Improvement of human cell line activation test (h-CLAT) using short-time exposure methods for prevention of false-negative results

Abstract

Recently, animal testing has been affected by increasing ethical, social, and political concerns regarding animal welfare. Several in vitro safety tests for evaluating skin sensitization, such as the human cell line activation test (h-CLAT), have been proposed. However, similar to other tests, the h-CLAT has produced false-negative results, including in tests for acid anhydride and water-insoluble chemicals. In a previous study, we demonstrated that the cause of false-negative results from phthalic anhydride was hydrolysis by an aqueous vehicle, with IL-8 release from THP-1 cells, and that short-time exposure to liquid paraffin (LP) dispersion medium could reduce false-negative results from acid anhydrides. In the present study, we modified the h-CLAT by applying this exposure method. We found that the modified h-CLAT is a promising method for reducing false-negative results obtained from acid anhydrides and chemicals with octanol–water partition coefficients (LogK_ow) greater than 3.5. Based on the outcomes from the present study, a combination of the original and the modified h-CLAT is suggested for reducing false-negative results. Notably, the combination method provided a sensitivity of 95% (overall chemicals) or 93% (chemicals with LogK_ow > 2.0), and an accuracy of 88% (overall chemicals) or 81% (chemicals with LogK_ow > 2.0). We found that the combined method is a promising evaluation scheme for reducing false-negative results seen in existing in vitro skin-sensitization tests. In the future, we expect a combination of original and modified h-CLAT to be applied in a newly developed in vitro test for evaluating skin sensitization.

INTRODUCTION

Skin sensitization, such as allergic contact dermatitis (ACD), is a phenomenon in which rashes occur on the skin owing to complex T-cell-mediated immune responses. ACD is induced by repeated contact with external low-molecular-weight chemicals (e.g., poison ivy/oak and metal allergens).

Recently, animal testing has been affected by increasing ethical, social, and political concerns regarding animal welfare. Several countries have banned safety testing of cosmetic products and their ingredients on animals (Adler et al., 2011). Accordingly, it is necessary to develop alternatives to animal experiments, particularly for in vitro safety testing and skin sensitization evaluation. Several in vitro safety tests have been proposed.

The human cell line activation test (h-CLAT) has been evaluated in a European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)-coordinated validation study and subsequent independent peer review by the EURL ECVAM Scientific Advisory Committee (OECD TG 442E, 2016). The h-CLAT is a mechanistically based in vitro assay that addresses the third key event of the skin sensitization adverse outcome pathway, namely, the activation of skin dendritic cells (DCs). Almost all skin sensitizers have electrophilic properties and covalently bind to nucleophilic sites in skin proteins (Goodwin and Roberts, 1986). Immature DCs recognize sensitizer-protein complexes and become activated. More specifically, sensitizers cause morphological and functional changes in DCs, altering the expression of CD40, CD80, CD83, CD86, and HLA-DR (Aiba et al., 1997; Aiba, 1998; Arrighi et al., 2001; De Smedt et al., 2001). Activated DCs migrate to local lymph nodes and present antigens to naive T cells, resulting in the proliferation of antigen-specific memory T cells (Weltzien et al., 1996; Banchereau and Steinman, 1998; Enk et al., 1993; Cumberbatch et al., 1997, 2005).

The h-CLAT uses THP-1 cells, a human monocytic leukemia cell line (Tsuchiya et al., 1980), as a replacement for DCs to evaluate changes in CD86 and CD54 expression. CD86 and CD54 are typical markers of monocytic THP-1 activation and may mimic DC activation by skin sensitizers, which plays a critical role in T-cell priming (Rothlein et al., 1986; Symington et al., 1993). Many studies have demonstrated the good predictive performance of the h-CLAT compared with Local Lymph Node Assay (LLNA) results or human data (Ashikaga et al., 2010; Nukada et al., 2011; Takenouchi et al., 2013). However, similar to other in vitro skin sensitization tests, the h-CLAT still shows false-negative results with some chemicals. Thus, for a testing method to fully substitute for animal tests, the problem of false-negative results should be solved.

