UNITED STATES ENVIRONMENTAL PROTECT\ON AGENCY 

WASHINGTON, D.C. 20460 

MEMORANDUM 

Date: 6/29/2015 

OFFICE OF CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

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

PC Code: 101101 

Decision No.: 460711, 463995 

Petition No.: NA 

DP Barcode: D398559, D4012 l 9 

Registration No.: NA 

Regulatory Action: NA 

Risk Assessment Type: NA 

TXR No.: 0056258 

MRID No.: See Table 

Case No.: NA 

CAS No.: 21087-64-9 

40 CFR: NA 

FROM: Greg Akerman, Ph.D. .£>-;y A--- Immediate Office 

Health Effects Division (7509P) 

THROUGH: Jess Rowland ~-C2 32 __ _ 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 metribuzin: Test Order# CON-101101-11. 

Page I of2 

Page 1 of 205
Page 2 of 2 

II. RESPONSE 

Attached are the EDSP Tier 1 assay DERs for metribuzin.

III. MRID Table 

Chemical: Metribuzin PC Code: 101101 

Guideline Assay MRID 

890.1100 Amphibian Metamorphosis Assay (Frog) NA 

890.1150 Androgen Receptor Binding (Rat Prostate) 48617905 

890.1200 Aromatase Assay (Human Recombinant) 48617901 

890.1250 Estrogen Receptor Binding 48617902 

890.1300 Estrogen Receptor Transcriptional Activation 

(Human Cell Line HeLa-9903) 48617903 

890.1350 Fish Short-Term Reproduction 48672202 

890.1400 Hershberger (Rat) 48617904 

890.1450 Female Pubertal (Rat) 48672203 

890.1500 Male Pubertal (Rat) 48672203 

890.1550 Steroidogenesis (Human Cell Line – H295R) 48717906 

890.1600 Uterotrophic (Rat) 48617907 

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

Page 2 of 205
DATA EVALUATION RECORD 

METRIBUZIN 

Study Type: OCSPP 890.1150, Androgen Receptor Binding (Rat Prostate Cytosol) 

EPA Contract No. EP10H001452 

Task Assignment No. 2-24-2012 (MRID 48617905) 

Prepared for 

Health Effects Division 

Office of Pesticide Programs 

U.S. Environmental Protection Agency 

2777 South Crystal Drive 

Arlington, VA 22202 

Prepared by 

CSS-Dynamac Corporation 

1910 Sedwick Road, 

Building 100, Suite B 

Durham, NC 27713 

Primary Reviewer: Signature:

Sandra Hastings Date: 3/06/2012 

Secondary Reviewer: Signature:

David A. McEwen, B.S. Date: 3/09/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 3/28/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 3/28/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 3 of 205
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 4 of 205
METRIBUZIN/ 10 11 01 

Androgen Recepto r Bind ing (Rat Prostate Cytosol) (20 11) I Page I of 14 

OCSPP 890. 11 50/ OECD None 

Primary Reviewer: Patience Browne, Ph.D. 

Office of Science and Coordination Pesticide Programs 

Secondary Reviewer: Greg Akerman, Ph.D. 

Health Effects Division 

SignDat

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Tern plate version 08/20 11 

DATA EVALUATION RECORD 

STUDY TYPE: Androgen Receptor Binding (Rat Prostate Cytosol); OCSPP 890.1150 

PC CODE: 101101 DP BARCODE: D398559 

TXR#: 0056258 CAS No.: 21087-64-9 

TEST MATERIAL (PURITY): Metribuzin (94.4% a.i.) 

SYNONYMS: 4-amino-6-tert-butyl-3-methylsulfanyl-1,2,4-triazin-5-one 

CIT A TI ON: Rouquie, D. (2011 ). Evaluation of Metribuzin in the Androgen Receptor Binding 

Assay. Bayer SAS, Sophia Antipolis Cedex, France. Laboratory Study No.: SA 

11191 , November 30, 2011. MRID 48617905. Unpublished. 

SPONSOR: Bayer CropScience AG, Alfred Nobel Str. 50, 40789 Monheim, Germany 

TEST ORDER#: CON-101101-11 

EXECUTIVE SUMMARY: In an androgen receptor (AR) binding assay (MRID 48617905), 

ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct a 

saturation binding experiment and a competitive binding experiment. The saturation binding 

experiment was conducted to demonstrate that the AR in the rat prostate cytosol was present in 

reasonable numbers and was functioning with appropriate affinity for the radio-labeled reference 

androgen (R 1881) prior to routinely conducting AR competitive binding experiments. The 

competitive binding assay was conducted to measure the binding of a single concentration of 

[3H]-Rl88 1 (1 nM) in the presence of increasing concentrations (10- 10 to 10-3 M) ofmetribuzin 

(94.4%, Batch# K285067). No precipitation of test substance was observed at any concentration 

and DMSO was used as the solvent vehicle at a final assay concentration of :S3 .3%. Three 

independent runs were performed, and each run inc luded dexamethasone as a weak positive 

contro l and Rl881 as the ligand reference standard. 

In the saturation binding experiment, the mean maximum binding capacity (Bmax) was 14 

fmol/100 μg protein and the mean dissociation constant (Kct) was 1.0 I nM. The Kct obtained 

from Run #2 (0.59 nM) was below recommended values for saturation binding, but the Kcts from 

the other two runs were within the recommended range (0.685 to 1.57 nM), and the Scatchard 

plots for the three Saturation assays were generally linear. The specific binding reached a 

plateau, and non-specific binding (NSB) was <20% of total binding ( 4.6-12 .6%). 

Page 5 of 205
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 2 of 14 

METRIBUZIN/ 101101 OCSPP 890.1150/ OECD None

In the competitive binding experiment, specific [3H]-R1881 binding was >75% at all 

concentrations of metribuzin tested in all three runs. An IC50 and relative binding affinity (RBA) 

could not be calculated for metribuzin. 

The estimated average log IC50s for R1881 and dexamethasone were −9.1 M and −4.4 M, 

respectively. Compared to the reference ligand, the mean RBA for dexamethasone was 

0.0021%. No precipitation of the test compound was visually observed at ≤10−3

 M. With the 

exception of the low Kd from run 2, all performance criteria were met.

Based on the results from the three runs, metribuzin is classified as a Non-Binder in the 

Androgen Receptor Binding Assay. 

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

Assay (OCSPP 890.1150). 

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

statements were provided. 

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Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 3 of 14 

METRIBUZIN/ 101101 OCSPP 890.1150/ OECD None

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Bayer S.A.S., Bayer CropScience 

Location: 355 Rue Dostoievski, BP 153, 06903 Sophia Antipolis Cedex, France 

Study Director: D. Rouquie 

Other Personnel: H. Tinwell (replacement Study Director); A. Arnaud (Technician); P. Almeras (Report 

Unit Assistant) 

Study Period: March 22, 2011 to November 30, 2011 

2. Test substance: Metribuzin 

 Description: Light grey powder 

 Source: Bayer CropScience 

 Batch #: K285067 (expiry date: May 7, 2012) 

 Purity: 94.4% a.i. 

 Solubility: Not reported 

 Volatility: Not reported 

 Stability: Not reported 

 Storage conditions: 25±5°C 

 CAS #: 21087-64-9 

 Molecular weight: 214.29 g/mol 

 Structure: 

3. Non-labeled ligand: R1881 

 Supplier: Fountain Limited (Malta)

 Catalog #: 

Batch #: 

E3164-000 

B1023 

 Purity: 98.8% 

 CAS #: 965-93-5 

4. Radioactive ligand: [

3

H]-R1881 

 Supplier: PerkinElmer, Inc. (Boston, MA, USA)

 Catalog and Batch #: NET590/ 653698 

 Date of production: February 24, 2011

 Date of use: May 17, 2011 to June 8, 2011 (Saturation Binding Experiment) 

July 4, 2011 to July 19, 2011 (Competitive Binding Experiment)

 Radiochemical purity: >97% 

 Specific activity: 85.1 Ci/mmol 

 Concentration of stock: 10 nM 

5. Positive control: Dexamethasone 

 Supplier: Sigma-Aldrich (Switzerland)

 Catalog # 

Batch #: 

D1756 

1419230 

 Purity: 98.9% 

 CAS # : 50-02-2 

6. Solvent/vehicle control: Dimethyl sulfoxide (DMSO) 

 Justification for choice of DMSO is one of the solvents recommended in the guideline (OPPTS 890.1150) and 

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METRIBUZIN/ 101101 OCSPP 890.1150/ OECD None

solvent: metribuzin is reasonably soluble in this solvent. 

 Final Concentration: ≤3.3% 

B. METHODS

1. Preparation of Rat Ventral Prostate Cytosol: The preparation of rat prostate cytosol for 

the AR binding assay was detailed under Study SA 11021, which included the saturation 

binding experiment (Attachment 4 of the subject study). A total of 50 male Sprague Dawley 

rats (Charles River Laboratories, France) were acclimatized for 8 days at the laboratory, 

castrated at 84 days of age and euthanized on the following day (~24 hours later). The 

ventral prostates were excised immediately after termination, weighed and minced, and 

placed in ice-cold low-salt TEDG pH 7.4 buffer [10 mM Tris/HCl, 1 mM sodium 

molybdate, 1.5 mM EDTA, 10% glycerol, 1 mM DTT, and 1 mM phenylmethylsulfonyl 

fluoride (PMSF)] at 10 mL/g of tissue. The tissues were homogenized and centrifuged for 

30 min at 30,000 × g at 4o C. The supernatant was pooled, and the cytosol was aliquoted (5 

mL) and stored at −74 ± 10o C until use. Protein concentration of the cytosol was 

determined to be 6.6 mg/mL using a commercially available protein kit compatible with 

DTT in the TEDG buffer (BioRad Protein Assay Kit). 

2. Saturation Radioligand Binding Experiment: A saturation binding experiment 

measuring total and non-specific binding of [3H]-R1881 was performed in a previous study 

(SA 11021) for the cytosol used in the subject competitive binding experiment. The details 

and results of the saturation binding experiment were included as an attachment to the 

subject study. The conditions for the saturation binding experiment are summarized in 

Table 1. 

TABLE 1. Summary of Conditions for Saturation Binding Experiment a

Source of receptor Rat prostate cytosol 

Concentration of radioligand (as serial dilutions) 0.25-10 nM 

Concentration of non-labeled ligand (100X [radioligand]) 25-1,000 nM 

Optimization of receptor concentration 0.72 mg protein/assay tube (binds 25-

35% of ligand) b

Temperature 5 ± 3oC (refrigerated) 

Incubation time 20 hours 

Composition of assay buffer 

(TEDG) 

Tris/HCl 10 mM (pH 7.4) 

EDTA 1.5 mM 

Glycerol 10% 

Sodium Molybdate 1 mM 

Phenylmethylsulfonyl fluoride 1 mM 

DTT 1 mM 

Protease inhibitor 0.5% 

 a Data were obtained from pages 72, 74, and 116-123 of the study report. 

b The optimal protein concentration was determined to be 0.72 mg protein/assay tube; however, the ligand 

depletion at 0.25 nM could not be determined from the raw data provided. 

On the day of the assay, the specific activity of the stock solution [3H]-R1881 was adjusted 

for decay over time, and serial dilutions in TEDG buffer were prepared to achieve the final 

Page 8 of 205
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 5 of 14 

METRIBUZIN/ 101101 OCSPP 890.1150/ OECD None

concentrations in cytosol of 0.25, 0.5, 0.7, 1, 1.5, 2.5, 5, and 10 nM to determine total 

binding. To determine NSB, solutions of non-labeled R1881 were prepared in a similar 

manner to achieve concentrations that were 100-fold greater than each respective 

radiolabeled concentration, resulting in final concentrations in cytosol of 25, 50, 70, 100, 

150, 250, 500, and 1,000 nM. In the absence of cytosol, the radiation found in 7.5, 15, 21, 

30, or 45 μL of 10 nM [3H]-R1881 and 7.5, 15, or 30 μL of 100 nM [3H]-R1881 was 

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

calculating specific binding to differing amounts of protein per tube, using 0.25 nM 

radiolabeled R1881. The optimal protein concentration was determined to be 0.72 mg 

protein/assay tube. Each run contained three concurrent replicates at each concentration, 

resulting in the 72 samples depicted in Table 2, plus three replicates of the solvent control. 

TABLE 2. Saturation Binding Experiment Run a, b

Total Binding Non-Specific Binding Radioligand alone 

Tubes 1-24 c Tubes 25-48 d Tubes 49-72 e 

[

3

H]-R1881 

Final conc. (nM) 

[

3

H]-R1881 

Final conc. (nM) 

R1881 

Final conc. (nM) 

[

3

H]-R1881 

Initial conc. (nM) 

[

3

H]-R1881 

(μL) 

0.25 0.25 25 10 7.5 

0.50 0.50 50 10 15 

0.70 0.70 70 10 21 

1.00 1.00 100 10 30 

1.50 1.50 150 10 45 

2.50 2.50 250 100 7.5 

5.00 5.00 500 100 15 

10.00 10.00 1000 100 30 

a Data were obtained from pages 116-123 of the study report. 

b Each concentration was run in triplicate for a total of 72 samples. 

c Tubes 1-24 contained 50 μL of triamcinolone acetonide and 7.5-45 μL [3

H]-R1881. Samples were dried, 

and 300 μL of prostate cytosol were added. 

d Tubes 25-48 contained 50 μL of triamcinolone acetonide and 7.5-45 μL [3

H]-R1881. R1881 was added in a 

100-fold molar excess of [3

H]-R1881 in a volume of 7.5-45 μL. Samples were dried, and 300 μL of prostate 

cytosol were added. 

e Tubes 49-72 contained only 7.5, 15, 21, 30, or 45 μL of 10 nM [3

H]-R1881 or 7.5, 15, or 30 μL of 100 nM 

[

3

H]-R1881 without cytosol or other components to determine the total counts added. 

Following addition of triamcinolone acetonide, [3

H]-R1881, and/or R1881, the tubes were 

dried, dissolved in diluted prostate cytosol (300 μL), and incubated in a rotor in a 

refrigerator (5 ± 3oC) for 20 hours. To separate bound from free R1881, an aliquot of each 

sample tube was incubated with hydroxyapatite (HAP) slurry in an ice water bath for 20 

minutes. The samples were then centrifuged, and the supernatant was discarded. The 

samples were washed 3 times in 50 mM TRIS buffer. Following the last wash and 

decanting of the Tris buffer, pellets were then extracted with 2 mL ethanol. The radiation 

was quantified by liquid scintillation counting of the ethanol extract. A total of three runs 

were performed. 

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

binding experiment is included in Table 3. 

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TABLE 3. Summary of Conditions for Competitive Binding Experiment a

Source of receptor Rat ventral prostate cytosol 

Concentration of radioligand 1 nM 

Optimization of receptor concentration 0.48 mg protein/assay tube (binds 10-15% 

of ligand) b 

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

Incubation Temperature 5 ± 3oC (refrigerated) 

Incubation time 20 ± 0.5 hours 

Composition of assay buffer Tris 10 mM (pH 7.4) 

EDTA 1.5 mM 

Glycerol 10% 

Phenylmethylsulfonyl fluoride 1 mM 

DTT 1 mM 

Protease Inhibitor 0.5% 

a Data were obtained from pages 12, 14 and 72 of the study report. 

b The protein concentration used was 0.48 mg protein/assay tube; however, the ligand depletion at 1 nM 

could not be determined from the raw data provided. 

The competitive binding experiment was performed according to the protocol provided in 

the EPA Test Guidelines OCSPP 890.1150. The competitive binding experiment measures 

the binding of a single concentration of [3H]-R1881 (the actual adjusted specific activity 

values used were not reported) to the AR in the presence of increasing concentrations of a 

test substance. 

DMSO was used as the vehicle, and no precipitation was visibly observed at any test 

substance concentration (≤10−3 M). Based on the results from the saturation binding 

experiment, the amount of cytosolic protein used for each incubation in the competitive 

binding experiments was 0.48 mg/assay tube, which was reported to bind no more than 10-

15% of the [3

H]-R1881 at 1 nM. 

Dilutions of the test substance (metribuzin), reference standard (R1881), weak positive 

control (dexamethasone), and solvent control (DMSO) were prepared to achieve the 

concentrations shown in Table 4. The assay consisted of three independent runs on three 

different days, and each run contained three replicates at each concentration, plus six 

replicates of solvent total binding, non-specific binding, and the hot mix resulting in a total 

of 81 samples per run. In addition, 3 replicates of the solvent blank were analyzed at the 

end of each run. 

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TABLE 4. Competitor Final Molar (M) Concentrations in Competitive Binding Assay a, b

Solvent Control Reference standard Weak positive control Test Chemical None 

DMSO R1881 Dexamethasone Metribuzin (Hot Mix) 

Tubes 1-3 and 70-72 Tubes 4-21 and 73-75 c Tubes 22-45 Tubes 46-69 Tubes 76-81

1×10−7

 1×10−3 1×10−3

1×10−8 1×10−4 1×10−4

1×10−9 1×10−5 1×10−5

1×10−10 1×10−6 1×10−6

1×10−11 1×10−7 1×10−7

-- 1×10−8 1×10−8

-- 1×10−9 1×10−9

-- 1×10−10 1×10−10 

a Data were obtained from pages 35-42 of the study report. 

b Each concentration of each chemical was run in triplicate for a total of 81 tubes per run. Tubes 1-75 contained 

50 μL of triamcinolone acetonide and 30 μL [3

H]-R1881. Samples were dried, and 300 μL of prostate cytosol 

were added. Tubes 1-75 also contained 10 μL of the solvent control, reference standard (non-radiolabeled 

R1881), weak positive control, or test substance. Tubes 76-81 contained only 30 μL of [3

H]-R1881. 

c Tubes 4-6 and 73-75 were used to evaluate non-specific binding by adding 100X of cold (non-radiolabeled) 

R1881. 

The procedures for assay incubation, separation of bound from free R1881 (washing with 

buffer and extraction with ethanol), followed by scintillation counting of bound [3H]-R1881 

were similar to those used in the saturation binding experiment 

4. Data Analysis: The maximal binding capacity (Bmax), dissociation constant (Kd), and 

inhibition concentration (IC50) were calculated using nonlinear regression analysis using 

Graph Pad Prism v. 5.04 (GraphPad Software, Inc.). Scatchard plots were also plotted for 

the binding data (GraphPad Software, Inc.). 

5. Definitions 

a. Classification of test material 

If the data fit a 4-parameter nonlinear regression model, the test chemical is classified as: 

Binder: The average curve for the test chemical across runs crosses 50% of radioligand 

bound. 

Equivocal: The average lowest portion of curves across runs is between 50% and 75% 

radioligand binding (i.e. radioligand displacement is at least 25% but less than 50%), or the 

curve falls outside the range for the weak positive control (−0.6 to −1.4). 

Non-Binder: The average lowest portion of curves across runs is greater than 75% activity 

(i.e. less than 25% displacement of radioligand), or the data do not fit the model. 

Untestable: If the test compound is not soluble above 1×10−6

 M and the binding curve does 

not cross 50%, the chemical is judged to be untestable. 

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b. Descriptors for receptor binding 

Bmax: maximal binding capacity

Kd: dissociation constants

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

by the competitor

Relative Binding Affinity (RBA): (IC50 of R1881 ÷ IC50 of test substance) × 100 

II. RESULTS 

A. SATURATION BINDING EXPERIMENT: Saturation binding experiment parameters 

are presented in Table 5. The dissociation constant (Kd) for [3H]-R1881 was 1.01 nM 

(±0.365), and the estimated Bmax was 14 fmol/100μg protein (±1.5) for the single batch of 

prostate cytosol that was prepared. The goodness of fit (r2

) was not reported and the Kd

obtained from Saturation Assay #2 was low (0.59 nM), but the Kds from the other two runs 

were within the recommended range (0.685 to 1.57 nM) and the Scatchard plot for the three 

saturation assays was linear demonstrating the androgen receptor was functioning with the 

appropriate affinity for the reference ligand R1881. 

TABLE 5. Saturation Binding Experiment of R-1881 with Androgen Receptor from Rat Prostate Cytosola

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

r

2

 NR NR NR NA 

Bmax (nM) 0.34 0.31 0.38 0.34 ± 0.02 

Bmax (fmol/100 μg protein) 14 13 16 14 ± 0.9 

Kd (nM) 1.19 0.59 1.25 1.01 ± 0.21 

a Data were obtained from page 76 of the study report. 

b The mean is reported for the concurrent replicates of each run. 

c Mean and standard error were calculated by the study reviewer. 

r

2

 Goodness of fit for curve calculated for specific binding 

NR Not reported 

NA Not applicable

The graphs in Figure 1 illustrate the NSB, specific, and total binding curves for [3H]-R1881 

to the androgen receptor. The specific binding reached a plateau, and non-specific binding 

was less than 20% of total binding (4.6-12.6%). Figure 2 is the Scatchard plots for each run 

that illustrate the binding of [3

H]-R1881 to the androgen receptor. Although r2 values were 

not reported for the plots, the data were generally linear. 

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FIGURE 1. Binding of [3H]-R1881 to the Androgen Receptor during the Saturation 

Binding Experiment. 

Saturation Assay #1 – NSB with Total binding and Specific Binding - Assay #1

 

Saturation Assay #1 – NSB with Total binding and Specific Binding - Assay #2

 

Saturation Assay #1 – NSB with Total binding and Specific Binding - Assay #3

 

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FIGURE 2. Scatchard Plots of the Binding of [3H]-R1881 to the Androgen Receptor. 

 

B. COMPETITIVE BINDING EXPERIMENT: The binding inhibition curves for the 

metribuzin and the reference compounds are shown in Figures 3 and 4, and the competitive 

binding experiment parameters are presented in Table 6. Specific [3H]-R1881 binding with 

metribuzin was >75% at all concentrations tested in all three runs. The estimated log IC50

for metribuzin could not be determined as it did not result in 50% inhibition (displacement) 

at any concentration. 

The estimated average log IC50s for R1881 and the weak positive control (dexamethasone) 

were −9.1 M and −4.4 M, respectively. The mean RBA for the positive control was 

0.0021%. No precipitation of the test compound was visually observed at ≤10−3 M. The 

solvent control responses indicated no drift in the study assay. The initial assay run #1 was 

discarded (and repeated) due to the use by error of an invalid control substance.

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TABLE 6. Competitive Binding Assay of Metribuzin with AR from Rat Prostate Cytosol a

Parameter Run 1 Run 2 Run 3 Mean ± SE b

r

2

 (unweighted) R1881 NR NR NR NA 

Dexamethasone NR NR NR NA 

Metribuzin NR NR NR NA 

Log IC50 (M) R1881 −9.1 −9.1 −9.1 −9.1 ± 0.0 

Dexamethasone −4.4 −4.4 −4.4 −4.4 ± 0.0 

Metribuzin NA NA NA NA 

IC50 (M) R1881 7.6 × 10−10 8.8 × 10−10 8.7 × 10−10 8.4 × 10−10 (±0.4)

Dexamethasone 4.0 × 10−5 4.3 × 10−5 4.0 × 10−5 4.1 × 10−5(±0.1) 

Metribuzin NA NA NA NA 

Log RBA b Dexamethasone −4.7 −4.7 −4.7 −4.7 ± 0.0 

Metribuzin NA NA NA NA 

RBA (%) Dexamethasone 0.0019 0.0021 0.0022 0.0021 ± 0.0001 

Metribuzin NA NA NA NA 

a Data were obtained from page 17 of the study report.

b Calculated by reviewer; for means expressed in scientific notation, the SE values in parentheses are presented 

in the same order of magnitude as the mean value. 

SE Standard error 

NR Not reported 

NA Not applicable 

r

2

 Goodness of fit 

RBA Relative binding affinity

FIGURE 3. Percentage R1881 Bound to the Androgen Receptor in the Presence of Test 

Compounds. 

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FIGURE 4. Mean Percentage R1881 Bound to the Androgen Receptor in the Presence of 

 Metribuzin in Three Competitive Binding Assays. 

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

functioning properly, each run was evaluated using the following criteria (Table 7): 

TABLE 7. Criterion a Tolerance Limit(s) b Value Yes No 

Ligand depletion is minimal. The recommended ratio of 

total binding in the absence of competitor to total amount 

of [3

H]-R1881 added per assay tube. 

≤15% Not reported c X 

Metribuzin Top (% binding) 80 to 115 101 to 111 X 

R1881 fitted curve parameters 

Top (% binding) 82 to 114 93 to 103 X 

Bottom (% binding) −2.0 to 2.0 −1.14 to 1.95 X 

Hill Slope −1.2 to −0.8 −1.11 to −0.89 X 

Weak positive control (dexamethasone) fitted curve parameters 

Top (% binding) 87 to 106 97 to 104 X 

Bottom (% binding) −12 to 12 −1.27 to 6.30 X 

Hill Slope −1.4 to −0.6 −1.07 to −0.93 X 

Saturation Binding Experiment Kd (nM) 0.685 to 1.57 0.59 to 1.25 d

 X 

Non-specific binding (%) ≤20.0 Not reported e X 

a Data were obtained from pages 17, 36, 39 and 42 of the study report.

b These values represent ranges from the validation study. 

c The protein concentration used was 0.48 mg protein/assay tube; however, the ligand depletion at 1 nM could not 

be determined from the raw data provided. 

d The Kd (0.59 nM) was below the lower tolerance limit in Run 2, but within the acceptable range in the other 

two runs (saturation binding experiment; SA 11021). 

e The non-specific binding was not reported and could not be calculated from the raw data provided. 

Page 17 of 205
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 14 of 14 

METRIBUZIN/ 101101 OCSPP 890.1150/ OECD None

Additionally, the curve for the reference material showed that increasing concentrations of 

unlabeled R1881 displaced [3H]-R1881 in a manner consistent with one-site binding, as 

indicated by a Hill slope of −1.1 to −0.89. Examination of the reference compounds across 

the runs indicated consistency of the Hill slope, placement along the X-axis, and top and 

bottom plateaus. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Based on the fact that data obtained in the three 

independent competitive assays or all data taken together did not fit the nonlinear regression 

model and that the average inhibition curve did not cross 75% activity, metribuzin was 

considered a non-binder with regards to its binding with AR.

B. AGENCY COMMENTS: The saturation binding experiment demonstrated that the 

androgen receptor was present in a reasonable concentration and functioning with 

appropriate affinity for the R1881 ligand. The mean Kd for [3

H]-R1881 was 1.01 nM 

(±0.365), and the estimated Bmax was 14 fmol/100μg protein (±1.5) for the single batch of 

prostate cytosol that was prepared. The Kd obtained from Saturation Assay #2 was low 

(0.59 nM), but the Kds from the other two runs were within the recommended range (0.685 

to 1.57 nM). Although the r2 values were not reported, the Scatchard plots for the three 

saturation runs generally were linear. The specific binding reached a plateau, and NSB was 

less than 20% of total binding (4.6-12.6%). 

In the competitive binding experiment, metribuzin is classified as a non-binder as specific 

[

3

H]-R1881 binding was >75% at all concentrations tested in all three runs. An IC50 and 

RBA could not be calculated for metribuzin. 

The estimated average log IC50s for R1881 and the weak positive control (dexamethasone) 

were −9.1 M and −4.4 M, respectively. The mean RBA for the positive control was 

0.0021%. No precipitation of metribuzin was visually observed at ≤10−3

 M. All 

performance criteria were met, with the exception that the ligand depletion and NSB were 

not reported, but the IC50s for R1881 and the weak positive control were within the expected 

ranges demonstrating that the ligand was available at an appropriate level. 

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 percent ligand depletion was not provided for the saturation and competitive 

binding experiments. 

 The NSB was not provided for the competitive binding experiment. 

 The adjusted specific activity values for the competitive assays were not reported. 

Page 18 of 205
DATA EVALUATION RECORD 

METRIBUZIN 

Study Type: OCSPP 890.1200, Aromatase Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-24-2012 (MRID 48617901) 

Prepared for 

Health Effects Division 

Office of Pesticide Programs 

U.S. Environmental Protection Agency 

2777 South Crystal Drive 

Arlington, VA 22202 

Prepared by 

CSS-Dynamac Corporation 

1910 Sedwick Road, 

Building 100, Suite B 

Durham, NC 27713 

Primary Reviewer Signature:

Rebecca L. Bryan, B.S. Date: 3/8/2012 

Secondary Reviewer Signature:

Michael E. Viana, Ph.D., D.A.B.T. Date: 3/16/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 3/29/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 3/29/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 19 of 205
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 20 of 205
METRIBVZIN / 101101 

Primary Reviewer: Deborah Smegal 

Health Effects Division 

Secondary Reviewer: Greg Akerman, Ph.D. 

Health Effects Division 

Aromatase (Human Recombinant) Assay (201 I) I Page I of 12 

OCSPP 890.1200 I OECD None 

DAT A EVALUATION RECORD 

STUDY TYPE: Aromatase (Human Recombinant); OCSPP 890.1200 

PC CODE: 101101 DP BARCODE: D398559 

TXR#: 0056258 CAS No.: 21087-64-9 

TEST MATERIAL (PURITY): Metribuzin (94.4%) 

SYNONYMS: 4-amino-6-(1, 1-dimethylethyl)-3-(methylthio )-1,2,4-triazin-5( 4H)-one 

CITATION: Tinwell, H. (2011) Evaluation ofmetribuzin in the aromatase assay. Bayer 

S.A.S., Bayer CropScience, Sophia Antipolis Cedex, France. Laboratory 

Report No.: SA 11062, November 10, 2011. MRID 48617901. Unpublished. 

SPONSOR: Bayer CropScience AG, Alfred Nobel Str. 50, Monheim, Germany 

TEST ORDER#: EDSP-101101-127 

'-

EXECUTIVE SUMMARY: In an in vitro aromatase (CYP 19) assay (MRID 48617901 ), 

Metribuzin (94.4% a.i., batch# AE-F055208-01-13) was incubated with human recombinant 

aromatase and tritiated androstenedione (1-~ [3H(N) ]-androst-4-ene-3, 17-dione; [3H]ASDN) in 

dimethyl sulfoxide (DMSO) at increasing logarithmic concentrations from 10- 10 M to 10-3 M for 

15 minutes to assess the potential of metribuzin to inhibit aromatase activity. 

Aromatase activity was determined by measuring the amount of tritiated water produced at the 

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

quantified using liquid scintillation counting (LSC). Three runs were conducted and each run 

included a full activity control, a background activity control, a positive control series (10- 10 to 

1 o-s M) using a known inhibitor ( 4-hydroxyandrostenedione; 4-0H ASDN), and the test 

metribuzin series (10- 10 to 10-3 M) with three repetitions per concentration. 

For the positive control (4-0H ASDN), aromatase activity averaged 0.356±0.0526 nmol/mgprotein/min at the lowest tested concentration (10- 10 M) and 0.0028±0.0005 nmol/mgprotein/min at the highest tested concentration ( 10- 5 M). These results were generally within the 

recommended ranges for the top of the curve, bottom curve, Hill slope, log ICsoand %CV. For 

4-0H ASDN, the estimated log ICso averaged -7.23 M, and the Hill slope was -0.79. Activity 

in the background controls ranged 2.9 to 3.9% and averaged 3.4% of the full activity control. 

For metribuzin, aromatase activity averaged 0.318±0.057 nmol/mg-protein/min at the lowest test 

concentration ( 10- 10 M) and 0.217±0.029 nmol/mg-protein/min at the highest tested 

Page 21 of 205
Aromatase (Human Recombinant) Assay (2011) / Page 2 of 12 

METRIBUZIN / 101101 OCSPP 890.1200 / OECD None

concentration (10−3 M). As a percent of control, aromatase activity was 91.4-103.3% at 10−10 to 

10−4 M metribuzin, but was 63% at 10−3 M. Although the data for metribuzin did not fit the 

4-parameter regression model, the average lowest portion of the inhibition curve across runs was 

between 50-75% activity, indicating an equivocal response. 

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

aromatase inhibition in this assay. 

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

890.1200). 

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

Assurance statements were provided. 

Page 22 of 205
Aromatase (Human Recombinant) Assay (2011) / Page 3 of 12 

METRIBUZIN / 101101 OCSPP 890.1200 / OECD None

I. MATERIALS AND METHODS

A. MATERIALS 

1. Test Substance: Metribuzin 

 Description: Light grey powder 

 Source: Bayer CropScience, Germany

 Lot/Batch #: AE-F055208-01-13 (expires May 7, 2012) 

 Purity: 94.4% 

 Volatility: Not provided 

 Storage conditions: In air-tight, light-resistant containers at room temperature 

 Stability: Not provided 

 Solvent: DMSO 

 Solubility (in test solvent): Soluble up to 10−3 M 

 Highest Concentration Tested: 10−3 M

 Stock Solution Preparation 

Methodology: Serial dilutions

 Molecular weight: 214.29 g/mol 

 CAS #: 21087-64-9 

 Structure: 

N

N

N

O

NH2

S

CH3

3

CH

3

CH

CH3

2. Non-Labeled Substrate: Androstenedione (ASDN) 

 CAS # : 63-05-8 

 Source: Molekula, Ltd. (product # 15237310)

 Lot/Batch #: 63779 (expiry January 2013) 

 Purity: 99.25% 

3. Radiolabeled Substrate: 1-β [3

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

 Source: Perkin Elmer (Catalog # NET-926)

 Lot/Batch #: 3615304 

 Radiochemical Purity (Supplier): 97%

 Specific activity: 20.7 Ci/mmol (0.766 TBq/mmol) 

 Radiochemical Purity (In-lab 

determination): 

Not provided 

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

 CAS #: 566-48-3

 Source: Sigma-Aldrich (Catalog # F2552)

 Lot/Batch #: 081K2133 

 Purity: 99.6 % 

5. Solvent (Vehicle Control): DMSO 

 Source: 

Lot/Batch #: 

Sigma-Aldrich (Ref. # 154938) 

76396EM 

 Justification for choice of solvent: Acceptable vehicle by OCSPP Guideline 890.1200 

 Concentration 

(% of total volume in assays): 1% 

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

Source: BD Biosciences 

Lot/Batch #: 74101 

Protein concentration: 7.95 mg/mL 

Cytochrome C reductase activity: Not provided

Aromatase activity: 0.1131 mmol/mg protein/min 

Page 23 of 205
Aromatase (Human Recombinant) Assay (2011) / Page 4 of 12 

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B. METHODS

1. Assay Components and Preparations: A mixture of non-labeled and radiolabeled 

[

3

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

approximately 4.1 nM, and the amount of tritium added to each incubation tube was 

approximately 0.07 μCi in a volume of 100 μL. 

Metribuzin stock solutions were prepared such that the total volume of each test chemical 

formulation used per assay was 1% of the total assay volume. A rationale for the selection 

of DMSO was not provided; however, metribuzin is soluble in DMSO at the tested 

concentrations and is a solvent recommended by the EPA guidelines. 

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

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

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

positive control substance (10–10 M to 10–5 M; Table 4) were achieved by the addition of 

20 μL of the dilution for a final assay volume of 2 mL. 

Human recombinant microsomes were purchased from BD Biosciences, and stored at −74 to 

−64oC for no longer than one year. Before use of the microsomes, the batch was thawed and 

sub-aliquoted at 0.004 mg/mL into individual vials before being refrozen, and stored at −74 

to −64oC for no more than six months. 

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

reported in Table 1.

TABLE 1. Assay Components and Conditions 

Assay Factor Values a

0.1M sodium phosphate buffer (pH 7.4) 

Microsomal Protein 0.004 mg/mL 

NADPH 0.3 mM 

[3H]ASDN 100 nM 

Propylene Glycol 5% 

Temperature 37°C 

Incubation Time 15 min 

a Data obtained from page 13 of the study report. 

2. Suitability Assessments: The protein concentration of the microsomes (Batch # 74101) 

was supplied by the vendor as 7.95 mg/mL. The aromatase activity of the microsomes was 

also provided by the vendor as 0.113 nmol/mg protein/min. The minimum aromatase 

activity in the full activity control samples of the proficiency test was determined to be 

0.098 nmol/mg protein/min, which was slightly less than the minimum recommended 

aromatase activity of 0.1 nmol/mg-protein/min. However, six other measurements reported 

for the proficiency test were >0.1 nmol/mg protein/min, and the average aromatase activity 

was 0.1131 or 0.1135 nmol/mg protein/min. 

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

background activity controls, respectively, and full concentration curves in the positive 

Page 24 of 205
Aromatase (Human Recombinant) Assay (2011) / Page 5 of 12 

METRIBUZIN / 101101 OCSPP 890.1200 / OECD None

control (duplicate) and metribuzin (triplicate). The aromatase assay was conducted 

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

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

aromatase activity was reported in units of nmol/mg-protein/min. 

4. Demonstration of Proficiency: Prior to performing the definitive assay, one run of the 

positive control was conducted to evaluate the responsiveness of the microsomes. This 

evaluation also served as a proficiency evaluation of the technician involved in the conduct 

of the definitive assays. It was stated that as the same technician was responsible for the 

development of the assay in-house and conducted all aromatase investigations, no further 

proficiency testing was considered necessary for the purpose of this study. 

a. Positive Control

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

following criteria: 

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

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

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

of 4-OH ASDN was <15%. 

 Performance criteria (Table 2) were generally met, and served as guidance in 

identifying runs that provided parameters in the preferred ranges. 

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

applicable): The positive control data met the criteria as listed in section (i) of OCSPP 

890.1200, except for the log IC50 of −6.7 M was slightly greater than the guideline range 

(−7.3 to −7.0).

TABLE 2. Performance Criteria for the Positive Control 

Parameter Lower Limit Criteria Upper Limit Criteria Observation 

Slope −1.2 −0.8 −0.94 

Top (%) 90 110 100.6% 

Bottom (%) −5 +6 1.8% 

Log IC50 (M) −7.3 −7.0 −6.7 

a Data obtained from page 16 of the study report.

b. Proficiency Chemicals: Assays to classify positive controls (econazole, fenarimol, 

nitrofen, and atrazine) were not reported. 

Page 25 of 205
Aromatase (Human Recombinant) Assay (2011) / Page 6 of 12 

METRIBUZIN / 101101 OCSPP 890.1200 / OECD None

TABLE 3. Proficiency Chemicals 

Compound CAS# Class Concentrations 

Econazole 24169-02-6 Inhibitor Not reported--- 

Fenarimol 60168-88-9 Inhibitor Not reported--- 

Nitrofen 1836-75-5 Inhibitor Not reported--- 

Atrazine 1912-24-9 Non-inhibitor Not reported--- 

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

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

tested during three independent runs. Solubility was visually assessed (presence of 

cloudiness or a precipitate), and no precipitation was observed. The full enzymatic activity 

defining the top of the concentration-response curve (≥95% for the means of each run) was 

obtained at concentrations of 10−7

 M to 10−4 M of metribuzin in Run 1, at concentrations of 

10−9

 M to 10−4 M of metribuzin in Run 2, and at the concentration of 10−7 M of metribuzin 

in Run 3. 

TABLE 4. Test Chemical Study Design for each Test Run

Sample Type Repetitions (Tubes) Description Reference or 

Chemical (M) 

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

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

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

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

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

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

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

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

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

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

Metribuzin Conc 1b 3 All test components plus metribuzin 1×10−3

Metribuzin Conc 2b 3 All test components plus metribuzin 1×10−4

Metribuzin Conc 3b 3 All test components plus metribuzin 1×10−5

Metribuzin Conc 4b 3 All test components plus metribuzin 1×10−6

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

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

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

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

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

b Proficiency chemicals or tests chemical(s). 

