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

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION



### MEMORANDUM

Date: 6/29/2015

SUBJECT: Cypermethrin: Data Evaluation Records (DERs) for EDSP Tier 1 

### Assays

PC Code: 109702


### Decision No.: 461805, 461850, 464613


### Petition No.: NA 

### DP Barcode: D 399304, D399338, D401652 

### Registration No.: NA



### Risk Assessment Type: NA


### TXR No.: 0052288


### MRID No.: See Table 

### Regulatory Action: NA


### Case No.: NA


### CAS No.: 52315-07-8
40 

### CFR: NA



### FROM: Greg Akerman, Ph

. D.

## ~**~** Immediate Office
Health Effects Division (7509P)



### THROUGH: Jess Rowland

## ~ Deputy Director
Health Effects Division



### TO: Jolene Trujillo Biologist / Chemical Review Manager
Risk Management and Implementation Branch V
Pesticide Re-evaluation Division (7505P)

I. 

### ACTION REQUESTED Yer.Apr. 20 I 0



### The Pesticide Re-evaluation Division (PRD) of OPP has requested that the Health Effects
Division (HED) review the Endocrine Disruptor Screening Pro gram (EDSP) Tier 1 assays submitted in response to the agency ' s Test Order for cypermethrin : Test Order# CON-109702-8.

Page I of2Page 2 of 2 

### II. RESPONSE

Attached are the EDSP Tier 1 assay DERs for cypermethrin.

III. MRID Table

Chemical: Cypermethrin PC Code: 109702
Guideline Assay MRID
890.1100 Amphibian Metamorphosis Assay (Frog) 49013801
890.1150 Androgen Receptor Binding (Rat Prostate) NA
890.1200 Aromatase Assay (Human Recombinant) 48670202
890.1250 Estrogen Receptor Binding NA
890.1300 Estrogen Receptor Transcriptional Activation (Human Cell Line HeLa-9903) 48670201

890.1350 Fish Short-Term Reproduction 48683001
890.1400 Hershberger (Rat) NA
890.1450 Female Pubertal (Rat) 48683002
890.1500 Male Pubertal (Rat) 48683003
890.1550 Steroidogenesis (Human Cell Line – H295R) 48670203
890.1600 Uterotrophic (Rat) 48615701 Range finding study 48615702 Range finding study 48683004 NA= Not Applicable (requirement satisfied by othe r scientifically relevant information (OSRI))Data Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis Assay

EPA MRID Number 49013801

Page 1 of 42

DER Template Version: 22 September 2011 Data Requirement: EPA DP Barcode 408187

OECD Data Point 231

EPA MRID 49013801

EPA Guideline 890.1100

Amphibian Metamorphosis Assay (Frog)

Test Material: Cypermethrin Purity (%): 95.2%

Common Name Cypermethrin

Chemical Name IUPAC Not reported

CAS Name Not reported

CAS No. 52315-07-8

Synonyms FMC 30980; (+/-)-a-cyano(3-phenoxyphenyl)methyl (+/-)

cis, trans-3-(2,2-dichloroethenyl)-2,2-

dimethylcylclopropanecarboxylate

EPA PC Code 109702

Primary Reviewer: Elizabeth Krupka Date: 6/26/2013

Environmental Scientist, CDM Smith

Secondary Reviewer: Teri S. Myers, Ph.D. Date: 7/01/2013

Environmental Scientist, CDM Smith

Primary Reviewer: Elizabeth Donovan Date: 8/1/2013

Biologist, EFED/ERB VI

Additional Reviewer: Elyssa Arnold Date: 11/12/13

Biologist, EFED/ERB II

Additional Reviewer: Amy Blankinship Date: Digitally signed by Elizabeth
Donovan DN: cn=Elizabeth Donovan, o=EPA, ou=EFED, email=donovan.elizabeth@epa .gov, c=US
Date: 2015.06.11 08:50:42
-04'00'



### AMY
BLANKINSHIP Digitally signed by AMY BLANKINSHIP DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=AMY BLANKINSHIP, dnQualifier=0000040917
Date: 2015.06.15 17:31:53 -04'00'Data Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis Assay

EPA MRID Number 49013801

Page 2 of 42

DER Template Version: 22 September 2011 Biologist, EFED/ERBVI

Date Evaluation Completed: 6/6/15

CITATION: Lee, M.R. 2012. Cypermethrin: Amphibian Metamorphosis Assay with African Clawed Frog ( Xenopus

laevis) . Unpublished study performed by Smithers Viscient, Inc., Wareham, MA. Laboratory report number

1781-6765. Study sponsored by Syngenta Crop Protection, LLC, Greensboro, NC. Study completed

November 28, 2012.

Note: 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).

Guideline recommendations are provided in italics; these recommendations should remain visible in the

completed DER.

Disclaimer: The guideline recommendations in this DER template are offered as a general reference to aid

in preparation of the DER. The purpose of these recommendations is not to serve as substitute for the Test

Guidelines, nor to provide any guidance on how the study should be conducted.Data Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis Assay

EPA MRID Number 49013801

Page 3 of 42

DER Template Version: 22 September 2011 EXECUTIVE SUMMARY

The 21-day assay of cypermethrin (95.2% purity) on amphibian metamorphosis of African Clawed Frog ( Xenopus

laevis) was conducted under flow-through conditions. Amphibi an larvae at Nieuwkoop-Faber (NF) stage 51 (80

larvae per group) were exposed to a negative control and test chemical nominal concentrations of 0.0000030,

0.000030, and 0.00030 mg a.i./L. The time-weighted average (TWA) concentrations were <0.0000010

(<LOQ, control), 0.0000019, 0.0000109, and 0.0000867 mg a.i/L; the %CV exceeded 20% for all treated

levels. The test system was maintained at 22 to 23 o C and a pH of 7.2 to 8.2.

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

Two mortalities occurred at the low treatment level and one mo rtality occurred at the mid treatment level. No other

mortalities were observed. Clinical signs including spinal deformities were observed in 27 tadpoles at the negative

control and low treatment levels and in 22 tadpoles at the mid and high treatment levels.

Cypermethrin caused no significant increase or decreas e (p>0.05) in 7-day hind-limb length (HLL) at any

treatment level. There was no significant acceleration or delay (p>0.05) of median NF developmental stage

observed at Day 7 or 21 at any treatment level relative to the negative control. Further, no asynchronous

development was observed. More than half of all individuals developed beyond NF stage 60 by Day 21 ( i.e. , NF

stage 61-62), with 53%, 59%, 54%, and 52% of living frogs exhibiting late stage (NF>60) development in the

control, low, mid, and high groups, respectively. There was no indication of a dose response pattern for occurrence

of late-stage development and both the reviewer and study author conducted separate analysis of only late-stage

individuals. The reviewer’s analysis indicated a significant dose-dependent reduction (p<0.05;

Jonckheere-Terpstra) in both Day 7 snout-vent length (SVL) (4%) and wet weight ( 12%) of tadpoles exposed

to the high treatment group, relative to the control. No other growth or developmental effects were noted in this

study, including for the additional analysis of late-stage frogs on Day 21. No effects on thyroid gland histopathology

were observed at any treatment level.

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 all test treatments. The CVs were 38.5%,

24.1%, and 35.2% in the low, medium, and high test levels, respectively. Mean percent recovery relative to nominalData Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis Assay

EPA MRID Number 49013801

Page 4 of 42

DER Template Version: 22 September 2011 concentrations was low, at 71%, 39%, and 32% in the low, medium, and high test levels, 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, the high CVs are not judged to sufficiently interfere with the interpretation of test

results. However, given the low recoveries and relative lack of findings, there is some uncertainty in whether the

organisms were sufficiently exposed to cypermethrin in this study.

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 reported

Test type: flow-throughData Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis Assay

EPA MRID Number 49013801

Page 5 of 42

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 Cypermethrin.

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 4 Day 7 Day 21 Day 21

0.0000019 No No No No No No No

0.0000109 No No No No No No No

0.0000867 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).

4 No significant effects (p>0.05) were detected for analyses excluding late-stage individuals or for the analysis

of only late-stage frogs.



### 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 Guidelines for Testing of Chemicals,

Guideline 231: Amphibian Metamorphosis Assay . The following deviation(s)

were noted:Data Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis Assay

EPA MRID Number 49013801

Page 6 of 42

DER Template Version: 22 September 2011

1. The reported flow through rate (50 mL/min) exceeded the recommended

(25 mL/min).

2. Hardness of the dilution water (160-180 mg/L as CaCO 3 ) exceeded the

recommended range (40-48 mg/L as CaCO 3 ).

3. Elevated temperature deviations of 0.1 to 0.2°C were noted on exposure

days 1, 4, 11 and 17.

4. The %CV for measured test concentrations exceeded 20% for all treated

levels. As a result, the reviewer based toxicity values on calculated

time-weighted average concentrations.

Aside from these deviations, all other performance and validity

requirements were met.

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:

routine food and water analyses were not performed according to GLP

Standards, but were performed using a certified laboratory and standard US

EPA analytical methods.

A. Test Material Cypermethrin

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.Data Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis Assay

EPA MRID Number 49013801

Page 7 of 42

DER Template Version: 22 September 2011 Lot No./Batch No. : PL10-0492

Purity: 95.2%

Impurities: None reported.

Stability of Compound: Mean measured concentrations yielded overall recoveries of 32-71% of

nominal. The coefficient of variation (CV) ranged from 24 to 39%.

Storage Conditions of

Test Chemicals: Stored at room temperature in the original container in a dark, ventilated cabinetData Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 49013801Page8of42 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 ReportedWere parents maintained asYesAdults were obtained from Nasco in Fort EPA recommends that larvae used in the1U.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 of Cypermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 49013801Page9of42 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline Recommendationsin-house stock?Atkinson, Wisconsin and were maintainedas in-house breeders for at least onemonth prior to generating tadpoles. assay be derived from in-house adults.Were parental acclimationconditions same as definitivetest?YesAcclimation period for parentalfrogs (if applicable):30 daysDetails on parental feeding:Not reportedDetails on parental health:Parental stock did notshow any signs ofsickness, disease,injuries or abnormalitiesprior to testing.Data Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 49013801Page10of42 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):ThreeNumber of eggs sampled perspawn:All eggs were sampledon a given day.Embryonic viability was checked daily.NF stage at test initiation51 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:13 days post-fertilization(dpf)Mean total length at test initiation(if reported):Not reportedRange of total length at testinitiation (if reported):Not reportedWas the optional size selectionmethod used?Not reportedData Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 49013801Page11of42 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on larval selection:When a sufficientnumberof tadpolesreached NF stage 51,the larvae weretransferred from arearing vessel to apooling tank containingdilution water at 22qC.The tadpoles that metthe developmentalcriteria based on hindlimb morphology weredistributed to four testvessels four at atimeuntil each vesselcontained 20 tadpoles.Abnormal tadpoles wereremoved and replacedData Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 49013801Page12of42 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline Recommendationswith healthy ones.Loading rate (rearing density):10 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:Xenopus ExpressSuitable for normal growth anddevelopment of X. laevistadpoles; fineparticulate that maintains water columnsuspension; stock solution prepared indilution water 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 d iet should demonstrate equal performance to Sera

Micron ® .Source of food:Xenopus Express,Brooksville, FloridaIodide concentration in diet (ifknown):Not providedFrequency of feeding:2 times/day EPA recommends that feeding occur at least

twice per day.Data Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 49013801Page13of42 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on feeding regime:Same as EPAGuidelines 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 of Cypermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 49013801Page14of42 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):Intermittent-flowproportionaldiluter Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 2Flow-through rate (ifapplicable):50 mL/min, based on a6.5L fill volume and6.8volume additions per day Recommended flow-through rate is 25 mL/min (complete volume replacement ca.
every 2.7 hrs).Details on toxicant mixing forflow-through systems (ifapplicable):Stock solution wasdelivered to the mixing cellat a rate of6.94 mL/min (0.0555mL/cycle). Stock was 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 is2Additional 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 of Cypermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 49013801Page15of42 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline Recommendationsprepared and replaceddaily during exposure viaa glass bead saturatorcolumn. A Fluid MeteringInc.(FMI)pump was usedto deliver a flow of dilutionwater through the column.The FMI pump, inconjunction with a 12-Lgraduated glass carboy,was calibrated to deliver0.0666 L/cycle of the0.005 mg/L stocksolution into the diluter'schemical mixing chamber.The mixing chambercontained the highestnominal test concentration completely mixed before introduced into the test system is recommended; 4) The recommended flow splitting accuracy is

within 10%.Data Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 49013801Page16of42 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline Recommendations(0.0003 mg/L) and wasproportionally diluted(10%) to provide theremaining nominalconcentrations (0.00003and 0.000003 mg/L).Renewal period for staticrenewal (if applicable):NA If static renewal is used, EPA recommends

24 -hr renewal; renewal period is recommended not to exceed 72 hours.Aeration?Yes 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:Laboratory well water wasused to prepare FETAXFETAX solution consisted of a blend of waterfrom an on-site 100-meter bedrock well and EPA recommends natural or reconstituted water; it is recommended that natural waterData Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 49013801Page17of42 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline Recommendations(Frog EmbryoTeratogenesisAssay– Xenopus)solutionde-chlorinated Town of Wareham well water.Solutions wereprepared in 3,400-L batchesand mixedfor approximately 48 hours. 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.Was dilution water analyzed forpesticides, heavy metals, andother contaminants?Yes (pesticides, PCBsand toxic metals)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

suppl ementation is in addition to the recommended dietary source of iodideData Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 49013801Page18of42 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline Recommendations (e.g.. in Sera Micron).Test vessel type/materials:Glass, silicone sealant,stainless steel andTeflon® 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:30 x 15 x 20 cm;2.5-gallonsFill volume:6.5 LAdditional details on exposuresystem:NAData Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 49013801Page19of42 DER Template Version: 22 September 2011Table 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsHardness (mg/L as CaCO3)160 180Not reportedDaily EPA recommends hardness 40 to 48 mg/L as CaCO 3.pH7.28.27.7Daily EPA recommends pH 7.5 ± 1,

inter -replicate and inter-treatment differentials should not exceed 0.5.Dissolved oxygen (mg/L)3.9 8.5 6.6Daily EPA recommends dissolved oxygen (DO) >3.5 mg/L (>40% air saturation). OECD recommends DO concentration >3.5 mg/L (>40% air saturation).Temperature21°C23qC 22qCDaily EPA recommends temperature 22±1 o C;

inter -replicate and inter-treatment differentials should not exceed 0.5 o C.Iodide(μg/L)Not reportedNot reported9.6Biannually EPA recommends aquatic iodide range 0.5

– 10 μ g/L (supplemental iodide should not

exceed 2 μ g/L).Data Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 49013801Page20of42 DER Template Version: 22 September 2011ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsAmmoniaNot reported General recommendations for frequency of measurements: EPA recommends that water quality parameters be measured in a control and at one test item concentration at least weekly. In static renewal systems, 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.FluorideNot reportedPerchlorateNot reportedChlorateNot reportedTotal Alkalinity (mg/L asCaCO3)Specific Conductance(μmS/cm)681800741900Not reportedNot reportedDailyDailyData Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 49013801Page21of42 DER Template Version: 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?YesIf yes, what was the method fordetermining the highest testconcentration in therange-finder?Solubility limit (7.6 mg/L) andconcentration at whichmortalityis<10% 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:51Test duration:14 daysData Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 49013801Page22of42 DER Template Version: 22 September 2011Additional details:Two 14-day range-finding studieswere conducted, with 10 tadpolesper replicate and two replicatesper treatment. Tadpoles wereexposed to nominal cypermethrinconcentrations of 0.00063,0.0013, 0.0025, 0.005 and0.01mg/L during the initial study andto concentrations of 0.000038,0.000075, 0.00015, 0.0003and0.00060 mg/L during the secondstudy.In the first range finding study,lethal or sublethal effects wereobserved at all treatment levels.In the second rangefindingstudy, neither lethal norsublethal effects were observedbelow 0.00060 mg/L. The EC10was 0.00028 mg/L (95% CI:0.00016-0.00035). Based onthese results, the maximumconcentration in the definitivestudy was set to 0.0003 mg/L.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.Data Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 49013801Page23of42 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsMethod for selecting the highesttest concentration in the definitivetest:Range-finding study 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):NASeparation of test concentrations:0.1 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?NoIndicate the type of valuespresented for measuredTime-weighted averageData Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 49013801Page24of42 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline Recommendationsconcentrations:Limit of quantification (LOQ):0.0000010 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:7 days 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:Four replicates EPA recommends 4 replicates.Number of replicates in solventcontrol (if applicable):NA 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 at80Data Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 49013801Page25of42 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline Recommendationstest initiation:Was a solvent used?NoSolvent type (if applicable):NAMaximum solvent concentration(if applicable):NA 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):NAPositive control concentration(s)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 ofDifficult Substances and Mixtures. Environmental Health and Safety Publications. Series onTesting and Assessment. No. 23. Paris, France.Data Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 49013801Page26of42 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline Recommendations(if applicable):Photoperiod:12 hrs light :12 hrs dark EPA recommends photoperiod 12:12

(light:dark).Light intensity at water’s surface:0.68 – 1.3 Klux EPA recommends light intensity 0.6 – 2 Klux (at water’s surface).Additional details:NAData Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 49013801Page27of42 DER Template Version: 22 September 2011Table 8: Summary of Treatment Concentrations in the Amphibian Metamorphosis Assay with Cypermethrin.Treatment IDNominalConcentration(mg a.i./L)TWA-MeasuredConcentration(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsNegative Control0.00<LOQNANA EPA and OECD recommend that test item concentrations be maintained at a coefficient of

var iation (CV)≤ 20%.Treatment 10.0000030.000001938.5NATreatment 20.000030.000010924.1NATreatment 30.00030.000086735.2NAAbbreviations:CVCoefficient of variation.E. ObservationsBiological Endpoints:SVL, total length, HLL, normalized HLL body weight, and developmental stage (Days 7 and 21), mortality and sub-lethal effects(daily), thyroid histology (Day 21)Were raw (individual) data provided?YesData Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 49013801Page28of42 DER Template Version: 22 September 2011 EPA recommends that observations of mortality and clinical signs occur daily, at a minimum; other observations are recommended as follows: NF developmental stage (days 7 and 21); any asynchronous development, indicated by tadpoles that cannot be assigned an NF stage (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 Cypermethrin to Amphibians, Metamorphosis Assay

EPA MRID Number 49013801

Page 29 of 42

DER Template Version: 22 September 2011 

### II. RESULTS AND DISCUSSION

A. Results

By test termination, two mortalities were observed in the low dose treatment group and a single mortality

was observed in the middle dose treatment group.

Table 9: Larval Mortality in Xenopus laevis

.

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

0.0000019 80 0 0 60 2 2 3.3

0.0000109 80 0 0 60 1 3 1.7

0.0000867 80 0 0 60 0 0

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

2 Both mortalities occurred in replicate D.

3 The single mortality occurred in replicate D.

On Day 7, all replicate and treatment median values for developmental stage were 54 (Table 10). By

test termination, the median development stage was 61 for the negative control and 60 for the highest

dose group. The mean 10 th and 90 th percentiles of the control’s development stage at test termination

were 59.8 and 61.8, respectively. No asynchronous development was observed.Data Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis Assay

EPA MRID Number 49013801

Page 30 of 42

DER Template Version: 22 September 2011 Table 10: Larval Development in Xenopus 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 # Asyn chronous

Negative Control 4 54 0 4 61 0

0.0000019 4 54 0 4 61 0

0.0000109 4 54 0 4 61 0

0.0000867 4 54 0 4 60 0

Day 7 normalized HLL averaged 0.141 in the negative control and 0.137, 0.134, and 0.140 in the

TWA-measured 0.0000019, 0.0000109, and 0.0000867 mg a.i./L treatment groups, respectively

(Table 11). The mean normalized HLL on Day 21 for individuals at NF stage ≤ 60 was 0.716 in the negative

control and 0.716, 0.754, and 0.740 in the TWA-measured 0.0000019, 0.0000109, and 0.0000867

mg a.i./L treatment groups, respectively.Data Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis Assay

EPA MRID Number 49013801

Page 31 of 42

DER Template Version: 22 September 2011 Table 11: Larval Development in Xenopus 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 2.92 0. 27 0.141 4 19.9 1.58 0.716

0.0000019 4 2.78 0.11 0.137 4 20.3 1.19 0.716

0.0000109 4 2.67 0.10 0.134 4 20.4 1.75 0.754

0.0000867 4 2.77 0.10 0.140 4 20.3 1.07 0.740

Abbreviations: SD Standard deviation.

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

Day 7 SVL averaged 20.7 mm in the negative control and 20.1, 20.0, and 19.8 mm in the TWA-measured

0.0000019, 0.0000109, and 0.0000867 mg a.i./L treatment groups, respectively (Table 12). By Day

21, mean SVL for individuals at NF stage ≤ 60 measured 28.1 mm in the negative control and 28.4, 27.5,

and 27.7 mm in the TWA-measured 0.0000019, 0.0000109, and 0.0000867 mg a.i./L treatment

groups, respectively.

Day 7 wet weight averaged 0.619 g in the negative control and 0.585, 0.569, and 0.546 g in the

TWA-measured 0.0000019, 0.0000109, and 0.0000867 mg a.i./L treatment groups, respectively

(Table 12). Day 21 wet weight for individuals at NF stage ≤ 60 averaged 1.74 g in the negative control

and 1.79, 1.70, and 1.70 g in the TWA-measured 0.0000019, 0.0000109, and 0.0000867 mg a.i./L

treatment groups, respectively.Data Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis Assay

EPA MRID Number 49013801

Page 32 of 42

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

Treatment

(mg a.i./L)

[ TWA- measured] 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 20.7 0.86 4 28.1 0.67 4 0.619 0.067 4 1. 74 0. 15

0.0000019 4 20.1 0.50 4 28.4 0.37 4 0.585 0.047 4 1.79 0.05

0.0000109 4 20.0 0.54 4 27.5 0.87 4 0.569 0.043 4 1.70 0.11

0.0000867 4 19.8 0.19 4 27.7 0.91 4 0.546 0.044 4 1.70 0.13

Abbreviations: SD Standard deviation.

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

No thyroid gland hypertrophy, atrophy, or follicular cell hyperplasia were observed in any control or

treatment group tadpoles (Table 13). No cases of follicular cell hypertrophy were observed in the negative

control or any of the treatment groups.Data Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis Assay

EPA MRID Number 49013801

Page 33 of 42

DER Template Version: 22 September 2011 Table 13: Gross Pathology and Histopathology of the Thyroid Gland in Xenopus 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 20 20 20 20 20 20 20

1 20 0 20 0 20 0 20 0

2 20 0 20 0 20 0 20 0

3 20 0 20 0 20 0 20 0

0.0000019 0 20 20 20 20 20 20 20 20

1 20 0 20 0 20 0 20 0

2 20 0 20 0 20 0 20 0

3 20 0 20 0 20 0 20 0

0.0000109 0 20 20 20 20 20 20 20 20

1 20 0 20 0 20 0 20 0

2 20 0 20 0 20 0 20 0

3 20 0 20 0 20 0 20 0

0.0000867 0 20 20 20 20 20 20 20 20

1 20 0 20 0 20 0 20 0

2 20 0 20 0 20 0 20 0

3 20 0 20 0 20 0 20 0

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 of Cypermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 49013801Page34of42 DER Template Version: 22 September 2011Table 14: Additional Thyroid Gland Histopathology Observations in Xenopus 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 Control020--20--20--20--20--120--20--20--20--20--220--20--20--20--20--320--20--20--20--20--0.0000019020--20--20--20--20--120--20--20--20--20--220--20--20--20--20--320--20--20--20--20--0.0000109020--20--20--20--20--120--20--20--20--20--220--20--20--20--20--320--20--20--20--20--0.0000867020--20--20--20--20--Data Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 49013801Page35of42 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 CellShapenIncidencenIncidencenIncidencenIncidencenIncidence120--20--20--20--20--220--20--20--20--20--320--20--20--20--20--1Thyroid histopathology is graded 0 – 3 based on severity: 0=Not remarkable, 1=Mild, 2=Moderate, 3=Severe. See OECD No. 82 forreference.At test termination, spinal deformities were observed in27 of the tadpoles in the negative control and 27, 22, and 22 tadpolesin the TWA-measured0.0000019, 0.0000109, and 0.0000867 mg a.i./L treatment groups, respectivelyData Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 49013801Page36of42 DER Template Version: 22 September 2011Table 15: Clinical Signs in Xenopus laevis.Treatment(mg a.i./L)[TWA-measured]Clinical Signs1TypenIncidenceNegative ControlSpinal deformities60270.0000019Spinal deformities60270.0000109Spinal deformities60220.0000867Spinal deformities60221Note that asynchronous development (unable to stage) is reported previously inTable 10.Data Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis Assay

EPA MRID Number 49013801

Page 37 of 42

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

For all continuous endpoints (HLL, SVL, wet weight) that followed a monotonic concentration-response,

the Jonckheere-Terpstra test was applied in a step-down manner to establish significant treatment effects.

For continuous endpoints that were not consistent with a monotonic response, the data were evaluated

for normality (Shapiro-Wilk’s test) and homogeneity (Bartlett’s test). If the data conformed to assumptions

of normality and equal variance, data were tested using Dunnett’s test. If the data were normally distributed

with unequal variance, the Tamhane-Dunnett (T3 test) was used to evaluate the data. If the data were

not normally distributed and had unequal variance, the Mann-Whitney-Wilcoxon U test using a

Bonferroni-Holm adjustment to the p-values was used to evaluate the data sets.

Concentration-response monotonicity was assessed visually from the replicate and treatment medians

or means. If some of these procedures were not appropriate for the respective data set, then the OECD

Current Approaches in the Statistical Analysis of Ecotoxicity Data: A Guidance to Application was

consulted (OECD, 2006). CETIS-Comprehensive Environmental Toxicity Information System TM (Ives,

2009) Version 1.8.4.20 software was used to perform all statistical computations.

One-sided Dunnett’s tests were ultimately used to analyze all continuous endpoints (HLL, SVL, wet

weight). The multi-quantal Jonckheere-Terpstra test was used to analyze development stage data. For

the mortality data, the Fisher’s Exact test with a Bonferroni-Holm adjustment was used.

All statistical conclusions were made at the 95% level of certainty except in the case of the basic

assumption tests such as Shapiro-Wilks’ Test and Bartlett’s Test in which the 99% level of certainty was

applied.

C. Reviewer’s Analysis and ConclusionsData Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis Assay

EPA MRID Number 49013801

Page 38 of 42

DER Template Version: 22 September 2011 Statistical Methods: All data were tested to determine if they satisfied the assumptions of normality and

homogeneity of variances using the Shapiro-Wilk’s and Bartlett’s tests at α =0.01, respectively. With the

exception of Day 7 developmental stage, all data satisfied these assumptions. Decreasing

dose-dependent trends were noted for Day 7 SVL, Day 7 wet weight, and Day 21 developmental stage

and an increasing dose-dependent trend was noted for Day 21 normalized HLL data (non-late stage only);

the Jonckheere-Terpstra test was used to analyze these endpoints. Data for Day 7 median developmental

stage were analyzed using the Mann-Whitney test, while all other non-trending endpoints were analyzed

using Dunnett’s test. The reviewer analyzed Day 21 endpoints excluding late-stage individuals (NF>60)

and using only late stage individuals.

No asynchronous development was observed. Thyroid histopathology findings were examined visually.

Unless otherwise indicated, effects were considered statistically significant at p <0.05

Conclusions:

No significant treatment-related effects were detected for any Day 21 endpoint (p>0.05) for non-late

stage frogs. The reviewer’s analysis detected a significant increase in body weight of Day 21 late stage

(only) frogs at the low dose, relative to control (p<0.05), but this isolated effect was not considered to

be treatment-related.

On Day 7, the reviewer’s analysis detected a significant reduction in SVL and wet weight at the highest

treatment level (Jonckheere-Terpstra, p<0.05), relative to the control, but no effects were detected on

developmental endpoints at Day 7.Data Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis AssayEPA MRID Number 49013801Page39of42 DER Template Version: 22 September 2011Table 16: Developmental and Thyroid Gross Pathology/HistopathologyEndpoints1,2in the AMA with Cypermethrin.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).4Day 21 excluding late-stage individualsTreatment(mg a.i./L)[TWA-measured]NF Developmental StageHind Limb Length3AsynchronousDevelopmentThyroid Gross andHistopathologyDay 7Day 21Day 7Day 214Day 7Day 21Day 21MedianpMedianp%Diff.p% Diff.p%Diff.p%Diff.pTreatment-RelatedEffects? (Yes/No)Negative Control54NA61NANANANANA----------0.0000019541.0610.50-2.140.0.00.56--------No0.0000109541.0610.50-4.980.225.380.31--------No0.0000867541.0600.13-0.530.993.390.25--------NoStatistical TestMann-WhitneyJonckheereTerpstraDunnett Jonckheere-Terpstra-- -- --Data Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis Assay

EPA MRID Number 49013801

Page 40 of 42

DER Template Version: 22 September 2011 Table 17: Growth Endpoints 1,2 in the AMA with Cypermethrin.

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.

a Day 21 excluding late-stage individuals

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 validity criteria. Performance criteria were met; with the exception that CV exceeded

20% in measured concentrations over 21 days (CV ranged from 24 - 39% at all treated levels). Mean

percent recovery relative to nominal concentrations was low, at 71%, 39%, and 32% in the low, medium,

and high test levels, 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, the high CVs

are not judged to interfere with the interpretation of test results. However, given the low recoveries, it is

not clear whether the organisms were adequately challenged in this study. Treatment

(mg a.i./L)

[TWA

- measured] Snout-Vent Length Body Weight

Day 7 Day 21 a Day 7 Day 21

% Diff. p %

Diff. p %

Diff. p % Diff. p

Negative Control NA NA NA NA NA NA NA NA

0.0000019 -2.74 0.17 1.14 0.87 -5.5 0.24 3.19 0.83

0.0000109 -3.6 0.08 -1.93 0.61 -8.14 0.08 -1.83 0.96

0.0000867 -4.2 0.02 -1.34 0.81 -11.9 0.04 -1.97 0.95

Statistical Test JonckheereTerpstra Dunnett JonckheereTerpstra DunnettData Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis Assay

EPA MRID Number 49013801

Page 41 of 42

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

The study author collected additional measurements on Day 7. Though the study protocol dictates that

samples from one test chamber of each concentrati on and control for determination of test substance

concentration will be taken at test initiation (Day 0) and weekly thereafter, on Day 7, samples were taken

from 2 replicates of each concentration and all four replicates from the control.

The reviewer’s results are reported in the Executive Summary and Conclusions sections of this DER.

Significant effects determined using the OCSPP 890.1100 flowchart were identical to those determined

using the historical EFED method (i.e., using William’s test to determine NOAEC/LOAEC values for

parametric, dose-dependent effects).

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)

Deviations in temperature of 0.1 to 0.2°C higher than the specified range were noted on exposure days

1, 4, 11, and 17.Data Evaluation Record on the Toxicity of Cypermethrin to Amphibians, Metamorphosis Assay

EPA MRID Number 49013801

Page 42 of 42

DER Template Version: 22 September 2011 Measured concentrations were based on the time-weighted average concentrations because

concentrations showed variability, with %CV ranging from 24 to 39% at all treated levels; concentrations

generally declined from nominal on Days 7 and 14, and were notably higher by Day 21.



### III. REFERENCES

Grim, KC et al. Thyroid Histopathology Assessments for the Amphibian Metamorphosis Assay to Detect

Thyroid Active Substances. Toxicol. Pathol. 37: 415-424, 2009.

Ives, Michael, 2009. CETIS, Comprehensive Environmental Toxicity Information System TM . User’s Guide.

Tidepool Scientific Software, McKinleyville, California.

Nieuwkoop, P.D. and Faber, J. Normal Tables of Xenopus laevi (Daudin). Garland Publishing, London, 1994.

# DATA EVALUATION RECORD

CYPERMETHRIN

Study Type: OCSPP 890.1200, Aromatase Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-43-2012 (MRID 48670202)

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: 5/09/2012

Secondary Reviewer: Signature: David A. McEwen, B.S. Date: 5/18/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 5/23/2012

Quality Assurance: Signature: Steven Brecher, Ph.D., D.A.B.T. Date: 5/23/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., it s potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustn ess 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 th e 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 wi th the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay re sults, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).CYPERMETHRIN I 109702



### Primary Reviewer: Jaime D ' Agostino, Ph.D.


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


### Health Effects Division Aromatase (Human Recombinant) Assay (2012) I Page I of 12
OCSPP 890

. 1 200 I OECD None



### S i g n a tu re:**___::.,~~~:::d..-=:::'::::::::..__** 

### Date:**--,,+-~-=------** 

### Signat ur e:**__,£_~~~=:..__** __ 

### Date:**--~ f**  

## ....

1

### C 

## __ Template version 08/2011



### DATA EVALUATION RECORD

STUDY TYPE: Aromatase (Human Recombinant); OCSPP 890.1200



### PC CODE: 109702 

### DP BARCODE: D399304



### TXR#: 0052288 

### CAS No.: 52315-07-8



### TEST MATERIAL (PURITY): Cypermethrin (95.2% a.i.)



### SYNONYMS: cyano(3-phenoxyphenyl)methyl 3-(2,2-dichloroethenyl)-2,2- dimethylcyclopropanecarboxylate



### CITATION: Wilga, P. (2012). Cypermethrin: Human Recombinant Aromatase Assay. Final Report. CeeTox, Inc

., Kalamazoo, MI. Laboratory Report No.: 9047V100353AROM, January 17

, 2012. MRID 48670202. Unpublished.



### SPONSOR: FMC Corporation, 1735 Market Street

, Philadelphia, PA; Syngenta Crop Protection, LLC, 410 Swing Road

, Greensboro, NC; and United Phosphorus

, Inc., 630 Freedom Business Center, King of Prussia

, PA



### TEST ORDER#: CON-109702-8



### EXECUTIVE SUMMARY: In an 

### in vitro aromatase (CYP 19) assay (MRID 48670202)

, cypermethrin (95.2% a.i. Lot# PL 10-0492) was incubated with human recombinant aromatase
and tritiated androstenedione**(1-~** 

### [3H(N)]-androst-4-ene-3, 17-dione; [3H]ASDN) in dimethyl
sulfoxide (DMSO) at log concentrations of 10- 1 0 

### to 10- 4 

### M for 15 minutes to assess the potential
of cypermethrin to inhibit aromatase activity.

Aromatase activity was determined by measuring the amount of tritiated water produced at the
end of a 15-minute incubation for each concentration of chemical. Tritiated water was quantified using liquid scintillation counting (LSC). Three independent runs of the aromatase assay were conducted and each test concentration of cypermethrin was tested in triplicate. Each run included a full activity control, a background activity control, a positive control series (10- 10 

### to
10- 5 

### M) using a known inhibitor ( 4-hydroxyandrostenedione; 4-0H ASDN). The highest
concentrations of cypermethrin tested in Run 1 ( 10- 3 

### M) and in Run 2 ( 10- 3 5 

### M) showed marked precipitation during the 15-minute incubation period and these data were not evaluated. The highest concentration for all three runs used for data interpretation was 10- 4 

### M because it did not
result in marked precipitation

.

Aromatase activity in the full activity controls averaged 0 .222 ± 

### 0.049 nmol/mg-protein/min.
The response of each full activity control within a run was between 90 to 110% of the averageAromatase (Human Recombinant) A ssay (2012) / Page 2 of 12
CYPERMETHRIN / 109702 OCSPP 890.1200 / OECD None



### full activity. Activity in the background cont rols ranged from 0.45 to 0.78% and averaged 0.64% of the full activity control.

For the positive control substance (4-OH ASDN), aromatase activity results were within the recommended ranges for the performance criteria. The estimated log IC 50 

### for 4-OH ASDN averaged -7.17 M and the slope was -0.94.

For cypermethrin, aromatase activity averaged 0.170 ± 0.018 nmol/mg-protein/min at the lowest
tested concentration 10

− 10 

### M and 0.193 ± 0.042 nmol/mg-protein/min at the highest tested concentration, 10

− 4 

### M, used for data interpretation. The average dose-res ponse curve indicated that the aromatase activity at all concentrations of cypermethrin te sted was essentially equivalent to the activity observed in the full activity control (>75% activ ity= non-inhibitor).

Based on the data from the average response curve, cypermethrin is classifi ed as a Non-inhibitor of aromatase activity in this assay.

The assay satisfies the EDSP Tier 1 Test Order requireme nts for an Aromatase assay (OCSPP 890.1200).

