## UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON

, D.C

. 20460

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION



## MEMORANDUM

Date: 6/29/2015

SUBJECT: Permethrin : Data Evaluation Records (DERs) for EDSP Tier l 

## Assays

PC Code: 10970 I



## Decision No.: 460377

, 464766

, 470225


## Petition No.: NA 

## DP Barcode: 0 3983 01

, D40179 3, 0405670 

## Registration No

. : NA



## Risk Assessment Type: NA

TXR 

## No. : 0056246


## MRID 

## No.: See Tab l e 

## Regulatory Action: NA



## CaseNo.: NA


## CAS No

. : 52645

- 53- 1

40 

## CFR: NA



## FROM: Gre g Akerman

, Ph.D. 

# 0-;y 

# g_--

Imm ed i ate Office

Health Effects Di v ision (7509P)



## THROUGH : J ess Rowland

# cr=-~.D~ Deputy Dir ector
Health Effects Divi sion



## TO: Jol e ne Tru jill o BioJogist/Chemical R ev i ew Manager

Risk Management and Impl ementa tion Branch 

## V

P estic id e Re-evaluation Division (7505 P )



## I. 

## ACTION REQUESTED Ver.Apr. 20 1 0



## The Pesticide R e-eva l uation Division (PRD) of OPP has requested that the H e alth E ffect s Division ( HED) review the E n docrine Disruptor Scree nin g Program (EDS P ) Tie r I assays



## s ubmitted in r es pon se to 

## the agency 's Test Order for permethrin: Test Order# CON -109701 -7.

Pa ge I of2

### Page 2 of 2 

## II. RESPONSE

Attached are the EDSP Tier 1 assay DERs for permethrin

.

III

. MRID Table

Chemical: Permethrin PC Code: 109701

Guideline Assay MRID

890.1100 Amphibian Metamorphosis Assay (Frog) 48988601

890.1150 Androgen Receptor Binding (Rat Prostate) NA

890.1200 Aromatase Assay (Human Recombinant) 48618601

890.1250 Estrogen Receptor Binding NA

890.1300 Estrogen Receptor Transcriptional Activation (Human Cell Line HeLa

- 9903) NA

890.1350 Fish Short

- Term Reproduction 48702301

890.1400 Hershberger (Rat) 486 1 8602

890.1450 Female Pubertal (Rat) 48672901

890.1500 Male Pubertal (Rat) 48687101

890.1550 Steroidogenesis (Human Cell Line

– H295R) 48618603

890.1600 Uterotrophic (Rat) NA
NA= Not Applicable (requirement satisfied by other scientifically relevant information (OSRI))

### Data Evaluation Record on the Toxicity of Permethrin to Amphibians, Metamorphosis Assay

EPA MRID Number 48988601

Page 1 of 41

DER Template Version: 22 September 2011 Data Requirement: EPA DP Barcode 407365

OECD Data Point 231

EPA MRID 48988601

EPA Guideline 890.1100

Amphibian Metamorphosis Assay (Frog)

Test Material: Permethrin Purity (%): 97.7%

Common Name Permethrin

Chemical Name IUPAC Not Reported

CAS Name Not Reported

CAS No. Not Reported

Synonyms None Reported

EPA PC Code 109701

Primary Reviewer: John Marton, Ph.D. Date: 6/8/13

Environmental Scientist, CDM Smith

Secondary Reviewer(s): Teri S. Myers, Ph.D. Date: 6/10/13

Environmental Scientist, CDM Smith

Primary Reviewer: Keith Sappington, M.S.

Senior Science Advisor, USEPA/OPP/EFED / ERB5 Date: 9/19/13

Additional Reviewer: Elyssa Arnold

Biologist, USEPA/OPP/EFED/ERB2 Date: 11/6/13

Additional (and final) Reviewer: Justin Housenger

Biologist, USEPA/OPP/EFED/ERB5 Date: 5/14/15

Date Evaluation Completed: 29-MAY-2015 Digitally signed by JUSTIN

HOUSENGER
DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=JUSTIN
HOUSENGER, dnQualifier=0000044455

Date: 2015.06.03 11:23:43 -04'00' 

# Keith Sappington Digitally signed by Keith Sappington DN: cn=Keith Sappington, o, ou, email=sappington.keith@epa.gov
, c=US

Date: 2015.06.10 13:59:17 -04'00'

### Data Evaluation Record on the Toxicity of Permethrin to Amphibians, Metamorphosis Assay

EPA MRID Number 48988601

Page 2 of 41

DER Template Version: 22 September 2011 CITATION: Schneider, S.Z., T.Z. Kendall, and H.O. Kruger. 2012. Permethrin: Amphibian Metamorphosis Assay

for the Detection of Thyroid Active Substances. Unpublished study performed by Wildlife International, Ltd.,

Easton, MD 21601. Laboratory report number 701A-109A. Study sponsored by Consumer Specialty Products

Association, For the Permethrin Data Group II Task Force Steering Committee/Joint Venture, Washington,

DC 20006. Study completed November 6, 2012.

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

screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact 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 within 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).

### Data Evaluation Record on the Toxicity of Permethrin to Amphibians, Metamorphosis Assay

EPA MRID Number 48988601

Page 3 of 41

DER Template Version: 22 September 2011 EXECUTIVE SUMMARY

The 21-day assay of permethrin (97.7% purity) on amphibian metamorphosis of African clawed frogs ( Xenopus

laevis ) was conducted under flow-through conditions. Amphibian larvae at developmental Nieuwkoop-Faber Stage

(NF) 51 (80; 20/replicate) were exposed to control (water only; negative), a solvent control (0.020 mL/L

dimethylformamide; DMF), and permethrin at nominal concentrations of 0.00011, 0.00033, and 0.0010 mg a.i./L;

mean-measured concentrations were <0.0000600 (<LOQ; controls), 0.000052, 0.00022, and 0.00063 mg

a.i./L. The reviewer-calculated time-weighted average concentrations were <0.0000600 (<LOQ; controls),

0.000054, 0.00022, and 0.00068 mg a.i./L. The test system was maintained at 21.5 to 23.0°C and a pH of

7.9 to 8.2.

Unless otherwise indicated, all effects are reported based on comparison to the negative (clean water) control.

Two non-incidental mortalities were observed in the test, one each in the low and high treatment groups. Clinical

signs included tail curvature, which was observed in both controls and all treatment groups (12-28% of individuals),

with no indication of a dose-dependent pattern.

Compared to the negative control, permethrin significantly promoted Day 21 normalized hind-limb length (HLL)

at the low and mid treatment groups (by 21% and 23% respectively). There were no effects on normalized HLL

on Day 7. However, there was an apparent solvent effect (21% stimulation of normalized HLL, p=0.01) relative

to the negative control at Day 21. When Day 21 normalized HLL results were compared to the solvent control,

no significant differences were observed. There was no significant acceleration or delay of median NF

developmental stage at Day 7 or 21 for any treatment level. Further, no asynchronous development was observed.

Compared to the negative control, there was a statistically significant increase in Day 21 wet weight and

snout-to-vent length (SVL) at the low treatment group. No solvent effect was seen for SVL.

Thyroid gland histopathology observations included thyroid gland atrophy and hypertrophy, follicular cell height

increases and decreases, and/or follicular cell asymmetry in both controls and all treatment groups. These effects

were of low frequency and did not exhibit a concentration-dependent response; therefore, they are not considered

to be treatment-related.

### Data Evaluation Record on the Toxicity of Permethrin to Amphibians, Metamorphosis Assay

EPA MRID Number 48988601

Page 4 of 41

DER Template Version: 22 September 2011 The study met all validity and performance criteria except for %CV in analytical measurements. The CV of

measured test concentrations exceeded the recommended  20% in the lowest and highest test treatments (50.5%

and 33.0%, respectively). Given the very low concentrations used (<1 ppb), the tendency for pyrethroids to sorb

onto glassware, and that the treatment concentrations did not overlap, these CVs are not judged to interfere with

the interpretation of test results.

The assay satisfies the EDSP Tier 1 Test Order requirements for an Amphibian Metamorphosis Assay (OCSPP

Guideline 890.1100).

Results Synopsis:

Test organism NF stage at test initiation: 51

Test organism total length at test initiation (optional): Not measured

Test type: Flow-through

### Data Evaluation Record on the Toxicity of Permethrin to Amphibians, Metamorphosis Assay

EPA MRID Number 48988601

Page 5 of 41

DER Template Version: 22 September 2011 Table 1: Summary of Developmental and Thyroid Pathology/Histopathology Effects 1,2 

### in the Amphibian

Metamorphosis Assay (AMA) with Permethrin.

Treatment

(mg a.i./L)

[TWA-measured] NF Developmental

Stage Hind Limb

Length 3 

### Asynchronous

Development Thyroid Gross

and

Histopathology

Day 7 Day 21 Day 7 Day 21 Day 7 Day 21 Day 21

0.000054 No No No Yes No No No

0.00022 No No No Yes No No No

0.00068 No No No No No No No

1 

### A “yes” indicates a significant difference based on comparison to the negative (clean water) control, unless

otherwise specified.

2 

### The 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 length (SVL).



## I. MATERIALS AND METHODS

Guideline Followed: This study was conducted following guidelines outlined in the U.S.

Environmental Protection Agency Series 890- Endocrine Disruptor Screening

Program Test Guidelines, OPPTS Number 890.1100, Amphibian

Metamorphosis (Frog) ; and the OECD Guideline for Testing of Chemicals,

Guideline 231, Amphibian Metamorphosis Assay . The following deviations were

noted:

1. The reported light intensity (0.137-0.602 Klux) was lower than recommended (0.6-2.0 Klux).

### Data Evaluation Record on the Toxicity of Permethrin to Amphibians, Metamorphosis Assay

EPA MRID Number 48988601

Page 6 of 41

DER Template Version: 22 September 2011 2. Hardness of the dilution water (140-144 mg/L as CaCO 3 

### ) exceeded the recommended range

(40-48 mg/L as CaCO 3 

### ).

3. The CVs in the lowest and highest test treatments, 50.5% and 33.0%, respectively, exceeded the

recommended  20%.

These deviations do not impact the interpretation of the results of the study.

Compliance: Signed and dated No Data Confidentiality, GLP, and Quality Assurance

statements were provided. This study was conducted in compliance with Good

Laboratory Practice Standards as published by the U.S. Environmental

Protection Agency (40 CFR Parts 160 and 792); and OECD Principles of Good

Laboratory Practice (ENV/MC/CHEM(98) 17), with the following exceptions:

periodic screening analyses of water for potential contaminants were not

performed according to Good Laboratory Practice Standards, but were

performed using a certified laboratory and standard US EPA analytical methods;

preliminary analyses of water iodide concentrations were not performed

according to Good Laboratory Practice Standards; and the characterization and

stability of the test substance under the storage conditions of the test site was

not determined in accordance with Good Laboratory Practice Standards.

A. Test Material Permethrin

Description: Liquid

OECD recommends describing water solubility, melting/boiling point stability in water and light, pKa, Pow or Kow,

vapor pressure of test compound, expiration date.



### Lot No./Batch No. : PMPS000014 (Lot No.)

Purity: 97.7%

### Data Evaluation Record on the Toxicity of Permethrin to Amphibians, Metamorphosis Assay

EPA MRID Number 48988601

Page 7 of 41

DER Template Version: 22 September 2011 Impurities: Not Reported

Stability of Compound: The reviewer-calculated TWA concentrations during the study yielded

recoveries of 49.5 to 68.1% of nominal with coefficients of variation of 18.4 to

45.1%. Measured concentrations in stock solutions were 90.7 to 92.6% of

nominal.

Storage Conditions of

Test Chemicals: Stored under ambient conditionsData Evaluation Record on the Toxicity ofPermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 48988601Page8of41 DER Template Version: 22 September 2011B. Test OrganismTable 2: General Information About the Test Species and Parental Care.ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies common name: African clawed frog 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: Xenopus laevisSpecies 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).Data Evaluation Record on the Toxicity ofPermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 48988601Page9of41 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsWere parents maintained asin-house stock?Yes Adults were originally obtained fromXenopus I of Dexter, Michigan. EPA recommends that larvae used in the assay be derived from in-house adults.Were parental acclimationconditions same as definitivetest?YesAcclimation period for parentalfrogs (if applicable):At least 11 daysDetails on parental feeding: Frogs were fed an adult

Xenopusdiet suppliedby Zeigler Brothers, Inc.(Gardners, PA) at a ratesufficient to maintain thehealth of the culture.Details on parental health: Culture frogs showed nosign of disease or stressprior to the test.Data Evaluation Record on the Toxicity ofPermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 48988601Page10of41 DER Template Version: 22 September 2011Table 3: Larval Selection and Care.ParameterValue(s)Details or RemarksGuideline RecommendationsBest single spawn?Yes EPA and OECD recommend that the best 2 – 3 individual spawns, with a minimum of 1500 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 spawns evaluated (ifapplicable):3Number of eggs sampled perspawn:250 Eggs were collected and evaluated on theday of egg deposition.NF stage at test initiation 51 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:17 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?NoData Evaluation Record on the Toxicity ofPermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 48988601Page11of41 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on larval selection: The best viability ofthree separate spawnswas selected forobtaining larvae for usein the definitive testThe selected spawn had a reportedviability of 99.6%.Loading rate (rearing density): 4 larvae/L EPA recommends that rearing density (loading rate) not exceed approximately 10 larvae/L culturing system 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 North America(Montgomeryville, PA)Iodide concentration in diet (ifknown):Not ReportedData Evaluation Record on the Toxicity ofPermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 48988601Page12of41 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsFrequency of feeding:3 times/day EPA recommends that feeding occur at least

twice per day.Details on feeding regime: Feeding regime was halfof the recommendeddaily rates. It is recommended that food rations during the pre-exposure period be increased along with larval growth to approximately 30 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).Data Evaluation Record on the Toxicity ofPermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 48988601Page13of41 DER Template Version: 22 September 2011C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:Flow-through EPA recommends the use of a flow-through

system.Type of flow-through dilutionsystem (if applicable):Continuous-flow diluter Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 2Flow-through rate (ifapplicable):48.6 mL/minThe calculated flow rate providedapproximately 10 volume additionsevery 25 hours providing a completevolume replacement every 2.4 hours. Recommended flow-through rate is 25 mL/min (complete volume replacement ca.

every 2.7 hrs).2Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.Data Evaluation Record on the Toxicity ofPermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 48988601Page14of41 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on toxicant mixing forflow-through systems (ifapplicable):A syringe pump was used todeliver volumes of testsubstance stock solutions andDMF to mixing chambers. Stocksolutions or solvent were dilutedwith well water in the mixingchambers to obtain the desiredconcentrations. Flow into eachmixing chamber usingrotameters. Recommended toxicant mixing for

flow- through systems: 1) Mixing chamber is recommended but not required; 2) Aeration is not recommended for mixing;3) A 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 If static renewal is used, EPA recommends 24-hr renewal; renewal period is recommended not to exceed 72 hours.Data Evaluation Record on the Toxicity ofPermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 48988601Page15of41 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsAeration? No 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: Freshwater obtained from a well~40 meters deep location onWLI property. Water was passedthrough a sand filter to removeparticles greater than ~25μmand pumped into a storage tankwhere it was aerated with spraynozzles. Prior to use, water wasfiltered to 0.45μm to removefine particles and passedthrough a UV sterilizer. EPA recommends natural or reconstituted water; it is recommended that natural water be sterilized with UV and tested for 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.Data Evaluation Record on the Toxicity ofPermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 48988601Page16of41 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsWas dilution water analyzed forpesticides, heavy metals, andother contaminants?Yes. See Reviewer’sComments.Iodide supplementation inwater?No If reconstituted water is used or if background levels of iodide in natural water are less than 0.5 μ g/L, iodide supplementation is recommended. This supplementation is in addition to the recommended dietary source of iodide (e.g.. in Sera Micron).Test vessel type/materials: Glass 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:9 LFill volume:7 LData Evaluation Record on the Toxicity ofPermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 48988601Page17of41 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details on exposuresystem:NoneTable 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsHardness (mg/L as CaCO3) 140 144 134Weekly in onereplicate of thenegative control andhigh-dose treatment. EPA recommends hardness 40 to 48 mg/L as CaCO 3.pH 7.98.28.0Weekly in each testvessel. EPA recommends pH 7.5 ± 1, inter-replicate and inter-treatment differentials should not exceed 0.5.Data Evaluation Record on the Toxicity ofPermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 48988601Page18of41 DER Template Version: 22 September 2011Dissolved oxygen (mg/L)5.38.46.7Weekly in each testvessel. EPA recommends dissolved oxygen (DO) >3.5 mg/L (>40% air saturation). OECD recommends DO concentration >3.5 mg/L (>40% air saturation).Temperature 21.522.322.0Measured in eachtest chamber weeklyand continuously inone negative controlreplicate. EPA recommends temperature 22±1 o C; inter-replicate and inter-treatment differentials should not exceed 0.5 o C.Iodide (μg/L) 3 6 N/APreliminary analysisof dilution water EPA recommends aquatic iodide range 0.5

– 10 μ g/L (supplemental iodide should not

exceed 2 μ g/L).Ammonia NotReported 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,Fluoride NotReportedPerchlorate NotReportedChlorate NotReportedData Evaluation Record on the Toxicity ofPermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 48988601Page19of41 DER Template Version: 22 September 2011Alkalinity (mg/L as CaCO3)Specific Conductance (μS/cm)172329184403179377Weekly in onereplicate of thenegative control andhigh-dose treatment. 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 temperature be measured at least weekly and that pH and hardness be measured at least at the beginning and end of the test.D. Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details or RemarksGuideline RecommendationsWas a range-finder conducted?YesData Evaluation Record on the Toxicity ofPermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 48988601Page20of41 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsIf yes, what was the method fordetermining the highest testconcentration in therange-finder?Solubility and reported LC50values for amphibians, whichranged from 100 to 700μg/L. 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.Species: Xenopus laevisLife stage:Not reportedTest duration:14 daysAdditional details:Nominal concentrations rangedfrom 0.02 to 1000μg a.i./L andwas conducted underflow-through conditions with theuse of a solvent. No mortalitiesor sub-lethal effects wereobserved in the controls or 0.02and 0.20μg a.i./L treatmentgroups. Complete mortality wasSigns of toxicity observed in the2.0μg a.i./L treatment groupincluded lethargy, loss ofequilibrium, lying on the bottom ofthe test chamber, and appearingsmall in comparison to thecontrols.Data Evaluation Record on the Toxicity ofPermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 48988601Page21of41 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline Recommendationsobserved in the 8.1 to 1000μga.i./L treatment groups.Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:21 days EPA recommends that the duration of the definitive test be 21 days.Method for selecting the highesttest concentration in the definitivetest:Range-finder; highconcentration wasapproximately 1/3 of theLC50value. 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):N/AData Evaluation Record on the Toxicity ofPermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 48988601Page22of41 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsSeparation of test concentrations:0.33 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?YesIndicate the type of valuespresented for measuredconcentrations:Study authors reportedmean-measuredconcentrations.Due to high analytical variability anddrastic changes in recoveries betweensampling periods, the reviewer calculatedtime-weighted average (TWA)concentrations.Limit of quantification (LOQ): 0.00006 mg a.i./L EPA recommends that for chemical test concentrations below the LOQ, analyses be conducted on the stock solutions.Level of detection (LOD): Not ReportedData Evaluation Record on the Toxicity ofPermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 48988601Page23of41 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsFrequency of measurement: Days 0, 7, 14, and 21 It is recommended that test item concentration be measured in one tank at each treatment level at test initiation and every week

thereafter.Number of replicates in control: 4 EPA recommends 4 replicates.Number of replicates in solventcontrol (if applicable):4 EPA and OECD recommend the use of a concurrent solvent control when a solubilizing agent is used. EPA recommends 4 replicates.Number of replicates per test itemtreatment level:4 EPA recommends 4 replicates.Number of larvae per treatment attest initiation:80Was a solvent used?YesSolvent type (if applicable): DMFData Evaluation Record on the Toxicity ofPermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 48988601Page24of41 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsMaximum solvent concentration(if applicable):0.020 mL/L EPA recommends that the solvent not exceed

0.02 ml/L3 . OECD recommends that solvent have no effect on survival nor produce any other adverse effects and that concentration not be greater than 0.1 ml/L4 .Was a positive control used? NoPositive control (if applicable): N/APositive control concentration(s)(if applicable):N/APhotoperiod:12 hrs light :12 hrs dark EPA recommends photoperiod 12:12

(light:dark).Light intensity at water’s surface: 0.137-0.602 Klux EPA recommends light intensity 0.6 – 2 Klux3Hutchinson 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.69–92.4OECD (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.Data Evaluation Record on the Toxicity ofPermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 48988601Page25of41 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline Recommendations (at water’s surface).Additional details:The stock solutions weremixed by inversion, andappeared clear andcolorless, with no visibleprecipitates.Data Evaluation Record on the Toxicity ofPermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 48988601Page26of41 DER Template Version: 22 September 2011Table 8: Summary of Treatment Concentrations in the Amphibian Metamorphosis Assay with Permethrin.Treatment IDNominalConcentration(mg a.i./L)Measured/TWAConcentration(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsControl (dilutionwater only)0.00 <LOQ N/A N/A EPA and OECD recommend that test item concentrations be maintained at a coefficient
of variation (CV) 20%.Solvent control (ifapplicable)0.00 <LOQ N/A N/ATreatment 1 0.00011 0.000052/0.000054 50.5N/ATreatment 2 0.00033 0.00022/0.00022 19.9N/ATreatment 3 0.00100 0.00063/0.00068 33.0N/AAbbreviations:CVCoefficient of variation.E. ObservationsBiological Endpoints:Day 7- NF stage, wet weight, HLL, SVL, normalized HLLDay 21- NF stage, wet weight, HLL, SVL, normalized HLL, thyroid histopathologyDaily- mortality, clinical signs of toxicityData Evaluation Record on the Toxicity ofPermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 48988601Page27of41 DER Template Version: 22 September 2011Were raw (individual) data provided?Yes 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 (d ays 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 lar val selection); and thyroid gland gross pathology and histopathology (day 21). Note the histopathology section of the test guideline also includes thyroid gross pathology observations.

### Data Evaluation Record on the Toxicity of Permethrin to Amphibians, Metamorphosis Assay

EPA MRID Number 48988601

Page 28 of 41

DER Template Version: 22 September 2011 

## II. RESULTS AND DISCUSSION

A. Results

After 7 days of exposure a single mortality was observed in the low-dose treatment group, in addition

to two incidental mortalities during siphoning (Table 9). By test termination, a single mortality was

observed in the high-dose treatment group, in addition to two more incidental mortalities during siphoning.

Overall, four of six mortalities observed were due to siphoning incidents.

Table 9: Larval Mortality in X enopus laevis

.

1 

### Sample size and cumulative mortality values at Day 7 prior to interim sacrifice.

2 

### Tadpole killed during siphoning in solvent control replicate A on Day 4.

3 

### 1 tadpole killed during siphoning in replicate D on Day 4. Second death occurred in replicate D on Day 7.

4 

### Tadpole killed during siphoning in replicate A on Day 11.

5 

### 1 tadpole killed during siphoning in replicate A on Day 11. Second death occurred in replicate A on Day 12.

Median developmental stage on Day 7 was 54 in the negative and solvent controls and in the low- and

high-dose treatment groups, and 54.5 in the mid-dose treatment group (Table 10). Median developmental Treatment (mg a.i./L)

[TWA-measured] Larval Mortality

Day 7 1 

### Day 21

n Mortality # Mortality % n Mortality # Mortality %

Negative Control 80 0 0 60 0 0

Solvent Control 80 1 2 

### 1.3 59 1 4 

### 1.7

0.000054 80 2 3 

### 2.5 58 0 0

0.00022 80 0 0 60 0 0

0.00068 80 0 0 60 2 5 

### 3.3

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DER Template Version: 22 September 2011 stage was 57 in the controls and all treatment groups at test termination. No asynchronous development

was detected. Two late-stage tadpoles were found at test termination, one in the solvent control and one

in the mid-dose treatment group. The 10 th 

### and 90 th 

### percentiles of NF stage in the negative control were

56 and 57, respectively.

Table 10: Larval Development in X enopus laevis – Developmental Stage and Asynchronous Development.

Treatment (mg a.i./L)

[TWA-measured] Developmental Stage

Day 7 Day 21

n Median

Stage # Asynchronous n Median

Stage # Asynchronous

Negative Control 4 54 0 4 57 0

Solvent Control 4 54 0 4 57 0

0.000054 4 54 0 4 57 0

0.00022 4 54.5 0 4 57 0

0.00068 4 54 0 4 57 0

Mean Day 7 normalized HLL was 0.15 in the negative control and mid-dose group and 0.14 in the solvent

control, low-dose group, and high-dose group (Table 11). Mean Day 21 normalized HLL was 0.34, 0.43,

0.41, 0.42, and 0.39 in the negative control, solvent control, low-dose, mid-dose, and high-dose groups,

respectively.

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DER Template Version: 22 September 2011 Table 11: Larval Development in X enopus laevis – Hind Limb Length.

Treatment

(mg a.i./L)

[TWA-measured] Hind Limb Length (HLL)

Day 7 Day 21

n Mean

(mm) ±SD HLL:

SVL 1 n Mean

(mm) ±SD HLL:

SVL 1



### Negative Control 4 2.25 0.08 0.15 4 8.10 0.67 0.34

Solvent Control 4 2.13 0.16 0.14 4 9.81 0.74 0.43

0.000054 4 2.30 0.24 0.14 4 10.3 0.53 0.41

0.00022 4 2.28 0.09 0.15 4 9.95 0.99 0.42

0.00068 4 2.23 0.13 0.14 4 9.25 0.76 0.39

Abbreviations: SD 

### Standard deviation.

1 

### Summary results for snout-vent length (SVL) are presented in the next table ( Table 12 ).

Day 7 SVL averaged 15.8 and 15.5 mm in the negative and solvent controls, respectively, and ranged

from 15.5 to 16.6 mm in the treatment groups (Table 12). By Day 21, mean SVL was 23.4 and 23.6

mm in the negative and solvent controls, respectively, and ranged from 23.7 to 24.8 mm in the treatment

groups.

Day 7 wet weight averaged 0.283 and 0.272 g in the negative and solvent controls, respectively, and

ranged from 0.290 to 0.300 g in the treatment groups (Table 12). By Day 21, mean wet weight was

0.855 and 0.969 g in the negative and solvent controls, respectively, and ranged from 0.952 to 1.078

g in the treatment groups.

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DER Template Version: 22 September 2011 Table 12: Larval Growth in X enopus laevis

.

Treatment

(mg a.i./L)

[TWAmeasured] Snout-Vent Length (SVL) Body Weight 1



### Day 7 Day 21 Day 7 Day 21

n Mean

(mm) ±SD n Mean

(mm) ±SD n Mean

(g) ±SD n Mean

(g) ±SD



### Negative Control 4 15.8 0.39 4 23.4 0.74 4 0.283 0.010 4 0.855 0.087

Solvent Control 4 15.5 0.44 4 23.6 0.74 4 0.272 0.024 4 0.969 0.031

0.000054 4 16.6 0.83 4 24.8 0.42 4 0.313 0.043 4 1.078 0.035

0.00022 4 15.5 0.53 4 23.7 0.24 4 0.290 0.016 4 0.952 0.050

0.00068 4 15.8 0.51 4 23.7 0.34 4 0.300 0.022 4 0.952 0.051

Abbreviations: SD 

### Standard deviation.

1 

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

Thyroid gland hypertrophy and atrophy, follicular cell height increases and decreases, and follicular cell

asymmetry were detected in both controls and all treatment groups (Tables 13 and 14). Thyroid gland

asymmetry was also assessed and similarly detected in both controls and all treatment groups. There

were no apparent treatment-related patterns in frequency or severity of these observations. Tadpoles

selected for histopathology were staged match to the median stage of the control at test termination (NF

stage 57), to the extent possible.

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DER Template Version: 22 September 2011 Table 13: Gross Pathology and Histopathology of the Thyroid Gland in X enopus laevis

.

Treatment

(mg a.i./L)

[TWA-measured] Diagnostic Observations 1

Severity 

### Thyroid Gland

Hypertrophy Thyroid Gland

Atrophy Follicular Cell

Hypertrophy Follicular Cell

Hyperplasia

n Incidence n Incidence n Incidence n Incidence



### Negative Control 0 20 18 20 17 20 -- 20 --

1 20 2 20 1 20 -- 20 --

2 20 0 20 2 20 -- 20 --

3 20 0 20 0 20 -- 20 --

Solvent Control 0 20 15 20 20 20 -- 20 --

1 20 3 20 0 20 -- 20 --

2 20 2 20 0 20 -- 20 --

3 20 0 20 0 20 -- 20 --

0.000054 0 20 15 20 20 20 -- 20 --

1 20 3 20 0 20 -- 20 --

2 20 2 20 0 20 -- 20 --

3 20 0 20 0 20 -- 20 --

0.00022 0 20 13 20 18 20 -- 20 --

1 20 5 20 1 20 -- 20 --

2 20 1 20 1 20 -- 20 --

3 20 1 20 0 20 -- 20 --

0.00068 0 20 16 20 20 20 -- 20 --

1 20 1 20 0 20 -- 20 --

2 20 2 20 0 20 -- 20 --

3 20 1 20 0 20 -- 20 --

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.Data Evaluation Record on the Toxicity ofPermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 48988601Page33of41 DER Template Version: 22 September 2011Table 14: Additional Thyroid Gland Histopathology Observations in X enopus laevis.Treatment(mg a.i./L)[TWA-measured]Additional Qualitative Observations1SeverityFollicular LumenArea (Increase)Follicular LumenArea (Decrease)Follicular CellHeight (Increase)Follicular Cell Height(Decrease)Follicular CellShapenIncidencenIncidencenIncidencenIncidencenIncidenceNegative Control 0 20 -- 20 -- 20 17 20 16 20 201 20 -- 20 -- 20 2 20 2 20 02 20 -- 20 -- 20 1 20 2 20 03 20 -- 20 -- 20 0 20 0 20 0Solvent Control 0 20 -- 20 -- 20 15 20 19 20 191 20 -- 20 -- 20 3 20 1 20 02 20 -- 20 -- 20 1 20 0 20 13 20 -- 20 -- 20 1 20 0 20 00.000054 0 20 -- 20 -- 20 14 20 17 20 161 20 -- 20 -- 20 3 20 3 20 22 20 -- 20 -- 20 3 20 0 20 23 20 -- 20 -- 20 0 20 0 20 00.00022 0 20 -- 20 -- 20 12 20 19 20 16Data Evaluation Record on the Toxicity ofPermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 48988601Page34of41 DER Template Version: 22 September 2011Treatment(mg a.i./L)[TWA-measured]Additional Qualitative Observations1SeverityFollicular LumenArea (Increase)Follicular LumenArea (Decrease)Follicular CellHeight (Increase)Follicular Cell Height(Decrease)Follicular CellShapenIncidencenIncidencenIncidencenIncidencenIncidence1 20 -- 20 -- 20 4 20 1 20 32 20 -- 20 -- 20 2 20 0 20 03 20 -- 20 -- 20 2 20 0 20 10.00068 0 20 -- 20 -- 20 13 20 19 20 171 20 -- 20 -- 20 3 20 1 20 22 20 -- 20 -- 20 3 20 0 20 13 20 -- 20 -- 20 1 20 0 20 01Thyroid histopathology is graded 0 – 3 based on severity: 0=Not remarkable, 1=Mild, 2=Moderate, 3=Severe. See OECD No. 82 forreference.By test termination, tail curvature was observed in both controls and all treatment groups (Table 15). The study authors believed tail curvature to be relatedto feeding rates during acclimation. With the exception of the tail curvature, all surviving tadpoles appeared normal and healthy.Data Evaluation Record on the Toxicity ofPermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 48988601Page35of41 DER Template Version: 22 September 2011Table 15: Clinical Signs in X enopus laevis.Treatment(mg a.i./L)[TWA-measured]Clinical Signs1TypenIncidenceNegative ControlTail curvature6012%Solvent ControlTail curvature6022%0.000054Tail curvature6021%0.00022Tail curvature6028%0.00068 Tailcurvature 6016%1Note that asynchronous development (unable to stage) is reported previously inTable 10.

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DER Template Version: 22 September 2011 B. Study Author’s Analysis and Conclusions

For each endpoint, data from the negative and solvent controls were compared using the appropriate

statistical test. No differences were detected ( p > 0.05) for any endpoint on Day 7 or 21 and all

subsequent analyses were conducted by comparing treatment data to the pooled control. Unless otherwise

noted, the unit of statistical analysis was the replicate test chamber. Most endpoints were analyzed using

two complementary statistical approaches. If responses appeared to be monotonic a step-down

Jonckheere-Terpstra trend test was used to evaluate trends in the ranks of replicate means to determine

possible concentration responsive trends among the treatment groups. All endpoints except for survival,

developmental stage, and histopathology severity scores also were analyzed by performing pair-wise

comparisons using Dunnett’s multiple comparison test or the Wilcoxon rank-sum test to determine which

treatment groups differed statistically from the control group. Data for endpoints analyzed by Dunnett’s

test were evaluated for normality using Shapiro-Wilk’s test and for homogeneity of variance using

Levene’s test ( Į = 0.01). When the data did not satisfy the assumptions, a log transformation was applied

to correct the data for non-normality or heterogeneous variances.

Survival, developmental stage, and histopathology severity scores were not amenable to the statistical

methods used for analysis of other endpoints. In particular, the most suitable unit of statistical analysis

for survival and histopathology severity scores was the individual animal. Therefore, survival was analyzed

using the Fishers Exact test and the Jonckheere-Terpstra trend test, and histopathology severity scores

of individuals were analyzed using the step-down Jonckheere-Terpstra trend tests only. In accordance

with recommendations of OECD TG 231 and OPPTS 890.1100, analysis of metamorphic stage was

performed using the multi-quantile analysis developed by T. Springer and J. Green, and using the

step-down Jonckheere-Terpstra trend test on stage quantiles in the replicate test chambers. Statistical

tests used to evaluate treatment effects were performed at a confidence level of Į = 0.05 with SAS

software.

There were no treatment-related effects on survival, developmental stage, normalized HLL, or

histopathology in any of the treatment groups relati ve to the pooled control. There was an increase in

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DER Template Version: 22 September 2011 wet weight on Day 21 and an increase in SVL on Days 7 and 21 in the low-dose treatment group, however,

these measurements fell within the ranges of historical control data. Additionally, because of the lack of

effects on normalized HLL and thyroid histopathology, the study author did not feel that these increases

were treatment-related.

C. Reviewer’s Analysis and Conclusions

Statistical Methods: No monotonic trends were indentified for any endpoint. Negative and solvent control

data for each endpoint were compared using an Equal Variance Two-Sample t-test. Solvent control data

were significantly greater for Day 21 wet weight, HLL, and normalized HLL. All analyses were conducted

by comparing treatment data to the negative control only. Data were tested for normality and homogeneity

of variances using the Shapiro-Wilk and Bartlett tests, respectively ( Į = 0.01). Day 7 and Day 21

developmental stage were analyzed using the Mann-Whitney U Two-Sample test ( Į = 0.05); all

remaining endpoints on Day 7 and Day 21 were analyzed using Dunnett’s Multiple Comparison test. For

Day 21 growth endpoints, all late-stage tadpoles (NF > 60) were excluded from calculation of replicate

means. Two late-stage tadpoles were identified at test termination, one in the solvent control and one

in the mid-dose treatment group. No asynchronous development was observed. Unless otherwise

indicated, effects were considered statistically significant at p < 0.05. All analyses were conducted using

CETIS v. 1.8.7.7. and the backend settings implemented by EFED on 1/30/13.

Conclusions:

No developmental or growth endpoints were significantly different between the treatment groups and the

negative control on Day 7. Further, there were no differences between the negative and solvent controls

for Day 7 endpoints. On Day 21, HLL and normalized HLL were significantly greater in the low- and

mid-dose treatment groups relative to the negative control and SVL and wet weight were significantly

greater in the low-dose treatment group. The solvent control was significantly greater than the negative

control for HLL, normalized HLL, and wet weight.Data Evaluation Record on the Toxicity ofPermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 48988601Page38of41 DER Template Version: 22 September 2011Table 16: Developmental and Thyroid Gross Pathology/HistopathologyEndpoints1,2in the AMA with Permethrin.Abbreviations: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 considered statistically significant at p< 0.05.3Hind-limb length is normalized to snout-vent length (SVL).4Difference and p value were determined from the comparison of negative and solvent control data (Equal Variance Two-Sample t-Test).Treatment(mg a.i./L)[TWA-measured]NF Developmental StageHind Limb Length3AsynchronousDevelopmentThyroid Gross andHistopathologyDay 7Day 21Day 7Day 21Day 7Day 21Day 21Median pMedian p%Diff. p%Diff. p%Diff. p%Diff. pTreatment-RelatedEffects? (Yes/No)Negative Control54 NA 57 NA NA NA NA NA NA NA NA NA NoSolvent Control454 1.0 57 1.0 -5.45 0.32 21.40.014NA NA NA NANo0.00005454 1.0 57 1.0 -3.45 0.47 21.30.011NA NA NA NANo0.0002254.5 0.57 57 1.0 3.45 0.47 22.80.007NA NA NA NANo0.0006854 0.42 57 1.0 -1.72 0.86 14.0 0.097 NA NA NA NANoStatistical Test Mann-Whitney Mann-Whitney Dunnett Dunnett NA NA--

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DER Template Version: 22 September 2011 Table 17: Growth Endpoints 1,2 

### in the AMA with Permethrin.

Abbreviations: Diff. 