C. DATA ANALYSIS 

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

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

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

(Table 5). 

Page 26 of 205
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METRIBUZIN / 101101 OCSPP 890.1200 / OECD None

TABLE 5. Raw and Calculated Data

Raw/Calculated Data Included (X) 

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

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

Total DPM for each aqueous portion (after extraction) X 

The total DPM present in the assay tube at initiation X 

The percentage of substrate converted to product X 

Total DPM after extraction corrected for background X 

Aromatase activity expressed in nmol/mg protein/min X 

Average aromatase activity in the full activity control tubes X 

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

DPM = Disintegrations per minute

2. Statistical Methods: Statistical analyses and graphical displays were conducted using 

Prism 5, v 5.04 (GraphPad Software Inc., La Jolla, CA). Basic statistical analyses were 

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

(SD), standard error of the mean (SEM), and coefficient of variation (CV). Analysis of 

variance (ANOVA) was not performed.

The response curves were fitted by a non-weighted least squares nonlinear regression 

analysis with Prism 5 software. For each run, the individual percent of control values were 

plotted versus logarithm of the corresponding concentration. The fitted concentration 

response curve was superimposed on the plot, with individual plots prepared for each run. 

The average percent of control values versus logarithm of the concentration for the 

individual runs for each chemical (with different symbols for each run) were included on the 

same graph with their respective fitted response curves. In addition, the average percent of 

control values for each run versus the logarithm of the concentration were plotted on a 

separate graph along with the average concentration response curve across runs were 

superimposed on the same plot. 

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

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

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

three runs, using the categories presented in Table 6. 

TABLE 6. Interpretation of Results 

Criteria Interpretation 

Data fit 4-parameter nonlinear 

regression model 

Average curve across runs crossed 50%a Inhibitor 

Average lowest portion of curves across runs is 

between 50% and 75% activityb

Equivocal 

Average lowest portion of curves across runs is greater 

than 75% activityb

Non-inhibitor 

Data do not fit model --- 

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

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

equivocal. 

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

determined to be untestable in the aromatase assay. 

Page 27 of 205
Aromatase (Human Recombinant) Assay (2011) / Page 8 of 12 

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II. RESULTS 

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

0.316 to 0.407 nmol/mg-protein/min for the three test runs, with a mean and standard 

deviation of 0.347±0.052 nmol/mg-protein/min. Activity in the background controls ranged 

2.9 to 3.9% and averaged 3.4% of the full activity control. The % change for the average 

background controls at the beginning and end of each run fell between the guideline 

recommendations of −5% and +6% of the overall average full activity. However, for the 

full activity data, a slight assay drift was observed in the first run, as the % changes at the 

beginning and end of each run were between 87.9 and 112.1% instead of the recommended 

guideline range of between 90 and 110%. Overall, the response of the full activity controls 

and background controls were acceptable for each run. 

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

activity averaged 0.356±0.0526 nmol/mg-protein/min at the lowest tested concentration 

(10−10 M) and 0.0028±0.0005 nmol/mg-protein/min at the highest tested concentration 

(10−5 M). The mean aromatase activity of the positive control (expressed as % full control 

activity) for each concentration tested across all three runs is presented in Table 7, along 

with the overall SD, SEM, and %CV. Inhibition response curves for the positive control 

from each run are shown in Figure 1. These results were generally within the recommended 

ranges for the %CV, top of the curve, bottom curve, Hill slope, and log IC50. 

TABLE 7. Effect of Metribuzin on Aromatase Activity (as percent of control) from Independent Runs a

Chemical 

Concen. 

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

4-OH ASDN 

(positive control) 

−5 3 0.8 0.00 0.00 0 

−6 3 7.1 0.55 0.32 8 

−6.5 3 18.4 1.17 0.68 6 

−7 3 39.2 2.15 1.24 6 

−7.5 3 65.3 6.51 3.76 10 

−8 3 78.8 6.53 3.77 8 

−9 3 99.3 11.78 6.80 12 

−10 3 102.9 7.99 4.61 8 

Metribuzin −3 3 62.9 7.41 4.28 12 

−4 3 93.1 14.60 8.43 16 

−5 3 101.5 11.30 6.52 11 

−6 3 99.4 9.54 5.51 10 

−7 3 103.3 8.18 4.72 8 

−8 3 90.5 17.45 10.07 19 

−9 3 95.6 10.04 5.80 11 

−10 3 91.4 4.16 2.40 5 

a Data were obtained from text tables on pages 17 and 18 of the study report. 

SD Standard Deviation 

SEM Standard Error of the Mean 

CV Coefficient of Variance 

Page 28 of 205
Aromatase (Human Recombinant) Assay (2011) / Page 9 of 12 

METRIBUZIN / 101101 OCSPP 890.1200 / OECD None

FIGURE 1. Inhibition Response Curves for 4-OH ASDN (Formestane). 

C. TEST SUBSTANCE: For metribuzin, aromatase activity averaged 0.318±0.057 nmol/mgprotein/min at the lowest tested concentration (10−10 M) and 0.217±0.029 nmol/mgprotein/min at the highest tested concentration (10−3 M). The mean aromatase activity of 

metribuzin (expressed as % full control activity) for each concentration tested across all 

three runs is presented in Table 7, along with the overall SD, SEM, and %CV. Inhibition 

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

inhibition response curve across all runs is shown in Figure 3. 

Page 29 of 205
Aromatase (Human Recombinant) Assay (2011) / Page 10 of 12 

METRIBUZIN / 101101 OCSPP 890.1200 / OECD None

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

FIGURE 3. Mean Inhibition Response Curve for Metribuzin. 

It was stated that analyses of the data for metribuzin indicated they did not fit the 

4-parameter regression model used for data interpretation. The mean aromatase activity of 

10–3 M metribuzin was determined to be 63% of control activity and the best fit mean 

inhibition response curve (Figure 3) was a straight line (essentially no inhibition was 

observed at other doses). The effect of the positive control on inhibition of aromatase 

Page 30 of 205
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METRIBUZIN / 101101 OCSPP 890.1200 / OECD None

activity is presented in Table 8. For 4-OH ASDN, the estimated log IC50 averaged −7.24 M, 

and the slope was −0.80. Confidence in these values is very high due to the small variations 

(<7% CV). 

TABLE 8. Effect of Metribuzin on Aromatase Activity (as Percent of Control) From Independent Runs a

Chemical Run 1 Run 2 Run 3 Mean b SD b %CV b

Log IC50 (M) 

Metribuzin NA NA NA NA NA NA 

4-OH ASDN −7.28 −7.17 −7.27 −7.24 0.061 −0.84 

Hill Slope 

Metribuzin NA NA NA NA NA NA 

4-OH ASDN −0.86 −0.78 −0.76 −0.80 0.053 −6.61 

a Data obtained from text tables on page 17 of the study report. 

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

SD Standard Deviation 

CV Coefficient of Variance 

NA Not Applicable 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS CONCLUSIONS: For each run the background activity controls 

were less than 15% of the corresponding full activity controls, which is in compliance with 

the guideline recommendations. Furthermore, the % change for the background controls at 

the beginning and end of each run was between recommended guideline values of −5 and 

+6%. A slight assay drift in two of the three runs was observed for the full activity data, but 

this did not affect the outcome or interpretation of the results. For the positive control, 

ASDN, the data confirmed the responsiveness of the aromatase enzyme to a reference 

inhibitor. The log IC50 value was between −7.17 and −7.28 M in each of the three 

independent evaluations, with an overall log IC50 value of −7.23 M. All guideline 

performance criteria were met for the positive control. For metribuzin, analyses of the data 

indicated that they did not fit the 4-parameter regression model used for data interpretation. 

Consequently, metribuzin is considered to be a non-inhibitor of the aromatase enzyme. 

B. AGENCY COMMENTS: Aromatase activity in the full activity controls ranged from 

0.316 to 0.407 nmol/mg-protein/min for the three test runs, with a mean (± SD) of 

0.347±0.052 nmol/mg-protein/min. Activity in the background controls ranged 2.9 to 3.9% 

and averaged 3.4% of the full activity control. The responses of the full activity controls 

and background controls were acceptable. 

For the positive control substance (4-OH ASDN), aromatase activity averaged 0.356±0.0526 

nmol/mg-protein/min at the lowest tested concentration (10−10 M) and 0.0028±0.0005 

nmol/mg-protein/min at the highest tested concentration (10−5 M). The estimated log IC50

for 4-OH ASDN averaged −7.23 M, and the Hill slope was −0.79. Confidence in these 

values is very high due to the small variations (<7% CV). 

 

For metribuzin, aromatase activity averaged 0.318±0.050 nmol/mg-protein/min at the lowest 

tested concentration (10−10 M) and 0.217±0.029 nmol/mg-protein/min at the highest tested 

concentration (10−3 M). The average aromatase activity was 91.4-103.3% at metribuzin 

concentrations from 10−10 to 10−4

 M, but was 63% at 10−3 M. Analyses of the metribuzin 

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Aromatase (Human Recombinant) Assay (2011) / Page 12 of 12 

METRIBUZIN / 101101 OCSPP 890.1200 / OECD None

data indicated that they did not fit the 4-parameter regression model, and the best fit mean 

inhibition response curve was essentially a straight line. However, because the average 

lowest portion of the response curve across runs was between 50 and 75% activity, 

metribuzin is classified as equivocal for inhibition of aromatase activity in this assay. 

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

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

 The required data for proficiency chemicals testing were not provided. 

 Information the stability of the test substance in the stock solution was not reported. 

 For the one run of the positive control conducted prior to performing the definitive 

assay, the log IC50 value (−6.70 M) was outside the guideline recommended range of 

−7.3 to −7.0 M. 

Page 32 of 205
DATA EVALUATION RECORD 

METRIBUZIN 

Study Type: OCSPP 890.1250, Estrogen Receptor Binding Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-24-2012 (MRID 48617902) 

Prepared for 

Health Effects Division 

Office of Pesticide Programs 

U.S. Environmental Protection Agency 

2777 South Crystal Drive 

Arlington, VA 22202 

Prepared by 

CSS-Dynamac Corporation 

1910 Sedwick Road, 

Building 100, Suite B 

Durham, NC 27713 

Primary Reviewer: Signature:

Sandra Hastings Date: 3/06/2012 

Secondary Reviewer: Signature:

David A. McEwen, B.S. Date: 3/08/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 3/28/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 3/28/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 33 of 205
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 34 of 205
METRIBUZIN/ 101101 

Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) I Page 1of18 

OCSPP 890.1250/ OECD None 

Primary Reviewer: Ray Kent, Ph.D. 

Health Effects Division 

Secondary Reviewer: Greg Akerman, Ph.D. 

Health Effects Division 

Signature: -~il'+-~-_,,...=-----,.._ 

Date: ----,-~"--'"'-¥-.1''-'"'<.-­

S ign a tu re: -="'~::p,.,._..,,,!J.==::::::_ 

Date: p- 1cJ._q1i .sTemplate version 08/2011 

DAT A EVALUATION RECORD 

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

OCSPP 890.1250 

PC CODE: 101101 DP BARCODE: D398559 

TXR#: 0056258 CAS No.: 21087-64-9 

TEST MATERIAL (PURITY): Metribuzin (94.4% a.i.) 

SYNONYMS: 4-amino-6-tert-butyl-3-methylsulfanyl-1 ,2,4-triazin-5-one 

CITATION: Rouquie, D. (2011 ). Evaluation of Metribuzin in the Estrogen Receptor Binding 

Assay. Bayer SAS, Sophia Antipolis Cedex, France. Laboratory Study No.: SA 

11213, December 2, 2011. MRID 48617902. Unpublished. 

SPONSOR: Bayer CropScience AG, Alfred Nobel Str. 50, 40789 Manheim, Germany 

TEST ORDER#: CON-101101-l l 

EXECUTIVE SUMMARY: In an estrogen receptor (ER) binding assay (MRID 48617902), 

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

and competitive 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 radiolabeled reference estrogen prior to conducting 

ER competitive binding experiments. The competitive binding experiment was conducted to 

measure the binding of a single concentration of [3H]-l 7p-estradiol ( 1 nM) in the presence of 

increasing concentrations (10- 10 to 10-3 M) of metribuzin (94.4%; Batch # K285067). DMSO 

was used as the vehicle at a final concentration of :S2%. A total of three independent runs were 

performed, and each run included 19-norethindrone as a weak positive control, 

octyltriethoxysilane as a negative control, and 17P-estradiol as the natural ligand reference 

material. 

For the saturation binding experiment, the appropriate protein concentration for the saturation 

binding assay was determined to be 0.5 mg/mL as it bound 25-35% of the [3H]-17P-estradiol. 

The mean Kd for [3H]- l 7P-estradiol was 0.070 nM, and the estimated Bmax was 0.034 nM for the 

prepared rat uterine cytosol. 

For the competitive binding experiment, the data for metribuzin did not fit the non-linear 

regression model and percent binding was >75% at all concentrations tested (:S l0-3 M) in all 

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Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 2 of 18 

METRIBUZIN/ 101101 OCSPP 890.1250/ OECD None

three runs. Therefore, a mean log IC50 and relative binding affinity (RBA) could not be 

calculated for metribuzin. 

The estimated mean log IC50 for 17β-estradiol and 19-norethindrone was −8.8 M and −5.0 M, 

respectively, and the mean RBA for 19-norethindrone was 0.017% compared to the natural 

ligand. Several performance criteria were not met for 17β-estradiol: the Hill slope was outside 

the recommended range (−2.0); the bottom % binding (2.1 to 2.5%) was just above the 

recommended range; and the top % binding (93%) was just below the recommended range. 

However, the responses for the weak positive control (19-norethindrone) and negative control 

(octyltriethoxysilane) were as expected. 

Metribuzin was tested over a concentration range that fully defined the top of the curve and the 

maximum percent binding was within approximately 25 percentage points of the lowest 

concentration of the estradiol standard. Each of the three runs was classified as not interactive. 

The lowest average percent radiolabeled estradiol binding to the ER in the presence of 

metribuzin at ≤10−3

 M was >75%. 

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

Binding Assay. 

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

Assay (OCSPP 890.1250). 

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

statements were provided. 

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I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Bayer S.A.S., Bayer CropScience 

Location: 355 Rue Dostoievski, BP 153, 06903 Sophia Antipolis Cedex, France 

Study Director: D. Rouquie 

Other Personnel: O. Blanck (replacement Study Director); M.P. Come (Technician); P. Almeras 

(Report Unit Assistant) 

Study Period: May 3, 2011 to December11, 2011 

2. Test substance: Metribuzin 

 Description: Light grey powder 

 Source: Bayer CropScience 

 Batch #: K285067 (expiry date: May 7, 2012) 

 Purity: 94.4% a.i. 

 Solubility: Not reported 

 Volatility: Not reported 

 Stability: Not reported 

 Storage conditions: 25±5°C 

 CAS #: 21087-64-9 

 Molecular weight: 214.29 g/mol 

 Structure: 

3. Non-labeled ligand: 17β-estradiol 

 Supplier: Sigma-Aldrich, St. Louis, MO

 Catalog #: E8875 

 Batch #: 010M0142 

 Purity: ≥98% 

 CAS #: 50-28-2 

4. Radioactive ligand: [2,4,6,7,16,17 - 3

H]-17β-estradiol 

 Supplier: Perkin Elmer, Boston, MA

 Catalog #: NET517 

 Batch#: 650702 

 Radiochemical purity: >97% 

 Specific activity: 130.2 Ci/mmol 

 Concentration of stock: 1.0 mCi/mL in ethanol 

5. Positive control: 19-norethindrone 

 Supplier: Sigma-Aldrich, St. Louis, MO

 Catalog #: N4128 

 Batch #: 019K1735 

 Purity: ≥98% 

 CAS #: 66-22-4 

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6. Negative control: Octyltriethoxysilane 

 Supplier: Alfa Aesar

 Catalog #: L04407 

 Batch #: 10153935 

 Purity: 97.3% 

 CAS #: 2943-75-1 

7. Solvent/vehicle control: Dimethyl sulfoxide (DMSO) 

 Justification for choice of 

solvent: 

DMSO is one of the solvents recommended in the guideline (OPPTS 890.1150) and 

metribuzin is reasonably soluble in this solvent 

 Final Concentration: ≤2% 

B. METHODS 

1. Preparation of Rat Uterine Cytosol (RUC): The preparation of rat uterine cytosol for the 

ER binding assay was detailed under Study SA 11020 which included the saturation binding 

experiment (Attachment 3 of the subject study). A total of 90 ovariectomized Sprague 

Dawley (Crl:CD(SD)) rats were obtained from Charles River Laboratories (St. Germain-surl’Abresle, France). Animals were 13 weeks old at the time of euthanasia, and rats had been 

ovariectomized 7 days prior to tissue harvest. The uteri were weighed, homogenized in 

TEDG (Tris, EDTA, DTT, glycerol) + PMSF (phenylmethylsulfonyl fluoride) buffer, and 

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

centrifuged for 60 minutes at 105,000 × g, discarding the resulting pellet. Protein 

concentration of the cytosol was determined to be 3.35 mg/mL using a DC protein assay kit 

(BioRad). The cytosol was divided into aliquots (volume not reported) for storage at −74 ± 

10o C until use.

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

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

study (SA 11020) for the cytosol used in the subject competitive binding experiment to 

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

affinity for the native ligand. The details and results of the saturation binding experiment 

were included as an attachment to the subject study. The conditions for the saturation 

binding experiment 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) 0.03-3.0 nM 

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

Concentration of receptor Sufficient to bind 25-35% of radioligand 

Temperature Refrigerated (4oC)

Incubation time 16-20 hours

Composition of assay buffer Tris 10 mM (Tris/HCl) 

EDTA 1.5 mM 

Glycerol 10% (v/v) 

Phenylmethylsulfonyl fluoride 1 mM 

DTT 1 mM 

a Data were obtained from pages 73-75 of the study report. 

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

adjusted for decay over time, 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, and 3 nM. Solutions of 

non-labeled 17β-estradiol were prepared in a similar manner to achieve concentrations that 

were 100-fold greater than each respective radiolabeled concentration to result in final 

concentrations of 3, 6, 8, 10, 30, 60, 100, and 300 nM. The optimal protein concentration 

for the cytosol batch was determined by testing serial amounts of protein per tube, using 

0.03 nM radiolabeled estradiol, until a concentration was reached that bound 25-35% of the 

total radioactivity added. Each assay consisted of three non-concurrent runs, and each run 

contained three concurrent replicates at each concentration (Table 2). 

TABLE 2. Saturation Binding Experiment Run a 

Total binding b Non-specific 

binding c Radioligand alone d

 Assay Components 

350 μL 300 μL --- TEDG + PMSF buffer 

50 μL 50 μL 50 μL [3H]-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 conc.) 

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

a Data were obtained from pages 119-126 of the study report. Tube numbers corresponding to assay 

conditions were not provided. 

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 16-20 hours at 4 C. To separate bound 

from free estradiol, hydroxyapatite (HAP) slurry was added to each tube and incubated in a 

water bath for 15 minutes, then centrifuged for 10 min at 1000 × g. The remaining pellet 

was washed three times with TEDG + PMSF buffer. Ethanol was added to the HAP pellet 

remaining in each tube to extract the [3

H]-17β-estradiol. An aliquot of supernatant was 

radioassayed by scintillation counting. The temperature was maintained at 4o C throughout 

the assay prior to extraction with ethanol. 

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

competitive binding experiment is included in Table 3. 

TABLE 3. Summary of Conditions for Competitive Binding Experiment a

Source of receptor Rat Uterine Cytosol 

Concentration of radioligand 1 nM 

Concentration of receptor Sufficient to bind ≤10-15% of radioligand

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

Temperature 4±3 C 

Incubation time 16-20 hours 

Composition of assay buffer Tris 10 mM 

EDTA 1.5 mM 

Glycerol 10% (v/v) 

Phenylmethylsulfonyl fluoride 1 mM 

DTT 1 mM 

a Data were obtained from pages 12, 15 and 18 of the study report. 

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

adjusted for decay over time, and diluted in TEDG+PMSF buffer to achieve the final 

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

determined by testing serial amounts of protein per tube, using 1.0 nM radiolabeled 

estradiol, until a concentration was reached that bound 10-15% of the total radioactivity 

added (125 μg protein per tube). Serial dilutions of metribuzin, the weak positive control 

(19-norethindrone), the negative control (octyltriethoxysilane), and the natural ligand (nonlabeled 17β-estradiol) were prepared to achieve the concentrations shown in Table 4. The 

test substance in DMSO was visually examined for precipitation. Each assay consisted of 

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

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

control) resulting in 105 samples/assay for a total of 315 samples. 

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TABLE 4. Molar (M) concentrations in Competitive Binding Assay Run a, b

Metribuzin 

Weak Positive control Negative control Reference Chemical 

19-Norethindrone Octyltriethoxysilane Non-labeled 

17β-estradiol 

Tubes 76-99 c Tubes 28-51 c Tubes 52-75 c Tubes 1-27 and 100-105 c

10−10 10−8.5 10−10 Solvent control d

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 10−4 10−3 10−7

a Data were obtained from pages 12-13 and 37-47 of the study report. 

b Each tube contains: 10μL of the test substance, positive control, negative control, solvent control, or nonlabeled 17β-estradiol; 390 μL of TEDG + PMSF buffer with [3

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

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

c Each concentration of each chemical was run in triplicate, for a total of 105 tubes per run. Tube numbers 

were not provided, but were assigned by the reviewer based on data spreadsheets on pages 37-47. 

d Solvent was DMSO (2%) in Tubes 1-3 and 100-102. 

The procedures for assay incubation, separation of bound from free estradiol (washing with 

buffer and extraction with ethanol), followed by scintillation counting of bound [3H]-17βestradiol were similar to those used in the saturation binding experiment. 

C. DATA ANALYSIS: For both the competitive binding and saturation binding assays, 

GraphPad Prism was used to generate nonlinear regression used to fit a curve (for 17βestradiol, 19-norethindrone, and metribuzin) to the Hill equation formula which 

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

saturation binding experiment (Kd and Bmax) and competitive binding experiment (log IC50

and RBA), the means were calculated for each run. 

1. Definitions 

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

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

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

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

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

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

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

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

concentration groups in the data is above 75%. 

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

test chemical concentration of 10−6

M and either a binding curve can be fit but 

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

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

concentration groups in the data is >50%. 

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

above. 

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

Run Classification Numerical Value 

Interactive 2 

Equivocal 1 

Not interactive 0 

Equivocal up to the limit of concentrations tested “missing” 

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

following ranges: 

Test Material Classification Numerical Range 

Interactive average ≥1.5 

Equivocal 0.5≥ average <1.5 

Not interactive average <0.5 

Equivocal up to the limit of concentrations tested “missing” 

b. Descriptors for receptor binding: 

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

concentration of active receptor sites

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

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

the receptor

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

II. RESULTS 

A. SATURATION BINDING EXPERIMENT: The appropriate protein concentration for the 

saturation binding assay was determined to be 0.5 mg/mL as it bound 25-35% of the [3H]-

17β-estradiol. The results from the three saturation binding assays are summarized in Table 

5 and shown graphically in Figures 1-9. The mean Kd for [3

H]-17β-estradiol was 0.070 nM, 

and the mean estimated Bmax was 34 fmol/100 ug protein for the prepared rat uterine cytosol. 

The Kd for each run was within the expected range of 0.03 to 1.5 nM. The data produced 

consistent and linear Scatchard plots, although the goodness of fit (r2

) was not reported. 

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TABLE 5. Saturation Binding Experiment of 17β-estradiol with Estrogen Receptor from Rat Uterine 

Cytosol a

Parameter Run 1 Run 2 Run 3 Mean ± SE b

r

2 (unweighted) NR NR NR NA 

Bmax (nM) 0.027 0.037 0.039 0.034 ± 0.004 

Bmax (fmol/100 μg protein) 27 37 39 34.3 ± 3.71 

Kd (nM) 0.089 0.073 0.049 0.070 ± 0.011 

a Data were obtained from page 77 of the study report. 

b Calculated by reviewers (Mean ± SE) 

r

2

 Goodness of fit 

NR Not reported 

NA Not applicable 

FIGURE 1. Total and Non-specific binding Run 1. 

FIGURE 2. Specific binding Run 1. 

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FIGURE 3. Scatchard plot for Specific binding Run 1. 

FIGURE 4. Total and Non-specific binding Run 2. 

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FIGURE 5. Specific binding Run 2. 

FIGURE 6. Scatchard plot for Specific binding Run 2. 

Page 45 of 205
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FIGURE 7. Total and Non-specific binding Run 3. 

FIGURE 8. Specific binding Run 3. 

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FIGURE 9. Scatchard plot for Specific binding Run 3. 

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

binding experiments are summarized in Table 6 and presented graphically in Figures 10-13. 

Precipitation was not observed at any concentration of metribuzin. In all three assay runs, 

the data for metribuzin did not fit the non-linear regression model and percent radiolabeled 

estradiol binding was >75% (i.e. not interactive) at all concentrations tested (≤10−3 M); 

therefore, log IC50 and RBA values could not be calculated for metribuzin. As the lowest 

average percent binding in the presence of metribuzin was >75% at ≤10−3

 M, metribuzin is 

classified as not interactive (0) in this assay (Table 7). It was noted that the third run was 

discarded (and repeated) due to a technical problem with the rotator. 

The estimated mean log IC50 for 17β-estradiol and 19-norethindrone was −8.8 M and −5.0 M, 

respectively. Compared to the natural ligand, the mean RBA was 0.017% for 

19-norethindrone. Confidence in these numbers is high due to the small variation. 

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TABLE 6. Competitive Binding Assay of Metribuzin with Estrogen Receptor from Rat Uterine Cytosol a

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

r

2

 (unweighted) 17β-estradiol NR NR NR NA 

19-norethindrone NR NR NR NA 

Metribuzin NR NR NR NA 

Log IC50 (M) 17β-estradiol −8.8 −8.8 −8.8 −8.8 ± 0.0 

19-norethindrone −5.0 −5.0 −5.0 −5.0 ± 0.0 

Metribuzin NA NA NA NA 

IC50 (M) 17β-estradiol 1.7 × 10−9 1.7 × 10−9 1.7 × 10−9 1.7 × 10−9 (±0.0)

19-norethindrone 0.9 × 10−5 1.1 × 10−5 1.0 × 10−5 1.0 × 10−5 (±0.1) 

Metribuzin NA NA NA NA 

Log (RBA) c 19-norethindrone −1.7 −1.8 −1.8 −1.77 ± 0.03 

Metribuzin NA NA NA NA 

RBA (%) 19-norethindrone 0.019 0.015 0.017 0.017 ± 0.001 

Metribuzin NA NA NA NA 

a Data were obtained from page 18 of the study report. 

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

c Calculated by reviewers; 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 

SE Standard Error 

RBA Relative binding affinity

NR Not reported 

NA Not applicable 

TABLE 7. Binding Classification of Metribuzin with Estrogen Receptor a

Run 1 2 3 Mean Binding Classification c

Classification category value b 0 0 0 0 Not Interactive 

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

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

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

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

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FIGURE 10. Percentage [3H]-E2 Bound to the Estrogen Receptor in the Presence of 

Metribuzin, Unlabeled E2, 19-Norethindrone or Octyltriethoxysilane. Run 1 

 

FIGURE 11. Percentage [3H]-E2 Bound to the Estrogen Receptor in the Presence of 

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

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FIGURE 12. Percentage [3H]-E2 Bound to the Estrogen Receptor in the Presence of 

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

FIGURE 13. Mean Percentage [3H]-E2 Bound to the Estrogen Receptor in the Presence of 

Metribuzin in Three Competitive Assays.

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

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

TABLE 8. Criterion a Tolerance Limit(s) Value Yes No 

17β-estradiol fitted curve parameters 

Loge residual SD ≤2.35 NR 

Top (% binding) 94 to 111 93 to 104 b X 

Bottom (% binding) −4 to 1 2.1 to 2.5 X 

Hill Slope (Log10(M)-1) −1.1 to −0.7 −2.0 X 

Weak Positive control (19-norethindrone) fitted curve parameters c

Loge residual SD NA NR 

Top (% binding) NA 105 to 112 X 

Bottom (% binding) NA −13.6 to −8.5 X 

Hill Slope (Log10(M)-1) NA −0.8 to −0.6 X 

Solvent concentration

DMSO ≤10% ≤2% X 

Negative control (octyltriethoxysilane) does not displace 

more than 25% of [3

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

across all concentrations 

≤25% 7.5 to 16% X 

a Data were obtained from pages 18, 38, 42 and 46 of the study report. 

b The top % binding was low in Run #2 (93%) but acceptable in the other two runs. 

c 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.

NA Not applicable 

The Hill slope was outside the recommended range, at −2.0, indicating that the receptors 

were behaving as though there was more than one binding site for 17β-estradiol. The 

bottom % binding for 17β-estradiol (2.1 to 2.5%) was also outside the recommended range 

in all three runs, and the top % binding (93%) in one of the runs was outside the 

recommended range. However, responses for the weak positive control (19-norethindrone)

and negative control (octyltriethoxysilane) were as expected. 

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

The maximum percent binding 109-122% was within approximately 25 percentage points of 

the lowest concentration of the estradiol standard (91.5-107%). Data for metribuzin did not 

fit the non-linear regression model. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: A total of three independent competitive binding 

experiments were conducted. The amount of cytosolic protein used in each competitive 

binding experiment was 125 μg/assay tube. The IC50 value for 17β-estradiol was 

determined to be 1.7×10−9

 M in each run. The IC50 values for 19-norethindrone were 

determined to be 0.9×10−5

 to 1.1×10−5 M. The RBA values for 19-norethindrone, in 

comparison to 17β-estradiol, were 0.015-0.019%. Analyses of the data generated for each 

evaluation for metribuzin in the ER binding assay indicated that they did not fit the 

Page 51 of 205
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 18 of 18 

METRIBUZIN/ 101101 OCSPP 890.1250/ OECD None

4-parameter regression model used for data interpretation. The lowest average binding 

value was greater than 75%. Consequently, metribuzin was considered a non-binder with 

ER.

B. AGENCY COMMENTS: For the saturation binding experiment, the appropriate protein 

concentration for the saturation binding assay was determined to be 0.5 mg/mL (50 ug/tube) 

as it bound 25-35% of the [3H]-17β-estradiol. The mean Kd for [3H]-17β-estradiol was 

0.070 nM, and the mean estimated Bmax was 34 fmol/100 μg protein for the prepared rat 

uterine cytosol. The Kd for each run was within the expected range of 0.03 to 1.5 nM. The 

data produced consistent and linear Scatchard plots, although the goodness of fit (r2) was not 

reported. 

For the competitive binding experiment, the data for metribuzin did not fit the non-linear 

regression model and percent radiolabeled ligand binding to the ER was >75% at all 

concentrations tested (≤10−3 M) in all three runs. Precipitation was not observed at any 

concentration. The estimated mean log IC50 and RBA was not calculated for metribuzin as 

the percent binding did not reach 50% for any run. 

The estimated mean log IC50 for 17β-estradiol and 19-norethindrone was −8.8 M and 

−5.0 M, respectively. Compared to the natural ligand, the mean RBA was 0.017% for 

19-norethindrone. Confidence in these numbers is high due to the small variation. The 

following performance criteria for 17β-estradiol were not met: (i) the Hill slope was outside 

the recommended range, at −2.0, indicating that the receptors were behaving as though there 

was more than one binding site for 17β-estradiol; (ii) the bottom % binding of 2.1 to 2.5% 

was also outside the recommended range (−4 to 1) in all three runs; and (iii) the top % 

binding (93%) in one of the runs was outside the recommended range (94 to 111%). 

However, responses for the weak positive control (19-norethindrone) and negative control 

(octyltriethoxysilane) were as expected demonstrating the assay was adequate. 

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

The maximum percent binding 109-122% was within approximately 25 percentage points of 

the lowest concentration of the estradiol standard (91.5-107%). As the lowest average 

percent labeled estradiol binding to the ER in the presence of metribuzin was >75% at 

concentrations up to 10−3

 M, metribuzin is classified as not interactive (score:0) in this 

assay. 

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

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

 Several performance criteria were not met for 17β-estradiol. 

 The criterion of Loge residual SD for the competitive binding assay was not reported. 

Page 52 of 205
DATA EVALUATION RECORD 

METRIBUZIN 

Study Type: OCSPP 890.1300, Estrogen Receptor Transcriptional Activation 

EPA Contract No. EP10H001452 

Task Assignment No. 2-24-2012 (MRID 48617903) 

Prepared for 

Health Effects Division 

Office of Pesticide Programs 

U.S. Environmental Protection Agency 

2777 South Crystal Drive 

Arlington, VA 22202 

Prepared by 

CSS-Dynamac Corporation 

1910 Sedwick Road, 

Building 100, Suite B 

Durham, NC 27713 

Primary Reviewer Signature:

Michelle Sharpe-Kass, M.S. Date: 3/7/2012 

Secondary Reviewer Signature:

Michael E. Viana, Ph.D., D.A.B.T. Date: 3/19/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 3/29/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 3/29/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 53 of 205
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 54 of 205
Estrogen Receptor Transcriptional Activation (2011) I Page I of IO 

METRIBUZIN/101 IOI OCSPP 890.1300/ OECD 455 

Primary Reviewer: Ray Kent PhD 

Health Effects Division 

Secondary Reviewer: Minerva Mercado PhD DABT 

Health Effects Division 

Template version 08/201 t 

DATA EVALUATION RECORD 

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

OCSPP 890.1300; OECD 455. 

PCCODE: 101101 DP BARCODE: D398559 

TXR#: 0056258 CAS No.: 21087-64-9 

TEST MATERIAL (PURITY): Metribuzin (94.4% a.i.) 

SYNONYMS: 4-amino-6-(1, l-dimethylethyl)-3-(methylthio )-1,2,4-triazin-5( 4H)-one 

CITATION: Blanck, 0. (20 11) Metribuzin: Evaluation in the in vitro (HeLa-9903) estrogen 

receptor transcriptional activation assay. Bayer S.A.S, Sophia Antipolic Cedex, 

France. Laboratory Report No. : SA 11157, October 21, 2011. MRID 

48617903. Unpublished. 

SPONSOR: Bayer CropScience Ag, Alfred Nobel Str. 50, Manheim, Germany 

TEST ORDER#: CON-101101-11 

EXECUTIVE SUMMARY: In an estrogen receptor transcriptional activation assay (MRJD 

48617903), hERa-HeLa-9903 cells cultured in vitro were exposed to metribuzin (94.4% a.i. 

Batch # K285067) at logarithmically increasing concentrations from 10- 10 M to 10-4 Min DMSO 

(0.1 %) for 24 hours, in two independent runs conducted on separate days. The experiments were 

performed using 96-well plates and each metribuzin concentration was tested in triplicate (3 

wells/plate). Cells were exposed to the test agent for 23± 1 h to induce reporter (luciferase) gene 

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

was measured upon addition of a luciferase substrate and detection with a luminometer with 

acceptable sensitivity. 

The laboratory proficiency assays using the required reference compounds demonstrated that the 

assay was functioning properly. The only deviation was a weak positive response in one run to 

corticosterone (negative control compound), otherwise the performance criteria were met for 

17~-estradiol and corticosterone. For l 7a-estradiol (weak agonist), the Log PC10 was lower than 

the expected range in the first assay, as was the Hill Slope. For l 7a-methyltestosterone (very 

weak agonist), the Log PCso was lower than the expected range in run 2, and the Log PC1 0 was 

below the excepted range in both runs. These results suggest increased sensitivity of the assay. 

Metribuzin was tested up to the limit of solubility, 10-4 M. The mean RPCMax for metribuzin 

was 0. 7% for the first run and 14.1 % for the second run, and the associated PCMax was 10- 4 M 

Page 55 of 205
Estrogen Receptor Transcriptional Activation (2011) / Page 2 of 10 

METRIBUZIN/101101 OCSPP 890.1300/ OECD 455 

for the first run and 10−6 M for the second run. Although the RPCMax ≥PC10 in run 2 (14.1%), 

this high value was due to only one of the three samples for that run, and the response was not 

dose-dependent. Therefore, methomyl was considered negative for estrogen receptor 

transcriptional activation in this test system. 

This assay satisfies the EDSP Tier 1 Test Order requirement for an Estrogen Receptor 

Transcriptional Activation assay (OCSPP 890.1300). 

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

statements were provided. 

 

Page 56 of 205
Estrogen Receptor Transcriptional Activation (2011) / Page 3 of 10 

METRIBUZIN/101101 OCSPP 890.1300/ OECD 455 

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: Metribuzin 

 Description: Light grey powder

 Source (Catalog #): Bayer CropScience Ag, Germany (not provided) 

 Lot/Batch # (Exp date): K285067 (May 7, 2012) 

 Purity: 94.4% 

 Solubility: NR 

 Volatility: NR 

 Stability: 2 years 

 Storage conditions: 25±5°C 

 CAS #: 21087-64-9 

 Structure: 

N

N

N

O

NH2

S

CH3

3CH

3CH

CH3

2. Reference substances 

 17β-estradiol (strong estrogen; positive control)

 Supplier: Sigma-Aldrich, St. Louis, MO 

 Catalogue and Batch #: 010M0142 

 Purity: 100% 

 CAS # : 50-28-2 

 

 17α-estradiol (weak estrogen) 

 Supplier: Sigma-Aldrich, St. Louis, MO 

 Catalogue and Batch #: 029K4124 

 Purity: ≥99.5% 

 CAS # : 57-91-0 

 

 Corticosterone (negative compound) 

 Supplier: Sigma-Aldrich, St. Louis, MO 

 Catalogue and Batch #: BCBB5955 

 Purity: 99.6% 

 CAS # : 50-22-6 

 

 17α-methyltestosterone (very weak agonist) 

 Supplier: Sigma-Aldrich, St. Louis, MO 

 Catalogue and Batch #: 039K0268 

 Purity: 100% 

 CAS # : 58-18-4 

3. Vehicle(s) 

Solvent: DMSO (Sigma Aldrich, Batch # 76396EM) 

Solvent control 

(final concentration): 

0.1% 

Page 57 of 205
Estrogen Receptor Transcriptional Activation (2011) / Page 4 of 10 

METRIBUZIN/101101 OCSPP 890.1300/ OECD 455 

B. METHODS

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

Collection of Research Bioresources Cell Bank and were verified by the supplier to be free 

of mycoplasma infection using PCR and DNA staining. Cells were maintained in Eagles 

Minimum Essential Medium (EMEM) without phenol red, supplemented with kanamycin 

(60 mg/L) and 10% dextran-coated charcoal-treated fetal bovine serum (DCC-FBS; Thermo 

Scientific, Batch ATF 32351), in an incubator under 5% CO2 at 37o C. Upon reaching 75-

90% confluence, cells were subcultured 6 times prior to exposure to the test material. 

2. Transcriptional Activation Assays: For each test, cells were plated at a density of 

1104 cells/100 μL medium/well in a 96-well plate and allowed to attach for 3 hours. The 

growth media was replaced with media containing serial log dilutions of metribuzin in 

DMSO. Cells were incubated for 22-24 hours at 37±1° C. The total final concentration of 

DMSO was 0.1%. Cytotoxicity was determined using a commercial kit (ATPlite 1Step, 

Perkin Elmer, Waltham, MA). 

