Tuesday, 26 July 2011 09:57

Working group - a Portrait: "Short-term Toxicology" of the department “Experimental Toxicology and Ecology”, BASF Featured

InvitroJobs presents scientists and their innovative research in a regular feature called “Working Group – a Portrait”. The focus is on newly developed methods, their evaluation and their potential for reducing and, where possible, replacing animal experimentation according to the “three Rs” of Russell & Burch (reduce, refine, replace).
Our third article presents selected examples from the research group “Short Term Toxicology” in the department “Experimental Toxicology and Ecology” at BASF SE. It is necessary to define the scope of this article this narrowly as there are over 50 staff members in five laboratories, with more than 20 methods being implemented and nearly as many again in development.


Working group - a Portrait: "Short-term Toxicology"
of the department “Experimental Toxicology and Ecology”, BASF

BASF SE (Societas Europaea) is the world's leading chemical company1. Before BASF places products on the market, it is necessary to test them for potential toxicological hazards. The basis for this are the OECD guidelines for toxicological tests of chemicals, which in most cases prescribe the use of animal experiments2. At BASF, all toxicological tests are performed by the Toxicology and Ecology department headed by Dr. Ben van Ravenzwaay.

Scientists are striving to meet the increasing demand for alternative methods to animal experiments using the “three Rs” of Russell & Burch, “reduction, refinement and replacement”, by reducing the number of animals used in experiments, refining test methods and replacing animal tests with animal-free methods. For more than 20 years BASF has sought to promote the development and evaluation of such methods, developing new replacement methods and state of the art measuring instruments7, and cooperates with institutes and universities. BASF enjoys international recognition for the development and validation of alternative methods and participates in numerous validation studies promoted by the EU and the German Federal Ministry of Education and Research (BMBF) in collaboration with other companies and institutes. The laboratories also serve as validation labs for toxicological methods (cell transformation, skin irritation effects, eye irritation effects, sensitisation).

In 2004 the concern founded its own laboratory for the development of alternative methods, further in 2009 the “Laboratory for Applied Alternative Methods”, where replacement methods for a range of toxicological tests are developed, such as in the fields of local tolerance3, reproduction toxicology4, genetic toxicology5 and systemic toxicology6. In addition devices have been and continue to be developed in order to be able to conduct the test methods approved the OECD guidelines.

The research group “Short Term Toxicology”, with more than 50 staff members and headed by Dr. Robert Landsiedel, comprises five labs (inhalation toxicology, genetic toxicology, applied alternative methods, bio-kinetics and development of alternative methods) and is part of the department “Experimental Toxicology and Ecology”. The group develops and validates alternative methods to animal tests, as well as implementing them in routines. It also develops measuring instruments and provides information on the development of replacement methods, e.g. in seminars as part master’s studies in toxicology. Some of the methods are already validated and implemented in the OECD guidelines, making them internationally applicable for regulatory purposes.

With some of the developed methods, which are have now been implemented in testing specifi-cations, the scientists were able to participate in validation procedures, for instance for replace-ment methods in the field of skin und eye irritation (see the following). In addition, state of the art instruments7 were developed for the purpose of conducting test methods compliant to regulations, such as an opacitometer for measuring eye irritants. In collaboration with Prof. Dr. Günter Vollmer, Chair of Molecular Cell Biology and Endocrinology at the Technical University of Dresden, BASF has developed a test system that uses yeasts instead of animals for reproductive toxicity tests (see below).
The following text introduces five important methods which the BASF labs have worked on, and that already have contributed to replacing animal experiments or may yet do so. The scientists have:

- developed and certified a device for testing eye irritating effects of substances (opacitometer), reproduced a cornea model using human cells and suggested both methods as a phased test system for the complete replacement of the Draize test on rabbits’ eyes for testing severe and mild ocular irritants.

- used human dermal tissue from cosmetic surgery to investigate substances’ dermal penetration in diffusion cells, as well as continuing to work on growing special human skin models.

