Experimental modification of photocarcinogenesis. I. Fluorescent whitening agents and short-wave UVR

Fd Cosmrr.

Tooxicol. Vol. 13. pp. 335-337.

EXPERIMENTAL FLUORESCENT

Pergamon

Press 1975. Printed

MODIFICATION WHITENING P. D.

Photobiology

in Greal

FORBES

Britain

OF PHOTOCARCINOGENESIS. AGENTS AND SHORT-WAVE and F.

Program, The Skin and Cancer Hospital, 3322 N. Broad Street, Philadelphia, (Received

UVR

URBACH

Temple University Health Pennsylvania 19140. USA

8 December

I.

Sciences

Center.

1974)

Abstract-Erythema was produced on the skin of hairless mutant mice by a single exposure to lowpressure mercury-vapour (germicidal) ultraviolet lamps. The acute reaction was not affected by pretreatment of the skin with 2Opg of a fluorescent whitening agent (FWA), disodium 4,4’-bis-(4,6-dianilino1,3,5-triazin-2-yl)-aminostilbene-2,2’-disulphonate. applied topically in 20 /II methanol. Skin tumours were produced in hairless mice during several months of daily exposure to the same lamps. Slightly fewer turnours, with a slightly longer latent period, were produced in mice similarly irradiated but pretreated daily with FWA as above. Thus, under the test conditions used, the FWA was not phototoxic, nor did it enhance photocarcinogenesis.

INTRODUCTION

genic potency of ultraviolet light (Bingham& Falk, 1970;Falk & Bingham,1973).Bingham(1972& 1973) hasconfirmedtheseobservations.In view of the fact that FWAs are added to a variety of domesticproducts and have becomea widely distributed component of the environment, any photobiological or promotingeffectof FWAs, includingthe enhancement chemicalswhich are phototoxic. of photocarcinogenesis, could have significantimpact. Photochemicalsensitizationof human skin is a Conceptsof chemicalinteraction with UVR-photocarcinogenesis are of more recentorigin. Blum (1959) clinical problem of considerablesignificance(Harber and Emmett (1973) reviewed a number of reports & Baer, 1972).Phototoxicity and photoallergy can dealing with the influenceof phototoxic substances beproducedexperimentallyin humanand laboratory on photocarcinogenesis. The resultsfrequentlyappear animalsby the useof natural or simulatedsunlight to be in disagreement, a situation possiblyreflecting (Harber 8c Baer, 1972).However, the laboratory condifferencesin technique,including solvents,routes of ditionsof studiescited above(Bingham& Falk, 1970) administration,light sourcesand criteria for tumour werequalitatively different from any naturally occurrecognition,and in statisticalevaluation(Blum, 1959). ring conditions. For example,the FWAs were susIn addition, characteristicsof somecompounds(toxi- pendedin a photoactive solvent(dimethylsulphoxide), city, carcinogenicity,instability) render their interac- applied to the densely-hairedback of mice, and the tions with light complex and their analysisdifficult. animalswere exposedto a germicidal lamp whose Interest in the photobiology of furocoumarinsgrew principal ultraviolet emission(UV-C; 1 < 280nm) is rapidly in the 1950s(Journal of Investigative Dermatonot found in earth-levelsunlight.The potential signifilogy, 1959).A memberof this family of compounds, cance of the results made it imperative to study where it was possibleto 8-methoxypsoralen@-MOP) wasshownto be photo- FWAs under circumstances toxic and to enhancethe ability of UVR to produce reduce the sourcesof ambiguity, while employing skin tumoursin mice(Hakim, Griffin & Knox, 1960; conditions reflecting those of human exposure to Urbach, 1959). chemicalsand light. Sincethe activation spectrumof Photocarcinogenesis can also involve non-photo- an unknown photosensitizercannot be predicted, biological promotion. Epstein & Roth (1968) and light sourcesusedfor photobiologicalstudiesshould Pound (1970)exposedmice to a singledoseof UVR contain the UVR spectrumin ratios approximating a which by itself did not produce tumours;subsequent those found in sunlight. Conversely, there is no repeatedpainting with croton oil elicited tumours in obvious reason for including portions of the solar the irradiated mice. spectrumthat do not ordinarily reach the surfaceof Fluorescentwhitening agents (FWAs) have been the earth (UV-C, X-ray etc.). This is the first of a seriesof studiesreporting the produced in increasingamounts for many years. in mice. The purExtensive toxicological testing in man and animals alteration of photocarcinogenesis has shownmost of thesematerialsto be innocuous poseof the seriesis twofold. First, it documentsthe and non-sensitizing(Keplinger, Fancher, Lyman 8~ developmentof methodsfor evaluating the interacCalandra, 1974; Snyder, Opdyke & Rubenkoenig, tion of UVR and chemicalsin skin carcinogenesis, 1963; Stensby, 1967; Swedish Natural Science utilizing methods that incorporate several recent ResearchCouncil, 1973).However,other investigators developmentsin the production and measurement of have concluded that FWAs enhancedthe carcino- light. Second,the seriescomparesthe ability of repreThe association of skin cancer and long-term exposure to ultraviolet radiation (UVR) has long been recognized (Blum, 1959; Emmett, 1973; Epstein, 1970; Johnson, Daniels & Magnus, 1968; Kopf & Gordon, 1969). The degree of acute skin damage as well as the chronic changes can be modified by certain

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P. D.

