In Vitro Toxicology

The use of in vitro technologies to evaluate the safety of drugs and industrial chemicals

[invitrotox]

What is ICCVAM?

ICCVAM is 'The Interagency Coordinating Committee on the Validation of Alternative Methods' was created under the ICCVAM Authorization Act of 2000.

Objective: To facilitate the research, development, translation, validation, and regulatory acceptance of alternative test methods that reduce, refine, and replace the use of animals in testing.

ICCVAM  is a permanent interagency committee administered by the NIEHS under NICEATM (NICEATM: the National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods)

ICCVAM is composed of members designated by the heads of 15 federal agencies.

Some ICCVAM Activities:

Links:

ICCVAM-NICEATM: http://ntp.niehs.nih.gov/pubhealth/evalatm/index.html NIEHS: http://www.niehs.nih.gov/

ICCVAM-NTP: http://ntp.niehs.nih.gov/pubhealth/evalatm/iccvam/index.html

[invitrotox]

[invitrotox]

Timeline: Regulation and In Vitro Toxicity Testing

•1959: Russell and Burch book that launched the 3Rs, The Principles of Humane Experimental Technique

•1969: Founding of the Fund for the Replacement of Animals in Medical Experiments (FRAME) in the UK

•1981: Founding of the Johns Hopkins University Center for Alternatives to Animal Testing (CAAT)

•1986: EU Directive 86/609 for the protection of animals used for experimentation and other scientific purposes, which stipulates that: "An experiment shall not be performed if another scientifically satisfactory method of obtaining the result sought, not entailing the use of an animal, is reasonably and practicably available"

•1989: Founding of the German Centre for the Documentation and Evaluation of Alternatives to Animal Experiments (ZEBET)

•1991: Establishment of the ECVAM as part of the European Commission

•1993: The US National Institutes of Health Revitalization Act calls for emphasis on alternatives

•1996: OECD convenes the first international validation conference

•1997: ICCVAM is established as an ad hoc standing committee; ECVAM endorses first cell-based toxicity test method, the 3T3 neutral red uptake phototoxicity test

•1998: Three in vitro skin corrosion test methods endorsed by ECVAM

•2000: Passage of ICCVAM Authorization Act; ICCVAM endorses its first in vitro method, the Corrositex® assay for assessing skin corrosion

•2001: Congress directs the US Environmental Protection Agency (EPA) to spend $4 million on alternatives; OECD Test Guideline 401 (oral lethal dose) is deleted from international guidelines

•2002: The OECD Test Guidelines Program adopts the first formally validated in vitro tests; OECD establishes a Validation Management Group dedicated to non-animal methods; the fourth World Congress on Alternatives & Animal Use in the Life Sciences is held in New Orleans

•2003: The 7th Amendment of the EU Cosmetics Directive creates deadlines for banning animal testing of cosmetic products and their raw ingredients

•2004: The UK National Centre for the 3Rs (NC3Rs) is established; EU ban on animal testing of finished cosmetic products as of September 11, 2004; OECD test guidelines for in vitro 3T3 NRU Phototoxicity Test and for in vitro skin dermal penetration test methods

•2005: The US National Toxicology Program (NTP) adopts a 21st Century Roadmap emphasizing mechanistic, non-animal studies; EU regulators and industry launch the European Partnership for Alternative Approaches to Animal Testing (EPAA)

•2006: The EU provides more than 80 million Euros for targeted, multiyear 3Rs research projects; an international task force of pesticide producers and regulators proposes a testing strategy that could reduce animal use in reproductive and developmental toxicity studies by up to 70 percent

•2007: A US National Academy of Sciences (NAS) panel calls for a fundamental paradigm shift in regulatory toxicology in its report, Toxicity Testing in the 21st Century: a Vision and a Strategy; ECVAM endorses the EPISKINTM skin irritation test as a full replacement for rabbit skin irritation tests; ICCVAM and ECVAM endorse two enucleated eye methods for classifying ocular severe/corrosive materials

