microbiological spoilage in pharmaceuticals and cosmetics

J. Soc. Cosmet. Chem. 23 721-737 (1972) ̧ 1972 Society of Cosmetic Chemists of Great Britain

Microbiological spoilage in pharmaceuticals and cosmetics

R. SMART and D. F. SPOONER*

Presented on 29th September 1971 in London, at the Symposium on 'Microbial control', organized by the Pharmaceutical Society of Great Britain and the Society of Cosmetic Citemists of Great Britain.

Synopsis--Manifestations of SPOILAGE by bacteria, yeasts and fungi are described. TOXIC visible, olfactory and audible effects and changes in texture and taste may be found and susceptible products are reviewed. LIQUIDS, including aqueous solutions and suspensions, syrups, emulsions and creams are particularly at risk. Spoilage of ointments and oils, solid raw materials, powders, tablets and solid COSMETICS also occurs. The involvement of PACKAGING materials and the CONTROL of MICROBIOLOGICAL spoilage is briefly discussed.

INTRODUCTION

A spoiled product may be described as one that has been rendered unfit for use. As pharmaceuticals and cosmetics are consumed by, or applied to, the user, manifestations of spoilage are essentially subjective, spoiled products becoming objectionable or perhaps even therapeutically inactive.

Microbial spoilage can be caused by bacteria, yeasts or fungi which are all extremely versatile in their metabolic activities. This capacity for variation, whether due to mutation in genetic composition followed by selection or to changes in behaviour unaccompanied by genetic change, allows adaptation to a very broad range of environmental conditions. As a result, all classes of natural organic compounds are susceptible to * Quality Control--Microbiology, The Boots Company Ltd, Nottingham, NG2 3AA.

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degradation and synthetic compounds are also attacked, although often less readily. In spite of this, relatively few accounts of the microbial degradation of pharmaceuticals or cosmetics have been published (1). This is partly due, no doubt, to the careful control measures exercised by responsible manu- facturers. It is also due to the difficulties of elucidating the mechanisms involved in a spoilage event. Each tends to be a unique encounter between a specific organism and substrate and often there is only time to record the happening and remedy the situation. In contrast, much has been written on the contamination of pharmaceuticals and cosmetics by micro-organisms (2). If the contaminants are potential pathogens then the products are obviously rendered unfit for use and are therefore spoiled. However, in this paper we are mainly concerned with degradation caused by contaminating micro-organisms. Their clinical significance is dealt with in detail elsewhere in the Symposium.

MANIFESTATIONS AND MECHANISMS OF MICROBIAL SPOILAGE

Before spoilage can occur organisms which are capable of altering the components of a product in situ must first be introduced via raw materials, the processing plant, packaging materials, operatives or elsewhere in the environment. Although spoilage does not necessarily depend upon the growth of these contaminants it is generally facilitated if the formulation and the ambient conditions of temperature and humidity encourage their multiplication. When these criteria are satisfied changes in the product will occur and may ultimately manifest themselves to the user in one or more of the following ways:

Toxic effects Microbial toxins

Several species of micro-organisms produce toxic molecules and may render a product dangerous if they grow in it under conditions supporting toxin production. Endotoxins, produced by Gram-negative bacteria such as Escherichia coli, are intimately bound to the cell, lipopolysaccharide in nature and are not necessarily inactivated by sterilization as they are heat stable. Toxins of this type are poorly absorbed by the oral route but are very important in connection with injectable products, particularly per- fusion fluids. Exotoxins are much more highly lethal and are bound less rigidly to the cell so that they are readily liberated into the growth medium. The outstanding example, of course, is that produced by Clostridium

MICROBIOLOGICAL SPOILAGE IN PHARMACEUTICALS AND COSMETICS 723

botulinum which is lethal to mice in doses of the order of 0.1 ng. Fortunately conditions for growth and toxin production are quite strict; anaerobiosis, the presence of suitable pH and nutrients and of few competing bacteria is required. Such conditions are not often attained in pharmaceuticals and cosmetics and we know of no case of botulism arising from their use. Certain strains of Staphylococcus aureus produce a toxin, characterized as a specific polysaccharide, but the organism must grow to a density of several million cells per gram before its toxin becomes a problem. The evidence in connection with other bacteria, e.g. Clostridium perfringens, Bacillus cereus, Strepto- coccusfaecalis, Proteus and Pseudomonas species is less clear, but poisonous metabolites are certainly produced by a variety of fungi. Over the last decade there has been much interest shown in the aflatoxins produced by Aspergillus flayus (3). These heat-stable compounds exhibit potent toxic and carcinogenic properties in animals. A. flayus commonly infects peanuts, cotton seed and grain which are all components of animal foods. Under poor storage conditions mould growth occurs and toxic doses of aflatoxin accumulate in the food stuff. While it is difficult to visualize this occurring with cosmetics or pharmaceuticals, it is wise to ensure that ingredients such as talc, kaolin or starch are not stored for long periods under conditions supporting mould growth.

