### 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 of2Page 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 ot her scientifically relevant information (OSRI))

# DATA EVALUATION RECORD

METRIBUZIN

Study Type: OCSPP 890.1150, Androgen Recep tor 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 alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended 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 re dundancy 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 potent ial 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).METRIBUZIN/ 1 0 11 01 Androgen R ecepto r B ind ing (Rat Prostate Cytoso l ) (20 1 1) I Page I of 14 OCSP P 890. 11 50 / OECD None


### Primary Reviewer: Patience Browne, Ph.D. 

### Office of Science and Coor din a tion P es ticid e Pro g ram s Secondary Reviewer: Greg Akerman, Ph.D. 

### Health Effects D i vision

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D a t e :

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### D a t e: 511 5112**~--=~~'--'-"'------** Tern p l ate version 08 / 20 1 1



### D ATA EVALUA TIO N R EC ORD

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



### PC CODE: 101101 

### DP B A R C OD E: D398559



### TXR#: 0056258 

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



### TEST MATER I AL (PU RI TY ) : Metribuzin (94.4% a.i.)



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



### CIT A T I 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: Ba ye r CropScience AG, Alfred Nobel Str. 50, 40789 Monheim

, Germany



### TEST O RD ER#: CON-101101-11



### EXECUT I VE SUMMA R Y: In an androgen receptor (AR) binding assay (MRID 48617905)

, ventra l 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
exper im e nt 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
compe titive 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

- 1 0 

### to 

### 10

- 3 

### M) ofmetribuzin (94.4%, Batch # K285067)

. No precipitation of test substance was observed at any concentration and DMSO was u se d as the solvent vehicle at a final assay concentration of :S3 .3 %. Three independent run s were performed, and each run inc l uded dexamethasone as a weak positive

contro l and Rl881 as the ligand reference s tandard

.

In the sa tur a tion binding experiment, the me a n 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 va l ues for saturation binding, but the Kcts from the ot h er two run s were within the recomm e nded range (0.685 to 1

. 57 nM)

, and the Scatchard plots for the thr ee Saturation assays were ge nerally linear. The specific binding reached a plateau

, and non- s pecific binding (NSB) was < 20% of total binding ( 4 .6 -12

. 6 %).Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 2 of 14 METRIBUZIN/ 101101 OCSPP 890.1150/ OECD None


### In the competitive binding experiment, specific [ 3 

### H]-R1881 binding was >75% at all concentrations of metr ibuzin tested in all three runs. An IC 50 

### and relative binding affinity (RBA) could not be calculated for metribuzin.

The estimated average log IC 50 

### s 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 K d 

### from run 2, all performance criteria were met.

Based on the results from the three runs, metri buzin is classified as a Non-Binder in the Androgen Receptor Binding Assay.

The assay satisfies the EDSP Tier 1 Test Orde r requirements for an A ndrogen Receptor Binding Assay (OCSPP 890.1150).

COMPLIANCE: Signed and dated GLP, Data Confidentiality, and Quality Assurance statements were provided.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) andAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 4 of 14 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 S tudy SA 11021, which included the saturation binding experiment (Attachment 4 of the subjec t study). A total of 50 male Sprague Dawley rats (Charles River Laboratorie s, 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 excise d immediately after termina tion, 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 pool ed, and the cytosol was aliquoted (5 mL) and stored at

− 74 ± 10o C until use. Protein concentratio n of the cytosol was determined to be 6.6 mg/mL using a commercia lly 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 [ 3 

### H]-R1881 was performed in a previous study (SA 11021) for the cytosol used in the subjec t 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 satu ration 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 specif ic activity of the stock solution [ 3 

### H]-R1881 was adjusted for decay over time, and serial dilutions in TEDG buffer were prepared to achieve the finalAndrogen 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 pr epared in a similar manner to achieve concentrations that we re 100-fold greater than each respective radiolabeled concentration, resulting in fi nal 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 [ 3 

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

### H]-R1881 was measured. For each batch of cytosol, the op timal protein concentration was determined by calculating specific binding to differing am ounts of protein per tube, using 0.25 nM radiolabeled R1881. The optimal protein co ncentration 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 tr iamcinolone 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 tr iamcinolone acetonide and 7.5-45 μL [ 3 H]-R1881. R1881 was added in a 100-fold mola r 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 cytoso l (300 μL), and incuba ted 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 centrifuge d, 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 scintillat ion counting of the ethanol extr act. 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.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 6 of 14 METRIBUZIN/ 101101 OCSPP 890.1150/ OECD None

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 singl e concentration of [ 3 

### H]-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 preci pitation 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, wh ich was reported to bind no more than 10- 15% of the [ 3 

### H]-R1881 at 1 nM.

Dilutions of the test substa nce (metribuzin), reference standard (R1881), weak positive control (dexamethasone), and solvent contro l (DMSO) were prepared to achieve the concentrations shown in Table 4. The assay consisted of three inde pendent 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.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 7 of 14 METRIBUZIN/ 101101 OCSPP 890.1150/ OECD None

TABLE 4. Competitor Final Molar (M) Con centrations in Competitive Binding Assay a, b
Solvent Control Reference standard We ak positive control Test Chemical None (Hot Mix) DMSO R1881 Dexamethasone Metribuzin 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 triplicat e 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 substan ce. 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, separa tion of bound from free R1881 (washing with buffer and extraction with ethanol), fo llowed by scintillation counting of bound [ 3 

### H]-R1881 were similar to those used in the saturation binding experiment

4. Data Analysis: The maximal binding capacity (B max 

### ), dissociation constant (K d 

### ), and inhibition concentration (IC 50 

### ) were calculated using nonlin ear 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 non linear 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 le ast 25% but less than 50%), or the curve falls outside the range fo r the weak positive control (

− 0.6 to

− 1.4).

Non-Binder: The average lowest portion of curves acr oss runs is greate r than 75% activity ( i.e. less than 25% displacement of radioliga nd), 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.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 8 of 14 METRIBUZIN/ 101101 OCSPP 890.1150/ OECD None


### b. Descriptors for receptor binding

B max 

### : maximal binding capacity K d 

### : dissociation constants IC 50 

### : Concentration of the test substance at which 50% of radioligand is displaced from the AR by the competitor 

### Relative Binding Affinity (RBA): ( IC 50 

### of R1881 ÷ IC 50 

### of test substance) × 100

II. RESULTS

A. SATURATION BINDING EXPERIMENT: Saturation binding experiment parameters are presented in Table 5. The dissociation constant (K d 

### ) for [ 3 

### H]-R1881 was 1.01 nM (±0.365), and the estimated B max 

### was 14 fmol/100 μ g protein (±1.5) for the single batch of prostate cytosol that was pr epared. The goodness of fit (r 2 

### ) was not reported and the K d 

### obtained from Saturation Assay #2 was low (0.59 nM), but the K d 

### s from the other two runs were within the recommended range (0.685 to 1.5 7 nM) and the Scatchard plot for the three saturation assays was linear de monstrating the androgen recept or was functioning with the appropriate affinity for th e reference ligand R1881.

TABLE 5. Saturation Binding Experiment of R-1881 wi th Androgen Receptor from Rat Prostate Cytosol a
Parameter Run 1 b Run 2 b Run 3 b Mean ± SE c

r 2 NR NR NR NA B max (nM) 0.34 0.31 0.38 0.34 ± 0.02 B max (fmol/100 μ g protein) 14 13 16 14 ± 0.9
K d (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 c oncurrent 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 [ 3 

### H]-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 r 2 

### values were not reported for the plots, the data were generally linear.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 9 of 14 METRIBUZIN/ 101101 OCSPP 890.1150/ OECD None


### FIGURE 1. Binding of [ 3 

### H]-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 #3Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 10 of 14 METRIBUZIN/ 101101 OCSPP 890.1150/ OECD None



### FIGURE 2. Scatchard Pl ots of the Binding of [ 3 

### H]-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 pr esented in Table 6. Specific [ 3 

### H]-R1881 binding with metribuzin was >75% at all concentrations te sted in all three runs. The estimated log IC 50 

### for metribuzin could not be determined as it did not result in 50% inhibition (displacement) at any concentration.

The estimated average log IC 50 

### s 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 indica ted 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.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 11 of 14 METRIBUZIN/ 101101 OCSPP 890.1150/ OECD None

TABLE 6. Competitive Binding Assay of Metrib uzin 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 IC 50 (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 IC 50 (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 A ndrogen Receptor in the Presence of Test Compounds.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 12 of 14 METRIBUZIN/ 101101 OCSPP 890.1150/ OECD NoneAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 13 of 14 METRIBUZIN/ 101101 OCSPP 890.1150/ OECD None


### FIGURE 4. Mean Percentage R1881 Bound to th e 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 evaluate d 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 K d (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 lim it in Run 2, but w ithin 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.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 [ 3 

### H]-R1881 in a manner consiste nt 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 consistenc y 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 take n 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 experi ment demonstrated that the androgen receptor was present in a reasona ble concentration and functioning with appropriate affinity for the R1881 ligand. The mean K d 

### for [ 3 

### H]-R1881 was 1.01 nM (±0.365), and the estimated B max 

### was 14 fmol/100 μ g protein (±1.5) for the single batch of prostate cytosol that was prepared. The K d 

### obtained from Saturation Assay #2 was low (0.59 nM), but the K d 

### s from the other two runs were w ithin the recommended range (0.685 to 1.57 nM). Although the r 2 

### values were not reported, th e Scatchard plots for the three saturation runs generally were linear. The specific binding re ached 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 concentratio ns tested in all three runs. An IC 50 

### and RBA could not be calculat ed for metribuzin.

The estimated average log IC 50 

### s for R1881 and the weak positiv e 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 excep tion that the ligand depletion and NSB were not reported, but the IC 50 

### s 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 provi ded for the saturation and competitive binding experiments.

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

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

# 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 alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended 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 re dundancy 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 potent ial 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).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

- 1 0 

### 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, lo g ICs o and %CV. For 4-0H ASDN, the estimated log ICs o 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

- 1 0 

### M) and 0

. 217±0

. 029 nmol / mg-protein/min at the highest testedAromatase (Human Recombinant) A ssay (2011) / Page 2 of 12 METRIBUZIN / 101101 OCSPP 890.1200 / OECD None


### concentration (10

− 3 

### M). As a percent of control, ar omatase 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, indica ting 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 requireme nts for an Aromatase assay (OCSPP 890.1200).

COMPLIANCE: Signed and dated Data Confiden tiality, GLP Compliance, and Quality Assurance statements were provided.Aromatase (Human Recombinant) A ssay (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 cont ainers 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

NH 2

S CH 3 CH

3 CH

3 CH 3



### 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; ([ 3 

### H]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/minAromatase (Human Recombinant) A ssay (2011) / Page 4 of 12 METRIBUZIN / 101101 OCSPP 890.1200 / OECD None



### B. METHODS

1. Assay Components and Preparations: A mixture of non-labe led 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 tri tium added to each incubation tube was approximately 0.07 μ Ci in a volume of 100 μ L.

Metribuzin stock solutions were prepared such that the total vo lume of each test chemical formulation used per assay was 1% of the tota l assay volume. A rationale for the selection of DMSO was not provided; however, metribuz in 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 prepar ed in the same solvent as the stock solution on the day of use. Dilutions were prepared such that the target concentrations of the positive control substance (10 –10 

### M to 10 –5 

### M; Table 4) were achieved by the addition of 20 μ L of the dilution for a final assay volume of 2 mL.

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

− 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 vi als 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 [ 3 H]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. Th e aromatase activity of the microsomes was also provided by the vendor as 0.113 nmo l/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 slight ly 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 pr otein/min, and the average aromatase activity was 0.1131 or 0.1135 nmol/mg protein/min.

3. Aromatase Assay: Each assay run contained four t ubes for the full enzyme activity and background activity controls, respectively, and full concentration curves in the positiveAromatase (Human Recombinant) A ssay (2011) / Page 5 of 12 METRIBUZIN / 101101 OCSPP 890.1200 / OECD None


### control (duplicate) and metr ibuzin (triplicate). The aromatase assay was conducted according to the procedures describe d in OCSPP 890.1200 (Section h, pp. 9-10).

The amount of 3 

### H 2 

### O in the aqueous fraction was quantified for each assay tube by LSC, and aromatase 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 th e responsiveness of the microsomes. This evaluation also served as a pr oficiency evaluation of the tech nician involved in the conduct of the definitive assays. It wa s 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 ne cessary 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 inhibito r 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 ge nerally met, and served as guidance in identifying runs that provided pa rameters in the preferred ranges.

