Animal, Human, and Microbiological Safety Testing of

J. Soc. Cosmet. Chem., 24, 135-146 (February 2, 1973)
Animal,Human,and Microbiological
SafetyTestingof Cosmetic
Products
M. J. THOMAS, Ph.D., and P. A. MAJORS,M.S.*
PresentedOctober27, 1971,beforethe CaliforniaChapter
Synopsis-Thethreeprincipalareasof TESTING pertinentto COSMETICS are discussed.
These include SAFETY determination
in ANIMALS, safety and efficacystudiesin
HUMANS, and MICROBIOLOGICAL STUDIES for safetyand productstability.Products must be shownsafeto animalsprior to use on humans.Most animal studiesinclude,
for the most part, acute and subacutestudies.The former are done in accordancewith
the FederalHazardousSubstances
Act (FHSA) requirements.
There are a numberof patchtestsapplicableto humansafetytestingof products.The
modifiedDraize techniqueappearsto be the preferredprocedure.It servesas a good
predictorof sensitizing
potentialand suppliesinformationon the primaryirritant characteristicsand cmnulativeprimaryirritantpropertiesof the test materials.A test program
is also presentedfor determinationof adequacyof PRESERVATIVES in new and current cosmetic products.
INTRODUCTION
The increasing
emphasis
placedby our Government
on the safetyof foods,
drugs,andsimilarproducts
hasbeenexpanded
intothe areaof cosmetic
productsin the last few years.Thosein the independentresearchand testinglab-
oratoryfieldsare verymuchawareof thischangebecausetheyhavebeenan
integralpart of the developingprogramand have seenthe variousregulations
evolve.Just as the regulatoryrequirementsfor safetyin food additivesand
drug productsbecamemoredefinitiveas time passed,so will the regulatory
requirementsfor cosmeticproductsbe more specificand morepronouncedas
time goeson.
The objectivehere is to summarizethe presenttrendsand methodsused in
testingfor the safetyand efficacyof cosmeticproducts.Recent action of the
*Hill Top Research, Inc., Miamiville, Ohio 45147.
135
136
JOURNAL
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CHEMISTS
FDA relative to voluntary disclosureof productingredientswill not be discussedin detail. Accompanyingthe publicationof the ingredientdisclosure
regulationswas the unexpectedchangein the classificationof 13 cosmetic
product categoriesas also being drugs. This reclassification
does not necessarily mean that additionaltestswill be required. Many cosmeticmanufacturers have productsafety standardswhich exceedthe standardswhich apply
to new drug approvals.
The one area of testing which will be affectedis the evaluationof these
productson humans.Before this can be done, proper investigationalforms
mustbe filed xviththe Food and Drug Administration.This requiressubmissionof detailedprotocolsfor review, and review of theseprotocolsby peer
committees.
The three ,principalaras of testingpertinentto the cosmeticfield include
(a) safetydeterminationsin animals,(b) safetyand efficacystudiesin human panelists,and (c) safetyand productstabilityfrom the microbiological
standpoint.
When a productis submittedfor humanpanelisttesting,it is mandatorythat sufficientanimaltoxicological
data existto showthat the product
is relativelynontoxicand will not induceexcessive
reactionsin the testingon
humans.After the producthasbeenshownto be safein animaland human
panelisttesting,it is readyfor the market.However,there are still microbiologicalcontamination
factorswhich shouldbe considered.
During the manufacturingprocessthe product may be inadvertentlyexposedto unsanitary
conditions
in the plant or aroundequipment,or to contaminatedair entering
the plant, or to obiectionable
microorganisms
in the basic raw materials.
ANIMAL
TOXICOLOGY
It is usuallyrecommended
that the animaltoxicologyprogramsfollow the
protocols
specified
by the FederalHazardousSubstances
Act (FHSA) and its
subsequent
Regulations,
publishedin the FederalRegister(1), and thoseof
additionalstudieswhich are frequentlyrequired.