FORBES

and F. URBACH

sentativesof two classesof compounds(FWAs and aslandmarks.A tumour could be distinguishedfrom psoralens)to influenceUVR photocarcinogenesis. cysts and other surface features by the time the growth was approximately 0.5mm in height or diameter. EXPERlMENTAL Tumoursand other lesionswereremovedfor histoHairlessmutant (HRS/J)micewerepurchasedfrom logical examination from mice dying during the exthe JacksonLaboratory, Bar Harbor, Maine. Animals periment,and from other animalsat the end of the were housedthroughout the experiment in special study (40wk). radiation cages(Forbes & Urbach, 1969)with free accessto mousechow and tap-water.The light source RESULTS wasa bank of three germicidal(G15T8) low-pressure mercurylamps,suspended 40cm abovethe irradiated Phototoxicity experiment surface.The UVR flux was 4W/m2(J-225 meter, By itself,neithersolutionprovokeda visible change Ultraviolet Products, San Gabriel, Cal.). Chemical in the skin. Areas exposedonly to the vehicle and treatment consistedof pipetting 204 reagent-grade UVR developedbarely perceptibleerythema by 8 hr methanol,with or without 2Opg FWA on to 2cm2 after exposure.The erythema had disappearedfrom of dorsalskin.The FWA, disodium4,4’-bis-(4,6-diani- somemiceby 24hr and from the remainderby 48hr. lino-1,3,5-triazin-2-yl)-aminostilbene-2,2’-disulphonate The reactionof FWA-treated areasdid not differ from (Fig. l), from CIBA-GEIGY, Inc., Ardsley, New York, the reaction of areaspretreated with vehicle only. wasapproximately90%pure,assuppliedto detergent Thus, the FWA was neither an irritant nor a photomanufacturers.UVR exposurebegan 30 min after toxic agentunder the test conditions. chemicaltreatment. For phototoxicity testing, ten animalspretreated Carcinogenesis experiment with FWA and an equalnumberpretreatedwith veHalf of each irradiated group (three mice out of hicleonly were immobilizedunder the light source.A six) survived to the end of the 40-wk observation l-cm diametercircular area of skin centred in each treatedareawasexposed,the remainderof the animal period. No tumours appeared in the unirradiated being covered with aluminium foil. These animals mice.A total of 40 tumoursappearedin the irradiated receiveda 5-minexposureonce(1200J/m2)and were mice. Most tumours tended to enlargeand become examinedat 4, 8, 24, 48 and 72hr after expsoure. increasinglycornified or necrotic, or both; very few For the carcinogenesis experiment,the mice were proved to be invasive or metastatic. Of the 40 exposed without restraint in the cages described tumours analysedhistologically,one was diagnosed above(Forbes& Urbach, 1969).Twelve femalemice asa haemangiomaand two were fibrosarcomas;the weretreatedwith FWA and an equal numberof mice remainderwere squamous-cellcarcinomas.Tumour weretreated with vehiclealone and half of the mice production in irradiated mice was measuredby in each group were exposeddaily, on Mondays to severalparameters(Table 1). Cumulative incidence Fridays, to a flux of 4W/m2, starting 30min after refersto the proportion of mice that developedone chemicaltreatment.The animalswere irradiated for or more tumours during the stated observation 5 min (1200J/m’) for eachof 45 exposures,then for period.Latent period is a measureof tumour develop1Omin for each of the next 30 exposuresand for ment time, and tumour yield is expressedboth as 20min for eachof the final 125exposures.The largest total turnoutsproducedand as the averagetumours singlepossibledosewas thus 72.5 x lo6 J/m’. With per affected animal. On each count, carcinogenesis eachmousefreeto moveaboutin an8 x 8 x 8 cmcubi- wasslightly greater in the vehicle-treatedgroup than cle, the actual dose received by each area of skin in the FWA-treated group. There is thus no evidence varied somewhatwith the animal’sorientation during that this FWA enhancesthe carcinogenicpotency of exposure,but the two UVR-exposedgroupsweresub- short-waveUVR in hairlessmice. ject to the samevariability. Each animal was examinedweekly. One sheetof paperwasmadeup for eachmouse,the sheetbearing Table1. Tumour induction in mice exposed to G15T8 @erimagesof the mouseviewed dorsally, ventrally and micidal) UV lampsjollowing topical pretreatment with FWA from each side.Any changein skin appearancewas in methanol or with methanol only noted at the appropriatelocation on the animal diagram,physical featuresand tattoo spotsbeing used Values for mice treated with Tumour-induction

Fig. 1. Structureof fluorescentwhiteningagentusedin the study.

parameter

Latcnl period to first turnour (wk) Latent period to 50% incidence (wk) Mean latcnl period lor all turnours (wk) Cumulative incidence at 24 wk. (7)0 Cumulatiw incidence at 40 wk (%I Total twnoun at 40 v/k Mean no. of tumours/atTectcd mouse Survival 81 40 wk (%) *Time of first death.

