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Els Adriaens Lab Pharmaceutical Technology Ghent University Harelbekestraat 72 9000 Gent, Belgium
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Els Adriaens

The Slug Mucosal Irritation assay: an alternative assay for local tolerance testing
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Depending on the intended purpose of the material, industrial, pharmaceutical, cosmetic and consumer products need to be evaluated for their local tolerance during the regulatory safety evaluation. Currently, animal studies are usually performed to assess this, using laboratory mammal species. However, several studies have shown that the Slug Mucosal Irritation assay can be used as an alternative test to predict the mucosal and ocular tolerance of new pharmaceuticals early in the research and development phase, thereby replacing use of laboratory mammals. In this article the current validation status and different applications of the Slug Mucosal Irritation assay are described. Keywords: local tolerance, Slug Mucosal Irritation assay, alternative Medicinal Products (EMEA), the local tolerance of medicinal products should be performed in laboratory animals prior to human exposure of the product (1). The methods described in this guidance note all use mammals (most frequently rabbits, rats and dogs). However, in some cases, these animal studies can be replaced by validated in vitro tests (1) and the development of such alternatives needs to be encouraged. For example, the Draize rabbit eye test (2) is still the only worldwide regulatory accepted method for eye irritation testing (3). The use of this method has been questioned for decades, based on ethical and scientific arguments (4). Several alternatives for eye irritation testing have been developed using diverse human and animal cells and tissues, but none of these methods are currently accepted by the international regulatory authorities (5). Before an alternative test can be considered as an accepted method, its relevance and reliability has to be investigated during a robust validation process (6). Recently, the validation process was made more flexible by breaking down the process into independent modules (7).

Local tolerance testing

The development of mucosal drug delivery systems as an alternative route for the delivery of peptide and protein drugs other than via the gastro-intestinal tract, and the need for drugs to prevent sexually transmitted diseases often to be administered via the vaginal route, has resulted in an increase of the local tolerance testing of these drugs. The local tolerance testing of products to the skin, eye or mucous membranes represents part of the safety assessment of industrial, pharmaceutical, cosmetic or consumer products. According to a guidance note from the European Agency for the Evaluation of

The Slug Mucosal Irritation assay

The Slug Mucosal Irritation (SMI) assay was developed at the University of Ghent (Belgium) in the Laboratory of Pharmaceutical Technology where one of the major interests is the mucosal drug delivery (e.g. nasal and

NC3Rs #8 The Slug Mucosal Irritation assay Sept 2006

buccal) of therapeutic peptides or proteins, avoiding degradation in the gastro-intestinal tract. Since the bioavailability of peptides and proteins is low, absorption enhancers and bioadhesive powders are often used to increase the bioavailability. Those formulations remain in contact with the mucosal surface for a longer time period, therefore it is important to assess their mucosal irritation potency. To replace use of laboratory mammals, an alternative mucosal irritation test was developed using slugs (Arion lusitanicus), which have a highly mucosal surface, as a test organism. Several studies have shown that the SMI assay can be used as an early screen in the research and development (R&D) phase of new pharmaceutical formulations to evaluate their local tolerance without the use of laboratory mammals (8-14). The SMI assay was validated previously in our laboratory for screening the eye irritation potency of chemicals (15-17). The SMI assay was also nominated as one of the available alternatives to the Draize rabbit eye test (5). Recently, an inter-laboratory study was performed to investigate the transferability of the SMI assay, involving four laboratories. Principle and applications of the SMI assay The body wall of slugs consists of a mucosal surface that contains cells with cilia, cells with microvilli and mucus secreting cells covering a sub-epithelial connective tissue. Slugs that are placed on an irritant substance will produce mucus. Additionally, tissue damage can be induced which results in the release of proteins and enzymes. The SMI assay also allows single and repeated exposure studies and can predict clinical discomfort such as stinging and burning sensations. This is a major advantage of the assay since stinging and burning sensations cannot be predicted in laboratory mammal studies or in vitro.

