j. Soc.Cosmet. (5hem., 47, 41-48 (January/February1996)

Petrolalumis not comedogenicin rabbitsor humans: A critical reappraisalof the rabbit ear assayand the conceptof "acne cosmetica"

ALBERT M. KLIGMAN, Departmentof Dermatology,University of PennsylvaniaSchool of Medicine,Philadelphia, PA 19104.

AcceptedMarch 8, •996.

Synopsis The objectivewas to find out whetherpetrolatum, previously reported to be comedogenicin the rabbit ear model,would worsenacne. Vaseline petrolatum jelly (Vaseline©, Chesebrough-Ponds)and Vaselinepe- trolatumjelly cream(Creamy Vaseline ©, Chesebrough-Ponds)were applied to separategroups of ten acne patientseach twice daily for eight weeks. No evidenceof comedogenicpotential was found by the follicular biopsytechnique. Papulo-pustules significantlydecreased in both groups. Greasinesscannot be equatedwith comedogenicity.The admonitionfor acnepatients to seek"oil-free" skin careproducts has no scientificmerit with regardto comedogenicity.Cosmetics are an unlikelycause of the relativelyhigh prevalenceof post-adolescentfemale acne.

INTRODUCTION

In a 1972 paperentitled "Acnecosmetica," Kligman and Mills reportedthat asmany as 50% of commercialcosmetics were comedogenic in the rabbit earmodel (1). Sincethen, this assayhas been extensively used by producersof drugsand cosmeticsto assesstheir potentialfor inducingacneform eruptions (2). Recently,manufacturers have added the human back test to appraiseacnegenicity. Reputable manufacturers of cosmeticsrou- tinely screentheir productsfor comedogenicityin order to satisfythe consumerthat their productis "non-comedogenic,"a usefulclaim in a highly competitivemarket. However, neither the rabbit nor the human assayis entirely satisfactory.Serious con- troversieshave sprung up regardingthe reliability andrelevance of the rabbit earmodel. A voluminousliterature reveals remarkably divergent results among laboratories and investigators.Substances deemed to be stronglycomedogenic by someare declaredby othersto be innocuous.Reviewers have not failed to notice the high frequencyof strikingly contradictoryresults. Long ago Frank questionedwhether the rabbit ear modelhad any relevancefor humans(3). The mostextreme example of the controversies generatedby the rabbitear assay is the publicationsof Fulton,a prominentacneologist 41 42 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

(4). He has classifieda large number of substancesaccording to their comedogenic potential.He statescategorically that manufacturersshould automatically exclude in- gredientsthat arecomedogenic at anyconcentration. This caveathas brought to the fore a vexing problemfor manufacturers,namely, the substantialnumber of desirableand seeminglysafe substances that havebeen "blacklisted" by Fulton. Nelsonand Rumsfield, too, havepublished lists of "unacceptableingredients" (5). They go so far as to recom- mendproducts that do not contain"unacceptable ingredients." Draelos, too, hassharply commentedon the complexitiesand uncertainties of evaluatingcosmetic components by the rabbit ear model (6). Especiallynotable in Fulton'smultitudinous list of come- dogenicsubstances are D & C red dyes and a large variety of fatty substancesthat contributeto the aestheticand functionalproperties of cosmetics.These have not been demonstratedto be acnegenicin humans.Because of theseconflicts, I updatedthe rabbit ear assayin a 1990 publication,adding specifications that, hopefully,would substan- tially reduceinter-laboratory disagreements (7).

