Conservanti - Artic Vechi

J. Soc. Cosmet. Chem. 23 703-720 (1972) ̧ 1972 Society of Cosmetic Chemists of Great Britain

Preservatives for pharmaceuticals H. S. BEAN*

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 Chemists of Great Britain.

Synopsis--Examination of the literature strongly indicates that PRESERVATIVE activity should be biocidal rather than biostatic and underlines the necessity of establishing STAN-

DARDS for preservative activity, such standards to include requirements for the rate of kill

or time to sterilize specified standard infections in a product and a specification for the capacity

of the preservative to deal with successive infections of the product. The chemical availability of a preservative in a product may alter during STORAGE as

a result of absorption by the package, in-use infection, or change in storage temperature and the microbiological significance of the loss may be estimated from a knowledge of the concen-

tration exponent and/or temperature coefficient of the preservative. The preservative activity of a substance may be markedly influenced by product environ-

ment, the more complex the product the greater the number of factors influencing the activity with the result that the preservative activity of a simple AQUEOUS SOLUTION can usually be stated with greater confidence than that of an EMULSION. Nevertheless, with the aid of

mathematical models and a knowledge of the appropriate parameters it is often possible to

predict the activity of a fluid containing SURFACE ACTIVE AGENTS or an emulsified preparation with acceptable accuracy.

For several decades pharmacists have been aware of the need to protect their products against microbial contamination but it is only during the last one or perhaps two decades that serious thought has been applied to the science of preservation.

A search through pharmaceutical compendia and texts reveals very few 'traditional' preservatives including ethyl alcohol at a concentration of not less than 20•o, chloroform at 0.25•o, sucrose at 67•o and benzoic acid at 0.1 •o. That they are still used is testimony to their effectiveness but

*Department of Pharmacy, Chelsea College of Science and Technology, London, S.W.3. 703

704 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

severe limitations in their applicability restrict their use in modern products. Alcohol was traditionally the preservative of tinctures and other galenicals the use of which has rapidly declined. Chloroform has been used both as a flavouring agent and preservative in extemporaneously prepared oral preparations with short storage life. It is volatile and readily lost from such preparations and is now losing favour because of possible toxicity. Syrups are still in vogue but temperature cycling during storage can produce a surface layer deficient in sucrose and deficient in antimicrobial activity. Indeed, recommendations to include preservatives in syrups (1) focus attention on possible failures in the use of syrup as a preservative. The physico-chemical properties of benzoic acid place severe restriction on its use. It has only moderate water-solubility (ca 0.28•) but high lipid solubility, for effectiveness must be employed at about half-saturation concentration which, as will be shown later, detracts from its usefulness and has a pKa of 4.2 which really restricts its employment to acidic preparations.

Several reports (2-5) have indicated the widespread nature of contamination in pharmaceuticals and stressed the necessity of controlling it Unfortunately, except for sterile products which are outside the purview of this survey, there are no universally recognized standards of what constitutes an acceptable number of micro-organisms in a product, the effectiveness of a preservation or even methods of determining preservative activity. Let it be first established that the writer strongly supports the hypothesis that preservation must never be a substitute for good manufacturing practice which can lead to a substantially reduced number of micro- organisms in a product at completion of manufacture. It is rather a safe- guard either to destroy or inhibit the growth of those organisms which cannot be eliminated by good manufacturing practice or which gain access to the product during use.

Hitherto, the effectiveness of preservation has usually been assessed by a so-called challenge test. These involve inoculating a product with known organisms and incubating perhaps for many months. The prolonged storage or incubation is a confession of the recognition of the possibility of failure of the preservative effectively to fulfil its role and a number of containers are put on storage in an attempt to quantify the probability of failure. The test is really a measure of the ability of contaminating organisms to destroy the product. I submit that more desirable would be a performance test--a measure of the ability of the product to destroy invading organisms. Such a test would be more positive, more rapidly performed and possibly more easily definable by regulatory bodies.

PRESERVATIVES FOR PHARMACEUTICALS 705

REQUIREMENTS OF PRESERVATIVES

'What constitutes satisfactory preservation?' is a question that has been asked by many and indeed was the subject of a paper presented in 1958 (6). The inclusion in a product of a bacteriostatic concentration of a preservative has often been regarded as adequate and many examples can be found of recommended concentrations of preservatives being at the level indicated in tests for minimum inhibitory concentrations. One does not have to seek far for the reason. It is that because the fundamental meta-

bolism of all cells is essentially similar, a compound which shows marked toxicity to microbial cells is likely to show at least some toxicity to human cells, depending upon the mode of application. This philosophy, though understandable, does not necessarily make for adequate preservation.

