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Critical Reviews in Toxicology

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Manuscript Type:

Complete List of Authors:

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Date Submitted by the Author:


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Manuscript ID:

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Assaying embryotoxicity in the test tube: Current limitations of the embryonic stem cell test (EST) challenging its applicability domain

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Riebeling, Christian; Federal Institute for Risk Assessment, ZEBET Hayess, Katrin; Federal Institute for Risk Assessment, ZEBET Peters, Annelieke; Astellas Europe B.V Steemans, Margino; Johnson & Johnson PRD, Janssen Pharmaceuticals Spielmann, Horst; Freie Universität Berlin, Faculty of Biology, Chemistry, Pharmacy Luch, Andreas; Federal Institute for Risk Assessment, ZEBET Seiler, Andrea; Federal Institute for Risk Assessment, ZEBET

Keywords:

: reproductive toxicology, teratogenicity, embryotoxicity, applicability domain, embryonic stem cell test (EST), in vitro methods, alternatives to animal testing

URL: http://mc.manuscriptcentral.com/btxc Email: [email protected]

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Assaying embryotoxicity in the test tube: Current limitations of the embryonic stem cell test (EST) challenging its applicability domain

Christian Riebeling*, Katrin Hayess*, Annelieke K. Peters†‡, Margino Steemans†, Horst Spielmann*¶, Andreas Luch* and Andrea E.M. Seiler*

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* German Federal Institute for Risk Assessment (BfR), Center for Alternative Methods to Animal Experiments – ZEBET, 12277 Berlin, Germany †

Johnson & Johnson PRD, Janssen Pharmaceuticals, Inc., 2340 Beerse, Belgium

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present address: Astellas Europe B.V., 2350 AC Leiderdorp, The Netherlands

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present address: Faculty of Biology, Chemistry, and Pharmacy, The Free University of Berlin, 14195 Berlin, Germany

To whom correspondence should be addressed:

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Andrea Seiler, PhD

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German Federal Institute for Risk Assessment, Center for Alternative Methods to Animal Experiments – ZEBET, Diedersdorfer Weg 1, 12277 Berlin, Germany. Tel.: +49 30 8412 2278

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Fax: +49 30 8412 2958 E-mail: [email protected]

Key Words: reproductive toxicology, teratogenicity, embryotoxicity, applicability domain, in vitro methods, alternatives to animal testing, embryonic stem cell test (EST)


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Abstract

Testing for embryotoxicity in vitro is an attractive alternative to animal experimentation. The embryonic stem cell test (EST) is such a method, and it has been formally validated by the European Centre for the Validation of Alternative Methods. A number of recent studies have underscored the power of this method. However, the EST performed well below expectation

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using a new set of chemicals and pharmaceutical compounds, and also of toxicity criteria, tested to enlarge the database of the validated EST as part of the Work Package III of the ReProTect Project funded within the 6th Framework Programme of the European Union. To improve the performance and applicability domain of the EST we present a detailed

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review of the substances and their effects in the EST being nitrofen, ochratoxin A, D-

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penicillamine, methylazoxymethanol, lovastatin, papaverine, warfarin, β-aminopropionitrile, dinoseb, furosemide, doxylamine, pravastatin, and metoclopramde. By delineation of the

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molecular mechanisms of the substances we identify six categories of reasons for misclassifications. Some of these limitations might also affect other in vitro methods assessing

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embryotoxicity. Substances that fall into these categories need to be included in future

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validation sets and in validation guidelines for embryotoxicity testing. Most importantly, we suggest conceivable improvements and additions to the EST which will resolve most of the limitations.

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Table of Contents

Abstract.......................................................................................................................................2 Introduction .................................................................................................................................5 Review of the substances ...........................................................................................................8 ReProTect WPIII class 1, strongly teratogenic .................................................................8

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Nitrofen ................................................................................................................8 Ochratoxin A ......................................................................................................10

D-Penicillamine...................................................................................................12

Methylazoxymethanol acetate ............................................................................14

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ReProTect WPIII class 2, moderately teratogenic..........................................................16

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Lovastatin...........................................................................................................16 Papaverine .........................................................................................................17

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Warfarin .............................................................................................................19 ReProTect WPIII class 3, mildly teratogenic ..................................................................20

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β-Aminopropionitrile fumarate.............................................................................20

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Dinoseb..............................................................................................................22 Furosemide ........................................................................................................23

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ReProTect WPIII class 4, non-teratogenic .....................................................................24 Doxylamine succinate ........................................................................................24

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Pravastatin .........................................................................................................26 Metoclopramide..................................................................................................27 Substances that were misclassified in the validation study ............................................28 Diphenhydramine ...............................................................................................28 Dimethadione .....................................................................................................28 Methylmercury....................................................................................................29


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Discussion ................................................................................................................................32 Conclusion ................................................................................................................................39 Acknowledgements...................................................................................................................40 Declarations of Interest ............................................................................................................41 References ...............................................................................................................................42 Table 1......................................................................................................................................57

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Table 2......................................................................................................................................58 Table 3......................................................................................................................................59 Figure Legends .........................................................................................................................61

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Introduction

The embryonic stem cell test (EST) is an alternative method to testing for embryotoxic potency of chemicals in animals (Seiler and Spielmann, 2011). It exploits the propensity of the pluripotent mouse embryonic stem cell line D3 to spontaneously differentiate into cardiac tissue upon removal of the cytokine leukemia inhibitory factor. Cardiomyocytes in attached embryoid

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body (EB) outgrowths cause visible beating, which is used as an endpoint for differentiation. Together with viability of D3 cells and of the embryonic (differentiated) fibroblast cell line 3T3 these three endpoints are used in a prediction model (PM) to calculate a classification of the embryotoxic potency of a substance. Briefly, D3 cells are treated throughout their differentiation

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for 10 days, and differentiation is assessed as the number of beating EB outgrowths compared

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to vehicle control. D3 cells and 3T3 cells are treated in parallel for 10 days to test effects on viability using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Mosmann,

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1983). From the concentration-response relationships, the EC50 values (ID50(D3), IC50(D3), and IC50(3T3) values, respectively) are derived as the concentration of 50 % reduction in the

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endpoint parameter compared to solvent controls. To this end curve fittings are performed using

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a three-parameter logistic function as described previously (Seiler and Spielmann, 2011). Logarithmic means of EC50 values of all valid experiments are used to calculate the prediction model (Seiler and Spielmann, 2011).

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The EST and its PM have been formally validated for use in assessing the embryotoxic

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potency of chemicals for regulatory purposes in a study coordinated by ZEBET at the German Federal Institute for Risk Assessment and funded by the European Centre for the Validation of Alternative Methods (ECVAM) (Genschow et al., 2004). In a follow-up study, as part of the Work Package III, early prenatal development, of the ReProTect Project funded within the 6th Framework Programme of the European Union (Marx-Stoelting et al., 2009; Pazos et al., 2010; Schenk et al., 2010), hereafter referred to as ReProTect WPIII study, the objective was the


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enlargement of the database of the validated EST with selected compounds covering additional chemical classes previously not tested in the EST. The report of an associated ECVAM/ReProTect Workshop concluded that the EST identified only two out of thirteen substances, or 15 %, correctly (Marx-Stoelting et al., 2009). This is in strong contrast to the validation study where the substances were identified correctly in 78 % of the experiments (Genschow et al., 2004). Moreover, it has been demonstrated that the EST is a powerful

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method to assess the embryotoxicity of sets of related substances of different teratogenic potency, such as glycol ethers (de Jong et al., 2009), and congeners of valproic acid (Riebeling et al., 2011a). The EST has also been embraced by the pharmaceutical industry for application during research and development of new therapeutic agents (Augustine-Rauch et al., 2010;

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Paquette et al., 2008; Whitlow et al., 2007), and several modifications have been made to

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improve the EST for this purpose (Paquette et al., 2008; Peters et al., 2008b; Seiler and Spielmann, 2011).

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An obvious difference between the validation of the EST and the ReProTect WPIII study is the use of a different classification system of embryotoxic potencies in vivo (Table 1). The

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former used three categories for the classification of substance potencies (Brown, 2002),

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whereas the latter uses four categories (Marx-Stoelting et al., 2009, and Table 1). To compensate for this difference and to be able to use the results of the PM of the validated EST,

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the four classes of the ReProTect WPIII study were simplified to three classes by combining the mildly and moderately teratogenic categories (Category 3 and 2) and comparing it to the weakly

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embryotoxic class of the EST PM (class 2, Brown, 2002). The strongly teratogenic category (ReProTect WPIII Category 1, Marx-Stoelting et al., 2009) was equated to the strongly embryotoxic class (EST PM class 3), and the non-teratogenic category (ReProTect WPIII Category 4) to the non-embryotoxic class (EST PM class 1). In respect of the Registration, Evaluation and Authorization of Chemicals (REACH) legislation adopted in 2003 by the European Union, and for ethic as well as economic reasons


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chemical testing on animals has to be restricted to an absolutely unavoidable level (European Chemicals Agency, 2009; Höfer et al., 2004; Rovida and Hartung, 2009). Moreover, in the USA the National Research Council recommended an overhaul to regulatory toxicity testing with the vision to ultimately cease animal testing (National Research Council, 2007). It is therefore becoming an urgent necessity to develop methods such as the EST into full replacement methods to animal testing.

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To understand the shortcomings of the EST in the ReProTect WPIII study and the limits to its applicability domain we here present a detailed review of the substances and their proposed mechanism of action of embryotoxicity. In addition, we included the two substances that were misclassified in the validation study, diphenhydramine and dimethadion, as well as

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methylmercury which led to ambiguous results (Genschow et al., 2004). Based upon this data

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we highlight several issues that might also affect other in vitro methods assessing embryotoxicity, and suggest how the EST could be further developed to overcome these

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shortcomings and thereby broaden its applicability domain.

