## 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 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))

# 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 / 2 4 /201 2

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.

## The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is

comprised of eleven screening assays intended to identify a chemical’s likely endocrine

bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T)

pathways. The robustness of the Tier 1 battery is based on the strengt hs of each individual

assay to identify potential endocrine bioactivity with complementary endpoints within the

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

individual assay should not be considered in isolation but rather should be considered in

the context of other assays in the battery as well as Other Scientifically Relevant

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

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

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

Document).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-ene**  

## 3, 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.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 2 of 13

LINURON / 035506 O CSPP 89 0. 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 IC 50 

## , and coefficient of variation for replicates of each

concentration within runs. Also, t he 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 IC 50 

## averaged –7.27 M, and th e 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 (high est 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 6 8.1% 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

conce ntrations.

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

aromatase inhibition in this assay.

The assay satisfies the EDSP Tier 1 Test Order requirement s for an Aromatase a ssay (OCSPP

890.120 0).

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

Assurance statements were provided.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 3 of 13

LINURON / 035506 O CSPP 89 0. 1200 / OECD None



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test S ubstance : Linuron technical

Description: Powder

Source: Tessenderlo Kerley, Inc.

Batch # (Expiration D ate) : 1012A3876 ( May 3 , 201 3 )

Purity: 9 9.4 % a.i.

Storage C onditions: Room temperature

Stability: Not reported

Solvent : DMSO

Solubility: Visually s oluble at 10

− 3.5 M in 2 of 3 test runs.

Highest C oncentration T ested: 10

− 3 M ; however, precipitation was observed at this dose in Run 1

Stock S olution P reparation : Not reported

Molecular W eight: 249.09 g/mol

CAS #: 330

- 55

- 2

Structure:



## 2. Non

- L abeled S ubstrate : Androstenedione (ASDN)

CAS #: 63

- 05

- 8

Source: Steraloids, Inc. (Cat. # A6030

- 100 )

Batch # (Expiration D ate) : L1712 ( April 2016 )

Purity: 99.8 %



## 3

. Radiolabeled S ubstrate : [ 1 β

- 3 H(N)]

- Androst

- 4

- ene

- 3,17

- dione ; ( [ 3 H]

- ASDN)

Source: Perkin Elmer (Cat. # NET

- 926 )

Batch # (Expiration D ate) : 619344 ( January 10, 2012 )

Radiochemical P urity ( S upplier): > 97 %

Specific A ctivity: 26.3 Ci/mmol

Radiochemical P urity ( In

- L ab

D etermination): Not reported



## 4. Positive C ontrol : 4

- hydroxyandrostenedione (4

- OH ASDN)

CAS # : 566

- 48

- 3

Source: Sigma

- Aldrich (Cat. # F2552 )

Batch # (Expiration D ate) : 081K2133 ( March 2015 )

Purity: 99.6%



## 5. Solvent (Vehicle Control) : DMSO

Source: Sigma

- Aldrich (Cat. # D2650)

Batch # (Expiration D ate): RNBB7886 (expiry date not provided)

Justification for Choice of S olvent: Acceptable vehicle by OCSPP guideline 890.1200

Concentration (% of Total Volume in

A ssays): ≤1% v/v



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

Source: BD Biosciences Gentest TM (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

CH

3

CH

3Aromatase (Human Recombinant) Assay ( 2011 ) / Page 4 of 13

LINURON / 035506 O CSPP 89 0. 1200 / OECD None



## B. METHODS

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

[ 3 

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

approximately 0.1 μM, and the amount of triti ated material a dded 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 O CS PP 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 μ L 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.

T ABLE 1. Assay Components and Conditions

Assay Factor Values

S odium phosphate buffer (pH 7.4) 0.1M

Microsomal p rotein 0.004 mg/mL

NADPH 0.3 mM

[ 3 H]ASDN 100 n M

Propylene g lycol 5%

Temperature 37 ±2 °C

Incubation t ime 15 min



## 2. Suitability Ass es s ments : The protein concentration of the microsomes (Lot # 19701 ) w as

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 n mol/ mg

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

nmol/mg

- protein/min.

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

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

beginning of the assay, and two of each control were run at the end of the assay. A fullAromatase (Human Recombinant) Assay ( 2011 ) / Page 5 of 13

LINURON / 035506 O CSPP 89 0. 1200 / OECD None



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

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

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

The amount of 3 

## H 2 

## O in the aqueous fraction was quantified for each assay tube by LSC, and

aromatase activ ity was reported in units of n mol∙mg

- protein –1 

## ∙min –1



## .

4. Demonstration of P roficiency : 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 guide line 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 P roficiency : The positive control data

(new/historical data for laboratory) w ere not reported in regards to the following criteria:

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

protein/min.

• Mean background control activi ty 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 IC 50 

## me t the criteria as listed in section (i) of OCSPP 890.1200.

TABLE 2 . Performance Criteria for the Positive Control a

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 IC 50 (M)

– 7.3

– 7.0

- 7.3

- 7.2

a Data were obtained from page 27 of the study report.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 6 of 13

LINURON / 035506 O CSPP 89 0. 1200 / OECD None



## 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 A ctivity with T est C hemical : 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 t he 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.

T ABLE 4. Test Chemical Study Design for Each Test Run

Sample Type Repe titions (Tubes) Description Reference or Chemical (M)

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

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

4

- OH ASDN Conc 1 2 All test components plus 4

- OH ASDN 1×10

– 5

4

- OH ASDN Conc 2 2 All test components plus 4

- OH ASDN 1×10

– 6

4

- OH ASDN Conc 3 2 All test components plus 4

- OH ASDN 1×10

– 6.5

4

- OH ASDN Conc 4 2 All test components plus 4

- OH ASDN 1×10

– 7

4

- OH ASDN Conc 5 2 All test components plus 4

- OH ASDN 1×10

– 7.5

4

- OH ASDN Conc 6 2 All test components plus 4

- OH ASDN 1×10

– 8

4

- OH ASDN Conc 7 2 All test components plus 4

- OH ASDN 1×10

– 9

4

- OH ASDN Conc 8 2 All test components plus 4

- OH ASDN 1×10

– 10

L inuron Conc 1 b 3 All test components plus linuron 1×10

– 3

Linuron Conc 2 b 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, [ 3 H]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. D ATA ANALYSIS

1. Raw D ata : Raw data were converted to aromatase activity ( n mol/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).Aromatase (Human Recombinant) Assay ( 2011 ) / Page 7 of 13

LINURON / 035506 O CSPP 89 0. 1200 / OECD None



### T ABLE 5. Raw and Calculated Data

Raw/Calculated Data Included (X)

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

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

Total DPM for each aqueous portion (after extraction) X

The total DPM present in the assay tube at initiation X

The percentage of substrate converted to product X

Total DPM after extraction corrected for background X

Aromatase activity expressed in n mol/mg protein/ min X

Average aromatase activity in the full activity control tubes X

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

DPM Disintegrations per minute



## 2. S tatistical M ethods : 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 w as 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 ass ay 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

equi vocal.

b If the test compound was not soluble above 10 –6 M and the inhibition curve does not cross 50%, the chemical is typically

determined to be untestable in the aromatase assay.



## II

. RESULTS

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

0.494 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 ), a romatase

activity averaged 0.585 ± 0.070 nmol

∙ mg -protein

− 1



## ∙ min

− 1 

## at the lowest tested concentrationAromatase (Human Recombinant) Assay ( 2011 ) / Page 8 of 13

LINURON / 035506 O CSPP 89 0. 1200 / OECD None



## (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 o verall 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, H ill Slope, log

IC 50 

## , and co efficient 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 VarianceAromatase (Human Recombinant) Assay ( 2011 ) / Page 9 of 13

LINURON / 035506 O CSPP 89 0. 1200 / OECD None



## 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 (highestAromatase (Human Recombinant) Assay ( 2011 ) / Page 10 of 13

LINURON / 035506 O CSPP 89 0. 1200 / OECD None



## non- precipitating dose ). P recipitation 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

c oncentration 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 acr o ss al l runs is shown in Figure 3.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 11 of 13

LINURON / 035506 O CSPP 89 0. 1200 / OECD None



## FIGURE 2

. Inhibition Response Curves for Linuron (Runs 1 -3).Aromatase (Human Recombinant) Assay ( 2011 ) / Page 12 of 13

LINURON / 035506 O CSPP 89 0. 1200 / OECD None



## F IGURE 3. Mean Inhibition Response Curve for linuron

.

The data for linuron were not suit able for modeling. T he 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 ar omatase activity is presented in Table 8.

