Els Adriaens, Ph.D.

Contact info

Laboratory of Pharmaceutical Technology
Harelbekestraat 72, 9000 Gent (Belgium)
Tel: +32 (0)9 2648016
e-mail : Els.Adriaens@UGent.be

PhD. Thesis :

Title: "Development of an alternative mucosal irritation test" (May 2000)

Summary

At the laboratory of Pharmaceutical Technology (Faculty of Pharmaceutical Sciences - Ghent University) research has been directed to the development of nasal and buccal drug delivery systems. Because the formulations remain in contact with the mucosal surface for some time, their irritation potential has to be evaluated. Different models have been developed to assess the potential for irritation of the mucosal tissue caused by the molecules and most of them use vertebrates as test organism. Toxicity testing using vertebrate animals to evaluate the safety of xenobiotics to humans has been severely criticised based on ethical and financial considerations. In the workshop: "The three Rs (refinement, replacement and reduction): the way forward" some alternative methods and approaches were proposed. So, the use of in vitro methods and the use of 'lower' organisms with limited sentience that are not protected by legislation controlling animal experiments (invertebrates, plants and micro-organisms) is promoted. The objective of this study was to develop a simple alternative test using invertebrates as a model organism for screening the toxicity of chemicals on mucosal surfaces. The rotifer Brachionus calicyflorus and the slug Arion lusitanicus were selected as test organism based on their characteristics.

In the first study we investigated if the rotifer B. calicyflorus could be used as a test organism for screening the toxicity of absorption enhancers on ciliated epithelium. Rotifers are microscopic aquatic invertebrates with their length ranging from 40-2000µm. The ciliated corona, located at the anterior end of the animal, serves as a locomotory and food-collecting organ. Morphological deformations of the cilia of the corona might effect the swimming and feeding behaviour and consequently influence the survival and the reproduction of the rotifers. When comparing the results of the acute, the chronic and the morphological damage following exposure to the absorption enhancers we can conclude that the same rank order of increasing toxicity was found except for DDPC. The rank order is in good agreement with published data on the toxic effect of absorption enhancers in other models. It was concluded that the use of acute and chronic toxicity testing, in combination with scanning electron microscopy could be considered as an interesting alternative for screening the toxicity of absorption enhancers on ciliated epithelium. There are several advantages of conducting the rotifer tests: rotifers are easy to culture, they produce resting eggs that are commercial available and the described tests are easy to conduct.

In a second study we investigated if the mucosa of the slug A. lusitanicus could be used for evaluating the effect of absorption enhancers on mucosal tissue such as human nasal epithelium. For this purpose the same absorption enhancers as with the rotifers were used. The terrestrial slug A. lusitanicus was used as a test organism. The foot of slugs consist of a single-layered epithelium containing ciliated cells, cells with microvilli, and mucus secreting cells. The body wall is particularly vulnerable to mechanical and chemical damage, and mucus secretions serve to lubricate and to protect the skin against damage. The effects of the molecules on the mucosal tissue was determined from the release of protein and enzymes (LDH and ALP) from the foot of the slug after treatment. Additionally, the mucus production and the reduction in body weight of the slugs were measured during the treatment period. The mucus secreted by the slugs is a measure for irritation. Reduction of the body weight is caused by mucus secretion, substances that cause severe damage result in the leakage of blood. This can cause an increased protein and enzyme release from the body wall. According to their effects on the mucosa of slugs the absorption enhancers can be classified into four different categories with increasing toxicity: (1) HP-ß-CD and ß-CD; (2) DDPC; (3) STDHF and (4) SDC. The results of the present study are in agreement with other studies using the same compounds but on other models.

In a comparative study we investigated the effect of different bioadhesive powders on the nasal mucosa of rabbits and on the mucosa of the slugs. The observed effects in the two models were comparable. We also showed that we could measure the effect of the bioadhesive powders on the nasal mucosa of the rabbits with a simple lavage technique.

In the next study we investigated if the mucosa of slugs could be used as a model for screening the potential buccal irritation induced by chemicals. Research on the development of buccal absorption of ß-blocking agents, also conducted at the laboratory of Pharmaceutical Technology, showed a remarkable difference in the irritation potential of ß-blocking agents with different lipophilicity. The relation between lipophilicity and toxicity has been shown already different times. In this study the slugs were treated with different ß-blocking agents and local anaesthetics with different lipophilicity. Based on the mucus production alone, it was possible to detect the molecules with in vivo irritation potency. A positive relation between the lipophilicity and the irritation could be detected for the local anaesthetics, alprenolol HCl and propranolol HCl.

If an alternative method has been developed, the relevance and the reliability of the test should be investigated. For this purpose reference standards should be used. A reference standard is a substance which has an known degree of toxicity in vivo, and which can be used to determine the degree of toxicity of the substance in the alternative method. Because there exists no list of reference standards for screening the mucosal toxicity of chemicals, a list with 15 substances was selected which was also used during a prevalidation study of the BCOP-test (Bovine Corneal Opacity and Permeability). The BCOP-test as one of the in vitro alternatives for the controversial Draize test for eye irritation. From this study it was shown that the mucus production was the most important endpoint for screening the eye irritation potential of chemicals in comparison with the Draize test. A single treatment of the slugs with the testsubstance resulted in a significant correlation (rs = 0.80) between the amount of mucus produced and the in vivo score (MMAS) for eye irritation. An alternative method is considered to be a valuable screening test when the occurrence of false positives is low (high specificity). Furthermore if a low incidence of false negatives is found (high sensitivity), the alternative method can be considered to be a good replacement assay. The sensitivity, the specificity and the accuracy of the mucosal irritation test was respectively, 83%, 100% and 87%. Testing all non-irritating substances at a 5% concentration (or higher concentrations) could further refine the method. This can probably reduce the incidence of false negatives. When additionally the ALP and LDH release will be measured, the reversibility of the damage can be estimated.

