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The chorioallantoic membrane in the prediction of eye irritation potential
Toxic. in Vitro Vol. 4, No. 4/5, pp. 321-323, 1990 Printed in Great Britain. All rights reserved
0887-2333/90 $3.00 + 0.00 Copyright © 1990 Pergamon Press plc
THE CHORIOALLANTOIC M E M B R A N E IN THE PREDICTION OF EYE IRRITATION POTENTIAL R. S. LAWRENCE,D. M. ACKROYD and D. L. WILLIAMS Unilever Research, Colworth House, Sharnbrook, Bedford MK44 1LQ, UK Abstract--lt has been suggested that the chick chorioallantoic membrane (CAM) could be used as an alternative to the rabbit eye test for irritation. Various authors have examined different methods for investigating the irritant potential of materials to the CAM. We used the CAM method as described by Luepke, to assess the effect of 34 materials of known in vivo eye irritancy, including fully formulated products ranging from skin creams to industrial detergents. Test materials were applied undiluted to the CAM on day I0 of incubation with rinsing after 20 seconds of contact. Effects to the CAM including vasodilation and haemorrhage were assessed for up to 5 min after application. A comparison was made between the rank order of effect on the CAM and the known ranking for in vivo eye irritancy. There was in general only a limited correlation between in vitro and in vivo results but a reasonable distinction between irritant materials and those of only very slight irritancy was observed. Among products of a similar type there was a better correlation between results from the CAM and in vivo eye results than when all materials were considered together. These data indicate that this method would only have a limited application as a screening procedure for eye irritation potential or as part of a battery of in vitro tests but so far has not been found to be superior to cell culture tests in our laboratory.
Introduction
Materials and Methods
There is much current interest in, and a proliferation of, in vitro and e x vivo techniques designed to replace or reduce live animal tests. Many of these potential alternatives have been directed towards replacement of the Draize rabbit eye test. We first developed a whole-eye organ technique to assess the potential of materials to damage the cornea (Burton et al., 1981; Y o r k et al., 1982) and also investigated the chorioallantoic membrane ( C A M ) of the hen's egg, which has been proposed as a possible replacement for the Draize rabbit eye test. Initially the C A M was examined to determine if this system could be used either as an indicator of conjunctival inflammation, or as a total replacement for assessment of eye irritation potential. This work was based on the method described by Leighton et al. (1985), in which test materials were applied to the C A M at 14 days' incubation and assessment was over a period of 72 hr after treatment. In these experiments we found that although this method may have some relevance as a screening test for potential eye irritants it was not suitable as either a model for conjunctival irritation (Friend et al., 1990; Parish, 1985) or to test eye irritation potential as a whole (Lawrence, 1987; Lawrence et al., 1986). Therefore we examined another C A M test method as described by Luepke (1985) and Luepke and Kemper (1986). This method has a short treatment time and short assessment period. We report here the macroscopic findings on C A M s from treatment with 34 products and chemicals. The C A M results are compared with historical in vivo eye irritation data from either our own databank or that reported in the literature.
Test materials. Thirty four materials were tested. The materials ranged from simple chemicals such as ethanol and acetone to fully formulated products including shampoos and industrial detergent cleaners. The eye irritation potential of each test product or chemical was assessed by reference to either literature sources or our own historical database. Thus, the in vivo eye irritancy is an assessment of available data but quoted in terms of the standard Draize eye test (Draize et al., 1944). Test system. The fertile hens' eggs (ISA Poultry Services, Peterborough, U K ) were incubated in a Marsh Roll-X automatic incubator (Robin Haigh Incubators, Surrey, UK). Access to the C A M followed the method described by Luepke (1985) and is shown in Fig. 1. The egg shell over the air space was removed with the aid of a small rotary cutter (Drillmaster Junior A400, Microflame (UK) Ltd) on the tenth day of incubation. A small drop of water was then placed and spread over the shell membrane, which aided the careful removal of this membrane without damaging the underlying C A M . Treatment. The test materials were all applied undiluted to the C A M with the sole exception of sodium hydroxide, which was applied as a molar solution (number 34). Two hundred microlitres of liquids were applied or 100 mg of solids. All materials were rinsed from the C A M 20 sec after instillation with 5 ml distilled water. Replicates of six eggs were used for each test material. Assessment. The effects of test substances on the C A M were scored as described by Luepke (1985) and are shown in Table 1. In addition, all effects were carefully recorded and also assessed on an alternative scoring system. This alternative system is not presented here since it adds little to the overall interpretation of results.