Theseprogramswfil require one or moretestsaccordingto the type of
productinvolved.Theseare:
1. Acute oral toxicity-rats
2. Acute eye irritation-rabbits
3. Primaryskinirritation and corrosivity-rabbits
4. Acute dermaltoxicity-rabbits
5. Acute inhalationtoxicity-rats
6. Subacutedermal (21-day) toxicity-rabbits
7. Landsteinerskinsensitizationin guineapigs
Acute Oral Toxicity
These studiesare conductedwith six groupsof five male albino rats (30
rats).A singleoraldoseof the testmaterialis administered
at graduated
dos-
SAFETY
TESTING
OF COSMETICS
137
agelevels.The ratsare observed
for grosssignsof systemic
toxicity,pharmacological
effects,
andmortalityat frequentintervals
duringthedayof dosage,
andat leastoncedailythereafterfor 14 days.Grossautopsies
are performed
on any animalsxvhiehdie.
Acute Eye Irritation
Sixalbinorabbitsareusedin thesetests.One-tenthmilliliterof a liquidtest
material,or 100 mg of solidsor pastes,is placedin one eye of eachof the
rabbits.The oppositeeye is untreatedand servesas a control.Readingsfor
eyeirritationare madeat 24, 48, and 72 hoursfollowingapplication.The scoring scalefor recordingeye irritationis that of Draize (2).
Primary SkinIrritation and Corrosivity
The test material is applied under a 1-in.2 gauzepatch to both an intact
and an abraded skin area on each of six albino rabbits, whose trunks have
beenclippedfree of fur. Four patchescan be applied,allowingfor two samplesto be testedon eachanimal.
Acute Derreal Toxicity
This studyusesfour groupsof four albinorabbits.A singlederrealapplicationof the testmaterialis madeto eachgroupat graduateddosagelevels.
The skinof two rabbitsper groupis abraded;the skinof the two remaining
rabbitsper groupremainsintact.The test materialis appliedunder a rubber
dental dam and remains in contact with the skin for 24 hours while the animals are restrained in stocks.
Acute Inhalation Toxicity
Ten male albinorats are exposedin an inhalationchamberfor onehour to
an atmosphericconcentration
.of20,000ppm of gas.orvapor or 200 mg/1. of
mist or dust.The rats are observedduringthe exposureperiod and daily for
14 daysfor grosssignsof systemictoxicity.Grossautopsiesare performedon
rats which die.
Standard criteria are used to evaluate the results of 'these various animal
studies as shown in Table I.
SubacuteDerreal (21-Day) Toxicity
Thisstudyevaluates
the percutaneous
absorption
andirritativepotentialof
the testmaterialfollowingsubacutederrealapplicationto rabbits.The testis
not specifiedby the regulationbut is frequentlycarriedout. Eighteenyoung
adult albino rabbits are divided randomlyinto three groupsof six rabbits
each.
Group1 is the controlgroupand receivesrepeatedskinapplicationsof dis-
tilledwater.Group2 receives
repeatedskinapplications
of the testmaterial
JOURNAL OF TIlE SOCIETY OF COSMETIC CHEMISTS
Table
I
Evaluation of Toxicity in Animal Studies
Route of
Administration
Oral
Derreal
Inhalation
Highly Toxic
Toxic
Nontoxic
LD5o of 50 mg/kg or less LD,o >50 mg/kg but LD•,o> g/kg
not >5 g/kg
LD5oof 200 mg/kg or less LD,o >200 mg/kg but LD•o >2 g/kg
not >2 g/kg
LD5o of 200 ppm of gas or LD5o >200 ppm of gas LD,o >20,000 ppm
vapor (2 rag/1. for mist or or vapor (2 mg/]. for of gas or vapor
rag/1. of
dust) or less during one- mist or dust) but not (>200
hour exposure
more than 20,000 ppm mist or dust)
(200 rag/1. of mist or
dust)
at a low dosagelevel. Group3 receivesrepeatedskin applications
of the test
material at a high dosagelevel ( 10 timesthat of Group 2).
Three rabbitsin eachgroupare abradedat the beginningof the first, second, and third weeksof the study.The skin of the remainingrabbitsis left
intact. The controland test materialsare applied oncedaily on a 5-day per
weekbasisovera 3-weekperiod,for a total of 15 applications.
Eachdaily ex-
posureperiodis for 6 to 8 hours.Followingthe 15 applications,
the rabbits
areobserved
for 2 weeks,thensacrificed,
andgrossnecropsies
areperformed.
During the studyeachanimalis weighedweeklyand observed
daily for
grosssignsof derrealirritationandsystemic
toxicity.A completebloodcount,
hemoglobindetermination,
and urinalysisare performedinitially and at terminationof the study.At necropsy,
representative
tissues
from eachcontrol
andtestanimalare preservedin 10%formaldehyde.
