Poster presented at ACT 2020: Applicability of GARD™skin for Accurate Assessment of Challenging Substances in the Context of Skin Sensitization Testing

J. Schmidt, A. Forreryd, H. Johansson, J. Li, A. Johansson
SenzaGen, Inc., Raleigh, NC, USA, SenzaGen AB, Lund, Sweden


Link to the poster



  • GARDskin demonstrated an overall high applicability for the evaluated challenging substances with 80% predictive accuracy compared to existing human data.
  • GARDskin demonstrated excellent applicability for pre/pro-haptens and low water solubility substances, correctly classifying all such compounds in the herein investigated dataset.
  • GARDskin also showed high applicability for assessment of surfactants with 89% predictive accuracy compared to existing human data, correctly classifying 8 out of 9 internally tested surfactants, including well known challenging ones such as Sodium Dodecyl Sulphate (SDS) and Benzalkonium chloride.


Current legislations and trends in predictive toxicology advocate a transition from in vivo methods for hazard and risk assessments to non-animal alternatives. However, certain groups of chemicals, including substances with severe membrane-damaging properties, pre- and pro-haptens, and those with high log P ratios, have been shown to be challenging to assess using cell-based assays in the context of skin sensitization testing. The aim of this study was to evaluate the applicability of GARDskin for such challenging substances, using an overlapping subset of chemicals previously tested in an integrated tested strategy (ITS) based on validated, aqueous in vitro assays, as well as in a series of Reconstructed Human Epidermis (RHE)-based assays.

The GARDskin assay (Genomic Allergen Rapid Detection) is a robust in vitro assay for identification of potential chemical skin sensitizers with over 90% prediction accuracy and broad applicability. The assay is included in the OECD Test Guideline Program (OECD TGP 4.106) and has gone through a formal validation study. The assay evaluates the gene expression of endpoint-specific genomic biomarkers in a human dendritic-like cell line following exposure to the test substance. Exposure-induced gene expression patterns are analysed using pattern recognition and machine-learning technology, providing classifications of each test item as a skin sensitizer or a non-sensitizer.

The applicability of GARDskin for a total of twelve challenging substances, including pre- and pro-haptens, low water-soluble substances, two surfactants and three additional substances known to have conflictive results when comparing in vitro and in vivo data were evaluated in this study. All twelve substances were selected from the Mehling et al. 2019 publication which reported results from three OECD validated in vitro methods, the “2 out of 3” Integrated Testing Strategy, three RHE-based models and the murine local lymph node assay (LLNA). Human potency classification was available for ten out of the twelve substances.

The GARDskin prediction results were reported from previously published studies, or from in house validation studies. Predictive accuracies were calculated by comparing skin sensitization classifications from different test methods to the available human data of each substance respectively. (N=10). To further explore and substantiate the GARDskin applicability for surfactants, additional GARDskin data for a total of nine surfactants are presented in order to complement the Mehling dataset with respect to the availability of human data.

The GARDskin assay demonstrated overall high applicability for the evaluated challenging substances, with 80% predictive accuracy compared to existing human data. GARDskin correctly classified all pre-and pro-haptens and low water-soluble substances in the data set. Furthermore, high applicability of GARDskin for severe membrane disruptive substances such as surfactants was demonstrated, with 89% predictive accuracy compared to existing human data.


Poster presented at ACT 2020: Dose-Response Analysis in GARD™ for Assessment of Skin Sensitizer Potency

J. Schmidt, A. Forreryd, R. Gradin2, H. Johansson.
SenzaGen Inc., Raleigh, NC., SenzaGen AB, Lund, Sweden.


Link to the poster



  • As an adaptation from the GARDskin assay, GARDskin Dose-Response is suitable for quantitative skin sensitizing potency assessment of chemicals.
  • The experimental readout, referred to as cDV0, corresponds to the lowest dose required to elicit a positive response in GARDskin. As such, experimental protocols are analogous to the LLNA, in which the cDV0 corresponds to the EC3-value.
  • The cDV0 may be used to directly monitor sensitizing potency, or further used to extrapolate LLNA EC3-values, estimation of Human Potency categories, or CLP 1A/1B classifications.


Several non-animal methods for identifying skin sensitizers have been developed with acceptable prediction performance. However, advancement of alternative methods for skin sensitizing potency assessment is still missing although a highly sought-after endpoint. The GARDskin assay is a genomics-based in vitro assay for hazard assessment of skin sensitizers, currently progressing towards regulatory acceptance. Here, we introduce GARDskin Dose-Response (DR), in which test chemicals are evaluated by the GARDskin assay in an extended range of concentrations, in order to investigate the dose-response relationship between GARDskin classifications and test chemical concentration.

