j. Soc.Cosmet.

Chem.,46, 199-220 (July/August1995)

Cosmeticpreservation
DANIEL K. BRANNAN, Department
of Biology,AbileneChristian
University,
Abilene,TX 79699.
Received
FebruaryI995.

Synopsis

The properuseof preservatives

to preventmicrobialcontaminationof cosmeticsis often viewedas an art
rather than a science.This view is a resultof the multifactorialthinking that hasto go into preservative
selection.In this generalarticle, an historicaland critical reviewof preservativeefficacytests(PETs) is
providedto understandthe assumptions
inherent in designingPETs. A conceptualframeworkof microorganismsexisting as communitiesin associationwith each other is also promoted, which providesa
differentunderstandingof how microorganisms
contaminatecosmeticsand why PETs are often misinterpreted.In addition, the modeof actionof preservatives
is discussed
andcontrasted
with the modeof action
of antibiotics.Finally, the role of the microbiologistis betterdefinedin light of the factthat he or shemust
haveexpertisein far more than microbiologyalone.

INTRODUCTION

Microbialcontaminationof cosmetics
did not becomean issueuntil about50 yearsago
(1). The first microbialcontamination
observed
wasprobablymold spoilage.Parabens
providedadequateprotection.During the 1960s, contaminationof consumerproducts
by Escherichia,
Klebsiella,Enterobacter,
Serratia,and Pseudomonas
spp. occurred(2-5),
demandingmore effectiveand responsible
preservation
practices.By the mid-1970s,
severalcases
of blindness
dueto Pseudomonas-contaminated
mascaras
caused
eyecosmetics
to be closelyscrutinized(6-8). Most productsreachedthe consumerin goodmicrobiologicalcondition,but they couldnot withstandcontaminationduring use(9-14).

The main issueaddressed

overthe next few yearswasto developpreservative
efficacy
tests(PETs) that predictedthe risk of consumercontamination.In 1975 and in 1985 the
Foodand Drug Administration(FDA) gavecontractsto developsuchPETs. The FDA
neverpublishedthe data from thesestudies,and no FDA nethodswere developed.In
1990, the Cosmetics,ToiletriesandFragranceAssociation
(CTFA) publishedthe results
of a surveyto determine if companieshad alreadycorrelatedtheir PET data with
consumeruse data (15). Nearly all the companiesclaimedthey alreadyhad correlation
programsin place, thus validatingthe ability of their company'sPET to predict consumercontaminationpotential(16). Inherentin this validationprocessis the question,

"What level of consumerabusemust a manufacturer

anticipatefor his product?"This
questionis usually answeredwith a legal definition: "To a level that is safe under
199

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JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

ordinaryuseand foreseeable
misuseconditions."Unfortunately,what constitutesforeseeablemisuseand ordinaryusehasneverbeendefined.It is left up to the courtsto
decidewhen prosecutingspecificcases.In the meantime,the companyis left to decide
for itself what "ordinary useand foreseeable
misuse"really means.
A joint programwith the FDA, the CTFA, and the Association
of Official Analytical
Chemists(AOAC) wasestablishedto developa standardPET in order "to demonstrate
the ability of productsto withstandmicrobialinsultwhichmay occurduringintended
use." However, the CTFA/AOAC/FDA collaborativestudyconductedno consumeruse
studiesto correlatewith PETs; consequently,the collaborativestudy (publicationexpectedin 1996) was not validatedby in-usetesting. Two PET methodshave been
publishedwith data allowingpredictionof the in-usepotentialfor consumercontamination (17-20). However, neither of thesemethodsis availablefor replicationsince
they both usedchallengeorganismsunique to the investigatorsconductingthe PET.

THE

IMPORTANCE

OF PRESERVATION

The cosmeticsthat most needpreservatives

are thosethat containwater. Productswith
low wateractivity(non-waterbasedlipsticks,rouges,talcs,andantiperspirants)
usually
needlittle morethan methylor ethylparabens
to protectagainstfungi. Table I provides
the water activity and pH limits for microorganisms
and relatestheseto producttypes
in general(21,22). The only limit to microbiallife is the availabilityof liquid water,
with microbesbeing found to grow at extremesof temperaturesand pH (23,24).
However, most organismsof concernto the cosmeticmicrobiologistare not extremoTable

Water Activity and the Potentialfor Growth

Water activity
0.98-1.00

Problemorganisms
capableof growth

pH
pH 5-9

Most Gram positivesand

Examplesof
cosmeticproducts
Shampoos
and emulsionproducts

negatives

0.95-0.97

pH 5-9

Most Gram positivesand

negatives(Pseudomonas
begins

Below 5.5

SomeGram negativesand most

Gram positives(Pseudomonas

Liquid make-upsand eyearea

products

to be limited)
Some hair conditioners

limited)
0.92-0.95

Above 5.5
Below 5.5

o. 90-0.92

pH 5-9

0.80-0.90
0.70-0.80
0.65-0.70

pH 5-9
pH 5-9
pH 5-9

0.60-0.70

pH 5-9

Few
Gram
negatives
and
most
}
Gram positives
Most Gram positives

Somepressedpowders

Gram positiveLactobacilliand
Staph.
Staph.,molds,yeasts
Molds, yeasts

Somerouges(non-waterbased)

pH 5-9

Some talcs

Osmotolerant
yeasts
}
Osmotolerantand xerophilic
molds

Below 0.60

Lipsticks(non-waterbased)

None

Someantiperspirants

COSMETIC

PRESERVATION

201

philes, and thus extremesof pH and Aw can be usedto controlthem. Where these
extremescannot be met, a biocide is used to control growth.

Microorganisms
metabolizeproductingredientsusinga varietyof hydrolyticenzymesto
causeadversechangesin productodor, color,andviscosity.Eventhoughhealth-related
contamination incidencesrelated to cosmeticsare rare, a few have occurred and include

infectionfrom a handlotion (3), eyeinfectionsfrom useof eyeareacosmetics

(25), and
the deathof one immunocompromised
individual(26).

