# UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION



# MEMORANDUM

Date: 6/29/2015
SUBJECT: Atrazine

- Amphibian Metamorphosis Assay



# PC Code: 0808803 

# Decision No.: Petition No.: NA 

# Risk Assessment Type: NA TXR 

# No.: 0057284 

# MRID No.: 48614801 

# DP Barcode: D399650 Registration No.: NA 

# Regulatory Action: NA 

# Case No.: NA 

# CAS No.: 27314-13-2 40 

# CFR : NA



# FROM: Greg Akerman

# 'J-.c.~<2-A-**~-< ~**  Immediate Office Health Effects Division (7509P)


# THROUGH: Jess Rowland

# '6c-~--~s;;> 

# __ Deputy Director Hea l th Effects Division


# TO: Jo l e ne Trujillo Bio l ogist/Chemical Review Manager Risk Management and Implem entatio n Branch V Pesticide Re-evaluation Division (75 05P )



# CONCLUSION

A Data Eva luation Record (DER) for this study i s attached. Ver.Apr

. 2010



# C IT ATION: Wolf , J.C. 2012. Atrazine: Study to Satisfy the EPA Test Ord er # EDSP-080803- 1 ,2,3,4 G uideline Study 890. 11 00 Amphibian Metamorphosis Assay (AMA) Data Requirement for Atrazine Using Ex isting Frog Tissues Availab l e from Kloas et al. Studies. Unpub li shed study performed b y Experimental Pathology Laboratories, Inc. , S t er lin g , VA. Laboratory report numb er 140-1 2 1. Study spo n sored by Syngenta Crop Protection , LLC , Greensboro, NC. Study completed February I 0, 2012.

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# Page 2 of 2 

# The objective of the current study was to evaluate preserved tissues from Xenopus laevis frogs for potential effects of atrazine on thyroid-dependent endpoints, in response to a test order for an Amphibian Metamorphosis Assay (OCSPP 890.1100). The frogs in this study were archived specimens, preserved at Nieuwkoop and Faber (NF) stage 66 (complete metamorphosis, 43-75 days post fertilization), that were originally utilized for gonad histopathology investigations. Specimens (100 frogs from each treatment and control) were evaluated from the original treatment groups of 0 (negative control), 0.0010, 0.025, and 0.100 mg a.i./L. The test system in the original study was conducted under flow-through conditions and was maintained at 22±1°C. As this study was originally conducted under a different protocol than the 890.1100 and the data report submitted under MRIDs 47046201-4 and 47153501 , there are differences in the collection of data and reporting endpoints. The differences are outlined below in the Methods and Material section under Guideline followed. Prior to submission of these additional thyroid- dependent endpoints, the registrant contacted the Agency to requesting to use frogs from the previous assay for evaluating the th yroid

- dependent endpoints. There were no significant effects (p>0.05) detected on snout- to -vent length or dry weight endpoints in the original study. No effects were seen on time to metamorphosis (p>0.05), but results were not reported for effects on medi an Nieuwkoop

- Faber (NF) developmental stage because this parameter was not relevant to the original, stage-matched experimental design. No developmental abnormalities were reported, although asynchronous development was not specifically observed. Thyroid histological findings in preserved NF stage 66 specimens included follicular cell hypertrophy, follicular cell hyperplasia, and decreased colloid, but there was no indication that these findings were more severe or occurring with greater incidence in the atrazine -treated groups, relative to the control group. Other histological endpoints such as changes in thyroid follicular lumen, cell height, or shape were not reported. Atrazine exposure was slightly (3 -5%), but significantly (p<0.05), associated with greater normalized hind- limb length (HLL) values in the NF stage 66 frogs at all treated concentrations, relative to the control. When considered in conjunction with other Tier 1 EDSP data available for atrazine, this submission satisfies the Test Order requir ement for an Amphibian Metamorphosis Assay (OCSPP Guideline 890.1100).    



  



    

     



 







 

  

















 

     

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# EPA DP Barcode D399657

OECD Data Point 231 EPA MRID 48614801 EPA Guideline 890.1100 Amphibian Metamorphosis Assay (Frog)

  



 

 

# Atrazine
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# Not Reported in this MRID, but was 97.1% in original report

Common Name : Atrazine Chemical Name IUPAC Not Reported

CAS Name Not Reported CAS No. Not Reported Synonyms None Reported EPA PC Code 080803

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# John Marton, Ph.D. 



# 5/9/2012 &  

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# Teri S. Myers, Ph.D. 



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# : Amy Blankinship 



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# 6/12/2015 EPA/OPP/EFED/ERB4

   



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# 6/12/15 AMY BLANKINSHIP Digitally signed by AMY BLANKINSHIP DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=AMY BLANKINSHIP, dnQualifier=0000040917 Date: 2015.06.15 05:44:47 -04'00'    



  



    

     



 







 

  

















 

     

# EPA MRID Number 48614801

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# DER Template Version: 22 September 2011 ' )    ) ,

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# Wolf, J.C. 2012. Atrazine: Study to Satisfy the EPA Test Order # EDSP-080803-1,2,3,4 Guideline Study 890.1100 Amphibian Metamorphosis Assay (AMA) Data Requirement for Atrazine Using Existing Frog Tissues Available from Kloas et al. Studies. Unpublished study performed by Experimental Pathology Laboratories, Inc., Sterling, VA. Laboratory report number 140-121. Study sponsored by Syngenta Crop Protection, LLC, Greensboro, NC. Study completed February 10, 2012.

Note: The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven

s creening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to intera ct with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints w ithin the assay, where available, and redundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potential to interact with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).

. / 0 1 2 3 / 4 5 6 7 

# The guideline recommendations in this DER template are offered as a general reference to aid in preparation of the DER. The purpose of these recommendations is not to serve as substitute for the Test Guidelines, nor to provide any guidance on how the study should be conducted.    



  



    

     



 







 

  

















 

     

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# DER Template Version: 22 September 2011 9 

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# The objective of the current study was to evaluate preserved tissues from 

# Xenopus laevis frogs for potential effects of atrazine on thyroid-dependent endpoints, in response to a test order for an Amphibian Metamorphosis Assay (OCSPP 890.1100). The frogs in this study were archived specimens, preserved at Nieuwkoop and Faber (NF) stage 66 (complete metamorphosis, 43-75 days post fertilization), that were originally utilized for gonad histopathology investigations. Specimens (100 frogs from each treatment and control) were evaluated from the original treatment groups of 0 (negative control), 0.0010, 0.025, and 0.100 mg a.i./L. The test system in the original study was conducted under flow-through conditions and was maintained at 22±1°C. As this study was originally conducted under a different protocol than the 890.1100 and the data report submitted under MRIDs 47046201-4 and 47153501 , there are differences in the collection of data and reporting endpoints. The differences are outlined below in the Methods and Material section under Guideline followed. Prior to submission of these additional thyroid-dependent endpoints, the registrant contacted the Agency to requesting to use frogs from the previous assay for evaluating the thyroid-dependent endpoints.