Microscopic
examination
of tissues
is madeon eachcontrolandhigh-level
rabbit,includingthe skin,heart,liver,kidney,spleen,adrenal,stomach,
small
intestine,gonad,and bonemarrow.From the low-levelgroup,sectionsof
skin,liver, and kidneyare examined.
All remainingtissues
from all animals
are held for the possibilityof further micrscopieexaminations.
An evaluation
whichfrequentlyis carriedoutin guineapigsis for detection
of allergenic
potentialof testmaterials.
This procedure
shouldbe applied
solelyin the evaluationof allergenicpotentialof proposed
productcomponents.The evaluation
of proposed
marketformulations
isusuallyrestricted
to
testson humanpanelists.
In reviewingthe applications
of the guineapig testfor sensitization,
the
generalopinionis that the productionof positivereactionsin guinea pigs
shouldexcludethe material from further considerationfor usein preparations
whichwill be topicallyappliedto the skin,or it shouldindicatecautionin
subsequent
testson humanswhichmayfollowin orderto determineconcen-
SAFETY
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139
trationswhich can be e•nployedwithout inducing sensitizationby finished
formulations.
Experiencesof someinvestigators,
however,have shownsomefalse positive
results;that is, sensitization
seeminglyinducedin the guineapig that was not
confirmedby subsequent
testsin humans.The possibilityof suchdifferences
in opinionsis readily apparentwhen oneconsidersthe variationsin procedure
which have been employedin the testson guineapigs and on humans.There
are asmany,probablymore,variationsin the predictivepatch test on humans
asthereare in the testsemployingguineapigs.
Recentreviewsof the importanceof predictivetestingand of the use of
guinea pigs and humansin predictive testing include one by Griffith and
Buehler (3). In this review, someof the findingsof Buehlerwere analyzed.
The studieswere carriedout to determinethe relativesensitivityof variations
in proceduresutilizingguineapigs.
The techniquescomparedwere the intradermalinjectionmethodof Landsteinerand Jacobs(4) and a new procedurewhich employeda closedpatch.
In thisseriesof studies,it was determinedthat the closed-patch
proceduredetectedsensitizing
potentialof eightmaterialsknownto be contactallergens,
while onlytwo of the eightweredetectedby the intradermalprocedure.The
applicationscheduleand test materialconcentration
were the samein both
series.This differencein sensitivitywas confirmedby additionalstudiesin
which sensitizationof 100% of the animalswas obtainedby the patch procedure with concentrations
of dinitroehlorobenzene
(DNCB) which failed to
inducesensitization
of any animalstestedby the intradermaltechnique.
The value of the intradermaltechniquecannotbe questioned,but it has
limitations.With manymaterials,the concentrations
whichcanbe employed
withoutproducingsevereprimaryirritation,includingnecrosis,
are limitedto
levelssomewhatbelowthe practicaluse level of the material.An equally
importantfactoris the dittleultyin interpretingthe reactions
to the intraderreal injections.Unfortunately,sensitizationreactionsin guinea pigs are not
characterized
by the typicalgrossly
visiblepapularor vesieularreactionscommonlyobservedin humans.Judgmentmustbe madeon the basisof the extent
of erythematous
or mfi•imaledematous
reactions
andcomparing
the challenge
reactionsto similar but less extensivereactionsto initial injections.When
properlyapplied,however,it is a valuabletoolfor the detectionof strong
sensitizers.
HUMAN CLINICAL STUDIES
After the animaltoxicologicalstudiesare completedand the producthas
been shown to be safe for human clinical studies,the latter are then initiated.
Clinical studiesperformedwith cosmetics
on humansare referredto by our
laboratoriesas proprietaryclinicalstudiesto distinguishthem from clinical
studieswhich are carried out on ethical drugs.
140
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
Patch Tests
Two basicprocedureshave been mostfrequently employedand numerous
variationsof thesehave been used.One of the basicprocedures,the Schwartz
and Peck (5), has limited value sinceit will detect only very strongsensitiz-
ers.Presentconsideration
of the humanpatchtestwill primarilybe limited to
the repeatedinsult procedureand its variousmodifications.Two variations
were originallypresented-oneby Shelanskiand Shelanski(6) and the other
by Draize (2). Both of fl•eseproceduresemployrepeatedapplicationslnade
over a period of 4 or 5 weeks.They differ in that 10 sensitizingapplications
are madein the Draize procedureand 15 are made in the procedureof Shelanski.They alsodifferin that challengeapplications
are madeto the original
testsitesonly in the Draize procedureand are madeto the originaltestsite
andto previouslyunpatchedsitesin the procedureof Shelanski.