Asidefrom spoilagepreventionand health-relatedconcerns,cosmetics

alsoneedto be
adequately
preserved
to withstandconsumer
use.Manufacturing
contamination
canbe
controlledwith good sanitation.But consumeruse and abusecannotbe controlled.
Consumers
mayrepeatedly
challengethe cosmetic
with microorganisms.
The bathroom,
wheremostcosmetics
and toiletry articlesareused,providesheatand humidity needed
for microbialgrowth (27,28).
During use, cosmetics
canbe contaminatedwith a varietyof spoilageorganismsfound
in the householdenvironment(29, 30). Table II lists someof the microorganisms
that
contaminateshampoos
and skin lotionsafter consumeruse (30). A few of thesemay
invadeand createdisease(31). With more and more immunocompromised
individuals
in the populationfrom the pandemicof AIDS, evenspoilageorganismsmay be opportunistic pathogens.The biggestcontaminationconcernsare pathogensthat presenta
frankhealthrisk suchasthe pseudomonads
(32). Cosmetics
intendedfor eyeareauseare
particularlysuspectsincethe cornea,when compromised,
is highly vulnerableto infection,and severalinstances
of mascaracontaminationfrom Pseudomonas
spp. havebeen
reported(6-10). Thus, choosingthe properpreservative
and packageis critical to
providingappropriateprotectionto the product.
Table

II

Typesand Percentages
of Microorganisms
ContaminatingCosmetics
After Use (30)
Organisms

Isolatedfrom shampoo

Citrobacter
freundii
Enterobacter
sppa

18
37

Isolatedfrom skin lotion

0
9

Klebsiella
spp.b

Pseudomonas
spp.c

21

Serratia
spp.d

18

GNR e (nonfermentative)
GNR (fermentative)

0
9

4
0

CDC serotype
IVC2
Bacillusspp.
Staphylococcus
epidermidis
Propionibacterium
sp.
Sarcinasp.
Diphtheroid

0
0
0
0
0
0

4
4
4
4
4
4

Yeasts and molds

29

E. aerogenes,
E. agglomerans,
and E. cloacae.

b K. pneumoniae
andK. oxytoca.
c p. putida,P. fluorescens,
P. paucimobilis,
P. aeruginosa,
andP. maltophilia.

S. liquefaciens,
S. odorifera,
andS. rubidaea.
e GNR, Gram-negativerod.
Table adaptedfrom Brannanand Dille (30).

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JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

However, selectionof preservatives

for a cosmeticis complex.The ideal characteristics
of a biocideare that it be safe,stable,and compatiblewith both the productand the

container,be inexpensive,
readilyavailable,approvedby appropriateregulatoryagencies,havea positiveconsumerperception,and be environmentally
friendly. Raw material quality, containerandcapdesign,expectedshelflife andexposure
conditions,and
evenhow the consumerwill useand misusethe productareadditionalconsiderations
in
choosingthe preservativesystem(30,33).

Compatibilityof the biocidewith otheringredientsin the productrequiresthe microbiologistto haveknowledgeof the art of formulation.Suspended
solidsin a formulation
(e.g., carbonates,
silicates,talc, metal oxides,cellulose,and starch)may adsorbpreservatives
(34). Minor pH changes
inactivateotherpreservatives
(35-37). Minor shifts
in ionic strengthor changesin the bufferingsystemin a productcan also alter a
bacterium'ssusceptibilityto a biocideor affecthowa preservative
partitionsbetweenthe
water matrix and the microbialcell (38,39). Parabensprovideunique formulation
challenges
for water-in-oilemulsions
because
theyhavean affinityfor the oil phasewhile
the microbeslive in the water phase(40). Eventhe surfactantsystemusedcan affect
biocideperformance
(41-43). In fact, nonionicsurfactants
are usedto neutralizesome
preservatives
(44-46). However,thesesamesurfactants
enhancequaternaryammonium
compounds
(47). Finally,protein(oftenusedin conditioner
andlotions)mayalsoreduce
the antimicrobialactivity of manypreservatives
(48-50); the presence
of hydrophilic
polymerswill affectothers(51).

Evensimplychoosing
a containerrequiresa microbiologist
to checkcompatibilitywith
the preservative(52,53). The preservativemay either be absorbedinto the container
materialin the caseof lipid-solublepreservatives,
inactivatedbecause
of complexation
of the preservative
with the dyesusedin the plastic,or lostbecause
of the volatility of
the preservative
(e.g., phenoxyethanol,
formaldehyde,
and ethanol).When considering
containers,one shouldalsonot overlookthe impactthat dispensingclosureshavein
preventingmicrobial contamination,especiallyduring consumeruse. Someclosures
providemoreprotectionof productsthan others(30). Alternatively,someclosures
may
inactivatethe preservative
(54).

PRESERVATIVE
DEFINING

THE

EFFICACY
PURPOSE

OF THE

TESTING
PET

Testprotocols
for determiningpreservation
efficacyin cosmetics
vary(55-58). The logic
and argumentsthat go into establishing
theseprotocols
areprimarilybasedon consensus.Thesecompendialefforts,suchas thosedevelopedby the CTFA, are "state-of-theart," but they are not rigidly controlledprotocolssubjectedto multiple laboratory
replicationand statisticalanalysis.Nevertheless,they have been useful. The CTFA/
AOAC/FDA collaborative
programmentionedpreviouslymay fill this gap despitenot
beinga methodthat hasbeenvalidatedto predictconsumer
contamination
potential.To
developsuchpredictivetests,a companymust employa microbiologistwho conducts
a validated "in-houseprotocol"that is specificfor the company'sproducts.Alternatively, the protocoldevelopedby the companymay be contractedout to laboratories
capableof conductingPETs.

