Evaluation of skin sensitization potential of chemicals by local lymph node assay using 5-bromo-2-deoxyuridine with flow cytometry

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Highlights

  • ā€¢

    LLNA: BrdU-FCM is a modified non-radioisotopic method to identify skin sensitizers.

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    This method is as accurate, sensitive, and specific as variant LLNA methods.

  • ā€¢

    This study can identify skin sensitization potentials of 20 test substances which is not listed in OECD TG 429.

Abstract

The local lymph node assay using 5-bromo-2-deoxyuridine with flow cytometry (LLNA: BrdU-FCM) is a modified LLNA used to identify skin sensitizers. This assay measures the proliferation of auricular lymph node cells (LNCs) during the induction phase of skin sensitization and the number of BrdU-positive LNCs using flow cytometry. We determined if LLNA: BrdU-FCM can evaluate the skin sensitization potential of 20 substances, including 16 sensitizers and 4 non-sensitizers, that were tested using LLNA: DA and LLNA: BrdU-ELISA but not listed in OECD TG 429. After selecting appropriate vehicles and conducting pre-screen tests in 2 phases, solvents and test concentrations for the main test were determined. In the main study, we measured changes in LN weight, the number of LNCs, and the proportion of BrdU incorporated into LNCs to calculate stimulation indexes (SI). SI was calculated based on the total number of LNCs and BrdU incorporation in LNCs. We found that all substances were correctly classified as sensitizers or non-sensitizers. Overall, we confirmed that the LLNA: BrdU-FCM can evaluate skin sensitization potential of the 20 substances. Additionally, our results of combining 22 reference substances listed in OECD TG 429 and 20 additional substances showed that concordance of LLNA: BrdU-FCM with the LLNA was higher than before.

Introduction

The murine local lymph node assay (LLNA) for evaluating the skin sensitization potential of substances has been used worldwide as an alternative test method to the conventionally used guinea pig test (OECD TG 406, 1992); it was adopted as OECD TG 429 (Skin Sensitization: Local Lymph Node Assay) in 2002 (OECD, 2010a). The LLNA is a skin sensitization test in mice that measures the proliferation of murine local auricular lymph node cells (LNCs) after topical exposure to test substances. However, the use of LLNA has been limited due to the employment of radioisotope-labeled 3H-methyl thymidine and the resultant difficulty in the disposal of radioactive waste in some countries. For this reason, LLNA was modified to replace 3H-methyl thymidine, and two non-radio isotopic LLNA methods (LLNA: DA as OECD TG 442A and LLNA: BrdU-ELISA as OECD TG 442B) were adopted in 2010. LLNA: DA quantifies the ATP content of LNCs and LLNA: BrdU-ELISA measures the incorporation of BrdU, an analogue of thymidine, into lymph nodes (OECD TG 442A, 2010b; OECD TG 442B, 2010c,d) to evaluate LNC proliferation. In Chemico Skin Sensitization (DPRA) as OECD TG 442C and In Vitro Skin Sensitization (KeratinoSens) as OECD TG 442D were adopted in 2015 and In Vitro Skin Sensitization (h-CLAT) as OECD TG 442E was adopted in 2017 (OECD TG 442C, 2015a; OECD TG 442D, 2015b; OECD TG 442E, 2017).