There were no significant effects (p>0.05) detected on snout-to-vent length or dry weight endpoints in the original study. No effects were seen on time to metamorphosis (p>0.05), but results were not reported for effects on median Nieuwkoop-Faber (NF) developmental stage because this parameter was not relevant to the original, stage-matched experimental design. No developmental abnormalities were reported, although asynchronous development was not specifically observed. Thyroid histological findings in preserved NF stage 66 specimens included follicular cell hypertrophy, follicular cell hyperplasia, and decreased colloid, but there was no indication that these findings were more severe or occurring with greater incidence in the atrazine-treated groups, relative to the control group. Other histological endpoints such as changes in thyroid follicular lumen, cell height, or shape were not reported. Atrazine exposure was slightly (3-5%), but significantly (p<0.05), associated with greater normalized hind-limb length (HLL) values in the NF stage 66 frogs at all treated concentrations, relative to the
control.

When considered in conjunction with other Tier 1 EDSP data available for atrazine, this submission satisfies the Test Order requirement for an Amphibian Metamorphosis Assay (OCSPP Guideline 890.1100).    



  



    

     



 







 

  

















 

     

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# Test organism NF stage at test initiation: Not Reported [reported as NF stage 46-48 in original study report, 8 dpf] Test organism total length at test initiation (optional): Not Reported Test type: flow-through

  

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# Abbreviations: Diff. 

# Difference. NA 

# Not applicable. DPF 

# Days post fertilization.

1 

# A “yes” indicates a signific ant difference based on comparison to the negative (clean water) control, unless otherwise specified.

2 

# Th e criteria for significance are described in the Reviewer’s Analysis and Statistical Verification sections of the DER. Conclusions regarding histopathology may be heavily weighted by the expert opinion of a board-certified pathologist.

3 

# Hind-limb length is normalized to snout-vent lengt h (SVL).

4 

# Frogs were binned by sacrifice date, between days 4 3 and 75 post-fertilization (dpf).  

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# 0.0010 NA NA NA Yes NA NA No
0.025 NA NA NA Yes NA NA No
0.100 NA NA NA Yes NA NA No    



  



    

     



 







 

  

















 

     

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# This study was not conducted following OCSPP 890.1100. Rather, preserved specimens from a previous study on the effects of atrazine on the gonad histopathology of 

# Xenopus laevis tadpoles were analyzed for thyroid pathology and growth parameters (hind-limb length, snout-vent length, and wet weight) were measured by image analysis. All pertinent details about the conduct of the original study were not included in this report, however the details of many of the identified deviations below were discussed in the original report (MRID 47153501). The following deviations from OCSPP 890.1100 were noted:

1. Based on the report, the left hind limb was not always measured as in some cases it was not available for analysis, and in cases were both left and right were available a coin toss was used to determine
which one was measured. 2. The percent mortality in one replicate of the negative control was 20% (5 tadpoles out of 25 exposed) and was greater than the 10% recommended in the 890.1100 guideline. 3. NF stage at initiation of the original study was not reported. However, it was specified that tadpoles were 8 days post fertilization at test initiation and the original report reported stage at NF 46-48 at initiation. Further, NF stage was not recorded at any point throughout the original study. 4. The purity, description, lot/batch number and associated impurities of the test material were not reported in this report. Purity reported as 97.1% atrazine in original report. 5. Stability of the test material was not reported. Only nominal concentrations were reported. Test concentrations were measured and results reported in original report. 6. Larval selection methods for use in the definitive test, were not specified. In original report, ten pairs of adult frogs used for embryo selection and larvae were combined during acclimation for eventual
exposure. 7. While detailed descriptions of the type of flow-through system, or source of dilution water (or contaminant screen) were not reported in this study, these details were presented in the original report. 8. Supplemental aeration was provided to the test vessels, though the aeration rate was not reported.    



  



    

     



 







 

  

















 

     

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# DER Template Version: 22 September 2011 9. Hardness (90-130 mg/L as CaCO 3 

# ) and pH (7.9-8.3) of the dilution water exceeded th e recommended ranges (40-48 mg/L as CaCO 3 

# , 7.5 ± 0.1). Actual temperature and DO

m easurements were not specified. 10. Original study was conducted for 75 days (8-83 dpf). In original reported tested from 8 to 83 dpf or until frogs reached NF stage 66. 11. The reported light intensity (0.1-0.5 Klux) was lower than recommended (0.6-2 Klux). 12. While in this study, the study authors did not specify when SVL and wet weight measurements were taken in the original study. However, in the original report, SVL and wet weight were measured after frogs reached stage 66.

The original study was conducted in accordance to its specific protocol and was determined to be scientifically valid. The results in the current submission provide additional information regarding amphibian larval development and growth through metamorphosis.

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# Signed and dated No Data Confidentiality, GLP, and Quality Assurance statements

were provided. This study was conducted in compliance with the Good Laboratory Practice Regulations of the Environmental Protection Agency (FIFRA) as stipulated by 40 CFR Part 160; and all applicable amendments.

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# Atrazine

     

# Not Reported

OECD recommends describing water solubility, melting/boiling point stability in water and light, pKa, Pow or Kow, vapor pressure of test compound, expiration date.

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# Not specified. Only nominal concentrations were reported. The concentrations of atrazine did decline during the first part of the test and as animals completed metamorphosis and were removed from the tanks, the measured concentrations increased and better approximated nominal concentrations. The stocks were measured and were consistent with nominal concentrations and the diluter system was monitored, therefore it was thought that the loss of atrazine in the system was due to uptake in the animals.

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# Not specifiedV W X W Y Z W [ \ W X ] ^ _ ` a b ^ c d ^ _ X e a f ^ g ] b ] X h ^ i j X c W k ] _ a X ^ j l m e ] n ] W _ o p q a X W l ^ c m e ^ o ] o j o o W hEPA MRID Number 48614801Page rof s t 

# DER Template Version: 22 September 2011

u v f a o X w c x W _ ] o l

f W n [ a y z { a _ a c W [ | _ i ^ cl W X ] ^ _ j n ^ \ X X e a f a o X } m a b ] a o W _ d ~ W c a _ X W [  W c a vAs stated before, details about the test species and care are reported in thein original study report (MRID 47153501).