### Difference. NA 

### Not applicable.

1 

### Unless otherwise indicated, effects are reported based on comparison to the negative (clean water) control.

2 

### Unless otherwise specified, effects are considered statistically significant at p < 0.05.

3 

### Difference and p value were determined from the comparison of negative and solvent control data (Equal Variance

Two-Sample t-Test).

E. Study Deficiencies

There were deviations from the guideline, as noted in the Materials and Methods section of the DER.

Except for %CV in analytical measurements, the study met all validity and performance criteria.

Specifically, the CV of measured test concentrations exceeded the recommended  20% in the lowest

and highest test treatments (50.5% and 33.0%, respectively). Given the very low concentrations used

(<1 ppb), the tendency for pyrethroids to sorb onto glassware, and that the treatment concentrations did

not overlap with other treatments, these CVs are not judged to interfere with the interpretation of test

results. Treatment

(mg a.i./L)

[TWA-measured] Snout-Vent Length Body Weight

Day 7 Day 21 Day 7 Day 21

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

Negative Control NA NA NA NA NA NA NA NA

Solvent Control 3 

### -1.94 0.34 0.95 0.68 -4.04 0.43 11.8 0.048

0.000054 5.24 0.16 6.1 0.003 10.4 0.30 26.2 0.0005

0.00022 -1.75 0.85 1.5 0.61 2.29 0.97 11.4 0.091

0.00068 0.16 1.0 1.18 0.75 5.83 0.70 11.4 0.090

Statistical Test Dunnett Dunnett Dunnett Dunnett

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DER Template Version: 22 September 2011 F. Reviewer’s Comments

The reviewer compared all treatment data to the negative control only, whereas the study author compared

treatment data to the pooled control. Therefore, the reviewer’s results are reported in the Executive

Summary section of this DER.

Because a statistically significant solvent effect was noted at Day 21 for HLL, normalized HLL and wet

weight, these endpoints were also analyzed compared to the solvent control by the reviewer. Dunnett’s

test revealed no significant differences in these endpoints for any of the test groups compared to the

solvent control.

The reviewer calculated the time-weighted average (TWA) concentrations using the following equation:

n nn nn

n n

TWA 

## t tt CC tt CC tt CC tt CC

C 1 1

21 21

12 12
01 01 

## 2 2 2 2  

 



##   ̧ 1 ·  ̈ © §    ̧ 1 ·  ̈ © §    ̧ 1 ·  ̈ © §    ̧ 1 ·  ̈ © § 

where:

C TWA is the time-weighted average concentration,

C j is the concentration measured at time interval j (j = 0, 1, 2,...n)

t j is the number of hours (or days or weeks, units used just need to be consistent in the equation) of

the test at time interval j

(e.g., t 0 = 0 hours (test initiation), t 1 =24 hours, t 2 =96 hours)

Results from the periodic screening analysis of the dilution water indicated the presence of the following

analytes: calcium (34.9 mg/L), chloride (4.5 mg/L), fluoride (0.85 mg/L), magnesium (13.2 mg/L),

potassium (7.00 mg/L), sodium (19.0 mg/L), and sulfate (5.7 mg/L).

The in-life portion of the definitive toxicity test was conducted from June 8 to 29, 2012.

### Data Evaluation Record on the Toxicity of Permethrin to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 

## III. REFERENCES

Nieuwkoop, P.D., Faber, J. (eds.). 1994. Normal Table of Xenopus laevis (Daudin): A Sytematical and

Chronological Survey of the Development from the Fertilized Egg till the End of Metamorphosis . Garland

Publishing Co, New York, NY USA.

Personal Communication Dr. Timothy Springer and Dr. John Green. November 30, 2010. Dr. Timothy

Springer, Wildlife International, Ltd., Easton, MD 21601, USA. Dr. John Green, E.I. DuPont de Nemours

and Company, Wilmington, DE 19898, USA.

Personal Communication Dr. Henry O. Krueger. 2011. Wildlife International, Ltd., Easton, MD 21601, USA.Report Date: 03 Jun-15 07:36 (p 1 of 12)

Test Code: 109701 48988601 | 05-4864-1330 

### CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 06 Nov-13 16:26 Endpoint: 07d Developmental Stage CETIS Version: CETISv1.8.7 Analysis ID: 17-3225-3301
Analysis: Nonparametric-Two Sample Official Results: Yes

Batch ID: 12-5938-9168

Start Date: 08 Jun-12

Ending Date: 29 Jun-12 Test Type: EDSP AMA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Brine: Not Applicable Species: Xenopus laevis

Source: Lab In-House Culture Analyst: K. Sappington

Age: NF51

Test Stat Critical P-Value Decision( α :5%) Ties vs Control C-mg ai/L Mann-Whitney U Two-Sample Test

DF P-Type

8 NA 2 1.0000 Non-Significant Effect Negative Control 0.000054 6 Exact

10 NA 2 1.0000 Non-Significant Effect 0.0002 6 Exact

11.5 NA 1 0.5714 Non-Significant Effect 0.0007 6 Exact NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.0007 >0.0007 1.66% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 1.1875 0.3958333 3 1.46 0.2743 Non-Significant Effect

Error 3.25 0.2708333 12

4.4375 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

0.0856 11.3 0.9935 Equal Variances Bartlett Equality of Variance Variances

0.91 0.841 0.1154 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 07d Developmental Stage Summary

54.3 54 55 54 0 4 Negative Control 0.25 0.92% 0.0% 53.5 55

54.3 54 55 54 0.000054 4 0.25 0.92% 0.0% 53.5 55

54.5 54 55 54.5 0.0002 4 0.289 1.06% -0.46% 53.6 55.4

53.8 53 54 54 0.0007 4 0.25 0.93% 0.92% 53 54.5CenteredUntransformed

Rankits -0.1

-0.2

-0.3

-0.4

-0.5

-0.6

-0.7

-0.8 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.007d Developmental Stage

C-mg ai/L 0 10 20 30 40 50 60

0 N 0.000054 0.0002 0.0007 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 03 Jun-15 07:36 (p 2 of 12)

Test Code: 109701 48988601 | 05-4864-1330 

### CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 19 Sep-13 10:52 Endpoint: 07d HLL CETIS Version: CETISv1.8.7 Analysis ID: 14-8403-4926
Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 12-5938-9168

Start Date: 08 Jun-12

Ending Date: 29 Jun-12 Test Type: EDSP AMA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Brine: Not Applicable Species: Xenopus laevis

Source: Lab In-House Culture Analyst: K. Sappington

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test

DF P-Type

0.462 2.68 0.279 0.9379 Non-Significant Effect Negative Control 0.000054 6 CDF

0.293 2.68 0.279 0.9822 Non-Significant Effect 0.0002 6 CDF

0.26 2.68 0.279 0.9874 Non-Significant Effect 0.0007 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.0007 >0.0007 12.4% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 0.01320077 0.004400257 3 0.204 0.8919 Non-Significant Effect

Error 0.259291 0.02160759 12

0.2724918 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

4.69 11.3 0.1962 Equal Variances Bartlett Equality of Variance Variances

0.91 0.841 0.1160 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 07d HLL Summary

2.25 2.17 2.35 2.25 0 4 Negative Control 0.0387 3.44% 0.0% 2.13 2.38

2.3 2.14 2.65 2.21 0.000054 4 0.121 10.5% -2.13% 1.92 2.69

2.29 2.16 2.35 2.31 0.0002 4 0.0415 3.64% -1.35% 2.15 2.42

2.23 2.08 2.36 2.24 0.0007 4 0.0617 5.54% 1.2% 2.03 2.42CenteredUntransformed

Rankits -0.05

-0.10

-0.15

-0.20 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.007d HLL

C-mg ai/L Reject Null

Reject Null

0.0 0.5 1.0 1.5 2.0 2.5 3.0

0 N 0.000054 0.0002 0.0007 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 03 Jun-15 07:36 (p 3 of 12)

Test Code: 109701 48988601 | 05-4864-1330 

### CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 19 Sep-13 10:52 Endpoint: 07d Normalized HLL CETIS Version: CETISv1.8.7 Analysis ID: 11-5716-5640
Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 12-5938-9168

Start Date: 08 Jun-12

Ending Date: 29 Jun-12 Test Type: EDSP AMA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Brine: Not Applicable Species: Xenopus laevis

Source: Lab In-House Culture Analyst: K. Sappington

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test

DF P-Type

0.866 2.68 0.013 0.7244 Non-Significant Effect Negative Control 0.000054 6 CDF

1.02 2.68 0.013 0.6277 Non-Significant Effect 0.0002 6 CDF

0.467 2.68 0.013 0.9360 Non-Significant Effect 0.0007 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.0007 >0.0007 8.93% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 0.0001787964 5.959879E-05 3 1.32 0.3145 Non-Significant Effect

Error 0.0005432247 4.526872E-05 12

0.000722021 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

5.17 11.3 0.1595 Equal Variances Bartlett Equality of Variance Variances

0.982 0.841 0.9770 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 07d Normalized HLL Summary

0.143 0.136 0.152 0.142 0 4 Negative Control 0.00395 5.52% 0.0% 0.13 0.155

0.139 0.128 0.151 0.138 0.000054 4 0.00456 6.56% 2.88% 0.124 0.153

0.148 0.146 0.15 0.148 0.0002 4 0.00094 1.27% -3.38% 0.145 0.151

0.141 0.136 0.147 0.14 0.0007 4 0.00284 4.04% 1.55% 0.132 0.15CenteredUntransformed

Rankits -0.002

-0.004

-0.006

-0.008

-0.010

-0.012

-0.014 0.000 0.002 0.004 0.006 0.008 0.010 0.012 0.014

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.007d Normalized HLL

C-mg ai/L Reject Null

Reject Null

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16

0 N 0.000054 0.0002 0.0007 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 03 Jun-15 07:36 (p 4 of 12)

Test Code: 109701 48988601 | 05-4864-1330 

### CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 19 Sep-13 10:52 Endpoint: 07d SVL CETIS Version: CETISv1.8.7 Analysis ID: 05-0978-4307
Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 12-5938-9168

Start Date: 08 Jun-12

Ending Date: 29 Jun-12 Test Type: EDSP AMA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Brine: Not Applicable Species: Xenopus laevis

Source: Lab In-House Culture Analyst: K. Sappington

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test

DF P-Type

1.97 2.68 1.11 0.1694 Non-Significant Effect Negative Control 0.000054 6 CDF

0.713 2.68 1.11 0.8184 Non-Significant Effect 0.0002 6 CDF

0.0846 2.68 1.11 0.9995 Non-Significant Effect 0.0007 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.0007 >0.0007 7.04% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 2.701876 0.9006252 3 2.63 0.0980 Non-Significant Effect

Error 4.1097 0.342475 12

6.811576 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

2.16 11.3 0.5405 Equal Variances Bartlett Equality of Variance Variances

0.984 0.841 0.9867 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 07d SVL Summary

15.8 15.3 16.2 15.8 0 4 Negative Control 0.178 2.26% 0.0% 15.2 16.3

16.6 15.6 17.6 16.6 0.000054 4 0.427 5.15% -5.17% 15.2 17.9

15.5 14.7 15.9 15.6 0.0002 4 0.264 3.42% 1.87% 14.6 16.3

15.8 15.2 16.4 15.8 0.0007 4 0.242 3.06% -0.22% 15 16.6CenteredUntransformed

Rankits -0.2

-0.4

-0.6

-0.8

-1.0

-1.2

-1.4 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.007d SVL

C-mg ai/L Reject Null

Reject Null

0 2 4 6 8 10 12 14 16 18

0 N 0.000054 0.0002 0.0007 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 03 Jun-15 07:36 (p 5 of 12)

Test Code: 109701 48988601 | 05-4864-1330 

### CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 19 Sep-13 10:52 Endpoint: 07d Wet Weight CETIS Version: CETISv1.8.7 Analysis ID: 13-2206-0011
Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 12-5938-9168

Start Date: 08 Jun-12

Ending Date: 29 Jun-12 Test Type: EDSP AMA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Brine: Not Applicable Species: Xenopus laevis

Source: Lab In-House Culture Analyst: K. Sappington

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test

DF P-Type

1.6 2.68 0.049 0.3008 Non-Significant Effect Negative Control 0.000054 6 CDF

0.356 2.68 0.049 0.9694 Non-Significant Effect 0.0002 6 CDF

0.912 2.68 0.049 0.6947 Non-Significant Effect 0.0007 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.0007 >0.0007 17.5% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 0.001975446 0.0006584821 3 0.971 0.4383 Non-Significant Effect

Error 0.00813649 0.0006780408 12

0.01011194 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

5.83 11.3 0.1202 Equal Variances Bartlett Equality of Variance Variances

0.92 0.841 0.1712 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 07d Wet Weight Summary

0.283 0.27 0.293 0.285 0 4 Negative Control 0.0049 3.46% 0.0% 0.267 0.299

0.312 0.278 0.375 0.299 0.000054 4 0.0216 13.8% -10.4% 0.244 0.381

0.29 0.268 0.305 0.293 0.0002 4 0.00808 5.58% -2.31% 0.264 0.315

0.3 0.279 0.329 0.295 0.0007 4 0.0111 7.43% -5.94% 0.264 0.335CenteredUntransformed

Rankits -0.01

-0.02

-0.03

-0.04 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.007d Wet Weight

C-mg ai/L Reject Null

Reject Null

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40

0 N 0.000054 0.0002 0.0007 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 03 Jun-15 07:36 (p 6 of 12)

Test Code: 109701 48988601 | 05-4864-1330 

### CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 19 Sep-13 10:52 Endpoint: 21d Developmental Stage CETIS Version: CETISv1.8.7 Analysis ID: 04-6216-0594
Analysis: Nonparametric-Two Sample Official Results: Yes

Batch ID: 12-5938-9168

Start Date: 08 Jun-12

Ending Date: 29 Jun-12 Test Type: EDSP AMA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Brine: Not Applicable Species: Xenopus laevis

Source: Lab In-House Culture Analyst: K. Sappington

Age: NF51

Test Stat Critical P-Value Decision( α :5%) Ties vs Control C-mg ai/L Mann-Whitney U Two-Sample Test

DF P-Type

8 NA 1 1.0000 Non-Significant Effect Negative Control 0.000054 6 Exact

10 NA 1 1.0000 Non-Significant Effect 0.0002 6 Exact

8 NA 1 1.0000 Non-Significant Effect 0.0007 6 Exact NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.0007 >0.0007 1.52% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 0.75 0.25 3 1 0.4262 Non-Significant Effect

Error 3 0.25 12

3.75 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

1 5.95 0.4262 Equal Variances Mod Levene Equality of Variance Variances

9 5.95 0.0021 Unequal Variances Levene Equality of Variance Variances

0.565 0.841 <0.0001 Non-normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 21d Developmental Stage Summary

57 57 57 57 0 4 Negative Control 0 0.0% 0.0% 57 57

57 57 57 57 0.000054 4 0 0.0% 0.0% 57 57

57.5 57 59 57 0.0002 4 0.5 1.74% -0.88% 55.9 59.1

57 57 57 57 0.0007 4 0 0.0% 0.0% 57 57CenteredUntransformed

Rankits -0.2

-0.4

-0.6

-0.8 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.021d Developmental Stage

C-mg ai/L 0 10 20 30 40 50 60

0 N 0.000054 0.0002 0.0007 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 03 Jun-15 07:36 (p 7 of 12)

Test Code: 109701 48988601 | 05-4864-1330 

### CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 19 Sep-13 10:52 Endpoint: 21d No LS HLL CETIS Version: CETISv1.8.7 Analysis ID: 20-9302-2925
Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 12-5938-9168

Start Date: 08 Jun-12

Ending Date: 29 Jun-12 Test Type: EDSP AMA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Brine: Not Applicable Species: Xenopus laevis

Source: Lab In-House Culture Analyst: K. Sappington

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test

DF P-Type

4.11 2.68 1.43 0.0039 Significant Effect Negative Control 0.000054* 6 CDF

3.46 2.68 1.43 0.0123 Significant Effect 0.0002* 6 CDF

2.17 2.68 1.43 0.1211 Non-Significant Effect 0.0007 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed

<0.000054 0.000054 17.7% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 11.26975 3.756584 3 6.57 0.0071 Significant Effect

Error 6.856555 0.5713796 12

18.12631 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

1.01 11.3 0.7982 Equal Variances Bartlett Equality of Variance Variances

0.967 0.841 0.7856 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 21d No LS HLL Summary

8.1 7.32 8.95 8.06 0 4 Negative Control 0.338 8.34% 0.0% 7.02 9.17

10.3 9.57 10.8 10.4 0.000054 4 0.267 5.19% -27.1% 9.44 11.1

9.95 9.03 11.2 9.76 0.0002 4 0.492 9.89% -22.9% 8.38 11.5

9.26 8.2 9.95 9.44 0.0007 4 0.38 8.21% -14.3% 8.05 10.5CenteredUntransformed

Rankits -0.2

-0.4

-0.6

-0.8

-1.0

-1.2

-1.4 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.021d No LS HLL

C-mg ai/L Reject Null

Reject Null

0 2 4 6 8 10 12

0 N 0.000054 0.0002 0.0007 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 03 Jun-15 07:36 (p 8 of 12)

Test Code: 109701 48988601 | 05-4864-1330 

### CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 19 Sep-13 10:53 Endpoint: 21d No LS HLL CETIS Version: CETISv1.8.7 Analysis ID: 04-5657-9563
Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 12-5938-9168

Start Date: 08 Jun-12

Ending Date: 29 Jun-12 Test Type: EDSP AMA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Brine: Not Applicable Species: Xenopus laevis

Source: Lab In-House Culture Analyst: K. Sappington

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test

DF P-Type

0.877 2.68 1.46 0.7177 Non-Significant Effect Solvent Blank 0.000054 6 CDF

0.245 2.68 1.46 0.9893 Non-Significant Effect 0.0002 6 CDF

1.02 2.68 1.46 0.6251 Non-Significant Effect 0.0007 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.0007 >0.0007 14.9% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 2.224006 0.7413353 3 1.24 0.3371 Non-Significant Effect

Error 7.151991 0.5959992 12

9.375997 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

0.942 11.3 0.8152 Equal Variances Bartlett Equality of Variance Variances

0.96 0.841 0.6569 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 21d No LS HLL Summary

9.81 8.95 10.6 9.87 0 4 Solvent Blank 0.372 7.59% 0.0% 8.63 11

10.3 9.57 10.8 10.4 0.000054 4 0.267 5.19% -4.88% 9.44 11.1

9.95 9.03 11.2 9.76 0.0002 4 0.492 9.89% -1.36% 8.38 11.5

9.26 8.2 9.95 9.44 0.0007 4 0.38 8.21% 5.67% 8.05 10.5CenteredUntransformed

Rankits -0.2

-0.4

-0.6

-0.8

-1.0

-1.2

-1.4 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.021d No LS HLL

C-mg ai/L Reject Null

Reject Null

0 2 4 6 8 10 12

0 S 0.000054 0.0002 0.0007 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 03 Jun-15 07:36 (p 9 of 12)

Test Code: 109701 48988601 | 05-4864-1330 

### CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 19 Sep-13 10:52 Endpoint: 21d No LS Normalized HLL CETIS Version: CETISv1.8.7 Analysis ID: 15-7586-3070
Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 12-5938-9168

Start Date: 08 Jun-12

Ending Date: 29 Jun-12 Test Type: EDSP AMA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Brine: Not Applicable Species: Xenopus laevis

Source: Lab In-House Culture Analyst: K. Sappington

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test

DF P-Type

3.31 2.68 0.056 0.0161 Significant Effect Negative Control 0.000054* 6 CDF

3.52 2.68 0.056 0.0111 Significant Effect 0.0002* 6 CDF

2.14 2.68 0.056 0.1276 Non-Significant Effect 0.0007 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed

<0.000054 0.000054 16.3% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 0.01364582 0.004548606 3 5.21 0.0156 Significant Effect

Error 0.01048606 0.0008738383 12

0.02413188 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

1.47 11.3 0.6891 Equal Variances Bartlett Equality of Variance Variances

0.973 0.841 0.8864 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 21d No LS Normalized HLL Summary

0.343 0.324 0.375 0.337 0 4 Negative Control 0.0113 6.58% 0.0% 0.307 0.379

0.413 0.384 0.433 0.417 0.000054 4 0.011 5.35% -20.2% 0.377 0.448

0.417 0.38 0.471 0.408 0.0002 4 0.0207 9.94% -21.4% 0.351 0.483

0.388 0.348 0.411 0.397 0.0007 4 0.014 7.2% -13.0% 0.344 0.433CenteredUntransformed

Rankits -0.01

-0.02

-0.03

-0.04 0.00 0.01 0.02 0.03 0.04 0.05 0.06

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.021d No LS Normalized HLL

C-mg ai/L Reject Null

Reject Null

0.0 0.1 0.2 0.3 0.4 0.5

0 N 0.000054 0.0002 0.0007 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 03 Jun-15 07:36 (p 10 of 12)

Test Code: 109701 48988601 | 05-4864-1330 

### CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 19 Sep-13 10:53 Endpoint: 21d No LS Normalized HLL CETIS Version: CETISv1.8.7 Analysis ID: 12-0818-3320
Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 12-5938-9168

Start Date: 08 Jun-12

Ending Date: 29 Jun-12 Test Type: EDSP AMA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Brine: Not Applicable Species: Xenopus laevis

Source: Lab In-House Culture Analyst: K. Sappington

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test

DF P-Type

0.728 2.68 0.069 0.8094 Non-Significant Effect Solvent Blank 0.000054 6 CDF

0.56 2.68 0.069 0.8977 Non-Significant Effect 0.0002 6 CDF

1.68 2.68 0.069 0.2654 Non-Significant Effect 0.0007 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.0007 >0.0007 16.0% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 0.00386035 0.001286783 3 0.978 0.4356 Non-Significant Effect

Error 0.01579626 0.001316355 12

0.01965661 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

1.84 11.3 0.6056 Equal Variances Bartlett Equality of Variance Variances

0.963 0.841 0.7239 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 21d No LS Normalized HLL Summary

0.431 0.371 0.487 0.433 0 4 Solvent Blank 0.0239 11.1% 0.0% 0.355 0.507

0.413 0.384 0.433 0.417 0.000054 4 0.011 5.35% 4.33% 0.377 0.448

0.417 0.38 0.471 0.408 0.0002 4 0.0207 9.94% 3.33% 0.351 0.483

0.388 0.348 0.411 0.397 0.0007 4 0.014 7.2% 10.0% 0.344 0.433CenteredUntransformed

Rankits -0.01

-0.02

-0.03

-0.04

-0.05

-0.06

-0.07 0.00 0.01 0.02 0.03 0.04 0.05 0.06

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.021d No LS Normalized HLL

C-mg ai/L Reject Null

Reject Null

0.0 0.1 0.2 0.3 0.4 0.5 0.6

0 S 0.000054 0.0002 0.0007 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 03 Jun-15 07:36 (p 11 of 12)

Test Code: 109701 48988601 | 05-4864-1330 

### CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 19 Sep-13 10:52 Endpoint: 21d No LS SVL CETIS Version: CETISv1.8.7 Analysis ID: 19-6248-5274
Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 12-5938-9168

Start Date: 08 Jun-12

Ending Date: 29 Jun-12 Test Type: EDSP AMA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Brine: Not Applicable Species: Xenopus laevis

Source: Lab In-House Culture Analyst: K. Sappington

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test

DF P-Type

4.22 2.68 0.902 0.0032 Significant Effect Negative Control 0.000054* 6 CDF

0.985 2.68 0.902 0.6473 Non-Significant Effect 0.0002 6 CDF

0.767 2.68 0.902 0.7865 Non-Significant Effect 0.0007 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed

<0.000054 0.000054 3.86% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 4.732066 1.577355 3 6.98 0.0057 Significant Effect

Error 2.713268 0.2261056 12

7.445334 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

3.67 11.3 0.2995 Equal Variances Bartlett Equality of Variance Variances

0.916 0.841 0.1460 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 21d No LS SVL Summary

23.4 22.3 23.9 23.7 0 4 Negative Control 0.375 3.21% 0.0% 22.2 24.6

24.8 24.3 25.3 24.8 0.000054 4 0.202 1.63% -6.07% 24.2 25.4

23.7 23.4 23.9 23.8 0.0002 4 0.122 1.03% -1.42% 23.3 24.1

23.6 23.2 24 23.7 0.0007 4 0.173 1.46% -1.1% 23.1 24.2CenteredUntransformed

Rankits -0.2

-0.4

-0.6

-0.8

-1.0

-1.2

-1.4 0.0 0.2 0.4 0.6 0.8

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.021d No LS SVL

C-mg ai/L Reject Null

Reject Null

0 5 10 15 20 25 30

0 N 0.000054 0.0002 0.0007 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 03 Jun-15 07:36 (p 12 of 12)

Test Code: 109701 48988601 | 05-4864-1330 

### CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 19 Sep-13 10:52 Endpoint: 21d No LS Wet Weight CETIS Version: CETISv1.8.7 Analysis ID: 15-7425-9361
Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 12-5938-9168

Start Date: 08 Jun-12

Ending Date: 29 Jun-12 Test Type: EDSP AMA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Brine: Not Applicable Species: Xenopus laevis

Source: Lab In-House Culture Analyst: K. Sappington

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test

DF P-Type

5.36 2.68 0.112 0.0005 Significant Effect Negative Control 0.000054* 6 CDF

2.34 2.68 0.112 0.0915 Non-Significant Effect 0.0002 6 CDF

2.34 2.68 0.112 0.0909 Non-Significant Effect 0.0007 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed

<0.000054 0.000054 13.1% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 0.1011469 0.03371564 3 9.66 0.0016 Significant Effect

Error 0.04188577 0.003490481 12

0.1430327 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

2.35 11.3 0.5023 Equal Variances Bartlett Equality of Variance Variances

0.892 0.841 0.0594 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 21d No LS Wet Weight Summary

0.855 0.725 0.913 0.89 0 4 Negative Control 0.0437 10.2% 0.0% 0.715 0.994

1.08 1.04 1.13 1.07 0.000054 4 0.0175 3.25% -26.2% 1.02 1.13

0.952 0.885 0.99 0.967 0.0002 4 0.0248 5.21% -11.4% 0.873 1.03

0.952 0.895 1 0.956 0.0007 4 0.0256 5.39% -11.4% 0.871 1.03CenteredUntransformed

Rankits -0.02

-0.04

-0.06

-0.08

-0.10

-0.12

-0.14

-0.16 0.00 0.02 0.04 0.06

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.021d No LS Wet Weight

C-mg ai/L Reject Null

Reject Null

0.0 0.2 0.4 0.6 0.8 1.0 1.2

0 N 0.000054 0.0002 0.0007 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________

# D ATA EVALUATION RECORD

PERMETHRIN

Study Type: OCSPP 890.1200, Aromatase Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-29-2012 (MRID 48618601)

Prepared for

Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by

CSS

- Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer Signature:

Joan Harlin , M.S. Date: 3 / 25/ 2012

Secondary Reviewer Signature:

Scott D. Studenberg, Ph.D. , D.A.B.T. Date: 4/1/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 4/10/ 20 12

Quality Assurance: Signature: Jack D. Early, M.S. Date: 4/10/ 20 12

This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by CSS

- Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengt hs of each individual assay to identify potential endocrine bioactivity with complementary endpoints within 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).

### PERMETHRIN / 109701 

### Aromatase ( Human Recombinant) Assay (20 11 ) 

### I Page 1 of 1 5

OCSPP 890.1200 

### I 

### OECD None



## Primary Reviewer: Edward Scollon. Ph.D.



## Health Effects Division Secondary Reviewer: Greg Akerman. Ph.D.


## Health Effec t s Division

# Si"'tlJe~~~**Date:~** Signature:

Date: 

## c Ii -r**I~** 

## -s

- - Template vers

. ion 08/2011



## DATA EVALUATION RECORD

STUDY TYPE: Aromatase ( Human Recombinant); OCSPP 890.1200



## PC CODE: 109701 

## DP BARCODE: 0398301

TXR#: 0056246



## TEST MATERIAL (PURITY}: Permethrin (97 .7% a.i

. )



## SYNONYMS: (3-phenoxyphenyl)methyl 3-(2

, 2 -dichl oroetheny l )-2

, 2- dimethylcyclopropanecarboxy lat e 

## CAS No.: 52645-53-1



## CITATION: Wilga, P.C. (20 11). Permethrin: Human Recombinant Aromatase Assay.

CeeTox

, I nc.
, Kalamazoo

, 

## Ml. Laboratory Report No.: 9146V100383AROM

, November 17

, 2011. MRID 48618601. Unpublished.



## SPONSOR: Permethrin Data Group II, c/o Consumer Specialty Products Association, Washington

, DC

TEST 

## ORDER #: CON- I 09701- 7



## EXECUTIVE SUMMARY: In an 

## in vitro aromatase (CYP 19) assay (MRID 48618601 ), permethrin (97.7% a.i.

, Lot# PMPSOOOOI4) was incubated w ith human recombinant aromatase

and tritiated androstenedione (1-P [3H(N)]-androst-4-ene-3,l 7-dione; [3H]ASDN) in dimethyl

sulfoxide ( DMSO) at log concentrations of I 0

- 1 0 

## to 10

- 3 

## M for 1 5 minutes to assess the potential

of permethrin to inhibit aromatase activity.

Aromatase activity was determined by m eas uring the amount of tritiated water produced at the

end of a 1 5-minute incubation for each concentration of chemica l. Tritiated water was quantified using liquid scintillation counting (LSC). Three runs were conducted and each run included a full activity control

, a background activity control

, a positive control series ( I 0

- 1 0 

## to 10- 5 

## M) using a known inhibitor (4 -h y droxyandro stene dione ; 4-0H ASDN)

, and the p e rmethrin series 

# c

10

- 10 

## to 10

- 3 

## M) with three repetitions per concentration.

Aromatase activity in the full activ ity contro l s was 0.588 ± 

## 0.079 nmol

· mg-protein

- 1 

## ·min

- 1
• 

## The

response of each full activity control within a run was between 90 to 110% of the average full activity, and the act i vity 

## in th e background contro l s ranged from 0.21 to 0.31 % and averaged

0.28% of the full activity. For the positive co ntrol su bstanc e (4- 0H ASDN)

, aromatase activity

results were within the recommended ranges for the performance criteria. The estimated log I C so for 4-0H ASDN averaged

- 7.3 Mand the H ill s l ope was

- l .O.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 2 of 15

PERMETHRIN / 109701 O CSPP 89 0. 1200 / OECD None



## Precipitation was noted at the two highest concentrations of permethrin tested (10 −4 

## and 10 −3 

## M); therefore, the maximum concentration used for data analysis was 10 −4.5 

## M. The lowest portion of the response curve was greater than 75% activity at all soluble concentrations of permethrin.

A romatase activity averaged 101.3% and 101.9% activity at concentrations of 10

- 10 

## and 10 −4.5 

## M permethrin

, respectively, indicating that permethrin did not inhibit aromatase activity in this

assay.

B ased on the data from the average response curve , permethrin is classified as a Non-inhibitor of aromatase activity in this assay.

The assay satisfies the EDSP Tier 1 Test Order requirement s for an Arom atase a ssay (OCSPP 890.1200).

COMPLIANCE : Signed and dated Data Confidentiality, GLP Compliance, and Quality

Assurance statements were provided.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 3 of 15

PERMETHRIN / 109701 O CSPP 89 0. 1200 / OECD None



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test S ubstance : Permethrin

Description: Liquid

Source: AMVAC

Lot # (expiration date) : PMPS000014 ( January 201 3 ) Purity: 9 7

. 7 % Volatility: N ot reported Storage conditions: Room temperature (e.g. ambient) Stability: N ot reported

Solvent: DMSO Solubility (in test solvent): Not reported Highest Concentration Tested: 10

− 3 M Stock Solution Preparation : Not reported Molecular weight: 391.28

CAS #: 52645

- 53

- 1

Structure:

O O

O Cl

CH

3 Cl

CH

3



## 2. Non

- L abeled S ubstrate : Androstenedione (ASDN)

CAS # : 63

- 05

- 8

Source: Steraloids, Inc. (Catalog # A6030

- 100)

Batch # (expiration date) : L1712 (April 2016) Purity: 99. 8 %



## 3

. Radiolabeled S ubstrate : 1

- β [ 3 

## H(N)]

- Androst

- 4

- ene

- 3,17

- dione ; [ 3 

## H] ASDN

Source: Perkin Elmer (Catalog # NET

- 926)

Batch # (expiration date) : 619344 ( January 10, 2012 ) Radiochemical Purity (Supplier): >97 % Specific activity: 26.3 mCi/mmol Radiochemical Purity (In

- lab determination): Not determined



## 4. Positive C ontrol : 4

- hydroxyandrostenedione (4

- OH ASDN)

CAS # 566

- 48

- 3

Source: Sigma

- Aldrich ( Cat alog # F2552 )

Batch # (expiration date) : 081K2133 ( March 2015 ) Purity: 99. 6 %



## 5. Solvent (V ehicle C ontrol ) : DMSO

Source:

Batch # (expiration date) : Not reported Not reported

Justification for choice of solvent One of the solvents of choice detailed in the EPA guideline.

Concentration (% of total volume in assays) ≤ 1%Aromatase (Human Recombinant) Assay ( 2011 ) / Page 4 of 15

PERMETHRIN / 109701 O CSPP 89 0. 1200 / OECD None



## 6. Test M icrosomes : Human recombinant aromatase ( CYP19 ) microsomes

Source : BD Gentest TM , Woburn, MA ( Cat alog # 456260 )

Lot # (expiration date) : 19701 ( July 201 4 )

Protein concentration : 3.7 mg / mL
Cytochrome C reductase activity: 5 4 0 nmol /mg protein/min
Aromatase activity: 5.7 p mol/ pmol P450 /min



## B. METHODS

1. Assay C omponent s and Preparations : A mixture of non- labeled and radiolabeled [ 3 

## H]ASDN was prepared such that the final concentration of ASDN in the assay was approximately 0.1 μM, and the amount of tritium added to each incubation tube was

0.1 μCi.

The test chemical(s) were formulated in DMSO such that the volume of DMSO used per assay was no more than 1% v/v of the total assay volume. DMSO was selected because it is listed as one of the solvents of choice detailed in the EPA guideline ; it not as volatile as ethanol and so evaporation was less of a concern in the assay, and is more accurate to pipette because of its density and viscosity.

A stock solution of the positive control substance, 4- OH ASDN, was formulat ed in DMSO

. Dilutions wer e prepared such that the target concent rations of the positive control substance (1 0

− 10 

## to 10

− 5 

## M; Table 4) were achieved by the addition of 20 μL of the dilution for a final assay volume of 2 mL.

Human recombinant microsomes were purchased from BD Gentest TM 

## , and stored at

− 80 ±

10oC. Before use of the microsomes, the batch was thawed and sub -aliquoted into individual vials before being refrozen to minimize freeze-thaw cycles to no more than one.

Other assay components sodium phosphate buffer, propylene glycol, and NADPH are reported in Table 1.

T ABLE 1. Assay Components and Conditions
Assay Factor Values
sodium phosphate buffer (pH 7.4) 0.1M

Microsomal Protein 0.00 4 mg/mL

NADPH 0.3 mM

[ 3 H]ASDN 100 nM Propylene Glycol 5%
Temperature 37°C

Incubation Time 15 min

a Data were obtained from pages 16 and 17 of the study report.



## 2. Suitability Ass es s ments : The protein concentration of the microsomes (Lot #19701 ) was supplied by the vendor as 3.7 mg/m L . Aromatase activity of the microsomes was also provided by the vendor as 5.7 pmol/min/ pmol P450. The mean aromatase activity in the full activity control samples over the three test runs was determined to be 0.509 nmol/min/mg protein, which was greater than the minimum recommended aromatase activity of 0.1 nmol/mg-p rotein/min.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 5 of 15

PERMETHRIN / 109701 O CSPP 89 0. 1200 / OECD None



## 3. Aromatase A ssay : Each assay run contained 4 tubes for the full enzyme activity and 4 tubes for the background activity controls. Two tubes of each control were run at the beginning of the assay, and two of each control were run at the end of the assay. A full concentration curve in duplicate for the positive control, and a full concentration curve in triplicate for the test substance were established. The aromatase assay was conducted generally according to the procedures described in OCSPP 890.1200 (Section h, pp. 9-10).

The amount of 3 

## H 2 

## O in the aqueous fraction was quantified for each assay tube by LSC, and aromatase activity was reported in units of p mol∙mg

- protein

− 1 

## ∙min

− 1



## .

4. Demonstration of P roficiency : The report stated that proficiency testing of th e CYP19 aromatase assay was conducted. Each of the proficiency chemicals (atrazine, econazole, fenarimol and nitrofen) were incubated alongside the positive control inhibitor (4- OH

ASDN ) on three separate occasions using the same methods employed in the current study.
However, t he actual data from the proficiency testing were not included in the study report.

Positive Control

(1) Initial Demonstration of Laboratory P roficiency : The positive control data for laboratory met the following criteria:

• Mean aromatase activity in the absence of an inhibitor was at least 0.1 nmol/mg

protein/min.

• Mean background control activity was ≤ 15 % of the full activity control.

