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The Slug Mucosal Irritation (SMI) assay: A tool for the evaluation of nasal discomfort
Toxicology in Vitro 27 (2013) 1954–1961
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The Slug Mucosal Irritation (SMI) assay: A tool for the evaluation of nasal discomfort Joke Lenoir a,⇑, Claus Bachert b, Jean-Paul Remon a, Els Adriaens c a
Laboratory of Pharmaceutical Technology, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium Department of Rhino-Otolaryngology, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium c InvertTox, Bellemdorpweg 95, 9881 Aalter, Belgium b
a r t i c l e
i n f o
Article history: Received 28 February 2013 Accepted 27 June 2013 Available online 9 July 2013 Keywords: Slug Mucosal Irritation assay Human Nose Irritation Test Nasal discomfort NaCl Benzalkonium chloride
a b s t r a c t In this research project, the Slug Mucosal Irritation (SMI) assay was applied to predict nasal discomfort, investigating the correlation between responses in slugs and humans. Several SMI experiments and a Human Nose Irritation Test (HNIT) were performed with five NaCl solutions (0.4%, 1.3%, 2.6%, 5.4% and 10.4%) and two benzalkonium chloride solutions (BAC 0.02% and BAC 0.05%). In the HNIT, subjective evaluation of clinical discomfort was performed by 24 participants at several time points. Analyzes reveal that (1) a significant positive association existed between immediate stinging reaction reported by the participants and the mean total mucus production of the slugs (Spearman’s Rank correlation = 0.963, p < 0.001); (2) NaCl 0.4% was best tolerated in both tests; (3) a concentration-response effect was observed for NaCl and BAC solutions; (4) NaCl 10.4% induced the highest mucus production in the slugs and received higher sting scores for immediate discomfort in the HNIT; (5) stinging sensations decreased rapidly in time and (6) based on these results a new classification prediction model for nasal applications was established. In conclusion, the SMI assay is a promising evaluation method for clinical nasal discomfort. Screening (prototype) formulations with this assay allows formula optimization prior to a clinical trial. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The olfactory receptors are important for the perception of smell, but the nose also serves as an efficient humidifier, heater and filter for inhaled air, thereby protecting the lower airways (Bousquet et al., 2001). Consequently, the mucosa lining the sinonasal cavity is constantly exposed to unconditioned and occasionally polluted inhaled air which may cause discomfort, irritation, sneezing, reflex-mediated hyper-secretion and nasal blockage (Bousquet et al., 2001). The protection against invading microorganisms by the nasal mucosa includes an intact mucosal barrier, mucociliary transport and clearance, and mucosal immunity (Mygind et al., 1990). Defects in any of these defense mechanisms may increase the susceptibility to infection (Bjerknes and Steinsvåg, 1993). Nasal spray formulations may be applied in the treatment of many nasal diseases. Unfortunately adverse events may occur, in some cases affecting patient’s compliance, which describes the ⇑ Corresponding author. Tel.: +32 92648016; fax: +32 92228236. E-mail addresses: [email protected] (J. Lenoir), [email protected] (C. Bachert), [email protected] (J.-P. Remon), [email protected] (E. Adriaens). 0887-2333/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tiv.2013.06.018
degree to which a patient correctly follows medical advice, a prescribed treatment or preventive procedure. Certain nasal pathologies require a prolonged therapy with pharmaceutical formulations. Non-compliance of the medications, which may be assigned to the pH or osmolality of the formulation, a certain active ingredients or preservative, affects the outcome of the treatment. Evaluating the irritation potential of (nasal medical) formulations is essential to their safety assessment. The concept of the three Rs (refinement, reduction and replacement of laboratory animals) (Russell and Burch, 1959) strongly stimulates the development of alternative testing methods, such as in vitro methods and the use of ‘‘lower’’ organisms as test species (e.g. invertebrates, plants and microorganisms) (Balls et al., 1995). Keeping this concept in mind, the Slug Mucosal Irritation (SMI) assay was developed and has proven its value for different applications. It has also demonstrated a relation between an increase in mucus production in slugs and an elevated incidence of Stinging, Itching and Burning (SIB) sensations in humans (Adriaens and Remon, 2008; Lenoir et al., 2011, 2012). In the past, different in vitro tests have been applied to investigate the effect of for example saline solutions (e.g. Boek et al., 1999a,b; Harkema, 1990), but none was able to predict clinical discomfort. The SMI assay might be a
J. Lenoir et al. / Toxicology in Vitro 27 (2013) 1954–1961
possible method to pretest nasal spray and drop formulations to screen for adverse events. The objective and subjective efficacy of saline irrigation for the treatment of sinonasal disease has been demonstrated and saline irrigations are increasingly used (Rabago et al., 2006). This procedure has the advantages to be simple to execute, inexpensive and is generally well tolerated (Osguthorpe and Hadley, 1999; Tomooka et al., 2000; Shoseyov et al., 1998). By flushing the nasal cavity with a saline solution, it provides short-term relief of congestion by moisturizing the nasal cavity, reducing dryness, and helping to clear encrusted material and secretions (Fagnan, 1998; Osguthorpe and Hadley, 1999). All these effects improve the mucociliary clearance (Benninger et al., 1997; Talbot et al., 1997), which is essential as a transport mechanism for foreign particles entrapped in the mucus layer lining the sinonasal cavity. Moreover, the use of hypertonic solution may reduce edema of the nasal mucosa (Fagnan, 1998), through diffusion of osmolar gradients (Talbot et al., 1997). Saline solutions are available on the market in different tonicities and pH levels. It has been well-described that hypertonic saline causes clinical nasal discomfort to a greater extent than physiological saline (Baraniuk et al., 1999; Hauptman and Ryan, 2007; Adam et al., 1998; Shoseyov et al., 1998). Benzalkonium chloride (BAC) is often used as a preservative in multi-dose topical aqueous nasal drop and spray formulations. It prevents bacterial contamination and maintains the safety of the preparations (Graf, 2005; Graf et al., 1995; Graf, 1999; Marple et al., 2004; Steinsvåg et al., 1996). However, it has been suggested that the presence of BAC in a formulation may induce severe morphological and histological changes, both in vitro and in vivo (e.g. Berg et al., 1995; Cüreog˘lu et al., 2002; Kuboyama et al., 1997; Lebe et al., 2004; Riechelmann et al., 2004), and in vivo nasal irritation, hypersecretion and a burning sensation (Riechelmann et al., 2004). In this research several saline and BAC solutions were first extensively tested with the 1-day repeated exposure protocol of the SMI assay, focusing on the prediction of short term sensations (Stinging, Itching and Burning (SIB) test). Later, the same formulations were clinically evaluated in a Human Nose Irritation Test (HNIT). By this means, we were able to investigate whether this protocol can be applied to evaluate the clinical irritation potential of nasal formulations. The main goal was to investigate the correlation between the slugs and humans for nasal applications and set up a new classification prediction model for nasal formulations. 2. Materials and methods 2.1. Materials A phosphate buffered saline (PBS; isotonic and pH neutral; 0.4% NaCl, 1.0% Na2HP042H20, 0.2% KH2PO4), as well as four NaCl solutions (1.3%, 2.6%, 5.4%, 10.4% (w/v)) and two benzalkonium chloride (BAC) solutions (0.02% and 0.05% (w/v)) were prepared in PBS and tested with the SIB protocol and the HNIT. All these solutions were formulated by the Ghent University Hospital pharmacy. In the SMI experiments a 1% (w/v) dilution of BAC in PBS (delivered by Sigma, St. Louis, MO, USA) was added as a positive control. 2.2. Methods 2.2.1. Osmolality measurements The osmolality of all the formulations was determined using an Advanced Micro Osmometer (Model 3300 Advanced Instruments Inc., Norwood, MA, US) by the freezing-point method. Clinitrol™ 290 was used as reference solution (Advanced Instruments). The measurements were performed in triplicate (on 20 lL aliquots) and mean values were used for analysis. Results are represented as mOsm/kg.
