UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

OFFICE OF CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

MEMORANDUM 

Date: 6/29/2015 

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

PC Code: 113501 

Decision No.: 461740, 468748 

Petition No.: NA 

Risk Assessment Type: NA 

TXR No.: 0054219 

DP Barcode: D399227, D404681 

Registration No.: NA 

Regulatory Action: NA 

CaseNo.: NA 

MRID No.: See Table 

CAS No.: 57837-19-1 

40CFR: NA 

FROM: Greg Akerman, Ph.D. A _Ab----

Immediate Office ; ~ Health Effects Division (7509P) 

THROUGH: 

Deputy 

Health 

Jess Rowland 

Effects 

Director 

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-evaluatipn 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 metalaxyl: Test Order# CON-113501-18. 

Page 1 of2 

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II. RESPONSE 

Attached are the EDSP Tier 1 assay DERs for metalaxyl.

III. MRID Table 

Chemical: Metalaxyl PC Code: 113501 

Guideline Assay MRID 

890.1100 Amphibian Metamorphosis Assay (Frog) 48685801 

890.1150 Androgen Receptor Binding (Rat Prostate) 48617601 

890.1200 Aromatase Assay (Human Recombinant) 48617602 

890.1250 Estrogen Receptor Binding 48617607 

890.1300 Estrogen Receptor Transcriptional Activation 

(Human Cell Line HeLa-9903) 48617603 

890.1350 Fish Short-Term Reproduction 48685802 

890.1400 Hershberger (Rat) 48617604 

890.1450 Female Pubertal (Rat) 48701301 

890.1500 Male Pubertal (Rat) 48701301 

890.1550 Steroidogenesis (Human Cell Line – H295R) 48617605 

890.1600 Uterotrophic (Rat) 48617606 

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

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DER Template Version: 22 September 2011 

Data Requirement: EPA DP Barcode 401220 

OECD Data Point 231 

EPA MRID 48685801

EPA Guideline 890.1100 

 Amphibian Metamorphosis Assay (Frog) 

Test Material: Metalaxyl Purity (%): 98.2% 

Common Name Metalaxyl 

Chemical Name IUPAC methyl N-(methoxyacetyl)-N-2,6-xylyl)-DL-alaninate; methyl 

 2-{[2,6-dimethylphenyl)methoxyacetyl]amino}propionate 

 CAS Name N-(2,6-dimethylphenyl)-N- (methyoxyacetyl)- alanine methyl ester 

CAS No. 57837-19-1 

Synonyms Metalaxyl Technical, N-(2,6-dimethylphenyl)-N-(methyoxyacetyl)-

alanine methyl ester [CAS# 57837-19-1] 

 methyl N-((methoxyacetyl)-N-2,6-xylyl)-DL-alaninate 

 methyl 2-([2,6-(dimethylphenyl)methoxyacetyl]amino)propionate 

 2-[(2,6-dimethylphenyl)-(2-methoxy-1-oxoethyl) imino]propanoic acid 

methyl ester 

 N-(2,6-dimethylphenyl)-N-(mehtoxyacetyl)-DL-alanine methyl ester

EPA PC Code 113501

Primary Reviewer: John Marton Signature: 

Staff Scientist, Cambridge Environmental, Inc. Date: 10/16/2012 

Secondary Reviewer: Teri S. Myers Signature:

Senior Scientist, Cambridge Environmental, Inc. Date: 12/20/2012 

Primary and Final Additional Reviewer: Robin Sternberg Signature: 

Wildlife Biologist, USEPA/OCSPP/OPP/EFED/ERB1 Date: 05/17/2013, 05/27/2015

Digitally signed by ROBIN STERNBERG 

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

ou=USEPA, ou=Staff, cn=ROBIN 

STERNBERG, dnQualifier=0000039126 

Date: 2015.06.01 16:01:22 -04'00'

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

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DER Template Version: 22 September 2011 

Additional Reviewer: Anita Ullagaddi Signature: No longer with EPA 

Biologist, USEPA/OCSPP/OPP/EFED/ERB1 Date: 01/11/2013

Additional Reviewer: Amy Blankinship Signature: 

Biologist, USEPA/OCSPP/OPP/EFED/ERB6 Date: 

Date Evaluation Completed: 05/27/2015 

CITATION: Lee, M.R. 2012. Metalaxyl- Amphibian Metamorphosis Assay with African Clawed Frog (Xenopus 

laevis). Unpublished study performed by Smithers Viscient, Wareham, Massachusetts. Laboratory report number 

13694.6146. Study sponsored by Metalaxyl EDSP Consortium, Burr Ridge, Illinois. Study completed February 

29, 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.

AMY 

BLANKINSHIP

Digitally signed by AMY 

BLANKINSHIP 

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

ou=USEPA, ou=Staff, cn=AMY 

BLANKINSHIP, 

dnQualifier=0000040917 

Date: 2015.06.05 08:57:02 -04'00'

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

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EXECUTIVE SUMMARY

The 21-day assay of metalaxyl (98.2% purity) on amphibian metamorphosis of the African clawed frog 

(Xenopus laevis) was conducted under flow-through conditions. Amphibian larvae at developmental NieuwkoopFaber Stage (NF) 51 (80/control and treatment group; 20/replicate) were exposed to nominal concentrations 

of 0 (negative control), 1.0, 10, and 100 mg a.i./L; mean-measured concentrations were <0.069 (<LOQ; 

negative control), 0.98, 10, and 95 mg a.i./L. The test system was maintained at 22 to 24°C and a pH of 

7.8 to 8.2. 

By test termination, mortality averaged 8.75% in the negative control and 7.5, 0, and 3.75% in the low, mid, 

and high treatment groups, respectively. Spinal curvature was observed in between 15 and 50% of surviving 

tadpoles in the negative control and all treatment groups and was not considered to be treatment related. The 

clinical sign of lethargy in all tadpoles of the high treatment group was observed on the first two days of 

exposure. 

Tadpoles at NF stage >60 were not included in analyses of growth and hind-limb length (HLL). Day 7 body 

weight was marginally significantly decreased (p=0.06) by 19% at the high treatment level. Day 21 body weight 

was significantly increased (p<0.05) by 15% in the high treatment group and was marginally significantly 

decreased (p=0.057) by 9% in the low treatment group. 

Metalaxyl significantly decreased (P<0.05) Day 7 and Day 21 normalized (for snout-vent length) HLL at the 

high treatment level. There was no significant (p>0.05) acceleration or delay of Day 7 or Day 21 median NF 

developmental stage at any treatment level. Although the number of late-stage tadpoles did not warrant a 

separate analysis (<20% observed), the reviewer noted a concentration-dependent reduction in the number 

of late-stage individuals (>NF 60); there were 16 individuals staged NF 61 and 62 in the negative control, 13 

in the low treatment group, 11 in the mid treatment group, and only 3 NF 61 tadpoles in the high treatment 

group. No asynchronous development was observed. Thyroid gland histopathology observations included mild 

follicular cell hyperplasia and hypertrophy in the negative control and all treatment groups and were not 

considered to be treatment-related. 

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The following performance criteria were not met: (1) negative control Replicate D and 0.98 mg a.i./L 

Replicate B both had a total of 3 mortalities by Day 7 which is greater than the guideline criterion of no more 

than two mortalities per replicate; and (2) temperature of the test solutions (22-24°C) exceeded the acceptable 

limits of 22±1°C. On Day 7, the maximum temperature was 23.6°C. Inter-replicate and inter-treatment 

differentials did not exceed 0.5 °C for any given day. However, these deviations did not impact the interpretation 

of the study. 

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

Guideline 890.1100). 

Results Synopsis:

Test organism NF stage at test initiation: 51 

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

Test type: Flow-through 

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

Metamorphosis Assay (AMA) with Metalaxyl. 

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 

0.98 No No No No No No No 

10 No No No No No No No 

95 No No Yes 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 boardcertified 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 the guidelines outlined in the U.S. 

Environmental Protection Agency’s Endocrine Disruptor Screening Program 

Test Guidelines 890.1100, Amphibian Metamorphosis (Frog); and OECD 

Guideline for Testing of Chemicals No. 231, Amphibian Metamorphosis 

Assay. The following deviations were noted: 

1. Details on parental feeding were not provided. 

2. Negative Control Replicate D and 0.98 mg a.i./L Replicate B both had a total of 3 mortalities by 

Day 7 which is greater than the criteria of no more than two mortalities per replicate.

3. Temperature of the test solutions (22-24°C) exceeded the recommended values (22±1°C). On 

Day 7, the maximum temperature was 23.6°C. Inter-replicate and inter-treatment differentials did 

not exceed 0.5 oC for any given day 

4. Results from the periodic screening analysis of the dilution water were not provided. However, the 

study author reported that no pesticides, PCBs, or toxic metals were detected at concentrations 

deemed harmful in accordance with ASTM (2005) standard practices. 

 These deviations do not impact the interpretation of the study. 

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

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

pertinent OECD and U.S. EPA Good Laboratory Practice regulations with the 

following exception: routine food and water contaminant screening analyses 

were conducted using standard U.S. EPA procedures by GeoLabs, Inc., 

Braintree, Massachusetts and are considered facility records under Smithers 

Viscient’s Standard Operating Procedures. 

A. Test Material Metalaxyl 

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DER Template Version: 22 September 2011 

Description: Solid

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. : 20100540 (Batch No.) 

Purity: 98.2% 

 For the first 14 days of exposure, nominal concentrations of exposure and 

stock solution were calculated based on the initial purity (100%). From Day 

15 through test termination, nominal concentrations and stock solutions were 

calculated based on the updated purity (98.2%), recertified on exposure day 

14 (20 July 2011). 

Impurities: None reported 

Stability of Compound: Stable. Mean-measured concentrations yielded recoveries of 95 to 100% of 

nominal with coefficients of variation (CV) of 2.6 to 4.3%.

Storage Conditions of 

Test Chemicals: Stored at room temperature in the dark. 

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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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DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Were parents maintained as inhouse stock? Yes Brood stock was originally obtained from Nasco, Fort Atkinson, Wisconsin. 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): 

>14 days

Details on parental feeding: Not specified Details on parental health: The brood stock did 

not show any sign of sickness, disease, injuries, or abnormalities from the day of receipt to the day of pre-exposure initiation. 

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Table 3: Larval Selection and Care.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Best single spawn? Yes 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 comixed)

Number of spawns evaluated (if applicable): 

3 

Number of eggs sampled per spawn:

At least 900 Embryos were assessed approximately 

5 days post-fertilization. Criteria were embryo survival and the quality of the embryos. 

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: 13 days postfertilization (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: Larvae were selected 

based on survival and overall quality with respect to the three spawns (~1500 eggs/spawn). 

Loading rate (rearing density): 10 larvae/L 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: Xenopus Express 

Tadpole Food

Xenopus Express is a commercially available diet that is suitable for normal growth and development of X. laevistadpoles. 

EPA recommends Sera Micron® throughout 

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

during the entire 21-d definitive study. If 

another diet is used, the study report should 

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DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Source of food: Xenopus Express, 

Brooksville, Florida

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: 2 times/day EPA recommends that feeding occur at 

least twice per day.

Details on feeding regime: Feeding regime 

conformed to guideline recommendations.

It is recommended that food rations during 

the pre-exposure period be increased along 

with larval growth to approximately 30

mg/larva/day by test initiation. EPA and 

OECD recommend that food rations 

increase from 30 mg/larva/day at test 

initiation (Study Day 0-4) to 80 

mg/larva/day in the last week of the test 

(Study Day 15-21).

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

Type of exposure: Flow-through EPA recommends the use of a flowthrough system.Type of flow-through dilution system (if applicable): 

intermittent flow proportional diluter

Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

Flow-through rate (if applicable):

~27.1 mL/min The flow rate was calculated based on a 

volume of 6.5 L and 6 volume replacements per day. This rate allowed complete volume replacement every 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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DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

Pumps delivered the stock solution into the diluter’s chemical mixing chamber. The appropriate concentrations were delivered to the test vessels using flowsplitting cells. 

Flow splitting accuracy of the diluter cells was within ±5% of the nominal value. The exposure system was operating properly for 11 days prior to test initiation. All test chambers were brushed and siphoned daily to remove detritus and uneaten food during the test; cleaning equipment were dedicated to treatment and controls to avoid cross contamination. 

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%.

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 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

recommended to set bubblers at levels 

that do not cause stress on the tadpoles.

Source of dilution water: Dilution water was 

FETAX solution, prepared by fortifying laboratory well water based on the formulation presented in “Standard Guide for Conducting the Frog Embryo Teratogenesis AssayXenopus (FETAX). 

Laboratory well water originated from an on-site 100 m bedrock well. Water was pumped to a concrete reservoir where it was supplemented on demand with untreated, de-chlorinated, Town of Wareham well water. The water was aerated prior to use to ensure proper equilibration of dissolved gases.

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. See Reviewer’s Comments 

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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 

սg/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: Each aquarium was 

constructed of glass, silicone sealant, stainless steel, and Teflon®.

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: 30 x 14.5 x 20 cmFill volume: 6.5 L Volume was maintained by a 14.5 cm high 

side drain. 

Additional details on exposure system: 

NA 

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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) 150 160 NA weekly 

EPA recommends hardness 40 to 48 

mg/L as CaCO3.

pH 7.4 8.3 7.79 

Daily in alternating 

replicate test 

vessels 

EPA recommends pH 7.5 ± 1, interreplicate and inter-treatment differentials 

should not exceed 0.5.

Dissolved oxygen (mg/L) 3.8 7.7 5.84 

Daily in alternating 

replicate 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 22 24 22.2 

Daily in alternating 

replicate test 

vessels and 

continuously in one 

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 7 

bi-annual samples 

of well water 

EPA recommends aquatic iodide range 

0.5 – 10 

սg/L (supplemental iodide 

should not exceed 2 

սg/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 

Fluoride <0.50 

bi-annual samples 

of well water 

Perchlorate Not Reported Chlorate Not Reported Alkalinity (mg/L as CaCO3)Conductivity (սmhos/cm)

74 

1700 

80 

1900 

Not Reported Not Reported 

Measured in 

replicate A of the 

control, low, and 

high test 

concentrations on 

Day 0 and in 

alternating replicates 

weekly thereafter. 

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Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

and hardness be measured at least at 

the 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 rangefinder? 

LC50 value of >100 

mg/L for rainbow trout, carp, and bluegill sunfish

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 

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Life stage: NF 51 Test duration: 4 daysAdditional details: The test was 

conducted under staticrenewal conditions with nominal concentrations of 6.3, 13, 25, 50, and 100 mg/L. 

No concentration results in more 

50% 

mortality and the LC50 was therefore 

estimated to be >100 mg/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. 

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

Estimated LC50 from 

the range-finding study 

(>100 mg/L).

EPA recommends that the highest test 

concentration is either the solubility limit of 

the test compound, 100 mg/L, or 

demonstrates adequate evidence of toxicity 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

(e.g., <10% mortality), whichever concentration is lowest.

Reference study citation (if applicable):

NA 

Separation of test concentrations:

0.10 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 

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): 0.061-0.069 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, 13, and 20.

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; a solvent control was not used

EPA and OECD recommend the use of a 

concurrent solvent control when a 

solubilizing agent is used. EPA 

recommends 4 replicates.

Number of replicates per test item treatment level: 

4 EPA recommends 4 replicates.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Number of larvae per treatment at test initiation: 

80 

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

NA EPA recommends that the solvent not 

exceed 0.02 ml/L3. OECD recommends 

that solvent have no effect on survival nor 

produce any other adverse effects and that 

concentration not be greater than 0.1 ml/L4

.

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

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

Aquatic Toxicology, 76, pp.69–92. 

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

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Positive control concentration(s) (if applicable): 

NA 

Photoperiod: 12 hrs light : 

12 hrs dark 

EPA recommends photoperiod 12:12 

(light:dark).

Light intensity at water’s surface: 

0.720 to 0.920 Klux EPA recommends light intensity 0.6 – 2 

Klux (at water’s surface).

Additional details: NA During the solubility trial, all stocks were 

observed to be clear and colorless with no visible undissolved test substance following overnight mixing. The same 

procedures were used during testing. 

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

Treatment ID

Nominal 

Concentration 

(mg a.i./L) 

Measured 

Concentration 

(mg a.i./L) 

Mean CV (%) Details or Remarks Guideline Recommendations 

Negative Control 0.00 <0.061-0.069 NA 

NA 

EPA and OECD recommend that test item 

concentrations be maintained at a coefficient 

of variation (CV) 

20%.

Treatment 1 1.0 0.98 4.3 Treatment 2 10 10 2.6 Treatment 3 100 95 3.9 

Abbreviations: CV Coefficient of variation. 

 E. Observations

Biological Endpoints: Day 7- NF stage, wet weight, snout-vent length (SVL), hind-limb length (HLL), normalized HLL 

 Day 21- NF stage, wet weight, snout-vent length (SVL), hind-limb length (HLL), normalized HLL, thyroid histopathology  Daily- mortality, clinical observations/sub-lethal effects 

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 test termination, % mortality averaged 8.75% in the negative control and 7.50, 0, and 3.75% in 

the mean-measured 0.98, 10, and 95 mg a.i./L treatment groups, respectively (Table 9). Replicate 

D in the negative control and Replicate B in the 0.98 mg a.i./L group had a total of three mortalities 

which is greater than the performance criteria of no more than 2 mortalities per replicate. 

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 72 8.75 60 0 0 

0.98 80 43 5 60 24 3.3 

10 80 0 0 60 0 0 

95 80 15 1 60 26 3.3 

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

2

 Replicate A and B, one mortality; Replicate C, two mortalities; Replicate D, three mortalities 

3

 Replicate A, one mortality; Replicate B, three mortalities 

4 Replicate C, two mortalities 

5 Replicate D, one mortality 

6 Replicate A, one mortality; Replicate C, one mortality 

Median developmental stage on Day 7 was 54 in the control and all treatment groups (Table 10). 

On Day 21, median developmental stage was 60 in the control and all treatment groups. The 10th

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and 90th percentiles did not differ by more than 4 NF stages. No asynchronous development was 

observed throughout the test. 

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 60 0 

0.98 4 54 0 4 60 0 

10 4 54 0 4 60 0 

95 4 54 0 4 60 0 

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Day 7 HLL ranged from 2.77 mm at 95 mg a.i./L to 0.31 mm at 0.98 mg a.i./L. Day 7 normalized HLL 

ranged from 0.14 at 95 mg a.i./L to 0.16 in the negative control and 0.98 mg a.i./L treatment group. Day 

21 HLL ranged from 16.95 mm at 0.98 mg a.i./L to 18.39 mm in the negative control. Day 21 normalized 

HLL ranged from 0.62 at 95 mg a.i./L to 0.71 in the negative control. 

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

Treatment 

(mg a.i./L) 

[measured]

Hind Limb Length (HLL) 

Day 7 Day 211

n Mean

(mm) 

±SD 

HLL: 

SVL2 n Mean(mm) 

±SD

HLL: 

SVL2

Negative Control 4 3.17 0.22 0.16 4 18.39 0.76 0.71 

0.98 4 3.31 0.22 0.16 4 16.95 1.70 0.68 

10 4 3.14 0.27 0.15 4 18.07 0.78 0.69 

95 4 2.77 0.11 0.14 4 17.03 0.81 0.62 

Abbreviations: SD Standard deviation. 

1

 NF Stage > 60 excluded 

2

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

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Day 7 SVL ranged from 19.35 mm at 95 mg a.i./L to 21.09 mm at 10 mg a.i./L. Day 7 body weight ranged 

from 0.44 g at 95 mg a.i./L to 0.58 g at 10 mg a.i./L. Day 21 SVL ranged from 25.38 at 0.98 mg a.i./L 

to 27.82 mm at 95 mg a.i./L. Day 21 body weight ranged from 1.13 g at 0.98 mg a.i./L to 1.43 g at 95 

mg a.i./L. 

 

Table 12: Larval Growth in Xenopus laevis.

Treatment 

(mg a.i./L) 

[measured]

Snout-Vent Length (SVL) Body Weight2

Day 7 Day 211 Day 7 Day 211

n Mean

(mm) 

±SD n Mean

(mm) 

±SD n Mean

(g) 

±SD n Mean

(g) 

±SD 

Negative 

Control 

4 20.30 0.72 4 26.19 0.18 4 0.54 0.05 4 1.24 0.02 

0.98 4 20.45 0.61 4 25.38 0.48 4 0.53 0.04 4 1.13 0.10 

10 4 21.09 1.13 4 26.28 0.28 4 0.58 0.09 4 1.22 0.04 

95 4 19.35 0.53 4 27.82 0.45 4 0.44 0.03 4 1.43 0.04 

Abbreviations: SD Standard deviation. 

1

 NF Stage > 60 excluded 

2

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

No thyroid gland hypertrophy or hyperplasia were observed in the control or treatment group tadpoles 

(Table 13). Mild follicular cell hypertrophy and hyperplasia were observed in the control and all 

treatment groups, though no treatment-related relationship was detected. 

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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 20 20 20 20 18 20 17 

1 20 0 20 0 20 2 20 3 

2 20 0 20 0 20 0 20 0 

3 20 0 20 0 20 0 20 0 

0.98 0 182 18 18 18 18 16 18 17 

1 182 0 18 0 18 2 18 1 

2 182 0 18 0 18 0 18 0 

3 182 0 18 0 18 0 18 0 

10 0 192 19 19 19 19 16 19 17 

1 192 0 19 0 19 3 19 2 

2 192 0 19 0 19 0 19 0 

3 192 0 19 0 19 0 19 0 

95 0 20 20 20 20 20 19 20 17 

1 20 0 20 0 20 1 20 3 

2 20 0 20 0 20 0 20 0 

3 20 0 20 0 20 0 20 0 

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. 

2

 Insufficient tissue was available. Therefore, the sample size was appropriately adjusted.

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

Treatment 

(mg a.i./L) 

[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 20 20 20 20 NA NA NA NA NA NA 

1 20 0 20 0 NA NA NA NA NA NA 

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

0.98 0 18 18 18 18 NA NA NA NA NA NA 

1 18 0 18 0 NA NA NA NA NA NA 

2 18 0 18 0 NA NA NA NA NA NA 3 18 0 18 0 NA NA NA NA NA NA 

10 0 19 19 19 19 NA NA NA NA NA NA 

1 19 0 19 0 NA NA NA NA NA NA 

2 19 0 19 0 NA NA NA NA NA NA 3 19 0 19 0 NA NA NA NA NA NA 

95 0 20 20 20 20 NA NA NA NA NA NA 

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Treatment 

(mg a.i./L) 

[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 20 0 20 0 NA NA NA NA NA NA 

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

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

Treatment 

(mg a.i./L) 

[measured]

Additional Qualitative Observations1

Severity

Colloid Depletion

Changes in Colloid 

Quality 

n Incidence n Incidence 

Negative Control 0 20 20 20 20 

1 20 0 20 0 

2 20 0 20 0 

3 20 0 20 0 

0.98 0 18 18 18 18 

1 18 0 18 0 

2 18 0 18 0 

3 18 0 18 0 

10 0 19 19 19 19 

1 19 0 19 0 

2 19 0 19 0 

3 19 0 19 0 

95 0 20 20 20 20 

1 20 0 20 0 

2 20 0 20 0 

3 20 0 20 0 

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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Beginning on Day 7 and continuing throughout the remainder of the exposure period, spinal curvature 

was observed among tadpoles in the control and treatment groups. At test termination, spinal curvature 

was observed in 37% of control tadpoles and 40, 50, and 15% of tadpoles in the 0.98, 10, and 95 

mg a.i./L treatment groups, respectively (Table 15). There was no clear treatment-related response, 

and the study author reported that for previous studies conducted at Smithers Viscient, there was no 

correlation between the presence of spinal deformities and the increase in CV% for potentially affected 

endpoints (e.g., SVL, HLL, and wet weight). 

In an amended study report, the study author added that: “Shortly following exposure initiation, all 

tadpoles within the 95 mg/L treatment level began to exhibit a sublethal response (e.g., lethargy). 

These observations continued the following day (day 1). After day 1, these observations were no 

longer observed and the tadpoles appeared to behave normally. All other tadpoles at the remaining 

treatment levels and control appeared to behave normally during the entire exposure period.” 

Table 15: Clinical Signs in Xenopus laevis.

Treatment 

(mg a.i./L) 

[mean- measured] 

Clinical Signs1

Type n Incidence2

Negative Control Spinal curvature 60 37% 

0.98 Spinal curvature 58 40% 

10 Spinal curvature 60 50% 

95 

Spinal curvature 

Lethargy 

58 

80 

15% 

100%3 

1 Note that asynchronous development (unable to stage) is reported previously in Table 10. 

2

 Represents observations on Day 21.

3 Observed on Day 0 and 1.

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B. Study Author’s Analysis and Conclusions

Larval mortality was analyzed using the step-down Cochran-Armitage test when a monotonic trend 

was present. When a there was not a monotonic trend, mortality data were analyzed using Fisher’s 

Exact test with the Bonferroni-Holm adjustment. Developmental stage, SVL, normalized HLL, and wet 

weight were analyzed using the Jonckheere-Terpstra test (step-down) when there was a monotonic 

trend. Otherwise, these variables were tested for normality using the Shapiro-Wilks (Į = 0.01) test 

and for homogeneity of variance using the Bartlett’s test (Į = 0.01). If the data met these assumptions, 

analyses were conducted using Dunnett’s Multiple Comparison test. When the data did not meet the 

assumptions of normality and homogeneity of variance, analyses were performed using TamhaneDunnett test or the Mann-Whitney test with Bonferroni-Holm adjustment. All analyses were conducted 

using CETIS-Comprehensive Environmental Toxicity Information SystemTM Verion 1.8.1.1. Unless 

otherwise noted, all tests were conducted at Į = 0.05. 

On Day 7, HLL, normalized HLL, and wet weight were all significantly reduced at the mean-measured 

95 mg a.i./L treatment group relative to the negative control. By test termination (Day 21), SVL and 

wet weight were significantly promoted at the mean-measured 95 mg a.i./L treatment group, whereas 

HLL, normalized HLL, and developmental stage were significantly reduced at this level relative to the 

negative control. Based on the significant reductions in normalized HLL at the highest treatment group, 

Metalaxyl can be considered “likely thyroid active” to Xenopus laevis larvae under conditions used in 

the current test. 

In an amended study report, the study author added: “The impacts observed at the high dose (95 

mg/L) appear to be indicative of a general toxic response. The increases observed at the 21-day wet 

weight and 21-day snout-vent lengths indicate that the tadpoles are delayed in reaching 

metamorphosis. When approaching metamorphosis, tadpoles begin to reduce in overall size and weight 

by resorbing tissue and reducing absolute water content. The decreases in 7-day hind limb length, 7-

day hind limb length (normalized by SVL), 7-day wet weight, 21-day developmental stage, 21-day 

hind limb length and 21-day hind limb length (normalized by SVL) also indicate a delay in development. 

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These developmental delays are likely attributed to the sublethal responses observed during the first 

two days. The animals exposed at this dose were unable to adapt to the exposure concentrations 

within a reasonable timeframe, resulting in a two-day developmental delay. These results coupled with 

the lack of findings with the thyroid gland histology support this reasoning.” 