~ W c W l a X a c  W [ \ a  o  V a X W ] [ o ^ c ` a l W c  o { \ ] d a [ ] _ a ` a b ^ l l a _ d W X ] ^ _ oSpecies common name: 

# Xenopus laevis EPA recommends African clawed frog (Xenopus laevis). Western [Africa] clawed frog Silurana (Xenopus) tropicalis may be

used as

an alternate species1 

# ; however, a list of all of the necessary protocol deviations to accommodate this species is recommended for inclusion in the study report. The guideline recommends that the performance criteria used to support the reliability of the test be

identified.Species scientific name:African clawed frogSpecies strain (if stated):Not reported1U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the Endocrine DisruptorScreening Program Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention (OCSPP), Washington,D.C. (http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf).V W X W Y Z W [ \ W X ] ^ _ ` a b ^ c d ^ _ X e a f ^ g ] b ] X h ^ i j X c W k ] _ a X ^ j l m e ] n ] W _ o p q a X W l ^ c m e ^ o ] o j o o W hEPA MRID Number 48614801Page of s t 

# DER Template Version: 22 September 2011

~ W c W l a X a c  W [ \ a  o  V a X W ] [ o ^ c ` a l W c  o { \ ] d a [ ] _ a ` a b ^ l l a _ d W X ] ^ _ oWere parents maintained asin-house stock?No 

# EPA recommends that larvae used in the assay be derived from in-house adults.Were parental acclimationconditions same as definitivetest?Not specifiedCulture conditions reported in originalreportAcclimation period for parentalfrogs (if applicable):Not specifiedTadpoles were received 3 dayspost-fertilization (dpf) and wereacclimated during dpf 4-7.Details on parental feeding:None providedDetails on parental health:None provided

f W n [ a z  W c Z W [ } a [ a b X ] ^ _ W _ d  W c a v

~ W c W l a X a c  W [ \ a  o  V a X W ] [ o ^ c ` a l W c  o { \ ] d a [ ] _ a ` a b ^ l l a _ d W X ] ^ _ oBest single spawn?Not specified 

# EPA and OECD recommend that the best 2 – 3 individual spawns, with a minimum of 1500Number of spawns evaluated (ifNot specifiedEggs collected from 10 pairs of adults andV W X W Y Z W [ \ W X ] ^ _ ` a b ^ c d ^ _ X e a f ^ g ] b ] X h ^ i j X c W k ] _ a X ^ j l m e ] n ] W _ o p q a X W l ^ c m e ^ o ] o j o o W hEPA MRID Number 48614801Page  of s t 

# DER Template Version: 22 September 2011

~ W c W l a X a c  W [ \ a  o  V a X W ] [ o ^ c ` a l W c  o { \ ] d a [ ] _ a ` a b ^ l l a _ d W X ] ^ _ oapplicable):embryos combined in original report 

# larvae/spawn, be evaluated to identify the best single spawn, and that the larvae selected for testing originate from the best single spawn

(i.e.,



# the spawns are not co-mixed)Number of eggs sampled perspawn:Not specifiedNF stage at test initiationNot reportedIn original report, stage reported as 46-48at initiation 

# EPA recommends that the definitive study be initiated with larvae at Nieuwkoop – Faber (NF) developmental stage 51 (  

# 17 days

post -fertilization).Age at test initiation:8 days post-fertilization(dpf)Mean total length at test initiation(if reported):Not reportedRange of total length at testinitiation (if reported):Not reportedWas the optional size selectionmethod used?Not specifiedDetails on larval selection:None providedLoading rate (rearing density):~3.6 larvae/L25 larvae per tank (7 L) 

# EPA recommends that rearing densityV W X W Y Z W [ \ W X ] ^ _ ` a b ^ c d ^ _ X e a f ^ g ] b ] X h ^ i j X c W k ] _ a X ^ j l m e ] n ] W _ o p q a X W l ^ c m e ^ o ] o j o o W hEPA MRID Number 48614801Page  of s t 

# DER Template Version: 22 September 2011

~ W c W l a X a c  W [ \ a  o  V a X W ] [ o ^ c ` a l W c  o { \ ] d a [ ] _ a ` a b ^ l l a _ d W X ] ^ _ o 

# (loading rate) not exceed approximately 10 larvae/L culturing syste m for flow- through systems or 4 tadpoles/L in static - renewal exposure systems.Type of food:Sera Micron®



# EPA recommends Sera Micron ® 

# throughout

pre - exposure (after NF stage 45/46) and during the entire 21 - d definitive study. If another diet is used, the study report should provide analysis of iodide content and potential contaminants, and the diet should demonstrate equal performance to Sera

Micron ® 

# .Source of food:Sera MicronIodide concentration in diet (ifknown):Not reportedFrequency of feeding:three times/day 

# EPA recommends that feeding occur at least twice per day.Details on feeding regime:300 mg/tank at 8 dpf,increasing to 900mg/tank at 33 dpf, thenThere were 25 tadpoles/tank at testinitiation.



# It is recommended that food rations during the

pre - exposure period be increased along with larval growth to approximately 30V W X W Y Z W [ \ W X ] ^ _ ` a b ^ c d ^ _ X e a f ^ g ] b ] X h ^ i j X c W k ] _ a X ^ j l m e ] n ] W _ o p q a X W l ^ c m e ^ o ] o j o o W hEPA MRID Number 48614801Page  yof s t 

# DER Template Version: 22 September 2011

~ W c W l a X a c  W [ \ a  o  V a X W ] [ o ^ c ` a l W c  o { \ ] d a [ ] _ a ` a b ^ l l a _ d W X ] ^ _ oadjusted downwardproportionately as frogswere removed from thetanks. 

# mg/larva/day by test initiation. EPA and OECD recommend that food rations increase from 30 mg/larva/day at test initiation (Study

Day 0

- 4) to 80 mg/larva/day in the last week of the test (Study Day 15-21).V W X W Y Z W [ \ W X ] ^ _ ` a b ^ c d ^ _ X e a f ^ g ] b ] X h ^ i j X c W k ] _ a X ^ j l m e ] n ] W _ o p q a X W l ^ c m e ^ o ] o j o o W hEPA MRID Number 48614801Page of s t 

# DER Template Version: 22 September 2011

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f W n [ a  z } \ l l W c h ^ i | _ i ^ c l W X ] ^ _ ^ _ X e a Y g m ^ o \ c a } h o X a l W _ d f a o X  a o oa [  e W c W b X a c ] o X ] b o. Again, details about exposure provided in original report(MRID 47153501)

~ W c W l a X a c  W [ \ a  o  V a X W ] [ o ^ c ` a l W c  o { \ ] d a [ ] _ a ` a b ^ l l a _ d W X ] ^ _ oType of exposure:Flow-through 

# EPA recommends the use of a flow-through

system.Type of flow-through dilutionsystem (if applicable):Not reported



# Intermittent flow proportional diluters or continuous flow serial diluters are recommended.

2Flow-through rate (ifapplicable):~35 mL/minCalculated from tank volume (7 L) and dailyflow rate (50 L/day). 

