Abstract
This study was conducted to evaluate the accuracy and reliability of the Spectro-DPRA, an enhanced method for the OECD TG 442C (direct peptide reactivity assay, DPRA), serving as an alternative to animal testing for skin sensitization. The validation of Spectro-DPRA was executed across four participating laboratories (Lab 1, Lab 2, Lab 3, and Lab 4, the latter only participating in the proficiency test) adhering to GLP principles. It covered transferability, proficiency, within laboratory and between laboratory reproducibility tests, and a predictive capacity test using 40 additional substances. In tests with 10 proficiency substances, results from all participating laboratories fell within the respective reference ranges for 8 of the 10 substances for each peptide. The within laboratory reproducibility for 12 test substances demonstrated success rates of 83.3% for both Lab 1 and Lab 3, and 100% for Lab 2. The between laboratory reproducibility for 20 test substances showed an 85% concordance rate. The predictive capacity for these substances did not align with the test method similarity criteria stipulated in the OECD Series on Testing and Assessment No. 303 (performance standards for the assessment of proposed similar or modified in vitro skin sensitisation DPRA and ADRA test methods, OECD PS No.303), with accuracy, sensitivity, and specificity rates at 74.6%, 70.9%, and 81.4%, respectively. However, the predictive capacity for an additional 40 substances revealed high accuracy (85.2%), sensitivity (82.2%), and specificity (97.0%). Although the Spectro-DPRA did not completely conform to the OECD PS No.303 criteria for test method similarity, it showed high predictive accuracy of over 80% for the 40 additional substances, confirming its reliability as a test method for skin sensitization evaluation.
Keywords: Validation, Spectro-DPRA, In chemico, Skin sensitization
Introduction
Advances in scientific technology and increased ethical awareness about animal testing have spurred the development of non-animal alternative testing methods [1, 2]. Currently, cutting-edge technologies such as 3D tissue models and artificial intelligence (AI) are being employed to predict toxicity levels, while organ-on-a-chip systems that mimic human tissues are utilized for studying drug effects and toxicity [3, 4]. The accuracy and reliability of these alternative methods are typically confirmed through rigorous scientific validation processes. The creation and optimization of new testing methods, aiming to address their limitations, significantly promote the progress of alternative testing strategies [5].
The test method known as DPRA (Direct Peptide Reactivity Assay), adopted by the Organization for Economic Cooperation and Development (OECD), was developed to predict allergic reactions caused by compounds contacting the skin [6]. This method quantifies the binding of chemicals to cysteine or lysine residues within skin peptides, offering predictions on chemical-induced skin sensitization [7]. Haptenation, a process where chemical compounds bind to amino acids in peptides through oxidation–reduction reactions, is central to this method [8, 9]. The interaction between compounds and peptides is assessed by measuring the peptide depletion relative to controls through high-performance liquid chromatography (HPLC) and ultraviolet (UV) detection [1]. However, such measurements necessitate costly equipment and encounter limitations when processing large numbers of samples within a restricted timeframe.
The Spectro-DPRA, developed by the Amorepacific R&I center, a Korean cosmetic company, improved the conventional DPRA by employing a 96-well plate for simultaneous multiple sample measurement [7, 10, 11]. Following the reaction between the test substance and peptides, the absorbance (for cysteine reactions) and fluorescence (for lysine reactions) are determined using the colorimetric agent 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB) and fluorescamine respectively. In the cysteine peptide response, the residual thiol groups of cysteine peptides react with DTNB to form TNB derivatives, whose absorbance is further measured. Likewise, the remaining free amino groups in lysine peptides react with fluorescamine to form fluorescent products that are subsequently measured [11, 12]. The Spectro-DPRA assesses the extent of peptide depletion by comparing absorbance and fluorescence measurements before and after the reaction [13]. To be acknowledged as a credible alternative improving upon DPRA for accuracy and reliability, Spectro-DPRA must undergo validation studies as prescribed by the OECD guidelines for development and validation of alternative animal testing methods.
This validation study was overseen by the Validation Management Team (VMT) of the Korean Center for the Validation of Alternative Methods (KoCVAM), with collaborations from four laboratories compliant with Good Laboratory Practice (GLP). The evaluation followed a sequence of transferability and proficiency testing with standard substances, leading to assessments of within and between laboratory reproducibilities. Lastly, the model's predictive capability for skin sensitization was tested on an additional 40 substances.
Materials and methods
Test substances
Cysteine (Ac-RFAACAA-COOH, Purity ≥ 95%, MW: 750.35 g/mol) and lysine (Ac-RFAAKAA-COOH, Purity ≥ 95%, MW: 775.45 g/mol) peptides were synthesized by Peptron Co., Ltd. (Daejeon, South Korea). The test substances were chosen based on results from the Local Lymph Node Assay (LLNA) and OECD TG 442C. For the assessment of within and between laboratory reproducibilities, substances were identified using OECD PS No.303 [14]. An additional 40 test substances were selected by examining their chemical properties and the presence of specific functional groups (–NH2, –SH) among those tested for skin sensitization, including OECD TG 442C. The substances employed in the validation study are listed in Table 1. They were dissolved in isopropanol at a concentration of 40 mM. If insoluble in isopropanol, they were dissolved in triple-distilled water. Positive control groups (DNCB for cysteine, Glutaric dialdehyde for lysine) and a negative control group (Glycerol) were also dissolved in isopropanol at the same concentration. All test substances were coded by KoCVAM, and distributed to each participating laboratory. Test substances were freshly prepared on the day of the experiment.
Table 1.
List of substances tested in this study
| No. | Test substance | Chemical information | In vivo prediction (LLNA) | DPRA predictionc | Used | |||
|---|---|---|---|---|---|---|---|---|
| CAS No | M.W | Physical state | EC3 (%)a | Resultsb | ||||
| 1 | Oxazolone | 15646-46-5 | 217.22 | Solid | 0.003 | Extreme | Sensitizer | Transferability and proficiency test |
| 2 | Trimellitic anhydride | 552-30-7 | 192.13 | Solid | 0.6 | Strong | Sensitizer | |
| 3 | Benzylideneacetone | 122-57-6 | 146.186 | Solid | 3.7 | Moderate | Sensitizer | |
| 4 | 1-Butanol | 71-36-3 | 74.122 | Liquid | NCd | Non-sensitizer | Non-sensitizer | |
| 5 | 6-Methylcoumarin | 92-48-8 | 160.169 | Solid | NC | Non-sensitizer | Non-sensitizer | |
| 6 | 2,4-Heptadienal | 4313-03-05 | 110.15 | Liquid | 4 | Moderate | Sensitizer | Proficiency test |
| 7 | Glyoxal solution | 107-22-2 | 58.04 | Liquid | 1.4 | Moderate | Sensitizer | |
| 8 | Ethylene glycol dimethacrylate | 97-90-5 | 198.216 | Liquid | 28 | Weak | Sensitizer | |
| 9 | Lactic Acid | 50-21-5 | 90.078 | Liquid | -0.65 | NC | Non-sensitizer | |
| 10 | 4-Methoxyacetophenone | 100-06-1 | 150.174 | Solid | 1.75 | NC | Non-sensitizer | |
| 11 | Lauryl gallate(dodecyl gallate) | 1166-52-5 | 338.44 | Solid | 0.6 | Strong | Sensitizer | Within and between laboratories test |
| 12 | Chloramine T trihydrate | 127-65-1 (7080-50-4) | 227.64 (281.69) | Solid | 0.4 | Strong | Sensitizer | |
| 13 | 4-(Methylamino)phenol sulfate | 55-55-0 | 344.38 (172.19) | Solid | 0.8 | Strong | Sensitizer | |
| 14 | 2-Mercaptobenzothiazole | 149-30-4 | 167.25 | Solid | 1.7 | Moderate | Sensitizer | |
| 15 | Benzyl salicylate | 118-58-1 | 228.25 | Liquid | 2.9 | Moderate | S/NSe | |
| 16 | Cinnamaldehyde | 14371-10-9 | 132.16 | Liquid | 3 | Moderate | Sensitizer | |
| 17 | Imidazolidinyl urea | 39236-46-9 | 388.293 | Solid | 24 | Weak | Sensitizer | |
| 18 | Ethyl acrylate | 140-88-5 | 100.117 | Liquid | 32.8 | Weak | Sensitizer | |
| 19 | Salicylic acid | 69-72-7 | 138.12 | Solid | NC | Non-sensitizer | Sensitizer | |
| 20 | Benzyl alcohol | 100-51-6 | 108.14 | Liquid | NC | Non-sensitizer | S/NS | |
| 21 | Glycerol | 56-81-5 | 92.09 | Liquid | NC | Non-sensitizer | Non-sensitizer | |
| 22 | Isopropanol | 67-63-0 | 60.1 | Liquid | NC | Non-sensitizer | Non-sensitizer | |
| 23 | p-Benzoquinone | 106-51-4 | 108.095 | Solid | 0.0099 | Extreme | Sensitizer | Between laboratory Test |
| 24 | Dihydroeugenol | 2785-87-7 | 166.217 | Liquid | 6.8 | Moderate | S/NS | |
| 25 | Palmitoyl chloride | 112-67-4 | 274.87 | Liquid | 8.8 | Moderate | Sensitizer | |
| 26 | Farnesal | 19317-11-4 | 220.35 | Liquid | 12 | Weak | Sensitizer | |
| 27 | Benzyl cinnamate | 103-41-3 | 238.28 | Solid | 18.4 | Weak | Non-sensitizer | |
| 28 | Dimethyl isophthalate | 1459-93-4 | 194.19 | Solid | NC | Non-sensitizing | Non-sensitizer | |