 C. Reviewer’s Analysis and Conclusions 

Statistical Methods: All growth and developmental variables were tested for normality using the 

Shapiro-Wilks test (Į = 0.01) and for homogeneity of variance using Levene’s test (Į = 0.01). If the 

data exhibited a monotonic trend, analyses were conducted using the Jonckheere-Terpstra test. If the 

data were not monotonic but met the assumptions of parametric statistics, Dunnett’s test was used to 

test for significant differences between control and treatment means. If the data were not monotonic 

and did not meet the assumptions of normality and homogeneity of variance, means were tested using 

the Mann-Whitney test. Day 7 wet weight, SVL, and HLL were analyzed using Dunnett’s test, whereas 

Day 7 normalized HLL was analyzed using the Jonckheere-Terpstra test. Day 7 developmental stage 

was not analyzed because all replicate medians were the same. Day 21 means and medians were 

calculated twice, once with including all tadpoles and once excluding tadpoles at NF stage  61. Day 

21 developmental stage and Day 21 wet weight (excluding NF > 60) were analyzed using the MannWhitney test. Day 21 wet weight (all tadpoles), Day 21 SVL, Day 21 HLL, and Day 21 normalized 

HLL (excluding NF >60) were analyzed using Dunnett’s test. Day 21 normalized HLL including all 

tadpoles was analyzed using the Jonckheere-Terpstra test. No asynchronous development was 

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

 Conclusions: 

On Day 7, normalized HLL was the only endpoint significantly reduced (8% reduction, JonckheereTerpstra, p = 0.008); all other endpoints were comparable between the treatment groups and control. 

Day 21 developmental stage was comparable between the control and treatment groups (MannWhitney, p > 0.05). The reviewer, however, noted a dose-dependent reduction in the appearance of 

late stage individuals (>NF 60); the abundance of late stage tadpoles did not warrant a separate 

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analysis, but there were 16 individuals staged NF 61 and 62 (50:50; 61:62) in the control, 13 (54:46) 

in the 0.98 mg a.i./L level, 11 (73:27) in the 10 mg a.i./L level, and only 3 NF 61 tadpoles in the 

95 mg a.i./L level. When NF >60 tadpoles were excluded from Day 21 endpoints (other than NF 

stage), no differences were found in Day 21 wet weight (Mann-Whitney, p > 0.05). 

Day 21 SVL was significantly increased at the mean-measured 95 mg a.i./L treatment group (6% , 

Dunnett’s, p < 0.001). Further, when NF > 60 tadpoles were excluded, Day 21 SVL was significantly 

reduced (3.1% reduction) at the mean-measured 0.98 mg a.i./L treatment group (Dunnett’s, p = 

0.024). Day 21 HLL was unaffected by treatment (Dunnett’s, p > 0.05). Body weight at Day 21 was 

significantly increased by 15% in the high treatment (95 mg a.i./L; Mann-Whitney p<0.05) and was 

marginally significantly (p=0.057) decreased by 9% in the lowest treatment group (0.98 mg a.i./L). 

Day 21 normalized HLL was significantly reduced (13%; p=0.028) at the mean-measured 95 mg 

a.i./L treatment group. The clinical sign of lethargy on days 0 and 1 in all of the tadpoles of the 

mean-measured 95 mg a.i./L treatment group may be indicative of overt toxicity. 

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

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 214 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 -- 60 -- -- -- -- -- -- -- -- -- No 

0.98 54 -- 60 1.0 3.7 0.88 -4.08 0.66 -- -- -- -- No 

10 54 -- 60 1.0 -4.3 0.15 -1.74 0.96 -- -- -- -- No 

95 54 -- 60 0.48 -8.4 0.008 -12.8 0.028 -- -- -- -- No 

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

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). 4 NF > 60 excluded 

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

Treatment 

(mg a.i./L) 

[measured]

Snout-Vent Length Body Weight 

Day 7 Day 213 Day 7 Day 213

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

% 

Diff. 

p 

Negative Control -- -- -- -- -- -- -- -- 

0.98 0.73 0.99 -3.08 0.024 -2.26 0.98 -8.92 0.057 

10 3.89 0.38 0.33 0.97 7.48 0.63 -1.65 0.63 

95 -4.66 0.26 6.22 <0.001 -19.21 0.06 15.07 0.029 

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

Abbreviations: Diff. Difference. 

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

 NF > 60 excluded 

E. Study Deficiencies 

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

The following performance criteria were not met: 

1. Negative Control Replicate D and 0.98 mg a.i./L Replicate B both had a total of 3 mortalities by 

Day 7 which is greater than the criteria of no more than two mortalities per replicate.

2. Temperature of the test solutions (22-24°C) exceeded the recommended values (22±1°C). On 

Day 7, the maximum temperature was 23.6°C. Inter-replicate and inter-treatment differentials did 

not exceed 0.5 oC for any given day. 

Although the mortality in two replicates exceeded the criteria of no more than 2 mortalities, the reported 

measurements for these replicates were similar to measurements in the other replicates. Therefore, 

these replicates were retained in the analysis and did not impact the interpretation of the results. 

These deviations do not impact the interpretation of the study. 

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F. Reviewer’s Comments 

Prior to the range-finding and definitive exposure assays, a solubility trial was conducted using nominal 

concentrations of 100, 200, 500, and 1000 mg a.i./L in FETAX solution. All solutions were observed 

to be clear and colorless with no visible undissolved test substance following overnight mixing. Solubility 

samples were handled according to the same procedures using during definitive testing and analytical 

recoveries were 100% in all four treatment levels. 

Representative samples of the well water source were analyzed bi-annually for the presence of 

pesticides, PCBs, and toxic metals by GeoLabs, Inc., Braintree, Massachusetts using U.S. EPA 

standard methods. None of these compounds were detected at concentrations considered toxic in any 

of the water samples analyzed, in agreement with ASTM (2005) standard practices. 

 

III. REFERENCES 

Ives, M. 2009. CETIS, Comprehensive Environmental Toxicity Information SystemTM. User’s Guide. 

Tidepool Scientific Software, McKinleyville, California. 

Niuewkoop, P.D. and J. Faber. 1994. Normal Table of Xenopus laevis. Garland Publishing, New York. 

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

Research 1: 21-29. 

Sprague, J.B. 1969. Measurement of pollutant toxicity to fish. 1. Bioassay methods for acute toxicity. Water 

Research 3: 793-821. 

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APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION: 

test for amphib metamorph screen study - Metalaxyl 

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 

 0.975 0.909 2.523 0.107 USE PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

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

 Ctrl 4 0.54 0.05 0.02 8.71 0.47, 0.62 

 Dose1 4 0.53 0.04 0.02 6.67 0.47, 0.59 

 Dose2 4 0.58 0.09 0.05 15.59 0.44, 0.73 

 Dose3 4 0.44 0.03 0.02 7.46 0.39, 0.49 

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

 Ctrl 0.54 0.50 0.60 . . 

 Dose1 0.54 0.48 0.56 97.74 2.26 

 Dose2 0.59 0.47 0.67 107.48 -7.48 

 Dose3 0.43 0.41 0.48 80.79 19.21 

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

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 4.66 0.022 

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 0.54 . 0.55 . . . . . . 

 Dose1 0.53 0.980 0.55 0.679 . . . . . 

 Dose2 0.58 0.629 0.55 0.714 0.568 . . . . 

 Dose3 0.44 0.058 0.44 0.014 0.154 0.016 . . . 

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

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 7.92 0.048 

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 0.54 . . 

 Dose1 0.54 0.678 0.282 

 Dose2 0.59 0.678 0.721 

 Dose3 0.43 0.067 0.046 

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

 EPA MRID Number 48685801

Page 43 of 67

DER Template Version: 22 September 2011 

 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 4.66 0.022 

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 -0.54 . -0.52 . . . . . . 

 Dose1 -0.53 0.980 -0.52 0.764 . . . . . 

 Dose2 -0.58 0.629 -0.52 0.797 0.568 . . . . 

 Dose3 -0.44 0.058 -0.52 0.814 0.154 0.016 . . . 

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

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 7.92 0.048 

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 -0.54 . . 

 Dose1 -0.54 0.678 0.718 

 Dose2 -0.59 0.678 0.279 

 Dose3 -0.43 0.067 0.954 

 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 - Metalaxyl 

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 

 . . . . NO DATA FOR TEST 

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

BASIC SUMMARY STATISTICS 

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

 Ctrl 4 54.00 0.00 0.00 0.00 . , . 

 Dose1 4 54.00 0.00 0.00 0.00 . , . 

 Dose2 4 54.00 0.00 0.00 0.00 . , . 

 Dose3 4 54.00 0.00 0.00 0.00 . , . 

Page 45 of 292
Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

 EPA MRID Number 48685801

Page 44 of 67

DER Template Version: 22 September 2011 

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

 Ctrl 54.00 54.00 54.00 . . 

 Dose1 54.00 54.00 54.00 100.00 0.00 

 Dose2 54.00 54.00 54.00 100.00 0.00 

 Dose3 54.00 54.00 54.00 100.00 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 

 . . . 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 54.00 . . . . . . . . 

 Dose1 54.00 . . . . . . . . 

 Dose2 54.00 . . . . . . . . 

 Dose3 54.00 . . . . . . . . 

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

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 54.00 . . 

 Dose1 54.00 1.000 . 

 Dose2 54.00 1.000 . 

 Dose3 54.00 1.000 . 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams Dose1 

 Jonckheere Dose1 

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

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 -54.00 . . . . . . . . 

 Dose1 -54.00 . . . . . . . . 

 Dose2 -54.00 . . . . . . . . 

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

 EPA MRID Number 48685801

Page 45 of 67

DER Template Version: 22 September 2011 

 Dose3 -54.00 . . . . . . . . 

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

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 -54.00 . . 

 Dose1 -54.00 1.000 . 

 Dose2 -54.00 1.000 . 

 Dose3 -54.00 1.000 . 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams Dose1 

 Jonckheere Dose1 

test for amphib metamorph screen study - Metalaxyl 

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 

 0.950 0.485 0.690 0.576 USE PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

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

 Ctrl 4 20.30 0.72 0.36 3.55 19.15, 21.44 

 Dose1 4 20.45 0.61 0.30 2.98 19.48, 21.41 

 Dose2 4 21.09 1.13 0.57 5.37 19.29, 22.89 

 Dose3 4 19.35 0.53 0.27 2.75 18.50, 20.20 

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

 Ctrl 20.47 19.31 20.95 . . 

 Dose1 20.43 19.73 21.19 100.73 -0.73 

 Dose2 21.39 19.50 22.08 103.89 -3.89 

 Dose3 19.29 18.78 20.06 95.34 4.66 

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

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 3.34 0.056 

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 20.30 . 20.61 . . . . . . 

 Dose1 20.45 0.987 20.61 0.793 . . . . . 

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

 EPA MRID Number 48685801

Page 46 of 67

DER Template Version: 22 September 2011 

 Dose2 21.09 0.383 20.61 0.824 0.662 . . . . 

 Dose3 19.35 0.256 19.35 0.073 0.251 0.038 . . . 

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

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 7.17 0.067 

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 20.47 . . 

 Dose1 20.43 0.889 0.614 

 Dose2 21.39 0.235 0.906 

 Dose3 19.29 0.156 0.152 

 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 3.34 0.056 

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 -20.30 . -20.30 . . . . . . 

 Dose1 -20.45 0.987 -20.30 0.585 . . . . . 

 Dose2 -21.09 0.383 -20.30 0.619 0.662 . . . . 

 Dose3 -19.35 0.256 -20.30 0.638 0.251 0.038 . . . 

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

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 7.17 0.067 

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 -20.47 . . 

 Dose1 -20.43 0.889 0.386 

 Dose2 -21.39 0.235 0.094 

 Dose3 -19.29 0.156 0.848 

 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 - Metalaxyl 

ANALYSIS RESULTS FOR VARIABLE VAR04 ( 7-d hind-limb length (mm) ) 

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

 EPA MRID Number 48685801

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DER Template Version: 22 September 2011 

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 

 0.974 0.905 1.176 0.360 USE PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

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

 Ctrl 4 3.17 0.22 0.11 7.05 2.81, 3.52 

 Dose1 4 3.31 0.22 0.11 6.55 2.97, 3.65 

 Dose2 4 3.14 0.27 0.13 8.48 2.72, 3.56 

 Dose3 4 2.77 0.11 0.05 3.95 2.59, 2.94 

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

 Ctrl 3.24 2.84 3.34 . . 

 Dose1 3.33 3.05 3.53 104.58 -4.58 

 Dose2 3.09 2.90 3.49 99.21 0.79 

 Dose3 2.77 2.63 2.89 87.36 12.64 

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

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 4.81 0.020 

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 3.17 . 3.24 . . . . . . 

 Dose1 3.31 0.659 3.24 0.767 . . . . . 

 Dose2 3.14 0.997 3.14 0.545 0.676 . . . . 

 Dose3 2.77 0.051 2.77 0.012 0.016 0.110 . . . 

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

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 8.69 0.034 

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 3.24 . . 

 Dose1 3.33 0.494 0.807 

 Dose2 3.09 0.889 0.385 

 Dose3 2.77 0.103 0.010 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams Dose3 

 Jonckheere Dose3 

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

Page 49 of 292
Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

 EPA MRID Number 48685801

Page 48 of 67

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 4.81 0.020 

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 -3.17 . -3.10 . . . . . . 

 Dose1 -3.31 0.659 -3.10 0.762 . . . . . 

 Dose2 -3.14 0.997 -3.10 0.795 0.676 . . . . 

 Dose3 -2.77 0.051 -3.10 0.812 0.016 0.110 . . . 

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

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 8.69 0.034 

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 -3.24 . . 

 Dose1 -3.33 0.494 0.193 

 Dose2 -3.09 0.889 0.615 

 Dose3 -2.77 0.103 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 - Metalaxyl 

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 

 0.949 0.475 0.403 0.754 USE PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

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

 Ctrl 4 0.16 0.01 0.00 4.12 0.15, 0.17 

 Dose1 4 0.16 0.01 0.00 4.06 0.15, 0.17 

 Dose2 4 0.15 0.01 0.00 5.95 0.13, 0.16 

 Dose3 4 0.14 0.00 0.00 3.51 0.13, 0.15 

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

 Ctrl 0.16 0.15 0.16 . . 

 Dose1 0.16 0.15 0.17 103.70 -3.70 

 Dose2 0.15 0.14 0.16 95.66 4.34 

 Dose3 0.14 0.14 0.15 91.64 8.36 

Page 50 of 292
Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

 EPA MRID Number 48685801

Page 49 of 67

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 5.66 0.012 

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 0.16 . 0.16 . . . . . . 

 Dose1 0.16 0.518 0.16 0.802 . . . . . 

 Dose2 0.15 0.398 0.15 0.119 0.096 . . . . 

 Dose3 0.14 0.050 0.14 0.012 0.010 0.585 . . . 

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

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 8.98 0.030 

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 0.16 . . 

 Dose1 0.16 0.343 0.877 

 Dose2 0.15 0.335 0.151 

 Dose3 0.14 0.101 0.008 

 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 5.66 0.012 

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 -0.16 . -0.15 . . . . . . 

 Dose1 -0.16 0.518 -0.15 0.837 . . . . . 

 Dose2 -0.15 0.398 -0.15 0.865 0.096 . . . . 

 Dose3 -0.14 0.050 -0.15 0.880 0.010 0.585 . . . 

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

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 8.98 0.030 

Page 51 of 292
Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

 EPA MRID Number 48685801

Page 50 of 67

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 -0.16 . . 

 Dose1 -0.16 0.343 0.123 

 Dose2 -0.15 0.335 0.849 

 Dose3 -0.14 0.101 0.992 

 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 - Metalaxyl 

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 

 0.565 <.001 9.000 0.002 USE NON-PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

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

 Ctrl 4 60.00 0.00 0.00 0.00 . , . 

 Dose1 4 60.00 0.00 0.00 0.00 . , . 

 Dose2 4 60.00 0.00 0.00 0.00 . , . 

 Dose3 4 59.75 0.50 0.25 0.84 58.95, 60.55 

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

 Ctrl 60.00 60.00 60.00 . . 

 Dose1 60.00 60.00 60.00 100.00 0.00 

 Dose2 60.00 60.00 60.00 100.00 0.00 

 Dose3 60.00 59.00 60.00 99.58 0.42 

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

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 1.00 0.426 

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 60.00 . 60.00 . . . . . . 

 Dose1 60.00 1.000 60.00 0.583 . . . . . 

 Dose2 60.00 1.000 60.00 0.618 1.000 . . . . 

 Dose3 59.75 0.389 59.75 0.119 0.515 0.515 . . . 

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

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 3.00 0.392 

Page 52 of 292
Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

 EPA MRID Number 48685801

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DER Template Version: 22 September 2011 

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 60.00 . . 

 Dose1 60.00 1.000 . 

 Dose2 60.00 1.000 . 

 Dose3 60.00 0.478 0.090 

 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 1.00 0.426 

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 -60.00 . -59.94 . . . . . . 

 Dose1 -60.00 1.000 -59.94 0.723 . . . . . 

 Dose2 -60.00 1.000 -59.94 0.757 1.000 . . . . 

 Dose3 -59.75 0.389 -59.94 0.775 0.515 0.515 . . . 

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

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 3.00 0.392 

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 -60.00 . . 

 Dose1 -60.00 1.000 . 

 Dose2 -60.00 1.000 . 

 Dose3 -60.00 0.478 0.910 

 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 - Metalaxyl 

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 

 0.969 0.821 4.866 0.019 USE NON-PARAMETRIC TESTS 

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

 EPA MRID Number 48685801

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DER Template Version: 22 September 2011 

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

BASIC SUMMARY STATISTICS 

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

 Ctrl 4 1.24 0.02 0.01 1.81 1.21, 1.28 

 Dose1 4 1.13 0.10 0.05 8.45 0.98, 1.29 

 Dose2 4 1.22 0.04 0.02 2.95 1.17, 1.28 

 Dose3 4 1.43 0.04 0.02 2.94 1.36, 1.50 

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

 Ctrl 1.25 1.22 1.27 . . 

 Dose1 1.15 1.02 1.22 91.08 8.92 

 Dose2 1.24 1.17 1.24 98.35 1.65 

 Dose3 1.42 1.39 1.49 115.07 -15.07 

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

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 19.69 <.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 1.24 . 1.26 . . . . . . 

 Dose1 1.13 0.042 1.26 0.722 . . . . . 

 Dose2 1.22 0.918 1.26 0.756 0.161 . . . . 

 Dose3 1.43 0.001 1.26 0.774 <.001 0.001 . . . 

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

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 11.42 0.010 

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 1.25 . . 

 Dose1 1.15 0.103 0.022 

 Dose2 1.24 0.676 0.330 

 Dose3 1.42 0.067 0.975 

 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 19.69 <.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 

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

 EPA MRID Number 48685801

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DER Template Version: 22 September 2011 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl -1.24 . -1.19 . . . . . . 

 Dose1 -1.13 0.042 -1.19 0.950 . . . . . 

 Dose2 -1.22 0.918 -1.22 0.809 0.161 . . . . 

 Dose3 -1.43 0.001 -1.43 <.001 <.001 0.001 . . . 

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

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 11.42 0.010 

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 -1.25 . . 

 Dose1 -1.15 0.103 0.978 

 Dose2 -1.24 0.676 0.670 

 Dose3 -1.42 0.067 0.025 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams Dose3 

 Jonckheere Dose3 

test for amphib metamorph screen study - Metalaxyl 

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 

 0.959 0.643 2.663 0.095 USE PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

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

 Ctrl 4 26.19 0.18 0.09 0.69 25.90, 26.48 

 Dose1 4 25.38 0.48 0.24 1.90 24.61, 26.15 

 Dose2 4 26.28 0.28 0.14 1.05 25.84, 26.72 

 Dose3 4 27.82 0.45 0.22 1.61 27.11, 28.53 

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

 Ctrl 26.21 25.95 26.39 . . 

 Dose1 25.40 24.82 25.92 96.92 3.08 

 Dose2 26.23 26.02 26.63 100.33 -0.33 

 Dose3 27.90 27.26 28.22 106.22 -6.22 

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

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 30.58 <.001 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing negative trend 

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 EPA MRID Number 48685801

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DER Template Version: 22 September 2011 

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 26.19 . 26.42 . . . . . . 

 Dose1 25.38 0.024 26.42 0.872 . . . . . 

 Dose2 26.28 0.974 26.42 0.897 0.022 . . . . 

 Dose3 27.82 <.001 26.42 0.910 <.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 12.75 0.005 

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 26.21 . . 

 Dose1 25.40 0.066 0.010 

 Dose2 26.23 0.889 0.558 

 Dose3 27.90 0.066 0.990 

 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 30.58 <.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 -26.19 . -25.79 . . . . . . 

 Dose1 -25.38 0.024 -25.79 0.964 . . . . . 

 Dose2 -26.28 0.974 -26.28 0.469 0.022 . . . . 

 Dose3 -27.82 <.001 -27.82 <.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 12.75 0.005 

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 -26.21 . . 

 Dose1 -25.40 0.066 0.990 

 Dose2 -26.23 0.889 0.442 

 Dose3 -27.90 0.066 0.010 

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 EPA MRID Number 48685801

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DER Template Version: 22 September 2011 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams Dose3 

 Jonckheere Dose3 

test for amphib metamorph screen study - Metalaxyl 

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 

 0.935 0.290 1.260 0.332 USE PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

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

 Ctrl 4 18.39 0.76 0.38 4.15 17.17, 19.60 

 Dose1 4 16.95 1.70 0.85 10.03 14.25, 19.66 

 Dose2 4 18.07 0.78 0.39 4.34 16.82, 19.32 

 Dose3 4 17.03 0.81 0.41 4.76 15.74, 18.32 

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

 Ctrl 18.40 17.61 19.14 . . 

 Dose1 16.43 15.55 19.39 92.20 7.80 

 Dose2 18.09 17.10 19.01 98.29 1.71 

 Dose3 17.16 16.08 17.74 92.65 7.35 

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

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 1.78 0.205 

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 18.39 . 18.39 . . . . . . 

 Dose1 16.95 0.201 17.51 0.166 . . . . . 

 Dose2 18.07 0.955 17.51 0.177 0.493 . . . . 

 Dose3 17.03 0.237 17.03 0.067 1.000 0.553 . . . 

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

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 4.46 0.216 

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 18.40 . . 

 Dose1 16.43 0.346 0.124 

 Dose2 18.09 0.889 0.442 

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

 EPA MRID Number 48685801

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DER Template Version: 22 September 2011 

 Dose3 17.16 0.156 0.131 

 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 1.78 0.205 

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 -18.39 . -17.61 . . . . . . 

 Dose1 -16.95 0.201 -17.61 0.899 . . . . . 

 Dose2 -18.07 0.955 -17.61 0.920 0.493 . . . . 

 Dose3 -17.03 0.237 -17.61 0.931 1.000 0.553 . . . 

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

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 4.46 0.216 

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 -18.40 . . 

 Dose1 -16.43 0.346 0.876 

 Dose2 -18.09 0.889 0.558 

 Dose3 -17.16 0.156 0.869 

 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 - Metalaxyl 

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 

 0.983 0.984 0.912 0.464 USE PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

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

 Ctrl 4 0.71 0.03 0.01 4.16 0.66, 0.75 

 Dose1 4 0.68 0.06 0.03 9.28 0.58, 0.78 

 Dose2 4 0.69 0.03 0.02 4.35 0.64, 0.74 

 Dose3 4 0.62 0.04 0.02 6.26 0.55, 0.68 

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

 EPA MRID Number 48685801

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DER Template Version: 22 September 2011 

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

 Ctrl 0.70 0.67 0.74 . . 

 Dose1 0.67 0.61 0.76 95.92 4.08 

 Dose2 0.70 0.65 0.72 98.26 1.74 

 Dose3 0.62 0.58 0.66 87.23 12.77 

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

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 3.55 0.048 

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 0.71 . 0.71 . . . . . . 

 Dose1 0.68 0.664 0.68 0.303 . . . . . 

 Dose2 0.69 0.955 0.68 0.324 0.945 . . . . 

 Dose3 0.62 0.028 0.62 0.007 0.226 0.095 . . . 

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

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 7.17 0.067 

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 0.70 . . 

 Dose1 0.67 0.494 0.193 

 Dose2 0.70 0.889 0.385 

 Dose3 0.62 0.067 0.016 

 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 3.55 0.048 

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 -0.71 . -0.67 . . . . . . 

 Dose1 -0.68 0.664 -0.67 0.913 . . . . . 

 Dose2 -0.69 0.955 -0.67 0.933 0.945 . . . . 

 Dose3 -0.62 0.028 -0.67 0.942 0.226 0.095 . . . 

Page 59 of 292
Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

 EPA MRID Number 48685801

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DER Template Version: 22 September 2011 

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

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 7.17 0.067 

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 -0.70 . . 

 Dose1 -0.67 0.494 0.807 

 Dose2 -0.70 0.889 0.615 

 Dose3 -0.62 0.067 0.984 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

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

 EPA MRID Number 48685801

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DER Template Version: 22 September 2011 

21 Day Body Weight (Mann-Whitney exact test) 

Obs conc endpoint 

1 0 1.234 

2 0 1.218 

3 0 1.260 

4 0 1.266 

5 1 1.197 

6 1 1.098 

7 1 1.224 

8 1 1.015 

9 2 1.170 

10 2 1.244 

11 2 1.244 

12 2 1.238 

13 3 1.418 

14 3 1.430 

15 3 1.390 

16 3 1.490 

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

 EPA MRID Number 48685801

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DER Template Version: 22 September 2011 

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 35.0 34.0 8.240146 8.750 

1 4 13.0 34.0 8.240146 3.250 

2 4 30.0 34.0 8.240146 7.500 

3 4 58.0 34.0 8.240146 14.500 

Average scores were used for ties. 

Kruskal-Wallis Test 

Chi-Square 11.4212 

DF 3 

Asymptotic Pr > Chi-Square 0.0097 

Exact Pr >= Chi-Square 8.605E-04 

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

 EPA MRID Number 48685801

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DER Template Version: 22 September 2011 

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

 EPA MRID Number 48685801

Page 62 of 67

DER Template Version: 22 September 2011 

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 25.0 18.0 3.464102 6.250 

1 4 11.0 18.0 3.464102 2.750 

Wilcoxon Two-Sample Test 

Statistic (S) 25.0000 

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 

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

 EPA MRID Number 48685801

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DER Template Version: 22 September 2011 

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

 EPA MRID Number 48685801

Page 64 of 67

DER Template Version: 22 September 2011 

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 20.0 18.0 3.443420 5.0 

2 4 16.0 18.0 3.443420 4.0 

Average scores were used for ties. 

Wilcoxon Two-Sample Test 

Statistic (S) 20.0000 

Normal Approximation 

Z 0.4356 

One-Sided Pr > Z 0.3316 

Two-Sided Pr > |Z| 0.6631 

t Approximation 

One-Sided Pr > Z 0.3381 

Two-Sided Pr > |Z| 0.6762 

Exact Test 

One-Sided Pr >= S 0.3143 

Two-Sided Pr >= |S - Mean| 0.6286 

Z includes a continuity correction of 0.5. 