# Recommended flow-through rate is 25 mL/min (complete volume replacement ca. every 2.7 hrs).Details on toxicant mixing forflow-through systems (ifapplicable):None provided



# Recommended toxicant mixing for

flow - through systems: 1) Mixing chamber is recommended but not required; 2) Aeration is not recommended for mixing;3) A2Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.V W X W Y Z W [ \ W X ] ^ _ ` a b ^ c d ^ _ X e a f ^ g ] b ] X h ^ i j X c W k ] _ a X ^ j l m e ] n ] W _ o p q a X W l ^ c m e ^ o ] o j o o W hEPA MRID Number 48614801Page  of s t 

# DER Template Version: 22 September 2011

~ W c W l a X a c  W [ \ a  o  V a X W ] [ o ^ c ` a l W c  o { \ ] d a [ ] _ a ` a b ^ l l a _ d W X ] ^ _ o 

# demonstration that the test solution is completely mixed before introduced into the test system is recommended; 4) The recommended flow splitting accuracy is within 10%.Renewal period for staticrenewal (if applicable):N/A- test was conductedunder flow-throughconditions



# If static renewal is used, EPA recommends

24 -hr renewal; renewal period is recommended not to exceed 72 hours.Aeration?Yes; rate not reported



# EPA recommends maintaining dissolved oxygen concentrations > 40% air saturation

(> 3.5 mg/L) . Aeration may be maintained through bubblers. It is recommended to set bubblers at levels that do not cause stress on the tadpoles.Source of dilution water:Not specified. Water wasfiltered (0.45 μm),UV-sterilized, and



# EPA recommends natural or reconstituted water; it is recommended that natural water be sterilized with UV and tested forV W X W Y Z W [ \ W X ] ^ _ ` a b ^ c d ^ _ X e a f ^ g ] b ] X h ^ i j X c W k ] _ a X ^ j l m e ] n ] W _ o p q a X W l ^ c m e ^ o ] o j o o W hEPA MRID Number 48614801Page  sof s t 

# DER Template Version: 22 September 2011

~ W c W l a X a c  W [ \ a  o  V a X W ] [ o ^ c ` a l W c  o { \ ] d a [ ] _ a ` a b ^ l l a _ d W X ] ^ _ oscreened forcontaminants. 

# pesticides, heavy metals, and other possible contaminants, including known substrates of the iodine transporter of the thyroid gland (e.g.,



# fluoride, chlorate, perchlorate). OECD accepts any water in which the test species show control survival at least as good as indicated in the test
guideline.Was dilution water analyzed forpesticides, heavy metals, andother contaminants?Yes. Results not provided.Iodide supplementation inwater?Not specified 

# If reconstituted water is used or if background levels of iodide in natural water are less than 0.5 μg/L, iodide supplementation is recommended. This
suppleme

ntation is in addition to the recommended dietary source of iodide (e.g.. in Sera Micron).V W X W Y Z W [ \ W X ] ^ _ ` a b ^ c d ^ _ X e a f ^ g ] b ] X h ^ i j X c W k ] _ a X ^ j l m e ] n ] W _ o p q a X W l ^ c m e ^ o ] o j o o W hEPA MRID Number 48614801Page  of s t 

# DER Template Version: 22 September 2011

~ W c W l a X a c  W [ \ a  o  V a X W ] [ o ^ c ` a l W c  o { \ ] d a [ ] _ a ` a b ^ l l a _ d W X ] ^ _ oTest vessel type/materials:Not specified 

# EPA and OECD recommend that

water - contact portions of the system not compromise the study (e.g., 

# all glass vessels or glass vessels with stainless steel frames are acceptable examples).Test vessel size:Not reportedFill volume:7 LAdditional details on exposuresystem:None provided

f W n [ a s z } \ l l W c h ^ i  W X a c  \ W [ ] X h  e W c W b X a c ] o X ] b o ] _ X e a f a o X } h o X a l v

~ W c W l a X a c q ] _ ] l \ l q W g ] l \ l q a W _ q a W o \ c a l a _ X

| _ X a c Z W [ { \ ] d a [ ] _ a ` a b ^ l l a _ d W X ] ^ _ oHardness (mg/L as CaCO3)90130Not ReportedNot Reported 

# EPA recommends hardness 40 to 48 mg/L as CaCO 3.V W X W Y Z W [ \ W X ] ^ _ ` a b ^ c d ^ _ X e a f ^ g ] b ] X h ^ i j X c W k ] _ a X ^ j l m e ] n ] W _ o p q a X W l ^ c m e ^ o ] o j o o W hEPA MRID Number 48614801Page  tof s t 

# DER Template Version: 22 September 2011pH7.98.3Not ReportedNot Reported 

# EPA recommends pH 7.5 ± 1,

inter -replicate and inter-treatment differentials should not exceed 0.5.Dissolved oxygen (mg/L)---- 60% Not Reported 

# EPA recommends dissolved oxygen (DO) >3.5 mg/L (>40% air saturation) . OECD recommends DO concentration >3.5 mg/L (>40% air saturation).Temperature (°C)----22±1 Not Reported



# EPA recommends temperature 22±1 o 

# C;

inter -replicate and inter-treatment differentials should not exceed 0.5 o 

# C.IodideNot Reported 

# EPA recommends aquatic iodide range 0.5

– 10 μg/L (supplemental iodide should not exceed 2 μg/L).Ammonia (mg/L)0.00.35Not ReportedNot Reported



# General recommendations for frequency of measurements: EPA recommends that water quality parameters be measured in a control and at one test item concentration at least weekly. In static renewal systems,FluorideNot ReportedPerchlorateNot ReportedChlorateNot ReportedV W X W Y Z W [ \ W X ] ^ _ ` a b ^ c d ^ _ X e a f ^ g ] b ] X h ^ i j X c W k ] _ a X ^ j l m e ] n ] W _ o p q a X W l ^ c m e ^ o ] o j o o W hEPA MRID Number 48614801Page  rof s t 

# DER Template Version: 22 September 2011Nitrate (mg/L)Alkalinity (mg/L as CaCO3)Conductivity (μS/cm)0.041337262.29172817Not ReportedNot Reported 

# water quality parameters, including ammonia, should be measured just prior to renewal. In addition, EPA recommends that DO be measured at each concentration at least weekly and that temperature be measured continuously.

OECD recommends that DO and t emperature be
measured at least weekly and that pH and

hardness be measured at least at the beginning and end of the test.