For this work, 29 chemicals of various sensitizing potencies were used to evaluate the efficacy of applying the assay in this manner. Each chemical was analyzed at several concentrations using a slightly modified GARDskin protocol. At each concentration, a decision-value was produced and a classification prediction (sensitizing or non-sensitizing) was made by the GARDskin algorithm. Afterwards, the lowest concentration where a test item would provide a positive GARDskin prediction was found using linear interpolation. This concentration (cDV0) was then hypothesized to reflect the test items skin sensitizing potency. Furthermore, when comparing cDV0 to LLNA EC3 values, a statistically significant correlation was realized between the values (correlation coefficient =0.74, p-value=4.1*10-4).

These results suggest that modifying the GARDskin protocol to accommodate dose-response measurements can provide sensitizing potency information analogous to the gold-standard in vivo methods. This presentation will further explain the testing process, expand on results, and demonstrate how this method can be used for decision-making throughout all stages of product development, without having to use animal experimentation.

Inter-laboratory ring trial of the GARD™air assay for assessment of respiratory sensitizers

Andy Forreryd1, Joshua Schmidt1, Robin Gradin1, Florence Burleson, Helge Gehrke3, Henrik Johansson1.
1SenzaGen AB, Lund, Sweden. 2Burleson Research Technologies, Morrisville, NC, USA. 3Eurofins BioPharma Product Testing Munich GmbH, Planegg, Germany.

Sensitization of the respiratory tract by sensitizing chemicals may lead to severe bronchoconstriction and asthma-like symptoms with a potentially fatal outcome. However, proactive identification and characterization of respiratory sensitization hazards is currently hampered by the lack of validated or even widely used predictive assays.

The GARD™ platform utilizes exposure-induced gene expression profiles of a dendritic cell-like cell line in combination with machine learning as a basis for predictive in vitro assays for different immunotoxicity endpoints (Johansson et al., 2020). Functional evidence of the ability to accurately identify respiratory sensitizers using the GARD platform has previously been demonstrated (Forreryd et al., 2015). Here, we present GARDair, an adaptation of previous findings on a gene expression analysis platform suited for routine acquisition, based on an optimized predictive biomarker signature. Furthermore, we present the initial results of an inter-laboratory ring-trial, demonstrating the functionality, transferability and reproducibility of the assay.


  • GARDair is a novel in vitro assay for assessment of respiratory sensitizers, based on evaluation of exposure-induced gene expression of genomic biomarkers in a Dendritic Cell-like cell and machine-learning assisted classification.
  • GARDair is the first in its class to be subjected to an inter-laboratory ring trial.
  • The method is highly specific, with high PPV, making it suitable for opt-out applications within product development and candidate prioritization.

Link to poster

Poster abstract accepted at SOT 2020. 

The GARD Skin Assay: A New In Vitro Testing Strategy for Skin Sensitization

E. Schmidt, V. Zuckerstätter, H. Gehrke | Eurofins BioPharma Product Testing Munich GmbH

A skin sensitiser refers to a substance that will lead to an allergic response following skin contact as defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS). The potential to induce skin sensitisation is an important consideration included in procedures for the safe handling, packaging and transports of chemicals.

The assessment of skin sensitisation typically involves the use of laboratory animals. Classical methods comprise the Magnusson Kligman Guinea Pig Maximisation Test, the Buehler Test (TG 406) as well as the local lymph node assay, in its radioactive and non-radioactive form (TG 429, TG 442A/B). In order to replace in vivo experiments validation studies on alternative, mechanistically based in chemico and in vitro test methods on skin sensitisation were conducted under the auspices of ECVAM and have been considered scientifically valid for the evaluation of the skin sensitisation hazard of chemicals.

Genomic Allergen Rapid Detection (GARDTM) is an in vitro assay designed to predict the ability of chemical substances to induce skin sensitisation based on the analysis of the relative expression levels of a biomarker signature of 196 genes using a human myeloid leukaemia cell line called SenzaCells. The GARDTM assay is based on chemical stimulation of the SenzaCells, acting as an in vitro model of human Dendritic Cells (DCs). The readout of the assay is a transcriptional quantification of the genomic predictors, collectively termed the GARDTM Prediction Signature (GPS), using Nanostring nCounter technology.

