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: D399304, 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 

Immediate 

Akerman, 

Office 

Ph.D. ~ ~ 

Health Effects Division (7509P) 

THROUGH: Jess 

Deputy 

Health 

Rowland 

Effects 

Director 

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 Program (EDSP) Tier 1 assays 

submitted in response to the agency's Test Order for cypermethrin: Test Order# CON-109702-8. 

Page I of2 

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Page 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 other scientifically relevant information (OSRI)) 

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

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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'

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

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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.

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

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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. Amphibian 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 23o

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 mortality 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 decrease (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 nominal 

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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-through 

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Table 1: Summary of Developmental and Thyroid Pathology/Histopathology Effects1,2 in the Amphibian 

Metamorphosis Assay (AMA) with Cypermethrin. 

Treatment 

(mg a.i./L) 

[TWA-measured]

NF Developmental 

Stage 

Hind Limb 

Length3

Asynchronous 

Development 

Thyroid Gross 

and 

Histopathology 

Day 7 Day 21 Day 7 

Day 

214 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: 

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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 CaCO3) exceeded the 

recommended range (40-48 mg/L as CaCO3). 

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.

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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 cabinet 

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B. Test Organism

Table 2: General Information About the Test Species and Parental Care.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Species 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 Reported Were parents maintained as Yes Adults were obtained from Nasco in Fort EPA recommends that larvae used in the 

1 U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the Endocrine Disruptor 

Screening 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). 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

in-house stock? Atkinson, Wisconsin and were maintained 

as in-house breeders for at least one month prior to generating tadpoles.

assay be derived from in-house adults.

Were parental acclimation conditions same as definitive test? 

Yes

Acclimation period for parental frogs (if applicable): 

30 days

Details on parental feeding: Not reported Details on parental health: Parental stock did not 

show any signs of sickness, disease, injuries or abnormalities prior to testing. 

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Table 3: Larval Selection and Care.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Best 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 (if applicable): 

Three 

Number of eggs sampled per spawn:

All eggs were sampled on a given day.

Embryonic viability was checked daily.

NF stage at test initiation 51 EPA recommends that the definitive study be 

initiated with larvae at Nieuwkoop – Faber 

(NF) developmental stage 51 (≤17 days 

post-fertilization).

Age at test initiation: 13 days post-fertilization 

(dpf)

Mean total length at test initiation (if reported): 

Not reported 

Range of total length at test initiation (if reported): 

Not reported

Was the optional size selection method used? 

Not reported

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on larval selection: When a sufficient 

number of tadpoles reached NF stage 51, 

the larvae were transferred from a rearing vessel to a pooling tank containing dilution water at 22qC. 

The tadpoles that met the developmental criteria based on hind limb morphology were distributed to four test vessels four at a time 

until each vessel 

contained 20 tadpoles. Abnormal tadpoles were removed and replaced 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

with 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 Express Suitable for normal growth and 

development of X. laevis tadpoles; fine 

particulate that maintains water column suspension; stock solution prepared in dilution 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 diet should 

demonstrate equal performance to Sera 

Micron® .

Source of food: Xenopus Express, 

Brooksville, Florida

Iodide concentration in diet (if known): 

Not provided 

Frequency of feeding: 2 times/day EPA recommends that feeding occur at least 

twice per day.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on feeding regime: Same as EPA 

Guidelines

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).

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C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Type of exposure: Flow-through EPA recommends the use of a flow-through 

system.

Type of flow-through dilution system (if applicable):

Intermittent-flow proportional diluter 

Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

Flow-through rate (if applicable):

50 mL/min, based on a 6.5L fill volume and 6.8 

volume additions per day

Recommended flow-through rate is 25 

mL/min (complete volume replacement ca. 

every 2.7 hrs).

Details on toxicant mixing for flow-through systems (if applicable):

Stock solution was delivered to the mixing cell at a rate of6.94 mL/min (0.0555 mL/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 is 

2 Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

prepared and replaced daily during exposure via a glass bead saturator column. A Fluid Metering Inc. (FMI) pump was used 

to deliver a flow of dilution water through the column. The FMI pump, in conjunction with a 12-L 

graduated glass carboy, 

was calibrated to deliver 0.0666 L/cycle of the 0.005 mg/L stock solution into the diluter's chemical mixing chamber. The mixing chamber contained the highest nominal test concentration 

completely mixed before introduced into the 

test system is recommended; 4) The 

recommended flow splitting accuracy is 

within 10%.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

(0.0003 mg/L) and was proportionally diluted (10%) to provide the remaining nominal concentrations (0.00003 

and 0.000003 mg/L). 

Renewal period for static renewal (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 was 

used to prepare FETAX 

FETAX solution consisted of a blend of water from an on-site 100-meter bedrock well and 

EPA recommends natural or reconstituted 

water; it is recommended that natural water 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

(Frog Embryo TeratogenesisAssay – Xenopus) solution

de-chlorinated Town of Wareham well water. Solutions were prepared in 3,400-L batches 

and mixed for 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 for pesticides, heavy metals, and other contaminants? 

Yes (pesticides, PCBs and toxic metals)

Iodide supplementation in water?

No If reconstituted water is used or if 

background levels of iodide in natural water 

are less than 0.5 

μg/L, iodide 

supplementation is recommended. This 

supplementation is in addition to the 

recommended dietary source of iodide 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

(e.g.. in Sera Micron).

Test vessel type/materials: Glass, silicone sealant, 

stainless steel and Teflon®

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-gallons

Fill volume: 6.5 LAdditional details on exposure system: 

NA 

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Table 5: Summary of Water Quality Characteristics in the Test System. 

Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Hardness (mg/L as CaCO3) 160 180 Not reported 

Daily 

EPA recommends hardness 40 to 48 

mg/L as CaCO3.

pH 7.2 8.2 7.7 Daily 

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 6.6 Daily 

EPA recommends dissolved oxygen (DO) 

>3.5 mg/L (>40% air saturation). OECD 

recommends DO concentration >3.5 mg/L

(>40% air saturation).

Temperature 21°C 23qC 22qC Daily 

EPA recommends temperature 22±1oC; 

inter-replicate and inter-treatment 

differentials should not exceed 0.5oC.

Iodide (μg/L) Not reported Not reported 9.6 Biannually 

EPA recommends aquatic iodide range 0.5 

– 10 

μg/L (supplemental iodide should not 

exceed 2 

μg/L).

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Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Ammonia Not 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.

Fluoride Not reported Perchlorate Not reported Chlorate Not reported Total Alkalinity (mg/L as CaCO3) Specific Conductance (μmS/cm)

68 

1800 

74 

1900 

Not reported Not reported 

Daily Daily 

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 D. Study Design and Additional Experimental Conditions

Table 6: Range-Finding Study Conditions (if Applicable).

Parameter Value(s) Details or Remarks Guideline Recommendations 

Was a range-finder conducted? YesIf yes, what was the method for determining the highest test concentration in the range-finder? 

Solubility limit (7.6 mg/L) and concentration at which mortality is 

<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: 51 Test duration: 14 days

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Additional details: Two 14-day range-finding studies 

were conducted, with 10 tadpoles per replicate and two replicates per treatment. Tadpoles were exposed to nominal cypermethrin concentrations of 0.00063, 0.0013, 0.0025, 0.005 and 0.01 

mg/L during the initial study and to concentrations of 0.000038, 

0.000075, 0.00015, 0.0003 and 

0.00060 mg/L during the second 

study.

In the first range finding study, lethal or sublethal effects were observed at all treatment levels. In the second range finding study, neither lethal nor sublethal effects were observed below 0.00060 mg/L. The EC10

was 0.00028 mg/L (95% CI: 0.00016-0.00035). Based on 

these results, the maximum concentration in the definitive study was set to 0.0003 mg/L. 

Table 7: Definitive Study Conditions.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Test duration: 21 days EPA recommends that the duration of the 

definitive test be 21 days. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Method for selecting the highest test concentration in the definitive test:

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 (if applicable):

NA 

Separation 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 concentrations adjusted for purity? 

No 

Indicate the type of values presented for measured 

Time-weighted average 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

concentrations: 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 reported Frequency 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 solvent control (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 item treatment level: 

4 EPA recommends 4 replicates.

Number of larvae per treatment at 80 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

test initiation: Was a solvent used? No Solvent type (if applicable): NA Maximum 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? No Positive control (if applicable): NA Positive control concentration(s) NA 

3 Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review. Aquatic 

Toxicology, 76, pp.69–92. 

4 OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Series on 

Testing and Assessment. No. 23. Paris, France. 

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Parameter Value(s) Details or Remarks Guideline 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: NA 

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Table 8: Summary of Treatment Concentrations in the Amphibian Metamorphosis Assay with Cypermethrin. 

Treatment ID

Nominal 

Concentration 

(mg a.i./L) 

TWA-Measured 

Concentration 

(mg a.i./L) 

Mean CV (%) Details or Remarks Guideline Recommendations 

Negative Control 0.00 <LOQ NA NA EPA and OECD recommend that test item 

concentrations be maintained at a coefficient of 

variation (CV) 

≤20%.

Treatment 1 0.000003 0.0000019 38.5 NATreatment 2 0.00003 0.0000109 24.1 NA Treatment 3 0.0003 0.0000867 35.2 NA 

Abbreviations: CV Coefficient of variation. 

 E. Observations

Biological 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? Yes

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EPA recommends that observations of mortality and clinical signs occur daily, at a minimum; other observations are recommended as follows: NF 

developmental stage (days 7 and 21); any asynchronous development, indicated by tadpoles that cannot be assigned an NF stage (days 7 and 21); 

hind limb length (days 7 and 21); snout-vent length (days 7 and 21); body weight (test initiation, for optional size-based larval selection); and thyroid 

gland gross pathology and histopathology (day 21). Note the histopathology section of the test guideline also includes thyroid gross pathology observations.

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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 71 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 10th and 90th percentiles of the control’s development stage at test termination 

were 59.8 and 61.8, respectively. No asynchronous development was observed. 

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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 

# Asynchronous 

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. 

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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: 

SVL1 n Mean(mm) 

±SD

HLL: 

SVL1

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. 

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Table 12: Larval Growth in Xenopus laevis

Treatment 

(mg a.i./L) 

[TWA-measured] 

Snout-Vent Length (SVL) Body Weight1

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. 

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Table 13: Gross Pathology and Histopathology of the Thyroid Gland in Xenopus laevis.

Treatment 

(mg a.i./L) 

[TWA-measured] 

Diagnostic Observations1

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.

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Table 14: Additional Thyroid Gland Histopathology Observations in Xenopus laevis. 

Treatment 

(mg a.i./L) 

[TWA-measured]

Additional Qualitative Observations1

Severity

Follicular Lumen 

Area (Increase) 

Follicular Lumen 

Area (Decrease) 

Follicular Cell 

Height (Increase) 

Follicular Cell Height 

(Decrease) 

Follicular Cell 

Shape 

n Incidence n Incidence n Incidence n Incidence n Incidence 

Negative Control 0 20 -- 20 -- 20 -- 20 -- 20 -- 

1 20 -- 20 -- 20 -- 20 -- 20 -- 

2 20 -- 20 -- 20 -- 20 -- 20 -- 3 20 -- 20 -- 20 -- 20 -- 20 -- 

0.0000019 0 20 -- 20 -- 20 -- 20 -- 20 -- 

1 20 -- 20 -- 20 -- 20 -- 20 -- 

2 20 -- 20 -- 20 -- 20 -- 20 -- 3 20 -- 20 -- 20 -- 20 -- 20 -- 

0.0000109 0 20 -- 20 -- 20 -- 20 -- 20 -- 

1 20 -- 20 -- 20 -- 20 -- 20 -- 

2 20 -- 20 -- 20 -- 20 -- 20 -- 3 20 -- 20 -- 20 -- 20 -- 20 -- 

0.0000867 0 20 -- 20 -- 20 -- 20 -- 20 -- 

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DER Template Version: 22 September 2011 

Treatment 

(mg a.i./L) 

[TWA-measured]

Additional Qualitative Observations1

Severity

Follicular Lumen 

Area (Increase) 

Follicular Lumen 

Area (Decrease) 

Follicular Cell 

Height (Increase) 

Follicular Cell Height 

(Decrease) 

Follicular Cell 

Shape 

n Incidence n Incidence n Incidence n Incidence n Incidence 

1 20 -- 20 -- 20 -- 20 -- 20 -- 

2 20 -- 20 -- 20 -- 20 -- 20 -- 3 20 -- 20 -- 20 -- 20 -- 20 -- 

1 Thyroid histopathology is graded 0 – 3 based on severity: 0=Not remarkable, 1=Mild, 2=Moderate, 3=Severe. See OECD No. 82 for reference. 

At test termination, spinal deformities were observed in 27 of the tadpoles in the negative control and 27, 22, and 22 tadpoles in the TWA-measured 

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

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Table 15: Clinical Signs in Xenopus laevis.