For 4

- OH ASDN , t he estimated log IC 50 

## averaged –7.27 M, and the Hill S lope was –1.00.

Confidence in the se 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 IC 50 (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 suit able 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.Aromatase (Human Recombinant) Assay ( 2011 ) / Page 13 of 13

LINURON / 035506 O CSPP 89 0. 1200 / OECD None



## 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, H ill Slope, log IC 50 

## , 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 IC 50 

## averaged –7.27 M, and the Hill S lope 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 inhibitio n 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.

# 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 /201 2

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

.

## 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 in dividual

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 shoul d 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).

### 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.Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 2 of 17

LINURON/ 035506 O CSPP 89 0.1250 / OECD None



## Precipitat ion 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 c ompetitive 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 IC 50 

## was not calculated for linuron as 50% binding displacement was not

achieved in any run. The mean l og IC 50 

## 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 I nteractive in the Estrogen

Receptor Binding Assay

.

The assay satisf ies the EDSP Tier 1 Test Order requirement s for an Estrogen Receptor Binding

As say (OCSPP 890.1250).

COMPLIANCE : Signed and dated GLP and Quality Assurance statements were provided.Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 3 of 17

LINURON/ 035506 O CSPP 89 0.1250 / 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 M c Colley, M.S.; Scientist

Colleen Toole, Ph.D.; Director of Project ma na gement

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 , 201 3 )

Purity: 9 9. 4 % a.i.

Solubility : N ot 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

CH

3

CH

3



## 3

. Non

- labeled ligand : 17 β

- estradiol

Supplier : Sigma

- Aldrich

, St. Louis, MO

Catalog # : E8875

Batch #: 110M0138V

Purity: 100 %

CAS #: 50

- 28

- 2



## 4

. Radioactive ligand : [ 3 

## H]

- 17 β

- estradiol

Supplier : Perkin

- Elmer, Boston, MA

Catalog #: NET517

Batch#: 650702

Radiochemical p urity: 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 #: 6 8

- 22

- 4



## 6

. Negative control : OctyltriethoxysilaneEstrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 4 of 17

LINURON/ 035506 O CSPP 89 0.1250 / 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 R at U terine Cytosol ( RUC) : Frozen Sprague Dawley uteri were purchased

from Harlan Laboratories. F emale 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) + P MSF

(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 s upernatant 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) B inding E xperiment : A saturation binding e xperiment

measuring total and non-specific binding of [ 3 

## H] -17 β -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 Experiment a



### 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 nM a



### 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 [ 3 

## H] -17 β

- 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,Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 5 of 17

LINURON/ 035506 O CSPP 89 0.1250 / OECD None



## and 3 nM. Solutions of non- labeled 17 β

- 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: typicall y 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 depicte d

in Table 2.

TABLE 2. Saturation Binding Experiment Run a



### Total binding b 

### Non

- specific

binding c 

### Radioligand alone d 

### 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

- min ute intervals). Subsequently, the contents of each tube were

washed three times as f ollows: 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 extrac t the [ 3 

## H] -17 β

- 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 B inding E xperiment : A summary of the experimental conditions for the

Competitive Bind ing Experiment is presented in Table 3

.Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 6 of 17

LINURON/ 035506 O CSPP 89 0.1250 / OECD None



### TABLE 3 . Summary of Conditions for Competitive Binding Experiment a



### Source of receptor Rat U terine C ytosol

Concentration of radioligand 1 nM

Concentration of receptor Sufficient to bind 17.4

- 23.6 % of

radioligand b



### 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 ( P I )

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 C alculated by reviewer from data on pages 58, 61, and 65 of the study report

.

c Th e 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] -17 β

- estradi ol was adjusted for

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

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

by testing serial amounts of protein per tube, using 1.0 nM rad iolabeled 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 ach ieve the concentrations shown in Table 4 . E ach assay co nsisted of three

runs, and each run contained three replicates at each co ncentration, six replicates measuring

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

resulting in a total of 306 samples.Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 7 of 17

LINURON/ 035506 O CSPP 89 0.1250 / OECD None



### TABLE 4. Molar (M) concentrations in Co mpetitive 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 page s 26 , 2 8, 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

- labele d 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 cont ents of each tube were washed three times as

follows: TEDG+ PI buffer was added, vortexed, centrifuged for 10 min utes 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] -17 β

- estradiol

, allowed to sit at room temperature for 15-20

min utes 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. D ATA 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 l og

IC 50 

## as a parame ter to be estimated. For parameters reported from the saturation b inding

experiment ( 

## K d 

## and B

max 

## ) and c ompetitive binding e xperiment ( l og IC 50 

## and RBA), m ean 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 regressi on: 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.Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 8 of 17

LINURON/ 035506 O CSPP 89 0.1250 / OECD None



## 1. Definitions

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

three runs. Each run wa s 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 follo ws:

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”Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 9 of 17

LINURON/ 035506 O CSPP 89 0.1250 / OECD None



## b. Descriptors for receptor binding :

B

max 

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

concentration of active receptor sites

K d 

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

IC 50 

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

the receptor

Relative Binding Affinity ( RBA % ): ( IC 50 

## of 17 β

- estradiol ÷ IC 50 

## of test substance) × 100

Log RBA: Log 10 

## ( IC 50 

## of 17 β

- estradiol ÷ IC 50 

## of test substance)

II. RESULTS

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

specific, and total binding curves for [ 3 

## H] -17 β

- 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] -17 β

- estradiol to the est rogen receptor.

Saturation binding e xperiment parameters are presented in Table 5. The K d 

## for [ 3 

## H]

- 17β

estradiol was 0.303 nM (± 0.090), and the estimated B

max 

## was 33.59 fmol/100 μg protein

(± 0.70) for the prepared rat uterine cytosol. The K d 

## 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]

- 17 β

- estradiol with Estrogen Receptor from Rat Uterine

Cytosol a



### Parameter Run 1 Run 2 Run 3 Mean ± SE b



### R 2 

### (unweighted) 0.992 0.993 0.955 0.955

- 0.992

B

max 

### (nM) 0.029 0.029 0.027 0.029 ± 0.001

B

max 

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

K d 

### (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 R 2 

### is reported and the mean ± SEM is reported for the other parameters.

R 2 

### Goodness of fit for curve calculated for specific bindingEstrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 10 of 17

LINURON/ 035506 O CSPP 89 0.1250 / OECD None



## FIGURE 1. Binding of [ 3 

## H] -17 β

- estradiol to the Estrogen Receptor during the Saturation

Binding Experiment

.Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 11 of 17

LINURON/ 035506 O CSPP 89 0.1250 / OECD None



## FIGURE 2. Scatchard Plot s of the Binding of [ 3 

## H] -17 β

- estradiol to the Estrogen Receptor.

B. COMPETITIVE BINDING EXPERIMENT : The results from the three c ompetitiveEstrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 12 of 17

LINURON/ 035506 O CSPP 89 0.1250 / OECD None



## binding e xperiment s 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 IC 50 

## 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 l og IC 50 

## 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 no n- interactive (0) in this assay

(Table 7).

TABLE 6

. Competitive Binding Assay of Linuron with Estrogen Receptor from Rat Uterine Cytosol a



### Parameter Run 1 b 

### Run 2 b 

### Run 3 b 

### Mean

R 2 

### (unweighted) 1 7 β

- estradiol NR NR NR NR

19

- norethindrone NR NR NR NR

Linuron NR NR NR NR

Log IC 50 

### ( M) 17 β

- 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

IC 50 

### (M) c 

### 17 β

- estradiol 1 .00 × 10

− 9 

### 1 .00 × 10

− 9 

### 1.58 × 10

− 9 

### 1

. 2 0 × 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. 0 32 0.0 32 0. 0 63 0.0 4 2 ±0.010

Linuron NA NA NA NA

a Data were obtained from page s 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 IC 50 

### 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.

R 2 

### Goodness of fit

RBA R elative binding affinity

NR N ot reported

NA N ot 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 No t 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 <0.5Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 13 of 17

LINURON/ 035506 O CSPP 89 0.1250 / OECD None



## F IGURE 3. Percentage [ 3 

## H]

- E2 Bound to the Estrogen Receptor in the Presence of

Linuron , Unlabeled E 2 , 19 -Norethindrone or Octyltriethoxysilane, Run 1Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 14 of 17

