3.1. Results
As there were no reference standards for screening respiratory mucosa irritation and toxicity, the reference chemicals used were selected from the ECETOC data bank for eye irritation. Twenty-eight compounds that cover entire irritancy range were selected [
12], but eighteen (9 irritants and 9 non-irritants) were used mainly due to solubility problems. The target maximum solubility for our studies was 1.0
w/
v% and compounds with lower solubility in Dulbecco’s Modified Eagle’s Medium–Ham’s F-12 (DMEM/F-12) were excluded. The test chemicals included seven alcohols (6 non-irritants and 1 irritant), four surfactants (1 non-irritant and 3 irritants), two heterocyclic compounds (1 non-irritant and 1 irritant), two ketones (1 non-irritant and 1 irritant), one inorganic compound (irritant), one carboxylic acid salt (irritant), and one amine (irritant) (
Table 1).
The result obtained after 60 min exposure of the Calu-3 cells to 0.2% test solutions showed that all the alcohols had viabilities greater than 50% (
Table 2). The values were PEG 400 (91.5 ± 4.6); 3-methoxy-1,2-propanediol (92.6 ± 4.1); glycerol (85.6 ± 8.7); PEG 600 (85.9 ± 13.4); 2-methyl-1-pentanol (95.3 ± 13.1); anhydrous ethanol (99.1 ± 3.9); and cyclohexanol (89.9 ± 14.3). Most of the tested surfactants were remarkably toxic to the Calu-3 cells. Other than Tween 20 (NI) that resulted in 57.1% ± 8.1% viability, 0.2%
w/
v of sodium dodecyl sulphate (R36), Triton X-100 (R36) and cetylpyridinium bromide (R41) killed more than 90% of the cells. The heterocyclic compounds: Toluene (NI) and imidazole (R41) had no significant effect on the cell viability. Similar results were observed for the ketones (methyl isobutyl ketone (NI) and acetone (R36)). Whereas the 0.2%
w/
v of the inorganic compound (sodium hydroxide, R41) killed about 95% of the cells, the tested carboxylic acid (sodium oxalate, R41) and amine (4-fluoroaniline, R41) resulted in 65.4 ± 4.0 and 114.9% ± 5.9% viability, respectively.
Table 2.
Effect of test compounds on Calu-3 cells viability after incubation with 0.2% test solutions for 60 min.
Table 2.
Effect of test compounds on Calu-3 cells viability after incubation with 0.2% test solutions for 60 min.
S/N | Test Compounds | Chemical Class | European Union Class | Calu-3 %Viability ± SD |
---|
1 | PEG400 | Alcohol | NI | 91.5 ± 4.6 |
2 | 3-Methoxy-1,2-propanediol | Alcohol | NI | 92.6 ± 4.1 |
3 | Glycerol | Alcohol | NI | 85.6 ± 8.7 |
4 | PEG600 | Alcohol | NI | 85.9 ± 13.4 |
5 | 2-Methyl-1-pentanol | Alcohol | NI | 95.0 ± 13.1 |
6 | Anhydrous ethanol | Alcohol | NI | 99.1 ± 3.9 |
7 | Cyclohexanol | Alcohol | R41 | 89.9 ± 14.3 |
8 | Tween20 | Surfactant | NI | 57.1 ± 8.1 |
9 | Sodium dodecyl sulphate USP | Surfactant | R36 | 4.3 ± 0.1 |
10 | Triton X-100 | Surfactant | R36 | 3.5 ± 0.5 |
11 | Cetylpyridinium bromide | Surfactant | R41 | 8.1 ± 1.1 |
12 | Toluene | Heterocyclic | NI | 85.7 ± 13.0 |
13 | Imidazole | Heterocyclic | R41 | 106.9 ± 10.8 |
14 | Methyl isobutyl ketone | Ketone | NI | 73.2 ± 7.4 |
15 | Acetone | Ketone | R36 | 100.4 ± 13.7 |
16 | Sodium hydroxide | Inorganic chemical | R41 | 4.6 ± 0.4 |
17 | Sodium oxalate | Carboxylic acid salt | R41 | 65.4 ± 4.0 |
18 | 4-Fluoroaniline | Amine | R41 | 114.9 ± 5.9 |
The result obtained after the cells were incubated with 1.0%
