UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

MEMORANDUM 

Date: 6/29/2015 

OFFICE OF CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

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

PC Code: 035506 

Decision No.: 462133, 467022 

Petition No.: NA 

Risk Assessment Type: NA 

TXRNo.: 0052347 

DP Barcode: D399669, D403535 

Registration No.: NA 

Regulatory Action: NA 

CaseNo.: NA 

CAS No.: 330-55-2 

MRID No.: See Table 40CFR: NA 

Greg Akerman, Ph.D . . ~- .tY~ . .,.._ ___ _ Immediate Office , - -0 

FROM: 

Health Effects Division (7509P) 

THROUGH: Jess Rowland ~ "Dc::;:252----

Deputy Director 

Health Effects Division 

TO: Jolene Trujillo 

Biologist/Chemical Review Manager 

Risk Management and Implementation Branch V 

Pesticide Re-evaluation Division (7505P) 

I. ACTION REQUESTED 

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 1 assays 

submitted in response to the agency's Test Order for linuron: Test Order# EDSP-035506-122. 

Page 1 of2 

Page 1 of 124
Page 2 of 2 

II. RESPONSE

Attached are the EDSP Tier 1 assay DERs for linuron.

III. MRID Table

Chemical: Linuron PC Code: 035506

Guideline Assay MRID 

890.1100 Amphibian Metamorphosis Assay (Frog) NA

890.1150 Androgen Receptor Binding (Rat Prostate) NA

890.1200 Aromatase Assay (Human Recombinant) 48617401

890.1250 Estrogen Receptor Binding 48617402

890.1300 Estrogen Receptor Transcriptional Activation 

(Human Cell Line HeLa-9903) NA

890.1350 Fish Short-Term Reproduction 48671701

890.1400 Hershberger (Rat) NA

890.1450 Female Pubertal (Rat) 48685501

890.1500 Male Pubertal (Rat) NA

890.1550 Steroidogenesis (Human Cell Line – H295R) NA

890.1600 Uterotrophic (Rat) 48617403

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

Page 2 of 124
DATA EVALUATION RECORD

LINURON

Study Type: OCSPP 890.1200; Aromatase Assay

EPA Contract No. EP10H001452 

Task Assignment No. 2-21-2012 (MRID 48617401) 

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:

David A McEwen, B.S. Date: 2/24/2012

Secondary Reviewer: Signature:

Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 2/28/2012

Program Manager: Signature:

Jack D. Early, M.S. Date: 3/02/2012

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 3/02/2012

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

signing by CSS-Dynamac Corporation personnel. 

Page 3 of 124
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 4 of 124
LINURON I 035506 

Primary Reviewer: Vincent Chen, M.S. 

Health Effects Division (7509P) 

Secondary Reviewer: Greg Akerman. Ph.D. 

Health Effects Division (7509P) 

Aromatase (Human Recombinant) Assay (2011) I Page l of 13 

OCSPP 890.1200 I OECD None 

Signature~~ Date: z.y -Sv tJ "Z-OJ.._$ 

Signature: ~ /?--. 

Date: -. ~ J 1 S"' 

Template version 08/2011 

DATA EVALUATION RECORD 

STUDY TYPE: Aromatase (Human Recombinant); OCSPP 890.1200 

PC CODE: 035506 DP BARCODE: D399669 

TXR: 0052347 CAS: 330-55-2 

TEST MATERIAL (PURITY): Linuron (99.4% a.i.) 

SYNONYMS: 

IUPAC: 

CAS: 

3-(3,4-dichlorophenyl)-1-methoxy-1-methylurea 

N'-(3,4-dichlorophenyl)-N-methoxy-N-methylurea 

CITATION: Wilga, P.C. (2011) Linuron: Human Recombinant Aromatase Assay, CeeTox, 

Inc [4717 Campus Drive, Kalamazoo, MI 49009, US]. Laboratory Study No.: 

9138V-100325AROM. 07 December 2011. MRID 48617401. Unpublished. 

SPONSOR: Tessenderlo Kerley, Inc.; 2255 N. 44th Street, Suite 300, Phoenix, AZ 85008, USA 

TEST ORDER: EDSP-035506-122 

EXECUTIVE SUMMARY: In an in vitro aromatase (CYP 19) assay (MRID 48617401), 

linuron (99.4% a.i., Batch#: 1012A3876) in dimethyl sulfoxide (DMSO; $1 %) was incubated 

with human recombinant aromatase and tritiated androstenedione ([1~- H(N)]-androst-4-ene3, 17-dione; [

3

H]-ASDN) for 15 minutes at 37°C to assess the potential oflinuron to inhibit 

aromatase activity. Linuron was tested at logarithmic concentrations from 10-10 M to 10-3 Min 

Run 1 and from 10-10 M to 10-3.

5 Min Runs 2 and 3, due to precipitation observed at 10-3 Min 

Run 1. 

Aromatase activity was determined by measuring the amount oftritiated water produced at the 

end of a 15-minute incubation period for each concentration of chemical. Tritiated water was 

quantified using liquid scintillation counting (LSC). Three independent runs were conducted, 

and each run included a full activity control, a background activity control, a positive control 

series (10-10 M to 10-5 M) using a known inhibitor (4-hydroxyandrostenedione; 4-0H ASDN), 

and the test chemical series (10-10 M to 10-3 or 10-3.

5 M) with 3 repetitions per concentration. 

Aromatase activity in the full activity controls ranged from 0.494 to 0.726 nmol·mgprotein-1·min-1 for the three test runs, with a mean and standard deviation of 0.598 ± 0.086 

nmol·mg-protein-1·min-1. Activity in the background controls ranged from 0.20% to 0.31 % and 

averaged 0.27% of the full activity controls. The response of the full activity controls and 

background controls was acceptable for each run. 

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Aromatase (Human Recombinant) Assay (2011) / Page 2 of 13

LINURON / 035506 OCSPP 890.1200 / OECD None

Results for the positive control were generally within the recommended ranges for the top of the 

curve, bottom curve, Hill slope, log IC50, and coefficient of variation for replicates of each 

concentration within runs. Also, the coefficients of variations (%CVs) for replicates of each 

concentration of linuron within a run were generally within the 15% guideline. For 4-OH 

ASDN, the estimated log IC50 averaged –7.27 M, and the slope was –1.00. 

For linuron, aromatase activity averaged 0.607 ± 0.071 nmol∙mg-protein−1

∙min−1 at the lowest 

tested concentration of 10–10 M and 0.372 ± 0.023 nmol∙mg-protein–1∙min−1 at the highest 

successfully tested concentration of 10−3.5 M (highest non-precipitating dose; precipitation was 

observed in Run 1 at 10−3 M). The data for linuron were not suitable for modeling. The mean 

aromatase activity of 10–3.5 M linuron was 68.1% of control activity, and the best fit mean 

inhibition response curve was a straight line (14% inhibition at 10–4 M and essentially no 

inhibition was observed at other doses). The average lowest portion of the response curve across 

runs was between 50 and 75% activity, indicating an equivocal response at soluble 

concentrations. 

Based on the data from the average response curve, linuron is classified as Equivocal for 

aromatase inhibition 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 6 of 124
Aromatase (Human Recombinant) Assay (2011) / Page 3 of 13

LINURON / 035506 OCSPP 890.1200 / OECD None

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: Linuron technical

Description: Powder

Source: Tessenderlo Kerley, Inc.

Batch # (Expiration Date): 1012A3876 (May 3, 2013)

Purity: 99.4% a.i.

Storage Conditions: Room temperature

Stability: Not reported

Solvent: DMSO

Solubility: Visually soluble at 10−3.5 M in 2 of 3 test runs.

Highest Concentration Tested: 10−3 M; however, precipitation was observed at this dose in Run 1

Stock Solution Preparation: Not reported

Molecular Weight: 249.09 g/mol

CAS #: 330-55-2

Structure:

2. Non-Labeled Substrate: Androstenedione (ASDN)

CAS #: 63-05-8

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

Batch # (Expiration Date): L1712 (April 2016)

Purity: 99.8%

3. Radiolabeled Substrate: [1β-3H(N)]-Androst-4-ene-3,17-dione; ([3H]-ASDN)

Source: Perkin Elmer (Cat. # NET-926)

Batch # (Expiration Date): 619344 (January 10, 2012)

Radiochemical Purity (Supplier): >97%

Specific Activity: 26.3 Ci/mmol

Radiochemical Purity (In-Lab

Determination): Not reported

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

CAS #: 566-48-3

Source: Sigma-Aldrich (Cat. # F2552)

Batch # (Expiration Date): 081K2133 (March 2015)

Purity: 99.6%

5. Solvent (Vehicle Control): DMSO

Source: Sigma-Aldrich (Cat. # D2650)

Batch # (Expiration Date): RNBB7886 (expiry date not provided)

Justification for Choice of Solvent: Acceptable vehicle by OCSPP guideline 890.1200

Concentration (% of Total Volume in 

Assays): ≤1% v/v

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

Source: BD Biosciences GentestTM (Woburn, MA; Cat. # 456260)

Lot # (Expiration Date): 19701 (July 2014)

Protein Concentration: 3.7 mg/mL

Cytochrome C Reductase Activity: 540 nmol/min/mg

Aromatase Activity: 5.7 pmol/min/pmol P450

N

H

Cl

Cl

O

N

O CH3

CH3

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LINURON / 035506 OCSPP 890.1200 / OECD None

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 tritiated material added to each incubation tube 

was approximately 0.1 μCi.

Test chemical(s) were formulated in DMSO such that the total volume of DMSO used per 

assay was no more than 1% v/v of the total assay volume to minimize the potential for the 

solvent to inhibit the enzyme. The report specified that DMSO was chosen over ethanol as 

the solvent of choice for the following reasons: 1) DMSO was listed as one of the vehicles 

acceptable for use in OCSPP 890.1200 guideline; 2) DMSO was not as volatile as ethanol 

and so evaporation was less of a concern in the assay, and 3) DMSO was more accurate to 

pipette because of density and viscosity. 

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

Fresh dilutions of the stock solution were prepared in the same solvent as the stock solution 

on the day of use. Dilutions were prepared such that the target concentrations of the positive 

control substance (10–10 M 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 Biosciences, and stored at –80 ± 

10 oC for no longer than 5 months for this study. Before use of the microsomes, the batch 

was thawed and sub-aliquoted at 0.008 mg/mL 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±2°C

Incubation time 15 min

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 minimum aromatase activity in 

the full activity control samples was determined by the laboratory to be 0.494 nmol/ mg

protein/min, which was greater than the minimum recommended aromatase activity of 0.1 

nmol/mg-protein/min.

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

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

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

Page 8 of 124
Aromatase (Human Recombinant) Assay (2011) / Page 5 of 13

LINURON / 035506 OCSPP 890.1200 / OECD None

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 nmol∙mg-protein–1∙min–1. 

4. Demonstration of Proficiency: No information was provided concerning demonstration of 

proficiency. However, the report stated that prior to conducting this assay using linuron; a 

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

control (4-OH ASDN), econazole, fenarimol, nitrofen, and atrazine. Proficiency was 

demonstrated when the positive control met the performance criteria from the guideline and 

by the correct classification of the four proficiency chemicals in Table 6 of the guideline. 

Data was generated by CeeTox and is maintained at CeeTox.

a. Positive Control: Data were not provided. 

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

(new/historical data for laboratory) were not reported in regards to 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 were reported for the test runs only (Table 2), 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): Data were not provided. The positive control data for slope, top and bottom 

percent, and log IC50 met the criteria as listed in section (i) of OCSPP 890.1200. 

TABLE 2. Performance Criteria for the Positive Controla

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

Slope –1.2 –0.8 -1.03 -0.97

Top (%) 90 110 96.6 100.8

Bottom (%) –5 +6 0.1 1.0

Log IC50 (M) –7.3 –7.0 -7.3 -7.2

a Data were obtained from page 27 of the study report.

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b. Proficiency Chemicals: Data were not provided. 

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---

5. Determination of Aromatase Activity with Test Chemical: The response of aromatase 

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

during three independent runs. Solubility was visually assessed (presence of cloudiness or a 

precipitate). Precipitation was observed in Run 1 (moderate amount) at 10–3 M, but the test 

compound was soluble at 10–3.5 M in Runs 2 and 3. The full enzymatic activity (≥95% for 

the means of each run) was obtained at the two lowest concentrations of linuron, defining 

the top of the concentration-response curve. 

TABLE 4. Test Chemical Study Design for Each Test Run

Sample Type Repetitions (Tubes) Description Reference or Chemical (M)

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

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

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

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

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

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

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

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

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

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

Linuron Conc 1b 3 All test components plus linuron 1×10–3

Linuron Conc 2b 3 All test components plus linuron 1×10–3.5

Linuron Conc 3 3 All test components plus linuron 1×10–4

Linuron Conc 4 3 All test components plus linuron 1×10–5

Linuron Conc 5 3 All test components plus linuron 1×10–6

Linuron Conc 6 3 All test components plus linuron 1×10–7

Linuron Conc 7 3 All test components plus linuron 1×10–8

Linuron Conc 8 3 All test components plus linuron 1×10–9

Linuron Conc 9 3 All test components plus linuron 1×10–10

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

b The 10−3 M concentration of linuron was used only in Run 1 due to observed precipitation, and Runs 2 and 3 included a 

concentration of 10−3.5 M.

C. DATA ANALYSIS

1. Raw Data: Raw data were converted to aromatase activity (nmol/mg protein/min) and 

percent control for the positive control and test chemical. The following raw data and 

calculated endpoints for each run were included in the report (Table 5). 

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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: Statistical analyses and graphical displays were conducted using 

IDBS XLfit v5.2.0.0 (Guildford, UK). Basic statistical analyses were 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 least squares nonlinear regression analysis with 

weights equal to 1/Y. Model fits were carried out using a 4-parameter regression model 

(XLfit v5.2.0.0, Fit Model 208) and Tukey’s Bi-Weight statistical analysis for outlier 

analysis. For each run, percent of control activity was plotted versus the log (base 10) or 

half-log of the linuron concentration. Each run was plotted with the data’s best fit curve. 

Additionally, the average inhibition response curve across all runs was also plotted. 

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

three runs, using the categories presented in Table 6. 

TABLE 6. Interpretation of Results

Criteria Interpretation

Data fit 4-parameter 

nonlinear regression 

model

Average curve across runs crossed 50% a Inhibitor

Average lowest portion of curves across runs is between 50% and 75% activity b Equivocal

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

Data do not fit model ---

a Ordinarily, an inhibition curve will fall from 90% to 10% over 2 log units with a slope near –1. Unusually steep curves 

may indicate protein denaturing or solubility issues. If the slope of the curve is steeper than –2.0, the result is classified as 

equivocal.

b If the test compound was not soluble above 10–6 M and the inhibition curve does not 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 to 0.726 nmol∙mg-protein−1∙min−1 for the three test runs, with a mean and standard 

deviation of 0.598 ± 0.086 nmol∙mg-protein−1∙min−1. Activity in the background controls 

ranged from 0.20% to 0.31% and averaged 0.27% of the full activity controls. One of the 

background activity controls in Run 2 was considered an outlier and was excluded from 

statistical analysis. 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 0.585 ± 0.070 nmol∙mg-protein−1∙min−1 at the lowest tested concentration 

Page 11 of 124
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(10−10 M) and 0.004 ± 0.000 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 three runs is presented in Table 7, along 

with the overall standard deviation, standard error of the mean, and %CV. Inhibition 

response curves for the positive control from each run are shown in Figure 1. These results 

were within the recommended ranges for the top of the curve, bottom curve, Hill Slope, log 

IC50, and coefficient of variation for replicates of each concentration within runs. The 

coefficient of variations (CVs) for replicates within each concentration of 4-OH ASDN and 

linuron were within the 15% guideline. 

TABLE 7. Effect of linuron on Aromatase Activity (as Percent of Control) from Independent Runs a

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

4-OH ASDN 

(positive control)

–5 3 0.66 0.06 0.02 8.8

–6 3 5.80 0.49 0.20 8.5

–6.5 3 15.39 1.38 0.56 9.0

–7 3 34.29 2.16 0.88 6.3

–7.5 3 62.08 2.30 0.94 3.7

–8 3 83.39 3.84 1.57 4.6

–9 3 97.65 1.14 0.47 1.2

–10 3 98.07 3.34 1.36 3.4

Linuron –3 1 52.71 0.76 0.31 1.4

–3.5 2 68.11 2.35 0.96 3.5

–4 3 85.65 2.29 0.93 2.7

–5 3 100.68 1.42 0.58 1.4

–6 3 100.83 3.69 1.51 3.7

–7 3 100.74 3.54 1.44 3.5

–8 3 101.96 2.93 1.20 2.9

–9 3 99.26 9.99 4.08 10.1

–10 3 101.61 3.12 1.27 3.1

a Values were calculated by the reviewers based on data provided on pages 40-54 of the study report. 

SD Standard Deviation

SEM Standard Error of the Mean

CV Coefficient of Variance

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FIGURE 1. Inhibition Response Curves for 4-OH ASDN. 

C. TEST SUBSTANCE: For linuron, aromatase activity averaged 0.607 ± 0.071 

nmol∙mg-protein−1∙min−1 at the lowest tested concentration of 10–10 M and 0.372 ± 0.023 

nmol∙mg-protein–1∙min−1 at the highest successfully tested concentration of 10−3.5 M (highest 

Page 13 of 124
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non-precipitating dose). Precipitation was observed in Run 1 at 10−3 M. In Run 2, 10–9 M 

linuron resulted in aromatase levels with a CV of 18.8% within plate, exceeding the 

guideline of 15%. All other linuron and ASDN samples had CVs within guideline. The 

mean aromatase activity of linuron (expressed as % full control activity) for each 

concentration tested across all three runs is provided in Table 7 (presented above), along 

with the overall standard deviation, standard error of the mean, and %CV. Inhibition 

response curves for linuron from each run are shown in Figure 2, and the average inhibition 

response curve across all runs is shown in Figure 3. 

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FIGURE 2. Inhibition Response Curves for Linuron (Runs 1-3). 

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FIGURE 3. Mean Inhibition Response Curve for linuron. 

The data for linuron were not suitable for modeling. The mean aromatase activity of 10–3.5 M 

linuron was 68.11% of control activity, and the best fit mean inhibition response curve 

(Figure 3) was a straight line (14% inhibition at 10–4 M and essentially no inhibition was 

observed at other doses). The average lowest portion of the response curve across runs was 

between 50 and 75% activity, indicating an equivocal response at soluble concentrations. 

The effect of the positive control on inhibition of aromatase activity is presented in Table 8. 

For 4-OH ASDN, the estimated log IC50 averaged –7.27 M, and the Hill Slope was –1.00. 

Confidence in these values is very high due to the small variation (<4% CV). 

TABLE 8. Effect of linuron and 4-OH ASDN on Aromatase Activity (as Percent of Control) from Independent Runs a

Chemical Run 1 Run 2 Run 3 Mean SD %CV

Log IC50 (M)

Linuron NA NA NA NA NA NA

4-OH ASDN –7.30 –7.30 –7.20 –7.27 0.06 –0.79

Hill Slope

Linuron NA NA NA NA NA NA

4-OH ASDN –0.99 –0.97 –1.03 –1.00 0.03 –3.07

a Data were provided on page 27 of the study report. Mean, SD, and %CV were calculated by the reviewers based on the 

data from the individual runs. 

SD Standard Deviation

CV Coefficient of Variance

NA Not applicable. Values for linuron were not suitable for modeling.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: Linuron was classified as “equivocal” in its 

response as defined by the Guideline OCSPP 890.1200. Although a decrease in aromatase 

activity was identified at the very highest concentration of linuron tested (e.g., 10-3 M) in the 

first run, solubility issues were observed so this concentration was not included in the data 

interpretation. In subsequent runs of the assay, 10–3.5 M linuron reduced aromatase activity, 

but not to a level that would classify this compound as an inhibitor of aromatase activity. 

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

activity averaged 0.585 ± 0.070 nmol∙mg-protein−1∙min−1 at the lowest tested concentration 

(10−10 M) and 0.004 ± 0.000 nmol∙mg-protein−1∙min−1 at the highest tested concentration 

(10−5 M). These results were within the recommended ranges for the top of the curve, 

bottom curve, Hill Slope, log IC50, and %CV for replicates of each concentration within 

runs. The CVs for replicates within each concentration of 4-OH ASDN and linuron were

generally within the 15% guideline. 

For linuron, aromatase activity averaged 0.607 ± 0.071 nmol∙mg-protein−1∙min−1 at the 

lowest tested concentration of 10–10 M and 0.372 ± 0.023 nmol∙mg-protein–1∙min−1 at the 

highest successfully tested concentration of 10−3.5 M (highest non-precipitating dose). 

Precipitation was observed in Run 1 at 10−3 M. For 4-OH ASDN, the estimated log IC50

averaged –7.27 M, and the Hill Slope was –1.00. Confidence in these values is very high 

due to the small variation (<4% CV). 

The data for linuron were not suitable for modeling. The mean aromatase activity of 

10–3.5 M linuron was 68.1% of control activity, and the best fit mean inhibition response 

curve was a straight line (14% inhibition at 10–4 M and essentially no inhibition was 

observed at other doses). Because the lowest portion of the curves across the three runs was 

between 50 and 75% activity, linuron was determined to be equivocal for aromatase 

inhibition in the assay.

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

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

• Proficiency chemical data were not provided.

• Information on the stability of the stock solution was not reported. 

Page 17 of 124
DATA EVALUATION RECORD

LINURON

Study Type: OCSPP 890.1250, Estrogen Receptor Binding Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 3-06-2012 

(Revisions to MRID 48617402 to include Saturation binding data; 

Main study was originally reviewed under TA 2-21-2012) 

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: 2/23/2012

Secondary Reviewer: Signature:

Scott D. Studenberg, Ph.D., D.A.B.T. Date: 7/20/2012

Program Manager: Signature:

Jack D. Early, M.S. Date: 7/27/2012

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 7/27/2012

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

signing by CSS-Dynamac Corporation personnel. 

