Permanent embryonic germ cell lines of BALB/cJ mice--an in vitro alternative for in vivo germ cell mutagenicity tests.

To offer a sensitive and predictive in vitro method to assess germ cell mutagenicity, we established primordial germ (PG) cell-derived permanent female and male embryonic germ (EG) cell lines of the mouse (strain BALB/cJ). The differences in developmental sensitivity of EG cells and differentiated fibroblast cells of the mouse cell line 3T3 to genotoxicants were tested comparatively under identical test conditions. Cytotoxicity assay was measured by the MTT test and genotoxic effects were determined by sister chromatid exchanges (SCE) rates induced by standard reference mutagens. Both methods are used to assign the chemicals to two classes of in vivo reproductive toxicity, non- and strongly genotoxic to germ cells. Applying linear discriminant analysis, a biostatistical prediction model (PM) was developed for the female cell line EG(3). This procedure identified a single variable, the Ig(SCE(200)EG(3)) as the statistically significant concentration related increase of 200% in the mean number of SCEs per metaphase spread after 3 h of exposure to be sufficient for separation into the classes: non- and strongly genotoxic to germ cells. Applying this PM to the training set of five genotoxic and three non-genotoxic test chemicals, 100% correct classifications were obtained.

Prevalidation of the Embryonic Stem Cell Test (EST)-A New In Vitro Embryotoxicity Test.

Pluripotent embryonic stem cells (ES cells) of the mouse (cell-line D3) can be maintained in the undifferentiated state in the presence of LIF (Leukaemia Inhibitory Factor). Upon withdrawal of LIF, these cells differentiate into various cell types under appropriate conditions. This property of ES cells allowed us to develop an in vitro embryotoxicity test, the Embryonic Stem Cell Test (EST; In Vitro Toxicology 1997, 10, 119-127), which does not require taking embryonic cells or tissues from pregnant animals. In the EST, the effect of test chemicals on three endpoints is assessed: inhibition of the differentiation of ES cells into contracting myocard, cytotoxicity in ES cells and cytotoxicity in mouse 3T3 fibroblasts, which are serving as differentiated cells in the test. The results of a prevalidation study of the EST are described, which was conducted according to the ECVAM prevalidation scheme. In the first stage of the study (Phase I), a standard operating procedure (SOP) was elaborated. In the second phase (Phase II), the interlaboratory transferability of the EST was assessed using three test chemicals representing three classes of embryotoxicity (a strong, a weak and a non-embryotoxic chemical) in two European laboratories (ZEBET at the BgVV in Berlin, Germany; ECVAM at the JRC in Ispra, Italy) and one US laboratory (Institute for In Vitro Sciences (IIVS) in Gaithersburgh, MA, USA). In the final stage of prevalidation (Phase III), nine test chemicals and a positive control were tested under blind conditions at ZEBET and ECVAM. The statistical evaluation of the results led to the development of an improved prediction model for the EST.

[Report on the ICCVAM workshop on in vitro methods for assessing acute systemic toxicity]. / Ergebnisse des ICCVAM Workshop über in vitro Methoden zur Bewertung der akuten systemischen Toxizität.

It was suggested in the ICCVAM workshop that the Register of Cytotoxicity (RC), using in vitro cytotoxicity data to predict the in vivo starting doses, should be implemented into acute toxicity testing as soon as possible. The validity of the in vitro cytotoxicity data to establish appropriate starting doses for acute toxicity testing will be assessed experimentally. Secondly, in order to replace the use of animals in acute lethality testing a formal validation will be conducted in which the ability to predict rodent LD50 values and toxicity classes from cytotoxicity data will be evaluated.

Dermal and inhalation acute toxic class methods: test procedures and biometric evaluations for the Globally Harmonized Classification System.

