Sarah J. HiomResearch and Development, St. Marys Pharmaceutical Unit, Cardiff and Vale University Health Board, Cardiff, Wales, UK
17
Nature of medicines and cosmetics
Medicines are formulated to assist in the administration of drugs to treat or prevent diseases or to alleviate symptoms in patients. Medicines can be delivered by a wide variety of routes, from rela-tively non-invasive topical applications to highly invasive injec-tions. Cosmetics, however, are designed to deliver agents that enhance personal appearance, modify body odor or assist in body cleansing. Application is largely restricted to the skin, although such products as toothpaste or those for “feminine hygiene” may come into contact with mucous membranes. Eye-area cosmetics may also come into secondary contact with the cornea and conjunctiva.
Although the intended outcomes for medicines and cosmetics are fundamentally different, there are many similarities in the nature of the formulations created and the uses (and abuses) to which both can be subjected, including common microbiological problems. In order to create elegant products that are also effica-cious, stable and safe to use throughout their intended shelf-life, it is often necessary to include several other ingredients in addi-tion to the specific therapeutic agent or that producing the cos-metic effect. While a few formulations may be simple aqueous
solutions or dry powders, many are extremely complex, both in the number of ingredients used and in their physicochemical complexity. Some indications of this variation and complexity of medicinal and cosmetic formulations can be obtained from the reviews of Frick [1] and Lund [2].
The possibility that microorganisms might contaminate medi-cines and cosmetics during manufacture, storage or use must be addressed to ensure the continued stability and safety of the product. The complex chemical and physicochemical nature of many formulations is often found to be conducive to the survival and even multiplication of such contaminants, unless specific precautions are taken to prevent it. Such survival, and even growth, may result in appreciable damage to the product as spoil-age and/or the user as infection. Good manufacturing practices should provide adequate control of contamination from raw materials and processing activities [3–5]. This includes attention to the packaging, which should minimize the access of microor-ganisms, so that the quality of the product is maintained through-out the stresses of manufacturing, distribution, storage and use [6].The ideal approach to reduce contamination risks would be to present the product as a sterile single-dose unit in robust packaging. However, this is only economically practical for high-risk dosage forms such as eye drops for hospital use or where
Russell, Hugo & Ayliffe’s: Principles and Practice of Disinfection, Preservation and Sterilization, Fifth Edition. Edited by Adam P. Fraise, Jean-Yves Maillard,
Nature of medicines and cosmetics, 388Consequences of microbial contamination, 390Effect of formulation parameters on microbial contamination and spoilage, 391Use of preservatives in medicines and cosmetics, 385Regulatory aspects of the preservation of medicines and cosmetics, 399Prediction of preservative efficacy, 400Adverse reactions of users to preservatives, 401References, 402
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often a case of “best choice under current circumstances”. However, the selection process will include consideration of the properties of the formulation, preservative and likely challenge microbes, together with an evaluation of the intended use of the product and the associated contamination risks.
While there is little in the manner of official lists of recom-mended preservatives for medicines, there are bodies of unofficial regulatory beliefs which consider that certain preservatives would or would not be suitable for particular medicines. Thus, the use of bronopol and chloroform in new applications for oral medicines is most unlikely to be permitted, despite any data submitted to support its by-mouth usage. Since public disclosure of the pre-servative content of medicines is not generally mandatory, it is difficult to get a detailed pattern of usage. However, examination of partial disclosures in the British National Forumulary (BNF) [10] and by some manufacturers does provide an indication of UK usage. From anecdotal information and experience, it would appear that a limited range is suitable for use in medicines, and that parabens are by far the most commonly selected. Detailed mono-graphs on the preservatives commonly used in medicines have
preservatives cannot be used due to overriding toxicity concerns. One procedure adopted to limit the establishment of microbial contamination after manufacture is to include antimicrobial pre-servatives in the formulations. Preservatives, however, must never be added to mask contamination that arises from unsatisfactory manufacturing procedures or inadequate packaging.
The selection of a preservative system is a complex issue. It is essential to understand and fully evaluate the preservative needs and problems of individual products and be aware of how poten-tial antimicrobial agents may behave in that formula.
The ideal properties of a preservative have been described [7–9] and include: being non-toxic and non-allergenic, having a broad spectrum of activity, being effective and stable over the range of pH values encountered in cosmetics and pharmaceuti-cals, be compatible with packaging and other ingredients in the formulation, be free from strong odor or color and not affect the physical properties of the product, be available at effective con-centrations in the aqueous phase of the product, have an ability to meet regulatory requirements and be cost effective. Very few preservatives are able to meet all the required criteria and it is
Table 17.1 Selected preservatives used in pharmaceutical formulations showing some typical in-use concentrations (%w/v) and optimal pH ranges.
(+) now generally regarded as being unsuitable; +* generally for vaccines.
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included severe eye infections from contaminated ophthalmic solutions [28] and tetanus infection of newborn children from contaminated talc dusting powders [29]. During the 1960s, a number of key investigations demonstrated the existence of a much wider problem. Ayliffe et al. [30] reported on an extensive UK outbreak of severe eye infections, traced to traditional but wholly inadequate official guidelines for the preservation and manufacture of ophthalmic solutions. The “Evans Medical disas-ter”, in which contaminated infusion fluids caused serious injury and contributed to some deaths, precipitated public awareness and led to an official inquiry [31]. In Sweden, Kallings et al. [32] linked an outbreak of salmonellosis to contaminated thyroid tablets and eye and other infections to a range of contaminated pharmaceuticals. Bruch [33] in the USA similarly reported links between microbial contaminants in medicines and cosmetics and infections. Wilson [34] implicated contaminated eye-area cosmetics with severe eye infections. The more general role of opportunistic pathogens, such as the pseudomonads, and their implication in nosocomial infections was also becoming more recognized at this time. These reports stimulated an appreciable tightening of regulatory controls in many countries, and it is generally believed that the present situation is greatly improved. Comprehensive reviews of this topic can be found by Spooner [27], Beveridge [7], Fassihi [35] and Sharpell and Manowitz [26].
There are still difficulties in preventing the build-up of patho-genic contaminants in multidose eye drop containers during use [19].The limited range of preservatives that are not damaging to the eye is also creating problems in controlling microbial prolif-eration, for example in contact lens maintenance [36, 37]. Addi-tionally, there are currently small but serious outbreaks of protozoal infections by Acanthamoeba, for which effective and safe preservatives are difficult to find [38, 39].
Contaminated injections can result in the most serious infec-tion outbreaks, as demonstrated by the administration of con-taminated dextrose solution with the subsequent death of six UK hospital patients [40]. Despite major advances in the quality of large-volume parenteral infusions, high numbers of localized and systemic infections occur which are directly attributable to the administration devices themselves, such as catheters and cannu-lae [41].Total parenteral nutrition infusions, compounded asepti-cally from sterile components, are conducive to microbial growth but cannot contain preservatives, due to their large volume [42]; they are therefore currently considered an area of great concern. Cases of fatal infections from contaminated units indicate a con-tinuous need for improved systems for dispensing and protecting them from contamination [43–47].
Patients whose resistance has been weakened by trauma, chem-otherapy, tissue damage or other disease often succumb to infec-tion by opportunist contaminants that are unlikely to cause harm to “normal” patients [48]. The infection of haemophiliacs with human immunodeficiency virus (HIV) from human-derived factor VIII [49] and hepatitis C from blood-derived products [50] has stimulated action on possible virus contamination of other products derived from human or biotechnology-derived origin,
been produced [9–12]. A selection are presented in Table 17.1, showing the common types of formulations they are used in together with some in-use concentrations and optimal pH ranges.
Consequences of microbial contamination
Microorganisms possess diverse metabolic activities and are likely to present a variety of hazards (e.g. infection, toxicity, degradation of the formulation) both to the user and to the stability of the products, if allowed to persist. The European Pharmacopoeia Commission [13] sets limits for the presence of microorganisms in medicines, which vary depending on the product and its intended use. However, microbial contamination over and above these pharmacopoeial levels are still reported in distributed UK medicines [14, 15], although stricter regulatory controls have improved the situation compared with that of the pre-1970 period [16]. Other indications that the risk of microbial contami-nation is still a problem include reports that 13% of the UK drug alerts between January 2009 and January 2010 were due to an inability to provide microbial assurance to the required level [17] and that 4.9% of European Medicines Agency inspection defi-ciency reports (1995–2005) [18] were associated with the poten-tial for microbial contamination. In-use contamination hazards also continue to be a problem, particularly for multidose eye drops [19]and multidose injections [20]. In the USA, concern currently centers on the microbial hazards that accumulate during the use of cosmetics [21, 22]. Few recent published data have been found for cosmetic contamination in the UK, although anecdotal evidence suggests a similar situation to that in the USA.