• Coefficient of variation (CV) for replicates within each sample type and concentration

of 4

- OH ASDN was <15%.

• Performance criteria (Table 2) were met, and served as guidance in identifying runs that provided parameters in the preferred ranges.

(2) Demonstration o f P roficiency of N ew T echnician for C onducting Assay (when applicable) : The positive control data for slope, top and bottom percent met the criteria as

listed in section (i ) of OCSPP 890.1200.

TABLE 2. Performance Criteria for the Positive Control

Parameter Lower Limit Criteria Upper Limit Criteria Actual Lower Limit Actual Upper Limit
Slope

− 1.2

− 0.8

− 1.0 3

− 0. 97 Top (%) 90 110 9 9.6 10 1 .2
Bottom (%)

− 5 +6 0.1 1.1 Log IC 50 (M)

− 7.3

− 7.0

− 7. 3

− 7. 2

a Data were obtained from pages 18 and 23 of the study report.



## 5. Proficiency C hemicals : The proficiency of the laboratory was demonstrated prior to running any test chemicals by conducting three full scale test runs on each of the proficiency

chemical s as listed in Table 3. The data were not included in the study report. It was stated

that a trazine, econazole, fenarimol, and nitrofen were correctly classified as non -inhibitor or

inhibitor.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 6 of 15

PERMETHRIN / 109701 O CSPP 89 0. 1200 / OECD None



### TABLE 3 . Proficiency Chemicals
Compound CAS# Class Concentrations

Econazole 24169

- 02

- 6 Inhibitor Not reported

Fenarimol 60168

- 88

- 9 Inhibitor Not reported

Nitrofen 1836

- 75

- 5 Inhibitor Not reported

Atrazine 1912

- 24

- 9 Non

- inhibitor Not reported



## 6. Determination of Aromatase A ctivity with T est C hemical(s) : T he response of aromatase activity to the presence of eight concentrations of permethrin per run , in triplicate, was

tested during three independent runs (Table 4)

. Solubility was assessed (presence of cloudiness or a precipitate). If insolubility was observed at the highest test concentration (10 -3 

## M) for the first run, then the highest test concentration would be adjusted for the second and third runs at the highest test concentration that appeared soluble using log or

half -log concentrations.

The full enzymatic activity ( >98 % for the means of each run) was obtained at the two lowest

co ncentrations of p ermethrin , defining the top of the concentration-response curve.

T ABLE 4. Test Chemical Study Design for each Test Run a

Sample Type Repe titions (Tubes) Description Reference or Chemical (M)
Full Activity Control 4 All test components b plus solvent vehicle N/A Bkgd Activity Control 4 Same as above without NADPH N/A

4

- OH ASDN Conc 1 2 All test components plus 4

- OH ASDN 1×10

− 5

4

- OH ASDN Conc 2 2 All test components plus 4

- OH ASDN 1×10

− 6

4

- OH ASDN Conc 3 2 All test components plus 4

- OH ASDN 1×10

− 6.5

4

- OH ASDN Conc 4 2 All test components plus 4

- OH ASDN 1×10

− 7

4

- OH ASDN Conc 5 2 All test components plus 4

- OH ASDN 1×10

− 7.5

4

- OH ASDN Conc 6 2 All test components plus 4

- OH ASDN 1×10

− 8

4

- OH ASDN Conc 7 2 All test components plus 4

- OH ASDN 1×10

− 9

4

- OH ASDN Conc 8 2 All test components plus 4

- OH ASDN 1×10

− 10

Permethrin Conc 1 c,d 3 All test components plus Permethrin 1×10

- 3

Permethrin Conc 2 c,e 3 All test components plus Permethrin 1×10

− 4

Permethrin Conc 3 c,f 3 All test components plus Permethrin 1×10 −4.5

Permethrin Conc 4 c 3 All test components plus Permethrin 1×10 −5

Permethrin Conc 5 c 3 All test components plus Permethrin 1×10 −6

Permethrin Conc 6 c 3 All test components plus Permethrin 1×10 −7

Permethrin Conc 7 c 3 All test components plus Permethrin 1×10 −8

Permethrin Conc 8 c 3 All test components plus Permethrin 1×10 −9

Permethrin Conc 9 c 3 All test components plus Permethrin 1×10 −10

a Data we re obtained from pages 19 of the study report.

b The complete assay contained buffer, propylene glycol, microsomal protein, [ 3 H]ASDN, and NADPH

. c T est chemical

. d Highest concentration in Run #1; not used in Runs #2 and 3 due to precipitation.

e Highest concentration in Runs #2 and 3.

f Concentration added to Runs #2 and 3.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 7 of 15

PERMETHRIN / 109701 O CSPP 89 0. 1200 / OECD None



## C. D ATA ANALYSIS

1. Raw D ata : Raw data were converted to aromatase activity (nmol/mg protein/min) and percent control for the positive control, proficiency chemicals and test chemical(s) . The following raw data and calculated endpoints for each run were included in the report (Table 5).

T ABLE 5. Raw and Calculated Data

Raw/Calculated Data Included (X)
DPM/mL for each portion of extracted aqueous incubation mixture X Average DPM/mL for each aqueous portion (after extraction) X

Total DPM for each aqueous portion (after extraction) X The total DPM present in the assay tube at initiation X The percentage of substrate converted to product X Total DPM after extraction corrected for background X Aromatase activity expressed in nmol/mg protein/ min X Average aromatase activity in the full activity control tubes X Percentage of control activity remaining in the presence of various inhibitor concentrations X

DPM Disintegrations per minute



## 2. S tatistical M ethods : For data generated at CeeTox, basic statistical analysis was performed on the data, which included means of replicates, standard deviation of the mean, standard error of the mean, and coefficient of variation. Analysis of variance (ANOVA) was not performed.



## The response curve was fitted by weighted nonlinear regression analysis using a 4-parameter regression model (XLfit; IDBS; Version 5.2.0.0; Fit Model 208) and Tukey’s BiWeight statistical analysis for outlier analysis. For each independent run of the test substance, the individual percent of control values were plotted versus logarithm of the test chemical or positive control concentration. The fitted concentration response curves were superimposed on the plot, with individual plots prepared for each run (Figures 1-3), and with plotted means (Figure 4).



## Assay drift was assessed by comparing the % full activity and background activity at values at the beginning of each run to the end of each run.

3. Interpretation of Results : Interpretation of the assay results was based on the average of three runs, using the categories presented in Table 6.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 8 of 15

PERMETHRIN / 109701 O CSPP 89 0. 1200 / OECD None



### TABLE 6 . Interpretation of Results

Criteria Interpretation

Data fit 4

- parameter nonlinear regression model Average curve across runs crossed 50% a Inhibitor Average lowest portion of curves across runs is between 50% and 75% activity b Equivocal

Average lowest portion of curves across runs is greater than 75% activity b Non

- inhibitor

Data do not fit model

---

a Ordinarily, an inhibition curve will fall from 90% to 10% over 2 log units with a slope near

− 1. Unusually steep curves may indicate protein denaturing or solubility issues. If the slope of the curve is steeper than

− 2.0, the result is classified as equi vocal.

b If the test compound was not soluble above 10

− 6 M and the inhibition curve does not cross 50%, the chemical is typically

determined to be untestable in the aromatase assay

.



## II

. RESULTS

A. CONTROL ACTIVITY : Aromatase activity in the full activity controls ranged from

0.494-0.714 nmol

∙ mg -protein

− 1



## ∙ min

− 1 

## for the 3 test runs, with a mean and standard deviation

of 0.588 ± 0.079 nmol

∙ mg

- protein

− 1



## ∙ min

− 1 

## . Activity in the background controls ranged from 0.21 to 0.31% and averaged 0.28% of the full activity controls. The response of each full activity control within a run was between 90 to 110% of the average full activity.

B. POSITIVE CONTROL : For the positive control substance (4- OH A S DN), aromatase activity averaged 0.513-0.666 nmol∙mg -protein −1 

## ∙min −1 

## at the lowest tested concentration

10 −10 

## M and 0.004 nmol∙mg -protein −1 

## ∙min −1 

## at the highest tested concentration 10 −5 

## M. The mean aromatase activity of the positive control (expressed as % full control activity) for

each concentration tested across all 3 runs is presented in Table 7, along with the overall

standard deviation , SEM, and % CV. Inhibition response curves for the positive control from each run are shown in Figures 1-3, and the average inhibition response curve across all runs is shown in Figure 4.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 9 of 15

PERMETHRIN / 109701 O CSPP 89 0. 1200 / OECD None



### TABLE 7 . Effect of Permethrin on Aromatase Activity (as percent of control) from Independent Runs a

Chemical Concen. ( Log M ) # Runs Overall Mean b Overall SD b Overall SEM b Overall % CV b

4

- OH AS D N (positive control) −5 3 0. 6 7 0. 05 0.0 3 7 %

−6 3 5. 8 9 0.5 0 0.29 9%

−6.5 3 15.6 1.3 3 0.77 8 %

−7 3 34.8 2.0 3 1.17 6%

−7.5 3 63.1 1.99 1.15 3%

−8 3 84.7 3.5 2 2.03 4%

−9 3 99.2 0.7 6 0.44 1%

−10 3 99.6 1.8 4 1.06 2%

Permethrin

- 4 .5 c 2 101.9 3. 87 2. 7 4 4%

−5 3 96.4 9.6 5 5. 5 7 10 %

−6 3 101.7 5.00 2.8 8 5%

−7 3 101.1 2.6 3 1.52 3%

−8 3 102.7 5.3 0 3.06 5%

−9 3 102.6 3. 59 2.0 7 4 %

−10 3 101.3 3. 76 2.17 4%

a Data were obtained from Appendix 1, pp. 41, 43, and 45 of the study report

b Calculated by the reviewers from data presented in this table.

c Data obtained for Runs 2 and 3; test concentration was not used for Run 1.

SD Standard Deviation

SEM Standard error of the mean

CV Coefficient of Variance



## C. TEST SUBSTANCE : For p ermethrin, aromatase activity averaged 0.532–0.674 nmol

∙ mg

protein

− 1



## ∙ min

− 1 

## at the lowest tested concentration 10

− 10 

## M and 0.528–0.572 nmol

∙ mg

protein

− 1



## ∙ min

− 1 

## at the highest tested concentration used for study interpretation, 10

− 4.5 

## M. In the first test run of the assay, permethrin was found to be soluble at concentrations of ≤ 10

-

4.5 

## M; therefore, for Runs 2 and 3 of the assay, the 10

- 4.5 

## M concentration was the highest tested concentration used for study interpretation. The mean aromatase activity of

p ermethrin (expressed as % full control activity) for each concentration tested across all 3 runs is presented in Table 7 (presented above) , along with the overall standard deviation, SEM, and % CV. Inhibition response cur ves for p ermethrin f rom each run are shown in Figure s 1-3 , and the average inhibition response curve across all runs is shown in Figure 4.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 10 of 15

PERMETHRIN / 109701 O CSPP 89 0. 1200 / OECD None



## F IGURE 1. Inhibition R esponse C urves for Permethrin and 4

- OH ASDN F rom Run 1.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 11 of 15

PERMETHRIN / 109701 O CSPP 89 0. 1200 / OECD None



## F IGURE 2. Inhibition Response Curves for Permethrin and 4

- OH ASDN From Run 2.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 12 of 15

PERMETHRIN / 109701 O CSPP 89 0. 1200 / OECD None



## F IGURE 3. Inhibition Response Curves for Permethrin and 4

- OH ASDN From Run 3.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 13 of 15

PERMETHRIN / 109701 O CSPP 89 0. 1200 / OECD None



## F IGURE 4. Mean Inhibition Response Curves for Permethrin and 4

- OH ASDN.

The average dose- response curve indicated that the aromatase activity of p ermethrin at all

soluble test concentrations tested was essentially equivalent to the activity observed in the full activity control. These data could not be fit to the 4- parameter nonlinear regression model; therefore, log IC 50 

## and Hill slope estimates were not determined for permethrin. For

4- OH ASDN, the estimated log IC 50 

## average d

− 7.3 M and the Hill slope was

− 1.0 (Table 8)

. The variation in the positive control values was acceptable (<1 5% CV).

TABLE 8 . Effect of Permethrin on Aromatase Activity (as P ercent of C ontrol) F rom Independent Runs a

Chemical Run 1 Run 2 Run 3 Mean b SD b % CV b

Log IC 50 ( M )

Permethrin N A NR NR NR NR NR

4

- OH A SD N

- 7.3

- 7.3

- 7.2

- 7.3 0.06 0.80 Slope

Permethrin NR NR NR NR NR NR

4

- OH A SD N

- 0.99

- 0.97

- 1.03

- 1.0 0.03 3 .0

a D ata were obtained from Table 13, page 29 of the study report

b Calculated by the reviewers from data presented in this table.

NR No Result

SD Standard Deviation

CV Coefficient of VarianceAromatase (Human Recombinant) Assay ( 2011 ) / Page 14 of 15

PERMETHRIN / 109701 O CSPP 89 0. 1200 / OECD None



## Based on the data from the average response curve and the criteria listed above in Table 8, the results support the conclusion that permethrin does not inhibit aromatase activity in the
aromatase assay . The lowest portion of the response curve across all runs was greater than 75% activity at all concentrations of permethrin, indicating that permethrin is not an inhibitor of aromatase activity in this assay. The m ean aromatase activity for permethrin was 101.9 ± 3.9% at the highest soluble test concentration of 10 −4.5 

## M.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS CONCLUSIONS : Permethrin was tested at final concentrations of

10 −10 

## to 10 −4.5 

## M. Aromatase activity, as a percent of control, was determined to be 101.9% (+ 3.9% SD) at the highest soluble concentration of permethrin tested (10 −4.5 

## M)

, classifying

it as a non -inhibitor of aromatase according to EDSP guideline O CS PP 890.1200.

B. AGENCY COMMENTS : Although data demonstrating proficiency with the positive control or recommended proficiency chemicals were not included, a statement was included

that proficiency of the laboratory was demonstrated prior to analysis of permethrin by the

conduct of three full scale test runs on each of the recommended proficiency chemicals. Although t he data were n ot included, i t was stated that the proficiency chemicals were correctly classified as non -inhibitor or inhibitor.

Aromatase activity in the full activity controls was 0.588 ± 0.079 nmol

∙ mg -protein −1



## ∙ min −1



## . The response of each full activity control within a run was between 90 to 110% of the average full activity , and the activity in the background controls ranged from 0.21 to 0.31%

and averaged 0.28 % of the full activity controls.

For the positive control substance (4

- OH A S DN), aromatase activity was within the recommended ranges for the top of the curve, bottom of the curve, H ill slope, log IC 50 

## , and

%CV. T he estimated log IC 50 

## for 4

- OH ASDN averaged − 7.3 M and the Hill slope was

−1.0.

For p ermethrin, aromatase activity at the lowest and the highest tested concentrations, 10

− 10



## and 10 −4.5 

## M, were 101.3% and 101.9% of control , respectively

. The two highest test

concentration of permethrin (10

− 3 

## and 10

− 4 

## M) showed precipitation during the 15- minute incubation period; therefore, the results of these concentrations were not used for data analysis

. The average dose

- response curve indicated that the aromatase activity of

p ermethrin at concentrations from 10 −10 

## to 10 −4.5 

## M was essentially equiv alent to the activity observed in the full activity control. Therefore, these data could not be fit to the 4- parameter nonlinear regression model. Because the mean aromatase activity for permethrin

was greater than 75% ( 101.9 ± 3.9% at the highest soluble test concentration of 10 −4.5 

## M), permethrin was determined to be a “non-inhibitor” of aromatase activity in this assay.

C. STUDY DEFICIENCIES : The following deficiencies were noted that are not considered to have had an adverse impact on the results, interpretation or conclusions of this study:Aromatase (Human Recombinant) Assay ( 2011 ) / Page 15 of 15

PERMETHRIN / 109701 O CSPP 89 0. 1200 / OECD None



## • For permethrin, only seven test concentrations were used for data interpretation because permethrin was insoluble at the 10

− 3 

## M and 10

− 4 

## M test concentrations for Run 1 . It was

determined that the highest concentration to be tested in Runs 2 and 3 would be 10

− 4.5


## M, as p ermethrin was soluble at this concentration . A second mid- log concentration (i.e. 10

− 6.5 

## M) should have been added in order to obtain the required eight test concentrations, as noted in section ( j ) of OCSPP 890.1200.

• For permethrin, the coefficient of variation for replicates within the 10

− 5 

## M test concentration was 21.8%, which is outside the 15% guideline.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Permethrin

EPA MRID Number 48702301

Page 1 R f 49

Version: 22 September 2011 Data Requirement: EPA DP Barcode 406018

OECD Data Point

EPA MRID 48702301

EPA Guideline 890.1350

Fish Short-Term Reproduction Assay

Test Material: Permethrin Purity (%): 97.7%

Common Name Permethrin

Chemical Name IUPAC Not Reported

CAS Name Not Reported

CAS No. Not Reported

Synonyms Not Reported

EPA PC Code 109701

Primary Reviewer: John M. Marton, Ph.D. Date: 6/8/13

Environmental Scientist, CDM Smith

Secondary Reviewer(s): Teri S. Myers, Ph.D. Date: 6/10/13

Environmental Scientist, CDM Smith

Primary Reviewer: Keith G. Sappington, M.S.

Senior Science Advisor, USEPA/OPP/EFED/ERB5 Date: 9/17/2013

Additional (and final) Reviewer: Justin Housenger

Biologist, USEPA/OPP/EFED/ERB5 Date: 5/27/15

Date Evaluation Completed: 29-MAY-2015 3

Digitally signed by JUSTIN

HOUSENGER
DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=JUSTIN
HOUSENGER, dnQualifier=0000044455

Date: 2015.06.03 11:55:10 -04'00' 

# Keith Sappington Digitally signed by Keith Sappington DN: cn=Keith Sappington, o, ou, email=sappington.keith@epa.gov,

c=US

Date: 2015.06.10 13:58:15 -04'00'

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Permethrin

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Version: 22 September 2011 CITATION: Schneider, S.Z., K.H. Martin, T.Z. Kendall, and H.O. Krueger. 2012. Permethrin: Fish Short-Term

Reproduction Assay with the Fathead Minnow ( Pimephales promelas ). Unpublished study performed by

Wildlife International, Ltd., Easton, Maryland. Laboratory report number 701A-108. Study sponsored by

Consumer Specialty Products Association for the Permethrin Data Group II Task Force Steering

Committee/Joint Venture, Washington, DC. Study completed September 11, 2012.

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

screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact 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

within 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).

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Permethrin

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Version: 22 September 2011 EXECUTIVE SUMMARY

The 21-day short-term reproduction assay of permethrin with fathead minnow ( Pimephales promelas ) was

conducted under flow-through conditions. Adult fish (6 months old, 2 males and 4 females per replicate) were

exposed to permethrin (97.7% purity) at nominal concent rations of 0 (negative and solvent controls), 0.00033,

0.00099, and 0.0030 mg a.i./L. Time-weighted average (TWA) concentrations were <0.000150 (<LOQ;

controls), 0.00022, 0.00075, and 0.0026 mg a.i./L. The solvent control consisted of dimethylformamide

(DMF) at a final concentration of 0.02 mL/L. The test system was maintained at 24.6 to 25.8°C and a pH

of 7.9 to 8.1.

Unless otherwise indicated, all effects are reported based on comparison to the negative (clean water) control.

Survival in the negative control and the low and mid treatment groups was 100%, but there was 63% survival

for males (5 of 8) and 50% survival for females (8 of 16) at the high treatment concentration. Additionally,

clinical signs of toxicity occurring in the high treatment group included erratic swimming (5 fish), loss of

equilibrium (2 fish), and surfacing (2 fish). There were also observations of excess feed noted in the test

chambers of the high treatment group on Day 10 and Days 17-19. These observations indicate that overt

toxicity was present during the course of this study at the high treatment concentration.

Fecundity in the negative control averaged 19.8 eggs/female/reproductive day with all replicates having at

least 15 eggs/female/reproductive day; spawning occurred at least every 4 days. Fertilization success in the

negative control averaged 95.4%. Permethrin had no significant effect on fecundity or fertility in any treatment

group relative to the negative control (p>0.05). While not statistically significant (p>0.05), there was a 42%

decrease on fecundity at the high treatment concentration.

Plasma vitellogenin (VTG) concentrations were comparable between the negative control and treatment groups

for both sexes. While not statistically significant (p>0.05), there was a 37.8% reduction in VTG as compared

to the negative control at the high treatment concentration. The data from this treatment group were based on

three replicates and not four (as the rest of the treatment groups) as 3 of 4 female fish from Replicate A died

during the course of the study.

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Version: 22 September 2011 Effects on gonadal histopathology were observed in female fish at the high treatment level, including increased

post-atretic follicles, decreased post-ovulatory follicles, and granulomatous inflammation. Microsporidia were

observed in one fish each of the control and treatment concentrations that showed a minimal to mild severity

of granulomatous inflammation. There were no effects of treatment on gonadal histopathology in male fish.

A significant increase (p<0.05) in gonado-somatic index (GSI) was detected at the high treatment level for

both sexes ( Ĺ 48.9% for males and 28.4% for females). Nuptial tubercle score was significantly decreased in

males at the high concentration (<0.05). Permethrin exposure was not associated with any significant changes

(p>0.05) in body weight or length for either sex.

Other clinical signs ( i.e. , behavioral and other sublethal effects) including bloody lower jaw and weakness were

observed in 1 to 3 of the 8 surviving females at the high treatment level.

All validity and performance criteria were met for this study.

This assay satisfies the EDSP Tier 1 Test Order requirements for a Fish Short-Term Reproduction Assay

(OCSPP Guideline 890.1350).

Results Synopsis:

Test organism age at test initiation: 6 months

Mean body weight at test initiation (if measured): 2.5 g (males), 1.0 g (females); individuals were

within ±20% of the mean for each sex

Mean length at test initiation (if measured): Not measured

Test type: Flow-throughData Evaluation Record on the Fish Short-Term Reproduction Assay with PermethrinEPA MRID Number 48702301Page5Rf49Version: 22 September 2011Table 1: Summary of Reproductive and HPG Effects1,2in the Fish Short-Term Reproduction Assay (FSTRA) with Permethrin.Treatment(mg a.i./L)[TWAmeasured]FecundityFert.SuccessTubercle ScoreGSIGonadal Histo.Plasma VTGPlasma TPlasma E2MFMFMFMFMFMF0.00022 No No No -- No No No No No No -- -- -- --0.00075 No No No -- No No No No No No -- -- -- --0.0026No3No Yes -- Yes Yes No Yes No No -- -- -- --Abbreviations:Conc.Concentration.Diff.Difference.E217ȕ-estradiol.FFemale.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.MMale.NANot applicable.TTestosterone.VTGVitellogenin.1A “yes” indicates a significant difference based on comparison to the negative (clean water) control, unless otherwise specified.2The criteria for significance are described in the Reviewer’s Analysis and Statistical Verification sections of the DER. Conclusions regarding histopathology may be heavilyweighted by the expert opinion of a board-certified pathologist.3While not statistically significant (p>0.05), fecundity was reduced at highest treatment concentration 42% as compared to thenegative control.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Permethrin

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Version: 22 September 2011 I. MATERIALS AND METHODS

Guideline Followed: This study was conducted following guidelines outlined in the U.S. EPA series

890- Endocrine Disruptor Screening Program Test Guidelines, OCSPP Number

890.1350: Fish Short-Term Reproduction Assay ; and the OECD Guidelines for

Testing of Chemicals, Guideline 229: Fish Short Term Reproduction Assay . The

following deviations were noted:

1. The unionized ammonia and residual chlorine concentrations of the dilution water were not

reported.

2. Age of the test organisms used in the range-finder was not specified.

3. Light intensity (163-760 lux) ranged lower than specified (540-1080 lux).

These deviations do not have an impact on the interpretation of the results of this study.

Compliance: Signed and dated No Data Confidentiality, GLP, and Quality Assurance

statements were provided. This study was conducted in compliance with Good

Laboratory Practice Standards as published by the U.S. Environmental

Protection Agency (40 CFR Parts 160 and 792); and OECD Principles of

Good Laboratory Practice (ENV/MC/CHEM (98) 17), with the following

exceptions: periodic analyses of water for potential contaminants were not

performed according to Good Laboratory Practice Standards, but were not

performed using a certified laboratory and standard US EPA analytical methods;

and the characterization of the test substance, and the stability of the test

substance under the conditions at the test site, were not determined in

accordance with Good Laboratory Practice Standards.

A. Test Material Permethrin

Description: A liquid

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Permethrin

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Version: 22 September 2011 OECD recommends describing water solubility, melting/boiling point stability in water and light, pKa,

Pow or Kow, vapor pressure of test compound, expiration date.

Lot No./Batch No. : PMPS000014 (Lot No.)

Purity: 97.7%

Impurities: Not reported

Stability of Compound: Stable. Mean-measured concentrations yielded recoveries of 70 to 87% of

nominal with coefficients of variation of 7 to 16%.

Storage Conditions of

Test Chemicals: Stored under ambient conditionsData Evaluation Record on the Fish Short-Term Reproduction Assay with PermethrinEPA MRID Number 48702301Page8Rf49Version: 22 September 2011B. Test OrganismTable 2: General Information About the Test Species and Acclimation.ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies common name:Fathead minnow EPA recommends fathead minnow (Pimephales

promelas).Species scientific name: Pimephales promelasSpecies strain (if stated):Not ReportedWere fish obtained from a singlelaboratory stock?Yes Adult fish were laboratory-raised fishobtained from Osage Catfisheries, Inc.(Osage Beach, Missouri). EPA recommends that fish be from a single

laboratory stock.Were acclimation conditions sameas definitive test?Yes EPA recommends that fish be acclimated under water quality and illumination conditions that are similar to the definitive test.Acclimation period:Two months prior to preexposure period (14days) EPA recommends a minimum two-week acclimation period. Note that the acclimation period is different from the subsequent, in situ pre-exposure phase.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PermethrinEPA MRID Number 48702301Page9Rf49Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on health:Fish showed no signs ofdisease or stress, andmortality was <5% duringthe 7 days prior to thetest.Fish did not receive treatment for diseaseduring the acclimation period, the two-weekpre-exposure period, or the exposure period. EPA recommends that mortality during the 7 days prior to the pre-exposure phase be less than 5% of the culture population. If mortality during these 7 days is greater than 10%, EPA recommends that the fish be rejected. If mortality is between 5-10%, EPA recommends that fish be held another 7 days. If mortalities greater than 5% occur during this extended acclimation period, EPA recommends that the fish not be

used.Type of food:Commercial flake food(Sera Vipan) and brineshrimp nauplii ( Artemiasp.). EPA recommends that fish be fed frozen brine shrimp twice per day to promote active reproduction and maintain body condition.Source of food:-Sera North America-Brine Shrimp Direct,Ogden, UtahFrequency of feeding:2 times/dayData Evaluation Record on the Fish Short-Term Reproduction Assay with PermethrinEPA MRID Number 48702301Page10Rf49Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on feeding:Fish were fed twice a dayduring the 14-day perexposure period and 21-day exposure period.Feeding was suspendedfor at least 12 hours priorto test termination.Feeding rates in each replicate wereadjusted approximately weekly, if necessary,to ensure that the feeding rate per fishremained constant. Uneaten food and fecalmaterial were removed from test chambersdaily by siphoning or by replacement withclean tanks.Table 3: Fish Selection and Pre-Exposure Performance.ParameterValue(s)Details or RemarksGuideline RecommendationsAge at test initiation:6 months EPA recommends reproductively mature (sexually dimorphic) fish, 4.5 - 6 months old.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PermethrinEPA MRID Number 48702301Page11Rf49Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsMean weight of males at testinitiation (if determined):2.5 g Based on sub-sample (n=10). EPA recommends that a subsample of fish be weighed before the test to estimate the mean weight for each sex. It is recommended that the individual weight of each fish selected for the test be within ±20% of the estimated mean for

each sex.Range of individual weights(males) at test initiation (ifdetermined):Not reportedIndividual weights were within ±20% of themean.Mean weight of females at testinitiation (if determined):1.0 g Based on sub-sample (n=10).Range of individual weights(females) at test initiation (ifdetermined):Not reportedIndividual weights were within ±20% of themean.Mean length of males at testinitiation (if determined):Not determinedMean length of females at testinitiation (if determined):Not determinedDuration of pre-exposure phase: 14 days EPA recommends a minimum of 14 days.Were pre-exposure conditionsidentical to the definitive test?Yes EPA recommends that pre-exposure conditions, including temperature, photoperiod, feeding, etc., be identical to definitive test conditions.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PermethrinEPA MRID Number 48702301Page12Rf49Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of pre-exposure tanks: 32Almost twice the number of breeding groupswas maintained in the pre-exposure periodas in the exposure period so that breedinggroups with a proven history of spawningcould be selected for inclusion in theexposure phase of the test. EPA recommends that additional tanks set up at the beginning of pre-exposure will ensure that sufficient replicates with the correct sex ratio are available for the definitive test.Number of males per tank: 2Number of females per tank: 4Pre-exposure fecundity:>17.6 eggs/female/reproductive day/replicate EPA recommends that pre-exposure fecundity in each replicate (tank) selected for use in the definitive test be at least 15 eggs/female/reproductive day/replicate during the 7 days prior to the definitive test.Number of spawns during preexposure:>2 times in 7 days EPA recommends that spawning occur at least twice in the 7 days prior to the definitive test.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PermethrinEPA MRID Number 48702301Page13Rf49Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on pre-exposure:Any pre-exposure aquariawith mortalities, incorrectsex ratio, or poorperformance wereexcluded from use in thetest.C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:Flow-through EPA recommends the use of a flow-through system. As noted in the Corrections and Clarifications document 1 , the use of a static renewal system is not recommended for this

assay.1U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the Endocrine Disruptor ScreeningProgram Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention (OCSPP), Washington, D.C.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PermethrinEPA MRID Number 48702301Page14Rf49Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsType of flow-through dilutionsystem:Continuous-flow diluter Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 2Flow-through rate:~62.5 mL/minFlow rate (reviewer-calculated)provided approximately 9 volumeadditions per day. Recommended flow-through rate is 45 mL/min (2.7 L/hr), or at least 6 total volume exchanges per day.Details on toxicant mixing forflow-through systems:A syringe pump was used to delivervolumes of test substance stocksolutions and solvent (DMF) tomixing chambers where they weremixed with well water to obtain thedesired concentration. Flow wascontrolled using rotameters andsolutions in each mixing chamberwere pumped to chambers using aperistaltic pump.Recommended toxicant mixing for flowthrough systems: 1) Mixing chamber is recommended but not required; 2) Aeration is not recommended for mixing; 3) A 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%.(http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf).2Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PermethrinEPA MRID Number 48702301Page15Rf49Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsAeration? No EPA recommends aeration if dissolved oxygen reaches <4.9 mg/L (<60% saturation).Source of dilution water:Well water located on WLI property.Water was passed through a sandfilter to remove particles greater than~25μm and aerated. Prior to use,water was filtered to 0.45μm toremove fine particles and was passedthrough a UV sterilizer. EPA recommends natural or reconstituted water; it is recommended that natural water be sterilized with UV and tested for pesticides, heavy metals, and other possible contaminants. 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. See Reviewer’s Comments.Test vessel type/materials: GlassEPA and OECD recommend that watercontact portions of the system not compromise the study (e.g., all glass vessels or glass vessels with stainless steel frames are acceptable examples).Data Evaluation Record on the Fish Short-Term Reproduction Assay with PermethrinEPA MRID Number 48702301Page16Rf49Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsTest vessel size:12 L EPA recommends the use of 18 L test chambers (e.g., 40 x 20 x 20 cm).Fill volume:10 L EPA recommends 10 L solution per tank.Spawning substrate material: PVC EPA recommends that each tank contain three semi-circular spawning substrates, e.g., aged PVC pipe, 10 - 20 cm in length, split

lengthwise.Spawning substrate size: 10 cm (3 rep test chamber)Additional details on exposuresystem:NoneTable 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurement IntervalGuideline RecommendationsTemperature (°C)24.625.825.3Weekly in each testchamber andcontinuously in onenegative controlreplicate. EPA recommends temperature 25±1 oC; interreplicate and inter-treatment differentials should not exceed 1 o C.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PermethrinEPA MRID Number 48702301Page17Rf49Version: 22 September 2011ParameterMinimumMaximumMeanMeasurement IntervalGuideline RecommendationspH 7.98.18.0Weekly in each testchamber. EPA recommends pH 6.5 to 9.0.Dissolved oxygen (mg/L)4.78.36.5Weekly in each testchamber andperiodically inalternating replicates. EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air saturation)Total alkalinity (mg/L as CaCO3) 166 178 174Measured in onereplicate of thenegative control andhighest treatment levelweekly. EPA recommends total alkalinity >20 mg/L

as CaCO 3.Hardness (mg/L as CaCO3) 132 144 140Measured in onereplicate of thenegative control andhighest treatment levelweekly.Total organic carbon (mg/L)<1Measured during the4-week periodpreceding the test. EPA recommends that total organic carbon in dilution water be <2 mg/L.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PermethrinEPA MRID Number 48702301Page18Rf49Version: 22 September 2011ParameterMinimumMaximumMeanMeasurement IntervalGuideline RecommendationsUnionized ammonia (Pg/L) Not Reported EPA recommends that unionized ammonia in the dilution water be <1 P g/L.Residual chlorine (Pg/L) Not Reported EPA recommends that residual chlorine in dilution water be <10 P g/L.Specific Conductance (μS/cm) 359 389 374Measured in onereplicate of thenegative control andhighest treatment levelweekly. General recommendations for frequency of measurements: EPA recommends that temperature, pH, and dissolved oxygen be measured in all test tanks at least weekly and that hardness and alkalinity be measured in controls and in one tank at the highest test concentration at least weekly. In addition, continuous temperature monitoring of at least one tank is encouraged.Abbreviations:NANot applicable.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PermethrinEPA MRID Number 48702301Page19Rf49Version: 22 September 2011D. Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details or RemarksGuideline RecommendationsWas a range-finder conducted?Yes EPA recommends conducting a range-finder if

96-hour LC 50 data for the fathead minnow are

unavailable.If yes, what was the method fordetermining the highest testconcentration in the range-finder?Not specified EPA recommends that the highest test concentration be selected based on toxicity data for other fish studies or species, if available. Otherwise, either the solubility limit of the test compound or 100 mg/L (whichever is lower) is

appropriate.Species: Pimephales promelasLife stage:Not specified EPA recommends that range-finding tests be performed with fish of similar age and size to those that would be utilized in the test.Test duration:14 days EPA recommends a 96-hour exposure.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PermethrinEPA MRID Number 48702301Page20Rf49Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details:Range-finder conducted at nominalconcentrations 0 (negative andsolvent controls), 0.00081,0.0027, 0.0090, 0.030, and0.100 mg a.i./L. 100% Mortality intwo highest treatment groups threedays after initiation. Because ofmortality, two additional lowerlevels added to range-finder onDay 2 (0.000073 and 0.00024mg a.i./L). All fish in controls and0.000073-0.0027 mg a.i./Ltreatment groups appeared normalwith no mortalities. By test end,100% mortality at 0.0090 mga.i./L.The resulting LC50 rangedbetween 0.0030 and 0.060 mga.i./L.All test solutions were clear andcolorless, though an oily layer wasnoted in the mixing chamber of thehighest treatment group. Analyticalrecoveries ranged from 73 to 113%of nominal. EPA recommends conducting a range-finder with five test concentrations plus a control (six total treatment levels), with four females and two males per exposure tank (36 fish total). The number of mortalities that occur may be used to develop a concentration-response curve. Based upon the results, the highest concentration that does not result in increased mortality or signs of overt morbidity compared to controls, or 1/3 the derived 96-hr LC 50, may be selected as the highest exposure concentration in the 21- day

test.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PermethrinEPA MRID Number 48702301Page21Rf49Version: 22 September 2011Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:21 days EPA recommends that the duration of the definitive

test be 21 days.Method for selecting the highesttest concentration in the definitivetest:Range-finder 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., 1/3 the 96-

hour LC 50 ), whichever concentration is lowest.Reference study citation (ifapplicable):NASeparation of test concentrations:0.33 EPA s uggests that a concentration separation of between 0.33 (or three-fold) and 0.1 (or tenfold) is scientifically acceptable 1 .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?YesData Evaluation Record on the Fish Short-Term Reproduction Assay with PermethrinEPA MRID Number 48702301Page22Rf49Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsIndicate the type of valuespresented for measuredconcentrations:Mean-measuredReview also calculated the time-weightedaverage (TWA) concentrations.Limit of quantification (LOQ): 0.000150 mg a.i./L 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: Samples were collected foranalysis on Days 0, 7, 14,and 21 It is recommended that test item concentration be measured prior to the addition of fish in all tanks and at least weekly thereafter in two replicates per treatment level.Was the randomized completeblock design used?Yes EPA recommends that all fish be randomly assigned to tanks during pre-exposure. Tanks are then ranked according to pre-exposure fecundity, and the tanks with the highest fecundity are randomly assigned to a definitive test treatment and block first. Each block contains one replicate of each treatment, including controls.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PermethrinEPA MRID Number 48702301Page23Rf49Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of replicates in control: 4 EPA recommends 4 replicates.Number of replicates in solventcontrol (if applicable):4 EPA recommends the use of a concurrent solvent control when a solubilizing agent is used. EPA recommends 4 replicates.Number of replicates per test itemtreatment level:4 EPA recommends 4 replicates.Number of male fish per replicateat test initiation:2 EPA recommends 2 males per replicate.Number of female fish per replicateat test initiation:4 EPA recommends 4 females per replicate.Was a solvent used?YesSolvent type (if applicable): DMFData Evaluation Record on the Fish Short-Term Reproduction Assay with PermethrinEPA MRID Number 48702301Page24Rf49Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsMaximum solvent concentration (ifapplicable):0.020 mL/L EPA recommends that the solvent not exceed