Kruskal-Wallis Test 

Chi-Square 0.3373 

DF 1 

Pr > Chi-Square 0.5614 

Page 66 of 292
Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

 EPA MRID Number 48685801

Page 65 of 67

DER Template Version: 22 September 2011 

Page 67 of 292
Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

 EPA MRID Number 48685801

Page 66 of 67

DER Template Version: 22 September 2011 

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 10.0 18.0 3.464102 2.50 

3 4 26.0 18.0 3.464102 6.50 

Wilcoxon Two-Sample Test 

Statistic (S) 10.0000 

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 

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

 EPA MRID Number 48685801

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DER Template Version: 22 September 2011 

Page 69 of 292
DATA EVALUATION RECORD 

METALAXYL 

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

EPA Contract No. EP10H001452 

Task Assignment No. 3-06-2012 

(Revisions to MRID 48617601 to include laboratory proficiency data; 

Main study was originally reviewed under TA 2-46-2012) 

Prepared for 

Health Effects Division 

Office of Pesticide Programs 

U.S. Environmental Protection Agency 

2777 South Crystal Drive 

Arlington, VA 22202 

Prepared by 

CSS-Dynamac Corporation 

1910 Sedwick Road 

Building 100, Suite B 

Durham, NC 27713 

Primary Reviewer: Signature:

Daniel R. Hunt, M.S. Date: 5/22/2012 

Secondary Reviewer Signature:

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

Program Manager: Signature:

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

Quality Assurance: Signature:

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

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

signing by CSS-Dynamac Corporation personnel. 

Page 70 of 292
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 71 of 292
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) I Page 1 of 17 

METALAXYL/ 113501 OCSPP 890.1150/0ECD None 

Primary Reviewer: Vincent Chen. M.S. Signature~~ Risk Assessment Branch III, Health Effects Division (7509P) Date: 2 '-i "Ji) N zo,J...S 

Secondary Reviewer: Greg Akerman, Ph.D. Signature: 4. /2--= 

Health Effects Division (7509P) Date: - J,.-f, r 

Template version 08/2011 

DATA EVALUATION RECORD 

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

PC CODE: 113501 DP BARCODE: D399227 

TXR#: 0054219 CAS No.: 57837-19-1 

TEST MATERIAL (PURITY): Metalaxyl (98.2%) 

SYNONYMS: Methyl N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alaninate 

CITATION: Willoughby, J.A. (2011) Metalaxyl: Androgen Receptor Binding Assay (Rat 

Prostate Cytosol). CeeTox, Inc., Kalamazoo, MI. Laboratory Study No: 9143V100326ARB, December 7, 2011. MRID 48617601. Unpublished. 

SPONSOR: 

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

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

Assays for Assorted Chemicals. CeeTox, Inc., Kalamazoo, MI. July 19, 2011. 

MRID 48843501. Unpublished. 

Metalaxyl EDSP Consortium, 20 Ambriance Drive, Burr Ridge, IL 

TEST ORDER#: CON-113501-18 

EXECUTIVE SUMMARY: In an androgen receptor (AR) binding assay (MRID 48617601), 

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

competitive binding experiment, which measured the binding of a single concentration of 

[

3H]-R1881 (1 nM) in the presence of increasing concentrations (10- 10 to 10-3 M) ofmetalaxyl 

(98.2% purity, Batch #20100540). DMSO was used as the solvent vehicle at a final assay 

concentration of ca. 3.2%. 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. 

A saturation binding experiment was conducted to demonstrate that the AR in the rat prostate 

cytosol was present in reasonable numbers and was functioning with appropriate affinity for the 

radiolabeled reference androgen (RI 881) prior to routinely conducting AR competitive binding 

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

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

submitted following a request by the Agency. The mean dissociation constant (Kd) for [3H]-

Rl 881 was 1.080±0.690 nM, and the estimated Bmax was 1.071±0.167 fmol/100 μg protein for 

the single batch of prostate cytosol that was prepared. Only one of the three~ values was 

within the range reported in the EPA validation program (0.685 to 1.57 nM), and all Bmax values 

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were below the recommended range (7 to 16 fmol/100 μg protein). Confidence in these numbers 

is low based on the large variation in Kd estimates among runs. 

In the competitive binding experiment, the estimated log IC50s for R1881 (−9.0 M, Runs 2 and 3) 

and the weak positive control, dexamethasone (average of −4.4 M), were within the expected 

ranges; however, the log IC50 for R1881 was low in Run 1 (−9.9 M). Due to a laboratory error, 

the R1881 concentrations used in the Run 1 assay differed from the ranges reported in the EPA 

Test Guidelines for AR binding. The relative binding affinity (RBA) for the weak positive 

control averaged 0.0022% across all three runs. All performance criteria were generally met, 

with the exception that bottom % binding could not be determined for dexamethasone for Run 1. 

The deviation was considered minor. No precipitation of the test compound was observed. 

Metalaxyl had no significant effect on specific binding of the [3H]-ligand (>75% bound) over the 

concentration range of 10−10 to 10−3 M in all runs; therefore, an IC50 and RBA could not be 

calculated for metalaxyl. 

Based on the results of the three assays, metalaxyl is classified as a Non-Binder in the Androgen 

Receptor Binding Assay. 

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

Assay (OCSPP 890.1150). 

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

and Certification of Authenticity statements were provided. All phases of the study were 

conducted in compliance with FIFRA GLP regulations except that dose concentrations of test 

chemical and control chemicals were not verified using analytical methods.

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I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: CeeTox, Inc. 

Location: Kalamazoo, MI 

Study Director: J.A. Willoughby 

Other Personnel: K. Rutherford, Director of Laboratory Operations 

D. Blakeman, Senior Scientist/Endocrine Group Leader 

C. Haines, Scientist 

S. McColley, Scientist 

C. Toole, Director of Project Management 

Study Period: May 26, 2011 to December 7, 2011 

2. Test substance: Metalaxyl 

 Description: Solid 

 Source: Midwest Research Institute 

 Batch #: 20100540 

 Purity: 98.2 ± 0.3% 

 Solubility: Not reported (NR) 

 Volatility: NR 

 Stability: NR 

 Storage conditions: Ambient 

 CAS #: 57837-19-1 

 Molecular weight: 279.3 g/mol 

 Structure: 

3

CH

N

O

CH3

O

3CH CH3

O O

3CH

3. Non-labeled ligand: R1881 

 Supplier: Sigma Aldrich, St. Louis, MO 

 Catalog #: 

Lot #: 

R0908 

060M4638 

 Purity: 98% 

 CAS #: 965-93-5 

4. Radioactive ligand: [

3

H]-R1881 

 Supplier: Perkin-Elmer, Boston, MA 

 Catalog and Lot #: NET590 (653698) 

 Date of production: February 24, 2011 

 Date of use: September 10, 2011 to September 17, 2011 

 Radiochemical purity: >97% 

 Specific activity: 85.1 Ci/mmol 

 Concentration of stock: 10 nM 

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5. Positive control: Dexamethasone 

 Supplier: Sigma Aldrich, St. Louis, MO 

 Catalog # 

Lot #: 

D1756 

077K1050 

 Purity: 99% 

 CAS # : 50-02-2 

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

 Justification for choice of 

solvent: 

Issues with pipetting and volatilization prevented the use of ethanol as the solvent. 

DMSO is one of the recommended solvents according to the EPA Guideline (OPPTS 

890.1150) 

 Final Concentration: ca. 3.2% 

B. METHODS

1. Preparation of Rat Ventral Prostate Cytosol: Male Sprague Dawley rats (number not 

specified) from Charles River Laboratories were castrated at 90 days of age and the ventral 

prostates were excised <1 day following castration, weighed, and placed in ice-cold TEDG 

(Tris, EDTA, DTT, glycerol) buffer (inclusion of protease inhibitor not reported), 

homogenized, and centrifuged for 30 min at 30,000 × g at 4oC. The supernatants were 

pooled, and the protein concentration of the cytosol was determined to be 6.2 mg/mL using 

a commercially available protein kit compatible with DTT in the TEDG buffer (Bradford 

Method). The cytosol was divided into aliquots and stored frozen at −80°C until use.

2. Saturation Radioligand Binding Experiment: A saturation binding experiment 

measuring total and non-specific binding of [3H]-R1881 was performed to demonstrate that 

the AR was present in reasonable concentrations and had the appropriate affinity for the 

R1881 ligand (MRID 48843501). The conditions for the saturation binding experiment are 

summarized in Table 1. 

TABLE 1. Summary of Conditions for Saturation Binding Experiment a

Source of receptor Rat ventral prostate cytosol 

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

Concentration of non-labeled ligand (100X [radioligand]) 2 to 1000 nM 

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

radioligand at 0.25 nM

Temperature 4o C 

Incubation time 16 to 20 hours 

Composition of assay buffer 

(TEDG) 

Tris 10 mM (pH 7.4) 

EDTA 1.5 mM 

Glycerol 10% 

Phenylmethylsulfonyl fluoride (PMSF) 1 mM 

DTT 1 mM 

Sodium Molybdate 1 mM 

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

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

85.1 Ci/mmol as manufactured on February 24, 2011) was adjusted for decay over time 

(adjusted specific activities were not reported), and serial dilutions in low-salt 

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TEDG+PMSF buffer were prepared to achieve the final concentrations in cytosol of 0.25, 

0.50, 0.70, 1.0, 1.5, 2.5, 5.0, and 10 nM to determine total binding. To determine nonspecific binding, 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 1000 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 with 0.25 nM radiolabeled R1881. 

The optimal protein concentration was determined to be 1.86 mg protein/assay tube, which 

resulted in the binding of approximately 25-35% of the total radioactivity added. Cytosolic 

protein used in this assay was thawed fresh for this experiment at 4°C, and maintained at 

4°C during the binding assay. Each saturation binding experiment consisted of three noncurrent runs (conducted on September 9, 11, and 16, 2011, respectively). Each run 

contained three replicates at each concentration, resulting in the 72 samples depicted in 

Table 2. 

TABLE 2. Saturation Binding Experiment Runa,b

Total Binding Non-Specific Binding Radioligand alone 

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

[

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 

1.0 1.0 100 10 30 

1.5 1.5 150 10 45 

2.5 2.5 250 100 7.5 

5.0 5.0 500 100 15 

10 10 1000 100 30 

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

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

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

H]-R1881. Samples were dried, 

and 300 μl of prostate cytosol were added. 

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

H]-R1881. R1881 was added in a 

100-fold molar excess of [3

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

cytosol were added. 

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

H]-R1881 or 7.5, 15, 21, or 30 μL of 100 

nM [3

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

Following addition of triamcinolone acetonide, [3

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

dried, dissolved in diluted prostate cytosol (300 μL), and incubated in a rotor for 20 hours at 

approximately 4°C. Samples were maintained at temperatures of approximately 4°C except 

during whole rack vortexing. To separate bound from free R1881, hydroxyapatite (HAP) 

slurry was added to each tube and vortexed 5 times with 4-minute intervals over 20 minutes. 

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

samples were washed 4 times in 2 mL of ice cold TEDG+PMSF buffer, followed by 

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vortexing and centrifugation for 3 minutes at 700 x g. Following the last wash and 

decanting of the Tris buffer pellets were then extracted by additional of 2 mL of ethanol. 

The samples were vortexed and centrifuged for 10 minutes at 1000 x g. Samples were 

maintained on ice at all times between vortexing. After the final centrifugation, the ethanol 

supernatants were decanted into scintillation vials that each contained 14-mL portions of 

scintillation cocktail, and the radiation was quantified by liquid scintillation counting. A 

total of three runs were performed. 

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

binding experiment is included in Table 3. 

TABLE 3. Summary of Conditions for Competitive Binding Experimenta

Source of receptor Rat ventral prostate cytosol 

Concentration of radioligand 1 nM 

Optimization of receptor concentration Sufficient to bind 10-15% of radioligandb

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

Incubation Temperature 4 oC 

Incubation time 16-20 hours 

Composition of assay buffer Tris 10 mM (pH 7.4) 

EDTA 1.5 mM 

Glycerol 10% 

DTT 1 mM 

Sodium molybdate 1 mM 

Protease Inhibitor (PI)c 0.5% 

a Data were obtained from pages 9, and 14-16 of the study report. 

b The protein concentration was not reported. 

c The recommended PI in the Guideline is 1 mM PMSF. The PI was identified in the study protocol as 

Calbiochem Protease Inhibitor Cocktail, Set III, EDTA free. It is a mixture of six PIs that includes 4-(2-

aminoethyl)benzenesulfonyl fluoride (AEBSF). AEBSF has similar specificity to PMSF with greater 

stability at lower pH. 

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

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

the binding of a single concentration of [3

H]-R1881 (adjusted specific activity of 

82.6 Ci/mmol for Run 1; and 82.5 Ci/mmol for Runs 2 and 3) to the AR in the presence of 

increasing concentrations of a test substance. 

DMSO was used as the solvent vehicle, and no precipitation of metalaxyl was visually 

observed at concentrations ≥10−3 M. The protein concentration used in the competitive 

binding experiment was not reported; however, it was reported to be sufficient to bind 10-

15% of the [3

H]-R1881. 

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

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

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

and each run contained three replicates at each concentration plus six replicates of the 

[

3

H]-R1881, non-specific binding (NSB), and solvent controls, resulting in a total of 81 

samples per run. 

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

Solvent Control Reference standard Weak positive control Test Chemical None DMSO R1881 Dexamethasone Metalaxyl 

Tubes 7-12 Tubes 13-33 c

 Tubes 37-60 Tubes 61-84 Tubes 1-6 Run 1 Runs 2 & 3d Run 1 Runs 2 & 3d

1×10−7.5 1×10−6

 1×10−4 1×10−3 1×10−3

1×10−8.5 1×10−7 1×10−5 1×10−4 1×10−4

1×10−9.5 1×10−8 1×10−6 1×10−5 1×10−5

1×10−10.5 1×10−9 1×10−7 1×10−6 1×10−6

1×10−11.5 1×10−10 1×10−8 1×10−7 1×10−7

1×10−12.5 1×10−11 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 12-14, 16, and 20 of the study report. 

b Each concentration of each chemical was run in triplicate for a total of 81 tubes per run (tubes were numbered 

1-84,but tubes 34-36 were excluded from the sequence). Tubes 1-84 contained 50 μL of triamcinolone 

acetonide and 30 μL [3

H]-R1881. Samples were dried, and 300 μL of prostate cytosol were added. Tubes 7-

84 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 13-18 that contained 30 μL of non-radiolabeled R1881 

(used to evaluate non-specific binding). Tubes 1-6 contained only 30 μL of [3

H]-R1881. 

c Tubes 13-18 were used to evaluate non-specific binding by adding 100X of cold (non-radiolabeled) R1881. 

d The reason cited for the deviation from the EPA Test Guideline (890.1150) was the accidental prepping of the 

wrong stock concentration (pp. 65-66). 

To separate bound from free R1881, 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 discarded. The samples were washed 4 times in 50 mM TRIS buffer. Following the 

last wash and decanting of the Tris buffer, the pellets were then extracted by addition of 2-

mL portions of ethanol. The samples were vortexed at 5 minute intervals for ca. 15-20 

minutes. Samples were centrifuged for 10 minutes at 700 × g. Supernatants were 

radioassayed by scintillation counting. 

4. Data Analysis: For the saturation binding experiment, total binding and non-specific 

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

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

according to the method of Motulsky and Brown (2006)1

 implemented by using the ROUT 

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

ligand depletion with the method of Swillens (1995)2

. For the competitive binding assay, 

 

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 

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similar methods of nonlinear regression (XLfitTM Model 204, IDBS, Guilford, UK) were 

used to fit a curve (for R1881, the positive control, and the test substance) to the Hill 

equation formula which incorporated log IC50 as a parameter to be estimated. For parameters 

reported from the saturation binding experiment (Kd and Bmax) and competitive binding 

experiment (log IC50 and RBA), mean and standard deviation were calculated for each run and 

mean and standard error were calculated for the composite three runs using Microsoft Excel 2007 

(v. 12.0.6557.5000; Microsoft Corporation, Redmond, WA), and mean and standard error were 

calculated for the composite three runs with Microsoft Excel 2010.

5. Definitions 

a. Classification of test material 

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

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

bound, 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 

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

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

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

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

 M and the binding curve does 

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

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 

Log RBA: Log10(IC50 of R1881 ÷ IC50 of test substance) 

II. RESULTS 

A. SATURATION BINDING EXPERIMENT: Saturation binding experiment parameters 

are presented in Table 5. The mean Kd for [3H]-R1881 was 1.080±0.690 nM, and the 

estimated Bmax was 1.071±0.167 fmol/100 μg protein for the single batch of prostate cytosol 

that was prepared. Only one of the three Kd values was within the range reported in the 

EPA validation program (0.685 to 1.57 nM); the mean value was above the acceptable 

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

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range. Confidence in these numbers is low based on the large variation in Kd estimates 

among runs.

TABLE 5. Saturation Binding Experiment of [3H]-R1881 with Androgen Receptor from Rat Prostate 

Cytosola

Parameter Run 1 Run 2 Run 3 Mean ± SEb

R2 (unweighted) 0.880 0.986 0.893 0.880-0.986 

Bmax (nM) 0.010 0.008 0.011 0.010±0.002 

Bmax (fmol/100 μg protein) 1.087 0.896 1.229 1.071±0.167 

Kd (nM) 0.855 0.531 1.855 1.080±0.690 

a Data were obtained from page 4 of the study report (MRID 48843501). 

b The range of R2

 is reported and the mean ± SE is reported for the other parameters. 

R2

 Goodness of fit for curve calculated for specific binding

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

H]-R1881 to the 

androgen receptor for the three independent runs. The specific binding reached a plateau in 

each run, and non-specific binding was less than 20% of total binding. Figure 2 contains the 

Scatchard plots that illustrate the binding of [3H]-R1881 to the androgen receptor. The data 

fits generally resulted in linear plots. 

 

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

Binding Experiment. 

 

 

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

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B. COMPETITIVE BINDING EXPERIMENT: Competitive binding experiment parameters 

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

The estimated average log IC50 was −9.3 M for R1881 and −4.4 M for the weak positive 

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

control was 0.0022%. Confidence in these numbers is high; however it is noted that the 

variations between Run 1 compared to Runs 2 and 3 are likely due to the concentration 

differences used for R1881 and dexamethasone. The curve for dexamethasone was not fully 

defined in the first run and therefore, the calculation for the IC50 may be affected. The range 

of concentrations for R1881 in the first run fully defined the curve, but the concentration 

range was off by more than an order of magnitude. In addition, all solvent control tubes 

were run at the beginning of each experiment, preventing the reviewer from determining 

drift in the assay from the beginning to the end of the run. 

Metalaxyl had no effect on specific binding (>75% binding) of the [3

H]-ligand at 

concentrations up to 10−3 M in all three runs. An IC50 and RBA for metalaxyl could not be 

determined. Based on the results of the three valid assays, metalaxyl is classified as a “nonbinder” with the androgen receptor under the conditions of this assay. 

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

Parameter Run 1 Run 2 Run 3 Mean ± SEb

R2 (unweighted) R1881 NR NR NR NA 

 Dexamethasone NR NR NR NA 

 Metalaxyl NR NR NR NA 

Log IC50 (M) R1881 −9.9 −9.0 −9.0 −9.3 ± 0.30 

 Dexamethasone −4.3 −4.4 −4.6 −4.4 ± 0.09 

 Metalaxyl NA NA NA NA 

IC50 (M) b R1881 1.26 × 10−10 1.00 × 10−9 1.00 × 10−9 7.09 × 10−10 (± 2.91) 

 Dexamethasone 5.01 × 10−5 3.98 × 10−5 2.51 × 10−5 3.83 × 10−5 (± 0.73) 

 Metalaxyl NA NA NA NA 

Log RBAb Dexamethasone −5.60 −4.60 −4.40 −4.87 ± 0.37 

 Metalaxyl NA NA NA NA 

RBA (%)b Dexamethasone 0.0003 0.0025 0.0040 0.0022 ± 0.0011 

 Metalaxyl NA NA NA NA 

a Data were obtained from page 21 of the study report. 

b All values are reviewer-calculated from the log IC50 values reported in the study report. For means 

expressed in scientific notation, the SE values in parentheses are presented in the same order of 

magnitude as the mean value. 

SE Standard Error 

NA Not applicable. 

NR Not reported 

R2

 Goodness of fit 

RBA (%) relative binding affinity

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FIGURE 3. Percentage [3H]-R1881 Bound to the Androgen Receptor in the Presence of 

Radioinert R1881, Dexamethasone, or Metalaxyl (Run 1). 

 

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FIGURE 4. Percentage [3H]-R1881 Bound to the Androgen Receptor in the Presence of 

Radioinert R1881, Dexamethasone, or Metalaxyl (Run 2). 

 

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FIGURE 5. Percentage [3H]-R1881 Bound to the Androgen Receptor in the Presence of 

Radioinert R1881, Dexamethasone, or Metalaxyl (Run 3). 

 

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

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

TABLE 7. Criterion a Tolerance 

Limit(s) b Value Yes No 

Ligand depletion is minimal. The recommended ratio of total 

binding in the absence of competitor to total amount of [3

H]-R1881 

added per assay tube. 

≤15% ≤12.7 X 

Test chemical Top (% binding)c 80 to 115 95.1 to 107.5 X 

R1881 fitted curve parameters 

Top (% binding) 82 to 114 96 to 103 X 

Bottom (% binding) −2.0 to 2.0 0 X 

Hill Slope −1.2 to −0.8 −1.0 X 

Weak positive control (dexamethasone) fitted curve parameters 

Top (% binding) 87 to 106 101 to 105 X 

Bottom (% binding) −12 to 12 NA; -1 to 7 Xd

Hill Slope −1.4 to −0.6 −1.0 to −0.6 X 

Saturation Binding Experiment Kd (nM) 0.685 to 1.57 0.531 to 

1.855e

 Xf

Non-specific binding (%) ≤10.0 0.28 to 0.50g

 X 

a Data were obtained from pages 23-28, and 52-57 of the study report. 

b These values represent ranges from the validation study. 

c 10−9

 to 10−10 M 

d A value could not be determined for Run 1 due to the aforementioned laboratory error with the prepared 

concentration of dexamethasone. Bottom % binding was within the acceptable range in the other two runs. 

e MRID 48843501 

f One value was below the limit, one value was within the limit, and one value was above the limit. 

g Calculated by the study reviewer. 

NR Not reported 

All performance criteria were met, with the exception that bottom % binding could not be 

determined for dexamethasone for Run 1 due to a laboratory error in the preparation of the 

concentration used in the assay (resulting in dexamethasone concentrations ranging from 

10−11 to 10−4

 M instead of 10−10 to 10−3 M). The resulting curve for dexamethasone fit two of 

the three criteria (Hill Slope and top plateau) and the study author stated that the log IC50 for 

dexamethasone performed within CeeTox historical data. The deviation was considered 

minor. 

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

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

indicated by a descent from 84.4-91.9% to 4.9-8.2% binding over approximately an 81-fold 

increase in concentration of R1881 (i.e., covering approximately 2 log units). Examination 

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

and bottom plateaus. 

 

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III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Metalaxyl was classified as a "non-binder" in all 

three valid independent runs of the assay. Thus, metalaxyl has a final classification of "nonbinder" to the AR. 

B. AGENCY COMMENTS: In the saturation binding assay, the mean Kd for [3H]-R1881 

was 1.080 nM, and the mean estimated Bmax was 1.071 fmol/100 μg protein for the single 

batch of prostate cytosol that was prepared. Only one of the three Kd values was within the 

range reported in the EPA validation program (0.685 to 1.57 nM); the mean value was 

above the acceptable range. 

In the competitive binding experiment, metalaxyl was classified as a non-binder in each run 

as there were no test substance-related effects on the [3H]-R1881 binding to the AR at 

concentrations up to 10−3

 M under the conditions of this assay. 

In the competitive binding experiment, the estimated average log IC50 was −9.3 M for 

R1881 and −4.4 M for the weak positive control (dexamethasone). Compared to R1881, the 

mean RBA for dexamethasone was 0.0022%. Confidence in these numbers is generally 

high due to the small variation between the runs. However, it is noted that variations 

between Run 1 compared to Runs 2 and 3 are likely due to the concentration differences 

used for R1881 and dexamethasone. The bottom of the curve for dexamethasone was not 

fully defined in the first run. The range of concentrations for R1881 in the first run fully 

defined the curve, but the concentration range was off by more than an order of magnitude. 

The concentrations prepared for R1881 and dexamethasone were lower than the targeted 

(guideline) levels because of an error in the stock preparation. Therefore, non-specific 

binding was not evaluated with 100X of cold (non-radiolabeled) R1881 but with 30X cold 

R1881, and the resulting curve for dexamethasone fit only two of the three criteria (Hill 

slope and Top plateau) in the first run. All performance criteria were met for the other two 

runs. 

The bottom percent binding for dexamethasone was not achieved in the first run, leaving 

only two valid runs instead of the required three. However, as the performance was 

consistent between the other two runs, it is unlikely that information from an additional run 

would change the interpretation of this study. 

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

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

 The bottom percent binding for dexamethasone was not achieved in the first run. The 

concentrations prepared for R1881 and dexamethasone were lower than the targeted 

(guideline) levels because of an error in the stock preparation. 

 Curves were not provided showing the average binding of each test substance across all 

three runs. 

Page 88 of 292
DATA EVALUATION RECORD 

METALAXYL 

Study Type: OCSPP 890.1200, Aromatase Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-46-2012 (MRID 48617602) 

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: 4/27/2012 

Secondary Reviewer: Signature:

David A. McEwen, B.S. Date: 5/14/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 6/05/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 6/05/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 89 of 292
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 90 of 292
Aromatase (Human Recombinant) Assay (2011) I Page 1 of 13 

METALAXYL / 113501 OCSPP 890.1200 I OECD None 

Risk 

Primary 

Assessment 

Reviewer: 

Branch 

Vincent 

III, Health 

Chen, M.S. 

Effects Division (7509P) 

Signature: 

Date: 

~ 

-Z'i -;J'-J.rJ "Z-O.L-5 

Secondary Reviewer: Greg Akerman, Ph.D. Signature: .4 ~ 

Health Effects Division (7509P) Date: a. /.J.o- hr= 

Template version 08/2011 

DATA EVALUATION RECORD 

STUDY TYPE: Aromatase (Human Recombinant); OCSPP 890.1200 

PC CODE: 113501 DP BARCODE: D399227 

TXR#: 0054219 CAS No.: 57837-19-1 

TEST MATERIAL (PURITY): Metalaxyl (100.4% a.i.) 

SYNONYMS: Alanine, N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-, methyl ester 

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

CeeTox, Inc., Kalamazoo, MI. Study ID: 9143V-100326AROM, December 7, 

2011. MRID 48617602. Unpublished. 

SPONSOR: Metalaxyl EDSP Consortium; 20 Ambriance Drive, Burr Ridge, IL 60527, USA 

TEST ORDER#: CON-113501-18 

EXECUTIVE SUMMARY: In an in vitro aromatase (CYP 19) assay (MRID 48617602), 

metalaxyl (100.4% a.i., Batch# 20100540) in 1 % dimethyl sulfoxide (DMSO) was incubated 

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

3

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

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

three independent runs. 

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). 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 (10-10 to 10-3 M) with three 

repetitions per concentration. 

Aromatase activity in the full activity controls was 0.240 ± 0.070 nmol·mg-protein-1

·min-1

• The 

response of each full activity control within a run was between 90 to 110% of the average full 

activity, with two exceptions likely due to an unevenly split sample. Activity in the background 

controls ranged 0.43 to 0.87% and averaged 0.59% of the full activity controls. 

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

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

replicates of each concentration within runs, except that the %CV for the 1 o-s M ( 19%) and 10-9 

Page 91 of 292
Aromatase (Human Recombinant) Assay (2011) / Page 2 of 13 

METALAXYL / 113501 OCSPP 890.1200 / OECD None

M (34%) replicates for Run 2 exceeded the recommended limit of 15%. The estimated log IC50

averaged −7.18 M and the Hill slope averaged −1.03 for 4-OH ASDN. 

