UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

OFFICE OF CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

MEMORANDUM 

Date: 6/29/2015 

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

PC Code: 10970 I 

Decision No.: 460377, 464766, 470225 

Petition No.: NA 

DP Barcode: 0 398301 , D401793, 0405670 

Registration No.: NA 

Risk Assessment Type: NA 

TXR No.: 0056246 

MRID No.: See Table 

Regulatory Action: NA 

CaseNo.: NA 

CAS No.: 52645-53-1 

40 CFR: NA 

FROM: Greg Akerman, Ph.D. 0-;y g_--

Immediate Office 

Health Effects Division (7509P) 

THROUGH: Jess Rowland cr=-~.D~ Deputy Director 

Health Effects Division 

TO: Jolene Trujillo 

BioJogist/Chemical Review Manager 

Risk Management and Implementation Branch V 

Pesticide Re-evaluation Division (7505P) 

I. ACTION REQUESTED 

Ver.Apr. 2010 

The Pesticide Re-evaluation Division (PRD) of OPP has requested that the Health Effects 

Division (HED) review the Endocrine Disruptor Screening Program (EDSP) Tier I assays 

submitted in response to the agency's Test Order for permethrin: Test Order# CON-109701 -7. 

Page I of2 

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II. RESPONSE

Attached are the EDSP Tier 1 assay DERs for permethrin.

III. MRID Table

Chemical: Permethrin PC Code: 109701

Guideline Assay MRID 

890.1100 Amphibian Metamorphosis Assay (Frog) 48988601

890.1150 Androgen Receptor Binding (Rat Prostate) NA

890.1200 Aromatase Assay (Human Recombinant) 48618601

890.1250 Estrogen Receptor Binding NA

890.1300 Estrogen Receptor Transcriptional Activation 

(Human Cell Line HeLa-9903) NA

890.1350 Fish Short-Term Reproduction 48702301

890.1400 Hershberger (Rat) 48618602

890.1450 Female Pubertal (Rat) 48672901

890.1500 Male Pubertal (Rat) 48687101

890.1550 Steroidogenesis (Human Cell Line – H295R) 48618603

890.1600 Uterotrophic (Rat) NA

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

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

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DER Template Version: 22 September 2011 

Data Requirement: EPA DP Barcode 407365 

OECD Data Point 231 

EPA MRID 48988601

EPA Guideline 890.1100 

 Amphibian Metamorphosis Assay (Frog) 

Test Material: Permethrin Purity (%): 97.7% 

 

Common Name Permethrin 

Chemical Name IUPAC Not Reported 

 CAS Name Not Reported 

CAS No. Not Reported 

Synonyms None Reported

EPA PC Code 109701 

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

Environmental Scientist, CDM Smith 

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

Environmental Scientist, CDM Smith 

Primary Reviewer: Keith Sappington, M.S. 

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

Additional Reviewer: Elyssa Arnold 

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

Additional (and final) Reviewer: Justin Housenger 

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

Date Evaluation Completed: 29-MAY-2015 

Digitally signed by JUSTIN 

HOUSENGER 

DN: c=US, o=U.S. Government, 

ou=USEPA, ou=Staff, cn=JUSTIN 

HOUSENGER, 

dnQualifier=0000044455 

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

Keith 

Sappington

Digitally signed by Keith 

Sappington 

DN: cn=Keith Sappington, o, ou, 

email=sappington.keith@epa.gov

, c=US 

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

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

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CITATION: Schneider, S.Z., T.Z. Kendall, and H.O. Kruger. 2012. Permethrin: Amphibian Metamorphosis Assay 

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

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

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

DC 20006. Study completed November 6, 2012. 

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

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

estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths 

of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, 

where available, and redundancy across the battery. Thus, the results of each individual assay should not be 

considered in isolation but rather should be considered in the context of other assays in the battery as well as 

Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potential to interact 

with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in combination with 

the findings in the OSRI, should be undertaken (refer to the WoE Document).

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EXECUTIVE SUMMARY

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

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

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

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

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

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

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

7.9 to 8.2. 

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

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

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

with no indication of a dose-dependent pattern. 

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

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

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

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

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

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

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

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

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

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

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

to be treatment-related. 

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The study met all validity and performance criteria except for %CV in analytical measurements. The CV of 

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

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

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

the interpretation of test results. 

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

Guideline 890.1100). 

Results Synopsis:

Test organism NF stage at test initiation: 51 

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

Test type: Flow-through 

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

Metamorphosis Assay (AMA) with Permethrin. 

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 Day 7 Day 21 Day 21 

0.000054 No No No Yes No No No 

0.00022 No No No Yes No No No 

0.00068 No No No No No No No 

1

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

otherwise specified. 

2

 The criteria for significance are described in the Reviewer’s Analysis and Statistical Verification sections of the 

DER. Conclusions regarding histopathology may be heavily weighted by the expert opinion of a board-certified 

pathologist. 

3

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

I. MATERIALS AND METHODS 

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

Environmental Protection Agency Series 890- Endocrine Disruptor Screening 

Program Test Guidelines, OPPTS Number 890.1100, Amphibian 

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

Guideline 231, Amphibian Metamorphosis Assay. The following deviations were 

noted: 

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

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2. Hardness of the dilution water (140-144 mg/L as CaCO3) exceeded the recommended range 

(40-48 mg/L as CaCO3). 

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

recommended 20%. 

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

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

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

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

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

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

periodic screening analyses of water for potential contaminants were not 

performed according to Good Laboratory Practice Standards, but were 

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

preliminary analyses of water iodide concentrations were not performed 

according to Good Laboratory Practice Standards; and the characterization and 

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

not determined in accordance with Good Laboratory Practice Standards. 

A. Test Material Permethrin 

Description: Liquid

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

vapor pressure of test compound, expiration date.

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

Purity: 97.7% 

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Impurities: Not Reported 

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

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

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

nominal.

Storage Conditions of 

Test Chemicals: Stored under ambient conditions 

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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 

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 

Were parents maintained as in-house stock? 

Yes Adults were originally obtained from 

Xenopus I of Dexter, Michigan. 

EPA recommends that larvae used in the 

assay be derived from in-house adults.

Were parental acclimation conditions same as definitive test? 

Yes

Acclimation period for parental frogs (if applicable): 

At least 11 days

Details on parental feeding: Frogs were fed an adult 

Xenopus diet supplied 

by Zeigler Brothers, Inc. (Gardners, PA) at a rate sufficient to maintain the 

health of the culture. 

Details on parental health: Culture frogs showed no 

sign of disease or stress prior to the test. 

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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): 

3 

Number of eggs sampled per spawn: 

250 Eggs were collected and evaluated on the 

day of egg deposition. 

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

initiated with larvae at Nieuwkoop – Faber 

(NF) developmental stage 51 (17 days 

post-fertilization).

Age at test initiation: 17 days post-fertilization 

(dpf) 

Mean total length at test initiation (if reported): 

Not reported 

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

Not reported 

Was the optional size selection method used? 

No 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on larval selection: The best viability of 

three separate spawns was selected for obtaining larvae for use in the definitive test 

The selected spawn had a reported viability of 99.6%. 

Loading rate (rearing density): 4 larvae/L EPA recommends that rearing density 

(loading rate) not exceed approximately 10 

larvae/L culturing system for flow-through 

systems or 4 tadpoles/L in static-renewal 

exposure systems.

Type of food: Sera Micron® EPA recommends Sera Micron® throughout 

pre-exposure (after NF stage 45/46) and 

during the entire 21-d definitive study. If 

another diet is used, the study report should 

provide analysis of iodide content and potential 

contaminants, and the diet should 

demonstrate equal performance to Sera 

Micron® .

Source of food: Sera North America 

(Montgomeryville, PA)

Iodide concentration in diet (if known): 

Not Reported 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Frequency of feeding: 3 times/day EPA recommends that feeding occur at least 

twice per day.

Details on feeding regime: Feeding regime was half 

of the recommended daily rates. 

It is recommended that food rations during the 

pre-exposure period be increased along with 

larval growth to approximately 30 

mg/larva/day by test initiation. EPA and 

OECD recommend that food rations increase 

from 30 mg/larva/day at test initiation (Study 

Day 0-4) to 80 mg/larva/day in the last week 

of the test (Study Day 15-21).

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C. Exposure System

Table 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): 

Continuous-flow diluter Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

Flow-through rate (if applicable): 

48.6 mL/min The calculated flow rate provided 

approximately 10 volume additions every 25 hours providing a complete volume replacement every 2.4 hours. 

Recommended flow-through rate is 25 

mL/min (complete volume replacement ca. 

every 2.7 hrs).

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 

Details on toxicant mixing for flow-through systems (if 

applicable): 

A syringe pump was used to deliver volumes of test substance stock solutions and DMF to mixing chambers. Stock solutions or solvent were diluted with well water in the mixing chambers to obtain the desired concentrations. Flow into each mixing chamber using rotameters. 

Recommended toxicant mixing for 

flow-through systems: 1) Mixing chamber is 

recommended but not required; 2) Aeration 

is not recommended for mixing;3) A 

demonstration that the test solution is 

completely mixed before introduced into the 

test system is recommended; 4) The 

recommended flow splitting accuracy is 

within 10%.

Renewal period for static renewal (if applicable): 

N/A If static renewal is used, EPA recommends 

24-hr renewal; renewal period is 

recommended not to exceed 72 hours.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Aeration? No EPA recommends maintaining dissolved 

oxygen concentrations >40% air saturation 

(>3.5 mg/L). Aeration may be maintained through bubblers. It is recommended to set 

bubblers at levels that do not cause stress 

on the tadpoles.

Source of dilution water: Freshwater obtained from a well 

~40 meters deep location on WLI property. Water was passed through a sand filter to remove particles greater than ~25 μm and pumped into a storage tank where it was aerated with spray nozzles. Prior to use, water was filtered to 0.45 μm to remove fine particles and passed through a UV sterilizer. 

EPA recommends natural or reconstituted 

water; it is recommended that natural water 

be sterilized with UV and tested for 

pesticides, heavy metals, and other 

possible contaminants, including known 

substrates of the iodine transporter of the 

thyroid gland (e.g., fluoride, chlorate, 

perchlorate). OECD accepts any water in 

which the test species show control survival 

at least as good as indicated in the test 

guideline.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Was dilution water analyzed for pesticides, heavy metals, and other contaminants? 

Yes. See Reviewer’s Comments. 

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 

(e.g.. in Sera Micron).

Test vessel type/materials: Glass EPA and OECD recommend that 

water-contact portions of the system not 

compromise the study (e.g., all glass 

vessels or glass vessels with stainless steel 

frames are acceptable examples).

Test vessel size: 9 LFill volume: 7 L

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Additional details on exposure system: 

None 

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

Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Hardness (mg/L as CaCO3) 140 144 134 

Weekly in one 

replicate of the 

negative control and 

high-dose treatment. 

EPA recommends hardness 40 to 48 

mg/L as CaCO3.

pH 7.9 8.2 8.0 

Weekly in each test 

vessel. 

EPA recommends pH 7.5 ± 1, 

inter-replicate and inter-treatment 

differentials should not exceed 0.5.

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Dissolved oxygen (mg/L) 5.3 8.4 6.7 

Weekly in each test 

vessel. 

EPA recommends dissolved oxygen (DO) 

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

recommends DO concentration >3.5 mg/L

(>40% air saturation).

Temperature 21.5 22.3 22.0 

Measured in each 

test chamber weekly 

and continuously in 

one negative control 

replicate. 

EPA recommends temperature 22±1oC; 

inter-replicate and inter-treatment 

differentials should not exceed 0.5oC.

Iodide (μg/L) 3 6 N/A 

Preliminary analysis 

of dilution water 

EPA recommends aquatic iodide range 0.5 

– 10 μg/L (supplemental iodide should not 

exceed 2 μg/L).

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, 

Fluoride Not Reported Perchlorate Not Reported Chlorate Not Reported 

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Alkalinity (mg/L as CaCO3) 

Specific Conductance (μS/cm) 

172 

329 

184 

403 

179 

377 

Weekly in one 

replicate of the 

negative control and 

high-dose treatment. 

water quality parameters, including 

ammonia, should be measured just prior to 

renewal. In addition, EPA recommends that 

DO be measured at each concentration at 

least weekly and that temperature be 

measured continuously. OECD 

recommends that DO and temperature be 

measured at least weekly and that pH and 

hardness be measured at least at the 

beginning and end of the test.

 D. Study Design and Additional Experimental Conditions

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

Was a range-finder conducted? Yes

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Parameter Value(s) Details or Remarks Guideline Recommendations 

If yes, what was the method for determining the highest test concentration in the range-finder? 

Solubility and reported LC50

values for amphibians, which ranged from 100 to 700 μg/L.

EPA recommends that the highest test 

concentration is either the solubility limit of the 

test compound, 100 mg/L, or demonstrates 

adequate evidence of toxicity (e.g., <10% 

mortality), whichever concentration is lowest.

Species: Xenopus laevis Life stage: Not reported Test duration: 14 daysAdditional details: Nominal concentrations ranged 

from 0.02 to 1000 μg a.i./L and was conducted under flow-through conditions with the 

use of a solvent. No mortalities or sub-lethal effects were observed in the controls or 0.02 and 0.20 μg a.i./L treatment 

groups. Complete mortality was 

Signs of toxicity observed in the 2.0 μg a.i./L treatment group included lethargy, loss of equilibrium, lying on the bottom of the test chamber, and appearing small in comparison to the controls. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

observed in the 8.1 to 1000 μg 

a.i./L treatment groups. 

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:

Range-finder; high concentration was approximately 1/3 of the LC50 value.

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):

N/A 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Separation of test concentrations: 0.33 EPA recommends that the maximum 

concentration separation be 0.1 and the 

minimum be 0.33. 

Number of test concentrations: 3 EPA recommends a minimum of 3 

concentrations and a control, plus solvent 

control if appropriate.

Are nominal concentrations adjusted for purity? 

Yes 

Indicate the type of values presented for measured concentrations: 

Study authors reported mean-measured concentrations. 

Due to high analytical variability and drastic changes in recoveries between sampling periods, the reviewer calculated time-weighted average (TWA) concentrations. 

Limit of quantification (LOQ): 0.00006 mg a.i./L EPA recommends that for chemical test 

concentrations below the LOQ, analyses be 

conducted on the stock solutions.

Level of detection (LOD): Not Reported 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Frequency of measurement: Days 0, 7, 14, and 21 It is recommended that test item concentration 

be measured in one tank at each treatment 

level at test initiation and every week 

thereafter.

Number of replicates in control: 4 EPA recommends 4 replicates.Number of replicates in solvent control (if applicable): 

4 EPA and OECD recommend the use of a 

concurrent solvent control when a solubilizing 

agent is used. EPA recommends 4 replicates.

Number of replicates per test item treatment level: 

4 EPA recommends 4 replicates.

Number of larvae per treatment at test initiation: 

80 

Was a solvent used? Yes Solvent type (if applicable): DMF 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Maximum solvent concentration (if applicable): 

0.020 mL/L EPA recommends that the solvent not exceed 

0.02 ml/L3. OECD recommends that solvent 

have no effect on survival nor produce any 

other adverse effects and that concentration 

not be greater than 0.1 ml/L4.

Was a positive control used? No Positive control (if applicable): N/A Positive control concentration(s) (if applicable): 

N/A 

Photoperiod: 12 hrs light : 

12 hrs dark 

EPA recommends photoperiod 12:12 

(light:dark).

Light intensity at water’s surface: 0.137-0.602 Klux EPA recommends light intensity 0.6 – 2 Klux 

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 

(at water’s surface).

Additional details: The stock solutions were 

mixed by inversion, and appeared clear and colorless, with no visible precipitates. 

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

Treatment ID 

Nominal 

Concentration 

(mg a.i./L) 

Measured/TWA 

Concentration 

(mg a.i./L) 

Mean CV (%) Details or Remarks Guideline Recommendations 

Control (dilution water only) 

0.00 <LOQ N/A N/A 

EPA and OECD recommend that test item 

concentrations be maintained at a coefficient 

of variation (CV) 20%. Solvent control (if 

applicable) 

0.00 <LOQ N/A N/A 

Treatment 1 0.00011 0.000052/0.000054 50.5 N/A Treatment 2 0.00033 0.00022/0.00022 19.9 N/A Treatment 3 0.00100 0.00063/0.00068 33.0 N/A 

Abbreviations: CV Coefficient of variation. 

 E. Observations

Biological Endpoints: Day 7- NF stage, wet weight, HLL, SVL, normalized HLL 

Day 21- NF stage, wet weight, HLL, SVL, normalized HLL, thyroid histopathology 

 Daily- mortality, clinical signs of toxicity 

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Were raw (individual) data provided? Yes

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

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

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

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

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

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

Table 9: Larval Mortality in Xenopus laevis.

1

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

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

3 1 tadpole killed during siphoning in replicate D on Day 4. Second death occurred in replicate D on Day 7. 

4

 Tadpole killed during siphoning in replicate A on Day 11. 

5 1 tadpole killed during siphoning in replicate A on Day 11. Second death occurred in replicate A on Day 12. 

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

high-dose treatment groups, and 54.5 in the mid-dose treatment group (Table 10). Median developmental 

Treatment (mg a.i./L) 

[TWA-measured]

Larval Mortality 

Day 71 Day 21 

n Mortality # Mortality % n Mortality # Mortality % 

Negative Control 80 0 0 60 0 0 

Solvent Control 80 1 2 1.3 59 1 4 1.7 

0.000054 80 2 3 2.5 58 0 0 

0.00022 80 0 0 60 0 0 

0.00068 80 0 0 60 2 5 3.3 

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stage was 57 in the controls and all treatment groups at test termination. No asynchronous development 

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

in the mid-dose treatment group. The 10th and 90th percentiles of NF stage in the negative control were 

56 and 57, respectively. 

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 57 0 

Solvent Control 4 54 0 4 57 0 

0.000054 4 54 0 4 57 0 

0.00022 4 54.5 0 4 57 0 

0.00068 4 54 0 4 57 0 

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

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

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

respectively.

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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 2.25 0.08 0.15 4 8.10 0.67 0.34 

Solvent Control 4 2.13 0.16 0.14 4 9.81 0.74 0.43 

0.000054 4 2.30 0.24 0.14 4 10.3 0.53 0.41 

0.00022 4 2.28 0.09 0.15 4 9.95 0.99 0.42 

0.00068 4 2.23 0.13 0.14 4 9.25 0.76 0.39 

Abbreviations: SD Standard deviation. 

1

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

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

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

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

groups. 

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

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

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

g in the treatment groups. 

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Table 12: Larval Growth in Xenopus laevis.

Treatment 

(mg a.i./L) 

[TWAmeasured]

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 15.8 0.39 4 23.4 0.74 4 0.283 0.010 4 0.855 0.087 

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

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

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

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

Abbreviations: SD Standard deviation. 

1

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

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

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

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

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

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

stage 57), to the extent possible. 

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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 18 20 17 20 -- 20 -- 

1 20 2 20 1 20 -- 20 -- 

2 20 0 20 2 20 -- 20 -- 

3 20 0 20 0 20 -- 20 -- 

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

1 20 3 20 0 20 -- 20 -- 

2 20 2 20 0 20 -- 20 -- 

3 20 0 20 0 20 -- 20 -- 

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

1 20 3 20 0 20 -- 20 -- 

2 20 2 20 0 20 -- 20 -- 

3 20 0 20 0 20 -- 20 -- 

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

1 20 5 20 1 20 -- 20 -- 

2 20 1 20 1 20 -- 20 -- 

3 20 1 20 0 20 -- 20 -- 

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

1 20 1 20 0 20 -- 20 -- 

2 20 2 20 0 20 -- 20 -- 

3 20 1 20 0 20 -- 20 -- 

1

 Thyroid gland gross pathology and histopathology are graded 0 – 3 based on severity: 0=Not remarkable, 1=Mild, 

2=Moderate, 3=Severe. See OECD No. 82 for reference.

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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 17 20 16 20 20 

1 20 -- 20 -- 20 2 20 2 20 0 

2 20 -- 20 -- 20 1 20 2 20 0 3 20 -- 20 -- 20 0 20 0 20 0 

Solvent Control 0 20 -- 20 -- 20 15 20 19 20 19 

1 20 -- 20 -- 20 3 20 1 20 0 

2 20 -- 20 -- 20 1 20 0 20 1 3 20 -- 20 -- 20 1 20 0 20 0 

0.000054 0 20 -- 20 -- 20 14 20 17 20 16 

1 20 -- 20 -- 20 3 20 3 20 2 

2 20 -- 20 -- 20 3 20 0 20 2 3 20 -- 20 -- 20 0 20 0 20 0 

0.00022 0 20 -- 20 -- 20 12 20 19 20 16 

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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 4 20 1 20 3 

2 20 -- 20 -- 20 2 20 0 20 0 3 20 -- 20 -- 20 2 20 0 20 1 

0.00068 0 20 -- 20 -- 20 13 20 19 20 17 

1 20 -- 20 -- 20 3 20 1 20 2 

2 20 -- 20 -- 20 3 20 0 20 1 3 20 -- 20 -- 20 1 20 0 20 0 

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. 

By test termination, tail curvature was observed in both controls and all treatment groups (Table 15). The study authors believed tail curvature to be related to feeding rates during acclimation. With the exception of the tail curvature, all surviving tadpoles appeared normal and healthy. 

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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 Tail curvature 60 12% Solvent Control Tail curvature 60 22% 0.000054 Tail curvature 60 21% 0.00022 Tail curvature 60 28% 0.00068 Tail curvature 60 16% 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

software. 

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

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

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wet weight on Day 21 and an increase in SVL on Days 7 and 21 in the low-dose treatment group, however, 

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

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

were treatment-related. 

 C. Reviewer’s Analysis and Conclusions 

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

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

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

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

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

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

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

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

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

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

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

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

 Conclusions: 

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

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

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

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

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

control for HLL, normalized HLL, and wet weight. 

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

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 Difference and p value were determined from the comparison of negative and solvent control data (Equal Variance Two-Sample t-Test). 

Treatment 

(mg a.i./L) 

[TWA-measured]

NF Developmental Stage Hind Limb Length3 Asynchronous 

Development 

Thyroid Gross and 

Histopathology 

Day 7 Day 21 Day 7 Day 21 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 57 NA NA NA NA NA NA NA NA NA No Solvent Control4 54 1.0 57 1.0 -5.45 0.32 21.4 0.014 NA NA NA NA No 0.000054 54 1.0 57 1.0 -3.45 0.47 21.3 0.011 NA NA NA NA No 0.00022 54.5 0.57 57 1.0 3.45 0.47 22.8 0.007 NA NA NA NA No 0.00068 54 0.42 57 1.0 -1.72 0.86 14.0 0.097 NA NA NA NA No Statistical Test Mann-Whitney Mann-Whitney Dunnett Dunnett NA NA -- 

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Table 17: Growth Endpoints1,2 in the AMA with Permethrin. 

Abbreviations: Diff. Difference. NA Not applicable.

1

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

2

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

3

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

Two-Sample t-Test). 

E. Study Deficiencies 

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

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

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

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

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

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

results. 

Treatment 

(mg a.i./L) 

[TWA-measured]

Snout-Vent Length Body Weight 

Day 7 Day 21 Day 7 Day 21 

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

Negative Control NA NA NA NA NA NA NA NA 

Solvent Control3 -1.94 0.34 0.95 0.68 -4.04 0.43 11.8 0.048 

0.000054 5.24 0.16 6.1 0.003 10.4 0.30 26.2 0.0005 

0.00022 -1.75 0.85 1.5 0.61 2.29 0.97 11.4 0.091 

0.00068 0.16 1.0 1.18 0.75 5.83 0.70 11.4 0.090 

Statistical Test Dunnett Dunnett Dunnett Dunnett 

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F. Reviewer’s Comments 

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

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

Summary section of this DER. 

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

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

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

solvent control. 

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

       

n

nn

nn

n

n

TWA t

tt

CC

tt

CC

tt

CC

tt

CC

C

1

1

21

21

12

12

01

01

2 2 2 2 





  ̧  1

·  ̈

©

§   ̧  1

·  ̈

©

§   ̧  1

·  ̈

©

§   ̧  1

·  ̈

©

§ 

 

where: 

C TWA is the time-weighted average concentration, 

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

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

the test at time interval j 

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

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

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

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

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

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III. REFERENCES 

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

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

Publishing Co, New York, NY USA. 

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

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

and Company, Wilmington, DE 19898, USA. 

Personal Communication Dr. Henry O. Krueger. 2011. Wildlife International, Ltd., Easton, MD 21601, USA.

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Report Date: 03 Jun-15 07:36 (p 1 of 12)

Test Code: 109701 48988601 | 05-4864-1330

CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 06 Nov-13 16:26

Analysis ID: 17-3225-3301 Endpoint: 07d Developmental Stage CETIS Version: CETISv1.8.7

Analysis: Nonparametric-Two Sample Official Results: Yes

Batch ID: 12-5938-9168

Start Date: 08 Jun-12

Ending Date: 29 Jun-12

Test Type: EDSP AMA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst: K. Sappington

Age: NF51

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

Mann-Whitney U Two-Sample Test

DF P-Type

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

0.0002 10 NA 2 6 1.0000 Exact Non-Significant Effect

0.0007 11.5 NA 1 6 0.5714 Exact Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

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

ANOVA Table

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

Between 1.1875 0.3958333 3 1.46 0.2743 Non-Significant Effect

Error 3.25 0.2708333 12

Total 4.4375 15

Distributional Tests

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

Variances Bartlett Equality of Variance 0.0856 11.3 0.9935 Equal Variances

Distribution Shapiro-Wilk W Normality 0.91 0.841 0.1154 Normal Distribution

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

07d Developmental Stage Summary

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

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

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

0.0007 4 53.8 53 54.5 54 53 54 0.25 0.93% 0.92% Centered Untransformed

 Rankits

-0.1

-0.2

-0.3

-0.4

-0.5

-0.6

-0.7

-0.8

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

07d Developmental Stage

 C-mg ai/L

0

10

20

30

40

50

60

0 N 0.000054 0.0002 0.0007

Graphics

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

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Report Date: 03 Jun-15 07:36 (p 2 of 12)

Test Code: 109701 48988601 | 05-4864-1330

CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 19 Sep-13 10:52

Analysis ID: 14-8403-4926 Endpoint: 07d HLL CETIS Version: CETISv1.8.7

Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 12-5938-9168

Start Date: 08 Jun-12

Ending Date: 29 Jun-12

Test Type: EDSP AMA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst: K. Sappington

Age: NF51

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

Dunnett Multiple Comparison Test

DF P-Type

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

0.0002 0.293 2.68 0.279 6 0.9822 CDF Non-Significant Effect

0.0007 0.26 2.68 0.279 6 0.9874 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

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

ANOVA Table

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

Between 0.01320077 0.004400257 3 0.204 0.8919 Non-Significant Effect

Error 0.259291 0.02160759 12

Total 0.2724918 15

Distributional Tests

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

Variances Bartlett Equality of Variance 4.69 11.3 0.1962 Equal Variances

Distribution Shapiro-Wilk W Normality 0.91 0.841 0.1160 Normal Distribution

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

07d HLL Summary

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

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

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

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

 Rankits

-0.05

-0.10

-0.15

-0.20

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

07d HLL

 C-mg ai/L

Reject Null

Reject Null

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 N 0.000054 0.0002 0.0007

Graphics

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

Page 45 of 206
Report Date: 03 Jun-15 07:36 (p 3 of 12)

Test Code: 109701 48988601 | 05-4864-1330

CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 19 Sep-13 10:52

Analysis ID: 11-5716-5640 Endpoint: 07d Normalized HLL CETIS Version: CETISv1.8.7

Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 12-5938-9168

Start Date: 08 Jun-12

Ending Date: 29 Jun-12

Test Type: EDSP AMA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst: K. Sappington

Age: NF51

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

Dunnett Multiple Comparison Test

DF P-Type

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

0.0002 1.02 2.68 0.013 6 0.6277 CDF Non-Significant Effect

0.0007 0.467 2.68 0.013 6 0.9360 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

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

ANOVA Table

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

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

Error 0.0005432247 4.526872E-05 12

Total 0.000722021 15

Distributional Tests

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

Variances Bartlett Equality of Variance 5.17 11.3 0.1595 Equal Variances

Distribution Shapiro-Wilk W Normality 0.982 0.841 0.9770 Normal Distribution

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

07d Normalized HLL Summary

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

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

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

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

 Rankits

-0.002

-0.004

-0.006

-0.008

-0.010

-0.012

-0.014

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

07d Normalized HLL

 C-mg ai/L

Reject Null

Reject Null

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0 N 0.000054 0.0002 0.0007

Graphics

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

Page 46 of 206
Report Date: 03 Jun-15 07:36 (p 4 of 12)

Test Code: 109701 48988601 | 05-4864-1330

CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 19 Sep-13 10:52

Analysis ID: 05-0978-4307 Endpoint: 07d SVL CETIS Version: CETISv1.8.7

Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 12-5938-9168

Start Date: 08 Jun-12

Ending Date: 29 Jun-12

Test Type: EDSP AMA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst: K. Sappington

Age: NF51

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

Dunnett Multiple Comparison Test

DF P-Type

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

0.0002 0.713 2.68 1.11 6 0.8184 CDF Non-Significant Effect

0.0007 0.0846 2.68 1.11 6 0.9995 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

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

ANOVA Table

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

Between 2.701876 0.9006252 3 2.63 0.0980 Non-Significant Effect

Error 4.1097 0.342475 12

Total 6.811576 15

Distributional Tests

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

Variances Bartlett Equality of Variance 2.16 11.3 0.5405 Equal Variances

Distribution Shapiro-Wilk W Normality 0.984 0.841 0.9867 Normal Distribution

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

07d SVL Summary

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

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

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

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

 Rankits

-0.2

-0.4

-0.6

-0.8

-1.0

-1.2

-1.4

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

07d SVL

 C-mg ai/L

Reject Null

Reject Null

0

2

4

6

8

10

12

14

16

18

0 N 0.000054 0.0002 0.0007

Graphics

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

Page 47 of 206
Report Date: 03 Jun-15 07:36 (p 5 of 12)

Test Code: 109701 48988601 | 05-4864-1330

CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 19 Sep-13 10:52

Analysis ID: 13-2206-0011 Endpoint: 07d Wet Weight CETIS Version: CETISv1.8.7

Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 12-5938-9168

Start Date: 08 Jun-12

Ending Date: 29 Jun-12

Test Type: EDSP AMA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst: K. Sappington

Age: NF51

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

Dunnett Multiple Comparison Test

DF P-Type

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

0.0002 0.356 2.68 0.049 6 0.9694 CDF Non-Significant Effect

0.0007 0.912 2.68 0.049 6 0.6947 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

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

ANOVA Table

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

Between 0.001975446 0.0006584821 3 0.971 0.4383 Non-Significant Effect

Error 0.00813649 0.0006780408 12

Total 0.01011194 15

Distributional Tests

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

Variances Bartlett Equality of Variance 5.83 11.3 0.1202 Equal Variances

Distribution Shapiro-Wilk W Normality 0.92 0.841 0.1712 Normal Distribution

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

07d Wet Weight Summary

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

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

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

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

 Rankits

-0.01

-0.02

-0.03

-0.04

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

07d Wet Weight

 C-mg ai/L

Reject Null

Reject Null

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0 N 0.000054 0.0002 0.0007

Graphics

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

Page 48 of 206
Report Date: 03 Jun-15 07:36 (p 6 of 12)

