This document summarizes alternative in vitro and in silico test methods developed by VITO to reduce animal testing. It describes cytotoxicity, inflammation, and genotoxicity assays using cell lines, as well as assays for ocular and dermal irritation, corrosion, and sensitization using 3D tissue models. The document also mentions use of the zebrafish embryo model to assess embryotoxicity, developmental neurotoxicity, and provides examples of validation studies conducted by VITO to replace animal testing.

1. MODE OF ACTION AND SAFETY TESTING:
ALTERNATIVE AND IN VITRO METHODS TO
REDUCE ANIMAL EXPERIMENTS
Overview of our test systems and related endpoints
»» Cytotoxicity assessment with cell lines for safety or biocompatibility testing (OECD
or ISO guidelines). Biological model systems (*) include 3T3 mouse fibroblasts or tar-
get-organ specific human cell lines (e.g. lung epithelial cells Beas-2B and A549, mono-
cytic cell line THP-1,..). Multiple assays are available to assess cellular functions,
such as membrane integrity, proliferation of cells, metabolic activity … and rank com-
pounds based on their toxicity profile.
»» Inflammation responses by measurement of cytokine production (e.g. IL-6, IL-8, IL-
1β, TNF-α) in cellular models (*)
»» Endocrine disrupting properties using human reporter cell lines (OECD 455,OECD
457) or hEr transformed yeast cells
»» Genotoxicity assessment: bacterial Ames II-test, in vitro micronucleus test (OECD
487) and comet assay in human blood cells
»» Ocular irritation: bovine cornea model - BCOP to classify compounds for
irritating potential (OECD 437) and human corneal epithelium model – HCE, which is
recently validated.
»» Dermal corrosion: 3D skin corrosion test with RHE model (OECD 431) and Corrositex®
(OECD 435) to assess damage to the skin.
»» Dermal irritation: 3D reconstituted skin model - RHE to assess irritating
potential of individual ingredients and finished products (OECD 439)
Environmental Risk & Health
What can we offer
VITO-ABS provides robust toxicity screening approaches, designed to improve and support the early discovery, efficacy testing and safe
design of new drugs, chemicals, nano­materials and nano-objects and related products by the pharmaceutical, biotechnology, nanotech-
nology, chemical and cosmetic industries.
Multi-endpoint bioassays, based on in vitro cellular assays or zebrafish embryo tests are employed to understand the mechanisms of
general or organ specific toxicity and allow predicting human toxicity in compliance with 3R principles (reduction, refinement or re-
placement of experimental animals).
The ABS team has expertise in the development and validation of these alternative assays, and is familiar with regulations on safety
testing of chemicals or nanomaterials (OECD), and biocompatibility testing of medical devices (ISO). The laboratory is certified for Good
Laboratory Practice (GLP) which is required to perform OECD safety tests for registration of chemicals and nanomaterials as defined by
REACH legislation. Activities in the cell culture facility are performed in line with principles of Good Cell Culture Practice (GCCP).
Furthermore, the team has access to cutting-edge technology (-omics approaches) and is open to joint tailor-made research projects
which contribute to the new vision on mechanism-based toxicity testing and elucidate Adverse Outcome Pathways (AOP).
(*): upon request specific cell lines or primary cultures available: to be selected for species, target
organ site and endpoint Dermal irritation RHE model
3T3 cells stained with neutral red

2. VITO NV | Boeretang 200 | 2400 MOL | Belgium | Tel. + 32 14 33 55 11 | www.vito.be
© 2015 VITO NV – All rights reserved
Although care has been taken to ensure the accuracy, completeness and reliability of the information provided, VITO assumes no responsibility therefore. The user of the information agrees that the information is subject to change without notice.
VITO assumes no responsibility for the consequences of use of such information, nor for any infringement of third party intellectual property rights which may result from its use. In no event shall VITO be liable for any direct, indirect, special or
incidental damage resulting from, arising out of or in connection with the use of the information.
TN-MRG-023-E/1502
»» Dermal sensitization using the VITOSENS®, a proprietary method based on gene ex-
pression profiles in human dendritic cells. The method allows to classify compounds
and assess their potency, and is shown to correlate with the LLNA test1
»» Embryotoxicity and developmental neurotoxicity using the zebrafish embryo by
evaluation of early developmental stages2
and locomotor activity as indicator of neu-
robehavioral dysfunction3
.
Examples of VITO projects:
»» EU-Reprotect: validation of hEr4
and hAr5
reporter cell lines for agonists and antagonists for endocrine disruptore (www.reprotect.eu);
»» validation study of Human Corneal Epithelial model (HCE) for ocular irritation;
»» collaboration with CEETOX on validation of their sensitization model;
»» US-CAAT research grants for the development of assays for Developmental Neurotoxicity (DNT) with zebrafish;
»» validation of Open Source Reconstructed Epidermis (OS-REP) model for skin irritation.
References
1. Hooyberghs J. et al. (2008). A cell-based in vitro alternative to identify skin sensitizers by gene expression. Toxicol. Appl. Pharmacol.
231, 103–111.
2. Selderslaghs I.W.T. et al. (2012). Feasibility study of the zebrafish assay as an alternative method to screen for developmental toxicity
and embryotoxicity using a training set of 27 compounds. Reprod Toxicol. 33 (2), 142-154.
3. Selderslaghs I.W.T. et al. (2013). Assessment of the developmental neurotoxicity of compounds by measuring locomotor activity in
zebrafish embryos and larvae. Neurotoxicology and Teratology 37, 44-56.
4. Witters H et al. (2010). The assessment of estrogenic or anti-estrogenic activity of chemicals by the human stably transfected estrogen
sensitive MELN cell line: Results of test performance and transferability. Reprod Toxicol. 30 (1), 60-72.
5. van der Burg B et al. (2010). Optimization and prevalidation of the in vitro AR-CALUX method to test androgenic and antiandrogenic
activity of compounds. Reprod Toxicol. 30 (1), 18-24.
An Van Rompay
Tel. +32 14 33 52 46
an.vanrompay@vito.be
Jef Hooyberghs
Tel. +32 14 33 52 77
jef.hooyberghs@vito.be
Contact
Microscopic analysis of developmental
characteristics in Zebrafish embryo