| 29 | Methyl salicylate | 119-36-8 | 152.15 | Liquid | NC | Non-sensitizing | S/NS | |
| 30 | 4-Aminobenzoic acid | 150-13-0 | 137.136 | Solid | NC | Non-sensitizing | Non-sensitizer | |
| 31 | Diphenylcyclopropenone | 886-38-4 | 206.24 | Solid | 0.0003 | Extreme | Sensitizer | Further evaluation of predictive tests |
| 32 | 4-Nitrobenzyl bromide | 100-11-8 | 216 | Solid | 0.05 | Extreme | Sensitizer | |
| 33 | Benzoyl peroxide | 94-36-0 | 242.2 | Solid | 0.06 | Extreme | Sensitizer | |
| 34 | Potassium dichromate | 7778-50-9 | 294.2 | Solid | 0.08 | Extreme | Sensitizer | |
| 35 | Tetrachlorosalicylanilide | 1154-59-2 | 351.01 | Solid | 0.04 | Extreme | Sensitizer | |
| 36 | 2-Methyl-2H-isothiazol-3-one(Methylisothiazolinone) | 2682-20-4 | 115.15 | Solid | 0.4 | Strong | Sensitizer | |
| 37 | Phthalic anhydride | 85-44-9 | 148.1 | Solid | 0.36 | Strong | Sensitizer | |
| 38 | Benzyl bromide | 100-39-0 | 171 | Liquid | 0.2 | Strong | Sensitizer | |
| 39 | Iodopropynyl butylcarbamate | 55406-53-6 | 281.1 | Solid | 0.87 | Strong | Sensitizer | |
| 40 | Fluorescein-5-isothiocyanate | 3326-32-7 | 389.38 | Solid | 0.14 | Strong | Sensitizer | |
| 41 | Toluene-2,5-Diamine sulphate | 615-50-9 | 220.25 | Solid | 0.31 | Strong | Sensitizer | |
| 42 | 1,4-Phenylenediamine | 106-50-3 | 108.1 | Solid | 0.16 | Strong | Sensitizer | |
| 43 | 1,4-Dihydroquinone (Hydroquinone) | 123-31-9 | 110.1 | Solid | 0.11 | Strong | Sensitizer | |
| 44 | m-Aminophenol | 591-27-5 | 109.1 | Solid | 3.2 | Moderate | Non-sensitizer | |
| 45 | Methyl-2-nonynoate | 111-80-8 | 168.2 | Liquid | 2.5 | Moderate | Sensitizer | |
| 46 | Safranal | 116-26-7 | 150.2 | Solid | 7.5 | Moderate | Sensitizer | |
| 47 | Methyldibromo glutaronitrile | 35691-65-7 | 265.9 | Solid | 5.2 | Moderate | Sensitizer | |
| 48 | trans-2-Hexenal | 6728-26-3 | 98.1 | Liquid | 5.5 | Moderate | Sensitizer | |
| 49 | Bisphenol A-diglycidyl ether | 1675-54-3 | 340.41 | Liquid | 1.5 | Moderate | Sensitizer | |
| 50 | Cinnamic aldehyde | 104-55-2 | 132.2 | Liquid | 3.1 | Moderate | Sensitizer | |
| 51 | 2-Hydroxyethyl acrylate | 818-61-1 | 116.1 | Liquid | 1.4 | Moderate | Sensitizer | |
| 52 | 2-Phenylpropionaldehyde | 93-53-8 | 134.18 | Liquid | 6.3 | Moderate | Sensitizer | |
| 53 | Propyl gallate | 121-79-9 | 212.2 | Solid | 0.32 | Moderate | Sensitizer | |
| 54 | Isoeugenol | 97-54-1 | 164.2 | Liquid | 1.3 | Moderate | Sensitizer | |
| 55 | 4-Allylanisole | 140-67-0 | 148.2 | Liquid | 20.15 | Weak | Sensitizer | |
| 56 | 5-Methyl-2,3-hexanedione | 13706-86-0 | 128.17 | Liquid | 26 | Weak | Sensitizer | |
| 57 | Lilial | 80-54-6 | 204.3 | Liquid | 18.7 | Weak | Sensitizer | |
| 58 | 2,3-Butanedione | 431-03-8 | 86.1 | Liquid | 11 | Weak | Sensitizer | |
| 59 | Eugenol | 97-53-0 | 164.2 | Liquid | 13 | Weak | Sensitizer | |
| 60 | Citral | 5392-40-5 | 152.2 | Liquid | 13 | Weak | Sensitizer | |
| 61 | Abietic acid | 514-10-3 | 302.5 | Solid | 11 | Weak | Sensitizer | |
| 62 | Cyclamen aldehyde | 103-95-7 | 190.3 | Liquid | 21 | Weak | Sensitizer | |
| 63 | Propyl paraben | 94-13-3 | 180.2 | Solid | NC | Non-sensitizing | Non-sensitizer | |
| 64 | Propylene glycol | 57-55-6 | 76.1 | Liquid | NC | Non-sensitizing | Non-sensitizer | |
| 65 | Diethyl phthalate | 84-66-2 | 222.2 | Liquid | NC | Non-sensitizing | Non-sensitizer | |
| 66 | Coumarin | 91-64-5 | 146.14 | Solid | NC | Non-sensitizing | Non-sensitizer | |
| 67 | Chlorobenzene | 108-90-7 | 112.6 | Liquid | NC | Non-sensitizing | Non-sensitizer | |
| 68 | Hexane | 110-54-3 | 86.18 | Liquid | NC | Non-sensitizing | Non-sensitizer | |
| 69 | Streptomycin sulfate | 3810-74-0 | 728.7 | Solid | NC | Non-sensitizing | Non-sensitizer | |
| 70 | Clofibrate | 637-07-0 | 242.7 | Liquid | NC | Non-sensitizing | Non-sensitizer | |
For the assessment of between and within laboratory reproducibility, substances were identified using OECD PS No. 303 [14]. An additional 40 test substances were selected by examining their chemical properties and the presence of specific functional groups (–NH2, –SH) among those tested for skin sensitization, including OECD TG 442C (Table 6)
a,bIn vivo predictive capacity relies on the LLNA data and the classification criteria proposed by ECETOC
cThe predictive capacity of DPRA is based on data derived from tests conducted in accordance with OECD test guideline 442C
dNC: not classified
eS/NS: Sensitizer/Non-sensitizer
Measurement of cysteine peptide depletion
Peptide solutions were freshly prepared on the day of the experiment. Cysteine peptide was dissolved in DMSO (Sigma Aldrich, St. Louis, USA) to a concentration of 10 mM. A final concentration of 400 µM was achieved by adding the 100 mM phosphate buffer solution (with 1 mM EDTA, pH 8) to the 10 mM cysteine peptide stock. Peptides and test substances were dispensed at 90 µL per well in a 96-well plate (Falcon, Cat. No. 353072), and the plate was incubated at room temperature (22 ± 2℃) for 24 h.
After 24 h, to assess cysteine reactivity, 5,5'-dithiobis 2-nitrobenzoic acid (DTNB; Dojindo laboratories, Kumamoto, Japan), a detection reagent, was prepared in 100 mM phosphate buffer solution (with EDTA, pH 8) at a concentration of 5 mM, and 20 µL was added per well. The reaction proceeded for 3 min. Absorbance measurements before and after DTNB addition were conducted at 412 nm using a spectrophotometer (SPECTRAMAX M2e Multi-Mode Microplate Reader, MOLECULAR DEVICE, California, USA).
Measurement of lysine peptide depletion
The lysine peptide was dissolved in triple distilled water to a concentration of 10 mM. A final concentration of 200 µM was achieved by adding the 100 mM phosphate buffer solution (containing 1 mM EDTA, pH 10) to the 10 mM lysine peptide stock. Peptides and test substances were dispensed at 110 µL per well in a polyethylene 96-well plate (Falcon, Cat. no 351190, NY, USA), and the plate was incubated at room temperature (22 ± 2℃) for 24 h.
After 24 h, to evaluate lysine reactivity, lysine peptide and test substances were transferred to a black polyethylene 96-well plate (Falcon, Cat. no 351190, NY, USA), with 180 µL per well. Subsequently, 10 mM fluorescamine (Sigma-Aldrich) dissolved in DMSO was added at 20 µL per well, and the reaction was allowed to proceed for 3 min. The RFU (Relative Fluorescence Units) before and after the addition of fluorescamine were measured using a fluorometer (SPECTRAMAX M2e Multi-Mode Microplate Reader) at an excitation wavelength of 390 nm and an emission wavelength of 465 nm.
Calculation of peptide depletion ratio
Some substances may have inherent colors or react with peptides and detection reagents, potentially affecting absorbance (OD) or fluorescence (RFU). Thus, the values before the reaction were corrected and calculated. The peptide depletion rate was determined as depicted in Fig. 1.
Fig. 1.
Calculating the depletion ratio in the spectrophotometric direct peptide reactivity assay. The spectrophotometric value change (SV) was calculated by measuring optical density (OD) or fluorescence intensity (Fl) immediately before and after the addition of the detection reagent. SVC,D and SVCD,DD represent the spectrophotometric values before and after addition of the detection reagent to the peptide-only and solvent-only controls, respectively. SVA and SVAD denote the spectrophotometric values before and after addition of the detection reagent to reaction solutions involving peptide and chemical, respectively. SVB and SVBD indicate the spectrophotometric values before and after addition of the detection reagent to the chemical-only control, respectively
The calculated peptide depletion rates were represented as the mean ± SD derived from three validated tests. Each validation was determined through more than 2 repeated runs.
If the peptide depletion rate exceeded 100, it was adjusted to 100, and if negative values occurred, they were corrected to 0. A result was considered positive if the cysteine peptide depletion rate was equal to or greater than 8.85%, or if the lysine peptide depletion rate was equal to or greater than 14.44%. These specific ranges were determined through statistical analysis of preliminary validation study results (data not shown).
Data acceptance criteria
The validity of the data was established based on the following criteria:
If the positive and negative judgments of the first and second tests match, the test is terminated. If they do not match, a third test is conducted to finalize the judgment.
The coefficient (r2) on the peptide standard curve should be > 0.99.
The peptide depletion rates (%) for the positive controls (DNCB for cysteine, Glutaric dialdehyde for lysine) and negative control (glycerol) should meet the following criteria. (a) Positive controls; DNCB (93.82–100), Glutaric dialdehyde (97.62–100), (b) negative control; glycerol (0.0–8.34 for cysteine and 0.0–13.07 for lysine).
In repeated experiments, the standard deviation should be ≤ 10.75% for cysteine and ≤ 14.96% for lysine.
If the value of each peptide depletion rate falls on the borderline between positive and negative, a retest is necessary for a definitive positive or negative judgment. a) 7.23 ≤ average % cysteine peptide depletion rate ≤ 12.02. b) 13.67 ≤ average % lysine peptide depletion rate ≤ 35.54.