LINURON/ 035506 O CSPP 89 0.1250 / OECD None



## FIGURE 4. Percentage [ 3 

## H]

- E2 Bound to the Estrogen Receptor in the Presence of

Linuron , Unlabeled E 2 , 19 -Norethindrone or Octyltriethoxysilane, Run 2

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

LINURON/ 035506 O CSPP 89 0.1250 / OECD None



## F IGURE 5. Percentage [ 3 

## H]

- E2 Bound to the Estrogen Receptor in the Presence of

Linuron , Unlabeled E 2 , 19 -N orethindrone or Octyltriethoxysilane, Run 3

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

LINURON/ 035506 O CSPP 89 0.1250 / OECD None



## C. P ERFORMANCE CRITERIA : To ensure that the competitive binding assay function ed

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

T ABLE 8. Criterion a 

### Tolerance

Limit(s) Value Yes No

17 β

- estradiol fitted curve parameters

Log

e 

### residual SD ≤2.35 0. 84 to 1. 5 4 X

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

Bottom (% binding)

- 4 to 1

- 5 to 0 X

Hill Slope (log 10 

### (M)

- 1 

### )

- 1.1 to

- 0.7

- 1. 1 to

- 0.8 X

Weak Positive control ( 19

- norethindrone ) b 

### fitted curve parameters

Log

e 

### residual SD ` NA 0. 76 to 1.08

Top (% binding) NA 93 to 10 0

Bottom (% binding) NA

- 8 to

- 1

Hill Slope (log 10 

### (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]

- 17 β

- estradiol from the ER on average across all

concentrations c 

### ≤25% 15.1

- 54.2 % X

a Data were obtained from page s 2 7, 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 (oct yltriethoxysilane)

displacements of [ 3 

## H] -17 β

- 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, t hese 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.Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 17 of 17

LINURON/ 035506 O CSPP 89 0.1250 / 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 , t he K d 

## for [ 3 

## H]

- 17β

- estradiol

was 0.303 nM, and the estimated B

max 

## was 33.59 fmol/100 μg protein. The K d 

## for each run

was within the expected range of 0.03 to 1.5 nM.

For the c ompetitive binding experiment, t he estimated mean IC 50 

## was not calculated for

linuron as it did not displace the radiolabeled ligand from the ER. The mean IC 50 

## and RBA

for the weak positive control w ere 2.95 × 10

− 6 

## M and 0.042%, respectively.

The performance criteria were generally met for the assay . T he 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 H ill slope s 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 n egative 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]-17 β

- 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.

### 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'

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

EPA MRID Number 48671701

Page 2 of 54 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.

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

EPA MRID Number 48671701

Page 3 of 54 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-mea sured 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 25 o 

### 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

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

EPA MRID Number 48671701

Page 4 of 54 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 23.4 q C 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-throughData Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 5 of 54Table 1: Summary of Reproductive and HPG Effects1,2in the Fish Short-Term Reproduction Assay (FSTRA) with Linuron.Treatment(mg a.i./L)[meanmeasured]FecundityFert.SuccessTubercle ScoreGSIGonadal Histo.Plasma VTGPlasma TPlasma E2MFMFMFMFMFMF0.099NoNoNoNANoNoNoNoNoNoNANANANA0.92YesYesNoNANoNoNoNoNoNoNANANANA9.1Yes3Yes3YesNAYesNoYesYesYesNoNANANANAAbbreviations:Conc.Concentration.Diff.Difference.E217β-estradiol.FFemale.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.MMale.NANot applicable.TTestosterone.VTGVitellogenin.1A “yes” indicates a significant difference based on comparison to the negative (clean water) control, unless otherwise specified.2The criteria for significance are described in the Reviewer’s Analysis and Statistical Verification sections of the DER. Conclusions regardinghistopathology may be heavily weighted by the expert opinion of a board-certified pathologist.3No eggs were produced at the high treatment level. Therefore, fecundity was 0; there were no fertility data to analyze; and the high treatment level wasexcluded from data analysis for the endpoints of fecundity and fertility.

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

EPA MRID Number 48671701

Page 6 of 54 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 23.4 q C; 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.

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

EPA MRID Number 48671701

Page 7 of 54 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 cabinetData Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 8 of 54B. Test organism:Table 2: General Information About the Test Species and Acclimation.ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies common name:Fathead Minnow EPA recommends fathead minnow (Pimephales promelas).Species scientific name: Pimephales promelasSpecies strain (if stated):Not reportedWere fish obtained from asingle laboratory stock?Yes EPA recommends that fish be from a single

laboratory stock.Were acclimation conditionssame as definitive test?Yes EPA recommends that fish be acclimated under water quality and illumination conditions that are similar to the definitive

test.Acclimation period: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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 9 of 54ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on health:Less than 1% mortalities were reportedduring the7 days prior to pre-exposureperiod.Fish did not receive any treatment fordiseasein the two weeks prior tospawning (pre-exposure period).Behavioral abnormalities or clinical signswere not reported. EPA recommends that mortality during the 7

days p rior 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 main tain body

condition.Source of food:Not reportedSMV Lot No. 082110Frequency of feeding:2 times/dayDetails on feeding:NoneData Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 10 of 54Table 3: Fish Selection and Pre-Exposure Performance.ParameterValue(s)Details or RemarksGuideline RecommendationsAge at test initiation: Ca. 4.25 months17 weeks EPA recommends reproductively mature (sexually dimorphic) fish, 4.5 - 6 months

old.Mean weight of males at testinitiation (if determined):3.3 gBased on a representative sample of 20malesfrom the test populationused tostock 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 (ifdetermined):2.9 to 3.8Individual weights within ±20% of theestimated meanMean weight of females at testinitiation (if determined):1.8 gBased on a representative sample of 20femalesfrom the test populationused tostock aquaria for pre-exposure phaseRange of individual weights(females) at test initiation (ifdetermined):1.4 to 2.2Individual weights within ±22% of theestimated meanMean length of males at testinitiation (if determined):Not reportedData Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 11 of 54ParameterValue(s)Details or RemarksGuideline RecommendationsMean length of females at testinitiation (if determined):Not reportedDuration of pre-exposure phase:15 days EPA recommends a minimum of 14 days.Were pre-exposure conditionsidentical to the definitive test?Yes EPA recommends that pre-exposure conditions, including temperature, photoperiod, feeding, etc., be identical to definitive test conditions.Number of pre-exposure tanks:33Extra replicates were established toaccount for a potential lack of spawningin some chambers and/or mortalityduring this phase. EPA recommends that additional tanks set up at the beginning of pre -exposure will ensure that sufficient replicates with the correct sex ratio are available for the

definitive test.Number of males per tank:2Number of females per tank:4Data Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 12 of 54ParameterValue(s)Details or RemarksGuideline RecommendationsPre-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-dayperiod precedingexposure initiation EPA recommends that spawning occur at

least twic e in the 7 days prior to the

definitive test.Details on pre-exposure:NoneData Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 13 of 54C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:Flow-throughEPA recommends the use of a flowthrough system. As noted in the Corrections and Clarifications document 1 , the use of a static renewal system is not recommended for this assay.Type of flow-through dilutionsystem:Intermittent flow proportionaldiluters Intermittent flow proportional diluters or continuous flow serial diluters are

recommended. 2Flow-through rate: Ca. 46 mL/minute or 2.8L/hr and 6.6 volumereplacements/dayCalculated by the reviewer based on 0.5L/cycle,132 cycles/24 hrs and 60minutes/hr Recommended flow-through rate is 45 mL/min (2.7 L/hr), or at least 6 total volume exchanges per day.1U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the Endocrine DisruptorScreening Program Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention (OCSPP), Washington, D.C.(http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf).2Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 14 of 54ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on toxicant mixing forflow-through systems:A mixing chamber was used.Flow-splitting chambers were usedbetween the diluter cells and the fourreplicate test vessels to promote mixing ofthe test substance solution and diluentwaterand to equally split the test solutionbetween the test vessels.Treatment recoveries measured prior totest initiationrangedfrom 110 to 130% ofnominal concentrations.Flow splitting accuracy not reportedRecommended toxicant mixing for flowthrough systems: 1) Mixing chamber is recommended but not required; 2) Aeration is not recommended for mixing; 3) A demonstration that the test solution is completely mixed before introduced into the test system is recommended; 4) The recommended flow splitting accuracy

is within 10%.Aeration?None reported EPA recommends aeration if dissolved

oxygen reaches <4.9 mg/L (<60%

saturation).Data Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 15 of 54ParameterValue(s)Details or RemarksGuideline RecommendationsSource of dilution water:Well water EPA recommends natural or reconstituted water; it is recommended that natural water be sterilized with UV and tested for pesticides, heavy metals, and other possible contaminants. OECD accepts any water in which the test species show control survival at least as good as indicated in the test guideline.Was dilution water analyzedfor pesticides, heavy metals,and other contaminants?YesTest vessel type/materials:Glass aquaria withsilicone adhesiveEPA 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 20cm 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).Data Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 16 of 54ParameterValue(s)Details or RemarksGuideline RecommendationsFill volume:10 L EPA recommends 10 L solution per tank.Spawning substrate material:appropriately aged PVCpipe EPA recommends that each tank contain

three semi -circular spawn ing substrates, e.g., aged PVC pipe, 10 - 20 cm in length, split lengthwise.Spawning substrate size:4 inch diameter cut inhalf (arches) and into7cm sectionsAdditional details on exposuresystem:NoneTable 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMean1MeasurementIntervalGuideline RecommendationsTemperature (°C)23 25 24 Continuously EPA recommends temperature 25±1 o C;

inter -replicate and inter-treatment differentials should not exceed 1 o C.pH7.07.67.3Daily EPA recommends pH 6.5 to 9.0.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 17 of 54ParameterMinimumMaximumMean1MeasurementIntervalGuideline RecommendationsDissolved 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 asCaCO3)26 30 28Weekly EPA recommends total alkalinity >20

mg/L as CaCO 3.Hardness (mg/L as CaCO3)648072WeeklyTotal 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 reportedNot reportedNot reportedNot reported EPA recommends that unionized ammonia in the dilution water be <1