Page 18 of 124
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 19 of 124
LINURON/ 035506 

Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) I Pagel of 17 

OCSPP 890.1250/ OECD None 

Primary Reviewer: Anwar Y. Dunbar, Ph.D. 

Risk Assessment Branch 1, Health Effects Division 

Secondary Reviewer: Greg Akerman, Ph.D. 

Risk Assessment Branch 1, Health Effects Division 

Signature: ~ tJ/.:. 

Date: 4 ~7-t i;,- Signature: ~~ 

Date: <Zlr.:rJr:- Temptate version 0812011 

DATA EVALUATION RECORD 

STUDY TYPE: Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (ER-RUC); 

OCSPP 890.1250 

PC CODE: 035506 DP BARCODE: D399699 

TXR#: 005234 7 CAS No.: 330-55-2 

TEST MATERIAL (PURITY): Linuron (99.4% a.i.) 

SYNONYMS: Not provided 

CITATION: Willoughby, J.A., Sr. (2011). Linuron: Estrogen receptor binding (rat uterine 

cytosol). CeeTox, Inc., Kalamazoo, Ml. Laboratory Study No.: 9138V100325ERB, December 8, 2011. MRID 48617402. Unpublished. 

Willoughby, J.A. (2012). Supplemental Information - Laboratory Proficiency 

Data for ERTA assays and Saturation Binding Data for AR and ER Binding 

Assays for Assorted Chemicals. CeeTox, Inc., Kalamazoo, MI. July 19 2012. 

MRID 48843501. Unpublished. 

SPONSOR: Tessenderlo Kerley, Inc., 2255 N. 44th Street, Suite 300, Phoenix, AZ 

TEST ORDER#: EDSP-035506-122 

EXECUTIVE SUMMARY: In an estrogen receptor (ER) binding assay (MRID 48617402), 

uterine cytosol from Sprague-Dawley rats was used as the source of ER to conduct binding 

experiments. A saturation binding experiment was conducted to demonstrate that the ER in the 

rat uterine cytosol was present in reasonable numbers and was functioning with appropriate 

affinity for the radio-labeled reference estrogen prior to conducting ER competitive binding 

experiments. The competitive binding experiment measured the binding of a single concentration 

of [

3

H]-l 7P-estradiol ( 1 nM) in the presence of increasing concentrations of linuron (logarithmic 

increase from 10-10 to 10-3 M). DMSO was used as a vehicle at a final concentration of2%. 

The assay included 19-norethindrone as a weak positive control, octyltriethoxysilane as a 

negative control, and l 7P-estradiol as the natural ligand reference material. 

Saturation binding data were not originally provided in the study report; however, summarized 

saturation binding data (MRID 48843501) from the performing laboratory were submitted 

following a request by the Agency. The Ka for [

3

H]-l 7P-estradiol was 0.303±0.090 nM, and the 

estimated Bmax was 33.59±0.70 fmol/100 μg protein. The Ka for each run was within the 

expected range of 0.03 to 1.5 nM. 

Page 20 of 124
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 2 of 17

LINURON/ 035506 OCSPP 890.1250/ OECD None

Precipitation was observed at 10−3 M for all runs; therefore, the data from the 10−3 M linuron 

concentration were not included in the analysis. In the competitive binding experiment, linuron 

had no effect on the specific binding of radiolabeled estradiol to ER at concentrations of ≤10−4 M 

in all runs. A mean log IC50 was not calculated for linuron as 50% binding displacement was not 

achieved in any run. The mean log IC50 was −8.9 M for the natural ligand, 17β-estradiol, and 

−5.5 M for the weak positive control, which also had a relative binding affinity (RBA) of 

0.042% compared to the natural ligand. 

Based on the results of the three runs, linuron is classified as Not Interactive in the Estrogen 

Receptor Binding Assay. 

The assay satisfies the EDSP Tier 1 Test Order requirements for an Estrogen Receptor Binding 

Assay (OCSPP 890.1250). 

COMPLIANCE: Signed and dated GLP and Quality Assurance statements were provided. 

Page 21 of 124
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 3 of 17

LINURON/ 035506 OCSPP 890.1250/ OECD None

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: CeeTox, Inc.

Location: Kalamazoo, MI

Study Director: Jamin A. Willoughby, Sr., Ph.D.

Other Personnel: Karen Rutherford, B.S.; Director of Laboratory Operations

David Blakeman, B.S.; Senior Scientist/ Endocrine Group Leader

Cameron Haines, B.S.; Scientist

Steven McColley, M.S.; Scientist

Colleen Toole, Ph.D.; Director of Project management

Study Period: June 17, 2011 – December 8, 2011

2. Test substance: Linuron

Description: White crystalline solid

Source: Tessenderlo Kerley, Inc.

Batch #: 1012A3876 (expiry date: May 3, 2013)

Purity: 99.4% a.i.

Solubility: Not reported (NR)

Volatility: NR

Stability: NR

Storage conditions: Ambient

CAS #: 330-55-2

Molecular weight: 249.09 g/mol

Structure:

N

H

Cl

Cl

O

N

O

CH3

CH3

3. Non-labeled ligand: 17β-estradiol

Supplier: Sigma-Aldrich, St. Louis, MO

Catalog #: E8875

Batch #: 110M0138V

Purity: 100%

CAS #: 50-28-2

4. Radioactive ligand: [

3

H]-17β-estradiol

Supplier: Perkin-Elmer, Boston, MA

Catalog #: NET517

Batch#: 650702

Radiochemical purity: NR

Specific activity: 130.2 Ci/mol

Concentration of stock: 1 nM

5. Positive control: 19-norethindrone

Supplier: Sigma-Aldrich, St. Louis, MO

Catalog #: N4128

Batch #: 030M1359V

Purity: 99% 

CAS #: 68-22-4

6. Negative control: Octyltriethoxysilane

Page 22 of 124
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 4 of 17

LINURON/ 035506 OCSPP 890.1250/ OECD None

Supplier: Sigma-Aldrich, St. Louis, MO

Catalog #: 440213

Batch #: 24996KK

Purity: 99.34%

CAS #: 2943-75-1

7. Solvent/vehicle control: DMSO

Justification for choice of solvent: Recommended solvent according to guideline, and linuron is soluble up to 

10-4 M

Final Concentration: 2%

B. METHODS 

1. Preparation of Rat Uterine Cytosol (RUC): Frozen Sprague Dawley uteri were purchased 

from Harlan Laboratories. Female Sprague Dawley rats were ovariectomized 7 days prior to 

being euthanized. Animals were 84-91 days old at the time of euthanasia. The uteri were 

weighed, placed in ice-cold TEDG (Tris, EDTA, DTT, glycerol) + PMSF

(phenylmethylsulfonyl fluoride) buffer and used immediately. Uteri were homogenized in 

buffer and then centrifuged for 10 min at 2500 × g at 4 oC. The resulting supernatant was 

transferred and centrifuged for 60 minutes at 105,000 × g, discarding the resulting pellets. 

Protein concentration of the cytosol was determined to be 1.10 mg/mL using a protein kit 

compatible with DTT in the TEDG buffer (BioRad Protein Assay Kit). Cytosol was divided 

into aliquots (1 to 6 mL) for immediate use or storage at −80oC.

2. Saturation (Radioligand) Binding Experiment: A saturation binding experiment 

measuring total and non-specific binding of [3H]-17β-estradiol was performed to 

demonstrate that the ER was present in reasonable concentrations and had the appropriate 

affinity for the native ligand (MRID 48843501). The conditions for the saturation binding 

are summarized in Table 1.

TABLE 1. Summary of Conditions for Saturation Binding Experimenta

Source of receptor Rat uterine cytosol 

Concentration of radioligand (as serial dilutions) 0.03 to 3 nM

Concentration of non-labeled ligand (100X [radioligand]) 3 to 300 nM

Concentration of receptor Sufficient to bind approximately 25 

to 35% of radioligand at 0.03 nMa 

Temperature 4oC 

Incubation time 16 to 20 hours 

Composition of assay buffer Tris 10 mM (pH 7.4)

EDTA 1.5 mM 

Glycerol 10% 

Phenylmethylsulfonyl fluoride (PMSF) 1 mM 

DTT 1 mM 

a Data were obtained from page 1 of the study report (MRID 48843501). 

On the day of the assay, the specific activity of the stock solution [3H]-17β-estradiol

(originally 130.2 Ci/mmol as manufactured on May 6, 2011) was adjusted for decay over 

time (adjusted specific activities were not reported), and serial dilutions in TEDG + PMSF 

buffer were prepared to achieve the final concentrations of 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1, 

Page 23 of 124
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 5 of 17

LINURON/ 035506 OCSPP 890.1250/ OECD None

and 3 nM. Solutions of non-labeled 17β-estradiol were prepared in a similar manner to 

achieve concentrations that were 100-fold greater than each respective radiolabeled 

concentration to result in final concentrations of 3, 6, 8, 10, 30, 60, 100, and 300 nM. For 

each batch of cytosol, the optimal protein concentration was determined by testing serial 

amounts of protein per tube, using 0.03 nM radiolabeled estradiol, until a concentration was 

reached that bound approximately 25 to 35% of the total radioactivity added. The protein 

concentration was 127.5 μg per assay tube for the three saturation binding experiments 

(Note: typically 50 ± 10 μg protein per tube). Each assay consisted of three non-concurrent 

runs (conducted on July 15, 16, and 18, 2011, respectively). Each run included three 

replicates of each test substance at each concentration, resulting in the 72 samples depicted 

in Table 2. 

TABLE 2. Saturation Binding Experiment Runa

Total bindingb Non-specific 

bindingc

Radioligand aloned Assay Components

Tubes 1-24 Tubes 25-48 Tubes 49-72

350 μL 300 μL --- TEDG + PMSF buffer

50 μL 50 μL 50 μL [

3

H]-17β-estradiol (8 serial dilutions)e

--- 50 μL --- Non-labeled 17β-estradiol (8 serial dilutions, 

100x each respective labeled concentration)f

100 μL 100 μL --- Uterine cytosol (diluted to appropriate 

concentration)

500 μL 500 μL 50 μL Total volume in each assay tube

a Data were obtained from page 2 of the study report (MRID 48843501). 

b Total binding = [

3

H]-17β-estradiol bound to ER

c Non-specific binding = [3

H]-17β-estradiol and 100-fold greater non-labeled bound to ER

d Total [

3

H]-17β-estradiol alone for dpm determination at each concentration

e Final concentrations of [3

H]-17β-estradiol = 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1, and 3 nM.

f Final concentrations of non-labeled 17β-estradiol = 3, 6, 8, 10, 30, 60, 100, and 300 nM.

Tubes were incubated with gentle vortexing for 17-21 hours at approximately 4°C. To 

separate bound from free estradiol, hydroxyapatite (HAP) slurry was added to each tube and 

vortexed (3 times with 5-minute intervals). Subsequently, the contents of each tube were 

washed three times as follows: 2-mL portions of TEDG +PMSF buffer were added, 

vortexed, centrifuged for 10 min at 1000 x g, and the supernatant decanted and discarded. 

After the final centrifugation, ethanol (1.5 mL) was added to the HAP pellet remaining in 

each tube to extract the [

3

H]-17β-estradiol, followed by vortexing, and centrifugation for 

10 min at 1000 x g. Aliquots (1 mL) of supernatant were radioassayed by scintillation 

counting. The temperature was maintained at approximately 4oC throughout the assay prior 

to extraction with ethanol.

3. Competitive Binding Experiment: A summary of the experimental conditions for the 

Competitive Binding Experiment is presented in Table 3. 

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TABLE 3. Summary of Conditions for Competitive Binding Experimenta

Source of receptor Rat Uterine Cytosol 

Concentration of radioligand 1 nM 

Concentration of receptor Sufficient to bind 17.4-23.6% of 

radioligandb 

Concentration of test substance (as serial dilutions) 10−10 to 10−3 mM

Temperature 4±2 °C 

Incubation time 16-20 hours 

Composition of assay buffer Tris 10 mM (pH 7.4)

EDTA 1.5 mM 

Glycerol 10% (v/v)

Protease Inhibitor (PI)

w/PMSF (unspecified)c 0.5% (v/v)

DTT 1 mM 

a Data were obtained from page 17 of the main study report (MRID 48617402). 

b Calculated by reviewer from data on pages 58, 61, and 65 of the study report. 

c The recommended PI in the Guideline is 1mM PMSF, but the PI was not identified (although it was 

described as containing PMSF).

A solubility test was performed to determine the appropriate solvent (DMSO) using visual 

observation to examine for precipitation (use of magnification as not reported). On the day 

of the assay, the specific activity of the stock solution [3

H]-17β-estradiol was adjusted for 

decay over time, and diluted in TEDG+PI buffer to achieve the final appropriate 

concentrations. For each batch of cytosol, the optimal protein concentration was determined 

by testing serial amounts of protein per tube, using 1.0 nM radiolabeled estradiol, until a 

concentration was reached that bound 17.4-23.6% of the total radioactivity added (80 μg 

protein per tube). Serial dilutions of linuron, the positive control (19-norethindrone), the 

negative control (octyltriethoxysilane), and the reference material (non-labeled 17β-estradiol) 

were prepared to achieve the concentrations shown in Table 4. Each assay consisted of three 

runs, and each run contained three replicates at each concentration, six replicates measuring 

total activity (50 μL master mix) plus three measuring total binding (solvent control) 

resulting in a total of 306 samples.

Page 25 of 124
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TABLE 4. Molar (M) concentrations in Competitive Binding Assay Run a b

Linuron Weak Positive control Negative control Reference Chemical

19-norethindrone Octyltriethoxysilane Non-labeled 17β-estradiol

Tubes 79-102 Tubes 31-54 Tubes 55-78 Tubes 1-30

10−10 10−8.5 10−10 Solvent control/master mix c

10−9 10−7.5 10−9 10−11

10−8 10−7 10−8 10−10

10−7 10−6.5 10−7 10−9.5

10−6 10−6 10−6 10−9

10−5 10−5.5 10−5 10−8.5

10−4 10−4.5 10−4 10−8

10−3 d 10−4 10−3 d 10−7

a Data were obtained from pages 26, 28, 30, and 58-66 of the study report. 

b Each tube contains: 10μL of either the test substance, positive control, negative control, solvent control, or 

non-labeled 17β-estradiol; 390 μL of TEDG+PI buffer with [3

H]-17β-estradiol; and 100 μL of uterine 

cytosol (with ER), for a total of 500 μL.

c Solvent is DMSO (2%) in Tubes 7-9.

d Precipitate observed at this concentration, and data were not included in evaluation. 

Tubes were incubated with gentle vortexing for 16-20 hours at 4±2 °C. To separate bound 

from free estradiol, HAP slurry was added to each tube and the tubes were vortexed (3 times 

with 5-minute intervals). Subsequently, the contents of each tube were washed three times as 

follows: TEDG+PI buffer was added, vortexed, centrifuged for 10 minutes at 1000×g, and 

the supernatant decanted and discarded. Ethanol was added to the HAP pellet remaining in 

each tube to extract the [

3

H]-17β-estradiol, allowed to sit at room temperature for 15-20 

minutes with vortexing, and centrifugation for 10 min at 1000×g. An aliquot of supernatant 

was radioassayed by scintillation counting. The temperature was maintained at 4±2 °C 

throughout the assay prior to extraction with ethanol.

C. DATA ANALYSIS: For the saturation binding experiment, total binding and non-specific 

binding data were modeled via non-linear regression by using Graph Pad Prism v. 5 

(GraphPad Software, Inc., La Jolla, CA)], incorporating automatic outlier elimination 

according to the method of Motulsky and Brown (2006)1 implemented by using the ROUT 

procedure in Prism v. 5 with a Q value of 1.0. Receptor binding data plots were corrected for 

ligand depletion with the method of Swillens (1995)2

. For the competitive binding assay, 

similar methods of nonlinear regression were used to fit a curve (for 17β-estradiol, the 

positive control, and the test substance) to the Hill equation formula which incorporated log 

IC50 as a parameter to be estimated. For parameters reported from the saturation binding 

experiment (Kd and Bmax) and competitive binding experiment (log IC50 and RBA), mean and 

standard deviation were calculated for each run, and mean and standard error were calculated for 

the composite three runs using Microsoft Excel 2007 (v. 12.0.6557.5000; Microsoft Corporation, 

Redmond, WA), and mean and standard error were calculated for the composite three runs with 

Microsoft Excel 2010. 

 

1 Motulsky, H.J. and Brown, R.E. (2006) Detecting outliers when fitting data with nonlinear regression: a new 

method based on robust nonlinear regression and the false discovery rate. BMC Bioinformatics, Vol 7, pp 123-142. 

2 Swillens, S. (1995) Interpretation of binding curves obtained with high receptor concentrations: practical aid for 

computer analysis. Molec. Pharmacol. 47(6):1197-1203.

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1. Definitions

a. Classification of test material: Classification of the test material is based on the average of 

three runs. Each run was first individually classified as follows:

Interactive = lowest point on the fitted curve within the range of the data is less than 50% 

(i.e., >50% of the radiolabeled estradiol has been displaced from the ER). 

Not interactive = there are usable data points at or above 10-6 M and either the lowest point 

on the fitted response curve within the range of the data is above 75% (i.e., 

<25% of the radiolabeled estradiol has been displaced from the ER) or a 

binding curve cannot be fitted and the lowest average percent binding among 

concentration groups in the data is above 75%. 

Equivocal up to the limit of concentrations tested = there are no data points at or above a 

test chemical concentration of 10-6 M and either a binding curve can be fit but 

≤50% of the radiolabeled estradiol has been displaced from the ER or a binding 

curve cannot be fit and the lowest average percent binding among 

concentration groups in the data is >50%. 

Equivocal = A run is classified as equivocal if it does not fall into any of the categories 

above. 

The categorical classification of each run was assigned a numerical value as follows:

Run Classification Numerical Value

Interactive 2

Equivocal 1

Not interactive 0

Equivocal up to the limit of concentrations tested “missing”

The values for each run were then averaged across runs and the chemical classified using the 

following ranges: 

Test Material Classification Numerical Range

Interactive average ≥1.5

Equivocal 0.5≥ average <1.5

Not interactive average <0.5

Equivocal up to the limit of concentrations tested “missing”

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LINURON/ 035506 OCSPP 890.1250/ OECD None

b. Descriptors for receptor binding: 

Bmax: maximum specific binding number (fmol ER/100 μg cytosol protein) measures the 

concentration of active receptor sites

Kd: dissociation constant (nM), measures the affinity of the receptor for its natural ligand

IC50: concentration of the test substance at which 50% of the radioligand is displaced from 

the receptor

Relative Binding Affinity (RBA %): (IC50 of 17β-estradiol ÷ IC50 of test substance) × 100 

Log RBA: Log10(IC50 of 17β-estradiol ÷ IC50 of test substance)

II. RESULTS

A. SATURATION BINDING EXPERIMENT: Figure 1 illustrates the non-specific, 

specific, and total binding curves for [3H]-17β-estradiol to the estrogen receptor for the three 

independent runs. The specific binding reached a plateau in each run, and non-specific 

binding was less than 20% of total binding. Figure 2 contains the Scatchard plots that 

illustrate the binding of [

3

H]-17β-estradiol to the estrogen receptor. 

Saturation binding experiment parameters are presented in Table 5. The Kd for [

3

H]-17βestradiol was 0.303 nM (± 0.090), and the estimated Bmax was 33.59 fmol/100 μg protein 

(± 0.70) for the prepared rat uterine cytosol. The Kd for each run was within the expected 

range of 0.03 to 1.5 nM. Although the Scatchard plots fit straight lines to the data, the 

concavity observed in the data sets may indicate issues with ligand depletion. Confidence in 

these numbers is high due to the goodness of fit and the small variation among runs. 

TABLE 5. Saturation Binding Experiment of [

3

H]-17β-estradiol with Estrogen Receptor from Rat Uterine 

Cytosola

Parameter Run 1 Run 2 Run 3 Mean ± SEb

R2 (unweighted) 0.992 0.993 0.955 0.955-0.992

Bmax (nM) 0.029 0.029 0.027 0.029±0.001

Bmax (fmol/100 μg protein) 34.09 34.46 32.21 33.59±0.70

Kd (nM) 0.265 0.475 0.169 0.303±0.090

a Data were obtained from page 3 of the study report (MRID 48843501). 

b The range of R2 is reported and the mean ± SEM is reported for the other parameters.

R2 Goodness of fit for curve calculated for specific binding

Page 28 of 124
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FIGURE 1. Binding of [3H]-17β-estradiol to the Estrogen Receptor during the Saturation 

Binding Experiment. 

Page 29 of 124
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FIGURE 2. Scatchard Plots of the Binding of [3H]-17β-estradiol to the Estrogen Receptor.

B. COMPETITIVE BINDING EXPERIMENT: The results from the three competitive 

Page 30 of 124
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binding experiments are summarized in Table 6 and presented graphically in Figures 3-5. 

Precipitate was observed at 10−3 M linuron for all runs; therefore, the data from the 10–3 M 

concentration were not evaluated. Linuron had no effect on specific binding at 

concentrations of ≤10−4 M in all runs. An estimated IC50 and RBA were not calculated for 

linuron did not displace 50% or more of the radiolabeled ligand from the ER in any run. The 

mean log IC50 was −8.9 M for 17β-estradiol and −5.5 M for 19-norethindrone., and the RBA 

for norethindrone was 0.042% 

Additional runs were unnecessary. As linuron showed <25% competitive binding at 

concentrations up to 10−4

 M for all runs, it is classified as non-interactive (0) in this assay 

(Table 7).

