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Thyroid Histopathology Assessments for the Amphibian Metamorphosis Assay to Detect Thyroid-active Substances K. Christiana Grim, Marilyn Wolfe, Thomas Braunbeck, Taisen Iguchi, Yasuhiko Ohta, Osamu Tooi, Les Touart, Douglas C. Wolf and Joe Tietge Toxicol Pathol 2009; 37; 415 originally published online Apr 22, 2009; DOI: 10.1177/0192623309335063 The online version of this article can be found at: http://tpx.sagepub.com/cgi/content/abstract/37/4/415

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Articles Toxicologic Pathology, 37: 415-424, 2009 Copyright # 2009 by The Author(s) ISSN: 0192-6233 print / 1533-1601 online DOI: 10.1177/0192623309335063

Thyroid Histopathology Assessments for the Amphibian Metamorphosis Assay to Detect Thyroid-active Substances K. CHRISTIANA GRIM,1 MARILYN WOLFE,2 THOMAS BRAUNBECK,3 TAISEN IGUCHI,4 YASUHIKO OHTA,5 OSAMU TOOI,6 LES TOUART,7 DOUGLAS C. WOLF,8 AND JOE TIETGE8 1

Smithsonian National Zoological Park, Conservation and Research Center, Center for Species Survival, Front Royal, VA U.S.A. and U.S. Environmental Protection Agency, Office of Science Coordination and Policy, Washington, DC U.S.A. 2 Experimental Pathology Laboratories, Inc., Sterling, Virgina U.S.A. 3 Aquatic Ecology and Toxicology Group, Department of Zoology, University of Heidelberg, Germany 4 Division of Bio-Environmental Science, Department of Bio-Environmental Science, Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, Aichi, Japan 5 Laboratory of Experimental Animals, Department of Veterinary Medicine, Faculty of Agriculture, Tottori University, Koyama, Japan 6 Institute of Environmental Ecology, IDEA Consultants, Inc., Shizuoka, Japan 7 U.S. Environmental Protection Agency, Office of Science Coordination and Policy, Washington, DC U.S.A. 8 National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina and Duluth, Minnesota, U.S.A. ABSTRACT In support of an Organization for Economic Cooperation and Development (OECD) Amphibian Metamorphosis Assay (AMA) Test Guideline for the detection of substances that interact with the hypothalamic-pituitary-thyroid axis, a document was developed that provides a standardized approach for evaluating the histology/histopathology of thyroid glands in metamorphosing Xenopus laevis tadpoles. Here, a consolidated description of histology evaluation practices, core diagnostic criteria and severity grading schemes for the AMA, an atlas of the normal architecture of amphibian thyroid glands over the course of metamorphosis, and the core diagnostic criteria with examples of severity grades is provided. Core diagnostic criteria include thyroid gland hypertrophy/atrophy, follicular cell hypertrophy, and follicular cell hyperplasia. The severity grading scheme is semiquantitative and employs a four-grade approach describing ranges of variation within assigned ordinal classes: not remarkable, mild, moderate, and severe. The purpose of this severity grading approach is to provide an efficient, semi-objective tool for comparing changes (compound-related effects) among animals, treatment groups, and studies. Proposed descriptions of lesions for scoring the four core criteria are also given. Keywords:

amphibia; metamorphosis; thyroid; histopathology; histology; Xenopus laevis.

INTRODUCTION

endocrine active substances (EAS). As part of this effort, the development and validation of the Amphibian Metamorphosis Assay (AMA) was undertaken (OECD 2004a, 2004b, 2007a, 2007b, 2007c, 2008a, 2008b). The AMA is intended to empirically identify substances that may interfere with the normal function of the hypothalamic-pituitary-thyroid (HPT) axis. It represents a generalized vertebrate model to the extent that it is based on the conserved structure and functions of thyroid systems within vertebrate classes. Additionally, amphibian metamorphosis is a well-studied, thyroid-dependent process that responds to substances active within the HPT axis, and it is currently the only available assay that assesses thyroid activity in an animal undergoing morphological development (Kloas et al. 1999; Lutz and Kloas 1999). For an in-depth description of this assay, please see the OECD Detailed Review Paper on the Amphibian Metamorphosis Assay (OECD 2004a), and for a description of the full histological method, see the Guidance Document on Amphibian Thyroid Histology (OECD 2007b).