V v } X \ d h V a o ] x _ W _ d j d d ] X ] ^ _ W [ Y g m a c ] l a _ X W [  ^ _ d ] X ] ^ _ o

f W n [ a  z ` W _ x a   ] _ d ] _ x } X \ d h  ^ _ d ] X ] ^ _ o  ] i j m m [ ] b W n [ a  v

~ W c W l a X a c  W [ \ a  o  V a X W ] [ o ^ c ` a l W c  o { \ ] d a [ ] _ a ` a b ^ l l a _ d W X ] ^ _ oWas a range-finder conducted?Not reportedIf yes, what was the method forN/AThe study from which preserved



# EPA recommends that the highest testV W X W Y Z W [ \ W X ] ^ _ ` a b ^ c d ^ _ X e a f ^ g ] b ] X h ^ i j X c W k ] _ a X ^ j l m e ] n ] W _ o p q a X W l ^ c m e ^ o ] o j o o W hEPA MRID Number 48614801Page  of s t 

# DER Template Version: 22 September 2011determining the highest testconcentration in therange-finder?specimens were collected includednominal concentrations of 0.01, 0.1, 1.0,25, and 100 μg/L. The three highesttreatment groups were selected foranalysis in the current study. 

# concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of toxicity (e. g., < 10% mortality), whichever concentration is lowest.Species:



# Xenopus laevisLife stage:Not ReportedTest duration:Not ReportedAdditional details:Tadpoles used in thecurrent study werearchived specimens thatwere originally utilizedfor gonad histopathologyinvestigations ofatrazine as reported inKloas et al. 2008.Original test was conducted from 8 dpfuntil complete metamorphosis (83 dpf).Defined as NF 46-48 at initiation to NFstage 66 in original report.V W X W Y Z W [ \ W X ] ^ _ ` a b ^ c d ^ _ X e a f ^ g ] b ] X h ^ i j X c W k ] _ a X ^ j l m e ] n ] W _ o p q a X W l ^ c m e ^ o ] o j o o W hEPA MRID Number 48614801Page y of s t 

# DER Template Version: 22 September 2011

f W n [ a t z V a i ] _ ] X ] Z a } X \ d h  ^ _ d ] X ] ^ _ o vDetails about study conduct were submitted under MRID 47153501

~ W c W l a X a c  W [ \ a  o  V a X W ] [ o ^ c ` a l W c  o { \ ] d a [ ] _ a ` a b ^ l l a _ d W X ] ^ _ oTest duration:75 days (original study) 

# EPA recommends that the duration of the definitive test be 21 days.Method for selecting the highesttest concentration in the definitivetest:Not reported



# EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of toxicity (e.g.,

< 10% mortality), whichever concentration is lowest.Reference study citation (ifapplicable):Hosmer, A, T Springer,W Kloas, I Lutz, J Wolf,and L Holden (2007)Response of LarvalXenopus laevis toAtrazine Exposure:Assessment ofMetamorphosis andGonadal Morphology:Overview andData from the original study also appearedin Kloas, W, I Lutz, T Springer, H Krueger,J Wolf, L Holden, and A Hosmer (2009)Does atrazine influence larvaldevelopment and sexual differentiation in 

# Xenopus laevis? Toxicological Sciences,107(2): 376-384.Data also submittedunder MRID 47153501.V W X W Y Z W [ \ W X ] ^ _ ` a b ^ c d ^ _ X e a f ^ g ] b ] X h ^ i j X c W k ] _ a X ^ j l m e ] n ] W _ o p q a X W l ^ c m e ^ o ] o j o o W hEPA MRID Number 48614801Page y of s t 

# DER Template Version: 22 September 2011

~ W c W l a X a c  W [ \ a  o  V a X W ] [ o ^ c ` a l W c  o { \ ] d a [ ] _ a ` a b ^ l l a _ d W X ] ^ _ oComparison of Sudies.Response to AtrazineGeneric and ProductSpecific Data Call-InNotice, March 17, 2004.Syngenta Study NumberT002536-05.Separation of test concentrations:0.04-0.25 

# EPA recommends that the maximum concentration separation be 0.1 and the minimum be 0.33.Number of test concentrations:3



# EPA recommends a minimum of 3 concentrations and a control , plus solvent control if appropriate.Are nominal concentrationsadjusted for purity?Not reportedIndicate the type of valuespresented for measuredNo measuredconcentrations wereV W X W Y Z W [ \ W X ] ^ _ ` a b ^ c d ^ _ X e a f ^ g ] b ] X h ^ i j X c W k ] _ a X ^ j l m e ] n ] W _ o p q a X W l ^ c m e ^ o ] o j o o W hEPA MRID Number 48614801Page y yof s t 

# DER Template Version: 22 September 2011

~ W c W l a X a c  W [ \ a  o  V a X W ] [ o ^ c ` a l W c  o { \ ] d a [ ] _ a ` a b ^ l l a _ d W X ] ^ _ oconcentrations:reported.Limit of quantification (LOQ):Not reported 

# EPA recommends that for chemical test concentrations below the LOQ, analyses be conducted on the stock solutions.Level of detection (LOD):Not reportedFrequency of measurement:Not reported



# It is recommended that test item concentration be measured in one tank at each treatment level at test initiation and every w eek thereafter.Number of replicates in control:16 replicate tanks in 4clustersOne cluster was randomly chosen so that4 replicates (maximum 25 tadpoles/rep)were selected for image analysis andthyroid histopathology.



# EPA recommends 4 replicates.Number of replicates in solventcontrol (if applicable):N/A; a solvent was notused



# EPA and OECD recommend the use of a concurrent solvent control when a solubilizing agent is used. EPA recommends 4 replicates.V W X W Y Z W [ \ W X ] ^ _ ` a b ^ c d ^ _ X e a f ^ g ] b ] X h ^ i j X c W k ] _ a X ^ j l m e ] n ] W _ o p q a X W l ^ c m e ^ o ] o j o o W hEPA MRID Number 48614801Page yof s t 

# DER Template Version: 22 September 2011

~ W c W l a X a c  W [ \ a  o  V a X W ] [ o ^ c ` a l W c  o { \ ] d a [ ] _ a ` a b ^ l l a _ d W X ] ^ _ oNumber of replicates per test itemtreatmentlevel:8 replicate tanks, 2clusters for each groupOne cluster was randomly chosen so that4 replicates (maximum 25 tadpoles/rep)were selected for image analysis andthyroid histopathology. 

