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ENDOCRINE DISRUPTORS (ED): The Challenge of Accessing the ED Regulatory Framework
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  • 1ENDOCRINE DISRUPTORS

    ENDOCRINE DISRUPTORS (ED): The Challenge of Accessing the ED Regulatory Framework

  • 2ENDOCRINE DISRUPTORS

    Introduction 3

    The Basics of Endocrine Disruption 4

    The Long History of ED Evaluation 7

    The Current Regulatory Environment for EDs 10

    US EPA 10

    Integrated Bioactivity Exposure Ranking (IBER): The Strategy Behind HTP Screening 13

    The EU Approach to ED Assessment 14

    Level 1: Existing Data and Non-test Information 15

    Level 2: Mechanistic In Vitro Tests 18

    Level 3: Mechanistic In Vivo Testing 20

    Level 4 And 5: Some Insights on the Application of the Latest Updated TGs 21

    The Big Challenge in Assessing ED Endpoints: Assessing and Interpreting Thyroid Hormones 23

    Summary 27

    References 28

    CONTENTS

  • 3ENDOCRINE DISRUPTORS

    This e-book is designed to provide you with an overview of how endocrine disruption became recognized as an issue and how regulatory authorities around the world have approached the assessment and regulation of chemicals with endocrine disruption potential. Given the complexity of the endocrine system, the e-book also explains the scientific approaches that have been used to describe the actions of ‘endocrine disruptors’ (EDs) and how they are assessed through a myriad of different tests and studies.

    Our endocrine system controls many basic body functions and plays a key role in reproduction and development. Disruption of this system can have profound effects on individuals, but also on whole populations and ecosystems. It is for this reason that there is such concern over the use of chemicals with the potential to disrupt the endocrine system and cause adverse toxic effects on animal and human health.

    INTRODUCTION

    In addition, the e-book explains how Covance has overcome the current big challenge in assessing EDs, which is how to reliably and reproducibly assay thyroid hormones and interpret the results in light of historical control data.

  • 4ENDOCRINE DISRUPTORS

    Endocrine disruption occurs when a chemical interferes with the body’s endocrine system to produce adverse effects. These adverse effects may impact development, reproduction, neurological function or the immune system of the animal.1

    THE BASICS OF ENDOCRINE DISRUPTION

    The endocrine system controls the production and release of hormones. The system is highly conserved biologically, so similar hormones are found in vertebrates and invertebrates. Consequently, a substance that disrupts the endocrine system in one species may also disrupt that of other species, although the detailed effect can vary.

    There are many different hormones that act on multiple body systems, and when considering EDs, the most extensively investigated are the estrogen, androgen, thyroid hormone and steroidogenesis pathways: these are known as EATS modalities.

  • 5ENDOCRINE DISRUPTORS

    FIGURE 1. EATS MODALITIES

    Note: EDs may also act via non-EATS modalities, such as the hypothalamus-pituitary-adrenocortical (HPA) axis, but as yet no validated and accepted assays exist to specifically monitor non-EATS effects.2

    Graphic

    Estrogens

    These are a group of female sex hormones, which

    govern the development and maintenance of female sex

    characteristics, the reproductive cycle, pregnancy, lactation

    and normal female reproductive function

    Androgens

    These are male sex hormones,which govern the development

    and maintenance of malesex characteristics and normal

    male reproductive function

    Thyroid

    The thyroid produces a rangeof hormones important

    for metabolism, growth anddevelopment, including

    thyroxine (T4) andtriiodothryonine (T3).

    The production of T3 and T4is itself controlled by the

    hormones thyrotrophin-releasinghormone (TRH) andthyroid-stimulating

    hormone (TSH)

    Steroidogenesis

    This describes the productionof steroid hormones, likeestrogens and androgens,

    from cholesterol.

    Disruption of this pathwayimpacts reproduction

    and normal development

    E A T S

  • 6ENDOCRINE DISRUPTORS

    To be classed as an ED, a chemical must meet three specific criteria:FIGURE 2. CRITERIA DEFINING AN ED