P g/L.Residual chlorine (Pg/L)Not reportedNot reportedNot reportedNot reported EPA recommends that residual chlorine in dilution water be <10 P g/L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 18 of 54ParameterMinimumMaximumMean1MeasurementIntervalGuideline RecommendationsConductivity (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:NANot applicable.1Average of minimum and maximumData Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 19 of 54D. Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details or RemarksGuideline RecommendationsWas a range-finder conducted?Yes EPA recommends conducting a range-finder

if 96 -hour LC

50 data for the fathead minnow

are unavailable.If yes, what was the method fordetermining the highest testconcentration in the rangefinder?96-hr LC50rangefinding studiesRange-finding study #1:xNominal test concentrations: 0.95,1.9, 3.8, 7.5 and 15 mg/Lxn = 2 males & 4 females in onetest vessel per test concentrationxResults: no mortality; 1 male in the15 mg/L group was observed tohave complete loss of equilibriumxLC50> 15 mg/LRange-finding study #2:xNominal test concentrations: 2.5,5.0, 10, 20 and 40 mg/Lxn = 2 males & 4 females in onetest 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 20 of 54ParameterValue(s)Details or RemarksGuideline RecommendationsxResults: 100% mortality at 40mg/L; 100% complete loss ofequilibrium at 20 mg/LxLC50(95% C.I.) = 28 (21 to 30)mg/LSpecies: Pimephales promelasLife stage:Not reportedApproximately the same age and sizeasthe 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 21 of 54ParameterValue(s)Details or RemarksGuideline 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 LC

50, may be selected as the highest exposure concentration in the 21-

day test.Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:21 days EPA recommends that the duration of the definitive test be 21 days.Method for selecting the highesttest concentration in thedefinitive test:The acute toxicity 96-hr LC50value wasestimated to be28mg/L. EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of toxicity

(e.g., 1/3 the 96-hour LC

50 ), whichever concentration is lowest.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 22 of 54ParameterValue(s)Details or RemarksGuideline RecommendationsReference study citation (ifapplicable):NASeparation of testconcentrations:0.1Step down factor of 10 from highest testconcentration. EPA suggests that a concentration separation of between 0.33 (or three- fold)

and 0.1 (or ten -fold) is scientifically

acceptable 1 .Number of test concentrations:3 EPA recommends a minimum of 3 concentrations and a control, plus solvent control if appropriate.Are nominal concentrationsadjusted for purity?YesIndicate the type of valuespresented for measuredconcentrations:Mean-measuredLimit of quantification (LOQ):<0.0029 to 0.0032mg a.i./L EPA recommends that for chemical test concentrations below the LOQ, analyses be conducted on the stock solutions.Level of detection (LOD):Not reportedData Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 23 of 54ParameterValue(s)Details or RemarksGuideline RecommendationsFrequency of measurement:0, 7, 15, and 21 daysin 2 reps for eachtreatment at each timepoint It is recommended that test item concentration be measured prior to the addition of fish in all tanks and at lea st weekly thereafter in two replicates per

treatment level.Was the randomized completeblock design used?Yes EPA recommends that all fish be randomly assigned to tanks during pre -exposure. Tanks are then ranked according to 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 solventcontrol (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 testitem treatment level:4 EPA recommends 4 replicates.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 24 of 54ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of male fish perreplicate at test initiation:2 EPA recommends 2 males per replicate.Number of female fish perreplicate at test initiation:4 EPA recommends 4 females per replicate.Was a solvent used?NoSolvent type (if applicable):NAMaximum solvent concentration(if applicable):NA EPA recommends that the solvent not

exceed 0.02 ml/L3 . OECD recommends that solvent have no effect on survival nor produce any other adverse effects and that concentration not be greater than 0.1 ml/L4 .Was a positive control used?NoPositive control (if applicable):NA3Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review. AquaticToxicology, 76, pp.69–92.4OECD (2000). Guidance Document on Aquatic Toxicity Testing ofDifficult Substances and Mixtures. Environmental Health and Safety Publications. Series onTesting and Assessment. No. 23. Paris, France.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 25 of 54ParameterValue(s)Details or RemarksGuideline RecommendationsPositive control concentration(s)(if applicable):NAPhotoperiod:16 hrs light:8 hrs dark EPA recommends photoperiod 16:8

(light:dark).Light intensity at water’ssurface:630-1000 luxFluorescent bulbs EPA recommends light intensity 540 – 1080 lux (at water’s surface).Additional details:NoneAll exposure solutions were reported asclear and colorless with no undissolvedmaterial present. A stock solution (15mg/L)was prepared daily and wasreported as clear and colorless with asmall amount ofundissolved materialsettled at the bottom of the mixingvessel.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 26 of 54Table 8: Summary of Treatment Concentrations in the Fish Short-Term Reproduction Assay with Linuron.Treatment IDNominalConcentration(mg a.i./L)Mean MeasuredConcentration(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsNegative Control0<LOQNA EPA recommends that test item concentrations be maintained at a coefficient

of variat ion (CV)≤ 20%.Treatment 10.0900.09917.3Treatment 20.900.925.0Treatment 39.09.11.2Abbreviations:CVCoefficient of variation;NANot ApplicableLOQ = 0.0029 to 0.0032 mg a.i./L1Study 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 27 of 54E. ObservationsBiological Endpoints:Survival, fecundity, fertilization success, and clinical signs were observed daily. At test termination (Day 21), secondary sexcharacterization (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 clinic al signs occur at least daily. At test termination (Day 21), additional observations include body weight and length, nuptial tubercle score, gonadal staging and histopathology, plasma vitellogenin,

and plasma sex steroids (testosterone and 17β -estradiol, if measured). Gonado-somatic 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 beha vior.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 28 of 54II. RESULTS AND DISCUSSIONA.ResultsMean 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]MalesFemalesn1# Surviving% Survivaln1# Surviving% SurvivalNegative Control (<LOQ)8810016161000.0998810016161000.928810016161009.18675161594Abbreviations:NANot applicable.LOQ=0.0032 mg a.i./L1Total number of fish at test initiationData Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 29 of 54Median 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 1.10g 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 9.1mg 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 WeightLengthMalesFemalesMalesFemalesn1Median(g)±SDn1Mean(g)±SDn1Mean(mm)±SDn1Mean(mm)±SDNegative Control(<LOQ)43.41 0.6041.55 0.16454.73.2442.61.10.09943.090.1141.520.07454.11.8442.20.50.9242.670.1841.520.10450.31.5442.70.89.142.530.4141.100.07453.41.7440.00.8Abbreviations:NANot applicable.NDNot determined.SDStandard deviation.LOQ=0.0032 mg a.i./L1Number of replicate tanks

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Page 30 of 54 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] Fecundity 1 