COSMETIC

PRESERVATION

203

A majordifferencebetweenPETsis dueto a lackof understanding

of the purposeof the
PET. Defining the purposeof the test is critical. The entire experimentaldesignfor
validatingthe PET will differ dependingon the definitionof purpose.The experimental
designfor validating useof a PET asa predictorof the potentialfor consumercontamination is differentfrom that for validatinguseof a PET to demonstratethe presenceof
the preservativeor as a predictorof potential for manufacturingcontamination.To
validatea PET asa predictorof consumercontaminationrequiresprospectivecorrelative
consumerstudiesor retrospective
validation, basedon lack of consumercomplaints,to
corroboratethe PET laboratoryresults.Regardless
of which philosophyone adoptsto
definethe purposeof a PET, at a minimum the goal shouldbe to developa data base
to rank the antimicrobialhostility of the company'sproducts.
The purposeof a PET as viewed by the FDA is to predict consumercontamination
potential(59,60). The FDA hastried severaltimesto developa PET for this purpose
without success.Productsfailing sucha test would be subjectto recall. Despite the
collaborativework betweenCTFA, AOAC, and FDA to developa standardizedPET
completewith multi-lab comparisons
and statisticalanalysis,the method has not yet
beendemonstratedto have the ability to predict a product'sability to withstand microbialinsult that may occurduring intendeduse,sinceno correlativeconsumerstudies
were conductedusing the sameproductsfor which the PET was conducted.Such an
omissionis fortunate. If sucha standardPET were developedthat was predictive of
consumercontamination,then it couldbe usedto enforcea recallon thoseproductsthat
fail it. One could counter the recall by pointing out that a PET doesnot accountfor
consumeruseand packagingparameters.One might alsocounterthis argumentwith
the observationthat if the PET is done on freshlymade product, then the PET data
would only apply to freshly made product. Sincemost companiesconductPETs on
shelf-agedproduct, sucha statementwould be admitting that they are out of line with
the majority of reputablecompaniesand haveproductsthat becomea risk over time.
Severalpublicationshavealreadyshownthat a modifiedversionof the CTFA preservative efficacytest is a valid predictivemodel of the risk of consumercontamination
(17-20), but these all used proprietary "in-house" organismsunavailable to others.
Thus, the CTFA test describedby thesepublicationsdoesnot provide a standardPET
that could be usedto enforcea recallas describedabove.Another study hascompared
severalPETsfor the ability to predict"in-use"contamination(61). The majorcriticism
of this work is that the in-usetest wasmerelysimulated.The subjectsdabbledwith the
productafter rubbing their underarmswith their fingers.The significanceof ranking
PETs against their ability to predict how well a product can withstand simulated
consumerusedoesnot representvalidationagainsttrue in-useconditions.Nevertheless,
sinceall the PETswererankedagainsta singlestandard,onecanstill deriveconsiderably
usefulinformation. For example,nearly all the compendialtestsadequatelyseparated
poorly preservedfrom well-preservedproducts.Some of the more conservativetests
classifiedmarginallypreservedproductsthe sameas poorly preservedones,while the
more liberal testsallowed marginallypreservedproductsto rank with well preserved
ones.The CTFA testexhibitedthe tendencyto rank all three(poor,marginal,andwell)
correctlyagainstthe flawed but usefulstandardof a simulatedin-usetest. This study
does not, however, support the use of the CTFA test to enforcerecalls, since the
comparisonwas againstan invalid simulatedin-usetest.

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COMMON

JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

ISSUES SHARED

BY PETS

PETs are conceptuallysimple. All the protocolsinvolve introducingmicroorganisms

into the cosmetic.All the protocolshavethe samegeneralsteps:productpreparation,
inoculumselection
andpreparation,inoculation,incubation,platingandestimatingthe
survivingmicroorganisms,
and interpretingthe data. The differencesare in how these
stepsare conducted.Thesedifferences
are describedbelow.
Product
preparation.
The productsampleusedin a PET shouldreplicateall the parametersfor nationaldistribution, includingformulation,packaging,and manufacturing
conditions.Raw materialsshouldbe of the samequality and from the samesourceas
expectedfor nationaldistribution.Evenminor changesfor adjustingviscosity,color,
perfume, pH--even changesin processwater--may adverselyaffect a product'sPET
results.Scale-upfrom lab to plant alsoprovidesopportunityfor variablesthat needto
be identifiedand controlledsothe final nationallydeliveredproductwill be adequately
preserved.

Most publishedpreservative
methodstestfull-strength(100%) product(15). However,
Brannanetal. (17, 18) andCookeetal. (62) addeddilutedproductto the challengetest
as well. The dilutions stressthe product and also provide a meansof ranking the
product.For example,productsthat canbe diluted and continuekilling the inoculum
may be classifiedaswell-preserved,whereasproductsthat kill the inoculumonly when
the productis at full strengthmay be adequatelypreservedif the packagingprevents
consumercontaminationduring use. In addition, this approachis another way of
mimickingexpectedusepatterns.For example,productsthat are diluted duringforeseeablemisuse,suchas shampoos,shouldbe able to continuekilling microbialchallenges.Finally, dilution mimicks potential manufacturingerrors,particularlythose
involvingwashoutswherediluted productis accidentlyleft in a line. If a productcan
remain hostile when diluted, then microorganisms
are lesslikely to be selectedto be
resistantin the biocide. If not, then the organismsare selectedfor survivalat diluted
biocideconcentrations
and are just a minor stepawayfrom being selectedfor growth in
full-strength product.
Inoculum:
Selection
andmaintaining
resistance.
An appropriatemicrobiological
challengeof
the product is the most critical factor in determiningthe validity of a preservative
efficacytest. All the testscurrentlyspecifya setof inoculummicroorganisms.
Someof the
methodslist specificstrainsfromATCC, whileothersalsoallowinclusionof otherorganisms
the microbiologistchooses.These choicesoften include preservative-resistant
strains
from consumer-used
productsamples,raw materials,or manufacturingsites.However,
useof theseresistantorganismsmay be considered
a form of abusetestingby some.
Use of thesespecialstrains,however,shouldbe reevaluatedif one has not maintained
a rigorousprogramof preservingthe originally isolatedculture. More often than not,
onemaintainsa culturecollectionby putting up an originalsetof vials. When the last
remainingvial is subcultured,an isolatedcolony(obtainedby streakingfor isolation)is
selectedto grow up and harvest.This culture is preservedin anotherset of lyophilized
vials. Unfortunately,this processrepresents
a departurefrom the originallydeposited
culture becausethe progenyof only a single individual was selectedto representthe
original population.
Routine subculturingon nonselective
growth media will alsocausethe lossof preservativeresistance
sincethe selectivepressureof the preservative
is no longerpresent.The

COSMETIC

PRESERVATION

205

likelihoodof selectingorganismswithout the resistantfactoris high when using traditional"streakfor isolation"purecultureconcepts.

