# Comments regarding the Amphibian Metamorphosis Assay (AMA)
John M. Brausch, Ph.D. BASF Corporation
US EPA Science Advisory Panel Meeting

– Tier 1 May 21

- 23, 2013∗ Maintaining test concentrations most difficult performance criterion

∗ 7 of 18 studies did not maintain appropriate test concentrations

∗ Increased flows generally improved performance

∗ No guidance on incorporating difficult materials in feed

∗ Use of solvents presented additional issues

∗ Synchrony among tadpoles leading to tadpoles > Stage 60

∗ Occurred in 16 of 18 reviewed studies (4 with > 20% of control tadpoles)

∗ Reduced feed and increased flow generally helped 

# Difficulties with Performance Criteria

### ∗ Dose setting was very difficult

∗ Guidance indicates 1/3 of LC 50 

### (maximum tolerated concentration )

∗ 12 of 18 studies non

- thyroid dependent effect ( Snout

vent length, body weight)

∗ Lack of historical data on amphibians

∗ Necessitated a 96h Lc50 study to estimate high dose

∗ Prolonged range

- finders were sometime necessary


### ∗ 1/10 of LC 50 

### could be more appropriate



# Complications of Dose Setting

### ∗ Most animal intense study requiring 8,000

– 16,000 tadpoles

∗ Only 320 tadpoles needed for definitive study



### ∗ Cost of study is approx. $100,000

∗ Labor intensive and great degree of specialty needed



### ∗ 21 day study with minimum 4 day range finding study 

# Animal Usage, Cost, Time

### ∗ 12 of 16 thyroid studies (male/female pubertal, AMA) demonstrated thyroid activity

∗ 7 of 16 demonstrated thyroid activity in AMA

∗ Only 1 instance where AMA suggested thyroid activity that was not observed in pubertal rat assays

∗ Supported by Pickford et al. (2010)



### ∗ 5 pubertal studies observed activity when AMA did not 

# Detecting Thyroid Activity in the AMA

### ∗ Histopathology was most sensitive endpoint and specific for thyroid activity

∗ But, current decision tree only indicates needed for thyroid antagonists



### ∗ Only assay that examines developing thyroid

∗ Examines effects in an ecological receptor

∗ Incorporates multiple routes of exposure

∗ Could be important for compounds that get metabolized

∗ Ex. Gill uptake bypasses first

- pass metabolism 

# Strengths of AMA

### ∗ Thyroid histopathology

∗ Stage matched thyroids



### ∗ Normalized Hind Limb Length

∗ Statistical analysis of NF Stage > 60 tadpoles

∗ Large amount of tissue remodeling/apoptosis



### ∗ Spinal curvature

∗ Appeared in 15 of 18 reviewed studies

∗ Diet is at least partially responsible 

# Limitations of AMA

### ∗ Staged

- fixed design vs. time

- fixed design

∗ Similar to proposed LAGDA



### ∗ 8d amphibian assay proposed by Tietge et al. (2010)

∗ Focuses primarily on biochemical endpoints



### ∗ 14d metamorphosis assay

∗ Initial EPA recommendation 

# Alternatives to AMA∗ AMA did not significantly add to battery

∗ Rat pubertal identified 6 of 7 compounds that indicated thyroid activity in AMA

∗ AMA not more sensitive than rat assays

∗ Definitive assessment cannot be made until all data is available

∗ Modifications of the study design could increase utility

∗ Eliminate growth issues, statistical issues, increase power for histology, greater sensitivity, etc. 

# Overall Conclusions