Transcriptional activation of the estrogen receptor was determined using a commercial kit 

neolite Reporter Gene Assay System, Perkin Elmer, Waltham, MA). Luminescence was 

measured using a Wallac 11450 MicroBeta Windows Workstation, V 4.01.014 (Perkin 

Elmer, Waltham, MA), with a detection limit of 32 fg luciferase/well. 

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

10−6 to 10−4 M was conducted to determine the appropriate concentration range and to 

determine concentrations resulting in insolubility and/or cytotoxicity. 

b. Proficiency Chemicals: The responsiveness of the test system was confirmed for each 

newly prepared batch of cell stocks taken from the frozen stock by testing the following set 

of proficiency chemicals. The details of the proficiency test were not provided, only the 

results.

Compound CAS No. Concentration

Range (M) 

Expected 

Response a Notes 

Diethylstilbestrol (DES) 56-53-1 10−14 to 10−8 Positive --- 

17α-Ethynyl estradiol (EE) 57-63-6 10−14 to 10−8 Positive --- 

Hexestrol 84-16-2 10−13 to 10−7 Positive --- 

Genistein 446-72-0 10−12 to 10−5 Positive Cytotoxic at 0.01b, 0.1, and 1 mM 

Estrone 53-16-7 10−12 to 10−6 Positive --- 

Butyl paraben 94-26-8 10−11 to 10−4 Positive Cytotoxic at 0.1b and 1 mM 

1,3,5-Tris(4-

hydroxyphenyl)benzene c

15797-52-1 10−12 to 10−5

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

Binds hERα and has ER antagonistic activity 

Dibutyl phthalate (DBP) 84-74-2 10−11 to 10−4 Negative d Cytotoxic at 1 mM 

Atrazine 1912-24-9 10−11 to 10−4 Negative Cytotoxic at 1 mM b

Corticosterone 50-22-6 10−10 to 10−4 Negative If not cytotoxic at 1 mM, then that should be 

the highest tested concentration 

a Positive = RPCMax ≥10% of the response of the positive control in at least 2 of 2 (or 2 of 3) runs 

 Negative = RPCMax fails to achieve at least 10% of the response of the positive control in 2 of 2 (or 2 of 3) runs 

b Cytotoxicity is expected to be close to 80% at this concentration. 

c Compound selected to challenge solubility and cytotoxicity 

d DBP is negative for ERα mediated transcriptional activation, but may not be negative for non-ERβ mediated transcriptional 

activation. A positive result would indicate that the system is detecting activity other than that due to pure ERα, and is 

therefore unacceptable. 

Page 58 of 205
Estrogen Receptor Transcriptional Activation (2011) / Page 5 of 10 

METRIBUZIN/101101 OCSPP 890.1300/ OECD 455 

c. Reference Chemicals: To ensure the stability of the response from the cell line, six 

concentrations of each of the following reference chemicals were included in each plate in 

the current assay, along with the test chemical: 

Reference Chemical CAS No. Concentration Range Class 

17β-estradiol 50-28-2 10−14 to 10−8 Strong estrogen 

17α-estradiol 57-91-0 10−12 to 10−6 Weak estrogen 

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

17α-methyltestosterone 58-18-4 10−11 to 10−5 Very weak agonist 

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

(PC), the luminescence signals from the concurrent plate were analyzed by subtracting the 

mean value of the vehicle control from each well value to normalize the data; each 

normalized value was then divided by the mean value of the normalized PC. The resulting 

value was multiplied by 100 in order to express relative transcriptional activity as a 

percentage of the PC. Graph Pad Prism v. 5.04 (GraphPad Software, Inc., La Jolla, CA) was 

used to calculate the EC50, PC10, PC50, RPCMax, and PCMax for metribuzin when applicable. 

The test material was defined as negative for inducing estrogen receptor transcriptional 

activation if the RPCMax < PC10 in at least 2 of 2 (or 2 of 3) runs. Log EC50 and Hill slope 

values are calculated only if a positive response is observed. Coefficients of variation (CV) 

were calculated for the luminescence data triplicates. Concentrations showing >20% 

cytotoxicity or evidence of insolubility were excluded from analyses. 

4. Definitions 

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

(bottom) and maximum (top) response 

PC10 = concentration of a test chemical at which the response is 10% of the response 

induced by the positive control (E2 at 1 nM) in each plate 

PC50 = concentration of a test chemical at which the response is 50% of the response 

induced by the positive control (E2 at 1 nM) in each plate 

RPCMax = maximum level of response induced by a test chemical, expressed as a percentage 

of the response induced by the positive control (1 nM E2) on the same plate 

PCMax = concentration of a test chemical inducing the RPCmax

II. RESULTS 

A. PRELIMINARY TEST: In order to identify a suitable top concentration for use in the 

transcriptional activation assays, preliminary cytotoxicity and precipitation assays were 

conducted. Due to the presence of cloudiness at the concentration of 1 mM metribuzin in 

DMSO, the definite runs were performed for metributzin at log serial dilutions starting at 

10−4 M in culture medium. The results of the cytotoxicity test are presented in Table 1. No 

cytotoxicity or precipitation was observed at concentration ≤10-4 M. Based on these results, 

logarithmically increasing concentrations from 10−10 to 10−4

 M were selected for the assay. 

Page 59 of 205
Estrogen Receptor Transcriptional Activation (2011) / Page 6 of 10 

METRIBUZIN/101101 OCSPP 890.1300/ OECD 455 

TABLE 1. Preliminary Test for Solubility, Cytotoxicity, and Concentration-Selection for metribuzin

Concentration (M) % Viability b Commentsc

10−4 123.1 

10−5 115.3 

10−6 112.2 

SDS 1% 0 Positive control for cytotoxicity 

VC b 100 

a Data were obtained from page 17 of the study report. 

b VC = Vehicle control 

B. Positive and Negative Reference Chemicals 

1. Proficiency Chemicals: The responsiveness of cells to the required proficiency chemicals 

was performed in duplicate on different days. The data are reported in Table 2. The 

responses demonstrated proficiency of the laboratory. The only minor deviation was in the 

first run, corticosterone produced an RPCMax of 16%, indicating a weakly positive response. 

In the second run, the RPCMax was −14%, indicating a negative response.

TABLE 2. Proficiency Chemicals a

Compound Expected Response Lab Response 

Diethylstilbestrol Positive Positive 

17α-Ethynyl estradiol Positive Positive 

Hexestrol Positive Positive 

Genistein Positive Positive 

Estrone Positive Positive 

Butyl paraben Positive Positive 

1, 3, 5-Tris(4-hydroxyphenyl)benzene Positive Positive 

Dibutyl phthalate Negative Negative 

Atrazine Negative Negative 

Corticosterone Negative Positive/Negative 

a Data were obtained from page 36 of the study report.

2. Reference Chemicals: Values derived from the concentration response curve (e.g., log

PC50, log PC10, log EC50, Hill slope) for the four concurrently run reference materials are 

included in Table 3. Performance criteria were met for 17β-estradiol and corticosterone. 

The log PC10 for 17α-estradiol was lower than the acceptable range in the first assay, as was 

the Hill Slope. For methyltestosterone, the log PC50 was lower than the acceptable range for 

run 2, and the log PC10 was lower than the acceptable range for both runs. These results 

indicate an increased sensitivity of the assay to weak agonists. 

Page 60 of 205
Estrogen Receptor Transcriptional Activation (2011) / Page 7 of 10 

METRIBUZIN/101101 OCSPP 890.1300/ OECD 455 

TABLE 3. Performance Criteria for Reference Chemicals

Reference Chemical 

Parameter Acceptable Range Values Acceptable 

Run 1 Run 2 Yes No 

17β-estradiola

 Log PC50 −11.4 to −10.1 −10.5 −10.7 X 

 Log PC10 <−11 −11.7 −11.4 X 

 Log EC50 −11.3 to −10.1 −10.5 −10.7 X 

 Hill Slope 0.7 to 1.5 0.77 1.27 X 

 Test range 10−14 to 10−8 M 10−14 to 10−8 M 10−14 to 10−8 M X 

17α-estradiola

 Log PC50 −9.6 to −8.1 −9.1 −8.9 X 

 Log PC10 −10.7 to −9.3 −11.9 −10.1 X 

 Log EC50 −9.6 to −8.4 −9.1 −8.9 X 

 Hill Slope 0.9 to 2.0 0.35 0.83 X 

 Test range 10−12 to 10−6 M 10−12 to 10−6 M 10−12 to 10−6 M X 

Corticosteronea

 Test range 10−10 to 10−4 M 10−10 to 10−4 M 10−10 to 10−4 M X 

17α-methyltestosteroneb

 Log PC50 −6.0 to −5.1 −5.83 −6.08 X 

 Log PC10 −8.0 to −6.2 −9.66 −8.33 X 

 Test range 10−11 to 10−5 M 10−11 to 10−5 M 10−11 to 10−5 M X 

a Data were obtained from pages 15 and 18 of the study report. 

b Data were calculated by the reviewer, based on data from pages 42 and 55 of the study report 

C. DEFINITIVE ASSAY 

1. Vehicle and Positive Controls: Data for the vehicle and positive controls are included in 

Table 4. The mean TA, expressed as arbitrary light units for the vehicle control was 444-

586 for the first run and 453-679 for the second run. The mean normalized value for the 

positive control was 1554-1796 for the first run and 1245-1601 for the second run. The 

PC50 (50% of the maximum response) for E2 in this assay is 777-898 for the first assay, 

623-801 for the second assay, and the PC10 (10% of the maximum response) is 155-180 for 

the first assay and 125-160 for the second assay. 

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

Sample Vehicle Control Positive Control b 

Runs Mean SD Mean SD Fold Induction c

1 444-586 36-69 1554-1796 NR 4.1-4.5 

2 453-679 30-62 1245-1601 NR 2.8-4.5 

a Data were obtained from page 38 of the study report. 

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

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

2. Test Material: Relative (to the PC) transcriptional activation at each concentration of the 

metribuzin during the two assay runs is presented in Table 5. The concentration-response 

curves depicting fold induction of relative transcriptional activation is presented in Figure 1 

and Figure 2 below. The mean RPCMax was 0.67% for the first run and 14.07% for the 

second run, and the associated PCMax was 10−4

 M for the first run and 10−6 M for the second 

run. The RPCMax ≥PC10 in Run 2, however, this happened only in one of the three samples 

for that run, and it was not at the highest concentration. Therefore, metribuzin is still 

considered negative for estrogen receptor transcriptional activation in this test system. 

Page 61 of 205
Estrogen Receptor Transcriptional Activation (2011) / Page 8 of 10 

METRIBUZIN/101101 OCSPP 890.1300/ OECD 455 

TABLE 5. Relative Transcriptional Activation (RTA) of Metribuzina

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

Run 1 Run 2 

Conc. (M) Mean SD Mean SD 

10−4 0.67 5.65 0.27 5.91 

10−5 −7.03 4.58 −7.20 6.33 

10−6 −6.27 3.45 14.07 31.60 

10−7 −6.53 3.75 −7.63 3.23 

10−8 −7.53 3.68 −11.13 3.59 

10−9 −6.93 2.84 −12.23 1.01 

10−10 −8.50 3.68 −9.87 5.83 

Log EC50 NA NA 

Hill Slope NA NA 

RPCmax 0.67 14.07 

PCmax 10−4 10−6

PC50 NA NA 

PC10 NA NA 

a Data were calculated by the reviewers from obtained on pages 52 and 65 of the study report.

NA = Not Applicable

FIGURE 1. Fold Induction of Relative Transcription Activation (RTA) of Metribuzin 

(Run 1) 

Page 62 of 205
Estrogen Receptor Transcriptional Activation (2011) / Page 9 of 10 

METRIBUZIN/101101 OCSPP 890.1300/ OECD 455 

FIGURE 2. Fold Induction of Relative Transcription Activation (RTA) of Metribuzin 

(Run 2) 

3. Performance Criteria: The laboratory proficiency assays using the required reference 

compounds adequately demonstrated that the assay was functioning properly. The only 

deviation was a weak positive response in one run in response to corticosterone. 

Performance criteria were met for 17β-estradiol and corticosterone. The log PC10 for 

17α-estradiol was lower than the acceptable range in the first assay, as was the Hill Slope. 

For 17α-methyltestosterone, the mean RPCMax was 79.0% for the first run and 70.9% for 

the second run. For methyltestosterone, the logPC50 was lower than the acceptable range 

for run 2, and the logPC10 was lower than the acceptable range for both runs. The results 

show an increased sensitivity to weak agonists. Mean luciferase activity was >4-fold for 

both plates in Run 1, and one plate in Run 2, but was only 2.8-fold for the second plate in 

Run 2. The fold-induction corresponding to the PC10 of the concurrent PC was greater 

than 1+2 standard deviations of the vehicle control on all plates, except for the plate with 

only 2.8-fold inductions, where those values were equal. Variability was minimal and 

results were reproducible among runs. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: The two independent runs gave similar results 

and the reference compounds gave the expected results. The maximum response for 

metribuzin was less than 10% of the positive control 17β-estradiol in both runs. Therefore 

metribuzin was negative in this human estrogen receptor transcriptional activation assay. 

B. AGENCY COMMENTS: The laboratory proficiency tests using the required reference 

compounds demonstrated that the assay was functioning properly. The only deviation was a 

weak positive response in one run in response to corticosterone. Performance criteria were 

met for 17β-estradiol and corticosterone. For 17α-estradiol, the Log PC10 was lower than 

the acceptable range in the first assay, as was the Hill Slope. In addition, for 17αPage 63 of 205
Estrogen Receptor Transcriptional Activation (2011) / Page 10 of 10 

METRIBUZIN/101101 OCSPP 890.1300/ OECD 455 

methyltestosterone, the Log PC50 was lower than the acceptable range in Run 2, and the Log 

PC10 was lower than the acceptable range in both runs. However, these results indicate that 

the assay had an increased sensitivity to weak agonists. 

 Metribuzin was tested up to the limit of solubility, 10−4

M. The mean RPCMax was 0.67% for 

the first run and 14.07% for the second run, and the associated PCMax was 10−4

 M for the 

first run and 10−6 M for the second run. Although the RPCMax ≥PC10 in Run 2 (14.1%), this 

high value was due to only one of the three samples for that run, and the response was not 

dose-dependent. Therefore, metribuzin is considered negative for estrogen receptor 

transcriptional activation in this test system 

C. STUDY DEFICIENCIES: No major deficiencies were noted.

Page 64 of 205
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin 

EPA MRID Number 48672202 

Page 1 of 70 

Data Requirement: EPA DP Barcode 401218 

OECD Data Point 229 

EPA MRID 48672202 

EPA Guideline 890.1350, Fish Short-Term Reproduction Assay 

Test material: Metribuzin Purity: 94.4% 

Common name Metribuzin 

Chemical name: IUPAC: 4-Amino-6-tert-butyl-3-(methylsulfanyl)-1,2,4-triazin-5(4H)-one 

 CAS name: 1,2,4-Triazin-5(4H)-one, 4-amino-6-(1,1-dimethylethyl)-3-(methylthio)- 

 (9CI) 

 CAS No.: 21087-64-9 

 Synonyms: 

 EPA PC Code: 101101 

Primary Reviewer: Joan Gaidos Signature: 

Senior Scientist, Cambridge Environmental, Inc. Date: 11/21/2012 

Secondary Reviewer: Teri S. Myers Signature: 

Senior Scientist, Cambridge Environmental, Inc Date: 11/26/2012 

Primary Reviewer: Michael Wagman Signature: 

USEPA/OCSPP/OPP/EFED/ERB6 Date:

Additional Reviewer: Amy Blankinship Signature:

USEPA/OCSPP/OPP/EFED/ERB3 Date:

Final Additional Reviewer: Robin Sternberg Signature:

USEPA/OCSPP/OPP/EFED/ERB1 Date: 06/05/2015

Date Evaluation Completed: 06/05/2015 

Wagman

, Michael

Digitally signed by Wagman, 

Michael 

DN: cn=Wagman, Michael, 

email=Wagman.Michael@ep

a.gov 

Date: 2015.06.11 13:44:54 

-04'00'

Digitally signed by ROBIN STERNBERG 

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

ou=USEPA, ou=Staff, cn=ROBIN 

STERNBERG, dnQualifier=0000039126 

Date: 2015.06.11 14:32:17 -04'00'

AMY 

BLANKINSHIP

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

EPA MRID Number 48672202 

Page 2 of 70 

CITATION: Braune, C.B. and E. Bruns (2012). Short-term Reproduction Assay with the Fathead Minnow 

(Pimephales promelas) Exposed to Metribuzin Technical Under Flow-Through Conditions. 

Performed by Bayer CropScience AG, Monheim, Germany, Report ID.: EBSEY003. Submitted by 

Bayer CropScience AG, Monheim, Germany. Completion date March 9, 2012. 

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

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

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

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

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

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

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

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

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

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

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

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

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EXECUTIVE SUMMARY:

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

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

group; ca. 6.5 months old) were exposed to metribuzin (94.4% purity) at nominal concentrations of 0 (negative 

control), 0.200, 1.00, and 5.00 mg a.i./L; mean-measured concentrations were <LOQ (<0.02; negative 

control), 0.199, 1.03, and 5.40 mg a.i./L. The test system was maintained at 25.2 to 26.0o

C and a pH of 

6.7 to 7.3. 

Overall survival was 100, 96.9, 96.9, and 90.6% in the negative control and low, mid, and high treatment 

groups, respectively; all mortalities in the treatment groups were female (one, one, and three female mortalities 

in the low, mid, and high treatment groups, respectively). There were no notable observations with regards to 

behavior or changes in appearance (specifically color/banding, ovipositor appearance, or size of dorsal nape 

pad) in the negative control or treatment groups. There were no significant differences (p>0.05) in male or 

female body weight or length compared to the negative control. 

In the negative control replicates, spawning occurred at least every 4 days and mean fecundity was 8.84 

eggs/female/day/replicate (range: 6.74-13). Fertilization success in the negative control averaged 96.9% 

(range: 95-98.5%). Fecundity was not significantly reduced (p>0.05) in any treatment group compared to the 

negative control. There was no significant difference (p>0.05) in fertilization success between the negative 

control and treatment groups. 

Male nuptial tubercle scores were not significantly different (p>0.05) in any treatment group compared to the 

negative control. None of the surviving females were found to have tubercles. For males and females, gonadosomatic index (GSI) in the treatment groups was not significantly different (p>0.05) from the negative control. 

Plasma vitellogenin (VTG) was not significantly different (p>0.05) in males or females. For males, it is noted 

that at the low test concentration, VTG was increased 195% compared to the control. However, this percent 

difference is due to one fish for which the VTG value was substantially greater than the control values and 

this increase does not reflect an overall increase in the fish exposed at this concentration. Sex steroids were 

not measured in this study. 

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In males, there was a higher incidence of advanced maturation (stage 3) at the high treatment level compared 

with the negative control and other treatment groups. The study author reported that while a relationship to 

exposure could not be excluded, the high treatment level did have increased female mortality, and therefore, 

the maturation could be related to overt toxicity or due to the lower density of females in the replicates. 

However, the reviewer did not discern a trend between the males with the advanced maturation and replicates 

that contained dead females. Overall, no substantial female histopathological results that could be correlated 

with exposure to the test substance were noted. 

The performance and validity criteria were met for this study. While the study met the control spawning 

performance criterion (at least 15 eggs/female/day/replicate or spawning occurring at least every 4 days), 

mean fecundity in the negative control was 8.84 eggs/female/day (range: 6.74-13.0); however, spawning 

was observed in all negative control replicates at least once every four days (each replicate should attain either 

15 eggs/female/day or spawn every four days). 

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

(OCSPP Guideline 890.1350). 

Results Synopsis

Test Organism age at test initiation: 6.5 months 

Mean body weight at test initiation: Male 4.69 g; Female 2.74 g 

Mean length at test initiation: Not reported 

Test Type: Flow-through 

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

Treatment 

(mg a.i./L) 

[meanmeasured]

Fecundity 

Fert. 

Success 

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

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

0.199 No No No No No No No No No No NA NA NA NA 

1.03 No No No No No No No No No No NA NA NA NA 

5.40 No No No No No No Yes No No No NA NA NA NA 

Abbreviations: E2 17β-estradiol. F Female. Fert. Fertilization. GSI Gonado-Somatic Index. Histo. Histopathology. M Male. NA Not applicable. T Testosterone. VTG Vitellogenin. 1 A “yes” indicates a significant difference based on comparison to the negative (clean water) control, unless otherwise specified. A solvent was not used in the conduct of 

this study. 

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

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

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I. MATERIALS AND METHODS

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

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

following deviations were noted: 

 

1. The study author’s indicated the transfer of four spawning groups from the pre-exposure tanks to the 

exposure tanks was not successful with one or two males showing reduced fitness. It was decided to 

exchange these spawning groups for the next best spawning groups which did not meet the minimum 

fecundity requirement; however, the study author indicated that regular spawning occurred at least 2 

times in the immediate 7 days prior to exposure.

2. The unionized ammonia, residual chlorine and total organic carbon 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. However, the study authors included information 

from an 8/30/2011 examination of the laboratory’s dilution water (ca. 5 weeks prior to study initiation) 

that indicated TOC < 2 mg/L and residual chlorine < 0.01 mg/L. This water sample was not analyzed 

for unionized ammonia, but ionized ammonia was < 0.03 mg/L. 

3. The age of fish at study initiation was slightly (ca. 2 weeks) older than the OCSPP 890.1350 guideline 

recommendation (4.5—6 months old). 

 These deviations do not have an impact on 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 

were conducted following standard validated methods, this exception has no 

impact on the study results. 

A. TEST MATERIAL: Metribuzin; CAS# 21087-64-9.

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Description: Light grey powder

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. : K285067 

Purity: 94.4% 

Impurities: None reported

Stability of Compound: Analytical verification of the test material from Days 0, 7, 14, and 21 yielded 

recoveries of 100% to 108% of nominal concentrations. Stock solutions 

fortified with metribuzin at concentrations of 575 mg a.i./L and sampled 

immediately and after 19 days yielded recoveries of 102 to 105%. 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. 

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B. Test organism: 

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

Species common name: Fathead Minnow EPA recommends fathead minnow (Pimephales 

promelas). Species scientific name: Pimephales promelas

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

Yes EPA recommends that fish be from a single 

laboratory stock.

Were acclimation conditions same as definitive test?

Yes EPA recommends that fish be acclimated under 

water quality and illumination conditions that are 

similar to the definitive test. 

Acclimation period: 14 days EPA recommends a minimum two-week 

acclimation period. Note that the acclimation 

period is different from the subsequent, in situ 

pre-exposure phase. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on health: No mortalities were 

observed

No mortalities during the 14 days prior to pre-exposure period were observed. Fish did not receive any treatment for disease during acclimation period.Behavioral abnormalities or clinical signs were not reported.

EPA recommends that mortality during the 7 

days prior to the pre-exposure phase be less 

than 5% of the culture population. If mortality 

during these 7 days is greater than 10%, EPA 

recommends that the fish be rejected. If 

mortality is between 5-10%, EPA recommends 

that fish be held another 7 days. If mortalities 

greater than 5% occur during this extended 

acclimation period, EPA recommends that the 

fish not be used. 

Type of food: Live brine shrimp 

(Artemia salina)

EPA recommends that fish be fed frozen brine 

shrimp twice per day to promote active 

reproduction and maintain body condition. Source of food: Not reported Representative samples of the food source 

were periodically analyzed for pesticides, PCBs, and toxic metals by Eurofins GeneScan GmbH. 

Frequency of feeding: 2-3 times/day Details on feeding: None 

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Table 3: Fish Selection and Pre-Exposure Performance.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Age at test initiation: 6.5 months EPA recommends reproductively mature 

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

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

4.69 g Based on 15 males used to stock aquaria 

for pre-exposure phase. 

EPA recommends that a subsample of fish be 

weighed before the test to estimate the mean 

weight for each sex. It is recommended that the 

individual weight of each fish selected for the 

test be within ±20% of the estimated mean for 

each sex.

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

3.84 to 5.59 Individual weights within ± 20% of the 

estimated mean.

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

2.74 g Based on 15 females used to stock aquaria 

for pre-exposure phase. 

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

2.19 to 3.24 Individual weights within ±20% of the 

estimated mean.

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

Not reported 

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

Not reported 

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Were pre-exposure conditions identical to the definitive test?

Yes EPA recommends that pre-exposure conditions, 

including temperature, photoperiod, feeding, etc., 

be identical to definitive test conditions. 

Number of pre-exposure tanks: 32 EPA recommends that additional tanks set up at 

the beginning of pre-exposure will ensure that 

sufficient replicates with the correct sex ratio are 

available for the definitive test. 

Number of males per tank: 2 Number of females per tank: 4 Pre-exposure fecundity: >10.2-27.4 

eggs/female/reproductive day/replicate

Three replicates did not meet the minimum pre-exposure fecundity of at least 15 eggs/female/reproductive day/replicate during the 7 days prior to definitive test. 

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: > two times in 7 days EPA recommends that spawning occur at least twice in the 7 days prior to the definitive test. Details on pre-exposure: None. 

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

system. As noted in the Corrections and 

Clarifications document1 , the use of a static

renewal system is not recommended for this 

assay.

Type of flow-through dilution system:

Flow-through diluters Not further described Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

Flow-through rate: Ca. 14 L/hr. Recommended flow-through rate is 45 

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

volume exchanges per day.

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

Program Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention (OCSPP), Washington, D.C. 

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

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on toxicant mixing for flow-through systems: 

Flow-splitting chambers were used between diluter cells and the 4 replicate test vessels. Treatment recoveries prior to test initiation (-1 

day) were 103-109%, indicating the diluter 

had reached equilibrium.Flow splitting accuracy was ±10%. 

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

recommended but not required; 2) Aeration is 

not recommended for mixing; 3) A 

demonstration that the test solution is 

completely mixed before introduced into the 

test system is recommended; 4) The 

recommended flow splitting accuracy is within 

10%.

Aeration? Yes, beginning on exposure 

Day 7. 

Aeration began after D7 sampling revealed DO concentrations ranging from 66—80% 

EPA recommends aeration if dissolved oxygen 

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

Source of dilution water: Reconstituted 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.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

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

Yes 

Test vessel type/materials: Glass aquaria with silicone 

adhesive

EPA and OECD recommend that watercontact portions of the system not 

compromise the study (e.g., all glass vessels 

or glass vessels with stainless steel frames 

are acceptable examples).

Test vessel size: 43 x 19.8 x 21 cm; solution 

depth 16.5 cm

EPA recommends the use of 18 L test 

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

Fill volume: 14 L EPA recommends 10 L solution per tank. Spawning substrate material: Three 1-mm thickness 

stainless steel pipes sitting on a round (1250 mm diameter) stainless screen (hole diameter 8 mm) covered in glass fiber mesh. 

EPA recommends that each tank contain 

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

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

lengthwise.

Spawning substrate size: Half pipes sectioned into 110 

x 75 x 85 mm pieces

Each substrate placed in lipped glass tray with petri dishes on the bottom (1350 mm diameter). 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Additional details on exposure system: 

None. 

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

Parameter Minimum Maximum Mean Measurement Interval Guideline Recommendations 

Temperature (°C) 25.1 25.7 25.4 Weekly 

EPA recommends temperature 25±1oC; interreplicate and inter-treatment differentials should not exceed 1oC.

pH 6.7 7.3 7.0 Weekly EPA recommends pH 6.5 to 9.0.Dissolved oxygen (%) 66 107 92 Weekly EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air saturation)Total alkalinity (mg/L as CaCO3) 35.2 43.4 37.9 Weekly EPA recommends total alkalinity >20 mg/L 

as CaCO3.

Hardness (mg/L as CaCO3) 40.8 52.9 45.6 Weekly Total organic carbon (mg/L) Not reported Not reported <2 Once EPA recommends that total organic carbon in 

dilution water be <2 mg/L. 

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Parameter Minimum Maximum Mean Measurement Interval Guideline Recommendations 

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

EPA recommends that unionized ammonia in 

the dilution water be <1 Pg/L. 

Residual chlorine (Pg/L) Not reported Not reported <10 Once 

EPA recommends that residual chlorine in 

dilution water be <10 Pg/L. 

Conductivity (PS/cm) 125 140 131 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. 

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D. Study Design and Additional Experimental Conditions

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

96-hour LC50 data for the fathead minnow are 

unavailable. 

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

Between the NOEC (13 mg/L) on a fathead minnow ELS study and 1/3 the derived 96-hour 

LC50 of the range finder test (>100 mg/L). 

EPA recommends that the highest test 

concentration be selected based on toxicity data 

for other fish studies or species, if available. 

Otherwise, either the solubility limit of the test 

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

appropriate.

Species: Pimephales promelasLife stage: Not reported EPA recommends that range-finding tests be 

performed with fish of similar age and size to 

those that would be utilized in the test.

Test duration: 96 hours EPA recommends a 96-hour exposure. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Additional details: EPA recommends conducting a range-finder with 

five test concentrations plus a control (six total 

treatment levels), with four females and two 

males per exposure tank (36 fish total). The 

number of mortalities that occur may be used to 

develop a concentration-response curve. 

Based upon the results, the highest concentration 

that does not result in increased mortality or 

signs of overt morbidity compared to controls, or 

1/3 the derived 96-hr LC50, may be selected as 

the highest exposure concentration in the 21-day 

test.

Table 7: Definitive Study Conditions.

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

test be 21 days. 

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

The acute toxicity 96-hr LC50 value with fathead 

minnows was reported 

A spacing factor of 5 was selected for the test concentrations. 

EPA recommends that the highest test 

concentration is either the solubility limit of the 

test compound, 100 mg/L, or demonstrates 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

determined to be >100 mg a.i./L. 

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

hour LC50), whichever concentration is lowest.

Reference study citation (if applicable):

Not reported 

Separation of test concentrations: 0 (control), 0.2, 1.0, and 

5.0 mg a.i./L 

EPA suggests that a concentration separation of 

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

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

concentrations and a control, plus solvent control 

if appropriate.

Are nominal concentrations adjusted for purity? 

Yes 

Indicate the type of values presented for measured concentrations: 

Mean measured 

Limit of quantification (LOQ): 0.02 mg a.i./L EPA recommends that for chemical test 

concentrations below the LOQ, analyses be 

conducted on the stock solutions.

Level of detection (LOD): Not reported 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

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

be measured prior to the addition of fish in all 

tanks and at least weekly thereafter in two 

replicates per treatment level.

Was the randomized complete block design used?

Yes EPA recommends that all fish be randomly 

assigned to tanks during pre-exposure. Tanks 

are then ranked according to pre-exposure 

fecundity, and the tanks with the highest 

fecundity are randomly assigned to a definitive 

test treatment and block first. Each block 

contains one replicate of each treatment, 

including controls. 

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

Not applicable EPA recommends the use of a concurrent 

solvent control when a solubilizing agent is used. 

EPA recommends 4 replicates.

Number of replicates per test item treatment level: 

4 EPA recommends 4 replicates.

Number of male fish per replicate at test initiation: 

2 EPA recommends 2 males per replicate. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Number of female fish per replicate at test initiation: 

4 EPA recommends 4 females per replicate. 

Was a solvent used? No Solvent type (if applicable): Not applicable Maximum solvent concentration (if applicable):

Not applicable EPA recommends that the solvent not exceed 

0.02 ml/L3. OECD recommends that solvent 

have no effect on survival nor produce any other 

adverse effects and that concentration not be 

greater than 0.1 ml/L4.

Was a positive control used? No Positive control (if applicable): Not applicable Positive control concentration(s) (if applicable): 

Not applicable 

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

Toxicology, 76, pp.69–92. 

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

and Assessment. No. 23. Paris, France. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Photoperiod: 16 hrs light: 

8 hrs dark 

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

Light intensity at water’s surface: 720-1080 lux Cool white fluorescents EPA recommends light intensity 540 – 1080 lux 

(at water’s surface).

Additional details: There was no evidence of precipitates during 

exposure. 

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

Treatment ID 

Nominal 

Concentration

(mg a.i./L) 

Mean Measured 

Concentration

(mg a.i./L) 

Mean CV (%) Details or Remarks Guideline Recommendations 

Negative Control 0 <LOQ NA EPA recommends that test item concentrations 

be maintained at a coefficient of variation (CV) 

≤20%.

Treatment 1 0.2 0.199 1.9 

Treatment 2 1.0 1.03 3.4 Treatment 3 5.0 5.40 0.35 

Abbreviations: CV Coefficient of variation. NA Not applicable. 

LOQ=0.02 mg a.i./L. 

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E. Observations

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

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

Were raw (individual) data provided? Yes

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

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

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

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

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

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II. RESULTS AND DISCUSSION A. Results 

Male survival values were 100% at all treatment levels, and female survival values were 100, 93.8, 93.8 and 81.3% in the negative control and 

mean-measured 0.199, 1.03, and 5.40 mg a.i./L treatment groups, respectively (Table 9). Overall survival values were 100, 96.9, 96.9, and 90.6% in the negative control, 0.199, 1.03, and 5.40 mg a.i./L treatment levels, respectively. 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Males Females 

N1 # Surviving % Survival N1 # Surviving % Survival 

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

0.199 8 8 100 16 15 93.8 

1.03 8 8 100 16 15 93.8 

5.40 8 8 100 16 13 81.3 

 LOQ=0.02 mg a.i./L. 1 Total number of fish at test initiation. 

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Mean male body weight values were 4.26, 4.38, 4.25 and 4.40 g and mean female body weight values were 1.99, 2.03, 2.00 and 2.05 g in the negative control and mean-measured 0.199, 1.03 and 5.40 mg a.i./L treatment groups, respectively (Table 10). Mean male body length values were 57.6, 57.3, 57.1, and 57.2 mm and mean female body length values were 44.8, 44.7, 45.0, and 44.1 mm in the negative control and meanmeasured 0.199, 1.03 and 5.40 mg a.i./L treatment groups, respectively. 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Body Weight Length 

Males Females Males Females 

n

Mean

(g) 

±SD n 

Mean

(g) 

±SD n

Mean

(mm) 

±SD N

Mean

(mm) 

±SD

Negative Control 

(<LOQ) 

4 4.26 0.41 4 1.99 0.25 4 57.6 1.98 4 44.8 1.27

0.199 4 4.38 0.63 4 2.03 0.17 4 57.3 3.16 4 44.7 0.57 

1.03 4 4.25 0.43 4 2.00 0.10 4 57.1 1.22 4 45.0 1.23 

5.40 4 4.40 0.61 4 2.05 0.18 4 57.2 1.65 4 44.1 0.92 

Abbreviations: SD Standard deviation. 

LOQ=0.02 mg a.i./L. 

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Mean fecundity values were 8.84, 6.36, 7.49 and 6.47 eggs/female/day/replicate and fertilization success 

was 96.9, 98.0, 97.4 and 98.1% in the negative control and mean-measured 0.199, 1.03 and 5.40 mg 

a.i./L treatment groups, respectively (Table 11). 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Fecundity1 Fertilization Success (%)2

Mean ± SD Mean ± SD 

Negative Control (<LOQ) 8.84 2.82 96.9 1.44 

0.199 6.36 5.04 98.0 1.00 

1.03 7.49 5.66 97.4 0.29 

5.40 6.47 5.06 98.1 0.84 

LOQ=0.02 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. 

 

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Median male tubercle scores were 40, 41, 39 and 43 in the negative control and mean-measured 0.199, 

1.03 and 5.40 mg a.i./L treatment groups, respectively (Table 12). None of the surviving females were 

found to have tubercles. 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Males Females 

n Median Tubercle 

Score1 N 

Median Tubercle 

Score 

Negative Control (<LOQ) 4 40 4 0 

0.199 4 41 4 0 

1.03 4 39 4 0 

5.40 4 43 4 0 

LOQ=0.02 mg a.i./L. 

1

 Mean tubercle scores: 39, 42, 39, 41 for the negative control and mean-measured 0.199, 1.03, and 5.04 mg a.i./L 

treatment levels, respectively. 

 

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Mean male GSI was 1.32, 1.56, 1.37 and 1.64% and mean female GSI was 15.9, 17.6, 15.4 and 18.6% 

in the negative control and mean-measured 0.199, 1.03 and 5.40 mg a.i./L treatment groups, respectively 

(Table 13). 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Males Females 

n Mean GSI1

(%) 

±SD n Mean GSI1

(%) 

±SD 

Negative Control (<LOQ) 4 1.32 0.45 4 15.9 2.01 

0.199 4 1.56 0.14 4 17.6 3.68 

1.03 4 1.37 0.16 4 15.4 1.64 

5.40 4 1.64 0.16 4 18.6 3.53 

Abbreviations: NA Not applicable. 

LOQ=0.02 mg a.i./L.

1 Gonado-somatic index (%) is calculated as gonad weight (to the nearest 0.1 mg) / body weight (mg) x 100. 

 

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Median male gonadal stages were 2, 2, 2 and 3 and female gonadal stages were 3, 3, 2 and 3 in the 

negative control and mean-measured 0.199, 1.03 and 5.40 mg a.i./L treatment groups, respectively (Table 

14). 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Males Females 

n Median Stage1 n Median Stage2

Negative Control (<LOQ) 4 2 4 3 

0.199 4 2 4 3 

1.03 4 2 4 2 

5.40 4 3 4 3 

LOQ=0.02 mg a.i./L.

1

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

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

2

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

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

In males, there was a higher incidence of advanced maturation (stage 3) at the high test concentration (5 

incidences; Table 15). According to the study report, in females, no significant histopathological results that 

could be correlated to exposure to the test substance were noted (Tables 17-18). 

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

Treatment 

(mg a.i./L) 

[meanmeasured] 

Diagnostic Observations1

Severity 

Increased 

Proportion of 

Spermatogonia 

Presence of 

Testis-Ova 

Increased 

Testicular 

Degeneration

Duct 

Mineralization

Interstitial cell 

(Leydig) 

hypertrophy / 

hyperplasia 

n Incidence n Incidence n Incidence n Incidence n Incidence 

Negative 

Control 

(<LOQ)

0 8 7 8 8 NA NA 8 5 NA NA 

1 8 0 8 0 NA NA 8 1 NA NA 

2 8 1 8 0 NA NA 8 2 NA NA 

3 8 0 8 0 NA NA 8 0 NA NA 

4 8 0 8 0 NA NA 8 0 NA NA 

0.199 0 8 8 8 7 NA NA 8 4 NA NA 

1 8 0 8 1 NA NA 8 4 NA NA 

2 8 0 8 0 NA NA 8 0 NA NA 

3 8 0 8 0 NA NA 8 0 NA NA 

4 8 0 8 0 NA NA 8 0 NA NA 

1.03 0 8 7 8 8 NA NA 8 3 NA NA 

1 8 1 8 0 NA NA 8 4 NA NA 

2 8 0 8 0 NA NA 8 1 NA NA 

3 8 0 8 0 NA NA 8 0 NA NA 

4 8 0 8 0 NA NA 8 0 NA NA 

5.40 0 8 7 8 8 NA NA 8 5 NA NA 

1 8 0 8 0 NA NA 8 2 NA NA 

2 8 1 8 0 NA NA 8 1 NA NA 

3 8 0 8 0 NA NA 8 0 NA NA 

4 8 0 8 0 NA NA 8 0 NA NA 

Abbreviation: NA Not applicable. 