The Organization for Economic Cooperation and Development (OECD) has initiated a high-priority activity to develop test guidelines for the screening and testing of potential Address correspondence to: Dr. K. Christiana Grim, Smithsonian National Zoological Park, Conservation Research Center, Center for Species Survival, 1500 Remount Road, Front Royal, VA 22630, U.S.A.; U.S. Environmental Protection Agency, Office of Science Coordination Policy, 1200 Pennsylvania Ave., NW, Ariel Rios (7203M), Washington, DC 20460, U.S.A.; e-mail addresses: [email protected], [email protected]. The OECD Guidance Document on Amphibian Thyroid Histology is published in the series on Testing and Assessment (No.82). This paper is an abbreviated version of the OECD guidance document, and is the sole responsibility of its authors. Competing interests: The authors have not declared any competing interests. Abbreviations: AMA, amphibian metamorphosis assay; EAS, endocrine active substances; H&E, hematoxylin and eosin; HPT, hypothalamicpituitary-thyroid; N&F, Nieuwkoop and Faber; OECD, Organization for Economic Cooperation and Development; TSH, thyroid stimulating hormone. 415


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The premise of the AMA is that metamorphic development in the African clawed frog (Xenopus laevis) is dependent upon proper synthesis and regulation of thyroid hormone and that xenobiotic perturbation of the HPT axis can lead to measurable developmental effects. The general experimental design of the AMA exposes Nieuwkoop and Faber (Nieuwkoop and Faber 1994) (N&F) stage 51 X. laevis tadpoles to a minimum of three different test chemical concentrations and a dilution water control for twenty-one days. The primary endpoints of the assay are hind limb length, developmental stage, and thyroid histology/histopathology. Hind limb length and developmental stage are morphological indicators of metamorphic development, as described by Nieuwkoop and Faber (1994). Effects on morphological development in treated organisms, in comparison to control organisms, suggest perturbation of normal thyroid hormone homeostasis. Thyroid gland histology is an important diagnostic end-point with several histomorphological features that are modulated by regulatory components of the HPT, most notably thyroid stimulating hormone (TSH), the pituitary hormone that regulates synthesis and release of thyroid hormone. Other endpoints, such as snout-to-vent length, wet weight, and daily observations of mortality and clinical manifestations of overt toxicity, are also measured to help distinguish between thyroid specific effects and generalized toxicity. During the validation process for the AMA, it was recognized that thyroid histology served as a valuable and sensitive diagnostic end point for detecting a chemical’s ability to interact with the HPT axis, particularly for thyroid system antagonism. Since this assay potentially will be used in various countries, it is important to standardize the histologic analysis of thyroid tissues from X. laevis to maximize comparability among pathologists, thereby reducing inconsistency and bias. Therefore, an OECD guidance document was developed that addresses histopathology reading practices, diagnostic criteria, severity grading, and data reporting and presents an atlas of illustrative reference photomicrographs. This guidance defines the diagnostic criteria for the histologic analysis of the AMA. The criteria are based on pathologists’ experience with the AMA and documented changes of the thyroid glands in response to chemical exposure. This report provides a consolidated description of the OECD histological guidance provided in the test guideline. It presents an approach to standardized reading practices for the AMA, a description of the core diagnostic criteria and severity grading schemes, and updated photomicrographic examples of the normal thyroid gland architecture during the course of metamorphosis and core diagnostic criteria with representative severity grades. Technical information on morphological sampling and histological preparation procedures is not reviewed here; however, that information can be obtained in the OECD guidance document on thyroid histology (OECD 2007c). GENERAL RECOMMENDATIONS FOR HISTOLOGICAL EVALUATION AMA STUDIES