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2.2.2. Stinging, Itching and Burning (SIB) test procedure The slug species used in this assay is Arion lusitanicus. The slugs are bred and kept under laboratory conditions in an air-conditioned room (18–20 °C). The effect of the selected formulations, negative (PBS – NaCl 0.4%) and positive controls (BAC 1%) on the mucosal tissue of the slugs was investigated with this protocol, described into detail in Lenoir et al. (2011). For each treatment group, three slugs are used. The specimens are not previously used in other experiments and are checked beforehand for any damage. They are brought into contact with 100 ll of the test substances during three 15-min contact periods (CPs). The body weight of the slugs and amount of mucus secreted by the slugs are quantified by weighing the Petri dishes at the beginning and end of each CP. In between the CPs there is one hour rest/recovery period during which the slugs are transferred to a fresh Petri dish and 1.5 ml PBS is added to prevent dehydration. Classification of the test substances is established calculating the average of the total mucus production (MP) during the three CPs of the three replicates, expressed as % (w/w) of the initial body weight of the slugs. 2.2.3. Human Nose Irritation Test The clinical trial was set up as a single-blind cross-over study with 24 participants and was organized in cooperation with the Department of Rhino-otolaryngology of the Ghent University Hospital. The protocol was reviewed and approved by an independent Commission for Medical Ethics, associated with the Ghent University Hospital, Belgium. Prior to enrollment, all participants read, understood and voluntary signed the informed consent document. 2.2.3.1. Study population. Twenty-four adult participants were recruited in the region of Ghent, Belgium. The study group consisted of 12 males and 12 females between 18 and 37 years, with an average age of 27 years. 2.2.3.2. Methodology. Seven sessions were organized. In every session approximately 10 ll (two sprays) of one of the test materials was administered at once in each nostril by the participants themselves, using a disposable nose spray bottle, which was only used once and then discarded. There were at least 2 days in between sessions to minimize the carry-over effect from previous sessions. 2.2.3.3. Measurements and observations. All subjects were instructed to report any nasal stinging, itching or burning sensation or reaction. A self evaluation was carried out by the participants, by means of a scoring table. The participants were able to express the incidence and score the intensity of nasal discomfort, starting 30 s after administration and continuing 1, 2, 5, 10, 15 and 30 min later. A grading scale from 0 to 5 was used to indicate the SIB potency of the tested substances (0 = no; 1 = very mild; 2 = mild; 3 = moderate; 4 = severe; 5 = very severe). 2.2.4. Data analyses The reproducibility of the SMI test was evaluated with a twoway ANOVA to investigate the run effect and test substance effect on the total mucus production. The normality of the residuals was investigated with a qq-plot. The homogeneity of variances was tested with the Levene’s test. If no significant interaction was present, the main effects were interpreted separately from the interaction and a multiple comparison among pairs of means was performed using a Scheffé post hoc test. If a significant interaction between both factors was present, a Bonferroni post hoc test was used to investigate if there were significant differences between the runs for each test substance. The effect of the different NaCl and BAC solutions on the intensity of the discomfort score and scores given by the participants was evaluated by means of descriptive statistics. The Spearman
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Rank correlation coefficient was calculated as a measure of association between the immediate discomfort observed by the participants and the endpoint of the SMI test. In order to develop a prediction model for the nasal stinging potency of formulations based on the total mucus production observed in the SMI assay, classes were assigned to the test formulations based on the discomfort scores observed in the volunteers. In a next step, classification tree analyses with bootstrap resampling was used to determine cut-off values for the total mucus production that distinguish between the different discomfort classes (rpart package running under R version 2.15.2). A value of p < 0.05 was considered statistically significant. All analyses were performed in SPSS version 21.0 and R version 2.15.2. 3. Results 3.1. Stinging, Itching and Burning (SIB) test The SIB procedure was used to evaluate the relative SIB potential of five NaCl and two BAC formulations in PBS. For this part of the study, a positive control was added in each run as a reference (BAC 1%). Experiments were repeated five times to investigate the reproducibility of this testing method. The total MP per test substance per experiment is presented in Table 1. A two-way ANOVA showed a significant treatment effect (p < 0.001), but no significant run effect (p = 0.563) and no significant interaction between these two factors (p = 0.947). The main effect of the treatment was interpreted and the Scheffé post hoc test suggested five subsets for the total amount of mucus secreted: (1) NaCl 0.4%, NaCl 1.3% and BAC 0.02% (p = 0.492), (2) NaCl 1.3%, NaCl 2.6%, BAC 0.02% and BAC 0.05% (p = 0.523), (3) NaCl 5.4%, (4) NaCl 10.4% and (5) BAC 1% (positive control). As an overall trend we could observe that with increasing concentrations of NaCl, also resulting in increasing osmolality of the formulations, resulted in a higher total mucus production of the slugs. Increasing concentrations of BAC resulted in an increased mucus production unrelated to osmolality, but due to its cytotoxic effect (Adriaens et al., 2001). 3.2. Human Nose Irritation Test The clinical stinging potential of the formulations was determined by spraying the same formulation twice in each nostril of the participants and scoring the occurrence and intensity of the experienced discomfort. In Fig. 1 the proportion of participants for the different scores of discomfort is represented for the
different NaCl solutions in function of time. It was observed that all reported SIB sensations decreased over time and that the higher the scores directly after administration, the higher the scores were in the course of time post administration. However, after 30 min, all participants only gave a score 0 or 1 to all tested formulations. Of all NaCl solutions tested, the participants tolerated NaCl 0.4% the best. Seventeen participants ranked it as least discomfort inducing NaCl formulation. Thirteen participants (54.2%) never felt discomfort when it was administered. Nine participants (37.5%) reported very mild or mild stinging sensations (score 1 or 2), while two (8.5%) experienced moderate stinging after 30 s. Five minutes post administration, it still received a score 2 from one participant, while all others did not feel any or only very mild sensations. After 30 min, none of the participants reported discomfort anymore. Directly after spraying NaCl 1.3% into the nose, the majority of the participants did not feel any discomfort (15/24 – 62.5%), while nine participants reported very mild or mild stinging (37.5%). After 5 min, the scores were even lower, with all participants reporting either no (20/24 – 83.5%) or very mild (4/24 – 16.5%) sensations. After 10 min, two participants (8.5%) still gave a score 1 (very mild stinging), while 30 min after administration this was only the case for one participant. Remarkably this participant did not indicate any stinging sensations at the earlier time points. All other participants did not report any discomfort at all after 15 min. For NaCl 2.6% a different scoring behavior was observed. Directly after administration only eight participants (33.5%) gave it a score 0, while 12 scored it as causing very mild or mild discomfort (50%). Three (12.5%) reported moderate sensations, while one participant even sensed severe discomfort. After 5 min, 13 participants (54%) did not feel anything anymore, while ten (41.5%) now reported either very mild or mild sensations, one participant still experienced moderate discomfort. Five minutes later, 19 people (79%) did not sense any stinging anymore, while five participants (21%) still felt very mild or mild sensations. After 30 min, 22 participants (91.5%) scored 0, while two participant (8.5%) still experienced very mild stinging. The participant reporting severe discomfort directly after administration, did not feel any discomfort after 30 min. Higher stinging scores were given to NaCl 5.4% in comparison to the lower NaCl concentrations. Directly after spraying it into the nose, all of the participants reported some degree of discomfort. Nine people (37.5%) experienced very mild or mild sensations, six (25%) indicated moderate discomfort, and nine (37.5%) felt severe or very severe sensations. After 5 min the distribution of the scores shifted substantially: already five participants (21%) did not report any stinging anymore, the majority (16/24 – 66.5%) reported only very mild or mild discomfort, while three participants
Table 1 Effect of a repeated exposure (three 15-min contact periods on the same day) to 100 ll of several NaCl and BAC solutions in PBS, with indication of the osmolality of the formulations. Test substance (%)
Osmolality (mOsm/kg)
NaCl 0.4 NaCl 1.3 NaCl 2.6 NaCl 5.4 NaCl 10.4 BAC 0.02 BAC 0.05 BAC 1 Run effectc
287 ± 1.7 552 ± 3.5 995 ± 4.0 1794 ± 1.2 3347 ± 28.3 293 ± 0.6 292 ± 1.7 290 ± 1.2
Total MP (%)a
Treatment effectb
Run 1
Run 2
Run 3
Run 4
Run 5
2.8 ± 0.5 5.0 ± 1.0 7.5 ± 2.2 13.1 ± 1.7 21.5 ± 3.2 3.9 ± 1.1 7.9 ± 0.6 30.5 ± 6.7 11.5 ± 9.8
3.1 ± 0.4 5.5 ± 0.5 6.0 ± 1.6 13.0 ± 2.0 24.5 ± 2.3 4.7 ± 0.6 6.9 ± 2.6 26.9 ± 8.3 11.3 ± 9.4
1.4 ± 0.3 3.2 ± 1.4 6.0 ± 1.5 12.1 ± 1.8 22.2 ± 1.8 4.6 ± 1.6 6.9 ± 0.6 27.6 ± 7.5 10.5 ± 9.5
1.2 ± 0.5 3.9 ± 0.6 6.6 ± 1.5 12.0 ± 2.0 19.8 ± 2.3 3.9 ± 0.4 5.3 ± 2.4 31.7 ± 4.3 10.6 ± 10.4
2.2 ± 1.5 5.4 ± 1.9 9.0 ± 0.6 14.9 ± 2.4 25.8 ± 5.1 6.0 ± 1.1 7.5 ± 2.2 28.9 ± 1.1 12.5 ± 9.9
MP: mucus production; PBS: phosphate buffered saline; BAC: benzalkonium chloride. a Total MP presented as the mean ± standard deviation (n = 3). b Total MP presented as the mean ± standard deviation (n = 15). c Total MP presented as the mean ± standard deviation (n = 24).
2.1 ± 0.8 4.6 ± 1.0 7.0 ± 1.2 13.0 ± 1.2 22.7 ± 2.4 4.6 ± 1.2 6.9 ± 1.8 29.1 ± 2.0 11.3 ± 9.7
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No discomfort (score 0)
80
80
60
% Participants
100
% Participants
100
NaCl 0.4% NaCl 1.3%
40
NaCl 2.6% NaCl 5.4%
20
(Very) Mild discomfort (score 1+2)
60 40 20
NaCl 10.4% 0
0 0
5
10
15 20 Time (min)
25
0
30
Moderate discomfort (score 3)
100
10
15 20 Time (min)
25
30
(Very) Severe discomfort (score 4+5)
100
80
80 % Participants
% Participants
5
60 40 20
60 40 20
0
0 0
5
10
15 20 Time (min)
25
30
0
5
10
15 20 Time (min)
25
30
Fig. 1. Percentage of the participants reporting the different classes of discomfort (n = 24).