LOQ=0.02 mg a.i./L.

1

 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 

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

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

Treatment 

(mg a.i./L) 

[mean-measured] 

Additional Diagnostic Observations1

Severity 

Decreased Proportion 

of Spermatogonia

Increased Vascular or 

Interstitial 

Proteinaceous Fluid 

Asynchronous 

Gonad 

Development 

Altered Proportions of 

Spermatocytes or 

Spermatids 

Granulomatous 

Inflammation 

n Incidence n Incidence n Incidence n Incidence n Incidence 

Negative Control 

(<LOQ) 

0 NA NA NA NA NA NA NA NA NA NA 

1 NA NA NA NA NA NA NA NA NA NA 

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

0.199 0 NA NA NA NA NA NA NA NA NA NA 

1 NA NA NA NA NA NA NA NA NA NA 

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

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Treatment 

(mg a.i./L) 

[mean-measured] 

Additional Diagnostic Observations1

Severity 

Decreased Proportion 

of Spermatogonia

Increased Vascular or 

Interstitial 

Proteinaceous Fluid 

Asynchronous 

Gonad 

Development 

Altered Proportions of 

Spermatocytes or 

Spermatids 

Granulomatous 

Inflammation 

n Incidence n Incidence n Incidence n Incidence n Incidence 

1.03 0 NA NA NA NA NA NA NA NA NA NA 

1 NA NA NA NA NA NA NA NA NA NA 

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

5.40 0 NA NA NA NA NA NA NA NA NA NA 

1 NA NA NA NA NA NA NA NA NA NA 

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

Abbreviation: NA Not applicable. 

LOQ=0.02 mg a.i./L.

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E of 

the test guideline for reference. 

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

Treatment 

(mg a.i./L) 

[meanmeasured] 

Additional Diagnostic Observations1

Severity 

Increased Oocyte 

Atresia 

Perifollicular Cell 

Hyperplasia/ 

Hypertrophy 

Decreased Yolk 

Formation 

Aggregates of 

macrophages, 

multifocal 

n Incidence n Incidence n Incidence n Incidence 

Negative Control 

(<LOQ) 

0 16 10 NA NA NA NA NA NA 

1 16 5 NA NA NA NA NA NA 

2 16 0 NA NA NA NA NA NA 

3 16 1 NA NA NA NA NA NA 

4 16 0 NA NA NA NA NA NA 

0.199 0 15 13 NA NA NA NA NA NA 

1 15 1 NA NA NA NA NA NA 

2 15 1 NA NA NA NA NA NA 

3 15 0 NA NA NA NA NA NA 

4 15 0 NA NA NA NA NA NA 

1.03 0 15 10 NA NA NA NA NA NA 

1 15 5 NA NA NA NA NA NA 

2 15 0 NA NA NA NA NA NA 

3 15 0 NA NA NA NA NA NA 

4 15 0 NA NA NA NA NA NA 

5.40 0 13 5 NA NA NA NA NA NA 

1 13 6 NA NA NA NA NA NA 

2 13 2 NA NA NA NA NA NA 

3 13 0 NA NA NA NA NA NA 

4 13 0 NA NA NA NA NA NA 

Abbreviation: NA Not applicable. 

LOQ=0.02 mg a.i./L.

1

 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 

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

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

promelas).

Treatment 

(mg a.i./L)

[meanmeasured]

Additional Diagnostic Observations1

Severity 

Interstitium/multifo

cal inflammation 

Egg Debris in 

Oviduct 

Granulomatous 

Inflammation 

Increased PostOvulatory Follicles 

n Incidence n Incidence n Incidence n Incidence 

Negative 

Control 

(<LOQ) 

0 NA NA 16 9 16 16 16 7 

1 NA NA 16 3 16 0 16 6 

2 NA NA 16 2 16 0 16 3 

3 NA NA 16 2 16 0 16 0 

4 NA NA 16 0 16 0 16 0 

0.199 0 NA NA 15 9 15 15 15 7 

1 NA NA 15 0 15 0 15 2 

2 NA NA 15 1 15 0 15 6 

3 NA NA 15 4 15 0 15 0 

4 NA NA 15 1 15 0 15 0 

1.03 0 NA NA 15 5 15 13 15 2 

1 NA NA 15 3 15 1 15 5 

2 NA NA 15 2 15 1 15 8 

3 NA NA 15 5 15 0 15 0 

4 NA NA 15 0 15 0 15 0 

5.40 0 NA NA 13 3 13 12 13 2 

1 NA NA 13 3 13 0 13 7 

2 NA NA 13 2 13 0 13 4 

3 NA NA 13 2 13 1 13 0 

4 NA NA 13 3 13 0 13 0 

Abbreviation: NA Not applicable. 

LOQ=0.02 mg a.i./L.

1

 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 

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

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Mean male VTG was 3040, 8952, 3129 and 1091 and mean female VTG was 1.89 x 106, 3.21 x 106, 

2.43 x 106 and 2.25 x 106 in the negative control and mean-measured 0.199, 1.03 and 5.40 mg a.i./L 

treatment groups, respectively (Table 19). 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Plasma Vitellogenin (VTG) 

Males Females 

n Mean

(ng/mL plasma) 

±SD1 n Mean

(ng/mL plasma) 

±SD1

Negative Control (<LOQ) 4 3040 5888 4 1.89 x 106 0.845 x 106

0.199 4 8952 14478 4 3.21 x 106 3.51 x 106

1.03 4 3129 4556 4 2.43 x 106 0.728 x 106

5.40 4 1091 1108 4 2.25 x 106 0.270 x 106

Abbreviations: SD Standard deviation. 

LOQ=0.02 mg a.i./L. 

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

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

Treatment 

 (mg a.i./L) 

[mean-measured]

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

Males Females Males Females 

n

Mean

(ng/mL 

plasma) 

±SD n 

Mean

(ng/mL 

plasma) 

±SD N

Mean

(ng/mL 

plasma) 

±SD n

Mean

(ng/mL 

plasma) 

±SD

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

0.199 NA NA NA NA NA NA NA NA NA NA NA NA 

1.03 NA NA NA NA NA NA NA NA NA NA NA NA 

5.40 NA NA NA NA NA NA NA NA NA NA NA NA 

 Abbreviations: NA Not applicable. SD Standard deviation. 

 LOQ=0.02 mg a.i./L. 

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There were no notable observations with regards to behavior or changes in appearance, specifically color/banding, ovipositor appearance, or size of dorsal nape pad in the control or treated groups (Table 21), although 2 females had only one eye in the 1.03 mg a.i./L treatment group and 2 females had exophthalmia in one eye in the 5.40 mg a.i./L treatment group. 

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

Treatment 

(mg a.i./L) 

[mean-measured] 

Secondary Sex Characteristics and Clinical Signs1

Males Females 

Type n Incidence Type n Incidence 

Negative Control (<LOQ) None 8 0 None 16 0 

0.199 None 8 0 None 15 0 

1.03 None 8 0 Only one eye 15 2 

5.40 None 8 0 Exophthalmia in one eye 13 2 

LOQ=0.02 mg a.i./L. 

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B. Study Author’s Analysis and Conclusions

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

Data were gender specific and analyzed in comparison to the controls. 

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

effects were detected for pd0.05 (TOXRAT, Version 2.10). Survival data were analyzed using a 

William’s test (Cochran-Armitage Trend Step-down test proposed in the guideline was not available in 

the current version of the statistical program used). For normality the Shapiro-Wilk’s test or the R/Stest was used, while for homogeneity of variance the Levene Test was used. If the endpoints met the 

assumptions for normality and homogeneity and the results were not consistent with a monotonic 

concentration-response, Dunnett’s Multiple Comparison test was used. Tubercle score effects were 

also analyzed using Dunnett’s (Jonckheere-Terpstra Step-Down test proposed in the guideline was 

not available). Gonadal histopathology results were evaluated in a semi-quantitative manner. 

Adult survival in males was 100% for all test levels therefore, no statistical analysis was performed. 

Adult survival in females was 100, 93.8, 93.8 and 81.3% and overall survival was 100, 96.9, 96.9, 

and 90.6% in the control, 0.199, 1.03, and 5.40 mg a.i./L (Table 9). There was a statistically 

significant difference in the 5.40 mg a.i./L treatment group compared to the negative control (p<0.05; 

William’s test) in both the female and overall survival. There was no statistically significant differences 

compared to the negative control for any other endpoint (p>0.05; Dunnett’s Multiple Comparison test). 

None of the surviving females were found to have tubercles. 

In males, there was a higher incidence of advanced maturation (stage 3) at the highest test 

concentration (5 incidences; Table 15). There was also an increase in female mortality at the highest 

test concentration therefore, the advanced maturation in males would be the result of overt toxicity or 

an indirect effect caused by the lower density of fish/females/replicate. In females, no significant 

histopathological results that could be correlated to exposure to the test substance were noted (Tables 

17-18). 

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C. Reviewer’s Analysis and Conclusions 

Statistical Methods: The reviewer analyzed survival (mortality) data with Fisher’s exact test via 

Toxstat 3.5. Male and female body weight and length, male and female mean vitellogenin (VTG), 

male and female GSI, male tubercle score, fertility and fecundity data were not consistent with a 

monotonic concentration-response. All data were tested for normality using Shapiro-Wilks test and for 

homogeneity of variance using Levene’s test. Data which met the assumptions of normality and 

homogeneity of variance were then analyzed using the parametric Dunnett’s test. Data which did not 

meet the parametric assumptions were analyzed using the non-parametric Mann-Whitney U test. 

Female and male mean VTG were neither monotonic nor parametric and were analyzed using Mann 

Whitney as recommended by the OCSPP 890.1350 guideline. None of the surviving females were 

found to have tubercles. Unless otherwise indicated, effects were considered statistically significant at 

p<0.05. These analyses were conducted using SAS® (SAS Institute, Cary, NC; version 8.1). Both the 

study author’s and reviewer relied on the mean-measured concentrations to discuss effects in this 

study. 

 

Conclusions: There were no significant differences in any endpoint evaluated. In the high treatment 

group, three out of 16 females had died by test termination whereas survival was 100% in the control. 

Adult survival in males was 100% for all test levels. Adult survival in females was 100, 93.8, 93.8 

and 81.3% and overall survival was 100, 96.9, 96.9, and 90.6% in the control, 0.199, 1.03, and 5.40 

mg a.i./L treatment groups (Table 9). There was no statistically significant difference in overall survival 

for a treatment group compared to the negative control. There was no statistically significant differences 

compared to the negative control for any other endpoint (p>0.05; Dunnett’s Multiple Comparison test 

and Mann Whitney test). None of the surviving females were found to have tubercles. 

In males, there was a higher incidence of advanced maturation (stage 3) at the high test concentration 

(5 incidences; Table 14). There was also an increase in female mortality at the high test concentration 

therefore, it was reported that the advanced maturation in males could be the result of overt toxicity 

or an indirect effect caused by the lower density of fish/females/replicate. 

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In females, no significant histopathological results that could be correlated to exposure to the test 

substance were noted. There were no notable observations with regards to behavior or changes in 

appearance, specifically color/banding, ovipositor appearance, or size of dorsal nape pad in the control 

or treated groups. There were no notable observations of secondary sex characteristics or clinical 

signs for any treatment group compared to the negative control. 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Tubercle Score GSI 

Gonadal 

Staging and 

Histo. 

Plasma VTG Plasma T Plasma E2 

Median p % Diff. p 

Effect? 

(Yes/No) 

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

Negative Control 

(<LOQ) 

40 NA 0 NA No 0 NA NA NA NA NA

0.199 41 0.79 18.6 0.44 No 1953 0.24 NA NA NA NA 

1.03 39 >0.99 3.80 0.99 No 2.93 0.68 NA NA NA NA 

5.40 43 0.95 24.5 0.25 Yes -64.1 0.68 NA NA NA NA 

Statistical Test Dunnett’s4 Dunnett’s NA Mann-Whitney NA NA 

Abbreviations: Diff. Difference. E2 17β-estradiol. GSI Gonado-Somatic Index. Histo. Histopathology. NA Not applicable. T Testosterone. VTG Vitellogenin. NA Not applicable. LOQ=0.02 mg a.i./L.

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

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

2 Unless otherwise specified, effects are considered statistically significant at p<0.05. 3 This percent difference is due to one fish for which the VTG value was substantially greater than the control values; this increase does not reflect an overall increase in 

the fish exposed at this concentration. 

4 Mean tubercle scores: 39, 42, 39, 41 for the negative control and mean-measured 0.199, 1.03, and 5.04 mg a.i./L treatment levels, respectively. 

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Table 23: Reproductive and HPG Endpoints1,2 for Female Fathead Minnow (Pimephales promelas) in the FSTRA with Metribuzin. Abbreviations: Diff. Difference. E2 17β-estradiol. Fert. Fertilization. GSI Gonado-Somatic Index. Histo. Histopathology. NA Not applicable. T Testosterone. VTG Vitellogenin. 

LOQ=0.02 mg a.i./L.

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

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

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

Treatment 

(mg a.i./L) 

[meanmeasured]

Fecundity Fert. Success Tubercle Score GSI 

Gonadal 

Staging 

and Histo. 

Plasma VTG Plasma T Plasma E2 

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

Effect? 

(Yes/No) 

% Diff. p 

% 

Diff. 

p

% 

Diff. 

p

Negative Control 

(<LOQ) 

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

0.199 -28.1 0.81 1.11 0.33 0 NA 10.7 0.74 No 69.5 >0.99 NA NA NA NA 

1.03 -15.4 0.96 0.57 0.77 0 NA -3.14 0.99 No 28.4 0.24 NA NA NA NA 

5.40 -26.8 0.83 1.29 0.22 0 NA 17.1 0.42 No 18.5 0.35 NA NA NA NA 

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

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

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 

0.199 2.88 0.98 1.88 0.98 -0.65 0.99 -0.28 1.0 

1.03 -0.18 >0.99 0.25 >0.99 -0.87 0.98 0.39 0.99 

5.40 3.29 0.97 2.89 0.94 -0.69 0.99 -1.51 0.69 

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

Abbreviations: Diff. Difference. NA Not applicable. 

LOQ=0.02 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.

 

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E. Study Deficiencies 

The study authors indicated the transfer of four spawning groups from the pre-exposure tanks to the 

exposure tanks was not successful with one or two males showing reduced fitness. It was decided to 

exchange these spawning groups for the next best spawning groups which did not meet the minimum 

fecundity requirement; however, the study author indicated that regular spawning occurred at least 2 

times in the immediate 7 days prior to exposure. 

The performance and validity criteria were met for this study. While the study met the control spawning 

performance criterion (at least 15 eggs/female/day/replicate or spawning occurring at least every 4 

days), the mean fecundity in the negative control was 8.84 eggs/female/day (range: 6.74-13.0); 

however, spawning was observed in all negative control replicates at least once every four days. 

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. 

F. Reviewer’s Comments 

The reviewer’s and the study authors’ results were in disagreement regarding effects on survival. While 

the study author’s analysis detected statistically significant reductions (p<0.05) in female and overall 

survival at the 5.04 mg a.i./L treatment level, the reviewer’s analysis did not. This is because the 

study author used Williams’ test whereas the reviewer used Fisher’s exact test. There was no 

statistically significant differences compared to the negative control for any other endpoint (p>0.05; 

Dunnett’s Multiple Comparison test and Mann-Whitney test). The reviewer’s conclusions based on the 

OCSPP 890.1350 flowchart are presented in the Executive Summary and Conclusions sections of this 

DER. 

For the VTG analysis, in some cases re-measurements were done using a second aliquot to verify 

the first measurement or repeat a failed measurement (absorbance didn’t fall within standard curve) 

because no concentration response relationship was noted. On assay Day 3, neither the standard 

male plasma nor the fortified male/female standard plasma (dilution factor 1:500) resulted in any 

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absorbance and on assay Day 4, standard male plasma resulted in 40% and fortified male/female 

standards resulted in 38% of expected concentrations. However, on both of these days most of the 

re-measured samples resulted in high accordance between the two measured aliquots therefore, the 

results measured on assay Day 3 and 4 were deemed to be reliable. 

The study authors reported that the first run of the study was cancelled during pre-exposure (after 23 

days) due to poor egg production over all spawning groups. The study was restarted using a new 

group of fish. 

In males, there was a higher incidence of advanced maturation (stage 3) at the high test concentration 

compared with the controls and other treatment groups. According to the study report, there was also 

an increase in female mortality at the high test concentration therefore, the advanced maturation in 

males could be the result of overt toxicity or an indirect effect caused by the lower density of 

fish/replicate. However, according to the reviewer, no trend was easily discernible between males with 

advanced maturation and replicates containing dead females (i.e. advanced maturation occurred even 

in replicates with no mortality). 

The reviewer noted some histopathological effects (Tables 17-18) following a concentration-response 

relationship in female fishes including prevalence and severity for incidence of egg debris in the oviduct. 

There were 7 (44%), 6 (40%), 10 (67%), and 10 (77%) findings of egg debris in the oviduct (Table 

18) in the control, 0.199, 1.03 and 5.40 mg a.i./L treatment groups, respectively. Although not 

following a concentration-response relationship, there was substantially more prevalence of increased 

oocyte atresia in fish in the 5.40 mg a.i./L treatment level compared to fish in the control group. 

However, the results in this DER reflect the conclusions of the study report. 

There were 0, 0, 2 (13%) and 1 (8%) findings of granulomatous inflammation (Table 18) in the 

control, 0.199, 1.03 and 5.40 mg a.i./L treatment groups, respectively. The incidences in the 1.03 

mg a.i./L treatment level were at the minimal to mild severity while the only incidence in the 5.40 mg 

a.i./L treatment group was at the moderate severity ranking. No reports of the appearance of protozoal 

spores were included in the report. 

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III. REFERENCES 

American Public Health Association, 1998. Standard Methods for the Examination of Water and 

Wastewater, 17th edition. Washington, D. C. 

American Society for Testing and Materials (ASTM), 2007. Standard Guide for Conducting Acute 

Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians. ASTM Standard 

E729. West Conshohocken, PA. 

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

of a short-term reproduction test with the fathead minnow (Pimephales promelas). Environ Toxicol 

Chem 20: 1276-1290. 

Ankley, G.T., K.M. Jensen, E.A. Makynen, M.D. Kahl, J.J. Korte, M.W. Hornung, T.R. Henry, J.S. 

Denny, R.L. Leino, V.S. Wilson, M.C. Cardon, P.C. Hartig and L.E. Gray. 2003. Effects of the 

androgenic growth promoter 17-B-trenbolone on fecundity and reproductive endocrinology of the 

fathead minnow (Pimephales promelas). Environ. Toxicol. Chem. 22, 1350-1360. 

OECD, 1992, Test Guideline 203: Fish, Acute Toxicity Test. 

ToxRat Solutions GmbH, 52477 Alsdorf, Germany, ToxRat Professional V2.10 7. USEPA, 1975. 

Methods for Acute Toxicity Tests with Fish, Macroinvertebrates and Amphibians. EPA-660/3-75-009. 

Office of Research and Development, Corvallis, OR. 

USEPA, 1975. Acquisition and Culture of Research Fish: Rainbow Trout, Fathead Minnows, Channel 

Catfish and Bluegills. EPA-660/3-75-011. Office of Research and Development, Corvallis, Oregon. 

45 pp. 

USEPA, 1982. Pesticide Assessment Guidelines, Subdivision E - Hazard Evaluation: Wildlife and 

Aquatic Organisms. EPA 540/9-82-024. Washington, D.C. 86 pp. 10. USEPA, 1985. Standard 

Evaluation Procedure. Fish Early Life Stage. EPA-540/9-86-138. Office of Pesticide Programs, 

Washington, D.C. 

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USEPA, 1989. Pesticide Programs; Good Laboratory Practice Standards; Final Rule (40 CFR, Part 

160). Federal Register, Vol. 54, No. 158: 34067-34074. 

USEPA, 1996. Draft OCSPP 850.1400: Fish Early-Life Stage Toxicity Test. 

USEPA, 2002. Short Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving 

Waters to Freshwater Organisms, 4th Edition. EPA821-R-02-013. Office of Water, Washington, D.C. 

USEPA, 2009. Endocrine Disruptor Screening Program Test Guidelines. OCSPP 890.1350. Fish 

Short-Term Reproduction Assay. 

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APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION:

 Fisher's Exact Test 

 ============================================================================ 

 NUMBER OF 

 ---------------------------------------- 

 IDENTIFICATION ALIVE DEAD TOTAL ANIMALS 

 -------------------------- ----- ----- --------------- 

 CONTROL 24 0 24 

 0.199 23 1 24 

 ---------------------------------------------------------------------------- 

 TOTAL 47 1 48 

 ============================================================================ 

 Critical Fisher's value (24,24,24) (alpha=0.05) is 19.0. b value is 23. 

 Since b is greater than 19.0 there is no significant difference 

 between CONTROL and TREATMENT at the 0.05 level. 

 

 

 

 Fisher's Exact Test 

 ============================================================================ 

 NUMBER OF 

 ---------------------------------------- 

 IDENTIFICATION ALIVE DEAD TOTAL ANIMALS 

 -------------------------- ----- ----- --------------- 

 CONTROL 24 0 24 

 1.03 23 1 24 

 ---------------------------------------------------------------------------- 

 TOTAL 47 1 48 

 ============================================================================ 

 Critical Fisher's value (24,24,24) (alpha=0.05) is 19.0. b value is 23. 

 Since b is greater than 19.0 there is no significant difference 

 between CONTROL and TREATMENT at the 0.05 level. 

 

 

 

 Fisher's Exact Test 

 ============================================================================ 

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 NUMBER OF 

 ---------------------------------------- 

 IDENTIFICATION ALIVE DEAD TOTAL ANIMALS 

 -------------------------- ----- ----- --------------- 

 CONTROL 24 0 24 

 5.40 21 3 24 

 ---------------------------------------------------------------------------- 

 TOTAL 45 3 48 

 ============================================================================ 

 Critical Fisher's value (24,24,24) (alpha=0.05) is 19.0. b value is 21. 

 Since b is greater than 19.0 there is no significant difference 

 between CONTROL and TREATMENT at the 0.05 level. 

 

 

 

 Summary of Fisher's Exact Tests 

 ---------------------------------------------------------------------------- 

 NUMBER NUMBER SIG 

 GROUP IDENTIFICATION EXPOSED DEAD 0.05 

 ----- ------------------------- ----------- ----------- ----- 

 CONTROL 24 0 

 1 0.199 24 1 

 2 1.03 24 1 

 3 5.40 24 3 

 ---------------------------------------------------------------------------- 

 

 

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test for fish screen study - Metribuzin 

ANALYSIS RESULTS FOR VARIABLE VAR01 ( F body weight (g) ) 

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS 

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 

Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 

Use parametric analyses if neither test rejected, otherwise non-parametric analyses. 

 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 0.916 0.148 2.016 0.166 USE PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 1.99 0.25 0.12 12.46 1.60, 2.38 

 Dose1 4 2.03 0.18 0.09 8.65 1.75, 2.31 

 Dose2 4 2.00 0.11 0.05 5.28 1.83, 2.16 

 Dose3 4 2.05 0.18 0.09 8.57 1.77, 2.33 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 1.97 1.76 2.26 . . 

 Dose1 1.98 1.88 2.27 101.88 -1.88 

 Dose2 1.96 1.92 2.15 100.25 -0.25 

 Dose3 2.00 1.90 2.29 102.89 -2.89 

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

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 0.09 0.965 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing negative trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl 1.99 . 2.02 . . . . . . 

 Dose1 2.03 0.983 2.02 0.662 . . . . . 

 Dose2 2.00 1.000 2.02 0.697 0.994 . . . . 

 Dose3 2.05 0.944 2.02 0.716 0.999 0.976 . . . 

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

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 0.51 0.916 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing negative trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 1.97 . . 

 Dose1 1.98 0.678 0.718 

 Dose2 1.96 0.889 0.696 

 Dose3 2.00 0.678 0.773 

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 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

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

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 0.09 0.965 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing INCREASING trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl -1.99 . -1.99 . . . . . . 

 Dose1 -2.03 0.983 -2.01 0.513 . . . . . 

 Dose2 -2.00 1.000 -2.01 0.546 0.994 . . . . 

 Dose3 -2.05 0.944 -2.05 0.437 0.999 0.976 . . . 

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

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 0.51 0.916 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -1.97 . . 

 Dose1 -1.98 0.678 0.282 

 Dose2 -1.96 0.889 0.304 

 Dose3 -2.00 0.678 0.227 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

test for fish screen study - Metribuzin 

ANALYSIS RESULTS FOR VARIABLE VAR02 ( M body weight (g) ) 

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS 

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 

Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 

Use parametric analyses if neither test rejected, otherwise non-parametric analyses. 

 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 0.950 0.489 0.265 0.849 USE PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 4.26 0.41 0.21 9.69 3.60, 4.91 

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 Dose1 4 4.38 0.63 0.32 14.40 3.37, 5.38 

 Dose2 4 4.25 0.43 0.21 10.07 3.57, 4.93 

 Dose3 4 4.40 0.61 0.30 13.79 3.43, 5.36 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 4.26 3.83 4.67 . . 

 Dose1 4.42 3.57 5.11 102.88 -2.88 

 Dose2 4.29 3.78 4.63 99.82 0.18 

 Dose3 4.45 3.67 5.01 103.29 -3.29 

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

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 0.09 0.965 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing negative trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl 4.26 . 4.32 . . . . . . 

 Dose1 4.38 0.976 4.32 0.653 . . . . . 

 Dose2 4.25 1.000 4.32 0.688 0.985 . . . . 

 Dose3 4.40 0.965 4.32 0.707 1.000 0.978 . . . 

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

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 0.26 0.967 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing negative trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 4.26 . . 

 Dose1 4.42 1.000 0.500 

 Dose2 4.29 1.000 0.500 

 Dose3 4.45 0.678 0.646 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

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

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 0.09 0.965 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing INCREASING trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

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Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl -4.26 . -4.26 . . . . . . 

 Dose1 -4.38 0.976 -4.31 0.518 . . . . . 

 Dose2 -4.25 1.000 -4.31 0.550 0.985 . . . . 

 Dose3 -4.40 0.965 -4.40 0.468 1.000 0.978 . . . 

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

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 0.26 0.967 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -4.26 . . 

 Dose1 -4.42 1.000 0.500 

 Dose2 -4.29 1.000 0.500 

 Dose3 -4.45 0.678 0.354 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

test for fish screen study - Metribuzin 

ANALYSIS RESULTS FOR VARIABLE VAR03 ( F body length (mm) ) 

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS 

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 

Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 

Use parametric analyses if neither test rejected, otherwise non-parametric analyses. 

 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 0.962 0.696 0.762 0.537 USE PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 44.80 1.28 0.64 2.86 42.76, 46.84 

 Dose1 4 44.68 0.56 0.28 1.24 43.79, 45.56 

 Dose2 4 44.98 1.22 0.61 2.72 43.03, 46.92 

 Dose3 4 44.13 0.94 0.47 2.12 42.64, 45.61 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 44.75 43.30 46.40 . . 

 Dose1 44.70 44.10 45.20 99.72 0.28 

 Dose2 44.80 43.80 46.50 100.39 -0.39 

 Dose3 44.35 42.80 45.00 98.49 1.51 

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

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 0.50 0.690 

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Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing negative trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl 44.80 . 44.82 . . . . . . 

 Dose1 44.68 0.996 44.82 0.593 . . . . . 

 Dose2 44.98 0.990 44.82 0.627 0.976 . . . . 

 Dose3 44.13 0.691 44.13 0.249 0.876 0.664 . . . 

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

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 0.98 0.806 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing negative trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 44.75 . . 

 Dose1 44.70 1.000 0.500 

 Dose2 44.80 0.889 0.615 

 Dose3 44.35 0.413 0.287 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

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

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 0.50 0.690 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing INCREASING trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl -44.80 . -44.64 . . . . . . 

 Dose1 -44.68 0.996 -44.64 0.670 . . . . . 

 Dose2 -44.98 0.990 -44.64 0.705 0.976 . . . . 

 Dose3 -44.13 0.691 -44.64 0.724 0.876 0.664 . . . 

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

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 0.98 0.806 

MannWhit - testing each trt median signif. different from control 

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Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -44.75 . . 

 Dose1 -44.70 1.000 0.500 

 Dose2 -44.80 0.889 0.385 

 Dose3 -44.35 0.413 0.713 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

test for fish screen study - Metribuzin 

ANALYSIS RESULTS FOR VARIABLE VAR04 ( M body length (mm) ) 

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS 

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 

Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 

Use parametric analyses if neither test rejected, otherwise non-parametric analyses. 

 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 0.970 0.845 0.636 0.606 USE PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 57.65 2.01 1.00 3.48 54.46, 60.84 

 Dose1 4 57.28 3.20 1.60 5.59 52.18, 62.37 

 Dose2 4 57.15 1.24 0.62 2.17 55.18, 59.12 

 Dose3 4 57.25 1.64 0.82 2.87 54.64, 59.86 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 58.05 55.10 59.40 . . 

 Dose1 57.05 53.60 61.40 99.35 0.65 

 Dose2 57.70 55.30 57.90 99.13 0.87 

 Dose3 57.65 55.00 58.70 99.31 0.69 

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

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 0.04 0.988 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing negative trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl 57.65 . 57.65 . . . . . . 

 Dose1 57.28 0.989 57.28 0.478 . . . . . 

 Dose2 57.15 0.975 57.20 0.487 1.000 . . . . 

 Dose3 57.25 0.987 57.20 0.504 1.000 1.000 . . . 

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

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

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 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 0.35 0.950 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing negative trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 58.05 . . 

 Dose1 57.05 0.889 0.386 

 Dose2 57.70 0.889 0.500 

 Dose3 57.65 0.678 0.500 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

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

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 0.04 0.988 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing INCREASING trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl -57.65 . -57.33 . . . . . . 

 Dose1 -57.28 0.989 -57.33 0.669 . . . . . 

 Dose2 -57.15 0.975 -57.33 0.704 1.000 . . . . 

 Dose3 -57.25 0.987 -57.33 0.723 1.000 1.000 . . . 

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

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 0.35 0.950 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -58.05 . . 

 Dose1 -57.05 0.889 0.614 

 Dose2 -57.70 0.889 0.500 

 Dose3 -57.65 0.678 0.500 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

test for fish screen study - Metribuzin 

ANALYSIS RESULTS FOR VARIABLE VAR05 ( F vitellogenin (ng/mL) ) 

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TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS 

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 

Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 

Use parametric analyses if neither test rejected, otherwise non-parametric analyses. 

 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 0.779 0.001 5.781 0.011 USE NON-PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 1895000 843899.7 421949.8 44.53 552167.3, 3237833 

 Dose1 4 3212500 3507396 1753698 109.18 -2368549, 8793549 

 Dose2 4 2432500 728943.3 364471.6 29.97 1272589, 3592411 

 Dose3 4 2245000 269382.0 134691.0 12.00 1816353, 2673647 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 1545000 1340000 3150000 . . 

 Dose1 1550000 1280000 8470000 169.53 -69.53 

 Dose2 2365000 1770000 3230000 128.36 -28.36 

 Dose3 2205000 1960000 2610000 118.47 -18.47 

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

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 0.36 0.780 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing negative trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl 1895000 . 2553750 . . . . . . 

 Dose1 3212500 0.631 2553750 0.774 . . . . . 

 Dose2 2432500 0.954 2432500 0.777 0.931 . . . . 

 Dose3 2245000 0.986 2245000 0.745 0.878 0.999 . . . 

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

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 3.20 0.362 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing negative trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 1545000 . . 

 Dose1 1550000 1.000 0.500 

 Dose2 2365000 0.235 0.906 

 Dose3 2205000 0.346 0.944 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams >highest dose (no sign. differences) 

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 Jonckheere >highest dose (no sign. differences) 

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

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 0.36 0.780 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing INCREASING trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl -1895000 . -1895000 . . . . . . 

 Dose1-3212500 0.631 -2630000 0.348 . . . . . 

 Dose2-2432500 0.954 -2630000 0.372 0.931 . . . . 

 Dose3-2245000 0.986 -2630000 0.385 0.878 0.999 . . . 

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

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 3.20 0.362 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -1545000 . . 

 Dose1 -1550000 1.000 0.500 

 Dose2 -2365000 0.235 0.094 

 Dose3 -2205000 0.346 0.056 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

test for fish screen study - Metribuzin 

ANALYSIS RESULTS FOR VARIABLE VAR06 ( M vitellogenin (ng/mL) ) 

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS 

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 

Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 

Use parametric analyses if neither test rejected, otherwise non-parametric analyses. 

 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 0.811 0.004 4.070 0.033 USE NON-PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 3039.65 5887.36 2943.68 193.69 -6328.45,12407.75 

 Dose1 4 8952.50 14478.52 7239.26 161.73 -14086.1,31991.05 

 Dose2 4 3128.84 4555.84 2277.92 145.61 -4120.52,10378.20 

 Dose3 4 1091.00 1108.25 554.12 101.58 -672.47, 2854.47 

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 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 141.65 6.30 11869.00 . . 

 Dose1 2494.00 332.00 30490.00 294.52 -194.52 

 Dose2 1303.00 8.35 9901.00 102.93 -2.93 

 Dose3 900.00 146.00 2418.00 35.89 64.11 

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

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 0.69 0.573 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing negative trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl 3039.65 . 5996.08 . . . . . . 

 Dose1 8952.50 0.622 5996.08 0.777 . . . . . 

 Dose2 3128.84 1.000 3128.84 0.624 0.747 . . . . 

 Dose3 1091.00 0.974 1091.00 0.485 0.544 0.984 . . . 

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

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 2.05 0.562 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing negative trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 141.65 . . 

 Dose1 2494.00 0.235 0.926 

 Dose2 1303.00 0.678 0.810 

 Dose3 900.00 0.678 0.646 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

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

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 0.69 0.573 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing INCREASING trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

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EPA MRID Number 48672202 

Page 60 of 70 

 Ctrl -3039.65 . -3039.65 . . . . . . 

 Dose1-8952.50 0.622 -4390.78 0.483 . . . . . 

 Dose2-3128.84 1.000 -4390.78 0.515 0.747 . . . . 

 Dose3-1091.00 0.974 -4390.78 0.532 0.544 0.984 . . . 

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

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 2.05 0.562 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -141.65 . . 

 Dose1 -2494.00 0.235 0.074 

 Dose2 -1303.00 0.678 0.190 

 Dose3 -900.00 0.678 0.354 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

test for fish screen study - Metribuzin 

ANALYSIS RESULTS FOR VARIABLE VAR07 ( F GSI ) 

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS 

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 

Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 

Use parametric analyses if neither test rejected, otherwise non-parametric analyses. 

 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 0.972 0.869 1.939 0.177 USE PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 15.90 1.97 0.99 12.40 12.76, 19.04 

 Dose1 4 17.60 3.68 1.84 20.92 11.74, 23.46 

 Dose2 4 15.40 1.64 0.82 10.64 12.79, 18.01 

 Dose3 4 18.63 3.53 1.76 18.95 13.01, 24.24 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 16.40 13.20 17.60 . . 

 Dose1 17.00 14.30 22.10 110.69 -10.69 

 Dose2 15.50 13.30 17.30 96.86 3.14 

 Dose3 19.60 13.80 21.50 117.14 -17.14 

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

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 1.10 0.387 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing negative trend 

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Page 61 of 70 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl 15.90 . 16.88 . . . . . . 

 Dose1 17.60 0.740 16.88 0.768 . . . . . 

 Dose2 15.40 0.989 16.88 0.801 0.702 . . . . 

 Dose3 18.63 0.423 16.88 0.818 0.956 0.416 . . . 

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

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 2.45 0.485 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing negative trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 16.40 . . 

 Dose1 17.00 0.678 0.718 

 Dose2 15.50 0.678 0.385 

 Dose3 19.60 0.235 0.772 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

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

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 1.10 0.387 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing INCREASING trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl -15.90 . -15.90 . . . . . . 

 Dose1 -17.60 0.740 -16.50 0.457 . . . . . 

 Dose2 -15.40 0.989 -16.50 0.487 0.702 . . . . 

 Dose3 -18.63 0.423 -18.63 0.132 0.956 0.416 . . . 

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

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 2.45 0.485 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

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 Ctrl -16.40 . . 

 Dose1 -17.00 0.678 0.282 

 Dose2 -15.50 0.678 0.615 

 Dose3 -19.60 0.235 0.228 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

test for fish screen study - Metribuzin 

ANALYSIS RESULTS FOR VARIABLE VAR08 ( M GSI ) 

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS 

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 

Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 

Use parametric analyses if neither test rejected, otherwise non-parametric analyses. 

 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 0.949 0.475 2.500 0.109 USE PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 1.32 0.45 0.23 34.32 0.60, 2.04 

 Dose1 4 1.56 0.14 0.07 9.24 1.33, 1.79 

 Dose2 4 1.37 0.17 0.08 12.07 1.10, 1.63 

 Dose3 4 1.64 0.16 0.08 9.60 1.39, 1.89 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 1.22 0.90 1.93 . . 

 Dose1 1.54 1.44 1.74 118.60 -18.60 

 Dose2 1.41 1.15 1.51 103.80 -3.80 

 Dose3 1.60 1.50 1.86 124.48 -24.48 

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

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 1.37 0.299 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing negative trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl 1.32 . 1.47 . . . . . . 

 Dose1 1.56 0.442 1.47 0.863 . . . . . 

 Dose2 1.37 0.986 1.47 0.889 0.726 . . . . 

 Dose3 1.64 0.246 1.47 0.901 0.975 0.487 . . . 

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

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 4.43 0.218 

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EPA MRID Number 48672202 

Page 63 of 70 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing negative trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 1.22 . . 

 Dose1 1.54 0.346 0.876 

 Dose2 1.41 0.678 0.615 

 Dose3 1.60 0.346 0.932 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

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

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 1.37 0.299 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing INCREASING trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl -1.32 . -1.32 . . . . . . 

 Dose1 -1.56 0.442 -1.47 0.265 . . . . . 

 Dose2 -1.37 0.986 -1.47 0.283 0.726 . . . . 

 Dose3 -1.64 0.246 -1.64 0.069 0.975 0.487 . . . 

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

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 4.43 0.218 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -1.22 . . 

 Dose1 -1.54 0.346 0.124 

 Dose2 -1.41 0.678 0.385 

 Dose3 -1.60 0.346 0.068 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

test for fish screen study - Metribuzin 

ANALYSIS RESULTS FOR VARIABLE VAR09 ( F tubercle score (median) ) 

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS 

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 

Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 

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Page 64 of 70 

Use parametric analyses if neither test rejected, otherwise non-parametric analyses. 

 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 . . . . NO DATA FOR TEST 

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

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 0.00 0.00 0.00 . . , . 

 Dose1 4 0.00 0.00 0.00 . . , . 

 Dose2 4 0.00 0.00 0.00 . . , . 

 Dose3 4 0.00 0.00 0.00 . . , . 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 0.00 0.00 0.00 . . 

 Dose1 0.00 0.00 0.00 . . 

 Dose2 0.00 0.00 0.00 . . 

 Dose3 0.00 0.00 0.00 . . 

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

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 . . . 1 . . . . . . . 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing negative trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl 0.00 . . . . . . . . 