Treatment 

(mg a.i./L) 

[TWA-measured] 

Clinical Signs1

Type n Incidence 

Negative Control Spinal deformities 60 27 0.0000019 Spinal deformities 60 27 0.0000109 Spinal deformities 60 22 0.0000867 Spinal deformities 60 22 

1 Note that asynchronous development (unable to stage) is reported previously in Table 10. 

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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 SystemTM (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 Conclusions 

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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. 

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Table 16: Developmental and Thyroid Gross Pathology/Histopathology Endpoints1,2 in the AMA with Cypermethrin.

Abbreviations: Diff. Difference. NA Not applicable.

1 Unless otherwise indicated, effects are reported based on comparison to the clean water control. Conclusions regarding histopathology may be heavily weighted 

by the expert opinion of a board-certified pathologist. 

2 Unless otherwise specified, effects are considered statistically significant at p<0.05. 3 Hind-limb length is normalized to snout-vent length (SVL). 4 Day 21 excluding late-stage individuals 

Treatment 

(mg a.i./L) 

[TWA-measured]

NF Developmental Stage Hind Limb Length3 Asynchronous 

Development 

Thyroid Gross and 

Histopathology 

Day 7 Day 21 Day 7 Day 21 4 Day 7 Day 21 Day 21 

Median p Median p

% 

Diff. p % Diff. p 

% 

Diff. 

p 

% 

Diff. 

p 

Treatment-Related 

Effects? (Yes/No) 

Negative Control 54 NA 61 NA NA NA NA NA -- -- -- -- --0.0000019 54 1.0 61 0.50 -2.14 0. 0.0 0.56 -- -- -- -- No 0.0000109 54 1.0 61 0.50 -4.98 0.22 5.38 0.31 -- -- -- -- No 0.0000867 54 1.0 60 0.13 -0.53 0.99 3.39 0.25 -- -- -- -- No Statistical Test Mann-Whitney 

JonckheereTerpstra Dunnett Jonckheere-Terpstra -- -- -- 

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DER Template Version: 22 September 2011 

Table 17: Growth Endpoints1,2 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 

Dunnett 

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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 concentration 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. 

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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 SystemTM. User’s Guide. 

Tidepool Scientific Software, McKinleyville, California. 

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

 

Page 44 of 223
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 45 of 223
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).

Page 46 of 223
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. 1200 I OECD None 

S igna tu re: ___::.,~~~:::d..-=:::'::::::::..__ 

Date: --,,+-~-=------

Signature: __,£_~~~=:..__ __ 

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- 10 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 average 

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Aromatase (Human Recombinant) Assay (2012) / Page 2 of 12 

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full activity. Activity in the background controls 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 IC50 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-response curve indicated 

that the aromatase activity at all concentrations of cypermethrin tested was essentially equivalent 

to the activity observed in the full activity control (>75% activity= non-inhibitor). 

Based on the data from the average response curve, cypermethrin is classified as a Non-inhibitor 

of aromatase activity in this assay. 

The assay satisfies the EDSP Tier 1 Test Order requirements for an Aromatase assay (OCSPP 

890.1200). 

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

and Certification of Authenticity statements were provided. 

 

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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 precipitation was observed at concentrations of 10-3.5 M 

and above. A small amount of precipitation 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 3

CH CH3

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; ([3H]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 pipette because of density and viscosity. 

 Concentration 

(% of total volume in assays): ≤1% 

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

Supersomes 

Source: GentestTM, 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-labeled and radiolabeled 

[

3

H]ASDN was prepared such that the final concentration of ASDN in the assay was 

approximately 0.1 μM, and the amount of tritium added to each incubation tube was 

0.1 μCi. 

The test chemical(s) were formulated in DMSO such that the volume of DMSO used per 

assay was no more than 1% v/v of the total assay volume. DMSO was selected because it is 

listed as one of the solvents of choice detailed in the EPA guideline; it 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 positive control substance 

(10−10 to 10−5 M; Table 4) were achieved by the addition of 20 μL of the dilution for a final 

assay volume of 2 mL and that the final DMSO concentration was no more than 1% v/v of 

the total assay volume. 

Human recombinant microsomes were purchased from GentestTM, 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 being 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 

[3H]ASDN 100 nM 

Propylene Glycol 5% 

Temperature 37±2°C 

Incubation Time 15 min 

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2. Suitability Assessments: The protein concentration of the microsomes (Lot #54390) was 

supplied by the vendor as 6.9 mg/mL. Aromatase activity of the microsomes was also 

provided by the vendor as 2.8 pmol/min/pmol P450. The protein concentration of the 

microsomes (Lot #03897) was supplied by the vendor as 7.4 mg/mL. Aromatase activity of 

the microsomes was also provided by the vendor as 6.0 pmol/min/pmol P450. The 

minimum aromatase activity in the full activity control samples was determined to be 0.166 

nmol/mg-protein/min, which was greater than the minimum acceptable aromatase activity of 

0.1 nmol/mg-protein/min. 

3. Aromatase Assay: Each assay run contained 4 tubes for the full enzyme activity and 4 

tubes for the background activity controls. Two tubes of each control were run at the 

beginning of the assay, and two of each control were run at the end of the assay. A full 

concentration curve in duplicate for the positive control, and a full concentration curve in 

triplicate for the test substance were established. The aromatase assay was conducted 

generally according to the procedures described in OCSPP 890.1200 (Section h, pp. 9-10). 

The amount of 3

H2O 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, fenarimol and nitrofen) 

were incubated alongside the positive control inhibitor (4-OH ASDN) on three separate 

occasions using the same methods employed in the current study. 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 inhibitor 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 the preferred ranges. 

(2) Demonstration of Proficiency of New Technician for Conducting Assay (when 

applicable): The positive control data for slope, top and bottom percent met the criteria as 

listed in section (i) of OCSPP 890.1200. 

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Aromatase (Human Recombinant) Assay (2012) / Page 6 of 12 

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TABLE 2. Performance Criteria for the Positive Controla

Parameter Lower Limit Criteria Upper Limit Criteria 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 IC50 (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 laboratory was demonstrated prior to 

running any test chemicals by conducting three full scale test runs on each of the proficiency 

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

that atrazine, econazole, fenarimol, and nitrofen were correctly classified 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 Activity with Test Chemical(s): The response of aromatase 

activity to the presence of eight concentrations of cypermethrin 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 highest 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 concentrations of cypermethrin, defining the top of the concentrationresponse curve. 

Page 52 of 223
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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 componentsa 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 1b,c 3 All test components plus Cypermethrin 1×10-3

Cypermethrin Conc 2b,d 3 All test components plus Cypermethrin 1×10−3.5

Cypermethrin Conc 3b,e 3 All test components plus Cypermethrin 1×10−4

Cypermethrin Conc 4b,f 3 All test components plus Cypermethrin 1×10−4.5

Cypermethrin Conc 6b 3 All test components plus Cypermethrin 1×10−5

Cypermethrin Conc 7b 3 All test components plus Cypermethrin 1×10−6

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

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

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

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

a The complete assay contained buffer, propylene glycol, microsomal protein, [3H]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 aromatase activity (nmol/mg protein/min) and 

percent control for the positive control, proficiency chemicals and test chemical(s). The 

following raw data and calculated endpoints for each run were included in the report 

(Table 5). 

TABLE 5. Raw and Calculated Data

Raw/Calculated Data Included (X) 

DPM/mL for each portion of extracted aqueous incubation mixture X 

Average DPM/mL for each aqueous portion (after extraction) X 

Total DPM for each aqueous portion (after extraction) X 

The total DPM present in the assay tube at initiation X 

The percentage of substrate converted to product X 

Total DPM after extraction corrected for background X 

Aromatase activity expressed in nmol/mg protein/min X 

Average aromatase activity in the full activity control tubes X 

Percentage of control activity remaining in the presence of various inhibitor concentrations X 

DPM Disintegrations per minute

2. Statistical Methods: For data generated at CeeTox, basic statistical analysis was performed 

on the data, which included means of replicates, standard deviation of the mean, standard error 

of the mean, and coefficient of variation. Analysis of variance (ANOVA) was not performed. 

Page 53 of 223
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The response curve was fitted by weighted least 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 analysis. For each run, the individual percent of 

control values was plotted versus logarithm of the test chemical concentration. The fitted 

concentration response curve was superimposed on the plot, with individual plots prepared 

for each run. The average percent of control values versus logarithm of test chemical 

concentration for the individual runs for each test chemical (with different symbols for each 

run) were included on the same graph with their respective fitted 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 % full 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 categories presented in Table 6. 

TABLE 6. Interpretation of Results 

Criteria Interpretation 

Data fit 4-parameter nonlinear 

regression model 

Average curve across runs crossed 50%a Inhibitor 

Average lowest portion of curves across runs is 

between 50% and 75% activityb

Equivocal 

Average lowest portion of curves across runs is greater 

than 75% activityb

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 cross 50%, the chemical is typically 

determined to be untestable in the aromatase assay. 

II. RESULTS 

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

0.166-0.293 nmol/mg-protein/min for the 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 activity. 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/min 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 the positive control are shown in Figure 1. 

Page 54 of 223
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CYPERMETHRIN / 109702 OCSPP 890.1200 / OECD None

TABLE 7. Effect of Cypermethrin on Aromatase Activity (as percent of control) from Independent Runsa

Chemical 

Concen. 

(Log M) # Runs 

Overall 

Meanb Overall SDb Overall SEMb Overall % CVb

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 

 -4.5c 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 used for Run 3 only. 

SD Standard Deviation 

SEM Standard error of the mean 

CV Coefficient of Variance 

FIGURE 1. Inhibition Response 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 concentration 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-minute incubation period and were not used for 

Page 55 of 223
Aromatase (Human Recombinant) Assay (2012) / Page 10 of 12 

CYPERMETHRIN / 109702 OCSPP 890.1200 / OECD None

data interpretation. The highest concentration 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 activity) for each concentration tested across all 3 

runs is presented in Table 7 (presented above), along with the overall standard deviation, 

SEM, and % CV. Inhibition response curves for cypermethrin from each run are shown in 

Figure 2, and the average inhibition 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 average dose-response curve indicated that the aromatase activity 

of cypermethrin at all soluble test concentrations tested was essentially equivalent to the 

activity observed in the full activity control. These data could not be fit to the 4-parameter 

nonlinear regression model; therefore, log IC50 and Hill slope estimates were not 

Page 56 of 223
Aromatase (Human Recombinant) Assay (2012) / Page 11 of 12 

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determined for cypermethrin. For 4-OH ASDN, the estimated log IC50 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 Activity (as Percent of Control) From Independent 

Runsa

Chemical Run 1 Run 2 Run 3 Meanb SDb %CVb

Log IC50 (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 curve and the criteria listed above in Table 6, 

the results support the conclusion that cypermethrin does not 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 validated 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 demonstrated 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 were correctly classified as non-inhibitor or 

inhibitor. 

Aromatase activity in the full activity controls averaged 0.222 ± 0.049 nmol/mgprotein/min. The response of each full activity control within a run was between 90 to 

110% of the average full activity, and activity in the background controls averaged 0.6 ± 

0.2% of the full activity 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 IC50, and 

%CV. The estimated log IC50 for 4-OH ASDN averaged −7.17 M and the slope was −0.94. 

For cypermethrin, aromatase activity at the lowest and the highest tested concentrations, 

10-10 and 10−4 M, respectively, used for interpretation was 91.79% and 87.37% of the full 

activity control. The two highest test concentrations of cypermethrin (10-3 and 10-3.5 M) 

showed precipitation in the incubation during the 15-minute incubation period; therefore, 

Page 57 of 223
Aromatase (Human Recombinant) Assay (2012) / Page 12 of 12 

CYPERMETHRIN / 109702 OCSPP 890.1200 / OECD None

these data were not evaluated. The average dose-response curve indicated that the 

aromatase activity of cypermethrin at all soluble test concentrations tested was essentially 

equivalent to the activity observed in the full activity control; therefore, these data could not 

be fit to the 4-parameter nonlinear regression model. Because the mean aromatase activity 

for cypermethrin was 87.37 ± 5.11% at the highest soluble test concentration of 10−4

 M, 

cypermethrin was determined to be a non-inhibitor of aromatase activity in this assay. 

C. STUDY DEFICIENCIES: The following deficiencies were noted that are not considered 

to have had an adverse impact on the results, interpretation or conclusions of this study: 

 Proficiency data were not included in the study report. However, it was stated that a 

full-scale assay consisting of three independent runs was conducted using the positive 

control (4OH-ASDN) and the four proficiency chemicals outlined in the OCSPP 

890.1200 guideline was conducted and that proficiency 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 Supersomes was not determined on each day of the 

assay as indicated in the guidelines. 