TABLE 6. Competitive Binding Assay of Linuron with Estrogen Receptor from Rat Uterine Cytosola

Parameter Run 1b Run 2b Run 3b Mean

R2 (unweighted) 17β-estradiol NR NR NR NR

19-norethindrone NR NR NR NR

Linuron NR NR NR NR

Log IC50 (M) 17β-estradiol −9.0 −9.0 −8.8 −8.9±0.07

19-norethindrone −5.5 −5.5 −5.6 −5.5±0.03

Linuron NA NA NA NA

IC50 (M)c 17β-estradiol 1.00 × 10−9 1.00 × 10−9 1.58 × 10−9 1.20 × 10−9 (±0.20)

19-norethindrone 3.16 × 10−6 3.16 × 10−6 2.51 × 10−6 2.95 × 10−6 (±0.22)

Linuron NA NA NA NA

Log (RBA)c 19-norethindrone −3.50 −3.50 −3.20 −3.4±0.1

Linuron NA NA NA NA

RBA (%)c 19-norethindrone 0.032 0.032 0.063 0.042±0.010

Linuron NA NA NA NA

a Data were obtained from pages 23 and 24 of the study report (MRID 48617402). 

b The mean are reported for the concurrent replicates within each run.

c Values are reviewer-calculated from the Log IC50 values reported in the study report. For means expressed in 

scientific notation, the SE values in parentheses are presented in the same order of magnitude as the mean 

value.

R2 Goodness of fit

RBA Relative binding affinity

NR Not reported

NA Not applicable

TABLE 7. Binding Classification of Linuron with Estrogen Receptor a

Run 1 2 3 Mean Binding Classification c

Classification category value b 0 0 0 0 Not interactive

a Data were obtained from page 23 of the study report (MRID 48617402). 

b Classification category value: Interactive = 2; Equivocal = 1; Not interactive = 0; Equivocal up to the limit of 

concentrations tested (“missing”, i.e., not included in calculation of mean).

c Interactive = mean ≥1.5; Equivocal = 0.5≤ mean <1.5; Not interactive = mean <0.5

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FIGURE 3. Percentage [

3H]-E2 Bound to the Estrogen Receptor in the Presence of 

Linuron, Unlabeled E2, 19-Norethindrone or Octyltriethoxysilane, Run 1

Page 32 of 124
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LINURON/ 035506 OCSPP 890.1250/ OECD None

FIGURE 4. Percentage [

3H]-E2 Bound to the Estrogen Receptor in the Presence of 

Linuron, Unlabeled E2, 19-Norethindrone or Octyltriethoxysilane, Run 2. 

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Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 15 of 17

LINURON/ 035506 OCSPP 890.1250/ OECD None

FIGURE 5. Percentage [

3H]-E2 Bound to the Estrogen Receptor in the Presence of 

Linuron, Unlabeled E2, 19-Norethindrone or Octyltriethoxysilane, Run 3. 

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Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 16 of 17

LINURON/ 035506 OCSPP 890.1250/ OECD None

C. PERFORMANCE CRITERIA: To ensure that the competitive binding assay functioned

properly, each run was evaluated using the following criteria (Table 8): 

TABLE 8. Criterion a Tolerance 

Limit(s) Value Yes No

17β-estradiol fitted curve parameters

Loge residual SD ≤2.35 0.84 to 1.54 X

Top (% binding) 94 to 111 93 to 96 X

Bottom (% binding) -4 to 1 -5 to 0 X

Hill Slope (log10(M)-1) -1.1 to -0.7 -1.1 to -0.8 X

Weak Positive control (19-norethindrone)

b fitted curve parameters

Loge residual SD` NA 0.76 to 1.08

Top (% binding) NA 93 to 100

Bottom (% binding) NA -8 to -1

Hill Slope (log10(M)-1) NA -1.1 to -0.7

Solvent concentration

DMSO ≤10% 2% X

Negative control (octyltriethoxysilane) does not displace more 

than 25% of [

3

H]-17β-estradiol from the ER on average across all 

concentrations c 

≤25% 15.1-54.2% X 

a Data were obtained from pages 27, 29 and 31 of the study report (MRID 48617402). 

b The EPA Guideline does not define a set of tolerance limits for 19-norethindrone. Acceptance criteria were only 

defined for norethynodrel, which cannot be obtained commercially. The values reported were considered 

acceptable as they show 19-norethindrone to be an acceptable weak positive control.

c Calculated by reviewer from data obtained from pages 26, 28 and 30 of the study report. Precipitation was 

observed at the 10-3 M concentration. Displacement was 6.0-12.7% at ≤ 10-4 M concentrations.

NA Not applicable

 

For unlabeled 17β-estradiol, the top % binding in Run 2 (93%) and the bottom % binding in 

Run 3 (-5%) were slightly less than the tolerance limits (94 to 111% and -5 to 0% for top 

and bottom % binding, respectively) The negative control (octyltriethoxysilane) 

displacements of [

3

H]-17β-estradiol from the ER were 51.8-54.2% at 10−3 M for two of the 

three runs, which were greater than the guideline requirement of ≤25%. It was stated that 

similar decreases in specific binding have been observed at 10−3 M octyltriethoxysilane at 

this facility, and precipitation was observed at 10−3

 M during all three runs. Because the 

other components of this study appeared to function properly, these findings were all 

considered minor deficiencies. 

Linuron was tested over a concentration range that fully defined the top of the curve. The 

maximum percent binding of 82.3-106.8% was within 25 percentage points of the lowest 

concentration of the estradiol standard 90.2-95.5%. Placements along the X-axis, and top 

and bottom plateaus were consistent across runs. 

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Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 17 of 17

LINURON/ 035506 OCSPP 890.1250/ OECD None

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: In all three valid independent runs, the mean 

specific binding was >75% at every concentration tested for linuron; therefore the 

classification for linuron is "non-interacting" for the estrogen receptor. 

 

B. AGENCY COMMENTS: In the saturation binding assay, the Kd for [

3

H]-17β-estradiol

was 0.303 nM, and the estimated Bmax was 33.59 fmol/100 μg protein. The Kd for each run 

was within the expected range of 0.03 to 1.5 nM. 

For the competitive binding experiment, the estimated mean IC50 was not calculated for 

linuron as it did not displace the radiolabeled ligand from the ER. The mean IC50 and RBA 

for the weak positive control were 2.95 × 10−6 M and 0.042%, respectively. 

The performance criteria were generally met for the assay. The curve for the reference 

material showed that increasing concentrations of unlabeled 17β-estradiol displaced [3

H]-

17β-estradiol in a manner consistent with one-site binding, as indicated by Hill slopes of −1.1 

to −0.8 in all runs. 

Linuron was tested over a concentration range that fully defined the top of the curve. The 

maximum percent binding of 82.3-106.8% was within 25 percentage points of the lowest 

concentration of the estradiol standard 90.2-95.5%. 

Based on the results of the three valid runs, linuron is classified as not interactive with the ER

as specific radiolabeled estradiol binding was >75% at all concentrations tested. 

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

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

• The radioligand depletion in the competitive assay was 17.4-23.6%, which is greater than 

the guideline recommendation of <15%. It was stated that values were within historical 

CeeTox values (data not provided). 

• The specific binding for the negative control (octyltriethoxysilane) at 10−3

M 

concentrations were 45.8% (Run 2) and 48.2% (Run 3), and precipitation was observed in 

all runs at 10−3 M. The negative control displacements of [3

H]-17β-estradiol from the ER

were 51.8-54.2%, which are greater than the guideline recommended value of ≤25%. 

Displacement was 6.0-12.7% at ≤ 10-4 M concentrations. 

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

EPA MRID Number 48671701 

Page 1 of 54 

Data Requirement: EPA DP Barcode 401285 

OECD Data Point 229 

EPA MRID 48671701 

EPA Guideline 890.1350, Fish Short-Term Reproduction Assay 

Test material: Linuron Purity: 99.4% 

Common name 

Chemical name: IUPAC: 3-(3,4-Dichlorophenyl)-1-methoxy-1-methylurea 

 CAS name: 

 CAS No.: 330-55-2 

 Synonyms: 

 EPA PC Code: 035506 

Primary Reviewer: Joan Gaidos Signature: 

Senior Scientist, Cambridge Environmental, Inc. Date: 12/08/2012 

Secondary Reviewer: Teri S. Myers Signature: 

Senior Scientist, Cambridge Environmental, Inc Date: 12/17/2012 

Primary and Final Additional Reviewer: Robin Sternberg Signature: 

Wildlife Biologist, EPA/OCSPP/OPP/EFED/ERB1 Date: 01/4/2013, 05/27/2015 

Additional Reviewer: Edward Scollon Signature: No longer with EPA 

Toxicologist, EPA/OCSPP/OPP/EFED/ERB4 Date: 01/15/2014 

Date Evaluation Completed: 05/27/2015 

 

Digitally signed by ROBIN 

STERNBERG 

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

ou=USEPA, ou=Staff, cn=ROBIN 

STERNBERG, 

dnQualifier=0000039126 

Date: 2015.06.01 16:22:56 -04'00'

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

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

promelas). Performed by Smithers Viscient, Wareham, Massachusetts, Laboratory Project No. 13694.6149. Submitted 

by Tessenderlo Kerley, Inc., Phoenix, Arizona. Completion date April 3, 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). 

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

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

nor to provide any guidance on how the study should be conducted.

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EXECUTIVE SUMMARY:

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

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

group; 4.25 months old) were exposed to linuron (99.4% purity) at nominal concentrations of 0 (negative 

control), 0.090, 0.90, and 9.0 mg a.i./L; mean-measured concentrations were <0.0032 (<LOQ, negative 

control), 0.099, 0.92, and 9.1 mg a.i./L. The test system was maintained at 23 to 25o

C and a pH of 7.0 to 

7.6. 

Overall mean survival values were 100% for both males and females in the negative control, mid, and low 

treatment groups; 2 males and 1 female died in the high treatment group (88% survival). There were changes 

in coloration and lethargy observed at the high treatment level. In addition, the reviewer noted the presence of 

dark coloration and partial/complete loss of equilibrium in males and females of the high treatment group. 

Male body weight exhibited significant reductions (p<0.05) of 22 and 26% at the mid and high treatment 

levels, respectively, compared to the negative control; and female body weight was significantly reduced 

(p<0.05) by 29% at the high treatment level compared to the negative control. Female body length was 

significantly reduced (p<0.05) by 6% at the high treatment level, and male length was significantly reduced 

(p<0.05) by 8% at the mid treatment level compared to the negative control. 

In the negative control, spawning occurred at least every 4 days in three of the four control replicates; mean 

fecundity was 16 eggs/female/day/replicate (range 13-20 E/F/D); and fertilization success averaged 98%. 

There were no eggs available for assessment at the high treatment level; therefore, the high treatment level 

was excluded from data analysis for the endpoints of fecundity and fertility. There were significant reductions 

in fecundity and fertility (p<0.05) of 50% and 5%, respectively, at the mid treatment level. 

Male vitellogenin (VTG) and gonadal-somatic index (GSI) were significantly reduced (p<0.05) by 97 and 

40%, respectively, at the high treatment level. Male tubercle score was also significantly reduced (p<0.05) at 

the high treatment level compared to the negative control. Tubercles were not observed in female fish. 

Although not analyzed statistically, in male fish, there was an increase in incidence and severity of increased 

proportion of spermatogonia at the high treatment level compared to the negative control. In female fish, there 

were changes in gonadal staging, decreased yolk synthesis, and increased oocyte atresia of predominantly 

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

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pre-vitellogenic follicles at the high treatment level compared to the negative control. Plasma sex steroids were 

not measured. 

All performance and validity criteria were met in this study with the following exceptions: (1) The temperature 

on Days 0 and 15 briefly decreased to a minimum of 23.4qC which is below the guideline criterion of 25±1 

°C. (2) For negative control replicate B, spawning did not occur at least every four days, and average fecundity 

was 13 eggs/female/day which is less than the guideline criterion of ≥15 eggs/female/day. However, the 

fecundity rates in the other negative control replicates ranged from 16 to 20 eggs/female/day indicating that 

fecundity was adequate in the negative control. These deviations did not impact the interpretation of the study. 

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

(OCSPP Guideline 890.1350). 

Results Synopsis

Test Organism age at test initiation: ca. 4.25 months 

Mean body weight at test initiation: Male 3.3 g; Female 1.8 g 

Mean length at test initiation: Not reported 

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

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

Treatment 

 (mg a.i./L) 

[meanmeasured]

Fecundity 

Fert. 

Success 

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

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

0.099 No No No NA No No No No No No NA NA NA NA 

0.92 Yes Yes No NA No No No No No No NA NA NA NA 

9.1 Yes3 Yes3 Yes NA Yes No Yes Yes Yes No NA NA NA NA 

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

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

2 The criteria for significance are described in the Reviewer’s Analysis and Statistical Verification sections of the DER. Conclusions regarding 

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

3 No eggs were produced at the high treatment level. Therefore, fecundity was 0; there were no fertility data to analyze; and the high treatment level was 

excluded from data analysis for the endpoints of fecundity and fertility. 

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I. MATERIALS AND METHODS 

GUIDELINE FOLLOWED: This study was conducted according to the U.S. EPA OCSPP 890.1350: 

“Fish Short-Term Reproductive Assay” and OECD 229 (2009). The 

following deviations were noted: 

 

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

2. Unionized ammonia and residual chlorine in the test water were not reported. The OCSPP 890.1350 

performance criteria establish maximum levels for these values, and it is unclear if the maximum 

recommendations were exceeded.

3. The temperature on Days 0 and 15 briefly decreased to a minimum of 23.4qC; however this slight 

deviation in temperature was not considered to have a negative impact on the results.

4. The fish were 17 weeks old (4.25 months) at the beginning of the exposure period. EPA recommends 

reproductively mature (sexually dimorphic) fish, 4.5 - 6 months old.

5. Female body weights (n=20) varied by ±22%. EPA recommends that a subsample of fish be weighed 

before the test to estimate the mean weight for each sex. It is recommended that the individual weight 

of each fish selected for the test be within ±20% of the estimated mean for each sex.

6. In replicate B of the negative control, spawning did not occur at least every four days nor was the 

average fecundity 15 eggs/female/day or greater. However, the average fecundity for that replicate 

was 13 eggs/female/day which was slightly less than the recommended rate, and the fecundity rates 

in the other replicates ranged from 16 to 20 eggs/female/day indicating that fecundity was adequate 

in the control treatment.

The deviations do not impact the interpretation of the study.

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

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

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

exceptions: routine dilution water and food contaminant screening analyses. 

Since the analyses were conducted following standard validated methods, 

this exception has no impact on the study results. 

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A. TEST MATERIAL: Linuron; CAS# 330-55-2

Description: Not reported

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

Kow, vapor pressure of test compound, expiration date.

Lot No./Batch No. : 1012A3876 

Purity: 99.4% 

Impurities: None reported

Stability of Compound: Analytical verification of the test material from Days 0, 7, 15, and 21 yielded 

mean-measured concentrations of 100% to 110% of nominal concentrations. 

AAP medium (a freshwater algal medium) fortified with linuron at 

concentrations of 0.0008, 0.010, and 1.0 mg a.i./L yielded recoveries of 

84.9 to 99.7%. There was no pattern of decline during the study period, 

indicating the test material was stable under the test conditions. 

Storage Conditions of 

Test Chemicals: Stored at room temperature in dark, ventilated cabinet 

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B. Test organism: 

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

Species common name: Fathead Minnow EPA recommends fathead minnow 

(Pimephales promelas). Species scientific name: Pimephales promelas

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

Yes EPA recommends that fish be from a single 

laboratory stock.

Were acclimation conditions same as definitive test?

Yes EPA recommends that fish be acclimated 

under water quality and illumination 

conditions that are similar to the definitive 

test. 

Acclimation period: 15 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: Less than 1% mortalities were reported 

during the 7 days prior to pre-exposure 

period. 

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

EPA recommends that mortality during the 7 

days prior to the pre-exposure phase be 

less than 5% of the culture population. If 

mortality during these 7 days is greater than 

10%, EPA recommends that the fish be 

rejected. If mortality is between 5-10%, 

EPA recommends that fish be held another 

7 days. If mortalities greater than 5% occur 

during this extended acclimation period, 

EPA recommends that the fish not be used. 

Type of food: Live brine shrimp EPA recommends that fish be fed frozen 

brine shrimp twice per day to promote 

active reproduction and maintain body 

condition.

Source of food: Not reported SMV Lot No. 082110 Frequency of feeding: 2 times/day Details on feeding: None 

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Table 3: Fish Selection and Pre-Exposure Performance.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Age at test initiation: Ca. 4.25 months 17 weeks EPA recommends reproductively mature 

(sexually dimorphic) fish, 4.5 - 6 months 

old.

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

3.3 g Based on a representative sample of 20 

males from the test population used to 

stock aquaria for pre-exposure phase 

EPA recommends that a subsample of fish be weighed before the test to 

estimate the mean weight for each sex. It 

is recommended that the individual weight 

of each fish selected for the test be within 

±20% of the estimated mean for each sex.

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

2.9 to 3.8 Individual weights within ±20% of the 

estimated mean

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

1.8 g Based on a representative sample of 20 

females from the test population used to 

stock aquaria for pre-exposure phase 

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

1.4 to 2.2 Individual weights within ±22% of the 

estimated mean

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

Not reported 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

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

Not reported 

Duration of pre-exposure phase: 15 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. 

Number of pre-exposure tanks: 33 Extra replicates were established to 

account for a potential lack of spawning in some chambers and/or mortality during this phase.

EPA recommends that additional tanks set 

up at the beginning of pre-exposure will 

ensure that sufficient replicates with the 

correct sex ratio are available for the 

definitive test. 

Number of males per tank: 2 Number of females per tank: 4 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Pre-exposure fecundity: >15 eggs/female/ 

reproductive day/replicate

EPA recommends that pre-exposure 

fecundity in each replicate (tank) selected 

for use in the definitive test be at least 15 

eggs/female/reproductive day/replicate 

during the 7 days prior to the definitive test. 

Number of spawns during preexposure: at least twice in the 7- day period preceding 

exposure initiation 

EPA recommends that spawning occur at 

least twice in the 7 days prior to the 

definitive test. 

Details on pre-exposure: None 

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

Corrections and Clarifications document1 , 

the use of a static renewal system is not 

recommended for this assay.

Type of flow-through dilution system:

Intermittent flow proportional diluters

Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

Flow-through rate: Ca. 46 mL/minute or 2.8 

L/hr and 6.6 volume 

replacements/day 

Calculated by the reviewer based on 0.5 L/cycle, 132 cycles/24 hrs and 60 minutes/hr

Recommended flow-through rate is 45 

mL/min (2.7 L/hr), or at least 6 total 

volume exchanges per day.

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

Screening Program Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention (OCSPP), Washington, D.C. (http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf). 

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on toxicant mixing for flow-through systems: 

A mixing chamber was used. Flow-splitting chambers were used between the diluter cells and the four replicate test vessels to promote mixing of the test substance solution and diluent water and to equally split the test solution 

between the test vessels. 

Treatment recoveries measured prior to test initiation ranged from 110 to 130% of 

nominal concentrations.

Flow splitting accuracy not reported 

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

recommended but not required; 2) 

Aeration is not recommended for mixing; 

3) A demonstration that the test solution 

is completely mixed before introduced 

into the test system is recommended; 4) 

The recommended flow splitting accuracy 

is within 10%.

Aeration? None reported EPA recommends aeration if dissolved 

oxygen reaches <4.9 mg/L (<60% 

saturation).

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Source of dilution water: Well water EPA recommends natural or reconstituted 

water; it is recommended that natural 

water be sterilized with UV and tested for 

pesticides, heavy metals, and other 

possible contaminants. OECD accepts 

any water in which the test species show 

control survival at least as good as 

indicated in the test guideline.

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

Yes 

Test vessel type/materials: Glass aquaria with 

silicone adhesive

EPA and OECD recommend that watercontact portions of the system not 

compromise the study (e.g., all glass 

vessels or glass vessels with stainless 

steel frames are acceptable examples).

Test vessel size: 20 L: 39 cm long x 20 

cm wide x 25 cm high; solution depth 13 cm. 

EPA recommends the use of 18 L test 

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Fill volume: 10 L EPA recommends 10 L solution per tank. Spawning substrate material: appropriately aged PVC 

pipe 

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: 4 inch diameter cut in 

half (arches) and into 7

cm sections 

Additional details on exposure system: 

None 

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

Parameter Minimum Maximum Mean1

Measurement 

Interval 

Guideline Recommendations 

Temperature (°C) 23 25 24 Continuously 

EPA recommends temperature 25±1oC; 

inter-replicate and inter-treatment 

differentials should not exceed 1oC.

pH 7.0 7.6 7.3 Daily EPA recommends pH 6.5 to 9.0.

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Parameter Minimum Maximum Mean1

Measurement 

Interval 

Guideline Recommendations 

Dissolved oxygen (mg/L) 5.1 (60%) 8.6 (100%) 6.9 (80%) Daily EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air saturation)Total alkalinity (mg/L as CaCO3) 

26 30 28 Weekly 

EPA recommends total alkalinity >20 

mg/L as CaCO3.

Hardness (mg/L as CaCO3) 64 80 72 Weekly Total organic carbon (mg/L) 0.43 1.2 0.82 Monthly EPA recommends that total organic 

carbon in dilution water be <2 mg/L. 

Unionized ammonia (Pg/L) Not reported Not reported Not reported Not reported 

EPA recommends that unionized 

ammonia in the dilution water be <1 

Pg/L. 

Residual chlorine (Pg/L) Not reported Not reported Not reported Not reported 

EPA recommends that residual chlorine 

in dilution water be <10 Pg/L. 