•2008: ICCVAM releases its five-year plan which identifies four priority areas for alternatives test method development; US Federal agencies announce collaboration on new high throughput toxicity screening initiative; two additional in vitro methods endorsed for skin irritation testing

•2009: EU ban on animal-based acute testing of cosmetic ingredients for all human health effects as of March 11, 2009; US Environmental Protection Agency (EPA) adopts NAS vision for evaluating the toxicity of chemicals in its new strategic plan; new international agreement to coordinate recommendations on alternative methods should speed their adoption and reduce animal testing; 50th anniversary of the Russell and Burch book that launched the 3Rs, The Principles of Humane Experimental Technique.

Some References:

http://www.frame.org.uk/timeline/

http://caat.jhsph.edu/about/timeline.html

http://www.nc3rs.org.uk/the-3rs

http://altweb.jhsph.edu/pubs/books/humane_exp/het-toc

[invitrotox]

Compounds For Validating an In Vitro Hepatotoxicity Screen

In validating in vitro screens for potential hepatotoxicity it is important to to evaluate each proposed test system with a set of compounds that range from highest to lowest risk of hepatotoxicity. Typically we would bin the compounds into severe, moderate (sometimes called variable) and minimal or no toxicity according to the following criteria:

A useful set of 60 compounds (20 in each category) is as follows:

[invitrotox]

ANIMAL RIGHTS - A MORE PRODUCTIVE APPROACH

Today we read of attacks by an animal rights group on a German neuroscientist   According to a report in Nature1 the Tierversuchsgegner Bundesrepublik Deutschland (Opponents of Animal Experiments Federal Republic of Germany) placed an aggressive full-page advertisement in two national quality newspapers and three regional newspapers. The personal attack on Dr Kreiter (Univ. of Bremen) puts his safety and that of his family at risk. These actions show a disappointing lack of imagination by the animal rights activists. Only small children believe they can get their way by sitting in the corner and screaming their anger. Small groups of protesters have to learn there are other ways to win arguments than by terrorizing individuals and frightening the public at large.

Towards the end of the 1980’s a wealthy member of the Berlin animal rights group, the Berliner Arbeitskreis gegen Tierversuche e. V., bought shares in Schering AG, the Berlin-based pharmaceutical and industrial chemical company (not to be confused with Schering-Plough, the company that was separated from its German parent during and after World War II). He used his stock ownership to attend the company’s annual meeting where he asked one question, “what was the company planning to do about alternatives to animal testing?” The company responded by setting up a meeting in 1987 to which I was invited by the animal rights group. We discussed the possibility that Schering would fund a program of research at a German university and Schering eventually agreed to establish a fund and ask for proposals. I was one of the reviewers.

None of the first round of proposals grasped the point that the motivation was to seek alternatives to animals. In response to the problem I wrote a series of proposals that I hoped would give the committee a better idea of what the animal rights group was looking for. To my surprise the representatives from Schering on the committee suggested that we select one of my proposals for funding if I could find an appropriate laboratory in Germany where the research could be done. The proposal we selected was on dermal irritancy testing. The idea was to compare measurements of cytotoxicity and interleukin release by human skin cell cultures. Cytokine release at non-cytotoxic concentrations would be a possible indicator of potential to induce dermal contact irritancy in vivo. I had gone into this topic in considerable depth since I thought it had the most potential for immediate results and so had good familiarity with the literature and the laboratories where the work could be conducted. It was relatively easy to identify Prof Friedrich Mark’s laboratory at the German Cancer Research Center in the University of Heidelberg as the most likely laboratory. After his initial surprise at this ‘out of the blue’ offer of research funding Prof Marks became an enthusiastic collaborator. He identified Dr Karin Müller-Decker in his laboratory as the scientist who would do this work.

Here is an excerpt from the report (2001)2 of the Deutsches Krebsforschungzentrum Heidelberg laboratory of Prof Friedrich Marks for the year 1999-2000.