As far as the preservation of ophthalmic solutions is concerned a very definite shift in attitude is detectable. Crompton (7) originally suggested that ophthalmic solutions should incorporate a bacteriostatic and indeed reconunended 0.005•o chlorhexidine which is certainly bacteriostatic rather than bactericidal (7). He later declared (8) that reliance on bacteriostatics can be hazardous and maintained that ophthalmic solutions should contain a quickly acting bactericide, and Williams and Boehm (9) criticized the Solution for Eye Drops (B.P.C. 1959) on the grounds that it was not bactericidal. Perhaps the most categorical statement is due to Kohn, Gershenfeld and Barr (10) who believe that any substance having a sterilizing time greater than 1 h is too slow-acting for use as a preservative in ophthalmic solutions. I am happy to observe that the preservatives cur- rently recommended by the British Pharmaceutical Codex for eye drops appear in most situations to meet this requirement and furthermore they do so without there having been criticisms of their use on the grounds of toxicity or irritancy. There are, however, exceptions to this general statement. For example, Ridley (11) found 0.0045/0 phenylmercuric nitrate to be necessary in eye drops to ensure freedom from contamination; a concentration four times that recommended by the Codex.

There is nothing like the weight of authoritative advice for products other than ophthalmic solutions but it is instructive to consider what are regarded as adequate preservatives by several pharmacopoeias (Table I).

Of the preservatives shown there is agreement only on the recommended concentration for phenol. In the case of cresol the specified concentrations range from 0.30 to 0.50•o and since the concentration exponent for cresol is about six the activity of the more concentrated solution is about (1.66) 6


Table I

Some preservatives recommended by pharmacopoeias

Pharmacopoeia

French Pharm.

Preservative B.P. Codex U.S.P. Helv.

Phenol 0.5 • 0.5 •o 0.5 • -- Cresol 0.3 % 0.3 % 0.5 % 0.3 •o Chlorocresol 0.1 •o 0.3 % -- 0.2 % Organomercurial 0.001% -- 0.1% -- Methylparaben -- -- -- O. 1%

or 20 times that of the weaker. For chlorocresol, the concentration range is 0.1-0.3• and the most concentrated will have an activity about 30 or 725 times that of the weakest. What is clear is that pharmacopoeias reflect the uncertainty of precisely what constitutes an adequate preservative.

If the activities of the various preservatives recommended by any single pharmacopoeia is examined an equally wide variation is obvious (Table Ii), those of the British Pharmacopoeia ranging from about 10 min to 10 h.

Table II

The activities of preservatives recommended by the British Pharmacopoeia

Preservative %w/v Time to kill

10 a ml-1 E. coli

Chlorocresol 0.1 10 min

Cresol 0.3 30 min

Phenylmercuric NO a 0.001 2 h* Phenol 0.5 10 h

*Using thioglycolate recovery medium.

The only point common to all pharmacopoeias and all preservatives recommended by them is that they are bactericidal rather than bacteriostatic and even markedly bactericidal with the possible exception of 0.1•o methyl parahydroxybenzoate. It is interesting to note that a working party report (12) recently published by the Society of Cosmetic Chemists of Great Britain recommends that cosmetic products should be self sterilizing.

It is my belief that the most satisfactory requirement to date is that of the U.S.P. XVIII (13) which requires that a satisfactorily preserved ophthalmic solution will reduce the viable count of any contamination to 0.1}/o of the original and not permit any subsequent multiplication of the survivors during a 7 day period of the 28 day test period. The latter clause is all important. Dr Walters and I (14) found, as long ag• a• 1)55,


that the last survivors in any initially bactericidal solution may multiply, utilizing as nutrient the purines and pyrimidines released by dead and/or dying cells. Several reports of similar observations have since been published (15-17) showing that the multiplication is not peculiar to the use of any one preservative and suggesting that it may occur generally with preservatives having high intrinsic activity and therefore used in low concentration. The phenomenon has, to my knowledge, never been associated with preservatives used at concentrations of 0.l•o or higher, unless the product provided considerable nutrient.