0.02 ml/L3 . OECD recommends that solvent have no effect on survival nor produce any other adverse effects and that concentration not be greater than 0.1 ml/L4 .Was a positive control used? NoPositive control (if applicable): NoPositive control concentration(s) (ifapplicable):NA; a positive control wasnot usedPhotoperiod:16 hrs light :8 hrs dark EPA recommends photoperiod 16:8 (light:dark).Light intensity at water’s surface: 163-760 lux EPA recommends light intensity 540 – 1080 lux (at water’s surface).Additional details:NA3Hutchinson 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.69–92.4OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Series on Testing andAssessment. No. 23. Paris, France.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PermethrinEPA MRID Number 48702301Page25Rf49Version: 22 September 2011Table 8: Summary of Treatment Concentrations in theFish Short-Term Reproduction Assay with Permethrin.Treatment IDNominalConcentration(mg a.i./L)MeasuredConcentration(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsNegative Control0.00<0.000150NANA EPA recommends that test item concentrations be maintained at a coefficient of variation (CV) 20%.Solvent Control0.00<0.000150NANATreatment 10.000330.0002316TWA=0.00022CV=17%Treatment 20.000990.000756TWA=0.00075CV=6%Treatment 30.00300.002610TWA=0.0026CV=10%Abbreviations:CVCoefficient of variation.LOQ=0.000150 mg a.i./LE. ObservationsBiological Endpoints:Survival, fecundity, fertilization success, length, body weight, tubercle score, GSI, VTG, gonad histopathology, secondarysex characteristics, clinical signs of toxicityData Evaluation Record on the Fish Short-Term Reproduction Assay with PermethrinEPA MRID Number 48702301Page26Rf49Version: 22 September 2011Were raw (individual) data provided?Yes EPA recommends that observations of survival, fecundity, fertilization success, secondary sex characteristics, and other clinic al signs occur at least daily. At test termination (Day 21), additional observations include body weight and length, nuptial tubercle score, gonadal staging and histopathology, plasma
vitellogenin, and plasma sex steroids (testosterone and 17 ȕ -estradiol, if measured). GSI is calculated using a ratio of gonad weight to body weight (gonad weight to the nearest 0.1 mg / body weight in mg x 100) at test termination. Clinical signs of overt toxicity may include (but are not limited to) hemorrhage, cessation of feeding, and other abnormal beha vior.II. RESULTS AND DISCUSSIONA. ResultsNo mortalities were observed in the controls or TWA 0.00022 and 0.00075 mg a.i./L treatment groups (Table 9). Male and female survival in themean-measured 0.0026 mg a.i./L treatment group was 63 and 50%, respectively. Survival in the negative control and the two lowertreatmentgroups (0.00022 and 0.00075 mg a.i/L) was 100%, but there was 63% survival for males (5 of 8) and 50% survival for females (8 of 16) in thehighest treatment concentration. Additionally, clinical sings of toxicity (occurring in the highest treatment group) includederratic swimming (6 fish),loss of equilibrium (2 fish),and surfacing (2 fish). There were also observations of excess feed notedin the test chambers ofthe highest treatmentData Evaluation Record on the Fish Short-Term Reproduction Assay with PermethrinEPA MRID Number 48702301Page27Rf49Version: 22 September 2011group on Day 10 and Days 17-19. These observations suggest that overt toxicity was present during the course of this study in the highest treatmentconcentration.Table 9: Adult Fish Survival in Fathead Minnow ( P imephales promelas).Treatment(mg a.i./L)[TWA-measured]MalesFemalesn# Surviving% Survivaln# Surviving% SurvivalNegative Control (<LOQ)881001616100Solvent Control (<LOQ)8810016161000.00022 8810016161000.00075 8810016161000.00268 5 63 16 8 50Abbreviations:NANot applicable.LOQ=0.000150 mg a.i./LData Evaluation Record on the Fish Short-Term Reproduction Assay with PermethrinEPA MRID Number 48702301Page28Rf49Version: 22 September 2011In the controls and treatment groups, male wet weight and total length ranged from 2.63-2.99 g and 56.7-58.8 mm, respectively (Table 10).Female wet weight and total length ranged from 1.06-1.16 g and 45.3-46.5 mm, respectively.Table 10: Size at Test Termination in Fathead Minnow ( P imephales promelas).Treatment(mg a.i./L)[TWA-measured]Body WeightLengthMalesFemalesMalesFemalesnMean(g)±SDnMean(g)±SDnMean(mm)±SDnMean(mm)±SDNegative Control(<LOQ)4 2.99 0.265 4 1.06 0.065 4 57.1 1.44 4 45.3 0.84Solvent Control(<LOQ)4 2.70 0.223 4 1.11 0.066 4 56.3 1.76 4 45.7 0.520.00022 4 2.85 0.317 4 1.16 0.019 4 58.8 1.76 4 46.5 0.350.00075 4 2.63 0.194 4 1.13 0.069 4 58.3 1.19 4 46.0 0.540.0026 4 2.86 0.120 4 1.09 0.155 4 57.1 1.84 4 45.5 1.44Abbreviations:NANot applicable.NDNot determined.SDStandard deviation.LOQ=0.000150 mg a.i./L

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Version: 22 September 2011 Fecundity averaged 19.9, 18.9, 22.1, 18.8, and 11.5 eggs/female/reproductive day in the negative control,

solvent control, 0.00022, 0.00075, and 0.0026 mg a.i./L treatment groups, respectively (Table 11). Fecundity

in the negative control averaged 19.8 eggs/female/reproductive day with all replicates having at least 15

eggs/female/reproductive day and spawning frequency occurring at least every 4 days. Fertilization success

in the negative control averaged 95.4%.

Table 11: Fecundity and Fertilization Success in Fathead Minnow ( P imephales promelas ).

Treatment

(mg a.i./L)

[TWA-measured] Fecundity 1 ± SD Fertilization Success

(%) 2 ± SD

Negative Control (<LOQ) 19.9 0.71 95.4 1.49

Solvent Control (<LOQ) 18.9 ND 96.4 ND

0.00022 22.1 3.56 97.5 0.62

0.00075 18.8 4.22 93.9 1.71

0.0026 11.5 7.19 94.7 3.09

Abbreviations: NA Not applicable. ND Not determined.

1 Fecundity is calculated as the number of eggs per surviving female per reproductive day per replicate.

2 Fertilization success (%) is calculated as the number of embryos divided by the number of eggs, multiplied by 100.

LOQ=0.000150 mg a.i./L



### Median male tubercle scores were 35, 30, 29, 28, and 16 for the negative control, solvent control, and low,

mid, and high treatment groups, respectively. No nuptial tubercles were observed in females.

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Version: 22 September 2011 Table 12: Nuptial Tubercle Score in Fathead Minnow ( P imephales promelas ).

Treatment

(mg a.i./L)

[TWA-measured] Males Females

n Median Tubercle

Score n Median Tubercle

Score

Negative Control (<LOQ) 4 35 4 0

Solvent Control (<LOQ) 4 30 4 0

0.00022 4 29 4 0

0.00075 4 28 4 0

0.0026 4 16 4 0

Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation.

LOQ=0.000150 mg a.i./L



### Male GSI averaged 1.56, 1.84, 1.59, 1.74, and 2.32% in the negative control, solvent control, and TWA

0.00022, 0.00075, and 0.0026 mg a.i./L treatment groups, respectively (Table 13). Female GSI averaged

14.0 and 14.3% in the negative and solvent controls, respectively, and 13.9, 14.0, and 18.0% in the TWA

0.00022, 0.00075, and 0.0026 mg a.i./L treatment groups, respectively.

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Version: 22 September 2011 Table 13: Gonado-Somatic Index (GSI) in Fathead Minnow ( P imephales promelas ).

Treatment

(mg a.i./L)

[TWA-measured] Males Females

n Mean GSI 1

(%) ±SD n Mean GSI 1

(%) ±SD

Negative Control (<LOQ) 4 1.56 0.293 4 14.0 1.23

Solvent Control (<LOQ) 4 1.84 0.378 4 14.3 0.54

0.00022 4 1.59 0.345 4 13.9 1.28

0.00075 4 1.74 0.253 4 14.0 1.97

0.0026 4 2.32 0.384 4 18.0 2.63

Abbreviations: NA Not applicable.

1 Gonado-somatic index (%) is calculated as gonad weight (to the nearest 0.1 mg) / body weight (mg) x 100.

LOQ=0.000150 mg a.i./L



### The median gonad stage in males and females ranged from 2.3 to 3.5 (Table 14).

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Version: 22 September 2011 Table 14: Gonadal Staging in Fathead Minnow ( P imephales promelas ).

Treatment

(mg a.i./L)

[TWA-measured] Males Females

n Median Stage 1 n Median Stage 2

Negative Control (<LOQ) 4 2.3 4 2.3

Solvent Control (<LOQ) 4 2.5 4 3.0

0.00022 4 2.5 4 3.0

0.00075 4 2.5 4 2.8

0.0026 4 3.0 4 3.5

Abbreviations: J Juvenile. NA Not applicable. ND Not determined. UTS Unable to stage.

1 The guideline recommends the following gonadal staging scale for male fathead minnow: 0=undeveloped, 1=early

spermatogenic, 2=mid-spermatogenic, 3=late spermatogenic, 4=spent.

2 The guideline recommends the following gonadal staging scale for female fathead minnow: 0=undeveloped, 1=early

development, 2=mid-development, 3=late development, 4=late development/hydrated, 5=post-ovulatory.

LOQ=0.000150 mg a.i./L



### There were no apparent gonad histopathological findings in any males (Tables 15 and 16). There were,

however, treatment-related effects in female gonad histopathology (Table 17 and 18). The highest treatment

group showed a tendency toward later developmental stage with a severe increase in oocyte atresia in 4 of

the 8 fish, as well as an increase in the average number of post-atretic follicles. These findings suggest a

disturbance in the production of mature/spawning oocytes. Since there was significant mortality in females at

this level, the study authors suggest that the effects at this level were due to overt toxicity.

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Version: 22 September 2011 Table 15: Gonadal Histopathology in Male Fathead Minnow ( P imephales promelas ).

Treatment

(mg a.i./L)

[TWA-measured] Diagnostic Observations 1

Severity Increased

Proportion of

Spermatogonia Presence of

Testis-Ova Increased

Testicular

Degeneration Interstitial Cell

Hypertrophy/

Hyperplasia

n Incidence n Incidence n Incidence n Incidence

Negative Control

(<LOQ) 0 8 8 8 8 8 8 8 8

1 8 0 8 0 8 0 8 0

2 8 0 8 0 8 0 8 0

3 8 0 8 0 8 0 8 0

4 8 0 8 0 8 0 8 0

Solvent Control

(<LOQ) 0 8 8 8 8 8 8 8 8

1 8 0 8 0 8 0 8 0

2 8 0 8 0 8 0 8 0

3 8 0 8 0 8 0 8 0

4 8 0 8 0 8 0 8 0

0.00022 0 8 8 8 8 8 8 8 8

1 8 0 8 0 8 0 8 0

2 8 0 8 0 8 0 8 0

3 8 0 8 0 8 0 8 0

4 8 0 8 0 8 0 8 0

0.00075 0 8 8 8 8 8 7 8 8

1 8 0 8 0 8 1 8 0

2 8 0 8 0 8 0 8 0

3 8 0 8 0 8 0 8 0

4 8 0 8 0 8 0 8 0

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Version: 22 September 2011 Treatment

(mg a.i./L)

[TWA-measured] Diagnostic Observations 1

Severity Increased

Proportion of

Spermatogonia Presence of

Testis-Ova Increased

Testicular

Degeneration Interstitial Cell

Hypertrophy/

Hyperplasia

n Incidence n Incidence n Incidence n Incidence

0.0026 0 5 5 5 5 5 5 5 5

1 5 0 5 0 5 0 5 0

2 5 0 5 0 5 0 5 0

3 5 0 5 0 5 0 5 0

4 5 0 5 0 5 0 5 0

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild,

3=Moderate, 4=Severe. See Appendix E of the test guideline for reference.

LOQ=0.000150 mg a.i./LData Evaluation Record on the Fish Short-Term Reproduction Assay with PermethrinEPA MRID Number 48702301Page35Rf49Version: 22 September 2011Table 16: Additional Gonadal Histopathology Observations in Male Fathead Minnow ( P imephales promelas).Treatment(mg a.i./L)[TWA-measured]Additional Diagnostic Observations1SeverityDecreasedProportion ofSpermatogoniaIncreased Vascularor InterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportions ofSpermatocytes orSpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidenceNegative Control (<LOQ) 0 8 8 8 8 8 8 8 8 8 71 8 0 8 0 8 0 8 0 8 12 8 0 8 0 8 0 8 0 8 03 8 0 8 0 8 0 8 0 8 04 8 0 8 0 8 0 8 0 8 0Solvent Control (<LOQ) 0 8 8 8 8 8 8 8 8 8 61 8 0 8 0 8 0 8 0 8 02 8 0 8 0 8 0 8 0 8 13 8 0 8 0 8 0 8 0 8 04 8 0 8 0 8 0 8 0 8 10.000220 8 8 8 8 8 8 8 8 8 81 8 0 8 0 8 0 8 0 8 02 8 0 8 0 8 0 8 0 8 03 8 0 8 0 8 0 8 0 8 04 8 0 8 0 8 0 8 0 8 0Data Evaluation Record on the Fish Short-Term Reproduction Assay with PermethrinEPA MRID Number 48702301Page36Rf49Version: 22 September 2011Treatment(mg a.i./L)[TWA-measured]Additional Diagnostic Observations1SeverityDecreasedProportion ofSpermatogoniaIncreased Vascularor InterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportions ofSpermatocytes orSpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidence0.000750 8 8 8 8 8 8 8 7 8 71 8 0 8 0 8 0 8 1 8 12 8 0 8 0 8 0 8 0 8 03 8 0 8 0 8 0 8 0 8 04 8 0 8 0 8 0 8 0 8 00.00260 5 5 5 5 5 5 5 5 5 51 5 0 5 0 5 0 5 0 5 02 5 0 5 0 5 0 5 0 5 03 5 0 5 0 5 0 5 0 5 04 5 0 5 0 5 0 5 0 5 01Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E of the testguideline for reference.LOQ=0.000150 mg a.i./L

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Version: 22 September 2011 Table 17: Gonadal Histopathology in Female Fathead Minnow ( P imephales promelas ).

Treatment

(mg a.i./L)

[TWA-measured] Diagnostic Observations 1

Severity Increased Oocyte

Atresia Perifollicular Cell

Hyperplasia/

Hypertrophy Decreased Yolk

Formation

n Incidence n Incidence n Incidence

Negative Control (<LOQ) 0 16 14 16 16 16 16

1 16 1 16 0 16 0

2 16 0 16 0 16 0

3 16 1 16 0 16 0

4 16 0 16 0 16 0

Solvent Control (<LOQ) 0 16 12 16 16 16 16

1 16 4 16 0 16 0

2 16 0 16 0 16 0

3 16 0 16 0 16 0

4 16 0 16 0 16 0

0.00022 0 16 14 16 16 16 16

1 16 2 16 0 16 0

2 16 0 16 0 16 0

3 16 0 16 0 16 0

4 16 0 16 0 16 0

0.00075 0 16 13 16 16 16 16

1 16 2 16 0 16 0

2 16 1 16 0 16 0

3 16 0 16 0 16 0

4 16 0 16 0 16 0

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Version: 22 September 2011 Treatment

(mg a.i./L)

[TWA-measured] Diagnostic Observations 1

Severity Increased Oocyte

Atresia Perifollicular Cell

Hyperplasia/

Hypertrophy Decreased Yolk

Formation

n Incidence n Incidence n Incidence

0.0026 0 8 4 8 8 8 8

1 8 0 8 0 8 0

2 8 0 8 0 8 0

3 8 0 8 0 8 0

4 8 4 8 0 8 0

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild,

3=Moderate, 4=Severe. See Appendix E of the test guideline for reference.

LOQ=0.000150 mg a.i./L



### Table 18: Additional Gonadal Histopathology Observations in Female Fathead Minnow ( P imephales

promelas ).

Treatment

(mg a.i./L)

[TWAmeasured] Additional Diagnostic Observations 1

Severity Interstitial Fibrosis Egg Debris in

Oviduct Granulomatous

Inflammation Decreased PostOvulatory Follicles

n Incidence n Incidence n Incidence n Incidence

Negative

Control

(<LOQ) 0 16 16 16 16 16 13 16 7

1 16 0 16 0 16 2 16 1

2 16 0 16 0 16 0 16 2

3 16 0 16 0 16 1 16 1

4 16 0 16 0 16 0 16 5

Solvent Control

(<LOQ) 0 16 16 16 16 16 13 16 5

1 16 0 16 0 16 2 16 1

2 16 0 16 0 16 1 16 1

3 16 0 16 0 16 0 16 3

4 16 0 16 0 16 0 16 6

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Version: 22 September 2011 Treatment

(mg a.i./L)

[TWAmeasured] Additional Diagnostic Observations 1

Severity Interstitial Fibrosis Egg Debris in

Oviduct Granulomatous

Inflammation Decreased PostOvulatory Follicles

n Incidence n Incidence n Incidence n Incidence

0.00022 0 16 16 16 16 16 11 16 4

1 16 0 16 0 16 4 16 0

2 16 0 16 0 16 1 16 2

3 16 0 16 0 16 0 16 0

4 16 0 16 0 16 0 16 10

0.00075 0 16 16 16 16 16 15 16 5

1 16 0 16 0 16 1 16 3

2 16 0 16 0 16 0 16 0

3 16 0 16 0 16 0 16 1

4 16 0 16 0 16 0 16 7

0.0026 0 8 8 8 8 8 7 8 1

1 8 0 8 0 8 1 8 0

2 8 0 8 0 8 0 8 0

3 8 0 8 0 8 0 8 2

4 8 0 8 0 8 0 8 5

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild,

3=Moderate, 4=Severe. See Appendix E of the test guideline for reference.

LOQ=0.000150 mg a.i./L



### Plasma VTG concentrations were highly variable in both males and females. Male VTG averaged 243,

380, 180, and 249 while female VTG averaged 1.66x10 6

, 

### 1.39x10 6 

### , 1.64x10 6 

### , and 1.03x10 6 

### for the

negative control, 0.00022, 0.00075, and 0.0026 mg a.i/L treatment concentrations, respectively.

(Table 19).

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Version: 22 September 2011 Table 19: Plasma Vitellogenin in Fathead Minnow ( P imephales promelas ).

Treatment

(mg a.i./L)

[TWA-measured] Plasma Vitellogenin (VTG)

Males Females

n Mean

(ng/mL plasma) ±SD n Mean

(ng/mL plasma) ±SD

Negative Control (<LOQ) 4 243 167.8 4 1.66x10 6 5.14x10 5

Solvent Control (<LOQ) 4 272 148.1 4 1.53x10 6 2.48x10 5

0.00022 4 380 602.3 4 1.39x10 6 4.37x10 5

0.00075 4 180 203.7 4 1.64x10 6 4.80x10 5

0.0026 4 249 320.5 4 1.03x10 6 6.90x10 5

Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation.

LOQ=0.000150 mg a.i./LData Evaluation Record on the Fish Short-Term Reproduction Assay with PermethrinEPA MRID Number 48702301Page41Rf49Version: 22 September 2011Table 20: Plasma Sex Steroids in Fathead Minnow ( P imephales promelas). (Not Measured)Treatment(mg a.i./L)[TWA-measured]Plasma Testosterone (T)Plasma 17ȕ-estradiol (E2)MalesFemalesMalesFemalesnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDNegative Control (<LOQ) -- -- -- -- -- -- -- -- -- -- -- --Solvent Control (<LOQ) -- -- -- -- -- -- -- -- -- -- -- --0.00022-- -- -- -- -- -- -- -- -- -- -- --0.00075-- -- -- -- -- -- -- -- -- -- -- --0.0026-- -- -- -- -- -- -- -- -- -- -- --Abbreviations:NANot applicable.NDNot determined.SDStandard deviation.LOQ=0.000150 mg a.i./LData Evaluation Record on the Fish Short-Term Reproduction Assay with PermethrinEPA MRID Number 48702301Page42Rf49Version: 22 September 2011By test termination, a single male in the solvent control was missing its left eye; all other males appeared normal (Table 21).Females in thecontrols and two lower treatment groups all appeared normal. Clinical signs were restricted to females in the highest treatmentlevel and includedsurfacing, erratic swimming, loss of equilibrium, weakness, bloody lower jaws, and fin-pecked.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PermethrinEPA MRID Number 48702301Page43Rf49Version: 22 September 2011Table 21: Secondary Sex Characteristics and Clinical Signs in Fathead Minnow ( P imephales promelas).Treatment(mg a.i./L)[TWA-measured]Secondary Sex Characteristics and Clinical SignsMalesFemalesTypenIncidenceTypenIncidenceNegative Control (<LOQ)None8 --None16 --Solvent Control (<LOQ)Missing left eye8 1None16 --0.00022 None8-- None16--0.00075 None8-- None16--0.0026None5 --Loss of equilibrium8 2Bloody lower jaw8 3Erratic swimming8 5Surfacing 82Weak 81Fin-pecked 81LOQ=0.000150 mg a.i./L

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Version: 22 September 2011 B. Study Author’s Analysis and Conclusions

Data from the negative and solvent control groups for each parameter were compared using an

appropriate statistical test. No differences were detected between the two control groups ( p >0.05) for

any parameter. Therefore, the control data were pooled for comparisons among treatment groups, with

the exception of histopathological endpoints. Those endpoints were compared to the negative control.

Measurements of vitellogenin are inherently variable, and boxplots of log-transformed VTG values were

used to identify potential outliers that might need special handling in the analyses. No outliers were

excluded from analyses in this study.

Unless otherwise noted, the unit of statistical analysis was the replicate test chamber. Males and

females were analyzed separately for each endpoint when appropriate. Most endpoints were analyzed

using two complementary statistical approaches. If responses appeared to be monotonic a step-down

Jonckheere-Terpstra trend test was used to evaluate possible trends in the ranks of replicate means

to determine concentration responsive trends among the treatment groups. All endpoints except for

survival and histopathology scores and stages also were analyzed by performing pair-wise comparisons

using Dunnett’s multiple comparison test to determine which treatment groups differed statistically from

the control.

Survival and histopathology severity scores and stages were not amenable to the statistical methods

used for analysis of other endpoints. In particular, the most suitable unit of statistical analysis for these

endpoints was the individual animal. Therefore, survival was analyzed using the step-down JonckheereTerpstra trend test and Fishers Exact test, and histopathology severity scores and stages of individuals

were analyzed using step-down Jonckheere-Terpstra trend tests.

Statistical tests used to evaluate treatment effects were performed at a confidence level of Į =0.05

with SAS software.

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Version: 22 September 2011 Survival and GSI (both sexes) were both significantly reduced at the highest treatment level relative

to the pooled control. Also, fecundity was significantly reduced at the same level and females exhibited

greater severity and frequency of gonad histopathological findings.

C. Reviewer’s Analysis and Conclusions

Statistical Methods: For each endpoint, data from the negative and solvent controls were compared

using the equal variance two-sample t-test. Median male tubercle score was significantly greater

(p=0.0458) in the negative control than solvent control; no other differences were detected between

the negative and solvent controls and all subsequent analyses were conducted by comparing

treatment data to the negative control only. Given the lack of apparent solvent effects for any other

endpoint and the marginal statistical significance of the tubercle score results, the solvent used in

this study was not considered as interfering with the integrity of the test results. All endpoints were

tested for normality (Shapiro-Wilks, Į =0.01) and homogeneity of variance (Bartlett, Į =0.01). Male

GSI, female GSI, and median male tubercle scores did not satisfy parametric assumptions and all

exhibited monotonic trends, so these endpoints were analyzed using the Jonckheere-Terpstra trend

test. Male body weight and male VTG also did not meet parametric assumptions, but did not display

concentration-dependency, so these endpoints were analyzed using the Mann-Whitney U TwoSample test. The remaining endpoints satisfied parametric assumptions, did not exhibit

concentration-dependency, and were analyzed using Dunnett’s Multiple Comparison Test. Unless

otherwise noted, all analyses were conducted at Į =0.05 using CETIS v.1.8.7.7. and the backend

settings implemented by EFED on 1/30/13.

Conclusions:

Male tubercle score was significantly reduced at the TWA 0.0026 mg a.i./L treatment level relative

to the negative control, and male and female GSI were significantly increased at the same level. No

other significant effects were detected by the reviewer. High mortality (37% males, 50% females)

and clinical signs of toxicity occurred in the highest test treatment (TWA 0.0026 mg a.i./L).Data Evaluation Record on the Fish Short-Term Reproduction Assay with PermethrinEPA MRID Number 48702301Page46Rf49Version: 22 September 2011Table 22: Reproductive and HPG Endpoints1,2for Male Fathead Minnow ( P imephales promelas) in the FSTRA with Permethrin.Treatment(mg a.i./L)[TWA-measured]Tubercle ScoreGSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2Median p% Diff. pEffect?(Yes/No)% Diff. p% Diff. p% Diff. pNegative Control(<LOQ)34.5 NA NA NA No NA NA NA NA NA NASolvent Control3(<LOQ)30.3 0.046 15.0 0.29 No 10.8 0.80 NA NA NA NA0.00022 28.5 0.17 1.76 0.44 No 56.9 0.49 NA NA NA NA0.0075 28 0.14 11.4 0.18 No -25.6 0.34 NA NA NA NA0.0026 15.5 0.01148.90.0056 No 2.78 0.74 NA NA NA NAStatistical TestJonckheere Jonckheere NA Mann-WhitneyNANAAbbreviations:Conc.Concentration.Diff.Difference.E217ȕ-estradiol.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water)control. Conclusions regardinghistopathology may be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p< 0.05.3 pvalues for solvent control were based on output from equal variance two-sample t-test.LOQ=0.000150 mg a.i./LData Evaluation Record on the Fish Short-Term Reproduction Assay with PermethrinEPA MRID Number 48702301Page47Rf49Version: 22 September 2011Table 23: Reproductive and HPG Endpoints1,2for Female Fathead Minnow ( P imephales promelas) in the FSTRA with Permethrin.Treatment(mg a.i./L)[TWAmeasured]FecundityFert. SuccessTubercle ScoreGSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2% Diff. p% Diff. pMedian p% Diff. pEffect?(Yes/No)% Diff. p% Diff. p% Diff. pNegative Control(<LOQ)NA NA NA NA NA NA NA NA No NA NA NA NA NA NASolvent Control3(<LOQ)-5.02 0.76 1.04 0.62 NA NA 2.44 0.62 No -8.22 0.67 NA NA NA NA0.00022 10.9 0.84 2.20 0.33 NA NA -0.71 0.56 No -16.5 0.81 NA NA NA NA0.00075 -5.28 0.98 -1.57 0.58 NA NA 0.18 0.41 No -1.1 1.0 NA NA NA NA0.0026 -42.0 0.06 -0.66 0.94 NA NA 28.4 0.009 Yes -37.8 0.32 NA NA NA NAStatistical Test Dunnett Dunnett NA Jonckheere NA Dunnett NANAAbbreviations:Conc.Concentration.Diff.Difference.E217ȕ-estradiol.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water)control. Conclusions regarding histopathologymay be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p< 0.05.3 pvalues for solvent control were based on output from equal variance two-sample t-test.LOQ=0.000150 mg a.i./L

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Permethrin

EPA MRID Number 48702301

Page 48 R f 49

Version: 22 September 2011 Table 24: Growth Endpoints 1,2 

### in the Fish Short-Term Reproducti on Assay (FSTRA) with Permethrin.

Treatment

(mg a.i./L)

[TWA-measured] Body Weight Length

Males Females Males Females

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

Negative Control (<LOQ) NA NA NA NA NA NA NA NA

Solvent Control 3 (<LOQ) -10.7 0.15 4.50 0.31 -1.33 0.51 0.0 1.0

0.00022 -4.52 0.69 9.20 0.34 3.06 0.32 2.20 0.37

0.00075 -28.9 0.11 7.08 0. 54 2.18 0.57 1.65 0.58

0.0026 -4.18 0.49 3.07 0.92 0.00 1.00 0.55 0.97

Statistical Test Mann-Whitney Dunnett Dunnett Dunnett

Abbreviations: Diff. Difference. NA Not applicable. ND Not determined.

1 Unless otherwise indicated, percent (%) differences are reported based on comparison to the negative (clean water)

control.

2 Unless otherwise specified, effects are considered statistically significant at p <0.05.

3 p values for solvent control were based on output from equal variance two-sample t-test.

LOQ=0.000150 mg a.i./L



### E. Study Deficiencies

Although there were deviations from the guideline, as noted in the Materials and Methods section of

the DER, the study met all other validity and performance criteria.

F. Reviewer’s Comments

The reviewer conducted all statistical analyses by comparing treatment data to the negative control,

whereas the study author compared treatment data to the pooled control. Therefore, the reviewer’s

results are reported in the Executive Summary section of this DER.

The reviewer calculated the time-weighted average (TWA) concentrations using the following equation:

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Permethrin

EPA MRID Number 48702301

Page 49 R f 49

Version: 22 September 2011 where:

C TWA is the time-weighted average concentration,

C j is the concentration measured at time interval j (j = 0, 1, 2,...n)

t j is the number of hours (or days or weeks, units used just need to be consistent in the equation)

of the test at time interval j

(e.g., t0 = 0 hours (test initiation), t1 = 24 hours, t2 = 96 hours)

Results from the periodic screening analysis of the dilution water indicated the presence of the following

analytes: calcium (34.9 mg/L), chloride (4.5 mg/L), fluoride (0.85 mg/L), magnesium (13.2 mg/L),

potassium (7.00 mg/L), sodium (19.0 mg/L), and sulfate (5.7 mg/L).

The in-life portion of the definitive toxicity test was conducted from February 28 to March 20, 2012.

III. REFERENCES

Tukey, J.W. 1977. Exploratory Data Analysis. Reading, MA. Addison-Wesley.

The SAS System for Windows. 1999-2001. Version 8.2. SAS Institute, Inc. Cary, North Carolina. n nn nn

n n

TWA 

## t tt CC tt CC tt CC tt CC

C 1 1

21 21

12 12
01 01 

## 2 2 2 2  

 



##   ̧ 1 ·  ̈ © §    ̧ 1 ·  ̈ © §    ̧ 1 ·  ̈ © §    ̧ 1 ·  ̈ © § Report Date: 06 Dec-13 13:02 (p 1 of 24)

Test Code: 109701 48702301 | 04-7869-4595 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:06 Endpoint: Fecundity CETIS Version: CETISv1.8.7 Analysis ID: 08-0652-0584
Analysis: Parametric-Control vs Treatments Official Results: Yes

Sample ID: 20-8846-4684
Sample Date: 28 Feb-12

Receive Date: Code: 48702301

Sample Age: NA Source: Permethrin Data Group II Task Force

Station: Client: EPA OCSPP EFED

Project: Insecticide Material: Permethrin Batch ID: 06-6597-1328

Start Date: 28 Feb-12

Ending Date: 20 Mar-12 Test Type: EDSP FSTRA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Well Water

Brine: Not Applicable Species: Pimephales promelas

Source: Osage Catfisheries, Osage Beach, MI Analyst: J. Housenger

Age: 6mo

flow-through 109701 48702301

109701 48702301, DMF Batch Note:

Sample Note:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test

DF P-Type

0.6765 2.683 8.626 0.8389 Non-Significant Effect Negative Control 0.00023 6 CDF

0.3266 2.683 8.626 0.9759 Non-Significant Effect 0.00075 6 CDF

2.597 2.683 8.626 0.0582 Non-Significant Effect 0.0026 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.0026 >0.0026 43.4% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 250.0219 83.34062 3 4.031 0.0338 Significant Effect

Error 248.0925 20.67438 12

498.1144 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

9.058 11.34 0.0285 Equal Variances Bartlett Equality of Variance Variances

0.9607 0.8408 0.6742 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL Fecundity Summary

19.88 19 20.5 20 0 4 Negative Control 0.3544 3.57% 0.0% 18.75 21

22.05 17.9 26.4 21.95 0.00023 4 1.779 16.13% -10.94% 16.39 27.71

18.83 14.6 24.2 18.25 0.00075 4 2.112 22.44% 5.28% 12.1 25.55

11.53 4.3 21 10.4 0.0026 4 3.595 62.39% 42.01% 0.08414 22.97

Control Type C-mg ai/L Fecundity Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 20.5 19.6 19 20.4

0.00023 21 17.9 22.9 26.4

0.00075 24.2 14.6 20 16.5

0.0026 21 12.7 4.3 8.1

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Dec-13 13:02 (p 2 of 24)

Test Code: 109701 48702301 | 04-7869-4595 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:06 Endpoint: Fecundity CETIS Version: CETISv1.8.7 Analysis ID: 08-0652-0584
Analysis: Parametric-Control vs Treatments Official Results: YesCenteredUntransformed

Rankits -2

-4

-6

-8 0 2 4 6 8 10

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0Fecundity

C-mg ai/L Reject Null

Reject Null

0 5 10 15 20 25 30

0 N 0.00023 0.00075 0.0026 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Dec-13 13:02 (p 3 of 24)

Test Code: 109701 48702301 | 04-7869-4595 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:27 Endpoint: Fecundity CETIS Version: CETISv1.8.7 Analysis ID: 19-1665-3394
Analysis: Nonparametric-Control vs Ord. Treatments Official Results: Yes

Sample ID: 20-8846-4684
Sample Date: 28 Feb-12

Receive Date: Code: 48702301

Sample Age: NA Source: Permethrin Data Group II Task Force

Station: Client: EPA OCSPP EFED

Project: Insecticide Material: Permethrin Batch ID: 06-6597-1328

Start Date: 28 Feb-12

Ending Date: 20 Mar-12 Test Type: EDSP FSTRA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Well Water

Brine: Not Applicable Species: Pimephales promelas

Source: Osage Catfisheries, Osage Beach, MI Analyst: J. Housenger

Age: 6mo

flow-through 109701 48702301

109701 48702301, DMF Batch Note:

Sample Note:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Jonckheere-Terpstra Step-Down Test

DF P-Type

4 NA 0.9000 Non-Significant Effect Negative Control 0.00023 -2 Exact

26 NA 0.4156 Non-Significant Effect 0.00075 -2 Exact

1.636 1.645 1 0.0509 Non-Significant Effect 0.0026 -2 Asymp NOEL LOEL TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.0026 >0.0026 NA C > T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 250.0219 83.34062 3 4.031 0.0338 Significant Effect

Error 248.0925 20.67438 12

498.1144 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

9.058 11.34 0.0285 Equal Variances Bartlett Equality of Variance Variances

0.9607 0.8408 0.6742 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL Fecundity Summary

19.88 19 20.5 20 0 4 Negative Control 0.3544 3.57% 0.0% 18.75 21

22.05 17.9 26.4 21.95 0.00023 4 1.779 16.13% -10.94% 16.39 27.71

18.83 14.6 24.2 18.25 0.00075 4 2.112 22.44% 5.28% 12.1 25.55

11.53 4.3 21 10.4 0.0026 4 3.595 62.39% 42.01% 0.08414 22.97

Control Type C-mg ai/L Fecundity Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 20.5 19.6 19 20.4

0.00023 21 17.9 22.9 26.4

0.00075 24.2 14.6 20 16.5

0.0026 21 12.7 4.3 8.1

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Dec-13 13:02 (p 4 of 24)

Test Code: 109701 48702301 | 04-7869-4595 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:27 Endpoint: Fecundity CETIS Version: CETISv1.8.7 Analysis ID: 19-1665-3394
Analysis: Nonparametric-Control vs Ord. Treatments Official Results: YesCenteredUntransformed

Rankits -2

-4

-6

-8 0 2 4 6 8 10

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0Fecundity

C-mg ai/L 0 5 10 15 20 25 30

0 N 0.00023 0.00075 0.0026 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Dec-13 13:02 (p 5 of 24)

Test Code: 109701 48702301 | 04-7869-4595 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:06 Endpoint: FemaleBodyWt CETIS Version: CETISv1.8.7 Analysis ID: 00-3475-5096
Analysis: Parametric-Control vs Treatments Official Results: Yes

Sample ID: 20-8846-4684
Sample Date: 28 Feb-12

Receive Date: Code: 48702301

Sample Age: NA Source: Permethrin Data Group II Task Force

Station: Client: EPA OCSPP EFED

Project: Insecticide Material: Permethrin Batch ID: 06-6597-1328

Start Date: 28 Feb-12

Ending Date: 20 Mar-12 Test Type: EDSP FSTRA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Well Water

Brine: Not Applicable Species: Pimephales promelas

Source: Osage Catfisheries, Osage Beach, MI Analyst: J. Housenger

Age: 6mo

flow-through 109701 48702301

109701 48702301, DMF Batch Note:

Sample Note:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test

DF P-Type

1.506 2.683 0.174 0.3427 Non-Significant Effect Negative Control 0.00023 6 CDF

1.158 2.683 0.174 0.5365 Non-Significant Effect 0.00075 6 CDF

0.5019 2.683 0.174 0.9227 Non-Significant Effect 0.0026 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.0026 >0.0026 16.4% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 0.02272499 0.007574997 3 0.9031 0.4682 Non-Significant Effect

