EDSPwebinar 4: The amphibian metamorphosis assay

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Information about EDSPwebinar 4: The amphibian metamorphosis assay
Education

Published on February 4, 2014

Author: JimBRegan

Source: slideshare.net

Description

Amphibians are considered as being exceptionally vulnerable to endocrine disrupters, as they exhibit obvious effects on limb development and metamorphosis in wild populations following exposure. They have a high degree of sensitivity, whether in the tadpole stage or as adults, and respond to seemingly minimal changes in the environment.

This webinar discusses the metamorphosis assay, the selection of Xenopus Laevis, some aspects of the study design and where improvements could be made. We also discuss what the study means for you and how you can ensure that your contractor has all they need to conduct the study successfully.

More info at http://www.huntingdon.com/Chemical/Endocrinedisruptorscreeningprogram/Webinars

Endocrine Disruptor Screening Program Webinar week 20-23 January 2014 www.huntingdon.com

Amphibian metamorphosis assay for the EPA’s EDSP Carole Jenkins BSc www.huntingdon.com

Amphibian Metamorphosis Assay Carole A Jenkins 23 January 2014 www.huntingdon.com

Definitions European Commission (EC) asked the Scientific Committee (SC) of European Food Safety Authority (EFSA) to review information relating to the testing and assessment of Endocrine Active Substances (EAS) and Endocrine Disrupters (ED) 1  Endocrine Disrupters (ED) “There must be reasonable evidence for a biologically plausible causal relationship between the endocrine activity and the induced adverse effect(s) seen in an intact organism or a (sub)population for a substance to be identified as an ED.” ie.     1 adverse effect endocrine activity relationship between the two Published in EFSA Journal 2013;11(3):3132. www.huntingdon.com

Definitions  “Endocrine Active Substance (EAS) = as a substance having the inherent ability to interact or interfere with one or more components of the endocrine system resulting in a biological effect, but need not necessarily cause adverse effects”. www.huntingdon.com

Regulatory requirements  EU    Plant Protection Products Regulation (1107/2009) Biocidal Products Regulation (528/2012) REACH Regulation (1907/2006) => Substance with endocrine disrupting properties are subject to evaluation and have special properties distinct from other chemicals but   Currently no agreement on the guidance on how to identify and evaluate endocrine activity and disruption Member States have been preparing schemes and evolving approaches to define the issues and make recommendations aim  Same criteria to apply to all EU legislation www.huntingdon.com

OECD Conceptual Framework  Framework for the testing and assessment of Endocrine Disrupters (revised 2011)      Level 1 = Existing data and non-test information Level 2 = In vitro assays providing data about selected endocrine mechanism(s)/pathway(s) Level 3 = In vivo assays providing data about selected endocrine mechanism(s)/pathway(s) => AMA TG 231 Level 4 = In vivo assays providing data on adverse effects on endocrine relevant endpoint Level 5 = In vivo assays providing more comprehensive data on adverse effects on endocrine relevant endpoints over more extensive parts of the life cycle of the organisms www.huntingdon.com

OECD Conceptual Framework Non mammalian toxicology Level 3 In vivo assays providing data about selected endocrine mechanism(s)/ pathway(s)1 Xenopus embryo thyroid signalling assay (when/if TG is available) Amphibian Metamorphosis assay (OECD TG 231) – (anti-)Thyroid Fish Reproductive Screening Assay (OECD TG 229) – estrogens, androgens, aromatose inhibitors, Fish Screening Assay (OECD TG 230) -– estrogens, androgens, anit-androgens, aromatose inhibitors, Androgenized female stickleback screen (GD 140) www.huntingdon.com

EDSP Progam  Tier 1    Screening to identify substances that have the potential to interact with the Estrogen, Androgen or Thyroid System (EATS) in vitro & in vivo screens = 11 assays  Amphibian Metamorphosis (Frog) – 890.1100 Tier 2  Testing – longer-term / multi-generational studies www.huntingdon.com

Thyroid Hormonal System  Hypothalamus-Pituitary-Thyroid (HPT) axis  controls metabolic processes in the body        in fish   thermo-regulation generation of energy growth development of the central nervous system control of the cardio-vascular system (heart beat) reproduction smoltification in amphibians  larval development & metamorphosis www.huntingdon.com

Metamorphosis in Amphibians  Metamorphosis is the most dramatic example of extensive morphological, biochemical and cellular changes occurring during postembryonic development  Amphibian metamorphosis is a thyroid-dependent process which responds to substances active within the hypothalamic-pituitarythyroid (HPT) axis  Thyroid Hormones (TH) :T3 (triiodothyronine) and T4 (Thyroxine)  Temperature (rate) and iodine dependant (to synthesis TH) www.huntingdon.com