(2) Demonstration of Proficiency of Ne w 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 IC 50 

### 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 IC 50 (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.Aromatase (Human Recombinant) A ssay (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 Ac tivity with Test Chemical(s): The response of aromatase activity to the presence of eight concentrations of metri buzin per run, in triplicate, was tested during three independent runs. Sol ubility was visually assessed (presence of cloudiness or a precipitate), and no precipitati on was observed. The full enzymatic activity defining the top of the con centration-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, a nd 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 components a plus solvent vehicle N/A Bkgd Activity Control 4 Same as above without NADPH N/A 4-OH ASDN Conc 1 2 All test components plus 4-OH ASDN 1×10

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

− 6
4-OH ASDN Conc 3 2 All test components plus 4-OH ASDN 1×10

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

− 7
4-OH ASDN Conc 5 2 All test components plus 4-OH ASDN 1×10

− 7.5
4-OH ASDN Conc 6 2 All test components plus 4-OH ASDN 1×10

− 8
4-OH ASDN Conc 7 2 All test components plus 4-OH ASDN 1×10

− 9
4-OH ASDN Conc 8 2 All test components plus 4-OH ASDN 1×10

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

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

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

− 5 Metribuzin Conc 4 b 3 All test components plus metribuzin 1×10

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

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

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

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

− 10 a The complete assay contained buffer, propylene glycol, microsomal protein, [ 3 H]ASDN, and NADPH. b Proficiency chemicals or tests chemical(s).



### C. DATA ANALYSIS

1. Raw Data: Raw data were converted to aromatas e 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 fo r each run were included in the report (Table 5).Aromatase (Human Recombinant) A ssay (2011) / Page 7 of 12 METRIBUZIN / 101101 OCSPP 890.1200 / OECD None

TABLE 5. Raw and Calculated Data
Raw/Calculated Data Included (X) DPM/mL for each portion of extract ed 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 graphica l displays were conducted using Prism 5, v 5.04 (GraphPad Software Inc., La Joll a, CA). Basic statistical analyses were performed on the data, which included means of replicates, standard de viation of the mean (SD), standard error of the mean (SEM), and coefficient of variati on (CV). Analysis of variance (ANOVA) was not performed.

The response curves were fitted by a non-we ighted least squares nonlinear regression analysis with Prism 5 software. For each run, the individual percent of control values were plotted versus logarithm of the correspondi ng concentration. The fitted concentration response curve was superimposed on the plot, wi th individual plots prepared for each run. The average percent of control values vers us logarithm of the concentration for the individual runs for each chemical (with differe nt symbols for each run) were included on the same graph with their respect ive fitted response curves. In addition, the av erage percent of control values for each run versus the logar ithm of the concentration were plotted on a separate graph along with the average concen tration response curve across runs were superimposed on the same plot.

Assay drift was assessed by comparing the % fu ll 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 categor ies presented in Table 6.

TABLE 6. Interpret ation of Results Criteria Interpretation Data fit 4-parameter nonlinear regression model Average curve across runs crossed 50% a Inhibitor Average lowest portion of curves across runs is between 50% and 75% activity b Equivocal

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

Data do not fit model --- a Ordinarily, an inhibition curve will fall from 90% to 10% over 2 log units with a slope near

− 1. Unusually steep curves may indicate protein denaturing or solubility issues. If the slope of the curve is steeper than

− 2.0, the result is classified as equivocal. b If the test compound was not soluble above 10

− 6 M and the inhibition curve does not cr oss 50%, the chemical is typically determined to be untestabl e in the aromatase assay.Aromatase (Human Recombinant) A ssay (2011) / Page 8 of 12 METRIBUZIN / 101101 OCSPP 890.1200 / OECD None



### 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. Ac tivity 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 e nd 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 assa y drift was observed in the firs t 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%. Overa ll, 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/m g-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 a ll 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 re sults were generally within the recommended ranges for the %CV, top of the curve, bottom curve, Hill slope, and log IC 50



### .

TABLE 7. Effect of Metribuzin on Aromatase Activi ty (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 VarianceAromatase (Human Recombinant) Assay (2011) / Page 9 of 12 METRIBUZIN / 101101 OCSPP 890.1200 / OECD None


### FIGURE 1. Inhibition Response Curv es 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 w ith 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.Aromatase (Human Recombinant) A ssay (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 Resp onse Curve for Metribuzin.

It was stated that analyses of the data fo r metribuzin indicated they did not fit the 4-parameter regression model used for data interpretation. Th e mean aromatase activity of 10 –3 

### M metribuzin was determined to be 63% of control activity and the best fit mean inhibition response curve (Fi gure 3) was a straight line (essentially no inhibition was observed at other doses). The effect of th e positive control on i nhibition of aromataseAromatase (Human Recombinant) A ssay (2011) / Page 11 of 12 METRIBUZIN / 101101 OCSPP 890.1200 / OECD None


### activity is presented in Table 8. For 4-OH ASDN, the estimated log IC 50 

### averaged

− 7.24 M, and the slope was

− 0.80. Confidence in these values is ve ry high due to the small variations (<7% CV).

TABLE 8. Effect of Metribuzin on Aromatase Activi ty (as Percent of Control) From Independent Runs a
Chemical Run 1 Run 2 Run 3 Mean b SD b %CV b Log IC 50 (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 bac kground activity controls were less than 15% of the corr esponding 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 IC 50 

### value was between

− 7.17 and

− 7.28 M in each of the three independent evaluations, with an overall log IC 50 

### 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 c onsidered to be a non-inhib itor 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 thre e test runs, with a mean (± SD) of 0.347±0.052 nmol/mg-protein/min. Activity in th e 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 IC 50 

### for 4-OH ASDN averaged

− 7.23 M, and the Hill slope was

− 0.79. Confidence in these values is very high due to th e 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 activ ity 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 metribuzinAromatase (Human Recombinant) A ssay (2011) / Page 12 of 12 METRIBUZIN / 101101 OCSPP 890.1200 / OECD None


### data indicated that they did not fit the 4-para meter regression model, and the best fit mean inhibition response curve was e ssentially a straight line. However, because the average lowest portion of the res ponse curve across runs was between 50 and 75% activity, metribuzin is classified as equivocal for inhi bition 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 ch emicals testing were not provided.

 Information the stability of the test substa nce in the stock solu tion was not reported.

 For the one run of the positive control c onducted prior to performing the definitive assay, the log IC 50 

### value (

− 6.70 M) was outside the guideline recommended range of

− 7.3 to

− 7.0 M.

# 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 alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended 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 re dundancy 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 potent ial 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).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-methylsulfany l-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 E R 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

- 1 0 

### to 

### 10

- 3 

### M) of metribuzin (94.4% ; Batch # K285067). DMSO

w as used as the vehicle at a final concentration of :S 2%. 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 Bma x 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 allEstrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 2 of 18 METRIBUZIN/ 101101 OCSPP 890.1250/ OECD None


### three runs. Therefore, a mean log IC 50 

### and relative binding affi nity (RBA) could not be calculated for metribuzin.

The estimated mean log IC 50 

### for 17 β -estradiol and 19-norethindrone was

− 8.8 M and

− 5.0 M, respectively, and the mean RBA for 19-noret hindrone was 0.017% compared to the natural ligand. Several performance criteria were not met for 17 β -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 c oncentration range that fully defi ned the top of the curve and the maximum percent binding was within approximate ly 25 percentage points of the lowest concentration of the estradiol standa rd. Each of the three runs was classified as not interactive. The lowest average percent radiolabeled estr adiol 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 requireme nts for an Estrogen Receptor Binding Assay (OCSPP 890.1250).

COMPLIANCE: Signed and dated GLP, Data Confidentiality, and Quality Assurance statements were provided.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 3 of 18 METRIBUZIN/ 101101 OCSPP 890.1250/ 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: 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: 17 β -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]-17 β -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 #: 66-22-4Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 4 of 18 METRIBUZIN/ 101101 OCSPP 890.1250/ OECD None


### 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 recomme nded 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 st udy). 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 harves t. 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 resulti ng pellet. Protein concentration of the cytosol wa s determined to be 3.35 mg/mL using a DC protein assay kit (BioRad). The cytosol was divided into al iquots (volume not repor ted) for storage at

− 74 ± 10o C until use.

2. Saturation (radioligand) Binding Experiment: A saturation binding experiment measuring total and non-specific binding of [ 3 

### H]-17 β -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 reason able 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 subj ect 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.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 5 of 18 METRIBUZIN/ 101101 OCSPP 890.1250/ OECD None



### On the day of the assay, the specif ic activity of the stock solution [ 3 

### H]-17 β -estradiol was adjusted for decay over time, and serial dilu tions 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 17 β -estradiol were prepared in a similar manner to achieve concentrations that were 100-fold greater than each respective radi olabeled 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 testi ng serial amounts of protein per tube, using 0.03 nM radiolabeled estradiol, until a concen tration was reached that bound 25-35% of the total radioactivity added. Each assay consisted of three non-concurre nt 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 [ 3 H]-17 β -estradiol (8 se rial dilutions) e

--- 50 μL --- Non-labeled 17 β -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]-17 β -estradiol bound to ER c Non-specific binding = [ 3 H]-17 β -estradiol and 100-fold greater non-labeled bound to ER d Total [ 3 H]-17 β -estradiol alone for dpm determination at each concentration e Final concentrations of [ 3 H]-17 β -estradiol = 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1, and 3 nM. f Final concentrations of non-labeled 17 β -estradiol = 3, 6, 8, 10, 30, 60, 100, and 300 nM.



### Tubes were incubated with gen tle 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]-17 β -estradiol. An aliquot of supernatant was radioassayed by scintillation counting. The temp erature was maintained at 4o C throughout the assay prior to extraction with ethanol.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 6 of 18 METRIBUZIN/ 101101 OCSPP 890.1250/ OECD None


### 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 specif ic activity of the stock solution [ 3 

### H]-17 β -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 pr otein 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 dilu tions of metribuzin, the weak positive control (19-norethindrone), the negative control (oct yltriethoxysilane), and the natural ligand (nonlabeled 17 β -estradiol) were prepared to achieve th e concentrations shown in Table 4. The test substance in DMSO was visually examined for precipitation. E ach assay consisted of three runs, and each run contained three repli cates at each concentr ation, six replicates measuring total activity (50 μ L master mix) plus six meas uring total binding (solvent control) resulting in 105 samples/as say for a total of 315 samples.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 7 of 18 METRIBUZIN/ 101101 OCSPP 890.1250/ OECD None

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 17 β -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, pos itive control, negative control, solvent control, or nonlabeled 17 β -estradiol; 390 μL of TEDG + PMSF buffer with [ 3 H]-17 β -estradiol; and 100 μL of uterine cytosol (with ER), for a total of 500 μL. c Each concentration of each chemical was run in tripli cate, 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, separati on of bound from free estradiol (washing with buffer and extraction with ethanol), fo llowed by scintillation counting of bound [ 3 

### H]-17 β

estradiol 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 nonlin ear regression used to fit a curve (for 17 β

estradiol, 19-norethindrone, and metribuzin) to the Hill equation formula which incorporated IC 50 

### as a parameter to be estimated. For parameters reported from the saturation binding experiment (K d 

### and B max 

### ) and competitive binding experiment (log IC 50 

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

1. Definitions

a. Classification of test material: Classification of the test mate rial is based on the average of three runs. Each run was first in dividually 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 estrad iol 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 da ta 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 butEstrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 8 of 18 METRIBUZIN/ 101101 OCSPP 890.1250/ OECD None



### ≤ 50% of the radiolabeled estradiol has been displaced from the ER or a binding curve cannot be f it and the lowest averag e 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 acro ss runs and the chemical classified using the following ranges:

Test Material Classifi cation 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:

B max 

### : maximum specific binding number (fmol ER/ 100 μg cytosol protein) measures the concentration of active receptor sites K d 

### : dissociation constant (nM), measures the a ffinity of the receptor for its natural ligand IC 50 

### : concentration of the test substance at wh ich 50% of the radioligand is displaced from the receptor Relative Binding Affinity (RBA %): ( IC 50 

### of 17 β -estradiol ÷ IC 50 

### of test substance) × 100

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 [ 3 

### H]- 17 β -estradiol. The results from the three satu ration binding assays are summarized in Table 5 and shown graphically in Figures 1-9. The mean K d 

### for [ 3 

### H]-17 β -estradiol was 0.070 nM, and the mean estimated B max 

### was 34 fmol/100 ug protein for the prepared rat uterine cytosol. The K d 

### for each run was within the expected ra nge of 0.03 to 1.5 nM. The data produced consistent and linear Scatchard pl ots, although the goodness of fit (r 2 

### ) was not reported.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 9 of 18 METRIBUZIN/ 101101 OCSPP 890.1250/ OECD None

TABLE 5. Saturation Bi nding Experiment of 17 β -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 B max (nM) 0.027 0.037 0.039 0.034 ± 0.004 B max (fmol/100 μ g protein) 27 37 39 34.3 ± 3.71 K d (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.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 10 of 18 METRIBUZIN/ 101101 OCSPP 890.1250/ OECD None


### FIGURE 3. Scatchard plot for Specific binding Run 1.

FIGURE 4. Total and Non-specific binding Run 2.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 11 of 18 METRIBUZIN/ 101101 OCSPP 890.1250/ OECD None


### FIGURE 5. Specific binding Run 2.

FIGURE 6. Scatchard plot for Specific binding Run 2.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 12 of 18 METRIBUZIN/ 101101 OCSPP 890.1250/ OECD None


### FIGURE 7. Total and Non-specific binding Run 3.

FIGURE 8. Specific binding Run 3.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 13 of 18 METRIBUZIN/ 101101 OCSPP 890.1250/ OECD None


### 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 IC 50 

### and RBA values could not be calcula ted 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 te chnical problem with the rotator.

