Optimization and validation of a method to identify skin sensitization hazards using IL-1 α and IL-6 secretion from HaCaT

Byoungjun Jeon, Mi Ok Kim, Yun soon Kim, Hyo youn Han, Jang Hyuk Yun, Jungah Kim, Yan Huang, Yunhee Choi, Chung-Hyun Cho, Byeong Cheol Kang, Sungwan Kim, Tae Hyun Choi

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Although many methods to assess sensitization have been investigated to replace animal testing, it is still imperative to develop an in vitro method to minimize the use of animals and to classify sensitizers. Recently, an assay using the human keratinocyte cell line (HaCaT) was developed as an alternative method. Our aim was to optimize this method and validate its ability to assess sensitization. The highest dose that resulted in 75% cell viability was determined for each test substance. Then, serial dilutions of the dose were applied to measure the levels of secreted proinflammatory cytokines. To optimize the assay, statistical analyses were performed to determine whether all of the doses tested were necessary to maintain the predictive values. Exclusion of the 0.5× dose did not change the predictive values drastically. To validate the optimized method, 22 substances were evaluated without the 0.5× dose, resulting in overall predictive values of 83.3% for sensitivity, 80.0% for specificity, and 81.8% for accuracy, which are comparable to results from other validated assays. These results suggest that statistical analysis can assist in development of alternative in vitro methods and that the optimized HaCaT cell assay is reproducible.

Original languageEnglish
Article number104589
JournalToxicology in Vitro
Volume61
DOIs
StatePublished - 1 Dec 2019

Fingerprint

Interleukin-1
Interleukin-6
Assays
Hazards
Skin
Animals
Cells
Dilution
Statistical methods
Cytokines
Keratinocytes
Cell Survival
Testing
Sensitivity and Specificity
Cell Line

Keywords

  • Alternative to animal testing
  • HaCaT
  • LLNA
  • Proinflammatory cytokine
  • Sensitization
  • Validation study

Cite this

Jeon, Byoungjun ; Kim, Mi Ok ; Kim, Yun soon ; Han, Hyo youn ; Yun, Jang Hyuk ; Kim, Jungah ; Huang, Yan ; Choi, Yunhee ; Cho, Chung-Hyun ; Kang, Byeong Cheol ; Kim, Sungwan ; Choi, Tae Hyun. / Optimization and validation of a method to identify skin sensitization hazards using IL-1 α and IL-6 secretion from HaCaT. In: Toxicology in Vitro. 2019 ; Vol. 61.
@article{5a736a13e5714c04a27476e93cda8f02,
title = "Optimization and validation of a method to identify skin sensitization hazards using IL-1 α and IL-6 secretion from HaCaT",
abstract = "Although many methods to assess sensitization have been investigated to replace animal testing, it is still imperative to develop an in vitro method to minimize the use of animals and to classify sensitizers. Recently, an assay using the human keratinocyte cell line (HaCaT) was developed as an alternative method. Our aim was to optimize this method and validate its ability to assess sensitization. The highest dose that resulted in 75{\%} cell viability was determined for each test substance. Then, serial dilutions of the dose were applied to measure the levels of secreted proinflammatory cytokines. To optimize the assay, statistical analyses were performed to determine whether all of the doses tested were necessary to maintain the predictive values. Exclusion of the 0.5× dose did not change the predictive values drastically. To validate the optimized method, 22 substances were evaluated without the 0.5× dose, resulting in overall predictive values of 83.3{\%} for sensitivity, 80.0{\%} for specificity, and 81.8{\%} for accuracy, which are comparable to results from other validated assays. These results suggest that statistical analysis can assist in development of alternative in vitro methods and that the optimized HaCaT cell assay is reproducible.",
keywords = "Alternative to animal testing, HaCaT, LLNA, Proinflammatory cytokine, Sensitization, Validation study",
author = "Byoungjun Jeon and Kim, {Mi Ok} and Kim, {Yun soon} and Han, {Hyo youn} and Yun, {Jang Hyuk} and Jungah Kim and Yan Huang and Yunhee Choi and Chung-Hyun Cho and Kang, {Byeong Cheol} and Sungwan Kim and Choi, {Tae Hyun}",
year = "2019",
month = "12",
day = "1",
doi = "10.1016/j.tiv.2019.104589",
language = "English",
volume = "61",
journal = "Toxicology in Vitro",
issn = "0887-2333",
publisher = "Elsevier Ltd",