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Parameter Minimum Maximum Mean1

Measurement 

Interval 

Guideline Recommendations 

Conductivity (Pmhos/cm) 350 530 440 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. 1 Average of minimum and maximum 

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D. Study Design and Additional Experimental Conditions

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

if 96-hour LC50 data for the fathead minnow 

are unavailable. 

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

96-hr LC50 rangefinding studies Range-finding study #1: x Nominal test concentrations: 0.95, 

1.9, 3.8, 7.5 and 15 mg/L 

x n = 2 males & 4 females in one 

test vessel per test concentration

x Results: no mortality; 1 male in the 

15 mg/L group was observed to have complete loss of equilibrium

x LC50 > 15 mg/L 

Range-finding study #2: 

x Nominal test concentrations: 2.5, 

5.0, 10, 20 and 40 mg/L 

x n = 2 males & 4 females in one 

test vessel per test concentration 

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.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

x Results: 100% mortality at 40 

mg/L; 100% complete loss of equilibrium at 20 mg/L 

x LC50 (95% C.I.) = 28 (21 to 30) 

mg/L 

Species: Pimephales promelasLife stage: Not reported Approximately the same age and size 

as the definitive study. 

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: 96-hr EPA recommends a 96-hour exposure. Additional details: NA 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. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

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.

Table 7: Definitive Study Conditions.

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

definitive test be 21 days. 

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

The acute toxicity 96-hr LC50 value was estimated to be 28 mg/L. 

EPA recommends that the highest test 

concentration is either the solubility limit of 

the test compound, 100 mg/L, or 

demonstrates adequate evidence of toxicity 

(e.g., 1/3 the 96-hour LC50), whichever 

concentration is lowest.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Reference study citation (if applicable):

NA 

Separation of test concentrations:

0.1 Step down factor of 10 from highest test 

concentration. 

EPA suggests that a concentration 

separation of between 0.33 (or three-fold) 

and 0.1 (or ten-fold) is scientifically 

acceptable1 . 

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: 

Mean-measured 

Limit of quantification (LOQ): <0.0029 to 0.0032 

mg a.i./L

EPA recommends that for chemical test 

concentrations below the LOQ, analyses be 

conducted on the stock solutions.

Level of detection (LOD): Not reported 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Frequency of measurement: 0, 7, 15, and 21 days 

in 2 reps for each treatment at each time point

It is recommended that test item 

concentration be measured prior to the 

addition of fish in all tanks and at least 

weekly thereafter in two replicates per 

treatment level.

Was the randomized complete block design used?

Yes EPA recommends that all fish be randomly 

assigned to tanks during pre-exposure. 

Tanks are then ranked according to preexposure fecundity, and the tanks with the 

highest fecundity are randomly assigned to 

a definitive test treatment and block first. 

Each block contains one replicate of each 

treatment, including controls. 

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

NA EPA recommends the use of a concurrent 

solvent control when a solubilizing agent is 

used. EPA recommends 4 replicates.

Number of replicates per test item treatment level: 

4 EPA recommends 4 replicates.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Number of male fish per replicate at test initiation: 

2 EPA recommends 2 males per replicate. 

Number of female fish per replicate at test initiation: 

4 EPA recommends 4 females per replicate. 

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

NA EPA recommends that the solvent not 

exceed 0.02 ml/L3. OECD recommends 

that solvent have no effect on survival nor 

produce any other adverse effects and that 

concentration not be greater than 0.1 ml/L4.

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

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

Toxicology, 76, pp.69–92. 

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

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Positive control concentration(s) (if applicable): 

NA 

Photoperiod: 16 hrs light: 

8 hrs dark 

EPA recommends photoperiod 16:8 

(light:dark).

Light intensity at water’s surface: 

630-1000 lux Fluorescent bulbs EPA recommends light intensity 540 – 

1080 lux (at water’s surface).

Additional details: None All exposure solutions were reported as 

clear and colorless with no undissolved material present. A stock solution (15 mg/L) was prepared daily and was reported as clear and colorless with a small amount of undissolved material settled at the bottom of the mixing vessel. 

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

Treatment ID

Nominal 

Concentration 

(mg a.i./L) 

Mean Measured 

Concentration 

(mg a.i./L) 

Mean CV (%) Details or Remarks Guideline Recommendations 

Negative Control 0 <LOQ NA EPA recommends that test item 

concentrations be maintained at a coefficient 

of variation (CV) ≤20%.

Treatment 1 0.090 0.0991 7.3 

Treatment 2 0.90 0.92 5.0 Treatment 3 9.0 9.1 1.2 

Abbreviations: CV Coefficient of variation; NA Not Applicable LOQ = 0.0029 to 0.0032 mg a.i./L 1 Study author-calculated value using the actual analytical results and not the rounded values (two significant figures) as presented in this study report. 

The reviewer-calculated mean-measured concentration using rounded values was 0.097 mg a.i./L. 

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E. Observations

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

characterization (body color, pattern, body shape), body weight, length, tubercle score, gonadal staging and histopathology, and plasma vitellogenin were evaluated.

Were raw (individual) data provided? Yes

EPA recommends that observations of survival, fecundity, fertilization success, secondary sex characteristics, and other clinical signs occur at least daily. At 

test termination (Day 21), additional observations include body weight and length, nuptial tubercle score, gonadal staging and histopathology, plasma vitellogenin, 

and plasma sex steroids (testosterone and 17β-estradiol, if measured). Gonado-somatic index (GSI) is calculated using a ratio of gonad weight to body 

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

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

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II. RESULTS AND DISCUSSION A. Results 

Mean male survival values were 100, 100, 100 and 75%, and mean female survival values were 100, 100, 100, and 94% in the mean-measured 0 (control), 0.099, 0.92, and 9.1 mg a.i./L treatment levels, respectively (Table 9). 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Males Females 

n1 # Surviving % Survival n1 # Surviving % Survival 

Negative Control (<LOQ) 8 8 100 16 16 100 

0.099 8 8 100 16 16 100 

0.92 8 8 100 16 16 100 

9.1 8 6 75 16 15 94 

Abbreviations: NA Not applicable. LOQ=0.0032 mg a.i./L 1 Total number of fish at test initiation 

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Median male body weight values were 3.41, 3.09, 2.67, and 2.53 g, and female body weight values were 1.55, 1.52, 1.52 and 1.10 g in the meanmeasured 0 (control), 0.099, 0.92 and 9.1 mg a.i./L treatment levels, respectively (Table 10). Mean male body length values were 54.7, 54.1, 

50.3, and 53.4 mm and female body length values were 42.5, 42.2, 42.7 and 40.0 mm in the mean-measured 0 (control), 0.099, 0.92 and 9.1 mg a.i./L treatment levels, respectively. 

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

Treatment 

 (mg a.i./L) 

[mean-measured]

Body Weight Length 

Males Females Males Females 

n1

Median

(g) 

±SD n1

Mean

(g) 

±SD n1

Mean

(mm) 

±SD n1

Mean

(mm) 

±SD 

Negative Control 

(<LOQ) 

4 3.41 0.60 4 1.55 0.16 4 54.7 3.2 4 42.6 1.1

0.099 4 3.09 0.11 4 1.52 0.07 4 54.1 1.8 4 42.2 0.5 

0.92 4 2.67 0.18 4 1.52 0.10 4 50.3 1.5 4 42.7 0.8 

9.1 4 2.53 0.41 4 1.10 0.07 4 53.4 1.7 4 40.0 0.8 

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

 LOQ=0.0032 mg a.i./L

1 Number of replicate tanks

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Mean fecundity values were 16, 15, 8.2 and 0 eggs/female/day, and fertilization success was 98, 99, 93 

and 0% in the mean-measured 0 (control), 0.099, 0.92 and 9.1 mg a.i./L treatment levels, respectively 

(Table 11). There were no eggs available for assessment at the highest treatment level. 

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

Treatment 

(mg a.i./L) 

[mean-measured] 

Fecundity1 Fertilization Success (%)2

Mean SD Mean SD 

Negative Control (<LOQ) 16.3 2.9 98 1.8 

0.099 15.3 2.9 98.8 0.5 

0.92 8.15 3.0 93 4.4 

9.1 03 NA NA3 NA 

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

LOQ=0.0032 mg a.i./L 

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. 

3

 No eggs available for assessment; therefore, treatment level was excluded from data analysis. 

 

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Median male tubercle scores were 32, 33, 27 and 2 in the mean-measured 0 (control), 0.099, 0.92 and 

9.1 mg a.i./L treatment levels, respectively (Table 12). None of the surviving females were found to have 

tubercles. 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Males Females 

n1 Median 

Tubercle Score2 n1 Median Tubercle Score 

Negative Control (<LOQ) 4 32 4 0 

0.099 4 34 4 0 

0.92 4 28 4 0 

9.1 4 2 4 0 

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

LOQ=0.0032 mg a.i./L 

1 Number of replicate tanks 

2

 Mean tubercle scores: 32, 33, 28, and 2 for the negative control and mean-measured 0.099, 0.92, and 9.1 mg 

a.i./L treatment levels, respectively. 

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Mean male GSI was 1.6, 1.5, 1.7 and 0.9% and mean female GSI was 12, 13, 13 and 4.3% in the 

mean-measured 0 (control), 0.099, 0.92 and 9.1 mg a.i./L treatment levels, respectively (Table 13). 

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

Treatment 

 (mg a.i./L) 

[mean-measured]

Males Females 

n1 Mean GSI2

(%) 

±SD n1 Mean GSI2

(%) 

±SD

Negative Control (<LOQ) 4 1.55 0.10 4 12.1 3.1 

0.099 4 1.47 0.30 4 12.5 1.3 

0.92 4 1.67 0.21 4 13.0 1.4 

9.1 4 0.932 0.30 4 4.28 0.8 

Abbreviations: NA Not applicable. 

LOQ=0.0032 mg a.i./L 

1

 Number of replicate tanks 

2

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

100. 

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Median male gonadal stage was 2, 2, 2 and 3 and median female gonadal stage was 3, 3, 3 and 2 in the 

mean-measured 0 (control), 0.099, 0.92 and 9.1 mg a.i./L treatment levels, respectively (Table 14). The 

study authors reported exposure-related decrease in median female gonadal staging, but no significant findings 

were reported in median male gonadal staging. 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Males Females 

n1 Median Stage2 n1 Median Stage3

Negative Control (<LOQ) 4 2 4 3 

0.099 4 2 4 3 

0.92 4 2 4 3 

9.1 4 3 4 2 

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

LOQ=0.0032 mg a.i./L 

1

 Number of replicate tanks 

2

 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. 

3

 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. 

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In male fish, there were an increased percentage of spermatogonia at the 9.1 mg a.i./L treatment levels compared to the control (Tables 15-16). In female fish, there were changes in gonadal staging, decreased yolk synthesis, and increased oocyte atresia of predominantly pre-vitellogenic follicles in the 9.1 mg a.i./L treatment compared to the control (Tables 17-18). Table 15: Gonadal Histopathology in Male Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[meanmeasured] 

Diagnostic Observations1

Severity

Increased Proportion 

of Spermatogonia

Presence of TestisOva 

Increased 

Testicular 

Degeneration

Duct 

Mineralization

Interstitial cell 

(Leydig) 

hypertrophy/ 

hyperplasia 

n Incidence n Incidence n Incidence n Incidence n Incidence 

Negative 

Control 

(<LOQ) 

0 8 8 NA NA 8 6 NA NA NA NA 

1 8 0 NA NA 8 2 NA NA NA NA 

2 8 0 NA NA 8 0 NA NA NA NA 3 8 0 NA NA 8 0 NA NA NA NA 4 8 0 NA NA 8 0 NA NA NA NA 

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Treatment 

(mg a.i./L) 

[meanmeasured] 

Diagnostic Observations1

Severity

Increased Proportion 

of Spermatogonia

Presence of TestisOva 

Increased 

Testicular 

Degeneration

Duct 

Mineralization

Interstitial cell 

(Leydig) 

hypertrophy/ 

hyperplasia 

n Incidence n Incidence n Incidence n Incidence n Incidence 

0.099 0 8 8 NA NA 8 8 NA NA NA NA 

1 8 0 NA NA 8 0 NA NA NA NA 

2 8 0 NA NA 8 0 NA NA NA NA 3 8 0 NA NA 8 0 NA NA NA NA 4 8 0 NA NA 8 0 NA NA NA NA 

0.92 0 8 8 NA NA 8 7 NA NA NA NA 

1 8 0 NA NA 8 1 NA NA NA NA 

2 8 0 NA NA 8 0 NA NA NA NA 3 8 0 NA NA 8 0 NA NA NA NA 4 8 0 NA NA 8 0 NA NA NA NA 

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Treatment 

(mg a.i./L) 

[meanmeasured] 

Diagnostic Observations1

Severity

Increased Proportion 

of Spermatogonia

Presence of TestisOva 

Increased 

Testicular 

Degeneration

Duct 

Mineralization

Interstitial cell 

(Leydig) 

hypertrophy/ 

hyperplasia 

n Incidence n Incidence n Incidence n Incidence n Incidence 

9.1 0 6 0 NA NA 6 6 NA NA NA NA 

1 6 2 NA NA 6 0 NA NA NA 

2 6 1 NA NA 6 0 NA NA NA 3 6 2 NA NA 6 0 NA NA NA 4 6 1 NA NA 6 0 NA NA NA 

Abbreviations: NA Not applicable. 

LOQ=0.0032 mg a.i./L 

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. 

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

Treatment 

(mg a.i./L) 

[mean-measured] 

Additional Diagnostic Observations1

Severity 

Decreased 

Proportion of 

Spermatogonia 

Increased Vascular 

or Interstitial 

Proteinaceous Fluid 

Asynchronous 

Gonad 

Development 

Altered Proportions 

of Spermatocytes 

or Spermatids 

Granulomatous 

Inflammation

n Incidence n Incidence n Incidence n Incidence n Incidence 

Negative Control 

(<LOQ) 

0 NA NA NA NA 8 7 NA NA 8 8 

1 NA NA NA NA 8 1 NA NA 8 0 

2 NA NA NA NA 8 0 NA NA 8 0 3 NA NA NA NA 8 0 NA NA 8 0 4 NA NA NA NA 8 0 NA NA 8 0 

0.099 0 NA NA NA NA 8 8 NA NA 8 8 

1 NA NA NA NA 8 0 NA NA 8 0 

2 NA NA NA NA 8 0 NA NA 8 0 3 NA NA NA NA 8 0 NA NA 8 0 4 NA NA NA NA 8 0 NA NA 8 0 

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Treatment 

(mg a.i./L) 

[mean-measured] 

Additional Diagnostic Observations1

Severity 

Decreased 

Proportion of 

Spermatogonia 

Increased Vascular 

or Interstitial 

Proteinaceous Fluid 

Asynchronous 

Gonad 

Development 

Altered Proportions 

of Spermatocytes 

or Spermatids 

Granulomatous 

Inflammation

n Incidence n Incidence n Incidence n Incidence n Incidence 

0.92 0 NA NA NA NA 8 8 NA NA 8 8 

1 NA NA NA NA 8 0 NA NA 8 0 

2 NA NA NA NA 8 0 NA NA 8 0 3 NA NA NA NA 8 0 NA NA 8 0 4 NA NA NA NA 8 0 NA NA 8 0 

9.1 0 NA NA NA NA 6 5 NA NA 6 5 

1 NA NA NA NA 6 1 NA NA 6 1 

2 NA NA NA NA 6 0 NA NA 6 0 3 NA NA NA NA 6 0 NA NA 6 0 4 NA NA NA NA 6 0 NA NA 6 0 

Abbreviations: NA Not applicable. 

LOQ=0.0032 mg a.i./L 

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. 

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

Treatment 

(mg a.i./L) 

[meanmeasured] 

Additional Diagnostic Observations1

Severity 

Increased Oocyte 

Atresia 

Perifollicular Cell 

Hyperplasia/ 

Hypertrophy 

Decreased Yolk 

Formation 

Aggregates of 

macrophages, 

multifocal 

n Incidence n Incidence n Incidence n Incidence 

Negative 

Control 

(<LOQ) 

0 16 7 NA NA 16 16 NA NA

1 16 6 NA NA 16 0 NA NA

2 16 2 NA NA 16 0 NA NA

3 16 1 NA NA 16 0 NA NA

4 16 0 NA NA 16 0 NA NA

0.099 0 16 12 NA NA 16 16 NA NA

1 16 4 NA NA 16 0 NA NA

2 16 0 NA NA 16 0 NA NA

3 16 0 NA NA 16 0 NA NA

4 16 0 NA NA 16 0 NA NA

0.92 0 16 11 NA NA 16 16 NA NA

1 16 3 NA NA 16 0 NA NA

2 16 2 NA NA 16 0 NA NA

3 16 0 NA NA 16 0 NA NA

4 16 0 NA NA 16 0 NA NA

9.1 0 15 4 NA NA 15 9 NA NA

1 15 4 NA NA 15 3 NA NA

2 15 6 NA NA 15 2 NA NA

3 15 1 NA NA 15 1 NA NA

4 15 0 NA NA 15 0 NA NA

Abbreviations: NA Not applicable. 

LOQ=0.0032 mg a.i./L 

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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. 

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

promelas).

Treatment 

(mg a.i./L) 

[meanmeasured]

Additional Diagnostic Observations1

Severity 

Interstitial fibrosis 

Egg Debris in 

Oviduct 

Granulomatous 

Inflammation 

Decreased PostOvulatory Follicles 

n Incidence n Incidence n Incidence n Incidence 

Negative 

Control 

(<LOQ) 

0 NA NA 16 14 16 3 NA NA

1 NA NA 16 2 16 13 NA NA

2 NA NA 16 0 16 0 NA NA

3 NA NA 16 0 16 0 NA NA

4 NA NA 16 0 16 0 NA NA

0.099 0 NA NA 16 13 16 4 NA NA

1 NA NA 16 3 16 12 NA NA

2 NA NA 16 0 16 0 NA NA

3 NA NA 16 0 16 0 NA NA

4 NA NA 16 0 16 0 NA NA

0.92 0 NA NA 16 13 16 8 NA NA

1 NA NA 16 1 16 8 NA NA

2 NA NA 16 2 16 0 NA NA

3 NA NA 16 0 16 0 NA NA

4 NA NA 16 0 16 0 NA NA

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Treatment 

(mg a.i./L) 

[meanmeasured]

Additional Diagnostic Observations1

Severity 

Interstitial fibrosis 

Egg Debris in 

Oviduct 

Granulomatous 

Inflammation 

Decreased PostOvulatory Follicles 

n Incidence n Incidence n Incidence n Incidence 

9.1 0 NA NA 15 14 15 1 NA NA

1 NA NA 15 1 15 14 NA NA

2 NA NA 15 0 15 0 NA NA

3 NA NA 15 0 15 0 NA NA

4 NA NA 15 0 15 0 NA NA

LOQ = 0.0032 mg a.i./L 

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. 

 

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Mean male VTG was 1650, 1790, 535 and 42 and female VTG was 1.43x106

, 2.06x106, 2.43x106 and 

0.399x106

 ng/mL in the mean-measured 0 (control), 0.099, 0.92 and 9.1 mg a.i./L treatment levels, 

respectively (Table 19). 

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

Treatment 

 (mg a.i./L) 

[mean-measured]

Plasma Vitellogenin (VTG) 

Males Females 

n1 Mean

(ng/mL plasma) 

±SD n1 Mean

(ng/mL plasma) 

±SD 

Negative Control 

(<LOQ) 

4 1650 2970 4 1.43x106 0.337x106

0.099 4 1780 2300 4 2.06x106 0.735x106

0.92 4 535 970 4 2.43x106 0.167x106

9.1 4 42 73 4 0.399x106 0.255x106

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

LOQ=0.0032 mg a.i./L 

1

 Number of replicate tanks 

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Mean plasma testosterone and plasma 17β-estradiol in male and females was not measured (Table 20). 

Table 20: Plasma Sex Steroids in Fathead Minnow (Pimephales promelas). Not measured.

Treatment 

(mg a.i./L) 

[mean-measured]

Plasma Testosterone (T) Plasma 17β-estradiol (E2) 

Males Females Males Females 

n

Mean

(ng/mL 

plasma) 

±SD n 

Mean

(ng/mL 

plasma) 

±SD n

Mean

(ng/mL 

plasma) 

±SD n

Mean

(ng/mL 

plasma) 

±SD

Negative Control 

(<LOQ) 

NA NA NA NA NA NA NA NA NA NA NA NA

0.099 NA NA NA NA NA NA NA NA NA NA NA NA 

0.92 NA NA NA NA NA NA NA NA NA NA NA NA 

9.1 NA NA NA NA NA NA NA NA NA NA NA NA 

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

LOQ=0.0032 mg a.i./L 

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According to the study author, there were changes in coloration and lethargy observed at the 9.1 mg a.i./L treatment level. Also according to the author, there were no other notable observations with regards to behavior or changes in appearance (coloration/banding, presence of ovipositor and dorsal nape pad), including secondary sex characteristics in the control, 0.099, and 0.92 mg a.i./L treatment groups. Upon examination of the raw data tables, the reviewer additionally noted the following which occurred at least once during the exposure: the presence of colors bars in some males of the control and all treatment groups; dark coloration and partial/complete loss of equilibrium in males and females of the 9.1 mg a.i./L treatment group; and 1 bloated female and 1 female with an edema in the 0.099 m a.i./L treatment group. 