Instead,oneshouldrely on assessing the purity of the populationby its homogenous
appearance
on a lawnedagarplate.
Preferablythis shouldbe doneon a mediumthat hasthe preservative
in an activestate
(not neutralized)incorporated
into the agar.Oncethe populationis grownup asa lawn,
the entire lawn shouldbe harvestedfor freezingor lyophilization.
An areaneedingmore researchis the effectof growing the inoculumin broth or on solid
media. Greater resistanceto preservedproduct has been describedfor broth-grown
culturescomparedto culturesgrownon solidmedium. However,this resultmay have
beendue to the carryoverof broth into the productactingasa neutralizingagentof the
preservative
in the productratherthan to any intrinsicresistance
gainedby the bacteria

by growingin broth (63,64).

Another area needingfurther researchis the investigationof the importanceof the
growth phaseof the challengeinoculum.The growth phaseaffectsthe physiological
stateof the organismsusedas the inoculum.For example,Holm-Hansenfound that
ATP per cell is decreased
ascellsreachstationaryphase(65). This physiological
change
and potentially other changesmay affectan organism'sresistance
to preservatives.
Inocu/um.'
Concentration
and recha//enge.
In the CTFA's PET, the recommendedinoculum

levelforbacteria
is 1 x 108colony-forming
unitspermilliliter(CFU/ml).If 20 grams
of product are inoculatedwith 0.2 ml (a 100:1 ratio), then the final CTFA recom-

mended
concentration
of 1 x 106colony-forming
unitspergram(CFU/g)
ofproduct
is
obtained.Other PETsmay usedifferentinoculumlevels.The key issueis to keepthe
dilution of productby the inoculumto a minimum; a goodrule is to not dilute the
productover 1% with the inoculum.In like manner,fungi andyeastareintroducedinto

theproduct.
However,
theirconcentration
is only1 X 104 CFU/gofproduct
in the
CTFA method. The assumptionthat thesecountsrepresentfungal sporesmay not be
valid sincehyphaecan alsogive rise to fungal colonies.
How to standardizethe concentration
of the inoculumis left up to the microbiologist
in the CTFA method.A transmittance
of 30-40% at 425 nm of bacteriasuspended
in

bufferwill usuallyyield 1.0 x 108CFU/ml.However,

anyreference
to standard
microbiologicalmethodswill providethe specificsfor determiningmicrobialconcentrations (66).

Rechallengeis the additionof freshinoculumto a productthat hasalreadykilled off the

first challengeafter an appropriatetime. CTFA providesfor a rechallengeif desiredbut
doesnot require it. Somestudiessuggestthis practicedoesnot provide any more
informationthan singlechallenges
(67). The manufacturer,however,may be able to
makea casefor multiple challenges.
For example,mascaras
arecommonlysubjectedto
repeatinsultsby the consumer.In this case,the microbiologistshouldselecta challenge

levelthatisreasonable
andlikelyfromconsumer
use(1 x 102CFU/gm)ratherthanthe
high levelsrecommendedin the compendialmethods.Theselevelscould be determined
by allowing peopleto useunpreserved
productsand analyzingthe level of organisms
introducedinto the product after that use.

Anotherareaof concernregardingthe inoculumis whetheror not to usepure or mixed

challenges.This question refersto the use of severalpure cultures that are mixed
togetherafter they weregrownup and harvested.Use of this mixed inoculummay be
more representative
of actualconditionsof contaminationsincemicroorganisms
do not

206

JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

exist as pure culturesin naturebut as interactingpopulationswithin communitiesof

microorganisms.
If one assumesthat co-metabolismor synergismoccurswithin a community biologydynamic,mixed culturesmay providegreaterstressto the preservative
systemthan pure challenges(68). In fact, cometabolism
and vitamin and cofactor
synthesishelp stimulatemixed interactionswithin communitiesof microorganisms
(69,70). The idea that such interactionsoccur during a PET is supportedby the
observations
of Henriette et al. (71), who describeda mixed-bacterialcommunitythat
developedin disinfectants
andantibiotics.None of the individualspecies
wereresistant
to the antimicrobials.Only the communityshowedresistance.
In contrastto the above,however,it is the ideathat mixed populationsaremorerobust
that formsoneof the objectionsto their use.The claim is that it introducesthe variable
of microbialpopulationdynamicsinto the challengetest. Alternatively,somefeel that
the mixed culturesmay be lessstringentthan pure challenges
because
oneorganismmay
producemetabolicfactorsthat are antagonisticagainstother microorganisms
in the
challenge(72) or that the organisms
will competewith eachotherfor limiting substrates
and growth factorssuchas iron (73). Resolutionof the issuewill take more research.
Platecounts
and otherassumptions.
There are two assumptions
that microbiologistsmake
that arefalseregardingplate counts. . . andyet we still rely on them: 1) oneorganism
givesriseto one colony,and 2) organismsare evenlydistributedas singlecellsand do
not existasclumps.A newparadigmof organisms
existingasnonuniformlydistributed
clumpsthat later break up into individual cellsmay help to explain the anomalous
resultsoneoccasionally
getsin preservative
efficacytesting.It must be emphasized
that
the followingis only a modelas it appliesto preservative
testing. It is, however,a valid
model since it is basedon a historicallywell known fact that organismsdo exist
predominantlyin clumpsratherthan assingleindividuals,evenin shakeflaskcultures
(74,75). It is also basedon reportsabout the clumping nature of bacteriadue to
hydrophobicity
(76) and on the newerreportsaboutbiofilm and aggregateformation,
particularlywhen exposedto biocides(77).
The followingenigmaticscenariois sometimesseenduring a PET. An initial kill occurs
at 7 days(seenasa decrease
in CFU) but is followedby an increase
in CFU at 14 days,
followedby anotherdecrease
at 21 days.Usuallythis is passedoff asexperimentalerror
suchas use of the wrong culture conditionsor recoverysystemor incorrectdilution/
pipetting techniques.Occasionallyone gets theseresultsdespitecontrollingall these
factors.When this happens,the experimenter
may passoff the resultasan anomalyof
biologicalsystems.However, all theseexplanationsassumethat a CFU comesfrom
singleorganismsthat are evenlydispersedthroughoutthe sample.
Let'sexplorethe newparadigmthat providesat leasta hypotheticalmodelthat may help
explaintheseresultsbetter. Most peopleworkingwith bacteriaexposed
to disinfectants
and antibioticsare very well awarethat bacteriado not exist as uniformly distributed
individualsbut as biofilms and as Poisson-distributed
or negativebinomial-distributed
clumpsor aggregates
(66,78-80). If one usesthe paradigmof microbesexistingin
aggregates
(or clumps),the enigmamay be explainedwithout havingto claim "experimentalerror"(Figure 1). The initial kill at 7 daysmay havebeendue to killing of the
cellsin smallerclumps,wherethe entire clump of cellsis killed but the largerclumps
havea few cellswithin them that remainalive because
they wereprotected.Our model
is that CFU are reallyderivedfrom clumpsratherthan individualcells.The surviving