(12.5%) experienced moderate stinging. No one experienced severe sensations anymore. Five minutes later, the scores even dropped more. Fourteen (58.5%) did not feel any adverse sensation anymore. Nine participants (37.5%) experienced very mild to mild sensation, while one still reported moderate stinging. Thirty min post administration, 21 participants (87.5%) reported no discomfort at all, while three (12.5%) still had very mild stinging sensations. These latter had indicated severe sensations earlier on. As expected, NaCl 10.4% received the highest scores of all tested formulations. As for NaCl 5.4%, all participants experienced stinging sensations. Directly after administration, majority indicated severe (7/24 – 29%) or even very severe (9/24 – 37.5%) discomfort, three particpants (12.5%) experienced moderate sensations, while five participants (21%) only felt very mild to mild sensations. After 5 min, the number of participants not feeling any discomfort increased to two (8.5%). The majority (16/24 – 66.5%) now sensed very mild to mild stinging, two (8.5%) had moderate sensations, while four (16.5%) still reported severe or very severe sensations. Ten min after application, the majority either felt no discomfort at all (11/24 – 46%) or very mild to mild sensations (10/24 – 41.5%). Two people (8.5%) reported moderate sensations, while one still experienced severe stinging. After 30 min, 21 participants (87.5%) scored 0, while three (12.5%) still had very mild stinging sensations. These latter indicated very severe sensations earlier on. Total sting scores were higher than for NaCl 5.4% in 19/24 participants. In Fig. 2 the percentage of participants reporting either no discomfort or (very) mild sensations in the course of time is presented for the same formulations. Directly after administration BAC 0.02% induced very mild to mild sensations in seven participants (29%), while all others reported no discomfort at all. After 5 min, all participants reported either no (22/24 – 91.5%) or very
mild (2/24 – 8.5%) discomfort, while after 10 min only one participant experienced very mild discomfort. After 30 min, nobody indicated any stinging anymore. For BAC 0.05%, 11 participants (46%) experienced no discomfort, while 12 (50%) had very mild to mild sensations, and one indicated moderate stinging directly after administration. After 5 min, 13 participants (54%) did not report any sensations, while the others (11/24 – 46%) sensed either very mild or mild stinging. Ten min post administration, 15 participants did not experience any discomfort anymore (62.5%) while nine had very mild or mild sensations (37.5%). At the last scoring time, 20 participants (83.5%) did not feel any discomfort, but four (16.5%) still experienced very mild sensations. 3.3. Correlation SMI – HNIT The results of the clinical trial indicated that none of the formulations induced discomfort for longer than 30 min post-instillation or only very mild sensations were reported by the participants. The clearest differentiation between the tested formulations was observed immediately after instillation (30 s and 1 min post-instillation). Therefore, the data for immediate stinging were used to investigate the correlation with the results in slugs after three 15 min contact periods. A significant positive association was found between the immediate stinging reaction observed by the panelists and the total amount of mucus produced by the slugs (Spearman’s Rank correlation = 0.963, p < 0.001). Classes were assigned to the test formulations if at least 50% of participants indicated a certain score: no = score 0, mild = score 1 and 2, moderate = score 3, severe = score 4 and 5. In the clinical trial, NaCl 0.4% was generally well tolerated by the participants, with the majority of the participants scoring 0
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No discomfort (score 0)
80
80
% Participants
100
% Participants
100
60 NaCl 0.4% 40
NaCl 1.3%
(Very) Mild SIB (score 1 + 2)
60 40
BAC 0.02% BAC 0.05%
20
20
0
0 0
5
10
15
20
25
30
0
5
10
15
20
25
30
Time (min) Fig. 2. Percentage of the participants reporting no, very mild or mild discomfort at the different time points (n = 24).
immediately after administration (score 0 in 13 and 19 (54% and 79%) participants after 30 s and 1 min respectively) (Fig. 1). Severe or very severe discomfort was never observed. Based on the scores given by the participants this formulation was assigned the class of causing no discomfort. Slugs only showed on average a low MP (total MP = 2.1 ± 0.8%). For NaCl 1.3% a similar reaction was seen, although more participants experienced very mild to mild discomfort (score 1 or 2) after administration compared to NaCl 0.4%. Nine and ten participants scored a 1 or 2 after 30 s and 1 min respectively compared to nine and four at the same time points for NaCl 0.4%. However, the majority scored 0 immediately after administration (62.5%), being assigned the class of no discomfort as well. The slugs showed on average a more than doubled total MP compared to NaCl 0.4% (4.6 ± 1.0% compared to 2.1 ± 0.8% for NaCl 0.4%). When administrating NaCl 2.6% in their nose, the majority of the participants reported very mild to mild sensations, score 1 or 2 in 12 and 13 participants after 30 s and 1 min (50% and 54% respectively); score 3 in three participants at these time points (12.5%) and even one participant with a score 4 at both time points (4%). Based on this scoring behavior the class mild was assigned, since 50% or more indicated either a score 1 or 2 immediately after application. In the SIB test exposure to this formulation resulted on average in a total MP of 7.0 ± 1.2%. The effect of exposure to higher concentrations of NaCl was more pronounced in both slugs and humans. When NaCl 5.4% was tested, all the participants experienced some kind of discomfort shortly after administration (none of the participants scored 0). The majority of the participants either reported mild, moderate, or severe discomfort (9 – 6 – 9 (37.5% – 25% – 37.5% respectively) at 30 s and 11 – 4 – 9 (46% – 17% – 37.5% respectively) at 1 min). This formulation was assigned to the class of moderate discomfort, because the scores were distributed quite evenly around this score. In the slugs this formulation induced on average a total MP of 13.0 ± 1.2% after three CPs. As for NaCl 5.4%, NaCl 10.4% induced discomfort in all participants (score 0 by none of the participants), with the majority indicating severe or even very severe stinging directly after administration (score 4 in seven participants and score 5 in nine after 30 s; score 4 and 5 both by seven participants after 1 min). Not only more high scores (scores 4 or 5) were given directly after administration compared to any other formulation tested, they also last longer (up to 10 min after administration for NaCl 10.4%, up to 2 min after administration for NaCl 5.4%). Based on these results the class of inducing severe discomfort was assigned to this solution. Testing it in slugs resulted in a highly increased total MP of 22.7 ± 2.4%, confirming the trend observed in humans.
The lowest BAC concentration tested (0.02%) was generally well tolerated, with 17 and 21 participants (71% and 87% respectively) reporting no discomfort after 30 s and 1 min respectively. Based on these results BAC 0.02% was assigned to the class of causing no discomfort. Total MP of the slugs was slightly increased compared to the NaCl 0.4% formulation (4.6 ± 1.2% compared to 2.1 ± 0.8%), but appeared not to be statistically significant (p = 0.492). For BAC 0.05% more than 50% of the participants reported some kind of discomfort immediately after administration (score 0 by only 11 participants, score 1 or 2 by 12 participants, score 3 by one participant after 30 s; score 0 by only 12 participants, score 1 or 2 by 11 participants, score 3 by one participant after 1 min). A statistically significant higher total MP was observed in the slugs exposed to BAC 0.05% compared to the lower concentration of BAC (6.9 ± 1.8% compared to 4.6 ± 1.2% for BAC 0.02%). Based on the scores given by the participants, this formulation was assigned to the class of causing mild discomfort. In order to determine threshold values for the total MP to distinguish between the four irritation categories, a classification tree with bootstrap resampling was used. This resulted in the following threshold values: total MP 6 5.5% corresponds with no discomfort, between 5.5% and 610% corresponds with mild discomfort, between 10% and 617.5% corresponds with moderate discomfort and a total MP > 17.5% is indicative for severe discomfort. Fig. 3 nicely illustrates the correspondence between the results in slugs and humans, with indication of the new threshold values for the different categories. 4. Discussion Previous research of our lab about nasal discomfort described a correlation between results in slugs and clinical results found in literature (Lenoir et al., 2011). Since comparative studies between in vivo and in vitro tests on mucosal discomfort are scarce, this research project focused on the comparison of the mucosal tolerance towards several nasal NaCl and BAC solutions with the 1-day protocol of the SMI assay vs. the human nasal irritation test in 24 participants. By this means, we aimed to investigate whether the SIB test can replace the HNIT to evaluate and predict clinical nasal discomfort to different irritants. Analyzes indicated a good correlation between results of the SIB-test and subjective human nasal responses from the HNIT, especially for the immediate sensation (scores given after 30 s and 1 min, Spearman’s Rank correlation = 0.963, p < 0.001). The general approach in product development when screening with the SIB test should be to deliver a product with a mean total MP as low as possible.
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Fig. 3. Relationship between the results in slugs and in humans induced by exposure to several NaCl and BAC (w/v) solutions (Spearman’s Rank correlation = 0.963, p < 0.001). Representing: (A) sting scores given by the participants 30 s and 1 min after administration of the formulations (n = 24); (B) total mucus production (MP) of the slugs after three 15-min contact periods (all data of five different experiments, each with n = 3) (cut-off between no and mild discomfort = 5.5%); cut-off between mild and moderate discomfort = 10%; cut-off between moderate and severe discomfort = 17.5%).