Transferability test
Lab 1 (AP, Amorepacific R&I center), a participating and leading laboratory, conducted method transfer training according to the Standard Operating Procedure (ver. 1.3) of Spectro-DPRA for the Korea Testing and Research Institute for Chemical Convergence Lab 2 (KTR, Korea Testing & Research Institute), Lab 3 (CorestemChemon Corporation), and Lab 4 (KCL, Korea Conformity Laboratories, only participating in proficiency test). Transferability was considered complete if the predicted results from the peptide depletion rates of cysteine and lysine peptides matched 100% with those of the LLNA (in vivo predictive capacity) using three sensitizers and two non-sensitizers. This assessment was conducted in a non-blind manner.
Proficiency test
Laboratories that passed the transferability test can proceed with the proficiency test. The test substances involve six sensitizers and four non-sensitizers. It is considered complete if the predicted results from the peptide depletion rates of cysteine and lysine peptides align with the LLNA (in vivo prediction) for eight or more substances and if the depletion rate of test substances falls within the specific ranges provided. These specific ranges were determined through statistical analysis of preliminary validation study results. Proficiency evaluations were conducted in a non-blind manner.
Evaluation of within and between laboratory reproducibility
The within laboratory reproducibility assessment used 12 substances, and was considered complete if the results were consistent for at least 10 out of these 12 substances (at least 80.0% of the predictions based on the performance standard criteria). And, between laboratory reproducibility was evaluated using 20 substances (including the 12 substances used in within laboratory reproducibility), and deemed complete if results were consistent for more than 16 out of 20 (at least 80.0% of the predictions). Both within and between laboratory reproducibility assessments were conducted in a blinded manner. Statistical analyses of reproducibility assessments were performed using SAS version 9.4 (SAS Institute) for Kappa statistics.
Additional evaluation of predictive capacity
Furthermore, 32 sensitizers and 8 non-sensitizers were selected to evaluate the predictive capacity of the Spectro-DPRA in blind methods. At least two laboratories assessed each test substance to ensure data reliability, with test substances evenly distributed among the labs.
Result
Transferability and proficiency test
The transferability test was conducted with 5 substances across 4 laboratories through a non-blind method. All 4 laboratories showed r2 > 0.99 from peptide standard curves. All participating laboratories showed 100% concordance with the in vivo sensitization data for the 5 proficiency substances (data not shown). For the proficiency test, 10 substances were evaluated using 2 qualified tests in 4 laboratories. The criteria range for each peptide’s depletion rate for 10 proficiency substances were established as mean ± 2SD values of raw data, obtained from pre-validation test and subsequent additional experiments. The results indicated 100% concordance with the in vivo sensitization data for all 10 substances. Regarding agreement on the criteria range of each peptide depletion rate, all laboratories demonstrated over 80% consistency (Table 2).
Table 2.
Criteria ranges of substances and results for proficiency tests
| No. | Chemical name | LLNA result | Criteria ranges (%) | Lab 1 | Lab 2 | Lab 3 | Lab 4 | |||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1st Run 2nd Run | 1st Run 2nd Run | 1st Run 2nd Run | 1st Run 2nd Run | |||||||||
| CysteineLysine | Cysteine Lysine |
Cysteine Lysine |
Cysteine Lysine |
Cysteine Lysine |
Cysteine Lysine |
Cysteine Lysine |
Cysteine Lysine |
|||||
| 1 | Oxazolone | S |
42.8 ≤ CDR ≤ 78.1 |
95.8 ≤ LDR ≤ 100 |
62.0 | 57.7 | 49.2 | 51.7 | 69.8 | 61.5 | 43.9 | 44.4 |
| 99.1 | 96.4 | 98.5 | 98.0 | 99.5 | 99.4 | 99.7 | 99.7 | |||||
| 2 | Trimellitic anhydride | S |
0.0 ≤ CDR ≤ 0.6 |
90.4 ≤ LDR ≤ 100 |
0.0 | 0.0 | 0.7 | 0.0 | 0.0 | 0.0 | 5.6* | 10.4* |
| 98.9 | 99.9 | 99.8 | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 | |||||
| 3 | Benzylideneacetone | S |
24.0 ≤ CDR ≤ 100 |
0.0 ≤ LDR ≤ 23.0 |
88.9 | 87.5 | 74.2 | 76.6 | 86.5 | 87.9 | 83.3 | 83.2 |
| 1.1 | 6.6 | 9.8 | 0.0 | 0.0 | 0.0 | 10.2 | 13.9 | |||||
| 4 | 2,4-Heptadienal | S |
92.8 ≤ CDR ≤ 100 |
67.6 ≤ LDR ≤ 100 |
91.3* | 90.6* | 99.9 | 98.1 | 98.9 | 99.4 | 97.5 | 96.4 |
| 69.0 | 40.2* | 96.3 | 76.5 | 80.9 | 89.6 | 80.6 | 77.4 | |||||
| 5 | Glyoxal solution | S |
38.5 ≤ CDR ≤ 100 |
36.6 ≤ LDR ≤ 79.7 |
60.7 | 68.4 | 81.0 | 77.7 | 74.4 | 73.5 | 76.5 | 75.3 |
| 61.1 | 63.3 | 66.9 | 66.8 | 43.7 | 58.8 | 62.5 | 70.1 | |||||
| 6 | Ethylene glycol dimethacrylate | S |
71.5 ≤ CDR ≤ 99.8 |
0.0 ≤ LDR ≤ 7.9 |
74.2 | 75.6 | 82.6 | 85.2 | 83.0 | 84.2 | 93.8 | 94.1 |
| 0.0 | 2.8 | 3.0 | 5.3 | 0.0 | 0.0 | 21.0* | 7.3 | |||||
| 7 | 1-Butanol | NS |
0.0 ≤ CDR ≤ 4.6 |
0.0 ≤ LDR ≤ 9.4 |
0.2 | 4.1 | 0.0 | 1.6 | 1.1 | 2.5 | 4.1 | 4.5 |
| 0.0 | 5.3 | 3.4 | 0.3 | 0.0 | 0.0 | 6.5 | 3.0 | |||||
| 8 | 6-Methylcoumarin | NS |
0.0 ≤ CDR ≤ 8.0 |
0.0 ≤ LDR ≤ 13.6 |
5.7 | 3.4 | 0.0 | 6.1 | 0.0 | 2.0 | 1.5 | 4.9 |
| 5.9 | 4.6 | 5.3 | 2.0 | 0.0 | 0.0 | 8.6 | 10.0 | |||||
| 9 | Lactic Acid | NS |
0.0 ≤ CDR ≤ 8.7 |
0.0 ≤ LDR ≤ 11.3 |
2.1 | 3.4 | 0.7 | 0.0 | 0.8 | 4.5 | 4.8 | 7.6 |
| 3.5 | 9.5 | 8.2 | 2.0 | 0.0 | 0.0 | 10.5 | 2.2 | |||||
| 10 | 4-Methoxyacetophenone | NS |
0.0 ≤ CDR ≤ 8.0 |
0.0 ≤ LDR ≤ 13.6 |
3.1 | 0.3 | 4.0 | 8.7* | 2.3 | 5.1 | 4.5 | 7.1 |
| 3.0 | 2.3 | 1.4 | 0.0 | 0.0 | 0.0 | 10.3 | 0.0 | |||||
CDR cysteine depletion rate, LDR lysine depletion rate, *data out of the criteria ranges
Most of the six positive substances were deemed positive due to high cysteine peptide depletion rates. In the case of trimellitic anhydride, it was negative in the cysteine peptide assay but positive in the lysine peptide assay, ultimately being judged as positive. All 4 non-sensitizers were negative, exhibiting depletion rates below the established criteria (cysteine: 8.85% or lysine: 14.44%) for both peptides.
Evaluation of reproducibility within and between laboratories
The within laboratory reproducibility was assessed using 12 test substances through a non-blind method, with peptide depletion rates verified in at least 2 qualified tests across 3 participating laboratories (Lab 1, Lab 2, and Lab 3). Among the 12 substances, Lab 1 confirmed consistent results for 10 substances (excluding lauryl gallate and salicylic acid) across 3 qualified tests (6 repeated experiments). Lauryl gallate was excluded due to ineffective outcomes. Lab 2 consistently showed positive results for all 12 substances across 3 qualified tests (6 repetitions). Lab 3 demonstrated consistent results for 10 substances (excluding glycerol and isopropanol) in 3 qualified tests (6 repetitions). In comparison with in vivo results, Lab 1 showed consistency for 10 substances (83.3%), Lab 2 for 11 substances (91.7%), and Lab 3 for 10 substances (83.3%), excluding 2 (lauryl gallate and benzyl salicylate), 1 (benzyl salicylate), and 2 (benzyl salicylate and glycerol), respectively (Table 3).
Table 3.