 Dose1 0.00 . . . . . . . . 

 Dose2 0.00 . . . . . . . . 

 Dose3 0.00 . . . . . . . . 

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

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 0.00 1.000 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing negative trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 0.00 . . 

 Dose1 0.00 1.000 . 

 Dose2 0.00 1.000 . 

 Dose3 0.00 1.000 . 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams Dose1 

 Jonckheere Dose1 

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EPA MRID Number 48672202 

Page 65 of 70 

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

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 . . . 1 . . . . . . . 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing INCREASING trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl 0.00 . . . . . . . . 

 Dose1 0.00 . . . . . . . . 

 Dose2 0.00 . . . . . . . . 

 Dose3 0.00 . . . . . . . . 

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

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 0.00 1.000 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 0.00 . . 

 Dose1 0.00 1.000 . 

 Dose2 0.00 1.000 . 

 Dose3 0.00 1.000 . 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams Dose1 

 Jonckheere Dose1 

test for fish screen study - Metribuzin 

ANALYSIS RESULTS FOR VARIABLE VAR10 ( M tubercle score (median) ) 

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS 

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 

Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 

Use parametric analyses if neither test rejected, otherwise non-parametric analyses. 

 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 0.962 0.697 0.412 0.747 USE PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 38.75 6.95 3.47 17.93 27.70, 49.80 

 Dose1 4 42.38 6.66 3.33 15.72 31.77, 52.98 

 Dose2 4 39.13 4.59 2.29 11.73 31.82, 46.43 

 Dose3 4 40.75 8.21 4.11 20.15 27.68, 53.82 

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 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 40.00 30.50 44.50 . . 

 Dose1 40.75 36.50 51.50 109.35 -9.35 

 Dose2 39.00 34.00 44.50 100.97 -0.97 

 Dose3 43.25 29.00 47.50 105.16 -5.16 

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

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 0.24 0.864 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing negative trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl 38.75 . 40.56 . . . . . . 

 Dose1 42.38 0.790 40.56 0.733 . . . . . 

 Dose2 39.13 1.000 39.94 0.719 0.902 . . . . 

 Dose3 40.75 0.952 39.94 0.738 0.986 0.986 . . . 

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

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 0.91 0.823 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing negative trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 40.00 . . 

 Dose1 40.75 0.676 0.719 

 Dose2 39.00 1.000 0.500 

 Dose3 43.25 0.676 0.680 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

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

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 0.24 0.864 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing INCREASING trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl -38.75 . -38.75 . . . . . . 

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Page 67 of 70 

 Dose1 -42.38 0.790 -40.75 0.405 . . . . . 

 Dose2 -39.13 1.000 -40.75 0.432 0.902 . . . . 

 Dose3 -40.75 0.952 -40.75 0.448 0.986 0.986 . . . 

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

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 0.91 0.823 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -40.00 . . 

 Dose1 -40.75 0.676 0.281 

 Dose2 -39.00 1.000 0.500 

 Dose3 -43.25 0.676 0.320 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

test for fish screen study - Metribuzin 

ANALYSIS RESULTS FOR VARIABLE VAR11 ( fecundity ) 

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS 

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 

Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 

Use parametric analyses if neither test rejected, otherwise non-parametric analyses. 

 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 0.939 0.339 0.638 0.605 USE PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 8.85 2.82 1.41 31.89 4.36, 13.34 

 Dose1 4 6.36 5.04 2.52 79.28 -1.66, 14.38 

 Dose2 4 7.48 5.66 2.83 75.62 -1.52, 16.49 

 Dose3 4 6.48 5.06 2.53 78.06 -1.57, 14.53 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 7.83 6.74 13.00 . . 

 Dose1 6.10 0.85 12.40 71.88 28.12 

 Dose2 6.99 1.35 14.60 84.57 15.43 

 Dose3 6.29 0.55 12.80 73.24 26.76 

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

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 0.23 0.871 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing negative trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Page 131 of 205
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin 

EPA MRID Number 48672202 

Page 68 of 70 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl 8.85 . 8.85 . . . . . . 

 Dose1 6.36 0.804 6.92 0.345 . . . . . 

 Dose2 7.48 0.956 6.92 0.369 0.987 . . . . 

 Dose3 6.48 0.825 6.48 0.328 1.000 0.990 . . . 

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

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 1.04 0.792 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing negative trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 7.83 . . 

 Dose1 6.10 0.678 0.282 

 Dose2 6.99 0.889 0.442 

 Dose3 6.29 0.346 0.288 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

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

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 0.23 0.871 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing INCREASING trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl -8.85 . -7.29 . . . . . . 

 Dose1 -6.36 0.804 -7.29 0.760 . . . . . 

 Dose2 -7.48 0.956 -7.29 0.793 0.987 . . . . 

 Dose3 -6.48 0.825 -7.29 0.810 1.000 0.990 . . . 

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

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 1.04 0.792 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -7.83 . . 

Page 132 of 205
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin 

EPA MRID Number 48672202 

Page 69 of 70 

 Dose1 -6.10 0.678 0.718 

 Dose2 -6.99 0.889 0.558 

 Dose3 -6.29 0.346 0.712 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

test for fish screen study - Metribuzin 

ANALYSIS RESULTS FOR VARIABLE VAR12 ( fertility ) 

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS 

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 

Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 

Use parametric analyses if neither test rejected, otherwise non-parametric analyses. 

 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 0.960 0.657 1.260 0.332 USE PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 96.90 1.44 0.72 1.49 94.60, 99.20 

 Dose1 4 97.98 1.00 0.50 1.02 96.38, 99.57 

 Dose2 4 97.45 0.29 0.14 0.30 96.99, 97.91 

 Dose3 4 98.15 0.84 0.42 0.86 96.81, 99.49 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 97.05 95.00 98.50 . . 

 Dose1 97.70 97.20 99.30 101.11 -1.11 

 Dose2 97.45 97.10 97.80 100.57 -0.57 

 Dose3 98.50 96.90 98.70 101.29 -1.29 

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

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 1.31 0.316 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing negative trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl 96.90 . 97.62 . . . . . . 

 Dose1 97.98 0.325 97.62 0.903 . . . . . 

 Dose2 97.45 0.773 97.62 0.924 0.873 . . . . 

 Dose3 98.15 0.224 97.62 0.934 0.994 0.750 . . . 

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

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 2.85 0.415 

Page 133 of 205
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin 

EPA MRID Number 48672202 

Page 70 of 70 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing negative trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 97.05 . . 

 Dose1 97.70 0.335 0.882 

 Dose2 97.45 0.494 0.770 

 Dose3 98.50 0.283 0.913 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

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

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 1.31 0.316 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing INCREASING trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl -96.90 . -96.90 . . . . . . 

 Dose1 -97.98 0.325 -97.71 0.158 . . . . . 

 Dose2 -97.45 0.773 -97.71 0.169 0.873 . . . . 

 Dose3 -98.15 0.224 -98.15 0.062 0.994 0.750 . . . 

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

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 2.85 0.415 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -97.05 . . 

 Dose1 -97.70 0.335 0.118 

 Dose2 -97.45 0.494 0.230 

 Dose3 -98.50 0.283 0.087 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

Page 134 of 205
DATA EVALUATION RECORD 

METRIBUZIN 

Study Type: OCSPP 890.1400, In vivo Hershberger Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-24-2012 (MRID 48617904) 

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:

Kelly Luck, M.S. Date: 3/08/2012 

Secondary Reviewer: Signature:

David A. McEwen, B.S. Date: 3/12/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 3/29/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 3/29/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 135 of 205
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 136 of 205
METRIBUZIN/ 101101 

EPA Primary Reviewer: Yung G. Yang, Ph.D. 

Health Effects Division 

EPA Secondary Reviewer: Greg Akerman, Ph.D. 

Health Effects Division 

Jn vivo Hershberger Assay (2011) I Page 1 of 13 

OCSPP 890.1400/ OECD 441 

Signature: lj'-'? b-. :J~ Date: {/ s-/z.1 /--,.,) 1~ 

Signature: .b__. = A-= 

b' Date: __ ___;G5........,l_,_1,,__ r __,/+1 ..... r_ Template version 10/2011 

DATA EVALUATION RECORD 

STUDY TYPE: In Vivo Hershberger Assay (Rat); OCSPP 890.1400; OECD 441 

PC CODE: 101101 DP BARCODE: D398559 

TXR#: 0056258 CAS#: 21087-64-9 

TEST MATERIAL (PURITY): Metribuzin (94.4% a.i.) 

SYNONYMS: 4-amino-6-tert-4,5-dihydro-3-methylthio-1,2,4-triazin-5-one 

CITATION: Totis, M. (2011) Metribuzin evaluation in the Hershberger bioassay. Bayer 

S.A.S., Sophia Antipolis Cedex, France. Laboratory Study No.: SA 10342, 

November 10, 2011. MRID 48617904. Unpublished. 

SPONSOR: Bayer CropScience AG, Alfred Nobel Str. 50, Monheim, Germany 

TEST ORDER#: CON-101101-11 

EXECUTIVE SUMMARY: In a Hershberger Assay (MRID 48617904) screening for 

androgenic activity, metribuzin (94.4% a.i., batch/lot K285067) in an aqueous solution of 0.5% 

methylcellulose 400 + 0.4% Tween 80 was administered daily via oral gavage to groups of 58-

day old, castrated male Sprague Dawley rats (6/group) at dose levels of 0 (vehicle), 40, or 80 

mg/kg/day. An androgenic positive control group consisted of six castrated rats exposed to 0.4 

mg/kg/day of testosterone propionate (TP) by subcutaneous (s.c.) injection. 

To screen for potential anti-androgenic activity, metribuzin in an aqueous solution of 0.5% 

methylcellulose 400 + 0.4% Tween 80 was also administered daily via oral gavage to groups of 

58-day old, castrated male Sprague Dawley rats (6/group) at dose levels of 0 (vehicle), 20, 40, or 

80 mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.4 mg/kg/day by s.c. 

injection. The anti-androgenic positive control group consisted of six castrated rats were dosed 

orally with flutamide (FT) in com oil at 3 mg/kg/day for 10 days in conjugation with a daily s.c. 

dose of TP (0.4 mg/kg/day). Animals received TP alone was used as the anti-androgenic 

negative control. 

For both components of the assay, body weights were determined daily. The animals were dosed 

for 10 consecutive days and terminated approximately 24 hours after the final dose 

administration. At necropsy, the five androgen-dependent tissues were collected and weighed. 

In the androgen agonist assay, body weights throughout the study and body weight gains from 

Days 1 to 10 were similar across all groups. At 80 mg/kg/day, there was an increase in glans 

penis weight (j28%) which was statistically significant (p:S0.01, ANCOVA using terminal body 

Page 137 of 205
In vivo Hershberger Assay (2011) / Page 2 of 13 

METRIBUZIN/ 101101 OCSPP 890.1400/ OECD 441

weight as the co-variable). There were no treatment-related effects on seminal vesicles, ventral 

prostate, levator ani-bulbocavernosus (LABC), or Cowper’s glands in either dose group. 

Animals in the TP positive control group had increased (p≤0.01) accessory sex organ weights as 

follows: 1280% in seminal vesicles; 2200% in ventral prostate; 197% in LABC; 570% in 

Cowper’s gland; and 89% in glans penis. Five of the ten possible individual CVs in the control 

and high dose treatment groups exceeded the guideline performance criteria values. 

In the anti-androgen assay, body weights throughout the study and body weight gains from Days 

1 to 10 were similar across all groups. There were no treatment-related effects on accessory sex 

organ weights in either dose group. Animals dosed with TP + FT (positive control) had 

decreased (p≤0.01) accessory sex organ weights as follows: 78% in seminal vesicles; 67% in 

ventral prostate; 49% in LABC; 61% in Cowper’s gland; and 27% in glans penis. The 

performance criteria indicated that this assay was performing as expected. 

The high dose used in this study (80 mg/kg/day) was considered adequate based on data from the 

dose range-finding study which showed decreases in total body weight gain of 21% (compared to 

control) at this dose. 

Statistically significant organ weight changes were not seen in two or more of the five androgen 

sensitive issue weights. Metribuzin was negative for androgenicity and anti-androgenicity in the 

Hershberger assay. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Hershberger assay (OCSPP 

890.1400). 

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

provided. 

Page 138 of 205
In vivo Hershberger Assay (2011) / Page 3 of 13 

METRIBUZIN/ 101101 OCSPP 890.1400/ OECD 441

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Bayer S.A.S. 

Location: Sophia Antipolis Cedex, France 

Study Director: M. Totis 

Other Personnel: M. Blanck (Replacement Study Director), J.P. Kocwin (Laboratory Animal 

Resources), C. Jaussely (In-Life Supervisor), B. Trela (Responsible 

Technician), C. Pallen (Statistician), L. A. Tahmasseb (Senior Chemist), M. 

Vagner (Report Unit Assistant) 

Study Period: April 12 - November 10, 2011 

2. Test Substance: Metribuzin 

 Description: Light grey powder 

 Source: Bayer CropScience AG (Frankfurt Am Main, Germany) 

 Lot/Batch #: K285067 (expiration date 5/07/2012) 

 Purity: 94.4% a.i. 

 Stability: Stable in vehicle (aqueous solution of 0.5% methylcellulose 400 + 0.4% 

Tween 80) for up to 21 days at 5  3 °C. 

 CAS #: 21087-64-9 

 Structure: 

3. Reference Androgen: Testosterone propionate (TP) 

 Supplier: Sigma Aldrich-Fluka (Buchs, Switzerland) 

 Lot/Batch #: 436674/1 (expiration date 9/6/2011) 

 Purity: 99.8% 

 CAS #: 57-85-2 

4. Reference Anti-androgen: Flutamide (FT) 

 Supplier: Sigma Aldrich (St. Louis, MO) 

 Lot/Batch #: 099K1112 (expiration date 9/2/2011) 

 Purity: >99% 

 CAS #: 13311-84-7 

5. Solvent/Vehicle Control 

(metribuzin): 

Methylcellulose 400 + Tween 80 

 Supplier: Sigma-Aldrich/ Merck 

 Lot/Batch #: 060M0123V/ S5264887.914 

 Rationale (if other than water): Not provided 

 Final concentration: 0.5% + 0.4% 

 Solvent/Vehicle Control 

(FT): 

Methylcellulose 400 

 Supplier: Sigma-Aldrich 

 Lot/Batch #: 060M0123V 

 Rationale (if other than water): Not provided 

 Final concentration: 0.5% 

N

N

N

O

NH2

S

CH3

3CH

3CH

CH3

Page 139 of 205
In vivo Hershberger Assay (2011) / Page 4 of 13 

METRIBUZIN/ 101101 OCSPP 890.1400/ OECD 441

 Solvent/Vehicle Control 

(TP): 

Corn oil 

 Supplier: Sigma-Aldrich 

 Lot/Batch #: MKBD2427 

 Rationale (if other than water): Not applicable 

 Final concentration: Not applicable 

6. Test Animals: 

 Species: Rat (castrated males only) 

 Strain: Sprague Dawley [Crl:CD (SD)] 

 Age/weight at dose initiation: Post-natal day (PND) 58/ 236.1-306.6 g 

 Source: Charles River Laboratories(St Germain-sur-l’Arbresle, France) 

 Housing: Rats were housed three per cage in makrolon cages containing heat-treated 

soft wood bedding 

 Diet: A04C-10 pelleted rodent diet, ad libitum 

Phytoestrogen content <350 μg of genistein equivalents/gram diet 

 Water: Filtered tap water, softened by ion exchange resins, ad libitum

 Environmental conditions: Temperature: 

Humidity: 

Air changes: 

Photoperiod: 

20-24 oC 

40-70% 

target of 10-15/hr 

12 hrs light/12 hrs dark 

 Acclimation period: Rats were castrated at the supplier on PND 43, received at the study facility 

on PND 48-49, and allowed to acclimate at the facility for 9-10 days prior to 

initiation of dosing 

B. STUDY DESIGN

1. In-life dates: Start: April 22, 2011 End: May 3, 2011 

2. Study Design: In a Hershberger Assay conducted to screen for potential androgenic 

activity, the test substance was administered daily via oral gavage to castrated male rats. 

Positive androgenic activity is defined as a significant increase in two or more target organ 

weights compared to the vehicle control. To screen for the potential anti-androgenic 

activity, the test substance was also administered daily via oral gavage to castrated male rats 

in conjunction with a daily dose of TP (0.4 mg/kg/day) by s.c. injection. Anti-androgenic 

activity is indicated by a statistically significant decrease in two or more target organ 

weights of the treated groups (test substance + TP) compared to the TP-only control group. 

For both assays, the animals were dosed for 10 consecutive days and necropsied 

approximately 24 hours after the final dose administration for organ weight measurements. 

3. Study Schedule: Rats were castrated at the supplier on PND 43 and allowed at least 4 days 

for recovery before shipment to the study facility. Animals were received at the study 

facility on PND 48-49 and allowed to acclimate for 9-10 days. The dose administration 

period was from PND 58 through PND 67. Rats were euthanized on PND 68 approximately 

24 hours after the last dose and necropsied for organ weight measurements. 

4. Animal Assignment: Animals were assigned to the test groups noted in Table 1 using 

computerized randomization procedures which selected animals from the middle of the 

weight range of the available animals. Statistical analysis indicated that there were no 

significant differences in group means at study initiation. Furthermore, the body weight of 

each animal was within 20% of the overall mean. 

Page 140 of 205
In vivo Hershberger Assay (2011) / Page 5 of 13 

METRIBUZIN/ 101101 OCSPP 890.1400/ OECD 441

TABLE 1. Study Designa

Test group Dose (mg/kg/day) # of Males 

Androgen Agonist Assay 

Vehicle control (negative control) 0 6 

Low 40 6 

High 80 6 

Testosterone propionate (TP), (positive control)b 0.4 6 

Anti-Androgen Assay 

Vehicle control (+TP) (negative control) 0 6 

Low (+TP) 20 + 0.4 6 

Mid (+TP) 40 + 0.4 6 

High (+TP) 80 + 0.4 6 

Flutamide + TP, (positive control) 3 + 0.4 6 

a Data were obtained from pages 17 and 18 of the study report. 

b This dose group served as the positive control for the androgen agonist assay and the negative control for the 

anti-androgen assay. 

5. Dose Selection Rationale: The dose levels used in this study were selected based on the 

results of a dose range-finding study in Sprague Dawley rats. A summary of this study is 

included as an Appendix to this DER.

6. (a) Dose Preparation: Dose formulations of the test substance were prepared by mixing 

appropriate amounts of metribuzin with the vehicle, an aqueous solution of 0.5% 

methylcellulose 400 + 0.4% Tween 80. Dose formulations of TP were prepared by mixing 

the appropriate amount of TP in corn oil, and dose formulations of FT were prepared by 

mixing appropriate amounts of FT with an aqueous solution of 0.5% methylcellulose 400. 

Dose formulations were prepared once during the study and stored at 5  3 °C when not in 

use. Stability was tested in a separate study;1 the test substance in the vehicle was found to 

be stable at concentrations of 1 and 100 g/L (which bracketed those used in the current 

study) for up to 21 days under similar conditions of usage and storage to those of the current 

study. Homogeneity was tested at two concentrations of metribuzin in vehicle (4 and 16 

g/L) by sampling the surface, middle, and bottom of the suspension after stirring for 15 

minutes. Achieved concentration was tested separately at 8 g/L; in addition, the mean 

results of the homogeneity analyses were used for achieved concentrations. The 

homogeneity and achieve concentration of the TP and FT dose formulations were also tested 

at one dose level each. 

 

1 Vincent, M. (2011, on going): Metribuzin, Stability in methylcellulose 400 + 0.4% Tween 80, Bayer 

CropScience, Sophia Antipolis, France (SA 11089). 

Page 141 of 205
In vivo Hershberger Assay (2011) / Page 6 of 13 

METRIBUZIN/ 101101 OCSPP 890.1400/ OECD 441

(b) Dose Analysis: 

Results 

Homogeneity (%CV): 0.1% and 0.4% 

Stability: No data provided 

Concentration (% of nominal): 98.6-101%

The analytical data indicated that the mixing procedure was adequate and that the variation 

between nominal and actual dosage to the animals was acceptable. The study referenced 

above should be submitted for verification of the stability findings. 

For TP, the %CV for homogeneity analyses was 1.2% and the average concentration was 

found to be 99.1% of nominal. For FT, the %CV for homogeneity analyses was 1.6% and 

the average concentration was found to be 95.1% of nominal. 

7. Dosage administration: Test formulations were administered to the animals daily via oral 

gavage (5 mL/kg bw) for 10 days. TP was given via s.c. injection at 0.5 mL/kg bw, and FT 

was administered via oral gavage at 5 mL/kg bw. Dose volumes were adjusted daily based 

on the concurrent body weight measurement. 

8. Statistics: The following parameters were analyzed statistically: body weights, body 

weight gains, terminal body weights, and organ weights. For body weight gain, the data 

were first analyzed using Bartlett’s test. If the data were not significant, an ANOVA test 

was performed; a two-sided Dunnett’s test was conducted if the results of the ANOVA were 

significant. If the results of Bartlett’s test were significant, a Kruskal-Wallis test was 

performed, followed by a two-sided Dunn’s test for significant data. The same procedures 

were used for body weight (except terminal body weight), except that data were transformed 

(log transformation) and a second Bartlett’s test was conducted if the data were significant 

after the first Bartlett’s test. 

For organ weights and terminal body weight, the data were first analyzed using Levene’s 

test; if the data were significant, a data transformation (log transformation) was conducted. 

If the data were not significant, an ANOVA test was performed; a one-sided (organ weights) 

or two-sided (terminal body weights) Dunnett’s test was conducted if the results of the 

ANOVA were significant. If the results of Levene’s test (after transformation) showed 

significance, a Kruskal-Wallis test was performed, followed by a one-sided (organ weights) 

or two-sided (terminal body weights) Dunn’s test for significant data.

In addition, analysis of covariance (ANCOVA) was conducted on organ weights using 

terminal body weight as a covariable; adjusted means were compared using a one-sided 

Dunnett’s test. For comparison of data from the positive control with data from the negative 

control in the androgen agonist and anti-androgen assays, the data were analyzed using an F 

test; for body weights and organ weights, the data were log-transformed and reanalyzed if 

significant after the F test. Data that were not significant were analyzed using a one- or twoPage 142 of 205
In vivo Hershberger Assay (2011) / Page 7 of 13 

METRIBUZIN/ 101101 OCSPP 890.1400/ OECD 441

sided T-test, and data that were significant were analyzed using a modified one- or twosided T-test. Significance was denoted at p≤0.05. 

C. METHODS

1. Clinical Examinations: All animals were examined twice daily (once daily on weekends 

and public holidays) for mortality and moribundity. Clinical examinations were conducted 

daily. 

2. Body Weight: Animals were weighed at randomization, daily throughout the dosing 

period, and just before termination. 

3. Food Consumption (Optional): Food consumption was not measured. 

4. Serum Hormone Measurements (Optional): Serum hormone measurements were not 

conducted. 

5. Dissection and Measurement of Tissue and Organ Weights: On PND 68 (approximately 

24 hours after the final administration of the test substance), all animals were anesthetized 

by isoflurane inhalation, exsanguinated, and subjected to a gross necropsy. The five 

mandatory androgen-dependent organs (ventral prostate, seminal vesicles, LABC, Cowper’s 

gland, and glans penis) were excised, trimmed free of adhering tissue, and weighed fresh 

(unfixed) according to the standard operating procedures detailed in the U.S. EPA Guideline 

(OCSPP 890.1400). 

II. RESULTS

A. OBSERVATIONS

1. Mortality: All animals survived until scheduled termination. 

2. Clinical signs of toxicity: No clinical signs of toxicity were observed in animals for any 

dose groups. 

B. BODY WEIGHT AND WEIGHT GAIN: Selected body weight and body weight gain 

data for the androgen agonist assay are presented in Table 2. Body weights throughout the 

study and body weight gains from Days 1 to 10 were similar across all groups. At 80 

mg/kg/day, there was a statistically significant (p≤0.01) loss in mean body weight from 

Days 1 to 2 (loss of 5.7 g compared to a mean gain of 10.4 g in the vehicle control). The 

mean body weight gain (Days 1-10) in this group was decreased 24% compared to the 

control; however, this decrease was not statistically significant. At 40 mg/kg/day, mean 

body weight gain from Days 1 to 2 was decreased by 31% (not statistically significant) 

compared to the vehicle control; mean body weights were not affected by the end of the 

study.

Page 143 of 205
In vivo Hershberger Assay (2011) / Page 8 of 13 

METRIBUZIN/ 101101 OCSPP 890.1400/ OECD 441

TABLE 2. Selected Group Mean Body Weights and Cumulative Body Weight Gains (g) in the Androgen 

Agonist Assaya

Study Day # 

Dose (mg/kg/day) 

Vehicle Control Positive Control 

Vehicle + TP (0.4) Metribuzin (40) Metribuzin (80) 

N Mean SD N Mean SD N Mean SD N Mean SD 

1 6 274.1 22.6 6 273.7 14.8 6 267.0 13.6 6 275.2 28.2 

4 6 298.4 22.7 6 301.7 16.5 6 288.6 16.0 6 289.5 29.1 

7 6 315.5 23.7 6 322.6 16.6 6 306.7 13.9 6 302.5 34.1 

11 6 340.7 24.9 6 354.4 19.7 6 333.1 12.2 6 326.8 37.0 

Body Weight Gain 

(Days 1-10)b 

6 58.0 13.6 6 70.2 6.8 6 56.4 14.6 6 44.1 9.8 

a Data were obtained from Tables 2a, 2b, 4a, 4b, 5a, and 5b on pages 44, 46, 62, 64, 71, and 73 of the study 

report. 

b The study authors did not report total body weight gain, only body weight gain through the final day of dosing. 

N Number of animals in the group 

SD Standard Deviation

Selected body weight and body weight gain data for the anti-androgen assay are presented in 

Table 3. Body weights throughout the study and body weight gain from Days 1 to 10 were 

similar across all groups. At 80 mg/kg/day, there was a statistically significant (p≤0.01) loss 

in mean body weight from Days 1 to 2 (loss of 0.87 g compared to a mean gain of 8.09 g in 

the vehicle control). The mean body weight gain (Days 1-10) in this group was decreased 

16% compared to the control; however, this decrease was not statistically significant. At 40 

mg/kg/day, mean body weight gain from Days 1 to 2 was decreased (p≤0.05) by 66% 

compared to the vehicle control; mean body weights were not affected by the end of the 

study. 

TABLE 3. Selected Group Mean Body Weights and Cumulative Body Weight Gains (g) in the Anti-Androgen 

Assaya

Study Day # 

Dose (mg/kg/day) 

Vehicle Control + 

TP (0.4) 

Positive Control 

TP (0.4) + FT (3) Metribuzin (20) Metribuzin (40) Metribuzin (80) 

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 

1 6 273.7 14.8 6 269.1 22.5 6 281.2 18.2 6 271.4 15.9 6 277.3 14.7

4 6 301.7 16.5 6 293.2 24.3 6 304.2 18.3 6 295.5 18.3 6 297.3 17.9

7 6 322.6 16.6 6 311.8 29.0 6 324.5 20.2 6 317.0 19.4 6 318.5 18.1

11 6 354.4 19.7 6 342.2 32.4 6 356.4 22.7 6 348.3 20.6 6 347.1 22.4

Body Weight 

Gain (Days 1-

10)b 

6 70.2 6.8 6 63.7 9.8 6 64.5 7.8 6 67.0 10.9 6 59.3 11.1

a Data were obtained from Tables 2c, 2d, 4c, 4d, 5c, and 5d on pages 48, 50, 66, 68, 75, and 77 of the study report. 

b The study authors did not report total body weight gain, only body weight gain through the final day of dosing. 

N Number of animals in the group 

SD Standard Deviation 

Page 144 of 205
In vivo Hershberger Assay (2011) / Page 9 of 13 

METRIBUZIN/ 101101 OCSPP 890.1400/ OECD 441

C. FOOD CONSUMPTION (Optional): Food consumption was not measured. 

D. SERUM HORMONE CONCENTRATIONS (Optional): Serum hormone 

measurements were not conducted. 

E. ORGAN WEIGHTS: Accessory sex organ weights for the androgen agonist assay are 

presented in Table 4. At 80 mg/kg/day, there was an increase in glans penis weight (↑28%) 

which was statistically significant (p≤0.01) using ANCOVA analysis (with terminal body 

weight as the co-variable). There were no treatment-related effects on seminal vesicles, 

ventral prostate, LABC, or Cowper’s glands in either dose group. Animals in the TP 

positive control group had increased (p≤0.01) accessory sex organ weights as follows: 

1280% in seminal vesicles; 2200% in ventral prostate; 197% in LABC; 570% in Cowper’s 

gland; and 89% in glans penis. 

Percent CVs for the vehicle control and the 80 mg/kg/day group were compared to the 

performance criteria in the Guidelines. The %CVs for the following organs were outside 

the performance criteria: seminal vesicles in the control and treated group (49% CV for 

both, compared to maximum 40% CV); ventral prostate in the control and treated group 

(94% and 78% CV, compared to maximum 45% CV); and glans penis in the treated group 

(28% CV, compared to maximum 22% CV). The %CV values for the other organs were 

below the maximum permissible values. The study author noted that the mean organ 

weights in animals from the 80 mg/kg/day group were within the range of historical control 

data for the facility; the historical data were included in the submission (the number of 

animals represented in the historical control data was not reported). 

The Guidelines specify that the study should be repeated when (i) three or more of the ten 

possible individual CVs in the control and high dose treatment groups exceed the 

maximums designated for agonist and antagonist studies, and (ii) at least two target tissues 

were marginally insignificant, i.e., p values between 0.05 and 0.10. The study author did 

not report the p values for organ weights. The p values for adjusted organ weights (control 

and high dose group) were calculated by the reviewers; the p values were >0.10 for ventral 

prostate (0.499), LABC (0.899), Cowper’s glands (0.214), and seminal vesicles (0.629). 

Page 145 of 205
In vivo Hershberger Assay (2011) / Page 10 of 13 

METRIBUZIN/ 101101 OCSPP 890.1400/ OECD 441

TABLE 4. Accessory Sex Organ Weights (mg) from Androgen Agonist Assay in Sprague Dawley Ratsa

Organ 

Dose (mg/kg/day) 

Vehicle Control Positive Control 

Vehicle + TP (0.4) Metribuzin (40) Metribuzin (80) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

Seminal 

vesicles 

6 39.7 19.4 48.9 6 547.5** 

(↑1280) 

160.0 29.2 6 41.7 13.9 33.3 6 44.3 21.9 49.4

Ventral 

prostate 

6 8.7 8.1 94.1 6 199.8** 

(↑2200) 

30.6 15.3 6 24.1 21.3 88.4 6 16.6 13.0 78.4

LABC 6 204.2 18.5 9.1 6 606.8** 

(↑197) 

89.6 14.8 6 197.3 24.9 12.6 6 198.8 39.6 19.9

Cowper’s 

glands 

6 6.5 3.6 55.3 6 43.3** 

(↑570) 

11.9 27.4 6 5.9 2.4 40.3 6 7.0 2.0 28.4

Glans penis 6 40.4 5.0 12.4 6 76.2** 

(↑89) 

5.8 7.6 6 39.3 7.2 18.4 6 51.7b 14.7 28.4

a Data were obtained from Tables 6a and 6b on pages 80 and 83 of the study report. Percent differences from control are 

included in parentheses. 

b Using ANCOVA with terminal body weight as the co-variable, glans penis weights was increased (p≤0.01) 28% compared to 

the control. 

N Number of animals in the group 

SD Standard Deviation 

CV Coefficient of Variation 

** Significantly different from controls at p≤0.01 

Accessory sex organ weights for the anti-androgen assay are presented in Table 5. The 

weights of the accessory sex organs in the metribuzin treatment groups were comparable to 

the controls. Animals dosed with TP + FT (positive control) had decreased (p≤0.01) 

accessory sex organ weights as follows: 78% in seminal vesicles; 67% in ventral prostate; 

49% in LABC; 61% in Cowper’s gland; and 27% in glans penis. 

Percent CVs for the vehicle control and the 80 mg/kg/day group were compared to the 

performance criteria in the Guidelines. All %CV values were less than the maximum 

permissible values. 

The study author did not report whether there were any gross pathological findings at 

necropsy for either assay. 

Page 146 of 205
In vivo Hershberger Assay (2011) / Page 11 of 13 

METRIBUZIN/ 101101 OCSPP 890.1400/ OECD 441

TABLE 5. Accessory Sex Organ Weights (mg) from Anti-Androgen Agonist Assay in Sprague Dawley Ratsa

Organ

Dose (mg/kg/day) 

Vehicle Control + 

TP (0.4) 

Positive Control 

TP (0.4) + FT (3) Metribuzin (20) Metribuzin (40) Metribuzin (80) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

Seminal 

vesicles 

6 547.5 160.0 29.2 6 121.6** 

(↓78) 

10.4 8.6 6 512.4 116.2 22.7 6 521.4 90.9 17.4 6 532.6 136.3 25.6 

Ventral 

prostate 

6 199.8 30.6 15.3 6 65.6** 

(↓67) 

19.6 29.9 6 186.6 27.8 14.9 6 197.1 47.0 23.8 6 194.1 41.2 21.2 

LABC 

6 606.8 89.6 14.8 6 307.9** 

(↓49) 

66.3 21.5 6 605.2 69.7 11.5 6 614.5 82.7 13.5 6 557.0 75.7 13.6 

Cowper’s 

glands 

6 43.3 11.9 27.4 6 17.1** 

(↓61) 

5.1 30.0 6 37.4 4.0 10.7 6 41.7 6.4 15.2 6 35.2 7.5 21.3 

Glans penis 6 76.2 5.8 7.6 6 55.6** 

(↓27) 

7.9 14.2 6 76.5 7.1 9.3 6 84.7 12.0 14.2 6 87.1 10.4 11.9 

a Data were obtained from Tables 6c and 6d on pages 86 and 89 of the study report. Percent differences from controls are included in 

parentheses. 

N Number of animals in the group 

SD Standard Deviation 

CV Coefficient of Variation 

** Significantly different from controls at p≤0.01 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: The investigator concluded that the only effect 

observed on the five androgen dependent tissue weights measured following administration 

of up to 80 mg/kg/day of metribuzin alone for 10 days to castrated Sprague Dawley male 

rats was a slight glans penis weight increase noted at 80 mg/kg/day. However, a statistical 

increase in one tissue does not meet the criteria for a positive androgen agonist, i.e. a 

statistically-significant increase (p≤0.05) in any two or more of the five required androgendependent tissue weights. 

B. AGENCY COMMENTS: All animals survived until scheduled termination. There were 

no signs of clinical toxicology. The study author did not report whether there were any 

gross pathological findings at necropsy. 

In the androgen agonist assay, body weights throughout the study and body weight gains 

from Days 1 to 10 were similar across all groups. At 80 mg/kg/day, there was an increase in 

glans penis weight (↑28%) which was statistically significant (p≤0.01) only using 

ANCOVA analysis (with terminal body weight as the co-variable). There were no 

treatment-related effects on seminal vesicles, ventral prostate, LABC, or Cowper’s glands in 

either dose group. Animals in the TP positive control group had increased (p≤0.01) 

accessory sex organ weights as follows: 1280% in seminal vesicles; 2200% in ventral 

prostate; 197% in LABC; 570% in Cowper’s gland; and 89% in glans penis. 

In the anti-androgen assay, body weights throughout the study and body weight gains from 

Days 1 to 10 were similar across all groups. There were no treatment-related effects on 

accessory sex organ weights in either dose group. Animals dosed with TP + FT (positive 

control) had decreased (p≤0.01) accessory sex organ weights as follows: 78% in seminal 

vesicles; 67% in ventral prostate; 49% in LABC; 61% in Cowper’s gland; and 27% in glans 

penis. The performance criteria indicated that this assay was performing as expected. 

Page 147 of 205
In vivo Hershberger Assay (2011) / Page 12 of 13 

METRIBUZIN/ 101101 OCSPP 890.1400/ OECD 441

The high dose used in this study (80 mg/kg/day) was considered adequate based on data 

from the dose range-finding study which showed decreases in total body weight gain of 21% 

(compared to control) at this dose. 

No statistically significant organ weight changes were seen in two or more of the five 

androgen sensitive tissue weights. Metribuzin was negative for androgenicity and antiandrogenicity in the Hershberger assay. 

C. STUDY DEFICIENCIES: None 

Page 148 of 205
In vivo Hershberger Assay (2011) / Page 13 of 13 

METRIBUZIN/ 101101 OCSPP 890.1400/ OECD 441

APPENDIX

Since this is a range-finding study, only a summary is provided to confirm the adequacy of the 

dose selection rationale used in the Hershberger assay (MRID 48617904). 

In a preliminary range-finding assay, metribuzin (94.4%; batch# K285067) in an aqueous 

solution of 0.5% methylcellulose 400 + 0.4% Tween 80 was administered via daily oral gavage 

in a dose volume of 5 mL/kg body weight to groups of 4 adult male Sprague Dawley 

[Crl:CD(SD)] rats at doses of 0, 20, 50, or 80 mg/kg/day. The animals were dosed for 10 

consecutive days and necropsied approximately 24 hours after the final dose administration. 

All animals survived until scheduled termination. At 80 mg/kg/day, treatment-related clinical 

signs consisted of reduced motor activity and half closed eyes in all animals between Days 1 and 

3. When compared to controls, the mean body weight was decreased by 6.5% on Day 2, 

corresponding to a body weight loss of 19 g compared to a body weight gain of 2 g for controls. 

The terminal body weight was decreased slightly compared to the controls (↓2.2%). The mean 

cumulative body weight gain between Days 1 and 11 was decreased by 21%, compared to the 

control group. 

At 50 mg/kg/day, treatment-related clinical signs consisted of reduced motor activity and half 

closed eyes in all animals between Days 1 and 3. When compared to controls, the mean body 

weight was reduced by 6.5% on Day 2, corresponding to a body weight loss of 18 g compared to 

a body weight gain of 2 g for controls. The terminal body weight was decreased slightly 

compared to the controls (↓1.4%). The mean cumulative body weight gain between Days I and 

11 was decreased by 11%, compared to the control group. 

At 20 mg/kg/day, there were not treatment-related clinical signs throughout the study and body 

weight parameters were not affected by treatment. 

It did not appear that statistical analyses were conducted. 

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

that it was not subjected to specific Quality Assurance inspections. 

Page 149 of 205
DATA EVALUATION RECORD 

METRIBUZIN 

Study Type: OCSPP 890.1450, Female Pubertal Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-66-2012 (MRID 48672203) 

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:

Kelly Luck, M.S. Date: 6/04/2012 

Secondary Reviewer: Signature:

Rebecca L. Bryan, B.S. Date: 6/07/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 6/29/2012 

Quality Assurance: Signature:

Steven Brecher, Ph.D., D.A.B.T. Date: 6/28/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 150 of 205
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 151 of 205
METRIBUZIN/ IOl 101 

Primary Reviewer: Deborah Smegal, MPH 

Health Effects Division 

Secondary Reviewer: Elizabeth Mendez, Ph.D. 

Health Effects Division 

Female Pubertal Assay (2011) I Page 1 of 15 

OCSPP 890.1450/ OECD None 

Sigtfi.2~ Date: / . 