w/
v of test solutions for 60 min (
Table 3) showed that with the exception of 2-methyl-1-pentanol (2.9% ± 0.2%) all the alcohols including PEG400 (92.2% ± 15.9%); 3-methoxy-1,2-propanediol (90.7% ± 4.3%); glycerol (86.5% ± 7.0%); PEG600 (81.7% ± 10.8%); anhydrous ethanol (90.3% ± 8.0%); cyclohexene (78.7% ± 11.7%) maintained viabilities greater than 50%. All the surfactants, Tween 20 (43.9% ± 2.5%); sodium dodecyl sulphate USP (9.0% ± 0.1%); Triton X-100 (4.5% ± 0.1%); cetylpyridinium bromide (5.4% ± 0.6%) had viabilities less than 50%. Both heterocyclic compounds, toluene (109.8% ± 6.6%) and imidazole (95.4% ± 2.9%), as well as the ketones, methyl isobutyl ketone (90.3% ± 7.1%) and acetone (95.4% ± 16.5%) had viability values of more than 50%. For sodium hydroxide (14.9% ± 1.2%), 1.0%
w/
v of the compounds reduced the cell viability of the cells more than 0.2% solutions following 60 min exposure while for 4-fluoroaniline (75.6% ± 10.9%), 0.2%
w/
v of the compounds reduced the cell viability of the cells more than 1.0% solutions following 60 min exposure.
Table 3.
Effect of test compounds on Calu-3 cells viability after incubation with 1.0% test solutions for 60 min.
Table 3.
Effect of test compounds on Calu-3 cells viability after incubation with 1.0% test solutions for 60 min.
S/N | Chemical | Chemical Class | European Union Class | Calu-3 %Viability ± SD |
---|
1 | PEG400 | Alcohol | NI | 92.2 ± 15.9 |
2 | 3-Methoxy-1,2-propanediol | Alcohol | NI | 90.7 ± 4.3 |
3 | Glycerol | Alcohol | NI | 86.5 ± 7.0 |
4 | PEG600 | Alcohol | NI | 81.7 ± 10.8 |
5 | 2-Methyl-1-pentanol | Alcohol | NI | 2.9 ± 0.2 |
6 | Anhydrous ethanol | Alcohol | NI | 90.3 ± 8.0 |
7 | Cyclohexanol | Alcohol | R41 | 78.7 ± 11.7 |
8 | Tween20 | Surfactant | NI | 43.9 ± 2.5 |
9 | Sodium dodecyl sulphate USP | Surfactant | R36 | 9.0 ± 0.1 |
10 | Triton X-100 | Surfactant | R36 | 4.5 ± 0.1 |
11 | Cetylpyridinium bromide | Surfactant | R41 | 5.4 ± 0.6 |
12 | Toluene | Heterocyclic | NI | 109.8 ± 6.6 |
13 | Imidazole | Heterocyclic | R41 | 95.4 ± 2.9 |
14 | Methyl isobutyl ketone | Ketone | NI | 90.3 ± 7.1 |
15 | Acetone | Ketone | R36 | 95.4 ± 16.5 |
16 | Sodium hydroxide | Inorganic chemical | R41 | 14.9 ± 1.2 |
17 | Sodium oxalate | Carboxylic acid salt | R41 | 64.5 ± 11.5 |
18 | 4-Fluoroaniline | Amine | R41 | 75.6 ± 10.9 |
Table 4 summarized the results of cells exposed to 0.2% and 1.0%
w/
v test solutions after 60 min exposure according to the ECVAM protocol. At 0.2%
w/
v solution, there were 5 false negatives (irritants falsely predicted as non-irritants). These compounds include cyclohexanol, imidazole, acetone, sodium oxalate, and 4-fluoroaniline. Four of these compounds were neither alcohol nor surfactant. There was no false positive (non-irritant falsely predicted as irritant) recorded. The major difference between the results of incubations of test solutions (0.2% and 1.0%
w/
v) for 60 min was two false positive results (Tween 20 and 2-Methyl-1-pentanol) obtained at 1.0%
w/
v.
Table 4 shows that 0.2% test solutions gave a better prediction than 1.0%. Cells exposed to 1.0% solutions for 60 min resulted in five false negatives (cyclohexanol, imidazole, acetone, sodium oxalate, 4-fluoroaniline) (
Table 4). Two false positives (Tween 20 and 2-Methyl-1-pentanol) were also observed.
