UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

OFFICE OF CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

MEMORANDUM 

Date: 6/29/2015 

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

PC Code: 108801 

Decision No.: 461243 

Petition No.: NA 

Risk Assessment Type: NA 

TXR No.: 0052848 

MRID No.: See Table 

DP Barcode: D398926 

Registration No.: NA 

Regulatory Action: NA 

Case No.: NA 

CAS No.: 51218-45-2 

40 CFR: NA 

FROM: Greg Akerman, Ph.D. b~ tr----

Immediate Office 

Health Effects Division (7509P) 

THROUGH: Jess Rowland ~-z.-Q-.C2.--­

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 metolachlor: Test Order# CON-108801-12. 

Page I of2 

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II. RESPONSE 

Attached are the EDSP Tier 1 assay DERs for metolachlor.

III. MRID Table 

Chemical: Metolachlor PC Code: 108801 

Guideline Assay MRID 

890.1100 Amphibian Metamorphosis Assay (Frog) 48617810 

890.1150 Androgen Receptor Binding (Rat Prostate) 48617806 

890.1200 Aromatase Assay (Human Recombinant) 48617805 

890.1250 Estrogen Receptor Binding 48617807 

890.1300 Estrogen Receptor Transcriptional Activation 

(Human Cell Line HeLa-9903) 48617808 

890.1350 Fish Short-Term Reproduction 48617811 

890.1400 Hershberger (Rat) 48617802 

890.1450 Female Pubertal (Rat) 48617804 

890.1500 Male Pubertal (Rat) 48617803 

890.1550 Steroidogenesis (Human Cell Line – H295R) 48617809 

890.1600 Uterotrophic (Rat) 48617801 

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

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DER Template Version: 22 September 2011 

CITATION: Palmer, S.J., S.Z. Schneider, T.Z. Kendall, and H.O. Krueger. 2012. Metolachlor: Amphibian 

Metamorphosis Assay for the Detection of Thyroid Active Substances. Unpublished study performed by Wildlife 

International, Ltd., Easton, MD. Laboratory report number 568A-103. Study sponsored by Metolachlor Task 

Force, Stilwell, KS. Study completed January 24, 2012. 

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

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

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

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

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

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

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

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

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

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

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

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

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

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EXECUTIVE SUMMARY

The 21-day assay of metolachlor (97.3% purity) on amphibian metamorphosis of Xenopus laevis (African clawed 

frog) was conducted under flow-through conditions. Amphibian larvae at developmental Nieuwkoop-Faber (NF) 

Stage 51 (80/control and treatment group; 20/replicate) were exposed to nominal concentrations of 0 (negative 

control), 0.09, 0.9, and 9.0 mg a.i./L. Mean-measured concentrations were <0.0500 (<LOQ; negative control), 

0.075, 0.79, and 8.4 mg a.i./L. The test system was maintained at 22 ± 1 °C and a pH of 8.1 to 8.3. 

On Day 21, mortality was 3.3 and 6.6% in the mid and high treatment groups, respectively. Crooked tails were 

observed in 27 to 57 tadpoles in the negative control and all treatment groups 

Metolachlor significantly reduced (p<0.05) Day 7 wet weight by 19% and Day 7 snout-vent length by 7% at the 

high treatment group relative to the negative control. Day 21 normalized (for snout-vent length) hind leg length 

(HLL) was also significantly reduced (p<0.05) by 23% at the high treatment level relative to the negative control. 

Metolachlor exposure had no effect on the NF developmental stage in all treatment groups relative to the negative 

control on Days 7 and 21; no asynchronous development was observed. Day 21 normalized HLL at the high 

treatment level was significantly reduced (p<0.05) by 23% relative to the negative control group. 

Thyroid gland histopathology observations included thyroid gland hypertrophy, thyroid gland atrophy, and/or 

follicular cell hypertrophy in the negative control and all treatment groups. 

Analysis of Day 21 wet weight, SVL, HLL, and normalized HLL excluded late stage tadpoles (i.e., NF stage 

=61-63); there was no pattern in the development of late stage individuals across treatment groups, with the 

proportion of late stage tadpoles ranging from 2 to 6% of all tadpoles tested in the negative control and all treatment 

groups. 

The study met all of the performance and validity criteria. 

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

Guideline 890.1100). 

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

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Results Synopsis:

Test organism NF stage at test initiation: 51 

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

Test type: Flow-through 

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

Metamorphosis Assay (AMA) with Metolachlor. 

Treatment 

(mg a.i./L) 

[measured]

NF Developmental 

Stage 

Hind Limb 

Length3

Asynchronous 

Development 

Thyroid Gross 

and 

Histopathology 

Day 7 Day 21 Day 7 Day 21 Day 7 Day 21 Day 21 

0.075 No No No No No No No 

0.79 No No No No No No No 

8.40 No No No Yes No No No 

1

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

otherwise specified. 

2

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

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

pathologist. 

3

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

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I. MATERIALS AND METHODS 

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

Environmental Protection Agency Series 890- Endocrine Disruptor Screening 

Program Test Guidelines, OPPTS Number 890.1100: Amphibian 

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

Guideline 231: Amphibian Metamorphosis Assay. The following deviations were 

noted: 

1. The flow rate (~69 mL/min) was greater than recommended (25 mL/min), though there was 

complete volume replacement approximately every 2.4 hours, which is in agreement with guideline 

recommendations (complete volume replacement every 2.7 hours). 

2. The reported pH (8.1-8.3) was greater than recommended (7.5 ± 0.1). 

 These deviations do not impact the interpretation of the study. 

 Compliance: Signed and dated Good Laboratory Practice and Quality Assurance statements 

were provided. A No Data Confidentiality statement was not included. This 

study was conducted in compliance with Good Laboratory Practice Standards 

as published by the U.S. Environmental Protection Agency (40 CFR Parts 160 

and 792); and OECD Principles of Good Laboratory Practice 

(ENV/MC/CHEM(98) 17), with the following exceptions: periodic analyses of 

water for potential contaminants were not performed according to Good 

Laboratory Practice Standards; the characterization of the test substance, and 

the stability of the test substance under conditions of storage at the test site, 

were not determined in accordance with GLP standards; and, preliminary 

analyses of water iodide concentrations were not performed according to GLP 

standards. 

A. Test Material Metolachlor 

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Description: Liquid

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

vapor pressure of test compound, expiration date.

Lot No./Batch No. : 20081115 (Lot #) 

Purity: 97.3% 

Impurities: None Reported 

Stability of Compound: Stable. Mean-measured concentrations over the 21-day exposure yielded 

recoveries of 83 to 93% of nominal.

Storage Conditions of 

Test Chemicals: Stored under ambient conditions. 

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B. Test Organism

Table 2: General Information About the Test Species and Parental Care.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Species common name: African clawed frog EPA recommends African clawed frog 

(Xenopus laevis). Western [Africa] clawed 

frog Silurana (Xenopus) tropicalis may be 

used as an alternate species1; however, a list 

of all of the necessary protocol deviations to 

accommodate this species is recommended 

for inclusion in the study report. The guideline 

recommends that the performance criteria 

used to support the reliability of the test be 

identified.

Species scientific name: Xenopus laevisSpecies strain (if stated): Not Reported 

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

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Were parents maintained as in-house stock? 

Yes Adult frogs for the culture were 

originally obtained from Xenopus I of Dexter, Michigan. 

EPA recommends that larvae used in the 

assay be derived from in-house adults.

Were parental acclimation conditions same as definitive test? 

Yes

Acclimation period for parental frogs (if applicable): 

~13 weeks

Details on parental feeding: Adults were fed an adult 

Xenopus diet supplied by Zeigler Brothers, Inc. of Gardners, PA at a rate sufficient to maintain the health of the culture. 

Details on parental health: Culture frogs showed no sign 

of disease or stress prior to the test. 

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Table 3: Larval Selection and Care.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Best single spawn? Yes Best single spawn had 98% viability. EPA and OECD recommend that the best 2 – 

3 individual spawns, with a minimum of 1500 

larvae/spawn, be evaluated to identify the 

best single spawn, and that the larvae selected 

for testing originate from the best single spawn 

(i.e., the spawns are not co-mixed)

Number of spawns evaluated (if applicable): 

2 Based on use of two breeding groups 

Number of eggs sampled per spawn:

250 eggs from the best spawn were evaluated for viability. 

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

initiated with larvae at Nieuwkoop – Faber 

(NF) developmental stage 51 (≤17 days 

post-fertilization).

Age at test initiation: 15 days post-fertilization 

(dpf)

Mean total length at test initiation (if reported): 

Not Reported 

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

Not Reported 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Was the optional size selection method used? 

No 

Details on larval selection: All tadpoles were derived 

from eggs spawned on the same day. To produce eggs, two male and female adults were injected with human chorionic gonadotropin and each pair was placed in a separate spawning tank overnight. 

Loading rate (rearing density): 2 larvae/L Estimated from 20 larvae per test 

vessel (10 L fill volume).

EPA recommends that rearing density 

(loading rate) not exceed approximately 10 

larvae/L culturing system for flow-through 

systems or 4 tadpoles/L in static-renewal 

exposure systems.

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Source of food: Sera North America, 

Montgomeryville, PA

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

during the entire 21-d definitive study. If 

another diet is used, the study report should 

provide analysis of iodide content and potential 

contaminants, and the diet should 

demonstrate equal performance to Sera 

Micron® .

Iodide concentration in diet (if known):

Not Reported 

Frequency of feeding: 3 times/day and once on the 

last day of the test

EPA recommends that feeding occur at least 

twice per day.

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

Type of exposure: Flow-through EPA recommends the use of a flow-through 

system.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Type of flow-through dilution system (if applicable):

Continuous-flow diluter Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

Flow-through rate (if applicable):

~69 mL/min The flow-through rate was 

calculated based on the volume of the test chambers (10 L) and 10 volume additions per day (10). The flow-through rate resulted in complete volume replacement approximately every 2.4 hours. 

Recommended flow-through rate is 25 

mL/min (complete volume replacement ca. 

every 2.7 hrs).

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on toxicant mixing for flow-through systems (if applicable):

The stock solution was delivered to mixing chambers using fluid metering pumps, and then delivered to the test vessels using rotameters and peristaltic pumps.

Mean flow rate did not vary by more than ±10% during the test. 

Recommended toxicant mixing for 

flow-through systems: 1) Mixing chamber is 

recommended but not required; 2) Aeration 

is not recommended for mixing;3) A 

demonstration that the test solution is 

completely mixed before introduced into the 

test system is recommended; 4) The 

recommended flow splitting accuracy is 

within 10%.

Renewal period for static renewal (if applicable):

NA If static renewal is used, EPA recommends 

24-hr renewal; renewal period is 

recommended not to exceed 72 hours.

Aeration? No EPA recommends maintaining dissolved 

oxygen concentrations 

>40% air saturation 

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

bubblers at levels that do not cause stress 

on the tadpoles.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Source of dilution water: Dilution water was freshwater 

obtained from a well approximately 40 meters deep located on the Wildlife International, Ltd. Site. Water was passed through a sand filter to remove particles greater than ~25 μm and aerated with spray nozzles. Prior to use, the water was filtered (0.45 μm) and passed through a UV sterilizer. 

EPA recommends natural or reconstituted 

water; it is recommended that natural water 

be sterilized with UV and tested for 

pesticides, heavy metals, and other 

possible contaminants, including known 

substrates of the iodine transporter of the 

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

perchlorate). OECD accepts any water in 

which the test species show control survival 

at least as good as indicated in the test 

guideline.

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

Yes TOC was <1 mg/L. No 

pesticides or organic were detected. See Reviewer’s Comments section for results on metals. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Iodide supplementation in water?

No If reconstituted water is used or if 

background levels of iodide in natural water 

are less than 0.5 

μg/L, iodide 

supplementation is recommended. This 

supplementation is in addition to the 

recommended dietary source of iodide 

(e.g.. in Sera Micron).

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

water-contact portions of the system not 

compromise the study (e.g., all glass 

vessels or glass vessels with stainless steel 

frames are acceptable examples).

Test vessel size: 12 LFill volume: 10 LAdditional details on exposure system: 

None 

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

Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Hardness (mg/L as CaCO3) 136 144 142 

Weekly in the control 

and highest 

treatment group. 

EPA recommends hardness 40 to 48 

mg/L as CaCO3.

pH 8.1 8.2 8.1 

Weekly in all test 

vessels. 

EPA recommends pH 7.5 ± 1, 

inter-replicate and inter-treatment 

differentials should not exceed 0.5.

Dissolved oxygen (mg/L) 7.2 8.4 7.7 Weekly in all test 

vessels. 

EPA recommends dissolved oxygen (DO) 

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

recommends DO concentration >3.5 mg/L

(>40% air saturation).

Temperature 21.1 22.2 21.9 

Weekly in all test 

vessels. 

Continuously in one 

negative control 

replicate. 

EPA recommends temperature 22±1oC; 

inter-replicate and inter-treatment 

differentials should not exceed 0.5oC.

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Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Iodide (μg/L) 3 6 Not Reported Period 

EPA recommends aquatic iodide range 0.5 

– 10 

μg/L (supplemental iodide should not 

exceed 2 

μg/L).

Ammonia Not reported General recommendations for frequency of 

measurements: EPA recommends that 

water quality parameters be measured in a 

control and at one test item concentration 

at least weekly. In static renewal systems, 

water quality parameters, including 

ammonia, should be measured just prior to 

renewal. In addition, EPA recommends that 

DO be measured at each concentration at 

least weekly and that temperature be 

measured continuously. OECD recommends that DO and temperature be 

measured at least weekly and that pH and 

hardness be measured at least at the 

Fluoride Not reported Perchlorate Not reported Chlorate Not reported Alkalinity (mg/L as CaCO3)Conductance (μS/cm)

174 

357 

182 

380 

180 

368 

Weekly in the 

negative control and 

highest treatment 

group. 

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Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

beginning and end of the test.

 D. Study Design and Additional Experimental Conditions

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

Was a range-finder conducted? YesIf yes, what was the method for determining the highest test concentration in the range-finder? 

Not specified EPA recommends that the highest test 

concentration is either the solubility limit of the 

test compound, 100 mg/L, or demonstrates 

adequate evidence of toxicity (e.g., 

<10% 

mortality), whichever concentration is lowest.

Species: Xenopus laevis Life stage: NF 51 Test duration: 96 hours

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Additional details: A 96-hr range-finding 

study was conducted under flow-through conditions with nominal concentrations of 0.13, 0.41, 1.4, 4.5, 15, and 50 mg a.i./L with NF 51 tadpoles. 

The resulting LC50 was 27 mg a.i./L and 

the highest does in the AMA was set as one-third of the LC50 to approximate the 

maximum tolerated dose (9.0 mg a.i./L). Two additional test concentrations were selected with a 10X separation factor (0.9 and 0.09 mg a.i./L).

Table 7: Definitive Study Conditions.

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

definitive test be 21 days. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

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

One-third of the calculated LC50 from the 

preliminary range-finding study.

EPA recommends that the highest test 

concentration is either the solubility limit of the 

test compound, 100 mg/L, or demonstrates 

adequate evidence of toxicity (e.g., 

<10% 

mortality), whichever concentration is lowest.

Reference study citation (if applicable):

None provided 

Separation of test concentrations: 0.1 EPA recommends that the maximum 

concentration separation be 0.1 and the 

minimum be 0.33. 

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

concentrations and a control, plus solvent 

control if appropriate.

Are nominal concentrations adjusted for purity? 

Yes 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Indicate the type of values presented for measured concentrations: 

Mean-measured 

Limit of quantification (LOQ): 0.0500 mg a.i./L EPA recommends that for chemical test 

concentrations below the LOQ, analyses be 

conducted on the stock solutions.

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

for analysis on Days 0, 7, 14, and 21

It is recommended that test item concentration 

be measured in one tank at each treatment 

level at test initiation and every week 

thereafter.

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

NA EPA and OECD recommend the use of a 

concurrent solvent control when a solubilizing 

agent is used. EPA recommends 4 replicates.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Number of replicates per test item treatment level: 

4 EPA recommends 4 replicates.

Number of larvae per treatment at test initiation: 

80 (20/rep) 

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

NA EPA recommends that the solvent not exceed 

0.02 ml/L3. OECD recommends that solvent 

have no effect on survival nor produce any 

other adverse effects and that concentration 

not be greater than 0.1 ml/L4

.

Was a positive control used? No 

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

Toxicology, 76, pp.69–92. 

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

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Positive control (if applicable): NA Positive control concentration(s) (if applicable): 

NA 

Photoperiod: 12 hrs light : 

12 hrs dark 

A 30-minute transition period was used between light and dark cycles. 

EPA recommends photoperiod 12:12 

(light:dark).

Light intensity at water’s surface: 0.801.68 Klux EPA recommends light intensity 0.6 – 2 Klux 

(at water’s surface).

Additional details: Test solutions reported as clear and 

colorless with no evidence of precipitate 

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

Treatment ID

Nominal 

Concentration 

(mg a.i./L) 

Measured 

Concentration 

(mg a.i./L) 

Mean CV (%) Details or Remarks Guideline Recommendations 

Negative Control 0 <LOQ NA None EPA and OECD recommend that test item 

concentrations be maintained at a coefficient of 

variation (CV) 

≤20%.

Treatment 1 0.09 0.075 3.4 None Treatment 2 0.90 0.79 3.3 None Treatment 2 9.0 8.4 1.6 None 

Abbreviations: CV Coefficient of variation. 

 E. Observations

Biological Endpoints: Development stage, hind-limb length (HLL), normalized HLL, snout-vent length (SVL), and wet weight were assessed on Days 

7 and 21. Histopathology was performed on tadpoles collected on Day 21. Observations for mortality and sub-lethal effects were performed daily. 

Were raw (individual) data provided? Yes

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

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

hind limb length (Days 7 and 21); snout-vent length (Days 7 and 21); body weight (test initiation, for optional size-based larval selection); and thyroid 

gland gross pathology and histopathology (Day 21). Note the histopathology section of the test guideline also includes thyroid gross pathology observations.

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II. RESULTS AND DISCUSSION 

A. Results

By Day 7, a single mortality occurred in the negative control and complete survival was observed in the 

remaining treatment groups (Table 9). By Day 21, no additional mortalities occurred in the control or 

mean-measured 0.075 mg a.i./L treatment group. Mortality was 3.3 and 6.6% in the mean-measured 

0.79 and 8.4 mg a.i./L treatment groups, respectively, on Day 21. 

Table 9: Larval Mortality in Xenopus laevis.

Treatment (mg a.i./L) 

[Mean-Measured]

Larval Mortality 

Day 71 Day 21 

n Mortality # Mortality % n Mortality # Mortality % 

Negative Control 80 1 1.3 60 0 0 

0.075 80 0 0 60 0 0 

0.79 80 0 0 60 2 3.3 

8.4 80 0 0 60 42 6.6 

1

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

2

 Two mortalities in both replicates A and D. 

Median NF stage was 54 and 57 on Day 7 and 21, respectively, in the control and all treatment groups. 

Individual tadpole stage ranged from 52 to 55 on Day 7 and 55 to 63 on Day 21 (Table 10). No 

asynchronous development was observed in any treatment group. The 10th and 90th percentiles were 53 

and 54, respectively, on Day 7, and 56 and 60, respectively, on Day 21. 

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Table 10: Larval Development in Xenopus laevis – Developmental Stage and Asynchronous Development.

Treatment (mg a.i./L) 

[mean-measured]

Developmental Stage 

Day 7 Day 21 

n 

Median 

Stage 

# Asynchronous n 

Median 

Stage 

# Asynchronous 

Negative Control 4 54 0 4 57 0 

0.075 4 54 0 4 57 0 

0.79 4 54 0 4 57 0 

8.4 4 54 0 4 57 0 

Normalized HLL on Day 7 averaged 0.14 in the negative control and 0.16, 0.15, and 0.16 in the 

mean-measured 0.075, 0.79, and 8.4 mg a.i./L treatment groups, respectively (Table 11). Normalized 

HLL on Day 21 averaged 0.36 in the negative control and 0.33, 0.36, and 0.28 in the mean-measured 

0.075, 0.79, and 8.4 mg a.i./L treatment groups, respectively. 

Table 11: Larval Development in Xenopus laevis – Hind Limb Length.

Treatment 

(mg a.i./L) 

[mean-measured] 

Hind Limb Length (HLL) 

Day 7 Day 21 

n Mean

(mm) 

±SD 

HLL: 

SVL1 n Mean(mm) 

±SD

HLL: 

SVL1

Negative Control 4 2.30 0.33 0.14 4 7.90 0.47 0.36 

0.075 4 2.55 0.32 0.16 4 7.53 0.39 0.33 

0.79 4 2.47 0.08 0.15 4 8.00 0.97 0.36 

8.4 4 2.49 0.27 0.16 4 6.10 0.84 0.28 

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Abbreviations: SD Standard deviation. 

1

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

On Day 7, SVL ranged from 15.6 to 16.8 mm (Table 12). By Day 21, SVL ranged from 22.0 to 22.5 

mm. On Day 7, wet weight ranged from 0.262 to 0.336 g (Table 12). By Day 21, wet weight ranged 

from 0.797 to 0.898 g. 

Table 12: Larval Growth in Xenopus laevis.

Treatment 

(mg a.i./L) 

[mean-measured] 

Snout-Vent Length (SVL) Body Weight1

Day 7 Day 21 Day 7 Day 21 

n Mean

(mm) 

±SD n Mean

(mm) 

±SD n Mean

(g) 

±SD n Mean

(g) 

±SD 

Negative Control 4 16.8 0.24 4 22.0 0.71 4 0.322 0.02 4 0.807 0.06 

0.075 4 16.1 0.31 4 22.5 0.49 4 0.291 0.02 4 0.872 0.04 

0.79 4 16.8 0.52 4 22.4 0.60 4 0.336 0.04 4 0.898 0.07 

8.4 4 15.6 0.20 4 22.0 0.70 4 0.262 0.01 4 0.797 0.06 

Abbreviations: SD Standard deviation. 

1

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

There were incidences of hypertrophy and atrophy of the thyroid gland, as well as follicular cell hypertrophy 

in the control and all treatment groups (Table 13). However, incidence of observation was comparable 

between the control and all treatment groups. 

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Table 13: Gross Pathology and Histopathology of the Thyroid Gland in Xenopus laevis.

Treatment 

(mg a.i./L)

[meanmeasured]

Diagnostic Observations1

Severity

Thyroid Gland 

Hypertrophy 

Thyroid Gland 

Atrophy 

Follicular Cell 

Hypertrophy 

Follicular Cell 

Hyperplasia 

n Incidence n Incidence n Incidence n Incidence 

Negative Control 0 20 17 20 17 20 17 NA NA 

1 20 1 20 3 20 2 NA NA

2 20 1 20 0 20 1 NA NA

3 20 1 20 0 20 0 NA NA

0.075 0 20 15 20 17 20 19 NA NA 

1 20 5 20 3 20 1 NA NA

2 20 0 20 0 20 0 NA NA

3 20 0 20 0 20 0 NA NA

0.79 0 20 16 20 19 20 20 NA NA 

1 20 3 20 1 20 0 NA NA

2 20 1 20 0 20 0 NA NA

3 20 0 20 0 20 0 NA NA

8.4 0 20 14 20 20 20 15 NA NA 

1 20 3 20 0 20 3 NA NA

2 20 1 20 0 20 2 NA NA

3 20 2 20 0 20 0 NA NA

Abbreviations: NA Not applicable. 

1

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

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

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Table 14: Additional Thyroid Gland Histopathology Observations in Xenopus laevis. 

Treatment 

(mg a.i./L) 

[mean-measured] 

Additional Qualitative Observations1

Severity

Follicular Lumen 

Area (Increase) 

Follicular Lumen 

Area (Decrease) 

Follicular Cell 

Height (Increase) 

Follicular Cell Height 

(Decrease) 

Follicular Cell 

Shape 

n Incidence n Incidence n Incidence n Incidence n Incidence 

Negative Control 0 NA NA NA NA NA NA NA NA 20 18 

1 NA NA NA NA NA NA NA NA 20 2 

2 NA NA NA NA NA NA NA NA 20 0 3 NA NA NA NA NA NA NA NA 20 0 

0.075 0 NA NA NA NA NA NA NA NA 20 20 

1 NA NA NA NA NA NA NA NA 20 0 

2 NA NA NA NA NA NA NA NA 20 0 3 NA NA NA NA NA NA NA NA 20 0 

0.79 0 NA NA NA NA NA NA NA NA 20 20 

1 NA NA NA NA NA NA NA NA 20 0 

2 NA NA NA NA NA NA NA NA 20 0 3 NA NA NA NA NA NA NA NA 20 0 

8.4 0 NA NA NA NA NA NA NA NA 20 16 

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Treatment 

(mg a.i./L) 

[mean-measured] 

Additional Qualitative Observations1

Severity

Follicular Lumen 

Area (Increase) 

Follicular Lumen 

Area (Decrease) 

Follicular Cell 

Height (Increase) 

Follicular Cell Height 

(Decrease) 

Follicular Cell 

Shape 

n Incidence n Incidence n Incidence n Incidence n Incidence 

1 NA NA NA NA NA NA NA NA 20 4 

2 NA NA NA NA NA NA NA NA 20 0 3 NA NA NA NA NA NA NA NA 20 0 

Abbreviations: NA Not applicable. 1 Thyroid histopathology is graded 0 – 3 based on severity: 0=Not remarkable, 1=Mild, 2=Moderate, 3=Severe. See OECD No. 82 for reference. 

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Occasional observations of a few tadpoles that appeared lethargic or were surfacing were noted in the high treatment group through Day 8 of the test (Table 15). However, no sublethal effects were noted throughout the remainder of the test. Beginning on Day 4, an increasing number of tadpoles in the control and all treatment groups had curved tails. By test termination, 66, 85, 98. And 48% of the surviving tadpoles had crooked tails in the negative control and mean-measured 0.075, 0.79, and 8.4 mg a.i./L treatment groups, respectively. At test termination, tail curvature was scored (from 0-3) for each tadpole, and there were 32, 28, 45 and 4% of tadpoles in the control, 0.075, 0.79 and 8.4 mg a.i./L that were considered to have tail curvature with a score 

≥2. 

Table 15: Clinical Signs in Xenopus laevis.

Treatment 

(mg a.i./L) 

[meanmeasured] 

Clinical Signs1

Type n Incidence2

Negative Control Crooked tail 59 39 

0.075 Crooked tail 60 51 

0.79 Crooked tail 58 57 

8.4 Crooked tail 56 27 

1 Note that asynchronous development (unable to stage) is reported previously in Table 102 Based on Day 21 observations 

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B. Study Author’s Analysis and Conclusions

The study authors analyzed survival, developmental stage, body weight, whole body length, SVL, 

normalized HLL, and incidence and severity of thyroid abnormalities. If responses appeared to be 

monotonic, a step-down Jonckheere-Terpstra trend test was used to evaluate trends in the ranks of 

replicate means to determine possible concentration-responsive trends among the treatment groups. All 

endpoints, except for survival, development stage, and histopathology severity scores also were analyzed 

by performing pair-wise comparisons using Dunnett’s multiple comparison test to determine which 

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

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

(α = 0.01). When the data did not satisfy the assumptions, a log transformation was applied to correct 

the data for non-normality or heterogeneous variances. 

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

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

for these endpoints was the individual animal. Therefore, survival was analyzed using the step-down 

Jonckheere-Terpstra trend test and Fisher’s Exact test, and histopathology severity scores of individuals 

were analyzed using step-down Jonckheere-Terpstra trend tests only. In accordance with 

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

using the multi-quantile analyses developed by T. Springer and J. Green, and using the step-down 

Jonckheere-Terpstra trend test on stage quantiles in the replicate test chambers. Statistical tests used 

to evaluate treatment effects were performed at α of 0.05 with SAS software. 

Treatment-related effects were limited to the mean-measured 8.4 mg a.i./L treatment group with 

significant reductions in SVL on Day 7 and reductions in normalized HLL on Day 21. There were no other 

significant differences in developmental or growth parameters. Further, there were no apparent 

treatment-related trends in histopatholgoical observations. 

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 C. Reviewer’s Analysis and Conclusions 

Statistical Methods: Mortality did not exceed 7% in any of the treatment groups. Therefore, the reviewer 

visually determined that the test material had no effects on larval survival. All Day 7 endpoints, except 

developmental stage, were non-monotonic with normal distributions and homogenous variances and were 

therefore analyzed with Dunnett’s test. Day 7 developmental stage was non-monotonic but did not meet 

the assumptions of parametric statistics and was analyzed using the Mann-Whitney test. All Day 21 growth 

parameters (i.e., wet weight, SVL, HLL) were non-monotonic but met the assumptions of parametric 

statistics and these variables were analyzed using Dunnett’s test. Both Day 21 development parameters 

(i.e., NF stage, normalized HLL) were analyzed using the Mann-Whitney test based on a lack of 

monotonicity and the failure to meet the assumptions of parametric statistics. There was no asynchronous 

development. There were late stage tadpoles present by Day 21 in the control and all metolachlor-treated 

groups, comprising 2-6% of the total sample size, with NF stages ranging from 61-63; there was no 

apparent pattern in late stage development or evidence of dose-dependency. Late stage tadpoles were 

excluded from the analysis of 21-day wet weight, SVL, HLL, and normalized HLL. Unless otherwise 

indicated, effects were considered statistically significant at p < 0.05. 

 Conclusions: 

Throughout the test, no effects were observed on NF developmental stage. For the 8.4 mg a.i./L 

treatment, there was a significant 23% reduction in Day 21 normalized HLL (p<0.05) from the negative 

control (Table 16). Growth parameters, SVL and wet weight, were significantly reduced (p<0.05) 7 and 

19%, respectively, on Day 7 and HLL was significantly reduced (p<0.05) 23% on Day 21 in the highest 

treatment group relative to the negative control (Table 17). There were no clear treatment-related effects 

in the histopathological observations. 

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Table 16: Developmental and Thyroid Gross Pathology/Histopathology Endpoints1,2 in the AMA with Metolachlor 4. 

Treatment 

(mg a.i./L) 

[mean-measured]

NF Developmental Stage Hind Limb Length3 Asynchronous 

Development 

Thyroid Gross and 

Histopathology 

Day 7 Day 21 Day 7 Day 21 Day 7 Day 21 Day 21 

Median p Median p 

% 

Diff. 

p 

% 

Diff. 

p 

% 

Diff

. 

p 

% 

Diff. 

p 

Treatment-Related 

Effects? (Yes/No) 

Negative Control 54 NA 57 NA NA NA NA NA NA NA NA NA No 

0.075 54 >0.99 57 >0.99 15 0.25 -8.3 0.12 NA NA NA NA No 

0.79 54 0.48 58 0.23 7.3 0.73 -2.1 0.48 NA NA NA NA No 

8.4 54 0.48 57 >0.99 16 0.18 -23 0.029 NA NA NA NA No 

Statistical Test Mann-Whitney Mann-Whitney Dunnett’s Mann-Whitney NA NA NA 

Abbreviations: Diff. Difference. NA Not applicable.

1 Unless otherwise indicated, effects are reported based on comparison to the clean water control. Conclusions regarding histopathology may be heavily weighted 

by the expert opinion of a board-certified pathologist. 

2 Unless otherwise specified, effects are considered statistically significant at 

p < 0.05. 

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

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4 Several tadpoles were excluded from the analysis of Day 21 endpoints for wet weight, SVL, HLL, and norm HLL. Excluded individuals comprised 2, 4, 6, and 

3% of the control, 0.075, 0.79, and 8.4 mg ai/L treatment group total sample sizes, respectively. Late stage individuals ranged from NF 61 to 63 with no 

apparent pattern of occurrence or dose-dependency. 

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 Table 17: Growth Endpoints1,2 in the AMA with Metolachlor3. 

Treatment 

(mg a.i./L) 

[mean-measured]

Snout-Vent Length Body Weight 

Day 7 Day 21 Day 7 Day 21 

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

Negative Control NA NA NA NA NA NA NA NA 

0.075 -3.9 0.052 2.0 0.63 -9.6 0.25 8.0 0.31 

0.79 0.2 0.999 1.8 0.71 4.2 0.80 11.3 0.11 

8.4 -7.0 0.001 -0.5 0.99 -18.7 0.02 -1.2 0.99 

Statistical Test Dunnett’s Dunnett’s Dunnett’s Dunnett’s 

 Abbreviations: Diff. Difference. NA Not applicable.

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

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

 3 Several tadpoles were excluded from the analysis of Day 21 endpoints for wet weight, SVL, HLL, and norm 

HLL. Excluded individuals comprised 2, 4, 6, and 3% of the control, 0.075, 0.79, and 8.4 mg ai/L treatment 

group total sample sizes, respectively. Late stage individuals ranged from NF 61 to 63 with no apparent 

pattern of occurrence or dose-dependency. 

E. Study Deficiencies 

The high prevalence of crooked tail/tail curvature across treatments raises some concerns about the 

general health and husbandry of the test specimens, although environmental conditions and feeding were 

generally consistent with the guideline recommendations. 

Deviations are listed in Section I. Materials and Methods of this DER. These deviations do 

not impact the interpretation of the study. 

F. Reviewer’s Comments 

The reviewer’s analyses were similar to the study authors, however, the study author reported significant 

decreases in SVL (p<0.05) at the highest treatment level on Day 21 whereas the reviewer’s analysis 

did not. 

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Results from the periodic screening analysis of the dilution water detected the following analytes: calcium 

(35.1 mg/L), chloride (3.5 mg/L), magnesium (13.0 mg/L), nitrate (0.55 mg N/L), potassium (6.77 

mg/L), sodium (18.5 mg/L), and sulfate (5.6 mg/L). 

Day 21 normalized HLL was significantly reduced at the top dose (23%), relative to the negative control. 

III. REFERENCES 

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

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

Publishing Co, New York, NY USA. 

Organization for Economic Cooperation and Development. 2006. Current Approaches in the Statistical 

Analysis of Ecotoxicity Data: A Guidance to Application. OECD ENV/JM/MONO(2008)18. 

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

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

and Company, Wilmington, DE 19898, USA. 

The SAS System for Windows. 1999-2001. Version 8.2. SAS Institute, Inc., Cary, North Carolina. 

 

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APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION: 

Endpoint Monotonic? Parametric? 890.1100 EFED Comments 7-day wet weight No Yes Dunnett’s: 

Dose 3 p=0.015 

Dunnett’s: Dose 3 p=0.015 

890.1100 and EFED same conclusions, significant 19% reduction (p>0.05) at the 8.4 mg ai/L level 

7-stage median No No Mann-Whitney: 

n.s. p>0.05 

Mann-Whitney: n.s. p>0.05 

890.1100 and EFED same conclusions, no effect (p>0.05) 

7-day SN-vent length No Yes Dunnett’s: 

Dose 3 p=0.001 

Dunnett’s: Dose 3 p=0.001 

890.1100 and EFED same conclusions, significant 7% reduction (p>0.05) at the 8.4 mg ai/L level 

7-day hind-limb length 

No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

890.1100 and EFED same conclusions, no effect (p>0.05) 

7-day normalized HLL 

No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

890.1100 and EFED same conclusions, no effect (p>0.05) 

21-day stage median No No Mann-Whitney: 

n.s. p>0.05 

Mann-Whitney: n.s. p>0.05 

890.1100 and EFED same conclusions, no effect (p>0.05) 

21-day wet weight No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

890.1100 and EFED same conclusions, no effect (p>0.05) 

21-day SN-vent length 

No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

890.1100 and EFED same conclusions, no effect (p>0.05) 

21-day HLL No Yes Dunnett’s: 

Dose 3 p=0.010 

Dunnett’s: Dose 3 p=0.010 

890.1100 and EFED same conclusions, significant 23% reduction (p>0.05) at the 8.4 mg ai/L level 

21-day normalized HLL 

No No Mann-Whitney: 

Dose 3 p=0.029 

Mann-Whitney: Dose 3 p=0.029 

890.1100 and EFED same conclusions, significant 23% reduction (p>0.05) at the 8.4 mg ai/L level 

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test for amphib metamorph screen study - metolachlor 

ANALYSIS RESULTS FOR VARIABLE VAR01 ( 7-d wet 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 

 0.954 0.555 2.475 0.112 USE PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 0.32 0.02 0.01 5.29 0.30, 0.35 

 Dose1 4 0.29 0.02 0.01 6.31 0.26, 0.32 

 Dose2 4 0.34 0.04 0.02 12.62 0.27, 0.40 

 Dose3 4 0.26 0.01 0.01 4.72 0.24, 0.28 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 0.32 0.31 0.35 . . 

 Dose1 0.29 0.28 0.32 90.38 9.62 

 Dose2 0.33 0.29 0.39 104.19 -4.19 

 Dose3 0.26 0.25 0.28 81.30 18.70 

******************************************************************************** 

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 

 3 12 6.76 0.006 

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 0.32 . 0.32 . . . . . . 

 Dose1 0.29 0.248 0.31 0.378 . . . . . 

 Dose2 0.34 0.795 0.31 0.404 0.114 . . . . 

 Dose3 0.26 0.015 0.26 0.003 0.399 0.007 . . . 

******************************************************************************** 

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 9.92 0.019 

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 0.32 . . 

 Dose1 0.29 0.154 0.041 

 Dose2 0.33 0.780 0.559 

 Dose3 0.26 0.067 0.025 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams Dose3 

 Jonckheere Dose3 

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******************************************************************************** 

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 6.76 0.006 

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 -0.32 . -0.30 . . . . . . 

 Dose1 -0.29 0.248 -0.30 0.911 . . . . . 

 Dose2 -0.34 0.795 -0.30 0.931 0.114 . . . . 

 Dose3 -0.26 0.015 -0.30 0.941 0.399 0.007 . . . 

******************************************************************************** 

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 9.92 0.019 

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 -0.32 . . 

 Dose1 -0.29 0.154 0.959 

 Dose2 -0.33 0.780 0.441 

 Dose3 -0.26 0.067 0.975 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

test for amphib metamorph screen study - metolachlor 

ANALYSIS RESULTS FOR VARIABLE VAR02 ( 7-d stage (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 

 0.677 <.001 6.000 0.010 USE NON-PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 53.75 0.50 0.25 0.93 52.95, 54.55 

 Dose1 4 53.75 0.50 0.25 0.93 52.95, 54.55 

 Dose2 4 54.00 0.00 0.00 0.00 . , . 

 Dose3 4 54.00 0.00 0.00 0.00 . , . 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 54.00 53.00 54.00 . . 

 Dose1 54.00 53.00 54.00 100.00 0.00 

 Dose2 54.00 54.00 54.00 100.47 -0.47 

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 Dose3 54.00 54.00 54.00 100.47 -0.47 

******************************************************************************** 

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 0.67 0.588 

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 53.75 . 53.88 . . . . . . 

 Dose1 53.75 1.000 53.88 0.773 . . . . . 

 Dose2 54.00 0.638 53.88 0.805 0.752 . . . . 

 Dose3 54.00 0.638 53.88 0.822 0.752 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 2.14 0.543 

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 54.00 . . 

 Dose1 54.00 1.000 0.500 

 Dose2 54.00 0.478 0.818 

 Dose3 54.00 0.478 0.905 

 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 0.67 0.588 

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 -53.75 . -53.75 . . . . . . 

 Dose1 -53.75 1.000 -53.75 0.583 . . . . . 

 Dose2 -54.00 0.638 -54.00 0.215 0.752 . . . . 

 Dose3 -54.00 0.638 -54.00 0.222 0.752 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 

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

 EPA MRID Number 48617810

Page 4 of 61

DER Template Version: 22 September 2011 

 3 2.14 0.543 

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 -54.00 . . 

 Dose1 -54.00 1.000 0.500 

 Dose2 -54.00 0.478 0.182 

 Dose3 -54.00 0.478 0.095 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

test for amphib metamorph screen study - metolachlor 

ANALYSIS RESULTS FOR VARIABLE VAR03 ( 7-d sn-vent 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 

 0.955 0.574 0.939 0.452 USE PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 16.78 0.25 0.13 1.49 16.38, 17.17 

 Dose1 4 16.13 0.33 0.17 2.05 15.60, 16.65 

 Dose2 4 16.80 0.51 0.25 3.04 15.99, 17.61 

 Dose3 4 15.60 0.22 0.11 1.38 15.26, 15.94 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 16.90 16.40 16.90 . . 

 Dose1 16.10 15.80 16.50 96.13 3.87 

 Dose2 16.70 16.30 17.50 100.15 -0.15 

 Dose3 15.65 15.30 15.80 93.00 7.00 

******************************************************************************** 

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 

 3 12 11.08 <.001 

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 16.78 . 16.78 . . . . . . 

 Dose1 16.13 0.052 16.46 0.134 . . . . . 

 Dose2 16.80 0.999 16.46 0.143 0.071 . . . . 

 Dose3 15.60 0.001 15.60 <.001 0.194 0.002 . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

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

 EPA MRID Number 48617810

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DER Template Version: 22 September 2011 

 Degrees of Freedom TestStat P-value 

 3 11.50 0.009 

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 16.90 . . 

 Dose1 16.10 0.096 0.019 

 Dose2 16.70 0.671 0.356 

 Dose3 15.65 0.062 0.007 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams Dose3 

 Jonckheere Dose3 

******************************************************************************** 

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 11.08 <.001 

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 -16.78 . -16.33 . . . . . . 

 Dose1 -16.13 0.052 -16.33 0.980 . . . . . 

 Dose2 -16.80 0.999 -16.33 0.987 0.071 . . . . 

 Dose3 -15.60 0.001 -16.33 0.989 0.194 0.002 . . . 

******************************************************************************** 

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 11.50 0.009 

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 -16.90 . . 

 Dose1 -16.10 0.096 0.981 

 Dose2 -16.70 0.671 0.644 

 Dose3 -15.65 0.062 0.993 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

test for amphib metamorph screen study - metolachlor 

ANALYSIS RESULTS FOR VARIABLE VAR04 ( 7-d hind-limb 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 

Page 46 of 288
Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

 EPA MRID Number 48617810

Page 6 of 61

DER Template Version: 22 September 2011 

 Test Stat P-value Test Stat P-value 

 0.946 0.435 1.065 0.400 USE PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 2.30 0.33 0.16 14.20 1.78, 2.82 

 Dose1 4 2.55 0.32 0.16 12.53 2.04, 3.06 

 Dose2 4 2.47 0.08 0.04 3.16 2.34, 2.59 

 Dose3 4 2.49 0.27 0.14 10.97 2.06, 2.93 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 2.35 1.86 2.65 . . 

 Dose1 2.64 2.09 2.82 110.76 -10.76 

 Dose2 2.47 2.37 2.56 107.28 -7.28 

 Dose3 2.42 2.25 2.88 108.37 -8.37 

******************************************************************************** 

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 0.63 0.611 

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 2.30 . 2.45 . . . . . . 

 Dose1 2.55 0.451 2.45 0.856 . . . . . 

 Dose2 2.47 0.715 2.45 0.882 0.974 . . . . 

 Dose3 2.49 0.630 2.45 0.895 0.991 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 2.26 0.520 

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 2.35 . . 

 Dose1 2.64 0.346 0.876 

 Dose2 2.47 0.346 0.721 

 Dose3 2.42 0.494 0.663 

 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 0.63 0.611 

Dunnett - testing each trt mean signif. different than control 

Page 47 of 288
Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

 EPA MRID Number 48617810

Page 7 of 61

DER Template Version: 22 September 2011 

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 -2.30 . -2.30 . . . . . . 

 Dose1 -2.55 0.451 -2.50 0.184 . . . . . 

 Dose2 -2.47 0.715 -2.50 0.196 0.974 . . . . 

 Dose3 -2.49 0.630 -2.50 0.203 0.991 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 2.26 0.520 

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 -2.35 . . 

 Dose1 -2.64 0.346 0.124 

 Dose2 -2.47 0.346 0.279 

 Dose3 -2.42 0.494 0.337 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

test for amphib metamorph screen study - metolachlor 

ANALYSIS RESULTS FOR VARIABLE VAR05 ( 7-d norm hind-limb ) 

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 

 0.938 0.325 0.414 0.746 USE PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 0.14 0.02 0.01 14.99 0.10, 0.17 

 Dose1 4 0.16 0.02 0.01 12.02 0.13, 0.19 

 Dose2 4 0.15 0.01 0.00 6.49 0.13, 0.16 

 Dose3 4 0.16 0.01 0.01 8.84 0.14, 0.18 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 0.14 0.11 0.16 . . 

 Dose1 0.17 0.13 0.17 114.55 -14.55 

 Dose2 0.15 0.14 0.16 107.27 -7.27 

 Dose3 0.16 0.15 0.18 116.36 -16.36 

******************************************************************************** 

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 1.57 0.247 

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

 EPA MRID Number 48617810

Page 8 of 61

DER Template Version: 22 September 2011 

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 0.14 . 0.15 . . . . . . 

 Dose1 0.16 0.248 0.15 0.919 . . . . . 

 Dose2 0.15 0.727 0.15 0.938 0.824 . . . . 

 Dose3 0.16 0.177 0.15 0.947 0.996 0.709 . . . 

******************************************************************************** 

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 4.00 0.262 

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 0.14 . . 

 Dose1 0.17 0.278 0.907 

 Dose2 0.15 0.555 0.701 

 Dose3 0.16 0.185 0.892 

 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 1.57 0.247 

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 -0.14 . -0.14 . . . . . . 

 Dose1 -0.16 0.248 -0.15 0.131 . . . . . 

 Dose2 -0.15 0.727 -0.15 0.139 0.824 . . . . 

 Dose3 -0.16 0.177 -0.16 0.048 0.996 0.709 . . . 

******************************************************************************** 

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 4.00 0.262 

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 -0.14 . . 

 Dose1 -0.17 0.278 0.093 

Page 49 of 288
Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

 EPA MRID Number 48617810

Page 9 of 61

DER Template Version: 22 September 2011 

 Dose2 -0.15 0.555 0.299 

 Dose3 -0.16 0.185 0.108 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams Dose3 

 Jonckheere >highest dose (no sign. differences) 

test for amphib metamorph screen study - metolachlor 

ANALYSIS RESULTS FOR VARIABLE VAR06 ( 21-d stage (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 

 0.654 <.001 13.500 <.001 USE NON-PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 57.00 0.00 0.00 0.00 . , . 

 Dose1 4 57.00 0.00 0.00 0.00 . , . 

 Dose2 4 57.75 0.96 0.48 1.66 56.23, 59.27 

 Dose3 4 57.00 0.00 0.00 0.00 . , . 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 57.00 57.00 57.00 . . 

 Dose1 57.00 57.00 57.00 100.00 0.00 

 Dose2 57.50 57.00 59.00 101.32 -1.32 

 Dose3 57.00 57.00 57.00 100.00 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 

 3 12 2.45 0.113 

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 57.00 . 57.25 . . . . . . 

 Dose1 57.00 1.000 57.25 0.841 . . . . . 

 Dose2 57.75 0.113 57.25 0.869 0.174 . . . . 

 Dose3 57.00 1.000 57.00 0.636 1.000 0.174 . . . 

******************************************************************************** 

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 6.40 0.094 

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 57.00 . . 

Page 50 of 288
Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

 EPA MRID Number 48617810

Page 10 of 61

DER Template Version: 22 September 2011 

 Dose1 57.00 1.000 . 

 Dose2 57.50 0.227 0.965 

 Dose3 57.00 1.000 0.743 

 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 2.45 0.113 

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 -57.00 . -57.00 . . . . . . 

 Dose1 -57.00 1.000 -57.00 0.583 . . . . . 

 Dose2 -57.75 0.113 -57.38 0.184 0.174 . . . . 

 Dose3 -57.00 1.000 -57.38 0.191 1.000 0.174 . . . 

******************************************************************************** 

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 6.40 0.094 

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 -57.00 . . 

 Dose1 -57.00 1.000 . 

 Dose2 -57.50 0.227 0.035 

 Dose3 -57.00 1.000 0.257 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

test for amphib metamorph screen study - metolachlor 

ANALYSIS RESULTS FOR VARIABLE VAR07 ( 21-d wet 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 

 0.897 0.073 0.266 0.848 USE PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 0.81 0.06 0.03 7.17 0.71, 0.90 

 Dose1 4 0.87 0.04 0.02 4.08 0.81, 0.93 

Page 51 of 288
Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

 EPA MRID Number 48617810

Page 11 of 61

DER Template Version: 22 September 2011 

 Dose2 4 0.90 0.07 0.03 7.52 0.79, 1.01 

 Dose3 4 0.80 0.06 0.03 8.15 0.69, 0.90 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 0.80 0.75 0.89 . . 

 Dose1 0.87 0.84 0.91 107.99 -7.99 

 Dose2 0.89 0.83 0.99 111.31 -11.31 

 Dose3 0.77 0.75 0.89 98.76 1.24 

******************************************************************************** 

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 2.90 0.079 

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 0.81 . 0.86 . . . . . . 

 Dose1 0.87 0.310 0.86 0.937 . . . . . 

 Dose2 0.90 0.110 0.86 0.953 0.912 . . . . 

 Dose3 0.80 0.989 0.80 0.527 0.311 0.115 . . . 

******************************************************************************** 

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 5.85 0.119 

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 0.80 . . 

 Dose1 0.87 0.154 0.959 

 Dose2 0.89 0.101 0.972 

 Dose3 0.77 0.889 0.574 

 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 2.90 0.079 

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 -0.81 . -0.81 . . . . . . 