4.1. Effect of osmolality According to several authors, nasal irrigation with (hypertonic) saline decongests the swollen membranes and may also improve mucociliary transport (Druce, 1990; Parsons, 1996; Slavin, 1991; Talbot et al., 1997). Several saline solutions with concentrations up to 2.3% NaCl are available as over the counter nasal sprays. Generally, these are well tolerated, although the use of hypertonic nasal saline elicits more burning and irritation compared with physiological nasal saline (Adam et al., 1998; Baraniuk et al., 1999; Hauptman and Ryan, 2007). Our results confirm this trend. In the study of Adam et al. (1998) for example, a clinical trial was performed with a normal and a hypertonic (2%; buffered) saline solution. In the group receiving the latter formulation, 32% reported nasal irritation, compared to 13% in the control group. In the current clinical trial these percentages were very similar (38% and 13% respectively). Additionally, in the study of Shoseyov et al. (1998), a 3.5% saline solution was tested in 30 children with chronic sinusitis three times daily for four weeks. Regarding tolerability of the treatment, the patients in the hypertonic saline group reported burning and itching in the nose more than those in the normal saline group, but these sensations vanished over time (reported only during the first 3–4 days). After that period, there was no difference between the groups and no problems were reported anymore. Although the current study only tested the effects of a single application, similar trends were observed: the higher the NaCl concentration, the higher the scores for discomfort, but these reduced quickly in time. After spraying NaCl 10.4% into their nose, all participants reported some degree of discomfort, while for NaCl
0.4% this was 8%, for NaCl 1.3% this was 13%, for NaCl 2.6% this was 39% and for NaCl 5.4% this was 88%. No substantial stinging was reported anymore after 2 min for NaCl 1.31%, after 10 min for NaCl 0.4%, after 15 min for NaCl 2.6% and 5.4%. For NaCl 10.4% this was the case only after 30 min. Baraniuk et al. (1999) tested concentrations of NaCl up to 21.6% against a control (NaCl 0.9%). The hypertonic saline solutions induced a burning sensation within the first ten sec after application, but faded in time, as was observed in the current study. Pain intensity became significantly different from NaCl 2.7% and increased in a dose-dependent fashion. Comparing our results with results found in literature, we may conclude that although generally safe, (daily) hypertonic nasal irrigation may be associated with some minor clinical side effects. Also, the higher the concentration of NaCl in the formulation, the higher the risk will be for clinical discomfort. 4.2. Effect of BAC Benzalkonium chloride (BAC) is often used as a preservative in multi-dose topical aqueous nose drop and spray formulations. It prevents bacterial contamination and maintains the safety of the preparations (Graf, 1999, 2005; Graf et al., 1995; Marple et al., 2004; Steinsvåg et al., 1996), by damaging the cell wall of microorganisms and altering its permeability (Richards and Cavill, 1976). In marketed multi-dose nasal products, BAC is usually added in concentrations of 0.005–0.02% (w/v) (Riechelmann et al., 2004; Verse et al., 2003), while the American College of Toxicology concluded that BAC can be used safely in a concentration
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up to 0.1% (w/v). In the current study, BAC concentration of 0.02% and 0.05% were selected because they fall within this range. It has been suggested that the presence of BAC in a formulation may induce severe morphological and histological changes, both in vitro (human epithelial cell studies) and in vivo in animal studies (e.g. Berg et al., 1995; Cüreog˘lu et al., 2002; Kuboyama et al., 1997; Lebe et al., 2004; Riechelmann et al., 2004), although other in vivo studies in human patients have demonstrated that BAC-preserved intranasal products were safe and well-tolerated for long- and short-term clinical use (Marple et al., 2004). Results from the SMI tests reveal a statistically significant concentration-response effect, which has also been described by Berg et al. (1995), Lebe et al. (2004) and Marple et al. (2004). In slugs, high concentrations of BAC result in a very high MP (positive control of BAC 1% induced a mean total MP of 29.1 ± 2.0%), while for lower concentrations of BAC the MP was much lower (average total MP of BAC 0.02% = 4.6 ± 1.2%; BAC 0.05% = 6.9 ± 1.8%). Compared to the negative control, this increased MP in lower concentrations of BAC is presumably induced by stinging or burning sensations. In the study of Riechelmann et al. (2004) the adverse effects of BAC on human nasal mucosa was evaluated, both in vitro and in vivo. In vitro, 0.01% and 0.025% BAC appeared to be ciliotoxic for human nasal respiratory epithelia from healthy, non-allergic volunteers. In vivo however, this ciliotoxic effect was not observed: there was no interference with nasal mucus transport and it did not induce nasal inflammatory alterations compared to the placebo. However, directly after application, participants exposed to the BAC 0.05% solution reported nasal irritation, hypersecretion and a burning sensation. Moreover, symptom scores for persistent nasal irritation were higher for BAC than the placebo (Ringer’s solution). In the current study, we also found higher stinging scores for BAC 0.05% compared to the 0.02% solution, and the duration of the sensations was also longer (10 min compared to 1 min). 4.3. Sensitivity of participants Substantial variation in the subjective self-evaluation by the participants was observed, as was the case in previous research of our group (Lenoir et al., 2012). The scoring behavior is amongst other factors influenced by the participants’ sensitivity. Despite the small number of participants in the current clinical trial, it is possible to categorize people into different sensitivity groups. Seven participants (5 males + 2 females) were clearly less sensitive than the others. However, they were able to differentiate between the higher concentration of NaCl (NaCl 5.4% and 10.4%) and the lower concentrations NaCl and BAC (NaCl 1.3% and 2.6%; BAC 0.02% and 0.05%). Three of them (2 males + 1 female) only reported stinging sensations for NaCl 5.4% and 10.4%, but scored 0 for all other formulations at all time points. The other four gave low scores overall, but scores slightly increased with increasing NaCl or BAC concentration, except one who gave the BAC 0.02% a total sting score of 1 and BAC 0.05% a total sting score of 0. Another group (2 males + 3 females) was able to make a clearer difference between the tested formulations, with much higher scores for NaCl 5.4% and 10.4% in comparison with the first group. The rest (12/24 – 50%) showed an intermediate scoring behavior: they were generally able to differentiate between lower and higher concentrations of NaCl, but some had difficulties distinguishing between NaCl 5.4% and 10.4% and also between BAC 0.02% and 0.05% (4/24 and 5/24 respectively). 4.4. New cut-off values for nasal discomfort A good correlation was obtained between results of the SIB test and subjective human nose responses from the HNIT, especially for the immediate sensations (scores given after 30 s and 1 min). In
Table 2 New cut-off values for the different categories of nasal discomfort. Degree of discomfort
Total mean MP (%) Ocular (%)
Nasal (%)
No Mild Moderate Severe
<3 3–8 8–15 >15
<5.5 5.5–10 10–17.5 >17.5
Classification formulations in current study
NaCl NaCl NaCl NaCl
0.4%, NaCl 1.3%, BAC 0.02% 2.4%, BAC 0.05% 5.4% 10.4%, BAC 1%
MP: mucus production expressed as a % of the initial body weight of the slugs; BAC = benzalkonium chloride.
the past, this correlation was already found for ocular discomfort induced by surfactant-based personal care product and a classification prediction model was developed (Lenoir et al., 2012; Table 2). However, results of the HNIT were not in agreement with these. Since a difference in sensitivity exists between the ocular tissue and nasal mucosae, we suggest new cut-off values for nasal applications taking into account the results of the current clinical trial. By this means, a more accurate prediction of clinical reactions can be made. The general approach in product development when screening with the SIB protocol should be to deliver a nasal formulation with a mean total MP less than 5.5%. 5. Conclusions This study has shown that the recently developed SIB protocol is a quick, accurate and reproducible screening method for the prediction of immediate nasal stinging, itching and/or burning responses in the human nose containing the excipients NaCl and benzalkonium chloride. Since a strong positive association was found between the results in slugs and in humans, the test allows formula optimization prior to product development investment in clinical trials. It is recommended that when a nasal formulation is tested with this protocol, the total amount of mucus secreted by the slugs after three CPs should be lower than 5.5%. The higher the MP of the slugs, the higher the potential for clinical SIB sensations. When exceeding 10%, it is suggested to reduce the irritability of the formulation, in which active ingredients, preservatives and osmolality play an important role. Conflict of Interest All authors state that there is no actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations within 3 years of beginning the submitted work that could inappropriately influence, or be perceived to influence, their work. References Adam, P., Stiffman, M., Blake, R., 1998. A clinical trial of hypertonic saline nasal spray in subjects with the common cold or rhinosinusitis. Arch. Fam. Med. 7, 39–43. Adriaens, E., Remon, J.P., 2008. Mucosal irritation potency of personal lubricants relates to product osmolality as detected by the slug mucosal irritation assay. Sex. Transm. Dis. 35 (5), 512–516. Adriaens, E., Dierckens, K., Bauters, T.G.M., Nelis, H.J., van Goethem, F., Vanparys, Ph., Remon, J.P., 2001. The mucosal toxicity of different benzalkonium chloride analogues evaluated with an alternative test using slugs. Pharm. Res. 18 (7), 937–942. Balls, M., Goldberg, A., Fentem, J., Broadhead, C., Burch, R., et al., 1995. The three Rs: the way forward. The report and recommendations of ECVAM workshop 11. ATLA 23, 838–866. Baraniuk, J., Ali, M., Yuta, A., Fang, S., Naranch, K., 1999. Hypertonic saline nasal provocation stimulates nociceptive nerves, substance P release, and glandular
J. Lenoir et al. / Toxicology in Vitro 27 (2013) 1954–1961 mucous exocytosis in normal humans. Am. J. Respir. Crit. Care Med. 160, 655– 662. Benninger, M., Anon, J., Mabry, R., 1997. The medical management of rhinosinusitis. Otolaryngol. Head Neck Surg. 117, S41–S49. Berg, Ø., Henriksen, R., Steinsvåg, S., 1995. The effect of a benzalkonium chloridecontaining nasal spray on human respiratory mucosa in vitro as a function of concentration and time of action. Pharmacol. Toxicol. 76, 245–249. Bjerknes, R., Steinsvåg, S., 1993. Inhibition of human neutrophil actin polymerization, phagocytosis and oxidative burst by components of decongestive nosedrops. Pharmacol. Toxicol. 73, 41–45. Boek, W., Keles, N., Graamans, K., Huizing, E., 1999a. Physiologic and hypertonic saline solutions impair ciliary activity in vitro. Laryngoscope 109 (3), 396–399. Boek, W., Romeijn, S., Graamans, K., Verhoef, C., Merkus, F., Huizing, E., 1999b. Validation of animal experiments on ciliary function in vitro II. The Influence of absorption enhancers, preservatives and physiologic Saline. Acta Otolaryngol. 119, 98–101. Bousquet, J., van Cauwenberge, P., Khaltaev, N., Aït-Khaled, N., Annesi-Maesano, I., et al., 2001. Allergic rhinitis and its impact on asthma. J. Allergy Clin. Immunol. 108 (Suppl. 5), S147–S334. Cüreog˘lu, S., Akkusß, M., Osma, Ü., Yaldiz, M., Oktay, F., et al., 2002. The effect of benzalkonium chloride on rabbit nasal mucosa in vivo: an electron microscopy study. Eur. Arch. Otorhinolaryngol. 259, 362–364. Druce, H.M., 1990. Adjuncts to medical management of sinusitis. Otolaryngol. Head Neck Surg. 103, 880–883. Fagnan, L.J., 1998. Acute sinusitis: a cost-effective approach to diagnosis and treatment. Am. Fam. Physician 15, 1795–1802. Graf, P., 1999. Adverse effects of benzalkonium chloride on the nasal mucosa: allergic rhinitis and rhinitis medicamentosa. Clin. Ther. 21 (10), 1749–1755. Graf, P., 2005. Rhinitis medicamentosa. A review of causes and treatment. Treat. Respir. Med. 4 (1), 21–29. Graf, P., Hallén, H., Juto, J.E., 1995. Benzalkonium chloride in a decongestant nasal spray aggravates rhinitis medicamentosa in healthy volunteers. Clin. Exp. Allergy 25, 395–400. Harkema, J., 1990. Comparative pathology of the nasal mucosa in laboratory animals exposed to inhaled irritants. Environ. Health Perspect. 85, 231–238. Hauptman, G., Ryan, M., 2007. The effect of saline solutions on nasal patency and mucociliary clearance in rhinosinusitis patients. Otolaryngol. Head Neck Surg. 137, 815–821. Kuboyama, Y., Suzuki, K., Hara, T., 1997. Nasal lesions induced by intranasal administration of benzalkonium chloride in rats. J. Toxicol. Sci. 22 (2), 153–160. Lebe, E., Baka, M., Yavasßog˘lu, A., Aktug˘, H., Atesß, U., et al., 2004. Effects of preservatives in nasal formulations on the mucosal integrity: an electron microscopic study. Pharmacology 72, 113–120.