In the h-CLAT, hydrolyzable chemicals such as phthalic anhydride (PAH) and water-insoluble chemicals tend to produce false-negative results (Ashikaga et al., 2010; Takenouchi et al., 2013). Negative results for test chemicals with octanol-water partition coefficients (LogK_ow) greater than 3.5 should not be considered valid (OECD TG 442E, 2016). Therefore, evaluation of water-insoluble chemicals is a crucial issue that must be solved in the development of in vitro skin sensitization tests. Limited studies have focused on technical aspects important for reducing false-negative results in the h-CLAT. It is important to elucidate these causes and to improve testing to minimize such results.

In our previous study, we demonstrated that the cause of false-negative results from PAH was hydrolysis by an aqueous vehicle by measuring IL-8 release in THP-1 cells (Narita et al., 2017). We also demonstrated that short-term exposure to liquid paraffin (LP) dispersion medium may be a promising method for evaluating the skin sensitization hazard of acid anhydrides. Although IL-8 protein release by THP-1 cells has been suggested as a promising biomarker of skin sensitization, it is not measured in internationally accepted in vitro skin sensitization tests. Therefore, the method of short-term exposure to LP medium was applied to the h-CLAT, and the effectiveness of this method was evaluated.

In this study, based on the results of our previous study (Narita et al., 2017), we test the hypothesis that LP medium can improve false-negative results of acid anhydrides and water-insoluble chemicals in the h-CLAT. Furthermore, we demonstrated that a combination of the original h-CLAT and our modified h-CLAT reduced false-negative results.

MATERIALS AND METHODS

Cells

THP-1 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI1640 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Thermo Fisher Scientific), 100 units/mL penicillin (Wako Pure Chemical Industries, Ltd., Tokyo, Japan), 100 μg/mL streptomycin (Wako Pure Chemical Industries, Ltd.), and 0.05 mM 2-mercaptoethanol (Thermo Fisher Scientific) at 37°C with 5% CO₂. THP-1 cells were routinely passaged and maintained at densities of 1-8 × 10⁵ cells/mL. Cells were used before passage 30 for all experiments.

Chemicals

The test chemicals are listed in Table 1 along with their Chemical Abstracts Service (CAS) numbers, LogK_ow values calculated with KOWWIN ver. 1.68 in EPI Suite (Environmental Protection Agency, Washington, DC, USA), skin sensitization potencies based on EC3 LLNA values, and results from clinical case reports, patch tests, and human maximization tests. Nickel sulfate (NiSO₄; > 98%), PAH (> 99%), maleic anhydride (MAH; > 99%), benzoic acid (> 99.5%), and cyclamen aldehyde (CA; > 95%) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Citral (CI; > 95.0%), phthalic acid (PA; > 99.0%), dimethyl sulfoxide, and LP were purchased from Wako Pure Chemical Industries, Ltd. Phenyl glycidyl ether (PGE; > 99.0%), butyl glycidyl ether (BGE; > 98.0%), benzyl benzoate (BB; > 99.0%), benzyl cinnamate (BC; > 98.0%), 10-undecenal (10-Un; > 95.0%), N,N-dibutylaniline (DBA; > 98.0%), hexyl cinnamic aldehyde (HCA; > 90.0%), and dibutyl phthalate (DBP; > 97.0%) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).

Table 1. List of chemicals tested.