COMPLIANCE: Signed and dated Data Confidentiali ty, GLP Compliance, Quality Assurance, and Certification of Authenticity statements were provided.Aromatase (Human Recombinant) A ssay (2012) / Page 3 of 12
CYPERMETHRIN / 109702 OCSPP 890.1200 / OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: Cypermethrin
Description: Amber liquid
Source: FMC Corporation Lot # (expiration date): PL10-0492 (December 15, 2012) Purity: 95.2% Volatility: Not reported Storage conditions: Room temperature (e.g. ambient) Stability: Not reported
Solvent: DMSO Solubility (in test solvent): A moderate amount of prec ipitation was observed at concentrations of 10 -3.5 M and above. A small amount of precipitati on was observed at a concentration of
10 -4 M but was considered acceptable. Highest Concentration Tested: 10

− 3 M Stock Solution Preparation: Not reported Molecular weight: 416.3 g/mol
CAS #: 52315-07-8
Structure:

CN O

O Cl O

Cl CH 3 CH

3



### 2. Non-Labeled Substrate: Androstenedione (ASDN)

CAS # : 63-05-8
Source: Steraloids, Inc. (Catalog # A6030-100) Batch # (expiration date): L1712 (April 2016) Purity: 99.8%



### 3. Radiolabeled Substrate: 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 reported



### 4. Positive Control: 4-hydroxyandrostenedione (4-OH ASDN)

CAS # : 566-48-3
Source: Sigma-Aldrich (Catalog # F2552) Batch # (expiration date): 081K2133 (March 2015) Purity: 99.6%



### 5. Solvent (Vehicle Control): DMSO

Source: Lot # (expiration date): Not reported RNBB7017 (expires Feb. 2013)
Justification for choice of solvent One of the solvents of choice detailed in the EPA guideline, less volatile than ethanol, and more accurate to pipett e because of densit y and viscosity.
Concentration (% of total volume in assays): ≤ 1%Aromatase (Human Recombinant) A ssay (2012) / Page 4 of 12
CYPERMETHRIN / 109702 OCSPP 890.1200 / OECD None



### 6. Test Microsomes: Human CYP19 (Aromatase) and P450 reductase Supersomes

Source: Gentest TM , Woburn, MA (Catalog # 456260) Lot # (expiration date): 54390 (September 2012)

Protein concentration: 6.9 mg/mL Cytochrome C reductase activity: 450 nmole /mg protein/minute Aromatase activity: 2.8 pmol/pmol P450/min Lot # (expiration date): 03897 (February 2014)

Protein concentration: 7.4 mg/mL Cytochrome C reductase activity: 290 nmole /mg protein/minute Aromatase activity: 6.0 pmol/pmol P450/min



### B. METHODS

1. Assay Components and Preparations: A mixture of non-labe led 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 tri tium added to each incubation tube was
0.1 μ Ci.

The test chemical(s) were formulated in DM SO such that the volume of DMSO used per assay was no more than 1% v/v of the total as say volume. DMSO was selected because it is
listed as one of the solvents of choice detailed in the EPA guideline; it is 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 formulated in DMSO. Dilutions were prepared such that the target concentrations of the pos itive control substance (10

− 10 

### to 10

− 5 

### M; Table 4) were achieved by the additi on of 20 μL of the dilution for a final assay volume of 2 mL and that the final DMSO concentration was no more than 1% v/v of the total assay volume.

Human recombinant microsomes were purchased from Gentest TM 

### , and stored at

− 80 ± 10oC (length of storage not reported). Before use of the microsomes, the batch was thawed and sub-aliquoted into individual vials before bei ng refrozen to minimize freeze-thaw cycles to no more than one (length of storage not reported).

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

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

Microsomal Protein 0.004 mg/mL

NADPH 0.3 mM [ 3 H]ASDN 100 nM Propylene Glycol 5% Temperature 37±2°C
Incubation Time 15 minAromatase (Human Recombinant) A ssay (2012) / Page 5 of 12
CYPERMETHRIN / 109702 OCSPP 890.1200 / OECD None



### 2. Suitability Assessments: The protein concentration of the microsomes (Lot #54390) was supplied by the vendor as 6.9 mg/mL. Aromat ase activity of the microsomes was also provided by the vendor as 2.8 pmol/min/pmol P450. The protein co ncentrati on of the microsomes (Lot #03897) was supplied by the vendor as 7.4 mg/mL. Aromatase activity of the microsomes was also provided by th e vendor as 6.0 pmol/min/pmol P450. The minimum aromatase activity in the full activity control samp les was determined to be 0.166 nmol/mg-protein/min, which was greater than th e minimum acceptable aromatase activity of 0.1 nmol/mg-protein/min.

3. Aromatase Assay: Each assay run contained 4 tube s 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 positiv e control, and a full concentration curve in triplicate for the test substa nce 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 nmol/mg-protein/min.

4. Demonstration of Proficiency: Proficiency testing of the CYP19 aromatase assay was conducted. Each of the proficiency chemicals (atrazine, econazole, fe narimol 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. The data were not included in the study report.

Positive Control

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

 Mean aromatase activity in the absence of an inhibito r was at least 0.1 nmol/mgprotein/min.

 Mean background control activity (0.6%) 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 th e preferred ranges.

(2) Demonstration of Proficiency of Ne w Technician for Conducting Assay (when applicable): The positive control data for slope, t op and bottom percent met the criteria as listed in section (i) of OCSPP 890.1200.Aromatase (Human Recombinant) A ssay (2012) / Page 6 of 12
CYPERMETHRIN / 109702 OCSPP 890.1200 / OECD None

TABLE 2. Performance Criteria for the Positive Control a
Parameter Lower Limit Criteria Upper Limit Crit eria Actual Lower Limit Actual Upper Limit Slope

− 1.2

− 0.8

− 1.00

− 0.89 Top (%) 90 110 96.89 104.28 Bottom (%)

− 5 +6

− 0.19 0.66 Log IC 50 (M)

− 7.3

− 7.0

− 7.19

− 7.14 a Data were obtained from page 23 of the study report.



### b. Proficiency Chemicals: The proficiency of the laborat ory was demonstrated prior to running any test chemicals by c onducting three full scal e test runs on each of the proficiency
chemicals as listed in Table 3. The data were not included in the study re port. It was stated that atrazine, econazole, fenarimol, and nitrofen were correctly classifi ed as non-inhibitor or
inhibitor.

TABLE 3. Proficiency Chemicals Compound CAS# Class Concentrations
Econazole 24169-02-6 Inhibitor No data
Fenarimol 60168-88-9 Inhibitor No data
Nitrofen 1836-75-5 Inhibitor No data
Atrazine 1912-24-9 Non-inhibitor No data



### 5. Determination of Aromatase Ac tivity with Test Chemical(s): The response of aromatase activity to the presence of eight concentrations of cyperm ethrin per run, in triplicate, was tested during three independent runs (Table 4). Solubility was assessed (presence of cloudiness or a precipitate). Precipitation was observed as follows: at 10 -3 

### M in Run 1; at
10 -3.5 

### and 10 -4 

### M in Run 2; and at 10 -4 

### M in Run 3. The precipitation observed at 10 -4 

### M was minimal; therefore, the hi ghest concentration evaluated in all three runs was 10 -4 

### M. The lowest concentration to be tested was 10 -10 

### M. The full enzymatic activity was obtained
at the two lowest concentrati ons of cypermethrin, defining the top of the concentrationresponse curve.Aromatase (Human Recombinant) A ssay (2012) / Page 7 of 12
CYPERMETHRIN / 109702 OCSPP 890.1200 / OECD None

TABLE 4. Test Chemical Study Design for each Test Run

Sample Type Repetitions (Tubes) Description Reference or Chemical (M) Full Activity Control 4 All test components a 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 Cypermethrin Conc 1 b ,c 3 All test components plus Cypermethrin 1×10 -3 Cypermethrin Conc 2 b

, d 3 All test components plus Cypermethrin 1×10

− 3.5 Cypermethrin Conc 3 b ,e 3 All test components plus Cypermethrin 1×10

− 4 Cypermethrin Conc 4 b

, f 3 All test components plus Cypermethrin 1×10

− 4.5 Cypermethrin Conc 6 b 3 All test components plus Cypermethrin 1×10

− 5 Cypermethrin Conc 7 b 3 All test components plus Cypermethrin 1×10

− 6 Cypermethrin Conc 8 b 3 All test components plus Cypermethrin 1×10

− 7 Cypermethrin Conc 9 b 3 All test components plus Cypermethrin 1×10

− 8 Cypermethrin Conc 7 b 3 All test components plus Cypermethrin 1×10

− 9 Cypermethrin Conc 8 b 3 All test components plus Cypermethrin 1×10

− 10 a The complete assay contained buffer, propylene glycol, microsomal protein, [ 3 H]ASDN, and NADPH. b Proficiency chemicals or tests chemical(s). c Test concentration used in Run 1 only; determined to be insoluble and was not used in data interpretation by study
author. d Test concentration used in Run 2 only; determined to be insoluble and was not used in data interpretation by study
author. e Highest test concentration used in Runs 1-3 determined to be soluble and used in data interpretation by study author. f Test concentration used in Run 3 only.



### C. DATA ANALYSIS

1. Raw Data: Raw data were converted to aromatas e 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 fo r each run were included in the report (Table 5).

TABLE 5. Raw and Calculated Data

Raw/Calculated Data Included (X) DPM/mL for each portion of extract ed 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. Statistical Methods: For data generated at CeeTox, b asic 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.Aromatase (Human Recombinant) A ssay (2012) / Page 8 of 12
CYPERMETHRIN / 109702 OCSPP 890.1200 / OECD None



### The response curve was fitted by weighted leas t squares nonlinear regression analysis using a 4-parameter regression model (XLfit; IDBS ; Version 5.2.0.0; Fit Model 208) and Tukey’s Bi-Weight statistical analysis for outlier anal ysis. For each run, the individual percent of control values was plotted versus logarithm of the test chemical c oncentration. The fitted concentration response curve was superimposed on the plot, with indivi dual plots prepared for each run. The average percent of control values versus logarithm of test chemical
concentration for the individual runs for each te st chemical (with different symbols for each run) were included on the same graph with their respective fi tted response curves. In addition, the average percent of control values for each run versus the logarithm of test chemical concentration was plotted on a separate graph along with the average concentration response curve across runs superimposed on the same plot.

Assay drift was assessed by comparing the % fu ll activity and background activity 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 categor ies presented in Table 6.

TABLE 6. Interpret ation 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 equivocal. b If the test compound was not soluble above 10

− 6 M and the inhibition curve does not cr oss 50%, the chemical is typically
determined to be untestabl e in the aromatase assay.



### II. RESULTS

A. CONTROL ACTIVITY: Aromatase activity in the full activity controls ranged from 0.166-0.293 nmol/mg-protein/min for th e 3 test runs, with a mean and standard deviation of 0.222 ± 0.049 nmol/mg-protein/min. The response of each full activity control within a run was between 90 to 110% of the average full acti vity. Activity in the background controls ranged from 0.45 to 0.78% and averaged 0.64% of the full activity control.

B. POSITIVE CONTROL: For the positive control substance (4-OH ASDN), aromatase activity averaged 0.224 ± 0.042 nmol/mg-protein/mi n at the lowest tested concentration,
10

− 10 

### M and 0.002 ± 0.000 nmol/mg-protein/min at the highest non-precipitating concentration in all runs, 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. As a percent of the control, aromatase activity for 4-OH ASDN was 0.94% at 10 -5 

### and 102.02%
at 10 -10 

### M. The inhibition response curves for th e positive control are shown in Figure 1.Aromatase (Human Recombinant) Assay (2012) / Page 9 of 12
CYPERMETHRIN / 109702 OCSPP 890.1200 / OECD None

TABLE 7. Effect of Cypermethrin 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 ASDN (positive control) − 5 3 0.94 0.12 0.07 12.7

− 6 3 7.65 0.91 0.52 11.8

− 6.5 3 20.07 2.02 1.16 10.1

− 7 3 40.57 2.99 1.72 7.4

− 7.5 3 68.48 2.49 1.44 3.6

− 8 3 87.12 2.64 1.52 3.0

− 9 3 97.76 2.35 1.36 2.4

− 10 3 102.02 4.83 2.79 4.7 Cypermethrin -4 3 87.37 5.11 2.95 5.9

-4.5 c 1 94.93

− 5 3 93.33 5.01 2.89 5.4

− 6 3 94.08 2.01 1.16 2.1

− 7 3 94.62 3.31 1.91 3.5

− 8 3 95.73 2.37 1.37 2.5

− 9 3 94.65 3.86 2.23 4.1

− 10 3 91.79 4.77 2.75 5.2

a Data were obtained from Appendix 1, pp. 42-45, 47-50, 52-55 of the study report b Calculated by the reviewers from data presented in this table.
c Test concentration us ed for Run 3 only.
SD Standard Deviation
SEM Standard error of the mean
CV Coefficient of Variance



### FIGURE 1. Inhibition Respon se Curves for 4-OH ASDN.

Blue = run1, Red = run 2, Green = run 3



### C. TEST SUBSTANCE: For cypermethrin, aromatase activity averaged 0.170 ± 0.018 nmol/mg-protein/min at the lowest tested concentration 10

− 10 

### M and 0.193 ± 0.042 nmol/mg-protein/min

− 1 

### at the highest tested concentra tion used for study interpretation, 10

− 4 

### M. The highest concentrations of cypermethrin tested in Run 1 (10 -3 

### M) and in Run 2 (10

-

3.5 

### M) showed precipitation during the 15-minut e incubation period and were not used forAromatase (Human Recombinant) A ssay (2012) / Page 10 of 12
CYPERMETHRIN / 109702 OCSPP 890.1200 / OECD None



### data interpretation. The highestconcentration for all three runs used for data interpretation
was 10 -4 

### M since it did not result in marked precipitation. The mean aromatase activity of cypermethrin (expressed as % full control activit y) 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 curves for cypermethrin from each run are shown in Figure 2, and the average inhi bition response curve across all runs is shown in Figure 3.

FIGURE 2. Inhibition Response Curves for Cypermethrin From Each Test Run.

Blue = run1, Red = run 2, Green = run 3



### FIGURE 3. Mean Inhibition Response Curve for Cypermethrin.

Blue = run1, Red = run 2, Green = run 3, Black = mean



### The effects of the positive control and cypermethrin on inhibition of aromatase activity are presented in Table 8. The av erage dose-response curv e indicated that the aromatase activity of cypermethrin at all soluble test concentra tions tested was essentially equivalent to the activity observed in the full activity control. Th ese data could not be fit to the 4-parameter nonlinear regression model; therefore, log IC50 and Hill slope estimates were notAromatase (Human Recombinant) A ssay (2012) / Page 11 of 12
CYPERMETHRIN / 109702 OCSPP 890.1200 / OECD None



### determined for cypermethrin. For 4-OH ASDN, the estimated log IC 50 

### averaged

− 7.17 M and the slope was

− 0.94. Confidence in these numbers is high due to the small variation.

TABLE 8. Effect of Cypermethrin on Aromatase Ac tivity (as Percent of Co ntrol) From Independent
Runs a

Chemical Run 1 Run 2 Run 3 Mean b SD b %CV b
Log IC 50 (M) Cypermethrin NA NA NA NA NA NA

4-OH ASDN -7.19 -7.18 -7.14 -7.17 0.03 -0.37 Slope Cypermethrin NA NA NA NA NA NA

4-OH ASDN -1.00 -0.89 -0.93 -0.94 0.06 -5.92

a Data were obtained from Table 14, page 30 of the study report b Calculated by the reviewers from data presented in this table.
SD Standard Deviation
CV Coefficient of Variance NA Not applicable



### Based on the data from the average response curv e and the criteria listed above in Table 6, the results support the c onclusion that cypermethrin does no t inhibit aromatase activity in this assay.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS CONCLUSIONS: Cypermethrin was determined to be a noninhibitor of aromatase activity in this validat ed assay as defined by EDSP guideline OPPTS (OCSPP) 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 demonstrat ed prior to analysis of cypermethrin by the
conduct of three full scale test runs on each of the recommended proficiency chemicals. It was stated that the proficiency chemicals we re correctly classified as non-inhibitor or
inhibitor.

Aromatase activity in the full activitycontrols averaged 0.222 ± 0.049 nmol/mgprotein/min. The response of each full activ ity control within a run was between 90 to 110% of the average full activity, and activity in the background cont rols averaged 0.6 ±
0.2% of the full act ivity control.

For the positive control substance (4-OH ASDN), aromatase activity results were within the recommended ranges for the top of the curve, bottom of the curve, Hill slope, log IC 50 

### , and %CV. The estimated log IC 50 

### for 4-OH ASDN averaged

− 7.17 M and the slope was

− 0.94.

For cypermethrin, aromatase activity at the lowe st and the highest tested concentrations,
10 -10 

### and 10

− 4 

### M, respectively, used for interpreta tion was 91.79% and 87.37% of the full activity control. The two highest testconcentrations of cypermethrin (10 -3 

### and 10 -3.5 

### M) showed precipitation in the incubation during the 15-minute in cubation period; therefore,Aromatase (Human Recombinant) A ssay (2012) / Page 12 of 12
CYPERMETHRIN / 109702 OCSPP 890.1200 / OECD None



### these data were not evaluated. The averag e dose-response curve indicated that the aromatase activity of cypermethrin at all solubl e test concentrations tested was essentially equivalent to the activity observed in the full acti vity control; therefore, these data could not be fit to the 4-parameter nonlin ear regression model. Because the mean aromatase activity for cypermethrin was 87.37 ± 5.11% at the hi ghest soluble test concentration of 10

− 4 

### M, cypermethrin was determined to be a non-inhi bitor 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:

 Proficiency data were not included in the st udy report. However, it was stated that a full-scale assay consisting of three indepe ndent runs was conducte d using the positive control (4OH-ASDN) and the four profic iency chemicals outlined in the OCSPP 890.1200 guideline was conducted and that pr oficiency was demonstrated and the
results maintained at CeeTox.

 The radiochemical purity was not measured in laboratory as indicated in the guideline.

 The protein concentration of the Supersom es was not determined on each day of the assay as indicated in the guidelines.

# DATA EVALUATION RECORD

CYPERMETHRIN

Study Type: OCSPP 890.1300, Estrogen Receptor Transcriptional Activation

EPA Contract No. EP10H001452 Task Assignment No. 2-43-2012 (MRID 48670201)

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: 5/03/2012

Secondary Reviewer: Signature: Scott D. Studenberg, Ph.D.,
D.A.B.T. Date: 5/21/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 5/24/2012

Quality Assurance: Signature: Steven Brecher, Ph.D., D.A.B.T.. Date: 5/24/2012

This Data Evaluation Record may have been alte red 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 in tended 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 re dundancy 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 potent ial 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).Estrogen Receptor Transcriptional Activation (2012) I Page I of 16
CYPERMETHRIN/109702 OCSPP 890.1300/ OECD 455



### Primary Reviewer: Patience Browne Ph.D.

# Signature**~** Health Effects Division Date: 

## c; JU/ 15 Secondary Reviewer: Minerva Mercado-Feliciano, Ph.D., DABT

### Signat~ Health Effects Division

· Date:**~** Template version 08/2011



### DATA EVALUATION RECORD

STUDY TYPE: Estrogen Receptor Transcriptional Activation (Human cell Line, HeLa-9903) ; OCSPP 890

. 1300 ; OECD 455.



### PC CODE: 109702 

### DP BARCODE: D399304



### TXR#: 0052288 

### CAS No.: 52315-07-8



### TEST MATERIAL (PURITY): Cypermethrin (95.2% a.i.)



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

, 2-dichloroethenyl)-2,2- dimethylcyclopropanecarboxylate



### CIT A TI ON: Willoughby

, 

### J.A. (2012) Cypermethrin: Estrogen Receptor Transcriptional

Activation (Human Cell Line (HeLa-9903) Final Report. CeeTox, Inc

, Kalamazoo

, MI. Laboratory Report No

. : 9047V-100353ERTA

, January 16

, 2012. MRID 48670201. Unpublished.



### SPONSO RS: FMC Corporation

, 1735 Market Street

, Philadelphia

, PA; United Phosphorous

, Inc.

, 630 Freedom Business Center

, Suite 402, King of Prussia, PA ; and Syngenta Crop Protection

, LLC

, 410 Swing Road, Greensboro

, NC



### TEST ORDER#: CON-109702-8



### EXECUTIVE SUMMARY: In an estrogen receptor transcriptional activation assay (MRID

48670201 ), h E Ra-HeLa-9903 cells cultured 

### in vitro were exposed to cypermethrin (95.2% a.i

., Batch PL 10-0492) at logarithmically increasing concentrations from 10

- 10 3 to 10

- 33 Min DMSO ( 0

. 1 % ) for approximately 24 hours in three independent runs. A total of 8 runs were conducted

, but onl y the data from three runs were reported. The experiments were performed using 96-well plates and each cypermethrin concentration was tested in replicates of 6 wells / plate in each run. Ce lls were exposed to the test agent for 24 ± 2 hours at 37 ± 1 ° C to induce reporter (luciferase)

g ene products. Luciferase expression in response to activation of the estrogen receptor by

c y permethrin was measured using a proprietary luciferase assay

.

Cy permethrin was tested up to the limit of solubility

, I 0 -4 M. The RPCM ax was 16.3% at l 0

- 4 

### M
and the PC10 was 10- 4 6 

### Min Run 1, indicating a positive response to the assay. However

, in

Runs 2 and 3 the RPCMax for cypermethrin was :S 0.9% at concentrations up to 10- 3 3 M, indicating

that cypermethrin was negative for estrogenic activity under th.e conditions of the assay

. Review

of the performance criteria parameters for Run 1 (Table 3) shows that Run 1 was unusually

s e nsitive

, with all parameters for all reference chemica l s at lo wer l eve l s than expected according

to the guidance.Estrogen Receptor Transc riptional Activation (2012) / Page 2 of 16
CYPERMETHRIN/109702 OCSPP 890.1300/ OECD 455



### In all runs, cypermethrin response was similar to that of the negative control (corticosterone),
and in no run did it reach levels similar to the weak estrogen 17 α -methyltestosterone. For Run 2, Log PC 50 

### values could not be determined for 17 α -methyltestosterone as it did not reach 50% activation at concentrations up to 10 -5 

### M. This indicates a decreased sensitivity of cells. However in that same run, responses were clos e to zero at all cypermethrin concentrations.

Although the conditions of this a ssay were not optimal, in general cypermethrin responses were similar to those of the negative control corticos terone and not comparable to the responses of
17 α -methyltestosterone, which was able to r each maximums of 124.3, 16.5, and 52.8% PC in Runs 1, 2, and 3, respectively. Cypermethrin wa s only able to reach maximums of 16.3, 0.9, and 0.6% PC in Runs 1, 2, and 3, respectively, when tested up to the highest concentration possible based on cytotoxicity. Because the RPC Max 

### < PC 10 

### in both assay runs, cypermethrin was considered negative for estrogen receptor tran scriptional activation in this test system.

This assay satisfies the EDSP Tier 1 Test Order requirement for an Estrogen Receptor Transcriptional Activation assay (OCSPP 890.1300).

COMPLIANCE: Signed and dated GLP Compliance a nd Quality Assurance statements were provided.Estrogen Receptor Transc riptional Activation (2012) / Page 3 of 16
CYPERMETHRIN/109702 OCSPP 890.1300/ OECD 455



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: Cypermethrin
Description: Brown viscous liquid

Source: Syngenta Crop Protection, LLC Batch # (Exp. Date): PL10-0492 (Dec 15, 2012) Purity: 95.2% Solubility: Not Reported (Soluble in DMSO) Volatility: Not Reported Stability: Not Reported Storage conditions: Ambient

CAS #: 52315-07-8

Structure:

CN O

O Cl O

Cl CH 3 CH

3



### 2. Reference substances
17 β -estradiol (strong estrogen; positive control)
Supplier: Not Reported Catalog and Batch #: Not Reported Purity: Not Reported

CAS # : 50-28-2



### 17 α -estradiol (weak estrogen)
Supplier: Not Reported Catalog and Batch #: Not Reported Purity: Not Reported

CAS # : 57-91-0



### Corticosterone (negative compound)
Supplier: Not Reported Catalog and Batch #: Not Reported Purity: Not Reported

CAS # : 50-22-6



### 17 α -methyltestosterone (very weak agonist)
Supplier: Not Reported Catalog and Batch #: Not Reported Purity: Not Reported

CAS # : 58-18-4



### 3. Vehicle(s)

Solvent: DMSO

Solvent control (final concentration): 0.1%Estrogen Receptor Transc riptional Activation (2012) / Page 4 of 16
CYPERMETHRIN/109702 OCSPP 890.1300/ OECD 455



### B. METHODS

1. Cell Culture: Stably-transfected hER α -HeLa-9903 cells were obtained from the Japanese
Collection of Research Bioresour ces Cell Bank and were verified to be free of mycoplasma infection by a DNA fluorochrome assay. Cells were maintained in Eagles Minimum Essential Medium (EMEM) without phenol re d, supplemented with kanamycin (60 mg/L)
and 10% dextran-coated charcoal-treated fetal bovine serum (DCC-FBS; source not reported), in an in cubator under 5% CO 2 

### at 37±2 oC. Upon reaching 75-90% confluence, cells were subcultured up to 34 times prior to ex posure to the test material. A total of 8 runs were conducted and cell passage information was not provided for all 8 runs. Assays 1, 2, 5,
6 and 7 were invalid. The cells used in this study were Passage 32 for the range-finder, Passage 34 for the third assay (Run 1), passage 17 for the fourth assay (Run 2 and passage 23 for the eighth assay (Run 3). Based on the passage numbers, the Reviewers believe that cells were brought out of cryopreservati on for each of the reported runs.

2. Transcriptional Activation Assays: For each test, cells were plated in a 96-well plate (estrogenic activity of plastic not reported) at a density of approximately 1×10 4 

### cells/100 μL
medium/well and allowed to attach for 3 hours. The growth medium was replaced with medium containing serial log dilutions of c ypermethrin in DMSO. In addition to the chemical being tested, each plate included 6 blank replicates, 6 positive control (1 nM 17 β

estradiol) replicates and 12 ve hicle control replicates. Cell s were incubated for 24±2 hours
at 37±2 °C. The total final c oncentration of DMSO was 0.1%. Cytotoxicity was determined by two-read propidium iodide uptake.

It was stated that transcrip tional activation of th e estrogen receptor was determined as described in CeeTox Standard Operating Pr otocol (SOP) 2041. A list of reagents was provided, but the assay reagent was classified as proprietary information, and the SOP was not provided.

a. Preliminary Test: A preliminary test evaluating concentrations ranging from 10

− 10 

### to 10

− 3 

### M was conducted to determine the appropri ate concentration range and to determine concentrations resulting in insolu bility and/or cy totoxicity.

b. Proficiency Chemicals: The responsiveness of the test system was evaluated on March 5, 2010 and April 12 and 28, 2011. Cells used for th e proficiency tests were at passage 15, 25 and 28, respectively; it is likely these cells are not from the same frozen stock as the cells used in the current assay. Cells were tested with the following set of proficiency chemicals. Each chemical was tested in duplicate on separate days (MRID 48843501):Estrogen Receptor Transc riptional Activation (2012) / Page 5 of 16
CYPERMETHRIN/109702 OCSPP 890.1300/ OECD 455

Compound CAS No. Concentration Range (M) Expected Response a Notes

Diethylstilbestrol (DES) 56-53-1 10

− 14 to 10

− 8 Positive
---

17 α -Ethynyl estradiol (EE) 57-63-6 10

− 14 to 10

− 8 Positive
---

Hexestrol 84-16-2 10

− 13 to 10

− 7 Positive
---

Genistein 446-72-0 10

− 12 to 10

− 5 Positive Cytotoxic at 0.01 b , 0.1, and 1 mM

Estrone 53-16-7 10

− 12 to 10

− 6 Positive
---
Butyl paraben 94-26-8 10

− 11 to 10

− 4 Positive Cytotoxic at 0.1 b and 1 mM

1,3,5-Tris(4- hydroxyphenyl)benzene c 15797-52-1 10

− 12 to 10

− 5 Positive Cytotoxic at 100μM. PC max approx. 50% of PC.
Binds to hER α and has ER antagonistic activity Dibutyl phthalate (DBP) 84-74-2 10

− 11 to 10

− 4 Negative d Cytotoxic at 1 mM

Atrazine 1912-24-9 10

− 11 to 10

− 4 Negative Cytotoxic at 1 mM b

Corticosterone 50-22-6 10

− 10 to 10

− 4 Negative If not cytotoxic at 1 mM, then that should be the highest tested concentration

a Positive = RPC Max ≥ 10% of the response of the positive control in at least 2 of 2 (or 2 of 3) runs Negative = RPC Max fails to achieve at least 10% of the response of the positive control in 2 of 2 (or 2 of 3) runs b Cytotoxicity is expected to be close to 80% at this concentration. c Compound selected to challenge solubility and cytotoxicity d DBP is negative for ER α mediated transcriptional activation, but may not be negative for non-ER β mediated transcriptional activation. A positive result would indicate that the sy stem is detecting activity other than that due to pure ER α , and is therefore unacceptable.



### c. Reference Chemicals: To ensure the stability of the response from the cell line, eight concentrations of each of the following refere nce chemicals were included in each run, in separate plates:

Reference Chemical CAS No. Concentration Range Class

17 β -estradiol (E2) 50-28-2 10

− 15 to 10

− 8 Strong estrogen

17 α -estradiol 57-91-0 10

− 13 to 10

− 6 Weak estrogen

Corticosterone 50-22-6 10

− 11 to 10

− 4 Negative compound

17 α -methyltestosterone 58-18-4 10

− 12 to 10

− 5 Very weak agonist



### 3. Data analysis: To obtain the relative transcriptiona l activity to the 1 nM E2 positive control (PC), the luminescence signals from the concur rent plate were analy zed by subtracting the
mean value of the vehicle control from each well value to normalize the data; each normalized value was then divided by the mean value of the normalized PC. The resulting value was multiplied by 100 in order to expres s relative transcriptional activity as a percentage of the PC. The test material was defined as negative for inducing estrogen receptor transcriptional activation if the RPC Max 

### < PC 10 

### in at least 2 of 2 runs. Log EC 50 

### and Hill slope values are calculated only if a positive response is observed. Coefficients of variation (CV) were calculated for the lumi nescence data triplicates. Concentrations showing >20% cytotoxicity or evidence of in solubility were excluded from analyses.

4. Definitions

EC 50 

### = concentration of agonist that induces a response ha lfway between the baseline (bottom) and maximum (top) responseEstrogen Receptor Transc riptional Activation (2012) / Page 6 of 16
CYPERMETHRIN/109702 OCSPP 890.1300/ OECD 455



### PC 10 

### = concentration of a test chemical at which the response is 10% of the response induced by the positive control (E2 at 1 nM) in each plate

PC 50 

### = concentration of a test chemical at which the response is 50% of the response induced by the positive control (E2 at 1 nM) in each plate

RPC Max 

### = maximum level of response induced by a test chemical, expressed as a percentage of the response induced by the positive control (1 nM E2) on the same plate

PC Max 

### = concentration of a test chemical inducing the RPC Max



### II. RESULTS

A. PRELIMINARY TEST: In order to identify a suitable top concentration cypermethrin for use in the transcriptional activation assays, pr eliminary cytotoxicity and precipitation assays were conducted. Precipitation was observed at 10 –3 

### M cypermethrin in all three runs; the results of the cytotoxicity test are presented in Table 1. No cytotoxicity was observed at any concentration. Based on these results, logar ithmically increasing c oncentrations from 10

− 7.5



### to 10

− 4 

### M were selected for Run 1, and 10

− 10.3 

### to 10

− 3.3 

### M were selected for Runs 2 and 3 of the assay.
TABLE 1. Preliminary Test for Solubility, Cy totoxicity, and Concentration-Selection for Cypermethrin a

Concentration (M) % Viability b Comments c Run 1 Run 2 Run 3

10

− 3 95 97 107 Precipitation observed (all 3)

10

− 4 93 99 98

10

− 5 98 102 103

10

− 6 96 99 101

10

− 7 97 101 100

10

− 8 94 101 104

10

− 9 96 101 99

10

− 10 98 102 103 a Data were obtained from page 20 of the study report. b If viability is <80%, the concentration is considered cytotoxic. c Include comments related to solubility issues, if applicable.



### B. POSITIVE AND NEGATIVE REFERENCE CHEMICALS

1. Proficiency Chemicals: The responsiveness of cells to the proficiency chemicals was conducted in duplicate on differe nt days for each chemical. The reported responses are
summarized in Table 2.Estrogen Receptor Transc riptional Activation (2012) / Page 7 of 16
CYPERMETHRIN/109702 OCSPP 890.1300/ OECD 455

TABLE 2. Proficiency Chemicals

Compound Expected Response Lab Response

Run 1 Run 2 Run 3 Diethylstilbestrol Positive Positive Positive NA

17 α -Ethynyl estradiol Pos itive Positive Positive NA

Hexestrol Positive Positive Positive NA

Genistein Positive Positive Positive NA
Estrone Positive Positive Positive NA Butyl paraben Positive Positive Positive NA 1, 3, 5-Tris(4-hydroxyphenyl)benzene Positive NA Positive Positive Dibutyl phthalate Negativ e Negative Negative NA Atrazine Negative Ne gative NA Negative

Corticosterone Negative Negative NA Negative NA = not applicable. The chemical was not tested at this time.



### 2. Reference Chemicals: The response curves for the th ree estrogenic reference chemicals are shown in Figures 1-3, and the values de rived from the concentr ation response curves ( e.g ., Log PC 50 

### , Log PC 10 

### , Log EC 50 

### , Hill slope) for the concurre ntly run reference materials are presented in Table 3.

Although performance criteria we re generally met, the data were extremely variable between runs. In Run 1, cytotoxicity wa s observed at the high concentration (10 -4 

### ) for corticosterone, the maximum responses of 17 β -estradiol, 17 α -estradiol and 17 α

methyltestosterone (124-150%) exceeded the response of the PC (1 nM 17 β -estradiol), and performance criteria parameters for 17 α -methyltestosterone were lower than the guideline range, indicating an increased sensitivity to w eak agonists (does not negatively impact the validity of Run 1). In Run 2, cytotoxicity wa s observed at the high c oncentrations of both
corticosterone and 17 α -methyltestosterone, and Log PC 50 

### values could not be determined for
17 α -methyltestosterone as it did not reach 50 % activation at concentrations up to 10 -5 

### M. In Run 3, the maximum response of both 17 β -estradiol and 17 α -estradiol exceeded (124-144%) the response of the PC.Estrogen Receptor Transc riptional Activation (2012) / Page 8 of 16
CYPERMETHRIN/109702 OCSPP 890.1300/ OECD 455

TABLE 3. Performance Criteria for Reference Chemicals a

Reference Chemical
Parameter Acceptable Range Values Acceptable

Run 1 Run 2 Run 3 Yes No

17 β -estradiol Log PC 50 − 11.4 to

− 10.1 -11.7 b -10.2 -10.6 Run 1 Log PC 10 <

− 11 -12.6 -11.7 -11.5 X Log EC 50 − 11.3 to

− 10.1 -11.7 b -10.3 -10.5 Run 1 Hill Slope 0.7 to 1.5 1.1 0.8 1.3 X Test range 10

− 14 to 10

− 8 M 10

− 15 to 10

− 8 10

− 15 to 10

− 8 10

− 15 to 10

− 8 X

17 α -estradiol Log PC 50 − 9.6 to

− 8.1 -9.4 -8.6 -8.7 X Log PC 10 − 10.7 to

− 9.3 -10.4 -9.8 -9.7 X Log EC 50 − 9.6 to

− 8.4 -9.3 -8.7 -8.7 X Hill Slope 0.9 to 2.0 1.0 1.2 1.2 X Test range 10

− 12 to 10

− 6 M 10

− 13 to 10

− 6 10

− 13 to 10

− 6 10

− 13 to 10

− 6 X

Corticosterone Test range 10

− 10 to 10

− 4 M 10

− 11 to 10

− 4 10

− 11 to 10

− 4 10

− 11 to 10

− 4 X

17 α -methyltestosterone Log PC 50 − 6.0 to

− 5.1 -8.8 b NC c -5.1 Run 1,2 Log PC 10 − 8.0 to

− 6.2 -9.7 b -6.6 -6.2 Run 1 Test range 10

− 11 to 10

− 5 M 10

− 12 to 10

− 5 10

− 12 to 10

− 5 10

− 12 to 10

− 5 X a Data were obtained from pages 12 and 24 of the study report. b Log PC 50 , EC 50, and PC 10 estimates were below the expected range in Run 1. c Log PC 50 estimates was not reached in Run 2.
NC Not CalculatedEstrogen Receptor Transcriptional Activation (2012) / Page 9 of 16
CYPERMETHRIN/109702 OCSPP 890.1300/ OECD 455



### FIGURE 1. Relative Transcription Acti vation (RTA) of the Natural Ligand, 17 β

estradiol, Compared to the Positive Control, 1 nM 17 β estradiol.

Mean ± SD are presented (n=6).Estrogen Receptor Transcriptional Activation (2012) / Page 10 of 16
CYPERMETHRIN/109702 OCSPP 890.1300/ OECD 455



### FIGURE 2. Relative Transcription Acti vation (RTA) of the Weak Estrogen, 17 α

estradiol, Compared to the Positive Control, 1 nM 17 β estradiol.

Mean ± SD are presented (n=6).Estrogen Receptor Transcriptional Activation (2012) / Page 11 of 16
CYPERMETHRIN/109702 OCSPP 890.1300/ OECD 455



### FIGURE 3. Relative Trans cription Activation (RTA) of the Very Weak Estrogen, 17 α

methyltestosterone, Compared to the Positive Control, 1 nM 17 β estradiol.