For metalaxyl, aromatase activity averaged 0.250 ± 0.069 nmol·mg-protein−1·min−1 at the lowest 

tested concentration 10−10 M and 0.232 ± 0.073 nmol·mg-protein−1

·min−1 at the highest tested 

concentration 10−3

 M. The average response curve indicated that the aromatase activity of 

metalaxyl at concentrations from 10−10 to 10−3 M was essentially equivalent (i.e. >75% activity) 

to the activity observed in the full activity control. 

Based on the data from the average response curve, metalaxyl is classified as a Non-inhibitor of 

aromatase activity in this assay. 

The study satisfies the Test Order requirement for an Aromatase Assay (OCSPP 890.1200). 

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 Substance: Metalaxyl 

 Description: White, sugar like 

 Source: Nation’s Ag, LLC (Burr Ridge, IL) 

 Lot/Batch #: 20100540 (expiry date: July 26, 2011) 

 Purity: 100.4% a.i. 

 Volatility: Not reported 

 Storage conditions: Room temperature (ambient) 

 Stability: Not reported 

 Solvent: DMSO 

 Solubility (in test solvent): Not reported 

 Highest Concentration Tested: 10−3 M in all runs 

 Stock Solution Preparation: Dissolved in DMSO to obtain a stock concentration of 100 mM 

 Molecular weight: 279.3 

 CAS #: 57837-19-1 

 Structure: 

3

CH

N

O

CH3

O

3CH CH3

O O

3

CH

2. Non-Labeled Substrate: Androstenedione (ASDN) 

 CAS # : 63-05-8 

 Source (catalog #): Steraloids, Inc. (catalog # L1712) 

 Lot/Batch # (expiration date): L1712 (expiry date: April 2016) 

 Purity: 99.8% 

3. Radiolabeled Substrate: 1-β [3

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

 Source (catalog #): Perkin Elmer (catalog # NET-926) 

 Lot/Batch # (expiration date): 619344 (expiry date: January 10, 2012) 

 Radiochemical Purity (Supplier): >97% 

 Specific activity: 26.3 Ci/mmol 

 Radiochemical Purity (In-lab 

determination): Not reported 

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

 CAS # : 566-48-3

 Source (catalog #): Sigma-Aldrich (catalog # F2552) 

 Batch # (expiration date): 081K2133 (expiry date: March 2015) 

 Purity: 99.6% 

5. Solvent (Vehicle Control): Dimethyl sulfoxide (DMSO) 

 Source: 

Lot # (expiration date): 

Sigma-Aldrich (Catalog # D2650) 

RNBB7617 (expiry date: February 2013) 

 Justification for choice of solvent DMSO was listed as one of the vehicles acceptable for use in OCSPP 890.1200 

guideline; DMSO was not as volatile as ethanol and so evaporation was less of a 

concern in the assay, and DMSO was more accurate to pipette because of density

and viscosity 

 Concentration 

(% of total volume in assays): ≤1% 

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

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

Source (catalog #): BD GentestTM, Woburn, MA (catalog # 456260) 

Lot/Batch # (expiration date): 03897 (expiry date: February 2014) 

Protein concentration: 7.4 mg protein per mL 

Cytochrome C reductase activity: 290 nmole/mg protein/minute 

Aromatase activity: 6.0 pmol/pmol P450/min 

B. METHODS

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

[

3

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

approximately 0.1 μM, and the amount of tritium added to each incubation tube was 

0.1 μCi. 

The test chemical(s) were formulated in DMSO such that the volume of DMSO used per 

assay was no more than 1% v/v of the total assay volume. DMSO was selected because it is 

listed as one of the solvents of choice detailed in the EPA guideline; it not as volatile as 

ethanol and so evaporation was less of a concern in the assay, and is more accurate to 

pipette because of its density and viscosity. 

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

Fresh dilutions of the stock solution were prepared in the same solvent 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 2 mL. 

Human recombinant microsomes were purchased from BD GentestTM, and stored at −80 ± 

10oC. Before use of the microsomes, the batch was thawed, diluted with sodium phosphate 

pH 7.4 buffer to 0.008 mg/mL microsomal protein (to achieve a final concentration of 

approximately 0.004 mg/mL microsomal protein per assay tube), and sub-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 a

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 

a Data were obtained from page 16 of the study report. 

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2. Suitability Assessments: The protein concentration of the microsomes (Lot # 03897) was 

supplied by the vendor as 7.4 mg/mL. Aromatase activity of the microsomes was also 

provided by the vendor as 6.0 pmol/min/pmol P450. The minimum aromatase activity in 

the full activity control samples was determined to be 0.160 nmol·mg-protein−1

·min−1, which 

was greater than the minimum recommended aromatase activity of 0.1 nmol·mgprotein−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 tubes 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 3H2O in the aqueous fraction was quantified for each assay tube by LSC, and 

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

4. Demonstration of Proficiency: It was stated that proficiency testing of the CYP19 

aromatase assay was conducted. Each of the proficiency chemicals (atrazine, econazole, 

fenarimol and nitrofen) were incubated alongside the positive control inhibitor (4-OH 

ASDN) on three separate occasions using the same methods employed in the current study. 

Proficiency records were archived at CeeTox, Inc. and the actual data from the proficiency 

testing were not included in the study report. 

a. Positive Control

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

laboratory 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. 

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

provided parameters in the preferred ranges. 

The coefficient of variation (CV) for replicates within each sample type and concentration 

of 4-OH ASDN was generally <15%, except for Run 2 in which the CV was >15% for the 

10−8

 M duplicate samples (19%), and for the 10−9 M duplicate samples (34%). The 15% CV 

maximum is a suggested limit, and the positive control met all other criteria. 

(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.

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TABLE 2. Performance Criteria for the Positive Control a

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

Slope −1.2 −0.8 −1.12 −0.98 

Top (%) 90 110 97.42 101.05 

Bottom (%) −5 +6 0.46 1.63 

Log IC50 (M) −7.3 −7.0 −7.29 −7.10 

a Data were obtained from pages 23 and 27 of the study report. 

b. Proficiency Chemicals: It was stated that the proficiency of the laboratory was 

demonstrated prior to running any test chemicals by conducting three full scale test runs on 

each of the proficiency chemicals as listed in Table 3. The data were not included in the 

study report, but it was reported that all assays were performed by CeeTox personnel that 

have demonstrated proficiency performing the assay as outlined in the test guideline. 

TABLE 3. Proficiency Chemicals 

Compound CAS# Class Concentrations 

Econazole 24169-02-6 Not reported Not reported 

Fenarimol 60168-88-9 Not reported Not reported 

Nitrofen 1836-75-5 Not reported Not reported 

Atrazine 1912-24-9 Not reported Not reported 

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

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

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

precipitate). No precipitation was observed at any concentration in the first run. The 

response of each full activity control within a run was between 91% to 108% of the average 

full activity, with the exception of the duplicate full activity control responses (129% and 

62%) from the end of Run 2 that were outside of the guideline recommended range. The 

laboratory reported that it was likely the sample was unevenly split between the two tubes, 

and had no impact on the study. 

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TABLE 4. Test Chemical Study Design for each Test Run a

Sample Type Repetitions 

(Tubes) Description Reference or 

Chemical (M) 

Full Activity Control 4 All test componentsb plus solvent vehicle (DMSO) 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

Metalaxyl Conc 1 3 All test components plus Metalaxyl 1×10−3

Metalaxyl Conc 2 3 All test components plus Metalaxyl 1×10−4

Metalaxyl Conc 3 3 All test components plus Metalaxyl 1×10−5

Metalaxyl Conc 4 3 All test components plus Metalaxyl 1×10−6

Metalaxyl Conc 5 3 All test components plus Metalaxyl 1×10−7

Metalaxyl Conc 6 3 All test components plus Metalaxyl 1×10−8

Metalaxyl Conc 7 3 All test components plus Metalaxyl 1×10−9

Metalaxyl Conc 8 3 All test components plus Metalaxyl 1×10−10

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

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

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 and metalaxyl. The following raw data and 

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

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: Basic statistical analysis was performed on the data, which included 

means of replicates, standard deviation of the mean, standard error of the mean, and 

coefficient of variation. 

The response curves were fitted by weighted least squares nonlinear regression analysis with 

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

(XLfit; IDBS; Version 5.2.0.0; Fit Model 208) and Tukey’s Bi-Weight statistical analysis 

Page 97 of 292
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METALAXYL / 113501 OCSPP 890.1200 / OECD None

for outlier analysis. Concentration response trend curves were fitted to the percent of 

control activity values within each of the replicate tubes at each test chemical concentration. 

Concentration was expressed on the log or half-log scale. For each run the individual 

percent of control values were plotted versus logarithm of the test chemical concentration. 

The fitted concentration response curves were superimposed on the plot. Individual plots 

were prepared for each run. Additional plots were prepared to compare the percent of 

control activity values across runs. For each run the average percent of control values 

versus logarithm of test chemical concentration were plotted on the same plot. The fitted 

concentration response curves for each run were superimposed on the plots. On a separate 

plot the average percent of control values for each run were plotted versus logarithm of test 

substance concentration. The average concentration response curve across runs was 

superimposed on the same plot. 

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

three runs, using the categories presented in Table 6. 

TABLE 6. Interpretation of Results 

Criteria Interpretation 

Data fit 4-parameter nonlinear 

regression model 

Average curve across runs crossed 50%a Inhibitor 

Average lowest portion of curves across runs is 

between 50% and 75% activityb

Equivocal 

Average lowest portion of curves across runs is greater 

than 75% activityb

Non-inhibitor 

Data do not fit model --- 

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

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

equivocal. 

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

determined to be untestable in the aromatase assay. 

II. RESULTS 

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

0.160 to 0.331 nmol·mg-protein−1·min−1 for the three test runs, with a mean and standard 

deviation of 0.240 ± 0.070 nmol·mg-protein−1

·min−1. Activity in the background controls 

ranged 0.43 to 0.87% and averaged 0.59% of the full activity controls. The response of each 

full activity control within a run was between 90 to 110% of the average full activity, with 

the exception of the duplicate full activity control responses (129% and 62%) from the end 

of Run 2 that were outside of the guideline recommended range. The laboratory reported 

that it was likely the sample was unevenly split between the two tubes, and had no impact 

on the study. The response of the full activity controls and background controls were 

considered acceptable for each run. 

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

activity averaged 0.249 ± 0.065 nmol·mg-protein−1·min−1 at the lowest tested concentration 

10−10 M and 0.002 ± 0.001 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 

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

response curves for the positive control from each run and the average of all runs are shown 

in Figure 1. These results were generally within the recommended ranges for the top of the 

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

concentration, with the following exceptions: the %CV for the 10−8

 M (19%) and 10−9 M 

(34%) replicates for Run 2 exceeded the recommended limit of 15%. 

TABLE 7. Effect of 4-OH ASDN and Metalaxyl on Aromatase Activity (as percent of control) from 

Independent Runs a

Chemical 

Concen. 

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

4-OH ASDN 

(positive control) 

−5 3 0.77 0.08 0.03 10.0 

−6 3 7.10 1.29 0.53 19.0 

−6.5 3 18.76 2.93 1.20 16.3 

−7 3 38.62 4.79 1.96 12.9 

−7.5 3 68.07 7.57 3.09 11.6 

−8 3 88.54 10.65 4.35 12.5 

−9 3 92.69 14.45 5.90 16.2 

−10 3 102.62 9.80 4.00 10.0 

Metalaxyl −3 3 95.45 14.46 4.82 15.6 

−4 3 102.10 4.13 1.36 4.2 

−5 3 99.79 3.06 1.02 3.1 

−6 3 99.93 3.68 1.23 3.8 

−7 3 101.07 4.96 1.65 5.0 

−8 3 104.54 11.23 3.74 11.0 

−9 3 104.03 10.45 3.98 10.3 

−10 3 103.33 10.71 3.57 10.6 

a Values were calculated by the reviewers based on data provided on pages 41-44, 46-49, and 51-54. 

SD Standard Deviation 

SEM Standard Error of the Mean 

CV Coefficient of Variance 

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

C. TEST SUBSTANCE: For metalaxyl, aromatase activity averaged 0.250 ± 0.069 nmol·mgprotein−1

·min−1 at the lowest tested concentration 10−10 M and 0.232 ± 0.073 nmol·mgprotein−1

·min−1 at the highest tested concentration 10−3 M. The mean aromatase activity of 

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

runs is presented in Table 7 (above), along with the overall standard deviation, standard 

error of the mean, and % CV. Inhibition response curves for metalaxyl from each run are 

shown in Figure 2, and the average inhibition response curve across all runs is shown in 

Figure 3. 

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

FIGURE 3. Mean Inhibition Response Curve for Metalaxyl. 

The effect of the positive control and metalaxyl on inhibition of aromatase activity is 

presented in Table 8. The response curve indicated that the aromatase activity of metalaxyl 

at all test concentrations tested was essentially equivalent (i.e. activity was >75% across all 

Page 101 of 292
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runs) to the activity observed in the full activity control. These data could not be fit to the 4-

parameter nonlinear regression model; therefore, log IC50 and Hill slope estimates were not 

determined for metalaxyl. For 4-OH ASDN, the estimated log IC50 averaged −7.18 M and 

the mean Hill slope was −1.03. Confidence in these values is high due to the small variation 

(<8% CV). 

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

Chemical Run 1 Run 2 Run 3 Mean SD %CV 

IC50 (M) 

Metalaxyl NA NA NA NA NA NA 

4-OH ASDN −7.14 −7.10 −7.29 −7.18 0.10 −1.51 

 Hill Slope 

Metalaxyl NA NA NA NA NA NA 

4-OH ASDN −1.00 −1.12 −0.98 −1.03 0.08 −7.94 

a Data were obtained from Table 12 on page 27 of the study report. The Mean, SD and %CV were calculated by the 

reviewers based on these data. 

SD Standard Deviation 

CV Coefficient of Variance 

NA Not applicable; values for metalaxyl were not suitable for modeling. 

Based on the data from the average response curve and the criteria listed above in Table 8, 

the results support the conclusion that metalaxyl is a non-inhibitor of aromatase activity. 

Mean aromatase activity for metalaxyl was 95.45 ± 14.97% at the highest test concentration 

of 10−3 M. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS CONCLUSIONS: In three independent runs of the assay, metalaxyl 

tested at concentrations of 10−10 to 10−3 M resulted in essentially no decrease in aromatase 

activity. Based on the data interpretation criteria established for the assay, metalaxyl was 

classified as a non-inhibitor of the aromatase enzyme. 

B. AGENCY COMMENTS: Although data demonstrating proficiency with the positive 

control or recommended proficiency chemicals were not included, a statement was included 

that proficiency of the laboratory was demonstrated prior to analysis of metalaxyl by the 

conduct of three full scale test runs on each of the recommended proficiency chemicals and 

that all assays were performed by CeeTox personnel that have demonstrated proficiency 

performing the assay as outlined in the test guideline. 

Aromatase activity in the full activity controls was 0.240 ± 0.070 nmol·mg-protein−1·min−1. 

The response of each full activity control within a run was between 90 to 110% of the 

average full activity, with the exception of the duplicate full activity control responses 

(129% and 62%) from the end of Run 2 that were outside of the guideline recommended 

range. The laboratory reported that it was likely the sample was unevenly split between the 

two tubes, and had no impact on the study. Activity in the background controls ranged 0.43 

to 0.87% and averaged 0.59% of the full activity controls. The response of the full activity 

controls and background controls were considered acceptable for each run. 

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

METALAXYL / 113501 OCSPP 890.1200 / OECD None

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, except that the %CV for the 10−8 M (19%) 

and 10−9

 M (34%) replicates for Run 2 exceeded the recommended limit of 15%. The 

estimated log IC50 averaged −7.18 M and the mean slope was −1.03 for 4-OH ASDN. 

For metalaxyl, aromatase activity averaged 0.250 ± 0.069 nmol·mg-protein−1

·min−1 at the 

lowest tested concentration 10−10 M and 0.232 ± 0.073 nmol·mg-protein−1·min−1 at the 

highest tested concentration 10−3 M. The lowest portion of the response curve was greater 

than 75% activity at all concentrations of metalaxyl evaluated (10−10 to 10−3 M). Based on 

the data from the average response curve, metalaxyl is classified as a Non-inhibitor of 

aromatase activity in this assay. 

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

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

 The required proficiency data were not provided. 

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

Page 103 of 292
DATA EVALUATION RECORD 

METALAXYL 

Study Type: OCSPP 890.1250, Estrogen Receptor Binding Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 3-06-2012 

(Revisions to MRID 48617607 to include Saturation binding data; 

Main study was originally reviewed under TA 2-46-2012) 

Prepared for 

Health Effects Division 

Office of Pesticide Programs 

U.S. Environmental Protection Agency 

2777 South Crystal Drive 

Arlington, VA 22202 

Prepared by 

CSS-Dynamac Corporation 

1910 Sedwick Road, 

Building 100, Suite B 

Durham, NC 27713 

Primary Reviewer: Signature:

Daniel R. Hunt, M.S. Date: 05/24/2012 

Secondary Reviewer: Signature:

Scott D. Studenberg, Ph.D., D.A.B.T. Date: 7/17/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 7/30/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 7/30/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 104 of 292
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 105 of 292
MET ALAXYL I 113501 

Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) I Page 1 of 18 

OCSPP 890.1250/ OECD None 

Primary Reviewer: Minerva Mercado, Ph.D. 

Health Effects Division 

Secondary Reviewer: Greg Akerman, Ph.D. 

Health Effects Division 

SignalDate: ~ · · 

Signature:==: 

Date: <.; h rt 1 S---

Template version 08/2011 

DATA EVALUATION RECORD 

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

OCSPP 890.1250 

PC CODE: 113501 DP BARCODE: D399227 

TXR#: 0054219 CAS No.: 57837-19-1 

TEST MATERIAL (PURITY): Metalaxyl (98.2% a.i.) 

SYNONYMS: Methyl N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alaninate 

CITATION: Willoughby, J.A. (2011) Metalaxyl: Estrogen Receptor Binding (Rat Uterine 

Cytosol). CeeTox, Inc., Kalamazoo, MI. Laboratory Study No: 9143V100326ERB, December 8, 2011. MRID 48617607. Unpublished. 

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

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

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

MRID 48843501. Unpublished. 

SPONSOR: Metalaxyl EDSP Consortium, 20 Ambriance Drive, Burr Ridge, IL 

TEST ORDER#: CON-113501-18 

EXECUTIVE SUMMARY: In an estrogen receptor (ER) binding assay (MRID 48617607) for 

metalaxyl (98.2% purity, Batch #20100540), uterine cytosol from Sprague Dawley rats was used 

as the source of ER for a competitive binding experiment, which measured the binding of a 

single concentration of [3H]-17~-estradiol (1 nM) in the presence of increasing concentrations 

(10- 10 to 10-3 M) of metalaxyl. DMSO was used as the solvent vehicle at a final concentration of 

ca. 4%. A total of three 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. 

A saturation binding experiment was conducted to demonstrate that the ER in the rat uterine 

cytosol was present in reasonable numbers and was functioning with appropriate affinity for the 

radio-labeled reference estrogen prior to conducting ER competitive binding experiments. 

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

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

following a request by the Agency. The protein concentrations used in the saturation binding 

runs varied between each run, and were approximately 3- to 6-fold greater than recommended 

Page 106 of 292
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 2 of 18 

METALAXYL / 113501 OCSPP 890.1250/ OECD None

(160 to 320 μg versus 50±10 μg). The Kd for [3H]-17β-estradiol was 0.331±0.061 nM and the 

estimated Bmax was 74.55±3.03 fmol/100 μg protein for the prepared rat uterine cytosol. The Kd

for each run was within the expected Guideline range of 0.03 to 1.5 nM. 

In the competitive binding experiment, the estimated mean log IC50 was −9.0 M for 17β-estradiol 

and −5.6 M for the weak positive control. The mean RBA was 0.0344% for the weak positive 

control. All performance criteria were generally met; however, octyltriethoxysilane displaced 

more than 25% of [3H]-17β-estradiol from the ER at the highest concentration tested (10−3 M) in 

Run 3, and bottom % binding and Hill slope were slightly outside of the tolerance limits for 17βestradiol in Run 2. 

Metalaxyl reduced the mean specific binding of the radiolabeled ligand in a concentrationdependent manner in one of the three runs (declining to <46.8% in Run 2); however, metalaxyl 

had <25% inhibition of binding at concentrations up to 10−3 M in the other two runs. The 

estimated log IC50 was −3.8 M for metalaxyl in Run 2, with a relative binding affinity (RBA) of 

0.0006%. 

Based on the results from the three runs (interactive in one run and not interactive in the other 

two runs), metalaxyl is classified as Equivocal in the Estrogen Receptor Binding Assay. 

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

Assay (OCSPP 890.1250). 

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

and Certification of Authenticity statements were provided. All phases of the study were 

conducted in compliance with FIFRA GLP regulations except that dose concentrations of the test 

chemical and control chemicals were not verified using analytical methods. 

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I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: CeeTox, Inc. 

Location: Kalamazoo, MI 

Study Director: J.A. Willoughby 

Other Personnel: K. Rutherford, Director of Laboratory Operations 

D. Blakeman, Senior Scientist/Endocrine Group Leader 

C. Haines, Scientist 

S. McColley, Scientist 

B. Meyer, Scientist 

C. Toole, Director of Project Management 

Study Period: May 26, 2011 to December 8, 2011 

2. Test substance: Metalaxyl 

 Description: Solid 

 Source: Nation’s AG, LLC 

 Batch #: 20100540 

 Purity: 98.2 ± 0.3% 

 Solubility: Not reported (NR) 

 Volatility: NR 

 Stability: NR 

 Storage conditions: Ambient 

 CAS #: 57837-19-1 

 Molecular weight: 279.3 g/mol 

 Structure: 

3CH

N

O

CH3

O

3CH CH3

O O

3CH

3. Non-labeled ligand: 17β-estradiol 

 Supplier: Sigma-Aldrich, St. Louis, MO 

 Catalog #: E8875 

 Lot #: 110M0138V 

 Purity: 100% 

 CAS #: 50-28-2 

4. Radioactive ligand: [

3

H]-17β-estradiol 

 Supplier: Perkin-Elmer, Boston, MA 

 Catalog #: NET517 

 Lot#: 650702 

 Radiochemical purity: >97% 

 Specific activity: 130.2 Ci/mmol 

 Concentration of stock: 50 nM 

Page 108 of 292
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 4 of 18 

METALAXYL / 113501 OCSPP 890.1250/ OECD None

5. Positive control: 19-Norethindrone 

 Supplier: Sigma-Aldrich, St. Louis, MO 

 Catalog #: N4128 

 Lot #: 030M1359V 

 Purity: 99% 

 CAS #: 68-22-4 

6. Negative control: Octyltriethoxysilane 

 Supplier: Sigma-Aldrich, St. Louis, MO 

 Catalog #: 440213 

 Lot #: 24996KK 

 Purity: 99.34% 

 CAS #: 2943-75-1 

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

 Justification for choice of 

solvent: 

DMSO is one of the recommended solvents according to the EPA Guideline (OPPTS 

890.1250) 

 Final Concentration: ca. 4% 

B. METHODS 

1. Preparation of Rat Uterine Cytosol (RUC): Female Sprague Dawley rats (number not 

specified) from Harlan Laboratories were ovariectomized 7 days prior to being euthanized. 

Animals were 84-91 days old at the time of euthanasia. The uteri were weighed, and placed 

in ice-cold TEDG (Tris, EDTA, DTT, glycerol) buffer + PMSF (phenylmethylsulfonyl 

fluoride), homogenized, and centrifuged for 10 min at 2,500 × g at 4oC. The supernatants 

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

pellets. Protein concentration of the cytosol was determined to be 3.22 mg/mL using a 

protein kit compatible with DTT in the TEDG buffer (Bradford Method). Cytosol was 

divided into 1- to 6-mL portions for immediate use or storage at −80oC until use. 

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

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

demonstrate that the estrogen receptor (ER) was present in reasonable concentrations and 

had the appropriate affinity for the native ligand (MRID 48843501). The conditions for the 

saturation binding are summarized in Table 1. 

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TABLE 1. Summary of Conditions for Saturation Binding Experimenta

Source of receptor Rat uterine cytosol 

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

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

Concentration of receptor Sufficient to bind approximately 25 

to 35% of radioligand at 0.03 nM

Temperature 4oC 

Incubation time 16 to 20 hours 

Composition of assay buffer Tris 10 mM (pH 7.4) 

EDTA 1.5 mM 

Glycerol 10% 

Phenylmethylsulfonyl fluoride (PMSF) 1 mM 

DTT 1 mM 

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

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

H]-17β-estradiol 

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

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

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

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 (isolated July 20, 2011), the optimal protein concentration was 

determined by testing serial amounts of protein per tube, using 0.03 nM radiolabeled 

estradiol, until a concentration was reached that bound approximately 25 to 35% of the total 

radioactivity added. The final protein concentrations were 320 μg, 192 μg and 160 μg per 

assay tube for the first, second and third saturation binding experiments, respectively (Note: 

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

(conducted on August 5, 6, and 7, 2011, respectively). Each run included three replicates of 

each test substance at each concentration, resulting in the 72 samples depicted in Table 2. 

TABLE 2. Saturation Binding Experiment Runa

Total bindingb Non-specific 

bindingc

Radioligand aloned

 Assay Components 

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

350 μL 300 μL --- TEDG+PMSF buffer 

50 μL 50 μL 50 μL [

3

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

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

100x each respective labeled concentration)f

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

concentration) 

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

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

b Total binding = [3

H]-17β-estradiol bound to ER 

c Non-specific binding = [3

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

d Total [3

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

e Final concentrations of [3

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

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

Page 110 of 292
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Tubes were incubated with gentle vortexing for 17.5-19 hours at approximately 4C. To 

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

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

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

vortexed, centrifuged for 10 minutes at 1000 x g, and the supernatant decanted and 

discarded. After the final centrifugation, ethanol (1.5 mL) was added to the HAP pellet 

remaining in each tube to extract the [3

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

centrifugation for 10 minutes at 1000 x g. Aliquots (1 mL) of supernatant were 

radioassayed by scintillation counting. The temperature was maintained at approximately 

4oC throughout the assay prior to extraction with ethanol. 

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

competitive binding experiment is 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 mM 

Temperature 4 ± 2 C 

Incubation time 16-20 hours 

Composition of assay buffer Tris 10 mM (pH 7.4) 

EDTA 1.5 mM 

Glycerol 10% 

Protease Inhibitor (PI) w/PMSF 

(unspecified)c 0.5% 

DTT 1 mM 

a Data were obtained from pages 17-18 of the study report. 

b The protein concentration was 80.0 μg/assay tube (pp. 61, 64, and 67). 

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

described as containing PMSF). 

No precipitation of test chemical was detected by visual observation in 100% DMSO at any 

of the concentrations tested. On the day of the assay, the specific activity of the stock 

solution [3

H]-17β-estradiol was adjusted for decay over time (adjusted specific activity of 

128.2 Ci/mmol for Run 1; 127.8 Ci/mmol for Run 2, and 126.3 Ci/mmol for Run 3), and 

diluted in TEDG+PI buffer to achieve a final concentration of 1 nM. The protein 

concentration used in the competitive binding experiment was 80.0 μg/assay tube, and was 

reported to be sufficient to bind 10-15% of the radioligand. 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. Each assay consisted of three independent runs on three different days. 

Each run contained three replicates at each concentration plus six replicates of the [3

H]-17βestradiol only control (total radioactivity) and triplicate replicates of the non-specific binding 

(NSB) and solvent controls at the beginning of each run, resulting in a total of 102 samples 

per run. 