### 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 0 3 

### NA NA 3 

### 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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Page 31 of 54 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

n 1 

### Median

Tubercle Score 2 

### n 1 

### 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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Page 32 of 54 Mean male GSI was 1.6, 1.5, 1.7 and 0.9% and me an 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

n 1 

### Mean GSI 2



### (%) ±SD n 1 

### Mean GSI 2



### (%) ±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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Page 33 of 54 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

n 1 Median Stage 2 n 1 Median Stage 3



### 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 34 of 54In 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 femalefish, there were changes in gonadal staging, decreased yolk synthesis, and increased oocyte atresia of predominantly pre-vitellogenic follicles in the 9.1 mga.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 Observations1SeverityIncreased Proportionof SpermatogoniaPresence of TestisOvaIncreasedTesticularDegenerationDuctMineralizationInterstitial cell(Leydig)hypertrophy/hyperplasianIncidencenIncidencenIncidencenIncidencenIncidenceNegativeControl(<LOQ)088NANA86NANANANA180NANA82NANANANA280NANA80NANANANA380NANA80NANANANA480NANA80NANANANAData Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 35 of 54Treatment(mg a.i./L)[meanmeasured]Diagnostic Observations1SeverityIncreased Proportionof SpermatogoniaPresence of TestisOvaIncreasedTesticularDegenerationDuctMineralizationInterstitial cell(Leydig)hypertrophy/hyperplasianIncidencenIncidencenIncidencenIncidencenIncidence0.099088NANA88NANANANA180NANA80NANANANA280NANA80NANANANA380NANA80NANANANA480NANA80NANANANA0.92088NANA87NANANANA180NANA81NANANANA280NANA80NANANANA380NANA80NANANANA480NANA80NANANANAData Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 36 of 54Treatment(mg a.i./L)[meanmeasured]Diagnostic Observations1SeverityIncreased Proportionof SpermatogoniaPresence of TestisOvaIncreasedTesticularDegenerationDuctMineralizationInterstitial cell(Leydig)hypertrophy/hyperplasianIncidencenIncidencenIncidencenIncidencenIncidence9.1060NANA66NANANANA162NANA60NANANA261NANA60NANANA362NANA60NANANA461NANA60NANANAAbbreviations:NANot applicable.LOQ=0.0032 mg a.i./L1Gonadal 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 37 of 54Table 16: Additional Gonadal HistopathologyObservations in Male Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[mean-measured]Additional Diagnostic Observations1SeverityDecreasedProportion ofSpermatogoniaIncreased Vascularor InterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportionsof Spermatocytesor SpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidenceNegative Control(<LOQ)0NANANANA87NANA881NANANANA81NANA802NANANANA80NANA803NANANANA80NANA804NANANANA80NANA800.0990NANANANA88NANA881NANANANA80NANA802NANANANA80NANA803NANANANA80NANA804NANANANA80NANA80Data Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 38 of 54Treatment(mg a.i./L)[mean-measured]Additional Diagnostic Observations1SeverityDecreasedProportion ofSpermatogoniaIncreased Vascularor InterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportionsof Spermatocytesor SpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidence0.920NANANANA88NANA881NANANANA80NANA802NANANANA80NANA803NANANANA80NANA804NANANANA80NANA809.10NANANANA65NANA651NANANANA61NANA612NANANANA60NANA603NANANANA60NANA604NANANANA60NANA60Abbreviations:NANot applicable.LOQ=0.0032 mg a.i./L1Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E ofthe test guideline for reference.

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Page 39 of 54 Table 17: Gonadal Histopathology in Female Fathead Minnow ( Pimephales promelas)

.

Treatment

(mg a.i./L)

[meanmeasured ] Additional Diagnostic Observations 1



### Severity Increased Oocyte

Atresia Perifollicular Cell

Hyperplasia/

Hypertrophy Decreased Yolk

Formation Aggregates of

macrophages,

multifocal

n Incidence n Incidence n Incidence n Incidence

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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Page 40 of 54 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 Observations 1

Severity Interstitial fibrosis Egg Debris in

Oviduct Granulomatous

Inflammation Decreased PostOvulatory Follicles

n Incidence n Incidence n Incidence n Incidence

Negative

Control

(<LOQ) 0 

### 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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EPA MRID Number 48671701

Page 41 of 54 Treatment

(mg a.i./L)

[meanmeasured ] Additional Diagnostic Observations 1

Severity Interstitial fibrosis Egg Debris in

Oviduct Granulomatous

Inflammation Decreased PostOvulatory Follicles

n Incidence n Incidence n Incidence n Incidence

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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Page 42 of 54 Mean male VTG was 1650, 1790, 535 and 42 and female VTG was 1.43x10 6 

### , 2.06x10 6 

### , 2.43x10 6 

### and

0.399x10 6 

### 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

n 1 

### Mean

(ng/mL plasma) ±SD n 1 

### Mean

(ng/mL plasma) ±SD

Negative Control

(<LOQ) 4 1650 2970 

### 4 1.43x10 6 0.337 

### x10 6



### 0.099 4 1780 2300 

### 4 2.06x10 6 0.735 

### x10 6



### 0.92 4 535 970 

### 4 2.43x10 6 0.167 

### x10 6



### 9.1 4 42 73 4 0.399x10 6 0.255 

### x10 6

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

LOQ=0.0032 mg a.i./L

1 Number of replicate tanksData Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 43 of 54Mean 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)MalesFemalesMalesFemalesnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDNegative Control(<LOQ)NANA NANANA NANANANANANANA0.099NANANANANANANANANANANANA0.92NANANANANANANANANANANANA9.1NANANANANANANANANANANANAAbbreviations:NANot applicable.NDNot determined.SDStandard deviation.LOQ=0.0032 mg a.i./LData Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 44 of 54According 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 theauthor, there were no other notable observations with regards to behavior or changes in appearance (coloration/banding, presence of ovipositor anddorsal nape pad), including secondary sex characteristics in the control, 0.099, and 0.92 mg a.i./L treatment groups. Upon examination of the rawdata tables, the reviewer additionally noted the following which occurred at least once during the exposure: the presence of colors bars in some malesof the control and all treatment groups; darkcoloration and partial/complete loss ofequilibrium in males and females of the 9.1 mg a.i./L treatmentgroup; and 1 bloated female and 1 female with an edema in the 0.099 m a.i./L treatment group.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 45 of 54Table 21: Secondary Sex Characteristicsand Clinical Signs inFathead minnow ( Pimephales promelas).Treatment(mg a.i./L)[mean-measured]Secondary Sex Characteristics and Clinical SignsMalesFemalesTypen1IncidenceTypen1IncidenceNegative Control(<LOQ)None 80 None 1600.099None80Edema1610.92None80None1609.1Dark6Not specified1Dark 15Notspecified1Lethargic6Not specified1Complete loss of equilibrium61LOQ=0.0032 mg a.i./L1 Number of affected fish not specified; noted for all replicates

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

EPA MRID Number 48671701

Page 46 of 54 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

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

EPA MRID Number 48671701

Page 47 of 54 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:

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

EPA MRID Number 48671701

Page 48 of 54 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 nega tive 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 49 of 54Table 22: Reproductive and HPG Endpoints1,2for Male Fathead Minnow ( Pimephales promelas)in the FSTRA with Linuron.Treatment(mg a.i./L)[mean-measured]Tubercle ScoreGSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2Medianp% Diff.pEffect?(Yes/No)% Diff.P% Diff.p% Diff.pNegative Control(<LOQ)32 NA 0 NANo0 NA NA NA NANA0.099340.88-4.840.57No8.300.56NANANANA0.92280.178.060.93No-67.50.22NANANANA9.12<0.0001-39.840.005Yes-97.50.017NANANANAStatistical TestDunnett’s3Dunnett’sNAJonckheereNANAAbbreviations:Conc.Concentration.Diff.Difference.E217β-estradiol.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.NANot applicable.LOQ=0.0032 mg a.i./L.1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water)control. Conclusions regardinghistopathology may be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.3Mean 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with LinuronEPA MRID Number 48671701Page 50 of 54Table 23: Reproductive and HPG Endpoints1,2for Female Fathead Minnow ( Pimephales promelas)in the FSTRA with Linuron.Treatment(mg a.i./L)[meanmeasured]FecundityFert. SuccessTubercleScoreGSIGonadalStagingand Histo.Plasma VTGPlasma TPlasma E2% Diff.p% Diff.PMedianp%Diff.pEffect?(Yes/No)%Diff.p%Diff.p%Diff.pNegative Control(<LOQ)0 NA0 NA0NA0 NA No 0 NANANANANA0.099-6.150.240.770.680NA3.520.85No44.20.96NANANANA0.92-49.850.003-5.100.0360NA7.660.93No70.10.99NANANANA9.1--3--3--3--30NA-64.60<0.0001Yes-72.10.16NANANANAStatistical TestJonckheere4Jonckheere5NADunnett’sNADunnett’sNANAAbbreviations:Conc.Concentration.Diff.Difference.E217β-estradiol.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.LOQ=0.0032 mg a.i./L1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water)control. Conclusions regarding histopathologymay be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.3No 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 fromdata 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.

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

EPA MRID Number 48671701

Page 51 of 54 Table 24: Growth Endpoints 1,2 

### 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 23.4 q C;

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

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

EPA MRID Number 48671701

Page 52 of 54 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.

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

EPA MRID Number 48671701

Page 53 of 54 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 System TM 

### . User’s

Guide. Tidepool Scientific Software, McKinleyville, California.