Although I believe the U.S.P. requirement to be basically satisfactory ! would be even happier if the required mortality level was increased to 100•o (admittedly a difficult condition to demonstrate in practice) and the latter requirement associated with a maximum time limit--perhaps 3 h because this represents the approximate interval at which glaucoma patients apply their ophthalmic drops. Again, pharmacopoeial preservatives would often meet this requirement and it therefore represents the type of standard which could be imposed.

ACTIVITY AND CAPACITY OF PRESERVATIVES

Having postulated possible requirements for preservatives a selection has to be made of a possible compound. A useful guide seems to be that if a saturated aqueous solution of the candidate-compound fails to sterilize an inoculum of about 108 ml -• vegetative organisms within 10 min, it is unlikely to prove suitable. Phenol, cresol, chlorocresol benzalkonium, chloride and chlorhexidine acetate amongst others would meet this requirement, chloroxylenol, benzylchlorophenol and the parabens would not. The first group have relatively high water solubility and relatively low intrinsic activity. Again, the acceptable compounds are recognized by the pharmaceutical compendia, the unacceptable usually are not.

The concentration of the chosen compound to produce the required activity is next determined, particular consideration being given to the probability of the preserved solution being able to maintain its activity against a heavy infection or even a succession of infections. The destruction of organisms by preservatives involves a chemical or physical reaction between the micro-organism and the preservative resulting in a loss of preservative to the cells from the solution, the heavier the infection the greater the loss of preservative from the solution. The more active preservatives are used in lowest concentration and it is these that suffer the


greatest depletion {,Table III) and greatest loss of activity. Phenylmercuric nitrate 0.001 }/o will sterilize an inoculum of 10 a ml -• Pseudomonas aeruginosa in 10 min, 105 ml 4 in 25 min and l0 s ml -• in 180 min. Solutions which suffer a marked loss of activity as the level of infection is increased or when they are subjected to a succession of infections may be described as possessing œoor (antimicrobial) capacity and those which suffer little loss of activity have good capacity and are obviously preferable.

Table III

Loss in activity of preservative solutions after infection by 109 E. coli ml -x

Preservative Phenol Initial Proportion Initial Sterilization

coefficient concn of of preserv- sterilization time of solution ative time for residual

(Yoo w/v) removed by E. coli solution cells (•) (rain) (rain)

Phenol 1 1.0 0.5 10 10

m-Cresol 2 0.33 0.6 10 10 Chlorocresol 12 0.08 2.5 10 11.5

Benzylchlorophenol 160 0.006 33.0 10 115.0

ABSORPTION OF PRESERVATIVES BY CONTAINERS

Preservatives are also liable to be removed from solutions through absorption by plastics containers or rubber closures of glass containers. In general, the less water-soluble and more lipid-soluble preservatives tend to be more soluble in organic phases and are absorbed to a greater extent by containers and their closures. Thus, phenol is not absorbed to any great extent but chlorocresol may suffer considerable depletion and phenylmercuric nitrate may suffer almost complete removal by rubber caps (Table IV), but because of its low concentration exponent the loss of preservative activity may be less serious than in the case of chlorocresol.

Table IV

Loss of preservative through absorption into rubber caps

Preservative

Initial Concentration Loss as Increase

concentration after percentage in time for (% w/v) absorption of initial residual

(%) concentration concentration to sterilize

Phenol 0.5 0.39 22 x 4.5 Cresol 0.3 0.21 30 x 8.5

Chlorocresol 0.1 0.04 60 x 250

Phenylmercuric NO a 0.001 0.00005 95 x 42


As an example of absorption by plastics, Hamdi (18) has shown that cellulose acetate absorbs 23 mg g-• phenol from a 0.1 •o solution, suggesting a plastics/water partition coefficient of about 30 and 19 mg g-• chlorocresol from a 0.025•o solution, indicating a partition coefficient of about 475. These figures indicate that the loss of chlorocresol into a plastics container is more likely seriously to reduce the activity of a preservative than is the loss of phenol.

