British Journal of Dermatology (1978) 99, 357.
Mouse screening test for evaluating protection to longwave ultraviolet radiation J.BARTH Department of Dermatology, Karl-Marx University, GDR-701 Leipzig, Liebigstrasse 2t,East Germany Accepted for publication 19 April 1978
SUMMARY
Albino mice photosensitized to psoralens were used for evaluating a sun-barrier preparation for use against longwave ultraviolet radiation. The difference in thickness of the treated and control ears at various times after irradiation was used as a measure of photoprotection.
Light protection in the UVA range (320-400 nm) is of importance, not only for preventive therapy of photodermatoses, but also in the avoidance of side effects in phototherapy, e.g. in PUVA treatment. Normally investigation of the photoprotective potency of a sun-barrier preparation might be studied initially on animals; humans are tested only later. Suitable methods on humans have been described by Maibach, Sams & Epstein (1967) and Dablen et al. (1970). Using the mouse method of Sawa (1968) we have developed a screening method for evaluating the protective properties of topically applied sunscreens using the ear. One ear is treated, the other untreated; thus the animal acts as its own contTol. As we wished to study photoprotection to UVA, the mice were photosensitized with psoralen. MATERIALS AND METHODS
Albino mice of both sexes, weighing approximately 30 g, were sensitized to UVA radiation by 02 ml subcutaneous 70% alcoholic 5-MOP and 8-MOP each 0 25%. The irradiation was started 30 min after the injection with a fluorescent lamp, LS 40 W UVA 70 (VEB Narva GDR-923 Brand-Erbisdorf). This lamp has a continuous spectrum between 290 and 400 nm, maximal at about 345 nm. The output ofthe lamp in the UVA spectrum, i m away, is 80/iW/cm^. The animals, immobilized during irradiation, which lasted 5 h, were 5 cm away from the lamp, and wavelengths shorter than 320 nm were restricted by 4 mm-thick window glass. Sixty animals, photosensitized and irradiated as above, but without any sunbarrier preparation on the ears, were used to test the validity ofthe method. The thickness ofthe ears was determined with a special instrument (Fig. i); measurements were done before, and 24,48 and 72 h after irradiation. The Correspondence: Dr J.Barth, Department of Dermatology, Karl-Marx University, GDR-701 Leipzig, Liebigstrasse 21, East Germany, 0007-0963/78/1000-0357502.00 ©1978 British Association of Dermatologists
357
358
J.Barth
FIGURE I, Instrument for measuring ear thickness of mice; a displacement dial gauge, slightly modified; ear placed between two platens to spread compression.
thickness ofthe ears was determined using a dial guage for measuring displacement. The instrument was modified by adding a counterbalance spring as so to reduce pressure on the ear. The gauge very lightly compressed the skin of the ear between two platens which spread the load on the ear. The compression gives a slight inaccuracy to the absolute measurement of thickness but reproducibiUty of relative thickness in the oedematous ear was estimated to be better than ± i o % . Six days after irradiation, the ears were also examined for naked eye changes. Another 36 mice, each photosensitized as above, were used for assessing tbe sunbarrier. To each of these animals, 60 mg of sunbarrier preparation cream (Contralum, Hermal-Chemie, FGR-Reinbeck) was applied to one ear; the active ingredient is a benzophenone. To the other ear a water-in-oil emulsion of wool fat alcohols 6-o mg was applied as a control. The thickness ofthe cream to each ear was estimated to be approximately 30 }tm. RESULTS
Tbe thickness of the normal ear in our mice lies between n o and 220 /im. In the studies of the 60 photosensitized control mice, with no sunbarrier preparation, no significant difference in the thickness
Mouse screening test for UVA protection
359
TABLE I. Effects of irradiation on mouse ear thickness (//m) mean and range After irradiation
Ears pretreated with sunbarrier preparation Control ears
Before irradiation
24 h
48 h
72 h
I30(ti0-I5o) 130 (110-150)
150(130-160) 155 (130-180)
220(130-400) 285 (t3O-47o)
275(150-470) 335 (270-490)
ofthe ears was found between the two sides at 24, 48 and 72 h after irradiation ( 7 ' > o i ) ; the mean values for thickness (with ranges) at these times were respectively 191 (150-240), 367 (360-450) and 475 (350-550)/'m. Ofthe 36 mice where the sunbarrier preparation was applied to one ofthe ears, 4 died within 2 days of irradiation, but we have complete data from 32 pairs of cars. Table i shows the course ofthe change ofthe thickness ofthe ears during the experiment and compares the ears pretreated with those with the active sunbarrier preparation. Because ofthe relatively small number of animals and uncertainty as to data being normally distributed, we used the Wilcoxon test for paired values (Weber, 1972) as the statistical method for assessment. There was a significantly increased thickness on control sides 48 and 72 h after irradiation (JP
Mouse screening test for evaluating protection to longwave ultraviolet radiation J.BARTH Department of Dermatology, Karl-Marx University, GDR-701 Leipzig, Liebigstrasse 2t,East Germany Accepted for publication 19 April 1978
SUMMARY
Albino mice photosensitized to psoralens were used for evaluating a sun-barrier preparation for use against longwave ultraviolet radiation. The difference in thickness of the treated and control ears at various times after irradiation was used as a measure of photoprotection.