- developed a test battery comprising multiple in vitro test systems, allowing the identify allergens (skin sensitising substances).

- worked on and continue to work on establishing lung slice models and 3D lung models from human cells in order to test certain inhalation toxicological parameters and evaluate these with regard to toxicological issues.

- developed a test system to reduce the number of animals used in preliminary tests (screening) for testing the endocrine disruptive potential of test substances, by employing yeast cells with human hormone receptors.

1.) Alternative to eye irritation test: the Opacitometer OP3.0 from BASF
Until now, eye irritating effects of industry chemicals have routinely been tested on rabbits’ eyes (Draize test or modifications thereof in accordance with OECD Guideline No. 405). However, in September 2009, the OECD approved an in vitro test with isolated bovine cornea (BCOP test using bovine cornea from slaughtered cattle) which can replace the Draize assay for testing substances for severe ocular irritation. The new assay has been incorporated into the guidelines as No. 437. During the in-house validation carried out by BASF, postdoctoral research assistant Dr. Arnhild Schrage from the “Laboratory for Applied Alternative Methods” (headed by Dr. Susanne Kolle) tested several devices no longer available on the international market in comparison to BASF’s own prototype8. The devices were tested in the laboratory for compliance to OECD guideline No. 437. In addition, a certification study was conducted in collaboration with the IIVS (Institute for In vitro Sciences), Gaithersburg / Maryland (USA). The BASF Opacitometer OP3.0 has approved and can be used. The certified device contains components that allow precise and reproducible tests. In addition the device includes calibration standard (grey glass filter), cornea holder and electronic data aquisition.

The BCOP was developed for recognising eye irritants. However, there is currently no approved test capable of distinguishing the effects of mild irritants and non-irritant substances. For this purpose the laboratory suggested to the OECD a graded test strategy consisting of two combined methods, the BCOP assay and a reconstructed cornea model, with the goal of fully replacing the Draize test. Results obtained from an in-house validation study with 60 test substances indicate that the combination of the reconstructed cornea model with the BCOP assay is a suitable testing strategy for identifying strong/severe eye irritants (GHS category 1)9, moderate and mild eye irritants (GHS category 2), and non-irritants in routine testing.

2.)  Dermal penetration studies in vitro: an established alternative method with potential
Dermal penetration studies in vitro are performed within the research group “Experimental Toxicology” in the laboratory of biokinetics under the direction of Dr. Eric Fabian. Currently, 9 technical employees with experience in biology, medicine and chemistry work in this lab team. In addition, the post-doc Dr. Christine Jäckh works on research oriented questions in the biokinetics laboratory. She investigates the activities of xenobiotic metabolising enzymes in human skin models and focuses on the question of how these enzymes differ from those in native human skin samples10, 11. In the lab, the research oriented work is supported by the two doctoral candidates Katharina Guth and Veronika Blatz.

Images: BASF

When investigating a test substance it is of interest which fractions can be washed off after a certain period of exposure and which fractions penetrate the skin during the period of exposure or remain in the skin after washing it off. The data from the investigation of a substance’s dermal penetration can be used to calculate detrimental systemic effects. Taking the product’s application conditions into account, these effects can play a considerable part in risk assessment. Until five years ago, most tests were performed in vivo on rats in accordance with OECD guideline No. 427. Since then this type of study has almost entirely been replaced in BASF laboratories by the approved alternative method OECD No. 428.

Because of the great potential variability of this new experiment type (due to the physical/chemical properties of the test substance, physical / chemical properties of the donor and receptor media, the quality of the skin specimen or the kind of diffusion cell), numerous tests are conducted in the lab in addition to the routine experiments, in order to systematically investigate the influence of selected parameters on dermal penetration. The tests also have the purpose of ascertaining whether future dermal penetration tests will require ex vivo skin samples or skin models produced from cell cultures can also be used.