Test chemical	Abbreviation	CAS No.	Physical state	Estimated LogKow *	EC3 value (% w/v) **	Potency category **	Human data*****	h-CLAT results******
Nickel (II) sulfate	NiSO4	10101-97-0	Solid	2.04	4.80	Moderatea	Positivei	Positivep
Phthalic anhydride	PAH	85-44-9	Solid	2.07	0.16	Strongb	Positivej	Negativep
Maleic anhydride	MAH	108-31-6	Solid	1.62	0.16	Strongb	Positivek	Positivep
Pyromellitic dianhydride	PMDAH	89-32-7	Solid	n.c. ***	n.f.****	n.f.****	n.f.****	n.f.****
Phenyl glycidyl ether	PGE	122-60-1	Liquid	1.61	0.46	Strongc	Positivel	n.f.****
Butyl glycidyl ether	BGE	2426-08-6	Liquid	1.08	28	Weakc	Positivei	Negativep
Citral	CI	5392-40-5	Liquid	3.45	13.2	Weakd	Positivem	Positivep
Benzyl benzoate	BB	120-51-4	Liquid	3.54	17	Weake	Negativen	Negativeq
Benzyl cinnamate	BC	103-41-3	Solid	4.06	18.4	Weakf	n.f.****	Negativeq
Cyclamen aldehyde	CA	103-95-7	Liquid	3.91	20.5	Weakd	n.f.****	Negativep
10-Undecenal	10-Un	112-45-8	Liquid	4.12	6.8	Moderated	n.f ****	Negativeq
N,N-Dibutylaniline	DBA	613-29-6	Liquid	5.12	19.6	Weakg	n.f.****	Negativeq
Hexyl cinnamic aldehyde	HCA	101-86-0	Liquid	4.82	12	Weakd	Positiveo	Negativep
Benzoic acid	BA	65-85-0	Solid	1.87	n.c.***	Non-sensitizerh	n.f.****	Negativep
Phthalic acid	PA	88-99-3	Solid	1.07	n.f.****	n.f.****	n.f.****	n.f.****
Dibutyl phthalate	DBP	84-74-2	Liquid	4.61	n.c.***	Non-sensitizerg	n.f.****	Negativeq

* LogK_ow value calculated with KOWWIN ver. 1.68 in EPI Suite (Environmental Protection Agency, Washington, DC, USA).

** The potency of skin sensitization was estimated using LLNA (^aRyan et al., 2002; ^bDearman et al., 2000; ^cNiklasson et al., 2009; ^dPatlewicz et al., 2002; ^eGerberick et al., 2005; ^fNatsch and Emter, 2008; ^gPatlewicz et al., 2007; ^hGildea et al., 2006).

*** n.f. not found.

**** n.c. not calculated.

***** Human data were from ⁱKligman, (1966), ^jMoffitt and Sansom, (2002), ^kMotolese et al. (1993), ^l IRAC (1989), ^mOpdyke, (1979), ⁿRIFM, (2007) and ^oBasketter et al., (2015).

****** h-CLAT results were from ^pAshikaga et al., (2010), ^qTakenouchi et al., (2013).

Preparation of chemicals and chemical exposure of THP-1 cells

The LP medium 5-min exposure method was performed as we reported previously (Narita et al., 2017). Briefly, test chemicals were first dispersed in LP and diluted. THP-1 cells were then directly exposed to these suspensions (1 × 10⁶ cells in 100 μL suspension) and mixed using a vortex mixer for 5 min at room temperature. After exposure, the cells were washed and resuspended in 1 mL of culture medium. Cell suspensions were transferred to individual wells of a 24-well plate, and the plates were then incubated for 24 hr at 37°C in an atmosphere containing 5% CO₂.

Cytotoxicity assay and detection of CD86 and CD54 expression

The following procedure was conducted according to the h-CLAT protocol (OECD TG 442E, 2016). After chemical exposure and incubation, cells were washed twice with phosphate-buffered saline containing 0.1% (w/v) bovine serum albumin. Cells were then treated with 0.01% globulins and Cohn fraction II, III (Sigma-Aldrich) to block Fc receptors. After blocking, the cells were stained with 0.625 μg/mL propidium iodide (PI, Sigma-Aldrich) and antibodies. The cells were analyzed by flow cytometry (FACSCalibur; Becton Dickinson, Franklin Lakes, NJ, USA), and CELLQUEST was used for analysis. In cell viability measurements, PI-negative cells were defined as living cells. Dead cells were gated out by PI staining. Cell were stained using a fluorescein isothiocyanate (FITC)-labeled mouse monoclonal anti-human CD86 antibody (clone Fun-1; PharMingen, San Diego, CA, USA) and a FITC-labeled mouse monoclonal anti-human CD54 antibody (clone 6.5B5; PharMingen). A FITC-labeled mouse IgG₁ was purchased from DAKO (clone: DAK-G01, Glostrup, Denmark) and used as an isotype control. Ten thousand live cells were analyzed for each sample. The relative fluorescence intensity (RFI) was used as an indicator of CD86 and CD54 expression and was calculated from the mean fluorescence intensity (MFI) as follows:

