Perfume Phototoxicity - Society of Cosmetic Chemists
Transcription
Perfume Phototoxicity - Society of Cosmetic Chemists
J. Soc.Con, met. Chem.,21,695-715 (Sept.17, 1970) PerfumePhototoxicity* FRANCIS N. MARZULLI, Ph.D.,* and HOWARD I. MAIBACH, M.D.* Presentedbe[oretheNew YorhChapter,April 1, 1970, Clifton,N.J. $ynopsis--A studyof BERGAMOTPHOTOTOXICITY in man and animalsis presented. A reviewof pertinentliteratureis includedand techniquesare describedwhich lead to conclusions regarding the phototoxiccomponentof bergamot, the concentrationsrequired for phototoxiceffects,and other importantexperimentalconditionsinvolvingthe light source,the skin site, and characteristics of phototoxicchemicals. Resnitsof a survey of phototoxicpotential of currently marketed commercialPERFUMES are discussed. Details of the methods of chemical preparation and the histopathologicfindings arc given in separate appendixes. INTRODUCTION During the past decade,the Food and Drug Administration has receivedconsumercomplaintsof skin damageby commercialperfumes; the peak number wasreceivedin 1965. The most frequently involved perfumeswere found to containbergamotoil. The declinein complaints in recentyearsis thoughtto coincidewith the useof tiurocoumarin-free bergamot,the useof artificial bergamot,or a reduction in the concentration of natural bergamotoil in perfumes. * Animal studiesconductedat FDA laboratorics. ttuman work performed at the California Medical Facilities of Vacaville, Calif., under Contract CPF-69-28: Stndy of Skin Sensitizationand Penetrationby Known Chemicals;Regentsof the University of California, San Francisco Medical Center, San Francisco, Calif. *Derreal Toxicity Branch, Division of Toxicology, Food and Drug Administration, Washington, D.C. 20204. $ University of California, San FranciscoMedical Center, San Francisco,Calif. 91422. 695 696 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Despitea substantial literature,certainunclearaspects of the nature of perfume-induced dermatitispromptedthis work. As bergamotappearedto be the principaloffender,it wasdecidedto startwith an investigation of oil of bergamotand to identifythosechemicalcomponents responsible for phototoxicity.This isa non-immunologic, light-induced skinresponse to a photoactive chemicallikenedto an exaggerated sunburn. The fact that complainants sometimes useda perfumefor a considerabletime beforesufferingadverseskin effectssuggested the possibility that allergenicor photoallergenic effectsmight alsobe involved (1, 2). This aspect,however,wasnot investigatedhere. Bergamotwas first suspectedof being capableof producingphototoxicityby Freund,who reportedin 1916 that four of his patientsdevelopederythemaand pigmentationfrom eau de colognecontaining bergamot (3). The term berlock dermatitis,spelledberloque-dermatitisin France and Berlockdermatitisin Germany (French breloque,German Betlocke ineaningpendant),hasbeen given to the skin syndromeproducedwhen bergamotoil, which is derived from the rind of Ciirus bergamia,an orange-likeMediterraneanfruit, is applied to the skin and followedby exposureto sunlight. The name Berlockdermatitisor dermatitis in Betlock-Form(4) was coined by Rosenthalin 1924 (published1925). Kuske (5) in 1938appearsto be the first to report that the furocoumarin componentsof plants are capableof producingphotodermatitis. He isolatedthe furocoumarinbergaptenfrom oil of bergamotand found it photoactive. In Germany,bergaptenor 5-methoxypsoralen wasknown to be a componentof oil of bergamotat least since 1839, when it was extractedfrom oil of bergamotby Ohme (6). In this work, often overlookedby investigatorstoday,Ohme describedthe solubility characteristicsand molecular weight of bergaptenand correctly estimatedthe bergaptencontentof crudebergamot(0.37%). We now know that many plantscontainphotosensitizing furocoumarins(7). METHODS A llanovia "Inspectolite"* provided the radiation sourcein both buman and animal