OF

Generally, tissues are prepared for histological examination by fixation in Davidson’s solution, paraffin embedding,

TOXICOLOGIC PATHOLOGY

transverse or frontal sectioning at the largest diameter of the glands, and staining with hematoxylin and eosin (OECD 2007b). Thyroid histology is evaluated using standard approaches for toxicologic pathology, as have been described in the Society of Toxicologic Pathology Best Practices document (Crissman et al. 2004) (e.g., nonblinded). It is recommended that the histological evaluation be performed by experienced toxicological pathologists who are familiar with normal X. laevis thyroid histology, with thyroid gland physiology, and with general responses of the thyroid glands to agonism or antagonism. Over the course of metamorphosis in X. laevis, the needs of developing and remodeling tissues are dynamic. Therefore, the normal histomorphology of the thyroid glands changes over time with the developmental stage of the tadpole. These normal changes must be considered when thyroid histology of tadpoles in different developmental stages is compared. NORMAL PROGRESSION OF THE AMPHIBIAN THYROID GLANDS OVER THE COURSE OF METAMORPHOSIS Over the course of metamorphosis, the architecture of the thyroid glands of X. laevis progressively changes to accommodate physiological needs of the developing amphibian (Dodd et al. 1976). The thyroid glands increase in size and volume from proliferation of follicles and an increase in follicular cell size and number. The thyroid glands achieve maximal size at approximately N&F stage 63, and maximum follicular cell height between stages 62 and 64 (Dodd et al. 1976). These changes must be taken into consideration when evaluating relative differences between treatments, so as not to confound developmental stage–specific effects. To provide a reference atlas of the normal architecture of the developing amphibian thyroid glands as it relates specifically to the AMA, representative examples, by developmental stage, of the histological appearance of the glands in developing X. laevis tadpoles are provided in Figures 1 and 2. Examples were taken from tadpoles sequentially sacrificed beginning with N&F stage 51 and ending with N&F stage 66. The tissues were processed as described above, sectioned at the level of the largest glandular diameter (approximately mid-gland) in the frontal plane, and stained with H&E. Consistent with Dodd et al. (1976), the thyroid glands during this developmental period increase in area, and follicular cells increase in height. Regression of thyroid gland size occurs after metamorphic climax. The maximum area of the glands is achieved close to N&F stage 64, whereas the maximum cell height is achieved between N&F stages 62 and 63. GENERAL SEVERITY GRADING SCHEME LESIONS

FOR

HISTOPATHOLOGICAL

For the AMA, quantitative approaches to scoring the grades of severity of diagnostic criteria are not suitable. The core criteria are most effectively evaluated using a semiquantitative, fourgrade severity scoring system, ranging between zero and three (Table 1). The descriptors are based on relative differences from


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FIGURE 1.—A-O. The normal microanatomy of thyroid glands from X. laevis undergoing metamorphosis between N&F stages 51 and 66; N&F stage 65 is not represented. Photomicrographs are taken from frontal sections at the largest cross-sectional gland area. Gland size reaches its maximum area at N&F stage 64, then regresses after metamorphic climax. H&E.


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FIGURE 2.—A-O. High-magnification images of the normal microanatomy of thyroid glands from X. laevis undergoing metamorphosis between N&F stages 51 and 66; N&F stage 65 is not represented. Photomicrographs are taken from frontal sections at the largest cross-sectional gland area. Follicular cell height progressively increases from cuboidal to columnar, reaching its maximum height between N&F stages 62 and 63. By N&F stage 66, the follicular cells have become squamous to cuboidal. H&E.


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TABLE 1.—General severity grading scheme for histological lesions of the thyroid gland.