Statistics

The assays were performed at least three times. The data are expressed as the mean ± standard deviation (S.D.). One-way analysis of variance followed by Dunnett’s post-hoc test was used to evaluate statistical significance using EZR (Kanda, 2013), a graphical user interface for R (The R Foundation for Statistical Computing, version 3.3.0). For comparisons with data from vehicle-treated cells, differences with p values less than 0.05 (*) and 0.01 (**) were considered statistically significant. The predictive performance of the test was calculated according to Cooper statistics as the sensitivity, specificity, and accuracy (Cooper et al., 1979). Sensitivity is the percentage of sensitizers predicted as positive, specificity is the percentage of non-sensitizers predicted as negative, and accuracy is the overall percentage of correct predictions.

RESULTS

Effect of LP on CD86 and CD54 expression in THP-1 cells

The effects of LP on cell viability, CD86 expression, CD54 expression, and non-specific antibody adsorption to THP-1 cells were studied. THP-1 cells were treated with LP for 5 min. After exposure to LP, the cells were washed and cultured for 24 hr in culture medium. The results showed no significant differences between the levels of these biomarkers in LP-exposed and unexposed cells in any of the seven replicate experiments (Fig. 1).

Fig. 1

Effect of LP on CD86 expression, CD54 expression, and non-specific antibody adsorption. THP-1 cells were cultured in medium for 24 hr (control) or for an additional 24 hr after a 5-min exposure to LP (LP). After incubation, the expression of cell-surface antigens (CD86 [black bars], CD54 [white bars], and non-specific antibody adsorption [gray bars]) was analyzed by flow cytometry, and dead cells were gated out by PI staining as a measure of cell viability (black dots). The results are expressed as the MFI and are presented as means ± S.D. (N = 7). The results are representative of seven independent experiments. Statistical significance was calculated by Student’s t-test (n.s.: not significant).

Next, the effects of LP dispersion medium on CD86 and CD54 expression in THP-1 cells were examined. THP-1 cells were treated with NiSO_4, which was judged positive by the h-CLAT, and benzoic acid, which was judged negative by the h-CLAT in LP dispersion medium. RFI values for CD86 and CD54 expression were then calculated, and the results showed that exposure to 75 μg/mL of NiSO₄ significantly augmented the RFI values of CD86 (161 ± 32%) and CD54 (1049 ± 283%) expression (Fig. 2A). Cell viability decreased dose-dependently with benzoic acid treatment at three concentrations (1.4, 1.7, and 2.0 mg/mL); however, the RFIs of CD86 and CD54 still did not meet the positive criteria for the h-CLAT (150% and 200%, respectively; Fig. 2B).

Fig. 2

Evaluation of water-soluble chemicals. THP-1 cells were cultured in medium for an additional 24 hr after a 5-min exposure to 75 μg/mL of NiSO₄ (A) and various concentrations of BA (B) in LP dispersion medium. After incubation, the expression of cell-surface antigens (CD86 [black bars] and CD54 [white bars]) was analyzed by flow cytometry, and dead cells were gated out by staining with PI (white diamonds). The assay was conducted according to the h-CLAT protocol. The results are expressed as RFI and presented as means ± S.D. (N = 3). The results are representative of three independent experiments. The lines across the graph indicate RFI values of 150 and 200.