Amphibian Metamorphosis Assay  OECD TG 231 adopted 7 September 2009  The Amphibian Metamorphosis Assay (AMA) is a screening assay intended to empirically identify substances which may interfere with the normal function of the hypothalamic-pituitary-thyroid (HPT) axis.  The AMA represents a generalized vertebrate model to the extent that it is based on the conserved structures and functions of the HPT axis.  Amphibian Metamorphosis is a well-studied, thyroid-dependent process. www.huntingdon.com

Test species  Xenopus laevis, African Clawed Frog      Name → small, black curved claws on inner three toes of hind feet Found → stagnant ditches and lakes in the southern areas of the African continent, ranging to Nigeria and Sudan Entirely aquatic, absorb oxygen through skin and rise to the water surface to breathe Live for up to 25 years → sexually mature at ca.1 year in males and 2 years in females Nostrils on top of head and no tongue www.huntingdon.com

Adults  Laboratory conditions      Group housed in same sex tanks (15-20 L) Quiet secluded environment hide in pipes and under lily pads 12:12 light cycle at 18 - 22°C Recirculating system with UV, mechanical and biological filtration to maintain water quality Feed three times a week - varied diet of specially developed pellets, frozen bloodworm and live earthworms www.huntingdon.com

Adults Each adult has a unique pigmentation pattern - used for identification in the laboratory Males = 100 g, more streamlined body shape and have black nuptial pads on the forearms Females = up to 300g, round body shape, obvious ovipositor www.huntingdon.com

Breeding - induction and egg production      Breeding is induced by injecting the adults with Human Chorionic Gonadotrophin (hCG) the evening before eggs are required Set up 3 to 5 pairs for breeding Male and female placed into each breeding tank - perforated false bottom to allow fertilised egg masses to sink to bottom Females produce between 1000 - 5000 eggs Need >1500 eggs from a single spawn www.huntingdon.com

Pre-exposure - larval development      Each spawn is transferred to a clean tank, held under static conditions 12:12 light:dark cycle at 22±1°C 4 days after spawn, best hatch is selected Transfer 800 tadpoles to hatching tanks, maintained using a flow through system: flows at 50 mL/minute per 100 tadpoles Herbivores -> fed several times daily initially on algal suspension (Spirulina), then as they develop, change to Sera Micron®, and gradually increase the ration Day 1 www.huntingdon.com Day 4

Stages of development  Stages - development of Xenopus was classified by Nieuwkoop and Faber1 and is used worldwide to determine the progression of the embryo  Metamorphosis → before stage 46 = no need for thyroid hormones = tadpole → stage 46 to 53 (pre-metamorphosis) = hind limb visible → stage 57/58 (post-metamorphosis) = front limbs visible → stage 66 (climax) = tail and gills absorbed = froglet  AMA covers the stages from 51 to 60  Tadpoles must reach stage 51 within 17 days post-fertilisation for use in the study 1 Nieuwkoop, P. D., and Faber, J. (1994). Normal Table of Xenopus laevis. Garland Publishing, New York. www.huntingdon.com

Stages of development Feeding begins Day 7 Exposure begins www.huntingdon.com Optimal Day 21 stage

AMA – test design      Duration of 21 days (controls from stage 51 to 60) Minimum 3 test concentration plus control (s) with 4 replicates and 20 tadpoles/vessel Concentrations separated by factor between 0.1 (max) to 0.33 (min) over at least one order of magnitude Highest test level = maximum tolerated concentration (MTC; 10% acute mortality), limit of solubility or 100 mg/L; whichever is lowest if no relevant data, range finding test is recommended    wide spaced concentrations 1 replicate/concentration with 10 tadpoles 7 to 14 days duration www.huntingdon.com

AMA – test design     Flow- through exposure regime preferred Avoid use of solvents 22±1°C with 12:12 hour light cycle at 600-2000 lux. Diluent water = natural water or dechlorinated tap water;    pH 6.5-8.5 and D.O.> 40% ASV and hardness 50-180 mg/L as CaCO3 and iodide 0.5-10 µg/L characterisation data for supply water Analytical verification of exposure levels  validated method, LOD / LOA www.huntingdon.com