Error 0.10065 0.008387499 12

0.123375 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

8.959 11.34 0.0298 Equal Variances Bartlett Equality of Variance Variances

0.8919 0.8408 0.0598 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL FemaleBodyWt Summary

1.06 0.98 1.12 1.07 0 4 Negative Control 0.03162 5.97% 0.0% 0.9594 1.161

1.157 1.13 1.17 1.165 0.00023 4 0.009465 1.64% -9.2% 1.127 1.188

1.135 1.07 1.22 1.125 0.00075 4 0.03428 6.04% -7.08% 1.026 1.244

1.093 0.86 1.2 1.155 0.0026 4 0.07825 14.32% -3.07% 0.8435 1.342

Control Type C-mg ai/L FemaleBodyWt Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 1.12 0.98 1.04 1.1

0.00023 1.13 1.17 1.16 1.17

0.00075 1.22 1.07 1.09 1.16

0.0026 1.16 1.15 0.86 1.2

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Dec-13 13:02 (p 6 of 24)

Test Code: 109701 48702301 | 04-7869-4595 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:06 Endpoint: FemaleBodyWt CETIS Version: CETISv1.8.7 Analysis ID: 00-3475-5096
Analysis: Parametric-Control vs Treatments Official Results: YesCenteredUntransformed

Rankits -0.05

-0.10

-0.15

-0.20

-0.25 0.00 0.05 0.10 0.15 0.20

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0FemaleBodyWt

C-mg ai/L Reject Null

Reject Null

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

0 N 0.00023 0.00075 0.0026 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Dec-13 13:02 (p 7 of 24)

Test Code: 109701 48702301 | 04-7869-4595 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:28 Endpoint: FemaleGSI CETIS Version: CETISv1.8.7 Analysis ID: 00-8481-6560
Analysis: Nonparametric-Control vs Ord. Treatments Official Results: Yes

Sample ID: 20-8846-4684
Sample Date: 28 Feb-12

Receive Date: Code: 48702301

Sample Age: NA Source: Permethrin Data Group II Task Force

Station: Client: EPA OCSPP EFED

Project: Insecticide Material: Permethrin Batch ID: 06-6597-1328

Start Date: 28 Feb-12

Ending Date: 20 Mar-12 Test Type: EDSP FSTRA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Well Water

Brine: Not Applicable Species: Pimephales promelas

Source: Osage Catfisheries, Osage Beach, MI Analyst: J. Housenger

Age: 6mo

flow-through 109701 48702301

109701 48702301, DMF Batch Note:

Sample Note:

Test Stat Critical P-Value Decision( α :5%) Ties vs Control C-mg ai/L Jonckheere-Terpstra Step-Down Test

DF P-Type

-0.1459 1.645 1 0.5580 Non-Significant Effect Negative Control 0.00023 -2 Asymp

0.2202 1.645 1 0.4129 Non-Significant Effect 0.00075 -2 Asymp

2.384 1.645 1 0.0086 Significant Effect 0.0026* -2 Asymp NOEL LOEL TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.00075 0.0026 0.001396 C < T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 48.035 16.01167 3 4.628 0.0226 Significant Effect

Error 41.515 3.459583 12

89.55 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

2.12 11.34 0.5478 Equal Variances Bartlett Equality of Variance Variances

0.9249 0.8408 0.2025 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL FemaleGSI Summary

14 12.4 15 14.3 0 4 Negative Control 0.6096 8.71% 0.0% 12.06 15.94

13.9 12.4 15.2 14 0.00023 4 0.6416 9.23% 0.71% 11.86 15.94

14.03 11.8 15.8 14.25 0.00075 4 0.9716 13.85% -0.18% 10.93 17.12

17.97 16.3 21.9 16.85 0.0026 4 1.316 14.64% -28.39% 13.79 22.16

Control Type C-mg ai/L FemaleGSI Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 14.9 13.7 15 12.4

0.00023 14.7 13.3 12.4 15.2

0.00075 15.5 13 11.8 15.8

0.0026 17 16.7 21.9 16.3

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Dec-13 13:02 (p 8 of 24)

Test Code: 109701 48702301 | 04-7869-4595 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:28 Endpoint: FemaleGSI CETIS Version: CETISv1.8.7 Analysis ID: 00-8481-6560
Analysis: Nonparametric-Control vs Ord. Treatments Official Results: YesCenteredUntransformed

Rankits -0.5

-1.0

-1.5

-2.0

-2.5

-3.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0FemaleGSI

C-mg ai/L 0 5 10 15 20 25

0 N 0.00023 0.00075 0.0026 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Dec-13 13:02 (p 9 of 24)

Test Code: 109701 48702301 | 04-7869-4595 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:06 Endpoint: FemaleLength CETIS Version: CETISv1.8.7 Analysis ID: 00-2496-0096
Analysis: Parametric-Control vs Treatments Official Results: Yes

Sample ID: 20-8846-4684
Sample Date: 28 Feb-12

Receive Date: Code: 48702301

Sample Age: NA Source: Permethrin Data Group II Task Force

Station: Client: EPA OCSPP EFED

Project: Insecticide Material: Permethrin Batch ID: 06-6597-1328

Start Date: 28 Feb-12

Ending Date: 20 Mar-12 Test Type: EDSP FSTRA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Well Water

Brine: Not Applicable Species: Pimephales promelas

Source: Osage Catfisheries, Osage Beach, MI Analyst: J. Housenger

Age: 6mo

flow-through 109701 48702301

109701 48702301, DMF Batch Note:

Sample Note:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test

DF P-Type

1.445 2.683 1.857 0.3729 Non-Significant Effect Negative Control 0.00023 6 CDF

1.083 2.683 1.857 0.5838 Non-Significant Effect 0.00075 6 CDF

0.3612 2.683 1.857 0.9681 Non-Significant Effect 0.0026 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.0026 >0.0026 4.08% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 2.5 0.8333333 3 0.8696 0.4837 Non-Significant Effect

Error 11.5 0.9583333 12

14 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

3.961 11.34 0.2657 Equal Variances Bartlett Equality of Variance Variances

0.9155 0.8408 0.1430 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL FemaleLength Summary

45.5 45 47 45 0 4 Negative Control 0.5 2.2% 0.0% 43.91 47.09

46.5 46 47 46.5 0.00023 4 0.2887 1.24% -2.2% 45.58 47.42

46.25 46 47 46 0.00075 4 0.25 1.08% -1.65% 45.45 47.05

45.75 44 47 46 0.0026 4 0.75 3.28% -0.55% 43.36 48.14

Control Type C-mg ai/L FemaleLength Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 45 45 45 47

0.00023 46 47 47 46

0.00075 47 46 46 46

0.0026 47 47 44 45

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Dec-13 13:02 (p 10 of 24)

Test Code: 109701 48702301 | 04-7869-4595 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:06 Endpoint: FemaleLength CETIS Version: CETISv1.8.7 Analysis ID: 00-2496-0096
Analysis: Parametric-Control vs Treatments Official Results: YesCenteredUntransformed

Rankits -0.2

-0.4

-0.6

-0.8

-1.0

-1.2

-1.4

-1.6

-1.8 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0FemaleLength

C-mg ai/L Reject Null

Reject Null

0 10 20 30 40 50

0 N 0.00023 0.00075 0.0026 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Dec-13 13:02 (p 11 of 24)

Test Code: 109701 48702301 | 04-7869-4595 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:06 Endpoint: FemaleVTG CETIS Version: CETISv1.8.7 Analysis ID: 09-3678-4332
Analysis: Parametric-Control vs Treatments Official Results: Yes

Sample ID: 20-8846-4684
Sample Date: 28 Feb-12

Receive Date: Code: 48702301

Sample Age: NA Source: Permethrin Data Group II Task Force

Station: Client: EPA OCSPP EFED

Project: Insecticide Material: Permethrin Batch ID: 06-6597-1328

Start Date: 28 Feb-12

Ending Date: 20 Mar-12 Test Type: EDSP FSTRA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Well Water

Brine: Not Applicable Species: Pimephales promelas

Source: Osage Catfisheries, Osage Beach, MI Analyst: J. Housenger

Age: 6mo

flow-through 109701 48702301

109701 48702301, DMF Batch Note:

Sample Note:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test

DF P-Type

0.7389 2.724 1009 0.8074 Non-Significant Effect Negative Control 0.00023 6 CDF

0.04926 2.724 1009 0.9999 Non-Significant Effect 0.00075 6 CDF

1.569 2.724 1090 0.3203 Non-Significant Effect 0.0026 5 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.0026 >0.0026 65.6% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 863048 287682.7 3 1.048 0.4099 Non-Significant Effect

Error 3019971 274542.8 11

3883019 14 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

0.5151 11.34 0.9156 Equal Variances Bartlett Equality of Variance Variances

0.9759 0.8328 0.9334 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL FemaleVTG Summary

1661 905 2050 1845 0 4 Negative Control 257.1 30.95% 0.0% 843.1 2479

1388 1030 2020 1250 0.00023 4 218.5 31.49% 16.48% 692.2 2083

1643 992 2140 1720 0.00075 4 240.7 29.3% 1.1% 876.9 2409

1033 481 1810 809 0.0026 3 399.7 67.0% 37.8% -686.5 2753

Control Type C-mg ai/L FemaleVTG Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 1880 1810 905 2050

0.00023 2020 1310 1030 1190

0.00075 2140 1800 992 1640

0.0026 1810 809 481

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Dec-13 13:02 (p 12 of 24)

Test Code: 109701 48702301 | 04-7869-4595 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:06 Endpoint: FemaleVTG CETIS Version: CETISv1.8.7 Analysis ID: 09-3678-4332
Analysis: Parametric-Control vs Treatments Official Results: YesCenteredUntransformed

Rankits -200

-400

-600

-800

0 200 400 600 800

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0FemaleVTG

C-mg ai/L Reject Null

Reject Null

0 500 1000 1500 2000 2500 3000

0 N 0.00023 0.00075 0.0026 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Dec-13 13:02 (p 13 of 24)

Test Code: 109701 48702301 | 04-7869-4595 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:06 Endpoint: Fertility CETIS Version: CETISv1.8.7 Analysis ID: 13-7395-8017
Analysis: Parametric-Control vs Treatments Official Results: Yes

Sample ID: 20-8846-4684
Sample Date: 28 Feb-12

Receive Date: Code: 48702301

Sample Age: NA Source: Permethrin Data Group II Task Force

Station: Client: EPA OCSPP EFED

Project: Insecticide Material: Permethrin Batch ID: 06-6597-1328

Start Date: 28 Feb-12

Ending Date: 20 Mar-12 Test Type: EDSP FSTRA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Well Water

Brine: Not Applicable Species: Pimephales promelas

Source: Osage Catfisheries, Osage Beach, MI Analyst: J. Housenger

Age: 6mo

flow-through 109701 48702301

109701 48702301, DMF Batch Note:

Sample Note:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test

DF P-Type

1.53 2.683 3.683 0.3311 Non-Significant Effect Negative Control 0.00023 6 CDF

1.093 2.683 3.683 0.5778 Non-Significant Effect 0.00075 6 CDF

0.4553 2.683 3.683 0.9401 Non-Significant Effect 0.0026 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.0026 >0.0026 3.86% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 28.20183 9.400609 3 2.495 0.1096 Non-Significant Effect

Error 45.21749 3.768125 12

73.41932 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

5.59 11.34 0.1334 Equal Variances Bartlett Equality of Variance Variances

0.9468 0.8408 0.4400 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL Fertility Summary

95.35 93.8 97.3 95.15 0 4 Negative Control 0.7467 1.57% 0.0% 92.97 97.73

97.45 96.8 98.2 97.4 0.00023 4 0.3122 0.64% -2.2% 96.46 98.44

93.85 91.3 94.8 94.65 0.00075 4 0.853 1.82% 1.57% 91.14 96.56

94.73 90.5 97.4 95.5 0.0026 4 1.545 3.26% 0.66% 89.81 99.64

Control Type C-mg ai/L Fertility Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 95.6 97.3 94.7 93.8

0.00023 98.2 97.7 96.8 97.1

0.00075 94.5 91.3 94.8 94.8

0.0026 96.6 94.4 90.5 97.4

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Dec-13 13:02 (p 14 of 24)

Test Code: 109701 48702301 | 04-7869-4595 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:06 Endpoint: Fertility CETIS Version: CETISv1.8.7 Analysis ID: 13-7395-8017
Analysis: Parametric-Control vs Treatments Official Results: YesCenteredUntransformed

Rankits -1

-2

-3

-4

-5 0 1 2 3

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0Fertility

C-mg ai/L Reject Null

Reject Null

0 20 40 60 80 100

0 N 0.00023 0.00075 0.0026 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Dec-13 13:02 (p 15 of 24)

Test Code: 109701 48702301 | 04-7869-4595 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:06 Endpoint: MaleBodyWt CETIS Version: CETISv1.8.7 Analysis ID: 06-2395-2162
Analysis: Nonparametric-Two Sample Official Results: Yes

Sample ID: 20-8846-4684
Sample Date: 28 Feb-12

Receive Date: Code: 48702301

Sample Age: NA Source: Permethrin Data Group II Task Force

Station: Client: EPA OCSPP EFED

Project: Insecticide Material: Permethrin Batch ID: 06-6597-1328

Start Date: 28 Feb-12

Ending Date: 20 Mar-12 Test Type: EDSP FSTRA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Well Water

Brine: Not Applicable Species: Pimephales promelas

Source: Osage Catfisheries, Osage Beach, MI Analyst: J. Housenger

Age: 6mo

flow-through 109701 48702301

109701 48702301, DMF Batch Note:

Sample Note:

Test Stat Critical P-Value Decision( α :5%) Ties vs Control C-mg ai/L Mann-Whitney U Two-Sample Test

DF P-Type

10 NA 0 0.6857 Non-Significant Effect Negative Control 0.00023 6 Exact

14 NA 0 0.1143 Non-Significant Effect 0.00075 6 Exact

11 NA 0 0.4857 Non-Significant Effect 0.0026 6 Exact NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.0026 >0.0026 35.7% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 1.862675 0.6208917 3 1.633 0.2338 Non-Significant Effect

Error 4.5623 0.3801917 12

6.424975 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

13.4 11.34 0.0038 Unequal Variances Bartlett Equality of Variance Variances

0.8057 0.8408 0.0032 Non-normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL MaleBodyWt Summary

2.99 2.65 3.25 3.03 0 4 Negative Control 0.1325 8.86% 0.0% 2.568 3.412

2.855 2.46 3.22 2.87 0.00023 4 0.158 11.07% 4.52% 2.352 3.358

2.125 0.4 2.79 2.655 0.00075 4 0.5781 54.41% 28.93% 0.2851 3.965

2.865 2.76 3.02 2.84 0.0026 4 0.05867 4.1% 4.18% 2.678 3.052

Control Type C-mg ai/L MaleBodyWt Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 3.14 3.25 2.65 2.92

0.00023 2.94 2.46 2.8 3.22

0.00075 2.78 2.79 0.4 2.53

0.0026 2.79 2.76 3.02 2.89

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Dec-13 13:02 (p 16 of 24)

Test Code: 109701 48702301 | 04-7869-4595 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:06 Endpoint: MaleBodyWt CETIS Version: CETISv1.8.7 Analysis ID: 06-2395-2162
Analysis: Nonparametric-Two Sample Official Results: YesCenteredUntransformed

Rankits -0.2

-0.4

-0.6

-0.8

-1.0

-1.2

-1.4

-1.6

-1.8 0.0 0.2 0.4 0.6 0.8

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0MaleBodyWt

C-mg ai/L 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

0 N 0.00023 0.00075 0.0026 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Dec-13 13:02 (p 17 of 24)

Test Code: 109701 48702301 | 04-7869-4595 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:28 Endpoint: MaleGSI CETIS Version: CETISv1.8.7 Analysis ID: 02-8831-9340
Analysis: Nonparametric-Control vs Ord. Treatments Official Results: Yes

Sample ID: 20-8846-4684
Sample Date: 28 Feb-12

Receive Date: Code: 48702301

Sample Age: NA Source: Permethrin Data Group II Task Force

Station: Client: EPA OCSPP EFED

Project: Insecticide Material: Permethrin Batch ID: 06-6597-1328

Start Date: 28 Feb-12

Ending Date: 20 Mar-12 Test Type: EDSP FSTRA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Well Water

Brine: Not Applicable Species: Pimephales promelas

Source: Osage Catfisheries, Osage Beach, MI Analyst: J. Housenger

Age: 6mo

flow-through 109701 48702301

109701 48702301, DMF Batch Note:

Sample Note:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Jonckheere-Terpstra Step-Down Test

DF P-Type

9 NA 0.4429 Non-Significant Effect Negative Control 0.00023 -2 Exact

31 NA 0.1756 Non-Significant Effect 0.00075 -2 Exact

75 NA 0.0056 Significant Effect 0.0026* -2 Exact NOEL LOEL TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.00075 0.0026 0.001396 C < T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 1.518619 0.5062062 3 4.898 0.0190 Significant Effect

Error 1.240225 0.1033521 12

2.758844 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

0.5057 11.34 0.9176 Equal Variances Bartlett Equality of Variance Variances

0.9048 0.8408 0.0959 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL MaleGSI Summary

1.56 1.18 1.87 1.595 0 4 Negative Control 0.1465 18.78% 0.0% 1.094 2.026

1.588 1.12 1.94 1.645 0.00023 4 0.1721 21.68% -1.76% 1.04 2.135

1.738 1.4 1.95 1.8 0.00075 4 0.1262 14.53% -11.38% 1.336 2.139

2.322 1.79 2.64 2.43 0.0026 4 0.1907 16.42% -48.88% 1.716 2.929

Control Type C-mg ai/L MaleGSI Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 1.51 1.18 1.87 1.68

0.00023 1.94 1.59 1.12 1.7

0.00075 1.95 1.91 1.4 1.69

0.0026 1.79 2.31 2.55 2.64

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Dec-13 13:02 (p 18 of 24)

Test Code: 109701 48702301 | 04-7869-4595 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:28 Endpoint: MaleGSI CETIS Version: CETISv1.8.7 Analysis ID: 02-8831-9340
Analysis: Nonparametric-Control vs Ord. Treatments Official Results: YesCenteredUntransformed

Rankits -0.1

-0.2

-0.3

-0.4

-0.5

-0.6 0.0 0.1 0.2 0.3 0.4

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0MaleGSI

C-mg ai/L 0.0 0.5 1.0 1.5 2.0 2.5 3.0

0 N 0.00023 0.00075 0.0026 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Dec-13 13:02 (p 19 of 24)

Test Code: 109701 48702301 | 04-7869-4595 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:06 Endpoint: MaleLength CETIS Version: CETISv1.8.7 Analysis ID: 11-1502-5161
Analysis: Parametric-Control vs Treatments Official Results: Yes

Sample ID: 20-8846-4684
Sample Date: 28 Feb-12

Receive Date: Code: 48702301

Sample Age: NA Source: Permethrin Data Group II Task Force

Station: Client: EPA OCSPP EFED

Project: Insecticide Material: Permethrin Batch ID: 06-6597-1328

Start Date: 28 Feb-12

Ending Date: 20 Mar-12 Test Type: EDSP FSTRA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Well Water

Brine: Not Applicable Species: Pimephales promelas

Source: Osage Catfisheries, Osage Beach, MI Analyst: J. Housenger

Age: 6mo

flow-through 109701 48702301

109701 48702301, DMF Batch Note:

Sample Note:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test

DF P-Type

1.552 2.683 3.024 0.3207 Non-Significant Effect Negative Control 0.00023 6 CDF

1.109 2.683 3.024 0.5676 Non-Significant Effect 0.00075 6 CDF

0 2.683 3.024 1.0000 Non-Significant Effect 0.0026 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.0026 >0.0026 5.28% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 9.5 3.166667 3 1.246 0.3364 Non-Significant Effect

Error 30.5 2.541667 12

40 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

1.635 11.34 0.6514 Equal Variances Bartlett Equality of Variance Variances

0.969 0.8408 0.8227 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL MaleLength Summary

57.25 56 59 57 0 4 Negative Control 0.6292 2.2% 0.0% 55.25 59.25

59 57 61 59 0.00023 4 0.9129 3.09% -3.06% 56.09 61.91

58.5 57 59 59 0.00075 4 0.5 1.71% -2.18% 56.91 60.09

57.25 55 60 57 0.0026 4 1.031 3.6% 0.0% 53.97 60.53

Control Type C-mg ai/L MaleLength Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 59 57 56 57

0.00023 61 58 57 60

0.00075 59 59 57 59

0.0026 55 57 60 57

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Dec-13 13:02 (p 20 of 24)

Test Code: 109701 48702301 | 04-7869-4595 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:06 Endpoint: MaleLength CETIS Version: CETISv1.8.7 Analysis ID: 11-1502-5161
Analysis: Parametric-Control vs Treatments Official Results: YesCenteredUntransformed

Rankits -0.5

-1.0

-1.5

-2.0

-2.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0MaleLength

C-mg ai/L Reject Null

Reject Null

0 10 20 30 40 50 60 70

0 N 0.00023 0.00075 0.0026 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Dec-13 13:02 (p 21 of 24)

Test Code: 109701 48702301 | 04-7869-4595 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:28 Endpoint: MaleMedianTubercleScore CETIS Version: CETISv1.8.7 Analysis ID: 20-1835-4249
Analysis: Nonparametric-Control vs Ord. Treatments Official Results: Yes

Sample ID: 20-8846-4684
Sample Date: 28 Feb-12

Receive Date: Code: 48702301

Sample Age: NA Source: Permethrin Data Group II Task Force

Station: Client: EPA OCSPP EFED

Project: Insecticide Material: Permethrin Batch ID: 06-6597-1328

Start Date: 28 Feb-12

Ending Date: 20 Mar-12 Test Type: EDSP FSTRA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Well Water

Brine: Not Applicable Species: Pimephales promelas

Source: Osage Catfisheries, Osage Beach, MI Analyst: J. Housenger

Age: 6mo

flow-through 109701 48702301

109701 48702301, DMF Batch Note:

Sample Note:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Jonckheere-Terpstra Step-Down Test

DF P-Type

12 NA 0.1714 Non-Significant Effect Negative Control 0.00023 -2 Exact

32 NA 0.1404 Non-Significant Effect 0.00075 -2 Exact

2.293 1.645 2 0.0109 Significant Effect 0.0026* -2 Asymp NOEL LOEL TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.00075 0.0026 0.001396 C > T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 487.5469 162.5156 3 3.357 0.0553 Non-Significant Effect

Error 580.9375 48.41146 12

1068.484 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

1.281 11.34 0.7338 Equal Variances Bartlett Equality of Variance Variances

0.8786 0.8408 0.0369 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL MaleMedianTubercleScore Summary

34.25 28.5 39.5 34.5 0 4 Negative Control 2.332 13.62% 0.0% 26.83 41.67

29.75 24.5 37.5 28.5 0.00023 4 3.01 20.24% 13.14% 20.17 39.33

29.88 23.5 40 28 0.00075 4 3.538 23.68% 12.77% 18.62 41.13

19.25 13 33 15.5 0.0026 4 4.626 48.06% 43.8% 4.529 33.97

Control Type C-mg ai/L MaleMedianTubercleScore Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 33 36 28.5 39.5

0.00023 37.5 24.5 25.5 31.5

0.00075 40 28 28 23.5

0.0026 13 15 33 16

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Dec-13 13:02 (p 22 of 24)

Test Code: 109701 48702301 | 04-7869-4595 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:28 Endpoint: MaleMedianTubercleScore CETIS Version: CETISv1.8.7 Analysis ID: 20-1835-4249
Analysis: Nonparametric-Control vs Ord. Treatments Official Results: YesCenteredUntransformed

Rankits -2

-4

-6

-8 0 2 4 6 8 10 12 14

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0MaleMedianTubercleScore

C-mg ai/L 0 5 10 15 20 25 30 35 40

0 N 0.00023 0.00075 0.0026 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Dec-13 13:02 (p 23 of 24)

Test Code: 109701 48702301 | 04-7869-4595 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 30 Oct-13 10:10 Endpoint: MaleVTG CETIS Version: CETISv1.8.7 Analysis ID: 09-7815-4018
Analysis: Nonparametric-Two Sample Official Results: Yes

Sample ID: 20-8846-4684
Sample Date: 28 Feb-12

Receive Date: Code: 48702301

Sample Age: NA Source: Permethrin Data Group II Task Force

Station: Client: EPA OCSPP EFED

Project: Insecticide Material: Permethrin Batch ID: 06-6597-1328

Start Date: 28 Feb-12

Ending Date: 20 Mar-12 Test Type: EDSP FSTRA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Well Water

Brine: Not Applicable Species: Pimephales promelas

Source: Osage Catfisheries, Osage Beach, MI Analyst: J. Housenger

Age: 6mo

flow-through 109701 48702301

109701 48702301, DMF Batch Note:

Sample Note:

Test Stat Critical P-Value Decision( α :5%) Ties vs Control C-mg ai/L Mann-Whitney U Two-Sample Test

DF P-Type

11 NA 0 0.4857 Non-Significant Effect Negative Control 0.00023 6 Exact

12 NA 0 0.3429 Non-Significant Effect 0.00075 6 Exact

9.5 NA 1 0.7429 Non-Significant Effect 0.0026 6 Exact NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.0026 >0.0026 261.0% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 0.08488669 0.02829556 3 0.2115 0.8865 Non-Significant Effect

Error 1.605654 0.1338045 12

1.69054 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

5.261 11.34 0.1537 Equal Variances Bartlett Equality of Variance Variances

0.831 0.8408 0.0072 Non-normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL MaleVTG Summary

0.2425 0.099 0.481 0.195 0 4 Negative Control 0.08395 69.24% 0.0% -0.02468 0.5097

0.3805 0.038 1.283 0.1005 0.00023 4 0.3012 158.3% -56.91% -0.5781 1.339

0.1805 0.067 0.485 0.085 0.00075 4 0.1017 112.7% 25.57% -0.1432 0.5042

0.2493 0.015 0.708 0.137 0.0026 4 0.1603 128.6% -2.78% -0.2608 0.7593

Control Type C-mg ai/L MaleVTG Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 0.481 0.099 0.159 0.231

0.00023 0.038 0.107 0.094 1.283

0.00075 0.485 0.098 0.067 0.072

0.0026 0.708 0.231 0.043 0.015

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Dec-13 13:02 (p 24 of 24)

Test Code: 109701 48702301 | 04-7869-4595 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 30 Oct-13 10:10 Endpoint: MaleVTG CETIS Version: CETISv1.8.7 Analysis ID: 09-7815-4018
Analysis: Nonparametric-Two Sample Official Results: YesCenteredUntransformed

Rankits -0.2

-0.4

-0.6 0.0 0.2 0.4 0.6 0.8 1.0

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0MaleVTG

C-mg ai/L 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

0 N 0.00023 0.00075 0.0026 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________

# DATA EVALUATION RECORD

PERMETHRIN

Study Type: O CSPP 890.1400, In vivo Hershberger Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-29-2012 (MRID 48618602)

Prepared for

Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by

CSS

- Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer : Signature: Rebecca L. Bryan, B.S. Date: 3/ 23 /2012

Secondary Reviewer : Signature:
David A. McEwen, B.S. Date: 3 / 29 /2012

Program Manager: Signature: Jack D. Early, M.S. Date: 4 / 10 /2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 4 / 10 /2012

This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by CSS -Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengt hs of each individual assay to identify potential endocrine bioactivity with complementary endpoints within 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).

### PERM ET HRIN/ 

### 109701



## Primary Reviewer: Edward Sca llon. Ph.D.



## Health Effects Division Secondary Reviewer: Greg Akerman, Ph

. D

. 

## Health Effects Division In vivo 

### Her s hberger Assay 

### (2011)/ P age I of 

### 11

# Si"~S!~CSPP890.1~400/£

Date: 

# ZC Signature:

__ ._...._ ..... ,_,,_,,,_ __
Date :**~h.r** 

### /, )
.- 

# I DATA EVALUATION RECORD Template version 

### 1012011



## STUDY TYPE: In Vivo 

## Hershberger Assay (Ra t) ; OCSPP 890.1400; OECD 441



## PC CODE: 1 09701 

## DP BARCO DE : D398301



## TXR# : 0056246



## TEST MATERIAL (PURITY): Permethrin (97.7% a.i. )



## SYNONYMS: (3-p h enoxypheny l )methy l 3-(2,2 -di chJoroethe n y l )-2

, 2

- dimethylcyclopropanecarboxy l ate 

## CAS#: 52645

- 53- 1



## CITATION: Zorri ll a, L. (2011 )

. The Hershberger Bioassay for Permethrin. Int egrated Laboratory Systems

, Inc.

, Durham

, NC. Laboratory Project Study ID: C l 95-
200, Novembe r 15

, 20 11. MRID 48618602

. Un publi s h e d

.



## SPONSOR: Consumer Specialty Products Association

, Inc. for the Pe rmethrin Data Group

II

, 1 667 K Street NW

, Suite 300

, Washington, DC 20006



## TEST ORDER#: CON- 10 970 1-7



## EXECU TIVE SUMM .ARY: In a Hershberger assay (MRID 48618602) screening fo r androgenic activity

, permethrin (97.7% a. i.

, Lot# PMPS000014) in com oil was adm i nistered daily v i a oral gavage to g roup s of 59 to 60-day old, castrate d m a l e Sp r ag ue Dawl ey rats (8/group) at dose l eve l s of 0 (ve hicle ), 37.5

, or 1 20 mg/kg/day. An an dr ogen i c positive control

group co n sisting of e i ght castrated rats exposed to 0.4 mg/kg/day testosterone propionate {TP) by

subcutaneous (s.c. ) injection was also included as a positive co n tro l.

To scree n for potential anti-androgenic activ i ty, permethrin in corn oi l was also administered

d ai l y via ora l gavage to 59 to 60-day old

, castrated male Sprague Dawley rats (8 / gro u p) at dose

l eve l s of 12

, 37.5

, 120 mg/kg/day in conjunction with a daily dose of reference androgen TP at
0.4 mg/kg/day by s.c. inject i on. The contro l gro up consisted of e i ght castrated male rats d osed daily wit h TP (0.4 mg/kg/day) b y s. c

. injection in corn o il

, and the ant i- androge ni c po s itive

cont r o l group co n s i sted of e i g h t castrated male rats dosed orally wit h flutamide (FT) 

## in com o il

at 3 mg/kg/day in con j unction with a dai l y s.c. dose ofTP (0.4 mg/k g/ day).

Fo r both components of the assay, the animals were dosed for 10 consecutive days and necropsied approximately 24 hours after the final d ose a dmini stration to determin e weights of

the five androgen-depen dent t i ssues.

All an i mals survived until scheduled termination. No clinical signs of toxicity were obse r ved i n the pe rm et hrin dose g roup s of the androgen ago ni st or antago ni st assays. Bod y we i g hts andIn vivo Hershberger Assay (2011 ) / Page 2 of 11

PERMETHRIN/ 109701 O CS PP 8 9 0. 1400/ OECD 441



## overall body weight gains in the treated groups were similar to controls in both the androgen agonist and anti-androgen assays.

In the androgen agonist assay, animals dosed with TP had increased (p<0.05) body weights ( ↑ 12%) on Day 11, and increased (p<0.05) overall body weight gains ( ↑ 110%) compared to controls. No significant increases in the organ weights were observed in any of the five accessory sex tissues of animals dosed with permethrin . Animals in the TP group had increases (p≤0.05) in accessory sex organ weights as follows: 1002% in seminal vesicles; 898% in ventral prostate; 187% in levator ani -bulbocavernosus ( LABC) ; 486% in Cowper’s gland; and 51% in glans penis. For the vehicle control, the maximum guideline

- specified %CV was exceeded for the LABC (45% vs. 30%). All other %CV values were less than the maximum permissible

values.

In the anti-androgen assay positive control group (TP + FT), overall body weight gains were decreased (p≤0.05) by 35%. No significant decreases in the organ weights were observed in any of the five accessory sex tissues of animals dosed with permethrin+TP

. Animals dosed with TP + FT (positive contro l) had decreased (p≤0.05) accessory sex organ weights as follows: 84% in seminal vesicles; 77% in ventral prostate; 60% in LABC; 62% in Cowper’s gland; and 20% in glans penis. The maximum guideline

- specified %CV was exceeded for the Cowper’s gland in the 120 mg/kg/day permethrin group (22% vs. 20%). All other %CV values were less than the maximum permissible values.

The dosing for this study (high-dose of 120 mg/kg/day) was considered adequate based on data from two earlier studies showing either mortal ity at doses ≥200 mg/kg/day or clinical signs of neurotoxicity at 150 mg/kg/day.

S tatistically significant changes were not seen in two or more of the five androgen sensitive

tissue weight s. Permethrin was negative for androgenicity and anti -androgenicity in the Hershberger assay.

Th e assay satisfies the EDSP Tier 1 Test Order requirement s for a Hershberger a ssay (OCSPP 890.1400).

COMPLIANCE : Signed and dated GLP Compliance an d Quality Assurance statements were

pro vided.In vivo Hershberger Assay (2011 ) / Page 3 of 11

PERMETHRIN/ 109701 O CS PP 8 9 0. 1400/ OECD 441



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility : Integrated Laboratory Systems, Inc.

Location: Durham, NC Study Director: L. Zorilla

Other Personnel: S. Borghoff (Study Toxicologist) Study Period: July 25 , 2011 to November 1 5 , 2011



## 2. Test Substance : Permethrin

Description: L iquid

Source: AMVAC Chemical Corporation

Lot/Batch #: PMPS000014 , expires J anuary 2013 Purity: 97.7 % a.i. Stability: Stable in corn oil for up to 2 0 days at 1

- 10 ° C

CAS #: 52645

- 53

- 1

Structure:

O O

O Cl

CH

3 Cl

CH

3



## 3

. Reference Androgen : Testosterone propionate (TP)

Supplier : Sigma

- Aldrich Company (St. Louis, MO)

Batch #: 048K1328 , expires March 17, 2012 Purity: 100%

CAS #: 57

- 85

- 2



## 4

. Reference Anti

- androgen : Flutamide ( FT )

Supplier : Sigma

- Aldrich Company (St. Louis, MO)

Lot #: 107K1293 , expires February 15, 2012 Purity: 99.5%

CAS #: 13311

- 84

- 7



## 5

. Solvent/Vehicle Control : Corn Oil

Supplier : MP Biomedicals, LLC (Solon, OH)

Lot/Batch #: 7862K Rationale (if other than water) : Vehicle used in ILS range

- finding gavage study.

Final concentration : Not applicable



## 6. Test Animals : Species: Rat (castrated males)

Strain: Sprague Da w ley (Crl:CD[SD] IGS ) Age/weight at dose initiation: Post

- natal day (PND) 59/60 ; 239.1

- 323.9 g Source : Charles River Laboratories International, Inc., Raleigh, NC Housing: Rats were housed up to 2 /cage polycarbonate cages with absorbent heat

treated hardwood bedding (Northeastern Products Corp., Warrensburg, NY)

. Diet: Teklad Global 16% Protein Rodent Diet (Teklad Diets, Madison, WI), ad

libitum ; low phytoestrogen with total gen i stein equivalents = 8.0 μg/g diet.

Water: T ap water , ad libitum

Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 22

- 24 oC

33

- 58 %

Not reported 12 hrs light/12 hrs dark Acclimation period: 7 daysIn vivo Hershberger Assay (2011 ) / Page 4 of 11

PERMETHRIN/ 109701 O CS PP 8 9 0. 1400/ OECD 441



## B. STUDY DESIGN

1. In

- life dates : Start: 8/1/11 End: 8/12/11

2. Study Design : Th is Hershberger Assay was conducted to screen for both potential androgenic and anti

- androgenic a ctivity. In the test for androgenic activity, p ermethrin was administered daily via oral gavage to groups of castrated male rats. Positive androgenic activity is defined as a significant increase in two or more target organ weights compared to

the vehicl e control group. The positive control group for androgenic activity consisted of

castrated male rats dosed by s.c. injection with TP at 0.4 mg/kg/day. In the test for antiandrogenic activity, p ermethrin was administered daily via oral gavage to groups of castrated male rats in conjunction with TP at 0.4 mg/kg/day by s .c. injection.

Anti

- androgenic activity is indicated by a statistically significant decrease in two or more target organ weights of the treated groups (test substance + TP) compared to the TP -only control group. The positive control group for anti- androgenic activity consisted of castrated male rats dosed by s.c. injection with TP (0.4 mg/kg/day) in conjunction with an oral dose of FT (3.0 mg/kg/day). For both assays, the animals were dosed for 10 consecutive days and necropsied approximately 24 hours after the final dose administration for measurement of target organ weights.

3. Study Schedule : Following the initial 7-day acclimation period, castrated male rats (surgical mani pulation performed by Supplier at PND 45 ) were administered the test

substance from PND 59-60 to PND 68-69. Rats were euthanized on PND 69-70, approximately 24 hours after the last dose and necropsied for organ weight measurements.

4. Animal Assignment : Animals were randomly assigned, stratified by body weight, to the test groups noted in Table 1. Statistical analysis indicated that there were no significant differences in group means at study initiation.