 Dose1 -0.87 0.310 -0.86 0.154 . . . . . 

Page 52 of 288
Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

 EPA MRID Number 48617810

Page 12 of 61

DER Template Version: 22 September 2011 

 Dose2 -0.90 0.110 -0.86 0.164 0.912 . . . . 

 Dose3 -0.80 0.989 -0.86 0.169 0.311 0.115 . . . 

******************************************************************************** 

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 5.85 0.119 

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 -0.80 . . 

 Dose1 -0.87 0.154 0.041 

 Dose2 -0.89 0.101 0.028 

 Dose3 -0.77 0.889 0.426 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

test for amphib metamorph screen study - metolachlor 

ANALYSIS RESULTS FOR VARIABLE VAR08 ( 21-d sn-vent 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 

 0.917 0.149 0.121 0.946 USE PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 22.05 0.70 0.35 3.20 20.93, 23.17 

 Dose1 4 22.50 0.49 0.24 2.18 21.72, 23.28 

 Dose2 4 22.45 0.58 0.29 2.58 21.53, 23.37 

 Dose3 4 21.95 0.72 0.36 3.27 20.81, 23.09 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 21.95 21.30 23.00 . . 

 Dose1 22.40 22.10 23.10 102.04 -2.04 

 Dose2 22.40 21.80 23.20 101.81 -1.81 

 Dose3 21.70 21.40 23.00 99.55 0.45 

******************************************************************************** 

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 0.78 0.529 

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 22.05 . 22.33 . . . . . . 

Page 53 of 288
Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

 EPA MRID Number 48617810

Page 13 of 61

DER Template Version: 22 September 2011 

 Dose1 22.50 0.632 22.33 0.813 . . . . . 

 Dose2 22.45 0.705 22.33 0.844 0.999 . . . . 

 Dose3 21.95 0.992 21.95 0.536 0.619 0.684 . . . 

******************************************************************************** 

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 3.54 0.316 

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 21.95 . . 

 Dose1 22.40 0.233 0.927 

 Dose2 22.40 0.494 0.880 

 Dose3 21.70 0.780 0.389 

 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 0.78 0.529 

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 -22.05 . -22.05 . . . . . . 

 Dose1 -22.50 0.632 -22.30 0.349 . . . . . 

 Dose2 -22.45 0.705 -22.30 0.373 0.999 . . . . 

 Dose3 -21.95 0.992 -22.30 0.386 0.619 0.684 . . . 

******************************************************************************** 

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 3.54 0.316 

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 -21.95 . . 

 Dose1 -22.40 0.233 0.073 

 Dose2 -22.40 0.494 0.120 

 Dose3 -21.70 0.780 0.611 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

test for amphib metamorph screen study - metolachlor 

Page 54 of 288
Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

 EPA MRID Number 48617810

Page 14 of 61

DER Template Version: 22 September 2011 

ANALYSIS RESULTS FOR VARIABLE VAR09 ( 21-d hind-limb 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 

 0.904 0.094 1.359 0.302 USE PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 7.90 0.47 0.23 5.93 7.16, 8.65 

 Dose1 4 7.53 0.39 0.19 5.12 6.91, 8.14 

 Dose2 4 8.00 0.97 0.49 12.18 6.45, 9.55 

 Dose3 4 6.10 0.84 0.42 13.83 4.76, 7.44 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 7.92 7.36 8.41 . . 

 Dose1 7.47 7.12 8.05 95.25 4.75 

 Dose2 7.63 7.32 9.44 101.27 -1.27 

 Dose3 5.90 5.35 7.24 77.16 22.84 

******************************************************************************** 

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 6.12 0.009 

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 7.90 . 7.90 . . . . . . 

 Dose1 7.53 0.800 7.77 0.467 . . . . . 

 Dose2 8.00 0.994 7.77 0.497 0.783 . . . . 

 Dose3 6.10 0.010 6.10 0.002 0.062 0.012 . . . 

******************************************************************************** 

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 8.81 0.032 

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 7.92 . . 

 Dose1 7.47 0.343 0.123 

 Dose2 7.63 0.889 0.385 

 Dose3 5.90 0.067 0.010 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams Dose3 

 Jonckheere Dose3 

Page 55 of 288
Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

 EPA MRID Number 48617810

Page 15 of 61

DER Template Version: 22 September 2011 

******************************************************************************** 

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 6.12 0.009 

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 -7.90 . -7.38 . . . . . . 

 Dose1 -7.53 0.800 -7.38 0.903 . . . . . 

 Dose2 -8.00 0.994 -7.38 0.924 0.783 . . . . 

 Dose3 -6.10 0.010 -7.38 0.934 0.062 0.012 . . . 

******************************************************************************** 

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 8.81 0.032 

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 -7.92 . . 

 Dose1 -7.47 0.343 0.877 

 Dose2 -7.63 0.889 0.615 

 Dose3 -5.90 0.067 0.990 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

test for amphib metamorph screen study - metolachlor 

ANALYSIS RESULTS FOR VARIABLE VAR10 ( 21-d norm hind-limb ) 

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 

 0.820 0.005 1.177 0.359 USE NON-PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 0.36 0.03 0.01 7.26 0.32, 0.40 

 Dose1 4 0.33 0.01 0.01 3.78 0.31, 0.35 

 Dose2 4 0.36 0.04 0.02 10.41 0.30, 0.41 

 Dose3 4 0.28 0.04 0.02 12.95 0.22, 0.33 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 0.36 0.34 0.40 . . 

 Dose1 0.33 0.32 0.35 91.72 8.28 

 Dose2 0.34 0.33 0.41 97.93 2.07 

 Dose3 0.27 0.25 0.33 76.55 23.45 

Page 56 of 288
Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

 EPA MRID Number 48617810

Page 16 of 61

DER Template Version: 22 September 2011 

******************************************************************************** 

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 6.73 0.006 

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 0.36 . 0.36 . . . . . . 

 Dose1 0.33 0.379 0.34 0.232 . . . . . 

 Dose2 0.36 0.969 0.34 0.248 0.711 . . . . 

 Dose3 0.28 0.004 0.28 <.001 0.090 0.014 . . . 

******************************************************************************** 

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 9.71 0.021 

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 0.36 . . 

 Dose1 0.33 0.123 0.029 

 Dose2 0.34 0.484 0.206 

 Dose3 0.27 0.067 0.005 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams Dose3 

 Jonckheere Dose3 

******************************************************************************** 

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 6.73 0.006 

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 -0.36 . -0.33 . . . . . . 

 Dose1 -0.33 0.379 -0.33 0.957 . . . . . 

 Dose2 -0.36 0.969 -0.33 0.969 0.711 . . . . 

 Dose3 -0.28 0.004 -0.33 0.974 0.090 0.014 . . . 

******************************************************************************** 

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 9.71 0.021 

Page 57 of 288
Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

 EPA MRID Number 48617810

Page 17 of 61

DER Template Version: 22 September 2011 

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 -0.36 . . 

 Dose1 -0.33 0.123 0.971 

 Dose2 -0.34 0.484 0.794 

 Dose3 -0.27 0.067 0.995 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

Page 58 of 288
Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

 EPA MRID Number 48617810

Page 18 of 61

DER Template Version: 22 September 2011 

21 Day NHLL (Mann-Whitney exact test) 

Obs conc endpoint 

1 0 0.34 

2 0 0.40 

3 0 0.35 

4 0 0.36 

5 1 0.33 

6 1 0.33 

7 1 0.32 

8 1 0.35 

9 2 0.41 

10 2 0.34 

11 2 0.33 

12 2 0.34 

13 3 0.27 

14 3 0.33 

15 3 0.25 

16 3 0.26 

Page 59 of 288
Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

 EPA MRID Number 48617810

Page 19 of 61

DER Template Version: 22 September 2011 

The NPAR1WAY 

Procedure

Wilcoxon Scores (Rank Sums) for Variable endpoint 

Classified by Variable conc 

conc N 

Sum of 

Scores 

Expected 

Under H0 

Std Dev 

Under H0 

Mean 

Score 

0 4 51.50 34.0 8.154753 12.8750 

1 4 29.50 34.0 8.154753 7.3750 

2 4 42.50 34.0 8.154753 10.6250 

3 4 12.50 34.0 8.154753 3.1250 

Average scores were used for ties. 

Kruskal-Wallis Test 

Chi-Square 9.7105 

DF 3 

Asymptotic Pr > Chi-Square 0.0212 

Exact Pr >= Chi-Square 0.0065 

Page 60 of 288
Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

 EPA MRID Number 48617810

Page 20 of 61

DER Template Version: 22 September 2011 

The NPAR1WAY 

Procedure

Wilcoxon Scores (Rank Sums) for Variable endpoint 

Classified by Variable conc 

conc N 

Sum of 

Scores 

Expected 

Under H0 

Std Dev 

Under H0 

Mean 

Score 

0 4 24.50 18.0 3.422614 6.1250 

1 4 11.50 18.0 3.422614 2.8750 

Average scores were used for ties. 

Wilcoxon Two-Sample Test 

Statistic (S) 24.5000 

Normal Approximation 

Z 1.7530 

One-Sided Pr > Z 0.0398 

Two-Sided Pr > |Z| 0.0796 

t Approximation 

One-Sided Pr > Z 0.0615 

Two-Sided Pr > |Z| 0.1230 

Exact Test 

One-Sided Pr >= S 0.0429 

Two-Sided Pr >= |S - Mean| 0.0857 

Z includes a continuity correction of 0.5. 

Kruskal-Wallis Test 

Chi-Square 3.6067 

DF 1 

Pr > Chi-Square 0.0575 

Page 61 of 288
Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

 EPA MRID Number 48617810

Page 21 of 61

DER Template Version: 22 September 2011 

The NPAR1WAY 

Procedure

Wilcoxon Scores (Rank Sums) for Variable endpoint 

Classified by Variable conc 

conc N 

Sum of 

Scores 

Expected 

Under H0 

Std Dev 

Under H0 

Mean 

Score 

0 4 21.0 18.0 3.380617 5.250 

2 4 15.0 18.0 3.380617 3.750 

Average scores were used for ties. 

Wilcoxon Two-Sample Test 

Statistic (S) 21.0000 

Normal Approximation 

Z 0.7395 

One-Sided Pr > Z 0.2298 

Two-Sided Pr > |Z| 0.4596 

t Approximation 

One-Sided Pr > Z 0.2418 

Two-Sided Pr > |Z| 0.4837 

Exact Test 

One-Sided Pr >= S 0.2571 

Two-Sided Pr >= |S - Mean| 0.5143 

Z includes a continuity correction of 0.5. 

Kruskal-Wallis Test 

Chi-Square 0.7875 

DF 1 

Pr > Chi-Square 0.3749 

Page 62 of 288
Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

 EPA MRID Number 48617810

Page 22 of 61

DER Template Version: 22 September 2011 

The NPAR1WAY 

Procedure

Wilcoxon Scores (Rank Sums) for Variable endpoi

nt 

Classified by Variable conc 

conc N 

Sum of 

Scores 

Expected 

Under H0 

Std Dev 

Under H0 

Mean 

Score 

0 4 26.0 18.0 3.464102 6.50 

3 4 10.0 18.0 3.464102 2.50 

Wilcoxon Two-Sample Test 

Statistic (S) 26.0000 

Normal Approximation 

Z 2.1651 

One-Sided Pr > Z 0.0152 

Two-Sided Pr > |Z| 0.0304 

t Approximation 

One-Sided Pr > Z 0.0336 

Two-Sided Pr > |Z| 0.0671 

Exact Test 

One-Sided Pr >= S 0.0143 

Two-Sided Pr >= |S - Mean| 0.0286 

Z includes a continuity correction of 0.5. 

Kruskal-Wallis Test 

Chi-Square 5.3333 

DF 1 

Pr > Chi-Square 0.0209 

Page 63 of 288
DATA EVALUATION RECORD 

METOLACHLOR 

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

EPA Contract No. EP10H001452 

Task Assignment No. 2-49-2012 (MRID 48617806) 

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:

Daniel R. Hunt, M.S. Date: 05/04/2012 

Secondary Reviewer: Signature:

Scott D. Studenberg, Ph.D., D.A.B.T. Date: 5/10/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 5/21/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 5/21/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 64 of 288
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 65 of 288
METOLACHLOR/108801 

Androgen Receptor Binding (Rat Prostate Cytosol) (2012) I Page 1of16 

OCSPP 890.1150/ OECD None 

Primary Reviewer: Ayaad Assaad, D.V.M, Ph.D. 

Health Effects Division 

Signature: A -'/}0h cJ 

Signature: Date: b;~ 

Date: c:;;(t.:- /, rSecondary Reviewer: _G_re_.,g..._A_k_er_m_a_n~,_P_h_.D_. ___ _ 

Health Effects Division 

Template version 08/2011 

DATA EVALUATION RECORD 

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

PC CODE: 108801 DP BARCODE: D398926 

TXR#: 0052848 CAS No.: 51218-45-2 

TEST MATERIAL (PURITY): Metolachlor (97.3%) 

SYNONYMS: 2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide 

CITATION: Thomas, I.A. (2012). In Vitro Rat Prostate Androgen Receptor Competitive 

Binding Assay. WIL Research Laboratories, LLC, Ashland, OH. Laboratory 

Study No.: WIL-786007, January 4, 2012. MRID 48617806. Unpublished. 

SPONSOR: Metolachlor Task Force, 16160 Nall, Stilwell, KS 66085 

TEST ORDER#: CON108801-12 

EXECUTIVE SUMMARY: In an androgen receptor (AR) binding assay (MRID 48617806), 

ventral prostate cytosol from previously castrated (23-24 hours prior to collection) Sprague 

Dawley rats, was used as the source of AR to conduct saturation and competitive binding 

experiments. The saturation binding experiment was conducted to demonstrate that the AR in 

the rat prostate was present in reasonable numbers and was functioning with appropriate affinity 

for the radio-labeled reference androgen ([3H]-Rl 881) prior to conducting the competitive 

binding experiments. The competitive binding assay measured the binding of a single 

concentration of [3H]-Rl881 (1 nM) in the presence of increasing concentrations (10- 10 to 10- 3 

M) of metolachlor (97.3% purity, Lot# 20081115). Ethanol was used as the solvent vehicle at a 

final assay concentration of 2.6%. A total of three runs were performed, and each run included 

dexamethasone as a weak positive control, and RI 881 as the ligand reference standard. 

In the saturation binding experiment, the maximum binding capacity (Bmax) was 14.1 fmol/l 00 

μg protein and the dissociation constant (Kct) was 0.83 nM. The Kct and Bmax values were within 

the range reported in the EPA validation program. Confidence in these numbers is high 

according to the goodness of fit (R2 = 0.985); however, only one run was performed. The 

specific binding reached a plateau, and non-specific binding (NSB) was :S8.8% of total binding 

The Scatchard plot for the saturation assay was not linear for the entire [3H]-Rl 881 concentration 

range tested; however, it was linear over the range from 0.5 to 10 nM, which encompasses the 

ligand concentration used in the competition binding experiment. 

Page 66 of 288
Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 2 of 16 

METOLACHLOR/108801 OCSPP 890.1150/ OECD None

In the competitive binding experiment, the estimated mean log IC50s for R1881 (−8.8 M) and the 

weak positive control, dexamethasone (−4.2 M), were within the expected ranges. The mean 

relative binding affinity (RBA) for the weak positive control was 0.0026%. The solvent control 

responses indicated no drift in the study assay. All performance criteria were generally met. 

In each run, there were no apparent reduction in average [3H]-R1881 binding at metolachlor 

concentrations from 10−10 to 10−4 M (87.7-106.8%), but average binding of the radiolabeled 

ligand declined to 48.96-50.99% at 10−3 M. The average binding was approximately 50% in 

Runs 2 and 3 (50.94 and 50.99%), the average binding curve for metolachlor across all three runs 

indicates that ~50% displacement (competitive binding to the AR) occurred at 10−3 M. The 

average log IC50 and RBA for metolachlor were −2.994 M and 0.00016%, respectively. 

Based on the results from the three runs, metolachlor is classified as a Binder in the Androgen 

Receptor Binding Assay. 

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

Assay (OCSPP 890.1150).

COMPLIANCE: An unsigned Data Confidentiality statement, partially signed and dated GLP 

Compliance statement, and signed and dated Quality Assurance statements were provided. 

Page 67 of 288
Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 3 of 16 

METOLACHLOR/108801 OCSPP 890.1150/ OECD None

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: WIL Research Laboratories, LLC 

Location: Ashland, OH 

Study Director: Jennifer A. Thomas 

Other Personnel: Justin L. Godsey, Group Supervisor, Metabolism 

Janet M. House, Data Coordinator, Metabolism 

Stephanie L. Ledyard, Quality Control Coordinator, Metabolism 

Celeste M. MacElrevey, Research Scientist, Metabolism 

Aimee P. Mahoney, Project Specialist, Metabolism 

Timothy B. Riehl, Jr., Group Supervisor, Metabolism 

Katie A. Alonso, Publishing Specialist, Reporting & Technical Support Services 

Melissa A. Hull, Group Manager, Reporting & Technical Support Services 

Robert A. Wally, Operations Manager, Reporting & Technical Support Services 

Study Period: October 26, 2010 to January 4, 2012 

2. Test substance: Metolachlor 

 Description: Dark brown clear liquid 

 Source: Sipcam, Valdosta, GA 

 Lot #: 20081115 

 Purity: 97.3% 

 Solubility: Not reported (NR) 

 Volatility: NR 

 Stability: NR 

 Storage conditions: Room temperature 

 CAS #: 51218-45-2 

 Molecular weight: 283.8 g/mol 

 Structure: 

CH3

CH3

N

O

CH3

O

Cl

CH3

3. Non-labeled ligand: R1881 

 Supplier: PerkinElmer, Waltham, MA 

 Catalog #: 

Lot #: 

NLP005020MG 

626128 

 Purity: >97% 

 CAS #: 965-93-5 

4. Radioactive ligand: [

3

H]-R1881 

 Supplier: PerkinElmer, Waltham, MA 

 Catalog (Lot #): NET-590 (614814) 

 Date of production: NR 

 Date of use: December 8, 2010 to December 20, 2010 

 Radiochemical purity: >97% 

 Specific activity: 85.1 Ci/mmol 

 Concentration of stock: 1 mCi/mL 

Page 68 of 288
Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 4 of 16 

METOLACHLOR/108801 OCSPP 890.1150/ OECD None

5. Positive control: Dexamethasone 

 Supplier: Sigma-Aldrich 

 Catalog # 

Lot #: 

 Purity: ≥98% 

 CAS # : 50-02-2 

6. Solvent/vehicle control: Ethanol 

 Justification for choice of solvent: Not provided 

 Final Concentration: 2.6% (reviewer-calculated from the raw data for the solvent control tubes; the solvent 

concentration in the test substance and control tubes were not reported but typically 

would be equivalent to the solvent control tubes) 

B. METHODS

1. Preparation of Rat Ventral Prostate Cytosol: A total of 30 male Sprague Dawley rats 

(Charles River Laboratories, Raleigh, NC) were castrated at ca. 79 days of age and were 

euthanized after ca. 23-24 hours. The ventral prostates were excised immediately after 

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

(phenylmethylsulfonyl fluoride) buffer, homogenized, and centrifuged for 30 min at 30,000 

× g at 4oC. Supernatant fractions were pooled, and the resulting pellets were discarded. 

Protein concentration of the cytosol was determined to be 4.33 mg/mL initially with a 

protein kit compatible with DTT in the TEDG buffer (Quick Start Bradford Protein Assay 

kit, Bio-Rad Laboratories, Inc.). Protein concentrations ranged from 3.67 to 4.60 mg/mL 

over the course of the assays. Cytosol was divided into 5-mL aliquots and stored at −70o C 

until use.

2. Saturation Radioligand Binding Experiment: A saturation binding experiment 

measuring total and NSB of [3H]-R1881 was performed to demonstrate that the AR was 

present in reasonable concentrations and had the appropriate affinity for the R1881 ligand. 

The conditions for the saturation binding experiment are summarized in Table 1. 

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TABLE 1. Summary of Conditions for Saturation Binding Experiment a

Source of receptor Rat ventral prostate cytosol 

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

Concentration of non-labeled ligand (100X [radioligand]) 25-1000 nM 

Optimization of receptor concentration Sufficient to bind 25-35% of radioligand 

at 0.25 nM b

Temperature 4o C 

Incubation time 20 hours 

Composition of assay buffer 

(TEDG) 

Tris 10 mM (pH 7.4) 

EDTA 1.5 mM 

Glycerol 10% 

Sodium molybdate 1 mM 

Phenylmethylsulfonyl fluoride 1 mM 

DTT 1 mM 

a Data were obtained from pages 16-19, of the study report. Triamcinolone acetonide was added to all assay 

tubes to saturate the progesterone receptor present in the rat prostate homogenate. 

b The protein concentration used was 0.88 mg/tube (2.6 mg/mL). 

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

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

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

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

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

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

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

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

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

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

radiolabeled R1881. The optimal protein concentration was determined to be 2.6 mg 

protein/mL, which resulted in the binding of 34.30% of the total radioactivity added. A 

single run contained three concurrent replicates at each concentration, resulting in the 72 

samples depicted in Table 2. 

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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 19, and 92-94 of the study report. 

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

c Tubes 25-48 contained 50 μL of triamcinolone acetonide and 7.5-45 μL of 10 nM [3

H]-R1881 or 7.5-30 μL 

of 100 nM of [3

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

d Tubes 1-24 contained 50 μL of triamcinolone acetonide and 7.5-45 μL of 10 nM [3

H]-R1881 or 7.5-30 μL 

of 100 nM of [3

H]-R1881. R1881 was added in a 100-fold molar excess of [3H]-R1881 in a volume of 7.5-

45 μL of 1 μM R1881 or 7.5 to 30 μL of 100 μM R1881. 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 samples 

were dried, dissolved in diluted prostate cytosol (300 μL), and incubated in the refrigerator 

for 20 hours at 4°C. Samples were maintained on ice except during whole rack vortexing. 

To separate bound from free R1881, hydroxyapatite (HAP) slurry was added to each tube 

and vortexed once every 5 minutes for 20 minutes. The samples were then centrifuged, and 

the supernatant was aspirated and discarded. The samples were washed 3 times in 50 mM 

Tris buffer. Following the last wash and decanting of the Tris buffer, the pellets were then 

extracted by additional of 2 ml ethanol. The samples were vortexed 3 times at 5 minute 

intervals. Portions of each ethanol supernatant were then decanted into scintillation vials, 

and the radiation was quantified by liquid scintillation counting. A total of 3 runs were 

performed. 

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

binding experiment is included in Table 3. 

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TABLE 3. Summary of Conditions for Competitive Binding Experiment a

Source of receptor Rat ventral prostate cytosol 

Concentration of radioligand 1 nM 

Optimization of receptor concentration Sufficient to bind 25-35% of 1 nM 

radioligand b 

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

Incubation Temperature 4o C 

Incubation time 20 hours 

Composition of assay buffer Tris 10 mM (pH 7.4) 

EDTA 1.5 mM 

Glycerol 10% 

Sodium molybdate 1 mM 

Phenylmethylsulfonyl fluoride 1 mM 

DTT 1 mM 

a Data were obtained from pages 16-17, 20, of the study report. Triamcinolone acetonide was added to all 

assay tubes to saturate the progesterone receptor present in the rat prostate homogenate. 

b The protein concentration used was 0.77-0.84 mg protein/tube (2.6 mg/mL). 

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

the EPA Test Guidelines OCSPP 890.1150, except that the receptor concentration was 

higher than specified in the guideline (sufficient to bind 10-15% of 1 nM radioligand). The 

competitive binding experiment measures the binding of a single concentration of [3H]-

R1881 (adjusted specific activity of 82.9 Ci/mmol) to the AR in the presence of increasing 

concentrations of a test substance. 

Ethanol was used as a vehicle. Solubility of metolachlor in ethanol and assay buffer was not 

reported. Results from the preliminary activity assay demonstrated that 2.6 mg/mL (0.8 

mg/tube) of cytosolic protein contains enough receptor to bind 21.68% of the [3

H]-R1881 at 

1 nM. 

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

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

shown in Table 4. Each assay consisted of three independent runs on three different days. 

Each run included triplicate sets of [3

H]-R1881 only (for total binding calculations) at the 

beginning of each run, duplicate sets of solvent blank and 1μM R1881 (NSB) at the 

beginning and end of each run, along with duplicate tubes of the reference standard and the 

weak positive control at each concentration and triplicate tubes of the test chemical at each 

concentration, resulting in a total of 61 samples per run. 

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

Solvent Control Reference standard Weak positive control Test Chemical None Ethanol R1881 Dexamethasone Metolachlor 

Tubes 4-5 and 58-59 Tubes 6-17 and 60-61 c Tubes 18-33 Tubes 34-57 Tubes 1-3

-- 1×10−3

 1×10−3

-- 1×10−4 1×10−4

-- 1×10−5 1×10−5

1×10−7 1×10−6 1×10−6

1×10−8 1×10−7 1×10−7

1×10−9 1×10−8 1×10−8

1×10−10 1×10−9 1×10−9

1×10−11 1×10−10 1×10−10 

a Data were obtained from pages 20, and 95-106 of the study report. 

b Each concentration of each chemical was run in duplicate (R1881 and dexamethasone) or triplicate 

(metolachlor) for a total of 61 tubes per run. Tubes 4-61 contained 50 μL of triamcinolone acetonide and 30 μL 

[

3

H]-R1881. Samples were dried, and 300 μL of prostate cytosol were added. Tubes 4-61 also contained 10 μL 

of the solvent control, reference standard (non-radiolabeled R-1881), weak positive control, or test substance, 

with the exception of Tubes 6-7 and 60-61 that contained 30 μL of non-radiolabeled R1881 (used to evaluate 

NSB). Tubes 1-3 contained only 30 μL of [3

H]-R1881. 

c Tubes 6-7 and 60-61 were used to evaluate NSB by adding 100X of cold (non-radiolabeled) R1881. 

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

buffer and extraction with ethanol), followed by scintillation counting of bound [3

H]-R1881 

were similar to those used in the saturation binding experiment. A total of three runs were 

performed as described. 

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

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

Graph Pad Prism v. 5.02 (GraphPad Software, Inc., La Jolla, CA). A Scatchard plot was 

also plotted for the binding data. Mean and standard error were calculated (IC50) for the 

composite three runs of the competitive binding experiment with Microsoft Excel. 

5. Definitions 

a. Classification of test material 

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

follows: 

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

bound, and the Hill slope is between −0.6 to −1.4. 

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

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

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

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

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

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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. 

b. Descriptors for receptor binding 

Bmax: maximal binding capacity

Kd: dissociation constants

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

by the competitor 

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

II. RESULTS 

A. SATURATION BINDING EXPERIMENT: Saturation binding experiment parameters 

are presented in Table 5. The Kd for [3H]-R1881 was 0.83 nM, and the estimated Bmax was 

14.1 fmol/100 μg protein for the single batch of prostate cytosol that was prepared. Only 

one saturation binding run was performed, rather than the three runs recommended in the 

guideline. The Kd and Bmax were within the range reported in the EPA validation program. 

The Scatchard plot for the single saturation assay was not linear for the entire [3

H]-R1881 

concentration range tested; however, it was linear over the range from 0.5 to 10 nM, which 

encompasses the ligand concentration used in the competition binding experiment and 

demonstrates that the androgen receptor was functioning with the appropriate affinity for the 

reference ligand R1881 at the ligand concentration used in the assay. 

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

Parameter Run 1 Run 2 Run 3 Mean ± SEM

R2 0.985 NA NA NA 

Bmax (nM) 0.377 NA NA NA 

Bmax (fmol/100 μg protein) 14.1 NA NA NA 

Kd (nM) 0.83 NA NA NA 

a Data were obtained from pages 27, and 45-46 of the study report. Only one run was performed.

R2

 Goodness of fit for curve calculated for specific binding 

NA Not applicable

Figure 1 illustrates the non-specific, specific, and total binding curves for [3

H]-R1881 to the 

androgen receptor. The specific binding reached a plateau, and NSB was less than 20% of 

total binding (2.5-8.8%). Figure 2 is a Scatchard plot that illustrates the binding of 

[

3

H]-R1881 to the androgen receptor. The data fit results in a linear plot over a test 

concentration of 0.5 to 10 nM [3H]-R1881. 

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

Binding Experiment.

 

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

 

B. COMPETITIVE BINDING EXPERIMENT: Competitive binding experiment 

parameters are presented in Table 6 and shown graphically in Figures 3 through 5. 

The estimated average log IC50 was −8.803 M for R1881 and −4.194 M for the weak 

positive control (dexamethasone). Compared to R1881, the mean RBA for dexamethasone 

was 0.0026%. Confidence in these numbers is high due to the small variation. No 

precipitation of the test compound was reported. The solvent control responses indicated no 

drift in the study assay. 

 There were no apparent alterations in radiolabeled R1881 binding at metolachlor 

concentrations of 10−10 to 10−4

 M (81.73-124.73%), with the specific binding of the 

radiolabeled ligand declining to 47.82-52.43% at a concentration of 10−3

 M. The estimated 

log IC50 was -3.013 M for metolachlor in Run 1. The study authors calculated the log IC50

and RBA for metolachlor in these runs as the mean binding in both runs was essentially at 

Page 75 of 288
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METOLACHLOR/108801 OCSPP 890.1150/ OECD None

50% (50.94% for Run 2 and 50.99% for Run 3) at 10−3 M. The estimated log IC50 in Runs 2 

and 3 were −2.982 and −2.987 M, respectively. Across all three runs the estimate log IC50

and RBA for metolachlor were −2.994 M and 0.00016%, respectively. Confidence in these 

numbers is high due to the small variation. 

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

Parameter Run 1 Run 2 Run 3 Mean ± SEMb

R2 (unweighted) R1881 1.000 0.9978 0.9990 0.9978-1.000 

 Dexamethasone 0.9958 0.9988 0.9926 0.9926-0.9988 

 Metolachlor 0.9682 0.9907 0.9697 0.9682-0.9907 

Log IC50 (M) R1881 −8.918 −8.604 −8.888 −8.803 ± 0.100 

 Dexamethasone −4.165 −4.208 −4.209 −4.194 ± 0.015 

 Metolachlor −3.013 −2.982c −2.987c −2.994 ± 0.010 

IC50 (M) R1881 1.208 x 10−9 2.489 x 10−9 1.294 x 10−9 1.664 x 10−9 (± 0.413) 

 Dexamethasone 6.839 x 10−5 6.194 x 10−5 6.180 x 10−5 6.405 x 10−5 (± 0.217) 

 Metolachlor 9.705 x 10−4 1.042 x 10−3 c 1.030 x 10−3 c 1.014 x 10−3 (± 0.022) 

Log %RBAb Dexamethasone −2.753 −2.396 −2.679 −2.609 ± 0.109 

 Metolachlor −3.905 −3.622 −3.901 −3.809 ± 0.094 

RBA (%)b Dexamethasone 0.0018 0.0040 0.0021 0.0026 ± 0.0007 

 Metolachlor 0.00012 0.00024 0.00013 0.00016 ± 0.00004 

a Data were obtained from page 42 of the study report. 

b The range of R2

 is reported and the mean ± SE is reported for the other parameters. %RBA, log %RBA, and 

mean ± SEM values are reviewer-calculated. For means expressed in scientific notation, the SE values in 

parentheses are presented in the same order of magnitude as the mean value. 

c The Hill equation could not be fit to the data, therefore the reported IC50 was interpolated from the formula for 

estimating log(EC50): Y=Bottom + (Top-Bottom)/(1+10^((LogEC50-X)*HillSlope)) 

NR Not reported 

NA Not applicable 

R2

 Goodness of fit 

RBA Relative binding affinity

 

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METOLACHLOR/108801 OCSPP 890.1150/ OECD None

FIGURE 3. Percentage [3H]-R1881 Bound in the Presence of Radioinert R1881, 

Dexamethasone, and Metolachlor.

 

 

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METOLACHLOR/108801 OCSPP 890.1150/ OECD None

FIGURE 4. Mean % [3H]-R1881 Bound in the Presence of Metolachlor in Three 

Competitive Binding Assays. 

 

 

 

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Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 14 of 16 

METOLACHLOR/108801 OCSPP 890.1150/ OECD None

FIGURE 5. Mean % [3H]-R1881 in the Presence of Metolachlora. 

a Average curve was calculated by the reviewers using GraphPad Prism. 

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

functioning properly, each run was evaluated using the criteria shown in Table 7. The 

assays generally met the performance criteria, although the % ligand depletion (20-23%) 

was above the guideline specified 15% for a 1 nM concentration of [3H]-R1881, and the Hill 

slope was out of range for R1881 in Run 2. 

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

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

indicated by a Hill slope of -1.2027. Examination across the runs indicated consistency of 

the Hill slope, placement along the X-axis, and top and bottom plateaus. 

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METOLACHLOR/108801 OCSPP 890.1150/ OECD None

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% 20.23-23.20% X 

Test chemical Top (% binding) 80 to 115 95.6 to 101.5 X 

R1881 fitted curve parameters 

Top (% binding) 82 to 114 89.49 to 101.5 X 

Bottom (% binding) −2.0 to 2.0 -1.18 to 0.671 X 

Hill Slope −1.2 to −0.8 -1.588 to -1.003 Xc

Weak positive control (dexamethasone) fitted curve parameters 

Top (% binding) 87 to 106 96.0 to 101.1 X 

Bottom (% binding) −12 to 12 -3.219 to 4.265 X 

Hill Slope −1.4 to −0.6 -1.057 to -0.9262 X 

Saturation Binding Experiment Kd (nM) 0.685 to 1.57 0.83 Xd

 

Non-specific binding (%) ≤10.0 2.5 to 8.8% X 

a Data were obtained from pages 42, 97-98, 101-102, and 105-106 of the study report.

b These values represent ranges from the validation study. 

c The Hill Slope was low (-1.588) in Run 2, but within the recommended range in the other two runs. 

d Based on a single run. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Rat prostate cytosol was successfully prepared 

and characterized, then employed in competition assays with the goal of investigating the 

potential of metolachlor to act as an endocrine disruptor. Results from these experiments 

suggest that metolachlor is not a viable substrate for the rat AR and does not bind to the 

active site in a competitive manner with R1881. However, metolachlor displaced <50% of 

the reference androgen when present in million-fold excess versus the radiolabeled ligand 

and must therefore be classified as an equivocal binder. 

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

androgen receptor was present in a reasonable concentration and functioning with 

appropriate affinity for the R1881 ligand. The Kd for [3H]-R1881 was 0.83 nM, and the 

estimated Bmax was 14.1 fmol/100 μg protein for the single batch of prostate cytosol that was 

prepared. The Kd and Bmax values were within the range reported in the EPA validation 

program. Confidence in these numbers is high according to the goodness of fit (R2

 = 0.985); 

however, only one run was performed. The specific binding reached a plateau, and NSB 

was ≤8.8% of total binding. The Scatchard plot for the single saturation assay was not 

linear for the entire [3

H]-R1881 concentration range tested; however, it was linear over the 

range from 0.5 to 10 nM, which encompasses the ligand concentration used in the 

competitive binding experiment.

 In the competitive binding experiment, the estimated mean log IC50s for R1881 (−8.8 M) 

and the weak positive control, dexamethasone (−4.2 M), were within the expected ranges. 

The mean RBA for the weak positive control was 0.0026%. Confidence in these numbers is 

high due to the small variation. The solvent control responses indicated no drift in the study 

assay. The performance criteria were generally met. 

 

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 The percent [3H]-R1881 binding was approximately 50% in Runs 2 and 3 (50.94% and 

50.99%, respectively), and the average binding curve for metolachlor across all three runs 

indicates that ~50% displacement of the radioligand occurred at 10−3 M which is the 

threshold for indicating a competitive binder to the AR; therefore, metolachlor is considered 

to be an AR binder under the conditions of this assay. The average log IC50 and RBA for 

metolachlor were −2.994 M and 0.00016%, respectively. 

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:

 Only a single run was performed in the saturation binding experiment. Test Guidelines 

recommend that a series of saturation radioligand binding assays be performed (typically 

three) prior to conducting competitive binding assays. 

 The amount of cytosol used in the competitive binding assay bound 20.2-23.2% of the 

radioactive R1881, which is higher than the Guideline specified 10-15%. 

 Tubes of unlabeled R1881 and dexamethasone were prepared in duplicate at each 

concentration. EPA’s protocol specifies that there are three replicates assayed 

simultaneously at each concentration. 

Page 81 of 288
DATA EVALUATION RECORD 

METOLACHLOR 

Study Type: OCSPP 890.1200, Aromatase Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-49-2012 (MRID 48617805) 

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: 5/1/2012 

Secondary Reviewer: Signature:

Michael E. Viana, Ph.D., D.A.B.T. Date: 5/2/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 5/2/2012 

Quality Assurance: Signature:

Steven Brecher, Ph.D., D.A.B.T. Date: 5/2/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 82 of 288
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 83 of 288
METOLACHLOR/108801 

Aromatase (Human Recombinant) Assay (2012) I Page I of 11 

OCSPP 890.1200 I OECD None 

Primary Reviewer: Ayaad Assaad, D.V.M., Ph.D. 

Health Effects Division 

Secondary Reviewer: Greg Akerman, Ph.D. 

Health Effects Division 

Signature: £;z · (j?za-=» J. 

Signa~:::~ ~J? Date: __ _....co'-"/-'-,.=;_ > _,,/.._,1'-"a=«---

Tem plate version 08/2011 

DATA EVALUATION RECORD 

STUDY TYPE: Aromatase (Human Recombinant); OCSPP 890.1200 

PC CODE: 108801 DP BARCODE: D398926 

TXR#: 0052848 CAS No.: 51218-45-2 

TEST MATERIAL (PURITY): Metolachlor (97.3% a.i.) 

SYNONYMS: 2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-l-methylethyl)acetamide 

CITATION: Thomas, J.A. (2012) Jn vitro inhibition assay with human recombinant 

aromatase. WIL Research Laboratories, LLC, Ashland, OH. Laboratory Report 

No. : WIL-786005 , January 4, 2012. MRID 48617805. Unpublished 

SPONSOR: Metolachlor Task Force, Chemical Consultants International, Inc., 16160 Nall, 

Stilwell, KS 

TEST ORDER#: CON-108801-12 

EXECUTIVE SUMMARY: In an in vitro aromatase (CYP19) assay (MRID 48617805), 

metolachlor (97.3% a.i., Lot# 20081115) was incubated with human recombinant aromatase and 

tritiated androstenedione (1-P [3H(N)]-androst-4-ene-3, 17-dione; [3H]ASDN) in 1 % ethanol at 

log concentrations from 10- 10 to 10-3 M for 15 minutes to assess the potential of metolachlor to 

inhibit aromatase activity. 

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

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

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

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

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

(I 0- 10 to 10-3 M) with three repetitions per concentration. 

Aromatase activity in the full activity controls ranged from 0.2747 to 0.4910 nmol·mgprotein- 1·min- 1 for the 3 test runs, with a mean and standard deviation of 0.3924±0.0224 

nmol·mg-protein- 1·min- 1

• Activity in the background controls ranged from 0.23 to 1.17% and 

averaged 0.49% of the full activity controls. The responses of the full activity controls and 

background controls were acceptable for each run. 

For the positive control substance (4-0H ASDN) aromatase activity was within the 

recommended ranges for the top and bottom of the curve, Hill slope, log IC so and %CV for 

Page 84 of 288
Aromatase (Human Recombinant) Assay (2012) / Page 2 of 11 

METOLACHLOR/108801 OCSPP 890.1200 / OECD None

replicates of each concentration within runs. The estimated log IC50 averaged −7.3 M and the 

Hill slope averaged −0.93. 

For metolachlor, aromatase activity averaged 87.86±6.2% of the full activity control at the 

lowest tested concentration of 10−10 M and 41.63±3.0% of the full activity control at the highest 

tested concentration of 10−3

 M. Aromatase activity at the second highest tested concentration 

(10−4 M) averaged 84.53±2.9% of the full activity control. For Runs 1 and 3, the report indicated 

that the log IC50 values were "ambiguous" but values of −3.79 and −3.81 M, respectively, as well 

as Hill slope values of –8.05 and –8.31, respectively, were reported. A log IC50 value and Hill 

slope were not determined for Run 2 as the concentration response curve did not fit the 4-

parameter model. 

Although >50% inhibition was observed at the highest test concentration (10−3 M) in all three 

runs, the Hill slope (-8.4) for the average response curve was steeper than -2. The unusually 

steep slope may indicate an issue with protein denaturation or solubility at this test concentration. 

Therefore, metolachlor is classified as Equivocal for aromatase inhibition in this assay. 

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

890.1200).

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

Assurance statements were provided.

Page 85 of 288
Aromatase (Human Recombinant) Assay (2012) / Page 3 of 11 

METOLACHLOR/108801 OCSPP 890.1200 / OECD None

I. MATERIALS AND METHODS

A. MATERIALS 

1. Test Substance: Metolachlor 

 Description: Dark brown clear liquid, technical 

 Source: Sipcam (Valdosta, GA) 

 Lot/Batch # (Exp. Date): 20081115 (Nov 15, 2013) 

 Purity: 97.3% 

 Volatility: Vapor pressure: 1.3×10-5 mm Hg at 20 °C 

 Storage conditions: Room temperature 

 Stability: Stable after storage at 0±1°C for 7 days, and stable after storage at 54±2°C for 

14 day 

 Solvent: Ethanol 

 Solubility (in test solvent): At least 0.1 M 

 Highest Concentration Tested: 0.001 M 

 Stock Solution Preparation: Not reported 

 Molecular weight: MW 283.8 

 CAS #: 51218-45-2 

 Structure: 

CH3

CH3

N

O

CH3

O

Cl

CH3

2. Non-Labeled Substrate: Androstenedione (ASDN) 

 CAS # : 63-05-8 

 Source (catalog #): AK Scientific (H320) 

 Lot/Batch # (expiration date): 90330HC (not reported) 

 Purity: 98% 

3. Radiolabeled Substrate: 1-β [3

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

 Source (catalog #): Perkin Elmer Life Sciences (NET926001MC) 

 Lot/Batch # (expiration date): 619344 (not reported) 

 Radiochemical Purity (Supplier): >97% 

 Specific activity: 26.3 Ci/mmol (on August 6, 2010) 

 Radiochemical Purity (In-lab 

determination): 

94.1% (recovery via radio-HPLC) 

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

 CAS # 566-48-3 

 Source (catalog #): Sigma-Aldrich (not reported) 

 Lot/Batch # (expiration date): 081K2133 

 Purity: Not reported 

5. Solvent (Vehicle Control): Ethanol 

 Lot/Batch # (expiration date): Not reported 

 Justification for choice of solvent Not reported 

 Concentration 

(% of total volume in assays) 

1% 

Page 86 of 288
Aromatase (Human Recombinant) Assay (2012) / Page 4 of 11 

METOLACHLOR/108801 OCSPP 890.1200 / OECD None

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

Source (catalog #): BD Biosciences(not reported) 

Lot/Batch # (expiration date): Not reported 

Protein concentration: 0.598-0.776 mg protein per mL 

Cytochrome C reductase activity: Not reported 

Aromatase activity: Not reported 

B. METHODS

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

[

3

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

approximately 100 nM, and the amount of tritium added to each incubation tube was 0.1 μCi 

in a volume of 100 μL. 

Test chemical(s) stock solutions were prepared such that the total volume of each test 

chemical formulation used per assay was no more than 1% of the total assay volume to 

minimize the potential for the solvent to inhibit the enzyme. Rationale for selection of the 

solvent (ethanol) was not reported. 

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

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

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

positive control substance (10−10 to 10−5 M; Table 4) were achieved by the addition of 20 μL 

of the dilution for a final assay volume of 1 mL. 

Three vials of human recombinant microsomes were purchased from BD Biosciences, and 

stored at ≤−70oC. Before use of the microsomes, the vials were thawed, pooled, and 

aliquoted into individual vials before being refrozen to minimize freeze-thaw cycles to no 

more than one. 

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

reported in Table 1. 

TABLE 1. Assay Components and Conditions 

Assay Factor Values 

0.1M sodium phosphate buffer (pH 7.4) 

Microsomal Protein 0.004 mg/mL 

NADPH 0.3 mM 

[3H]ASDN 100 nM 

Propylene Glycol 5% 

Temperature 37°C 

Incubation Time 15 min 

2. Suitability Assessments: The nominal protein concentration of the microsomes (lot # not 

reported) was supplied by the vendor as 7.0 mg/mL. A six-point standard curve prepared 

with bovine serum albumin (BSA) was analyzed with a standard protein assay kit, and the 

Page 87 of 288
Aromatase (Human Recombinant) Assay (2012) / Page 5 of 11 

METOLACHLOR/108801 OCSPP 890.1200 / OECD None

protein concentration of the microsome preparation was determined on each day of use of 

the microsomes in the aromatase assay. 

Aromatase activity in each lot of human recombinant microsomes was determined to 

demonstrate the presence of sufficient activity for analysis of metolachlor. The minimum 

aromatase activity in the three acceptable runs was determined to be 0.2747 nmol·mgprotein−1

·min−1, which was greater than the minimum recommended aromatase activity of 

0.1 nmol·mg-protein−1

·min−1. 

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

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

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

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

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

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

The amount of 3

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

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

4. Demonstration of Proficiency: No data were provided on proficiency testing of the CYP19 

aromatase assay. 

a. Positive Control

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

following criteria: 

 Mean aromatase activity in the absence of an inhibitor was at least 0.1 nmol·mgprotein−1

·min−1. 

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

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

of 4-OH ASDN was <15%. 

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

provided parameters in the preferred ranges. 

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

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

890.1200. 

TABLE 2. Performance Criteria for the Positive Controla

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

Slope −1.2 −0.8 −0.96 −0.90 

Top (%) 90 110 93.1 97.5 

Bottom (%) −5 +6 −0.3 0.21 

Log IC50 (M) −7.3 −7.0 −7.36 −7.31 

a Data were obtained from pages 24 and 56 of the study report. 

b. Proficiency Chemicals: No data were provided on testing of proficiency chemicals 

Page 88 of 288
Aromatase (Human Recombinant) Assay (2012) / Page 6 of 11 

METOLACHLOR/108801 OCSPP 890.1200 / OECD None

TABLE 3. Proficiency Chemicals 

Compound CAS# Class Concentrations 

Econazole 24169-02-6 Inhibitor Not reported 

Fenarimol 60168-88-9 Inhibitor Not reported 

Nitrofen 1836-75-5 Inhibitor Not reported 

Atrazine 1912-24-9 Non-inhibitor Not reported 

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

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

tested during three independent runs. No problems with insolubility were reported. 

The full enzymatic activity was obtained at the two lowest concentrations of metolachlor to 

define the top of the concentration-response curve. 

TABLE 4. Test Chemical Study Design for each Test Run

Sample Type Repetitions 

(Tubes) Description Reference or 

Chemical (M) 

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

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

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

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

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

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

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

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

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

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

Metolachlor Conc 1b 3 All test components plus metolachlor 1×10−3

Metolachlor Conc 2b 3 All test components plus metolachlor 1×10−4

Metolachlor Conc 3b 3 All test components plus metolachlor 1×10−5

Metolachlor Conc 4b 3 All test components plus metolachlor 1×10−6

Metolachlor Conc 5b 3 All test components plus metolachlor 1×10−7

Metolachlor Conc 6b 3 All test components plus metolachlor 1×10−8

Metolachlor Conc 7b 3 All test components plus metolachlor 1×10−9

Metolachlor Conc 8b 3 All test components plus metolachlor 1×10−10

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

b Data were obtained from pages 15-16 of the study report. 

C. DATA ANALYSIS 

1. Raw Data: Raw data were converted to aromatase activity (nmol·mg protein−1

·min−1) and 

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

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

(Table 5). 

Page 89 of 288
Aromatase (Human Recombinant) Assay (2012) / Page 7 of 11 

METOLACHLOR/108801 OCSPP 890.1200 / OECD None

TABLE 5. Raw and Calculated Data

Raw/Calculated Data Included (X) 

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

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

Total DPM for each aqueous portion (after extraction) X 

The total DPM present in the assay tube at initiation X 

The percentage of substrate converted to product X 

Total DPM after extraction corrected for background X 

Aromatase activity expressed in nmol·mg protein−1·min−1 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 displays such as summary tables, graphical 

displays, analysis of variance, and multiple comparisons were conducted using Microsoft 

Excel® spreadsheets using full floating decimal point calculations. Analysis of variance 

(ANOVA) was not performed. 

Response curves were fit using Prism 5.02 (GraphPad Software, Inc., San Diego, CA). Data 

from each assay were recorded into an assay-specific worksheet for the analysis of 

aromatase inhibition data. Data recorded included the assay date, technician, chemical and 

log chemical concentration, total DPM minus background DPM, and percent activity. The 

spreadsheet calculated DPM/mL for each aliquot of extracted aqueous incubation mixture 

and average DPM/mL and total DPM for each aqueous portion after extraction. Total initial 

DPM was calculated by the multiplication of the volume of substrate solution added to the 

incubation by the average substrate solution radiochemical content (DPM/mL) and yielded 

the total DPM present in the assay tube. Background DPM was calculated as the average of 

the DPMs present in the aqueous portion for the background tubes and was subtracted from 

Total DPMs for all samples to provide DPMs for calculating aromatase activity. This 

corrected DPM was then converted to nmol product formed by dividing by the substrate 

specific activity (DPM/nmol). The activity of the enzyme reaction is expressed in nmol (mg 

protein)−1

 min−1 and is calculated by dividing the amount of estrogen formed (nmol) by the 

product of mg microsomal protein used and the incubation time. 