TABLE 2.—Severity grading scheme for thyroid gland atrophy. Grade

Grade 0

Descriptor

Criterion

Not remarkable

Ranging from inconspicuous to barely noticeable but so minor, small, or infrequent as to warrant no more than the least assignable grade. For multifocal or diffusely distributed alterations, this grade is used for processes where less than 20% of the tissue in the section is involved. A noticeable feature of the tissue. For multifocal or diffusely distributed alterations, this grade is used for processes where 30%-50% of the tissue in the section is involved. A dominant feature of the tissue. For multifocal or diffusely distributed alterations, this grade is used for processes where 60%-80% of the tissue in the section is involved. An overwhelming feature of the tissue. For multifocal or diffusely distributed alterations, this grade is used for processes where greater than 80% of the tissue in the section is involved.

1

Mild

2

Moderate

3

Severe

thyroid glands in control animals, and/or on the percentage of cells or tissue affected. In addition to the severity grade, qualitative changes associated with the lesions should be documented. Several validation studies for the AMA were conducted using chemicals that interact with the HPT axis through different modes of action, including iodine uptake inhibition, iodine deficiency, and altered thyroxine metabolism (OECD 2004b, 2007a, 2008b; Opitz et al. 2005; Opitz et al. 2006). Using the information on the typical effects on thyroid gland histology that was collected during these studies, four core diagnostic criteria were determined: thyroid gland atrophy, thyroid gland hypertrophy, follicular cell hypertrophy, and follicular cell hyperplasia. Following are descriptions and photomicrographs of these lesions and approaches to grading the severity of the lesions. The examples used in the photomicrographs are taken from midluminal sections at the widest cross-sectional area of the thyroid glands. The plane of section is either transverse or frontal. After several histological analyses were performed using the AMA protocol, it was found that information from both planes of section was equivalent.

419

Descriptor

Criterion

0

Not remarkable

1

Mild

2

Moderate

3

Severe

Less than a 20% reduction in size in comparison to controls. Gland size is 30%-50% reduced from the size of control glands. Gland size is 60%-80% reduced from the size of control glands. Gland size is over 80% reduced from the size of control glands.

TABLE 3.—Severity grading scheme for thyroid gland hypertrophy. Grade

Descriptor

Criterion

0

Not remarkable

1

Mild

2

Moderate

3

Severe

Less than 20% enlargement of glands in comparison to controls. Diffuse enlargement of glands that exceeds the size of control glands by 30%-50%. Diffuse enlargement of glands that exceeds the size of control glands by 60%-80%. Diffuse enlargement of glands that exceeds the size of control glands by over 80%. There is contact of both glands at the midline, and they exceed normal boundaries into surrounding tissue space.

TABLE 4.—Severity grading scheme for follicular cell hypertrophy. Grade 0 1 2 3

Descriptor

Criterion

Not remarkable Mild Moderate Severe

Fewer than 20% of the cells exhibit hypertrophy. 30%-50% of follicular cells exhibit hypertrophy. 60%-80% of follicular cells exhibit hypertrophy. Over 80% of follicular cells exhibit hypertrophy.

the glands. See Tables 2 and 3 for severity grading schemes specific to thyroid gland atrophy and hypertrophy and Figures 3 and 4 for photomicrographs of examples of these lesions.

FOLLICULAR CELL HYPERTROPHY THYROID GLAND ATROPHY/HYPERTROPHY Decreases (atrophy) or increases (hypertrophy) in the overall size of the thyroid glands are a consequence of changes in follicular cell size and number, and/or in the size and number of follicles. The severity of either thyroid atrophy or hypertrophy is graded on an overall, general appearance of the thyroid glands. Because the diagnosis of hypertrophy or atrophy is dependent on a comparison to thyroid glands from control animals, it is necessary to establish the normal variability of thyroid gland sizes in control tadpoles prior to making determinations on thyroid gland size in treatment/dose groups. Additionally, to make comparable observations, evaluated sections must be representative of the greatest cross-sectional area of