REASSESSMENT OF THE COMEDOGENICITY OF OILS

This wordy introductionbrings us to the issuethat is centralto the focusof this paper. Using the originalmodel, Kligman and Mills statedthat petrolatumsand mineraloils from different sourceswere uniformly comedogenic(1). With the updatedmodel, it turnsout that thesewere "falsepositives" (see below). This is far morethan an academic controversysince it stronglyimpacts on the credibility of safetyclaims for skin care products. Seborrhoea,the excessiveproduction of sebum,is a prerequisitefor the developmentof acne(8). Personswith seborrhoeaare also greatly discomforted and try to removeexcess oil by frequentwashing or by oil-absorbingpapers. It is understandablethat acnepatients intuitively wish to avoid oily and greasycos- metics. Indeed, it has become the universal mantra for cosmeticmanufacturers to claim that their products are "oil-free." Dermatologists, too, routinely proscribe oil- containingfacial products for acnesufferers. Patients are advisedto readthe labelsand avoid medicamentsand cosmeticsthat contain "oils." The assumptionis that oily substancesare intrinsically comedogenic. A justificationfor this beliefstemmed from an earlierpublication that describedpomade acne in Afro-Americanmen (9). Adult black men usea varietyof greasyproducts daily to groomcurly, kinky hair. Densecrops of open and closedcomedones sometimes develop on the glabrousskin adjacentto the scalp. The casewould seemto have been made for a generalwarning againstproducts that containgreases and oils. As a matter of fact, the shelvesare now loadedwith a wide varietyof productsthat claim that theyare "oil-free.""Oils," however,comprise a great variety of chemicallyunrelated materials. The implicationis that viscosity(greasiness) alonedetermines comedogenicity. Morris and Kwan have also becomevexed by contradictoryreports and questionthe usefulnessof the rabbit ear test for formulatingnon-comedogenic cosmetics (10). The presentstudy was undertaken to seeka resolutionof thesedisputes. Petrolatum was selectedas the centerpieceof this investigationfor two reasons:(1) Petrolatumis the PETROLATUM AND COMEDOGENICITY 43 archetypicalhydrophobic grease, and (2) it is widely usedin a great variety of topical productsfor varied purposes.It behoovesus to have accurateinformation about its potential for producingadverse effects.

METHODS

SUBJECTSAND TREATMENTS

Paid volunteerswere recruitedfrom a singledistrict in SouthPhiladelphia. Each par- ticipant had persistent,moderate acne of at least two yearsduration. The criteria for entry wasat leastten papulo-pustulesand 15 comedones.Both mild and severecases of acne were excluded. There were two groupsof ten each.In eachgroup therewere six malesand four females, with an averageage of about 18 (range14 to 22). A varietyof OTC productshad been usedby most subjectswith varyingefficacy. Anti-acne medications were stoppedone month beforestarting the study. One groupreceived a productconsisting of a highly refinedpetrolatum, twice daily, to the entire facefor eight weeks.The secondgroup receiveda creamproduct that is an aqueousemulsion containing 30% petrolatum. The identity of the productswas masked to the subjectsby putting the contentsinto newunmarked jars. The volunteerswere told that the treatments,though greasy, might be beneficialin soothingacne. We indicatedthat the useof oily formulationsin acnewas a new approachand that this wasa preliminarystudy to evaluateefficacy. The subjects were paid a modestfee for their participation. We showedeach subject how to applya pea-sizedamount to both sidesof the forehead, followedby spreadingwith the fingersover the entire face. The facewas washedwith a mild soapprior to eachapplication. The studywas carried out in the winter months, when the greasinessof the productswould be lessdiscomforting.

OBSERVATIONS

Lesioncounts of the entire facewere madeat baselineand after eight weeks.A derma- tologistexperienced in the methodcounted all comedones,open and closed,recording thesetogether as a singlecategory. Likewise, papules were not separatedfrom pustules, and were simply categorizedas papulo-pustules. Microcomedoneswere estimatedby the cyanoacrylatefollicular biopsy technique at 0 and 8 weeks, following the quantitative image analysismethodology of Pagnoni and Kligman (11,12). Briefly, a drop of cyanoacrylate(Krazy Glue) was applied to the mid-foreheadand spreadout under a plasticslide. After hardeningof the polymer in abouttwo minutes, the slidewas gently lifted off, carryingwith it a thin sheetof the outerhorny layer with attachedveilus hairs. Those hairs that wereencased by hornycasts as observedunder the stereomicroscopewere consideredto be microcomedones.The densityof microcomedoneswas expressedas the numberper squarecentimeter. Global estimatesof improvementwere made by the subjectsand by the dermatologist 44 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS at 0, 4, and 8 weeks, using the followinggrading system:worse, no change,slight improvement,moderate improvement.

STATISTICS

Changesin lesioncounts from baselinewere assessedby Student'st-test.

RESULTS

Changesfrom baselineare shownin Figure 1 for microcomedones,visible comedones, andpapulo-pustules. A slightbut insignificantdecrease in visiblecomedones was noted in both groups.Likewise, the slightdecrease in the densityof microcomedoneswas not statisticallysignificant. By contrast, in both groupspapulo-pustules decreased significantly (p = <0.05). Global self-assessmentsyielded the followingdata:

PETROLEUM JELLY: ß Three recordedno change. ß Six recordedslight improvement. ß One recordedmoderate improvement.

20 Week0 T Week8

Mean 10

Comedones Papulo-pustules Microcomedones 20- Vaseline Petroleum • Week0 15- • Week8

Mean 10- 5 T 0 Comedones Papulo-pustules Microcomedones 'Jr P=<.05 Figure 1. Effect of petrolatumson acne lesion counts after twice-daily applicationsfor eight weeks. Coinedoneswere not affected.Both productssignificantly reduced papulo-pustules. PETROLATUM AND COMEDOGENICITY 45

PETROLEUM JELLY CREAM: ß Two recordedno change. ß Six recordedmild improvement. ß Two recordedmoderate improvement.