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Review of the substances

ReProTect WPIII class 1, strongly teratogenic

Nitrofen

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Nitrofen (2,4-dichlorphenyl-4'-nitrophenylether, CAS RN 1836-75-5) is a herbicide for the control of annual broad-leaved and grass weeds. It interferes with protoporphyrin biosynthesis and thereby affects chloroplast and mitochondrial electron transport (Moreland, 1999; van Assche and Carles, 1982). The amount of cuticular wax on leaves dictates the rate of its

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absorption and hence its selectivity (Kearney and Kaufman, 1975).

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Nitrofen has been described as a teratogen in rats and mice, but not rabbit (Hurt et al., 1983). Abnormal development of the heart, kidney, lung, and diaphragmatic hernia with

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resulting neonatal mortality have been described as effects of prenatal exposure to nitrofen at doses without maternal toxicity (Greer et al., 2000; Manson, 1986). Nitrofen is metabolized

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mainly to 5-hydroxy-nitrofen in the rat. However, the parent compound appears to be the major

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teratogen, as it is the compound that primarily accumulates in embryo tissue (Brown and Manson, 1986). Little data is published on effects of human exposure, and no reports of birth

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defects are available. The exact molecular mechanism of nitrofen teratogenicity is unclear, but probably involves retinoic acid signaling (see below). Nitrofen is listed in the REACH legislature,

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Appendix 6 Point 30 as toxic to reproduction (Category 2). It has a solubility in water of about 0.6 µg/ml (Herzel and Murty, 1984), and all concentrations tested exhibited visible precipitates in the cell culture medium resulting in uncertainty about the actual free concentrations in the assay. Nevertheless, reproducible response curves were generated and nitrofen was included in the ECVAM/ReProTect report where it was classified as a strongly embryotoxic compound in


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vivo (Category 1), and the EST prediction model (PM) result was weakly embryotoxic (Class 2) (Marx-Stoelting et al., 2009). In mammals, nitrofen inhibits the rate-limiting enzymes of retinoic acid synthesis (Kling et al., 2010; Mey et al., 2003; Noble et al., 2007). Retinoic acid has a crucial role in organogenesis (Duester, 2008). It has also been suggested that metabolites of nitrofen bind to thyroid hormone receptors and thus interfere with thyroid hormone signaling (Manson, 1986). Nitrofen induces

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pulmonary hypoplasia associated with congenital diaphragmatic hernia and indeed is used to generate an animal model for the latter disease (Kling and Schnitzer, 2007). This characteristic developmental effect of nitrofen occurs also in a vitamin A deficiency model developed in rats (Wilson et al., 1953). Diaphragmatic hernia also occurs in stra6—/— knock-out mice, a gene

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encoding a membrane receptor for retinal binding protein which mediates cellular uptake of

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vitamin A (Pasutto et al., 2007). Moreover, the retinoic acid receptor α/β2 compound knock-out mouse shows similar malformations (Mendelsohn et al., 1994). Nitrofen induces apoptosis

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preferentially in undifferentiated cells (Aidlen et al., 2007). This is accompanied by generation of reactive oxygen species, and it was shown that supplementation of antioxidants such as

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vitamins A, C and E decrease cell death (González-Reyes et al., 2005). However, only vitamin

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A was able to rescue the effect of nitrofen on a retinoic acid response element reporter system (Noble et al., 2007).

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All concentrations of nitrofen tested produced visible crystals in the cell culture medium. Limited solubility of hydrophobic substances is an issue with many in vitro assays as they

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usually require an aqueous environment. Nevertheless, reproducible response curves were produced in the assays. Since the medium composition in the two assay components involving D3 cells is identical and the medium used for 3T3 cells is very similar, it could be argued that the free concentrations of nitrofen at a given total concentration were the same in all three assay components of the EST. Therefore, there are no concentration values that can be used to compute the PM but the relative distance between the curves can be evaluated qualitatively. All


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curves fall into a narrow concentration range and the cytotoxicity of 3T3 cells occurs at the lowest concentrations, followed by inhibition of differentiation of D3 cells at somewhat lower concentrations than cytotoxicity on D3 cells (Fig. 1). This is the characteristic of a substance with embryotoxic potency at maternally toxic concentrations, a weakly embryotoxic substance according to Brown (Brown, 2002). However, at sufficiently low concentrations this would lead to a classification as strongly embryotoxic substance. Nitrofen was classified as a strongly

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embryotoxic compound in the ReProTect WPIII study. Strongly embryotoxic compounds (Class 3) were defined by Brown as “developmentally toxic in all species tested” (Brown, 2002). Nitrofen is not a teratogen in rabbits and teratogenic only at high concentrations in hamster (Hurt et al., 1983). It was not included in the list by Brown (Brown, 2002), but according to this

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definition would be weakly embryotoxic (Class 2), as it was correctly predicted by the PM from the response curves.

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Ochratoxin A

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Ochratoxin A (N-{[(3R)-5-chloro-8-hydroxy-3-methyl-1-oxo-3,4-dihydro-1H-isochromen-

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7-yl]carbonyl}-L-phenylalanine, CAS RN 1836-75-5) is a mycotoxin produced by Aspergillus and Penicilium species (Huffman et al., 2010). It is found in many foodstuffs, especially those

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derived from cereals (Duarte et al., 2010). Teratogenicity has been observed in a number of animal models including rats, mice, hamsters, chick embryos, quail, and rabbits (O'Brien and

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Dietrich, 2005; Patil et al., 2006). Ochratoxin A causes malformations of the eye, the central nervous system, the axial skeleton, and of craniofacial and soft tissue at concentrations that also show maternal toxicity (Patil et al., 2006). The parent compound is metabolized to a small extend and appears to be the major source of teratogenic activity (O'Brien and Dietrich, 2005). In humans, nephropathy, especially the Balkan endemic nephropathy has been ascribed to the effects of ochratoxin A (Pfohl-Leszkowicz, 2009), but no reports of birth defects are available.


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The molecular mechanism of ochratoxin A teratogenicity is unclear and the effects on the many suggested targets either require a high concentration and/or are similarly affected by a much less toxic metabolite (see below). Ochratoxin A was classified as a strongly embryotoxic compound in vivo (Category 1) in the ReProTect WPIII study and the EST PM result was weakly embryotoxic (Class 2, Marx-Stoelting et al., 2009). Many adverse effects have been described for ochratoxin A, such as apoptosis and

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necrosis of several tissue types, increased frequency of micronuclei, and the formation of DNA adducts (O'Brien and Dietrich, 2005). There are also reports that ochratoxin A might interfere with mitogen-activated protein kinase (Rumora and Grubisic, 2009) or Aurora kinase (Adler et al., 2009) signaling pathways. Moreover, the expression of the transcription factor dlx5, which is

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involved in bone development, was reduced in response to ochratoxin A exposure in mice

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(Napoletano et al., 2010). These effects require high concentrations of the mycotoxin and some are equally affected by ochratoxin α, the metabolite resulting from phenylalanine elimination via

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amidolysis, but which is a much less potent toxin (Ringot et al., 2006). Folic acid supplementation was able to significantly rescue ochratoxin A-induced neural tube defects in

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pdn/pdn mice (Katagiri et al., 2007). Interestingly, ochratoxin A synergizes with fumonisin B1

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(Ringot et al., 2006), another mycotoxin, the effect of which on neural tube closure can also be rescued by folic acid supplementation (Gelineau-van et al., 2009). In general, depletion of folic

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acid increases the frequency of chromosomal aberrations including chromosome breakage, sister chromatid exchanges and expression of fragile sites (Everson et al., 1988; Heath, Jr.,

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1966; MacGregor et al., 1990; Reidy et al., 1983; Sutherland, 1979). Hence, it is conceivable that ochratoxin A interferes with folate transport or a folate-dependent enzyme and thereby causes its embryotoxic effects. The concentration-response curves of the three assay components of the EST fall closely together around 10 µg/ml, with cytotoxicity of 3T3 cells being the most sensitive endpoint (Fig. 1 and Table 2). High cytotoxicity is most likely the reason for the classification as weakly


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embryotoxic by the PM. In addition, cell culture media, such as Dulbecco’s Modified Essential Medium, contain a variety of vitamins, including folic acid. Folic acid levels in human serum have been found to be 2.3–18.4 ng/ml (Gorgojo Martínez et al., 2006) and DMEM contains 4 mg/l which is a 200-1700-fold excess. In light of the finding that folic acid supplementation can partially rescue ochratoxin A-induced embryotoxicity, the available level of folic acid in DMEM might attenuate the effects of ochratoxin A thus resulting in a lower classification.