Test Code: 109701 48988601 | 05-4864-1330

CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 19 Sep-13 10:52

Analysis ID: 04-6216-0594 Endpoint: 21d Developmental Stage CETIS Version: CETISv1.8.7

Analysis: Nonparametric-Two Sample Official Results: Yes

Batch ID: 12-5938-9168

Start Date: 08 Jun-12

Ending Date: 29 Jun-12

Test Type: EDSP AMA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst: K. Sappington

Age: NF51

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

Mann-Whitney U Two-Sample Test

DF P-Type

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

0.0002 10 NA 1 6 1.0000 Exact Non-Significant Effect

0.0007 8 NA 1 6 1.0000 Exact Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

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

ANOVA Table

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

Between 0.75 0.25 3 1 0.4262 Non-Significant Effect

Error 3 0.25 12

Total 3.75 15

Distributional Tests

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

Variances Mod Levene Equality of Variance 1 5.95 0.4262 Equal Variances

Variances Levene Equality of Variance 9 5.95 0.0021 Unequal Variances

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

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

21d Developmental Stage Summary

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

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

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

0.0007 4 57 57 57 57 57 57 0 0.0% 0.0% Centered Untransformed

 Rankits

-0.2

-0.4

-0.6

-0.8

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

21d Developmental Stage

 C-mg ai/L

0

10

20

30

40

50

60

0 N 0.000054 0.0002 0.0007

Graphics

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

Page 49 of 206
Report Date: 03 Jun-15 07:36 (p 7 of 12)

Test Code: 109701 48988601 | 05-4864-1330

CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 19 Sep-13 10:52

Analysis ID: 20-9302-2925 Endpoint: 21d No LS HLL CETIS Version: CETISv1.8.7

Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 12-5938-9168

Start Date: 08 Jun-12

Ending Date: 29 Jun-12

Test Type: EDSP AMA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst: K. Sappington

Age: NF51

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

Dunnett Multiple Comparison Test

DF P-Type

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

0.0002* 3.46 2.68 1.43 6 0.0123 CDF Significant Effect

0.0007 2.17 2.68 1.43 6 0.1211 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

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

ANOVA Table

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

Between 11.26975 3.756584 3 6.57 0.0071 Significant Effect

Error 6.856555 0.5713796 12

Total 18.12631 15

Distributional Tests

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

Variances Bartlett Equality of Variance 1.01 11.3 0.7982 Equal Variances

Distribution Shapiro-Wilk W Normality 0.967 0.841 0.7856 Normal Distribution

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

21d No LS HLL Summary

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

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

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

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

 Rankits

-0.2

-0.4

-0.6

-0.8

-1.0

-1.2

-1.4

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

21d No LS HLL

 C-mg ai/L

Reject Null

Reject Null

0

2

4

6

8

10

12

0 N 0.000054 0.0002 0.0007

Graphics

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

Page 50 of 206
Report Date: 03 Jun-15 07:36 (p 8 of 12)

Test Code: 109701 48988601 | 05-4864-1330

CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 19 Sep-13 10:53

Analysis ID: 04-5657-9563 Endpoint: 21d No LS HLL CETIS Version: CETISv1.8.7

Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 12-5938-9168

Start Date: 08 Jun-12

Ending Date: 29 Jun-12

Test Type: EDSP AMA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst: K. Sappington

Age: NF51

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

Dunnett Multiple Comparison Test

DF P-Type

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

0.0002 0.245 2.68 1.46 6 0.9893 CDF Non-Significant Effect

0.0007 1.02 2.68 1.46 6 0.6251 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

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

ANOVA Table

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

Between 2.224006 0.7413353 3 1.24 0.3371 Non-Significant Effect

Error 7.151991 0.5959992 12

Total 9.375997 15

Distributional Tests

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

Variances Bartlett Equality of Variance 0.942 11.3 0.8152 Equal Variances

Distribution Shapiro-Wilk W Normality 0.96 0.841 0.6569 Normal Distribution

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

21d No LS HLL Summary

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

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

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

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

 Rankits

-0.2

-0.4

-0.6

-0.8

-1.0

-1.2

-1.4

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

21d No LS HLL

 C-mg ai/L

Reject Null

Reject Null

0

2

4

6

8

10

12

0 S 0.000054 0.0002 0.0007

Graphics

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

Page 51 of 206
Report Date: 03 Jun-15 07:36 (p 9 of 12)

Test Code: 109701 48988601 | 05-4864-1330

CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 19 Sep-13 10:52

Analysis ID: 15-7586-3070 Endpoint: 21d No LS Normalized HLL CETIS Version: CETISv1.8.7

Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 12-5938-9168

Start Date: 08 Jun-12

Ending Date: 29 Jun-12

Test Type: EDSP AMA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst: K. Sappington

Age: NF51

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

Dunnett Multiple Comparison Test

DF P-Type

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

0.0002* 3.52 2.68 0.056 6 0.0111 CDF Significant Effect

0.0007 2.14 2.68 0.056 6 0.1276 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

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

ANOVA Table

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

Between 0.01364582 0.004548606 3 5.21 0.0156 Significant Effect

Error 0.01048606 0.0008738383 12

Total 0.02413188 15

Distributional Tests

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

Variances Bartlett Equality of Variance 1.47 11.3 0.6891 Equal Variances

Distribution Shapiro-Wilk W Normality 0.973 0.841 0.8864 Normal Distribution

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

21d No LS Normalized HLL Summary

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

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

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

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

 Rankits

-0.01

-0.02

-0.03

-0.04

0.00

0.01

0.02

0.03

0.04

0.05

0.06

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

21d No LS Normalized HLL

 C-mg ai/L

Reject Null

Reject Null

0.0

0.1

0.2

0.3

0.4

0.5

0 N 0.000054 0.0002 0.0007

Graphics

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

Page 52 of 206
Report Date: 03 Jun-15 07:36 (p 10 of 12)

Test Code: 109701 48988601 | 05-4864-1330

CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 19 Sep-13 10:53

Analysis ID: 12-0818-3320 Endpoint: 21d No LS Normalized HLL CETIS Version: CETISv1.8.7

Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 12-5938-9168

Start Date: 08 Jun-12

Ending Date: 29 Jun-12

Test Type: EDSP AMA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst: K. Sappington

Age: NF51

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

Dunnett Multiple Comparison Test

DF P-Type

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

0.0002 0.56 2.68 0.069 6 0.8977 CDF Non-Significant Effect

0.0007 1.68 2.68 0.069 6 0.2654 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

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

ANOVA Table

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

Between 0.00386035 0.001286783 3 0.978 0.4356 Non-Significant Effect

Error 0.01579626 0.001316355 12

Total 0.01965661 15

Distributional Tests

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

Variances Bartlett Equality of Variance 1.84 11.3 0.6056 Equal Variances

Distribution Shapiro-Wilk W Normality 0.963 0.841 0.7239 Normal Distribution

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

21d No LS Normalized HLL Summary

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

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

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

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

 Rankits

-0.01

-0.02

-0.03

-0.04

-0.05

-0.06

-0.07

0.00

0.01

0.02

0.03

0.04

0.05

0.06

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

21d No LS Normalized HLL

 C-mg ai/L

Reject Null

Reject Null

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0 S 0.000054 0.0002 0.0007

Graphics

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

Page 53 of 206
Report Date: 03 Jun-15 07:36 (p 11 of 12)

Test Code: 109701 48988601 | 05-4864-1330

CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 19 Sep-13 10:52

Analysis ID: 19-6248-5274 Endpoint: 21d No LS SVL CETIS Version: CETISv1.8.7

Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 12-5938-9168

Start Date: 08 Jun-12

Ending Date: 29 Jun-12

Test Type: EDSP AMA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst: K. Sappington

Age: NF51

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

Dunnett Multiple Comparison Test

DF P-Type

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

0.0002 0.985 2.68 0.902 6 0.6473 CDF Non-Significant Effect

0.0007 0.767 2.68 0.902 6 0.7865 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

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

ANOVA Table

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

Between 4.732066 1.577355 3 6.98 0.0057 Significant Effect

Error 2.713268 0.2261056 12

Total 7.445334 15

Distributional Tests

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

Variances Bartlett Equality of Variance 3.67 11.3 0.2995 Equal Variances

Distribution Shapiro-Wilk W Normality 0.916 0.841 0.1460 Normal Distribution

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

21d No LS SVL Summary

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

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

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

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

 Rankits

-0.2

-0.4

-0.6

-0.8

-1.0

-1.2

-1.4

0.0

0.2

0.4

0.6

0.8

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

21d No LS SVL

 C-mg ai/L

Reject Null

Reject Null

0

5

10

15

20

25

30

0 N 0.000054 0.0002 0.0007

Graphics

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

Page 54 of 206
Report Date: 03 Jun-15 07:36 (p 12 of 12)

Test Code: 109701 48988601 | 05-4864-1330

CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Wildlife International

Analyzed: 19 Sep-13 10:52

Analysis ID: 15-7425-9361 Endpoint: 21d No LS Wet Weight CETIS Version: CETISv1.8.7

Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 12-5938-9168

Start Date: 08 Jun-12

Ending Date: 29 Jun-12

Test Type: EDSP AMA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1100 Tier I AMA Diluent: Well Water

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst: K. Sappington

Age: NF51

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

Dunnett Multiple Comparison Test

DF P-Type

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

0.0002 2.34 2.68 0.112 6 0.0915 CDF Non-Significant Effect

0.0007 2.34 2.68 0.112 6 0.0909 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

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

ANOVA Table

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

Between 0.1011469 0.03371564 3 9.66 0.0016 Significant Effect

Error 0.04188577 0.003490481 12

Total 0.1430327 15

Distributional Tests

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

Variances Bartlett Equality of Variance 2.35 11.3 0.5023 Equal Variances

Distribution Shapiro-Wilk W Normality 0.892 0.841 0.0594 Normal Distribution

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

21d No LS Wet Weight Summary

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

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

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

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

 Rankits

-0.02

-0.04

-0.06

-0.08

-0.10

-0.12

-0.14

-0.16

0.00

0.02

0.04

0.06

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

21d No LS Wet Weight

 C-mg ai/L

Reject Null

Reject Null

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 N 0.000054 0.0002 0.0007

Graphics

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

Page 55 of 206
DATA EVALUATION RECORD

PERMETHRIN

Study Type: OCSPP 890.1200, Aromatase Assay 

EPA Contract No. EP10H001452 

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

Prepared for

Health Effects Division

Office of Pesticide Programs

U.S. Environmental Protection Agency 

2777 South Crystal Drive 

Arlington, VA 22202 

Prepared by

CSS-Dynamac Corporation

1910 Sedwick Road, 

Building 100, Suite B 

Durham, NC 27713 

Primary Reviewer Signature:

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

Secondary Reviewer Signature:

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

Program Manager: Signature:

Jack D. Early, M.S. Date: 4/10/2012

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 4/10/2012

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

signing by CSS-Dynamac Corporation personnel. 

Page 56 of 206
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 57 of 206
PERMETHRIN / 109701 

Aromatase (Human Recombinant) Assay (2011) I Page 1 of 15 

OCSPP 890.1200 I OECD None 

Primary Reviewer: Edward Scollon. Ph.D. 

Health Effects Division 

Secondary Reviewer: Greg Akerman. Ph.D. 

Health Effects Division 

Si"'tlJe~~~ Date:~ 

Signature: 

Date: c Ii -r I~ -s - - Template vers.ion 08/2011 

DATA EVALUATION RECORD 

STUDY TYPE: Aromatase (Human Recombinant); OCSPP 890.1200 

PC CODE: 109701 DP BARCODE: 0398301 

TXR#: 0056246 

TEST MATERIAL (PURITY}: Permethrin (97.7% a.i.) 

SYNONYMS: (3-phenoxyphenyl)methyl 3-(2,2-dichloroethenyl)-2,2-

dimethylcyclopropanecarboxy late 

CAS No.: 52645-53-1 

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

CeeTox, Inc., Kalamazoo, Ml. Laboratory Report No.: 9146V100383AROM, November 17, 2011. MRID 48618601. Unpublished. 

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

Washington, DC 

TEST ORDER #: CON-I 09701-7 

EXECUTIVE SUMMARY: In an in vitro aromatase (CYP 19) assay (MRID 48618601 ), 

permethrin (97.7% a.i., Lot# PMPSOOOOI4) was incubated with human recombinant aromatase 

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

sulfoxide (DMSO) at log concentrations of I 0- 10 to 10-3 M for 15 minutes to assess the potential 

of permethrin 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 runs were conducted and each run included a 

full activity control, a background activity control, a positive control series (I 0- 10 to 10-5 M) 

using a known inhibitor (4-hydroxyandrostenedione; 4-0H ASDN), and the permethrin series 

c10-10 to 10-3 M) with three repetitions per concentration. 

Aromatase activity in the full activity controls was 0.588 ± 0.079 nmol·mg-protein- 1

·min- 1

• The 

response of each full activity control within a run was between 90 to 110% of the average full 

activity, and the activity in the background controls ranged from 0.21 to 0.31 % and averaged 

0.28% of the full activity. For the positive control substance (4-0H ASDN), aromatase activity 

results were within the recommended ranges for the performance criteria. The estimated log ICso 

for 4-0H ASDN averaged - 7.3 Mand the Hill slope was - l .O. 

Page 58 of 206
Aromatase (Human Recombinant) Assay (2011) / Page 2 of 15

PERMETHRIN / 109701 OCSPP 890.1200 / OECD None

Precipitation was noted at the two highest concentrations of permethrin tested (10−4 and 10−3 M); 

therefore, the maximum concentration used for data analysis was 10−4.5 M. The lowest portion of 

the response curve was greater than 75% activity at all soluble concentrations of permethrin. 

Aromatase activity averaged 101.3% and 101.9% activity at concentrations of 10-10 and 10−4.5 M 

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

assay. 

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

aromatase activity in this assay.

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

890.1200). 

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

Assurance statements were provided. 

Page 59 of 206
Aromatase (Human Recombinant) Assay (2011) / Page 3 of 15

PERMETHRIN / 109701 OCSPP 890.1200 / OECD None

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: Permethrin

Description: Liquid

Source: AMVAC

Lot # (expiration date): PMPS000014 (January 2013)

Purity: 97.7%

Volatility: Not reported

Storage conditions: Room temperature (e.g. ambient)

Stability: Not reported

Solvent: DMSO

Solubility (in test solvent): Not reported

Highest Concentration Tested: 10−3 M

Stock Solution Preparation: Not reported

Molecular weight: 391.28

CAS #: 52645-53-1

Structure:

O

O

O

Cl

3CH

Cl

3CH

2. Non-Labeled Substrate: Androstenedione (ASDN)

CAS # : 63-05-8

Source: Steraloids, Inc. (Catalog # A6030-100)

Batch # (expiration date): L1712 (April 2016)

Purity: 99.8%

3. Radiolabeled Substrate: 1-β [

3

H(N)]-Androst-4-ene-3,17-dione; [

3

H]ASDN

Source: Perkin Elmer (Catalog #NET-926)

Batch # (expiration date): 619344 (January 10, 2012)

Radiochemical Purity (Supplier): >97%

Specific activity: 26.3 mCi/mmol

Radiochemical Purity (In-lab 

determination):

Not determined

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:

Batch # (expiration date):

Not reported

Not reported

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

Concentration 

(% of total volume in assays)

≤1%

Page 60 of 206
Aromatase (Human Recombinant) Assay (2011) / Page 4 of 15

PERMETHRIN / 109701 OCSPP 890.1200 / OECD None

6. Test Microsomes: Human recombinant aromatase (CYP19) microsomes

Source: BD GentestTM, Woburn, MA (Catalog # 456260)

Lot # (expiration date): 19701 (July 2014)

Protein concentration: 3.7 mg/mL 

Cytochrome C reductase activity: 540 nmol /mg protein/min 

Aromatase activity: 5.7 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 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.

Human recombinant microsomes were purchased from BD GentestTM, and stored at −80 ± 

10oC. Before use of the microsomes, the batch was thawed and sub-aliquoted into 

individual vials before being refrozen to minimize freeze-thaw cycles to no more than one. 

Other assay components sodium phosphate buffer, propylene glycol, and NADPH are 

reported in Table 1. 

TABLE 1. Assay Components and Conditions

Assay Factor Values

sodium phosphate buffer (pH 7.4) 0.1M

Microsomal Protein 0.004 mg/mL

NADPH 0.3 mM

[3H]ASDN 100 nM

Propylene Glycol 5%

Temperature 37°C

Incubation Time 15 min

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

2. Suitability Assessments: The protein concentration of the microsomes (Lot #19701) was 

supplied by the vendor as 3.7 mg/mL. Aromatase activity of the microsomes was also 

provided by the vendor as 5.7 pmol/min/ pmol P450. The mean aromatase activity in the 

full activity control samples over the three test runs was determined to be 0.509 

nmol/min/mg protein, which was greater than the minimum recommended aromatase 

activity of 0.1 nmol/mg-protein/min.

Page 61 of 206
Aromatase (Human Recombinant) Assay (2011) / Page 5 of 15

PERMETHRIN / 109701 OCSPP 890.1200 / OECD None

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 3H2O in the aqueous fraction was quantified for each assay tube by LSC, and 

aromatase activity was reported in units of pmol∙mg-protein−1∙min−1. 

4. Demonstration of Proficiency: The report stated that 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. 

However, the actual data from the proficiency testing were not included in the study report. 

Positive Control

(1) Initial Demonstration of Laboratory Proficiency: The positive control data for 

laboratory met the following criteria:

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

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

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

of 4-OH ASDN was <15%.

• Performance criteria (Table 2) were met, and served as guidance in identifying runs that 

provided parameters in the preferred ranges. 

(2) Demonstration 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. 

TABLE 2. Performance Criteria for the Positive Control

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

Slope −1.2 −0.8 −1.03 −0.97

Top (%) 90 110 99.6 101.2

Bottom (%) −5 +6 0.1 1.1

Log IC50 (M) −7.3 −7.0 −7.3 −7.2

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

5. 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. 

Page 62 of 206
Aromatase (Human Recombinant) Assay (2011) / Page 6 of 15

PERMETHRIN / 109701 OCSPP 890.1200 / OECD None

TABLE 3. Proficiency Chemicals

Compound CAS# Class Concentrations

Econazole 24169-02-6 Inhibitor Not reported

Fenarimol 60168-88-9 Inhibitor Not reported

Nitrofen 1836-75-5 Inhibitor Not reported

Atrazine 1912-24-9 Non-inhibitor Not reported

6. Determination of Aromatase Activity with Test Chemical(s): The response of aromatase 

activity to the presence of eight concentrations of permethrin per run, in triplicate, was 

tested during three independent runs (Table 4). Solubility was assessed (presence of 

cloudiness or a precipitate). If insolubility was observed at the highest test concentration 

(10-3 M) for the first run, then the highest test concentration would be adjusted for the 

second and third runs at the highest test concentration that appeared soluble using log or 

half-log concentrations. 

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

concentrations of permethrin, defining the top of the concentration-response curve. 

TABLE 4. Test Chemical Study Design for each Test Runa

Sample Type Repetitions 

(Tubes) Description Reference or Chemical (M)

Full Activity Control 4 All test componentsb plus solvent vehicle N/A

Bkgd Activity Control 4 Same as above without NADPH N/A

4-OH ASDN Conc 1 2 All test components plus 4-OH ASDN 1×10−5

4-OH ASDN Conc 2 2 All test components plus 4-OH ASDN 1×10−6

4-OH ASDN Conc 3 2 All test components plus 4-OH ASDN 1×10−6.5

4-OH ASDN Conc 4 2 All test components plus 4-OH ASDN 1×10−7

4-OH ASDN Conc 5 2 All test components plus 4-OH ASDN 1×10−7.5

4-OH ASDN Conc 6 2 All test components plus 4-OH ASDN 1×10−8

4-OH ASDN Conc 7 2 All test components plus 4-OH ASDN 1×10−9

4-OH ASDN Conc 8 2 All test components plus 4-OH ASDN 1×10−10

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

Permethrin Conc 2c,e 3 All test components plus Permethrin 1×10−4

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

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

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

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

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

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

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

a Data were obtained from pages 19 of the study report.

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

c Test chemical. 

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

e Highest concentration in Runs #2 and 3.

f Concentration added to Runs #2 and 3.

Page 63 of 206
Aromatase (Human Recombinant) Assay (2011) / Page 7 of 15

PERMETHRIN / 109701 OCSPP 890.1200 / OECD None

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. 

The response curve was fitted by weighted nonlinear regression analysis using a 4-

parameter regression model (XLfit; IDBS; Version 5.2.0.0; Fit Model 208) and Tukey’s BiWeight statistical analysis for outlier analysis. For each independent run of the test substance, 

the individual percent of control values were plotted versus logarithm of the test chemical or 

positive control concentration. The fitted concentration response curves were superimposed on 

the plot, with individual plots prepared for each run (Figures 1-3), and with plotted means 

(Figure 4).

Assay drift was assessed by comparing the % full activity and background activity at values 

at the beginning of each run to the end of each run. 

3. Interpretation of Results: Interpretation of the assay results was based on the average of 

three runs, using the categories presented in Table 6. 

Page 64 of 206
Aromatase (Human Recombinant) Assay (2011) / Page 8 of 15

PERMETHRIN / 109701 OCSPP 890.1200 / OECD None

TABLE 6. Interpretation of Results

Criteria Interpretation

Data fit 4-parameter nonlinear 

regression model

Average curve across runs crossed 50%a Inhibitor

Average lowest portion of curves across runs is 

between 50% and 75% 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 

0.494-0.714 nmol∙mg-protein−1∙min−1 for the 3 test runs, with a mean and standard deviation 

of 0.588 ± 0.079 nmol∙mg-protein−1∙min−1. Activity in the background controls ranged from 

0.21 to 0.31% and averaged 0.28% of the full activity controls. The response of each full 

activity control within a run was between 90 to 110% of the average full activity. 

B. POSITIVE CONTROL: For the positive control substance (4-OH ASDN), aromatase 

activity averaged 0.513-0.666 nmol∙mg-protein−1∙min−1 at the lowest tested concentration 

10−10 M and 0.004 nmol∙mg-protein−1∙min−1 at the highest tested concentration 10−5 M. The 

mean aromatase activity of the positive control (expressed as % full control activity) for 

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

standard deviation, SEM, and % CV. Inhibition response curves for the positive control 

from each run are shown in Figures 1-3, and the average inhibition response curve across all 

runs is shown in Figure 4. 

Page 65 of 206
Aromatase (Human Recombinant) Assay (2011) / Page 9 of 15

PERMETHRIN / 109701 OCSPP 890.1200 / OECD None

TABLE 7. Effect of Permethrin 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 0.67 0.05 0.03 7%

−6 3 5.89 0.50 0.29 9%

−6.5 3 15.6 1.33 0.77 8%

−7 3 34.8 2.03 1.17 6%

−7.5 3 63.1 1.99 1.15 3%

−8 3 84.7 3.52 2.03 4%

−9 3 99.2 0.76 0.44 1%

−10 3 99.6 1.84 1.06 2%

Permethrin -4.5c 2 101.9 3.87 2.74 4%

−5 3 96.4 9.65 5.57 10%

−6 3 101.7 5.00 2.88 5%

−7 3 101.1 2.63 1.52 3%

−8 3 102.7 5.30 3.06 5%

−9 3 102.6 3.59 2.07 4%

−10 3 101.3 3.76 2.17 4%

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

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

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

SD Standard Deviation

SEM Standard error of the mean

CV Coefficient of Variance

C. TEST SUBSTANCE: For permethrin, aromatase activity averaged 0.532–0.674 nmol∙mgprotein−1

∙min−1 at the lowest tested concentration 10−10 M and 0.528–0.572 nmol∙mgprotein−1

∙min−1 at the highest tested concentration used for study interpretation, 10−4.5 M. In 

the first test run of the assay, permethrin was found to be soluble at concentrations of ≤10-

4.5 M; therefore, for Runs 2 and 3 of the assay, the 10-4.5 M concentration was the highest 

tested concentration used for study interpretation. The mean aromatase activity of 

permethrin (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 permethrin from each run are shown in 

Figures 1-3, and the average inhibition response curve across all runs is shown in Figure 4. 

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Aromatase (Human Recombinant) Assay (2011) / Page 10 of 15

PERMETHRIN / 109701 OCSPP 890.1200 / OECD None

FIGURE 1. Inhibition Response Curves for Permethrin and 4-OH ASDN From Run 1.

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PERMETHRIN / 109701 OCSPP 890.1200 / OECD None

FIGURE 2. Inhibition Response Curves for Permethrin and 4-OH ASDN From Run 2.

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PERMETHRIN / 109701 OCSPP 890.1200 / OECD None

FIGURE 3. Inhibition Response Curves for Permethrin and 4-OH ASDN From Run 3.

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PERMETHRIN / 109701 OCSPP 890.1200 / OECD None

FIGURE 4. Mean Inhibition Response Curves for Permethrin and 4-OH ASDN.

The average dose-response curve indicated that the aromatase activity of permethrin 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 determined for permethrin. For 

4-OH ASDN, the estimated log IC50 averaged −7.3 M and the Hill slope was −1.0 (Table 8). 

The variation in the positive control values was acceptable (<15% CV).

TABLE 8. Effect of Permethrin on Aromatase Activity (as Percent of Control) From Independent Runsa

Chemical Run 1 Run 2 Run 3 Meanb SDb % CVb

Log IC50 (M)

Permethrin NA NR NR NR NR NR

4-OH ASDN -7.3 -7.3 -7.2 -7.3 0.06 0.80

Slope

Permethrin NR NR NR NR NR NR

4-OH ASDN -0.99 -0.97 -1.03 -1.0 0.03 3.0

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

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

NR No Result

SD Standard Deviation

CV Coefficient of Variance

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PERMETHRIN / 109701 OCSPP 890.1200 / OECD None

Based on the data from the average response curve and the criteria listed above in Table 8, 

the results support the conclusion that permethrin does not inhibit aromatase activity in the 

aromatase assay. The lowest portion of the response curve across all runs was greater than 

75% activity at all concentrations of permethrin, indicating that permethrin is not an 

inhibitor of aromatase activity in this assay. The mean aromatase activity for permethrin 

was 101.9 ± 3.9% at the highest soluble test concentration of 10−4.5 M.

III. DISCUSSION AND CONCLUSIONS

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

10−10 to 10−4.5 M. Aromatase activity, as a percent of control, was determined to be 101.9% 

(+ 3.9% SD) at the highest soluble concentration of permethrin tested (10−4.5 M), classifying 

it as a non-inhibitor of aromatase according to EDSP guideline 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 permethrin by the 

conduct of three full scale test runs on each of the recommended proficiency chemicals. 

Although the data were not included, it was stated that the proficiency chemicals were 

correctly classified as non-inhibitor or inhibitor. 

Aromatase activity in the full activity controls was 0.588 ± 0.079 nmol∙mg-protein−1∙min−1. 

The response of each full activity control within a run was between 90 to 110% of the 

average full activity, and the activity in the background controls ranged from 0.21 to 0.31% 

and averaged 0.28% of the full activity controls. 

For the positive control substance (4-OH ASDN), aromatase activity was 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 −7.3 M and the Hill slope was 

−1.0. 

For permethrin, aromatase activity at the lowest and the highest tested concentrations, 10−10

and 10−4.5 M, were 101.3% and 101.9% of control, respectively. The two highest test 

concentration of permethrin (10−3 and 10−4 M) showed precipitation during the 15-minute 

incubation period; therefore, the results of these concentrations were not used for data 

analysis. The average dose-response curve indicated that the aromatase activity of 

permethrin at concentrations from 10−10 to 10−4.5 M 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 permethrin 

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

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

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

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

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PERMETHRIN / 109701 OCSPP 890.1200 / OECD None

• For permethrin, only seven test concentrations were used for data interpretation because 

permethrin was insoluble at the 10−3

 M and 10−4 M test concentrations for Run 1. It was 

determined that the highest concentration to be tested in Runs 2 and 3 would be 10−4.5

M, as permethrin was soluble at this concentration. A second mid-log concentration 

(i.e. 10−6.5 M) should have been added in order to obtain the required eight test 

concentrations, as noted in section (j) of OCSPP 890.1200. 

• For permethrin, the coefficient of variation for replicates within the 10−5 M test 

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

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

EPA MRID Number 48702301

Page 1Rf49 

Version: 22 September 2011 

Data Requirement: EPA DP Barcode 406018

OECD Data Point 

 EPA MRID 48702301

EPA Guideline 890.1350 

 Fish Short-Term Reproduction Assay 

Test Material: Permethrin Purity (%): 97.7% 

Common Name Permethrin 

Chemical Name IUPAC Not Reported 

 CAS Name Not Reported 

CAS No. Not Reported 

Synonyms Not Reported

EPA PC Code 109701 

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

Environmental Scientist, CDM Smith 

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

Environmental Scientist, CDM Smith 

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

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

Additional (and final) Reviewer: Justin Housenger 

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

Date Evaluation Completed: 29-MAY-2015 

3

Digitally signed by JUSTIN 

HOUSENGER 

DN: c=US, o=U.S. Government, 

ou=USEPA, ou=Staff, cn=JUSTIN 

HOUSENGER, 

dnQualifier=0000044455 

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

Keith 

Sappington

Digitally signed by Keith Sappington 

DN: cn=Keith Sappington, o, ou, 

email=sappington.keith@epa.gov, 

c=US 

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

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

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

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

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

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

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

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

the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on 

the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints 

within the assay, where available, and redundancy across the battery. Thus, the results of each individual 

assay should not be considered in isolation but rather should be considered in the context of other assays in 

the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical 

has the potential to interact with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 

assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE 

Document).

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EXECUTIVE SUMMARY

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

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

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

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

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

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

of 7.9 to 8.1. 

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

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

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

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

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

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

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

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

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

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

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

decrease on fecundity at the high treatment concentration. 

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

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

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

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

during the course of the study. 

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Effects on gonadal histopathology were observed in female fish at the high treatment level, including increased 

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

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

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

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

both sexes (Ĺ48.9% for males and 28.4% for females). Nuptial tubercle score was significantly decreased in 

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

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

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

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

All validity and performance criteria were met for this study. 

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

(OCSPP Guideline 890.1350). 

Results Synopsis:

Test organism age at test initiation: 6 months 

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

within ±20% of the mean for each sex 

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

Test type: Flow-through 

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

Treatment 

(mg a.i./L) 

[TWAmeasured]

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 

0.00022 No No No -- No No No No No No -- -- -- -- 0.00075 No No No -- No No No No No No -- -- -- -- 0.0026 No3 No Yes -- Yes Yes No Yes No No -- -- -- -- 

Abbreviations: Conc. Concentration. Diff. Difference. E2 17 ȕ-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. 

3 While not statistically significant (p>0.05), fecundity was reduced at highest treatment concentration 42% as compared to the negative control. 

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I. MATERIALS AND METHODS 

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

890- Endocrine Disruptor Screening Program Test Guidelines, OCSPP Number 

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

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

following deviations were noted: 

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

reported. 