Metabolic products In addition to microbial toxins, which are complex molecules and may

be looked upon as biosynthetic products, simpler catabolic products such as organic acids and amines, which can be toxic to man, may be produced. Indeed, many microbial metabolites exhibit pharmacological activity (4). As these compounds are considerably less toxic than are the classic bacterial toxins, relatively high concentrations have to be attained before a spoiled product causes illness and the senses often detect that something is wrong before food spoiled to this extent is swallowed. This may not apply to medicines, as they are expected to be unpleasant and, indeed, frequently contain a flavouring agent in order to mask an unpleasant taste. However, well-documented examples incriminating specific metabolic products in pharmaceuticals are not easy to find.

Irritancy Incidents of irritation following the application of cosmetics occasion-

ally occur, and the offending preparation may subsequently be shown to contain a high level of microbial contamination. Direct evidence that irritation is caused by the presence of the micro-organisms is lacking but it

724 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

is reasonable to suppose that, on some occasions, the contaminants provide a source of foreign protein evoking an allergic contact dermatitis reaction or that high levels of a microbial metabolite will cause a primary irritant reaction. The eye, of course, is particularly susceptible to infection from contaminated cosmetics and it is also at risk from the direct effect of

irritant metabolites left in a product even after the organisms producing them have been eradicated.

Change of activity

An interesting aspect, but perhaps not one of great significance, is the inactivation of biologically active molecules by organisms contaminating a. formulation. Several examples have now been demonstrated in the laboratory and in some cases have been observed to occur in practice. A classic example is the destruction of penicillins by penicillinases, enzymes produced by a broad range of micro-organisms. Microbial enzymes which inactivate chloramphenicol are also known (5) and the destruction of preservatives and disinfectants is established (6). Pharmacologically active substances can also be degraded. For instance Kedzia, Lewon and Wis- niewski (7) found that a loss of atropine of up to 20•o in eye drops could be caused by Corynebacterium and œseudomonas spp. isolated from the eye drops and atropine itself. Recently, Grant, de Szors and Wilson (8) have shown that in the laboratory, a strain of Acinetobacter lwoffi, obtained from distilled water, utilized aspirin as a sole carbon source in a mineral salt solution. The same organism metabolized other active esters; for instance it could degrade heroin to morphine. Another organism, Corynebacterium hoffnaii, which was isolated from laboratory dust, metabolized the anal- gesics aspirin, phenacetin and paracetamol.

Loss of useful activity is not restricted to pharmaceutical products. For instance emphasis on the need for detergents which are biodegradable has had some repercussion and shampoos have been known to lose their surface-active properties due to degradation of the surfactants by contaminating bacteria.

Visible effects Visible growth

When micro-organisms can actually be observed in or on a product then there is obviously no doubt that microbial spoilage has occurred. In fact, this is probably the most common way in which it is manifest. In liquid


formulations contaminants may be seen as a sediment, turbidity or a pellicle while on more solid preparations colonies, often coloured, of bacteria, yeasts or moulds may form.

Colour changes Sometimes visible spoilage is more striking, particularly if a colour

change is involved. Colour changes due to alterations in the components of a product may result from pH, redox or other changes caused by the metabolic activities of an organism, or to pigment production by the contaminants themselves.

Members of the Pseudomonas genus are often implicated in spoilage of this type. These organisms metabolize a very broad range of compounds, and can also produce soluble pigments ranging in colour from blue-green to brown. In addition, they can render conditions suitable for less adaptable spoilage organisms; for example they can create conditions favouring the growth of anaerobes. Similarly, in an acidic product, oxidative yeasts can cause a rise in pH by utilizing organic acids and this will encourage bacterial growth.