Improvement in false-negative h-CLAT results by a 5-min exposure to hydrolyzable chemicals in LP dispersion medium

Previously, we observed that PAH significantly increased IL-8 release in LP dispersion medium (Narita et al., 2017). Because PAH also showed false-negative h-CLAT results, we examined whether hydrolyzable chemicals such as PAH augment CD86 and CD54 expression, using LP dispersion medium. The results showed that exposure to 625 μg/mL of MAH, which showed a true-positive h-CLAT result, significantly augmented the RFI values of both CD86 (205 ± 39%) and CD54 (427 ± 39%; Fig. 3A). Notably, 156 μg/mL and 313 μg/mL of PAH, which showed a false-negative h-CLAT result, augmented the RFI value of CD54 (281 ± 82% and 303 ± 133%) (Fig. 3B). Similarly, PMDAH, which showed a false-negative h-CLAT result (data not shown), significantly augmented the RFI values of both CD86 (maximum value: 170 ± 68%; exposure concentration: 625 μg/mL) and CD54 (maximum value: 1,033 ± 371%; exposure concentration: 625 μg/mL; Fig. 3C).

Fig. 3

Evaluation of acid anhydrides and epoxides. THP-1 cells were cultured in medium for an additional 24 hr after a 5-min exposure to various concentrations of MAH (A), PAH (B), PMDAH (C), PA (D), PGE (E), and BGE (F) in LP dispersion medium. After incubation, the expression of cell-surface antigens (CD86 [black bars] and CD54 [white bars]) was analyzed by flow cytometry, and dead cells were gated out by staining with PI (white diamonds). The assay was conducted according to the h-CLAT protocol. The results expressed as the RFI and are presented as means ± S.D. (N = 3). The results are representative of three independent experiments. The lines across the graph indicate RFI values of 150 and 200.

However, although cell viability decreased dose-dependently with PA (Fig. 3D), PGE (Fig. 3E), and BGE (Fig. 3F) treatment, the RFIs of CD86 and CD54 still did not meet the positive criteria for the h-CLAT (150% and 200%, respectively).

Reduction of false-negative h-CLAT results after a 5-min exposure to water-insoluble chemicals in LP medium

To evaluate water-insoluble chemicals in the h-CLAT, we optimized the 5-min exposure of LP medium method. We investigated the optimal exposure concentration using CI (Fig. 4A). Even though CI is a water-insoluble chemical (LogK_ow = 3.45), CI was classified as a skin sensitizer by the LLNA and h-CLAT. The results showed that when cell viability was more than 50%, the maximum RFI value of CD86 was 203 ± 52% (exposure concentration: 15 mg/mL; cell viability: 73 ± 7.4%), and the maximum RFI value of CD54 was 282 ± 49% (exposure concentration: 13 mg/mL; cell viability: 81 ± 0.2%). Similar to the treatment with 13 mg/mL of CI, 15 mg/mL of CI augmented the RFI value of CD54 (277 ± 66%). Next, we investigated the optimal post-culture time of THP-1 cells after a 5-min exposure in LP medium using 15 mg/mL CI (Fig. 4B). The results showed that there was no obvious change in cytotoxicity, CD86 expression, or CD54 expression immediately after exposure. CI induced cytotoxicity and augmentation of CD86 expression in THP-1 cell cultures in a time-dependent manner for up to 24 hr. Notably, CI augmented CD54 expression in a time-dependent manner for up to 48 hr. At 48 hr, the maximum RFI value of CD54 (587 ± 142%) was observed.

Fig. 4

Determination of the optimal exposure concentration (A) and post-culture time (B) for increasing CD86 and CD54 expression when evaluating water-insoluble chemicals. THP-1 cells were cultured in medium for an additional 24 hr after a 5-min exposure to various concentrations of CI (A) or for various post-culture times after a 5-min exposure to 15 mg/mL CI (B) in LP medium. After incubation, the expression of cell-surface antigens (CD86 [black bars] and CD54 [white bars]) was analyzed by flow cytometry, and dead cells were gated out by staining with PI (white diamonds). The assay was conducted according to the h-CLAT protocol. The results are expressed as the RFI and are presented as means ± S.D. (N = 3). Statistical significance was calculated by one-way ANOVA followed by Dunnett’s post-hoc test, compared with the control group (*p < 0.05, **p < 0.01). The results are representative of three independent experiments. The lines across the graph indicate RFI values of 150 and 200.