studies. The emissionspectrum of one of these -- * H•novia lnspectolite withNo.16125, TypeEH-4bulbandredpurple filter[Corning 7-39 (5874)]. Tfiis filter transmits0% at 300 rim, 55% at 360 nm, and 0% at 410 nm as a more or less bell-shapedcurve. It also has a frosted glasscut-off at 290 nm. Hanovia Izm H) Co., 100 Chestnut Street, Newark, N.J. PERFUME PHOTOTOXICITY 697 radiation sourceswas •neasured*using a Perkin-Elmer monochromator with stripchartand taperecorders(8). Over 90t•oof the ultraviolet (UV) radiationoutput (between300and400nm) of the Inspectolite was found to be at 365 ----q5 nm. The total UV output was about 3000 /•watts/cm2 at a distanceof 10 cm from the sourceand 1900/•watts/cm2 at 15 cm.* The first step was to isolate coumarinsand [urocoumarinsfrom bcrgamotand testthem for phototoxicityon human subjects(Appendix I). As thesefirst preparationswere not entirely pure, this was considereda screeningstep. Phototoxicitytestswere then performed on nine subjectsusing oil of bergamot, the coumarin-psoralcnnonvolatile fraction, coumarin-psoralen componentsseparatedby thin-layer chromatography(TLC), and an ethyl ether-precipitatedportion of the coumarin-psoralcns. Thus, in addition to oil of bergamot, which contains about 2% coumarin-psoralens, testswere performedon TLC band 1 consistingmainly of bcrgamottin,band 2 which was mainly 7-mcthoxy-5geranoxycoumarin,band 3 which was about 96:4 limettin-bergapten, band 4 which containedelementsof band 3 plus bergaptol,and an ethyl ether-precipitatedportion which was recrystallizedthree times with methanol and wasmainly bergapten. The chemicalformulasare shown in Fig. 1. The human studies were performed according to the method of Burdick (10). The skin of the forearm wastape-strippedto glistening. The agent (0.05 ml) was applied to the strippedarea (5 X 12 cm), allowed to remain undisturbed for 5 minutes, and then irradiated with the Inspectolitefor 40 minutes at a distanceof 8-10 cm. The arm was examined at 24 and 48 hours. RESULTS Results (Table I) showthat phototoxicity,consistingof erythema, edema,and sometimes vesiculation,was easilyobtainedon stripped • Measuremcntsby Dr. M.P. Thekaehara, Goddard SpaceFlight Center, Greenbelt, Md. t Inverse square law is applicable if the source is assumed to be 8.2 cm behind the front of filter. The Inspectolite was calibrated with a G.E. Spectral Irradiance Standard lamp, a 1000-wattquartz-iodinelamp standardizedby Eppley Laboratories,Newport, R.I. After the Inspectolite has been calibrated, it can be checked from time to time with a Jagger UV dose-rate meter fitted with a diaphragm over the photocell to reduce the UV output intensity (9). An important factor to note, especiallyin biologic experiments involving different exposuretimes, is that the output increasesas the instrument warms up. 698 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS CI H• CH• (•H• CH• OCH;CH=CCH•CH•CH:C •' OC2CH:C-CH2CH•CH= C'•CH• '•'C H• BEROAMOTTIN 7-Methoxy t •eranoxy coumar•n geranylether 5% (0.05% ,nbergareal) OCH• PSORALEN 5•7-Dimethaxycaumorin (0.3ø/8 ,nbergoreo,) BERGAPTEN OCH• 5 - Melhoxypsoralen XANTHOTOXIN (033% •nbergarno 0 8-Me•haxypsaralen OH O[••O O••"O BERGAPTOL OH 5-Hydroxypsoralen XANTHOTOXOL (hydrolysis product ofbergapten) 8-Hydroxypsaralen Figure 1. Structural formulas of test psoralensand coumarins skin with oil of bergamot,the complete coumarin-psoralenfraction, and thoseportionswhich containedbergapten. Figure 2 showsthe skin reactionsobtained on one of the subjectstested with these materials. Figure 3 showshyperpigrnentationat the application site 23 days after a positive reaction to 10% bergamot. In this subject, the pigmentation effect lasted about 1 month' in some individuals it is reported to lastmuch longer (11). Limettin To evaluate limettin more preciselyas a possiblephototoxic agent, and to define further the limits of bergaptenphototoxicity,testswere conductedon pure samplesof