Abbreviation: CAM = chorioallantoic membrane.
321
R. S. LAWRENCEet al.
322 LUEPKE METHOD 10 Days Old
Airspace ~
Shell~
Chorioallantoic Membrane
Shell
Membrane
Embryo&Yolk
Treat Shell ~
V~,.~l~m-Embryo&Yolk
%#
Chorioallantoic Membrane
Fig. 1. Diagrammatic representation of procedure used to gain access to the chorioallantoic membrane.
Results The effects o f 34 test materials o n the C A M are c o m p a r e d with their respective in vivo eye irrita'don potential expressed in terms o f anticipated effects in the s t a n d a r d Draize eye test (Table 2). Test p r o d u c t s or chemicals have been r a n k e d f r o m 1 to 34, with 1 referring to t h a t p r o d u c t which p r o d u c e d least effect o n the C A M a n d 34 the m o s t severe. W h e n two or m o r e materials have been allocated identical scores then the high or low n u m b e r i n g within such a g r o u p is arbitrary. The score (Table 1) for each substance is the m e a n o f six eggs.
Table I. Method of scoring effects on chorioallantoic membrane as described by Luepke (1985) Luepke scoring method* Score Effect 0.5 min 2 min 5 rain Injection 5 3 1 Haemorrhage 7 5 3 Coagulation 9 7 5 *0-0.9 practically non-irritant; 1-4.9 slight irritant; 5-8.9 moderate irritant; >9 strong irritant. M o s t substances induced ' s t r o n g ' effects a n d it should be n o t e d t h a t m o r e t h a n 20 o f the materials b a d m e a n scores between l0 a n d 12 (Table 2). There is some correlation between effects o n the C A M a n d in vivo eye irritation as s h o w n by materials 1-6 being at m o s t very slightly irritant to the eye. However, the majority o f materials are graded in the strong category with scores ranging from 9.8 to 12. W i t h i n this b r o a d b a n d o f materials the in vivo eye irritancy ranges f r o m virtually n o n - i r r i t a n t to severe.
Discussion The substances e x a m i n e d here represent b o t h fully f o r m u l a t e d p r o d u c t s a n d single chemicals with b o t h groups covering a wide range o f in vivo eye irritancy. The data reported indicate t h a t overall there is not a good correlation between the C A M results a n d in vivo eye irritation potential. The fact t h a t m a n y o f the materials induced very similar effects on the C A M indicates t h a t the C A M could n o t be used to differentiate between such a b r o a d spectrum o f chemicals a n d products. These findings tend to c o n t r a d i c t those reported by Luepke (1985) a n d Luepke a n d K e m p e r (1986) but are m o r e in agreement with those o f Price et al. (1986). However, o n e x a m i n a t i o n o f the effects p r o d u c e d by p r o d u c t s of a similar type, for example formulations based o n anionic surfactants, we f o u n d some potential for r a n k i n g such materials within a closely related p r o d u c t category. This particular property o f the test does not offer any a d v a n t a g e over m a n y simple cytotoxicity tests a n d is in fact p r o b a b l y only
Table 2. Effects of test materials on the CAM with reference to their effects in the Draize eye test In vivo
In vivo