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Lenoir, J., Adriaens, E., Remon, J.P., 2011. New aspects of the slug mucosal irritation (SMI) assay: predicting nasal stinging, itching and burning sensations. J. Appl. Toxicol. 31, 640–648. Lenoir, J., Claerhout, I., Kestelyn, Ph., Klomp, A., Remon, J.P., Adriaens, E., 2012. The slug mucosal irritation (SMI) assay: development of a screening tool for the evaluation of ocular discomfort caused by shampoos. Toxicol. in Vitro 25, 1919– 1925. Marple, B., Roland, P., Benninger, M., 2004. Safety review of benzalkonium chloride used as a preservative in intranasal solutions: an overview of conflicting data and opinions. Otolaryngol. Head Neck Surg. 130, 131–141. Mygind, N., Pipkorn, U., Dahl, R., 1990. Rhinitis and Asthma: Similarities and Differences. Munksgaard, Copenhagen. Osguthorpe, J., Hadley, J., 1999. Rhinosinusitis: current concepts in evaluation and management. Med. Clin. N. Am. 83 (1), 27–41. Parsons, D.S., 1996. Chronic sinusitis: a medical or surgical disease? Otolaryngol. Clin. N. Am. 29, 1–9. Rabago, D., Barrett, B., Marchand, L., Maberry, R., Mundt, M., 2006. Qualitative aspects of nasal irrigation use by patients with chronic sinus disease in a multimethod study. Ann. Fam. Med. 2006 (4), 295–301. Richards, R., Cavill, R., 1976. Electron microscope study of effect of benzalkonium chloride and edetate disodium on cell envelope of Pseudomonas aeruginosa. J. Pharm. Sci. 65 (1), 76–80. Riechelmann, H., Deutsche, T., Stuhlmiller, A., Gronau, S., Brüner, H., 2004. Nasal toxicity of benzalkonium chloride. Am. J. Rhinol. 18 (5), 291–299. Russell, W.M.S., Burch, R.L., 1959. The Principles of Humane Experimental Technique. Methuen, London, United Kingdom. Shoseyov, D., Bibi, H., Shai, P., Shoseyov, N., Shazberg, G., et al., 1998. Treatment with hypertonic saline versus normal saline nasal wash of pediatric chronic sinusitis. J. Allergy Clin. Immunol. 101, 602–605. Slavin, G., 1991. Management of sinusitis. J. Am. Geriatr. Soc. 39, 212–217. Steinsvåg, S., Bjerknes, R., Berg, Ø., 1996. Effects of topical nasal steroids on human respiratory mucosa and human granulocytes in vitro. Acta Otolaryngol. (Stockh) 116, 868–875. Talbot, A.R., Herr, T.M., Parsons, D.S., 1997. Mucociliary clearance and buffered hypertonic saline solution. Laryngoscope 107, 500–503. Tomooka, L., Murphy, C., Davidson, T., 2000. Clinical study and literature review of nasal irrigation. Laryngoscope 110, 1189–1193. Verse, T., Sikora, C., Rudolph, P., Klöcker, N., 2003. The tolerability of nasal drugs with special regard to preservatives and physico-chemical parameters. Laryngo-Rhino-Otologie 82, 782–789.
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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
The Slug Mucosal Irritation (SMI) assay: A tool for the evaluation of nasal discomfort Joke Lenoir a,⇑, Claus Bachert b, Jean-Paul Remon a, Els Adriaens c a
Laboratory of Pharmaceutical Technology, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium Department of Rhino-Otolaryngology, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium c InvertTox, Bellemdorpweg 95, 9881 Aalter, Belgium b
a r t i c l e
i n f o
Article history: Received 28 February 2013 Accepted 27 June 2013 Available online 9 July 2013 Keywords: Slug Mucosal Irritation assay Human Nose Irritation Test Nasal discomfort NaCl Benzalkonium chloride
a b s t r a c t In this research project, the Slug Mucosal Irritation (SMI) assay was applied to predict nasal discomfort, investigating the correlation between responses in slugs and humans. Several SMI experiments and a Human Nose Irritation Test (HNIT) were performed with five NaCl solutions (0.4%, 1.3%, 2.6%, 5.4% and 10.4%) and two benzalkonium chloride solutions (BAC 0.02% and BAC 0.05%). In the HNIT, subjective evaluation of clinical discomfort was performed by 24 participants at several time points. Analyzes reveal that (1) a significant positive association existed between immediate stinging reaction reported by the participants and the mean total mucus production of the slugs (Spearman’s Rank correlation = 0.963, p < 0.001); (2) NaCl 0.4% was best tolerated in both tests; (3) a concentration-response effect was observed for NaCl and BAC solutions; (4) NaCl 10.4% induced the highest mucus production in the slugs and received higher sting scores for immediate discomfort in the HNIT; (5) stinging sensations decreased rapidly in time and (6) based on these results a new classification prediction model for nasal applications was established. In conclusion, the SMI assay is a promising evaluation method for clinical nasal discomfort. Screening (prototype) formulations with this assay allows formula optimization prior to a clinical trial. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The olfactory receptors are important for the perception of smell, but the nose also serves as an efficient humidifier, heater and filter for inhaled air, thereby protecting the lower airways (Bousquet et al., 2001). Consequently, the mucosa lining the sinonasal cavity is constantly exposed to unconditioned and occasionally polluted inhaled air which may cause discomfort, irritation, sneezing, reflex-mediated hyper-secretion and nasal blockage (Bousquet et al., 2001). The protection against invading microorganisms by the nasal mucosa includes an intact mucosal barrier, mucociliary transport and clearance, and mucosal immunity (Mygind et al., 1990). Defects in any of these defense mechanisms may increase the susceptibility to infection (Bjerknes and Steinsvåg, 1993). Nasal spray formulations may be applied in the treatment of many nasal diseases. Unfortunately adverse events may occur, in some cases affecting patient’s compliance, which describes the ⇑ Corresponding author. Tel.: +32 92648016; fax: +32 92228236. E-mail addresses: [email protected] (J. Lenoir), [email protected] (C. Bachert), [email protected] (J.-P. Remon), [email protected] (E. Adriaens). 0887-2333/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tiv.2013.06.018
degree to which a patient correctly follows medical advice, a prescribed treatment or preventive procedure. Certain nasal pathologies require a prolonged therapy with pharmaceutical formulations. Non-compliance of the medications, which may be assigned to the pH or osmolality of the formulation, a certain active ingredients or preservative, affects the outcome of the treatment. Evaluating the irritation potential of (nasal medical) formulations is essential to their safety assessment. The concept of the three Rs (refinement, reduction and replacement of laboratory animals) (Russell and Burch, 1959) strongly stimulates the development of alternative testing methods, such as in vitro methods and the use of ‘‘lower’’ organisms as test species (e.g. invertebrates, plants and microorganisms) (Balls et al., 1995). Keeping this concept in mind, the Slug Mucosal Irritation (SMI) assay was developed and has proven its value for different applications. It has also demonstrated a relation between an increase in mucus production in slugs and an elevated incidence of Stinging, Itching and Burning (SIB) sensations in humans (Adriaens and Remon, 2008; Lenoir et al., 2011, 2012). In the past, different in vitro tests have been applied to investigate the effect of for example saline solutions (e.g. Boek et al., 1999a,b; Harkema, 1990), but none was able to predict clinical discomfort. The SMI assay might be a
J. Lenoir et al. / Toxicology in Vitro 27 (2013) 1954–1961
possible method to pretest nasal spray and drop formulations to screen for adverse events. The objective and subjective efficacy of saline irrigation for the treatment of sinonasal disease has been demonstrated and saline irrigations are increasingly used (Rabago et al., 2006). This procedure has the advantages to be simple to execute, inexpensive and is generally well tolerated (Osguthorpe and Hadley, 1999; Tomooka et al., 2000; Shoseyov et al., 1998). By flushing the nasal cavity with a saline solution, it provides short-term relief of congestion by moisturizing the nasal cavity, reducing dryness, and helping to clear encrusted material and secretions (Fagnan, 1998; Osguthorpe and Hadley, 1999). All these effects improve the mucociliary clearance (Benninger et al., 1997; Talbot et al., 1997), which is essential as a transport mechanism for foreign particles entrapped in the mucus layer lining the sinonasal cavity. Moreover, the use of hypertonic solution may reduce edema of the nasal mucosa (Fagnan, 1998), through diffusion of osmolar gradients (Talbot et al., 1997). Saline solutions are available on the market in different tonicities and pH levels. It has been well-described that hypertonic saline causes clinical nasal discomfort to a greater extent than physiological saline (Baraniuk et al., 1999; Hauptman and Ryan, 2007; Adam et al., 1998; Shoseyov et al., 1998). Benzalkonium chloride (BAC) is often used as a preservative in multi-dose topical aqueous nasal drop and spray formulations. It prevents bacterial contamination and maintains the safety of the preparations (Graf, 2005; Graf et al., 1995; Graf, 1999; Marple et al., 2004; Steinsvåg et al., 1996). However, it has been suggested that the presence of BAC in a formulation may induce severe morphological and histological changes, both in vitro and in vivo (e.g. Berg et al., 1995; Cüreog˘lu et al., 2002; Kuboyama et al., 1997; Lebe et al., 2004; Riechelmann et al., 2004), and in vivo nasal irritation, hypersecretion and a burning sensation (Riechelmann et al., 2004). In this research several saline and BAC solutions were first extensively tested with the 1-day repeated exposure protocol of the SMI assay, focusing on the prediction of short term sensations (Stinging, Itching and Burning (SIB) test). Later, the same formulations were clinically evaluated in a Human Nose Irritation Test (HNIT). By this means, we were able to investigate whether this protocol can be applied to evaluate the clinical irritation potential of nasal formulations. The main goal was to investigate the correlation between the slugs and humans for nasal applications and set up a new classification prediction model for nasal formulations. 2. Materials and methods 2.1. Materials A phosphate buffered saline (PBS; isotonic and pH neutral; 0.4% NaCl, 1.0% Na2HP042H20, 0.2% KH2PO4), as well as four NaCl solutions (1.3%, 2.6%, 5.4%, 10.4% (w/v)) and two benzalkonium chloride (BAC) solutions (0.02% and 0.05% (w/v)) were prepared in PBS and tested with the SIB protocol and the HNIT. All these solutions were formulated by the Ghent University Hospital pharmacy. In the SMI experiments a 1% (w/v) dilution of BAC in PBS (delivered by Sigma, St. Louis, MO, USA) was added as a positive control. 2.2. Methods 2.2.1. Osmolality measurements The osmolality of all the formulations was determined using an Advanced Micro Osmometer (Model 3300 Advanced Instruments Inc., Norwood, MA, US) by the freezing-point method. Clinitrol™ 290 was used as reference solution (Advanced Instruments). The measurements were performed in triplicate (on 20 lL aliquots) and mean values were used for analysis. Results are represented as mOsm/kg.