COSMETIC

PRESERVATION

207

cellsin clumpsthen disperseto resultin singlecellsthat give a higherCFU at 14 days

but now are more susceptibleto the biocide,and so a reductionfollowsat 21 days.
Although this model needsfurther testing, it is satisfyingthat it logically describes
what hasheretoforebeenpassedoff asexperimentalerroror biologicalvariabilityor the
developmentof resistance.
In doingplate counts,onecanuseeitherpourplatesor spreadplatesto determinehow
manyCFU/gm survive.In pourplates,thedilutedproduct(about1 ml) isvortexedinto

a testtubeof about15 ml of meltedagarat 46 -+ 2C.The agaris thenpouredinto a

Petri dish. Alternatively,the dilution may be placedinto the Petri dish andagarpoured
on top of it while the experimenterswirlsthe plate in a "figure8" motion. With spread
plates,the diluted product(about0.1 ml) is spreadontoprepouredsolidifiedagarplates
using a bent glassrod. Spreadplating allowseasyprocessingof samples.The main
advantageis that it avoidsexposingmicroorganisms
to heatedmedia. However, pour
plating allows for more exposureto neutralizingagentsin the agar. Somepublished
informationfinds that the two methodsgive similar results(81,82).

One can also performenrichmentsof the productto detect low levelsof potentially

T=Od;

T=7d;
10 CFU/ml

18 CFU/ml

T=14d;

21 CFU/rnl

T=21d;
6 CFU/rnl

Figure 1. Bacteriaexist asclumpsin Poissondistribution.This model helpsexplainanomalousresultsin

PET testing. Eachclump givesrise to a colonyratherthan eachindividualdoing so. After 7 days, the
CFU/ml is reduceddue to the deathof organisms
existingoutsideof the protectionof the clump. A few of
the organisms
at the peripheryof the clumparekilled, but the clumpstill formsan individualcolony.The
fact that someof the organisms
in the clumpdiedis not detecteduponplating. After 14 days,the clumps
breakup to providemoreCFU. Now the individualsareno longerwithin the protectionof the clumpand
are more susceptibleto exposureto the biocide.Therefore,at day 21 the total CFU is decreased.

208

JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

recoverableorganisms(83). The sensitivityor detectionlimit of typical dilution and

plate count methodsis usuallyfrom 10 to 20 CFU/gm of product. In enrichment,at
least10 g of productis put into 1 liter of brothand incubated.Any turbidity (or color
changeif one useseither a pH or redoxindicator)indicatesat least one organismwas
presentin the 10-g sample.This approachmakesthe detectionlimit 1 CFU/10 gm of
product(theoretically0.1 CFU/gm). Thus the sensitivityis increasedby 100X comparedto traditional plate count methods.It is most usefulin determiningif, after the
28-day period, low levelsof inoculumstill exist that may still be capableof growing
later

on.

Adaptationand resistance.
All of the recognizedtests require long incubationperiods
(28-56 days). These long periodsare supposedto accountfor the phenomenonof
adaptation.After somelag phasethe microbes"growback"to high enoughlevelsto be
detectedagain. The mechanism(s)
for this regrowthare not well understood.Perhapsit
is due to survivaland adaptation.Perhapsit is due to in situ recoveryof injured
organisms
(84). It may be dueto container-associated
organisms
that sloughoff into the
product(85,86). It may even be due to inadequatemixing and inconsistentplating
methods,sincebacteriadisplayPoissondistributionin the sample.The paradigmof
organisms
existingasclumpsis alsoa possibleexplanationto help explain"grow-back,"
without needing to claim microbial adaptation, or recoveryof injured cells or the
"PhoenixPhenomenon"(87,88). Theselatter two explanationsneednot be the soleor
even primary explanations;the clumping paradigm also explainswhat appearsto be
anomalousresultswhen cellsdie off but then "recover"during a PET. Whether or not
the clumping paradigm is a more valid explanationfor theseanomalousresults than
adaptationor the "PhoenixPhenomenon"remainsto be shownempirically.
Certainlytherearecaseswhereadaptationoccurs.However,whereadaptationis claimed
for preservatives
that havemultiple modesof action,resistance
is rarelyvia an individual
occurrenceof plasmid acquisition,mutation, or lifting of repression(89), as is often
found with antibiotics but rather is a result of enhancementof the expressionof a
characteristic
within a populationdue to geneticdrift. This may occuras a shift in the
amountof capsuleproduction,clumping, stimulationof productionof glutathione, or
evenphysicalcommunitydevelopments
within biofilms wherecertainorganismsexist
asprotectorguildsfor otherorganisms(90-92). Suchresistance
is typified by whole-cell
poisons
suchaschlorine(93,94). Fewcases
of true chlorineresistance
occur(e.g., point
mutationsby a singlemutant cell that survives).Instead,any "resistance"
seenis really
a populationor communityeffectof cellsexistingwithin the protectionof a biofilm or
surrounded
with a capsulecomposed
of extracellularpolymericsubstances
that excludes
the chlorineor useof cellular energyto producehigher levelsof glutathione(90,95).
Perhapsin theseexamplesa moreproperterm to usewouldbe biocide"tolerance"rather
than resistance.The establishmentof a biofilm or clumps of organismsprovidesan
adaptationmechanismfor toleranceto biocidesusingextracellularpolymersin the form
of a capsule.This biofilm then leadsto an inoculumsourcethat is constantlybeing
exposed
to sublethalor subinhibitorylevelsof biocide.Onceestablished,
adaptationvia
increasedproductionof glutathioneor a slowdownof metabolism(or even perhaps
mutation) can result in a resistantphenotype(or even genotype),and the problem
becomescompounded(personalcommunication,J. S. Chapman, Rohm, and Haas).