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D-Penicillamine

D-Penicillamine

((2S)-2-amino-3-methyl-3-sulfanyl-butanoic acid, CAS RN 52-67-5) is a

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pharmaceutical compound used in the treatment of Wilson's disease (Brewer, 2006) and heavy

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metal intoxication (Sinicropi et al., 2010). It is also used to reduce cystine excretion in cystinuria (Joly et al., 1999), and to treat patients with severe, active rheumatoid arthritis unresponsive to

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conventional therapy (Suarez-Almazor et al., 2000). It has been found teratogenic in rats, mice, and guinea pigs (Rosa, 1986). Skeletal defects, cleft palate and fetal toxicity have been

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reported, and in hamster neural tube lesions have been found (Myint, 1984; Rosa, 1986; Wiley

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and Joneja, 1978). Birth defects in humans have been found on rare occasions that cannot be fully ascribed to D-penicillamine alone; it is therefore considered safe to be applied in pregnant

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women suffering from Wilson’s disease (Pinter et al., 2004). D-Penicillamine can be abiotically oxidized to the relatively stable penicillamine disulfide (Joyce, 1989). Disulfide formation with

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itself, cysteine, homocysteine and serum proteins is the major metabolic fate of penicillamine; in addition some metabolism to S-methyl-D-penicillamine occurs in the liver (Joyce, 1989). DPenicillamine chelates heavy metals, and its most prominent effect as a consequence of copper depletion is inhibition of lysyl oxidase, accounting for its teratogenic effect in rat (Köçtürk et al., 2006). D-Penicillamine was classified as a strongly embryotoxic compound in vivo (Category 1)


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in the ReProTect WPIII study, and the EST PM result was non-embryotoxic (Class 1, MarxStoelting et al., 2009). D-Penicillamine

can be described as the β-dimethyl derivative of D-cysteine. Its

stereoisomer L-penicillamine is toxic because of its higher reactivity toward aldehydes and ketones, resulting in thiazolidine formation with pyridoxal and subsequent vitamin B6 depletion (Joyce, 1989). Similar to cysteine, D-penicillamine is a general chelator of heavy metals,

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including lead, mercury, copper and zinc (Joyce, 1989; Sinicropi et al., 2010). Copperdependent enzymes include some monooxygenases (Torres Pazmino et al., 2010), copper-zinc superoxide dismutase (Liochev and Fridovich, 2010), and lysyl oxidase (Molnar et al., 2003).

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Lysyl oxidase is responsible for the formation of crosslinks in elastin and collagen, an important part of the maturation of the extracellular matrix (Hornstra et al., 2003). Copper deficiency as

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well as lysyl oxidase knock-out mice show similar developmental effects to D-penicillamine exposure (Hornstra et al., 2003; Uriu-Adams et al., 2010). Prominently, effects on bone

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formation and on the formation of the vasculature have been reported (Hornstra et al., 2003; Rosa, 1986; Uriu-Adams et al., 2010). It can be concluded that cardiovascular defects seen in D-penicillamine

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treatment are due to reduced vascularization most likely resulting from lysyl

oxidase deficiency.

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The most prominent developmental effects of D-penicillamine on vascularization and

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bone formation occur in later prenatal development, which is not covered by the endpoints of the validated EST. In addition, cell culture media contain free O2 in contrast to blood. The typical

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O2 concentration dissolved in cell culture media is 0.24 mM, which is being constantly replenished by exchange with the 20 Vol-% O2 containing incubator atmosphere on the large surface of the cell culture dish. D-Penicillamine contains a functionally indispensible sulfhydryl group that is prone to inactivation by oxidation. It affects D3 cells only at high concentrations and cytotoxicity of 3T3 cells is the most sensitive endpoint, albeit at high concentrations resulting in a non-embryotoxic classification by the EST PM (Fig. 1 and Table 2). These effects


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are most probably due to the general chelation of essential metal ions in the media, and a specific low concentration response is not visible since the activity of lysyl oxidase is not affecting the endpoints measured in the EST.

Methylazoxymethanol acetate

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Methylazoxymethanol (CAS RN 590-96-5) is the aglycone metabolite of cycasin (CAS RN 14901-08-7), a plant toxin of cycads (Schneider et al., 2002). Cycads have been used by the food industry and have been part of the diet of pacific islanders and native americans (Morgan and Hoffmann, 1983). Cycasin is found in incompletely washed starch from the stem of

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cycads and in fat of animals that consumed cycad seeds (Morgan and Hoffmann, 1983).

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Methylazoxymethanol has been shown to be teratogenic in several species including mouse, rat, hamster and ferret (Fischer et al., 1972; Haddad et al., 1972). Prenatally exposed embryos

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exhibit structural brain abnormalities, most prominently microencephaly (Bassanini et al., 2007; Cattabeni and Di, 1997). Consequently, cycad toxins are the suspected cause of Western

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Pacific amyotrophic lateral sclerosis and parkinsonism-dementia complex in humans (Kisby et

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al., 2011). However, no clear evidence for birth defects is available. Methylazoxymethanol decomposes abiotically to a mixture of methanol (CAS RN 67-56-1), formaldehyde (CAS RN 50-

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00-0) and nitrogen (CAS RN 7727-37-9) (Nagasawa et al., 1972). Methanol and formaldehyde are known teratogens at high concentrations (Hansen et al., 2005). In the ReProTect WPIII

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study (Marx-Stoelting et al., 2009), the stable acetyl ester of methylazoxymethanol (acetyloxymethylimino-methyl-oxidoazanium, CAS RN 592-62-1) was used. The probable active metabolite is the aldehyde formylimino-methyl-oxidoazanium that is generated by alcohol dehydrogenase

(Morgan

and

Hoffmann,

1983).

The

molecular

mechanism

of

methylazoxymethanol teratogenicity is unclear (see below). Methylazoxymethanol is listed in the REACH legislature,

Appendix 6 Point

30 as toxic

to reproduction (Category 2).


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Methylazoxymethanol was classified as a strongly embryotoxic compound in vivo (Category 1) in the ReProTect WPIII study and the EST PM result was non-embryotoxic (Class 1) (MarxStoelting et al., 2009). Cycasin is toxic to a number of organs, depending on the availability of β-glucosidase in the tissue (Morgan and Hoffmann, 1983), but no effects on heart have been reported. Methylazoxymethanol acetate is being used for a chemically induced model of schizophrenia

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(Moore et al., 2006). There is some indication that the regular consumption of starch derived from cycads is a factor in the development of Lytico-Bodig disease, a neurological disease with symptoms similar to those of Parkinson's disease and amyotrophic lateral sclerosis (Esclaire et

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al., 1999; Trojanowski et al., 2002). The effect of methylazoxymethanol acetate also depends on the timing of the exposure prenatally, postnatally or during adulthood (Bejar et al., 1985;

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Cattabeni and Di, 1997). The generation of the probable active aldehyde metabolite requires alcohol dehydrogenase (Morgan and Hoffmann, 1983). This makes methylazoxymethanol a

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locally cytotoxic compound, by acting only on cells that express the required enzyme. Most alcohol dehydrogenases are expressed late in embryonal development (Crabb et al., 2004;

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Duester, 1998). It is therefore likely that D3 embryonic stem cells do not express the alcohol

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dehydrogenase required for the activation of methylazoxymethanol acetate to the extend and/or for the time required to convert substantial amounts of methylazoxymethanol to its active metabolite.

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It has been discussed by Marx-Stoelting et al. (2009) that the EST lacks metabolizing

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Page 16 of 63

capacity and that this is the likely cause of the misclassification of methylazoxymethanol. In the specific case of methylazoxymethanol a liver-mediated metabolism is dispensable since alcohol dehydrogenases are expressed in several tissues. The concentration-response curves show a much higher sensitivity of the 3T3 cells toward methylazoxymethanol compared to D3 cells and inhibition of differentiation is the least sensitive endpoint (Fig. 1 and Table 2). The higher sensitivity of the 3T3 cells is probably due to endogenous alcohol dehydrogenase expression.


15

Page 17 of 63

Differentiation of D3 cells into the neuronal lineage and the accompanying expression of alcohol dehydrogenases would confer the necessary metabolic capacity to detect the embryotoxic potency of methylazoxymethanol.

ReProTect WPIII class 2, moderately teratogenic

Lovastatin

rP Fo

Lovastatin ([1S-[1α(R),3α,7β,8β(2S,4S),8aβ]]-1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8-[2tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl]-1-naphthalenyl 2-methylbutanoate, CAS RN

ee

75330-75-5) is a cholesterol lowering drug isolated from a strain of Aspergillus terreus (Casas

rR

López et al., 2003). Prenatal treatment with lovastatin results in skeletal defects in rat but not rabbit (Minsker et al., 1983). Cases of birth defects in humans have been reported, however,

ev

their significance is debated (Taguchi et al., 2008). Lovastatin itself is an inactive lactone that is converted to mevinolinic acid in the liver (Halpin et al., 1993) as well as by broadly expressed

ie

nonspecific intracellular esterases. The pharmacologically active β-hydroxy acid is the principal

w

metabolite and an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (Endo, 1980), the rate limiting enzyme in the biosynthesis of isoprenoids. The exact molecular

On

mechanism of lovastatin teratogenicity is unclear, but probably involves inhibition of farnesylation and geranylgeranylation of signaling proteins (see below). Lovastatin was

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classified as a moderately embryotoxic compound in vivo (Category 2) in the ReProTect WPIII study and the EST PM result was weakly embryotoxic (Class 2) in three experiments and strongly embryotoxic (Class 3) in two experiments (see figure 2 in Marx-Stoelting et al., 2009). As an inhibitor of HMG-CoA reductase, lovastatin inhibits the biosynthesis of isoprenoids. The products of this pathway in mammals include, besides cholesterol, dolichol, ubiquinone, as well as geranylgeranyl and farnesyl diphosphate (Goldstein and Brown, 1990).