The estimated mean log IC 50 

### for 17 β -estradiol and 19-norethindrone was

− 8.8 M and

− 5.0 M, respectively. Compared to the natura l ligand, the mean RBA was 0.017% for 19-norethindrone. Confidence in these numbers is high due to the small variation.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 14 of 18 METRIBUZIN/ 101101 OCSPP 890.1250/ OECD None

TABLE 6. Competitive Binding Assay of Metribuzin with Estrogen Receptor fr om Rat Uterine Cytosol a Parameter Run 1 b Run 2 b Run 3 b Mean ± SE c

r 2 (unweighted) 17 β -estradiol NR NR NR NA 19-norethindrone NR NR NR NA Metribuzin NR NR NR NA Log IC 50 (M) 17 β -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 IC 50 (M) 17 β -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 concu rrent replicates within each run. c Calculated by reviewers; for means ex pressed 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 Es trogen 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: Interac tive = 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 inte ractive = mean <0.5Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 15 of 18 METRIBUZIN/ 101101 OCSPP 890.1250/ OECD None


### FIGURE 10. Percentage [ 3 

### H]-E2 Bound to the Estrogen R eceptor in the Presence of Metribuzin, Unlabeled E2, 19-Norethindr one or Octyltriethoxysilane. Run 1

FIGURE 11. Percentage [ 3 

### H]-E2 Bound to the Estrogen R eceptor in the Presence of Metribuzin, Unlabeled E2, 19-Norethindr one or Octyltriethoxysilane. Run 2Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 16 of 18 METRIBUZIN/ 101101 OCSPP 890.1250/ OECD None


### FIGURE 12. Percentage [ 3 

### H]-E2 Bound to the Estrogen R eceptor in the Presence of Metribuzin, Unlabeled E2, 19-Norethindr one or Octyltriethoxysilane. Run 3

FIGURE 13. Mean Percentage [ 3 

### H]-E2 Bound to the Estrogen Receptor in the Presence of Metribuzin in Three Competitive Assays.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 17 of 18 METRIBUZIN/ 101101 OCSPP 890.1250/ OECD None



### C. PERFORMANCE CRITERIA: To ensure that the compe titive binding assay functioned properly, each run was evaluated using the following criteria (Table 8):

TABLE 8. Criterion a Tolerance Limit(s) Value Yes No 17 β -estradiol fitted curve parameters Log e 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 (Log 10 (M) -1 )

− 1.1 to

− 0.7

− 2.0 X Weak Positive control (19-norethindrone) fitted curve parameters c Log e residual SD NA NR Top (% binding) NA 105 to 112 X Bottom (% binding) NA

− 13.6 to

− 8.5 X Hill Slope (Log 10 (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]-17 β -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 (9 3%) but acceptable in the other two runs. c The EPA Guideline does not define a set of tolerance li mits for 19-norethindrone. A cceptance criteria were only defined for norethynodrel, which cannot be obtained commercially. The values reported were considered acceptable as they show 19-nor ethindrone 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 17 β -estradiol. The bottom % binding for 17 β -estradiol (2.1 to 2.5%) was also outside the recommended range in all three runs, and the t op % binding (93%) in one of the runs was outside the recommended range. However, responses fo r the weak positive control (19-norethindrone ) 

### and negative control (octyltriethoxysilane ) 

### were as expected.

Metribuzin was tested over a c oncentration range that fully de fined the top of the curve. The maximum percent binding 109-122% was within approximately 25 percentage points of the lowest concentration of the estradiol sta ndard (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 μ g/assay tube. The IC 50 

### value for 17 β -estradiol was determined to be 1.7×10

− 9 

### M in each run. The IC 50 

### 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 17 β -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 theEstrogen 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 expe riment, 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 [ 3 

### H]-17 β -estradiol. The mean K d 

### for [ 3 

### H]-17 β -estradiol was 0.070 nM, and the mean estimated B max 

### was 34 fmol/100 μg protein for the prepared rat uterine cytosol. The K d 

### for each run was within the exp ected range of 0.03 to 1.5 nM. The data produced consistent and linear Scat chard plots, although the goodness of fit (r 2 

### ) was not reported.

For the competitive binding experiment, the da ta 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. Precip itation was not observed at any concentration. The estimated mean log IC 50 

### and RBA was not calculated for metribuzin as the percent binding did not reach 50% for any run.

The estimated mean log IC 50 

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

− 4 to 1) in all three r uns; and (iii) the top % binding (93%) in one of the runs was outsi de the recommended range (94 to 111%). However, responses for the weak positive control (19-norethindrone ) 

### and negative control (octyltriethoxysilane ) 

### were as expected demonstra ting the assay was adequate.

Metribuzin was tested over a c oncentration range that fully de fined the top of the curve. The maximum percent binding 109-122% was within approximately 25 percentage points of the lowest concentration of the estradiol st andard (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 17 β -estradiol.

 The criterion of Log e 

### residual SD for the competitive binding assay was not reported.

# 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 alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended 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 re dundancy 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 potent ial 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).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: Eva luation 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. Unpublis h ed.



### 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 lucifera se 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 

### MEstrogen Receptor Transc riptional 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 RPC Max 

### ≥ PC 10 

### 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 c onsidered 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.Estrogen Receptor Transc riptional 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

NH 2

S CH 3 CH

3 CH

3 CH 3



### 2. Reference substances 17 β -estradiol (strong estrogen; positive control) Supplier: Sigma-Aldrich, St. Louis, MO Catalogue and Batch #: 010M0142 Purity: 100% CAS # : 50-28-2



### 17 α -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



### 17 α -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%Estrogen Receptor Transc riptional Activation (2011) / Page 4 of 10 METRIBUZIN/101101 OCSPP 890.1300/ OECD 455



### B. METHODS

1. Cell Culture: Stably-transfected hER α -HeLa-9903 cells were obtained from the Japanese Collection of Research Biores ources Cell Bank and were verifi ed by the supplier to be free of mycoplasma infection using PCR and DNA st aining. 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% CO 2 

### at 37o C. Upon reaching 75- 90% confluence, cells were subcultured 6 time s prior to exposure to the test material.

2. Transcriptional Activation Assays: For each test, cells were plated at a density of 1  10 4 

### cells/100 μL medium/well in a 96-well plate and allowed to attach for 3 hours. The growth media was replaced with media containi ng 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 determin ed 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 me tribuzin concentrations ranging from 10
− 6 

### to 10
− 4 

### M was conducted to determine the appr opriate 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 st ock by testing the following set of proficiency chemicals. The details of th e 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 --- 17 α -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 0.01 b , 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 0.1 b and 1 mM 1,3,5-Tris(4- hydroxyphenyl)benzene c 15797-52-1 10

− 12 to 10

− 5 Positive Cytotoxic at 100μM. PC max ~. 50% of PC. Binds hER α 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 = RPC Max ≥ 10% of the response of the positive control in at least 2 of 2 (or 2 of 3) runs Negative = RPC Max 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 ER α mediated transcriptional activation, but may not be negative for non-ER β mediated transcriptional activation. A positive result would indicate that the sy stem is detecting activity other than that due to pure ER α , and is therefore unacceptable.Estrogen Receptor Transc riptional Activation (2011) / Page 5 of 10 METRIBUZIN/101101 OCSPP 890.1300/ OECD 455



### c. Reference Chemicals: To ensure the stability of th e response from the cell line, six concentrations of each of the following reference chemicals were included in each plate in the current assay, along w ith the test chemical:

Reference Chemical CAS No. Concentration Range Class 17 β -estradiol 50-28-2 10

− 14 to 10

− 8 Strong estrogen 17 α -estradiol 57-91-0 10

− 12 to 10

− 6 Weak estrogen Corticosterone 50-22-6 10

− 10 to 10

− 4 Negative compound 17 α -methyltestosterone 58-18-4 10

− 11 to 10

− 5 Very weak agonist



### 3. Data analysis: To obtain the relative transcriptiona l activity to the 1 nM E2 positive control (PC), the luminescence signals from the concur rent plate were analy zed 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 expres s 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 EC 50 

### , PC 10 

### , PC 50 

### , RPC Max 

### , and PC Max 

### for metribuzin when applicable. The test material was defined as negative for inducing estrogen receptor transcriptional activation if the RPC Max 

### < PC 10 

### in at least 2 of 2 (o r 2 of 3) runs. Log EC 50 

### and Hill slope values are calculated only if a positive respons e is observed. Coefficien ts of variation (CV) were calculated for the luminescence data tr iplicates. Concentr ations showing >20% cytotoxicity or evidence of insol ubility were excluded from analyses.

4. Definitions

EC 50 

### = concentration of agonist that induces a response ha lfway between the baseline (bottom) and maximum (top) response PC 10 

### = 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

PC 50 

### = 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

RPC Max 

### = 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

PC Max 

### = concentration of a test chemical inducing the RPC max



### 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 pe rformed for metributzin at log serial dilutions starting at 10
− 4 

### M in culture medium. The results of the cyto toxicity test are pres ented 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.Estrogen Receptor Transc riptional Activation (2011) / Page 6 of 10 METRIBUZIN/101101 OCSPP 890.1300/ OECD 455

TABLE 1. Preliminary Test for Solubility, Cytoto xicity, and Concentration-Selection for metribuzin Concentration (M) % Viability b Comments c 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 profic iency chemicals was performed in duplicate on different days. The data are reported in Table 2. The responses demonstrated proficie ncy of the laboratory. The only minor deviation was in the first run, corticostero ne produced an RPC Max 

### of 16%, indicating a w eakly positive response. In the second run, the RPC Max 

### was

− 14%, indicating a negative response.

TABLE 2. Proficiency Chemicals a Compound Expected Response Lab Response Diethylstilbestrol Positive Positive 17 α -Ethynyl estradio l 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 Nega tive 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 PC 50, 

### log PC 10, 

### log EC 50, 

### Hill slope) for the four concurrently run reference materials are included in Table 3. Performance criteria were met for 17 β -estradiol and corticosterone. The log PC 10 

### for 17 α -estradiol was lower than the acceptable range in the first assay, as was the Hill Slope. For methyltestosterone, the log PC 50 

### was lower than the acceptable range for run 2, and the log PC 10 

### was lower than the acceptable range for both runs. These results indicate an increased sensitivity of the assay to weak agonists.Estrogen Receptor Transc riptional 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
17 β -estradiol a Log PC 50 − 11.4 to

− 10.1

− 10.5

− 10.7 X Log PC 10 <

− 11

− 11.7

− 11.4 X Log EC 50 − 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
17 α -estradiol a Log PC 50 − 9.6 to

− 8.1

− 9.1

− 8.9 X Log PC 10 − 10.7 to

− 9.3

− 11.9

− 10.1 X Log EC 50 − 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
Corticosterone a Test range 10

− 10 to 10

− 4 M 10

− 10 to 10

− 4 M 10

− 10 to 10

− 4 M X
17 α -methyltestosterone b Log PC 50 − 6.0 to

− 5.1

− 5.83

− 6.08 X Log PC 10 − 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 pos itive controls are included in Table 4. The mean TA, expressed as arbitrar y light units for the ve hicle 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 PC 50 

### (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 PC 10 

### (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 17 β -estradiol (E2) at 1 nM. c Fold-induction = (mean TA of PC)/(mean TA of VC)



### 2. Test Material: Relative (to the PC) transcriptional ac tivation at each concentration of the metribuzin during the two assay runs is presen ted in Table 5. The concentration-response curves depicting fold induction of relative tran scriptional activation is presented in Figure 1 and Figure 2 below. The mean RPC Max 

### was 0.67% for the first run and 14.07% for the second run, and the associated PC Max 

### was 10
− 4 

### M for the first run and 10

− 6 

### M for the second run. The RPC Max 

### ≥ PC 10 

### in Run 2, however, this happened on ly in one of the three samples for that run, and it was not at the highest conc entration. Therefore, metribuzin is still considered negative for estrogen receptor tran scriptional activation in this test system.Estrogen Receptor Transcriptional Activation (2011) / Page 8 of 10 METRIBUZIN/101101 OCSPP 890.1300/ OECD 455

TABLE 5. Relative Tran scriptional Activation (RTA) of Metribuzin a

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 EC 50 NA NA Hill Slope NA NA RPC max 0.67 14.07 PC max 10

− 4 10

− 6
PC 50 NA NA PC 10 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 Tr anscription Activation (RTA) of Metribuzin (Run 1)Estrogen Receptor Transcriptional Activation (2011) / Page 9 of 10 METRIBUZIN/101101 OCSPP 890.1300/ OECD 455



### FIGURE 2. Fold Induction of Relative Tr anscription Activation (RTA) of Metribuzin (Run 2)

3. Performance Criteria: The laboratory proficiency assays using the required reference compounds adequately demonstrated that th e assay was functioning properly. The only deviation was a weak positive response in one run in response to corticosterone. Performance criteria were met for 17 β -estradiol and corticosterone. The log PC 10 

### for 17 α -estradiol was lower than the acceptable range in the first a ssay, as was the Hill Slope. For 17 α -methyltestosterone, the mean RPC Max 

### was 79.0% for the first run and 70.9% for the second run. For methyltestosterone, the logPC 50 

### was lower than the acceptable range for run 2, and the logPC 10 

### was lower than the acceptable range for both runs. The results show an increased sensitivity to weak agonist s. 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 PC 10 

### 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 17 β -estradiol in both runs. Therefore metribuzin was negative in this human estrogen receptor transcriptional activation assay.