Geneticadaptationto biocidesat the individualratherthan populationlevel is a possibility in somecases

(96). However,severalpapersclaimingto havedemonstrated
this

COSMETIC

PRESERVATION

209

phenomenon
areeithera caseof neutralizationof the biocide(by carryoverof the growth
medium) or a caseof saturatingthe biocidewith more organismsthan availablebiocide
to the point of inactivatingit (97,98). Specificgeneticmechanisms
(e.g., point mutations, plasmidacquisition,lifting repression)
or the expression
of formaldehyde
dehydrogenase
do existin somecases(99,100). The hallmarkof whetheror not a permanent
geneticadaptationhas occurredis the stability of the resistancein the absenceof
selectivepressure
from the presence
of preservative,
asapparentlyis the casefor parabens
(101). However, resistanceto all biocidesby permanentgenotypicchangemust not
alwaysbe assumed.The most naYveidea is that the resistancemechanismsagainst
biocides are similar to those mechanisms found in antibiotic resistance. Whereas anti-

biotic resistancecan be describedbasedon specificmolecularactivity at specificsites,

the resistance to biocides cannot. Often the resistance to biocides must be maintained

at a populationlevel by continuouslyculturing the organismin the presenceof the

preservativeto maintain the selectivepressureon the population.This selectivepressure
causesthe populationto develophigher capsuleproduction,which enhancesclumping
associations,
and the productionof biofilms. Alternatively, enhancementof the expression of glutathionesynthetasecould alsooccurwithin the populationto provideresistance to some biocides(102). Take the selectivepressureaway, however, and this
expression
stops,indicatingthat a permanentgeneticchangewithin individualsdid not
take placebut rather that populationshiftsoccurred.
Useof neutralizers.
Appropriateuseof neutralizersis often overlookedwhen conducting
PETs. Some preservativesonly require dilution in buffer to be inactivated. Others
requirechemicalneutralizersused in the diluent or the plating medium, or both.
Filtrationis anotherapproachbut is limited to thoseproductsthat canbe filtered. The
work of Suttonand othersdescribes
a numberof neutralizationmethodsfor preservatives
as well as a scientific basis for their evaluation (103-107).

The goal of a neutralizeris to inactivatethe biocidebeforethe biocideinactivatesthe

microorganism
in orderto provideuninhibitedmicrobialgrowth.Failureto inactivate
the biocideimmediatelyuponsamplingcauses
oneto overestimate
the killing potential
of the biocide. This failure is actuallya measureof the kill that continueswithin the
plating medium becausethe activebiocideis carriedover into the medium (108). A
fairly effectiveall-purpose(universal)neutralizingmedium is Dey-Engleybroth (109).
Dey-Engleybroth is described
furtherin Atlas'Handbook
ofMicrobio/ogica/
Media(110)
andthe DifcoManual(111). A thoroughreviewof thisandmanyotherneutralizers
may
be foundin the articlesby Russell(84) and Sutton(112).
The ASTM providesa methodto determineif a neutralizeris nontoxicand effective,
usingmicroorganisms
asbiologicalindicators(113). This methodis a retroactivecheck
for neutralization.It is doneby streakingplatesshowingnogrowthwith testorganisms.
The streakis done48 hoursor moreafterthe inoculatedproductwasoriginallyplated.
Sincethis streakis donesolong after the initial plating, the retroactivetest only proves
that neutralizationfinally occursafter allowingthe biocideto incubatein the medium
for somelength of time; it doesnot provethat neutralizationoccurredinstantaneously
when the product containingthe biocidewas mixed into the medium. Retroactive
checks of neutralization, and thus the ASTM method of neutralizer validation, are
invalid.

Interpretation
ofdata. Interpretationof the datausingthe criteriasetby the compendial

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JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

testsis basedon anecdotalevidenceand opinionregardinghow long a productshould

take to reducethe numbersof the challengeinocula.The bestway of interpretingthe
data, however,is to comparehow the testproductperformsagainsthow well-preserved
and poorlypreserved
controlproductsperform.Well-preserved
productsarethosethat
do not becomecontaminatedduring consumeruse, and poorly preservedproductsare
thosethat do becomecontaminatedwhen usedby consumers.
OTHER

PET METHODS

D-valuemethods.
Rapid testsare sometimes
usedfor quick impressions
of whichpreservativesto usein a product.One suchmethodis the D-valuemethod.Asidefrom one
author, no one else claims D-value methodsare valid for final testing of nationally
distributedproduct(114). In fact, D-valuemethodsareinappropriate
for at leastsome
consumerproducts(115). This methodis actuallyan adaptationfrom foodmicrobiology'sheat or radiationdestructionD-values.
Heat and radiation kills do indeed follow first-order rate kinetics, and therefore the

D-valuesdeterminedfor them are quite valid. However, biocidekills follow secondorderrate kinetics(115,116). The only casewherea second-order
reactioncanapproach
pseudo-first-order
ratekineticsis whenthe secondreactant(biocide)is presentin such
largeexcess
that it is virtuallyin constant
concentration.
Preserved
productsdo not have
an excessof preservativesuchthat the biocideremainsin constantconcentrationwhen
contaminationoccurs(117). A biocide-organismreactionis stoichiometric;the biocide
doesnot act like an enzymethat catalyzesa reactionwherelive organismgoesto dead
organism,but the biocideis not spent.Therefore,sincethe biocide-organism
reaction
is secondorder, with the biocideservingas the limiting reactant,D-value testsbased
on first-order rate kinetics are invalid (115,117).