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

Treatment 

(mg a.i./L) 

[mean-measured] 

Secondary Sex Characteristics and Clinical Signs 

Males Females 

Type n1 Incidence Type n1 Incidence 

Negative Control 

(<LOQ) 

None 8 0 None 16 0 

0.099 None 8 0 Edema 16 1 

0.92 None 8 0 None 16 0 

9.1 

Dark 6 Not specified1

Dark 15 Notspecified1 Lethargic 6 Not specified1

Complete loss of equilibrium 6 1 

LOQ=0.0032 mg a.i./L 1 Number of affected fish not specified; noted for all replicates 

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B. Study Author’s Analysis and Conclusions

The study author analyzed survival, weight, tubercle score, GSI, fertility, fecundity, and VTG. Data 

were gender specific and analyzed in comparison to the negative control. 

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

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

based on p<0.01 (CETIS, version 1.8.4.20, 2011). Survival data were analyzed using a CochranArmitage Trend Step-Down Test. Fertility, female weight, male and female GSI, and male and female 

VTG were analyzed using ANOVA and a Dunnett’s Multiple Comparison test. Fecundity, male tubercle 

scores, and median male weight were analyzed by ANOVA and Jonckheere-Terpstra Step-Down test. 

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

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

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

was performed. If non-normality or unequal variance were indicated (p<0.01), a non-parametric 

analysis was performed on the ranks of the data. These methods appear to be consistent with the 

methods recommended in the guideline. 

There was a significant difference in male and combined sex survival at the 9.1 mg a.i./L treatment 

level compared to the control (p<0.05; Cochran-Armitage Trend Step-Down test). There was no 

significant difference in female survival at any treatment levels (Table 9). At the 9.1 mg a.i./L treatment 

level, fecundity was 0 eggs/female/reproductive day, and fertility was not reported since there was 

no spawning. Therefore this treatment level was excluded from statistical analysis for the endpoints of 

fecundity and fertility (Table 11). With the highest treatment removed, there was a significant difference 

in the fecundity (p<0.05; Jonckheere-Terpstra Step-Down test) and fertility (p<0.05; Dunnett’s 

Multiple Comparison Test) at the 0.92 mg a.i./L treatment level compared to the negative control. 

There were significant reductions in median male tubercle scores at the 9.1 mg a.i./L treatment level 

compared to the negative control (p<0.05; Jonckheere-Terpstra Step-Down test; Table 12). There 

were no tubercles observed in females. There was a significant decrease in median male weight at 

the 0.92 and 9.1 mg a.i./L treatment levels compared to the negative control (p<0.05; JonckheereTerpstra Step-Down test; Table 10). There was also a significant decrease in mean female body 

weight at the 9.1 mg a.i./L treatment level compared to the negative control (p<0.05; Dunnett’s 

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Multiple Comparison test). There was a significant decrease in mean male and female GSI score at 

the 9.1 mg a.i./L treatment level compared to the negative control (p<0.05; Dunnett’s Multiple 

Comparison test; Table 13). There was no significant difference in mean male or female VTG at any 

treatment level compared to the control (p>0.05; Dunnett’s test; Table 19). 

In male fish, there were an increased percentage of spermatogonia at the 9.1 mg a.i./L treatment 

levels (Tables 15-16). In female fish, there were changes in gonadal staging, decreased yolk synthesis, 

and increased oocyte atresia of predominantly pre-vitellogenic follicles in the 9.1 mg a.i./L treatment 

(Tables 17-18). There were no significant findings observed in the male gonadal staging. 

C. Reviewer’s Analysis and Conclusions 

Statistical Methods: All data were tested for normality using Shapiro-Wilks test and for homogeneity 

of variance using Levene’s test. Non-monotonic data which met the assumptions of normality and 

homogeneity of variance were then analyzed using the parametric Dunnett’s test. Non-monotonic data 

which did not meet the parametric assumptions were analyzed using the non-parametric Mann-Whitney 

U test. Monotonic decreasing data were analyzed using the non-parametric Jonckheere-Terpstra test. 

Male and female body weight, male VTG, and fertilization success and fecundity were consistent with 

a monotonic concentration-response and analyzed using the non-parametric Jonckheere-Terpstra test, 

as recommended by the OCSPP 890.1350 guideline. Male tubercle score, male and female GSI, 

female VTG, and male and female length were parametric and not monotonic and analyzed using 

Dunnett’s. None of the surviving females were found to have tubercles. Unless otherwise indicated, 

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

CETIS. 

Both the study author and reviewer relied on the mean-measured concentrations to discuss effects in 

this study. 

 Conclusions: 

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There was a significant decrease in mean male and combined sex survival at the 9.1 mg a.i./L 

treatment level compared to the control (p>0.05; Fisher’s Exact Test). There was no difference in 

female survival at any treatment levels compared to the negative control (Table 9). Male body weight 

(Table 24) exhibited a significant (p<0.05) reduction of 22 and 26% at 0.92 and 9.1 mg a.i./L 

treatment levels, respectively, compared to the negative control; and female body weight was reduced 

29% at the 9.1 mg a.i./L compared to the negative control. Female body length was significantly 

reduced 6% at the 9.1 mg a.i./L treatment level, and male length was significantly reduced 8% at the 

0.92 mg a.i./L treatment level compared to the negative control. Male VTG and GSI (Table 22) was 

significantly reduced 97 and 40%, respectively, at the 9.1 mg a.i./L treatment level. Median male 

tubercle score was also significantly reduced (p<0.05) 95% compared to the control at the 9.1 mg 

a.i./L treatment level. There were no eggs available for assessment at the highest treatment level. 

Therefore, the highest treatment level was excluded from data analysis for the endpoints of fecundity 

and fertility. There was a significant reduction in fecundity and fertility (Table 23) of 50% and 5%, 

respectively, at the 0.92 mg a.i./L treatment level. There were no other significant effects on 

endpoints. 

Although not analyzed statistically, in male fish, there were an increased percentage of spermatogonia 

at the 9.1 mg a.i./L treatment levels. In female fish, there were changes in gonadal staging, decreased 

yolk synthesis, and increased oocyte atresia of predominantly pre-vitellogenic follicles in the 9.1 mg 

a.i./L treatment. 

There were changes in coloration and lethargy observed at the 9.1 mg a.i./L treatment level attributed 

to overt toxicity. In addition, the reviewer noted partial/complete loss of equilibrium in males and 

females of the 9.1 mg a.i./L treatment group. Mild granulomatous inflammation was observed in 1 of 

6 males (17%) from the 9.1 mg a.i./L treatment group and was not diagnosed in fish from the control 

or other treatment groups. In females, minimal granulomatous inflammation was observed in ovaries 

from all groups; 13 of 16 (81% from the control group, 12 of 16 (75%) from the 0.09 mg a.i./L group, 

8 of 16 from the (50%) from the 0.92 mg a.i./L treatment, and 14 of 15 (93%) from the 9.1 mg 

a.i./L group. 

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

 Treatment 

(mg a.i./L) 

[mean-measured]

Tubercle Score GSI 

Gonadal 

Staging and 

Histo. 

Plasma VTG Plasma T Plasma E2 

Median p % Diff. p 

Effect? 

(Yes/No) 

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

Negative Control 

(<LOQ) 

32 NA 0 NA No 0 NA NA NA NA NA

0.099 34 0.88 -4.84 0.57 No 8.30 0.56 NA NA NA NA 

0.92 28 0.17 8.06 0.93 No -67.5 0.22 NA NA NA NA 

9.1 2 <0.0001 -39.84 0.005 Yes -97.5 0.017 NA NA NA NA 

Statistical Test Dunnett’s3 Dunnett’s NA Jonckheere NA NA 

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

LOQ=0.0032 mg a.i./L. 

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 Mean tubercle scores: 32, 33, 28, and 2 for the negative control and mean-measured 0.099, 0.92, and 9.1 mg a.i./L treatment levels, respectively. 

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

Treatment 

(mg a.i./L) 

[meanmeasured]

Fecundity Fert. Success 

Tubercle 

Score 

GSI 

Gonadal 

Staging 

and Histo. 

Plasma VTG Plasma T Plasma E2 

% Diff. p % Diff. P Median p 

% 

Diff. 

p 

Effect? 

(Yes/No) 

% 

Diff. 

p

% 

Diff. 

p 

% 

Diff. 

p 

Negative Control 

(<LOQ) 

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

0.099 -6.15 0.24 0.77 0.68 0 NA 3.52 0.85 No 44.2 0.96 NA NA NA NA 

0.92 -49.85 0.003 -5.10 0.036 0 NA 7.66 0.93 No 70.1 0.99 NA NA NA NA 

9.1 --3 --3 --3 --3 0 NA -64.60 <0.0001 Yes -72.1 0.16 NA NA NA NA 

Statistical Test Jonckheere4 Jonckheere5 NA Dunnett’s NA Dunnett’s NA NA 

Abbreviations: Conc. Concentration. Diff. Difference. E2 17β-estradiol. Fert. Fertilization. GSI Gonado-Somatic Index. Histo. Histopathology. NA Not applicable. T Testosterone. VTG Vitellogenin.LOQ=0.0032 mg a.i./L 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 No eggs were produced at the highest test concentration. Therefore fecundity was 0, there were no fertility data to analyze, and the highest treatment was excluded from 

data analysis for fecundity. 

4 Using Dunnett’s, p-values for 0.099 and 0.92 mg/L are 0.46 and 0.003, respectively. 5 Using Dunnett’s, p-values for 0.099 and 0.92 mg/L are 0.80 and 0.027, respectively.

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Table 24: Growth Endpoints1,2 in the Fish Short-Term Reproduction Assay (FSTRA) with Linuron.

Treatment 

(mg a.i./L) 

[mean-measured]

Body Weight Length 

Males Females Males Females 

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

Negative Control 

(<LOQ) 

0 NA 0 NA 0 NA 0 NA 

0.099 -9.52 0.24 -2.19 0.44 -1.10 0.59 -0.82 0.50 

0.92 -21.67 0.004 -1.68 0.42 -8.18 0.017 0.35 0.83 

9.1 -25.76 0.0007 -28.91 0.008 -2.38 0.40 -5.88 0.002 

Statistical Test Jonckheere Jonckheere Dunnett’s Dunnett’s 

Abbreviations: Diff. Difference. NA Not applicable. 

LOQ=0.0032 mg a.i./L 

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. 

E. Study Deficiencies 

There were deviations from the guideline as noted in Section I. Materials and Methods of the DER which 

did not impact the interpretation of the study. All performance criteria were met in this study with the 

following exceptions: (1) The temperature on Days 0 and 15 briefly decreased to a minimum of 23.4qC; 

however this slight deviation in temperature was not considered to have a negative impact on the results. 

(2) For control replicate B, spawning did not occur at least every four days, and average fecundity was 

not 15 eggs/female/day or greater. However, the average fecundity for that replicate was 13 

eggs/female/day which is slightly less than the recommended rate, and the fecundity rates in the other 

replicates ranged from 16 to 20 eggs/female/day indicating that fecundity was adequate in the control. 

 

F. Reviewer’s Comments 

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

reviewer’s analysis detected statistically significant reductions (p<0.05) in male and combined sex survival, 

male tubercle scores, male and female GSI, and female body weight at the 9.1 mg a.i./L treatment level. 

They also both found a significant decrease (p<0.05) in fecundity and fertilization success at the 0.92 

mg a.i./L, and a decrease in male body weight at both the 0.92 and 9.1 mg a.i./L treatment levels. 

Statistical analyses by the reviewer additionally detected a significant decrease (p<0.05) in male VTG 

and female length at the 9.1 mg a.i./L treatment level and a significant decrease in male length at the 

0.92 mg a.i./L treatment level. The reviewer’s conclusions based on the OCSPP 890.1350 flowchart are 

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

In regards to the histopathology, mild granulomatous inflammation was observed in 1 of 6 males (17%) 

from the high treatment group and was not diagnosed in fish from the control or other treatment groups. 

The granulomatous inflammation was characterized by multifocal accumulations of macrophages within the 

testes in which approximately 2 micron ovoid, eosinophilic structures were occasionally observed. In 

females, minimal granulomatous inflammation was observed in ovaries from all groups; 13 of 16 (81%) 

from the control group, 12 of 16 (75%) from the low treatment group, 8 of 16 from the (50%) from the 

mid treatment group, and 14 of 15 (93%) from the high treatment group. The granulomatous inflammation 

was characterized by focal to multifocal accumulations of macrophages within ovaries and was occasionally 

associated with approximately 2 micron ovoid, eosinophilic structures. Additionally, oocyte atresia was 

reported in the control and treatment groups with 9 of 16 (56%) from the control group, 4 of 16 (25%) 

from the low treatment group, 5 of 16 from the (31%) from the mid treatment treatment, and 11 of 15 

(73%) from the high treatment group. The findings were reported as mild to moderate severity, and with 

the exception of the high treatment, the atresia was in predominantly vitellogenic and/or mature/spawning 

follicles; in the high treatment group, the finding was most often associated in pre-vitellogenic follicles. In 

the control, low and mid treatments, the atresia was frequently associated with granulomatous inflammation 

(89, 100, and 60% of the time for the control, low and mid treatment groups). Granulomatous inflammation 

was associated with oocyte atresia in 10/11 cases in the high treatment group. While oocyte atresia and 

inflammation was observed in the control fish, the fecundity rates for three of the four replicates were 

greater than the minimum rate recommended by the guideline (16 or 20 eggs/female/day) and the fourth 

replicate was close (13 eggs/female/day) to the minimum recommendation of 15 eggs/female/day 

indicating that fecundity in the control was adequate for evaluation in this study. 

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III. REFERENCES 

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

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

Toxicol Chem 20:1276-1290. 

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

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

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

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

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

Pennsylvania. 

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

Guide. Tidepool Scientific Software, McKinleyville, California. 

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

Research 1: 21-29. 

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

Water Res. 3: 793-821. 

OECD (2009). Fish Short Term Reproduction Assay. OECD Guideline for the testing Chemicals: Test 

No. 229. Paris, France. 40 pp 

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

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

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

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U.S. EPA. 1996. Office of Prevention, Pesticides and Toxic Substances. Ecological Effects Guideline, 

OPPTS 850.1000. Special Consideration for Conducting Aquatic Laboratory Studies. “Public Draft”. 

EPA 712-C-96-113. April 1996. U.S. Environmental Protection Agency, Washington, D.C. 

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

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

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

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

Program Test Guideline, OPPTS 890.1350. Fish Short-Term Reproduction Assay. EPA 740-C-09-

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

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CETIS Summary Report Report Date: 10 Jan-14 12:39 (p 1 of 5)

Test Code: 035506 48671701 | 18-0850-2597

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Smithers Viscient

Batch ID: 14-8617-8800

Start Date: 24 Jan-12

Ending Date: 16 Feb-12

Test Type: EDSP FSTRA Tier 1

Duration: 23d 0h

Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Well Water

Species: Pimephales promelas Brine:

Source:

Analyst: E. Scollon

Age: 17w

Sample ID: 19-4222-5604

Sample Date: 24 Jan-12

Receive Date: 16 Feb-12

Code: 035506

Sample Age: NA

Source: Tessenderlo Kerley, Inc.

Station:

Client: EPA OCSPP EFED

Material: Linuron Project: Herbicide

Batch Note: Flow Through

Comparison Summary

Analysis ID Endpoint NOEL LOEL TOEL PMSD TU Method

10-8236-3077 Fecundity 0.099 0.92 0.3018 NA Jonckheere-Terpstra Step-Down Test

11-1767-8906 Fecundity 0.099 0.92 0.3018 27.6% Dunnett Multiple Comparison Test

15-7199-5369 FemaleBodyWt 0.92 9.1 2.893 NA Jonckheere-Terpstra Step-Down Test

04-4721-1928 FemaleBodyWt 0.92 9.1 2.893 11.1% Dunnett Multiple Comparison Test

17-8672-4863 FemaleGSI 0.92 9.1 2.893 NA Jonckheere-Terpstra Step-Down Test

02-7102-8473 FemaleGSI 0.92 9.1 2.893 24.8% Dunnett Multiple Comparison Test

04-4027-9771 FemaleLength 0.92 9.1 2.893 NA Jonckheere-Terpstra Step-Down Test

08-1927-4794 FemaleLength 0.92 9.1 2.893 3.15% Dunnett Multiple Comparison Test

07-5969-5726 FemaleMedianTubercleSco 9.1 >9.1 NA NA Jonckheere-Terpstra Step-Down Test

19-5475-5994 FemaleMedianTubercleSco 9.1 >9.1 NA NA Mann-Whitney U Two-Sample Test

20-5084-9852 FemaleVTG 9.1 >9.1 NA NA Jonckheere-Terpstra Step-Down Test

11-1492-9456 FemaleVTG 9.1 >9.1 NA 106.0% Dunnett Multiple Comparison Test

16-1264-8919 Fertility 0.099 0.92 0.3018 NA Jonckheere-Terpstra Step-Down Test

19-6698-6644 Fertility 0.099 0.92 0.3018 4.35% Dunnett Multiple Comparison Test

03-5527-0519 MaleBodyWt 0.099 0.92 0.3018 NA Jonckheere-Terpstra Step-Down Test

12-3821-5103 MaleBodyWt 0.099 0.92 0.3018 18.0% Dunnett Multiple Comparison Test

09-7606-5995 MaleGSI 0.92 9.1 2.893 NA Jonckheere-Terpstra Step-Down Test

13-9185-5081 MaleGSI 0.92 9.1 2.893 25.1% Dunnett Multiple Comparison Test

00-1534-2468 MaleLength 9.1 >9.1 NA NA Jonckheere-Terpstra Step-Down Test

15-2218-6203 MaleLength 9.1 >9.1 NA 6.44% Dunnett Multiple Comparison Test

12-3845-2165 MaleMedianTubercleScore 0.92 9.1 2.893 NA Jonckheere-Terpstra Step-Down Test

11-6239-3516 MaleMedianTubercleScore 0.92 9.1 2.893 20.0% Dunnett Multiple Comparison Test

14-2658-7275 MaleVTG 0.92 9.1 2.893 NA Jonckheere-Terpstra Step-Down Test

12-3145-2825 MaleVTG 9.1 >9.1 NA 162.0% Mann-Whitney U Two-Sample Test

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

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OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Smithers Viscient

Group Control Type Count Mean Min Max Std Err Std Dev CV%

Fecundity Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 16.3 11.7 20.8 13 20 1.44 2.87 17.7% 0.0%

0.099 4 15.3 10.7 19.8 12 19 1.44 2.87 18.8% 6.15%

0.92 4 8.15 3.39 12.9 4.7 12 1.49 2.99 36.7% 49.8%

Group Control Type Count Mean Min Max Std Err Std Dev CV%

FemaleBodyWt Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 1.55 1.29 1.81 1.37 1.74 0.0822 0.164 10.6% 0.0%

0.099 4 1.52 1.41 1.62 1.45 1.59 0.0326 0.0653 4.31% 2.19%

0.92 4 1.52 1.37 1.68 1.43 1.63 0.0489 0.0977 6.41% 1.68%

9.1 4 1.1 0.997 1.21 1.02 1.18 0.0332 0.0664 6.02% 28.9%

Group Control Type Count Mean Min Max Std Err Std Dev CV%

FemaleGSI Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 12.1 7.21 16.9 9.6 16 1.53 3.06 25.3% 0.0%

0.099 4 12.5 10.4 14.6 11 14 0.645 1.29 10.3% -3.52%

0.92 4 13 10.7 15.3 11 14 0.707 1.41 10.9% -7.66%

9.1 4 4.28 2.93 5.62 3.4 5.3 0.423 0.846 19.8% 64.6%

Group Control Type Count Mean Min Max Std Err Std Dev CV%

FemaleLength Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 42.6 40.8 44.3 41.1 43.7 0.552 1.1 2.59% 0.0%

0.099 4 42.2 41.4 43 41.8 42.8 0.245 0.49 1.16% 0.82%

0.92 4 42.7 41.5 43.9 41.8 43.4 0.389 0.779 1.82% -0.35%

9.1 4 40 38.7 41.4 39.1 41.1 0.411 0.823 2.05% 5.88%

Group Control Type Count Mean Min Max Std Err Std Dev CV%

FemaleMedianTubercleScore Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 0 0 0 0 0 0 0

0.099 4 0 0 0 0 0 0 0

0.92 4 0 0 0 0 0 0 0

9.1 4 0 0 0 0 0 0 0

Group Control Type Count Mean Min Max Std Err Std Dev CV%

FemaleVTG Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 1.43E+6 8.93E+5 1.97E+6 1.18E+6 1.92E+6 1.69E+5 3.37E+5 23.6% 0.0%

0.099 4 2.06E+6 8.92E+5 3.23E+6 1.37E+6 3.10E+6 3.68E+5 7.35E+5 35.7% -44.2%

0.92 4 2.43E+6 -2.28E+5 5.09E+6 4.48E+5 3.88E+6 8.36E+5 1.67E+6 68.7% -70.1%

9.1 4 3.99E+5 -7.21E+3 8.04E+5 8.72E+4 7.11E+5 1.28E+5 2.55E+5 64.0% 72.1%

Group Control Type Count Mean Min Max Std Err Std Dev CV%

Fertility Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 98 95.1 101 96 100 0.913 1.83 1.86% 0.0%

0.099 4 98.8 98 99.5 98 99 0.25 0.5 0.51% -0.77%

0.92 4 93 86 100 88 98 2.2 4.4 4.73% 5.1%

Group Control Type Count Mean Min Max Std Err Std Dev CV%

MaleBodyWt Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 3.41 2.46 4.37 2.87 4.27 0.3 0.599 17.6% 0.0%

0.099 4 3.09 2.92 3.26 2.96 3.22 0.0539 0.108 3.49% 9.52%

0.92 4 2.67 2.38 2.96 2.47 2.87 0.091 0.182 6.81% 21.7%

9.1 4 2.53 1.88 3.19 1.94 2.9 0.206 0.413 16.3% 25.8%

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

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OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Smithers Viscient

Group Control Type Count Mean Min Max Std Err Std Dev CV%

MaleGSI Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 1.55 1.39 1.71 1.4 1.6 0.05 0.1 6.45% 0.0%