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

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

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

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

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

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

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

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

exceptions: periodic analyses of water for potential contaminants were not 

performed according to Good Laboratory Practice Standards, but were not 

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

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

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

accordance with Good Laboratory Practice Standards. 

A. Test Material Permethrin 

Description: A liquid

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

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

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

Purity: 97.7% 

Impurities: Not reported 

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

nominal with coefficients of variation of 7 to 16%.

Storage Conditions of 

Test Chemicals: Stored under ambient conditions 

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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 Adult fish were laboratory-raised fish 

obtained from Osage Catfisheries, Inc. (Osage Beach, Missouri). 

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: Two months prior to preexposure period (14 

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: Fish showed no signs of 

disease or stress, and mortality was <5% during the 7 days prior to the test. 

Fish did not receive treatment for disease during the acclimation period, the two-week pre-exposure period, or the exposure period. 

EPA recommends that mortality during the 7 days prior to the pre-exposure phase be less 

than 5% of the culture population. If mortality during these 7 days is greater than 10%, EPA 

recommends that the fish be rejected. If mortality

is between 5-10%, EPA recommends that fish 

be held another 7 days. If mortalities greater 

than 5% occur during this extended acclimation period, EPA recommends that the fish not be 

used. 

Type of food: Commercial flake food 

(Sera Vipan) and brine shrimp nauplii (Artemia 

sp.).

EPA recommends that fish be fed frozen brine shrimp twice per day to promote active 

reproduction and maintain body condition. 

Source of food: -Sera North America 

-Brine Shrimp Direct, Ogden, Utah 

Frequency of feeding: 2 times/day 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on feeding: Fish were fed twice a day 

during the 14-day perexposure period and 21-day exposure period. Feeding was suspended for at least 12 hours prior to test termination. 

Feeding rates in each replicate were adjusted approximately weekly, if necessary, to ensure that the feeding rate per fish remained constant. Uneaten food and fecal material were removed from test chambers daily by siphoning or by replacement with clean tanks. 

Table 3: Fish Selection and Pre-Exposure Performance.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Age at test initiation: 6 months 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): 

2.5 g Based on sub-sample (n=10). EPA recommends thata 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): 

Not reported Individual weights were within ±20% of the 

mean. 

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

1.0 g Based on sub-sample (n=10). 

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

Not reported Individual weights were within ±20% of the 

mean. 

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

Not determined 

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

Not determined 

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Number of pre-exposure tanks: 32 Almost twice the number of breeding groups 

was maintained in the pre-exposure period as in the exposure period so that breeding groups with a proven history of spawning could be selected for inclusion in the exposure phase of the test. 

EPA recommends that additional tanks set up at the beginning of pre-exposure will ensure that 

sufficient replicates with the correct sex ratio are available for the definitive test. 

Number of males per tank: 2 Number of females per tank: 4 Pre-exposure fecundity: >17.6 eggs/female/ 

reproductive day/ replicate 

EPA recommends that pre-exposure fecundity in each replicate (tank) selected for use in the 

definitive test be at least 15 eggs/female/reproductive day/replicate during the 7 days prior to the definitive test. 

Number of spawns during preexposure: >2 times in 7 days EPA recommends that spawning occur at least twice in the 7 days prior to the definitive test. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on pre-exposure: Any pre-exposure aquaria 

with mortalities, incorrect sex ratio, or poor performance were excluded from use in the test. 

C. Exposure System

Table 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. As noted in the Corrections and Clarifications document1 , the use of a static 

renewal system is not recommended for this assay.

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. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Type of flow-through dilution 

system: 

Continuous-flow diluter Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

Flow-through rate: ~62.5 mL/min Flow rate (reviewer-calculated) 

provided approximately 9 volume additions per day. 

Recommended flow-through rate is 45 mL/min (2.7 L/hr), or at least 6 total 

volume exchanges per day.

Details on toxicant mixing for flow-through systems: 

A syringe pump was used to deliver volumes of test substance stock solutions and solvent (DMF) to mixing chambers where they were mixed with well water to obtain the desired concentration. Flow was controlled using rotameters and solutions in each mixing chamber were pumped to chambers using a peristaltic pump. 

Recommended toxicant mixing for flowthrough systems: 1) Mixing chamber is 

recommended but not required; 2) Aeration is 

not recommended for mixing; 3) A demonstration that the test solution is 

completely mixed before introduced into the test system is recommended; 4) The recommended flow splitting accuracy is within 10%.

(http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf). 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 

Aeration? No EPA recommends aeration if dissolved oxygen 

reaches <4.9 mg/L (<60% saturation).

Source of dilution water: Well water located on WLI property. 

Water was passed through a sand filter to remove particles greater than ~25 μm and aerated. Prior to use, water was filtered to 0.45 μm to remove fine particles and was passed through a UV sterilizer. 

EPA recommends natural or reconstituted 

water; it is recommended that natural water be sterilized with UV and tested for 

pesticides, heavy metals, and other possible 

contaminants. OECD accepts any water in which the test species show control survival 

at least as good as indicated in the test 

guideline.

Was dilution water analyzed for pesticides, heavy metals, and other contaminants? 

Yes. See Reviewer’s Comments. 

Test vessel type/materials: Glass 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: 12 L EPA recommends the use of 18 L test 

chambers (e.g., 40 x 20 x 20 cm).

Fill volume: 10 L EPA recommends 10 L solution per tank. Spawning substrate material: PVC EPA recommends that each tank contain 

three semi-circular spawning substrates, e.g., 

aged PVC pipe, 10 - 20 cm in length, split 

lengthwise. 

Spawning substrate size: 10 cm (3 rep test chamber) Additional details on exposure system: 

None 

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

Parameter Minimum Maximum Mean Measurement Interval Guideline Recommendations 

Temperature (°C) 24.6 25.8 25.3 

Weekly in each test 

chamber and 

continuously in one 

negative control 

replicate. 

EPA recommends temperature 25±1oC; interreplicate and inter-treatment differentials 

should not exceed 1oC.

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Parameter Minimum Maximum Mean Measurement Interval Guideline Recommendations 

pH 7.9 8.1 8.0 

Weekly in each test 

chamber. 

EPA recommends pH 6.5 to 9.0.

Dissolved oxygen (mg/L) 4.7 8.3 6.5 

Weekly in each test 

chamber and 

periodically in 

alternating replicates. 

EPA recommends dissolved oxygen (DO) 

>4.9 mg/L (>60% air saturation)

Total alkalinity (mg/L as CaCO3) 166 178 174 

Measured in one 

replicate of the 

negative control and 

highest treatment level 

weekly. 

EPA recommends total alkalinity >20 mg/L 

as CaCO3.

Hardness (mg/L as CaCO3) 132 144 140 

Measured in one 

replicate of the 

negative control and 

highest treatment level 

weekly. 

Total organic carbon (mg/L) <1 

Measured during the 

4-week period 

preceding the test. 

EPA recommends that total organic carbon in 

dilution water be <2 mg/L. 

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Parameter Minimum Maximum Mean Measurement Interval Guideline Recommendations 

Unionized ammonia (Pg/L) Not Reported 

EPA recommends that unionized ammonia in the dilution water be <1 

Pg/L. 

Residual chlorine (Pg/L) Not Reported 

EPA recommends that residual chlorine in 

dilution water be <10 

Pg/L. 

Specific Conductance (μS/cm) 359 389 374 

Measured in one 

replicate of the 

negative control and 

highest treatment level 

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 range-finder? 

Not specified EPA recommends that the highest test 

concentration be selected based on toxicity data for other fish studies or species, if available. 

Otherwise, either the solubility limit of the test 

compound or 100 mg/L (whichever is lower) is 

appropriate.

Species: Pimephales promelas Life stage: Not specified EPA recommends that range-finding tests be 

performed with fish of similar age and size to those that would be utilized in the test.

Test duration: 14 days EPA recommends a 96-hour exposure. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Additional details: Range-finder conducted at nominal 

concentrations 0 (negative and solvent controls), 0.00081, 0.0027, 0.0090, 0.030, and 0.100 mg a.i./L. 100% Mortality in two highest treatment groups three days after initiation. Because of mortality, two additional lower levels added to range-finder on Day 2 (0.000073 and 0.00024 mg a.i./L). All fish in controls and 0.000073-0.0027 mg a.i./L treatment groups appeared normal with no mortalities. By test end, 100% mortality at 0.0090 mg 

a.i./L. 

The resulting LC50 ranged between 0.0030 and 0.060 mg a.i./L. All test solutions were clear and colorless, though an oily layer was noted in the mixing chamber of the highest treatment group. Analytical recoveries ranged from 73 to 113% of nominal. 

EPA recommends conducting a range-finder with five test concentrations plus a control (six total 

treatment levels), with four females and two males per exposure tank (36 fish total). The 

number of mortalities that occur may be used to 

develop a concentration-response curve. Based upon the results, the highest concentration 

that does not result in increased mortality or signs of overt morbidity compared to controls, or 

1/3 the derived 96-hr 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:

Range-finder EPA recommends that the highest test 

concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates 

adequate evidence of toxicity (e.g., 1/3 the 96-

hour LC50), whichever concentration is lowest.

Reference study citation (if applicable):

NA 

Separation of test concentrations: 0.33 EPA suggests that a concentration separation of 

between 0.33 (or three-fold) and 0.1 (or tenfold) is scientifically acceptable1 . 

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? 

Yes 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Indicate the type of values presented for measured concentrations: 

Mean-measured Review also calculated the time-weighted 

average (TWA) concentrations. 

Limit of quantification (LOQ): 0.000150 mg a.i./L EPA recommends that for chemical test 

concentrations below the LOQ, analyses be 

conducted on the stock solutions.

Level of detection (LOD): Not reported Frequency of measurement: Samples were collected for 

analysis on Days 0, 7, 14, and 21 

It is recommended that test item concentration 

be measured prior to the addition of fish in all tanks and at least weekly thereafter in two 

replicates per treatment level.

Was the randomized complete block design used? 

Yes EPA recommends that all fish be randomly 

assigned to tanks during pre-exposure. Tanks 

are then ranked according to pre-exposure fecundity, and the tanks with the highest 

fecundity are randomly assigned to a definitive test treatment and block first. Each block 

contains one replicate of each treatment, 

including controls. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Number of replicates in control: 4 EPA recommends 4 replicates.Number of replicates in solvent control (if applicable): 

4 EPA recommends the use of a concurrent 

solvent control when a solubilizing agent is used. 

EPA recommends 4 replicates.

Number of replicates per test 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? Yes Solvent type (if applicable): DMF 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Maximum solvent concentration (if applicable): 

0.020 mL/L EPA recommends that the solvent not exceed 

0.02 ml/L3. OECD recommends that solvent 

have no effect on survival nor produce any other adverse effects and that concentration not be 

greater than 0.1 ml/L4

.

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

NA; a positive control was not used 

Photoperiod: 16 hrs light : 

8 hrs dark 

EPA recommends photoperiod 16:8 (light:dark).

Light intensity at water’s surface: 163-760 lux EPA recommends light intensity 540 – 1080 lux 

(at water’s surface).

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

Treatment ID 

Nominal 

Concentration 

(mg a.i./L) 

Measured 

Concentration 

(mg a.i./L) 

Mean CV (%) Details or Remarks Guideline Recommendations 

Negative Control 0.00 <0.000150 NA NA EPA recommends that test item concentrations 

be maintained at a coefficient of variation (CV) 

20%.

Solvent Control 0.00 <0.000150 NA NA Treatment 1 0.00033 0.00023 16 

TWA=0.00022 

CV=17% 

Treatment 2 0.00099 0.00075 6 

TWA=0.00075 

CV=6% 

Treatment 3 0.0030 0.0026 10 

TWA=0.0026 

CV=10% 

Abbreviations: CV Coefficient of variation. 

LOQ=0.000150 mg a.i./L 

E. Observations

Biological Endpoints: Survival, fecundity, fertilization success, length, body weight, tubercle score, GSI, VTG, gonad histopathology, secondary 

sex characteristics, clinical signs of toxicity 

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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 17

ȕ-estradiol, if measured). GSI is calculated using a ratio of gonad weight to body weight (gonad 

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

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

II. RESULTS AND DISCUSSION A. Results

No mortalities were observed in the controls or TWA 0.00022 and 0.00075 mg a.i./L treatment groups (Table 9). Male and female survival in the 

mean-measured 0.0026 mg a.i./L treatment group was 63 and 50%, respectively. Survival in the negative control and the two lower treatment groups (0.00022 and 0.00075 mg a.i/L) was 100%, but there was 63% survival for males (5 of 8) and 50% survival for females (8 of 16) in the highest treatment concentration. Additionally, clinical sings of toxicity (occurring in the highest treatment group) included erratic swimming (6 fish), loss of equilibrium (2 fish), and surfacing (2 fish). There were also observations of excess feed noted in the test chambers of the highest treatment 

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group on Day 10 and Days 17-19. These observations suggest that overt toxicity was present during the course of this study in the highest treatment 

concentration. 

Table 9: Adult Fish Survival in Fathead Minnow (

Pimephales promelas).

Treatment 

(mg a.i./L) 

[TWA-measured]

Males Females 

n # Surviving % Survival n # Surviving % Survival 

Negative Control (<LOQ) 8 8 100 16 16 100 Solvent Control (<LOQ) 8 8 100 16 16 100 0.00022 8 8 100 16 16 100 0.00075 8 8 100 16 16 100 0.0026 8 5 63 16 8 50 

Abbreviations: NA Not applicable. 

LOQ=0.000150 mg a.i./L 

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In the controls and treatment groups, male wet weight and total length ranged from 2.63-2.99 g and 56.7-58.8 mm, respectively (Table 10). Female wet weight and total length ranged from 1.06-1.16 g and 45.3-46.5 mm, respectively. 

Table 10: Size at Test Termination in Fathead Minnow (

Pimephales promelas).

Treatment 

(mg a.i./L) 

[TWA-measured]

Body Weight Length 

Males Females Males Females 

n 

Mean

(g) 

±SD n 

Mean

(g) 

±SD n 

Mean

(mm) 

±SD n 

Mean

(mm) 

±SD 

Negative Control (<LOQ) 

4 2.99 0.265 4 1.06 0.065 4 57.1 1.44 4 45.3 0.84 

Solvent Control (<LOQ) 

4 2.70 0.223 4 1.11 0.066 4 56.3 1.76 4 45.7 0.52 

0.00022 4 2.85 0.317 4 1.16 0.019 4 58.8 1.76 4 46.5 0.35 0.00075 4 2.63 0.194 4 1.13 0.069 4 58.3 1.19 4 46.0 0.54 0.0026 4 2.86 0.120 4 1.09 0.155 4 57.1 1.84 4 45.5 1.44 

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

LOQ=0.000150 mg a.i./L 

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Fecundity averaged 19.9, 18.9, 22.1, 18.8, and 11.5 eggs/female/reproductive day in the negative control, 

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

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

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

in the negative control averaged 95.4%. 

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

Treatment 

(mg a.i./L) 

[TWA-measured]

Fecundity1 ±SD 

Fertilization Success 

(%)2 ±SD 

Negative Control (<LOQ) 19.9 0.71 95.4 1.49 

Solvent Control (<LOQ) 18.9 ND 96.4 ND 

0.00022 22.1 3.56 97.5 0.62 

0.00075 18.8 4.22 93.9 1.71 

0.0026 11.5 7.19 94.7 3.09 

Abbreviations: NA Not applicable. ND Not determined. 

1

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

2

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

LOQ=0.000150 mg a.i./L 

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

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

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

Treatment 

(mg a.i./L) 

[TWA-measured]

Males Females 

n Median Tubercle 

Score n Median Tubercle Score 

Negative Control (<LOQ) 4 35 4 0 

Solvent Control (<LOQ) 4 30 4 0 

0.00022 4 29 4 0 

0.00075 4 28 4 0 

0.0026 4 16 4 0 

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

LOQ=0.000150 mg a.i./L 

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

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

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

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

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

Treatment 

(mg a.i./L) 

[TWA-measured]

Males Females 

n Mean GSI1

(%) 

±SD n Mean GSI1

(%) 

±SD 

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

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

0.00022 4 1.59 0.345 4 13.9 1.28 

0.00075 4 1.74 0.253 4 14.0 1.97 

0.0026 4 2.32 0.384 4 18.0 2.63 

Abbreviations: NA Not applicable. 

1

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

LOQ=0.000150 mg a.i./L 

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

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

Treatment 

(mg a.i./L) 

[TWA-measured]

Males Females 

n Median Stage1 n Median Stage2

Negative Control (<LOQ) 4 2.3 4 2.3 

Solvent Control (<LOQ) 4 2.5 4 3.0 

0.00022 4 2.5 4 3.0 

0.00075 4 2.5 4 2.8 

0.0026 4 3.0 4 3.5 

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

1

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

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

2

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

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

LOQ=0.000150 mg a.i./L 

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

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

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

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

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

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

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

Treatment 

(mg a.i./L) 

[TWA-measured] 

Diagnostic Observations1

Severity

Increased 

Proportion of 

Spermatogonia 

Presence of 

Testis-Ova 

Increased 

Testicular 

Degeneration 

Interstitial Cell 

Hypertrophy/ 

Hyperplasia 

n Incidence n Incidence n Incidence n Incidence 

Negative Control 

(<LOQ) 

0 8 8 8 8 8 8 8 8 

1 8 0 8 0 8 0 8 0 

2 8 0 8 0 8 0 8 0 

3 8 0 8 0 8 0 8 0 

4 8 0 8 0 8 0 8 0 

Solvent Control 

(<LOQ) 

0 8 8 8 8 8 8 8 8 

1 8 0 8 0 8 0 8 0 

2 8 0 8 0 8 0 8 0 

3 8 0 8 0 8 0 8 0 

4 8 0 8 0 8 0 8 0 

0.00022 0 8 8 8 8 8 8 8 8 

1 8 0 8 0 8 0 8 0 

2 8 0 8 0 8 0 8 0 

3 8 0 8 0 8 0 8 0 

4 8 0 8 0 8 0 8 0 

0.00075 0 8 8 8 8 8 7 8 8 

1 8 0 8 0 8 1 8 0 

2 8 0 8 0 8 0 8 0 

3 8 0 8 0 8 0 8 0 

4 8 0 8 0 8 0 8 0 

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Treatment 

(mg a.i./L) 

[TWA-measured] 

Diagnostic Observations1

Severity

Increased 

Proportion of 

Spermatogonia 

Presence of 

Testis-Ova 

Increased 

Testicular 

Degeneration 

Interstitial Cell 

Hypertrophy/ 

Hyperplasia 

n Incidence n Incidence n Incidence n Incidence 

0.0026 0 5 5 5 5 5 5 5 5 

1 5 0 5 0 5 0 5 0 

2 5 0 5 0 5 0 5 0 

3 5 0 5 0 5 0 5 0 

4 5 0 5 0 5 0 5 0 

1

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

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

LOQ=0.000150 mg a.i./L

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Table 16: Additional Gonadal Histopathology Observations in Male Fathead Minnow (

Pimephales promelas).

Treatment 

(mg a.i./L) 

[TWA-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 

Negative Control (<LOQ) 0 8 8 8 8 8 8 8 8 8 7 

1 8 0 8 0 8 0 8 0 8 1 

2 8 0 8 0 8 0 8 0 8 0 3 8 0 8 0 8 0 8 0 8 0 4 8 0 8 0 8 0 8 0 8 0 

Solvent Control (<LOQ) 0 8 8 8 8 8 8 8 8 8 6 

1 8 0 8 0 8 0 8 0 8 0 

2 8 0 8 0 8 0 8 0 8 1 3 8 0 8 0 8 0 8 0 8 0 4 8 0 8 0 8 0 8 0 8 1 

0.00022 0 8 8 8 8 8 8 8 8 8 8 

1 8 0 8 0 8 0 8 0 8 0 

2 8 0 8 0 8 0 8 0 8 0 3 8 0 8 0 8 0 8 0 8 0 4 8 0 8 0 8 0 8 0 8 0 

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Treatment 

(mg a.i./L) 

[TWA-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 

0.00075 0 8 8 8 8 8 8 8 7 8 7 

1 8 0 8 0 8 0 8 1 8 1 

2 8 0 8 0 8 0 8 0 8 0 3 8 0 8 0 8 0 8 0 8 0 4 8 0 8 0 8 0 8 0 8 0 

0.0026 0 5 5 5 5 5 5 5 5 5 5 

1 5 0 5 0 5 0 5 0 5 0 

2 5 0 5 0 5 0 5 0 5 0 3 5 0 5 0 5 0 5 0 5 0 4 5 0 5 0 5 0 5 0 5 0 

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test 

guideline for reference. 

LOQ=0.000150 mg a.i./L

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

Treatment 

(mg a.i./L) 

[TWA-measured] 

Diagnostic Observations1

Severity

Increased Oocyte 

Atresia 

Perifollicular Cell 

Hyperplasia/ 

Hypertrophy 

Decreased Yolk 

Formation 

n Incidence n Incidence n Incidence 

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

1 16 1 16 0 16 0 

2 16 0 16 0 16 0 

3 16 1 16 0 16 0 

4 16 0 16 0 16 0 

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

1 16 4 16 0 16 0 

2 16 0 16 0 16 0 

3 16 0 16 0 16 0 

4 16 0 16 0 16 0 

0.00022 0 16 14 16 16 16 16 

1 16 2 16 0 16 0 

2 16 0 16 0 16 0 

3 16 0 16 0 16 0 

4 16 0 16 0 16 0 

0.00075 0 16 13 16 16 16 16 

1 16 2 16 0 16 0 

2 16 1 16 0 16 0 

3 16 0 16 0 16 0 

4 16 0 16 0 16 0 

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Treatment 

(mg a.i./L) 

[TWA-measured] 

Diagnostic Observations1

Severity

Increased Oocyte 

Atresia 

Perifollicular Cell 

Hyperplasia/ 

Hypertrophy 

Decreased Yolk 

Formation 

n Incidence n Incidence n Incidence 

0.0026 0 8 4 8 8 8 8 

1 8 0 8 0 8 0 

2 8 0 8 0 8 0 

3 8 0 8 0 8 0 

4 8 4 8 0 8 0 

1

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

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

LOQ=0.000150 mg a.i./L

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

promelas).

Treatment 

(mg a.i./L) 

[TWAmeasured] 

Additional Diagnostic Observations1

Severity

Interstitial Fibrosis 

Egg Debris in 

Oviduct 

Granulomatous 

Inflammation 

Decreased PostOvulatory Follicles 

n Incidence n Incidence n Incidence n Incidence 

Negative 

Control 

(<LOQ) 

0 16 16 16 16 16 13 16 7 

1 16 0 16 0 16 2 16 1 

2 16 0 16 0 16 0 16 2 

3 16 0 16 0 16 1 16 1 

4 16 0 16 0 16 0 16 5 

Solvent Control 

(<LOQ) 

0 16 16 16 16 16 13 16 5 

1 16 0 16 0 16 2 16 1 

2 16 0 16 0 16 1 16 1 

3 16 0 16 0 16 0 16 3 

4 16 0 16 0 16 0 16 6 

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Treatment 

(mg a.i./L) 

[TWAmeasured] 

Additional Diagnostic Observations1

Severity

Interstitial Fibrosis 

Egg Debris in 

Oviduct 

Granulomatous 

Inflammation 

Decreased PostOvulatory Follicles 

n Incidence n Incidence n Incidence n Incidence 

0.00022 0 16 16 16 16 16 11 16 4 

1 16 0 16 0 16 4 16 0 

2 16 0 16 0 16 1 16 2 

3 16 0 16 0 16 0 16 0 

4 16 0 16 0 16 0 16 10 

0.00075 0 16 16 16 16 16 15 16 5 

1 16 0 16 0 16 1 16 3 

2 16 0 16 0 16 0 16 0 

3 16 0 16 0 16 0 16 1 

4 16 0 16 0 16 0 16 7 

0.0026 0 8 8 8 8 8 7 8 1 

1 8 0 8 0 8 1 8 0 

2 8 0 8 0 8 0 8 0 

3 8 0 8 0 8 0 8 2 

4 8 0 8 0 8 0 8 5 

1

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

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

LOQ=0.000150 mg a.i./L

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

380, 180, and 249 while female VTG averaged 1.66x106

, 1.39x106

, 1.64x106, and 1.03x106 for the 

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

(Table 19). 

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

Treatment 

(mg a.i./L) 

[TWA-measured]

Plasma Vitellogenin (VTG) 

Males Females 

n Mean

(ng/mL plasma) 

±SD n Mean

(ng/mL plasma) 

±SD 

Negative Control (<LOQ) 4 243 167.8 4 1.66x106 5.14x105

Solvent Control (<LOQ) 4 272 148.1 4 1.53x106 2.48x105

0.00022 4 380 602.3 4 1.39x106 4.37x105

0.00075 4 180 203.7 4 1.64x106 4.80x105

0.0026 4 249 320.5 4 1.03x106 6.90x105

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

LOQ=0.000150 mg a.i./L

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Table 20: Plasma Sex Steroids in Fathead Minnow (

Pimephales promelas). (Not Measured)

Treatment 

(mg a.i./L) 

[TWA-measured]

Plasma Testosterone (T) Plasma 17ȕ-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 

Negative Control (<LOQ) -- -- -- -- -- -- -- -- -- -- -- -- Solvent Control (<LOQ) -- -- -- -- -- -- -- -- -- -- -- -- 0.00022 -- -- -- -- -- -- -- -- -- -- -- -- 0.00075 -- -- -- -- -- -- -- -- -- -- -- -- 0.0026 -- -- -- -- -- -- -- -- -- -- -- -- 

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

LOQ=0.000150 mg a.i./L 

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By test termination, a single male in the solvent control was missing its left eye; all other males appeared normal (Table 21). Females in the 

controls and two lower treatment groups all appeared normal. Clinical signs were restricted to females in the highest treatment level and included surfacing, erratic swimming, loss of equilibrium, weakness, bloody lower jaws, and fin-pecked. 

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

EPA MRID Number 48702301

Page 43Rf 49 

Version: 22 September 2011 

Table 21: Secondary Sex Characteristics and Clinical Signs in Fathead Minnow (

Pimephales promelas).

Treatment 

(mg a.i./L) 

[TWA-measured] 

Secondary Sex Characteristics and Clinical Signs 

Males Females 

Type n Incidence Type n Incidence 

Negative Control (<LOQ) None 8 -- None 16 -- Solvent Control (<LOQ) Missing left eye 8 1 None 16 -- 0.00022 None 8 -- None 16 -- 0.00075 None 8 -- None 16 -- 0.0026 None 5 -- Loss of equilibrium 8 2 

Bloody lower jaw 8 3 

Erratic swimming 8 5 

Surfacing 8 2 

Weak 8 1 

Fin-pecked 8 1 

LOQ=0.000150 mg a.i./L

Page 115 of 206
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Permethrin

EPA MRID Number 48702301

Page 44Rf49 

Version: 22 September 2011 

B. Study Author’s Analysis and Conclusions

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

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

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

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

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

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

excluded from analyses in this study. 

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

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

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

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

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

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

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

the control. 

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

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

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

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

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

with SAS software. 

Page 116 of 206
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Permethrin

EPA MRID Number 48702301

Page 45Rf49 

Version: 22 September 2011 

Survival and GSI (both sexes) were both significantly reduced at the highest treatment level relative 

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

greater severity and frequency of gonad histopathological findings. 

 C. Reviewer’s Analysis and Conclusions 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

settings implemented by EFED on 1/30/13. 

 Conclusions: 

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

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

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

and clinical signs of toxicity occurred in the highest test treatment (TWA 0.0026 mg a.i./L). 

Page 117 of 206
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Permethrin

EPA MRID Number 48702301

Page 46Rf 49 

Version: 22 September 2011 

Table 22: Reproductive and HPG Endpoints1,2 for Male Fathead Minnow (

Pimephales promelas) in the FSTRA with Permethrin. 

Treatment 

(mg a.i./L) 

[TWA-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 

Negative Control (<LOQ) 

34.5 NA NA NA No NA NA NA NA NA NA 

Solvent Control3 (<LOQ) 

30.3 0.046 15.0 0.29 No 10.8 0.80 NA NA NA NA 

0.00022 28.5 0.17 1.76 0.44 No 56.9 0.49 NA NA NA NA 0.0075 28 0.14 11.4 0.18 No -25.6 0.34 NA NA NA NA 0.0026 15.5 0.011 48.9 0.0056 No 2.78 0.74 NA NA NA NA Statistical Test Jonckheere Jonckheere NA Mann-Whitney NA NA 

Abbreviations: Conc. Concentration. Diff. Difference. E2 17 ȕ-estradiol. 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. 

3 p values for solvent control were based on output from equal variance two-sample t-test. 

LOQ=0.000150 mg a.i./L 

Page 118 of 206
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Permethrin

EPA MRID Number 48702301

Page 47Rf 49 

Version: 22 September 2011 

Table 23: Reproductive and HPG Endpoints1,2 for Female Fathead Minnow (

Pimephales promelas) in the FSTRA with Permethrin. 

Treatment 

(mg a.i./L) 

[TWAmeasured]

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 

Negative Control (<LOQ) 

NA NA NA NA NA NA NA NA No NA NA NA NA NA NA

Solvent Control3

(<LOQ) 

-5.02 0.76 1.04 0.62 NA NA 2.44 0.62 No -8.22 0.67 NA NA NA NA

0.00022 10.9 0.84 2.20 0.33 NA NA -0.71 0.56 No -16.5 0.81 NA NA NA NA0.00075 -5.28 0.98 -1.57 0.58 NA NA 0.18 0.41 No -1.1 1.0 NA NA NA NA0.0026 -42.0 0.06 -0.66 0.94 NA NA 28.4 0.009 Yes -37.8 0.32 NA NA NA NAStatistical Test Dunnett Dunnett NA Jonckheere NA Dunnett NA NA 

Abbreviations: Conc. Concentration. Diff. Difference. E2 17 ȕ-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. 

3 p values for solvent control were based on output from equal variance two-sample t-test.LOQ=0.000150 mg a.i./L

Page 119 of 206
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Permethrin

EPA MRID Number 48702301

Page 48Rf49 

Version: 22 September 2011 

Table 24: Growth Endpoints1,2 in the Fish Short-Term Reproduction Assay (FSTRA) with Permethrin.

Treatment 

(mg a.i./L) 

[TWA-measured]

Body Weight Length 

Males Females Males Females 

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

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

Solvent Control3 (<LOQ) -10.7 0.15 4.50 0.31 -1.33 0.51 0.0 1.0 

0.00022 -4.52 0.69 9.20 0.34 3.06 0.32 2.20 0.37 

0.00075 -28.9 0.11 7.08 0.54 2.18 0.57 1.65 0.58 

0.0026 -4.18 0.49 3.07 0.92 0.00 1.00 0.55 0.97 

Statistical Test Mann-Whitney Dunnett Dunnett Dunnett 

Abbreviations: Diff. Difference. NA Not applicable. ND Not determined. 

1

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

control. 

2

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

3 p values for solvent control were based on output from equal variance two-sample t-test.

LOQ=0.000150 mg a.i./L 

E. Study Deficiencies 

Although there were deviations from the guideline, as noted in the Materials and Methods section of 

the DER, the study met all other validity and performance criteria. 