Signature: }=~ (j.a;...br===~ 

Date: b f~ ur 

Template version 08/2011 

DATA EVALUATION RECORD 

STUDY TYPE: Female Pubertal Assay; OCSPP 890.1450; OECD None. 

PC CODE: 101101 DP BARCODE: D401219 

TXR#: 0056258 CAS#: 21087-64-9 

TEST MATERIAL (PURITY): Metribuzin (94.4% a.i.) 

SYNONYMS: 4-Amino-6-( I, l-dimethylethyl)-3-(methylthio )-1,2,4-triazin-5( 4H)-one 

CITATION: Blanck, M. (2011) Metribuzin assessment of pubertal development and thyroid 

function in juvenile/peripubertal male and female rats. Bayer S.A.S., Sophia 

Antipolis Cedex, France. Laboratory Study No.: SA 10341, December 1, 2011. 

MRID 48672203. Unpublished. 

SPONSOR: Bayer CropScience AG, Alfred Nobel Str. 50, Monheim, Germany 

TEST ORDER#: CON-101101-127 

EXECUTIVE SUMMARY: In a Female Pubertal Assay (MRID 48672203), 15 Sprague 

Dawley rats/dose group were treated daily via oral gavage with metribuzin (94.4% a.i., 

Batch/lot# K285067) in aqueous 0.5% methylcellulose 400 + 0.4% Tween 80 (vehicle) at doses 

of 0 (vehicle), 60, or 120 mg/kg/day from post-natal day (PND) 22 to 42. Animals were 

examined for vaginal opening (VO) daily beginning on PND 22, and age and weight at day of 

attainment were recorded. Following sacrifice on PND 42, serum thyroxine (T 4) and thyroid 

stimulating hormone (TSH) levels were determined using enzyme immunoassay kits. Adrenals, 

liver, pituitary, thyroid, and urogenital organ weights were recorded and microscopic 

examination of the ovaries, uterus, thyroid, and kidneys were performed. In addition, any livers 

found to be enlarged at necropsy were subjected to microscopic examination. 

There were no effects of treatment on mean age at first vaginal estrus, mean cycle length, percent 

cycling, or percent regularly cycling; on the weights of kidneys, pituitary, adrenals, ovaries, or 

uterus (wet or blotted); or on levels of creatinine, blood urea nitrogen, total protein, total 

bilirubin, aspartate aminotransferase, or alkaline phosphatase. There were no treatment-related 

histopathological findings in the thyroid gland, ovaries, uterus, or kidneys. 

At 120 mg/kg/day, increased salivation was observed in 7/15 animals on one or several 

occasions throughout the study. The age (PND 36.47 treated vs. PND 35.00 controls) and body 

weight (j6%) at VO were slightly increased (not significant; NS) compared to the controls. 

Overall body weight gains were comparable to the control. Absolute, adjusted (for body weight 

Page 152 of 205
Female Pubertal Assay (2011) / Page 2 of 15 

METRIBUZIN/ 101101 OCSPP 890.1450/ OECD None

on PND 21), and relative liver weights were increased (p≤0.01) by 21-22% and adjusted thyroid 

gland weights were increased (p≤0.05) by 15% in the 120 mg/kg/day group compared to 

controls. At necropsy, enlarged thyroid was observed in 1/15 rats and enlarged liver was 

observed in 5/15 rats in this group; there were increased numbers of mitoses in 4/5 examined 

livers. Serum T4 levels were decreased (p≤0.01) by 44% in rats dosed at 120 mg/kg/day; mean 

TSH levels were increased (NS) by 33% and alanine aminotransferase was increased (p≤0.05) in 

this group. 

At 60 mg/kg/day, increased salivation was observed in 6/15 rats on several occasions throughout 

the study. Final body weight and body weight gain, and age and body weight at VO in this 

group were comparable to the control. Relative liver weights were increased (p≤0.05) by 10%; 

thyroid gland weights were comparable to the controls at this dose level. At necropsy, enlarged 

liver was observed in 1/15 rats and increased numbers of mitoses were observed in this liver. 

Mean TSH levels were increased (NS) by 29% at 60 mg/kg/day; however, serum T4 and alanine 

aminotransferase levels in this group were comparable to the controls. 

The dose levels tested in this assay are adequate based on the results of a dose range-finding 

study. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Female Pubertal Assay 

(OCSPP 890.1450). 

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

Assurance statements were provided. 

Page 153 of 205
Female Pubertal Assay (2011) / Page 3 of 15 

METRIBUZIN/ 101101 OCSPP 890.1450/ OECD None

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Bayer S.A.S, Bayer CropScience 

Location: Sophia Antipolis Cedex, France 

Study Director: M. Blanck 

Other Personnel: S. Lautraite (Replacement Study Director), J.P. Kocwin (Laboratory Animal Resources), C. 

Jaussely (In-Life Supervisor), H. Vincent (Responsible Technician), C. Pallen (Statistician), 

L. Amir Tahmasseb (Senior Chemist), D. De Barbeyrac (Clinical Pathology Supervisor), L. 

Elies (Pathologist), M. Vagner (Report Unit Assistant) 

Study Period: March 10 - December 01, 2011 

2. Test Substance: Metribuzin 

 Description: Light grey powder 

 Source: Bayer CropScience AG (Frankfurt Am Main, Germany) 

 Lot/Batch #: K285067 (expiration date 5/07/2012) 

 Purity: 94.4% a.i. 

 Stability: Stable in vehicle (aqueous solution of 0.5% methylcellulose 400 + 0.4% Tween 80) for up to 

21 days at 5  3 °C. 

 CAS #: 21087-64-9 

 Structure: 

3. Vehicle: 0.5% methylcellulose 400 + 0.4% Tween 80

4. Test Animals:

 Species: Rat (females only) 

 Strain: Sprague Dawley [Crl:CD(SD)] 

 Age/Weight at 

Study Initiation: 

PND 22/ 49.3 – 67.5 g 

 Source: Charles River Laboratories(St Germain-sur-l’Arbresle, France) 

 Housing: Rats were housed 2-3 per cage in makrolon cages containing heat-treated laboratory-grade 

wood shavings 

 Diet: A04C-10 pelleted rodent diet (Scientific Animal Food and Engineering, Augy, France), ad 

libitum; 

Dietary level of phytoestrogens (including genistein, genistin, daidzin, daidzein and glycitin) 

were ≤300 μg of phytoestrogens equivalents/gram of diet. 

 Water: Filtered tap water, softened by ion exchange resins, ad libitum

 Environmental 

Conditions: 

Temperature: 

Humidity: 

Air changes: 

Photoperiod: 

20-24 °C 

40-70% 

10-15/hr 

14 hrs light/ 10 hrs dark 

B. STUDY DESIGN

1. In-Life Dates: Start: April 15, 2011 End: May 6, 2011 

2. Mating: Time-mated pregnant dams were received from the supplier on GD 7 or 8. Any 

litters with fewer than 8 pups and any litters not delivered by GD 23 were excluded. Litters 

N

N

N

O

NH2

S

CH3

3CH

3CH

CH3

Page 154 of 205
Female Pubertal Assay (2011) / Page 4 of 15 

METRIBUZIN/ 101101 OCSPP 890.1450/ OECD None

with more than 8 pups were standardized by culling, on PND 4, to 8 pups per litter, with, if 

possible, an equal number of pups of each sex. 

3. Animal Assignment: Offspring were weaned on PND 21 and weighed. Animals were 

randomly assigned to the test groups noted in Table 1 using a procedure that ensured a 

similar body weight distribution among groups. Littermates were not assigned to the same 

treatment group. 

TABLE 1. Study Design a

Test group Dose (mg/kg/day) # of Females 

Control 0 15 

Low (Metribuzin) 60 15 

High (Metribuzin) 120 15 

a Data were obtained from page 18 of the study report. 

4. Dose Selection Rationale: The dose levels were selected based on the results from a dose 

range-finding study, which was included as an appendix in the current study report. The 

dose range-finding study was also included with a previously submitted uterotrophic study 

and was evaluated at that time (refer to the DER for MRID 48617907). In the study, 

metribuzin in 0.5% methylcellulose 400 + 0.4% Tween 80 was administered via oral gavage 

to 4 female Sprague Dawley rats/dose group at doses of 0, 5, 20, 50, 80 or 150 mg/kg/day 

for 10 consecutive days. Rats were at PND 21 on the first day of dosing. All animals 

survived until scheduled termination. At 150 mg/kg/day, treatment-related clinical signs 

included reduced motor activity between Day 1 and 3. The mean overall body weight gain 

(Days 1-11) was decreased 7% compared to the controls. At 80 and 50 mg/kg/day, mean 

overall body weight gain was decreased (↓10% and ↓6%, respectively). At 20 mg/kg/day, 

body weight gain was decreased from Days 1-4 (↓13%); no other effects on body weight 

were observed during the study. No treatment-related effects were observed at 5 mg/kg/day. 

5. Dose Preparation and Analysis: Dose formulations were prepared twice during the study 

by mixing appropriate amounts of test substance with aqueous 0.5% methylcellulose 400 + 

0.4% Tween 80; formulations were stored at 53 °C when not in use. Stability was tested in 

a separate study;1

 it was stated that the test substance in the vehicle was found to be stable at 

concentrations of 1 and 100 g/L for up to 21 days under similar conditions of usage and 

storage to those of the current study. Homogeneity was tested at two concentrations (12 and 

24 g/L) of the first formulations of metribuzin in vehicle by sampling the surface, middle, 

and bottom of the suspension. Achieved concentration was tested separately in both 

formulations from the second preparation; in addition, the mean results of the homogeneity 

analyses were used for achieved concentrations. 

 

1

 Amir Tahmasseb, L. (2011): Metribuzin, Stability in aqueous 0.5% methylcellulose 400 + 0.4% tween 80, Bayer 

CropScience, Sophia Antipolis, France (SA 11089). 

Page 155 of 205
Female Pubertal Assay (2011) / Page 5 of 15 

METRIBUZIN/ 101101 OCSPP 890.1450/ OECD None

Results 

Homogeneity (%CV): 0.0-0.2% 

Stability: Provided in SA 11089 

Concentration (% of nominal): 98-100% 

The analytical data indicated that the mixing procedure was adequate and that the variation 

between nominal and actual dosage to the animals was acceptable. 

6. Dosage Administration: All doses were administered once daily by gavage, from PND 22 

through PND 42, in a volume of 5 mL/kg. Dose volume was adjusted based on the 

individual daily animal body weight recorded. Dosing was performed between 0700 and 

0900 hours daily.

7. Statistics: The following parameters were analyzed statistically: body weights, body 

weight gains, terminal body weights, organ weights, VO, clinical chemistry and hormonal 

analysis, and estrous cyclicity parameters. Clinical chemistry parameters, hormonal 

parameters, and body weight parameters were analyzed using Path/Tox System V4.2.2. 

Estrous cyclicity parameters, age at VO, body weight at VO, organ weight, body weight at 

PND 21, and terminal body weight parameters were analyzed using statistical procedures 

from SAS (V 9.2). Significance was denoted at p≤0.05. 

For body weight gain and clinical chemistry parameters, the data were first analyzed using 

Bartlett’s test. If the data were not significant, an ANOVA test was performed; a two-sided 

Dunnett’s test was conducted if the results of the ANOVA were significant. If the results of 

Bartlett’s test were significant, a Kruskal-Wallis test was performed, followed by a twosided Dunn’s test for significant data. The same procedures were used for body weights 

(except PND 21 and terminal body weight) and hormonal parameters, except that data were 

transformed (log transformation) and a second Bartlett’s test was conducted if the data were 

significant after the first Bartlett’s test. 

For age and body weight at VO, organ weights, organ to body weight ratio, body weight at 

PND 21, terminal body weight, mean cycle length, and mean age at first vaginal estrus, the 

data were first analyzed using Levene’s test; if the data were significant, a data 

transformation (log transformation) was conducted and a second Levene’s test was 

conducted. If the data were not significant, an ANOVA test was performed; a two-sided 

Dunnett’s test was conducted if the results of the ANOVA were significant. If the results of 

Levene’s test (after transformation) showed significance, a Kruskal-Wallis test was 

performed, followed by a two-sided Dunn’s test for significant data. 

In addition, analysis of covariance (ANCOVA) was conducted, using body weight at PND 

21 as the covariable, on the means of the treated and control groups except for terminal body 

weight, mean age at first estrus, and mean cycle length. Adjusted means were compared 

using a two-sided Dunnett’s test. For cycling status (cycling/not cycling and regular 

cycling/irregular cycling), the control group and treated groups were compared using the 

Chi-squares test. The statistical analyses were considered adequate. 

Page 156 of 205
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C. METHODS

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

on weekends and public holidays) for mortality and moribundity. Clinical examinations 

were conducted daily. 

2. Body Weight: Animals were weighed daily and prior to termination. 

3. Vaginal Opening (VO): Beginning on PND 22, all animals were examined daily for onset 

of VO. Age and weight on the day of completion of VO were recorded. 

4. Estrous Cyclicity: Beginning on the day of complete VO, up to and including the day of 

necropsy, daily vaginal smears were obtained and evaluated for the presence of leukocytes, 

nucleated epithelial cells, or cornified epithelial cells. The vaginal smears were classified as 

diestrus, proestrus, or estrus (predominance of cornified epithelial cells). The age at first 

vaginal estrus was recorded. 

The overall estrous cycle pattern of each female was characterized as regular (recurring 4-5 

day cycles), irregular (having cycles with diestrus for a period >3 days or cornification >2 

days), or not cycling (having prolonged period of either vaginal cornification or leukocyte 

smears). Mean cycle length was defined as the number of days from the first day of diestrus 

to the first day of the next diestrus. Incomplete cycles were not used in calculating mean 

cycle length. In cases when the time between VO and euthanasia did not allow observation 

of more than 1 complete cycle, classification was based on the available data with the 

assumption that animals are cycling regularly if the partial data fit the definition, or cycling 

irregularly if the study ends without being able to distinguish between irregular and not 

cycling. 

5. Sacrifice and Pathology: On PND 42, all surviving animals were anesthetized using 

isoflurane and blood was collected from the abdominal aorta into serum separation tubes. 

The length of time between final dose and exsanguination was not specified; however, all 

blood collections were completed by 1300 hours. Serum separation tubes were stored at 

−74 °C for subsequent hormone and clinical chemistry evaluations. 

a. Hormone Analysis: Serum T4 and TSH levels were determined using enzyme 

immunoassay kits. 

Page 157 of 205
Female Pubertal Assay (2011) / Page 7 of 15 

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

 ELECTROLYTES OTHER 

 Calcium Albumin 

 Chloride X Creatinine* 

 Magnesium X Urea nitrogen* 

 Phosphorus Total cholesterol 

 Potassium Globulins 

 Sodium Glucose 

ENZYMES X Total bilirubin 

X Alkaline phosphatase X Total protein 

 Cholinesterase Triglycerides 

 Creatine phosphokinase Serum protein electrophoresis 

 Lactic acid dehydrogenase 

X Alanine aminotransferase 

X Aspartate aminotransferase 

 Sorbitol dehydrogenase 

 Gamma glutamyl transferase 

 Glutamate dehydrogenase 

* Recommended for the pubertal assay in female rats based on guideline 890.1450. 

c. Organ Weights and Histopathology: The following CHECKED (X) tissues were 

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

examination. 

 UROGENITAL OTHER 

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

XX Uterus*+ X Liver* 

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

 X Pituitary* 

 XX Significant macroscopic findings 

* Weights required based on guideline 890.1450 

+ Histopathological examination required based on guideline 890.1450 

All organs collected were weighed prior to fixation. Paired organs (kidneys, adrenals, and 

ovaries) were weighed together. The uterus and cervix were separated from the vagina and 

weighed. The uterus was weighed again following removal of the fluid in the lumen 

(blotted weight). All collected organs were preserved in 10% neutral buffered formalin, 

processed and embedded in paraffin wax, sectioned, stained with hematoxylin and eosin, 

and examined microscopically. 

Three random sections of thyroid gland were processed according to the facility’s 

procedures (not described) and subjectively evaluated for follicular cell height and colloid 

area. The incidences of colloid alteration, follicular cell hypertrophy, follicular cell height 

decrease, and follicular cell shape were reported, graded on a six-point scale based on 

severity. 

Five random sections of ovary were processed according to the facility’s procedures (not 

described) and subjected to histology including a semi-qualitative evaluation of follicle 

development (including presence/absence of tertiary/antral follicles, presence/absence of 

corpora lutea, changes in corpus luteum development, or changes in the number of primary 

Page 158 of 205
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and atretic follicles). It was not reported which ovary (left or right) was examined for each 

rat. 

II. RESULTS

A. Mortality: All animals survived until scheduled termination. 

B. Clinical Signs of Toxicity: The incidence of clinical observations is summarized in Table 

2. Increased salivation was observed on one or several occasions throughout the study in 

6/15 rats in the 60 mg/kg/day group and in 7/15 rats in the 120 mg/kg/day group. 

TABLE 2. Incidence of Clinical Observations a

Observation 

Dose Level 

Vehicle Control Metribuzin

(60 mg/kg/day) 

Metribuzin

(120 mg/kg/day) 

# Observed # Examined # Observed # Examined # Observed # Examined 

Increased salivation 0 15 6 15 7 15 

a Data were extracted from page 30 of the study report 

C. General Growth and Vaginal Opening: Initial and final body weights, body weight gains, 

age and weight at day of attainment of VO, and proportion of rats that did not attain VO are 

presented in Table 3. At 120 mg/kg/day, the age (PND 36.47 treated vs. PND 35.00 

controls) and body weight (↑6%) at VO were slightly increased (NS) compared to the 

controls. Final body weight and body weight gain in the 60 and 120 mg/kg/day groups, and 

age and body weight at VO in the 60 mg/kg/day group were comparable to the controls. All 

rats achieved VO before study termination. 

At 60 and 120 mg/kg/day, mean body weight gain/day (not reported in Table 3) was reduced 

(p≤0.01) by 33% and 31% respectively on Day 2; thereafter, mean body weight gain was 

comparable to the controls. Mean cumulative body weight gain was slightly reduced 

(p≤0.01) at 60 and 120 mg/kg/day (↓11% and 18%, respectively) between Days 1 and 8 but 

was comparable to the controls at the end of the study. 

The mean age at VO, body weight at VO, and final body weight in the control group were 

within the acceptable range of the performance criteria provided in the Guideline 

(890.1450), however, the CV for age at VO was outside the acceptable range (7.5%; 

maximum 6.52%). 

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TABLE 3. General Growth and Vaginal Opening (VO)a

Parameter Evaluated 

Vehicle Control Metribuzin

(60 mg/kg/day) 

Metribuzin 

(120 mg/kg/day) 

N Mean 

SD/ 

SEb 

CV 

(%) N Mean 

SD/ 

SEb 

CV 

(%) N Mean 

SD/ 

SEb 

CV 

(%) 

Initial body weight 

(PND 22; g) 

U 15 59.2 4.6 7.8 15 58.0 5.7 9.8 15 58.1 4.8 8.3 

A NA NA NA NA NA NA NA NA NA NA NA NA 

Body weight at vaginal 

opening (g) 

U 15 128.80 17.8 13.8 15 128.06 16.1 12.6 15 136.57 

(↑6) 

14.1 10.4 

A 15 128.29 3.9 NA 15 128.60 3.9 NA 15 136.54 3.9 NA 

Final body weight 

(g) 

U 15 168.1 12.6 7.5 15 164.4 10.1 6.2 15 168.9 12.7 7.5 

A NR NR NR NR NR NR NR NR NR NR NR NR 

Final body weight 

(% of control) 

U NA NA NA NA NA 97.8 NA NA NA 100.5 NA NA 

A NA NA NA NA NA NA NA NA NA NA NA NA 

Body weight gain 

(final – initial; g) 

U 15 106.9 10.0 9.4 15 107.2 6.1 5.7 15 111.4 12.9 11.6 

A NR NR NR NR NR NR NR NR NR NR NR NR 

Age at vaginal opening 

(PND) 

U 15 35.00 2.6 7.5 15 35.47 2.4 6.9 15 36.47 2.2 6.1 

A 15 35.00 0.6 NA 15 35.46 0.6 NA 15 36.47 0.6 NA 

Proportion unopened (#/N) 0/15 0/15 0/15 

a Data were obtained from page 32 of the study report. 

b SD = Standard Deviation; SE = Standard Error (for covariance analysis) 

U Unadjusted for body weight on PND 21 

A Adjusted for body weight on PND 21 

N Number of animals examined 

CV Coefficient of Variation 

NA Not applicable 

NR Not reported 

D. Organ Weights: Organ weights at necropsy are presented in Table 4. At 120 mg/kg/day, 

absolute, adjusted (for body weight on PND 21), and relative liver weights were increased 

(p≤0.01) by 21-22% and adjusted thyroid weights were increased (p≤0.05) by 15% 

compared to controls. At 60 mg/kg/day, relative liver weights were increased (p≤0.05) by 

10%; thyroid glands weights were comparable to the controls at this dose level. There were 

no treatment-related effects on the weights of kidneys, pituitary, adrenals, ovaries, or uterus 

(wet or blotted). 

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 (33.9 mg; acceptable range 38.34-48.84 mg). 

Page 160 of 205
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TABLE 4. Organ Weights at Necropsya

Organ 

Vehicle Control Metribuzin

(60 mg/kg/day) 

Metribuzin 

(120 mg/kg/day) 

N Mean 

SD/ 

SEb 

CV 

(%) N Mean 

SD/ 

SEb 

CV 

(%) N Mean 

SD/ 

SEb 

CV 

(%) 

Liver 

(g) 

U 15 7.10 0.76 10.7 15 7.67 0.75 9.8 15 8.66** 

(↑22) 

1.15 13.3 

A 15 7.07 0.220 NA 15 7.70 0.220 NA 15 8.66** 

(↑22) 

0.219 NA 

R 15 4.2173 0.2269 5.4 15 4.6593** 

(↑10) 

0.2972 6.4 15 5.1127** 

(↑21) 

0.3773 7.4 

Kidneys 

(g) 

U 15 1.44 0.12 8.6 15 1.49 0.13 8.8 15 1.53 0.15 9.9 

A 15 1.44 0.033 NA 15 1.49 0.033 NA 15 1.53 0.033 NA 

R 15 0.8600 0.0594 6.9 15 0.9053 0.0648 7.2 15 0.9080 0.0537 5.9 

Pituitary 

(mg) 

U 15 7.77 1.24 16.0 15 8.03 1.43 17.8 15 7.96 1.44 18.1 

A 15 7.76 0.358 NA 15 8.04 0.358 NA 15 7.96 0.358 NA 

R 15 0.0046 0.0006 13.9 15 0.0049 0.0010 19.8 15 0.0047 0.0007 14.5 

Adrenals 

(mg) 

U 15 33.89 5.27 15.5 15 33.54 4.16 12.4 15 33.49 4.39 13.1 

A 15 33.87 1.211 NA 15 33.55 1.211 NA 15 33.49 1.209 NA 

R 15 0.0202 0.0032 15.8 15 0.0205 0.0027 13.2 15 0.0199 0.0028 14.2 

Ovaries 

(mg) 

U 15 54.7 7.0 12.7 15 53.6 9.9 18.5 15 52.5 9.6 18.2 

A 15 54.5 2.21 NA 15 53.9 2.21 NA 15 52.5 2.20 NA 

Uterus, wet 

(mg) 

U 15 321.6 173.3 53.9 15 359.6 143.7 40.0 15 290.2 166.3 57.3 

A 15 321.9 42.27 NA 15 359.3 42.27 NA 15 290.2 42.22 NA 

Uterus, blotted 

(mg) 

U 15 261.8 85.0 32.5 15 277.1 65.7 23.7 15 246.0 92.3 37.5 

A 15 261.4 21.36 NA 15 277.5 21.36 NA 15 246.0 21.33 NA 

Thyroid 

(mg) 

U 15 12.2 1.7 13.7 15 12.3 2.3 18.5 15 14.0 2.5 17.9 

A 15 12.2 0.56 NA 15 12.3 0.56 NA 15 14.0* 

(↑15) 

0.56 NA 

a Data were obtained from page 35 of the study report. Percent differences from controls, calculated by the reviewers, are 

included in parentheses. 

b SD = Standard Deviation; SE = Standard Error (for covariance analysis) 

U Unadjusted for body weight on PND 21 

A Adjusted for body weight on PND 21 

N Number of animals examined 

CV Coefficient of Variation 

NA Not applicable 

R Organ-to-body weight ratio (relative to body weight) 

* Significantly different from controls at p≤0.05. 

** Significantly different from controls at p≤0.01. 

E. Estrous Cyclicity: Estrous cycle data are provided in Table 5. There were no effects of 

treatment on mean age at first vaginal estrus, mean cycle length, percent cycling, or percent 

regularly cycling. 

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TABLE 5. Estrous Cyclicitya

Treatment 

Groups N

Mean Age at 

First Vaginal 

Estrus (PND) 

Mean Cycle 

Length 

(days) 

Cycling 

(%) 

Regularly 

Cycling 

(%) 

Cycle Status at Necropsy (# Females) 

Diestrus Proestrus Estrus

Not 

Cycling 

Vehicle 15 35.08 4.75 93.3 100 11 1 3 1 

Metribuzin 

(60 mg/kg/day) 

15 36.07 4.94 100 93.3 5 5 5 0 

Metribuzin 

(120 mg/kg/day) 

15 36.67 4.90 100 100 10 2 3 0 

a Data were obtained from page 36 of the study report. 

N Number of animals

F. Clinical Chemistry and Hormone Levels: Mean hormone and clinical chemistry levels 

are presented in Table 6. Serum T4 levels were decreased (p≤0.01) by 44% in rats dosed at 

120 mg/kg/day. Mean TSH levels were slightly increased (NS) by 29% and 33% at 60 and 

120 mg/kg/day, respectively. Alanine aminotransferase was increased (p≤0.05) by 20% at 

120 mg/kg/day. There were no treatment-related effects on creatinine, blood urea nitrogen, 

total protein, total bilirubin, aspartate aminotransferase, or alkaline phosphatase. Historical 

control data were not provided for either hormone or clinical chemistry parameters. In 

addition, the facility’s criteria for evaluating the performance of the enzyme immunoassay 

kits used for determination of hormone levels were not described. 

The results for serum T4 in the control group were within the acceptable range of the 

performance criteria provided in the Guideline (890.1450). 

Page 162 of 205
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TABLE 6. Hormone Levels and Clinical Chemistry a

Parameter 

Evaluated 

Vehicle Control Metribuzin

(60 mg/kg/day) 

Metribuzin 

(120 mg/kg/day) 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) 

Hormones 

Serum T4, Total 

(μg/dL) 

15 3.2 0.9 28 15 2.8 0.7 25 15 1.8** 

(↓44) 

0.5 28 

Serum TSH 

(ng/mL) 

15 1.71 0.76 44 15 2.21 

(↑29) 

0.79 36 14 2.27 

(↑33) 

0.67 30 

Clinical Chemistry 

Creatinine 

(μmol/L) 

15 47 2 4 15 46 3 7 15 47 3 6 

Blood urea nitrogen 

(mmol/L) 

15 5.28 0.78 15 15 5.30 0.82 15 15 5.90 1.29 22 

Total protein (g/L) 15 54 2 4 15 56 2 4 15 56 2 4 

Total bilirubin 

(μmol/L) 

15 0.4 0.2 50 15 0.4 0.2 50 15 0.6 0.3 50 

Aspartate 

aminotransferase 

(IU/L) 

15 97 21 22 15 114 28 25 15 115 22 19 

Alanine 

aminotransferase 

(IU/L) 

15 35 6 17 15 39 5 13 15 42* 

(↑20) 

7 17 

Alkaline 

phosphatase (IU/L) 

15 308 54 18 15 311 74 24 15 311 67 22 

a Data were obtained from page 38 of the study report. Percent differences from controls, calculated by the reviewers, are 

included in parentheses. 

N Number of animals examined 

SD Standard Deviation 

CV Coefficient of Variation 

* Significantly different from controls at p≤0.05. 

** Significantly different from controls at p≤0.01. 

G. Gross Pathology: At necropsy, enlarged thyroid was observed in 1/15 rats in the 120 

mg/kg/day group. Enlarged liver was observed in 5/15 rats in the 120 mg/kg/day group and 

in 1/15 rats in the 60 mg/kg/day group. No other gross observations were noted in female 

rats surviving to the scheduled termination. 

H. Histopathology: The incidences of histopathological findings of the thyroid gland are 

presented in Table 7. There were no treatment-related findings in the thyroid gland. 

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

Treatment 

Groups 

Parameter Evaluated 

Severity Incidence of 

Colloid Alteration

Incidence of 

Follicular Cell 

Hypertrophy 

Incidence of 

Follicular Cell 

Height (Decrease) 

Incidence of 

Follicular Cell 

Shape 

Vehicle Control 

N = 15 

0 15 15 15 15 

1 0 0 0 0 

2 0 0 0 0 

3 0 0 0 0 

4 0 0 0 0 

5 0 0 0 0 

Metribuzin

(60 mg/kg/day) 

N = 15 

0 15 15 15 15 

1 0 0 0 0 

2 0 0 0 0 

3 0 0 0 0 

4 0 0 0 0 

5 0 0 0 0 

Metribuzin

(120 mg/kg/day) 

N = 15 

0 15 15 15 15 

1 0 0 0 0 

2 0 0 0 0 

3 0 0 0 0 

4 0 0 0 0 

5 0 0 0 0 

a Data were obtained from page 41 of the study report. 

b Thyroid histopathology was graded 0 – 5 based on severity: 0=Not remarkable, 1=Minimal, 2=Slight, 3=Moderate, 

4=Marked, 5=Severe. 

N Number examined 

The incidence of histopathological findings of the liver, ovaries, uterus, and kidneys are 

presented in Table 8. The liver was examined only in the cases in which it was found to be 

enlarged at necropsy. There were increased numbers of mitoses in the one examined liver in 

the 60 mg/kg/day group and 4/5 examined livers in the 120 mg/kg/day group. 

None of the findings in the ovaries, uterus, or kidneys were treatment related. 

TABLE 8. Incidence of Histopathological Findings of the Liver, Ovaries, Uterus, and Kidneya

Findings 

Dose Level 

Vehicle Control Metribuzin

(60 mg/kg/day) 

Metribuzin

(120 mg/kg/day) 

# Observed # Examined # Observed # Examined # Observed # Examined

Liver 

Increased number of mitoses 0 0 1 1 4 5 

Glycogen accumulation: diffuse 0 0 1 1 5 5 

Interstitial mononuclear cell infiltrate: focal 0 0 1 1 1 5 

Extramedullary hematopoiesis: focal 0 0 1 1 5 5 

Ovaries 

Presence of tertiary/antral follicles 15 15 15 15 15 15 

Presence of corpora lutea 15 15 15 15 15 15 

Decreased number of primary follicles 0 15 0 15 0 15 

Increased number of atretic follicles 0 15 0 15 0 15 

Sertoliform tubules: focal 3 15 2 15 2 15 

Cyst(s) 0 15 1 15 0 15 

Uterus 

Dilatation of horn(s) 2 15 5 15 3 15 

Page 164 of 205
Female Pubertal Assay (2011) / Page 14 of 15 

METRIBUZIN/ 101101 OCSPP 890.1450/ OECD None

TABLE 8. Incidence of Histopathological Findings of the Liver, Ovaries, Uterus, and Kidneya

Findings 

Dose Level 

Vehicle Control Metribuzin

(60 mg/kg/day) 

Metribuzin

(120 mg/kg/day) 

# Observed # Examined # Observed # Examined # Observed # Examined

Congestion: diffuse 0 15 0 15 0 15 

Kidney 

Collecting duct hyperplasia: unilateral: 

focal 2 15 0 15 1 15 

Collecting duct hyperplasia: bilateral: focal 0 15 0 15 1 15 

Transitional cell hyperplasia: unilateral: 

focal 1 15 1 15 0 15 

Transitional cell hyperplasia: bilateral: 

focal 1 15 2 15 2 15 

Basophilic tubules: unilateral: focal 7 15 5 15 7 15 

Basophilic tubules: bilateral: focal 2 15 3 15 3 15 

Interstitial mononuclear cell infiltrate: focal 4 15 3 15 3 15 

Interstitial mixed cell infiltrate: focal 1 15 0 15 3 15 

Suburothelial mixed cell infiltrate: focal 1 15 3 15 1 15 

Medullary mineralization: focal 7 15 4 15 4 15 

Papillary mineralization: focal 1 15 0 15 1 15 

Pelvic dilatation: unilateral 1 15 2 15 3 15 

Pelvic dilatation: bilateral 0 15 1 15 1 15 

Atrophy/fibrosis/scar: unilateral 4 15 2 15 2 15 

Atrophy/fibrosis/scar: bilateral 0 15 0 15 1 15 

Cyst(s) 2 15 1 15 0 15 

a Data were obtained from Table 11 on pages 97 and 98 of the study report. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: The effect of metribuzin was characterized by 

effects on the thyroid, as evidenced by hormonal changes and increased thyroid weight. A 

modest increase in age of vaginal patency and body weight when VO occurred was 

observed at the high dose, but these differences from the control group were not statistically 

significant. The regularity of estrous cycling was unaffected by treatment. 

B. AGENCY COMMENTS: There were no effects of treatment on mean age at first vaginal 

estrus, mean cycle length, percent cycling, or percent regularly cycling; on the weights of 

kidneys, pituitary, adrenals, ovaries, or uterus (wet or blotted); or on levels of creatinine, 

blood urea nitrogen, total protein, total bilirubin, aspartate aminotransferase, or alkaline 

phosphatase. There were no treatment-related histopathological findings in the thyroid 

gland, ovaries, uterus, or kidneys. 

At 120 mg/kg/day, increased salivation was observed in 7/15 animals on one or several 

occasions throughout the study. The age (PND 36.47 treated vs. PND 35.00 controls) and 

body weight (↑6%) at VO were slightly increased (NS) compared to the controls. Overall 

body weight gains were comparable to the control. Absolute, adjusted (for body weight on 

PND 21), and relative liver weights were increased (p≤0.01) by 21-22% and adjusted 

thyroid gland weights were increased (p≤0.05) by 15% in the 120 mg/kg/day group 

compared to controls. At necropsy, enlarged thyroid was observed in 1/15 rats and enlarged 

liver was observed in 5/15 rats in this group; there were increased numbers of mitoses in 4/5 

examined livers. Serum T4 levels were decreased (p≤0.01) by 44% in rats dosed at 120 

Page 165 of 205
Female Pubertal Assay (2011) / Page 15 of 15 

METRIBUZIN/ 101101 OCSPP 890.1450/ OECD None

mg/kg/day; mean TSH levels were slightly increased (NS) by 33% and alanine 

aminotransferase was increased (p≤0.05) in this group. 

At 60 mg/kg/day, increased salivation was observed in 6/15 rats on several occasions 

throughout the study. Final body weight and body weight gain, and age and body weight at 

VO in this group were comparable to the control. Relative liver weights were increased 

(p≤0.05) by 10%; thyroid gland weights were comparable to the controls at this dose level. 

At necropsy, enlarged liver was observed in 1/15 rats; increased numbers of mitoses were 

observed in this liver. Mean TSH levels were slightly increased (NS) by 29% at 60 

mg/kg/day; serum T4 and alanine aminotransferase levels in this group were comparable to 

the controls. 

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

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

study: 

 Control %CV for age at VO (7.5%) was greater than the performance criteria maximum 

of 6.52% 

 Control mean adrenal weight (33.9 mg) was higher than the performance criteria 

acceptable range (38.34-48.84 mg) 

Page 166 of 205
DATA EVALUATION RECORD 

METRIBUZIN 

Study Type: OCSPP 890.1500, Male Pubertal Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-66-2012 (MRID 48672203) 

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:

Kelly Luck, M.S. Date: 6/06/2012 

Secondary Reviewer: Signature:

Rebecca L. Bryan, B.S. Date: 6/11/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 6/29/2012 

Quality Assurance: Signature:

Steven Brecher, Ph.D., D.A.B.T. Date: 6/28/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 167 of 205
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 168 of 205
METRIBUZIN/ 101101 

Male Pubertal Assay (2011) I Page 1 of 13 

OCSPP 890.1500 I OECD None 

Primary Reviewer: Deborah Smegal Sig""~ ) ~ · Health Effects Divis-io-n-=--=-"-"'-'--'=''-=-"==-------- b':.::~~ t;;Y7}): 

Secondary 

Health Effects 

Reviewer: 

Division 

John Liccione, Ph.D. Signature:

Date: . ~~ 

~ 

Template version 08/2011 

DATA EVALUATION RECORD 

STUDY TYPE: Male Pubertal Assay; OCSPP 890.1500 

PC CODE: 101101 DP BARCODE: D40121 9 

TXR#: 0056258 CAS#: 21087-64-9 

TEST MATERIAL (PURITY): Metribuzin (94.4% a.i.) 

SYNONYMS: 4-Amino-6-( 1, 1-dimethylethyl)-3-(methylthio )-1,2,4-triazin-5( 4H)-one 

CITATION: Blanck, M. (20 11 ) Metribuzin assessment of pubertal development and thyroid 

function in juvenile/peripubertal male and female rats. Bayer S.A.S., Sophia 

Antipolis Cedex, France. Laboratory Study No.: SA 10341, December 1, 2011. 

MRID 48672203. Unpublished. 

SPONSOR: Bayer CropScience AG, Alfred Nobel Str. 50, Monheim, Germany 

TEST ORDER#: CON- 101101-127 

EXECUTIVE SUMMARY: In a Male Pubertal Assay (MRID 48672203), 15 Sprague Dawley 

rats/dose group were treated daily via oral gavage with metribuzin (94.4% a.i., Batch/lot# 

K285067) in aqueous 0.5% methylcellulose 400 + 0.4% Tween 80 (vehicle) at doses of 0 

(vehicle), 60, or 120 mg/kg/day from post-natal day (PND) 23 to 53. Animals were examined 

for preputial separation (PPS) dail y beginning on PND 30 and age and weight at day of 

attainment were recorded . Following sacrifice on PND 53, total serum testosterone, thyroxine 

(T4), and thyroid stimulating hormone (TSH) levels were analyzed using enzyme immunoassay 

kits. Adrenals, liver, pituitary, thyroid, and urogenital organ weights were recorded and 

microscopic examinations of the testes, epididymides, thyroid, and kidneys were performed. In 

addition, any livers found to be enlarged at necropsy were subjected to microscopic examination. 

There were no effects of treatment on final body weights, overall body weight gain, or age and 

body weight at PPS; on the weights of kidneys, pituitary, adrenals, seminal vesicles plus 

coagulating gland (with or without fluid), ventral prostate, dorsolateral prostate, levator anibulbocavernosus muscle complex (LABC), epididymides, or testes; or on levels of testosterone, 

creatinine, blood urea nitrogen, total bilirubin, aspartate aminotransferase, alanine 

aminotransferase, or alkaline phosphatase. There were no treatment-related histopathological 

findings in the testes, epididymides, or kidneys. 