0.099 4 1.47 1 1.95 1.1 1.8 0.149 0.299 20.2% 4.84%

0.92 4 1.67 1.35 2 1.5 1.9 0.103 0.206 12.3% -8.06%

9.1 4 0.932 0.455 1.41 0.57 1.3 0.15 0.3 32.2% 39.8%

Group Control Type Count Mean Min Max Std Err Std Dev CV%

MaleLength Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 54.7 49.5 59.9 52 59.4 1.63 3.27 5.97% 0.0%

0.099 4 54.1 51.3 57 53 56.8 0.901 1.8 3.33% 1.1%

0.92 4 50.3 47.9 52.6 48.4 52 0.746 1.49 2.97% 8.18%

9.1 4 53.4 50.7 56.1 50.9 54.4 0.844 1.69 3.16% 2.38%

Group Control Type Count Mean Min Max Std Err Std Dev CV%

MaleMedianTubercleScore Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 31.8 23.7 39.8 26 38 2.53 5.06 15.9% 0.0%

0.099 4 33 25.8 40.2 27 38 2.27 4.55 13.8% -3.94%

0.92 4 27.5 21.3 33.7 23 32 1.94 3.87 14.1% 13.4%

9.1 4 1.63 0.863 2.39 1 2 0.239 0.479 29.5% 94.9%

Group Control Type Count Mean Min Max Std Err Std Dev CV%

MaleVTG Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 1650 -3080 6380 61 6100 1490 2970 180.0% 0.0%

0.099 4 1780 -1880 5450 35 4920 1150 2300 129.0% -8.3%

0.92 4 535 -1010 2080 20 1990 485 970 181.0% 67.5%

9.1 4 41.8 -74.1 158 5 151 36.4 72.8 174.0% 97.5%

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

Page 93 of 124
CETIS Summary Report Report Date: 10 Jan-14 12:39 (p 4 of 5)

Test Code: 035506 48671701 | 18-0850-2597

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Smithers Viscient

Group Control Type

Fecundity Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 16 13 20 16

0.099 15 12 19 15

0.92 8 12 4.7 7.9

9.1

Group Control Type

FemaleBodyWt Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 1.63 1.37 1.74 1.46

0.099 1.48 1.45 1.54 1.59

0.92 1.58 1.45 1.43 1.63

9.1 1.18 1.1 1.02 1.1

Group Control Type

FemaleGSI Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 9.7 13 16 9.6

0.099 12 11 14 13

0.92 13 11 14 14

9.1 3.4 4.6 3.8 5.3

Group Control Type

FemaleLength Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 43.7 41.1 43 42.4

0.099 41.8 42.8 42.4 41.8

0.92 43.4 42.3 41.8 43.3

9.1 41.1 40.1 39.1 39.9

Group Control Type

FemaleMedianTubercleScore Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 0 0 0 0

0.099 0 0 0 0

0.92 0 0 0 0

9.1 0 0 0 0

Group Control Type

FemaleVTG Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 1.92E+6 1.24E+6 1.38E+6 1.18E+6

0.099 1.83E+6 1.95E+6 1.37E+6 3.10E+6

0.92 1.65E+6 3.88E+6 4.48E+5 3.75E+6

9.1 7.11E+5 4.14E+5 8.72E+4 3.82E+5

Group Control Type

Fertility Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 97 96 99 100

0.099 99 99 98 99

0.92 98 91 95 88

9.1

Group Control Type

MaleBodyWt Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 3.3 4.27 2.87 3.22

0.099 3.11 2.96 3.07 3.22

0.92 2.77 2.87 2.58 2.47

9.1 2.9 2.64 2.65 1.94

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

Page 94 of 124
CETIS Summary Report Report Date: 10 Jan-14 12:39 (p 5 of 5)

Test Code: 035506 48671701 | 18-0850-2597

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Smithers Viscient

Group Control Type

MaleGSI Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 1.6 1.6 1.4 1.6

0.099 1.1 1.4 1.8 1.6

0.92 1.5 1.9 1.5 1.8

9.1 0.97 0.57 1.3 0.89

Group Control Type

MaleLength Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 54.4 59.4 52 53.1

0.099 53.6 53 53.1 56.8

0.92 50 52 50.6 48.4

9.1 54.4 54.3 54.1 50.9

Group Control Type

MaleMedianTubercleScore Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 38 33 30 26

0.099 34 33 27 38

0.92 23 26 32 29

9.1 2 1 2 1.5

Group Control Type

MaleVTG Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 71 61 6100 359

0.099 83 2100 4920 35

0.92 92 20 39 1990

9.1 5 6 151 5

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

Page 95 of 124
DATA EVALUATION RECORD

LINURON

Study Type: OCSPP 890.1450, Female Pubertal Assay

EPA Contract No. EP10H001452 

Task Assignment No. 3-08-2012 (MRID 48685501) 

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: 7/31/2012

Primary Reviewer: Signature:

David A. McEwen, B.S. Date: 8/3/2012

Program Manager: Signature:

Jack D. Early, M.S. Date: 8/08/2012

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 8/08/2012

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

signing by CSS-Dynamac Corporation personnel. 

Page 96 of 124
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 97 of 124
Female Pubertal Assay (2012)/ Page 1 of 16 

LINURON I 35506 

Primary Reviewer: Anwar Y. Dunbar, Ph.D. 

Risk Assessment B.ranch 1, Health Effects Division 

Secondary Reviewer: Elizabeth Mendez, Ph.D. 

Risk Assessment Branch 1, Health Effects Division 

DATA EVALUATION RECORD 

OCSPP 890.1450/ OECD None 

STUDY TYPE: Female Pubertal Assay; OCSPP 890.1450; OECD None. 

PC CODE: 35506 DP BARCODE: D403535 

TXR#: 005234 7 CAS No: 330-55-2 

TEST MATERIAL (PURITY): Linuron Technical (99.4% a.i.) 

SYNONYMS: N'-(3,4-dichlorophenyl)-N-methoxy-N-methylurea 

CITATION: Beck, M. J. (2012) A Pubertal Development and Thyroid Function Assay of 

Linuron Administered Orally in Intact Juvenile/Peripubertal Female Rats. WIL 

Research Laboratories, LLC, Ashland, OH, Laboratory Project ID: WIL851004, June 5, 2012. MRID 48685501. Unpublished. 

SPONSOR: Tessenderlo Kerley, Inc., 2255 North 44th Street, Suite 300, Phoenix AZ 

TEST ORDER#: EDSP-033506-122 

EXECUTIVE SUMMARY: In a Female Pubertal Assay (MRID 48685501), 15 Crl:CD(SD) 

Sprague-Dawley rats/dose group were treated daily via oral gavage with linuron technical 

(99.4% a.i., Lot# 1012A3876) in corn oil at doses ofO, 50 or 100 mg/kg/day from post-natal day 

(PND) 22 to 42 or 43. Animals were examined for vaginal opening (VO) daily beginning on 

PND 22, and the age and weight at day of attainment of VO was recorded. Following sacrifice 

on PND 42 or 43, blood was collected for clinical chemistry analyses, including total thyroxine 

(T 4) and thyroid stimulating hormone (TSH), which were analyzed using an 

electrochemiluminescent immunoassay or radioimmunoassay, respectively. Kidney, liver, 

adrenal gland, thyroid, pituitary, uterus, and ovary weights were recorded, and microscopic 

examinations were performed on the thyroid, kidney, ovary, and uterus. 

All females survived to the scheduled euthanasia on Days 42 or 43. No test substance-related 

clinical findings were noted at either dose level at 3 hours post-dose or at the daily examinations 

(-24 hours post-dose). 

There were no significant differences in mean age at VO, body weight at VO, or final body 

weights in the treated groups relative to controls. There were no treatment-related effects on the 

weights ofliver, adrenals, uterus (wet or blotted), or thyroid. There were no dose-related effects 

on cycle status at necropsy, mean age at first vaginal estrous, mean cycle length, percent of 

females cycling, or percent of females regularly cycling. No treatment-related microscopic 

Page 98 of 124
Female Pubertal Assay (2012)/ Page 2 of 16

LINURON / 35506 OCSPP 890.1450/ OECD None

lesions were observed on the thyroid, ovary, uterus, or kidney. Serum TSH was not affected by 

treatment. 

At 100 mg/kg/day, mean body weights were slightly reduced (p≤0.05) by 5-8% for Days 26-29, 

31-39, and 43. Mean cumulative body weight gain was comparable to the controls at the end of 

the study. No effects on bodyweights were observed at 50 mg/kg/day. 

The unadjusted and adjusted kidney weights were decreased (p≤0.05) by 7%, the unadjusted, 

adjusted and relative pituitary weights were decreased (p≤0.05) by 20-22%, and the adjusted 

ovary weights were decreased (p≤0.05) by 12% compared to controls. At 50 mg/kg/day, the 

unadjusted, adjusted, and relative pituitary weights were decreased (p≤0.05) by 13-15%. 

There was a dose-dependent decrease (p≤0.05) in serum T4 levels of 27% and 48% at 50 and 100 

mg/kg/day, respectively. However, there was no associated change in TSH levels or in thyroid 

weight or histology. 

At 100 mg/kg/day, A/G ratio was increased (p≤0.05) by 13%, glucose was decreased (p≤0.05) by 

8%, sodium was decreased (p≤0.05) by 1%, total bilirubin was increased (p≤0.05) by 33%, and 

total protein was decreased (p≤0.05) by 5% compared to the controls. At 50 and 100 mg/kg/day, 

mean AST levels were decreased (p≤0.05) by 29% and 25%, bile acids were increased (p≤0.05) 

by 120% and 92%, mean globulin levels were decreased (p≤0.05) by 7% and 13%, and serum 

urea nitrogen levels were decreased (p≤0.05) by 7% and 13%, respectively. At 50 mg/kg/day, 

calcium was increased (p≤0.05) by 1.7%, chloride was decreased (p≤0.05) by 1%, and potassium 

was decreased (p≤0.05) by 7% compared to the controls. 

The dosing levels tested in this assay are adequate based on the results of a dose range-finding 

study showing body weight loss at 100 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 GLP Compliance, Data Confidentiality, Flagging, and 

Quality Assurance statements were provided. 

Page 99 of 124
Female Pubertal Assay (2012)/ Page 3 of 16

LINURON / 35506 OCSPP 890.1450/ OECD None

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: WIL Research Laboratories, LLC

Location: Ashland, OH

Study Director: M. J. Beck

Other Personnel: G.M. Maginnis (Clinical Veterinarian)

L. Freshwater (Statistical Analysis)

R. E. Boes (Analytical Chemist)

C. A. Picut (Histolopathology) 

Study Period: August 10, 2011 through June 5, 2012

2. Test Substance: Linuron

Description: Off-white powder

Source: MRI Global

Lot #: 1012A3876 (expires April 8, 2016)

Purity: 99.4%

Stability: Stable in corn oil for 15 days at room temperature and refrigerated conditions

CAS #: 330-55-2

Structure:

N

H

Cl

Cl

O

N

O

CH3

CH3

3. Vehicle: Corn oil

4. Test Animals:

Species: Rat (juvenile females)

Strain: Sprague-Dawley (Crl:CD[SD])

Age/Weight at 

Study Initiation: 22 days old/ 42.3-54.1 g

Source: Charles River Laboratories, Inc., Raleigh, NC

Housing: Two-three/cage in plastic cages with Aspen bedding as nesting material.

Diet: Harlan Laboratories 2016CM Teklad Global Certified 16% protein rodent diet, ad libitum

Phytoestrogen content average 11.0 ppm total isoflavones (genistein+daidzein+glycitein)

Water: Reverse-osmosis filtered (on site), ad libitum

Environmental 

Conditions:

Temperature:

Humidity:

Air changes:

Photoperiod:

20.3-22.6°C

35.6-50.7% 

≥10 fresh air changes/hour

14 hrs light/ 10 hrs dark 

B. STUDY DESIGN

1. In-Life Dates: Start: 9/19/2011 End: 10/10/2011 

2. Mating: Time-mated pregnant dams were received from the supplier on gestation day 5 

and were allowed to deliver naturally and rear their pups to PND 21. Litter size was 

standardized to 10 pups (5 males/5 females) on PND 4, using a computer randomization 

procedure. 

Page 100 of 124
Female Pubertal Assay (2012)/ Page 4 of 16

LINURON / 35506 OCSPP 890.1450/ OECD None

3. Animal Assignment: Following weaning on PND 21, the rats from the extreme ends of the 

weight range were eliminated. Animals were assigned to the test groups noted in Table 1

using a randomization procedure designed to ensure equal stratification of body weights. 

Littermates were not assigned to the same treatment group. 

TABLE 1. Study Design a

Test group Dose (mg/kg/day) # of Females

Control 0 15

Low 50 15

High 100 15

a Data were obtained from page 24 of the study report.

4. Dose Selection Rationale: Linuron dose levels were selected based on the body weight

results from previous 14-day range finding study1. Body weight gains were reduced at 75, 

100, and 150 mg/kg/day. The mean body weight reduction was 12.4% at 100 mg/kg/day. 

Based on these results, the dose levels selected for this study were ≤100 mg/kg/day. 

5. Dose Preparation and Analysis: The test substance and solvent control formulations were 

prepared approximately weekly as single formulations for each dose level, divided into 

aliquots for daily dispensation, and stored at room temperature. The formulations were 

stirred continuously throughout the preparation, sampling, and dose administration 

procedures. 

Analyses to demonstrate homogeneity and stability of the test substance formulations 

prepared at 1 and 200 mg/mL following 15 days of refrigerated and room temperature 

storage were conducted in a previous study2. Homogeneity and resuspension homogeneity 

was established at concentrations of 6 and 245 mg/mL following 8 days of refrigerated 

storage during a previous uterotrophic study3 (MRID 48617403). Therefore, stability and 

homogeneity analyses were not conducted in the current study. Samples for concentration 

analysis were collected from the middle stratum of each dosing formulation (including the 

control group) prepared during the in-life phase of the study.

Results of Dose Analysis

Homogeneity (% RSD): Not provided

Stability (% of initial): Provided in report WIL Study Nos. WIL-851005 and WIL851003) 

Concentration (% of nominal): 99.6-105%.

 

1 Sawhney Coder, P. (2012) A 14-Day Dose Range-Finding Oral (Gavage) Toxicity Study of Linuron in Juvenile Female Rats 

for the Endocrine Disruptor Screening Program (Study No. WIL-851002). WIL Research Laboratories, LLC, Ashland, OH

2 Bodle, E.S. (2011). Analytical Validation and Stability Study of Linuron in Aqueous Formulations. Study No. WIL-851005. 

WIL Research Laboratories, LLC, Ashland, OH.

3 Sawhney Coder, P. (2011). A Uterotrophic Assay of Linuron Administered Orally in Ovariectomized Rats. WIL Research 

Laboratories, LLC, Ashland, OH. Study No. WIL-851003, WIL Research Laboratories, LLC, Ashland, OH. MRID 48617403

Page 101 of 124
Female Pubertal Assay (2012)/ Page 5 of 16

LINURON / 35506 OCSPP 890.1450/ OECD None

The analytical data indicated that the variation between nominal and actual dosage to the 

animals was acceptable. 

6. Dosage Administration: All doses were administered once daily by oral gavage, from

PND 22 through PND 42 or 43, in a volume of 5 mL/kg body weight. Individual dosages

were based on the most recently recorded body weights to provide the correct mg/kg/day

dose. Dosing was performed between 0700 and 0900 hours daily.

7. Statistics: Cycling status (cycling vs. non-cycling) and percent of animals cycling were 

assessed using Chi-square analysis. Estrous cycle length and day of first estrus were 

analyzed using a one-way analysis of variance (ANOVA) followed by Dunnett’s test when 

intergroup differences were indicated by ANOVA. Dichotomous pathology data were 

analyzed with pairwise Fisher’s exact test. Tests were conducted at the p≤0.05 significance 

level.

The data that were statistically analyzed included organ weights (liver, kidneys, pituitary 

and adrenals), organ weight to final body weight ratios, daily body weights, weight gains, 

serum chemistries, hormones and vaginal opening data. Each endpoint was tested for 

homogeneity of variance using Levene’s test. If this was positive (p≤0.01) then log or 

square root transformations were applied to the data; if these were positive then a nonparametric test was used. Organ weights, age at vaginal opening and weight at vaginal 

opening data, when parametric, were analyzed using analysis of covariance (ANCOVA) 

followed by Dunnett’s test using PND 21 body weights as the covariate. Other parameters 

were analyzed with ANOVA followed by Dunnett’s test. Linear trend tests were used in 

conjunction with both ANOVA and ANCOVA. For non-parametric data the Kruskal-Wallis 

test was used followed by Dunn’s test. Pathology data presented as a graded response were 

analyzed with a pairwise Mann-Whitney U test. Dichotomous histopathology data were 

analyzed using a Fisher’s exact test.

Mean values were presented with standard deviations and coefficient of variation. Analyses 

were conducted using SAS® (Cary, NC) version 9.2 (or higher) software. 

Page 102 of 124
Female Pubertal Assay (2012)/ Page 6 of 16

LINURON / 35506 OCSPP 890.1450/ OECD None

Classification Procedure

Cycling status

Percent of animals cycling 

regularly

Chi-square analysis was performed.

Estrous cycle length

Day of first estrus

One-way ANOVA was conducted, followed by Dunnett’s test when ANOVA 

demonstrated significant intergroup variance.

Pathology (dichotomous 

response)

One-sided Fisher’s exact test was performed.

Organ weights

Organ weight to terminal body 

weight ratio

Body weight

Cumulative body weight gain

Clinical chemistry

Hormone levels

Vaginal opening data

Each endpoint was tested for homogeneity of variance using Levene’s test. If that test was 

significant at p=0.01, then a log transform was applied and Levene’s test conducted on the 

transformed data. If that test was still significant then the square root transformation was 

applied to the raw data and Levene’s test conducted again. If the test was still significant 

then a nonparametric test, as described below, was used to analyze the data. 

If the variances were homogeneous then an analysis of variance (ANOVA) was conducted,

on the raw or transformed data, as appropriate. The statistical model contained a factor for 

treatment group and a blocking factor based on necropsy date. A 2-sided Dunnett’s test 

was conducted, regardless of the outcome of the ANOVA, looking for significant 

differences in the test substance groups when compared with the vehicle control. If the 

transformations were unsuccessful in making the variances homogeneous then the 

nonparametric Kruskal-Wallis test was used, ignoring the blocking factor, followed by 

Dunn’s test, to compare each of the test substance groups with the vehicle control. Since 

these are pre-planned pairwise comparisons, Dunn’s test was conducted regardless of the 

outcome of the Kruskal-Wallis test. The tests were 2-sided, at the 0.05 significance level, 

looking for significant differences from the vehicle control.

In addition, organ weights and vaginal opening data were subject to the following analyses 

if they met the homogeneity of variance criteria: 1) Analysis of covariance (ANCOVA) 

with Dunnett’s test. The model was as described above for ANOVA with the exception 

that PND 21 body weight was included as a covariate; 2) linear trend test using the 

ANOVA model; and 3) linear trend test using the ANCOVA model.

When an animal did not attain vaginal opening, PND 43 or 44 (last study day + 1) and 

body weight at necropsy were used as the age and body weight at vaginal opening, 

respectively. Gamma glutamyltransferase values under range were assigned a value of 0.1 

(half the lower limit of quantitation) for statistical analysis and reporting.

Graded histopathology findings The pairwise Mann-Whitney U test was used to compare each treated group with the

control. The tests were 1-sided at the 0.05 significance level testing for increased severity. 

The tests were 2-sided for graded responses presented on a Grade 1-5 scale at the 0.05 

significance level testing for increased or decreased severity. Exact p-values were

calculated for the Mann-Whitney U test.

The statistical analyses were considered appropriate. 

C. METHODS

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

mortality and moribundity. Clinical examinations were conducted on the day of 

randomization and daily through the day of euthanasia. Additional observations for signs of 

toxicity were conducted approximately 3 hours after dosing.

2. Body Weight: Animals were weighed on the day of randomization, daily prior to dosing 

and the day of euthanasia. Mean body weight changes were calculated for each 

corresponding interval and also for PND 22-42. 

Page 103 of 124
Female Pubertal Assay (2012)/ Page 7 of 16

LINURON / 35506 OCSPP 890.1450/ OECD None

3. Vaginal Opening: Beginning on PND 22, all animals were examined daily for onset of 

vaginal opening. The appearance of a small “pin hole”, a vaginal thread, and complete 

vaginal opening were recorded. The age and body weight on the day at which the vaginal 

lumen was first observed to open was recorded for each animal. Examination of the females 

was continued daily until vaginal opening was present or until euthanasia.

4. Estrous Cyclicity: Beginning on the day vaginal opening was observed, vaginal lavages 

were performed daily, and the slides were evaluated microscopically to determine the stage 

of estrus of each female until necropsy. The age of first vaginal estrus after vaginal opening 

was recorded. The average cycle length was calculated and reported for complete estrous 

cycles. At the end of the study, the overall pattern of each female was characterized as 

regularly cycling, irregularly cycling, not cycling, or insufficient data. 

5. Sacrifice and Pathology: On the day prior to sacrifice, all animals were moved to a 

holding room separate from where the necropsies were performed so as not to induce stressrelated responses in other animals which may affect hormone measurements. On PND 42 or 

43, all females were euthanized randomly across all groups by decapitation without 

anesthesia, and the time of euthanasia was recorded. Animals were euthanized beginning at 

least 2 hours following dose administration that day, and all sacrifices were completed by 

1300 hours. Immediately following decapitation, trunk blood samples were taken for T4, 

TSH and serum chemistry analyses. The samples were stored at ≤−20° C. Necropsies were 

performed on all females found dead or that survived to the scheduled sacrifice. Any tissues 

with gross lesions were saved in 10% neutral-buffered formalin for possible future 

histopathologic examination.

a. Hormone Analysis: T4 was analyzed using an electrochemiluminescent assay, and TSH 

levels were analyzed using the [125I]rTSH kit obtained from Izotop, Institute of Isotopes Ltd. 

