`Preservation
`
`PRINCIPLES AND PRACTICE
`
`Edited by
`JON J. KABARA
`Department of Biomechanics
`Michigan State University
`East Lansing, Michigan
`
`
`
`MARCEL DEKKER, INC.
`
`'
`
`New York and Basel
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`Library of Congress Cataloging in Publication Data
`Main entry under title:
`
`Cosmetic and drug preservation.
`
`(Cosmetic science and technology ; V. 1)
`Includes index.
`1. Cosmetics--Preservation.
`
`2. Cosmetics--
`
`4. Drugs--
`3. Drugs-—Preservation.
`Microbiology.
`Microbiology. 1. Kabara, Jon J., [date].
`11 . Series.
`TP983.C774 1984
`ISBN 0-8247- 7104-4
`
`84-15603
`
`668'.55
`
`.5)
`6\ i
`fix
`
`COPYRIGHT © 1984 by MARCEL DEKKER, INC.
`
`ALL RIGHTS RESERVED
`
`Neither this book nor any part may be reproduced or transmitted in
`any form or by any means, electronic or mechanical, including photo-
`copying, microfilming, and recording, or by any information storage
`and retrieval system, without permission in writing from the publisher
`
`MARCEL DEKKER, INC.
`270 Madison Avenue, New York, New York 10016
`
`Current printing (last digit):
`10
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`
`PRINTED IN THE UNITED STATES OF AMERICA
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`This material may be protected by Copyright law (Title 17 U.S. Code)
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`54
`
`INTRODUCTION
`
`Haag and Loncrini
`
`The alkyl esters of p-hydroxybenzoic acid, more commonly known in the United
`States as the parabens, are the most widely used preservatives in the cosmetic
`industry today [1] . They were first introduced by Sabalitschka in 1924 [2] ,
`when he presented a series of reports on their use as antimicrobial agents.
`Sabalitschka's primary goal was to find replacements for salicylic and benzoic
`acids, both of which are effective preservatives only in the highly acid pH
`range. His research was rewarding when he found that by esterifying p—
`hydroxybenzoic acid with various alkyl and aryl alcohols, the resulting prod-
`ucts were effective over a wide pH range.
`Official recognition of the parabens came in 1934 with the adoption of the
`methyl ester in the fifth edition of the Swiss Pharmacopeia.
`In the United
`States the methyl ester was first adopted in the National Formulary VII in
`1942; both the methyl and propyl esters were admitted to the U.S. Pharma—
`copeia XIII in 1947. Methylparaben and propylparaben are also contained on
`the "generally recognized as safe" list (Code of Federal Regulations, Title 21
`§184.1490 and 184.1670), with a maximum permissible use quantity of 0.1%.
`In many ways the parabens are ideal cosmetic preservatives. They are
`essentially colorless, odorless, nonvolatile, stable, effective over a wide pH
`range, and relatively active against a broad spectrum of microorganisms.
`Their cost is low in relation to their use concentration. Numerous studies
`
`have indicated that the parabens have a low order of acute and chronic toxic7
`ity [3] , although in some isolated cases contact dermatitis has been reported
`[4] . Therefore, considering the widespread usage of the parabens and the
`low incidence of adverse reactions, it is generally accepted that the parabens
`are among the safest of all cosmetic preservatives.
`
`II. PRODUCTION
`
`The esters of p-hydroxybenzoic acid are prepared by reacting the corre—
`sponding alcohols with p—hydroxybenzoic acid in the presence of an acid cat—
`alyst. The reaction products are formed in high yields, especially if the by-
`product water is removed azeotropically according to the following equation:
`
`CI-OH
`
`IC-OR
`
`ROH
`
`+
`
`H2O
`
`Generally , sulfuric acid or p-toluenesulfonic acid is recommended as the catalyst.
`p—Hydroxybenzoic acid is produced commercially by treating the dry
`potassium salt of phenol with carbon dioxide at temperatures above 200°C
`under 4—7 atm of pressure. The resulting salt is then treated with acid to
`yield p—hydroxybenzoic acid :
`
`co2
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`Esters of para—Hydroxybenzoic Acid
`
`65
`
`III. PHYSICAL AND CHEMICAL PROPERTIES
`
`The methyl, ethyl, propyl, and butyl esters of p-hydroxybenzoic acid (shown
`below) are the most widely used by the cosmetic industry [1].