AMA – test design  Day 0       Tadpoles pooled and individually staged = 51 Measure whole body length of a sample of 20 tadpoles ± 3 mm (mean: 24-28 mm for stage 51) Verify test concentrations achieved Water quality in all vessels = temperature, dissolved oxygen, pH Water quality in control(s), low and high concentrations = hardness, alkalinity and TOC Randomly distributed to control & test vessels : 20 in each www.huntingdon.com

AMA – test design  Daily   Checks on performance of dosing systems & temperature in 2 vessels Observation of tadpoles      mortality sub-lethal = morphological & behavioural effects Feed = twice/daily on Sera Micron (weight per animal), increased during the test: 30 to 80 mg/animal/day Cleaning = twice daily ca. 1 hours after feeding Weekly    Water quality in all vessels = temperature, dissolved oxygen & pH Water quality in control, low & high concentrations = hardness Chemical analysis – in each vessel (optional, stock solutions) www.huntingdon.com

AMA – test design  Apparatus   Continuous flow Dilution of a concentrated stock solution at each level     aqueous = 1:10 solvent = 100 µL/L; 20 µL/L Delivered at 25 mL/minute Vessels = glass (9 L), 4 L medium; depth of 10-15 cm www.huntingdon.com

Assessments  Day 7  5 tadpoles/vessel – euthanised, measured and discarded      wet weight (mm) developmental stage snout-vent length (SVL) hind limb length (HLL; left) Day 21  Remaining tadpoles/vessel – euthanised, measured and placed in fixative and retained   as for Day 7 plus select 5 tadpoles/vessel for thyroid gland histology www.huntingdon.com

SVL and HLL Measurements Scale marker = 0.1cm www.huntingdon.com

Measurements  All animals = > adverse effect   Wet weight (mm) Developmental stage against median stage of control(s)        advanced/accelerated: > controls on D.7 & 21 for HL & SVL delayed: < controls (antagonistic) but no signs of overt toxicity asynchronous: disruption of the timing of the development of different tissues in a single animal but tissues are not abnormal = unable to stage unaffected: same as controls Snout-vent length (SVL; 0.1 mm) Hind limb length (HLL; 0.1 mm) – normalised by SVL 5 animals/vessel = > cause (endocrine activity?)  histopathology assessment of thyroid Relationship between the endocrine activity and the induced adverse effect(s) www.huntingdon.com

Day 7 - Thyroxine Body Weight Stage 0.700 60 59 58 57 56 55 54 53 52 51 50 0.600 p = <0.05* 0.500 p = <0.001*** p = <0.001*** (g) p = <0.001*** 0.400 0.300 0.200 0.100 0.000 CONTROL 0.25 ug/L 0.5 ug/L 1.0 ug/L 2.0 ug/L CONTROL 0.5 ug/L 1.0 ug/L 2.0 ug/L Hind Limb Length Snout-Vent Length 600 2.00 500 Length (um) 2.10 Length (cm) 0.25 ug/L 1.90 1.80 p = <0.001*** 400 300 200 1.70 100 1.60 0 CONTROL 0.25 ug/L 0.5 ug/L www.huntingdon.com 1.0 ug/L 2.0 ug/L CONTROL 0.25 ug/L 0.5 ug/L 1.0 ug/L 2.0 ug/L

Day 21- Thyroxine Stage Body Weight 65 2.00 p = <0.05* 64 1.50 p = <0.001*** 62 1.00 p = <0.001*** 61 p = <0.001*** (g) 63 60 0.50 59 58 0.00 CONTROL 0.25 ug/L 0.5 ug/L 1.0 ug/L 2.0 ug/L CONTROL 0.5 ug/L 1.0 ug/L 2.0 ug/L Hind Limb Length Snout-Vent Length 2.30 2.50 p = <0.01** 2.20 2.30 p = <0.05* 2.20 p = <0.001*** 2.10 2.00 Length (cm) 2.40 Length (cm) 0.25 ug/L 2.10 2.00 1.90 1.80 1.90 CONTROL 0.25 ug/L 0.5 ug/L www.huntingdon.com 1.0 ug/L 2.0 ug/L 1.70 CONTROL 0.25 ug/L 0.5 ug/L 1.0 ug/L 2.0 ug/L

Results - Thyroxine Day 21: 2.0 µg/L Thyroxine Scale marker = 0.1cm Day 21: Control www.huntingdon.com

Day 21 - Sodium perchlorate p < 0.05* p <0.05* www.huntingdon.com

Day 21 – Sodium perchlorate Control Treatment Scale marker = 0.1cm www.huntingdon.com