At 120 mg/kg/day, increased sali vation was observed in all rats on one or several occasions 

throughout the study. In addition, one animal in this group had reduced motor activity and 

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labored respiration on Day 13 and another rat had ocular discharge on Days 20-25. All rats in 

this group achieved PPS before study termination. Absolute, adjusted (for body weight on PND 

21), and relative liver weights were increased (p≤0.01) by 21-23% and absolute and adjusted 

thyroid weights were increased (p≤0.01) by 45-46% compared to controls. Serum T4 levels were 

decreased (p≤0.01) by 72% in rats dosed at 120 mg/kg/day; mean TSH levels were increased 

(p≤0.01) by 236% and total protein was increased (p≤0.01) by 5% in this group. At necropsy, 

enlarged thyroid was observed in 6/15 rats and enlarged liver was observed in 11/15 rats. 

Minimal to slight follicular cell hypertrophy and colloid alteration were observed in the thyroid 

glands; an increased number of mitoses was noted in 2/15 males in this group. There were 

increased numbers of mitoses in 7/11 examined livers in the 120 mg/kg/day group, as well as 

minimal centrilobular hepatocellular hypertrophy (4/11 rats). 

At 60 mg/kg/day, increased salivation was observed on several occasions throughout the study in 

5/15 rats. All but one rat in this group achieved PPS before study termination. Relative liver 

weights were increased (p≤0.01) by 12%; thyroid gland weights were comparable to the controls 

at this dose level. Serum T4 levels were decreased (p≤0.01) by 43% and mean TSH levels were 

increased (p≤0.01) by 109% in rats dosed at 60 mg/kg/day; total protein levels in this group were 

comparable to controls. At necropsy, enlarged liver was observed in 1/15 rats. 

The high dose of metribuzin selected for this study (120 mg/kg/day) was adequate based on the 

increased liver weights observed at this dose level. 

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

890.1500). 

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

Assurance statements were provided. 

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I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Bayer S.A.S, Bayer CropScience 

Location: Sophia Antipolis Cedex, France 

Study Director: M. Blanck 

Other Personnel: S. Lautraite (Replacement Study Director), J.P. Kocwin (Laboratory Animal Resources), C. 

Jaussely (In-Life Supervisor), H. Vincent (Responsible Technician), C. Pallen (Statistician), 

L. Amir Tahmasseb (Senior Chemist), D. De Barbeyrac (Clinical Pathology Supervisor), L. 

Elies (Pathologist), M. Vagner (Report Unit Assistant) 

Study Period: March 10 - December 01, 2011 

2. Test Substance: Metribuzin 

 Description: Light grey powder 

 Source: Bayer CropScience AG (Frankfurt Am Main, Germany) 

 Lot/Batch #: K285067 (expiration date 5/07/2012) 

 Purity: 94.4% a.i. 

 Stability: Stable in vehicle (aqueous solution of 0.5% methylcellulose 400 + 0.4% Tween 80) for up to 

21 days at 5  3 °C. 

 CAS #: 21087-64-9 

 Structure: 

3. Vehicle: 0.5% methylcellulose 400 + 0.4% Tween 80

4. Test Animals:

 Species: Rat (males only) 

 Strain: Sprague Dawley [Crl:CD(SD)] 

 Age/Weight at Study 

Initiation: 

PND 23/ 50.0 – 77.1 g 

 Source: Charles River Laboratories(St Germain-sur-l’Arbresle, France) 

 Housing: Rats were housed 2-3 per cage in makrolon cages containing heat-treated laboratory-grade 

wood shavings 

 Diet: A04C-10 pelleted rodent diet (Scientific Animal Food and Engineering, Augy, France), ad 

libitum; 

Dietary level of phytoestrogens (including genistein, genistin, daidzin, daidzein and glycitin) 

were ≤300 μg of phytoestrogens equivalents/gram of diet. 

 Water: Filtered tap water, softened by ion exchange resins, ad libitum

 Environmental 

Conditions: 

Temperature: 

Humidity: 

Air changes: 

Photoperiod: 

20-24 °C 

40-70% 

10-15/hr 

14 hrs light/ 10 hrs dark 

B. STUDY DESIGN

1. In-Life Dates: Start: April 16, 2011 End: May 17, 2011 

N

N

N

O

NH2

S

CH3

3CH

3CH

CH3

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2. Mating: Time-mated pregnant dams were received from the supplier on GD 7 or 8. Any 

litters with fewer than 8 pups and any litters not delivered by GD 23 were excluded. Litters 

with more than 8 pups were standardized by culling, on PND 4, to 8 pups per litter, with, if 

possible, an equal number of pups of each sex. 

3. Animal Assignment: Offspring were weaned on PND 21 and weighed. Animals were 

randomly assigned to the test groups noted in Table 1 using a procedure that ensured a 

similar body weight distribution among groups. Littermates were not assigned to the same 

treatment group. 

TABLE 1. Study Design a

Test group Dose (mg/kg/day) # of Males 

Control 0 15 

Low 60 15 

High 120 15 

a Data were obtained from page 18 of the study report. 

4. Dose Selection Rationale: The dose levels were selected based on the results from a dose 

range-finding study, which was included as an appendix in the current study report. In the 

study, metribuzin in 0.5% methylcellulose 400 + 0.4% Tween 80 was administered via oral 

gavage to 4 peripubertal male Sprague Dawley rats/dose group at doses of 0, 5, 20, 50, or 80 

mg/kg/day for 10 consecutive days beginning on PND 21. All animals survived until 

scheduled termination. There were no treatment-related effects observed at any dose level. 

5. Dose Preparation and Analysis: Dose formulations were prepared twice during the study 

by mixing appropriate amounts of test substance with aqueous 0.5% methylcellulose 400 + 

0.4% Tween 80; formulations were stored at 53 °C when not in use. Stability was tested in 

a separate study;1

 it was stated that the test substance in the vehicle was found to be stable at 

concentrations of 1 and 100 g/L for up to 21 days under similar conditions of usage and 

storage to those of the current study. Homogeneity was tested at two concentrations (12 and 

24 g/L) of the first formulations of metribuzin in vehicle by sampling the surface, middle, 

and bottom of the suspension. Achieved concentration was tested separately in both 

formulations from the second preparation; in addition, the mean results of the homogeneity 

analyses were used for achieved concentrations. 

Results of Dose Analysis 

Homogeneity (%CV): 0.0% and 0.2% 

Stability: Not provided 

Concentration (% of nominal): 98-100% 

The analytical data indicated that the mixing procedure was adequate and that the variation 

between nominal and actual dosage to the animals was acceptable. 

 

1

 Amir Tahmasseb, L. (2011): Metribuzin, Stability in aqueous 0.5% methylcellulose 400 + 0.4% tween 80, Bayer 

CropScience, Sophia Antipolis, France (SA 11089). 

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

through PND 53, in a volume of 5 mL/kg. Dose volume was adjusted based on the 

individual daily animal body weight recorded. Dosing was performed between 0700 and 

0900 hours daily.

7. Statistics: The following parameters were analyzed statistically: body weights, body 

weight gains, terminal body weights, organ weights, PPS, clinical chemistry, and hormonal 

analysis. Clinical chemistry parameters, hormonal parameters, and body weight parameters 

were analyzed using Path/Tox System V4.2.2. Age at PPS, body weight at PPS, organ 

weight, body weight at PND 21, and terminal body weight parameters were analyzed using 

statistical procedures from SAS (V 9.2). Significance was denoted at p≤0.05. 

For body weight gain and clinical chemistry parameters, the data were first analyzed using 

Bartlett’s test. If the data were not significant, an ANOVA test was performed; a two-sided 

Dunnett’s test was conducted if the results of the ANOVA were significant. If the results of 

Bartlett’s test were significant, a Kruskal-Wallis test was performed, followed by a twosided Dunn’s test for significant data. The same procedures were used for body weights 

(except PND 21 and terminal body weight) and hormonal parameters, except that data were 

transformed (log transformation) and a second Bartlett’s test was conducted if the data were 

significant after the first Bartlett’s test. 

For age and body weight at PPS, organ weights, organ to body weight ratio, body weight at 

PND 21, and terminal body weight, the data were first analyzed using Levene’s test; if the 

data were significant, a data transformation (log transformation) was conducted and a 

second Levene’s test was conducted. If the data were not significant, an ANOVA test was 

performed; a two-sided Dunnett’s test was conducted if the results of the ANOVA were 

significant. If the results of Levene’s test (after transformation) showed significance, a 

Kruskal-Wallis test was performed, followed by a two-sided Dunn’s test for significant data. 

In addition, analysis of covariance (ANCOVA) was conducted, using body weight at PND 

21 as the covariable, on the means of the treated and control groups except for terminal body 

weight. The statistical analyses were considered adequate. 

C. METHODS

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

on weekends and public holidays) for mortality and moribundity. Clinical examinations 

were conducted daily. 

2. Body Weight: Animals were weighed daily and prior to termination. 

3. Preputial Separation (PPS): Beginning on PND 30, all animals were examined daily for 

onset of PPS. Age and weight at on the day of completion of PPS were recorded. In cases 

where PPS or complete PPS had not occurred prior to necropsy, age at necropsy plus one 

(PND 54) and weight at necropsy (on PND 53) were used for those animals when 

calculating the group means. 

4. Sacrifice and Pathology: On PND 53, all surviving animals were anesthetized using 

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isoflurane and blood was collected from the abdominal aorta into serum separation tubes. 

The length of time between final dose and exsanguination was not specified; however, all 

blood collections were completed by 1300 hours. Serum separation tubes were stored at 

−74 °C for subsequent hormone and clinical chemistry evaluations. 

a. Hormone Analysis: Total testosterone, T4, and TSH levels were determined using enzyme 

immunoassay kits. 

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

 ELECTROLYTES OTHER 

 Calcium Albumin 

 Chloride X Creatinine* 

 Magnesium X Urea nitrogen* 

 Phosphorus Total cholesterol 

 Potassium Globulins 

 Sodium Glucose 

ENZYMES X Total bilirubin 

X Alkaline phosphatase X Total protein 

 Cholinesterase Triglycerides 

 Creatine phosphokinase Serum protein electrophoresis 

 Lactic acid dehydrogenase 

X Alanine aminotransferase 

X Aspartate aminotransferase 

 Sorbitol dehydrogenase 

 Gamma glutamyl transferase 

 Glutamate dehydrogenase 

* Recommended based on guideline 890.1500. 

c. Organ Weights and Histopathology: The following CHECKED (X) tissues were 

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

examination. 

 UROGENITAL OTHER 

XX Testes (left and right separately)*+ XX Thyroid*+

XX Epididymides (left and right separately)*+ X Liver* 

X Seminal vesicle plus coagulating glands (with and without fluid)* X Adrenals (paired)* 

X Ventral prostate* X Pituitary* 

X Dorsolateral prostate* XX Significant 

macroscopic findings 

X Levator ani-bulbocavernosus (LABC) muscle complex* 

XX Kidneys (paired)*+ 

* Weights required based on guideline 890.1500 

+ Histopathological examination required based on guideline 890.1500 

All organs collected were weighed prior to fixation. Paired organs (kidneys and adrenals) 

were weighed together. All collected organs, with the exception of testis and epididymis, 

were preserved in 10% neutral buffered formalin, processed and embedded in paraffin wax, 

sectioned, stained with hematoxylin and eosin, and examined microscopically. The testis 

and epididymis were preserved in modified Davidson’s fixative. 

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Three random sections of thyroid gland were processed according to the facility’s 

procedures (not described) and subjectively evaluated for follicular cell height and colloid 

area. The incidences of colloid alteration, follicular cell hypertrophy, follicular cell height 

decrease, and follicular cell shape were reported, graded on a six-point scale based on 

severity. 

Testis histology included but was not limited to retained spermatids, missing or degenerated 

germ cell layers or types, multinucleated giant cells, or sloughing of spermatogenic cells 

into the lumen. Examination of epididymis was conducted in order to identify such lesions 

as sperm granulomas, leukocytic infiltration (inflammation), aberrant cell types within the 

lumen, or the absence of clear cells in the cauda epididymal epithelium. It was not reported 

which testis or epididymis (left or right) was examined for each rat. 

II. RESULTS

A. Mortality: All animals survived until scheduled termination. 

B. Clinical Signs of Toxicity: The incidence of clinical observations is summarized in Table 

2. At 120 mg/kg/day, increased salivation was observed in all rats on one or several 

occasions throughout the study. In addition, one animal in this group had reduced motor 

activity and labored respiration on Day 13 and another rat had ocular discharge on Days 20-

25. At 60 mg/kg/day, increased salivation was observed in 5/15 rats on one or several 

occasions throughout the study. 

TABLE 2. Incidence of Clinical Observations a

Observation 

Dose Level 

Vehicle Control Metribuzin

(60 mg/kg/day) 

Metribuzin

(120 mg/kg/day) 

# Observed # Examined # Observed # Examined # Observed # Examined 

Ocular discharge 0 15 0 15 1 15 

Labored respiration, 

reduced motor activity 0 15 0 15 1 15 

Increased salivation 0 15 5 15 15 15 

a Data were extracted from page 30 of the study report 

C. General Growth and Preputial Separation: Body weights, body weight gains, age and 

weight at attainment of PPS, and proportion of rats that did not achieve PPS by study 

termination are presented in Table 3. There were no treatment-related effects on final body 

weights, overall body weight gain, or age or body weight at attainment of PPS. All rats, 

with the exception of one in the 60 mg/kg/day group, attained PPS before study termination. 

At 120 mg/kg/day, mean body weight gain/day (not reported in Table 3) was reduced 

(p≤0.01) by 34% on Day 2; thereafter, mean body weight gain was comparable to the 

controls. Mean cumulative body weight gain between Days 1 and 7 was reduced (p≤0.05) at 

120 mg/kg/day (↓16%) but was comparable to the controls at the end of the study. At 60 

mg/kg/day, mean body weight gain/day was reduced (p≤0.01) by 27% on Day 2, and 

thereafter, mean body weight gain was slightly reduced compared to the controls. Mean 

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cumulative body weight gain was reduced (p≤0.05) by 12% between Days 1 and 6 and was 

reduced (not significant; NS) by 6% between Days 1 and 31. 

The mean age at PPS and final body weight in the control group were within the acceptable 

range of the performance criteria provided in the Guideline (890.1500), however, the body 

weight at PPS (258.07 g; acceptable range 188.277-256.169 g) and the % CV for age at PPS 

(6.4%; maximum 5.67%), body weight at PPS (10.3%; maximum 7.57%), and final body 

weight (7.7%; maximum 7.47%) were outside the acceptable range. 

TABLE 3. General Growth and Preputial Separation (PPS)a

Parameter Evaluated 

Vehicle Control Metribuzin

(60 mg/kg/day) 

Metribuzin 

(120 mg/kg/day) 

N Mean 

SD/ 

SEb 

CV 

(%) N Mean

SD/ 

SEb 

CV 

(%) N Mean 

SD/ 

SEb 

CV 

(%) 

Initial body weight 

(PND 23; g) 

U 15 68.3 5.8 8.5 15 65.9 7.8 11.8 15 67.2 5.1 7.6 

A NA NA NA NA NA NA NA NA NA NA NA NA 

Body weight at PPS 

(g) 

U 15 258.07 26.5 10.3 15 249.88 34.5 13.8 15 252.42 33.6 13.3 

A 15 258.09 8.3 NA 15 249.85 8.3 NA 15 252.43 8.3 NA 

Final body weight 

(g) 

U 15 322.2 24.7 7.7 15 302.2 25.7 8.5 15 317.6 23.7 7.4 

A NR NR NR NR NR NR NR NR NR NR NR NR 

Final body weight 

(% of control) 

U NA NA NA NA NA 93.8 NA NA NA 98.6 NA NA 

A NA NA NA NA NA NA NA NA NA NA NA NA 

Body weight gain 

(final – initial; g) 

U 15 253.7 20.4 8.0 15 239.3 21.8 9.1 15 256.1 21.3 8.3 

A NR NR NR NR NR NR NR NR NR NR NR NR 

Age at PPS 

(PND) 

U 15 46.00 3.0 6.4 15 46.60 3.2 6.9 15 45.67 3.1 6.8 

A 15 46.09 0.8 NA 15 46.47 0.8 NA 15 45.70 0.8 NA 

Proportion unseparated 

(#/N) 0/15 1/15 0/15 

a Data were obtained from page 31 of the study report. 

b SD = Standard Deviation; SE = Standard Error (for covariance analysis) 

U Unadjusted for body weight on PND 21 

A Adjusted for body weight on PND 21 

N Number of animals examined 

CV Coefficient of Variation 

NA Not applicable 

NR Not reported 

D. Organ Weights: Organ weights at necropsy are presented in Table 4. At 120 mg/kg/day, 

absolute, adjusted (for body weight on PND 21), and relative liver weights were increased 

(p≤0.01) by 21-23% and absolute and adjusted thyroid weights were increased (p≤0.01) by 

45-46% compared to controls. At 60 mg/kg/day, relative liver weights were increased 

(p≤0.01) by 12%; thyroid weights were comparable to the controls at this dose level. There 

were no dose-related effects on the weights of kidneys, pituitary, adrenals, seminal vesicles 

plus coagulating gland (with or without fluid), ventral prostate, dorsolateral prostate, LABC, 

epididymides, or testes. 

The unadjusted values for all organ weights in the control group were within the acceptable 

range of the performance criteria provided in the Guideline (890.1500), with the exception 

of the CVs for epididymis weight (16.8 and 19.8%; maximum 16.39%). 

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TABLE 4. Organ Weights at Necropsya

Organ 

Vehicle Control Metribuzin

(60 mg/kg/day) 

Metribuzin 

(120 mg/kg/day) 

N Mean 

SD/ 

SEb 

CV 

(%) N Mean 

SD/ 

SEb 

CV 

(%) N Mean 

SD/ 

SEb 

CV 

(%) 

Liver 

(g) U 15 14.24 1.28 9.0 15 14.91 1.47 9.9 15 17.28** 

(↑21) 

1.89 10.9 

A 15 14.19 0.385 NA 15 14.98 0.385 NA 15 17.26** 

(↑22) 

0.384 NA 

R 15 4.4207 0.2256 5.1 15 4.9347** 

(↑12) 

0.2539 5.1 15 5.4307** 

(↑23) 

0.3031 5.6 

Kidneys 

(g) 

U 15 2.32 0.21 9.2 15 2.27 0.32 14.3 15 2.35 0.18 7.5 

A 15 2.31 0.056 NA 15 2.29 0.056 NA 15 2.34 0.056 NA 

R 15 0.7193 0.0410 5.7 15 0.7500 0.0601 8.0 15 0.7407 0.0440 5.9 

Pituitary 

(mg) 

U 15 9.92 1.12 11.3 15 9.88 1.39 14.1 15 10.08 1.56 15.5 

A 15 9.92 0.359 NA 15 9.88 0.359 NA 15 10.08 0.358 NA 

R 15 0.0031 0.0003 8.5 15 0.0033 0.0005 13.7 15 0.0032 0.0005 15.5 

Adrenals 

(mg) 

U 15 40.57 7.45 18.4 15 39.19 5.58 14.2 14 35.52 5.96 16.8 

A 15 40.57 1.672 NA 15 39.18 1.678 NA 14 35.53 1.732 NA 

R 15 0.0126 0.0021 16.9 15 0.0130 0.0019 14.6 14 0.0113 0.0024 21.1 

Seminal vesicle + 

coagulating gland, 

with fluid (mg) 

U 15 519.7 106.9 20.6 15 496.0 135.0 27.2 15 542.2 136.1 25.1 

A 15 517.8 32.67 NA 15 498.6 32.71 NA 15 541.5 32.63 NA 

Seminal vesicle + 

coagulating gland, 

without fluid (mg) 

U 15 309.4 61.3 19.8 15 292.5 52.4 17.9 15 312.8 64.6 20.6 

A 15 308.3 15.26 NA 15 293.9 15.28 NA 15 312.4 15.24 NA 

Ventral prostate 

(mg) 

U 15 255.7 44.5 17.4 15 250.3 43.6 17.4 15 253.8 44.1 17.4 

A 15 255.3 11.48 NA 15 250.8 11.50 NA 15 253.6 11.47 NA 

Dorsolateral prostate 

(mg) 

U 15 139.6 28.2 20.2 15 139.6 35.4 25.4 15 136.2 35.4 26.0 

A 15 139.1 8.54 NA 15 140.3 8.55 NA 15 136.0 8.53 NA 

LABC 

(mg) 

U 15 621.4 100.5 16.2 15 570.2 108.1 19.0 15 594.9 87.1 14.6 

A 15 618.6 24.44 NA 15 574.0 24.47 NA 15 593.9 24.41 NA 

Epididymis, left 

(mg) 

U 15 231.0 38.8 16.8 15 235.2 31.4 13.4 15 237.5 30.7 12.9 

A 15 229.7 7.95 NA 15 237.0 7.96 NA 15 237.0 7.94 NA 

Epididymis, right 

(mg) 

U 15 241.9 47.9 19.8 15 239.7 34.0 14.2 15 252.7 32.2 12.7 

A 15 240.5 9.19 NA 15 241.6 9.20 NA 15 252.1 9.18 NA 

Testis, left 

(mg) 

U 15 1423.1 113.7 8.0 15 1410.2 131.6 9.3 15 1444.3 142.7 9.9 

A 15 1418.1 30.26 NA 15 1417.2 30.29 NA 15 1442.4 30.22 NA 

Testis, right 

(mg) 

U 15 1432.0 116.3 8.1 15 1407.9 136.4 9.7 15 1440.5 127.7 8.9 

A 15 1427.7 30.54 NA 15 1413.9 30.57 NA 15 1438.9 30.50 NA 

Thyroid 

(mg) U 15 17.5 3.7 21.3 15 19.7 3.0 15.1 15 25.4** 

(↑45) 

5.6 22.0 

A 15 17.4 1.09 NA 15 19.7 1.09 NA 15 25.4** 

(↑46) 

1.09 NA 

a Data were obtained from pages 33-34 of the study report. Percent differences from controls, calculated by the reviewers, 

are included in parentheses. 

b SD = Standard Deviation; SE = Standard Error (for covariance analysis) 

U Unadjusted for body weight on PND 21 

A Adjusted for body weight on PND 21 

N Number of animals examined 

CV Coefficient of Variation 

R Organ-to-body weight ratio (relative to body weight) 

NA Not applicable

** Significantly different from controls at p≤0.01 

E. Clinical Chemistry and Hormone Levels: Mean hormone levels are presented in Table 5. 

Serum T4 levels were decreased (p≤0.01) by 72% in rats dosed at 120 mg/kg/day and by 

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43% in rats dosed at 60 mg/kg/day. Mean TSH levels were increased (p≤0.01) by 109% and 

236% at 60 and 120 mg/kg/day, respectively. Total protein was increased (p≤0.01) by 5% 

at 120 mg/kg/day. There were no treatment-related effects on testosterone, creatinine, blood 

urea nitrogen, total bilirubin, aspartate aminotransferase, alanine aminotransferase, or 

alkaline phosphatase. The facility’s criteria for evaluating the performance of the enzyme 

immunoassay kits used for determination of hormone levels were not described. 

The results for serum T4 and testosterone in the control group were within the acceptable 

range of the performance criteria provided in the Guideline (890.1500). The results for 

serum TSH were outside the performance criteria, as follows: mean concentration of 2.66 

ng/mL (acceptable range 4.212-24.112 ng/mL) and CV of 67% (maximum 58.29%). 

TABLE 5. Hormone Levels and Clinical Chemistry a

Parameter 

Evaluated

Vehicle Control Metribuzin

(60 mg/kg/day) 

Metribuzin 

(120 mg/kg/day) 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) 

Hormones 

Serum T4, Total 

(μg/dL) 

15 4.7 0.7 15 15 2.7** 

(↓43) 

1.0 37 15 1.3** 

(↓72) 

0.5 38 

Serum TSH 

(ng/mL) 

15 2.66 1.79 67 15 5.55** 

(↑109)

3.65 66 15 8.93** 

(↑236) 

2.92 33 

Serum testosterone 

(pg/mL) 

15 1452 623 43 15 2226 2277 102 15 1789 2122 119 

Clinical Chemistry 

Creatinine 

(μmol/L) 

15 48 5 10 15 47 2 4 15 47 3 6 

Blood urea nitrogen 

(mmol/L) 

15 5.27 0.54 10 15 5.66 0.73 13 15 5.39 0.81 15 

Total protein (g/L) 15 58 2 3 15 60 2 3 15 61** 

(↑5) 

2 3 

Total bilirubin 

(μmol/L) 

15 0.4 0.3 75 15 0.3 0.3 100 15 0.5 0.3 60 

Aspartate 

aminotransferase 

(IU/L) 

15 136 60 44 15 126 31 25 15 119 26 22 

Alanine 

aminotransferase 

(IU/L) 

15 50 32 64 15 44 15 34 15 42 9 21 

Alkaline phosphatase 

(IU/L) 

15 332 73 22 15 317 55 17 15 345 68 20 

a Data were obtained from page 37 of the study report. Percent differences from controls, calculated by the reviewers, are 

included in parentheses. 

N Number of animals examined 

SD Standard Deviation 

CV Coefficient of Variation 

** Significantly different from controls at p<0.01

F. Gross Pathology: At necropsy, enlarged thyroid was observed in 6/15 rats in the 120 

mg/kg/day group and enlarged liver was observed in 11/15 rats in the 120 mg/kg/day group 

and in 1/15 rats in the 60 mg/kg/day group. In addition, one rat in the 120 mg/kg/day group 

was found to have only one adrenal gland; this organ was not weighed. No other gross 

observations were noted in male rats surviving to the scheduled termination.

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G. Histopathology: The incidences of histopathological findings of the thyroid gland are 

presented below in Table 6. At 120 mg/kg/day, minimal to slight follicular cell hypertrophy 

and colloid alteration were observed. In addition, an increased number of mitoses was noted 

in 2/15 males in this group (not shown in Table 6). 

TABLE 6. Incidence of Histopathological Lesions of the Thyroid Gland a

Treatment 

Groups 

Parameter Evaluated 

Severityb Incidence of 

Colloid Alteration

Incidence of 

Follicular Cell 

Hypertrophy 

Incidence of 

Follicular Cell 

Height (Decrease) 

Incidence of 

Follicular Cell 

Shape 

Vehicle Control 

N = 15 

0 15 15 15 15 

1 0 0 0 0 

2 0 0 0 0 

3 0 0 0 0 

4 0 0 0 0 

5 0 0 0 0 

Metribuzin

(60 mg/kg/day) 

N = 15 

0 15 15 15 15 

1 0 0 0 0 

2 0 0 0 0 

3 0 0 0 0 

4 0 0 0 0 

5 0 0 0 0 

Metribuzin

(120 mg/kg/day) 

N = 15 

0 13 5 15 15 

1 1 9 0 0 

2 1 1 0 0 

3 0 0 0 0 

4 0 0 0 0 

5 0 0 0 0 

a Data were obtained from page 39 of the study report. 

b Thyroid histopathology was graded 0 – 5 based on severity: 0=Not remarkable, 1=Minimal, 2=Slight, 3=Moderate, 

4=Marked, 5=Severe. 

N Number examined 

The incidence of histopathological findings of the adrenal glands, liver, testes, 

epididymides, and kidneys are presented in Table 7. The liver was examined in the cases in 

which macroscopic findings were noted at necropsy. There were increased numbers of 

mitoses in 7/11 examined livers in the 120 mg/kg/day group, as well as minimal 

centrilobular hepatoceullular hypertrophy (4/11 rats). It was noted that the evaluation of 

hepatocellular hypertrophy was difficult due to the presence of abundant glycogen 

accumulation in the cytoplasm (due to rats not being fasted). 

None of the findings in the testes, epididymides, or kidneys were treatment related. 

TABLE 7. Incidence of Histopathological Findings of the Adrenal Glands, Liver, Testes, Epididymides, and 

Kidneya

Findings 

Dose Level 

Vehicle Control Metribuzin

(60 mg/kg/day) 

Metribuzin

(120 mg/kg/day) 

# Observed # Examined # Observed # Examined # Observed # Examined

Adrenal Glands 

Page 179 of 205
Male Pubertal Assay (2011) / Page 12 of 13 

METRIBUZIN/ 101101 OCSPP 890.1500 / OECD None

TABLE 7. Incidence of Histopathological Findings of the Adrenal Glands, Liver, Testes, Epididymides, and 

Kidneya

Findings 

Dose Level 

Vehicle Control Metribuzin

(60 mg/kg/day) 

Metribuzin

(120 mg/kg/day) 

# Observed # Examined # Observed # Examined # Observed # Examined

Cortical atrophy: unilateral: diffuse 0 0 0 0 1 1 

Liver 

Hepatocellular hypertrophy: centrilobular: 

diffuse

0 2 0 1 4 11

Increased number of mitoses 0 2017 11

Glycogen accumulation: diffuse 1 2 1 1 11 11

Extramedullary hematopoiesis 1 2 1 1 10 11

Hepatocellular necrotic focus(i) 1 2012 11

Hepatocellular necrosis: centribular 1 2010 11

Interstitial mononuclear cell infiltrate: focal 0 2016 11

Interstitial mixed cell infiltrate: focal 1 2011 11

Subcapsular angiectasis: focal 0 2011 11

Testes 

Tubular degeneration: focal: unilateral 1 15 0 15 0 15 

Sloughing of germ cells: focal 0 15 1 15 1 15 

Sertoli cell vacuolation: focal 1 15 0 15 0 15 

Epididymides 

Oligospermia: tail: bilateral 5 15 6 15 6 15 

Luminal/sloughed germ cells/debris: tail: 

focal

15 15 15 15 15 15 

Interstitial mononuclear cell infiltrate: focal 5 15 10 15 7 15 

Peripheral mixed cell infiltrate: focal 1 15 0 15 0 15 

Interstitial hemorrhage: focal 0 15 0 15 1 15 

Kidney 

Basophilic tubules: unilateral: focal 8 15 7 15 5 15 

Basophilic tubules: bilateral: focal 3 15 5 15 4 15 

Collecting duct hyperplasia: focal 1 15 0 15 0 15 

Atrophy/fibrosis/scar: unilateral 0 15 2 15 0 15 

Interstitial mononuclear cell infiltrate: focal 8 15 4 15 5 15 

Papillary mineralization: focal 0 15 1 15 0 15 

Pelvic dilatation: unilateral 5 15 4 15 2 15 

Cyst(s) 2 15 0 15 2 15 

a Data were obtained from Table 11 on pages 95-96 of the study report. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: The effect of metribuzin was characterized by 

effects on the thyroid, as evidenced by hormonal changes, increased thyroid weight, and/or 

treatment-related microscopic findings. The preputial separation examinations and 

testosterone measurement were unaffected by treatment. 

B. AGENCY COMMENTS: There were no effects of treatment on final body weights, 

overall body weight gain, or age and body weight at PPS; on the weights of kidneys, 

pituitary, adrenals, seminal vesicles plus coagulating gland (with or without fluid), ventral 

prostate, dorsolateral prostate, LABC, epididymides, or testes; or on levels of testosterone, 

creatinine, blood urea nitrogen, total bilirubin, aspartate aminotransferase, alanine 

aminotransferase, or alkaline phosphatase. There were no treatment-related 

histopathological findings in the testes, epididymides, or kidneys. 

Page 180 of 205
Male Pubertal Assay (2011) / Page 13 of 13 

METRIBUZIN/ 101101 OCSPP 890.1500 / OECD None

At 120 mg/kg/day, increased salivation was observed in all rats on one or several occasions 

throughout the study. In addition, one animal in this group had reduced motor activity and 

labored respiration on Day 13 and another rat had ocular discharge on Days 20-25. All rats 

in this group achieved PPS before study termination. Absolute, adjusted (for body weight 

on PND 21), and relative liver weights were increased (p≤0.01) by 21-23% and absolute and 

adjusted thyroid weights were increased (p≤0.01) by 45-46% compared to controls. Serum 

T4 levels were decreased (p≤0.01) by 72% in rats dosed at 120 mg/kg/day; mean TSH levels 

were increased (p≤0.01) by 236% and total protein was increased (p≤0.01) by 5% in this 

group. At necropsy, enlarged thyroid was observed in 6/15 rats and enlarged liver was 

observed in 11/15 rats. Minimal to slight follicular cell hypertrophy and colloid alteration 

were observed in the thyroid glands; an increased number of mitoses was noted in 2/15 

males in this group. There were increased numbers of mitoses in 7/11 examined livers in 

the 120 mg/kg/day group, as well as minimal centrilobular hepatoceullular hypertrophy 

(4/11 rats). 

At 60 mg/kg/day, increased salivation was observed on several occasions throughout the 

study in 5/15 rats. All but one rat in this group achieved PPS before study termination. 

Relative liver weights were increased (p≤0.01) by 12%; thyroid weights were comparable to 

the controls at this dose level. Serum T4 levels were decreased (p≤0.01) by 43% and mean 

TSH levels were increased (p≤0.01) by 109% in rats dosed at 60 mg/kg/day; total protein 

levels in this group were comparable to controls. At necropsy, enlarged liver was observed 

in 1/15 rats. 

The increased liver weights at 120 mg/kg/day indicated that the high dose was an adequate 

dose. 

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: 

 Mean body weight at PPS was 258.07 g (acceptable range 188.277-256.169 g) 

 CV for age at PPS was 6.4% (maximum 5.67%) 

 CV for body weight at PPS was 10.3% (maximum 7.57%) 

 CV for final body weight was 7.7% (maximum 7.47%) 

 CVs for epididymis weight were 16.8 and 19.8% (maximum 16.39%) 

 Mean serum TSH was 2.66 ng/mL (acceptable range 4.212-24.112 ng/mL) 

 CV for serum TSH was 67% (maximum 58.29%) 

Page 181 of 205
DATA EVALUATION RECORD 

METRIBUZIN 

Study Type: OCSPP 890.1550, Steroidogenesis Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-24-2012 (MRID 48617906) 

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:

Scott D. Studenberg, Ph.D., D.A.B.T. Date: 3/16/2012 

Secondary Reviewer Signature:

Michael E. Viana, Ph.D., D.A.B.T. Date: 3/27/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 3/30/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 3/30/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 182 of 205
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 183 of 205
METRIBUZIN I IOI IOI 

Primary Reviewer: Patience Browne, Ph.D. ____ _ 

Health Effects Division 

Secondary Reviewer: John Liccione Ph.D. 

Health Effects Division 

Sign a tu re: ~~--7'>-+--."'9-f ..... _ -_ -_ -_ -_ -----

Date: -----l'-4-.-4-~:.____.z:,---

DATA EVALUATION RECORD 

STUDY TYPE: Steroidogenesis Assay (H295R Cells); OCSPP 890.1550 

PC CODE: 101101 DP BARCODE: 0398559 

TXR#: 0056258 CAS No.: 21087-64-9 

TEST MATERIAL (PURITY): Metribuzin (94.4% a.i .) 

SYNONYMS: 4-Amino-6-( 1, l-dimethylethyl)-3-(methylthio )-1 ,2,4-triazin-5( 4H)-one 

CIT A TI ON: Tinwell, H. (2011) Evaluation of metribuzin in the H295R steroidogenesis 

assay. Bayer S.A.S. , Bayer CropScience, Sophia Antipolis Cedex, France. 

Laboratory Study No.: SA 11268, December 09, 2011. MRID 48617906. 

Unpublished. 

SPONSOR: Bayer CropScience AG, Alfred Nobel Str. 50, Manheim, Germany 

TEST ORDER#: CON-101101-11 

EXECUTIVE SUMMARY: In a steroidogenesis assay (MRID 48617906), H295R cells 

cultured in vitro in 24-well plates were incubated with metribuzin (94.4% purity, Lot # AE 

F055208-01-13) at serial log concentrations of 10- 10 to 10- 4 M for 48 hours in triplicate in three 

independent tests. Dimethyl sulfoxide (DMSO) was used as a vehicle, at a final concentration of 

0.1%. 

Testosterone and estradiol levels were measured by using EIA. The hormone assay interference 

test indicated no interference in the measurement of testosterone and estradiol concentrations in 

the presence of metribuzin. A Quality Control (QC) plate was run concurrently with each 

independent run of the test chemical plate to demonstrate that the assay responded properly to 

positive control agents at two concentration levels. The positive controls included a known 

inhibitor (prochloraz) and a known inducer (forskolin) of estradiol and testosterone production. 

Precipitation of the test compound, and cytotoxicity, were not observed at any concentration 

tested (:'.S l 0-4 M). Guideline acceptability recommendations and requirements were generally 

met, including lack of cytotoxicity, adequate production of testosterone and estradiol, acceptable 

reproducibility (low coefficients of variation, %CV), and appropriate induction and inhibition 

with positive controls. Exceptions included that the induction of testosterone on the QC plates in 

all assay runs (1 .50- to 1.86-fold) was below the 2-fold Guideline criteria, and the inter-plate % 

CV of the solvent control (SC) for estradiol (33%) in the QC plates exceeded the Guideline 

criteria (:'.S30%). 

Page 184 of 205
Steroidogenesis Assay (2011) / Page 2 of 11 

METRIBUZIN / 101101 OCSPP 890.1550/ OECD None 

No significant effects of metribuzin on testosterone production (0.81- to 0.97-fold change) were 

noted between 10−10 and 10−4

 M. No effects of metribuzin were noted on estradiol production at 

concentrations of 10−10 to 10−6 M; however, dose-dependent increases of 1.56- and 2.20-fold 

were observed for estradiol at metribuzin concentrations of 10−5

 and 10−4 M, respectively. 

Based on hormone responses in each of the independent runs, metribuzin treatment resulted in 

statistically significant and reproducible increases in estradiol production, but did not result in 

statistically significant and reproducible alterations in testosterone production. 

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

(OCSPP 890.1550). 

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

Confidentiality statements were provided. 

 

Page 185 of 205
Steroidogenesis Assay (2011) / Page 3 of 11 

METRIBUZIN / 101101 OCSPP 890.1550/ OECD None 

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Bayer S.A.S. 

 Location: Sophia Antipolis Cedex, France 

 Study Director: H. Tinwell 

 Other Personnel: O. Blank, Replacement Study Director 

D. Cespedes, Responsible Technician 

D. De Barbeyrac, Clinical Pathology Laboratory 

M. Vagner, Report Unit Assistant 

 Study Period: September 09, 2011 through December 09, 2011 

2. Test Substance: Metribuzin 

 Description: Light grey powder 

 Lot # (expiration date): AE F055208-01-13 (May 07, 2012) 

 Purity: 94.4% 

 Solubility: Soluble in DMSO at 10−1

 M 

 Vapor pressure: Not reported 

 Stability: 2 years 

 Storage conditions: Air-tight, light-resistant container at room temperature 

 CAS #: 21087-64-9 

 Molecular weight: 214.28

 Structure: 

N

N

N

O

NH2

S

CH3

3

CH

3CH

CH3

3. Positive Control: Forskolin 

 Description (molecular weight): White powder (410.50) 

 Source: Sigma-Aldrich (St. Louis, MO) 

 Batch # (expiration date): 109K5057V (Retest December, 2014) 

 Purity: 98% 

 Solubility (in solvent): Soluble in DMSO up to 0.01 M 

 Storage conditions: Air-tight, light-resistant container at room temperature 

 CAS #: 66575-29-9 

4. Negative Control: Prochloraz 

 Description (molecular weight): White powder (376.67) 

 Source: Sigma-Aldrich (Seelze, GmbH) 

 Batch # (expiration date): SZE6220X (August 8, 2012) 

 Purity: 99.1% 

 Solubility (in solvent): Soluble in DMSO up to 0.001 M 

 Storage conditions: Air-tight, light-resistant container at room temperature 

 CAS #: 67747-09-5 

Page 186 of 205
Steroidogenesis Assay (2011) / Page 4 of 11 

METRIBUZIN / 101101 OCSPP 890.1550/ OECD None 

5. Solvent/Vehicle Control: Dimethyl Sulfoxide (DMSO) 

 Description (molecular weight): Clear liquid (78.13) 

 Source: Sigma-Aldrich 

 Batch # (expiration date): 76396EM (not provided) 

 Purity: Not provided 

 Storage conditions: Not provided 

 CAS #: 67-68-5 

 Justification for choice of 

solvent: 

DMSO is a Guideline recommended solvent in which metribuzin is reasonably 

soluble. 