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

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

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

three runs, using the categories presented in Table 6. 

Page 90 of 288
Aromatase (Human Recombinant) Assay (2012) / Page 8 of 11 

METOLACHLOR/108801 OCSPP 890.1200 / OECD None

TABLE 6. Interpretation of Results 

Criteria Interpretation 

Data fit 4-parameter nonlinear 

regression model 

Average curve across runs crossed 50%a Inhibitor 

Average lowest portion of curves across runs is 

between 50% and 75% activityb

Equivocal 

Average lowest portion of curves across runs is greater 

than 75% activityb

Non-inhibitor 

Data do not fit model --- 

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

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

equivocal. 

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

determined to be untestable in the aromatase assay. 

II. RESULTS 

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

0.2747 to 0.4910 nmol·mg-protein−1·min−1 for the 3 test runs, with a mean and standard 

deviation of 0.3924±0.0224 nmol·mg-protein−1

·min−1. Activity in the background controls 

ranged from 0.23 to 1.17% and averaged 0.49% of the full activity controls. The responses 

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 93.76±1.97 nmol·mg-protein−1·min−1 at the lowest tested concentration 

(10−10 M) and 0.71±0.22 nmol·mg-protein−1·min−1 at the highest tested concentration 

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

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

with the overall standard deviation and % CV. Individual inhibition response curves for the 

positive control and for metolachlor from all three runs are shown in Figure 1. 

TABLE 7. Effect of Metolachlor on Aromatase Activity (as percent of control) from Independent Runsa

Chemical 

Concent. 

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

4-OH ASDN 

(positive control) 

−5 3 0.71 0.22 0.13 31.0 

−6 3 6.09 0.07 0.04 1.1 

−6.5 3 15.50 0.19 0.11 1.2 

−7 3 31.69 1.93 1.12 6.1 

−7.5 3 59.72 1.49 0.86 2.5 

−8 3 78.81 2.11 1.22 2.7 

−9 3 93.17 1.48 0.85 1.6 

−10 3 93.76 1.97 1.14 2.1 

Metolachlor −3 3 41.63 3.04 1.75 7.3 

−4 2 84.53 2.87 2.03 3.4 

−5 3 93.31 4.98 2.88 5.3 

−6 3 91.64 3.80 2.20 4.1 

−7 3 90.23 7.31 4.22 8.1 

−8 3 90.23 6.78 3.91 7.5 

−9 3 88.49 3.72 2.15 4.2 

−10 3 87.86 6.20 3.58 7.1 

a Data were obtained from Tables 1 and 2 on pages 35-36 of the study report. Overall means, SD, SEM, and %CV were 

calculated by the reviewers. 

SD Standard Deviation 

SEM Standard Error of the Mean 

CV Coefficient of Variance 

Page 91 of 288
Aromatase (Human Recombinant) Assay (2012) / Page 9 of 11 

METOLACHLOR/108801 OCSPP 890.1200 / OECD None

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

C. TEST SUBSTANCE: For metolachlor, aromatase activity averaged 87.86±6.2% of full 

activity control at the lowest tested concentration (10−10 M) and 41.63±3.0% of full activity 

control at the highest tested concentration (10−3 M). The mean aromatase activity of 

metolachlor (expressed as % full activity) for each concentration is presented in Table 7, 

along with the overall standard deviation and % CV. Average inhibition response curve 

across all runs is shown in Figure 2. 

FIGURE 2. Mean Inhibition Response Curves for 4-OH ASDN and Metolachlor From 

Each Test Run. 

The effect of metolachlor on inhibition of aromatase activity is presented in Table 8. As the 

data for Run 2 could not be fit into the 4-parameter nonlinear regression model, a log IC50

and Hill slope were not determined for this run. For 4-OH ASDN, the estimated log IC50

Page 92 of 288
Aromatase (Human Recombinant) Assay (2012) / Page 10 of 11 

METOLACHLOR/108801 OCSPP 890.1200 / OECD None

averaged −7.3 M and the Hill slope averaged −0.93. The variation in the positive control 

values was acceptable (<15 % CV). 

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

Chemical Run 1 Run 2 Run 3 Meanb SEMb %CVb

Log IC50 (M) 

Metolachlor –3.79 NC –3.81 −3.8 NA NA 

4-OH ASDN −7. 36 −7.32 −7.31 −7.3 0.03 −0.01 

 Hill Slope 

Metolachlor –8.05 NC –8.31 −8.4 NA NA 

4-OH ASDN −0.90 −0.92 −0.96 −0.93 0.02 1.00 

a Data were obtained from Figure 4 on page 41 of the study report. 

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

SD Standard Deviation 

NC Not calculable 

NA Not applicable 

Based on the data from the average response curve and the criteria listed above in Table 7, 

the results support the conclusion that metolachlor is equivocal as an inhibitor of aromatase 

activity in this assay. Although mean aromatase activity was 41.63% at the highest tested 

concentration of 10−3

 M, the calculated Hill slope (-8.4) was steeper than -2. The unusually 

steep slope indicates a potential issue with protein denaturation or solubility at this 

concentration. The overall response is considered equivocal. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS CONCLUSIONS: Enzyme activity was above the minimum 

acceptable level. The test system correctly responded to the positive control compound, 

4-OH ASDN. Evaluation of the effect of metolachlor on aromatase activity indicated an 

effect only at the highest concentration of metolachlor, 10−3 M, but the overall evaluation of 

the data indicated that the effect was not a result of competitive binding. In accordance with 

the Test Guidelines, the interaction between metolachlor and aromatase is labeled equivocal. 

B. AGENCY COMMENTS: Data demonstrating proficiency with the positive control and 

recommended proficiency chemicals were not included, and no statement was included as to 

the proficiency of the laboratory with recommended proficiency chemicals. 

Aromatase activity in the full activity controls ranged from 0.2747 to 0.4910 nmol·mgprotein−1

·min−1 for the 3 test runs, with a mean and standard deviation of 0.3924±0.0224 

nmol·mg-protein−1·min−1. Activity in the background controls ranged from 0.23 to 1.17% 

and averaged 0.49% of the full activity controls. The responses of the full activity controls 

and background controls were acceptable for each run. 

For the positive control substance (4-OH ASDN) aromatase activity was within the 

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

for replicates of each concentration within runs. The estimated log IC50 averaged −7.3 M 

and the Hill slope averaged −0.93. 

Page 93 of 288
Aromatase (Human Recombinant) Assay (2012) / Page 11 of 11 

METOLACHLOR/108801 OCSPP 890.1200 / OECD None

For metolachlor, aromatase activity averaged 87.86±6.2% of the full activity control at the 

lowest tested concentration of 10−10 M and 41.63±3.0% of the full activity control at the 

highest tested concentration of 10−3

 M. Aromatase activity at the second highest tested 

concentration (10−4 M) averaged 84.53±2.9% of the full activity control. For Runs 1 and 3, 

the report indicated that the log IC50 values were "ambiguous" but values of −3.79 and −3.81 

M, respectively, as well as Hill slope values of –8.05 and –8.31, respectively, were reported. 

Values for the log IC50 and Hill slope could not be calculated for Run 2 as the concentration 

response curve did not fit the 4-parameter model. 

Although there appears to be an effect on aromatase activity at 10−3 M metolachlor, the Hill 

slope was steeper than −2 (average −8.4) indicating a potential issue with protein 

denaturation or solubility at this test concentration. Therefore the classification for 

metolachlor in this aromatase assay is equivocal. 

C. STUDY DEFICIENCIES: The following deficiency was noted that is not considered to 

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

 No data were provided on testing of proficiency chemicals 

Page 94 of 288
DATA EVALUATION RECORD 

METOLACHLOR 

Study Type: OCSPP 890.1250, Estrogen Receptor Binding Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-49-2012 (MRID 48617807) 

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:

Daniel R. Hunt, M.S. Date: 05/02/2012 

Secondary Reviewer: Signature:

Scott D. Studenberg, Ph.D., 

D.A.B.T. Date: 05/07/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 5/17/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 5/17/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 95 of 288
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 96 of 288
METOLACHLOR/108801 

Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) I Page I of 17 

OCSPP 890.1250/ OECO None 

Primary Reviewer: Ayaad Assaad, D.V.M., Ph.D. 

Health Effects Division 

Secondary Reviewer: Greg Akerman, Ph.D. 

Health Effects Division 

Signature: fJ fj 55 & J Date: ,:_~ Signature: =~ Date: G- '' .. [J ,,--

Template version 08/2011 

DATA EVALUATION RECORD 

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

OCSPP 890.1250 

PC CODE: 108801 DP BARCODE: D398926 

TXR#: 0052848 CAS No.: 51218-45-2 

TEST MATERIAL (PURITY): Metolachlor (97.3%) 

SYN 0 NYMS: 2-chloro-N-(2-ethyl-6-methy 1 phenyl )-N-(2-methoxy- l -methy I ethyl )acetamide 

CITATION: Thomas, J.A. (2012). In Vitro Rat Uterine Estrogen Receptor Competitive 

Binding Assay. WIL Research Laboratories, LLC, Ashland, OH. Laboratory 

Study No.: WIL-786006, January 4, 2012. MRID 48617807. Unpublished. 

SPONSOR: Metolachlor Task Force, 16160 Nall, Stilwell, KS 66085 

TEST ORDER#: CON-108801-12 

EXECUTIVE SUMMARY: In an estrogen receptor (ER) binding assay (MRID 48617807) for 

metolachlor (97.3% purity, Lot# 20081115), uterine cytosol from previously ovariectomized 

Sprague Dawley rats was used as the source of ER to conduct saturation binding and competitive 

binding experiments. The 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 the competitive 

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

concentration of[3

H]- l n-estradiol (I nM) in the presence of increasing concentrations 

(logarithmic increase from 10- 10 to 10-3 M) of metolachlor. Ethanol was used as the solvent 

vehicle at a final concentration of 2%. A total of 5 runs were performed, and each run included 

19-norethindrone as a weak positive control, octyltriethoxysilane as a negative control, and 17-~­

estradiol as the natural ligand reference material. 

In the saturation binding experiments, the mean maximum binding capacity (Bmax) was 31. 7 

fmol/l 00 μg protein and the mean dissociation constant (Kct) was 0.075 nM. The Bmax value was 

within the recommended range and demonstrated that the estrogen receptor was present in a 

reasonable concentration. The Scatchard plots for the three Saturation assays were linear. The 

specific binding appeared to reach a plateau, and NSB was generally Jess than 20% of total 

binding (1.97 to 21.51%). 

Page 97 of 288
Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 2 of 17 

METOLACHLOR/108801 OCSPP 890.1250/ OECD None

For the competitive binding experiment, the estimated mean log IC50 was −8.675 M for 17βestradiol and −5.254 M for the weak positive control. The mean RBA was 0.045% for the weak 

positive control. Confidence in these numbers is high due to the small variation. All 

performance criteria were generally met; however, parameters for 19-norethindrone were outside 

of the tolerance limits for a weak positive control in Run 5, and a technical error occurred with 

the negative control, octyltriethoxysilane, in Run 2. 

Metolachlor had no significant effect on specific binding of the [3H]-ligand to the ER in runs 2-5; 

therefore, a log IC50 and RBA could not be calculated for metolachlor (specific binding of [3H]-

17β-estradiol was ≥72.28% in Run 1). 

Based on the results of the five runs, metolachlor is classified as Not Interactive in the Estrogen 

Receptor Binding Assay. 

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

Assay (OCSPP 890.1250). 

COMPLIANCE: An unsigned Data Confidentiality statement, partially signed and dated GLP 

Compliance statement, and signed and dated Quality Assurance statements were provided. 

Page 98 of 288
Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 3 of 17 

METOLACHLOR/108801 OCSPP 890.1250/ OECD None

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: WIL Research Laboratories, LLC 

Location: Ashland, OH 

Study Director: Jennifer A. Thomas 

Other Personnel: Justin L. Godsey, Group Supervisor, Metabolism 

Janet M. House, Data Coordinator, Metabolism 

Stephanie L. Ledyard, Quality Control Coordinator, Metabolism 

Celeste M. MacElrevey, Research Scientist, Metabolism 

Aimee P. Mahoney, Project Specialist, Metabolism 

Timothy B. Riehl, Jr., Group Supervisor, Metabolism 

Gregory A. Hawks, Group Supervisor, Reporting & Technical Support Services 

Melissa A. Hull, Group Manager, Reporting & Technical Support Services 

Robert A. Wally, Operations Manager, Reporting & Technical Support Services 

Study Period: October 20, 2010 to January 4, 2012 

2. Test substance: Metolachlor 

 Description: Dark brown clear liquid 

 Source: Sipcam (Valdosta, GA) 

 Lot #: 20081115 

 Purity: 97.3% 

 Solubility: Not reported (NR) 

 Volatility: NR 

 Stability: NR 

 Storage conditions: Stored at room temperature 

 CAS #: 51218-45-2 

 Molecular weight: 283.793 g/mol 

 Structure: 

CH3

CH3

N

O

CH3

O

Cl

CH3

3. Non-labeled ligand: 17β-estradiol 

 Supplier: Sigma-Aldrich 

 Catalog #: E8875 

 Lot #: 060M0149V 

 Purity: ≥98% 

 CAS #: 50-28-2 

4. Radioactive ligand: [

3

H]-17β-estradiol 

 Supplier: American Radiolabeled Chemicals, St. Louis, MO 

 Catalog #: ART 1549 

 Lot #: 110114 

 Radiochemical purity: 99% 

 Specific activity: 162.9 Ci/mmol 

 Concentration of stock: Not reported 

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5. Positive control: 19-Norethindrone 

 Supplier: Sigma-Aldrich 

 Catalog #: NR 

 Lot #: NR 

 Purity: ≥98% 

 CAS #: 68-22-4 

6. Negative control: Octyltriethoxysilane 

 Supplier: Sigma-Aldrich 

 Catalog #: NR 

 Lot #: NR 

 Purity: ≥97.5% 

 CAS #: 2943-75-1 

7. Solvent/vehicle control: Ethanol (EtOH) 

 Justification for choice of 

solvent: 

Not provided 

 Final Concentration: 2% (reviewer-calculated from the raw data for the solvent control tubes; the solvent 

concentration in the test substance and control tubes were not reported but typically 

would be equivalent to the solvent control tubes) 

B. METHODS 

1. Preparation of Rat Uterine Cytosol (RUC): A total of 25 female Sprague Dawley rats 

(Charles River Laboratories, Stone Ridge, NY) were ovariectomized 9 days prior to being 

euthanized. Animals were 87-99 days old at the time of euthanasia. The uteri were 

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

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

buffer, centrifuged for 30 min at 2500 × g at 4oC. Supernatant was transferred and 

centrifuged for 60 minutes at 100,000 × g, and the resulting pellets were discarded. Protein 

concentration of the cytosol was determined with a protein kit compatible with DTT in the 

TEDG buffer (Quick Start Bradford Protein Assay kit, Bio-Rad Laboratories, Inc.). The 

protein concentration of the cytosol preparation was determined to be 3.276 mg/mL initially, 

and the cytosol protein concentration ranged from 2.57 to 3.33 mg/mL over the course of the 

assay runs. Cytosol was divided into 2-mL aliquots and stored at approximately −70oC until 

use.

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

measuring total and NSB of [3H]-17β-estradiol was performed to demonstrate that the 

estrogen receptor (ER) was present in reasonable concentrations and had the appropriate 

affinity for the native ligand. The conditions for the saturation binding are summarized in 

Table 1. 

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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 

at 0.03 nMb 

Temperature 4oC 

Incubation time 16-20 hours 

Composition of assay buffer Tris 10 mM (pH 7.4) 

EDTA 1.5 mM 

Glycerol 10% 

Phenylmethylsulfonyl fluoride 1 mM 

DTT 1 mM 

a Data were obtained from pages 11 and 16-19 of the study report. 

b The protein concentration used was 40 μg protein/tube (0.341-0.391 mg/mL, measured; p. 42). 

On the day of the assay, the specific activity of the stock solution [3H]-17β-estradiol was not 

adjusted for decay over time, and serial dilutions in TEDG + PMSF buffer were prepared to 

achieve the final concentrations of 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1, and 3 nM. Solutions of 

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

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

concentrations of 3, 6, 8, 10, 30, 60, 100, and 300 nM. For each batch of cytosol, the 

optimal protein concentration was determined by testing serial amounts of protein per tube, 

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

of the total radioactivity added (40 μg protein per tube). Each assay consisted of three nonconcurrent runs. Each run included three replicates of each test substance at each 

concentration, resulting in the 72 samples depicted in Table 2. 

TABLE 2. Saturation Binding Experiment Run a

Total binding b Non-specific 

binding c

Radioligand alone d

 Assay Components 

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

350 μL 300 μL --- TEDG + PMSF buffer 

50 μL 50 μL 50 μL [

3

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

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

100x each respective labeled concentration) f

100 μL 100 μL --- Uterine cytosol (diluted to appropriate conc.) 

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

a Data were obtained from pages 104-112 of the study report. 

b Total binding = [3

H]-17β-estradiol bound to ER 

c NSB = [3

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

d Total [3

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

e Final concentrations of [3

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

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

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

from free estradiol, hydroxyapatite (HAP) slurry was added to each tube and vortexed once 

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every 5 minutes for 15 minutes. Subsequently, the contents of each tube were washed three 

times as follows: TEDG + PMSF buffer was added, vortexed, centrifuged for 10 min at 

1000 x g, and the supernatant decanted and discarded. EtOH was then added to the HAP 

pellet remaining in each tube to extract the [3

H]-17β-estradiol, followed by vortexing three 

times (once every five minutes), and centrifugation for 10 min at 1000 x g. An aliquot of 

each supernatant was radioassayed by scintillation counting. The temperature was 

maintained at 4oC throughout the assay prior to extraction with EtOH. 

 

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 b

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

Temperature 4C 

Incubation time 16-20 hours 

Composition of assay buffer Tris 10 mM (pH 7.4) 

EDTA 1.5 mM 

Glycerol 10% 

Phenylmethylsulfonyl fluoride 1 mM 

DTT 1 mM 

a Data were obtained from pages 11, 16-18, and 22 of the study report. 

b The protein concentration used was 90 μg protein/tube (0.725-0.891 mg/mL, measured; p. 42). 

Solubility of metolachlor in EtOH and assay buffer was not reported specifically but the 

metolachlor stock solution concentration (in EtOH) was 100 mM and the highest assay 

concentration was 10−3

 M. On the day of the assay, the specific activity of the stock solution 

[

3

H]-17β-estradiol was not adjusted for decay over time, and diluted in TEDG + PMSF 

buffer to achieve a final concentration of 1 nM. For each batch of cytosol, the optimal 

protein concentration was determined by testing serial amounts of protein per tube, using 1.0 

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

radioactivity added (90 μg protein per tube). Serial dilutions of the test substance, positive 

control (19-norethindrone), negative control (octyltriethoxysilane), and reference material 

(non-labeled 17β-estradiol) were prepared to achieve the concentrations shown in Table 4. 

The competitive binding experiment consisted of five independent runs on five different 

days. Each run included duplicate sets of 0.1 μM estradiol (NSB) at the beginning and 

triplicate sets at the end of each run, along with duplicate tubes of the reference standard, the 

weak positive control, and negative control at each concentration, and triplicate sets of 

metolachlor at each concentration, resulting in a total of 81 samples per run (including three 

replicates of [3

H]-17β-estradiol only, for total binding calculations). 

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

Metolachlor Positive control Negative control Reference Chemical 

19-norethindrone Octyltriethoxysilane Non-labeled 

17β-estradiol 

Tubes 52-75 c Tubes 36-51 c Tubes 20-35 c Tubes 4-19 and 76-81 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 21-22, and 113-142 of the study report. 

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

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

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

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

c Each concentration of each chemical was run in duplicate except metolachlor (triplicate), for a total of 81 

tubes per run. 

d Solvent is EtOH in tubes 4-5 and 76-78. 

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

buffer and extraction with EtOH), followed by scintillation counting of bound [3

H]-17βestradiol were similar to those used in the saturation binding experiment. 

C. DATA ANALYSIS: For the saturation binding experiment, total binding and NSB data 

were modeled via non-linear regression using Graph Pad Prism v. 5.02 (GraphPad Software, 

Inc., La Jolla, CA), incorporating automatic outlier elimination according to the method of 

Motulsky and Brown (2006)1 with a Q value of 1.0. Receptor binding data plots were 

corrected for ligand depletion using the method of Swillens (1995)2

. For the competitive 

binding assay, similar methods of nonlinear regression were used to fit a curve (for 17βestradiol, the positive control, and the test substance) to the Hill equation formula which 

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

saturation binding experiment (Kd and Bmax), mean values were calculated for the composite 

three runs using Microsoft Excel. For the competitive binding experiment, the composite 

mean was reported for RBA and the composite mean and standard error was reported for log 

IC50 using Microsoft Excel.

1. Definitions 

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

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

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

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

 

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

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

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

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

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

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

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

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

concentration groups in the data is above 75%. 

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

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

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

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

concentration groups in the data is >50%. 

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

above. 

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

Run Classification Numerical Value 

Interactive 2 

Equivocal 1 

Not interactive 0 

Equivocal up to the limit of concentrations tested “missing” 

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

following ranges: 

Test Material Classification Numerical Range 

Interactive average ≥1.5 

Equivocal 0.5≥ average <1.5 

Not interactive average <0.5 

Equivocal up to the limit of concentrations tested “missing” 

b. Descriptors for receptor binding: 

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

concentration of active receptor sites

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

IC50: concentration of the test substance (M) at which 50% of the radioligand is displaced 

from the receptor

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

II. RESULTS 

A. SATURATION BINDING EXPERIMENT: Saturation binding experiment parameters 

are presented in Table 5. The mean Kd for [3H]-17β-estradiol was 0.075 nM, and the mean 

estimated Bmax was 31.7 fmol/100 μg protein for the prepared rat uterine cytosol. The Kd for 

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

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linear Scatchard plots. Confidence in these numbers is high due to the small variation among 

runs. The goodness of fit (R2) was not reported. 

Figure 1 illustrates the non-specific and total binding curves for [3H]-R1881 to the estrogen 

receptor (specific binding was not presented graphically). Based on the raw data, the specific 

binding reached a plateau in Run 1 and Run 3, and NSB was generally less than 20% of total 

binding (1.97 to 21.51%). Figure 2 contains the Scatchard plots that illustrate the binding of 

[3H] R1881 to the estrogen receptor across the three runs: the data fits resulted in linear plots. 

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

Cytosol a

Parameter Run 1 Run 2 Run 3 Mean ± SEM b

R2 (unweighted) NR NR NR NA 

Bmax (nM) 0.023 0.028 0.025 0.025 ± 0.001 

Bmax (fmol/100 μg protein) 29.2 34.8 31.2 31.7 ± 1.64 

Kd (nM) 0.063 0.083 0.078 0.075 ± 0.006 

a Data were obtained from page 43 of the study report. 

b SEM values were calculated by the study reviewer. 

R2

 Goodness of fit 

NR Not reported 

NA Not applicable

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

Binding Experiment. 

 

 

 

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

Receptor. 

 

 

 

 

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

experiments are presented in Table 6 and shown graphically in Figures 3 to 4. A total of 

five independent assay runs were conducted. The three reference chemicals generally 

performed as expected; however, parameters for 19-norethindrone were outside of the 

tolerance limits for a weak positive control (developed for norethynodrel) in Run 5, and the 

negative control octyltriethoxysilane displaced >25% of [3H]-17β-estradiol at the highest 

concentration tested in Run 1 and the two highest concentrations in Run 2. 

Octyltriethoxysilane had no effect on specific binding of the [3H]-ligand in Runs 3-5 (77.95 

to 116.13%, excluding one outlier at 20.61%); however, minimum total binding of [3H]-

17β-estradiol in the negative control assay was 47.24% in Run 1 at the highest concentration 

tested, indicating that octyltriethoxysilane appeared to have some interaction with the ER in 

Run 1 (data for Run 2 were compromised due to a technical error). The estimated mean log 

IC50 was −8.675 M for 17β-estradiol and −5.254 M for the weak positive control, and the 

mean RBA was 0.045% for the weak positive control. Confidence in these numbers is high 

due to the small variation. Overall, the results for 17β-estradiol were acceptable for all five 

runs, with four and three acceptable runs for 19-norethindrone and octyltriethoxysilane, 

respectively. 

Metolachlor had no significant effect on specific binding (74.86-114.57%, excluding one 

replicate at 62.24% in Run 1) of the [3

H]-ligand; therefore, a log IC50 and RBA could not be 

calculated for metolachlor. Based on the results of the five runs, metolachlor is classified as 

“not interactive” with the estrogen receptor under the conditions of this assay (Table 7).

Page 108 of 288
Estrogen Receptor Binding (Rat Uterine Cy

METOLACHLOR/108801 OC

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

Parameter Run 1 Run 2 Run 3 Run 4 Run 5 Mean R2 (unweighted) 17β-estradiol 0.9988 0.9899 0.9980 0.9985 0.9994 N 19-norethindrone 0.9980 0.9680 0.9970 0.9994 0.9657 N Metolachlor NA NA NA NA NA NLog IC50 (M) 17β-estradiol 

−8.954 

−8.524 

−8.607 

−8.643 

−8.647 

−8.675 

 19-norethindrone 

−5.05 

−4.97 

−5.282 

−5.535 

−5.435 

−5.254 

 Metolachlor NA NA NA NA NA NIC50 (M) 17β-estradiol 1.11 x 10−9 2.99 x 10−9 2.47 x 10−9 2.28 x 10−9 2.25 x 10−9 2.22 x 10

19-norethindrone 8.913 x 10−6 1.0715 x 10−5 5.224 x 10−6 2.917 x 10−6 3.673 x 10−6 6.288 x 10

 Metolachlor NA NA NA NA NA NLog %RBAb 19-norethindrone 

−1.905 

−1.554 

−1.325 

−1.107 

−1.213 

−1.421 

 Metolachlor NA NA NA NA NA NRBA (%)b 19-norethindrone 0.012 0.028 0.047 0.078 0.061 0.045 ± 0.0 Metolachlor NA NA NA NA NA N

a Data were obtained from page 44 of the study report. b Values are reviewer-calculated. For mean values reported in scientific notation, the SEM are reported in the same exponenti

R2 Goodness of fit 

RBA (%) Relative binding affinity NA Not applicable
Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 13 of 17 

METOLACHLOR/108801 OCSPP 890.1250/ OECD None

TABLE 7 Binding Classification of Metolachlor with Estrogen Receptor a

Run 1 2 3 4 5 Mean Binding Classification c

Classification category value b 1 0 0 0 0 0.2 Not interactive 

a Binding classifications were determined by the reviewer. 

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

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

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

FIGURE 3. Percentage E2 Bound to the Estrogen Receptor in the Presence of Unlabeled 

E2, 19-Norethindrone, Octyltriethoxysilane or Metolachlor.

 

 

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Page 111 of 288
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FIGURE 4. Mean Percentage [3H]-E2 Bound to the Estrogen Receptor in the Presence of 

Metolachlor in Five Competitive Assays.

 

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

properly, each run was evaluated using the criteria shown in Table 8. 

TABLE 8. Criterion a Tolerance 

Limit(s) Value Yes No 

17β-estradiol fitted curve parameters 

Loge residual SD ≤2.35 NR 

Top (% binding) 94 to 111 98.5 to 106.6 X 

Bottom (% binding) −4 to 1 −3.600 to −1.060 X 

Hill Slope (Log10(M)−1) −1.1 to −0.7 −1.1260 to −0.9208 Xc

Weak Positive control (19-norethindrone) fitted curve parametersb

Loge residual SD NA NR 

Top (% binding) NA 92.63 to 103.8 Xd 

Bottom (% binding) NA −10.41 to 2.589 Xd 

Hill Slope (Log10(M)−1) NA −1.13 to −0.79 Xd 

Solvent concentration

Ethanol ≤3% 2% X 

Negative control (octyltriethoxysilane) does not displace 

more than 25% of [3

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

across all concentrations 

≤25% ≤25% Xe

 

a Data were obtained from page 44 of the study report. 

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

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

acceptable for Runs 1-4 as they show 19-norethindrone to be an acceptable weak positive control. 

c The Hill slope was low (−1.1260) in Run 3, but within the recommended range in the other four runs. 

d Excludes Run 5, which exhibited an ambiguous curve that was not consistent with one-site competitive binding 

behavior. 

e The minimum total binding of [3

H]-17β-estradiol in the negative control assays was 47.24% in Run 1 and 

5.01% in Run 2, however, the average binding across all concentrations was >75% for all runs.

NA Not applicable 

NR Not reported 

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METOLACHLOR/108801 OCSPP 890.1250/ OECD None

The curve for the reference material showed that increasing concentrations of unlabeled 

17β-estradiol displaced [3H]-17β-estradiol in a manner consistent with one-site binding, as 

indicated by a mean Hill slope of −1.0091. 

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

The percent binding at this top plateau 76.14-107.70% was within 25 percentage points of 

the value for the lowest concentration of the estradiol standard 95.43-109.03%. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Rat uterine cytosol was successfully prepared and 

characterized, then employed in competition assays with the goal of investigating the 

potential of metolachlor to act as an endocrine disruptor. Results from these experiments 

demonstrate that metolachlor was not a viable substrate for the rat ER and did not bind to 

the receptor in a manner that disrupts 3

H-E2 binding. 

B. AGENCY COMMENTS: For the saturation binding experiment, the mean maximum 

binding capacity (Bmax) was 31.7 fmol/100 μg protein and the mean dissociation constant 

(Kd) was 0.075 nM. The Bmax value was within the recommended range (10 to 150 

fmol/100 μg) and demonstrated that the estrogen receptor was present in a reasonable 

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

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

plateau in Run 1 and Run 3, and NSB was generally less than 20% of total binding (1.97 to 

21.51%). 

For the competitive binding experiment, the estimated mean log IC50 was −8.675 M for 17βestradiol and −5.254 M for the weak positive control. The mean RBA was 0.045% for the 

weak positive control. Performance criteria were generally met; however, parameters for 19-

norethindrone were outside of the tolerance limits for a weak positive control in Run 5 and 

the negative control, octyltriethoxysilane, appeared to show some activity against the 

estrogen receptor at the highest concentrations in Run 1 (activity at the two highest 

concentrations in Run 2 was attributed to a technical error). 

The estimated mean IC50 and RBA were not calculated for metolachlor as it did not show 

competitive binding (i.e. displacement of the radiolabeled estradiol from the ER). 

Metolachlor is classified as non-interactive in this assay as the mean binding classification 

from the five runs was <0.5 (mean specific radiolabeled ligand ER binding was >75% at 

metolachlor concentrations up to 10–3 M in Runs 2-5, and ≥72.28% in Run 1).

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:

 Specific binding of [3H]-17β-estradiol to the ER during the saturation binding experiment 

was not presented graphically. Analysis of the raw data indicated that specific binding 

reached a plateau in Run 1 and Run 3 (pp. 105, 108, and 111). 

 The amount of cytosol used in the competitive binding assay bound 2.20-10.73% of the 

radioactive estradiol, which is lower than the guideline 10-15%. 

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METOLACHLOR/108801 OCSPP 890.1250/ OECD None

 Tubes of unlabeled 17β-estradiol, 19-norethindrone, and octyltriethoxysilane were 

prepared in duplicate at each concentration. EPA’s protocol specifies that there should be 

three replicates assayed simultaneously at each concentration. 

Page 114 of 288
DATA EVALUATION RECORD 

METOLACHLOR 

Study Type: OCSPP 890.1300, Estrogen Receptor Transcriptional Activation 

EPA Contract No. EP10H001452 

Task Assignment No. 2-49-2012 (MRID 48617808) 

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: 5/2/2012 

Secondary Reviewer: Signature:

Michael E. Viana, Ph.D., D.A.B.T. Date: 5/11/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 5/15/2012 

Quality Assurance: Signature:

Steven Brecher, Ph.D., D.A.B.T. Date: 5/15/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 115 of 288
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 116 of 288
METOLACHLOR/108801 

Estrogen Receptor Transcriptional Activation (2012) I Page I of 13 

OCSPP 890.1300/ OECD 455 

Primary Reviewer: Ayaad Assaad, D.V.M., Ph.D. 

Health Effects Division 

Secondary Reviewer: Minerva Mercado Ph.D., DABT 

Health Effects Division 

Signature: 4 tf ~, 

~ z~cJ Date: ~/7,%]6 

Signature Date. ~ · ./sTemplate version 08/20 11 

DAT A EVALUATION RECORD 

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

OCSPP 890.1300; OECD 455 . 

PC CODE: 108801 DP BARCODE: D398926 

TXR#: Not Provided CAS No.: 51218-45-2 

TEST MATERIAL (PURITY): Metolachlor (97.3% a.i.) 

SYNONYMS: 2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide 

CITATION: Willoughby, I.A. (2012) Metolachlor: estrogen receptor transcriptional 

activation (human cell line (HeLa-9903)). CeeTox, Inc., Kalamazoo, MI. 

Laboratory Report No.: 9179V-100537ERTA, January 25, 2012. MRID 

48617808. Unpublished 

SPONSOR: Metolachlor Task Force, 16160 Nall, Stillwell, KS 

TEST ORDER#: CON-108801-12 

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

48617808), hERa-HeLa-9903 cells cultured ;n vUro were exposed to metolachlor (97.3% a.i. , 

Lot #20081115) at logarithmically increasing concentrations from 10- 11 to 10-

4 M in DMSO 

(0.1 %) for approximately 24 hours in two independent runs. The experiments were performed 

using 96-well plates and each metolachlor concentration was tested in 6 wells/plate in each run. 

Cells were exposed to the test agent for 24±2 hours at 37±2°C to induce reporter (luciferase) 

gene products. Luciferase expression in response to activation of the estrogen receptor was 

measured upon using a proprietary luciferase assay 

Metolachlor was tested up to a cytotoxic level, which was observed at concentrations 2:10-

4 M. 

The mean RPCMax was 3.3% for the first run and 0.6% for the second, and the associated PCMax 

was 10- 7 M for both. 

In the main assays, the responsiveness of the cells to the very weak positive control l 7amethyltestosterone was lower than the expected values, indicating a decreased sensitivity of the 

assay to very weak agonists. Although the conditions of this assay were not optimal to detect 

very weak activity, metolachlor responses were similar to those of the negative control 

corticosterone and not comparable to the responses of l 7a-methyltestosterone, which was able to 

reach a maximum of 36.5-46.3% PC. Metolachlor was only able to reach a maximum of 0.6-

3 .3% PC when tested up to the highest concentration possible based on cytotoxicity. Because 

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Estrogen Receptor Transcriptional Activation (2012) / Page 2 of 13 

METOLACHLOR/108801 OCSPP 890.1300/ OECD 455 

the RPCMax < PC10 in both assay runs, metolachlor was considered negative for estrogen receptor 

transcriptional activation in this test system. 

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

Transcriptional Activation assay (OCSPP 890.1300). 

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

Confidentiality statements were provided. 

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Estrogen Receptor Transcriptional Activation (2012) / Page 3 of 13 

METOLACHLOR/108801 OCSPP 890.1300/ OECD 455 

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: Metolachlor 

 Description: Technical, liquid, MW 283.79 g/mol 

 Source (Cat #): WIL Research (not reported0 

 Lot/Batch # (Exp Date): 20081115 (Nov 15, 2013) 

 Purity: 97.3% 

 Solubility: Not reported 

 Volatility: Vapor pressure: 1.3 X 10 to the minus 5 mm Hg at 20 degrees C (added by 

reviewer) 

 Stability: Stable after storage at 0±1Ԩ for 7 days, and stable after storage at 54±2 Ԩ for 14 

days (added by reviewer). 

 Storage conditions: Ambient 

 CAS #: 51218-45-2 

 Structure: 

CH3

CH3

N

O

CH3

O

Cl

CH3

2. Reference substances 

 17β-estradiol (strong estrogen; positive control)

 Supplier: NR 

 Catalog and Batch #: NR 

 Purity: NR 

 CAS # : 50-28-2 

 

 17α-estradiol (weak estrogen) 

 Supplier: NR 

 Catalog and Batch #: NR 

 Purity: NR 

 CAS # : 57-91-0 

 

 Corticosterone (negative compound) 

 Supplier: NR 

 Catalog and Batch #: NR 

 Purity: NR 

 CAS # : 50-22-6 

 

 17α-methyltestosterone (very weak agonist) 

 Supplier: NR 

 Catalog and Batch #: NR 

 Purity: NR 

 CAS # : 58-18-4 

3. Vehicle(s) 

Solvent: DMSO 

Solvent control 

(final concentration): 

0.1% 

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Estrogen Receptor Transcriptional Activation (2012) / Page 4 of 13 

METOLACHLOR/108801 OCSPP 890.1300/ OECD 455 

B. METHODS

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

Collection of Research Bioresources Cell Bank and were verified to be free of mycoplasma 

infection by a DNA fluorochrome assay. Cells were maintained in Eagles Minimum 

Essential Medium without phenol red, supplemented with 60 mg/L kanamycin and 10% 

dextran-coated charcoal-treated fetal bovine serum (source not reported), in an incubator 

under 5% CO2 at 37 oC. Upon reaching 75-90% confluence, cells were subcultured prior to 

exposure to the test material. The cells used in this study were passage 26 (range-finder), 

passage 27 (first run, 9/22/2011), and passage 16 (new from cryopreservation for second 

run, 10/20/2011). 

2. Transcriptional Activation Assays: For each test, cells were plated in a 96-well plate 

(estrogenic activity of plastic not reported) at a density of approximately 1×104 cells/100 μL 

medium/well and allowed to attach for 3 hours. The growth medium was replaced with 

medium containing serial log dilutions of metolachlor in DMSO. Cells were incubated for 

24±2 hours at 37±2 °C. The total final concentration of DMSO was 0.1%. Cytotoxicity was 

determined by two-read propidium iodide uptake. It was stated that transcriptional 

activation of the estrogen receptor was determined as described in CeeTox Standard 

Operating Protocol (SOP) 2041. A list of reagents was provided, but the assay reagent was 

classified as proprietary information, and the SOP was not provided.

a. Preliminary Test: A preliminary test evaluating concentrations ranging from 10−6.5 to 

10−3 M was conducted to determine the appropriate concentration range and to determine 

concentrations resulting in insolubility and/or cytotoxicity. 

b. Proficiency Chemicals: The responsiveness of the test system was tested on March 5, 

April 12, and April 28, 2011 (MRID 48843501), using cells at passage 15, 25 and 28, 

respectively. Based on the assay dates and cell passage numbers, it is unlikely that the cells 

used for proficiency testing are from the same frozen stock as the cells used in the main 

assay. The cells were tested using the following set of proficiency chemicals, and each 

chemical was tested in duplicate on separate days: 

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Estrogen Receptor Transcriptional Activation (2012) / Page 5 of 13 

METOLACHLOR/108801 OCSPP 890.1300/ OECD 455 

Compound CAS No. Concentration 

Range (M) 

Expected 

Response a Notes 

Diethylstilbestrol (DES) 56-53-1 10−14 to 10−8 Positive --- 

17α-Ethynyl estradiol (EE) 57-63-6 10−14 to 10−8 Positive --- 

Hexestrol 84-16-2 10−13 to 10−7 Positive --- 

Genistein 446-72-0 10−12 to 10−5 Positive Cytotoxic at 0.01b, 0.1, and 1 mM 

Estrone 53-16-7 10−12 to 10−6 Positive --- 

Butyl paraben 94-26-8 10−11 to 10−4 Positive Cytotoxic at 0.1b and 1 mM 

1,3,5-Tris(4-

hydroxyphenyl)benzene c

15797-52-1 10−12 to 10−5

 Positive Cytotoxic at 100μM. PCmax approx. 50% of PC. 

Binds to hERα and has ER antagonistic activity 

Dibutyl phthalate (DBP) 84-74-2 10−11 to 10−4 Negative d Cytotoxic at 1 mM 

Atrazine 1912-24-9 10−11 to 10−4 Negative Cytotoxic at 1 mM b

Corticosterone 50-22-6 10−10 to 10−4 Negative If not cytotoxic at 1 mM, then that should be the 

highest tested concentration 

a Positive = RPCmax ≥10% of the response of the positive control in at least 2 of 2 (or 2 of 3) runs 

Negative = RPCmax fails to achieve at least 10% of the response of the positive control in 2 of 2 (or 2 of 3) runs 

b Cytotoxicity is expected to be close to 80% at this concentration. 

c Compound selected to challenge solubility and cytotoxicity. 

d DBP is negative for ERα mediated transcriptional activation, but may not be negative for non-ERβ mediated transcriptional 

activation. A positive result would indicate that the system is detecting activity other than that due to pure ERα, and is 

therefore unacceptable. 

 

c. Reference Chemicals: To ensure the stability of the response from the cell line, eight 

concentrations of each of the following reference chemicals were included in the current 

assay, along with the test chemical: 

3. Data analysis: To obtain the relative transcriptional activity to the 1 nM E2 positive control 

(PC), the luminescence signals from the concurrent plate were analyzed by subtracting the 

mean value of the vehicle control from each well value to normalize the data; each 

normalized value was then divided by the mean value of the normalized PC. The resulting 

value was multiplied by 100 in order to express relative transcriptional activity as a 

percentage of the PC. The test material was defined as negative for inducing estrogen 

receptor transcriptional activation if the RPCMax < PC10 in at least 2 of 2 runs. Log EC50

and Hill slope values are calculated only if a positive response is observed. Coefficients of 

variation (CV) were calculated for the luminescence data triplicates. Concentrations 

showing >20% cytotoxicity or evidence of insolubility were excluded from analyses. 

4. Definitions 

EC50 = concentration of agonist that induces a response halfway between the baseline 

(bottom) and maximum (top) response 

PC10 = concentration of a test chemical at which the response is 10% of the response 

induced by the positive control (E2 at 1 nM) in each plate 

PC50 = concentration of a test chemical at which the response is 50% of the response 

induced by the positive control (E2 at 1 nM) in each plate 

Reference Chemical CAS No. Concentration Range Class 

17β-estradiol (E2) 50-28-2 10−15 to 10−8 Strong estrogen 

17α-estradiol 57-91-0 10−13 to 10−6 Weak estrogen 

Corticosterone 50-22-6 10−11 to 10−4 Negative compound 

17α-methyltestosterone 58-18-4 10−12 to 10−5 Very weak agonist 

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METOLACHLOR/108801 OCSPP 890.1300/ OECD 455 

RPCMax = maximum level of response induced by a test chemical, expressed as a percentage 

of the response induced by the positive control (1 nM E2) on the same plate 

PCMax = concentration of a test chemical inducing the RPCMax

II. RESULTS 

A. PRELIMINARY TEST In order to identify a suitable top concentration for use in the 

transcriptional activation assays, a preliminary cytotoxicity and precipitation assay was 

conducted (Table 1). Cytotoxicity (<80% cell viability) was noted at 10−3

 M, and 

precipitation was observed at ≥10−4

 M metolachlor. Based on these results, logarithmically 

increasing concentrations from 10−11 to 10−4 M were selected for the assay. 

TABLE 1. Preliminary Test for Solubility, Cytotoxicity, and Concentration-Selection for Metolachlora

Concentration (M) % Viability b Commentsc

10−3 52 Precipitation observed, Cytotoxic 

10−3.5 92 Precipitation observed 

10−4 89 Precipitation observed 

10−4.5 97 

10−5 99 

10−5.5 100 

10−6 103 

10−6.5 100 

a Data were obtained from page 20 of the study report. 

B. POSITIVE AND NEGATIVE REFERENCE CHEMICALS 

1. Proficiency Chemicals: The laboratory proficiency assays using the required reference 

compounds were not included in the original study report, but were provided to the Agency 

at a later date (MRID 48843501). The responsiveness of cells to the required proficiency 

chemicals was performed in duplicate on different days for each chemical. The reported 

responses are in Table 2. 

TABLE 2. Proficiency Chemicals 

Compound Expected Response Lab Response 

Run 1 Run 2 Run 3 

Diethylstilbestrol Positive Positive Positive NA 

17α-Ethynyl estradiol Positive Positive Positive NA 

Hexestrol Positive Positive Positive NA 

Genistein Positive Positive Positive NA 

Estrone Positive Positive Positive NA 

Butyl paraben Positive Positive Positive NA 

1, 3, 5-Tris(4-hydroxyphenyl)benzene Positive NA Positive Positive 

Dibutyl phthalate Negative Negative Negative NA 

Atrazine Negative Negative NA Negative 

Corticosterone Negative Negative NA Negative 

NA = not applicable. The chemical was not tested at this time. 

2. Reference Chemicals: The response curves for the three estrogenic reference chemicals 

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Estrogen Receptor Transcriptional Activation (2012) / Page 7 of 13 

METOLACHLOR/108801 OCSPP 890.1300/ OECD 455 

are shown in Figures 1 – 3, and the values derived from the concentration response curves 

(e.g., Log PC50, Log PC10, Log EC50, Hill slope) for the concurrently run reference materials 

are presented in Table 3. 

Although all four performance criteria were met for 17β-estradiol and corticosterone, and 

three of the four criteria were met for 17α-estradiol, the available data indicate that both runs 

were invalid due to the lack of responsiveness of the cells to the very weak agonist, 

17α-methyltestosterone. Additionally, in Run 2, the maximum responsiveness of both 

17β-estradiol and 17α-estradiol exceeded (122%-163%) the response of the PC. 

TABLE 3. Performance Criteria for Reference Chemicalsa

Reference Chemical 

Parameter Acceptable Range Values Acceptable 

Run 1 Run 2 Yes No 

17β-estradiol

 Log PC50 −11.4 to −10.1 −10.4 −10.6 X 

 Log PC10 <−11 −11.4 −11.4 X 

 Log EC50 −11.3 to −10.1 −10.3 −10.5 X 

 Hill Slope 0.7 to 1.5 1.2 1.5 X 

 Test range 10−14 to 10−8 M 10−15 to 10−8 M 10−15 to 10−8 M X 

17α-estradiol

 Log PC50 −9.6 to −8.1 −8.6 −8.7 X 

 Log PC10 −10.7 to −9.3 −9.6 −9.6 X 

 Log EC50 −9.6 to −8.4 −8.6 −8.8 X 

 Hill Slope 0.9 to 2.0 1.6 2.2 X 

 Test range 10−12 to 10−6 M 10−13 to 10−6 M 10−13 to 10−6 M X 

Corticosterone

 Test range 10−10 to 10−4 M 10−11 to 10−4 M 10−11 to 10−4 M X 

17α-methyltestosterone

 Log PC50 −6.0 to −5.1 NC NC X 

 Log PC10 −8.0 to −6.2 −5.9 −5.9 X 

 Test range 10−11 to 10−5 M 10−12 to 10−5 M 10−12 to 10−5 M X 

a Data were obtained from pages 12 and 23 of the study report. 

NC = not calculable 

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Estrogen Receptor Transcriptional Activation (2012) / Page 8 of 13 

METOLACHLOR/108801 OCSPP 890.1300/ OECD 455 

FIGURE 1. Relative Transcription Activation (RTA) of the Natural Ligand, 17βestradiol, Compared to the Positive Control, 1 nM 17β estradiol. 

Mean ± SD are presented (n=6). 

 

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Estrogen Receptor Transcriptional Activation (2012) / Page 9 of 13 

METOLACHLOR/108801 OCSPP 890.1300/ OECD 455 

FIGURE 2. Relative Transcription Activation (RTA) of the Weak Estrogen, 17αestradiol, Compared to the Positive Control, 1 nM 17β estradiol. 

Mean ± SD are presented (n=6). 

 

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Estrogen Receptor Transcriptional Activation (2012) / Page 10 of 13 

METOLACHLOR/108801 OCSPP 890.1300/ OECD 455 

FIGURE 3. Relative Transcription Activation (RTA) of the Very Weak Agonist, 17αmethyltestosterone, Compared to the Positive Control, 1 nM 17β estradiol. 

Mean ± SD are presented (n=6). 

C. DEFINITIVE ASSAY 

1. Vehicle and Positive Controls: Data for the vehicle and positive controls are included in 

Table 4. The overall mean TA value for the vehicle control was 18092 for the first run and 

8938 for the second run; the overall mean TA value for the positive control was 516575 for 

the first run and 1023950 for the second run. The induction for the positive control ranged 

from 28.6- to 114.6-fold. The mean normalized value for the positive control was 498483 

for the first run and 1015013 for the second run. The PC50 (50% of the maximum response) 

for E2 in this assay is 249242 for the first run and 507507 for the second run; the PC10 (10% 

of the maximum response) is 49848 for the first run and 101501 for the second. 