both bergaptenand limettin, in addition PERFUME PHOTO [O\ ICI'I'• 699 F•AC •3A / COeW•ZN Figure 2. Phototoxic responsesin one subject tested with 8 different materials Figure8. Hyperpigmentationin a subject23 daysafter positiveresponse to bcrgamol 700 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table I Phototoxicityof Oil of Bcrgamot, Coumarin-psoralcn(C-P) Nonvolatile Fraction, TLCSeparated C-P Components,and Ethyl Ether-Precipitated Portion of C-P• Fraction Concentration Substance Components About 2% C-P Bergapten,limettin, bergamottin, bergaptol, methoxygeranoxycoumarin C-P, TLC band 1 Mainly bergamottin C-P, TLC band 2 Mainly 7-methoxy-5-geranoxycoumarin C-P, TLC band 3 Limettin-bergapten C-P, TLC band 4 Band3 to origin (includesbergaptol) C-P (ethyl ether-pptd. Mainly bergapten(ca. 90%) portion recrystallized Oil of bergamot C-P fraction (%) 100 1 0.5 O. 5 0.5 0.5 0.5 Showing Positive Skin Response 9/9 9/9 1/9 3,/9 9/9 2/9 9/9 3X with methanol), fraction 3A • Test materials (diluted with 95% alcohol) were applied to a 5 X 12-cm area of "stripped" forearm skin of nine subjects. The areaswere irradiated for 40 minuteswith UV light (principally 360-370 nm) emittedfrom an Inspectolite(Hanovia) at a distanceof 8-10 cm. to the limettin-bergaptenmixture obtainedfrom the TLC band g. The results(Table II) indicate that pure bergaptenis phototoxicdown to 0.002% concentration;pure limettin on the other hand, thoughstrongly fluorescentunder UV, is not phototoxic. CommercialSamples Fifteen commercialperfumeswere purchasedin the San Francisco area in 1968 for human photopatch tests by the methods described earlier. An old (1964) sample of "Shalimar" Perfume (el) was includedaswell as a new sample(½t2). Resultsshowthat "Shalimar" "Replique," and "Park 8cTilford No. 3" producedphototoxicityin some test subjects(Table III). On chemicalanalysis,our sampleof "Shallmar" el wasfound to contain 0.1% bergapten. This is equivalentto 30% bergamot,usingCieri'srecentanalyticaldata,i.e., 0.33,%bergapten in bergamot(12). "Shalimar"•t2, on the other hand, containedno bergaptenand producedno phototoxicityin four subjectstested. Samples o15"Replique," subsequentlyprocuredin the Washingtonarea in 1969 and testedon hairlessmice, as describedlater, showedno phototoxiceffects. Furthermore, "Park and Tilford No. 3" is no longer manufactured. PERFUME PHOTOTOXICITY Table 701 II Effects of Concentration on Phototoxic Effects of Bergamot and Some of Its Components• Concentration Substance (•) Test 1; 6 subjects Oil of bergamot Bergapten,pure, mp 187øC Limettin-bergapten(96: 4) Test 2; 6 subjects Limettin, pure, mp 147øC Oil of bergamot Fraction Showing Positive Skin Response 100 20 o. 05 o. 02 o. 002 o.ool 0.5 0.2 O. 02 O. O1 5/6 3/5 4/4 4/4 1/2 0/2 4/4 1/4 1/2 0/2 1.0 0.1 100.0 0/6 0/6 6,/6 Materials were tested as describedin Table I ("stripped" human skin). Table III Phototoxicityof Commercial Perfumes• Fraction Showing Positive Perfume Manufac turcr Skin Response Shalimar #1 Shalimar/•2 L' interdit Arpege Replique Guerlain, Paris Guerlain, Paris H. de Givenchy, Montvale, N.J. Lanvin Perfumes,Inc., New York Raphael, Paris 3/9 0,/4 0/6 0/13 1/13 No. 3 Park & Tilford L' aimant Park & Tilford, New York Coty, Inc., New York Houbigant Ondine Bellodgia Houbigant, Inc., New York SuzanneThierry, Miami, Fla. Caron, Paris Ma Griffe Chanel No. 5 Crepe de Chine Miss Dior M. Rochas Cabochard Carven, Paris Chanel, Inc., New York F. Millot, Paris Christian Dior, Paris Marcel Rochas, Paris Gres, Paris 1/6 0/5 0/5 0/4 0/2 0/2 0/6 0/2 0/2 0/2 0/2 a Perfumes were applied to a 5 X 12-cm area of "stripped" forearm skin (20 subjects). Areaswere irradiated for 40-60 minuteswith Inspectolite (principally 360-370 nm) at 8-10 cm distance from skin. 702 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table IV PhototoxicityTest Resultswith 100% Oil of Bergamoton Different Areas of "Unstripped" Skins of Human Subjects Positive