Page 58 of 223
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 59 of 223
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).

Page 60 of 223
Estrogen Receptor Transcriptional Activation (2012) I Page I of 16 

CYPERMETHRIN/109702 OCSPP 890.1300/ OECD 455 

Health 

Primary 

Effects 

Reviewer: 

Division 

Patience Browne Ph.D. Signature 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. 

SPONSORS: 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 ), hERa-HeLa-9903 cells cultured in vitro were exposed to cypermethrin (95.2% a.i., 

Batch PL 10-0492) at logarithmically increasing concentrations from 10- 103 to 10-33 Min DMSO 

(0.1 %) for approximately 24 hours in three independent runs. A total of 8 runs were conducted, 

but only 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. 

Cells were exposed to the test agent for 24±2 hours at 37± 1°C to induce reporter (luciferase) 

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

cypermethrin was measured using a proprietary luciferase assay. 

Cypermethrin was tested up to the limit of solubility, I 0-4 M. The RPCMax 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 :S0.9% at concentrations up to 10-3 3M, 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 

sensitive, with all parameters for all reference chemicals at lower levels than expected according 

to the guidance. 

Page 61 of 223
Estrogen Receptor Transcriptional 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 17α-methyltestosterone. For Run 2, 

Log PC50 values could not be determined for 17α-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 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 

17α-methyltestosterone, which was able to reach maximums of 124.3, 16.5, and 52.8% PC in 

Runs 1, 2, and 3, respectively. 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 

based on cytotoxicity. Because the RPCMax < PC10 in both assay runs, cypermethrin was 

considered negative for estrogen receptor transcriptional 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 and Quality Assurance statements were 

provided. 

 

Page 62 of 223
Estrogen Receptor Transcriptional 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 3CH CH3

2. Reference substances 

 17β-estradiol (strong estrogen; positive control)

 Supplier: Not Reported 

 Catalog and Batch #: Not Reported 

 Purity: Not Reported 

 CAS # : 50-28-2 

 

 17α-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 

 

 17α-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% 

Page 63 of 223
Estrogen Receptor Transcriptional Activation (2012) / Page 4 of 16 

CYPERMETHRIN/109702 OCSPP 890.1300/ OECD 455 

B. METHODS

1. Cell Culture: Stably-transfected hERα-HeLa-9903 cells were obtained from the Japanese 

Collection of Research Bioresources 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 red, supplemented with kanamycin (60 mg/L) 

and 10% dextran-coated charcoal-treated fetal bovine serum (DCC-FBS; source not 

reported), in an incubator under 5% CO2 at 37±2 oC. Upon reaching 75-90% confluence, 

cells were subcultured up to 34 times prior to exposure 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 cryopreservation 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 1×104 cells/100 μL 

medium/well and allowed to attach for 3 hours. The growth medium was replaced with 

medium containing serial log dilutions of cypermethrin in DMSO. In addition to the 

chemical being tested, each plate included 6 blank replicates, 6 positive control (1 nM 17βestradiol) replicates and 12 vehicle control replicates. Cells were incubated for 24±2 hours 

at 37±2 °C. The total final concentration of DMSO was 0.1%. Cytotoxicity was determined 

by two-read propidium iodide uptake. 

It was stated that transcriptional activation of the estrogen receptor was determined as 

described in CeeTox Standard Operating Protocol (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 appropriate concentration range and to determine 

concentrations resulting in insolubility and/or cytotoxicity. 

b. Proficiency Chemicals: The responsiveness of the test system was evaluated on March 5, 

2010 and April 12 and 28, 2011. Cells used for the 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): 

Page 64 of 223
Estrogen Receptor Transcriptional 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 --- 

17α-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 0.01b, 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 0.1b and 1 mM 

1,3,5-Tris(4-

hydroxyphenyl)benzene c

15797-52-1 10−12 to 10−5

 Positive Cytotoxic at 100μM. PCmax approx. 50% of PC. 

Binds to hERα 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 = RPCMax ≥10% of the response of the positive control in at least 2 of 2 (or 2 of 3) runs 

Negative = RPCMax 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 ERα mediated transcriptional activation, but may not be negative for non-ERβ mediated transcriptional 

activation. A positive result would indicate that the system is detecting activity other than that due to pure ERα, 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 reference chemicals were included in each run, in 

separate plates: 

Reference Chemical CAS No. Concentration Range Class 

17β-estradiol (E2) 50-28-2 10−15 to 10−8 Strong estrogen 

17α-estradiol 57-91-0 10−13 to 10−6 Weak estrogen 

Corticosterone 50-22-6 10−11 to 10−4 Negative compound 

17α-methyltestosterone 58-18-4 10−12 to 10−5 Very weak agonist 

3. Data analysis: To obtain the relative transcriptional activity to the 1 nM E2 positive control 

(PC), the luminescence signals from the concurrent plate were analyzed 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 express relative transcriptional activity as a 

percentage of the PC. The test material was defined as negative for inducing estrogen 

receptor transcriptional activation if the RPCMax < PC10 in at least 2 of 2 runs. Log EC50 and 

Hill slope values are calculated only if a positive response is observed. Coefficients of 

variation (CV) were calculated for the luminescence data triplicates. Concentrations 

showing >20% cytotoxicity or evidence of insolubility were excluded from analyses. 

4. Definitions 

EC50 = concentration of agonist that induces a response halfway between the baseline 

(bottom) and maximum (top) response 

Page 65 of 223
Estrogen Receptor Transcriptional Activation (2012) / Page 6 of 16 

CYPERMETHRIN/109702 OCSPP 890.1300/ OECD 455 

PC10 = 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 

PC50 = 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 

RPCMax = 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 

PCMax = concentration of a test chemical inducing the RPCMax

II. RESULTS 

A. PRELIMINARY TEST: In order to identify a suitable top concentration cypermethrin for 

use in the transcriptional activation assays, preliminary 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, logarithmically increasing concentrations 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, Cytotoxicity, and Concentration-Selection for 

Cypermethrina 

Concentration (M) % Viability b

Commentsc

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 different days for each chemical. The reported responses are 

summarized in Table 2. 

Page 66 of 223
Estrogen Receptor Transcriptional 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 

17α-Ethynyl estradiol Positive 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 Negative Negative Negative NA 

Atrazine Negative Negative 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 three estrogenic reference chemicals 

are shown in Figures 1-3, and the values derived from the concentration response curves 

(e.g., Log PC50, Log PC10, Log EC50, Hill slope) for the concurrently run reference materials 

are presented in Table 3. 

Although performance criteria were generally met, the data were extremely variable 

between runs. In Run 1, cytotoxicity was observed at the high concentration (10-4) for 

corticosterone, the maximum responses of 17β-estradiol, 17α-estradiol and 17αmethyltestosterone (124-150%) exceeded the response of the PC (1 nM 17β-estradiol), and 

performance criteria parameters for 17α-methyltestosterone were lower than the guideline 

range, indicating an increased sensitivity to weak agonists (does not negatively impact the 

validity of Run 1). In Run 2, cytotoxicity was observed at the high concentrations of both 

corticosterone and 17α-methyltestosterone, and Log PC50 values could not be determined for 

17α-methyltestosterone as it did not reach 50% activation at concentrations up to 10-5 M. In 

Run 3, the maximum response of both 17β-estradiol and 17α-estradiol exceeded (124-144%) 

the response of the PC. 

Page 67 of 223
Estrogen Receptor Transcriptional Activation (2012) / Page 8 of 16 

CYPERMETHRIN/109702 OCSPP 890.1300/ OECD 455 

TABLE 3. Performance Criteria for Reference Chemicalsa

Reference Chemical 

 Parameter Acceptable Range Values Acceptable 

Run 1 Run 2 Run 3 Yes No 

17β-estradiol

 Log PC50 −11.4 to −10.1 -11.7b -10.2 -10.6 Run 1 

 Log PC10 <−11 -12.6 -11.7 -11.5 X 

 Log EC50 −11.3 to −10.1 -11.7b -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 

17α-estradiol

 Log PC50 −9.6 to −8.1 -9.4 -8.6 -8.7 X 

 Log PC10 −10.7 to −9.3 -10.4 -9.8 -9.7 X 

 Log EC50 −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 

17α-methyltestosterone

 Log PC50 −6.0 to −5.1 -8.8b NCc -5.1 Run 1,2 

 Log PC10 −8.0 to −6.2 -9.7b -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 PC50, EC50, and PC10 estimates were below the expected range in Run 1. 

c Log PC50 estimates was not reached in Run 2. 

NC Not Calculated 

 

Page 68 of 223
Estrogen Receptor Transcriptional Activation (2012) / Page 9 of 16 

CYPERMETHRIN/109702 OCSPP 890.1300/ OECD 455 

FIGURE 1. Relative Transcription Activation (RTA) of the Natural Ligand, 17βestradiol, Compared to the Positive Control, 1 nM 17β estradiol. 

Mean ± SD are presented (n=6). 

 

Page 69 of 223
Estrogen Receptor Transcriptional Activation (2012) / Page 10 of 16 

CYPERMETHRIN/109702 OCSPP 890.1300/ OECD 455 

FIGURE 2. Relative Transcription Activation (RTA) of the Weak Estrogen, 17αestradiol, Compared to the Positive Control, 1 nM 17β estradiol. 

Mean ± SD are presented (n=6). 

 

Page 70 of 223
Estrogen Receptor Transcriptional Activation (2012) / Page 11 of 16 

CYPERMETHRIN/109702 OCSPP 890.1300/ OECD 455 

FIGURE 3. Relative Transcription Activation (RTA) of the Very Weak Estrogen, 17αmethyltestosterone, Compared to the Positive Control, 1 nM 17β estradiol. 

Mean ± SD are presented (n=6). 

Page 71 of 223
Estrogen Receptor Transcriptional 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 positive controls are included in 

Table 4. The overall mean TA value for 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-fold. The mean normalized value for the positive 

control was 974775-1973392. The PC50 (50% of the maximum response) for E2 in this 

assay is 487397-986696 and the PC10 (10% of the maximum 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 data obtained on pages 35-37of the study report. 

b Positive control was 17β-estradiol (E2) at 1 nM. 

c Fold-induction = (mean TA of PC)/(mean TA of VC) 

2. Test Material: Relative (to the PC) transcriptional activation 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 RPCMax was 16.3%, and the associated PCMax was 

10-4 M. For Runs 2 and 3, each conducted with a different batch of frozen cells, the RPCMax 

was <PC10 in both runs. Given that a new cell line was brought out of frozen stock after a 

positive response, the performance of those cells indicate inadequate response to the 

reference chemicals in general, and a weaker than acceptable response to the very weak 

agonist, 17α-methyltestosterone, Runs 2 and 3 are not valid. The PC10 for Run 1 was 

10-4.6 M. 

Page 72 of 223
Estrogen Receptor Transcriptional Activation (2012) / Page 13 of 16 

CYPERMETHRIN/109702 OCSPP 890.1300/ OECD 455 

TABLE 5. Relative Transcriptional Activation (RTA) of Cypermethrina

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 EC50b NA NA NA 

Hill Slopeb NA NA NA 

RPCMax 16.3 0.9 0.6 

PCMax 10-4 10−4.3 10−6.3

PC50 NA NA NA 

PC10 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 Applicable

Page 73 of 223
Estrogen 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. 

Page 74 of 223
Estrogen Receptor Transcriptional 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 the study report. While the Log PC50, Log PC10, Log EC50, 

and Hill slope values for the concurrent reference 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 17α-methyltestosterone, the mean 

RPCMax 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 cytotoxicity observed at any of the exposure 

concentrations tested. In three independent runs of the transcriptional activation assay, 

cypermethrin resulted in an increase in luciferase activity in the third run to 12.7% at 

10-4.5 M and 16.3 % at 10-4 M. This results in a PC10 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 RPCMax was 16.3% at 

10-4 M and the PC10 was 10-4.6 M in Run 1, indicating a positive response to the assay. 

However, in Runs 2 and 3 the RPCMax for cypermethrin was ≤0.9% at concentrations up to 

10-3.3 M, indicating that cypermethrin was negative for estrogenic activity under the 

conditions of the assay. Review of the performance criteria parameters for Run 1 (Table 3) 

shows that Run 1 was unusually sensitive, with all parameters for all reference chemicals at 

lower levels than expected according to the guidance. 

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 17αmethyltestosterone. For Run 2, Log PC50 values could not be determined for 17αmethyltestosterone 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 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 17α-methyltestosterone, which was able to reach maximums of 124.3, 16.5, 

and 52.8% PC in Runs 1, 2, and 3, respectively. 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 based on cytotoxicity. Because the RPCMax < PC10 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 17α-methyltestosterone) in two of the three runs. Cytotoxicity 

should not be observed at any level for the reference chemicals. 