Table 4.
Comparison of validation data, based on test compound concentration and 60 min exposure to Calu-3 cells.
Table 4.
Comparison of validation data, based on test compound concentration and 60 min exposure to Calu-3 cells.
European Union Classification | Calu-3 Result |
---|
0.2% Test Solutions | 1.0% Test Solutions |
---|
Irritants | Non-Irritants | Irritants | Non-Irritants |
---|
Non-Irritants (9) | 0 | 9 | 2 | 7 |
Irritants (9) | 4 | 5 | 4 | 5 |
The specificity, concordance and sensitivity for 0.2%
w/
v and 60 min incubation were 100%, 72% and 44%, respectively (
Table 5). All non-irritants were correctly predicted and had cell viabilities beyond 85% except for Tween 20, which had a viability of 57.1% ± 8.1%. The irritants (R36, R41) that were correctly predicted had viabilities less than 9.0%, while irritants falsely predicted as non-irritants (false negatives) had percent viabilities beyond 65%. Six (PEG400, 3-Methoxy-1,2-propanediol, glycerol, PEG600, 2-Methyl-1-pentanol, anhydrous ethanol) out of the seven alcohols tested were correctly predicted. A similar observation was seen within the surfactant group where all four compounds (1 non-irritant and 3 irritants) were correctly predicted. The specificity, concordance and sensitivity for 1.0%
w/
v test compounds exposed to the Calu-3 cells for 60 min were 78%, 61% and 44% respectively (
Table 5). Most (78%) of the non-irritants were correctly predicted. The two false positives have cell viabilities of 43.9 ± 2.5 and 2.9 ± 0.2 for Tween 20 and 2-Methyl-1-pentanol, respectively. The irritants (R36, R41) that were correctly predicted (sodium dodecyl sulphate USP, Triton X-100, cetylpyridinium bromide, sodium hydroxide) had viabilities less than 17% while irritants falsely predicted as non-irritants (false negatives) had viabilities of between 64% and 96%.
Table 5.
Comparison of validation parameters (sensitivity, specificity, concordance) based on test compound concentration and 60 min exposure to Calu-3 cells. Comparison of results of Calu-3 cells treated with 0.2% and 1.0% test solutions for 60 min.
Table 5.
Comparison of validation parameters (sensitivity, specificity, concordance) based on test compound concentration and 60 min exposure to Calu-3 cells. Comparison of results of Calu-3 cells treated with 0.2% and 1.0% test solutions for 60 min.
Groups | Sensitivity * | Specificity ** | Concordance *** |
---|
1.0% at 60 min treatment | 44% | 78% | 61% |
0.2% at 60 min treatment | 44% | 100% | 72% |
3.2. Discussion
The bovine corneal opacity and permeability (BCOP), Hen’s Egg Test on chorio-allantoic membrane (HET-CAM), chorioallantoic membrane vascular assay (CAMVA), isolated rabbit eye (IRE), isolated chicken eye (ICE), slug mucosal irritation (SMI) [
12], and the reconstituted human corneal epithelial (HCE) methods [
27,
28] have been investigated as experimental models for mucosal irritation and toxicity screening. The BCOP assay method is one of the leading alternative assays to the Draize test and has been accepted by many regulatory agencies since 2009 [
5]. The SMI was developed as a general test method for the nasal, buccal, and vaginal mucosal surfaces irritation and has been used for mucosal tolerance testing of pharmaceutical formulations and ingredients [
10].
In this study we demonstrated that the Calu-3 cell culture model is a potentially useful cell line for investigating respiratory mucosa irritation. The sensitivity, specificity and concordance of our data compared favorably well to BCOP and SMI irritation models (
Table 6). Our data showed that the range of values for each of the investigated assay parameter (sensitivity, specificity, concordance) were comparable to other methods: 75%–81% (BCOP), 68%–100% SMI (mucus endpoint), and 44%–100% (Calu-3 cell line; 0.2% at 60 min exposure), respectively. Furthermore, our model showed a concordance of 72%, which is slightly higher than that of the SMI model (68%), but lower than BCOP method (79%–81%).
Table 6.
Comparison of sensitivity, specificity and concordance of different assay methods used in prediction of mucosal toxicity.