# EPA recommends 4 replicates.Number of larvae per treatment attest initiation:400 larvae in negativecontrol, 200 larvae pergroup in the treatmentgroups.Was a solvent used?NoSolvent type (if applicable):N/AMaximum solvent concentration(if applicable):N/A 

# EPA recommends that the solvent not exceed
0.02 ml/L3 

# . OECD recommends that solvent have no effect on survival nor produce any3Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB(2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review. AquaticToxicology, 76, pp.69–92.V W X W Y Z W [ \ W X ] ^ _ ` a b ^ c d ^ _ X e a f ^ g ] b ] X h ^ i j X c W k ] _ a X ^ j l m e ] n ] W _ o p q a X W l ^ c m e ^ o ] o j o o W hEPA MRID Number 48614801Page y of s t 

# DER Template Version: 22 September 2011

~ W c W l a X a c  W [ \ a  o  V a X W ] [ o ^ c ` a l W c  o { \ ] d a [ ] _ a ` a b ^ l l a _ d W X ] ^ _ o 

# other adverse effects and that concentration not be greater than 0.1 ml/L4 

# .Was a positive control used?YesThough a positive control was included, nomortality, growth, or development datawere provided for this treatment.Positive control (if applicable):17 -estrdial [E2]Positive control concentration(s)(if applicable):0.0002 mg a.i./L8 replicate tanks, 2 clustersPhotoperiod:12 hrs light :12 hrs dark 

# EPA recommends photoperiod 12:12 (light:dark).Light intensity at water’s surface:0.1-0.5 Klux



# EPA recommends light intensity 0.6 – 2 Klux (at water’s surface).Additional details:None provided4OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Series onTesting and Assessment. No. 23. Paris, France.V W X W Y Z W [ \ W X ] ^ _ ` a b ^ c d ^ _ X e a f ^ g ] b ] X h ^ i j X c W k ] _ a X ^ j l m e ] n ] W _ o p q a X W l ^ c m e ^ o ] o j o o W hEPA MRID Number 48614801Page y sof s t 

# DER Template Version: 22 September 2011

f W n [ a r z } \ l l W c h ^ i f c a W X l a _ X  ^ _ b a _ X c W X ] ^ _ o ] _ X e a j l m e ] n ] W _ q a X W l ^ c m e ^ o ] o j o o W h  ] X e j X c W k ] _ a v

f c a W X l a _ X | V  ^ l ] _ W [

 ^ _ b a _ X c W X ] ^ _

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 l x W v ] v    q a W _      V a X W ] [ o ^ c ` a l W c  o { \ ] d a [ ] _ a ` a b ^ l l a _ d W X ] ^ _ oNegative Control0.00Not Reported. Inoriginal report,mean measuredwere 0.000957,0.0234, and0.0867Not Reported;Not reported inoriginal reportIn original report rangeof % nominal was68-110, 72-116 and67-109, respectively 

# EPA and OECD recommend that test item concentrations be maintained at a coefficient of

varia tion (CV)  

# 20%.Treatment 10.001Treatment 20.025Treatment 30.100Abbreviations:CVCoefficient of variation.

Y v w n o a c Z W X ] ^ _ o

u ] ^ [ ^ x ] b W [ Y _ d m ^ ] _ X o zthyroid histopathology and hind limb length; body weight and snout-vent length were measured in the original study

 a c a c W   ] _ d ] Z ] d \ W [  d W X W m c ^ Z ] d a d YesV W X W Y Z W [ \ W X ] ^ _ ` a b ^ c d ^ _ X e a f ^ g ] b ] X h ^ i j X c W k ] _ a X ^ j l m e ] n ] W _ o p q a X W l ^ c m e ^ o ] o j o o W hEPA MRID Number 48614801Page y of s t 

# DER Template Version: 22 September 2011 EPA recommends that observations of mortality and clinical signs occur daily, at a minimum; other observations are recommended as follows: NF developmental stage (days 7 and 21); any asynchronous development, indicated by tadpoles that cannot be assigned an NF stage (days 7 and 21); hind limb length (days 7 and 21); snout-vent length (days 7 and 21); body weight (test initiation, for optional size-based larval selection); and thyroid gland gross pathology and histopathology (day 21). Note the histopathology section of the test guideline also includes thyroid gross pathology observations.    



  



    

     



 







 

  

















 

     

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# No mortality results from the original study were reported. In the original study, the percent mortality at test termination, by replicate is summarized below

  

  





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# Abbreviations: NA 

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1 

# As there were 8 tanks (1-4 and 5-8) per treatment an d only one cluster of four was used in this study, the actual tank identification for the negative control was Negative control1 tanks 5-8 and Tanks 1-4 for the treatment groups. Therefore, replicate A corresponds to either Tank 5 for the NC or tank 1 for the atrazine treatment group, and Rep B corresponds to Tank 6 for the NC and Tank 2 for the atrazine groups and so on.

2 

# 25/rep for replicate A,C,D. One suspected mishap in Rep B, so % mortality based on 24/rep.

No results pertaining to NF stage or asynchronous dev elopment were reported in this study. In the original ¡ ¢

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# DER Template Version: 22 September 2011 study, frogs were exposed till complete metamorophosis NF stage 66 or until day 75 of exposure (83 days post fertilization). No statistical differences in time to metamorphosis was observed between the control and treatment groups. Average time to metamorphosis ranged from 50.9 to 52.9 days. Only two frogs in the study that survived until test termination did not complete metamorphosis, with one in the 0.1 mg a.i./L group.

  

 

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Normalized hind limb length was calculated by the re viewer and averaged 1.22 in the negative control and 1.26, 1.26, and 1.28 in the nominal 0.0010, 0.025, and 0.100 mg a.i./L treatment groups, respectively, with increases of 2.9 to 4.6% relative to the negative control. The study authors did not analyze normalized hind limb length.

  

 

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# Negative Control NA NA NA NA 4 22.7 0.48 1.22

0.0010 NA NA NA NA 4 23.6 0.59 1.26

0.025 NA NA NA NA 4 23.2 0.77 1.26

0.100 NA NA NA NA 4 23.1 0.89 1.28

Abbreviations: NA 

# Not applicable. SD 

# Standard deviation.

1 

# The normalized value (HLL: SVL) reported in the tabl e should be the treatment mean of individual ratios of hind    



  



    

     



 







 

  

















 

     

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# DER Template Version: 22 September 2011 limb length (HLL) to snout-vent length (SVL).

2 

# Summary results for snout-vent length (SVL) are pr esented in the next table (   

 

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The study authors reported that all growth parameters (i.e., HLL, SVL, wet weight) were comparable between the three treatment groups and the negative control (p ì 

# 0.071). HLL ranged from 22.7 mm in the negative control to 23.6 mm in the 0.0010 mg a.i./L treatment group, with inhibitions ranging -4.1 to -1.9% relative to the control. Wet weight ranged from 549.2 mg in the negative control to 591.0 mg in the 0.0010 mg a.i./L treatment group with increases of 2.2 to 7.6% in all groups relative to the negative control. SVL ranged from 18.1 mm in the 0.100 mg a.i./L treatment group to 18.7 mm in the 0.0010 mg a.i./L treatment group. Inhibitions in SVL were -0.9, 0.5, and 2.5% in the 0.0010, 0.025, and 0.100 mg a.i./L treatment groups, respectively, relative to the negative control.