Due to the great potential variability of this type of test (for instance because of physical / chemical properties of the test substance, physical / chemical properties of the donor and receptor media, condition of the skin specimens, or the kind of diffusion cell), numerous experiments are performed in the laboratory to systematically investigate the influence of selected parameters on dermal penetration. Additionally, experiments will have to find out whether such ex vivo skin samples will be needed for future dermal penetration studies or whether tests of this kind might be conducted using skin models especially derived from cell cultures.

Construction and process of a dermal in vitro penetration study
The in vitro approach uses ex vivo skin, usually obtained from cosmetic surgery. As the differences in the penetration characteristics of human and animal skin are known, scientists routinely use human skin. For this purpose the skin is dermatomised (peeled off in thin slices using a dermatome) to a thickness of 400 micrometers. The cross section of the skin distinctly displays the skin layers, stratum corneum, epidermis, basal membrane and dermis (from top to bottom). The stratum corneum is the main penetration barrier.

In the experiment, five skin specimens per dose are clamped into so-called Franz diffusion cells (see figure 1-3). The samples are equilibrated in a physiological salt solution and brought to body temperature. The samples’ quality is then first checked by measuring their resistance, determining their transepidermal water loss or testing the barrier function with titrated water. In the test itself, the test substance is applied to the skin. After different time intervals, aliquots (samples) are taken from the receptor medium in the receptor chamber of the Franz diffusion cell.

After the period of exposure, the skin is rinsed. In order to evaluate the substance residues on the skin, a further observation of the system is conducted, normally for up 24 hours. Since C14 marked substances are often used in this kind of tests, the analysis can be performed by determining activity via measurements of liquid scintillation. To this purpose, all the samples obtained (donor chamber, receptor chamber, skin specimens, rinsing solutions etc.) are measured. The fractions in the receptor medium can be used to determine the fraction of the applied dose absorbed by the skin, whereas kinetic parameters such as absorption rate and permeability constants can be calculated from the receptor samples taken at various times. A crucial parameter for the evaluation of the test’s validity is the recovery of the applied activity, which should be between 90 and 110 per cent for each cell.

Setup of a static Franz diffusion cell:
Fig. 1: Schematic setup of a static Franz diffusion cell.
Fig. 2: Individual components of the diffusion cell (1 = receptor chamber; 2 = donor chamber,
3 = stainless steel clamp, 4 = 3-way check valve, 5 = Heidelberger extensions for water jacket).
Fig. 3: Assembled Franz diffusion cell.
Diagram: BASF
Photos: BASF


3.) In vitro test strategies suitable for predicting the skin sensitisation12 potential of chemicals
Skin sensitisation is an important health problem when a person has repeatedly been exposed to a sensitising substance and has for instance developed an allergy or some other skin condition. Until now, tests for substances’ skin sensitising characteristics have completely relied on animal testing in accordance with REACH stipulations, such as the local lymph node assay (LLNA) using mice13, and there is no in vitro method available which has been validated and approved. As the seventh amendment of the Cosmetics Directive will prohibit the marketing of cosmetic products tested on animals as of March 2013, BASF is working on developing and establishing an in vitro test system for skin sensitisation as a replacement method for the LLNA, and has performed an evaluation study with several laboratories for four in vitro test procedures14.

The methods are being developed in the Laboratory for Applied Alternative Methods under the direction of Ms Dr. Tzutzuy Ramirez by post-doc Dr. Tobias Eltze together with doctoral candidates Caroline Bauch and Britta Wareing (BSc). Since the mechanism of skin sensitisation is a complex procedure that takes place in several stages, an in vitro test system has to be made up of several individual tests. Therefore the following three test combinations have been suggested:

a) Direct Peptide Reactivity Assay (DPRA)
The protocol was developed by Procter & Gamble in collaboration with the University Louis Pasteur in Strasbourg15. Here scientists make use of the fact that skin sensitisation reactions begin with the reaction of chemical allergens with proteins. Most chemical allergens are electrophilic and therefore react with nucleophilic amino acids such as cysteine or lysine. The DPRA is performed using two synthetic peptides which carry either lysine or cysteine residues for interaction with the test substance. The mean peptide degradation is measured by HPLC-UV. The property of the conversion into electrophilic metabolism products is a common feature of skin sensitising substances.