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

Research 1: 21-29.

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.

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

EPA MRID Number 48671701

Page 54 of 54 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. Environm ental 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.

### 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

Brine: Species: Pimephales promelas

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

Project: Herbicide Material: Linuron

Flow Through Batch Note:

Comparison Summary

NOEL LOEL TOEL

Endpoint Analysis ID Method PMSD TU

0.099 0.92 0.3018 10-8236-3077 Jonckheere-Terpstra Step-Down Test NA Fecundity

0.099 0.92 0.3018 11-1767-8906 Dunnett Multiple Comparison Test 27.6% Fecundity

0.92 9.1 2.893 15-7199-5369 Jonckheere-Terpstra Step-Down Test NA FemaleBodyWt

0.92 9.1 2.893 04-4721-1928 Dunnett Multiple Comparison Test 11.1% FemaleBodyWt

0.92 9.1 2.893 17-8672-4863 Jonckheere-Terpstra Step-Down Test NA FemaleGSI

0.92 9.1 2.893 02-7102-8473 Dunnett Multiple Comparison Test 24.8% FemaleGSI

0.92 9.1 2.893 04-4027-9771 Jonckheere-Terpstra Step-Down Test NA FemaleLength

0.92 9.1 2.893 08-1927-4794 Dunnett Multiple Comparison Test 3.15% FemaleLength

9.1 >9.1 NA 07-5969-5726 Jonckheere-Terpstra Step-Down Test NA FemaleMedianTubercleSco

9.1 >9.1 NA 19-5475-5994 Mann-Whitney U Two-Sample Test NA FemaleMedianTubercleSco

9.1 >9.1 NA 20-5084-9852 Jonckheere-Terpstra Step-Down Test NA FemaleVTG

9.1 >9.1 NA 11-1492-9456 Dunnett Multiple Comparison Test 106.0% FemaleVTG

0.099 0.92 0.3018 16-1264-8919 Jonckheere-Terpstra Step-Down Test NA Fertility

0.099 0.92 0.3018 19-6698-6644 Dunnett Multiple Comparison Test 4.35% Fertility

0.099 0.92 0.3018 03-5527-0519 Jonckheere-Terpstra Step-Down Test NA MaleBodyWt

0.099 0.92 0.3018 12-3821-5103 Dunnett Multiple Comparison Test 18.0% MaleBodyWt

0.92 9.1 2.893 09-7606-5995 Jonckheere-Terpstra Step-Down Test NA MaleGSI

0.92 9.1 2.893 13-9185-5081 Dunnett Multiple Comparison Test 25.1% MaleGSI

9.1 >9.1 NA 00-1534-2468 Jonckheere-Terpstra Step-Down Test NA MaleLength

9.1 >9.1 NA 15-2218-6203 Dunnett Multiple Comparison Test 6.44% MaleLength

0.92 9.1 2.893 12-3845-2165 Jonckheere-Terpstra Step-Down Test NA MaleMedianTubercleScore

0.92 9.1 2.893 11-6239-3516 Dunnett Multiple Comparison Test 20.0% MaleMedianTubercleScore