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Estrogen Receptor Transcriptional Activation (2012) / Page 11 of 13 

METOLACHLOR/108801 OCSPP 890.1300/ OECD 455 

 TABLE 4. Transcriptional Activation (TA) Response of Vehicle and Positive Control a 

Sample Vehicle Control Positive Control b Normalized Positive Control b

Runs Mean SD Mean SD Fold Induction c Mean SD 

1 18092 3417 516575 129778 28.6 498483 129778 

2 8938 2194 1023950 254690 114.6 1015013 254690 

a Data were calculated by the reviewers from data obtained on pages 31-32 of the study report. 

b Positive control was 17β-estradiol (E2) at 1 nM. 

c Fold-induction = (mean TA of PC)/(mean TA of VC) 

2. Test Material: Relative (to the PC) transcriptional activation at each concentration of the 

test chemical during the two assay runs is presented in Table 5. The concentration-response 

curves depicting fold induction of relative transcriptional activation is presented in Figure 4 

below. The mean RPCMax was 3.3% for the first run and 0.6% for the second, and the 

associated PCMax was 10−7 M for both. Because the RPCMax < PC10 in both runs, 

metolachlor was considered negative for estrogen receptor transcriptional activation in this 

test system. However, due to the failed responsiveness of these cells to the very weak 

agonist 17α-methyltestosterone, the possibility that metalochlor is a very weak estrogen 

agonist could not be evaluated. 

TABLE 5. Relative Transcriptional Activation (RTA) of Metolachlor a

Parameter RTA (mean ± SD); % of Positive Control (PC) 

Run 1 Run 2 

Conc. (M) Mean SD Mean SD 

10−4 -- -- -- -- 

10−5 1.0 0.5 0.2 0.3 

10−6 1.2 0.5 0.5 0.3 

10−7 3.3 3.2 0.6 0.2 

10−8 1.1 1.8 0.0 0.1 

10−9 1.3 1.6 0.2 0.3 

10−10 1.3 1.9 0.0 0.2 

10−11 0.9 1.9 0.1 0.3 

Log EC50 NA NA 

Hill Slope NA NA 

RPCMax 3.3 0.6 

PCMax 10−7 10−7

PC50 NA NA 

PC10 NA NA 

a Data were obtained from pages 21-22 of the study report. 

NA = Not Applicable

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Estrogen Receptor Transcriptional Activation (2012) / Page 12 of 13 

METOLACHLOR/108801 OCSPP 890.1300/ OECD 455 

FIGURE 4. Fold Induction of Relative Transcription Activation (RTA) of Metolachlor 

Compared to the Positive Control. 

3. Performance Criteria: While the Log PC50, Log PC10, Log EC50, and Hill slope values for 

most of the concurrent reference chemicals fell within or near the acceptable ranges (Table 

3), the response of the cells to the reference chemical 17α-methyltestosterone was not 

adequate; the log PC10 for 17α-methyltestosterone was higher than the validated range in the 

both runs and the log PC50 for 17α-methyltestosterone was not calculable in either run. For 

17α-methyltestosterone, the mean RPCMax was 46.3% for the first run and 36.5% for the 

second run. 

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Estrogen Receptor Transcriptional Activation (2012) / Page 13 of 13 

METOLACHLOR/108801 OCSPP 890.1300/ OECD 455 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: The suitable top concentration for metolachlor for 

use in both independent runs was 10−5 M because of excessive cytotoxicity observed at 

nominal concentrations of 10−4 M and higher. Precipitation was also observed with 

metolachlor in the initial rangefinder at concentrations ≥10−4

 M. In both runs, there was 

slight precipitation of metolachlor observed at 10−4 M. In two independent runs of the 

transcriptional activation assay, metolachlor did not result in an increase in luciferase 

activity at any of the viable concentrations tested; metolachlor is not an agonist of human 

estrogen receptor alpha (hERα) in the HeLa-9903 model system. 

B. AGENCY COMMENTS: Metolachlor was tested up to a cytotoxic level, which was 

observed at concentrations ≥10−4 M. The mean RPCMax was 3.3% for the first run and 0.6% 

for the second, and the associated PCMax was 10−7 M for both. Because the RPCMax < PC10

in both runs, metolachlor was considered negative for estrogen receptor transcriptional 

activation in this test system. 

Although the conditions of this assay were not optimal to detect very weak activity, 

metolachlor responses were similar to those of the negative control corticosterone and not 

comparable to the responses of 17α-methyltestosterone, which was able to reach a maximum 

of 36.5-46.3% PC. Metolachlor was only able to reach a maximum of 0.6-3.3% PC when 

tested up to the highest concentration possible based on cytotoxicity. 

C. STUDY DEFICIENCIES: The following deficiencies were noted: 

 Responsiveness of the cells to the very weak positive control 17α-methyltestosterone 

was lower than the expected values, indicating a decreased sensitivity of the assay to 

very weak agonists. 

 Certificates of Analyses were not provided for the reference chemicals. 

 The source of the fetal bovine serum was not provided. 

Page 129 of 288
AMY 

BLANKINSHIP

Digitally signed by AMY BLANKINSHIP 

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

ou=USEPA, ou=Staff, cn=AMY 

BLANKINSHIP, dnQualifier=0000040917 

Date: 2015.06.04 12:14:32 -04'00'

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

EPA MRID Number 48617811 

Page 2 of 82 

CITATION: Palmer, S.J., K.H. Martin, T.Z. Kendall, and H.O. Krueger. 2012. Metolachlor: Fish Short-term 

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

Wildlife International, Ltd., Easton, Maryland. Lab Study No.: 568A-102. Study sponsored by Metolachlor 

Task Force, Stilwell, Kansas. Study completed January 23, 2012. 

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

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

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

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

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

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

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

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

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

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

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

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

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

EPA MRID Number 48617811 

Page 3 of 82 

EXECUTIVE SUMMARY:

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

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

group; 5.5 months old) were exposed to metolachlor (97.3% purity) at nominal concentrations of 0 (negative 

control), 0.050, 0.50, and 5.0 mg a.i./L; mean-measured concentrations were <LOQ (<0.0300, negative 

control), 0.046, 0.48, and 4.7 mg a.i./L. The test system was maintained at 24.1 to 25.2o

C and a pH of 7.0 

to 8.2. 

Overall survival was significantly reduced (p<0.05) by 66.7% for males, 73.3% for females, and 70.8% for 

the combined sexes at the high treatment level compared to the negative control. Additionally, male and female 

fish in the high treatment level showed signs of erratic swimming, loss of equilibrium, lethargic swimming, and 

curved spine; excess feed was observed in high treatment tanks. There were no significant differences 

(p>0.05) for male or female growth parameters (weight and length); however, there was a non-statistically 

significant decrease (p>0.05) of 23% in male body weight at the high treatment compared to the negative 

control. 

Mean spawning frequency and fecundity in the negative control were at least every 4 days and 28 

eggs/female/day (range 20.2-33.9), respectively; fertilization success in the negative control was 97.9%. 

There was a significant reduction (p<0.05) in fecundity of 98% at the high treatment level relative to the 

negative control; only one replicate in the high treatment level produced eggs, and there was 100% fertilization 

success in that replicate. 

Female vitellogenin (VTG) levels were significantly reduced (p<0.05) by 77% at the high treatment level 

relative to the negative control. Male VTG levels were not significantly different (p>0.05) from the negative 

control. There were no significant differences in female gonado-somatic index (GSI) compared to the negative 

control, but there was a marginally significant increase (p=0.057; Mann-Whitney) of 29% in male GSI at the 

high treatment level compared to the negative control. There was a significant reduction (p<0.05, MannWhitney) in male tubercle score at the high treatment level (median tubercle score: 5) compared to the 

negative control (median tubercle score: 22). No tubercles were observed in female fish. The study authors 

also reported a statistically-significant decrease (p<0.05; Jonckheere-Terpstra trend test) in mean male fatpad 

scores in the mid and high treatment groups compared to the negative control. Sex steroids were not measured. 

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

EPA MRID Number 48617811 

Page 4 of 82 

Gonadal histopathologic changes in females included increased oocyte atresia related to increases in granulosa 

cell hypertrophy and hyperplasia and increased granulosa cell phagocytosis as well as decreased early and 

late vitellogenic oocytes and ovary size at the high treatment level. In males of the high treatment group, 

germinal epithelium showed an increased presence of diploid spermatogonia with a decreased presence of 

spermatocysts containing spermatocytes and spermatids. One in six males in the high treatment group showed 

moderate alterations in these ratios and could not be staged under normal criteria. These treatment-related 

changes appeared to result from an inability of spermatogonia in the testes of affected fish to enter into 

spermatogenesis. 

All performance and validity criteria were met in this study. 

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

(OCSPP Guideline 890.1350). 

Results Synopsis

Test Organism age at test initiation: 5.5 months 

Mean body weight at test initiation (if measured): male 2.5 g; females 1.3 g. 

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

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

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

EPA MRID Number 48617811 

Page 5 of 82 

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

 Treatment 

(mg a.i./L)

[meanmeasured]

Fecundity 

Fert. 

Success 

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

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

0.046 No No No No No No No No No No NA NA NA NA 0.48 No No No No No No No No No No NA NA NA NA 4.7 Yes No Yes No Yes3 No Yes Yes No Yes NA NA NA NA 

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

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

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

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

3 The p value for this treatment was 0.057 (Mann-Whitney) suggesting a significant increase in male GSI compared to the control.

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

EPA MRID Number 48617811 

Page 6 of 82 

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 total organic carbon, unionized ammonia, and residual chlorine in the dilution water were not 

provided; EPA recommends that total organic carbon in dilution water be <2 mg/L; unionized ammonia 

in the dilution water be <1 Pg/L; and residual chlorine in dilution water be <10 Pg/L. 

2. Male and female body weight ranges for a subsample of 10 males and 10 females were ±30% of the 

mean weight of each sex. EPA recommends that the individual weight of each fish selected for the 

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

 These deficiencies/deviations do not have an impact on the interpretation of the study.

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

statements were provided. This study was conducted in accordance with GLP 

Standards as published by the U.S. EPA (40 CFR Parts 160), and OECD 

Principles of GLP [ENV/MC/CHEM(98)17]. 

A. TEST MATERIAL: Metolachlor, CAS No. 51218-45-2

Description: The test material was a liquid; insoluble in acetone (0.05%).

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. : 20081115 (Lot No.) 

Purity: 97.3% (originally determined June 15, 2010). 

Impurities: None reported

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Stability of Compound: Analytical verification of diluter stock fortified with 4.0, 40 and 400 mg a.i./L 

yielded recoveries ranging from 92.4 to 99.6% of nominal concentrations; 

and pre-test (Day -1) verification of samples fortified at 0.05, 0.50 and 5.0 

mg a.i./L yielded recoveries ranging from 77.6 to 89.1% of nominal 

concentrations. Analytical verification of the test solutions at Days 0, 7, 14, 

and 21 yielded recoveries ranging from 81.7% to 104% of nominal 

concentrations. The %CV for the 0.05, 0.50 and 5.0 mg a.i./L treatment 

levels were 10, 6.3 and 3.5%. These data indicate the test material was 

stable under the test conditions. 

Storage Conditions of 

Test Chemicals: The stock solution was stored at ambient temperature in the dark. Storage 

stability was determined after 7, 14, and 21 days; recoveries ranged from 

81.7 to 104%. 

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B. Test organism: 

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

Species common name: Fathead Minnow EPA recommends fathead minnow (Pimephales 

promelas). Species scientific name: Pimephales promelas

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

Yes Osage Catfisheries, Inc., Osage Beach, 

Missouri. 

EPA recommends that fish be from a single 

laboratory stock.

Were acclimation conditions same as definitive test?

Yes EPA recommends that fish be acclimated under 

water quality and illumination conditions that are 

similar to the definitive test. 

Acclimation period: 2 weeks EPA recommends a minimum two-week 

acclimation period. Note that the acclimation 

period is different from the subsequent, in situ 

pre-exposure phase. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on health: Mortalities during 7 days prior to preexposure period were not reported. 

Fish did not receive any treatment for disease during acclimation period.No behavioral abnormalities or clinical signs were noted. 

EPA recommends that mortality during the 7 

days prior to the pre-exposure phase be less 

than 5% of the culture population. If mortality 

during these 7 days is greater than 10%, EPA 

recommends that the fish be rejected. If 

mortality is between 5-10%, EPA recommends 

that fish be held another 7 days. If mortalities 

greater than 5% occur during this extended 

acclimation period, EPA recommends that the 

fish not be used. 

Type of food: Commercial flake food 

(Sera Vipan) and brine shrimp

EPA recommends that fish be fed frozen brine 

shrimp twice per day to promote active 

reproduction and maintain body condition.

Source of food: Sera, North AmericaFrequency of feeding: 2 times/day Details on feeding: 

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Table 3: Fish Selection and Pre-Exposure Performance.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Age at test initiation: 5-6 months EPA recommends reproductively mature 

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

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

2.5 g Based on 10 males used to stock aquaria 

for pre-exposure phase. 

EPA recommends that a subsample of fish be 

weighed before the test to estimate the mean 

weight for each sex. It is recommended that the 

individual weight of each fish selected for the 

test be within ±20% of the estimated mean for 

each sex.

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

Individual weights within ±30% of the estimated mean.

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

1.3 g Based on 10 females used to stock aquaria 

for pre-exposure phase. 

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

Individual weights within ±30% of the estimated mean.

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

Not reported 

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

Not reported 

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Were pre-exposure conditions identical to the definitive test?

Yes EPA recommends that pre-exposure conditions, 

including temperature, photoperiod, feeding, etc., 

be identical to definitive test conditions. 

Number of pre-exposure tanks: 32 tanks EPA recommends that additional tanks set up at 

the beginning of pre-exposure will ensure that 

sufficient replicates with the correct sex ratio are 

available for the definitive test. 

Number of males per tank: 2 males/tank Number of females per tank: 4 females/tank Pre-exposure fecundity: >15 eggs/female/ 

reproductive day/replicate

EPA recommends that pre-exposure fecundity in 

each replicate (tank) selected for use in the 

definitive test be at least 15 

eggs/female/reproductive day/replicate during 

the 7 days prior to the definitive test. 

Number of spawns during preexposure: >2 times in 7 days EPA recommends that spawning occur at least twice in the 7 days prior to the definitive test. Details on pre-exposure: None. 

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

Type of exposure: Flow-through EPA recommends the use of a flow-through 

system. As noted in the Corrections and 

Clarifications document1 , the use of a static 

renewal system is not recommended for this 

assay.

Type of flow-through dilution system:

Continuous flow diluter Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

Flow-through rate: 200 mL/min Recommended flow-through rate is 45 

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

volume exchanges per day.

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

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

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on toxicant mixing for flow-through systems: 

Solution from mixing chamber split equally among replicate test vessels. Treatment recoveries prior to test initiation ranged from 44.6-89.1%. The flow splitting accuracy was within ±10%. The test solutions were clear and colorless during the test with no evidence of precipitation 

or surface films in any control or test solution. 

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? No EPA recommends aeration if dissolved oxygen 

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

Source of dilution water: Well water Prior to use, the water was sand-filtered, 

aerated, and UV-irradiated. 

EPA recommends natural or reconstituted 

water; it is recommended that natural water 

be sterilized with UV and tested for 

pesticides, heavy metals, and other possible 

contaminants. OECD accepts any water in 

which the test species show control survival 

at least as good as indicated in the test 

guideline.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

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

Yes All potential contaminants were confirmed to be 

below the detection limit.

Test vessel type/materials: Glass aquaria with stainless 

steel screen drains

EPA and OECD recommend that watercontact portions of the system not 

compromise the study (e.g., all glass vessels 

or glass vessels with stainless steel frames 

are acceptable examples).

Test vessel size: 19L aquaria; solution depth 

10 cm.

Dimensions not reported. EPA recommends the use of 18 L test 

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

Fill volume: 10 L EPA recommends 10 L solution per tank. Spawning substrate material: PVC pipe cut and sectioned 

lengthwise and inverted to form a semicircular arch. 

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: Dimensions not reported. 

Additional details on exposure system: 

None. 

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

Parameter Minimum Maximum Mean1 Measurement Interval Guideline Recommendations 

Temperature (°C) 24.1 25.2 25.0 Continuous 

EPA recommends temperature 25±1oC; interreplicate and inter-treatment differentials should not exceed 1oC.

pH 8.0 8.2 8.1 Weekly EPA recommends pH 6.5 to 9.0.Dissolved oxygen (mg/L) 6.1 7.7 6.9 Weekly EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air saturation)Total alkalinity (mg/L as CaCO3) 175 178 176.5 Weekly EPA recommends total alkalinity >20 mg/L 

as CaCO3.

Hardness (mg/L as CaCO3) 136 148 142 Weekly Total organic carbon (mg/L) Not reported Not reported Not reported Not reported EPA recommends that total organic carbon in 

dilution water be <2 mg/L. 

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

EPA recommends that unionized ammonia in 

the dilution water be <1 Pg/L. 

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

EPA recommends that residual chlorine in 

dilution water be <10 Pg/L. 

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Parameter Minimum Maximum Mean1 Measurement Interval Guideline Recommendations 

Conductivity (Pmhos/cm) 306 461 182 Weekly 

General recommendations for frequency of 

measurements: EPA recommends that 

temperature, pH, and dissolved oxygen be 

measured in all test tanks at least weekly and 

that hardness and alkalinity be measured in 

controls and in one tank at the highest test 

concentration at least weekly. In addition, 

continuous temperature monitoring of at least 

one tank is encouraged. 

Abbreviations: NA Not applicable. 1 Means were calculated by the reviewer as the average of the minima and maxima for the ranges provided across control and treated levels. D. Study Design and Additional Experimental Conditions

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

if 96-hour LC50 data for the fathead minnow 

are unavailable. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

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

Not reported. 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: Fathead Minnow Pimephales promelas Life stage: Not reported Sexually mature 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-hrs EPA recommends a 96-hour exposure. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Additional details: NA EPA recommends conducting a range-finder 

with five test concentrations plus a control 

(six total treatment levels), with four 

females and two males per exposure tank 

(36 fish total). The number of mortalities 

that occur may be used to develop a 

concentration-response curve. 

Based upon the results, the highest 

concentration that does not result in 

increased mortality or signs of overt 

morbidity compared to controls, or 1/3 the 

derived 96-hr LC50, may be selected as the 

highest exposure concentration in the 21-

day test.

Table 7: Definitive Study Conditions.

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

test be 21 days. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

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

The acute toxicity 96-hour LC50 for metolachlor 

with fathead minnow is 15 mg/L. 

Selected based on 1/3 of the 96-hour LC50

(15 mg a.i./L). 

EPA recommends that the highest test 

concentration is either the solubility limit of the 

test compound, 100 mg/L, or demonstrates 

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

hour LC50), whichever concentration is lowest.

Reference study citation (if applicable):

Not reported. 

Separation of test concentrations: Ten-fold EPA suggests that a concentration separation of 

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

Number of test concentrations: 4 (3 treated, and a 

negative control)

EPA recommends a minimum of 3 

concentrations and a control, plus solvent control 

if appropriate.

Are nominal concentrations adjusted for purity? 

Yes 

Indicate the type of values presented for measured concentrations: 

Mean-measured 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Limit of quantification (LOQ): 0.0300 mg a.i./L EPA recommends that for chemical test 

concentrations below the LOQ, analyses be 

conducted on the stock solutions.

Level of detection (LOD): Not reported. Frequency of measurement: 0, 7, 14 and 21 days It is recommended that test item concentration 

be measured prior to the addition of fish in all 

tanks and at least weekly thereafter in two 

replicates per treatment level.

Was the randomized complete block design used?

Yes EPA recommends that all fish be randomly 

assigned to tanks during pre-exposure. Tanks 

are then ranked according to pre-exposure 

fecundity, and the tanks with the highest 

fecundity are randomly assigned to a definitive 

test treatment and block first. Each block 

contains one replicate of each treatment, 

including controls. 

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

NA EPA recommends the use of a concurrent 

solvent control when a solubilizing agent is used. 

EPA recommends 4 replicates.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Number of replicates per test item treatment level: 

4 EPA recommends 4 replicates.

Number of male fish per replicate at test initiation:

2 One fish in replicate C of the 4.7 mg a.i./L 

group was sexed incorrectly during the preexposure period (immature male considered a female). The error was determined during 

dissection due to the presence of testis in this fish. Therefore, this tank actually had 3 males and 3 females at the start of the exposure period. 

EPA recommends 2 males per replicate. 

Number of female fish per replicate at test initiation: 

4 EPA recommends 4 females per replicate. 

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Maximum solvent concentration (if applicable):

NA EPA recommends that the solvent not exceed 

0.02 ml/L3. OECD recommends that solvent 

have no effect on survival nor produce any other 

adverse effects and that concentration not be 

greater than 0.1 ml/L4.

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

NA 

Photoperiod: 16 hrs light: 

8 hrs dark 

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

Light intensity at water’s surface: 432 to 636 lux EPA recommends light intensity 540 – 1080 lux 

(at water’s surface).

Additional details: The test solutions and stock solutions 

appeared clear and colorless with no evidence of precipitate 

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

Toxicology, 76, pp.69–92. 

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

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

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

Treatment ID 

Nominal 

Concentration

(mg a.i./L) 

Mean Measured 

Concentration

(mg a.i./L) 

Mean CV (%) Details or Remarks Guideline Recommendations 

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

be maintained at a coefficient of variation (CV) 

20%.

Treatment 1 0.050 0.046 10 

Treatment 2 0.50 0.48 6.3 Treatment 3 5.0 4.7 3.5 

Abbreviations: CV Coefficient of variation. 

LOQ=0.0300 mg a.i./L E. Observations

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

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

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Were raw (individual) data provided? Yes. It appears that one fish in the 4.7 mg a.i./L treatment group was mis-sexed (male classified as female); however, the occurrence of three males and three females in the replicate does not appear to be discussed in the study report. Data from this fish 

appear to be included in statistical analysis by the study author.

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

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

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

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

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

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II. RESULTS AND DISCUSSION 

A. Results 

One male died in the 0.48 mg a.i./L treatment group and two died in the 4.7 mg a.i./L treatment 

group during the study. One female died in each of the negative control, 0.046 and 0.48 mg a.i./L 

treatment groups. Five females died in the 4.7 mg a.i./L treatment group. Overall mean survival 

values were 95.8, 95.8, 91.7 and 70.8% in the mean-measured 0 (control), 0.046, 0.48, and 4.7 

mg a.i./L treatment levels, respectively. 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Males Females 

n1 # Surviving % Survival2 n # Surviving % Survival2

Negative Control (<LOQ) 8 8 100 16 15 93.8 

0.046 8 8 100 16 15 93.8 

0.48 8 7 87.5 16 15 93.8 

4.7 93 63 66.7 153 11 73.3 

Abbreviations: NA Not applicable. 

LOQ=0.0300 mg a.i./L 

1

 Total number of fish at test initiation. 

2

 The percent survival for each sex was calculated by the reviewer based on the survival data. 

3 Based on the study report, there appeared to be three females and three males present at test termination in one 

replicate; therefore, the initial number of males and females in this treatment were 9 and 15, respectively. 

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Mean male body weight values were 3.12, 2.97, 3.28 and 2.39 g and female body weight values 

were 1.34, 1.41, 1.39 and 1.21 g in the mean-measured 0 (control), 0.046, 0.48, and 4.7 mg a.i./L 

treatment levels, respectively. Mean male body length values were 61, 60, 63, and 58 mm and female 

body length values were 49, 49, 49 and 48 mm in the mean-measured 0 (control), 0.046, 0.48, 

and 4.7 mg a.i./L treatment levels, respectively. 

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

Treatment 

(mg a.i./L) 

[meanmeasured]

Body Weight Length 

Males Females Males Females 

n 

Mean

(g) 

±SD n

Mean

(g) 

±SD n 

Mean

(mm) 

±SD n 

Mean

(mm) 

±SD 

Negative 

Control 

(<LOQ) 

4 3.12 0.08 4 1.34 0.11 4 61 1.0 4 49 0.8 

0.046 4 2.97 0.47 4 1.41 0.13 4 60 2.5 4 49 0.8 

0.48 4 3.28 0.53 4 1.39 0.13 4 63 3.4 4 49 1.7 

4.7 4 2.39 0.53 4 1.21 0.10 4 58 1.4 4 48 2.1 

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

LOQ=0.0300 mg a.i./L 

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Mean fecundity values were 27.9, 29.9, 22.0 and 0.5 and fertilization success was 97.9, 98.6, 97.9 

and 100% in the mean-measured 0 (control), 0.046, 0.48, and 4.7 mg a.i./L treatment levels, 

respectively. Fertilization success ranged from 97.9 to 100%. 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Fecundity1 Fertilization Success (%)2

mean ± SD mean ± SD 

Negative Control (<LOQ) 27.9 6.15 97.9 1.08 

0.046 29.9 5.86 98.6 0.39 

0.48 22.0 4.76 97.9 0.89 

4.7 0.5 1.00 1003 NA 

Abbreviations: NA Not applicable. ND Not determined. 

LOQ=0.0300 mg a.i./L

1

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

2

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

3

 Only one replicate produced eggs. 

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Median male tubercle scores were 22, 20, 22 and 5 in the mean-measured 0 (control), 0.046, 0.48, and 

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

tubercles. 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Males Females 

n Median Tubercle 

Score n Median Tubercle Score 

Negative Control (<LOQ) 4 22 4 0 

0.046 4 20 4 0 

0.48 4 22 4 0 

4.7 4 5 4 0 

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

LOQ=0.0300 mg a.i./L 

The study author reported fatpad scores (Table 12b). Mean male fatpad scores were 3.5, 3.4, 2.8 and 1.9 

in the mean-measured 0 (control), 0.046, 0.48, and 4.7 mg a.i./L treatment levels, respectively. Mean 

female fatpad scores were 1.0 at all treatment levels. 

Table 12b: Fatpad Score in Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[mean-measured]

Males Females 

n Mean Fatpad 

Score n Mean Fatpad Score 

Negative Control (<LOQ) 4 3.5 4 1.0 

0.046 4 3.4 4 1.0 

0.48 4 2.8 4 1.0 

4.7 4 1.9 4 1.0 

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

LOQ=0.0300 mg a.i./L. 

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Mean male GSI was 1.45, 1.78, 1.53 and 1.87 and mean female GSI was 12.6, 12.8, 12.8 and 12.0 in 

the mean-measured 0 (control), 0.046, 0.48, and 4.7 mg a.i./L treatment levels, respectively (Table 13). 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Males Females 

n Mean GSI1

(%) 

±SD n Mean GSI1

(%) 

±SD 

Negative Control (<LOQ) 4 1.45 0.14 4 12.6 1.1 

0.046 4 1.78 0.48 4 12.8 1.4 

0.48 4 1.53 0.26 4 12.8 0.15 

4.7 4 1.87 0.32 4 12.0 2.6 

Abbreviations: NA Not applicable. 

LOQ=0.0300 mg a.i./L

1

 Gonado-somatic index (%) is calculated as gonad weight (to the nearest 0.1 mg) / body weight (mg) x 100. 

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

the mean-measured 0 (control), 0.046, 0.48, and 4.7 mg a.i./L treatment levels, respectively (Table 14). 

The study authors did indicate that testes development was clearly disrupted in one fish in the 4.7 mg 

a.i./L treatment. 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Males Females 

n Median Stage1 n Median Stage2

Negative Control (<LOQ) 8 3 15 3 

0.046 8 2 15 3 

0.48 7 2 15 3 

4.7 5 3 11 2 

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

LOQ=0.0300 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, germinal epithelium showed an increased presence of diploid spermatogonia with decreased 

presence of spermatocysts containing spermatocytes and spermatids. One in six male fish showed moderate 

alterations in these ratios and could not be staged under normal criteria. These changes appeared to result 

from a treatment-related inability of spermatogonia in the testes of affected fish to enter into 

spermatogenesis. These results indicate an inability of immature germ cells in the testes that had developed 

into spermatogonia to enter into spermatogenesis in the 4.7 mg a.i./L treatment group. This directly reduces 

the number of spermatocysts containing primary spermatocytes, leading to a concomitant decrease in 

spermatocysts containing secondary spermatocytes and spermatids. 

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In females, an increase in atresia was related to increases in granulose cell hypertrophy and hyperplasia 

and granulose cell phagocytosis, as well as decreases in late vitellogenic oocytes and ovary size. Moderate 

atresia was observed in one fish in the 0.48 mg a.i./L treatment group. These results indicate mild to 

marked increase in atresia that was treatment related. 

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Table 15: Gonadal Histopathology in Male Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[mean-measured] 

Diagnostic Observations1

Severity

Increased Proportion of 

Spermatogonia 

Presence of Testis-Ova 

Increased Testicular 

Degeneration 

Interstitial Cell 

Fibrosis 

Germinal Epithelium, 

Atrophy 

n Incidence n Incidence n Incidence n Incidence n Incidence 

Negative Control 

(<LOQ) 

0 8 8 NA NA 8 7 NA NA 8 8 

1 8 0 NA NA 8 0 NA NA 8 0 

2 8 0 NA NA 8 1 NA NA 8 0 3 8 0 NA NA 8 0 NA NA 8 0 4 8 0 NA NA 8 0 NA NA 8 0 

0.046 0 8 8 NA NA 8 8 NA NA 8 8 

1 8 0 NA NA 8 0 NA NA 8 0 

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

0.48 0 7 7 NA NA 7 7 NA NA 7 3 

1 7 0 NA NA 7 0 NA NA 7 0 

2 7 0 NA NA 7 0 NA NA 7 4 3 7 0 NA NA 7 0 NA NA 7 0 4 7 0 NA NA 7 0 NA NA 7 0 

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Treatment 

(mg a.i./L) 

[mean-measured] 

Diagnostic Observations1

Severity

Increased Proportion of 

Spermatogonia 

Presence of Testis-Ova 

Increased Testicular 

Degeneration 

Interstitial Cell 

Fibrosis 

Germinal Epithelium, 

Atrophy 

n Incidence n Incidence n Incidence n Incidence n Incidence 

4.7 0 6 2 NA NA 6 3 NA NA 6 6 

1 6 0 NA NA 6 0 NA NA 6 0 

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

Abbreviations: NA Not applicable. 

LOQ=0.0300 mg a.i./L 

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E of 

the test guideline for reference. 

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

Treatment 

(mg a.i./L) 

[mean-measured] 

Additional Diagnostic Observations1

Severity 

Decreased Proportion 

of Spermatogonia

Increased Vascular or 

Interstitial 

Proteinaceous Fluid 

Asynchronous 

Gonad 

Development 

Altered Proportions of 

Spermatocytes or 

Spermatids 

Granulomatous 

Inflammation 

n Incidence n Incidence n Incidence n Incidence n Incidence 

Negative Control 

(<LOQ) 

0 8 NA NA NA NA NA 8 8 8 8 

1 8 NA NA NA NA NA 8 0 8 0 

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

0.046 0 8 NA NA NA NA NA 8 8 8 8 

1 8 NA NA NA NA NA 8 0 8 0 

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

0.48 0 7 NA NA NA NA NA 7 6 7 6 

1 7 NA NA NA NA NA 7 0 7 0 

2 7 NA NA NA NA NA 7 0 7 0 3 7 NA NA NA NA NA 7 0 7 0 4 7 NA NA NA NA NA 7 1 7 1 

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Treatment 

(mg a.i./L) 

[mean-measured] 

Additional Diagnostic Observations1

Severity 

Decreased Proportion 

of Spermatogonia

Increased Vascular or 

Interstitial 

Proteinaceous Fluid 

Asynchronous 

Gonad 

Development 

Altered Proportions of 

Spermatocytes or 

Spermatids 

Granulomatous 

Inflammation 

n Incidence n Incidence n Incidence n Incidence n Incidence 

4.7 0 6 NA NA NA NA NA 6 6 6 6 

1 6 NA NA NA NA NA 6 2 6 1 

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

Abbreviations: NA Not applicable. 

LOQ=0.0300 mg a.i./L 

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E of 

the test guideline for reference. 

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

Treatment 

(mg a.i./L) 

[meanmeasured] 

Additional Diagnostic Observations1

Severity 

Increased Oocyte 

Atresia 

Granulosa Cell 

Hyperplasia/ 

Hypertrophy 

Increased Granulosa 

Cell Phagocytosis 

Infection, 

Microsporidia 

n Incidence n Incidence n Incidence n Incidence 

Negative 

Control 

(<LOQ) 

0 15 15 15 15 15 15 NA NA 

1 15 0 15 0 15 0 NA NA 

2 15 0 15 0 15 0 NA NA 

3 15 0 15 0 15 0 NA NA 

4 15 0 15 0 15 0 NA NA 

0.046 0 15 15 15 15 15 15 NA NA 

1 15 0 15 0 15 0 NA NA 

2 15 0 15 0 15 0 NA NA 

3 15 0 15 0 15 0 NA NA 

4 15 0 15 0 15 0 NA NA 

0.48 0 15 15 15 15 15 15 NA NA 

1 15 0 15 0 15 0 NA NA 

2 15 0 15 0 15 0 NA NA 

3 15 0 15 0 15 0 NA NA 

4 15 0 15 0 15 0 NA NA 

4.7 0 11 3 11 8 11 5 NA NA 

1 11 0 11 0 11 0 NA NA 

2 11 2 11 3 11 6 NA NA 

3 11 0 11 0 11 0 NA NA 

4 11 6 11 0 11 0 NA NA 

Abbreviations: NA Not applicable. 

LOQ=0.0300 mg a.i./L 

1

 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 

2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference. 

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

promelas).

Treatment 

(mg a.i./L)

[meanmeasured]

Additional Diagnostic Observations1

Severity 

Early Vitellogenic 

Oocytes Decrease 

Late Vitellogenic 

Oocytes 

Decrease 

Granulomatous 

Inflammation 

Decreased PostOvulatory Follicles 

n Incidence n Incidence n Incidence n Incidence 

Negative 

Control 

(<LOQ) 

0 15 15 15 15 15 15 NA NA 

1 15 0 15 0 15 0 NA NA 

2 15 0 15 0 15 0 NA NA 

3 15 0 15 0 15 0 NA NA 

4 15 0 15 0 15 0 NA NA 

0.046 0 15 14 15 14 15 14 NA NA 

1 15 0 15 0 15 0 NA NA 

2 15 0 15 0 15 0 NA NA 

3 15 0 15 0 15 0 NA NA 

4 15 1 15 1 15 1 NA NA 

0.48 0 15 15 15 15 15 15 NA NA 

1 15 0 15 0 15 0 NA NA 

2 15 0 15 0 15 0 NA NA 

3 15 0 15 0 15 0 NA NA 

4 15 0 15 0 15 0 NA NA 

4.7 0 11 7 11 6 11 11 NA NA 

1 11 0 11 0 11 0 NA NA 

2 11 0 11 1 11 0 NA NA 

3 11 2 11 0 11 0 NA NA 

4 11 2 11 4 11 0 NA NA 

Abbreviations: NA Not applicable. 

LOQ=0.0300 mg a.i./L 

1

 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 

2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference. 

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Mean male VTG was 442.5, 527.5, 765.0 and 822.5 ng/mL and female VTG was 3260750, 3294750, 

3371500 and 765750 ng/mL in the mean-measured 0 (control), 0.046, 0.48, and 4.7 mg a.i./L 

treatment levels, respectively (Table 19). 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Plasma Vitellogenin (VTG) 

Males Females 

n Mean

(ng/mL plasma) 

±SD n Mean

(ng/mL plasma) 

±SD 

Negative Control (<LOQ) 4 442.5 286.5 4 3260750 486506 

0.046 4 527.5 349.0 4 3294750 597410 

0.48 4 765.0 1139 4 3371500 839708 

4.7 4 822.5 1028 4 765750 460589 

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

LOQ=0.0300 mg a.i./L. 

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Male and female plasma testosterone and plasma 17ȕ-estradiol were not measured. 

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

Treatment 

(mg a.i./L) 

[mean-measured]

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

Males Females Males Females 

n 

Mean

(ng/mL 

plasma) 

±SD n 

Mean

(ng/mL 

plasma) 

±SD n

Mean

(ng/mL 

plasma) 

±SD n

Mean

(ng/mL 

plasma) 

±SD 

Negative Control (<LOQ) NA NA NA NA NA NA NA NA NA NA NA NA 

0.046 NA NA NA NA NA NA NA NA NA NA NA NA 

0.48 NA NA NA NA NA NA NA NA NA NA NA NA 

4.7 NA NA NA NA NA NA NA NA NA NA NA NA 

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

LOQ=0.0300 mg a.i./L 

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There appeared to be an increase in clinical signs of overt toxicity at the 4.7 mg a.i./L treatment level in both males and females that were interpreted 

by the study authors as treatment-related (Table 21). Also, excess feed was noted in the bottom of the test chamber at 4.7 mg a.i./L compared to the control. All other fish reported as appearing normal at test termination. 

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

Treatment 

(mg a.i./L) 

[mean-measured] 

Secondary Sex Characteristics and Clinical Signs at Test Termination 

Males Females 

Type n Incidence Type n Incidence 

Negative Control (<LOQ) Redness behind operculum, dead 8 1 None 15 0 

0.046 Hemorrhage in or missing eye 8 1 Hemorrhage in or missing eye 15 1 

0.48 None 7 0 None 15 2 

4.7 

Erratic swimming, loss of equilibrium, 

curved spine 

6 1 

Erratic swimming and lethargic swimming 

11 

1 

 Loss of equilibrium and lethargic swimming 1 

LOQ=0.0300 mg a.i./L 

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B. Study Author’s Analysis and Conclusions

The study author analyzed survival, weight, length, tubercle score, GSI, fertility (% eggs that were 

fertile), fecundity (#eggs/female/reproductive day), and VTG. Data for growth, tubercle score, GSI, 

and VTG were analyzed separately by sex. For all parameters, metolachlor-treated conditions were 

compared to the control. The study author’s performed statistical analyses using SAS software (Version 

8.2). Data from the one mis-sexed fish in the high treatment group was used in the data analysis. 

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

effects were detected for p<0.05 unless otherwise noted (statistical program used was not reported). 

Effects on survival were determined using a Fisher’s exact test. All endpoints except survival, fertility 

and histopathology severity scores and stages were determined by ANOVA and a pair-wise Dunnett’s 

test or Wilcoxon rank-sum test. For endpoints showing a monotonic response, a step-down 

Jonckheere-Terpstra trend test was used. Prior to Dunnett’s, data were analyzed by Shapiro-Wilk’s 

test and Levene’s to test for normality and homogeneity of variances, respectively. If normality and 

homogeneity tests passed (p>0.01), a parametric analysis was performed. If non-normality or unequal 

variance were indicated (p<0.01), a non-parametric analysis was performed on the ranks of the data. 

These methods appear to be consistent with the methods recommended in the guideline. 

Adult survival (range 70.8-95.8%) was significantly different compared to the controls (95.8%) at the 

4.7 mg a.i./L treatment level (p<0.05; Fisher’s Exact test). There was also a statistically significant 

decrease in fecundity in the 4.7 mg a.i./L treatment group compared to the control (p<0.05; 

Jonckheere-Terpstra test and Dunnett’s test). There were no statistically differences in the growth 

parameter (weight and length) in either sex compared to the control (p>0.05). There was also no 

statistically significant effect on male GSI or VTG. There was a statistically significant decrease in male 

tubercle score at the 4.7 mg a.i./L treatment level compared to the control (p<0.05; Dunnett’s test), 

and a significant decrease in Fatpad score at the 0.48 mg a.i./L (p<0.05; Jonckheere-Terpstra test) 

treatment level and the 4.7 mg a.i./L treatment levels compared to the control (p<0.05; JonckheereTerpstra trend test and Dunnett’s test). There was also a statistically significant decrease in female 

VTG at the 4.7 mg a.i./L (p<0.05; Jonckheere-Terpstra and Dunnett’s test). Although not analyzed 

statistically, there were generalized overt signs of toxicity in both males and females (i.e., dead, erratic 

swimming, loss of equilibrium, lethargic swimming, curved spine). Histopathology results were 

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statistically analyzed and significant effects were determined in both males and females at the 4.7 mg 

a.i./L treatment level. In males, there were statistically significant increase in proportion of 

spermatogonia, altered proportion of spermatocytes and spermatids, and deceased gonad size, sertoli 

cells, germinal epithelium thickness and milt at the 4.7 mg a.i./L treatment compared to the control 

(p=0.001 to p=0.02; Jonckheere-Terpstra test). In females, there were statistically significant increase 

in oocyte atresia, granulose cell hyperplasia/hypertrophy, and granulose cells phagocytosis, and 

decreases in early and late vitellogenic oocytes at the 4.7 mg a.i./L treatment compared to the control 

(p<0.0001 to p=0.002; Jonckheere-Terpstra test). 

 C. Reviewer’s Analysis and Conclusions 

Statistical Methods: The reviewer analyzed survival (mortality) data using Fisher’s Exact test via 

Toxstat 3.5. Female and male weight, male length, female mean VTG, male GSI, and fertility 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 that failed parametric assumptions were analyzed using the non-parametric MannWhitney U test. Female body weight, male VTG, female GSI, male tubercle score and fecundity data 

exhibited monotonic trends and were analyzed using the non-parametric Jonckheere-Terpstra test, as 

recommended by the OCSPP 890.1350 guideline. There was no response for fertility; however, only 

one replicate at the high concentration produced eggs, of which 100% survived. The reviewer compared 

all metolachlor-treated groups to the negative control group. Female VTG exhibited a significant 

reduction of 77% compared to the negative control, at the 4.7 mg a.i./L treatment levels (p<0.05; 

Dunnett’s test). Fecundity was significantly reduced 98% at the 4.7 mg a.i./L treatment, compared to 

the negative control (p<0.05; Jonckheere-Terpstra). Growth was unaffected in male and female fish 

relative to the negative control. Survival (combined sexes) was significantly different at the 4.7 mg 

a.i./L treatment level (p<0.05; Fisher’s Exact test) compared to the control. 

Histopathology and secondary sex characteristics were adversely indicated at the 4.7 mg a.i./L level, 

but these effects were not statistically analyzed by the reviewer. Male and female fish at the 4.7 mg 

a.i./L level showed signs of erratic swimming, loss of equilibrium, lethargic swimming, and curved 

spine. In males, there was an increase in the proportion of spermatogonia, altered proportion of 

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spermatocytes and spermatids, and deceased gonad size, sertoli cells, germinal epithelium thickness 

and milt at the 4.7 mg a.i./L treatment compared to the control or lower treatment levels. One in six 

male fish showed could not be staged under normal criteria at the 4.7 mg a.i./L treatment. In females, 

there was an increase in atresia, granulose cell hypertrophy and hyperplasia and granulose cell 

phagocytosis, as well as decreases in early and late vitellogenic, oocytes and ovary size in the 4.7 

mg a.i./L treatment compared to the control and lower metolachlor-treated levels. 

The reviewer did not re-analyze the fat pad score data and relied on the study author’s conclusion for 

this endpoint. 

It appears that the there was a mis-sexed fish in replicate C of the high treatment group, based on 

visual observation of the data for the fish; however, this occurrence of three males and three females 

does not appear to be discussed in the study report. Based on the raw data, the mis-sexed fish 

appears to be fish #4 in that replicate, but the study report does not confirm this. In the analysis of 

the data, all male endpoints for this replicate were included in the analysis; however, if fish #4 from 

that replicate were removed from the analysis it doesn’t appear that it would make a difference in the 

interpretation of the overall response and conclusion for that treatment group for any endpoint. 

 Conclusions: 

Survival (combined sexes) was significantly reduced at the 4.7 mg a.i./L treatment level (p<0.05; 

Fisher’s Exact test) compared to the control. Additionally, male and female fish at the 4.7 mg a.i./L 

level showed signs of erratic swimming, loss of equilibrium, lethargic swimming, and curved spine. 

Female VTG was reduced 77% and fecundity by 98% at the highest treatment level, compared to the 

negative control. However, there was no effect on fertility due to only one replicate producing eggs 

and 100% fertilization success in that replicate. There was also a significant increase in oocyte atresia 

related to increases in granulose cell hypertrophy and hyperplasia and granulose cell phagocytosis, as 

well as decreases in late vitellogenic oocytes and ovary size at the highest treatment level. There were 

no significant differences in female GSI compared to the control; the p value for male GSI at the 4.7 

mg a.i./L was 0.057, suggesting a significant increase compared to the control. A significant 76% 

reduction in male tubercle score at the 4.7 mg a.i./L treatment was observed compared to the control. 

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No tubercles were observed in female fish. The study authors found a statistically significant decrease 

in male Fatpad score at the 0.48 mg a.i./L (p<0.05; Jonckheere-Terpstra test) treatment level and 

the 4.7 mg a.i./L treatment levels compared to the control (p<0.05; Jonckheere-Terpstra trend test 

and Dunnett’s test). In males, germinal epithelium showed an increased presence of diploid 

spermatogonia with decreased presence of spermatocysts containing spermatocytes and spermatids. 

One in six male fish showed moderate alterations in these ratios and could not be staged under normal 

criteria. These changes appeared to result from a relative inability of spermatogonia in the testes of 

affected fish to enter into spermatogenesis that was treatment-related. These results indicate an inability 

of immature germ cells in the testes that had developed into spermatogonia to enter into 

spermatogenesis in the 4.7 mg a.i./L treatment group. This directly reduces the number of 

spermatocysts containing primary spermatocytes, leading to a concomitant decrease in spermatocysts 

containing secondary spermatocytes and spermatids. 

 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Tubercle Score3 GSI 

Gonadal 

Staging and 

Histo. 

Plasma VTG Plasma T Plasma E2 

Median p % Diff. p 

Effect? 

(Yes/No) 

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

Negative Control 

(<LOQ) 

22 NA 0 NA NA 0 NA 0 NA 0 NA

0.046 20 0.094 23.14 0.486 No 19.21 0.282 NA NA NA NA 

0.48 22 0.500 5.87 0.486 No 72.884 0.696 NA NA NA NA 

4.7 5 0.041 29.19 0.057 Yes 85.884 0.663 NA NA NA NA 

Statistical Test Jonckheere-Terpstra MannWhitney NA Jonckheere-Terpstra NA NA 

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

LOQ=0.0300 mg a.i./L 

1 Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusions regarding 

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

2 Unless otherwise specified, effects are considered statistically significant at p<0.05. 3 The study authors reported a statistically significant decrease in mean male fatpad scores at the 0.48 and 4.7 mg a.i./L treatment group compared to the control (p<0.05; 

Jonckheere-Terpstra trend test). 

4 These differences were due to VTG values in one fish for each group being substantially greater than the control VTG values. 

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

Treatment 

(mg a.i./L) 

[meanmeasured]

Fecundity Fert. Success Tubercle Score GSI 

Gonadal 

Staging and 

Histo. 

Plasma VTG Plasma T Plasma E2

% Diff. p % Diff. P Median p 

% 

Diff. 

p

Effect? 

(Yes/No) 

% Diff. p 

% 

Diff. 

p % Diff. p

Negative Control 

(<LOQ) 

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

0.046 7.26 0.614 0.71 0.343 0 NA 1.39 0.718 No 1.04 0.614 NA NA NA NA 

0.48 -21.08 0.094 -0.08 >0.99 0 NA 1.59 0.441 No 3.40 0.670 NA NA NA NA 

4.7 -98.21 <0.001 2.1 --3 0 NA -4.37 0.425 Yes -76.52 0.025 NA NA NA NA 

Statistical Test Jonckheere-Terpstra Mann Whitney U NA 

JonckheereTerpstra NA Jonckheere- Terpstra NA NA 

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

LOQ=0.0300 mg a.i./L. 

1 Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusions regarding 

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

2 Unless otherwise specified, effects are considered statistically significant at p<0.05. 3 Only one replicate in this treatment produced eggs and fertilization success was 100%. 

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

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 

0.046 -4.96 0.927 4.66 0.801 -2.45 0.684 0.00 0.500 

0.48 4.88 0.930 3.35 0.909 2.04 0.782 -0.51 0.322 

4.7 -23.38 0.096 -9.87 0.311 -5.31 0.158 -2.55 0.155 

Statistical Test Dunnett’s Dunnett’s Dunnett’s Jonckheere-Terpstra 

Abbreviations: Diff. Difference. NA Not applicable. 

LOQ=0.0300 mg a.i./L 

1

 Unless otherwise indicated, percent (%) differences are reported based on comparison to the negative (clean water) 

control. 

2

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

E. Study Deficiencies 

There were deviations/deficiences 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 generally in agreement. The reviewer and study authors 

agreed on the subjectively-determined effects on secondary sex characteristics, gonad development, and 

histopathology. 

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III. REFERENCES 

U.S. Environmental Protection Agency. 2009. Series 890-Endocrine Disruptor Screening Program Test 

Guidelines, OCSPP Number 890.1350: Fish Short-Term Reproduction Assay. 

Organization for Economic Cooperation and Development. 2009. OECD Guidelines for Testing of 

Chemicals, Guideline 229. Fish Short Term Reproduction Assay. Adopted 7 September 2009. 

APHA, AWWA, WEF. 1998. Standard Methods for the Examination of Water and Wastewater. 20th Edition, 

American Public Health Association. American Water Works Association. Water Environment 

Federation, Washington, D.C. 

Organization for Economic Cooperation and Development. 2006. Current Approaches in the Statistical 

Analysis of Ecotoxicity Data: A Guidance to Application. OECD ENV/JM/MONO(2008)18. 

The SAS System for Windows. 1999 - 2001. Version 8.2. SAS Institute, Inc. Cary, North Carolina. 

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APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION:

Reviewer’s statistical verification table 

Endpoint Monotonic? Parametric? OCSPP 

890.1350 

Historical EFED Comments 

Female body weight 

No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Male body weight No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Female body length 

Yes No Jonckheere: 

n.s. p>0.05 

Jonckheere: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Male body length No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Female VTG No Yes Dunnett’s: 

Dose 3 p=<0.001 

Dunnett’s: Dose 3 p=<0.001 

EDSP and EFED same conclusions; a significant 77% reduction from control at 4.7 mg a.i./L, relative to control. 