B. AGENCY COMMENTS: The laboratory proficiency te sts using the required reference compounds demonstrated that the assay was func tioning properly. The only deviation was a weak positive response in one r un in response to corticosterone . Performance criteria were met for 17 β -estradiol and corticosterone. For 17 α -estradiol, the Log PC 10 

### was lower than the acceptable range in the first assay, as was the Hill Slope. In addition, for 17 α

Estrogen Receptor Transc riptional Activation (2011) / Page 10 of 10 METRIBUZIN/101101 OCSPP 890.1300/ OECD 455


### methyltestosterone, the Log PC 50 

### was lower than the acceptable range in Run 2, and the Log PC 10 

### was lower than the acceptable range in both r uns. However, these results indicate that the assay had an increased sens itivity to weak agonists.

Metribuzin was tested up to the limit of solubility, 10

− 4 

### M. The mean RPC Max 

### was 0.67% for the first run and 14.07% for the s econd run, and the associated PC Max 

### was 10
− 4 

### M for the first run and 10

− 6 

### M for the second run. Although the RPC Max 

### ≥ 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.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-amin o-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 Digitally signed by AMY BLANKINSHIP DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=AMY BLANKINSHIP, dnQualifier=0000040917 Date: 2015.06.15 17:56:51 -04'00'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.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

Page 3 of 70 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 26.0 o 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.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

Page 4 of 70 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-throughData Evaluation Record onthe Fish Short-Term Reproduction Assay with MetribuzinEPA MRID Number 48672202Page 5 of 70Table 1: Summary of Reproductive and HPG Effects1,2in the Fish Short-Term Reproduction Assay (FSTRA) with Metribuzin.Treatment(mg a.i./L)[meanmeasured]FecundityFert.SuccessTubercle ScoreGSIGonadal Histo.Plasma VTGPlasma TPlasma E2MFMFMFMFMFMF0.199NoNoNoNoNoNoNoNoNoNoNANANANA1.03NoNoNoNoNoNoNoNoNoNoNANANANA5.40NoNoNoNoNoNoYesNoNoNoNANANANAAbbreviations:E217β-estradiol.FFemale.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.MMale.NANot applicable.TTestosterone.VTGVitellogenin.1A “yes” indicates a significant difference based on comparison to the negative (clean water) control, unless otherwise specified. A solvent was not used in the conduct ofthis study.2The criteria for significance are described in the Reviewer’s Analysis and Statistical Verification sections of the DER. Conclusions regarding histopathology may beheavily weighted by the expert opinion of a board-certified pathologist.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

Page 6 of 70 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.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

Page 7 of 70 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetribuzinEPA MRID Number 48672202Page 8 of 70B. Test organism:Table 2: General Information About the Test Species and Acclimation.ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies common name:Fathead Minnow EPA recommends fathead minnow (Pimephales

promelas).Species scientific name: Pimephales promelasSpecies strain (if stated):Not reportedWere fish obtained from a singlelaboratory stock?Yes EPA recommends that fish be from a single
laboratory stock.Were acclimation conditions sameas 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetribuzinEPA MRID Number 48672202Page 9 of 70ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on health:No mortalities wereobservedNo mortalities during the 14 days prior topre-exposure period were observed.Fish did not receive any treatment fordisease during acclimation period.Behavioral abnormalities or clinical signswere 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

mortalit y 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 reportedRepresentative samples of the food sourcewere periodically analyzed for pesticides,PCBs, and toxic metals byEurofinsGeneScan GmbH.Frequency of feeding:2-3 times/dayDetails on feeding:NoneData Evaluation Record onthe Fish Short-Term Reproduction Assay with MetribuzinEPA MRID Number 48672202Page 10 of 70Table 3: Fish Selection and Pre-Exposure Performance.ParameterValue(s)Details or RemarksGuideline RecommendationsAge at test initiation:6.5 months EPA recommends reproductively mature (sexually dimorphic) fish, 4.5 - 6 months old.Mean weight of males at testinitiation (if determined):4.69 gBased on 15 males used to stock aquariafor pre-exposure phase. EPA recommends that a subsample of fish be weighed before the test to estimate the mean weight for each sex. It is recommended that the individual weight of each fish selected for the test be within ±20% of the estimated mean for

each sex.Range of individual weights(males) at test initiation (ifdetermined):3.84 to 5.59Individual weights within ± 20% of theestimated mean.Mean weight of females at testinitiation (if determined):2.74 gBased on 15 females used to stock aquariafor pre-exposure phase.Range of individual weights(females) at test initiation (ifdetermined):2.19 to 3.24Individual weights within ±20% of theestimated mean.Mean length of males at testinitiation (if determined):Not reportedMean length of females at testinitiation (if determined):Not reportedDuration of pre-exposure phase:14 days EPA recommends a minimum of 14 days.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetribuzinEPA MRID Number 48672202Page 11 of 70ParameterValue(s)Details or RemarksGuideline RecommendationsWere pre-exposure conditionsidentical to the definitive test?Yes EPA recommends that pre-exposure conditions, including temperature, photoperiod, feeding, etc., be identical to definitive test conditions.Number of pre-exposure tanks: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:2Number of females per tank:4Pre-exposure fecundity:>10.2-27.4eggs/female/reproductive day/replicateThree replicates did not meet the minimumpre-exposure fecundity of at least 15eggs/female/reproductive day/replicateduring the 7days 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetribuzinEPA MRID Number 48672202Page 12 of 70C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:Flow-through EPA recommends the use of a flow-through system. As noted in the Corrections and Clarifications document 1 , the use of a static renewal system is not recommended for this

assay.Type of flow-through dilutionsystem:Flow-through dilutersNot further described Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 2Flow-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.1U.S. Environmental Protection Agency (EPA). (2011). Correctionsand Clarifications on Technical Aspects of the Test Guidelines for the Endocrine Disruptor ScreeningProgram Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention (OCSPP), Washington, D.C.(http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf).2Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetribuzinEPA MRID Number 48672202Page 13 of 70ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on toxicant mixing forflow-through systems:Flow-splitting chambers were used betweendiluter cells and the 4 replicate test vessels.Treatment recoveries prior to test initiation (-1day)were 103-109%, indicating the diluterhad reached equilibrium.Flow splitting accuracywas ±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 exposureDay 7.Aeration began after D7 sampling revealed DOconcentrations 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetribuzinEPA MRID Number 48672202Page 14 of 70ParameterValue(s)Details or RemarksGuideline RecommendationsWas dilution water analyzed forpesticides, heavy metals, andother contaminants?YesTest vessel type/materials:Glass aquaria with siliconeadhesiveEPA 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; solutiondepth 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 thicknessstainless steel pipes sittingon a round (1250 mmdiameter) 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 110x 75 x 85 mm piecesEach substrate placed in lipped glass tray withpetri dishes on the bottom (1350 mmdiameter).Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetribuzinEPA MRID Number 48672202Page 15 of 70ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details on exposuresystem:None.Table 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurement IntervalGuideline RecommendationsTemperature (°C)25.1 25.7 25.4 Weekly EPA recommends temperature 25±1 oC; interreplicate and inter -treatment differentials should not exceed 1 o C.pH6.77.37.0Weekly 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 CaCO 3.Hardness (mg/L as CaCO3)40.852.945.6WeeklyTotal organic carbon (mg/L)Not reportedNot reported<2 Once EPA recommends that total organic carbon in dilution water be <2 mg/L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetribuzinEPA MRID Number 48672202Page 16 of 70ParameterMinimumMaximumMeanMeasurement IntervalGuideline RecommendationsUnionized ammonia (Pg/L)Not reportedNot reportedNot reportedOnce EPA recommends that unionized ammonia in the dilution water be <1 P g/L.Residual chlorine (Pg/L)Not reportedNot reported<10 Once EPA recommends that residual chlorine in dilution water be <10 P g/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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetribuzinEPA MRID Number 48672202Page 17 of 70D. Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details or RemarksGuideline RecommendationsWas a range-finder conducted?Yes EPA recommends conducting a range-finder if

96 -hour LC

50 data for the fathead minnow ar e

unavailable.If yes, what was the method fordetermining the highest testconcentration in the range-finder?Between the NOEC (13mg/L) on a fatheadminnow ELS study and1/3 the derived 96-hourLC50 of the range findertest (>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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetribuzinEPA MRID Number 48672202Page 18 of 70ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional 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 LC

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

test.Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:21 days EPA recommends that the duration of the definitive
test be 21 days.Method for selecting the highesttest concentration in the definitivetest:The acute toxicity 96-hrLC50valuewith fatheadminnows was reportedA spacing factor of 5 was selected for thetest concentrations. EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstratesData Evaluation Record onthe Fish Short-Term Reproduction Assay with MetribuzinEPA MRID Number 48672202Page 19 of 70ParameterValue(s)Details or RemarksGuideline Recommendationsdetermined to be >100mg a.i./L. adequate evidence of toxicity (e.g., 1/3 the 96-

hour LC 50 ), whichever concentration is lowest.Reference study citation (ifapplicable):Not reportedSeparation of test concentrations:0 (control), 0.2, 1.0, and5.0mg a.i./L EPA suggests that a concentration separation of between 0.33 (or three-fold) and 0.1 (or tenfold) is scientifically acceptable 1 .Number of test concentrations:3 EPA recommends a minimum of 3 concentrations and a control, plus solvent control

if appropriate.Are nominal concentrationsadjusted for purity?YesIndicate the type of valuespresented for measuredconcentrations:Mean measuredLimit of quantification (LOQ):0.02 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 reportedData Evaluation Record onthe Fish Short-Term Reproduction Assay with MetribuzinEPA MRID Number 48672202Page 20 of 70ParameterValue(s)Details or RemarksGuideline RecommendationsFrequency 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 completeblock design used?Yes EPA recommends that all fish be randomly assigned to tanks during pre - exposure. Tanks

are the n 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 solventcontrol (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 itemtreatment level:4 EPA recommends 4 replicates.Number of male fish per replicateat test initiation:2 EPA recommends 2 males per replicate.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetribuzinEPA MRID Number 48672202Page 21 of 70ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of female fish per replicateat test initiation:4 EPA recommends 4 females per replicate.Was a solvent used?NoSolvent type (if applicable):Not applicableMaximum solvent concentration (ifapplicable):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?NoPositive control (if applicable):Not applicablePositive control concentration(s) (ifapplicable):Not applicable3Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review. AquaticToxicology, 76, pp.69–92.4OECD (2000). Guidance Document on Aquatic Toxicity Testing ofDifficult Substances and Mixtures. Environmental Health and Safety Publications. Series on Testingand Assessment. No. 23. Paris, France.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetribuzinEPA MRID Number 48672202Page 22 of 70ParameterValue(s)Details or RemarksGuideline RecommendationsPhotoperiod:16 hrs light:8 hrs dark EPA recommends photoperiod 16:8 (light:dark).Light intensity at water’s surface:720-1080 luxCool white fluorescents EPA recommends light intensity 540 – 1080 lux (at water’s surface).Additional details:There was no evidence of precipitates duringexposure.Table 8: Summary of Treatment Concentrations in the Fish Short-Term Reproduction Assay with Metribuzin.Treatment IDNominalConcentration(mg a.i./L)Mean MeasuredConcentration(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsNegative Control0<LOQNA EPA recommends that test item concentrations be maintained at a coefficient of variation (CV)≤ 20%.Treatment 10.20.1991.9Treatment 21.01.033.4Treatment 35.05.400.35Abbreviations:CVCoefficient of variation.NANot applicable.LOQ=0.02 mg a.i./L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetribuzinEPA MRID Number 48672202Page 23 of 70E. ObservationsBiological Endpoints:Survival, fecundity, fertilization success, and clinical signs were observed daily. At test termination (Day 21), secondary sexcharacterization (body color, pattern, body shape), body weight, length, tubercle score, gonadal staging and histopathology,and plasma vitellogenin were evaluated.Were raw (individual) data provided?Yes EPA recommends that observations of survival, fecundity, fertilization success, secondary sex characteristics, and other clinic al signs occur at least daily. At test termination (Day 21), additional observations include body weight and length, nuptial tubercle score, gonadal staging and histopathology, plasma vitellogenin, and plasma sex steroids (testosterone and 17β -estradiol, if measured). Gonado-somatic index (GSI) is calculated using a ratio of gonad weight to body weight (gonad weight to the nearest 0.1 mg / body weight in mg x 100) at test termination. Clinical signs of overt toxicity may include (but are not limited to) hemorrhage, cessation of feeding, and other abnormal beha vior.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetribuzinEPA MRID Number 48672202Page 24 of 70II.RESULTS AND DISCUSSIONA.ResultsMale 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 andmean-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]MalesFemalesN1# Surviving% SurvivalN1# Surviving% SurvivalNegative Control (<LOQ)8810016161000.19988100161593.81.0388100161593.85.4088100161381.3LOQ=0.02 mg a.i./L.1Total number of fish at test initiation.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetribuzinEPA MRID Number 48672202Page 25 of 70Mean 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 2.05g in thenegative 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 were57.6, 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 andmeanmeasured 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 WeightLengthMalesFemalesMalesFemalesnMean(g)±SDnMean(g)±SDnMean(mm)±SDNMean(mm)±SDNegative Control(<LOQ)44.26 0.414 1.99 0.25457.61.98444.81.270.19944.380.6342.030.17457.33.16444.70.571.0344.250.4342.000.10457.11.22445.01.235.4044.400.6142.050.18457.21.65444.10.92Abbreviations:SDStandard deviation.LOQ=0.02 mg a.i./L.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

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Page 26 of 70 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 cont rol 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] Fecundity 1 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.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

Page 27 of 70 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

Score 1 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.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

Page 28 of 70 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 GSI 1
(%) ±SD n Mean GSI 1
(%) ±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.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

Page 29 of 70 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 Stage 1 n Median Stage 2

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).Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

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Page 30 of 70 Table 15: Gonadal Histopathology in Male Fathead Minnow ( Pimephales promelas)

.