(Budapest, Hungary); the assay system relies on magnetic separation for quantitative 

measurement of TSH using a GAMMA-C12 gamma counter. For both assays, multiple 

quality control samples were run prior to analysis.

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

Page 104 of 124
Female Pubertal Assay (2012)/ Page 8 of 16

LINURON / 35506 OCSPP 890.1450/ OECD None

ELECTROLYTES OTHER

X Calcium X Albumin

X Chloride X Creatinine*

Magnesium X Urea nitrogen*

X Phosphorus X Total cholesterol

X Potassium X Globulins

X Sodium X Glucose

ENZYMES X Total bilirubin

X Alkaline phosphatase (ALP) X Total protein

Cholinesterase (ChE) X Triglycerides

Creatine phosphokinase Serum protein electrophoresis

Lactic acid dehydrogenase (LDH) X Albumin globulin ratio (A/G Ratio, by calculation)

X Alanine aminotransferase (ALT/also SGPT) X Bile acids

X Aspartate aminotransferase (AST/also SGOT)

Sorbitol dehydrogenase

X Gamma glutamyl transferase (GGT)

Glutamate dehydrogenase

* Recommended for the pubertal assay in female rats based on Guideline 890.1450.

c. Organ Weights and Histopathology: The following CHECKED (X) tissues were 

collected and weighed. The (XX) organs, in addition, were subjected to histological 

examination.

UROGENITAL OTHER

XX Ovaries (paired, without oviducts)*+ XX Thyroid*+

XX Uterus*+ X Liver*

XX Kidneys (paired)*+ X Adrenals (paired)*

XX Pituitary*

* Weights required based on guideline 890.1450

+ Histopathological examination required based on Guideline 890.1450

All organs except the thyroid/trachea were weighed prior to fixation. Paired organs 

(kidneys, adrenals, and ovaries) were weighed together. The uterus was weighed “wet” and 

then again following removal of the fluid in the lumen (blotted weight). 

The ovaries (left or right not specified), uterus, and kidneys were fixed in 10% buffered 

formalin, rinsed, and stored in 70% ethanol prior to embedding. The thyroid was fixed in 

10% buffered formalin for at least 24 hours. The thyroid gland was dissected from the 

trachea, blotted, weighed, and placed in 70% ethanol until histopathological processing. 

The liver, adrenals, and pituitary were placed in 10% neutral-buffered formalin for potential 

future histopathologic examination.

After fixation, protocol-specified tissues were shipped to Biotechnics, LLC (Hillsborough, 

NC) for processing. Tissue samples from the thyroid, kidney, ovary, and uterus were 

routinely processed into paraffin blocks, sectioned, stained with hematoxylin and eosin, and 

examined microscopically.

Page 105 of 124
Female Pubertal Assay (2012)/ Page 9 of 16

LINURON / 35506 OCSPP 890.1450/ OECD None

Thyroid sections were subjectively evaluated for follicular epithelial cell height and colloid 

area, using a five point grading scale (1 = shortest/smallest; 5 = tallest/largest), and any 

abnormalities/lesions were noted. At least two sections of the thyroid were examined. Five 

random sections of an ovary were evaluated for follicular development and any 

abnormalities/lesions (such as atrophy). The ovary sections were evaluated on a five point 

grading scale (1=markedly reduced number or absent; 3=normal; 5=markedly increased 

number) for primordial, primary, secondary, tertiary, and atretic follicles and growth phase, 

mature, and regressive corpora lutea. Uterine histology documented cases of uterine hyperor hypotrophy as characterized by changes in uterine horn diameter and myometrial stromal, 

or endometrial gland development. The stage of the estrous cycle of the female at the time 

of necropsy was taken into account in the final histological assessment, as ovarian and 

uterine cellular changes are dependent upon endocrine status. 

II. RESULTS 

A. MORTALITY: All females survived to the scheduled euthanasia on Days 42 or 43. 

B. CLINICAL SIGNS OF TOXICITY: No test substance-related clinical findings were 

noted at 3 hours post-dose or at the daily examinations (~24 hours post-dosing) at any dose 

level. Incidental findings noted in the treated groups, including scabbing and clear or red 

material around the mouth, occurred infrequently, at similar frequencies in the control 

group, and/or in a manner that was not dose-related.

C. GENERAL GROWTH AND VAGINAL OPENING: Body weights, body weight gains, 

age at attainment of vaginal opening and weight at day of attainment are presented in Table 

2. There were no significant differences in mean age at vaginal opening, body weight at 

vaginal opening, or final body weights in the treated groups relative to controls. 

At 100 mg/kg/day, mean body weights were slightly reduced (p≤0.05) by 5-8% for Days 26-

29, 31-39, and 43. However, mean cumulative body weight gain was comparable to the 

controls at the end of the study. No effects on bodyweights were observed at 50 mg/kg/day.

Guideline criteria in the controls for body weight at VO and final body weight were met. 

The mean age at VO in the control group (36.6 days) was slightly greater than the maximum 

acceptable value in the performance criteria (35.62 days). 

Page 106 of 124
Female Pubertal Assay (2012)/ Page 10 of 16

LINURON / 35506 OCSPP 890.1450/ OECD None

TABLE 2. General Growth and Vaginal Opening (VO)a

Parameter Evaluated

Vehicle Control Linuron

50 mg/kg/day

Linuron

100 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)

Initial body weight

(PND 22; g) U 15 49.3 3.33 6.76 15 49.4 2.12 4.29 15 49.3 1.96 3.98

Body weight at vaginal opening (g) U 15 125.9 11.73 9.32 15 120.2 11.31 9.41 15 122.9 9.28 7.55

A 15 126.1 11.73 9.32 15 120.1 11.31 9.41 15 122.8 9.28 7.55

Final body weight (g) U 15 153.5 12.58 8.19 15 151.1 9.54 6.32 15 146.2 10.17 6.96

Final body weight (% of control) U 15 NA 15 98.4 15 95.2

Body weight gain

(final – initial; g) U 15 104.2 10.8 10.37 15 101.7 8.53 8.38 15 96.9 9.2 9.5

Age at vaginal opening (PND) U 15 36.6 2.29 6.26 15 36.7 1.71 4.65 15 38 1.93 5.07

A 15 36.6 2.29 6.26 15 36.7 1.71 4.65 15 38 1.93 5.07

Proportion unopened (#/N) 0/15 0/15 1/15

a Data were obtained from Table 1 on page 53 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. At 100 mg/kg/day, 

the unadjusted and adjusted kidney weights were decreased (p≤0.05) by 7%, the unadjusted, 

adjusted, and relative pituitary weights were decreased (p≤0.05) by 20-22%, and the 

adjusted ovary weights were decreased (p≤0.05) by 12% compared to controls. At 50 

mg/kg/day, the unadjusted, adjusted, and relative pituitary weights were decreased (p≤0.05) 

by 13-15%. There were no treatment-related effects on the weights of liver, adrenals, uterus 

(wet or blotted), or thyroid. 

The lower kidney, pituitary, and ovary weights fell within the WIL historical control 

database. The lower mean ovary weight at 100 mg/kg/day was not considered treatmentrelated because one animal was observed with no corpora lutea.

The unadjusted values for all organ weights in the control group were within the acceptable 

range of the performance criteria provided in the Guideline (890.1450), with the exception 

of mean adrenal glands weight (32.8 mg; acceptable range 38.34-48.84 mg).

Page 107 of 124
Female Pubertal Assay (2012)/ Page 11 of 16

LINURON / 35506 OCSPP 890.1450/ OECD None

TABLE 3. Organ Weights at Necropsya

Organ Vehicle Control Linuron

50 mg/kg/day

Linuron

100 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)

Liver

(g)

U 15 7.32 0.895 12.23 15 7.13 0.776 10.89 15 7.08 0.849 11.99

A 15 7.32 0.895 12.23 15 7.14 0.776 10.89 15 7.06 0.849 11.99

R 15 4.71 0.292 6.19 15 4.65 0.299 6.44 15 4.77 0.32 6.71

Kidneys

(g)

U 15 1.21 0.098 8.06 15 1.16 0.112 9.64 15 1.12* 

(↓7) 0.125 11.08

A 15 1.21 0.098 8.06 15 1.16 0.112 9.64 15 1.12* 

(↓7) 0.125 11.08

R 15 0.78 0.041 5.27 15 0.76 0.045 5.92 15 0.76 0.05 6.54

Pituitary

(mg)

U 15 9.4 1.32 14.01 15 8* 

(↓15) 1.46 18.14 15 7.3* (↓22) 1.48 20.14

A 15 9.4 1.32 14.01 15 8* 

(↓15) 1.46 18.14 15 7.3* (↓22) 1.48 20.14

R 15 6.1 0.71 11.62 15 5.3* 

(↓13) 1 19.04 15 4.9* (↓20) 0.79 16.07

Adrenals

(mg)

U 14 32.8 3.76 11.48 15 32.1 5.81 18.1 15 31.8 4.94 15.52

A 14 32.7 3.76 11.48 15 32.1 5.81 18.1 15 31.7 4.94 15.52

R 14 21.1 1.88 8.92 15 21 3.61 17.19 15 21.5 2.81 13.09

Ovaries

(mg)

U 15 69.2 10.83 15.65 15 68.5 9.27 13.54 15 60.6 12.25 20.22

A 15 69.3 10.83 15.65 15 68.5 9.27 13.54 15 60.3* 

(↓12) 12.25 20.22

Uterus, wet

(mg)

U 15 326.6 187.0 57.24 15 306.3 130 42.44 15 269.2 147.2 54.67

A 15 323.8 187.0 57.24 15 307.7 130 42.44 15 269.2 147.2 54.67

Uterus, blotted

(mg)

U 15 252.8 73.94 29.25 15 245.1 63.01 25.71 15 221.6 77.82 35.12

A 15 252.4 73.94 29.25 15 245.4 63.01 25.71 15 221.3 77.82 35.12

Thyroid, fixed

(mg)

U 15 7.74 1.38 17.77 15 7.68 1.63 21.18 15 7.45 1.31 17.63

A 15 7.73 1.38 17.77 15 7.69 1.63 21.18 15 7.44 1.31 17.63

a Data were obtained from Table 2 on page 54 of the study report. Percent differences from controls, calculated by the 

reviewers, are included in parentheses.

U Unadjusted for body weight on PND 22

A Adjusted for 100 g body weight on PND 22

N Number of animals examined

SD Standard Deviation

CV Coefficient of Variation

R Organ-to-body weight ratio (relative to body weight) * Significantly different from controls at p<0.05.

E. ESTROUS CYCLICITY: Estrous cycle data are provided in Table 4. There were no 

dose-related effects on cycle status at necropsy, mean age at first vaginal estrous, mean 

cycle length, percent of females cycling, or percent of females regularly cycling. For the 

dietrus females, one vehicle control female and one 50 mg/kg/day female were not cycling. 

The percent of females regularly cycling were 73% and 20% for the 50 and 100 mg/kg/day 

females, respectively, compared to 43% of control females.

Page 108 of 124
Female Pubertal Assay (2012)/ Page 12 of 16

LINURON / 35506 OCSPP 890.1450/ OECD None

TABLE 4. Estrous Cyclicitya

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 15 38.2 4.8 100.0 42.9 9b 0 6 1b

Linuron

50 mg/kg/day 15 38.1 4.8 93.3 66.7 10b 1 4 1b

Linuron

100 mg/kg/day 14 38.7 5.0 100.0 20.0 11 1 2 0 

a Data were obtained from Tables 2 and 3 on pages 54 and 55 of the study report. 

b One diestrus female was not cycling (no estrus).

N Number of animals

** Significantly different from controls at p<0.01.

E. HORMONE LEVELS AND CLINICAL CHEMISTRY: Mean hormone levels and 

clinical chemistry values are presented in Table 5. Serum T4 levels were decreased 

(p≤0.05) by 27% and 48% at 50 and 100 mg/kg/day, respectively, but serum TSH levels 

were not affected by treatment.

At 100 mg/kg/day, A/G ratio was increased (p≤0.05) by 13%, glucose was decreased 

(p≤0.05) by 8%, sodium was decreased (p≤0.05) by 1%, total bilirubin was increased 

(p≤0.05) by 33%, and total protein was decreased (p≤0.05) by 5% compared to the controls. 

At 50 and 100 mg/kg/day, mean AST levels were decreased (p≤0.05) by 29% and 25%, bile 

acids were increased (p≤0.05) by 120% and 92%, mean globulin levels were decreased 

(p≤0.05) by 7% and 13%, and serum urea nitrogen levels were decreased (p≤0.05) by 7% 

and 13%, respectively. 

At 50 mg/kg/day, calcium was increased (p≤0.05) by 1.7%, chloride was decreased (p≤0.05) 

by 1%, and potassium was decreased (p≤0.05) by 7% compared to the controls. 

All other evaluated clinical chemistry parameters in the treated groups were similar to the 

control group. In addition, the study report provided normal ranges for the clinical 

chemistry measures.

The Guideline criterion was met for serum T4 level in the controls. Thirteen of the 20 

clinical chemistry control values were outside the historical range for the performing 

laboratory.

Page 109 of 124
Female Pubertal Assay (2012)/ Page 13 of 16

LINURON / 35506 OCSPP 890.1450/ OECD None

TABLE 5. Hormone Levels and Clinical Chemistrya

Parameter

Evaluated

Vehicle Control Linuron

50 mg/kg/day

Linuron

100 mg/kg/day

N Mean SD

CV 

(%) N Mean SD

CV 

(%) N Mean SD

CV 

(%)

Hormones

Serum T4, Total

(μg/dL)

15 4.47 0.693 15.50 15 3.26*

(↓27)

0.47 14.42 15 2.32*

(↓48)

0.33 14.18

Serum TSH

(ng/mL)

15 5.19 1.61 31.07 15 4.79 2.22 46.22 15 4.83 1.79 37.09

Clinical Chemistry

A/G ratio 15 2.64 0.408 15.45 15 2.84 0.38 13.37 15 2.99*

(↑13)

0.365 12.21

Albumin (g/dL) 15 4 0.21 5.3 15 3.9 0.28 7.04 15 3.9 0.22 5.57

Alkaline phosphatase (U/L) 15 336 44.2 13.2 15 325 42.5 13.1 15 310 40.6 13.1

ALT (U/L) 15 95 20.8 21.9 15 90 8.3 9.2 15 103 19.5 19

AST (U/L) 15 318 156 49 15 227*

(↓29)

66.4 29.3 15 239*

(↓25)

92.8 38.9

Bile acids (μmol/L) 15 26.2 13.6 51.91 15 57.8*

(↑120)

33.5 57.92 15 50.2*

(↑92)

24.96 49.67

Calcium (mg/dL) 15 11.8 0.49 4.19 15 12*

(↑1.7)

0.62 5.15 15 12 0.6 4.98

Chloride (mEq/L) 15 101 1.6 1.6 15 100*

(↓1.0)

1.3 1.3 15 101 1.5 1.5

Cholesterol (mg/dL) 15 97 16 16.4 15 91 10.4 11.4 15 91 10.3 11.3

Creatinine

(mg/dL)

15 0.1 0.06 56.47 15 0.1 0.05 42.91 15 0.1 0.04 31.05

GGT (U/L) 15 0.6 0.51 88.32 15 0.6 0.55 91.94 15 0.7 0.59 89.97

Globulin (g/dL) 15 1.5 0.19 12.25 15 1.4*

(↓7)

0.12 8.69 15 1.3*

(↓13)

0.15 11.46

Glucose (mg/dL) 15 146 11.7 8 15 139 15.6 11.2 15 134*

(↓8)

11.6 8.7

Phosphorus (mg/dL) 15 9.9 1.13 11.46 15 10.2 0.91 8.84 15 9.7 0.72 7.41

Potassium (mEq/L) 15 7.14 1.139 15.95 15 6.65*

(↓7)

0.367 5.51 15 6.97 0.642 9.22

Sodium (mEq/L) 15 139 2.3 1.6 15 138 2.1 1.5 15 138*

(↓1)

1.4 1

Total bilirubin (mg/dL) 15 0.06 0.029 44.65 15 0.06 0.04 62.73 15 0.08*

(↑33)

0.032 38.39

Total protein (g/dL) 15 5.5 0.24 4.46 15 5.3 0.25 4.73 15 5.2*

(↓5)

0.26 5.02

Triglyceride (mg/dL) 15 143 65.8 46.1 15 182 105.6 58.1 15 149 105.9 71.2

Serum urea nitrogen

(mg/dL)

15 13.2 1.58 12.04 15 10.6*

(↓20)

2.05 19.38 15 10*

(↓24)

2.14 21.38

a Data were obtained from Tables 4 and 5 on pages 56 and 57, and Tables S8 and S9 on pages 81-91 of the study report. 

b Percent difference from controls, calculated by the reviewers, is included in parentheses. 

N Number of animals examined

SD Standard Deviation

CV Coefficient of Variation

* Significantly different from controls at p<0.05.

Page 110 of 124
Female Pubertal Assay (2012)/ Page 14 of 16

LINURON / 35506 OCSPP 890.1450/ OECD None

F. GROSS PATHOLOGY: One 100 mg/kg/day female was observed with no corpora lutea 

in the ovary and one ovary was not present. One 50 mg/kg/day female and two 100 

mg/kg/day females were observed with a relative increase in the number of acidophils in the 

pars distalis of the pituitary. These findings were reported as unrelated to treatment and 

caused by biologic variability. 

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

are presented in Table 6. There were no treatment-related findings in the colloid area or 

follicular cell height. 

TABLE 6. Incidence of Histopathological Lesions of the Thyroid Gland a

Treatment 

Groups

Parameter Evaluated

Colloid Area Follicular Cell Height

Gradeb Incidence Gradeb Incidence

O E O E

Vehicle Control

1 0 15 1 6 15

2 0 15 2 9 15

3 1 15 3 0 15

4 10 15 4 0 15

5 4 15 5 0 15

Linuron

50 mg/kg/day

1 0 15 1 9 15

2 0 15 2 6 15

3 0 15 3 0 15

4 10 15 4 0 15

5 5 15 5 0 15

Linuron

100 mg/kg/day

1 0 15 1 10 15

2 0 15 2 5 15

3 0 15 3 0 15

4 9 15 4 0 15

5 6 15 5 0 15

a Data were obtained from Table S12 on page 102 of the study report.

b Thyroid histopathology is graded on a 1 – 5 scale: Follicular cell height,1 = lowest, 5 = highest; and Colloid area, 

1 = most colloid, 5 = least colloid.

O Number Observed

E Number Examined

The incidence of histopathological findings of the ovaries, uterus, and kidneys are presented 

in Table 7. There were no treatment-related findings in the ovaries, uterus, or kidneys.

Page 111 of 124
Female Pubertal Assay (2012)/ Page 15 of 16

LINURON / 35506 OCSPP 890.1450/ OECD None

TABLE 7. Incidence of Histopathological Lesions of the Ovaries, Uterus and Kidneya

Findings

Dose Level (mg/kg/day)

Vehicle Control Linuron

50 mg/kg/day

Linuron

100 mg/kg/day

# Observed # Examined # Observed # Examined # Observed # Examined

Ovaries

Unremarkable 15 15 14 15 15 15

Cyst 0 15 1 15 0 15

Uterus

Unremarkable 15 15 15 15 15 15

Kidney

Unremarkable 9 15 14 15 11 15

Basophilic tubules, minimal 1 15 1 15 2 15

Dilatation, tubular, minimal 1 15 0 15 0 15

Dilatation, tubular, mild 1 15 0 15 0 15

Fibrosis, minimal 3 15 0 15 2 15

Mineralization, minimal 1 15 0 15 0 15

Mineralization, mild 1 15 0 15 0 15

a Data were obtained from Table S12 on pages 99-102 of the study report.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: Effects of linuron in this study were limited to 

lower body weights and body weight gains at the high dosage level of 100 mg/kg/day and 

lower total T4 levels at dose levels of 50 and 100 mg/kg/day. The significance of the lower 

T4 is not clear, since there was no statistically significant change in mean serum TSH levels, 

no change in thyroid gland weights, and no test substance-related microscopic findings in 

the thyroid gland. 

However, based on the lack of effects on the attainment of vaginal patency, estrous 

cyclicity, organ weights, serum chemistry, and histopathology of the ovary, thyroid, uterus, 

pituitary, and kidney, linuron was not considered to be a pubertal developmental toxicant 

when administered orally by gavage to juvenile/peripubertal female Crl:CD(SD) rats at 

dosage levels of 50 and 100 mg/kg/day.

In the dose-range finding study, a dosage level of 100 mg/kg/day resulted in lower mean 

body weights and body weight gains that approached the maximum tolerated dose. Based 

on the effects observed in the range-finding study, a high dosage level of 100 mg/kg/day 

was considered appropriate for the definitive pubertal development study.

B. AGENCY COMMENTS: All females survived to the scheduled euthanasia on Days 42 or 

43. No test substance-related clinical findings were noted at 3 hours post-dose or at the 

daily examinations (ca. 24 hours post-dose) at any dose level. 

There were no significant differences in mean age at vaginal opening, body weight at 

vaginal opening, or final body weights in the treated groups relative to controls. There were 

no treatment-related effects on the weights of liver, adrenals, uterus (wet or blotted), or 

thyroid. There were no dose-related effects on cycle status at necropsy, mean age at first 

vaginal estrous, mean cycle length, percent of females cycling, or percent of females 

regularly cycling. It should be noted, however, that less than half of the females in the 

Page 112 of 124
Female Pubertal Assay (2012)/ Page 16 of 16

LINURON / 35506 OCSPP 890.1450/ OECD None

control group were cycling regularly (≈ 43%) thus confounding the interpretation of the 

estrus cyclicity data. No treatment-related microscopic lesions were observed on the 

thyroid, ovary, uterus, or kidney. Serum TSH was not affected by treatment. 