 Final concentration in assay: 0.1% v/v 

6. Stock Medium: Dulbecco’s modified Eagle’s medium/Ham’s F12 nutrient 

mixture 

Source: LGC (Ref: 30-2006) 

Lot # (expiration date): 30001173 (April 2012) 

Sodium bicarbonate: Component of stock medium 

Nu-Serum: 2.5%, BD Biosciences (Ref: 355100); Lot # 81515; not reported if background 

hormone concentrations were measured by performing laboratory 

ITS+premix: VWR (Ref: 734-1315); Lot # 84339 

Other components: 0.1% Penicillin/streptomycin; GIBCO (15070-063); Lot # 847134. 

7. Test Cells: H295R human adrenocortical carcinoma cells (ATCC CLR-2128; Batch # 

58660579) at passage 5.5 – 8.5 were incubated in the stock medium. Incubation conditions 

were at 37±1° C with 5% CO2 for approximately 24 hours prior to exposure. 

 The following performance criteria were met (indicated by an “x”): 

x Cell passage identifier. Cell Passage #: 5.5 – 8.5 

x Cells frozen down at passage 5 

x Frozen cells cultured for 4 additional passages 

x Total number of passages does not exceed 10 

B. METHODS

1. Pre-Test Information 

a. Hormone Assay Interference Test: Analysis of testosterone and estradiol by EIA kits 

indicated mean variation of samples with metribuzin were 10% and 22%, respectively, both 

within the 30% specified by the test guidelines. 

b. Hormone Extraction: Hormone extraction was not reported. 

Page 187 of 205
Steroidogenesis Assay (2011) / Page 5 of 11 

METRIBUZIN / 101101 OCSPP 890.1550/ OECD None 

c. Laboratory Proficiency Test: A laboratory proficiency determination (SA 11281; 

non-GLP) was previously conducted by the technician who conducted the present study. 

The test conditions were the same as the current assay. The concentrations used and the 

EC50 values for each positive control are given below: 

TABLE 1. Results of Laboratory Proficiency Test with Forskolin and Prochloraz.a 

Compound Concentrations tested (μM) Estimated EC50 (μM) b

Testosterone Estradiol 

Forskolin 0.03, 0.1, 0.3, 1, 3, and 10 ~0.2 ~0.55 

Prochloraz 0.01, 0.03, 0.1, 0.3, 1, and 3 ~0.08 ~0.25 

a Data were obtained from page 16 of the study report. 

b The Guideline (890.1550) specified range for EC50s for forskolin are 0.2-2.0 μM for testosterone and 0.3-3.0 μM for 

estradiol, and the specified range for ED50s for prochloraz are 0.01-0.1 μM for testosterone and 0.03-0.3 μM for 

estradiol. 

2. Test Solutions: Metribuzin, forskolin, and prochloraz were dissolved in DMSO and diluted 

1:1000 in the final treatment medium. When added to the cell culture plates, these dilutions 

yielded final concentrations of 1 μM and 10 μM for forskolin, 0.1 μM and 1 μM for 

prochloraz, and 10−4 to 10−10 M for metribuzin. The final concentration of DMSO in the 

medium was held constant at 0.1% (v/v). 

3. Cell Plating and Preincubation: Cells were maintained in the stock medium described 

above. The hormone concentrations in undiluted Nu-Serum were not reported. H295R cells 

were grown for five passages, frozen in liquid nitrogen, then thawed and cultured for five to 

eight additional passages prior to use in the assay. The cells were plated into wells of a 

24-well cell culture plate at a density of approximately 300,000 cells in 1 mL. The cells 

were then placed into an incubator (37±1°C/5% CO2) for approximately 24 hours prior to 

chemical exposure. After a 24-hour pre-incubation, the cells were checked microscopically 

for good attachment and proper morphology. 

4. Exposure: The medium was removed from the cells and replaced with 1 mL of medium 

containing concentrations of metribuzin (or solvent) in triplicate, according to the schematic 

presented in Table 2. 

TABLE 2. Dosing Schematic for the Exposure of H295R Cells to Metribuzin (Final Concentrations in 

M).a

 1 2 3 4 5 6 

A DMSO DMSO DMSO 10−4 10−4 10−4

B 10−5 10−5 10−5 10−6 10−6 10−6

C 10−7

 10−7 10−7 10−8 10−8 10−8

D 10−9 10−9 10−9 10−10 10−10 10−10

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

A concurrent QC plate was included with each of the three independent runs of the test 

chemical plates to demonstrate the assay’s response to forskolin (an inducer of testosterone 

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

Page 188 of 205
Steroidogenesis Assay (2011) / Page 6 of 11 

METRIBUZIN / 101101 OCSPP 890.1550/ OECD None 

production). The QC plate was prepared and dosed in the same manner with either forskolin 

or prochloraz, according to the schematic presented in Table 3. 

TABLE 3. Dosing Schematic for the QC Plate for Positive Controls (Final Concentrations in μM).a

 1 2 3 4 5 6 

A Blankb Blank Blank Blank Blank Blank 

B DMSO DMSO DMSO DMSO DMSO DMSO 

C Forskolin 

10 μM 

Forskolin 

10 μM 

Forskolin 

10 μM 

Forskolin 

1 μM 

Forskolin 

1 μM 

Forskolin 

1 μM 

D Prochloraz 

1 μM 

Prochloraz 

1 μM 

Prochloraz 

1 μM 

Prochloraz 

0.1 μM 

Prochloraz 

0.1 μM 

Prochloraz 

0.1 μM 

a Data were obtained from page 17 and 18 of the study report. 

b Blank designates wells containing only cells and culture medium. 

Following dosing, the plates were incubated for approximately 48 hours under the 

conditions previously described. After the incubation period, three or four portions of 

medium were collected and frozen at −74±10°C until hormone analysis was conducted. 

5. Cell Viability/Cytotoxicity Assay: Prior to the main study, the cytotoxicity of metribuzin, 

at all concentrations (10−4 to 10−10 M), was assessed with the XTT (2,3-Bis(2-methoxy-4-

nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) method, which measures the 

mitochondrial activity of living cells. The cells were first exposed to metribuzin for 48 h 

under the same conditions of the test. During the last four hours of incubation the cells were 

exposed to XTT (supplied by Sigma, ref. number: X4251; batch number: 038K1592)/ 

phenazine methosulfate (supplied by Sigma, ref. number: P9625; batch number: 058K1537). 

The resulting orange formazan derivative was measured at OD 450 nm. No cytotoxicity 

was observed at any concentration (10−4 to 10−10 M). In addition, at the end of each test 

evaluation, the treated cells were stained with trypan blue to evaluate cell viability 

6. Hormone Measurement System: The concentration of each hormone (testosterone and 

estradiol) was evaluated in triplicate in the culture medium using specific EIA kits supplied 

by Enzo Life Sciences Inc. (Plymouth Meeting, PA) for testosterone and Cayman Chemical 

Company (Ann Arbor, MI) for estradiol. The limit of detection for testosterone was 95.7 

pg/mL and for estradiol it was 5.7 pg/mL. 

The following performance criteria were met (indicated by an “x”): 

X Method detection limit (100 pg/mL testosterone; 10 pg/mL estradiol) 

X Spiked sample recovery acceptable for two concentrations of testosterone and estradiol (mean measured amount 

from triplicate samples within 30% of nominal concentration) 

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

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

X Test compound tested for interference with measurement system 

C. DATA ANALYSIS: To evaluate the relative increase or decrease of hormone production 

after test chemical exposure, the results were normalized to the mean SC value for each 

assay and results were expressed as fold-change relative to the SC in each exposure plate. 

All data were expressed as mean ± standard deviation (SD). Relative changes were 

calculated as follows: 

Page 189 of 205
Steroidogenesis Assay (2011) / Page 7 of 11 

METRIBUZIN / 101101 OCSPP 890.1550/ OECD None 

Relative Change = (Hormone concentration in each well) ÷ (Mean hormone concentration 

of SC) 

For each hormone, the Bartlett test was performed to compare the homogeneity of group 

variances. If the Bartlett test was not significant, means were compared by using the 

analysis of variance (ANOVA). If the ANOVA was not significant, the group means were 

considered to be homogeneous and no further analysis was performed. If the ANOVA was 

significant, means of the exposed groups were compared to the mean of the control group 

with the Dunnett test (2-sided). If the Bartlett test was significant, group means were 

compared by using the non-parametric Kruskal-Wallis test. If the Kruskal-Wallis test was 

not significant, the group means were considered to be homogeneous and no further analysis 

was performed. If the Kruskal-Wallis test was significant, means of the exposed groups 

were compared to the mean of the control group with the Dunn test (2-sided). The level of 

significance for each statistical comparison was performed at p = 0.05. These statistical 

analyses were considered appropriate. 

II. RESULTS

A. TEST COMPOUND: Precipitation of the test compound was not reported at any 

concentration. The variability (% CV) between the runs for the SC based on absolute 

hormone concentrations (calculated by the reviewer) was 21% for testosterone and 26% 

for estradiol, and within the Guideline recommendation (≤30%). The % CV within the 

runs for the SC was <10% for testosterone and <25% for estradiol. 

The hormone concentrations following exposure to metribuzin are presented in Table 4, 

and the fold changes in hormone production for metribuzin-treated cells compared to the 

solvent control are shown graphically in Figures 1 and 2. No effects of metribuzin on 

testosterone production were noted between 10−10 and 10−4

 M with the percent changes 

for the individual evaluations varying between −19% (0.81-fold) and −3% (0.97-fold). 

No concentration dependent effects of metribuzin were noted on estradiol production at 

concentrations of 10−10 to 10−6

 M. Increases of 1.56- and 2.20-fold were observed for 

estradiol at metribuzin concentrations of 10−5 and 10−4 M, respectively. 

Page 190 of 205
Steroidogenesis Assay (2011) / Page 8 of 11 

METRIBUZIN / 101101 OCSPP 890.1550/ OECD None 

TABLE 4. Mean (±SD) Hormone Concentrations Following Treatment with Metribuzin for 48 Hours.a

Nominal 

Concentration 

(M) 

Run 1 Run 3 Run 4 Run 1 Run 3 Run 4 Mean ± SD Statistical 

Significance

Testosterone (pg/mL) Fold Difference 

DMSO 5868.7 4096.3 6208.3 — — — — — — 

10−10 4901.0 4126.7 4113.0 0.84 1.01 0.66 0.81 0.18 — 

10−9 6323.0 4519.0 4900.3 1.08 1.10 0.79 0.97 0.17 — 

10−8 5094.3 4018.7 4392.3 0.87 0.98 0.71 0.84 0.14 — 

10−7 5619.3 4182.3 5292.0 0.96 1.02 0.85 0.93 0.09 — 

10−6 5333.7 4088.3 5610.3 0.91 1.00 0.90 0.93 0.06 — 

10−5 5533.0 4311.7 5313.7 0.94 1.05 0.86 0.94 0.10 — 

10−4 5600.3 4133.0 5806.3 0.95 1.01 0.94 0.96 0.04 — 

Estradiol (pg/mL) Fold Difference 

DMSO 146.3 246.3 187.0 — — — — — — 

10−10 185.3 317.7 161.3 1.27 1.29 0.86 1.15 0.24 — 

10−9 211.7 285.0 154.7 1.45 1.16 0.83 1.12 0.31 — 

10−8 133.0 247.3 242.7 0.91 1.00 1.30 1.08 0.20 — 

10−7 213.0 294.0 190.7 1.46 1.19 1.02 1.20 0.22 — 

10−6 179.3 254.3 208.3 1.23 1.03 1.11 1.11 0.10 — 

10−5 196.3 322.0 386.0 1.34 1.31 2.06 1.56 0.42 ** 

10−4 399.7 441.3 433.0 2.73 1.79 2.32 2.20 0.47 ** 

a Data (rounded up) were obtained from pages 27–28 of the study report. The lower limit of quantification (LLQ) was 

95.7 pg/mL for testosterone and 5.7 pg/mL for estradiol. Statistical significance was determined for the mean of the three 

runs. 

** p≤0.01 

 

Page 191 of 205
Steroidogenesis Assay (2011) / Page 9 of 11 

METRIBUZIN / 101101 OCSPP 890.1550/ OECD None 

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

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

B. CYTOTOXICITY: No cytotoxicity was observed for metribuzin at any concentration 

(10−4 to 10−10 M) by the XTT method. In addition, at the end of each evaluation, no toxicity 

was noted in any of the metribuzin-treated cells (10−4

 to 10−10 M) when evaluated with the 

trypan blue exclusion method. 

C. QC PLATE: The hormone concentrations after exposure to the reference chemicals, SC, 

and blank samples, as well as the fold difference change relative to SC (individual and mean 

± SD) for the three assay runs are presented in Table 5. The minimum basal hormone 

production levels (500 pg/mL for testosterone, 40 pg/mL for estradiol) were met in both 

blank and SC wells for testosterone. Guideline requirements for basal hormone production, 

induction, and inhibition were met for testosterone and estradiol, except for induction of 

testosterone with 10 μM forskolin in all three test runs (average 1.70-fold induction) which 

were below the Guideline recommendation of ≥2-fold induction. 

Page 192 of 205
Steroidogenesis Assay (2011) / Page 10 of 11 

METRIBUZIN / 101101 OCSPP 890.1550/ OECD None 

The variability (%CV) between the runs based on the absolute hormone concentrations in 

the SC was 6.4% for testosterone, which meets guideline criteria of ≤30%. The variability 

(%CV) between the runs in the SC was 33% for estradiol which is greater than the guideline 

requirement. The variability (CV) in the SC within each run in the testosterone and 

estradiol assays was <22%, which was within the guideline recommendation of ≤30%. 

TABLE 5. Mean (±SD) Hormone Concentrations Following Treatment with Forskolin or Prochloraz for 

48 Hours.a

Concentration (μM) 

Run 1 Run 3 Run 4 Run 1 Run 3 Run 4 Mean ± SD 

Testosterone (pg/mL) Fold Difference (Relative to DMSO) 

Blank 5891.8 5115.0 5456.2 — — — — — 

DMSO 5646.3 5267.3 4968.3 — — — — — 

1 μM Forskolin 8250.0 5947.3 6992.7 1.46 1.13 1.41 1.33 0.18 

10 μM Forskolin 9809.3 7876.3 9247.7 1.74 1.50 1.86 1.70 0.18 

0.1 μM Prochloraz 1735.7 1368.7 1269.7 0.31 0.26 0.26 0.28 0.03 

1 μM Prochloraz 350.0 315.0 154.0b

 0.06 0.06 0.03b 0.05c 0.02 

Estradiol (pg/mL) Fold Difference (Relative to DMSO) 

Blank 211.2 362.5 272.3 — — — — — 

DMSO 168.3 325.0 232.5 — — — — — 

1 μM Forskolin 3982.0 4759.0 4063.3 23.7 14.6 17.5 17.6 4.65 

10 μM Forskolin 5718.7 7432.0 7705.7 34.0 22.9 33.1 28.7 6.17 

0.1 μM Prochloraz 134.0d

 202.7 I 0.80d 0.62 I 0.77e 0.13 

1 μM Prochloraz I I I I I I I — 

a Data were obtained from pages 23-25 of the study report. The lower limit of quantification (LLQ) was 95.7 pg/mL for 

testosterone and 5.7 pg/mL for estradiol. 

b n = 2 instead of 3 

c overall n = 8 instead of 9 

d n = 1 instead of 3 

e overall n = 4 instead of 9 

I Complete inhibition (< 100 pg/mL) 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Overall, metribuzin was not cytotoxic to the 

H295R adrenal cells at any of the concentrations tested, and metribuzin did not interfere 

with the EIA analysis of testosterone and estradiol concentrations. Metribuzin did not affect 

testosterone secretion at any concentration tested (fold change ranged from 0.81 to 0.97) in 

the H295R steroidogenesis assay. Statistically significant, dose-dependent increases in 

estradiol secretion were observed at 10−5 and 10−4 M (1.56-fold and 2.20-fold, respectively), 

indicating that metribuzin is capable of interfering with steroidogenesis at high 

concentrations. 

B. AGENCY COMMENTS: In the QC plate, the minimum basal hormone production levels 

(500 pg/mL for testosterone, 40 pg/mL for estradiol) were met in both blank and SC wells 

for testosterone. Guideline requirements for basal hormone production, induction, and 

inhibition were met for testosterone and estradiol, except for induction of testosterone with 

10 μM forskolin in all assay runs (1.50- to 1.86-fold induction) which were below the 

Guideline recommendation (≥2-fold). Intra- and interplate variability in both the test and 

QC plates met the Guideline recommendation (≤30% CV), except for the between plate % 

CV for estrogen in QC plates (33%) which was slightly above the Guideline limit. 

Page 193 of 205
Steroidogenesis Assay (2011) / Page 11 of 11 

METRIBUZIN / 101101 OCSPP 890.1550/ OECD None 

 Precipitation of the test compound, and cytotoxicity, were not reported at any concentration 

tested, though detailed cytotoxicity data were not provided. No effects of metribuzin on 

testosterone production were noted between 10−10 and 10−4 M with the percent changes for 

the individual evaluations varying between −19% (0.81-fold) and −3% (0.97-fold). The 

variability in testosterone concentrations within the runs for the SC wells was within the 

Guideline recommendation for the testosterone assays (≤22% compared to the Guideline 

limit of <30%). No effects of metribuzin were noted on estradiol production at 

concentrations of 10−10 to 10−6 M. However, dose-dependent increases of 1.56- and 2.20-

fold were observed in estradiol at metribuzin concentrations of 10−5 and 10−4 M, 

respectively. The variability in estradiol concentrations between the runs for the SC wells 

was within the Guideline recommendation for the estradiol assays (≤19% compared to the 

guideline limit of 30%). 

Based on hormone responses in each of the independent runs, metribuzin treatment resulted 

in a statistically significant and reproducible alteration in estradiol production. 

 

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

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

 Stability of metribuzin in the test system was not reported. 

 Induction of testosterone in Runs 1, 3, and 4 on the QC plates (1.50- to 1.86-fold) was 

below the ≥2-fold Guideline criteria. 

 The range for inter-plate % CVs for estradiol in the QC plates (33%) exceeded the 

Guideline criteria (≤30%).

Page 194 of 205
DATA EVALUATION RECORD 

METRIBUZIN 

Study Type: OCSPP 890.1600, In vivo Uterotrophic Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-24-2012 (MRID 48617907) 

Prepared for 

Health Effects Division 

Office of Pesticide Programs 

U.S. Environmental Protection Agency 

2777 South Crystal Drive 

Arlington, VA 22202 

Prepared by 

CSS-Dynamac Corporation 

1910 Sedwick Road, 

Building 100, Suite B 

Durham, NC 27713 

Primary Reviewer Signature:

Rebecca L. Bryan, B.S. Date: 3/12/2012 

Secondary Reviewer Signature:

Steven Brecher, Ph.D. Date: 3/13/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 3/28/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 3/28/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 195 of 205
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 196 of 205
METRIBUZIN / 101101 

In vivo Uterotrophic Assay (201 I) I Page I of9 

OCSPP 890.1600/ OECD 440 

Primary Reviewer: _Y~u_n...,.g_Y_a_n_..g~,_P_h_.D_. ________ Signature: ~ G-. lfc---a 

Health Effects Division Date: -~fJ __ .a ...... z_:J.._f~/_iN_ ~-- Secondary Reviewer: Jess Rowland Signature: ,}=~a-~ 

Health Effects Division Date: --~:,.,."'""if-1---"-L_.Z ...... I_-,...._> __ 

Template version 09/2011 

DATA EVALUATION RECORD 

STUDY TYPE: Uterotrophic Assay (Rat); OCSPP 890.1600; OECD 440 

PC CODE: 101101 DP BARCODE: D398559 

TXR#: 0056258 CAS#: 21087-64-9 

TEST MATERIAL (PURITY): Metribuzin (94.4% purity) 

SYNONYMS: 4-amino-6-(1, 1-dimethylethyl)-3-(methylthio )-1,2,4-triazin-5( 4H)-one 

CITATION: Totis, M. (2011). Metribuzin Evaluation in the Immature Rat Uterotrophic Assay. 

Bayer SAS, France. Laboratory report number SA 10345, November 10, 2011. 

MRID 48617907. Unpublished. 

SPONSOR: Bayer CropScience AG, Alfred Nobel Str. 50, 40789 Monheim, Germany 

TEST ORDER#: CON-101101-11 

EXECUTIVE SUMMARY: In an Uterotrophic Assay (MRID 48617907) conducted to screen 

for potential estrogenic activity, metribuzin (94.4%, Batch # K285067) in 0.5% methylcellulose 

400 + 0.4% Tween 80 was administered daily via oral gavage to groups of 6 immature female, 

Sprague-Dawley rats at dose levels of 0 (vehicle), 10, 40, or 120 mg/kg/day on post-natal days 

(PND) 19-21. A positive control group was treated with 17a-ethynyl estradiol (EE) in corn oil 

by gavage at a dose level of 1 μg/kg/day. All animals were terminated and necropsied on PND 

22 approximately 24 hours after the final dose to determine wet and blotted uterine weights. 

All animals survived until scheduled termination. No clinical signs of toxicity were observed in 

animals for any dose groups. Body weights in the metribuzin treated groups were comparable to 

the controls throughout the study; however, the body weight gains for Days 1-3 were decreased 

in all metribuzin treated groups. The body weight gains were decreased (p<0.01) by 33, 40, and 

46% at 10, 40, and 120 mg/kg/day, respectively. Uterine weights in the metribuzin treated 

groups were comparable to the controls. Absolute wet and blotted uterus weights for the EE 

group were increased (p<O. 01) by 1 09% and 110%, respectively, as expected. 

The doses tested were determined to be adequate based on the dose-dependent decreases in body 

weight gains (33-46%). 

No statistically significant changes were seen in uterine weight in this assay. Metribuzin is 

negative in the uterotrophic assay. 

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

Page 197 of 205
In vivo Uterotrophic Assay (2011) / Page 2 of 9 

METRIBUZIN / 101101 OCSPP 890.1600/ OECD 440

890.1600). 

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

provided. 

Page 198 of 205
In vivo Uterotrophic Assay (2011) / Page 3 of 9 

METRIBUZIN / 101101 OCSPP 890.1600/ OECD 440

I. MATERIALS AND METHODS

A. MATERIALS 

1. Test Facility: Bayer S.A.S. 

Location: 355 Rue Dostoievski, BP 153, 06903 Sophia Antipolis Cedex, France

 Study Director: M. Totis 

 Other Personnel: M. Blanck (Replacement Study Director); J.P Kocwin (Laboratory Animal Resources); 

C. Jaussely (Toxicology Supervisor); B. Trela (Technician); L. Amir Tahmasseb 

(Senior Chemist); C. Pallen (Statistician); M. Vagner (Report Unit Assistant) 

 Study Period: May 10, 2011 to November 10, 2011 

2. Test Substance: Metribuzin 

 Description: Light grey powder

 Source: Bayer CropScience

 Lot/Batch #: K285067 (expiry date May 7, 2012)

 Purity: 94.4% 

 Stability: Stable in 0.5% methylcellulose 400 + 0.4% Tween 80 at 1 and 100 g/L 

under conditions similar to the study for at least 21 days. 

 CAS #: 21087-64-9

 Structure: 

N

N

N

O

NH2

S

CH3

3

CH

3CH

CH3

3. Reference Estrogen: 17α-ethynyl estradiol (EE) 

 Supplier Sigma-Aldrich (U.S.A.)

 Lot/Batch #: 090M1241V 

 Purity: 99% 

 CAS # : 57-63-6 

4a. Solvent/Vehicle Control 

for Test Chemical: Methylcellulose 400 

 Supplier: Sigma-Aldrich 

 Batch #: 060M123V 

 Rationale (if other than water): Not reported 

 Final concentration: 

Additional Solvent: 

0.5% aqueous 

Tween 80 at 0.4% aqueous (Batch # S5264887.914; Merck) 

4b. Solvent/Vehicle Control 

for Positive Control: Corn Oil 

Supplier: Sigma Aldrich 

Batch #: MKBD2427 

Rationale (if other than 

water): 

Not reported 

Final concentration: Not applicable 

Page 199 of 205
In vivo Uterotrophic Assay (2011) / Page 4 of 9 

METRIBUZIN / 101101 OCSPP 890.1600/ OECD 440

5. Test Animals: 

 Species: Rats (immature, female only) 

 Strain: Sprague Dawley (Crl:CD[SD]) 

 Age/weight at dose initiation: 19 days old/ 36.2-46.6 g 

 Source: Charles River Laboratories (France); shipped with dam

 Housing: Rats were housed 6 per cage in makrolon cages with heat-treated soft wood 

bedding (S.A.F.E.; Scientific Animal Food and Engineering, Augy, France). 

 Diet: Certified rodent pelleted and irradiated diet A04C-10 (S.A.F.E.; Augy France) with 

phytoestrogens <350 μg/g diet, ad libitum. 

 Water: Filtered tap water, ad libitum 

 Environmental conditions: Temperature: 

Humidity: 

Air changes: 

Photoperiod: 

20-24oC 

40-70% 

10-15/hr 

12 hrs light/ 12 hrs dark 

 Acclimation period: 5 days; immature females housed with their dam 

B. STUDY DESIGN 

1. In Life Dates: Start: May 20, 2011 End: May 23, 2011 

2. Study Design: Following an initial 5-day acclimation period, immature, intact female rats 

were administered the test substances from PND 19 to 21. Rats were euthanized 

approximately 24 hours after the last dose and necropsied for uterine weight measurements. 

The pups were housed with their dam prior to weaning. 

3. Animal Assignment: Animals were randomly assigned, stratified by body weight, to the 

test groups noted in Table 1. A computerized randomization procedure (XMS Path/Tox 

V4.2.2) was used to select animals for the study from the middle of the weight range of the 

available animals, which ensured a similar body weight distribution among groups. Thirty of 

the 40 female pups received were selected for the study. The body weight of each animal 

was within 20% of the overall mean. 

TABLE 1. Study Designa

Test Group Dose (mg/kg/day) # of Females 

Estrogen Agonist Assay 

Vehicle Control 0 6 

Low Metribuzin 10 6 

Mid Metribuzin 40 6 

High Metribuzin 120 6 

17α-ethynyl estradiol (EE), Reference Estrogen 0.001 6 

a Data were obtained from page 16 of the study report. 

4. Dose Selection Rationale: The dose levels used in this study were chosen based on the 

results of a preliminary range-finding study in Sprague Dawley rats. A summary of this 

study is included as an Appendix to this DER.

5. Dose Preparation and Analysis: Dose formulations were prepared once prior to initiation 

of treatment by suspending the appropriate amounts of metribuzin in 0.5% methylcellulose 

400 + 0.4% Tween 80. The positive control, EE, was prepared once prior to initiation of 

treatment by diluting in corn oil. Doses were adjusted daily based on individual body 

Page 200 of 205
In vivo Uterotrophic Assay (2011) / Page 5 of 9 

METRIBUZIN / 101101 OCSPP 890.1600/ OECD 440

weight measurements. Homogeneity of the test substance formulation was checked at the 

beginning of the study for the lowest and the highest concentrations (2 and 24 g/L) at the 

surface and bottom levels. The mean values obtained from the homogeneity check were 

used as measured concentrations. In addition, the intermediate concentration (8 g/L) was 

checked. Data were recorded and analyzed using Empower 2 (Build 2154). It was reported 

that a separate study (SA 110891) demonstrates that metribuzin in 0.5% methylcellulose 400 

+ 0.4% Tween 80 is stable at concentrations of 1 and 100 g/L for at least 21 days under 

similar conditions to those of the current study. Dose formulations were stored at 5 ± 30C 

when not in use. 

Results of Dose Analysis 

Homogeneity: 98-100% of nominal 

Stability: Stable in 0.5% methylcellulose 400 + 0.4% Tween 80 for up to 21 days under 

conditions similar to the subject study. 

Concentration: 98-100% of nominal

Although the middle layer of the test solution was not tested for homogeneity, the analytical 

data indicated that the mixing procedure was adequate and that the variation between 

nominal and actual dosage to the animals was acceptable. 

6. Dosage Administration: Animals were administered the metribuzin formulations, positive 

control (EE) or vehicle control daily via oral gavage for three consecutive days in a dose 

volume of 5 mL/kg body weight. Dose volumes were adjusted daily based on the 

concurrent body weight measurement.

7. Statistics: Statistical analyses of body weight and body weight changes were carried out 

using Path/Tox System V4.2.2. Statistical analyses of terminal body weight and uterine 

weights were carried out using SAS system. Means and standard deviations were calculated 

for each group and percent coefficient of variation was calculated for terminal body weight 

and uterine weight parameters. In addition, a covariance analysis (ANCOVA) was 

performed on wet and blotted uterus weights using the terminal body weight as a covariable; 

adjusted means were compared using the Dunnett test. Group means were compared at the 

5% and 1% levels of significance. The statistical analyses were considered appropriate.

C. METHODS

1. Clinical Examinations: Animals were observed for clinical signs of toxicity at least once 

daily, and observed for mortality and moribundity twice daily, except on weekends and 

holidays when they were checked once a day. 

2. Body Weight: Animals were weighed at randomization, study initiation, daily throughout 

the dosing period and at termination. 

 

1

 Vincent, M. (2011, on going). Metribuzin, Stability in methylcellulose 400 + 0.4% Tween 80, SA 11089, Bayer 

CropScience, Sophia Antipolis, France. 

Page 201 of 205
In vivo Uterotrophic Assay (2011) / Page 6 of 9 

METRIBUZIN / 101101 OCSPP 890.1600/ OECD 440

3. Food Consumption (Optional): Food consumption was not measured. 

4. Necropsy and Measurement of Uterine Weight: On PND 22 (approximately 24 hours 

after final administration of the test substance), all animals were anesthetized with 

isoflurane, exsanguinated, and subjected to a gross necropsy. Dissection of the uterus was 

performed according to the U.S. EPA Guideline. Briefly, the vagina was removed just below 

the cervix in order to retain the luminal fluid in the uterus. The “wet” uterus (i.e., containing 

the luminal fluid) was weighed. Subsequently, the uterus was pierced and blotted to remove 

the luminal fluid and the blotted uterus was weighed. 

5. Microscopic Examination (Optional): Microscopic examinations were not conducted.

II. RESULTS

A. OBSERVATIONS

1. Mortality: All animals survived until scheduled termination. 

2. Clinical Signs of Toxicity: No clinical signs of toxicity were observed in animals for any 

dose groups. 

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

presented in Table 2. Body weights in the metribuzin-treated groups were comparable to 

the controls throughout the study; however, the overall body weight gains for Days 1-3 were 

decreased in all metribuzin treated groups. The body weight gains were decreased (p<0.01) 

by 33, 40, and 46% at 10, 40, and 120 mg/kg/day, respectively. In the positive control 

group (EE), the body weight gains decreased (p<0.05) by 17% for Days 1-3. 

TABLE 2. Group Body Weights and Cumulative Body Weight Gains (g) in the Estrogen Agonist Assay a

Study Day 

#

Dose Group (mg/kg/day) 

Vehicle Control Metribuzin 

(10) 

Metribuzin 

(40) 

Metribuzin 

(120) 

Estrogen, EE 

(0.001) 

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 

1 6 42.0 1.6 6 41.8 3.1 6 42.8 2.6 6 42.7 2.2 6 42.5 1.7 

2 6 46.2 2.2 6 44.4 3.3 6 44.5 3.0 6 44.8 3.3 6 46.0 1.6 

3 6 52.7 2.1 6 49.0 2.9 6 49.2 3.0 6 48.5 3.5 6 51.4 2.3 

BWG 

Days 1-3 6 10.7 1.1 6 7.2** (↓33) 0.9 6 6.4** (↓40) 0.9 6 5.8** (↓46) 1.4 6 8.9* (↓17) 1.2 

a Data were obtained from Tables 2a , 2b, 3a, and 3b on pages 34, 36, 39, and 41 of the study report. 

N No. of animals in the group 

SD Standard Deviation 

* Significantly different from vehicle control at p < 0.05 (Days 1-3). 

** Significantly different from vehicle control at p < 0.01 (Days 1-3). 

C. FOOD CONSUMPTION (Optional): Food consumption was not measured.

D. PATHOLOGY 

Page 202 of 205
In vivo Uterotrophic Assay (2011) / Page 7 of 9 

METRIBUZIN / 101101 OCSPP 890.1600/ OECD 440

1. Uterine Weights: Uterine weight data are presented in Table 3. Uterine weights in the 

metribuzin treated groups were comparable to the controls. 

Absolute wet and blotted uterus weights for the positive EE group were increased (p<0.01) 

by 109% and 110%, respectively. These wet and blotted uterine weights increased as 

expected. 

TABLE 3. Uterine Weights (mg) from Estrogen Agonist Assay in SD Rats a

Parameter 

Dose Group (mg/kg/day) 

Vehicle Control Metribuzin 

(10) 

Metribuzin 

(40) 

Metribuzin 

(120) 

Estrogen, EE 

(0.001) 

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 

Terminal 

BW (g) 6 57.1 1.8 6 55.8 2.8 6 54.6 3.3 6 52.6 3.3 6 55.4 2.9 

Wet, 

absolute (g) 5 b 0.0288 0.0056 6 0.0288 0.0027 6 0.0295 0.0037 6 0.0280 0.0048 6 0.0601** 

(↑109) 0.0134 

Wet, 

relative (%) 5 0.050 0.008 6 0.052 0.004 6 0.054 0.005 6 0.053 0.009 6 0.108 0.020 

Blotted, 

absolute (g) 5 0.0260 0.0058 6 0.0257 0.0029 6 0.0252 0.0044 6 0.0238 0.0026 6 0.0546** 

(↑110) 0.0124 

Blotted, 

relative (%) 5 0.045 0.008 6 0.046 0.004 6 0.046 0.007 6 0.045 0.006 6 0.098 0.019 

a The means and standard deviations for terminal body weights, and wet and blotted uterine weights (absolute and relative) 

were calculated by the study reviewer from the individual data (Appendix D, page 63 of the study report). The calculated 

percent difference from controls is presented in parentheses. 

b The wet and blotted uterine weights of one vehicle control animal were “not tested.” 

BW Body weight 

N No. of animals in the group 

SD Standard Deviation 

** Significantly different from vehicle control at p < 0.01. 

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

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Individual uterine weights in the control group 

met the acceptance criteria defined in the US EPA guideline. Ethynyl estradiol, the 

reference positive control, administered daily by oral gavage for 3 days at 1 μg/kg/day 

increased absolute uterine weights (wet and blotted), (p≤0.01) as expected. Metribuzin had 

no effect on terminal body weight or uterine weights at any dose level. At 120, 40 and 10 

mg/kg/day of metribuzin, the mean cumulative body weight gain was reduced by 46, 40 and 

33% (p≤0.01), respectively, between Study Day 1 and 3 compared to the controls. In 

conclusion, these data indicate that administration of up to 120 mg/kg/day of metribuzin for 

3 days to immature Sprague Dawley female rats had no effect on the uterus weights (wet 

and blotted). Therefore, no evidence of an estrogenic potential was detected. 

B. AGENCY COMMENTS: All animals survived until scheduled termination. No clinical 

signs of toxicity were observed in animals for any dose groups. Body weights in the 

metribuzin treated groups were comparable to the controls throughout the study; however, 

the body weight gains for Days 1-3 were decreased (p<0.01) by 33, 40, and 46% at 10, 40, 

and 120 mg/kg/day, respectively. Uterine weights in the metribuzin treated groups were 

Page 203 of 205
In vivo Uterotrophic Assay (2011) / Page 8 of 9 

METRIBUZIN / 101101 OCSPP 890.1600/ OECD 440

comparable to the controls. Absolute wet and blotted uterus weights for the EE group were 

increased (p<0.01) by 109% and 110%, respectively, as expected. 

 

The dose levels were determined to be adequate based on the dose-dependent decreases in 

body weight gains (33-46%). No statistically significant changes were seen in the uterine 

weight in this study. Metribuzin was negative in the uterotrophic assay 

C. STUDY DEFICIENCIES: None 

Page 204 of 205
In vivo Uterotrophic Assay (2011) / Page 9 of 9 

METRIBUZIN / 101101 OCSPP 890.1600/ OECD 440

APPENDIX

Since this is a range-finding study, only a summary is provided to confirm the adequacy of the 

dose selection rationale used in the uterotrophic assay (MRID 48617907). 

In a preliminary range-finding assay (Appendix F of MRID 48617907) with peripubertal rats, 

metribuzin (purity not reported; batch# K285067) in 0.5% methylcellulose 400 + 0.4% Tween 80 

was administered via daily oral gavage in a dose volume of 5 mL/kg body weight to 4 female 

Sprague Dawley [Crl:CD(SD)] rats/dose group at doses of 0, 5, 20, 50, 80 or 150 mg/kg/day. 

Rats were PND 21 on the first day of dosing. The animals were dosed for 10 consecutive days 

and necropsied approximately 24 hours after the final dose administration. 

All animals in the 150 mg/kg/day dose group survived until scheduled termination. Treatmentrelated clinical signs included reduced motor activity from Day 1 and 3. On Day 2, body weight 

and body weight gain were decreased (↓7% and ↓60%, respectively). On Day 4, body weight 

and body weight gain were decreased (↓9% and ↓33%, respectively). At 150 mg/kg/day, the 

mean overall body weight gain (Days 1-11) was decreased 7% compared to the controls. 

All animals in the 80 mg/kg/day dose group survived until scheduled termination and no clinical 

signs of toxicity were observed. On Day 2, body weight and body weight gain were decreased 

(↓6% and ↓56%, respectively). On Day 4, body weight and body weight gain were decreased 

(↓7% and ↓26%, respectively). At 80 mg/kg/day, mean final body weight and mean overall body 

weight gain were decreased slightly (↓6% and ↓10%, respectively). 

All animals in the 50 mg/kg/day dose group survived until scheduled termination and no clinical 

signs of toxicity were observed. On Day 2, body weight and body weight gain were decreased 

(↓3% and ↓42%, respectively). On Day 4, body weight and body weight gain were decreased 

(↓3% and ↓18%, respectively). At 50 mg/kg/day, mean overall body weight gain (Days 1-11) 

was decreased 6% compared to the controls. 

All animals in the 20 mg/kg/day dose group survived until scheduled termination and no clinical 

signs of toxicity were observed. Body weight gain was decreased 36% compared to the controls 

between Days 1 and 2. The body weight gain (Days 1-4) was decreased 13% compared to the 

controls. No other effects on body weight were observed during the study. 

No treatment-related effects were observed at 5 mg/kg/day. 

It did not appear that statistical analyses were conducted. 

Page 205 of 205