}

Optimization and validation of a method to identify skin sensitization hazards using IL-1 α and IL-6 secretion from HaCaT. / Jeon, Byoungjun; Kim, Mi Ok; Kim, Yun soon; Han, Hyo youn; Yun, Jang Hyuk; Kim, Jungah; Huang, Yan; Choi, Yunhee; Cho, Chung-Hyun; Kang, Byeong Cheol; Kim, Sungwan; Choi, Tae Hyun.

In: Toxicology in Vitro, Vol. 61, 104589, 01.12.2019.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Optimization and validation of a method to identify skin sensitization hazards using IL-1 α and IL-6 secretion from HaCaT

AU - Jeon, Byoungjun

AU - Kim, Mi Ok

AU - Kim, Yun soon

AU - Han, Hyo youn

AU - Yun, Jang Hyuk

AU - Kim, Jungah

AU - Huang, Yan

AU - Choi, Yunhee

AU - Cho, Chung-Hyun

AU - Kang, Byeong Cheol

AU - Kim, Sungwan

AU - Choi, Tae Hyun

PY - 2019/12/1

Y1 - 2019/12/1

N2 - Although many methods to assess sensitization have been investigated to replace animal testing, it is still imperative to develop an in vitro method to minimize the use of animals and to classify sensitizers. Recently, an assay using the human keratinocyte cell line (HaCaT) was developed as an alternative method. Our aim was to optimize this method and validate its ability to assess sensitization. The highest dose that resulted in 75% cell viability was determined for each test substance. Then, serial dilutions of the dose were applied to measure the levels of secreted proinflammatory cytokines. To optimize the assay, statistical analyses were performed to determine whether all of the doses tested were necessary to maintain the predictive values. Exclusion of the 0.5× dose did not change the predictive values drastically. To validate the optimized method, 22 substances were evaluated without the 0.5× dose, resulting in overall predictive values of 83.3% for sensitivity, 80.0% for specificity, and 81.8% for accuracy, which are comparable to results from other validated assays. These results suggest that statistical analysis can assist in development of alternative in vitro methods and that the optimized HaCaT cell assay is reproducible.

AB - Although many methods to assess sensitization have been investigated to replace animal testing, it is still imperative to develop an in vitro method to minimize the use of animals and to classify sensitizers. Recently, an assay using the human keratinocyte cell line (HaCaT) was developed as an alternative method. Our aim was to optimize this method and validate its ability to assess sensitization. The highest dose that resulted in 75% cell viability was determined for each test substance. Then, serial dilutions of the dose were applied to measure the levels of secreted proinflammatory cytokines. To optimize the assay, statistical analyses were performed to determine whether all of the doses tested were necessary to maintain the predictive values. Exclusion of the 0.5× dose did not change the predictive values drastically. To validate the optimized method, 22 substances were evaluated without the 0.5× dose, resulting in overall predictive values of 83.3% for sensitivity, 80.0% for specificity, and 81.8% for accuracy, which are comparable to results from other validated assays. These results suggest that statistical analysis can assist in development of alternative in vitro methods and that the optimized HaCaT cell assay is reproducible.

KW - Alternative to animal testing

KW - HaCaT

KW - LLNA

KW - Proinflammatory cytokine

KW - Sensitization

KW - Validation study

UR - http://www.scopus.com/inward/record.url?scp=85069740887&partnerID=8YFLogxK

U2 - 10.1016/j.tiv.2019.104589

DO - 10.1016/j.tiv.2019.104589

M3 - Article

VL - 61

JO - Toxicology in Vitro

JF - Toxicology in Vitro

SN - 0887-2333

M1 - 104589

ER -