the SMI assay for assessing the local tolerance of pharmaceuticals. The irritation potency of the test substances is evaluated by placing the slugs (n=5) on the undiluted test item for contact periods of 30 minutes for five successive days and measuring the amount of mucus produced. After each 30-min contact period, the protein and enzyme release (lactate dehydrogenase [LDH] and alkaline phosphatase [ALP]) from the body wall of the slugs is measured (Figure 1). Subsequently, 1 ml of phosphate buffered saline (PBS, pH 7.4) is added near the foot of the slug. Test substances that cause tissue damage will result in the release of biomarkers into the PBS and the proteins and enzymes can be measured in these PBS samples. The SMI assay can predict the local tolerance of solids, semi-solids or liquids. Based on the results of several formulations a prediction model was built that distinguishes between irritation potency and tissue damage (Figure 2). The irritation potency is predicted based on the total amount of mucus produced (total MP) during the repeated 30-min contact periods. The mucus production is expressed as a percentage of the body weight of the slugs. For each slug the total MP is calculated by adding up the mucus produced during each 30-min contact; then the mean of the five slugs is calculated. The percentages shown in Figure 1 correspond with the cut-off values for the total MP. The amount of mucus produced by the slugs depends also on the physical state of the test substance; however the physical state does not affect the release of the biomarkers. Tissue damage is predicted by the number of slugs (max 5) that show ALP release, the mean LDH release of all the samples and the mean protein release excluding the samples taken on day 1. The buccal and vaginal tolerance of some commercial or experimental formulations will be discussed briefly.

Mucosal tolerance testing

To date more than 200 raw materials, experimental and commercial formulations with known animal and clinical irritation data have been used to optimise and validate

NC3Rs #8 The Slug Mucosal Irritation assay Sept 2006

Test procedure (5-day)

Daily during 5 successive days 30 CP



Prediction Irritation

30 contact period with the test substance Transfer slug to new petri dish. Add 1 ml PBS and remove this after 1 h.

Measure mucus production

Calculate total mucus production

Cut-off values for the total mucus production are used to classify the compounds into non-irritant, mild,

moderate and severe irritants


Tissue damage
Measure protein, LDH and ALP release in samples


Transfer slug to new petri dish. Add 1 ml PBS and remove this after 1 h.

Calculate mean protein, mean LDH release, and count number of slugs inducing ALP release

A decision tree combining the results of the mean protein, mean LDH, and number of slugs showing ALP release, is used to convert the results into tissue damage grades:

no, mild, moderate and severe tissue damage

Figure 1. Overview of the test procedure; this procedure is repeated during five successive days.

Figure 2. Prediction model that distinguishes between irritation potency and tissue damage.

Buccal tolerance tablets




tested in volunteers as slow-release tablets that were placed on the buccal mucosa. The 5% Carbopol 974P containing formulation was well tolerated in human and none of the tablets were removed by volunteers, whereas the 50% and pure Carbopol 974P tablets induced severe irritation resulting in small mucosal lesions. Respectively, 28% and 44% of the volunteers (n = 14 to 18) removed the tablets due to irritation (18). These results correspond with the SMI data, since the 5% Carbopol 974P powder formulation caused no irritation or tissue damage whereas the 50% Carbopol 974P powder formulation induced moderate irritation and slight tissue damage and pure Carbopol 974P resulted in severe irritation with moderate tissue damage.

The mucosal tolerance of several bioadhesive powder formulations increasing in Carbopol 974P concentration, a polyacrylic acid that is used to enhance the bioadhesive properties of the powder formulations, were evaluated with the SMI assay using the 5-day repeated test procedure. The irritation potency of the mixtures increased with increasing Carbopol 974P concentration (Figure 3). Mixtures containing 20% and more Carbopol

974P induced mild to severe irritation in slugs. Mixtures containing 15% or less Carbopol 974P can be considered as safe. Two of the formulations, 5% Carbopol 974P and 50% Carbopol 974P, were also

NC3Rs #8 The Slug Mucosal Irritation assay Sept 2006


160 LDH ALP 140 120 100 80

Total MP (%) LDH (U/l.g)


Protein total MP



60 40

5 20 0 A/C 90/10 A/C 85/15 A/C 80/20 A/C 75/25 A/C 50/50 A/C 95/5 A/C 60/40 Carbopol 0

Figure 3. Effect of increasing Carbopol 974P concentration on the endpoints of the SMI assay. Data points represent the mean of

5 slugs. A/C cospray-dried Amiocastarch/Carbopol974P bioadhesive powder mixture.

