Amphibian Metamorphosis Assay (AMA)

OCSPP 890.1100

Catherine B. Aubee, MPA

Environmental Fate and Effects Division

Office of Chemical Safety and Pollution Prevention

U.S. Environmental Protection Agency
Overview

Assay Purpose and Design

Endpoints

Validity Elements and Performance Criteria

Observations from Case Study Chemicals

2
Assay Purpose

Screen for potential interaction with the 

hypothalamic-pituitary-thyroid (HPT) axis,

using frog metamorphosis as a model system

Results will be ultimately be considered in 

concert with other results from the Tier 1 

battery

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Study Design

(per guideline recommendations)

21-day aqueous exposure

Day 0: NF stage 51 X. laevis tadpoles

 ≤17 days post-fertilization (dpf)

≥3 concentrations (plus control/s)

≥4 replicates each

≥20 tadpoles per replicate

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X. laevis at NF Stage 51

5

Image: Hind limb morphology of 

Xenopus laevis at NF Stage 51 

(AMA test initiation). Image in 

OCSPP 890.1100 test guideline; 

original source unknown.
Summary Data in the DER

Parameter Values (Day 7 and Day 21)

Mortality sample size, number and % mortality

Nieuwkoop-Faber (NF) 

developmental stage

sample size, treatment median, and number of 

tadpoles unable to stage → asynchronous 

development

Hind limb length (HLL) sample size, treatment mean, std. dev., and HLL 

normalized to SVL

SVL and body weight sample size, treatment mean, std. dev.

6

Parameter Values (Day 21)

Thyroid histopathology for each diagnostic observation: sample size 

and incidence, for each severity grade 0-3

Clinical signs sample size and incidence by type at test 

termination, unless otherwise specified
Validity Elements

7

Element Reference Value(s)

Mortality 

(each treatment, including controls)

<10%

Mortality (each replicate)

 If positive for thyroid interaction

 If negative for thyroid interaction

<2 tadpoles/replicate (<10%)

<3 tadpoles/replicate (<15%)

Uncompromised treatment levels

(all 4 replicates available for analysis)

>2, in addition to controls

Treatment levels without overt toxicity 

(mortality)

>2, in addition to controls
General Performance Criteria

8

Criterion Reference Value(s)

Measured test concentrations CV <20% over 21 days

Control mortality <2 tadpoles/each replicate (<10%)

Median NF stage in controls at test 

termination >57

Difference between 10th and 90th

percentile of NF stage distribution in 

controls at test termination

<4 stages

Dissolved oxygen >40% air saturation

pH 6.5–8.5 (<0.5 difference between 

replicates, treatments)

Water temperature 22±1°C (<0.5°C difference between 

replicates, treatments)
Performance Across Studies

9

0 3 6 9 12 15 18 

Control and treatment mortality ≤ 10%

Replicate mortality ≤ 10%

Replicate mortality ≤ 15%

Uncompromised treatments ≥ 2

Treatments without overt toxicity ≥ 2

Test concentration CV ≤ 20% over 21 days

Median Day 21 NF stage in controls ≥ 57

Control Day 21 NF stage (10th - 90th %ile) ≤ 4 ...

Dissolved oxygen ≥ 40%

pH 6.5-8.5 (≤ 0.5 difference)

Water temperature 22±1°C (≤0.5°C difference)

Met Not Met Not reported 

Figure. The majority of AMA study submissions reviewed to date have met the

 validity elements and performance criteria outlined in the test guideline.
Day 21 NF Stage: A Balancing Act

Minimum recommended stage: NF 57 (median)

Late stage tadpole exclusion: >NF 60 

(individuals) 

Some labs reducing feed by 50% to minimize late 

stage 
Spinal Curvature (“Bent Tail”)

Color Key: Prevalence Up to 25% Up to 50% Up to 100%

Lab Test 

Chemical Feeding Add’l Sourcesof Iodide

Prevalence of Spinal 

Curvature (%)

ID ID Product Reduced

Iodide 

(ug/g)

Wateri

(ug/L) Supplem Control(s) Trtmnts

2 O Sera Micron® No 54 <50 No NR NR

E 40 <10 NR NR

I 40 <10 “few” “few”

N Yes 54 <50 NR 17 17-29

J No 40 7 No 27 8-19

F 50 <50 2-5 2-13

3 M Nasco Frog 

Brittle®

Yes 48 

(Nasco)

1.2 No 29 16-26

5 H Sera Micron® No 40 3.6 Yes 22-28 13-23

6 C Sera Micron® Yes NR 4.5 No 43-53 36-40

U Yes 74 10-59

P No 66 48-98

B Yes 74-87 60-92

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Lab Test 

Chemical Feeding Add’l Sourcesof Iodide

Prevalence of Spinal 

Curvature (%)