`
`Ou
`C-O-R
`
`OH
`
`Methyl
`Ethyl
`Propyl
`Butyl
`
`R = CH3
`R = 02H5
`R = C3H7
`R = C4H9
`
`These esters may be described as small colorless crystals or as white crystal—
`line powders, slightly to very slightly soluble in water, and soluble to freely
`soluble in nonpolar vehicles. Their water solubility decreases with increasing
`molecular weight, while their lipid solubility increases with increasing mole—
`cular weight (see Table 1). A linear relationship exists between the alkyl
`chain length and the log of the water solubility, and also between chain length
`and the log of the partition coefficient [5] .
`The paraben esters are colorless in solution. The methyl ester has been
`reported to have a slight characteristic odor, whereas the other esters are
`practically odorless in their purified form [5] . No particular taste is notice—
`able in solutions of the esters, but in saturated aqueous solution these esters
`exhibit a slight tang or weak burning sensation in the mouth [5] .
`Both methylparaben and propylparaben undergo acid—catalyzed and
`base—catalyzed ester hydrolysis. Maximum stability for the paraben esters is
`in the pH range of 4—5 [6] . For example, negligible hydrolysis occurred
`after several hours of boiling an aqueous solution of paraben esters at neutral
`or slightly acidic pH levels, while slight hydrolysis occurred after the same
`length of time at pH 8 or above [5] . At higher temperatures and at higher
`pH ranges, hydrolysis occurred rapidly. To exemplify this, Raval and
`Parrott [7] found that after autoclaving (121°C) a methylparaben solution for
`30 min at pH 6 and 9, there remained 94. 5 and 58.0% of the initial paraben con—
`centrations , respectively.
`
`IV. ANTIMICROBIAL ACTIVITY
`
`Table 2 shows the minimal inhibitory concentrations of methyl- , ethyl— , pro—
`py1—, and butylparaben against a variety of molds, yeasts, and bacteria come
`mon in cosmetic spoilage.
`In general, the parabens are more effective
`against yeast and molds than they are against bacteria, and more effective
`against gram—positive bacteria than against gram-negative bacteria. Because
`of their limited water solubility, parabens are not particularly effective
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`68
`
`Haag and Loncrini
`
`against Pseudomonas aeruginosa, since more preservative is required for in-
`hibition than would be soluble in water at room temperature.
`The antimicrobial activity of the p-hydroxybenzoic acid esters increases
`with increasing chain length of the alkyl moiety up to and including the butyl
`ester. The benzyl ester has been reported to have even higher activity [8] ,
`but it has not gained widespread acceptance in the cosmetic industry. The
`limitations of use imposed on the longer—chain esters are due to their poor
`aqueous solubilities. Thus, in practice, the lower esters (i.e. , methyl,
`ethyl, propyl, butyl) are most commonly used. The problem of effectiveness
`versus aqueous solubility can be overcome somewhat by combining two or more
`paraben esters in the product formulation. Aalto et a1.
`[5] found that As-
`pergillus niger was inhibited by 0.05% methylparaben or 0.015% propylparaben.
`By combining these two, one can reduce the paraben concentration required
`for complete inhibition of this microorganism to 0.025% methylparaben and
`0.01% propylparaben. This is an additive antimicrobial effect. According to
`Gottfried [9], the main advantage of using paraben mixtures is that the solu—
`bilities of the individual esters are independent of each other. Thus a
`higher total concentration of preservative may be incorporated into the pro—
`duct.
`
`[10] compared the preservative efficacy of methyl- , ethyl—,
`O'Neill et al.
`propyl—, and butylparaben in "problem" products.
`(Problem products were
`defined as products containing high levels of vegetable oils emulsified with
`nonionic surfactants and thickened with weak acid polyelectrolytes at pH
`values above 7.0). Methylparaben was found to be the most effective ester
`in terms of preservative capabilities. The authors did not show any examples
`of better preservation with a mixed paraben system than with an equal weight
`of the methyl ester. Thus they concluded that the least soluble paraben in
`the product is the most efficient in initial potency and capacity. The general
`greater efficacy of methylparaben in such systems was attributed in part to
`its lower solubility in vegetable oils when compared to the longer—chain esters.