F. Reviewer’s Comments 

The reviewer conducted all statistical analyses by comparing treatment data to the negative control, 

whereas the study author compared treatment data to the pooled control. Therefore, the reviewer’s 

results are reported in the Executive Summary section of this DER. 

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

Page 120 of 206
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Permethrin

EPA MRID Number 48702301

Page 49Rf49 

Version: 22 September 2011 

where: 

C TWA is the time-weighted average concentration, 

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

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

of the test at time interval j 

(e.g., t0 = 0 hours (test initiation), t1 = 24 hours, t2 = 96 hours) 

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

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

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

The in-life portion of the definitive toxicity test was conducted from February 28 to March 20, 2012. 

III. REFERENCES 

Tukey, J.W. 1977. Exploratory Data Analysis. Reading, MA. Addison-Wesley. 

The SAS System for Windows. 1999-2001. Version 8.2. SAS Institute, Inc. Cary, North Carolina. 

       

n

nn

nn

n

n

TWA t

tt

CC

tt

CC

tt

CC

tt

CC

C

1

1

21

21

12

12

01

01

2 2 2 2 





  ̧  1

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©

§   ̧  1·  ̈

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·  ̈

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§ 

 

Page 121 of 206
Report Date: 06 Dec-13 13:02 (p 1 of 24)

Test Code: 109701 48702301 | 04-7869-4595

CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:06

Analysis ID: 08-0652-0584 Endpoint: Fecundity CETIS Version: CETISv1.8.7

Analysis: Parametric-Control vs Treatments Official Results: Yes

Sample ID: 20-8846-4684

Sample Date: 28 Feb-12

Receive Date:

Code: 48702301

Sample Age: NA

Source: Permethrin Data Group II Task Force

Station:

Client: EPA OCSPP EFED

Material: Permethrin Project: Insecticide

Batch ID: 06-6597-1328

Start Date: 28 Feb-12

Ending Date: 20 Mar-12

Test Type: EDSP FSTRA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Well Water

Species: Pimephales promelas Brine: Not Applicable

Source: Osage Catfisheries, Osage Beach, MI

Analyst: J. Housenger

Age: 6mo

flow-through 109701 48702301

109701 48702301, DMF

Batch Note:

Sample Note:

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

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 0.00023 0.6765 2.683 8.626 6 0.8389 CDF Non-Significant Effect

0.00075 0.3266 2.683 8.626 6 0.9759 CDF Non-Significant Effect

0.0026 2.597 2.683 8.626 6 0.0582 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 43.4% 0.0026 >0.0026 NA

ANOVA Table

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

Between 250.0219 83.34062 3 4.031 0.0338 Significant Effect

Error 248.0925 20.67438 12

Total 498.1144 15

Distributional Tests

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

Variances Bartlett Equality of Variance 9.058 11.34 0.0285 Equal Variances

Distribution Shapiro-Wilk W Normality 0.9607 0.8408 0.6742 Normal Distribution

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

Fecundity Summary

0 Negative Control 4 19.88 18.75 21 20 19 20.5 0.3544 3.57% 0.0%

0.00023 4 22.05 16.39 27.71 21.95 17.9 26.4 1.779 16.13% -10.94%

0.00075 4 18.83 12.1 25.55 18.25 14.6 24.2 2.112 22.44% 5.28%

0.0026 4 11.53 0.08414 22.97 10.4 4.3 21 3.595 62.39% 42.01%

C-mg ai/L Control Type

Fecundity Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 20.5 19.6 19 20.4

0.00023 21 17.9 22.9 26.4

0.00075 24.2 14.6 20 16.5

0.0026 21 12.7 4.3 8.1

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

Page 122 of 206
Report Date: 06 Dec-13 13:02 (p 2 of 24)

Test Code: 109701 48702301 | 04-7869-4595

CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:06

Analysis ID: 08-0652-0584 Endpoint: Fecundity CETIS Version: CETISv1.8.7

Analysis: Parametric-Control vs Treatments Official Results: Yes Centered Untransformed

 Rankits

-2

-4

-6

-8

0

2

4

6

8

10

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

Fecundity

 C-mg ai/L

Reject Null

Reject Null

0

5

10

15

20

25

30

0 N 0.00023 0.00075 0.0026

Graphics

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

Page 123 of 206
Report Date: 06 Dec-13 13:02 (p 3 of 24)

Test Code: 109701 48702301 | 04-7869-4595

CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:27

Analysis ID: 19-1665-3394 Endpoint: Fecundity CETIS Version: CETISv1.8.7

Analysis: Nonparametric-Control vs Ord. Treatments Official Results: Yes

Sample ID: 20-8846-4684

Sample Date: 28 Feb-12

Receive Date:

Code: 48702301

Sample Age: NA

Source: Permethrin Data Group II Task Force

Station:

Client: EPA OCSPP EFED

Material: Permethrin Project: Insecticide

Batch ID: 06-6597-1328

Start Date: 28 Feb-12

Ending Date: 20 Mar-12

Test Type: EDSP FSTRA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Well Water

Species: Pimephales promelas Brine: Not Applicable

Source: Osage Catfisheries, Osage Beach, MI

Analyst: J. Housenger

Age: 6mo

flow-through 109701 48702301

109701 48702301, DMF

Batch Note:

Sample Note:

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

Jonckheere-Terpstra Step-Down Test

DF P-Type

Negative Control 0.00023 4 NA -2 0.9000 Exact Non-Significant Effect

0.00075 26 NA -2 0.4156 Exact Non-Significant Effect

0.0026 1.636 1.645 1 -2 0.0509 Asymp Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed NOEL LOEL TOEL TU

Untransformed NA C > T NA NA 0.0026 >0.0026 NA

ANOVA Table

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

Between 250.0219 83.34062 3 4.031 0.0338 Significant Effect

Error 248.0925 20.67438 12

Total 498.1144 15

Distributional Tests

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

Variances Bartlett Equality of Variance 9.058 11.34 0.0285 Equal Variances

Distribution Shapiro-Wilk W Normality 0.9607 0.8408 0.6742 Normal Distribution

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

Fecundity Summary

0 Negative Control 4 19.88 18.75 21 20 19 20.5 0.3544 3.57% 0.0%

0.00023 4 22.05 16.39 27.71 21.95 17.9 26.4 1.779 16.13% -10.94%

0.00075 4 18.83 12.1 25.55 18.25 14.6 24.2 2.112 22.44% 5.28%

0.0026 4 11.53 0.08414 22.97 10.4 4.3 21 3.595 62.39% 42.01%

C-mg ai/L Control Type

Fecundity Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 20.5 19.6 19 20.4

0.00023 21 17.9 22.9 26.4

0.00075 24.2 14.6 20 16.5

0.0026 21 12.7 4.3 8.1

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

Page 124 of 206
Report Date: 06 Dec-13 13:02 (p 4 of 24)

Test Code: 109701 48702301 | 04-7869-4595

CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:27

Analysis ID: 19-1665-3394 Endpoint: Fecundity CETIS Version: CETISv1.8.7

Analysis: Nonparametric-Control vs Ord. Treatments Official Results: Yes Centered Untransformed

 Rankits

-2

-4

-6

-8

0

2

4

6

8

10

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

Fecundity

 C-mg ai/L

0

5

10

15

20

25

30

0 N 0.00023 0.00075 0.0026

Graphics

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

Page 125 of 206
Report Date: 06 Dec-13 13:02 (p 5 of 24)

Test Code: 109701 48702301 | 04-7869-4595

CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:06

Analysis ID: 00-3475-5096 Endpoint: FemaleBodyWt CETIS Version: CETISv1.8.7

Analysis: Parametric-Control vs Treatments Official Results: Yes

Sample ID: 20-8846-4684

Sample Date: 28 Feb-12

Receive Date:

Code: 48702301

Sample Age: NA

Source: Permethrin Data Group II Task Force

Station:

Client: EPA OCSPP EFED

Material: Permethrin Project: Insecticide

Batch ID: 06-6597-1328

Start Date: 28 Feb-12

Ending Date: 20 Mar-12

Test Type: EDSP FSTRA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Well Water

Species: Pimephales promelas Brine: Not Applicable

Source: Osage Catfisheries, Osage Beach, MI

Analyst: J. Housenger

Age: 6mo

flow-through 109701 48702301

109701 48702301, DMF

Batch Note:

Sample Note:

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

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 0.00023 1.506 2.683 0.174 6 0.3427 CDF Non-Significant Effect

0.00075 1.158 2.683 0.174 6 0.5365 CDF Non-Significant Effect

0.0026 0.5019 2.683 0.174 6 0.9227 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 16.4% 0.0026 >0.0026 NA

ANOVA Table

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

Between 0.02272499 0.007574997 3 0.9031 0.4682 Non-Significant Effect

Error 0.10065 0.008387499 12

Total 0.123375 15

Distributional Tests

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

Variances Bartlett Equality of Variance 8.959 11.34 0.0298 Equal Variances

Distribution Shapiro-Wilk W Normality 0.8919 0.8408 0.0598 Normal Distribution

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

FemaleBodyWt Summary

0 Negative Control 4 1.06 0.9594 1.161 1.07 0.98 1.12 0.03162 5.97% 0.0%

0.00023 4 1.157 1.127 1.188 1.165 1.13 1.17 0.009465 1.64% -9.2%

0.00075 4 1.135 1.026 1.244 1.125 1.07 1.22 0.03428 6.04% -7.08%

0.0026 4 1.093 0.8435 1.342 1.155 0.86 1.2 0.07825 14.32% -3.07%

C-mg ai/L Control Type

FemaleBodyWt Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 1.12 0.98 1.04 1.1

0.00023 1.13 1.17 1.16 1.17

0.00075 1.22 1.07 1.09 1.16

0.0026 1.16 1.15 0.86 1.2

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

Page 126 of 206
Report Date: 06 Dec-13 13:02 (p 6 of 24)

Test Code: 109701 48702301 | 04-7869-4595

CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:06

Analysis ID: 00-3475-5096 Endpoint: FemaleBodyWt CETIS Version: CETISv1.8.7

Analysis: Parametric-Control vs Treatments Official Results: Yes Centered Untransformed

 Rankits

-0.05

-0.10

-0.15

-0.20

-0.25

0.00

0.05

0.10

0.15

0.20

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

FemaleBodyWt

 C-mg ai/L

Reject Null

Reject Null

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 N 0.00023 0.00075 0.0026

Graphics

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

Page 127 of 206
Report Date: 06 Dec-13 13:02 (p 7 of 24)

Test Code: 109701 48702301 | 04-7869-4595

CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:28

Analysis ID: 00-8481-6560 Endpoint: FemaleGSI CETIS Version: CETISv1.8.7

Analysis: Nonparametric-Control vs Ord. Treatments Official Results: Yes

Sample ID: 20-8846-4684

Sample Date: 28 Feb-12

Receive Date:

Code: 48702301

Sample Age: NA

Source: Permethrin Data Group II Task Force

Station:

Client: EPA OCSPP EFED

Material: Permethrin Project: Insecticide

Batch ID: 06-6597-1328

Start Date: 28 Feb-12

Ending Date: 20 Mar-12

Test Type: EDSP FSTRA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Well Water

Species: Pimephales promelas Brine: Not Applicable

Source: Osage Catfisheries, Osage Beach, MI

Analyst: J. Housenger

Age: 6mo

flow-through 109701 48702301

109701 48702301, DMF

Batch Note:

Sample Note:

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

Jonckheere-Terpstra Step-Down Test

DF P-Type

Negative Control 0.00023 -0.1459 1.645 1 -2 0.5580 Asymp Non-Significant Effect

0.00075 0.2202 1.645 1 -2 0.4129 Asymp Non-Significant Effect

0.0026* 2.384 1.645 1 -2 0.0086 Asymp Significant Effect

Data Transform Zeta Alt Hyp Trials Seed NOEL LOEL TOEL TU

Untransformed NA C < T NA NA 0.00075 0.0026 0.001396

ANOVA Table

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

Between 48.035 16.01167 3 4.628 0.0226 Significant Effect

Error 41.515 3.459583 12

Total 89.55 15

Distributional Tests

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

Variances Bartlett Equality of Variance 2.12 11.34 0.5478 Equal Variances

Distribution Shapiro-Wilk W Normality 0.9249 0.8408 0.2025 Normal Distribution

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

FemaleGSI Summary

0 Negative Control 4 14 12.06 15.94 14.3 12.4 15 0.6096 8.71% 0.0%

0.00023 4 13.9 11.86 15.94 14 12.4 15.2 0.6416 9.23% 0.71%

0.00075 4 14.03 10.93 17.12 14.25 11.8 15.8 0.9716 13.85% -0.18%

0.0026 4 17.97 13.79 22.16 16.85 16.3 21.9 1.316 14.64% -28.39%

C-mg ai/L Control Type

FemaleGSI Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 14.9 13.7 15 12.4

0.00023 14.7 13.3 12.4 15.2

0.00075 15.5 13 11.8 15.8

0.0026 17 16.7 21.9 16.3

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

Page 128 of 206
Report Date: 06 Dec-13 13:02 (p 8 of 24)

Test Code: 109701 48702301 | 04-7869-4595

CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:28

Analysis ID: 00-8481-6560 Endpoint: FemaleGSI CETIS Version: CETISv1.8.7

Analysis: Nonparametric-Control vs Ord. Treatments Official Results: Yes Centered Untransformed

 Rankits

-0.5

-1.0

-1.5

-2.0

-2.5

-3.0

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

FemaleGSI

 C-mg ai/L

0

5

10

15

20

25

0 N 0.00023 0.00075 0.0026

Graphics

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

Page 129 of 206
Report Date: 06 Dec-13 13:02 (p 9 of 24)

Test Code: 109701 48702301 | 04-7869-4595

CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:06

Analysis ID: 00-2496-0096 Endpoint: FemaleLength CETIS Version: CETISv1.8.7

Analysis: Parametric-Control vs Treatments Official Results: Yes

Sample ID: 20-8846-4684

Sample Date: 28 Feb-12

Receive Date:

Code: 48702301

Sample Age: NA

Source: Permethrin Data Group II Task Force

Station:

Client: EPA OCSPP EFED

Material: Permethrin Project: Insecticide

Batch ID: 06-6597-1328

Start Date: 28 Feb-12

Ending Date: 20 Mar-12

Test Type: EDSP FSTRA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Well Water

Species: Pimephales promelas Brine: Not Applicable

Source: Osage Catfisheries, Osage Beach, MI

Analyst: J. Housenger

Age: 6mo

flow-through 109701 48702301

109701 48702301, DMF

Batch Note:

Sample Note:

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

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 0.00023 1.445 2.683 1.857 6 0.3729 CDF Non-Significant Effect

0.00075 1.083 2.683 1.857 6 0.5838 CDF Non-Significant Effect

0.0026 0.3612 2.683 1.857 6 0.9681 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 4.08% 0.0026 >0.0026 NA

ANOVA Table

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

Between 2.5 0.8333333 3 0.8696 0.4837 Non-Significant Effect

Error 11.5 0.9583333 12

Total 14 15

Distributional Tests

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

Variances Bartlett Equality of Variance 3.961 11.34 0.2657 Equal Variances

Distribution Shapiro-Wilk W Normality 0.9155 0.8408 0.1430 Normal Distribution

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

FemaleLength Summary

0 Negative Control 4 45.5 43.91 47.09 45 45 47 0.5 2.2% 0.0%

0.00023 4 46.5 45.58 47.42 46.5 46 47 0.2887 1.24% -2.2%

0.00075 4 46.25 45.45 47.05 46 46 47 0.25 1.08% -1.65%

0.0026 4 45.75 43.36 48.14 46 44 47 0.75 3.28% -0.55%

C-mg ai/L Control Type

FemaleLength Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 45 45 45 47

0.00023 46 47 47 46

0.00075 47 46 46 46

0.0026 47 47 44 45

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

Page 130 of 206
Report Date: 06 Dec-13 13:02 (p 10 of 24)

Test Code: 109701 48702301 | 04-7869-4595

CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:06

Analysis ID: 00-2496-0096 Endpoint: FemaleLength CETIS Version: CETISv1.8.7

Analysis: Parametric-Control vs Treatments Official Results: Yes Centered Untransformed

 Rankits

-0.2

-0.4

-0.6

-0.8

-1.0

-1.2

-1.4

-1.6

-1.8

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

FemaleLength

 C-mg ai/L

Reject Null

Reject Null

0

10

20

30

40

50

0 N 0.00023 0.00075 0.0026

Graphics

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

Page 131 of 206
Report Date: 06 Dec-13 13:02 (p 11 of 24)

Test Code: 109701 48702301 | 04-7869-4595

CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:06

Analysis ID: 09-3678-4332 Endpoint: FemaleVTG CETIS Version: CETISv1.8.7

Analysis: Parametric-Control vs Treatments Official Results: Yes

Sample ID: 20-8846-4684

Sample Date: 28 Feb-12

Receive Date:

Code: 48702301

Sample Age: NA

Source: Permethrin Data Group II Task Force

Station:

Client: EPA OCSPP EFED

Material: Permethrin Project: Insecticide

Batch ID: 06-6597-1328

Start Date: 28 Feb-12

Ending Date: 20 Mar-12

Test Type: EDSP FSTRA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Well Water

Species: Pimephales promelas Brine: Not Applicable

Source: Osage Catfisheries, Osage Beach, MI

Analyst: J. Housenger

Age: 6mo

flow-through 109701 48702301

109701 48702301, DMF

Batch Note:

Sample Note:

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

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 0.00023 0.7389 2.724 1009 6 0.8074 CDF Non-Significant Effect

0.00075 0.04926 2.724 1009 6 0.9999 CDF Non-Significant Effect

0.0026 1.569 2.724 1090 5 0.3203 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 65.6% 0.0026 >0.0026 NA

ANOVA Table

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

Between 863048 287682.7 3 1.048 0.4099 Non-Significant Effect

Error 3019971 274542.8 11

Total 3883019 14

Distributional Tests

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

Variances Bartlett Equality of Variance 0.5151 11.34 0.9156 Equal Variances

Distribution Shapiro-Wilk W Normality 0.9759 0.8328 0.9334 Normal Distribution

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

FemaleVTG Summary

0 Negative Control 4 1661 843.1 2479 1845 905 2050 257.1 30.95% 0.0%

0.00023 4 1388 692.2 2083 1250 1030 2020 218.5 31.49% 16.48%

0.00075 4 1643 876.9 2409 1720 992 2140 240.7 29.3% 1.1%

0.0026 3 1033 -686.5 2753 809 481 1810 399.7 67.0% 37.8%

C-mg ai/L Control Type

FemaleVTG Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 1880 1810 905 2050

0.00023 2020 1310 1030 1190

0.00075 2140 1800 992 1640

0.0026 1810 809 481

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

Page 132 of 206
Report Date: 06 Dec-13 13:02 (p 12 of 24)

Test Code: 109701 48702301 | 04-7869-4595

CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:06

Analysis ID: 09-3678-4332 Endpoint: FemaleVTG CETIS Version: CETISv1.8.7

Analysis: Parametric-Control vs Treatments Official Results: Yes Centered Untransformed

 Rankits

-200

-400

-600

-800

0

200

400

600

800

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

FemaleVTG

 C-mg ai/L

Reject Null

Reject Null

0

500

1000

1500

2000

2500

3000

0 N 0.00023 0.00075 0.0026

Graphics

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

Page 133 of 206
Report Date: 06 Dec-13 13:02 (p 13 of 24)

Test Code: 109701 48702301 | 04-7869-4595

CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:06

Analysis ID: 13-7395-8017 Endpoint: Fertility CETIS Version: CETISv1.8.7

Analysis: Parametric-Control vs Treatments Official Results: Yes

Sample ID: 20-8846-4684

Sample Date: 28 Feb-12

Receive Date:

Code: 48702301

Sample Age: NA

Source: Permethrin Data Group II Task Force

Station:

Client: EPA OCSPP EFED

Material: Permethrin Project: Insecticide

Batch ID: 06-6597-1328

Start Date: 28 Feb-12

Ending Date: 20 Mar-12

Test Type: EDSP FSTRA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Well Water

Species: Pimephales promelas Brine: Not Applicable

Source: Osage Catfisheries, Osage Beach, MI

Analyst: J. Housenger

Age: 6mo

flow-through 109701 48702301

109701 48702301, DMF

Batch Note:

Sample Note:

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

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 0.00023 1.53 2.683 3.683 6 0.3311 CDF Non-Significant Effect

0.00075 1.093 2.683 3.683 6 0.5778 CDF Non-Significant Effect

0.0026 0.4553 2.683 3.683 6 0.9401 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 3.86% 0.0026 >0.0026 NA

ANOVA Table

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

Between 28.20183 9.400609 3 2.495 0.1096 Non-Significant Effect

Error 45.21749 3.768125 12

Total 73.41932 15

Distributional Tests

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

Variances Bartlett Equality of Variance 5.59 11.34 0.1334 Equal Variances

Distribution Shapiro-Wilk W Normality 0.9468 0.8408 0.4400 Normal Distribution

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

Fertility Summary

0 Negative Control 4 95.35 92.97 97.73 95.15 93.8 97.3 0.7467 1.57% 0.0%

0.00023 4 97.45 96.46 98.44 97.4 96.8 98.2 0.3122 0.64% -2.2%

0.00075 4 93.85 91.14 96.56 94.65 91.3 94.8 0.853 1.82% 1.57%

0.0026 4 94.73 89.81 99.64 95.5 90.5 97.4 1.545 3.26% 0.66%

C-mg ai/L Control Type

Fertility Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 95.6 97.3 94.7 93.8

0.00023 98.2 97.7 96.8 97.1

0.00075 94.5 91.3 94.8 94.8

0.0026 96.6 94.4 90.5 97.4

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

Page 134 of 206
Report Date: 06 Dec-13 13:02 (p 14 of 24)

Test Code: 109701 48702301 | 04-7869-4595

CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:06

Analysis ID: 13-7395-8017 Endpoint: Fertility CETIS Version: CETISv1.8.7

Analysis: Parametric-Control vs Treatments Official Results: Yes Centered Untransformed

 Rankits

-1

-2

-3

-4

-5

0

1

2

3

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

Fertility

 C-mg ai/L

Reject Null

Reject Null

0

20

40

60

80

100

0 N 0.00023 0.00075 0.0026

Graphics

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

Page 135 of 206
Report Date: 06 Dec-13 13:02 (p 15 of 24)

Test Code: 109701 48702301 | 04-7869-4595

CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:06

Analysis ID: 06-2395-2162 Endpoint: MaleBodyWt CETIS Version: CETISv1.8.7

Analysis: Nonparametric-Two Sample Official Results: Yes

Sample ID: 20-8846-4684

Sample Date: 28 Feb-12

Receive Date:

Code: 48702301

Sample Age: NA

Source: Permethrin Data Group II Task Force

Station:

Client: EPA OCSPP EFED

Material: Permethrin Project: Insecticide

Batch ID: 06-6597-1328

Start Date: 28 Feb-12

Ending Date: 20 Mar-12

Test Type: EDSP FSTRA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Well Water

Species: Pimephales promelas Brine: Not Applicable

Source: Osage Catfisheries, Osage Beach, MI

Analyst: J. Housenger

Age: 6mo

flow-through 109701 48702301

109701 48702301, DMF

Batch Note:

Sample Note:

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

Mann-Whitney U Two-Sample Test

DF P-Type

Negative Control 0.00023 10 NA 0 6 0.6857 Exact Non-Significant Effect

0.00075 14 NA 0 6 0.1143 Exact Non-Significant Effect

0.0026 11 NA 0 6 0.4857 Exact Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 35.7% 0.0026 >0.0026 NA

ANOVA Table

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

Between 1.862675 0.6208917 3 1.633 0.2338 Non-Significant Effect

Error 4.5623 0.3801917 12

Total 6.424975 15

Distributional Tests

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

Variances Bartlett Equality of Variance 13.4 11.34 0.0038 Unequal Variances

Distribution Shapiro-Wilk W Normality 0.8057 0.8408 0.0032 Non-normal Distribution

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

MaleBodyWt Summary

0 Negative Control 4 2.99 2.568 3.412 3.03 2.65 3.25 0.1325 8.86% 0.0%

0.00023 4 2.855 2.352 3.358 2.87 2.46 3.22 0.158 11.07% 4.52%

0.00075 4 2.125 0.2851 3.965 2.655 0.4 2.79 0.5781 54.41% 28.93%

0.0026 4 2.865 2.678 3.052 2.84 2.76 3.02 0.05867 4.1% 4.18%

C-mg ai/L Control Type

MaleBodyWt Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 3.14 3.25 2.65 2.92

0.00023 2.94 2.46 2.8 3.22

0.00075 2.78 2.79 0.4 2.53

0.0026 2.79 2.76 3.02 2.89

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

Page 136 of 206
Report Date: 06 Dec-13 13:02 (p 16 of 24)

Test Code: 109701 48702301 | 04-7869-4595

CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:06

Analysis ID: 06-2395-2162 Endpoint: MaleBodyWt CETIS Version: CETISv1.8.7

Analysis: Nonparametric-Two Sample Official Results: Yes Centered Untransformed

 Rankits

-0.2

-0.4

-0.6

-0.8

-1.0

-1.2

-1.4

-1.6

-1.8

0.0

0.2

0.4

0.6

0.8

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

MaleBodyWt

 C-mg ai/L

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0 N 0.00023 0.00075 0.0026

Graphics

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

Page 137 of 206
Report Date: 06 Dec-13 13:02 (p 17 of 24)

Test Code: 109701 48702301 | 04-7869-4595

CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:28

Analysis ID: 02-8831-9340 Endpoint: MaleGSI CETIS Version: CETISv1.8.7

Analysis: Nonparametric-Control vs Ord. Treatments Official Results: Yes

Sample ID: 20-8846-4684

Sample Date: 28 Feb-12

Receive Date:

Code: 48702301

Sample Age: NA

Source: Permethrin Data Group II Task Force

Station:

Client: EPA OCSPP EFED

Material: Permethrin Project: Insecticide

Batch ID: 06-6597-1328

Start Date: 28 Feb-12

Ending Date: 20 Mar-12

Test Type: EDSP FSTRA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Well Water

Species: Pimephales promelas Brine: Not Applicable

Source: Osage Catfisheries, Osage Beach, MI

Analyst: J. Housenger

Age: 6mo

flow-through 109701 48702301

109701 48702301, DMF

Batch Note:

Sample Note:

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

Jonckheere-Terpstra Step-Down Test

DF P-Type

Negative Control 0.00023 9 NA -2 0.4429 Exact Non-Significant Effect

0.00075 31 NA -2 0.1756 Exact Non-Significant Effect

0.0026* 75 NA -2 0.0056 Exact Significant Effect

Data Transform Zeta Alt Hyp Trials Seed NOEL LOEL TOEL TU

Untransformed NA C < T NA NA 0.00075 0.0026 0.001396

ANOVA Table

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

Between 1.518619 0.5062062 3 4.898 0.0190 Significant Effect

Error 1.240225 0.1033521 12

Total 2.758844 15

Distributional Tests

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

Variances Bartlett Equality of Variance 0.5057 11.34 0.9176 Equal Variances

Distribution Shapiro-Wilk W Normality 0.9048 0.8408 0.0959 Normal Distribution

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

MaleGSI Summary

0 Negative Control 4 1.56 1.094 2.026 1.595 1.18 1.87 0.1465 18.78% 0.0%

0.00023 4 1.588 1.04 2.135 1.645 1.12 1.94 0.1721 21.68% -1.76%

0.00075 4 1.738 1.336 2.139 1.8 1.4 1.95 0.1262 14.53% -11.38%

0.0026 4 2.322 1.716 2.929 2.43 1.79 2.64 0.1907 16.42% -48.88%

C-mg ai/L Control Type

MaleGSI Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 1.51 1.18 1.87 1.68

0.00023 1.94 1.59 1.12 1.7

0.00075 1.95 1.91 1.4 1.69

0.0026 1.79 2.31 2.55 2.64

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

Page 138 of 206
Report Date: 06 Dec-13 13:02 (p 18 of 24)

Test Code: 109701 48702301 | 04-7869-4595

CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:28

Analysis ID: 02-8831-9340 Endpoint: MaleGSI CETIS Version: CETISv1.8.7

Analysis: Nonparametric-Control vs Ord. Treatments Official Results: Yes Centered Untransformed

 Rankits

-0.1

-0.2

-0.3

-0.4

-0.5

-0.6

0.0

0.1

0.2

0.3

0.4

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

MaleGSI

 C-mg ai/L

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 N 0.00023 0.00075 0.0026

Graphics

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

Page 139 of 206
Report Date: 06 Dec-13 13:02 (p 19 of 24)

Test Code: 109701 48702301 | 04-7869-4595

CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:06

Analysis ID: 11-1502-5161 Endpoint: MaleLength CETIS Version: CETISv1.8.7

Analysis: Parametric-Control vs Treatments Official Results: Yes

Sample ID: 20-8846-4684

Sample Date: 28 Feb-12

Receive Date:

Code: 48702301

Sample Age: NA

Source: Permethrin Data Group II Task Force

Station:

Client: EPA OCSPP EFED

Material: Permethrin Project: Insecticide

Batch ID: 06-6597-1328

Start Date: 28 Feb-12

Ending Date: 20 Mar-12

Test Type: EDSP FSTRA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Well Water

Species: Pimephales promelas Brine: Not Applicable

Source: Osage Catfisheries, Osage Beach, MI

Analyst: J. Housenger

Age: 6mo

flow-through 109701 48702301

109701 48702301, DMF

Batch Note:

Sample Note:

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

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 0.00023 1.552 2.683 3.024 6 0.3207 CDF Non-Significant Effect

0.00075 1.109 2.683 3.024 6 0.5676 CDF Non-Significant Effect

0.0026 0 2.683 3.024 6 1.0000 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 5.28% 0.0026 >0.0026 NA

ANOVA Table

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

Between 9.5 3.166667 3 1.246 0.3364 Non-Significant Effect

Error 30.5 2.541667 12

Total 40 15

Distributional Tests

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

Variances Bartlett Equality of Variance 1.635 11.34 0.6514 Equal Variances

Distribution Shapiro-Wilk W Normality 0.969 0.8408 0.8227 Normal Distribution

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

MaleLength Summary

0 Negative Control 4 57.25 55.25 59.25 57 56 59 0.6292 2.2% 0.0%

0.00023 4 59 56.09 61.91 59 57 61 0.9129 3.09% -3.06%

0.00075 4 58.5 56.91 60.09 59 57 59 0.5 1.71% -2.18%

0.0026 4 57.25 53.97 60.53 57 55 60 1.031 3.6% 0.0%

C-mg ai/L Control Type

MaleLength Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 59 57 56 57

0.00023 61 58 57 60

0.00075 59 59 57 59

0.0026 55 57 60 57

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

Page 140 of 206
Report Date: 06 Dec-13 13:02 (p 20 of 24)

Test Code: 109701 48702301 | 04-7869-4595

CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:06

Analysis ID: 11-1502-5161 Endpoint: MaleLength CETIS Version: CETISv1.8.7

Analysis: Parametric-Control vs Treatments Official Results: Yes Centered Untransformed