Mean ± SD are presented (n=6).Estrogen Receptor Transc riptional Activation (2012) / Page 12 of 16
CYPERMETHRIN/109702 OCSPP 890.1300/ OECD 455



### C. DEFINITIVE ASSAY

1. Vehicle and Positive Controls: Data for the vehicle and pos itive controls are included in
Table 4. The overall mean TA value fo r the vehicle control was 16379-400558, and the overall mean TA value for the positive control was 1010892-2373950. The induction for the positive control ranged from 5.9- to 73-fol d. The mean normalized value for the positive
control was 974775-1973392. The PC 50 

### (50% of the maximum response) for E2 in this assay is 487397-986696 and the PC 10 

### (10% of the maximu m response) is 97477-197339.

TABLE 4. Transcriptional Activation (TA) Response of Vehicle and Positive Control a

Sample Vehicle Control Positive Control b Normalized Positive Control b Positive Control b

Fold Induction c Runs Mean SD Mean SD Mean SD

1 400558 44937 2373950 302859 1973392 302859 4.9

2 16379 5100 1188858 165141 1172479 165141 71.6

3 36117 10435 1010892 220760 974775 220760 27 a Data were calculated by the reviewers from da ta obtained on pages 35-37of the study report.
b Positive control was 17 β -estradiol (E2) at 1 nM. c Fold-induction = (mean TA of PC)/(mean TA of VC)



### 2. Test Material: Relative (to the PC) transcriptional ac tivation at each concentration of the test chemical during the three assay runs is presented in Table 5. The concentrationresponse curves depicting fold induction of relative transcriptional activation are presented in Figure 4 below. For Run 1, the mean RPC Max 

### was 16.3%, and the associated PC Max 

### was 10 -4 

### M. For Runs 2 and 3, each conducted with a different batch of frozen cells, the RPC Max 

### was <PC 10 

### in both runs. Given that a new cell line was brought out of frozen stock after a positive response, the performance of those ce lls indicate inadequate response to the reference chemicals in general, and a weaker than acceptable response to the very weak agonist, 17 α -methyltestosterone, Runs 2 and 3 are not valid. The PC 10 

### for Run 1 was
10 -4.6 

### M.Estrogen Receptor Transc riptional Activation (2012) / Page 13 of 16
CYPERMETHRIN/109702 OCSPP 890.1300/ OECD 455

TABLE 5. Relative Transcriptiona l Activation (RTA) of Cypermethrin a

Parameter RTA (mean ± SD); % of Positive Control (PC)

Run 1 Run 2 Run 3 Conc. (M) Mean SD Mean SD Mean SD

10

− 3.3 -- -- 0.4 0.4 -0.2 0.6

10

− 4 16.3 2.4 -- -- -- --

10

− 4.3 -- -- 0.9 1.0 -0.3 1.0

10

− 4.5 12.7 1.2 -- -- -- --

10

− 5 3.4 2.5 -- -- -- --

10

− 5.3 -- -- 0.4 0.3 0.4 0.6

10

− 5.5 -0.1 3.0 -- -- -- --

10

− 6 -6.4 2.3 -- -- -- --

10

− 6.3 -- -- 0.7 0.6 0.6 0.4

10

− 6.5 -6.5 2.5 -- -- -- --

10

− 7 -8.1 2.6 -- -- -- --

10

− 7.3 -- -- 0.0 0.7 -0.2 0.5

10

− 7.5 -6.0 3.3 -- -- -- -- 10

− 8.3 -- -- -0.2 0.3 0.6 0.6

10

− 9.3 -- -- -0.2 0.4 0.1 0.7

10

− 10.3 -- -- -0.1 0.2 -0.1 0.8
Log EC 50 b NA NA NA Hill Slope b NA NA NA

RPC Max 16.3 0.9 0.6

PC Max 10 -4 10

− 4.3 10

− 6.3

PC 50 NA NA NA

PC 10 10 -4.6 NA NA a Data were obtained from pages 18 and 21-23 of the study report. b Report if appropriate NA = Not ApplicableEstrogen Receptor Transcriptional Activation (2012) / Page 14 of 16
CYPERMETHRIN/109702 OCSPP 890.1300/ OECD 455



### Figure 4. Fold Induction of Relative Transcription Activation (RTA) of Cypermethrin Compared to the Positive Control.Estrogen Receptor Transc riptional Activation (2012) / Page 15 of 16
CYPERMETHRIN/109702 OCSPP 890.1300/ OECD 455



### 3. Performance Criteria: Laboratory proficiency assays using the required reference compounds were not included in th e study report. While the Log PC 50 

### , Log PC 10 

### , Log EC 50, 

### and Hill slope values for the concurrent re ference chemicals fell within or near the acceptable ranges (Table 3), the full response of the cells to the reference chemicals was not satisfactory, as detailed above in Section B.2. For 17 α -methyltestosterone, the mean
RPC Max 

### was 124.3% for the first run, 16.5% for the second run, and 52.8% for the third run.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: The suitable top concentration of cypermethrin for use in the transcriptional activation assays was 10 -4 

### M identified in the range finder and all three independent runs. There was no cy totoxicity observed at any of the exposure
concentrations tested. In th ree independent runs of the tr anscriptional activation assay, cypermethrin resulted in an increase in luci ferase activity in the third run to 12.7% at
10 -4.5 

### M and 16.3 % at 10 -4 

### M. This results in a PC 10 

### value of -4.6 Log[M]. The fourth and eighth runs showed no increase in luciferase activity at any of the concentrations tested.

B. AGENCY COMMENTS: Cypermethrin was tested up to the limit of solubility, 10 -4 

### M, as precipitation was observed at 10 -3 

### M in the range finding study. The RPC Max 

### was 16.3% at
10 -4 

### M and the PC 10 

### was 10 -4.6 

### M in Run 1, indicating a positive response to the assay. However, in Runs 2 and 3 the RPC Max 

### for cypermethrin was ≤ 0.9% at concentrations up to
10 -3.3 

### M, indicating that cypermethrin was ne gative for estrogenic activity under the conditions of the assay. Review of the perfor mance criteria parameters for Run 1 (Table 3) shows that Run 1 was unusually sensitive, with a ll parameters for all re ference chemicals at lower levels than expected according to the guidance.

In all runs, cypermethrin response was si milar to that of the negative control (corticosterone), and in no run did it reach levels similar to the weak estrogen 17 α

methyltestosterone. For Run 2, Log PC 50 

### values could not be determined for 17 α

methyltestosterone as it di d not reach 50% activation at concentrations up to 10 -5 

### M. This indicates a decreased sensitivity of cells. Howeve r in that same run, responses were close to zero at all cypermethrin concentrations.

Although the conditions of this assay were not optimal, in general cypermethrin responses were similar to those of the negative control corticosterone and not comparable to the responses of 17 α -methyltestosterone, which was able to reach maximums of 124.3, 16.5, and 52.8% PC in Runs 1, 2, and 3, respectivel y. Cypermethrin was only able to reach maximums of 16.3, 0.9, and 0.6% PC in Runs 1, 2, and 3, respectively, when tested up to the highest concentration possible base d on cytotoxicity. Because the RPC Max 

### < PC 10 

### in both assay runs, cypermethrin was considered negative for estrogen receptor transcriptional activation in this test system.

C. STUDY DEFICIENCIES: The following deficiencies were noted:

 Cytotoxicity was observed in cells treated with two of the reference compounds (corticosterone and 17 α -methyltestosterone) in two of the three runs. Cytotoxicity should not be observed at any leve l for the reference chemicals.Estrogen Receptor Transc riptional Activation (2012) / Page 16 of 16
CYPERMETHRIN/109702 OCSPP 890.1300/ OECD 455



###  The maximum response of 17 β -estradiol, 17 α -estradiol, and 17 α -methyltestosterone in Runs 1 and/or 3 exceeded the response of the PC (1 nM 17 β -estradiol). The maximum responsiveness should be observed at 1 nM 17 β -estradiol in both the PC samples and reference compound samples.

 The cells responded inadequately (<50% RTA) to the very weak estrogen,
17 α -methyltestosterone in Run 2.

 Certificates of Analysis or information re garding supplier, Catalog #, Lot/Batch #, and purity, were not pr ovided for the Reference Chemicals.

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

EPA MRID Number 48683001

Page 1 of 82 Data Requirement: EPA DP Barcode 401651

OECD Data Point 229

EPA MRID 48683001

EPA Guideline 890.1350, Fish Sh ort-Term Reproduction Assay

Test material: Cypermethrin Purity: 95.2%

Common name

Chemical name: IUPAC: mixture of the stereoisomers (S)- α -cyano-3-phenoxybenzyl (1RS,3RS;1RS,3SR)-3-

(2,2-dichlorovinyl)-2,2-dimethylcyclopr opanecarboxylate where the ratio of the

(S);(1RS,3RS) isomeric pair to the (S);(1RS ,3SR) isomeric pair lies in the ratio range

45-55 to 55-45 respectively

CAS name: (S)-cyano(3-phenoxypheny l)methyl 3-(2,2-dichloroethenyl)-2,2-

dimethylcyclopropanecarboxylate.

CAS No.: 52315-07-8

Synonyms: FMC 30980

EPA PC Code: 109703

Primary Reviewer: Joan Gaidos Signature:

Senior Scientist, Cambridge Environmental, Inc. Date: 1/07/13

Secondary Reviewer: Teri S. Myers Signature:

Program Manager, CDM Smith Date: 2/25/13

Primary Reviewer: Elizabeth Donovan Date: 6/17/13

EPA/EFED/ERB6

Additional Reviewer(s): Justin Housenger, Biologist Date: 12/19/13

EPA/EFED/ERB5 Digitally signed by Elizabeth
Donovan DN: cn=Elizabeth Donovan, o=EPA, ou=EFED, email=donovan.elizabeth@epa.g ov, c=US
Date: 2015.06.11 08:51:27 -04'00'

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

Date: 2015.06.11 11:05:33
-04'00'

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

EPA MRID Number 48683001

Page 2 of 82 Additional Reviewer(s): Amy Blankinship, Chemist Date:

EPA/EFED/ERB6

Date Evaluation Completed: 6/6/15

CITATION: York, D.O. 2012. Cypermethrin – Fish Short-term Reproduction Assay with the Fathead Minnow ( Pimephales

promelas ). Performed by Smithers Viscient, Wareham, Massachu setts, Laboratory Project No. 1781.6766. Submitted

by Syngenta Ltd, Jealott’s Hill International Research C entre, Bracknell, Berkshire RG42 6EY, United Kingdom; FMC

Corporation, Philadelphia, Pennsylvania; United Phosphorus , Inc., King of Prussia, Pennsylvania. Completion date

April 18, 2012

.

Note: The US EPA Endocrine Disruptor Screening Program (E DSP) Tier 1 screening batte ry is comprised of eleven

screening assays intended to identify a chemic al’s likely endocrine bioactivity, i.e., its potential to interact with the estro gen,
androgen, or thyroid (E, A, or T) pathways. The robustness of t he Tier 1 battery is based on the strengths of each individual

assay to identify potential endocrine bioacti vity with complementary endpoints with in the assay, where available, and

redundancy across the battery. Thus, the re sults 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 Ot her Scientifically Relevant Information

(OSRI). In order to determine if a chemical has the potential to interact with the E, A or T pat hways, 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).

Guideline recommendations are provided in italics; th ese recommendations should remain visible in the

completed DER.

Disclaimer: The guideline recommendations in this DER template are offered as a general reference to aid in

preparation of the DER. The purpose of these recommenda tions is not to serve as substitute for the Test

Guidelines, nor to provi de any guidance on how the study should be conducted. 

## AMY
BLANKINSHIP Digitally signed by AMY BLANKINSHIP DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=AMY BLANKINSHIP, dnQualifier=0000040917
Date: 2015.06.15 17:32:30 -04'00'

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

EPA MRID Number 48683001

Page 3 of 82 EXECUTIVE SUMMARY:

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

conducted under flow-through conditi ons. Adult fish (16 spawning groups , 2 males and 4 females in each

group; 5 months old) were exposed to cypermethrin (95.2% purity) at nominal concentrations of 0 (control),

0.000030, 0.00030, and 0.003 mg a.i./L; mean-m easured concentrations were <LOQ (<0.000001),

0.000013, 0.00012, and 0.0014 mg a.i./L. The test syst em was maintained at 25 to 26°C and a pH of 7.0

to 7.7.

There were no significant differences in either male or female survival at any treatment levels compared to the

negative control. Percent survival was 100% for all groups of males and was 10 0, 94, 94, and 88% for the

negative control and low, mid, and high treatment concent rations, respectively, for females. During the in-life

exposure, there were no notable observations that oc curred with regards to behavior, coloration/banding,

changes in ovipositor appearance, or size of the dorsa l nape pad, and no clinical signs of toxicity were

observed. There were no treatment-related effect s on male and female body weight and length.

Spawning occurred at least every 4 days in thee of the four control replicates and mean fecundity in control

averaged 14 eggs/female/day/replicate (range: 13-18 eggs /female/day among the replicates); fertilization

success in the control group averaged 96%. Fecundity was decreased (p<0.05) by 42% and 84% in the midand high-treatment females, respectively. There were no treatment-related effects (p>0.05) on fertilization

success.

Male gonadal somatic index (GSI) was increased (p<0 .05) by 22 and 27% at the mid and high treatment

concentrations, respectively, compared to the negative control. There were no treatment-related effects on

female GSI (p>0.05) or male and female plasma vitello genin (VTG). Additionally, there were no significant

effects on median male tubercle score, and no tubercles were observed on any females.

Although not analyzed statistically, in male fish , there were only background or sporadic gonadal

histopathological findings noted that were not consider ed related to treatment. In female fish, there was an

increased incidence of mature oocyte atresia at the high treatment concentration, which was associated with

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

EPA MRID Number 48683001

Page 4 of 82 granulomatous inflammation and the presence of micros poridia in half the observed cases across all treatment

levels. There were no significant findings observed in the gonadal stage for either males or females, and no

notable observations in secondary sex characteristics for any treatment group compared to the controls.

The performance criteria for fecundity was not met. There were one replicate in the control group (Replicate

C) where there was a period of 4 days in which no spaw ning occurred and fecundity was 13 eggs/female/day.

Therefore, this replicate did not meet the criteria of either at least 15 eggs/female/day or spawning at least

every 4 days. However, it is noted that the overall cont rol mean fecundity value (14) was just slightly below

15 eggs/female/day, and the other two replicates did achieve 15 or greater eggs/female/day and the

remaining replicate spawned at least every 4 days (11 e ggs/female/day). Also, the %CV values for the mean

recoveries at the two highest treatment concentrations were 45.6 and 51.2%, which did not meet the validity

criteria of <20% over the 21-day test. There was no pattern of decline during the study period and no undissolved test substance was observed in the dilution system , as such, the recoveries indicate the test material

was generally poorly recovered in so lution under the te st conditions. Given the physicochemical properties of

cypermethrin, these results may be considered consist ent with expectations for the chemical and previous

experience with the test item. The remaining validity cr iteria for OCSPP 890.1350 were met. Therefore, while

these deviations are noted, they did not sufficiently impact the interpretation of the assay results.

There was one fish that was inadvertently counted as a female but later discovered to be a male. This occurred

in one of the control replicates and data from that replicate were subsequently reanalyzed for the gender

specific endpoints of male GSI, ma le VTG, body weight, and length. Th is did not have an impact on the

conclusions of the study as male GSI remained the only ma le gender specific endpoint with significant effects.

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: ca . 5 months

Mean body weight at test initiation (if m easured): Male 3.2 g; Female 1.7 g

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

EPA MRID Number 48683001

Page 5 of 82 Mean length at test initiation (if measured): Not reported.

Test Type (Flow-through, Static, Static Renewal): Flow-throughData Evaluation Record on the Fish Short-Term Reproduction Assay with CypermethrinEPA MRID Number 48683001Page 6 of 82Table 1: Summary of Reproductive and HPG Effects1,2in the Fish Short-Term Reproduction Assay (FSTRA) with Cypermethrin.Treatment(mg a.i./L)[meanmeasured]FecundityFert.SuccessTubercle ScoreGSIGonadal Histo.Plasma VTGPlasma TPlasma E2MFMFMFMFMFMF0.000013NoNoNoNANoNoNANANoNoNANANANA0.00012YesNoNoNAYesNoNANANoNoNANANANA0.0014YesNoNoNAYesNoNANANoNoNANANANAAbbreviations:Conc.Concentration.Diff.Difference.E217β-estradiol.FFemale.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.MMale.NANot applicable.TTestosterone.VTGVitellogenin.1A “yes” indicates a significant difference based on comparison tothe negative (clean water) control, unless otherwise specified.2The criteria for significance are described in the Reviewer’sAnalysis and Statistical Verification sections of the DER. Conclusions regardinghistopathology may be heavily weighted by the expert opinion of a board-certified pathologist.

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

EPA MRID Number 48683001

Page 7 of 82 I. MATERIALS AND METHODS

GUIDELINE FOLLOWED: This study was conducted according to the U.S. EPA OCSPP

890.1350: “Fish Short-Term Repr oductive Assay” and OECD 229

(2009). The following deviations were noted:

1. There were one replicate in the control group (Replicate C) where there was a

period of 4 days in which no spawni ng occurred, and fecundity was 13

eggs/female/day, respectively. Therefore, th is replicate did not meet the criteria

of either at least 15 eggs/female/day or spawning at least every 4 days.

However, it is noted that the overall control mean fecundity value (14) was just

slightly below 15 eggs/female/day, and the other two replicates did achieve 15

or greater eggs/female/day and the rema ining replicate spawned at least every

4 days (11 eggs/female/day).

2. Analytical verification of the test material from Days 0, 6, 11, 19 and 21 yielded

recoveries of 44% to 67% of nominal concentrations. Results of these analyses

indicate measured concentrations where generally lower than nominal, but

according to the study author were c onsistent with expectations for the

physicochemical properties and previous experience with the test item. Therefore,

although there was no pattern of decline during the study period and no undissolved test substance was observed in the dilution system, the recoveries

indicate the test material was generally poorly recovered in solution under the

test conditions. The %CV values for th e mean recoveries at the two highest

treatment concentrations were 45.6 and 51 .2%, which did not meet the validity

criteria of <20% over the 21-day test. The test material was detected on Days

11 (one replicate) and 19 (two replicat es) in the control samples. The study

author’s concluded the contamination was likely a result of processing samples

after being removed from the exposure system and that the control fish were not

exposed to cypermethrin.

3. The physico-chemical properties of the test material were not reported.

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

EPA MRID Number 48683001

Page 8 of 82 4. The unionized ammonia and resi dual chlorine in the test water were not reported.

The OCSPP 890.1350 performance criteria establish maximum levels for these

values and it is unclear if the ma ximum recommendations were exceeded.

5. Incorrect sexing of individuals in one rep licate from the contro l group resulted in

there being three males and three females in this replicate instead of the two

males and four females specified in the OCSPP 890.1350 guideline.

These deviations do not sufficiently im pact the acceptability of the study.

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

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

pertinent U.S. EPA Good Laboratory Practice regulations with the following

exceptions: routine dilution water and food contaminant screening analyses

were conducted following standard validat ed methods. This exception has no

impact on the study results.

A. TEST MATERIAL: Cypermethrin; CAS# 52315-07-8.

Description: Amber liquid.

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

Kow, vapor pressure of test compound, expiration date.

Lot No./Batch No. : PL10-0492

Purity: 95.2%

Impurities: None reported

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

EPA MRID Number 48683001

Page 9 of 82 Stability of Compound: Analytical verification of the test material from Days 0, 6, 11, 19 and 21

yielded recoveries of 44% to 67% of nominal concentr ations. Stock solution (FETAX) fortified with

cypermethrin at concentrations of 0.000003, 0.00005, and 0.0005 mg a.i./L yielded recoveries of

80.9 to 96.8% of nominal concen trations. Quality control samples (n=17) fortified at 0.000015,

0.0003, and 0.003 mg a.i. /L yielded recoveries of 83.0 to 118% of nominal concentrations.

Results of these analyses indicate measured concen trations where generally lower than nominal, but

according to the study author were consistent with expectations for the physicochemical properties

and previous experience with the test item. Theref ore, although there was no pattern of decline

during the study period and no un-dissolved test su bstance was observed in the dilution system, the

recoveries indicate the test material was genera lly poorly recovered in solution under the test

conditions. Additionally, the %CV values were 45 .6 and 51.2% for the 0.00012 and 0.0014 mg

a.i./L treatment concentrations, respectively, and di d not meet the validation criteria of <20%.

Storage Conditions of Test Chemicals: Stored at room temperature in a dark, ventilated cabinet.Data Evaluation Record on the Fish Short-Term Reproduction Assay with CypermethrinEPA MRID Number 48683001Page 10 of 82B. Test organism:Table 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 asingle laboratory stock?Yes EPA recommends that fish be from a single
laboratory stock.Were acclimation conditionssame as definitive test?Yes EPA recommends that fish be acclimated under water quality and illumination conditions that are similar to the definitive

test.Acclimation period:16 days 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 CypermethrinEPA MRID Number 48683001Page 11 of 82ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on health:No mortalities were reported during the7 days prior to pre-exposure period.Fish did not receive any treatment fordisease in the two weeks prior tospawning (pre-exposure period).Behavioral abnormalities or clinical signswere not reported. 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.Data Evaluation Record on the Fish Short-Term Reproduction Assay with CypermethrinEPA MRID Number 48683001Page 12 of 82ParameterValue(s)Details or RemarksGuideline RecommendationsType of food:Frozen brine shrimp EPA recommends that fish be fed frozen brine shrimp twice per day to promote active reproduction and maintain body

condition.Source of food:Not reportedRepresentative samples of the food sourcewere periodically analyzed for pesticides,PCBs, and toxic metals by GeoLabs, Inc.and analyte levels were below LOC specifiedby ASTM (2005).Frequency of feeding:2 times/dayDetails on feeding:None.Table 3: Fish Selection and Pre-Exposure Performance.ParameterValue(s)Details or RemarksGuideline RecommendationsAge at test initiation: Ca. 5 monthsCa. 20 weeks old EPA recommends reproductively mature (sexually dimorphic) fish, 4.5 - 6 months

old.Data Evaluation Record on the Fish Short-Term Reproduction Assay with CypermethrinEPA MRID Number 48683001Page 13 of 82ParameterValue(s)Details or RemarksGuideline RecommendationsMean weight of males at testinitiation (if determined):3.2 gBased on 20 males used to stockaquaria for pre-exposure phase. 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):2.8 to 3.7Individual weights within ±20% of theestimated mean.Mean weight of females at testinitiation (if determined):1.7 gBased on 20 females used to stockaquaria for pre-exposure phase.Range of individual weights(females) at test initiation (ifdetermined):1.3 to 2.0Individual weights within ±20% of theestimated mean.Mean length of males at testinitiation (if determined):Not reportedMean length of females at testinitiation (if determined):Not reportedDuration of pre-exposure phase:16 days EPA recommends a minimum of 14 days.Data Evaluation Record on the Fish Short-Term Reproduction Assay with CypermethrinEPA MRID Number 48683001Page 14 of 82ParameterValue(s)Details or RemarksGuideline RecommendationsWere 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.Number of pre-exposure tanks:36Extra replicates were established toaccount for a potential lack of spawninginsome chambers and/or mortalityduring this phase. 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:>15 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.Data Evaluation Record on the Fish Short-Term Reproduction Assay with CypermethrinEPA MRID Number 48683001Page 15 of 82ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of spawns during preexposure:>two times in 7 days EPA recommends that spawning occur at least twice in the 7 days prior to the
definitive test.Details on pre-exposure:None.Data Evaluation Record on the Fish Short-Term Reproduction Assay with CypermethrinEPA MRID Number 48683001Page 16 of 82C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:Flow-throughEPA recommends the use of a flowthrough system. As noted in the Corrections and Clarifications document 1 , the use of a static renewal system is not recommended for this assay.Type of flow-through dilutionsystem:Intermittent flow proportionaldiluters Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 21U.S. Environmental Protection Agency (EPA).(2011). Corrections and Clarificationson Technical Aspects of the Test Guidelines for the Endocrine DisruptorScreening Program Tier 1 Assays (OCSPP TestGuideline 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).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 CypermethrinEPA MRID Number 48683001Page 17 of 82ParameterValue(s)Details or RemarksGuideline RecommendationsFlow-through rate: Ca. 45 mL/minute or 2.7L/hr and 6.6 volumereplacements/day.Calculated by the reviewer based on 0.5L/cycle, 131 cycles/24 hrs and 60minutes/hr. 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:Flow-splitting chambers were usedbetween diluter cellsand the 4 replicatetest vessels.Treatment recoveries were not measuredprior to test initiation to indicate if diluterhad reached equilibrium.Flow splitting accuracy not reported.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%.Aeration?None reported. EPA recommends aeration if dissolved oxygen reaches <4.9 mg/L (< 60%

saturation).Data Evaluation Record on the Fish Short-Term Reproduction Assay with CypermethrinEPA MRID Number 48683001Page 18 of 82ParameterValue(s)Details or RemarksGuideline RecommendationsSource of dilution water:Well water 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 analyzedfor pesticides, heavy metals,and other contaminants?YesTest vessel type/materials:Glass aquaria withsilicone adhesive.EPA 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 CypermethrinEPA MRID Number 48683001Page 19 of 82ParameterValue(s)Details or RemarksGuideline RecommendationsTest vessel size:20 cm wide x 39 cmlong x 25 cm high 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:4 inch diameter agedPVC cutlengthwise inhalf (arches) and into 7cm sections. 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:Aged PVC cut in half(arches) and into 7 cmsections.Additional details on exposuresystem:None.Data Evaluation Record on the Fish Short-Term Reproduction Assay with CypermethrinEPA MRID Number 48683001Page 20 of 82Table 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsTemperature (°C)25 26 25.5 Continuously EPA recommends temperature 25±1 o C;

inter -replicate and inter-treatment differentials should not exceed 1 o C.pH7.07.77.35Weekly EPA recommends pH 6.5 to 9.0.Dissolved oxygen (mg/L)4.9 (61%)8.2 (98%)6.6 (79.5%)Weekly EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air saturation)Total alkalinity (mg/L asCaCO3)20 26 23Weekly EPA recommends total alkalinity >20 mg/L as CaCO 3.Hardness (mg/L as CaCO3)486054WeeklyTotal organic carbon (mg/L)0.30 0.42 0.36 Twice EPA recommends that total organic carbon in dilution water be <2 mg/L.Unionized ammonia (Pg/L)Not reportedNot reportedNot reportedNot reported EPA recommends that unionized ammonia in the dilution water be <1

P g/L.Data Evaluation Record on the Fish Short-Term Reproduction Assay with CypermethrinEPA MRID Number 48683001Page 21 of 82ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsResidual chlorine (Pg/L)Not reportedNot reportedNot reportedNot reported EPA recommends that residual chlorine in dilution water be <10 P g/L.Conductivity (Pmhos/cm) 260 340 300 Weekly 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 CypermethrinEPA MRID Number 48683001Page 22 of 82D. 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 conducti ng 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 rangefinder?14 day LC50valuerange finding study.Nominal test concentrations of 0(negative and acetone controls),0.00063, 0.0013, 0.0025, 0.0050 and0.010 mg/L. 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 reported EPA recommends that range-finding tests be performed with fish of similar age and size to those that w ould be utilized in the

test.Data Evaluation Record on the Fish Short-Term Reproduction Assay with CypermethrinEPA MRID Number 48683001Page 23 of 82ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:14 day EPA recommends a 96-hour exposure.Additional details:The test wasconducted under flowthrough conditions. OnDays 12 and 13, twofemale fish at thehighest treatment levelwere observed atsolution’s surface. Attest termination, 40%combined male andfemale mortality wasobserved at the highestlevel. No othermortalities or sub-lethaleffects were observed. EPA recommends conducti ng 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

concentrati on-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 CypermethrinEPA MRID Number 48683001Page 24 of 82Table 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 highesttestconcentration in thedefinitive test:The toxicity 14 dayLC50value wasestimated to be>0.010 mg/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., 1/3 the 96 -hour LC

50 ), whichever concentration is lowest.Reference study citation (ifapplicable):NASeparation of testconcentrations:0 (control), 0.00003,0.0003, and 0.003mg a.i./LStep down factor of 10 from highest testconcentration. EPA suggests that a concentration separation of between 0.33 (or three - fold)
and 0.1 (or ten -fold) is scientifically

acceptable 1 .Data Evaluation Record on the Fish Short-Term Reproduction Assay with CypermethrinEPA MRID Number 48683001Page 25 of 82ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of test concentrations:4 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:Mean measuredLimit of quantification (LOQ):<0.000001 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 Fish Short-Term Reproduction Assay with CypermethrinEPA MRID Number 48683001Page 26 of 82ParameterValue(s)Details or RemarksGuideline RecommendationsFrequency of measurement:0, 4, 6, 11, and 19days 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 topreexposure 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.Number of replicates in control:4 EPA recommends 4 replicates.Number of replicates in solventcontrol (if applicable):NA EPA recommends the us e of a concurrent solvent control when a solubilizing agent is used. EPA recommends 4 replicates.Data Evaluation Record on the Fish Short-Term Reproduction Assay with CypermethrinEPA MRID Number 48683001Page 27 of 82ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of replicates per testitem treatment level:4 EPA recommends 4 replicates.Number of male fish perreplicate at test initiation:2 EPA recommends 2 males per replicate.Number of female fish perreplicate at test initiation:4 EPA recommends 4 females per replicate.Was a solvent used?NoSolvent type (if applicable):NAMaximum solvent concentration(if applicable):NA 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 .3Hutchinson 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 Documenton Aquatic Toxicity Testing ofDifficult Substances and Mixtures. Environmental Health and Safety Publications. Series onData Evaluation Record on the Fish Short-Term Reproduction Assay with CypermethrinEPA MRID Number 48683001Page 28 of 82ParameterValue(s)Details or RemarksGuideline RecommendationsWas a positive control used?NoPositive control (if applicable):NAPositive control concentration(s)(if applicable):NAPhotoperiod:16 hrs light:8 hrs dark EPA recommends photoperiod 16:8

(light:dark).Light intensity at water’ssurface:550-1000 luxFluorescent bulbs EPA recommends light intensity 540 – 1080 lux (at water’s surface).Additional details:None.Testing and Assessment. No. 23. Paris, France.Data Evaluation Record on the Fish Short-Term Reproduction Assay with CypermethrinEPA MRID Number 48683001Page 29 of 82Table 8: Summary of Treatment Concentrations in theFish Short-Term Reproduction Assay with Cypermethrin.Treatment IDNominalConcentration(mg a.i./L)Mean MeasuredConcentration1(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsControl (dilutionwater only)<0.000001<LOQ NA EPA recommends that test item concentrations be maintained at a coefficient of variation (CV)≤ 20%.Solvent control (ifapplicable)NA NA NATreatment 10.000030.00001314.2± 0.0.0016Treatment 20.00030.0001245.6± 0.0546Treatment 30.0030.001451.2± 0.7096Diluter StockNANANAAbbreviations:CVCoefficient of variation.1 The mean measured values obtained by thestudy author are presented in the table andwere calculated using the actual analytical results and notthe rounded valuespresented. These values are different thanthe mean measured values obtained by the reviewer of 0.0000142, 0.00012 and 0.0014mg a.i./L.LOQ=0.000001 mg a.i./L.Data Evaluation Record on the Fish Short-Term Reproduction Assay with CypermethrinEPA MRID Number 48683001Page 30 of 82E. ObservationsBiological Endpoints: Survival, fecundity, fertilization success, andclinical signs were observed daily. At test termination(Day 21),secondary sex characterization (body color, pattern, body shape), body weight, length, tubercle score, gonadal stagingand histopathology, and plasma vitellogenin were evaluated.Were raw (individual) data provided?Yes EPA recommends that observations of surviv al, 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 17β -estradiol, if measured). Gonado-somatic in dex (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 limi ted to) hemorrhage, cessation of feeding, and other abnormal beha vior.Data Evaluation Record on the Fish Short-Term Reproduction Assay with CypermethrinEPA MRID Number 48683001Page 31 of 82II. RESULTS AND DISCUSSIONA.ResultsMean male survival values were 100% at all test levels, and female survival valueswere 100, 94, 94, and 88% in the mean-measured 0 (control),0.000013, 0.00012, and 0.0014 mg a.i./L treatment levels, respectively (Table 9). The mortalityrate in the water control was0%, therefore, thesurvival rate in the control group satisfied the minimum acceptable control value criteria of≥90% according to the USEPA OCSPP 890.1350guideline. During the in-life exposure, no notable observations occurred with regards to behavior, coloration/banding, changesin ovipositorappearance or size of dorsal nape pad.Data Evaluation Record on the Fish Short-Term Reproduction Assay with CypermethrinEPA MRID Number 48683001Page 32 of 82Table 9: Adult Fish Survival in Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[mean-measured]MalesFemalesN1# Surviving% Survivaln# Surviving% SurvivalControl (<LOQ)9910015151000.000013881001615940.00012881001615940.001488100161488Abbreviations:NANot applicable.1 Total number of fish at test initiation.LOQ=0.000001 mg a.i./L.Mean male body weight values were 3.10, 3.57, 3.60, and 3.18 g andfemale body weight values were1.45, 1.47, 1.26 and 1.28 g in the meanmeasured 0 (control), 0.000013, 0.00012 and0.0014 mg a.i./L treatment levels, respectively (Table 10). Mean male body lengthvalues were62.2, 64.1, 64.3, and 62.7 mm and female body length values were 50.5, 49.5, 48.5 and 50.5 mm in the mean-measured 0 (control),0.000013, 0.00012 and 0.0014 mg a.i./Ltreatment levels, respectively.Data Evaluation Record on the Fish Short-Term Reproduction Assay with CypermethrinEPA MRID Number 48683001Page 33 of 82Table 10: Size at Test Termination in Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[mean-measured]Body WeightLengthMalesFemalesMalesFemalesnMean(g)±SDnMean(g)±SDnMean(mm)±SDnMean(mm)±SDControl (<LOQ)43.100.3941.450.50462.22.44450.52.990.00001343.571.0441.470.33464.14.76449.52.520.0001243.601.2441.260.24464.35.89448.52.980.001443.181.2441.280.36462.73.66450.52.99Abbreviations:NANot applicable.NDNot determined.SDStandard deviation.LOQ=0.000001 mg a.i./L.

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

EPA MRID Number 48683001

Page 34 of 82 Mean fecundity values were 14, 13, 8.3 and 2.2 eg gs/female/day and fertilization success was 96, 97,

96 and 92% in the mean-measured 0 (control), 0. 000013, 0.00012 and 0.0014 mg a.i./L treatment

levels, respectively (Table 11). Replicate B of th e control group inadvertently contained three male and

three female fish due to a mis-sexed fish.

Table 11: Fecundity and Fertilization Success in Fathead Minnow ( Pimephales promelas)

.

Treatment

(mg a.i./L)

[meanmeasured] Fecundity 1 ± SD Fertilization

Success (%) 2 ± SD

Control

(<LOQ) 14 2.99 96 4.36

0.000013 13 3.00 97 0.5

0.00012 8.3 4.73 96 1.73

0.0014 2.2 0.88 92 6.65

Abbreviations: NA Not applicable. ND Not determined.

1 Fecundity is calculated as the number of eggs per su rviving female per reproduc tive day per replicate.

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

LOQ=0.000001 mg a.i./L.

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

EPA MRID Number 48683001

Page 35 of 82 Median male tubercle scores were 32.5, 37, 33 and 25 in the mean-measured 0 (control), 0.000013,

0.00012 and 0.0014 mg a.i./L treatment levels, resp ectively (Table 12). None of the surviving females

were found to have tubercles.

Table 12: Nuptial Tubercle Score in Fathead Minnow ( Pimephales promelas)

.

Treatment

(mg a.i./L)

[mean-measured] Males Females

n Median

Tubercle Score n Median

Tubercle Score

Control (<LOQ) 4 32.5 4 0

0.000013 4 37 4 0

0.00012 4 33 4 0

0.0014 4 25 4 0

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

LOQ=0.000001 mg a.i./L.

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

EPA MRID Number 48683001

Page 36 of 82 Median male GSI was 1.1, 1.2, 1.4 and 1.4% and mean female GSI was 12, 11, 11 and 14% in the

mean-measured 0 (control), 0.000013, 0.00012 and 0.0014 mg a.i./L treatment levels,

respectively (Table 13).

Table 13: Gonado-Somatic Index (GSI) in Fathead Minnow ( Pimephales promelas)

.

Treatment

(mg a.i./L)

[mean-measured] Males Females

n Median

GSI 1 (%) ±SD n Mean GSI 1

(%) ±SD

Control (<LOQ) 4 1.1 0.25 4 12 4.3

0.000013 4 1.2 0.19 4 11 2.2

0.00012 4 1.4 0.07 4 11 2.4

0.0014 4 1.4 0.22 4 14 1.6

Abbreviations: NA Not applicable.

1 Gonado-somatic index (%) is calcul ated as gonad weight (to the nearest 0. 1 mg) / body weight (mg) x 100.

LOQ=0.000001 mg a.i./L.

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

EPA MRID Number 48683001

Page 37 of 82 Median male gonadal stage was 2, 2, 2 and 3 and medi an female gonadal stage wa s 3, 3, 3 and 3 in the

mean-measured 0 (control), 0.000013, 0. 00012 and 0.0014 mg a.i./L treatment levels, respectively (Table

14).

Table 14: Gonadal Staging in Fathead Minnow ( Pimephales promelas)

.

Treatment

(mg a.i./L)

[mean-measured] Males Females

n Median Stage 1 n Median Stage 2

Control (<LOQ) 4 2 4 3

0.000013 4 2 4 3

0.00012 4 2 4 3

0.0014 4 3 4 3

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

1 The guideline recommends the following gonadal stagi ng 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 developm ent/hydrated, 5=post-ovulatory.

LOQ=0.000001 mg a.i./L.

In male fish, there were only background or sporad ic findings noted and were not related to treatment

(Tables 15-16). In female fish, there was an increased incidence of mature oocyte atresia at the 0.0014 mg

a.i./L treatment, which was associated with granulomatous inflammation and the presence of microsporidia in

half the observed cases across all treat ment levels (Tables 17-18).