The most commonly reported microbial hazards found in liquid medicines and cosmetics are pseudomonads and their related Gram-negative rods, with spores (bacterial and fungal) predominating in dry tablets, capsules and cosmetic powders. Shared-use cosmetics accumulate human microflora, such as Sta-phylococcus epidermidis, Staphylococcus aureus and corynebacte-ria, as well as pathogenic fungi, yeasts and bacterial spores. Those that contain water or become wet during use reveal pseudomon-ads and related bacteria. The clinical and pharmaceutical signifi-cance of such contamination of medicines has been reviewed by Ringertz and Ringertz [23], Martone et al. [24] and Denyer [25] and for cosmetics by Sharpell and Manowitz [26]. The implica-tions for product spoilage of both have been discussed by Spooner [27] and Beveridge [7].
The risk (likelihood of harm actually occurring) associated with delivery of contaminated products is less clearly determined. It will depend upon the type of microorganism present, the infec-tive dose (dependent on the ability of the formulation to encour-age microbial survival and the level of preservative protection built into it), the route of administration of the product and the host’s resistance to infection (including immune status or degree of tissue damage at site of application). Prior to the 1960s, inci-dents of infection attributed to contaminated products seemed to be regarded as unfortunate but isolated occurrences, these
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biodegradable, and even the trace residues of non-specific chemi-cal contaminants present in most commercial ingredients are likely to provide ample nutrients to permit growth. For example, even standard distilled and demineralized water contains suffi-cient trace nutrients to permit ample growth of pseudomonads and related species [68]. For microorganisms to multiply and/or survive there must also be appropriate physicochemical condi-tions that allow them to maintain adequate homeostasis. From the formulator’s point of view the main conditions that can be modified include the amount of available water present and con-ducive temperatures and pH. Such manipulations form the basis for the preservation and protection of many foodstuffs, where the ability to add antimicrobial preservatives is strictly limited by law. A wealth of basic and applied food-protection research is available for those who wish to assess these principles for application to medicines and cosmetics, and the reviews of Chirife and Favetto [69], Dillon and Board [70], Gould [71] and Roberts [72] are recommended. Their application to pharmaceuticals and cosmet-ics has been considered briefly by Orth [73] and Beveridge [7].
Available waterMost microorganisms require over 70% water to grow [74], which necessitates that, if they are to replicate, they must success-fully compete for ready access to water against the other ingredi-ents of a formulation – which the microorganisms may interact with often in strong and complex ways [75]. The amount of water available to microorganisms in a product is given by:
Aw
Vapor pressure of product
Vapor pressure of water=
Certain organisms will only grow at specific available water (Aw) levels (Table 17.2)
The manipulation of product water activity to a level below the minimum essential for growth offers a major potential for the protection of some products. It is possible to reduce the Aw of tablets, pastilles, capsules and powders sufficiently by drying to provide their major mode of spoilage protection, although some contaminants may continue to survive in a senescent state for a
as has the contraction of Creutzfeld–Jakob disease by patients treated with human growth hormone products from human origin [51]. More recently, a report links contamination from ultrasound gel to postoperative infections with Burkholderia cepacia [52].
Despite many well-publicised incidents, infection of patients with burned or otherwise damaged skin caused by using antisep-tic cleaning solutions contaminated with Pseudomonas spp. con-tinues [53–55], as does infection from contaminated nebulizer solutions [56, 57].The liberation of endotoxins by growth of Gram-negative contaminants in large-volume intravenous infu-sions and peritoneal dialysis fluids remains a problem [58, 59]. More recent are incidents of algal toxins, such as mycocystins, surviving in process water and causing damage and even death when used for the dilution of kidney dialysates [60]. The implica-tions of aflatoxin contamination in cosmetics has become of interest [61], with the suggestion that these toxins could penetrate the epidermis [62].
With the link between infection and contaminated cosmetics long established [33, 34], current concerns center on the practice of in-store cosmetic multiuser testers. These have been shown to accumulate appreciable levels of contamination, including a variety of hazardous bacteria, yeasts and fungi, which are able to initiate severe eye infections and infections associated with the use of contaminated hand creams and lotions [21, 22].
The weight of published evidence, both past and present, on the implications of microbial contamination for medicines and cosmetics demands that a careful and specific microbiological risk assessment is made for each individual product at its design and validation stages, using conventional risk-assessment tech-niques [63–66]. These must take into account worst-case sce-narios, such as the possibility that eye cosmetics may be applied while driving, where an applicator might scratch the cornea [67], or that multidose eye drop units may well receive varied and appreciable contamination during use by the lay public. Such assessments should take into account the highly critical expecta-tions of the public concerning standards for medicines and other consumer products, which are usually far greater than those for their food.
Effect of formulation parameters on microbial contamination and spoilage
The formulation may have an effect on microbial contamination in one of two ways. Firstly, through modification of the physico-chemical environment of any contaminant, which will challenge its ability to multiply and/or survive; and secondly, by having an effect on the efficacy of any preservative present.
Formulation effects on microbial growthThe nutritional requirements of most saprophytic, non-fastidious spoilage contaminants are likely to be well met in almost all pharmaceuticals and cosmetics, since many ingredients are easily
Table 17.2 Available water (Aw) requirements of common microbial species.
that might be initiated, although this itself may result in a pH change, allowing other contaminants to take over. Thus, the low pH of fruit juice-flavored medicines may aid fungal spoilage but suppress bacterial growth, while the slightly alkaline antacid mix-tures would favor the growth of pseudomonads and related bac-teria. It is unlikely that the redox potential of most pharmaceuticals or cosmetics is likely to be low enough to favor anaerobic spoilage, as is seen in some foodstuffs.
TemperatureLow temperatures will slow growth and rising temperatures will increase growth. Above optimum temperatures, growth is inhib-ited and microbes will eventually be killed, however most micro-organisms can survive at low temperatures even if they do not grow. Most fungi considered to be contaminants to pharmaceu-ticals and cosmetics are recovered at 25°C and bacteria at 30°C.
Low-temperature storage (8–12°C) is used to improve the short-term stability of some unpreserved, or weakly preserved, medicines, such as unpreserved eye drops [85] and reconstituted antibiotic syrups. Where dispensed products are to be “stored in a cool place” during use, the growth-inhibitory effect of the reduced temperature needs to be balanced against the conse-quent, and often significant, reduction in efficacy of any preserva-tive present (see below).
The response of contaminants to the physicochemically complex environment of many pharmaceutical and cosmetic for-mulations will differ significantly from that in simple laboratory media [7], being markedly influenced by their spatial arrange-ment [86] and the phase status of the systems [87]. There are many reports of modified behavior of microorganisms at solid–liquid and liquid–liquid interfaces, but the evidence for increased resistance or longevity compared with freely planktonic situations is far from clear [88]. An increasing understanding of the survival strategies of microorganisms in sparse ecosystems [89] may provide an insight into the longevity of vegetative contaminants in seemingly unlikely products. For example Salmonella spp. in chocolate [90] and thyroid tablets [91] or vegetative bacteria in “dry” powdered ingredients such as plant material, starch, hydroxymethyl cellulose and gels such as aluminum hydroxide [92, 93].