16


The latter two are functionally important lipid anchors for the γ-subunit of most heterotrimeric Gproteins and some small G-proteins of the RAS superfamily, and inhibition of their biosynthesis has profound effects on cellular signaling (Morris and Malbon, 1999; Takai et al., 2001). HMGCoA reductase inhibition reduces myogenic differentiation in vitro (Martini et al., 2009). Moreover, statins have been shown to inhibit stem cell renewal (Lee et al., 2007). The EST uses 15-20 % fetal bovine serum during maintenance and differentiation of cells (Seiler and

rP Fo

Spielmann, 2011), and serum is the only source of lipids for the cells in this protocol. The level of isoprenoids that could bypass the effects of HMG-CoA reductase inhibition on farnesylation and geranylgeranylation in serum is not known and might depend on the diet. This has to be

ee

compared to blood where serum represents the entire non-cellular liquid phase. Hence, any isoprenoid precursors would be in low availability in vitro which would exaggerate the effects of intracellular isoprenoid depletion.

rR

For the ReProTect WPIII study, results of individual experiments were analyzed to get a

ev

better understanding of the performance of the EST. Here we report mean values of five or more independent experiments performed in two separate laboratories, and the PM places

ie

lovastatin in class 3, strongly embryotoxic. The concentration-response curves show the typical

w

profile with inhibition of differentiation as the most sensitive endpoint at low concentrations and cytotoxicity in D3 cells and 3T3 cells at higher, similar concentrations (Fig. 1 and Table 2). The

On

low effective concentration and hence the classification could be due to low isoprenoid content in the cell culture medium.

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Page 18 of 63

Papaverine

Papaverine (1-(3,4-dimethoxybenzyl)-6,7-dimethoxyisoquinoline, CAS RN 61-25-6) is an alkaloid originally obtained from opium that exhibits spasmolytic and vasodilatory activity (Han et al., 2010). As such, it is used for the counteraction of some forms of cerebral, peripheral, and


17

Page 19 of 63

myocardial ischemic attacks (Priebe, 2007). Teratogenic effects have been reported in amphibians (Moran and Rice, 1976), chicken (Lee and Nagele, 1979), and rodents (Smedley and Stanisstreet, 1986; Waterman, 1979). Increased fetal mortality, retardation, and neural tube defects have been reported as a consequence of prenatal exposure of rats (Smedley and Stanisstreet, 1986). No reports of birth defects in humans are available. Metabolism of papaverine occurs in the liver by O-demethylation to yield predominantly the 4’-phenol, followed

rP Fo

by the 6-, 7-, 3’-, and 4’,6-demethylated products (Belpaire et al., 1975). These metabolites are further converted to glucuronides and sulfates in mammals (Belpaire and Bogaert, 1975). Papaverine itself is a general inhibitor of cyclic phosphodiesterase (PDE) and has been demonstrated to be selective for the PDE10A subtype (Siuciak et al., 2006), which probably

ee

accounts for its teratogenic potency (see below). It was classified as a moderately embryotoxic

rR

compound in vivo (Category 2) in the ReProTect WPIII study and the EST PM result was weakly embryotoxic (Class 2, Marx-Stoelting et al., 2009).

ev

Papaverine is a smooth muscle relaxant. It was correctly identified as weakly embryotoxic in the EST. However, given its mechanism of action it is possible that the observed

ie

effect (Fig. 1 and Table 2) is due to its muscle relaxant action. Another muscle relaxant,

w

diphenhydramine has been tested as moderately embryotoxic in the EST (Spielmann et al., 1997). The effects of diphenhydramine were reversible, indicating that the effects were due to

On

its muscle relaxant activity (Peters et al., 2008a). It is suggested that such false positive results could be detected by testing for acute and reversible effects on beating EBs.

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The main target of papaverine, PDE10A, is found mainly in the striatum of the brain (Lakics et al., 2010). It is conceivable that the inhibition of this isoform causes the developmental neural defects, whereas the effect on heart muscle is reversible. In this case, testing for reversibility in the EST would identify this drug as a false positive. Hence, only testing for developmental neurotoxicity could identify this compound appropriately.


18


Warfarin

Warfarin (4-hydroxy-3-(3-oxo-1-phenylbutyl)-2H-chromen-2-one, CAS RN 81-81-2) is a rodenticide and also used medicinally as an anticoagulant (Wardrop and Keeling, 2008). No developmental effects were reported for rats, mice and rabbits at doses without maternal toxicity (Howe and Webster, 1990), whereas teratogenic effects were reported in humans (Hall et al.,

rP Fo

1980). Mostly skeletal defects were reported, specifically nasal hypoplasia and stippled epiphyses (Hall et al., 1980; van Driel et al., 2002). Warfarin is metabolized to inactive dehydrowarfarin and hydroxylated metabolites, such as 10-hydroxy-warfarin (Kaminsky and Zhang, 1997), which are subsequently subject to glucuronidation (Jones et al., 2010). As an acute oral

ee

toxicant, warfarin inhibits blood coagulation by depletion of vitamin K through inhibition of

rR

vitamin K epoxide reductase (Li et al., 2004). As such it indirectly inhibits all vitamin Kdependent enzymes, including γ-glutamyl carboxylases which are important in bone and

ev

cartilage development (Wallin and Hutson, 2004). This inhibition probably accounts for its teratogenic potency (see below). Warfarin is listed in the REACH legislature, Appendix 5 Point

ie

30 as toxic to reproduction (Category 1). Warfarin was classified as a moderately embryotoxic

w

compound in vivo (Category 2) in the ReProTect WPIII study and the EST PM result was weakly embryotoxic (Class 2) in three experiments and non-embryotoxic (Class 1) in two experiments (see figure 2 in Marx-Stoelting et al., 2009).

On

Vitamin K is a cofactor of γ-glutamyl carboxylases (Berkner, 2005). These enzymes

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Page 20 of 63

catalyze the carboxylation of certain glutamate residues in proteins to form γ-carboxy glutamic acid residues. The interaction of coagulation proteins such as factors VII, IX, X, and II (prothrombin) with membrane phospholipids is achieved through a domain containing γ-carboxy glutamic acid residues and calcium. In addition to proteins in the blood coagulation cascade, proteins involved in bone development and homeostasis, the bone γ-carboxy glutamyl protein osteocalcin, the calcification inhibiting matrix γ-carboxy glutamyl protein MGP, and the matrix URL: http://mc.manuscriptcentral.com/btxc Email: [email protected]

19

Page 21 of 63

protein periostin exhibit this functionally important protein modification (Coutu et al., 2008; Murshed et al., 2004). Moreover, this modification is found in the growth arrest-specific protein 6 (Gas6) which is involved in endothelial development and homeostasis (Hafizi and Dahlback, 2006), the recently identified proline-rich γ-carboxy glutamyl proteins (PRGPs) 1 and 2, and transmembrane γ-carboxy glutamyl proteins (TMGs) 3 and 4, which are broadly expressed and are involved in cellular signaling (Kulman et al., 2007). Some of the more recently discovered γ-

rP Fo

carboxy glutamic acid-containing proteins have unknown functions and are expressed in heart. In addition, warfarin also inhibits arylsulfatase E, another enzyme important in bone and cartilage

development

(Franco

et

al.,

1995).

Mutations

in

arylsulfatase

E

cause

ee

chondrodysplasia punctata, carriers of which exhibit skeletal changes and stripped epiphyses (Parenti et al., 1997). Laboratory animals typically have a 50- to 300-fold higher vitamin K

rR

plasma level than humans and hence their relative resistance to embryotoxicity by warfarin (Howe and Webster, 1990). Fetal bovine serum should similarly contain sufficient vitamin K

ev

levels to counteract warfarin, even though serum levels in vitro are 3- to 7-fold lower than in vivo.

ie

When mean values are computed from five or more independent experiments,

w

cytotoxicity of 3T3 cells was the most sensitive endpoint at high concentrations of test compound, and D3 cell differentiation and viability was affected at a slightly higher

On

concentration. This places warfarin into class 1, non-embryotoxic, according to the PM of the EST; probably in part because animal serum provides enough vitamin K to attenuate vitamin K-

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dependent effects of warfarin. Moreover, most of the known targets of warfarin play a role in later developmental stages and tissues not present in the EST.

ReProTect WPIII class 3, mildly teratogenic

β-Aminopropionitrile fumarate


20


β-Aminopropionitrile fumarate (3-aminopropanenitrile (E)-butenedioic acid salt, CAS RN 2079-89-2) is the salt of 3-aminopropanenitrile (CAS RN 151-18-8), a substance found in the food plant grass pea (Lathyrus sativus). It is used for the treatment of equine tendonitis (Alves et al., 2001). β-Aminopropionitrile is teratogenic in many species (Joneja and Wiley, 1982), and causes mostly skeletal abnormalities including deformities of the ribs, fibula, and scapula (Wiley

rP Fo

and Joneja, 1976). In addition, exencephaly and encephalocele was observed in hamsters (Wiley and Joneja, 1976). One case report in humans suggests an association of βaminopropionitrile with the so called Cantrell-sequence syndrome (Dembinski et al., 1997). β-

ee

Aminopropionitrile is metabolized to cyanoacetic acid (CAS RN 372-09-8) (Sievert et al., 1960), a substance without reported embryotoxic potential. It is an irreversible inhibitor of lysyl oxidase

rR

and in addition is able to chelate copper from its active sites (Tang et al., 1983), which accounts for its teratogenic potency (see below). β-Aminopropionitrile was classified as a mildly

ev

embryotoxic compound in vivo (Category 3) in the ReProTect WPIII study and the EST PM result was non-embryotoxic (Class 1, Marx-Stoelting et al., 2009).

ie

β-Aminopropionitrile fumarate is found in grass pea, a legume that is commonly grown

w

for human consumption and livestock feed in Asia and East Africa. An unbalanced diet

On

dominated by this crop due to drought or famine leads to osteolathyrism. Osteolathyrism is characterized by hernias, aortic dissection, exostoses, and kyphoscoliosis and other skeletal

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Page 22 of 63

deformities, apparently as the result of defective maturation of collagen tissue. The inhibition of lysyl oxidase causes loss of cross-linking of procollagen and proelastin in bone homeostasis. This property is exploited in the treatment of tendonitis. β-Aminopropionitrile fumarate had some effects at similarly high concentrations on D3 cell differentiation and 3T3 cell viability (Fig. 1 and Table 2). Similar to D-penicillamine this drug inhibits lysyl oxidase, and in both cases this appears to be the main mechanism for the embryotoxic potency in vivo. Consequently, the reasons for classification in vitro as nonURL: http://mc.manuscriptcentral.com/btxc Email: [email protected]

21

Page 23 of 63

embryotoxic are the same as for D-penicillamine, namely the absence of a functional significance of the target protein.