`The mechanism of action of the p—hydroxybenzoates corresponds in prin—
`ciple to that of phenol. One proposed mechanism involves the destruction of
`the cell membrane and protein denaturation in the interior of the cell. An-
`other involves competitive reactions with coenzymes [11] .
`..
`Development of resistance was shown not to occur by Luck and Rickerl
`(Resistance is defined here as an increase in the minimum inhibitory
`[12] .
`concentrations under the influence of subthreshold concentrations of p—
`hydroxybenzoic acid esters.) However, more recent work by Peterson et al.
`[13] indicated that Escherichia coli has the ability to adapt to paraben solu—
`tions. Similarly O'Neill and Mead [14] stated that the rate of adaptation in—
`creased markedly with increasing molecular weight; E. coli adapted readily
`to propylparaben and hardly at all to the methyl analog. As a result, O'Neill
`ranked the parabens in the order methyl > ethyl > propyl > butyl > benzyl in
`their antimicrobial capacities.
`Various agents have been reported to potentiate the antimicrobial activity
`of the parabens. Prickett et a1.
`[15] demonstrated that 2—5% propylene glycol
`enhanced the antimicrobial activity of the parabens against vegetative cells,
`but not against bacterial spores. Phenylethanol was reported by Richards and
`McBride [16] to enhance paraben antimicrobial activity in ophthalmic prepara—
`tions. Similarly, ethylenediaminetetraacetate (EDTA) increased the activity
`of the p-hydroxybenzoic acid esters against gram—negative organisms, espec-
`ially P. aeruginosa [17] .
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`Esters of para-Hydroxybenzoic Acid
`
`69
`
`V. FACTORS INFLUENCING ANTIMICROBIAL ACTIVITY
`
`The antimicrobial action of the parabens is inactivated by several known
`mechanisms. The more notable ones that can significantly influence activity
`are the type and concentration of surfactant, the oil—to—water ratio, pH, and
`the presence of certain macromolecules or other potentially binding solids.
`
`A . Surfactants
`
`Paraben inactivation by nonionic surfactants has been well known for several
`decades. Their inactivating effects are of greater significance than those of
`anionic and cationic systems because nonionic surfactants have no growth—
`inhibiting properties. Thus the necessity for adequate preservation of sys-
`tems containing them is much greater.
`Belle and Mirimanoff [18] were the first to emphasize the reduced effect
`of methylparaben in the presence of several structurally different nonionic
`surfactants. Their findings stimulated a widespread interest in the pharma-
`ceutical and cosmetic industries.
`Investigations by de Navarre [19] and
`deNavarre and Bailey [20] led to the conclusion that any soluble ethoxylated
`nonionic surfactant would inactivate the paraben esters at use concentrations in
`an agar medium -, For example, deNavarre found that O. 1% paraben was ineffective
`in the presence of 1% nonionic surfactant. Similar studies by Blaug and Ahsan
`[21] dealing with the interaction of the methyl, ethyl, propyl, and butyl
`esters with several nonionic macromolecules indicated that the higher mole-
`cular weight esters were inactivated to a greater extent than those of lower
`molecular weight. Other studies and reviews in the literature have confirmed
`the ability of nonionic surfactants to reduce paraben activity [22—25] .
`The mechanism of paraben inactivation by nonionic surfactants has also
`received considerable study. Aoki et a1. [26] attributed the decrease of
`paraben activity to the envelopment of the paraben in a micelle formed by the
`nonionic surfactant. Evans [27] explained inactivation on the basis of solu—
`bilization of the preservative in the nonionic micelles. Thus compounds with
`high micellar solubility would be present in insufficient quantities in the
`aqueous phase to inhibit microbial growth.
`Patel and Kostenbauder [28] employed a semipermeable membrane to
`evaluate the degree of binding or intermolecular association between paraben
`esters and Tween 80. Both the methyl and propyl esters were investigated,
`and a relatively high degree of interaction with Tween 80 was observed for
`both. These authors concluded that the binding was a function of both the
`concentration of unbound p—hydroxybenzoate and the concentration of Tween
`80. Patel and Kostenbauder also noted that the propyl ester had a far greater
`affinity for Tween 80 than the methyl ester. At a concentration of 5% Tween
`80, 22% of the total methylparaben existed as a free preservative, whereas in
`the case of the propyl ester, only 4. 5% existed as free propylparaben.