    DEFINING AN ED

    ADVERSEEFFECT

    i.e. adversity in anintact organism

    or o�spring

    ENDOCRINEACTIVITY

    The adversee�ect is a

    CONSEQUENCEof the endocrine

    activity

    This is a change in themorphology, physiology, growth, development, reproduction or life

    span of an organism, system or (sub)population that results in impairment

    of functional capacity, impairment of the

    capacity to compensate for additional stress,

    or an increase in susceptibility to other influences

    The substance has an endocrine mode of

    action – in other words, thereis a biologically plausible link

    between the endocrine activity and the adverse effect

    The substance has thecapacity to alter the function

    of the endocrine system

  • 7ENDOCRINE DISRUPTORS

    About the same time, all over the world, ecologists were starting to observe reproductive changes in a number of animal species; for example, in the Great Lakes in the USA, an almost complete reproductive failure was observed in domesticated mink, which was later ascribed to the use of polychlorinated biphenyls (PCBs), while in England, severe reproductive abnormalities were observed in fish.3

    The effects of compounds that interfere with the endocrine system were observed as long ago as the 1960s, when scientists realized that synthetic steroids could be used to manipulate the reproductive cycles of animals.

    THE LONG HISTORY OF ED EVALUATION

    Evidence of a growing environmental impact from chemicals continued to build through the 1970s and 1980s, resulting in the early 1990s in an international conference – the Wingspread Conference – being convened in the USA to review the science and debate the issues.5

    Wingspread was a turning point in the field of EDs, and the conference was, in fact, where the term ‘endocrine disruptor’ was first used.

  • 8ENDOCRINE DISRUPTORS

    FIGURE 3. THE WINGSPREAD CONFERENCE 1991

    The consensus and pressure generated by Wingspread resulted in action on EDs from the Organization for Economic Co-operation and Development (OECD), focusing on Europe, and the Environmental Protection Agency (EPA) in the US.

    As you can see from the timeline (Figure 4), it took some time for the testing frameworks and assays to be developed and validated by the EPA and OECD. Despite this void in regulation, commercial companies were exploring endocrine assays to help them get ahead of the game and answer some of the questions that were of concern to clients and the public. Covance was an early adopter of these industry-led techniques and has continued to lead the field in the adoption and validation of testing approaches from both the EPA and OECD.

    Purpose

    Integrate and evaluate findingsfrom diverse disciplines on themagnitude of the problem ofendocrine disruptors to theenvironments, establish conclusions and clarify remaining uncertainties.

    Key Finding

    We are certain of the following:A large number of man-made

    chemicals that have been releasedinto the environment, as well

    as a few natural ones, havethe potential to disrupt

    the endocrine system ofanimals, including humans.

    WINGSPREADCONFERENCE 1991

    21 internationalscientists from

    15 di�erentdisciplines

  • 9ENDOCRINE DISRUPTORS

    FIGURE 4. A TIMELINE OF KEY ED REGULATORY EVENTS AND COVANCE’S ROLE IN ED ASSAY DEVELOPMENT

    Endocrine DisruptorScreening and Testing AdvisoryCommittee (EDSTAC) establishedby the EPA

    Glaxo develop recombinant yeast screening tool for estrogen/androgen

    Covance validate each five Tier 1 EPA in vitro screening assays

    1995 1996 1997

    Endocrine DisruptorsTesting and Assessment(EDTA) task forceestablished by OECD

    1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018

    EDSP – publication of the ED testing orders for industry - the Tier 1 screening program. This covered a total of 67 chemicals

    Covance joins EUNETVAL as first UK member

    Covance validate their thyroid hormone assay

    EDSP 21: EPA announces high throughput screening programme as an alternative to Tier 1 in vivo test battery

    EPA finalizes Tier 2 testing guidelines

    Endocrine Disruptor Screening Program (EDSP) initiated – identified the ED endpoints which chemicals were to be screened for and outlined the tests required

    Conceptual Framework published by the OECD as Guidance Document 150 which provides a roadmap for ED testing

    EU publisheshazard-basedcriteria foridentifyingEDs

    OECD GuidanceDocument150 revised

    EU ED testingrequirementcomes intoforce for PPPand BPs

    Selection, development and validation of a batteryof tests needed for the ED screening programme

    Development of the ED testing and assessment approach for the OECD

    Covance adopt and use the recombinant yeast screening toolCovance labs and staff validated for using the screening assays

    Validation of an in vitro androgentranscriptional assay

    Collection of historic thyroid hormone data

    Eur

    ope

    Cov

    ance

    USA

  • 10ENDOCRINE DISRUPTORS

    The regulatory environment for testing for EDs varies between the US and Europe. In the EU, a hazard-based approach has been taken, which focuses on testing for ED potential through approaches outlined in the OECD conceptual framework. This hazard-based approach does not take into account exposure to a chemical.

    The US EPA has taken a risk-based approach, which evolved subsequently to the Tier 1 test analysis. For risk-based approaches, both the ED properties of a substance and the exposure are taken into account.