Mean Irrit. eye irrit. Mean Irrit. eye irrit. Code score assess, pot. Code score assess, pot. 1 4 Slight VIRT NI 18 10.3 Strong MOD 2 4.6 Slight NI 19 10.3 Strong MOD 3 4.7 Slight VIRT NI 20 I 1.0 Strong MOD 4 5 Moderate VIRT NI 21 11.2 Strong SEV 5 5.5 Moderate V. SL 22 11.3 Strong MOD 6 7.16 Moderate V. SL 23 11.3 Strong MOD/SEV 7 9.8 Strong MOD 24 11.6 Strong SEV 8 10.0 Strong MOD/SEV 25 11.6 Strong SL/MOD 9 10.0 Strong MOD/SEV 26 11.6 Strong SL/MOD 10 10.0 Strong VIRT NI 27 11.6 Strong SEV I1 10.0 Strong SL 28 11.6 Strong MOD 12 10.0 Strong MOD 29 11.6 Strong SEV 13 10.0 Strong VIRT NI 30 11.7 Strong SEV 14 10.0 Strong SL 31 12.0 Strong SL/MOD 15 10.3 Strong MOD/SEV 32 12.0 Strong MOD 16 10.3 Strong MOD/SEV 33 12.0 Strong V. SL 17 10.3 Strong V. SL 34 15.16 Strong SEV NI = non-irritant VIRT NI = virtually non-irritant V. SL = very slight SL = slight SL/MOD = slight to moderate MOD = moderate MOD/SEV= moderate to severe SEV = severe
CAM in prediction of eye irritation a reflection of differing levels of surfactant, which could be determined from a close e x a m i n a t i o n o f the formulation. Thus, a l t h o u g h d a t a for only 34 materials are presented here there is a clear indication t h a t the C A M would not be a technique suited to differentiating between the eye irritation potential of a wide range of materials. It would therefore not be a suitable m e t h o d to a d o p t for replacing in vivo eye irritation assessments.
REFERENCES Burton A. B. G., York M. and Lawrence R. S. (1981) The in vitro assessment of severe eye irritants. Fd Cosmet. Toxicol. 19, 471-480. Draize J. H., Woodard G. and Calvery H. O. (1944) Methods for the study of irritation and toxicity of substances applied to the skin and mucous membranes. J. Pharmac. exp. Ther. 82, 377-390. Friend J. V., Crevel R. W. R., Williams T. C. and Parish W. E. (1990) Immaturity of the inflammatory response of the chick chorioallantoic membrane. Toxic. in Vitro 4, 324-326.
323
Lawrence R. S. (1987) The chorioallantoic membrane in irritancy testing. In In Vitro Methods in Toxicology. Edited by C. K. Atterwill and C. E. Steele. pp. 263-278. Cambridge University Press. Lawrence R. S., Groom M. H., Ackroyd D. M. and Parish W. E. (1986) The chorioallantoic membrane in irritation testing. Fd Chem. Toxic. 23, 497-502. Leighton J., Nassauer J. and Tchao R. (1985) The chick embryo in toxicology: an alternative to the rabbit eye. Fd Chem. Toxic. 23, 293-298. Luepke N. P. (1985) Hen's egg chorioallantoic membrane test for irritation potential. Fd Chem. Toxic. 23, 287-291. Luepke N. P. and Kemper F. H. (I 986) The HET-CAM test: an alternative to the Draize eye test. Fd Chem. Toxic. 24, 495-496. Parish W. E. (1985) Ability of in vitro (corneal injury---eye organ--and chorioallantoic membrane) tests to represent histopathological features of acute eye inflammation. Fd Chem. Toxic. 23, 215-227. Price J. B., Barry M. P. and Andrews I. J. (1986) The use of the chick chorioallantoic membrane to predict eye irritants. Fd Chem. Toxic. 24, 503-505. York M., Lawrence R. S. and Gibson G. B. (1982) An in vitro test for the assessment of eye irritancy in consumer products--preliminary findings. Int. J. cosmet. Sci. 4, 223-234.