Hypertrophic follicular cells are graded based on the percentage of the cells exhibiting this feature. For accurate determination of follicular cell size, the tissue section must be in the proper location (ideally mid-luminal). Evaluation of cells that are tangentially sectioned can be misleading. Follicular cells in appropriately sectioned follicles exhibit uniformity in nuclear size and shape. It is recognized that follicular cell hypertrophy may present as a generalized lesion and interpreted as such. Since normal amphibian thyroid glands show heterogeneity in follicular cell shape, ranging from squamous to tall columnar, severity is determined by the change in percentage of cells exhibiting tall columnar structure. See Table 4 for a severity grading scheme specific to follicular cell


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FIGURE 3.—Glandular atrophy. A. Grade 0. Thyroid glands from an N&F stage 58 X. laevis tadpole. The thyroid follicles contain eosinophilic homogeneous colloid and are lined by simple cuboidal follicular cells. There is minimal follicular cell hyperplasia and hypertrophy, which may be present in normal thyroid glands. The thyroid follicles are variable in size. H&E. B. Grade 1. Thyroid glands from an N&F stage 59 X. laevis tadpole exposed to 1 mg/L thyroxine for twenty-one days. There is minimally decreased quantity of colloid and a minimal decrease in the size of thyroid follicles as compared to control thyroid glands. There is mild atrophy of the thyroid glands. H&E. C. Grade 2. Thyroid glands from an N&F stage 60 X. laevis tadpole exposed to 1 mg/L thyroxine for twenty-one days. There is a moderate decrease in the size of the thyroid follicles and moderately decreased colloid. The glands are moderately atrophied. H&E. D. Grade 3. Thyroid glands from an N&F stage 60 X. laevis tadpole exposed to 1 mg/L thyroxine for twenty-one days. There is severe thyroid gland atrophy, and the thyroid follicles are uniformly small. There is a moderately severe decease in amount of colloid, but what little colloid is present is eosinophilic and homogeneous. H&E.

hypertrophy and Figure 5 for photomicrographs of examples of this lesion. FOLLICULAR CELL HYPERPLASIA Follicular cell hyperplasia is diagnosed when there is follicular cell crowding, stratification (multiple layers), and/ or papillary infolding of single or multiple layers of follicular cells. The severity grading scheme for follicular cell hyperplasia is based on the percentage of follicles that exhibit

hyperplasia, and/or the percentage of tissue that is affected. As with follicular cell size, the section plane should ideally be mid-luminal. See Table 5 for a severity grading scheme specific to follicular cell hyperplasia and Figure 6 for photomicrographs of examples of this lesion. Additional descriptive criteria include follicular lumen area and colloid quality. Luminal area can be reduced or increased and is indicative of a steady state and/or instantaneous physiological condition of the thyroid gland, such as the release of T4. Severity of effects on luminal area is


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FIGURE 4.—Glandular hypertrophy. A. Grade 0. Thyroid glands from a control, N&F stage 59 X. laevis tadpole. The thyroid follicles are colloid filled and are lined by simple cuboidal follicular cells. There is minimal follicular cell hyperplasia. H&E. B. Grade 1. Thyroid glands from an N&F stage 59 X. laevis tadpole exposed to 62.5 mg/L perchlorate for twenty-one days. There is moderate hypertrophy of the follicular cells and mild follicular cell hyperplasia. There is mild glandular hypertrophy, and there is mild decreased colloid in the thyroid follicles. H&E. C. Grade 2. Thyroid glands from an N&F stage 59 X. laevis tadpole exposed to 62.5 mg/L perchlorate for twenty-one days. Moderate follicular cell hypertrophy and moderate follicular cell hyperplasia are present. The thyroid glands are moderately enlarged overall, and there is mildly decreased colloid in the thyroid follicles. H&E. D. Grade 3. Thyroid glands from an N&F stage 59 X. laevis tadpole exposed to 250 mg/L perchlorate for twenty-one days. There is severe hypertrophy of the thyroid glands with contact at the midline. There is moderate follicular cell hypertrophy and moderately severe follicular cell hyperplasia. There is mildly decreased colloid in the thyroid follicles. H&E.