Page 75 of 223
Estrogen Receptor Transcriptional Activation (2012) / Page 16 of 16 

CYPERMETHRIN/109702 OCSPP 890.1300/ OECD 455 

 The maximum response of 17β-estradiol, 17α-estradiol, and 17α-methyltestosterone in 

Runs 1 and/or 3 exceeded the response of the PC (1 nM 17β-estradiol). The maximum 

responsiveness should be observed at 1 nM 17β-estradiol in both the PC samples and 

reference compound samples. 

 The cells responded inadequately (<50% RTA) to the very weak estrogen, 

17α-methyltestosterone in Run 2. 

 Certificates of Analysis or information regarding supplier, Catalog #, Lot/Batch #, and 

purity, were not provided for the Reference Chemicals. 

Page 76 of 223
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 Short-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-dimethylcyclopropanecarboxylate 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-phenoxyphenyl)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'

Page 77 of 223
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, Massachusetts, Laboratory Project No. 1781.6766. Submitted 

by Syngenta Ltd, Jealott’s Hill International Research Centre, 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 (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.

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'

Page 78 of 223
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 conditions. 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-measured concentrations were <LOQ (<0.000001), 

0.000013, 0.00012, and 0.0014 mg a.i./L. The test system 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 100, 94, 94, and 88% for the 

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

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

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

observed. There were no treatment-related effects 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 vitellogenin (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 considered related to treatment. In female fish, there was an 

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

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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 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 spawning 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 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 

remaining replicate spawned at least every 4 days (11 eggs/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 solution under the test conditions. Given the physicochemical properties of 

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

experience with the test item. The remaining validity criteria 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, male VTG, body weight, and length. This did not have an impact on the 

conclusions of the study as male GSI remained the only male 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 measured): Male 3.2 g; Female 1.7 g 

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

Test Type (Flow-through, Static, Static Renewal): Flow-through 

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Table 1: Summary of Reproductive and HPG Effects1,2 in the Fish Short-Term Reproduction Assay (FSTRA) with Cypermethrin. 

Treatment 

(mg a.i./L) 

[meanmeasured]

Fecundity 

Fert. 

Success 

Tubercle Score GSI Gonadal Histo. Plasma VTG Plasma T Plasma E2 

M F M F M F M F M F M F

0.000013 No No No NA No No NA NA No No NA NA NA NA

0.00012 Yes No No NA Yes No NA NA No No NA NA NA NA

0.0014 Yes No No NA Yes No NA NA No No NA NA NA NA

Abbreviations: Conc. Concentration. Diff. Difference. E2 17β-estradiol. F Female. Fert. Fertilization. GSI Gonado-Somatic Index. Histo. Histopathology. M Male. NA Not applicable. T Testosterone. VTG Vitellogenin.

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. 

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I. MATERIALS AND METHODS

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

890.1350: “Fish Short-Term Reproductive 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 spawning occurred, and fecundity was 13 

eggs/female/day, respectively. 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 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 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. Results of these analyses 

indicate measured concentrations 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. 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 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. The test material was detected on Days 

11 (one replicate) and 19 (two replicates) 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.

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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 replicate 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 do not sufficiently impact 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 validated 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 solubility, 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

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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 concentrations. 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 concentrations. 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 concentrations 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. 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. 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 did not meet the validation criteria of <20%. 

Storage Conditions of Test Chemicals: Stored at room temperature in a dark, ventilated cabinet. 

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B. Test organism: 

Table 2: General Information About the Test Species and Acclimation.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Species common name: Fathead Minnow EPA recommends fathead minnow 

(Pimephales promelas). Species scientific name: Pimephales promelas

Species strain (if stated): Not reported Were fish obtained from a single laboratory stock? 

Yes EPA recommends that fish be from a single 

laboratory stock.

Were acclimation conditions same 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. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on health: No mortalities were reported during the 

7 days prior to pre-exposure period. Fish did not receive any treatment for disease in the two weeks prior to spawning (pre-exposure period). Behavioral abnormalities or clinical signs were 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. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Type 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 reported Representative samples of the food source 

were periodically analyzed for pesticides, PCBs, and toxic metals by GeoLabs, Inc. and analyte levels were below LOC specified by ASTM (2005).

Frequency of feeding: 2 times/day Details on feeding: None. 

Table 3: Fish Selection and Pre-Exposure Performance.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Age at test initiation: Ca. 5 months Ca. 20 weeks old EPA recommends reproductively mature 

(sexually dimorphic) fish, 4.5 - 6 months 

old.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Mean weight of males at test initiation (if determined): 

3.2 g Based on 20 males used to stock 

aquaria 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 (if determined): 

2.8 to 3.7 Individual weights within ±20% of the 

estimated mean.

Mean weight of females at test initiation (if determined): 

1.7 g Based on 20 females used to stock 

aquaria for pre-exposure phase. 

Range of individual weights (females) at test initiation (if determined): 

1.3 to 2.0 Individual weights within ±20% of the 

estimated mean.

Mean length of males at test initiation (if determined): 

Not reported 

Mean length of females at test initiation (if determined): 

Not reported 

Duration of pre-exposure phase: 16 days EPA recommends a minimum of 14 days. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Were pre-exposure conditions identical 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: 36 Extra replicates were established to 

account for a potential lack of spawning in some chambers and/or mortality during 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: 2 Number of females per tank: 4 Pre-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. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Number 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. 

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C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics. 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Type of exposure: Flow-through EPA recommends the use of a flowthrough system. As noted in the 

Corrections and Clarifications document1 , 

the use of a static renewal system is not 

recommended for this assay.

Type of flow-through dilution system:

Intermittent flow proportional diluters

Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

1 U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the Endocrine Disruptor 

Screening 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). 

2 Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Flow-through rate: Ca. 45 mL/minute or 2.7 

L/hr and 6.6 volume replacements/day. 

Calculated by the reviewer based on 0.5 L/cycle, 131 cycles/24 hrs and 60 minutes/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 for flow-through systems: 

Flow-splitting chambers were used between diluter cells and the 4 replicate test vessels.Treatment recoveries were not measured prior to test initiation to indicate if diluter had 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).

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Source 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 analyzed for pesticides, heavy metals, and other contaminants? 

Yes 

Test vessel type/materials: Glass aquaria with 

silicone 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).

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Test vessel size: 20 cm wide x 39 cm 

long 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 aged 

PVC cut lengthwise in half (arches) and into 7 cm 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 cm sections. 

Additional details on exposure system: 

None. 

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Table 5: Summary of Water Quality Characteristics in the Test System.

Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Temperature (°C) 25 26 25.5 Continuously 

EPA recommends temperature 25±1oC; 

inter-replicate and inter-treatment 

differentials should not exceed 1oC.

pH 7.0 7.7 7.35 Weekly 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 as CaCO3) 

20 26 23 Weekly 

EPA recommends total alkalinity >20 

mg/L as CaCO3.

Hardness (mg/L as CaCO3) 48 60 54 Weekly Total 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 reported Not reported Not reported Not reported 

EPA recommends that unionized 

ammonia in the dilution water be <1 

Pg/L. 

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Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Residual chlorine (

Pg/L) Not reported Not reported Not reported Not reported 

EPA recommends that residual chlorine 

in dilution water be <10 

Pg/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: NA Not applicable. 

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D. Study Design and Additional Experimental Conditions

Table 6: Range-Finding Study Conditions (if Applicable).

Parameter Value(s) Details or Remarks Guideline Recommendations 

Was a range-finder conducted? Yes EPA recommends conducting a range-finder 

if 96-hour LC50 data for the fathead minnow 

are unavailable. 

If yes, what was the method for determining the highest test concentration in the rangefinder? 

14 day LC50 value 

range finding study.

Nominal test concentrations of 0 (negative and acetone controls), 0.00063, 0.0013, 0.0025, 0.0050 and 0.010 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 would be utilized in the 

test.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Test duration: 14 day EPA recommends a 96-hour exposure. Additional details: The test was 

conducted under flowthrough conditions. On Days 12 and 13, two female fish at the highest treatment level were observed at solution’s surface. At test termination, 40% combined male and female mortality was observed at the highest level. No other mortalities or sub-lethal 

effects were observed. 

EPA recommends conducting a range-finder 

with five test concentrations plus a control 

(six total treatment levels), with four 

females and two males per exposure tank 

(36 fish total). The number of mortalities 

that occur may be used to develop a 

concentration-response curve. 

Based upon the results, the highest 

concentration that does not result in 

increased mortality or signs of overt 

morbidity compared to controls, or 1/3 the 

derived 96-hr LC50, may be selected as the 

highest exposure concentration in the 21-

day test.

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Table 7: Definitive Study Conditions.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Test duration: 21 days EPA recommends that the duration of the 

definitive test be 21 days. 

Method for selecting the highest test concentration in the definitive test:

The toxicity 14 day LC50 value was estimated 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 LC50), whichever 

concentration is lowest.

Reference study citation (if applicable):

NA 

Separation of test concentrations:

0 (control), 0.00003, 

0.0003, and 0.003 

mg a.i./L 

Step down factor of 10 from highest test concentration.

EPA suggests that a concentration 

separation of between 0.33 (or three-fold) 

and 0.1 (or ten-fold) is scientifically 

acceptable1 . 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Number of test concentrations:

4 EPA recommends a minimum of 3 

concentrations and a control, plus solvent 

control if appropriate.

Are nominal concentrations adjusted for purity? 

Yes 

Indicate the type of values presented for measured concentrations: 

Mean measured 

Limit 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 reported 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Frequency of measurement: 0, 4, 6, 11, and 19 

days 

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 complete block design used?

Yes EPA recommends that all fish be randomly 

assigned to tanks during pre-exposure. 

Tanks are then ranked according to preexposure 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 solvent control (if applicable):

NA EPA recommends the use of a concurrent 

solvent control when a solubilizing agent is 

used. EPA recommends 4 replicates.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Number of replicates per test item treatment level: 

4 EPA recommends 4 replicates.

Number of male fish per replicate at test initiation: 

2 EPA recommends 2 males per replicate. 

Number of female fish per replicate at test initiation: 

4 EPA recommends 4 females per replicate. 

Was a solvent used? No Solvent type (if applicable): NA Maximum 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

.

3 Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review. Aquatic 

Toxicology, 76, pp.69–92. 

4 OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Series on 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Was a positive control used? No Positive control (if applicable): NA Positive control concentration(s) (if applicable): 

NA 

Photoperiod: 16 hrs light: 

8 hrs dark 

EPA recommends photoperiod 16:8 

(light:dark).

Light intensity at water’s surface: 

550-1000 lux Fluorescent bulbs EPA recommends light intensity 540 – 

1080 lux (at water’s surface).

Additional details: None. 

Testing and Assessment. No. 23. Paris, France. 

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Table 8: Summary of Treatment Concentrations in the Fish Short-Term Reproduction Assay with Cypermethrin. 

Treatment ID

Nominal 

Concentration 

(mg a.i./L) 

Mean Measured 

Concentration1

(mg a.i./L) 

Mean CV (%) Details or Remarks Guideline Recommendations 

Control (dilution water only) 

<0.000001 <LOQ NA 

EPA recommends that test item 

concentrations be maintained at a coefficient 

of variation (CV) 

≤20%. Solvent control (if 

applicable) 

NA NA NA 

Treatment 1 0.00003 0.000013 14.2 ± 0.0.0016 Treatment 2 0.0003 0.00012 45.6 ± 0.0546 Treatment 3 0.003 0.0014 51.2 ± 0.7096 Diluter Stock NA NA NA 

Abbreviations: CV Coefficient of variation. 

1 The mean measured values obtained by the study author are presented in the table and were calculated using the actual analytical results and not the rounded values 

presented. These values are different than the mean measured values obtained by the reviewer of 0.0000142, 0.00012 and 0.0014 mg a.i./L. 

LOQ=0.000001 mg a.i./L. 

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E. Observations

Biological Endpoints: Survival, fecundity, fertilization success, and clinical signs were observed daily. At test termination (Day 21), 

secondary sex characterization (body color, pattern, body shape), body weight, length, tubercle score, gonadal staging 

and histopathology, and plasma vitellogenin were evaluated. 

Were raw (individual) data provided? Yes

EPA recommends that observations of survival, fecundity, fertilization success, secondary sex characteristics, and other clinical 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 index (GSI) is calculated using a ratio of gonad 

weight to body weight (gonad weight to the nearest 0.1 mg / body weight in mg x 100) at test termination. 

Clinical signs of overt toxicity may include (but are not limited to) hemorrhage, cessation of feeding, and other abnormal behavior. 

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II. RESULTS AND DISCUSSION A. Results 

Mean male survival values were 100% at all test levels, and female survival values were 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 mortality rate in the water control was 0%, therefore, the 

survival rate in the control group satisfied the minimum acceptable control value criteria of 

≥90% according to the USEPA OCSPP 890.1350 

guideline. During the in-life exposure, no notable observations occurred with regards to behavior, coloration/banding, changes in ovipositor 

appearance or size of dorsal nape pad. 