ID ID Product Reduced

Iodide 

(ug/g)

Wateri

(ug/L) Supplem Control(s) Trtmnts

9 V Sera Micron® No NR 13 No 5-25 5-25

S 13 NR NR

10 A Xenopus 

Express®

No NR 7 No 12 17-29

Q 7.4 41 20-37

K Yes 7.8 41 30-49

G No 7 “several” “several”

Spinal Curvature (“Bent Tail”)

Color Key: Prevalence Up to 25% Up to 50% Up to 100%

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Thyroid Histopathology

Treatment 

(mg a.i./L)

[measured]

Diagnostic Observations1

Severit

y

Follicular Cell 

Hypertrophy

n Incidence

Negative 

Control

0

20

3

1 10

2 6

3 1

Solvent 

Control

0

20

1

1 12

2 7

3 0

0.019 0

20

5

1 9

2 6

3 0

0.19 0

20

2

1 10

2 7

3 1

1.9 0

20

13

1 6

2 1

3 0

Table: Example of a treatment-related shift toward decreasing incidence

 and severity of follicular cell hypertrophy.

1 Thyroid gland gross pathology and histopathology are graded 0 – 3 based on severity:

 0=Not remarkable, 1=Mild, 2=Moderate, 3=Severe. See OECD No. 82 for reference.

Apparently lower incidence and severity of 

follicular cell hypertrophy in high treatment.

From the histopathology report:

As thyroid follicular cells normally 

increase in height during tadpole 

development, this finding suggests 

some retardation of metamorphosis at 

the high treatment level.

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Solvent Effects

Treatment

(mg a.i./L)

[TWA-measured]

NF Developmental Stage Hind Limb Length Thyroid

Histo

Day 7 Day 21 Day 7 Day 21 Day 21

Median p Median p % Diff. p % Diff. pd Effects? 

(Yes/No)

Negative Control 54 -- 58 -- N/A -- N/A -- --

Solvent Control 54 -- 59 -- 0.80 0.84 16.1 0.05 No

0.003 54 >0.99 59 0.10 3.05 0.77 25.4 0.01 No

0.03 54 >0.99 59 0.10 -0.76 0.99 17.9 0.04 No

0.23 54 0.48 57 0.10 -1.53 0.96 -19.6 0.02 No

Statistical Test MannWhitney

MannWhitney Dunnett’s Dunnett’s N/A

Table: Developmental and Thyroid Gross Pathology/Histopathology Endpoints[1],[2]

 in the AMA with Chemical F.

Color Key Increase Decrease

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Solvent Effects

Treatment

(mg a.i../L)

[TWA-measured]

Snout-Vent Length Body Weight

Day 7 Day 21 Day 7 Day 21

% Diff. pd % Diff. pd % Diff. pd % 

Diff. pd

Negative Control 0 N/A 0 N/A N/A -- N/A --

Solvent Control 6.90 0.04 1.00 0.35 18.6 0.08 10.0 0.05

0.003 5.63 0.22 1.80 0.24 14.8 0.31 12.6 0.18

0.03 8.79 0.04 -0.17 >0.99 23.3 0.07 -5.23 0.77

0.23 -16.9 <0.01 -5.76 0.08 -38.5 <0.01 14.4 0.11

Statistical Test Dunnett’s Mann-Whitney Dunnett’s Dunnett’s

Color Key Increase Decrease

Table: Growth Endpoints in the AMA with Chemical F.

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Absence of Effect with Difficult-to-Test Substances

0

100

200

300

400

500

0 7 14 21

Measured Concentration 

(ng ai/L) 

Sample Day (Exposure Phase) 

Low Treatment (Nominal: 90 ng ai/L)

Medium Treatment (Nominal: 300 ng ai/L)

High Treatment (Nominal: 1000 ng ai/L)

Figure. Measured concentrations of Chemical S showed poor and variable

 recovery throughout the test. The study failed to meet the performance

 criteria for %CV <20% across 21 days.

No significant effects were 

observed on any endpoint.

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AMA Conclusions

Performance of the AMA generally meets 

expectations

The assay provides useful and sometimes 

unique information 

The test guideline and other materials provide 

sufficient information to successfully perform 

the test in most cases

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AMA Conclusions

Additional, targeted guidance could further 

improve study conduct and interpretation

• Difficult-to-test substances (e.g., lipophilic)

• Prevalence of spinal curvature

• Histologic indicators of mild developmental delay

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