`paraben.
`Later studies by Pisano and Kostenbauder [29] demonstrated that the
`activity of p—hydroxybenzoates in the presence of nonionic surfactants is a
`function of the unbound preservative. They suggested that the total con-
`centration of preservative required to protect a nonionic formulation could
`be estimated from predetermined binding data. These investigations suggest
`that the required concentration of p—hydroxybenzoates can be estimated by
`multiplying the preservative use level by the ratio of total to free preservative
`at any given Tween 80 concentration. This approach to emulsion preservation
`assumes, however, that other factors such as the oil—water ratio or pH do
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`70
`
`Haag and Loncrini
`
`not contribute to the efficacy of the preservative. Since it is known that
`these factors can exert powerful influences, it would be wrong to rely on this
`kind of prediction to the exclusion of other considerations.
`While most nonionic inactivation studies were conducted in relatively sim—
`ple aqueous systems, Charles and Carter [30] determined the activity of p—
`hydroxybenzoates in 18 typical nonionic cosmetic emulsions. They found the
`preservatives to be more effective in these products than would be antici—
`pated from results obtained with simpler systems. Their results indicated
`the need for testing the final finished product with all components present.
`Propylene glycol has been shown to potentiate the activity of the p-hy—
`droxybenzoates in the presence of nonionic surfactants. According to Poprzan
`and deNavarre [31] , 10% propylene glycol prevented the interaction between
`methylparaben and Tween 80.
`
`B . Partition Coefficients
`
`Owing to their high oil solubility, p—hydroxybenzoates have relatively unfav-
`orable partition coefficients between the oil and water phases of emulsions.
`Protection of these emulsions requires that lethal levels of the preservative
`contact the microorganisms in the aqueous phase.
`In an emulsion containing
`50% or more oil, more preservative may be present in the oil phase than in the
`aqueous phase after equilibrium is achieved. Thus a considerable proportion
`of the preservative can become inactivated by migration into the wrong
`phase. This migration can occur over a considerable period of time, which is
`one more reason why prolonged microbiological testing of formulated products
`is required.
`Hibbot and Monks [32] determined the oil—water partition coefficient of
`methylparaben in a number of oils and waxes. They then tested the activity
`of this preservative in a series of creams prepared from these oils and waxes
`covering a range of calculated partition coefficients. Results of their tests
`showed good correlation between activity and partition coefficients, indicating
`that this property can be used to estimate the performance of a preservative
`in an emulsion.
`
`[33] determined the parition coefficients of methylparaben
`Bean et al.
`in aqueous mixtures and emulsions of mineral oil, safflower oil, isopropyl pal-
`mitate, and lanolin. The oil—water coefficient for the preservative in mineral
`oil was 0.11 because of its limited solubility in this vehicle. The partition co—
`efficients in the other oils, on the other hand, were about 100—150 times
`greater than in mineral oil.
`
`The partition coefficient of many preservatives can be reduced by the
`addition to the aqueous phase of a substance in which the preservative is
`preferentially soluble. Propylene glycol, glycerin, sorbitol, and hexylene
`glycol are examples which can have advantageous effects in certain systems
`[34] . Humectants can, of course, provide an added benefit by increasing
`the osmotic pressure of the aqueous phase and thus increasing the inhibitory
`effect of the preservative. Propylene glycol, in addition, appears to have
`toxic effects on bacteria and fungi in its own right if used in the aqueous
`phase at a concentration of 15%.
`
`C.
`
`pH
`
`The parabens are effective over a pH range of 4—8. As the pH rises above 8,
`the parabens become less effective because of disassociation of the molecule.
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`Esters of para—Hydroxybenzoic Acid
`
`71
`
`.
`
`At a pH of 8.5, 50% of the compound is disassociated, and considerable loss
`of antimicrobial activity occurs. At pH 7 the parabens are 60— 65% effective
`[35] .
`
`D. Other lnactivating Agents
`
`Binding of parabens by other macromolecular materials has been reported.