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Table 9: Adult Fish Survival in Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[mean-measured]

Males Females 

N1 # Surviving % Survival n # Surviving % Survival 

Control (<LOQ) 9 9 100 15 15 100 

0.000013 8 8 100 16 15 94 

0.00012 8 8 100 16 15 94 

0.0014 8 8 100 16 14 88 

Abbreviations: NA Not 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 and female body weight values were 1.45, 1.47, 1.26 and 1.28 g in the meanmeasured 0 (control), 0.000013, 0.00012 and 0.0014 mg a.i./L treatment levels, respectively (Table 10). Mean male body length values were 

62.2, 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./L treatment levels, respectively. 

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Table 10: Size at Test Termination in Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[mean-measured]

Body Weight Length 

Males Females Males Females 

n 

Mean

(g) 

±SD n

Mean

(g) 

±SD n

Mean

(mm) 

±SD n

Mean

(mm) 

±SD 

Control (<LOQ) 4 3.10 0.39 4 1.45 0.50 4 62.2 2.44 4 50.5 2.99 

0.000013 4 3.57 1.04 4 1.47 0.33 4 64.1 4.76 4 49.5 2.52 

0.00012 4 3.60 1.24 4 1.26 0.24 4 64.3 5.89 4 48.5 2.98 

0.0014 4 3.18 1.24 4 1.28 0.36 4 62.7 3.66 4 50.5 2.99 

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

LOQ=0.000001 mg a.i./L. 

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Mean fecundity values were 14, 13, 8.3 and 2.2 eggs/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 the 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]

Fecundity1 ±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 surviving female per reproductive day per replicate. 

2

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

LOQ=0.000001 mg a.i./L.

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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, respectively (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. 

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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 

GSI1 (%) 

±SD n 

Mean GSI1

(%) 

±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 calculated as gonad weight (to the nearest 0.1 mg) / body weight (mg) x 100. 

LOQ=0.000001 mg a.i./L. 

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Median male gonadal stage was 2, 2, 2 and 3 and median female gonadal stage was 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 Stage1 n Median Stage2

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 staging scale for male fathead minnow: 0=undeveloped, 1=early 

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

2

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

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

LOQ=0.000001 mg a.i./L. 

In male fish, there were only background or sporadic 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 treatment levels (Tables 17-18). 

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Table 15: Gonadal Histopathology in Male Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[meanmeasured] 

Diagnostic Observations1

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.

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Table 16: Additional Gonadal Histopathology Observations in Male Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[mean-measured] 

Additional Diagnostic Observations1

Severity 

Decreased 

Proportion of 

Spermatogonia 

Increased Vascular 

or Interstitial 

Proteinaceous Fluid 

Asynchronous 

Gonad 

Development 

Altered Proportions 

of Spermatocytes 

or Spermatids 

Granulomatous 

Inflammation

n Incidence n Incidence n Incidence n Incidence n Incidence 

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

1 NA NA NA NA NA NA NA NA NA NA 

2 NA NA NA NA NA NA NA NA NA NA 3 NA NA NA NA NA NA NA NA NA NA 4 NA NA NA NA NA NA NA NA NA NA 

0.000013 0 NA NA NA NA NA NA NA NA NA NA 

1 NA NA NA NA NA NA NA NA NA NA 

2 NA NA NA NA NA NA NA NA NA NA 3 NA NA NA NA NA NA NA NA NA NA 4 NA NA NA NA NA NA NA NA NA NA 

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Treatment 

(mg a.i./L) 

[mean-measured] 

Additional Diagnostic Observations1

Severity 

Decreased 

Proportion of 

Spermatogonia 

Increased Vascular 

or Interstitial 

Proteinaceous Fluid 

Asynchronous 

Gonad 

Development 

Altered Proportions 

of Spermatocytes 

or Spermatids 

Granulomatous 

Inflammation

n Incidence n Incidence n Incidence n Incidence n Incidence 

0.00012 0 NA NA NA NA NA NA NA NA NA NA 

1 NA NA NA NA NA NA NA NA NA NA 

2 NA NA NA NA NA NA NA NA NA NA 3 NA NA NA NA NA NA NA NA NA NA 4 NA NA NA NA NA NA NA NA NA NA 

0.0014 0 NA NA NA NA NA NA NA NA NA NA 

1 NA NA NA NA NA NA NA NA NA NA

2 NA NA NA NA NA NA NA NA NA NA3 NA NA NA NA NA NA NA NA NA NA4 NA NA NA NA NA NA 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. 

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Table 17: Gonadal Histopathology in Female Fathead Minnow (Pimephales promelas). 

Treatment 

(mg a.i./L)

[meanmeasured] 

Additional Diagnostic Observations1

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 

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Treatment 

(mg a.i./L)

[meanmeasured] 

Additional Diagnostic Observations1

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 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. 

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

promelas).

Treatment 

(mg a.i./L) 

[meanmeasured]

Additional Diagnostic Observations1

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 

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Treatment 

(mg a.i./L) 

[meanmeasured]

Additional Diagnostic Observations1

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 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. 

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Mean male VTG was 41, 53, 39 and 35 and female VTG was 1.2x106

, 8.4x105, 1.1x106 and 1.1x106

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 106 2.553 x 105

0.000013 4 53 72 4 8.4 x 105 2.34 x 105

0.00012 4 39 25 4 1.1 x 106 3.64 x 105

0.0014 4 35 29 4 1.1 x 106 3.01 x 105

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

LOQ=0.000001 mg a.i./L.

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Mean plasma testosterone and plasma 17β-estradiol in male and females was 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) 

Males Females Males Females 

n

Mean

(ng/mL 

plasma) 

±SD n 

Mean

(ng/mL 

plasma) 

±SD n

Mean

(ng/mL 

plasma) 

±SD n

Mean

(ng/mL 

plasma) 

±SD

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

0.000013 NA NA NA NA NA NA NA NA NA NA NA NA

0.00012 NA NA NA NA NA NA NA NA NA NA NA NA

0.0014 NA NA NA NA NA NA NA NA NA NA NA NA

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

LOQ=0.000001 mg a.i./L. 

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There were no notable observations with regards to behavior or changes in appearance (coloration/banding, presence of ovipositor and dorsal nape 

pad), including secondary sex characteristics in the control or treated groups (Table 21). Replicate B in the control group contained three males 

and three females due to mis-sexed fish. 

Table 21: Secondary Sex Characteristics and Clinical Signs in Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[mean-measured] 

Secondary Sex Characteristics and Clinical Signs 

Males Females 

Type n Incidence Type n Incidence 

Control (<LOQ) None 9 0 None 15 0 

0.000013 None 8 0 None 15 0 

0.00012 None 8 0 None 15 0 

0.0014 None 8 0 None 14 0 

LOQ=0.000001 mg a.i./L. 

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B. Study Author’s Analysis and Conclusions

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

specific and analyzed in comparison 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, fertility, male and female VTG, and female GSI were 

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

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

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

data were analyzed by Shapiro-Wilk’s test and Bartlett’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 non-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 fertilization success at any 

treatment level compared to the control (p>0.05; Dunnett’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 levels 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 gonadal histopathology findings noted which 

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

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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 treatment 

levels (Tables 17-18). There were no significant findings observed in the gonadal 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 response. All data were tested for normality using 

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

assumptions of normality and homogeneity of variance were then analyzed using the parametric 

Dunnett’s test. Data which did not meet the parametric assumptions were analyzed using the nonparametric Mann-Whitney U test. Male GSI and fecundity exhibited monotonic trends and was 

analyzed using the non-parametric Jonckheere-Terpstra 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 the 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 

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effects on all other endpoints (female GSI, male and 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 treatment, 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. 

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Table 22: Reproductive and HPG Endpoints1,2 for Male Fathead Minnow (Pimephales promelas) in the FSTRA with Cypermethrin. 

Treatment 

(mg a.i./L) 

[mean-measured]

Tubercle Score GSI 

Gonadal 

Staging and 

Histo. 

Plasma VTG Plasma T Plasma E2 

Median p % Diff. p 

Effect? 

(Yes/No) 

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

Control (<LOQ) 32.5 NA 0 NA No 0 NA NA NA NA NA 

0.000013 37 0.914 10.41 0.231 No 59.4 0.859 NA NA NA NA 

0.00012 33 0.600 22.17 0.041 No 18.05 0.994 NA NA NA NA 

0.0014 25 0.071 26.70 0.018 No 5.26 1.00 NA NA NA NA 

Statistical Test Mann-Whitney Jonckheere’s NA Dunnett’s NA NA 

Abbreviations: Conc. Concentration. Diff. Difference. E2 17β-estradiol. GSI Gonado-Somatic Index. Histo. Histopathology. NA Not applicable. T Testosterone. VTG Vitellogenin. NA

Not applicable. 

1 Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusions regarding 

histopathology may be heavily weighted by the expert opinion of a board-certified pathologist. 

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

LOQ=0.000001 mg a.i./L. 

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Table 23: Reproductive and HPG Endpoints1,2 for Female Fathead Minnow (Pimephales promelas) in the FSTRA with Cypermethrin. 

Treatment 

(mg a.i./L) 

[meanmeasured]

Fecundity Fert. Success 

Tubercle 

Score 

GSI 

Gonadal 

Staging 

and Histo. 

Plasma VTG Plasma T Plasma E2 

% Diff. p 

% 

Diff. 

P Median p 

% 

Diff. p

Effect? 

(Yes/No) 

% 

Diff. p

% 

Diff. 

p 

% 

Diff. 

p 

Control (<LOQ) 

0 NA 0 NA 0 NA 0 NA No 0 NA NA NA NA NA 

0.000013 -5.26 0.328 1.30 0.9140 0 NA -5.05 0.979 No -28.30 0.302 NA NA NA NA 

0.00012 -41.93 0.032 0.00 >0.05 0 NA -5.68 0.971 No -5.32 0.981 NA NA NA NA 

0.0014 -84.39 <0.001 -4.40 0.659 0 NA 20.00 0.492 No -10.64 0.879 NA NA NA NA 

Statistical Test Jonckheere’s Dunnett’s T3 NA Dunnett’s NA Dunnett’s NA NA 

Abbreviations: Conc. Concentration. Diff. Difference. E2 17β-estradiol. Fert. Fertilization. GSI Gonado-Somatic Index. Histo. Histopathology. NA Not applicable. T Testosterone. VTG Vitellogenin.

1 Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusions regarding histopathology 

may be heavily weighted by the expert opinion of a board-certified pathologist. 

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

LOQ=0.000001 mg a.i./L. 

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Table 24: Growth Endpoints1,2 in the Fish Short-Term Reproduction 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 comparison to the negative (clean water) 

control. 

2

 Unless otherwise specified, effects are considered 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 regarding 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 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 

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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. Additionally, 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 concentrations 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 (one replicate) and 19 (two replicates) in the 

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

among replicates and not consistent throughout exposure. The study author concluded 

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

exposure system, indicating the control fish 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 replicate 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. 

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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 conclusions 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 (Pimephales promelas). Environ 

Toxicol Chem 20:1276-1290. 

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

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

ASTM, 2005. Committee E-47 on Biological Effects 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 Harbor Drive, PO Box C700, West Conshohocken, 

Pennsylvania. 

Ives, Michael, 2011. CETIS, Comprehensive Environmental Toxicity Information SystemTM. 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. 

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Sprague, J.B., 1969. Measurement of pollutant toxicity to fish. 1. Bioassay methods for acute toxicity. 

Water Res. 3: 793-821. 

OECD (2009). Fish Short Term Reproduction 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, Pesticides 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. Environmental 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 edition. Office of Water, U. S. Environmental 

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

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

Screening Program Test Guideline, OPPTS 890.1350. Fish Short-Term Reproduction Assay. EPA 

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

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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 

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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) ) 

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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 

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 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 

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

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) 

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

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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 

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 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 

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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 

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

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 . . . . . 

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 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 

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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 

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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 

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 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 . . 

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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 

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 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 

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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 

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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 

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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 

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 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 

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

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 

Page 151 of 223
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 

Page 152 of 223
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 

Page 153 of 223
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 . . . . . 

Page 154 of 223
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 . . . . . 

Page 155 of 223
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 . . . . . , . 

Page 156 of 223
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 

Page 157 of 223
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 

Page 158 of 223
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 159 of 223
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).

Page 160 of 223
CYPERMETHRJN I 109702 

Primary Reviewer: Karlyn Middleton 

Health Effects Division (7509P) 

Secondary Reviewer: Elizabeth 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- Template version 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/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 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. 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 Female Pubertal Assay (MRID 48683002), 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 (corn oil), 15 or 30 

mg/kg/day from post-natal day (PND) 22 to 42/43 . Animals were examined for vaginal opening 

(VO) daily beginning on PND 22, and weight at day of attainment was recorded. Following 

sacrifice on PND 42/43, total 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 uterus, ovaries, thyroid, and kidney were 

performed. 