To evaluate water-insoluble chemicals, we used the exposure concentration corresponding to 75% cell viability, and 24 hr and 48 hr post-culture times for CD86 and CD54, respectively. Under these conditions, we examined whether water-insoluble chemicals, which produced false negative results in the h-CLAT, augmented CD86 and CD54 expression using LP medium (Fig. 5). The results showed that 10-Un (76 mg/mL) and CA (62.5 mg/mL) augmented the RFI value of CD54 (209 ± 78% and 256 ± 25%, respectively). Similarly, BB (62.5 mg/mL) and BC (125 mg/mL) augmented the RFI value of CD86 (151 ± 27% and 235 ± 59%, respectively). When we stimulated THP-1 cells with 250 mg/mL of DBP as a negative control, the RFIs of CD86 and CD54 did not meet the positive criteria for the h-CLAT (150% and 200%, respectively). On the other hand, after treatment with DBA (Fig. 5B) and HCA (Fig. 5C), which were judged as false negatives by the h-CLAT, the RFIs of CD86 and CD54 did not meet the positive criteria for the h-CLAT (150% and 200%, respectively).

Fig. 5

Evaluation of water-insoluble chemicals. THP-1 cells were cultured in medium for an additional 24 hr after a 5-min exposure to 15 mg/mL of CI, 62.5 mg/mL of BB, 125 mg/mL of BC, 62.5 mg/mL of CA, 76 mg/mL of 10-Un, or 250 mg/mL of DBP (A), or various concentrations of DBA (B) or HCA (C) in LP medium. After incubation, the expression of cell-surface antigens (CD86 [black bars] and CD54 [white bars]) was analyzed by flow cytometry, and dead cells were gated out by staining with PI (white diamonds). The assay was conducted according to the h-CLAT protocol. The results are expressed as the RFI and are presented as means ± S.D. (N = 3). The results are representative of three independent experiments. The lines across the graph indicate RFI values of 150 and 200.

Performance of the combination of h-CLAT and modified h-CLAT

Based on the above findings, we demonstrated that combining the original and modified h-CLATs reduced false-negative results (Fig. 6), as follows.

Fig. 6

Combination of h-CLAT and modified h-CLAT. The modified h-CLAT involved a 5-min exposure using LP medium methods applied to the h-CLAT.

1. 1.    The modified h-CLAT was performed with chemicals judged negative by the h-CLAT, considering the possibility of false-negative results.
2. 2.    A hydrolyzability check for each test chemical was performed. If the test chemical was hydrolyzable under the conditions of the h-CLAT, then the modified h-CLAT was performed regardless of the solubility in LP.
3. 3.    If the modified h-CLAT gave a positive result, then hydrolysis was considered the cause of the false negative, and the test chemical was considered a sensitizer.
4. 4.    If the test chemical was not hydrolyzable under the h-CLAT conditions or if its hydrolyzability was unknown, then the LogK_ow value of the test chemical was investigated.
5. 5.    If the LogK_ow value was over 2.0, then a solubility check in LP was performed.
6. 6.    If the chemical was soluble at 500 mg/mL in LP, then a modified h-CLAT was performed.
7. 7.    If the modified h-CLAT gave a positive result, then water-insolubility was considered the cause of the false negative, and the test chemical was considered a sensitizer.
8. 8.    If the modified h-CLAT gave a negative result, then the test chemical was not considered a sensitizer.
9. 9.    If the chemical was not soluble at 500 mg/mL in LP, then it was unevaluable by this testing approach.