0.92 9.1 2.893 14-2658-7275 Jonckheere-Terpstra Step-Down Test NA MaleVTG

9.1 >9.1 NA 12-3145-2825 Mann-Whitney U Two-Sample Test 162.0% MaleVTG

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

### CETIS Summary Report Report Date: 10 Jan-14 12:39 (p 2 of 5)

Test Code: 035506 48671701 | 18-0850-2597

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Smithers Viscient

Control Type Mean Min Max Std Dev

Count CV% Std Err Group Fecundity Summary

95% LCL 95% UCL %Effect

0 Negative Control 16.3 13 20 2.87 4 17.7% 1.44 11.7 20.8 0.0%

0.099 15.3 12 19 2.87 4 18.8% 1.44 10.7 19.8 6.15%

0.92 8.15 4.7 12 2.99 4 36.7% 1.49 3.39 12.9 49.8%

Control Type Mean Min Max Std Dev

Count CV% Std Err Group FemaleBodyWt Summary

95% LCL 95% UCL %Effect

0 Negative Control 1.55 1.37 1.74 0.164 4 10.6% 0.0822 1.29 1.81 0.0%

0.099 1.52 1.45 1.59 0.0653 4 4.31% 0.0326 1.41 1.62 2.19%

0.92 1.52 1.43 1.63 0.0977 4 6.41% 0.0489 1.37 1.68 1.68%

9.1 1.1 1.02 1.18 0.0664 4 6.02% 0.0332 0.997 1.21 28.9%

Control Type Mean Min Max Std Dev

Count CV% Std Err Group FemaleGSI Summary

95% LCL 95% UCL %Effect

0 Negative Control 12.1 9.6 16 3.06 4 25.3% 1.53 7.21 16.9 0.0%

0.099 12.5 11 14 1.29 4 10.3% 0.645 10.4 14.6 -3.52%

0.92 13 11 14 1.41 4 10.9% 0.707 10.7 15.3 -7.66%

9.1 4.28 3.4 5.3 0.846 4 19.8% 0.423 2.93 5.62 64.6%

Control Type Mean Min Max Std Dev

Count CV% Std Err Group FemaleLength Summary

95% LCL 95% UCL %Effect

0 Negative Control 42.6 41.1 43.7 1.1 4 2.59% 0.552 40.8 44.3 0.0%

0.099 42.2 41.8 42.8 0.49 4 1.16% 0.245 41.4 43 0.82%

0.92 42.7 41.8 43.4 0.779 4 1.82% 0.389 41.5 43.9 -0.35%

9.1 40 39.1 41.1 0.823 4 2.05% 0.411 38.7 41.4 5.88%

Control Type Mean Min Max Std Dev

Count CV% Std Err Group FemaleMedianTubercleScore Summary

95% LCL 95% UCL %Effect

0 Negative Control 0 0 0 0 4 0 0 0

0.099 0 0 0 0 4 0 0 0

0.92 0 0 0 0 4 0 0 0

9.1 0 0 0 0 4 0 0 0

Control Type Mean Min Max Std Dev

Count CV% Std Err Group FemaleVTG Summary

95% LCL 95% UCL %Effect

0 Negative Control 1.43E+6 1.18E+6 1.92E+6 3.37E+5 4 23.6% 1.69E+5 8.93E+5 1.97E+6 0.0%

0.099 2.06E+6 1.37E+6 3.10E+6 7.35E+5 4 35.7% 3.68E+5 8.92E+5 3.23E+6 -44.2%

0.92 2.43E+6 4.48E+5 3.88E+6 1.67E+6 4 68.7% 8.36E+5 -2.28E+5 5.09E+6 -70.1%

9.1 3.99E+5 8.72E+4 7.11E+5 2.55E+5 4 64.0% 1.28E+5 -7.21E+3 8.04E+5 72.1%

Control Type Mean Min Max Std Dev

Count CV% Std Err Group Fertility Summary

95% LCL 95% UCL %Effect

0 Negative Control 98 96 100 1.83 4 1.86% 0.913 95.1 101 0.0%

0.099 98.8 98 99 0.5 4 0.51% 0.25 98 99.5 -0.77%

0.92 93 88 98 4.4 4 4.73% 2.2 86 100 5.1%

Control Type Mean Min Max Std Dev

Count CV% Std Err Group MaleBodyWt Summary

95% LCL 95% UCL %Effect

0 Negative Control 3.41 2.87 4.27 0.599 4 17.6% 0.3 2.46 4.37 0.0%

0.099 3.09 2.96 3.22 0.108 4 3.49% 0.0539 2.92 3.26 9.52%

0.92 2.67 2.47 2.87 0.182 4 6.81% 0.091 2.38 2.96 21.7%

9.1 2.53 1.94 2.9 0.413 4 16.3% 0.206 1.88 3.19 25.8%

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

### CETIS Summary Report Report Date: 10 Jan-14 12:39 (p 3 of 5)

Test Code: 035506 48671701 | 18-0850-2597

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Smithers Viscient

Control Type Mean Min Max Std Dev

Count CV% Std Err Group MaleGSI Summary

95% LCL 95% UCL %Effect

0 Negative Control 1.55 1.4 1.6 0.1 4 6.45% 0.05 1.39 1.71 0.0%

0.099 1.47 1.1 1.8 0.299 4 20.2% 0.149 1 1.95 4.84%

0.92 1.67 1.5 1.9 0.206 4 12.3% 0.103 1.35 2 -8.06%

9.1 0.932 0.57 1.3 0.3 4 32.2% 0.15 0.455 1.41 39.8%

Control Type Mean Min Max Std Dev

Count CV% Std Err Group MaleLength Summary

95% LCL 95% UCL %Effect

0 Negative Control 54.7 52 59.4 3.27 4 5.97% 1.63 49.5 59.9 0.0%

0.099 54.1 53 56.8 1.8 4 3.33% 0.901 51.3 57 1.1%

0.92 50.3 48.4 52 1.49 4 2.97% 0.746 47.9 52.6 8.18%

9.1 53.4 50.9 54.4 1.69 4 3.16% 0.844 50.7 56.1 2.38%

Control Type Mean Min Max Std Dev

Count CV% Std Err Group MaleMedianTubercleScore Summary

95% LCL 95% UCL %Effect

0 Negative Control 31.8 26 38 5.06 4 15.9% 2.53 23.7 39.8 0.0%

0.099 33 27 38 4.55 4 13.8% 2.27 25.8 40.2 -3.94%

0.92 27.5 23 32 3.87 4 14.1% 1.94 21.3 33.7 13.4%

9.1 1.63 1 2 0.479 4 29.5% 0.239 0.863 2.39 94.9%

Control Type Mean Min Max Std Dev

Count CV% Std Err Group MaleVTG Summary

95% LCL 95% UCL %Effect

0 Negative Control 1650 61 6100 2970 4 180.0% 1490 -3080 6380 0.0%

0.099 1780 35 4920 2300 4 129.0% 1150 -1880 5450 -8.3%

0.92 535 20 1990 970 4 181.0% 485 -1010 2080 67.5%

9.1 41.8 5 151 72.8 4 174.0% 36.4 -74.1 158 97.5%

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

### 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

Control Type Group 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

Control Type Group 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

Control Type Group 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

Control Type Group 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

Control Type Group 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

Control Type Group 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

Control Type Group 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

Control Type Group 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

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

### 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

Control Type Group 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

Control Type Group 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

Control Type Group 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

Control Type Group 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

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

# DATA EVALUATION RECORD

LINURON

Study Type: O CSPP 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

.

## 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 in dividual

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 shoul d 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).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 microscopicFemale Pubertal Assay ( 2012) / Page 2 of 16

LINURON / 35506 O CSPP 89 0. 1 45 0/ 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 cumulat ive 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.

T he unadjusted and adjusted kidney weights were decreased (p≤0.05) by 7%, the unadjusted,

adjusted and relative pituita ry 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, t he

unadjusted, adjusted , and relative pituitary weights were decreased (p≤0.05) by 13

- 15%.

There was a d ose- dependent decrease ( p ≤ 0.05) in s erum T 4 

## 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 th is 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 requirement s for a Female Pubertal Assay

(OCSPP 890.1450).

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

Quality Assurance statements were provided.Female Pubertal Assay ( 2012) / Page 3 of 16

LINURON / 35506 O CSPP 89 0. 1 45 0/ 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 S ubstance : 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

CH

3



### CH

3



## 3

. Vehicle : Corn oil

4

. Test A nimals :

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: R everse

- 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. ST UDY DESIGN

1. In -L ife 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.Female Pubertal Assay ( 2012) / Page 4 of 16

LINURON / 35506 O CSPP 89 0. 1 45 0/ OECD None



## 3. Animal A ssignment : Following weaning on PND 21, the rats from the extreme ends of the

weight range were eliminated. A nimals 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 Fem ales

Control 0 15

Low 50 15

High 100 15

a Data were obtained from page 24 of the study report.



## 4. Dose S election Rationale : L inuron dose levels were selected based on the body weight

results from previous 14- day range finding stud y 1



## . 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 resu lts, the dose levels selected for this study were ≤ 100 mg/kg/day

.

5. Dose P reparation and A nalysis : 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 thr oughout 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 co nducted in a previous study 2



## . Homogeneity and resuspension homogeneity

was established at concentrations of 6 and 245 mg/mL following 8 days of refrigerated

storage during a previous uterotrophic study 3 

## (MRID 48617403). Therefore, stability and

homogenei ty 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 Analys is

Homogeneity (% RSD): Not provided

Stability (% of initial): Provided in report WIL Study No s . WIL

- 851005 and WIL

851003)

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). An alytical 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 U terotrophic A ssay of L inuron A dministered O rally in O variectomized R ats. WIL Research

Laboratories, LLC, Ashland, OH. Study No. WIL

- 851003, WIL Research Laboratories, LLC, Ashland, OH. MRID 48617403Female Pubertal Assay ( 2012) / Page 5 of 16

LINURON / 35506 O CSPP 89 0. 1 45 0/ OECD None



## The analytical data indicated that the variation between nominal and actual dosage to the

animals was acceptable.

6. Dosage A dministration : 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. Di chotomous pathology data were

analyzed with pairwise Fisher’s exact test. Tests were conducted at the p ≤ 0.05 significance

level.

T he 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 transformation s were applied to the data; if these were positive then a non

parametric 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 te st 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 followe d 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. A nalyses

were conducted using SAS ® 

## (Cary, NC) version 9.2 (or higher) software

.Female Pubertal Assay ( 2012) / Page 6 of 16

LINURON / 35506 O CSPP 89 0. 1 45 0/ 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 e xact 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 Lev ene ’ 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 compar isons, 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 da ta 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 co variate; 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 p airwise Mann

- Whitne y 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 le vel 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 C linical 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 ho urs after dosing.

2. Body W eight : 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.Female Pubertal Assay ( 2012) / Page 7 of 16

LINURON / 35506 O CSPP 89 0. 1 45 0/ OECD None



## 3. Vaginal O pening : Beginning on PND 22 , all animals were examined daily for onset of

vaginal opening

. T he 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. Estr o us C yclicity : Beginning on the day vaginal opening was observed, vaginal lavages

were perf ormed 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 comple te 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 stress

related responses in other animals which may affect hormone measurements. On PND 42 or

43, all females were euthanize d 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. Immediat ely following decapitation , trunk blood samples were taken for T 4 

## , TSH and serum chemistry analyses. The sample s were stored at ≤− 20° C. Necropsies were

performed on all females found dead or that survived to the scheduled sacrifice . Any tissues

with gr oss lesions were saved in 10% neutral

- buffered formalin for possible future

histopathologic examination.

a. Hormone A nalysis : T 4 

## was analyzed using an electrochemiluminescent assay , and TSH

levels were analyzed using the [ 125 

## I]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. C linical C hemistry : The following CHECKED (X) parameters were examined.Female Pubertal Assay ( 2012) / Page 8 of 16

LINURON / 35506 O CSPP 89 0. 1 45 0/ 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 G uideline 890.1 45 0.



## c. Organ W eights and Histopathology : The following CHECKED (X) tissues were

collected and weighed. The (XX) organs, in addition, were subjecte d to histological

examination.

UROGENITAL OTHER

XX Ovaries ( paired, without oviducts) * + XX Thyroid * +

XX Uterus* + X Liver *

XX Kidneys (paired)*+ X Adrenals (paired) *

X

X Pituitary *

* Weights required based on guideline 890.1 4 50

+ Histopathological examination required based on G uideline 890.1 4 50



## 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 hou rs. 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 tissu es were shipped to Biotechnics, LLC (Hillsborough,

NC) for processing. T issue samples from the thyroid, kidney, ovary, and uterus were

routinely processed into paraffin blocks, sectioned, stained with hematoxylin and eosin, and

examined microscopically.Female Pubertal Assay ( 2012) / Page 9 of 16

LINURON / 35506 O CSPP 89 0. 1 45 0/ 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 thyr oid 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 document ed cases of uterine hyper

or hypotrophy as characterized by changes in uterine horn diameter and myome trial 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 depe ndent upon endocrine status.