 Rankits

-0.5

-1.0

-1.5

-2.0

-2.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

MaleLength

 C-mg ai/L

Reject Null

Reject Null

0

10

20

30

40

50

60

70

0 N 0.00023 0.00075 0.0026

Graphics

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

Page 141 of 206
Report Date: 06 Dec-13 13:02 (p 21 of 24)

Test Code: 109701 48702301 | 04-7869-4595

CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:28

Analysis ID: 20-1835-4249 Endpoint: MaleMedianTubercleScore CETIS Version: CETISv1.8.7

Analysis: Nonparametric-Control vs Ord. Treatments Official Results: Yes

Sample ID: 20-8846-4684

Sample Date: 28 Feb-12

Receive Date:

Code: 48702301

Sample Age: NA

Source: Permethrin Data Group II Task Force

Station:

Client: EPA OCSPP EFED

Material: Permethrin Project: Insecticide

Batch ID: 06-6597-1328

Start Date: 28 Feb-12

Ending Date: 20 Mar-12

Test Type: EDSP FSTRA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Well Water

Species: Pimephales promelas Brine: Not Applicable

Source: Osage Catfisheries, Osage Beach, MI

Analyst: J. Housenger

Age: 6mo

flow-through 109701 48702301

109701 48702301, DMF

Batch Note:

Sample Note:

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

Jonckheere-Terpstra Step-Down Test

DF P-Type

Negative Control 0.00023 12 NA -2 0.1714 Exact Non-Significant Effect

0.00075 32 NA -2 0.1404 Exact Non-Significant Effect

0.0026* 2.293 1.645 2 -2 0.0109 Asymp Significant Effect

Data Transform Zeta Alt Hyp Trials Seed NOEL LOEL TOEL TU

Untransformed NA C > T NA NA 0.00075 0.0026 0.001396

ANOVA Table

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

Between 487.5469 162.5156 3 3.357 0.0553 Non-Significant Effect

Error 580.9375 48.41146 12

Total 1068.484 15

Distributional Tests

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

Variances Bartlett Equality of Variance 1.281 11.34 0.7338 Equal Variances

Distribution Shapiro-Wilk W Normality 0.8786 0.8408 0.0369 Normal Distribution

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

MaleMedianTubercleScore Summary

0 Negative Control 4 34.25 26.83 41.67 34.5 28.5 39.5 2.332 13.62% 0.0%

0.00023 4 29.75 20.17 39.33 28.5 24.5 37.5 3.01 20.24% 13.14%

0.00075 4 29.88 18.62 41.13 28 23.5 40 3.538 23.68% 12.77%

0.0026 4 19.25 4.529 33.97 15.5 13 33 4.626 48.06% 43.8%

C-mg ai/L Control Type

MaleMedianTubercleScore Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 33 36 28.5 39.5

0.00023 37.5 24.5 25.5 31.5

0.00075 40 28 28 23.5

0.0026 13 15 33 16

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

Page 142 of 206
Report Date: 06 Dec-13 13:02 (p 22 of 24)

Test Code: 109701 48702301 | 04-7869-4595

CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 29 Oct-13 14:28

Analysis ID: 20-1835-4249 Endpoint: MaleMedianTubercleScore CETIS Version: CETISv1.8.7

Analysis: Nonparametric-Control vs Ord. Treatments Official Results: Yes Centered Untransformed

 Rankits

-2

-4

-6

-8

0

2

4

6

8

10

12

14

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

MaleMedianTubercleScore

 C-mg ai/L

0

5

10

15

20

25

30

35

40

0 N 0.00023 0.00075 0.0026

Graphics

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

Page 143 of 206
Report Date: 06 Dec-13 13:02 (p 23 of 24)

Test Code: 109701 48702301 | 04-7869-4595

CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 30 Oct-13 10:10

Analysis ID: 09-7815-4018 Endpoint: MaleVTG CETIS Version: CETISv1.8.7

Analysis: Nonparametric-Two Sample Official Results: Yes

Sample ID: 20-8846-4684

Sample Date: 28 Feb-12

Receive Date:

Code: 48702301

Sample Age: NA

Source: Permethrin Data Group II Task Force

Station:

Client: EPA OCSPP EFED

Material: Permethrin Project: Insecticide

Batch ID: 06-6597-1328

Start Date: 28 Feb-12

Ending Date: 20 Mar-12

Test Type: EDSP FSTRA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Well Water

Species: Pimephales promelas Brine: Not Applicable

Source: Osage Catfisheries, Osage Beach, MI

Analyst: J. Housenger

Age: 6mo

flow-through 109701 48702301

109701 48702301, DMF

Batch Note:

Sample Note:

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

Mann-Whitney U Two-Sample Test

DF P-Type

Negative Control 0.00023 11 NA 0 6 0.4857 Exact Non-Significant Effect

0.00075 12 NA 0 6 0.3429 Exact Non-Significant Effect

0.0026 9.5 NA 1 6 0.7429 Exact Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 261.0% 0.0026 >0.0026 NA

ANOVA Table

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

Between 0.08488669 0.02829556 3 0.2115 0.8865 Non-Significant Effect

Error 1.605654 0.1338045 12

Total 1.69054 15

Distributional Tests

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

Variances Bartlett Equality of Variance 5.261 11.34 0.1537 Equal Variances

Distribution Shapiro-Wilk W Normality 0.831 0.8408 0.0072 Non-normal Distribution

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

MaleVTG Summary

0 Negative Control 4 0.2425 -0.02468 0.5097 0.195 0.099 0.481 0.08395 69.24% 0.0%

0.00023 4 0.3805 -0.5781 1.339 0.1005 0.038 1.283 0.3012 158.3% -56.91%

0.00075 4 0.1805 -0.1432 0.5042 0.085 0.067 0.485 0.1017 112.7% 25.57%

0.0026 4 0.2493 -0.2608 0.7593 0.137 0.015 0.708 0.1603 128.6% -2.78%

C-mg ai/L Control Type

MaleVTG Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 0.481 0.099 0.159 0.231

0.00023 0.038 0.107 0.094 1.283

0.00075 0.485 0.098 0.067 0.072

0.0026 0.708 0.231 0.043 0.015

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

Page 144 of 206
Report Date: 06 Dec-13 13:02 (p 24 of 24)

Test Code: 109701 48702301 | 04-7869-4595

CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Wildlife International

Analyzed: 30 Oct-13 10:10

Analysis ID: 09-7815-4018 Endpoint: MaleVTG CETIS Version: CETISv1.8.7

Analysis: Nonparametric-Two Sample Official Results: Yes Centered Untransformed

 Rankits

-0.2

-0.4

-0.6

0.0

0.2

0.4

0.6

0.8

1.0

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

MaleVTG

 C-mg ai/L

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 N 0.00023 0.00075 0.0026

Graphics

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

Page 145 of 206
DATA EVALUATION RECORD

PERMETHRIN

Study Type: OCSPP 890.1400, In vivo Hershberger Assay

EPA Contract No. EP10H001452 

Task Assignment No. 2-29-2012 (MRID 48618602) 

Prepared for

Health Effects Division

Office of Pesticide Programs

U.S. Environmental Protection Agency 

2777 South Crystal Drive 

Arlington, VA 22202 

Prepared by

CSS-Dynamac Corporation

1910 Sedwick Road, 

Building 100, Suite B 

Durham, NC 27713 

Primary Reviewer: Signature:

Rebecca L. Bryan, B.S. Date: 3/23/2012

Secondary Reviewer: Signature:

David A. McEwen, B.S. Date: 3/29/2012

Program Manager: Signature:

Jack D. Early, M.S. Date: 4/10/2012

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 4/10/2012

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

signing by CSS-Dynamac Corporation personnel. 

Page 146 of 206
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 147 of 206
PERMETHRIN/ 109701 

Primary Reviewer: Edward Scallon. Ph.D. 

Health Effects Division 

Secondary Reviewer: Greg Akerman, Ph.D. 

Health Effects Division 

In vivo Hershberger Assay (2011)/ Page I of 11 

Si"~S!~CSPP890.1~400/£ 

Date: ZC Signature: __ ._...._ ..... ,_,,_,,,_ __ 

Date: ~h.r /, ).-

I DATA EVALUATION RECORD 

Template version 1012011 

STUDY TYPE: In Vivo Hershberger Assay (Rat); OCSPP 890.1400; OECD 441 

PC CODE: 109701 DP BARCO DE: D398301 

TXR#: 0056246 

TEST MATERIAL (PURITY): Permethrin (97.7% a.i.) 

SYNONYMS: (3-phenoxyphenyl)methyl 3-(2,2-dichJoroethenyl)-2,2-

dimethylcyclopropanecarboxylate 

CAS#: 52645-53-1 

CITATION: Zorrilla, L. (2011 ). The Hershberger Bioassay for Permethrin. Integrated 

Laboratory Systems, Inc., Durham, NC. Laboratory Project Study ID: Cl 95-

200, November 15, 2011. MRID 48618602. Unpublished. 

SPONSOR: Consumer Specialty Products Association, Inc. for the Permethrin Data Group 

II, 1667 K Street NW, Suite 300, Washington, DC 20006 

TEST ORDER#: CON-109701-7 

EXECUTIVE SUMM.ARY: In a Hershberger assay (MRID 48618602) screening for 

androgenic activity, permethrin (97.7% a.i., Lot# PMPS000014) in com oil was administered 

daily via oral gavage to groups of 59 to 60-day old, castrated male Sprague Dawley rats 

(8/group) at dose levels of 0 (vehicle), 37.5, or 120 mg/kg/day. An androgenic positive control 

group consisting of eight castrated rats exposed to 0.4 mg/kg/day testosterone propionate {TP) by 

subcutaneous (s.c.) injection was also included as a positive control. 

To screen for potential anti-androgenic activity, permethrin in corn oil was also administered 

daily via oral gavage to 59 to 60-day old, castrated male Sprague Dawley rats (8/group) at dose 

levels of 12, 37.5, 120 mg/kg/day in conjunction with a daily dose of reference androgen TP at 

0.4 mg/kg/day by s.c. injection. The control group consisted of eight castrated male rats dosed 

daily with TP (0.4 mg/kg/day) by s.c. injection in corn oil, and the anti-androgenic positive 

control group consisted of eight castrated male rats dosed orally with flutamide (FT) in com oil 

at 3 mg/kg/day in conj unction with a daily s.c. dose ofTP (0.4 mg/kg/day). 

For both components of the assay, the animals were dosed for 10 consecutive days and 

necropsied approximately 24 hours after the final dose administration to determine weights of 

the five androgen-dependent tissues. 

All animals survived until scheduled termination. No clinical signs of toxicity were observed in 

the permethrin dose groups of the androgen agonist or antagonist assays. Body weights and 

Page 148 of 206
In vivo Hershberger Assay (2011)/ Page 2 of 11

PERMETHRIN/ 109701 OCSPP 890.1400/ OECD 441

overall body weight gains in the treated groups were similar to controls in both the androgen 

agonist and anti-androgen assays. 

In the androgen agonist assay, animals dosed with TP had increased (p<0.05) body weights 

(↑12%) on Day 11, and increased (p<0.05) overall body weight gains (↑110%) compared to 

controls. No significant increases in the organ weights were observed in any of the five 

accessory sex tissues of animals dosed with permethrin. Animals in the TP group had increases 

(p≤0.05) in accessory sex organ weights as follows: 1002% in seminal vesicles; 898% in ventral 

prostate; 187% in levator ani-bulbocavernosus (LABC); 486% in Cowper’s gland; and 51% in 

glans penis. For the vehicle control, the maximum guideline-specified %CV was exceeded for 

the LABC (45% vs. 30%). All other %CV values were less than the maximum permissible 

values.

In the anti-androgen assay positive control group (TP + FT), overall body weight gains were 

decreased (p≤0.05) by 35%. No significant decreases in the organ weights were observed in any 

of the five accessory sex tissues of animals dosed with permethrin+TP. Animals dosed with TP 

+ FT (positive control) had decreased (p≤0.05) accessory sex organ weights as follows: 84% in 

seminal vesicles; 77% in ventral prostate; 60% in LABC; 62% in Cowper’s gland; and 20% in 

glans penis. The maximum guideline-specified %CV was exceeded for the Cowper’s gland in 

the 120 mg/kg/day permethrin group (22% vs. 20%). All other %CV values were less than the 

maximum permissible values.

The dosing for this study (high-dose of 120 mg/kg/day) was considered adequate based on data 

from two earlier studies showing either mortality at doses ≥200 mg/kg/day or clinical signs of 

neurotoxicity at 150 mg/kg/day. 

Statistically significant changes were not seen in two or more of the five androgen sensitive 

tissue weights. Permethrin was negative for androgenicity and anti-androgenicity in the 

Hershberger assay. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Hershberger assay (OCSPP 

890.1400). 

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

provided. 

Page 149 of 206
In vivo Hershberger Assay (2011)/ Page 3 of 11

PERMETHRIN/ 109701 OCSPP 890.1400/ OECD 441

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Integrated Laboratory Systems, Inc.

Location: Durham, NC

Study Director: L. Zorilla

Other Personnel: S. Borghoff (Study Toxicologist)

Study Period: July 25, 2011 to November 15, 2011

2. Test Substance: Permethrin

Description: Liquid

Source: AMVAC Chemical Corporation

Lot/Batch #: PMPS000014, expires January 2013

Purity: 97.7% a.i.

Stability: Stable in corn oil for up to 20 days at 1-10°C

CAS #: 52645-53-1

Structure:

O

O

O

Cl

3CH

Cl

3CH

3. Reference Androgen: Testosterone propionate (TP)

Supplier: Sigma-Aldrich Company (St. Louis, MO)

Batch #: 048K1328, expires March 17, 2012

Purity: 100%

CAS #: 57-85-2

4. Reference Anti-androgen: Flutamide (FT)

Supplier: Sigma-Aldrich Company (St. Louis, MO)

Lot #: 107K1293, expires February 15, 2012

Purity: 99.5%

CAS #: 13311-84-7

5. Solvent/Vehicle Control: Corn Oil 

Supplier: MP Biomedicals, LLC (Solon, OH)

Lot/Batch #: 7862K

Rationale (if other than water): Vehicle used in ILS range-finding gavage study.

Final concentration: Not applicable

6. Test Animals:

Species: Rat (castrated males)

Strain: Sprague Dawley (Crl:CD[SD] IGS)

Age/weight at dose initiation: Post-natal day (PND) 59/60; 239.1-323.9 g 

Source: Charles River Laboratories International, Inc., Raleigh, NC

Housing: Rats were housed up to 2/cage polycarbonate cages with absorbent heattreated hardwood bedding (Northeastern Products Corp., Warrensburg, NY).

Diet: Teklad Global 16% Protein Rodent Diet (Teklad Diets, Madison, WI), ad 

libitum; low phytoestrogen with total genistein equivalents = 8.0 μg/g diet.

Water: Tap water, ad libitum

Environmental conditions: Temperature:

Humidity:

Air changes:

Photoperiod:

22-24oC

33-58% 

Not reported

12 hrs light/12 hrs dark

Acclimation period: 7 days

Page 150 of 206
In vivo Hershberger Assay (2011)/ Page 4 of 11

PERMETHRIN/ 109701 OCSPP 890.1400/ OECD 441

B. STUDY DESIGN

1. In-life dates: Start: 8/1/11 End: 8/12/11 

2. Study Design: This Hershberger Assay was conducted to screen for both potential 

androgenic and anti-androgenic activity. In the test for androgenic activity, permethrin was 

administered daily via oral gavage to groups of castrated male rats. Positive androgenic 

activity is defined as a significant increase in two or more target organ weights compared to 

the vehicle control group. The positive control group for androgenic activity consisted of 

castrated male rats dosed by s.c. injection with TP at 0.4 mg/kg/day. In the test for antiandrogenic activity, permethrin was administered daily via oral gavage to groups of 

castrated male rats in conjunction with TP at 0.4 mg/kg/day by s.c. injection. 

Anti-androgenic activity is indicated by a statistically significant decrease in two or more 

target organ weights of the treated groups (test substance + TP) compared to the TP-only 

control group. The positive control group for anti-androgenic activity consisted of castrated 

male rats dosed by s.c. injection with TP (0.4 mg/kg/day) in conjunction with an oral dose 

of FT (3.0 mg/kg/day). For both assays, the animals were dosed for 10 consecutive days 

and necropsied approximately 24 hours after the final dose administration for measurement 

of target organ weights. 

3. Study Schedule: Following the initial 7-day acclimation period, castrated male rats 

(surgical manipulation performed by Supplier at PND 45) were administered the test 

substance from PND 59-60 to PND 68-69. Rats were euthanized on PND 69-70, 

approximately 24 hours after the last dose and necropsied for organ weight measurements. 

4. Animal Assignment: Animals were randomly assigned, stratified by body weight, to the 

test groups noted in Table 1. Statistical analysis indicated that there were no significant 

differences in group means at study initiation.

TABLE 1. Study design a

Test group Dose (mg/kg/day) # of Males

Androgen Agonist Assay

Vehicle control (corn oil) 0 8

Low (Permethrin) 37.5 8

High (Permethrin) 120 8

Positive control (corn oil) +TP b 0.4 8

Anti-Androgen Assay

Vehicle control (corn oil) +TP b 0 8

Low (Permethrin+TP) 12 8

Mid (Permethrin+TP) 37.5 8

High (Permethrin+TP) 120 8

Flutamide (oral gavage) + TP (s.c.), positive control 3 8

a Data were obtained from Tables 1 and 2 on page 15 of the study report.

b Same animals dosed for both androgen agonist (serves as positive control) and anti-androgen (served as vehicle control) 

assays. This did not affect the outcome or conclusions of this study.

5. Dose Selection Rationale: The permethrin dose levels were selected based on the results of 

a range-finding study (ILS Inc., Study No. C195-500) and a previous Hershberger study 

Page 151 of 206
In vivo Hershberger Assay (2011)/ Page 5 of 11

PERMETHRIN/ 109701 OCSPP 890.1400/ OECD 441

(Kunimatsu, T., 2002, Regul Toxicol Pharmacol). In the range-finding study, male rats 

were administered permethrin for 14 days at concentrations of 100, 200, 300, and 400 

mg/kg/day (four rats/group). All rats at 300 and 400 mg/kg/day died during testing, and 

only one 200 mg/kg/day rat survived to study termination. No effects were observed on 

body weights, liver or kidney weights of surviving rats. In the previous Hershberger study, 

male rats were administered permethrin for 5 days at concentrations of 150 and 300 

mg/kg/day. At 150 mg/kg/day, clinical signs included tremors and hypersensitivity. Based 

on these results, doses of ≤120 mg/kg/day were used for both the androgen agonist and antiandrogen assays. 

6. (a) Dose Preparation: Dose formulations were prepared once prior to initiation of 

treatment by mixing the appropriate amounts of test substance in corn oil. Flutamide and 

TP were prepared once prior to dosing by mixing in corn oil. Homogeneity (top, middle, 

and bottom samples) and concentration of permethrin were determined for all dose 

formulations. 

In a previous storage stability study1, permethrin (lot number not reported) was determined 

to be stable in corn oil for up to 20 days at 1-10°C. 

(b) Dose Analysis

Results

Homogeneity (%CV): 0.853-2.55%

Concentration (% of nominal): 95-106% 

The analytical data indicated that the mixing procedure was adequate and that the variation 

between nominal and actual dosage to the animals was acceptable. 

7. Dosage administration: Test formulations of permethrin were administered to the animals 

daily via oral gavage (5 mL/kg bw) for 10 days. TP was given via s.c. injection at 

0.5 mL/kg bw, and FT was administered via oral gavage at 5 mL/kg bw. Dose volumes 

were adjusted daily based on the concurrent body weight measurement. 

8. Statistics: Body weights, body weight gains and accessory sex organ weights were 

analyzed using studentized residual plots for outliers and Levene’s test for homogeneity of 

variance. Heterogeneous data were transformed by logarithm (glans penis) or multiplicative 

inverse (LABC weights) and re-analyzed. Body weights, body weight gains, and accessory 

sex organ weights were analyzed using one-way ANOVA followed by Dunnett’s t-tests. 

The positive controls were compared to the vehicle controls using the t-test. Significance 

was denoted at p<0.05. Statistical analyses were performed using MS EXCEL and SAS®

Version 9.2. The statistical analyses were considered adequate.

 

1 Smith, R. 2011a. Storage Stability of Permethrin in Corn Oil. Unpublished study report prepared by Smithers 

Viscient Inc., Study No. 14010-6105. 

Page 152 of 206
In vivo Hershberger Assay (2011)/ Page 6 of 11

PERMETHRIN/ 109701 OCSPP 890.1400/ OECD 441

C. METHODS

1. Clinical Examinations: Animals were checked for mortality or moribundity twice daily on 

weekdays and once daily on weekends. Rats were also checked for clinical signs of toxicity 

at randomization, daily prior to dosing throughout the dosing period, and at termination. 

2. Body Weight: Animals were weighed at randomization, daily prior to dosing throughout 

the dosing period, and at termination. 

3. Food Consumption (Optional): Food consumption was not determined. 

4. Serum Hormone Measurements (Optional): Serum hormone measurements were not 

conducted. 

5. Dissection and Measurement of Tissue and Organ Weights: On PND 69-70 

(approximately 24 hours after the final administration of the test substance), all rats were 

weighed, anesthetized by CO2 inhalation followed by cervical dislocation and subjected to a 

gross necropsy. The five mandatory androgen-dependent organs (ventral prostate, seminal 

vesicles with coagulating glands and fluid, LABC, Cowper’s gland, and glans penis) were 

excised, trimmed free of adhering tissue, and weighed fresh (unfixed) according to the 

standard operating procedures detailed in the U.S. EPA Guideline (OCSPP 890.1400). 

Page 153 of 206
In vivo Hershberger Assay (2011)/ Page 7 of 11

PERMETHRIN/ 109701 OCSPP 890.1400/ OECD 441

II. RESULTS

A. OBSERVATIONS

1. Mortality: All animals survived until scheduled termination. 

2. Clinical signs of toxicity: No clinical signs of toxicity were observed in the permethrin 

dose groups of the androgen agonist or antagonist assays. 

B. BODY WEIGHT AND WEIGHT GAIN: Selected body weight and body weight gains 

for the androgen agonist assay are presented in Table 2. Body weights and overall body 

weight gains for the 37.5 and 120 mg/kg/day treatment groups were comparable to controls 

throughout the duration of the assay. Animals dosed with TP had increased (p<0.05) body 

weights (↑12%) on Day 11, and increased (p<0.05) overall body weight gains (↑110%)

compared to controls. 

TABLE 2. Selected Group Mean (±SD) Body Weights and Cumulative Body Weight Gains (g) in the 

Androgen Agonist Assay a

Study Day #

Dose (mg/kg/day)

Vehicle Control Positive Control

Vehicle + TP (0.4) b Permethrin (37.5) Permethrin (120) 

N Mean SD N Mean SD N Mean SD N Mean SD 

1 8 283.1 27.9 8 276.5 25.4 8 286.4 22.7 8 287.6 25.4

4 8 298.4 29.1 8 312.0 31.2 8 303.1 23.8 8 305.3 27.5

7 8 311.2 31.9 8 336.5 33.3 8 317.5 19.9 8 319.9 29.0

11 8 324.8 34.0 8 364.1*

(↑12)

36.3 8 332.0 20.9 8 338.4 33.3

BWG

(Days 1-11)

8 41.7 11.3 8 87.7*

(↑110)

35.2 8 45.6 6.2 8 50.8 14.1

a Data were obtained from Table 5 on page 21 and Appendix V on page 78 of the study report. Percent differences from 

controls were calculated by the reviewers and included in parentheses.

b The positive control group for the androgen agonist is same as the vehicle control for the anti-androgen. This did not affect 

the outcome or conclusions of this study.

N Number of animals in the group

SD Standard Deviation

* Significantly different from controls at p≤0.05

Body weight and body weight gain data for the anti-androgen assay are presented in Table 

3. Body weights and overall body weight gains for permethrin-treated groups were 

comparable to controls throughout the duration of the assay. For the positive control group 

(TP + FT), overall body weight gains were decreased (p≤0.05) by 35%.

Page 154 of 206
In vivo Hershberger Assay (2011)/ Page 8 of 11

PERMETHRIN/ 109701 OCSPP 890.1400/ OECD 441

TABLE 3. Selected Group Mean (±SD) Body Weights and Cumulative Body Weight Gains (g) in the AntiAndrogen Assay a

Study 

Day #

Dose (mg/kg/day)

Vehicle Control

+ TP (0.4) b

Positive Control

TP + FT 

(0.4 + 3.0)

Permethrin+TP 

(12 + 0.4)

Permethrin +TP 

(37.5 + 0.4)

Permethrin +TP 

(120 + 0.4)

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 

1 8 276.5 25.4 8 285.9 25.6 8 279.7 23.2 8 284.9 22.4 8 284.4 24.9

4 8 312.0 31.2 8 304.5 25.9 8 304.3 23.1 8 309.1 23.8 8 306.5 25.7

7 8 336.5 33.3 8 322.2 25.9 8 326.1 25.5 8 332.8 26.9 8 327.2 29.0

11 8 364.1 36.3 8 343.0 26.1 8 348.9 27.4 8 358.1 28.7 8 354.5 28.2

BWG

(days 1-11)

8 87.7 35.2 8 57.2*

(↓35)

4.9 8 69.3 21.4 8 73.2 10.2 8 70.1 4.9

a Data were obtained from Table 6 on page 22 and Appendix V on pages 78 and 79 of the study report. Percent differences 

from vehicle controls (calculated by the reviewers) are included in parentheses.

b The vehicle control for the anti-androgen is same as the positive control group for the androgen agonist. This did not affect 

the outcome or conclusions of this study.

N Number of animals in the group

SD Standard Deviation

* Significantly different from vehicle controls at p<0.05.

C. FOOD CONSUMPTION (Optional): Food consumption was not determined. 

D. SERUM HORMONE CONCENTRATIONS (Optional): Serum hormone concentrations 

were not determined.

E. ORGAN WEIGHTS: Accessory sex organ weights for the androgen agonist assay are 

presented in Table 4. There were no treatment-related effects in any of the accessory sex 

organs of animals dosed with permethrin. Animals in the TP group had increases (p≤0.05) 

in accessory sex organ weights as follows: 1002% in seminal vesicles; 898% in ventral 

prostate; 187% in LABC; 486% in Cowper’s gland; and 51% in glans penis. In the vehicle 

control group, the maximum guideline-specified %CV (30%) was exceeded for the LABC

(35%). All other %CV values were less than the maximum permissible values.

Page 155 of 206
In vivo Hershberger Assay (2011)/ Page 9 of 11

PERMETHRIN/ 109701 OCSPP 890.1400/ OECD 441

TABLE 4. Accessory Sex Organ Weights (mg) from Androgen Agonist Assay in Sprague-Dawley Rats a

Organ

Dose (mg/kg/day)

Vehicle Control Permethrin (37.5) Permethrin (120) Positive Control

Vehicle + TP (0.4) b

N Mean SD CV N Mean SD CV N Mean SD CV N Mean SD CV

Seminal 

vesicles

8 56.0 20.4 36 8 45.6 13.5 30 8 46.7 11.1 24 8 616.9*

(↑1002)

190.2 31

Ventral 

prostate

8 14.9 6.6 44 8 12.3 3.4 28 8 14.0 5.6 40 8 148.7*

(↑898)

39.6 27

LABC 8 211.5 75.0 35c 8 165.4 32.0 19 8 190.4 30.6 16 8 606.9*

(↑187) 

90.8 15

Cowper’s 

glands

8 8.4 3.4 41 8 7.6 3.9 51 8 8.0 2.5 31 8 49.2*

(↑486)

8.5 17

Glans penis 8 63.0 13.6 22 8 64.4 6.1 9 8 65.3 5.7 9 8 95.0*

(↑51) 

13.3 14

a Data were obtained from Table 7 on page 24 of the study report. Percent differences from controls were calculated by the 

reviewers and included in parentheses.

b The positive control group for the androgen agonist is same as the vehicle control for the anti-androgen. This did not affect 

the outcome or conclusions of this study.

c The %CV exceeds the maximum acceptable %CV.

N Number of animals in the group

SD Standard Deviation

CV Coefficient of Variation

* Significantly different from vehicle controls at p<0.05.

Accessory sex organ weights for the anti-androgen assay are presented in Table 5. There 

were no treatment-related effects in any of the accessory sex organs of animals dosed with 

permethrin+TP. Animals dosed with TP + FT (positive control) had decreased (p≤0.05) 

accessory sex organ weights as follows: 84% in seminal vesicles; 77% in ventral prostate; 

60% in LABC; 62% in Cowper’s gland; and 20% in glans penis. The maximum guidelinespecified %CV (20%) was exceeded for the LABC in the 120 mg/kg/day permethrin group 

(22%). All other %CV values were less than the maximum permissible values.

Page 156 of 206
In vivo Hershberger Assay (2011)/ Page 10 of 11

PERMETHRIN/ 109701 OCSPP 890.1400/ OECD 441

TABLE 5. Accessory Sex Organ Weights (mg) from Anti-Androgen Agonist Assay in Sprague-Dawley Rats a

Organ

Dose (mg/kg/day)

Vehicle Control

+TP (0.4) b 

Permethrin+TP

(12 + 0.4)

Permethrin +TP

(37.5 + 0.4)

Permethrin +TP

(120 + 0.4)

TP + FT

Positive Control

N Mean SD CV N Mean SD CV N Mean SD CV N Mean SD CV N Mean SD CV

Seminal 

vesicles

8 616.9 190.2 31 8 669.8 172.1 26 8 614.7 188.5 31 8 638.9 125.7 20 8 100.2*

(↓84)

40.3 40

Ventral 

prostate

8 148.7 39.6 27 8 154.8 41.7 27 8 173.7 40.6 23 8 180.3 37.6 21 8 34.7*

(↓77)

10.3 30

LABC

8 606.9 90.8 15 8 570.9 76.8 13 8 556.7 62.0 11 8 532.2 119.5 22c 8 240.0*

(↓60) 

80.7 34

Cowper’s 

glands

8 49.2 8.5 17 8 45.5 6.6 15 8 42.8 7.5 17 8 47.1 13.4 29 8 18.6*

(↓62)

7.7 41

Glans 

penis

8 95.0 13.3 14 8 98.0 11.9 12 8 90.9 14.2 16 8 95.5 15.1 16 8 76.3*

(↓20)

10.7 14

a Data were obtained from Tables 8 on page 25 of the study report. Percent differences from controls were calculated by the reviewers 

and included in parentheses.

b The vehicle control for the anti-androgen is same as the positive control group for the androgen agonist. This did not affect the 

outcome or conclusions of this study.

c The %CV exceeds the maximum acceptable %CV.

N Number of animals in the group

SD Standard Deviation

CV Coefficient of Variation

* Significantly different from vehicle controls at p<0.05.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: In the agonist assay, dose levels of 37.5 or 120 

mg/kg/day permethrin did not increase androgen-dependent tissue weights compared to the 

weights of these tissues in the vehicle control animals. In the antagonist assay, permethrin 

co-administered with TP at dose levels of 12, 37.5, or 120 mg/kg/day did not decrease 

androgen dependent tissue weights. Based on these findings using the Hershberger Assay 

(OCSPP 890.1400), oral administration of permethrin at up to 120 mg/kg/day did not show 

any androgen agonist/antagonist activity, or 5α-reductase inhibition. 