Gas production If microbial metabolism produces gas in a sufficient amount to exceed

its solubility in a product, visible bubbles, frothing and other manifestations of an increase in pressure occur. Products containing carbohydrates or other fermentable substrates are particularly susceptible to this type of spoilage. Of the latter, glycerol, an essential ingredient in many cosmetic preparations, is fermented particularly readily by some common waterborne organisms. Other changes

Microbial metabolism can result in the composition of a homogeneous product becoming visibly heterogeneous. Emulsions, for instance, are notoriously susceptible to changes in physicochemical conditions; hydrolysis of the oil phase or changes in the pH of the aqueous phase will upset the equilibrium and thus cause visible changes. In liquid products changes of viscosity can be seen to occur when contaminants have broken down large molecules, utilized sugars or caused the aggregation of particles in suspension.

Olfactory effects

It has long been known that many micro-organisms produce characteristic odours and as early as 1923 a variety of aroma-producing bacteria had been listed (9). These aromas include the highly characteristic ones of sulphur-containing metabolites such as hydrogen sulphide, the sickly


smells of the fatty acids, the 'fishy' odours of the amines and the astringency of ammonia. Often these are combined to produce the 'off' odours of a spoiled product. Changes in the aroma of a product due to contaminants vary from the production of a nauseating smell to a slight change in the bouquet but all can be disastrous, particularly to cosmetic and toiletry preparations which depend so much upon their specific perfumes. One of the most common olfactory warnings of spoilage is the typical smell of mould. The responsible aromatic elements have not been clearly identified but some actinomycetes which taint water with undesirable earthy odours have recently been shown to produce geosmin, a strongly earth-smelling, neutral oil (10). An alcoholic odour, produced from fermentable substrates, is typical of spoilage by yeasts.

Taste

Reports that products taste 'peculiar' are often the first indications that they may be spoiled. The sense of taste varies widely between individuals and these reports do not invariably indicate microbial contamination. For this reason, and because of the hazards involved, taste is not a practicable control procedure with which to detect spoilage at an early stage. Never- theless, the combined senses of smell and taste are highly perceptive to changes in flavour, particularly in bland, unflavoured, preparations where the presence of microbial metabolites is not masked. Margalith and Schwartz (11) have listed over 100 organic compounds involved in the production of flavour by micro-organisms. These consist mainly of alcohols, aldehydes, ketones, acetals, acids, amines, esters and phenols.

Texture

The feel of topical preparations, particularly cosmetic and toiletry ones, is vital to their acceptability but texture may be marred by contaminants. For instance, creams can become lumpy or 'gritty' and changes in viscosity of liquid preparations, which can be detected when applied to the skin, may occur.

Audible effects

Apart from immediate manifestations of toxicity, which happily appear to be rare, audible manifestations of spoilage are the most dramatic. If visible effects of spoilage are obscured by the pack, an explosion can be the


first indication that a gas-producing micro-organism has successfully adapted itself to what may have been considered inimical conditions.

TYPES OF SUSCEPTIBLE PRODUCT

The range and composition of pharmaceutical and cosmetic products is so varied and the species, and even strains, of micro-organism capable of causing spoilage are so multifarious that, as we have already emphasized, each spoilage incident tends to be unique. Generalizations about susceptible products are therefore likely to be inaccurate and are made more difficult today because of the inclusion, particularly in cosmetics, of increasingly sophisticated and often highly. biodegradable ingredients. Never- theless some types of product are more susceptible than others to spoilage by specific organisms and those of which we have experience are described below.

Liquids Water

Water is a major constituent of living material and participates in many metabolic reactions. Bacteria, in particular, require high concentrations of water in their immediate environment and may be regarded as aquatic organisms. Hence, all products containing large amounts of free water can be particularly susceptible to spoilage by bacteria.

Water supplied by water undertakings in this country is of high microbiological quality and is generally suitable for the manufacture of pharmaceuticals and cosmetics. Low-conductivity water, whether prepared by distillation or deionization, may be chemically purer but can constitute a greater microbiological hazard. Distilled water leaving the still can readily pick up organisms from pipes and tubing and ion-exchange columns may actually serve as a reservoir of organisms because nutrient organic residues are not removed by the process. Without effective treatment to minimize contamination, water can, within a few days, contain large numbers of initially Gram-negative and Gram-positive bacteria, and subsequently a wide variety of bacteria, moulds and yeasts. At this stage visible and olfactory spoilage occurs and a foul taste may develop. Indeed contaminated deionized water has often been incriminated as the original source of spoilage in a formulation. Often the responsible organisms are pseudomonads which are not only highly resistant to preservatives but are also able to use the widest range of organic compounds as substrates.