Formulation effects on preservative efficacyThe choice of a preservative depends on many factors, including the intrinsic properties of the agent (see Chapters 2 and 5), the extrinsic environment (product formulation) and the nature of product usage. The factors influencing the efficacy of antimicro-bial preservatives has been dealt with in detail elsewhere in this book (see Chapter 3) and include consideration of the concen-tration, temperature coefficients and environmental pH. These areas will be briefly reviewed in relation to formulation effects, together with a more detailed discussion on additional areas such as water availability, chemical interaction and stability, adsorption, partitioning in multiphase formulations and inactivation.
considerable time [76]. Friedl [77] has proposed reducing the number of microbiological attribute tests carried out on pharma-ceuticals based on information collated on the Aw content of different types of products and the ideal Aw growth requirements of the likely contaminating microorganisms [78]. These figures may also be used to assist decisions concerning the need to include preservatives in a product. For example, rectal ointments have an Aw of approximately 0.26 and as no organisms are expected to proliferate below an Aw of 0.6, preservatives are not recommended in these type of preparations. The proposed reduc-tion in Aw must be maintained throughout the life of the product, possibly by using water vapor-resistant bottles or film-strip packing, otherwise protection will be lost. The use of vapor-repellent film coatings [79] has been suggested to assist in the control of spoilage of bulk tablets intended for distribution to humid climates [80]. The Aw of some aqueous systems can be lowered sufficiently to give useful protection by the addition of quite large amounts of water-binding low molecular weight solutes: sucrose (66% w/v, approximate Aw = 0.86) in, for example, reconstituted antibiotic syrups; sorbitol (c. >35% w/v) for denti-frice pastes [81]; glycerol (c. >40%) for cosmetic lotions and urea (10–20%) for some cosmetics [82]. Limtner [83] described the use of polyacrylamide hydrogels in cosmetic creams to enhance formulation robustness, presumably by very effectively lowering Aw. Ointments have a low Aw and it is not usual to add preserva-tives to these formulations. Strongly alcoholic formulations, such as perfumes, also have a low Aw, as well as being antimicrobial in their own right.
Condensed-moisture films can develop with sufficiently high Aw on the surfaces of waxy cosmetics, such as lipsticks, or highly viscous formulations, such as toothpastes, or on compressed cos-metic powders to permit localized fungal growth. This can happen when they are persistently exposed to humid environments, such as steamy bathrooms, or if moisture is regularly applied by mouth or applicator during use. Growth of contaminants has occurred in condensed films under the tops of containers formed when hot aqueous products have been packed into cold containers. This effect can also result in appreciable dilution and loss of localized efficiency of any preservative present [84]. Should sparse fungal growth be initiated on marginally dried products or those which have become damp during storage, such as tablets packed in bulk, water generated by respiration will create locally raised Aw levels and initiate a cycle of enhanced rates of growth, leading to appre-ciable levels of spoilage [75].
pHThe optimum pH for bacterial growth is normally between 7.4 and 7.8. However, fungi prefer more acidic conditions of 6.5 to 7.0. At extremes outside the range pH 3 to 11, growth of most species is inhibited. The food industry’s wide use of pH reduction or gas environment modification of redox potentials to control spoilage is more limited in application to medicines and cosmet-ics due to physiological acceptability and formulation stability. However, the pH of a product will influence the type of spoilage
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partition coefficients, oil : water ratios, solubilization constants and polymer-binding constants, which have been reviewed by Attwood and Florence [96]. For example, Mitchell and Kamzi [100] proposed equations such as the following for estimating the free or available preservative in the aqueous phase of an emulsi-fied system:
C C nK M KC K= + + + +w w wo/[ ( ) ( ) ]( )1 1 1φ φ
where C is the gross concentration of preservative in the system; Cw is the “unbound” preservative in the aqueous phase; n is the number of binding sites on the surfactant; K is the association constant for the preservative and surfactant; M is the preserva-tive : surfactant ratio for a given surfactant concentration; Kw
o is the oil : water partition coefficient; and ϕ is the oil : water phase ratio.
Mitchell and Kamzi were able to produce some correlation of experimentally determined measurements of bactericidal activity for chlorocresol in cetomacrogol emulsified systems with esti-mates obtained using their equations [101]. While these equa-tions have been of value in resolving theoretical concerns and in providing some limited practical information, in general there is difficulty in obtaining adequate values for commercially available ingredients for them to be of general practical application. Another approach has been to estimate the unbound preservative in the aqueous phase by dialysis and measurement of the agent in the dialysate [96, 102], with some formulators believing that useful practical information can be obtained. Kurup et al. [103] compared estimates of “unbound” preservative in the aqueous phases of emulsified systems as obtained by equilibrium dialysis with direct measurements of antimicrobial activity. They reported that the efficacy of the emulsified systems was greater than that of the corresponding simple aqueous solutions containing those concentration of preservatives estimated from the dialysis deter-minations to be “available” in the aqueous phase of these systems.
pHEnvironmental pH has an effect on the efficacy of many preserva-tives (see Table 17.1), predominantly through ionic changes and the impact this has on preservative potency and/or direct interac-tion with cellular target sites. Depending on the preservative’s mode of action (see Chapter 5) some are active in the non-ionized state (benzoic and sorbic acid) and will have greatest potency at or below their pKa. It is possible to determine the extent of ioniza-tion using the equation below and figures for pKa can be found in various monographs [12, 42]:
Undissociated fraction of weak acid preservative
/ antil= +1 1 oog pH p a( )− K
TemperatureThe effect of temperature on preservative efficacy may be described by the equation,
ConcentrationThe exponential effect of change in preservative concentration on microbial death is given by the equation Cnt = k, where C is the preservative concentration, n is the concentration exponent, t is time to achieve a certain kill effect and k is a constant. The con-centration exponent is a measure of the effect of change in con-centration or dilution on microbial death and will depend on the type of preservative being used [94, 95]. The activity of a pre-servative depends on the free concentration of the active form of the molecule in the aqueous phase and will be affected by parti-tioning in complex multiphase formulations and pH effects.
PartitioningWhen preservatives are incorporated into multiphase formula-tions, their efficacy is generally markedly attenuated by a variety of competing interactive possibilities, which have been reviewed by Attwood and Florence [96], van Doorne [97] and Dempsey [98]. The partitioning of preservatives between oil and water phases will occur, in line with their partition coefficients, which may be different for commercial-grade systems from those of simple laboratory-devised oil–water mixtures of purified ingre-dients. Appreciable migration into oil phases of lipophilic pre-servatives, such as the parabens and phenolic agents, is likely, with less effect on the more hydrophilic preservatives, such as imida-zolidinyl urea or the isothiazolinones. The oil : water ratio will also significantly influence the extent of overall migration, and there is the probability that localized preservative concentration will occur at the oil–water interface. Since microbes also concentrate at interfaces, there are reasonable theoretical grounds for antici-pating enhanced activity here [96]. Some evidence for this was provided by Bean et al. [99], but was discounted by Dempsey [98].
When preservatives are added to complex formulations, the above phenomena are likely to interact competitively and the eventual distribution of preservative molecules by the different phenomena between the different ingredients and phases will be determined by the relative affinities of each for the other and the relative proportions of each. In highly viscous and complex systems, full equilibration might never happen within the life of the product. As contaminant microorganisms will represent a minute mass in proportion to the vast amounts of the other components, their “share” could be expected to be dramatically restricted in many situations. It is generally believed that the proportion of total added preservative responsible (“available”) for any inhibition of contaminants is principally that which remains “free” or unbound to other ingredients in the aqueous phase [96–98]. Preservative molecules in the oil or micellar phases, or bound to other ingredients, are considered not to con-tribute directly, except that slow back-migration should occur as preservative concentrations become depleted in the aqueous phase. Experimental evidence for this is limited and might be difficult to obtain. However, there is very good indirect physico-chemical evidence to support this contention.
Attempts have been made to develop equations to calculate probable free, or unbound, preservative concentrations, based on
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The aqueous solubility of some commonly used preservatives is low and some form salts or complexes with very low-solubility products, which precipitate from solution. Thus, the usefulness of chlorhexidine is restricted by its ability to form insoluble prod-ucts with chloride, sulfate, phosphate or citrate ions and anionic surfactants. Quaternary ammonium preservatives form insoluble complexes with anionic surfactants and a range of anionic inor-ganic ions. Benzoates and parabens form insoluble and discolor-ing complexes with iron salts, chlorocresol precipitates with phosphates, and phenylmercuric nitrate precipitates with chlo-ride ions. Bronopol has formed complexes with unprotected alu-minum in flexible tubes.