Results of the within laboratory validation study
| No. | Test chemicals | Lab 1 | Lab 2 | ||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1st qualified test | 2nd qualified test | 3rd qualified test | 1st qualified test | 2nd qualified test | 3rd qualified test | ||||||||||||||
| CDR | LDR | Result | CDR | LDR | Result | CDR | LDR | Result | CDR | LDR | Result | CDR | LDR | Result | CDR | LDR | Result | ||
| 1 | Lauryl gallate | 3.8 | 58.1 | S | 4.6 | 73.2 | S | - | - | - | 16.2 | 91.6 | S | 12.7 | 94.5 | S | 12.3 | 96.2 | S |
| 2 | Chloramine T trihydrate | 96.3 | 97.5 | S | 97.6 | 97.4 | S | 98 | 96.6 | S | 92.9 | 96.8 | S | 97.1 | 94.2 | S | 97.2 | 95.1 | S |
| 3 | 4-(Methylamino)phenol sulfate | 97.2 | 88.8 | S | 99.4 | 81.3 | S | 94.4 | 83.7 | S | 97.7 | 99.9 | S | 99.8 | 99.5 | S | 97.0 | 99.6 | S |
| 4 | 2-Mercaptobenzothiazole | 35 | 9.3 | S | 33.6 | 10.9 | S | 24.7 | 6.5 | S | 35.5 | 4.9 | S | 33.6 | 6.0 | S | 43.5 | 7.0 | S |
| 5 | Benzyl salicylate | 4.4 | 3.4 | NS | 1.9 | 4.4 | NS | 0.7 | 4.2 | NS | 4.0 | 1.1 | NS | 0.7 | 4.2 | NS | 3.2 | 4.6 | NS |
| 6 | Cinnamaldehyde | 62 | 18.9 | S | 59.3 | 13.5 | S | 60.2 | 14.6 | S | 25.2 | 1.1 | S | 51.5 | 5.5 | S | 49.6 | 6.7 | S |
| 7 | Imidazolidinyl urea | 0 | 53.6 | S | 0 | 39.5 | S | 0 | 37.8 | S | 0.0 | 42.7 | S | 2.2 | 37.2 | S | 0.0 | 41.7 | S |
| 8 | Ethyl acrylate | 99.7 | 16.2 | S | 100 | 10.9 | S | 98.6 | 6 | S | 85.5 | 91.5 | S | 24.3 | 0.8 | S | 30.9 | 3.7 | S |
| 9 | Salicylic acid | 2.4 | 7.1 | NS | 1 | 17.1 | S | 1.2 | 4.7 | NS | 3.3 | 8.9 | NS | 6.4 | 2.6 | NS | 6.8 | 2.9 | NS |
| 10 | Benzyl alcohol | 1.6 | 3.3 | NS | 4.6 | 2 | NS | 1 | 2.9 | NS | 1.9 | 4.8 | NS | 0.0 | 4.5 | NS | 3.3 | 5.5 | NS |
| 11 | Glycerol | 0.4 | 0.6 | NS | 2.7 | 8 | NS | 0.9 | 1.3 | NS | 0.1 | 4.6 | NS | 2.9 | 2.1 | NS | 1.9 | 4.4 | NS |
| 12 | Isopropanol | 3.8 | 1.1 | NS | 5.1 | 0.8 | NS | 1.2 | 2 | NS | 2.0 | 5.2 | NS | 4.2 | 3.5 | NS | 5.8 | 3.9 | NS |
| Reproducibility | 83.3% | 100% | |||||||||||||||||
| No. | Test chemicals | Lab 3 | LLNA result | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 1st qualified test | 2nd qualified test | 3rd qualified test | |||||||||
| CDR | LDR | Result | CDR | LDR | Result | CDR | LDR | Result | |||
| 1 | Lauryl gallate | 51.5 | 87.2 | S | 48.7 | 88.3 | S | 56.9 | 83.8 | S | S |
| 2 | Chloramine T trihydrate | 99.2 | 93.6 | S | 99.2 | 96.8 | S | 99.3 | 95.8 | S | S |
| 3 | 4-(Methylamino)phenol sulfate | 99.4 | 99.3 | S | 96.5 | 99.8 | S | 93.5 | 99.9 | S | S |
| 4 | 2-Mercaptobenzothiazole | 43.2 | 2.5 | S | 36.9 | 0 | S | 30.4 | 0 | S | S |
| 5 | Benzyl salicylate | 2.4 | 1.3 | NS | 0.9 | 0 | NS | 1.3 | 4.2 | NS | S |
| 6 | Cinnamaldehyde | 56.1 | 0 | S | 49.9 | 0 | S | 57.3 | 6.3 | S | S |
| 7 | Imidazolidinyl urea | 0 | 31.5 | S | 0 | 39.3 | S | 0 | 37.2 | S | S |
| 8 | Ethyl acrylate | 30.9 | 0 | S | 30 | 0 | S | 22.8 | 0 | S | S |
| 9 | Salicylic acid | 3.7 | 3.7 | NS | 0.2 | 0 | NS | 2.3 | 0 | NS | NS |
| 10 | Benzyl alcohol | 0.1 | 1.4 | NS | 2 | 0 | NS | 0.5 | 0 | NS | NS |
| 11 | Glycerol | 25.1 | 0.8 | S | 26.7 | 0 | S | 0.4 | 0 | NS | NS |
| 12 | Isopropanol | 0.3 | 0.3 | NS | 17.8 | 0 | NS | 0.1 | 0 | NS | NS |
| Reproducibility | 83.3% | ||||||||||
CDR cysteine depletion rate, LDR lysine depletion rate, S sensitizer, NS non-sensitizer
The between laboratory reproducibility was assessed with 20 test substances through a non-blind method, with peptide depletion rates validated through 2 qualified tests in 3 participating laboratories (Lab 1, Lab 2, and Lab 3). Among the 20 substances, 12 were also used in the within laboratory test. Two substances (dihydroeugenol and benzyl salicylate) recorded false negatives, and 4-aminobenzoic acid was a false positive. The reproducibility results from the three laboratories indicated that 17 out of 20 test substances displayed consistency, achieving an 85% reproducibility rate (Table 4).
Table 4.
Results of the between laboratory validation study
| No. | Test substance | Lab 1 | Lab 2 | Lab 3 | Concordance | LLNA result | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| CDR | LDR | Result | CDR | LDR | Result | CDR | LDR | Result | ||||||||||
| Mean | SD | Mean | SD | Mean | SD | Mean | SD | Mean | SD | Mean | SD | |||||||
| 1 | p-Benzoquinone | 100.0 | 0.0 | 90.2 | 4.0 | S | 98.2 | 1.9 | 96.0 | 0.0 | S | 100.0 | 0.0 | 81.8 | 8.7 | S | Yes | S |
| 2 | Lauryl gallate | – | – | – | – | - | 12.7 | 1.5 | 94.5 | 0.4 | S | 48.7 | 37.6 | 88.3 | 2.0 | S | No | S |
| 3 | Chloramine T trihydrate | 96.3 | 0.9 | 97.5 | 0.1 | S | 97.1 | 0.9 | 94.2 | 0.1 | S | 99.2 | 0.6 | 96.8 | 0.5 | S | Yes | S |
| 4 | 4-(Methylamino)phenol sulfate | 97.2 | 0.2 | 88.8 | 2.3 | S | 99.8 | 0.3 | 99.5 | 0.4 | S | 96.5 | 2.4 | 99.8 | 0.2 | S | Yes | S |
| 5 | 2-Mercaptobenzothiazole | 35.0 | 1.5 | 9.3 | 4.3 | S | 33.6 | 0.8 | 6.0 | 6.0 | S | 36.9 | 7.7 | 0.0 | 0.0 | S | Yes | S |
| 6 | Dihydroeugenol | 1.1 | 0.8 | 3.9 | 0.8 | NS | 6.0 | 0.2 | 3.5 | 2.4 | NS | 0.7 | 0.4 | 0.0 | 0.0 | NS | Yes | S |
| 7 | Palmitoyl chloride | 7.6 | 5.1 | 8.5 | 1.9 | NS | 23.2 | 2.4 | 40.7 | 1.3 | S | 7.0 | 4.6 | 0.0 | 0.0 | NS | No | S |
| 8 | Benzyl salicylate | 4.4 | 0.1 | 3.4 | 0.4 | NS | 0.7 | 0.7 | 4.2 | 4.2 | NS | 0.9 | 0.9 | 0.0 | 0.0 | NS | Yes | S |
| 9 | Cinnamaldehyde | 62.0 | 0.3 | 18.9 | 5.0 | S | 51.5 | 0.9 | 5.5 | 1.4 | S | 49.9 | 1.2 | 0.0 | 0.0 | S | Yes | S |
| 10 | Farnesal | 56.6 | 30.8 | 10.3 | 3.7 | S | 97.4 | 2.7 | 9.1 | 2.7 | S | 92.8 | 4.6 | 0.0 | 0.0 | S | Yes | S |
| 11 | Imidazolidinyl urea | 0.0 | 0.0 | 53.6 | 2.0 | S | 2.2 | 2.2 | 37.2 | 0.6 | S | 0.0 | 0.0 | 26.6 | 17.9 | S | Yes | S |
| 12 | Ethyl acrylate | 99.7 | 0.1 | 16.2 | 2.9 | S | 24.3 | 2.8 | 0.8 | 0.8 | S | 30.0 | 9.8 | 0.0 | 0.0 | S | Yes | S |
| 13 | Benzyl cinnamate | 1.8 | 0.3 | 4.4 | 1.9 | NS | 5.0 | 1.6 | 2.0 | 2.0 | NS | 0.0 | 0.0 | 0.0 | 0.0 | NS | Yes | S |
| 14 | Salicylic acid | 2.4 | 1.0 | 7.1 | 3.3 | NS | 6.4 | 0.1 | 2.6 | 2.6 | NS | 0.2 | 0.1 | 0.0 | 0.0 | NS | Yes | NS |
| 15 | Benzyl alcohol | 1.6 | 1.5 | 3.3 | 0.8 | NS | 0.0 | 0.0 | 4.5 | 4.5 | NS | 2.0 | 0.8 | 0.0 | 0.0 | NS | Yes | NS |
| 16 | Glycerol | 0.4 | 0.1 | 0.6 | 0.4 | NS | 2.9 | 0.7 | 2.1 | 2.1 | NS | 18.2 | 13.9 | 0.0 | 0.0 | S | No | NS |
| 17 | Isopropanol | 3.8 | 0.6 | 1.1 | 1.1 | NS | 4.2 | 0.8 | 3.5 | 3.5 | NS | 0.1 | 0.1 | 0.0 | 0.0 | NS | Yes | NS |
| 18 | Dimethyl isophthalate | 3.0 | 1.4 | 4.0 | 4.0 | NS | 2.1 | 2.1 | 10.8 | 2.1 | NS | 0.1 | 0.1 | 0.0 | 0.0 | NS | Yes | NS |
| 19 | Methyl salicylate | 6.4 | 0.6 | 3.9 | 1.6 | NS | 3.0 | 1.1 | 7.3 | 6.3 | NS | 1.7 | 1.6 | 0.9 | 0.9 | NS | Yes | NS |
| 20 | 4-Aminobenzoic acid | 2.5 | 0.5 | 91.1 | 5.1 | S | 2.1 | 2.1 | 62.0 | 25.7 | S | 1.2 | 0.5 | 95.4 | 4.7 | S | No | NS |
| Reproducibility (%) | 85.0 | |||||||||||||||||
CDR cysteine depletion rate, LDR lysine depletion rate
This test demonstrated greater than 80% reproducibility in both within and between laboratory evaluations, thereby meeting the criteria of the proposed performance standard for the evaluation of similar or modified in vitro skin sensitization DPRA and ADRA assays (OECD PS No.303) [14].
Evaluation of predictive capacity within and between laboratories
The predictive capacity results for the 20 test substances (Table 1) revealed that one substance (lauryl gallate), which could not be evaluated by Lab 1 due to precipitation and turbidity, resulted in a positive classification. Among the 20 test substances, 3 substances (dihydroeugenol, benzyl salicylate, benzyl cinnamate) were classified as false negatives, while 1 substance (4-aminobenzoic acid) was classified as a false positive. Of the remaining substances (palmitoyl chloride, imidazolidinylurea, salicylic acid, isopropanol), palmitoyl chloride was classified as false negative by both Lab 1 and Lab 3, whereas imidazolidinylurea and isopropanol were classified as false positive by Lab 2 and Lab 3, respectively. The overall predictive evaluation results from between-laboratory studies were as follows: 74.6% (91/122) accuracy, 70.9% (56/79) sensitivity, 81.4% (35/43) specificity across all labs; Lab 1 had 74.4% accuracy, 68.0% sensitivity, 85.7% specificity; Lab 2 had 80.0% accuracy, 76.9% sensitivity, 85.7% specificity; and Lab 3 had 69.8% accuracy, 67.9% sensitivity, 73.3% specificity, as shown in Table 5.