B. AGENCY COMMENTS: All animals survived until scheduled termination. No clinical 

signs of toxicity were observed in the permethrin dose groups of the androgen agonist or 

antagonist assays.

Although no toxicity was observed in the current study at doses of permethrin up to 120 

mg/kg/day, the high dose is adequate based on data from two earlier studies showing either 

mortality at doses ≥200 mg/kg/day or clinical signs of neurotoxicity at 150 mg/kg/day. 

 In the androgen agonist assay, body weights and overall body weight gains for the 37.5 and 

120 mg/kg/day treatment groups were comparable to controls throughout the duration of the 

assay. No significant increases in the organ weights were observed in any of the five 

accessory sex tissues of animals dosed with permethrin. Animals dosed with TP had 

increased (p<0.05) body weights (↑12%) on Day 11, and increased (p<0.05) overall body 

weight gains (↑110%) compared to controls. Animals in the TP group showed the expected 

increases (p≤0.05) in accessory sex organ weights. For the vehicle control, the maximum 

guideline-specified %CV was exceeded for the LABC (35% vs. 30%). All other %CV 

values were less than the maximum permissible values.

Page 157 of 206
In vivo Hershberger Assay (2011)/ Page 11 of 11

PERMETHRIN/ 109701 OCSPP 890.1400/ OECD 441

In the anti-androgen assay, body weights and overall body weight gains for permethrintreated groups were comparable to controls throughout the duration of the assay. No 

significant decreases in the organ weights were observed in any of the five accessory sex 

tissues of animals dosed with permethrin+TP. For the positive control group (TP + FT), 

overall body weight gains were decreased (p≤0.05) by 35%. Animals dosed with TP + FT 

(positive control) showed the expected decreases in accessory sex organ weights. The 

maximum guideline-specified %CV was exceeded for the LABC in the 120 mg/kg/day 

permethrin group (22% vs. 20%). All other %CV values were less than the maximum 

permissible values.

No statistically significant changes were seen in two or more of the five androgen sensitive 

tissues. Permethrin was negative for androgenicity and anti-androgenicity in the 

Hershberger assay. 

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

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

• In the androgen agonist assay, the maximum guideline-specified %CV was exceeded 

for the LABC in the vehicle control (35% vs. 30%). 

• The maximum guideline-specified %CV was exceeded for the LABC in the 120 

mg/kg/day permethrin group (22% vs. 20%) in the anti-androgen assay. 

Page 158 of 206
DATA EVALUATION RECORD

PERMETHRIN

Study Type: OCSPP 890.1450, Female Pubertal Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-75-2012 (MRID 48672901) 

Prepared for

Health Effects Division

Office of Pesticide Programs

U.S. Environmental Protection Agency 

2777 South Crystal Drive 

Arlington, VA 22202 

Prepared by

CSS-Dynamac Corporation

1910 Sedwick Road, 

Building 100, Suite B 

Durham, NC 27713 

Primary Reviewer: Signature:

Michelle Sharpe-Kass, M.S. Date: 7/10/2012

Secondary Reviewer: Signature:

David A. McEwen, B.S. Date: 7/16/2012

Program Manager: Signature:

Jack D. Early, M.S. Date: 7/18/2012

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 7/18/2012

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

signing by CSS-Dynamac Corporation personnel. 

Page 159 of 206
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 206
PERMETHRIN / 109701 

Primary Reviewer: Edward Scollon, Ph.D. 

Health Effects Division 

Secondary Reviewer: Elizabeth Mendez, Ph.D. 

Health Effects Division 

Female Pubertal Assay (2012) I Page I of 14 

OCSPP 890.1450/ OECD None 

Template version 08/2011 

DATA EVALUATION RECORD 

STUDY TYPE: Female Pubertal Assay; OCSPP 890.1450; OECD None. 

PC CODE: 109701 DP BARCO DE: D401793 

TXR#: 0056246 CAS No: 52645-:53-1 

TEST MATERIAL (PURITY): Permethrin (97.7% a.i.) 

SYNONYMS: 3-Phenoxybenzyl (1 RS)-cis,trans-3-(2,2-dichlorovinyl)-2,2-

dimethylcyclopropanecarboxylate 

CITATION: Zorilla, L. (2012) Pubertal Development and Thyroid Function in Intact 

Juvenile/Peripubertal Female Rats; Permethrin. Integrated Laboratory Systems, 

Inc., Durham NC. Laboratory Report No.: Cl 95-300, April 13, 2012. MRID 

48672901. Unpublished. 

SPONSOR: Consumer Specialty Products Association for the Permethrin Data Group 

II/Joint Venture. 1667 K Street NW, Suite 300, Washington, DC 

TEST ORDER#: CON-109701-7 

EXECUTIVE SUMMARY: ln a Female Pubertal Assay (MRID48672901), 16 SpragueDawley (Crl:CD® (SD] IGS) female rats/dose group were treated daily via oral gavage with 

permethrin (Lot# PMPS00014, 97.7% a.i.)at doses ofO (com oil) 75, or 150 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 age and weight at attainment of VO were recorded. Following 

sacrifice on PND 42/43, blood was collected for clinical chemistry analyses, including analysis 

of total thyroxine (T4) and thyroid stimulating hormone (TSH) using a radioimmunoassay (RIA). 

Adrenal, liver, thyroid, pituitary and urogenital organ weights were recorded, and microscopic 

examinations were performed on the ovaries, uterus, thyroid, and kidneys. 

All rats administered permethrin survived until schedule termination. From PND 34 onward, 

tremors were observed in 4 animals administered 75 mg/kg/day and all animals administered 150 

mg/kg/day at 4-6 hours post-dosing on at least one day. These findings did not persist to the 

daily examinations, which were conducted just prior to dosing. 

No treatment-related effects were observed on body weights, body weight gains, age of 

attainment of VO, or weight at day of attainment at any dose. All surviving animals attained 

complete VO. 

Page 161 of 206
Female Pubertal Assay (2012) / Page 2 of 14

PERMETHRIN / 109701 OCSPP 890.1450/ OECD None

Absolute liver weights (adjusted and unadjusted) were increased (p<0.05) by 10% at 150 

mg/kg/day. Relative (to body weight) liver weights were increased (p<0.05) by 4 and 8% at 75 

and 150 mg/kg/day. Both unadjusted and adjusted thyroid weights were decreased (p<0.05) by 

22% at 150 mg/kg/day. 

Administration of permethrin did not change the day of first estrus compared to the vehicle 

control group. Animals exhibited similar cycle lengths and regularity. The majority of females 

in the 150 mg/kg/day group were in diestrus at necropsy. 

Serum T4 levels were decreased (p<0.05) by 26 and 42% at 75 and 150 mg/kg/day, respectively, 

and, serum TSH levels were increased (p<0.05) by 40% at 150 mg/kg/day. Additionally, at 150 

mg/kg/day, increases (p<0.05) were observed in sodium (↑3%), chloride (↑4%), and sorbitol 

dehydrogenase (SDH; ↑20%), alkaline phosphatase (ALP) was decreased (↓16%). 

There were no effects of treatment in the thyroid on either colloid area or follicular cell height in 

any treated group. No effects of treatment were observed microscopically in the ovaries, uterus 

or kidney. 

The dose levels tested were determined to be adequate since they were based on the results of the 

range finding study where mortality was seen at doses greater than 200 mg/kg/day and tremors at 

150 mg/kg/day. 

 

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 206
Female Pubertal Assay (2012) / Page 3 of 14

PERMETHRIN / 109701 OCSPP 890.1450/ OECD None

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Integrated Laboratory Services, Inc.

Location: Durham, NC

Study Director: Leah Zorilla

Other Personnel: Thomas G. Osimits - Study Monitor

Study Period: June 30, 2011 - April 13, 2012

2. Test Substance: Permethrin

Description: Yellow Liquid

Source: AMVAC Corporation

Lot/Batch #: PMPS00014 (exp January 2013)

Purity: 97.7%

Stability: It was stated that dose formulations of permethrin in corn oil held from 1 to 10°C for up to 20 

days were considered stable.

CAS #: 52645-53-1

Structure:

O

O

O

Cl

3CH

Cl

3CH

3. Vehicle: Corn Oil

4. Test Animals:

Species: Rat

Strain: Sprague Dawley CRL: CD®(SD) IGS

Age/Weight at 

Study Initiation:

PND 22/44.5-69.3 g females only

Source: Charles River Laboratories International, Inc. (Raleigh, NC)

Housing: 2/cage in polycarbonate cages with micro-isolator tops and absorbent, heat-treated hardwood 

bedding.

Diet: Teklad Global 16% protein rodent diet (while at ILS) and Autoclaved Purina 5L9 Rat and 

Mouse Diet (while at Charles River), ad libitum. Total genistein equivalents of genistein plus 

daidzein were 8.0 and 8.6 μg/g feed. 

Water: Reverse-osmosis filtered tap water, ad libitum

Environmental 

Conditions:

Temperature:

Humidity:

Air changes:

Photoperiod:

19-26 °C

36-67% 

Not reported

14 hrs light/ 10 hrs dark 

B. STUDY DESIGN

1. In-Life Dates: Start: August 5, 2011 End: August 26, 2011 

2. Mating: Time-mated pregnant dams were received from the supplier on Gestation Day 8. 

Dams with their litters were kept together until weaning (PND 21). Litters with more than 8 

pups were standardized between PND 3 and 5 to 8 pups per litter with equal numbers of 

males and females. 

Page 163 of 206
Female Pubertal Assay (2012) / Page 4 of 14

PERMETHRIN / 109701 OCSPP 890.1450/ OECD None

3. Animal Assignment: Animals were assigned to the test groups noted in Table 1 using a 

procedure that stratifies animals across groups by body weight such that mean body weight 

of each croup was not statistically different from any other group using ANOVA. 

Littermates were not assigned to the same treatment group. 

TABLE 1. Study Design a

Test group Dose (mg/kg/day) # of Females

Control 0 16

Low 75 16

High 150 16

a Data were obtained from page 14 of the study report.

4. Dose Selection Rationale: The dose levels were selected based on the results from a dose 

range finding study1. Four immature female rats per group were administered either corn oil 

or permethrin at dose levels of 50, 100, 150, 200, or 250 mg/kg for 14 days. Tremors and 

seizures were noted within 1-4 hours post-dosing in all animals on Day 1 and in all 

surviving animals on Day 2. All animals administered 200 and 250 mg/kg permethrin did 

not survive past Day 2. Beginning on Day 4, mild tremors were noted in 3 of 4 animals 

administered 150 mg/kg permethrin. All rats administered 50-150 mg/kg permethrin were 

observed as normal 24-hours post-dose for 14 days. No statistically significant group mean 

body weight differences or differences in body weight gain were observed as compared to 

control. 

Based on this study and the guideline requirements of dosing immature rats, a dose of 150 

mg/kg/day was selected for the female pubertal assay based on deaths, moribundity or 

adverse clinical signs observed at up to 24-hours post-dose at ≥200 mg/kg/day. 

5. Dose Preparation and Analysis: Dose formulations were prepared daily by mixing 

appropriate amounts of test substance with corn oil. Homogeneity (top, middle, and bottom) 

and achieved concentrations were tested for all dose formulations by an off-site analytical 

laboratory (Smithers Viscient, LLC, Aareham, MA). It was stated that permethrin was 

previously demonstrated to be stable in corn oil when stored refrigerated (1-10 °C) for up to 

10 days (Smithers Viscient Study No. 1410-6105; no data provided).2

 

Results of Dose Analysis 

Homogeneity (% CV): 0.297-1.38% 

Concentration (% of nominal): 96-107% 

Stability (% of initial): No data provided 

 

1 . Zorilla, L. (2011) Range Finder Study for In Vitro Mammalian Assays for Permethrin. (Draft). Unpublished 

study report prepared by ILS, Inc. Study No. C195-500

2 . Smith, R (2011) Storage Stability of Permethrin in Corn Oil. Unpublished study report prepared by Smithers 

Viscient Inc. Study No. 1410-6105

Page 164 of 206
Female Pubertal Assay (2012) / Page 5 of 14

PERMETHRIN / 109701 OCSPP 890.1450/ OECD None

The analytical data indicated that the mixing procedure was adequate and that the variation 

between nominal and actual dosage to the animals was acceptable. The storage stability 

data (Smithers Viscient Study No. 1410-6105) should be submitted for independent review.

6. Dosage Administration: All doses were administered once daily by gavage, from PND 22 

through PND 42/43, in a volume of 5 mL/kg of body weight. Dosing was performed 24 ± 2 

hours from the previous dose. Dose volume was adjusted daily based on individual body 

weights.

7. Statistics: The following statistical analyses were performed. Statistical significance was 

reported when p<0.05. The analyses were considered appropriate.

Parameter Statistical test

Initial body weight

Final body weight

Final body weight gain

Age and body weight at VO

Age at first estrus

Cycle length

Ovarian counts (follicles, corpora lutea)

Homogeneous data sets were analyzed using a oneway analysis of variance (ANOVA), followed by 

pairwise comparisons using Dunnett’s two-tailed t 

tests.

Tissue weights

Relative tissue weights (liver, kidney, pituitary, adrenal)

Hormone levels

Clinical chemistry

Homogeneous data sets were analyzed using a twoway ANOVA with treatment and necropsy day as 

the main effects, followed by pairwise comparisons 

using Dunnett’s two-tailed t tests.

Initial body weight

Final body weight

Final body weight gain

Age and body weight at VO

Tissue weights

Homogeneous data sets were analyzed using a twoway analysis of covariance (ANCOVA) with PND 

21 body weight as the covariable, followed by 

pairwise comparisons using Dunnett’s two-tailed t 

tests.

Heterogeneous data sets Chi-square analysis

Thyroid scoring Fisher’s exact test

C. METHODS

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

weekdays, once daily on weekends/holidays for mortality and moribundity. Clinical 

examinations were conducted daily prior to dosing throughout the study. Beginning on 

PND 34, an additional examination was also performed at 4-6 hours post-dosing. 

2. Body Weight: Body weights were recorded prior to allocation of the animals to study 

groups, daily prior to dosing, and prior to scheduled termination. Overall (PND 22 to 42/43) 

body weight gain was also calculated.

3. Vaginal Opening: Beginning on PND 22, all animals were examined daily for onset of 

VO. Age and weight on the day of completion of VO were recorded.

4. Estrous Cyclicity: Beginning on the day of complete VO through to euthanasia, vaginal 

smears were prepared daily and evaluated. Animals were classified as diestrus, proestrus, or 

estrus; metestrus was classified as diestrus. The overall cycling pattern for each female was 

characterized as regular, irregular, or not cycling. Regular cycling was defined as having 

recurring 4 to 5 day cycles, while irregular cycling was defined as having cycles with 

Page 165 of 206
Female Pubertal Assay (2012) / Page 6 of 14

PERMETHRIN / 109701 OCSPP 890.1450/ OECD None

diestrus for a period >3 days or estrus ≥ 3 days. Animals were not considered to be cycling 

if there were ≥3 consecutive days of estrus or ≥5 consecutive days of diestrus. Mean cycle 

length was defined as the number of days from one diestrus to the next diestrus. Incomplete 

cycles were not used in calculating mean cycle length. In instances where the time between 

VO and humane euthanasia did not allow observation of more than 1 complete cycle, 

classification was based on data available and the assumption that animals were cycling 

regularly if the partial data fit the definition, and cycling irregularly if the study ends 

without being able to distinguish between irregular and not cycling. 

5. Sacrifice and Pathology: On the day of termination, animals were moved to the necropsy 

holding room approximately 2 hours prior to sacrifice. All surviving animals were 

sacrificed by decapitation on PND 42/43 approximately 2 hours post-dosing. Blood from 

the trunk of the animals was collected immediately into serum separation tubes and stored 

on wet ice until processing. Samples were centrifuged at 3000 g for 30 min at 4°C and 

stored at ≤−70°C for subsequent hormone and clinical chemistry evaluations. 

a. Hormone Analysis: Total T4 and TSH levels were determined using radioimmunoassays 

by a commercial analytical laboratory (AniLytics, Gaithersburg, MD). Samples were 

assayed in duplicate with multiple quality control samples.

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

ELECTROLYTES OTHER

X Calcium X Albumin

X Chloride X Creatinine*

Magnesium X Urea nitrogen*

X Phosphorus Total cholesterol

X Potassium Globulins

X Sodium Glucose

ENZYMES X Total bilirubin

X Alkaline phosphatase (ALP) X Total protein

Cholinesterase (ChE) Triglycerides

Creatine phosphokinase Serum protein electrophoresis

Lactic acid dehydrogenase (LDH)

X Alanine aminotransferase (ALT/also SGPT)

X Aspartate aminotransferase (AST/also SGOT)

X Sorbitol dehydrogenase (SDH)

X Gamma glutamyl transferase (GGT)

Glutamate dehydrogenase

* Recommended for the pubertal assay in female rats based on guideline 890.1450.

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*

Page 166 of 206
Female Pubertal Assay (2012) / Page 7 of 14

PERMETHRIN / 109701 OCSPP 890.1450/ OECD None

* Weights required based on guideline 890.1450

+ Histopathological examination required based on guideline 890.1450

The uterus, ovaries, and kidneys were weighed prior to fixation. The thyroid (with 

parathyroid) was collected with the trachea and fixed in 10% neutral buffered formalin for at 

least 24 hours. Following fixation, the thyroid was dissected free of the trachea and 

weighed. The uterus (without fluid), left ovary, and left kidney were fixed in 10% neutral 

buffered formalin for at least 24 hours. Following fixation, the fixed tissues were 

transferred to 70% histology grade alcohol, routinely processed into paraffin blocks, 

sectioned, stained with hematoxylin and eosin, and examined microscopically. Five random 

sections of the ovary were evaluated microscopically for follicular development (including 

the presence or absence of tertiary/antral follicles, presence or absence of corpora lutea, 

changes in corpus luteum development, and changes in the number of both primary and 

atretic follicles) in addition to any abnormalities or lesions. Two sections of each of the two 

lobes of the thyroid were examined and evaluated for follicular cell height and colloid area, 

using a five point grading scale (1 = shortest/smallest; 5 = tallest/largest), and any 

abnormalities/lesions noted.

II. RESULTS 

A. MORTALITY: All rats administered permethrin survived until scheduled termination. 

One animal administered vehicle control was moribund and euthanized on PND 34 due to 

clinical signs (piloerection, lethargy, pale appearance, labored breathing) and weight loss. 

These findings were attributed to gavage error.

B. CLINICAL SIGNS OF TOXICITY: From PND 34 onward, tremors were observed in 4 

animals administered 75 mg/kg/day and all animals administered 150 mg/kg/day at 4-6 

hours post-dosing on at least one day. These findings did not persist to the daily 

examinations conducted just prior to dosing on the next day. 

C. GENERAL GROWTH AND VAGINAL OPENING: No treatment-related effects were 

observed on body weights, body weight gains, age of attainment of VO, or weight at day of 

attainment at any dose (Table 2). All surviving animals attained complete VO. All control 

group performance criteria for means and CVs were within the acceptable ranges.

Page 167 of 206
Female Pubertal Assay (2012) / Page 8 of 14

PERMETHRIN / 109701 OCSPP 890.1450/ OECD None

TABLE 2. General Growth and Vaginal Opening (VO)a

Parameter Evaluated

Vehicle Control Permethrin

(75 mg/kg/day)

Permethrin

(150 mg/kg/day)

N Mean SD

CV 

(%) N Mean SD

CV 

(%) N Mean SD

CV 

(%)

Initial body weight

(PND 22; g)

U 15 56.5 4.3 7.7 16 55.9 5.5 9.9 16 56.3 5.4 9.6

A 15 56.2 ― ― 16 56.2 ― ― 16 56.2 ― ―

Body weight at vaginal 

opening (g)

U 15 124.7 7.4 5.9 16 125.9 10.9 8.6 16 120.4 14.3 11.9

A 15 124.5 ― ― 16 126.2 ― ― 16 120.4 ― ―

Final body weight

(g)

U 15 169.8 12.7 7.5 16 171.4 10.2 5.9 16 171.7 12.8 7.4

A 15 169.3 ― ― 16 171.9 ― ― 16 171.6 ― ―

Final body weight 

(% of control)

U 15 NA 16 100.9 ― ― 16 101.1 ― ―

A 15 16 101.5 ― ― 16 101.4 ― ―

Body weight gain

(final – initial; g)

U 16 113.1 10.5 9.3 16 115.5 9.0 7.8 16 115.4 10.1 8.8

A 16 113.0 ― ― 16 115.6 ― ― 16 115.3 ― ―

Age at vaginal opening 

(PND)

U 15 33.9 1.8 5.2 16 34.0 1.3 3.9 16 33.2 1.2 3.5

A 15 33.9 ― ― 16 34.0 ― ― 16 33.2 ― ―

Proportion unopened (#/N) 0/15 0/16 0/16

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

U Unadjusted for body weight on PND 22

A Adjusted for body weight on PND 22

N Number of animals examined

SD Standard Deviation

CV Coefficient of Variation

NA Not applicable

D. ORGAN WEIGHTS: Organ weights at necropsy are presented in Table 3. Absolute liver 

weights (adjusted and unadjusted) were increased (p<0.05) by 10% at 150 mg/kg/day. 

Relative (to body weight) liver weights were increased (p<0.05) by 4 and 8% at 75 and 150 

mg/kg/day, respectively. Both unadjusted and adjusted thyroid weights were decreased 

(p<0.05) by 22% each at 150 mg/kg/day. 

 All control group performance criteria for absolute weights and CVs were within the 

acceptable ranges.

Page 168 of 206
Female Pubertal Assay (2012) / Page 9 of 14

PERMETHRIN / 109701 OCSPP 890.1450/ OECD None

TABLE 3. Organ Weights at Necropsya

Organ

Vehicle Control Permethrin

(75 mg/kg/day)

Permethrin

(150 mg/kg/day)

N Mean SD

CV 

(%) N Mean SD

CV 

(%) N Mean SD

CV 

(%)

Liver (g) 

U 15 7.64 0.70 9.1 16 8.11 0.70 8.6 16 8.39* 

(↑10) 0.73 8.7

A ― 7.62 ― ― ― 8.14 ― ― ― 8.39* 

(↑10) ― ―

R 15 4.49 0.21 4.6 16 4.69* 

(↑4) 0.25 5.2 16 4.84* (↑8) 0.25 5.1 

Kidneys (g) 

U 15 1.30 0.11 8.7 16 1.32 0.08 5.9 16 1.32 0.12 9.2

A ― 1.30 ― ― ― 1.32 ― ― ― 0.32 ― ―

R 15 0.77 0.05 6.0 16 0.76 0.05 6.1 16 0.76 0.05 7.1

Pituitary (mg)

U 15 9.7 1.1 11.3 16 9.4 0.8 8.7 16 9.9 1.8 17.8

A ― 9.6 ― ― ― 9.4 ― ― ― 9.9 ― ―

R 15 5.7 0.6 11.6 16 5.4 0.5 9.6 16 5.7 1.0 17.1

Adrenals (mg)

U 15 39.0 6.4 16.4 16 38.7 4.1 10.6 16 41.2 5.8 14.1

A ― 38.9 ― ― ― 38.9 ― ― ― 41.1 ― ―

R 15 22.9 3.2 13.9 16 22.5 2.8 12.6 16 23.7 2.4 10.0

Ovaries (mg) U 15 57.3 9.3 16.2 16 57.4 7.4 12.9 16 57.9 8.7 15.1

A ― 57.1 ― ― ― 57.6 ― ― ― 57.8 ― ―

Uterus, wet

(mg)

U 15 333.6 164.9 49.4 16 281.0 84.1 29.9 16 379.7 174.8 46.0

A ― 330.4 ― ― ― 284.5 ― ― ― 379.2 ― ―

Uterus, blotted

(mg)

U 15 285.7 67.8 23.7 16 265.3 69.2 26.1 16 319.7 108.2 33.9

A ― NR ― ― ― NR ― ― ― NR ― ―

Thyroid, fixed

(mg)

U 15 14.4 2.8 19.2 16 12.6 2.4 19.2 16 11.3* 

(↓22) 2.3 20.7

A ― 14.4 ― ― ― 12.7 ― ― ― 11.3* 

(↓22) ― ―

a Data were obtained from page 25 of the study report. Percent differences from controls, calculated by the reviewers, are 

included in parentheses.

U Unadjusted for body weight on PND 21 

A Adjusted for body weight on PND 21

N Number of animals examined

SD Standard Deviation

CV Coefficient of Variation

R Organ-to-body weight ratio (relative to body weight)

NR No value given for adjusted ANCOVA mean

* Significantly different from controls at p<0.05.

E. ESTROUS CYCLICITY: Estrous cycle data are provided in Table 4. Administration of 

permethrin did not change the day of first estrus compared to the vehicle control group. 

Animals exhibited similar cycle lengths and regularity. The majority of females in the 150 

mg/kg/day group were in diestrus at necropsy. 

Page 169 of 206
Female Pubertal Assay (2012) / Page 10 of 14

PERMETHRIN / 109701 OCSPP 890.1450/ OECD None

TABLE 4. Estrous Cyclicitya (mean±SD)

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 (corn oil) 15 35.3±2.5 4.2±0.9 100 100 5 2 7 0

Permethrin

(75 mg/kg/day) 16 36.2±2.0 4.2±1.0 100 81.25 6 3 4 0 

Permethrin

(150 mg/kg/day) 16 35.6±1.4 4.3±0.7 93.75 81.25 10 3 1 0 

a Data were obtained from pages 25 and 26 of the study report. Percent differences from controls, calculated by the 

reviewers, are included in parentheses.

N Number of animals

* Significantly different from controls at p<0.05.

F. CLINICAL CHEMISTRY AND HORMONE LEVELS: Mean hormone levels are 

presented in Table 5. Serum T4 levels were decreased (p<0.05) by 26 and 42% at 75 and 

150 mg/kg/day, respectively. At 150 mg/kg/day, serum TSH levels were increased (p<0.05) 

by 40%. The control group performance criteria for T4 level and CV were within the 

acceptable ranges. Laboratory historical control data were provided for the hormones and 

clinical chemistry parameters on page 29 of the study report. 

Additionally, at 150 mg/kg/day, increases (p<0.05) were observed in sodium (↑3%), 

chloride (↑4%), and SDH (↑20%), and ALP was decreased (↓16%). It was noted that 8 of 

the 15 clinical chemistry parameters in the control group were outside the historical control 

ranges provided. 

Page 170 of 206
Female Pubertal Assay (2012) / Page 11 of 14

PERMETHRIN / 109701 OCSPP 890.1450/ OECD None

TABLE 5. Hormone Levels and Clinical Chemistry a

Parameter

Evaluated (Historical 

Control Data)

Vehicle Control Permethrin

(75 mg/kg/day)

Permethrin

(150 mg/kg/day)

N Mean SD

CV 

(%) N Mean SD

CV 

(%) N Mean SD

CV 

(%)

Hormones

Serum T4, Total (μg/dL)

(3-7) 15 3.70 0.97 26.16 15 2.73* (↓26) 0.56 20.66 16 2.13* (↓42) 0.69 32.34

Serum TSH (ng/mL)

(0.57-3.41) 15 1.24 0.56 44.80 15 1.37 0.29 21.24 16 1.73* (↑40) 0.51 29.34

Clinical Chemistry

NA+ (MEQ/L)

(141-151) 15 137 6 4 15 139 3 2 16 141* 

(↑3) 2 2 

K+(MEQ/L)

(5-7.6) 15 8.2 0.7 8.7 15 8.3 0.6 7.3 16 8.4 0.6 6.7

Cl-(MEQ/L)

(84-109) 15 104 4 4 15 106 2 2 16 108* 

(↑4) 2 2 

Calcium (mg/dL)

(9.4-13.8) 15 9.9 0.6 5.6 15 10.2 0.5 4.5 16 10.3 0.4 3.7

Phosphorous (mg/dL)

(4.7-9.5) 15 9.8 0.8 7.7 15 10.0 0.7 7.0 16 9.6 0.9 9.4

AST (U/L)

(68-192) 15 250 63 25 15 259 87 33 16 253 59 23

ALT (U/L)

(20-56) 15 91 16 17 15 93 21 22 16 90 16 18

GGT (U/L)

(0-4) 15 0.73 1.28 175 15 0.67 1.05 157 16 0.44 1.03 236

ALP (U/L)

(17-183) 15 369 44 12 15 331 71 22 16 310* 

(↓16) 57 18

Blood urea nitrogen

(mmol/L) (12-23) 15 11.73 1.83 16 15 11.27 0.96 9 16 10.88 2.28 21

Creatinine (μmol/L)

(0.5-1.1) 15 0.31 0.05 14.9 15 0.33 0.06 18.5 16 0.32 0.05 17.1

Total Bilirubin

(0-0.5) 15 0.05 0.07 139.4 15 0.06 0.08 138.0 16 0.11 0.13 121.4

SDH (U/L)

(13-37) 15 9 3 39 15 11 4 34 16 12* 

(↑20) 3 29

Total protein (g/dL)

(4.8-9.2) 15 5.3 0.3 4.8 15 5.3 0.2 4.0 16 5.4 0.2 4.4

Albumin (g/dL)

(2.9-6) 15 4.2 0.2 4.3 15 4.2 0.2 5.2 16 4.2 0.2 5.6

a Data were obtained from pages 26 and 28 of the study report. Percent differences from controls, calculated by the 

reviewers, are included in parentheses.

N Number of animals examined

SD Standard Deviation

CV Coefficient of Variation

* Significantly different from controls at p<0.05.

Historical Control Data on page 29 of study report

Page 171 of 206
Female Pubertal Assay (2012) / Page 12 of 14

PERMETHRIN / 109701 OCSPP 890.1450/ OECD None

G. HISTOPATHOLOGY: There were no effects of treatment observed on either colloid area 

or follicular cell height in the thyroid in any group (Table 6). 

 

a Data were obtained from Table 11 on page 31 of the study report. 

b A 5-point grading scale (1=shortest/smallest; 5=tallest/largest) was used.

O No. Observed

E No. Examined

There were no effects of treatment observed microscopically in the ovaries, uterus or kidney 

(Table 7). 