0887-2333/90 $3.00 + 0.00 Copyright © 1990 Pergamon Press plc
THE CHORIOALLANTOIC M E M B R A N E IN THE PREDICTION OF EYE IRRITATION POTENTIAL R. S. LAWRENCE,D. M. ACKROYD and D. L. WILLIAMS Unilever Research, Colworth House, Sharnbrook, Bedford MK44 1LQ, UK Abstract--lt has been suggested that the chick chorioallantoic membrane (CAM) could be used as an alternative to the rabbit eye test for irritation. Various authors have examined different methods for investigating the irritant potential of materials to the CAM. We used the CAM method as described by Luepke, to assess the effect of 34 materials of known in vivo eye irritancy, including fully formulated products ranging from skin creams to industrial detergents. Test materials were applied undiluted to the CAM on day I0 of incubation with rinsing after 20 seconds of contact. Effects to the CAM including vasodilation and haemorrhage were assessed for up to 5 min after application. A comparison was made between the rank order of effect on the CAM and the known ranking for in vivo eye irritancy. There was in general only a limited correlation between in vitro and in vivo results but a reasonable distinction between irritant materials and those of only very slight irritancy was observed. Among products of a similar type there was a better correlation between results from the CAM and in vivo eye results than when all materials were considered together. These data indicate that this method would only have a limited application as a screening procedure for eye irritation potential or as part of a battery of in vitro tests but so far has not been found to be superior to cell culture tests in our laboratory.
Introduction
Materials and Methods
There is much current interest in, and a proliferation of, in vitro and e x vivo techniques designed to replace or reduce live animal tests. Many of these potential alternatives have been directed towards replacement of the Draize rabbit eye test. We first developed a whole-eye organ technique to assess the potential of materials to damage the cornea (Burton et al., 1981; Y o r k et al., 1982) and also investigated the chorioallantoic membrane ( C A M ) of the hen's egg, which has been proposed as a possible replacement for the Draize rabbit eye test. Initially the C A M was examined to determine if this system could be used either as an indicator of conjunctival inflammation, or as a total replacement for assessment of eye irritation potential. This work was based on the method described by Leighton et al. (1985), in which test materials were applied to the C A M at 14 days' incubation and assessment was over a period of 72 hr after treatment. In these experiments we found that although this method may have some relevance as a screening test for potential eye irritants it was not suitable as either a model for conjunctival irritation (Friend et al., 1990; Parish, 1985) or to test eye irritation potential as a whole (Lawrence, 1987; Lawrence et al., 1986). Therefore we examined another C A M test method as described by Luepke (1985) and Luepke and Kemper (1986). This method has a short treatment time and short assessment period. We report here the macroscopic findings on C A M s from treatment with 34 products and chemicals. The C A M results are compared with historical in vivo eye irritation data from either our own databank or that reported in the literature.
Test materials. Thirty four materials were tested. The materials ranged from simple chemicals such as ethanol and acetone to fully formulated products including shampoos and industrial detergent cleaners. The eye irritation potential of each test product or chemical was assessed by reference to either literature sources or our own historical database. Thus, the in vivo eye irritancy is an assessment of available data but quoted in terms of the standard Draize eye test (Draize et al., 1944). Test system. The fertile hens' eggs (ISA Poultry Services, Peterborough, U K ) were incubated in a Marsh Roll-X automatic incubator (Robin Haigh Incubators, Surrey, UK). Access to the C A M followed the method described by Luepke (1985) and is shown in Fig. 1. The egg shell over the air space was removed with the aid of a small rotary cutter (Drillmaster Junior A400, Microflame (UK) Ltd) on the tenth day of incubation. A small drop of water was then placed and spread over the shell membrane, which aided the careful removal of this membrane without damaging the underlying C A M . Treatment. The test materials were all applied undiluted to the C A M with the sole exception of sodium hydroxide, which was applied as a molar solution (number 34). Two hundred microlitres of liquids were applied or 100 mg of solids. All materials were rinsed from the C A M 20 sec after instillation with 5 ml distilled water. Replicates of six eggs were used for each test material. Assessment. The effects of test substances on the C A M were scored as described by Luepke (1985) and are shown in Table 1. In addition, all effects were carefully recorded and also assessed on an alternative scoring system. This alternative system is not presented here since it adds little to the overall interpretation of results.