Male VTG Yes No Jonckheere: 

n.s. p>0.05 

Jonckheere: n.s. p>0.05 

EDSP and EFED same conclusions, no effect for increasing trend 

Female GSI No No Jonckheere: 

n.s. p>0.05 

Jonckheere: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Male GSI No No MannWhitney: 

n.s. p=0.057 

MannWhitney: n.s. p=0.057 

EDSP and EFED same conclusions, no effect for increasing trend 

Page 180 of 288
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor 

EPA MRID Number 48617811 

Page 52 of 82 

Endpoint Monotonic? Parametric? OCSPP 

890.1350 

Historical EFED Comments 

Female tubercle score 

NA NA NA NA No score 

Male tubercle score 

No No Jonckheere: 

Dose 3 p=0.041 

Jonckheere: Dose 3 p=0.041 

EDSP and EFED same conclusions, no effect 

Fecundity Yes Yes Jonckheere: 

Dose 3 p=<0.001 

William’s: Dose 3 p=<0.001 

EDSP and EFED same conclusions; significant reduction at 4.7 mg a.i./L (p<0.001; 98% lower than control) 

Fertility Yes No MannWhitney: 

n.s. p>0.05

MannWhitney: n.s. p>0.05

EDSP and EFED same conclusions, no effect; at the top dose, only one replicate produced eggs of which 100% survived. 

F testosterone NA NA NA NA NA M testosterone NA NA NA NA NA F estradiol NA NA NA NA NA M estradiol NA NA NA NA NA 

Page 181 of 288
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EPA MRID Number 48617811 

Page 53 of 82 

 Fisher's Exact Test 

 

============================================================================ 

 NUMBER OF 

 --------------------------------------- 

 IDENTIFICATION ALIVE DEAD TOTAL ANIMALS 

 -------------------------- ----- ----- -------------- 

 CONTROL 8 0 8 

 0.046 8 0 8 

 --------------------------------------------------------------------------- 

 TOTAL 16 0 16 

 

============================================================================ 

 Critical Fisher's value (8,8,8) (alpha=0.05) is 4.0. b value is 8. 

 Since b is greater than 4.0 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 8 0 8 

 0.48 7 1 8 

 --------------------------------------------------------------------------- 

 TOTAL 15 1 16 

 

============================================================================ 

 Critical Fisher's value (8,8,8) (alpha=0.05) is 4.0. b value is 7. 

 Since b is greater than 4.0 there is no significant difference 

 between CONTROL and TREATMENT at the 0.05 level. 

 

 

 

 

Page 182 of 288
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EPA MRID Number 48617811 

Page 54 of 82 

 Fisher's Exact Test 

 

============================================================================ 

 NUMBER OF 

 --------------------------------------- 

 IDENTIFICATION ALIVE DEAD TOTAL ANIMALS 

 -------------------------- ----- ----- -------------- 

 CONTROL 8 0 8 

 4.7 6 2 8 

 --------------------------------------------------------------------------- 

 TOTAL 14 2 16 

 

============================================================================ 

 Critical Fisher's value (8,8,8) (alpha=0.05) is 4.0. b value is 6. 

 Since b is greater than 4.0 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 8 0 

 1 0.046 8 0 

 2 0.48 8 1 

 3 4.7 8 2 

 --------------------------------------------------------------------------- 

 

 Fisher's Exact Test 

 

============================================================================ 

 NUMBER OF 

 --------------------------------------- 

 IDENTIFICATION ALIVE DEAD TOTAL ANIMALS 

 -------------------------- ----- ----- -------------- 

 CONTROL 15 1 16 

 0.046 15 1 16 

 --------------------------------------------------------------------------- 

 TOTAL 30 2 32 

 

============================================================================ 

 Critical Fisher's value (16,16,15) (alpha=0.05) is 10.0. b value is 15. 

 Since b is greater than 10.0 there is no significant difference 

 between CONTROL and TREATMENT at the 0.05 level. 

Page 183 of 288
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor 

EPA MRID Number 48617811 

Page 55 of 82 

 

 

 Fisher's Exact Test 

 

============================================================================ 

 NUMBER OF 

 --------------------------------------- 

 IDENTIFICATION ALIVE DEAD TOTAL ANIMALS 

 -------------------------- ----- ----- -------------- 

 CONTROL 15 1 16 

 0.48 15 1 16 

 --------------------------------------------------------------------------- 

 TOTAL 30 2 32 

 

============================================================================ 

 Critical Fisher's value (16,16,15) (alpha=0.05) is 10.0. b value is 15. 

 Since b is greater than 10.0 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 15 1 16 

 4.7 11 5 16 

 --------------------------------------------------------------------------- 

 TOTAL 26 6 32 

 

============================================================================ 

 Critical Fisher's value (16,16,15) (alpha=0.05) is 10.0. b value is 11. 

 Since b is greater than 10.0 there is no significant difference 

 between CONTROL and TREATMENT at the 0.05 level. 

 

 

 

 

Page 184 of 288
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EPA MRID Number 48617811 

Page 56 of 82 

Summary of Fisher's Exact Tests 

 --------------------------------------------------------------------------- 

 NUMBER NUMBER SIG 

 GROUP IDENTIFICATION EXPOSED DEAD 0.05 

 ----- ------------------------- ----------- ----------- ----- 

 CONTROL 16 1 

 1 0.046 16 1 

 2 0.48 16 1 

 3 4.7 16 5 

---------------------------------------------------------------------------- 

 

 Fisher's Exact Test 

 

============================================================================ 

 NUMBER OF 

 --------------------------------------- 

 IDENTIFICATION ALIVE DEAD TOTAL ANIMALS 

 -------------------------- ----- ----- -------------- 

 CONTROL 23 1 24 

 0.046 23 1 24 

 --------------------------------------------------------------------------- 

 TOTAL 46 2 48 

 

============================================================================ 

 Critical Fisher's value (24,24,23) (alpha=0.05) is 18.0. b value is 23. 

 Since b is greater than 18.0 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 23 1 24 

 0.48 22 2 24 

 --------------------------------------------------------------------------- 

 TOTAL 45 3 48 

 

============================================================================ 

 Critical Fisher's value (24,24,23) (alpha=0.05) is 18.0. b value is 22. 

 Since b is greater than 18.0 there is no significant difference 

 between CONTROL and TREATMENT at the 0.05 level. 

Page 185 of 288
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor 

EPA MRID Number 48617811 

Page 57 of 82 

 

 Fisher's Exact Test 

 

============================================================================ 

 NUMBER OF 

 --------------------------------------- 

 IDENTIFICATION ALIVE DEAD TOTAL ANIMALS 

 -------------------------- ----- ----- -------------- 

 CONTROL 23 1 24 

 4.7 17 7 24 

 --------------------------------------------------------------------------- 

 TOTAL 40 8 48 

 

============================================================================ 

 Critical Fisher's value (24,24,23) (alpha=0.05) is 18.0. b value is 17. 

 Since b is less than or equal to 18.0 there is a 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 1 

 1 0.046 24 1 

 2 0.48 24 2 

 3 4.7 24 7 * 

 --------------------------------------------------------------------------- 

 

 

Page 186 of 288
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor 

EPA MRID Number 48617811 

Page 58 of 82 

108801 Metolachlor 48617811 890.1350 

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 

 0.961 0.673 0.252 0.859 USE PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 1.34 0.11 0.06 8.35 1.16, 1.52 

 Dose1 4 1.41 0.13 0.06 8.97 1.20, 1.61 

 Dose2 4 1.39 0.13 0.07 9.64 1.17, 1.60 

 Dose3 4 1.21 0.10 0.05 8.43 1.05, 1.37 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 1.29 1.28 1.51 . . 

 Dose1 1.43 1.23 1.53 104.66 -4.66 

 Dose2 1.40 1.23 1.53 103.35 -3.35 

 Dose3 1.23 1.07 1.31 90.13 9.87 

******************************************************************************** 

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 2.19 0.142 

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 1.34 . 1.38 . . . . . . 

 Dose1 1.41 0.801 1.38 0.748 . . . . . 

 Dose2 1.39 0.909 1.38 0.781 0.997 . . . . 

 Dose3 1.21 0.311 1.21 0.091 0.149 0.206 . . . 

******************************************************************************** 

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 4.63 0.201 

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 1.29 . . 

 Dose1 1.43 0.676 0.719 

 Dose2 1.40 0.676 0.722 

 Dose3 1.23 0.233 0.112 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

Page 187 of 288
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EPA MRID Number 48617811 

Page 59 of 82 

******************************************************************************** 

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 2.19 0.142 

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 -1.34 . -1.34 . . . . . . 

 Dose1 -1.41 0.801 -1.34 0.614 . . . . . 

 Dose2 -1.39 0.909 -1.34 0.649 0.997 . . . . 

 Dose3 -1.21 0.311 -1.34 0.668 0.149 0.206 . . . 

******************************************************************************** 

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 4.63 0.201 

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 -1.29 . . 

 Dose1 -1.43 0.676 0.281 

 Dose2 -1.40 0.676 0.278 

 Dose3 -1.23 0.233 0.888 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

108801 Metolachlor 48617811 890.1350 

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 

 0.945 0.419 1.582 0.245 USE PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 3.12 0.08 0.04 2.61 2.99, 3.25 

 Dose1 4 2.97 0.47 0.24 16.00 2.21, 3.72 

 Dose2 4 3.28 0.53 0.27 16.33 2.42, 4.13 

 Dose3 4 2.39 0.53 0.27 22.16 1.55, 3.24 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 3.12 3.03 3.22 . . 

 Dose1 2.99 2.39 3.51 95.04 4.96 

 Dose2 3.28 2.62 3.93 104.88 -4.88 

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EPA MRID Number 48617811 

Page 60 of 82 

 Dose3 2.26 1.97 3.09 76.62 23.38 

******************************************************************************** 

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 2.98 0.074 

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 3.12 . 3.12 . . . . . . 

 Dose1 2.97 0.927 3.12 0.582 . . . . . 

 Dose2 3.28 0.930 3.12 0.616 0.767 . . . . 

 Dose3 2.39 0.096 2.39 0.024 0.312 0.068 . . . 

******************************************************************************** 

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 6.17 0.104 

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 3.12 . . 

 Dose1 2.99 0.678 0.282 

 Dose2 3.28 0.346 0.768 

 Dose3 2.26 0.125 0.104 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams Dose3 

 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 2.98 0.074 

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 -3.12 . -2.94 . . . . . . 

 Dose1 -2.97 0.927 -2.94 0.797 . . . . . 

 Dose2 -3.28 0.930 -2.94 0.828 0.767 . . . . 

 Dose3 -2.39 0.096 -2.94 0.844 0.312 0.068 . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

Page 189 of 288
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor 

EPA MRID Number 48617811 

Page 61 of 82 

 3 6.17 0.104 

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 -3.12 . . 

 Dose1 -2.99 0.678 0.718 

 Dose2 -3.28 0.346 0.232 

 Dose3 -2.26 0.125 0.896 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

108801 Metolachlor 48617811 890.1350 

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 

 0.931 0.254 3.600 0.046 USE NON-PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 49.00 0.82 0.41 1.67 47.70, 50.30 

 Dose1 4 49.00 0.82 0.41 1.67 47.70, 50.30 

 Dose2 4 48.75 1.71 0.85 3.50 46.03, 51.47 

 Dose3 4 47.75 2.06 1.03 4.32 44.47, 51.03 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 49.00 48.00 50.00 . . 

 Dose1 49.00 48.00 50.00 100.00 0.00 

 Dose2 48.50 47.00 51.00 99.49 0.51 

 Dose3 47.50 46.00 50.00 97.45 2.55 

******************************************************************************** 

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 0.67 0.588 

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 49.00 . 49.00 . . . . . . 

 Dose1 49.00 1.000 49.00 0.583 . . . . . 

 Dose2 48.75 0.990 48.75 0.511 0.995 . . . . 

 Dose3 47.75 0.503 47.75 0.163 0.631 0.768 . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

Page 190 of 288
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EPA MRID Number 48617811 

Page 62 of 82 

 Degrees of Freedom TestStat P-value 

 3 1.04 0.791 

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 49.00 . . 

 Dose1 49.00 1.000 0.500 

 Dose2 48.50 0.775 0.322 

 Dose3 47.50 0.568 0.155 

 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 0.67 0.588 

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 -49.00 . -48.63 . . . . . . 

 Dose1 -49.00 1.000 -48.63 0.726 . . . . . 

 Dose2 -48.75 0.990 -48.63 0.761 0.995 . . . . 

 Dose3 -47.75 0.503 -48.63 0.779 0.631 0.768 . . . 

******************************************************************************** 

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 0.791 

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 -49.00 . . 

 Dose1 -49.00 1.000 0.500 

 Dose2 -48.50 0.775 0.678 

 Dose3 -47.50 0.568 0.845 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

108801 Metolachlor 48617811 890.1350 

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 

Page 191 of 288
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor 

EPA MRID Number 48617811 

Page 63 of 82 

 Test Stat P-value Test Stat P-value 

 0.963 0.716 1.579 0.246 USE PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 61.25 0.96 0.48 1.56 59.73, 62.77 

 Dose1 4 59.75 2.50 1.25 4.18 55.77, 63.73 

 Dose2 4 62.50 3.42 1.71 5.47 57.06, 67.94 

 Dose3 4 58.00 1.41 0.71 2.44 55.75, 60.25 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 61.50 60.00 62.00 . . 

 Dose1 59.50 57.00 63.00 97.55 2.45 

 Dose2 62.00 59.00 67.00 102.04 -2.04 

 Dose3 57.50 57.00 60.00 94.69 5.31 

******************************************************************************** 

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 2.90 0.079 

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 61.25 . 61.25 . . . . . . 

 Dose1 59.75 0.684 61.13 0.550 . . . . . 

 Dose2 62.50 0.782 61.13 0.584 0.363 . . . . 

 Dose3 58.00 0.158 58.00 0.042 0.705 0.068 . . . 

******************************************************************************** 

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 6.58 0.086 

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 61.50 . . 

 Dose1 59.50 0.410 0.153 

 Dose2 62.00 0.779 0.616 

 Dose3 57.50 0.079 0.066 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams Dose3 

 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 2.90 0.079 

Dunnett - testing each trt mean signif. different than control 

Page 192 of 288
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor 

EPA MRID Number 48617811 

Page 64 of 82 

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 -61.25 . -60.38 . . . . . . 

 Dose1 -59.75 0.684 -60.38 0.786 . . . . . 

 Dose2 -62.50 0.782 -60.38 0.818 0.363 . . . . 

 Dose3 -58.00 0.158 -60.38 0.834 0.705 0.068 . . . 

******************************************************************************** 

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 6.58 0.086 

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 -61.50 . . 

 Dose1 -59.50 0.410 0.847 

 Dose2 -62.00 0.779 0.384 

 Dose3 -57.50 0.079 0.934 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

108801 Metolachlor 48617811 890.1350 

ANALYSIS RESULTS FOR VARIABLE VAR05 ( F 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 

 0.934 0.285 2.381 0.121 USE PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 3260750 486506.2 243253.1 14.92 2486610, 4034890 

 Dose1 4 3294750 597410.2 298705.1 18.13 2344137, 4245363 

 Dose2 4 3371500 839708.1 419854.0 24.91 2035337, 4707663 

 Dose3 4 765750.0 460589.7 230294.9 60.15 32848.93, 1498651 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 3122500 2875000 3923000 . . 

 Dose1 3531500 2423000 3693000 101.04 -1.04 

 Dose2 3359000 2618000 4150000 103.40 -3.40 

 Dose3 703000.0 318000.0 1339000 23.48 76.52 

******************************************************************************** 

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 17.15 <.001 

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Page 65 of 82 

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 3260750 . 3309000 . . . . . . 

 Dose1 3294750 1.000 3309000 0.629 . . . . . 

 Dose2 3371500 0.988 3309000 0.664 0.998 . . . . 

 Dose3765750.0 <.001 765750.0 <.001 <.001 <.001 . . . 

******************************************************************************** 

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 8.54 0.036 

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 3122500 . . 

 Dose1 3531500 0.889 0.614 

 Dose2 3359000 1.000 0.670 

 Dose3 703000.0 0.067 0.025 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams Dose3 

 Jonckheere Dose3 

******************************************************************************** 

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 17.15 <.001 

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 -3260750 . -2673188 . . . . . . 

 Dose1-3294750 1.000 -2673188 0.946 . . . . . 

 Dose2-3371500 0.988 -2673188 0.961 0.998 . . . . 

 Dose3 -765750 <.001 -2673188 0.967 <.001 <.001 . . . 

******************************************************************************** 

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 8.54 0.036 

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 -3122500 . . 

 Dose1 -3531500 0.889 0.386 

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 Dose2 -3359000 1.000 0.330 

 Dose3 -703000 0.067 0.975 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

108801 Metolachlor 48617811 890.1350 

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 

 0.824 0.006 2.424 0.116 USE NON-PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 442.50 286.52 143.26 64.75 -13.41, 898.41 

 Dose1 4 527.50 349.03 174.52 66.17 -27.89, 1082.89 

 Dose2 4 765.00 1138.79 569.39 148.86 -1047.06, 2577.06 

 Dose3 4 822.50 1028.12 514.06 125.00 -813.46, 2458.46 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 320.00 260.00 870.00 . . 

 Dose1 560.00 110.00 880.00 119.21 -19.21 

 Dose2 245.00 100.00 2470.00 172.88 -72.88 

 Dose3 430.00 120.00 2310.00 185.88 -85.88 

******************************************************************************** 

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 0.21 0.888 

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 442.50 . 639.38 . . . . . . 

 Dose1 527.50 0.997 639.38 0.721 . . . . . 

 Dose2 765.00 0.893 639.38 0.755 0.974 . . . . 

 Dose3 822.50 0.841 639.38 0.773 0.952 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 0.59 0.899 

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 320.00 . . 

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EPA MRID Number 48617811 

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 Dose1 560.00 0.678 0.718 

 Dose2 245.00 0.413 0.304 

 Dose3 430.00 0.889 0.337 

 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 0.21 0.888 

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 -442.50 . -442.50 . . . . . . 

 Dose1 -527.50 0.997 -527.50 0.519 . . . . . 

 Dose2 -765.00 0.893 -765.00 0.369 0.974 . . . . 

 Dose3 -822.50 0.841 -822.50 0.340 0.952 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 0.59 0.899 

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 -320.00 . . 

 Dose1 -560.00 0.678 0.282 

 Dose2 -245.00 0.413 0.696 

 Dose3 -430.00 0.889 0.663 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

108801 Metolachlor 48617811 890.1350 

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 

 0.965 0.757 12.955 <.001 USE NON-PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 12.58 1.06 0.53 8.45 10.88, 14.27 

 Dose1 4 12.75 1.41 0.71 11.06 10.51, 14.99 

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 Dose2 4 12.78 0.15 0.08 1.17 12.54, 13.01 

 Dose3 4 12.03 2.59 1.29 21.50 7.91, 16.14 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 13.00 11.00 13.30 . . 

 Dose1 12.85 11.10 14.20 101.39 -1.39 

 Dose2 12.80 12.60 12.90 101.59 -1.59 

 Dose3 12.40 9.10 14.20 95.63 4.37 

******************************************************************************** 

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 0.20 0.895 

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 12.58 . 12.70 . . . . . . 

 Dose1 12.75 0.997 12.70 0.630 . . . . . 

 Dose2 12.78 0.996 12.70 0.665 1.000 . . . . 

 Dose3 12.03 0.925 12.03 0.414 0.912 0.904 . . . 

******************************************************************************** 

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 0.22 0.974 

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 13.00 . . 

 Dose1 12.85 0.678 0.718 

 Dose2 12.80 0.484 0.441 

 Dose3 12.40 1.000 0.425 

 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 0.20 0.895 

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 -12.58 . -12.53 . . . . . . 

 Dose1 -12.75 0.997 -12.53 0.600 . . . . . 

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Page 69 of 82 

 Dose2 -12.78 0.996 -12.53 0.635 1.000 . . . . 

 Dose3 -12.03 0.925 -12.53 0.653 0.912 0.904 . . . 

******************************************************************************** 

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 0.22 0.974 

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 -13.00 . . 

 Dose1 -12.85 0.678 0.282 

 Dose2 -12.80 0.484 0.559 

 Dose3 -12.40 1.000 0.575 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

108801 Metolachlor 48617811 890.1350 

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 

 0.973 0.883 3.718 0.042 USE NON-PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 1.45 0.14 0.07 9.76 1.22, 1.67 

 Dose1 4 1.78 0.48 0.24 26.93 1.02, 2.55 

 Dose2 4 1.53 0.26 0.13 17.22 1.11, 1.95 

 Dose3 4 1.87 0.32 0.16 16.93 1.37, 2.37 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 1.43 1.30 1.64 . . 

 Dose1 1.83 1.24 2.23 123.14 -23.14 

 Dose2 1.64 1.14 1.71 105.87 -5.87 

 Dose3 1.78 1.62 2.31 129.19 -29.19 

******************************************************************************** 

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 1.53 0.257 

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 1.45 . 1.66 . . . . . . 

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EPA MRID Number 48617811 

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 Dose1 1.78 0.364 1.66 0.882 . . . . . 

 Dose2 1.53 0.966 1.66 0.906 0.701 . . . . 

 Dose3 1.87 0.207 1.66 0.917 0.980 0.482 . . . 

******************************************************************************** 

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 3.93 0.270 

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 1.43 . . 

 Dose1 1.83 0.494 0.807 

 Dose2 1.64 0.494 0.721 

 Dose3 1.78 0.103 0.944 

 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 1.53 0.257 

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 -1.45 . -1.45 . . . . . . 

 Dose1 -1.78 0.364 -1.66 0.226 . . . . . 

 Dose2 -1.53 0.966 -1.66 0.241 0.701 . . . . 

 Dose3 -1.87 0.207 -1.87 0.057 0.980 0.482 . . . 

******************************************************************************** 

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 3.93 0.270 

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 -1.43 . . 

 Dose1 -1.83 0.494 0.193 

 Dose2 -1.64 0.494 0.279 

 Dose3 -1.78 0.103 0.056 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

108801 Metolachlor 48617811 890.1350 

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EPA MRID Number 48617811 

Page 71 of 82 

ANALYSIS RESULTS FOR VARIABLE VAR09 ( F tubercle score (median) ) 

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS 

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 

Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 

Use parametric analyses if neither test rejected, otherwise non-parametric analyses. 

 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 . . . . NO DATA FOR TEST 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 0.00 0.00 0.00 . . , . 

 Dose1 4 0.00 0.00 0.00 . . , . 

 Dose2 4 0.00 0.00 0.00 . . , . 

 Dose3 4 0.00 0.00 0.00 . . , . 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 0.00 0.00 0.00 . . 

 Dose1 0.00 0.00 0.00 . . 

 Dose2 0.00 0.00 0.00 . . 

 Dose3 0.00 0.00 0.00 . . 

******************************************************************************** 

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 . . . 1 . . . . . . . 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing negative trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl 0.00 . . . . . . . . 

 Dose1 0.00 . . . . . . . . 

 Dose2 0.00 . . . . . . . . 

 Dose3 0.00 . . . . . . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 0.00 1.000 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing negative trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 0.00 . . 

 Dose1 0.00 1.000 . 

 Dose2 0.00 1.000 . 

 Dose3 0.00 1.000 . 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams Dose1 

 Jonckheere Dose1 

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EPA MRID Number 48617811 

Page 72 of 82 

******************************************************************************** 

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 

108801 Metolachlor 48617811 890.1350 

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 

 0.903 0.089 5.164 0.016 USE NON-PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 21.13 2.50 1.25 11.81 17.15, 25.10 

 Dose1 4 19.63 1.49 0.75 7.61 17.25, 22.00 

 Dose2 4 22.00 4.30 2.15 19.55 15.16, 28.84 

 Dose3 4 7.88 10.05 5.03 127.66 -8.12, 23.87 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 22.00 17.50 23.00 . . 

 Dose1 20.00 17.50 21.00 92.90 7.10 

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 Dose2 22.25 16.50 27.00 104.14 -4.14 

 Dose3 5.25 0.00 21.00 37.28 62.72 

******************************************************************************** 

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 5.43 0.014 

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 21.13 . 21.13 . . . . . . 

 Dose1 19.63 0.965 20.81 0.550 . . . . . 

 Dose2 22.00 0.992 20.81 0.584 0.932 . . . . 

 Dose3 7.88 0.016 7.88 0.004 0.053 0.019 . . . 

******************************************************************************** 

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 6.79 0.079 

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 22.00 . . 

 Dose1 20.00 0.281 0.094 

 Dose2 22.25 1.000 0.500 

 Dose3 5.25 0.101 0.041 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams Dose3 

 Jonckheere Dose3 

******************************************************************************** 

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 5.43 0.014 

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 -21.13 . -17.66 . . . . . . 

 Dose1 -19.63 0.965 -17.66 0.871 . . . . . 

 Dose2 -22.00 0.992 -17.66 0.896 0.932 . . . . 

 Dose3 -7.88 0.016 -17.66 0.908 0.053 0.019 . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

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Page 74 of 82 

 Degrees of Freedom TestStat P-value 

 3 6.79 0.079 

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 -22.00 . . 

 Dose1 -20.00 0.281 0.906 

 Dose2 -22.25 1.000 0.500 

 Dose3 -5.25 0.101 0.959 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

108801 Metolachlor 48617811 890.1350 

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 

 0.973 0.890 2.644 0.097 USE PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 27.88 6.15 3.07 22.06 18.09, 37.66 

 Dose1 4 29.90 5.86 2.93 19.59 20.58, 39.22 

 Dose2 4 22.00 4.76 2.38 21.63 14.43, 29.57 

 Dose3 4 0.50 1.00 0.50 200.00 -1.09, 2.09 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 28.70 20.20 33.90 . . 

 Dose1 28.70 24.20 38.00 107.26 -7.26 

 Dose2 22.65 16.60 26.10 78.92 21.08 

 Dose3 0.00 0.00 2.00 1.79 98.21 

******************************************************************************** 

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 30.31 <.001 

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 27.88 . 28.89 . . . . . . 

 Dose1 29.90 0.886 28.89 0.700 . . . . . 

 Dose2 22.00 0.259 22.00 0.072 0.157 . . . . 

 Dose3 0.50 <.001 0.50 <.001 <.001 <.001 . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

Page 203 of 288
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor 

EPA MRID Number 48617811 

Page 75 of 82 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 10.27 0.016 

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 28.70 . . 

 Dose1 28.70 0.889 0.614 

 Dose2 22.65 0.346 0.094 

 Dose3 0.00 0.062 <.001 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams Dose3 

 Jonckheere Dose3 

******************************************************************************** 

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 30.31 <.001 

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 -27.88 . -20.07 . . . . . . 

 Dose1 -29.90 0.886 -20.07 0.992 . . . . . 

 Dose2 -22.00 0.259 -20.07 0.995 0.157 . . . . 

 Dose3 -0.50 <.001 -20.07 0.996 <.001 <.001 . . . 

******************************************************************************** 

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 10.27 0.016 

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 -28.70 . . 

 Dose1 -28.70 0.889 0.386 

 Dose2 -22.65 0.346 0.906 

 Dose3 0.00 0.062 0.999 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

108801 Metolachlor 48617811 890.1350 

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. 

Page 204 of 288
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor 

EPA MRID Number 48617811 

Page 76 of 82 

 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 0.587 <.001 8.722 0.002 USE NON-PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 97.93 1.08 0.54 1.11 96.20, 99.65 

 Dose1 4 98.63 0.39 0.19 0.39 98.01, 99.24 

 Dose2 4 97.85 0.89 0.44 0.91 96.44, 99.26 

 Dose3 4 25.00 50.00 25.00 200.00 -54.56, 104.56 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 98.30 96.40 98.70 . . 

 Dose1 98.70 98.10 99.00 100.71 -0.71 

 Dose2 97.75 96.90 99.00 99.92 0.08 

 Dose3 0.00 0.00 100.00 25.53 74.47 

******************************************************************************** 

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 8.55 0.003 

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 97.93 . 98.28 . . . . . . 

 Dose1 98.63 1.000 98.28 0.592 . . . . . 

 Dose2 97.85 1.000 97.85 0.616 1.000 . . . . 

 Dose3 25.00 0.004 25.00 <.001 0.006 0.007 . . . 

******************************************************************************** 

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 3.34 0.342 

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 98.30 . . 

 Dose1 98.70 0.343 0.877 

 Dose2 97.75 1.000 0.471 

 Dose3 0.00 0.332 0.120 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams Dose3 

 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 8.55 0.003 

Page 205 of 288
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor 

EPA MRID Number 48617811 

Page 77 of 82 

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 -97.93 . -79.85 . . . . . . 

 Dose1 -98.63 1.000 -79.85 0.901 . . . . . 

 Dose2 -97.85 1.000 -79.85 0.923 1.000 . . . . 

 Dose3 -25.00 0.004 -79.85 0.933 0.006 0.007 . . . 

******************************************************************************** 

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 3.34 0.342 

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 -98.30 . . 

 Dose1 -98.70 0.343 0.123 

 Dose2 -97.75 1.000 0.529 

 Dose3 0.00 0.332 0.880 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

Page 206 of 288
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor 

EPA MRID Number 48617811 

108801 48617811 890.1350 all fish Male GSI 

Page 78 of 82 

Obs conc endpoint 

1 0 1.42 

2 0 1.64 

3 0 1.30 

4 0 1.43 

5 1 1.24 

6 1 1.52 

7 1 2.14 

8 1 2.23 

9 2 1.14 

10 2 1.65 

11 2 1.71 

12 2 1.63 

13 3 2.31 

14 3 1.89 

15 3 1.62 

16 3 1.66 

Page 207 of 288
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor 

EPA MRID Number 48617811 

108801 48617811 890.1350 all fish Male GSI 

The NPAR1WAY 

Procedure 

Page 79 of 82 

Wilcoxon Scores (Rank Sums) for Variable endpoint 

Classified by Variable conc 

conc N 

Sum of 

Scores 

Expected 

Under H0 

Std Dev 

Under H0 

Mean 

Score 

0 4 21.0 34.0 8.246211 5.250 

1 4 37.0 34.0 8.246211 9.250 

2 4 31.0 34.0 8.246211 7.750 

3 4 47.0 34.0 8.246211 11.750 

Kruskal-Wallis Test 

Chi-Square 3.9265 

DF 3 

Asymptotic Pr > Chi-Square 0.2695 

Exact Pr >= Chi-Square 0.2845 

Page 208 of 288
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor 

EPA MRID Number 48617811 

108801 48617811 890.1350 all fish Male GSI 

The NPAR1WAY 

Procedure 

Page 80 of 82 

Wilcoxon Scores (Rank Sums) for Variable endpoint 

Classified by Variable conc 

conc N 

Sum of

Scores 

Expecte

d

Under H

0 

Std Dev

Under H0 

Mean

Score 

0 4 15.0 18.0 3.464102 3.750 

1 4 21.0 18.0 3.464102 5.250 

Wilcoxon Two-Sample Test 

Statistic (S) 15.0000 

Normal Approximation 

Z -0.7217 

One-Sided Pr < Z 0.2352 

Two-Sided Pr > |Z| 0.4705 

t Approximation 

One-Sided Pr < Z 0.2469 

Two-Sided Pr > |Z| 0.4939 

Exact Test 

One-Sided Pr <= S 0.2429 

Two-Sided Pr >= |S - Mean| 0.4857 

Z includes a continuity correction of 0.5. 

Kruskal-Wallis Test 

Chi-Square 0.7500 

DF 1 

Pr > Chi-Square 0.3865 

Page 209 of 288
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor 

EPA MRID Number 48617811 

108801 48617811 890.1350 all fish Male GSI 

The NPAR1WAY 

Procedure 

Page 81 of 82 

Wilcoxon Scores (Rank Sums) for Variable endpoint 

Classified by Variable conc 

conc N 

Sum of

Scores 

Expecte

d

Under H

0 

Std Dev

Under H0 

Mean

Score 

0 4 15.0 18.0 3.464102 3.750 

2 4 21.0 18.0 3.464102 5.250 

Wilcoxon Two-Sample Test 

Statistic (S) 15.0000 

Normal Approximation 

Z -0.7217 

One-Sided Pr < Z 0.2352 

Two-Sided Pr > |Z| 0.4705 

t Approximation 

One-Sided Pr < Z 0.2469 

Two-Sided Pr > |Z| 0.4939 

Exact Test 

One-Sided Pr <= S 0.2429 

Two-Sided Pr >= |S - Mean| 0.4857 

Z includes a continuity correction of 0.5. 

Kruskal-Wallis Test 

Chi-Square 0.7500 

DF 1 

Pr > Chi-Square 0.3865 

Page 210 of 288
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor 

EPA MRID Number 48617811 

108801 48617811 890.1350 all fish Male GSI 

The NPAR1WAY 

Procedure 

Page 82 of 82 

Wilcoxon Scores (Rank Sums) for Variable endpoint 

Classified by Variable conc 

conc N 

Sum of

Scores 

Expecte

d

Under H

0 

Std Dev

Under H0 

Mean

Score 

0 4 11.0 18.0 3.464102 2.750 

3 4 25.0 18.0 3.464102 6.250 

Wilcoxon Two-Sample Test 

Statistic (S) 11.0000 

Normal Approximation 

Z -1.8764 

One-Sided Pr < Z 0.0303 

Two-Sided Pr > |Z| 0.0606 

t Approximation 

One-Sided Pr < Z 0.0514 

Two-Sided Pr > |Z| 0.1027 

Exact Test 

One-Sided Pr <= S 0.0286 

Two-Sided Pr >= |S - Mean| 0.0571 

Z includes a continuity correction of 0.5. 

Kruskal-Wallis Test 

Chi-Square 4.0833 

DF 1 

Pr > Chi-Square 0.0433 

Page 211 of 288
DATA EVALUATION RECORD 

METOLACHLOR 

Study Type: OCSPP 890.1400, In vivo Hershberger Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-49-2012 (MRID 48617802) 

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: 05/01/2012 

Secondary Reviewer: Signature:

David A. McEwen, B.S. Date: 5/07/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 5/09/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 5/09/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 212 of 288
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 213 of 288
METOLACHLOR/ 108801 

Primary Reviewer: Ayaad Assaad, D.V.M., Ph.D. 

Health Effects Division 

Secondary Reviewer: Greg Akerman, Ph.D. 

Health Effects Division 

In vivo Hershberger Assay (2012) I Page 1 of 13 

OCSPP 890.1400/ OECD 441 

Signature: fj A?,?~ 

Sig ~:::~ &~;? Date: ___ _..cc,,__t...,_1....._r_.,._. / 1'-.L1---- _ _-

Template version 10/201 I 

DATA EVALUATION RECORD 

STUDY TYPE: Jn Vivo Hershberger Assay (Rat); OCSPP 890.1400; OECD 441 

PC CODE: 108801 DP BARCODE: D398926 

TXR#: 0052848 CAS#: 51218-45-2 

TEST MATERIAL (PURITY): Metolachlor (97.3% a.i.) 

SYNONYMS: 2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-l-methylethyl)acetamide 

CITATION: Sloter, E. D. (2012). A Hershberger Assay of Metolachlor Administered 

Subcutaneously in Peripubertal Orchidoepididymectomized Rats. WIL 

Research Laboratories, LLC, Ashland, OH. Laboratory project ID: WIL786010, January 25, 2012. MRID 48617802. Unpublished. 

SPONSOR: Metolachlor Task Force, 16160 Nall, Stilwell, KS 

TEST ORDER#: CON-108801-12 

EXECUTIVE SUMMARY: In a Hershberger Assay (MRID 48617802) screening for 

androgenic activity, metolachlor (97.3% a.i., Lot #20081115) in corn oil was administered daily 

via subcutaneous (s.c.) injection to groups of six 54- to 56-day old, castrated male Sprague 

Dawley rats at dose levels of 0 (vehicle), 100, 300, or 500 mg/kg/day. An androgenic positive 

control group consisted of six castrated rats exposed to 0.2 mg/kg/day of testosterone propionate 

(TP) by s.c. injection. 

To screen for potential anti-androgenic activity, metolachlor (97.3% a.i.) in com oil was 

administered daily via s.c. injection to groups of six 54- to 56-day old, castrated male Sprague 

Dawley rats at dose levels of 0 (vehicle), 100, 300, or 500 mg/kg/day in conjunction with a daily 

dose of reference androgen TP at 0.2 mg/kg/day by s.c. injection. The anti-androgenic positive 

control group consisted of six castrated rats exposed to 0.2 mg/kg/day TP (s.c. injection) and 

3 mg/kg/day flutamide (FT, via oral gavage). 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 I 0 consecutive days and terminated approximately 24 hours after the final dose 

administration. At necropsy, the five androgen-dependent tissues, adrenal glands, kidney, and 

liver were collected and weighed. One animal in the 100 mg/kg/day group in the androgen 

agonist assay was inadvertently given FT on the last day of dose administration; therefore, this 

animal was removed from the study. 

Page 214 of 288
In vivo Hershberger Assay (2012) / Page 2 of 13 

METOLACHLOR/ 108801 OCSPP 890.1400/ OECD 441

One animal in the 300 mg/kg/day group in the anti-androgen assay was found dead on Day 10, 

within one hour of dosing; no macroscopic findings were noted at necropsy. All other rats 

survived until scheduled sacrifice. No animals exhibited any dose-related clinical signs of 

toxicity and there were no treatment-related gross pathological findings. 

In the androgen agonist assay, overall body weight gains were decreased compared to controls 

by 18% (not significant [NS]) and 45% (p<0.01) in the 300 and 500 mg/kg/day groups, 

respectively. Body weights were also decreased on Days 6-11 in the 500 mg/kg/day group; 

however, the decreases were not statistically significant. Body weights in the 100 and 300 

mg/kg/day dose groups and the positive control group were comparable to controls throughout 

the duration of the assay. There were no treatment related effects in accessory sex organs, 

adrenal glands, kidneys, or liver for any metolachlor treatment group. Animals in the positive 

control group had increased (p<0.01) accessory sex organ weights as follows: 327% in seminal 

vesicles; 381% in ventral prostate; 132% in levator ani-bulbocavernosus (LABC); 322% in 

Cowper’s gland; and 53% in glans penis. Adrenal glands, kidney, and liver weights in the 

positive control group were comparable to the vehicle control. 

Percent CVs for the vehicle control and the 500 mg/kg/day treatment groups were compared to 

the performance criteria in the Guidelines. All %CV values were less than the maximum 

permissible values, with the exception of the %CV for ventral prostate for the 500 mg/kg/day 

group (CV = 48.7%; maximum permissible CV = 45%). 

In the anti-androgen assay, overall body weight gains were decreased by 16% (NS) and 33% 

(p<0.01) in the 300 and 500 mg/kg/day groups, compared to controls. Body weights were also 

decreased on Days 6-11 in the 500 mg/kg/day group; however, the decreases were not 

statistically significant. Body weights in the 100 and 300 mg/kg/day dose groups and the 

positive control group were comparable to controls throughout the duration of the assay. At 500 

mg/kg/day, the weights for ventral prostate and LABC were decreased (p<0.05) by 25% and 

22%, respectively. LABC weights were also decreased (p<0.05) in the 300 mg/kg/day group 

(↓17%). There were no treatment related effects in seminal vesicles, Cowper’s glands, glans 

penis, adrenal glands, kidneys, or liver for any metolachlor group. Animals dosed with TP + FT 

(positive control) had decreased (p<0.01) accessory sex organ weights as follows: 74% in 

seminal vesicles; 74% in ventral prostate; 58% in LABC; 75% in Cowper’s gland; and 33% in 

glans penis. Adrenal glands, kidney, and liver weights in the positive control group were 

comparable to the vehicle control. The performance criteria indicated that this assay was 

performing as expected. 

The high dose of metolachlor selected for this study (500 mg/kg/day) is appropriate because it 

induced anti-androgenic effects but did not cause significant toxicity. In addition, reductions in 

body weight grains of 16-18% (NS) at 300 mg/kg/day and 33-45% (p<0.01) at 500 mg/kg/day 

indicate that the doses selected were adequate. 

Metolachlor was negative for androgenic activity in the Hershberger assay as no statistically 

significant increases in organ weights were seen in two or more of the five androgen sensitive 

tissues in the androgen agonist assay. Metolachlor was positive for potential anti-androgenic 

activity in this Hershberger assay as statistically significant decreases in organ weights were seen 

in two or more of the androgen sensitive tissues in the androgen antagonist assay. 

Page 215 of 288
In vivo Hershberger Assay (2012) / Page 3 of 13 

METOLACHLOR/ 108801 OCSPP 890.1400/ OECD 441

The assay satisfies the EDSP Tier 1 Test Order requirements for a Hershberger assay (OCSPP 

890.1400). 

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

Assurance statements were provided. 

Page 216 of 288
In vivo Hershberger Assay (2012) / Page 4 of 13 

METOLACHLOR/ 108801 OCSPP 890.1400/ OECD 441

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: WIL Research Laboratories, LLC 

Location: Ashland, OH 

Study Director: E. D. Sloter 

Other Personnel: E. S. Bodle (Assistant Director, Analytical Chemistry), S. A. Keets (Senior 

Operations Manager, Vivarium), C. A. Kopp (Manager, Gross Pathology and 

Developmental Toxicology Laboratory), G. M. Maginnis (Clinical 

Veterinarian), T. M. Rafeld (Group Manager, Formulations Laboratory), M. 

J. Schlosser (Vice President, Chemistry Services), B. J. Varsho (Director, 

Operations), E. C. Vedaa (Manager, Clinical Pathology), R. A. Wally 

(Operations Manager, Reporting & Technical Support Services), S. T. Coffee 

(Contributing Scientist, Analyses of Dosing Formulations), L. Freshwater 

(BioSTAT Consultants, Statistical Analysis) 

Study Period: January 4, 2011 - January 25, 2012 

2. Test Substance: Metolachlor 

 Description: Dark brown, clear liquid 

 Source: Sipcam (Valdosta, GA) 

 Lot #: 20081115 (expiration date 11/15/2013) 

 Purity: 97.3% 

 Stability: Stable in corn oil for 13 days at room temperature 

 CAS #: 51218-45-2 

 Structure: 

3. Reference Androgen: Testosterone propionate (TP) 

 Supplier: AK Scientific, Inc. (Mountain View, CA) 

 Lot/Batch #: 7032J (expiration date 8/9/2012) 

 Purity: 98.3% 

 CAS #: 57-85-2 

4. Reference Anti-androgen: Flutamide (FT) 

 Supplier: Spectrum Chemical Manufacturing Corporation (New Brunswick, NJ) 

 Lot/Batch #: YV0734 (March 1, 2011) 

 Purity: 100% 

 CAS #: 1311-84-7 

5. Solvent/Vehicle Control: Corn oil 

 Supplier: Spectrum Chemical Manufacturing Corporation (New Brunswick, NJ) 

 Lot/Batch #: ZT1301 (expiration date 7/1/2012) 

 Rationale (if other than water): Metolachlor technical is insoluble in water 

 Final concentration: Not applicable 

3CH

N

O

Cl

CH3

O

3CH

CH3

Page 217 of 288
In vivo Hershberger Assay (2012) / Page 5 of 13 

METOLACHLOR/ 108801 OCSPP 890.1400/ OECD 441

6. Test Animals: 

 Species: Rat (castrated males only) 

 Strain: Sprague Dawley 

 Age/weight at dose initiation: Post-natal day (PND) 54-56/ 227.4 – 301.4 g 

 Source: Charles River Laboratories, Inc. (Raleigh, NC) 

 Housing: Rats were individually housed in stainless steel wire-mesh cages suspended 

above cage board. 

 Diet: Certified Rodent LabDiet® 5002, PMI International, ad libitum 

Phytoestrogen content was not reported 

 Water: Reverse-osmosis purified drinking water, ad libitum

 Environmental conditions: Temperature: 

Humidity: 

Air changes: 

Photoperiod: 

21.3-21.7 oC (mean daily temperature) 

34.6-48.8% (mean daily humidity) 

10/hr (minimum) 

12 hrs light/12 hrs dark 

 Acclimation period: Rats were castrated at the supplier and received at the facility approximately 

one week post-castration; rats were then allowed a 6-day acclimation period 

at the facility 

B. STUDY DESIGN

1. In life dates: Start: January 10, 2011 End: January 21, 2011 

2. Study Design: In a Hershberger Assay conducted to screen for potential androgenic 

activity, the test substance was administered daily via s.c. injection to castrated male rats. 

Positive androgenic activity is defined as a significant increase in two or more target organ 

weights compared to the vehicle control. To screen for the potential anti-androgenic 

activity, the test substance was also administered daily via s.c. injection to castrated male 

rats in conjunction with a daily dose of TP (0.2 mg/kg/day) by s.c. injection. 

Anti-androgenic activity is indicated by a statistically significant decrease in two or more 

target organ weights of the treated groups (test substance + TP) compared to the TP-only 

control group. For both assays, the animals were dosed for 10 consecutive days and 

necropsied approximately 24 hours after the final dose administration for organ weight 

measurements. 

3. Study Schedule: Male rats were castrated on PND 42 by the supplier and were received at 

the test facility approximately one week following castration, at PND 48-49 (rats were born 

over two days but were not kept separate after receipt at facility; therefore, exact age of each 

rat was not known). The animals were acclimated at the facility for at least 6 days prior to 

initiation of dosing. The dose administration period was from PND 54/55 through PND 

63/64 for half the rats in each group, and from PND 55/56 through PND 64/65 for the 

remaining rats in each group. Rats were euthanized approximately 24 hours after the last 

dose and necropsied for organ weight measurements. 

4. Animal Assignment: Animals were randomly assigned, stratified by body weight, to the 

test groups noted in Table 1. Statistical analysis indicated that there were no significant 

differences in group mean weights at study initiation. Furthermore, the body weight of each 

animal was within 20% of the group mean. 

Page 218 of 288
In vivo Hershberger Assay (2012) / Page 6 of 13 

METOLACHLOR/ 108801 OCSPP 890.1400/ OECD 441

TABLE 1. Study Designa

Test group Dose (mg/kg/day) # of Males 

Androgen Agonist Assay 

Vehicle control (negative control) 0 6 

Low (Metolachlor) 100 6 

Mid (Metolachlor) 300 6 

High (Metolachlor) 500 6 

Testosterone propionate (TP), positive controlb 0.2 6 

Anti-Androgen Assay 

Low (Metolachlor + TP) 100 + 0.2 6 

Mid (Metolachlor + TP) 300 + 0.2 6 

High (Metolachlor + TP) 500 + 0.2 6 

Flutamide + TP, positive control 3 + 0.2 6 

a Data were obtained from page 24 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 chosen based on the 

results of published toxicity data (Reregistration Eligibility Decision Metolachlor, U.S. 

EPA, 1995) and a previous study.1 No discussion of the results of the previous study was 

provided. 

6. (a) Dose Preparation: Dose formulations were prepared twice during the study as single 

formulations for each dose level, by mixing appropriate amounts of test substance with corn 

oil. Formulations of TP and FT were prepared once by dissolving the material in a small 

amount of 95% ethanol and diluting to volume with corn oil. Analyses to demonstrate 

homogeneity and stability were conducted with a concurrent uterotrophic study (MRID 

48617801, concurrently reviewed) for dose formulations at 20 and 200 mg/mL following up 

to 13 days of room temperature storage. During the study, samples of each test substance 

dosing formulation (middle stratum of each) prepared during the in-life phase were analyzed 

for achieved concentration. 

(b) Dose Analysis: 

Results 

Homogeneity (% CV): 1.1-2.5% 

Stability (% of Day 0): 92.5% and 97.9% after 13 days of storage at room temperature 

Concentration (% of nominal): 109-112% 

The analytical data indicated that the mixing procedure was adequate and that the variation 

between nominal and actual dosage to the animals was acceptable. 

 

1

 Sloter, E.D. A Hershberger Assay of Metolachlor Administered Subcutaneously in Peripubertal 

Orchidoepididymectomized Rats (Study No. WIL-786002). WIL Research Laboratories, LLC, Ashland, OH, Draft 

Page 219 of 288
In vivo Hershberger Assay (2012) / Page 7 of 13 

METOLACHLOR/ 108801 OCSPP 890.1400/ OECD 441

7. Dosage administration: Test formulations were administered to the animals daily via s.c. 

injection (5 mL/kg bw) for 10 days. TP was given via s.c. injection at 0.5 mL/kg bw, and 

FT was administered via oral gavage at 10 mL/kg bw. Dose volumes were adjusted daily 

based on the concurrent body weight measurement. 

8. Statistics: Statistical analyses were conducted for organ weights, daily body weights, and 

body weight gains. Each endpoint was tested for homogeneity of variance using Levene's 

test. If that test was significant at p=0.01, then a log transformation was applied and 

Levene's test conducted on the transformed data. If that test was still significant, then the 

square root transformation was applied to the raw data (except cumulative body weight 

gain) and Levene's test conducted again. If the test was still significant, then a 

nonparametric test, as described below, was used to analyze the data. One-sided tests were 

conducted for organ weight data and two-sided tests were conducted for in-life data. 