Simple aqueous solutions Some moulds will grow on such unlikely media as strong solutions of

copper sulphate or sulphuric acid and simple solutions of inorganic compounds will support the growth of many sorts of microbe. The presence of organic material greatly increases the chance of growth occurring as it not only provides a utilizable substrate but may serve to introduce contaminants into the solutions. We have detected deposits of turbidity due to algae, moulds, bacteria or yeasts in a multiplicity of different solutions including ammonium carbonate, neutral ammonium tartrate, calcium digluconate, potassium citrate and Amaranth Solution B.P., even when apparently preserved with chloroform.

Many solutions are included in the great diversity of mixtures in the B.P.C. and other formularies. These are often prepared from aqueous con- centrates which are themselves adequately preserved. However, the preservative can be diluted out on mixing and the resulting preparation is then at risk. An example of recent interest is peppermint water which has been implicated as a source of contaminants in formulations containing this ingredient. In at least one case there is evidence that potential pathogens have been transmitted to patients from it (12).

Suspensions Aqueous suspensions of inorganic material for pharmaceutical use fre-

quently support microbial growth, particularly as added preservatives tend to be absorbed and inactivated by the suspended matter. Unless growth is at the surface, as with mould contaminants, it is not easily detected visually because of the opacity of these products. When the lid is removed spoilage is sometimes manifested by an offensive odour or an unpleasant taste, but otherwise large numbers of bacteria may unwittingly be taken with the preparation. Thus, a medicament for the treatment of an intestinal disorder may exacerbate, rather than alleviate, the condition. However, apart from visible growth, a variety of other changes in appear- ance may be seen and preparations of this type can thin, separate, de- colourize, or change colour. The sequential growth of an aerobic organism, particularly a pseudomonad, followed by an anaerobe can cause striking changes in pharmaceutical products. The sequence occurring in cutting oil emulsions, used for cooling during drilling operations in the engineering industry, has been studied by several workers (13). The oils often become blackened due to bacterial action, the first organism lowering the redox potential and allowing the proliferation of anaerobic sulphate-reducing


bacteria, Desulphovibrio or Desulphotomaculum species, which oxidize simple organic compounds and reduce sulphates to hydrogen sulphide. This then deposits iron sulphide due to the abundance of iron present in the environment where the oil is used.

Emulsions

O/w emulsions are particularly susceptible to spoilage as the water in the continuous phase allows contaminants to spread throughout the product. Preservatives generally exert their influence only within this phase, and at its boundaries, but their concentration depends on their relative solubilities in the particular oil and in water and on the oil water ratio in the emulsion (14). In addition to partition effects the activity of preservatives may be further diminished due to inactivation by compounds such as the nonionic emulgents. These possess little bactericidal adtivity and may even be utilized by pseudomonads (15, 16). In a comprehensive review Wedder- burn (17) pointed out that many other materials used in emulsions are susceptible to microbial degradation.

Spoilage in emulsions can be manifest by changes in rheological properties, including separation or 'breaking down'. Discolouration, decolouriza- tion, changes in odour and taste and signs of visible growth also occur.

Creams and lotions

A wide variety of complex cosmetic and toiletry preparations are included under this heading. Apart from the usual ingredients there is a current tendency to employ substances of natural origin including animal proteins and vitamins. These materials are not only highly nutrient to micro-organisms but may inactivate preservatives and even serve as a source of contamination. Glycerol is commonly used in both pharmaceutical and cosmetic formulations and may be metabolized by organisms frequently found in water. For instance, Klebsiella species will frequently produce gas in poorly preserved products when used to challenge creams and lotions containing this component.

Mould growth is one of the most common causes of spoilage of creams of all types and can occur in products as varied as antifungal, calamine, baby and hair creams and a number of other cosmetic formulations including moisture and cleansing creams. The difficulties of preserving these products against mould growth are enhanced by the risk of contamination from containers, the presence of large air spaces and poor storage conditions.


Ointments and oils

As these are anhydrous materials, in theory they do not support the growth of micro-organisms. However, in practice they are often filled into jars or other containers with large air-spaces and this introduces the possibility of mould spoilage, as for creams, because these organisms can utilize atmospheric moisture. Whereas creams can supply moisture by evaporation, oils and ointments require the accidental ingress of water or the presence of a humid atmosphere. Fortunately a much smaller incidence of this kind of spoilage therefore occurs but we have seen a few examples of mould colonies on the surfaces of ointments, including white petroleum jelly, and ironically, fungicidal ointment.