A shortreview of theseand otherproceduresemployinghumanpanelists
hasbeenpresentedby Kligman (7), who suggested
the maximizationprocedure as an alternative.Variousmethodsfor achievingthe objectiveof Kligman'smaximization
procedurehavebeenemployed.We useda procedurein
whichthe skinis abradedprior to sampleapplication.The testmaterialsare
appliedto abradedsitesunderpatcheswhich are appliedto the upper arms.
Reactions
to the testapplications
arescoredon eachWednesday,
Friday,and
Mondayfollowingapplications
on the preceding
Monday,Wednesday,
and
Friday.The patchesare removedby the panelists
9,4hoursafterapplication.
Nineapplications
aremadein 3 successive
weeks,andthechallenge
application is madeon Mondayof the sixthweek.The challengeapplications
are
madeto previouslyunpatchedsites.
Abrasions
of the testsitesare madejustpriorto sampleapplications
on test
days1, 4, 7, and11, or preferably,
priorto eachapplication.
Thismorefrequentschedule
of abradingcanbe donewhenup to fourmaterials
areevaluatedon abradedskinonly.The abrasions
are in the form of two concentric
circleswith diametersof % in. and "%in. and are inflictedby a speciallycon-
structed
apparatus.
The skinareasarecleansed
with isopropyl
alcoholjust
priorto abrading.
A separate
sterileabraderisusedfor eachpanelist.
All adhesive
patches
andabsorbent
padsappliedoverabraded
areasaresterilized
byexposure
to ethylene
oxidepriortouse.Theusualgrading
scaleisutilized.
Anotherprocedure
employed
is to stripthe skinby repeatedapplication
andremovalof adhesive
tapefromthetestsitespriorto sampleapplications.
All of theseprocedures
havevaluein certainapplications.
The onechosen
should
providetherequired
degree
of sensitivity
aswellasdatafromwhich
valid decisions can be derived.
A variation
of therepeated
insultpatchtesthasprovedto be themostvaluableprocedure
available.
Mostof the criticisms
of the basicprocedure
shouldhavebeencriticism
of thewayin whichtheprocedurc
•vascarriedout.
Products
arefrequently
formulated
onthepremise
thatthiscanbe safely
SAFETY TESTING
OF COSMETICS
141
doneprovidedthat all components
havea significant
historyof safeuse.This
is trueonlyif thereis nosignificant
deviation
frmnprovenuseof all components,bothasto typeof productandingredientconcentration.
It mustalsobe
recognizedthat there alwaysis the possibilityof synergisticactionbetween
components.
The importanceof evaluatingthe productin its finishedform is e•nphasizedby observations
of manyevaluators.
Oneof our •noststrikingpersonal
experiences
was with triehlorosalieylanilide
(TCSA). At the ti•ne of its intro-
ductionand prmnotion
as an antibacterial
agentfor topicalapplications,
it
was not recognizedthat it was a potent contactas well as a photoallergen.
We evaluateda cleansing
lotioncontaining1% TCSA in a repeatedinsult
patchtest.About5% of the panelistsdevelopedstrongcontactsensitization
to
the .product.
It wasestablished,
in subsequent
reehallenges,
that TCSA was
the sensitizingagent.A surprisingresultof thesereehallenges
wasthat the responseto TCSA was evokedwhen dispersions
were preparedin water by
means of a low concentrationof a surfaetant,but was not evoked frmn these
samereactivepanelistswhen they were challengedwith solutionsof TCSA in
diethylphthalate.
The originaltestsfor sensitizing
potentialwere carriedout
on solutionsin thissolvent.We frequentlyare proneto assrune
that the preferredsolventis onein whichthe •naterialunderinvestigation
is highlysoluble. The above instance,as well as otherswhich have been encountered,indi-
catesthe necessityof any candidateproduct additivebeing evaluatedin finishedformulationsor in producttypessi•nilarto finishedformulations.
The failure to detectthe sensitization
potentialof TCSA in initial testswas
not due to the inabilityof the procedureto detectthis property.It wasdue to
the failureto insultthe skinwith preparations
havingthe properskinpenetration characteristics.