If variances were homogeneous, the one- or two-sided t-test (for comparing positive control 

data to data for negative control) or an ANOVA (for comparing dose groups to the negative 

control) was performed on data; the ANOVA test was followed by a one- or two-sided 

Dunnett’s test. If the transformations were unsuccessful in making the variances 

homogeneous, the nonparametric one- or two-sided Wilcoxon rank sum test was used to 

compare data for the positive control to the negative control. For comparison of dose 

groups to the negative control, if the transformations were unsuccessful in making the 

variances homogeneous, the nonparametric Kruskal-Wallis test was used, followed by a 

one- or two-sided Dunn's test. Significance was denoted at p<0.05. Statistical analyses 

were performed using SAS software (version 9.2 or higher). The statistical analyses were 

considered adequate. 

C. METHODS

1. Clinical Examinations: Cage-side checks for mortality and moribundity were conducted 

twice daily. Individual clinical observations (hand-held physical examinations) were 

recorded daily through termination. Each rat was also observed for signs of toxicity at the 

time of dosing and approximately 1 hour following dosing. 

2. Body Weight: Animals were weighed at randomization, daily throughout the dosing 

period, and on the day of termination. Mean body weight changes were calculated for each 

corresponding interval and also for the overall dosing period (Days 1-11). 

3. Food Consumption (Optional): Food consumption was not measured. 

4. Serum Hormone Measurements (Optional): At study termination, each animal was 

anesthetized with isoflurane, and blood was collected from the vena cava for potential future 

serum hormone analyses; however, serum hormone analyses were not conducted for this 

study. 

5. Dissection and Measurement of Tissue and Organ Weights: On PND 64 to 66 

(approximately 24 hours after the final administration of the test substance), all animals 

were anesthetized with isoflurane, exsanguinated, and examined. The five mandatory 

androgen-dependent organs (ventral prostate, seminal vesicles with coagulating glands, 

Page 220 of 288
In vivo Hershberger Assay (2012) / Page 8 of 13 

METOLACHLOR/ 108801 OCSPP 890.1400/ OECD 441

LABC, Cowper’s gland, and glans penis) were excised and weighed fresh (unfixed) 

according to the standard operating procedures detailed in the U.S. EPA Guideline (OCSPP 

890.1400). Additionally, the liver, kidneys, and adrenals were weighed. The tissues and 

organs were placed in 10% neutral buffered formalin. 

II. RESULTS

A. OBSERVATIONS

1. Mortality: One animal in the 300 mg/kg/day group in the anti-androgen assay (Animal No. 

92315) was found dead on Day 10, within one hour of dosing. Within 15 minutes of dosing 

on that day, the rat was prostrate, gasping, convulsing, and had labored respiration. No 

macroscopic findings were noted at necropsy. All other rats survived until scheduled 

sacrifice. The death of this animal does not affect the validity of this study as a screening 

assay because no other rats were found dead or exhibited similar clinical findings. 

One animal in the 100 mg/kg/day group in the androgen agonist assay (Animal No. 92317) 

was inadvertently given FT on the last day of dose administration; therefore, this animal was 

removed from the study resulting in only 5 males in that dose group. 

2. Clinical signs of toxicity: No treatment-related clinical signs of toxicity were observed in 

animals for any dose groups. Any clinical findings noted in the dose groups occurred 

infrequently and in a manner that was not dose related. 

B. BODY WEIGHT AND WEIGHT GAIN: Selected body weight and body weight gain 

data for the androgen agonist assay are presented in Table 2. Overall body weight gains 

(Days 1-11) were decreased by 20% (NS), 18% (NS) and 45% (p<0.01) in the 100, 300 and 

500 mg/kg/day groups, respectively, compared to controls. Body weights in the 500 

mg/kg/day group were also decreased by 6% and 10% on Days 6 and 11; however, the 

decreases were not statistically significant. Body weights in the 100 and 300 mg/kg/day 

dose groups and the positive control group were comparable to controls throughout the 

duration of the assay. 

Page 221 of 288
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METOLACHLOR/ 108801 OCSPP 890.1400/ OECD 441

TABLE 2. Selected Group Mean Body Weights and Cumulative Body Weight Gains (g) in the 

Androgen Agonist Assaya

Study Day # 

Dose (mg/kg/day) 

Vehicle Control Positive Control 

Vehicle + TP (0.2) Metolachlor (100) Metolachlor (300) Metolachlor (500) 

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 

1 6 263.4 22.4 6 263.8 26.5 5 267.7 23.8 6 261.9 26.4 6 262.7 27.4 

4 6 283.2 24.4 6 282.5 23.2 5 288.6 25.2 6 279.1 25.1 6 275.2 28.0 

7 6 310.1 22.4 6 309.7 21.0 5 309.4 24.0 6 300.9 24.1 6 290.1 28.2 

11 6 336.9 23.5 6 339.0 18.2 5 326.8 37.2 6 322.2 22.9 6 302.8 33.4 

Body Weight 

Gain (Days 1-11) 

6 73.5 12.8 6 75.1 11.0 5 59.1 

(↓20) 

29.8 6 60.4 

(↓18) 

14.6 6 40.1** 

(↓45) 

9.4 

a Data were obtained from Tables S3, S4, and S6 on pages 49-51, 54, and 60 of the study report. Percent differences from 

controls, calculated by the reviewers, are included in parentheses. 

N Number of animals in the group 

SD Standard Deviation 

** Significantly different from controls at p<0.01 

Selected body weight and body weight gain data for the anti-androgen assay are presented in 

Table 3. Overall body weight gains (Days 1-11) were decreased by 16% (NS) and 33% 

(p<0.01) in the 300 and 500 mg/kg/day groups, respectively, compared to controls. Body 

weights were also decreased by on Days 6-11 in the 500 mg/kg/day group (↓5% on Day 6 to 

↓7% on Day 11); however, the decreases were not statistically significant. Body weights 

and body weight gains in the 100 and 300 mg/kg/day dose groups and the positive control 

group were comparable to controls throughout the duration of the assay. 

TABLE 3. Selected Group Mean Body Weights and Cumulative Body Weight Gains (g) in the 

Anti-Androgen Assaya

Study Day # 

Dose (mg/kg/day) 

Vehicle Control + 

TP (0.2) 

Positive Control 

TP + FT (0.2 + 3) 

Metolachlor + TP 

(100 + 0.2) 

Metolachlor + TP 

(300 + 0.2) 

Metolachlor + TP 

(500 + 0.2) 

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 

1 6 263.8 26.5 6 258.6 24.6 6 262.5 26.4 6 260.7 24.1 6 262.5 23.4 

4 6 282.5 23.2 6 273.8 26.7 6 284.4 26.1 6 277.4 24.1 6 272.8 22.4 

7 6 309.7 21.0 6 297.0 24.3 6 308.3 24.7 6 300.3 22.7 6 294.0 20.9 

11 6 339.0 18.2 6 323.7 26.2 6 339.2 25.3 5 328.6 25.1 6 312.9 20.2 

Body Weight 

Gain (Days 1-11) 

6 75.1 11.0 6 65.1 5.2 6 76.7 

(↑2) 

6.6 5 63.2 

(↓16) 

7.4 6 50.4** 

(↓33) 

14.8 

a Data were obtained from Tables S3, S4, and S8 on pages 49-51, 54, and 66 of the study report. Percent differences from 

controls, calculated by the reviewers, are included in parentheses. 

N Number of animals in the group 

SD Standard Deviation 

** Significantly different from controls at p<0.01 

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

D. SERUM HORMONE CONCENTRATIONS (Optional): Serum hormone concentrations 

were not measured. 

E. ORGAN WEIGHTS: Accessory sex organ, adrenal glands, kidney, and liver weights for 

the androgen agonist assay are presented in Table 4. There were no treatment related effects 

in accessory sex organs, adrenal glands, kidneys, or liver for any metolachlor treatment 

Page 222 of 288
In vivo Hershberger Assay (2012) / Page 10 of 13 

METOLACHLOR/ 108801 OCSPP 890.1400/ OECD 441

group. Animals in the TP group had increased (p<0.01) accessory sex organ weights as 

follows: 327% in seminal vesicles; 381% in ventral prostate; 132% in LABC; 322% in 

Cowper’s gland; and 53% in glans penis. Adrenal glands, kidney, and liver weights in the 

positive control group were comparable to the vehicle control. 

Percent CVs for the vehicle control and the 500 mg/kg/day treatment groups were compared 

to the performance criteria in the Guidelines. All %CV values were less than the maximum 

permissible values, with the exception of the %CV for ventral prostate for the 500 

mg/kg/day group (CV = 48.7%; maximum permissible CV = 45%). 

There were no treatment-related gross pathological findings. 

TABLE 4. Accessory Sex Organ, Adrenal Glands, Kidney, and Liver Weights from Androgen Agonist Assay in 

Sprague Dawley Ratsa

Organ 

Dose (mg/kg/day) 

Vehicle Control Positive Control 

Vehicle + TP (0.2) Metolachlor (100) Metolachlor (300) Metolachlor (500) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

Seminal 

vesicles 

(mg) 

6 100.4 11.1 11.0 6 429.0** 

(↑327) 42.0 9.8 5 100.0 12.3 12.3 6 98.0 15.4 15.7 6 89.6 9.8 11.0

Ventral 

prostate 

(mg) 

6 23.6 4.7 19.8 6 113.4** 

(↑381) 7.0 6.2 5 19.2 8.6 44.6 6 21.5 5.1 23.5 6 19.1 9.3 48.7

LABC 

(mg) 6 195.7 33.4 17.1 6 455.0** 

(↑132) 48.9 10.8 5 167.8 23.0 13.7 6 152.8 22.6 14.8 6 151.2 24.8 16.4

Cowper’s 

glands 

(mg) 

6 7.8 1.6 20.3 6 32.9** 

(↑322) 4.8 14.7 5 8.1 0.7 9.2 6 6.2 1.1 17.8 6 6.8 0.9 13.0

Glans 

penis (mg) 6 94.1 9.1 9.7 6 144.1** (↑53) 16.5 11.5 5 102.2 24.1 23.5 6 88.9 10.1 11.4 6 82.6 30.0 36.4

Adrenal 

glands 

(mg) 

6 66.8 13.3 19.9 6 59.8 5.3 8.8 5 57.6 10.1 17.4 6 57.9 7.3 12.6 6 54.8 9.6 17.5

Kidneys 

(g) 6 2.062 0.259 12.6 6 2.058 0.133 6.5 5 2.002 0.115 5.7 6 2.17 0.259 11.9 6 2.101 0.302 14.4

Liver (g) 6 12.9 1.19 9.2 6 13.3 0.92 6.9 5 12.9 1.67 13.0 6 14.2 0.94 6.7 6 13.2 2.54 19.2

a Data were obtained from Tables S15 and S18 on pages 81, 82, and 89-91 of the study report. Percent differences from controls, 

calculated by the reviewers, are included in parentheses. 

N Number of animals in the group 

SD Standard Deviation 

CV Coefficient of Variation 

** Significantly different from controls at p<0.01 

 

Page 223 of 288
In vivo Hershberger Assay (2012) / Page 11 of 13 

METOLACHLOR/ 108801 OCSPP 890.1400/ OECD 441

Accessory sex organ, adrenal glands, kidney, and liver weights for the anti-androgen assay 

are presented in Table 5. At 500 mg/kg/day, the weights for ventral prostate and LABC 

were decreased (p<0.05) by 25% and 22%, respectively. LABC weights were also 

decreased (p<0.05) in the 300 mg/kg/day group (↓17%). Adrenal glands weights were 

decreased (p<0.05) in the 300 mg/kg/day group (↓19%); however, because significant 

decreases were not observed in the 500 mg/kg/day group, these differences were not 

considered to be dose related. There were no treatment related effects in seminal vesicles, 

Cowper’s glands, glans penis, kidneys, or liver for any metolachlor treatment group. 

Animals dosed with TP + FT (positive control) had decreased (p<0.01) accessory sex organ 

weights as follows: 74% in seminal vesicles; 74% in ventral prostate; 58% in LABC; 75% 

in Cowper’s gland; and 33% in glans penis. Adrenal glands, kidney, and liver weights in the 

positive control group were comparable to the vehicle control. 

Percent CVs for the vehicle control and the 500 mg/kg/day treatment groups were compared 

to the performance criteria in the Guidelines. All %CV values were less than the maximum 

permissible values. 

There were no treatment-related gross pathological findings. 

TABLE 5. Accessory Sex Organ, Adrenal Glands, Kidney, and Liver Weights from Anti-Androgen Assay in 

Sprague Dawley Ratsa

Organ

Dose (mg/kg/day) 

Vehicle Control + TP 

(0.2) 

Positive Control TP + 

FT (0.2 + 3) 

Metolachlor + TP 

(100 + 0.2) 

Metolachlor + TP 

(300 + 0.2) 

Metolachlor + TP 

(500 + 0.2) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%)

Seminal 

vesicles 

(mg) 

6 429.0 42.0 9.8 6 113.0** 

(↓74) 14.6 12.9 6 392.2 49.5 12.6 5 376.4 95.9 25.5 6 355.8 51.5 14.5

Ventral 

prostate 

(mg) 

6 113.4 7.0 6.2 6 29.4** 

(↓74) 9.5 32.3 6 106.3 17.2 16.1 5 90.6 17.6 19.5 6 84.8* (↓25) 24.9 29.4

LABC (mg) 6 455.0 48.9 10.8 6 189.0** 

(↓58) 34.8 18.4 6 410.9 65.7 16.0 5 376.0* 

(↓17) 59.1 15.7 6 354.3** 

(↓22) 45.3 12.8

Cowper’s 

glands (mg) 6 32.9 4.8 14.7 6 8.1** 

(↓75) 1.7 20.7 6 29.1 8.2 28.3 5 27.4 6.0 22.0 6 26.6 6.8 25.5

Glans penis 

(mg) 6 144.1 16.5 11.5 6 96.1** 

(↓33) 7.5 7.8 6 141.9 12.1 8.5 5 144.1 8.5 5.9 6 134.3 7.9 5.9 

Adrenal 

glands (mg) 6 59.8 5.3 8.8 6 56.2 10.8 19.2 6 59.2 7.7 13.0 5 48.5* 

(↓19) 9.0 18.5 6 51.2 6.2 12.2

Kidneys (g) 6 2.058 0.133 6.5 6 1.974 0.128 6.5 6 2.130 0.080 3.8 5 2.057 0.100 4.8 6 2.143 0.074 3.5 

Liver (g) 6 13.3 0.92 6.9 6 13.7 1.94 14.2 6 13.5 0.99 7.4 5 13.6 1.32 9.8 6 13.8 0.94 6.8 

a Data were obtained from Tables S15, S17, and S19 on pages 81, 82, 86-88, and 92-94 of the study report. Percent differences 

from controls, calculated by the reviewers, are included in parentheses. 

N Number of animals in the group 

SD Standard Deviation 

CV Coefficient of Variation 

* Significantly different from controls at p<0.05 

** Significantly different from controls at p<0.01 

Page 224 of 288
In vivo Hershberger Assay (2012) / Page 12 of 13 

METOLACHLOR/ 108801 OCSPP 890.1400/ OECD 441

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Based on the results of this study, statistically 

significant lower weights of ≥2 androgen-dependent organs in males given 500 mg/kg/day 

with TP suggested an anti-androgenic response when administered by subcutaneous 

injection to peripubertal, castrated male Crl:CD(SD) rats. No anti-androgenic effects were 

noted at 100 and 300 mg/kg/day. No androgenic activity was demonstrated at metolachlor 

dosage levels up to 500 mg/kg/day, the highest dosage level of metolachlor tested, as 

evidenced by the lack of increase in the weights of the androgen-dependent organs relative 

to the vehicle control group. The anti-androgenic positive control substance, flutamide, 

generally elicited the expected reduction in androgen-dependent organ weights. The 

androgenic positive control substance, TP, elicited the expected increase in androgendependent organ weights. 

B. AGENCY COMMENTS: One animal in the 300 mg/kg/day group in the anti-androgen 

assay was found dead on Day 10, within one hour of dosing; no macroscopic findings were 

noted at necropsy. All other rats survived until scheduled sacrifice. The death of this 

animal does not affect the validity of this study as a screening assay because no other rats 

were found dead or exhibited similar clinical findings. One animal in the 100 mg/kg/day 

group in the androgen agonist assay was inadvertently given FT on the last day of dose 

administration; therefore, this animal was removed from the study. No animals exhibited 

any dose-related clinical signs of toxicology and there were no treatment-related gross 

pathological findings. 

In the androgen agonist assay, overall body weight gains were decreased by 18% (NS) and 

45% (p<0.01) in the 300 and 500 mg/kg/day groups, respectively, compared to controls. 

Body weights were also decreased on Days 6-11 in the 500 mg/kg/day group (↓6% on Day 6 

to ↓10% on Day 11); however, the decreases were not statistically significant. Body 

weights in the 100 and 300 mg/kg/day dose groups and the positive control group were 

comparable to controls throughout the duration of the assay. There were no treatment 

related effects in accessory sex organs, adrenal glands, kidneys, or liver for any metolachlor 

treatment group. Animals in the positive control group had the expected increases accessory 

sex organ weights. Adrenal glands, kidney, and liver weights in the positive control group 

were comparable to the vehicle control. 

Percent CVs for the vehicle control and the 500 mg/kg/day treatment groups were compared 

to the performance criteria in the Guidelines. All %CV values were less than the maximum 

permissible values, with the exception of the %CV for ventral prostate for the 500 

mg/kg/day group (CV = 48.7%; maximum permissible CV = 45%). 

In the anti-androgen assay, overall body weight gains were decreased by 16% (NS) and 33% 

(p<0.01) in the 300 and 500 mg/kg/day groups, respectively, compared to controls. Body 

weights were also decreased by on Days 6-11 in the 500 mg/kg/day group (↓5% on Day 6 to 

↓7% on Day 11); however, the decreases were not statistically significant. Body weights in 

the 100 and 300 mg/kg/day dose groups and the positive control group were comparable to 

controls throughout the duration of the assay. At 500 mg/kg/day, the weights for ventral 

prostate and LABC were decreased (p<0.05) by 25% and 22%, respectively. LABC weights 

were also decreased (p<0.05) in the 300 mg/kg/day group (↓17%). There were no treatment 

Page 225 of 288
In vivo Hershberger Assay (2012) / Page 13 of 13 

METOLACHLOR/ 108801 OCSPP 890.1400/ OECD 441

related effects in seminal vesicles, Cowper’s glands, glans penis, adrenal glands, kidneys, or 

liver for any metolachlor group. Animals dosed with TP + FT (positive control) had the 

expected decreases in accessory sex organ weights. Adrenal glands, kidney, and liver 

weights in the positive control group were comparable to the vehicle control. The 

performance criteria indicated that this assay was performing as expected. 

The high dose level tested in the study (500 mg/kg/day) is appropriate because it induced 

anti-androgenic effects but did not cause significant toxicity. Additional support for the 

high dose selected includes reductions in body weight grains of 16-18% (NS) at 300 

mg/kg/day and 33-45% (p<0.01) at 500 mg/kg/day. 

 No statistically significant increases in organ weights were seen in two or more of the five 

androgen responsive tissues in the androgen agonist assay. Metolachlor was negative for 

androgenic activity. Statistically significant decreases in tissue weights were seen in LABC 

and ventral prostate in the androgen antagonist assay. Metolachlor was positive for potential 

anti-androgenic activity in this Hershberger assay 

C. STUDY DEFICIENCIES: None 

Page 226 of 288
DATA EVALUATION RECORD 

METOLACHLOR 

Study Type: OCSPP 890.1450, Female Pubertal Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-49-2012 (MRID 48617804) 

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:

Joseph Rieth, Ph.D., D.A.B.T. Date: 5/9/2012 

Secondary Reviewer: Signature:

David A. McEwen, B.S. Date: 5/11/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 5/16/2012 

Quality Assurance: Signature:

Steven Brecher, Ph.D., D.A.B.T. Date: 5/16/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 227 of 288
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 228 of 288
METOLACHLOR/ 108801 

Fema le Puberta l Assay (2012)/ Page I of 15 

OCSPP 890.1450/ OECD None 

Primary Reviewer: Ayaad Assaad, D.V.M, Ph.D 

Health Effects Division 

Secondary Reviewer: Elizabeth Mendez, Ph.D. 

Health Effects Division 

DAT A EVALUATION RECORD 

STUDY TYPE: Female Pubertal Assay; OCSPP 890.1450; OECD None. 

PC CODE: 108801 DP BARCODE: D398926 

TXR#: 0052848 CAS No: 51218-45-2 

TEST MATERIAL (PURITY): Metolachlor Technical (97.3% a.i.) 

SYN 0 NYMS: 2-chloro-N-(2-ethyl-6-methyl pheny l)-N-(2-methoxy- l -methylethy l)acetamide 

CITATION: Sloter, E.D. (2012) Pubertal Development and Thyroid Function in Intact 

Juvenile/Peripubertal Female Rats Following Oral Administration of 

Metolachlor. WIL Research Laboratories, LLC, Ashland, OH, Laboratory 

Project ID: WIL-786003 , January 25, 2012. MRID 48617804. Unpublished. 

SPONSOR: Metolachlor Task Force, 16160 Nall, Stilwell, KS 

TEST ORDER#: CON 108801-112 

EXECUTIVE SUMMARY: In a Female Pubertal Assay (MRID 48617804), 15 Crl:CD(SD) 

Sprague-Dawley rats/dose group were treated daily via oral gavage (5 mL/kg) with metolachlor 

technical (97.3% a.i., Lot #20081115) in com oil at doses ofO, 300 or 600 mg/kg/day from postnatal day (PND) 22 to 42. Animals were examined for vaginal opening daily beginning on PND 

22, and weight at day of attainment was recorded. Following sacrifice on PND 42, total 

thyroxine (T4) and thyroid stimulating hormone (TSH) levels were analyzed using an 

electrochemiluminescent immunoassay (T4) and a magnetic [1 25

I]rTSH gamma counter 

immunoassay (TSH). Kidneys, liver, adrenal glands, thyroid, pituitary, and ovary weights were 

recorded, and microscopic examination of the thyroid, kidneys, ovary and uterus were 

performed. 

One animal in the control group was sacrificed in extremis on PND 25 due to impairment of the 

left forelimb. All other rats survived until scheduled sacrifice. No treatment-related clinical 

findings were noted during the daily examinations at any dose level. There were no significant 

differences in body weight at vaginal opening or final body weights in the treated groups relative 

to controls. Adjusted age at VO was significantly earlier at 300 mg/kg/day (p<0.05) but not at 

600 mg/kg/day. One female in the control group failed to attain vaginal opening. There were no 

statistically significant differences in mean age at first vaginal estrous or mean cycle length. The 

cycle status at necropsy was similar among groups. Serum TSH was not affected by treatment. 

Page 229 of 288
Female Pubertal Assay (2012)/ Page 2 of 15 

METOLACHLOR/ 108801 OCSPP 890.1450/ OECD None

Unadjusted, adjusted and relative liver weights were increased at 600 mg/kg/day (p<0.05, ↑20-

22%); only relative liver weight was significantly increased at 300 mg/kg/day (p<0.05, ↑9%). 

Unadjusted, adjusted and relative kidney weights were increased at 600 mg/kg/day (p<0.05, ↑8-

10%). No other effects on organ weights were reported. The percent of females cycling (93.3%) 

and regularly cycling (77.8%) were lower at 600 mg/kg/day (p<0.05) than the controls (100% for 

both). Percent regularly cycling was also reduced at 300 mg/kg/day (p<0.05, 53.8%). There 

were dose-related increases in serum T4 levels of 14% and 25% in the 300 and 600 mg/kg/day 

groups, respectively; the increase was significant (p<0.05) at 600 mg/kg/day only. Mean 

globulin levels were significantly higher in the 300 mg/kg/day (p<0.05, ↑7%) and 600 mg/kg/day 

(p<0.05, ↑7%) groups; there were corresponding reductions in A/G ratios of 8% and 12%, 

respectively, with only the high dose being significant (p<0.05). GGT was significantly elevated 

at 600 mg/kg/day (p<0.05, ↑700%). 

There was an increased incidence of Grade 2 follicular cell height at 600 (11/14) mg/kg/day 

relative to control (4/14); also, one animal at the high dose was observed with Grade 3. There 

was an increased incidence of Grade 4 colloid area at 600 mg/kg/day (2/14, 4/15 and 11/14 in the 

control, 300 and 600 mg/kg/day respectively). The differences in follicular cell height and 

colloid area were significant at 600 mg/kg/day (p=0.005). The ovaries appeared normal 

histopathologically. There were no microscopic findings in the uterus related to treatment with 

the test material. Renal tubular cysts were increased in the 300 and 600 mg/kg/day groups (1/14, 

2/15 and 4/15 in the 300 and 600 mg/kg/day groups, respectively). 

No effects were observed on body weights or body weight gain in the study. Increased liver 

weights accompanied by elevated GGT levels and increased kidney weights demonstrate organ 

toxicity as a result of treatment. However, histopathology was not conducted on the liver and 

microscopic findings in the kidney were minimal, so the degree of toxicity in these organs is 

unclear. Age at vaginal opening was not affected by treatment. There was a dose-related 

increase in serum T4 levels of 14% and 25% in the 300 and 600 mg/kg/day groups, respectively; 

the increase was significant (p<0.05) at 600 mg/kg/day only. Serum TSH was unaffected by 

treatment. The dosing levels tested in this assay are adequate based on systemic toxicity 

(increased liver and kidney weights) at the high dose. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Female Pubertal Assay 

(OCSPP 890.1450). 

COMPLIANCE: Signed and dated GLP Compliance and Quality Assurance statements were 

provided. 

 

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I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: WIL Research Laboratories, LLC 

Location: Ashland, OH 

Study Director: E. D. Sloter 

Other Personnel: J. B. Nold, Pathologist 

L. Freshwater, Statistics 

Study Period: July 29, 2010 – January 25, 2012 

2. Test Substance: Metolachlor 

 Description: Dark brown, clear liquid, MW 283.79 

 Source: Sipcam (Valdosta, GA) 

 Lot/Batch #: 20081115 (exp. November 15, 2013) 

 Purity: 97.3% 

 Stability: Stable in corn oil for 13 days at room temperature 

 CAS #: 51218-45-2 

 Structure: 

CH3

CH3

N

O

CH3

O

Cl

CH3

3. Vehicle: Corn oil 

4. Test Animals:

 Species: Rat 

 Strain: Crl:CD(SD) Sprague-Dawley 

 Age/Weight at 

Study Initiation: PND [22]/47.0 – 61.6 g females only 

 Source: Charles River Laboratories; Raleigh, NC 

 Housing: Two-three/cage in plastic cages with heat treated laboratory-grade pine shavings for bedding.

 Diet: Harlan Laboratories 2016CM Teklad Global 16% protein rodent diet, ad libitum 

Phytoestrogen content average 10 ppm total isoflavones (genistein+daidzein+glycitein)

 Water: Municipal water, reverse-osmosis filtered (on site), ad libitum

 Environmental 

Conditions: 

Temperature: 

Humidity: 

Air changes: 

Photoperiod: 

22 ± 3 °C 

50 ± 20% 

10/hr 

14 hrs light/ 10 hrs dark 

B. STUDY DESIGN

1. In-Life Dates: Start: August 28, 2010 End: September 18, 2010 

2. Mating: Time-mated pregnant dams were received from the supplier on gestation days 

(GD) 11 or 12. The day an animal was found to be sperm positive was designated GD 0. 

Litters with more than 8 pups were standardized by culling on PND 4 to 8 pups per litter. 

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3. Animal Assignment: Following weaning on PND 21, animals were assigned to the test 

groups noted in Table 1 using a randomization procedure designed to ensure equal 

stratification of body weights. Littermates were not assigned to the same treatment group. 

TABLE 1. Study Design a

Test group Dose (mg/kg/day) # of Females 

Control 0 15 

Low 300 15 

High 600 15 

a Data were obtained from page 26 of the study report. 

4. Dose Selection Rationale: It was stated that the dose levels were selected based on the 

results of published toxicity studies (no further details were provided). 

5. Dose Preparation and Analysis: Studies on the homogeneity and stability of the test 

substance in formulations were conducted in a previous study (MRID 48617801, reviewed 

concurrently)1 and the results are reported below. Concentration of the test material as 

administered to the study animals was assessed pre-study and once during the in-life phase 

of the study.

Results of Dose Analysis 

Homogeneity (% RSD): 1.8-2.5% 

Stability (% of initial): 92.5-97.9% following storage for 13 days at room temperature. 

Concentration: The formulated concentration of the test material ranged from 101% to 

103% of nominal.

The analytical data indicated that the mixing procedure was adequate and that the variation 

between nominal and actual dosage to the animals was acceptable. 

6. Dosage Administration: All doses were administered once daily by oral gavage, from 

PND 22 through PND 42, in a volume of 5 mL/kg of body weight. Dosing was performed 

between 0700 and 0900 hours daily.

7. Statistics: Statistics were conducted at WIL Research and at BioSTAT Consultants, Inc. 

At WIL, Cycling status (cycling vs. non-cycling) and percent of animals cycling were 

assessed using Chi-square analysis. Estrous cycle length and day of first estrus were 

analyzed using a one-way analysis of variance (ANOVA) followed by Dunnett’s test when 

intergroup differences were indicated by ANOVA. Dichotomous pathology data were 

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

significance level. 

 

1 Sloter, E.D. (2012) A Uterotrophic Assay of Metolachlor Administered Subcutaneously in Ovariectomized Rats 

Study No. WIL-786001. WIL Research Laboratories, LLC, Ashland, OH. 

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At BioSTAT, the data that were statistically analyzed included organ weights (liver, 

kidneys, pituitary and adrenals), organ weight to final body weight ratios, daily body 

weights, weight gains, serum chemistries, hormones and vaginal opening data. Each 

endpoint was tested for homogeneity of variance using Levene’s test. If this was positive 

(p≤ 0.01) then log or square root transformations were applied to the data; if these were 

positive then a non-parametric test was used. Organ weights, age at vaginal opening and 

weight at vaginal opening data, when parametric, were analyzed using analysis of 

covariance (ANCOVA) followed by Dunnett’s test using PND 21 body weights as the 

covariate. Other parameters were analyzed with ANOVA followed by Dunnett’s test. 

Linear trend tests were used in conjunction with both ANOVA and ANCOVA. For nonparametric data the Kruskal-Wallis test was used followed by Dunn’s test. Pathology data 

presented as a graded response were analyzed with a pairwise Mann-Whitney U test. 

Dichotomous histopathology data were analyzed using a Fisher’s exact test. 

C. METHODS

1. Mortality and Clinical Examinations: All animals were examined twice daily for 

mortality and moribundity. Clinical examinations were conducted on the day of 

randomization and daily through the day of euthanasia. 

2. Body Weight: Animals were weighed on the day of randomization, daily prior to dosing 

and the day of euthanasia. 

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

vaginal opening. The appearance of a small “pin hole”, a vaginal thread, and complete 

vaginal opening were recorded for all days they were observed. Age and weight on the day 

of completion of vaginal opening were recorded. 

4. Estrous Cyclicity: Beginning on the day of vaginal opening, up to and including the day of 

necropsy, daily vaginal smears were obtained and evaluated to determine the stage of estrus 

for each female. Cycle length was determined for a complete estrous cycle by counting the 

number of days from one diestrus to the next diestrus. The overall pattern for each female 

was characterized as regular, irregular, non-cycling or insufficient data. The definitions for 

these terms are as follows: 

 Regular cycling (RC): the animal has at least 6 days of data collected and displays 1 

complete cycle with no cycles greater than 5 days of duration, no cycles with 3 or more 

days of proestrus (P) and/or estrus (E), and no cycles less than 4 days in duration. 

 Irregular cycling (IC): 1) the animal does not display 1 complete cycle but has at least 1 E 

and/or P and a partial cycle greater than 5 days, 2) the animal has at least 1 E and either at 

least 1 cycle (complete or partial) greater than 5 days in duration or at least 1 cycle with 3 

or more consecutive days of P and/or E, 3) the animal has at least 1 cycle less than 4 days 

in duration, or 4) the animal has 1 irregular cycle and 1 regular cycle. 

 Non-cycling (NC): no E or P present on any days of estrous cycle determination and at 

least 5 days of data collected. 

 Insufficient data (ID): 1) the animal does not display at least 1 complete cycle but has at 

least 1 E and/or P and a partial cycle of 5 days or fewer, 2) no E is present on any days of 

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estrous cycle determination and 4 or fewer days of data collected, or 3) at least 1 E or P 

present and only 1-4 days of data collected. 

Percent cycling and percent regularly cycling were calculated as follows: 

Percent cycling: [(RC + IC + ID) / (RC + IC + ID + NC)] X 100 

Percent cycling regularly: [RC / (RC + IC)] X 100 

Note that, 1) as long as there are no animals classified as NC, the % cycling will be 100, and 

2) when no animals are classified as IC, the % cycling regularly will be 100. Also, the 

equation presented on page 26 of the report results in a ratio, not a percentage. 

5. Sacrifice and Pathology: On the day before euthanasia, rats were moved to a holding room 

separate from where the necropsies were to be performed. Dosing was performed in the 

holding room on the day of termination and the animals were moved to the necropsy room 

one at a time for terminal procedures. Approximately 2 hours following dosing, animals 

were sacrificed by decapitation without anesthesia and all sacrifices were completed by 

1300 hours to minimize hormonal variability due to normal diurnal fluctuation. 

Immediately following decapitation, trunk blood samples were taken for thyroxine (T4), 

thyroid stimulating hormone (TSH) and serum chemistry analyses. The sample for TSH 

analysis was stored at ≤ 20° C and the sample for T4 was analyzed fresh. 

a. Hormone Analysis: Total thyroxine (T4) was analyzed using an electrochemiluminescent 

assay and thyroid stimulating hormone (TSH) levels were analyzed using an [125 I]

radioimmunoassay. For both assays, multiple quality control samples were run prior to 

analysis. 

b. Clinical Chemistry: The following CHECKED (X) parameters were examined. 

 ELECTROLYTES OTHER 

X Calcium X Albumin 

X Chloride X Creatinine* 

 Magnesium X Urea nitrogen* 

X Phosphorus X Total cholesterol 

X Potassium X Globulins 

X Sodium X Glucose 

ENZYMES X Total bilirubin 

X Alkaline phosphatase (ALP) X Total protein 

 Cholinesterase (ChE) X Triglycerides 

 Creatine phosphokinase Serum protein electrophoresis 

 Lactic acid dehydrogenase (LDH) X Albumin globulin ratio (A/G Ratio, by calculation) 

X Alanine aminotransferase (ALT/also SGPT) X Bile acids 

X Aspartate aminotransferase (AST/also SGOT) 

 Sorbitol dehydrogenase 

X Gamma glutamyl transferase (GGT) 

 Glutamate dehydrogenase 

* Recommended for the pubertal assay in female rats based on guideline 890.1450. 

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c. Organ Weights and Histopathology: The following CHECKED (X) tissues were 

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

examination. 

 UROGENITAL OTHER 

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

XX Uterus*+ X Liver* 

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

 X Pituitary* 

* Weights required based on guideline 890.1450 

+ Histopathological examination required based on guideline 890.1450 

All organs except the thyroid/trachea were weighed prior to fixation. Paired organs 

(kidneys, adrenals, and ovaries) were weighed together. The uterus was separated from the 

vagina and weighed. The uterus was weighed “wet” and then again following removal of 

the fluid in the lumen (blotted weight). 

The ovaries (left or right not specified), uterus and kidneys were fixed in 10% buffered 

formalin, rinsed and stored in 70% ethanol prior to embedding. The thyroid was fixed in 

10% buffered formalin for at least 24 hrs. Following fixation, the thyroid was dissected 

from the trachea. All collected tissues were routinely processed into paraffin blocks, 

sectioned, stained with hematoxylin and eosin, and examined microscopically. 

Thyroid sections were subjectively evaluated for follicular cell height and colloid area, using 

a five point grading scale (1 = shortest/smallest; 5 = tallest/largest), and any 

abnormalities/lesions were noted. At least two sections of the thyroid were examined. Five 

random sections of an ovary were evaluated for follicular development and any 

abnormalities/lesions (such as atrophy). Uterine histology documented cases of uterine 

hyper- or hypotrophy as characterized by changes in uterine horn diameter and myometrial, 

stromal, or endometrial gland development. The stage of the estrous cycle of the female at 

the time of necropsy was evaluated. 

II. RESULTS 

A. Mortality: One animal in the control group (Animal No. 79829-10) was sacrificed in 

extremis on PND 25 due to impairment of the left forelimb; this animal lost 5.9 g body 

weight during PND 24-25. All other rats survived until scheduled sacrifice. 

B. Clinical Signs of Toxicity: No treatment-related clinical findings were noted during the 

daily examinations at any dose level. Clinical signs infrequently noted in the study included 

clear material around the mouth, salivation, vocalization, and red material on the eye, nose 

or mouth.

C. General Growth and Vaginal Opening: Body weights, body weight gains, age of 

attainment of vaginal opening and weight at day of attainment are presented in Table 2. 

There were no significant differences in body weight at vaginal opening or final body 

weights in the treated groups relative to controls. Adjusted age at VO was significantly 

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earlier (p<0.05) at 300 mg/kg/day but not at 600 mg/kg/day. One female in the control 

group failed to attain vaginal opening. 

The control CV for the mean age at vaginal opening (9.39%) exceeded the performance 

criteria maximum (6.52%). 

TABLE 2. General Growth and Vaginal Opening (VO)a

Parameter Evaluated 

Vehicle Control Metolachlor

300 mg/kg/day 

Metolachlor 

600 mg/kg/day 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) 

Initial body weight 

(PND 22; g) U 

15 54.6 3.97 7.27 15 54.6 3.88 7.10 15 54.9 3.67 6.69 

Body weight at vaginal 

opening (g) 

U 14 121.0 14.78 12.22 15 109.8 15.29 13.92 15 116.6 17.36 14.89

A 14 122.0 14.78 12.22 15 110.4 15.29 13.92 15 117.0 17.36 14.89

Final body weight(g) U 14 153.8 10.38 6.75 15 156.0 9.80 6.28 15 157.4 8.99 5.71 

Final body weight 

(% of control) U 15 101 15 102 

Body weight gain 

(final – initial; g) U 

14 98.7 9.7 9.83 15 101.4 8.55 8.43 15 102.5 7.91 7.72 

Age at vaginal opening 

(PND) 

U 14 35.4 3.32 9.39 15 33.4 2.29 6.86 15 34.5 2.53 7.35 

A 14 35.6 3.32 9.39 15 33.4* 2.29 6.86 15 34.6 2.53 7.35 

Age at vaginal opening 

(incomplete) (PND) 

 14 34.7 2.49 7.18 15 33.4 2.29 6.86 15 34.5 2.53 7.35 

 14 34.9 2.49 7.18 15 33.4 2.29 6.86 15 34.6 2.53 7.35 

Proportion unopened (#/N) 1/14 0/15 0/15 

a Data were obtained from page 63 of the study report 

U Unadjusted for body weight on PND 22 

A Adjusted for body weight on PND 22 

N Number of animals examined 

SD Standard Deviation 

CV Coefficient of Variation 

* Significantly different from controls at p<0.05 

D. Organ Weights: Organ weights at necropsy are presented in Table 3. Unadjusted, adjusted 

and relative liver weights were increased at 600 mg/kg/day (p<0.05, ↑20-22%); only relative 

liver weight was significantly increased at 300 mg/kg/day (p<0.05, ↑9%). Unadjusted, 

adjusted and relative kidney weights were increased at 600 mg/kg/day (p<0.05, ↑8-10%). 

No other effects on organ weights were reported. 

The control group CV for liver weight (13.360%) was higher than the performance criteria 

maximum (13.13%). 

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TABLE 3. Organ Weights at Necropsya

Organ 

Vehicle Control Metolachlor

300 mg/kg/day 

Metolachlor 

600 mg/kg/day 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) 

Liver 

(g) 

U 14 6.91 0.923 13.360 15 7.62 0.670 8.786 15 8.45* 

(↑22) 

0.962 11.377

A 14 6.91 0.923 13.360 15 7.64 0.670 8.786 15 8.44* 

(↑22) 

0.962 11.377

R 14 4.48 0.349 7.777 15 4.89* 

(↑9) 

0.265 5.416 15 5.37* 

(↑20) 

0.436 8.125 

Kidneys 

(g) 

U 14 1.19 0.104 8.807 15 1.25 0.100 8.001 15 1.31* 

(↑10) 

0.093 7.129 

A 14 1.18 0.104 8.807 15 1.25 0.100 8.001 15 1.3* 

(↑10) 

0.093 7.129 

R 14 0.77 0.061 7.896 15 0.80 0.064 7.927 15 0.83* 

(↑8) 

0.059 7.074 

Pituitary 

(mg) 

U 14 8.1 1.26 15.46 15 7.6 0.820 10.74 15 8.2 0.82 10.07 

A 14 8.1 1.26 15.46 15 7.6 0.820 10.74 15 8.1 0.82 10.07 

R 14 5.3 0.83 15.60 15 4.9 0.390 8.03 15 5.2 0.49 9.52 

Adrenals 

(mg) 

U 14 32.5 4.44 13.65 15 31.8 3.74 11.73 15 33.0 4.76 14.4 

A 14 32.5 4.44 13.65 15 32.0 3.74 11.73 15 32.9 4.76 14.4 

R 14 21.2 3.13 14.73 15 20.5 2.91 14.18 15 21.0 2.91 13.86 

Ovaries 

(mg) 

U 14 72.5 11.96 16.50 15 70.0 6.25 8.93 15 70.7 8.30 11.75 

A 14 72.4 11.96 16.50 15 70.1 6.25 8.93 15 70.5 8.30 11.75 

Uterus, wet 

(mg) 

U 14 296.3 108.24 36.54 15 309.4 178.10 57.56 15 337.4 195.36 57.91 

A 14 297.6 108.24 36.54 15 309.3 178.10 57.56 15 338.7 195.36 57.91 

Uterus, blotted 

(mg) 

U 14 246.5 46.17 18.73 15 238.5 98.69 41.37 15 260.5 71.18 27.32 

A 14 245.4 46.17 18.73 15 238.2 98.69 41.37 15 259.9 71.18 27.32 

Thyroid, fixed

(mg) 

U 14 9.59 2.154 22.468 15 9.67 2.093 21.64 15 10.15 1.763 17.367

A 14 9.67 2.154 22.468 15 9.72 2.093 21.64 15 10.18 1.763 17.367

a Data were obtained from page 64 of the study report. Percent differences from controls, calculated by the reviewers, are 

included in parentheses. 

U Unadjusted for body weight on PND 22 

A Adjusted for 100 g body weight on PND 22 

N Number of animals examined 

SD Standard Deviation 

CV Coefficient of Variation 

R Organ-to-body weight ratio (relative to body weight) 

* Significantly different from controls at p<0.05. 

E. Estrous Cyclicity: Estrous cycle data are provided in Table 4. There were no statistically 

significant differences in mean age at first vaginal estrous or mean cycle length. The cycle 

status at necropsy was similar among groups. The percent of females cycling (93.3%) and 

regularly cycling (77.8%) was lower at 600 mg/kg/day (p<0.05) than the controls (100% for 

both). Percent regularly cycling was also reduced at 300 mg/kg/day (p<0.05, 53.8%). 

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TABLE 4. Estrous Cyclicitya

Treatment 

Groups N

Mean Age at 

First Vaginal 

Estrus (PND) 

Mean Cycle 

Length 

(days) 

Cycling 

(%) 

Regularly 

Cycling 

(%) 

Cycle Status at Necropsy (# Females) 

Diestrus Proestrus Estrus

Not 

Cycling 

Vehicle 14 35.8 4.7 100 100 12 0 1 0 

Metolachlor

300 mg/kg/day 15 36.1 4.6 100 53.8* 12 0 2 0 

Metolachlor

600 mg/kg/day 15 37.2 4.6 93.3* 77.8* 13 0 2 1 

a Data were obtained from pages 64 & 65 of the study report. 

N Number of animals 

* Significantly different from controls at p<0.05.

F. Clinical Chemistry and Hormone Levels: Mean clinical chemistry and hormone levels 

are presented in Table 5. In addition, the study report provided historical control ranges on 

pages 421-424 for the clinical chemistry, but not hormone measures. There was a doserelated increase in serum T4 levels of 14% and 25% in the 300 and 600 mg/kg/day groups, 

respectively; the increase was significant at 600 mg/kg/day (p<0.05) only. Serum TSH was 

not affected by treatment. 

Mean globulin levels were significantly higher in the 300 and 600 mg/kg/day groups 

(p<0.05, ↑7%); there were corresponding reductions in A/G ratios of 8% and 12%, 

respectively, with only the high dose being significant (p<0.05). GGT was significantly 

elevated at 600 mg/kg/day (p<0.05, ↑700%). 

It is noted that 12/20 of the control clinical chemistry means are outside the historical 

control range for the performing laboratory. 

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TABLE 5. Hormone Levels and Clinical Chemistrya

Parameter 

Evaluated 

Vehicle Control Metolachlor

300 mg/kg/day 

Metolachlor

600 mg/kg/day 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) 

Hormones 

Serum T4, Total 

(μg/dL) 

14 4.42 1.143 25.851 15 5.05 

(↑14) 

1.147 22.727 15 5.51* 

(↑25) 

1.399 25.41 

Serum TSH 

(ng/mL) 

13 8.76 5.686 64.901 15 9.02 3.712 41.153 15 10.41 5.893 56.594

Clinical Chemistry 

A/G ratio 13 2.97 0.305 10.289 15 2.73 

(↓8) 

0.219 8.030 15 2.61* 

(↓12) 

0.103 3.930 

Albumin (g/dL) 13 4.1 0.22 5.36 15 4.1 0.22 5.38 15 4 0.21 5.34 

Alkaline 

phosphatase (U/L) 

13 329 73.0 22.2 15 289 42.7 14.8 15 300 83.8 27.9 

Alanine amino 

transferase (U/L) 

13 96 19.0 19.8 15 92 13.7 14.9 15 96 13.4 14.0 

Aspartate 

aminotransferase 

(U/L) 

13 341 105.8 31.0 15 324 77.2 23.8 15 311 75.9 24.4 

Bile acids (μmol/L) 13 23.8 10.27 43.13 15 29.3 19.49 66.52 15 32.2 13.66 42.40 

Calcium (mg/dL) 13 11.9 0.77 6.48 15 12.1 0.46 3.77 15 12.0 0.23 1.93 

Chloride (mEq/L) 13 101 1.5 1.5 15 101 1.7 1.6 15 101 1.3 1.3 

Cholesterol 

(mg/dL) 

13 92 14.4 15.7 15 101 10.9 10.7 15 97 11.5 11.8 

Creatinine 

(mg/dL) 

13 0.2 0.03 14.42 15 0.2 0.05 22.15 15 0.2 0.05 24.21 

Gamma glutamyl 

transferase (U/L) 

13 0.2 0.30 121.44 15 0.3 0.39 115.95 15 1.6* 

(↑700) 

1.44 89.11 

Globulin (g/dL) 13 1.4 0.13 9.25 15 1.5* 

(↑7) 

0.15 9.95 15 1.5* 

(↑7) 

0.08 5.39 

Glucose (mg/dL) 13 145 12.7 8.8 15 142 8.0 5.6 15 142 7.5 5.3 

Phosphorus 

(mg/dL) 

13 10 0.78 7.82 15 10.3 0.75 7.32 15 10.2 0.85 8.38 

Potassium (mEq/L) 13 7.04 0.825 11.722 15 7.01 0.603 8.603 15 6.91 0.682 9.875 

Sodium (mEq/L) 13 139 2.4 1.7 15 139 1.9 1.3 15 140 1.4 1.0 

Total bilirubin 

(mg/dL) 

13 0.15 0.039 26.105 15 0.14 0.029 21.670 15 0.16 0.025 15.510

Total protein (g/dL) 13 5.5 0.25 4.61 15 5.6 0.31 5.63 15 5.5 0.29 5.14 

Triglyceride 

(mg/dL) 

13 132 71.7 54.20 15 142 60.2 42.5 15 117 25.7 22.0 

Serum urea 

nitrogen 

(mg/dL) 

13 12.7 1.81 14.20 15 14.1 2.26 16.05 15 13 3.12 24.04 

a Data were obtained from pages 66 & 67 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. 

G. Histopathology: The incidences of histopathological findings of the thyroid gland are 

presented in Table 6. There was an increased incidence of Grade 2 follicular cell height at 

300 (8/15) and 600 (11/14) mg/kg/day relative to control (4/14); also, one animal at the high 

dose was observed with a Grade 3. There was an increased incidence of Grade 4 colloid 

area at 600 mg/kg/day (2/14, 4/15 and 11/14 in the control, 300 and 600 mg/kg/day 

respectively. The differences in follicular cell height and colloid area were significant at 

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600 mg/kg/day (p=0.005) and, with the exception of the single female with Grade 3 

follicular cell height, all animals had values in the historical range. The only other 

significant difference in the thyroid gland was an increase in ultimobranchial cyst at 300 

mg/kg/day that was not dose related. 

TABLE 6. Incidence of Histopathological Lesions of the Thyroid Gland a

Treatment 

Groups 

Parameter Evaluated 

Colloid Area Follicular Cell Height 

Gradeb Incidence Gradeb Incidence 

O E O E 

Vehicle Control 

1 0 14 1 10 14 

2 0 14 2 4 14 

3 0 14 3 0 14 

4 2 14 4 0 14 

5 12 14 5 0 14 

Metolachlor

300 mg/kg/day 

1 0 15 1 7 15 

2 0 15 2 8 15 

3 0 15 3 0 15 

4 4 15 4 0 15 

5 11 15 5 0 15 

Metolachlor

600 mg/kg/day 

1 0 14 1 2 14 

2 0 14 2 11 14 

3 0 14 3 1 14 

4 11 14 4 0 14 

5 3 14 5 0 14 

a Data were obtained from pages 45 & 113 of the study report. 

b Thyroid histopathology is graded on a 1 – 5 scale: Follicular cell height,1 = lowest, 5 = highest; and Colloid area, 

1 = most colloid, 5 = least colloid. 