Following this tiered system, chemicals judged as negative by the h-CLAT were reevaluated by the modified h-CLAT following a hydrolyzable or lipophilicity check. Comparisons of the predictive performance of the h-CLAT alone and of this tiered system with that of LLNA using a previous data set (Ashikaga et al., 2010; Nukada et al., 2011; Takenouchi et al., 2013) are summarized in Tables 2 and 3. However, methylchloroisothiazolinone/methylisothiazolinone, NiSO₄, and dextran (whose LogK_ow values could not be calculated by KOWWIN) and tocopherol (for which false positive results in LLNA have been reported [Basketter et al., 2014]), were excluded. Notably, this tiered system provided a sensitivity of 95% (overall chemicals) or 93% (chemicals with a LogK_ow > 2.0) and an accuracy of 88% (overall chemicals) or 81% (chemicals with a LogK_ow >2.0). These data reflect a reduction in false-negative results. Moreover, no new produced false-positive results were observed using the tiered system.

Table 2. Predictive performance of the h-CLAT and tiered systems compared with LLNA.

		h-CLAT****			Tiered system
		All chemical			All chemical
		Positive	Negative		Positive	Negative
LLNA	Sensitizer	85	17		90	5*****
	Non-sensitizer	11	26		11	26
Sensitivity * (%)			83 (85/102)			95 (90/ 95)
Specificity** (%)			70 (26/ 37)			70 (26/ 37)
Accuracy*** (%)			80 (111/139)			88 (116/132)

* The percentage of chemicals correctly evaluated as sensitizers by the h-CLAT, compared to that by LLNA.

** The percentage of chemicals correctly evaluated as non-sensitizers by the h-CLAT, compared that by to LLNA.

*** The percentage of chemicals correctly evaluated by the h-CLAT, compared to that by LLNA.

**** h-CLAT data are based on Ashikaga et al., (2010), Nukada et al., (2012), and Takenouchi et al. (2013) for the chemicals listed, except for methylchloroisothiazolinone/methylisothiazolinone, NiSO₄, tocopherol, and dextran.

***** Seven chemicals unevaluable by the tiered testing approach were excluded from statistical analysis.

Table 3. Predictive performance comparison of the tiered system with LLNA for chemicals according to LogK_ow value.

		Tiered system
		Chemicals with LogKow > 2.0			Chemicals with LogKow ≤ 2.0
		Positive	Negative		Positive	Negative
LLNA	Sensitizer	42	3		48	2
	Non-sensitizer	8	5		3	21
Sensitivity (%)			93 (42/45)			96 (48/50)
Specificity (%)			38 (5/13)			88 (21/24)
Accuracy (%)			81 (47/58)			93 (69/74)

DISCUSSION

In our previous study, we reported that PAH significantly increased IL-8 release using LP dispersion medium (Narita et al., 2017). In this study, we aimed to prevent false-negative results in the h-CLAT using a short-term exposure method in LP medium.

Like other in vitro skin sensitization tests, the h-CLAT still produces false-negative results. It has been reported that the h-CLAT shows high predictive performance when analyzing chemicals with LogK_ow values less than 2.0 (Ashikaga et al., 2010; Nukada et al., 2011; Takenouchi et al., 2013). These results showed that the accuracy for 74 chemicals tested with the h-CLAT was 93%, the sensitivity was 96%, and the specificity was 88%. However, the test has tended to produce false-negative results in tests for water-insoluble chemicals, especially chemicals with LogK_ow values greater than 2.0, based on data from its developers. Therefore, we defined water-insoluble chemicals as those with LogK_ow values greater than 2.0.

We hypothesized that short-term exposure in LP medium could overcome the lingering problem of false-negative results for water-insoluble chemicals. Therefore, we applied a short-time exposure in LP medium method to the h-CLAT and established an in vitro skin sensitization test. Using this method, we correctly evaluated the skin sensitization potential of NiSO₄, CI, and BA. These results indicated that the biomarkers and testing criteria of the ordinary h-CLAT were applicable with the modified h-CLAT involving a 5-min exposure in LP medium. Furthermore, the modified h-CLAT may be a promising method for evaluating the skin sensitization potential of acid anhydrides; we conclude that the false-negative results for PAH and PMDAH in the h-CLAT were caused by hydrolysis by an aqueous vehicle.