Vaginal tolerance of OTCs The vagina offers a potential route for local and systemic drug delivery, such as the application of vaginal contraceptives or hormone replacement therapies, and the development of new microbicides for vaginal administration that can prevent the transmission of sexually transmitted diseases (19). Generally it is recommended that the active ingredient and the clinical formulation are tested in the rabbit vaginal irritation test (10-day repeated application) (20). The local tolerance of several over-the-counter (OTC) and experimental formulations for vaginal application were evaluated with the SMI assay (12, 13) to assess its potential to predict human irritation. A brief overview is presented in Table 1.

Table 1. Irritation potency of excipients and OTC formulations as assessed with the SMI assay (5-day repeated treatment): comparison with published data


Total MP (%)

Protein (g/ml.g)

LDH (IU/l.g) 0.5 0.4 1.0 1.1 2.7 2.2

# of slugs with ALP 0/5 0/5 3/5 5/5 5/5

Classification Irritation NI** Moderate Moderate Severe Severe Damage No No Mild Moderate Severe

HEC* K-Y jelly Gynol II Gynol II Extra strength Conceptrol

12.9 2.3 20.9 2.3 23.4 4.3 27.7 5.5 27.6 3.3

86 52 37 26 67 45 228 148

*HEC: hydroxyethyl cellulose; **NI: non-irritant

NC3Rs #8 The Slug Mucosal Irritation assay Sept 2006

Protein (g/ml.g)

A 7-day application of HEC gel resulted in genital heat or burning in only 5% of the women, whereas Gynol II








formulation (Gynol II ) was classified as a moderate irritant (Total MP = 23.4%) according to the results of the SMI assay, whilst 25% of the women reported genital symptoms. Gynol II Extra strength and Conceptrol, both classified as severe irritants by the SMI assay (Total MP = 27%), resulted in genital irritation in 87% of the women or colposcopic findings in 58% of the women respectively. Genital burning is clearly a limiting factor for several products for vaginal application. This cannot be measured in preclinical animal (mammal) or in vitro models (21). These data show that the SMI assay can predict genital burning; the increase in mucus production is related with an increased incidence of stinging and burning sensation. Generally there is a good agreement between the data obtained with the Slug Mucosal Irritation assay and published clinical data.

(containing 2% Nonoxynol-9) users experienced genital symptoms in 25% of the women (21). In a phase I study 15 women (17%) applying K-Y Jelly twice a day for seven consecutive days reported mild genital symptoms including bleeding, burning, itching, irritation, pain and vaginal discharge (22). Mild to moderate penile irritation (itching, tingling and burning) was reported by 41.7% (n=12) after seven days of K-Y Jelly use (23). Evidence of genital irritation was reported by 87% (n=15) of the women using Gynol II Extra strength (containing 3% N9) for seven consecutive days. Colposcopic findings were seen in 58% of the women (n-12) during a vaginal contraceptive (Conceptrol containing 4% N-9) use over six days (24). Several vaginal contraceptives contain Nonoxynol-9 (N-9) as active ingredient. Multiple studies reported that N-9 can disrupt the vaginal epithelium thereby increasing the risk of HIV transmission. All the N-9 containing formulations (Gynol II, Gynol II Extra strength and Conceptrol) caused tissue damage in the SMI assay whereas the other formulations (HEC and K-Y jelly) did not induce tissue damage. Furthermore, an increase in the number of patients complaining of genital heat, itching or burning is related to an increase in irritation (increase in mucus production), as shown in the SMI assay. For the HEC gel, only 5% of the women experience genital symptoms. This formulation also resulted in the lowest mucus production (12.9%) and was classified as non-irritant. K-Y jelly induced an increased mucus production (20.9%) and was classified as moderately irritating; 17% of the women using K-Y
Test procedure (1-day)
60 CP
60 CP