FWA + UV-C 17

Vehicle

+ UV-C

16

19

17

27

25

SO W4

83W)

67 (4/a) 16

83 (S/6) 24

4.0 SO (316)

4.8 50 O/6)

Modification

of phol tocarcinogenesis. I

DISCUSSION

Erythema and skin carcinomas were induced on hairless mice by exposure to UV-C. This radiation treatment did not cause skin ulceration. Neither the erythemal nor the carcinogenic potency of UV-C was enhanced by the presence of the FWA. The circumstances of testing, although not technically relevant to a potential human health problem, were similar to those used by Bingham & Falk (1970); the FWA was applied topically in an organic solvent, and the animals were then exposed to a germicidal lamp. In this system, we found no evidence that the FWA was photobiologically active or cocarcinogenic. In order to relate laboratory data to realistic conditions of human exposure, we have developed test systems utilizing more appropriate light sources (Forbes & Urbach, 1974). Experiments based on these test systems are presented in companion reports (Forbes & Urbach, 1975a,b) and elsewhere (Forbes & Urbach, 197%). Ackrlowledgement-This work was supported in part by NIH Grant ES 00269 from the National Institute of Environmental Health Sciences.

REFERENCES

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Falk. H. L. & Bingham. E. (1973). Interaction’of fluorescent whitening agents and ultraviolet radiation. Ambio 2, 21. Forbes, P. D. & Urbach. F. (1969). Vascular and neoplastic changes in mice following ultraviolet radiation. In The Biologic Eferts q/’ Ulrrauioler Rudiution. Edited by F. Urbach. p. 279. Pergamon Press, New York. Forbes, P. D. & Urbach. F. (1974). Modification of photocarcinogen&s by environmental chemicals. Proc. Am. Ass.

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Forbes. P. D. & Urbach. F. (1975~1).Experimental modification of photocarcinogenesis. II. Fluorescent whitening agents and simulated solar UVR. Fd Cosmet. Toxicol. 13, 339. Forbes, P. D. & Urbach. F. (1975b). Experimental modification of photocarcinogenesis. III. Simulation of exposure to sunlight and fluorescent whitening agents. Fd Cosmet. Tosicol. 13. 343. Forbes. P. D. & Urbach. F. (1975~). Photocarcinogenesis: Lack of enhancement by fluorescent whitening agents. In Encironmental Quality rind Safety: Chemisrry, To.sico/ogy rend Technology. Edited by 6. Coulston and F. Korte. Academic Press, New York. In mess. Hakim. R. E.. Griffin, A. C. & Knox. J. M: (1960). Erythema and tumor formation in methoxsalen-treated mice exposed to fluorescent light. Archs Derm. 82, 572. Harber. L. C. & Baer. R. L. (1972). Pathogenic mechanisms of drug induced photosensitivity. J. inuest. Derm. 58, 327. Johnson, B. E.. Daniels. F. & Magnus, I. A. (1968). Response of human skin to ultraviolet light. In Photophysiology. Vol. IV. Edited by A’. C. Giese. p. 139. Academic Press, New York. Journc~l o/ Irroestigatiue Dermalo/oc/ 1~(I 959). Psoralens and radiani energy-ihid (Suppl.) 32,-i32. Keolinaer. M. L.. Fancher. 0. E.. Lvman. F. L. & Calan&a, j. C. (1974). Toxicologic studies of four fluorescent whitening agents. Tosic. appl. Pharmac. 27, 494. Kopf, A. & Gordon, D. (Chairmen) (1969). Panel on pathologic effects of ultaviolet radiation (cancer). In The &ologic- Eficts of Ulrrauiolet Radiation. Edited by F. Urbach. D. 541. Peraamon Press. New York. Pound, A. ‘W. (1970).-Induced cell proliferation and the initiation of skin tumour formation in mice by ultraviolet light. Parhology 2, 269. Snyder, F. H.. Opdyke. D. L. & Rubenkoenig, H. (1963). Toxicologic studies on brighteners. Toxicol. nppl. Pharmat. 5, 176. Stensby, P. S. (1967). Optical brighteners and their evaluation. Soap chrm. Spec. 43, April, p. 41; May, p. 34; July, p. 80; August, p. 94; September, p. 96. Swedish Natural Science Research Council (1973). Fluorescellt Whitening Agents. Proceedings of a Symposium held at the Royal Institute of Technology, Stockholm. Sweden, April I I. 1973. MVC-Report 2. The Center for Environmental Sciences, Royal Institute of Technology, Stockholm. Urbach, F. (1959). Modification of ultraviolet carcinogenesis by photoactive agents. J. inuest. Derm. 32, 373.