Dinoseb

The herbicide dinoseb (2-sec-butyl-4,6-dinitrophenol, CAS RN 88-85-7) is used on field

rP Fo

crops for the selective control of grass and broadleaf weeds as well as an insecticide for grapes, and as a seed crop drying agent (Matsumoto et al., 2008). It interferes with chloroplast and mitochondrial electron transport (van Assche and Carles, 1982). Dinoseb has been shown to be teratogenic in mice and rats at maternally toxic doses, and in rabbit without maternal toxicity

ee

(Crawford, 1986; Matsumoto et al., 2008). Skeletal deformities and microphthalmia have been

rR

reported in newborn rats (Matsumoto et al., 2010). In mice skeletal defects, cleft palate, hydrocephalus and adrenal agenesis have been found (Preache and Gibson, 1975). No reports

ev

of birth defects in humans are available. Dinoseb is metabolized by reduction of a nitro group and oxidation of the aliphatic side chain followed by glucuronidation (Gibson and Rao, 1973).

ie

The exact molecular mechanism of dinoseb embryotoxicity is unclear, but probably involves

w

uncoupling of oxidative phosphorylation in mitochondria (see below). Dinoseb is listed in the REACH legislature, Appendix 6 Point 30 as toxic to reproduction (Category 2). Dinoseb was

On

classified as a mildly embryotoxic compound in vivo (Category 3) in the ReProTect WPIII study and the EST PM result was weakly embryotoxic (Class 2) (Marx-Stoelting et al., 2009).

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The effects of dinoseb in vivo suggest that cytotoxicity due to uncoupling of oxidative phosphorylation in mitochondria and the resulting maternal toxicity are in large parts responsible for the observed embryotoxicity (Matsumoto et al., 2010; Preache and Gibson, 1975). Similarly, in fish embryos a generally reduced growth, including of the heart was observed (Viant et al., 2006). In rat, the type of diet influences the embryotoxic outcome, but the dietetic factor responsible has not been identified yet (Giavini et al., 1989; Matsumoto et al., 2008).


22


Interestingly, microphthalmia with linear skin defects syndrome is associated with a mutation in a mitochondrial holocytochrome c-type synthetase (Schaefer et al., 1996). Dinoseb exhibits effects on all three endpoints of the EST at similar concentrations (Fig. 1 and Table 2). This reproduces its in vivo classification correctly as mildly embryotoxicant, as it acts “at approximately the same dosage as maternal toxicity”, which also corresponds to a class 2c substance according to Brown (Brown, 2002, and Table 1).

Furosemide

rP Fo

Furosemide (4-chloro-2-(furan-2-yl-methylamino)-5-sulfamoylbenzoic acid, CAS RN 54-

ee

31-9) is a potent diuretic drug used in the treatment of congestive heart failure and edema

rR

(Goldsmith, 2005). It inhibits reabsorption of sodium and chloride ions in the Henle loop of the nephron (Ho and Power, 2010). Little information on the teratogenicity of furosemide has been

ev

published in the peer reviewed literature, and those exclusively report wavy ribs in rat embryos as developmental effect (Bucher, 1989; Nakatsuka et al., 1993). No reports of birth defects in

ie

humans are available. Furosemide is primarily glucuronidated, but additional oxidized

w

metabolites may be responsible for its hepatotoxicity (Williams et al., 2007). The molecular mechanism of furosemide teratogenicity has been ascribed to its diuretic activity and the

On

resultant maternal alkalosis (Bucher, 1989; Nakatsuka et al., 1993). Furosemide was classified as a mildly embryotoxic compound in vivo (Category 3) in the ReProTect WPIII study and the

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Page 24 of 63

EST PM result was non-embryotoxic (Class 1, Marx-Stoelting et al., 2009).

Furosemide is an inhibitor of the Na+-K+-Cl— cotransporter NKCC2 (Carota et al., 2010; Krämer et al., 2008) and to a lesser extend of all Na+-K+-Cl— cotransporters (Blaesse et al., 2009; Russell, 2000). NKCC2 is specifically expressed in the kidney, whereas NKCC1 and other cotransporters are more widely expressed, including in muscle (Becker et al., 2003; Kristensen and Juel, 2010). NKCC2 knock-out mice are born with hydronephrosis and die before weaning


23

Page 25 of 63

(Takahashi et al., 2000). It has been suggested that malformations of the embryo after prenatal furosemide exposure are the result of maternal alkalosis, i.e. a secondary effect to maternal toxicity (Nakatsuka et al., 1993; Pazos et al., 2010). However, pups of rescued NKCC2 knockout mice were not investigated by Takahashi et al. (2000) and thus the results of the study could also argue for NKCC2-independent furosemide embryotoxicity, possibly by inhibition of other Na+-K+-Cl— cotransporters.

rP Fo

Furosemide showed a pronounced cytotoxicity in vitro toward 3T3 cells while differentiation of D3 cells was affected at about double the concentration, and cytotoxicity on D3 cells at about triple of that concentration (Fig. 1 and Table 2). This probably reflects the maternal toxicity in vivo which is responsible for its embryotoxic potency as a secondary effect,

ee

but causes the PM to predict it as a non-embryotoxic substance.

rR

ReProTect WPIII class 4, non-teratogenic

w

ie

Doxylamine succinate

ev

Doxylamine (N,N-dimethyl-2-(1-phenyl-1-pyridin-2-yl-ethoxy)-ethanamine, CAS RN 46921-6) is an antihistamine and a sedative (Brown and Werner, 1948), and is primarily used as

On

doxylamine succinate (N,N-dimethyl-2-(1-phenyl-1-pyridin-2-yl-ethoxy)-ethanamine butanedioic acid salt, CAS RN 562-10-7). It is usually formulated with other drugs in night-time cold and

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allergy relief drugs (Smith and Feldman, 1993). Its arguably best known and investigated formulation is bendectin (CAS RN 99007-20-2), a combination of doxylamine succinate with pyridoxine (CAS RN 65-23-6), which is used to prevent morning sickness in pregnant women (Bishai et al., 2000). No teratogenic effects have been reported in rats or rabbits (Brent, 2003; Brent, 1995). However, there is a continued controversy about possible teratogenicity of bendectin in humans (Gilboa et al., 2009), see also below), and it has been discontinued for that


24


reason in some markets (Culliton, 1983). Doxylamine is partially demethylated and glucuronidated to doxylamine O-glucuronide, N-desmethyl-doxylamine O-glucuronide, and N,Ndidesmethyldoxylamine O-glucuronide (Holder et al., 1990). Doxylamine succinate was classified as a non-embryotoxic compound in vivo (Category 4) in the ReProTect WPIII study and the EST PM result was weakly embryotoxic (Class 2) (Marx-Stoelting et al., 2009). In monkey, temporarily delayed closure of the ventricular septum, which spontaneously

rP Fo

corrected before birth, has been reported (Hendrickx and Peterson, 1997). In addition, some reduced ossification has been reported in rats (Tyl et al., 1988) and rabbit (Mcbride and Hicks, 1987). Doxylamine is a histamine receptor antagonist, but little is published on the molecular pharmacology of this compound. There are four isoforms of the histamine receptor in mammals,

ee

HRH1 – HRH4 (Jutel et al., 2009), and the relative affinities of doxylamine for the different

rR

isoforms is unknown although doxylamine is generally assumed to primarily bind to the H1 isoform. The sedative effect is associated with the ability to cross the blood-brain barrier, since

ev

second generation antihistamines which are not sedative lack this ability (Timmerman, 1999). Interestingly, HRH1 is also expressed in the heart (De Bakker et al., 1998).

ie

Diphenhydramine, another antihistaminic drug with anticholinergic activity and very

w

similar pharmacological properties (cf. below), was also classified as moderately embryotoxic by the PM (Spielmann et al., 1997), despite being classified as non-embryotoxic in vivo (Brown,

On

2002). Its effect on contractility of EB outgrowths was apparent immediately after exposure and reversible by removal of the drug (Peters et al., 2008a). This suggests that the direct effect of

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Page 26 of 63

diphenhydramine on muscle function caused the misclassification in the EST rather than a specific developmental effect (Peters et al., 2008a). Potassium voltage-gated channels of the subfamily H are inhibited by diphenhydramine and other antihistamines, and this inhibition is likely responsible for the cardiotoxic effect of second generation antihistamines (Taglialatela et al., 2000). No data is available on doxylamine and potassium channels, however, given the structurally and mechanistically similarities it is conceivable that doxylamine elicits the same


25

Page 27 of 63

effect as other antihistamines. This would explain the inhibition of differentiation of D3 cells being the most sensitive endpoint, and its cytotoxicity being more pronounced in D3 cells than on 3T3 cells (Fig. 1 and Table 2). Nevertheless, it can not be excluded that the reported delayed closure of the ventricular septum in baboon embryos is detected by the EST, which is corrected at later developmental stages.