One animal in the 30 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 until 

Page 161 of 223
Female Pubertal Assay (2012) / Page 2 of 17 

CYPERMETHRIN / 109702 OCSPP 890.1450/ OECD None

scheduled sacrifice. As there was no other evidence of toxicity in this study, this single death 

was considered incidental. There were no treatment-related clinical signs of toxicity at any dose 

level; findings noted in the treated groups 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, and food consumption in the treated groups were similar to 

the control group throughout the study. 

No treatment-related effects were observed on mean age or weight at vaginal opening (VO), and 

mean age at first estrus. The cycle status at necropsy (diestrus, proestrus, estrus, or not cycling) 

appeared similar across all groups. There were no treatment-related changes 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/peripubertal female rat at cypermethrin dose levels of 15 and 30 

mg/kg/day. 

No evidence of toxicity was observed in the study up to the highest dose tested; however, 

treatment-related clinical signs were seen in the preliminary 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, 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 administration 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 requirements for a Female Pubertal Assay 

(OCSPP 890.1450). 

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

Assurance statements were provided. 

Page 162 of 223
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 Manager, Vivarium); C.A. Kopp (Manager, Gross 

Pathology and Developmental Toxicology Laboratory); G.M. Maginnis (Clinical 

Veterinarian); J.S. Moore (Operations Manager, Developmental, Reproductive and 

Neurotoxicology); G.A. Parker (VP, Pathology); T.M. Rafeld (Group Manager, 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 (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 Laboratories, Inc. (Portage, MI)

 Housing: Female weanlings were housed three per cage in plastic cages containing nesting material 

(Aspen BedTM)

 Diet: 2016CM Teklad Global 16% Protein Rodent Diet (Harlan Laboratories), ad libitum; average 

29.0 and 11.0 ppm total isoflavones (genistein+daidzein+glycitein) for two lots of feed used.

 Water: Reverse-osmosis purified (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 3CH CH3

Page 163 of 223
Female Pubertal Assay (2012) / Page 4 of 17 

CYPERMETHRIN / 109702 OCSPP 890.1450/ OECD None

2. Mating: Timed-pregnant dams were received from the supplier on gestation day 7. The 

day when parturition was first observed was designated 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 rats were assigned to the test 

groups noted in Table 1 using a computer program which randomized the animals based on 

stratification of body weights in a block design; no littermates were assigned 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 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 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 dose level, divided into aliquots for daily 

dispensation, and stored refrigerated. The test substance formulations were stirred 

continuously throughout the preparation, sampling, and dose administration procedures. 

Analyses to demonstrate the homogeneity, stability, and resuspension homogeneity of the 

test substance formulations at concentrations 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 stability analyses were not conducted during the current study. 

The first dosing formulations were inspected and found to be visibly homogeneous. 

Samples for concentration analysis were collected from the middle stratum of each dosing 

formulation prepared during the 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 Study of Cypermethrin in Corn Oil Formulations (Study 

No. WIL-639153). WIL Research Laboratories, LLC, Ashland, OH.

Page 164 of 223
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 variation 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 treatment 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 determine 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/absent) were analyzed using 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 increase. The statistical analyses described 

above were conducted by the study facility. 

The remaining statistical analyses were conducted 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 weights, organ weights to necropsy body weight 

ratio (liver, kidneys, pituitary, and adrenal glands), daily body weights (including necropsy 

body weight), cumulative body weight gain, serum 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 statistical analysis and reporting. 

Each endpoint was tested for homogeneity of variance 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 Levene'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 statistical model contained a factor for treatment group and a 

blocking factor based 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 KruskalWallis test was used, ignoring the blocking factor, 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-Wallis 

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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 following analyses if they met 

the homogeneity of variance criteria: 1) analysis 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 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 increased severity. The tests were two-sided 

for graded responses presented on a Grade 1-5 scale at the 0.05 significance level testing for 

increased or decreased severity. Exact p-values were calculated for the Mann-Whitney U 

test. 

C. METHODS

1. Mortality and Clinical Examinations: All animals were examined twice daily for 

mortality and moribundity. Individual detailed physical examinations 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 allocation of the animals to study groups, 

daily prior to dosing, and prior to scheduled termination. 

3. Food Consumption: Food consumption was recorded daily 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 completion 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 first 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 or 

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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 than 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 consecutive days of P and/or E, 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 display 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 last day of observation + 1). If a female had no 

E, but had a P during the evaluation period, the age at first estrus 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 necropsies were performed. On the day of 

euthanasia, dosing was performed in the holding 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 surviving 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 collected, serum was separated, 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 analyses. The sample for TSH analysis was 

stored frozen (≤-20°C) for subsequent analysis and the sample for T4 was analyzed fresh. 

a. Hormone Analysis: Total T4 levels were determined using an electrochemiluminescent 

immunoassay and TSH levels were determined using a [125I]rTSH radioimmunoassay kit. 

b. Clinical Chemistry: The following CHECKED (X) parameters were examined. The study 

report provided historical control ranges for the clinical chemistry parameters in the results 

table. 

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 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 Serum protein electrophoresis 

 Lactic acid dehydrogenase (LDH) X Albumin/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 female 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 subjected 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 based on guideline 890.1450 

+ Histopathological examination required based on guideline 890.1450 

All organs collected, except the thyroid, were weighed prior to fixation. Paired organs 

(kidneys, adrenals, and ovaries) were weighed 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, kidney, adrenal, and pituitary weights. 

The ovaries, uterus, thyroid (with parathyroid), and kidneys were fixed in 10% buffered 

formalin for at least 24 hrs. Following fixation, 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 biochemical analysis. The remaining 

liver and remaining tissues were placed in 10% neutral-buffered formalin for potential 

future histopathologic examination. After fixation, tissues were shipped to WIL Research 

Hillsborough (Hillsborough, NC) for processing. Trimmed tissues were processed into 

paraffin blocks, sectioned and stained with hematoxylin and eosin, and examined 

microscopically. 

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Five random sections from ovary were evaluated microscopically for follicular development 

(including the level of primordial, primary, secondary, tertiary, and atretic follicles) and 

changes in the corpus luteum development (growth phase, maturity, and regression), in 

addition to any abnormalities or lesions. It was 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 grading 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 noted 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. Findings 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 signs 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 opening (VO) and weight at day of attainment are presented in 

Table 3. Mean body weights, daily body weight gains, and cumulative mean body weight 

gain in the 15 and 30 mg/kg/day groups were 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 throughout the treatment 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 of 

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incomplete opening. When a second analysis was performed adjusting the day of attainment 

for this 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 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 the control group in the second analysis. 

The mean body weight at VO and final body weight in the control group 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 above, the mean age at attainment 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 analysis 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 presented in Table 4. It is noted that 

no explanation was provided for missing pituitary gland weights from two animals in the 

control group and for missing ovary weights from 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, ovaries, uterus, and thyroid in the control group 

were within the range of the performance criteria provided in the Guideline (890.1450). The 

CVs for kidney (12.66%) and liver (14.14%) weights in the control group exceeded the 

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performance criteria (maximum 10.76% and 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 obtained 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 from 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 significant differences in the age at the first 

occurrence of estrus were noted when the test substance-treated groups were compared to 

the control group. The cycle status at necropsy (diestrus, proestrus, 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 15 

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mg/kg/day group compared to 80% in the control 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 pages 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 presented in Table 6. There were no cypermethrinrelated changes in serum hormone or clinical chemistry levels. The mean and CV values for 

serum T4 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 bilirubin, total protein, triglycerides, and urea nitrogen. However, 

the cypermethrin test groups had mean values similar to the control for each of these 

parameters. 

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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 T4, 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 pages 101-102 of the study report; no statistical differences. 

Bolded mean values were outside the performing 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 were observed on gross pathology. 

H. HISTOPATHOLOGY: The incidences of histopathological 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 from one animal in the 15 mg/kg/day group. 

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TABLE 7. Incidence of Histopathological Lesions of the Thyroid Gland a

Treatment Groups 

Parameter Evaluated 

Colloid Area Follicular Cell Height 

(Increase) 

Grade b Incidence Grade Incidence 

O E O E 

Vehicle Control 

1 0 

15 

1 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 observed microscopically in the uterus, ovaries, or 

kidney. 

TABLE 8. Incidence of Histopathological Lesions 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 3b 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. 

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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 was there any evidence of direct 

test substance-related effects on pubertal development or thyroid function in the 

juvenile/peripubertal female rat at cypermethrin 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 until scheduled sacrifice. As there was no other evidence of 

toxicity in this study, this single death was considered incidental. There were no treatmentrelated clinical signs of toxicity 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 control group throughout the study. 

No treatment-related effects were observed on mean age or weight at VO, and mean age at 

first estrus. The cycle status at necropsy (diestrus, proestrus, estrus, or not cycling) 

appeared similar across all groups. There were no cypermethrin-related changes in serum 

hormone or chemistry levels. In addition, there 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 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 the control group in the second analysis. This minor change was considered 

incidental in the absence of other effects 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 group. This was a minor change, related to 

body weight; adjusted and relative adrenal gland weights were similar to controls. 

There was no evidence of direct test substance-related effects on pubertal development or 

thyroid function in the juvenile/peripubertal female rat at cypermethrin dose levels of 15 and 

30 mg/kg/day. 

No evidence of toxicity was observed in the study up to the highest dose tested; however, 

treatment-related clinical signs were seen in the preliminary 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, 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 administration in the preliminary 

study, which is closer to the time-to-peak effect for cypermethrin. Therefore, the doses 

tested were considered to be adequate. 

Page 175 of 223
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 the 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 criteria (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 performance criteria (48.84 mg). 

Page 176 of 223
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), cypermethrin (95.2% a.i.; cis/trans ratio: 

49.8/50.2; Batch #PL10-0492) in corn oil was administered daily via oral 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 after the final dose administration. Adrenals, 

kidneys, and liver were collected and weighed. Only the results for 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 material around the mouth were noted 4 hours following 

dosing in the remaining females in the 65 mg/kg/day group. Two of these females also had 

splayed hindlimbs. It was concluded that a dose of 65 mg/kg/day would not be suitable for 

continued dosing; therefore, all remaining females 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 dose administration in the 30 mg/kg/day group 

consisted of slight to moderate tremors and clear material around the mouth and on the ventral 

neck, ventral thoracic area, and forelimbs (beginning as early as Day 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 loss 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 statistically significant) than the 

control group on Day 2. Markedly lower food consumption was noted in this group during Days 

1-2 compared to the control group. Slightly lower mean body weight gain and food consumption 

was noted in the 30 mg/kg/day group females during Days 1-2; mean body weight gains and 

food consumption in these females were generally similar to the control group for the remainder 

of the study and none of the differences from the control group were statistically significant. 

Mean body weights, body weight gains, and food consumption in the 5 and 15 mg/kg/day groups 

were unaffected by test substance 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 termination were discarded without examination. 

COMPLIANCE: It was stated that the study was not performed in compliance with GLPs. 

Page 177 of 223
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 178 of 223
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).

Page 179 of 223
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. Findings 

Page 180 of 223
Male Pubertal Assay (2012) / Page 2 of 15 

CYPERMETHRIN / 109702 OCSPP 890.1500 / OECD None

noted in the treated groups, including red and/or clear material 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 complete PPS, and mean body weight at the age of 

attainment in the 15 and 30 mg/kg/day groups were similar to the control group throughout the 

study. 

No treatment-related gross observations were noted at necropsy and no significant effects of 

treatment were observed on the weights of any of the organs and tissues collected. 

There were no test substance-related changes in serum T4, 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. However, decreases in bile 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 incidental finding. There were no other 

serum chemistry changes in the cypermethrin-dosed groups compared to the control group. 

There were no test substance-related histologic changes in the 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 the study up to the highest dose tested; however, 

treatment-related clinical signs were seen in the preliminary 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, 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 administration in the preliminary study, which is 

closer to the time to peak effect for cypermethrin. The dose levels used in the study are 

considered adequate based on the results of the range-finding study. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Male Pubertal Assay (OCSPP 

890.1500). 

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

Assurance statements were provided. 

Page 181 of 223
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 Manager, Vivarium); C.A. Kopp (Manager, Gross 

Pathology and Developmental Toxicology Laboratory); G.M. Maginnis (Clinical 

Veterinarian); J.S. Moore (Operations Manager, Developmental, Reproductive and 

Neurotoxicology); T.M. Rafeld (Group Manager, 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 (Principal 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 Laboratories, Inc. (Portage, MI)

 Housing: Male weanlings were housed three per cage in plastic cages containing nesting material (Aspen 

BedTM)

 Diet: 2016CM Teklad Global 16% Protein Rodent Diet (Harlan Laboratories), ad libitum; average 

29.0 and 11.0 ppm total isoflavones (genistein+daidzein+glycitein) for two lots of feed used.