Eye irritation of chemicals

The Slug Mucosal Irritation (SMI) assay was previously validated in house using a set of reference chemicals for eye irritation. This study showed that the SMI assay can be used as an alternative to the in vivo Draize eye irritation test (15-17). Recently, a multi-center study with four participating laboratories was performed to assess the transferability and inter-laboratory variability of the SMI assay. 20 reference chemicals covering the whole irritancy range were selected from the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) data bank (25). The eye irritation potency of the chemicals was assessed by measuring the amount of

Sample 1 n=5 Transfer slug to new Petri dish. Add 1 ml PBS and remove this after 1 h.

60 CP

Sample 2

60 contact period 1% w/v dilution

60 contact period 3.5% w/v dilution

Transfer slug to new Petri dish. Add 1 ml PBS and remove this after 1 h.

Measure mucus production

Measure protein and LDH release in samples

Measure mucus production

Measure protein and LDH release in samples

Figure 4. Overview of the test procedure for screening the eye irritation potential of chemicals.
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mucus produced during a 60-min contact period with a 1% dilution, and a second 60-min treatment with a 3.5% dilution. After each contact period the protein and enzyme release from the mucosal surface were measured (Figure 4). Applying linear discriminant analysis (LDA), a

damage to the eyes). The amount of mucus produced during the first 60-min contact period and the release of proteins from the body wall of the slugs after each 60min contact period were the best predictors to discriminate between the three irritation categories. The predictions were compared with the corresponding EU label of the chemicals. The results of this multi-centre study are presented in Table 2.

classification prediction model was built that classifies the chemicals into the corresponding EU classes (NI: nonirritant, R36: irritating to the eye, and R41: risk of serious

Table 2. Eye irritation potential of the reference chemicals as assessed with the SMI assay

Test compound 3-Methoxy-1,2-propanediol PEG 400 Potassium tetrafluoroborate Glycerol Methylcyclopentane Tween 20 Tetraaminopyrimidine sulf.salt Ethyl acetate Ammonium nitrate 1-Octanol 2-Ethyl-1-hexanol 1-Hexanol Acetone Triton X-100 Imidazole Sodium oxalate Promethazine hydrochloride Chlorhexidine Cetylpyridinium bromide Benzalkonium chloride

MMAS 0 0 0 1.7 3.7 4 10.3 15 18.3 41 51.3 64.8 65.8 68.7 59.3 61.3 71.7 82.3 89.7 108

EU/GHS NI NI NI NI NI NI NI NI R36/2B R36/2B R36/2B R36/2A R36/2A R36/2A R41/1 R41/1 R41/1 R41/1 R41/1 R41/1

SPL NI NI NI NI NI NI NI NI R36 R36 R36 R36 R36 R41 R41 R41 R41 R41 R36 R41

VITO NI NI NI NI NI NI NI NI NI R36 R36 R36 R36 R36 NI R41 R41 R41 NI R41

J&J PRD NI NI NI NI NI NI NI NI R36 R36 R36 R36 R36 R36 R41 R41 R41 R41 R36 R41

UGENT NI NI NI NI NI NI NI NI R36 R36 R36 R36 R36 R41 R41 R41 R41 R41 R41 R41

MMAS: Modified Maximum Average Score (25); EU classification (26); GHS classification (27); SPL: Safepharm Laboratories (UK); VITO (Belgium); J&JPRD: Johnson & Johnson Pharmaceutical Research & Development (Belgium); UGent: Ghent University (Belgium)