Oils are at a slightly greater risk than ointments as, being more fluid, they allow condensed water to carry organisms to the bottom of the container and remain trapped. Moulds have been reported to metabolize arachis oil and liquid paraffin (18, 19) and obnoxious odours, tastes and slimy deposits have been found in liquid paraffin while clumps of moulds, yeasts or bacteria have been seen in maize and olive oil. Without exception traces of water are found in these spoiled samples and the detection of foreign substances, such as food particles, often indicates the origin of the contamination.

Shampoos Shampoos are particularly susceptible to contamination by a range of

Gram-negative waterborne bacteria (20). Although they may support the growth of large numbers of bacteria without visible spoilage, slimy sedi- ments, pellicles, discolouration and odours are sometimes found and the loss of lathering properties has also been noted. Medicated shampoos are not immune from spoilage because the antimicrobial ingredients they contain often possess only a narrow spectrum of antimicrobial activity. It is almost impossible to keep shampoos away from the eyes of the user and contamination by invasive bateria such as pseudomonads must also render products of this type unfit for use, whether other spoilage has occurred or not.

Syrups The sugar content of syrups inhibits the growth of many micro-organ-

isms by virtue of its high osmotic pressure but osmotolerant moulds and yeasts are a source of trouble. Fermentation of the sugar by these organisms causes foul flavours due to the production of alcohol, lactic acid and other

MICROBIOLOGICAL SPOILAGE IN PHARMACEUTICALS AND COSMETICS 73l

organic acids while the production of carbon dioxide leads to gassing and troublesome pressure increases (21). In addition, these products, including syrup B.P. and various syrup-containing cough remedies, can be spoiled by the presence of suspended or deposited osmophilic moulds. As with other products spoilage in syrup can be accelerated by an excessive air space in the container. Fluctuating storage temperatures then cause sufficient condensation of water vapour to dilute the syrup at its surface so that growth can occur. Cork closures, now happily uncommon, have in the past provided the necessary inoculum of mould spores. Osmophilic organisms may also cause trouble in malt extracts which largely depend on their high concentration of low molecular weight saccharides for preservation. The effect of a number of environmental factors on fermentation caused by Sac- charomyces rouxii in malt extract has been studied (22).

Compounds formulated in syrup may be metabolized with the production of toxic substances. For instance Wills (23) isolated a species of Penicillium from a sample of Syrup of Tolu which smelled of toluene. He showed that this organism could grow on benzoic or cinnamic acid as a sole carbon source and that a toluene-like odour was produced from the latter. The toluene-like product was not characterized because of the presence of interfering substances but rupture of the unsaturated linkage in the cinnamic acid molecule could have yielded toluene itself.

Tinctures, elixirs and linctuses In general, these formulations do not allow microbial survival because

of their high concentrations of alcohol, sugars or glycerol. For instance, even in concentrations as low as 5•o, alcohol will kill most bacteria and moulds in time, while yeasts are generally killed at concentrations above 15}/o . Nevertheless isolated incidents of spoilage due to suspensions or deposits of dead mould have occurred. The organisms presumably grow in the container for a while before becoming immersed by the formulation and succumbing.

Solids

Raw materials

Solid raw materials may serve as a source of contaminants which will later spoil a formulated product. Natural earths such as kaolin, bentonite, Fuller's earth or french chalk contain anaerobic spore-bearing rods, moulds or Gram-negative bacteria which can render a product objectionable or

32 JOURNAL OF THE SOCIETY OF COSMETIC CHEM1STS

even dangerous. These materials are best sterilized by a gaseous or heating process before formulation. Solids of biological origin, including egg and milk products and dried animal and plant extracts, may also contain pathogens, including salmonellae, E. coli and staphylococci. Spoilage of the solid raw matehal itself is largely due to mould growth on the surface due to improper storage with inadequate coverings in a damp enviromnent or under conditions of fluctuating temperature.

Powders

Spoilage of products in powdered form due to visible mould growth also occurs under damp conditions. Again the possibility of illness due to microbial contamination is an important consideration and it is particularly necessary to ensure that topical preparations do not contain Clostridia spores. There is no clear evidence of a relationship between the presence of contaminants and irritation due to cosmetic powders but more than a few hundred organisms per gram is undesirable and powders for use on broken skin should be prepared from sterilized raw materials. This precaution also applies to those powders and other solid cosmetics which are intended to be applied in the region of the eyes.

