory methods to predict the in-use efficacy of antimicrobial preservatives concluded that these standard published test methods could all differentiate between well-preserved and poorly-preserved formulations.

Present status and future trends
The US personal care market for preservatives was estimated to be $55-65 million for 2005 with a growth rate of 3-5%.2 Parabens have a leading position accounting for 20% of the total US market share, while Imidazolidinyl urea and Isothiazolinones each accounted for 15%; DMDM Hydantoin and Diazolidiny each accounted for 10% with other preservatives accounted for the remaining 30% of the total market share. Among these traditional preservative systems, parabens, formaldehyde-donors and isothiazolinones have suffered adverse publicity in the media in recent years, which has created negative consumer perceptions and evolving regulations that limit their use. Some of these reports are founded on questionable science – they can nevertheless generate highly negative reactions from the media, which lead to adverse consumer perception.

In case of parabens, P. D. Darbre’s et al3 demonstrated the presence of parabens in breast tissue, which suggested a role of parabens in breast cancer. Other preservatives have also become less desirable due to changes in labelling requirements or classification. Formaldehyde has been reclassified from “probable” to “known” human carcinogen, which has made some consumer product companies avoid formaldehyde-releasing preservatives altogether. Isothiazolinones also have sensitisation concerns, which already made them lose market share.

In wake of the negative publicity on the traditional preservative systems discussed above, one important emerging trend is to develop new preservative systems without the controversial components. Another key trend for preservatives is to develop preservative systems that are acceptable globally, as more and more consumer product companies seek to do business globally in various regions, including the United States, Asia Pacific, Latin America and Europe. Many large consumer product companies operate their businesses in many countries, which require global approval for all ingredients used in their products including preservatives. Developing suitable preservative systems that are acceptable globally is quite challenging, given the divergent regulations by the United States, Europe and Japan. Most other regions or countries of the world base their regulations on one or a combination of these three main regulatory bodies.

Of these, Japan has the most restrictive policy on preservatives, which requires that the ingredients in the preservatives are on the Approved List of the Japanese Ministry of Health and Welfare (Japanese MHW). For a preservative system to be truly global requires that all components of the preservative system are on the approved list of the Japanese MHW and that all of these components are also acceptable in the United States and Europe.

In search of alternatives to the traditional preservatives with adverse publicity, the use of blends will continue to be an important trend. The challenge for cosmetic preservation is to develop globally approved preservative systems that are safe and effective. Most preservative suppliers will offer some form of blends to optimise preservation, lower usage level and save costs. The demand to reduce the total amount of preservatives used in consumer products is one driving force behind the growing popularity of blends.

Various new blends are expected to come out into the marketplace, although it is unlikely that new chemistry will be created for preservatives due to the high costs of complying with regulations around the world. The trend will be to take molecules registered in one application and try to fit them in other applications, and use blends to expand the spectrum of antimicrobial activity and possibly lower the overall concentration of preservatives in the product.

References

1 J. K. Farrinton, E. L. Martz, S. J. Wells, C.C. Ennis, J. Holder, J. W. Levchuk, K. E. Avis, P.S. Hoffman, A.D. Hitchins & J. M. Madden, Ability of Laboratory Methods to Predict In-Use Efficacy of Antimicrobial. Preservatives in an Experimental Cosmetic, Appl. Environ. Microbiol., 1994, 60 (12), 4553-4558.

2 D. De Guzman, Preservatives in the spotlight, Chemical Market Reporter, 12/5/2005, Vol. 268, Issue 19, p20-22.

3 P. D. Darbre, A. Aljarrah, W. R. Miller, N. C. Coldham, M. J. Sauer & G. S. Pope, Concentrations of Parabens in Human Breast Tumours, Journal of Applied Toxicology, 2004, 24, 5-13.