    THE CURRENT REGULATORY ENVIRONMENT FOR ENDOCRINE DISRUPTORS

    US EPA

    The US EPA uses a two-tier approach for screening pesticides, with Tier 1 focused on assessing the potential for a substance to interact with the endocrine system. Tier 2 then identifies the adverse effects associated with this interaction and establishes a quantitative relationship between the dose of the substance and the adverse effect.6

    The initial 67 substances put through the Tier 1 screening program generated a huge amount of data and made the EPA reconsider the original process and change direction to focus on a risk-based analysis. This was initiated in 2012 as the Endocrine Disruptor Screening Program (EDSP) in the 21st Century, or EDSP21, and aimed to use computational and in silico methodologies combined with high-throughput (HTP) screening to make testing more efficient and cost-effective (Figure 5).7

    The EPA’s intention in the short term is to use the in silico or HTP assays to prioritize chemicals for Tier 1 testing, with a long-term goal of replacing all the Tier 1 animal tests with in silico/HTP testing.8 The future direction of EPA screening is summarized in Table 1.

    Note on terminology The guidelines developed and accepted by the US EPA are labeled OPPTS, which stands for the Office of Prevention, Pesticides and Toxic Substances. The name changed in 2010 to the Office of Chemical Safety and Pollution Prevention or OCSPP. This name change does not otherwise affect the guidelines.5

  • 11ENDOCRINE DISRUPTORS

    FIGURE 5. THE EPA APPROACH TO ED TESTING

    EDSP21In silico/HTP

    assays

    Computational orin silico and molecularbased in vitro HTPscreening assays

    Weight of evidenceTier 1

    ScreeningBattery

    A range of complementary in vitro and in vivo assays designed to assess the potential for interaction with the endocrine system i.e. the mode of action

    Longer term the Tier 1 tests will be replaced by in silico or HTP assays

    Tier 2Testing

    Additional testing toidentify adverse eventsand dose relationship

    for adversity

    Weight of evidence

  • 12ENDOCRINE DISRUPTORS

    TABLE 1. THE FUTURE DIRECTION OF EPA SCREENING

    AR = androgen receptor; ER = estrogen receptor; STR = steroidogenesis; THY = thyroid

    ORIGINAL EDSP TIER 1 SCREENING ASSAY EDSP21 HTP SCREENING AND IN SILICO MODEL ALTERNATIVES

    IN VITRO

    ER transactivation OCSPP 890.1300ER binding OCSPP 890.1250AR binding TG OPPTS 890.1150Steroidogenesis OCSPP 890.1550Aromatase TG OPPTS 890.1200

    ToxCast ER modelToxCast ER modelAR model (future)STR model (future)STR model (future)

    IN VIVO MAMMALIAN

    Uterotrophic OCSPP 890.1600Hershberger OCSPP 890.1400Pubertal male OCSPP 890.1500Pubertal female OCSPP 890.1450

    ToxCast ER modelAR model (future)AR, STR and THY model (future)ER, STR and THY model (future)

  • 13ENDOCRINE DISRUPTORS

    FIGURE 6. THE IBER APPROACH9

    The EPA uses a risk-based approach to ED screening by combining biological information with exposure information:

    INTEGRATED BIOACTIVITY EXPOSURE RANKING (IBER): THE STRATEGY BEHIND HTP SCREENING

    The ToxCast modelingsystem is used to predictbioactivity of a substance

    The ExpoCast modelingsystem is used to determinethe exposure to a substance

    BIOACTIVITY EXPOSURE

    The amount of overlap determines the risk and prioritizes further testing

  • 14ENDOCRINE DISRUPTORS

    The EU’s approach to assessing EDs is based on the OECD conceptual framework (CF), which provides a way of organizing OECD test assays into levels related to their biological complexity and helps with the interpretation of results.

    THE EU APPROACH TO ED ASSESSMENT

    In addition, however, the European Food Safety Authority (EFSA) has also included information on the key events and postulated mode of action, and it is this combined approach that underlines the scheme used for considering lines of evidence supporting ED action.10

    Levels 1 and 2 of the CF are relevant to both mammalian and non-mammalian toxicology. Not surprisingly, there are separate Test Guidelines (TGs) for mammals and non-mammals for Levels 3, 4, and 5. Figure 7 shows how the different CF levels relate to mechanistic events at the different levels from the cell up to the whole organism or population.