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

EPA MRID Number 48683001

Page 38 of 82 Table 15: Gonadal Histopathology in Male Fathead Minnow ( Pimephales promelas)

.

Treatment

(mg a.i./L)

[meanmeasured] Diagnostic Observations 1

Severity Increased

Proportion of

Spermatogonia Presence of

Testis-Ova Increased

Testicular

Degeneration Duct

Mineralization Interstitial cell

(Leydig)

hypertrophy/hype

rplasia

n Incidence n Incidence n Incidence n Incidence n Incidence

Control

( <LOQ) 0 9 7 9 8 9 8 NA NA NA NA

1 9 1 9 1 9 1 NA NA NA NA

2 9 1 9 0 9 0 NA NA NA NA

3 9 0 9 0 9 0 NA NA NA NA

4 9 0 9 0 9 0 NA NA NA NA

0.000013 0 8 8 8 8 8 7 NA NA NA NA

1 8 0 8 0 8 0 NA NA NA NA

2 8 0 8 0 8 1 NA NA NA NA

3 8 0 8 0 8 0 NA NA NA NA

4 8 0 8 0 8 0 NA NA NA NA

0.00012 0 8 6 8 8 8 8 NA NA NA NA

1 8 1 8 0 8 0 NA NA NA NA

2 8 1 8 0 8 0 NA NA NA NA

3 8 0 8 0 8 0 NA NA NA NA

4 8 0 8 0 8 0 NA NA NA NA

0.0014 0 8 8 8 8 8 8 NA NA NA NA

1 8 0 8 0 8 0 NA NA NA NA

2 8 0 8 0 8 0 NA NA NA NA

3 8 0 8 0 8 0 NA NA NA NA

4 8 0 8 0 8 0 NA NA NA NA

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=0.000001 mg a.i./L.Data Evaluation Record on the Fish Short-Term Reproduction Assay with CypermethrinEPA MRID Number 48683001Page 39 of 82Table 16: Additional Gonadal HistopathologyObservations in Male Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[mean-measured]Additional Diagnostic Observations1SeverityDecreasedProportion ofSpermatogoniaIncreased Vascularor InterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportionsof Spermatocytesor SpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidenceControl (<LOQ)0NANANANANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANA4NANANANANANANANANANA0.0000130NANANANANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANA4NANANANANANANANANANAData Evaluation Record on the Fish Short-Term Reproduction Assay with CypermethrinEPA MRID Number 48683001Page 40 of 82Treatment(mg a.i./L)[mean-measured]Additional Diagnostic Observations1SeverityDecreasedProportion ofSpermatogoniaIncreased Vascularor InterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportionsof Spermatocytesor SpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidence0.000120NANANANANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANA4NANANANANANANANANANA0.00140NANANANANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANA4NANANANANANANANANANA1Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E ofthe test guideline for reference.LOQ=0.000001 mg a.i./L.

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

EPA MRID Number 48683001

Page 41 of 82 Table 17: Gonadal Histopathology in Female Fathead Minnow ( Pimephales promelas)

.

Treatment

(mg a.i./L)

[meanmeasured] Additional Diagnostic Observations 1

Severity Increased Oocyte

Atresia Perifollicular Cell

Hyperplasia/

Hypertrophy Decreased Yolk

Formation Aggregates of

macrophages,

multifocal

n Incidence n Incidence n Incidence n Incidence

Control

(<LOQ ) 0 15 15 NA NA NA NA NA NA

1 15 0 NA NA NA NA NA NA

2 15 0 NA NA NA NA NA NA

3 15 0 NA NA NA NA NA NA

4 15 0 NA NA NA NA NA NA

0.000013 0 15 13 NA NA NA NA NA NA

1 15 2 NA NA NA NA NA NA

2 15 0 NA NA NA NA NA NA

3 15 0 NA NA NA NA NA NA

4 15 0 NA NA NA NA NA NA

0.00012 0 15 14 NA NA NA NA NA NA

1 15 1 NA NA NA NA NA NA

2 15 0 NA NA NA NA NA NA

3 15 0 NA NA NA NA NA NA

4 15 0 NA NA NA NA NA NA

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

EPA MRID Number 48683001

Page 42 of 82 Treatment

(mg a.i./L)

[meanmeasured] Additional Diagnostic Observations 1

Severity Increased Oocyte

Atresia Perifollicular Cell

Hyperplasia/

Hypertrophy Decreased Yolk

Formation Aggregates of

macrophages,

multifocal

n Incidence n Incidence n Incidence n Incidence

0.0014 0 14 7 NA NA NA NA NA NA

1 14 1 NA NA NA NA NA NA

2 14 3 NA NA NA NA NA NA

3 14 3 NA NA NA NA NA NA

4 14 0 NA NA NA NA NA NA

1 Gonadal histopathology diagnostic observ ations are graded 0 – 4 based on seve rity: 0=Not remarkable, 1=Minimal,

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

LOQ=0.000001 mg a.i./L.

Table 18: Additional Gonadal Histopathology Observations in Female Fathead Minnow ( Pimephales

promelas)

.

Treatment

(mg a.i./L)

[meanmeasured] Additional Diagnostic Observations 1

Severity Interstitial fibrosis Egg Debris in

Oviduct Granulomatous

Inflammation Decreased PostOvulatory Follicles

n Incidence n Incidence n Incidence n Incidence

Control

( <LOQ) 0 NA NA 15 13 15 14 NA NA

1 NA NA 15 1 15 1 NA NA

2 NA NA 15 1 15 0 NA NA

3 NA NA 15 0 15 0 NA NA

4 NA NA 15 0 15 0 NA NA

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

EPA MRID Number 48683001

Page 43 of 82 Treatment

(mg a.i./L)

[meanmeasured] Additional Diagnostic Observations 1

Severity Interstitial fibrosis Egg Debris in

Oviduct Granulomatous

Inflammation Decreased PostOvulatory Follicles

n Incidence n Incidence n Incidence n Incidence

0.000013 0 NA NA 15 15 15 13 NA NA

1 NA NA 15 0 15 2 NA NA

2 NA NA 15 0 15 0 NA NA

3 NA NA 15 0 15 0 NA NA

4 NA NA 15 0 15 0 NA NA

0.00012 0 NA NA 15 15 15 13 NA NA

1 NA NA 15 0 15 2 NA NA

2 NA NA 15 0 15 0 NA NA

3 NA NA 15 0 15 0 NA NA

4 NA NA 15 0 15 0 NA NA

0.0014 0 NA NA 14 13 14 11 NA NA

1 NA NA 14 0 14 0 NA NA

2 NA NA 14 1 14 3 NA NA

3 NA NA 14 0 14 0 NA NA

4 NA NA 14 0 14 0 NA NA

1 Gonadal histopathology diagnostic observ ations are graded 0 – 4 based on seve rity: 0=Not remarkable, 1=Minimal,

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

LOQ=0.000001 mg a.i./L.

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

EPA MRID Number 48683001

Page 44 of 82 Mean male VTG was 41, 53, 39 and 35 and female VTG was 1.2x10 6 , 8.4x10 5 , 1.1x10 6 and 1.1x10 6

ng/mL in the mean-measured 0 (control), 0.000013, 0. 00012 and 0.0014 mg a.i./L treatment levels,

respectively (Table 19).

Table 19: Plasma Vitellogenin in Fathead Minnow ( Pimephales promelas)

.

Treatment

(mg a.i./L)

[mean-measured] Plasma Vitellogenin (VTG)

Males Females

n Mean

(ng/mL plasma) ±SD n Mean

(ng/mL plasma) ±SD

Control (<LOQ) 4 41 34 4 1.2 x 10 6 2.553 x 10 5

0.000013 4 53 72 4 8.4 x 10 5 2.34 x 10 5

0.00012 4 39 25 4 1.1 x 10 6 3.64 x 10 5

0.0014 4 35 29 4 1.1 x 10 6 3.01 x 10 5

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

LOQ=0.000001 mg a.i./L.Data Evaluation Record on the Fish Short-Term Reproduction Assay with CypermethrinEPA MRID Number 48683001Page 45 of 82Mean plasma testosterone and plasma 17β-estradiol in male and femaleswas not measured (Table 20).Table 20: Plasma Sex Steroids in Fathead Minnow ( Pimephales promelas). Not measured.Treatment(mg a.i./L)[mean-measured]Plasma Testosterone (T)Plasma 17β-estradiol (E2)MalesFemalesMalesFemalesnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDControl (<LOQ)NANANANANANANANANANANANA0.000013NANANANANANANANANANANANA0.00012NANANANANANANANANANANANA0.0014NANANANANANANANANANANANAAbbreviations:NANot applicable.NDNot determined.SDStandard deviation.LOQ=0.000001 mg a.i./L.Data Evaluation Record on the Fish Short-Term Reproduction Assay with CypermethrinEPA MRID Number 48683001Page 46 of 82There were no notable observations with regards to behavior orchanges in appearance (coloration/banding, presence of ovipositor and dorsal napepad), including secondary sex characteristics in the control ortreated groups (Table 21). Replicate B in the control group contained three malesand three females due to mis-sexed fish.Table 21: Secondary Sex Characteristicsand Clinical Signs in Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[mean-measured]Secondary Sex Characteristics and Clinical SignsMalesFemalesTypenIncidenceTypenIncidenceControl (<LOQ)None90None1500.000013None80None1500.00012None80None1500.0014None80None140LOQ=0.000001 mg a.i./L.

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

EPA MRID Number 48683001

Page 47 of 82 B. Study Author’s Analysis and Conclusions

The study author analyzed tubercle score, GSI, fertili ty, fecundity, and VTG. Data were gender

specific and analyzed in co mparison to the controls.

Descriptive statistics (mean, standard deviation, etc.) were determined for each endpoint. Significant

effects were detected for p<0.05 with the exception of Shapiro-Wilk’s and Bartlett’s Tests, which were

based on p<0.01 (CETIS, version 1.8.4, 2011). Survival, body weight and body length data were

recoded but not statistically analyzed. Fecundity, fert ility, male and female VT G, and female GSI were

analyzed using ANOVA and a one-taile d Dunnett’s Multiple Comparison test. Median male tubercle

scores and median male GSI were analyzed by AN OVA and Jonckheere-Terpstra Step–Down test.

Male and female length was recorded but statistica l analysis was not reported. Prior to Dunnett’s,

data were analyzed by Shapiro-Wilk’s test and Bart lett’s to test for normality and homogeneity of

variances, respectively. If normality and homogeneity tests passed (p>0.01), a parametric analysis

was performed using the transformed data. If no n-normality or unequal variance were indicated

(p<0.01), a non-parametric analysis was performed on the ranks of the data. These methods appear

to be consistent with the methods recommended in the guideline.

There were no differences in male or female survival at any treatment level based on visual inspection

of the data (Table 9). There was no statistically significant difference in fe rtilization success at any

treatment level compared to the control (p>0.05; Du nnett’s). There was a statistically significant

reduction in fecundity at the 0.00012 and 0.0014 mg a.i./L treatment levels compared to the control

(p<0.05; Dunnett’s). However, the mean fecundity in the control was 14 eggs/female/day; OCSPP

guidance requires fecundity in the controls of at least 15 eggs/female/day. There was a significant

decrease in median male GSI at the 0.00012 and 0.0014 mg a.i./L treatment leve ls compared to

the control (p<0.05; Jonckheere-Terpstra Step-Down test; Table 13). There were no significant

differences between treated groups and control for any other endpoint.

In male fish, there were only background or sporadic g onadal histopathology findings noted which

were not related to treatment (Tables 15-16). In female fish, there was an increased incidence of

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

EPA MRID Number 48683001

Page 48 of 82 mature oocyte atresia at the 0.00 14 mg a.i./L treatment, which was associated with granulomatous

inflammation and the presence of microsporidia in half the observed cases across all treatment

levels (Tables 17-18). There were no significant findings observed in the g onadal stage for either

males or females.

C. Reviewer’s Analysis and Conclusions

Statistical Methods: The reviewer analyzed survival (mortality) data based on visual observation.

Male GSI and fecundity were consistent with a monotonic concentration-response; no other

endpoints were associated with a monotonic respons e. All data were tested for normality using

Shapiro-Wilks test and for homogeneity of vari ance using Levene’s test. Data which met the

assumptions of normality and homogeneity of vari ance were then analyzed using the parametric

Dunnett’s test. Data which did not meet the par ametric assumptions were analyzed using the nonparametric Mann-Whitney U test. Male GSI a nd fecundity exhibited m onotonic trends and was

analyzed using the non-parametric Jonckheere-Te rpstra test, as recommended by the OCSPP

890.1350 guideline.

None of the surviving females were found to have tubercles. Unless otherwise indicated, effects

were considered statistically significant at p<0.05. These analyses were conducted using SAS ®

(SAS Institute, Cary, NC; version 8.1). The data was subsequently re-run for the gender-specific

endpoints (male GSI, male VTG, male length, male weight) when excluding the mis-sexed male fish

in the control group with CETIS (Version 1.8.7.12). Both the study author and reviewer relied on

the mean-measured concentrations to discuss effects in this study.

Conclusions:

There were no significant differences in either male or female survival at any treatment levels

compared to the negative control based on visual observation (Table 9). Male GSI exhibited a

significant (p<0.05) increase of 22 to 27% at t he 0.00012 and 0.0014 mg a.i./L treatment levels

compared to the negative control. Fecundity was significantly (p<0.05) reduced 42% and 84% of

control at the 0.00012 and 0.0014 mg a.i./L treatment level. There were no other significant

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

EPA MRID Number 48683001

Page 49 of 82 effects on all other endpoints (female GSI, male an d female plasma VTG, male and female weight

and length and male and female nuptial tubercle score).

Although not analyzed statistically, in male fish, there were only background or sporadic findings

noted and were not related to treatment. In female fish, there was an increased incidence of mature

oocyte atresia at the 0.0014 mg a.i./L treat ment, which was associated with granulomatous

inflammation and the presence of microsporidia in half the observed cases across all treatment

levels.

There were no significant findings observed in the gonadal stage for either males or females, and no

notable observations in secondary sex characteristics or clinical signs for any treatment group

compared to the controls. Sex steroids were not measured in this study.Data Evaluation Record on the Fish Short-Term Reproduction Assay with CypermethrinEPA MRID Number 48683001Page 50 of 82Table 22: Reproductive and HPG Endpoints1,2for Male Fathead Minnow ( Pimephales promelas)in the FSTRA with Cypermethrin.Treatment(mg a.i./L)[mean-measured]Tubercle ScoreGSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2Medianp% Diff.pEffect?(Yes/No)% Diff.P% Diff.p% Diff.pControl (<LOQ)32.5NA0NANo0NANANANANA0.000013370.91410.410.231No59.40.859NANANANA0.00012330.60022.170.041No18.050.994NANANANA0.0014250.07126.700.018No5.261.00NANANANAStatistical TestMann-WhitneyJonckheere’sNADunnett’sNANAAbbreviations:Conc.Concentration.Diff.Difference.E217β-estradiol.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.NANot applicable.1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison tothe 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.LOQ=0.000001 mg a.i./L.Data Evaluation Record on the Fish Short-Term Reproduction Assay with CypermethrinEPA MRID Number 48683001Page 51 of 82Table 23: Reproductive and HPG Endpoints1,2for Female Fathead Minnow ( Pimephales promelas)in the FSTRA with Cypermethrin.Treatment(mg a.i./L)[meanmeasured]FecundityFert. SuccessTubercleScoreGSIGonadalStagingand Histo.Plasma VTGPlasma TPlasma E2% Diff.p%Diff.PMedianp%Diff.pEffect?(Yes/No)%Diff.p%Diff.p%Diff.pControl(<LOQ)0 NA 0 NA 0NA0 NANo 0 NANANANANA0.000013-5.260.3281.300.91400NA-5.050.979No-28.300.302NANANANA0.00012-41.930.0320.00>0.050NA-5.680.971No-5.320.981NANANANA0.0014-84.39<0.001-4.400.6590NA20.000.492No-10.640.879NANANANAStatistical TestJonckheere’sDunnett’s T3NADunnett’sNADunnett’sNANAAbbreviations:Conc.Concentration.Diff.Difference.E217β-estradiol.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.1Unless otherwise indicated, effects and percent (%) differences are reported based oncomparison to the negative (clean water) control. Conclusions regarding histopathologymay be heavily weighted by the expertopinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.LOQ=0.000001 mg a.i./L.

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

EPA MRID Number 48683001

Page 52 of 82 Table 24: Growth Endpoints 1,2 in the Fish Short-Term Reproducti on Assay (FSTRA) with Cypermethrin.

Treatment

(mg a.i./L)

[mean-measured] Body Weight Length

Males Females Males Females

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

Control (<LOQ) 0 NA 0 NA 0 NA 0 NA

0.000013 15.22 0.497 5.83 0.808 2.97 0.651 -2.37 0.502

0.00012 16.16 0.435 -9.03 0.552 3.32 0.577 -4.43 0.102

0.0014 2.64 0.993 -9.29 0.531 0.75 0.989 -1.55 0.768

Statistical Test Dunnett’s Dunnett’s Dunnett’s Dunnett’s

Abbreviations: Diff. Difference. NA Not applicable.

1 Unless otherwise indicated, percent (% ) differences are reported based on com parison to the negative (clean water)

control.

2 Unless otherwise specified, effects are co nsidered statistically significant at p<0.05.

LOQ=0.000001 mg a.i./L.

E. Study Deficiencies

There were deviations in the validity and performance criteria and a couple of minor deviations described

in Section I. Materials and Methods of this DER regar ding failure to report certain water characteristics and

meet environmental criteria:

1. There were one replicate in the control group (Replicate C) where there was a period of

4 days in which no spawning occurred, and fecundity was 13 eggs/female/day,

respectively. Therefore, this replicate did no t meet the criteria of either at least 15

eggs/female/day or spawning at least every 4 day s. However, it is noted that the overall

control mean fecundity value (14) was just slightly below 15 eggs/female/day, and the

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

EPA MRID Number 48683001

Page 53 of 82 other two replicates did achieve 15 or greater eggs/female/day and the remaining

replicate spawned at least every 4 days (11 eggs/female/day).

2. Analytical verification of the test material from Days 0, 6, 11, 19 and 21 yielded recoveries

of 44% to 67% of nominal concentrations. Ad ditionally, the %CV values were 14.2, 45.6

and 51.2%, respectively, and did not meet the validation criteria of <20%. Results of

these analyses indicate measured concentratio ns were generally lower than nominal, but

according to the study author were consistent with expectations for the physicochemical

properties and previous experience with the test item. Therefore, although there was no

pattern of decline during the study period and no un-dissolved test substance was

observed in the dilution system, the recoveries indicate the test material was generally

poorly recovered in solution under the test conditions.

3. The test material was detected at Days 11 (o ne replicate) and 19 (two replicates) in the

control samples. The study author concluded that the contamination was not consistent

among replicates and not consistent through out exposure. The study author concluded

the contamination was likely a result of processing samples after being removed from the

exposure system, indicating the control fi sh were not exposed to cypermethrin.

4. The unionized ammonia and residual chlorine in the test water were not reported. The

OCSPP 890.1350 performance criteria establish maximum levels for these values and it

is unclear if the maximum recommendations were exceeded.

5. Incorrect sexing of individuals in one replic ate from the control group resulted in there

being three males and three females in this replicate instead of the two males and four

females specified in the OCSPP 890.1350 guideline.

These deviations did not sufficiently adversely impact interpretation of the assay results. The remaining

validity and performance criteria for OCSPP 890.1350 were met.

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

EPA MRID Number 48683001

Page 54 of 82 F. Reviewer’s Comments

The reviewer’s and the study author’s results were in agreement. Both the study author’s and reviewer’s

analysis detected a statistically significant reduction (p<0 .05) in fecundity and a statistically significant

(p<0.05) increase in male GSI score at the 0.00012 and 0.0014 mg a.i./L treatment levels compared

to the negative control. The reviewer’s conc lusions based on the OCSPP 890.1350 flowchart are

presented in the Executive Summary and Conclusions sections of this DER.

III. REFERENCES

Ankley, G.T., Jensen, K.M., Kahl, M.D., Korte, J.J., and Makynen E.A., 2001. Description and

evaluation of a short-term reproduction test with the fathead minnow (Pimep hales promelas). Environ

Toxicol Chem 20:1276-1290.

APHA, AWWA, WPCF, 1995. Standard Methods for t he Examination of Water and Wastewater. 19th

Edition, Washington, DC, 2-23-25.

ASTM, 2005. Committee E-47 on Biological Effe cts and Environmental Fate. Standard Guide for

Conducting Early Life-Stage Toxicity Tests with Fishes. ASTM Designation E: 1241-05. American

Society for Testing and Materials, 100 Barr Ha rbor Drive, PO Box C700, West Conshohocken,

Pennsylvania.

Ives, Michael, 2011. CETIS, Comprehensive Environmental Toxicity Information System TM . User’s

Guide. Tidepool Scientific Software, McKinleyville, California.

Mount, D. I. and W. A. Brungs. 1967. A simplified dosing apparatus for fish toxicology studies.

Water Research 1: 21-29.

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

EPA MRID Number 48683001

Page 55 of 82 Sprague, J.B., 1969. Measurement of pollutant toxici ty to fish. 1. Bioassay methods for acute toxicity.

Water Res. 3: 793-821.

OECD (2009). Fish Short Term R eproduction Assay. OECD Guideline for the testing Chemicals:

Test No. 229. Paris, France. 40 pp

U.S. EPA, 1989. 40 CFR, Part 160. Federal Insecticide, Fungicide, and Rodenticide Act. Good

Laboratory Practices Standards; Final Rule. Office of the Federal Register, National Archives and

Records Administration. U.S. Government Printing Office, Washington, D.C.

U.S. EPA. 1996. Office of Prevention, Pestici des and Toxic Substances. Ecological Effects

Guideline, OPPTS 850.1000. Special Consideration for Conducting Aquatic Laboratory Studies.

“Public Draft”. EPA 712-C- 96-113. April 1996. U.S. Environment al Protection Agency, Washington,

D.C.

U.S. EPA, 2002. Short-term methods for estimating the chronic toxicity of effluents and receiving

waters to marine and estuarine organisms. Third ed ition. Office of Water, U. S. Environmental

Protection Agency, Washington, DC 20460. EPA/821/R-02/014.

U.S. EPA, 2009. Office of Prevention, Pestici des and Toxic Substances. Endocrine Disruptor

Screening Program Test Guideline, OPPTS 890.1 350. Fish Short-Term Reproduction Assay. EPA

740-C-09-007. October 2009. U.S. Environment al Protection Agency. Washington, D.C.

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

EPA MRID Number 48683001

Page 56 of 82 APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION:

test for fish screen study - Cypermethrin ANALYSIS RESULTS FOR VARIABLE VAR01 ( F body weight (g) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value
0.913 0.132 1.115 0.381 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 1.39 0.20 0.10 14.28 1.08, 1.71 Dose1 4 1.48 0.13 0.07 8.84 1.27, 1.68 Dose2 4 1.27 0.11 0.05 8.29 1.10, 1.44 Dose3 4 1.26 0.18 0.09 13.92 0.98, 1.54

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 1.32 1.25 1.69 . .
Dose1 1.47 1.34 1.63 105.83 -5.83
Dose2 1.24 1.18 1.41 90.97 9.03
Dose3 1.27 1.09 1.44 90.71 9.29

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 1.71 0.218

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 1.39 . 1.43 . . . . . .
Dose1 1.48 0.808 1.43 0.727 . . . . .
Dose2 1.27 0.552 1.27 0.177 0.292 . . . .
Dose3 1.26 0.531 1.26 0.175 0.279 1.000 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 3.86 0.277

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

EPA MRID Number 48683001

Page 57 of 82 MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 1.32 . .
Dose1 1.47 0.494 0.807
Dose2 1.24 0.346 0.153
Dose3 1.27 0.678 0.081

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 1.71 0.218

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -1.39 . -1.35 . . . . . .
Dose1 -1.48 0.808 -1.35 0.737 . . . . .
Dose2 -1.27 0.552 -1.35 0.770 0.292 . . . .
Dose3 -1.26 0.531 -1.35 0.788 0.279 1.000 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 3.86 0.277

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -1.32 . .
Dose1 -1.47 0.494 0.193
Dose2 -1.24 0.346 0.847
Dose3 -1.27 0.678 0.919

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - Cypermethrin ANALYSIS RESULTS FOR VARIABLE VAR02 ( M body weight (g) )

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

EPA MRID Number 48683001

Page 58 of 82 TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value
0.936 0.305 3.162 0.064 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 3.02 0.37 0.19 12.34 2.43, 3.61 Dose1 4 3.57 0.78 0.39 21.91 2.33, 4.81 Dose2 4 3.60 0.62 0.31 17.10 2.62, 4.58 Dose3 4 3.18 0.17 0.09 5.41 2.91, 3.45

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 3.00 2.64 3.44 . .
Dose1 3.84 2.42 4.17 118.17 -18.17
Dose2 3.63 2.93 4.20 119.13 -19.13
Dose3 3.19 2.97 3.38 105.26 -5.26

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 1.14 0.373

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 3.02 . 3.40 . . . . . .
Dose1 3.57 0.374 3.40 0.895 . . . . .
Dose2 3.60 0.337 3.40 0.917 1.000 . . . .
Dose3 3.18 0.953 3.18 0.796 0.739 0.696 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 3.04 0.385

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 3.00 . .
Dose1 3.84 0.346 0.876
Dose2 3.63 0.235 0.928

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

EPA MRID Number 48683001

Page 59 of 82 Dose3 3.19 0.678 0.680

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 1.14 0.373

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -3.02 . -3.02 . . . . . .
Dose1 -3.57 0.374 -3.45 0.168 . . . . .
Dose2 -3.60 0.337 -3.45 0.179 1.000 . . . .
Dose3 -3.18 0.953 -3.45 0.186 0.739 0.696 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 3.04 0.385

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -3.00 . .
Dose1 -3.84 0.346 0.124
Dose2 -3.63 0.235 0.072
Dose3 -3.19 0.678 0.320

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - Cypermethrin ANALYSIS RESULTS FOR VARIABLE VAR03 ( F body length (mm) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value
0.912 0.126 1.347 0.306 USE PARAMETRIC TESTS

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

EPA MRID Number 48683001

Page 60 of 82 ********************************************************************************

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 50.81 0.98 0.49 1.93 49.25, 52.37 Dose1 4 49.61 1.15 0.57 2.32 47.78, 51.44 Dose2 4 48.56 0.91 0.46 1.88 47.11, 50.01 Dose3 4 50.02 2.18 1.09 4.36 46.56, 53.49

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 50.72 49.72 52.10 . .
Dose1 49.58 48.45 50.83 97.63 2.37
Dose2 48.17 47.99 49.92 95.57 4.43
Dose3 50.83 46.89 51.54 98.45 1.55

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 1.79 0.202

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 50.81 . 50.81 . . . . . .
Dose1 49.61 0.502 49.61 0.148 . . . . .
Dose2 48.56 0.102 49.29 0.094 0.719 . . . .
Dose3 50.02 0.768 49.29 0.097 0.975 0.480 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 5.05 0.168

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 50.72 . .
Dose1 49.58 0.346 0.124
Dose2 48.17 0.103 0.006
Dose3 50.83 0.889 0.131

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

********************************************************************************

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

EPA MRID Number 48683001

Page 61 of 82 PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 1.79 0.202

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -50.81 . -49.66 . . . . . .
Dose1 -49.61 0.502 -49.66 0.923 . . . . .
Dose2 -48.56 0.102 -49.66 0.941 0.719 . . . .
Dose3 -50.02 0.768 -50.02 0.895 0.975 0.480 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 5.05 0.168

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -50.72 . .
Dose1 -49.58 0.346 0.876
Dose2 -48.17 0.103 0.994
Dose3 -50.83 0.889 0.869

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - Cypermethrin ANALYSIS RESULTS FOR VARIABLE VAR04 ( M body length (mm) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value
0.910 0.118 0.652 0.597 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 61.64 2.27 1.13 3.68 58.03, 65.25 Dose1 4 64.07 3.62 1.81 5.65 58.31, 69.82 Dose2 4 64.28 2.64 1.32 4.10 60.08, 68.48

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

EPA MRID Number 48683001

Page 62 of 82 Dose3 4 62.68 1.69 0.84 2.69 60.00, 65.37

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 61.78 58.82 64.19 . .
Dose1 65.35 58.93 66.63 103.93 -3.93
Dose2 64.89 60.91 66.43 104.28 -4.28
Dose3 62.24 61.31 64.94 101.69 -1.69

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 0.88 0.481

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 61.64 . 63.33 . . . . . .
Dose1 64.07 0.455 63.33 0.878 . . . . .
Dose2 64.28 0.392 63.33 0.902 0.999 . . . .
Dose3 62.68 0.900 62.68 0.838 0.880 0.828 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 2.60 0.457

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 61.78 . .
Dose1 65.35 0.346 0.876
Dose2 64.89 0.346 0.810
Dose3 62.24 0.494 0.610

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 0.88 0.481

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend

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

EPA MRID Number 48683001

Page 63 of 82 Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -61.64 . -61.64 . . . . . .
Dose1 -64.07 0.455 -63.68 0.178 . . . . .
Dose2 -64.28 0.392 -63.68 0.190 0.999 . . . .
Dose3 -62.68 0.900 -63.68 0.197 0.880 0.828 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 2.60 0.457

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -61.78 . .
Dose1 -65.35 0.346 0.124
Dose2 -64.89 0.346 0.190
Dose3 -62.24 0.494 0.390

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - Cypermethrin ANALYSIS RESULTS FOR VARIABLE VAR05 ( F vitellogenin (ng/mL) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value
0.941 0.361 0.460 0.715 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 1175000 262995.6 131497.8 22.38 756515.4, 1593485 Dose1 4 842500.0 241436.7 120718.3 28.66 458320.4, 1226680 Dose2 4 1112500 371068.3 185534.1 33.35 522047.6, 1702952 Dose3 4 1050000 288675.1 144337.6 27.49 590653.4, 1509347

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 1250000 800000.0 1400000 . .
Dose1 875000.0 520000.0 1100000 71.70 28.30
Dose2 1030000 790000.0 1600000 94.68 5.32
Dose3 1050000 700000.0 1400000 89.36 10.64

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

EPA MRID Number 48683001

Page 64 of 82 ******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 0.96 0.445

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 1175000 . 1175000 . . . . . .
Dose1842500.0 0.302 1001667 0.252 . . . . .
Dose2 1112500 0.981 1001667 0.270 0.584 . . . .
Dose3 1050000 0.879 1001667 0.279 0.755 0.990 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 2.43 0.488

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 1250000 . .
Dose1 875000.0 0.235 0.074
Dose2 1030000 0.780 0.304
Dose3 1050000 0.580 0.372

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 0.96 0.445

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -1175000 . -1008750 . . . . . .
Dose1 -842500 0.302 -1008750 0.855 . . . . .

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

EPA MRID Number 48683001

Page 65 of 82 Dose2-1112500 0.981 -1081250 0.789 0.584 . . . .
Dose3-1050000 0.879 -1081250 0.807 0.755 0.990 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 2.43 0.488

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -1250000 . .
Dose1 -875000 0.235 0.926
Dose2 -1030000 0.780 0.696
Dose3 -1050000 0.580 0.628

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - Cypermethrin ANALYSIS RESULTS FOR VARIABLE VAR06 ( M vitellogenin (ng/mL) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value
0.857 0.017 2.309 0.128 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 41.00 33.34 16.67 81.31 -12.05, 94.05 Dose1 4 53.00 72.35 36.17 136.50 -62.12, 168.12 Dose2 4 39.25 25.38 12.69 64.67 -1.14, 79.64 Dose3 4 35.00 29.13 14.57 83.23 -11.36, 81.36

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 38.00 12.00 76.00 . .
Dose1 19.50 12.00 161.00 129.27 -29.27
Dose2 36.50 13.00 71.00 95.73 4.27
Dose3 25.00 13.00 77.00 85.37 14.63

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 0.12 0.946

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

EPA MRID Number 48683001

Page 66 of 82 Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 41.00 . 47.00 . . . . . .
Dose1 53.00 0.962 47.00 0.662 . . . . .
Dose2 39.25 1.000 39.25 0.593 0.970 . . . .
Dose3 35.00 0.995 35.00 0.551 0.938 0.999 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 0.49 0.921

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 38.00 . .
Dose1 19.50 0.887 0.384
Dose2 36.50 1.000 0.588
Dose3 25.00 0.780 0.664

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 0.12 0.946

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -41.00 . -41.00 . . . . . .
Dose1 -53.00 0.962 -42.42 0.564 . . . . .
Dose2 -39.25 1.000 -42.42 0.598 0.970 . . . .
Dose3 -35.00 0.995 -42.42 0.617 0.938 0.999 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value

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

EPA MRID Number 48683001

Page 67 of 82 3 0.49 0.921

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -38.00 . .
Dose1 -19.50 0.887 0.616
Dose2 -36.50 1.000 0.412
Dose3 -25.00 0.780 0.336

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - Cypermethrin ANALYSIS RESULTS FOR VARIABLE VAR07 ( F GSI )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value
0.910 0.117 1.388 0.294 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 11.88 4.21 2.11 35.46 5.17, 18.58 Dose1 4 11.28 2.18 1.09 19.37 7.80, 14.75 Dose2 4 11.20 2.25 1.12 20.06 7.63, 14.77 Dose3 4 14.25 1.50 0.75 10.53 11.86, 16.64

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 10.50 8.50 18.00 . .
Dose1 11.00 9.10 14.00 94.95 5.05
Dose2 10.85 9.10 14.00 94.32 5.68
Dose3 14.00 13.00 16.00 120.00 -20.00

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 1.10 0.386

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

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

EPA MRID Number 48683001

Page 68 of 82 Ctrl 11.88 . 12.15 . . . . . .
Dose1 11.28 0.979 12.15 0.642 . . . . .
Dose2 11.20 0.971 12.15 0.677 1.000 . . . .
Dose3 14.25 0.492 12.15 0.696 0.445 0.425 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 3.82 0.282

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 10.50 . .
Dose1 11.00 1.000 0.558
Dose2 10.85 1.000 0.500
Dose3 14.00 0.343 0.933

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 1.10 0.386

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -11.88 . -11.45 . . . . . .
Dose1 -11.28 0.979 -11.45 0.673 . . . . .
Dose2 -11.20 0.971 -11.45 0.708 1.000 . . . .
Dose3 -14.25 0.492 -14.25 0.158 0.445 0.425 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 3.82 0.282

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -10.50 . .

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

EPA MRID Number 48683001

Page 69 of 82 Dose1 -11.00 1.000 0.442
Dose2 -10.85 1.000 0.500
Dose3 -14.00 0.343 0.067

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - Cypermethrin ANALYSIS RESULTS FOR VARIABLE VAR08 ( M GSI )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value
0.976 0.923 2.555 0.104 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 1.11 0.25 0.12 22.28 0.71, 1.50 Dose1 4 1.22 0.18 0.09 14.72 0.93, 1.51 Dose2 4 1.35 0.06 0.03 4.28 1.26, 1.44 Dose3 4 1.40 0.18 0.09 13.04 1.11, 1.69

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 1.09 0.84 1.40 . .
Dose1 1.25 0.98 1.40 110.41 -10.41
Dose2 1.35 1.30 1.40 122.17 -22.17
Dose3 1.40 1.20 1.60 126.70 -26.70

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 2.18 0.143

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 1.11 . 1.27 . . . . . .
Dose1 1.22 0.700 1.27 0.939 . . . . .
Dose2 1.35 0.182 1.27 0.955 0.740 . . . .
Dose3 1.40 0.095 1.27 0.962 0.515 0.978 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests

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

EPA MRID Number 48683001

Page 70 of 82 Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 4.28 0.233

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 1.09 . .
Dose1 1.25 0.575 0.769
Dose2 1.35 0.222 0.959
Dose3 1.40 0.190 0.982

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 2.18 0.143

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -1.11 . -1.11 . . . . . .
Dose1 -1.22 0.700 -1.22 0.229 . . . . .
Dose2 -1.35 0.182 -1.35 0.048 0.740 . . . .
Dose3 -1.40 0.095 -1.40 0.024 0.515 0.978 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 4.28 0.233

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -1.09 . .
Dose1 -1.25 0.575 0.231
Dose2 -1.35 0.222 0.041
Dose3 -1.40 0.190 0.018

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL
Williams Dose2
Jonckheere Dose2

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

EPA MRID Number 48683001

Page 71 of 82 test for fish screen study - Cypermethrin ANALYSIS RESULTS FOR VARIABLE VAR09 ( F tubercle score (median) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value

. . . . NO DATA FOR TEST

********************************************************************************

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.00 0.00 0.00 . . , . Dose1 4 0.00 0.00 0.00 . . , . Dose2 4 0.00 0.00 0.00 . . , . Dose3 4 0.00 0.00 0.00 . . , .

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 0.00 0.00 0.00 . .
Dose1 0.00 0.00 0.00 . .
Dose2 0.00 0.00 0.00 . .
Dose3 0.00 0.00 0.00 . .

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value

. . . 1 . . . . . . .

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 0.00 . . . . . . . .
Dose1 0.00 . . . . . . . .
Dose2 0.00 . . . . . . . .
Dose3 0.00 . . . . . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 0.00 1.000

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

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

EPA MRID Number 48683001

Page 72 of 82 Level Median MannWhit p-value Jonckheere p-value
Ctrl 0.00 . .
Dose1 0.00 1.000 .
Dose2 0.00 1.000 .
Dose3 0.00 1.000 .