Treatment
(mg a.i./L)

[meanmeasured] Diagnostic Observations 1

Severity Increased
Proporti on 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetribuzinEPA MRID Number 48672202Page 31 of 70Table 16: Additional Gonadal HistopathologyObservations in Male Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[mean-measured]Additional Diagnostic Observations1SeverityDecreased Proportionof SpermatogoniaIncreased Vascular orInterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportions ofSpermatocytes orSpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidenceNegative Control(<LOQ)0NANANANANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANA4NANANANANANANANANANA0.1990NANANANANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANA4NANANANANANANANANANAData Evaluation Record onthe Fish Short-Term Reproduction Assay with MetribuzinEPA MRID Number 48672202Page 32 of 70Treatment(mg a.i./L)[mean-measured]Additional Diagnostic Observations1SeverityDecreased Proportionof SpermatogoniaIncreased Vascular orInterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportions ofSpermatocytes orSpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidence1.030NANANANANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANA4NANANANANANANANANANA5.400NANANANANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANA4NANANANANANANANANANAAbbreviation:NANot applicable.LOQ=0.02 mg a.i./L.1Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E ofthe test guideline for reference.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

Page 33 of 70 Table 17: Gonadal Histopathology in Female Fathead Minnow ( Pimephales promelas)

.

Treatment
(mg a.i./L)

[meanmeasured] Additional Diagnostic Observations 1

Severity Increased Oocyte

Atresia Perifollicular Cell
Hyperplasia/
Hypertrophy Decreased Yolk

Formation Aggregates of
macrophages,

multifocal

n Incidence n Incidence n Incidence n Incidence

Negative Control (<LOQ) 0 16 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

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

EPA MRID Number 48672202

Page 34 of 70 Table 18: Additional Gonadal Histopathology Observations in Female Fathead Minnow ( Pimephales
promelas)

.

Treatment
(mg a.i./L)

[meanmeasured] Additional Diagnostic Observations 1

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

EPA MRID Number 48672202

Page 35 of 70 Mean male VTG was 3040, 8952, 3129 and 1091 and mean female VTG was 1.89 x 10 6 , 3.21 x 10 6

, 2.43 x 10 6 and 2.25 x 10 6 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) ±SD 1 n Mean
(ng/mL plasma) ±SD 1

Negative Control (<LOQ) 4 3040 5888 4 1.89 x 10 6 0.845 x 10 6

0.199 4 8952 14478 4 3.21 x 10 6 3.51 x 10 6

1.03 4 3129 4556 4 2.43 x 10 6 0.728 x 10 6

5.40 4 1091 1108 4 2.25 x 10 6 0.270 x 10 6

Abbreviations: SD Standard deviation.
LOQ=0.02 mg a.i./L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetribuzinEPA MRID Number 48672202Page 36 of 70Plasma testosterone and plasma 17β-estradiol in male and females was not measured (Table 20).Table 20: Plasma Sex Steroids in Fathead Minnow ( Pimephales promelas). Not measured.Treatment(mg a.i./L)[mean-measured]Plasma Testosterone (T)Plasma 17β-estradiol (E2)MalesFemalesMalesFemalesnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDNMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDNegative Control (<LOQ)NANANANANANANANANANANANA0.199NANANANANANANANANANANANA1.03NANANANANANANANANANANANA5.40NANANANANANANANANANANANAAbbreviations:NANot applicable.SDStandard deviation.LOQ=0.02 mg a.i./L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetribuzinEPA MRID Number 48672202Page 37 of 70There were no notable observations with regards to behavior or changes in appearance, specifically color/banding, ovipositor appearance, or size ofdorsal 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 femaleshad exophthalmia in one eye in the 5.40 mg a.i./L treatment group.Table 21: Secondary Sex Characteristicsand Clinical Signs in Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[mean-measured]Secondary Sex Characteristics and Clinical Signs1MalesFemalesTypenIncidenceTypenIncidenceNegative Control (<LOQ)None80None1600.199None80None1501.03None80Only one eye1525.40None80Exophthalmia in one eye132LOQ=0.02 mg a.i./L.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

Page 38 of 70 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 p d 0.05 (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).Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

Page 39 of 70 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.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

Page 40 of 70 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetribuzinEPA MRID Number 48672202Page 41 of 70Table 22: Reproductive and HPG Endpoints1,2for Male Fathead Minnow ( Pimephales promelas)in the FSTRA with Metribuzin.Treatment(mg a.i./L)[mean-measured]Tubercle ScoreGSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2Medianp% Diff.pEffect?(Yes/No)% Diff.P% Diff.P% Diff.pNegative Control(<LOQ)40 NA 0 NANo 0 NA NA NA NANA0.199410.7918.60.44No19530.24NANANANA1.0339>0.993.800.99No2.930.68NANANANA5.40430.9524.50.25Yes-64.10.68NANANANAStatistical TestDunnett’s4Dunnett’sNAMann-WhitneyNANAAbbreviations:Diff.Difference.E217β-estradiol.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.NANot applicable.LOQ=0.02 mg a.i./L.1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water)control. Conclusions regarding histopathologymay be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.3This 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 inthe fish exposed at this concentration.4Mean 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetribuzinEPA MRID Number 48672202Page 42 of 70Table 23: Reproductive and HPG Endpoints1,2for Female Fathead Minnow ( Pimephales promelas)in the FSTRA with Metribuzin.Abbreviations:Diff.Difference.E217β-estradiol.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.LOQ=0.02 mg a.i./L.1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water)control. Conclusions regarding histopathologymay be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.Treatment(mg a.i./L)[meanmeasured]FecundityFert. SuccessTubercle ScoreGSIGonadalStagingand Histo.Plasma VTGPlasma TPlasma E2% Diff.p% Diff.PMedianp% Diff.PEffect?(Yes/No)% Diff.p%Diff.p%Diff.pNegative Control(<LOQ)0 NA 0 NA 0NA0 NANo 0 NANANANANA0.199-28.10.811.110.330NA10.70.74No69.5>0.99NANANANA1.03-15.40.960.570.770NA-3.140.99No28.40.24NANANANA5.40-26.80.831.290.220NA17.10.42No18.50.35NANANANAStatistical TestDunnett’sDunnett’sNADunnett’sNAMann-WhitneyNANAData Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

Page 43 of 70 Table 24: Growth Endpoints 1,2 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

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

EPA MRID Number 48672202

Page 44 of 70 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 revi ewer 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 Exec utive 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 anyData Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

Page 45 of 70 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.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

Page 46 of 70 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.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

Page 47 of 70 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.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

Page 48 of 70 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

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

EPA MRID Number 48672202

Page 49 of 70 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

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

EPA MRID Number 48672202

Page 50 of 70 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 0.773Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

Page 51 of 70 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, 4.91Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

Page 52 of 70 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 LOECData Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

Page 53 of 70 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 0.690Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

Page 54 of 70 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 controlData Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

Page 55 of 70 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 testsData Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

Page 56 of 70 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) )Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

Page 57 of 70 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)Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

Page 58 of 70 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, 2854.47Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

Page 59 of 70 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 Dose5Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

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 trendData Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

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-valueData Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

Page 62 of 70 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 0.218Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

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=0.05Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

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 Dose1Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

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, 53.82Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

Page 66 of 70 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 . . . . . .Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metribuzin

EPA MRID Number 48672202

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 LOECData 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 . .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 0.415Data 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)

# 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 alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended 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 re dundancy 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 potent ial 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).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: 

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### Date:

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### 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 bodyIn vivo Hershberger Assay (201 1) / Page 2 of 13 METRIBUZIN/ 101101 OCSPP 890.1400/ OECD 441


### weight as the co-variable). There were no trea tment-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 indivi dual CVs in the control and high dose treatment groups exceeded the guideline performan ce criteria values.

In the anti-androgen assay, body we ights 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 follo ws: 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 ad equate 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 wei ght changes were not seen in tw o or more of the five androgen sensitive issue weights. Metribuzin was negativ e for androgenicity and anti-androgenicity in the Hershberger assay.

The assay satisfies the EDSP Tier 1 Test Order requireme nts for a Hershberger assay (OCSPP 890.1400).

COMPLIANCE: Signed and dated GLP Compliance a nd Quality Assurance statements were provided.In vivo Hershberger Assay (201 1) / 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 S upervisor), 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

NH 2

S CH 3 CH

3 CH

3 CH 3In vivo Hershberger Assay (201 1) / 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 Ge rmain-sur-l’Arbresle, France) Housing: Rats were housed three per cage in ma krolon cages containi ng heat-treated soft wood bedding Diet: A04C-10 pelleted rodent diet, ad libitum Phytoestrogen content <350 μ g 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 pot ential androgenic activity, the test substance was administered daily via oral gavage to castrated male rats. Positive androgenic activity is defined as a signif icant increase in two or more target organ weights compared to the vehicle control. To screen for the po tential 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 signifi cant 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 admi nistration for organ weight measurements.

3. Study Schedule: Rats were castrated at the supplie r on PND 43 and allowed at least 4 days for recovery before shipment to the study faci lity. Animals were received at the study facility on PND 48-49 and allowed to acclimat e 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 necrops ied for organ weight measurements.

4. Animal Assignment: Animals were assigned to the te st groups noted in Table 1 using computerized randomization procedures which selected animals from the middle of the weight range of the available animals. Statis tical 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.In vivo Hershberger Assay (201 1) / Page 5 of 13 METRIBUZIN/ 101101 OCSPP 890.1400/ OECD 441

TABLE 1. Study Design a 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 th e vehicle, an aqueous solution of 0.5% methylcellulose 400 + 0.4% Tween 80. Dose fo rmulations of TP were prepared by mixing the appropriate amount of TP in corn oil, a nd 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 brackete d 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 concentr ations 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).In vivo Hershberger Assay (201 1) / 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 ad equate and that the variation between nominal and actual dosage to the animals was acceptable. The study referenced above should be submitted for verifi cation of the stability findings.

For TP, the %CV for homogeneity analyses was 1.2% and the averag e concentration was found to be 99.1% of nominal. For FT, the %CV for homogeneity analyses was 1.6% and the average concentration was f ound to be 95.1% of nominal.

7. Dosage administration: Test formulations were administ ered to the animals daily via oral gavage (5 mL/kg bw) for 10 days. TP was give n via s.c. injection at 0.5 mL/kg bw, and FT was administered via oral gava ge at 5 mL/kg bw. Dose volum es were adjusted daily based on the concurrent body weight measurement.

8. Statistics: The following parameters were anal yzed statistically: body weights, body weight gains, terminal body weights, and or gan 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 te st were significant, a Kruskal-Wallis test was performed, followed by a two-side d Dunn’s test for significant data. The same procedures were used for body weight (except terminal body we ight), 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 we ights) Dunnett’s test was cond ucted if the results of the ANOVA were significant. If the results of Le vene’s test (after transformation) showed significance, a Kruskal-Wallis test was perfor med, followed by a one-sided (organ weights) or two-sided (terminal body weights) Dunn’s test for significant data.

In addition, analysis of c ovariance (ANCOVA) was conduc ted on organ weights using terminal body weight as a covariable; adjust ed means were compared using a one-sided Dunnett’s test. For comparison of data from th e 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 twoIn vivo Hershberger Assay (201 1) / Page 7 of 13 METRIBUZIN/ 101101 OCSPP 890.1400/ OECD 441


### sided T-test, and data that we re 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 moribundit y. Clinical examinations were conducted daily.