II. RESULTS

A. MORTALITY : All females survived to the scheduled euthanasia on Day s 42 or 43.

B. CLINICAL SIGNS OF TOXICITY : No test substance

- related clinical findings were

n

oted at 3 hour s post-dose or at the daily examinations (~24 hours post

- dosing) at any dose

level. Incidental f indings 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, m ean body weight s w ere slightly reduced (p≤0.0 5 ) by 5 -8% for Days 26-

29, 31-39, and 43. However, m ean cumul ative 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).Female Pubertal Assay ( 2012) / Page 10 of 16

LINURON / 35506 O CSPP 89 0. 1 45 0/ 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 de creased (p≤0.0 5 ) 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 ov a ry weights fell within the WIL historical control

database. The lower mean ovary weight at 100 mg/kg/day was not considered treatment

related 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).Female Pubertal Assay ( 2012) / Page 11 of 16

LINURON / 35506 O CSPP 89 0. 1 45 0/ OECD None



### TABLE 3. Organ Weights at Necropsy a

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 7.1 4 0.776 10.89 15 7.0 6 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 : Estr o us 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, o n e 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.Female Pubertal Assay ( 2012) / Page 12 of 16

LINURON / 35506 O CSPP 89 0. 1 45 0/ OECD None



### TABLE 4. Estrous Cyclicity a

Treatment

Groups N Mean Age at

First Vaginal

Estrus (PND) Mean Cycle

Length

(days) Cycling

(%) Regularly

Cycling

(%) Cycle Status at Necropsy (# Females)

Diestrus Proestrus Estrus Not

Cycling

Vehicle 15 38.2 4.8 100.0 42.9 9 b 0 6 1 b

Linuron

50 mg/kg/day 15 38.1 4.8 93.3 66.7 10 b 1 4 1 b

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 C LINICAL CHEMISTRY : Mean hormone levels and

clinical chemistry values are presented in Table 5

. Serum T 4 

## levels were decreased

(p ≤ 0.05) by 27% and 48% at 50 and 100 mg/kg/day, respectively , but s erum 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, m ean AST levels were decreased (p ≤ 0.05) by 29% and 25%, b ile

acids were increased (p ≤ 0.05) by 120% and 92%, m ean globulin levels were decreased

(p ≤ 0.05) by 7% and 13% , and s erum 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 contr ols.

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 criteri on was met for serum T 4 

## level in the controls. Thirteen of the 20

clinical chemistry control values were outside the historical range for the performing

laboratory.Female Pubertal Assay ( 2012) / Page 13 of 16

LINURON / 35506 O CSPP 89 0. 1 45 0/ OECD None



### TABLE 5. Hormone Levels and Clinical Chemistry 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

(%)

H ormones

Serum T 4 , 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 page s 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.Female Pubertal Assay ( 2012) / Page 14 of 16

LINURON / 35506 O CSPP 89 0. 1 45 0/ OECD None



## F. GROSS PATHOLOGY : One 100 mg/kg/day female was observed with no corpora lutea

in the ovary and one ovary was n ot 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 incidence s of histopathological findings of the thyroi d gland

are presented in Table 6

. There were no treatment

- related find ings 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

Grade b Incidence Grade b 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 S1 2 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 N umber O bserved

E N umber 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.Female Pubertal Assay ( 2012) / Page 15 of 16

LINURON / 35506 O CSPP 89 0. 1 45 0/ OECD None



### TABLE 7. Incidence of Histopathological Lesions of the Ovaries, Uterus and Kidney a

Findings Dose Level (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 S1 2 on page s 99

- 102 of the study report.



## III. DISCUSSION AND CONCLUSIONS

A. I NVESTIGATORS’ CONCLUSIONS : Effects of l inuron 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 T 4 

## levels at dos e levels of 50 and 100 mg/kg/day. The significance of the lower

T 4 

## 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, l inuron was not considered to be a pubertal developmental toxicant

when administered orally by gavage to ju venile/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. Ba sed

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 theFemale Pubertal Assay ( 2012) / Page 16 of 16

LINURON / 35506 O CSPP 89 0. 1 45 0/ OECD None



## control group were cycling regularly (≈ 43%) thus confounding the interp retation 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, m ean cumulative body weight gain was comparable to the

controls at the end of the study. N o effects on body weights were observed at 50

mg/ kg/day

.

T he 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 contro ls. At 50

mg/kg/day, t he 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 T 4 

## 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 d ecreased (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 ur ea 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 range

finding study showing body weight loss at 100 mg/kg/day.

C. STUDY DEFICIENCIES : The following minor deficienc ies w ere 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 mea n for adrenal gland weight ( 32.8 mg ) was less than the

performance criteria minimum (38.34 mg ).

# DATA EVALUATION RECORD

LINURON

Study Type: O CSPP 890.1600, In V ivo 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 /201 2

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

.

## 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).

### 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.In Vivo Uterotrophic Assay ( 2011) / Page 2 of 9

LINURON / 035506 O CS PP 8 9 0. 1600/ OECD 440



## No statistically significant cha nges 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.In Vivo Uterotrophic Assay ( 2011) / Page 3 of 9

LINURON / 035506 O CS PP 8 9 0. 1600/ OECD 440



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility : WIL Research Laboratories, LLC

Location: Ashland, OH

Study Director: Pr ä gati 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 S ubstance : 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

CH

3

CH

3



## 3

. Reference E strogen : 17α

- ethynyl estradiol (EE)

Supplier : Sigma Aldrich (St. Louis, MO)

Lot #: 011M1252V (expires August 2, 2012)

Purity: 9 9.0 %

CAS # : 57

- 63

- 6



## 4

. Solvent/ V ehicle C ontrol : Corn Oil

Supplier : Spectrum Chemical Manufacturing Corporation ( New Brunswick, NJ or Gardena,

CA )

Lot #: ZT1301 and 2AE0415 (expire July 1 , 201 2 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)In Vivo Uterotrophic Assay ( 2011) / Page 4 of 9

LINURON / 035506 O CS PP 8 9 0. 1600/ OECD 440



## 5

. Test A nimals :

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 )

Housin g: Rats were housed individually in stainless

- steel wire

- meshed cages suspended

above cage

- board.

Diet : Harlan Laboratories Teklad Global 16% Protein Rodent Diet (2016 CM ) , 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 -L ife D ates : 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 measureme nts.

3. Animal A ssignment : 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.

T ABLE 1. Study Design a



### Test G roup Dose (mg/kg/day) # of Females b



### Estrogen A gonist A ssay

Vehicle C ontrol 0 6

Low (Linuron) 30 6

Mid (Linuron) 60 6

High (Linuron) 120 6

17 α

- ethynyl estradiol (EE), R eference

E strogen 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 range

finding study 1 

## 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/k g/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.In Vivo Uterotrophic Assay ( 2011) / Page 5 of 9

LINURON / 035506 O CS PP 8 9 0. 1600/ OECD 440



## 50 mg/kg/day there was a lower mean body weight gain. In a previous Hershberger assay 2



## 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. S amples

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, a fter mixi ng 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 D ose 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 A dministration : 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. S tatistics : Statistical analyses were conducted using SAS version 9.2, or higher, software.

Body weights, body weight gain, and uterine (b lotted 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,

Histopathologi cal, 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.In Vivo Uterotrophic Assay ( 2011) / Page 6 of 9

LINURON / 035506 O CS PP 8 9 0. 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 nonpar ametric 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 appro ximately 3 hours after

dosing. The injection sites for positive co ntrol, 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 W eight : 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 n ot conducted.

II. RESULTS

A. OBSERVATIONS

1. Mortality : All animals survived until scheduled termination.

2. Clinical S igns of Toxicity : Increased incidence of r ed material was observed around the

nose in the high- dose group (3 incidents and 3 animals) but not in the control group. ThisIn Vivo Uterotrophic Assay ( 2011) / Page 7 of 9

LINURON / 035506 O CS PP 8 9 0. 1600/ OECD 440



## 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 observa tions were noted.

B. BODY WEIGHT AND WEIGHT GAIN : B ody weight and body weight gain data are

presented in Table 2. The body weights were decreased in all linuron treated groups with

significant ly 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 %).

T ABLE 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 S D N Mean S D N Mean S D N Mean S D N Mean S D

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 Table s 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 W eights : U terine weight data are presented in Table 3. Absolute and

relative to body weight u terine 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<0.0 5 )

by 797 % and 300 %, respectively. The se wet and blotted uterine weights increased as

expected.In Vivo Uterotrophic Assay ( 2011) / Page 8 of 9

LINURON / 035506 O CS PP 8 9 0. 1600/ OECD 440



### T ABLE 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 0.0 23 0.00 4 6 0.0 24 0.00 3 6 0.0 24 0.00 4 6 0.0 27 0.00 5 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 0.0 20 0.00 3 6 0.0 21 0.00 2 6 0.0 21 0.00 4 6 0.0 23 0.00 5 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 B ody weight

N No. of animals in the group

SD Standard Deviation

* Significantly different from vehicle control at p < 0.0 5.



## 2. Microscopic Examination (Optional) : M icroscopic examinations were not conducted.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR ’ S CONCLUSIONS : Based on the absence of effects on uterine

weights (wet and blotted), l inuron did not demonstrate or mimic biological activities

consistent with agonism of natural estrogens or show evidence of interaction with the

estrogen pat hway 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 surviv ed 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 treat ed groups with significantly

decreased body weights at 120 mg/kg/day (↓10 -12%, D ays 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 linuronIn Vivo Uterotrophic Assay ( 2011) / Page 9 of 9

LINURON / 035506 O CS PP 8 9 0. 1600/ OECD 440



## 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