edspwebinar 4: the amphibian metamorphosis assay
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/WebinarsTRANSCRIPT
www.huntingdon.com
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
Carole A Jenkins
23 January 2014
Amphibian Metamorphosis
Assay
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.
adverse effect
endocrine activity
relationship between the two
1 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
thermo-regulation
generation of energy
growth
development of the central nervous system
control of the cardio-vascular system (heart beat)
reproduction
in fish
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-pituitary-
thyroid (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
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
Test species
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
Females = up to 300g, round
body shape, obvious ovipositor
Males = 100 g, more streamlined
body shape and have black
nuptial pads on the forearms
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 4 Day 1
www.huntingdon.com
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
Exposure begins
Day 7 Optimal Day 21 stage
www.huntingdon.com
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
50
51
52
53
54
55
56
57
58
59
60
CONTROL 0.25 ug/L 0.5 ug/L 1.0 ug/L 2.0 ug/L
Stage
p = <0.001***
p = <0.001***
0.000
0.100
0.200
0.300
0.400
0.500
0.600
0.700
CONTROL 0.25 ug/L 0.5 ug/L 1.0 ug/L 2.0 ug/L
(g)
Body Weight
p = <0.001***
p = <0.05*
1.60
1.70
1.80
1.90
2.00
2.10
CONTROL 0.25 ug/L 0.5 ug/L 1.0 ug/L 2.0 ug/L
Len
gth
(cm
)
Snout-Vent Length
0
100
200
300
400
500
600
CONTROL 0.25 ug/L 0.5 ug/L 1.0 ug/L 2.0 ug/L
Len
gth
(u
m)
Hind Limb Length
p = <0.001***
www.huntingdon.com
Day 21- Thyroxine
58
59
60
61
62
63
64
65
CONTROL 0.25 ug/L 0.5 ug/L 1.0 ug/L 2.0 ug/L
Stage
p = <0.001***
p = <0.001***
0.00
0.50
1.00
1.50
2.00
CONTROL 0.25 ug/L 0.5 ug/L 1.0 ug/L 2.0 ug/L
(g)
Body Weight
p = <0.001***
p = <0.05*
1.90
2.00
2.10
2.20
2.30
2.40
2.50
CONTROL 0.25 ug/L 0.5 ug/L 1.0 ug/L 2.0 ug/L
Len
gth
(cm
)
Snout-Vent Length
p = <0.001***
p = <0.05*
1.70
1.80
1.90
2.00
2.10
2.20
2.30
CONTROL 0.25 ug/L 0.5 ug/L 1.0 ug/L 2.0 ug/L
Len
gth
(cm
)
Hind Limb Length
p = <0.01**
www.huntingdon.com
Results - Thyroxine
Day 21: Control
Day 21: 2.0 µg/L Thyroxine
Scale marker = 0.1cm
www.huntingdon.com
Day 21 - Sodium perchlorate
p <0.05*
p < 0.05*
www.huntingdon.com
Day 21 – Sodium perchlorate
Scale marker = 0.1cm
Control
Treatment
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 thyroid gland
Toxicologic Pathology, 37: 415-424, 2009; K. Christiana Grim et al
www.huntingdon.com
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
Test concentration (g/L) 0 65 125 250 500
Thyroid gland hypertrophy
Minimal 0 7 8 7 3
Slight 0 1 4 10 8
Moderate 0 0 0 0 8
Total 0 8 12 17 19
Follicular cell hypertrophy
Minimal 0 7 7 2 1
Slight 0 1 6 13 11
Moderate 0 0 0 3 8
Total 0 8 13 18 20
Follicular cell hyperplasia
Minimal 0 5 8 12 6
Slight 0 0 3 3 12
Moderate 0 0 0 0 1
Total 0 5 11 15 19
Reduced colloid
Minimal 0 7 8 7 2
Slight 0 1 4 10 8
Moderate 0 0 1 2 7
Total 0 8 13 19 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
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
+44 (0) 1480 892031