Abbreviation: CAM = chorioallantoic membrane.
321
R. S. LAWRENCEet al.
322 LUEPKE METHOD 10 Days Old
Airspace ~
Shell~
Chorioallantoic Membrane
Shell
Membrane
Embryo&Yolk
Treat Shell ~
V~,.~l~m-Embryo&Yolk
%#
Chorioallantoic Membrane
Fig. 1. Diagrammatic representation of procedure used to gain access to the chorioallantoic membrane.
Results The effects o f 34 test materials o n the C A M are c o m p a r e d with their respective in vivo eye irrita'don potential expressed in terms o f anticipated effects in the s t a n d a r d Draize eye test (Table 2). Test p r o d u c t s or chemicals have been r a n k e d f r o m 1 to 34, with 1 referring to t h a t p r o d u c t which p r o d u c e d least effect o n the C A M a n d 34 the m o s t severe. W h e n two or m o r e materials have been allocated identical scores then the high or low n u m b e r i n g within such a g r o u p is arbitrary. The score (Table 1) for each substance is the m e a n o f six eggs.
Table I. Method of scoring effects on chorioallantoic membrane as described by Luepke (1985) Luepke scoring method* Score Effect 0.5 min 2 min 5 rain Injection 5 3 1 Haemorrhage 7 5 3 Coagulation 9 7 5 *0-0.9 practically non-irritant; 1-4.9 slight irritant; 5-8.9 moderate irritant; >9 strong irritant. M o s t substances induced ' s t r o n g ' effects a n d it should be n o t e d t h a t m o r e t h a n 20 o f the materials b a d m e a n scores between l0 a n d 12 (Table 2). There is some correlation between effects o n the C A M a n d in vivo eye irritation as s h o w n by materials 1-6 being at m o s t very slightly irritant to the eye. However, the majority o f materials are graded in the strong category with scores ranging from 9.8 to 12. W i t h i n this b r o a d b a n d o f materials the in vivo eye irritancy ranges f r o m virtually n o n - i r r i t a n t to severe.
Discussion The substances e x a m i n e d here represent b o t h fully f o r m u l a t e d p r o d u c t s a n d single chemicals with b o t h groups covering a wide range o f in vivo eye irritancy. The data reported indicate t h a t overall there is not a good correlation between the C A M results a n d in vivo eye irritation potential. The fact t h a t m a n y o f the materials induced very similar effects on the C A M indicates t h a t the C A M could n o t be used to differentiate between such a b r o a d spectrum o f chemicals a n d products. These findings tend to c o n t r a d i c t those reported by Luepke (1985) a n d Luepke a n d K e m p e r (1986) but are m o r e in agreement with those o f Price et al. (1986). However, o n e x a m i n a t i o n o f the effects p r o d u c e d by p r o d u c t s of a similar type, for example formulations based o n anionic surfactants, we f o u n d some potential for r a n k i n g such materials within a closely related p r o d u c t category. This particular property o f the test does not offer any a d v a n t a g e over m a n y simple cytotoxicity tests a n d is in fact p r o b a b l y only
Table 2. Effects of test materials on the CAM with reference to their effects in the Draize eye test In vivo
In vivo