    FIGURE 7. EUROPEAN SCHEME LINKING OECD CF TO KEY EVENTS AND POSTULATED ED MODE OF ACTION10

    Lines of evidence for adversity and/or endocrine activity

    In vitro mechanistic In vivo mechanistic Sensitive to butnot diagnostic of EATSEATS mediated

    Other scientific information e.g. (Q)SAR, ADME, read-across, epidemiology and field studies (OECD CF level 1)

    OECD CF level 2

    Early events

    MIE KE n KE n+1 Adverse effect

    Intermediate events Late events

    OECD CF level 3 OECD CF level 4 and 5

    Cell

    Tissue

    Organ

    Individual

    Population

  • 15ENDOCRINE DISRUPTORS

    LEVEL 1, THEREFORE, INVOLVES REVIEWING:

    ▶ The physical and chemical properties of the substance; for example, molecular weight reactivity, volatility, biodegradability

    ▶ All available (eco)toxicological data from standardized or non-standardized tests. In addition to the study data, this also includes literature searches in databases and scientific journals

    ▶ All available read-across, chemical categories, quantitative structure – activity relationship (QSAR) models and other in silico predictions, as well as absorption, distribution, metabolism and excretion (ADME) model predictions

    For many chemicals, huge amounts of data are available, so Level 1 is about reviewing and analyzing that data, looking specifically for effects from existing toxicology or ecotoxicology studies performed already in Level 4 and Level 5.

    LEVEL 1: EXISTING DATA AND NON-TEST INFORMATION

    QSAR MODELING: THE ART OF PREDICTIONQSAR models can be utilized for screening to help predict the ED activity of a substance. However, the correlation between a QSAR prediction and the actual ED effects (from Level 4 and 5 studies) of a chemical may not be very strong. The reason for such a discrepancy must be verified with additional screening from in vitro and in vivo studies, to support weight-of-evidence arguments for the presence or absence of an ED mode of action. In addition, in the absence of an ED mode of action, ED effects observed in Level 4 and Level 5 studies may result from secondary effects related to the general toxicity of the substance. ED effects are considered long-term and/or generation effects; therefore, in silico assessment for chemicals must take into account the ADME of the substance, in addition to the toxicokinetic effects.

  • 16ENDOCRINE DISRUPTORS

    Table 2 lists some of the models commonly used and the EATS modality for which they make predictions. The strategy for using these model systems is to run the test chemical in each model, then review the results to build a weight-of-evidence argument on the receptor activity by modality.

    TABLE 2. SOME QSAR SYSTEMS AND THEIR EATS MODALITIES

    THE QSAR MODELING FOR ED ACTIVITY IS AN EVOLVING AREA OF RESEARCH

    QSAR MODEL SYSTEM INSIGHT EATS MODALITY

    E A T S

    DEREK Nexus 6.0.1 Based on expert rules X X X

    VEGA NIC 1.1.4 Statistic-based model X

    OECD QSAR Toolbox 4.2Some limitations; e.g., molecular weight or functional groups

    X

    Endocrine DisruptomeFocuses on the receptors; good coverage of all modalities

    X X X X

    Danish QSAR Database Only useful if the chemical is already included X X X X

  • 17ENDOCRINE DISRUPTORS

    ALL THE INFORMATION GENERATED FROM THE LEVEL 1 DATA REVIEW AND MODELING IS ORGANIZED AND ANALYZED TO ESTABLISH IF:

    ▶ The data are sufficient to show that a substance does not meet the ED criteria

    ▶ Additional information is needed

    ▶ A mechanism-of-action analysis is required as a next step to conclude on the substance’s ED properties

    BUILDING A WEIGHT-OF-EVIDENCE ARGUMENT FOR NEXT STEPS

    AN ILLUSTRATIVE EXAMPLE FROM AN ECOTOXICOLOGY VIEWPOINT Non-primary endpoint data captured during previous in vivo studies may give insights into the possibility of ED. For example, in bird reproductive studies, the testes size at the end of the study may differ from the normal range and this indicates disruption to the endocrine system. What it does not indicate, however, is the mode of action, which could involve a variety of mechanisms related to the androgen or estrogen systems. Taking such individual pieces of information and piecing them together with expert scientific knowledge is key to crafting Level 1 assessments.

  • 18ENDOCRINE DISRUPTORS

    Table 3 lists all the OECD TGs for detecting ED chemicals currently validated and in use. Some of the tests outlined in the OECD CF overlap with the EPA Tier 1 screening battery. More information on the Level 2 mechanistic in vitro tests is provided in Table 4.