Response Site Fraction Scrotum Neck Forearm Forearma 4/6 4/6 1/4 0/2 • Agent applied and site occluded24 hours prior to irradiation. Regional Shin Variation To find out if the penetrantbarrier actionof skin may be a factor in the phototoxicreaction,testswere performedon highly permeablescrotal skin, moderately permeable neck skin, and lesspermeable forearm skin. Oil of bergamotwasapplied to the unstrippedskinsof theseareas of six subjects. Results (Table IV) showthat unstrippedforearm skin providedthe mostprotection. Animal Studies Phototoxicitystudieswere conductedon six animal speciesusingessentiallythe sameprocedureand irradiationequipmentaswasusedfor the humanstudiesexceptthat the hair wasremovedby clippingand the skin was generally not stripped. Rabbits, hairless mice, hamsters, squirrel monkeys,guinea pigs, and swine were tested. Five minutes after applicationof testmaterial(bergamotor bergapten),the sitewas irradiated for 20-,q0 minutes. The skin was observed at 24 and 48 hours andagainat 7 days. The irradiatedskinfiuoresced after topical(and evenintradermal)administration. A phototoxicskinreactionwaschar- acterized 1)yerythema andedemaduringthefirst48 hoursandscaling or necrosisby 7 days. Thus the animal skin reactionsresembledthoseof man except that severe effectsdid not include vesiculation. Further- more,hyperpigmentation wasnot observed in the mouse,rabbit,guinea pig (albinoor pi•mented),or hamster.Histopathologic findingsare reportedin Appendix II. Whentheintervalbetweenagentapplication andirradiationexceeded 1 hour,phototoxic effects werenotobtained on animals.This suggests •hattheagentwaschemically changed, or hadalreadybeenabsorbed into thebloodstream andwasnolongerpresent in skinin amounts capable of producingphototoxiceffects. PERFUME PHOTOTOXICITY Table 703 V PhototoxicEffects (PositiveResponseFraction) of Several Concentrationsof Oil of Bergamot on Various Animal Speciesat 48 Hours Concentration (%) Site Species 10 Back Back Back Hairless mouse Rabbit Hamster 2/2 2/2 1/2 Back Back Guinea pig Squirrel monkey 4/4 Ear Abdomen Swine Swine 1/ 1 1/3 Abdomen, stripped Swine 4/4 Table 5 1/1 o/1 1 0.5 2/2 2,/2 4/5 0/5 VI PhototoxicEffects (PositiveResponseFraction) of Several Concentrationsof Bergapten on Various Animal Speciesat 48 Hours Concentration (%) Species,Site 0.01 0. 001 0. 0001 0. 00001 Guinea pig, back Hairlessmouse,back Rabbit, back Squirrelmonkey,back 5/6 2/2 6/6 5/7 5/5 3/6 1/8 0/4 3/4 0/1 0/7 0/3 o/1 Hamster, back Swine, back Swine, abdomen, stripped 0/3 0/3 0/2 2/2 0/3 0/6 2/2 Bergamotwhich was irradiated prior to application was found ineffectivein producinga skin responsein the absenceof concurrentultraviolet irradiation.* - The hairlessmouse and rabbit gave the strongestphototoxic skin reactionsby grossobservation. They would therefore appear to be speciesof choice in phototoxicityscreeningstudies. Guinea pig and swineskin waslessreactive;nevertheless, asin human skin, strippingenhancedthe responsein swine. Monkeys and hamstersgave poor skin responses.Tables V and VI give somephototoxicityresultsobtained on thesespecieswith severalconcentrationsof oil of bergamotand bergapten. * Irradiation of 0.02% alcoholic bergapten for 1 hour in a stoppered quartz cuvette at a distanceof 2.5 cm resulted in photodecomposition,as indicated by the appearanceof five TLC spots. The phototoxiccapacityof the resultantsolutionwas only slightly reduced when testedon rabbits. Irradiation photolysishas been reportedby others (13). 