O Number Observed 

E Number Examined 

The incidence of histopathological findings of the ovaries, uterus and kidneys are presented 

in Table 7. The ovaries appeared normal histopathologically. There were no microscopic 

findings in the uterus related to treatment with the test material. Renal tubular cysts were 

increased in the 300 and 600 mg/kg/day groups (1/14, 2/15 and 4/15 in the 300 and 600 

mg/kg/day groups, respectively). Other observations were considered to be incidental and 

included renal tubular degeneration, mineralization, pelvis dilatation and acute 

inflammation. 

Page 240 of 288
Female Pubertal Assay (2012)/ Page 13 of 15 

METOLACHLOR/ 108801 OCSPP 890.1450/ OECD None

TABLE 7. Incidence of Histopathological Lesions of the Ovaries, Uterus and Kidneya

Findings 

Dose Level (mg/kg/day) 

Vehicle Control Metolachlor

300 mg/kg/day 

Metolachlor

600 mg/kg/day 

# Observed # Examined # Observed # Examined # Observed # Examined 

Ovaries 

Unremarkable 14 14 15 15 15 15 

Uterus 

Apoptosis minimal 1 14 3 15 1 15 

Apoptosis mild 4 14 2 15 1 15 

Glans, developing 

minimal 5 14 3 15 3 15 

Glans, developing mild 7 14 9 15 11 15 

Infiltrate, eosinophil 

minimal 2 14 5 15 6 15 

Infiltrate, eosinophil 

mild 11 14 10 15 9 15 

Infiltrate, eosinophil 

moderate 1 14 0 15 0 15 

Kidney 

Unremarkable 0 14 0 15 1 15 

Cyst, tubular 1 14 2 15 4 15 

Dilitation, pelvis 

minimal 0 14 2 15 1 15 

Dilitation, pelvis mild 1 14 0 15 0 15 

Inflammation,, acute 

mild 1 14 0 15 0 15 

Mineralization, minimal 3 14 2 15 2 15 

Mineralization, mild 0 14 1 15 0 15 

Regeneration, renal 

tubular minimal 13 14 15 15 14 15 

Regeneration, renal 

tubular mild 1 14 0 15 0 15 

a Data were obtained from pages 110 - 114 of the study report. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: No evidence of altered female pubertal 

development was noted at any dose level, based on the lack of effects on the mean age and 

body weight at attainment of vaginal opening, age at the first occurrence of estrus, estrous 

cycle length and regularity, uterine weights (wet and blotted), ovary weights, or test 

substance-related macroscopic and microscopic findings in the uterus and ovaries. This 

indicates that metolachlor technical does not demonstrate or mimic biological activities 

consistent with agonism of natural estrogens when administered orally to peripubertal female 

rats at dosage levels up to 600 mg/kg/day. 

At 600 mg/kg/day, increases in follicular cell height and colloid depletion in the thyroid were 

noted; however, the changes were generally minimal and some control animals also exhibited 

the same findings. These slight changes in the thyroid of the 300 and 600 mg/kg/day females 

are consistent with an adaptive response to test substance metabolism and most likely 

secondary to chemical metabolizing enzyme induction and increased hepatic clearance of 

thyroxine. Liver weights were significantly higher and this is consistent with hepatocellular 

hypertrophy; however, liver histopathology was not performed in this study. Serum gamma 

Page 241 of 288
Female Pubertal Assay (2012)/ Page 14 of 15 

METOLACHLOR/ 108801 OCSPP 890.1450/ OECD None

glutamyl transferase levels were also higher in the 600 mg/kg/day group, which may indicate 

an effect on hepatobiliary function. Mean T4 concentration was slightly, yet significantly 

higher; however, there were no corresponding effects on TSH levels or thyroid gland 

weights. Serum globulin levels were increased and thus A/G ratios were reduced. Test 

substance-related higher mean kidney weights were noted in the 600 mg/kg/day group. 

At 300 mg/kg/day, increases in follicular cell height and colloid depletion in the thyroid were 

noted; liver weights were significantly higher. Serum globulin levels were increased and 

thus A/G ratios were lower. 

In conclusion, a separate amphibian assay conducted as part of this same package of 

endocrine studies using metolachlor showed no effects on the thyroid at dose levels up to 8.4 

mg/L (Palmer et al., 2012, Wildlife International, Ltd. Project Number 568A-103). In 

addition, metolachlor has been shown to induce liver cytochrome P450 enzymes (Dalton, et 

al., 2003). Therefore, despite the larger proportion of females in the 300 and 600 mg/kg/day 

groups with minimal microscopic changes in the thyroid relative to the control group, the 

weight of evidence suggests that metolachlor is not a clear disrupter of thyroid homeostasis at 

oral dose levels up to 600 mg/kg/day in peripubertal female rats. 

B. AGENCY COMMENTS: One animal in the control group was sacrificed in extremis on 

PND 25 due to impairment of the left forelimb. All other rats survived until scheduled 

sacrifice. No treatment-related clinical findings were noted during the daily examinations at 

any dose level. There were no significant differences in body weight at vaginal opening or 

final body weights in the treated groups relative to controls. Adjusted age at VO was 

significantly earlier at 300 mg/kg/day (p<0.05) but not at 600 mg/kg/day. One female in the 

control group failed to attain vaginal opening. There were no statistically significant 

differences in mean age at first vaginal estrous or mean cycle length. The cycle status at 

necropsy was similar among groups. Serum TSH was not affected by treatment. 

Unadjusted, adjusted and relative liver weights were increased at 600 mg/kg/day (p<0.05, 

↑20-22%); only relative liver weight was significantly increased at 300 mg/kg/day (p<0.05, 

↑9%). Unadjusted, adjusted and relative kidney weights were increased at 600 mg/kg/day 

(p<0.05, ↑8-10%). No other effects on organ weights were reported. The percent of 

females cycling (93.3%) and regularly cycling (77.8%) were lower at 600 mg/kg/day 

(p<0.05) than the controls (100% for both). Percent regularly cycling was also reduced at 

300 mg/kg/day (p<0.05, 53.8%). There were dose-related increases in serum T4 levels of 

14% and 25% in the 300 and 600 mg/kg/day groups, respectively; the increase was 

significant (p<0.05) at 600 mg/kg/day only. Mean globulin levels were significantly higher 

in the 300 mg/kg/day (p<0.05, ↑7%) and 600 mg/kg/day (p<0.05, ↑7%) groups; there were 

corresponding reductions in A/G ratios of 8% and 12%, respectively, with only the high 

dose being significant (p<0.05). GGT was significantly elevated at 600 mg/kg/day (p<0.05, 

↑700%). 

There was an increased incidence of Grade 2 follicular cell height at 600 (11/14) mg/kg/day 

relative to control (4/14); also, one animal at the high dose was observed with Grade 3. 

There was an increased incidence of Grade 4 colloid area at 600 mg/kg/day (2/14, 4/15 and 

11/14 in the control, 300 and 600 mg/kg/day respectively). The differences in follicular cell 

height and colloid area were significant at 600 mg/kg/day (p=0.005). The ovaries appeared 

Page 242 of 288
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normal histopathologically. There were no microscopic findings in the uterus related to 

treatment with the test material. Renal tubular cysts were increased in the 300 and 600 

mg/kg/day groups (1/14, 2/15 and 4/15 in the 300 and 600 mg/kg/day groups, respectively). 

No effects were observed on body weights or body weight gain in the study. Increased liver 

weights accompanied by elevated GGT levels and increased kidney weights demonstrate 

organ toxicity as a result of treatment. However, histopathology was not conducted on the 

liver and microscopic findings in the kidney were minimal, so the degree of toxicity in these 

organs is unclear. Age at vaginal opening was not affected by treatment. There was a doserelated increase in serum T4 levels of 14% and 25% in the 300 and 600 mg/kg/day groups, 

respectively; the increase was significant (p<0.05) at 600 mg/kg/day only. Serum TSH was 

unaffected by treatment. 

STUDY DEFICIENCIES: The following minor deficiencies were noted that are not 

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

study: 

 The control CV for the mean age at vaginal opening (9.39%) exceeded the performance 

criteria maximum (6.52%). 

 The control CV for the mean TSH level (64.901%) exceeded the performance criteria 

maximum (58.29%). 

 The control CV for liver weight (13.360%) was higher than the performance criteria 

maximum (13.13%). 

Page 243 of 288
DATA EVALUATION RECORD 

METOLACHLOR 

Study Type: OCSPP 890.1500, Male Pubertal Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-49-2012 (MRID 48617803) 

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:

Joseph Rieth, Ph.D., DABT Date: 5/9/2012 

Secondary Reviewer: Signature:

David A. McEwen, B.S. Date: 5/16/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 5/17/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 5/17/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 244 of 288
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 245 of 288
Male Pubertal Assay (2012) I Page I of 16 

METOLACHLOR/108801 OCSPP 890.1500 I OECD None 

Primary Reviewer: Ayaad Assaad,D.V.M., Ph.D. 

Health Effects Division 

Secondary Reviewer: John Liccione Ph.D. 

Health Effects Division 

DATA EVALUATION RECORD 

STUDY TYPE: Male Pubertal Assay; OCSPP 890.1500 

PC CODE: 108801 DP BARCODE: D398926 

TXR#: 0052848 CAS No.: 51218-45-2 

TEST MATERIAL (PURITY): Metolachlor Technical (97.3% purity) 

SYNONYMS: 2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-l-methylethyl)acetamide 

CITATION: Sloter, E.D. (2012) Pubertal Development and Thyroid Function in Intact 

Juvenile/Peri pubertal Male Rats Following Oral Administration of Metolachlor. 

WIL Research Laboratories, LLC, Ashland Ohio. WIL-786004, January 25, 

2012. MRID 48617803. Unpublished. 

SPONSOR: Metolachlor Task Force, 16160 Nall, Stilwell, KS 

TEST ORDER#: CON-108801-112 

EXECUTIVE SUMMARY: In a Male Pubertal Assay (MRID 48617803), 15 Crl:CD(SD) 

Sprague-Dawley rats/dose group were treated daily via oral gavage with metolachlor technical 

(97.3% a.i., Lot #20081115) in corn oil at doses of 0, 400 or 800 mg/kg/day from post-natal day 

(PND) 23 to 53 . Animals were examined for preputial separation (PPS) daily beginning on PND 

30 and weight at day of attainment was recorded. Following sacrifice on PND 53, blood was 

taken for total thyroxine (T4), testosterone, thyroid stimulating hormone (TSH) and clinical 

chemistry analysis. Although the hormones were reportedly analyzed by radioimmunoassay 

(RIA) or chemiluminescence, the specific details of the methods were not provided. weights 

were recorded for kidneys, liver, adrenal glands, thyroid, pituitary, epididymides, ventral 

prostate, dorsal prostate, seminal vesicle, levator ani plus bulbocavernosus (LABC) muscles and 

testes, and microscopic examinations were performed on the thyroid, kidneys, testis and 

epididymides. 

One male in the 800 mg/kg/day group (Animal No. 79812-07) was found dead on PND 40; no 

significant clinical or macroscopic findings were observed in this animal. One male in the 

control group (Animal No. 79823-03) was sacrificed in extremis on PND 4 7 following clinical 

findings of a swollen right forelimb resulting in impaired use; gross examination of this animal at 

necropsy revealed a punctured esophagus probably due to a dosing error. All other rats survived 

until scheduled sacrifice. Clinical findings included salivation prior to dosing and clear material 

Page 246 of 288
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METOLACHLOR/108801 OCSPP 890.1500 / OECD None

around the mouth approximately 1 hour post-dosing in the 400 and 800 mg/kg/day dose groups. 

At 800 mg/kg/day, clear material was also observed around the ventral neck. 

No treatment-related changes in body weights, body weight gains, or age and weight at PPS 

occurred in either treatment group. Liver weights were increased (p<0.05) by 15-20% and 

kidney weights were increased (p<0.05) by 11% each at 400 and 800 mg/kg/day, respectively. A 

significant trend for increased thyroid weights was observed, although pairwise comparisons 

were not significant; the increasing trend in thyroid weights did not correlate with higher TSH 

levels or microscopic findings. Although a significant trend for decreasing right epididymis 

weights was observed, it was not significant by pairwise comparison and was not observed in the 

left epididymis. 

TSH levels were increased (p<0.05) by 54 and 134% at 400 and 800 mg/kg/day, respectively. 

There was no corresponding effect on T4 levels, and the increased TSH levels did not 

consistently correlate with thyroid gland weight or histopathological findings on an individual 

animal basis. 

Mean testosterone levels were decreased in a dose-related manner (↓45%, NS and ↓69%, p<0.05) 

in the 400 and 800 mg/kg/day groups, respectively. The control group had three animals with 

unusually high testosterone levels (>8.00 ng/mL), and the control mean was above the 

performance criteria maximum (3.96 ng/mL) when these animals are included in the analysis. 

When these animals were excluded from the analysis, the mean value for the control group was 

2.27 ng/mL, which is similar to the values for the treated groups (2.29 and 1.35 ng/mL) although 

a statistical analysis was not conducted using the adjusted mean. 

At the scheduled euthanasia, no treatment-related findings were noted. A slight treatment-related 

effect on thyroid morphology is considered possible based the larger number of animals with 

follicular height Grade 1 and colloid area Grade 5 in the control group relative to the 400 and 

800 mg/kg/day animals; this is consistent with a higher number of treated animals with follicular 

height and colloid area scores of 3. These findings are consistent with higher mean thyroid 

weights and elevated TSH levels in the treated animals; however, these endpoints were not 

correlated when evaluated on an individual animal basis. Additionally, increased follicular 

height was not necessarily correlated with decreased colloid area in individual animals. There 

were no treatment-related findings in the testes, epididymides or kidneys. 

The high dose tested was considered to be adequate based on the results of a study in which a 

metolachlor dose of 1000 mg/kg/day (route of administration not reported) resulted in overt 

toxicity (all of the males found dead, euthanized in extremis or terminated early). In addition, 

organ toxicity was observed in the current study with both liver and kidney weights significantly 

increased at 400 and 800 mg/kg/day. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Male Pubertal Assay (OCSPP 

890.1500). 

COMPLIANCE: Signed and dated GLP Compliance and Quality Assurance statements were 

provided. 

Page 247 of 288
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I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: WIL Research Laboratories, LLC 

Location: Ashland, OH 44805 

Study Director: E. D. Sloter 

Other Personnel: G. B. Marit, Pathologist 

L. Freshwater, Statistics 

Study Period: July 27, 2010 – January 25, 2010 

2. Test Substance: Metolachlor 

 Description: Dark brown, clear liquid, MW 283.79 

 Source: Sipcam (Valdosta, GA) 

 Lot/Batch #: 20081115, exp. 15 November 2013 

 Purity: 97.3% 

 Stability: Stable in corn oil for 13 days at room temperature 

 CAS #: 51218-45-2 

 Structure: 

CH3

CH3

N

O

CH3

O

Cl

CH3

3. Vehicle: Corn oil 

4. Test Animals

 Species: Rat 

 Strain: Crl:CD(SD) Sprague-Dawley 

 Age/Weight at Study 

Initiation: PND 23 /42.2 – 69.1 g males only 

 Source: Charles River Laboratories; Raleigh, NC 

 Housing: Two-three/cage in plastic cages with heat treated laboratory-grade pine shavings for bedding 

 Diet: Harlan Laboratories 2016CM Teklad Global 16% protein rodent diet, ad libitum 

Phytoestrogen content average 10 ppm total isoflavones (genistein+daidzein+glycitein)

 Water: Municipal water, reverse-osmosis filtered (on site), ad libitum

 Environmental 

Conditions: 

Temperature: 

Humidity:

Air changes: 

Photoperiod:

22 ± 3 °C 

50 ± 20% 

10/hr 

14 hrs light/ 10 hrs dark 

B. STUDY DESIGN

1. In-Life Dates: Start: 08/29/2012 End: 09/29/2010 

2. Mating: Time-mated pregnant dams were received from the supplier between gestation days 

(GD) 11 or 12. The day an animal was found to be sperm positive was designated GD 0. 

Litters with more than 8 pups were standardized by culling on PND 4 to 8 pups per litter. 

Page 248 of 288
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3. Animal Assignment: Animals were assigned to the test groups noted in Table 1 using a 

randomization procedure designed to ensure equal stratification of body weights. 

Littermates were not assigned to the same treatment group. 

TABLE 1. Study Design a

Test group Dose (mg/kg/day) # of Males 

Control 0 15 

Low 400 15 

High 800 15 

a Data were obtained from page 28 of the study report. 

4. Dose Selection Rationale: Dose levels were selected based on the results of published 

toxicity data and the results of a previously conducted Hershberger assay (WIL-786002). In 

Phase 1 of the Hershberger assay, a dose level of 1000 mg/kg/day (route of administration 

not reported) resulted in all of the males found dead, euthanized in extremis or terminated 

early. In a subsequent Phase, oral gavage dose levels of 400 and 800 mg/kg/day for 5 days 

were tolerated by the animals and these dose levels were selected for the current study. 

5. Dose Preparation and Analysis: Dose formulations were prepared every 10 days by 

mixing appropriate amounts of test substance with corn oil. Homogeneity and stability of 

the test substance in formulations were assessed in a previous uterotrophic study (MRID 

48617801, reviewed concurrently) and data from the uterotrophic study are reported below. 

During the current study, samples from all dose formulations prepared during the in-life 

phase were analyzed for achieved concentration.

Results of Dose Analysis 

Homogeneity (% RSD): 1.8-2.5% 

Stability (% of initial): 92.5-97.9% following storage for 13 days at room temperature. 

Concentration: Mean concentrations ranged from 101% to 106% of nominal.

The analytical data indicated that the mixing procedure was adequate and that the variation 

between nominal and actual dosage to the animals was acceptable. 

6. Dosage Administration: All doses were administered once daily by gavage from PND 23 

through PND 53, in a volume of 5mL/kg of body weight. Dosing was performed between 

0700 and 0900 hours daily. 

7. Statistics: Statistics were conducted by BioSTAT Consultants, Inc using SAS® software. 

Data statistically analyzed included the organ weights, organ weight to body weight ratio 

(liver, kidneys, pituitary, and adrenal glands), daily body weights, cumulative body weight 

gain, serum chemistries, hormones, and weight and age at PPS. When an animal did not 

attain PPS, PND 54 (last Study Day + 1) and terminal body weight were used as the age and 

body weight at PPS, respectively. Gamma glutamyltransferase values under range were 

assigned a value of 0.1 (half the LLQ) for statistical analysis and reporting. 

Page 249 of 288
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Endpoints were tested for homogeneity of variance using Levene's test. If that test was 

significant (p=0.01), then a log or square root transformation was applied to the raw data; if 

the test was still significant then a nonparametric test was used to analyze the data. If the 

variances were homogeneous then an analysis of variance (ANOVA) was conducted. The 

statistical model contained a factor for treatment group and a blocking factor based on the 

time of study start. A two-sided Dunnett's test was conducted, regardless of the outcome of 

the ANOVA, looking for significant differences in the test article groups when compared 

with the vehicle control. If the variances were not homogeneous then the nonparametric 

Kruskal-Wallis test was used, ignoring the blocking factor, followed by Dunn's test, to 

compare each of the test article groups with the vehicle control. Since these were preplanned pairwise comparisons, Dunn's test was conducted regardless of the outcome of the 

Kruskal-Wallis test. The tests were two-sided (p=0.05), looking for significant differences 

from the vehicle control. In addition, organ weights, age and weight at PPS were subject to 

the following analyses if they met the homogeneity of variance criteria: 1) Analysis of 

covariance (ANCOVA) with Dunnett's test, using body weight at PND 21 as the covariate; 

2) linear trend test using the ANOVA model; and 3) linear trend test using the ANCOVA 

model. 

Histopathology findings presented as a graded response were analyzed with pairwise MannWhitney U tests. The tests were one-sided at p=0.05 for increased severity. The tests were 

two sided for graded responses presented on a Grade 1-5 scale at p=0.05 for increased or 

decreased severity. 

C. METHODS

1. Mortality and Clinical Examinations: All animals were examined twice daily for 

mortality and moribundity. Clinical examinations were conducted on the day of 

randomization and daily through the day of euthanasia. 

2. Body Weight: Animals were weighed on the day of randomization, daily prior to dosing 

and the day of euthanasia. Mean body weight changes were calculated for each 

corresponding interval and for the overall treatment period (PND 23-53). 

3. Preputial Separation (PPS): Beginning on PND 30, all animals were examined daily for 

onset of PPS. The appearance of a partial or a persistent thread of tissue between the glans 

and prepuce was recorded. Age and weight at on the day of completion of PPS were 

recorded. 

4. Sacrifice and Pathology: On the day before euthanasia, rats were moved to a holding room 

separate from where the necropsies were to be performed. Dosing was performed in the 

holding room on the day of termination and the rats were moved to the necropsy room one 

at a time for terminal procedures. Approximately 2 hours following dosing, animals were 

sacrificed by decapitation without anesthesia and all sacrifices were completed by 1300 

hours to minimize hormonal variability due to normal diurnal fluctuation. Immediately 

following decapitation, trunk blood samples were taken for T4, TSH, testosterone, and 

Page 250 of 288
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serum chemistry analyses. The hormone samples were stored at ≤−20° C for subsequent 

analyses. 

a. Hormone Analysis: The study authors reported that hormones were analyzed using either 

RIA or chemiluminescence; however, specific details and supporting data (i.e. method 

recovery and specificity data) were not provided.

b. Clinical Chemistry: The following CHECKED (X) parameters were examined. 

 ELECTROLYTES OTHER 

X Calcium X Albumin 

X Chloride Creatinine* 

 Magnesium X Urea nitrogen* 

X Phosphorus X Total cholesterol 

X Potassium X Globulins (by calculation) 

X Sodium X Glucose 

ENZYMES X Total bilirubin 

X Alkaline phosphatase (ALK) X Total protein 

 Cholinesterase (ChE) X Triglycerides 

 Creatine phosphokinase Serum protein electrophoresis 

 Lactic acid dehydrogenase (LDH) X A/G ratio (by calculation) 

X Alanine aminotransferase (ALT/also SGPT) X Creatinine 

X Aspartate aminotransferase (AST/also SGOT) X Bile acids 

 Sorbitol dehydrogenase 

X Gamma glutamyl transferase (GGT) 

 Glutamate dehydrogenase 

* Recommended based on guideline 890.1500. 

c. Organ Weights and Histopathology: The following CHECKED (X) tissues were 

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

examination. 

 UROGENITAL OTHER 

XX Testes (left and right separately)*+ XX Thyroid*+

XX Epididymides (left and right separately)*+ X Liver* 

X Seminal vesicle plus coagulating glands (with fluid)* X Adrenals (paired)* 

X Ventral prostate* X Pituitary* 

X Dorsolateral prostate* 

X Levator ani-bulbocavernosus (LABC) muscle complex* 

XX Kidneys (paired)*+ 

* Weights required based on guideline 890.1500 

+ Histopathological examination required based on guideline 890.1500 

The right testis, right epididymis and kidneys were weighed prior to fixation. Following 

weighing, the testis and epididymis were fixed in Bouin’s solution (duration of fixation not 

specified). The thyroid (with parathyroid) was collected with the trachea and fixed in 10% 

buffered formalin for at least 24 hrs. Following fixation, the thyroid was dissected free of 

the trachea and weighed. All collected tissues were routinely processed into paraffin blocks, 

sectioned, stained with hematoxylin and eosin, and examined microscopically. Thyroid 

Page 251 of 288
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sections were subjectively evaluated for follicular cell height and colloid area using a five 

point grading scale (1 = shortest/smallest; 5 = tallest/largest), and any abnormalities/lesions 

noted. Two sections from the thyroid were examined in order to obtain representative 

samples of the thyroid tissue. 

II. RESULTS 

A. Mortality: One male in the 800 mg/kg/day group (Animal No. 79812-07) was found dead 

on PND 40; no significant clinical or macroscopic findings were observed in this animal. 

One male in the control group (Animal No. 79823-03) was sacrificed in extremis on PND 47 

following clinical findings of a swollen right forelimb resulting in impaired use; gross 

examination of this animal at necropsy revealed a punctured esophagus probably due to a 

dosing error. All other rats survived until scheduled sacrifice. 

B. Clinical Signs of Toxicity: Clinical findings included salivation prior to dosing and clear 

material around the mouth approximately 1 hour post-dosing in the 400 and 800 mg/kg/day 

dose groups (Table 2). At 800 mg/kg/day, clear material was also observed around the 

ventral neck. 

TABLE 2. Incidence of Clinical Observations a

Observation 

Dose Level (mg/kg/day) 

Vehicle Control Metolachlor 

400 mg/kg/day

Metolachlor 

800 mg/kg/day 

# Observed # Examined # Observed # Examined # Observed # Examined 

No clinical observations 472b 15 4801 15 450b 15 

Found dead 0 15 0 15 1 15 

Sacrificed in extremis 1 15 0 15 0 15 

Salivation prior to dosing 0 15 8 15 10 15 

Wet clear material ventral 

neck-1 hr post-dose 0 15 2 15 6 15 

Wet clear material, mouth1 hr post-dose 3 15 10 15 15 15 

a Data were extracted from pages 68-70 of the study report 

b Total number of observations. All other observations refer to total number of animals with that observation at least once. 

C. General Growth and Preputial Separation: Body weights, body weight gains, age of 

attainment of PPS and weight at day of PPS are presented in Table 3. No treatment-related 

changes in body weights or body weight gains occurred at 400 or 800 mg/kg/day. The 

control group CV values for body weight at PND 21 (10.94%) and PND 53 (11.27%) were 

above the performance criteria acceptable ranges of 10.25% and 7.47%, respectively. 

The mean ages of PPS were 43.4, 42.4 and 43.4 days in the control, 400 and 800 mg/kg/day 

groups, respectively; there was no effect of treatment on this developmental landmark. The 

CV value for the age at attainment of PPS (6.08%) was above the acceptable performance 

criteria range (5.67%). The elevated CV was attributed to one control animal (# 79813-01) 

that did not achieve separation until PND 52. 

Page 252 of 288
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TABLE 3. General Growth and Preputial Separation (PPS)a

Parameter Evaluated 

Vehicle Control Metolachlor 

400 mg/kg/day

Metolachlor 

800 mg/kg/day 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) 

Initial body weight 

(PND 23; g) 

 

U 15 60.7 6.69 11.02 15 61.2 4.73 7.73 14 61.8 5.4 8.75 

Body weight at PPS 

(g) 

U 15 198.2 13.8 6.96 15 194.4 13 6.69 14 196.2 15.63 7.97 

A 15 198.1 13.8 6.96 15 194.1 13 6.69 14 196.2 15.63 7.97 

Final body weight 

(g) 

 

U 15 276.7 31.18 11.27 15 279.9 27.57 9.85 14 272.4 22.81 8.37 

Body weight gain 

(final – initial; g) U 15 215.8 25.48 11.81 15 218.8 25.52 11.67 14 211 22.01 10.43 

Final body weight 

percent of control NA NA NA NA 15 101 NA NA 15 98 NA NA 

Age at PPS 

(PND) 

U 15 43.4 2.64 6.08 15 42.4 1.45 3.43 14 43.4 1.7 3.91 

A 15 43.4 2.64 6.08 15 42.5 1.45 3.43 14 43.5 1.7 3.91 

Proportion unseparated 

(#/N) 15 0 15 0 14 0 

a Data were obtained from page 63 of the study report. 

U Unadjusted for body weight on PND 23 

A Adjusted for body weight on PND 23 

N Number of animals examined 

SD Standard Deviation 

CV Coefficient of Variation 

NA Not applicable 

D. Organ Weights: Organ weights at necropsy are presented in Table 4. Liver weights were 

significantly increased by 15-20% at 400 and 800 mg/kg/day (p<0.05). Kidney weights 

were significantly increased by 11% each at 400 and 800 mg/kg/day (p<0.05). A significant 

trend for increased thyroid weights was observed, although pairwise comparisons were not 

significant. The increasing trend in thyroid weights did not correlate with the observed 

higher TSH levels or with microscopic findings when evaluated on an individual animal 

basis. A significant trend for decreasing right epididymis weights was observed, but this 

was not significant by pairwise comparison and was not observed in the left epididymis. 

The mean control absolute thyroid (11.62 mg) and kidney weights (1.84 g) were below the 

performance criteria minimum acceptable value (14 mg and 2.242 g, respectively) but were 

within the laboratory’s historical control ranges for rats of this age. The mean CV values for 

ventral prostate (24.18%), adrenal gland (24.51%) and pituitary gland (20.39%) weights 

were above the performance criteria maximums of 22.32%, 22.77% and 15.98%, 

respectively.

Page 253 of 288
Male Pubertal Assay (2012) / Page 9 of 15 

METOLACHLOR/108801 OCSPP 890.1500 / OECD None

TABLE 4. Organ Weights at Necropsya

Organ 

Vehicle Control Metolachlor 

400 mg/kg/day

Metolachlor 

800 mg/kg/day 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) 

Liver 

(g) U 14 13.13 1.891 14.404 15 15.11*(↑15) 

1.323 8.757 14 15.77* 

(↑20) 

1.499 9.508

A 14 13.11 1.891 14.404 15 15.06*

(↑15) 

1.323 8.757 14 15.79* 

(↑20) 

1.499 9.508

R 14 4.73 0.246 5.207 15 5.41* 

(↑14) 

0.23 4.25 14 5.79* 

(↑22) 

0.32 5.518

Kidneys 

(g) U 14 1.84 0.23 12.47 15 2.05* (↑11) 

0.16 7.806 14 2.05* 

(↑11) 

0.151 7.392

A 14 1.84 0.23 12.47 15 2.04* 

(↑11) 

0.16 7.806 14 2.04* 

(↑11) 

0.151 7.392

R 14 0.67 0.031 4.724 15 0.74* 

(↑10) 

0.043 5.868 14 0.75* 

(↑12) 

0.05 6.638

Pituitary 

(mg) 

U 14 8.9 1.82 20.39 15 9 2.04 22.6 14 8 1.34 16.81

A 14 9 1.82 20.39 15 9 2.04 22.6 14 8.1 1.34 16.81

R 14 3.1 0.36 11.55 15 3.3 0.8 24.45 14 3.1 0.47 15.45

Adrenals 

(mg) 

U 14 36.1 8.84 24.51 15 34.6 6.47 18.72 14 33.7 7.88 23.41

A 14 36.2 8.84 24.51 15 34.6 6.47 18.72 14 34.1 7.88 23.41

R 14 12.9 2.31 17.89 15 12.4 2.15 17.34 14 12.4 3.01 24.2 

Seminal vesicle + 

coagulating gland, 

with fluid (mg) 

U 14 493.9 84.24 17.06 15 488.1 84.22 17.26 14 509.4 94.4 18.53

A 14 493.8 84.24 17.06 15 487.2 84.22 17.26 14 510.4 94.4 18.53

Seminal vesicle + 

coagulating gland, 

without fluid (mg) 

U NR NR NR NR NR NR NR NR NR NR NR NR 

A NR NR NR NR NR NR NR NR NR NR NR NR 

Ventral prostate 

(mg) 

U 14 210.9 50.99 24.18 15 208.8 41.58 19.91 14 198.7 53.39 26.87

A 14 210.5 50.99 24.18 15 207.9 41.58 19.91 14 198.6 53.39 26.87

Dorsolateral prostate 

(mg) 

U 14 103.6 25.66 24.78 15 105.3 20.2 19.18 14 103 21.34 20.71

A 14 104.2 25.66 24.78 15 105.9 20.2 19.18 14 104.9 21.34 20.71

LABC 

(mg) 

U 14 478.8 78.63 16.42 15 485.8 50.2 10.33 14 450.8 76.26 16.92

A 14 477.5 78.63 16.42 15 483.1 50.2 10.33 14 449.4 76.26 16.92

Epididymis, left 

(mg) 

U 14 189.9 32.56 17.15 15 196.4 22.24 11.32 14 176.6 31.05 17.58

A 14 190 32.56 17.15 15 196.1 22.24 11.32 14 177.5 31.05 17.58

Epididymis, right 

(mg) 

U 14 193.4 31.18 16.13 15 192 12.09 6.29 14 176.7 17.74 10.04

A 14 193.1 31.18 16.13 15 191.3 12.09 6.29 14 176.7 17.74 10.04

Testis, left 

(mg) 

U 14 1360.7 165.05 12.13 15 1349.6 126.42 9.37 14 1339 155.25 11.59

A 14 1361.9 165.05 12.13 15 1348.4 126.42 9.37 14 1346.2 155.25 11.59

Testis, right 

(mg) 

U 14 1352.4 159.52 11.8 15 1372.5 132.73 9.67 14 1278.4 97.06 7.59 

A 14 1354.1 159.52 11.8 15 1372 132.73 9.67 14 1286.5 97.06 7.59 

Thyroid, fixed 

(mg) 

U 14 11.62 1.796 15.457 15 13.38 2.471 18.473 14 13.85 3.479 25.108

A 14 11.56 1.796 15.457 15 13.28 2.471 18.473 14 13.76 3.479 25.108

a Data were obtained from page 66 of the study report. Percent differences from controls, calculated by the reviewers, are 

included in parentheses. 

U Unadjusted for body weight on PND 23 

A Adjusted for body weight on PND 23 

R Organ-to-body weight ratio (relative to body weight) 

N Number of animals examined

SD Standard Deviation 

CV Coefficient of Variation 

NR Not recorded 

* Significantly different from controls at p<0.05. 

Page 254 of 288
Male Pubertal Assay (2012) / Page 10 of 15 

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E. Clinical Chemistry and Hormone Levels: Mean hormone levels are presented in Table 5. 

TSH levels were increased (p<0.05) by 54% and 134% at 400 and 800 mg/kg/day, 

respectively. However, there was no corresponding effect on T4 levels and the TSH levels 

did not consistently correlate with thyroid gland weight or histopathological findings when 

evaluated on an individual animal basis. 

Mean testosterone levels were decreased in a dose related manner; the levels were 4.2, 2.3 

(↓45%) and 1.3 (↓69%, p<0.05) ng/mL in the control, 400 and 800 mg/kg/day groups, 

respectively. The control group had three animals (Nos. 79826-02, 79828-02 & 79829-04) 

with unusually high testosterone levels (>8.00 ng/mL); the control mean was above the 

performance criteria maximum level (3.96 ng/mL) when these animals were included in the 

analysis. However, when these animals were excluded from the analysis, the mean value for 

testosterone in the control group was 2.273 ng/mL. Statistical analysis using the adjusted 

mean was not presented but the values between treated and control groups appear similar. 

The following study deficiencies in the testosterone data were noted: 1) the details for the 

analytical method used testosterone analysis were not presented in the report, 2) no method 

validation or concurrent quality control data were presented, and 3) no historical control 

data from the performing laboratory were presented. Because of these limitations, it is not 

possible to determine if the control animals with high testosterone levels should or should 

not be excluded from the analysis. Thus, it is not possible to definitively determine if there 

is an effect of the test material on testosterone levels and reliable interpretation of the 

testosterone results. 

Globulin was increased (p<0.05) by 13-25% at 400 and 800 mg/kg/day, resulting in the A/G 

ratio being decreased (p<0.05) at both dose levels. Higher mean total protein was observed 

at 800 mg/kg/day (↑7%, p<0.05). GGT was increased (↑600%, p<0.05) at 800 mg/kg/day. 

Lower mean ALP levels were reported at 400 and 800 mg/kg/day but the control mean was 

well above the laboratory’s historical range, and the treated groups were within the 

historical range, so the toxicological significance is doubtful. No other clinical chemistry 

findings were noted. 

 

Page 255 of 288
Male Pubertal Assay (2012) / Page 11 of 15 

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TABLE 5. Hormone Levels and Clinical Chemistrya

Parameter 

Evaluated 

Vehicle Control Metolachlor

400 mg/kg/day 

Metolachlor

800 mg/kg/day 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) 

Hormones 

Serum T4, Total 

(μg/dL) 

14 6.82 1.192 17.48 15 7.78 1.514 19.458 14 7.27 1.196 16.445

Serum TSH 

(ng/mL) 

14 9.76 3.994 40.938 15 15.01*

(↑54) 

6.912 46.057 14 22.87* 

(↑134) 

12.143 53.092

Testosterone (ng/mL) 11 4.2 3.52 84 15 2.3 1.58 69.18 14 1.3* 

(↓69) 

0.76 56.56 

Clinical Chemistry 

A/G ratio 14 2.39 0.267 11.142 15 2.15* 

(↓10) 

0.164 7.602 14 2.03* 

(↓15) 

0.145 7.132 

Albumin (g/dL) 14 3.9 0.19 4.79 15 3.9 0.21 5.4 14 4 0.22 5.55 

Alkaline phosphatase 

(U/L) 

14 405 75.6 18.7 15 361 76.7 21.3 14 291* 

(↓28) 

59.9 20.6 

Alanine amino 

transferase (U/L) 

14 69 11.8 17.1 15 70 11.2 16 14 71 9 12.8 

Aspartate 

aminotransferase (U/L) 

14 157 42.1 26.9 15 151 52.6 34.8 14 148 31.3 21.2 

Bile acids (μmol/L) 14 40.4 27.15 67.28 15 31 12.9 41.55 14 29.9 10.3 34.44 

Calcium (mg/dL) 14 11.9 0.43 3.67 15 12 0.37 3.06 14 12 0.4 3.34 

Chloride (mEq/L) 14 101 1.1 1.1 15 101 1.2 1.2 14 100 1.4 1.4 

Cholesterol (mg/dL) 14 94 13.2 14.1 15 100 14.8 14.7 14 94 8.2 8.7 

Creatinine 

(mg/dL) 

14 0.2 0.05 34.59 15 0.1 0.05 36.22 14 0.2 0.04 19.55 

Gamma glutamyl 

transferase (U/L) 

14 0.1 0.11 83.15 15 0.2 0.26 127.32 14 0.7* 

(↑600) 

0.87 128.76

Globulin (g/dL) 14 1.6 0.15 9.16 15 1.8* 

(↑13) 

0.18 9.81 14 2* 

(↑25) 

0.14 7.37 

Glucose (mg/dL) 14 138 7.7 5.6 15 137 7.5 5.5 14 139 9.5 6.9 

Phosphorus (mg/dL) 14 9.6 0.68 7.09 15 9.6 0.85 8.87 14 9.7 1.13 11.67 

Potassium (mEq/L) 14 6.29 0.419 6.671 15 6.29 0.356 5.669 14 6.16 0.755 12.268

Sodium (mEq/L) 14 141 1.5 1.1 15 141 2 1.4 14 142 1.5 1 

Total bilirubin (mg/dL) 14 0.15 0.03 20.331 15 0.16 0.045 28.224 14 0.16 0.02 12.377

Total protein (g/dL) 14 5.6 0.17 3.04 15 5.7 0.33 5.81 14 6* 

(↑7) 

0.3 4.96 

Triglyceride (mg/dL) 14 471 183.9 39 15 581 164 28.2 14 477 193 40.5 

Serum urea nitrogen 

(mg/dL) 

14 15.1 2.06 13.64 15 14.8 1.74 11.83 14 14.5 2.38 16.44 

a Data were obtained from pages 64 and 65 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. 

F. Macro- and Histopathology: At the scheduled euthanasia, no treatment-related 

macroscopic findings were noted. Dilated renal pelvis was reported in 1 male at 800 

mg/kg/day. The incidences of histopathological findings of the thyroid gland are presented 

below in Table 6. A slight treatment-related effect on thyroid morphology was noted 

Page 256 of 288
Male Pubertal Assay (2012) / Page 12 of 15 

METOLACHLOR/108801 OCSPP 890.1500 / OECD None

possible based the larger number of animals with follicular height Grade 1 and colloid area 

Grade 5 in the control group relative to the 400 and 800 mg/kg/day animals; this is 

consistent with a higher number of treated animals with follicular height and colloid area 

scores of 3. These findings are consistent with higher mean thyroid weights and elevated 

TSH levels in the treated animals; however, these endpoints were not correlated when 

evaluated on an individual animal basis. Further complicating the evaluation is that the 

animals with abnormal follicular heights were not necessarily the same animals observed 

with abnormal colloid area. There were no treatment-related findings in the testes, 

epididymides or kidneys. 

TABLE 6. Incidence of Histopathological Lesions of the Thyroid Gland a

Treatment 

Groups 

Parameter Evaluated 

Colloid Area Follicular Cell Height 

Gradeb Incidence Gradeb Incidence 

O E O E 

Vehicle Control 

1 0 14 0 4 14 

2 0 14 2 10 14 

3 1 14 3 0 14 

4 7 14 4 0 14 

5 6 14 5 0 14 

Metolachlor

400 mg/kg/day 

1 0 15 1 1 15 

2 1 15 2 11 15 

3 2 15 3 3 15 

4 10 15 4 0 15 

5 2 15 5 0 15 

Metolachlor

800 mg/kg/day 

1 0 14 1 1 14 

2 1 14 2 8 14 

3 5 14 3 5 14 

4 7 14 4 0 14 

5 1 14 5 0 14 

a Data were obtained from page 397 of the study report. 

b Thyroid histopathology is graded on a 1 – 5 scale: Follicular cell height,1 = lowest, 5 = highest; and Colloid area, 

1 = most colloid, 5 = least colloid. 

O Number Observed 

E Number Examined 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: There was no evidence of altered pubertal 

development at any dose level of metolachlor tested (400 or 800 mg/kg/day) based on the 

absence of effects on the attainment of PPS and male reproductive organs. Higher globulin 

levels, and consequently lower A/G ratios, and higher kidney weights were also noted at 400 

and 800 mg/kg/day, and increased total protein levels were noted at 800 mg/kg/day. Mean 

testosterone levels were lower in the 400 and 800 mg/kg/day groups due to extraordinarily 

high testosterone values in 3 control males, resulting in an atypically high mean testosterone 

concentration in the control group. However, there were no perturbations in male pubertal 

development or effects on the male reproductive organs in this study at oral dose levels up 

to 800 mg/kg/day, the highest dose level of metolachlor tested. 

Page 257 of 288
Male Pubertal Assay (2012) / Page 13 of 15 

METOLACHLOR/108801 OCSPP 890.1500 / OECD None

Although mean TSH levels were increased at dosage levels of 400 and 800 mg/kg/day; there 

was no corresponding effect on T4 levels and the higher TSH values did not consistently 

correlate on an individual animal basis with thyroid gland weights or the subtle microscopic 

changes observed in the thyroid (minimally increased follicular cell height or decreased 

colloid area). The thyroid changes observed in this study were consistent with an adaptive 

response to test substance metabolism, and most likely secondary to chemical metabolizing 

enzyme induction and increased hepatic clearance of thyroxine (Curran and DeGroot, 1991). 

Metolachlor has been shown to induce liver cytochrome P450 enzymes (Dalton, et. al., 

2003). This is consistent with the higher liver weights noted in the 400 and 800 mg/kg/day 

group animals and apparent hepatocellular hypertrophy; however, liver histopathology was 

not performed in this study. Serum GGT levels were also higher for the 800 mg/kg/day 

group animals, which may indicate an effect on hepatobiliary function. A separate 

amphibian metamorphosis assay conducted using metolachlor concentrations up to 8.4 mg/L 

for 21 days found no changes in the thyroid glands of treated tadpoles (Palmer, et al., 2012, 

Wildlife International, Ltd. Project Number 568A-103). Therefore, based on the overall 

weight of evidence from this and other studies, metolachlor does not appear to produce an 

endocrine effect on the thyroid gland or disrupt thyroid homeostasis in peripubertal male 

rats, especially at an oral dose level of 400 mg/kg/day. 

B. AGENCY COMMENTS: One male in the 800 mg/kg/day group (Animal No. 79812-07) 

was found dead on PND 40; no significant clinical or macroscopic findings were observed 

in this animal. One male in the control group (Animal No. 79823-03) was sacrificed in 

extremis on PND 47 following clinical findings of a swollen right forelimb resulting in 

impaired use; gross examination of this animal at necropsy revealed a punctured esophagus 

probably due to a dosing error. All other rats survived until scheduled sacrifice. Clinical 

findings included salivation prior to dosing and clear material around the mouth 

approximately 1 hour post-dosing in the 400 and 800 mg/kg/day dose groups. At 800 

mg/kg/day, clear material was also observed around the ventral neck. 

No treatment-related changes in body weights or body weight gains occurred at 400 or 800 

mg/kg/day, and there was no effect on the age or weight at PPS. Liver weights were 

increased (p<0.05) by 15 and 20% at 400 and 800 mg/kg/day, respectively. Kidney weights 

were increased (p<0.05) by 11% each at 400 and 800 mg/kg/day. A significant trend for 

increased thyroid weights was observed, although pairwise comparisons were not 

significant; the increasing trend in thyroid weights did not correlate with higher TSH levels 

or microscopic findings. A significant trend for decreasing right epididymis weights was 

observed, but this was not significant by pairwise comparison and was not observed in the 

left epididymis. 

TSH levels were increased (p<0.05) by 54-134% at 400 and 800 mg/kg/day. However, 

there was no corresponding effect on T4 levels and the TSH levels did not consistently 

correlate with thyroid gland weight or histopathological findings when evaluated on an 

individual animal basis. 

Mean testosterone levels were decreased in a dose related manner, by 45% and 69% 

(p<0.05) at 400 and 800 mg/kg/day groups, respectively. However, the control group had 

Page 258 of 288
Male Pubertal Assay (2012) / Page 14 of 15 

METOLACHLOR/108801 OCSPP 890.1500 / OECD None

three animals with unusually high testosterone levels (>8.00 ng/mL); the control mean for 

testosterone (4.2 ng/mL) was above the performance criteria maximum level (3.96 ng/mL) 

when these animals were included in the analysis. When these animals were excluded from 

the analysis, the mean value for the control group was 2.273 ng/mL; statistical analysis 

using the adjusted mean was not presented but the values between treated and control 

groups appear similar. 

Globulin was increased (p<0.05) by 13-25% at 400 and 800 mg/kg/day; this resulted in the 

A/G ratio being decreased (p<0.05) at both dose levels. Higher mean total protein was 

observed at 800 mg/kg/day (↑7%, p<0.05). GGT was increased (↑600%, p<0.05) at 800 

mg/kg/day. Lower mean ALP levels were reported at 400 and 800 mg/kg/day but the 

control mean was well above the laboratory’s historical range, and the treated groups were 

within the historical range, so the toxicological significance is doubtful. No other clinical 

chemistry findings were noted. 

At the scheduled euthanasia, no treatment-related findings were noted. Dilated renal pelvis 

was reported in 1 male at 800 mg/kg/day. A slight treatment-related effect on thyroid 

morphology is considered possible based the larger number of animals with follicular height 

Grade 1 and colloid area Grade 5 in the control group relative to the 400 and 800 mg/kg/day 

animals; this is consistent with a higher number of treated animals with follicular height and 

colloid area scores of 3. These findings seem consistent with higher mean thyroid weights 

and elevated TSH levels in the treated animals, however, these endpoints were not 

correlated when evaluated on an individual animal basis. Further complicating the 

evaluation is that the animals with abnormal follicular heights were not necessarily the same 

animals observed with abnormal colloid area. There were no treatment-related findings in 

the testes, epididymides or kidneys. 

The highest dose tested was considered to be adequate based on the results of a Phase 1 of 

the Hershberger assay in which a dose level of 1000 mg/kg/day (route of administration not 

reported) resulted in all of the males found dead, euthanized in extremis or terminated early. 

Also, in this pubertal study, organ toxicity was observed; both liver and kidney weights 

were significantly increased at 400 and 800 mg/kg/day. 

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 control group CV values for body weights at PND 21 (10.94%) and PND 53 

(11.27%) were above the performance criteria acceptable ranges of 10.25% and 7.47%, 

respectively. 

 The CV value for the age at attainment of PPS (6.08%) was above the acceptable 

performance criteria range (5.67%). The elevated CV was attributed to one control 

animal (# 79813-01) that did not achieve separation until PND 52. 

 The mean control absolute thyroid (11.62 mg) and kidney weights (1.84 g) were below 

the performance criteria acceptable ranges (14 mg and 2.242 g, respectively) but were 

within the laboratory’s historical control ranges for rats of this age. 

Page 259 of 288
Male Pubertal Assay (2012) / Page 15 of 15 

METOLACHLOR/108801 OCSPP 890.1500 / OECD None

 The mean CV values for ventral prostate (24.18%), adrenal gland (24.51%) and 

pituitary gland (20.39%) weights were above the performance criteria maximums of 

22.32%, 22.77% and 15.98%, respectively. 

 The testis and epididymis were fixed in Bouin’s solution (duration of fixation not 

specified). 

 8/20 of the clinical chemistry control values were out of historical range at the 

performing laboratory. 

 Seminal vesicle + coagulating gland, without fluid (mg) were not weighed as per the 

guideline. 