Table 5.
Predictive results from all runs of the between laboratory validation study
| No. | Test substance | Lab 1 | Result | Lab 2 | Result | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 QT | 1 QT | |||||||||||||||||
| 1st run | 2nd run | 3rd run | 1st run | 2nd run | ||||||||||||||
| CDR | LDR | Pre | CDR | LDR | Pre | CDR | LDR | Pre | CDR | LDR | Pre | CDR | LDR | Pre | ||||
| 1 | p-Benzoquinone | 100 | 94.2 | P | 100 | 86.2 | P | S | 100 | 96 | P | 96.3 | 95.9 | P | S | |||
| 2 | Lauryl gallate | – | – | – | – | 11.2 | 94.8 | P | 14.2 | 94.1 | P | S | ||||||
| 3 | Chloramine T trihydrate | 95.4 | 97.6 | P | 97.2 | 97.3 | P | S | 96.2 | 94 | P | 97.9 | 94.3 | P | S | |||
| 4 | 4-(Methylamino)phenol sulfate | 97.4 | 91.1 | P | 97 | 86.5 | P | S | 99.5 | 99.1 | P | 100 | 99.9 | P | S | |||
| 5 | 2-Mercaptobenzothiazole | 36.4 | 5 | P | 33.5 | 13.5 | P | S | 34.3 | 0 | P | 32.8 | 11.9 | P | S | |||
| 6 | Dihydroeugenol | 1.8 | 3.1 | N | 0.3 | 4.6 | N | NS | 6.1 | 1.1 | N | 5.8 | 5.9 | N | NS | |||
| 7 | Palmitoyl chloride | 14.6 | 6.6 | P | 5.3 | 11.1 | N | 2.8 | 3.1 | N | NS | 25.6 | 39.4 | P | 20.8 | 42 | P | S |
| 8 | Benzyl salicylate | 4.3 | 3 | N | 4.5 | 3.7 | N | NS | 0 | 0 | N | 1.3 | 8.4 | N | NS | |||
| 9 | Cinnamaldehyde | 61.7 | 13.9 | P | 62.2 | 23.8 | P | S | 52.4 | 6.8 | P | 50.5 | 4.1 | P | S | |||
| 10 | Farnesal | 87.3 | 14 | P | 25.8 | 6.6 | P | S | 100 | 11.7 | P | 94.7 | 6.4 | P | S | |||
| 11 | Imidazolidinyl urea | 0 | 51.6 | P | 0 | 55.6 | P | S | 4.4 | 37.7 | P | 0 | 36.6 | P | S | |||
| 12 | Ethyl acrylate | 99.6 | 13.3 | P | 99.8 | 19.1 | P | S | 27 | 1.6 | P | 21.5 | 0 | P | S | |||
| 13 | Benzyl cinnamate | 2 | 2.5 | N | 1.5 | 6.3 | N | NS | 6.5 | 0 | N | 3.4 | 3.9 | N | NS | |||
| 14 | Salicylic acid | 3.4 | 3.8 | N | 1.4 | 10.3 | N | NS | 6.3 | 0 | N | 6.4 | 5.1 | N | NS | |||
| 15 | Benzyl alcohol | 3.1 | 4 | N | 0.1 | 2.5 | N | NS | 0 | 0 | N | 0 | 8.9 | N | NS | |||
| 16 | Glycerol | 0.3 | 0.9 | N | 0.5 | 0.2 | N | NS | 2.2 | 0 | N | 3.5 | 4.2 | N | NS | |||
| 17 | Isopropanol | 3.2 | 2.1 | N | 4.3 | 0 | N | NS | 5 | 0 | N | 3.4 | 6.9 | N | NS | |||
| 18 | Dimethyl isophthalate | 4.3 | 8 | N | 1.6 | 0 | N | NS | 4.2 | 12.8 | N | 0 | 8.7 | N | NS | |||
| 19 | Methyl salicylate | 7 | 2.3 | N | 5.8 | 5.5 | N | NS | 4.1 | 1 | N | 1.9 | 13.6 | N | NS | |||
| 20 | 4-Aminobenzoic acid | 3 | 96.2 | P | 2 | 86 | P | S | 4.1 | 87.6 | P | 0 | 36.3 | P | S | |||
| No. | Test substance | Lab 3 | Result | LLNA result | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 QT | ||||||||||||
| 1 run | 2 run | 3 run | ||||||||||
| CDR | LDR | Pre | CDR | LDR | Pre | CDR | LDR | Pre | ||||
| 1 | p-Benzoquinone | 100 | 90.4 | P | 99.9 | 73.1 | P | S | S | |||
| 2 | Lauryl gallate | 86.3 | 90.3 | P | 11.1 | 86.3 | P | S | S | |||
| 3 | Chloramine T trihydrate | 98.6 | 97.3 | P | 99.7 | 96.3 | P | S | S | |||
| 4 | 4-(Methylamino)phenol sulfate | 98.8 | 99.6 | P | 94.1 | 100 | P | S | S | |||
| 5 | 2-Mercaptobenzothiazole | 29.2 | 0 | P | 44.6 | 0 | P | S | S | |||
| 6 | Dihydroeugenol | 1.1 | 0 | N | 0.3 | 0 | N | NS | S | |||
| 7 | Palmitoyl chloride | 7.4 | 0 | N | 12.5 | 0 | P | 1.2 | 0 | N | NS | S |
| 8 | Benzyl salicylate | 1.7 | 0 | N | 0 | 0 | N | NS | S | |||
| 9 | Cinnamaldehyde | 48.7 | 0 | P | 51.1 | 0 | P | S | S | |||
| 10 | Farnesal | 97.4 | 0 | P | 88.2 | 0 | P | S | S | |||
| 11 | Imidazolidinyl urea | 0 | 38.3 | P | 0 | 1.3 | N | 0 | 40.2 | p | S | S |
| 12 | Ethyl acrylate | 20.2 | 0 | P | 39.8 | 0 | P | S | S | |||
| 13 | Benzyl cinnamate | 0 | 0 | N | 0 | 0 | N | NS | S | |||
| 14 | Salicylic acid | 0.1 | 0 | N | 0.3 | 0 | N | NS | NS | |||
| 15 | Benzyl alcohol | 2.8 | 0 | N | 1.2 | 0 | N | NS | NS | |||
| 16 | Glycerol | 1.3 | 0 | N | 35.3 | 0 | P | 18.1 | 0 | p | S | NS |
| 17 | Isopropanol | 0.1 | 0 | N | 0 | 0 | N | NS | NS | |||
| 18 | Dimethyl isophthalate | 0 | 0 | N | 0.1 | 0 | N | NS | NS | |||
| 19 | Methyl salicylate | 0.1 | 1.8 | N | 3.3 | 0 | N | NS | NS | |||
| 20 | 4-Aminobenzoic acid | 0.7 | 100 | P | 1.6 | 90.7 | P | S | NS | |||
| All run-based results of Lab 1 | All run-based results of Lab 2 | All run-based results of Lab 3 | All run-based results of 3 laboratories | |
|---|---|---|---|---|
| Accuracy (%) | 74.4 | 80.0 | 69.8 | 74.6 |
| Sensitivity (%) | 68.0 | 76.9 | 67.9 | 70.9 |
| Specificity (%) | 85.7 | 85.7 | 73.3 | 81.4 |
QT qualified test, CDR cysteine depletion rate, LDR lysine depletion rate, Pre prediction, P positive, N negative, S sensitizer, NS non-sensitizer
Within- and Between-laboratory reproducibility
Kappa statistics was conducted to evaluate the within- and between-laboratory reproducibility consistency at three laboratories (Table 6). Fleiss' kappa is a measure of reliability used to determine the level of agreement between two or more raters when the assessment method is measured on a categorical scale [15, 16]. Kappa values ranging from 0.21–0.40, 0.41–0.60, 0.61–0.80, and 0.81–1.00 were classified as fair, moderate, good, and very good, respectively [17]. Kappa statistics for three reproducibility evaluations within laboratories were 0.876, 1.000 and 0.880 for Lab 1, 2, and 3, each, indicating very good levels of agreement, respectively, which were statistically significant (p < 0.001). Based on the results, the reproducibility of each laboratory was confirmed. Between-laboratory validation Kappa statistics was 0.860, indicating a very good agreement, and it was statistically significant (p < 0.001).
Table 6.
Within- and between-laboratory reproducibility
| Within laboratory | No. of samples | No. of trials | Fleiss' Kappa | 95% CI | Z-score | p value |
|---|---|---|---|---|---|---|
| Lab 1 | 11* | 3 | 0.876 | (0.647, 1.000) | 5.032 | < 0.001 |
| Lab 2 | 12 | 3 | 1.000 | (1.000, 1.000) | 6.000 | < 0.001 |
| Lab 3 | 12 | 3 | 0.880 | (0.650, 1.000) | 5.278 | < 0.001 |
| Between Laboratory | No. of samples | No. of trials | Fleiss' Kappa | 95% CI | Z-score | p value |
|---|---|---|---|---|---|---|
| 15* | 3 | 0.860 | (0.676, 1.000) | 6.490 | < 0.001 |
*Missing result is excluded from the sample count
Additional evaluation of predictive capacity
The predictive capacity of Spectro-DPRA for an additional 40 test substances (32 sensitizers and 8 non-sensitizers) was compared with ADRA and DPRA results based on LLNA data (Table 7). Six substances (4-Nitrobenzyl bromide, Benzyl bromide, Iodopropynyl butylcarbamate, Methyl-2-nonynoate, Eugenol, Cyclamen aldehyde) were classified as false positives by one of the two participating laboratories. Although the classification results were consistent, three substances (Bisphenol A-diglycidyl ether, 4-Allylanisole, Lilial) did not show agreement in in vivo predictability. The additional predictive assessment for the 40 substances demonstrated an accuracy of 85.2%, sensitivity of 82.2%, and specificity of 97.0%, according to data from all participating laboratory runs (Table 8).