TABLE 1. Study design a


### Test group Dose (mg/kg/day) # of Males
Androgen Agonist Assay Vehicle control (corn oil) 0 8 Low ( Permethrin ) 37.5 8 High ( Permethrin ) 120 8 Positive control (corn oil) +TP b 

### 0.4 8

Anti

- Androgen Assay Vehicle control (corn oil) +TP b 

### 0 8
Low ( Permethrin +TP) 12 8 Mid ( Permethrin +TP) 37.5 8 High ( Permethrin +TP) 120 8 Flutamide (oral gavage) + TP (s.c.), positive control 3 8

a Data were obtained from Tables 1 and 2 on page 15 of the study report.

b Same animals dosed for both androgen agonist (serves as positive control) and anti

- androgen (served as vehicle control) assays. This did not affect the outcome or conclusions of this study.



## 5. Dose Selection Rationale : The p ermethrin dose levels were selected based on the results of

a range -finding study (ILS Inc., Study No. C195-500) and a previous Hershberger studyIn vivo Hershberger Assay (2011 ) / Page 5 of 11

PERMETHRIN/ 109701 O CS PP 8 9 0. 1400/ OECD 441



## (Kunimatsu, T., 2002, Regul Toxicol Pharmacol ). In the range -finding study, male rats
were administered p ermethrin for 1 4 days at concentrations of 100, 200, 300, and 400 mg/kg/day (four rats/group) . All rats at 300 and 400 mg/kg/day died during testing, and only one 200 mg/kg/day rat survived to study termination. No effects were observed on body weights, liver or kidney weights of surviving rats. In the previous Hershberger study,

male rats were ad ministered permethrin for 5 days at concentrations of 150 and 300 mg/kg/day . At 150 mg/kg/day, clinical signs included tremors and hypersensitivity. Based on these results, doses of ≤ 120 mg/kg/day were used for both the androgen agonist and antiandrogen assays

.

6. (a) Dose Preparation : Dose formulations were prepared once prior to initiation of treatment by mixing the appropriate amounts of test substance in corn oil

. Flutamide and

TP w ere prepared once prior to dosing by mixing in corn oil. Homogeneity (top, middle, and bottom samples) and concentration of permethrin were determined for all dose

formulations.

In a previous storage stability study 1 

## , p ermethrin (lot number not reported) was determined to be stable in corn oil for up to 20 da ys at 1 -10°C.

( b) Dose Analysis

Results

Homogeneity (%CV) : 0.853-2.55%

Concentration (% of nominal): 95-106%

The analytical data indicated that the mixing procedure was adequate and that the variation between nominal and actual dosage to the animals was acceptable.

7. Dosage administration : Test formulations of p ermethrin were administered to the animals daily via oral gavage (5 mL/kg bw) for 10 days. TP was given via s.c. injection at 0.5 mL/kg bw, and FT was administered via oral gavage at 5 mL/kg bw. Dose volumes were adjusted daily based on the concurrent body weight measurement.

8. Statistics : Body wei ghts, body weight gains and accessory sex organ weights were analyzed using studentized residual plots for outliers and Levene’s test for homogeneity of variance. Heterogeneous data were transformed by logarithm (glans penis) or multiplicative inverse (LA BC weights) and re-analyzed. Body weights, body weight gains, and accessory sex organ weights were analyzed using one- way ANOVA followed by Dunnett’s t

- tests. The positive controls were compared to the vehicle controls using the t- test. Significance

was denoted at p<0.05. Statistical analyses were performed using MS EXCEL and SAS ®



## Version 9.2 . The statistical analyses were considered adequate.

1 

### Smith, R . 2011 a. Storage Stability of Permethrin in Corn Oil . Unpublished study report prepared by Smithers Viscient Inc., Study No. 14010

- 6105

.In vivo Hershberger Assay (2011 ) / Page 6 of 11

PERMETHRIN/ 109701 O CS PP 8 9 0. 1400/ OECD 441



## C. METHODS

1. Clinical Examinations : Animals were checked for mortality or moribundity twice daily on weekdays and once daily on weekends. Rats were also checked for clinical signs of toxicity at randomization, daily prior to dosing throughout the dosing period, and at termination.

2. Body Weight : Animals were weighed at randomization, daily prior to dosing throughout the dosing period, and at termination.

3. Food Consumption (Optional) : Food consumption was not determined.

4. Serum Hormone Measurements (Optional) : Serum hormone measurements were not

conducted.

5. Dissection and Measurement of Tissue and Organ Weights : On PND 69-70 (approximately 24 hours after the final administration of the test substance)

, all rats were weighed, anesthetized by CO 2 

## inhalation followed by cervical dislocation and subjected to a

gross necropsy. The five mandatory androgen-dependent organs (ventral prostate, seminal vesicles with coagulating glands and fluid, LABC, Cowper’s gland, and glans penis) were excised, trimmed free of adhering tissue, and weighed fresh (unfixed) according to the standard operating procedures detailed in the U.S. EPA Guideline (O CSPP 890.1400).In vivo Hershberger Assay (2011 ) / Page 7 of 11

PERMETHRIN/ 109701 O CS PP 8 9 0. 1400/ OECD 441



## II. RESULTS

A. OBSERVATIONS

1. Mortality : All animals survived until scheduled termination.

2. Clinical signs of toxicity : No clinical signs of toxicity were observed in the permethri n dose groups of the androgen agonist or antagonist assays.

B. BODY WEIGHT AND WEIGHT GAIN : Selected body weight and body weight gains for the androgen agonist assay are presented in Table 2. Body weights and overall body weight gains for the 37.5 and 120 mg/kg/day treatment group s were comparable to controls throughout the duration of the assay. Animals dosed with T P had increased (p<0.05) body weights ( ↑ 12%) on Day 11, and increased (p< 0.05) overall body weight gains ( ↑ 110 %) compared to controls.

TABLE 2. Selected Group Mean (±SD) Body Weights and Cumulative Body Weight Gains (g) in the Androgen Agonist Assay a

Study Day # Dose (mg/kg/day)

Vehicle Control Positive Control

Vehicle + TP (0.4) b Permethrin ( 37.5 ) Permethrin ( 120 )

N Mean SD N Mean SD N Mean SD N Mean SD

1 8 283.1 27.9 8 276.5 25.4 8 286.4 22.7 8 287.6 25.4

4 8 298.4 29.1 8 312.0 31.2 8 303.1 23.8 8 305.3 27.5

7 8 311.2 31.9 8 336.5 33.3 8 317.5 19.9 8 319.9 29.0

11 8 324.8 34.0 8 364.1 * (↑ 12 ) 36.3 8 332.0 20.9 8 338.4 33.3

BWG (Days 1

- 11) 8 41.7 11.3 8 87.7 * (↑ 110 ) 35.2 8 45.6 6.2 8 50.8 14.1

a D ata were obt ained from Table 5 on page 21 and Appendix V on page 78 of the study report. Percent differences from controls were calculated by the reviewers and included in parentheses.

b The positive control group for the androgen agonist is same as the vehicle control for the anti

- androgen. This did not affect

the outc ome or conclusions of this study.

N Number of animals in the group

SD Standard Deviation

* Significantly different from controls at p ≤ 0.05



## Body weight and body weight gain data for the anti- androgen assay are presented in Table 3. Body weights and overall body weight gains for p er methrin

- treated groups were comparable to controls throughout the duration of the assay. For the positive contro l group (TP + FT), overall body weight gains were decreased (p≤0.05) by 35%.In vivo Hershberger Assay (2011 ) / Page 8 of 11

PERMETHRIN/ 109701 O CS PP 8 9 0. 1400/ OECD 441



### TABLE 3. Selected Group Mean (±SD) Body Weights and Cumulative Body Weight Gains (g) in the Anti

Androgen Assay a

Study Day # Dose (mg/kg/day)

Vehicle Control

+ TP (0.4) b Positive Control

TP + FT (0.4 + 3.0) Permethrin +TP ( 12 + 0.4) Permethrin +TP ( 37.5 + 0.4) Permethrin +TP ( 120 + 0.4)

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD

1 8 276.5 25.4 8 285.9 25.6 8 279.7 23.2 8 284.9 22.4 8 284.4 24.9

4 8 312.0 31.2 8 304.5 25.9 8 304.3 23.1 8 309.1 23.8 8 306.5 25.7

7 8 336.5 33.3 8 322.2 25.9 8 326.1 25.5 8 332.8 26.9 8 327.2 29.0

11 8 364 .1 36.3 8 343.0 26.1 8 348.9 27.4 8 358.1 28.7 8 354.5 28.2

BWG (days 1

- 11) 8 87.7 35.2 8 57.2 * (↓ 35 ) 4.9 8 69.3 21.4 8 73.2 10.2 8 70.1 4.9

a Data were obtained from Table 6 on page 22 and Appendix V on pages 78 and 79 of the study report. Percent differences

from vehicle controls (calculated by the reviewers) are included in parentheses.

b The vehicle control for the anti

- androgen is same as the positive control group for the androgen agonist. This did not affect the outcome or conclusions of this study.

N Number of animals in the group

SD Standard Deviation

* Significantly different from vehicle controls at p<0.05.



## C. FOOD CONSUMPTION (Optional) : Food consumption was not determined.

D. SERUM HORMONE CONCENTRATIONS (Optional) : Serum hormone concentrations

were not determined.

E. ORGAN WEIGHTS : Accessory sex organ weights for the androgen agonist assay are presented in Table 4. There were no treatment

- related effects in any of the accessory sex organs of animals dosed with p ermethri n. Animals in the TP group had increases (p ≤ 0.05)

in accessory sex organ weight s as follows: 1002 % in seminal vesicles; 898% in ventral
prostate; 187 % in LABC; 486% in Cowper’s gland; and 51% in glans penis. In the vehicle control group, t he maximum guideline

- specified %CV (30%) w as exceeded for the LABC (35%). All other %CV values were less than the maximum permissible values.In vivo Hershberger Assay (2011 ) / Page 9 of 11

PERMETHRIN/ 109701 O CS PP 8 9 0. 1400/ OECD 441



### TABLE 4. Accessory Sex Organ Weights (mg) from Androgen Agonist Assay in Sprague

- Dawley Rats a

Organ Dose (mg/kg/day)

Vehicle Control Permethrin ( 37.5 ) Permethrin ( 120 ) Positive Control

Vehicle + TP (0.4) b

N Mean SD CV N Mean SD CV N Mean SD CV N Mean SD CV

Seminal

vesicles 8 56.0 20.4 36 8 45.6 13.5 30 8 46.7 11.1 24 8 616.9 * (↑ 1002 ) 190.2 31

Ventral
prostate 8 14.9 6.6 44 8 12.3 3.4 28 8 14.0 5.6 40 8 148.7 * (↑ 898 ) 39.6 27

LABC 8 211.5 75.0 35 c 8 165.4 32.0 19 8 190.4 30.6 16 8 606.9 * (↑ 187 ) 90.8 15

Cowper’s glands 8 8.4 3.4 41 8 7.6 3.9 51 8 8.0 2.5 31 8 49.2 * (↑ 486 ) 8.5 17

Glans penis 8 63.0 13.6 22 8 64.4 6.1 9 8 65.3 5.7 9 8 95.0 * (↑ 51 ) 13.3 14

a Data were obtained from Table 7 on page 2 4 of the study report. Percent differences from controls were calculated by the reviewers and included in parentheses.

b The positive control group for the androgen agonist is same as the vehicle control for the anti

- androgen. This did not affect the outcome or conclusions of this study.

c The %CV exceeds the maximum acceptable %CV.

N Number of animals in the group

SD Standard Deviation

CV Coefficient of Variation

* Significantly different from vehicle controls at p<0.05.



## Accessory sex organ weights for the anti -androge n assay are presented in Table 5

. There

were no treatment

- related effects in any of the accessory sex organs of animals dosed with

perme thrin +TP . Animals dosed with TP + FT (positive control) had decreased (p≤0.05) accessory sex organ weights as follows: 84 % in seminal vesicles; 77 % in ventral prostate;

60 % in LABC; 62% in Cowper’s gland; and 20% in glans penis. The maximum guidelinesp ecified %CV (20%) was exceeded for the LABC in the 120 mg/kg/day p ermethrin group (22 %). All other %CV values were less than the maximum permissible values.In vivo Hershberger Assay (2011 ) / Page 10 of 11

PERMETHRIN/ 109701 O CS PP 8 9 0. 1400/ OECD 441



### TABLE 5. Accessory Sex Organ Weights (mg) from Anti

- Androgen Agonist Assay in Sprague

- Dawley Rats a

Organ Dose (mg/kg/day)

Vehicle Control

+TP (0.4) b Permethrin +TP ( 12 + 0.4) Permethrin +TP ( 37.5 + 0.4) Permethrin +TP ( 120 + 0.4) TP + FT

Positive Control

N Mean SD CV N Mean SD CV N Mean SD CV N Mean SD CV N Mean SD CV

Seminal

vesicles 8 616.9 190.2 31 8 669.8 172.1 26 8 614.7 188.5 31 8 638.9 125.7 20 8 100.2 * (↓ 84 ) 40.3 40

Ventral
prostate 8 148.7 39.6 27 8 154.8 41.7 27 8 173.7 40.6 23 8 180.3 37.6 21 8 34.7 * (↓ 77 ) 10.3 30

LABC 8 606.9 90.8 15 8 570.9 76.8 13 8 556.7 62.0 11 8 532.2 119.5 22 c 8 240.0* (↓ 60 ) 80.7 34

Cowper’s glands 8 49.2 8.5 17 8 45.5 6.6 15 8 42.8 7.5 17 8 47.1 13.4 29 8 18.6 * (↓ 62 ) 7.7 41

Glans penis 8 95.0 13.3 14 8 98.0 11.9 12 8 90.9 14.2 16 8 95.5 15.1 16 8 76.3 * (↓ 20 ) 10.7 14

a Data were obtained from Tables 8 on page 25 of the study report. Percent differences from controls were calculated by the reviewers and included in parentheses.

b The vehicle control for the anti

- androgen is same as the positive control group for the androgen agonist. This did not affect the outcome or conclusions of this study.

c The %CV exceeds the maximum acceptable %CV.

N Number of animals in the group

SD Standard Deviation

CV Coefficient of Variation

* Significantly different from vehicle controls at p<0.05.



## III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS : In the agonist assay, dose levels of 37.5 or 120 mg/kg/day permethrin did not increase androgen -dependent tissue weights compared to the weights of these tissues in the vehicle control animals. In the antagonist assay, p ermethrin

co -administered with TP at dose levels of 12, 37.5, or 120 mg/kg/day did not decrease androgen dependent tissue weights. Based on these findings using the Hershberger Assay (O CS PP 890.1400), oral administration of permethrin at up to 120 mg/kg/day did not show any androgen agonist/ antagonist activity, or 5 α -reductase inhibition.

B. AGENCY COMMENTS : All animals survived until schedul ed termination. No clinical signs of toxicity were observed in the permethrin dose groups of the androgen agonist or antagonist assays.

Although no toxicity was observed in the current study at doses of permethrin up to 120 mg/kg/day, the high dose is adequate based on data from two earlier studies showing either mortality at doses ≥200 mg/kg/day or clinical signs of neurotoxicity at 150 mg/kg/day

.

In the androgen agonist assay, body weights and overall body weight gains for the 37.5 and 120 mg/kg/day treatment groups were comparable to controls throughout the duration of the

assay. No significant increases in the organ weights were observed in any of the five accessory sex tissues of animals dosed with permethrin . Animals dosed with TP had

increased (p<0.05) body weights ( ↑ 12%) on Day 11, and increased (p<0.05) overall body weight gains ( ↑ 110%) compared to controls. Animals in the TP group showed the expected increases (p≤0.05) in accessory sex organ weights

. For the vehicle control, the maximum

gu ideline

- specified %CV was exceeded for the LABC ( 35% vs. 30%). All other %CV values were less than the maximum permissible values.In vivo Hershberger Assay (2011 ) / Page 11 of 11

PERMETHRIN/ 109701 O CS PP 8 9 0. 1400/ OECD 441



## In the anti-androgen assay, body weights and overall body weight gains for permethrintreated groups were comparable to con trols throughout the duration of the assay. No significant de creases in the organ weights were observed in any of the five accessory sex

tissues of animals dosed with permethrin+TP . For the positive control group (TP + FT), overall body weight gains were decreased (p≤0.05) by 35%. Animals dosed with TP + FT (positive control) showed the expected decrease s in accessory sex organ weights

. The maximum guideline

- specified %CV was exceeded for the LABC in the 120 mg/kg/day permethrin group (22% vs. 20%). All other %CV values were less than the maximum permissible values.

No statistically significant changes were seen in two or more of the five androgen sensitive tissues. Permethrin was negative for androgenicity and anti-androgenicity in the Hershberger assay.

C. STUDY DEFICIENCIES : The following deficiencies were noted that are not considered to have had an adverse impact on the results, interpretation or conclusions of this study:

• In the androgen agonist assay, the maximum guideline

- specified %CV was exceeded for the LABC in the vehicle control (35% vs. 30%).

• The maximum guideline

- specified %CV was exceeded for the LABC in the 120 mg/kg/day permethrin group (22% vs. 20%) in the anti

- androgen assay

.

# DATA EVALUATION RECORD

PERMETHRIN

Study Type: O CSPP 890.1450, Female Pubertal Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-75-2012 (MRID 48672901)

Prepared for

Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by

CSS

- Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer : Signature: Michelle Sharpe

- Kass, M.S. Date: 7/10 / 2012

Secondary Reviewer : Signature:
David A. McEwen, B.S. Date: 7 / 16 /2012

Program Manager: Signature: Jack D. Early, M.S. Date: 7/18 /2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 7/18 /2012

This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by CSS

- Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of eac h individual assay to identify potential endocrine bioactivity with complementary endpoints within 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).

### PERMETHRIN / 109701



## Primary Reviewer: Edward Sco llon

, Ph.D.

Health Effects Division 

## Secondary Reviewer: Elizabeth M e nd ez
, Ph

. D.

Health Effects Division Female Pubertal Assay (2012) 

### I Page I of 

### 14

OCSPP 

### 890.1450/ OECD 

### None

Templa t e version 08 / 2011



## DATA EVALUATION RECORD

STUDY TYPE: Female Puberta l Assay ; OCS PP 890.1450; OE CD None.



## P C CODE: 109701 DP 

## BARCO DE: D40179 3



## TXR#: 0056246 

## CAS No: 52645 -: 53 -1



## TEST MATERIAL (PURITY): Perm et hrin (97 .7 % a. i. )



## SYNONYMS: 3 -Phenox y b e n zy l ( 1 RS)-cis,trans-3-(2

, 2 -di ch lo rovinyl)

- 2,2

- d imeth y lc yclopro pan ecarboxy l ate



## CITATION: Zori lla

, 

## L. (2012) Pub e rt a l Development and Thyroid Function in Intact

Juvenile/P e ripubertal Fe mal e Rats ; Permethrin. Int egrate d Labor a tor y Systems,

Inc

., Durham NC. Labor atory Report No .: Cl 95-300

, A pril 1 3

, 2012. MRID

48672901. U n pub li s h e d

.



## SPONSOR: Co n s umer Sp ec ialty Product s Association for the Permethrin Data Group

II /Jo int Venture. 1667 K Stree t N W , S uit e 300, Washington, D C



## TEST ORDER#: C O N

- 109701

- 7



## EXECUTIVE SUMMARY: ln a Female Pubertal Assay (MRID48672901), 16 SpragueD aw le y (C rl:CD ® (SD ] IG S) female rats/dose g roup we r e treated dai l y vi a oral gavage w ith permethrin (Lot# P MPS000 1 4

, 97.7% a .i .)at doses ofO (com oi l ) 75

, o r 15 0 mg/kg/day from

post -n a t al day (PND) 22 to 42 / 43. Animals were exam in ed for vagi n a l opening (VO) daily beg in ni n g on PND 22, and age and weight at attainment of VO were recorded. Fo ll owi n g

sac rific e on PND 42/43, blood was collected for clinical c hemi stry ana l yses, including anal ys i s of t o t al thyroxin e (T4) and thyroid st imula ting h ormone (TSH) using a radioimmunoassay (RIA).

Adre n a l

, li ve r
, thyro i d

, pituitary and uro genita l organ weig ht s were recorded, and microscopic

examinations were performed o n th e ovaries, uteru s
, thyroid

, and kidneys.

A ll r ats administe r ed permethrin survived unt il sc hedul e terminati on. Fro m P ND 34 onward,

tremors were observed 

## in 4 animals admini ste r ed 75 mg/kg/day and all animals admi nist e r ed 1 50 mg/kg/day at 4-6 h o ur s p ost-dosing on at l east o ne day. These findings did not persist to the

d a il y ex amination s
, w hich were cond u cted ju s t prior to dosing

.

No treatment-related effec t s were observed o n body weights

, body we i ght gai n s
, age of

attainment of VO

, or we i ght at day of attainme n t at any dose. All surv iving anima l s attained

com pl ete VO.Female Pubertal Assay ( 2012 ) / Page 2 of 14

PERMETHRIN / 109701 O CSPP 89 0. 1 45 0/ OECD None



## Absolute liver weight s (adjusted and unadjusted) were increased (p<0.05) by 10% at 150 mg/kg/day. R elative (to body weight) liver weight s w ere increased (p<0.05) by 4 and 8% at 75 and 150 mg/kg/day. Both unadjusted and adjusted thyroid weig hts were decreased (p<0.05) by 22% at 150 mg/kg/day.

Administration of permethrin did not change the day of first estrus compared to the vehicle control group. Animals exhibited similar cycle lengths and regularity. The majority of females in the 150 mg/kg/day group were in diestrus at necropsy.

S erum T 4 

## levels were decreased (p<0.05) by 26 and 42% at 75 and 150 mg/kg/day , respectively

, and, serum TSH levels were increased (p<0.05) by 40% at 150 mg/kg/day. Additionally, at 150 mg/kg/day, increases (p <0.05) were observed in sodium (↑3%), chloride (↑4%) , and sorbitol dehydrogenase ( SDH ; ↑20%) , alkaline phosphatase ( ALP ) was decreased (↓16%).

There were no effects of treatment in the thyroid on either colloid area or follicular cell height in

any treated group. No effects of treatment were observed microscopically in the ovaries, uterus or kidney.

The dose levels tested were determined to be adequate since they were based on the results of the range finding study where mortality was seen at doses gr eater than 200 mg/kg/day and tremors at 150 mg/kg/day.

The assay satisfies the EDSP Tier 1 Test Order requirement s for a Female Pubertal Assay (OCSPP 890.1450).

COMPLIANCE : Signed and dated Data Confidentiality, GLP Compliance, and Quality

Assurance statements were provided.Female Pubertal Assay ( 2012 ) / Page 3 of 14

PERMETHRIN / 109701 O CSPP 89 0. 1 45 0/ OECD None



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility : Integrated Laboratory Services, Inc.

Location: Durham, NC Study Director: Leah Zorilla

Other Personnel: Thomas G. Osimits

- Study Monitor Study Period: June 30, 2011

- April 13, 2012



## 2

. Test S ubstance : Permethrin

Description: Yellow Liquid

Source: AMVAC Corporation

Lot/Batch #: PMPS00014 (exp January 2013) Purity : 97.7 % Stability: It was stated that d ose formulations of permethrin in corn oil held from 1 to 10 ° C for up to 20 days were considered stable

. CAS #: 52645

- 53

- 1

Structure:

O O

O Cl

CH

3 Cl

CH

3



## 3

. Vehicle : Corn Oil

4

. Test A nimals : Species: Rat

Strain: Sprague Dawley CRL: CD ® (SD) IGS Age/Weight at Study Initiation: PND 22 / 44.5

- 69.3 g females only

Source: Charles River Laboratories International, I nc. (Raleigh, NC) Housing: 2/cage in polycarbonate cages with micro

- isolator tops and absorbent, heat

- treated hardwood bedding

. Diet: Teklad Global 16% protein rodent diet (while at ILS) and Autoclaved Purina 5L9 Rat and

Mouse Diet (while at Charles River), ad libitum

. Total genistein equivalents of genistein plus

daidzein were 8.0 and 8.6 μg/g feed

. Water: Reverse

- osmosis filtered tap water, ad libitum

Environmental

Conditions: Temperature: Humidity: Air changes: Photoperiod: 19

- 26 °C

36

- 67 % Not reported

14 hrs light/ 10 hrs dark



## B. ST UDY DESIGN

1. In -L ife D ates : Start: August 5, 2011 End: August 26, 2011

2. Mating : Time

- mated pregnant dams were received from the supplier on Gestation Day 8. Dams with their litters were kept together until weaning (PND 21). Litters with more than 8 pups were standardized between PND 3 and 5 to 8 pups per litter with equal numbers of

males and females

.Female Pubertal Assay ( 2012 ) / Page 4 of 14

PERMETHRIN / 109701 O CSPP 89 0. 1 45 0/ OECD None



## 3. Animal A ssignment : Animals were assigned to the test groups noted in Table 1 using a procedure that stratifies animals across groups by body weight such that mean body weight of each croup was not statistically different from any other group using ANOVA.

Littermates were not assigned to the same treatment group.

TABLE 1. Study Design a


### Test group Dose (mg/kg/day) # of Fem ales

Control 0 16

Low 75 16 High 150 16

a Data were obtained from page 14 of the study report.



## 4. Dose S election Rationale : The dose levels were selected based on the results from a dose range finding study 1

. 

## Four immature female rats per group were administered either corn oil or permethrin at dose levels of 50, 100, 150, 200, or 250 mg/kg for 14 days. Tremors and

seizure s were noted within 1-4 hours post-dosing in all animals on Day 1 and in all surviving animals on Day 2. All animals administered 200 and 250 mg/kg permethrin did not survive past Day 2. Beginning on Day 4, mild tremors were noted in 3 of 4 animals administered 150 mg/kg permethrin. All rats administered 50 -150 mg/kg permethrin were observed as normal 24-hours post-dose for 14 days. No statistically significant group mean body weight differences or differences in body weight gain were observed as compared to

control.

Based on th is study and the guideline requirements of dosing immature rats, a dose of 150 mg/kg/day was selected for the female pubertal assay based on deaths, moribundity or

adverse clinical signs observed at up to 24-hours post-dose at ≥ 200 mg/kg/day.

5. Dose P reparation and Analysis : Dose formulations were prepared daily by mixing appropriate amounts of test substance with corn oil. Homogeneity (top, middle, and bottom)

and achieved concentrations were tested for all dose formulations by an off- site analytical laboratory (Smithers Viscient, LLC, Aareham, MA). It was stated that permethrin was previously demonstrated to be stable in corn oil when stored refrigerated (1-10 ° C) for up to 10 days (Smithers Viscient Study No. 1410-6105; no data provided). 2



## Results of D ose Analysis

Homogeneity (% CV) : 0.297-1.38%

Concentration (% of nominal): 96-107%

Stability (% of initial) : No data provided

1 . Zorilla, L. (2011) Range Finder Study for In Vitro Mammalian Assays for Permethrin. (Draft). Unpublished study report prepared by ILS, Inc. Study No. C195

- 500

2 . Smith, R (2011) Storage Stability of Permethrin in Corn Oil. Unpublished study report prepared by Smithers Viscient Inc. Study No. 1410

- 6105Female Pubertal Assay ( 2012 ) / Page 5 of 14

PERMETHRIN / 109701 O CSPP 89 0. 1 45 0/ OECD None



## The analytical data indicated that the mixing procedure was adequate and that the variation between nominal and actual dosage to the animals was acceptable. The storage stability

data (Smithers Viscient Study No. 1410-6105) should be submitted for independent review.

6. Dosage A dministration : All doses were administered once daily by gavage, from PND 22 through PND 42/43, in a volume of 5 mL/kg of body weight. Dosing was performed 24 ± 2 hours from the previous dose. Dose volume was adjusted daily based on individual body weights.

7. Statist ics : The following statistical analyses were performed. Statistical significance was reported when p<0.05. The analyses were considered appropriate.

Parameter Statistical test Initial body weight Final body weight Final body weight gain Age and body weight at VO Age at first estrus Cycle length Ovarian counts (follicles, corpora lutea) Homogeneous data sets were analyzed using a one

way analysis of variance (ANOVA), followed by pairwise comparisons using Dunnett’s two

- tailed t

tests.

Tissue weights Relative tissue weights (liver, kidney, pituitary, adrenal)

Hormone levels Clinical chemistry Homogeneous data sets were analyzed using a two

way ANOVA with treatment and necropsy day as the main effects, followed by pairwise comparisons using Dunnett’s tw o

- tailed t tests. Initial body weight Final body weight Final body weight gain Age and body weight at VO Tissue weights Homogeneous data sets were analyzed using a two

way analysis of covariance (ANCOVA) with PND 21 body weight as the covariable, followed by pairwise comparisons using Dunnett’s two

- tailed t

tests. Heterogeneous data sets Chi

- square analysis Thyroid scoring Fisher’s exact test



## C. METHODS

1. Mortality and C linical Examinations : All animals were examined twice daily on weekdays, once daily on weekends/holidays for mortality and moribundity. Clinical

examinations were conducted daily prior to dosing throughout the study. Beginning on PND 34, an additional examination was also performed at 4-6 hours post-dosing.

2. Body W eight : Body weights were recorded prior to allocation of the animals to study groups, daily prior to dosing, and prior to scheduled termination. Overall (PND 22 to 42/43) body weight gain was also calculated.

3. Vaginal O pening : Beginning on PND 22 , all animals were examined daily for onset of

VO . Age and weight on the day of completion of VO were recorded.

4. Estr o us C yclicity : Beginning on the day of complete VO through to euthanasia, vaginal smears were prepared daily and evaluated . Animals were classified as diestrus, proestrus, or estrus; metestrus was classified as diestrus. The overall cycling pattern for each female was characterized as regular, irregular, or not cycling. Regular cycling was defined as having

recurr ing 4 to 5 day cycles, while irregular cycling was defined as having cycles withFemale Pubertal Assay ( 2012 ) / Page 6 of 14

PERMETHRIN / 109701 O CSPP 89 0. 1 45 0/ OECD None



## diestrus for a period >3 days or estrus ≥ 3 days. Animals were not considered to be cycling if there were ≥3 consecutive days of estrus or ≥5 consecutive days of diestrus. Mean cycle length was defined as the number of days from one diestrus to the next diestrus. Incomplete cycles were not used in calculating mean cycle length. In instances where the time between

VO and humane euthanasia did not allow observation of more t han 1 complete cycle, classification was based on data available and the assumption that animals were cycling regularly if the partial data fit the definition, and cycling irregularly if the study ends without being able to distinguish between irregular and not cycling.

5. Sacrifice and Pathology : On the day of termination, animals were moved to the necropsy holding room approximately 2 hours prior to sacrifice. All surviving animals were sacrificed by decapitation on PND 42/43 approximately 2 hours post-dosing. Blood from the trunk of the animals was collected immediately into serum separation tubes and stored on wet ice until processing. Samples were centrifuged at 3000 g for 30 min at 4 ° C and

stored at ≤− 70°C for subsequent hormone and clinical c hemistry evaluations.

a. Hormone Analysis : Total T 4 

## and TSH levels were determined using radioimmunoassays by a commercial analytical laboratory (AniLytics, Gaithersburg, MD). Samples were assayed in duplicate with multiple quality control samples.

b. Clinical C hemistry : The following CHECKED (X) parameters were examined

.

ELECTROLYTES OTHER

X Calcium X Albumin

X Chloride X Creatinine*
Magnesium X Urea nitrogen*

X Phosphorus Total cholesterol

X Potassium Globulins

X Sodium Glucose

ENZYMES X Total bilirubin

X Alkaline phosphatase ( ALP ) X Total protein

Cholinesterase (ChE) Triglycerides
Creatine phosphokinase Serum protein electrophoresis
Lactic acid dehydrogenase (LDH)

X Alanine aminotransferase (ALT/also SGPT)

X Aspartate aminotransferase (AST/also SGOT)

X Sorbitol dehydrogenase (SDH)

X Gamma glutamyl transferase (GGT)
Glutamate dehydrogenase

* Recommended for the pubertal assay in female rats based on guideline 890.1 45 0.



## c. Organ W eights and Histopathology : The following CHECKED (X) tissues were collected and weighed. The (XX) organs, in addition, were subjec ted to histological

examination

.

UROGENITAL OTHER

XX Ovaries ( paired, without oviducts) * + XX Thyroid * +

XX Uterus* + X Liver *

XX Kidneys (paired)*+ X Adrenals (paired) *

X Pituitary *Female Pubertal Assay ( 2012 ) / Page 7 of 14

PERMETHRIN / 109701 O CSPP 89 0. 1 45 0/ OECD None

* Weights required based on guideline 890.1 4 50

+ Histopathological examination required based on guideline 890.1 4 50



## The uterus, ovaries, and kidneys were weighed prior to fixation. The thyroid (with parathyroid) was collected with the trachea and fixed in 10% neutral buffered formalin for at least 24 hours. Following fixation, the thyroid was dissected free of the trachea and weighed. The uterus (without fluid), left ovary, and left kidney were fixed in 10% neutral buffered formalin for at least 24 hours. Following fixation, the fixed tissues were transferred to 70% histology grade alcohol, routinely processed into paraffin blocks, sectioned, stained with hematoxylin and eosin, and examined microscopically. Five random sections of the ovary were evaluated microscopically for follicular development (including the presence or absence of tertiary/antral follicles, presence or absence of corpora lutea, changes in corpus luteum development, and changes in the number of both primary and atretic follicles) in addition to any abnormalities or lesions. Two sections of each of the two lobes of the thyroid were examined and evaluated for follicular cell height and colloid area, using a five point grading scale (1 = shortest/smallest; 5 = tallest/largest), and any

abnormalities/lesions noted.

II. RESULTS

A. MORTALITY : All rats administered permethrin survived until scheduled termination.

One animal administered vehicle control was moribund and euthanized on PND 34 due to clinical signs ( piloerection, lethargy, pale appearance, labored breathing) and weight loss. These findings were attributed to gavage error.

B. CLINICAL SIGNS OF TOXICITY : From PND 34 onward, tremors were observed in 4 animals administered 75 mg/kg /day and all animals administered 150 mg/kg /day at 4-6 hours post-dosing on at least one day. These findings did not persist to the daily

examinations conducted just prior to dosing on the next day.

C. GENERAL GROWTH AND VAGINAL OPEN ING : No treatment

- related effects were observed on body weights, body weight gains, age of attainment of VO, or weight at day of

attainment at any dose (Table 2). All surviving animals attained complete VO. All control group performance criteria for means and CVs were within the acceptable ranges.Female Pubertal Assay ( 2012 ) / Page 8 of 14

PERMETHRIN / 109701 O CSPP 89 0. 1 45 0/ OECD None



### TABLE 2

. General Growth and Vaginal Opening (VO) a

Parameter Evaluated Vehicle Control Permethrin (75 mg/kg/day) Permethrin ( 150 mg/kg/day)

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)

Initial body weight (PND 22; g) U 15 56.5 4.3 7.7 16 55.9 5.5 9.9 16 56.3 5.4 9.6

A 15 56.2

― ― 16 56.2

― ― 16 56.2

― ― Body weight at vaginal opening (g) U 15 124.7 7.4 5.9 16 125.9 10.9 8.6 16 120.4 14.3 11.9

A 15 124.5

― ― 16 126.2

― ― 16 120.4

― ― Final body weight (g) U 15 169.8 12.7 7.5 16 171.4 10.2 5.9 16 171.7 12.8 7.4

A 15 169.3

― ― 16 171.9

― ― 16 171.6

― ― Final body weight (% of control) U 15 NA 16 100.9

― ― 16 101.1

― ― A 15 16 101.5

― ― 16 101.4

― ― Body weight gain (final

– initial; g) U 16 113.1 10.5 9.3 16 115.5 9.0 7.8 16 115.4 10.1 8.8

A 16 113.0

― ― 16 115.6

― ― 16 115.3

― ― Age at vaginal opening (PND) U 15 33. 9 1.8 5.2 16 3 4.0 1.3 3.9 16 33.2 1. 2 3.5

A 15 33.9

― ― 16 34.0

― ― 16 33.2

― ― Proportion unopened (#/N) 0/15 0/16 0/16

a Data were obtained from page 23 of the study report

. U Unadjusted for body weight on PND 22

A Adjusted for body weight on PND 22

N Number of animals examined

SD Standard Deviation

CV Coefficient of Variation

NA Not applicable



## D. ORGAN WEIGHTS : Organ weights at necropsy are presented in Table 3. Absolute liver weight s (adjusted and unadjusted) were increased (p<0.05) by 10% at 150 mg/kg/day.

R elative (to body weight) liver weight s w ere increased (p<0.05) by 4 and 8% at 75 and 150 mg/kg/day

, respectively . Both unadjusted and adjusted thyroid weights were decreased (p<0.05) by 22% each at 150 mg/kg/day.