701 .JOURNAL OF THE SOCIETY OF GOS\IETIC CHEX,IlS-lb Purified samplesof the two couxnarins,limettin and 7-methoxy-5gcranoxycoumarin,were testedfor phototoxiceffectsin tabhits at l and 0.5%, respectively. No adverseskin effectswere observed,and consequently, thesetwo coumarinsare not consideredto be phototoxic componentsof bergamot. Becausethe tluorescenceintensity of bergaptensoh•tionsvaric• with the ratio of alcohol to water, one experiment was conductedin which 70 and 95% alcoholicsolutionscontaining bergaptenat 0.008 aud 0.0008% were applied to four sitesin closeproximity on the clipped back of each of 12 rabbits. The more concentrated alcoholi, solution spreadover ahout twice as much skin area as the 705/0alcohol. Results (Table VII) showthat at 24 hours,9 of 12 animalsgavea positivereactionto 0.0008%hergaptenin 70% alcoholwhereasonly5 of 12reacted to the salneconcentrationof bergaptenin 95% alcohol. In addition, the reactionswere strongerwith 70 than 95% alcohol. The fact that the agentspreadoverconsiderably moreskin surfacewhenappliedin 95%alcoholsuggests thatthereislessactiveagentpertrait area,thereby accountingfor theseresults(Fig. •). Figure4. Areaandintensit•of phototoxic reactions to alcoholic bergapten in rabbits. Riglitside,95%alcohol; leftside,70%alcohol; upper,0.0008% bergapten; lower,0.008% bergapten PERFUME PHOTOTOXICITY Table 705 VII PhototoxicEffectsof Bergapten on Rabbits at 24 Hours Bergapten Concentration (%) PositiveResponse Fraction In 70%Mcohol O.OO8 0.0008 In 95%alcohol 0.008 0.0008 12/12 9/12 12/12 5/12 Table VIII Comparisonof Phototoxicity(ResponseFraction) of Psoralensin 95% Alcohol on Clipped Rabbits Concentration (%) Compound 0. 1000 0. 0040 0. 0008 0. 00008 Bergapten 4/4 6/6 3/5 0/5 Psoralen 8-Methoxypsoralen 8-Hydroxypsoralen 4/4 2/2 0/6 6/6 2/2 5/5 2/2 0/5 0/5 The phototoxicactivitiesof four related furocoumarins--bergapten, psoralen,8-methoxypsoralen (8-MOP), and 8-hydroxypsoralen (8-HOP) (Fig. 1)--werecomparedusingrabbitsastestanimals. The results,given in Table VIII, showthat psoralen,8-MOP, and bergaptenare quite comparable in phototoxicactivity. 8-Hydroxypsoralendoesnot appear to be phototoxic. Relation betweenFluorescenceCharacteristicsand Phototoxicity A studyof fluorescence intensity,emission,and excitationpeakswas conductedto find out if thesemight be useful measurementsfor predicting phototoxicity. The Aminco-Bowmanspectrophotofiuorometer was used. Data (Table IX) for variousconcentrationsof bergaptenin 70 and 95% alcohol show that the excitation peaks for bergapten range from 320 to 360 nm dependingon concentration. The emissionpeak was consistentat 480 nm. The fluorescenceintensity of bergapten is greaterin 95% than in 70% alcohol. However, the relative intensities of fluorescence(the product of the emissionpeak height in arbitrary units and the meter multiplier setting)showa relatively straightline relation when plotted againstthe log of the bergaptenconcentrations. 706 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table IX FluorescenceIntensity and Excitation and EmissionPeaksof Bergaptenin Alcoholic Solutions Relative Bergapten Concentration (%) Excitation Peak (nm) Emission Peak (nm) Intensity (units)• In 95%alcohol 0.0100 360 480 50 0.0080 360 480 41 0.0010 330 480 26 0.0008 330 480 25 0.0001 320 480 6 In 70%alcohol 0.0080 360 480 26 0.0007 332 484 16 • Product of the emissionpeak height in arbitrary unim and the spectrophotofluorometer meter multiplier setting. Table X Comparisonof PhotochemicalPropertiesand PhototoxicCapacity of Four Concen- Test Substance tration (%) Psoralens and One Coumarin Alcohol Relative Concen- Fluorescence tration (%) Excitation Peak (nm) Emission Peak (nm) Intensity (units) Photo- toxic Effects Bergapten 0. 001 95 330 480 26 Yes 8-MOP 8-MOP 0. 001 0. 001 95 70 320 320 480 480 I 2 Yes Yes Psoralen Psoralen Psoralen 0. 010 0. 001 0. 0001 95 95 95 395 389 389 460 460 460 65 5 1 Yes Yes Yes 8-HOP 0.100 0. 