Mean Irrit. eye irrit. Mean Irrit. eye irrit. Code score assess, pot. Code score assess, pot. 1 4 Slight VIRT NI 18 10.3 Strong MOD 2 4.6 Slight NI 19 10.3 Strong MOD 3 4.7 Slight VIRT NI 20 I 1.0 Strong MOD 4 5 Moderate VIRT NI 21 11.2 Strong SEV 5 5.5 Moderate V. SL 22 11.3 Strong MOD 6 7.16 Moderate V. SL 23 11.3 Strong MOD/SEV 7 9.8 Strong MOD 24 11.6 Strong SEV 8 10.0 Strong MOD/SEV 25 11.6 Strong SL/MOD 9 10.0 Strong MOD/SEV 26 11.6 Strong SL/MOD 10 10.0 Strong VIRT NI 27 11.6 Strong SEV I1 10.0 Strong SL 28 11.6 Strong MOD 12 10.0 Strong MOD 29 11.6 Strong SEV 13 10.0 Strong VIRT NI 30 11.7 Strong SEV 14 10.0 Strong SL 31 12.0 Strong SL/MOD 15 10.3 Strong MOD/SEV 32 12.0 Strong MOD 16 10.3 Strong MOD/SEV 33 12.0 Strong V. SL 17 10.3 Strong V. SL 34 15.16 Strong SEV NI = non-irritant VIRT NI = virtually non-irritant V. SL = very slight SL = slight SL/MOD = slight to moderate MOD = moderate MOD/SEV= moderate to severe SEV = severe
CAM in prediction of eye irritation a reflection of differing levels of surfactant, which could be determined from a close e x a m i n a t i o n o f the formulation. Thus, a l t h o u g h d a t a for only 34 materials are presented here there is a clear indication t h a t the C A M would not be a technique suited to differentiating between the eye irritation potential of a wide range of materials. It would therefore not be a suitable m e t h o d to a d o p t for replacing in vivo eye irritation assessments.
REFERENCES Burton A. B. G., York M. and Lawrence R. S. (1981) The in vitro assessment of severe eye irritants. Fd Cosmet. Toxicol. 19, 471-480. Draize J. H., Woodard G. and Calvery H. O. (1944) Methods for the study of irritation and toxicity of substances applied to the skin and mucous membranes. J. Pharmac. exp. Ther. 82, 377-390. Friend J. V., Crevel R. W. R., Williams T. C. and Parish W. E. (1990) Immaturity of the inflammatory response of the chick chorioallantoic membrane. Toxic. in Vitro 4, 324-326.
323
Lawrence R. S. (1987) The chorioallantoic membrane in irritancy testing. In In Vitro Methods in Toxicology. Edited by C. K. Atterwill and C. E. Steele. pp. 263-278. Cambridge University Press. Lawrence R. S., Groom M. H., Ackroyd D. M. and Parish W. E. (1986) The chorioallantoic membrane in irritation testing. Fd Chem. Toxic. 23, 497-502. Leighton J., Nassauer J. and Tchao R. (1985) The chick embryo in toxicology: an alternative to the rabbit eye. Fd Chem. Toxic. 23, 293-298. Luepke N. P. (1985) Hen's egg chorioallantoic membrane test for irritation potential. Fd Chem. Toxic. 23, 287-291. Luepke N. P. and Kemper F. H. (I 986) The HET-CAM test: an alternative to the Draize eye test. Fd Chem. Toxic. 24, 495-496. Parish W. E. (1985) Ability of in vitro (corneal injury---eye organ--and chorioallantoic membrane) tests to represent histopathological features of acute eye inflammation. Fd Chem. Toxic. 23, 215-227. Price J. B., Barry M. P. and Andrews I. J. (1986) The use of the chick chorioallantoic membrane to predict eye irritants. Fd Chem. Toxic. 24, 503-505. York M., Lawrence R. S. and Gibson G. B. (1982) An in vitro test for the assessment of eye irritancy in consumer products--preliminary findings. Int. J. cosmet. Sci. 4, 223-234.