b) Antioxidant Response Element (ARE) assay
A reporter keratocyte cell line (HaCaT cell strain, supplied by the RWTH Aachen University) serves as receptor cell for the verifying the activation of ARE-dependent genes. The molecular biological signal pathway is such that ARE-dependent genes are activated by the presence of a skin sensitising substance16. A particular repressor protein (Keap1) is modified by the test substance and dissociated from the so-called transcription factor, triggering the production of a luciferase reporter protein. The luminescence emitted by the luciferase activity serves as indirect proof of the expression of ARE-dependent genes17.

c) Dendritic Cell Like Line Activation Myeloid U937 Skin Sensitation assay (MUSST)
The protocol was developed by L’Oréal in collaboration with Procter & Gamble. The activation and alteration of the surface marker spectrum of dendritic cells play a central part in sensitisation. In the MUSST assay, the cell line U937 is used. After incubation of the test substance the cell surface molecule CD86 is measured by a flow cytometer. This test battery has shown a very good estimation of the skin sensitising effects of substances. The results were published just recently.

4.) In vitro methods for inhalation toxicology
Using precision cut lung slices (PCLS using rat or human donor organs), scientists can investigate the cellular and functional reactions of lung tissue to test substances in vitro, especially human tissue. This allows a greater number of test substances to be compared more easily with each other. Parameters are cytotoxicity, oxidative stress18, apoptosis (programmed cell death), immune reaction and histological alterations. The test is currently being pre-validated within the framework of a research project by the BASF lab, RWTH Aachen University and the Fraunhofer ITEM, Hannover, with funding from the German Federal Ministry of Education and Research (BMBF). At BASF, post-doc Dr. Sandra Vogel and doctoral candidate Annemarie Hess are working on the project alongside Ms. Dr. Lan Ma-Hock (director of the Laboratory for Inhalation Toxicology).

There are also 3D lung models already available that are produced using human cells. There are several lung models are available that differ morphologically and use different cell lines. Here test substances can reach the lung epithelia in a way similar to the natural in vivo situation. BASF’s Laboratory for Experimental Toxicology has established several lung models with the objective of comparing and evaluating which model is most suitable for in vivo predictions.

5.) In vitro reproduction toxicity test: Yeast Estrogen Screening (YES) and Yeast Androgen Screening (YAS)
Both tests were established in collaboration with the Technical University of Dresden, Chair of Molecular Cell Physiology and Endocrinology, Prof. Dr. Günter Vollmer. The tests were further developed by BASF under GLP conditions (GLP standard, see glossary) and validated by testing more than 100 substances.

The tests serve to determine the endocrine disruptive potential of test substances. Yeast cells were genetically modified to contain a plasmid with the sequences of human estrogen receptors and androgen receptors, as well as using β-galactosidase as a so-called reporter gene for detection purposes. The estrogen/androgen antagonistic and the estrogen/androgen agonistic potentials can be detected by colorimetric reaction using a plate reader. The tests were conducted in the Gene Toxicology Lab by Claudia Woitkowiak (BSc) under the direction of Dr. Markus Schulz.

Both tests are used as screening tests19, 20. The results of the YES/YAS tests help to identify endocrine disruptive substances and to prioritise further tests.

Workstation YES-Test. Image: BASF.