TABLE 5.—Severity grading scheme for follicular cell hyperplasia. Grade

Descriptor

0 1

Not remarkable Mild

2

Moderate

3

Severe

Criterion Focal or diffuse crowding of follicular cells affecting less than 20% of the tissue. Focal or diffuse crowding of follicular cells affecting 30%-50% of the tissue, and/or single or multiple papillary infoldings of the follicular cell layer. 60%-80% of the follicles exhibit focal hyperplasia characterized by pseudostratified or stratified follicular epithelium. Papillary infolding may be present. Over 80% of follicles exhibit extensive hyperplasia with stratification 2-3 cell layers thick. Papillary infolding may be present.


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FIGURE 5.—Follicular cell hypertrophy. A. Grade 0. Thyroid glands from a control, N&F stage 59 X. laevis tadpole. The thyroid follicles are colloid filled and are lined by simple cuboidal follicular cells. There is minimal follicular cell hyperplasia. H&E. B. Grade 1. Thyroid glands from an N&F stage 54 X. laevis tadpole exposed to 100 mg/L methimazole for ninety-six hours. There is mild follicular cell hypertrophy characterized by cells that are plump and columnar as compared to the usual cuboidal cells. There is also mild follicular cell hyperplasia. H&E. C. Grade 2. Thyroid glands from an N&F stage 57 X. laevis tadpole exposed to 125 mg/L perchlorate for twenty-one days. There is slight/mild follicular cell hyperplasia and moderate follicular cell hypertrophy. There is slight/mild decreased colloid in the thyroid follicles. H&E. D. Grade 3. Thyroid glands from an N&F stage 54 X. laevis tadpole exposed to 100 mg/L methimazole for 192 hours. There is severe hypertrophy of the thyroid glands. The amount of colloid in the follicles is severely reduced, and there is severe follicular cell hypertrophy and moderately severe hyperplasia. H&E.

graded based on the general grading scheme. Changes in colloid quality are generally considered in association with follicular luminal area. Typical descriptions include homogeneous, heterogeneous, lacy, or granular. If present, these findings are reported in a narrative format. The specific significance of changes in colloid quality is not always certain, possibly representing artifacts of tissue processing or reabsorption of thyroglobulin in cases of decreased circulating T4.

SUMMARY The diagnostic criteria and severity scheme presented are based on the current understanding of the relative changes that occur in the developing tadpole in response to thyroid-active substances. The diagnostic criteria presented in this document are associated with compensatory responses that are modulated by changes in circulating thyroid hormone and TSH concentrations, including, for example, follicular cell hypertrophy and hyperplasia, and colloid depletion. These alterations and


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FIGURE 6.—Follicular cell hyperplasia. A. Grade 0. Thyroid glands from an N&F stage 59 X. laevis control animal. The thyroid follicles are colloid filled, and most are lined by simple cuboidal to columnar follicular cells. H&E. B. Grade 1. Thyroid glands from an N&F stage 59 X. laevis control animal. The thyroid follicles are colloid filled, and most are lined by columnar follicular cells. Mild follicular cell hypertrophy and mild follicular cell hyperplasia are present, as demonstrated by crowding and the stratified/ pseudostratified appearance of the follicular cells, affecting less than 30% of the tissue. H&E. C. Grade 2. Thyroid glands from an N&F stage 59 X. laevis tadpole exposed to 62.5 mg/L perchlorate for twenty-one days. Moderate follicular cell hypertrophy and moderate follicular cell hyperplasia are present. The thyroid glands are moderately enlarged overall, and there is slight/ mild decreased colloid in the thyroid follicles. The hyperplasia affects 30%-50% of the tissue. H&E. D. Grade 3. Thyroid glands from an N&F stage 57 X. laevis tadpole exposed to 500 mg/L perchlorate for twenty-one days. There is severe hypertrophy of the thyroid glands, and the colloid is mildly decreased in the thyroid follicles. There is severe follicular cell hyperplasia demonstrated by stratification/pseudostratification of the follicular cells, affecting over 80% of the tissue, and there is moderate follicular cell hypertrophy. H&E.

hormone changes, when excessive or prolonged, can also result in a toxic developmental effect.

guidance for the AMA. We also thank the Smithsonian’s National Zoo for graciously hosting the expert group meeting that facilitated the development of this guidance.