2. Body Weight: Animals were weighed at rando mization, daily thr oughout 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 animal s were anesthetized by isoflurane inhalation, exsanguinated, and subjected to a gross necropsy. The five mandatory androgen-dependent organs (ventral prostate, seminal vesi cles, LABC, Cowper’s gland, and glans penis) were excised, trimme d free of adhering tissue, and weighed fresh (unfixed) according to the sta ndard operating procedures deta iled 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 pres ented 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 significan t. At 40 mg/kg/day, mean body weight gain from Days 1 to 2 was decreas ed by 31% (not statis tically significant) compared to the vehicle control; mean body we ights were not affected by the end of the study.In vivo Hershberger Assay (201 1) / Page 8 of 13 METRIBUZIN/ 101101 OCSPP 890.1400/ OECD 441

TABLE 2. Selected Group Mean Body Weights and Cumu lative Body Weight Gains (g) in the Androgen Agonist Assay a

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
B ody 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/da y, 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 ga in (Days 1-10) in this group was decreased 16% compared to the control; however, this decrease was not st atistically 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 we ights were not affected by the end of the study.

TABLE 3. Selected Group Mean Body Weights and Cumu lative Body Weight Gains (g) in the Anti-Androgen Assay a

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
B ody 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 DeviationIn vivo Hershberger Assay (201 1) / Page 9 of 13 METRIBUZIN/ 101101 OCSPP 890.1400/ OECD 441


### C. FOOD CONSUMPTION (Optional): Food consumption was not measured.

D. SERUM HORMONE CONCE NTRATIONS (Optional): Serum hormone measurements were not conducted.

E. ORGAN WEIGHTS: Accessory sex organ weights fo r the androgen agonist assay are presented in Table 4. At 80 mg/kg/day, there was an increas e 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. Th e %CVs for the following organs were outside the performance criteria: semi nal vesicles in the control a nd treated group (49% CV for both, compared to maximum 40% CV); ventral prostate in the cont rol 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 we re 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 a nd high dose treatment groups exceed the maximums designated for agonist and antagonist st udies, and (ii) at leas t two target tissues were marginally insignifican t, 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 revi ewers; the p values were >0.10 for ventral prostate (0.499), LABC (0.899), Cowper’s gla nds (0.214), and seminal vesicles (0.629).In vivo Hershberger Assay (201 1) / Page 10 of 13 METRIBUZIN/ 101101 OCSPP 890.1400/ OECD 441

TABLE 4. Accessory Sex Organ Weig hts (mg) from Androgen Agonis t Assay in Sprague Dawley Rats a

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 51.7 b 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-andr ogen assay are presented in Table 5. The weights of the accessory sex organs in the metr ibuzin 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. A ll %CV values were less than the maximum permissible values.

The study author did not report whether there were any gross pathol ogical findings at necropsy for either assay.In vivo Hershberger Assay (201 1) / Page 11 of 13 METRIBUZIN/ 101101 OCSPP 890.1400/ OECD 441

TABLE 5. Accessory Sex Organ Weig hts (mg) from Anti-Androgen Agonis t Assay in Sprague Dawley Rats a

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 inclu ded 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 tissu e 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 th e criteria for a positive androgen agonist, i.e. a statistically-significant increase (p ≤ 0.05) in any two or more ofthe five required androgendependent tissue weights.

B. AGENCY COMMENTS: All animals survived until scheduled termination. There were no signs of clinical toxicology. The study auth or did not report wh ether there were any gross pathological findings at necropsy.

In the androgen agonist assay, body weight s 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, ve ntral 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 throug hout 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 prosta te; 49% in LABC; 61% in Cowper’s gland; and 27% in glans penis. The performance criteria indicated th at this assay was perf orming as expected.In vivo Hershberger Assay (201 1) / Page 12 of 13 METRIBUZIN/ 101101 OCSPP 890.1400/ OECD 441



### The high dose used in this study (80 mg/kg/day) was consid ered 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 cha nges were seen in two or more of the five androgen sensitive tissue weights. Metribuz in was negative for androgenicity and antiandrogenicity in the Hershberger assay.

C. STUDY DEFICIENCIES: NoneIn vivo Hershberger Assay (201 1) / 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 th e Hershberger assay (MRID 48617904).

In a preliminary range-finding assay, metr ibuzin (94.4%; batch# K 285067) in an aqueous solution of 0.5% methylcellulose 400 + 0.4% Tween 80 was administ ered 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 sche duled 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 sl ightly 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 a nd 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-rela ted 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.

# 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 alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended 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 re dundancy 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 potent ial 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).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

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### 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 weightFemale 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 obs erved in 1/15 rats a nd enlarged liver was observed in 5/15 rats in this gr oup; there were increased numbers of mitoses in 4/5 examined livers. Serum T 4 

### 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 saliva tion was observed in 6/15 rats on several occasions throughout the study. Final body weight and body weight ga in, 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 comparab le to the controls at this dose level. At necropsy, enlarged liver was observed in 1/15 rats and increased numb ers of mitoses were observed in this liver. Mean TSH levels were increased (NS) by 29% at 60 mg/kg/day; however, serum T 4 

### and alanine aminotransferase levels in this group were comparable to the controls.

The dose levels tested in this assay are adequa te based on the results of a dose range-finding study.

The assay satisfies the EDSP Tier 1 Test Order require ments for a Female Pubertal Assay (OCSPP 890.1450).

COMPLIANCE: Signed and dated Data Confiden tiality, GLP Compliance, and Quality Assurance statements were provided.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 (Res ponsible Technician), C. Pallen (Statistician), L. Amir Tahmasseb (Senior Chemist), D. De Ba rbeyrac (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% me thylcellulose 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 Ge rmain-sur-l’Arbresle, France) Housing: Rats were housed 2-3 per cage in makrolon cag es 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 μ g 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

NH 2

S CH 3 CH

3 CH

3 CH 3Female 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 curren t study report. The dose range-finding study was also included w ith 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 gr oup 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-re lated 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-re lated effects were obser ved at 5 mg/kg/day.

5. Dose Preparation and Analysis: Dose formulations were prepared twice during the study by mixing appropriate amounts of test substan ce with aqueous 0.5% me thylcellulose 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 metribuz in in vehicle by sampling the surface, middle, and bottom of the suspension. Achieved con centration 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).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 ad equate 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 record ed. Dosing was performed between 0700 and 0900 hours daily.

7. Statistics: The following parameters were anal yzed statistically: body weights, body weight gains, terminal body we ights, 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). Signi ficance 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 te st was performed; a two-sided Dunnett’s test was conducted if th e results of the ANOVA were sign ificant. If the results ofBartlett’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 hor monal parameters, except that data were transformed (log transformation) and a second Bart lett’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 te st; if the data were significant, a data transformation (log transformation) was c onducted and a second Levene’s test was conducted. If the data were not significant, an ANOVA test was pe rformed; a two-sided Dunnett’s test was conducted if th e results of the ANOVA were sign ificant. 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 c ovariance (ANCOVA) was conducte d, using body weight at PND 21 as the covariable, on the means of the treat ed 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 cycli ng status (cycling/not cycling and regular cycling/irregular cycling), th e control group and treated groups were compared using the Chi-squares test. The statistical an alyses were considered adequate.Female Pubertal Assay (2011) / Page 6 of 15 METRIBUZIN/ 101101 OCSPP 890.1450/ OECD None



### C. METHODS

1. Mortality and Clin ical 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 an d evaluated for the pr esence 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 cy cles with diestrus for a period >3 days or cornification >2 days), or not cycling (having prolonged period of either vagi nal cornification or leukocyte smears). Mean cycle length was defined as the nu mber of days from the first day of diestrus to the first day of the next diestrus. Incomple te cycles were not used in calculating mean cycle length. In cases when the time betw een VO and euthanasia did not allow observation of more than 1 complete cycle, classifica tion was based on the available data with the assumption that animals are cycli ng regularly if the partial data fit the definition, or cycling irregularly if the study ends w ithout being able to distinguish between irregular and not cycling.

5. Sacrifice and Pathology: On PND 42, all surviving an imals were anesthetized using isoflurane and blood was collected from the a bdominal aorta into serum separation tubes. The length of time between final dose and exsa nguination was not spec ified; however, all blood collections were complete d by 1300 hours. Serum separation tubes were stored at

− 74 °C for subsequent hormone and clinical chemistry evaluations.

a. Hormone Analysis: Serum T 4 

### and TSH levels were determined using enzyme immunoassay kits.Female Pubertal Assay (2011) / Page 7 of 15 METRIBUZIN/ 101101 OCSPP 890.1450/ OECD None


### 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 Se rum protein electrophoresis Lactic acid dehydrogenase
X Alanine aminotransferase X Aspartate aminotransferase Sorbitol dehydrogenase Gamma glutamyl transferase Glutamate dehydrogenase * Recommended for the pubertal assay in fe male 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 subject ed 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 base d on guideline 890.1450 + Histopathological examination re quired based on guideline 890.1450



### All organs collected were weighed prior to fi xation. Paired organs (kidneys, adrenals, and ovaries) were weighed together. The uterus a nd cervix were separate d from the vagina and weighed. The uterus was weighed again fo llowing 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, sec tioned, 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 follic ular cell height and colloid area. The incidences of collo id alteration, follicular cell hyp ertrophy, follicular cell height decrease, and follicular cell shape were repo rted, graded on a six-point scale based on severity.

Five random sections of ovary were processed according to the facili ty’s procedures (not described) and subjected to histology including a semi-qualitative evaluation of follicle development (including presence/absence of te rtiary/antral follicles, presence/absence of corpora lutea, changes in corpus luteum develo pment, or changes in the number of primaryFemale Pubertal Assay (2011) / Page 8 of 15 METRIBUZIN/ 101101 OCSPP 890.1450/ OECD None


### and atretic follicles). It was not reported wh ich 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 obse rvations 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 we ights, body weight gains, age and weight at day of attainment of VO, a nd 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 incr eased (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 th e 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 reporte d 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 performa nce criteria provided in the Guideline (890.1450), however, the CV for age at VO was outside the acceptable range (7.5%; maximum 6.52%).Female Pubertal Assay (2011) / Page 9 of 15 METRIBUZIN/ 101101 OCSPP 890.1450/ OECD None

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/ SE b CV (%) N Mean SD/ SE b CV (%) N Mean SD/ SE b 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 = Sta ndard 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 necr opsy are presented in Ta ble 4. At 120 mg/kg/day, absolute, adjusted (for body weight on PND 21), and relative liver we ights were increased (p ≤ 0.01) by 21-22% and adjusted thyr oid 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 dos e level. There were no treatment-related effects on th e weights of kidneys, pituitar y, 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).Female Pubertal Assay (2011) / Page 10 of 15 METRIBUZIN/ 101101 OCSPP 890.1450/ OECD None

TABLE 4. Organ Weights at Necropsy a

Organ Vehicle Control Metribuzin (60 mg/kg/day) Metribuzin (120 mg/kg/day)

N Mean SD/ SE b CV (%) N Mean SD/ SE b CV (%) N Mean SD/ SE b 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 = Sta ndard 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.Female Pubertal Assay (2011) / Page 11 of 15 METRIBUZIN/ 101101 OCSPP 890.1450/ OECD None

TABLE 5. Estrous Cyclicity a

Treatment Groups N Mean Age at First Vaginal Estrus (PND) Mean Cycle Length (days) Cycling (%) Regularly Cycling

(%) Cycle Status at Necropsy (# Females)

Diestrus Proestrus Estrus Not Cycling Vehicle 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 T 4 

### 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-rela ted effects on creatinine, blood urea nitrogen, total protein, total bilirubin, as partate aminotransferase, or al kaline phosphatase. Historical control data were not provided for either hor mone or clinical chemistry parameters. In addition, the facility’s criteria for evaluati ng the performance of the enzyme immunoassay kits used for determination of hormone levels were not described.

The results for serum T 4 

### in the control group were with in the acceptable range of the performance criteria provid ed in the Guideline (890.1450).Female Pubertal Assay (2011) / Page 12 of 15 METRIBUZIN/ 101101 OCSPP 890.1450/ OECD None

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 T 4 , 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 wa s 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 ot her gross observations we re 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 treatmen t-related findings in the thyroid gland.Female Pubertal Assay (2011) / Page 13 of 15 METRIBUZIN/ 101101 OCSPP 890.1450/ OECD None

TABLE 7. Incidence of Histopathologi cal 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 sever ity: 0=Not remarkable, 1=Mini mal, 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 numbe rs 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, uter us, or kidneys were treatment related.

TABLE 8. Incidence of Histopathological Finding s of the Liver, Ovaries, Uterus, and Kidney a

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 m ononuclear 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 primar y 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 15Female Pubertal Assay (2011) / Page 14 of 15 METRIBUZIN/ 101101 OCSPP 890.1450/ OECD None

TABLE 8. Incidence of Histopathological Finding s of the Liver, Ovaries, Uterus, and Kidney a

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 m ononuclear cell infiltrate: focal 4 15 3 15 3 15 Interstitial mixed cell infi ltrate: 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 metribuz in was characterized by effects on the thyroid, as evidenced by hormona l changes and increased thyroid weight. A modest increase in age of vaginal pate ncy and body weight when VO occurred was observed at the high dose, but these differences from the control group we re 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 cyclin g; on the weights of kidneys, pituitary, adrenals, ovaries , or uterus (wet or blotted); or on levels of creatinine, blood urea nitrogen, total protein, total biliru bin, aspartate aminotra nsferase, or alkaline phosphatase. There were no treatment-relate d histopathological findi ngs in the thyroid gland, ovaries, uterus, or kidneys.