NC3Rs #8 The Slug Mucosal Irritation assay Sept 2006

All the NIs (n=8) were predicted correctly by the four laboratories resulting in a 100% specificity. Three irritants (ammonium nitrate, imidazole and cetylpyridinium bromide) were predicted NI by one laboratory resulting in a sensitivity of 75%. For the other laboratories no false negatives were present resulting in 100% sensitivity. However, cetylpyridinium bromide (R41) was underpredicted as R36 by two laboratories. Only one chemical (Triton X-100; R36) was over-predicted as R41 by two laboratories. Generally, 16 of the 20 chemicals were classified the same by the four laboratories. 85% to 95% of the chemicals were classified correctly into the three EU categories. Based on the results of this multi-center study we can conclude that the SMI assay is easily transferable and reproducible and has a high predictivity.







Development (1993) Test guideline 405: Acute eye irritation/corrosion.






Cooperation and Development 4. Wilhelmus KR (2001) The Draize eye test. Survey of

Ophthalmology 45, 493-515

5. Eskes C, Bessou S, Bruner LH, Curren RD, Harbell JW, Jones P, Kreiling R, Liebsch M, McNamee P, Pape W, Prinsen M, Seidle T, Vanparys P, Worth AP & Zuang V (2005) Eye irritation. In Alternative (Non-Animal)

Methods for Cosmetics Testing: Current Status and Future Prospects, A Report Prepared in the Context of the 7th Amendment to the Cosmetics Directive for Establishing the Timetable for Phasing Out Animal Testing (Eskes C & Zuang V, eds.) Alternatives to Laboratory Animals 33, Suppl. 1
6. Balls M, Blaauboer B, Brusick D, Frazier J, Lamb D, Pemberton M, Reinhardt C, Roberfroid M, Rosenkranz H, Schmid B, Spielmann H, Stammati A & Walum E (1990) Report and recommendations of the CAAT/ERGATT workshop on the validation of toxicity test procedures. Alternatives to Laboratory

The SMI assay offers a valuable alternative for predicting the mucosal irritation potency of raw materials and formulations. Heat and burning sensation is a limiting factor in terms of product acceptability and this cannot be assessed by preclinical in vitro or testing in laboratory mammals. Slugs are very sensitive to chemical induced irritation and react by producing mucus. The membrane damaging potency of formulations can be assessed by the protein and enzyme release from the mucosal surface of the slugs. The assay can be used early in the R&D phase of new pharmaceutical formulations, cosmetic and consumer products to evaluate their local tolerance and can replace the use of laboratory mammal species. 7.

Animals 18, 313-337

Hartung T, Bremer S, Casati S, Coecke S, Corvi R, Fortaner S, Gribaldo L, Halder M, Hoffmann S, Roi AJ, Prieto P, Sabbioni E, Scott L, Worth AP & Zuang V (2004) A modular approach to the ECVAM principles on test validity. Alternatives to Laboratory Animals 32, 467-472

1. EMEA (2001)


Adriaens E, Ameye D, Dhondt MMM, Foreman P & Remon JP (2003) Evaluation of the mucosal irritation potency of co-spray dried Amioca/Poly (Acrylic Acid) and Amioca/Carbopol 974P mixtures.

CPMP/SWP/2145/00: Note for

guidance on Non-clinical local tolerance testing of medicinal products. pp. 1-6

2. Draize JH, Woodward G & Calvery HO (1944) Methods for the study of irritation and toxicity of substances applied to the skin and mucous membranes. 9.

