# 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

 

# A ssay Purpose and Design

 

# Endpoints

 

# V alidity Elements and Performance Criteria

 

# Observations from Case Study Chemicals

2

# Assay Purpose

 

# S creen 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

3

# Study Design

(per guideline recommendations)



#  

# 2 1

- 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

4

# 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 10 th 

### and 90 th



### 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

F igure. 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 Sources**

**of Iodide Prevalence of Spinal** 

**Curvature (%)**

**ID ID**  **Product Reduced**  **Iodide**

**(ug/g) Water**  **i**

**(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 Sources** 

**of Iodide Prevalence of Spinal** 

**Curvature (%)**

**ID ID**  **Product Reduced**  **Iodide**

**(ug/g) Water**  **i**



### (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 Observations 1

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 l ower 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. p d 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 Mann

Whitney Mann

Whitney Dunnett’s Dunnett’s N/A T

able : Developmental and Thyroid Gross Pathology/Histopathology Endpoints [1]

, [2]



### in the AMA with Chemical F.

Color Key Increase Decrease

14

# Solvent Effects

Treatment

(mg a.i ../L)

[TWA

- measured] Snout

- Vent Length Body Weight

Day 7 Day 21 Day 7 Day 21

% Diff. p d % Diff. p d % Diff. p d %

Diff. p d

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 

### T

able : Growth Endpoints in the AMA with Chemical F.

15

# Absence of Effect with Difficult

- to

- Test Substances

0 100 200 300 400 500

0 7 14 21Measured Concentration(ngai/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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