Table 7.
Results of ADRA and LLNA for an additional 40 substances
| No | Test substance | ADRA | LLNA result | ||
|---|---|---|---|---|---|
| NAC | NAL | Result | |||
| 1 | Diphenylcyclopropenone | 28.7 [18] | 6.3 [18] | S | S |
| 2 | 4-Nitrobenzyl bromide | –* | – | – | S |
| 3 | Benzoyl peroxide | 100.0 [18] | 50.6 [18] | S | S |
| 4 | Potassium dichromate | – | – | – | S |
| 5 | Tetrachlorosalicylanilide | 43.4 [18] | 2.1 [18 | S | S |
| 6 | 2-Methyl-2H-isothiazol-3-one(Methylisothiazolinone) | – | – | – | S |
| 7 | Phthalic anhydride | – | – | – | S |
| 8 | Benzyl bromide | – | – | – | S |
| 9 | Iodopropynyl butylcarbamate | – | – | S | |
| 10 | Fluorescein-5-isothiocyanate | 100.0 [19] | 100.0 [19] | S | S |
| 11 | Toluene-2,5-diamine sulphate | – | – | – | S |
| 12 | 1,4-Phenylenediamine | – | – | – | S |
| 13 | 1,4-Dihydroquinone(Hydroquinone) | – | – | – | S |
| 14 | m-Aminophenol | – | – | – | S |
| 15 | Methyl-2-nonynoate | 15.0 [18] | 1.4 [18] | S | S |
| 16 | Safranal | – | – | – | S |
| 17 | Methyldibromo glutaronitrile | – | – | – | S |
| 18 | trans-2-Hexenal | – | – | – | S |
| 19 | Bisphenol A-diglycidyl ether | – | – | – | S |
| 20 | Cinnamic aldehyde | 95.5 [19] | 81.3 [19] | S | S |
| 21 | 2-Hydroxyethyl acrylate | 100.0 [19] | 81.5 [19] | S | S |
| 22 | 2-Phenylpropionaldehyde | 8.0 [18] | 4.6 [18] | S | S |
| 23 | Propyl gallate | 93.7 [19] | 75.2 [19] | S | S |
| 24 | Isoeugenol | – | – | – | S |
| 25 | 4-Allylanisole | 27.0 [18] | 3.9 [18] | S | S |
| 26 | 5-Methyl-2,3-hexanedione | 23.2 [19] | 98.9 [19] | S | S |
| 27 | Lilial | 7.0 [18] | 3.9 [18] | S | S |
| 28 | 2,3-Butanedione | 100.0 [19] | 73.1 [19] | S | S |
| 29 | Eugenol | – | – | – | S |
| 30 | Citral | – | – | – | S |
| 31 | Abieticacid | 32.3 [20] | 0.0 [20] | S | S |
| 32 | Cyclamen aldehyde | 3.1 [18] | 1.7 [18] | NS | S |
| 33 | Propyl paraben | –3.3 [19] | 1.0 [19] | NS | NS |
| 34 | Propylene glycol | –5.0 [19] | –0.2 [19] | NS | NS |
| 35 | Diethyl phthalate | 0.0 [18] | 1.7 [18] | NS | NS |
| 36 | Coumarin | 2.5 [19] | 1.5 [19] | NS | NS |
| 37 | Chlorobenzene | 0.0 [18] | 0.7 [18] | NS | NS |
| 38 | Hexane | 0.2 [18] | 0.9 [18] | NS | NS |
| 39 | Streptomycin sulfate | – | – | – | NS |
| 40 | Clofibrate | 0.2 [20] | 0.4 [20] | NS | NS |
NAC: N-(2-(1-naphthyl)acetyl)-L-cysteine, NAL: α-N-(2-(1-naphthyl)acetyl)-L-lysine
*No ADRA test results
Table 8.
Predictive capacity from all runs of an additional 40 substances
| No. | Test substance | In vivo prediction | Lab 1 | Lab 2 | |||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 qualified test | 1 qualified test | ||||||||||||||||||
| 1 run | Result | 2 run | Result | 3 run | Result | Prediction (Lab 1) | 1 run | Result | 2 run | Result | Prediction (Lab 2) | ||||||||
| CDR | LDR | CDR | LDR | CDR | LDR | CDR | LDR | CDR | LDR | ||||||||||
| 1 | Diphenylcyclopropenone | Extreme | 89.8 | 0.0 | POS | 100.0 | 2.4 | POS | NT | NT | NT | POS | 92.1 | 5.0 | POS | 97.9 | 7.3 | POS | POS |
| 2 | 4-Nitrobenzyl bromide | Extreme | 87.3 | 20.7 | POS | 1.3 | 5.1 | NEG | 2.1 | 9.0 | NEG | NEG | 12.9 | 45.0 | POS | 13.3 | 38.9 | POS | POS |
| 3 | Benzoyl peroxide | Extreme | 8.7 | 41.5 | POS | 5.5 | 39.9 | POS | NT | NT | NT | POS | NT | NT | NT | NT | NT | NT | NT |
| 4 | Potassium dichromate | Extreme | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT | 22.6 | 91.7 | POS | 31.1 | 92.4 | POS | POS |
| 5 | Tetrachlorosalicylanilide | Extreme | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT | -* | -* | -* | -* | -* | -* | -* |
| 6 | 2-Methyl-2H-isothiazol-3-one(Methylisothiazolinone) | Strong | 38.0 | 7.0 | POS | 34.3 | 0.4 | POS | NT | NT | NT | POS | 30.4 | 0.0 | POS | 30.1 | 3.1 | POS | POS |
| 7 | Phthalic anhydride | Strong | 0.0 | 99.6 | POS | 0.0 | 99.7 | POS | NT | NT | NT | POS | 0.0 | 99.8 | POS | 0.0 | 100.0 | POS | POS |
| 8 | Benzyl bromide | Strong | 7.0 | 4.0 | NEG | 1.5 | 4.0 | NEG | NT | NT | NT | NEG | 0.0 | 37.7 | POS | 0.0 | 35.7 | POS | POS |
| 9 | Iodopropynyl butylcarbamate | Strong | 97.3 | 20.1 | POS | 1.5 | 6.1 | NEG | 1.2 | 0.9 | NEG | NEG | NT | NT | NT | NT | NT | NT | NT |
| NT | NT | POS | |||||||||||||||||
| 10 | Fluorescein-5-isothiocyanate | Strong | 61.6 | 98.4 | POS | 36.4 | 97.7 | POS | NT | NT | NT | POS | NT | NT | NT | NT | NT | NT | NT |
| 11 | Toluene-2,5-diamine sulfate | Strong | 89.8 | 78.1 | POS | 91.2 | 87.5 | POS | NT | NT | NT | POS | NT | NT | NT | NT | NT | NT | NT |
| 12 | 1,4-Phenylenediamine | Strong | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT | 99.9 | 96.9 | POS | 98.2 | 98.4 | POS | POS |
| 13 | 1,4-Dihydroquinone(Hydroquinone) | Strong | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT | 100.0 | 97.5 | POS | 100.0 | 94.7 | POS | POS |
| 14 | m-Aminophenol | Moderate | 0.3 | 73.7 | POS | 1.0 | 84.9 | POS | NT | NT | NT | POS | 1.9 | 64.6 | POS | 0.0 | 71.5 | POS | POS |
| 15 | Methyl-2-nonynoate | Moderate | 11.6 | 5.9 | POS | 2.4 | 5.9 | NEG | 1.8 | 0.1 | NEG | NEG | 2.7 | 35.5 | POS | 4.9 | 38.6 | POS | POS |
| 16 | Safranal | Moderate | 96.4 | 33.8 | POS | 98.6 | 15.0 | POS | NT | NT | NT | POS | 99.5 | 13.6 | POS | 100.0 | 12.3 | POS | POS |
| 17 | Methyldibromo glutaronitrile | Moderate | 98.5 | 4.3 | POS | 98.7 | 8.0 | POS | NT | NT | NT | POS | NT | NT | NT | NT | NT | NT | NT |
| 18 | Trans-2-Hexenal | Moderate | 86.7 | 20.6 | POS | 87.0 | 21.1 | POS | NT | NT | NT | POS | NT | NT | NT | NT | NT | NT | NT |
| 19 | Bisphenol A-diglycidyl ether | Moderate | 15.9 | 11.9 | POS | 1.5 | 4.1 | NEG | 1.6 | 2.0 | NEG | NEG | NT | NT | NT | NT | NT | NT | NT |
| 20 | Cinnamic aldehyde | Moderate | 73.1 | 16.0 | POS | 65.0 | 16.6 | POS | NT | NT | NT | POS | NT | NT | NT | NT | NT | NT | NT |
| 21 | 2-Hydroxyethyl acrylate | Moderate | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT | 100.0 | 45.3 | POS | 100.0 | 36.1 | POS | POS |
| 22 | 2-Phenylpropionaldehyde | Moderate | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT | 88.4 | 13.4 | POS | 94.4 | 11.7 | POS | POS |
| 23 | Propyl gallate | Moderate | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT | 95.8 | 95.0 | POS | 95.4 | 94.7 | POS | POS |
| 24 | Isoeugenol | Moderate | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT | 99.9 | 37.7 | POS | 99.9 | 36.6 | POS | POS |
| 25 | 4-Allylanisole | Weak | 5.3 | 8.2 | NEG | 1.9 | 8.2 | NEG | NT | NT | NT | NEG | 6.0 | 4.3 | NEG | 2.3 | 7.8 | NEG | NEG |
| 26 | 5-Methyl-2,3-hexanedione | Weak | 13.8 | 40.6 | POS | 13.3 | 40.7 | POS | NT | NT | NT | POS | 16.1 | 85.4 | POS | 15.7 | 85.1 | POS | POS |
| 27 | Lilial | Weak | 0.0 | 4.4 | NEG | 1.8 | 0.0 | NEG | NT | NT | NT | NEG | 0.0 | 8.3 | NEG | 4.3 | 0.0 | NEG | NEG |
| 28 | 2,3-Butanedione | Weak | 26.9 | 2.0 | POS | 40.0 | 2.8 | POS | NT | NT | NT | POS | NT | NT | NT | NT | NT | NT | NT |
| 29 | Eugenol | Weak | 6.8 | 1.3 | NEG | 4.0 | 1.3 | NEG | NT | NT | NT | NEG | NT | NT | NT | NT | NT | NT | NT |
| 30 | Citral | Weak | 75.1 | 49.9 | POS | 66.4 | 49.8 | POS | NT | NT | NT | POS | NT | NT | NT | NT | NT | NT | NT |
| 31 | Abieticacid | Weak | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT | 84.2 | 8.5 | POS | 89.2 | 3.2 | POS | POS |
| 32 | Cyclamen aldehyde | Weak | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT | 4.9 | 0.0 | NEG | 3.0 | 1.5 | NEG | NEG |
| 33 | Propyl paraben | Non-sensitizer | 1.9 | 13.0 | NEG | 1.3 | 0.0 | NEG | NT | NT | NT | NEG | 5.9 | 4.6 | NEG | 4.3 | 8.6 | NEG | NEG |
| 34 | Propylene glycol | Non-sensitizer | 3.2 | 11.9 | NEG | 0.5 | 1.5 | NEG | NT | NT | NT | NEG | 0.0 | 0.6 | NEG | 1.0 | 0.0 | NEG | NEG |
| 35 | Diethyl phthalate | Non-sensitizer | 0.2 | 2.7 | NEG | 1.9 | 2.8 | NEG | NT | NT | NT | NEG | NT | NT | NT | NT | NT | NT | NT |
| 36 | Coumarin | Non-sensitizer | 0.4 | 3.1 | NEG | 0.0 | 0.0 | NEG | NT | NT | NT | NEG | NT | NT | NT | NT | NT | NT | NT |
| 37 | Chlorobenzene | Non-sensitizer | 5.6 | 6.2 | NEG | 1.2 | 0.0 | NEG | NT | NT | NT | NEG | NT | NT | NT | NT | NT | NT | NT |
| 38 | Hexane | Non-sensitizer | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT | 6.1 | 3.0 | NEG | 3.9 | 3.0 | NEG | NEG |
| 39 | Streptomycin sulfate | Non-sensitizer | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT | 1.0 | 1.0 | NEG | 0.2 | 8.3 | NEG | NEG |