`Interaction between methyl cellulose and the parabens was reported by Till—
`man and Kuramoto [36] . The methyl p—hydroxybenzoate showed the greatest
`binding effect, while the butyl ester showed the least. According to Miya—
`waki et a1. [37], methyl— and propylparaben are bound to some extent with
`polyethylene glycol, polyvinylpyrrolidone, and gelatin. No significant inter-
`action was noted with carboxymethylcellulose or tragacanth. These authors
`concluded that the concentrations of macromolecular polymers commonly used
`were not sufficient to interfere seriously with the use of the parabens.
`
`VI. APPLICATIONS
`
`The paraben esters are commonly used at levels of 0.1—0.8% in cosmetic pro—
`ducts. Combinations of these esters are generally found to be more effective
`against bacteria, yeasts, and molds than single esters. These combinations
`take advantage of the fact that each ester is independently soluble, and as a
`result, a higher combined concentration can effectively be introduced into a
`formulation.
`
`In many cosmetic formulations the paraben esters are used in combination
`with other preservations. Advantages of using preservative combinations
`include the following aspects:
`(1) The spectrum of activity can be increased,
`(2) the toxicological hazard can be reduced by using lower concentrations of
`component preservatives, (3) the development of resistance by an organism
`to one preservative alone may be prevented, and (4) additive or synergistic
`activities may result. The combination of Germall 115 (Sutton Laboratories,
`Inc.) and the paraben esters has been recommended [38] . Mixtures of the
`paraben esters with Glydant (Glyco Chemicals, Inc.) , Bronopol (The Boots
`Co. , Ltd.), and Dowicil 200 (Dow Chemical Co.) , among others, are also
`common throughout the cosmetic industry.
`
`VII. METHODS OF INCORPORATION
`
`The incorporation of the p—hydroxybenzoic acid esters into various formula—
`tions requires a familiarity with their solubility characteristics, particularly
`in water. Even though their water solubility is limited, they can be dis—
`solved in aqueous products at room temperature, provided that good agitation
`is maintained. The rate of solution is a function of particle size. With the
`fine powders that are normally available, aqueous concentrations of 0.20%
`methyl, 0.10% ethyl, 0.03% propyl, or 0.015% butyl can be achieved in 20 or
`30 min. A coarse powder, on the other hand, dissolves only after a long
`period of continuous stirring. Dissolution can be accomplished more readily
`by heating the medium to 70 or 75°C and stirring until solution is complete.
`This temperature should be kept about 20°C below the melting point of the
`paraben ester used to prevent agglomeration of the paraben particles.
`Another method of incorporation involves the preparation of stock solu-
`tions of the paraben esters in solvents such as alcohol or propylene glycol.
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`72
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`Haag and Loncrini
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`Such a solution must be added slowly to the aqueous phase of the formulation
`in order to prevent precipitation of the ester.
`
`Vlll. TOXICITY
`
`A. Acute Toxicity
`
`Sokol [3] reported the acute oral LD50 in mice to be greater than 8 g/kg of
`body weight for the methyl, ethyl, and propyl esters, and greater than 5
`g/kg for the butyl ester.
`In a similar study, Matthews and co-workers [39]
`determined the LD50 in mice of orally administered methylparaben to be 8
`g/kg of body weight, and the intraperitoneal LD50 to be 960 mg/kg of body
`weight. These authors observed the acute toxicity to increase with an in-
`crease in the molecular weight of the ester, and they judged the butyl ester
`to be approximately three times as toxic as the methyl ester. Symptoms of
`acute toxicity observed include ataxia, paralysis, hypotension, and general
`and myocardial depression. The animals that survived the test, however,
`recovered rapidly.
`
`B . Subchronic Toxicity
`
`[39] reported that dogs fed 500 and 1000 mg/kg methylparaben
`Matthews et a1.
`per day were unaffected after 1 year on the regimen. Sabalitschka and
`Neufeld—Crzellitzer [40] reported that a man was unaffected after taking
`2000 mg of methylparaben daily for 1 month. Similar studies by Braccesi [41]
`confirmed these observations.
`
`C . Chronic Toxicity
`
`Weanling Wistar rats, fed 0.9—1.2 g/kg of either methyl- or propylparaben
`for 96 weeks remained indistinguishable from the controls. Autopsies revealed
`no pathological changes in kidney, liver, heart, lung, spleen, or pancreas.