Depiction of anti-estrogen activity (left) and estrogen activity (right). Photo: BASF

Contents of the YES/YAS test plates:
Row A: vehicle controls (columns 1 to 4 and 9 to 12) and a hormonally active control plus an antagonistic control substance (column 5 to 8).
Rows B through H (in increasing concentrations from B to H): hormonally active positive control substance (columns 1 to 4), test substance plus hormonally active positive control substance (columns 5 to 8), and test substance alone (columns 9 to 10).
The culture medium contains a yellow ß-D-galactopyranoside (CPRG), the β-gal enzyme is metabolised to the pink CPR in the presence of hormonally active substances. The colour development as well as the optical density as an indicator for cytotoxicity are determined photometrically after approximately 48 hours.

We thank BASF, working group “Short Term Toxicology” of the department “Experimental Toxicology and Ecology” for providing information, depictions and photos.


stands for Registration, Evaluation and Authorisation of Chemicals. The European Directive requires that producers and importers determine possible harmful properties (for instance toxic, carcinogenic, harmful to the environment) of their produced or imported substances (chemicals and natural substances) and to evaluate their effects on health and the environment.

OECD guidelines
Organisation for Economic Cooperation and Development (OECD). There are test guidelines for almost all health effects of chemicals.

Local tolerance
In toxicology, local tolerance (local = at the contact site such as eye or skin) means the adaptation of an organism to the influence of a test substance by increasing biosynthesis of an enzyme which can degrade the toxic substance.

Toxic for reproduction
Substances which affect the reproduction ability or can induce undesirable foetal developments.

Gene toxicity
Effect of substances which induce alterations in genetic material.

Systemic toxicity
Systemic toxins often have an effect on a certain target structure or target organ, the reason for this being that the transformation of the toxin into its actual active form often only takes place in the specific organ, or that cell growth is particularly high in that organ.

Global Harmonised System.

Sensitising substances
Substances or preparations which can induce a hypersensitive reaction after inhalation or skin absorption, so that characteristic disorders such as allergies appear upon further exposure to the substance or preparation.

Assay approved within guidelines for testing skin sensitising effects on mice.

Specialised human epidermal cells that produce keratinous substances. Keratin has a water-repellent effect, and protects and stabilises skin24.

Chemical compound which prevents undesired oxidation of other substances.

Repressor protein
A repressor is a DNA-binding regulator protein which prevents transcription of a gene section by binding to a special site25.

Reference laboratory
Reference laboratories are labs which are appointed by the European Commission (“Community Reference Laboratory”) or a national government (“national reference laboratory”) on the basis of common or national legal provisions. These laboratories have especially qualified in their field, have a high degree of independence and can work on transnational level. National reference laboratories were founded with the objective of creating a basis for harmonizing the measures in the field of laboratory diagnostics on a European level23.

Validation delivers the proof that a process or system reproducible satisfies the previously specified requirements (acceptance criteria) in a practical application.

In-House validation
Methods are adapted to the company’s own requirements. A validation has to characterise a method by laboratory tests as well as possible and has to prove their suitability for the given purpose. It has to test the specificity, sensitivity, relative correctness, reproducibility (precision), comparability (robustness), detection limit, limit of determination, linearity, false-positive rate, false-negative rate, statistical correlation26.

GLP certification
Good laboratory practice. The OECD principles are a quality control programme for harmonising test methods. To create a foundation of trust for the mutual recognition of test data, it must be ensured that the laboratories conducting the tests have the same standards in all countries and work according to similar methods. A lab or a method can be GLP certified within this context.

Good laboratory standard

Oxidative stress
The amount of reactive oxygen compounds which exceeds physiological levels.