| 40 | Clofibrate | Non-sensitizer | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT | 0.0 | 0.0 | NEG | 2.6 | 0.2 | NEG | NEG |
| No. | Test substance | In vivo prediction | Lab 3 | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 qualified test | ||||||||||||
| 1 run | Result | 2 run | Result | 3 run | Result | Prediction (Lab 3) | ||||||
| CDR | LDR | CDR | LDR | CDR | LDR | |||||||
| 1 | Diphenylcyclopropenone | Extreme | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT |
| 2 | 4-Nitrobenzyl bromide | Extreme | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT |
| 3 | Benzoyl peroxide | Extreme | 2.8 | 34.3 | POS | 2.8 | 29.9 | POS | NT | NT | NT | POS |
| 4 | Potassium dichromate | Extreme | 38.6 | 94.8 | POS | 25.5 | 92.7 | POS | NT | NT | NT | POS |
| 5 | Tetrachlorosalicylanilide | Extreme | 16.7 | 100.0 | POS | 30.3 | 100.0 | POS | NT | NT | NT | POS |
| 6 | 2-Methyl-2H-isothiazol-3-one(Methylisothiazolinone) | Strong | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT |
| 7 | Phthalic anhydride | Strong | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT |
| 8 | Benzyl bromide | Strong | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT |
| 9 | Iodopropynyl butylcarbamate | Strong | 99.0 | 0.0 | POS | 98.9 | 0.0 | POS | NT | NT | NT | |
| NT | NT | |||||||||||
| 10 | Fluorescein-5-isothiocyanate | Strong | 82.3 | 99.9 | POS | 79.9 | 99.8 | POS | NT | NT | NT | POS |
| 11 | Toluene-2,5-diamine sulfate | Strong | 92.3 | 82.9 | POS | 93.3 | 66.8 | POS | NT | NT | NT | POS |
| 12 | 1,4-Phenylenediamine | Strong | 97.9 | 97.8 | POS | 89.1 | 96.2 | POS | NT | NT | NT | POS |
| 13 | 1,4-Dihydroquinone(Hydroquinone) | Strong | 99.7 | 95.8 | POS | 100.0 | 85.2 | POS | NT | NT | NT | POS |
| 14 | m-Aminophenol | Moderate | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT |
| 15 | Methyl-2-nonynoate | Moderate | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT |
| 16 | Safranal | Moderate | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT |
| 17 | Methyldibromo glutaronitrile | Moderate | 99.1 | 0.0 | POS | 97.3 | 0.0 | POS | NT | NT | NT | POS |
| 18 | Trans-2-Hexenal | Moderate | 68.1 | 0.0 | POS | 83.0 | 0.0 | POS | NT | NT | NT | POS |
| 19 | Bisphenol A-diglycidyl ether | Moderate | 0.6 | 0.0 | NEG | 0.0 | 0.0 | NEG | NT | NT | NT | NEG |
| 20 | Cinnamic aldehyde | Moderate | 68.3 | 12.7 | POS | 64.2 | 0.0 | POS | NT | NT | NT | POS |
| 21 | 2-Hydroxyethyl acrylate | Moderate | 99.8 | 0.0 | POS | 99.0 | 0.0 | POS | NT | NT | NT | POS |
| 22 | 2-Phenylpropionaldehyde | Moderate | 98.6 | 2.5 | POS | 93.2 | 0.0 | POS | NT | NT | NT | POS |
| 23 | Propyl gallate | Moderate | 96.3 | 82.2 | POS | 99.8 | 82.6 | POS | NT | NT | NT | POS |
| 24 | Isoeugenol | Moderate | 80.3 | 8.5 | POS | 88.3 | 0.0 | POS | NT | NT | NT | POS |
| 25 | 4-Allylanisole | Weak | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT |
| 26 | 5-Methyl-2,3-hexanedione | Weak | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT |
| 27 | Lilial | Weak | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT |
| 28 | 2,3-Butanedione | Weak | 52.0 | 19.6 | POS | 45.3 | 45.4 | POS | NT | NT | NT | POS |
| 29 | Eugenol | Weak | 12.7 | 0.0 | POS | 13.1 | 0.0 | POS | NT | NT | NT | POS |
| 30 | Citral | Weak | 72.6 | 5.7 | POS | 70.7 | 0.0 | POS | NT | NT | NT | POS |
| 31 | Abieticacid | Weak | 67.3 | 0.0 | POS | 76.2 | 0.0 | POS | NT | NT | NT | POS |
| 32 | Cyclamen aldehyde | Weak | 76.5 | 0.0 | POS | 86.4 | 0.0 | POS | NT | NT | NT | POS |
| 33 | Propyl paraben | Non-sensitizer | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT |
| 34 | Propylene glycol | Non-sensitizer | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT |
| 35 | Diethyl phthalate | Non-sensitizer | 1.4 | 0.0 | NEG | 2.6 | 0.0 | NEG | NT | NT | NT | NEG |
| 36 | Coumarin | Non-sensitizer | 2.1 | 0.0 | NEG | 1.0 | 0.0 | NEG | NT | NT | NT | NEG |
| 37 | Chlorobenzene | Non-sensitizer | 6.7 | 0.0 | NEG | 12.0 | 0.0 | POS | 0.0 | 0.0 | NEG | NEG |
| 38 | Hexane | Non-sensitizer | 1.6 | 0.0 | NEG | 1.4 | 0.0 | NEG | NT | NT | NT | NEG |
| 39 | Streptomycin sulfate | Non-sensitizer | 3.8 | 9.3 | NEG | 0.2 | 0.0 | NEG | NT | NT | NT | NEG |
| 40 | Clofibrate | Non-sensitizer | 3.8 | 0.0 | NEG | 2.7 | 0.0 | NEG | NT | NT | NT | NEG |
| Predictive capacity across all runs | Lab 1 (%) | Lab 2 (%) | Lab 3 (%) | Results from all runs of the three laboratories (%) |
|---|---|---|---|---|
| Accuracy (%) | 73.7 | 88.0 | 94.5 | 85.2 |
| Sensitivity (%) | 68.1 | 85.0 | 95.2 | 82.2 |
| Specificity (%) | 100.0 | 100.0 | 92.3 | 97.0 |
CDR cysteine depletion rate, LDR lysine depletion rate, NT not tested, *results cannot be calculated due to saturation
Discussion
To identify chemicals that induce skin sensitization, several traditional tests are used, including the guinea pig maximization test (GPMT), Buehler occluded patch test, and local lymph node assay (LLNA) [21, 22]. However, with advances in science and increasing ethical concerns about animal experimentation, international efforts to develop alternative testing methods that do not use animals have expanded [11].
The Direct Peptide Reactivity Assay (DPRA) is an alternative test method for evaluating protein reactivity, a key event in the Adverse Outcome Pathway (AOP). It predicts allergic reactions from chemical skin exposure [12, 23, 24]. However, the DPRA requires costly equipment, including High-Performance Liquid Chromatography (HPLC), and analytical expertise [25]. It also has limitations in sample throughput, posing challenges when managing large sample volumes within constrained times.
Spectro-DPRA is an enhanced test method designed to overcome these limitations. It measures the color intensity of remaining peptides post chemical reaction to evaluate early stages of skin sensitization. By using a 96-well plate, Spectro-DPRA allows for the simultaneous evaluation of multiple test chemicals, thereby reducing both time and costs.
The previous study [13] conducted proficiency testing and skill assessment using the optimized test method, confirming over 80% in vivo predictability across four participating laboratories. The within laboratory reproducibility results were 83.3% for Lab 1, 100% for Lab 2, and 83.3% for Lab 3. Benzyl salicylate consistently produced false negatives in three participating laboratories, attributed to precipitation and turbidity that hindered accurate positive results. Glycerol exhibited false positives only in Lab 3. The between laboratory reproducibility evaluation showed 85% consistency. In the between laboratory reproducibility, 17 out of 20 substances demonstrated agreement, excluding three substances: lauryl gallate, palmitoyl chloride, and glycerol, which exhibited precipitation and color change. Both within and between laboratory reproducibility results demonstrated over 80% reproducibility, thereby meeting the criteria for similarity testing evaluation. However, due to precipitation and turbidity affecting predictability evaluations, the standard operating procedures were revised. As a result, lauryl gallate, which initially could not be evaluated due to precipitation and turbidity, was ultimately identified as positive in the final confirmatory result. In assessing the predictive capacity of 40 additional substances, six substances (4-Nitrobenzyl bromide, Benzyl bromide, Iodopropynyl butylcarbamate, Methyl-2-nonynoate, Eugenol, Cyclamen aldehyde) showed inconsistent results in one of the two repeated tests, while three substances (Bisphenol A-diglycidyl ether, 4-Allylanisole, Lilial) yielded false negatives compared to in vivo predictability. The final predictability evaluation yielded high accuracy of 85.2%, sensitivity of 82.2%, and specificity of 97.0%. Although the Spectro-DPRA did not pass the verification study due to its failure to meet the similarity testing criteria in the OECD PS No. 303 predictability evaluation, the high predictability of over 80% for unknown substances in the additional predictability evaluation of 40 substances suggests that Spectro-DPRA holds promise for evaluating skin sensitization. Nevertheless, it is believed that the 20 substances specified in OECD PS No. 303 include some that are unsuitable for evaluation with this test method.