  

 

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# Negative Control NA NA NA 4 18.5 0.39 NA NA NA 4 549 20

0.0010 NA NA NA 4 18.7 0.24 NA NA NA 4 591 29

0.025 NA NA NA 4 18.4 0.56 NA NA NA 4 563 49

0.100 NA NA NA 4 18.1 0.47 NA NA NA 4 562 53

Abbreviations: NA 

# Not applicable. ND 

# Not determined. SD 

# Standard deviation.

1 

# A lso referred to as “wet weight” in the test guideline.    



  



    

     



 







 

  

















 

     

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# DER Template Version: 22 September 2011 No thyroid hypertrophy or atrophy was noted in control or treatment tadpoles. Further, only mild follicular cell hypertrophy and hyperplasia were noted, though the occurrence was comparable between treatment and control tadpoles. Decreased colloid was also observed, though like thyroid and follicular cell observations, was comparable between all groups and did not appear to be treatment-related. In evaluating histopathology, frogs were binned according to age at sacrifice (reported as dpf 43-50, dpf 51-60, and dpf 61-75) and comparisons between treated and control animals were made within each age group category. However, in the following tables the observed effects are combined for all binned
groups.    



  



    

     



 







 

  

















 

     

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# Negative Control 0 20 20 20 20 20 18 20 20

1 20 0 20 0 20 2 20 0
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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.V W X W Y Z W [ \ W X ] ^ _ ` a b ^ c d ^ _ X e a f ^ g ] b ] X h ^ i j X c W k ] _ a X ^ j l m e ] n ] W _ o p q a X W l ^ c m e ^ o ] o j o o W hEPA MRID Number 48614801Page yof s t 

# DER Template Version: 22 September 2011

f W n [ a   z j d d ] X ] ^ _ W [ f e h c ^ ] d { [ W _ d ú ] o X ^ m W X e ^ [ ^ x h w n oa c Z W X ] ^ _ o ] _ û ü ý þ ÿ   ü    Potential changes in follicular lumen area, cell height and cell shape were not reported and therefore Table 14 was deleted.No clinical signs or sub-lethal effects from the original assay were reported. In the original report, clinical signs of toxicity were reported. The most commonfinding was small size and was observed in the control and atrazine treatment tanks and was never for more than two frogs in a tank. Four frogs wereobserved with gross abnormalities including one in the NC (not the cluster used here) and one frog in the 0.001 mg a.i./L treatment group was observedwith reddened color around head and forelimbs. None of the findings reported were considered to be treatment related.

f W n [ a  s z  [ ] _ ] b W [ } ] x _ o ] _ û ü ý þ ÿ   ü    vTable 15 deleted    



  



    

     



 







 

  

















 

     

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# DER Template Version: 22 September 2011 B. 

 

    



   



  



 











# Differences in hind-limb length were analyzed using a two-sided Jonckheere-Terpstra non-parametric trend test in a step-down fashion. Significance was set at  

# = 0.05 and the FREQ procedure in SAS 9.2 was used to conduct the tests. Exact 

# p values were obtained using the Monte Carlo option with N = 10,000 replications.

The study authors found no significant treatment-rela ted effects on hind limb length ( 

# p = 0.5599), snout-vent length ( 

# p

= 0.9539), or body weight ( 

# p = 0.0708). Further, they reported that there were no treatment-related patterns in thyroid histopathology observations.

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# Replicate values for snout-to-vent length, dry weight, hind-to-limb length, and normalized hind-to-limb length were statistically analyzed. Snout-to-vent length, dry weight, and hind-to-limb length data did not exhibit a monotonic trend, but the data satisfied the assumptions of normality and homogeneity of variances, as determined using Shapiro- Wilk’s and Levene’s tests. The control group was compared to the
atrazine- treated groups using Dunnett’s test. Data for normalized hind -to-limb length exhibited a suggestive monotonically increasing trend, but data did not satisfy parametric assumptions. The control group was compared to the treated levels using the Jonckheere-Terpstra test. These analyses were conducted using SAS v. 8.1.

'  



  



# The reviewer’s analysis detected no significant effect on snout-to-vent length, dry weight, or hind-to-limb length (p>0.05). There was, however, a slight (3-5%) significant increase in the normalized hind-to-limb length values in the treated groups, relative to the negative control (p<0.05; Jonckheere-Terpstra). No effects were seen on thyroid histology. All observations were made on frogs at NF stage 66.V W X W Y Z W [ \ W X ] ^ _ ` a b ^ c d ^ _ X e a f ^ g ] b ] X h ^ i j X c W k ] _ a X ^ j l m e ] n ] W _ o p q a X W l ^ c m e ^ o ] o j o o W hEPA MRID Number 48614801Page of s t 

# DER Template Version: 22 September 2011

f W n [ a   z V a Z a [ ^ m l a _ X W [ W _ d f e h c ^ ] d { c ^ o o ~ W X e ^ [ ^ x h  ú ] o X ^ mW X e ^ [ ^ x h Y _ d m ^ ] _ X o1,2 ] _ X e a j q j  ] X e j X c W k ] _ a v

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Y i i a b X o    a o   ^ 0.0010NANANANANANA30.029NANANANANo0.025NANANANANANA30.044NANANANANo0.100NANANANANANA50.010NANANANANoStatistical testNANANAJonckheereTerpstraNAV W X W Y Z W [ \ W X ] ^ _ ` a b ^ c d ^ _ X e a f ^ g ] b ] X h ^ i j X c W k ] _ a X ^ j l m e ] n ] W _ o p q a X W l ^ c m e ^ o ] o j o o W hEPA MRID Number 48614801Page sof s t 

# DER Template Version: 22 September 2011Abbreviations:Diff.Difference.NANot applicable.1Unless otherwise indicated, effects are reported based on comparison to the clean water control. Conclusions regarding histopathology may be heavily weightedby the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are consideredstatistically significant at 

# p<0.05.3Hind-limb length is normalized to snout-vent length (SVL).4While not specifically analyzed in this study, in the original study, frogs were exposed until complete metamorphosis (NF stage 66) or until Day 75 of study.The original report did analyzed time to metamorphosis and no statistical differences between the control and treatment groups were observed. Discussions aboutasynchronous development were not specifically reported, but abnormal development, or lack of development was and with no mention of any treatment-relatedeffects on development was reported.    