TABLE 6. Incidence of Histopathological Lesions of the Thyroid Gland a

Treatment Groups

Parameter Evaluated

Colloid Area Follicular Cell Height 

(Increase)

Grade b Incidence Grade Incidence

O E O E

Vehicle Control

1 0

15

1 0

15

2 0 2 5

3 10 3 10

4 5 4 0

5 0 5 0

Permethrin

(75 mg/kg/day)

1 0

16

1 0

16

2 0 2 5

3 11 3 11

4 5 4 0

5 0 5 0

Permethrin

(150 mg/kg/day)

1 0

16

1 0

16

2 0 2 3

3 13 3 13

4 3 4 0

5 0 5 0

Page 172 of 206
Female Pubertal Assay (2012) / Page 13 of 14

PERMETHRIN / 109701 OCSPP 890.1450/ OECD None

TABLE 7. Incidence of Histopathological Lesions of the Ovaries, Uterus and Kidneya

Findings

Dose Level

Vehicle Control Permethrin

(75 mg/kg/day)

Permethrin

(150 mg/kg/day)

# Observed # Examined # Observed # Examined # Observed # Examined

Ovaries

[Mean ±SD (%CV)]

Small Follicles 25.3 ± 9.6 

(37.9) 15 21.8 ± 10.0 (45.9) 16 22.1 ± 15.8 (71.5) 16

Medium Follicles 14.1 ± 4.6 

(32.6) 15 13.4 ± 4.9 (36.6) 16 13.4 ± 3.5 (26.1) 16

Antral Follicles 10.1 ± 3.0 

(29.7) 15 9.6 ± 3.8 (39.6) 16 9.5 ± 3.1 (32.6) 16

Antretic Follicles 32.6 ± 7.6 

(23.3) 15 34.4 ± 11.6 (33.7) 16 29.3 ± 7.0 (23.9) 16

Corpora Lutea 8.0 ± 2..6 

(32.5) 15 7.6 ± 2.0 (26.3) 16 9.0 ± 2.8 (31.1) 16

Uterus

Dilation Minimal 8 15 13 16 13 16

Mild 6 15 3 16 3 16

Moderate 1 15 0 16 0 16

Eosinophilic material Minimal 12 15 10 16 9 16

Inflammatory cells/debris Minimal 6 15 2 16 3 16

Endometrial folding Minimal 9 15 5 16 9 16

Endometrial inflammatory cells

Minimal 1 15 1 16 3 16

Kidney

Nephropathy Minimal 13 15 12 16 12 16

Mild 1 15 0 16 0 16

Tubular dilation Minimal 1 15 2 16 2 16

Mineralization Minimal 1 15 2 16 2 16

Fibrosis Minimal 1 15 0 16 0 16

Cyst Minimal 0 15 1 16 1 16

a Data were obtained from Table 10 on page 30 and Appendix IX on pages 106 and 108 of the study report.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: Administration of permethrin to intact 

juvenile/peripubertal female rates did not affect pubertal development. Permethrin 

administration slightly increased liver weight and caused slight effects on thyroid weight 

and thyroid hormone levels. However, the thyroid effects may have been secondary to liver 

enzyme induction, and not a reflection of a direct effect on the thyroid. 

B. AGENCY COMMENTS: All rats administered permethrin survived until schedule 

termination. One animal in the control was euthanized on PND 34 for signs related to 

gavage error. From PND 34 onward, tremors were observed in 4 animals administered 75 

mg/kg/day and all animals administered 150 mg/kg/day at 4-6 hours post-dosing on at least 

one day. These findings did not persist to the daily examinations prior to daily dosing. 

No treatment-related effects were observed at any dose on body weights, body weight gains, 

or age and weight at attainment of VO attainment. All surviving animals attained complete 

VO.

Page 173 of 206
Female Pubertal Assay (2012) / Page 14 of 14

PERMETHRIN / 109701 OCSPP 890.1450/ OECD None

Absolute liver weights (adjusted and unadjusted) were increased (p<0.05) by 10% at 150 

mg/kg/day. Relative (to body weight) liver weights were increased (p<0.05) by 4 and 8% at 

75 and 150 mg/kg/day. Both unadjusted and adjusted thyroid weights were both decreased 

(p<0.05) by 22% at 150 mg/kg/day. 

Administration of permethrin did not change the day of first estrus compared to the vehicle 

control group. Animals exhibited similar cycle lengths and regularity. The majority of 

females in the 150 mg/kg/day group were in diestrus at necropsy. 

Serum T4 levels were decreased (p<0.05) by 26 and 42% at 75 and 150 mg/kg/day, 

respectively, and serum TSH levels were increased (p<0.05) by 40% at 150 mg/kg/day. 

Additionally, at 150 mg/kg/day, increases (p<0.05) were observed in sodium (↑3%), 

chloride (↑4%), and SDH (↑20%), and ALP was decreased (↓16%). However, it is 

noteworthy that values for 8 of the 15 clinical chemistry parameters measured in the control 

group were outside of historical control data. 

In the thyroid, there were no effects of treatment observed on either colloid area or follicular 

cell height in any treated group. No effects of treatment were observed microscopically in 

the ovaries, uterus or kidney. 

The dose levels tested were determined to be adequate based on the tremors and estrogenrelated effects seen at the high dose in this assay, as well as the results of the range findings 

study. 

C. STUDY DEFICIENCIES: None 

Page 174 of 206
DATA EVALUATION RECORD

PERMETHRIN

Study Type: OCSPP 890.1500, Male Pubertal Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 3-27-2012 (MRID 48687101) 

Prepared for

Health Effects Division

Office of Pesticide Programs

U.S. Environmental Protection Agency 

2777 South Crystal Drive 

Arlington, VA 22202 

Prepared by

CSS-Dynamac Corporation

1910 Sedwick Road, 

Building 100, Suite B 

Durham, NC 27713 

Primary Reviewer: Signature:

Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 10/22/2012

Secondary Reviewer: Signature:

Michael E. Viana, Ph.D., D.A.B.T. Date: 10/25/2012

Program Manager: Signature:

Jack D. Early, M.S. Date: 10/25/2012

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 10/25/2012

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

signing by CSS-Dynamac Corporation personnel. 

Page 175 of 206
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 176 of 206
PERMETHRIN / 109701 

Primary Reviewer: Gregory Akerman 

Health Effects Division 

Secondary Reviewer: John Liccione, Ph.D. 

Health Effects Division 

Male Pubertal Assay (2012) I Page I of IS 

OCSPP 890.1500/ OECD None 

Signature: __.....~~~.p.L.-­

Date;;-- : -~~~~~1:..--­

Signature.~-JE.~~~::::;;::::-

Dat S:O- Z-1/,1) 

Template version 08/20 11 

DATA EVALUATION RECORD 

STUDY TYPE: Male Pubertal Assay; OCSPP 890.1500; OECD None. 

PC CODE: 109701 DP BARCODE: D405670 

TXR #: 0056246 

TEST MATERIAL (PURITY): Permethrin (97.7% a.i.) 

SYNONYMS: (3-Phenoxyphenyl)methyl 3-(2,2-dichloroethenyl)-2,2-

dimethylcyclopropanecarboxylate 

CAS #: 52645-53-1 

CITATION: Zorrilla, L. (2012). Pubertal Development and Thyroid Function in Intact 

Juvenile/Peripubertal Male Rats with Pennethrin. Integrated Laboratory 

Systems, In~. , Durham, NC. Laboratory Study No.: Cl95-800, September 5, 

2012. MRID 4868710 l. Unpublished. 

SPONSOR: Consumer Specialty Product Association for the Permethrin Data Group 

II/Joint Venture, 1667 K Street NW, Suite 300, Washington, DC 

TEST ORDER#: CON-109701 -7 

EXECUTIVE SUMMARY: In a Male Pubertal Assay (MRID 48687101), 16 Sprague-Dawley 

[Crl:CD(SD) lGS] rats/dose group were treated daily via oral gavage with pennethrin (97.7% 

a.i., Lot# PMPS00014) in corn oil at doses ofO, 75, or 150 mg/kg/day from postnatal day (PND) 

23 to 53/54. However, three 150 mg/kg/day rats were found dead after two days of dosing. 

Therefore, the high dose was reduced to l 20 mg/kg/day on PND 25. Animals were examined for 

preputial separation (PPS) daily beginning on PND 30, and age and weight on day of attainment 

were recorded. Following sacrifice on PND 53/54, blood was collected for clinical chemistry 

and hormone analyses. Total serum testosterone, thyroxine (T4) and thyroid stimulating 

hormone (TSH) levels were determined using radioimmunoassays. Adrenal, liver, pituitary, 

thyroid, and urogenital organs were weighed and examined macroscopically. The left testes, left 

epididymides, left kidney, and thyroid were examined microscopically. 

No treatment-related related effects were observed on body weights, body weight gains, age or 

weight at attainment of PPS, organ weights (except liver), clinical chemistry, and gross or 

histopathology parameters. 

After reduction of the high dose to 120 mg/kg/day, all remaining animals survived to study 

termination. At 6 hours post-dose, tremors were observed at 75 and 120 mg/kg/day in a dosePage 177 of 206
tremors on two days. At 120 mg/kg/day, very mild to severe tremors were observed in all 

surviving animals on several days. Additionally, one 120 mg/kg/day rat was considered 

uncoordinated on one day. 

Serum testosterone levels in the treated groups were similar to controls. Mean serum T4 levels 

were decreased (p<0.001) by 28 and 42% at 75 and 120 mg/kg/day, respectively, and serum TSH 

level was increased (p<0.05) by 77% at 120 mg/kg/day. The T4 and TSH values were all within 

or just below their respective reference ranges. Additionally, there were no concomitant effects 

observed on thyroid weights or thyroid follicular cell height or colloid area. Absolute and 

relative liver weight were increased (p<0.05) by 6-11% at 75 and 120 mg/kg/day. There were no 

changes in clinical chemistry parameters that suggested hepatotoxicity due to permethrin 

administration. The increased liver weights were considered to be the result of hypertrophy and 

associated microsomal enzyme induction, a common finding with permethrin. 

The 150 mg/kg/day dose was an excessive dose level as three rats were found dead on Day 2. 

Therefore, the dose was lowered to 120 mg/kg/day which was an adequate high dose based on 

the clinical signs (tremor and in coordination) seen in these animals. Tremors were also noted at 

75 mg/kg/day. 

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

890.1500). 

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

Assurance statements were provided. 

Page 178 of 206
I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Integrated Laboratories Systems, Inc.

Location: Durham, NC

Study Director: Leah Zorilla, Ph.D.

Other Personnel: Susan Borghoff (Study Toxicologist), Lauren M. Staska (Study Pathologist), and Carol Swartz

(Dose Formulations)

Study Period: December 28, 2011 through September 5, 2012

2. Test Substance: Permethrin

Description: Yellow liquid

Source: AMVAC Corporation

Batch #: PMPS00014 (expires January 2013)

Purity: 97.7%

Stability: Report stated that the compound is stable in the vehicle at 1-10°C for up to 20 days

CAS #: 52645-53-1

Structure:

O

O

O

Cl

3CH

Cl

3CH

3. Vehicle: Corn oil

4. Test Animals:

Species: Rat

Strain: Sprague-Dawley [Crl:CD®(SD) IGS]

Age / Weight at 

Study Initiation: PND 23 / 54.0-68.5 g males only

Source: Charles River Laboratories International, Inc. (Raleigh, NC)

Housing: Parental dams were individually housed in polycarbonate cages with micro-isolator tops and 

absorbent, heat-treated, hardwood bedding; each dam was housed with her litter until weaning 

on PND 21. After weaning, males were housed two/cage.

Diet: Teklad Global 16% Protein Rodent Diet (Teklad Diets, Madison, WI), ad libitum. It was 

stated that the total genistein equivalents of genistein plus daidzein was determined to be 4.8

μg/g of feed.

Water: Reverse osmosis-purified tap water, ad libitum

Environmental 

Conditions:

Temperature:

Humidity:

Air changes:

Photoperiod:

12-23°C

19-63% 

Not reported

14 h light / 10 h dark

B. STUDY DESIGN

1. In-Life Dates: Start: February 4, 2012 Approximate End: March 7, 2012 

2. Mating: Timed pregnant, nulliparous dams (age not reported) were received from the 

supplier on gestation day 8, allowed to deliver naturally, and rear their pups to weaning on 

PND 21. Between postnatal days (PND) 3 and 5, litters with the same date of birth were 

standardized to 8 pups with equal numbers of males and females. 

Page 179 of 206
3. Animal Assignment: Following weaning, animals were assigned using a procedure that 

stratified animals across groups by body weight, such that the mean body weight of each 

group was not statistically different from any other group using analysis of variance. 

Littermates were not placed in the same dose group. The test groups are provided in Table 

1.

TABLE 1. Study Design a

Test group Dose (mg/kg/day)b # of Males

Control 0 16

Low 75 16

High 120c 16

a Data were obtained from Table 1 on page 15 of the study report.

b Dose adjusted for purity.

c Animals received 150 mg/kg/day on Days 1 and 2. 

4. Dose Selection Rationale: The doses were selected based upon three previously conducted 

studies and toxicity observed in the current study. In a range finder study1, intact adult male 

rats and juvenile female rats were administered up to 400 mg/kg/day and 250 mg/kg/day 

permethrin, respectively. In both sexes, animals administered ≥200 mg/kg/day permethrin 

exhibited severe clinical signs up to 24-hours post-dose and were euthanized prior to 

scheduled study termination. A Female Pubertal Assay (48672901.DER) was performed at 

ILS where permethrin was orally administered a high dose of 150 mg/kg/day for 21/22 days. 

Final body weights of the high dose group were 101.1% of control; however, 9 of 16 

animals exhibited tremors on 1 or more days approximately 6 hours after dose 

administration. No adverse clinical signs were noted 24 hours post-dose. A Hershberger 

Assay (48618602.DER) in castrated adult male rats was also performed at ILS at a high 

dose of 120 mg/kg/day permethrin. In the portion of the assay to assess agonist activity, 

final body weights were 104.2% of control body weights after 10 days of administration of 

120 mg/kg/day permethrin. No adverse clinical observations were noted 24 hours postdose. A high dose of 150 mg/kg/day was selected for the current Male Pubertal Assay 

based on deaths, moribundity, and adverse clinical signs up to 24 hours following dose 

administration at higher dose levels. However, after two days of dosing at 150 mg/kg/day 

permethrin, three animals in this group were found dead, and gavage errors were ruled out 

upon necropsy. Therefore to ensure animals survived until scheduled termination, the high 

dose group was changed on Day 3 (PND 25) to 120 mg/kg/day.

5. Dose Preparation and Analysis: Dose formulations were prepared in corn oil three times

during the study at a concentration of 15 mg/mL, once at a concentration of 30 mg/mL, and 

two times at a concentration of 24 mg/mL. Formulations were dispensed into vials to be 

used daily during the study. Dose concentrations were adjusted to correct for purity of 

permethrin. 

Samples of dose formulations prepared on February 2, 8, and 17, 2012 were collected from 

the top, middle, and bottom of the formulation and sent to Smithers Viscient for analysis. 

Smithers Viscient analyzed, in duplicate, samples received for concentration and

 

1 Zorrilla, L. (2011a). Range Finder Study for In Vivo Mammalian Assays for Permethrin. Unpublished Study 

Report Prepared by ILS, Inc. (draft) Study No. C195-500.

Page 180 of 206
homogeneity. The report stated that the compound is stable in the vehicle at 1-10°C for up 

to 20 days on the basis of a previously performed study2. 

Results

Homogeneity (CV): 0.260-6.42% 

Stability (% of time zero): Not reported

Concentration (% of nominal): 84.6-102% 

The analytical data indicated that the mixing procedure was adequate and the variation 

between nominal and actual dosage to the animals was acceptable. The low dose 

formulation was only 84.6-85.6% of nominal concentration in 2/3 formulations, and the 

homogeneity exceeded 5% CV in 1/3 formulations; however, these values were not 

considered to have adversely impacted study results. Stability data should be submitted for 

independent verification. 

6. Dosage Administration: Dose formulations were administered once daily by oral gavage 

in a dose volume of 5 mL/kg body weight. The dose formulations were administered for 31 

or 32 consecutive days from PND 23-53/54. Dosing occurred 24 hours (± 2 hours) from the 

previous dose. Dose volume was adjusted daily based on individual animal body weight. 

The daily dosing sequence was stratified across all dose groups; 1 animal from each group 

and then repeated until all animals were dosed. 

7. Statistics: Descriptive statistics (mean, standard deviation, coefficient of variance, and 

sample size) were calculated using Microsoft Excel 2003/2007 (Redmond, WA). Data sets 

listed below were analyzed using Statistical Analysis System version 9.2 (SAS Institute, 

Cary, NC). Studentized residual plots were used to detect possible outliers in the data and

Levene’s test was used to assess homogeneity of variance. Heterogeneous data were

transformed (logarithm, square root, and multiplicative inverse), and analyzed using the 

non-parametric Kruskal-Wallis and Dunn’s test if data remained heterogeneous. 

 

2 Smith, R. (2011). Storage Stability of Permethrin in Corn Oil. Unpublished study report prepared by Smithers 

Viscient Inc. Study No. 14010-6105.

Page 181 of 206
Classification Procedure

Initial body weights

Final body weight (last day all body weights collected)

Cumulative body weight gain (last day all body weights 

collected)

Age and body weight at PPS

Homogeneous data sets:

One-way analysis of variance (ANOVA) followed by pairwise comparisons performed using Dunnett’s two-tailed 

t-tests

Body weight

Organ weight

Tests for linear trend were performed and reported when 

significant (p<0.05) for endpoints that did not show any 

significant pair-wise comparisons.

Tissue weights

Relative tissue weights (liver, kidneys, pituitary, and 

adrenals)

Hormone levels

Clinical chemistry levels

Two-way ANOVA with treatment and necropsy day as 

main effects. 

Pair-wise comparisons were performed using Dunnett’s 

two-tailed t-tests.

Final body weight (last day all body weights collected)

Cumulative body weight gain (last day all body weights 

collected)

Age and body weight at PPS

Tissue weights

Analysis of covariance (ANCOVA) with PND 21 body 

weight (weight on day of allocation) as the covariable. 

Pair-wise comparisons were performed using Dunnett’s 

two-tailed t-tests.

Thyroid scoring (colloid area and follicular cell height) Fisher’s Exact test

Significance was reported as exact p-values, except no p-value was reported for Dunn's Test 

(p<0.05). The statistical analyses were considered appropriate.

C. METHODS

1. Mortality and Clinical Examinations: Animals were observed for mortality and 

moribundity twice daily on weekdays and once daily on weekends and holidays Rats were 

observed for clinical signs of toxicity prior to allocation of animals to study groups, daily 

prior to dose administration, 6 hours post-dosing, and on the day of euthanasia.

2. Body Weight: Animals were weighed weekly following birth, prior to at allocation of 

animals to study groups, daily prior to dose administration, and on the day of euthanasia.

3. Food Consumption: Food consumption was not measured. 

4. Preputial Separation (PPS): Beginning on PND 30, males were examined daily following 

dosing for onset of PPS. PPS was described as: progression not initiated, partial separation, 

a persistent thread of tissue between the glans and prepuce, or complete PPS. Age and 

weight on the day of completion of PPS were recorded.

5. Sacrifice and Pathology: Any animals found moribund or dead were necropsied to 

determine the cause, if possible. At least two hours before euthanasia, animals surviving to 

scheduled termination were moved to the necropsy holding room. In the same order as the

animals were dosed, they were humanely euthanized by decapitation with 2 hours of dosing. 

Prior to tissue collection, trunk blood was collected, and serum was obtained. All serum 

samples were stored at ≤−70°C. Serum samples were shipped overnight on dry ice to 

AniLytics Inc. (Gaithersburg, MD) for analysis.

a. Hormone Analysis: Testosterone, T4, and TSH levels were measured in serum using 

radioimmunoassays (RIAs). Samples were assayed in duplicate and in conjunction with 

multiple quality control samples.

Page 182 of 206
b. Clinical Chemistry: The following CHECKED (X) parameters were examined.

ELECTROLYTES OTHER

X Calcium X Albumin

X Chloride X Creatinine*

Magnesium X Urea nitrogen*

X Phosphorus Total cholesterol

X Potassium Globulins

X Sodium Glucose

ENZYMES X Total bilirubin

X Alkaline phosphatase (ALP) X Total protein

Cholinesterase (ChE) Triglycerides

Creatine phosphokinase Albumin/globulin ratio (calculated)

Lactic acid dehydrogenase (LDH) Bile acids

X Alanine aminotransferase (ALT/also SGPT)

X Aspartate aminotransferase (AST/also SGOT)

X Sorbitol dehydrogenase (SDH)

X Gamma glutamyl transferase (GGT)

Glutamate dehydrogenase

* Recommended for the pubertal assay in male rats based on guideline 890.1500.

c. Organ Weights and Histopathology: The following CHECKED (X) tissues were 

collected and weighed and grossly observed from all study animals. The (XX) organs, in 

addition, were subjected to histological examination. 

UROGENITAL OTHER

XX Testes (left and right separately)*+ XX Thyroid a*+

XX Epididymides (left and right separately)*+ X Liver*

X Seminal vesicle plus coagulating glands (with and without fluid)* X Adrenals (paired)*

X Ventral prostate* X Pituitary*

X Dorsolateral prostate*

X Levator ani/bulbocavernosus (LABC) muscle complex*

XX Kidneys (paired)*+

a Thyroid was weighed post-fixation.

* Weights required based on guideline 890.1500. 

+ Histopathological examination required based on guideline 890.1500. 

The left kidney and thyroid (with attached trachea) were fixed in 10% neutral buffered 

formalin for at least 24 hours. Following fixation, the thyroid was dissected free of the 

trachea and weighed. The left testis and left epididymis were each placed in Bouin’s 

fixative for 18-24 hours and then washed in 70% histology grade alcohol. After fixation, all 

tissues were transferred to 70% histology grade alcohol, routinely processed into paraffin 

blocks, sectioned, and stained with hematoxylin and eosin. All processed tissues (thyroid, 

left kidney, left epididymis, and left testis) from all animals were microscopically evaluated. 

Two sections of each of the 2 lobes of the thyroid were examined and evaluated for 

follicular cell height and colloid area. A 5-point grading scale (1 = shortest/smallest; 5 = 

tallest/largest) was used, and the thyroids were evaluated relative to photomicrographs 

supplied by the U.S. EPA. 

Page 183 of 206
II. RESULTS

A. MORTALITY: On Day 2, three 150 mg/kg/day rats were found dead; gavage error was 

ruled out as the cause of death. Starting the next day, this group was treated at 120 

mg/kg/day, and all remaining animals survived to study termination.

B. CLINICAL SIGNS OF TOXICITY: At 6 hours post-dosing, tremors were observed at 75 

and 120 mg/kg/day in a dose-dependent manner, with incidence, frequency, and severity 

increasing with dose. At 75 mg/kg/day, three animals exhibited mild tremors on one day 

and one animal exhibited mild tremors on two days. At 120 mg/kg/day, very mild to severe 

tremors were observed in all surviving animals on 2-12 days. Tremors were not reported for 

the animals that were found dead. One 120 mg/kg/day was considered uncoordinated on 

one day. All other findings, such as rough coat, fur discoloration, ungroomed, lethargic, or

red nasal/ocular discharge, were considered incidental during the daily and 6-hour post-dose 

observations. 

C. GENERAL GROWTH AND PREPUTIAL SEPARATION: Initial and final body 

weights, body weight gains, age and weight at attainment of PPS, and proportion/incidence 

of separated prepuce are presented in Table 2. All values in the treated groups were similar 

to the controls.

The Guideline performance criteria were met for age and weight at PPS and weaning and 

final body weight in the control group, except for the following: the CV for body weight at 

PPS (11.3%) was greater than the maximum acceptable value (7.57%), and the CV for final 

body weight (9.9%) was greater than the maximum acceptable value (7.47%). 

Page 184 of 206
TABLE 2. General Growth and Preputial Separation (PPS) a

Parameter Evaluated

Vehicle Control 75 mg/kg/day Permethrin 120 mg/kg/day Permethrin

N Mean SD

CV

(%) N b Mean SD

CV

(%) N c Mean SD

CV

(%)

Age at PPS 

(PND)

U 16 45.4 1.3 2.8 15 45.7 1.9 4.2 13 45.5 1.9 4.2

A 16 45.4 --- --- 15 45.6 --- --- 13 45.6 --- ---

Body weight at 

PPS (g)

U 16 229.0 25.8 11.3 15 240.3 28.0 11.7 13 236.2 21.5 9.1

A 16 229.2 --- --- 15 242.1 --- --- 13 233.9 --- ---

Age at partial 

PPS (PND)

U 16 41.8 1.1 2.7 15 41.5 1.1 2.7 16 41.8 1.5 3.6

A 16 41.7 --- --- 15 41.5 --- --- 16 41.9 --- ---

Initial body 

weight 

(PND 23; g)

U 16 61.8 4.0 6.4 15 60.7 3.4 5.5 16 61.2 3.2 5.2

A 16 61.6 --- --- 15 61.0 --- --- 16 61.1 --- ---

Final body 

weight (g)

U 16 290.4 28.8 9.9 15 304.3 36.7 12..1 13 298.6 19.0 6.4

A 16 290.7 --- --- 15 306.9 --- --- 13 295.2 --- ---

Final body 

weight 

(% of control)

U 16 --- --- --- 15 104.8 --- --- 13 102.8 --- ---

A 16 --- --- --- 15 --- --- --- 13 --- --- ---

Body weight 

gain

(final – initial; g)

U 16 228.7 26.3 11.5 15 243.6 34.1 14.0 13 237.1 18.4 7.8

A 16 228.9 --- --- 15 245.7 --- --- 13 234.5 --- ---

Proportion 

unseparated (#/N) 0/16 0/15 0/13

a Data were obtained from Table 5 on page 24 of the study report. No statistical differences were detected.

b One animal was female and was removed from the study; N = 15.

c Three animals were found dead; N = 13.

U Unadjusted for body weight on PND 21 

A Adjusted for body weight on PND 21

N Number of animals examined

SD Standard Deviation

CV Coefficient of Variation

 --- Not applicable

D. ORGAN WEIGHTS: Organ weights at necropsy are presented in Table 3, and all values 

in the treated groups were similar to controls, except for minor increases (p<0.05) in 

absolute and relative liver weight at 75 (↑6-11%) and 120 (↑8-11%) mg/kg/day. Adjusted 

weights for the liver were not provided. 

The unadjusted values for all organ weights in the control group were within the acceptable 

range of the Guideline performance criteria, with the following exceptions: (i) mean kidney 

weight was 1.92 g (acceptable range: 2.242-3.050 g); (ii) CV for pituitary weight was 18.9% 

(maximum acceptable value: 15.98%); and (iii) mean LABC weight was 0.350 g (acceptable 

range: 0.447-0.855 g). 

Page 185 of 206
TABLE 3. Organ Weights at Necropsy a

Organ

Vehicle Control 75 mg/kg/day Permethrin 120 mg/kg/day Permethrin

N Mean SD

CV

(%) N b Mean SD

CV

(%) N c Mean SD

CV

(%)

Liver

(g)

U 16 12.47 1.40 11.2 15 13.80* 

(↑11) 2.09 15.2 13 13.80* (↑11) 0.90 6.5

A 16 NP --- --- 15 NP --- --- 13 NP --- ---

R 16 4.29 0.18 4.2 15 4.53** 

(↑6) 0.24 5.2 13 4.62*** (↑8) 0.18 3.9

Kidneys

(g)

U 16 1.92 0.23 11.9 15 2.05 0.22 10.9 13 2.05 0.16 7.8

A 16 1.92 --- --- 15 2.06 --- --- 13 2.03 --- ---

R 16 0.66 0.05 7.8 15 0.68 0.04 6.5 13 0.69 0.04 6.0

Pituitary

(mg)

U 16 9.0 1.7 18.9 15 9.2 1.8 19.8 13 9.4 0.9 9.7

A 16 9.0 --- --- 15 9.2 --- --- 13 9.4 --- ---

R 16 3.1 0.6 19.1 15 3.0 0.5 15.9 13 3.2 0.3 8.8

Adrenals

(mg)

U 16 39.1 4.8 12.2 15 40.6 5.9 14.5 13 42.5 6.2 14.5

A 16 39.1 --- --- 15 40.7 --- --- 13 42.3 --- ---

R 16 13.6 2.0 14.8 15 13.4 1.9 14.2 13 14.3 2.1 14.8

Seminal vesicle 

+ coagulating 

gland, with fluid 

(mg)

U 16 535.0 77.7 14.5 14 537.9 106.4 19.8 13 542.6 98.8 18.2

A 16 535.0 --- --- 14 538.8 --- --- 13 541.7 --- ---

Seminal vesicle 

+ coagulating 

gland, without 

fluid (mg)

U 16 292.6 32.6 11.1 15 298.7 45.0 15.1 13 313.0 51.5 16.4

A 16 292.7 --- --- 15 299.6 --- --- 13 311.9 --- ---

Ventral 

prostate (mg)

U 16 202.3 22.5 11.1 15 219.6 46.6 21.1 13 215.8 44.5 20.6

A 16 202.4 --- --- 15 221.0 --- --- 13 214.0 --- ---

Dorsolateral 

prostate (mg)

U 16 129.6 14.7 11.3 15 137.1 21.9 15.9 13 120.0 16.1 12.9

A 16 129.6 --- --- 15 136.8 --- --- 13 120.4 --- ---

LABC 

(mg)

U 16 349.5 43.0 12.3 15 369.9 47.2 12.8 13 353.4 67.4 19.1

A 16 349.7 --- --- 15 372.0 --- --- 13 350.8 --- ---

Epididymis, left 

(mg)

U 16 219.3 19.0 8.7 15 220.9 17.7 8.0 13 214.3 11.3 5.3

A 16 219.3 --- --- 15 221.6 --- --- 13 213.4 --- ---

Epididymis, 

right (mg)

U 15 221.5 18.4 8.3 15 218.2 18.0 8.2 13 221.2 8.8 4.0

A 15 221.6 --- --- 15 218.6 --- --- 13 220.7 --- ---

Testis, left (mg) U 15 1455.9 132.9 9.1 14 1446.0 124.2 8.6 13 1426.7 99.4 7.0

A 15 1452.3 --- --- 14 1457.8 --- --- 13 1418.1 --- ---

Testis, right 

(mg)

U 15 1475.0 114.4 7.8 15 1452.1 113.8 7.8 12 1471.6 114.6 7.8

A 15 1465.0 --- --- 15 1459.1 --- --- 12 1459.7 --- ---

Thyroid, fixed

(mg)

U 16 14.6 2.3 15.4 15 14.9 2.1 14 13 13.6 1.6 11.8

A 16 14.6 --- --- 15 15.0 --- --- 13 13.4 --- ---

a Data were obtained from Table 6 on pages 26-27 of the study report. 

b One animal was female and was removed from the study; N = 15.

c Three animals were found dead; N = 13.

U 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) 

SD Standard Deviation

CV Coefficient of Variation

NP No value given for adjusted ANCOVA mean

 * Significantly different from controls at p<0.05

** Significantly different from controls at p<0.01

*** Significantly different from controls at p<0.001

Page 186 of 206
E. HORMONE LEVELS AND CLINICAL CHEMISTRY: Mean hormone levels and 

clinical chemistry values and are presented in Table 4. Nominal ranges3 for the hormone 

and clinical chemistry values were provided. 

Mean serum T4 levels were decreased (p<0.001) at 75 and 120 mg/kg/day by 28 and 42%, 

respectively, and serum TSH level was increased (p<0.05) by 77% at 120 mg/kg/day. The 

T4 and TSH values were all within or just below their respective historical control range, 

except T4 in high-dosed rats that was just below the historical range. Testosterone levels in 

the treated groups were similar to controls. Minor differences (p<0.05) of 1-2% were noted 

in serum sodium, chloride, and calcium at 120 mg/kg/day compared to controls. Serum 

phosphorus was increased (p<0.05) by 11% at 75 mg/kg/day, but the 120 mg/kg/day group 

was similar to the controls. All other clinical chemistry values in the treated groups were 

similar to control values.