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2.2.2. Stinging, Itching and Burning (SIB) test procedure The slug species used in this assay is Arion lusitanicus. The slugs are bred and kept under laboratory conditions in an air-conditioned room (18–20 °C). The effect of the selected formulations, negative (PBS – NaCl 0.4%) and positive controls (BAC 1%) on the mucosal tissue of the slugs was investigated with this protocol, described into detail in Lenoir et al. (2011). For each treatment group, three slugs are used. The specimens are not previously used in other experiments and are checked beforehand for any damage. They are brought into contact with 100 ll of the test substances during three 15-min contact periods (CPs). The body weight of the slugs and amount of mucus secreted by the slugs are quantified by weighing the Petri dishes at the beginning and end of each CP. In between the CPs there is one hour rest/recovery period during which the slugs are transferred to a fresh Petri dish and 1.5 ml PBS is added to prevent dehydration. Classification of the test substances is established calculating the average of the total mucus production (MP) during the three CPs of the three replicates, expressed as % (w/w) of the initial body weight of the slugs. 2.2.3. Human Nose Irritation Test The clinical trial was set up as a single-blind cross-over study with 24 participants and was organized in cooperation with the Department of Rhino-otolaryngology of the Ghent University Hospital. The protocol was reviewed and approved by an independent Commission for Medical Ethics, associated with the Ghent University Hospital, Belgium. Prior to enrollment, all participants read, understood and voluntary signed the informed consent document. 2.2.3.1. Study population. Twenty-four adult participants were recruited in the region of Ghent, Belgium. The study group consisted of 12 males and 12 females between 18 and 37 years, with an average age of 27 years. 2.2.3.2. Methodology. Seven sessions were organized. In every session approximately 10 ll (two sprays) of one of the test materials was administered at once in each nostril by the participants themselves, using a disposable nose spray bottle, which was only used once and then discarded. There were at least 2 days in between sessions to minimize the carry-over effect from previous sessions. 2.2.3.3. Measurements and observations. All subjects were instructed to report any nasal stinging, itching or burning sensation or reaction. A self evaluation was carried out by the participants, by means of a scoring table. The participants were able to express the incidence and score the intensity of nasal discomfort, starting 30 s after administration and continuing 1, 2, 5, 10, 15 and 30 min later. A grading scale from 0 to 5 was used to indicate the SIB potency of the tested substances (0 = no; 1 = very mild; 2 = mild; 3 = moderate; 4 = severe; 5 = very severe). 2.2.4. Data analyses The reproducibility of the SMI test was evaluated with a twoway ANOVA to investigate the run effect and test substance effect on the total mucus production. The normality of the residuals was investigated with a qq-plot. The homogeneity of variances was tested with the Levene’s test. If no significant interaction was present, the main effects were interpreted separately from the interaction and a multiple comparison among pairs of means was performed using a Scheffé post hoc test. If a significant interaction between both factors was present, a Bonferroni post hoc test was used to investigate if there were significant differences between the runs for each test substance. The effect of the different NaCl and BAC solutions on the intensity of the discomfort score and scores given by the participants was evaluated by means of descriptive statistics. The Spearman
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Rank correlation coefficient was calculated as a measure of association between the immediate discomfort observed by the participants and the endpoint of the SMI test. In order to develop a prediction model for the nasal stinging potency of formulations based on the total mucus production observed in the SMI assay, classes were assigned to the test formulations based on the discomfort scores observed in the volunteers. In a next step, classification tree analyses with bootstrap resampling was used to determine cut-off values for the total mucus production that distinguish between the different discomfort classes (rpart package running under R version 2.15.2). A value of p < 0.05 was considered statistically significant. All analyses were performed in SPSS version 21.0 and R version 2.15.2. 3. Results 3.1. Stinging, Itching and Burning (SIB) test The SIB procedure was used to evaluate the relative SIB potential of five NaCl and two BAC formulations in PBS. For this part of the study, a positive control was added in each run as a reference (BAC 1%). Experiments were repeated five times to investigate the reproducibility of this testing method. The total MP per test substance per experiment is presented in Table 1. A two-way ANOVA showed a significant treatment effect (p < 0.001), but no significant run effect (p = 0.563) and no significant interaction between these two factors (p = 0.947). The main effect of the treatment was interpreted and the Scheffé post hoc test suggested five subsets for the total amount of mucus secreted: (1) NaCl 0.4%, NaCl 1.3% and BAC 0.02% (p = 0.492), (2) NaCl 1.3%, NaCl 2.6%, BAC 0.02% and BAC 0.05% (p = 0.523), (3) NaCl 5.4%, (4) NaCl 10.4% and (5) BAC 1% (positive control). As an overall trend we could observe that with increasing concentrations of NaCl, also resulting in increasing osmolality of the formulations, resulted in a higher total mucus production of the slugs. Increasing concentrations of BAC resulted in an increased mucus production unrelated to osmolality, but due to its cytotoxic effect (Adriaens et al., 2001). 3.2. Human Nose Irritation Test The clinical stinging potential of the formulations was determined by spraying the same formulation twice in each nostril of the participants and scoring the occurrence and intensity of the experienced discomfort. In Fig. 1 the proportion of participants for the different scores of discomfort is represented for the
different NaCl solutions in function of time. It was observed that all reported SIB sensations decreased over time and that the higher the scores directly after administration, the higher the scores were in the course of time post administration. However, after 30 min, all participants only gave a score 0 or 1 to all tested formulations. Of all NaCl solutions tested, the participants tolerated NaCl 0.4% the best. Seventeen participants ranked it as least discomfort inducing NaCl formulation. Thirteen participants (54.2%) never felt discomfort when it was administered. Nine participants (37.5%) reported very mild or mild stinging sensations (score 1 or 2), while two (8.5%) experienced moderate stinging after 30 s. Five minutes post administration, it still received a score 2 from one participant, while all others did not feel any or only very mild sensations. After 30 min, none of the participants reported discomfort anymore. Directly after spraying NaCl 1.3% into the nose, the majority of the participants did not feel any discomfort (15/24 – 62.5%), while nine participants reported very mild or mild stinging (37.5%). After 5 min, the scores were even lower, with all participants reporting either no (20/24 – 83.5%) or very mild (4/24 – 16.5%) sensations. After 10 min, two participants (8.5%) still gave a score 1 (very mild stinging), while 30 min after administration this was only the case for one participant. Remarkably this participant did not indicate any stinging sensations at the earlier time points. All other participants did not report any discomfort at all after 15 min. For NaCl 2.6% a different scoring behavior was observed. Directly after administration only eight participants (33.5%) gave it a score 0, while 12 scored it as causing very mild or mild discomfort (50%). Three (12.5%) reported moderate sensations, while one participant even sensed severe discomfort. After 5 min, 13 participants (54%) did not feel anything anymore, while ten (41.5%) now reported either very mild or mild sensations, one participant still experienced moderate discomfort. Five minutes later, 19 people (79%) did not sense any stinging anymore, while five participants (21%) still felt very mild or mild sensations. After 30 min, 22 participants (91.5%) scored 0, while two participant (8.5%) still experienced very mild stinging. The participant reporting severe discomfort directly after administration, did not feel any discomfort after 30 min. Higher stinging scores were given to NaCl 5.4% in comparison to the lower NaCl concentrations. Directly after spraying it into the nose, all of the participants reported some degree of discomfort. Nine people (37.5%) experienced very mild or mild sensations, six (25%) indicated moderate discomfort, and nine (37.5%) felt severe or very severe sensations. After 5 min the distribution of the scores shifted substantially: already five participants (21%) did not report any stinging anymore, the majority (16/24 – 66.5%) reported only very mild or mild discomfort, while three participants
Table 1 Effect of a repeated exposure (three 15-min contact periods on the same day) to 100 ll of several NaCl and BAC solutions in PBS, with indication of the osmolality of the formulations. Test substance (%)
Osmolality (mOsm/kg)
NaCl 0.4 NaCl 1.3 NaCl 2.6 NaCl 5.4 NaCl 10.4 BAC 0.02 BAC 0.05 BAC 1 Run effectc
287 ± 1.7 552 ± 3.5 995 ± 4.0 1794 ± 1.2 3347 ± 28.3 293 ± 0.6 292 ± 1.7 290 ± 1.2
Total MP (%)a
Treatment effectb
Run 1
Run 2
Run 3
Run 4
Run 5
2.8 ± 0.5 5.0 ± 1.0 7.5 ± 2.2 13.1 ± 1.7 21.5 ± 3.2 3.9 ± 1.1 7.9 ± 0.6 30.5 ± 6.7 11.5 ± 9.8
3.1 ± 0.4 5.5 ± 0.5 6.0 ± 1.6 13.0 ± 2.0 24.5 ± 2.3 4.7 ± 0.6 6.9 ± 2.6 26.9 ± 8.3 11.3 ± 9.4
1.4 ± 0.3 3.2 ± 1.4 6.0 ± 1.5 12.1 ± 1.8 22.2 ± 1.8 4.6 ± 1.6 6.9 ± 0.6 27.6 ± 7.5 10.5 ± 9.5
1.2 ± 0.5 3.9 ± 0.6 6.6 ± 1.5 12.0 ± 2.0 19.8 ± 2.3 3.9 ± 0.4 5.3 ± 2.4 31.7 ± 4.3 10.6 ± 10.4
2.2 ± 1.5 5.4 ± 1.9 9.0 ± 0.6 14.9 ± 2.4 25.8 ± 5.1 6.0 ± 1.1 7.5 ± 2.2 28.9 ± 1.1 12.5 ± 9.9
MP: mucus production; PBS: phosphate buffered saline; BAC: benzalkonium chloride. a Total MP presented as the mean ± standard deviation (n = 3). b Total MP presented as the mean ± standard deviation (n = 15). c Total MP presented as the mean ± standard deviation (n = 24).