KINETICS OF PRESERVATIVES

When bacteria are introduced into a solution of preservative the rate at which they die can for practical purposes (there are theoretical objections) be represented by the first-order reaction equation

-dN - KN (I)

dt

where N is the number of viable organisms m1-1 at time t and K is the death-rate constant. Integrating the equation between viable number No ml 4 at time t =0 and viable number N ml -• at time t and converting to common logarithms, equation I becomes

2.303 No K - log

t N-

and the reaction may be represented by Fig. 1. Any reduction in the 1¸5 -

10 4

7

• I0• --K

.9

o• IC) •

t0 20 30 40 50 60

Time ( rain )

•i•u•e 1. Typical representation of the death of mi•m-o•ganisms


concentration of the preservative will increase the time needed to reach any specified mortality level and reduce the slope of the line in Fig. 1. Con- versely any increase in concentration will reduce the time and increase the slope.

The practical worker is likely to be concerned about the increase in time needed to reach the 99.9}/0 mortality level specified by U.S.P. XVIII for ophthalmic solutions resulting from a known concentration of preservative being absorbed by the container. This increase in time can be calculated from a knowledge of the concentration exponent of the preservative and

log K•- log K•. log C•- log Ca

( C• ) n K• or • =K-• where n -- concentration exponent of preservative

K• = death rate constant at concentration C• K•. = death rate constant at concentration

Now, at concentration C•

2.303 100

K•- t• 1øg 0.1 and at concentration

2.303 100

K•.- t•. log0.1 where t• -- time to reach specified mortality level at concentration C•

t•. = time to reach specified mortality level at concentration Ca

Equation H indicates that if the concentration of a preservative is plotted against the time taken to reach some specified level of mortality, such as 99.9• or even 100• or sterility, a straight line results with a slope numerically equal to n, the concentration exponent (Fig. 2). Thus, when the concentration exponent has once been determined for a named preservative it can be of great practical value and the writer regards it as the most useful parameter of a preservative. Preservatives belonging to a particular chemical group have approximately the same concentration exponent,


Log conch preservahve

Figure 2. The concentration exponent of a preservative.

some examples being shown in Table V and Table VI shows the theoretical change in activity with change in concentration for the preservative groups listed in Table V, and indicates that losses of preservatives of the order of 10•o may be of no significance and that even losses of up to 50• of

Table V

Concentration exponents of some commonly used preservatives

Preservative n Preservative n

Organomercurials 0.5 Chlorhexidine 2.0 Formaldehyde 1.0 Parabens 2.5 Quaternaries 1.0 Phenolics 6.0

Table VI

Relationship between concentration exponent, change in concentration ' and change in activity

Increase in killing time for concentration loss of: Concentration

exponent 50 % 40 • 20 % 10 %

0.5 x 1.4 x 1.3 x 1.11 x 1.05

1.0 x 2 x 1.66 x 1.25 x 1.11

2.0 x 4 x 2.75 x 1.56 x 1.23

2.5 x 5.6 x 3.55 x 1.75 x 1.30

6.0 x64 x20.9 x 3.8 x 1.90


organomercurials and quaternaries may not be of practical importance but 50•o loss of a phenolic can be very serious. Thus, when considering the loss of preservative from a solution, through no matter what cause, it is not sufficient to state the weight lost or percentage lost, but it is imperative that this information be linked with the concentration exponent because it is the loss of activity and not the loss in weight that is of concern.

Preservative activity is influenced by temperature, the higher the ambient temperature the more active is the preservative. It is usual to relate preservative temperature to the death-rate constant by the expression

0(50: - 50x ) - K•. (Ill) Ki

where 0 = temperature coefficient of the preservative Kx = death-rate constant at temperature Tx K,. = death-rate constant at temperature T1

and since by equation (II)

Kx tz K2 tl

0(50,- 50•) - tx (IV) ß

Thus, if the temperature coefficient of a preservative is known the change in sterilization time may be calculated for a stated temperature change. Unfortunately, temperature coefficients vary according to the temperature range over which they are measured and the test organism (19, 20). For the sake of an example, a representative 0 xø for phenol over the temperature range 10-20 ø would be 5.0 (0•ø=5-1.175øC -•) which means that at 20 • a solution of phenol would kill E. coli five times as fast as at l0 ø.

It is sometimes useful to be able to predict the effect on preservative activity of changing both the concentration and the temperature of a preservative. If from equation (II) is abstracted

C1 n

re-arranged as Clntl = Cs":

and this is combined with equation (IV) re-arranged as tl = t2 0(50:-501)


we have Cx•tx = C•.,,t•. 0(v:- Vl ) which is an equation incorporating all the terms necessary to predict the effect on extinction time of changing both concentration and temperature.