AdsorptionAdsorption on to the surfaces of suspended drug and cosmetic ingredients can also significantly reduce preservative efficacy. Examples include adsorption of parabens on to magnesium tris-ilicate [115] and other natural hydrocolloids [116], loss of chlo-rhexidine on to mineral earths, such as kaolin and calamine [117], parabens on to cosmetic pigments [118] and benzoic acid on to sulphadimidine particles [119] and various other incidences of adsorption [102]. In some cases, adsorption is followed by absorption into the solids to form a “solid” solution. This is a particular problem with preservatives and plastic containers. For example, phenolics and parabens absorb appreciably into nylon and plasticized PVC [120] and chlorobutanol is absorbed appre-ciably into polyethylene bottles during autoclaving [121]. Kakemi et al. [122] examined the absorption of parabens and benzalko-nium chloride into various container plastics. Aspinall et al. [123] found that the absorption of phenylmercuric acetate into low-density polythene eye drop bottles could be inhibited by the presence of phosphate ions. Methods for assessing possible preservative–plastic interactions are provided by Wang and Chien [124].
InactivationAs preservative molecules inactivate microorganisms, they them-selves become inactivated and are no longer available to inhibit subsequent additions of contaminants. For those preservatives with high-concentration exponents, such as the phenolic agents, this steady depletion of available agent can result in significant attenuation in preservative efficacy during repeated use and in the contamination of multidose formulations. The relative non-specificity of preservative reactivity will mean that appreciable preservative depletion will also occur from interaction with a significant amount of non-microbial detritus (“dirt”) also intro-duced during repeated and prolonged use. This must be allowed for when deciding upon the necessary preservative capacity of a formulation at the design stage.
It is clear that effective preservation of complex formulations is only likely to be successful if there is a good appreciation of these interactive problems. Even then, it may not be possible to provide highly effective preservation for many multiphase systems without recourse to more potent preservatives whose enhanced
Q t tT T10 10= +( ) ( )/
where Q10 is the change in activity per 10°C change in tempera-ture, t(T) is the death time at temperature T°C and t(T+10) is the death time at (T + 10)°C.
Preservatives respond differently to temperature changes and this must be taken into account when predicting activity and when recommended storage temperatures are different to those of preservative efficacy testing conditions.
Water availabilityIt is commonly believed that preservatives are usually far less effective at low Aw and virtually inactive at the very low Aw levels expected of powders, tablets or capsules, although there is only limited published work to support this. Low levels of various electrolytes have long been known to influence the activity of phenolic disinfectants, often with enhancement of effect [104]. Cooper [105, 106] and Anagnostopoulos and Kroll [107] gener-ally found marked reductions in the efficacy of phenolic and other disinfectants and preservatives in solutions with the Aw appreciably lowered by addition of higher concentrations of sucrose, glycerol and similar glycols. There is good anecdotal evi-dence that manufacturers generally find the efficacy of preserva-tives in syrups and cosmetics such as toothpastes to be appreciably weaker than in simple aqueous solutions with high Aw. Bos et al. [108] found that the incorporation of parabens and sorbate into lactose-starch tablets contaminated with Bacillus brevis spores did not reduce subsequent spore viability while maintained at a suit-ably low Aw. Ethylene oxide [109] and high temperatures [110] are markedly less effective as sterilizing agents for powders and “dry” products with very low Aw, when compared with medicines with high Aw. It might be extremely difficult to devise an experi-ment to evaluate fully the in situ efficacy of preservatives under the very low Aw conditions of powders on vegetative microbial cells, since attempts to assess survivors would generally raise Aw and activate the preservatives during the recovery phase. When the Aw of the external environment is lowered below the osmoreg-ulatory capacity of a microbial cell by non-permeant solutes, growth and other activities cease or are reduced to very low levels. This is due to the inability of the cell to accumulate sufficient intracellular water for the necessary metabolic reactions to occur [111]. It is possible that the apparent minimal efficacy of pre-servatives at very low Aw indicates a similar critical influence of intracellular water levels upon antimicrobial reactions.
Chemical interactionsPreservatives can react with polymeric suspending and thickening agents, such as tragacanth, alginates, starch mucilage and poly-ethylene glycols, by displacement of water of hydration, even to the extent of forming insoluble sticky complexes [102, 112]. Cyclodextrins have also been found to interact with a variety of preservatives [113]. However, there are also reports of en -hancement of activity for some preservative/polymer combina-tions [114].
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maintain organisms at an acceptable level to prevent spoilage and/or the delivery of high levels of microorganisms or specific pathogens to the recipient.
Concerns over preservative toxicity by medicine licensing authorities means that applicants for marketing authorizations must fully justify the inclusion or exclusion of any preservative in a formulation, and are expected to adopt alternative strate-gies for product protection where realistic. With cosmetics, reg-ulatory attitudes often differ, placing the emphasis upon the use of preservatives with acceptable levels of toxicity at specified concentrations, and placing exclusions on others. There must therefore be a balance between the presence of microorganisms with their potential infective risks and the toxicity of the preservative.
It is essential that adequate precautions be taken in the manu-facture and packaging of medicines and cosmetics to minimize the access of microorganisms. Awareness of the microbial quality of raw materials is essential, especially those of natural origin which may have a high initial microbial load. For UK medicines, the British Pharmacopoeia (BP) indicates microbial limits for raw materials both in terms of bioburden and absence of particular organisms (Table 17.3). Good manufacturing practice (GMP) also includes attention to atmospheric control, appropriate design of premises and equipment processes and training of staff [4, 5].
Sterile products must demonstrate, by the sterility test or vali-dation of the sterilization process, that when released for use they contain no viable microorganisms. Sterile products being used for multidose purposes and non-sterile products contain-ing a preservative must then demonstrate adequate preservation throughout their use by adherence to the efficacy of antimicro-bial preservation test (see below). Non-sterile finished products
antimicrobial potency is matched by unacceptable levels of irri-tancy. Provided the problem of preservation is fully explored at the earliest stages of formulation development, it is sometimes possible to reduce the worst interactive effects by knowledgeable selection of ingredients and preservative(s) to maximize the levels of “free” agent in the aqueous phase.
Chemical stabilityFinally, the chemical stability of the preservative must also be considered, for example with respect to the processing procedures of the product. Thus, isothiazolinones [125] and bronopol [102] deteriorate significantly if processing temperatures exceed 55°C. Chlorobutanol is unstable around neutral or alkaline pH and suffers appreciable destruction at autoclaving temperatures [121]. Unless light-proof containers are used, the photocatalyzed dete-rioration of preservatives such as the phenolics and quaternary ammonium and organomercurial agents may become significant. Paraben loss will occur by steam distillation at process tempera-tures approaching 100°C and has poor stability in slightly alkaline and above products [126]. Transesterification between parabens and polyols, such as sorbitol, may occur and result in significant loss of activity [127]. Alternatively, the formaldehyde-releasing agents depend on suitable conditions to provide a slow, steady decomposition and release of formaldehyde. This ensures pre-servative protection over the full life of the product, without too rapid a conversion early on in the product’s life leaving little reservoir for the later stages of use; an example is pH 5–6.5 for bronopol [128]. It cannot be presumed that preservative degra-dation products will be inert. Thus, an excessive rate of for-maldehyde release might create undue irritancy, and bronopol degradation releases nitrite ions, which might result in the forma-tion of potentially toxic nitrosamines if ingredients such as amine soaps are present in the formulation or if it comes into contact with dietary amines [21, 128].
Use of preservatives in medicines and cosmetics
The preceding sections of this chapter have indicated not only that the survival of contaminant microorganisms in medicines and cosmetics may present serious risks for both users and the formulations themselves, but also that the use of antimicrobial agents to limit these risks will introduce additional problems. This is due to the relatively non-specific interactive nature of preservatives, readily combining as they do with formulation ingredients and users, as well as microbial contaminants.
There is general acceptance that preservatives should only be included in formulations to deal with possible contamination during storage or use of a product. Formulations that require a preservative generally fall into two categories: (i) those that are required to be sterile but are delivered in multidose containers (e.g. injections, eye drops/ointments, applications to wounds) where the preservative is necessary to ensure safety throughout its use; and (ii) those that are not required to be sterile but must
Table 17.3 Microbiological test requirements for selected raw materials (adapted from European Pharmacopoeia 2010 texts).