Other
variations
have been encountered
in which
the
mnountsof test •naterial applied were much reduced-as much as tenfoldfrownthe 0.5-g amountsmostinvestigators
apply.
It is not possibleto considerthe •neritsof the variousmodificationsof the
repeatedinsult patch test procedure.This would require lengthy discussions
based on extensiveexperiencewith each modification.These •nodifieations
have mostfrequentlybeen e•nployedto satisfythe proposeduse or nature of
the product.We prefernot to deviatefrom our standardprocedurein which
we employclosedpatchesand follow basicallythe procedureof Draize with
the challengeprocedureof Shelanski.It is frequently necessaryto employ
se•ni-openor openpatchesor to resortto short exposuresachievedby panelistssittingwith the testmaterialon the flexorsurfaceof their forearms.These
variationshavebeenrequiredbecauseof the volatileor irritant natureof the
product.We haveinducedtypicalsensitization
in numerousinstances
by all
degreesof occlusionemployed.
A significantcauseof possibleerroneousresultsis that of eross-sensitization
reactions.It is •vell recognizedthat materialswhich in themselvesare not
sensitizers
canevoketypicalsensitization
reactionsfrom individualswho have
142
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
beensensitizedby chemicallysimilarmaterialswhich are sensitizers.
For this
reasonproductsfrom different clientsshouldnot be combinedon the same
panelists.The one exceptionis joint studieswhich are carried out for ingredient suppliersand formulatorsof finishedproducts.In suchstudiesthe proposedadditivemay be si•nultaneously
testedin severalformulatedproducts.
Regardless
of the procedureemployedin testson humanpanelists,minimal
reactions
will be frequentlyevokedduringthe serialapplications
and at challenge,whichrequirethe applicationof expertiseacquiredby yearsof observationsto make proper judgmentsas to whetherthe materialis a sensitizer
or not. It is frequentlynecessary
to conductrechallenges
severalweeksfollowingthe initial challengein orderto assess
the significance
of the observed
reactions.
The interpretationof test resultsof the repeatedinsult patch test procedureswill notbe discussed
hereat lengthsincethisbecomesa rathercomplex
endeavor.
Thereis markedvariationin the scoringsystems
employed.We employ a seeminglycomplexscoringscale(Table II) which has developed
throughthe additionof designations
asthe needfor thembecameevident.
Table
II
Patch Test Reaction Scoring Scale
0
No
irritation
1 Slight erythema
2 Marked erythema
3 Erythema and papules
E Erythema and edema
4
Erythema, papules and edema
5
Vesicles
6
Strong spreading reaction
A
B
C
F
G
Effect on Superficial Layers of Skin
Slight glaze
Marked glaze
Glazing with cracking and peeling
Moist fissures
Film of serous exudate
Whenproperlyapplied,therepeated
insultpatchtestis a severetestforthe
sensitizing
potentialof products.
Therearemanyproducts
whichwill induce
sensitization
in a low percentage
of panelists
testedby thisprocedure
which
can be usedwithoutuntowardreactions.The proposedusemustbe consid-
ered.Forexample,
a product
whichshows
minimal
sensitization
potential
and
is to be usedon the feet shouldnotbe marketed,while a productwhichwould
beinfrequently
usedundernonocclusive
conditions
couldprobably
besafely
marketed.
Someassurance
thatproducts
foundto be minimalsensitizers
canbe safely
marketedcanbe obtainedby conducting
product-use
challenges
of panelists
SAFETY
TESTING
OF COSMETICS
143
who have been sensitizedin the patch test procedure.If thesereactive individualsdo not respondto useapplications,it is mostunlikelythat sensitization
would be induced by normal use of the product. However, this reasoning
cannotbe appliedto productsusedfor whole-bodyapplications.Whole-body
exposures
by sensitizedpanelistscannotbe safelydone, and limited area applicationsmay be misleading.
In additionto the value of the repeatedinsult patch test as a predictorof
sensitizingpotential,it furnishesvaluableinformationon the primaryirritant
characteristicsand cumulativeprimary irritant propertiesof test materials.
The latter propertyis descriptivelyreferredto frequentlyas"skinfatigue."