 Water: Reverse-osmosis purified (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 from the supplier on gestation day 7. The 

CN

O

O

Cl O

Cl 3CH CH3

Page 182 of 223
Male Pubertal Assay (2012) / Page 4 of 15 

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day when parturition was first observed was designated 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 program which randomized the animals based on 

stratification of body weights in a block design; no littermates were assigned 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 dose level, divided into aliquots for daily 

dispensation, and stored refrigerated. The test substance formulations were stirred 

continuously throughout the preparation, sampling, and dose administration procedures. 

Analyses to demonstrate the homogeneity, stability, and resuspension homogeneity of the 

test substance formulations at concentrations 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 stability analyses were not conducted during the current study. 

The first dosing formulations were inspected and found to be visibly homogeneous. 

Samples for concentration analysis were collected from the middle stratum of each dosing 

formulation prepared during the 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 variation between nominal and actual dosage to the 

animals was acceptable. 

 

1 Bodle, E.S. (2012) Analytical Validation and Stability Study of Cypermethrin in Corn Oil Formulations (Study 

No. WIL-639153). WIL Research Laboratories, LLC, Ashland, OH.

Page 183 of 223
Male Pubertal Assay (2012) / Page 5 of 15 

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6. Dosage Administration: All doses were administered once daily by gavage from PND 23 

through PND 53 (seven animals in each treatment 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 presenting as a dichotomous response were analyzed with 

pairwise Fisher’s exact tests to compare each treated group with the control. The tests were 

one-sided at the 0.05 significance level testing for an increase and were conducted by the 

study facility. 

The remaining statistical analyses were conducted 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 weights, organ weights to necropsy body weight 

ratio (liver, kidneys, pituitary, and adrenal glands), daily body weights (including necropsy 

body weight), cumulative body weight gain, serum chemistries, and preputial separation 

data. Gamma glutamyl transferase values under range were assigned a value of 0.1 (half the 

lower limit of quantitation) for statistical analysis and reporting. 

Each endpoint was tested for homogeneity of variance 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 Levene'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 based 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 used, ignoring the blocking factor, 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-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 separation data were subject to the following 

analyses if they met the homogeneity of variance criteria: 1) analysis 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. 

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 increased severity. The tests were two-sided 

for graded responses presented on a Grade 1-5 scale at the 0.05 significance level testing for 

increased or decreased severity. Exact p-values were calculated for the Mann-Whitney U 

test. 

Page 184 of 223
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C. METHODS

1. Mortality and Clinical Examinations: All animals were examined twice daily for 

mortality and moribundity. Individual detailed physical examinations 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 allocation of the animals to study groups, 

daily prior to dosing, and prior to scheduled termination. 

 

3. Food Consumption: Food consumption was recorded daily prior to test substance 

administration on a per cage basis. Food intake was calculated and reported 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 between the glans and prepuce was recorded. The age and body 

weight on the day at which complete preputial separation was observed was recorded for 

each animal. However, if any animal within any group showed incomplete separation 

(persistent threads) for greater than 3 days, a second analysis was conducted using the age at 

which incomplete separation was first observed. 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 necropsies were performed. On the day of 

euthanasia, dosing was performed in the holding 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, beginning approximately 2 hours post-dosing, all surviving 

animals were sacrificed by decapitation without anesthesia; sacrifices 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: T4 and testosterone levels were determined using an 

electrochemiluminescent immunoassay and TSH levels were determined using a [125I]rTSH 

radioimmunoassay.

b. Clinical Chemistry: The following CHECKED (X) parameters were examined. The study 

report provided reference ranges, based on historical control data, for the clinical chemistry 

parameters. 

Page 185 of 223
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 Serum protein electrophoresis 

 Lactic acid dehydrogenase (LDH) X Albumin/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 subjected 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 (with and without fluid)* X Adrenals (paired)* 

X Ventral prostate* X Pituitary* 

X Dorsolateral prostate* 

X Levator ani-bulbocavernosus (LABC) muscle complex* 

XX Kidneys (paired)*+ 

* Weights required based on guideline 890.1500 

+ Histopathological examination required based on guideline 890.1500 

The testis and epididymis (left and right) and kidneys were weighed prior to fixation. 

Following weighing, the testis and epididymis were fixed in Bouin’s solution for 

approximately 24 hours, rinsed, and 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 fixation, 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 future histopathological examinations. After 

fixation, tissues were shipped to Biotechnics, LLC (a division of WIL Research; 

Hillsborough, NC) for processing. Trimmed tissues were processed into paraffin blocks, 

sectioned, and stained with hematoxylin and eosin, except testes and epididymides (right), 

which were stained with PAS and hematoxylin, 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 scale 

Page 186 of 223
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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 PND 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 necropsy. This death was likely 

due to dosing error, given the timing of death 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 (Table 2), including red 

and/or clear material around the nose and/or mouth, occurred infrequently, 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 and 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 cumulative mean body weight gain in the 15 and 

30 mg/kg/day groups were 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 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 administration. When a second analysis was performed, 

adjusting the day of attainment for those males with 3 or more consecutive days of 

Page 187 of 223
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incomplete separation (persistent threads), no significant delays in the mean age of 

attainment of PPS were noted in the treatment groups compared to the controls. 

The mean values for the weaning weight, final body weight, age at PPS, and body weight at 

PPS in the control group were within the range of the performance criteria provided in the 

Guideline (890.1500). The CV for the body weight at PPS (9.74%) and final body weight 

(9.01%) were greater than the maximum values 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 presented in Table 4. No significant 

effects of treatment were observed on the weights 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 within the range of the 

performance criteria provided in the Guideline (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). 

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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 14b 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 NA 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 NA NA 15 1481.3 NA NA 

Thyroid, fixed 

(mg) 

U 15 13.20 3.306 25.05 14 12.63 2.26 17.891 14c 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 fluid 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. There were no test substance-related changes in 

serum T4, TSH, and testosterone values at any dose level. The mean and CV values for 

serum T4, TSH, and testosterone values in the control group were within the range of the 

performance criteria provided in the Guideline (890.1500). 

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Urea nitrogen (↓17%) and bile acid (↓52%) were decreased (p<0.05) in the 30 mg/kg/day 

group compared to controls. Decreases in bile 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 incidental finding. There were no other serum 

chemistry changes in the cypermethrin-dosed groups compared to the control group. 

Mean values for the control group were outside the historical reference control data from the 

performing laboratory for ALT, AST, bile acid, 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 T4, 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 transferase 

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F. GROSS PATHOLOGY: No treatment-related effects were observed on gross pathology. 

G. HISTOPATHOLOGY: The incidences of histopathological findings of the thyroid gland 

are presented below in Table 6. There were no test substance-related histologic changes. 

The amount of colloid and follicular height of thyroid epithelial cells were similar between 

control and test substance-treated animals. 

TABLE 6. Incidence of Histopathological 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. 

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TABLE 7. Incidence of Histopathological Findings 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-androgenic effects, nor was there any evidence of any test 

substance-related effects on pubertal development or thyroid function in 

juvenile/peripubertal male rats at cypermethrin 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 PND 45, which was likely due to dosing error 

given the timing of death relative to dosing and the fact that no mortalities occurred at the 

higher dose. All other rats survived until scheduled sacrifice. There were no treatmentrelated clinical signs of toxicity 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 attainment of PPS in the 15 and 30 mg/kg/day 

groups were similar to the control group throughout the study. 

No treatment-related gross observations were noted at necropsy and no significant effects of 

treatment were observed on the weights of any of the organs and tissues collected. A doserelated 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. 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 T4, 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. Decreases in bile 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 incidental finding. There were no other 

serum chemistry changes in the cypermethrin-dosed groups compared to the control group. 

Page 192 of 223
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CYPERMETHRIN / 109702 OCSPP 890.1500 / OECD None

There were no test substance-related histologic changes in the 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 the study up to the highest dose tested; however, 

treatment-related clinical signs were seen in the preliminary 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, 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 administration 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 control 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 thyroid 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). 

 

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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), cypermethrin (95.2% a.i.; cis/trans ratio: 

49.8/50.2; Batch #PL10-0492) in corn oil was administered daily via oral 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 after the final dose administration. Adrenals, 

kidneys, and liver were collected and weighed. Only the results for the male rats are discussed in 

this appendix; the results for the female rats 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 hindlimbs; 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 Day 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 early 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-dose 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 were not observed in a dose-related manner. 

Mean body weights, body weight gains, and food consumption in the 5, 15, and 30 mg/kg/day 

groups were unaffected by test substance administration. 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 relative 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 groups 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 unaffected 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 without examination after euthanasia due to dosegroup termination. 

COMPLIANCE: It was stated that the study was not performed in compliance with GLPs. 

Page 194 of 223
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 195 of 223
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).

Page 196 of 223
Steroidogenesis Assay (2012) I Page 1 of 14 

CYPERMETHRIN 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) 

SYNONYMS: 

CITATION: 

SPONSOR: 

Cyano(3-phenoxyphenyl)methyl 3-(2,2-dichloroethenyl)-2,2-

dimethylcyclopropanecarboxylate 

Wagner, H. (2012). Cypermethrin - H295R Steroidogenesis Assay. 

CeeTox, Inc., Kalamazoo, MI. Laboratory Study No.: 9047VI 00353STER, January 17, 20 12. MRID 48670203 . Unpublished. 

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 (MRID 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 

evaluated 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, acceptable reproducibility (low %CV), and appropriate induction and 

inhibition with positive controls. Cytotoxicity greater than 20% was not observed. 

Page 197 of 223
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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 control (SC). All other values of 

testosterone and estradiol in the treated cells were similar to the SC. 

Based on the hormone responses in each of the three independent runs, cypermethrin treatment 

resulted in statistically significant and reproducible increases in estradiol production, but 

cypermethrin treatment did not result in statistically significant and reproducible alterations in 

testosterone production. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Steroidogenesis assay 

(OCSPP 890.1550). 

COMPLIANCE: Signed and dated GLP Compliance and Quality Assurance statements were 

provided. A Data Confidentiality statement was not provided. 

 

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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 Scientist/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 3

CH CH3

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 #: 67747-09-5 

Page 199 of 223
Steroidogenesis 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 solub

 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 concentration 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 Lakes, 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 carcinoma cells (ATCC CLR-2128; Lot # 

7635054) at passage 7.5–8.5 were incubated in the stock medium, supplemented with 10 μM 

22R-hydroxycholesterol in ethanol at the time of plating, dosing, and harvest. Incubation 

conditions were at 5% CO2 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 hormone extraction were provided. 

c. Laboratory Proficiency Test: No laboratory proficiency test 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 medium to a final concentration of 10 μM. 

Page 200 of 223
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 serially diluted in DMSO. Forskolin and 

prochloraz were then diluted in supplemented medium containing 10 μM 22R-hydroxycholesterol. When added to the cell culture plates, these dilutions yielded final 

concentrations of 1 μM and 10 μM for forskolin, 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). Precipitation 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 concentrations in undiluted Nu-Serum were 

3754 pg/mL testosterone and 3846 pg/mL estradiol. H295R cells were grown for five 

passages, frozen in liquid nitrogen, then thawed and cultured for seven or eight additional 

passages prior to use in the assay. The culture medium was supplemented with 10 uM 

22R-hydroxycholesterol at the time of plating, dosing, and harvest. The concentration of 

22R-hydroxycholesterol was chosen based on laboratory 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% CO2 incubator at 37 ± 2°C for approximately 24 hours prior to chemical 

exposure. Prior to dosing, the cells were checked 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 inspected for precipitation. The medium was 

removed from the cells and replaced with 1 mL of medium containing 10 μM 22Rhydroxycholesterol and concentrations of cypermethrin in triplicate, according to the 

schematic presented in Table 1. Dose concentrations 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 assay’s response to forskolin (an inducer of testosterone and 

estradiol production) and prochloraz (an inhibitor of testosterone and estradiol production). 

The QC plate was prepared and dosed in the same manner with either forskolin or 

prochloraz, according to the schematic presented in Table 2. 