 “Proinflammatory mediator release as an in vitro parameter of skin irritancy. In cooperation with the Schering AG we developed an in vitro test for skin irritants based on the release of arachidonic acid and interleukin-1α from a human keratinocyte line. This test was validated by a placebo-controlled, open, randomized patch test study with volunteers and shown to provide a suitable substitute for animal experiments. The responsible scientist, Dr. Karin Müller-Decker, has been awarded the Felix Wankel Tierschutzpreis 2000.”

Karin Müller-Decker has become a world recognized expert3 on cytokines and signal transduction and still works in Heidelberg at the Cancer Research Center. The work that begun in Heidelberg informed similar programs later organized by the European Center for Validation of Alternative Methods. Human cell cultures are widely used in studies of the role of cytokines in inflammation, skin irritancy, skin sensitization and cancer. Good science that avoids the use of animals in testing grew from the good intentions of a small animal rights group and the willing collaboration of a large chemical company.

The following publications by Dr Müller-Decker were a result of the Schering AG funding:

Müller-Decker, K., Heinzelmann, T., Fürstenberger, G., Marks, F. (1998) Entzündungsmediatoren aus einer humanen Keratinozytenlinie als Indikatoren für Hautreizung: Entwicklung einer Ersatzmethode für den Draize-Test. Krebsforschung heute, Steinkopf-Verlag, Darmstadt, pp 47-51. 

Müller-Decker, K., Heinzelmann, T., Fürstenberger, G., *Kecskes, A., Lehmann , W.D., Marks, F. (1998) Arachidonic acid metabolism in primary irritant dermatitis produced by patch testing of human skin with surfactants. Toxicol. Appl. Pharmacol. 153: 59-67. 

Marks, F., Fürstenberger, G., Müller-Decker, K. (1998) Arachidonic acid metabolism as a reporter of skin irritancy and target of cancer chemoprevention. Toxicol. Lett. 96: 111-118. [15] Müller-Decker, K., Heinzelmann, T., Fürstenberger, G., Marks, F. (1998) Entzündungs-mediatoren aus einer humanen Keratinocytenlinie als Indikatoren für Hautreizung: Entwicklung einer Ersatzmethode für den Draize-Test. In: Schöffl et al. (eds.) ‘Forschung ohne Tierversuche’. Springer Wien etc. (1998). 

Müller-Decker K, Fürstenberger G, Marks F., (1994) Keratinocyte-derived proinflammatory key mediators and cell viability as in vitro parameters of irritancy: a possible alternative to the Draize skin irritation test. Toxicol Appl Pharmacol. 127(1): 99-108. 

http://blogs.nature.com/news/2014/05/german-research-agencies-condemn-animal-rights-attack-on-neuroscientist.html?utm_source=feedburner&utm_medium=feed&utm_campaign=Feed%3A+news%2Frss%2Fnewsblog+%28News+Blog+-+Blog+Posts%29&WT.ec_id=NEWS-20140513

http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.122.2605&rep=rep1&type=pdf

Cellular Signal Processing: An Introduction to the Molecular Mechanisms of Signal Transduction by Friedrich Marks, Ursula Klingmüller and Karin Müller-Decker (Nov 14, 2008)

[invitrotox]

Is it safe?

Is it safe? Or, why was it not safe? These are the fundamental questions of toxicology. Superficial answers can often be found in animal studies. In one example, human exposure is not recommended after the drug inhibits the hERG channel in vitro and irregularities are later found in ECGs in monkeys. Or, a drug may not be safe to use in humans because it induces liver toxicity in rats and in monkeys. After cardiotoxicity, liver toxicity is the most frequent adverse effect found in human clinical trials.