Finally, the irritating potency of different BAC-homologues on the mucosa of the slugs was investigated. Homologues with a different alkyl chain length may have a different degree in toxicity. The mucosal irritation in the slugs increased with decreasing alkyl chain length: BAC-C16 < BAC-C14 < BAC-C12. These results were also confirmed with the BCOP-test. The anti-microbial activity of the different BAC-homologues against E. coli and S. aureus was also investigated. BAC-C16 showed a lower activity against E. coli and the same activity as BAC-C14 against S. aureus. Moreover, the anti-microbial activity was stronger than that of BAC-mix. From this study we can conclude that it should be assigned to use BAC-C16 as a conservative in pharmaceutical preparations.

We can conclude that the mucosal irritation test using slugs seems to be a reproducible and reliable method for screening the irritating potency of chemicals on mucosal tissue. By simply measuring the mucus production it was possible to detect the molecules with in vivo irritating potential. This demonstrates that rapid screening tests can be carried out using simpler toxicity endpoints, which have the advantage of not requiring complex test equipment or a long testing time. When additionally the ALP and LDH release will be measured, the reversibility of the damage can be estimated.

Keywords: Alternative screening test, mucosal irritation, slug

Publications:

M.M.M. Dhondt, E. Adriaens, J. Pinceel, K. Jordaens, T. Backeljau, J.P. Remon
Slug species- and population-specific effects on the end points of the slug mucosal irritation test
Toxicology in Vitro, in press - abstract
F. Van Goethem, E. Adriaens, N. Al鰩e, F. Straube, B. De Wever, M. Cappadoro, S. Catoire, E. Hansen, A. Wolf, P. Vanparys
Prevalidation of a new in vitro reconstituted human cornea model to assess the eye irritating potential of chemicals
Toxicology in Vitro, 20, 1-17 (2006) - abstract
M. Dhondt, E. Adriaens, J. Van Roey, J.P. Remon
The evaluation of the local tolerance of vaginal formulations containing dapivirine using the Slug Mucosal Irritation test and the rabbit vaginal irritation test
European Journal of Pharmaceutics and Biopharmaceutics, 60, 419-425 (2005) - abstract
E. Adriaens, M. Dhondt, J.P. Remon
Refinement of the Slug Mucosal Irritation test as an alternative screening test for eye irritation
Toxicology in vitro, 19, 79-89 (2005) - abstract
W. Weyenberg, A. Vermeire, M. Dhondt, E. Adriaens, P. Kestelyn, J.P. Remon, A. Ludwig
Ocular bioerodible minitablets as strategy for the management of microbial keratitis
Investigative Ophtalmology & Visual Science, 45 (9), 3229-3233 (2004) - abstract
M. Dhondt, E. Adriaens, J.P. Remon
The evaluation of the local tolerance of vaginal formulations, with or without nonoxynol-9, using the slug mucosal irritation test
Sexually Transmitted Diseases, 31 (4), 229-235 (2004)- abstract
E. Adriaens, D. Ameye, M. Dhondt, P. Foreman, J.P. Remon
Evaluation of the mucosal irritation potency of co-spray dried Amioca® /poly(acrylic acid) and Amioca® /Carbopol® 974P mixtures
J. Control. Rel., 88, 393-399 (2003) - abstract
E. Adriaens, J.P. Remon
Evaluation of an alternative mucosal irritation test using slugs
Toxicology and Applied Pharmacology, 185, 169-175 (2002) - abstract
J. Ceulemans, A. Vermeire, E. Adriaens, J.P. Remon, A. Ludwig
Evaluation of a mucoadhesive tablet for ocular use
Journal of Controlled Release, 77 (3), 333-344 (2001) - abstract
C. Callens, E. Adriaens, K. Dierckens, J.P. Remon
Toxicological evaluation of a bioadhesive nasal powder containing a starch and Carbopol 974 P on rabbit nasal mucosa and slug mucosa
J. Control. Rel., 76, 81-91 (2001) - abstract
E. Adriaens, K. Dierckens, T. Bauters, H. Nelis, F. van Goethem, P. Vanparys, J.P. Remon
The mucosal toxicity of different benzalkonium chloride analogues evaluated with an alternative test using slugs
Pharm Res, 18 (7), 937-942 (2001) - abstract
E. Adriaens, J.P. Remon
Gastropods as an evaluation tool for screening the irritating potency of absorption enhancers and drugs
Pharm Res, 16, 1240-1244 (1999) - abstract
E. Adriaens, J. Voorspoels, J. Mertens, J.P. Remon
Effect of absorption enhancers on ciliated epithelium: a novel in vivo toxicity screening method using rotifers
Pharm Res, 14(4), 541-545 (1997)