Pravastatin

rP Fo

Pravastatin ((3R,5R)-3,5-dihydroxy-7-((1R,2S,6S,8R,8aR)-6-hydroxy-2-methyl-8-{[(2S)2-methylbutanoyl]oxy}-1,2,6,7,8,8a-hexahydronaphthalen-1-yl)-heptanoic acid, CAS RN 81131-

ee

70-6) is structurally and functionally related to lovastatin. It was originally identified as a

rR

metabolite of mevastatin (CAS RN 73573-88-3) in dog urine and is derived from microbial conversion of mevastatin by the bacterium Nocardia autotrophica (Tsujita et al., 1986). No

ev

teratogenicity has been reported for pravastatin, probably due to its impermeability of the bloodplacenta barrier (Quion and Jones, 1994). It is primarily converted to the 6β-O-sulfate ester by

ie

sulfotransferases and subsequently oxidized (Kitazawa et al., 1993). Pravastatin is an inhibitor

w

of HMG-CoA reductase (Tsujita et al., 1986). Pravastatin was classified as a non-embryotoxic compound in vivo (Category 4) in the ReProTect WPIII study and the EST PM result was weakly embryotoxic (Class 2, Marx-Stoelting et al., 2009).

On

Similar to lovastatin, pravastatin is an inhibitor of HMG-CoA reductase (Tsujita et al.,

ly

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1986). Accordingly the FDA has assigned the same pregnancy Category X to lovastatin and pravastatin. As an inhibitor of HMG-CoA reductase it reduces isoprenoid biosynthesis and hence the same considerations of its embryotoxic potency have to be taken into account that have been mentioned above for lovastatin. The EST provides no barrier function as has been discussed by Marx-Stoelting et al. (2009), but the higher hydrophilicity of pravastatin compared to lovastatin affects also its plasma


26


membrane permeability. Hence, pravastatin reveals with similar profile of the three endpoints of the EST as lovastatin but acts at much higher concentrations (Fig. 1 and Table 2). Consequently, pravastatin was recognized as Class 2, weakly embryotoxic by the EST.

Metoclopramide

rP Fo

Metoclopramide methoxybenzamide

hydrochloride

hydrochloride

salt,

(4-amino-5-chloro-N-(2-(diethylamino)ethyl)-2CAS

RN

7232-21-5)

is

an

antiemetic

and

gastroprokinetic drug (Fraser and Bryant, 2010). No developmental toxicity has been found (Matok et al., 2009). Metoclopramide is primarily metabolized to monodeethylmetoclopramide

ee

(Desta et al., 2002), and subsequently glucuronidated and sulfated (Bakke and Segura, 1976).

rR

Metoclopramide was classified as a non-embryotoxic compound in vivo (Category 4) in the ReProTect WPIII study and the EST PM result was weakly embryotoxic (Class 2) (MarxStoelting et al., 2009).

ev

Metoclopramide binds to dopamine and serotonin (5-hydroxytryptamine, 5-HT) receptors

ie

(Tonini et al., 2004). It is an antagonist of dopamine D2 receptors (Farooqui et al., 1994).

w

Dopamine receptors are predominantly expressed in brain, but also found in heart, including the D2 receptor (Cavallotti et al., 2010). Metoclopramide is also an agonist of the 5-HT4 receptor

On

(Dumuis et al., 1989; Sanger, 2009), and an antagonist of the 5-HT3 receptor (Hirokawa et al., 2003; Tonini et al., 2004). These receptors are predominantly expressed in the brain.

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Page 28 of 63

Interestingly, the functional 5-HT4 receptor is found in newborn but not adult rat atrial cardiomyocytes (Derangeon et al., 2010). It is therefore likely that the effect of metoclopramide on dopamine and serotonin receptors causes reversible suppression of muscle contraction. In line with this suggestion, differentiation of D3 cells was the most sensitive endpoint, and cytotoxicity of metoclopramide was more pronounced in D3 cells than on 3T3 cells (Fig. 1 and Table 2).


27

Page 29 of 63

Substances that were misclassified in the validation study

Diphenhydramine

Diphenhydramine (2-(diphenylmethoxy)-N,N-dimethylethanamine, CAS RN 58-73-1),

rP Fo

and its hydrochloride salt (CAS RN 147-24-0), is an antihistaminic, antiemetic, sedative and a local anesthetic drug (Cirillo and Tempero, 1976). It is also in use as an antiarrhythmic drug with sodium channel blockage as one of the mechanisms of action (Khalifa et al., 1999; Sharma and Hamelin, 2003). No major developmental toxicity has been found (Gilboa et al., 2009), although

ee

some retardation of male sexual development has been reported in rats (Moraes et al., 2004).

rR

Diphenhydramine is primarily metabolized by N-demethylation to the secondary and subsequently

to

the

primary

amine.

These

products

are

further

oxidized

to

ev

diphenylmethoxyacetic acid which is conjugated with glycine or glutamine (Baldacci et al., 2004). Diphenhydramine was classified as a non-embryotoxic compound in vivo (Class 1) in the

ie

validation study (Brown, 2002), and the EST PM result was weakly embryotoxic (Class 2, Genschow et al., 2004).

w

The reversible action of diphenhydramine in the EST has been described (Peters et al.,

On

2008a). It acts selectively on the histamine H1 receptor and thereby results in muscle relaxation. This causes a reversible inhibition of contractions in EB outgrowths that does not reflect an

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effect on differentiation as has been described by Peters et al. (2008a) and discussed under doxylamine succinate above.

Dimethadione


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Dimethadione (5,5-dimethyl-1,3-oxazolidine-2,4-dione, CAS RN 695-53-4) is the active metabolite of trimethadione (3,5,5-trimethyl-1,3-oxazolidine-2,4-dione, CAS RN 127-48-0). Trimethadione is an oxazolidinedione anticonvulsant. Teratogenicity of trimethadione has been reported in rats, mice, monkeys, and chicken (Finnell and Dansky, 1991). The so called fetal trimethadione syndrome in humans manifests with growth retardation, microcephaly, heart defects, orofacial clefts, and limb defects (Finnell and Dansky, 1991; Goldman and Yaffe, 1978).

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Dimethadione is not further metabolized to any appreciable extend (Tanaka et al., 1996). It was classified as a weakly-embryotoxic compound in vivo (Class 2) in the validation study (Brown, 2002), and the EST PM result was non-embryotoxic (Class 1, Genschow et al., 2004). Dimethadione inhibits low-threshold calcium currents in the brain by inhibition of T-type

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calcium channels (Coulter et al., 1989). The T-type calcium channels are found in the brain and

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the heart (Mizuta et al., 2010; Vacher et al., 2008). In addition, it is an inhibitor of potassium voltage-gated channels of the subfamily H of the heart, causing arrhythmias (Danielsson et al.,

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2007). A significant effect on embryonic cardiac repolarization was only seen at concentrations of 20 mM (2580 µg/ml), and effects on cardiac rhythm were seen in some animals beginning at

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5 mM (646 µg/ml) (Danielsson et al., 2007). However, in the EST the beating rate is not

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recorded, only beating or non-beating areas in EB outgrowths are being distinguished. Arrhythmia or slow beating would therefore not be detected. The teratogenic effects of

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dimethadione as a consequence of potassium current perturbations are probably caused indirectly by hypoxia resulting from cardiac arrhythmia (Danielsson et al., 2007). The EST can

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not detect such an indirect systemic effect on the brain. Nevertheless, the inhibition of calcium channels would probably affect developing neurons.

Methylmercury


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The methylmercury cation (CAS RN 22967-92-6), here used as methylmercury chloride (CAS RN 115-09-3) has been produced as a fungicide for grains (Fitzgerald and Clarkson, 1991), and has been a by-product of several industrial processes such as the production of acetaldehyde (Sakamoto et al., 2010). It is also released as indirect consequence of the burning of wastes and fossil fuels, particularly coal (Bose-O'Reilly et al., 2010). In addition, inorganic mercury is used in large quantities, for instance during gold and silver ore amalgamation (Bose-

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O'Reilly et al., 2010). Methylmercury is formed from inorganic mercury by the action of anaerobic organisms that live in aquatic systems (Ullrich et al., 2001), and by that route enters the food chain. Its teratogenic effects are general growth and developmental retardation. The most common malformations in mammals are generalized edema and brain lesions (Kakita et

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al., 2000), and mental retardation and cerebral palsy have been reported (Elhassani, 1982). In

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addition, at high concentrations skeletal defects including wavy ribs, cleft palate, absence of vertebral centra, and defects of the sternum were seen (Lee and Han, 1995; Yasuda et al.,

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1985). In humans, severe behavioral and sensory deficits, including deafness and blindness, were reported after accidental prenatal exposure (Gilbert and Grant-Webster, 1995).

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Methylmercury readily binds sulfhydryl groups and selenium and as such is transported

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in vivo bound to proteins and accumulates in tissues, especially in the liver (Bridges and Zalups, 2010; Lee and Han, 1995). It is in part demethylated to inorganic mercury in the liver and by the

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intestinal microflora (Bridges and Zalups, 2010). A specific molecular target is not known for methylmercury. It has been concluded that methylmercury alters the normal migration of

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neurons to the cerebellar and cerebral cortices during brain development (Choi et al., 1978; Choi, 1986). Effects on the dopaminergic and GABAergic neurotransmitter systems appear to dominate (Newland et al., 2008). Methylmercury chloride was initially classified as a weaklyembryotoxic compound (Class 2b) and subsequently moved to strongly embryotoxic in vivo (Class 3) in the final list of test chemicals for the validation study (Brown, 2002). The result of the EST PM was ambiguous with about half of the experiments indicating non-embryotoxic


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(Class 1) and the other half indicating strongly embryotoxic (Class 3) (Genschow et al., 2004), while in all cases low effective inhibition concentrations indicated a strongly toxic compound. Methylmercury readily reacts with, and is neutralized by, sulfhydryl groups (LoPachin and Barber, 2006). The reaction with functionally important sulfhydryl groups in synaptic proteins is suggested as the reason for its strong neurotoxicity (LoPachin and Barber, 2006). Free sulfhydryl groups are available in cell culture media through serum proteins and by the

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addition of β-mercaptoethanol (Seiler et al., 2006). The latter is added at 0.1 mM only to media for D3 cells but not for the 3T3 cells, which might in part explain the much higher toxicity of methylmercury on 3T3 cells reported in the validation study (Genschow et al., 2004). The

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potency of methylmercury could therefore also depend on the oxidation state of the media as has been described under D-penicillamine above. However, the main reason for methylmercury

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being misclassified in the EST appears to be its high toxicity toward 3T3 cells in combination with a half-maximal inhibition of differentiation at a higher concentration than the cytotoxicity in

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D3 cells (Genschow et al., 2004; Stummann et al., 2007). Substances with such a profile in the EST had not been present in the prevalidation set that was used for developing the PM (Scholz

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et al., 1999), and consequently this situation is not covered by the PM. This can be seen by its

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position in the two linear discriminant representation of the PM (Fig. 2). The high cytotoxicity toward 3T3 cells is an indication of strong maternal toxicity.