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL
Williams Dose1

Jonckheere Dose1

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value

. . . 1 . . . . . . .

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 0.00 . . . . . . . .
Dose1 0.00 . . . . . . . .
Dose2 0.00 . . . . . . . .
Dose3 0.00 . . . . . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 0.00 1.000

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 0.00 . .
Dose1 0.00 1.000 .
Dose2 0.00 1.000 .
Dose3 0.00 1.000 .

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL
Williams Dose1

Jonckheere Dose1

test for fish screen study - Cypermethrin ANALYSIS RESULTS FOR VARIABLE VAR10 ( M tubercle score (median) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS

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

EPA MRID Number 48683001

Page 73 of 82 Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value
0.967 0.782 1.418 0.286 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 32.50 2.08 1.04 6.41 29.19, 35.81 Dose1 4 37.75 5.91 2.95 15.65 28.35, 47.15 Dose2 4 32.25 4.65 2.32 14.41 24.86, 39.64 Dose3 4 26.50 4.51 2.25 17.02 19.32, 33.68

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 32.50 30.00 35.00 . .
Dose1 37.00 32.00 45.00 116.15 -16.15
Dose2 33.00 26.00 37.00 99.23 0.77
Dose3 25.00 23.00 33.00 81.54 18.46

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 4.16 0.031

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 32.50 . 35.13 . . . . . .
Dose1 37.75 0.279 35.13 0.861 . . . . .
Dose2 32.25 1.000 32.25 0.584 0.353 . . . .
Dose3 26.50 0.194 26.50 0.053 0.019 0.318 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 6.34 0.096

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 32.50 . .
Dose1 37.00 0.283 0.904
Dose2 33.00 1.000 0.559
Dose3 25.00 0.190 0.055

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

EPA MRID Number 48683001

Page 74 of 82 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 4.16 0.031

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -32.50 . -32.25 . . . . . .
Dose1 -37.75 0.279 -32.25 0.616 . . . . .
Dose2 -32.25 1.000 -32.25 0.651 0.353 . . . .
Dose3 -26.50 0.194 -32.25 0.670 0.019 0.318 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 6.34 0.096

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -32.50 . .
Dose1 -37.00 0.283 0.096
Dose2 -33.00 1.000 0.441
Dose3 -25.00 0.190 0.945

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - Cypermethrin ANALYSIS RESULTS FOR VARIABLE VAR11 ( fecundity )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value
0.953 0.538 1.887 0.186 USE PARAMETRIC TESTS

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

EPA MRID Number 48683001

Page 75 of 82 ********************************************************************************

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 14.25 2.99 1.49 20.95 9.50, 19.00 Dose1 4 13.50 3.00 1.50 22.22 8.73, 18.27 Dose2 4 8.28 4.73 2.37 57.20 0.74, 15.81 Dose3 4 2.23 0.88 0.44 39.42 0.83, 3.62

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 14.00 11.00 18.00 . .
Dose1 13.00 11.00 17.00 94.74 5.26
Dose2 7.10 3.90 15.00 58.07 41.93
Dose3 2.20 1.30 3.20 15.61 84.39

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 12.07 <.001

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 14.25 . 14.25 . . . . . .
Dose1 13.50 0.975 13.50 0.442 . . . . .
Dose2 8.28 0.054 8.28 0.013 0.151 . . . .
Dose3 2.23 <.001 2.23 <.001 0.002 0.083 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 10.65 0.014

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 14.00 . .
Dose1 13.00 0.775 0.328
Dose2 7.10 0.190 0.032
Dose3 2.20 0.067 <.001

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL
Williams Dose2
Jonckheere Dose2

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests

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

EPA MRID Number 48683001

Page 76 of 82 Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 12.07 <.001

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -14.25 . -9.56 . . . . . .
Dose1 -13.50 0.975 -9.56 0.988 . . . . .
Dose2 -8.28 0.054 -9.56 0.992 0.151 . . . .
Dose3 -2.23 <.001 -9.56 0.994 0.002 0.083 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 10.65 0.014

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -14.00 . .
Dose1 -13.00 0.775 0.672
Dose2 -7.10 0.190 0.968
Dose3 -2.20 0.067 1.000

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - Cypermethrin ANALYSIS RESULTS FOR VARIABLE VAR12 ( fertility )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value
0.880 0.038 3.102 0.067 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 96.50 4.36 2.18 4.52 89.56, 103.44 Dose1 4 97.75 0.50 0.25 0.51 96.95, 98.55 Dose2 4 96.50 1.73 0.87 1.79 93.74, 99.26 Dose3 4 92.25 6.65 3.33 7.21 81.67, 102.83

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

EPA MRID Number 48683001

Page 77 of 82 Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 98.50 90.00 99.00 . .
Dose1 98.00 97.00 98.00 101.30 -1.30
Dose2 97.00 94.00 98.00 100.00 0.00
Dose3 94.00 83.00 98.00 95.60 4.40

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 1.39 0.292

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 96.50 . 97.13 . . . . . .
Dose1 97.75 0.947 97.13 0.672 . . . . .
Dose2 96.50 1.000 96.50 0.618 0.972 . . . .
Dose3 92.25 0.358 92.25 0.108 0.275 0.481 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 4.32 0.229

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl 98.50 . .
Dose1 98.00 0.555 0.219
Dose2 97.00 0.407 0.053
Dose3 94.00 0.281 0.010

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences)
Jonckheere Dose3

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 1.39 0.292

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

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

EPA MRID Number 48683001

Page 78 of 82 Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -96.50 . -95.75 . . . . . .
Dose1 -97.75 0.947 -95.75 0.688 . . . . .
Dose2 -96.50 1.000 -95.75 0.723 0.972 . . . .
Dose3 -92.25 0.358 -95.75 0.742 0.275 0.481 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 4.32 0.229

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value
Ctrl -98.50 . .
Dose1 -98.00 0.555 0.781
Dose2 -97.00 0.407 0.947
Dose3 -94.00 0.281 0.990

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - Cypermethrin ANALYSIS RESULTS FOR VARIABLE VAR13 ( F testosterone (ng/mL) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value

. . . . NO DATA FOR TEST

********************************************************************************

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 0 . . . . . , . Dose1 0 . . . . . , . Dose2 0 . . . . . , . Dose3 0 . . . . . , .

Level Median Min Max %of Control(means) %Reduction(means)

Ctrl . . . . .
Dose1 . . . . .
Dose2 . . . . .
Dose3 . . . . .

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

EPA MRID Number 48683001

Page 79 of 82 ******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 1.39 0.292

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl . . .
Dose1 . . .
Dose2 . . .
Dose3 . . .
Jonckheere Dose1

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 1.39 0.292

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl . . .
Dose1 . . .
Dose2 . . .
Dose3 . . .
Jonckheere Dose1

test for fish screen study - Cypermethrin ANALYSIS RESULTS FOR VARIABLE VAR14 ( M testosterone (ng/mL) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value

. . . . NO DATA FOR TEST

********************************************************************************

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 0 . . . . . , . Dose1 0 . . . . . , . Dose2 0 . . . . . , . Dose3 0 . . . . . , .

Level Median Min Max %of Control(means) %Reduction(means)

Ctrl . . . . .

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

EPA MRID Number 48683001

Page 80 of 82 Dose1 . . . . .
Dose2 . . . . .
Dose3 . . . . .

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 1.39 0.292

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl . . .
Dose1 . . .
Dose2 . . .
Dose3 . . .
Jonckheere Dose1

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 1.39 0.292

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl . . .
Dose1 . . .
Dose2 . . .
Dose3 . . .
Jonckheere Dose1

test for fish screen study - Cypermethrin ANALYSIS RESULTS FOR VARIABLE VAR15 ( F 17b-estradiol (ng/mL) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value

. . . . NO DATA FOR TEST

********************************************************************************

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 0 . . . . . , . Dose1 0 . . . . . , . Dose2 0 . . . . . , .

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

EPA MRID Number 48683001

Page 81 of 82 Dose3 0 . . . . . , .

Level Median Min Max %of Control(means) %Reduction(means)

Ctrl . . . . .
Dose1 . . . . .
Dose2 . . . . .
Dose3 . . . . .

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 1.39 0.292

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl . . .
Dose1 . . .
Dose2 . . .
Dose3 . . .
Jonckheere Dose1

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 1.39 0.292

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl . . .
Dose1 . . .
Dose2 . . .
Dose3 . . .
Jonckheere Dose1

test for fish screen study - Cypermethrin ANALYSIS RESULTS FOR VARIABLE VAR16 ( M 17b-estradiol (ng/mL) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value

. . . . NO DATA FOR TEST

********************************************************************************

BASIC SUMMARY STATISTICS

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

EPA MRID Number 48683001

Page 82 of 82 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 0 . . . . . , . Dose1 0 . . . . . , . Dose2 0 . . . . . , . Dose3 0 . . . . . , .

Level Median Min Max %of Control(means) %Reduction(means)

Ctrl . . . . .
Dose1 . . . . .
Dose2 . . . . .
Dose3 . . . . .

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 1.39 0.292

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl . . .
Dose1 . . .
Dose2 . . .
Dose3 . . .
Jonckheere Dose1

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test
Numerator df Denominator df F-stat P-value
3 12 1.39 0.292

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl . . .
Dose1 . . .
Dose2 . . .
Dose3 . . .
Jonckheere Dose1

# DATA EVALUATION RECORD

CYPERMETHRIN

Study Type: OCSPP 890.1450, Female Pubertal Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-69-2012 (MRID 48683002)

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: Sandra Hastings Date: 6/20/2012

Secondary Reviewer: Signature: Kelly Luck, M.S. Date: 6/26/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 7/11/2012

Quality Assurance: Signature: Steven Brecher, Ph.D., D.A.B.T. Date: 7/10/2012

This Data Evaluation Record may have been alte red 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 in tended 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 re dundancy 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 potent ial 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).CYPERMETH RJ N I 109702



### Primary Reviewer: Karlyn Middleton


### Health Effects Division (7509P) Secondary Reviewer: E lizabeth Mendez, Ph.D.


### Health Effects Division (7509P) Female Pubertal Assay (2012) I Page 1of17
OCSPP 890.1450/ OECD None



### Signature:

Date: Signature:

Date:

# 4=©~**l,;/,,-~ £M**  '5

/ 2 'f/u

- T e mpl a t e ve r s i o n 02 / 06



### DAT A EVALUATION RECORD

STUDY TYPE: Female Pubertal Assay ; OCSPP 890.1450 ; OECD None

.



### PC CODE: 109702 

### DP BARCODE: D401652



### TXR#: 0052288 

### CASNo: 52315-07-8



### TEST MATERIAL (PURITY): Cypermethrin (95

. 2 % a.i. ; 

### cis /

tran s ratio 49.8 / 50.2)



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

, 2-dichloroethenyl)-2

, 2- dimethylcyclopropanecarboxylate



### CITATION: Herberth

, M.T. (2012) Cypermethrin

- Pubertal Development and Thyroid
Function in Intact Juvenile / Peripubertal Female Rats. WIL Research
Laboratories

, LLC

, Ashland

, OH. Laboratory Project ID Number : WIL639152

, April 9

, 2012. MRID 48683002. Unpublished

.



### SPONSOR: Sawhney Coder

, P. (2011) Cypermethrin - A 14-Day Dose Range-Finding Oral ( Gavage) Toxicity Study in Juvenile Rats. WIL Research Laboratories, LLC

, Ashland

, OH

. Laborator y Project ID Number : WIL-639149

, December 19

, 2011. MRID 48683004. Unpublished.

S y ngenta Cop Protection

, LLC

, Greensboro

, NC; FMC Corporation

, Philadelphia

, PA ; and United Phosphorus

, Inc.

, King of Prussia

, PA



### TEST ORDER#: CON-109702-8



### EXECUTIVE SUMMARY: In a Female Pubertal Assay (MRID 48683002)

, 15 Sprague
Da w le y rats / dose group were treated dail y v ia oral gavage with cypermethrin (95

. 2 % a.i. ; 

### cis /

t r an s ratio : 49

. 8 / 50 .2; Batch # PLl0-0492 ) in com oil at doses ofO (corn oil)

, 15 or 30

mg / kg / da y from post-natal day (PND) 22 to 42 / 43

. Animals were examined for vaginal opening ( VO) dail y be g inning on PND 22

, and weight at day of attainment was recorded

. Following

s acrific e on PND 42 / 4 3, total th y roxine (T 4 ) levels were determined using an electrochemiluminescent immunoassa y and thyroid stimulating hormone (TSH) levels were determined using

a radioimmunoa ss a y

. Adrenal

, liver

, thyroid

, pituitary

, and urogenital organ weights were recorded

, and microscopic examinations of the uterus

, ovaries

, thyroid

, and kidney were performed.

On e animal in the 3 0 mg / kg / day group was found dead on PND 25

. No internal findings were noted for this animal during gross examination at necropsy. All other rats survived untilFemale Pubertal Assay (2012) / Page 2 of 17
CYPERMETHRIN / 109702 OCSPP 890.1450/ OECD None



### scheduled sacrifice. As there wa s no other evidence of toxicity in this study, this single death
was considered incidental. There were no treatmen t-related clinical signs of toxicity at any dose level; findings noted in the treated groups occurr ed infrequently in single animals and/or in a
manner that was not considered dose related. Mean body weights, daily body weight gains, cumulative mean body weight gain, and food consum ption in the treated groups were similar to the control group throughout the study.

No treatment-related effects were observed on m ean age or weight at vaginal opening (VO), and mean age at first estrus. The cycle status at necr opsy (diestrus, proestrus, estrus, or not cycling) appeared similar across all groups. There were no treatment-related cha nges in serum hormone or clinical chemistry levels. In addition, there were no effects of treatment observed in gross or microscopic pathology.

There was no evidence of direct test substance-related effects on pubertal development or thyroid function in the juvenile/peripube rtal female rat at cypermet hrin dose levels of 15 and 30 mg/kg/day.

No evidence of toxicity was observed in th e study up to the highest dose tested; however, treatment-related clinical signs were seen in the preliminar y range-finding study at 30 and 65 mg/kg/day. In the preliminary study, tremors and clear material around the mouth were seen at 30 mg/kg/day, 4 hours after administration. At 65 mg/kg/day (after 4 hours), tremors, splayed limbs, and clear material around the mouth, ventra l neck and thoracic area, and forelimbs were seen. It is possible that th e effects observed in the prelim inary study were missed in the definitive study since clinical signs were meas ured 1 hour after chemical administration. Clinical signs were measured 4 hours after ad ministration in the preliminary study, which is closer to the time-to-peak effect for cypermethrin. Therefore, dosing levels tested in this assay are considered appropriate.

The assay satisfies the EDSP Tier 1 Test Order require ments for a Female Pubertal Assay (OCSPP 890.1450).

COMPLIANCE: Signed and dated Data Confiden tiality, GLP Compliance, and Quality Assurance statements were provided.Female Pubertal Assay (2012) / Page 3 of 17
CYPERMETHRIN / 109702 OCSPP 890.1450/ OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: WIL Research Laboratories, LLC

Location: Ashland, OH Study Director: M.T. Herberth
Other Personnel: E.S. Bodle (Assistant Director, Analytical Chemistry); S.C. Haley (Operations Manager, Pathology); S.A. Keets (Senior Operations Ma nager, Vivarium); C.A. Kopp (Manager, Gross Pathology and Developmental Toxicology Laboratory); G.M. Maginnis (Clinical Veterinarian); J.S. Moore (Operations Ma nager, Developmenta l, Reproductive and Neurotoxicology); G.A. Parker (VP, Pathology) ; T.M. Rafeld (Group Ma nager, Formulations Laboratory); M.A. Safron (Manager, Histology); M.J. Schlosser (VP, Analytical, Metabolism, & In Vitro Services); B.J. Varsho (Director Operations); E.C. Vedaa (Manager, Clinical Pathology); R.A. Wally (Opera tions Manager, Reporting & Technical Support Se rvices); R. Green (Syngenta Study Monitor); L. Freshwater (Principal Investigator, Statistical Analysis) Study Period: May 11, 2011 to April 9, 2012



### 2. Test Substance: Cypermethrin
Description: Technical; dark yellow, very viscous liquid
Source: FMC Innovation Center (Ewing, NJ)
Batch #: PL10-0492 (expiry date: December 15, 2012) Purity: 95.2% a.i.; cis/trans ratio: 49.8/50.2 Stability: Formulations in corn oil are stable for 19 days of room temperature and refrigerated storage
CAS #: 52315-07-8
Structure:



### 3. Vehicle: Corn oil ( expiry date: July 1, 2012 & May 4, 2013)



### 4. Test Animals:
Species: Rat (female only)
Strain: Sprague Dawley [Crl:CD(SD)] Age/Weight at Study Initiation: PND 22 / 40.5–57.0 g

Source: Charles River Laboratorie s, Inc. (Portage, MI) Housing: Female weanlings were housed three per cage in plastic cages containi ng nesting material (Aspen BedTM)
Diet: 2016CM Teklad Global 16% Protein Rode nt Diet (Harlan Laboratories), ad libitum ; average 29.0 and 11.0 ppm total isoflavones (genistein+dai dzein+glycitein) for two lots of feed used.

Water: Reverse-osmosis purifie d (on-site) tap water , ad libitum
Environmental
Conditions: Temperature: Humidity: Air changes: Photoperiod: 21.2-22.0°C (mean daily temperature) 42.1-58.7% (mean daily humidity)

≥ 10/hr 14 hrs light/10 hrs dark



### B. STUDY DESIGN

1. In-Life Dates: Start: August 6, 2011 End: August 27, 2011 CN O

O Cl O

Cl CH 3 CH

3Female Pubertal Assay (2012) / Page 4 of 17
CYPERMETHRIN / 109702 OCSPP 890.1450/ OECD None



### 2. Mating: Timed-pregnant dams were received fro m the supplier on gestation day 7. The day when parturition was first observed was de signated as PND 0 for the offspring. On PND 4, litters were culled to 5 male and 5 female pups/litter.

3. Animal Assignment: Following weaning on PND 21, female ra ts were assigned to the test groups noted in Table 1 using a computer pr ogram which randomized the animals based on stratification of body weights in a block design; no littermates were a ssigned to the same dose group.

TABLE 1. Study Design a Test group Dose (mg/kg/day) # of Females Control (corn oil) 0 15 Low (cypermethrin) 15 15 High (cypermethrin) 30 15 a Data were obtained from page 21 of the study report.



### 4. Dose Selection Rationale: The dose levels were selected based on the results from a 14-day dose range-finding study (MRID 48683004) . Results at the HDT, 65 mg/kg/day, included one female death on Day 2 (splayed hindlimbs and clear material around the mouth 4 hours following dosing on Day 1), and slight to moderate tremors and wet or dried clear material around the mouth in the remaining 3 females at 4 hours post dosing. Two of these females also had splayed hindlimbs. At 4 hour s post-dose at 30 mg/kg/day, 4/4 females displayed tremors on 12/54 observations. Clin ical findings in the 5 and 15 mg/kg/day group occurred infrequently and were not observed in a dose-related manner. A summary of the study is included in the Appendix of this DER.

5. Dose Preparation and Analysis: Cypermethrin dose formulations were prepared in corn oil weekly as single formulations for each dos e level, divided into aliquots for daily dispensation, and stored refrigerated. The test substance formulations were stirred continuously throughout the preparation, sampli ng, and dose administration procedures.

Analyses to demonstrate the homogeneity, stab ility, and resuspension homogeneity of the
test substance formulations at concentra tions of 0.1 to 20 mg/mL following 4, 11, and 19 days of room temperature and refrigerated storage were conducted in a previous study. 1 

### Therefore, homogeneity and stab ility analyses were not condu cted during the current study. The first dosing formulations were inspect ed and found to be visibly homogeneous. Samples for concentration analysis were colle cted from the middle stratum of each dosing formulation prepared during th e in-life phase of the study. All analyses were conducted by the WIL Research Analytical Chemistry Department.

Results of Dose Analysis

Homogeneity: Not reported

1 Bodle, E.S. (2012) Analytical Validation and Stability St udy of Cypermethrin in Corn Oil Formulations (Study No. WIL-639153). WIL Research Laboratories, LLC, Ashland, OH.Female Pubertal Assay (2012) / Page 5 of 17
CYPERMETHRIN / 109702 OCSPP 890.1450/ OECD None



### Stability: Formulations are reportedly stable during 19 days of room temperature or refrigerated storage

Concentration (% of nominal): 100-114%

The analytical data indicated that the varia tion between nominal and actual dosage to the animals was acceptable.

6. Dosage Administration: All doses were administered once daily by oral gavage from PND 22 through PND 42 (eight animals in each treat ment group) or 43 (remaining animals in each group), at a dose volume of 5 mL/kg of body weight. Individual doses were based on the most recently recorded body weight. Dosing was performed between 0700 and 0900 hours daily.

7. Statistics: Chi-square analysis was used to de termine significant differences between the test substance groups and the control group for cycling status (cycling vs. not cycling) and percent of animals cycling regularly. Estrous cycle length and the day of first estrus were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test substance treated groups to the control group. Pathology findings presenting as a dichotomous response (present/abs ent) were analyzed us ing pairwise Fisher's exact tests to compare each treated group with the control group. The tests were one-sided at the 0.05 significance level testing for an incr ease. The statistical analyses described above were conducted by the study facility.

The remaining statistical analyses were c onducted by BioSTAT Consultants, Inc., using SAS® version 9.2 (SAS Institute, Inc., 2002-2008) , or higher, software. Data that were statistically analyzed included the organ wei ghts, organ weights to necropsy body weight ratio (liver, kidneys, pituitary, and adrenal gl ands), daily body weights (including necropsy body weight), cumulative body weight gain, seru m chemistries, hormones, and VO data. Gamma glutamyl transferase values under range were assigned a value of 0.1 (half the lower limit of quantitation) for statis tical analysis and reporting.

Each endpoint was tested for homogeneity of vari ance using Levene's test. If that test was significant at p=0.01, then a log transform was applied and Levene's test conducted on the
transformed data. If that test was still significant then the square root transformation was applied to the raw data and Leve ne's test conducted again. If the test was still significant then a nonparametric test, as described below, was used to analyze the data.

If the variances were homogeneous then ANOVA was conducted, on the raw or transformed data, as appropriate. The st atistical model contained a factor for treatment group and a blocking factor based on the study necrops y date. A two-sided Dunnett's test was conducted, regardless of the outcome of the ANO VA, looking for significant differences in the test substance groups when compared with the control group. If the transformationswere unsuccessful in making the variances homogeneous, then the nonparametric KruskalWallis test was used, ignoring the blocking f actor, followed by Dunn's test, to compare each of the test substance groups with the control group. Since these are pre-planned pairwise comparisons, Dunn's test was conducted regardless of the outcome of the Kruskal-WallisFemale Pubertal Assay (2012) / Page 6 of 17
CYPERMETHRIN / 109702 OCSPP 890.1450/ OECD None



### test. The tests were two-sided, at the 0.05 significance level, looking for significant
differences from the vehicle control.

In addition, organ weights and VO data were subject to the fo llowing analyses if they met the homogeneity of variance criteria: 1) anal ysis of covariance (ANCOVA) with Dunnett's test using PND 21 body weight as a covariate; 2) linear trend test using the ANOVA model; and 3) linear trend test using the ANCOVA model.

Pathology findings presented as a graded res ponse were analyzed with pairwise MannWhitney U tests to compare each treated group with the control group. The tests were onesided at the 0.05 significance level testing for in creased severity. The tests were two-sided for graded responses presented on a Grade 1-5 sc ale at the 0.05 significance level testing for increased or decreased severity. Exact p-va lues were calculated for the Mann-Whitney U

test.

C. METHODS

1. Mortality and Clin ical Examinations: All animals were examined twice daily for mortality and moribundity. Indivi dual detailed physical examin ations were recorded on the day of randomization and daily (prior to test substance administration during the treatment period) through the day of euthanasia. Animals were also observed for signs of toxicity approximately one hour following dose administration.

2. Body Weight: Body weights were recorded for allo cation of the animals to study groups, daily prior to dosing, and prio r to scheduled termination.

3. Food Consumption: Food consumption was recorded da ily prior to test substance administration on a per cage basis. Food intake was calculated and reported as g/cage/day for the corresponding body weight change intervals.

4. Vaginal Opening: Beginning on PND 22, all animals were examined daily for onset of VO. The appearance of a small “pin hole”, a vaginal thread, and complete VO were recorded. Age and weight on the day of comple tion of VO were recorded. When an animal did not complete VO by PND 42/ 43, PND 43/44 and body weight at necropsy were used as the age and body weight at VO, respectively, when determining the mean age and body weight at puberty onset for the experimental group.

5. Estrous Cyclicity: Beginning on the day of VO through scheduled termination, daily vaginal smears were obtained, microscopically evaluated, and classified as diestrus, estrus, or proestrus. The age at firs t vaginal estrus was recorded. Cycle length was determined for a complete estrous cycle by counting the number of days from one metestrus or diestrus to the next metestrus or diestrus. The overall cycling pattern for each female was characterized as regular, irregular, not cycling, or insufficient data. The definitions for these terms are as
follows:

 Regular cycling: the animal has at least 6 days of data collected and displays one complete cycle with no cycles greater than 5 days of duration, no cycles with 3 orFemale Pubertal Assay (2012) / Page 7 of 17
CYPERMETHRIN / 109702 OCSPP 890.1450/ OECD None



### more days of proestrus (P) and/or estrus (E), and no cycles less than 4 days in
duration.

 Irregular cycling: 1) the animal does not display one complete cycle but has at least one E and/or P and a partial cycle greater th an 5 days, 2) the animal has at least one E and either at least one cycle (complete or partial) greater than 5 days in duration or at least one cycle with 3 or more consecu tive days of P and/orE, 3) the animal has at least one cycle less than 4 days in duration, or 4) the animal has one irregular cycle and one regular cycle.

 Non-cycling: no E or P present on any days of estrous cycle determination and at least 5 days of data collected.

 Insufficient data: 1) the animal does not di splay at least one complete cycle but has at least one E and/or P and a partial cycle of 5 days or fewer, 2) no E is present on any days of estrous cycle determination and 4 or fewer days of data collected, or 3) at least one E or P present and only 1-4 days of data collected.

Mean age at first estrus, mean cycle length, percent cycling, percent regularly cycling, and estrus stage at termination were reported. For calculation of the age at the first occurrence of estrus, females (which had attained VO) that had no E or P during the evaluation period were assigned an age of 43 or 44 days (the la st day of observation + 1). If a female had no E, but had a P during the evalua tion period, the age at first estr us was considered the age on the day of P + 1 day.

6. Sacrifice and Pathology: On the day before euthanasia, all animals were moved to a holding room separate from where the necr opsies were performed. On the day of euthanasia, dosing was performed in the holdi ng room; the holding room was not disturbed except for the removal of the next animal to be euthanized. On PND 42/43, beginning approximately 2 hours post-dosing, all survivi ng animals were sacrificed randomly across all groups by decapitation without anesthesia ; sacrifices were completed by 1300 hours.
Blood from the trunk of the animals was colle cted, serum was sepa rated, and the serum divided equally into three tubes. One tube was used for serum chemistry evaluation, and the remaining two tubes were used for hormone an alyses. The sample for TSH analysis was stored frozen ( ≤ -20°C) for subsequent analysis and the sample for T 4 

### was analyzed fresh.

a. Hormone Analysis: Total T 4 

### levels were determined usi ng an electrochemiluminescent immunoassay and TSH levels were determined using a [ 125 

### I]rTSH radioimmunoassay kit.

b. Clinical Chemistry: The following CHECKED (X) parame ters were examined. The study report provided historical contro l ranges for the clinical chemistry parameters in the results
table

.Female Pubertal Assay (2012) / Page 8 of 17
CYPERMETHRIN / 109702 OCSPP 890.1450/ OECD None

ELECTROLYTES OTHER

X Calcium X Albumin

X Chloride X Creatinine* Magnesium X Urea nitrogen* X Phosphorus X Total cholesterol

X Potassium X Globulins (by calculation)

X Sodium X Glucose

ENZYMES X Total bilirubin

X Alkaline phosphatase (ALP) X Total protein Cholinesterase (ChE) X Triglycerides Creatine phosphokinase Se rum protein electrophoresis Lactic acid dehydrogenase (LDH) X Al bumin/globulin ratio (by calculation) X Alanine aminotransferase (ALT) X Bile acids X Aspartate aminotransferase (AST) Sorbitol dehydrogenase X Gamma glutamyl transferase (GGT) Glutamate dehydrogenase
* Recommended for the pubertal assay in fe male rats based on guideline 890.1450.



### c. Organ Weights and Histopathology: The following CHECKED (X) tissues were collected and weighed. The (XX) organs, in addition, were subject ed to histological
examination.

UROGENITAL OTHER XX Ovaries (paired, without oviducts)*+ XX Thyroid*+

XX Uterus*+ X Liver* XX Kidneys (paired)*+ X Adrenals (paired)* X Pituitary* * Weights required base d on guideline 890.1450 + Histopathological examination re quired based on guideline 890.1450



### All organs collected, except the thyroid, were weighed prior to fixa tion. Paired organs (kidneys, adrenals, and ovaries) were weighe d together. The uterus and cervix were separated from the vagina and weighed. The uterus was weighed again following removal of the fluid in the lumen (blotted weight). The ratios of organ weight to terminal body weight were calculated for liver, kidn ey, adrenal, and pituitary weights.

The ovaries, uterus, thyroid (w ith parathyroid), and kidneys were fixed in 10% buffered formalin for at least 24 hrs. Following fixa tion, the thyroid was dissected from the trachea and weighed. Fixed tissues were rinsed and placed in ethanol solutions. In addition, the right portion of the medial lobe of the liver was rinsed in chilled saline, wrapped in foil, flash frozen, and stored at - 70°C for potential future bioche mical analysis. The remaining liver and remaining tissues were placed in 10% neutral-buffered formalin for potential future histopathologic examination. After fi xation, tissues were shipped to WIL Research Hillsborough (Hillsborough, NC) for processing. Trimmed tissues were processed into paraffin blocks, sectioned and stained w ith hematoxylin and eosin, and examined microscopically.Female Pubertal Assay (2012) / Page 9 of 17
CYPERMETHRIN / 109702 OCSPP 890.1450/ OECD None



### Five random sections from ovary were evaluate d microscopically for follicular development (including the level of primordial, primary, s econdary, tertiary, and atretic follicles) and changes in the corpus luteum development (g rowth phase, maturity, and regression), in addition to any abnormalities or lesions. It wa s not reported which ovary (left or right) was
examined for each rat. Two sections of each of the two lobes of the thyroid were examined
and evaluated for follicular cell height and colloid area, and any abnormalities/lesions were noted. The thyroid and ovaries were evaluated using a five point grad ing scale. Uterine histology documented changes in myometrial, stromal, or endometrial gland development. The stage of the estrous cycle of the female at the time of necropsy was taken into account in the final histological assessment.

II. RESULTS

A. MORTALITY: One female in the high-dose group (Animal No. 18322-10) was found dead on PND 25. No internal findings were not ed for this animal during gross examination at necropsy. All other rats survived until scheduled sacrifice.

B. CLINICAL SIGNS OF TOXICITY: There were no treatment-related clinical signs of toxicity at any dose level. Fi ndings noted in the treated groups (Table 2), consisting of hair loss around the nose and dorsal head and neck, and red material around the eyes and nose, occurred infrequently in single animals and/or in a manner that was not considered dose related. No clinical sign s were reported for any animals one hour following dose
administration.

TABLE 2. Incidence of Clinical Observations a

Observation Dose Level (mg/kg/day)

Vehicle Control Cypermethrin (15) Cypermethrin (30)

# Observed # Examined # Observed # Examined # Observed # Examined

Hair loss dorsal head 0 15 0 15 1 14

Hair loss dorsal neck 0 15 0 15 1 14

Hair loss around nose 0 15 2 15 0 14 Right eye small 1 15 0 15 0 14 Wet red material around eye 1 15 1 15 0 14

Dried red material around eye 1 15 1 15 0 14

Dried red material around nose 0 15 3 15 0 14 a Data were extracted from Table S1 on page 38 of the study report



### C. GENERAL GROWTH AND VAGINAL OPENING: Body weights, body weight gains, age at attainment of vaginal ope ning (VO) and weight at day of attainment are presented in Table 3. Mean body weights, daily body wei ght gains, and cumulative mean body weight gain in the 15 and 30 mg/kg/day groups we re similar to the control group throughout the study. In addition, mean food consumption (g /cage/day) for both treatment groups was similar to the control group th roughout the trea tment period.

There were no effects of treatment on age or weight at VO. One female (#18313-09) in the control group did not attain complete VO by study termination and showed 11 days ofFemale Pubertal Assay (2012) / Page 10 of 17
CYPERMETHRIN / 109702 OCSPP 890.1450/ OECD None



### incomplete opening. When a second analysis was performed adjusting the day of attainment for this female to be the first day of incomple te VO, the mean age at attainment of VO in the 30 mg/kg/day group (37.1 days) wa s significantly (p<0.05) higher ( ↑ 4%) than the control group (35.6 days). The mean body weight at the age of attainment in the 30 mg/kg/day group was not significantly different from th e control group in the second analysis.

The mean body weight at VO and final body wei ght in the control gr oup were within the range of the performance criteria provided in the Guideline (890.1450). The mean age at attainment of VO in the control group (36.3 days) exceeded the maximum value in the performance criteria (35.62 days); however, the CV value was within the range. When a second analysis was performed as described abov e, the mean age at a ttainment of VO in the control group (35.6 days) was within the performance criteria range (30.67 to 35.62 days). The CV value in the second analys is was also within the range.

TABLE 3. General Growth and Vaginal Opening (VO) a

Parameter Evaluated Vehicle Control Cypermethrin (15 mg/kg/day) Cypermethrin (30 mg/kg/day)

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Initial body weight (PND 22; g) U 15 49.9 3.78 7.57 15 49.3 4. 37 8.87 15 51.0 3.85 7.55

A NA NA NA NA NA NA NA NA NA NA NA NA Body weight at vaginal opening (g) U 15 124.8 12.08 9.68 15 123.3 11. 75 9.53 14 132.9 15.80 11.89

A 15 125.3 NA NA 15 123.9 NA NA 14 132.0 NA NA Final body weight (g) U 15 154.1 11.50 7.46 15 155.4 12. 91 8.31 14 157.6 14.17 8.99

A NR NR NR NR NR NR NR NR NR NR NR NR Final body weight (% of control) U NA NA NA NA 15 0.84 NR NR 14 2.27 NR NR

A NA NA NA NA NR NR NR NR NR NR NR NR Body weight gain (final – initial; g) U 15 104.1 10.09 9.69 15 106.1 10. 48 9.87 14 106.4 12.93 12.15

A NR NR NR NR NR NR NR NR NR NR NR NR Age at vaginal opening (PND) U 15 36.3 2.13 5.85 15 35.7 1. 40 3.92 14 37.1 1.64 4.42

A 15 36.4 NA NA 15 35.7 NA NA 14 37.1 NA NA Proportion unopened (#/N) 1/15 0/15 0/14 a Data were obtained from Table 1 on page 98 of the study report; no statistical differences. 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 NA Not applicable NR Not reported



### D. ORGAN WEIGHTS: Organ weights at necropsy are pres ented in Table 4. It is noted that no explanation was provided for missing pituitar y gland weights from two animals in the control group and for missing ovary weights fr om one animal in the 15 mg/kg/day group.

The mean absolute adrenal gland weight was increased (p<0.05) by 12% for females in the 30 mg/kg/day group compared to the control group.

The mean and CV values for the pituitary, ova ries, uterus, and thyroi d in the control group were within the range of th e performance criteria provided in the Guideline (890.1450). The CVs for kidney (12.66%) and liver (14.14%) wei ghts in the control group exceeded theFemale Pubertal Assay (2012) / Page 11 of 17
CYPERMETHRIN / 109702 OCSPP 890.1450/ OECD None



### performance criteria (maximum 10.76% a nd 13.13%, respectively). However, the differences in the CV values were slight and the mean values of these organ weights were within the ranges noted in the performance criteria. In addition, the mean weight of the adrenal glands (33.6 mg) in the control group was below the minimum value in the performance criteria (48.84 mg); however, the CV value was within the range.