Raw material TAMC TYMC Absence of
Acacia 104 102 Escherichia coli, SalmonellaAgar 103 102 E. coli, SalmonellaAlginic acid 102 E. coli, SalmonellaAluminum hydroxide 103 102 E. coli, bile-tolerant
Gram-negative bacteriaBentonite 103
Gelatin 103 102 E. coli, SalmonellaKaolin 103 102
Lactose 102 E. coliPancreatin E. coli, SalmonellaSterculia E. coliTalc, purified (cutaneous use) 102
Talc, purified (oral use) 103 102
Tragacanth 104 102 E. coli, SalmonellaXanthan gum 103 102
TAMC, total aerobic microbial count (cfu/g or cfu/ml); TYMC, total yeast/mold count (cfu/g or cfu/ml).
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medicines [10]. Occasionally, a higher risk of infection for a product justifies the use of preservatives considered too toxic for general application, such as the allowance by the US Food and Drug Administration (FDA) of organomercurial preservatives for cosmetics to be used around the eye, but not for other body-area products [21].
The longer a product is in use, the greater the opportunity for contamination to accumulate and the chance of growth and spoilage to ensue. Medicines prepared extemporaneously under Section 10 exemption of the Medicines Act (1968) are dispensed with short shelf-lives in an attempt to reduce the risk of growth and contamination. Oldham and Andrews [85] found that con-tamination could be held at reasonable levels in many types of unpreserved eye drops for up to 7 days, provided they were also stored in a refrigerator.
MedicinesParenteralsDue to concerns over toxicity and the build-up of contamination in multidose vials [129], preserved multidose containers of injec-tions have been largely replaced by sterile single-dose units without the need for preservatives, leaving only multidose units for parenterals such as campaign vaccines and insulin [130]. Organomercurial preservatives are rarely used in new formula-tions due to concerns of toxicity and have been replaced mainly with benzyl alcohol, phenol, phenoxyethanol and occasionally parabens [131]. Thiomersal has long been used in the preparation and preservation of vaccines and there is some suggestion that it has a positive effect on the efficacy of the antigen. Although no evidence of harm is presented it is discouraged for use in infant and toddler preparations and a general move towards its removal from formulations is supported, although the issues of reformula-tion and validation are acknowledged. However “the balance of risks and benefits of thiomersal-containing vaccines is considered positive” by current government guidelines [132]. Benzyl alcohol is still used, but not for injections that might be used in children. Preservatives are not permitted in solutions for direct injection into spinal, cranial or ophthalmic tissues, or in doses of greater than 15 ml, where the risks of toxic damage become greater [133, 134]. As a consequence, these must always be supplied as single-use vials or ampoules. Preservatives are no longer included in oily injections, as they are considered to be ineffective in non-aqueous systems (see Chapter 3).
Eye dropsMultidose containers of eye drops are still widely supplied for domestic use, due the perceived high cost of single-dose units. These require good preservative protection to minimize the appreciable risk of infections from organisms such as Pseu-domonas and Staphylococcus [135], to which the damaged eye is particularly susceptible. Benzalkonium chloride, often in combi-nation with ethylenediamine tetraacetic acid (EDTA), now appears to be the most commonly used preservative, with chlo-rhexidine and organomercurials occasionally reported [10].
must also adhere to certain regulatory guidelines on total viable counts (TVC) and the absence of particular objectionable micro-bial species (Table 17.4). Total bioburden is an indication of the “cleanliness” of the material and objectional organisms are indicative of pathogenic or opportunistic pathogens. Unfortu-nately the requirements of various National Pharmacopoeias differ and limits applied “in-house” must often be based on a compilation of official critieria. The International Conference on Harmonization (ICH) was set up in the late 1990s to harmonize pharmacopoeial criteria between Europe (EP), USA (USP) and Japan (JP) and is currently still in progress. Within the UK, if a monograph is present in the EP it will take precedence over a BP monograph.
In general, the more potent antimicrobial agents are usually associated with problems of toxicity. There is only a limited range of materials with both reasonable preservative efficacy and acceptably low toxicity, and extremely few with sufficient poten-tial to kill bacterial spores. In complex multiphase formulations, attenuating preservative interactions are so appreciable that it can be very difficult to achieve more than weak antimicrobial efficacy. This is reflected in the low efficacy criteria set for creams by the BP, and other pharmacopoeias, efficacy test protocols (see below). The difficulty of adequately balancing efficacy with toxicity con-siderations has led to an almost complete shift from preserved multidose units to sterile single-dosage forms for parenteral
Table 17.4 Acceptance criteria for microbiological quality of non-sterile dosage forms (adapted from European Pharmacopoeia 2010 texts).
Route of administration
TAMC TYMC Specified microorganisms
Oral, non-aquous 103 102 Absence of Escherichia coliOral, aqueous 102 101 Absence of E. coliRectal 103 102
Oromucosa, nasal, auricular
102 101 Absence of Staphylococcus aureus, Pseudomonas aeruginosa
Vaginal 102 101 Absence of S. aureus, P. aeruginosa, Candida albicans
Transdermal patches 102 101 Absence of S. aureus, P. aeruginosaInhalation 102 101 Absence of S. aureus, P. aeruginosa,
bile-tolerant Gram-negative bacteriaOral, natural raw materialsa
104 102 Absence of E. coli, Salmonella, S. aureus, ≤102 cfu bile-tolerant Gram-negative bacteria
Herbal medicines, boiling water added
107 105 ≤102 cfu E. coli
Herbal medicines, no boiling water added
105 104 Absence of E. coli, Salmonella, ≤103 cfu bile-tolerant Gram-negative bacteria
TAMC, total aerobic microbial count (cfu/g or cfu/ml); TYMC, total yeast/mold count (cfu/g or cfu/ml).a Special EP provisions for oral dosage forms containing raw materials of natural origin where antimicrobial pretreatment is not feasible and where the TAMC of raw material exceeding 103 cfu is acceptable.
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recommended the use of water vapor-resistant film coatings to reduce vapor uptake and assist in the maintenance of low Aw for bulk-packed tablets. The main protection must, however, remain adequate Aw reduction during manufacture and the use of water vapor-resistant packaging. It is understood that the UK licensing authorities will not condone the incorporation of preservatives into new tablet formulations.
Some medicinal ingredients have an intrinsic capability to inactivate likely microbial contamination, and no additional pre-servative is then necessary. Thus, lindane cream, some alkaloid solutions, furosemide (frusemide) injection, some local anes-thetic injections and some broad-spectrum antibiotic creams are able to cope with contaminants adequately without the need for additional preservation. However, the mere presence of an anti-biotic should not be presumed automatically to provide an ade-quate spectrum of preservative cover; there is still the need for a full efficacy-testing program.
CosmeticsThere is some difference of approach to the preservation of cos-metics compared with that for medicines. It is generally accepted that cosmetics are for use on a more restricted range of body sites involving healthy skin, and occasionally membranes, or around undamaged eyes, with lower risks of infection from contaminants than for some medicinal routes of administration. Contamina-tion and spoilage possibilities for some cosmetics, however, may be high, due to their physicochemical complexity and the high potential for consumer abuse, such as regular fingering, repeated contact with saliva, repeated and communal application and pos-sibilities for in-use dilution of remaining product, such as for shampoos or soap in the shower [139]. Although some manufac-turers do not include preservatives in formulations such as dusting powders, block cosmetics, lipsticks, stick deodorants or alcohol-based perfumes with low Aw, many others do so for added reassurance and to cater for in-use abuse. Considerations of pre-servative toxicity, irritancy and sensitizing potential take into account the duration of contact and regularity of use on healthy skin for stay-on cosmetics, and the general levels of adverse reac-tions to other formulation ingredients. Higher levels of poten-tially more problematic preservatives may be used in rinse-off cosmetics, where the period of contact may be short and signifi-cant dilution will take place during application. Accordingly, many agents are used which would be considered too toxic for medicinal applications. Where the risk of infection by contami-nants is deemed to be higher than for most situations, preserva-tives with greater efficiency may be used, despite their increased toxicity potential, such as the use of organomercurial agents in eye-area cosmetics.