`When the dosage of either compound was increased to approximately four
`times these amounts, rats showed a slower rate of weight gain than the con—
`trols. These authors estimated that the toxic threshold for rats of both
`
`methyl— and propylparaben is at least 3000 mg/kg per day [39] .
`Sokol [3] reported that propylparaben stimulated growth in rats when fed
`at a dosage of 150 mg/kg per day for 18 months. On the other hand a dosage
`of 1500 mg/kg per day for the same period resulted in a growth depression
`without specific pathology.
`A survey by the U.S. Department of Commerce [42] revealed that there
`are no short—term toxicological consequences in the rat, rabbit, cat, dog, or
`man, and no long-term toxicological consequences in rats when the parabens
`are consumed in amounts greatly exceeding those currently consumed in the
`normal diet of the U.S. population.
`
`D. Biochemical Behavior
`
`p-Hydroxybenzoic acid esters are readily and completely absorbed from the
`gastrointestinal tract.
`In a study by Matthews et al.
`[39] in which dogs
`were orally administered 1.0 g of methyl— or propylparaben per kilogram
`body weight per day for 1 year, between 96 and 100% of the daily administered
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`Esters of para—Hydroxybenzoic Acid
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`73
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`[43]
`In a similar study Jones et a1.
`dose was accounted for in the urine.
`found no accumulation of methyl- or propylparaben or their metabolites in dogs
`which had been given 1.0 g/kg per day for 1 year. These authors were able
`to account for 96% of the daily administered dose in the urine in the form of
`free or conjugated p—hydroxybenzoic acid. By giving a fasted man 70 mg of
`methylparaben per kilogram body weight, they concluded that the paraben is
`handled similarly in human metabolism .
`
`E . Carcinogenicity
`
`A carcinogenic study reported by Homberger [44] which involved several
`techniques for ascertaining carcinogenicity, that is, subcutaneous injection—
`secondary host transfer, intravenous injection—observation of lung adenomas,
`and cocarcinogenesis, showed that methylparaben did not have carcinogenic
`activity. Boyland et a1.
`[45] dissolved methylparaben in polyethylene glycol
`and, by introducing it twice weekly into the vaginas of weanling mice for 18
`months, found no initiation of carcinomas.
`
`F. Teratogenicity
`
`Methylparaben elicited no teratogenic response in pregnant mice or rats when
`these were fed up to 550 mg/kg per day for 10 consecutive days, or in preg—
`nant hamsters when these were fed up to 300 mg/kg per day for 5 consecutive
`days [46] .
`
`G. Skin Irritation Studies
`
`[39] reported Shelanski‘s [47] results on human patch tests
`Matthews et a1.
`carried out with the paraben esters. The esters were dissolved in propylene
`glycol at 5, 7, 10, 12, and 15%. The highest percentages not causing irrita-
`tion were methyl, 5%; ethyl, 7%; propyl, 12%; and butyl, 5%. Repeated insult
`tests were also carried out on 50 subjects and no sensitization was observed.
`The parabens, when used in topical therapeutic agents, have been reported
`to sensitize approximately 1% of the population with eczematous dermatitis [4] .
`However, these identical parabens are generally considered "safe" in cosmetic
`preparations widely used by thousands of persons. The reason for this is
`that cosmetics are usually applied to normal skin, whereas therapeutic agents
`are applied to inflamed, eczematous, excoriated, or otherwise damaged skin,
`which is much more readily sensitized than even the thinnest normal skin.
`Simpson [48] reported a rare instance of a patient with allergic hypersen—
`sitivity to parabens in several cosmetic preparations. Similarly, Henry et a1.
`[49] reported a case of contact urticaria developed in a patient after topical
`application of several paraben—containing compounds. While occasional cases
`of paraben sensitivity occur, the incidence of sensitization is low when the
`extensive use of the material is considered.
`
`H. Eye Irritation Studies
`
`Methyl- and propylparaben as saturated aqueous solutions have been reported
`to be moderately irritating to the eye [50]. According to Foster [51] , nearly
`saturated solutions of parabens are too irritating to the eyes for inclusion in
`ophthalmic preparations .