TABLE 4. Organ Weights at Necropsy a

Organ Vehicle Control Cypermethrin (15 mg/kg/day) Cypermethrin (30 mg/kg/day)

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)

Liver (g) U 15 7.31 1.03 14.14 15 7.51 0. 74 9.80 14 7.44 0.83 11.12

A 15 7.32 NA NA 15 7.53 NA NA 14 7.38 NA NA

R 15 4.70 0.42 8.86 15 4.77 0. 27 5.69 14 4.65 0.25 5.41

Kidneys (g) U 15 1.23 0.16 12.66 15 1.26 0. 20 15.74 14 1.27 0.25 19.93

A 15 1.23 NA NA 15 1.26 NA NA 14 1.26 NA NA

R 15 0.79 0.10 12.87 15 0.80 0. 12 14.96 14 0.79 0.10 12.76

Pituitary (mg) U 13 9.3 2.06 22.07 15 9.2 1. 19 12.93 14 9.4 1.61 17.21

A 13 9.3 NA NA 15 9.2 NA NA 14 9.4 NA NA

R 13 6.1 1.29 21.32 15 5.9 0. 93 15.73 14 5.9 0.83 14.09

Adrenals (mg) U 15 33.6 5.12 15.24 15 36.2 4.42 12.21 14 37.5* ( ↑ 12) 4.50 12.03

A 15 33.7 NA NA 15 36.3 NA NA 14 37.2 NA NA

R 15 21.7 2.67 12.33 15 23.1 2. 69 11.65 14 23.4 2.05 8.74

Ovaries (mg) U 15 75.1 15.51 20.64 14 72.9 11. 44 15.68 14 68.2 10.32 15.14

A 15 75.1 NA NA 14 73.3 NA NA 14 67.7 NA NA
Uterus, wet (mg) U 15 280.2 88.88 31.72 15 262.2 87. 35 33.32 14 282.5 146.26 51.77

A 15 276.9 NA NA 15 258.4 NA NA 14 285.1 NA NA Uterus, blotted (mg) U 15 246.8 67.52 27.36 15 232.2 65. 40 28.16 14 226.0 63.65 28.17

A 15 245.5 NA NA 15 230.9 NA NA 14 226.4 NA NA Thyroid, fixed (mg) U 15 7.09 1.39 19.66 15 6.31 1. 58 25.04 14 6.58 1.48 22.51

A 15 7.11 NA NA 15 6.34 NA NA 14 6.51 NA NA a Data were obtained from Summary Table 2 on page 99 of the study report. Percent differences from controls, included in parentheses, and statistical significance were obtaine d from Table S13, on pages 79-84 of the study report. 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) NA Not applicable * Significantly different fro m controls at p<0.05.



### E. ESTROUS CYCLICITY: Estrous cycle data are provided in Table 5. Mean estrous cycle lengths in the 15 and 30 mg/kg/day groups (4.4 and 5.0 days, respectively) were similar to the control group (4.8 days). In addition, no sign ificant differences in the age at the first
occurrence of estrus were noted when the test substance-trea ted groups were compared to the control group. The cycle status at necropsy (diestrus, proest rus, estrus, or not cycling) appeared similar across all groups.

There were no test substance-related effects on normal estrous cycling, as determined by the number and percentage of females that were cycling and cycling regularly and the number of females with normal cycles. Only 44.4% of animals were regularly cycling in the 15Female Pubertal Assay (2012) / Page 12 of 17
CYPERMETHRIN / 109702 OCSPP 890.1450/ OECD None



### mg/kg/day group compared to 80% in the c ontrol group and 100% in the 30 mg/kg/day

group.

TABLE 5. Estrous Cyclicity a

Treatment Groups N Mean Age at First Vaginal Estrus (PND) Mean Cycle Length (days) Cycling (%) Regularly Cycling

(%) Cycle Status at Necropsy (# Females)

Diestrus Proestrus Estrus Not Cycling

Vehicle 14 38.0 4.8 92.9 80.0 9 0 5 1 Cypermethrin (15 mg/kg/day) 15 38.1 4.4 100.0 44.4 12 0 3 0

Cypermethrin (30 mg/kg/day) 14 37.9 5.0 100.0 100.0 11 0 3 0

a Data were obtained from Summary Tables 2 and 3 on page s 99-100 of the study report; no statistical differences.
N Number of animals



### F. CLINICAL CHEMISTRY AND HORMONE LEVELS: Mean serum hormone levels and clinical chemistry parameters are presen ted in Table 6. There were no cypermethrinrelated changes in serum hormone or clinical chemistry levels. The mean and CV values for
serum T 4 

### and TSH values in the control group were within the range of the performance criteria provided in the Guideline (890.1450).

It is noted that mean values in the control group were outside the historical control range for the performing laboratory for ALP, ALT, AST, bile acid, calcium, cholesterol, GGT, glucose, potassium, total bilir ubin, total protein, triglycerides, and urea nitrogen. However, the cypermethrin test groups had mean values similar to the control for each of these parameters.Female Pubertal Assay (2012) / Page 13 of 17
CYPERMETHRIN / 109702 OCSPP 890.1450/ OECD None

TABLE 6. Hormone Levels and Clinical Chemistry a

Parameter
Evaluated Vehicle Control Cypermethrin (15 mg/kg/day) Cypermethrin (30 mg/kg/day)

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)

Hormones

Serum T 4 , Total (μg/dL) 15 4.64 0.76 16.49 15 4.32 0.68 15.81 14 4.23 0.75 17.60 Serum TSH (ng/mL) 15 5.56 2.97 53.48 15 5.17 3.61 69.73 14 4.93 2.22 45.02 Clinical Chemistry
Albumin/Globulin Ratio 15 3.02 0.36 11.85 15 3.09 0.42 13.63 14 3.20 0.31 9.63 Albumin (g/dL) 15 4.2 0.16 3.82 15 4.2 0.25 6.04 14 4.4 0.18 4.10 ALP (U/L) 15 305 58.1 19.0 15 300 48.8 16.3 14 304 44.5 14.6 ALT (U/L) 15 66 13.0 19.6 15 67 6.9 10.4 14 67 10.0 14.8 AST (U/L) 15 174 58.8 33.8 15 173 22.6 13.0 14 167 33.8 20.3 Bile Acid (μmol/L) 15 39.3 42.37 107.73 15 35.0 28.05 80. 20 14 21.9 8.89 40.54 Calcium (mg/dL) 15 12.3 0.77 6.26 15 12.4 0.66 5.32 14 12.6 0.49 3.90 Chloride (mEq/L) 15 102 1.9 1. 9 15 102 1.9 1.9 14 103 1.8 1.8 Cholesterol (mg/dL) 15 90 17.1 19.0 15 86 14.3 16.6 14 95 15.1 15.9 Creatinine (mg/dL) 15 0.1 0.00 0.00 15 0.1 0.03 24.21 14 0.1 0.00 0.00 GGT (U/L) 15 0.1 0.00 0.00 15 0.1 0.00 0.00 14 0.1 0.00 0.00 Globulin (g/dL) 15 1.4 0.19 13.36 15 1.4 0.16 11.83 14 1.4 0.14 9.75 Glucose (mg/dL) 15 144 10.8 7.5 15 142 12.0 8.4 14 150 15.1 10.1 Phosphorus (mg/dL) 15 9.5 0.94 9.88 15 9.8 0.84 8.61 14 10.1 0.72 7.10 Potassium (mEq/L) 15 6.35 0.37 5.76 15 6.47 0.47 7.21 14 6.22 0.30 4.88 Sodium (mEq/L) 15 142 1.7 1. 2 15 142 1.8 1.2 14 143 1.9 1.3 Total Bilirubin (mg/dL) 15 0.05 0.02 34.62 15 0.04 0.02 43.04 14 0.04 0.02 53.27 Total Protein (g/dL) 15 5.6 0.30 5.30 15 5.6 0.31 5.61 14 5.7 0.24 4.19 Triglyceride (mg/dL) 15 94 43.6 46.3 15 128 107.4 83.8 14 111 64.1 57.7 Urea Nitrogen (mg/dL) 15 10.3 1.77 17.19 15 10.9 1.97 18.08 14 11.0 2.73 24.73 a Data were obtained from Summary Tables 4 and 5 on pa ges 101-102 of the study report; no statistical differences.
Bolded mean values were outside the perfo rming laboratory historical control ranges provided in the study report.
N Number of animals examined
SD Standard Deviation
CV Coefficient of Variation ALP Alkaline phosphatase
ALT Alanine aminotransferase AST Aspartate transferase GGT Gamma glutamyl transferase



### F. GROSS PATHOLOGY: No treatment-related effects we re observed on gross pathology.

H. HISTOPATHOLOGY: The incidences of histopathologi cal findings of the thyroid gland are presented below in Table 7. There were no effects of treatment observed on either colloid area or follicular cell height in any group. It is noted that no explanation was provided for the missing thyroid gland data fr om one animal in the 15 mg/kg/day group.Female Pubertal Assay (2012) / Page 14 of 17
CYPERMETHRIN / 109702 OCSPP 890.1450/ OECD None

TABLE 7. Incidence of Histopathol ogical 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 4

15 2 0 2 11

3 2 3 0

4 10 4 0

5 3 5 0

Cypermethrin (15 mg/kg/day) 1 0

14 1 2

14 2 0 2 12

3 0 3 0

4 10 4 0

5 4 5 0

Cypermethrin (30 mg/kg/day) 1 0

14 1 2

14 2 0 2 11

3 4 3 1

4 8 4 0

5 2 5 0 a Data were obtained from Table S14 on page 88 of the study report.
b A 5-point grading scale (1=shortest/smallest; 5=tallest/largest) was used.
O Number Observed
E Number Examined



### The incidence of histopathological findings of the ovaries, uterus, and kidneys are presented
in Table 8. No effects of treatment were obser ved microscopically in th e uterus, ovaries, or kidney.

TABLE 8. Incidence of Histopathological Le sions of the Ovaries, Uterus and Kidney a

Findings Dose Level (mg/kg/day) Vehicle Control Cypermethrin (15) Cypermethrin (30)

# Observed # Examined # Observed # Examined # Observed # Examined

Uterus Unremarkable 15 15 15 15 14 14

Ovaries Unremarkable 15 15 15 15 14 14 Corpora Lutea Growth Grade 3 b 15 15 15 15 14 14 Corpora Lutea, Mature Grade 3 15 15 15 15 14 14 Corpora, Lutea, Regressive Grade 3 15 15 15 15 14 14 Corpora Lutea, Atretic Grade 3 15 15 15 15 14 14 Follicles, Primary Grade 3 15 15 15 15 14 14 Follicles, Primordial Grade 3 15 15 15 15 14 14 Follicles, Secondary Grade 3 15 15 15 15 14 14 Follicles, Tertiary Grade 3 15 15 15 15 14 14 Kidney Basophilic tubules Minimal 3 15 0 15 0 14

Tubular dilatation Minimal 1 15 0 15 0 14

Fibrosis Minimal 0 15 1 15 0 14

Mild 1 15 1 15 0 14

Mineralization Minimal 0 15 1 15 0 14

a Data were obtained from Table S14 on pages 85-88 of the study report. b Grade 3 = Number of structures comparable to that found in a cycling female.Female Pubertal Assay (2012) / Page 15 of 17
CYPERMETHRIN / 109702 OCSPP 890.1450/ OECD None



### III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: There was no evidence of any direct test substance-related estrogenic or anti-estrogenic effects, nor wa s there any evidence of direct test substance-related effects on pubertal development or thyroid function in the juvenile/peripubertal female rat at cyperm ethrin dose levels of 15 and 30 mg/kg/day.

B. AGENCY COMMENTS: One animal in the 30 mg/kg/day group was found dead on PND
25. All other rats survived unt il scheduled sacrifice. As there was no other evidence of toxicity in this study, this single death was considered in cidental. There were no treatmentrelated clinical signs of toxi city at any dose level. Mean body weights, daily body weight gains, cumulative mean body weight gain, and food consumption in the 15 and 30 mg/kg/day groups were similar to the c ontrol group throughout the study.

No treatment-related effects were observed on m ean age or weight at VO, and mean age at first estrus. The cycle status at necropsy (d iestrus, proestrus, es trus, or not cycling) appeared similar across all groups. There were no cypermethrin-related changes in serum hormone or chemistry levels. In addition, th ere were no effects of treatment observed in gross or microscopic pathology.

When a second analysis was performed adjusting the day of attainment for one female to be the first day of incomplete VO, the mean age at attainment of VO in the 30 mg/kg/day group (37.1 days) was significan tly (p<0.05) higher ( ↑ 4%) than the control group (35.6 days). The mean body weight at the age of attainment in the 30 mg/kg/day group was not significantly different from the control group in the second an alysis. This minor change was considered
incidental in the absence of other e ffects on VO or anti-estrogenic effects.

The mean absolute adrenal gland weight was increased (p<0.05) by 12% for females in the 30 mg/kg/day group compared to the control gr oup. This was a minor change, related to body weight; adjusted and rela tive adrenal gland weights we re similar to controls.

There was no evidence of direct test substa nce-related effects on pube rtal development or thyroid function in the ju venile/peripubertal female rat at cy permethrin dose levels of 15 and 30 mg/kg/day.

No evidence of toxicity was observed in th e study up to the highest dose tested; however, treatment-related clinical signs were seen in the preliminar y range-finding study at 30 and 65 mg/kg/day. In the preliminar y study, tremors and clear material around the mouth were seen at 30 mg/kg/day, 4 hours after administ ration. At 65 mg/kg/ day (after 4 hours), tremors, splayed limbs, and clear material around the mouth, ventral neck and thoracic area, and forelimbs were seen. It is possible that the effects observed in the preliminary study were missed in the definitive study since clinical signs were measured 1 hour after chemical administration. Clinical signs were measured 4 hours after administratio n in the preliminary study, which is closer to the time-to-peak eff ect for cypermethrin. Therefore, the doses tested were considered to be adequate.Female Pubertal Assay (2012) / Page 16 of 17
CYPERMETHRIN / 109702 OCSPP 890.1450/ OECD None



### C. STUDY DEFICIENCIES: The following minor deficiencies were noted that are not considered to have had an adverse impact on th e results, interpretation or conclusions of this study:

 The mean age of attainment of VO in the control group (36.3 days) exceeded the maximum value in the performance criteria ( 35.62 days). When a second analysis was performed, adjusting the day of attainment to be the first day of incomplete VO for the female with 3 or more days of incomplete opening, the mean age of attainment of VO in the control group (35.6 days) was within the performance criter ia (30.67-35.62 days).

 The CVs for kidney (12.66%) and liver (14.14% ) weights in the control group exceeded the performance criteria maximum (10.76% and 13.13%, respectively).

 The mean weight of the adrenal glands in the control group (33.6 mg) was below the minimum value in the perfor mance criteria (48.84 mg).Female Pubertal Assay (2012) / Page 17 of 17
CYPERMETHRIN / 109702 OCSPP 890.1450/ OECD None



### APPENDIX

Since this is a range-finding study, only a summary is provided to confirm the adequacy of the
dose selection rationale used in the Female Pubertal Assay.

In a dose range-finding assay (MRID 48683004), cype rmethrin (95.2% a.i.; cis/trans ratio: 49.8/50.2; Batch #PL10-0492) in corn oil was admi nistered daily via or al gavage in a dose volume of 5 mL/kg to four juvenile female (PND 22) and male (PND 23) Sprague Dawley [Crl:CD(SD)] rats/dose group at doses of 0, 5, 15, 30, or 65 mg/kg/day. The animals were dosed for 14 consecutive days and necropsied one day af ter the final dose administration. Adrenals, kidneys, and liver were collected and weighed. Only the results fo r the female rats are discussed in this appendix; the results for the male rats are summarized in the DER for MRID 48683003 (concurrently reviewed).

One female in the 65 mg/kg/day group was found dead on Day 2; this female had splayed
hindlimbs and clear material around the mouth 4 hours following dosing on Day 1. Slight to
moderate tremors and wet or dried clear mate rial around the mouth were noted 4 hours following dosing in the remaining females in the 65 mg/k g/day group. Two of these females also had splayed hindlimbs. It was concluded that a do se of 65 mg/kg/day would not be suitable for continued dosing; therefore, all remaining fema les in the 65 mg/kg/day group were euthanized on Day 2. All other rats survived to scheduled termination.

Clinical findings noted 4 hours following dos e administration in the 30 mg/kg/day group consisted of slight to moderate tremors and cl ear material around the mouth and on the ventral neck, ventral thoracic area, and fo relimbs (beginning as early as Da y 3). At 4 hours post-dose at 30 mg/kg/day, 4/4 females displayed tremors on 12/ 54 observations. Clinical findings in the 5 and 15 mg/kg/day group occurred infrequently and were not observed in a dose-related manner.

A statistically significant, mean body weight lo ss of 0.7 g was noted in the 65 mg/kg/day group females compared to a mean body weight gain of 4.1 g in the control group during Days 1-2, resulting in a mean body weight that was 6.0% lower (not statis tically significant) than the control group on Day 2. Markedly lower food c onsumption was noted in this group during Days 1-2 compared to the control group. Slightly lo wer mean body weight gain and food consumption was noted in the 30 mg/kg/day group females dur ing Days 1-2; mean body weight gains and food consumption in these females were generally similar to the contro l group for the remainder of the study and none of the differences from the control group were sta tistically significant. Mean body weights, body weight gains, and food consumption in the 5 and 15 mg/kg/day groups were unaffected by test s ubstance administration.

Mean absolute and relative organ weights in the 5, 15, and 30 mg/kg/day group females were unaffected by test substance administration.

There were no macroscopic findings at necropsy in the 5, 15, or 30 mg/kg/day groups or in the rat in the 65 mg/kg/day group that was found dead on Day 2. The rats in the 65 mg/kg/day group that were euthanized due to dose-group termin ation were discarded without examination.

COMPLIANCE: It was stated that the study was not performed in comp liance with GLPs.

# DATA EVALUATION RECORD

CYPERMETHRIN

Study Type: OCSPP 890.1500, Male Pubertal Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-69-2012 (MRID 48683003)

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: Sandra Hastings Date: 6/20/2012

Secondary Reviewer: Signature: Kelly Luck, M.S. Date: 6/28/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 7/10/2012

Quality Assurance: Signature: Steven Brecher, Ph.D., D.A.B.T. Date: 7/9/2012

This Data Evaluation Record may have been alte red 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 in tended 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 re dundancy 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 potent ial 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).CYPERMETHRIN I 109702



### Primary Reviewer: Karlyn Middleton
Health Effects Division (7509P) Secondary Reviewer: John Liccione, Ph.D

. Health Effects Division (7509P) Male Pubertal Assay (2012) I Page J of 15
OCSPP 890.1500 I OECD None



### DATA EVALUATION RECORD

STUDY TYPE: Male Pubertal Assay; OCSPP 890.1500

PC CODE: 109702 DP BARCODE: D401652

TXR#: 0052288 CAS No.: 52315-07-8

TEST MATERIAL (PURITY): Cypermethrin (95.2% a.i

. ; cis / trans ratio 49.8 / 50.2)

SYNONYMS: Cyano(3-phenoxyphenyl)methyl 3-(2,2-dichloroethenyl)-2,2- dimethylcyclopropanecarboxylate

CITATION: Herberth

, M.T

. (2012) Cypermethrin

- Pubertal Development and Thyroid
Function in Intact Juvenile / Peripubertal Male Rats. WIL Research Laboratories, LLC

, Ashland

, OH. Laboratory Project ID Number: WIL639151

, April 9

, 2012

. MRID 48683003. Unpublished.

SPONSOR: Sawhney Coder

, P. (2011) Cypermethrin

- A 14-Day Dose Range-Finding Oral (Gavage) Toxicity Study in Juvenile Rats. WIL Research Laboratories, LLC

, Ashland

, OH

. Laboratory Project ID Number: WIL-639149, December 19,
2011. MRID 48683004. Unpublished

.

Syngenta Cop Protection

, LLC

, Greensboro, NC ; FMC Corporation, Philadelphia

, PA ; and United Phosphorus

, Inc., King of Prussia

, PA

TEST ORDER#: CON-109702-8

EXECUTIVE SUMMARY: In a Male Pubertal Assay (MRID 48683003)

, 15 Sprague Dawley

rats / dose group were treated daily via oral gavage with cypermethrin (95.2% a.i.; cis / trans ratio:
49

. 8 / 50.2 ; Batch #PLl0-0492) in com oil at doses ofO (com oil)

, 15

, or 30 mg / kg / day from postnatal day (PND) 23 to 53 / 54. Animals were examined for preputial separation (PPS) daily beginning on PND 30 and weight at day of attainment was recorded. Following sacrifice on PND 53 or 54

, total serum testosterone and thyroxine (T4) levels were determined using an electrochemiluminescent immunoassay

, and thyroid stimulating hormone (TSH) levels were determined using a radioimmunoassay. Adrenal

, liver

, thyroid

, pituitary

, and urogenital organ weights were recorded

, and microscopic examinations of the testis, epididymis

, thyroid, and kidney were performed

.

One male in the 15 mg / kg / day group was found dead approximately one hour following dosing

on PND 45

, which was likely due to dosing error. All other rats survived until scheduled
sacrifice. There were no treatment-related clinical signs of toxicity at any dose level. FindingsMale Pubertal Assay (2012) / Page 2 of 15
CYPERMETHRIN / 109702 OCSPP 890.1500 / OECD None



### noted in the treated groups, in cluding red and/or clear materi al around the nose and/or mouth, occurred infrequently in single animals and/or in a manner that was not considered dose related. Mean body weights, daily body weight gains, cumulative mean body weight gain, food consumption, mean age of attainment of comp lete PPS, and mean body weight at the age of attainment in the 15 and 30 mg/kg/day groups we re similar to the control group throughout the study.

No treatment-related gross observa tions were noted at necropsy and no significant effects of treatment were observed on the weights of a ny of the organs and tissues collected.

There were no test substance-related changes in serum T 4 

### , TSH and testosterone values at any dose level. Urea nitrogen ( ↓ 17%) and bile acid ( ↓ 52%) were decreased (p<0.05) in the 30 mg/kg/day group compared to controls. Howeve r, decreases in bile acids have not been associated with toxicological effects. The slig ht decrease in blood urea nitrogen was within the historical control reference range and considered an incidental finding. There were no other serum chemistry changes in the cypermethrin -dosed groups compared to the control group.

There were no test substance-rela ted histologic changes in the t hyroid, testes, epididymides, and kidneys. The amount of colloid and follicular height of thyroi d epithelial cells were similar
between control and test substance-treated animals.

No evidence of toxicity was observed in th e study up to the highest dose tested; however, treatment-related clinical signs were seen in the preliminar y range-finding study at 30 and 65 mg/kg/day. In the preliminary study, tremors and clear material around the mouth were seen at 30 mg/kg/day, 4 hours after administration. At 65 mg/kg/day (after 4 hours), tremors, splayed limbs, and clear material around the mouth, ventra l neck and thoracic area, and forelimbs were seen. It is possible that th e effects observed in the prelim inary study were missed in the definitive study since clinical signs were meas ured 1 hour after chemical administration. Clinical signs were measured 4 hours after ad ministration in the preliminary study, which is closer to the time to peak effect for cyperm ethrin. The dose levels used in the study are considered adequate based on the results of the range-findi ng study.

The assay satisfies the EDSP Tier 1 Test Or der requirements for a Male Pubertal Assay (OCSPP 890.1500).

COMPLIANCE: Signed and dated Data Confiden tiality, GLP Compliance, and Quality Assurance statements were provided.Male Pubertal Assay (2012) / Page 3 of 15
CYPERMETHRIN / 109702 OCSPP 890.1500 / OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: WIL Research Laboratories, LLC

Location: Ashland, OH Study Director: M.T. Herberth
Other Personnel: E.S. Bodle (Assistant Director, Analytical Chemistry); S.C. Haley (Operations Manager, Pathology); S.A. Keets (Senior Operations Ma nager, Vivarium); C.A. Kopp (Manager, Gross Pathology and Developmental Toxicology Laboratory); G.M. Maginnis (Clinical Veterinarian); J.S. Moore (Operations Ma nager, Developmenta l, Reproductive and Neurotoxicology); T.M. Rafeld (Group Manage r, Formulations Laboratory); M.A. Safron (Manager, Histology); M.J. Schlosser (VP, Analytical, Metabolism, & In Vitro Services); B.J. Varsho (Director, Operations); E.C. Vedaa (Manager, Clinical Pathology); R.A. Wally (Operations Manager, Reporting & Technical Support Services); R. Green (Syngenta Study Monitor); L. Freshwater (Princip al Investigator, Statistical Analysis); K.S. Marks (Principal Investigator, Histology Slide Preparation); C.A. Picut (Participating Scientist, Pathology); A.M. Stanton (Participating Scientist, Analytical Chemistry) Study Period: May 11, 2011 to April 9, 2012



### 2. Test Substance: Cypermethrin
Description: Technical; dark yellow, very viscous liquid
Source: FMC Innovation Center (Ewing, NJ)
Lot/Batch #: PL10-0492 (expiry date: December 15, 2012) Purity: 95.2% a.i.; cis/trans ratio: 49.8/50.2 Stability: Formulations in corn oil are stable for 19 days of room temperature and refrigerated storage
CAS #: 52315-07-8
Structure:



### 3. Vehicle: Corn oil ( expiry date: July 1, 2012 & May 4, 2013)



### 4. Test Animals:
Species: Rat (male only)
Strain: Sprague Dawley [Crl:CD(SD)] Age/Weight at Study
Initiation: PND 23 / 47.7–66.7 g

Source: Charles River Laboratorie s, Inc. (Portage, MI) Housing: Male weanlings were housed three per cage in pl astic cages containing ne sting material (Aspen BedTM)
Diet: 2016CM Teklad Global 16% Protein Rode nt Diet (Harlan Laboratories), ad libitum ; average 29.0 and 11.0 ppm total isoflavones (genistein+dai dzein+glycitein) for two lots of feed used.

Water: Reverse-osmosis purifie d (on-site) tap water , ad libitum
Environmental
Conditions: Temperature: Humidity: Air changes: Photoperiod: 21.2-22.0°C (mean daily temperature) 42.1-58.7% (mean daily humidity)
≥ 10/hr 14 hrs light/ 10 hrs dark



### B. STUDY DESIGN

1. In-Life Dates: Start: August 7, 2011 End: September 7, 2011

2. Mating: Timed-pregnant dams were received fro m the supplier on gestation day 7. The CN O

O Cl O

Cl CH 3 CH

3Male Pubertal Assay (2012) / Page 4 of 15
CYPERMETHRIN / 109702 OCSPP 890.1500 / OECD None



### day when parturition was first observed was de signated as PND 0 for the offspring. On PND 4, litters were culled to 5 male and 5 female pups/litter.

3. Animal Assignment: Following weaning on PND 21, juvenile male animals were weighed and examined in detail for physical abnormalities. Rats were then assigned to the test groups noted in Table 1 using a computer pr ogram which randomized the animals based on stratification of body weights in a block design; no littermates were a ssigned to the same dose group.

TABLE 1. Study Design a Test group Dose (mg/kg/day) # of Males Control (corn oil) 0 15 Low (cypermethrin) 15 15 High (cypermethrin) 30 15 a Data were obtained from page 21 of the study report.



### 4. Dose Selection Rationale: The dose levels were selected based on the results from a 14-day dose range-finding study (MRID 48683004). A summary of the study is included as an Appendix to this DER.

5. Dose Preparation and Analysis: Cypermethrin dose formulations were prepared in corn oil weekly as single formulations for each dos e level, divided into aliquots for daily dispensation, and stored refrigerated. The test substance formulations were stirred continuously throughout the preparation, sampli ng, and dose administration procedures.

Analyses to demonstrate the homogeneity, stab ility, and resuspension homogeneity of the
test substance formulations at concentra tions of 0.1 to 20 mg/mL following 4, 11, and 19 days of room temperature and refrigerated storage were conducted in a previous study. 1 

### Therefore, homogeneity and stab ility analyses were not condu cted during the current study. The first dosing formulations were inspect ed and found to be visibly homogeneous. Samples for concentration analysis were colle cted from the middle stratum of each dosing formulation prepared during th e in-life phase of the study. All analyses were conducted by the WIL Research Analytical Chemistry Department.

Results of Dose Analysis

Homogeneity: Not reported

Stability: Formulations are reportedly stable during 19 days of room temperature or refrigerated storage

Concentration (% of nominal): 96.5-114%

The analytical data indicated that the varia tion between nominal and actual dosage to the animals was acceptable.

1 Bodle, E.S. (2012) Analytical Validation and Stability St udy of Cypermethrin in Corn Oil Formulations (Study No. WIL-639153). WIL Research Laboratories, LLC, Ashland, OH.Male Pubertal Assay (2012) / Page 5 of 15
CYPERMETHRIN / 109702 OCSPP 890.1500 / OECD None



### 6. Dosage Administration: All doses were administered once daily by gavage from PND 23 through PND 53 (seven animals in each treatme nt group) or 54 (remaining animals in each treatment group), in a volume of 5.0 mL/kg of body weight. Individual dosages were based on the most recently recorded body weights. Dosing was performed between 0700 and 0900 hours daily.

7. Statistics: Pathology findings presen ting as a dichotomous response were analyzed with pairwise Fisher’s exact tests to compare each treated group with the cont rol. The tests were one-sided at the 0.05 significan ce level testing for an increas e and were conducted by the study facility.

The remaining statistical analyses were c onducted by BioSTAT Consultants, Inc., using SAS® version 9.2 (SAS Institute, Inc., 2002-2008) , or higher, software. Data that were statistically analyzed included the organ wei ghts, organ weights to necropsy body weight ratio (liver, kidneys, pituitary, and adrenal gl ands), daily body weights (including necropsy body weight), cumulative body weight gain, seru m chemistries, and preputial separation data. Gamma glutamyl transferase values unde r range were assigned a value of 0.1 (half the lower limit of quantitation) for st atistical analysis and reporting.

Each endpoint was tested for homogeneity of vari ance using Levene's test . If that test was significant at p=0.01, then a log transform was applied and Levene's test conducted on the
transformed data. If that test was still significant then the square root transformation was applied to the raw data and Leve ne's test conducted again. If the test was still significant then a nonparametric test, as described below, was used to analyze the data.

If the variances were homogeneous then an analysis of variance (ANOVA) was conducted, on the raw or transformed data, as appropriate. The statistical model contained a factor for treatment group and a blocking factor base d on the study necropsy date. A two-sided Dunnett's test was conducted, regardless of the outcome of the ANOVA, looking for significant differences in the test substance groups when compared with the control group. If the transformations were unsuccessful in making the variances homogeneous, then the nonparametric Kruskal-Wallis test was use d, ignoring the blocking factor, followed by Dunn's test, to compare each of the test s ubstance groups with the control group. Since these are pre-planned pairwise comparisons, D unn's test was conducted regardless of the
outcome of the Kruskal-Wallis test. The tests were two-sided, at the 0.05 significance level, looking for significant differences from the vehicle control.

In addition, organ weights and preputial sepa ration data were subject to the following analyses if they met the homogeneity of vari ance criteria: 1) analysis of covariance (ANCOVA) with Dunnett's test using PND 21 b ody weight as a covari ate; 2) linear trend test using the ANOVA model; and 3) linear trend test using the ANCOVA model.

Histopathology findings presented as a graded response were analyzed with pairwise MannWhitney U tests to compare each treated group with the control group. The tests were onesided at the 0.05 significance level testing for in creased severity. The tests were two-sided for graded responses presented on a Grade 1-5 sc ale at the 0.05 significance level testing for increased or decreased severity. Exact p-va lues were calculated for the Mann-Whitney U

test.Male Pubertal Assay (2012) / Page 6 of 15
CYPERMETHRIN / 109702 OCSPP 890.1500 / OECD None



### C. METHODS

1. Mortality and Clin ical Examinations: All animals were examined twice daily for mortality and moribundity. Indivi dual detailed physical examin ations were recorded on the day of randomization and daily (prior to test substance administration during the treatment period) through the day of euthanasia. Animals were also observed for signs of toxicity approximately one hour following dose administration.

2. Body Weight: Body weights were recorded for allo cation of the animals to study groups, daily prior to dosing, and prio r to scheduled termination.

3. Food Consumption: Food consumption was recorded da ily prior to test substance administration on a per cage basis. Food intake was calculated and repo rted as g/cage/day.

4. Preputial Separation (PPS): Beginning on PND 30, all animals were examined daily for onset of PPS. In addition, the appearance of partial and complete preputial separation or a persistent thread of tissue be tween the glans and prepuce wa s recorded. The age and body weight on the day at which complete preput ial separation was observed was recorded for each animal. However, if any animal with in any group showed incomplete separation (persistent threads) for greater than 3 days, a second analysis was c onducted using the age at which incomplete separation was first observe d. Examination of the males was continued daily until complete preputial separation was attained or until euthanasia.

5. Sacrifice and Pathology: On the day before euthanasia, all animals were moved to a holding room separate from where the necr opsies were performed. On the day of euthanasia, dosing was performed in the holdi ng room; the holding room was not disturbed except for the removal of the next animal to be euthanized. Only animals to be euthanized next were transferred from the holding room to the room where the euthanasia was performed. On PND 53/54, begi nning approximately 2 hours post-dosing, all surviving animals were sacrificed by decapitation without anesthesia; sacrific es were completed by approximately 1300 hours. Blood from the trunk of the animals was collected, serum was separated, and serum was divided equally into three tubes. One tube was used for serum chemistry evaluation, and the remaining two tubes were used for hormone analyses (analyzed fresh or stored frozen at ≤− 20 °C for subsequent analysis).

a. Hormone Analysis: T 4 

### and testosterone levels were determined using an electrochemiluminescent immunoassay and TS H levels were determined using a [ 125 

### I]rTSH radioimmunoassay.

b. Clinical Chemistry: The following CHECKED (X) para meters were examined. The study report provided reference ranges, based on histori cal control data, for the clinical chemistry parameters

.Male Pubertal Assay (2012) / Page 7 of 15
CYPERMETHRIN / 109702 OCSPP 890.1500 / OECD None

ELECTROLYTES OTHER

X Calcium X Albumin

X Chloride X Creatinine* Magnesium X Urea nitrogen* X Phosphorus X Total cholesterol

X Potassium X Globulins (by calculation)

X Sodium X Glucose

ENZYMES X Total bilirubin
X Alkaline phosphatase (ALP) X Total protein Cholinesterase (ChE) X Triglycerides Creatine phosphokinase Se rum protein electrophoresis Lactic acid dehydrogenase (LDH) X Al bumin/globulin ratio (by calculation) X Alanine aminotransferase (ALT) X Bile acids X Aspartate aminotransferase (AST) Sorbitol dehydrogenase X Gamma glutamyl transferase (GGT) Glutamate dehydrogenase
* Recommended based on guideline 890.1500.



### c. Organ Weights and Histopathology: The following CHECKED (X) tissues were collected and weighed. The (XX) organs, in addition, were subject ed to histological
examination

UROGENITAL OTHER XX Testes (left and right separately)* + XX Thyroid* + XX Epididymides (left and right separately)* + X Liver* X Seminal vesicle plus coagulating glands (w ith and without fluid)* X Adrenals (paired)* X Ventral prostate* X Pituitary* X Dorsolateral prostate* X Levator ani-bulbocavernosus (LABC) muscle complex* XX Kidneys (paired)* + * Weights required base d on guideline 890.1500 + Histopathological examination re quired based on guideline 890.1500



### The testis and epididymis (l eft and right) and kidneys were weighed prior to fixation. Following weighing, the testis and epididym is were fixed in Bouin’s solution for approximately 24 hours, rinsed, a nd stored in ethanol solution. The thyroid glands with attached trachea and the kidneys were fixed in 10% buffered formalin for at least 24 hours. The kidney was then placed in ethanol solution until histological processing. The thyroid was dissected from the trachea following fixatio n, blotted, weighed, and placed in ethanol solution until processing. In addition, the right portion of the medial lobe of the liver was rinsed in chilled saline, wrapped in foil, flash frozen, and stored at -70°C for potential future biochemical analysis. The remaining liver and remaining tissues were placed in 10% neutral-buffered formalin for potential futu re histopathological examinations. After fixation, tissues were shipped to Biotechni cs, LLC (a division of WIL Research; Hillsborough, NC) for processing. Trimmed tissues were processed into paraffin blocks, sectioned, and stained with hematoxylin and eo sin, except testes and epididymides (right),
which were stained with PAS and hema toxylin, and examined microscopically.

The thyroid sections (minimum of 2 sections from each of the 2 lobes) were subjectively evaluated for follicular epithelial height and colloid area using a five-point grading scaleMale Pubertal Assay (2012) / Page 8 of 15
CYPERMETHRIN / 109702 OCSPP 890.1500 / OECD None



### (1=shortest/smallest; 5=tallest/largest) and any abnormalities/lesions were noted.

II. RESULTS

A. MORTALITY: One male in the low-dose group (Animal No. 18307-04) was found dead approximately one hour following dosing on P ND 45. This animal expelled the dose and was noted as struggling during dosing on PND 45. Macroscopic findings of dark red discoloration and lungs not fully collapsed were noted at ne cropsy. This death was likely due to dosing error, given the timing of d eath relative to dosing and the fact that no mortalities occurred at the higher dose. All other rats survived until scheduled sacrifice.

B. CLINICAL SIGNS OF TOXICITY: There were no treatment-related clinical signs of toxicity at any dose level. Findings noted in the treated groups (T able 2), including red
and/or clear material around th e nose and/or mouth, occurred in frequently, in single animals,
and/or in a manner that was not considered dose related.

TABLE 2. Incidence of Clinical Observations a

Observation Dose Level (mg/kg/day)

Vehicle Control Cypermethrin (15) Cypermethrin (30)

# Observed # Examined # Observed # Examined # Observed # Examined

Detailed Examinations

Hair loss forelimbs 1 15 0 14 0 15

Dried red material
around nose 0 15 1 14 6 b 15

Dried red material
around mouth 0 15 1 14 1 15

At Dosing Salivation prior to dosing 0 15 2 14 1 15

One-Hour Post Dosing

Dried red material
around nose 0 15 2 14 0 15

Wet clear material
around mouth 4 15 1 14 6 15

Dried red material
around mouth 0 15 1 14 2 15

a Data were extracted from Tables S1 a nd S2 on pages 37-38 of the study report.
b Total occurrence was 8/479.



### C. GENERAL GROWTH AND PREPUTIAL SEPARATION: Body weights, body weight gains, age of attainment of PPS and weight at day of PPS are presented in Table 3. Mean body weights, daily body weight gains, and cumu lative mean body weight gain in the 15 and 30 mg/kg/day groups were similar to the cont rol group throughout the study. In addition, mean food consumption (g/cage/day) for both treatment groups was similar to the control group throughout the treatment period.