Voluntary disclosure to the FDA revealed the use of over 100 preservatives for cosmetics in the USA [140, 141]. Parabens were by far the most commonly used preservatives, followed by imidazolidinyl urea, isothiazolinones, Quaternium 15, formal-dehyde, phenoxyethanol and bronopol. The range of preserva-tives now in use in the EU is considerably less, but it is believed
However, concern at the appreciable damaging effect of quater-nary ammonium antimicrobial agents on the cornea and their involvement in “dry eye” syndrome has resulted in the widespread use of unpreserved eye drops and artificial tears, usually in single-dosage units, for people suffering from this and related problems [136].
CreamsThe complex distribution of preservatives in creams makes it difficult to obtain rapid inactivation of contamination. However, the major risk is seen as that of spoilage rather than of infec-tion, and the poor levels of inactivation achieved with those preservatives considered to be sufficiently non-irritant for medicinal use, is accepted by licensing authorities as the best that is possible. Parabens are again by far the most commonly used preservative, with chlorocresol and benzyl alcohol lagging well behind [10]. Formaldehyde-releasing agents are used, but not widely, and the isothiazolinones are not considered suitable for potentially damaged skin. Most non-aqueous ointments are unpreserved, as the risk of accumulation and replication of contaminants is considered to be low. For high-risk areas, such as the eye, sterile ointments are used. Many UK medicinal creams and ointments which might be used on damaged skin are supplied to microbial specifications approaching those for sterile products.
OralsParabens are also the most commonly used preservative for oral aqueous medicines, probably due to their long usage with appar-ent safety [137] and the need to perform expensive oral toxico-logical evaluation if replacement systems are used. Weakly alkaline medicines, such as antacid suspensions, are difficult to preserve as parabens are relatively unstable at these pH levels [138]. Chlo-roform has been an excellent preservative for oral products sup-plied in well-sealed containers and with a short use life, but it is now banned in some countries over fears of toxicity. Although it may still be used as a preservative in UK medicines it is unlikely to pass regulatory approval as a new product constituent. Oral medicines supplied as a dry powder for reconstitution prior to use usually require a preservative to cope with possible in-use contamination once dispensed. Many contain parabens, although the presence of large amounts of sugar or other solutes to provide a low Aw solution for additional protection against spoilage, as well as for taste, often reduces their efficacy.
TabletsThere are suggestions that the inclusion of preservatives into tablets would give protection should they become damp during storage or use [80]. Bos et al. [108] suggested that this might be appropriate for tablets for use in tropical and humid environ-ments. However, if tablets became damp, they would be inher-ently spoiled as the low Aw also offers protection against non-biological degradation, which is accelerated in the presence of water, as well as being physically damaged. Whiteman [79] has
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The effectiveness of some preservatives can be enhanced by the presence of a variety of materials which in themselves are not strongly antimicrobial. Thus, the chelating agent EDTA usefully potentiates the activity of quaternary ammonium agents, ethanol, parabens, phenolics and sorbic acid among others [151], and is now used to a significant extent in cosmetic formulations. It is also used to potentiate various preservative systems towards pseu-domonads and similar microorganisms in medicines, particularly those for use in and around the eyes [152]. Essential oils and fragrances [153] and humectants, such as glycerol and proplylene glycol, in modest concentration can assist in overall protection. The gallate ester antioxidants, butylated hydroxyanisole (BHT) and butylated hydroxytoluene (BHA) also reveal modest antimi-crobial activity, which can assist conventional antimicrobial pre-servatives [154].
Biochemical explanations for such enhancements of antimi-crobial activity are not always clear. However, in a number of cases, there is good evidence to indicate that enhancement is related to the ability of chaotropic agents, such as EDTA, to dis-organize lipopolysaccharides in the outer membranes of Gram-negative bacteria, a well-known barrier to the penetration of many agents [155]. Phenoxyethanol [156] and benzalkonium chloride [155] are known to disrupt the barrier properties of the cytoplasmic membrane. In both cases, this would then aid greater penetration of the main preservative to its site of lesion. In other cases, such as the synergy of chlorocresol with phenylethanol [157] and acetates with lactates or propionates [158], there are indications of more specific mechanistic interactions.
Synergy is the activity observed with combinations of agents greater than that anticipated from the sum of their activities when individually applied. It is often confused with cases where a widening of antimicrobial spectrum, or simple addition, has been observed. True synergism is often species-specific and most apparent at quite specific ratios of the agents involved. Practically useful examples of preservative synergy include mix-tures of parabens [159], parabens with imidazolidinyl ureas [149], parabens with phenoxyethanol [150], chlorocresol with phenoxyethanol [157], parabens with acrylic acid homopolymers and copolymers [159], and others [142]. Laboratory techniques, such as those described by Pons et al. [160] and Gilliland et al. [159], often demonstrate synergy between combinations of anti-microbial agents, although only a few prove to be of effective value in practical situations. It is therefore essential to confirm any apparent indications of synergy by full testing in complete formulations before placing any commercial reliance upon them.
Antagonism between preservatives is uncommon [160], but there are reports of antagonism between sorbic acid and parabens [161] and between benzalkonium chloride and chlorocresol [160].
The limited list of currently authorized preservatives is likely to increase the use of combinations of preservatives and enhanc-ers. This approach together with hurdle technology is also a likely way forward to exploit the use of low-level chemical preservation [162].
that those in most common usage are comparable to those in the USA, with parabens still topping the range. Sterile cosmetics are not in common use, except for eye conditioning, brighten-ing and coloring drops, which should be supplied sterile, and preserved if in multidose containers. The range of preservatives and their applicability to cosmetic protection is indicated in the new Cosmetic Product Regulation (see below) and references [142, 143].
Other attempts to reduce in-use contamination include the replacement of wide-mouthed jars (with ready access for fingers) by flexible tubes for creams and ointments, the redesign of bottles to reduce the accumulation of liquid residues around the mouth and neck and the introduction of plastic “squeezy” eye drop bottles instead of the conventional glass-dropper bottles [144]. Brannan and Dille [145] also found that slit-top and pump-action closures provided greater protection for shampoo and skin lotion than a conventional screw-cap closure. Wet in-use bars of soap have also been recognized as a source of microbes and there has been a move towards liquid soap dispensers in an attempt to reduce hand contamination.
Potentiation and synergyThere have been numerous attempts to enhance preservative effi-cacy by using preservatives in combination with each other or together with various potentiators [146]. For this strategy to be successfully applied, there must be clear evidence of enhancement for any selected combination, rather than the creation of multi-component preservative systems on the basis of wishful thinking.
It is generally believed that, by using combinations of parabens, each approaching its aqueous solubility maximum, greater levels of unbound paraben will remain in the aqueous phase of mul-tiphase systems, with some evidence that this offers enhanced preservative protection for such formulations [147]. It may also be possible to reduce the extent of such migration into the lipophilic regions by the addition of hydrophilic co-solvents to modify oil : water and micellar distribution coefficients more in favor of preservative retention in the aqueous phase. For example, Darwish and Bloomfield [148] were able to improve the efficacy of parabens in an emulsion by the incorporation of modest con-centrations of ethanol, propylene glycol or glycerol as hydrophilic co-solvents.
Individual preservatives are sometimes less effective against certain microbial species than others, and the careful selection of preservative combinations can offer protection from a wider range of likely contaminants. Thus, it is believed that mixtures of methyl, propyl and butyl parabens have a wider combined anti-microbial spectrum than each individual ester alone [147]. Imi-dazolidinyl ureas have weak antifungal properties and parabens are less effective against pseudomonads, but combinations of both yield usefully improved protection against both contami-nants [149]. Combinations of parabens with phenoxyethanol are also reported to provide a wider spectrum of activity than either alone [150].