All control group performance criteria for absolute weights and CVs were within the acceptable ranges.Female Pubertal Assay ( 2012 ) / Page 9 of 14

PERMETHRIN / 109701 O CSPP 89 0. 1 45 0/ OECD None



### TABLE 3

. Organ Weights at Necrops y a

Organ Vehicle Control Permethrin (75 mg/kg/day) Permethrin (150 mg/kg/day)

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)

Liver (g) U 15 7.64 0.70 9.1 16 8.11 0.70 8.6 16 8.39 * ( ↑ 10 ) 0.73 8.7

A

― 7.62

― ― ― 8.14

― ― ― 8.39 * ( ↑ 10 ) ― ―

R 15 4.4 9 0.21 4.6 16 4.69 * (↑4) 0.25 5.2 16 4.84 * ( ↑ 8) 0.25 5. 1

Kidneys (g) U 15 1.30 0.11 8.7 16 1.32 0.08 5.9 16 1.32 0.12 9.2

A

― 1.30

― ― ― 1.32

― ― ― 0.32

― ― R 15 0.77 0.05 6.0 16 0.76 0.05 6.1 16 0.76 0.05 7.1

Pituitary (mg) U 15 9.7 1.1 11.3 16 9.4 0.8 8. 7 16 9.9 1.8 17.8

A

― 9.6

― ― ― 9.4

― ― ― 9.9

― ― R 15 5.7 0.6 11.6 16 5.4 0.5 9.6 16 5.7 1.0 17.1

Adrenals (mg) U 15 39.0 6.4 16.4 16 38.7 4.1 10.6 16 41.2 5.8 14.1

A

― 38.9

― ― ― 38.9

― ― ― 41.1

― ― R 15 22.9 3.2 13.9 16 22.5 2.8 12.6 16 23.7 2.4 10.0

Ovaries (mg) U 15 57.3 9.3 16.2 16 57.4 7.4 12.9 16 57. 9 8. 7 15.1

A

― 57. 1

― ― ― 57.6

― ― ― 57.8

― ― Uterus, wet (mg) U 15 333.6 164.9 49.4 16 281.0 84.1 29.9 16 379.7 174.8 46. 0

A

― 330.4

― ― ― 284.5

― ― ― 379.2

― ― Uterus, blotted (mg) U 15 285.7 67.8 23.7 16 265.3 69.2 26.1 16 319.7 108.2 33.9

A

― NR

― ― ― NR

― ― ― NR

― ―

Thyroid, fixed (mg) U 15 14.4 2.8 19.2 16 12.6 2.4 19.2 16 11.3 * ( ↓ 22) 2.3 20.7

A

― 14.4

― ― ― 12.7

― ― ― 11.3 * ( ↓ 22) ― ―

a Data were obtained from page 25 of the study report . Percent differences from controls, calculated by the reviewers, are included in parentheses.

U Unadjusted for body weight on PND 21

A Adjusted for body weight on PND 21

N Number of animals examined

SD Standard Deviation

CV Coefficient of Variation

R Organ

- to

- body weight ratio (relative to body weight)

NR No value given for adjusted ANCOVA mean

* Significantly different from controls at p<0.05.



## E. ESTROUS CYCLICITY : Estr ous cycle data are provided in Table 4. Administration of permethrin did not change the day of first estrus compared to the vehicle control group. Animals exhibited similar cycle lengths and regularity. The majority of females in the 150 mg/kg/day group were in diestrus at necropsy.Female Pubertal Assay ( 2012 ) / Page 10 of 14

PERMETHRIN / 109701 O CSPP 89 0. 1 45 0/ OECD None



### TABLE 4

. Estrous Cyclicity a 

### (mean ±SD)

Treatment Groups N Mean A ge at

F irst V aginal

E strus (PND) Mean C ycle

L ength (days) Cycling (%) Regularly

C ycling

(%) Cycle S tatus at N ecropsy (# F emales)

Diestrus Proestrus Estrus Not

C ycling

Vehicle (corn oil) 15 35.3 ±2.5 4.2 ±0.9 100 100 5 2 7 0

Permethrin (75 mg/kg/day) 16 36.2 ±2.0 4.2 ±1.0 100 81.25 6 3 4 0

Permethrin (150 mg/kg/day) 16 35.6 ±1.4 4.3 ±0.7 93.75 81.2 5 10 3 1 0

a Data were obtained from page s 25 and 26 of the study report . Percent differences from controls, calculated by the reviewers, are included in parentheses.

N Number of animals

* Significantly different from controls at p<0.05.



## F. CLINICAL CHEMISTRY AND HORMONE LEVELS : Mean hormone levels are presented in Table 5. S erum T 4 

## levels were decreased (p<0.05) by 26 and 42% at 75 and 150 mg/kg/day , respectively . At 150 mg/kg/day, s erum TSH levels were increased (p<0.05) by 40%. The control group performance criteria for T 4 

## level and CV were within the acceptable ranges. Laboratory historical control data were provided for the hormones and clinical chemistry parameters on page 29 of the study report.

Additionally, at 150 mg/kg/day, increases (p<0.05) were observed in sodium ( ↑ 3%), chloride ( ↑ 4%), and SDH ( ↑ 20%), and ALP was decreased ( ↓ 16%). It was noted that 8 of

the 15 clinical chemistry parameters in the control group were outside the historical control ranges provided.Female Pubertal Assay ( 2012 ) / Page 11 of 14

PERMETHRIN / 109701 O CSPP 89 0. 1 45 0/ OECD None



### TABLE 5

. Hormone Levels and Clinical Chemistry a

Parameter

Evaluated (Historical Control Data) Vehicle Control Permethrin (75 mg/kg/day) Permethrin (150 mg/kg/day)

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)

Hormones

S erum T 4 , Total (μg/dL) (3

- 7) 15 3.70 0.97 26.16 15 2.73 * ( ↓ 26) 0.5 6 20.66 16 2.13 * ( ↓ 42 ) 0.69 32.34

Serum TSH (ng/mL) (0.57

- 3.41) 15 1.24 0.56 44.80 15 1.37 0.29 21.24 16 1.73 * ( ↑ 40) 0.51 29.34

Clinical Chemistry

NA + (MEQ/L) (141

- 151) 15 137 6 4 15 139 3 2 16 141 * ( ↑ 3) 2 2

K + (MEQ/L) (5

- 7.6) 15 8.2 0.7 8.7 15 8.3 0.6 7.3 16 8.4 0.6 6.7

Cl

- (MEQ/L) (84

- 109) 15 104 4 4 15 106 2 2 16 108 * ( ↑ 4) 2 2

Calcium (mg/dL) (9.4

- 13.8) 15 9.9 0.6 5.6 15 10.2 0.5 4.5 16 10.3 0.4 3.7

Phosphorous (mg/dL) (4.7

- 9.5) 15 9.8 0.8 7.7 15 10.0 0.7 7.0 16 9.6 0.9 9.4

AST (U/L) (68

- 192) 15 250 63 25 15 259 87 33 16 253 59 23

ALT (U/L) (20

- 56) 15 91 16 17 15 93 21 22 16 90 16 18

GGT (U/L) (0

- 4) 15 0.73 1.28 175 15 0.67 1.05 157 16 0.44 1.03 236

ALP (U/L) (17

- 183) 15 369 44 12 15 331 71 22 16 310 * ( ↓ 16) 57 18

Blood urea nitrogen (mmol/L) (12

- 23) 15 11.73 1.83 16 15 11.27 0.96 9 16 10.88 2.28 21

Creatinine (μmol/L) (0.5

- 1.1) 15 0.31 0.05 14.9 15 0.33 0.06 18.5 16 0.32 0.05 17.1

Total Bilirubin (0

- 0.5) 15 0.05 0.07 139.4 15 0.06 0.08 138.0 16 0.11 0.13 121.4

SDH (U/L) (13

- 37) 15 9 3 39 15 11 4 34 16 12 * ( ↑ 20 ) 3 29

Total protein (g/dL) (4.8

- 9.2) 15 5.3 0.3 4.8 15 5.3 0.2 4.0 16 5.4 0.2 4.4

Albumin (g/dL) (2.9

- 6) 15 4.2 0.2 4.3 15 4.2 0.2 5.2 16 4.2 0.2 5.6

a Data were obtained from pages 26 and 28 of the study report . Percent differences from controls, calculated by the reviewers, are included in parentheses.

N Number of animals examined

SD Standard Deviation

CV Coefficient of Variation

* Significantly different from controls at p<0.05. Historical Control Data on page 29 of study reportFemale Pubertal Assay ( 2012 ) / Page 12 of 14

PERMETHRIN / 109701 O CSPP 89 0. 1 45 0/ OECD None



## G. HISTOPATHOLOGY : There were no effects of treatment observed on either colloid area or follicular cell height in the thyroid in any group (Table 6)

.

a Data were obtained from Table 11 on page 31 of the study report.
b A 5

- point grading scale (1=shortest/smallest; 5=tallest/largest) was used.

O No. Observed

E No. Examined



## There were n o effects of treatment observed microscopically in the ovaries, uterus or kidney (Table 7)

. TABLE 6 . Incidence of Histopathological Lesions of the Thyroid Gland a

Treatment Groups Parameter Evaluated

Colloid Area Follicular Cell Height (Increase)

Grade b Incidence Grade Incidence

O E O E

Vehicle Control 1 0

15 1 0

15 2 0 2 5

3 10 3 10

4 5 4 0

5 0 5 0

Permethrin (75 mg/kg/day) 1 0

16 1 0

16 2 0 2 5

3 11 3 11

4 5 4 0

5 0 5 0

Permethrin (150 mg/kg/day) 1 0

16 1 0

16 2 0 2 3

3 13 3 13

4 3 4 0

5 0 5 0Female Pubertal Assay ( 2012 ) / Page 13 of 14

PERMETHRIN / 109701 O CSPP 89 0. 1 45 0/ OECD None



### TABLE 7

. Incidence of Histopathological Lesions of the Ovaries, Uterus and Kidney a

Findings Dose Level

Vehicle Control Permethrin (75 mg/kg/day) Permethrin (150 mg/kg/day)

# Observed # Examined # Observed # Examined # Observed # Examined

Ovaries [ Mean ±SD ( %CV) ]

Small Follicles 25.3 ± 9.6 (37.9) 15 21.8 ± 10.0 (45.9) 16 22.1 ± 15.8 (71.5) 16

Medium Follicles 14.1 ± 4.6 (32.6) 15 13.4 ± 4.9 (36.6) 16 13.4 ± 3.5 (26.1) 16

Antral Follicles 10.1 ± 3.0 (29.7) 15 9.6 ± 3.8 (39.6 ) 16 9.5 ± 3.1 (32.6) 16

Antretic Follicles 32.6 ± 7.6 (23.3) 15 34.4 ± 11.6 (33.7) 16 29.3 ± 7.0 (23.9) 16

Corpora Lutea 8.0 ± 2..6 (32.5) 15 7.6 ± 2.0 (26.3) 16 9.0 ± 2.8 (31.1) 16

Uterus

Dilation Minimal 8 15 13 16 13 16

Mild 6 15 3 16 3 16

Moderate 1 15 0 16 0 16
Eosinophilic m aterial Minimal 12 15 10 16 9 16
Inflammatory cells/debris Minimal 6 15 2 16 3 16
Endometrial folding Minimal 9 15 5 16 9 16
Endometrial inflammatory cells

Minimal 1 15 1 16 3 16
Kidney
Nephropathy Minimal 13 15 12 16 12 16

Mild 1 15 0 16 0 16

Tubular d ilation Minimal 1 15 2 16 2 16

Mineralization Minimal 1 15 2 16 2 16

Fibrosis Minimal 1 15 0 16 0 16
Cyst Minimal 0 15 1 16 1 16

a Data were obtained from Table 10 on page 30 and Appendix IX on page s 106 and 108 of the study report.



## III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS : Administration of permethrin to intact juvenile/peripubertal female rates did not affect pubertal development. Permethrin administration slightly increased liver weight and caused slight effects on thyroid weight and thyroid hormone levels. However, the thyroid effects may have been secondary to liver

enz yme induction, and not a reflection of a direct effect on the thyroid.

B. AGENCY COMMENTS : Al l rats administered permethrin survived until schedule

termination. One animal in the control was euthanized on PND 34 for signs related to

gavage error. From PND 34 onward, tremors were observed in 4 animals administered 75 mg/kg /day and all animals adm inistered 150 mg/kg/day at 4-6 hours post-dosing on at least one day. These findings did not persist to the daily examinations prior to daily dosing.

No treatment

- related effects were observed at any dose on body weights, body weight gains,

or age and weight at attainment of VO attainment. All surviving animals attained complete

VO.Female Pubertal Assay ( 2012 ) / Page 14 of 14

PERMETHRIN / 109701 O CSPP 89 0. 1 45 0/ OECD None



## Absolute liver weight s (adjusted and unadjusted) were increased (p<0.05) by 10% at 150 mg/kg/day. R elative (to body weight) liver weight s w ere increased (p<0.05) by 4 and 8% at 75 and 150 mg/kg/day. Both unadjusted and adjusted thyroid weights were both decreased (p<0.05) by 22% at 150 mg/kg/day.

Administration of permethrin did not change the day of first estrus compared to the vehicle control group. Animals exhibi ted similar cycle lengths and regularity. The majority of females in the 150 mg/kg/day group were in diestrus at necropsy.

S erum T 4 

## levels were decreased (p<0.05) by 26 and 42% at 75 and 150 mg/kg/day, respectively, and s erum TSH levels were increased (p<0.05) by 40% at 150 mg/kg/day. Additionally, at 150 mg/kg/day, increases (p<0.05) were observed in sodium (↑3%), chloride (↑4%)

, and SDH (↑20%)

, and ALP was decreased (↓16%). However, it is noteworthy that values for 8 of the 15 clinical chemistry para meters measured in the control group were outside of historical control data.

In the thyroid, there were no effects of treatment observed on either colloid area or follicular cell height in any treated group. No effects of treatment were observed microscopically in the ovaries, uterus or kidney.

The dose levels tested were determined to be adequate based on the tremors and estrogen

related effects seen at the high dose in this assay, as well as the results of the range findings study.

C. STUDY DEFICIENCIES : None

# DATA EVALUATION RECORD

PERMETHRIN

Study Type: OCSPP 890.1500, Male Pubertal Assay

EPA Contract No. EP10H001452 Task Assignment No. 3-27-2012 (MRID 48687101)

Prepared for

Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by

CSS

- Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer: Signature:
Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 10/ 2 2 /2012

Secondary Reviewer: Signature:
Michael E. Viana, Ph.D., D.A.B.T. Date: 10 / 2 5 /2012

Program Manager: Signature: Jack D. Early, M.S. Date: 10/ 25 /2012

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 10/ 25 /2012

This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by CSS -Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact 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 within 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).PERMETHRIN / 109701



## P rim ary Reviewer: Gregory Akerman

Health Effects Division Secondary Reviewer : John Liccione, Ph.D.

Health Effects Division Mal e Pubertal Assay ( 2012) I P age I of IS

OCSPP 890.1500 / OECD None



## Signature:**__.....~~~.p.L.-­** 

## Date ;;-- :**-~~~~~1:..--­ Signature.~-JE.~~~::::;;::::-**  

## Dat 

# S:O- Z-1/,1) Template version 08 /20 11



## DATA EVALUATION RECORD

STUDY TYPE: Ma l e Pubertal Assay; OCSP P 890.1500 ; OECD None.

PC C ODE: 109701 DP BARCODE : D405670

TXR #: 

## 0056246

T EST MATERIAL (PURITY): Permethrin (97.7% a.i.)

SYNONYMS: (3-P h e no xyphe n y l )methy l 3-(2,2 -di c hloro e thenyl)- 2,2

- dimethy l cyclopropanecarboxy l ate CAS #: 

## 52 6 45-53 -1

CITATION: Zorri ll a, L. (2012). Pubertal De velop m e nt and Thyroid Function in Intact Juvenile/Peripuberta l Male Rats w ith Pennethrin. Integrated Laboratory Systems,**In~. ,**  

## Durham

, NC. Laboratory S tud y No.: Cl95 -8 00, September 5

, 20 1 2. MRID 4868710 l

. U npubli s hed

.

SPONSOR: Cons um er Spec ialt y Product Association for the Permethrin Data Group

II/Joint Venture

, 1667 K Street NW, Suite 300, Washington

, DC

T EST ORDER# : CON-109701 -7

EX EC UTIVE SUMMARY: In a Male Pubertal Assay ( MRID 48687101)

, 16 Sprague-Dawley [Crl:CD(SD) l GS] rats / dose group were treated daily via oral gavage with pennethrin (97.7%

a.i.

, Lot# PMPS00014) in corn oil at dos es ofO, 75

, or 150 mg/kg/da y from po s tnatal day (PND)

23 to 53/54

. Howe ver, three 150 mg/kg/day rats were fo und dead after two days of dosing.

Th erefore, th e hi g h dose was reduce d to l 20 mg/kg/day o n PND 25. Ani mal s we r e exami n ed for preputial se p arat i on (P P S) daily beginning on PND 30, and age a nd weight on day of attainment

were recorded. Following sacr ifice on PND 53/54, blood was collected for cl inical chemistry

and hormo n e analyses. Total serum testosterone, thyroxine (T4) and thyroid stimu l ati n g hormone ( TSH) l eve ls we r e dete rmined using radioimmunoassays. Adrenal, li ver, pituitary

, thyroid

, and urogenital organs were weig h ed a nd examined m acrosco pi ca ll y. The l eft t estes, l eft epididymides, left kidney

, and thyroid were examined microscopically.

No treatment -r elate d related effects were observed on body weights, body weight gains, age o r weight a t attainment of PPS, orga n weights (except li ver), c linic a l chemis try , a nd gross or hi stopatho lo gy parameters.

After reduction of the high do se to 120 mg/k g/ day

, all remaining animals survived to study

termi n ation. At 

## 6 hours post-dose

, tremors were observed at 75 and 1 20 mg/kg/day in a d ose## tremors on two days. At 120 mg/kg/day, very mild to severe tremors were observed in all surviving animals on several days

. Additionally, o ne 120 mg/kg/day rat was considered uncoordinated on one day.

S erum testosterone levels in the treated groups were similar to controls. Mean serum T 4 

## levels were decreased (p<0.001) by 28 and 42% at 75 and 120 mg/kg/day, respectively, and serum TSH

level was increased (p<0.05) by 77% at 120 mg/kg/day. The T 4 

## and TSH values were all within or just below their respective reference range s . Additionally, there were no concomitant effects observed on thyroid weights or thyroid follicular cell height or colloid area. Absolute and relative liver weight were increased (p<0.05) by 6-11% at 75 and 120 mg/kg/day. There were no changes in clinical chemistry parameters that suggested hepatotoxicity due to permethrin administration. The increased liver weights w ere considered to be the result of hypertrophy and associated microsomal enzyme induction, a common finding with permethrin.

The 150 mg/kg/day dose was an excessive dose level as three rats were found dead on Day 2. Therefore, the dose was lowered to 120 mg/kg/day which was an adequate high dose based on the clinical signs ( tremor and in coordination ) seen in these animals

. Tremors were also noted at 75 mg/kg/day.

The assay satisfies the EDSP Tier 1 Test Order requirement s for a Male Pubertal Assay (OCSPP 890.1500).

COMPLIANCE : Signed and dated Data Confidentiality, GLP Compliance, and Quality Assurance statements were provided.

## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility : Integrated Laboratories Systems , Inc.

Location: Durham, NC Study Director: Leah Zorilla, Ph.D.

Other Personnel: Susan Borghoff ( Study Toxicologist ), Lauren M. Staska (S tudy Pathologist ), and Carol Swartz ( Dose Formulations ) Study Period: December 28, 2011 through September 5, 2012



## 2

. Test S ubstance : Permethrin

Description: Yellow liquid

Source: AMVAC Corporation

Batch #: PMPS00014 (expires January 2013) Purity: 97.7 % Stability: Report stated that the compound is stable in the vehicle at 1

- 10°C for up to 20 days

CAS #: 52645

- 53

- 1

Structure:

O O

O Cl

CH

3 Cl

CH

3



## 3

. Vehicle : Corn oil

4

. Test A nimals : Species: Rat

Strain: Sprague

- Dawley [ Crl:CD ® (SD) IGS ] Age / W eight at

S tudy I nitiation: PND 23 / 54.0

- 68.5 g males only

Source: Charles River Laboratories International, Inc. (Raleigh, NC) Housing: Parental dams were individually housed in polycarbonate cages with micro

- isolator tops and absorbent, heat

- treated, hardwood bedding; each dam was housed with her litter until weaning on PND 21. After weaning, males were housed two/ cage.
Diet: Teklad G lobal 16% Protein Rodent Diet (Teklad Diets, Madison, WI), ad libitum . It was

stated that t he total genistein equivalents of genistein plus daidzein was determined to be 4.8 μg/g of feed.

Water: Reverse osmosis

- purified tap water

, ad libitum

Environmental

C onditions: Temperature: Humidity: Air changes: Photoperiod: 12

- 23 °C

19

- 63 % Not reported

14 h light / 10 h dark



## B. ST UDY DESIGN

1. In -L ife D ates : Start: February 4, 2012 Approximate End: March 7 , 2012

2. Mating : Time d pregnant , nulliparous dams (age not reported) were received from the supplier on gestation day 8, allowed to deliver naturally, and rear their pups to weaning on

PND 21. Between postnatal days (PND) 3 and 5, litters with the same date of birth were standardized to 8 pups with equal numbers of males and females.

## 3. Animal A ssignment : Following weaning, animals were assigned using a procedure that

stratifie d animals across groups by body weight, such that the mean body weight of each group was not statistically different from any other group using analysis of variance.

L ittermates were not placed in the same dose group. The test groups are provided in Table

1.

TABLE 1. Study Design a

Test group Dose (mg/kg/day) b # of M ales

Control 0 16

Low 75 16

High 120 c 16

a Data were obtained from Table 1 on page 1 5 of the study report.

b Dose adjusted for purity.

c Animals received 150 mg/kg/day on Days 1 and 2

.



## 4. Dose Selection Rationale : The doses were selected based upon three previously conducted studies and toxicity observed in the current study. In a range finder study 1 

## , intact adult male rats and juvenile female rats were administered up to 400 mg/kg/day and 250 mg/kg/day permethrin, respectively. In both sexes , animals administered ≥ 200 mg/kg/day permethrin exhibited severe clinical signs up to 24-hours post-dose and were euthanized prior to scheduled study termination. A Female Pubertal Assay (48672901.DER) was performed at

ILS where permethrin was orally administered a high dose o f 150 mg/kg/day for 21/22 days. Final body weights of the high dose group were 101.1% of control; however, 9 of 16 animals exhibited tremors on 1 or more days approximately 6 hours after dose

administration. No adver se clinical signs were noted 24 hours post-dose. A Hershberger

A ssay (48618602.DER) in castrated adult male rats was also performed at ILS at a high dose of 120 mg/kg/day permethrin. In the portion of the assay to assess agonist activity, final body weights were 104.2% of control body weights after 10 days of administration of 120 mg/kg/day permethrin. No adverse clinical observations were no ted 24 hours postdose. A high dose of 150 mg/kg/day was selected for the current Male Pubertal Assay based on deaths, moribundity, and adverse clinica l signs up to 24 hours following dose administration at higher dose levels. However, after two days of dos ing at 150 mg/kg/day permethrin, three animals in this group were found dead, and gavage errors were ruled out

upon necropsy. Therefore to ensure animals survived until scheduled termination, the high dose group was changed on Day 3 (PND 25) to 120 mg/kg/day.

5. Dose P reparation and Analysis : Dose formulations were prepared in corn oil t hree times during the study at a concentration of 15 mg/mL , o nce at a concentration of 30 mg/mL, and two times at a concentration of 24 mg/mL. Formulations were dispensed into vials to be used daily during the study. Dose concentrations were adjusted to correct for purity of permethrin.

Samples of dose formulations prepared on F ebruary 2, 8, and 17, 2012 were collected from the top, middle, and bottom of the formulation and sent to Smithers Viscient for analysis. Smithers Viscient analyzed, in duplicate, samples received for concentration and

1 

### Zorrilla, L. (2011a). Range Finder Study for In Vivo Mammalian Assays for Permethrin. Unpublished Study

Report Prepared by ILS, Inc. (draft) Study No. C195

- 500.

## homogeneity . The report stated that the compoun d is stable in the vehicle at 1 -10°C for up

to 20 days on the basis of a previous ly performed study 2



## . Results

Homogeneity (CV): 0.260-6.42%

Stability (% of time zero): Not reported

Concentration (% of nominal): 84.6-102%

The analytical data indicated that the mixing procedure was adequate and the variation between nominal and actual dosage to the animals was acceptable. The low dose formulation was only 84.6-85.6% of nominal concentration in 2/3 formulations, and the homogeneity exceeded 5% CV in 1/3 formulations; however, these values were not

considered to have adversely impacted study results. Stability data should be submitted for independent verification.

6. Dosage A dministration : D ose formulations were administere d once daily by oral gavage in a dose volume of 5 mL/kg body weight. The dose formulations were administered for 31

or 32 consecutive days from PND 23 -53/54. Dosing occurred 24 hours (± 2 hours) from the previous dose. Dose volume was adjusted daily bas ed on individual animal body weight. The daily dosing sequence was stratified across all dose groups; 1 animal from each group and then repeated until all animals were dosed.

7. Statistics : Descriptive statistics (mean, standard deviation, coefficient of variance, and sample size) were calculated using Microsoft Excel 2003/2007 (Redmond, WA). Data sets

listed below were analyzed using Statistical Analysis System version 9.2 (SAS Institute, Cary, NC). Studentized residual plots were used to detect possib le outliers in the data and Levene’s test was used to assess homogeneity of variance. Heterogeneous data were transformed (logarithm, square root, and multiplicative inverse), and analyzed using the

non- parametric Kruskal

- Wallis and Dunn’s test if data remained heterogeneous.

2 

### Smith, R. (2011). Storage Stability of Permethrin in Corn Oil. Unpublished study report prepared by Smithers

Viscient Inc. Study No. 14010

- 6105.Classification Procedure

Initial body weights Final body weight (last day all body weights collected) Cumulative body weight gain (last day all body weights collected) Age and body weight at PPS Homogeneous data sets:

One

- way analysis of variance (ANOVA) followed by pair

wise comparisons performed using Dunnett’s two

- tailed

t- tests

Body weight Organ weight Tests for linear trend were performed and reported when significant (p<0.05) for endpoints that did not show any significant pair

- wise comparisons. Tissue weights Relative tissue weights (liver, kidneys, pituitary, and adrenals)

Hormone levels Clinical chemistry levels Two

- way ANOVA with treatment and necropsy day as

main effects.

Pair

- wise comparisons were perform ed using Dunnett’s

two

- tailed t

- tests.

Final body weight (last day all body weights collected) Cumulative body weight gain (last day all body weights collected) Age and body weight at PPS Tissue weights Analysis of covariance (ANCOVA) with PND 21 body

wei ght (weight on day of allocation) as the covariable.

Pair

- wise comparisons were performed using Dunnett’s

two

- tailed t

- tests.

Thyroid scoring (colloid area and follicular cell height) Fisher’s Exact test



## Significance was reported as exact p -values, except no p-value was reported for Dunn's Test (p<0.05). The statistical analyses were considered appropriate.

C. METHODS

1. Mortality and C linical Examinations : Animals were observed for mortality and moribundity twice daily on weekdays and once daily on weekends and holidays Rats were observed for clinical signs of toxicity prior to allocation of animals to study groups, daily prior to dose administration, 6 hours post-dosing, and on the day of euthanasia.

2. Body W eight : Animals were weighed weekl y following birth, prior to at allocation of animals to study groups, daily prior to dose administration, and on the day of euthanasia.

3. Food Consumption : Food consumption was not measured.

4. Preputial Separation (PPS) : Beginning on PND 30, males were examined daily following dosing for onset of PPS. PPS was described as: progression not initiated, partial separation, a persistent thread of tissue between the glans and prepuce, or complete PPS

. Age and weight on the day of completion of PPS were recorded.

5. Sacrifice and Pathology : Any animals found moribund or dead were necropsied to determine the cause, if possible. A t least two hours before euthanasia, a nimals surviving to scheduled termination were moved to the necropsy holding room. In the same order as the

animals were dosed , they were humanely euthanized by decapitation with 2 hours of dosing. Prior to tissue collection, trunk blood was collected, and serum was obtained. All serum samples were stored at ≤

− 70°C. Serum samples were shipped overnight on dry ice to AniLytics Inc. (Gaithersbu rg, MD) for analysis.

a. Hormone Analysis : Testosterone, T 4 

## , and TSH levels were measured in serum using radioimmunoassays (RIAs). Samples were assayed in duplicate and in conjunction with multiple quality control samples.

## b. Clinical C hemistry : The following CHECKED (X) parameters were examined.

ELECTROLYTES OTHER

X Calcium X Albumin

X Chloride X Creatinine*

Magnesium X Urea nitrogen*

X Phosphorus Total cholesterol

X Potassium Globulins

X Sodium Glucose

ENZYMES X Total bilirubin

X Alkali ne phosphatase (ALP ) X Total protein

Cholinesterase (ChE) Triglycerides

Creatine phosphokinase Albumin/globulin ratio (calculated)

Lactic acid dehydrogenase (LDH) Bile acids

X Alanine aminotransferase (ALT/also SGPT)

X Aspartate aminotransferase (AST/also SGOT)

X Sorbitol dehydrogenase (SDH)

X Gamma glutamyl transferase (GGT)

Glutamate dehydrogenase

* Recommended for the pubertal assay in male rats based on guideline 890.1 500.



## c. Organ Weights and Histopathology : The following CHECKED (X) tissues were collected and weighed and grossly observed from all study animals. The (XX) organs, in addition, were subjected to histological examination

.

UROGENITAL OTHER

XX Testes (left and right separately) *+ XX Thyroid a * +

XX Epididymides (left and right separately) *+ X Liver*

X Seminal vesicle plus coagulating glands (with and without fluid)* X Adrenals (paired)*

X Ventral prostate* X Pituitary*

X Dorsolateral prostate*

X Levator ani/bulbocavernosus (LABC) muscle complex*

XX Kidneys (paired)* +

a Thyroid was weighed post

- fixation.

* Weights required based on guideline 890.1 50 0.

+ Histopathological examination required based on guideline 890.1 50 0.



## The l eft kidney and thyroid (with attached trachea) were fixed in 10% neutral buffered

formalin for at least 24 hours. Following fixation, the thyroid was dissected free of the trachea and weighed. The l eft testis and left epididymis were each placed in Bouin’s

fixative for 18 -24 hours and then washed in 70% histology grade alcohol. After fixation, all

tissues were transferred to 70% histology grade alcohol, routinely processed into paraffin blocks, sectioned, and stained with hematoxylin and eosin. All processed tissues (thyroid, left kidney, left epididymis, and left testis) from all animals were microscopically evaluated.

Two sections of each of the 2 lobes of the thyroid were examined and evaluated for

follicular cell height and colloid area. A 5 -point grading scale (1 = shortest/smallest; 5 = tallest/largest) was used , and the thyroids were evaluated relative to photomicrographs supplied by the U.S. EPA.

## II. RESULTS

A. MORTALITY : On Day 2, three 150 mg/kg/day rats were found dead; g avage error was ruled out as the cause of death. Starting the next day, th is group was treated at 120 mg/kg/day , and a ll remaining animals survived to study termination.

B. CLINICAL SIGNS OF TOXICITY : At 6 hours post-dosing, tremors were observed at 75 and 120 mg/kg/day in a dose-dependent manner , with incidence, frequency, and severity

increas ing with dose. At 75 mg/kg/day, three animals exhibited mild tremors on one day and one animal exhibited mild tremors on two days. At 120 mg/kg/day, very mild to severe

tremors were observed in all surviving animals on 2-12 days. Tremors were not reported for the animals that were found dead. One 120 mg/kg/day was considered uncoordinated on one day. All other findings, such as rough coat, fur discoloration, ungroomed, lethargic, or red nasal/ocular discharge, were considered incidental during the daily and 6-hour post-dose

observations.

C. GENERAL GROWTH AND PREPUTIAL SEPARATION : Initial and final b ody

wei ghts, body weight gains, age and weight at attainment of PPS, and proportion/incidence of separated prepuce are presented in Table 2. All values in the treated groups were similar

to the controls.

The Guideline performance criteria were met for age and weight at PPS and weaning and final body weight in the control group, except for the following: the CV for body weight at PPS ( 11.3 %) was greater than the maximum acceptable value (7.57%) , and the CV for final body weight (9.9 %) was greater than the maximum acceptable value ( 7.4 7%)

.

### TABLE 2 . General Growth and Preputial Separation (PPS) a

Parameter Evaluated Vehicle Control 75 mg/kg/day Permethrin 120 mg/kg/day Permethrin

N Mean SD CV (%) N b Mean SD CV
(%) N c Mean SD CV
(%)

Age at PPS (PND) U 16 45.4 1.3 2.8 15 45.7 1.9 4.2 13 45.5 1.9 4.2

A 16 45.4

--- --- 15 45.6

--- --- 13 45.6

--- --- Body weight at PPS (g) U 16 229.0 25.8 11.3 15 240.3 28.0 11.7 13 236.2 21.5 9.1

A 16 229.2

--- --- 15 242.1

--- --- 13 233.9

--- ---

Age at partial PPS (PND) U 16 41.8 1.1 2.7 15 41.5 1.1 2.7 16 41.8 1.5 3.6

A 16 41.7

--- --- 15 41.5

--- --- 16 41.9

--- ---

Initial body weight
(PND 23; g) U 16 61.8 4.0 6.4 15 60.7 3.4 5.5 16 61.2 3.2 5.2

A 16 61.6

--- --- 15 61.0

--- --- 16 61.1

--- ---

Final body weight (g) U 16 290.4 28.8 9.9 15 304.3 36.7 12..1 13 298.6 19.0 6.4

A 16 290.7

--- --- 15 306.9

--- --- 13 295.2

--- ---
Final body weight

(% of control) U 16

--- --- --- 15 104.8

--- --- 13 102.8

--- ---

A 16

--- --- --- 15

--- --- --- 13

--- --- ---

Body weight gain

(final

– initial; g) U 16 228.7 26.3 11.5 15 243.6 34.1 14.0 13 237.1 18.4 7.8

A 16 228.9

--- --- 15 245.7

--- --- 13 234.5

--- ---

Proportion unseparated (#/N) 0/16 0/15 0/13

a Data were obtained from Table 5 on page 24 of the study report

. No statistical differences were detected.

b One animal was female and was removed from the study ; N = 15.

c Three animals were found dead ; N = 13.

U Unadjusted for body weight on PND 2 1

A Ad justed for body weight on PND 21

N Number of animals examined

SD Standard Deviation

CV Coefficient of Variation

--- Not applicable



## D. ORGAN WEIGHTS : Organ weights at necropsy are presented in Table 3, and all values in the treated groups were si milar to controls, except for minor increase s (p<0.05) in

absolute and relative liver weight at 75 (↑6

- 11%) and 120 (↑8 -11%) mg/kg/day. Adjusted weights for the liver were not provided.

The unadjusted values for all organ weights in the control group were within the acceptable range of the Guideline pe rformance criteria, with the following exceptions: (i) mean kidney weight was 1.92 g (acceptable range: 2.242-3.050 g); (ii) CV for pituitary weight was 18.9% ( maximum acceptable value : 15.98%); and (iii) mean LABC weight was 0.350 g (acceptable

range: 0.447-0.855 g).

### TABLE 3 . Organ Weights at Necropsy a

Organ Vehicle Control 75 mg/kg/day Permethrin 120 mg/kg/day Permethrin

N Mean SD CV (%) N b Mean SD CV (%) N c Mean SD CV (%)

Liver (g) U 16 12.47 1.40 11.2 15 13.80* (↑11) 2.09 15.2 13 13.80* (↑11) 0.90 6.5

A 16 NP

--- --- 15 NP

--- --- 13 NP

--- ---

R 16 4.29 0.18 4.2 15 4.53** (↑6) 0.24 5.2 13 4.62*** (↑8) 0.18 3.9

Kidneys (g) U 16 1.92 0.23 11.9 15 2.05 0.22 10.9 13 2.05 0.16 7.8

A 16 1.92

--- --- 15 2.06

--- --- 13 2.03

--- --- R 16 0.66 0.05 7.8 15 0.68 0.04 6.5 13 0.69 0.04 6.0

Pituitary (mg) U 16 9.0 1.7 18.9 15 9.2 1.8 19.8 13 9.4 0.9 9.7

A 16 9.0

--- --- 15 9.2

--- --- 13 9.4

--- --- R 16 3.1 0.6 19.1 15 3.0 0.5 15.9 13 3.2 0.3 8.8

Adrenals (mg) U 16 39.1 4.8 12.2 15 40.6 5.9 14.5 13 42.5 6.2 14.5

A 16 39.1

--- --- 15 40.7

--- --- 13 42.3

--- --- R 16 13.6 2.0 14.8 15 13.4 1.9 14.2 13 14.3 2.1 14.8

Seminal vesicle + coagulating gland, with fluid (mg) U 16 535.0 77.7 14.5 14 537.9 106.4 19.8 13 542.6 98.8 18.2

A 16 535.0

--- --- 14 538.8

--- --- 13 541.7

--- ---

Seminal vesicle + coagulating gland, without fluid (mg) U 16 292.6 32.6 11.1 15 298.7 45.0 15.1 13 313.0 51.5 16.4

A 16 292.7

--- --- 15 299.6

--- --- 13 311.9

--- ---

Ventral prostate (mg) U 16 202.3 22.5 11.1 15 219.6 46.6 21.1 13 215.8 44.5 20.6

A 16 202.4

--- --- 15 221.0

--- --- 13 214.0

--- ---
Dorsolateral prostate (mg) U 16 129.6 14.7 11.3 15 137.1 21.9 15.9 13 120.0 16.1 12.9

A 16 129.6

--- --- 15 136.8

--- --- 13 120.4

--- --- LABC (mg) U 16 349.5 43.0 12.3 15 369.9 47.2 12.8 13 353.4 67.4 19.1

A 16 349.7

--- --- 15 372.0

--- --- 13 350.8

--- --- Epididymis, left (mg) U 16 219.3 19.0 8.7 15 220.9 17.7 8.0 13 214.3 11.3 5.3

A 16 219.3

--- --- 15 221.6

--- --- 13 213.4

--- --- Epididymis, right (mg) U 15 221.5 18.4 8.3 15 218.2 18.0 8.2 13 221.2 8.8 4.0

A 15 221.6

--- --- 15 218.6

--- --- 13 220.7

--- --- Testis, left (mg) U 15 1455.9 132.9 9.1 14 1446.0 124.2 8.6 13 1426.7 99.4 7.0

A 15 1452.3

--- --- 14 1457.8

--- --- 13 1418.1

--- --- Testis, right (mg) U 15 1475.0 114.4 7.8 15 1452.1 113.8 7.8 12 1471.6 114.6 7.8

A 15 1465.0

--- --- 15 1459.1

--- --- 12 1459.7

--- --- Thyroid, fixed (mg) U 16 14.6 2.3 15.4 15 14.9 2.1 14 13 13.6 1.6 11.8

A 16 14.6

--- --- 15 15.0

--- --- 13 13.4

--- ---

a Data were obtained from Table 6 on page s 26

- 27 of the study report.

b One animal was female and was removed from the study ; N = 15.

c Three animals were found dead ; N = 13.