But what is the relevance of the animal studies to human safety? We make an inference from the preclinical drug safety studies but without establishing to the best of our ability that the mechanism will be similar in humans we cannot say that the inference is absolutely correct. Of course, our decision making will not be taken in isolation.  We may not need to go the extra mile to explore the relevance to humans of each safety issue. There may be more than one candidate for development and we can simply choose the candidate with least evidence of toxicity. Or, other issues such as poor bioavailability may trump the safety issue. But when we need to go that extra mile to understand the mechanism of toxicity we should be prepared to use all our experimental armamentarium to focus on the problem.

Among the available tools are in vitro studies. In the latter half of the twentieth century cell cultures, usually of cell lines developed from human and animal tumors, were used as screens to provide an initial idea of the cytotoxicity of drugs and chemicals. Some of these screens were used in regulatory testing, for example in the safety testing of chemicals to which our eyes and skin might be exposed. One scientist, Bjorn Ekwall, was convinced that there would be a direct correlation between lethal doses of drugs and their toxicity in cell cultures (1). To some extent this is true. For many chemicals, the greater the cytotoxicity the greater the toxicity in vivo. Examples we all know about are found in the first generations of anticancer drugs. Examples are 5-fluoruracil and adriamycin both of which are very cytotoxic and have serious side-effects when used in cancer chemotherapy. Cytotoxicity was a key objective in cancer discovery programs, following the theory that uncontrollably dividing cancer cells would be more susceptible than other cells in the body with slow rates of regulated cell division. Treatment-related neutropenia and hair loss are evidence that normal cells with higher division rates are adversely affected by these drugs. Unexpected toxicities that could not be explained by a cytostatic or cytotoxic mechanism were also found. For example adriamycin induces a severe cardiotoxicity if treatment is carried out beyond a certain number of doses (cardiotoxicity has also been a problem with some of the later anticancer drugs, for example the tyrosine kinase inhibitors, imatinib mesylate, dasatinib, nilotinib, sunitinib, sorafenib and lapatinib).

Towards the end of the twentieth century, primary cultures of several human tissues became more easily available in some countries (e.g. the USA) through organ donor networks. Hepatocytes were the most frequently available cells but cardiac muscle cells were never available, primarily because it has been extraordinarily difficult to culture adult animal or human cardiac muscle for extended periods of time. Hepatocytes could be cultured for several days but under conventional culture conditions rapidly lost their intrinsic ability to metabolize drugs. Much work has gone into either developing culture conditions that help maintain the differentiated state of hepatocytes and other primary cell cultures, including the use of 3-dimensional culture systems (3). Another approach has been the development of human stem cell cultures. For example, it has been possible to generate viable, beating human cardiac muscle cells. The commercial availability of these cells has now provided an opportunity to explore in some detail the biochemical mechanisms underlying drug-induced cardiotoxicity observed in the clinic (4, 5, 6, 7). Although screening for risk of toxicity will continue to be a significant use for in vitro systems, these recent studies and developments in in vitro technologies point the way to studies of mechanism that will add to our confidence when we make inferences about human safety from preclinical data.

(1)    http://www.bemf.eu/about

(2)    Albini et al. Cardiotoxicity of Anticancer Drugs: The Need for Cardio-Oncology and Cardio-Oncological Prevention. J Natl Cancer Inst. Jan 6, 2010; 102(1): 14–25.

(3)    http://www.promega.com/resources/webinars/worldwide/archive/overview-of-3d-cell-culture-model-systems/?__utma=1.1777481411.1399488768.1399488768.1399488768.1&__utmb=1.1.10.1399488768&__utmc=1&__utmx=-&__utmz=1.1399488768.1.1.utmcsr=google|utmccn=(organic)|utmcmd=organic|utmctr=(not%20provided)&__utmv=-&__utmk=177304101

(4)    Li eta al. BMS-986094: Investigations of In Vitro Metabolism, Reactive Metabolite Formation, and Oxidative Stress, in Human Liver Microsomes, Hepatocytes, and Cardiomyocytes. The Toxicologist, p 221, 2014

(5)    Kwagh et al. BMS-986094: Potential Cytotoxicity in Differentiated Human Cardiomyocytes. The Toxicologist, p 221, 2014