The LLNA: BrdU- FCM is a novel non-radioisotopic version of the LLNA that performs similar to existing LLNA methods. This test measures the proliferation of auricular LNCs during the induction phase of skin sensitization by determining the number of BrdU-positive LNCs by flow cytometry. The LLNA: BrdU-FCM has been optimized and validated (Yang et al., 2015; Kim et al., 2016; Ahn et al., 2016). LLNA: BrdU-FCM can be used to evaluate the skin sensitization potency of test substances in the same way as the conventional LLNA, LLNA: BrdU-ELISA, and LLNA: DA. The LLNA: BrdU-FCM has several additional advantages. First, the proliferation of living LNCs is quantitatively measured in the LLNA: BrdU-FCM, whereas cells are indirectly scored based on BrdU content, regardless of whether these cells are alive or dead, in the LLNA: BrdU-ELISA (in which the BrdU-content of LNCs is measured). Second, other endpoints, such as cell surface markers or intracellular cytokines, can be measured using a multi-color FACS machine along with specific antibodies. Our pre-validation study demonstrated the usefulness of the test method because it can analyze B cells, T cells, and cytokines by FCM (Jung et al., 2012). Third, the modified pre-screen tests in the LLNA: BrdU-FCM can allow for minimization of pain and distress by refined dose selection scheme. Fourth, BALB/c mice, instead of CBA/J mice, can be used in the LLNA: BrdU-FCM (Lee et al., 2017). BALB/c mice are widely used owing to their easier access and better cost-efficiency in some countries than those of CBA/J mice. Previously, we reported the predictive capacity of LLNA: BrdU-FCM with the 22 reference substances listed in OECD TG 429 using the final protocol version 1.3 (Kim et al., 2016; Ahn et al., 2016), and the results showed 87.5% sensitivity, 100% specificity, and 90.9% accuracy compared with the original LLNA. The data presented are considered to meet the criteria for the performance standard, and its predictive capacity was also sufficiently validated. In this study, we demonstrate an enhancement in the predictive capacity of LLNA: BrdU-FCM by evaluating 20 test substances whose skin sensitization potentials were evaluated by LLNA: DA and LLNA: BrdU-ELISA but not listed in OECD TG 429.

Section snippets

Test substances

A total of 20 test substances were selected to include 16 sensitizers and 4 non-sensitizers (Table 1). All of these were selected based on published information about the LLNA: DA and LLNA BrdU-ELISA Test Method Evaluation Report (ICCVAM, 2010a; ICCVAM, 2010b). The 20 test substances were purchased from Sigma-Aldrich (St. Louis, MO, USA). The details of the 20 test substances are presented in Table 1. Vehicles replaced in the original LLNA were selected (OECD TG 429, 2010a). Acetone, olive oil,

Results of the pre-screen tests for 20 substances

The 20 test substances (ICCVAM, 2010a; ICCVAM, 2010b) were selected to evaluate their predictive capacity and listed in Table 1. We selected 20 test substances, including 16 sensitizers and 4 non-sensitizers. First, we selected an appropriate solvent. In the vehicle selection test, AOO was chosen as the vehicle for p-benzoquinone (BQ), cinnamic aldehyde (CIN), cyclamen aldehyde (CA), diethyl maleate (DEM), hydroxycitronellal (HC), isopropyl myristate (IPM), linalool (LL), butyl glycidyl ether

Discussion

The LLNA is a validated method for evaluating the skin sensitization potential of chemicals. Similar to conventional LLNA, LLNA: DA, and LLNA: BrdU-ELISA, the LLNA: BrdU-FCM method was developed to evaluate the potential of skin sensitization. LLNA: BrdU-FCM measures the proliferation of auricular LNCs during the induction phase of skin sensitization and the number of BrdU-positive LNCs using flow cytometry.

The LLNA methods address refinement among the 3Rs (Russell and Burch, 1959) as it does

Acknowledgments

This work was supported by the Korean Ministry of Food and Drug Safety [grant numbers 16181MFDS386 (2016), 17181MFDS486 (2017)].

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      For completeness, it is appropriate to cite relatively recent studies in which MMA has been evaluated using the LLNA with non-radioisotopic endpoints. Employing the LLNA:BrdU-FCM (OECD TG 442B) MMA was found to be negative (Ahn et al., 2016; Han et al., 2019; OECD, 2018d). MMA was also negative, or a weak positive, in the LLNA:DA (OECD TG 442A)(Zhang et al., 2017; OECD, 2018d).

    1

    These authors contributed equally to do this work.

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