Tablets, pastilles and lozenges Visible spoilage of tablets, generally manifested as surface discoloura-

tion, may be caused by the growth of moulds. Spores from the environment, container or tablet itself may find sufficient moisture to initiate growth on the tablet surface even under apparently dry conditions. For instance, fluctuations in temperature or variations between those in different parts of the container can cause corresponding changes in rh. If an rh of around 70•o is exceeded where mould spores reside then spoilage is possible, although this may occur slowly as a high enough rh may be achieved only spasmodically. In experiments with Paracetamol Tablets, B.P. we have found that, while spoilage could be prevented by attention to storage conditions and the value of preservatives was limited, the incorporation of 0.1•o propyl p-hydroxybenzoate retarded the onset and extent of mould growth, even under damp storage conditions, in laboratory and large-scale trials (24).

Interest has recently been shown in the carriage of contaminating pathogens by products of this type. Synthetic drugs in tablet form usually carry less than 100 organisms per tablet but those compounded with natural drugs may contain up to 105 organisms, often Gram-positive spore-

MICROBIOLOGICAL SPOILAGE 1N PHARMACEUTICALS AND COSMETICS 733

bearing bacteria, per tablet. Large numbers of enteric pathogens such as salmonellae have also been found and tablets containing biological products have been incriminated in outbreaks of salmonellosis (25).

Pastilles and lozenges of the boiled sweet type are not generally found to suffer from microbial spoilage as heating during manufacture has a sterilizing action and often individual dry wrappings are used to prevent surface contamination. However, when pastilles are dusted with starch powder, moulds may be introduced and these can give rise to discolouration under poor storage conditions.

Solid cosmetics

Lipsticks often contain preservatives but some are still subject to mould 'blooms'. Mould grows on the lipstick while it is inside the lipstick case, often after the product has become moistened by breath during use. Moisture, perhaps from saliva, may also initiate growth in preparations of mascara which often contain many bacteria. There is also a danger of contamination via the brush which can pick up organisms from the skin during use and similar dangers are present with solid cakes of make-up. Preser- vatives have limited use in this situation, perhaps because they are ad- sorbed onto the solid material. In addition their concentration must be

limited for fear of irritation to the eyes.

Packaging materials

Containers for pharmaceuticals and cosmetics are becoming increasingly elegant and are now made from a large variety of materials, particularly plastics. This should result in a reduction in microbial spoilage because plastics are not biodegradable like the cellulose materials, paper and card. In addition the latter, being absorbent, soak up liquids thus providing a substrate for moulds. In any case, liners of paper and card and cork closures often contain many micro-organisms and therefore frequently are a source of mould contamination whereas plastic closures are free from this defect. However, plastics suffer from some disadvantages. They are porous to varying degrees and some allow the diffusion of oxygen and carbon-dioxide and may thus facilitate microbial growth in the packed product. They also encourage condensation of water and, if mould spores are present, can facilitate spoilage by these organisms. Soap, for instance, gives off moisture, and, if wrapped with an impermeable plastic over paper or card, may become discoloured due to mould growth on the damp paper. Sometimes


paper wrappings can be protected from spoilage by the incorporation of a preservative into them, but this is not entirely without difficulties.

CONCLUSIONS

Microbial spoilage of pharmaceuticals and cosmetics is significant both from health and economic viewpoints and obviously should be prevented. It is dear that disease-causing organisms must be excluded, although sometimes it may be difficult to decide if an opportunist pathogen will be troublesome in a specific product. The situation with regard to spoilage micro-organisms, in the strict sense, is less dear as much is still to be learnt about the biochemical and microbiological mechanisms involved; certainly there is room for further fundamental studies in this fascinating field.

Deleterious changes will be avoided, of course, if all microbes are excluded from a product but it is not practicable to treat most pharmaceuticals and cosmetics as sterile products. Indeed, neither may this be desirable as we do not live in a sterile world and products, like human beings, must also survive in the presence of some micro-organisms. This raises the difficult question of how many organisms of what sort are accept- able in a given product. Several millions of organisms per gram are usually present in foods before decomposition reaches the point where it is detect- able to the senses (26) and this is generally also true of liquid pharmaceuticals and cosmetics. It also applies to more solid products where, although organisms may be concentrated at a focus, nevertheless they have to be present in some millions before the resulting colonies become troublesome. Contamination of pharmaceuticals and cosmetics with fewer cells may herald populations of spoilage potential if the contaminants can multiply; this is a matter of experience and experiment. Preservatives cannot always be relied upon to prevent the multiplication of micro- organisms because the ideal universal preservative does not exist and therefore the choice for each product is an individual, and often not entirely successful, matter. In any case preservatives are no substitute for good hygiene during production.