00001 95 95 385 335 480 420 55 Limettin 0.1 No No The data of Table X show that there is no apparent connectionbetweenphototoxicactivity and fluorescence intensity,excitationor emission peaks. In further pursuitof the mechanism of the photobiologic activityof psoralens,exploratoryelectron spin resonancestudieswere conducted. Absorption curveswere obtained* on 0.02% solutionsof 8-MOP and 8-HOP, eachin 95% ethanol. The testmaterialswere first frozenwith liquidnitrogen in a quartztubeandthenirradiated withtheInspectolite for $0 minutes at a distance of 5 cm. The frozen solutions were scanned * Work performedby Dr. H. Kon, National Institutesof Health, Bethesda,Md. PERFUME PHOTOTOXICITY 707 and the resultingcurvesshowedone principal and three secondarypeaks. In both cases, the peakheightsand locationswere very similar sothat this analysisfailed to provide a clue to the differencein phototoxic activity of thesetwo compounds. DISCUSSION A satisfactoryresolution of this as well as most other biologic problems is dependentupon the availability of appropriate tools. When working with a mixture, bergamot included, it is essentialto know the compositionwith somedegree of precision. Thus, the first barrier to overcomein the resolution of bergamotphototoxicitywas the lack of simpletechniquesfor chemicalanalysisand preparationof components. Anothermoreseriousproblemwasthe lackof readilyavailableradiation sourceswhosequantitative output wasknown. When all the data on man and animalsare considered, it is apparent that bergaptenis the onlysignificantphototoxiccomponentof bergamot. Sincethis is the case,the photobiologic effectsof bergamotwould more appropriatelybe termedbergaptenphototoxicityrather than the more flamboyant but lessdescriptive "berloquedermatitis." The factthat the morepermeableskinsof animals,suchasrabbit and mouse,the strippedskinof humans,and the morepermeablescrotaland neckskinsareamongthemostsusceptible to bergapten phototoxicity indicatesthat individualswho developthe syndrome havea poorlydevelopedskinbarrier. This hasbeensuggested by others(10, 14). Since the productionof phototoxiceffectsis dependentupon the amountof activeingredient reaching targettissue, thesedatapermitoneto establish a safehumanconcentration of bergaptenand bergamotoil on skin. A humansafety factorisprovided byutilizingresults obtained on stripped skin. Thus, sincebergamot contains0.33% bergapten and the apparent "no-effect"level on strippedskin for bergaptenlies between 0.002and0.001%,thesafeconcentration of bergamot oil onmanisprojectedto be between 0.3 and0.6%. This is comparable to, although somewhat lowerthan,the 2% concentration givenin regulations issued undertheHazardous Substances Act (15). It is apparent fromthisthat unless bergamot is chemically treatedto removebergapten, it is likely tobephototoxic in some individuals if usedin practical amounts in perfumes. The resultsobtained on animals show that the rabbit and hairless mousearemoresensitiveto bergaptenthan man and are thereforeuseful 708 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS for screeningtests. Swineappearto duplicatemore closelythe human response andprobablyaremoresuitablefor studiesat the concentrations which affect humans. Whenphotoactive compounds areirradiatedtheyabsorbphotonsand becomeelectronicallyexcitedto a new energylevel. On returning to the groundstate,they fluoresce. The biologicallysignificantevent, that is, the productiono[ tissue-damaging chemicalreactivityo[ the molecule, probably occursin the fraction o[ a secondintervening between these two processes.The amount o[ this reactivityappearsfrom our observations to be independentof absorptionpeak, emissionpeak, or fluorescenceintensity. Thus, measurementof these parametersprovided no clue asto phototoxicpotentialof the compoundsstudied. While a compound which is phototoxicis photoactive,the reverseis not necessarily true. Usingirradiationtime asa criterion,othershaveconcludedthat psoralen is significantly more phototoxicthan bergaptenor 8-MOP (16-19). The presentstudies,basedon a comparison o[ minimum effectiveconcentration,showedthat the threecompounds had aboutthe samephototoxicity. In agreement with reportso[ others(18,20), the presence o[ a methoxygroupin the 5 or 8 positiono[ the psoralenmoleculewasassociated with phototoxicactivity,whereasthe hydroxygroupin thesepositions was not. CONCLUSIONS 1. Variouscomponents ot5bergamot weretested[or phototoxic effects on human skin. Resultssuggestthat Berlockdermatitisis due to a singleactivecomponent o[ bergamot, eitherbergapten or 5-methoxypsoralen.It is there[oreappropriate to call this syndrome bergapten phototoxicity. 