The Spectro-DPRA method is a promising testing technique, albeit with several limitations. It is an in chemico test that omits metabolic pathways related to skin sensitization. Consequently, chemicals that require enzymatic activity for their biological effects cannot be evaluated with this method. Similarly, metal compounds, which form non-covalent interactions with proteins, pose challenges for this test. Additionally, during the analysis of chemicals containing color interference, thiol, and amine groups, direct reactions with detection reagents may occur, necessitating careful data review post-testing. Further consideration may be needed to provide rationale for false negative and false positive in the future. Despite these constraints, the Spectro-DPRA offers a new approach that saves time and cost compared to traditional DPRA or ADRA methods. To improve predictability, it is recommended to conduct supplementary tests such as KeratinoSens™ and h-CLAT following Spectro-DPRA evaluation to confirm sensitization potential [26]. This strategy could provide a reliable and streamlined method for assessing sensitization potential.
Acknowledgements
This research was funded by a grant (21182MFD339) from the Ministry of Food and Drug Safety (MFDS) and by the KoCVAM project for ‘Establishment of national foundation for development of alternative test methods’ in 2023. The validation study was conducted under the guidance of KoCVAM, which facilitated the entire validation process, including organizing the VMT, selecting and distributing test chemicals, and collecting raw data.
Authors’ contribution
All authors contributed to the study design and progress, together. Material preparation, study progress and data production were performed by S-AC, MC, CH, CEP, J-JO, and SA. Data collection and analysis were performed by H-JS, J-YB and B-HK. The first draft of the manuscript was written by H-JS and J-YB, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Funding
This research was funded by a grant (21182MFD339) from the Ministry of Food and Drug Safety (MFDS) and by the KoCVAM project for ‘Establishment of national foundation for development of alternative test methods’ in 2023. The validation study was conducted under the guidance of KoCVAM, which facilitated the entire validation process, including organizing the VMT, selecting and distributing test chemicals, and collecting raw data.
Declarations
Conflict of interest
The authors have no relevant financial or non-financial interests to disclose.
Ethics approval
This is an in chemico study, so ethical approval is not required.
References
- 1.Gerberick F, Aleksic M, Basketter D, Casati S, Karlberg AT, Kern P, Kimber I, Lepoittevin JP, Natsch A, Ovigne JM, Rovida C, Sakaguchi H, Schultz T (2008) Chemical reactivity measurement and the predicitve identification of skin sensitisers. The report and recommendations of ECVAM Workshop 64. Altern Lab Anim ATLA 36:215–242. 10.1177/026119290803600210 [DOI] [PubMed] [Google Scholar]
- 2.Tannenbaum J, Bennett BT (2015) Russell and Burch’s 3Rs then and now: the need for clarity in definition and purpose. J Am Assoc Lab Anim Sci JAALAS 54:120–132 [PMC free article] [PubMed] [Google Scholar]
- 3.Lei Y, Schaffer DV (2013) A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation. Proc Natl Acad Sci USA 110:E5039–E5048. 10.1073/pnas.1309408110 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Shuler ML, Hickman JJ (2014) Toward in vitro models of brain structure and function. Proc Natl Acad Sci USA 111:13682–13683. 10.1073/pnas.1414484111 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Griesinger C, Desprez B, Coecke S, Casey W, Zuang V (2016) Validation of alternative in vitro methods to animal testing: concepts, challenges, processes and tools. Adv Exp Med Biol 856:65–132. 10.1007/978-3-319-33826-2_4 [DOI] [PubMed] [Google Scholar]
- 6.OECD. OECD Guidelines for the Testing of Chemicals (2021) Key event-based test guideline for in chemico skin senditisation assays addressing the adverse outcome pathway key event on covalent binding to proteins. Organization for Economic Co-Operation and Development, Paris
- 7.Cho SA, An S, Park JH (2019) High-throughput screening (HTS)-based spectrophotometric direct peptide reactivity assay (Spectro-DPRA) to predict human skin sensitization potential. Toxicol Lett 314:27–36. 10.1016/j.toxlet.2019.07.014 [DOI] [PubMed] [Google Scholar]
- 8.Ahlfors SR, Sterner O, Hansson C (2003) Reactivity of contact allergenic haptens to amino acid residues in a model carrier peptide, and characterization of formed peptide-hapten adducts. Skin Pharmacol Appl Skin Physiol 16:59–68. 10.1159/000068288 [DOI] [PubMed] [Google Scholar]
- 9.Lalko JF, Kimber I, Gerberick GF, Foertsch LM, Api AM, Dearman RJ (2012) The direct peptide reactivity assay: selectivity of chemical respiratory allergens. Toxicol Sci 129:421–431. 10.1093/toxsci/kfs205 [DOI] [PubMed] [Google Scholar]
- 10.Cho SA, Jeong YH, Kim JH, Kim S, Cho JC, Heo Y, Suh KD, Shin K, An S (2014) Method for detecting the reactivity of chemicals towards peptides as an alternative test method for assessing skin sensitization potential. Toxicol Lett 225:185–191. 10.1016/j.toxlet.2013.12.007 [DOI] [PubMed] [Google Scholar]
- 11.Jeong YH, An S, Shin K, Lee TR (2013) Peptide reactivity assay using spectrophotometric method for high-throughput screening of skin sensitization potential of chemical haptens. Toxicol In Vitro Int J Assoc BIBRA 27:264–271. 10.1016/j.tiv.2012.08.032 [DOI] [PubMed] [Google Scholar]
- 12.Udenfriend S, Stein S, Böhlen P, Dairman W, Leimgruber W, Weigele M (1972) Fluorescamine: a reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science 178:871–872. 10.1126/science.178.4063.871 [DOI] [PubMed] [Google Scholar]
- 13.Seo JA, Cho SA, Park CE, Seo DH, Choi M, An S, Kim BH (2022) Pre-validation study of spectrophotometric direct peptide reactivity assay (Spectro-DPRA) as a modified in chemico skin sensitization test method. Toxicol Res 38:531–544. 10.1007/s43188-022-00130-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.OECD (2023) Series on testing & assessment no. 303 second edition: Performance standards for the assessment of proposed similar or modified in vitro skin sensitization DPRA and ADRA test methods as described in TG 442C. Editions OECD, Paris
- 15.Fleiss JL (1971) Measuring nominal scale agreement among many raters. Psychol Bull 76:378–382. 10.1037/h0031619 [Google Scholar]
- 16.Fleiss JL, Levin B, Paik MC (2003) Statistical methods for rates and proportions, 3rd edn. Wiley, Hoboken [Google Scholar]
- 17.Landis JR, Koch GG (1977) The measurement of observer agreement for categorical data. Biometrics 33:159–174. 10.2307/2529310 [PubMed] [Google Scholar]
- 18.Yamamoto Y, Wanibuchi S, Sato A, Kasahara T, Fujita M (2019) Precipitation of test chemicals in reaction solutions used in the amino acid derivative reactivity assay and the direct peptide reactivity assay. J Pharmacol Toxicol Methods 100:106624. 10.1016/j.vascn.2019.106624 [DOI] [PubMed] [Google Scholar]
- 19.Fujita M, Yamamoto Y, Tahara H, Kasahara T, Jimbo Y, Hioki T (2014) Development of a prediction method for skin sensitization using novel cysteine and lysine derivatives. J Pharmacol Toxicol Methods 70:94–105. 10.1016/j.vascn.2014.06.001 [DOI] [PubMed] [Google Scholar]
- 20.Yamaga H, Watanabe S, Fujita M, Yamamoto Y, Kasahara T, Kataoka S (2021) Amino acid derivative reactivity assay–organic solvent reaction system: a novel alternative test for skin sensitization capable of assessing highly hydrophobic substances. J Appl Toxicol 41:1634–1648. 10.1002/jat.4152 [DOI] [PubMed] [Google Scholar]
- 21.OECD (2022) Test no. 406: skin sensitisation, OECD guidelines for the testing of chemicals, section 4, Éditions OECD, Paris. 10.1787/9789264070660-en
- 22.OECD (2010) Test no. 429: skin sensitisation : local lymph node assay, OECD guidelines for the testing of chemicals, Section 4, Éditions OECD, Paris. 10.1787/9789264071100-en
- 23.Reisinger K, Hoffmann S, Alépée N, Ashikaga T, Barroso J, Elcombe C et al (2015) Systematic evaluation of non-animal test methods for skin sensitisation safety assessment. Toxicol In Vitro 29:259–270. 10.1016/j.tiv.2014.10.018 [DOI] [PubMed] [Google Scholar]
- 24.Roberts DW (2018) Is a combination of assays really needed for non-animal prediction of skin sensitization potential? Performance of the GARD™(Genomic Allergen Rapid Detection) assay in comparison with OECD guideline assays alone and in combination. Regul Toxicol Pharmacol 98:155–160. 10.1016/j.yrtph.2018.07.014 [DOI] [PubMed] [Google Scholar]
- 25.Zhang F, Erskine T, Klapacz J, Settivari R, Marty S (2018) A highly sensitive and selective high pressure liquid chromatography with tandem mass spectrometry (HPLC/MS-MS) method for the direct peptide reactivity assay (DPRA). J Pharmacol Toxicol Methods 94:1–15. 10.1016/j.vascn.2018.07.004 [DOI] [PubMed] [Google Scholar]
- 26.Cho SA, Choi M, Park SR, An S, Park JH (2020) Application of Spectro-DPRA, KeratinoSens™ and h-CLAT to estimation of the skin sensitization potential of cosmetics ingredients. J Appl Toxicol 40:300–312. 10.1002/jat.3904 [DOI] [PubMed] [Google Scholar]