Table 6.
Comparison of sensitivity, specificity and concordance of different assay methods used in prediction of mucosal toxicity.
Methods | Sensitivity % | Specificity % | Concordance% | Source |
---|
BCOP | 75–84 | 79–81 | 79–81 | [29] |
SMI (mucus endpoint) | 75 | 100 | 68 | [4] |
Calu-3 cell model (0.2% at 60 min) | 44 | 100 | 72 | |
Regarding the various test compounds, results from alcohols in our study were comparable to the data from the SMI model using mucus production as irritation endpoint. The non-irritating alcohols were correctly predicted as non-irritants whereas the irritating alcohols were under-predicted. Historically, the irritation potential of alcohols is difficult to predict.
In vivo, irritating alcohols induced no increased mucus production in SMI model [
12]. Alcohols/polyols have a tendency to introduce false results in the BCOP assay [
29]. Results of validation studies conducted by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) for BCOP assay showed that false negative rates for alcohols and solids range from 42% to 100% depending on the hazard classification system [
27]. Furthermore, alcohols and esters (including volatile substances, such as isopropanol, ethanol, methyl acetate, or ethyl acetate) are reported to have a relatively low predictive capacity compared to that of other substances [
30].
In our studies, the four surfactants (Tween 20, sodium dodecyl sulfate, Triton
®-X 100 and cetylpyridinium bromide) that were investigated were correctly predicted (100% concordance). In the SMI model, Adriaens and Remon (2002) reported 2 correct predictions (Tween 20, cetylpyridinium bromide) and 2 over-predictions (sodium dodecyl sulfate, Triton-X 100). The result shows that our model performed well on surfactants just like Hen’s Egg Test Chorioallantoic Membrane (HET-CAM) [
29]. Similarly, methyl isobutyl ketone was wrongly classified in SMI but was correctly identified as non-irritant in our model. Furthermore, acetone gave a false negative both in SMI and in our model.
The same measure of specificity (100%), which was recorded both in SMI and Calu-3 cell models, means that both models are comparable when used to test non-irritants. The concordance of the BCOP test method with regard to each of the three classification systems (European Union (EU), EPA, and GHS) ranged from 79% to 81%. The false positive and false negative rates ranged from 19% to 21% and 16% to 25%, respectively [
29]. Compared with BCOP, our model recorded a higher specificity with lower sensitivity (
Table 6). A comparison of Draize eye test and BCOP reported 84.6% concordance with specificity and sensitivity being more than 84%. All the false negatives recorded were solids whereas most of the false positives were liquids, indicating that the physical state of the substance under investigation affects the result [
31].
The higher specificity (100%) observed for our model implies that unlike the BCOP test (79–81) method, we were able to identify non-irritant compounds correctly, irrespective of its class. For a compound to be irritating to the cells, the compound must diffuse into the cells. We used Log
p values comparison to assess which of the compounds had difficulty diffusing into the cells. The Log
p values range for the test compounds was +4.15 to −1.76. For optimal permeation, an ideal compound generally has a Log
p value of between 1 and 4 [
31]. The non-irritating compounds have optimal Log
p values; most of the non-irritating alcohols have negative values. All the non-irritating non-alcoholic compounds (cyclohexanol, sodium dodecyl sulphate USP, Triton X-100, cetylpyridinium bromide, toluene, methyl isobutyl ketone) with optimal Log
p were correctly predicted except 4-fluoroaniline. Most of the non-alcoholic compounds with false negative results (imidazole, acetone, sodium oxalate) have negative Log
p values. These suggest that the alcohols may not have penetrated the cell membranes in adequate quantities and it is possible that the false predictions associated with the alcohols may be due to their Log
p values.
The wide disparity in the sensitivity values between BCOP (75%–84%) and Calu-3 cell model (44%) suggest that more work is required to improve the sensitivity of the Calu-3 model. Sensitivity disparity may be related to the fact that the test compounds affected cells viability via different mechanisms such as physical or interfacial mechanisms, hypertonicity, solvent-based solubilization, chelation, and membrane fluidization. Better sensitivity may be achieved by altering the Calu-3 cell culture method (e.g., using air-liquid interface or 3-D culture methods) or by using other toxicity indices other than MTT for estimating toxicity endpoint.