The h-CLAT produced false-negative results for PGE (data not shown) and BGE (Ashikaga et al., 2010), and these results were not due to hydrolysis by an aqueous vehicle. The half-lives for PGE and BGE hydrolysis at 25°C and pH = 7 were 2.2 × 10⁶ hr and 5.4 × 10⁵ hr, respectively (HYDROWIN ver. 2.00 in EPI suite); thus, it is unlikely that PGE or BGE hydrolysis occurred during the h-CLAT procedure. PGE and BGE were still evaluated as false-negatives by the modified h-CLAT. Because the ARE-Nrf2 luciferase test correctly evaluates PGE and BGE as positives (Delaine et al., 2011), the cause of this difference for epoxides may be associated specifically with THP-1 cells or with CD86 and CD54.

Chemicals used in this study with high LogK_ow values were observed to have lower skin sensitization potentials because these chemicals precipitate or separate out from the aqueous vehicle in almost all in vitro skin sensitization tests. Notably, in the modified h-CLAT, we found that BB, BC, CA, and 10-Un augmented CD86 or CD54 expression in THP-1 cells. On the other hand, DBA and HCA were still evaluated as false-negatives by the modified h-CLAT. It is likely that DBA is a pro-hapten, because DBA was evaluated as positive by the ARE-Nrf2 luciferase test in the presence of rat liver S9 fractions (Natsch and Haupt, 2013). Therefore, LP medium exposure may be unsuitable for evaluating pro-haptens correctly. HCA was classified as a weaker skin sensitizer than predicted by LLNA (Basketter et al., 2014). It possesses a low capacity to induce skin sensitization under consumer-exposure conditions (Basketter et al., 2015). BB has been placed in the same category, as a rare cause of contact allergy except perhaps in special circumstances. This can be attributed to its very slight augmentation of CD86 expression on the BB-treated cells.

However, LP-insoluble chemicals, especially solid, water-insoluble chemicals (except for BC), cannot be evaluated by the modified h-CLAT. Accordingly, additional studies are needed to develop methods for the study of solid, water-insoluble chemicals.

In this study, the combination of the h-CLAT and modified h-CLAT provided high sensitivity and accuracy. We demonstrated the high predictive performance of the combined method, despite including some chemicals with LogK_ow values greater than 3.5. We believe that these results are due to the following three factors: the h-CLAT correctly evaluated chemicals with LogK_ow values less than 2.0 (Ashikaga et al., 2010; Nukada et al., 2011; Takenouchi et al., 2013), the application limits of the h-CLAT (e.g., on water-insoluble or hydrolyzable compounds) were clear (Ashikaga et al., 2010; Takenouchi et al., 2013), and the modified h-CLAT reduced false-negative results obtained for water-insoluble or hydrolyzable chemicals. When we defined water-insoluble chemicals as those with LogK_ow values greater than 3.5, this tiered system provided a sensitivity of 90% (24 chemicals with LogK_ow > 3.5). However, the sensitivity was slightly lower than those with logK_ow value = 2.0, even though the number of the test chemicals was increased.

In conclusion, we found that the combined use of the h-CLAT and modified h-CLAT is a promising evaluation scheme that reduces the false-negative results seen in existing in vitro skin sensitization tests. Our modified h-CLAT should be selected to test acid anhydrides and water-insoluble chemicals rather than original the h-CLAT. In the future, we expect that this 5-min exposure in LP medium method will be applied in new in vitro tests for skin sensitization evaluation. Moreover, we believe that these findings may help investigate cytotoxicity mechanisms of chemicals that cannot be evaluated by conventional, cell-based, in vitro methods.

ACKNOWLEDGMENTS

This research was partially supported by a Research on Regulatory Science of Pharmaceuticals and Medical Devices grant from the Japan Agency for Medical Research and development (AMED).

Conflict of interest

The authors declare that there is no conflict of interest.

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