(6)    Flint et al. BMS-986094: In Vitro Mitochondrial DNA Replication, Transcription, and Translation. The Toxicologist, p 221, 2014

(7)    The Toxicologist can be downloaded here: http://www.toxicology.org/AI/PUB/Tox/2014Tox.pdf

[invitrotox]

PANETH, STEM CELLS AND CANCER

On April 29 the IAS in Princeton, New Jersey (https://www.ias.edu/), hosted the latest in a series of annual meetings titled “The Governor’s Conference on Effective Partnering in Cancer Research” (http://www.cinj.org/education/2014-governors-conference-effective-partnering-cancer-research). The meeting is organized by Arnie Levine, director of The Simons Center for Systems Biology’ (http://www.sns.ias.edu/csb).

The first talk by Hans Clevers from the Hubrecht Institute (http://www.hubrecht.eu/research/clevers/) described the fascinating discovery that Lgr5 (GPR49), a signaling protein in the Wnt pathway (http://www.rndsystems.com/Pathway.aspx?p=15489&r=15433) is a marker of many, if not most, adult and tumor stem cells. Clevers took an historical approach showing how the Austrian biologist Joseph Paneth (1857–1890), first reported these interesting large cells in the base of the intestinal crypts and then a narrow, slim cell that lies between each Paneth cell. Bringing us up to date Clevers showed how the narrow slim cells are in fact Lgr5 positive stem cells supported by factors such as EGF, TGF-alpha, Wnt-3 and the Notch ligand DII4 secreted by the Paneth cells. He showed how it was possible to isolate a single Lgr5+ cell and grow it clonally. In 3-D matrigel culture this cell goes on to produce what is essentially a miniature gut, a small vesicular body budding structures that are similar to villi. The growth and differentiation of these structures, as in the gut in vivo, is regulated through Lgr5 by Wnt agonists (R-spondins) and stem cell-specific E3 ligases that downregulate Wnt receptors. Some very beautiful movies explaining the role of Lgr5 in gut epithelium differentiation, gut tumor formation and in stem cells in other locations, such as the liver, can be found here, http://www.hubrecht.eu/research/clevers/research.html.

So why is all this medically important? The single mantra ‘follow the money’ is an excellent place to begin rooting out the causes of corruption in society. We can invent a similar mantra, ‘follow the pathway’ for understanding cancer. Understanding the Wnt pathway may lead us to identify targets that are amenable to treatment, perhaps through chemical synthesis of new Lgr5 ligands – drugs that correct aberrations in downstream functioning of the Wnt5 pathway. One such aberration is already well known. The most common mutation in colon cancer is inactivation of APC, a component of the complex of proteins that downregulate the Wnt pathway.

NOTE: Why hold the meeting at the IAS? Although the IAS only supports theoretical science on its campus, Prof Levine, joint discoverer of the cell death regulator protein p53, also holds a professorship at the Robert Wood Johnson Medical School (Rutgers) where his research can be pursued at the bench. The Governor’s Conference is an excellent mixture of both the practical and the theoretical aspects of cancer biology.

[invitrotox]

IN VITRO TOXICOLOGY I - Some definitions

Toxicology: study of the adverse effects of chemicals on living organisms.

In vivo: In a living organism. Most often referring to studies of chemical safety in laboratory animals.

In vitro (literally 'in glass'): working with cells outside the organism - culture of living cells and tissues. Originally all tissue culture used sterilized glassware. Today, almost all culture is done using commercially available plastic dishes and flasks.

In vitro toxicology: The use of cultured cells and tissues to evaluate the safety of chemicals. Typically cells are used to either detect a potential safety issue or to understand a biochemical or molecular mechanism of an already identified toxicity. The most exciting recent development has been the availability of previously unavailable human cells, such as heart muscle, derived from stem cell cultures. These cells bring us closer to the ideal assessment of human toxicity in cells and tissues behaving as the do in vivo.