    LEVEL 2: MECHANISTIC IN VITRO TESTS

    TG NUMBER AND NAME CF LEVEL MODALITY ADDRESSED

    E A T STG 493: In Vitro Oestrogen Receptor Binding Assay 2 X

    TG 455: In Vitro Oestrogen Receptor Transactivation Assay 2 X

    TG 458: In Vitro Androgen Receptor Transactivation Assay 2 X

    TG 456: H295R Steroidogenesis Assay 2 X X X

    TG 440: Uterotrophic Bioassay 3 X

    TG 441: Hershberger Bioassay 3 X

    TG 229: Fish Short-Term Reproduction Test 3 X X X

    TG 230: Fish Screening Assay 3 X X X

    TG 231: Amphibian Metamorphosis Assay 3 X

    TG 407: 28-day Repeated Dose Toxicity Study in Rodents 4 X X

    TG 408: 90-day Repeated Dose Toxicity Study in Rodents 4 X X

    TG 414: Prenatal Developmental Toxicity Study 4 X X X X

    TG 421: Reproduction/Developmental Toxicity Screening Test 4 X X X X

    TG 422: Combined Repeated Dose Reproduction/Developmental Toxicity Screening Test 4 X X X X

    TG 426: Developmental Neurotoxicity Study 4 X X X X

    TG 451-3: Combined Chronic Toxicity/Carcinogenicity Study 4 X X X X

    TG 234: Fish Sexual Development Test 4 X X X

    TG 241: Larval Amphibian Growth and Development Assay 4 X

    TG 443: Extended One-Generation Reproductive Toxicity Study 5 X X X X

    TG 240: Medaka Extended One-Generation Reproductive Toxicity Study 5 X X X X

    TG 416: Two Generation Reproduction Toxicity Study 5 X X X X

    TABLE 3. OECD TGS, CF LEVEL AND MODALITIES1

  • 19ENDOCRINE DISRUPTORS

    TG OECD TG 455 493 458 456

    US EPA OPPTS 890.1300 890.1250 880.1150 890.1200

    Species/in vitro test system

    ER TA (human) cells expressing ERα

    Binding to rat (EPA) or human (OECD) estrogen receptor

    Binding to rat androgen receptor

    AR TA (human AR-Eco-Screen™) cell line

    Human recombinant microsomes

    Human H295R cells

    Indicative of:* E E A A S S

    Androgen receptor binding/transactivation X X

    Aromatase X

    Estrogen receptor binding/transactivation X X

    Steroidogenesis (estradiol and/or testosterone synthesis) X

    TABLE 4. LEVEL 2 IN VITRO TESTS1,5

    ER TA = estrogen receptor transcriptional activation; AR TA = androgen receptor transcriptional activation; * Based on OECD GD 150 indicative of EATS modalities

  • 20ENDOCRINE DISRUPTORS

    TABLE 5. LEVEL 3 IN VIVO MECHANISTIC TESTING1,5

    Level 3 assesses the mechanism of disruption in live animals and requires tests in rodents, fish and amphibians:

    LEVEL 3: MECHANISTIC IN VIVO TESTING

    OECD TG 440OCSPP 890.1600

    OECD TG 441OCSPP 890.1400

    Test duration 3 days 10 days

    Life stagesImmature females (after weaning and prior to puberty) or young adult females after ovariectomy

    Immature males (after weaning and prior to puberty) or young adult males after castration

    Species Rat Rat

  • 21ENDOCRINE DISRUPTORS

    TABLE 6. TGS RELEVANT TO LEVEL 4 AND 51,5

    Many of the TG studies relevant for Levels 4 and 5 have been in use for some time and have simply been updated to include or expand endpoints related to EDs. Table 6 lists the relevant OECD and EPA.

    LEVEL 4 AND 5: SOME INSIGHTS ON THE APPLICATION OF THE LATEST UPDATED TEST GUIDELINES

    TEST GUIDELINE DETAILS LEVEL

    OECD TG 407, OCSPP 870.3050 28-day Repeated Dose Toxicity Study in Rodents 4

    OECD TG 408, OCSPP 870.3100 90-day Repeated Dose Toxicity Study in Rodents 4

    OECD TG 414, OCSPP 870.3700 Prenatal Developmental Toxicity Study 4

    OECD TG 421, OCSPP 870.3550 Reproduction/Developmental Toxicity Screening Test 4

    OECD TG 422, OCSPP 870.3650 Combined Repeated Dose Reproduction/Developmental Toxicity Screening Test 4

    OECD TG 426 Developmental Neurotoxicity Study 4

    OECD TG 451-3, OCSPP 870.4100, 870.4200, 870.4300

    Combined Chronic Toxicity/Carcinogenicity Study 4

    OPPTS 890.1500, OCSPP 890.1450 Peripubertal male and female assays (rodents) Tier 1