Lower viable counts in a product may represent the survivors of a larger population which has already caused decomposition but this is often obvious. A few hundred viable organisms per gram are generally of little consequence. The presence of many thousands of organisms, however, is usually a matter of concern as a count of this magnitude frequently indicates that spoilage will occur. To attempt to define more precise numerical

MICROBIOLOGICAL SPO1LAGE IN 'PHARMACEUTICALS AND COSMETICS 735

limits which are meaningful and yet can be applied generally to pharmaceutical and cosmetic products is extremely difficult, if not impossible, in view of the multiplicity of organisms and products involved.

In practice, regular monitoring during development and manufacture establishes the type and minimum number of organisms which are achiev- able for each specific product. Providing, of course, that this level is compatible with microbiological stability it forms the best approximate guide-line for a product.

(Received: 13th June 1971)

REFERENCES

(1)

(2)

(3) (4)

(5)

(6)

(7)

(8)

(9) (1o)

(11)

(12)

(13)

(14)

(15)

(16)

(17)

(•8)

(19)

(20)

(21)

Butler, N.J. The microbiological deterioration of cosmetics and pharmaceutical products. In Biodeterioration of Materials 269 (1968) (Elsevier Publishing Co., Amsterdam, London and New York). Sykes, G. Microbial contamination in pharmaceutical preparations for oral and topical use. Indian J. Pharmacy 31 No. 2 33 (1969). Barnes, J. M. Aflatoxin as a health hazard. J. Appl. Bacteriol. 33 285 (1970). Perlman, D. and Peruzzotti, G. P. Microbiological metabolites as potentially useful pharmacologically active agents. In Advances in Applied Microbiology 12 277 (1970) (Academic Press, New York and London). Smith, G. N. and Worrel, Celia S. Studies on the action of chloramphenicol on enzymatic systems. Archives Blochem. 23 No. 1 341 (1949). Hugo, W. B. The degradation of preservatives by micro-organisms. Sci. Tech. Symp. 122 112th Ann. Mtg Am. Pharm. Assoc. C411, 1 (1965). Kedzia, W., Lewon, J. and Wisnienski, T. The breakdown of attopine by bacteria. J. Pharm. Pharmacol. 13 614 (1961). Grant, D. J. W., de Szors, J. and Wilson, J. V. Utilization of acetylsalicylic acid as sole carbon source and the induction of its enzymatic hydrolysis by an isolated strain of Acinetobacter lwoffi. J. Pharm. Pharmacol. 22 No. 6 461 (1970). Omelianski, V. C. Aroma-producing micro-organisms. J. Bacteriol. 8 No. 4 393 (1923). Gerber, N. N. and Lechevalier, H. A. Geosmin, an earth-smelling substance isolated from actinomycetes. Appl. Microbiol. 13 935 (1965). Margalith, P. and Schwartz, Y. Flavor and micro-organisms. In Advances in Applied Microbiology 12 35 (1970) (Academic Press, New York and London). Shooter, R. A., Cooke, Mary E., Gaya, H., Kumar, P. and Patel, N. Food and medica- ments as possible sources of hospital strains of Pseudomonas aeruginosa. Lancet i 1227 (1969). Guyne, C. J. and Bennet, E. O. Bacterial deterioration of emulsion oils. Appl. Microbiol. 7 117 No. 2 (1959). Bean, H. S. The microbiology of topical preparations in pharmaceutical practice. 2. Pharmaceutical aspects. Pharm. J. 199 No. 5 421,289 (1967). Bart, M. and Tice, L. F. The preservation of aqueous preparations containing non-ionic surfactants I. J. Am. Pharm. Assoc. Sci. Ed. 46 No. 7 442 (1957). Tice, L. F. and Barr, M. Factors to be considered in the preservation of cosmetic emulsions. J. Soc. Cosmet. Chem. 9 171 (1958). Wedderburn, D. L. Preservation of emulsions against microbial attack. In Advances in Pharmaceutical Sciences 195 (1966) (Academic Press, London). Rivers, S. M. and Waiters, V. The effect of benzoic acid, phenol and hydroxybenzoates on the oxygen uptake and growth of some lipolytic fungi. J. Pharm. Pharmacol. 18 supp. 45S (1966). Eggins, H. O. W. and Walters, V. The decomposition of pharmaceutical emulsions by fungi. W. African J. Biol. Appl. Chem. 7 No. 1 2 (1963). Bryce, D. M. and Smart, R. The preservation of shampoos. J. Soc. Cosmet. Chem. 16 187 (1965). Walker, H. W. and Ayres, J. C. Yeasts as spoilage organisms. In The Yeasts 3 463 (1969) (Academic Press, London and New York).