To illustrate the use of the equation, a multiple dose injection contained at the time of manufacture 0.1•o chlorocresol which provided an approximate sterilization time for an infection of vegetative cells of 10 min at 25øC. After several months storage and as a result of absorption by the closure the chlorocresol concentration in the solution had fallen to 0.04•o. If the container was stored in a cupboard at 10 ø the activity of the solution would have fallen as a result of both the drop in concentration and drop in temperature. A new estimate of the sterilization time would be provided by

Cxntl 0(•':- tz = C2 n

(0.1) ø x 10 x --

--

(0.04)0 = 27 470 min.

Such an interminable time may be without real meaning but it indicates a change from a solution possessing considerable preservative activity to one virtually devoid of preservative activity--a potentially dangerous solution.

INFLUENCE OF FORMULATION

The formulation of a product may substantially modify the chemical and biological availability of an included preservative and thereby modify the activity of the preservative. The major formulation factors affecting activity are pH, complexation with emulgents or dispersing agents and partitioning of the preservative between the components of an emulsified product.

Whilst pH is by no means unimportant as a factor influencing preservative activity, there have been very few systematic and comprehensive studies of the problem and it is very difficult to quantify because changes in pH may modify the ionization of chemical groupings on the bacterial surface, produce ionization of the preservative and influence partitioning of the preservative between the product and the microbial cell and all three factors may interact. In very general terms there is evidence that a rise in the pH of solution of a quaternary produces increased uptake of the quaternary leading to enhanced antimicrobial activity (21). On the other


hand there is ample evidence that a rise in the pH of a solution of a phenolic or an organic acid reduces cellular uptake and activity (22, 23).

Much more readily quantifiable is the effect of nonionic surfactants on the activity of preservatives. Kostenbauder and others (24-26) have assumed that interaction between surfactants and phenolics or organic acids involves complexation and a reduction in the concentration of 'free' preservative, the latter largely but not necessarily completely controlling the activity of the system. The amount of preservative bound to the surfactant is directly related to the concentration of surfactant, the relationship being representable by the equation

R = SC+I

where R = ratio of total/free preservative C -- constant

$ = surfactant concentration.

The constant C has a unique value for each surfactant-preservative pair and increases in value as the lipid solubility of the preservative increases (Fig. 3). R is the factor by which the total concentration of preservative

2O

c

40-

3o

B

A

0 I I I I I I I 2 4 6 8 I0 12 I•

Cetommcrogol conch (% w/v)

Figure 3. The binding of phenolics to Cetomacrogol (27). A, phenol; B, cresol; C, chlorocresol.


must be increased for a g•ven surfactant concentration to maintain a specified concentration free in the water.

Even the simplest emulsion contains oil, water and emulgent the latter being partially adsorbed at the o/w interfaces but the majority of it being dispersed throughout the water phase as emulgent micelies. A preservative incorporated in an emulsion is distributed between all three components or phases and the influence of each of the phases on preservative availability and activity is most easily understood by considering simplified models. The influence of the emulgent in the water on preservative availability has already been considered. If now the oil and water phases are considered without reference to the emulgent, the preservative will be distributed between the two phases so that

Co Cw- K?v

where Co = concentration of preservative in the oil at equilibrium Cw = concentration of preservative in the water at equilibrium Køw = oil/water partition coefficient, a constant at a specified

temperature for any oil, water preservative mixture. An enormous range of values for oil/water partition coefficients is recorded (28, 29) some values for the parabens being shown in Table VII and having

Table VII

Oil/water partition coefficients for parabens (25 ø)

Compound

Partition coefficient

Liquid paraffin/water Soya oil/water

Methyl hydroxybenzoate 0.02 Propyl hydroxybenzoate 0.50 Butyl hydroxybenzoate 3.0

7.5

80.0

280.0

a range of 14 000. The coefficients can be used in the equation below (30) for calculation of the way in which the relationship of the total concentration of paraben in the oil/water mixture to the concentration in the water depends on the proportion of oil and water (Table VIII).

Cw- c(o Koo+I

where C = total concentration of preservative Cw = concentration of preservative in the water 0 = oil' water ratio.