The dermatologists'assessments at eight weeksroughly corresponded to the patients' estimates,except that the gradeswere somewhatlower. No caseof worseningwas observed.

DISCUSSION

The salientfinding in this studyis that Vaselinepetroleum jelly was not comedogenic whenapplied twice daily to adolescentswith activeacne. The emulsioncontaining 30% petrolatumbehaved similarly.

It is well knownthat acnepatients will developcomedones fairly rapidlywhen potent comedogens,such as crude coal tar, areapplied (8). We havefound that evenmoderately comedogenicsubstances such as estersof fatty acids(for example,undiluted isopropyl myristate)will induce microcomedoneson the foreheadof patientswith active acne when applied twice daily for eight weeks(unpublished observations). The discussion that followsis an attempt to bring someperspective into what has been an area of frustrating contentiousness.

In the 1972 "acnecosmetica" study, the test formulationswere insertedinside the ear canal,based on the fact that the folliclesthere are larger and morenumerous. What was not appreciatedat the time was that in this protected site loose horny material is commonlyretained in the follicularlumens, mimicking comedones.We failed to dis- tinguishbetween the densecompact horn that comprisesa microcomedo,a pathologic impaction,and the normalaccumulation of loosehorny squames. A more seriouserror was our failure not to biopsythe oppositeuntreated ear. This is a necessarycontrol in orderto providea referencefor judgingthe amountof hornymaterial in the treatedear. Without a controlbiopsy, many materialswill be falselylabeled as comedogenic.

Our currentguidelines are as follows:The test substancesare appliedto the underside of the pinna in the concavearea just externalto the ear canal(7). An inch-longellipse is excisedfor histologicevaluation, followed by semi-serialsectioning. This allowsmany more folliclesto be visualized.It shouldbe noted that the follicular populationis heterogeneous.As in humans,individual follicles vary considerablyin sizeand respon- siveness.The final gradingis a globalestimate of all the follicleswithin the section.This is, of course,a subjectiveestimate. Quantification can be obtainedby preparingwhole, heat-separatedepidermal mounts (13). The diametersof all the folliclescan be scanned, enablingcalculation of the meandiameter. The daily exposureshave been extended from two to three weeks.Using this new procedure,including proper controls, petrolatum was reevaluatedfor comedogenicity.None of five samplesfrom different producers, includingyellow and white petrolatums,were found to be comedogenic.

It is reassuringfor the validity of the methodthat "red veterinarianpetrolatum," used as a sunscreenin World War II was found to be stronglycomedogenic. This is an 46 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS extremelycrude grade of petrolatum.It is alsoreassuring that certainlanolin derivatives suchas acetylated lanolin were also comedogenic. These examples validate the specificity of the revisedassay. Attention is calledto the reliability of the cyanoacrylatefollicular biopsy,assisted by imageanalysis, for identifyingcomedogenic substances in humans.When appliedtwice daily for eightweeks to the foreheadsof oily, acne-pronepersons, comedogenic materials suchas acetylated lanolin and isopropylmyristate increased both the densityand size of horny casts(unpublished observations). Thus, there is encouragingevidence that the resultsof rabbit and human testsare congruent.There is too little awarenessthat the risk of inducing comedonesis concentration-dependent.Substances that are strongly comedogenicwhen tested neat or in high concentrationsbecome non-comedogenic after sufficientdilution. Neat isopropylmyristate and neatacetylated lanolin are definitely comedogenic.However, the concentrationsin facialproducts are generallywell below 15%. At this level, neitheragent was found to be comedogenicin either the rabbit or humanmodel (unpublished observations). One cannotdetermine from a readingof the ingredientswhether a givenproduct will be acnegenicor not. What matterssolely is the behaviorof the product itself. In the current study both petrolatumand the 30% petrolatumemulsion product sig- nificantly reducedthe number of papulo-pustules.This is a fairly typical result for non-medicatedvehicles. It is of surpassinginterest that in double-blind, vehicle- controlled,anti-acne studies, the beneficialeffect of the vehicleoften approaches that of the active agent! Mills and Kligman reviewedworldwide reports on the efficacyof dozensof anti-acnemedicaments (14). Therewas not a singleinstance in whichthe test substancewas found to be without benefit.Acne nearly always improves under medical care,regardless of the agent. This is the reasonwhy somany marginally effective agents can be sold in large quantities. Sorenessand tendernessis a frequentcomplaint of patientswho havemany inflammatory lesions.Most of the patientsin this studyvolunteered that sorenesswas moderated by bothproducts. For overa centurypetrolatum has been extensively used to treata variety of skin ailmentssuch as burns, abrasions,rashes, and dry skin. Petrolatumalso has protectiveactions against soap and solventdamage and againstirritating drugs like tretinoin (unpublishedobservations). These uses probably reflect its ability to enhance the barrierfunction of the stratumcorneum and perhapsto promotehealing. It hasbeen shownthat petrolatumbecomes incorporated into the intercellularlipid domainsbe- tweencorneocytes, thereby becoming part of the structureof the hornylayer (15). In the original, "acnecosmetica" report, half of the cosmeticswere found to be come- dogenic.This was profferedas the explanationfor the relativelyhigh frequencyof acne in post-adolescentwomen. Severalstudies have shown that asmany as one-third of adult women suffer from "breakouts"especially premenstrually (16,17). "Breakouts"are typical acnelesions, mostly incited by the ruptureof microcomedones(18). It is now highly questionablewhether cosmetics are the chief causeof post-adolescent female acne. Reputablemanufacturers of a great variety of facial skin care products, moisturizers,sunscreens, foundations, lotions, and tonersroutinely screen their prod- uctsfor comedogenicity.Women havebeen taught to lookcarefully at the labelto make sure that the product is "non-comedogenic."Despite theseefforts, the prevalenceof post-adolescentfemale acne has, in my experience,increased rather than decreased(19). PETROLATUM AND COMEDOGENICITY 47