The secondcriticism of the D-value techniqueis that it extrapolatesbeyondthe measureddata by falselyassuminga linearrelationshipbetweenbiocideexposuretime and
the numberof survivingmicroorganisms.
In defenseof rapid D-valuemethods,however, one may find they allow a preliminaryscreeningof preservatives.
This approach
assumes
that appropriatereproducible
controlsarein placesuchthat onewill be ableto
rankthe variousD-valuesfor a widevarietyof productsandbe ableto correlatethat data
to full-scalePET resultson the sameproducts.

Capacitytests.A capacitytest determineshow many bacterialchallengesare needed

beforethe product begins growing microorganisms
(118). After each challenge,the
productsare sampledand challenged
againuntil the producteitherreceives15 challengeswithout showinggrowth(a well-preserved
product)or until threeconsecutive
positiveresultsoccur(a lesser-preserved
product).The goalis for the productto reduce
the numberof viableorganisms
by 3 logs(99.9%) in 48 hours.With eachsubsequent
challenge,this ability diminishesas a resultof dilution, neutralization,and reaction
with the inoculum.The claim, by somestudies,that multiple challengesprovideno
moreinformationthan singlechallenges
(67) may actuallybe morepragmaticallybased
than scientificallybased.The reasonwhy multiple challenges
with low levelsof organismsare not the sameasonechallengewith a high level is similar to the conceptof the
Danyszphenomenon
in immunology(119), wherewhen a high level of inoculumis
used, the biocide combineswith an equivalent amount of microbes,allowing the
challengeto be killed, but when challengingmultiple times, eachchallengecombines

COSMETIC

PRESERVATION

211

with more than its equivalentamountof biocide,leavinginsufficientbiocideto react

with subsequently
addedmicrobes
in the challenge
(120). The valuein sucha testmay
be for multi-useproducts.However,for a morepredictivetest of consumercontaminationpotential, one shouldlower the challengeinoculato levelslikely to be encounteredduring use.Oncedone,the capacitytest may be a quantitativeandvalid way to
understanda product'sability to handlecontaminationfrom use.

PRESERVATIVES
MODE

OF ACTION

AVAILABLE

FOR

USE

OF PRESERVATIVES

The modeof actionof antibioticsis knownat the molecularlevel sincethey act via
specificbiochemicalreactions.In contrast,the modesof actionof preservatives
and
biocidesare far moregeneralized,with
numerous
pointsof attack.Nearly all biocides
work by denaturingcellularproteinsor by affectingmembranepermeabilityso that
eithertransportor energygenerationis blocked.For example,chlorineoxidizesreduced
sitesof organiccompounds,includingproteins,throughoutthe bacterialcell. Protein
denaturantsalso include formaldehyde,formaldehydereleasers,isothiazolinones,and
brominecompounds.

The parabens
and weakacids(e.g., sorbic,benzoic,and dehydroacetic
acids)disrupt
controlof membraneelectricalpotentialto blockenergygenerationand nutrienttransport (121). Thus the parabensapparentlyinhibit nutrient uptakeby shuttingdown
permeases,
disruptingporin channels,or by disruptingthe membranepH gradientor
electricalchargepotentialacross
the membraneto preventsubstrate
transportandATP
generation.This inhibition is apparentlyreversibleand is consistentwith other observationsthat the modeof actionof parabensis by disruptionof the membraneelectrical
potential (122).

Organicacidsprobablywork in the samefashion(123); however,they may evenbe

enzymeinhibitorsas well (124,125). Typically, they are only biocidalat pH values
belowtheir pK. In this protonatedform, they passthroughthe membrane,and the
hydrogenion dissociates
from the weakacidto decrease
the cytoplasmic
pH. As a result,
both substrate
transportandoxidativephosphorylation
areuncoupledfrom the electron
transportsystem.This effectivelystarvesthe cell of neededsubstrateandenergyderived
from ATP synthetase
drivenby hydrogenions.
Phenolicsdisruptthe protonmotiveforceof the cell membrane(126,127). They also
havethe ability to non-specifically
denaturecytoplasm,cell walls, and cell membranes
(128). The more lipophilic phenolicshave the greaterantibacterialcapacityperhaps
becauseof a greaterability to partition out of the water phaseand into the lipid
membrane(129,130). Alcoholslikewisedisruptthe membrane,causingpermeability
loss(131), and they alsoappearto inhibit enzymes(132).
Perhapssomeof the mostwidely usedof the newerpreservatives
arethe isothiazolinones.
These are usually compoundedinto a single product composedof chloromethylisothiazolinone
andmethyl-isothiazolinone,
but theycanalsoincludebenzyl-typecompounds(133,134). The isothiazolinones
inhibit glucoseoxidationand activetransport
without significantlyaffectingmembraneintegrity (135). In fact, thesecompounds
denatureenzymesand other proteinscontainingthiol groups(e.g., ATPase, glyceral-

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JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

dehyde-3-phosphate
dehydrogenase,
and asparaginase).
Initially, the isothiazolinone
forms a disulfide link with the thiol group on the amino acid. Occasionally,the
chloromethyl-isothiazolinone
may facilitatelinkagewith anotherthiol group to establish a new disulfide linkage and releasethe biocide as a mercaptoacrylamide.
This
mercaptoacrylamide
can tautomerizeto a thioacyl chloride that may react again by
denaturingnucleicacids(136).