  



    

     



 







 

  

















 

     

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1 

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2 

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# p < 0.05.

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# This study was not conducted as a traditional AMA study. Rather, preserved specimens from a previous study were examined in the context of an AMA study. However, because this was not conducted according to OCSPP 890.1100 guidelines, there were several deviations and several pertinent details pertaining to experimental design were not included in this report, however, details on culturing, acclimation, and study conduct were provided in MRID 47153501. See Section 1. Materials and Methods for details.

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# The objective of the current study was to evaluate preserved tissues from metamorphosed 

# Xenopus laevis frogs at NF stage 66 for potential thyroid-dependent effects of atrazine in accordance with guidelines of Amphibian Metamorphosis Assay. The frogs in this study were archived specimens that were originally utilized for gonad histopathology investigations. The developmental stage of the archived specimens was  

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*  

# Negative Control NA NA 0 NA NA NA 0 NA
0.0010 NA NA 1 0.892 NA NA 8 0.359
0.025 NA NA -0.5 0.981 NA NA 2 0.926
0.100 NA NA -2 0.338 NA NA 2 0.948
Statistical test NA Dunnett’s NA Dunnett’s    



  



    

     



 







 

  

















 

     

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# DER Template Version: 22 September 2011 beyond that recommended for analysis in the Amphibian Metamorphosis Assay ( 

# i.e ., < NF stage 60). Thyroid-relevant endpoints recommended in the AMA are likely less sensitive by the time metemorphosis has completed; however, there were no effects on time to metamorphosis in the original study.

The reviewer’s conclusions did not agree with the study author’s. The study author concluded that there were no significant effects in this study (p>0.05 ). The reviewer’s analysis detected a significant increases of the normalized hind-to-limb length at all treated levels, relative to the control (p<0.05).

The original study from which preserved specimens were collected and analyzed was conducted at Wildlife International Laboratories, Ltd., and included eight replicate tanks per atrazine dose group (0.0010, 0.025, 0.100 mg a.i./L). The eight tanks were arranged in two clusters of four tanks each and each tank contained 25 tadpoles. For the present analysis, a single cluster of four tanks was selected randomly from each of the dose groups. The original study also included 16 replicate tanks for the negative control, which were arranged in four clusters of four tanks each. One negative control cluster was contaminated during the original study and was therefore excluded from the final analysis. Consequently, the single cluster of negative control tanks included in this AMA evaluation was selected randomly from among the three remaining negative control clusters. However, according to the original report, one cluster of tanks was contaminated with atrazine and was excluded from analysis and a microbial bloom (one tank) was observed in another cluster of control tanks and was also excluded, thereby leaving two clusters for final analysis. As this report indicates that only one cluster (contaminated, reviewer assumed the one with atrazine detected) was removed, it means that the other cluster with the microbial bloom was retained for potential analysis. However, based on this and the original report, the control animals used in this analysis were from a uncompromised control cluster.

For pathology examination, frogs were binned by age at sacrifice (i.e., 43-50 dpf, 51-60 dpf, and 61-75 dpf). However, it was not clear if hind limb length measurements were binned by age, but it does not appear so, as the data in the appendices of the report do not indicate a binning exercise..

Measurements of hind limb lengths were obtained via image analysis from color digital photographs of the entire preserved caudal segment. A single image was acquired for each frog, and saved as a    



  



    

     



 







 

  

















 

     

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# DER Template Version: 22 September 2011 high- resolution TIFF file with the animal’s identifying number. A calibration metric rule and identifying number were placed within the field of view next to each frog. Due to processing for histology, some hind limbs were removed from frogs. For frogs with only one hind limb attached to pelvis, only the attached limb was measured. For frogs were both limbs were attached, a coin toss was used to determine if right or left leg was measured; it is unclear why the choice of a coin toss was used to determine selection. For frogs with no attached limbs, measurements were not taken.

It was originally intended that hind limb lengths would be measured for each frog that had been selected for thyroid histopathology (20 frogs per treatment) plus an additional 40 frogs selected randomly from the tank clusters selected for the gonad histopathology evaluation (total hind limb measurements per treatment = 60). However, for the control and high dose groups, the numbers of frogs that were suitable for analysis (i.e., hind limbs still attached to the pelvic girdle) were <60. Numbers of frogs were 41, 64, 63, and 35 for the control, low, middle, and high dose treatments, respectively. It is unclear why for low and middle concentrations, the number of frogs analyzed is greater than 60.

In traditional AMA studies, tadpoles 13-15 dpf (NF stage 51) are used and after 21 days, tadpoles are 34-36 dpf. Tadpoles at NF stages greater than 60 are excluded from analysis of growth parameters due to rapid morphological changes in tadpoles at this stage. In the current study, frogs were collected at complete metamorphosis, NF 66 and according to the binning exercise in this study appear to have been exposed from 43-75 days. Therefore, all frogs should have been at the same stage making comparisons in growth appropriate. Time to metamorphosis was not significantly different across the treatment groups.

The frogs were exposed for 75 days which equated to 83 days post fertilization. As the study reported the binning ages as dpf (43-75), it is not sure if this is to indicate days of exposure till metamorphosis and not days post fertilization as animals were not exposed until 8 days post fertilization and according to raw data, some animals in the treatment groups used in this study were exposed until day 70 (however, it is unknown if those specific animals were used in this study).

G G G H

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# DER Template Version: 22 September 2011 Grim, KC, Wolfe, M, Braunbeck T, Iguchi T, Ohta Y, Tooi O, Touart L, Wolf DC, Tietge J (2009) Thyroid histopathology assessments for the amphibian metamorphosis assay to detect thyroid-active substances. Toxicologic Pathology, 37:415-424.

Hollander M, Wolfe DA (1999) Nonparametric Statistical Methods, Second Edition, New York: John Wiley & Sons.

Hosmer A, Springer T, Kloas W, Lutz I, Wolf J, Holden L (2007) Response of Larval Xenopus laevis to Atrazine Exposure: Assessment of Metamorphosis and Gonadal Morphology: Overview and Comparison of Studies. Response to Atrazine Generic and Product Specific Data Call-In Notice, March 17, 2004. Syngenta Study Number T002536-05.

Lutz I, Kloas W, Springer TA, Holden LR, Wolf JC, Krueger HO, Hosmer AJ (2008) Development, standardization, and refinement of procedures for evaluating effects of endocrine active compounds on development and sexual differentiation of 

# Xenopus laevis . Analytical and Bioanalytical Chemistry, 390: 2031-2048.

SAS (2009) Base SAS 9.2 Procedures Guide. Cary, NC: SAS Institute, Inc.    



  



    

     



 







 

  

















 

     

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