    OECD TG 234 Fish Sexual Development Test 4

    OECD TG 241 Larval Amphibian Growth and Development Assay 4

    OECD TG 443 Extended One-Generation Reproductive Toxicity Study 5

    OECD TG 240 Medaka Extended One-Generation Reproductive Toxicity Study 5

    OECD TG 416, OCSPP 870.3800 Two Generation Reproduction Toxicity Study 5

  • 22ENDOCRINE DISRUPTORS

    TABLE 7. NEW ED ASSESSMENTS IN TGS 408 AND 414

    The most recently updated OECD TGs for CF Level 4 are TG 414, which is a prenatal developmental toxicity study in rodents, and TG 408, which is a 90-day repeated-dose toxicity study, also in rodents (see Table 7). There are some complexities that need to be considered when running these studies according to the updated guidelines.

    Many of the additional assessments requested are commonplace; however, careful study planning is required to ensure the most efficient use of study resources.

    TG 414 NEW ASSESSMENTS TG 408 NEW ASSESSMENTS

    Fetal assessments:

    ▶ Anogenital distance (AGD) in all live fetuses

    ▶ External genital observation

    Dam assessments at about 14 weeks of age:

    ▶ Measure thyroid hormone (T3, T4) and TSH levels in serum on morning of the day of necropsy

    ▶ Thyroids to be weighed and examined microscopically for all dames on Day 20 of gestation

    ▶ Thyroid histopathology

    Assessment at about 19 weeks of age:

    ▶ T3, T4 and TSH to be measured in all animals ▶ Thryoids to be weighed an dexamined microspocially for all animal ▶ High- and low-density lipoprotein (HDL and LDL) levels

    Additional optional assessments:

    ▶ Histopathology of pancreatic islets ▶ Estrous cyclicity ▶ Circulating levels of testosterone, estradiol, follicle-stimulating hormone (FSH),

    luteinizing hormone (LH)

    ▶ Enumeration of cauda epididymis sperm reserves ▶ Sperm morphology, sperm motility and count

    Major challenges:

    ▶ Accurate assessment of T3, T4 and TSH ▶ Historic control data

    Major challenges:

    ▶ Accurate assessment of T3, T4 and TSH ▶ Historic control data

  • 23ENDOCRINE DISRUPTORS

    THE BIG CHALLENGE IN ASSESSING ED ENDPOINTS: ASSESSING AND INTERPRETING THYROID HORMONES

    Many of the data collection and assay requirements for ED endpoints are straightforward to measure and interpret; however, a major challenge that is proving hard to overcome for many laboratories is the reliable measurement of thyroid hormone levels and, perhaps more importantly, an informed interpretation of any results in the context of historical data.

    Thyroid endpoints required by regulators include the assessment of TSH, T4 and T3 in serum. These hormones are essential for normal growth and development, but they are very different types of molecules (see Figure 8). TSH is a large glycoprotein, which lends itself to immunoassays, whereas T4 and T3 are smaller molecules, often occurring at very low levels and difficult to detect using immunoassay.

    FIGURE 8. TSH AND THYROID HORMONES11

    Thyroid stimulatinghormone (TSH)

    Thyroid hormones

    T4 orthyroxine

    Thyroid hormones have a range of physiological effects on metabolism, growth and development. Normal levels of thyroid hormone are essential to the development of the fetal and neonatal brain.

    TSH controls the releaseof thyroid hormones

    from the thyroid gland.

    Thyroid hormones arederivatives of the amino

    acid tyrosine boundcovalently to iodine.

    The main thyroidhormone secreted

    by the thyroid is T4,although T3 is

    biologically more active.

    There are other inactivemolecules produced

    e.g. reverse T3.

    TSH is a largeglycoprotein.

    T3 ortriiodothyronine

    rT3 orreverse rT3

  • 24ENDOCRINE DISRUPTORS

    THE MAIN ISSUES WITH THYROID HORMONE ASSESSMENT ARE:

    ▶ Sensitivity – can you detect the thyroid hormones across the whole range of concentrations that you may need?

    ▶ Selectivity – are you sure you are measuring the right molecule with minimal interference from other substances?

    ▶ Stability – will your assay be impacted by long-term storage of samples (long-term storage can occur in studies that use triggers to initiate additional analysis)?

    ▶ Reproducibility – is your method sufficiently robust so your results can be replicated intra- and inter-laboratory?