Table VIII

Influence of proportion of oil on the concentration of methyl paraben in the water of an oil/water mixture

Preservative concentration in water

Total Oil: water ratio preservative

concentration Oil phase Kwø 0.11 0.25 0.43 0.66 1.00 in oil/water mixture • w/v oil phase

10 20 30 40 50

Methyl paraben Liquid paraffin 0.1 • (S.G. 0.85) 0.02 0.11 0.12 0.14 0.16 0.20

Methyl paraben Liquid paraffi 0.2% (S.G. 0.85) 0.02 0.22 0.24 0.24 0.24 0.24

Methyl paraben 0.2• Soya oil 80.0 0.027 0.012 0.008 0.006 0.005

In liquid paraffin/water mixtures the concentration of methyl paraben in the water is always higher than the total concentration in the mixture and increases as the proportion of oil is increased until water saturation (0.24•o w/v) is reached. With vegetable oils the water concentration is so far below the total concentration that the aqueous phase is virtually devoid of activity and the concentration falls as the proportion of oil is increased. The same general picture is obtained with many preservatives no matter what the vegetable oil.

The inclusion of an emulgent in the system results in a redistribution of the preservative between the total aqueous phase and the oil, the concentration in the total aqueous now being calculated using the equation

C.• = C(0 + 1) K0+I

where Ca = concentration of preservative in the total aqueous phase K = oil/aqueous phase partition coefficient.

Because the preservative in the water is partly associated with the emulgent, the concentration free in the water is

Cw = c^/J which can be shown (31) to be equivalent to

C(0+l) Cw = 0 0 + R) (v)

a more generally useful expression because Kw ø, the true oil/water partition


coefficient, is constant for any oil/preservative/water mixture, whereas K falls as the concentration of emulgent is increased.

If it is now assumed that the liquid paraffin/water mixtures of Table VIII are emulsified by the inclusion of 5.0•o Polysorbate 80 the concentration of methyl paraben free in the water can now be recalculated (Table IJO using equation (V) the appropriate value of R being 4.5.

Table IX

Influence of proportion of oil on the concentration of methylparaben free in the water of liquid paraffin/water emulsions emulsified with 5 % Polysorbate 80

Total

preservative concentration

in emulsion

o

Oil phase Kw

Preservative concentration in water

Oil: water ratio

0.11 0.25 0.43 0.66 1.00

% w/v oil phase

10 20 30 40 50

Methyl paraben Liquid paraffin 0.1% (S.G. 0.85) 0.02 0.025 0.027 0.032 0.037 0.044

Methyl paraben Liquid paraffin 0.2 % (S.G. 0.85) 0.02 0.05 0.055 0.063 0.074 0.088

Because in the emulsions the concentrations of methyl paraben in the water are so far below those required for preservation the total concentration would have to be increased. If it is accepted that 0.2•o is required in the water, the total concentrations that would be required in the emulsions containing 10, 20, 30, 40 and 50•o oil would be 0.81, 0.72, 0.67, 0.54 and 0.47•o respectively, which can be shown experimentally to produce an activity in the emulsion approximately equivalent to that produced by 0.2•o methyl paraben in water.

The writer has endeavoured to show that once the required activity of a preservative in a product has been established, it can be maintained in spite of predictable losses for the solution provided certain basic parameters of the preservative are known. Even in products which are more complex than an aqueous solution it is often possible to quantify changes in the chemical availability of a preservative with changes in formulation and to modify either the total concentration of preservative to meet the availability requirements or substitute another preservative with more satisfactory


characteristics. That is, provided the parameters of preservatives are adequately quantified it is possible to elevate preservation from a hit-and- miss strategy to a more precise science.

(Received: 15th June 1971)