2. Data obtained on different areas o[ human skin and on different animalspecies showa relationbetween skinpermeability andbergapten phototoxicity.In man,biologiceffects wereproduced moreeasilyon highlypermeable scrotal or strippedskin. In animals, the morepermeableclippedskinsot5rabbitsand hairlessmiceweremorereactivethan thoseo[ monkeys andswine. Thus,individuals mostsusceptible to bergaptenphototoxicity probablyhavea poorlydeveloped skinbarrier. 3. Bergapten producedphototoxicity in strippedhumanlorearmsat concentrations downto 0.002%. This effectwasproduced in clipped rabbitsat concentrations downto 0.0008%. PERFUME PHOTOTOXICITY 709 4. On the basisof data obtainedon strippedskin and estimatesof thebergapten contentof bergamot,it isexpectedthat perfumes are likely to producephototoxicity unlessthe furocoumarins are eliminated,the natural bergamotconcentration reducedto 0.3%, or the bergaptenreducedto 0.001 5. In phototoxicexperimentsconductedon clipped rabbits,fewer skineffectswereproducedby bergaptenin 955othan705• ethylalcohol. The differencewasthoughtto be due to reducedskin surfacespreading with the more aqueoussolution. APPENDIX I ISOLATION OF COUMARINS AND PSORALENS FROM PgERGAMOT OIL RONALD YATES, DIVISION OF COLORS& COSMETICS, FOOD AND DRUG ADMINISTRATION Bergamotoil wasfirst treatedto producea coumarin-psoralen(C-P) mixture containingtwo coumarins(limettin and 7-methoxy-5-geranoxycoumarin)and three psoralens(bergapten,bergaptol,and bergamottin). This mixture wasfurther separatedinto five fractions. Isolation of Coumarin-Psoralen(C-P) Mixture A 450-gsampleof oil o.fbergamot(N.F.)* wasplacedin a•flaskand the volatile fractionwasremovedat 35-65øC and 2 mm pressure. The C-P mixture wasthen isolatedfrom the nonvolatilematerial (ca.50 g) accordingto a methodgivenby Guenther(21). Yield: 10 g. Fractionation of C-P Mixture Five fractionswere separated from the C-P mixture by preparative thin-layerchromatography(TLC). The platesusedwere 20 X 20 cm coated withsilicagelG of anaverage thickness of 500v. Approximately 70 mg of the C-P mixturewasspottedalongthe bottomof eachplate. The TLC plateswere developedwith toluene-ethyl acetate-aceticacid (80:20:2). A totalof 1.5g of theC-Pmixturewaschromatographed by this procedure. The developedplateswere divided into four fractions accordingto the schemein Fig. 5. Eachfractionwasscrapedfrom the plate, and the correspondingfractionsfrom each plate were combined and extracted with a suitable solvent. The solvent was removed on the * Obtained from George Lueders and Co., New York, N.Y. 710 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS SOLVENT FRONT Bergomomn _•_FRAC 1 ........... 7-MethoxytS-gemnoxy FRAC2 L,mettm__•?_, FRAC 3 6or more 1 "repounds FRAC 4 ORIGIN Figure 5. TLC chromatogramof psoralen-coumarin mixture steambath and eachisolatedfractionwasrechromatographed asdescribed above. Eachfractionwasthen spottedon an analyticalTLC plate and developed. The plate was then examinedunder UV light to estimate the purity of eachfraction. Fraction/--Fraction 1, whenfirstisolated,weighed640mõ andsolidified upon standing. The solid material was dissolvedin an excessof hot methanoland cooled. Crystallinebergamottinwas removedby filtration (ca. 400 mg) and discarded.The mother liquor was concentratedand spottedon preparativeTLC platesin 50-mgportionsand developed.Fraction1wasrecovered fromtheplatesasabove;220mgof crystalline materialwasobtained. AnalyticalTLC showedthat Fraction 1 wasprimarilybergamottin with a smallamountot•methoxygeranoxycoumarin (est.1% or less). Fraction2--Fraction2 amountedto 29 mg. AnalyticalTLC indicatedthisfractionto consist of methoxygeranoxycoumarin