In summaryof our consideration
of the evaluationof sensitizingpotential,
guineapig sensitizationstudiesare valuablein the detectionof strongsensitizers.The closedpatch procedureof Buehler (3) is much more sensitivethan
the intradermalinjectionprocedure.Productsshowingdefinite evidenceof
sensitizationin guineapigs shouldbe droppedfrom further considerationunlessthey can be employedin concentrations
significantlybelow the minimal
sensitizinglevel for guineapigs.
Irrespectivcof negativeresultsobtainedin testson guineapigs, test materials with no historyof toleranceby humansshouldbe initially testedin the
repeatedinsultpatch test procedurein a pilot studyon 10-12panelists.Fullscalerepeatedinsultpatchtestsshouldbe carriedout on all productsprior to
release to market.
In approximately20 yearsof experiencethe presentauthorshave never encountereda severeincidenceof sensitizingpotentialin full-scale60-200panelisttestswhichwasnot predictedby the pilot study.A pilot repeatedinsult
patch test on 10-12 human panelistsis of much more value than guinea pig
testsas a screeningtest for finishedproducts.
The total number of human panelistswhich shouldbe employedin tests
shouldbe governedto someextentby the type and extentof exposureto the
product which will be encounteredin use. For materialswhich will be employedin nonocclusive
uses,suchasperfumes,hand and face lotions,etc., and
will be appliedto limited body areas,completelynegativeresultsin testson
60 panelistshaveprovedto be adequateassurance
that suchproductswill be
essentiallyinnocuousunder use conditions.Productswhich are used under
occlusivetestconditionsshouldbe evaluatedon largernumbersof panelists.
It is also desirableto carry out controlleduse studiesof such products.
Theseshouldbe precededby a repeatedinsultpatchteston 60 panelistsand
shouldbe designedso that one groupof panelistsuse the productsomewhat
in excessof recommended
use.Challengepatclaesof the test materialshould
be appliedat the end of the useperiod.
The discussion
of patchtestingabovehasbeenrestrictedto testsdesigned
primarily for the detectionof sensitizingpotentialof formulationsor ingredients.Many cosmetics
produceno primary irritationwhen evaluatedin the
aboveprocedures.
In a continuous
patch procedurepresentedby Lanman
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JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
(8), a total of asmanyas8 samplescanbe appliedto the backof eachpanelist.Six of theseare test samplesand two shouldbe referencesamplesof
knownirritativepotential.
The testpatchis a 1-in?Webrilswatchmoistened
with 0.5 ml of liquidor 0.2 ml of semisolid
samples.
The swatchis covered
with Blendermtape. The patchesare removedeachday and the sitesare
scoredfor irritation.Freshpatchesare applied immediately.This routineis
followedeachdayuntil the firstsignsof erythemaareseenor for a maximum
of 21 days.As eachsampleproduces
erythemaits application
to the affected
subjectis discontinued.
At the conclusion
the IT;o (numberof daysrequired
to produceerythemain 50%of the subjects)is calculated
for eachsample.By
this procedureminimaldifferences
in irritant potentialof essentiallyinnocuousformulations
canfrequentlybe demonstrated.
MICROBIAL STUDIES
The importance
of controlof the microbialqualityof formulations
needsno
emphasis
asthisis an areaof whichwe all are acutelyaware.In additionto
the healthhazardto consumers
from contaminated
products,the effectof microbialgrowthon productqualityis alsoan importantfactor.A comprehensivereviewof thisproblemwaspresented
by Bruch(9) givingin somedetail
the procedures
for evaluating
microbialqualityof cosmetics
andthe testing
programs
whichshouldbe employedto assurethe manufacturers
that their
productsare adequatelypreservedand that they are producedunderGMP
guidelines.
Sinceit is possiblethat preservativesmay act as sensitizers,or irritants to
humanskin,both animaltoxicologyand human tests should be conducted be-
fore cosmeticproductsare releasedfor sale. In addition to a recommended
testingprogramfor cosmeticproducts,it is highly desirableto monitorthe
of the plant manmicrobiological
qualityof raxvmaterialsand the cleanliness
ufacturing
facilities.
Thisis doneby the collection
of swabsamples
fromvariousareasof the plant,the processing
equipmentand containers
in orderto
evaluatesanitizing
procedures,
andby sampling
raw materials
for microbiologicalcontent.
It is of primeimportance
alsoto carefullyinspectfor other
contamination
possibfiities
duetothegeneral
location
andlayoutof thebuilding,andforair contaminants
originating
fromplantsin thenearbyarea.From
information
obtained
by theseprocedures,
properactioncanbe takento correct any factorscausingcontamination.