U Unad justed for body weight on PND 21

A Ad justed for body weight on PND 21

R Organ

- to

- body weight ratio (relative to body weight)

SD Standard Deviation

CV Coefficient of Variation

NP No value given for adjusted ANCOVA mean

* Significantly different from controls at p<0.05

** Significantly different from controls at p<0.01

** * Significantly different from controls at p<0. 0 01

## E. HORMONE LEVELS AND CLINICAL CHEMISTRY : Mean hormone levels and clinical chemistry val ues and are presented in Table 4. Nominal ranges 3 

## for the hormone

and clinical chemistry values were provided.

Mean serum T 4 

## levels were de creased (p <0.001) at 75 and 12 0 mg/kg/day by 28 and 42%, respectively, and serum TSH level was increased (p <0.05) by 77% at 120 mg/kg/day. The

T 4 

## and TSH values were all within or just below their respective historical control range, except T 4 

## in high-dosed rats that was just below the historical range. Testosterone levels in the treated groups were similar to controls. Minor differences (p<0.05) of 1 -2% were noted in serum sodium, chloride, and calcium at 120 mg/kg/day compared to controls. S erum phosphorus was increased (p<0.05) by 11% at 75 mg/kg/day , but the 120 mg/kg/day group

was similar to the controls

. All other clinical chemistry values in the treated groups were

similar to control value s.

The results for serum T 4 

## and testosterone in the control group were within the acceptable range of the Guideline performance criteria , but the mean TSH level of 1.67 ng /mL was

below the acceptable range of 4.212-24.112 ng/mL.

TABLE 4 . Hormone Levels and Clinical Chemistry a

Parameter

Evaluated Vehicle Control 75 mg/kg/day Permethrin 120 mg/kg/day Permethrin

N Mean SD CV
(%) N b Mean SD CV
(%) N c Mean SD CV
(%)

Hormones

Serum T 4 , Total (μg/dL) 16 5.11 0.60 11.7 15 3.70*** (↓28) 0.55 14.8 13 2.98*** (↓42) 0.43 14.4

Serum TSH (ng/mL) 16 1.67 0.89 53.1 15 2.63 1.51 57.4 13 2.95* (↑77) 1.84 62.2

Serum

testosterone (ng/ m L) 16 1.73 0.68 39.3 15 1.61 1.17 73.2 13 1.30 0.96 74.0

Clinical Chemistry

Sodium (mEq/L) 16 138 2 2 15 138 2 2 13 140* (↑1) 2 2

Potassium (mEq/L) 16 7.2 0.4 6.1 15 7.4 0.5 6.7 13 7.5 0.5 6.4

Chloride (mEq/L) 16 103 2 2 15 103.9 1.6 1.5 13 105* (↑2) 1 1

Calcium (mg/dL) 16 10.6 0.2 2.2 15 10.5 0.5 4.4 13 10.4* (↓2) 0.4 4.0

Phosphorus (mg/dL) 16 9.0 0.7 8.1 15 10.0* (↑11) 1.1 11.5 13 9.9 1.3 13.1

AST (U/L) 16 186 46 25 15 190 40 21 13 209 66 32

ALT (U/L) 16 84 12 14 15 83 12 14 13 88 11 12

GGT (U/L) 16 0.25 0.77 310 15 0.20 0.77 387 13 0.08 0.28 361

ALP (U/L) 16 563 108 19 15 550 120 22 13 501 120 24

3 

### Loeb, W.F. and F.W. Quimby, Eds. (1999). Clinical Chemistry of Laboratory Animals, 2nd ed. Taylor and Francis,

Philadelphia.

### TABLE 4 . Hormone Levels and Clinical Chemistry a

Parameter

Evaluated Vehicle Control 75 mg/kg/day Permethrin 120 mg/kg/day Permethrin

N Mean SD CV
(%) N b Mean SD CV
(%) N c Mean SD CV
(%)

BUN (mg/dL) 16 11 2 20 15 11 2 17 13 11 2 17

Creatinine (mg/dL) 16 0.41 0.05 12.1 15 0.41 0.08 19.6 13 0.41 0.05 12.1

Total

Bilirubin (mg/dL) 16 0.05 0.10 193 15 0.02 0.06 280 13 0.05 0.08 168

SDH (U/L) 16 4.13 4.62 112 15 3.93 1.28 33 13 3.31 1.11 34

Total Protein (g/dL) 16 5.7 0.2 4.1 15 5.9 0.4 6.4 13 5.9 0.2 4.0

Albumin (g/dL) 16 4.7 0.2 5.1 15 4.8 0.3 6.2 13 4.8 0.2 4.7

a Data were obtained from Tables 7

- 8 on pages 29

- 30 of the study report. Percent difference from controls (calculated by reviewers) is presented parenthetically.

b One animal was female and was removed from the study ; N = 15.

c Three animals were found dead ; N = 13.

SD Standard Deviation

CV Coefficient of Variation

* Significantly different from controls at p<0.05

* ** Significantly different from controls at p<0.001



## G. GROSS PATHOLOGY : There were no treatment -related macroscopic findings. In the controls, t he right testis and epididymis were moderately enlarged in one animal, and the left

testis was mildly enlarged in one animal. In the 120 mg/kg/day group, t he right testis was mildly enlarged in one animal . No other gross observations were noted in male rats surviving to the scheduled term ination.

H. HISTOPATHOLOGY : The incidences of histopathological findings of the thyroid gland are presented in Table 5; follicular cell shape was not reported. Thyroid colloid area and follicular cell height were similar in the treated groups to the controls; all animal samples were classified as Grade 3 or 4 for colloid area and Grade 2 or 3 for follicular height.TABLE 5 . Thyroid Colloid Area and Follicular Cell Height a

Treatment Groups Parameter Evaluated

Colloid Area Follicular Cell Height (Increase)

Grade b Incidence Grade b Incidence

O E O E

Vehicle Control 1 0 16 1 0 16

2 0 16 2 10 16

3 6 16 3 6 16

4 10 16 4 0 16

5 0 16 5 0 16

7 5 mg/kg/day

Permethrin 1 0 15 c 1 0 15

2 0 15 2 9 15

3 6 15 3 6 15

4 9 15 4 0 15

5 0 15 5 0 15

120 mg/kg/day

Permethrin 1 0 13 d 1 0 13

2 0 13 2 6 13

3 7 13 3 7 13

4 6 13 4 0 13

5 0 13 5 0 13

a Data were obtained from Table 10 on page 32 of the study report.

b A 5

- point grading scale (1 = shortest/smallest; 5 = tallest/largest) was used.

c One animal was female and was removed from the study ; N = 15.

d Three animals were found dead ; N = 13.

O No. Observed

E No. Examined



## The incidence of histopathological findings of the testes, epididymides, and kidneys are presented in Table 6 . N o treatment

- related histological lesion s were noted in these organs.

TABLE 6

. Incidence of Histopathological Lesions of the Testes, Epididymides, and Kidney a

Findings Vehicle Control 75 mg/kg/day Permethrin 120 mg/kg/day

Permethrin

# Observed # Examined # Observed # Examined

b # Observed # Examined c

Testis

Seminiferous tubule

dilatation , mild 1 16 1 15 0 13

Interstitium edema,

mild 1 16 1 15 0 13

Epididymidis
Inflammation, minimal 5 16 3 15 5 13
Kidney
Nephropathy, minimal 13 16 14 15 13 13 Cyst, minimal 2 16 0 15 1 13

a Data were obtained from Appendices 1, 5, and 10 on page s 98, 100, and 102, respectively, of the study report.

b One animal was female and was removed from the study ; N = 15.

c Three animals were found dead ; N = 13.



## III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS : Administration of permethrin to intact juvenile/

## peripubertal male rats did not affect pubertal development. Permethrin administration increased liver weight and caused a significant decrease in T 4 

## and increase in TSH serum

concentration s, without a corresponding change in thyroid weight or histopathology. This suggests that the thyroid effects may have been secondary to liver enzyme induction, and

not a reflection of a direct effect on the thyroid.

B. AGENCY COMMENTS : No treatment

- related related effects were observed on body weights, body weight gains, organ weights (except liver), clinical chemistry, or gross pathology. No adverse histological lesions were observed in the testes, epididymides, or kidneys. Age and body weight at attainment of PPS, and proportion/incidence of separated prepuce were similar in the treated groups to the controls. Also, serum testosterone levels in the treated gr oups were similar to controls.

A tre atment -related effect on mortality was observed at 150 mg/kg/day. On Day 2, three 150 mg/kg/day rats were found dead; gavage error was ruled out as the cause of death. Starting the next day, this group was treated at 120 mg/kg/day, and all remaining animals survived to study termination.

At 6 hours post-dosing, tremors were observed at 75 and 120 mg/kg/day in a dosedependent manner, with incidence, frequency, and severity increas ing with dose. At 75 mg/kg/day, three animals exhibited mild tremors on one day and one animal exhibited mild tremors on two days. At 120 mg/kg/day, very mild to severe tremors were observed in all surviving animals on several days. Tremors were not reported in animals that were found

dead . Additionally, one 120 mg/kg/day rat was considered uncoordinated on one day.

Mean serum T 4 

## levels were decreased at 75 and 120 mg/kg/day, and serum TSH level was increased at 120 mg/kg/day. The T 4 

## and TSH values were all within or just below their respective reference range s. Additionally, there were no concomitant effects observed on thyroid weights or thyroid follicular cell height or colloid area. Minor increases in absolute and relative liver weight s were observed at 75 and 120 mg/kg/day. There were no changes in clinical chemistry parameters that suggested hepatotoxicity due to permethrin administration. The increased liver weights w ere considered to be the result of liver hypertrophy and associated microsomal enzyme induction, a common finding with permethrin. Induction of hepatic enzyme s can result in increased metabolism of T 4



## .

The 150 mg/kg/day dose was an excessive dose level as three rats were found dead on Day 2. Therefore, the dose was lowered to 120 mg/kg/day which was an adequate high dose based on the clinical signs ( tremor and in coordination ) seen in these animals. Tremors were also noted at 75 mg/kg/day.

C. STUDY DEFICIENCIES : The following deficiencies were noted that are not considered to have had an adverse impact on the results, interpretation or conclusions of this study:

• CV for body weight at PPS was 11.3% (m aximum acceptable value of 7.57%)

• CV for final body weight was 9.9% (maximum acceptable value of 7.47%)

• Mean kidney weight was 1.92 g (acceptable range of 2.242- 3.050 g)

• CV for pituitary weight was 18.9% (maximum acceptable value of 15.98%)

• Mean LABC weight was 0.350 g (acceptable range of 0.447- 0.855 g)

• M ean TSH level was 1.67 ng/mL ( acceptable range of 4.212 -24.112 ng/mL)

# DATA EVALUATION RECORD

PERMETHRIN

Study Type: O CSPP 890.1550, Steroidogenesis Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-29-2012 (MRID 48618603)

Prepared for

Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by

CSS

- Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer : Signature: Michelle Sharpe

- Kass , M.S. Date: 3 /26 /2012

Secondary Reviewer : Signature:
Michael E. Viana, Ph.D., D.A.B.T. Date: 3/30/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 4 / 10 /2012

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 4 / 10 /2012

This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by CSS

- Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact 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 within 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).

### PERMETHRIN /109701



## Primary Reviewer: Patience Browne, Ph .D.



## Health Effects Division Secondary Reviewer: John Liccione, Ph

. D

. 

## Health Effects Division · Steroidogenesis Assay 

### (2011) I 

### Page I of 13

OCSPP 890.1550/ OECD None



## Sign a tu re:**:;~E:: :::::::~;c.:;=-;--:** **:::-** 

## Da 

## te:**~~tf::~~'iL,t.**



## DATA EVALUATION RECORD

STUDY TYPE: Steroidogenesis Assay (H295R Cells); OCSPP 890.1550



## PC CODE: 109701 

## DP BARCODE: 0398301



## TXR#: 0056246



## TEST MATERIAL (PURITY): Permethrin (97.7% a.i.)



## SYNONYMS: (3-phenoxyphenyl)methy I 3-(2,2-d ichloroethenyl)-2 ,2

- dimethylcyclopropanecarboxylate 

## CAS No.: 52645-53-1



## CITATION: Wagner

, H. (2011) Permethrin: 

## Hl95R steroidogenesis assay. CeeTox,

Kalamazoo

, 

## ML Laboratory Report No.: 9 l 

## 46V- I 003 83 STER

, November 17

, 201 I. MRID 48618603. Unpublished.



## SPONSOR: Permethrin Data Group

, II

, c/o Consumer Specialty Products Association, Inc.

, 1900

- 17 111 

## Street NW

, Suite 300

, Washington DC



## TEST ORDER#: CON-109701 -7



## EXECUTIVE SUMMARY: In a steroidogenesis assay (MRJD 48618603) H295R cells

cultured 

## in vitro in 24-well plates were incubated with permethrin (97.7% a.i.; Batch

PMPSOOOOl4 ) at concentrations of0.0001

, 0.001, 0.01

, 0.1

, 1

, 10 and 100 μMin triplicate for 48

hours. Dimethyl su lfo x id e (DMSO) was used as the vehicle

, at a final concentration of 0.05%.

Testosterone and estradiol levels were mea sured using HPLC / MS-MS. Three independent experiments were perfom1ed (Run #s 2-4). A Quality Control (QC) plate was run concurrently

with each independent run of a test chemical plate to demon s trate that the assay responded properl y to positive control agen ts at two concentra tion l eve l s; positive controls included the

known inhibitor (prochloraz) and inducer (forskolin) of estradiol and testosterone production.

Guideline acceptability recommendations and requirements were met, including lack of cytotoxicity

, adequate production of testosterone and estTadiol

, accep tabl e r ep roducibility (low %CV) and appropriate induction and inhibition with positive controls.

No cytotoxic it y was observed ove r the rang e of tested permethrin concentrations. However

, precipitation of the permethrin was observed at the l 00 μM concentration in each run and at

10 μM in Run # 2. Accordingly

, the data from these concentrations were not included in the analysis of the respective run s. For permethrin, there was a s tati st ically s ignificant (p<0.05)

increase in testosterone production at 10 μM in Runs #3 and # 4 (average l. I 4-fold)

, and aSteroidogenesis Assay ( 2011 ) / Page 2 of 13

PERMETHRIN /109701 O CSPP 89 0. 1550/ OECD None



## statistically significant increase in estradiol production at 1 and 10 μM in Runs #3 and #4 (average 1.16-fold for 1 μM and 1.51-fold for 10 μ M)

.

Based on hormone responses in two of the three independent runs, permethrin treatment resulted in statistically significant and reproducible increases in testosterone and estradiol production.

Th e assay satisfies the EDSP Tier 1 Test Order requirement s for a Steroidogenesis a ssay (OCSPP 890.1550).

COMPLIANCE : Signed and dated GLP Compliance, Quality Assurance, and Data Confidentiality statements were provided.Steroidogenesis Assay ( 2011 ) / Page 3 of 13

PERMETHRIN /109701 O CSPP 89 0. 1550/ OECD None



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility : CeeTox

Location: Kalamazoo, MI Study Director: Hilary Wagner

Other Personnel: Colleen Toole, Director of Project Management Karen Rutherford, Director of Laboratory Operations David Blakeman, Senior Scientist Briana Wallace, Lead Cell Culture Scientist Steven McColleg, Scientist Cameron Haines, Scientist Francisca Wong, Scientist Joey Burg am, Associate Scientist Lisa Blakeman, Associate Scientist Study Period: July 5, 2011 to November 17, 2011



## 2

. Test S ubstance : Permethrin

Description: Technical, yellowish crystallized gel

Lot / Batch #: (Expiration Date) PMPS000014 (Jan 2013) Purity: 97.7 % Solubility (in Solvent): E thanol (25 mg/ml), DMSO (40 mg/ml) Volatility: 3.4 x 10

- 7 mm Hg Stability: N ot reported (N R ) Storage conditions: Ambient

CAS #: 52645

- 53

- 1 Molecular weight: 391.3

Structure:



## 3

. Positive C ontrol (Inducer) : Forskolin

Description ( MW ) : White powder, ( 410.50 )

Source (catalog #) : Sigma

- Aldrich, St Louis, MO (not reported) Lot / Batch #: (Expiration Date) : 109K50571V (July 2016) Purity: 98% Solubility (in Solvent): NR Storage conditions: Room te mperature
CAS #: 66575

- 29

- 9



## 4. Positive C ontrol (Inhibitor) : Prochloraz

Description (MW) : White po wder (376.67) Source (catalog #): Sigma

- Aldrich, St Louis, MO (not reported) Lot / Batch #: (Expiration Date) SZE6220X (Aug 8, 2012 ) Purity: 99.1% Solubility (in Solvent): NR Storage conditions: Room te mperature
CAS #: 67747

- 09

- 5Steroidogenesis Assay ( 2011 ) / Page 4 of 13

PERMETHRIN /109701 O CSPP 89 0. 1550/ OECD None



## 5. Other Materials : 22R

- Hydroxycholesterol

Description: White powder

Source: Sigma Aldrich, St. Louis, MO Lot # (expiration date): 089K4132 (retest date July, 2012) and 060M4098 (retest date June, 2013) Purity: 99.0%

Solvent used: Ethanol Storage conditions: Room temperature

CAS: Molecular weight: 17954

- 98

- 2

402.65



## 6

. Solvent/ V ehicle C ontrol : DMSO

Description: NR Source (catalog #): Sigma

- Aldrich, St Louis, MO (not reported) Lot / Batch #: (Expiration Date) RNBB7617 Purity: NR Storage conditions: NR

CAS #: 67

- 68

- 5

Justification for choice of solvent: NR

Final concentration: (% volume in assay) 0.05% ( v/v )



## 7

. Stock M edium : Dulbecco’s modified Eagle’s medium/Ham’s F12 nutrient

mixture

Lot / Batch #: (Expiration Date) NR

Sodium bicarbonate : NR

Nu

- Serum : Lot number 81515 ; tested for background hormone concentrations by performing laborator y: Testosterone: 3754 pg/mL, Estradiol: 3846 pg/mL ITS+ premix : Lot number NR



## 8. Test Cells : H295R human adrenocortical carcinoma cells (ATCC CLR -2128; lot 7635054) were maintained in Dulbecco’s modified Eagle’s medium/Ham’s F12 nutrient mixture in a

5% CO 2 

## incubator at 17 ±2 ° C. Cells were grown for five passages, frozen in liquid nitrogen,

then thawed and cultured for seven or eight additional passages prior to use in the assay.

The following performance criteria were met (indicated by an “x”):

X Cell passage identifier . Cell Passage # : >5.8

X Cells frozen down at passage 5

X Frozen cells cultured for 4 additional passages

X Total number of passages does not exceed 10



## B. METHODS

1. Pre -T est Information

a. Hormone Assay I nterference T est : No details were provided on a separate chemical interference assay. Testosterone and estradiol levels were measured using HPLC/MS -MS,

with a detect ion limit of 100 pg/mL for testosterone an d 10 pg/mL for estradiol. No

interference would be anticipated us ing this analytical method.

b. Hormone Extraction : After the addition of internal standards, the samples were mixed with methyl tert

- butyl ether, centrifuged , evaporated to dryness, derivatized, and analyzed by HPLC

- MS/MS.

c. Laboratory P roficiency T est : No information was provided.Steroidogenesis Assay ( 2011 ) / Page 5 of 13

PERMETHRIN /109701 O CSPP 89 0. 1550/ OECD None



## 2. Test Solutions : Permethrin, forskolin and prochloraz w ere dissolved in DMSO to make 200 mM (permethrin) or 100 mM (forskolin or prochloraz) stocks, then serially diluted 1:10 in DMSO. 22R-Hydroxycholesterol was dissolved in ethanol to make a 40 mM stock and diluted in supplemented medium to a final concentration of 10 μM. Permethrin was diluted 1:2000 in supplemented medium containing 10 μM 22R-hydroxycholesterol to prepare mastermix solutions. When added to the cell culture plates, these dilutions yielded final concentrations of 1 μM and 10 μm forskolin, 0.1 μm and 1 μM prochloraz and 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 μM permethrin, with the final concentration of DMSO in the medium being held constant at 0.05% and the final concentration of ethanol at 0.025%.

3. Cell Plating and P reincubation : H295R cells (ATCC CLR -2128) were grown for five passages, frozen in liquid nitrogen, then thawed and cultured for seven or eight additional passages. The cells were then seeded in 24- well plates at a concentration of 300,000

cells/mL and incubated for 24 hours at 37°C in a 5% CO 2 

## atmosphere.

4. Exposure : The cells were checked microscopically for good attachment and proper morphology, and the medium was removed and replaced with 1 mL of medium containing 10 μM 22R-hydroxycholesterol and the concentrations of test chemicals indicated in triplicate according to the schematic presented in Table 1.

TABLE 1

. Dosing S chematic fo r the E xposure of H295R C ells to Permethrin ( F inal C oncentrations in μM) a

1 2 3 4 5 6

A DMSO DMSO DMSO 0.1 0.1 0.1

B 100 100 100 0.01 0.01 0.01

C 10 10 10 0.001 0.001 0.001

D 1 1 1 0.0001 0.0001 0.0001

a Data were obtained from page 15 of the study report. Dosing was calculated based on a total volume of 1 mL per well.



## A concurrent quality control (QC) plate was included with each of the three independent runs of the test chemical plates to demonstrate the assay’s response to forskolin (an inducer of testosterone and estradiol production) and prochloraz (an inhibitor of testosterone and estradiol production). The QC plate was prepared and dosed in the same manner with either

forskolin or prochloraz according to the schematic presented in Table 2.

TABLE 2

. Dosing S chematic for the QC P late for P ositive C ontrols ( F inal C oncentrations in μM) a

1 2 3 4 5 6

A Blank b Blank Blank Background c Background Background

B DMSO DMSO DMSO DMSO + MeOH d DMSO + MeOH DMSO + MeOH

C Forskolin 1μM Forskolin 1μM Forskolin 1μM Prochloraz 0. 1μM Prochloraz 0.1μM Prochloraz 0.1μM

D Forskolin 10μM Forskolin 10μM Forskolin 10μM Prochloraz 1μM Prochloraz 1μM Prochloraz 1μM

a Data were obtained from page 15 of the study report. Dosing was calculated based on a total volume of 1 mL per well.

b Blank wells received medium containi ng 10μM 22R

- hydroxycholesterol

.

c Background wells received media without 22R

- hydroxycholesterol.

d MeOH = 70% methanol was added to these wells for 30 minutes at room temperature following medium removal.Steroidogenesis Assay ( 2011 ) / Page 6 of 13

PERMETHRIN /109701 O CSPP 89 0. 1550/ OECD None



## Following dosing, the plates were incubated for 48 hours under the conditions previously described. The medium from each well was removed, split into two equal volume aliquots,

and frozen at −80°C until hormone measurements.

5. Cell V iability/ Cytotoxicity A ssay : Cell viability was determined using the MTT assay immediately after removal of the culture medium . Methanol was used as the positive

control for cell death. The plate wells were rinsed three times with PBS, and 0.5 ml of a 0.5 mg/mL MTT solution in supplemented medium containing 10 μM 22R-hydroxycholesterol was added to each well of the test chemical and QC plates. Plates were incubated for 3 hours, after which the MTT solution as removed and 0.5 mL of isopropanol was added to each well. Plates were incubated at room temperature for 20 min with shaking, and

absorbance at 570 nM and 650 nm were measured to calculate the MTT value for each well.

6. Hormone M easurement S ystem : The concentration of each hormone (testosterone and estradiol) was evaluated in triplicate in the culture medium. Hormones were extracted from the samples with MTBE, derivatized , and analyzed by HPLC/MS- MS. The lower limit of quantification (LLQ) for this measurement system was 100 pg/mL for testosterone and 10 pg/mL for estradiol.

The following performance criteria were met (indicated by an “x”) :

X Method detection limit (100 pg/mL testosterone; 10 pg/mL estradiol)

X Spiked sample recovery acceptable for two concentrations of testosterone and estradiol (mean measured amount from triplicate samples within 30% of nominal concentration)

NA Hormone cross

- reactivity (antibody

- based assays only; ≤30% of basal production of the respective hormone)

X Solvent control within 75% range below maximum response on standard curve

NA Test compound tested for interference with measurement system



## C. DATA ANALYSIS : To evaluate the relative increase or decrease of hormone production after test chemical exposure, the results were normalized to the mean solvent control (SC) value for each assay and results were expressed as fold

- change relative to the SC in each

exposur e plate. All data were expressed as mean ± standard deviation (SD). Relative changes were calculated as follows:

Relative Change = Hormone concentration in each well ÷ Hormone concentration of mean

solvent control

For forskolin induction of testostero ne, relative change was calculated both with and without subtracting the background hormone production from the forskolin- treated wells and blank and solvent control wells. This was done because of the high background production of testosterone and the small dynamic range for forskolin induction. Background hormone production was calculated from three wells with cells on the QC plate that received no 22R-hydroxycholesterol at the time of exposure.

Normality of the data was evaluated using Shapiro

- Wilk’s test. Homogeneity of the variances between the treatment groups was evaluated using Levene’s test. If the p

- values were greater than 0.05 in both tests, statistical significance between each treatment group and the control group was evaluated using Dunn ett’s test. If the p

- values were less than or equal to 0.05 in either the normality or the homogeneity test, a log transformation was performed on the data to attempt to approximate a normal distribution. If, following theSteroidogenesis Assay ( 2011 ) / Page 7 of 13

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## transformation, p- values were g reater than 0.05 in both the normality and homogeneity tests, Dunnett’s test was performed on the transformed data to evaluate statistical significancebetween each treatment group and the control group. If following the log transformation, pvalues were less than or equal to 0.05 in either the normality or the homogeneity test, the

non- transformed data set was analyzed using the nonparametric Kruskal

- Wallis test followed by Dunn’s test to evaluate statistical significance between each treatment group and the control group.

HPLC

- MS/MS data were acquired using proprietary software application MassHunter Workstation Acquisition (Version B.03.01 (B2065), Agilent Technologies, Inc.). Data were processed (integrated) using the software application MassHunter Quantitative Analysis for QQQ (version B.04.00/Build 4.0.225.0. Agilent Technologies, Inc.). Calibration plots of area ratio versus testosterone and estradiol concentrations were constructed and a weighted 1/x2 linear regression applied to the data using M assHunter Quantitative Analysis for QQQ.

II. RESULTS

A. TEST COMPOUND : The basal hormone production for the SC (DMSO) was greater than 500 and 40 pg/mL for testosterone and estrogen (in the presence of 22R-hydroxycholesterol. Precipitation of the permethrin was observed in one well at 10 μM in Run #2, and in all

three wells at 100 μM for all three runs. Cytotoxicity was not observed at any tested

concentration. The between plate coefficient of variation (CV) for the SC based on absolute

concentrations (calculated by the reviewer) was 19 % for testosterone and 26 % for estradiol

, meeting the guideline recommendation of ≤30% . The CV within each run for the SC was 2.36-3.08% for testosterone and 0.70-1.88 % for estradiol, both meet ing the guideline

recommendation of ≤ 30%.

The results from the test runs with permethrin are summarized in Table 3 and presented graphically in Figures 1 and 2. A statistically significant increase in testosterone production

was observed at 10 μM in Runs #3 and #4 (average 1.14-fold) , and a statistically significant increase in estradiol production as observed at 1μM and 10μM in Runs #3 and #4 (average 1.16-fold for 1 μM and 1.51-fold for 10 μ M)

.Steroidogenesis Assay ( 2011 ) / Page 8 of 13

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### TABLE 3

. Mean (±SD) Hormone Concentrations Following Treatment with Permethrin for 48 Hours. a

Nominal

Concentration (μM) Trial 2 Trial 3 Trial 4 Trial 2 Trial 3 Trial 4 Mean ± SD Statistical Significan

ce Testosterone (pg/mL) Fold Difference

DMSO 2577 1799 2516

— — — — — — 0.0001 2552 1853 2447 0.99 1.03 0.97 1.00 0.03

— 0.001 2453 1890 2368 0.95 1.05 0.94 0.98 0.06

— 0.01 2468 1853 2362 0.9 6 1.03 0.94 0.9 8 0.0 5

— 0.1 2584 1889 2344 1.00 1.05 0.93 0.99 0.06

— 1 2710 1979 2456 1.05 1.10 0.98 1.04 0.06

— 10 2967 2069 2845 NA 1.15 1.13 1.14 0.01 Run 3 , 4

100 2657 2075 2832 NA NA NA NA NA NA
Estradiol (pg/mL) Fold Difference

DMSO 258 151 211

— — — — — — 0.0001 242 163 218 0.94 1.08 1.03 1.02 0.07

— 0.001 247 165 212 0.96 1.09 1.00 1.02 0.07

— 0.01 245 1 61 21 3 0.95 1.06 1.01 1.01 0.06

— 0.1 254 159 214 0.99 1.06 1.01 1.02 0.04

— 1 287 186 239 1.11 1.23 1.13 1.16 0.06 Runs 3 , 4

10 402 232 314 NA 1.53 1.49 1.51 0.03 Runs 3 , 4

100 358 235 334 NA NA NA NA NA NA

a Data were obtained from page s 26

- 27 and 33

- 35 of the study report. The lower limit of quantification (LLQ) was 100 pg/mL for testosterone and 10 pg/mL for estradiol.

NA N ot applicable. Concentration not analyzed because of precipitation observed post

- exposure under the microscope.Steroidogenesis Assay ( 2011 ) / Page 9 of 13

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## FIGURE 1. Change in Testosterone Production Relative to 2,4- D Concentration.Steroidogenesis Assay ( 2011 ) / Page 10 of 13

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## FIGURE 2. Change in Estradiol Production Relative to 2,4- D Concentration.

B. CYTOTOXICITY : Permethrin was not cytotoxic to the H295R cells (Table 4 ). Viability in each run ranged from 91.0 -100.2% relative to solvent controls.Steroidogenesis Assay ( 2011 ) / Page 11 of 13

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### T ABLE 4 . Mean (±SD) MTT Cell Viabilit y Results after Treatment with F orskolin, Prochloraz, or

Permethrin for 48 Hours. a

Compound Concen. ( μ M) Cell Viability

– Run 2 Cell Viability

– Run 3 Cell Viability

– Run 4

Mean SD Mean SD Mean SD

Blank NA 104.3 2.69 104.3 2.81 100.7 3.98 Background NA 95.4 4.14 102.5 1.79 98.9 0.9 4

SC + Methanol NA 5.6 2.51 11.4 1.99 5.1 0.1 4

Forskolin 1 107.8 1.52 109.3 1.17 105.0 1.55

Forskolin 10 107.0 2.43 108.5 4.17 102.4 2.58

Prochloraz 0.1 98.3 2.20 101.2 0.47 100.7 0.86

Prochloraz 1 99.4 1.19 101.5 0.78 99.5 2.37

Permethrin 

### 0.0001 95.7 4.13 94.6 3.23 98.3 2.44

Permethrin 

### 0.001 95.9 1.74 97. 0 3.18 98.2 1.76

Permethrin 

### 0.01 95.9 1.62 98.4 2.11 100.2 0.47

Permethrin 

### 0.1 99.1 1.79 98.1 2.55 98.1 0.72

Permethrin 

### 1 95.9 2.43 91.0 3.08 97.3 0.72

Permethrin 

### 10 96.5 3.48 99.4 5.54 98.3 2.39

Permethrin 

### 100 95.3 2.35 94.8 6.90 99.4 1.16

a Data were obtained from page s 20

- 21 of the study report.

NA Not applicable

SC Solvent control



## C. QC PLATE : Concentrations of testosterone and estradiol, and the fold induction or inhibition in the presence of forskolin and prochloraz, on the QC plates are shown in Table 5. The minimum basal hormone production levels (500 pg/mL for testosterone, 40 pg/mL

for es tradiol) were met in both blank and SC wells for both testosterone and estradiol. Cells incubated with 10 μM forskolin induced testosterone production by 1.61- to 1.89-fold which

is below the Guideline criteria of ≤ 2-fold; however, when the background is subtracted, 10 μM forskolin induced testosterone an average of 2.47-fold. Estradiol production was induced with 10 μM forskolin by 9.3- to 11.1- fold. Cells incubated with 1 μM prochloraz

inhibited testosterone and estradiol production by ≤ 0.49-fold. Thus, the guideline requirements were met indicating that the assay was sensitive to induction and inhibition of

testosterone and estradiol.

The variability (%CV) between the runs (calculated by the reviewer) based on the absolute

hormone concentrations in the SC were 1 5% for testosterone and 11 % for estradiol, which

meets the G uideline criteria of ≤30% for the assay. The SC %CVs within each run for the QC plates were 1.61 to 8.28% for testosterone and 2.90 to 6.16 % for estradiol. Evaluation

of the in terplate and intraplate variability (%CV) indicated consistent performance of the

assays.Steroidogenesis Assay ( 2011 ) / Page 12 of 13

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### TABLE 5 . Hormone Concentrations Following Treatment with Forskolin or Prochloraz for 48 Hours. a

Compound Conc. (μM) Run 2 Run 3 Run 4 Run 2 Run 3 Run 4 Mean b ± SD b

Testosterone (pg/mL) Fold Difference

Background NA 1135 1073 1262 0.45 0.57 0.54 0.52 0.06

Blank NA 2581 1873 2304 1.03 1.00 0.99 1.01 0.02

DMSO NA 2511 1874 2339 1.00 1.00 1.00 1.00

— Forskolin 1 3764 2595 3120 1.50 c 1.38 c 1.33 c 1.40 c 0.09

Forskolin 10 4751 3019 3780 1.89 d 1.61 d 1.62 d 1.71 d 0.16

Prochloraz 0.1 1816 1149 1818 0.72 0 .61 0.78 0.70 0. 09

Prochloraz 1 1188 501 1153 0.47 0.27 0.49 0.41 0.12
Estradiol (pg/mL) Fold Difference
Background NA 41 56 77 0.21 0.31 0.35 0.29 0.07

Blank NA 208 182 228 1.04 1.01 1.02 1.02 0.02

DMSO NA 200 179 223 1.00 1.00 1.00 1.00

— Forskolin 1 1343 1254 1538 6.72 7.01 6.91 6.88 0.15

Forskolin c 10 2116 1661 2477 10.60 9.28 11.13 10.34 0.95

Prochloraz 0.1 133 104 171 0.67 0.58 0.77 0.67 0.10

Prochloraz 1 57 52 83 0.29 0.29 0.37 0.32 0.05

a Data were obtained from page 22.

- 23 of the study report.

. b Calculated by the reviewers.

c When the background is subtracted, the values are 1.91

- , 1.90

- , and 1.73

- fold difference for Runs 2, 3, and 4, respectively,

with a mean of 1.84

- fold change.

d When the background is subtracted, the values are 2.63

- , 2.43

- , and 2.34

- fold difference for Runs 2, 3, and 4, respectively

, with a mean of 2.47

- fold change.



## III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR ’ S CONCLUSIONS : Permethrin exposure resulted in statistically significant (13

- 15%) increases in testosterone at the highest concentration analyzed (10 μ M) in two of the three independent runs of the assay. The 10 μM concentration could not be analyzed in the remaining assay run because precipitation was observed. Statistically significant increases (13 -53%) increases in estradiol were also observed at the two highest concentrations (1 and 10 μM) analyzed in two of three independent runs. The 10 μ M concentration could not be analyzed in the remaining assay run because precipitation was

observed.

B. AGENCY COMMENTS : Guideline acceptability recommendations and requirements were met, including lack of cytotoxicity, adequate production of testosterone and estradi ol, acceptable reproducibility ( low %CV) and appropriate induction and inhibition with positive

controls.

A statistically significant increase in testosterone production was observed at 10 μM in Runs #3 and #4 (average 1.14-fold), and a statistically significant increase in estradiol production

as observed at 1 μM and 10 μM in Runs 3 and 4 (Average 1.16- fold for 1 μM and 1.51-fold for 10 μ M)

.Steroidogenesis Assay ( 2011 ) / Page 13 of 13

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## C. STUDY DEFICIENCIES : The following deficienc ies w ere noted that are not considered to have had an adverse impact on the results, interpretation or conclusions of this study:

• No information was provided on the l aboratory p roficiency testing

.

• Recoveries of radio ligands were not reported for MTBE extractions prior to HPLC/MS

MS determination of steroids