(22) English, M.P. The fermentation of malt extract by an osmophilic yeast. J. Gen. Microbiol. 9 No. 1 15 (1953).

(23) Wills, B. A. Fungal growth in Syrup of Tolu. J. lPharm. Pharmacol. 10 302 (1958). (24) Payne, M. and Smart, R. Unpublished. (25) Kallings, L. O., Ringertz, O., Silverstolpe, L. and Ernerfeldt, F. Microbiological con-

tamination of medical preparations. Acta lPharm. Suecica. 3 No. 3 249 (1966). (26) Elliot, R. P. and Michenar, H. D. Microbiological standards and handling codes for chilled

and frozen foods. A review. Appl. Microbiol. 9 No. 5 452 (1961).

DISCUSSION

D•.. H. S. BE^N: We have been talking about the levels of contamination and many people have been talking in terms of not more than 100 micro-organisms g-• product. Connected with this standard there should be a time factor. When must the product meet this requirement? Immediately after manufacture or when somebody has been around the country and bought some off pharmacists' shelves 2 months, 2 years later? My colleagues and I have examined perhaps many thousands of different types of systems in our laboratory and we have found that the count never remains constant for very long. If the product has some antimicrobial action itself the count goes down but it may go up. The product may pass the test today and fail next week, next month, or next year. The third possibility is that the count will initially go down, and then we shall suffer quite considerable subsequent multiplication. If the product is liable to contamination and good manufacturing practice will not, or cannot, keep the number down below the limit we must include a preservative. When we are talking in terms of standards for pharmacopoeias or some other compendia we have to be quite sure about the type of product to which we are applying these standards. If the product is capable of supporting growth then it is not sufficient to merely state that the product should contain not more than 100 micro-organisms g-• because inevitably sooner or later it will. If the product is capable of supporting growth then we have to state some measure of the activity of the product against the invading micro-organisms.

DR. J. R. GWILT: YOU referred to the need to train production people in microbiological techniques and a participant from Italy also referred, en passant, to the need to check production people, at least to ensure that the products were not contaminated where they had infections. One thing that concerns me very much is that there seems to be more stress upon the environment and particularly upon the raw materials, than upon the people concerned. It seems that there are two ways in which the people who are in production, hospitals, and so on are involved. The first one I would call a negative way-- the people who are the shedders and the carriers in production; some of these we can pick up through high plate counts in the area when they are actually working with, for instance, sterile products, or we can find some of the carriers by throat and nose swabs. The second instance is where the people are positive, i.e. they definitely contribute. There is a tendency, certainly in the U.S.A., to dub quality controllers as second class, and bacteriologists, I fear, sometimes as third class. They are the people who should be involved in plant hygiene and also consulted where there are any changes in process, procedure or in packaging because all these can have such an enormous influence upon the survival, and the persistence, of bacteria in the products.

DR. SPOONER: I agree and would like to applaud you when you say that people are important because I think that obviously they are the major factor in manufacturing.


1 would like to correct you if I may--I did not say that we wish to train people in microbiological techniques: I actually said that I think it creates a considerable impact if you show your manufacturing staff, be they operatives or supervisors, the organisms they carry, and the organisms in the manufacturing environment, and what these organisms can do to the product. For many years my predecessor trained the staff in our sterile product areas and now we are trying to extend this training to people who are manufacturing non-sterile products. When you show people what they are carrying around it creates such an impact that they become more careful. While we certainly do not want to delegate our quality control function to production, we do want to make production personnel keenly aware of the problems by means of realistic demonstrations, best done by two types of courses. The first one lasts for about 3• days and is for supervisors. They go into the subject in some depth, but perhaps not so much as do the sterile products staff. The other is for operatives, and one might have several hundred to deal with. This can be done by dividing them into small groups and using a carousel slide projector to show pictures, a taped commentary, and attendance by microbiology technicians who can swab participants, take samples, and show actual spoiled products. This takes perhaps 2 h. After a gap of 2 days the people are brought back and are shown the microbes isolated from the swabs.

microbiological spoilage in pharmaceuticals and cosmetics

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