Formaldehyde
alsodenaturesproteinbut by alkylatingaminoand sulfhydrilgroups;it
can also alkylate the nitrogensof purine rings to denatureDNA (137). Most of the
formaldehydedonors(e.g., DMDM hydantoin, imidazolidinyl urea, Quaterium 15,
polymethoxybicyclicoxazolidine,etc.) act in this basicmannersincethesecompounds
releaseformaldehydeinto the productor the microbialcell. Differencesseenbetweenthe
formaldehydedonorsmay exist as a result of when or what triggersthe compoundto
releaseor "donate"formaldehyde.For example,a compoundwith a long hydrophilic
chain connectedto the formaldehyde-donating
region(e.g., polymethoxybicyclicoxazolidine)may releaseformaldehydeonly when the long chainentersinto the lipopolysaccharide
portion of the membrane.
Brominatedcompoundssuchasbromo-nitropropanediol
and bromo-nitrodioxane
act by
oxidation of thiol groups (138-141) or by causingthioIs to convert to disulfides
(142,143) wherethe thiol groupfirst becomes
brominatedandthen reactswith another
thiol group to yield a disulfideand free bromide. As a result, enzymesinvolvedin
respiratoryactivity (e.g., dehydrogenases)
and nucleicacid synthesisare inhibited, cell
membraneintegrity is compromised,and the cell wall may evenbe affected(144).
One compoundthat is not technicallya biocidebut rathera biocideadjuvantis ethylenediamine-tetra-acetic
acid (EDTA). This and other chelatingagentsremovemagnesiumandcalciumdivalentcationsfromthe cellwall, whichis neededfor stability(145).
Once destabilized,they permit easieraccessof biocidesinto the cell.
SELECTION

OF PRESERVATIVE

The ideal preservativewould be broad-spectrum,safeand completelyfree of any sensitizationissues,completelywater-soluble,completelystableto all extremesof pH and
temperature,completelycompatiblewith all ingredientsand packages,and impart no
coloror odorto the product,be inexpensive,and complywith governmentregulations.
This idealdoesnot exist. One mustselecta preservative
basedon empiricaltesting.The
only approachborderingon a theoreticalbasisfor choosinga preservative
is a qualified
microbiologist's
intuition, finely honedby experience.Selectionof preservative
may also
be from publishedlistsof availablepreservatives
(146,147). Theseprovidegoodsources
for getting ideasof what might work in a formulation. Every formulationmust be
considered
unique. Factorssuchasthe physicalandchemicalnatureof the product,how
it is to be used, the containertype and closure,and the shelflife must be considered
when choosingthe preservative(30). Often the selectionof a preservativemust be a
compromise
betweenefficacy,stability, and safety.More detail on the selectionprocess
of preservatives
can be foundby referringto severalbooksand articleson the subject
(148-150).
SAFETY

CONSIDERATIONS

OF PRESERVATIVES

One mustalwaysbalancethe risk of microbialcontamination

with the risk that a biocide

COSMETIC

PRESERVATION

213

maygiveto a product.Forexample,manyeyeareaproducts
werepermittedto contain
phenylmercuricacetatebecause
theriskof infectionto theeyewasgreaterthanthe risk
of exposure
to the compound.The key consideration
is to judgewhetherthe product
will be safe for the consumer under normal use and foreseeable misuse conditions.

One of the first considerations

of a preservative
is its acutetoxicity. Ocular irritation and
subchronic
andchronictoxicitytestsareperformedvia the expectedconsumer
exposure
route to determineat what level the preservative
can exhibit any irritant, toxic, or

carcinogenic
properties.Perhaps
moreimportantthanthesetestsarethe skin responses
to biocides.Basicirritant responses
can be a result of corrosion,acute irritation, cumulative irritation, or photoirritation.

Skinsensitization
is anotherkeyconcernwhenusingbiocides.Nearlyall biocidesused
todaywill elicit sensitization.
Sensitization
testingis performedin muchthe sameway
as irritant patchtesting, exceptthat much lower concentrations
and repetitiveapplicationsare used.Anotherconcernfor biocidesis mutagenicitytestingto determineif the

biocidehasthepotentialformutatingsomaticor germcells.In additionto this testing,

embryological
(or developmental)
toxicitytestingis doneto determineif the biocide
maybe a teratogencapableof causingbirth defects.
In all thesetests, the resultsmust be comparedto the ordinary-useand foreseeablemisuseexposure
levelsto give us a reasonable
risk assessment.
The definitionof reasonablerisk must include considerations
basedon the benefitsfrom using the biocide,

the abilityto uselessriskybiocides

for the sameuse,the economic
benefitsfromusing
the biocide(canthe biocidehelppreventcostlyrecallsdueto contamination?),
evenhow
the biocidemay affectthe qualityof life, the environment,and publicopinionof the

company.Moredetailon thesafetyconsiderations
of cosmetic
ingredients
maybefound
in booksby Waggonerand Whittam (151,152).

CONCLUSION

This article doesnot detail or discussthe prosand consof the variousmethodsusedin

cosmeticmicrobiology.Thereareplentyof references
availablefrom whichthe serious
studentcanget thisinformation
(56,153-156). Regardless
of whichmethodsarein use
by anyparticularcompany,
thefactthat thecosmetic
industryhasbeensosuccessful
in
providingadequately
preserved
productsfor its consumers
is commendable
and reinforcesthe wisdom that we are capableof self regulation.

The cosmeticmicrobiologistmust balancea variety of factorsto provide for safe,

unspoiled
qualityproducts(157). In additionto knowingpreservatives,
he or shemust
understand
microbialphysiology,pathogenic
microbiology,and microbialecology.In
additionto microbiology,he or shemust understandorganicand physicalchemistry,
toxicology,engineering,manufacturingand processing,
sanitation,and regulatory/
environmentallaw. The cosmeticmicrobiologistmustuseall this educationandknowledgewithin the contextof the business
needsof the companyand be ableto balance
risk/benefitto the consumerusing the product. Finally and most importantly, this

person
musthavethe highestof ethicalstandards,
considering
himselfor herselfaspart
of the cadreof healthcareprovidersin the worlddedicatedto servinghumankindvia the
missionof providingmicrobiallysafeand efficacious
products.

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JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

ACKNOWLEDGMENTS

I thank the many collegueswho provided good insight: Phil Gels, Bill Apel, Scott
Sutton, Tom Sox,Jan Curry, StephenRichards,and Rich Hennessy.I thank Bonnie
Bailey and Pat Hernandezfor reviewingthe paperfor grammarand easeof reading.

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