plusan unidentifiedmaterialwith a deepblue,faint fluorescence. Fraction3--Fractiong consisted of 75 mg of a mixtureof limettin and bergapten. Fraction4--Fraction 4 consisted of all compounds present from Fraction gtotheorigin ofthechromatogram. Noneofthese compounds, with theexception of bergaptol, havebeenidentified. After Fraction4 wasrechromatographed, 20 mgof material wasobtained.Analytical TLCofthismaterial showed complete absence ofanyofthethree pre- vious fractions. PERFUME PHOTOTOXICITY 711 Fraction3A--This fractionwasnot obtainedby preparativeTLC but by direct crystallizationof the crude C-P mixture. To one volume of the crude mixture, two volumesof ethyl ether wasadded. A mixture of bergaptenand limetriMimmediatelyprecipitated,wasisolatedby filtration, and was recrystallizedthree times from hot methanol. This fraction is includedbecausebergaptenis believedto be the compoundprimarily responsiblefor photosensitization. APPENDIX II COMPARATIVE PATHOLOGY PRODUCED BY SKIN APPLICATION OF OIL OF BERGAMOTIN THE HUMAN, RABBIT, GUINEA PIG, HAIRLESSMOUSE, HAMSTER, AND PIG HOWARD L. RICHARDSON, M.D., ANDKENTJ. DAvis,D.V.M. DIVISION OF PATHOLOGY,FOOD AND DRUG ADMINISTRATION Hematoxylin-eosin stainedparaffinsectionsof skin taken 6, 16, 24, 48,and72 hoursaftertopicalapplication of 10% oil of bergamotin 70% alcoholwereexaminedby light microscopy to studyspecies variations in phototoxicreactions. Histopathologic findings,involvingfurocoumarin phototoxicity in animals,are summarized in Table XI. Exceptin the caseof hamsters, gross appearance of theskinprovidedtheclueto damage in underlying tissues. In severe involvement, theepidermis becameacanthotic, sometimes edematons,and occasionallynecrotic. The reticular dermis showed greaterinflammatory cellinfiltrationthanthe papillarydermis. The appendages showed slight,immediate involvement, but aftera periodof twomonths, therabbithadlittlehairgrowthin theareaof application andhairfollicles became sparse anddegenerated in appearance. The majorchanges in thehumanforearm skinwith72 hourspostbergamottreatmentwere epidermalnecrosisintermixed with areas of epidermal edemaand acanthosis, and with necrobiosisof sebaceous glands. The mostnearlycomparable change notedin skinsections from otheranimals examined occurred in thecorneum-stripped pigskin. Oil of bergamot application to normalpig skinproduced no significant microscopic lesions duringthe6- to 8-hourobservation period.Rabbit skinwasalsorelatively nonreactive microscopically; however, twomonths afterapplication of 0.1% bergapten solutionthereweredecreased num- bers ofhairfollicles andthefewhairfollicles remaining orregenerating '712 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS • o •o PERFUME PHOTOTOXICITY 713 714 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS displayeda hyperplasticbulbar epitheliumand werelocatedmuchdeeper in the dermis than normal. Glinically, hair had not regrown in the treatedareas. The guineapig skin wasalsorelatively nonreactive,with the principal lesionbeing slight (or slight to moderate)acanthosis. In the guinea pig skin taken 16 and 24 hours after treatment, this slight acanthosishad a zonal pattern suggestiveof reactionsalong corneum scratches causedby clipper blade teeth. In the hairlessmouse,applications of oil ot5bergamotresulted in a rather severemononuclear,and later granulocytic,cell infiltration into the dermisafter 24 hours. Despitethe lack of grosslesions,the hamster showedmarked derreal and moderate epidermallesionsafter 24 hours. Early resolutionof thesechangeswere noted, however, in the 72-hour section. ACKNOWLEDGMENTS Assistance is acknowledged to Dr. Millard Maienthal for preparing pure bergapten,psoralen,and hydroxypsoralen; to Mr. Ronald Yates for preparingpure limettin and 7-methoxy-5-geranoxycoumarin; to Miss SusanBarkan for spectrophotofiuorometry; to Mr. J. Jonesand Mr. U. 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