Mean ages of attainment of complete PPS and mean body weights at the age of attainment were unaffected by test substance administrati on. When a second an alysis was performed, adjusting the day of attainment for those males with 3 or more consecutive days ofMale Pubertal Assay (2012) / Page 9 of 15
CYPERMETHRIN / 109702 OCSPP 890.1500 / OECD None



### incomplete separation (persistent threads), no significant delays in the mean age of
attainment of PPS were noted in the treatment groups comp ared to the controls.

The mean values for the weaning weight, fina l body weight, age at PPS, and body weight at PPS in the control group were within the rang e of the performance cr iteria provided in the Guideline (890.1500). The CV for the body wei ght at PPS (9.74%) and final body weight (9.01%) were greater than the maximum valu es in the performance criteria (7.57% and 7.47%, respectively); the CVs for weaning weight and age at PPS were within the range.

TABLE 3. General Growth and Preputial Separation (PPS) a

Parameter Evaluated Vehicle Control Cypermethrin (15 mg/kg/day) Cypermethrin (30 mg/kg/day)

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Initial body weight (PND 23; g) U 15 57.1 4.43 7.75 15 57.4 4. 59 8.00 15 56.6 5.14 9.09

A NA NA NA NA NA NA NA NA NA NA NA NA Body weight at PPS (g) U 15 223.8 21.79 9.74 15 225.3 22. 04 9.78 15 221.3 30.91 13.97

A 15 215.8 NA NA 15 219.0 NA NA 15 214.7 NA NA Final body weight (g) U 15 287.2 25.86 9.01 14 282.9 24. 01 8.49 15 279.9 35.77 12.78

A NR NR NR NR NR NR NR NR NR NR NR NR Final body weight (% of control) U NA NA NA NA 14 -1.5 NR NR 15 -2.54 NR NR

A NA NA NA NA NR NR NR NR NR NR NR NR Body weight gain (final – initial; g) U 15 230.1 23.55 10.24 14 226.1 21. 35 9.45 15 223.3 32.82 14.70

A NA NA NA NA NA NA NA NA NA NA NA NA Age at PPS (PND) U 15 45.2 2.27 5.03 15 45.1 2. 34 5.2 15 45.4 2.80 6.16

A 15 44.2 NA NA 15 44.2 NA NA 15 44.3 NA NA Proportion unseparated (#/N) 0/15 0/15 0/15

a Data were obtained from Table 1 on page 106 of the study report; no statistical differences. 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 NA Not applicable NR Not reported



### D. ORGAN WEIGHTS: Organ weights at necropsy are pr esented in Table 4. No significant effects of treatment were observed on the weight s of any of the organs and tissues collected. However, a dose-related decrease (NS) was observed for the dorsolateral prostate weight at 15 mg/kg/day ( ↓ 7%) and 30 mg/kg/day ( ↓ 14%) compared to the controls.

The mean and CV values for organ weights in the control group were w ithin the range of the performance criteria provided in the Guid eline (890.1500) with the following exceptions: (i) the CV for the pituitary (17.35%) weight exceeded the performance criteria maximum (15.98%); (ii) the CV for thyroid (25.05%) weight exceeded the performance criteria maximum (23.63%); (iii) the mean of the thyroid weight (13.20 mg) was below the minimum value in the performance criteria ( 14 mg); and (iv) the mean kidney weight (2.03 g) was below the minimum value in the performance criteria (2.242 g).Male Pubertal Assay (2012) / Page 10 of 15
CYPERMETHRIN / 109702 OCSPP 890.1500 / OECD None

TABLE 4. Organ Weights at Necropsy a

Organ Vehicle Control Cypermethrin (15 mg/kg/day) Cypermethrin (30 mg/kg/day)

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)

Liver (g) U 15 13.59 1.413 10.399 14 13.33 2.025 15.192 15 12.91 2.061 15.973

A 15 13.51 NA NA 14 13.40 NA NA 15 12.94 NA NA

R 15 4.65 0.212 4.554 14 4.62 0.479 10.367 15 4.54 0.339 7.460 Kidneys (g) U 15 2.03 0.159 7.829 14 2.04 0.158 7.781 15 1.92 0.177 9.194

A 15 2.02 NA NA 14 2.04 NA NA 15 1.93 NA NA

R 15 0.70 0.042 6.091 14 0.71 0. 047 6.61 15 0.68 0.047 6.845 Pituitary (mg) U 15 11.5 2.00 17.35 14 10.9 1. 01 9.28 15 12.3 2.68 21.86

A 15 11.5 NA NA 14 10.8 NA NA 15 12.3 NA NA

R 15 3.9 0.59 15.01 14 3.8 0. 41 10.76 15 4.3 0.87 20.14
Adrenals (mg) U 15 42.2 7.25 17.19 14 44.5 5. 24 11.77 15 42.4 7.42 17.51

A 15 42.1 NA NA 14 44.2 NA NA 15 42.5 NA NA

R 15 14.4 2.00 13.83 14 15.5 2. 26 14.54 15 15.0 2.32 15.51
Seminal vesicle + coagulating gland, with fluid (mg) U 14 b 691.8 125.38 18.12 14 659.4 128. 39 19.47 15 652.7 148.86 22.81

A 14 687.4 NA NA 14 656.1 NA NA 15 654.8 NA NA

Seminal vesicle + coagulating gland, without fluid (mg) U 15 415.9 51.69 12.43 14 398.6 43. 85 11.00 15 386.8 73.07 18.89

A 15 414.1 NA NA 14 397.8 NA NA 15 388.0 NA NA

Ventral prostate (mg) U 15 227.4 36.37 15.99 14 205.8 32.4 15.74 15 223.0 39.88 17.89

A 15 226.6 NA NA 14 204.7 NA NA 15 223.7 NA NA Dorsolateral prostate (mg) U 15 135.7 35.15 25.91 14 126.0 ( ↓ 7) 23.79 18.87 15 117.2 ( ↓ 14) 35.55 30.33

A 15 134.4 NA NA 14 124.4 NA NA 15 118.3 NA NA

LABC (mg) U 15 514.1 60.48 11.76 14 507.9 57. 55 11.33 15 528.1 81.67 15.47

A 15 511.3 NA NA 14 507.5 NA NA 15 529.9 NA NA Epididymis, left (mg) U 15 197.6 18.61 9.42 14 192.5 21.37 11.10 15 191.6 23.92 12.48

A 15 197.3 NA NA 14 191.2 NA NA 15 192.1 NA NA Epididymis, right (mg) U 15 204.1 19.07 9.34 14 196.9 23.70 12.04 15 198.0 26.59 13.43

A 15 203.9 NA NA 14 195.5 NA NA 15 198.3 NA NA Testis, left (mg) U 15 1472.4 82.58 5.61 14 1506.3 139.41 9.26 15 1473.4 177.64 12.06

A 15 1470.9 NA NA 14 1508.4 N A NA 15 1473.9 NA NA Testis, right (mg) U 15 1495.3 120.81 8.08 14 1546.0 160.51 10.38 15 1481.5 146.78 9.91

A 15 1495.6 NA NA 14 1546.8 N A NA 15 1481.3 NA NA Thyroid, fixed (mg) U 15 13.20 3.306 25.05 14 12.63 2.26 17.891 14 c 13.29 2.033 15.293

A 15 13.17 NA NA 14 12.72 NA NA 14 13.26 NA NA a Data were obtained from Table 2 on page 107 and Table S12 on pages 87-95 of the study report. Percent differences from controls are included in parentheses. b For one animal, seminal vesicle fl uid was lost prior to weighing. c Thyroid gland weight for one rat was not included as one lobe of the thyroid gland was missing. U Unadjusted for body weight on PND 21 A Adjusted for body weight on PND 21 R Organ-to-body weight ratio (relative to body weight)
N Number of animals examined
SD Standard Deviation
CV Coefficient of Variation



### E. CLINICAL CHEMISTRY AND HORMONE LEVELS: Mean hormone and clinical chemistry levels are presented in Table 5. Th ere were no test substa nce-related changes in
serum T 4 

### , TSH, and testosterone values at any dose level. The mean and CV values for
serum T 4 

### , TSH, and testosterone values in the co ntrol group were within the range of the performance criteria provid ed in the Guideline (890.1500).Male Pubertal Assay (2012) / Page 11 of 15
CYPERMETHRIN / 109702 OCSPP 890.1500 / OECD None



### Urea nitrogen ( ↓ 17%) and bile acid ( ↓ 52%) were decreased (p< 0.05) in the 30 mg/kg/day group compared to controls. Decreases in bi le acids have not been associated with toxicological effects. The slight decrease in blood urea nitrogen was within the historical control reference range and considered an in cidental finding. Ther e were no other serum chemistry changes in the cypermethrin-dos ed groups compared to the control group.

Mean values for the control group were outside th e historical reference control data from the performing laboratory for ALT, AST, bile ac id, calcium, cholesterol, potassium, and triglycerides.

TABLE 5. Hormone Levels and Clinical Chemistry a

Parameter
Evaluated Vehicle Control Cypermethrin (15 mg/kg/day) Cypermethrin (30 mg/kg/day)

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)

Hormones

Serum T 4 , Total (μg/dL) 15 5.31 0.927 17.446 14 5.00 0.865 17.282 15 5.06 0.612 12.086 Serum TSH (ng/mL) 15 15.13 6.775 44.789 14 10.96 6.433 58.711 15 15.25 8.175 53.62 Serum testosterone (ng/mL) 15 2.8 1.54 54.74 14 2.1 1.47 71.60 15 2.4 2.03 84.14 Clinical Chemistry
Albumin/Globulin Ratio 15 2.29 0.269 11. 769 14 2.15 0.493 22.904 15 2.34 0.256 10.925 Albumin (g/dL) 15 4.0 0.18 4.44 14 3.9 0.28 7.09 15 4.1 0.17 4.26 ALP (U/L) 15 373 49.4 13.2 14 327 76.3 23.3 15 350 68.4 19.5 ALT (U/L) 15 75 10.7 14.3 14 69 13.4 19.5 15 73 10.0 13.8 AST (U/L) 15 175 51.4 29.3 14 176 41.6 23.6 15 157 36.6 23.3

Bile Acid (μmol/L) 15 44.9 33.52 74.61 14 35.2 ( ↓ 22) 24.09 68.51 15 21.7* ( ↓ 52) 11.18 51.58

Calcium (mg/dL) 15 12.6 0.56 4.45 14 12.5 0.33 2.65 15 12.6 0.3 2.42 Chloride (mEq/L) 15 102 1.5 1. 5 14 101 1.7 1.7 15 102 1.5 1.5 Cholesterol (mg/dL) 15 96 16.9 17.6 14 90 13.5 14.9 15 90 13.5 15.0 Creatinine (mg/dL) 15 0.1 0.05 36.60 14 0.1 0.05 44.30 15 0.1 0.04 34.50 GGT (U/L) 15 0.1 0.00 0.00 14 0. 1 0.00 0.00 15 0.1 0.00 0.00 Globulin (g/dL) 15 1.7 0.24 13.66 14 1.9 0.30 16.29 15 1.8 0.16 9.31 Glucose (mg/dL) 15 151 8.5 5. 6 14 153 8.1 5.3 15 157 11.0 7.0 Phosphorus (mg/dL) 15 10.3 0.74 7.22 14 10.4 0.76 7.34 15 10.6 0.72 6.78 Potassium (mEq/L) 15 6.63 0.555 8.378 14 6.30 0.292 4.627 15 6.39 0.412 6.447 Sodium (mEq/L) 15 143 1.4 1. 0 14 143 1.0 0.7 15 144 1.8 1.2 Total Bilirubin (mg/dL) 15 0.06 0.022 35. 329 14 0.06 0.020 35.854 15 0.04 0.016 36.755 Total Protein (g/dL) 15 5.7 0.35 6.10 14 5.8 0.30 5.14 15 5.8 0.20 3.51 Triglyceride (mg/dL) 15 200 113.3 56.5 14 235 144.5 61.4 15 244 253.0 103.8

Urea nitrogen (mg/dL) 15 12.2 1.98 16.19 14 10.9 ( ↓ 11) 2.31 21.12 15 10.1* ( ↓ 17) 2.68 26.58

a Data were obtained from Tables 3 and 4 on pages 108-109 and Tables S8 and S9 on pages 73-84 of the study report. Percent differences from controls are included in parentheses. Bolded mean values were outside the reference ranges provided in the study report.
N Number of animals examined
SD Standard Deviation
CV Coefficient of Variation * Significantly different from controls at p<0.05 ALP Alkaline phosphatase
ALT Alanine aminotransferase AST Aspartate aminotransferase GGT Gamma glutamyl transferaseMale Pubertal Assay (2012) / Page 12 of 15
CYPERMETHRIN / 109702 OCSPP 890.1500 / OECD None



### F. GROSS PATHOLOGY: No treatment-related effects we re observed on gross pathology.

G. HISTOPATHOLOGY: The incidences of histopathologi cal findings of the thyroid gland are presented below in Table 6. There were no test substance-relate d histologic changes. The amount of colloid and follicular height of thyroid epithelial cells were similar between
control and test substa nce-treated animals.

TABLE 6. Incidence of Histopathologi cal Findings of the Thyroid Gland 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

15 1 3

15 2 0 2 12

3 10 3 0

4 5 4 0

5 0 5 0

Cypermethrin 15 mg/kg/day 1 0

14 1 1

14 2 3 2 13

3 8 3 0

4 3 4 0

5 0 5 0

Cypermethrin 30 mg/kg/day 1 0

15 1 0

15 2 2 2 14

3 10 3 1

4 3 4 0

5 0 5 0 a Data were obtained from Table S13 on page 97 of the study report.
b A 5-point grading scale (1=shortest/smallest; 5=tallest/largest) was used.
O Number Observed
E Number Examined



### The incidence of histopathological findings of the testes, epididymides and kidneys are presented in Table 7. No effects of treatment were observed microscopically in the testes, epididymides, or kidney compared to the control group.Male Pubertal Assay (2012) / Page 13 of 15
CYPERMETHRIN / 109702 OCSPP 890.1500 / OECD None

TABLE 7. Incidence of Histopathological Findi ngs of the Testes, Epididymides and Kidney a

Findings Dose Level (mg/kg/day)

Vehicle Control Cypermethrin (15) Cypermethrin (30)

# Observed # Examined # Observed # Examined # Observed # Examined

Testes Dilation, tubular mild 0 15 2 14 0 15 Epididymides Hypospermia moderate 0 15 1 14 0 15 Infiltrate, lymphocyte
minimal 1 15 0 14 0 15

Kidney Basophilic tubules minimal 3 15 2 14 2 15 Cyst present 2 15 0 14 2 15 Dilatation, tubular mild 0 15 0 14 1 15 Infiltrate, lymphocyte
minimal 0 15 2 14 0 15

a Data were obtained from Table S13 on page 96 of the study report.



### III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: There was no evidence of any test substancerelated androgenic or anti-andr ogenic effects, nor was ther e any evidence of any test substance-related effects on pubertal development or thyroid function in juvenile/peripubertal male rats at cyperm ethrin dose levels of 15 and 30 mg/kg/day.

B. AGENCY COMMENTS: One male in the 15 mg /kg/day group was found dead approximately one hour following dosing on P ND 45, which was likely due to dosing error given the timing of death relativ e to dosing and the fact that no mortalities occurred at the higher dose. All other rats survived until sc heduled sacrifice. There were no treatmentrelated clinical signs of toxi city at any dose level. Mean body weights, daily body weight gains, cumulative mean body weight gain, food consumption, mean age of attainment of complete PPS, and mean body weight at atta inment of PPS in the 15 and 30 mg/kg/day groups were similar to the cont rol group throughout the study.

No treatment-related gross observa tions were noted at necropsy and no significant effects of treatment were observed on the weights of any ofthe organs and tissues collected. A doserelated decrease (NS) was observed for the dor solateral prostate weight at 15 mg/kg/day ( ↓ 7%) and 30 mg/kg/day ( ↓ 14%) compared to the controls. No other effect was noted on androgen-dependent tissues, and this slight decrease was considered incidental.

There were no test substance-related changes in serum T 4 

### , TSH and testosterone values at any dose level. Urea nitrogen ( ↓ 17%) and bile acid ( ↓ 52%) were decreased (p<0.05) in the 30 mg/kg/day group compared to co ntrols. Decreases in bile aci ds have not been associated with toxicological effects. The slight d ecrease in blood urea nitr ogen was within the historical control reference range and considered an incidental finding. There were no other serum chemistry changes in the cypermethrin -dosed groups compared to the control group.Male Pubertal Assay (2012) / Page 14 of 15
CYPERMETHRIN / 109702 OCSPP 890.1500 / OECD None



### There were no test substance-re lated histologic changes in th e thyroid, testes, epididymides, and kidneys. The amount of colloid and follicular height of thyroid epithelial cells were
similar between control and test substance-treated animals.

No evidence of toxicity was observed in th e study up to the highest dose tested; however, treatment-related clinical signs were seen in the preliminar y range-finding study at 30 and 65 mg/kg/day. In the preliminar y study, tremors and clear material around the mouth were seen at 30 mg/kg/day, 4 hours after administ ration. At 65 mg/kg/ day (after 4 hours), tremors, splayed limbs, and clear material around the mouth, ventral neck and thoracic area, and forelimbs were seen. It is possible that the effects observed in the preliminary study were missed in the definitive study since clinical signs were measured 1 hour after chemical administration. Clinical signs were measured 4 hours after administratio n in the preliminary study, which is closer to the time to peak effect for cypermethrin.

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:

 The CV for the body weight at PPS in the control group (9.74%) was greater than the maximum value in the performance criteria (7.57%).

 The CV for final body weight in the cont rol group (9.01%) was greater than the maximum value (7.47%).

 The CV for the pituitary weights in the control group (17.35%) exceeded the performance criteria maximum (15.98%).

 The CV for the thyroid weights in the control group (25.05%) exceeded the performance criteria maximum (23.63%) and the mean of the t hyroid weights (13.20 mg) was below the minimum value in the performance criteria (14 mg).

 The mean kidney weight in the control group (2.03 g) was below the minimum value in the performance criteria (2.242 g).Male Pubertal Assay (2012) / Page 15 of 15
CYPERMETHRIN / 109702 OCSPP 890.1500 / OECD None



### APPENDIX

Since this is a range-finding study, only a summary is provided to confirm the adequacy of the
dose selection rationale used in the Male Pubertal Assay.

In a dose range-finding assay (MRID 48683004), cype rmethrin (95.2% a.i.; cis/trans ratio: 49.8/50.2; Batch #PL10-0492) in corn oil was admi nistered daily via or al gavage in a dose volume of 5 mL/kg to four juvenile female (PND 22) and male (PND 23) Sprague Dawley [Crl:CD(SD)] rats/dose group at doses of 0, 5, 15, 30, or 65 mg/kg/day. The animals were dosed for 14 consecutive days and necropsied one day af ter the final dose administration. Adrenals, kidneys, and liver were collected a nd weighed. Only the results for the male rats are discussed in this appendix; the results for the female ra ts are summarized in the DER for MRID 48683002 (concurrently reviewed).

Slight to moderate tremors were noted 4 hours following dosing in all males in the 65 mg/kg/day group. Two of these males also had splayed hindli mbs; lacrimation, clear or red material around the mouth, and/or clear material on the ventral neck were noted in three of the males in this group. Due to these observations, and the fact that a female dosed at 65 mg/kg/day was found dead on Day 2, it was concluded that a dose of 65 mg/kg/day would not be suitable for continued dosing, and all males in the 65 mg/kg/day group were euthanized on Da y 1. All other rats
survived to scheduled termination.

Clinical findings noted 1 or 4 hours following dose administration in the 30 mg/kg/day group consisted of slight tremors (beginning as ear ly as Day 2), a single occurrence of splayed hindlimbs on Day 5, and clear material around the mouth and on the ventral neck and/or forelimbs (beginning as early as Day 1). At 4 hours post-dos e at 30 mg/kg/day, 3/4 animals displayed tremors on 5/55 observations compared to 0/4 controls. Clinical findings in the 5 and 15 mg/kg/day group occurred infrequently and we re not observed in a dose-related manner.

Mean body weights, body weight gains, and f ood consumption in the 5, 15, and 30 mg/kg/day groups were unaffected by test substance administ ration. Slightly higher mean kidney and liver weights (absolute and relative to final body weight) were noted in the 30 mg/kg/day group males ( ↑ 10% and 4% for absolute and relati ve kidney weight, respectively; and ↑ 15% and 8% for absolute and relative liver weight, respectively). In addition, mean liver weights (absolute and relative) in the 15 mg/kg/day group males were slightly higher than the control group ( ↑ 14% and 8%, respectively). The differences in relative liver weights in both gr oups were statistically significant compared to the control group. Mean absolute and relative adrenal gland weights in the 15 and 30 mg/kg/day group males were unaffect ed by test substance administration. Mean absolute and relative organ weights in the 5 mg/kg/day group males were unaffected by test
substance administration.

There were no macroscopic findings at necropsy in the 5, 15, or 30 mg/kg/day groups. The rats in the 65 mg/kg/day group were discarded wit hout examination after eu thanasia due to dosegroup termination.

COMPLIANCE: It was stated that the study was not performed in comp liance with GLPs.

# DATA EVALUATION RECORD

CYPERMETHRIN

Study Type: OCSPP 890.1550, Steroidogenesis Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-43-2012 (MRID 48670203)

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: 5/18/2012

Secondary Reviewer Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 5/22/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 5/24/2012

Quality Assurance: Signature: Steven Brecher, Ph.D., D.A.B.T. Date: 5/24/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended 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 re dundancy 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 potent ial 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).Steroidogenesis Assay (2012) I Page 1 of 14 CYPE RMETHRIN I 109702 0 SPP 890.1550/ OECD None



### Primary Reviewer: Sheila Healy, Ph.D

. 

### Health Effects Division Secondary Reviewer: John Liccione, Ph.D.


### Health Effects Division

DATA EVALUATION RECORD

STUDY TYPE: Steroidogenesis Assay (H295R Cells); OCSPP 890

. 1550



### PC CODE: 109702 

### DP BARCODE: D399304



### TXR#: 0052288 

### CASNo.: 52315-07-8



### TEST MATERIAL (PURITY): Cypermethrin (95

. 2% ; Cis / Trans ratio: 49.8 / 50.2)



### SYN ONYMS:

CITAT ION :

SP ONSOR: Cyano(3-phenoxyphenyl)methy l 3

- (2,2 -dichloro etheny l )-2,2- dimethylcyclopropanecarboxylate

Wagner

, H

. (2012). Cypermethrin

- H295R Steroidogenesis Assay

. CeeTox

, Inc., Kalamazoo, MI. Laboratory Study No.: 9047VI 00353STER, January 17

, 20 1 2. MRID 48670203

. Unpub li shed.

FMC Corporation

, 1735 Market Street, Philadelphia, PA; United Phosphorus

, Inc., 630 Freedom Business Center, Suite 402, King of Prussia

, PA; Syngenta Crop Protection

, LLC

., 410 Swing Road

, PO Box 18300,
Greensboro

, NC



### TEST ORDER#: CON-109702-8



### EXECUTIVE SUMMARY: In a steroidogenesis assay (MR ID 48670203), H295R cells
cultured 

### in vitro in 24-well plates were incubated with cypermethrin (95.2% purity

, Lot# PLl 0- 0492) at concentrations of 100

, 10

, 1, 0.1

, 0

. 01

, 0.001

, and 0.0001 μM for 48 hours in triplicate
for four independent experiments

. Dimethyl sulfoxide (DMSO) was used as the vehicle, at a final concentration of 0.05 %

. Because precipitation was observed at 100 μM

, this group was not

e v aluated statistically. Run 1 was not analyzed because one of the forsko lin concentrations on the quality control plate was incorrect (3.3 μM was used instead of 1 μM).

Testosterone and estradiol levels were measured using HPLC-MS / MS. A Quality Control (QC) plate was run concurrently with each independent run of a test chemical plate to demonstrate that
the assay responded properly to positive control agents at two concentration levels; positive
controls included the known inhibitor (prochloraz) and inducer (forskolin) of estradiol and

testosterone production.

Guideline recommendations and requirements were met, including adequate production of

testosterone and estradiol

, acceptab l e reproducibility (low %CV), and appropriate induction and
inhibition with positive controls. Cytotoxicity greater than 20% was not observed.Steroidogenesis Assay (2012) / Page 2 of 14
CYPERMETHRIN / 109702 OCSPP 890.1550/ OECD None



### Statistically significant increases in estradiol were noted at 10 μM in Runs 3 and 4, and estradiol levels in all three runs at 10 μM were 111-117% of the solvent cont rol (SC). All other values of
testosterone and estradiol in the tr eated cells were similar to the SC.

Based on the hormone responses in each of the th ree independent runs, cypermethrin treatment resulted in statistically si gnificant and reproducible increas es in estradiol production, but cypermethrin treatment did not result in statisti cally significant and reproducible alterations in testosterone production.

The assay satisfies the EDSP Tier 1 Test Order requi rements for a Steroidogenesis assay (OCSPP 890.1550).

COMPLIANCE: Signed and dated GLP Compliance a nd Quality Assurance statements were provided. A Data Confidentiality statement was not provided.Steroidogenesis Assay (2012) / Page 3 of 14
CYPERMETHRIN / 109702 OCSPP 890.1550/ OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: CeeTox

Location: Kalamazoo, MI Study Director: Hilary Wagner, M.S.
Other Personnel: David Blakeman, B.S., Senior Sc ientist/Endocrine Group Leader Briana Wallace, B.S., Lead Cell Culture Scientist Steven McColley, M.S., Scientist Cameron Haines, B.S., Scientist Francisca Wong, M.S., Scientist Joey Burgam, B.S., Associate Scientist Lisa Blakeman, B.S., Associate Scientist Study Period: May 13, 2011 through January 17, 2012



### 2. Test Substance: Cypermethrin
Description (molecular weight): Amber liquid (416.3) Lot # (expiration date): PL10-0492 (December 15, 2012) Purity: 95.2% (Cis/Trans ratio: 49.8/50.2) Solubility: Soluble up to 10 μM in DMSO under tested conditions Vapor pressure: Not reported; 5.1 x 10 -7 nPa at 70°C Stability: Not reported Storage conditions: Room temperature
CAS #: 52315-07-8
Structure:

CN O

O Cl O

Cl CH 3 CH

3



### 3. Positive Control (Inducer): Forskolin
Description (molecular weight): White powder (410.50)
Source: Sigma-Aldrich (St. Louis, MO) Lot # (expiration date): 109K50571V (July 2016) Purity: 98% Solubility (in solvent): Soluble at tested concentrations in DMSO Storage conditions: Room temperature
CAS #: 66575-29-9



### 4. Positive Control (Inhibitor): Prochloraz
Description (molecular weight): White powder (376.67)
Source: Sigma-Aldrich (St. Louis, MO) Lot # (expiration date): SZE6220X (August 8, 2012) Purity: 99.1% Solubility (in solvent): Soluble at tested concentrations in DMSO Storage conditions: Room temperature
CAS #: 67747-09-5Steroidogenesis Assay (2012) / Page 4 of 14
CYPERMETHRIN / 109702 OCSPP 890.1550/ OECD None



### 5. Solvent/Vehicle Control: Dimethyl Sulfoxide (DMSO)
Description (molecular weight): Clear liquid (78.13)
Source: Sigma-Aldrich (St. Louis, MO) Lot # (expiration date): RNBB7617 (Not reported) Purity: Not reported Storage conditions: Not reported
CAS #: 67-68-5
Justification for choice of solvent: Not reported, but is a Guideline solvent of choice in which cypermethrin is solu b Final concentration in assay: 0.05% v/v



### 6. Other Materials: 22R-Hydroxycholesterol
Description (molecular weight): White powder (402.65)
Source: Sigma-Aldrich (St. Louis, MO) Lot # (expiration date): 089K4132, 060M4098 (not provided) Purity: 99.0% Storage conditions: Room temperature
Solvent used: Ethanol, final concentratio n in assay, 0.025% v/v
CAS #: 17954-98-2



### 7. Stock Medium: Dulbecco’s modified Eagle’s medium/nutrient mixture
F12 Ham with 15 mM HEPES

Source: Sigma-Aldrich (St. Louis, MO) Lot # (expiration date): 021M8304 (not provided)

Sodium bicarbonate: Sigma-Aldrich (St. Louis, MO); Lot # 068K0105; purity not reported
Nu-Serum: 2.5%; Becton-Dickinson (Franklin La kes, NJ); Catalog # 355500; Lot # 81515; tested for background hormone concentrations by performing laboratory ITS+premix: Becton-Dickinson (Franklin Lakes, NJ); Catalog # 354352; Lot # 05245,
09233



### 8. Test Cells: H295R human adrenocortical carcin oma cells (ATCC CLR-2128; Lot # 7635054) at passage 7.5–8.5 were incubated in th e stock medium, supplemented with 10 μM 22R-hydroxycholesterol in ethanol at the time of plating, dosi ng, and harvest. Incubation
conditions were at 5% CO 2 

### and 37 ± 2°C for approximately 24 hours prior to exposure.

The following performance criteria were met (indicated by an “x”):
x Cell passage identifier. Cell Passage #: 7.5 or 8.5 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-Test Information

a. Hormone Assay Interference Test: A hormone assay interference test was not performed.

b. Hormone Extraction: No data from pre-test horm one extraction were provided.

c. Laboratory Proficiency Test: No laboratory proficiency te st data were provided.

2. Test Solutions: Cypermethrin was dissolved in DMSO to make a 200 mM stock and then serially diluted in DMSO. 22R-Hydroxycholesterol was dissolved in ethanol to make a 40 mM stock and then diluted in supplemented me dium to a final concentration of 10 μM.Steroidogenesis Assay (2012) / Page 5 of 14
CYPERMETHRIN / 109702 OCSPP 890.1550/ OECD None



### Cypermethrin was then diluted in supplemented medium containing 10 μM 22R-hydroxycholesterol to prepare mastermix solutions. Forskolin and prochloraz were dissolved in
DMSO to make 100 mM solutions and then se rially diluted in DMSO. Forskolin and prochloraz were then diluted in supple mented medium containing 10 μM 22R-hydroxycholesterol. When added to the cell cultu re plates, these dilu tions yielded final concentrations of 1 μM and 10 μM for fors kolin, 0.1 μM and 1 μM for prochloraz, and 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 μM for cypermethrin. The final concentration of
DMSO in the medium was held constant at 0.05 % (v/v). The final ethanol concentration in the supplemented medium was 0.025% (v/v). Pr ecipitation was noted microscopically in the test wells at 100 μM cypermethrin in all runs.

3. Cell Plating and Preincubation: Cells were maintained in the Stock Medium described above. It was reported that hormone con centrations in undiluted Nu-Serum were 3754 pg/mL testosterone and 3846 pg/mL estradio l. H295R cells were grown for five passages, frozen in liquid nitrogen, then thaw ed and cultured for seven or eight additional passages prior to use in the assay. The culture medium was supplemented with 10 uM 22R-hydroxycholesterol at the time of plating, do sing, and harvest. The concentration of 22R-hydroxycholesterol was chosen based on labora tory proficiency experiments previously conducted at the Performing Laboratory. The cells were plated into wells of a 24-well cell culture plate at a density of approximately 300,000 cells in 1 mL. The cells were then placed into a 5% CO 2 

### incubator at 37 ± 2°C for approxi mately 24 hours prior to chemical exposure. Prior to dosing, the cells were check ed microscopically for attachment and proper morphology. Four independent runs of the steroidogenesis assay were conducted. Run 1 was not analyzed because one of the forskolin concentrations on the quality control plate was incorrect (3.3 uM was used instead of 1 uM).

4. Exposure: Dosing solutions were visually insp ected for precipitation. The medium was removed from the cells and replaced with 1 mL of medium containing 10 μ M 22Rhydroxycholesterol and concentrations of cypermethrin in triplicate, according to the schematic presented in Table 1. Dose concentrat ions of the test substance were verified by Smithers Viscien (data not included in report).

TABLE 1. Dosing Schematic for the Exposure of H295R Cells to Cypermethrin (Final Concentrations 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.



### A concurrent QC plate was included with each of the independent runs of the test chemical plates to demonstrate the assa y’s response to forskolin (an inducer of testosterone and estradiol production) and prochloraz (an inhibito r 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.Steroidogenesis Assay (2012) / Page 6 of 14
CYPERMETHRIN / 109702 OCSPP 890.1550/ OECD None

TABLE 2. Dosing Schematic for the QC Plate for Positive Controls (Final Concentrations 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 b DMSO + MeOH b

C Forskolin 1 μM Forskolin 1 μM Forskolin 1 μM Prochloraz 0.1 μM Prochloraz 0.1 μM Prochloraz 0.1 μM

D Forskolin 10 μM Forskolin 10 μM Forskolin 10 μM Prochloraz 1 μM Prochloraz 1 μM Prochloraz 1 μM a Data were obtained from page 15 of the study report.
b Blank wells received medium contai ning 10μM 22R-hydroxycholesterol. c Background wells received media without 22R-hydroxycholesterol.
d MeOH = methanol was added to these wells for 30 mi nutes at room temperature following medium removal.



### Following dosing, the plates were incubated for 48 ± 2 hours under the conditions previously described. After the 48 ± 2 hour exposure, each well was examined under the microscope. Any precipitate was noted. Images were taken of the vehicle control wells, as well as the two highest non-cytotoxic concentr ations. The medium was collected from all wells in two equal portions and stored at -80 ± 10°C until shipment to the analytical laboratory for hormone measurement (samples were shipped on dry i ce and stored at the analytical laboratory at -80°C until analyzed).

5. Cell Viability/Cytotoxicity Assay: After medium removal, cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-di phenyltetrazolium bromide (MTT) assay. On the QC plates, wells designated to receive methanol (control wells for cell death measurements) were rinsed twice with phospha te buffered saline (PBS; Gibco; Cat. # 10010; Lot # 979431), and then incubated in methanol (Sigma; Cat. # 494437; Lot # SHBB0973V) for 30 minutes at room temperat ure. After the methanol incubation, the methanol-treated wells were rinsed again with PBS three times. Following medium removal and/or methanol treatment, 0.5 mL of 0.5 mg/mL MTT (Sigma; Cat. # M5655; Lot # MKBG1611V) solution in supplemente d medium containing 10 μM 22Rhydroxycholesterol was added to each well of the test chemical and QC plates. The plates
were incubated at 37±2°C in a 5% CO 2 

### incubator for 3 hours. Following the 3 hour incubation, the MTT solution was removed, and 0.5 mL of cypermethrin was added to each
well. Plates were incubated at room temperature for 20 minutes with shaking. Following this incubation, absorbance at 570 nm and 650 nm were measured on a Packard Fusion plate
reader. The absorbance at 650 nm was subt racted from the absorbance at 570 nm to
calculate the MTT value for each well. The change in cell viability was determined by comparing treated wells to the solvent control wells. A greater than 20% reduction in cell viability was considered evidence of cytotoxicity.

6. Hormone Measurement System: Testosterone and estradio l levels were measured at OpAns, LLC (Durham, NC). Testosterone and estradiol were extracted from H295R supplemented medium by liquid-liquid extract ion using methyl tert -butyl ether (MTBE) after the addition of [ 2 

### H 5 

### ]-testosterone and [ 2 

### H 5 

### ]-estradiol as internal standards. Extracts were analyzed by HPLC-MS/MS using positive ion multiple reaction monitoring. This method was validated over the range 100 to 100,000 pg/mL for testosterone and 10 to 10,000 pg/mL for estradiol (OpAns Document # OPR-OPP-0006). The lower limit of quantification (LLQ) was 100 pg/mL for testos terone and 10 pg/mL fo r estradiol using a 300 μL aliquot of H295R supplemented medium . Freshly prepared calibration standards (n = 7) were prepared in supplemented me dium for each run. QC samples spiked inSteroidogenesis Assay (2012) / Page 7 of 14
CYPERMETHRIN / 109702 OCSPP 890.1550/ OECD None



### supplemented medium at three concentrations (2 replicates per concentration), were
included with each run.

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 fo r two concentrations of testosterone and estradio l (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 be low maximum response on standard curve NA Test compound tested for interf erence with meas urement system



### C. DATA ANALYSIS: Mean values (pg/mL) and standard deviations for testosterone and
estradiol were calculated for each concentrati on of cypermethrin, reference chemicals, the solvent controls, blanks, and background wells . Relative changes in testosterone and estradiol production were calcula ted using the equation below:

Relative Change = Hormone concentration in each well ÷ Hormone c oncentration of mean
solvent control

For forskolin induction of testosterone, th e background hormone production was subtracted
from the forskolin-treated wells and blank and solvent control wells before calculating the relative change. Background hormone production was calculated from three wells with cells on the QC plate that receive d no 22R-hydroxycholesterol at the time of exposure. Concentrations where precipitation was observe d or cytotoxicity was >20% were excluded from further analysis.

Normality of the data was evaluated using Shapiro-Wilk's test. Homogeneity of the
variances between the tr eatment groups was evaluated using Le vene's test. If the p-values were greater than 0.05 in both tests, statisti cal significance between each treatment group and the control group was evaluated using Dunnett's test. If the p-values were less than or equal to 0.05 in either the normality or th e homogeneity test, a log transformation was performed on the data to attempt to approxi mate a normal distribution. If (following the transformation) p-values were greater than0.05 in both the normality and homogeneity tests, Dunnett's test was performed on the transformed data to evaluate statistical significance between eac h treatment group and the contro l group. If following the log transformation, p-values were less than or equal to 0.05 in either the normality or the homogeneity test, the non-tr ansformed data set was anal yzed using the nonparametric Kruskal-Wallis test followed by Dunn's test to evaluate statistical significance between each treatment group and the control gr oup. Data analysis at the an alytical laboratory (OpAns, LLC) was conducted in Microsoft Excel. No add itional software for statistical analyses or graphing was specified. The reviewer s consider these analyses acceptable.

