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%.
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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.
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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
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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.
Gieri for analyzingcertainbergaptensamples;and to Mr. PaigeYoder for
technicalassistance.Dr. John Epsteinprovidedhelpful suggestions
in
the conduct
of the human
studies.
(ReceivedApril 30, 1970)
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PERFUME
PHOTOTOXICITY
715
(12) Cieri, U. R., Characterizationof the steam nonvolatileresidue of bergamotoil and
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(13) Lerner, A. B., Denton, C. H., and Fitzpatrick, T. B., Clinical and experimental studies
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the photosensitizingpotency of furocoumarins(psoralens)and related compounds,
]. Invest. Dermatol., 48, 103-18 (1967).
(18) Musajo, L., Rodighiero, G., and Caporale, G., Activit• photodynamiquedes coumarins
naturelies,Bull. Soc. Chim. Biol., 36, 1213-24 (1954).
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