Page 201 of 223
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 Blankb

 Blank Blank Backgroundc 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 containing 10μM 22R-hydroxycholesterol. 

c Background wells received media without 22R-hydroxycholesterol. 

d MeOH = methanol was added to these wells for 30 minutes 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 concentrations. 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 ice 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-diphenyltetrazolium bromide (MTT) assay. On 

the QC plates, wells designated to receive methanol (control wells for cell death 

measurements) were rinsed twice with phosphate buffered saline (PBS; Gibco; Cat. # 

10010; Lot # 979431), and then incubated in methanol (Sigma; Cat. # 494437; Lot # 

SHBB0973V) for 30 minutes at room temperature. 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 supplemented 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% CO2 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 subtracted 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 estradiol levels were measured at 

OpAns, LLC (Durham, NC). Testosterone and estradiol were extracted from H295R 

supplemented medium by liquid-liquid extraction using methyl tert-butyl ether (MTBE) 

after the addition of [2

H5]-testosterone and [2H5]-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 testosterone and 10 pg/mL for estradiol using a 

300 μL aliquot of H295R supplemented medium. Freshly prepared calibration standards 

(n = 7) were prepared in supplemented medium for each run. QC samples spiked in 

Page 202 of 223
Steroidogenesis 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 for two concentrations of testosterone and estradiol (mean measured amount 

from triplicate samples within 30% of nominal concentration) 

NA Hormone cross-reactivity (antibody-based assays only; ≤30% of basal production of the respective hormone) 

X Solvent control within 75% range below maximum response on standard curve 

NA Test compound tested for interference with measurement system 

C. DATA ANALYSIS: Mean values (pg/mL) and standard deviations for testosterone and 

estradiol were calculated for each concentration of cypermethrin, reference chemicals, the 

solvent controls, blanks, and background wells. Relative changes in testosterone and 

estradiol production were calculated using the equation below: 

Relative Change = Hormone concentration in each well ÷ Hormone concentration of mean 

solvent control 

For forskolin induction of testosterone, the 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 received no 22R-hydroxycholesterol at the time of exposure. 

Concentrations where precipitation was observed 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 treatment groups was evaluated using Levene's test. If the p-values 

were greater than 0.05 in both tests, statistical significance between each treatment group 

and the control group was evaluated using Dunnett's test. If the p-values were less than or 

equal to 0.05 in either the normality or the homogeneity test, a log transformation was 

performed on the data to attempt to approximate a normal distribution. If (following the 

transformation) p-values were greater than 0.05 in both the normality and homogeneity 

tests, Dunnett's test was performed on the transformed data to evaluate statistical 

significance between each treatment group and the control 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-transformed data set was analyzed using the nonparametric 

Kruskal-Wallis test followed by Dunn's test to evaluate statistical significance between each 

treatment group and the control group. Data analysis at the analytical laboratory (OpAns, 

LLC) was conducted in Microsoft Excel. No additional software for statistical analyses or 

graphing was specified. The reviewers 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.

Page 203 of 223
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 analysis 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 testosterone and estradiol assays was ≤7.3%. Thus, inter- and 

intraplate variability were within the guideline recommendation of ≤30%. Basal hormone 

production in SCs met guideline 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 (Table 3). All other values of 

testosterone and estradiol in the treated cells were similar to the SC. 

TABLE 3. Mean (±SD) Hormone Concentrations Following 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 Appendices 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 precipitation

 

Page 204 of 223
Steroidogenesis Assay (2012) / Page 9 of 14 

CYPERMETHRIN / 109702 OCSPP 890.1550/ OECD None 

FIGURE 1. Change in Testosterone Production Relative to Cypermethrin Concentration. 

Page 205 of 223
Steroidogenesis Assay (2012) / Page 10 of 14 

CYPERMETHRIN / 109702 OCSPP 890.1550/ OECD None 

FIGURE 2. Change in Estradiol Production Relative to Cypermethrin Concentration. 

Page 206 of 223
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 graphically in Figure 3. 

TABLE 4. Mean (±SD) MTT Cell Viability (% SC) Results after Treatment with Forskolin, Prochloraz, or 

Cypermethrin for 48 Hours. a 

Compound Concen. 

(μM) 

Cell Viability – Run 2 Cell Viability – Run 3 Cell Viability – Run 4 

Mean SD Mean SD Mean SD 

Blank NA 104.3 2.69 104.3 2.81 100.7 3.98 

Background NA 95.4 4.14 102.5 1.79 98.9 0.94 

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 and 2 on pages 20-21 of the study report. 

NA Not applicable 

 

Page 207 of 223
Steroidogenesis Assay (2012) / Page 12 of 14 

CYPERMETHRIN / 109702 OCSPP 890.1550/ OECD None 

FIGURE 3. MTT cell viability results 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 levels (500 pg/mL for testosterone, 40 pg/mL 

for estradiol) were met in both blank and SC wells. OCSPP 890.1550 guideline 

requirements for basal hormone production in SC wells, induction by 10 μM forskolin, and 

Page 208 of 223
Steroidogenesis Assay (2012) / Page 13 of 14 

CYPERMETHRIN / 109702 OCSPP 890.1550/ OECD None 

inhibition by 1 μM prochloraz were met for testosterone 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 below the Guideline recommendation of ≥2-fold; 

however, when the background is subtracted, 10 μM forskolin induced testosterone an 

average of 2.47-fold. Estradiol 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 testosterone to 0.41-fold and estradiol to 0.32-fold. Thus, the 

guideline requirements were met indicating that the assay was sensitive to induction and 

inhibition of testosterone and estradiol. 

The variability (%CV) between the runs (calculated by the reviewer) based on the absolute 

hormone concentrations in the SC were 15% for testosterone and 11% for estradiol, within 

the recommended limit of ≤30%. The %CVs within each run 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 Meanb ± SDb

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 values 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 values 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 resulted in no statistically 

significant effects on testosterone in the steroidogenesis assay. 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 statistically significant, 

Page 209 of 223
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-fold 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 concentrations, and these samples 

were not analyzed. Cytotoxicity greater than 20% was not observed. Run 1 was not 

analyzed because one of the forskolin concentrations on the quality control plate was 

incorrect (3.33 μM was used instead of 1 μM). 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 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 SC. All other values of 

testosterone and estradiol in the treated 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-differences were not reported, but could be 

calculated from the data presented. 

Page 210 of 223
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 211 of 223
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).

Page 212 of 223
CYPERMETHRIN/ 109702 

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 EVALUATION 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-dichloroethenyl)-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 Range-Finding Oral 

(Gavage) Toxicity Study in Young Adult Rats. WIL Research Laboratories, 

LLC, Ashland. OH. Laboratory Report No.: WIL-639148, November JO, 

2011. MRID 48615702. Unpublished. 

SPONSORS: Syngenta 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) conducted to screen 

for potential estrogenic activity, cypermethrin (technical, 95.2% a.i .. Batch# PLl0-0492) in corn 

oil was administered daily via oral gavage (dosing volume 5 ml/kg bw) to groups of six 

ovariectomized female Sprague Dawley rats/dose at 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 

daily do e of l 7a-ethynyl estradiol (EE) in com oil at 0.3 mg/kg/day by oral gavage (do ·ing 

volume 5 ml/kg bw). The selected dose levels were based on toxicity observed in the dose 

range-finding study (MRID 48615702), in which clinical signs and body weight gain effects 

were ob:erved at 80 and I 00 mgfkg/day, and marginal effects at 65 mg/kg/day. Body weights 

and food consumption were determined daily. All animals were terminated and necropsied on 

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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 clinical 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 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%). The overall average food consumption (Days 1-4) was decreased 

(p<0.05) by 15% at 80 mg/kg/day. Overall average 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 control, 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<0.05) 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% decrease in overall body weight gains in the high-dose group 

(together with consistent findings in the range-finding study at doses of 65, 80 and 100 

mg/kg/day) indicated that the tested 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 requirements for a uterotrophic assay (OCSPP 

890.1600). 

COMPLIANCE: Signed and dated GLP Compliance and Quality Assurance statements were 

provided. 

 

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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 (Clinical 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: 17α-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 Corporation (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 Corporation (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 3CH CH3

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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 Laboratories (Portage, MI) 

 Housing: Rats were individually housed in stainless steel wire-mesh cages suspended above 

cage board. 

 Diet: 2016CM Teklad Global 16% Protein Rodent 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 the supplier. Animals were 

received approximately one week following ovariectomy (PND 55) and acclimated for 11 

days prior to initiation of dosing. Vaginal smears were taken daily for five consecutive days 

prior to assignment of animals to study, to verify that females were in persistent diestrus. 

The dose administration period was from PND 66 through 68. Rats were euthanized 

approximately 24 hours later on PND 69 and necropsied for uterine weight measurements 

3. Animal Assignment: Animals were randomly assigned, stratified by body weight, to the 

test groups noted in Table 1. Statistical analysis indicated that there were no significant 

differences between group mean weights at study initiation. Furthermore, the body weight 

of each animal was within 20% of the overall mean. 

TABLE 1. Study Designa

Test Group Dose (mg/kg/day) # of Females 

Estrogen Agonist Assay 

Vehicle Control 0 6 

Low Cypermethrin 25 6 

High Cypermethrin 80 6 

17α-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 adult 

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(69-77 days old) female Sprague 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 appropriate 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 corn oil; dosing formulations were prepared 

daily by diluting the stock solution. Analyses to demonstrate homogeneity, stability, and 

resuspension homogeneity were conducted previously 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 formulation (middle stratum of each) prepared 

during the in-life phase were analyzed 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 variation 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 consecutive 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 were conducted for wet and blotted uterine weights, daily 

body weights, cumulative body weight gains, and 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 significant, then the square 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 described below, was used to analyze the data. 

One-sided tests were conducted for uterine weights 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. 

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For uterine weights, if variances were homogeneous, the analysis of covariance 

(ANCOVA), using the body weight at termination as the covariate was performed; the two 

groups were compared using a one-sided Dunnett’s test (for comparing dose groups to the 

negative control) or a one-sided t-test (for comparing positive control data to data for 

negative control). For body weight and body weight gain data, if variances were 

homogeneous, an ANOVA was performed on data; the ANOVA test was followed by a twosided Dunnett’s test (for comparing 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 data 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 mortality and moribundity were conducted 

twice daily. Individual clinical observations (hand-held physical examinations) were 

recorded daily through termination. Each rat was also observed for signs of toxicity 

approximately 1 and 4 hours following dosing. 

2. Body Weight: Animals were weighed at randomization, daily throughout the dosing period, 

and at termination. Mean body weight changes were calculated for each corresponding 

interval and also for the overall 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 the test substance), all surviving animals were euthanized by 

carbon dioxide inhalation and subjected to a gross 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 the 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 preserved 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. 

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II. RESULTS

A. OBSERVATIONS

1. Mortality: All animals survived until scheduled termination. 

2. Clinical Signs of Toxicity: At 80 mg/kg/day, treatment-related clinical signs of toxicity 

observed 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. 

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 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%). 

In the positive control (EE) group, body weights were decreased (p<0.05) by 11% on Day 4 

compared to the vehicle control, 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 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. 

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In the positive control (EE) group, the overall average food consumption (Days 1-4) was 

decreased (p<0.05) by 46%. 

TABLE 3. Food Consumption (g/rat/day) in the Estrogen Agonist Assaya

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 control (EE) group were increased (p<0.05) by 370% 

and 171%, respectively. The increased wet and blotted uterine weights for the positive 

control animals were within or above the expected ranges as compared to the available inlab historical positive control data. 

The mean blotted vehicle control uterine weight 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 residual ovarian tissue was noted at necropsy. 

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TABLE 4. Uterine Weights from Estrogen Agonist Assay in Sprague Dawley Ratsa

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 reviewers, 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 cypermethrin 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, cypermethrin can be considered to be negative for 

estrogenicity in the uterotrophic assay. The positive control substance (17αethynylestradiol) elicited the expected increases in wet and blotted 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 animals with dried red material around 

the 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 

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 cypermethrin treatment groups were comparable to the 

vehicle controls. In addition, the findings are consistent with those of the dose rangefinding study, in which weight loss and clinical signs were observed in males and females at 

doses of 65, 80 or 100 mg/kg/day. 

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In the positive control (EE) group, body weights were decreased (p<0.05) by 11% on Day 4 

compared to the vehicle control, 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 the vehicle controls. Overall average food 

consumption in the positive control (EE) group was decreased (p<0.05) 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 decrease in overall body weight gains for the high-dose group 

indicates that the tested dose levels were adequate. No statistically significant changes were 

seen in uterine weight in this assay. Cypermethrin is negative in the uterotrophic assay. 

C. STUDY DEFICIENCIES: None 

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APPENDIX

Because this is a range-finding study, only a summary is provided to confirm the adequacy of the 

dose selection rationale used in the 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 dose 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 group initiated every 5 days. Animals were 

necropsied approximately 24 hours after the final dose administration. 

All animals survived to the scheduled termination. For females at 100 mg/kg/day, clinical signs 

for one animal included convulsions, vocalization upon handling, hypersensitivity to handling, 

and wet clear material around mouth, 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 swaying while ambulating and wet clear material around 

mouth. No treatment-related clinical signs were observed in females at 65 mg/kg/day. 

For males in the 65 mg/kg/day group, clinical signs in both animals included splayed hindlimbs 

or rocking, lurching, and swaying while ambulating, 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 had 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) during 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 average 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 average 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. 

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