2.1 ± 0.8 4.6 ± 1.0 7.0 ± 1.2 13.0 ± 1.2 22.7 ± 2.4 4.6 ± 1.2 6.9 ± 1.8 29.1 ± 2.0 11.3 ± 9.7
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No discomfort (score 0)
80
80
60
% Participants
100
% Participants
100
NaCl 0.4% NaCl 1.3%
40
NaCl 2.6% NaCl 5.4%
20
(Very) Mild discomfort (score 1+2)
60 40 20
NaCl 10.4% 0
0 0
5
10
15 20 Time (min)
25
0
30
Moderate discomfort (score 3)
100
10
15 20 Time (min)
25
30
(Very) Severe discomfort (score 4+5)
100
80
80 % Participants
% Participants
5
60 40 20
60 40 20
0
0 0
5
10
15 20 Time (min)
25
30
0
5
10
15 20 Time (min)
25
30
Fig. 1. Percentage of the participants reporting the different classes of discomfort (n = 24).
(12.5%) experienced moderate stinging. No one experienced severe sensations anymore. Five minutes later, the scores even dropped more. Fourteen (58.5%) did not feel any adverse sensation anymore. Nine participants (37.5%) experienced very mild to mild sensation, while one still reported moderate stinging. Thirty min post administration, 21 participants (87.5%) reported no discomfort at all, while three (12.5%) still had very mild stinging sensations. These latter had indicated severe sensations earlier on. As expected, NaCl 10.4% received the highest scores of all tested formulations. As for NaCl 5.4%, all participants experienced stinging sensations. Directly after administration, majority indicated severe (7/24 – 29%) or even very severe (9/24 – 37.5%) discomfort, three particpants (12.5%) experienced moderate sensations, while five participants (21%) only felt very mild to mild sensations. After 5 min, the number of participants not feeling any discomfort increased to two (8.5%). The majority (16/24 – 66.5%) now sensed very mild to mild stinging, two (8.5%) had moderate sensations, while four (16.5%) still reported severe or very severe sensations. Ten min after application, the majority either felt no discomfort at all (11/24 – 46%) or very mild to mild sensations (10/24 – 41.5%). Two people (8.5%) reported moderate sensations, while one still experienced severe stinging. After 30 min, 21 participants (87.5%) scored 0, while three (12.5%) still had very mild stinging sensations. These latter indicated very severe sensations earlier on. Total sting scores were higher than for NaCl 5.4% in 19/24 participants. In Fig. 2 the percentage of participants reporting either no discomfort or (very) mild sensations in the course of time is presented for the same formulations. Directly after administration BAC 0.02% induced very mild to mild sensations in seven participants (29%), while all others reported no discomfort at all. After 5 min, all participants reported either no (22/24 – 91.5%) or very
mild (2/24 – 8.5%) discomfort, while after 10 min only one participant experienced very mild discomfort. After 30 min, nobody indicated any stinging anymore. For BAC 0.05%, 11 participants (46%) experienced no discomfort, while 12 (50%) had very mild to mild sensations, and one indicated moderate stinging directly after administration. After 5 min, 13 participants (54%) did not report any sensations, while the others (11/24 – 46%) sensed either very mild or mild stinging. Ten min post administration, 15 participants did not experience any discomfort anymore (62.5%) while nine had very mild or mild sensations (37.5%). At the last scoring time, 20 participants (83.5%) did not feel any discomfort, but four (16.5%) still experienced very mild sensations. 3.3. Correlation SMI – HNIT The results of the clinical trial indicated that none of the formulations induced discomfort for longer than 30 min post-instillation or only very mild sensations were reported by the participants. The clearest differentiation between the tested formulations was observed immediately after instillation (30 s and 1 min post-instillation). Therefore, the data for immediate stinging were used to investigate the correlation with the results in slugs after three 15 min contact periods. A significant positive association was found between the immediate stinging reaction observed by the panelists and the total amount of mucus produced by the slugs (Spearman’s Rank correlation = 0.963, p < 0.001). Classes were assigned to the test formulations if at least 50% of participants indicated a certain score: no = score 0, mild = score 1 and 2, moderate = score 3, severe = score 4 and 5. In the clinical trial, NaCl 0.4% was generally well tolerated by the participants, with the majority of the participants scoring 0
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No discomfort (score 0)
80
80
% Participants
100
% Participants
100
60 NaCl 0.4% 40
NaCl 1.3%
(Very) Mild SIB (score 1 + 2)
60 40
BAC 0.02% BAC 0.05%
20
20
0
0 0
5
10
15
20
25
30
0
5
10
15
20
25
30
Time (min) Fig. 2. Percentage of the participants reporting no, very mild or mild discomfort at the different time points (n = 24).
immediately after administration (score 0 in 13 and 19 (54% and 79%) participants after 30 s and 1 min respectively) (Fig. 1). Severe or very severe discomfort was never observed. Based on the scores given by the participants this formulation was assigned the class of causing no discomfort. Slugs only showed on average a low MP (total MP = 2.1 ± 0.8%). For NaCl 1.3% a similar reaction was seen, although more participants experienced very mild to mild discomfort (score 1 or 2) after administration compared to NaCl 0.4%. Nine and ten participants scored a 1 or 2 after 30 s and 1 min respectively compared to nine and four at the same time points for NaCl 0.4%. However, the majority scored 0 immediately after administration (62.5%), being assigned the class of no discomfort as well. The slugs showed on average a more than doubled total MP compared to NaCl 0.4% (4.6 ± 1.0% compared to 2.1 ± 0.8% for NaCl 0.4%). When administrating NaCl 2.6% in their nose, the majority of the participants reported very mild to mild sensations, score 1 or 2 in 12 and 13 participants after 30 s and 1 min (50% and 54% respectively); score 3 in three participants at these time points (12.5%) and even one participant with a score 4 at both time points (4%). Based on this scoring behavior the class mild was assigned, since 50% or more indicated either a score 1 or 2 immediately after application. In the SIB test exposure to this formulation resulted on average in a total MP of 7.0 ± 1.2%. The effect of exposure to higher concentrations of NaCl was more pronounced in both slugs and humans. When NaCl 5.4% was tested, all the participants experienced some kind of discomfort shortly after administration (none of the participants scored 0). The majority of the participants either reported mild, moderate, or severe discomfort (9 – 6 – 9 (37.5% – 25% – 37.5% respectively) at 30 s and 11 – 4 – 9 (46% – 17% – 37.5% respectively) at 1 min). This formulation was assigned to the class of moderate discomfort, because the scores were distributed quite evenly around this score. In the slugs this formulation induced on average a total MP of 13.0 ± 1.2% after three CPs. As for NaCl 5.4%, NaCl 10.4% induced discomfort in all participants (score 0 by none of the participants), with the majority indicating severe or even very severe stinging directly after administration (score 4 in seven participants and score 5 in nine after 30 s; score 4 and 5 both by seven participants after 1 min). Not only more high scores (scores 4 or 5) were given directly after administration compared to any other formulation tested, they also last longer (up to 10 min after administration for NaCl 10.4%, up to 2 min after administration for NaCl 5.4%). Based on these results the class of inducing severe discomfort was assigned to this solution. Testing it in slugs resulted in a highly increased total MP of 22.7 ± 2.4%, confirming the trend observed in humans.
The lowest BAC concentration tested (0.02%) was generally well tolerated, with 17 and 21 participants (71% and 87% respectively) reporting no discomfort after 30 s and 1 min respectively. Based on these results BAC 0.02% was assigned to the class of causing no discomfort. Total MP of the slugs was slightly increased compared to the NaCl 0.4% formulation (4.6 ± 1.2% compared to 2.1 ± 0.8%), but appeared not to be statistically significant (p = 0.492). For BAC 0.05% more than 50% of the participants reported some kind of discomfort immediately after administration (score 0 by only 11 participants, score 1 or 2 by 12 participants, score 3 by one participant after 30 s; score 0 by only 12 participants, score 1 or 2 by 11 participants, score 3 by one participant after 1 min). A statistically significant higher total MP was observed in the slugs exposed to BAC 0.05% compared to the lower concentration of BAC (6.9 ± 1.8% compared to 4.6 ± 1.2% for BAC 0.02%). Based on the scores given by the participants, this formulation was assigned to the class of causing mild discomfort. In order to determine threshold values for the total MP to distinguish between the four irritation categories, a classification tree with bootstrap resampling was used. This resulted in the following threshold values: total MP 6 5.5% corresponds with no discomfort, between 5.5% and 610% corresponds with mild discomfort, between 10% and 617.5% corresponds with moderate discomfort and a total MP > 17.5% is indicative for severe discomfort. Fig. 3 nicely illustrates the correspondence between the results in slugs and humans, with indication of the new threshold values for the different categories. 4. Discussion Previous research of our lab about nasal discomfort described a correlation between results in slugs and clinical results found in literature (Lenoir et al., 2011). Since comparative studies between in vivo and in vitro tests on mucosal discomfort are scarce, this research project focused on the comparison of the mucosal tolerance towards several nasal NaCl and BAC solutions with the 1-day protocol of the SMI assay vs. the human nasal irritation test in 24 participants. By this means, we aimed to investigate whether the SIB test can replace the HNIT to evaluate and predict clinical nasal discomfort to different irritants. Analyzes indicated a good correlation between results of the SIB-test and subjective human nasal responses from the HNIT, especially for the immediate sensation (scores given after 30 s and 1 min, Spearman’s Rank correlation = 0.963, p < 0.001). The general approach in product development when screening with the SIB test should be to deliver a product with a mean total MP as low as possible.