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REFERENCES

Lord, C. F. and Husa, W. J. Antimolding agents for syrups. J. Am. Pharm. Assoc. Sci. Ed. 43 438 (1954). Report by the Royal Swedish Medical Board into microbiological contamination of medical preparations (1956). Report of Working Party of National Health Service Laboratories, Denmark. Dansk. Tidsskr. Farm. 42 1, 50, 71,125, 257 (1967). World Health Organization. Microbiological contamination of non-sterile drugs. Working paper WHO/Pharm./69.433 (1969). Dony, J. Condition microbiologique du m•dicament non sterile. XIX Journ•es pharma- ceutiques Franfaises, 25 (1969). Sykes, G. The basis for 'sufficient of a suitable bacteriostatic' in injections. J. Pharm. Pharmacol. 10 Suppl. 4OT (1958). Crompton, D. O. Ophthalmic prescribing. Austral. J. Pharm. 1020 (1962). Crompton, D. O. Sterility of eye medicaments. Lancet i 41 (1964). Williams, R. and Boehm, E. E. Sterility of eye medicaments. Lancet ii 790 (1963). Kohn, S. R., Gershenfeld, L. and Barr, M. Effectiveness of antibacterial agents presently employed in ophthalmic preparations as preservatives against Pseudomonas aeruginosa. J. Pharm. Sci. 52 967 (1963). Ridley, F. Sterile drops and lotions in ophthalmic practice. Brit. J. Ophthal. 42 641 (1968). Hygienic manufacture and preservation of toiletries and cosmetics. J. $oc. Cosmet. Chem. 21 719 (1970). U.S. Pharmacopoeia XVIII. p. 845. Antimicrobial agents--effectiveness. (1970) (Bethesda, Md). Bean, H. S. and Walters, V. The viability of Escherichia coli in aqueous solutions of benzylchlorophenol. J. Pharm. Pharmacol. 7 661 (1955). Brown, M. R. W. and Garrett, E. R. Kinetics and mechanisms of action of antibiotics on micro-organisms. J. Pharm. Sci. 53 179 (1964). Garrett, E. R. and Brown, M. R. W. The action of tetracycline and chloramphenicol alone and in admixture on the growth of E. coli. J. Pharm. Pharmacol. 15, 185T (1963). Bean, H. S. and Farrell, R. The persistence of Pseuabmonas aeruginosa in aqueous solutions of phenols. J. Pharm. Pharmacol. 19 Suppl. 183S (1967). Hamdi, F. J. Studies in the interaction of some phenols with plastics. M.Phil. Thesis, University of London (1968). Tilley, F. W. An experimental study of the influence of temperature on the bactericidal activities of alcohols and phenols. J. Bacteriol. 43 521 (1942). Cohen, B. Effect of temperature and hydrogen-ion concentration upon viability of E. coli and Ebethella typhi in water. J. Bacteriol. 7 183 (1922). Salton, M. R. J. The action of lytic agents on the surface structures of the bacterial cell. Proceedings of the Second International Congress on Surface Activity 274 (1957). (Butter- worth, London). Lundy, H. W. The effect of salts upon the germicidal action of phenol and secamyl- tricresol. J. Bacteriol. 35 633 (1938). Oka, S. Studies on transfer of antiseptics to microbes and their toxic effect. Bull. Agr. Chem. Soc. Japan 24 59 (1960). Patel, N. K. and Kostenbauder, H. B. Interaction of preservatives with macromolecules I. J. Am. Pharm. Assoc. Sci. Ed. 47 289 (1958). Pisano, F. D. and Kostenbauder, H. B. Interaction of preservatives with macromolecules II. J. Am. Pharm. Assoc. Sci. Ed. 48 310 (1959). Miyawaki, G. M., Patel, N. K. and Kostenbauder, H. B. Interaction of preservatives with macromolecules III. J. Am. Pharm. Assoc. $ci. Ed. 48 315 (1959). Sheikh, A. W. Studies on the influence of a surface active agent on the activity of some preservatives. Ph.D. Thesis, University of London (1971).


(28) Hibbott, H. W. and Monks, J. Preservation of emulsions--p-hydroxybenzoic ester partition coefficient. J. $oc. Cosmet. Chem. 12 2 (1961).

(29) Bean, H. W., Heman-Ackah, S. M. and Thomas, J. The activity of antibacterials in two- phase systems. J. $oc. Cosmet. Chem. 16 15 (1965).

(30) Bean, H. S. and Heman-Ackah, S. M. Influence of oil: water ratio on the activity of some bactericides against Escherichia cell in liquid paraffin and water dispersions. J. Pharm. Pharmacol. 16 Suppl. 58T (1964).

(31) Bean, H. S., Kenning, G. H. and Malcolm, S. A. A model for the influence of emulsion formulation on the activity of phenolic preservatives. J. Pharm. Pharmacol. 21 Suppl. 173S (1969).