1 BASF Website http://www.basf.com/group/corporate/de/about-basf/index?mid=0
2 http://www.bfr.bund.de/de/oecd_richtlinien_zur_toxikologischen_pruefungen_von_chemikalien-61575.html
3 http://www.uni-protokolle.de/Lexikon/Toleranz.html
4 http://de.wikipedia.org/wiki/Teratogen
5 http://de.wikipedia.org/wiki/Genotoxizit%C3%A4t
6 http://www.chemgapedia.de/vsengine/vlu/vsc/de/ch/11/toxikologie/kap_1/vlu/wirkung.vlu/Page/vsc/de/ch/11/toxikologie/kap_1/kap1_3/lokal.vscml.html
7 http://de.wikipedia.org/wiki/State_of_the_Art.
8 Schrage, A., Kolle, S. N., Rey Moreno, M. C., Norman, K., Raabe, H., Curren, R., van Ravenzwaay, B & Landsiedel, R. (2011): The Bovine Corneal Opacity and Permeability Test in Routine Ocular Irritation Testing and Its Improvement Within the Limits of OECD Test Guideline 437.  ATLA 39:37-53
9 http://www.bgchemie.de/REACH-GHS
10 Jäckh, Ch., Blatz, V., Fabian, E., Guth, K., Reisinger, K., van Ravenzwaay, B., Landsiedel, R.: Characterization of enzyme activities of Cytochromes P450, Flavin-dependent monooxygenases, N-Acetyltransferases and Glucuronyltransferases in human reconstructed epidermis and full-thickness skin models. Toxicology in Vitro (Epub ahead of print)
11 HYPERLINK "http://www.nature.com/nprot/journal/v6/n5/full/nprot.2011.316.html" \l "auth-1#auth-1" Landsiedel,  R., Fabian, E., Tralau, T. & Luch, A.: Chemical toxicity testing in vitro using cytochrome P450–expressing cell lines, such as human CYP1B1.  Nature Protocols 6: 677–688.
12 http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CELEX:31992L0032:DE:HTML
13 http://ecvam.jrc.it/ft_doc/ECVAM%20LLNA%20Performance%20Standards-Sec.pdf
14 Bauch, C., Kolle, S. N., Fabian, E., Pachel, Ch., Ramirez, T., Wiench, B., Wruck, Ch. J., van Ravenzwaay, B. & Landsiedel, R. (2011): In house validation of four in vitro assays for the prediction of the skin sensitizing potentials of chemicals. Toxicology in Vitro (Epub ahead of print)  http://www.sciencedirect.com/science/article/pii/S0887233311001664
15 Gerberick, G. F. (2004): Development of a Peptide Reactivity Assay for Screening Contact Allergens. Toxicological Sciences 81/2: 332-343. http://toxsci.oxfordjournals.org/content/81/2/332.full
16 Natsch, A. & Emter, R. (2007): Skin Sensitizers Induce Antioxidant Response Element Dependent Genes: Application to the In Vitro Testing of the Sensitization Potential of Chemicals. Toxicological Sciences 102/1: 110-119. : http://toxsci.oxfordjournals.org/content/102/1/110.full
17 Natsch, A. (2010): The Nrf2-Keap1-ARE. Toxicity Pathway as a Cellular Sensor for Skin Sensitizers-Functional Relevance and a Hypothesis on Innate Reactions to Skin Sensitizers. Toxicological Sciences 113: 284-292.
18 http://de.wikipedia.org/wiki/Oxidativer_Stress
19 http://www.bmu.de/chemikalien/doc/4056.php
20 Kolle, S. N., Kamp, H. G., Huener, H. A., Knicke, J., Verlohner, A., Woitkowiak, C., Landsiedel, R. & van Ravenzwaay, B. (2010): House Validation Of A Recombinant Yeast Estrogen And Androgen Receptor Agonist And Antagonist Screening Assays. Toxicology in Vitro 24: 2030-2040.
21 http://de.wikipedia.org/wiki/Validierung_%28Pharmatechnik%29
22 www.kantonslabor-bs.ch/.../In_house_Validierung_mikrobiologischer_Pruefverfahren.pdf -
23 http://de.wikipedia.org/wiki/Referenzlabor
24 http://flexikon.doccheck.com/Keratinozyt
25 http://de.wikipedia.org/wiki/Repressor.