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

Metalaxyl 

Positive control Negative control Reference Chemical 

19-Norethindrone Octyltriethoxysilane Non-labeled 

17β-estradiol 

Tubes 79-102 c Tubes 31-54 c Tubes 55-78 c Tubes 7-30 c

10−10 3.16 × 10−9 10−10 Solvent control d

10−9 3.16 × 10−8 10−9 10−11

10−8 10−7 10−8 10−10

10−7 3.16 × 10−7 10−7 3.16 × 10−10

10−6 10−6 10−6 10−9

10−5 3.16 × 10−6 10−5 3.16 × 10−9

10−4 3.16 × 10−5 10−4 10−8

10−3 10−4 10−3 10−7

a Data were obtained from pages 14-17, and 19-20 of the study report. 

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

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

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

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

c Each concentration of each chemical was run in triplicate, for a total of 102 tubes per run. 

d Solvent is DMSO (~4%) in tubes 7-9. 

Tubes were gently vortexed and incubated for 16-20 hours at 4 ± 2C. To separate bound 

from free estradiol, HAP slurry was added to each tube and vortexed once every 5 minutes 

for 15 minutes. Subsequently, the contents of each tube were washed three times as follows: 

TEDG buffer+PI was added, vortexed, centrifuged for 10 min at 1000 × g, and the 

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

tube to extract the bound [3H]-17β-estradiol, followed by vortexing, and centrifugation for 

10 min at 1000 × g. Aliquots of supernatant were radioassayed by liquid scintillation 

counting. The temperature was maintained at 4 ± 2 oC throughout the assay prior to 

extraction with ethanol.

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

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

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

according to the method of Motulsky and Brown (2006)1

 implemented by using the ROUT 

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

ligand depletion with the method of Swillens (1995)2

. For the competitive binding assay, 

similar methods of nonlinear regression (the Excel addition XLfit version 5.2.0.0 (IDBS, 

Guildford, Surrey, UK), linear regression model 204) 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) and competitive binding experiment (log IC50 and 

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

were calculated for the composite three runs using Microsoft Excel 2007 (v. 12.0.6557.5000; 

 

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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Microsoft Corporation, Redmond, WA), and mean and standard error were calculated for the 

composite three runs with Microsoft Excel 2010. 

1. Definitions 

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

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

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

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

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

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

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

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

concentration groups in the data is above 75%. 

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

test chemical concentration of 10−6

M and either a binding curve can be fit but 

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

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

concentration groups in the data is >50%. 

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

above. 

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

Run Classification Numerical Value 

Interactive 2 

Equivocal 1 

Not interactive 0 

Equivocal up to the limit of concentrations tested “missing” 

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

following ranges: 

Test Material Classification Numerical Range 

Interactive average ≥1.5 

Equivocal 0.5≥ average <1.5 

Not interactive average <0.5 

Equivocal up to the limit of concentrations tested “missing” 

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METALAXYL / 113501 OCSPP 890.1250/ OECD None

b. Descriptors for receptor binding: 

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

concentration of active receptor sites 

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

IC50: concentration of the test substance (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 

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

II. RESULTS 

A. SATURATION BINDING EXPERIMENT: Saturation binding experiment parameters 

are presented in Table 5. The Kd for [3H]-17β-estradiol was 0.331±0.061 nM, and the 

estimated Bmax was 74.55±3.03 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. Although the 

Scatchard plots fit straight lines to the data, the concavity observed in the data sets may 

indicate issues with ligand depletion. Confidence in these numbers is high due to the 

goodness of fit and the small variation among runs. 

TABLE 5. Saturation Binding Experiment of [3H]-17β-estradiol with Estrogen Receptor from Rat Uterine 

Cytosola

Parameter Run 1 Run 2 Run 3 Mean ± SEb

R2 (unweighted) 0.976 0.982 0.980 0.976-0.982 

Bmax (nM) 0.148 0.102 0.080 0.110±0.035 

Bmax (fmol/100 μg protein) 69.25 79.76 74.65 74.55±3.03 

Kd (nM) 0.453 0.286 0.255 0.331±0.061 

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

b The range of R2

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

R2

 Goodness of fit for curve calculated for specific binding

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

H]-17β-estradiol 

to the estrogen receptor for the three independent runs. The specific binding reached a 

plateau in each run, and non-specific binding was less than 20% of total binding. Figure 2 

contains the Scatchard plots that illustrate the binding of [3

H]-17β-estradiol to the estrogen 

receptor. 

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

Binding Experiment. 

Page 115 of 292
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FIGURE 2. Scatchard Plots of the Binding of [3H]-17β-estradiol to the Estrogen Receptor. 

Page 116 of 292
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B. COMPETITIVE BINDING EXPERIMENT: The results from the competitive binding 

experiments are presented graphically in Figures 3-5. The parameters determined from the 

competitive binding curves (IC50, log IC50, and RBA) are presented in Table 6. 

Metalaxyl exhibited >50% inhibition of binding at a concentration of 10−3 M in Run 2 

(classified as “interactive”), however, it had <25% inhibition of binding at concentrations up 

to 10−3

 M in Runs 1 and 3 (classified as “not interactive”); therefore it is classified as 

“equivocal” in this assay (with a mean value of 0.67 across the three runs; Table 7). There 

were no apparent alterations in [3H]-17β-estradiol binding at metalaxyl concentrations of 

10−10 to 10−3 M (90.8-110.8%), with the exception of Run 2 where the specific binding of 

the radiolabeled ligand declined to 85.5% at a concentration of 10−4

 M and to 46.8% at a 

concentration of 10−3

 M. The estimated log IC50 was −3.8 M for metalaxyl in Run 2, with 

an RBA of 0.0006%. No precipitation of metalaxyl was noted in the assay tubes, however, 

the study authors noted that precipitation in the assay tubes was difficult to assess visually 

due to the opaqueness of the cytosol solution. 

The three reference chemicals generally performed as expected. The estimated mean log 

IC50 was −9.0 M for 17β-estradiol and −5.6 M for the weak positive control. The mean 

RBA was 0.0344% for the weak positive control. Mean specific binding of [3H]-17βestradiol in the negative control assay was 44.6% in Run 3 at a concentration of 10−3

 M 

octyltriethoxysilane, which is lower than the tolerance limit (≥75%) for the negative control. 

The study author stated that binding of octyltriethoxysilane to [3H]-17β-estradiol at 10−3 M 

has been previously observed in other studies, although typically accompanied by 

precipitation (not observed). Excluding the highest concentration (10−3

 M) in Run 3, the 

negative control, octyltriethoxysilane, had no effect on specific binding of the [3

H]-ligand 

(90.9 to 106.4%). 

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

Parameter Run 1 Run 2 Run 3 Mean ± SE b

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

 19-norethindrone NR NR NR NA 

 Metalaxyl NR NR NR NA 

Log IC50 (M) 17β-estradiol −9.0 −9.0 −9.1 −9.0 ± 0.033 

 19-norethindrone −5.6 −5.5 −5.6 −5.6 ± 0.033 

 Metalaxyl NA −3.8 NA NA 

IC50 (M) b 17β-estradiol 1.00 × 10−9 1.00 × 10−9 7.94 × 10−10 9.31 × 10−10 (± 0.69) 

 19-norethindrone 2.51 × 10−6 3.16 × 10−6 2.51 × 10−6 2.73 × 10−6 (± 0.22) 

 Metalaxyl NA 1.58 × 10−4 NA NA 

Log RBAb 19-norethindrone −3.40 −3.50 −3.50 −3.47 ± 0.033 

 Metalaxyl NA −5.20 NA NA 

RBA (%)b 19-norethindrone 0.0398 0.0316 0.0316 0.0344 ± 0.0027 

 Metalaxyl NA 0.0006c NA NA 

a Data were obtained from pages 24-25 of the study report. 

b All values are reviewer-calculated from the log IC50 values reported in the study report. For means 

expressed in scientific notation, the SE values in parentheses are presented in the same order of 

magnitude as the mean value. 

c The study author incorrectly reported the RBA for metalaxyl in Run 2 as 0.4 on page 25 of the study 

report. 

SE Standard Error 

NA Not applicable. 

NR Not reported 

R2

 Goodness of fit 

RBA (%) Relative binding affinity

TABLE 7 Binding Classification of Metalaxyl with Estrogen Receptor a

Run 1 2 3 Mean c Binding Classification d

Classification category value b 0 2 0 0.67 Equivocal 

a Data were obtained from pages 24-25 of the study report. 

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

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

c Mean of three runs expressed to the tenths place 

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

 

Page 118 of 292
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 14 of 18 

METALAXYL / 113501 OCSPP 890.1250/ OECD None

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

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

 

Page 119 of 292
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 15 of 18 

METALAXYL / 113501 OCSPP 890.1250/ OECD None

FIGURE 4. Percentage [3H]-E2 Bound to the Estrogen Receptor in the Presence of 

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

 

Page 120 of 292
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 16 of 18 

METALAXYL / 113501 OCSPP 890.1250/ OECD None

FIGURE 5. Percentage [3H]-E2 Bound to the Estrogen Receptor in the Presence of 

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

Page 121 of 292
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 17 of 18 

METALAXYL / 113501 OCSPP 890.1250/ OECD None

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 ≤1.95 X 

Top (% binding) 94 to 111 97 to 109 X 

Bottom (% binding) −4 to 1 −2 to 5 Xc

Hill Slope (Log10(M)−1) −1.1 to −0.7 −1.7 to −0.9 Xd

Weak Positive control (19-norethindrone) fitted curve parametersb

Loge residual SD NA ≤1.59 NA 

Top (% binding) NA 98 to 100 NA 

Bottom (% binding) NA −2 to 1 NA 

Hill Slope (Log10(M)−1) NA −1.4 to −0.9 NA 

Solvent concentration

DMSO ≤10% 4% X 

Negative control (octyltriethoxysilane) does not displace more than 

25% of [3

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

concentrations 

≤25% ≤55.4 Xe

a Data were obtained from page 28-33 and 61-69 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 as they show 19-norethindrone to be an acceptable weak positive control. 

c Bottom % binding was high in Run 2 (5%) and within the tolerance limits in the other two runs.

d Hill Slope was low in Run 2 (−1.7) and within the tolerance limits in the other two runs. 

e Reviewer-calculated. The minimum specific binding of [3

H]-17β-estradiol in the negative control assays was 

44.6% in Run 3 at 10−3

 M; however, excluding this value at the highest test concentration, octyltriethoxysilane 

did not displace more than 25% of [3

H]-17β-estradiol from the ER (minimum specific binding of 90.9%).

NA Not applicable 

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

unlabeled 17β-estradiol displaced [3

H]-17β-estradiol in a manner consistent with one-site 

binding, as indicated by a descent from 90.4-103.8% to 7.4-9.4% binding over 

approximately an 81-fold increase in concentration of the test chemical (i.e., covering 

approximately 2 Log units). 

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

percent binding at this top plateau 99.8-110.8% was within 25 percentage points of the value 

for the lowest concentration of the estradiol standard 95.1-107.4%. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Metalaxyl was classified as “non-interactive” in 

the first and third independent valid runs, and was classified as “interactive” in the second 

independent valid run. Although this results in metalaxyl having a final classification of 

“equivocal,” it is important to note that the “interactive” categorization in the second valid 

independent run appears to be based on an anomaly in the data, as ER binding was >85% at 

Page 122 of 292
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 18 of 18 

METALAXYL / 113501 OCSPP 890.1250/ OECD None

all metalaxyl concentrations but 10−3 M in this run. Additionally, the test material was 

negative for binding in all other runs of the assay (including a run conducted on 01-

September-11 for which the weak positive control did not meet the assay acceptance 

criteria). Although no specific problems or issues could be identified with the second valid 

independent run to explain the inconsistency of this run with the others conducted for 

metalaxyl, it has been shown that when the competitive binding curve drops sharply over a 

single log increase in test substance concentration, as exhibited by metalaxyl in the second 

assay run, follow up Ki assays generally show that the test substance is typically not a true 

competitive inhibitor (Laws et al, 2006)3.

B. AGENCY COMMENTS: The protein concentrations used in the saturation binding runs 

varied between each run, and were approximately 3- to 6-fold greater than recommended. 

The Kd for [3

H]-17β-estradiol was 0.331 nM and the estimated Bmax was 74.55 fmol/100 μg 

protein for the prepared rat uterine cytosol. The Kd for each run was within the expected 

Guideline range of 0.03 to 1.5 nM. In the Scatchard plots, points below 20 fmol bound 

ligand /100 μg reached a plateau compatible with ligand depletion at less than 0.3 nM [3

H]-

17β-estradiol; this phenomena was correctly accounted for in the Scatchard analysis. 

For the competitive binding experiment, the estimated mean log IC50 was −9.0 M for 17βestradiol and −5.6 M for the weak positive control. The mean RBA was 0.0344% for the 

weak positive control. All performance criteria were generally met; however, 

octyltriethoxysilane displaced more than 25% of [3

H]-17β-estradiol from the ER at the 

highest concentration tested (10−3 M) in Run 3, and bottom % binding and Hill Slope were 

slightly outside of the tolerance limits for 17β-estradiol in Run 2. 

Metalaxyl reduced the mean specific binding of the radiolabeled ligand in a concentrationdependent manner in one of the three runs (declining to 46.8% in Run 2); however, had 

<25% inhibition of binding at concentrations up to 10−3 M in the other two runs and is 

therefore classified as “equivocal” in this assay. 

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

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

 The protein concentrations used in the saturation binding runs varied between each run, 

and were approximately 3- to 6-fold greater than recommended. 

 Cytosol preparation (July 30, 2011) and Last Saturation Assay run (August 7, 2011) 

occurred more than 90 days before last Competitive Assay run (November 17, 2011). 

 Performance criteria were not met for octyltriethoxysilane in Run 3 at 10−3

 M. 

 

3

 Laws, SC, Yavanhxay S, Cooper RL, Eldridge JC. Nature of the binding interaction for 50 structurally diverse 

chemicals with rat estrogen receptors. Toxicol Sci. 2006 Nov;94(1):46-56. 

Page 123 of 292
DATA EVALUATION RECORD 

METALAXYL 

Study Type: OCSPP 890.1300, Estrogen Receptor Transcriptional Activation 

EPA Contract No. EP10H001452 

Task Assignment No. 3-06-2012 

(Revisions to MRID 48617603 to include laboratory proficiency data; 

Main study was originally reviewed under TA 2-46-2012) 

Prepared for 

Health Effects Division 

Office of Pesticide Programs 

U.S. Environmental Protection Agency 

2777 South Crystal Drive 

Arlington, VA 22202 

Prepared by 

CSS-Dynamac Corporation 

1910 Sedwick Road, 

Building 100, Suite B 

Durham, NC 27713 

Primary Reviewer: Signature:

Michelle Sharpe-Kass, M.S. Date: 6/11/2012 

Secondary Reviewer: Signature:

Michael E. Viana, Ph.D., D.A.B.T. Date: 7/16/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 7/24/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 7/24/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 124 of 292
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 125 of 292
METALAXYL I 113501 

Estrogen Receptor Transcriptional Activation (2011) I Page 1 of 10 

OCSPP 890.1300/ OECD 455 

Primary Reviewer: Sheila Healy, Ph.D. 

Health Effects Division 

Secondary Reviewer: Minerva Mercado Ph.D. DABT 

Health Effects Division 

Sign~{~e· _____..~i.--;::.-=..,__ __ 

Date: -----=~~~-=--;;r--

Template version 08/2011 

DATA EVALUATION RECORD 

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

OCSPP 890.1300; OECD 455. 

PC CODE: 113501 DP BARCODE: D399227 

TXR#: 0054219 CAS No.: 57837-19-1 

TEST MATERIAL (PURITY): Metalaxyl (100.4% (first analysis); 98.2% (second analysis)) 

SYNONYMS: Methyl N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alaninate 

CITATION: Willoughby, J.A. (2011) Metalaxyl: Estrogen Receptor Transcriptional 

Activation (Human Cell Line (HeLa-9903)) Final Report. CeeTox, Inc, 

Kalamazoo, MI. Laboratory Project ID: 9143V-100326ERTA, December 7, 

2011. MRID 48617603. Unpublished. 

SPONSOR: 

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

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

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

MRID 48843501. Unpublished. 

Metalaxyl EDSP Consortium, 20 Ambriance Drive, Burr Ridge, IL 

TEST ORDER#: CON-113501-18 

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

48617603), hERa-HeLa-9903 cells cultured in vitro were exposed to metalaxyl (:::::98.2% a.i., Lot 

#20100540) at logarithmically increasing concentrations from 10-4 to 10-11 Min0.1% DMSO in 

two independent runs. The experiments were performed using 96-well plates and each metalaxyl 

concentration was tested in 6 wells/plate in each run. Cells were exposed to the test agent for 

24±2 hours to induce reporter (luciferase) gene products. Luciferase expression in response to 

activation of the estrogen receptor by metalaxyl was measured using a proprietary luciferase 

activity assay. 

Metalaxyl was tested up to the limit of cytotoxicity 10-4 M. Cytotoxicity of 17 amethyltestosterone in the first run prevented observation of its log PCso. The mean RPCMax for 

metalaxyl was 0.5% for the first run and 1.1 % for the second, and the associated PCMax was 10-5 

M for the first run and 10-7 M for the second run. 

Page 126 of 292
Estrogen Receptor Transcriptional Activation (2011) / Page 2 of 10 

METALAXYL / 113501 OCSPP 890.1300/ OECD 455 

In the main assays, the responsiveness of the cells to the very weak positive control 17αmethyltestosterone 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, metalaxyl 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 16.5-52.8% PC. Metalaxyl was only able to reach a maximum of 0.5-1.1% 

PC when tested up to the highest concentration possible based on cytotoxicity. Because the 

RPCMax < PC10 in both assay runs, metalaxyl was considered negative for estrogen receptor 

transcriptional activation in this test system. 

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

Transcriptional Activation assay (OCSPP 890.1300). 

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

statements were provided. 

 

Page 127 of 292
Estrogen Receptor Transcriptional Activation (2011) / Page 3 of 10 

METALAXYL / 113501 OCSPP 890.1300/ OECD 455 

I. MATERIALS AND METHODS

A. MATERIALS 

1. Test Facility: CeeTox, Inc. 

Location: Kalamazoo, MI

Study Director: J.A. Willoughby

Other Personnel: K. Rutherford (Director Laboratory Operations); D. Blakeman (Senior Scientist); C. 

Haines (Scientist); S. McColley (Scientist); B. Wallace (Lead Cell Culture Scientist); 

and C. Toole (Director of Project Management)

Study Period: May 26, 2011 to December 7, 2011 

2. Test Substance: Metalaxyl 

 Description: White solid, MW 279.33 

 Source (Cat#): Nation's Ag. L.L.C (not reported) 

 Lot/Batch # (Exp. Date): 20100540 (July 26, 2011 - first analysis) (June 13 2012 - second analysis) 

 Purity: 100.4% (first analysis) 98.2% (second analysis) 

 Solubility: Not Reported (NR) 

 Volatility & Stability: NR 

 Storage conditions: Ambient 

 CAS #: 57837-19-1 

 Structure: 

3CH

N

O

CH3

O

3CH CH3

O O

3CH

3. 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 

4. Vehicle(s) 

Solvent: DMSO 

Solvent control (final conc.): 0.1% 

Page 128 of 292
Estrogen Receptor Transcriptional Activation (2011) / Page 4 of 10 

METALAXYL / 113501 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 using DNA fluorochrome analysis. 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 37oC. Upon reaching 75-90% confluence, cells were subcultured at least 

twice prior to exposure to the test material. The cells used in this study were Passage 35 for 

the rangefinder, Passage 17 for the first run and new cells were brought up from 

cryopreservation and used at passage 23 for the second run prior to seeding into plates. 

2. Transcriptional Activation Assays: For each test, cells were plated at a density of 1104

cells/100 μL medium/well in a 96-well plate and allowed to attach for at least 3 hours. The 

growth media was replaced with media containing serial log dilutions of metalaxyl in 

DMSO (0.1% final concentration). Cells were incubated for 24±2 hours at 37±1°C. 

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 a description was not provided. Runs 

1 and 2 for the main assay were conducted on June 14 and September 8, 2011, respectively. 

3. Preliminary Test: A preliminary test evaluating concentrations ranging from 10−3 to 

10−10 M was conducted to determine the appropriate concentration range and to determine 

concentrations resulting in insolubility and/or cytotoxicity. Cytotoxicity was determined by 

PI uptake. An endoscope was used to assess precipitation in each sample.

4. Proficiency Chemicals: Responsiveness of the test system was evaluated on March 5, 

April 12 and April 28, 2011(MRID 48843501), using cells at passage 15, 25 and 28, 

respectively. Based on dates and passage number, it is unlikely these cells are from the 

same frozen stock as the cells used in the main assay. The cells were tested using the 

following proficiency chemicals, each chemical tested in duplicate on separate days: 

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. 

Page 129 of 292
Estrogen Receptor Transcriptional Activation (2011) / Page 5 of 10 

METALAXYL / 113501 OCSPP 890.1300/ OECD 455 

5. 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 each plate in 

the current assay, along with the test chemical: 

Reference Chemical CAS No. Concentration Range Class 

17β-estradiol 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 

6. Data analysis: To obtain the relative transcriptional activity compared 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 were calculated only if a positive response was observed. Coefficients of 

variation (CV) were calculated for the luminescence data triplicates. Concentrations 

showing >20% cytotoxicity or evidence of insolubility were excluded from analyses. 

7. Definitions 

EC50 = concentration of agonist that induces a response halfway between the baseline 

(bottom) and maximum (top) response 

PC10 = concentration of a test chemical at which the response is 10% of the response 

induced by the positive control (E2 at 1 nM) in each plate 

PC50 = concentration of a test chemical at which the response is 50% of the response 

induced by the positive control (E2 at 1 nM) in each plate 

RPCMax = maximum level of response induced by a test chemical, expressed as a percentage 

of the response induced by the positive control (1 nM E2) on the same plate 

PCMax = concentration of a test chemical inducing the RPCMax

II. RESULTS 

A. PRELIMINARY TEST: In order to identify a suitable top concentration for use in the 

transcriptional activation assays, preliminary cytotoxicity and precipitation assays were 

conducted. Cytotoxicity was observed at 10−3

 M of metalaxyl (Table 1). Based on this 

result, metalaxyl concentrations of 10−4

 to 10−11 M were selected for the main assays. 

Page 130 of 292
Estrogen Receptor Transcriptional Activation (2011) / Page 6 of 10 

METALAXYL / 113501 OCSPP 890.1300/ OECD 455 

TABLE 1. Preliminary Test for Solubility, Cytotoxicity, and Concentration-Selection for Metalaxyla

Concentration (M) % Viability Comments 

10−3 78 Cytotoxic 

10−4 90 

10−5 90 

10−6 91 

10−7 90 

10−8 93 

10−9 92 

10−10 93 

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 conducted in duplicate on different days for each chemical. The reported 

responses are summarized in Table 2a.

TABLE 2a. 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 

 From MRID 48843501 

 NA = not applicable. The chemical was not tested at this time. 

2. Reference Chemicals: Values derived from the concentration response curve (e.g., log 

PC50, log PC10, log EC50, Hill slope) for the four concurrently run reference materials are 

included in Table 3. The acceptance criteria were met for all reference chemicals with the 

exception of 17α-methyltestosterone in the first run, where the maximum response was 

16.5%. Additionally, cytotoxicity was noted at 10−5

 M of 17α-methyltestosterone and 

10−4

 M of corticosterone in the first run, and in the second run 17β-estradiol (1 nM) and 

17α-estradiol (100 nM) demonstrated responsiveness well above the PC (143.8% and 

123.6% RTA, respectively). 

Page 131 of 292
Estrogen Receptor Transcriptional Activation (2011) / Page 7 of 10 

METALAXYL / 113501 OCSPP 890.1300/ OECD 455 

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 −10.2 −10.6 X 

 Log PC10 <−11 −11.7 −11.5 X 

 Log EC50 −11.3 to −10.1 −10.3 −10.5 X 

 Hill Slope 0.7 to 1.5 0.8 1.3 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 −9.8 −9.7 X 

 Log EC50 −9.6 to −8.4 −8.7 −8.7 X 

 Hill Slope 0.9 to 2.0 1.2 1.2 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 −5.1 Run 1 

 Log PC10 −8.0 to −6.2 −6.6 −6.2 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 page 12 and 23 of the study report. 

C. DEFINITIVE ASSAY 

1. Vehicle and Positive Controls: Data for the vehicle and positive controls are included in 

Table 4. The mean TA values for the vehicle control were 16658 and 22954 for Runs 1 and 

2, respectively, and the mean TA values for the positive control were 1151283 and 619650 

for Runs 1 and 2, respectively. The induction for the positive control ranged from 27- to 69-

fold. The mean normalized values for the positive control were 1134625 and 596696 for 

Runs 1 and 2, respectively. The PC50 (50% of the maximum response) for E2 in this assay 

were 567313 and 298348 for Run 1 and 2, respectively, and the PC10 (10% of the maximum 

response) were 113463 and 59670 for Run 1 and 2, respectively. Both PC10 values are 

greater than respective vehicle control values + 2SD.

 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 16658 4730 1151283 353650 69 1134625 353650 

2 22954 4536 619650 55512 27 596696 55512 

a Data were calculated by the reviewer 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 

metalaxyl during the two assay runs is presented in Table 5. The concentration-response 

curves depicting fold induction of relative transcriptional activation are presented in 

Figure 1. The mean RPCMax was 0.5% for the first run and 1.1% for the second, and the 

associated PCMax was 10−5 M for the first run and 10−7 M for the second. Because the 

Page 132 of 292
Estrogen Receptor Transcriptional Activation (2011) / Page 8 of 10 

METALAXYL / 113501 OCSPP 890.1300/ OECD 455 

RPCMax < PC10 in both runs, metalaxyl was considered negative for estrogen receptor 

transcriptional activation in this test system. 

TABLE 5. Relative Transcriptional Activation (RTA) of Metalaxyla

Parameter RTA (mean ± SD); % of Positive Control (PC) 

Run 1 Run 3 

Conc. (M) Mean SD Mean SD 

10−4 0.4 0.2 −0.3 1.1 

10−5 0.5 0.4 0.9 1.0 

10−6 0.4 0.5 0.4 0.8 

10−7 0.5 0.6 1.1 0.9 

10−8 −0.2 0.2 0.1 1.3 

10−9 −0.2 0.4 0.8 1.2 

10−10 −0.1 0.2 −0.2 0.9 

10−11 −0.1 0.3 0.4 1.1 

Log EC50b NA NA 

Hill Slopeb NA NA 

RPCMax 0.5 1.1 

PCMax 10−5 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 (2011) / Page 9 of 10 

METALAXYL / 113501 OCSPP 890.1300/ OECD 455 

FIGURE 1. Fold Induction of Relative Transcription Activation (RTA) of Metalaxyl 

Compared to the Positive Control. 

3. Performance Criteria: The acceptance criteria were met for all reference chemicals with 

the exception of 17α-methyltestosterone in the first run, where the maximum response was 

16.5%. For 17α-methyltestosterone, the mean RPCMax was 16.5% for the first run and 

52.8% for the second run. Additionally, cytotoxicity was noted at 10−5

 M of 

17α-methyltestosterone and 10−4 M of corticosterone in the first run, and in the second run 

17β-estradiol and 17α-estradiol demonstrated responsiveness well above the PC 

(123.6-143.8%). Cytotoxicity should not be observed at any level for the reference 

chemicals, and the maximum responsiveness should be observed in the positive control, 

1 nM 17β-estradiol. Due to this, the ability of this test system to observe transcriptional 

changes in response to chemical exposure was not considered valid. 