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Regulatory bodies generally place the onus on applicants to fully justify the safety, effectiveness and stability of a proposed medicine, including the steps that have been taken to assess and minimize the risks of microbial contamination and spoilage by all relevant means. Where preservatives are deemed necessary, preservative efficacy tests must demonstrate adequate protec-tion throughout the life of the product, and success in the appropriate national pharmacopoeial efficacy test is usually taken as a minimum requirement. Evidence of the safety of any preservatives used is also required, together with reasons for inclusion, details of labeling and methods of control in the fin-ished product. Lists of approved preservatives are rarely issued, although some official compendia, such as pharmacopoeias, may give indications of possible preservatives for various pur-poses. Acceptance of the suitability of the proposals, including preservatives, usually depends on the panels of experts, who assess the choices in the light of the desirable balanced against the possible. When a preservative system is chosen which has been in common usage for similar medicines, the amount of toxicological data required by licensing authorities is usually considerably less than that for newer and less established pre-servatives. This tends to encourage applicants to go for the former, despite the possible advantages of the latter. The high cost of extensive toxicological testing for preservatives has mini-mized the likelihood of novel agents being brought into use. In both the EU and the UK, there is an obligation on producers of cosmetics to include microbiological risk assessments in the development process, to take steps to limit such risks and to record this in the PIP. The choice of preservatives is restricted by lists of banned, approved, provisionally approved and restricted-use preservatives. The FDA strongly recommends that preservative tests are carried out to prevent subsequent product failure and prosecution should defective products be offered for sale. Applications for cosmetic product licenses in Japan must include full risk assessments for microbiological problems, including details of preservative efficacy testing and a full toxic-ity evaluation. Restrictive lists of approved ingredients are published.
Various other regulations will have an impact on preserva-tive usage, such as the banning of chloroform as a preserva-tive, except for medicines in the UK (SI 1979 No. 382) and in all products in the USA, due to some reports of carcinogenic-ity in animals. Detailed environmental impact assessments will be required for preservatives (and other ingredients) under environmental protection legislation being brought into effect in most western countries, since cosmetic and medicinal components will eventually be disposed of into the biosphere. Increasingly strict direct product liability laws may offer a clearer route to compensation for users who believe they have suffered damage from a microbiologically inadequate medi-cine or cosmetic. International reviews of the legislation and impact relating to damage from contact dermatitis have been made by Frosch and Rycroft [168] and Hogan and Ledet [169].
Regulatory aspects of the preservation of medicines and cosmetics
European Community Directives are legislative instruments and must be implemented into national legislation within a defined period of time. The EC Directive 65/65/EEC (1965) laid down original standards and procedures for the safe and effective use of medicines within the EU and was reflected in the UK Medi-cines Act 1968. A marketing authorization from the relevant licensing authority must be obtained before a company can market a product. Detailed scrutiny of the quality, safety and efficacy data for the product will be examined and this will include any data on excipients such as preservatives. In the UK the regulatory authority is the Medicines and Healthcare Prod-ucts Regulatory Agency (MHRA), however European-wide approval may now also be obtained through relevant member state mutual recognition procedures or alternatively as a cen-tralized submission to the Committee for Medicinal Products for Human Use (CHMP) Scientific Committee, overseen by the European Medicine Agency (EMEA). An analogous system operates in the USA for medicines via the Federal Food, Drug, and Cosmetic Act, as Amended (Title 21 USC, 350 et seq.), enforced through the FDA. Similar control of medicines in Japan is made under the Pharmaceutical Affairs Law (No. 96, 2002), administered through the Pharmaceutical and Food Safety Bureau.
Specific formal control of cosmetic safety across the EU com-menced in 1976 with Council Directive 76/768/EEC, followed subsequently by the new Cosmetic Product Regulation 1223/2009, which aims to phase out animal testing by 2013. From January 1997, disclosure of cosmetic ingredients including preservatives had to be made on labels [163]. Cosmetics in the UK are con-trolled under the Cosmetic Products Regulation and are regu-lated via the Department of Industry. There are obligations for suitable qualified persons to carry out safety assessments for the manufacturer to maintain detailed product and processing information in a product information pack (PIP). However, regulatory action can only be taken once the product is offered for sale and believed to be defective. In the USA, cosmetics are regulated by the FDA through the Federal Food, Drug, and Cos-metic Act (2002); again action can only be taken once the product is offered for sale and is believed unsafe. There are only limited lists of banned substances and no formal ingredient rec-ommendations are made.
Japanese control of cosmetics is made through the same laws as for medicine (Pharmaceutical Affairs Law, established in 1960) where prior approval by the Pharmaceutical and Food Safety Bureau is required before a cosmetic may be placed on the Japa-nese market.
The following publications provide a wider insight into the legislative arena: EU and UK medicines [164, 165], EU cosmetics (Cosmetics Products Regulation 2009), USA medicines and cos-metics (http://fda.gov; [166]) and Japanese regulations [167].
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creams or gels are to be examined, it may be necessary first to disperse them in an aqueous medium; this can be done either directly or with the aid of a dispersant such as a surfactant. For some solid products (e.g. soap bars), however, direct surface inoculation is a more realistic option [172, 181]. Single-species inocula are more commonly used than mixed challenges, although there might be potential advantages to multiple-species inoculation. The latter is not generally used for medicines [170] but is often used for cosmetic testing [173, 181, 182]. Inocula are commonly added as a single large challenge (single-challenge testing) and monitored for inhibition over a specified period. Multiple challenges (repeat-challenge testing), which repeat the inoculation at set intervals for a specified number of cycles or until the product fails, are also used, particularly for cosmetics [177, 183, 184].
Preservative efficacy is estimated by cultivating and counting survivors from small aliquots of the inoculated formulation added to culture media, either by conventional colony formation or via membrane filtration or by using most probable number schemes with liquid media [185]. This presumes an initial uniform distribution of inoculum and any subsequent growth in the product, both assumptions being unlikely to be correct [171]. Neutralization of residual preservative must be ensured before recovery is undertaken. Appropriate neutralizers have been docu-mented [186], however work carried out by Johnston et al. [187] describes a rapid method to assist in the choice. Many of the standard laboratory media are somewhat stressful to damaged microorganisms [188]. Confirmation must be sought that the recovery procedures are capable of resuscitating viable but damaged microorganisms, and that they are not inhibitory.
For the purpose of preservative efficacy testing of medicines, products are divided into groups, each having their own compli-ance criteria (Table 17.5). The number of categories described in the BP [189], EP [190] and USP [191] monographs are now similar after changes to the USP (although the content of some groups vary), although compliance criteria are still less stringent in the USP. The basic test uses four stock cultures of Aspergillus niger, Candida albicans, Pseudomonas aeruginosa and S. aureus, which may be supplemented with other strains or species that may represent likely challenges for that product. Most mono-graphs will detail the “preparation of inoculum” as variation in this can have a considerable effect on preservative sensitivity. Studies have been carried out to validate alternative preparative methods [192]. A single challenge of 105–106 microorganisms/g (or ml) of formulation is used; the product is incubated at 20–25°C and aliquots are tested for survivors at specified intervals by conventional plate count or membrane-filtration techniques. Two levels of criteria, A and B, are given for acceptable performance in the test – level A being the recommended level of efficiency, except where this is not possible for reasons such as toxicity, and then level B applies. The relatively weak compliance criteria for some formulations are indicative of the problems in achieving adequate preservative efficacy in complex products and potential toxicity issues.
Prediction of preservative efficacy
Due to the many interactive possibilities for both microorganisms and preservatives in complex formulations (see above), it is almost impossible to predict, with any reasonable degree of preci-sion, the ultimate effectiveness of a preservative in all but the simplest solutions. It is therefore necessary to obtain some assur-ance of likely in-use and abuse performance, by conducting a direct microbiological preservative efficacy test on the complete formulation. Detailed reviews of such test procedures and the problems associated with them have been made by Baird [170] for medicines and by Brannan [171], Perry [172] and Leak et al. [173] for cosmetics, while Hopton and Hill [174] reviewed test methodology for a wider range of commercial materials.