    ▶ Historic control data – can you put your findings in the context of natural variability by looking at historic control data?

    THE ISSUES WITH THYROID HORMONE ASSESSMENT

    SENSITIVITY There are two main challenges with sensitivity: first, knowing how low a concentration you may need to detect and, second, having a wide enough dynamic range that your assay can detect and quantify both low and high thyroid hormone concentrations. This is necessary because some studies require measurement of T4 and T3 in fetuses and pups at Day 4 and Day 13, as well as in adults, so there could be a large variation in blood levels. To be able to estimate the range you will need to test for, you need to understand what the normal biological serum concentration range for T4 and T3 is and extend the range to cover down-regulation or up-regulation of the thyroid levels to sufficiently assess a drug- or chemical-induced effect. The method also needs to consider animal welfare and must be efficient in minimizing the amount of sample needed to provide reliable results.

    SELECTIVITY The assay must be able to differentiate between T4 and T3 and have no interference from other (inactive) thyroid hormones that are produced by the thyroid, such as reverse T3 (rT3) (Figure 8), or by breakdown of the main hormones or other endogenous entities.

  • 25ENDOCRINE DISRUPTORS

    STABILITYIn some cases, testing of certain samples is only initiated when triggered by another analysis; for example, for OECD TGs 421 and 422, Day 13 pup and adult male samples are analyzed for T4, and if thyroid effects are observed, then T4 assessment of the Day 4 pup and dam samples (or TSH for the Day 13 and adult male samples) will normally need to be triggered for analysis to confirm the effect.

    However, the review and decision process for the triggered analysis (normally by protocol amendment) will mean that the Day 4 samples could potentially have been in storage for over six months; therefore, a sufficient stability period of the thyroid hormones in serum samples for the relevant assay must be established prior to reporting data, in order to assure the integrity of the thyroid hormone data generated from these stored samples.

    REPRODUCIBILITYAny assay must be reproducible, both in your own laboratory and inter-company laboratories, if possible, as this demonstrates the reliability and robustness of the methodology.

    HISTORIC CONTROL DATAHistoric control data are important, as they provide a rich characterization of the normal background variability that can be found in T3 and T4 concentrations. Such insight is essential when drawing conclusions from studies, as you can see what may be the result of normal variation and what may be a clear thyroid effect.

  • 26ENDOCRINE DISRUPTORS

    Covance has pioneered an in-house method that is sensitive and accurate for assessing T4 and T3 in rat dams and fetuses, down to low picogram per milliliter concentrations. The technique involves the use of ultra high-pressure liquid chromatography coupled with tandem mass spectrometry12 (UHPLC-MS/MS) and has been widely applied to studies requiring assessment of thyroid function such as OECD TGs 421, 422 and 443, as well as some US EPA studies. Figure 9 explains how this assay overcomes the challenges of thyroid hormone assessment and delivers reliable and robust T3 and T4 endpoint data.

    THE COVANCE THYROID HORMONE ASSAY

    Sensitivity Covance established the required concentration range needed for T4 and T3 assessment by performing a propylthiouracil-positive control pre- and post-natal developmental thyroid study in rat dams, pups and fetuses. The study demonstrated that TSH levels could be quantified in serum from pregnant female rats and fetuses using a validated immunoassay method and although quantifiable levels of T3 and T4 could be detected in pregnant females, they could not be detected in fetuses or pups on Days 4 and 13. This showed that detection of low picogram per milliliter concentrations was needed, which was achieved with an optimized UHPLC-MS/MS technique.

    The UHPLC-MS/MS assay can measure rat serum concentrations of:

    ▶ T3 = 5–1,500 pg/mL ▶ T4 = 70–70,000 pg/mL.

    The sample volume is 50 μL, which is sufficient to detect and quantify concentrations in control samples and test samples that are subject to increases or reductions in hormone levels. This minimizes the use of animals and ensures efficient resource use.

    Selectivity Covance has assessed and assay for selectivity for T4 and T3. This has been confirmed through chromatographic assessment of total T3 and T4, and the detection of the inactive rT3, which is sufficiently chromatographically separated from both T3 and T4.

    Stability The assay has been tested for its stability under a number of different conditions that may occur during studies, including for: ▶ Freeze/thaw stability ▶ Short-term stability at room temperature ▶ Frozen storage stability, at both –20°C and –70°C ▶ Long-term frozen storage stability for over a year.

    Reproducibility The technique has been successfully validated for: ▶ Linearity ▶ Intra- and inter-batch precision and accuracy ▶ Recovery and dilution integrity.