Page 260 of 288
DATA EVALUATION RECORD 

METOLACHLOR 

Study Type: OCSPP 890.1550, Steroidogenesis Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-49-2012 (MRID 48617809) 

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: 5/3/2012 

Secondary Reviewer: Signature:

Michael E. Viana, Ph.D., D.A.B.T. Date: 5/7/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 5/8/2012 

Quality Assurance: Signature:

Steven Brecher, Ph.D., D.A.B.T. Date: 5/8/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 261 of 288
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 262 of 288
METOLACHLOR I 108801 

Primary Reviewer: Ayaad Assaad, D.V.M., Ph.D. 

Health Effects Division 

Secondary Reviewer: John Liccione, Ph.D. 

Health Effects Division 

Steroidogenesis Assay (2012) I Page 1 of 15 

OCSPP 890.1550/ OECD None 

S igna tu re: --1+--'Y---'+-..,f-iZ->--~ 

Date: ~r+-"7L~....::m----1£,;;--

Sign a tu r~~~~::!;g::;:=:::;;;~--­

Date: ---'"""""'-r-+-..!......-,'---

DATA EVALUATION RECORD 

STUDY TYPE: Steroidogenesis Assay (H295R Cells); OCSPP 890.1550 

PC CODE: 108801 DP BARCODE: D398926 

TXR#: 0052848 CAS No.: 51218-45-2 

TEST MATERIAL (PURITY): Metolachlor (97.3%) 

SYNONYMS: 2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-l-methylethyl)acetamide 

CITATION: Wagner, H. (2012) Metolachlor: H295R steroidogenesis assay. CeeTox, Inc., 

Kalamazoo, MI. Laboratory Study No.: 9179V-100537STER, January 26, 

2012. MRID 48617809. Unpublished. 

SPONSOR: Metolachlor Task Force, I 6 I 60 Nall, Stilwell, KS. 

TEST ORDER#: CON-108801-12 

EXECUTIVE SUMMARY: In a steroidogenesis assay (MRID 48617809), H295R cells 

cultured in vitro in 24-well plates were incubated with metolachlor, (97.3% a.i., Batch# 

2008 I 115) at log concentrations from 0.000 I to 100 μM in triplicate for 48 hours. Dimethyl 

sulfoxide (DMSO) was used as a vehicle, at a final concentration was 0.05%. 

Testosterone and estradiol concentrations were measured by HPLC/MS-MS with positive ion 

multiple reaction monitoring. Three independent experiments were performed. A Quality 

Control (QC) plate was run concurrently with each independent run of a test chemical plate to 

demonstrate that the assay responded properly to positive control agents at two concentration 

levels; positive controls included the known inhibitor (prochloraz) and inducer (forskolin) of 

estradiol and testosterone production. The highest suitable concentration of metolachlor in all 

three runs was 100 μM. 

Guideline recommendations were met, including adequate production of testosterone and 

estradiol, acceptable reproducibility (low %CV), and appropriate induction and inhibition with 

positive controls. Cytotoxicity greater than 20% was not observed. 

Statistically significant changes in testosterone production after incubation with metolachlor 

were not observed, although the mean fold-difference was decreased 17% for 100 μM 

metolachlor. Statistically significant increases in estradiol production were observed in two of 

the three runs at I 0 μM, and in all three runs at the 100 μM concentration. The mean foldPage 263 of 288
Steroidogenesis Assay (2012) / Page 2 of 15 

METOLACHLOR / 108801 OCSPP 890.1550/ OECD None

differences were approximately 1.2- and 1.3-fold, respectively, and showed a concentrationdependent response. 

Based on the hormone responses in each of the three independent runs, metolachlor treatment 

resulted in statistically significant and reproducible increases in estradiol production, but did not 

result in a statistically significant and reproducible alterations in testosterone production. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Steroidogenesis assay 

(OCSPP 890.1550).

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

Assurance statements were provided. 

 

Page 264 of 288
Steroidogenesis Assay (2012) / Page 3 of 15 

METOLACHLOR / 108801 OCSPP 890.1550/ OECD None

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: CeeTox, Inc. 

 Location: Kalamazoo, MI 

 Study Director: H. Wagner 

 Other Personnel: C. Toole, Director of Project Management 

K. Rutherford, Director of Laboratory Operations 

D. Blakeman, Senior Scientist 

B. Wallace, Lead Cell Culture Scientist 

F. Wong, Scientist 

L. Blakeman, Associate Scientist 

A. Cutter, Scientist 

 Study Period: August 31, 2011 to January 26, 2012 

2. Test Substance: Metolachlor 

 Description: Liquid 

 Batch #: (Expiration Date) 20081115 (November 15, 2013) 

 Purity: 97.3% 

 Solubility (in Solvent): Soluble in DMSO up to 200 mM 

 Volatility: Not reported 

 Stability: Stability in test preparations was not conducted. 

 Storage conditions: Room temperature 

 CAS #: 51218-45-2 

 Molecular weight: 283.8 

 Structure: 

CH3

CH3

N

O

CH3

O

Cl

CH3

3. Positive Control (Inducer): Forskolin 

 Description: White powder, MW 410.50 

 Source: Sigma Aldrich, St. Louis, MO (catalog # not reported) 

 Lot #: (Expiration Date): 109K50571V (July and August, 2016) 

 Purity: 98% 

 Solubility (in Solvent): Soluble in DMSO up to 100 mM 

 Storage conditions: Room temperature 

 CAS #: 66575-29-9 

4. Positive Control (Inhibitor): Prochloraz 

 Description: White powder, MW 376.67 

 Source: Sigma Aldrich, St. Louis, MO (catalog # not reported) 

 Lot #: (Expiration Date) SZE6220X (August 8, 2012) 

 Purity: 99.1% 

 Solubility (in Solvent): Soluble in DMSO up to 100 mM 

 Storage conditions: Room temperature 

 CAS #: 67747-09-5 

Page 265 of 288
Steroidogenesis Assay (2012) / Page 4 of 15 

METOLACHLOR / 108801 OCSPP 890.1550/ OECD None

5. Solvent/Vehicle Control: Dimethyl Sulfoxide (DMSO) 

 Description: Not reported 

 Source: Not reported 

 Lot #: (Expiration Date) Not reported 

 Purity: Not reported 

 Storage conditions: Not reported 

 CAS #: Not reported 

 Justification for choice of solvent: Not reported (Metolachlor, forskolin, and prochloraz soluble at test 

concentrations) 

 Final concentration: 

(% volume in assay) 0.05% (v/v) 

6. Other Materials: 22R-Hydroxycholesterol 

 Description (molecular weight): White powder (402.65) 

 Source: Sigma-Aldrich (St. Louis, MO) 

 Lot # (expiration date): 089K4132, 060M4098 (not provided) 

 Purity: 99.0% 

 Storage conditions: Room temperature 

 Solvent used: Ethanol, final concentration in assay, 0.025% v/v 

 CAS #: 17954-98-2 

7. Stock Medium: Dulbecco’s modified Eagle’s medium/Ham’s F12 

nutrient mixture (DMEM/Ham-F12) 

Description: Included 15 mM HEPES 

Source: Not reported 

Lot / Batch #: (Expiration Date) Not reported 

Sodium bicarbonate: Not reported 

Nu-Serum: Becton Dickinson, Catalog #355500, Lot #81515; tested for background 

hormone concentrations. 

ITS+ premix: Not reported 

8. Test Cells: H295R human adrenocortical carcinoma cells (ATCC, Manassas, VA, CLR2128; Lot #7635054). Cells were maintained in DMEM/Ham-F12 with 15 mM HEPES, 

sodium bicarbonate, ITS+Premix, and 2.5% Nu-Serum in a 5% CO2 incubator at 37±2oC. 

The background hormone concentrations present in the Nu-Serum were 3754 pg/mL for 

testosterone and 3846 pg/mL for estradiol. Cells were initially grown for five passages, and 

then frozen in liquid nitrogen. After thawing, cells were cultured additionally for seven or 

eight passages prior to use in the steroidogenesis assay. 22R-Hydroxycholesterol (SigmaAldrich, St. Louis, MO, Lot #060M4098, 99.0% pure, 10 μM) was added to the culture 

medium at plating, dosing, and harvesting. Cells were plated into wells of 24-well culture 

plates at a density of approximately 3 × 105

 cells/mL. Plates were incubated in a 5% CO2

atmosphere at 37±2oC for approximately 24 hours prior to exposure to the test chemical or 

the positive controls.

The following performance criteria were met (indicated by an “x”): 

x Cell passage identifier. Cell Passage #: ≥7.5 

x Cells frozen down at passage 5 

x Frozen cells cultured for 4 additional passages 

x Total number of passages does not exceed 10 (not reported) 

Page 266 of 288
Steroidogenesis Assay (2012) / Page 5 of 15 

METOLACHLOR / 108801 OCSPP 890.1550/ OECD None

B. METHODS

1. Pre-Test Information

a. Hormone Assay Interference Test: Hormone cross-reactivity or interference tests were 

not conducted because testosterone and estradiol concentrations were determined by 

HPLC/MS-MS analysis; no interference was expected. 

b. Hormone Extraction: Testosterone and estradiol were extracted from H295Rsupplemented medium by liquid/liquid extraction with methyl tert-butyl ether spiked with 

[

2

H5]-testosterone and [2H5]-estradiol as internal standards. The extracts were analyzed 

using HPLC/MS-MS with positive ion multiple reaction monitoring. The method MDL for 

testosterone and estradiol were 100 and 10 pg/mL, respectively, for a 300-μL portion of 

medium. 

c. Laboratory Proficiency Test: Laboratory proficiency testing was not conducted for the 

current study. 

2. Test Solutions: A 200-mM stock of metolachlor was prepared in DMSO, followed by 

serial dilutions (1:10) in DMSO. A 40-mM stock of 22R-hydroxycholesterol was dissolved 

in ethanol and further diluted in supplemented medium to achieve a final concentration of 

10 μM. Mastermix solutions with metolachlor were prepared by 1:2000 dilutions in 

supplemented medium containing 10 μM 22R-hydroxycholesterol. Forskolin and 

prochloraz solutions were prepared in a similar manner by preparing 100 mM solutions in 

DMSO, followed by serial dilutions in DMSO and 1:2000 dilutions in supplemented 

medium containing 10 μM 22R-hydroxycholesterol. Final concentrations after addition to 

the 24-well culture plates were 1 and 10 μM for forskolin, 0.1 and 1 μM for prochloraz, and 

0.0001 to 100 μM for metolachlor. Final concentrations of DMSO and ethanol in the 

medium were 0.05% and 0.025% (v/v), respectively.

3. Cell Plating and Preincubation: H295R cells (ATCC CLR-2128, Manassas, VA, Lot 

#7635054) were initially grown for five passages, and then frozen in liquid nitrogen. After 

thawing, cells were cultured additionally for seven or eight passages prior to use. 22RHydroxycholesterol (10 μM) was added to the culture medium at plating, dosing, and 

harvesting. Cells (1 mL) were seeded into wells of 24-well culture plates at a density of 

approximately 3 × 105

 cells/mL. Plates were incubated in a 5% CO2 atmosphere at 37±2oC 

for approximately 24 hours prior to exposure to the test chemical or the positive controls. 

The percent confluency after 24-hour incubation was not reported, but the protocol stated 

that 50-60% confluence was expected after plating 3 × 105

 cells/mL followed by a 24-hour 

incubation period. 

4. Exposure: The cells were checked microscopically for good attachment and proper 

morphology, and the medium was removed and replaced with 1 mL of supplemented 

medium containing 10 μM 22R-hydroxycholesterol. The cells were then exposed to 

identical volumes of either each serial dilution of the test compound or DMSO (solvent 

control; SC) in triplicate according to the schematic presented in Table 1. 

Page 267 of 288
Steroidogenesis Assay (2012) / Page 6 of 15 

METOLACHLOR / 108801 OCSPP 890.1550/ OECD None

TABLE 1. Dosing Schematic for the Exposure of H295R Cells to Metolachlor (Final Concentrations in 

μM)a

 1 2 3 4 5 6 

A DMSO DMSO DMSO 0.1 0.1 0.1 

B 100 100 100 0.01 0.01 0.01 

C 10 10 10 0.001 0.001 0.001 

D 1 1 1 0.0001 0.0001 0.0001 

a Data were obtained from page 13 of the study report. Dosing was calculated based on a total volume of 1 mL per well. 

A concurrent quality control (QC) plate was included with each of the four independent runs 

of the test chemical plates to demonstrate the assay’s response to forskolin (an inducer of 

testosterone and estradiol production) and prochloraz (an inhibitor of testosterone and 

estradiol production). The QC plate was prepared and dosed in the same manner with either 

forskolin or prochloraz according to the schematic presented in Table 2. 

TABLE 2. Dosing Schematic for the QC Plate for Positive Controls (Final Concentrations in μM)a

 1 2 3 4 5 6 

A Blankb

 Blank Blank Backgroundc Background Background 

B DMSO DMSO DMSO DMSO + MeOHd DMSO + MeOH DMSO + MeOH 

C Forskolin 1μM Forskolin 1μM Forskolin 1μM Prochloraz 0.1μM Prochloraz 0.1μM Prochloraz 0.1μM 

D Forskolin 10μM Forskolin 10μM Forskolin 10μM Prochloraz 1μM Prochloraz 1μM Prochloraz 1μM 

a Data were obtained from page 14 of the study report. Dosing was calculated based on a total volume of 1 mL per well. 

b Blank wells received medium containing 10 μM 22R-hydroxycholesterol. 

c Background wells received medium without 22R-hydroxycholesterol). 

d MeOH = 70% methanol was added to these wells for 30 minutes at room temperature following medium removal. 

Following dosing, the plates were incubated for 48±2 hours under the conditions described 

previously. After the incubation period, each well was examined microscopically for 

cytotoxicity and cell attachment. Precipitation, if present, was noted. The medium from 

each well was removed, split into two equal volumes, and frozen at −80±10°C until 

hormone measurements were conducted. Cell viability was determined after media removal 

was completed. 

5. Cell Viability/Cytotoxicity Assay: Cell viability was determined with the MTT [3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] test described by Mosman (1983)1

immediately after removal of the culture medium after the 48-hour incubation. The QC 

plate wells designated to receive methanol (MeOH) were rinsed twice with phosphate 

buffered saline (PBS), followed by an incubation with MeOH for 30 minutes at room 

temperature, and three additional rinses with PBS. Next, 0.5-mL portions of a 0.5 mg/mL 

MTT solution in supplemented medium containing 10 μM 22R-hydroxycholesterol were 

added to each well on the test chemical and QC plates. MTT-treated plates were incubated 

in a 5% CO2 atmosphere at 37±2oC for 3 hours, followed by removal of the MTT solutions 

and addition of 0.5 mL of isopropanol to each well. Plates were incubated for a further 20 

minutes at room temperature with shaking. After the 20-minute incubation, the absorbance 

values at 570 and 650 nm were determined with a Packard Fusion plate reader, and the 

 

1 Mosman T. (1983). Rapid colorimetric assay for growth and survival: application to proliferation and 

cytotoxicity. J. Immunol. Methods. 100:45-50. 

Page 268 of 288
Steroidogenesis Assay (2012) / Page 7 of 15 

METOLACHLOR / 108801 OCSPP 890.1550/ OECD None

absorbance at 650 nm was subtracted from the absorbance at 570 nm to calculate the MTT 

value. A reduction of ≥20% in cell viability was considered evidence of cytotoxicity.

6. Hormone Measurement System: Concentrations of testosterone and estradiol in the 

supplemented medium were determined by HPLC-MS/MS at a separate analytical facility 

(OpAns, LLC, Durham, NC). The extraction method (with spiked, labeled internal 

standards) was described previously (B.1.b.). All back-calculated calibration standard 

concentrations and QC concentrations were acceptable. The percent recovery across the test 

runs for the QC samples ranged from 87.1-111.9% for testosterone and 86.4-114.7% for 

estradiol.

The following performance criteria were met (indicated by an “x”): 

X Method detection limit (100 pg/mL testosterone; 10 pg/mL estradiol) 

X Spiked sample recovery acceptable for two concentrations of testosterone and estradiol (mean measured amount 

from triplicate samples within 30% of nominal concentration) 

NA Hormone cross-reactivity (antibody-based assays only; ≤30% of basal production of the respective hormone) 

X Solvent control within 75% range below maximum response on standard curve 

NA Test compound tested for interference with measurement system 

C. DATA ANALYSIS: Mean values (pg/mL) and standard deviations (SD) for testosterone 

and estradiol concentrations were calculated for each concentration of the reference 

chemicals and SC, as well as the blank and background wells, on the QC plates and for each 

concentration of metolachlor and SC on the test chemical plate. Relative changes in 

hormone production were calculated with the following equation:

Relative change = (hormone concentration in each well) ÷ (mean SC hormone 

concentration) 

For forskolin induction of testosterone, the background hormone production was subtracted 

from the forskolin-treated wells, and blank and SC wells, prior to calculation of the relative 

change. Background hormone production was determined from three wells on the QC plate 

that received H295R cells with no 22R-hydroxycholesterol. 

Concentrations that exhibited ≥20% cytotoxicity, or where precipitation was observed, were 

omitted from further analysis. 

Normality of the data was evaluated with the Shapiro-Wilk’s test. Homogeneity of the 

variances between the treatment groups was evaluated by using Levene’s test. Statistical 

significance between each treatment group and control was evaluated with Dunnett’s test if 

p>0.05 in both tests. If p≤0.05 in the normality or homogeneity tests, a log transformation 

was performed on the data to attempt to approximate a normal distribution. Dunnett’s test 

was conducted to evaluate statistical significance between the treatment and control groups 

if p>0.05 in both the normality and homogeneity tests after transformation. If p≤0.05 in 

either the normality or homogeneity tests after transformation, the non-transformed data 

were analyzed with the nonparametric Kruskal-Wallis test, followed by Dunn’s test, to 

evaluate statistical significance between the treatment and control groups. 

Data analysis was conducted with Microsoft Excel, and statistics were calculated with the 

Unistat 6.0 Light program for Excel. These analyses are considered to be appropriate. 

Page 269 of 288
Steroidogenesis Assay (2012) / Page 8 of 15 

METOLACHLOR / 108801 OCSPP 890.1550/ OECD None

II. RESULTS

A. TEST COMPOUND: Three of the four assay runs met the guideline criteria for further 

evaluation. The hormone concentrations after exposure to metolachlor, and the folddifference change relative to SC and mean ± SD for the three suitable runs (2 through 4), are 

presented in Table 3. The fold-changes in hormone concentration for metolachlor in the 

three runs are shown graphically in Figures 1 and 2 for testosterone and estradiol, 

respectively. The highest suitable concentration of metolachlor in all three runs was 

100 μM. 

The %CVs for absolute hormone concentrations for the SC replicates within the test plates 

were 3.41 to 8.82% for testosterone, and 1.66 to 4.68% for estradiol. The between-plate 

%CV for the absolute concentrations in the SC (calculated by the reviewer) was 12.4% for 

testosterone and 3.6% for estradiol. These data met the recommended performance criteria 

of ≤30%. 

Statistically significant changes in testosterone production after incubation with metolachlor 

were not observed, although the mean fold-difference was decreased 17% for 100 μM 

metolachlor. Statistically significant increases in estradiol production were observed in 

Runs 2 and 3 at 10 μM, and in all three runs at the 100 μM concentration. The mean folddifferences were approximately 1.2- and 1.3-fold, respectively, and showed a concentrationdependent response. 

Page 270 of 288
Steroidogenesis Assay (2012) / Page 9 of 15 

METOLACHLOR / 108801 OCSPP 890.1550/ OECD None

TABLE 3. Mean (±SD) Hormone Concentrations Following Treatment with Metolachlor for 48 Hours.a

Nominal 

Concentration 

(μM) 

Run 2 Run 3 Run 4 Run 2 Run 3 Run 4 Meanb ± SDb Statistical 

Significance

Testosterone (pg/mL) Fold Difference 

DMSO (SC) 1832 1706 2164 — — — — — — 

0.0001 1819 1710 2150 0.99 1.00 0.99 0.99 0.006 — 

0.001 1760 1743 2123 0.96 1.02 0.98 0.99 0.031 — 

0.01 1886 1768 2163 1.03 1.04 1.00 1.02 0.021 — 

0.1 1808 1810 2233 0.99 1.06 1.03 1.03 0.035 — 

1 1821 1705 2099 0.99 1.00 0.97 0.99 0.015 — 

10 1906 1803 2095 1.04 1.06 0.97 1.02 0.047 — 

100 1505 1336 1903 0.82 0.78 0.88 0.83 0.050 — 

Estradiol (pg/mL) Fold Difference 

DMSO (SC) 99 93 99 — — — — — — 

0.0001 95 97 105 0.96 1.04 1.06 1.02 0.053 — 

0.001 100 98 103 1.01 1.05 1.04 1.03 0.021 — 

0.01 96 97 104 0.97 1.04 1.05 1.02 0.044 — 

0.1 98 99 101 0.99 1.06 1.02 1.02 0.035 — 

1 95 94 99 0.96 1.01 1.00 0.99 0.026 — 

10 108 121 122 1.09* 1.30* 1.23 1.21 0.107 Run 2 and 3 

100 114 116 145 1.15* 1.24* 1.46* 1.28 0.159 Run 2, 3, and 4 

a Data were obtained from page 26-27 and 34-36 of the study report. 

b Calculated by the reviewers 

SC Solvent control 

* Statistically significant (p≤0.05) 

Page 271 of 288
Steroidogenesis Assay (2012) / Page 10 of 15 

METOLACHLOR / 108801 OCSPP 890.1550/ OECD None

FIGURE 1. Change in Testosterone Production Relative to Metolachlor Concentration in 

Test Runs #2 to #4. 

Page 272 of 288
Steroidogenesis Assay (2012) / Page 11 of 15 

METOLACHLOR / 108801 OCSPP 890.1550/ OECD None

FIGURE 2. Change in Estradiol Production Relative to Metolachlor Concentration in 

Test Runs #2 to #4. 

Page 273 of 288
Steroidogenesis Assay (2012) / Page 12 of 15 

METOLACHLOR / 108801 OCSPP 890.1550/ OECD None

B. CYTOTOXICITY: No evidence of cytotoxicity was present from the MTT cell viability 

assay conducted on the QC and metolachlor test plates (Table 4 and Figure 3), although 

viability at the 100 μM concentration was decreased approximately 10%. The % viability 

values ranged from 97.5-106.8% on the QC plates and 87.1-104.6% on the test chemical 

plates; methanol exposure reduced cell viability to 3.6-4.7%. 

TABLE 4. Mean (±SD) MTT Cell Viability Results after Treatment with Forskolin, Prochloraz, 

or Metolachlor for 48 Hours.a 

Compound Concen. 

(μM) 

Cell Viability – Run 2 Cell Viability – Run 3 Cell Viability – Run 4 

Mean SD Mean SD Mean SD 

Blank NA 100.4 2.85 98.3 1.14 101.6 4.49 

Background NA 100.8 1.16 99.6 0.8 101.1 3.65 

SC + Methanol NA 4.70 1.06 4.40 1.22 3.60 1.61 

Forskolinb 1 106.2 1.63 105 0.6 106.8 1.05 

Forskolin 10 105.1 1.63 104.4 3.65 106.4 2.52 

Prochloraz 0.1 100.3 1.52 97.5 2.87 101.9 2.55 

Prochloraz 1 102.5 5.39 99.3 2.61 101.8 5.07 

Metolachlor 0.0001 94.2 0.48 96.7 1.56 101.0 4.38 

Metolachlor 0.001 95.2 3.69 99.6 1.97 100.5 3.90 

Metolachlor 0.01 96.9 2.18 102.3 5.83 103.9 5.34 

Metolachlor 0.1 96.1 1.47 104.6 4.21 98.1 3.06 

Metolachlor 1 93.7 5.33 101.6 5.29 93.3 3.06 

Metolachlor 10 92.8 2.18 98.5 3.04 96.6 2.05 

Metolachlor 100 87.1 6.68 92.8 0.8 89.9 1.85 

a Data were obtained from page 20-21 of the study report. 

SC Solvent control 

Page 274 of 288
Steroidogenesis Assay (2012) / Page 13 of 15 

METOLACHLOR / 108801 OCSPP 890.1550/ OECD None

FIGURE 3. MTT cell cytotoxicity results for Test Runs 2 and 3. 

C. QC PLATE: The hormone concentrations after exposure to the reference chemical, SC, 

blank, and background samples, as well as the fold-difference change relative to SC 

(individual and mean ± SD) for the three suitable assay runs, are presented in Table 5. 

Minimum basal hormone production was met in all blank and SC wells (500 pg/mL for 

testosterone, 40 pg/mL for estradiol), and basal hormone production met the fold-increase 

criteria in SC of ≥5-fold for testosterone and ≥2.5-fold for estrogen above the MDL of the 

assay. Exposure to 10 μM forskolin induced testosterone production 1.4- to 1.7-fold, which 

is below the Guideline criteria of ≥2-fold; however, when the background is subtracted, 10 

μM forskolin induced testosterone an average of 2.3-fold. Estradiol production was induced 

Page 275 of 288
Steroidogenesis Assay (2012) / Page 14 of 15 

METOLACHLOR / 108801 OCSPP 890.1550/ OECD None

with 10 μM forskolin by 12.5- to 17.6-fold. Cells incubated with 1 μM prochloraz inhibited 

testosterone and estradiol production by ≤0.5-fold. Thus, the guideline requirements were 

met indicating that the assay was sensitive to induction and inhibition of testosterone and 

estradiol. 

The %CVs based on absolute hormone concentration in SC well replicates were 2.49-7.65% 

for testosterone, and 2.22-8.11% for estradiol. The between-plate variability (calculated by 

the reviewer) for the QC plates based on the absolute hormone concentrations in the SC was 

8.8% for testosterone and 8.3% for estradiol. These data met the performance criteria 

recommended by the Guideline (≤30%). 

TABLE 5. Mean (±SD) Hormone Concentrations Following Treatment with Forskolin or Prochloraz for 

48 Hours.a

Concentration (μM) Run 2 Run 3 Run 4 Run 2 Run 3 Run 4 Meanb ± SDb

Testosterone (pg/mL) Fold Difference (Relative to DMSO) 

Background 1213 1030 1081 — — — — — 

Blank 1736 1892 2102 — — — — — 

DMSO 1857 1844 2148 — — — — — 

1 μM Forskolin 2518 2539 3050 1.36c

 1.38 c 1.42 c 1.39 c 0.03 

10 μM Forskolin 2585 3011 3649 1.39 d

 1.63 d 1.70 d 1.57 d 0.16 

0.1 μM Prochloraz 1534 1503 1697 0.83 0.82 0.79 0.81 0.02 

1 μM Prochloraz 735 700 803 0.40 0.38 0.37 0.38 0.02 

Estradiol (pg/mL) Fold Difference (Relative to DMSO) 

Background 28 26 43 — — — — — 

Blank 95 104 92 — — — — — 

DMSO 97 95 83 — — — — — 

1 μM Forskolin 662 867 775 6.80 9.14 9.33 8.42 1.41 

10 μM Forskolin 1213 1534 1463 12.47 16.17 17.62 15.4 2.66 

0.1 μM Prochloraz 90 89 78 0.93 0.93 0.94 0.93 0.01 

1 μM Prochloraz 52 33 35 0.53 0.34 0.42 0.43 0.10 

a Data were obtained from page 22-23 of the study report. 

b Calculated by the reviewers. 

c When the background is subtracted, the values are 2.03-, 1.85-, and 1.84-fold difference for Runs 2, 3, and 4, respectively, 

with a mean of 1.91-fold change. 

d When the background is subtracted, the values are 2.13-, 2.43-, and 2.41-fold difference for Runs 2, 3, and 4, respectively, 

with a mean of 2.32-fold change. 

Page 276 of 288
Steroidogenesis Assay (2012) / Page 15 of 15 

METOLACHLOR / 108801 OCSPP 890.1550/ OECD None

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Metolachlor exposure did not result in increases 

or decreases in testosterone at any of the concentrations tested in any of the assays. 

Statistically significant increases in estradiol relative to the solvent control were observed in 

all three runs of the assay at nominal concentrations of 10 μM and/or 100 μM metolachlor. 

The increases were observed at the two highest metolachlor concentrations tested in Runs 2 

and 3 and at the highest metolachlor concentration tested in Run 4. 

B. AGENCY COMMENTS: Laboratory proficiency testing results were not conducted as 

detailed in a protocol amendment for the current study because laboratory proficiency data 

had previously been determined for this batch of cells. The details of this previous 

proficiency determination, including estimates of EC50s for prochloraz and forskolin were 

not reported in the current study report. No evidence of cytotoxicity was present from the 

MTT cell viability assay conducted on the QC and metolachlor test plates, although viability 

at the 100 μM concentration was decreased approximately 10%. The highest suitable 

concentration of metolachlor in all three runs was 100 μM. 

Minimum basal hormone production was met in all blank and SC wells, and basal hormone 

production met the fold-increase criteria in SC of ≥5-fold for testosterone and ≥2.5-fold for 

estrogen above the MDL of the assay. Exposure to 10 μM forskolin induced testosterone 

production 1.4- to 1.7-fold, which is below the Guideline criteria of ≥2-fold; however, when 

the background is subtracted, 10 μM forskolin induced testosterone an average of 2.3-fold. 

Estradiol production was induced with 10 μM forskolin by 12.5- to 17.6-fold. Cells 

incubated with 1 μM prochloraz inhibited testosterone and estradiol production by ≤0.5-

fold. These data generally met the performance criteria recommended by the Guideline. 

The %CVs based on the absolute hormone concentration in the SC met the Guideline 

performance criteria of ≤30% for testosterone and estradiol in the test and QC plates.. 

Statistically significant changes in testosterone production after incubation with metolachlor 

were not observed, although the mean fold-difference was decreased 17% for 100 μM 

metolachlor. Statistically significant increases in estradiol production were observed in 

Runs 2 and 3 at 10 μM, and in all three runs at the 100 μM concentration. The mean folddifferences were approximately 1.2- and 1.3-fold, respectively, and showed a concentrationdependent response. Based on the hormone responses in each of the three independent runs, 

metolachlor treatment resulted in a statistically significant and reproducible alteration in 

estradiol production, but not in testosterone production. 

C. STUDY DEFICIENCIES: The following deficiency was noted that is not considered to 

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

 Laboratory proficiency testing prior to the initiation of the current study was not 

conducted, and the details of a previous proficiency determination with the current 

batch of cells, including estimates of EC50s for prochloraz and forskolin, were not 

reported in the study report.

Page 277 of 288
DATA EVALUATION RECORD 

METOLACHLOR 

Study Type: OCSPP 890.1600, In vivo Uterotrophic Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-49-2012 (MRID 48617801) 

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: 5/1/2012 

Secondary Reviewer: Signature:

David A. McEwen, B.S. Date: 5/7/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 5/8/2012 

Quality Assurance: Signature:

Steven Brecher, Ph.D., D.A.B.T. Date: 5/8/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 278 of 288
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 279 of 288
METOLACHLOR / 108801 

Primary Reviewer: Ayaad Assad, D.V.M., Ph.D. 

Health Effects Division 

Secondary Reviewer: _J"""""'e=s-=-s -=-R=o--'-w=la=n=d::.._____ _____ _ 

Health Effects Division 

/11 vivo Vterotrophic Assay (2012) I Page I of9 

OCSPP 890.1600/ OECD 440 

Signature: t:Jb~d Date:~~_!5 Signature: ------1~'-'~--'-'----==-::--=---

Date: '=r'>(r 2 

Template version 09/2011 

DATA EVALUATION RECORD 

STUDY TYPE: Uterotrophic Assay (Rat); OCSPP 890.1600; OECD 440 

PC CODE: 108801 DP BARCODE: D398926 

TXR#: 0052848 CAS#: 51218-45-2 

TEST MATERIAL (PURITY): Metolachlor (97.3% a.i.) 

SYNONYMS: 2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-l-methylethyl)acetamide 

CITATION: Sloter, E.D. (2012) A Uterotrophic Assay ofMetolachlor Administered 

Subcutaneously in Ovariectomized Rats. WIL Research Laboratories, LLC, 

Ashland, OH. Laboratory Study No.: WIL-786001, January 3, 2012. MRID 

48617801 . Unpublished. 

SPONSOR: Metolachlor Task Force, 16160 Nall, Stilwell, KS 

TEST ORDER#: CON-108801-12 

EXECUTIVE SUMMARY: In an uterotrophic assay (MRID 48617801) conducted to screen 

for potential estrogenic activity, metolachlor (97.3% a.i., lot# 20081115) in com oil was 

administered daily via subcutaneous (s.c.) injection to groups of six ovariectomized Sprague 

Dawley rats at dose levels of 0 (vehicle), 100, 300, or 1000 mg/kg/day on post-natal days (PND) 

57-59. A positive control group was treated with a daily dose of 17a-ethynyl estradiol (EE) at 

0.5 μg/kg/day by s.c. injection. Body weights were determined daily. All animals were 

terminated and necropsied on PND 60 approximately 24 hours after the final dose administration 

to determine wet and blotted uterine weights. 

All animals survived until scheduled termination. No test substance-related clinical findings 

were noted in any of the animals during the study. Body weights were comparable to the 

controls throughout the duration of the assay, but overall (Days 1-4) body weight gains were 

decreased (p<0.01) in the 300 mg/kg/day (142%) and 1000 mg/kg/day (153%) metolachlor 

groups compared to the controls. Body weight gains in the positive control (EE) group were also 

decreased (p<0.01) by 57% compared to the controls. 

Uterine weights in the metolachlor treated groups were comparable to the controls. Absolute wet 

and blotted uterus weights for the positive control (EE) group were increased (p<0.05) as 

expected by 288% and 175%, respectively. 

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Based on the observed decreases (42% and 53%) in body weight gain at 300 and 1000 

mg/kg/day, adequate dose levels were achieved in this study. 

No statistically significant changes were seen in uterine weight in this assay. Metolachlor is 

negative in the uterotrophic assay. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a uterotrophic assay (OCSPP 

890.1600). 

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

Assurance statements were provided. 

 

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I. MATERIALS AND METHODS

A. MATERIALS 

1. Test Facility: WIL Research Laboratories, LLC 

Location: Ashland, OH 

 Study Director: E.D. Sloter 

 Other Personnel: S.T. Coffee (dose formulation analyses); L. Freshwater (BioSTAT Consultants, 

statistical analyses); E.S. Bodle (Assistant Director, Analytical Chemistry); C.A. 

Kopp (Manager, Gross Pathology and Developmental Toxicology Laboratory); 

T.M. Rafeld (Manager, Formulations Laboratory); 

 Study Period: July 28, 2010 to January 3, 2012 

2. Test Substance: Metolachlor 

 Description: Dark brown, clear liquid 

 Source: Sipcam (Valdosta, GA) 

 Lot #: 20081115 (expiry date: November 15, 2013) 

 Purity: 97.3% a.i. 

 Stability: Stable at room temperature (ambient) 

 CAS #: 51218-45-2 

 Structure: 

3. Reference Estrogen: 17α-ethynyl estradiol (EE) 

 Supplier: Sigma Aldrich, Inc. (St. Louis, MO) 

 Lot #: 028K1411 (expiry date: August 4, 2011) 

 Purity: 99.0% 

 CAS #: 57-63-6 

4. Solvent/Vehicle Control: Corn oil 

 Supplier: Spectrum Chemical Manufacturing Corporation (New Brunswick, NJ) 

 Lot #: ZR0032 and ZQ0791 (expiry dates: March 30, 2012 and May 3, 2012, respectively)

 Rationale (if other than water): Corn oil was selected as the vehicle due to the insolubility of metolachlor in water 

 Final concentration: Not applicable 

5. Test Animals: 

 Species: Rat (ovariectomized) 

 Strain: Sprague Dawley, Crl:CD(SD) 

 Age/weight at dose initiation: 57 days old/ 195.2 – 267.1 g females 

 Source: Charles River Laboratories (Portage, MI)

 Housing: Individually caged in stainless steel wire-mesh cages suspended above cage-boards

 Diet: Harlan Laboratories 2016CM Teklad Global 16% Protein Rodent Diet, ad libitum 

Average of 10.0 ppm total isoflavones (genistein + daidzein + glycitein) 

 Water: Reverse-osmosis filtered tap water, ad libitum 

 Environmental conditions: Temperature: 

Humidity: 

Air changes: 

Photoperiod: 

20.6-21.6 oC 

37.5-45% 

>10/hr 

12 hrs light/ 12 hrs dark 

 Acclimation period: 10 days 

3CH

N

O

Cl

CH3

O

3CH

CH3

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B. STUDY DESIGN 

1. In Life Dates: Start: August 27, 2010 End: August 30, 2010 

2. Study Design: Sexually mature ovariectomized rats (PND 47) were received from Charles 

River Laboratories (ovariectomized on PND 42). Animals were acclimated for 10 days 

prior to initiation of dosing. Vaginal lavage was conducted daily during the last five days of 

the acclimation period to verify complete removal of the ovaries as indicated by a lack of 

estrous cycling. The dose administration period was from PND 57 through 59. Rats were 

euthanized approximately 24 hours later on PND 60, and necropsied for uterine weight 

measurements. 

3. Animal Assignment: Animals were assigned to the test groups noted in Table 1, using a 

computer program which randomized the animals based on stratification of body weights in 

a block design. Statistical analysis indicated that there were no significant differences 

between group mean weights at study initiation. Furthermore, the body weight of each 

animal was within 20% of the overall mean. 

TABLE 1. Study Design a

Test Group Dose (mg/kg/day) # of Females 

Estrogen Agonist Assay 

Vehicle Control (Corn oil) 0 6 

Metolachlor Low 100 6 

Metolachlor Mid 300 6 

Metolachlor High 1000 6 

17α-ethynyl estradiol (EE), Reference Estrogen 0.0005 6 

a Data were obtained from page 22 of the study report. 

4. Dose Selection Rationale: The dose levels were selected based on published toxicity data 

in which a pilot range-finding study (U.S. EPA1) showed no effects on body weights at 800 

mg/kg/day metolachlor. Therefore, the limit dose of 1000 mg/kg/day was selected for the 

high-dosage level for the uterotrophic study. The dosage level for 17α-ethynylestradiol was 

based on the validation study (WIL-995792

).

5. Dose Preparation and Analysis: The metolachlor formulations were prepared once as 

single formulations for each dosage level, divided into sterile aliquots for daily dispensation, 

and stored at room temperature. The positive control substance (EE) was dissolved in a 

minimal amount of 95% v/v aqueous ethanol solution prior to addition of corn oil for a 0.05 

mg/mL stock solution. The stock solution was prepared once and stored at room 

 

1

 United States Environmental Protection Agency. (1995) Registration Eligibility Decision (RED): Metolachlor. 

(EPA 738-R-95-006), Washington, DC: Government Printing Office. 

2

 Kuhl, A.J. (2011) A Baseline Positive Control Uterotrophic Assay of 17α-Ethynylestradiol Administered 

Subcutaneously in Ovariectomized Sprague Dawley Rats (WIL-99579). WIL Research Laboratories, LLC, Ashland, 

OH. 

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temperature, protected from light. EE dosing formulations were prepared daily by dilution 

of the stock solution with corn oil. The test substance and positive control substance 

formulations were stirred continuously throughout the preparation, sampling, and 

administration. 

Prior to the initiation of dose administration, quadruplicate samples for homogeneity 

determination were collected from the top, middle, and bottom strata of the 20 and 200 

mg/mL dosing formulations (100 and 1000 mg/kg/day, respectively). For stability 

determinations, quadruplicate samples for resuspension homogeneity determinations were 

collected from the top and bottom strata of these same dosing suspensions following room 

temperature storage for 13 days. In addition, quadruplicate samples for concentration 

analysis were collected from the middle stratum of each dosing formulation. All analyses 

were conducted by the Analytical Chemistry Department at WIL Research. 

Results of Dose Analysis 

Homogeneity (% RSD): 1.8-2.5% 

Stability (% of initial): 92.5-97.9% following 13 days at room temperature. 

Concentration (% of nominal): 98.8-106% 

The analytical data indicated that the mixing procedure was adequate and that the variation 

between nominal and actual dosage to the animals was acceptable. 

6. Dosage Administration: Animals were administered the metolachlor formulations, EE, or 

vehicle (corn oil) daily via s.c. injection for three consecutive days in a dose volume of 5 

mL/kg body weight. Each daily dosage of the test substance was divided between two 

injection sites (two distinct regions on the scapular region of the rat). The dosage volume 

for all groups was 5 mL/kg and individual dosages were based on the most recently recorded 

body weights to provide the correct mg/kg/day dose. All animals were dosed at 

approximately the same time each day.

7. Statistics: Data statistically analyzed by WIL Laboratories included body weights and body 

weight gains. Analyses were two-tailed for significance levels of 5% and 1%. Two subset 

analyses were conducted. Body weights and body weight gains were analyzed using a twosample t-test to compare the positive control group to the vehicle control group. Body 

weights and body weight gains were analyzed using Dunnett’s test to compare the 

metolachlor-treated groups to the vehicle control group. 

Data statistically analyzed by BioSTAT Consultants, Inc. included wet uterine weight and 

blotted uterine weight. Two subset analyses were conducted. All analyses were conducted 

using SAS version 9.1 (SAS Institute, Inc., 2002-2003), or higher, software. 

Levene’s test was applied for uterine weight, at the 0.01 significance level, to test for 

homogeneity of variances. If that test was significant, then a log transform was applied to 

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the data and Levene’s test repeated. If that test was still significant, then the nonparametric 

Wilcoxon rank sum test (EE compared to vehicle control) or the nonparametric KruskalWallis test followed by Dunn’s test (test substance compared to vehicle control) was used. 

The tests were one-sided, at the 0.05 significance level, looking for significant increases 

from the vehicle control. If Levene’s test was not significant, then an analysis of covariance 

(ANCOVA) was conducted, on the raw or log-transformed data, as appropriate. The 

necropsy body weight was used as the covariate in the analysis of wet and blotted uterine 

weight. For the metolachlor groups, one-sided Dunnett’s test was conducted regardless of 

the outcome of the ANCOVA for significant increases (p<0.05) compared with the vehicle 

control. 

C. METHODS

1. Clinical Examinations: All rats were observed (cage-side) twice daily for moribundity and 

mortality. Individual clinical observations (hand-held physical examination) were recorded 

daily (prior to test substance administration during the treatment period) through the day of 

euthanasia. Animals were also observed for signs of toxicity at the time of dosing (within 

15 minutes) and approximately 1 hour following dose administration. 

2. Body Weight: Animals were weighed at randomization, daily throughout the dosing period 

and at termination. Mean body weight gains were calculated for each corresponding 

interval and for the overall (Days 1-4) study period. 

3. Food Consumption (Optional): Food consumption was not measured. 

4. Necropsy and Measurement of Uterine Weight: On PND 60 (approximately 24 hours 

after final administration of the test substance), all animals were euthanized by carbon 

dioxide inhalation and subjected to a gross necropsy. Animals were examined for residual 

ovarian tissue. Dissection of the uterus was performed according to the U.S. EPA 

Guideline. Briefly, the vagina was removed just below the cervix in order to retain the 

luminal fluid in the uterus. The “wet” uterus (i.e., containing the luminal fluid) was 

weighed. Subsequently, the uterine horns were cut longitudinally and blotted with filter 

paper to remove the luminal fluid while preventing desiccation. The blotted uterus was 

weighed. 

5. Microscopic Examination (Optional): Microscopic examinations were not conducted. 

II. RESULTS

A. OBSERVATIONS

1. Mortality: All animals survived until scheduled termination. 

2. Clinical Signs of Toxicity: No test substance-related clinical findings were noted at the 

daily examinations, at the time of dose administration, or 1 hour following dose 

administration at any dosage level. Findings noted in the metolachlor-treated groups (300 

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and 1000 mg/kg/day), including red or yellow material on various body surfaces occurred 

infrequently, on the day following administration of the last dose, and/or in a manner that 

was not considered treatment-related.

B. BODY WEIGHT AND WEIGHT GAIN: Selected body weight and body weight gain 

data are presented in Table 2. Body weights were comparable to the vehicle controls 

throughout the duration of the assay, but overall (Days 1-4) body weight gains were 

decreased (p<0.01) in the 300 mg/kg/day (↓42%) and 1000 mg/kg/day (↓53%) metolachlor 

groups compared to the vehicle controls. Body weight gains in the positive control (EE) 

group were also decreased (p<0.01) by 57% compared to the vehicle controls. 

TABLE 2. Selected Group Body Weights and Cumulative Body Weight Gains (g) In the Estrogen Agonist 

Assay a

Study Day 

Dose (mg/kg/day) 

Vehicle Control Metolachlor 

(100) 

Metolachlor 

(300) 

Metolachlor 

(1000) 

Reference 

Estrogen 

EE (0.0005) 

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 

1 6 227.3 16.9 6 226.1 18.6 6 226.4 16.1 6 232.4 23.0 6 225.6 17.6

 2 6 233.2 18.0 6 227.8 17.4 6 228.1 17.5 6 235.3 23.5 6 230.6 17.8

 3 6 239.3 17.6 6 234.0 17.9 6 233.8 16.8 6 238.9 22.0 6 231.4 16.6

4 (termination) 6 245.2 18.8 6 239.8 18.1 6 236.7 15.9 6 240.8 19.6 6 233.3 16.0

Body Weight 

Gain (Days 1-4) 

6 17.9 3.0 6 13.7 4.3 6 10.3**

(↓42) 

3.0 6 8.4** 

(↓53) 

4.0 6 7.7**

(↓57)

4.1 

a Data were obtained from Tables S3 and S4 on pages 42 and 43 of the study report; note that the study 

numbered days started with 0 (dose initiation), but the reviewer numbered the study days starting with 1 (dose 

initiation). BWG percent differences (calculated by the reviewers) are included in parentheses. 

N Number of animals in the group 

SD Standard Deviation 

** Significantly different from controls at p<0.01 

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

D. PATHOLOGY 

1. Uterine Weights: No residual ovarian tissue was noted for any of the animals at necropsy. 

Selected uterine weight data are presented in Table 3. Uterine weights in the metolachlor 

treated groups were comparable to the vehicle controls. Absolute wet and blotted uterus 

weights for the positive control (EE) group were increased (p<0.05) as expected by 288% 

and 175%, respectively. 

The mean blotted uterine weight for the vehicle control group was 0.03% of terminal body 

weight, which was within the performance criteria (<0.09%) of the test guideline. 

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TABLE 3. Uterine Weights from Estrogen Agonist Assay in SD Rats a

Parameters 

Dose (mg/kg/day) 

Vehicle Control Metolachlor 

(100) 

Metolachlor 

(300) 

Metolachlor 

(1000) 

Reference 

Estrogen 

EE (0.0005) 

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 

Terminal BW 

(g) 

6 245.2 18.8 6 239.8 18.1 6 236.7 15.9 6 240.8 19.6 6 233.3 16.0 

Wet, absolute 

(g) 

6 0.0817 0.0111 6 0.0764 0.0096 6 0.0733 0.0136 6 0.0754 0.0067 6 0.3171*

(↑288) 

0.0465

Wet, relative 

(%) 

6 0.030 0.004 6 0.032 0.005 6 0.031 0.006 6 0.031 0.002 6 0.136 0.019 

Blotted, 

absolute (g) 

6 0.0729 0.0088 6 0.0683 0.0089 6 0.0663 0.0135 6 0.0674 0.0064 6 0.2006*

(↑175) 

0.0325

Blotted, 

relative (%) 

6 0.030 0.004 6 0.029 0.005 6 0.028 0.006 6 0.028 0.002 6 0.086 0.0120

a Data were obtained from Tables S6, S7, and S8 on pages 45-47 of the study report. Percent differences from 

controls, calculated by the reviewers, are included in parentheses. 

BW Body weight 

N Number of animals in the group 

SD Standard Deviation 

* Significantly different from controls at p<0.05 

2. Microscopic Examination (Optional): No microscopic examinations were conducted. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Based on the lack of higher mean uterine weights 

(wet and blotted), metolachlor technical did not demonstrate or mimic biological activities 

consistent with agonism of natural estrogens when administered by subcutaneous injection 

to ovariectomized female rats at dosage levels of 100, 300, and 1000 mg/kg/day. The 

positive control substance (17α-ethynylestradiol) elicited the expected increases in uterine 

(wet and blotted) weights.

B. AGENCY COMMENTS: All animals survived until scheduled termination. No test 

substance-related clinical findings were noted in any of the animals during the study. 

However, minor clinical findings (red/yellow material on various body surfaces) were noted 

for the 300 and 1000 mg/kg/day groups; these occurrences were infrequent and not 

considered to be treatment related. 

Body weights were comparable to the vehicle controls throughout the duration of the assay, 

but overall (Days 1-4) body weight gains were decreased (p<0.01) in the 300 mg/kg/day 

(↓42%) and 1000 mg/kg/day (↓53%) metolachlor groups compared to the vehicle controls. 

Body weight gains in the positive control (EE) group were also decreased (p<0.01) by 57% 

compared to the vehicle controls. 

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No abnormal gross observations were seen at necropsy. Uterine weights in the metolachlor 

treated groups were comparable to the vehicle controls. Absolute wet and blotted uterus 

weights for the positive control (EE) group were increased (p<0.05) as expected by 288% 

and 175%, respectively. 

Based on the observed decreases (42% and 53%) in body weight gain at 300 and 1000 

mg/kg/day, adequate dose levels were achieved in this study. 

No statistically significant changes were seen in the uterine weight in this assay. 

Metolachlor was negative in the uterotrophic assay. 

C. STUDY DEFICIENCIES: None. 

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