The DPRA, KeratinoSensTM and h-CLAT are well known sensitization assays which address three different key events of the AOP. The GARDTM skin assay is a new procedure that analyses the sensitization potential based on almost 200 human genes. If a substance is a skin sensitiser with the GARDTM skin assay you have the benefit of measuring the potency on top with a different code set to make a 1A or 1B classification.
The GARDTM skin assay is especially for products that have a high log Pow (h-CLAT > 3.5, KeratinoSensTM > 7) because in those cases the classical sensitization tests are inconclusive if negative and there is no option for a replacing test method. Therefore, the GARDTM skin assay is not only an excellent alternative of the sensitization methods for these cases but it can furthermore predict the potency of a skin sensitiser, a unique feature, which makes it a testing method needed in the future.

Link to poster

Poster presented at Eurotox, Helsinki, Sep 9, 2019.


In vitro skin sensitization testing of Medical Devices using GARD®

Rose-Marie Jenvert, Angelica Johansson, Olivia Larne, Emelie Danefur, Emil Altonen, Anders Jerre, Robin Gradin, Gunilla Grundström.
SenzaGen, Lund, Sweden.

All medical devices need to be evaluated for the end point skin sensitization according to the Biological Evaluation of Medical Devices (ISO 10993-1:2018), today commonly involving in vivo assays. Here, we show that the in vitro assay GARD®skin Medical Devices can classify leachables as either skin sensitizers or non-sensitizers in polar and non-polar extraction of Medical Devices.

GARD®skin Medical Device

  • is an in vitro alternative for assessment of skin sensitization of Medical Devices
  • is compatible with the extraction vehicles salin, olive oil and sesame oil.

Link to poster

Poster presented at Eurotox, Helsinki, Sep 9, 2019.

Extended solvent selection for in vitro sensitization testing using GARD®

Olivia Larne, Ulrika I Torstensdotter Mattson, Rikard Alm, and Gunilla Grundström.
SenzaGen, Lund, Sweden.

The GARD®skin assay is an in vitro assay developed for the assessment of skin sensitizers. It is based on SenzaCells™, a human dendritic-like cell line, and a biomarker signature analyzed by a prediction model including pattern recognition and machine learning.

During the development of the GARD®skin platform, two solvents were used: DMSO (0.1%) and water. To increase the applicability domain of GARD®skin and the possibility to dissolve certain test items, for e.g. hard to dissolve substances and UVCBs, where show a broader range of solvents compatible with GARD®skin. Also, use of higher concentrations of the tested solvents were explored for the possibility to increase test item concentrations.

Concluding highlights
GARD®skin compatible solvents:

  • Acetone
  • DMF
  • DMF/Glycerol
  • DMSO
  • Ethanol
  • Glycerol
  • Isopropanol

Increased applicability domain.

Link to poster

Poster presented at Eurotox, Helsinki, Sep 9, 2019.


GARDair – A novel assay for assessment of chemical respiratory sensitizers based on a genomic platform

Henrik Johansson, Andy Forreryd, Robin Gradin, Angelica Johansson, Olivia Larne, Emil Aaltonen, Anders Jerre, Carl A.K. Borrebaeck and Malin Lindstedt
SenzaGen AB, Lund, Sweden. Department of Immunotechnology, Lund, Sweden.

Exposure to chemicals may induce allergic hypersensitivity reactions in skin or respiratory tract. To minimize exposure, chemicals are routinely screened for their sensitizing potential. Proactive identification has historically been performed using animal models, but the use of animals for safety assessment of cosmetics was recently banned within EU. Today, similar trends are spreading both globally and across industry and market segments. Methods for specific identification of respiratory sensitizers are greatly underdeveloped, with no validated, or even widely used assay readily available. Thus, there is an urgent need for development of non-animal-based methods for hazard classification of respiratory sensitizing chemicals.

GARD– Genomic Allergen Rapid Detection – is a state of the art technology platform for assessment of chemical sensitizers (Figure 1). It is based on a dendritic cell (DC)-like cell line, thus mimicking the cell type involved in the initiation of the response leading to sensitization. Following test chemical exposure, induced transcriptional changes are measured to study the activation state of the cells. These changes are associated with the immunological decision-making role of DCs in vivo and constitutes of e.g. up-regulation of co-stimulatory molecules, induction of cellular and oxidative stress pathways and an altered phenotype associated with recognition of xenobiotic matter. By using state-of-the-art gene expression technologies, high informational content data is generated, that allows the user to get a holistic view of the cellular response induced by the test substance.