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Discussion

In the ReProTect WPIII study, thirteen compounds were assessed for their embryotoxic potential in the EST in two independent laboratories. The outcome resulted in an unexpected misclassification. The current review aims to clarify the cause of this misclassification based upon detailed information on toxicological mechanism and mode of action of the test

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compounds.

The reviewed substances can be sorted into six broad categories of suspected reasons for their misclassification (Table 3). Most of the substances fall into the category of ‘acting on different tissues and/or at later developmental stages than what is represented by the validated

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EST’. This has been discussed by Marx-Stoelting et al. (2009), but it was not recognized that it

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accounts for up to 55 % of the misclassifications in the ReProTect WPIII study. The effects of these substances can be further divided into two major fields of embryotoxicity: neurotoxicity

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and osteotoxicity. Methylazoxymethanol requires the metabolic activation by alcohol dehydrogenases that are expressed in neurons (Crabb et al., 2004; Duester, 1998; Morgan and

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Hoffmann, 1983). Papaverine acts on neuronal cyclic PDE10A (Lakics et al., 2010; Siuciak et

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al., 2006), dimethadione on neuronal T-type calcium channels (Coulter et al., 1989), and methylmercury on sulfhydryl containing enzymes important for neuronal function and

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development (LoPachin and Barber, 2006). D-Penicillamine and β-aminopropionitrile inhibit lysyl oxidase that is important for bone formation (Köçtürk et al., 2006; Tang et al., 1983), albeit by

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different mechanisms and D-penicillamine might exhibit additional effects due to depletion of heavy metal cofactors. β-Aminopropionitrile therefore should be a useful substance to test the applicability domain and validate a developmental osteotoxicity testing setup. Warfarin indirectly inhibits the formation of γ-carboxy-glutamyl residues which are important for bone matrix proteins (Wallin and Hutson, 2004). On the other hand, furosemide causes malformations indirectly through maternal alkalosis (Bucher, 1989; Nakatsuka et al., 1993; Pazos et al., 2010),


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and dimethadione causes damage to the brain indirectly via cardiac arrhythmia-induced hypoxia (Danielsson et al., 2007). This is a situation the EST, even when including other embryonal developmental stages, can only detect as strong cytotoxicity on 3T3 cells. We demonstrated that using a molecular endpoint for cardiac differentiation allows for a shortening of the assay time (Genschow et al., 2004), and that the performance of the so called FACS-EST is identical to the validated EST (Buesen et al., 2009; Riebeling et al., 2011a).

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Moreover, using serum-free conditions we detected simultaneous differentiation into cardiomyocytes and neuronal cells by flow cytometry, suggesting that parallel measurement of developmental cardiotoxicity and neurotoxicity might be possible (Riebeling et al., 2011b). Initial studies have shown that it is possible to modify the EST for both the measurement of

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developmental neurotoxicity and osteotoxicity (Buesen et al., 2004; Theunissen et al., 2010; zur

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Nieden et al., 2010). Multiple parallel molecular endpoints enhance the predictivity of the EST (Groebe et al., 2010; Paquette et al., 2008; van Dartel et al., 2010) and will also allow for the

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identification of substances with muscle relaxant and embryotoxic potency such as papaverine while discriminating from substances with only muscle relaxant properties such as doxylamine,

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metoclopramide and diphenhydramine, the third biggest category of misclassifications according to our analysis (Table 3).

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The second most prevalent reason for misclassification is the nutrient composition of the

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medium, affecting up to 45 % of the misclassified substances in the ReProTect WPIII study. Traditionally, the major concern over media composition was the binding of lipophilic

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substances to serum albumin (Schmidt et al., 2010). Our analysis of the ReProTect WPIII study reveals that it is levels of nutrients that play a far greater role in the outcome of the EST. In respect to many amino acids and vitamins the cells are exposed to excess concentrations in cell culture. The excess of folic acid, at least compared to the human situation, was the probable cause of misclassification for ochratoxin A; and vitamin K levels might also have had an impact on the outcome of the EST with warfarin. Hence, for an EST humanized with respect to


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nutrients, to reduce the number of misclassifications in the EST, a closer look at how media compositions relate to the in vivo situation is necessary. Conversely, serum is in low supply in the medium relative to in vivo, and it contains a cornucopia of factors, such as growth factors and albumin, but also bioactive and nutritional lipids. Lovastatin and pravastatin are inhibitors of HMG-CoA reductase. We suspect that low levels of lipid precursors because of lower serum levels compared to blood cause a pronounced

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embryotoxicity of the statins in the EST. In addition, pravastatin is non-embryotoxic in vivo due to its barrier impermeability, and the EST has no such functionality (Marx-Stoelting et al., 2009; Pazos et al., 2010).

There is an ongoing development of cell culture media especially for stem cells and their

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differentiation, including serum-free and defined media (Riebeling et al., 2011b; van der Valk et

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al., 2010). Whereas it might be possible to adjust some vitamin levels to endogenous levels, serum protein bound lipid factors are not well defined and might act as apoptogens at higher

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concentrations, or depending on the cell type (Watterson et al., 2003). Current media compositions have been optimized for cell culture, which means growth in O2-rich environments

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compared to in vivo. This is an unfavorable condition for mammalian cells and also causes

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problems for the generation of embryonic stem cells (Lengner et al., 2010). The oxidation of media components including the test substances over time adds a

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factor of variability to the assay, albeit a negligible one for most experiments. Cells could be cultured and differentiated under low O2 conditions and the oxidation of test substances should

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be monitored. Free sulfhydryl groups, for instance in the form of β-mercaptoethanol or the less toxic monothioglycerol (Brielmeier et al., 1998) and dithiothreitol (Tarin et al., 1998), and some vitamins can be added to reduce reactive oxygen species in the medium. Methylmercury is sensitive to free sulfhydryl groups (LoPachin and Barber, 2006), and its high cytotoxicity on 3T3 cells compared to D3 cells could also be caused by the difference in free sulfhydryl groups in


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the media. These limits surely have to be taken into account when developing a defined media composition for an in vitro test. Related to the discussion of nutrients is also the case of maternal malnutrition which has embryotoxic effects, especially on the skeleton (Carney and Kimmel, 2007). Malnutrition is caused by a large number of substances; in the case of furosemide it appears to be the sole reason for its embryotoxicity (Bucher, 1989; Nakatsuka et al., 1993; Pazos et al., 2010).

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Substances that cause malformations because of maternal toxicity, especially malnutrition, are not specifically identified by the PM. The higher cytotoxicity of a substance on 3T3 cells relative to its cytotoxicity or inhibition of differentiation on D3 cells at low concentrations might be a useful additional parameter to take subsequent embryotoxicty into account as a potential

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secondary effect in the PM, or needs to be specifically recognized in a revised PM.

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A humanized EST, be it by adjusting medium composition or by use of human cells, comes with a challenge in the validation of such an assay. Most human data on embryotoxicity

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are based on epidemiology and case studies, which do not provide dose-effect relationships or other detailed data. Hence, there would be insufficient data to validate such a method. For

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example, of the ten teratogenic substances used in ReProTect WPIII only one, warfarin, has

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established embryotoxic effects in humans. Another four substances, D-penicillamine, methylazoxymethanol, lovastatin, and β-aminopropionitrile, have at least one suspected case

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associated. For the remaining five substances no associated human teratogenicity has been reported yet. Moreover, in line with these criteria one of the non-teratogenic substances,

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doxylamine succinate, has to be listed as associated with embryotoxic effects in humans. The routinely used animal models exhibit a predictivity for humans of around 60 % in the case of skin irritation (Hartung, 2008), and similar for carcinogenicity (Gaylor, 2005) and teratogenicity (Knight, 2007; Schardein and Keller, 1989). An estimated 20 % of bioactive chemicals undergo metabolic activation (Coecke et al., 2006). There are considerable and often crucial interspecies differences in metabolic pathways of chemicals, representing the major factor of interspecies


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difference in toxicological responses (Dorne, 2010; Voisin et al., 1990; Walton et al., 2001). The incorporation of liver metabolism into in vitro embryotoxicity assays is a major research interest (Hettwer et al., 2010; Uibel et al., 2010). All sets of substances used with the EST so far intentionally lack substances that require hepatic metabolism before they can elicit their embryotoxic potential (Brown, 2002; Pazos et al., 2010). However, as this is an oft-cited issue with the EST and other in vitro methods we will briefly discuss it here. Since species differences

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in metabolism are a major, if not the lone, reason for misclassifications in vivo, it would be important to employ human hepatocytes to represent maternal metabolism, such as human hepatocytes that have been generated from human induced pluripotency stem cells (Greenhough et al., 2010). Alternatively, primary hepatocytes from animals with a similar

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metabolic capacity and profile to humans such as swine derived from abattoirs could be

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employed to improve the predictability (Turpeinen et al., 2007). Subsequently, mouse embryonic stem cells can be used for testing the embryotoxicity of the metabolites. Mouse embryonic stem

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cells exhibit a faster development than human derived cells and hence allow for shorter protocols, a highly desirable trait. This is especially important when embryotoxic effects depend

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on expression of proteins that are induced at a late stage, such as lysyl oxidase and alcohol dehydrogenase.