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Estrogen Receptor Transcriptional Activation (2011) / Page 10 of 10 

METALAXYL / 113501 OCSPP 890.1300/ OECD 455 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: The suitable top concentration of metalaxyl for 

use in the transcriptional activation assays was 10−4 M. There was no significant 

cytotoxicity at any of the concentrations tested in both independent runs. In two 

independent runs of the transcriptional activation assay, metalaxyl did not result in an 

increase in luciferase activity at any other concentrations tested; metalaxyl is not an agonist 

of the human estrogen receptor alpha (hERα) in the HeLa-9903 model system. 

B. AGENCY COMMENTS: Metalaxyl was tested up to the limit of cytotoxicity 10−4 M. 

The mean RPCMax was 0.5% for the first run and 1.1% for the second, and the associated 

PCMax was 10−5

 M for the first run and 10−7 M for the second. Because the RPCMax < PC10

in both runs, metalaxyl was considered negative for estrogen receptor transcriptional 

activation in this test system. However cytotoxicity was observed at higher concentrations 

of 17α-methyltestosterone in the first run, preventing observation of its log PC50. 

Although the conditions of this assay were not optimal to detect very weak activity, 

metalaxyl 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 16.5-52.8% PC. Metalaxyl was only able to reach a maximum of 0.5-1.1% PC when 

tested up to the highest concentration possible based on cytotoxicity. Because the RPCMax < 

PC10 in both assay runs, metalaxyl was considered negative for estrogen receptor 

transcriptional activation in this test system. 

C. STUDY DEFICIENCIES: The following deficiencies were noted: 

 In both assays, cytotoxicity was observed in cells treated with corticosterone and 

17α-methyltestosterone. 

 Certificates of Analyses were not provided for the reference chemicals. 

 The source of the fetal bovine serum was not provided. 

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

EPA MRID Number 48685802 

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Data Requirement: EPA DP Barcode 401220 

OECD Data Point 229 

EPA MRID 48685802 

EPA Guideline 890.1350, Fish Short-Term Reproduction Assay 

Test material: Metalaxyl Purity: 100.4% 

Common name 

Chemical name: IUPAC: Methyl N-(methoxyacetyl)-N-2,6-xylyl)-DL-alaninate 

 CAS name: Methyl 2-{[2,6-dimethylphenyl)methoxyacetyl]amino}propionate 

 CAS No.: 57837-19-1 

 Synonyms: Metalaxyl TC. 

 EPA PC Code: 113501 

Primary Reviewer: Joan Gaidos Signature: 

Senior Scientist, Cambridge Environmental, Inc. Date: 12/6/2012 

Secondary Reviewer: Teri S. Myers Signature: 

Senior Scientist, Cambridge Environmental, Inc Date: 12/20/2012 

Primary and Final Additional Reviewer: Robin Sternberg Signature: 

Wildlife Biologist, USEPA/OCSPP/OPP/EFED/ERB1 Date: 02/27/2014, 05/27/2015

Additional Reviewer: Anita Ullagaddi Signature: No longer with EPA 

Biologist, USEPA/OCSPP/OPP/EFED/ERB1 Date: 

Date Evaluation Completed: 05/27/2015 

 

Digitally signed by ROBIN STERNBERG 

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

ou=USEPA, ou=Staff, cn=ROBIN 

STERNBERG, dnQualifier=0000039126 

Date: 2015.06.01 16:36:07 -04'00'

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

promelas). Performed by Smithers Viscient, Wareham, Massachusetts, Laboratory Project No. 13694.6143. 

Submitted by Metalaxyl EDSP Consortium, Burr Ridge, Illinois. Completion date March 2, 2012. 

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

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

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

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

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

should not be considered in isolation but rather should be considered in the context of other assays in the 

battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has 

the potential to interact with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay 

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

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

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

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

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EXECUTIVE SUMMARY:

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

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

group; 21 weeks old) were exposed to metalaxyl (100.4% purity) at nominal concentrations of 0 (negative 

control), 1.0, 10.0, and 100 mg a.i./L; mean-measured concentrations were <LOQ (<0.066, negative control), 

1.1, 10.0, and 104 mg a.i./L. The test system was maintained at 25 to 26o

C and a pH of 6.9 to 7.6. 

Adult male and female survival was unaffected by treatment with metalaxyl; survival in males was 100, 88, 

100, and 88% and survival in females was 100, 100, 100, and 94% in the negative control, low, mid, and 

high treatment groups, respectively. Male body weight exhibited a significant (p<0.05), generally concentrationdependent reduction of 7 to 16% across all treatment levels compared to the negative control. There were no 

notable differences in secondary sex characteristics or clinical signs for any treatment group compared to the 

negative control. 

In the negative control, fecundity was 19 eggs/female/day/replicate, and fertilization success averaged 96%. 

Fecundity and fertility were not significantly affected at the low and mid treatment levels. However, there were 

no eggs available for assessment at the high treatment level. 

At the high treatment level, there was a 96% decrease in female plasma vitellogenin (VTG) concentrations 

that was not statistically significant (p=0.092). However upon visual observation of the data, this reduction in 

VTG was considered to be biologically significant. Female gonado-somatic index (GSI) was significantly reduced 

(p<0.05) by 23% at the high treatment level compared to the negative control. Male tubercle score was 

significantly reduced (p<0.05) at the high treatment level compared to the negative control. 

There were no notable differences in gonadal stage. In male fish, there was an increased incidence of 

increased percentage of spermatogonia at the mid and high treatment levels and asynchronous gonad 

development at the high treatment level. In female fish, there was decreased yolk synthesis at the high 

treatment level. There was an increase in the incidence of atretic oocytes and granulomatous inflammation in 

females in the high treatment group. However, given that these findings were occasionally coincident with the 

presence of protozoal spores and were also observed in negative control females (at a lower incident rate), 

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these lesions were not considered to be treatment related. Plasma testosterone and 17ß-estradiol were not 

measured in this study. 

All of the performance criteria were met with the exception of the following: the dissolved oxygen concentrations 

fell to ca. 42% of saturation (3.3 mg/L) on Day 6 which is less than the recommended ≥4.9 mg/L (≥60% 

saturation); however, gentle aeration was added such that dissolved oxygen concentrations remained above 

60% of saturation for the remainder of the test. This deviation did not impact the interpretation of the 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: 21 weeks 

Mean body weight at test initiation (if measured): Male 3.4 g; Female 1.6 g 

Mean length at test initiation (if measured): Not reported. 

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

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

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

1.1 No No No No No No No No No No NA NA NA NA 

10 No No No No No No Yes No No No NA NA NA NA 

104 Yes3 Yes3 Yes No No Yes Yes Yes No No NA NA NA NA 

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

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

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

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

3 No eggs were produced at the highest test concentration. Therefore fecundity was 0, and there were no fertility data to analyze. 

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I. MATERIALS AND METHODS

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

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

following deviations were noted: 

 

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

2. The total organic carbon, unionized ammonia, and residual chlorine in the test water were not reported. 

The OCSPP 890.1350 performance criteria establish maximum levels for these values, and it is 

unclear if the maximum recommendations were exceeded.

3. Dissolved oxygen concentrations in the test solutions remained at or above 60% of saturation (4.9 

mg/L) for most of the exposure period. However, dissolved oxygen concentrations fell to ca. 42% of 

saturation (3.3 mg/L) on Day 6, and gentle aeration was added. Dissolved oxygen concentrations 

remained above 60% of saturation for the remainder of the test. OCSPP 890.1350 guideline 

requirements recommend dissolved oxygen (DO) ≥4.9 mg/L (≥60% air saturation).

 

 These deviations do not impact on the interpretation of the study. 

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

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

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

exceptions: routine dilution water and food contaminant screening analyses 

were conducted following standard validated methods, this exception has no 

impact on the study results. 

A. TEST MATERIAL: Metalaxyl; CAS# 57837-19-1

Description: Not reported

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

Kow, vapor pressure of test compound, expiration date.

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Lot No./Batch No. : 20100540 

Purity: 100.4% (tested as 100%) 

Impurities: None reported

Stability of Compound: Analytical verification of the test material from Days 0, 7, 14, and 21 yielded 

recoveries of 100% to 110% of nominal concentrations. Stock solution 

(FETAX) fortified with metalaxyl at concentrations of 0.005, 1.0, and 100 

mg a.i./L yielded recoveries of 94.3 to 105%. There was no pattern of 

decline during the study period, indicating the test material was stable under 

the test conditions. 

Storage Conditions of 

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

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B. Test organism: 

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

Species common name: Fathead Minnow EPA recommends fathead minnow 

(Pimephales promelas). Species scientific name: Pimephales promelas

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

Yes EPA recommends that fish be from a single 

laboratory stock.

Were acclimation conditions same as definitive test?

Yes EPA recommends that fish be acclimated 

under water quality and illumination 

conditions that are similar to the definitive 

test. 

Acclimation period: 16 days (pre-exposure) EPA recommends a minimum two-week 

acclimation period. Note that the 

acclimation period is different from the 

subsequent, in situ pre-exposure phase. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on health: No mortalities were reported during the 

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

EPA recommends that mortality during the 7 

days prior to the pre-exposure phase be 

less than 5% of the culture population. If 

mortality during these 7 days is greater than 

10%, EPA recommends that the fish be 

rejected. If mortality is between 5-10%, 

EPA recommends that fish be held another 

7 days. If mortalities greater than 5% occur 

during this extended acclimation period, 

EPA recommends that the fish not be used. 

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

brine shrimp twice per day to promote 

active reproduction and maintain body 

condition.

Source of food: Not reported Representative samples of the food source 

were periodically analyzed for pesticides, PCBs, and toxic metals by GeoLabs, Inc. and analyte levels were below LOC specified by ASTM (2005).

Frequency of feeding: 2 times/day Details on feeding: None 

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Table 3: Fish Selection and Pre-Exposure Performance.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Age at test initiation: Ca. 21 weeks EPA recommends reproductively mature 

(sexually dimorphic) fish, 4.5 - 6 months 

old.

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

3.4 g Based on 20 males from the test 

population used to stock aquaria for pre-exposure phase 

EPA recommends that a subsample of fish 

be weighed before the test to estimate the 

mean weight for each sex. It is 

recommended that the individual weight of 

each fish selected for the test be within 

±20% of the estimated mean for each sex.

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

3.0 to 4.0 Individual weights within ±20% of the 

estimated mean

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

1.6 g Based on 20 females from the test 

population used to stock aquaria for pre-exposure phase. 

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

1.4 to 1.8 Individual weights within ±20% of the 

estimated mean

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

Not reported 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

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

Not reported 

Duration of pre-exposure phase: 16 days EPA recommends a minimum of 14 days. Were pre-exposure conditions identical to the definitive test? 

Yes EPA recommends that pre-exposure 

conditions, including temperature, 

photoperiod, feeding, etc., be identical to 

definitive test conditions. 

Number of pre-exposure tanks: 36 Extra replicates were established to 

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

EPA recommends that additional tanks set 

up at the beginning of pre-exposure will 

ensure that sufficient replicates with the 

correct sex ratio are available for the 

definitive test. 

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Pre-exposure fecundity: >15 eggs/female/ 

reproductive day/replicate

EPA recommends that pre-exposure 

fecundity in each replicate (tank) selected 

for use in the definitive test be at least 15 

eggs/female/reproductive day/replicate 

during the 7 days prior to the definitive test. 

Number of spawns during preexposure: >two times in 7 days EPA recommends that spawning occur at least twice in the 7 days prior to the 

definitive test. 

Details on pre-exposure: None 

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

Corrections and Clarifications document1 , 

the use of a static renewal system is not 

recommended for this assay.

Type of flow-through dilution system:

Intermittent-flow proportional diluter

Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

Flow-through rate: Ca. 45 mL/minute or 2.7 

L/hr; 6.5 volume 

replacements/day 

Calculated by the reviewer based on 0.5 L/cycle, 130 cycles/24 hrs and 60 minutes/hr

Recommended flow-through rate is 45 

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

volume exchanges per day.

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

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

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on toxicant mixing for flow-through systems: 

The mixing chamber of the diluter was positioned over a magnetic stir plate and mixed with a magnetic stir bar. The continuous stirring aided the solubilization of the test substance with the dilution water.Flow-splitting chambers were used to promote further mixing and to equally distribute the test solutions through glass delivery tubes to 4 replicate test vessels. 

Flow splitting accuracy not reported. 

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

recommended but not required; 2) 

Aeration is not recommended for mixing; 

3) A demonstration that the test solution 

is completely mixed before introduced 

into the test system is recommended; 4) 

The recommended flow splitting accuracy 

is within 10%.

Aeration? Yes On test day 6, oxygen levels fell to 42% 

of saturation. Aeration was added and the dissolved oxygen levels increased to >60% of saturation within a few hours. 

EPA recommends aeration if dissolved 

oxygen reaches <4.9 mg/L (<60% 

saturation).

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Source of dilution water: Well water Aerated prior to use EPA recommends natural or reconstituted 

water; it is recommended that natural 

water be sterilized with UV and tested for 

pesticides, heavy metals, and other 

possible contaminants. OECD accepts 

any water in which the test species show 

control survival at least as good as 

indicated in the test guideline.

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

Yes 

Test vessel type/materials: Glass aquaria with 

silicone adhesive

EPA and OECD recommend that watercontact portions of the system not 

compromise the study (e.g., all glass 

vessels or glass vessels with stainless 

steel frames are acceptable examples).

Test vessel size: 20 L; 20 cm wide x 39 

cm long x 25 cm high; solution depth 13 cm

EPA recommends the use of 18 L test 

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Fill volume: 10 L EPA recommends 10 L solution per tank. Spawning substrate material: Aged PVC pipe EPA recommends that each tank contain 

three semi-circular spawning substrates, 

e.g., aged PVC pipe, 10 - 20 cm in 

length, split lengthwise.

Spawning substrate size: 4 inch diameter PVC cut 

lengthwise in half (arches) and into 7 cm sections 

Additional details on exposure system: 

None 

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

Parameter Minimum Maximum Mean1 Measurement Interval Guideline Recommendations 

Temperature (°C) 25 26 25.5 

Continuously (upper (control replicate B) 

and lower (control replicate D) water

baths). Weekly (each concentration and 

control aquarium at exposure initiation and 

subsequently in alternating replicates) 

EPA recommends temperature 25±1oC; 

inter-replicate and inter-treatment 

differentials should not exceed 1oC.

pH 6.9 7.6 7.25 EPA recommends pH 6.5 to 9.0.

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Parameter Minimum Maximum Mean1 Measurement Interval Guideline Recommendations 

Dissolved oxygen (mg/L)

3.3 (42%) 8.3 (100%) 

5.8 

(71%) 

Weekly (each concentration and control 

aquarium at exposure initiation and 

subsequently in alternating replicates) 

EPA recommends dissolved oxygen (DO) 

>4.9 mg/L (>60% air saturation)

Total alkalinity (mg/L as CaCO3) 

22 28 25 Weekly 

(one replicate of one treatment level and 

the control alternating between treatment 

levels and replicate aquaria) 

EPA recommends total alkalinity >20 

mg/L as CaCO3.

Hardness (mg/L as CaCO3) 

52 64 58

Total organic carbon (mg/L) 0.21 (July) 0.33 (June) 

Not 

reported 

Twice (June and July), dilution water EPA recommends that total organic carbon 

in dilution water be <2 mg/L. 

Unionized ammonia (Pg/L) 

Not 

reported 

Not reported 

Not 

reported 

Not reported 

EPA recommends that unionized ammonia 

in the dilution water be <1 Pg/L. 

Residual chlorine (Pg/L) 

Not 

reported 

Not reported 

Not 

reported 

Not reported 

EPA recommends that residual chlorine in 

dilution water be <10 Pg/L. 

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Parameter Minimum Maximum Mean1 Measurement Interval Guideline Recommendations 

Conductivity (Pmhos/cm) 

280 360 320

Weekly (each concentration and control 

aquarium at exposure initiation and 

subsequently in alternating replicates)

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 Calculated by reviewer as mean of minimum and maximum values.

D. Study Design and Additional Experimental ConditionsTable 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? 

96-hr LC50 range 

finding study

Nominal test concentrations of 6.3, 13, 25, 50 and 100 mg/L

EPA recommends that the highest test 

concentration be selected based on toxicity 

data for other fish studies or species, if 

available. Otherwise, either the solubility 

limit of the test compound or 100 mg/L 

(whichever is lower) is appropriate.

Species: Pimephales promelasLife stage: Approximately 

the same age and size as those used in the definitive exposure 

EPA recommends that range-finding tests 

be performed with fish of similar age and 

size to those that would be utilized in the 

test.

Test duration: 96-hr EPA recommends a 96-hour exposure. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Additional details: One test vessel, 

containing two males and four females, was established for each concentration and the control. 

At test termination (96 hours), no mortality or adverse effects were observed among fish exposed to any of the treatment levels tested or the control. The LC50 value for this exposure was empirically estimated to be >100 mg/L for combined survival. 

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-hr LC50 value was estimated to be >100 mg/L. 

EPA recommends that the highest test 

concentration is either the solubility limit of 

the test compound, 100 mg/L, or 

demonstrates adequate evidence of 

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

whichever concentration is lowest.

Reference study citation (if applicable):

NA 

Separation of test concentrations:

0.1 (ten-fold): 0 (negative control), 1.0, 10, and 100 mg 

a.i./L 

EPA suggests that a concentration 

separation of between 0.33 (or threefold) and 0.1 (or ten-fold) is scientifically 

acceptable1 . 

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

concentrations and a control, plus solvent 

control if appropriate.

Are nominal concentrations adjusted for purity? 

Yes 

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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): 0.053-0.066 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 Samples were collected from two 

alternating replicates (A, C and B, D, respectively) on each sampling day.

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.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Was the randomized complete block design used?

Yes EPA recommends that all fish be 

randomly assigned to tanks during preexposure. 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.

Number of replicates per test item treatment level: 

4 EPA recommends 4 replicates.

Number of male fish per replicate at test initiation: 

2 EPA recommends 2 males per replicate. 

Number of female fish per replicate at test initiation: 

4 EPA recommends 4 females per replicate. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

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

NA EPA recommends that the solvent not 

exceed 0.02 ml/L3. OECD recommends 

that solvent have no effect on survival nor 

produce any other adverse effects and 

that concentration not be greater than 0.1 

ml/L4.

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

NA 

Photoperiod: 16 hrs light: 

8 hrs dark 

EPA recommends photoperiod 16:8 

(light:dark).

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 

Light intensity at water’s surface: 

720-1100 lux Fluorescent bulbs EPA recommends light intensity 540 – 

1080 lux (at water’s surface).

Additional details: Following overnight 

mixing with an overhead mixer, the stock solutions were observed to be clear and colorless with no visible undissolved test substance following preparation. 

Throughout the study period, all exposure solutions were observed to be clear and colorless. No undissolved test substance was observed in the diluter system.

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

Treatment ID 

Nominal 

Concentration 

(mg a.i./L) 

Mean Measured 

Concentration

(mg a.i./L) 

Mean CV (%) Details or Remarks Guideline Recommendations

Negative Control 0 <LOQ NA EPA recommends that test item 

concentrations be maintained at a 

coefficient of variation (CV) ≤20%.

Treatment 1 1.0 1.1 6.6 

Treatment 2 10 10 5.1 Treatment 3 100 1041 4.6 

Abbreviations: CV Coefficient of variation. LOQ=0.066 mg a.i./L. 1 The study author reported a mean measured concentration of 100 mg a.i./L; however, this was not consistent with the data provided, and the 

reviewer’s mean-measured concentration was used. 

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E. Observations

 Biological Endpoints: Survival, fecundity, fertilization success, and clinical signs were observed 

daily. At test termination (Day 21), secondary sex characterization (body 

color, pattern, body shape), body weight, length, tubercle score, gonadal 

staging and histopathology, and plasma vitellogenin were evaluated. 

 Were raw (individual) data provided? Yes

 

EPA recommends that observations of survival, fecundity, fertilization success, secondary sex characteristics, 

and other clinical signs occur at least daily. At test termination (Day 21), additional observations include body 

weight and length, nuptial tubercle score, gonadal staging and histopathology, plasma VTG, and plasma sex 

steroids (testosterone and 17β-estradiol, if measured). GSI is calculated using a ratio of gonad weight to 

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

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

abnormal behavior. 

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II. RESULTS AND DISCUSSION A. Results 

Male survival values were 100, 88, 100 and 88% and female survival values were 100, 100, 100, and 94% in the mean-measured 0 (negative 

control), 1.1, 10, and 104 mg a.i./L treatment levels, respectively (Table 9). 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Males Females 

n1 # Surviving % Survival n1 # Surviving % Survival 

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

1.1 8 7 88 16 16 100 

10 8 8 100 16 16 100 

104 8 7 88 16 15 94 

Abbreviations: NA Not applicable. 

LOQ=0.066 mg a.i./L. 

1 Total number of fish at test initiation. 

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Mean male body weight values were 3.13, 2.89, 2.63, and 2.623 g and female body weight values were 1.28, 1.44, 1.34 and 1.16 g in the meanmeasured 0 (control), 1.1, 10 and 104 mg a.i./L treatment levels, respectively (Table 10). Mean male body length values were 63.0, 60.1, 60.1, and 61.4 mm and female body length values were 47.8, 49.2, 49.4 and 49.0 mm in the mean-measured 0 (control), 1.1, 10 and 104 mg a.i./L treatment levels, respectively. 

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

Treatment 

 (mg a.i./L) 

[mean-measured]

Body Weight Length 

Males Females Males Females 

n 

Mean

(g) 

±SD n

Mean

(g) 

±SD n

Mean

(mm) 

±SD n

Mean

(mm) 

±SD

Negative control (<LOQ) 4 3.13 0.12 4 1.28 0.09 4 63.0 1.3 4 47.8 0.90 

1.1 4 2.89 0.20 4 1.44 0.03 4 60.1 2.3 4 49.2 1.5 

10 4 2.63 0.30 4 1.34 0.09 4 60.1 1.7 4 49.4 1.2 

104 4 2.63 0.16 4 1.16 0.12 4 61.4 1.9 4 49.0 2.2 

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

LOQ=0.066 mg a.i./L. 

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Mean fecundity values were 19, 19, 18 and 0 eggs/female/day and fertilization success was 96, 96, 97 

and 0% in the mean-measured 0 (control), 1.1, 10 and 104 mg a.i./L treatment levels, respectively (Table 

11). 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Fecundity1 Fertilization Success (%)2

Negative control (<LOQ) 19 96 

1.1 19 96 

10 18 97 

104 0 0 

Abbreviations: NA Not applicable. ND Not determined. 

LOQ=0.066 mg a.i./L. 

1 Fecundity is calculated as the number of eggs per surviving female per reproductive day per replicate. 

2

 Fertilization success (%) is calculated as the number of embryos divided by the number of eggs, 

multiplied by 100. 

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Median male tubercle scores were 31.5, 34.5, 32 and 6.25 in the mean-measured 0 (control), 1.1, 10 and 

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

tubercles. 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Males Females 

n 

Median 

Tubercle Score1 n 

Median 

Tubercle Score 

Negative control (<LOQ) 4 32 4 0 

1.1 4 35 4 0 

10 4 32 4 0 

104 4 6 4 0 

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

LOQ=0.066 mg a.i./L. 

1 Mean tubercle scores: 32, 35, 32, and 9 for the negative control and mean-measured 1.1, 10, and 104 mg a.i./L 

treatment levels, respectively.

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Mean male GSI was 1.3, 1.3, 1.3 and 1.4% and mean female GSI was 11, 11, 12 and 8.4% in the 

mean-measured 0 (control), 1.1, 10 and 104 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 1.3 0.08 4 11 0.82 

1.1 4 1.3 0.32 4 11 1.8 

10 4 1.3 0.17 4 12 1.3 

104 4 1.4 0.49 4 8.4 0.75 

Abbreviations: NA Not applicable. 

LOQ=0.066 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 2, 2, 2 and 3 and median female gonadal stage was 3, 3, 3 and 3 in the 

mean-measured 0 (control), 1.1, 10 and 104 mg a.i./L treatment levels, respectively (Table 14). 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Males Females 

n Median Stage1 n Median Stage2

Negative control (<LOQ) 4 2 4 3 

1.1 4 2 4 3 

10 4 2 4 3 

104 4 3 4 3 

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

LOQ=0.066 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=postovulatory. 

In male fish, there was an increase in incidence of increased percentage of spermatogonia at the 10 and 104 

mg a.i./L treatment levels and asynchronous gonad development in the 104 mg a.i./L treatment (Tables 15-

16). In female fish, increased oocyte atresia was present in all groups, although a larger percentage of females 

from the 104 mg a.i./L treatment group were affected. Also, there was decreased yolk synthesis in females 

at the 104 mg a.i./L treatment (Tables 17-18). Females throughout the study had minimal to mild 

granulomatous inflammation associated with oocyte atresia and/or protozoal spores. There were no significant 

findings observed in the gonadal stage. 

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Table 15: Gonadal Histopathology in Male Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[meanmeasured] 

Diagnostic Observations1

Severity 

Increased 

Proportion of 

Spermatogonia 

Presence of 

Testis-Ova 

Increased 

Testicular 

Degeneration

Duct 

Mineralization

Interstitial cell 

(Leydig) 

hypertrophy/hype

rplasia 

n Incidence n Incidence n Incidence n Incidence n Incidence 

Negative 

control 

(<LOQ)

0 8 7 NA NA 8 8 NA NA NA NA 

1 8 0 NA NA 8 0 NA NA NA NA 

2 8 0 NA NA 8 0 NA NA NA NA 

3 8 1 NA NA 8 0 NA NA NA NA 

4 8 0 NA NA 8 0 NA NA NA NA 

1.1 0 7 7 NA NA 7 7 NA NA NA NA 

1 7 0 NA NA 7 0 NA NA NA NA 

2 7 0 NA NA 7 0 NA NA NA NA 

3 7 0 NA NA 7 0 NA NA NA NA 

4 7 0 NA NA 7 0 NA NA NA NA 

10 0 9 6 NA NA 9 8 NA NA NA NA 

1 9 2 NA NA 9 1 NA NA NA NA 

2 9 1 NA NA 9 0 NA NA NA NA 

3 9 0 NA NA 9 0 NA NA NA NA 

4 9 0 NA NA 9 0 NA NA NA NA 

104 0 7 1 NA NA 7 7 NA NA NA NA 

1 7 4 NA NA 7 0 NA NA NA NA 

2 7 2 NA NA 7 0 NA NA NA NA 

3 7 0 NA NA 7 0 NA NA NA NA 

4 7 0 NA NA 7 0 NA NA NA NA 

Abbreviations: NA Not applicable. 

LOQ=0.066 mg a.i./L.

1

 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 

2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference. 