ACKNOWLEDGMENTS We thank Dr. Leif Norrgren and Dr. Charles Sagoe for their expert input on the development of the histological guidance for the AMA. We thank Anne Gourmelon for her enduring support during the validation effort and development of histology

REFERENCES Crissman, J. W., Goodman, D. G., Hildebrandt, P. K., Maronpot, R. R., Prater, D. A., Riley, J. H., Seaman, W. J., and Thake, D. C. (2004). Best practices guideline: toxicologic histopathology. Toxicol Path 32, 126–31.


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Dodd, M. H. I., Dodd, J. M., and Lofts, B. (1976). The Biology of Metamorphosis. In Physiology of Amphibia, pp. 467–599. Academic Press, New York. Kloas, W., Lutz, I., and Einspanier, R. (1999). Amphibians as a model to study endocrine disruptors: II. Estrogenic activity of environmental chemicals in vitro and in vivo. Sci Total Environ 225, 59–68. Lutz, I., and Kloas, W. (1999). Amphibians as a model to study endocrine disruptors: I. Environmental pollution and estrogen receptor binding. Sci Total Environ 225, 49–57. Nieuwkoop, P. D., and Faber, J. (1994). Normal Table of Xenopus laevis. Garland Publishing, New York. Organization of Economic Cooperation and Development (OECD) (2004a). Detailed Review Paper on Amphibian Metamorphosis Assay for the Detection of Thyroid Active Substances. In Series on Testing and Assessment. Environmental Health and Safety Publications, Paris, France. OECD (2004b). Report of the Validation of the Amphibian Metamorphosis Assay for the detection of thyroid active substances: Phase 1 – Optimisation of the Test Protocol. In Series on Testing and Assessment. Environmental Health and Safety Publications, Paris, France. OECD (2007a). Final Report of the Validation of the Amphibian Metamorphosis Assay: Phase 2 – Multi-chemical Interlaboratory Study. In Series on Testing and Assessment. Environmental Health and Safety Publications, Paris, France.


OECD (2007b). Guidance Document on Amphibian Thyroid Histology. In Series on Testing and Assessment. Environmental Health and Safety Publications, Paris, France. OECD (2007c). Validation of the Amphibian Metamrophosis Assay as a Screen for Thyroid-Active Chemicals: Integrated Summary Report. In Series on Testing and Assessment. Environmental Health and Safety Publications, Paris, France. OECD (2008a). OECD Guideline for the Testing of Chemicals – The Amphibian Metamorphosis Assay. In Series on Testing and Assessment. Environmental Health and Safety Publications, Paris, France. OECD (2008b). Report of the Validation of the Amphibian Metamorphosis Assay (Phase 3). In Series on Testing and Assessment. Environmental Health and Safety Publications, Paris, France. Opitz, R., Braunbeck, T., Bogi, C., Pickford, D. B., Nentwig, G., Oehlmann, J., Tooi, O., Lutz, I., and Kloas, W. (2005). Description and initial evaluation of a Xenopus metamorphosis assay for detection of thyroid system-disrupting activities of environmental compounds. Environ Toxicol Chem 24, 653–64. Opitz, R., Hartmann, S., Blank, T., Braunbeck, T., Lutz, I., and Kloas, W. (2006). Evaluation of histological and molecular endpoints for enhanced detection of thyroid system disruption in Xenopus laevis tadpoles. Toxicol Sci 90, 337–48.

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