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There are currently a number of drawbacks to using a metabolic conversion system. The

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first is the incompatibility of the hepatocyte-conditioned medium with embryonic stem cells (Hettwer et al., 2010). Secondly, the toxicity of a substance and its metabolites toward

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hepatocytes limits the achievable concentrations. This plays into the third limitation, the unknown efficiency of the metabolic conversion. Therefore, at this stage a hepatocyte supernatant can be used for initial screening such as in research and development and as supplemental data to hazard assessment. The one misclassification because of lack of metabolism in the ReProTect WPIII study (Marx-Stoelting et al., 2009) was not due to the absence of liver metabolism but consequence of the missing alcohol dehydrogenases. These


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enzymes are expressed during later development, including in neuronal cells (Crabb et al., 2004). Methylazoxymethanol therefore should be a useful compound to test the applicability domain and to validate a developmental neurotoxicity testing setup. Pravastatin has been misclassified as weakly embryotoxic in vitro because it does not cross the blood-placenta barrier in vivo (Quion and Jones, 1994). It has the same mode of action as lovastatin and is located close to it in the two linear discriminant representation of the

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PM (Riebeling et al., 2011a), although on the other side of the class separator (Fig. 2). Another blood-tissue barrier, the blood-brain barrier has been modeled in vitro (Cecchelli et al., 2007; Stolper et al., 2005), suggesting that similar models could be developed for the blood-placenta barrier. However, as the properties of the placental barrier change throughout the different

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stages of pregnancy, this could require use of several models. An alternative approach is the

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computer-aided modeling of the systemic behavior of a substance using structure-activity relationships (SAR) to predict the barrier permeability (Mensch et al., 2009). Such modeling

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could allow for calculating physiologically relevant concentration ranges that reach the embryo to be tested. This would reduce false positive rates due to unrealistic exposure doses.

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Nitrofen was insoluble in the cell culture medium and no PM can be calculated from the

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endpoint results. Compound insolubility is a general limitation of in vitro as well as in vivo test systems. Mostly, the insolubility of the substances results from their hydrophobicity. More

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solvents have to be tested for their compatibility with the EST to address this problem. Experimental delivery of exogenous lipids to cells is facing a similar challenge, and some

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systems such as ethanol/dodecane, cyclodextrins, and complexation with albumin have proven useful. Moreover, the choice of solvent could be guided by the aforementioned SAR calculations. Results derived from nitrofen, furosemide and methylmercury indicate that the classification scheme and the PM need to be scrutinized. An unknown substance tested in an in vitro method, especially in a humanized system, can only be suspected as embryotoxic or non-


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embryotoxic to humans. When this classification is obtained at realistic concentrations calculated by SAR, further class differentiation or a detailed review of the data would give insights into the mechanism of the substance but would not add to a regulatory decision. To successfully apply a new set of criteria, and/or a changed number of categories of embryotoxicity would require reclassifying the substances previously tested by the EST accordingly. With this set of reclassified substances and their known half maximal effective

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concentration values a new PM can be developed that satisfies the new criteria. Only with the corresponding new PM conclusions can be drawn from results of previously untested substances classified by the new criteria on the performance and applicability of the tested method.

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Conclusion

The applicability domain of the validated EST is limited to substances that do not require metabolic conversion and act in early embryonic development. This is arguably the most crucial period, since at this point there can still be unawareness of pregnancy and hence continued medication or other chemical exposures which would be consciously avoided once pregnancy

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became aware. The applicability domain can be broadened to later developmental stages by including endpoints for developmental neurotoxicity and osteotoxicity, the lack of these two developmental stages was the cause of misclassification of many chemicals in this study. Also, care has to be taken in the composition of cell culture media if a more humanized system

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should be achieved. The major shortcoming, although mostly avoided in this set of chemicals, is

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the lack of metabolism of a substance. Ideally, a metabolizing system should be added to the assay components of the EST in order for the EST to take the embryotoxic potential of maternal

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as well as fetal metabolites into account. Furthermore, addition of a test of barrier permeability taking changes thoughout human pregnancy into account, and/or prediction of pharmacokinetic behavior should be considered.

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Research into adaptations and improvement of the technical aspects of the EST is ongoing. Lessons learned from the misclassification of a set of thirteen compounds in the

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ReProTect WPIII study include the necessity of inclusion of physiological relevance of both the mechanism of action as well as by humanizing the in vitro environment to more closely reflect

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the human situation. Further development of the EST and its modifications has the potency to yield a method with a predictivity toward humans superior to animal testing.


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Acknowledgements

We thank Katharina Schlechter and Birgitta Slawik for expert help in performing the embryonic stem cell test. The work described in the current manuscript was partly funded within the 6th Framework Programme of the European Union.

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Declarations of Interest

The author’s affiliation is as shown on the cover page. The authors have sole responsibility for the writing and content of the paper.

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Table 1: Comparison of the two classification schemes. Brown, 2002 Classification Description Class 3: Developmentally Strongly embryotoxic toxic in all species tested, inducing multiple developmental effects, and with a high adult to developmental (A/D) ratio Class 2: Subgroup (a) was Weakly embryotoxic defined as developmentally toxic in multiple (but not all) species, with a high A/D ratio. Subgroup (b) was defined as developmentally toxic in multiple species, inducing multiple effects, with exposures that are clearly less than maternally toxic exposures. Subgroup (c) was defined as developmentally toxic, inducing effects that are clearly unrelated to maternal toxicity, but with exposures that are close to maternally toxic exposures Class 1: Not developmentally Non-embryotoxic toxic at maternally toxic exposures, but which may show some minor embryo/fetal toxicity at high maternally toxic exposures, and which cannot be separated from maternal toxicity

Marx-Stoelting et al., 2009 Classification Description Category 1: Teratogenic in all Strongly teratogenic species tested, in the absence of maternal toxicity

Teratogenic in some species tested, in the absence of maternal toxicity

Category 2: Moderately teratogenic

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Category 3: Mildly teratogenic

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Teratogenic, but at approximately the same dosage as maternal toxicity

Category 4: Non-teratogenic

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Non-teratogenic


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Table 2: Results of the EST as means of all experiments (four or more independent experiments each) of both partners. Substance

In vivo classification Marx-

Mean EC50 [µg/ml]

Reclassification ID50 D3

Stoelting et

matching EST

al., 2009

PM

rP Fo

IC50 D3

PM

IC50 3T3

Strong

Strong

n.d.a

n.d.a

n.d.a

Weaka

Ochratoxin A

Strong

Strong

10

15

6.6

Weak

D-Penicillamine

Strong

Strong

519

596

208

Non

Methylazoxymethanol Strong

Strong

41

19

6.4

Non

Lovastatin

Weak

2.2

3.4

3.8

Strongb

Nitrofen

Moderate Moderate

Weak

3.1

9.3

5.2

Weak

Warfarin

Moderate

Weak

210

190

133

Nonb

β-Aminopropionitrile

Mild

Weak

659

909

674

Non

Dinoseb

Mild

Weak

12

9.7

10

Weak

Furosemide

Mild

Weak

421

595

202

Non

Doxylamine

Non

Non

54

173

399

Weak

Pravastatin

Non

Non

31

134

381

Weak

Metoclopramide

Non

Non

50

126

195

Weak

w

a

ie

succinate

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Papaverine

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Precipitates in all concentrations tested, a free concentration can not be determined. The PM

is the result of a qualitative assessment of the assay results. b

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Substances produced ambiguous results in single experiments as reported (Marx-Stoelting et

al., 2009).

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58


Table 3: Categories of misclassification. Suspected reasons for misclassification 1. Substance acts on a different tissue and/or at a later developmental stage Probably to be detected by developmental neurotoxicity testing Methylazoxymethanol acetate (Papaverine*)

rP Fo Dimethadione

Methylmercury chloride*

Probably to be detected by developmental osteotoxicity testing D-Penicillamine

Warfarin*

ee

β-Aminopropionitrile

Other (indirect developmental toxicity through maternal alkalosis) Furosemide*

rR

2. Different concentration of a factor (nutrient, vitamin etc.) in differentiation media compared to plasma Ochratoxin A

Furosemide* Pravastatin*

3. Substance acts as a muscle relaxant

Metoclopramide

ly

Doxylamine succinate

On

Methylmercury chloride*

w

Warfarin*

ie

Lovastatin

ev

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Diphenhydramine (Papaverine*) 4. Lack of barrier function Pravastatin* 5. Selection criteria differ between ReProTect WPIII study and validation study


59

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Nitrofen 6. Results lie outside the prediction model Methylmercury chloride* * denotes substances that appear in more than one category

w

ie

ev

rR

ee

rP Fo ly

On

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60


Figure Legends

Fig. 1: Representative concentration-response curves for the thirteen substances tested in the ReProTect WP III study. Experiments were performed according to Seiler and Spielmann (2011).

rP Fo

Fig. 2: Two-linear discriminant plot of the data of the substances discussed in this review. Data was processed and used in linear discriminant functions as described by Riebeling et al. (2011a).

w

ie

ev

rR

ee ly

On

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61

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ev

rR

ee

rP Fo 142x106mm (300 x 300 DPI)

w

ie ly

On

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rR

ee

rP Fo 59x40mm (300 x 300 DPI)

w

ie

ev

ly

On

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