At 120 mg/kg/day, increased salivation was obs erved in 7/15 animals on one or several occasions throughout the study. The age (P ND 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 cont rol. 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 t hyroid was observed in 1/15 rats and enlarged liver was observed in 5/15 rats in this group; th ere were increased numbers of mitoses in 4/5 examined livers. Serum T 4 

### levels were decreased (p ≤ 0.01) by 44% in rats dosed at 120Female 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 cont rol. Relative liver weights were increased (p ≤ 0.05) by 10%; thyroid gland weights were comparab le 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 T 4 

### and alanine aminotransferase levels in this group were comparable to the controls.

C. STUDY DEFICIENCIES: The following minor deficienci es were noted that are not considered to have had an adverse impact on th e 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) wa s higher than the performance criteria acceptable range (38.34-48.84 mg)

# 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 alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended 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 re dundancy 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 potent ial 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).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 Reviewer: John Liccione, Ph.D

.**Signature: ~**  

### Health Effects Division 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-dimeth y lethyl)-3-(meth y lthio )-1,2,4-triazin-5( 4H)-one



### CITATION: Blanck

, M. (20 11 ) Metribuzin assessment of pubertal de ve lopment and th yroi d function in juvenile / peripubertal male a nd female rats

. Ba yer S.A.S., Sophia Antipo li s Cedex, France. Laboratory Study No.: SA 10341, December 1

, 2011. MRID 48672203. U npubli s hed.



### SPONSOR: Bayer CropSc i e nc e AG, A lfr e d Nobel Str. 50, Monheim, Germany



### TEST ORDER#: CON- 101101-127



### EXECUT IVE SUMMARY: In a Male Pubertal Assay (MRID 48672203)

, 15 Sprague Da w le y rats / dose group were treated daily via oral gavage with m etr ibuzin (94.4% a.i., Batch/lot # K285067) in aqueo u s 0

. 5% methylcellulose 400 

## + 0.4 % Tween 80 (vehicle) at doses of 0 (vehicle)

, 60

, or 120 mg / kg / day from po st- natal da y (PND) 23 to 53. Animals were examined for preputial separation (P PS) d a il y beginning on PND 30 and age and weight at da y of attainment were recorded

. Following sacrifice on PND 53, total serum testosterone
, th yroxine (T 4 )

, 

### and thyroid stimu l ating hormone (TSH) le ve l s we re analyzed usin g enzyme immunoa ssay kits. Adrenals

, liver, pituitary

, thyroid, and uro ge nital organ weights were recorded and microscopic exam in ations of the testes
, epi did y mides

, th yro id

, and kidne ys were performed

. In addition

, any livers found to be en lar ged at necrop sy were s ubjected to micro sco pic exa mination

.

There were no effects of treatment on final bod y weights, overall bod y weight gain, or age and body weight at PPS; on the we i g ht s of kidneys

, pituitary, adrenals

, se minal ves icle s plus coagulating gland (with or witho ut fluid ), ventral prostate
, dorsolateral prostate
, levator anibulbocavernosus muscle comp l ex (LABC)

, epididymides

, or te s te s; or on l eve ls of testosterone
, creatinine, blood urea nitrogen, total bilirubin

, aspartate a minotran sferase, a lanine aminotransferase, or alka lin e phosphatase. There were no treatment-related histopatholo gica l findings in the testes, epididy mid es, or kidneys

.

At 120 mg / kg / day, increased sa li va tion was observed in all rats on one or several occasions throughout the study. In a ddition

, one animal in this group had r e duced motor activity andMale Pubertal Assay (2011) / Page 2 of 13 METRIBUZIN/ 101101 OCSPP 890.1500 / OECD None


### labored respiration on Day 13 and another rat had ocular discharge on Days 20-25. All rats in this group achieved PPS before study terminatio n. Absolute, adjusted (for body weight on PND 21), and relative liver we ights 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 T 4 

### levels were decreased (p ≤ 0.01) by 72% in rats dosed at 120 mg/kg/da y; 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 a nd enlarged liver was obs erved in 11/15 rats. Minimal to slight follicular cell hypertrophy and colloid alteration were observed in the thyroid glands; an increased num ber 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 hepatocellula r hypertrophy (4/11 rats).

At 60 mg/kg/day, increased sali vation 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 we re comparable to the controls at this dose level. Serum T 4 

### 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 metribuz in 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 requirement s for a Male Pubertal Assay (OCSPP 890.1500).

COMPLIANCE: Signed and dated Data Confiden tiality, GLP Compliance, and Quality Assurance statements were provided.Male Pubertal Assay (2011) / Page 3 of 13 METRIBUZIN/ 101101 OCSPP 890.1500 / 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 (Res ponsible Technician), C. Pallen (Statistician), L. Amir Tahmasseb (Senior Chemist), D. De Ba rbeyrac (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% me thylcellulose 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 Ge rmain-sur-l’Arbresle, France) Housing: Rats were housed 2-3 per cage in makrolon cag es 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 μ g 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

NH 2

S CH 3 CH

3 CH

3 CH 3Male Pubertal Assay (2011) / Page 4 of 13 METRIBUZIN/ 101101 OCSPP 890.1500 / OECD None


### 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 P ND 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 ap pendix 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 substan ce with aqueous 0.5% me thylcellulose 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 metribuz in in vehicle by sampling the surface, middle, and bottom of the suspension. Achieved con centration 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 ad equate 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).Male Pubertal Assay (2011) / Page 5 of 13 METRIBUZIN/ 101101 OCSPP 890.1500 / OECD None


### 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 record ed. Dosing was performed between 0700 and 0900 hours daily.

7. Statistics: The following parameters were anal yzed statistically: body weights, body weight gains, terminal body we ights, organ weights, PPS, c linical chemistry, and hormonal analysis. Clinical chemistry parameters, hormonal parameters, and body weight parameters were analyzed using Path/Tox System V 4.2.2. Age at PPS, body weight at PPS, organ weight, body weight at PND 21, and terminal body weight para meters 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 te st was performed; a two-sided Dunnett’s test was conducted if th e results of the ANOVA were sign ificant. If the results ofBartlett’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 hor monal parameters, except that data were transformed (log transformation) and a second Bart lett’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 transformati on (log transformation) was conducted and a second Levene’s test was conducted. If the da ta were not significant, an ANOVA test was performed; a two-sided Dunnett’ s test was conducted if th e results of the ANOVA were significant. If the resu lts of Levene’s test (after transformation) showed significance, a Kruskal-Wallis test was performed, followed by a two-sided Dunn’s test fo r significant data.

In addition, analysis of c ovariance (ANCOVA) was conducte d, using body weight at PND 21 as the covariable, on the means of the treat ed and control groups except for terminal body weight. The statistical analys es were considered adequate.

C. METHODS

1. Mortality and Clin ical 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 pr ior 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 an imals were anesthetized usingMale Pubertal Assay (2011) / Page 6 of 13 METRIBUZIN/ 101101 OCSPP 890.1500 / OECD None



### isoflurane and blood was collected from the a bdominal aorta into serum separation tubes. The length of time between final dose and exsa nguination was not spec ified; however, all blood collections were complete d by 1300 hours. Serum separation tubes were stored at

− 74 °C for subsequent hormone and clinical chemistry evaluations.

a. Hormone Analysis: Total testosterone, T 4 

### , 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 Se rum 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 subject ed 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 (w ith 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 base d on guideline 890.1500 + Histopathological examination re quired based on guideline 890.1500



### All organs collected were weighed prior to fi xation. Paired organs (kidneys and adrenals) were weighed together. All co llected organs, with the excepti on of testis and epididymis, were preserved in 10% neutral buffered forma lin, 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.Male Pubertal Assay (2011) / Page 7 of 13 METRIBUZIN/ 101101 OCSPP 890.1500 / OECD None


### Three random sections of thyroid gland were processed according to the facility’s procedures (not described) and subjectively evaluated for follic ular cell height and colloid area. The incidences of collo id alteration, follicular cell hyp ertrophy, follicular cell height decrease, and follicular cell shape were repo rted, 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 gi ant cells, or sloughing of spermatogenic cells into the lumen. Examination of epididymis wa s conducted in order to identify such lesions as sperm granulomas, leukocytic infiltration (i nflammation), 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 obse rvations 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, on e animal in this group had reduced motor activity and labored respirati on 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 P PS, and proportion of rats that did not achieve PPS by study termination are presented in Table 3. Ther e were no treatment-rela ted effects on final body weights, overall body weight gai n, 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 ga in between Days 1 and 7 was reduced (p ≤ 0.05) at 120 mg/kg/day ( ↓ 16%) but was comparable to the contro ls 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. MeanMale Pubertal Assay (2011) / Page 8 of 13 METRIBUZIN/ 101101 OCSPP 890.1500 / OECD None


### 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 th e control group were wi thin the acceptable range of the performance criteria provided in the Guideline (8 90.1500), 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/ SE b CV (%) N Mean SD/ SE b CV (%) N Mean SD/ SE b 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 = Sta ndard 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 necr opsy are presented in Ta ble 4. At 120 mg/kg/day, absolute, adjusted (for body weight on PND 21), and relative liver we ights 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 wei ghts 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 th e weights of kidneys, pituitar y, adrenals, seminal vesicles plus coagulating gland (with or without fluid) , ventral prostate, dorso lateral 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 wei ght (16.8 and 19.8%; maximum 16.39%).Male Pubertal Assay (2011) / Page 9 of 13 METRIBUZIN/ 101101 OCSPP 890.1500 / OECD None

TABLE 4. Organ Weights at Necropsy a

Organ Vehicle Control Metribuzin (60 mg/kg/day) Metribuzin (120 mg/kg/day)

N Mean SD/ SE b CV (%) N Mean SD/ SE b CV (%) N Mean SD/ SE b 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 = Sta ndard 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 T 4 

### levels were decreased (p ≤ 0.01) by 72% in rats dosed at 120 mg/kg/day and byMale Pubertal Assay (2011) / Page 10 of 13 METRIBUZIN/ 101101 OCSPP 890.1500 / OECD None


### 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, respectivel y. Total protein was increased (p ≤ 0.01) by 5% at 120 mg/kg/day. There were no treatment-relate d 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 T 4 

### 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 criteri a, 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 T 4 , 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 wa s observed in 6/15 rats in the 120 mg/kg/day group and enlarged liver was observe d 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; th is organ was not weighed. No other gross observations were noted in male rats surviving to the scheduled termination.Male Pubertal Assay (2011) / Page 11 of 13 METRIBUZIN/ 101101 OCSPP 890.1500 / OECD None



### 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 additio n, an increased number of mitoses was noted in 2/15 males in this group (not shown in Table 6).

TABLE 6. Incidence of Histopathologi cal Lesions of the Thyroid Gland a

Treatment Groups Parameter Evaluated

Severity b 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 sever ity: 0=Not remarkable, 1=Mini mal, 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 Tabl e 7. The liver was examined in the cases in which macroscopic findings were noted at n ecropsy. There were increased numbers of mitoses in 7/11 examined livers in th e 120 mg/kg/day group, as well as minimal centrilobular hepatoceullular hype rtrophy (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, epididym ides, or kidneys were treatment related.

TABLE 7. Incidence of Histopathological Findings of th e Adrenal Glands, Liver, Testes, Epididymides, and Kidney a

Findings Dose Level

Vehicle Control Metribuzin (60 mg/kg/day) Metribuzin (120 mg/kg/day)
# Observed # Examined # Observed # Examined # Observed # Examined Adrenal GlandsMale Pubertal Assay (2011) / Page 12 of 13 METRIBUZIN/ 101101 OCSPP 890.1500 / OECD None

TABLE 7. Incidence of Histopathological Findings of th e Adrenal Glands, Liver, Testes, Epididymides, and Kidney a

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 metri buzin was characterized by effects on the thyroid, as evidenced by hormona l changes, increased t hyroid weight, and/or treatment-related microscopic findings. The preputial separati on examinations and
testosterone measurement we re unaffected by treatment.

B. AGENCY COMMENTS: There were no effects of treatment on final body weights, overall body weight gain, or age and body we ight at PPS; on the weights of kidneys, pituitary, adrenals, seminal vesicles plus coag ulating gland (with or without fluid), ventral prostate, dorsolateral prostate, LABC, epididymid es, or testes; or on le vels of testosterone, creatinine, blood urea nitrogen, total bilirubin, aspartate aminotransferase, alanine aminotransferase, or alkaline phosphata se. There were no treatment-related histopathological findings in the te stes, epididymides, or kidneys.Male Pubertal Assay (2011) / Page 13 of 13 METRIBUZIN/ 101101 OCSPP 890.1500 / OECD None



### 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 labored respiration on Day 13 and another rat ha d ocular discharge on Days 20-25. All rats in this group achieved PPS before study termin ation. 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 T 4 

### 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 obser ved in 6/15 rats and enlarged liver was observed in 11/15 rats. Minima l to slight follicular cell hypertrophy and colloid alteration were observed in the thyroid glands; an incr eased number of mitoses was noted in 2/15 males in this group. There were increased num bers of mitoses in 7/ 11 examined livers in the 120 mg/kg/day group, as well as minima l 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 T 4 

### 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 contro ls. At necropsy, enla rged liver was observed in 1/15 rats.