Professionaladult women seem especiallyvulnerable to episodic"breakouts," often lasting for decadesinto menopause.By contrast,ache is uncommonin post-adolescent males.I haveventured the speculationthat post-adolescentfemale acne is due to release of androgeniccorticosteroids, induced by work-associatedstress, superimposed on householdduties and marital commitments.The work of Luckyet al. hasmade it clear that the onsetof acnein prepubertalgirls is closelycorrelated with the serumlevel of adrenalandrogens, notably dehydroepiandrosterone (20). Stressis a well known stim- ulus for the synthesisof adrenalandrogens. It must also be said that genuineache cosmeticahas not altogetherdisappeared. There are literally hundredsof producersof cosmeticproducts throughout the world, and many do not adequatelytest their wares. I haverandomly collected from the pocketbooksof my patientsa surprisingnumber of exotic skin care productsby unregisteredforeign manufacturers.Some of thesewere comedogenicin the rabbit assay. Petroleumjelly is an exceedinglycomplex mixture of hundredsof saturatedhydrocar- bons. It is producedby the fractionaldistillation of petroleum.During the process, petrolatumremains in the bottomportion as a semisolid,bluish-colored material. It is then refinedexhaustively to removecolor, aromatic hydrocarbons, odoriferous materials, and other"impurities." Petrolatum is knowngenerically as softwhite paraffin.Speci- ficationsfor acceptabilityare given in the U.S. Pharmacopea.Vaseline © is the trade- name of petrolatum made by Chesebrough-Ponds.Piewig et al. in their report on pomadeache in Afro-Americanmales, specifically incriminated Vaseline © amongother greasygrooming products(9). Herein lies an interestingstory that is worth recounting.It turns out that Vaselinehas becomea genericterm for almostany hydrophobicgrease. Recently, we bought"Vas- eline"at six localpharmacies whose customers are mainly Afro-Americans. In only three storeswas the productbona fide Vaseline©. The otherthree were Vaseline-like greases of unknowncomposition made by unfamiliarmanufacturers. We foundthese three to be comedogenicin the revisedrabbit model. Accordingly, in reportsthat incriminate Vaselineas comedogenic,one must checkthe source. This study is not intendedas a recommendationof petrolatumfor the treatmentof inflammatoryacne vulgaris. However, it doesshow the benefitsof moisturizationfor inflammatoryache. Dermatologistsroutinely inveigh against oily cosmeticsfor achesufferers. Patients are urgedto seekoil-free cosmetics. This advicehas not beensupported by scientificstudies. Comedogenicityhas nothing whatever to do with oiliness;the latter is a physical attribute and not a chemicalentity. Someof the most potent comedogenicsubstances, dioxin for example, are non-oily. On the other hand, many classicaloils, notably vegetableoils, are usually non-comedogenic. Chemical structuredetermines comedogenicity. As yet, acnegenicitycannot be pre- dicted by structure. The mechanismunderlying comedogenicityis unknown. Why corneocytesstick to eachother to form hornyimpactions is still a mystery.

REFERENCES

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