II. RESULTS

A. TEST COMPOUND: The hormone concentrations after exposure to cypermethrin, and the fold-difference change relative to SC and mean ±SD for the three assay runs are presented in Table 3, and the fold-difference change relative to SC for the three assay runs are shown in Figures 1 and 2.Steroidogenesis Assay (2012) / Page 8 of 14
CYPERMETHRIN / 109702 OCSPP 890.1550/ OECD None



### Precipitation of the test compound was observed microscopically in all test wells with 100 μM cypermethrin. Contamination was also noted in one 100 μM cypermethrin well, but this well was already excluded from statistical anal ysis because of the observed precipitation. Cytotoxicity greater than 20% was not observed. The variability (CV) between the runs for the solvent controls, based on absolute concentration, was 21% (testosterone) and 15% (estradiol). Based on absolute concentrations , the variability (CV) within each run for the
solvent controls in the testoste rone and estradiol assays was ≤ 7.3%. Thus, inter- and intraplate variability were within the guideline recommendation of ≤ 30%. Basal hormone production in SCs met guide line recommendations.

Statistically significant induction of estradiol was noted at 10 μM in Runs 3 and 4, and
estradiol levels in all runs at 10 μM were 111-117% of the SC (T able 3). All other values of
testosterone and estradiol in the treat ed cells were similar to the SC.

TABLE 3. Mean (±SD) Hormone Concentrations Follo wing Treatment with Cypermethrin for 48 Hours. a

Nominal
Concentration (μM) Run 2 Run 3 Run 4 Run 2 Run 3 Run 4 Mean b ± SD b Statistical Significance

Testosterone (pg/mL) Fold Difference

DMSO 2925 1940 2347 — — — — — —

0.0001 2862 1958 2432 0.98 1.01 1.04 1.01 0.030 —

0.001 2597 1973 2373 0.89 1.02 1.01 0.97 0.072 —

0.01 2750 2022 2372 0.94 1.04 1.01 1.00 0.051 —

0.1 2772 2084 2401 0.95 1.07 1.02 1.01 0.060 —

1 2890 2033 2437 0.99 1.05 1.04 1.03 0.032 —

10 2806 2050 2495 0.96 1.06 1.06 1.03 0.058 —

100 3001 1822 2033 NE NE NE — — — Estradiol (pg/mL) Fold Difference

DMSO 259 194 249 — — — — — —

0.0001 257 204 253 0.99 1.05 1.02 1.02 0.030 —

0.001 252 206 248 0.98 1.06 1.00 1.01 0.042 —

0.01 266 203 245 1.03 1.05 0.98 1.02 0.036 —

0.1 259 202 248 1.00 1.04 1.00 1.01 0.023 —

1 274 206 257 1.06 1.06 1.03 1.05 0.017 —

10 287 218 290 1.11 1.13* 1.17* 1.14 0.031 Runs 3 and 4

100 214 147 170 NE NE NE — — — a Data were obtained from Tables 7-8 on pages 26-27 and Ap pendices 1-3 on pages 33-35 of the study report. The lower limit of quantification (LLQ) was 100 pg/mL for testosterone and 10 pg/mL for estradiol. b Calculated by the reviewers. * p ≤ 0.05 NE Not examined because of precipitationSteroidogenesis Assay (2012) / Page 9 of 14
CYPERMETHRIN / 109702 OCSPP 890.1550/ OECD None



### FIGURE 1. Change in Testosterone Producti on Relative to Cypermethrin Concentration.Steroidogenesis Assay (2012) / Page 10 of 14
CYPERMETHRIN / 109702 OCSPP 890.1550/ OECD None



### FIGURE 2. Change in Estradiol Production Relative to Cypermethrin Concentration.Steroidogenesis Assay (2012) / Page 11 of 14
CYPERMETHRIN / 109702 OCSPP 890.1550/ OECD None



### B. CYTOTOXICITY: No cytotoxicity was noted for the cypermethrin-treated wells, positive controls, blank, and background wells; mean viability ranged from 92.7–109.3%. Methanol resulted in the expected response, less than 12% viability. Data are summarized in Table 4 and depicted graphica lly in Figure 3.

TABLE 4. Mean (±SD) MTT Cell Viability (% SC) Result s after Treatment with Forskolin, Prochloraz, or Cypermethrin for 48 Hours. a

Compound Concen. (μM) Cell Viability – Run 2 Cell Viabilit y – 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 0.94
SC + Methanol NA 5.6 2. 51 11.4 1.99 5.1 0.14

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 Cypermethrin 0.0001 100.0 3. 19 93.6 3.99 99.0 1.46 Cypermethrin 0.001 97.8 1.66 96.1 2.76 99.1 0.70 Cypermethrin 0.01 97.4 1.06 95.0 5.31 102.7 0.77 Cypermethrin 0.1 97.9 2.58 93.4 3.32 100.9 1.88 Cypermethrin 1 98.3 3.84 97.3 2.93 98.0 1.27 Cypermethrin 10 96.3 1.65 92.7 3.01 99.9 0.87 Cypermethrin 100 95.0 2.59 95.0 4.65 94.7 1.11

a Data were obtained from Tables 1 a nd 2 on pages 20-21 of the study report. NA Not applicableSteroidogenesis Assay (2012) / Page 12 of 14
CYPERMETHRIN / 109702 OCSPP 890.1550/ OECD None



### FIGURE 3. MTT cell viability resu lts for cypermethrin-treated cells.

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 le vels (500 pg/mL for testosterone, 40 pg/mL for estradiol) were met in both blan k and SC wells. OCSPP 890.1550 guideline requirements for basal hormone production in SC wells, induction by 10 μM forskolin, andSteroidogenesis Assay (2012) / Page 13 of 14
CYPERMETHRIN / 109702 OCSPP 890.1550/ OECD None



### inhibition by 1 μM prochloraz were met for test osterone and estradiol. Mean basal hormone production in SC wells was >18-fold minimum detection level (MDL) for testosterone and >17-fold MDL for estrodiol. Compared to SC, 10 μM forskolin on average induced testosterone by 1.71-fold, which is be low the Guideline recommendation of ≥ 2-fold; however, when the background is subtracted, 10 μM forskolin induced testosterone an average of 2.47-fold. Estradio l induction in the presence of 10 μM forskolin (10.34-fold) met the Guideline requirement ( ≥ 7.5-fold). Compared to SC, 1 μM prochloraz on average inhibited the synthesis of test osterone to 0.41-fold and estradiol to 0.32-fold. Thus, the guideline requirements were met indicating th at the assay was sensitive to induction and
inhibition of testoster one and estradiol.

The variability (%CV) between the runs (calculated by the re viewer) based on the absolute
hormone concentrations in the SC were 15% fo r testosterone and 11% for estradiol, within
the recommended limit of ≤ 30%. The %CVs within each ru n for the QC plates were 1.23-
7.33% for testosterone and 0.32-2.93% for estradiol.

TABLE 5. Mean (±SD) Hormone Concentrations Following Treatment with Forskolin or Prochloraz for 48
Hours. a

Compound Run 2 Run 3 Run 4 Run 2 Run 3 Run 4 Mean b ± SD b
Testosterone (pg/mL) Fold Difference (Relative to DMSO) Background 1135 1073 1262 0.45 0.57 0.54 0.52 0.062

Blank 2581 1873 2304 1.03 1.00 0.99 1.01 0.021

DMSO 2511 1874 2339 1.00 1.00 1.00 1.00 -- 1 μM Forskolin 3764 2595 3120 1.50 c 1.38 c 1.33 c 1.40 c 0.087 10 μM Forskolin 4751 3019 3780 1.89 d 1.61 d 1.62 d 1.71 d 0.159 0.1 μM Prochloraz 1816 1149 1818 0.72 0.61 0.78 0.70 0.086 1 μM Prochloraz 1188 501 1153 0.47 0.27 0.49 0.41 0.122 Estradiol (pg/mL) Fold Difference (Relative to DMSO) Background 41 56 77 0.21 0.31 0.35 0.29 0.072

Blank 208 182 228 1.04 1.01 1.02 1.02 0.015

DMSO 200 179 223 1.00 1.00 1.00 1.00 -- 1 μM Forskolin 1343 1254 1538 6.72 7.01 6.91 6.88 0.147 10 μM Forskolin 2116 1661 2477 10.60 9.28 11.13 10.34 0.953 0.1 μM Prochloraz 133 104 171 0.67 0.58 0.77 0.67 0.095 1 μM Prochloraz 57 52 83 0.29 0.29 0.37 0.32 0.046 a Data were obtained from Tables 3 and 4 on pages 22–23 of the study report. The lower limit of quantification (LLQ) was 100 pg/mL for testosterone and 10 pg/mL for estradiol. b Calculated by the reviewers. c When the background is subtracted, the valu es are 1.91, 1.90, and 1.73-fold difference for Runs 2, 3, and 4, respectively. The mean is a 1.85-fold change. d When the background is subtracted, the valu es are 2.63, 2.43, and 2.34-fold difference for Runs 2, 3, and 4, respectively. The mean is a 2.47-fold change.



### III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Cypermethrin exposure resu lted in no statistically significant effects on testosterone in the steroidogenesis assa y. Statistically significant increases (1.13-1.17 fold) in estradiol production were observed at the highest concentration evaluated (10 μM) in two of the three independent runs. Although statis tically significant,Steroidogenesis Assay (2012) / Page 14 of 14
CYPERMETHRIN / 109702 OCSPP 890.1550/ OECD None



### the increases were less than the 9.28 to 11.13-fo ld increases in estradiol observed with the forskolin positive control.

B. AGENCY COMMENTS: Guideline recommendations and requirements were met, including adequate production of testosterone and estradiol, acceptable reproducibility (low %CV), and appropriate induction and inhibition with positive controls. Precipitation of the test compound was observed at 100 μM cypermethrin concen trations, and these samples were not analyzed. Cytotoxicity greater than 20% was not observed. Run 1 was not analyzed because one of the forskolin con centrations on the quality control plate was incorrect (3.33 μ M was used instead of 1 μ M). The QC plate data was the same as that submitted for MRID 48618909, which had a study initiation date of April 18, 2011 vs May 13, 2011 for the current study.

Statistically significant increases in estradio l were noted at 10 μM in Runs 3 and 4, and
estradiol levels in all three r uns at 10 μM were 111-117% of the SC. All other values of
testosterone and estradiol in the tr eated cells were similar to the SC.

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:

 Stability of cypermethrin in the test system was not reported.

 The laboratory proficiency test data were not provided.

 Mean ± SD and %CV for the fold-differe nces were not reported, but could be calculated from the data presented.

# DATA EVALUATION RECORD

CYPERMETHRIN

Study Type: OCSPP 890.1600, In vivo Uterotrophic Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-43-2012 (MRIDs 48615701 and 48615702)

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: 5/2/2012

Secondary Reviewer: Signature: Kelly Luck, M.S. Date: 5/11/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 5/23/2012

Quality Assurance: Signature: Steven Brecher, Ph.D., D.A.B.T. Date: 5/23/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended 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 re dundancy 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 potent ial 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).CYPERMETHRIN/ 1097 02 In vivo Uterotruphic Assay (2011) I Pagel of 11
OCSPP 890.1600/ OECD 440

_,- \ :**'\~-/** / ·' 

### ' : 

### - 

## . Primary Reviewer: Linnea J

. Hansen, Ph.D. Signature: /.}

_, v. \ "-' 

### c 

### !

. 1 : (. 

# "'i' i 

### Risk Assessment Branch 

### II, Health Effects Division Date:

### ~ 

### \.;· \ Secondary Reviewer:

### ~J**~R=o~w~ n""'d"'--**  _________ Signature:

### ~s--0-')..,.. Health Effects Division Date: 

# C::.cc:ef**1~** Template version 09/2011



### DATA EVALUATI ON RECORD

STUDY TYPE: Uterotrophic Assay (Rat); OCSPP 890.1600: OECD 440

PC CODE: l 09702 DP BARCODE: D399338

TXR#: 0052288 CAS#: 52315-07-8

TEST MATERIAL (PURITY): Cypermethrin (95.2% a .i.)

SYNONYMS : Cyano(3-phenoxyphenyl)methyl 3 -(2 ,2-dich loroe t h eny l ) -2 ,2

- dimethylcyclopropanecarboxylate

CITATION: Herberth

, M. 

### T. (2011). Cypermethrin-Uterotrophic Assay in Ovariectomized

Rats. WIL Research Laboratories, LLC, Ashland, OH

. Laboratory Report No.: WIL-639150, November 10, 2011. MRID 48615701. Unpublished.

Sawhney Coder, P. (2011)

. Cyperrnethrin-A Dose R ange -Finding Oral (Gavage) Toxicity Study in Young Adult Rat s. WIL Research Laboratories, LLC, Ashla nd

. OH. Laboratory R eport No.: WIL-639148

, November 

### JO

, 2011. MRID 48615702. Unpu bli shed.

SPONSORS: Sy n genta Crop Protection, LLC. 410 Swing Road, PO Box 18300, Greensboro. NC; FMC Corporation. 1735 Market Street, Philadelphia, PA ; United
Pho phorus. Inc.

, 630 Freedom Business Center, Suite 402, King of Prussia, PA

TEST ORDER#: CON-109702-8

EXECUTIVE SUMMARY: In a Uterotrophic Assay (MRID 48615701) co ndu cted to screen for potential estrogenic activity, cypermethrin (technical, 95.2% a.i

.. Batch# PLl0-0492) in corn oil w a s administered daily via oral gavage (dos in g vo lume 5 ml/kg bw) to groups of s i x

ov a riectomized female Sprague Dawley rats /d ose a t dose levels of 0 (vehicle). 25. or 80 mg/kg/day on post-natal days (PND) 66 to 68

. The positive control group was treated with a d a ily do e of l 7a- e thynyl e s tradio l (EE) in com oil at 0. 3 mg/kg/day by oral gavage (do

· ing
volum e 5 ml/kg bw). The s elected dose levels were based on toxicity observed in the dose range-finding study (MRID 48615702)

, in which c lini cal sig n s and body weight gain effects

w e re ob:erv e d a t 80 and I 

### 00 mgfkg /d ay, a nd marginal effects a t 65 mg/kg/day. Body we ight s and food c onsumption were determined daily. A l l anima l s were terminated a nd necropsi ed onIn vivo Uterotrophic Assay (2011) / Page 2 of 11
CYPERMETHRIN/ 109702 OCSPP 890.1600/ OECD 440



### PND 69 approximately 24 hours after the final dose administration to determine wet and blotted uterine weights.

All animals survived until scheduled termination. Treatment-related clinic al signs of toxicity included one or two animals with dried red material around nose, hypersensitivity to handling, vocalization upon handling, salivation and/or wet clear material around mouth at 80 mg/kg/day. Body weights in the treatment groups were co mparable to vehicle controls throughout the duration of the study. At 80 mg/kg/day, the overall body weight gain was significantly decreased (p<0.05; ↓ 51%). The overall aver age food consumption (Day s 1-4) was decreased (p<0.05) by 15% at 80 mg/kg/day. Overall av erage food consumption in the 25 mg//kg/day group was comparable to the vehicle control. Uterine weights in the cypermethrin treatment groups were comparable to the vehicle controls.

In the positive control (EE) group, body weights were decreased (p<0.05) by 11% on Day 4 compared to the vehicle cont rol, leading to an overall body weight loss (p<0.05) of –14.9 g compared to a gain of 14.6 g in the vehicle controls. Overall average food consumption in the positive control (EE) group was decreased (p<0.0 5) by 46%. Absolute wet and blotted uterus weights for the positive control (EE) group were increased (p<0.05) by 370% and 171%, respectively, as expected.

The clinical signs and 51% d ecrease in overall body weight gains in the high-dose group (together with consistent findings in th e range-finding study at doses of 65, 80 and 100 mg/kg/day) indicated that the te sted dose levels were adequate.

No statistically significant changes were seen in uterine weight in this assay. Cypermethrin is negative in the uterotrophic assay.

The assay satisfies the EDSP Tier 1 Test Order require ments for a uterotrophic assay (OCSPP 890.1600).

COMPLIANCE: Signed and dated GLP Compliance a nd Quality Assurance statements were provided.In vivo Uterotrophic Assay (2011) / Page 3 of 11
CYPERMETHRIN/ 109702 OCSPP 890.1600/ OECD 440



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: WIL Research Laboratories, LLC

Location: Ashland, OH Study Director: M. T. Herberth
Other Personnel: E. S. Bodle (Assistant Director, Analytical Chemistry), S. A. Keets (Senior Operations Manager, Vivarium), C. A. Kopp (Manager, Gross Pathology and Developmental Toxicology Laboratory), G. Maginnis (Cli nical Veterinarian), T. M. Rafeld (Group Manager, Formulations Laboratory), R. A. Wally (Operations Manager, Reporting & Technical Support Services), L. Freshwater (Principal Investigator, Statistical Analysis) Study Period: May 11, 2011 – November 10, 2011



### 2. Test Substance: Cypermethrin
Description: Technical, dark yellow, very viscous liquid (Cis/Trans Ratio 49.8:50.2)
Source: FMC Innovation Center (Ewing, NJ)
Batch #: PL10-0492 (expiration date 12/15/2012) Purity: 95.2% Stability: Stable in corn oil for up to 19 days at room temperature or refrigerated storage
CAS #: 52315-07-8
Structure:



### 3. Reference Estrogen: 17 α -ethynyl estradiol (EE)
Supplier: Sigma Aldrich (St. Louis, MO)
Lot/Batch #: 028K1411 (expiration date 8/4/2011) Purity: 99.0%
CAS #: 57-63-6



### 4. Solvent/Vehicle Control (test substance): Corn oil

Supplier: Spectrum Chemical Manufacturing Co rporation (New Brunswick, NJ)
Lot #: ZT1301 (expiration date 7/1/2012) Rationale (if other than water): Not reported
Final concentration: Not applicable



### Solvent/Vehicle Control (EE): Ethanol/Corn oil

Supplier: Spectrum Chemical Manufacturing Co rporation (New Brunswick, NJ)
Lot #: Ethanol: ZT0426 (expiration date 8/2/2013) Corn oil: ZT1301 (expiration date 7/1/2012) Rationale (if other than water): Not applicable
Final concentration: EE was dissolved in minimal amounts of 95% ethanol and then diluted with corn oil (ratio of ethanol to corn oil was not reported) CN O

O Cl O

Cl CH 3 CH

3In vivo Uterotrophic Assay (2011) / Page 4 of 11
CYPERMETHRIN/ 109702 OCSPP 890.1600/ OECD 440



### 5. Test Animals:
Species: Rat (ovariectomized females only)
Strain: Sprague Dawley [Crl:CD(SD)] Age/weight at dose initiation: PND 66/ 229.8-280.1 g
Source: Charles River Laborat ories (Portage, MI) Housing: Rats were individually housed in stainless steel wire-m esh cages suspended above cage board.
Diet: 2016C M Teklad Global 16% Protein Rode nt Diet (Harlan Laboratories) , ad
libitum Genistein equivalent content = 29.0 ppm total isoflavones (genistein + daidzein + glycitein)
Water: Reverse-osmosis purified drinking water, ad libitum Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 21.2-21.6 oC (mean daily temperature) 34.9-63.8% (mean daily humidity)
10/hr 12 hrs light/12 hrs dark Acclimation period: 11 days



### B. STUDY DESIGN

1. In-Life Dates: Start: May 23, 2011 End: May 26, 2011

2. Study Design: Sexually mature ovariectomized female rats were received from Charles River Laboratories; rats were ovariectomized at PND 47 by th e supplier. Animals were received approximately one week followi ng ovariectomy (PND 55) and acclimated for 11 days prior to initiation of dosing. Vaginal smear s were taken daily for five consecutive days prior to assignment of animals to study, to veri fy that females were in persistent diestrus. The dose administration period was from P ND 66 through 68. Rats were euthanized approximately 24 hours later on PND 69 and necr opsied for uterine weight measurements

3. Animal Assignment: Animals were randomly assigned, stratified by body weight, to the test groups noted in Table 1. Statistical anal ysis indicated that th ere were no significant differences between group mean weights at study initiation. Furthermore, the body weight
of each animal was within  20% of the overall mean.

TABLE 1. Study Design a Test Group Dose (mg/kg/day) # of Females Estrogen Agonist Assay

Vehicle Control 0 6 Low Cypermethrin 25 6 High Cypermethrin 80 6

17 α -ethynyl estradiol (EE), Reference Estrogen 0.3 6 a Data were obtained from page 20 of the study report (MRID 48615701).



### 4. Dose Selection Rationale: The dose levels were selected based on the results from a rangefinding study (MRID 48615702, submitted concurrently ) in which cypermethrin in corn oil (5 mL/kg bw dosing volume) was administered via single daily oral gavage dose to 2 adultIn vivo Uterotrophic Assay (2011) / Page 5 of 11
CYPERMETHRIN/ 109702 OCSPP 890.1600/ OECD 440



### (69-77 days old) female Spra gue Dawley [Crl:CD(SD)] rats/dose group at doses of 0, 65, 80, or 100 mg/kg/day for 3 consecutive days. A summary of the study is included as an Appendix to this DER. Based on the clinical signs observed, the high-dose level of 80 mg/kg/day was selected for the current study.

5. Dose Preparation and Analysis: Dose formulations were prepared once as single formulations for each dose level by mixing appropr iate amounts of test substance with corn oil. A stock solution of EE was prepared once by dissolving the material in a small amount of 95% ethanol and diluting to volume with co rn oil; dosing formulations were prepared daily by diluting the stoc k solution. Analyses to demons trate homogeneity, stability, and resuspension homogeneity were conducted pr eviously for dose formulations at 0.1 to 20 mg/mL following up to 19 days of room temperature and refrigerated storage. 1 

### During the study, samples of each test substance dosing fo rmulation (middle stratum of each) prepared during the in-life phase were analyz ed for achieved concentration.

Results of Dose Analysis

Homogeneity: Not provided

Stability: It was stated that cypermethrin in corn oil at 0.1 to 20 mg/mL was stable at room temperature and refrigerated storage for 19 days.

Concentration (% of nominal): 98.3 and 105%

The analytical data indicated that the varia tion between nominal and actual dosage to the animals was acceptable. The study referenced above should be submitted for verification of the homogeneity and stability findings.

6. Dosage Administration: Animals were administered the test formulations and/or EE or vehicle daily via oral gavage for three cons ecutive days in a dose volume of 5 mL/kg body weight. Dose volumes were adjusted daily based on the concurrent body weight

measurement.

7. Statistics: Statistical analyses we re conducted for wet and blot ted uterine weights, daily body weights, cumulative body weight gains, an d overall average food consumption. Each endpoint was tested for homogeneity of variance using Levene's test. If that test was significant at p=0.01, then a log transformation was applied and Levene's test conducted on
the transformed data. If that test was still si gnificant, then the squa re root transformation was applied to the raw data and Levene's test conducted again. If the test was still significant, then a nonparametric test, as descri bed below, was used to analyze the data.
One-sided tests were conducted for uterine we ights and two-sided tests were conducted for
in-life data.

1 Bodle, E. S. Analytical Validation and Stability Study of Cypermethrin in Corn Oil Formulations. Syngenta Task No. TK0056162 (Study No. WIL-639153). WIL Research Laboratories, LLC, Ashland, OH, Draft.In vivo Uterotrophic Assay (2011) / Page 6 of 11
CYPERMETHRIN/ 109702 OCSPP 890.1600/ OECD 440



### For uterine weights, if variances were homogeneous, the analysis of covariance (ANCOVA), using the body weight at terminatio n as the covariate was performed; the two groups were compared using a one-sided Dunnett ’s test (for compari ng dose groups to the negative control) or a one-side d t-test (for comparing positiv e control data to data for negative control). For body weight and body weight gain data, if variances were homogeneous, an ANOVA was performed on da ta; the ANOVA test was followed by a twosided Dunnett’s test (for compari ng dose groups to the negative control) or a two-sided t-test (for comparing positive control to negative control). If the transformations were unsuccessful in making the variances homogeneous, the nonparametric one- or two-sided Wilcoxon rank sum test was used to compare da ta for the positive control to the negative control. For comparison of dose groups to the negative control, if the transformations were unsuccessful in making the variances homogeneous, the nonparametric Kruskal-Wallis test was used, followed by a one- or two-sided Dunn's test. Significance was denoted at p<0.05. Statistical analyses were performed using SAS software (version 9.2 or higher). The statistical analyses were considered adequate.

C. METHODS

1. Clinical Examinations: Cage-side checks for mortalit y and moribundity were conducted twice daily. Individu al clinical observati ons (hand-held physical examinations) were recorded daily through termination. Each ra t was also observed fo r signs of toxicity approximately 1 and 4 hours following dosing.

2. Body Weight: Animals were weighed at randomiza tion, daily throughout the dosing period, and at termination. Mean body weight cha nges were calculated for each corresponding
interval and also for the overa ll treatment period (Days 1-4).

3. Food Consumption: Food consumption was recorded daily during the dosing period and on the day of termination. Values were reported in terms of g/animal/day.

4. Necropsy and Measurement of Uterine Weight: On PND 69 (approximately 24 hours
after final administration of th e test substance), all surviving animals were euthanized by carbon dioxide inhalation and subjected to a gros s examination of the uterus. The vagina was removed just below the cervix in order to retain the luminal fluid in the uterus. The “wet” uterus (i.e., containing th e luminal fluid) was weighed. Subsequently, the uterus was opened longitudinally and gently blotted with filter paper to remove the luminal fluid and the blotted uterus was weighed. The uterus and vagina were pres erved in 10% neutral buffered formalin for possible future histopathologic examination. All females were
examined for residual ovarian tissue.

5. Microscopic Examination (Optional): Microscopic examinations were not conducted.In vivo Uterotrophic Assay (2011) / Page 7 of 11
CYPERMETHRIN/ 109702 OCSPP 890.1600/ OECD 440



### II. RESULTS

A. OBSERVATIONS

1. Mortality: All animals survived until scheduled termination.

2. Clinical Signs of Toxicity: At 80 mg/kg/day, treatment-rela ted clinical signs of toxicity
observed included one or two animals with dr ied red material around nose, hypersensitivity to handling, vocalization upon ha ndling, salivation, and/or wet clear material around mouth. Findings occurred on the second and third day of dosing. One vehicle control animal and one 25 mg/kg/day animal were observed with forelimb hair loss.

B. BODY WEIGHT AND WEIGHT GAIN: Body weight and body weight gain data are presented in Table 2. Body weights in the tr eatment groups were comparable to vehicle controls throughout the duration of the study. At 80 mg/kg/day, the overall body weight gain was significantly decreased (p<0.05; ↓ 51%).

In the positive control (EE) group, body weights were decreased (p<0.05) by 11% on Day 4 compared to the vehicle cont rol, leading to an overall body weight loss (p<0.05) of –14.9 g compared to a gain of 14.6 g in the vehicle controls.

TABLE 2. Group Body Weights and Cumulative Body Weight Gains (g) In the Estrogen Agonist Assay a

Study Day
# Dose (mg/kg/day)

Vehicle Control Cypermethrin (25) Cypermethrin (80) Reference Estrogen EE (0.3)

N Mean SD N Mean SD N Mean SD N Mean SD

1 6 256.6 11.35 6 256.1 16.43 6 257.6 12.61 6 256.8 16.57

2 6 263.9 11.52 6 261.0 17.24 6 258.1 15.49 6 251.0 17.56

3 6 265.6 11.64 6 262.0 16.41 6 259.2 13.54 6 246.3 18.47

4 6 271.2 10.54 6 268.2 16.78 6 264.8 9.84 6 241.9* ( ↓ 11) 18.18

Body Weight Gain (Days 1 - 4) 6 14.6 2.91 6 12.1 2.53 6 7.2* ( ↓ 51) 4.60 6 -14.9* ( ↓ 202) 5.52

a Data were obtained from Tables S4-S8 on pages 33-39 of the study report (MRID 48615701). Percent differences from controls, calculated by the reviewers, are included in parentheses. N Number of animals in the group
SD Standard Deviation * Significantly different from controls at p<0.05



### C. FOOD CONSUMPTION: Food consumption data are presented in Table 3. At 80 mg/kg/day, the overall average food consump tion (Days 1-4) was decreased (p<0.05) by 15%. Overall average food consumption in th e 25 mg//kg/day group was comparable to the
vehicle control.In vivo Uterotrophic Assay (2011) / Page 8 of 11
CYPERMETHRIN/ 109702 OCSPP 890.1600/ OECD 440



### In the positive control (EE) group, the overa ll average food consumption (Days 1-4) was decreased (p<0.05) by 46%.

TABLE 3. Food Consumption (g/rat/d ay) in the Estrogen Agonist Assay a

Study Day
# Dose (mg/kg/day)

Vehicle Control Cypermethrin (25) Cypermethrin (80) Reference Estrogen EE (0.3)

N Mean SD N Mean SD N Mean SD N Mean SD

1-2 6 20.5 0.85 6 19.2 2.63 6 16.2 3.25 6 11.2 1.93

2-3 6 21.8 2.15 6 20.4 1.66 6 18.6 2.03 6 13.4 1.55

3-4 6 21.4 0.79 6 20.7 2.14 6 19.2 0.67 6 9.9 2.59 Overall Average (Days 1-4) 6 21.2 0.98 6 20.1 1.93 6 18.0* ( ↓ 15) 1.43 6 11.5* ( ↓ 46) 1.32

a Data were obtained from Tables S9-S11 on pages 40-42 of (MRID 48615701). Percent differences from controls, calculated by the reviewers, are included in parentheses. N Number of animals in the group
SD Standard Deviation * Significantly different from controls at p<0.05



### D. PATHOLOGY

1. Uterine Weights: Uterine weight data are presented in Table 4. Uterine weights in the cypermethrin treatment groups were comparable to the vehicle controls, and were within the available in-lab historical control data range for untreated animals. Absolute wet and blotted uterus weights for the positive cont rol (EE) group were increased (p<0.05) by 370% and 171%, respectively. The increased wet a nd blotted uterine weights for the positive control animals were within or above the expe cted ranges as compared to the available inlab historical positive control data.

The mean blotted vehicle control uterine we ight was 0.030% of the mean vehicle control terminal body weight, which meets the Guideline requirement (<0.04%).

No macroscopic findings in the uterus were observed in the cypermethrin treatment groups or the positive control group. No residua l ovarian tissue was noted at necropsy.In vivo Uterotrophic Assay (2011) / Page 9 of 11
CYPERMETHRIN/ 109702 OCSPP 890.1600/ OECD 440

TABLE 4. Uterine Weights from Estrogen Agonist Assay in Sprague Dawley Rats a

Parameter Dose (mg/kg/day)

Vehicle Control Cypermethrin (25) Cypermethrin (80) Reference Estrogen EE (0.3)

N Mean SD N Mean SD N Mean SD N Mean SD Terminal BW (g) 6 271.2 10.54 6 268.2 16.78 6 264.8 9.84 6 241.9 18.18

Wet, absolute (mg) 6 89.6 11.38 6 93.7 9.29 6 94.3 7.58 5 b 421.4* ( ↑ 370) 75.59

Wet, relative (%) 6 0.033 0.0040 6 0.035 0.0052 6 0.036 0.0035 5 b 0.176 0.0293

Blotted, absolute (mg) 6 82.1 10.56 6 84.5 8.00 6 84.9 6.94 6 222.1* ( ↑ 171) 19.91

Blotted, relative (%) 6 0.030 0.0038 6 0.032 0.0042 6 0.032 0.0031 6 0.092 0.0092 a Data were obtained from Tables S14-S16 on pages 45-47 and Tables on pp. 166-173 of the study report (MRID 48615701). Percent differences from controls, calculated by the review ers, are included in parentheses. b Animal number 13776 excluded from analysis due to loss of luminal fluid prior to weighing. BW Body weight N Number of animals in the group
SD Standard Deviation * Significantly different from controls at p<0.05



### 2. Microscopic Examination (Optional): Microscopic examinations were not conducted.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Treatment with cypermethr in at dose levels of 25 or 80 mg/kg/day had no effect on either wet or blotted uterine weights of ovariectomized female Crl:CD(SD) rats. Therefore, cypermet hrin can be considered to be negative for estrogenicity in the uterotrophic assa y. The positive control substance (17 α

ethynylestradiol) elicited the e xpected increases in wet and bl otted uterine weights (4.7- and 2.7-fold, respectively).

B. AGENCY COMMENTS: All animals survived until scheduled termination. Treatmentrelated clinical signs of toxicity included one or two an imals with dried red material around the nose, hypersensitivity to handling, vo calization upon handling, salivation and/or wet clear material around mouth at 80 mg/kg/day. Body weights in the treatment groups were comparable to vehicle controls throughout the duration of the study. At 80 mg/kg/day, the overall body weight gain was significantly decreased (p<0.05; ↓ 51%). At 80 mg/kg/day, the overall average food consumption (Days 1-4) was decreased (p<0.05) by 15%. Overall average food consumption in the 25 mg//kg/ day group was comparable to the vehicle control. Uterine weights in the cypermethr in treatment groups were comparable to the vehicle controls. In addition, the findings ar e consistent with t hose of the dose rangefinding study, in which weight lo ss and clinical signs were obse rved in males and females at doses of 65, 80 or 100 mg/kg/day.In vivo Uterotrophic Assay (2011) / Page 10 of 11
CYPERMETHRIN/ 109702 OCSPP 890.1600/ OECD 440



### In the positive control (EE) group, body weights were decreased (p<0.05) by 11% on Day 4 compared to the vehicle cont rol, leading to an overall body weight loss (p<0.05) of –14.9 g (p<0.05) compared to a gain of 14.6 g in th e vehicle controls. Overall average food consumption in the positive control (EE) gr oup was decreased (p<0.05) by 46%. Absolute wet and blotted uterus weights for the positiv e control (EE) group we re increased (p<0.05) by 370% and 171%, respectively, as expected.

The clinical signs and decr ease in overall body weight gains for the high-dose group indicates that the tested dose levels were adeq uate. No statistically significant changes were seen in uterine weight in this assay. Cypermet hrin is negative in the uterotrophic assay.

C. STUDY DEFICIENCIES: NoneIn vivo Uterotrophic Assay (2011) / Page 11 of 11
CYPERMETHRIN/ 109702 OCSPP 890.1600/ OECD 440



### APPENDIX

Because this is a range-finding study, only a summary is provided to confirm the adequacy of the
dose selection rationale used in th e Uterotrophic Assay (MRID 48615701).

In a preliminary range-finding assay (MRID 48615702), cypermethrin (95.2% a.i.; batch# PL10- 0492) in corn oil was administered via daily oral gavage in a dos e volume of 5 mL/kg body weight to 2 adult (69-77 days old) female Sprague Dawley [Crl:CD(SD)] rats/dose group at doses of 0, 65, 80 or 100 mg/kg/day and to 2 adult (63-77 days old) male Sprague Dawley rats/dose group at 30, 45, and 65 mg/kg/day. The animals were dosed for 3 consecutive days using a staggered study design with one dose gr oup initiated every 5 days. Animals were necropsied approximately 24 hours afte r the final dose administration.

All animals survived to the scheduled terminatio n. For females at 100 mg /kg/day, clinical signs for one animal included convulsions, vocalizat ion upon handling, hypersensitivity to handling,
and wet clear material around mout h, urogenital, and abdominal areas . Changes in posture/gait were also observed in this animal. At 80 mg/ kg/day after the third dose, clinical signs for one female included rocking, lurching, or swayi ng while ambulating and wet clear material around mouth. No treatment-related clinical signs we re observed in females at 65 mg/kg/day.

For males in the 65 mg/kg/day group, clinical si gns in both animals included splayed hindlimbs or rocking, lurching, and swaying while ambulati ng, as well as clear material around the mouth, forelimbs, and/or ventral neck. No clinical signs were observed in males in the 30 and 45 mg/kg/day groups.

At 100 mg/kg/day, body weight losses were observed for both females (

− 0.1 g and

− 8.7 g) during the dosing period. At 80 mg/kg/day, one female ha d overall body weight loss (

− 8.3 g) and the other female had overall body weight gain (2.7 g). Both of the 65 mg/kg/day females gained body weight (10.0 g and 7.8 g) duri ng the dosing period. Overall average food consumption was 18.2 g for the 65 mg/kg/day group, 13.4 g for the 80 mg/kg/day/group, and 12.9 g for the 100 mg/kg/day group; overall averag e food consumption for the two females that were not dosed with cypermethrin was 19.1 g.

At 65 mg/kg/day, body weight losses were observed for both males (

− 1.5 g and

− 5.2 g) during the dosing period. Overall body weight gains were observed for the males in the 30 (14.4 g and 18.4 g) and 45 (12.5 g and 15.2 g) mg/kg/day groups during the dosing period. Overall average food consumption was 22.6 g for the 30 mg/kg/ day group, 22.3 g for the 45 mg/kg/day/group, and 19.6 g for the 65 mg/kg/day group; overall aver age food consumption for the two males that were not dosed with cypermethrin was 25.3 g.

No macroscopic findings were noted for males or females dosed with cypermethrin at any
concentration level.