DISCUSSION

MR. R. J. MCBRIDE: It is good to see you advocating 100K mortality level within a fixed time limit as a standard for a preservative suitable for use in preserving ophthalmic solutions. We believe that the time period of 3 h is too slow, however, especially for multidose solutions used in eye clinics, and we prefer the time of 1 h, suggested by Kohn et al (10). Our own work has shown that this 1 h sterilization time can be achieved against high inocula of Pseudomonas aeruginosa in preserved fluorescein sodium, pilocarpine hydrochloride, atropine sulphate, physostigmine sulphate, or salicylate and sodium bicarbonate ophthalmic solutions.

T•E LECTURER: I suppose for ophthalmic solutions it is a question of toxicity; I really do not know the optimal time. I would make the point that if you do not get 100•o mortality you are liable to have subsequent multiplication. For injections we have been obtaining sterilization times < 1 h by incorporating 0.1• chlorocresol, and there have been no criticisms. It was only when we had 0.1• chlorocresol which did produce roughly a 10 min kill in ophthalmic solutions, that the problem of toxicity arose; we now know that this was due to an unfortunate use in ophthalmic surgery. The major problem is one of toxicity rather than producing sterility.

DR. R. M. E. RICHARDS: The use of mixtures of antibacterial agents for the preservation of pharmaceuticals has not often been reported in the literature. Nevertheless mixtures of antibacterial agents are in frequent use in commercial preparations. Accord- ing to Remington (32) they now account for almost 50•o of the preservative systems in commercially available ophthalmic solutions produced in the U.S.A. Remington also predicts the increase of the use of mixtures of antibacterials in extemparaneous com- pounding. Would you please comment on the parameters to be observed in choosing a mixture of antibacterials in pharmaceutical preservation.

THE LECTURER: AS far as I see, the advantages are that a mixture broadens the spectrum of micro-organisms which may l•e dealt with and it may increase the capacity of the preservative system. For example, if one uses shall we say, two of the parabens-- methyl paraben, propyl paraben, the probability is that with the more active compound (propyl parabort) the cell-aqueous phase partition coefficient will be in fayour of much of the propyl compound going into the cell, leaving the system deficient in activity. If one would also include the methyl compound this would tend to increase the capacity. There are great theoretical difficulties in handling these mixtures of preservatives--if they work, use them. I think the reason that so little has appeared in the literature is the

(32) Remington, P. J. Pharmaceutical scieaces 14th edn. 1570 (1970) (Mack Publishing Co.. Easton, Pennsylvania).


difficulty of predicting precisely what they will do. Garratt and Brown (33) were able to produce a very simple kinetic explanation; if you try to apply that to most antibacterials it just does not work. One can get a long way with mixtures within one chemical class. For example, if you are going to mix phenol and if you like chloroxylenol, and if you know the activities of the separate compounds and their concentration exponents, you can get a long way towards calculating with reasonable accuracy (q-50•o) precisely what the activity of the mixture will be. For mixtures of different classes of compounds, for example phenyl ethyl alcohol together with a paraben or similar, heaven only knows where the mathematics goes. It is not only a question of mixture of the preservatives; I think Richards and McBride (34) made this point. If you have two preservatives in an ophthalmic solution you have three components in the system. Now attopine and, I think, pilocarpine will tend to enhance the activity of many of these preservatives whereas fluorescein will actimt•ro•posite direction. Until we have much more knowledge it is thus very difficult •to predicf• •Wrhat the activity of a product will be.

MR. G. S¾•cœs: I wonder w}i•Iher Remington (32) means a real 505/0 in the terms of mixtures in which we think of t :•:!• .,. or whether a large proportion of that 505/0 is not groups of homologues of the paralSL•,•hich can be called mixtures.

DR. R. M. E. RicH^v,r>s: The 505/0 do not include the parabens--these are all kinds of combinations of antibacterials probably with different modes of action and sometimes used at quite strange concentrations.

THœ Lœc'ruv,œv,: Many insulins contain a mixture of preservatives and, I think, this mixture is not always there for preservative reasons alone, e.g. there may be analgesic reasons.

(33) Garrett, E. R. and Brown, M. R. W. The action of tetracydine and chloranphenicol alone and in admixture on the growth of E. cell. J. Pharm. Pharma½ol. 15 185T (1963).

(34) Richards, R. M. E. and McBride, R. J. The preservation of ophthalmic solutions with antibacterial combinations. J. Pharm. Pharrna½ol. 23 Suppl. 235S (1971).

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