The increased liver weights at 120 mg/kg/day in dicated 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%)

# 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 alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended 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 re dundancy 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 potent ial 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).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



### EXECUT IVE 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 concurrent l y with each independent run of the test chemical plate to demonstrate that the assay responded properly to
po s itive control agents at two concentration levels

. The positive controls included a known inhibitor (prochloraz) and a known inducer (forsko lin ) 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

m e t
, including lack of cytotoxicity, adequate production of testosterone and estradiol, acceptable reproducibility (low coefficients of variation

, %CV)

, and appropriate induction and inhibition

w ith positive controls

. Exceptions included that the induction of testosterone on the QC plates in

a ll a s sa y runs (1

. 50- to 1.86-fold) was below the**2-fold** 

### Guide lin e cr it eria

, and the inter-plate % CV of the solvent control (SC) for estradiol (33%) in the QC plates exceeded the Guideline criteria ( :'.S 30%)

.Steroidogenesis Assay (2011) / Page 2 of 11 METRIBUZIN / 101101 OCSPP 890.1550/ OECD None



### No significant effects of metribuzin on testoste rone production (0.81- to 0.97-fold change) were noted between 10

− 10 

### and 10

− 4 

### M. No effects of metribuzin we re 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 inde pendent runs, metribuzin treatment resulted in statistically significant and repr oducible 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 Or der requirements for a Steroidogenesis assay (OCSPP 890.1550).

COMPLIANCE: Signed and dated GLP Compliance, Quality Assurance, and Data Confidentiality statements were provided.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, Respons ible 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 cont ainer at room temperature CAS #: 21087-64-9 Molecular weight: 214.28 Structure:

N N N O

NH 2

S CH 3 CH

3 CH

3 CH 3



### 3. Positive Control: Forskolin Description (molecular weight): White powder (410.50) Source: Sigma-Aldrich (St. Louis, MO) Batch # (expiration date): 109K5057V (Retest De cember, 2014) Purity: 98% Solubility (in solvent): Soluble in DMSO up to 0.01 M Storage conditions: Air-tight, light-resistant cont ainer 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 cont ainer at room temperature CAS #: 67747-09-5Steroidogenesis 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 solven t 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/streptomyci n; GIBCO (15070-063); Lot # 847134.



### 7. Test Cells: H295R human adrenocortical carcin oma 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% CO 2 

### 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.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 tech nician who conducted the present study. The test conditions were the same as the curr ent assay. The concentrations used and the EC 50 

### values for each positive control are given below:

TABLE 1. Results of Laboratory Proficienc y Test with Forskolin and Prochloraz. a Compound Concentrations tested ( μ M) Estimated EC 50 ( μ M) 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 EC 50 s for forskolin are 0.2-2.0 μ M for testosterone and 0.3-3.0 μ M for estradiol, and the specified range for ED 50 s for prochloraz are 0.01-0.1 μ M for testosterone and 0.03-0.3 μ M for estradiol.



### 2. Test Solutions: Metribuzin, forskolin, and prochloraz we re dissolved in DMSO and diluted 1:1000 in the final treatment medi um. When added to the cell cu lture plates, these dilutions yielded final concentrations of 1 μM a nd 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 consta nt at 0.1% (v/v).

3. Cell Plating and Preincubation: Cells were maintained in the stock medium described above. The hormone concentrations in undilute d 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% CO 2 

### ) for approximately 24 hours prior to chemical exposure. After a 24-hour pre-incuba tion, the cells were checked microscopically for good attachment and proper morphology.

4. Exposure: The medium was removed from the ce lls and replaced with 1 mL of medium containing concentrations of metribuzin (or solv ent) in triplicate, according to the schematic presented in Table 2.

TABLE 2. Dosing Schematic for the Exposure of H295 R Cells to Metribuzin (F inal 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 res ponse to forskolin (an inducer of testosterone and estradiol production) and prochloraz (an inhibitor of testoste rone and estradiolSteroidogenesis 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 Po sitive Controls (Final Concentrations in μM). a
1 2 3 4 5 6
A Blank b 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 incuba ted for approximately 48 hours under the conditions previously described. After the in cubation 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 th e XTT (2,3-Bis(2-methoxy-4- nitro-5-sulfophenyl)-2H-tetrazolium-5-carbox anilide) 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; ba tch 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 eval uate cell viability

6. Hormone Measurement System: The concentration of eac h hormone (testosterone and estradiol) was evaluated in triplicate in the cu lture medium using specific EIA kits supplied by Enzo Life Sciences Inc. (Plymouth Meeti ng, 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 estrad iol (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 be low maximum response on standard curve X Test compound tested for interfer ence with meas urement system



### C. DATA ANALYSIS: To evaluate the relative increas e 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 ± standa rd deviation (SD). Relative changes were calculated as follows: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 perf ormed to compare the homogeneity of group variances. If the Bartlett test was not si gnificant, 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 furthe r 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 wa s significant, group means were compared by using the non-parametric Kruskal-Wa llis test. If the Kruskal-Wallis test was not significant, the group means were considered to be homogen eous and no further analysis was performed. If the Kruskal-Wallis test wa s significant, means of the exposed groups were compared to the mean of the control group with the Dunn test (2-s ided). The level of significance for each statistical comparison wa s 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 (calcu lated 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 test osterone and <25% for estradiol.

The hormone concentrations following exposure to metribuzin are presented in Table 4, and the fold changes in hormone production fo r metribuzin-treated cel ls compared to the solvent control are shown graphically in Figur es 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 evalua tions varying between

− 19% (0.81-fold) and

− 3% (0.97-fold).

No concentration dependent effects of metr ibuzin 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.Steroidogenesis Assay (2011) / Page 8 of 11 METRIBUZIN / 101101 OCSPP 890.1550/ OECD None

TABLE 4. Mean (±SD) Hormone Concen trations 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 st udy report. The lower limit of quantification (LLQ) was 95.7 pg/mL for testosterone and 5.7 pg/mL fo r estradiol. Statistical significance was determined for the mean of the three

runs. ** p ≤ 0.01Steroidogenesis Assay (2011) / Page 9 of 11 METRIBUZIN / 101101 OCSPP 890.1550/ OECD None



### FIGURE 1. Change in Testosterone Produc tion Relative to Metribuzin Concentration.

FIGURE 2. Change in Estradiol Producti on Relative to Metri buzin 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 expos ure 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 presente d 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. Guide line requirements for basal hormone production, induction, and inhibition were met for testostero ne and estradiol, except for induction of testosterone with 10 μM forsko lin in all three test runs (a verage 1.70-fold induction) which were below the Guideline recommendation of ≥ 2-fold induction.Steroidogenesis Assay (2011) / Page 10 of 11 METRIBUZIN / 101101 OCSPP 890.1550/ OECD None


### The variability (%CV) between th e 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 Followi ng 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 350.0 315.0 154.0 b 0.06 0.06 0.03 b 0.05 c 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 134.0 d 202.7 I 0.80 d 0.62 I 0.77 e 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 estrad iol concentrations. Metr ibuzin 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 minimu m basal hormone production levels (500 pg/mL for testosterone, 40 pg/mL for estrad iol) were met in both blank and SC wells for testosterone. Guideline requirements for basal hormone production, induction, and inhibition were met for testosterone and estrad iol, 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 wa s slightly above the Guideline limit.Steroidogenesis Assay (2011) / Page 11 of 11 METRIBUZIN / 101101 OCSPP 890.1550/ OECD None



### Precipitation of the test compound, and cytotoxi city, were not reported at any concentration tested, though detailed cytotoxi city 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 th e testosterone assays ( ≤ 22% compared to the Guideline limit of <30%). No effects of metribuz in 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 conc entrations 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 i ndependent runs, metribuz in treatment resulted in a statistically significant and reproduc ible alteration in es tradiol 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 estradio l in the QC plates (33%) exceeded the Guideline criteria ( ≤ 30%).

# 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 alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended 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 re dundancy 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 potent ial 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).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 (OCSPPIn vivo Uterotrophic Assay (2011) / Page 2 of 9 METRIBUZIN / 101101 OCSPP 890.1600/ OECD 440


### 890.1600).

COMPLIANCE: Signed and dated GLP Compliance a nd Quality Assurance statements were provided.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 (Statisticia n); 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

NH 2

S CH 3 CH

3 CH

3 CH 3



### 3. Reference Estrogen: 17 α -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 applicableIn 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 (F rance); shipped with dam Housing: Rats were housed 6 per cage in makrol on cages with heat-treated soft wood bedding (S.A.F.E.; Scientific Animal Food and Engineering, Augy, France). Diet: Certified rodent pelleted and irradiated di et A04C-10 (S.A.F.E.; Augy France) with phytoestrogens <350 μ g/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 female s 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 fr om PND 19 to 21. Rats were euthanized approximately 24 hours after the last dose and ne cropsied 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 computer ized 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 fo r the study. The body weight of each animal was within 20% of the overall mean.

TABLE 1. Study Design a Test Group Dose (mg/kg/day) # of Females Estrogen Agonist Assay Vehicle Control 0 6 Low Metribuzin 10 6 Mid Metribuzin 40 6 High Metribuzin 120 6 17 α -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 pr epared once prior to initiation of treatment by suspending the appropriate amou nts of metribuzin in 0.5% methylcellulose 400 + 0.4% Tween 80. The positive control, EE, was prepared once prio r to initiation of treatment by diluting in corn oil. Doses were adjusted daily based on individual bodyIn vivo Uterotrophic Assay (2011) / Page 5 of 9 METRIBUZIN / 101101 OCSPP 890.1600/ OECD 440


### weight measurements. Homogeneity of the te st substance formulation was checked at the beginning of the study for the lowest and the hi ghest 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 additi on, the intermediate con centration (8 g/L) was checked. Data were recorded and analyzed us ing Empower 2 (Build 215 4). It was reported that a separate study (SA 11089 1 

### ) demonstrates that metribuz in 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 we re stored at 5 ± 3 0 

### C 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 wa s not tested for homogeneity, the analytical data indicated that the mixing procedure wa s 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 volum es 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. M eans and standard deviations were calculated for each group and percent coefficient of variation was calculated for terminal body weight and uterine weight parameters. In a ddition, a covariance an alysis (ANCOVA) was performed on wet and blotted uterus weights us ing the terminal body weight as a covariable; adjusted means were compared using the Dunne tt test. Group means were compared at the 5% and 1% levels of significance. The statistical analyses we re considered appropriate.

C. METHODS

1. Clinical Examinations: Animals were observed for clinical signs of toxicity at least once daily, and observed for mortality and mori bundity twice daily, except on weekends and holidays when they were checked once a day.

2. Body Weight: Animals were weighed at randomi zation, study initiati on, 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.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 gr oss necropsy. Dissecti on of the uterus was performed according to the U.S. EPA Guideline. Briefly, the vagina wa s 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 bl otted 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 me tribuzin-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 decr eased (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. PATHOLOGYIn 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 an d blotted uterine weights increased as expected.

TABLE 3. Uterine Weights (mg) from Estrogen Agonist A ssay 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 weight s, 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 ve hicle control animal were “not tested.” BW Body weight N No. of animals in the group SD Standard Deviation ** Significantly different from ve hicle control at p < 0.01.



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

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Individual uterine weight s 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 μ g/kg/day increased absolute uterine we ights (wet and blotted), (p ≤ 0.01) as expected. Metribuzin had no effect on terminal body weight or uterine weights at any dose le vel. At 120, 40 and 10 mg/kg/day of metribuzin, the mean cumulativ e body weight gain was reduced by 46, 40 and 33% (p ≤ 0.01), respectively, between Study Day 1 a nd 3 compared to the controls. In conclusion, these data indicate th at administration of up to 120 mg/kg/day of metribuzin for 3 days to immature Sprague Dawley female ra ts 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 anim als for any dose groups. Body weights in the metribuzin treated groups were comparable to the controls thr oughout the study; however, the body weight gains for Days 1-3 were d ecreased (p<0.01) by 33, 40, and 46% at 10, 40, and 120 mg/kg/day, respectively. Uterine weight s in the metribuzin treated groups wereIn vivo Uterotrophic Assay (2011) / Page 8 of 9 METRIBUZIN / 101101 OCSPP 890.1600/ OECD 440


### comparable to the controls. Absolute wet a nd blotted uterus weight s for the EE group were increased (p<0.01) by 109% and 11 0%, respectively, as expected.

The dose levels were determined to be ade quate 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: NoneIn 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 th e 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 af ter the final dose administration.

All animals in the 150 mg/kg/day dose group surv ived 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, m ean 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 (D ays 1-4) was decreased 13% compared to the controls. No other effects on body we ight 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.