    Covance developed this technique in its UK laboratory and it has been validated at its Spanish site, providing capacity and flexibility when running assays within regulatory timelines.

    Historical data Since its validation in 2016, Covance has run approximately 10–15 studies using this assay every month, allowing a wealth of historic control data to be amassed. This substantial dataset, combined with the experience of the scientists using it, means that Covance is well positioned to make informed interpretations of study results to support regulatory arguments.

    FIGURE 9. HOW COVANCE HAS OVERCOME THE CHALLENGES OF THYROID HORMONE ASSESSMENT12

  • 27ENDOCRINE DISRUPTORS

    SUMMARY

    The drive to address ED started in the 1960s; however, given the complexity of the endocrine system, development of validated and accepted tests for EDs have been slow to come into regulatory effect.

    The regulatory approach varies across jurisdictions, with the US EPA taking a risk-based approach that takes into account both the ED properties of a substance and the exposure to it, while the EU has taken a hazard-based approach, which focuses on testing for ED potential, contained in a conceptual framework.

    There is overlap in the testing approaches used by both the EPA and EU, with an increasing reliance on in silico methodologies to supply insights based on modeling of existing data. This field is still evolving, however, and regulators are still reliant on in vitro and in vivo studies to provide answers on some of the more challenging reproductive and developmental endpoints.

    One current major challenge is the assessment of thyroid hormones, which is required for Level 4 and 5 in vivo studies. Covance’s validated UHPLC-MS/MS assay for T4 and T3 has overcome the widespread challenges that have dogged this research and, thanks to the extensive historic control data amassed over multiple studies.

  • 28ENDOCRINE DISRUPTORS

    1 OECD. OECD work on endocrine disrupting chemicals. March 2018. Available from http://oe.cd/ endocrine-disrupters

    2 Day et al. 2018. Toxicology Letters 296:10-22.

    3 Schug et al. 2016. Molecular Endocrinology 30: 833–847.

    4 Wingspread: Chemically-induced alterations in sexual development: the wildlife/human connection. 1992. https://endocrinedisruption.org/ assets/media/documents/ wingspread_consensus_statement.pdf

    5 OCSPP Harmonized Test Guidelines - Master List. February 2018. Available at: https://www.epa.gov/sites/ production/files/2018-02/documents/ ocspp_test_guidelines_master_list_ february_2018.pdf

    REFERENCES

    6 EPA. Endocrine Disruptor Screening Program (EDSP) Overview. Available at https://www.epa.gov/endocrine- disruption/endocrine-disruptor- screening-program-edsp-overview#tab-2

    7 EPA. Endocrine Disruptor Screening Program (EDSP) in the 21st Century. Available at https://www.epa.gov/ endocrine-disruption/endocrine- disruptor-screening-program-edsp- 21st-century

    8 EPA. EDSP21 Work Plan. 2011 https://www.epa.gov/sites/production/ files/2015-07/documents/edsp21_work_ plan_summary_overview_final.pdf

    9 EPA. Dix D. Endocrine Disruptor Screening Program. EPA’s Computational Toxicology Communities of Practice. 2015. https://www.epa. gov/sites/production/files/2015-04/ documents/edsp_dix_ord_ communities_of_practice_ 04_23_15_f.pdf

    10 EFSA.Guidancefortheidentification of endocrine disruptors in the context of Regulations (EU) No 528/2012 and (EC) No 1107/2009. EFSA Journal 2018;16(6):5311. Available from: https://efsa.onlinelibrary.wiley.com/doi/ epdf/10.2903/j.efsa.2018.5311

    11 Chemistry of Thyroid Hormones. Vivo pathophysiology. Colorado State University. Available at http://www.vivo. colostate.edu/hbooks/pathphys/ endocrine/thyroid/chem.html

    12 Diaram, S. Stability evaluation of thyroid hormones 3,3,5’-Triiodo- L-Thyronine (T3) and Thyroxine (T4) in rat serum at low pg/mL concentrations using liquid chromatography coupled with tandem mass spectrometry (LC-MS/ MS). The Toxicologist: Late-Breaking Supplement 2018, Abstract number: 3627

  • 29ENDOCRINE DISRUPTORS

    Learn more about our crop protection solutions at

    www.covance.com/crop-protectionCovance is the drug, medical device and diagnostics business

    segment of LabCorp, a leading global life sciences company. COVANCE is a registered trademark and the marketing name

    for Covance Inc. and its subsidiaries around the world.

    The Americas +1.888.COVANCE (+1.888.268.2623) +1.609.452.4440

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