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Fig. 3. Relationship between the results in slugs and in humans induced by exposure to several NaCl and BAC (w/v) solutions (Spearman’s Rank correlation = 0.963, p < 0.001). Representing: (A) sting scores given by the participants 30 s and 1 min after administration of the formulations (n = 24); (B) total mucus production (MP) of the slugs after three 15-min contact periods (all data of five different experiments, each with n = 3) (cut-off between no and mild discomfort = 5.5%); cut-off between mild and moderate discomfort = 10%; cut-off between moderate and severe discomfort = 17.5%).
4.1. Effect of osmolality According to several authors, nasal irrigation with (hypertonic) saline decongests the swollen membranes and may also improve mucociliary transport (Druce, 1990; Parsons, 1996; Slavin, 1991; Talbot et al., 1997). Several saline solutions with concentrations up to 2.3% NaCl are available as over the counter nasal sprays. Generally, these are well tolerated, although the use of hypertonic nasal saline elicits more burning and irritation compared with physiological nasal saline (Adam et al., 1998; Baraniuk et al., 1999; Hauptman and Ryan, 2007). Our results confirm this trend. In the study of Adam et al. (1998) for example, a clinical trial was performed with a normal and a hypertonic (2%; buffered) saline solution. In the group receiving the latter formulation, 32% reported nasal irritation, compared to 13% in the control group. In the current clinical trial these percentages were very similar (38% and 13% respectively). Additionally, in the study of Shoseyov et al. (1998), a 3.5% saline solution was tested in 30 children with chronic sinusitis three times daily for four weeks. Regarding tolerability of the treatment, the patients in the hypertonic saline group reported burning and itching in the nose more than those in the normal saline group, but these sensations vanished over time (reported only during the first 3–4 days). After that period, there was no difference between the groups and no problems were reported anymore. Although the current study only tested the effects of a single application, similar trends were observed: the higher the NaCl concentration, the higher the scores for discomfort, but these reduced quickly in time. After spraying NaCl 10.4% into their nose, all participants reported some degree of discomfort, while for NaCl
0.4% this was 8%, for NaCl 1.3% this was 13%, for NaCl 2.6% this was 39% and for NaCl 5.4% this was 88%. No substantial stinging was reported anymore after 2 min for NaCl 1.31%, after 10 min for NaCl 0.4%, after 15 min for NaCl 2.6% and 5.4%. For NaCl 10.4% this was the case only after 30 min. Baraniuk et al. (1999) tested concentrations of NaCl up to 21.6% against a control (NaCl 0.9%). The hypertonic saline solutions induced a burning sensation within the first ten sec after application, but faded in time, as was observed in the current study. Pain intensity became significantly different from NaCl 2.7% and increased in a dose-dependent fashion. Comparing our results with results found in literature, we may conclude that although generally safe, (daily) hypertonic nasal irrigation may be associated with some minor clinical side effects. Also, the higher the concentration of NaCl in the formulation, the higher the risk will be for clinical discomfort. 4.2. Effect of BAC Benzalkonium chloride (BAC) is often used as a preservative in multi-dose topical aqueous nose drop and spray formulations. It prevents bacterial contamination and maintains the safety of the preparations (Graf, 1999, 2005; Graf et al., 1995; Marple et al., 2004; Steinsvåg et al., 1996), by damaging the cell wall of microorganisms and altering its permeability (Richards and Cavill, 1976). In marketed multi-dose nasal products, BAC is usually added in concentrations of 0.005–0.02% (w/v) (Riechelmann et al., 2004; Verse et al., 2003), while the American College of Toxicology concluded that BAC can be used safely in a concentration
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up to 0.1% (w/v). In the current study, BAC concentration of 0.02% and 0.05% were selected because they fall within this range. It has been suggested that the presence of BAC in a formulation may induce severe morphological and histological changes, both in vitro (human epithelial cell studies) and in vivo in animal studies (e.g. Berg et al., 1995; Cüreog˘lu et al., 2002; Kuboyama et al., 1997; Lebe et al., 2004; Riechelmann et al., 2004), although other in vivo studies in human patients have demonstrated that BAC-preserved intranasal products were safe and well-tolerated for long- and short-term clinical use (Marple et al., 2004). Results from the SMI tests reveal a statistically significant concentration-response effect, which has also been described by Berg et al. (1995), Lebe et al. (2004) and Marple et al. (2004). In slugs, high concentrations of BAC result in a very high MP (positive control of BAC 1% induced a mean total MP of 29.1 ± 2.0%), while for lower concentrations of BAC the MP was much lower (average total MP of BAC 0.02% = 4.6 ± 1.2%; BAC 0.05% = 6.9 ± 1.8%). Compared to the negative control, this increased MP in lower concentrations of BAC is presumably induced by stinging or burning sensations. In the study of Riechelmann et al. (2004) the adverse effects of BAC on human nasal mucosa was evaluated, both in vitro and in vivo. In vitro, 0.01% and 0.025% BAC appeared to be ciliotoxic for human nasal respiratory epithelia from healthy, non-allergic volunteers. In vivo however, this ciliotoxic effect was not observed: there was no interference with nasal mucus transport and it did not induce nasal inflammatory alterations compared to the placebo. However, directly after application, participants exposed to the BAC 0.05% solution reported nasal irritation, hypersecretion and a burning sensation. Moreover, symptom scores for persistent nasal irritation were higher for BAC than the placebo (Ringer’s solution). In the current study, we also found higher stinging scores for BAC 0.05% compared to the 0.02% solution, and the duration of the sensations was also longer (10 min compared to 1 min). 4.3. Sensitivity of participants Substantial variation in the subjective self-evaluation by the participants was observed, as was the case in previous research of our group (Lenoir et al., 2012). The scoring behavior is amongst other factors influenced by the participants’ sensitivity. Despite the small number of participants in the current clinical trial, it is possible to categorize people into different sensitivity groups. Seven participants (5 males + 2 females) were clearly less sensitive than the others. However, they were able to differentiate between the higher concentration of NaCl (NaCl 5.4% and 10.4%) and the lower concentrations NaCl and BAC (NaCl 1.3% and 2.6%; BAC 0.02% and 0.05%). Three of them (2 males + 1 female) only reported stinging sensations for NaCl 5.4% and 10.4%, but scored 0 for all other formulations at all time points. The other four gave low scores overall, but scores slightly increased with increasing NaCl or BAC concentration, except one who gave the BAC 0.02% a total sting score of 1 and BAC 0.05% a total sting score of 0. Another group (2 males + 3 females) was able to make a clearer difference between the tested formulations, with much higher scores for NaCl 5.4% and 10.4% in comparison with the first group. The rest (12/24 – 50%) showed an intermediate scoring behavior: they were generally able to differentiate between lower and higher concentrations of NaCl, but some had difficulties distinguishing between NaCl 5.4% and 10.4% and also between BAC 0.02% and 0.05% (4/24 and 5/24 respectively). 4.4. New cut-off values for nasal discomfort A good correlation was obtained between results of the SIB test and subjective human nose responses from the HNIT, especially for the immediate sensations (scores given after 30 s and 1 min). In
Table 2 New cut-off values for the different categories of nasal discomfort. Degree of discomfort
Total mean MP (%) Ocular (%)
Nasal (%)
No Mild Moderate Severe
<3 3–8 8–15 >15
<5.5 5.5–10 10–17.5 >17.5
Classification formulations in current study
NaCl NaCl NaCl NaCl
0.4%, NaCl 1.3%, BAC 0.02% 2.4%, BAC 0.05% 5.4% 10.4%, BAC 1%
MP: mucus production expressed as a % of the initial body weight of the slugs; BAC = benzalkonium chloride.
the past, this correlation was already found for ocular discomfort induced by surfactant-based personal care product and a classification prediction model was developed (Lenoir et al., 2012; Table 2). However, results of the HNIT were not in agreement with these. Since a difference in sensitivity exists between the ocular tissue and nasal mucosae, we suggest new cut-off values for nasal applications taking into account the results of the current clinical trial. By this means, a more accurate prediction of clinical reactions can be made. The general approach in product development when screening with the SIB protocol should be to deliver a nasal formulation with a mean total MP less than 5.5%. 5. Conclusions This study has shown that the recently developed SIB protocol is a quick, accurate and reproducible screening method for the prediction of immediate nasal stinging, itching and/or burning responses in the human nose containing the excipients NaCl and benzalkonium chloride. Since a strong positive association was found between the results in slugs and in humans, the test allows formula optimization prior to product development investment in clinical trials. It is recommended that when a nasal formulation is tested with this protocol, the total amount of mucus secreted by the slugs after three CPs should be lower than 5.5%. The higher the MP of the slugs, the higher the potential for clinical SIB sensations. When exceeding 10%, it is suggested to reduce the irritability of the formulation, in which active ingredients, preservatives and osmolality play an important role. Conflict of Interest All authors state that there is no actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations within 3 years of beginning the submitted work that could inappropriately influence, or be perceived to influence, their work. References Adam, P., Stiffman, M., Blake, R., 1998. A clinical trial of hypertonic saline nasal spray in subjects with the common cold or rhinosinusitis. Arch. Fam. Med. 7, 39–43. Adriaens, E., Remon, J.P., 2008. Mucosal irritation potency of personal lubricants relates to product osmolality as detected by the slug mucosal irritation assay. Sex. Transm. Dis. 35 (5), 512–516. Adriaens, E., Dierckens, K., Bauters, T.G.M., Nelis, H.J., van Goethem, F., Vanparys, Ph., Remon, J.P., 2001. The mucosal toxicity of different benzalkonium chloride analogues evaluated with an alternative test using slugs. Pharm. Res. 18 (7), 937–942. Balls, M., Goldberg, A., Fentem, J., Broadhead, C., Burch, R., et al., 1995. The three Rs: the way forward. The report and recommendations of ECVAM workshop 11. ATLA 23, 838–866. Baraniuk, J., Ali, M., Yuta, A., Fang, S., Naranch, K., 1999. Hypertonic saline nasal provocation stimulates nociceptive nerves, substance P release, and glandular
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