The acute toxic class (ATC) methods were developed for determining LD(50)/LC(50) estimates of chemical substances with significantly fewer animals than needed when applying conventional LD(50)/LC(50) tests. The ATC methods are sequential stepwise procedures with fixed starting doses/concentrations and a maximum of six animals used per dose/concentration. The numbers of dead/moribund animals determine whether further testing is necessary or whether the test is terminated. In recent years we have developed classification procedures for the oral, dermal and inhalation routes of administration by using biometric methods. The biometric approach assumes a probit model for the mortality probability of a single animal and assigns the chemical to that toxicity class for which the best concordance is achieved between the statistically expected and the observed numbers of dead/moribund animals at the various steps of the test procedure. In previous publications we have demonstrated the validity of the biometric ATC methods on the basis of data obtained for the oral ATC method in two-animal ring studies with 15 participants from six countries. Although the test procedures and biometric evaluations for the dermal and inhalation ATC methods have already been published, there was a need for an adaptation of the classification schemes to the starting doses/concentrations of the Globally Harmonized Classification System (GHS) recently adopted by the Organization for Economic Co-operation and Development (OECD). Here we present the biometric evaluation of the dermal and inhalation ATC methods for the starting doses/concentrations of the GHS and of some other international classification systems still in use. We have developed new test procedures and decision rules for the dermal and inhalation ATC methods, which require significantly fewer animals to provide predictions of toxicity classes, that are equally good or even better than those achieved by using the conventional LD(50)/LC(50) methods. In order to cope with rather narrow dose/concentration classes of the GHS we have, as in our previous publications, combined the outcome of all results that can be obtained during testing for the allocation to one of the defined toxicity classes of the GHS. Our results strongly recommend the deletion of the dermal LD(50) and the inhalation LC(50) test as regulatory tests and the adoption of the dermal and inhalation ATC methods as internationally accepted alternatives.

[Permanent embryonic mouse germ cell-lines, an in vitro alternative to in vivo germ cell mutagenicity tests]. / Permanente embryonale Keimzelllinien - eine in vitro Alternative zur Beurteilung von Keimzellmutagenität.

Germ cell mutagenesis is required by the 7th amendment of the directive 67/548 EEC into the national regulations on existing chemicals. Officially accepted in vivo test systems for stage specific mutagenicity are the dominant lethal (DL) test and the specific locus test (SLT) in mice. An acceptable in vitro alternative designed to address germ cell mutagenesis and discriminate between male and female specific effects is not available at present. In order to offer a sensitive and predictive in vitro method to assess the genotoxic potential of chemical agents on male and female reproduction, we established primordial germ (PG) cell-derived permanent embryonic germ (EG) cell lines of the mouse (strain BALB/cJ). The differences in developmental sensitivity of the EG(3) cell line and differentiated fibroblast cells 3T3 were comparatively tested with cytotoxicity assay (MTT test ) and genotoxic studies (SCE-assay) under identical test conditions. The concentration-response curves reflected the female cell line EG(3) to be extremely sensitive concerning cytotoxic and genotoxic endpoints. Therefore this cell line was used to classify in vivo genotoxic and non-genotoxic test substances with different potential endpoints. Applying linear discriminant analysis three endpoints were identified for the correct classification (100%) of all test chemicals, namely the SCE(200) value (increase of 200% in the mean number of SCEs per metaphase spread) for EG(3) (3 hrs and 24 hrs assay) and the IC(5)0 value for EG(3) after 3 hrs of exposure to test chemicals.

Embryotoxicity screening using embryonic stem cells in vitro: correlation to in vivo teratogenicity.

Blastocyst-derived pluripotent embryonic stem (ES) cells of the mouse can be induced to differentiate in culture into a variety of cell types, including cardiac muscle cells. The embryonic stem cell test that makes use of the differentiation of ES cells into cardiomyocytes in a standardized in vitro model was developed to offer an alternative method to comprehensive in vivo studies in reproductive toxicology about toxic effects of chemicals. ES cells of the mouse cell line D3 are investigated for their preserved capability to differentiate following drug exposure, and both ES cells and differentiated fibroblast cells of the mouse cell line 3T3 are comparatively analyzed for effects on viability. The following endpoints are used to classify the embryotoxic potential of chemicals into three classes of in vitro embryotoxicity (non-, weakly or strongly embryotoxic). These endpoints are: (1) the inhibition of differentiation of ES cells into cardiomyocytes after 10 days of treatment, and the decrease of viability (cytotoxicity) of (2) 3T3 cells and (3) ES cells after 10 days of treatment, determined by a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) test. 50% inhibition concentrations for differentiation (ID(50)) and cytotoxicity (IC(50)D3 and IC(50)3T3) are calculated from concentration-response curves. Applying linear analysis of discriminance, a biostatistical prediction model (PM) was developed. This procedure identified three variables, the lg(IC(50)D3), the lg(IC(50)3T3) and the relative distance between IC(50)3T3 and ID(50), that improved the separation of the three classes of embryotoxicity compared to the prediction model that was originally proposed after test development. Unlike the original PM, the improved PM incorporates as one variable the relative distance between IC(50)3T3 and ID(50), instead of the ratio ID(50)/IC(50)D3 that was used previously.

