July Blog 2022 - Immunotoxicity assessment criteria

July 26, 2022

Immunotoxicity assessment is a huge consideration when developing a new pharmaceutical intended for use in humans. Are 2D cell based systems enough to truly model immunotoxicity?

How do our assays compare with others available on the market?

2D OECD guideline tests

Currently the gold standard techniques to determine skin sensitisation rely on a best 2 of 3 approach from the following OECD assays;

  1. Key event 1 – Molecular initiating event. The DPRA assay is an in chemico method, using synthetic peptides to determine protein reactivity. (1)
  2. Key event 2 – Keratinocyte inflammatory responses. The KeratinoSens assay uses a genetically modified immortalised cell lineage in a 2D system. Increased fluorescence from a Luciferase reporter gene allows for quantification of inflammatory upregulation (2)
  3. Key event 3 – Activation of Dendritic Cells (DC). The h-CLAT assay uses a human leukaemia cell line to mimic DC activation measured through changes in surface marker expression. (3)

As a field do we need to move away from the use of genetically modified cancer cell 2D cultures for our sensitisation assessments?

Here at Alcyomics® we would argue yes, and for a number of key reasons;

  1. Cancer lineages contain variable genetic abnormalities which can be detrimental to cellular function.
  2. Cell lines are homogenous and do not represent the heterogeneity of sensitisation responses present at a population level. Would you trust the safety of a drug if it had only been tested on a single donor?
  3. 2D cell cultures have been shown to be less physiologically relevant at both the genetic and proteome level. Recent understandings have shown that a cells shape can have significant effects on its function and differentiative potential. (4)

What about animal studies?

Other animal based immunotoxicity assays such as the local lymph node assay (LLNA) have been used extensively in the past to determine immunotoxicity in human drugs. However, these assays have been demonstrated to suffer from several disadvantages such as false positive responses, variability due to the vehicle used, their overall predictivity (~72% against human data) and their inability to distinguish between type of hypersensitivity response. (5)

Skimune® assay 

What you may not have known is that Alcyomics’s® Skimune® assay is a human tissue explant assay which includes autologous dendritic cell and T-cell lineages to measure sensitisation and immunotoxicity both in the skin but also as a test for more general systemic immunoreactivity.

Benefits of Skimune® are:

  1. Primary human tissue-based from varied donors, allowing for a much more detailed understanding of immunotoxicity across age groups, sex and other demographics.
  2. Does not rely on 2D cultures of cells.
  3. Skimune® is a truly dynamic assay which is capable of a wide array of readouts. Specifically, Skimune® can cover both key event 2, 3 and 4 in the sensitisation pathway.
  4. Because Skimune® covers multiple sensitisation pathway key events, using Skimune® in your product development will save time and money compared to other available tests.
  5. Capable of distinguishing type of hypersensitivity reaction.
  6. Validated to be sensitive against: Chemicals, Complex formulations, Cosmetics, Biologicals, Cellular therapies and many more! (Read our papers 6, 7 & 8)

Written by Dr Matthew Freer – Scientific business development officer.


(1) Test No.442C-English.pdf (

(2) Test No. 442D: In Vitro Skin Sensitisation – ARE-Nrf2 Luciferase Test Method (

(3) Test No. 442E: In Vitro Skin Sensitisation – In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation (

(4) Kapałczyńska M, Kolenda T, Przybyła W, Zajączkowska M, Teresiak A, Filas V, Ibbs M, Bliźniak R, Łuczewski Ł, Lamperska K. 2D and 3D cell cultures – a comparison of different types of cancer cell cultures. Arch Med Sci. 2018 Jun;14(4):910-919. doi: 10.5114/aoms.2016.63743. Epub 2016 Nov 18. PMID: 30002710; PMCID: PMC6040128.

(5) Anderson SE, Siegel PD, Meade BJ. The LLNA: A Brief Review of Recent Advances and Limitations. J Allergy (Cairo). 2011;2011:424203. doi: 10.1155/2011/424203. Epub 2011 Jun 16. PMID: 21747867; PMCID: PMC3124934.

(6) Monnot AD, Towle KM, Ahmed SS, Dickinson AM, Fung ES. An in vitro human assay for evaluating immunogenic and sensitization potential of a personal care and cosmetic product. Toxicol Mech Methods. 2021 Mar;31(3):205-211. doi: 10.1080/15376516.2020.1868640. Epub 2021 Jan 13. PMID: 33371753.

(7) Ahmed SS, Whritenour J, Ahmed MM, Bibby L, Darby L, Wang XN, Watson J, Dickinson AM. Evaluation of a human in vitro skin test for predicting drug hypersensitivity reactions. Toxicol Appl Pharmacol. 2019 Apr 15;369:39-48. doi: 10.1016/j.taap.2019.02.005. Epub 2019 Feb 12. PMID: 30768973.

(8) Ahmed SS, Wang XN, Fielding M, Kerry A, Dickinson I, Munuswamy R, Kimber I, Dickinson AM. An in vitro human skin test for assessing sensitization potential. J Appl Toxicol. 2016 May;36(5):669-84. doi: 10.1002/jat.3197. Epub 2015 Aug 7. PMID: 26251951.

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