The results for serum T4 and testosterone in the control group were within the acceptable 

range of the Guideline performance criteria, but the mean TSH level of 1.67 ng/mL was 

below the acceptable range of 4.212-24.112 ng/mL. 

TABLE 4. Hormone Levels and Clinical Chemistry a

Parameter

Evaluated

Vehicle Control 75 mg/kg/day Permethrin 120 mg/kg/day Permethrin

N Mean SD

CV

(%) N b Mean SD

CV

(%) N c Mean SD

CV

(%)

Hormones

Serum T4, 

Total (μg/dL) 16 5.11 0.60 11.7 15 3.70*** (↓28) 0.55 14.8 13 2.98*** (↓42) 0.43 14.4

Serum TSH 

(ng/mL) 16 1.67 0.89 53.1 15 2.63 1.51 57.4 13 2.95* (↑77) 1.84 62.2

Serum 

testosterone 

(ng/mL)

16 1.73 0.68 39.3 15 1.61 1.17 73.2 13 1.30 0.96 74.0

Clinical Chemistry

Sodium 

(mEq/L) 16 138 2 2 15 138 2 2 13 140* 

(↑1) 2 2 

Potassium 

(mEq/L) 16 7.2 0.4 6.1 15 7.4 0.5 6.7 13 7.5 0.5 6.4

Chloride 

(mEq/L) 16 103 2 2 15 103.9 1.6 1.5 13 105* 

(↑2) 1 1 

Calcium 

(mg/dL) 16 10.6 0.2 2.2 15 10.5 0.5 4.4 13 10.4* 

(↓2) 0.4 4.0

Phosphorus 

(mg/dL) 16 9.0 0.7 8.1 15 10.0* 

(↑11) 1.1 11.5 13 9.9 1.3 13.1

AST (U/L) 16 186 46 25 15 190 40 21 13 209 66 32

ALT (U/L) 16 84 12 14 15 83 12 14 13 88 11 12

GGT (U/L) 16 0.25 0.77 310 15 0.20 0.77 387 13 0.08 0.28 361

ALP (U/L) 16 563 108 19 15 550 120 22 13 501 120 24

 

3 Loeb, W.F. and F.W. Quimby, Eds. (1999). Clinical Chemistry of Laboratory Animals, 2nd ed. Taylor and Francis, 

Philadelphia.

Page 187 of 206
TABLE 4. Hormone Levels and Clinical Chemistry a

Parameter

Evaluated

Vehicle Control 75 mg/kg/day Permethrin 120 mg/kg/day Permethrin

N Mean SD

CV

(%) N b Mean SD

CV

(%) N c Mean SD

CV

(%)

BUN 

(mg/dL) 16 11 2 20 15 11 2 17 13 11 2 17

Creatinine 

(mg/dL) 16 0.41 0.05 12.1 15 0.41 0.08 19.6 13 0.41 0.05 12.1

Total 

Bilirubin 

(mg/dL)

16 0.05 0.10 193 15 0.02 0.06 280 13 0.05 0.08 168

SDH (U/L) 16 4.13 4.62 112 15 3.93 1.28 33 13 3.31 1.11 34

Total Protein 

(g/dL) 16 5.7 0.2 4.1 15 5.9 0.4 6.4 13 5.9 0.2 4.0

Albumin 

(g/dL) 16 4.7 0.2 5.1 15 4.8 0.3 6.2 13 4.8 0.2 4.7

a Data were obtained from Tables 7-8 on pages 29-30 of the study report. Percent difference from controls (calculated by 

reviewers) is presented parenthetically.

b One animal was female and was removed from the study; N = 15.

c Three animals were found dead; N = 13.

SD Standard Deviation

CV Coefficient of Variation

* Significantly different from controls at p<0.05

*** Significantly different from controls at p<0.001

G. GROSS PATHOLOGY: There were no treatment-related macroscopic findings. In the 

controls, the right testis and epididymis were moderately enlarged in one animal, and the left 

testis was mildly enlarged in one animal. In the 120 mg/kg/day group, the right testis was 

mildly enlarged in one animal. No other gross observations were noted in male rats 

surviving to the scheduled termination. 

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

are presented in Table 5; follicular cell shape was not reported. Thyroid colloid area and 

follicular cell height were similar in the treated groups to the controls; all animal samples 

were classified as Grade 3 or 4 for colloid area and Grade 2 or 3 for follicular height. 

Page 188 of 206
TABLE 5. Thyroid Colloid Area and Follicular Cell Height a

Treatment Groups

Parameter Evaluated

Colloid Area Follicular Cell Height

(Increase)

Grade b Incidence Grade b Incidence

O E O E

Vehicle Control

1 0 16 1 0 16

2 0 16 2 10 16

3 6 16 3 6 16

4 10 16 4 0 16

5 0 16 5 0 16

75 mg/kg/day 

Permethrin

1 0 15c 1 0 15

2 0 15 2 9 15

3 6 15 3 6 15

4 9 15 4 0 15

5 0 15 5 0 15

120 mg/kg/day 

Permethrin

1 0 13d 1 0 13

2 0 13 2 6 13

3 7 13 3 7 13

4 6 13 4 0 13

5 0 13 5 0 13

a Data were obtained from Table 10 on page 32 of the study report.

b A 5-point grading scale (1 = shortest/smallest; 5 = tallest/largest) was used.

c One animal was female and was removed from the study; N = 15.

d Three animals were found dead; N = 13.

O No. Observed

E No. Examined

The incidence of histopathological findings of the testes, epididymides, and kidneys are 

presented in Table 6. No treatment-related histological lesions were noted in these organs. 

TABLE 6. Incidence of Histopathological Lesions of the Testes, Epididymides, and Kidneya

Findings

Vehicle Control 75 mg/kg/day Permethrin 120 mg/kg/day

Permethrin

# Observed # Examined # Observed # Examined b # Observed # Examined c 

Testis

Seminiferous tubule 

dilatation, mild 1 16 1 15 0 13

Interstitium edema, 

mild 1 16 1 15 0 13

Epididymidis

Inflammation, minimal 5 16 3 15 5 13

Kidney

Nephropathy, minimal 13 16 14 15 13 13

Cyst, minimal 2 16 0 15 1 13

a Data were obtained from Appendices 1, 5, and 10 on pages 98, 100, and 102, respectively, of the study report.

b One animal was female and was removed from the study; N = 15.

c Three animals were found dead; N = 13.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Administration of permethrin to intact juvenile/

Page 189 of 206
peripubertal male rats did not affect pubertal development. Permethrin administration 

increased liver weight and caused a significant decrease in T4 and increase in TSH serum 

concentrations, without a corresponding change in thyroid weight or histopathology. This 

suggests that the thyroid effects may have been secondary to liver enzyme induction, and 

not a reflection of a direct effect on the thyroid. 

B. AGENCY COMMENTS: No treatment-related related effects were observed on body 

weights, body weight gains, organ weights (except liver), clinical chemistry, or gross 

pathology. No adverse histological lesions were observed in the testes, epididymides, or 

kidneys. Age and body weight at attainment of PPS, and proportion/incidence of separated 

prepuce were similar in the treated groups to the controls. Also, serum testosterone levels in 

the treated groups were similar to controls. 

A treatment-related effect on mortality was observed at 150 mg/kg/day. On Day 2, three 

150 mg/kg/day rats were found dead; gavage error was ruled out as the cause of death. 

Starting the next day, this group was treated at 120 mg/kg/day, and all remaining animals 

survived to study termination. 

At 6 hours post-dosing, tremors were observed at 75 and 120 mg/kg/day in a dosedependent manner, with incidence, frequency, and severity increasing with dose. At 75 

mg/kg/day, three animals exhibited mild tremors on one day and one animal exhibited mild 

tremors on two days. At 120 mg/kg/day, very mild to severe tremors were observed in all 

surviving animals on several days. Tremors were not reported in animals that were found 

dead. Additionally, one 120 mg/kg/day rat was considered uncoordinated on one day. 

Mean serum T4 levels were decreased at 75 and 120 mg/kg/day, and serum TSH level was

increased at 120 mg/kg/day. The T4 and TSH values were all within or just below their 

respective reference ranges. Additionally, there were no concomitant effects observed on 

thyroid weights or thyroid follicular cell height or colloid area. Minor increases in absolute 

and relative liver weights were observed at 75 and 120 mg/kg/day. There were no changes 

in clinical chemistry parameters that suggested hepatotoxicity due to permethrin 

administration. The increased liver weights were considered to be the result of liver 

hypertrophy and associated microsomal enzyme induction, a common finding with 

permethrin. Induction of hepatic enzymes can result in increased metabolism of T4. 

The 150 mg/kg/day dose was an excessive dose level as three rats were found dead on Day 

2. Therefore, the dose was lowered to 120 mg/kg/day which was an adequate high dose 

based on the clinical signs (tremor and in coordination) seen in these animals. Tremors 

were also noted at 75 mg/kg/day. 

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

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

• CV for body weight at PPS was 11.3% (maximum acceptable value of 7.57%) 

• CV for final body weight was 9.9% (maximum acceptable value of 7.47%) 

• Mean kidney weight was 1.92 g (acceptable range of 2.242-3.050 g)

• CV for pituitary weight was 18.9% (maximum acceptable value of 15.98%) 

• Mean LABC weight was 0.350 g (acceptable range of 0.447-0.855 g)

• Mean TSH level was 1.67 ng/mL (acceptable range of 4.212-24.112 ng/mL) 

Page 190 of 206
Page 191 of 206
DATA EVALUATION RECORD

PERMETHRIN

Study Type: OCSPP 890.1550, Steroidogenesis Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-29-2012 (MRID 48618603) 

Prepared for

Health Effects Division

Office of Pesticide Programs

U.S. Environmental Protection Agency 

2777 South Crystal Drive 

Arlington, VA 22202 

Prepared by

CSS-Dynamac Corporation

1910 Sedwick Road, 

Building 100, Suite B 

Durham, NC 27713 

Primary Reviewer: Signature:

Michelle Sharpe-Kass, M.S. Date: 3/26/2012

Secondary Reviewer: Signature:

Michael E. Viana, Ph.D., D.A.B.T. Date: 3/30/2012

Program Manager: Signature:

Jack D. Early, M.S. Date: 4/10/2012

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 4/10/2012

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

signing by CSS-Dynamac Corporation personnel. 

Page 192 of 206
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 193 of 206
PERMETHRIN /109701 

Primary Reviewer: Patience Browne, Ph.D. 

Health Effects Division 

Secondary Reviewer: John Liccione, Ph.D. 

Health Effects Division · 

Steroidogenesis Assay (2011) I Page I of 13 

OCSPP 890.1550/ OECD None 

Sign a tu re: :;~E:::::::::~;c.:;=-;--::::-

Da te: ~~tf::~~'iL,t. 

DATA EVALUATION RECORD 

STUDY TYPE: Steroidogenesis Assay (H295R Cells); OCSPP 890.1550 

PC CODE: 109701 DP BARCODE: 0398301 

TXR#: 0056246 

TEST MATERIAL (PURITY): Permethrin (97.7% a.i.) 

SYNONYMS: (3-phenoxyphenyl)methy I 3-(2,2-dichloroethenyl)-2,2-

dimethylcyclopropanecarboxylate 

CAS No.: 52645-53-1 

CITATION: Wagner, H. (2011) Permethrin: Hl95R steroidogenesis assay. CeeTox, 

Kalamazoo, ML Laboratory Report No.: 9 l 46V-I 003 83 STER, November 17, 

201 I. MRID 48618603. Unpublished. 

SPONSOR: Permethrin Data Group ,II, c/o Consumer Specialty Products Association, Inc., 

1900 - 17111 Street NW, Suite 300, Washington DC 

TEST ORDER#: CON-109701 -7 

EXECUTIVE SUMMARY: In a steroidogenesis assay (MRJD 48618603) H295R cells 

cultured in vitro in 24-well plates were incubated with permethrin (97.7% a.i.; Batch 

PMPSOOOOl4) at concentrations of0.0001 , 0.001, 0.01 , 0.1 , 1, 10 and 100 μMin triplicate for 48 

hours. Dimethyl sulfoxide (DMSO) was used as the vehicle, at a final concentration of 0.05%. 

Testosterone and estradiol levels were measured using HPLC/MS-MS. Three independent 

experiments were perfom1ed (Run #s 2-4). A Quality Control (QC) plate was run concurrently 

with each independent run of a test chemical plate to 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 acceptability recommendations and requirements were met, including lack of 

cytotoxicity, adequate production of testosterone and estTadiol, acceptable reproducibility (low 

%CV) and appropriate induction and inhibition with positive controls. 

No cytotoxicity was observed over the range of tested permethrin concentrations. However, 

precipitation of the permethrin was observed at the l 00 μM concentration in each run and at 

10 μM in Run #2. Accordingly, the data from these concentrations were not included in the 

analysis of the respective runs. For permethrin, there was a statistically significant (p<0.05) 

increase in testosterone production at 10 μM in Runs #3 and #4 (average l. I 4-fold), and a 

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statistically significant increase in estradiol production at 1 and 10 μM in Runs #3 and #4 

(average 1.16-fold for 1 μM and 1.51-fold for 10 μM). 

Based on hormone responses in two of the three independent runs, permethrin treatment resulted 

in statistically significant and reproducible increases in testosterone and estradiol production. 

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

(OCSPP 890.1550). 

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

Confidentiality statements were provided. 

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I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: CeeTox

Location: Kalamazoo, MI

Study Director: Hilary Wagner

Other Personnel: Colleen Toole, Director of Project Management

Karen Rutherford, Director of Laboratory Operations

David Blakeman, Senior Scientist

Briana Wallace, Lead Cell Culture Scientist

Steven McColleg, Scientist

Cameron Haines, Scientist

Francisca Wong, Scientist

Joey Burgam, Associate Scientist

Lisa Blakeman, Associate Scientist

Study Period: July 5, 2011 to November 17, 2011

2. Test Substance: Permethrin

Description: Technical, yellowish crystallized gel

Lot / Batch #: (Expiration Date) PMPS000014 (Jan 2013)

Purity: 97.7%

Solubility (in Solvent): Ethanol (25 mg/ml), DMSO (40 mg/ml)

Volatility: 3.4 x 10-7 mm Hg

Stability: Not reported (NR)

Storage conditions: Ambient

CAS #: 52645-53-1

Molecular weight: 391.3

Structure:

3. Positive Control (Inducer): Forskolin

Description (MW): White powder, (410.50)

Source (catalog #): Sigma-Aldrich, St Louis, MO (not reported)

Lot / Batch #: (Expiration Date): 109K50571V (July 2016)

Purity: 98%

Solubility (in Solvent): NR

Storage conditions: Room temperature

CAS #: 66575-29-9

4. Positive Control (Inhibitor): Prochloraz

Description (MW): White powder (376.67)

Source (catalog #): Sigma-Aldrich, St Louis, MO (not reported)

Lot / Batch #: (Expiration Date) SZE6220X (Aug 8, 2012)

Purity: 99.1%

Solubility (in Solvent): NR

Storage conditions: Room temperature

CAS #: 67747-09-5

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5. Other Materials: 22R-Hydroxycholesterol

Description: White powder

Source: Sigma Aldrich, St. Louis, MO

Lot # (expiration date): 089K4132 (retest date July, 2012) and 060M4098 (retest date June, 2013)

Purity: 99.0%

Solvent used: Ethanol

Storage conditions: Room temperature

CAS:

Molecular weight:

17954-98-2

402.65

6. Solvent/Vehicle Control: DMSO 

Description: NR

Source (catalog #): Sigma-Aldrich, St Louis, MO (not reported)

Lot / Batch #: (Expiration Date) RNBB7617

Purity: NR

Storage conditions: NR

CAS #: 67-68-5

Justification for choice of solvent: NR

Final concentration:

(% volume in assay)

0.05% (v/v)

7. Stock Medium: Dulbecco’s modified Eagle’s medium/Ham’s F12 nutrient 

mixture

Lot / Batch #: (Expiration Date) NR

Sodium bicarbonate: NR

Nu-Serum: Lot number 81515; tested for background hormone concentrations by 

performing laboratory: Testosterone: 3754 pg/mL, Estradiol: 3846 pg/mL

ITS+ premix: Lot number NR

8. Test Cells: H295R human adrenocortical carcinoma cells (ATCC CLR-2128; lot 7635054) 

were maintained in Dulbecco’s modified Eagle’s medium/Ham’s F12 nutrient mixture in a 

5% CO2 incubator at 17±2°C. Cells were grown for five passages, frozen in liquid nitrogen, 

then thawed and cultured for seven or eight additional passages prior to use in the assay. 

The following performance criteria were met (indicated by an “x”):

X Cell passage identifier. Cell Passage #: >5.8

X Cells frozen down at passage 5

X Frozen cells cultured for 4 additional passages

X Total number of passages does not exceed 10

B. METHODS

1. Pre-Test Information

a. Hormone Assay Interference Test: No details were provided on a separate chemical 

interference assay. Testosterone and estradiol levels were measured using HPLC/MS-MS, 

with a detection limit of 100 pg/mL for testosterone and 10 pg/mL for estradiol. No 

interference would be anticipated using this analytical method.

b. Hormone Extraction: After the addition of internal standards, the samples were mixed 

with methyl tert-butyl ether, centrifuged, evaporated to dryness, derivatized, and analyzed 

by HPLC-MS/MS.

c. Laboratory Proficiency Test: No information was provided. 

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2. Test Solutions: Permethrin, forskolin and prochloraz were dissolved in DMSO to make 

200 mM (permethrin) or 100 mM (forskolin or prochloraz) stocks, then serially diluted 1:10 

in DMSO. 22R-Hydroxycholesterol was dissolved in ethanol to make a 40 mM stock and 

diluted in supplemented medium to a final concentration of 10 μM. Permethrin was diluted 

1:2000 in supplemented medium containing 10 μM 22R-hydroxycholesterol to prepare 

mastermix solutions. When added to the cell culture plates, these dilutions yielded final 

concentrations of 1 μM and 10 μm forskolin, 0.1 μm and 1 μM prochloraz and 0.0001, 

0.001, 0.01, 0.1, 1, 10 and 100 μM permethrin, with the final concentration of DMSO in the 

medium being held constant at 0.05% and the final concentration of ethanol at 0.025%. 

3. Cell Plating and Preincubation: H295R cells (ATCC CLR-2128) were grown for five

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

passages. The cells were then seeded in 24-well plates at a concentration of 300,000 

cells/mL and incubated for 24 hours at 37°C in a 5% CO2 atmosphere.

4. Exposure: The cells were checked microscopically for good attachment and proper 

morphology, and the medium was removed and replaced with 1 mL of medium containing 

10 μM 22R-hydroxycholesterol and the concentrations of test chemicals indicated in 

triplicate according to the schematic presented in Table 1. 

TABLE 1. Dosing Schematic for the Exposure of H295R Cells to Permethrin(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. Dosing was calculated based on a total volume of 1 mL per well.

A concurrent quality control (QC) plate was included with each of the three independent 

runs of the test chemical plates to demonstrate the assay’s response to forskolin (an inducer 

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

estradiol production). The QC plate was prepared and dosed in the same manner with either 

forskolin or prochloraz according to the schematic presented in Table 2. 

TABLE 2. Dosing 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 + MeOHd DMSO + MeOH DMSO + MeOH

C Forskolin 1μM Forskolin 1μM Forskolin 1μM Prochloraz 0.1μM Prochloraz 0.1μM Prochloraz 0.1μM

D Forskolin 10μM Forskolin 10μM Forskolin 10μM Prochloraz 1μM Prochloraz 1μM Prochloraz 1μM

a Data were obtained from page 15 of the study report. Dosing was calculated based on a total volume of 1 mL per well.

b Blank wells received medium containing 10μM 22R-hydroxycholesterol. 

c Background wells received media without 22R-hydroxycholesterol.

d MeOH = 70% methanol was added to these wells for 30 minutes at room temperature following medium removal.

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Following dosing, the plates were incubated for 48 hours under the conditions previously 

described. The medium from each well was removed, split into two equal volume aliquots, 

and frozen at −80°C until hormone measurements. 

5. Cell Viability/Cytotoxicity Assay: Cell viability was determined using the MTT assay

immediately after removal of the culture medium. Methanol was used as the positive 

control for cell death. The plate wells were rinsed three times with PBS, and 0.5 ml of a 0.5 

mg/mL MTT solution in supplemented medium containing 10 μM 22R-hydroxycholesterol 

was added to each well of the test chemical and QC plates. Plates were incubated for 3 

hours, after which the MTT solution as removed and 0.5 mL of isopropanol was added to 

each well. Plates were incubated at room temperature for 20 min with shaking, and 

absorbance at 570 nM and 650 nm were measured to calculate the MTT value for each well.

6. Hormone Measurement System: The concentration of each hormone (testosterone and 

estradiol) was evaluated in triplicate in the culture medium. Hormones were extracted from 

the samples with MTBE, derivatized, and analyzed by HPLC/MS-MS. The lower limit of 

quantification (LLQ) for this measurement system was 100 pg/mL for testosterone and 10 

pg/mL for estradiol. 

The following performance criteria were met (indicated by an “x”):

X Method detection limit (100 pg/mL testosterone; 10 pg/mL estradiol)

X Spiked sample recovery acceptable for two concentrations of testosterone and estradiol (mean measured amount 

from triplicate samples within 30% of nominal concentration)

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

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

NA Test compound tested for interference with measurement system

C. DATA ANALYSIS: To evaluate the relative increase or decrease of hormone production 

after test chemical exposure, the results were normalized to the mean solvent control (SC) 

value for each assay and results were expressed as fold-change relative to the SC in each 

exposure plate. All data were expressed as mean ± standard deviation (SD). Relative 

changes were calculated as follows:

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

solvent control 

For forskolin induction of testosterone, relative change was calculated both with and 

without subtracting the background hormone production from the forskolin-treated wells 

and blank and solvent control wells. This was done because of the high background 

production of testosterone and the small dynamic range for forskolin induction. 

Background hormone production was calculated from three wells with cells on the QC plate 

that received no 22R-hydroxycholesterol at the time of exposure. 

Normality of the data was evaluated using Shapiro-Wilk’s test. Homogeneity of the 

variances between the treatment groups was evaluated using Levene’s test. If the p-values 

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

and the control group was evaluated using 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 

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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, pvalues were less than or equal to 0.05 in either the normality or the homogeneity test, the 

non-transformed data set was analyzed using the nonparametric Kruskal-Wallis test 

followed by Dunn’s test to evaluate statistical significance between each treatment group 

and the control group. 

HPLC-MS/MS data were acquired using proprietary software application MassHunter 

Workstation Acquisition (Version B.03.01 (B2065), Agilent Technologies, Inc.). Data were 

processed (integrated) using the software application MassHunter Quantitative Analysis for 

QQQ (version B.04.00/Build 4.0.225.0. Agilent Technologies, Inc.). Calibration plots of 

area ratio versus testosterone and estradiol concentrations were constructed and a weighted 

1/x2 linear regression applied to the data using MassHunter Quantitative Analysis for QQQ.

II. RESULTS

A. TEST COMPOUND: The basal hormone production for the SC (DMSO) was greater than 

500 and 40 pg/mL for testosterone and estrogen (in the presence of 22R-hydroxycholesterol. 

Precipitation of the permethrin was observed in one well at 10 μM in Run #2, and in all 

three wells at 100 μM for all three runs. Cytotoxicity was not observed at any tested 

concentration. The between plate coefficient of variation (CV) for the SC based on absolute 

concentrations (calculated by the reviewer) was 19% for testosterone and 26% for estradiol, 

meeting the guideline recommendation of ≤30%. The CV within each run for the SC was 

2.36-3.08% for testosterone and 0.70-1.88% for estradiol, both meeting the guideline 

recommendation of ≤30%. 

The results from the test runs with permethrin are summarized in Table 3 and presented 

graphically in Figures 1 and 2. A statistically significant increase in testosterone production 

was observed at 10μM in Runs #3 and #4 (average 1.14-fold), and a statistically significant 

increase in estradiol production as observed at 1μM and 10μM in Runs #3 and #4 (average 

1.16-fold for 1 μM and 1.51-fold for 10 μM). 

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TABLE 3. Mean (±SD) Hormone Concentrations Following Treatment with Permethrin for 48 Hours.a

Nominal 

Concentration 

(μM)

Trial 2 Trial 3 Trial 4 Trial 2 Trial 3 Trial 4 Mean ± SD

Statistical 

Significan

ce

Testosterone (pg/mL) Fold Difference

DMSO 2577 1799 2516 — — — — — —

0.0001 2552 1853 2447 0.99 1.03 0.97 1.00 0.03 —

0.001 2453 1890 2368 0.95 1.05 0.94 0.98 0.06 —

0.01 2468 1853 2362 0.96 1.03 0.94 0.98 0.05 —

0.1 2584 1889 2344 1.00 1.05 0.93 0.99 0.06 —

1 2710 1979 2456 1.05 1.10 0.98 1.04 0.06 —

10 2967 2069 2845 NA 1.15 1.13 1.14 0.01 Run 3, 4

100 2657 2075 2832 NA NA NA NA NA NA

Estradiol (pg/mL) Fold Difference

DMSO 258 151 211 — — — — — —

0.0001 242 163 218 0.94 1.08 1.03 1.02 0.07 —

0.001 247 165 212 0.96 1.09 1.00 1.02 0.07 —

0.01 245 161 213 0.95 1.06 1.01 1.01 0.06 —

0.1 254 159 214 0.99 1.06 1.01 1.02 0.04 —

1 287 186 239 1.11 1.23 1.13 1.16 0.06 Runs 3, 4

10 402 232 314 NA 1.53 1.49 1.51 0.03 Runs 3, 4

100 358 235 334 NA NA NA NA NA NA

a Data were obtained from pages 26 - 27 and 33-35 of the study report. The lower limit of quantification (LLQ) was 100 

pg/mL for testosterone and 10 pg/mL for estradiol.

NA Not applicable. Concentration not analyzed because of precipitation observed post-exposure under the microscope.

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FIGURE 1. Change in Testosterone Production Relative to 2,4-D Concentration.

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FIGURE 2. Change in Estradiol Production Relative to 2,4-D Concentration. 

B. CYTOTOXICITY: Permethrin was not cytotoxic to the H295R cells (Table 4). Viability 

in each run ranged from 91.0-100.2% relative to solvent controls. 

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

Permethrin 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

Permethrin 0.0001 95.7 4.13 94.6 3.23 98.3 2.44

Permethrin 0.001 95.9 1.74 97.0 3.18 98.2 1.76

Permethrin 0.01 95.9 1.62 98.4 2.11 100.2 0.47

Permethrin 0.1 99.1 1.79 98.1 2.55 98.1 0.72

Permethrin 1 95.9 2.43 91.0 3.08 97.3 0.72

Permethrin 10 96.5 3.48 99.4 5.54 98.3 2.39

Permethrin 100 95.3 2.35 94.8 6.90 99.4 1.16

a Data were obtained from pages 20-21 of the study report.

NA Not applicable

SC Solvent control 

C. QC PLATE: Concentrations of testosterone and estradiol, and the fold induction or 

inhibition in the presence of forskolin and prochloraz, on the QC plates are shown in Table 

5. The minimum basal hormone production levels (500 pg/mL for testosterone, 40 pg/mL 

for estradiol) were met in both blank and SC wells for both testosterone and estradiol. Cells 

incubated with 10 μM forskolin induced testosterone production by 1.61- to 1.89-fold which 

is below the Guideline criteria of ≤2-fold; however, when the background is subtracted, 10 

μM forskolin induced testosterone an average of 2.47-fold. Estradiol production was 

induced with 10 μM forskolin by 9.3- to 11.1-fold. Cells incubated with 1 μM prochloraz 

inhibited testosterone and estradiol production by ≤0.49-fold. Thus, the guideline 

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

testosterone and estradiol. 

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

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

meets the Guideline criteria of ≤30% for the assay. The SC %CVs within each run for the 

QC plates were 1.61 to 8.28% for testosterone and 2.90 to 6.16% for estradiol. Evaluation 

of the interplate and intraplate variability (%CV) indicated consistent performance of the 

assays.

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TABLE 5. Hormone Concentrations Following Treatment with Forskolin or Prochloraz for 48 Hours.a

Compound Conc. 

(μM)

Run 2 Run 3 Run 4 Run 2 Run 3 Run 4 Mean b ± SD b

Testosterone (pg/mL) Fold Difference

Background NA 1135 1073 1262 0.45 0.57 0.54 0.52 0.06

Blank NA 2581 1873 2304 1.03 1.00 0.99 1.01 0.02

DMSO NA 2511 1874 2339 1.00 1.00 1.00 1.00 —

Forskolin 1 3764 2595 3120 1.50c 1.38 c 1.33 c 1.40 c 0.09

Forskolin 10 4751 3019 3780 1.89 d 1.61 d 1.62 d 1.71 d 0.16

Prochloraz 0.1 1816 1149 1818 0.72 0.61 0.78 0.70 0.09

Prochloraz 1 1188 501 1153 0.47 0.27 0.49 0.41 0.12

Estradiol (pg/mL) Fold Difference

Background NA 41 56 77 0.21 0.31 0.35 0.29 0.07

Blank NA 208 182 228 1.04 1.01 1.02 1.02 0.02

DMSO NA 200 179 223 1.00 1.00 1.00 1.00 —

Forskolin 1 1343 1254 1538 6.72 7.01 6.91 6.88 0.15

Forskolinc 10 2116 1661 2477 10.60 9.28 11.13 10.34 0.95

Prochloraz 0.1 133 104 171 0.67 0.58 0.77 0.67 0.10

Prochloraz 1 57 52 83 0.29 0.29 0.37 0.32 0.05

a Data were obtained from page 22.-23 of the study report. . 

b Calculated by the reviewers.

c When the background is subtracted, the values are 1.91-, 1.90-, and 1.73-fold difference for Runs 2, 3, and 4, respectively, 

with a mean of 1.84-fold change.

d When the background is subtracted, the values are 2.63-, 2.43-, and 2.34-fold difference for Runs 2, 3, and 4, respectively, 

with a mean of 2.47-fold change.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Permethrin exposure resulted in statistically 

significant (13-15%) increases in testosterone at the highest concentration analyzed (10 μM) 

in two of the three independent runs of the assay. The 10 μM concentration could not be 

analyzed in the remaining assay run because precipitation was observed. Statistically 

significant increases (13-53%) increases in estradiol were also observed at the two highest 

concentrations (1 and 10 μM) analyzed in two of three independent runs. The 10 μM 

concentration could not be analyzed in the remaining assay run because precipitation was 

observed. 

B. AGENCY COMMENTS: Guideline acceptability recommendations and requirements 

were met, including lack of cytotoxicity, adequate production of testosterone and estradiol, 

acceptable reproducibility (low %CV) and appropriate induction and inhibition with positive 

controls. 

A statistically significant increase in testosterone production was observed at 10 μM in Runs 

#3 and #4 (average 1.14-fold), and a statistically significant increase in estradiol production 

as observed at 1 μM and 10 μM in Runs 3 and 4 (Average 1.16-fold for 1 μM and 1.51-fold 

for 10 μM). 

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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: 

• No information was provided on the laboratory proficiency testing. 

• Recoveries of radioligands were not reported for MTBE extractions prior to HPLC/MSMS determination of steroids 

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