Histological assessment  Thyroid gland is located between the eyes, 200-250 µm length  Fixing of samples    10% neutral buffered formalin decapitated to provide the head tissue containing lower jaw Tissue sections      Locate Thyroid gland Discard first 25 to 30 µm Five 4-5 µm step sections taken ca. 25-30 µm apart from mid-region (widest) Stained with haematoxylin & eosin Viewed using light microscopy www.huntingdon.com

Thyroid gland during metamorphosis development of thyroid gland Toxicologic Pathology, 37: 415-424, 2009; K. Christiana Grim et al www.huntingdon.com thyroid gland

Thyroid histopathology  Diagnostic criteria are:     thyroid gland : atrophy / hypertrophy (decrease/increase in gland size) follicular cell : hypertrophy (change in cell shape – monitor number of tall columnar cells) follicular cell : hyperplasia (cell crowding, stratification or papillary infolding) Other (qualitative) : colloid quality, follicular lumen area and follicular cell height/shape Thyroid Histopathology Assessments for the Amphibian Metamorphosis Assay to Detect Thyroid-active Substances; Toxicologic Pathology, 37: 415-424, 2009; K. Christiana Grim et al www.huntingdon.com

Thyroid histopathology  4 severity grades for each criteria      0 = none to minimal (<20% effect) 1 = mild or slight (30 to 50% effect) 2 = moderate (60 to 80% effect) 3 = severe (>80% effect) Histological analysis is required when   no significant mortality or adverse effects no evidence of morphological or developmental delay www.huntingdon.com

Xenopus AMA Validation Day 21: 2.0 µg/L Thyroxine Day 21: Control x 10 x 100 www.huntingdon.com

Results – Day 21 Thyroxine www.huntingdon.com

Results – Sodium perchlorate Summary of treatment related findings in the thyroids for animals exposed to sodium perchlorate killed after 21 Days Group 1 2 3 4 5 0 65 125 250 500 Test concentration (g/L) Thyroid gland hypertrophy Minimal Slight Moderate Total 0 0 0 0 7 1 0 8 8 4 0 12 7 10 0 17 3 8 8 19 Minimal Slight Moderate Total 0 0 0 0 7 1 0 8 7 6 0 13 2 13 3 18 1 11 8 20 Minimal Slight Moderate Total 0 0 0 0 5 0 0 5 8 3 0 11 12 3 0 15 6 12 1 19 Minimal Slight Moderate Total 0 0 0 0 7 1 0 8 8 4 1 13 7 10 2 19 2 8 7 17 Apoptosis of follicular epithelial cells Minimal 0 1 1 3 8 Accumulation of eosinophilic material – Follicular cells Minimal 0 0 1 2 4 Number of animals examined 20 20 20 19 20 Follicular cell hypertrophy Follicular cell hyperplasia Reduced colloid www.huntingdon.com

Results – Sodium perchlorate www.huntingdon.com

Acceptance criteria     Measured concentrations: ≤ 20% CoV Mortality: ≤10% in control(s) / 2 tadpoles per vessel Development stage in controls: at least stage 57 on Day 21 and 10th & 90th percentile not differ by > 4 stages Water quality:      D.O. = >40% ASV; 22±1°C and 0.5°C/vessel or group pH = 6.5-8.5 ±0.5 unit/vessel or group Test groups: ≥2 with no overt toxicity Replicates: ≤2 compromised across the test Solvent: no statistical differences from water control www.huntingdon.com

Stengths/weaknesses:  Strengths    Clearly defined endpoints In-life phase is “relatively easy” to maintain once set up Weaknesses     Animal wastage – large numbers required Techniques - require skill & precision eg. egg production, staging, histopathology Sampling days - very labour intensive; large numbers of animals involved Feeding – excess food (developmental problems) www.huntingdon.com

Improvements      Pre-exposure husbandry techniques – temperature, feeding, cleaning Feeding regime in test – refined to minimise excess Animal processing – standardisation & automation Assessment techniques – standardisation & training Histopathology – processing & standardisation www.huntingdon.com

Conclusions  AMA is a good screening assay [but due to the diverse role of the thyroid gland we cannot assume that the effects observed are indicative of a substance with a endocrine disrupting properties without evidence from other assays] www.huntingdon.com

 Thank you for listening www.huntingdon.com

HLS EDSP expert team      Ephi Gur – Team lead and Regulatory Bob Parker – Toxicology Will Davies – Toxicology John Carter – In vitro technologies Carole Jenkins – Aquatic toxicology  Contact via me  reganj@ukorg.huntingdon.com  +44 (0) 1480 892031 www.huntingdon.com

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