Development of prediction models for three in vitro embryotoxicity tests in an ECVAM validation study.

Since 1997 the National Center for Documentation and Evaluation of Alternative Methods to Animal Experiments, ZEBET, in Berlin, has been coordinating a validation study aimed at prevalidation and validation of three in vitro embryotoxicity tests, funded by the European Center for the Validation of Alternative Methods (ECVAM) at the Joint Research Center (JRC, Ispra, Italy). The tests use the cultivation of postimplantation rat whole embryos (WEC test), cultures of primary limb bud cells of rat embryos (micromass or, MM, test), and cultures of a pluripotent mouse embryonic stem cell line (embryonic stem cell test or EST). Each of the tests was performed in four laboratories under blind conditions. In the preliminary phase of the validation study 6 out of 20 test chemicals comprising different embryotoxic potential (non, weakly, and strongly embryotoxic) were tested. The results were used to define biostatistically based prediction models (PMs) to identify the embryotoxic potential of test chemicals for the WEC test and the MM test. The PMs developed with the results of the preliminary phase of the validation study (training set) will be evaluated with the results of the remaining 14 test chemicals (definitive phase) by the end of the study. In addition, the existing, improved PM (iPM) for the EST, which had been defined previously, was evaluated using the results of the preliminary phase of this study. Applying the iPM of the EST to the results of this study, in 79% of the experiments, chemicals were classified correctly according to the embryotoxic potential defined by in vivo testing. For the MM and the WEC test, the PMs developed during the preliminary phase of this validation study provided 81% (MM test) and 72% (WEC test) correct classifications. Because the PM of the WEC test took into account only parameters of growth and development, but not cytotoxicity data, a second PM (PM2) was developed for the WEC test by incorporating cytotoxicity data of the differentiated mouse fibroblast cell line 3T3, which was derived from the EST. This approach, which has previously never been used, resulted in an increase to 84% correct classifications in the WEC test.

[Development of prediction models for three in vitro embryotoxicity tests which are evaluated in an ECVAM validation study] / Die Entwicklung von Prädiktionsmodellen für drei in vitro Embryotoxizitätstests im Rahmen einer ECVAM Validierungsstudie.

In 1997 ZEBET started the co-ordination of a study funded by the European Centre for the Validation of Alternative Methods (ECVAM) with the aim of pre-validation and validation of three in vitro embryotoxicity tests. These tests employ the cultivation of post-implantation whole rat embryos (WEC test), cultures of primary limb bud cells of rat embryos (micromass test, MM-Test), and cultures of a pluripotent mouse embryonic stem cell line (embryonic stem cell test, EST). In the current Validation Study each of the tests is evaluated in four laboratories under blind conditions. In an initial phase of the validation study six out of 20 test chemicals comprising different embryotoxic potential (non, weak and strong embryotoxic) were tested. The results were used to improve the prediction models (PM) for the WEC test and the MM-Test in order to identify the embryotoxic potential of test chemicals. In addition, the existing PM for the EST was evaluated using the results from testing of the initial six chemicals. The PM for the EST was developed using the results of a previous pre-validation study (Scholz et al.,1999), in which 94% of the learning sample were classified correctly. Applying this PM to the results of the initial phase of the current validation study, 80% of the experiments were classified correctly according to the embryotoxic potential of the tested chemicals in vivo. Applying the PM for the MM-Test and the WEC test that were developed during the current validation study in both tests, 79% correct classifications were achieved. Since the PM of the WEC-Test took into account only parameters of growth and development, but not cytotoxicity data, a second PM (PM2) for the WEC was developed that was improved by incorporating cytotoxicity data of the differentiated mouse fibroblast cell line 3T3 derived from the EST. This approach, which has previously never been used as an adjunct to the WEC test, resulted in an increase of correct classifications to 96%.