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

Treatment 

(mg a.i./L) 

[mean-measured] 

Additional Diagnostic Observations1

Severity 

Decreased 

Proportion of 

Spermatogonia 

Increased Vascular 

or Interstitial 

Proteinaceous Fluid 

Asynchronous 

Gonad 

Development 

Altered Proportions 

of Spermatocytes 

or Spermatids 

Granulomatous 

Inflammation

n Incidence n Incidence n Incidence n Incidence n Incidence 

Negative control 

(<LOQ) 

0 NA NA NA NA 8 8 NA NA NA NA 

1 NA NA NA NA 8 0 NA NA NA NA 

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

1.1 0 NA NA NA NA 7 7 NA NA NA NA 

1 NA NA NA NA 7 0 NA NA NA NA 

2 NA NA NA NA 7 0 NA NA NA NA 3 NA NA NA NA 7 0 NA NA NA NA 4 NA NA NA NA 7 0 NA NA NA NA 

10 0 NA NA NA NA 9 9 NA NA NA NA 

1 NA NA NA NA 9 0 NA NA NA NA 

2 NA NA NA NA 9 0 NA NA NA NA 3 NA NA NA NA 9 0 NA NA NA NA 4 NA NA NA NA 9 0 NA NA NA NA 

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Treatment 

(mg a.i./L) 

[mean-measured] 

Additional Diagnostic Observations1

Severity 

Decreased 

Proportion of 

Spermatogonia 

Increased Vascular 

or Interstitial 

Proteinaceous Fluid 

Asynchronous 

Gonad 

Development 

Altered Proportions 

of Spermatocytes 

or Spermatids 

Granulomatous 

Inflammation

n Incidence n Incidence n Incidence n Incidence n Incidence 

104 0 NA NA NA NA 7 2 NA NA NA NA 

1 NA NA NA NA 7 3 NA NA NA NA 

2 NA NA NA NA 7 2 NA NA NA NA 3 NA NA NA NA 7 0 NA NA NA NA 4 NA NA NA NA 7 0 NA NA NA NA 

Abbreviations: NA Not applicable. 

LOQ=0.066 mg a.i./L. 

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E of 

the test guideline for reference. 

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

Treatment 

(mg a.i./L)

[meanmeasured] 

Additional Diagnostic Observations1

Severity 

Increased Oocyte 

Atresia 

Perifollicular Cell 

Hyperplasia/ 

Hypertrophy 

Decreased Yolk 

Formation 

Aggregates of 

macrophages, 

multifocal 

n Incidence n Incidence n Incidence n Incidence 

Negative control 

(<LOQ) 

0 15 10 NA NA 15 13 NA NA 

1 15 4 NA NA 15 0 NA NA 

2 15 1 NA NA 15 2 NA NA 

3 15 0 NA NA 15 0 NA NA 

4 15 0 NA NA 15 0 NA NA 

1.1 0 16 12 NA NA 16 15 NA NA 

1 16 3 NA NA 16 1 NA NA 

2 16 1 NA NA 16 0 NA NA 

3 16 0 NA NA 16 0 NA NA 

4 16 0 NA NA 16 0 NA NA 

10 0 15 8 NA NA 15 13 NA NA 

1 15 6 NA NA 15 2 NA NA 

2 15 1 NA NA 15 0 NA NA 

3 15 0 NA NA 15 0 NA NA 

4 15 0 NA NA 15 0 NA NA 

104 0 14 1 NA NA 14 0 NA NA 

1 14 5 NA NA 14 8 NA NA 

2 14 8 NA NA 14 3 NA NA 

3 14 0 NA NA 14 2 NA NA 

4 14 0 NA NA 14 1 NA NA 

Abbreviations: NA Not applicable. 

LOQ=0.066 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 

Interstitial fibrosis 

Egg Debris in 

Oviduct 

Granulomatous 

Inflammation 

Decreased PostOvulatory Follicles 

n Incidence n Incidence n Incidence n Incidence 

Negative 

control 

(<LOQ) 

0 NA NA 15 14 15 10 NA NA 

1 NA NA 15 1 15 5 NA NA 

2 NA NA 15 0 15 0 NA NA 

3 NA NA 15 0 15 0 NA NA 

4 NA NA 15 0 15 0 NA NA 

1.1 0 NA NA 16 14 16 13 NA NA 

1 NA NA 16 1 16 2 NA NA 

2 NA NA 16 1 16 1 NA NA 

3 NA NA 16 0 16 0 NA NA 

4 NA NA 16 0 16 0 NA NA 

10 0 NA NA 15 11 15 13 NA NA 

1 NA NA 15 2 15 2 NA NA 

2 NA NA 15 1 15 0 NA NA 

3 NA NA 15 1 15 0 NA NA 

4 NA NA 15 0 15 0 NA NA 

104 0 NA NA 14 13 14 8 NA NA 

1 NA NA 14 0 14 6 NA NA 

2 NA NA 14 1 14 0 NA NA 

3 NA NA 14 0 14 0 NA NA 

4 NA NA 14 0 14 0 NA NA 

Abbreviations: NA Not applicable. 

LOQ=0.066 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 247, 99, 33 and 73 and female VTG was 1.97x106

, 1.14x106, 1.63x106 and 7.48x104

ng/mL in the mean-measured 0 (control), 1.1, 10 and 104 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) 

4 247 431 4 1.97 x 106 1.83 x 106

1.1 4 99 101 4 1.14 x 106 8.75 x 105

10 4 33 10 4 1.63 x 106 1.07 x 106

104 4 73 96 4 7.48 x 104 8.95 x 104

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

LOQ=0.066 mg a.i./L. 

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

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

Treatment 

 (mg a.i./L) 

[mean-measured]

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

Males Females Males Females 

n 

Mean

(ng/mL 

plasma) 

±SD n 

Mean

(ng/mL 

plasma) 

±SD n 

Mean

(ng/mL 

plasma) 

±SD n

Mean

(ng/mL 

plasma) 

±SD 

Negative control (<LOQ) NA NA NA NA NA NA NA NA NA NA NA NA 

1.1 NA NA NA NA NA NA NA NA NA NA NA NA 

10 NA NA NA NA NA NA NA NA NA NA NA NA 

100 NA NA NA NA NA NA NA NA NA NA NA NA 

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

LOQ=0.066 mg a.i./L. 

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There were no notable observations with regards to behavior or changes in appearance (coloration/banding, presence of ovipositor and dorsal nape 

pad), including secondary sex characteristics in the control or treated groups (Table 21). Two males were mis-sexed as females and inadvertently added to the control replicate B and 10 mg a.i./L replicate C. 

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

Treatment 

(mg a.i./L) 

[mean-measured] 

Secondary Sex Characteristics and Clinical Signs 

Males Females 

Type n

Incidence 

at Test 

Termination 

Type n 

Incidence 

at Test 

Termination 

Negative control (<LOQ) Deformed 8 1 

None 4 0 

Color bars 8 1 

1.1 Color bars 7 1 Bloated 16 2 

10 Color bars 8 1 None 4 0 

104 None 4 0 None 4 0 

LOQ=0.066 mg a.i./L. 

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B. Study Author’s Analysis and Conclusions

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

were gender specific and analyzed in comparison to the control. 

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

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

based on p<0.01 (CETIS, version 1.8.5, 2011). Survival data were analyzed using a Fisher’s Exact 

Test/Bonferroni-Holm Test. Fecundity, fertility, female weight and GSI, and male VTG were analyzed 

using ANOVA and Dunnett’s Multiple Comparison test. Male tubercle scores, male weight and GSI 

were analyzed by ANOVA and Jonckheere-Terpstra Step–Down test. Female VTG was analyzed by 

Tamhane-Dunnett Step-Down test. Male and female length was recorded but statistical analysis was 

not reported. Prior to Dunnett’s, data were analyzed by Shapiro-Wilk’s test and Bartlett’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. 

Statistical analysis (Fisher Exact/Bonferroni-Holm Test) determined no significant difference in male, 

female, or combined survival. At the 104 mg a.i./L treatment level, fecundity was 0 

eggs/female/reproductive day, and fertility was not reported since there was no spawning therefore 

this treatment level was excluded from statistical analysis. With the highest treatment removed, there 

were no significant differences in fecundity or fertility compared to the negative control (p>0.05; 

Dunnett Multiple Comparison Test). There was a significant reduction in median male tubercle score 

at the 104 mg a.i./L treatment level compared to the negative control (p<0.05; Jonckheere-Terpstra 

Step-Down test). There were no tubercles observed in females. There was a significant decrease in 

mean male weight at the 10 and 104 mg a.i./L treatment levels compared to the negative control 

(p<0.05; Jonckheere-Terpstra Step-Down test). There was also a significant increase in mean female 

body weight at the 1.1 mg a.i./L treatment level compared to the negative control (p<0.05; Dunnett’s 

Multiple Comparison test). Statistical analysis (Jonckheere-Terpstra Step-Down Test) determined no 

significant decrease (p>0.05) in mean male GSI score among fish exposed to any of the treatment 

levels tested, compared to the negative control. There was a significant decrease in mean female GSI 

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score at the 104 mg a.i./L treatment level compared to the negative control (p<0.05; Dunnett’s 

Multiple Comparison test). One male in the 10 mg/L concentration was empirically determined to be 

an outlier and was removed from the statistical analysis. Statistical analysis (Dunnett’s Multiple 

Comparison Test) determined no significant difference (p >0.05) for male vitellogenin concentration 

in any of the treatment levels compared to the negative control. There was also a significant difference 

in mean female VTG at the 104 mg a.i./L treatment level compared to the control (p<0.05; TamhaneDunnett Step-Down test). 

In male fish, there were an increased incidence of increased percentage of spermatogonia at the 10 

and 104 mg a.i./L treatment levels and asynchronous gonad development in the 104 mg a.i./L 

treatment level. In female fish, there was decreased yolk synthesis at the 104 mg a.i./L treatment 

level. There were no significant findings observed in the gonadal stage. Ninety-one out of 96 fish were 

examined. Four of the fish were not examined due to mortality. The fifth fish was not examined due 

to the size of the ovaries being too small for examination. 

C. Reviewer’s Analysis and Conclusions 

Statistical Methods: The reviewer analyzed survival (mortality) data using Fisher’s Exact test via 

Toxstat 3.5. Male body weight was consistent with a monotonic concentration-response; female 

vitellogenin no other endpoints were associated with a monotonic response. All data were tested for 

normality using Shapiro-Wilks test and for homogeneity of variance using Levene’s test. Data which 

met the assumptions of normality and homogeneity of variance were then analyzed using the parametric 

Dunnett’s test. Data which did not meet the parametric assumptions were analyzed using the nonparametric Mann-Whitney U test. 

Male VTG and fecundity were neither monotonic nor parametric and were analyzed using MannWhitney. Male body weight exhibited monotonic trends and was analyzed using the non-parametric 

Jonckheere-Terpstra test, as recommended by the OCSPP 890.1350 guideline. None of the surviving 

females were found to have tubercles. Unless otherwise indicated, effects were considered statistically 

significant at p<0.05. These analyses were conducted using SAS® (SAS Institute, Cary, NC; version 

8.1). Both the study author and reviewer relied on the mean-measured concentrations to discuss 

effects in this study. 

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 Conclusions: 

There were no significant differences in either male or female survival at any treatment levels compared 

to the negative control (Table 9). Male body weight (Table 24) exhibited a significant (p<0.05) 

reduction of 7 to 16% at all treatment levels compared to the negative control. Female GSI (Table 

23) was significantly (p<0.05) reduced 23% of control at the 104 mg a.i./L treatment level. Although 

the study author found a significant reduction in female plasma VTG at the 104 mg a.i./L treatment 

level, the reviewer did not find this effect to be significant using Dunnett’s test (SAS) or Dunnett’s T3 

(CETIS). Median male tubercle score was also significantly reduced (p<0.05) 80% compared to the 

control at the 104 mg a.i./L treatment level. There was no significant reduction in fecundity or fertility 

(Table 23) at the low and middle treatment levels; there were no eggs available for assessment at 

the highest treatment level. Therefore, that treatment level was excluded from data analysis, but the 

lack of fecundity at the highest treatment level is considered a significant effect. There were no other 

significant effects on other endpoints. Although not analyzed statistically, in male fish, there were an 

increased percentage of spermatogonia at the 10 and 104 mg a.i./L treatment levels and asynchronous 

gonad development in the 104 mg a.i/L treatment. In female fish, there was decreased yolk synthesis 

at the 104 mg a.i./L treatment. There were no significant findings observed in the gonadal stage, and 

no notable observations secondary sex characteristics or clinical signs for any treatment group 

compared to the controls. 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Tubercle Score GSI 

Gonadal 

Staging and 

Histo. 

Plasma VTG Plasma T Plasma E2 

Median p % Diff. p 

Effect? 

(Yes/No) 

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

Negative control 

(<LOQ) 

32 NA 0 NA No 0 NA NA NA NA NA

1.1 35 0.689 -1.92 0.999 No -59.72 0.780 NA NA NA NA 

10 32 1.000 -1.92 0.999 Yes -86.54 0.491 NA NA NA NA 

104 6 <0.001 10.96 0.847 Yes -70.65 0.780 NA NA NA NA 

Statistical Test Dunnett’s3 Dunnett’s NA Mann-Whitney NA NA 

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

LOQ=0.066 mg a.i./L. 

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

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

2 Unless otherwise specified, effects are considered statistically significant at p<0.05. 3 Mean tubercle scores: 32, 35, 32, and 9 for the negative control and mean-measured 1.1, 10, and 104 mg a.i./L treatment levels, respectively.

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

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 

1.1 4.19 0.400 -0.26 0.982 0 NA 0.00 1.00 No -42.04 0.625 NA NA NA NA 

10 -4.05 0.6571 0.78 0.854 0 NA 11.36 0.373 No -17.33 0.952 NA NA NA NA 

104 -100.03 0.0286 –3 –3 0 NA -23.41 0.030 Yes -96.20 0.092 NA NA NA NA 

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

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

LOQ=0.066 mg a.i./L. 

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

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

2 Unless otherwise specified, effects are considered statistically significant at p<0.05. 3 No eggs were produced at the highest test concentration; therefore fecundity was 0, and there were no fertility data to analyze. 

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

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

1.1 -7.44 0.042 12.08 0.071 -4.57 0.112 2.98 0.446 

10 -15.95 0.003 4.85 0.639 -4.57 0.112 3.51 0.327 

104 -15.92 0.001 -9.34 0.183 -2.50 0.501 2.72 0.514 

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

Abbreviations: Diff. Difference. NA Not applicable. 

LOQ=0.066 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 a couple of deviations described in Section I. Materials and Methods of this DER regarding 

failure to report certain water characteristics and meet environmental criteria. These deviations do not 

impact the interpretation of the study. The remaining validity and performance criteria for OCSPP 

890.1350 were met. 

F. Reviewer’s Comments 

The reviewer’s and the study authors’ results were in general agreement. Both the study author’s and 

reviewer’s analysis detected statistically significant reductions (p<0.05) in male tubercle scores and female 

GSI at the 104 mg a.i./L. The study author found a significant decrease (p<0.05) in mean male body 

weight at the 10 and 104 mg a.i./L, while the reviewer found significant decreases at all treatment levels 

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compared to the negative control. The study author also reported a significant decrease in female VTG at 

the 104 mg a.i./L treatment level and a significant increase in mean female weight at the 1.1 mg a.i./L 

treatment level compared to the negative control. The reviewer’s conclusions based on the OCSPP 

890.1350 flowchart are presented in the Executive Summary and Conclusions sections of this DER. 

During the study, two males were mis-sexed as females and inadvertently added to the negative control 

replicate B and the mid concentration (10 mg a.i./L) replicate C. The study author did not specify which 

males were mis-sexed and used the data for all three males to calculate descriptive statistics for the 

affected replicates. Since the study report did not explicitly identify the mis-sexed fish, the reviewer used 

data for all three males in the affected replicates for statistical analysis even though the mis-sexed males 

could probably be identified by their tubercles scores (i.e., 0). 

There is a discrepancy in the statistical test used to analyze mean male body weight. The data table 

(Table 5, p. 39) reports the significant differences in mean male body weight at the 10 and 104 mg a.i./L 

treatment levels compared to the control were determined using Dunnett’s Multiple Comparison test, while 

the “Results and Discussion” text reported the differences were determined using Jonckheere-Terpstra 

Step-Down test. The reviewer reported Jonckheere-Terpstra Step-Down test was used. 

III. REFERENCES 

Ankley, G.T., Jensen, K.M., Kahl, M.D., Korte, J.J., and Makynen E.A., 2001. Description and evaluation 

of a short-term reproduction test with the fathead minnow (Pimephales promelas). Environ Toxicol Chem 

20:1276-1290. 

APHA, AWWA, WPCF, 1995. Standard Methods for the Examination of Water and Wastewater. 19th 

Edition, Washington, DC, 2-23-25. 

ASTM, 2005. Committee E-47 on Biological Effects and Environmental Fate. Standard Guide for 

Conducting Early Life-Stage Toxicity Tests with Fishes. ASTM Designation E: 1241-05. American Society 

for Testing and Materials, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, Pennsylvania. 

Page 183 of 292
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Ives, Michael, 2011. CETIS, Comprehensive Environmental Toxicity Information SystemTM. User’s Guide. 

Tidepool Scientific Software, McKinleyville, California. 

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

Research 1: 21-29. 

Sprague, J.B., 1969. Measurement of pollutant toxicity to fish. 1. Bioassay methods for acute toxicity. 

Water Res. 3: 793-821. 

OECD (2009). Fish Short Term Reproduction Assay. OECD Guideline for the testing Chemicals: Test No. 

229. Paris, France. 40 pp 

U.S. EPA, 1989. 40 CFR, Part 160. Federal Insecticide, Fungicide, and Rodenticide Act. Good Laboratory 

Practices Standards; Final Rule. Office of the Federal Register, National Archives and Records 

Administration. U.S. Government Printing Office, Washington, D.C. 

U.S. EPA. 1996. Office of Prevention, Pesticides and Toxic Substances. Ecological Effects Guideline, 

OCSPP 850.1000. Special Consideration for Conducting Aquatic Laboratory Studies. “Public Draft”. EPA 

712-C-96-113. April 1996. U.S. Environmental Protection Agency, Washington, D.C. 

U.S. EPA, 2002. Short-term methods for estimating the chronic toxicity of effluents and receiving waters 

to marine and estuarine organisms. Third edition. Office of Water, U. S. Environmental Protection Agency, 

Washington, DC 20460. EPA/821/R-02/014. 

U.S. EPA, 2009. Office of Prevention, Pesticides and Toxic Substances. Endocrine Disruptor Screening 

Program Test Guideline, OCSPP 890.1350. Fish Short-Term Reproduction Assay. EPA 740-C-09-007. 

October 2009. U.S. Environmental Protection Agency. Washington, D.C. 

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APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION:

Reviewer’s statistical verification table 

Endpoint Monotonic? Parametric? 890.1350 EFED Comments Female body weight No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

890.1350 and EFED same conclusions, no effect 

Male body weight Yes Yes Jonckheere: 

Dose 1 p=0.042 Dose 2 p=0.003 Dose 3 p=0.001 

Jonckheere: Dose 1 p=0.042 Dose 2 p=0.003 Dose 3 p=0.001 

890.1350 and EFED same conclusions; Jonckheere’s reveals significant 7 to 16% reduction in male body weight at all treatment levels, relative to the negative control

Female body length No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

890.1350 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 

890.1350 and EFED same conclusions, no effect 

Female VTG No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

890.1350 and EFED same conclusions, no effect 

Male VTG No No MannWhitney: 

n.s. p>0.05 

MannWhitney: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Female GSI No Yes Dunnett’s: 

Dose 3 p=0.030 

Dunnett’s: Dose 3 p=0.030 

890.1350 and EFED same conclusions, significant 23% reduction at the highest level. 

Male GSI No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

890.1350 and EFED same conclusions, no effect 

Female tubercle score 

NA NA NA NA No score 

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Endpoint Monotonic? Parametric? 890.1350 EFED Comments Male tubercle score No Yes Dunnett’s: 

Dose 3 p<0.001 

Dunnett’s: Dose 3 p<0.001 

890.1350 and EFED same conclusions, significant 81 % reduction (median values) at the highest level. 

Fecundity No No MannWhitney: 

Dose 3 p=0.052 

MannWhitney: Dose 3 p=0.052 

890.1350 and EFED same conclusions; no eggs produced at the high dose level. 

Fertility No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

890.1350 and EFED same conclusions, no effect at low and middle doses (no data for high dose). 

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 

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test for fish screen study - Metalaxyl 

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 

 0.976 0.920 1.729 0.214 USE PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

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

 Ctrl 4 1.28 0.09 0.05 7.10 1.14, 1.43 

 Dose1 4 1.44 0.03 0.01 1.92 1.39, 1.48 

 Dose2 4 1.34 0.09 0.04 6.44 1.21, 1.48 

 Dose3 4 1.16 0.12 0.06 10.36 0.97, 1.35 

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

 Ctrl 1.29 1.18 1.37 . . 

 Dose1 1.44 1.40 1.47 112.08 -12.08 

 Dose2 1.33 1.27 1.45 104.85 -4.85 

 Dose3 1.18 1.00 1.28 90.66 9.34 

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

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 6.84 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 1.28 . 1.36 . . . . . . 

 Dose1 1.44 0.071 1.36 0.934 . . . . . 

 Dose2 1.34 0.639 1.34 0.919 0.473 . . . . 

 Dose3 1.16 0.183 1.16 0.050 0.004 0.054 . . . 

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

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 9.66 0.022 

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 1.44 0.067 0.990 

 Dose2 1.33 0.494 0.810 

 Dose3 1.18 0.346 0.112 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams Dose3 

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 Jonckheere >highest dose (no sign. differences) 

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

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

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 6.84 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 -1.28 . -1.28 . . . . . . 

 Dose1 -1.44 0.071 -1.31 0.365 . . . . . 

 Dose2 -1.34 0.639 -1.31 0.389 0.473 . . . . 

 Dose3 -1.16 0.183 -1.31 0.403 0.004 0.054 . . . 

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

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 9.66 0.022 

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 -1.44 0.067 0.010 

 Dose2 -1.33 0.494 0.190 

 Dose3 -1.18 0.346 0.888 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

test for fish screen study - Metalaxyl 

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 

 0.967 0.790 1.390 0.293 USE PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

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

 Ctrl 4 3.13 0.12 0.06 3.81 2.94, 3.31 

 Dose1 4 2.89 0.20 0.10 6.78 2.58, 3.20 

 Dose2 4 2.63 0.30 0.15 11.32 2.15, 3.10 

 Dose3 4 2.63 0.16 0.08 6.09 2.37, 2.88 

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

 Ctrl 3.12 3.01 3.25 . . 

 Dose1 2.95 2.62 3.06 92.56 7.44 

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 Dose2 2.62 2.29 2.98 84.05 15.95 

 Dose3 2.62 2.46 2.80 84.08 15.92 

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

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 5.53 0.013 

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 3.13 . 3.13 . . . . . . 

 Dose1 2.89 0.295 2.89 0.078 . . . . . 

 Dose2 2.63 0.012 2.63 0.003 0.301 . . . . 

 Dose3 2.63 0.013 2.63 0.003 0.305 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 9.64 0.022 

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 2.95 0.156 0.042 

 Dose2 2.62 0.067 0.003 

 Dose3 2.62 0.067 0.001 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams Dose2 

 Jonckheere Dose1 

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

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 5.53 0.013 

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 -3.13 . -2.82 . . . . . . 

 Dose1 -2.89 0.295 -2.82 0.989 . . . . . 

 Dose2 -2.63 0.012 -2.82 0.993 0.301 . . . . 

 Dose3 -2.63 0.013 -2.82 0.995 0.305 1.000 . . . 

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

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

 Kruskal-Wallis test - equality among treatment groups 

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 Degrees of Freedom TestStat P-value 

 3 9.64 0.022 

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 -2.95 0.156 0.958 

 Dose2 -2.62 0.067 0.997 

 Dose3 -2.62 0.067 0.999 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

test for fish screen study - Metalaxyl 

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 

 0.953 0.536 1.268 0.329 USE PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

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

 Ctrl 4 47.75 0.90 0.45 1.88 46.32, 49.18 

 Dose1 4 49.18 1.50 0.75 3.05 46.79, 51.56 

 Dose2 4 49.43 1.23 0.61 2.48 47.47, 51.38 

 Dose3 4 49.05 2.22 1.11 4.53 45.51, 52.59 

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

 Ctrl 47.95 46.50 48.60 . . 

 Dose1 49.50 47.10 50.60 102.98 -2.98 

 Dose2 49.70 47.70 50.60 103.51 -3.51 

 Dose3 49.35 46.20 51.30 102.72 -2.72 

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

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 0.95 0.448 

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 47.75 . 48.85 . . . . . . 

 Dose1 49.18 0.446 48.85 0.899 . . . . . 

 Dose2 49.43 0.327 48.85 0.921 0.995 . . . . 

 Dose3 49.05 0.514 48.85 0.931 0.999 0.985 . . . 

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

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

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 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 2.53 0.470 

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 47.95 . . 

 Dose1 49.50 0.235 0.926 

 Dose2 49.70 0.233 0.939 

 Dose3 49.35 0.494 0.897 

 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 0.95 0.448 

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 -47.75 . -47.75 . . . . . . 

 Dose1 -49.18 0.446 -49.18 0.128 . . . . . 

 Dose2 -49.43 0.327 -49.24 0.124 0.995 . . . . 

 Dose3 -49.05 0.514 -49.24 0.128 0.999 0.985 . . . 

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

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 2.53 0.470 

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 -47.95 . . 

 Dose1 -49.50 0.235 0.074 

 Dose2 -49.70 0.233 0.061 

 Dose3 -49.35 0.494 0.103 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

test for fish screen study - Metalaxyl 

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. 

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 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 0.950 0.496 0.303 0.823 USE PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

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

 Ctrl 4 62.98 1.29 0.65 2.06 60.92, 65.03 

 Dose1 4 60.10 2.29 1.15 3.81 56.46, 63.74 

 Dose2 4 60.10 1.68 0.84 2.79 57.43, 62.77 

 Dose3 4 61.40 1.92 0.96 3.13 58.34, 64.46 

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

 Ctrl 62.75 61.90 64.50 . . 

 Dose1 60.80 56.90 61.90 95.43 4.57 

 Dose2 59.75 58.70 62.20 95.43 4.57 

 Dose3 60.90 59.80 64.00 97.50 2.50 

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

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 2.22 0.138 

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 62.98 . 62.98 . . . . . . 

 Dose1 60.10 0.112 60.53 0.049 . . . . . 

 Dose2 60.10 0.112 60.53 0.052 1.000 . . . . 

 Dose3 61.40 0.501 60.53 0.053 0.750 0.750 . . . 

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

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

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 5.39 0.145 

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 62.75 . . 

 Dose1 60.80 0.096 0.019 

 Dose2 59.75 0.154 0.028 

 Dose3 60.90 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) 

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

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 2.22 0.138 

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

EPA MRID Number 48685802 

Page 58 of 75 

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 -62.98 . -61.06 . . . . . . 

 Dose1 -60.10 0.112 -61.06 0.958 . . . . . 

 Dose2 -60.10 0.112 -61.06 0.970 1.000 . . . . 

 Dose3 -61.40 0.501 -61.40 0.955 0.750 0.750 . . . 

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

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

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 5.39 0.145 

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 -62.75 . . 

 Dose1 -60.80 0.096 0.981 

 Dose2 -59.75 0.154 0.972 

 Dose3 -60.90 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) 

test for fish screen study - Metalaxyl 

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 

 0.875 0.033 3.372 0.055 USE PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

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

 Ctrl 4 1969500 1831612 915805.8 93.00 -945003, 4884003 

 Dose1 4 1141509 874488.3 437244.2 76.61 -249998, 2533015 

 Dose2 4 1628250 1071015 535507.3 65.78 -75973.2, 3332473 

 Dose3 4 74800.00 89463.85 44731.92 119.60 -67556.9,217156.9 

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

 Ctrl 1125000 918000.0 4710000 . . 

 Dose1 1343000 34.00 1880000 57.96 42.04 

 Dose2 1295000 753000.0 3170000 82.67 17.33 

 Dose3 37700.00 15800.00 208000.0 3.80 96.20 

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

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 2.07 0.158 

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

EPA MRID Number 48685802 

Page 59 of 75 

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 1969500 . 1969500 . . . . . . 