Journal of Controlled Release 88, 393-399

Adriaens E, Dierckens K, Bauters TGM, Nelis HJ, Van Goethem F, Vanparys P & Remon JP. (2001) The mucosal toxicity of different benzalkonium chloride analogues evaluated with an alternative test using slugs. Pharmaceutical Research 18, 937-942





Experimental Therapeutics 82, 377

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10. Adriaens E & Remon JP (1999) Gastropods as an evaluation tool for screening the irritating potency of absorption enhancers and drugs. Pharmaceutical

containing slow-release tablets for oral use. Journal

of Pharmacy and Pharmacology 43, 457-464

19. Vermani K & Sanjay G (2000) The scope and potential of vaginal drug delivery. Pharmaceutical

Research 16, 1240-1244

11. Ceulemans J, Vermeire A, Adriaens E, Remon JP & Ludwig A (2001) Evaluation of a mucoadhesive tablet for ocular use. Journal of Controlled Release 77, 333-344 12. Dhondt MMM, Adriaens E & Remon JP (2004) The evaluation of the local tolerance of vaginal formulations, with or without nonoxynol-9, using the slug mucosal irritation test. Sexually Transmitted

Science and Technology Today 10, 359-364

20. Alexander NJ, Alexander V, Allen S, Dorflinger L, Dommel WF, Duerr A, Elias C, Feigal D, Gollub E, Gabelnick H, Harris M, Hitchcock PJ, Karam M, Kazempour K, Lange J, Roddy R, Rowe P, Rosenberg Z, Perriens J, Stone A & Stratton P (1996) Recommendations for the development of vaginal microbicides. Aids 10, 1-6 21. Ballagh SA, Baker JM, Henry DM, Archer DF. 2002. Safety of single daily use for one week of C31G HEC gel in women. Contraception 66, 369-375 22. Malonza IM, Mirembe F, Nakabiito C, Odusoga LO, Osinupebi OA, Hazari K, Chitlange S, Ali MM, Callahan M & Van Damme L (2005) Expanded Phase I safety and acceptability study of 6% cellulose sulfate vaginal gel. Aids 19, 2157-2163 23. Schwartz JL, Poindexter A, Schmitz SW, Mauck C & Callahan MM (2005) Male tolerance of ACIDFORM gel. Contraception 71, 443-446 24. Mauck CK, Weiner DH, Ballagh SA, Creinin MD,

Diseases 31, 229-235

13. Dhondt MMM, Adriaens E, Van Roey J & Remon JP (2005) 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

14. Weyenberg W, Vermeire A, Dhondt MMM, Adriaens E, Kestelyn P, Remon JP & Ludwig A (2004) Ocular bioerodible minitablets as strategy for the management of microbial keratitis. Investigative

Opthalmology and Visual Science 45(9), 3229-3233

15. Adriaens E, Dhondt MMM & Remon JP (2005) Refinement of the Slug Mucosal Irritation test as an alternative screening test-for eye irritation.

Archer DF, Schwartz JL, Pymar HC, Lai JJ, Rencher WF &Callahan MM (2004) Single and multiple exposure tolerance study of polystyrene sulfonate gel: a Phase I safety and colposcopy study. Contraception 70, 77-83 25. European Centre for Ecotoxicology and Toxicology of Chemicals (1998) Eye irritation reference chemicals

Toxicology In Vitro 19, 79-89

16. Adriaens E & Remon JP (2002) The evaluation of an alternative mucosal irritation test using slugs.

Toxicology and Applied Pharmacology 182, 169-175

17. Dhondt MMM, Adriaens E, Pinceel J, Jordaens K, Backeljau T & Remon JP (2006) Slug species- and population-specific effects on the end points of the Slug Mucosal Irritation test. Toxicology In Vitro 20, 448-457 18. Bottenberg P, Cleymaet R, Demuynck C, Remon JP, Coomans D, Michotte Y & Slop D (1991) Development and testing of bioadhesive, fluoride-

data bank. Technical Report No. 48(2), Brussels,

ECETOC 26. European Commission (1993) Commission Directive 93/21/EEC of 27 April 1993 adapting to technical progress for the 18th time Council Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to

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the classification, packaging and labelling of dangerous substances. Official Journal L 110, 20-21 27. United Nations (2003) Globally Harmonised System







(GHS).New York and Geneva: United Nations Publications

All views or opinions expressed in this article are those of the author and do not necessarily reflect the views and opinions of the NC3Rs.

NC3Rs #8 The Slug Mucosal Irritation assay Sept 2006