Thetesting
program
weuseis designed
to determine
theadequacy
of preservatives
presentin newandcurrentcosmetic
products.
Its objectives
are:
1. To determine
if products
in currentproduction
aresufficiently
biostatic
to preventthe growthof microorganisms,
and to determine
for new
products
thelevelsof biostatic
agents
required
to rendersuchproducts
suitablybiostatic
for extendedperiodsof time.
2. To determine
if the products
in currentproduction
arefree of microorganisms
whicharepotentiallyhumanpathogens.
SAFETY
TESTING
OF
COSMETICS
14,5
Therefore,the procedureconsists
of two phases:
1. Evaluationof biostaticactivityof the completeformulations.
2. The examinationof productionsamplesfor the presenceof viable organisms.
Evaluation o1'BiostaticActivity
The efficacyof the preservativein a preparationis determinedin the laboratoryby the additionof pure or mixed culturesof microorganisms
to the
finishedproduct.The initial microbialpopulationof the inoculatedproductis
determinedby plating aliquotsof suitabledilutionsin agar media.Countsof
viable microorganisms
are redeterminedat selectedtime intervals.The initial
populationsin inoculatedproductsshouldbe about5 x 105 per ml or g. Aliquotsof inoculatedproductsare plated at varioustestingintervalsto determinethe microbialpopulation.Variousneutralizersfor the preservative
present in the formulationcan be addedto the plating media to insuremaximal
recoveryof the challengeorganisms.
The inoculumto be usedshouldl'•epreparedby mixing pure culturesof
organismsisolatedh'om spoiledor grosslycontaminatedproducts.In the
eventthisprocedureis not feasible,pure culturesof organisms
which are suspeckas contaminantsof suchproductscan be mixed to prepare the incoulum.
The most commonlyencounteredcontaminantsin industrial products are
membersof the genusPseudomonas.
This organislnis generallythe mostdifficult to controlin the productsto be tested.The inhibitory actionof the products shouldalsobe challengedby suchorganismsas Aspergillusniger, Bacillus subtilis,Staphylococcus
aureus,Escherichiacoli, and otherswhich are ca-
pable of causingdiseaseor productspoilage.Samplingintervalsshouldbe
such that rapid growth of contaminantsis detected,as well as very slow
growth.In somematerialscontaminants
will proliferatevery rapidly duringa
relativelyshorttime (one week). This rapid increasemay then be followedby
a gradualdecreasein numbersof viable organisms
to a level below that originally present.In both of thesegrowthpatterns,countsshouldbe performed
immediately,after one week, and again one month after inocualtion;and repeatedat 4-monthintervalsfor 12 months.If it is desiredto determinepreservative adequacyonly over a shortperiod of time, terminationof testing at
one month should suffice.
A secondchallengewith the inoculummay be indicatedwith someproducts.Productswhoseusagefrom onecontainermay be distributedoverlong
periods(severalmonths) would be in this category.The biostaticactivity
may diminishafter prolongedstorage.Productswhich are adequatelyprotected at the time of productionmay permit growth of contaminantsintroducedduringextendeduse.If the productis usedup shortlyafter opening,
this problemis eliminated.
146
JOURNAL OF THE SOCIETY OF COSMETIC
CHEMISTS
SUMMARY
A shortresumehasbeen presentedof the varioustestsfor productsafety
which are commonlycarried out on cosmetics.There is one very important
questionto which an answerhasnot been attempted.After one has carried
out all the requiredtests,what assurance
is therethat the productwill be safe
to use?This is very difficultto predict.A practice,whichshouldbe followed
andwhichwill facilitatesuchpredictions,
is to alwaysincludein anyseriesof
testson new productsa referencesample.This preferablyshouldbe one of
your own similarproductswhichhas an acceptablehistoryof freedomfrom
complaints.
Competitors'
samples
canalsobe used,but the complaintrecords
of suchsampleswill not be available.
( ReceivedApril 14, 1972)
REFERENCES
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(3) Griffith, J. E., and Buehler,E. V., Experimentalskin sensitizationin the guinea
pig and man, Presentedat the 28th Annual Meeting, AmericanAcademyof Dermatology, Bal Harbour, Fla., December9, 1969.
(4) Landsteiner,K., and Jacobs,J., Studieson sensitization
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