Light protection in the UVA range (320-400 nm) is of importance, not only for preventive therapy of photodermatoses, but also in the avoidance of side effects in phototherapy, e.g. in PUVA treatment. Normally investigation of the photoprotective potency of a sun-barrier preparation might be studied initially on animals; humans are tested only later. Suitable methods on humans have been described by Maibach, Sams & Epstein (1967) and Dablen et al. (1970). Using the mouse method of Sawa (1968) we have developed a screening method for evaluating the protective properties of topically applied sunscreens using the ear. One ear is treated, the other untreated; thus the animal acts as its own contTol. As we wished to study photoprotection to UVA, the mice were photosensitized with psoralen. MATERIALS AND METHODS
Albino mice of both sexes, weighing approximately 30 g, were sensitized to UVA radiation by 02 ml subcutaneous 70% alcoholic 5-MOP and 8-MOP each 0 25%. The irradiation was started 30 min after the injection with a fluorescent lamp, LS 40 W UVA 70 (VEB Narva GDR-923 Brand-Erbisdorf). This lamp has a continuous spectrum between 290 and 400 nm, maximal at about 345 nm. The output ofthe lamp in the UVA spectrum, i m away, is 80/iW/cm^. The animals, immobilized during irradiation, which lasted 5 h, were 5 cm away from the lamp, and wavelengths shorter than 320 nm were restricted by 4 mm-thick window glass. Sixty animals, photosensitized and irradiated as above, but without any sunbarrier preparation on the ears, were used to test the validity ofthe method. The thickness ofthe ears was determined with a special instrument (Fig. i); measurements were done before, and 24,48 and 72 h after irradiation. The Correspondence: Dr J.Barth, Department of Dermatology, Karl-Marx University, GDR-701 Leipzig, Liebigstrasse 21, East Germany, 0007-0963/78/1000-0357502.00 ©1978 British Association of Dermatologists
357
358
J.Barth
FIGURE I, Instrument for measuring ear thickness of mice; a displacement dial gauge, slightly modified; ear placed between two platens to spread compression.
thickness ofthe ears was determined using a dial guage for measuring displacement. The instrument was modified by adding a counterbalance spring as so to reduce pressure on the ear. The gauge very lightly compressed the skin of the ear between two platens which spread the load on the ear. The compression gives a slight inaccuracy to the absolute measurement of thickness but reproducibiUty of relative thickness in the oedematous ear was estimated to be better than ± i o % . Six days after irradiation, the ears were also examined for naked eye changes. Another 36 mice, each photosensitized as above, were used for assessing tbe sunbarrier. To each of these animals, 60 mg of sunbarrier preparation cream (Contralum, Hermal-Chemie, FGR-Reinbeck) was applied to one ear; the active ingredient is a benzophenone. To the other ear a water-in-oil emulsion of wool fat alcohols 6-o mg was applied as a control. The thickness ofthe cream to each ear was estimated to be approximately 30 }tm. RESULTS
Tbe thickness of the normal ear in our mice lies between n o and 220 /im. In the studies of the 60 photosensitized control mice, with no sunbarrier preparation, no significant difference in the thickness
Mouse screening test for UVA protection
359
TABLE I. Effects of irradiation on mouse ear thickness (//m) mean and range After irradiation
Ears pretreated with sunbarrier preparation Control ears
Before irradiation
24 h
48 h
72 h
I30(ti0-I5o) 130 (110-150)
150(130-160) 155 (130-180)
220(130-400) 285 (t3O-47o)
275(150-470) 335 (270-490)
ofthe ears was found between the two sides at 24, 48 and 72 h after irradiation ( 7 ' > o i ) ; the mean values for thickness (with ranges) at these times were respectively 191 (150-240), 367 (360-450) and 475 (350-550)/'m. Ofthe 36 mice where the sunbarrier preparation was applied to one ofthe ears, 4 died within 2 days of irradiation, but we have complete data from 32 pairs of cars. Table i shows the course ofthe change ofthe thickness ofthe ears during the experiment and compares the ears pretreated with those with the active sunbarrier preparation. Because ofthe relatively small number of animals and uncertainty as to data being normally distributed, we used the Wilcoxon test for paired values (Weber, 1972) as the statistical method for assessment. There was a significantly increased thickness on control sides 48 and 72 h after irradiation (JP