At 100 mg/kg/day, mean body weights were slightly reduced (p≤0.05) by 5-8% for Days 26-

29, 31-39, and 43. However, mean cumulative body weight gain was comparable to the 

controls at the end of the study. No effects on body weights were observed at 50 

mg/kg/day. 

The unadjusted and adjusted kidney weights were decreased (p≤0.05) by 7%, the 

unadjusted, adjusted and relative pituitary weights were decreased (p≤0.05) by 20-22%, and 

the adjusted ovary weights were decreased (p≤0.05) by 12% compared to controls. At 50 

mg/kg/day, the unadjusted, adjusted, and relative pituitary weights were decreased (p≤0.05) 

by 13-15%. 

For the dietrus females, one vehicle control female and one 50 mg/kg/day female were not 

cycling. The percent of females regularly cycling were 73% and 20% for the 50 and 100 

mg/kg/day females, respectively, compared to 43% of control females.

Serum T4 levels were decreased (p≤0.05) by 27% and 48% at 50 and 100 mg/kg/day, 

respectively, however, there was no corresponding change in TSH levels or in thyroid 

weights for histopathology.

 

At 100 mg/kg/day, A/G ratio was increased (p≤0.05) by 13%, glucose was decreased 

(p≤0.05) by 8%, sodium was decreased (p≤0.05) by 1%, total bilirubin was increased 

(p≤0.05) by 33%, and total protein was decreased (p≤0.05) by 5% compared to the controls. 

At 50 and 100 mg/kg/day, mean AST levels were decreased (p≤0.05) by 29% and 25%, bile 

acids were increased (p≤0.05) by 120% and 92%, mean globulin levels were decreased 

(p≤0.05) by 7% and 13%, and serum urea nitrogen levels were decreased (p≤0.05) by 7% 

and 13%, respectively. At 50 mg/kg/day, calcium was increased (p≤0.05) by 1.7%, chloride 

was decreased (p≤0.05) by 1%, and potassium was decreased (p≤0.05) by 7% compared to 

the controls.

The high dose of 100 mg/kg/day was considered adequate based on data from a dose rangefinding study showing body weight loss at 100 mg/kg/day.

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

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

study: 

• The mean age at VO in the control group (36.6 days) was greater than the performance 

criteria maximum (35.62 days). 

• The control group mean for adrenal gland weight (32.8 mg) was less than the 

performance criteria minimum (38.34 mg).

Page 113 of 124
DATA EVALUATION RECORD

LINURON

Study Type: OCSPP 890.1600, In Vivo Uterotrophic Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-21-2012 (MRID 48617403) 

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: 2/20/2012

Secondary Reviewer Signature:

Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 2/27/2012

Program Manager: Signature:

Jack D. Early, M.S. Date: 3/02/2012

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 3/02/2012

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

signing by CSS-Dynamac Corporation personnel. 

Page 114 of 124
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 115 of 124
LINURON/ 035506 

In Vivo Uterotrophic Assay (2011) I Page 1 of 9 

OCSPP 890.1600/ OECD 440 

Primary Reviewer: Sheila Healy, Ph.D. Sign,t"Dr''': ----1'--j.,..---+"7'"77-

Health Effects Division Date: -~~m:J~[9J5,, 

Secondary Reviewer: ~Je~s~s_R~o_w_l~an~d~ _________ Signature: -----~--

Health Effects Division Date: __:~~~~~ 

Template version 09/2011 

DATA EVALUATION RECORD 

STUDY TYPE: Uterotrophic Assay (Rat); OCSPP 890.1600; OECD 440 

PC CODE: 035506 DP BARCODE: D399669 

TXR#: 0052347 CAS#: 330-55-2 

TEST MATERIAL (PURITY): Linuron (99.4% a.i.) 

SYNONYMS: N'-(3,4-dichlorophenyl)-N-methoxy-N-methylurea 

CITATION: Coder, P.S. (2011). A uterotrophic assay of linuron administered orally in 

ovariectomized rats. WIL Research Laboratories, LLC, Ashland, OH. 

Laboratory Study ID: WIL-851003, December 8, 2011. MRID 48617403. 

Unpublished. 

SPONSOR: Tessenderlo Kerley, Inc., Phoenix, AZ 

TEST ORDER#: EDSP-035506-122 

EXECUTIVE SUMMARY: In a uterotrophic assay (MRID 48617403) conducted to screen for 

potential estrogenic activity, linuron (99.4% a.i., Lot# 1012A3876) in com oil was administered 

daily via oral gavage to groups of 6 ovariectomized Sprague-Dawley rats at dose levels of 0 

(vehicle), 30, 60 and 120 mg/kg/day on post-natal days (PND) 72-74. A positive control group 

was treated with 17a-ethynyl estradiol (EE) by daily subcutaneous (s.c.) injection at a dose level 

of 0.003 mg/kg/day. All animals were terminated and necropsied approximately 24 hours after 

the final dose on PND 75 to determine liver, and wet and blotted uterine weights. 

All animals survived until scheduled termination. Red material was observed around the nose at 

120 mg/kg/day on one day for 3/6 animals compared to 016 animals in the other groups. This 

finding remained in a single animal (same day) at 3 hours post-dose. Body weights were 

decreased in all linuron treated groups with significantly decreased body weights at 120 

mg/kg/day (!10-12%, Days 3-4). Overall body weight gains were decreased (p<0.05) for the 30, 

60 and 120 mg/kg/day groups by 66, 117, and 192%, respectively. Uterine and liver weights in 

the linuron-treated groups were comparable to the controls. Absolute wet and blotted uterus 

weights for the positive EE group were increased (p<0.05) by 797% and 300%, respectively, as 

expected. The high dose tested in this study was adequate based on the observed decreases in 

body weights. 

Page 116 of 124
In Vivo Uterotrophic Assay (2011) / Page 2 of 9 

LINURON/ 035506 OCSPP 890.1600/ OECD 440

No statistically significant changes were seen in uterine weight in this assay. Linuron is negative 

in the uterotrophic assay. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a uterotrophic assay (OCSPP 

890.1600). 

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

Quality Assurance statements were provided. 

Page 117 of 124
In Vivo Uterotrophic Assay (2011) / Page 3 of 9 

LINURON/ 035506 OCSPP 890.1600/ OECD 440

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: WIL Research Laboratories, LLC

Location: Ashland, OH

Study Director: Prägati S. Coder, PhD, DABT

Other Personnel: Eric S. Bodle, PhD 

Sally A. Keets, AS 

Carol A. Kopp, BS, LAT

Gwendalyn M. Maginnis, DVM

John S. Moore, BS, RLATG

Theresa M. Rafeld, CPhT

Michael J. Schlosser, PhD, DABT

Bennett J. Varsho, MPH, DABT

Robert A. Wally, BS

Assistant Director, Analytical Chemistry

Senior Operations Manager, Vivarium

Manager, Gross Pathology and

Developmental Toxicology Laboratory

Clinical Veterinarian

Operations Manager, Developmental,

Reproductive, and Neurotoxicology

Group Manager, Formulations Laboratory

Vice President, Chemistry Services

Director, Operations

Operations Manager, Reporting &

Technical Support Services

Study Period: August 21-24, 2011

2. Test Substance: Linuron

Description: Off white powder

Source: MRI Global (Kansas City, MO)

Lot #: 1012A3876 (expires May 3, 2013)

Purity: 99.4%

Stability: Stable in corn oil for up to 15 days at room temperature and refrigerated conditions.

CAS #: 330-55-2

Structure:

N

H

Cl

Cl

O

N

O

CH3

CH3

3. Reference Estrogen: 17α-ethynyl estradiol (EE)

Supplier: Sigma Aldrich (St. Louis, MO)

Lot #: 011M1252V (expires August 2, 2012)

Purity: 99.0%

CAS # : 57-63-6

4. Solvent/Vehicle Control: Corn Oil

Supplier: Spectrum Chemical Manufacturing Corporation (New Brunswick, NJ or Gardena, 

CA)

Lot #: ZT1301 and 2AE0415 (expire July 1, 2012 and May 4, 2013, respectively)

Rationale (if other than water): Selected due to the solubility properties of the test substance

Final concentration:

Other solvents:

Not applicable

Ethanol (used for positive control prior to addition to corn oil): 95% purity; expires 

August 2, 2013; Lot # ZT0426; Spectrum Chemical Manufacturing Corp. (New 

Brunswick, NJ)

Page 118 of 124
In Vivo Uterotrophic Assay (2011) / Page 4 of 9 

LINURON/ 035506 OCSPP 890.1600/ OECD 440

5. Test Animals:

Species: Rats (ovariectomized females)

Strain: Sprague-Dawley (Crl:CD[SD])

Age/weight at dose initiation: Approximately 72 days old; 251.1-319.4 g

Source: Charles River Laboratories, Inc. (Raleigh, NC)

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

above cage-board.

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

(geinstein-equivalent content averaged 11.0 and 29.0 ppm total isoflavones).

Water: Reverse osmosis-purified tap water, ad libitum 

Environmental conditions: Temperature:

Humidity: 

Air changes:

Photoperiod:

21.3-21.4 oC

49.2-63.3% 

≥10 fresh air changes/hour

12 hrs light/12 hrs dark

Acclimation period:

≥19 days 

B. METHODS 

1. In-Life Dates: Start: 8/21/2011 End: 8/24/2011 

2. Study Design: Following a 19-day acclimation period, ovariectomized female rats (surgery 

performed by Supplier at PND 49) were administered the test substances from PND 72 to 

PND 74. Rats were euthanized approximately 24 hours after the last dose (PND 75) and 

necropsied for uterine weight measurements. 

3. Animal Assignment: Animals were randomly assigned, stratified by body weight, to the 

test groups noted in Table 1. Statistical analysis indicated that there were no significant 

differences between group mean weights at study initiation. Furthermore, the body weight 

of each animal was within 20% of the overall mean. 

TABLE 1. Study Design a

Test Group Dose (mg/kg/day) # of Females b

Estrogen Agonist Assay

Vehicle Control 0 6

Low (Linuron) 30 6

Mid (Linuron) 60 6

High (Linuron) 120 6

17α-ethynyl estradiol (EE), Reference 

Estrogen 0.003 6 

a Data were obtained from page 24 of the study report.

4. Dose Selection Rationale: The dose levels were selected based on the results from a rangefinding study1 in which female rats were administered the test substance in corn oil via 

gavage at doses of 50, 150, 350, and 700 mg/kg/day for 3 days. Effects observed at 350 and 

700 mg/kg/day included morbidity, clinical findings, and body weight losses. At 150 

mg/kg/day, smaller overall body weight loss and similar clinical findings were observed. At 

 

1 Sawhney Coder, P. (2011). A Dose Range-Finding Oral (Gavage) Toxicity Study of Linuron in Young Adult 

Female Rats. Study No. WIL-851001. WIL Laboratories, LLC, Ashland, OH.

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50 mg/kg/day there was a lower mean body weight gain. In a previous Hershberger assay2

validation study for linuron in male Crl:CD(SD) rats, the highest dose tested was 100 

mg/kg/day. Based on these results, the dose levels selected for this study were ≤ 120 

mg/kg/day. 

5. Dose Preparation: Dose formulations were prepared once prior to initial dosing by mixing 

appropriate amounts of test substance with corn oil. The positive control was initially 

dissolved in 95% aqueous ethanol solution, and then added to corn oil. 

Prior to dose administration, samples of linuron dose formulations from the low and high 

dose formulations were analyzed for achieved concentration and homogeneity. Samples 

were taken from the top, middle, and bottom of the container. Additional resuspension 

homogeneity samples were collected from the top and bottom of the same containers 

following storage for 8 days at room temperature, after mixing for 30 minutes. In a previous 

study,3

 linuron was determined to be stable in corn oil for up to 15 days at concentrations of 

1 and 200 mg/mL at room temperature and refrigerated conditions. 

Results of Dose Analysis 

Homogeneity (%RSD): 0.62-0.85% (top, middle, and bottom); 

0.26-1.8% (top and bottom, resuspension samples) 

Stability: Stable in corn oil for up to 15 days at room temperature and refrigerated 

conditions (WIL Laboratories, LLC, 2011). 

Concentration (% of nominal): 103-105%

The analytical data indicated that the mixing procedure was adequate and that the variation 

between nominal and actual dosage to the animals was acceptable. Stability data should 

have been included for review. 

6. Dosage Administration: Animals were administered the test formulations or vehicle 

control daily via gavage for three consecutive days in a dose volume of 5 mL/kg body 

weight. The positive control, EE, was administered daily via s.c. injection (divided between 

3 injection sites) for three consecutive days. Dose volumes were adjusted daily based on the 

concurrent body weight measurement. 

7. Statistics: Statistical analyses were conducted using SAS version 9.2, or higher, software. 

Body weights, body weight gain, and uterine (blotted and wet) weights were analyzed by 

 

2 Tinwell, H.; Friry-Santini, C.; Rouquie, D.; Belluco, S.; Elies, L.; Pallen, C.; Bars, R. Evaluation of the 

Antiandrogenic Effects of Flutamide, DDE, and Linuron in the Weanling Rat Assay Using Organ Weight, 

Histopathological, and Proteomic Approaches. Toxicological Sciences, 2007, 100(1), 54-65. 3 Bodle, E.S. (2011). Analytical Validation and Stability Study of Linuron in Aqueous Formulations. Study No. 

WIL-851005. WIL Laboratories, LLC, Ashland, OH.

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LINURON/ 035506 OCSPP 890.1600/ OECD 440

Levene’s test for homogeneity of variance. If Levene’s test was significant, data were 

transformed using log or square root transformations. If data variance remained 

heterogeneous, data were analyzed using nonparametric test. For uterine (blotted and wet) 

weights, 1-sided analysis of covariance (ANCOVA) was performed with terminal body 

weight as the covariate or nonparametric 1-sided Wilcoxon rank sum test as appropriate. 

Data were analyzed further using either the Dunnett’s test or nonparametric Kruskal-Wallis 

and Dunn’s test, as appropriate. Significance was denoted at p≤0.05. The statistical 

analyses were considered adequate.

C. METHODS

1. Clinical Examinations: Cage-side checks for mortality and moribundity were conducted 

twice daily. Animals were observed (including hand-held physical examination) for signs 

of toxicity prior to treatment each day and were also examined approximately 3 hours after 

dosing. The injection sites for positive control, EE animals were examined daily from the 

initiation of dosing for erythema, swelling, and other dermal findings. Erythema and 

swelling were evaluated in accordance with the Draize 4-step grading system of very slight, 

slight, moderate, and severe.

2. Body Weight: Animals were weighed at randomization, daily throughout the dosing period 

and at termination.

3. Food Consumption (Optional): Food consumption was not measured.

4. Necropsy and Measurement of Uterine Weight: On PND 75 (approximately 24 hours 

after final administration of the test substance), all surviving animals were euthanized by 

carbon dioxide inhalation, and subjected to a gross necropsy. Dissection of the uterus was 

performed according to the U.S. EPA Guideline. Briefly, the uterus was dissected and 

trimmed, being careful to retain the luminal fluid. The “wet” uterus (i.e., containing the 

luminal fluid) was weighed. Subsequently, the uterine horns were cut longitudinally and 

blotted with moist filter paper to remove the luminal fluid. The blotted uterus was weighed. 

After uteri were weighed, the uteri and vagina were fixed in 10% neutral-buffered formalin 

for potential future examination. 

5. Microscopic Examination (Optional): Microscopic examinations were not conducted. 

II. RESULTS

A. OBSERVATIONS

1. Mortality: All animals survived until scheduled termination. 

2. Clinical Signs of Toxicity: Increased incidence of red material was observed around the 

nose in the high-dose group (3 incidents and 3 animals) but not in the control group. This 

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finding was still evident at the 3-hour post dose observation in only one animal. Hair loss 

was also observed, but not in a dose-dependent manner. For the positive control, no 

remarkable injection site observations were noted. 

B. BODY WEIGHT AND WEIGHT GAIN: Body weight and body weight gain data are 

presented in Table 2. The body weights were decreased in all linuron treated groups with 

significantly decreased body weights at 120 mg/kg/day (↓10-12%, days 3-4). Overall body 

weight gains were decreased (p<0.05) for the 30, 60 and 120 mg/kg/day groups by 66, 117, 

and 192%, respectively. 

In the positive control group (EE), body weights on Day 4 were decreased (p<0.05) by 8%, 

and overall body weight gains decreased (p<0.05) for Days 1-4 (↓123%).

TABLE 2. Group Body Weights and Cumulative Body Weight Gains (g) in the Estrogen Agonist Assay a

Study Day

#

Dose Group (mg/kg/day)

Vehicle Control Linuron

(30)

Linuron

(60)

Linuron

(120)

Estrogen, EE

(0.003)

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD

1 6 284.0 16.7 6 280.0 21.4 6 281.4 18.2 6 284.7 22.7 6 283.5 14.6

2 6 290.0 15.1 6 279.9 18.3 6 278.5 15.0 6 276.9 23.7 6 289.0 17.3

3 6 297.7 17.4 6 284.1 17.2 6 276.0 14.1 6 267.3

(↓10) 22.7 6 283.1 16.9

4 6 302.6 18.9 6 286.2 18.7 6 278.3 13.3 6 267.8*

(↓12) 26.9 6 279.3*(↓8) 13.8

BWG

Days 1-4

6 18.5 7.57 6 6.3*

(↓66)

3.66 6 −3.1*

(↓117)

7.37 6 −17.0*

(↓192)

7.49 6 −4.2*

(↓123)

3.96

a Data were obtained from Tables S6, S7, S8, and S9 on pages 49-54 of the study report. 

N No. of animals in the group

SD Standard Deviation

* Significantly different from vehicle control at p < 0.05.

C. FOOD CONSUMPTION (Optional): Food consumption was not measured. 

D. PATHOLOGY

1. Uterine and Liver Weights: Uterine weight data are presented in Table 3. Absolute and 

relative to body weight uterine and liver weights in the linuron treated groups were 

comparable to the controls. 

Absolute wet and blotted uterus weights for the positive EE group were increased (p<0.05) 

by 797% and 300%, respectively. These wet and blotted uterine weights increased as

expected.

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TABLE 3. Uterine (mg) and Liver Weights (g) from Estrogen Agonist Assay in SD Rats a

Parameter

Dose Group (mg/kg/day)

Vehicle Control Linuron

(30) 

Linuron 

(60) 

Linuron

(120) 

Estrogen, EE 

(0.003) 

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD

Terminal 

BW (g) 6 302.6 18.9 6 286.2 18.7 6 278.3 13.3 6 267.8*(↓12) 26.9 6 279.3*(↓8) 13.8

Wet, 

absolute 

(mg)

6 70.1 8.82 6 69.5 10.3 6 67.6 10.9 6 71.0 8.51 6 629.1* 

(↑797) 184.7

Wet, 

relative 

(%) b

6 0.023 0.004 6 0.024 0.003 6 0.024 0.004 6 0.027 0.005 6 0.226 0.066

Blotted, 

absolute 

(mg)

6 60.5 7.72 6 61.5 8.69 6 59.7 9.05 6 62.1 8.20 6 

242.0

* 

(↑300)

27.1

Blotted, 

relative 

(%) b

6 0.020 0.003 6 0.021 0.002 6 0.021 0.004 6 0.023 0.005 6 0.087 0.011

Liver (g) 6 13.8 1.36 6 12.7 0.85 6 12.4 1.05 6 12.9 1.91 6 12.0 0.61

a Data were obtained from Tables S12-S13 on pages 56-58 of the study report. Percent difference from controls (calculated 

by reviewer) is presented in parentheses.

b Relative wet and blotted uterine weights were calculated by the reviewer from the individual data (Appendix L, Table A7, 

pages 289-298). 

BW Body weight

N No. of animals in the group

SD Standard Deviation 

* Significantly different from vehicle control at p < 0.05. 

2. Microscopic Examination (Optional): Microscopic examinations were not conducted. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Based on the absence of effects on uterine 

weights (wet and blotted), linuron did not demonstrate or mimic biological activities 

consistent with agonism of natural estrogens or show evidence of interaction with the 

estrogen pathway when administered orally to ovariectomized female rats at dosage levels 

of 30, 60, and 120 mg/kg/day. The high dose in this study, 120 mg/kg/day, approached the 

MTD based on a statistically significant lower mean body weight (11.5%) compared to the 

control group. The positive control substance (EE) elicited the expected increases in uterine 

(wet and blotted) weights. 

B. AGENCY COMMENTS: All animals survived until scheduled termination. Red material 

was observed around the nose at 120 mg/kg/day on one day for 3/6 animals compared to 0/6 

animals in the control group. This finding remained in a single animal (same day) at 3 hours 

post-dose. Body weights were decreased in all linuron treated groups with significantly 

decreased body weights at 120 mg/kg/day (↓10-12%, Days 3-4). Overall body weight gains 

were decreased (p<0.05) for the 30, 60 and 120 mg/kg/day groups by 66, 117, and 192%, 

respectively. Absolute and relative to body weight uterine and liver weights in the linuronPage 123 of 124
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treated groups were comparable to the controls. Absolute wet and blotted uterus weights for 

the positive EE group were increased (p<0.05) by 797% and 300%, respectively, as expected. 

The high dose tested in this study was adequate based on the observed decreases in body 

weights. No statistically significant changes were seen in uterine weight in this assay. 

Linuron is negative in the uterotrophic assay. 

C. STUDY DEFICIENCIES: None 

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