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`74
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`IX . COMMERCIAL PRODUCTS
`
`Haag and Loncrini
`
`The methyl, ethyl, propyl, and butyl esters of p—hydroxybenzoic acid are
`generally sold directly by domestic producers, although some imported material
`is handled through distributors and agents. Blends of the methyl and propyl
`esters are also available to the industry.
`A liquid paraben is now commercially available to the cosmetic and toiletry
`industry. LiquaPar (Mallinckrodt, Inc.) , a 50% by weight paraben oil-in-
`water emulsion, consists of 20% isopropyl-p-hydroxybenzoic acid, 15% iso-
`butyl-p—hydroxybenzoic acid, and 15% n-butyl—p—hydroxybenzoic acid. The
`remaining 50% is water containing emulsion stabilizers. The principle behind
`the product is that the three solid paraben esters, when combined in the proper
`ratio, form a eutectic oil which has a freezing point near 0°C. This oil can
`then be easily emulsified.
`
`According to the manufacturer, LiquaPar offers the following advantages
`over conventional crystalline paraben esters:
`ease of incorporation into
`formulations, since additional solvents are not necessary; a decreased chance
`of error in formulation measurements; elimination of paraben particle size
`problems, thus eliminating the chance of grittiness in creams or lotions; sub—
`stantial savings in labor and product preparation time; and cost effectiveness
`in many formulations.
`
`X. METHODS OF DETECTION AND DETERMINATION
`
`The literature contains a number of papers on the detection and determina-
`tion of the pihydroxybenzoates in various formulations. A colorimetric
`method, described by Jones et a1.
`[43] , makes use of the reagent 4—aminoanti—
`pyrine. Higuchi and co-workers [52] described a method based on prelimin-
`ary extraction of the paraben from the product, separation of the component
`esters by partition chromatography, and subsequent determination of the in—
`dividual constituents by ultraviolet spectrophotometry.
`Liem [53] described a quantitative determination of the paraben esters
`using gas—liquid chromatography.
`In this procedure, the parabens are ex—
`tracted from the product with methanol, purified by thin—layer chromatography,
`and derivatized with N,O—bis-trimethy1-silyl acetamide prior to injection.
`The concentrations of the esters are calculated from a standard calibration
`curve.
`
`Methods of detection and determination have also been developed using
`high—performance liquid chromatography [54] .
`
`REFERENCES
`
`1. E. Richardson. Update-frequency of preservative use in cosmetic
`formulas as disclosed to FDA, Cosmet. Toiletr. , 96:91— 92 (1981).
`2. T. Sabalitschka. Chemische Konstitution und Conservierungsvermogen,
`Chem. Ztg., 48:703 (1924).
`3. H. Sokol. Recent developments in the preservation of pharmaceuticals,
`Drug Stand., 20:89—106 (1952).
`4. A. Fisher. Cosmetic dermatitis part 11, reactions to some commonly
`used preservatives, Curr. Contact News, 26:136—148 (1980).
`5. T. Aalto, M. Firman, and N. Rigler.
`p-Hydroxybenzoic acid esters
`as preservatives I, J. Am. Pharm. Assoc. Sci. Ed., 42:449—457 (1953).
`6. K. Connors, G. Amidon, and L. Kennon. Chemical Stability of Phar—
`maceuticals, Wiley, New York, (1979), pp. 260—265, 307—310.
`
`UCB Biopharma SPRL (IPR2019-00400)
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`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2027 Page 14
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`Esters of para—Hydroxybenzoic Acid
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`75
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`from art to
`
`N. Raval and E. Parrott. Hydrolysis of methylparaben, J. Pharm.
`Sci., 56:274—275 (1967).
`E. MacDonald. The preservative p—hydroxybenzoic acid esters, J.
`Am. Pharm. Assoc. Prac. Pharm. Ed., 3:181 (1942).
`N. Gottfried. Alkyl p—hydroxybenzoate esters as pharmaceutical pre-
`servatives. A review of the parabens, Am. J. Hosp. Pharm., 19:310—
`314 (1962).
`J. O'Neill, P. Peelor, A. Peterson, and C. Strube. Selection of para—
`bens as preservatives for cosmetics and toi