Most conventional preservative efficacy testing protocols share common features and intentions, although the fine details and interpretation of the results vary significantly. Aliquots of com-plete formulations should be tested in their final containers, where possible, as these can influence overall efficacy [145, 175]. The testing of diluted cosmetic products should also be consid-ered where dilution in use might occur, such as with shampoos in bottles or bars of soap taken into the shower or bath, as well as the addition of an organic load to simulate in-use soiling [143, 171, 176]. Formulations without preservative should be examined for possible inherent inhibitory activity. Samples should be tested within a general stability test program to determine whether pre-servative efficacy will remain throughout the intended life of the product. A limited range of test microorganisms, representative of likely contaminants, is usually selected from official culture collections, and often supplemented in individual companies with known problem strains such as osmophilic yeasts for sugary formulations, “wild” strains from the factory environment or those isolated from previously spoiled batches [177]. Usually, only elementary methods of cultivation and harvesting of inocula are used, despite much evidence that even minor variations here can dramatically influence the antimicrobial sensitivity of the result-ant test suspension [178, 179]. In general, routine cultivation is well known to attenuate survival and spoilage potential appreci-ably, and numerous attempts have been made to develop main-tenance systems that retain the aggressiveness of wild isolates, often with only limited success. Thus, Spooner and Croshaw [177] cultivated contaminant isolates in unpreserved product, and Flawn et al. [180] maintained the shampoo-degrading activ-ity of tapwater isolates by routine cultivation in mineral salt media containing anionic surfactants. The ability of pseudomon-ads and related environmental isolates to degrade parabens, phe-nolic preservatives and a variety of surfactants could be retained over considerable periods by routine cultivation in minimal liquid media containing the agents as the primary carbon source.
Formulations are usually challenged by intimate mixing with the microbial suspensions and incubation at temperatures rele-vant to likely use conditions. Where solid formulations (e.g. powders or cakes), oily or waxy products (e.g. lipsticks), viscous
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purposes, the short time of the test protocol necessitates addi-tional testing to check for possible regrowth phenomena after long delays.
Conventional preservative challenge test procedures are time consuming and expensive, so attempts have been made to develop and assess alternatives [186, 200]. Impedance changes during the growth and death of microorganisms can be detected and used for rapid preservative efficacy screening [201, 202]. Other methods to estimate cell viability such as direct epifluorescence (DEF) and adenosine triphosphate (ATP) bioluminescence have been exam-ined and considered unlikely alternatives to positron emission tomography (PET) [203]. However, more recent workers have included them together with flow cytometry, polymerase chain reaction (PCR) and immunoassays as rapid methods in use for the microbiological surveillance of pharmaceuticals, ultimately leading to real-time monitoring [204].
Adverse reactions of users to preservatives
The non-specific and reactive nature of preservatives not only results in interaction with many formulation ingredients (see above), but is also reflected by incidences of adverse reactions of users to preserved products. Significant incidences of senitization and dermatitis have been recorded to most of the commonly used preservatives at a frequency of approximately 0.5–1.0% of those tested. However, this needs to be seen in the context of overall levels of around 5% senitization to all cosmetic ingredients [205–208]. Regulatory activity exerts appreciable control to limit the risks of adverse reactions, by detailed specification of toxicity testing requirements, as well as attempting to allay public con-cerns over the use of animals for the purpose [209]. Screening studies of sensitivity are periodically carried out and highlight emerging allergens. For example, increased sensitivity to methyl-dibromoglutaronitrile was reported [210, 211], resulting in the
Although preservative efficacy evaluation with panels of vol-unteers, using test formulations under controlled conditions, is not generally realistic for medicines, this type of follow-up test is quite common for cosmetics [193, 194]. Thus, Farrington et al. [195] developed a panel test whereby volunteers applied the test products for a specified number of times to axillary areas, ensur-ing that the application fingers came into contact with residual product. Formulations were then examined for any accumulated contamination. There is some agreement that results obtained from in-use panel tests do show a reasonable correlation with estimates obtained from in vitro challenge testing and general in-use performance for cosmetics, including the ability to dif-ferentiate between products which subsequently perform well during use and those which do not [171, 194–196]. Spooner and Davison [197] compared the performance of an extensive array of medicines in the BP efficacy test with levels of contamination detected in used and returned medicines. They concluded that compliance in the official test generally indicated products that would perform adequately in the marketplace. Fels et al. [198] determined that a wide range of European preserved medicines found to be microbiologically reliable over many years gave pre-dictive indications of failure when submitted retrospectively to the BP efficacy test. Applicants for marketing authorization in the UK for a new medicine must normally demonstrate that, if a preservative is necessary, the product at least satisfies the basic compliance criteria of the BP test, as the licensing authority believes that this gives a reasonable estimate of likely microbial stability in use. Orth has promoted an alternative to the con-ventional challenge test, in that, although the methodology is comparable, formal decimal reduction times (D-values) are determined for the inactivation of inocula, and predictions on the efficacy of formulations are obtained by extrapolation of data to estimate times of contact necessary to yield prescribed log levels of reduction [199]. Although there is some evidence to show that reliable information can be obtained for preliminary screening
Table 17.5 Compliance criteria for the efficacy of antimicrobial preservation, European Pharmacopoeia 2010.
Type of product Type of inoculum Level criteria Required log10 reduction of inoculum by time shown
Topical preparations Bacteria A – – 2 3 – NIB – – – – 3 NI
Fungi A – – – – 2 NIB – – – – 1 NI
NI, no increase in numbers over previous count; NR, no organisms to be recovered.
Section 2 Practice
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absorption following the use of cord dusting powders containing hexachlorophene on neonates and its application to burned and damaged skin or mucous membranes [226]. More recently, reports relating to both parabens [227] and tricolsan [228] have been associated with antiandrogenic effects and the topical administration of parabens through underarm body care prod-ucts with breast cancer [229].
European Commission performing an expert review on its safety [212]. A more recent UK review of preservative sensitivity by patch testing identified formaldehyde and the isothiazolines as having the highest positive rates and chloroxylenol the lowest, with parabens having the highest irritancy rate [213].The risk of preservative damage will be related to the frequency and duration of product contact, the route and site of administration as well as the concentration of preservative used. Thus, preservatives in rinse-off shampoos might be expected to present lower risks of senitization than those in prolonged-contact products, such as stay-on creams. Direct injection into the central nervous system or ophthalmic tissue is far more likely to be damaging than administration by the oral or topical routes.
This section can only illustrate the problems with selected examples, and interested readers are directed to the reviews of D’Arcy [214], de Groot and White [205] and Goon [215] for a more detailed treatment of the topic.
Injections preserved with chlorocresol, chlorobutanol, benzyl alcohol and organomercurials have all induced appreciable hypersensitivity and severe adverse reactions [216, 217]. Benzyl alcohol has been of particular concern with small children, who are unable to metabolize it effectively, and a number of neonatal deaths have been attributed to its use [218, 219]. A variety of eye-damaging reactions have been reported due to preservatives in multidose eye drops, and the particularly distressing condition of “dry eye” has been related to their use [220]. Benzalkonium chloride and other quaternary ammonium preservatives have been found to be particularly damaging to the cornea, by interfer-ing with tear-film stability and direct toxic effects on the cells [221, 222]. Their use with local anesthetic eye drops (which reduce the blink reflex and therefore prolong contact time) is discouraged due to the risk of increased toxicity; single-use minims without preservatives are recommended instead. Neb-ulizers containing antimicrobials have also induced bronchocon-striction in asthmatic patients [57, 223, 224].
The parabens are by far the most commonly used preservatives in cosmetics and pharmaceuticals, reflecting their ability to meet many of the criteria associated with an ideal preservative, despite possessing only modest preservative efficacy [141, 208, 225]. Formaldehyde-releasing agents have been regarded as very effec-tive preservatives for rinse-off cosmetics, but fears over carcino-genicity and sensitivity [211, 213] have limited their use. Topical medicines are implicated as the cause of 14–40% of all allergic contact dermatitis reports, the majority of these, however, being related to the therapeutic agents. There is a rather limited range of preservatives used in medicines, parabens paradoxically are the most commonly used but are reported as the most common irritant [213] and are generally well tolerated [137]. The majority of contact dermatitis reactions recede once the offending product is identified and use ceases. However, re-exposure to the preserva-tive in another formulation will usually provoke further adverse effects [205].
Systemic damage from the topical application of preservatives is rare, but there are reports of serious to fatal reactions from skin
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Chapter 17 Preservation of Medicines and Cosmetics
42 British Pharmacopoeia Commission (2009) British Pharmacopoeia, The Sta-
tionary Office, London.
43 Freund, H.R. and Rimon, B. (1990) Sepsis during total parenteral nutrition.
Journal of Parenteral and Enteral Nutrition, 14, 39–41.
44 Anon. (1995) Accidental death verdict on children infected by TPN at a Man-