GARDair is a novel assay for assessment of respiratory sensitizers. It is an adaptation of the GARD platform, utilizing gene expression analysis of predictive biomarker signatures and state-of-the-art data analysis methodology. GARDair has been proven functional and is currently progressing towards industrial implementation and  regulatory acceptance with financial support from the EU programme Horizon 2020. This progress includes scientific verification of results, assay optimization, assay
transfer and formal validation by a blinded ring trial.

Link to poster

A mechanistic reinterpretation of the AOP for skin sensitisation

David W Roberts, Liverpool John Moores University, Liverpool

Introduction – Non-Animal Prediction: the 21st Century Consensus
Because of the biological complexity of the skin sensitisation process no single in chemico or in vitro assay will be an appropriate replacement for an animal-based assay such as LLNA or GPMT…
…to ensure a mechanistic basis and cover the complexity, multiple methods should be integrated into a testing strategy, in accordance with the adverse outcome pathway that describes all key events in skin sensitisation.

We need an ITS based on the KEs of the AOP…but
Is that what we really need?


A single assay, GARD™, predicts sensitisation potential and absence of sensitisation potential better than any of, or combinations of, the OECD guideline assays DPRA, KeratinosensTM (ARE-Nrf2 ) and h-CLAT.

We do not really need an ITS covering all KE’s of the AOP.

Link to poster

The Validation of GARD®skin

Sandberg P, Johansson A, Agemark M, Gradin R, Larne O, Appelgren H, Forreryd A, Jerre A, Edwards A, Hoepflinger V, Burleson F, Gehrke H, Roggen E, Johansson H
SenzaGen, Lund, Sweden, Burleson Research Technologies, Morrisville, US, Eurofins, Munich, Germany

The prevalence of allergic contact dermatitis (ACD) is estimated to >20% in the western world. Not only the individual is affected, but downstream socioeconomic effects are high. To minimize exposure, chemicals must be safety tested. Traditional testing strategies like the murine local lymph node assay (LLNA) comprise animals, but the regulatory authorities, public opinion and economic interests require animal-free models. The Genomic Allergen Rapid Detection skin (GARD®skin) is an in vitro assay addressing this need. Here, we present the results of the GARD®skin ring trial (OECD TGP 4.106) for validity of the assay. In addition, we show data for GARD®potency – a complementary assay developed to categorize identified senitizers as CLP 1A or 1B.



Transfer study
Transferability: 100%

Validation study
WLR: 82 – 89%BLR: 92% (92 – 100%)
Test performance
– Accuracy: 94%
– Sensitivity: 93%
– Specificity: 96%

A blinded ring trial was performed to assess the functionality of the GARDskin assay. The data demonstrates that GARDskin is a powerful tool for assessment of chemical skin sensitizers, with a predictive accuracy of 94% and excellent reproducibility between laboratories.
In addition, we show that GARDpotency accurately assesses potency of identified sensitizers.

Poster download

Predicting skin sensitizers with confidence – Using conformal prediction to determine applicability domain

GARD – Genomic Allergen Rapid Detection – is a state of the art non-animal based technology platform for classification of skin sensitizing chemicals. The assay has proven to be reliable and highly accurate for identification of skin sensitizing chemicals, and consistently reports predictive performances > 90% across external test sets. The aim of the current project is to complement assessments of average model performance with an estimate of uncertainty involved in each individual prediction, thus allowing for classification of skin sensitizers with confidence.

An Internal validation procedure was initially performed on samples in the GARD training set (n=38) using the strategy described in Fig. 3A. Results from this exercise is summarized in Fig. 4A. Conformal prediction by definition allows the user to determine a reasonable and acceptable significance level to guarantee a maximum error rate in predictions. The significance level was set to 15%, i.e. the model was allowed to make a maximum of 15% errors. Performance of the conformal predictor was measured by validity and efficiency. A model was valid if the number of prediction errors did not exceed the significance level, while efficiency corresponded to the percentage of single class predictions. Internal validation of the training data resulted in a valid and highly effective model (92% single classifications, 1 empty, 2 both), indicating that the ambitious significance level was at a reasonable level for the GARD® assay. Following internal validation, samples in a large external test set (n =70) was classified within the CP framework as described in Fig. 3B, which resulted in generation of a valid and highly efficient model (99% single classifications, 0 empty, 1 both) (Fig.4B). Additional data on model performance is illustrated in Table 1.

Poster download