 Dose1 1141509 0.625 1384879 0.290 . . . . . 

 Dose2 1628250 0.952 1384879 0.310 0.930 . . . . 

 Dose374800.00 0.092 74800.00 0.023 0.572 0.273 . . . 

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

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 6.42 0.093 

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 1125000 . . 

 Dose1 1343000 0.678 0.282 

 Dose2 1295000 1.000 0.442 

 Dose3 37700.00 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 2.07 0.158 

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 -1969500 . -1203515 . . . . . . 

 Dose1-1141509 0.625 -1203515 0.887 . . . . . 

 Dose2-1628250 0.952 -1203515 0.910 0.930 . . . . 

 Dose3-74800.0 0.092 -1203515 0.921 0.572 0.273 . . . 

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

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 6.42 0.093 

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 -1125000 . . 

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

EPA MRID Number 48685802 

Page 60 of 75 

 Dose1 -1343000 0.678 0.718 

 Dose2 -1295000 1.000 0.558 

 Dose3 -37700.0 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 fish screen study - Metalaxyl 

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 

 0.758 <.001 5.989 0.010 USE NON-PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

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

 Ctrl 4 247.00 430.96 215.48 174.48 -438.76, 932.76 

 Dose1 4 99.50 101.43 50.72 101.94 -61.91, 260.91 

 Dose2 4 33.25 9.84 4.92 29.61 17.59, 48.91 

 Dose3 4 72.50 95.66 47.83 131.95 -79.72, 224.72 

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

 Ctrl 41.00 13.00 893.00 . . 

 Dose1 64.00 22.00 248.00 40.28 59.72 

 Dose2 28.50 28.00 48.00 13.46 86.54 

 Dose3 31.00 13.00 215.00 29.35 70.65 

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

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 0.68 0.581 

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 247.00 . 247.00 . . . . . . 

 Dose1 99.50 0.689 99.50 0.224 . . . . . 

 Dose2 33.25 0.432 52.88 0.158 0.975 . . . . 

 Dose3 72.50 0.580 52.88 0.163 0.998 0.995 . . . 

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

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 1.80 0.616 

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 

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

EPA MRID Number 48685802 

Page 61 of 75 

 Ctrl 41.00 . . 

 Dose1 64.00 0.780 0.668 

 Dose2 28.50 0.491 0.209 

 Dose3 31.00 0.780 0.141 

 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 0.68 0.581 

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 -247.00 . -113.06 . . . . . . 

 Dose1 -99.50 0.689 -113.06 0.864 . . . . . 

 Dose2 -33.25 0.432 -113.06 0.890 0.975 . . . . 

 Dose3 -72.50 0.580 -113.06 0.903 0.998 0.995 . . . 

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

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 1.80 0.616 

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 -41.00 . . 

 Dose1 -64.00 0.780 0.332 

 Dose2 -28.50 0.491 0.791 

 Dose3 -31.00 0.780 0.859 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

test for fish screen study - Metalaxyl 

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 

 0.952 0.523 2.768 0.088 USE PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

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

 Ctrl 4 11.00 0.82 0.41 7.42 9.70, 12.30 

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EPA MRID Number 48685802 

Page 62 of 75 

 Dose1 4 11.00 1.78 0.89 16.19 8.17, 13.83 

 Dose2 4 12.25 1.26 0.63 10.27 10.25, 14.25 

 Dose3 4 8.43 0.75 0.38 8.90 7.23, 9.62 

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

 Ctrl 11.00 10.00 12.00 . . 

 Dose1 10.80 9.40 13.00 100.00 0.00 

 Dose2 12.00 11.00 14.00 111.36 -11.36 

 Dose3 8.80 7.30 8.80 76.59 23.41 

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

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 6.91 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 11.00 . 11.42 . . . . . . 

 Dose1 11.00 1.000 11.42 0.767 . . . . . 

 Dose2 12.25 0.373 11.42 0.799 0.497 . . . . 

 Dose3 8.43 0.030 8.43 0.007 0.049 0.004 . . . 

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

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 9.79 0.020 

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 11.00 . . 

 Dose1 10.80 1.000 0.442 

 Dose2 12.00 0.214 0.870 

 Dose3 8.80 0.061 0.059 

 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 6.91 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 -11.00 . -10.67 . . . . . . 

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EPA MRID Number 48685802 

Page 63 of 75 

 Dose1 -11.00 1.000 -10.67 0.733 . . . . . 

 Dose2 -12.25 0.373 -10.67 0.767 0.497 . . . . 

 Dose3 -8.43 0.030 -10.67 0.785 0.049 0.004 . . . 

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

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 9.79 0.020 

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 -11.00 . . 

 Dose1 -10.80 1.000 0.558 

 Dose2 -12.00 0.214 0.130 

 Dose3 -8.80 0.061 0.941 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

test for fish screen study - Metalaxyl 

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 

 0.944 0.404 2.603 0.100 USE PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

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

 Ctrl 4 1.30 0.08 0.04 6.28 1.17, 1.43 

 Dose1 4 1.28 0.31 0.15 24.28 0.78, 1.77 

 Dose2 4 1.28 0.17 0.09 13.39 1.00, 1.55 

 Dose3 4 1.44 0.49 0.24 33.91 0.66, 2.22 

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

 Ctrl 1.30 1.20 1.40 . . 

 Dose1 1.20 1.00 1.70 98.08 1.92 

 Dose2 1.25 1.10 1.50 98.08 1.92 

 Dose3 1.35 0.97 2.10 110.96 -10.96 

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

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 0.28 0.839 

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 

Page 198 of 292
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EPA MRID Number 48685802 

Page 64 of 75 

 Ctrl 1.30 . 1.32 . . . . . . 

 Dose1 1.28 0.999 1.32 0.628 . . . . . 

 Dose2 1.28 0.999 1.32 0.663 1.000 . . . . 

 Dose3 1.44 0.847 1.32 0.682 0.863 0.863 . . . 

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

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 0.38 0.945 

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 1.30 . . 

 Dose1 1.20 0.671 0.277 

 Dose2 1.25 0.775 0.354 

 Dose3 1.35 1.000 0.500 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

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

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

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 0.28 0.839 

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 -1.30 . -1.28 . . . . . . 

 Dose1 -1.28 0.999 -1.28 0.616 . . . . . 

 Dose2 -1.28 0.999 -1.28 0.650 1.000 . . . . 

 Dose3 -1.44 0.847 -1.44 0.344 0.863 0.863 . . . 

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

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 0.38 0.945 

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 -1.30 . . 

 Dose1 -1.20 0.671 0.723 

 Dose2 -1.25 0.775 0.646 

 Dose3 -1.35 1.000 0.500 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

Page 199 of 292
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EPA MRID Number 48685802 

Page 65 of 75 

test for fish screen study - Metalaxyl 

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 

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EPA MRID Number 48685802 

Page 66 of 75 

 Jonckheere Dose1 

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

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

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 . . . 1 . . . . . . . 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing INCREASING trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl 0.00 . . . . . . . . 

 Dose1 0.00 . . . . . . . . 

 Dose2 0.00 . . . . . . . . 

 Dose3 0.00 . . . . . . . . 

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

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

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 0.00 1.000 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 0.00 . . 

 Dose1 0.00 1.000 . 

 Dose2 0.00 1.000 . 

 Dose3 0.00 1.000 . 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams Dose1 

 Jonckheere Dose1 

test for fish screen study - Metalaxyl 

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 

 0.921 0.172 2.258 0.134 USE PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

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

 Ctrl 4 31.75 4.11 2.06 12.95 25.21, 38.29 

 Dose1 4 34.50 3.87 1.94 11.23 28.34, 40.66 

 Dose2 4 32.00 0.82 0.41 2.55 30.70, 33.30 

 Dose3 4 8.75 6.22 3.11 71.14 -1.16, 18.66 

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

 Ctrl 31.50 27.00 37.00 . . 

Page 201 of 292
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl 

EPA MRID Number 48685802 

Page 67 of 75 

 Dose1 34.50 30.00 39.00 108.66 -8.66 

 Dose2 32.00 31.00 33.00 100.79 -0.79 

 Dose3 6.25 4.50 18.00 27.56 72.44 

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

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 32.64 <.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 31.75 . 33.13 . . . . . . 

 Dose1 34.50 0.689 33.13 0.760 . . . . . 

 Dose2 32.00 1.000 32.00 0.653 0.836 . . . . 

 Dose3 8.75 <.001 8.75 <.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 9.29 0.026 

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 31.50 . . 

 Dose1 34.50 0.494 0.807 

 Dose2 32.00 0.775 0.559 

 Dose3 6.25 0.067 0.015 

 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 32.64 <.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 -31.75 . -26.75 . . . . . . 

 Dose1 -34.50 0.689 -26.75 0.972 . . . . . 

 Dose2 -32.00 1.000 -26.75 0.981 0.836 . . . . 

 Dose3 -8.75 <.001 -26.75 0.984 <.001 <.001 . . . 

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

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

Page 202 of 292
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl 

EPA MRID Number 48685802 

Page 68 of 75 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 9.29 0.026 

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 -31.50 . . 

 Dose1 -34.50 0.494 0.193 

 Dose2 -32.00 0.775 0.441 

 Dose3 -6.25 0.067 0.985 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

test for fish screen study - Metalaxyl 

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 

 0.845 0.012 5.092 0.017 USE NON-PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

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

 Ctrl 4 18.50 4.04 2.02 21.85 12.07, 24.93 

 Dose1 4 19.28 8.17 4.09 42.39 6.27, 32.28 

 Dose2 4 17.75 2.87 1.44 16.18 13.18, 22.32 

 Dose3 4 0.00 0.00 0.00 . . , . 

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

 Ctrl 18.50 15.00 22.00 . . 

 Dose1 23.00 7.10 24.00 104.19 -4.19 

 Dose2 18.00 14.00 21.00 95.95 4.05 

 Dose3 0.00 0.00 0.00 0.00 100.00 

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

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 15.07 <.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 18.50 . 18.89 . . . . . . 

 Dose1 19.28 0.991 18.89 0.631 . . . . . 

 Dose2 17.75 0.992 17.75 0.520 0.968 . . . . 

 Dose3 0.00 <.001 0.00 <.001 <.001 0.001 . . . 

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

Page 203 of 292
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl 

EPA MRID Number 48685802 

Page 69 of 75 

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 9.66 0.022 

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 18.50 . . 

 Dose1 23.00 0.478 0.816 

 Dose2 18.00 0.673 0.328 

 Dose3 0.00 0.052 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 15.07 <.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 -18.50 . -13.88 . . . . . . 

 Dose1 -19.28 0.991 -13.88 0.948 . . . . . 

 Dose2 -17.75 0.992 -13.88 0.962 0.968 . . . . 

 Dose3 0.00 <.001 -13.88 0.968 <.001 0.001 . . . 

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

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 9.66 0.022 

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 -18.50 . . 

 Dose1 -23.00 0.478 0.184 

 Dose1 -23.00 0.478 0.184 

 Dose1 -23.00 0.478 0.184 

 Dose2 -18.00 0.673 0.672 

 Dose2 -18.00 0.673 0.672 

 Dose2 -18.00 0.673 0.672 

 Dose3 0.00 0.052 0.995 

 Dose3 0.00 0.052 0.995 

 Dose3 0.00 0.052 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 204 of 292
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl 

EPA MRID Number 48685802 

Page 70 of 75 

test for fish screen study - Metalaxyl 

ANALYSIS RESULTS FOR VARIABLE VAR12 ( fertility ) 

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS 

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 

Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 

Use parametric analyses if neither test rejected, otherwise non-parametric analyses. 

 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 0.848 0.035 1.294 0.321 USE PARAMETRIC TESTS 

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

BASIC SUMMARY STATISTICS 

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

 Ctrl 4 96.00 2.71 1.35 2.82 91.69, 100.31 

 Dose1 4 95.75 0.96 0.48 1.00 94.23, 97.27 

 Dose2 4 96.75 2.63 1.31 2.72 92.57, 100.93 

 Dose3 0 . . . . . , . 

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

 Ctrl 97.00 92.00 98.00 . . 

 Dose1 95.50 95.00 97.00 99.74 0.26 

 Dose2 97.50 93.00 99.00 100.78 -0.78 

 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 

 . . . 9 . . 2 . . . . . 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing negative trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl 96.00 . . . . . . . . 

 Dose1 95.75 0.982 . . . . . . . 

 Dose2 96.75 0.854 . . 0.808 . . . . 

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

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

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 2 1.58 0.455 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing negative trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 97.00 . . 

 Dose1 95.50 . 0.186 

 Dose2 97.50 . 0.673 

 Dose3 . . . 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams Dose1 

 Jonckheere >highest dose (no sign. differences) 

Page 205 of 292
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl 

EPA MRID Number 48685802 

Page 71 of 75 

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

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 

 . . . 9 . . 2 . . . . . 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing INCREASING trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl -96.00 . . . . . . . . 

 Dose1 -95.75 0.982 . . . . . . . 

 Dose2 -96.75 0.854 . . 0.808 . . . . 

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

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

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 2 1.58 0.455 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -97.00 . . 

 Dose1 -95.50 . 0.814 

 Dose2 -97.50 . 0.327 

 Dose3 . . . 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams Dose1 

 Jonckheere >highest dose (no sign. differences) 

Page 206 of 292
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl 

EPA MRID Number 48685802 

Page 72 of 75 

Fecundity (Mann-Whitney exact test) 

Obs conc endpoint 

1 0 15.0 

2 0 22.0 

3 0 15.0 

4 0 22.0 

5 1 24.0 

6 1 24.0 

7 1 22.0 

8 1 7.1 

9 2 21.0 

10 2 14.0 

11 2 18.0 

12 2 18.0 

13 3 0.0 

14 3 0.0 

15 3 0.0 

16 3 0.0 

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 41.0 34.0 8.142481 10.250 

1 4 49.0 34.0 8.142481 12.250 

2 4 36.0 34.0 8.142481 9.000 

3 4 10.0 34.0 8.142481 2.500 

Average scores were used for ties. 

Kruskal-Wallis Test 

Chi-Square 9.6606 

DF 3 

Asymptotic Pr > Chi-Square 0.0217 

Exact Pr >= Chi-Square 0.0071 

Page 207 of 292
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl 

EPA MRID Number 48685802 

Page 73 of 75 

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 15.0 18.0 3.338092 3.750 

1 4 21.0 18.0 3.338092 5.250 

Average scores were used for ties. 

Wilcoxon Two-Sample Test 

Statistic (S) 15.0000 

Normal Approximation 

Z -0.7489 

One-Sided Pr < Z 0.2269 

Two-Sided Pr > |Z| 0.4539 

t Approximation 

One-Sided Pr < Z 0.2392 

Two-Sided Pr > |Z| 0.4783 

Exact Test 

One-Sided Pr <= S 0.2000 

Two-Sided Pr >= |S - Mean| 0.4000 

Z includes a continuity correction of 0.5. 

Kruskal-Wallis Test 

Chi-Square 0.8077 

DF 1 

Pr > Chi-Square 0.3688 

Page 208 of 292
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl 

EPA MRID Number 48685802 

Page 74 of 75 

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 20.0 18.0 3.401680 5.0 

2 4 16.0 18.0 3.401680 4.0 

Average scores were used for ties. 

Wilcoxon Two-Sample Test 

Statistic (S) 20.0000 

Normal Approximation 

Z 0.4410 

One-Sided Pr > Z 0.3296 

Two-Sided Pr > |Z| 0.6592 

t Approximation 

One-Sided Pr > Z 0.3363 

Two-Sided Pr > |Z| 0.6725 

Exact Test 

One-Sided Pr >= S 0.3286 

Two-Sided Pr >= |S - Mean| 0.6571 

Z includes a continuity correction of 0.5. 

Kruskal-Wallis Test 

Chi-Square 0.3457 

DF 1 

Pr > Chi-Square 0.5566 

Page 209 of 292
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl 

EPA MRID Number 48685802 

Page 75 of 75 

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 26.0 18.0 3.207135 6.50 

3 4 10.0 18.0 3.207135 2.50 

Average scores were used for ties. 

Wilcoxon Two-Sample Test 

Statistic (S) 26.0000 

Normal Approximation 

Z 2.3385 

One-Sided Pr > Z 0.0097 

Two-Sided Pr > |Z| 0.0194 

t Approximation 

One-Sided Pr > Z 0.0260 

Two-Sided Pr > |Z| 0.0520 

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 6.2222 

DF 1 

Pr > Chi-Square 0.0126 

Page 210 of 292
DATA EVALUATION RECORD 

METALAXYL 

Study Type: OCSPP 890.1400, In vivo Hershberger Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-46-2012 (MRID 48617604) 

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: 4/19/2012 

Secondary Reviewer: Signature:

Kelly Luck, M.S. Date: 5/15/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 6/05/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 6/05/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 211 of 292
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 212 of 292
METALAXYL I PC Code 113501 

In vivo Hershberger Assay (2011) I Page 1of16 

QCSPP 890.140Q/ OECD 441 

J --- Primary Reviewer: Vincent Chen. M.S. Signature: ~~~-=:::=V'~~Z::::=--

Risk Assessment Branch III, Health Effects Division (7509P) Date: z.4 :s'v'N Z.e>JS 

Secondary Reviewer: Greg Akerman, Ph.D. Signature: .aa .~

Health Effects Division (7509P) Date: Cb:r:J3 1 Template version 08/2011 

DATA EVALUATION RECORD 

STUDY TYPE: In Vivo Hershberger Assay (Rat); OCSPP 890.1400; OECD 441 

PC CODE: 113501 DP BARCODE: D399227 

TXR#: 0054219 CAS#: 57837-19-1 

TEST MATERIAL (PURITY): Metalaxyl (98.2% a.i.) 

SYNONYMS: Alanine, N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-, methyl ester 

CITATION: Carlson, M.B. (2011) Oral (Gavage) Hershberger Bioassay of Metalaxyl in 

Male Rats. Charles River Laboratories, Preclinical Services, Horsham, PA. 

Laboratory Project ID number: 20012407, December 9, 2011. MRID 

48617604. Unpublished. 

SPONSOR: Metalaxyl EDSP Consortium, 20 Ambriance Drive, Burr Ridge, IL 

TEST ORDER#: CON-113501-18 

EXECUTIVE SUMMARY: In a Hershberger Assay (MRID 48617604) screening for 

androgenic activity, metalaxyl (98.2% a.i., Batch/lot# 20100540) in 2.0% aqueous 

carboxymethylcellulose (CMC) was administered daily via oral gavage to groups of eight 57-day 

old, castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 125, or 375 mg/kg/day. An 

androgenic positive control group consisted of eight castrated rats exposed to 0.4 mg/kg/day of 

testosterone propionate (TP) by subcutaneous (s.c.) injection. 

To screen for potential anti-androgenic activity, metalaxyl (98.2% a.i.) in 2.0% aqueous CMC 

was administered daily via oral gavage to groups of eight 54- or 60-day old, castrated male 

Sprague Dawley rats at dose levels of 0 (vehicle), 40, 125, or 375 mg/kg/day in conjunction with 

a daily dose ofreference androgen TP at 0.4 mg/kg/day by s.c. injection. The anti-androgenic 

positive control group consisted of eight castrated rats exposed to 0.4 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 and food consumption were determined daily. 

The animals were dosed for 10 consecutive days and terminated approximately 24 hours after the 

final dose administration. At necropsy, the five androgen-dependent tissues, liver, and kidneys 

were collected and weighed. 

All animals survived until scheduled termination. No adverse clinical signs of toxicity were 

observed in the androgen agonist assay. In the anti-androgen assay, treatment related clinical 

Page 213 of 292
In vivo Hershberger Assay (2011) / Page 2 of 16 

METALAXYL / PC Code 113501 OCSPP 890.1400/ OECD 441

signs of toxicity (urine-stained abdominal fur) were observed in three rats from the 375 

mg/kg/day group. 

In the androgen agonist assay, mean body weights and body weight gains in the metalaxyl 

treatment groups were comparable to the vehicle controls throughout the assay. Animals in the 

positive control (TP) group had increased (p≤0.01) overall (Days 1-11) body weight gains 

(↑31%) compared to the vehicle controls. Overall average food consumption for both metalaxyl 

treatment groups were comparable to the vehicle controls; however, overall food consumption in 

the TP positive control group was increased (p≤0.01) by 10%. No treatment-related effects were 

observed in any of the accessory sex organs or kidneys of metalaxyl-dosed animals, metalaxyl. 

However, mean liver weights were increased (p<0.01) by 17% in the 375 mg/kg/day group 

compared to the vehicle controls. No gross lesions were observed at necropsy, but one animal in 

the 375 mg/kg/day group did not achieve preputial separation by necropsy. Animals in the TP 

group had increased (p≤0.01) accessory sex organ weights as follows: 500% in seminal vesicles; 

691% in ventral prostate; 177% in levator ani-bulbocavernosus (LABC); 645% in Cowper’s 

glands; and 71% in glans penis. The performance criteria indicated that this assay was 

performing as expected. 

In the anti-androgen assay, mean body weights and overall body weight gains of the metalaxyl 

treatment groups were comparable to the vehicle controls. Animals dosed with TP + FT 

(positive control) had mean body weights and overall mean body weight gains comparable to the 

corn oil vehicle controls. Overall average food consumption was decreased (p≤0.01) in animals 

treated with metalaxyl at 125 mg/kg/day (↓6%) compared to the vehicle controls. Overall 

average food consumption in the TP + FT group was decreased (p≤0.01) by 7% compared to the 

corn oil vehicle control. No treatment-related effects were observed in any of the accessory sex 

organs or kidneys of metalaxyl-dosed animals. However, mean liver weights were increased 

(p≤0.01) by 13% in the 375 mg/kg/day group compared to the vehicle controls. No gross lesions 

were observed at necropsy, but the left Cowper’s gland was not present in one animal in the 40 

mg/kg/day group. Animals in the TP + FT group had decreased (p≤0.01) accessory sex organ 

weights as expected: 78% in seminal vesicles; 70% in ventral prostate; 44% in LABC; 53% in 

Cowper’s gland; and 19% in glans penis. The %CV values for each accessory sex organ of the 

vehicle control and metalaxyl high dose group were less than the maximum guideline-specified 

%CV values, except for the % CV for glans penis of the vehicle control (CV = 18%; maximum 

permissible CV = 17%). 

The high dose of metalaxyl selected for this study (375 mg/kg/day) was appropriate based on 

data from earlier studies showing clinical signs of systemic toxicity at doses ≥500 mg/kg/day and 

the increased liver weights observed at 375 mg/kg/day in the current assay. 

Statistically significant changes were not seen in two or more of the five androgen sensitive 

tissue weights. Metalaxyl was negative for androgenicity and anti-androgenicity in the 

Hershberger assay. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Hershberger assay (OCSPP 

890.1400). 

Page 214 of 292
In vivo Hershberger Assay (2011) / Page 3 of 16 

METALAXYL / PC Code 113501 OCSPP 890.1400/ OECD 441

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

Assurance statements were provided. 

Page 215 of 292
In vivo Hershberger Assay (2011) / Page 4 of 16 

METALAXYL / PC Code 113501 OCSPP 890.1400/ OECD 441

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Charles River Laboratories, Preclinical Services, 

Pennsylvania (PCS-PA) 

Location: Horsham, PA 

Study Director: M.B. Carlson (June 29, 2011); A.M. Hoberman (previous) 

Other Personnel: K. Strauss (Principal Investigator) 

Study Period: June 24, 2011 to December 9, 2011 

2. Test Substance: Metalaxyl 

 Description: White to beige powder 

 Source: Nation’s Ag, LLC (Knoxville, TN) 

 Lot/Batch #: 20100540 (expiry date: June 13, 2012) 

 Purity: 98.2% a.i. 

 Stability: Stable in vehicle at room temperature for 25 hours and under refrigerated 

conditions for 10 days 

 CAS #: 57837-19-1 

 Structure: 

3CH

N

O

CH3

O

3CH CH3

O O

3

CH

3. Reference Androgen: Testosterone propionate (TP) 

 Supplier: PCCA USA (Houston, TX) 

 Item #: 

Lot/Batch #: 

23-1029 

C141154 (expiry date: May 19, 2013) 

 Purity: 99.7% 

 CAS #: 57-85-2 

4. Reference Anti-androgen: Flutamide (FT) 

 Supplier: 

Product #: 

Tokyo Chemical Industry (TCI), Co., LTD (Tokyo, Japan) 

F0663 

 Lot/Batch #: 7EDOD (expiry date: January 12, 2012) 

 Purity: 99.8% 

 CAS #: 1311-84-7 

5. Solvent/Vehicle Control 

(test substance): 

Carboxymethylcellulose (CMC) 

 Supplier: 

Product #: 

Sigma Aldrich (St. Louis, MO) 

C5678 (sodium salt, low viscosity) 

 Lot/Batch #: 060M0079V (expiry (retest) date: June 30, 2012) 

 Rationale (if other than water): Use of an aqueous solution/suspension is recommended and an analytical 

method is available. 

 Final concentration: 2.0% in reverse osmosis membrane-processed deionized water 

Page 216 of 292
In vivo Hershberger Assay (2011) / Page 5 of 16 

METALAXYL / PC Code 113501 OCSPP 890.1400/ OECD 441

 Solvent/Vehicle Control 

(reference substances):

Corn oil 

 Supplier: Charkit Chemical Corporation 

 Lot/Batch #: M-631 (expiry date: March 18, 2012) 

 Rationale (if other than water): Not applicable 

 Final concentration: Not applicable 

6. Test Animals: 

 Species: Rat (castrated peripubertal males) 

 Strain: Sprague Dawley, Crl:CD(SD) 

 Age/weight at dose initiation: Post-natal day (PND) 54-60; 213.4 – 271.8 g 

 Source: Charles River Laboratories (Portage, MI) 

 Housing: 2-3 rats/cage in clear plastic nesting boxes with heat-treated laboratory-grade 

pine shavings 

 Diet: Harlan’s Teklad 2018c meal food or Certified RodentDiet® 5002 (PMI® 

Nutrition International, St. Louis, MO), ad libitum; total genistein 

compounds were ≤213 ppm 

Chewable Nylabones® were supplied during the study.

 Water: Reverse-osmosis treated tap water, ad libitum

 Environmental conditions: Temperature: 

Humidity: 

Air changes: 

Photoperiod: 

19-25oC 

30-70% 

>10/hr 

12 hrs light/12 hrs dark 

 Acclimation period: 7 days at facility (androgen assay); 12 days post-castration 

6 days at facility (anti-androgen assay); 11 days post-castration 

B. STUDY DESIGN

1. In-life dates: Start: June 28, 2011 End: July 21, 2011 

2. Study Design: In a Hershberger Assay conducted to screen for potential androgenic 

activity, metalaxyl was administered daily via oral gavage to castrated male rats. A positive 

control group received testosterone propionate (TP). Positive androgenic activity is defined 

as a significant increase in two or more target organ weights compared to the vehicle control 

group. To screen for the potential anti-androgenic activity, metalaxyl was administered 

daily via oral gavage to groups of castrated male rats in conjunction with TP at 

0.4 mg/kg/day by s.c. injection. The positive control group received FT plus TP. 

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 measurement of 

target organ weights. 

3. Study Schedule: Castrated sexually mature male rats (castrated by the supplier on PND 43, 

45, or 49) were received at the laboratory at PND 50 (androgen assay) or PND 48 or 54 

(anti-androgen assay). Dosing was initiated following a 6- or 7-day acclimation period. 

The dose administration period was from PND 57 to 66 (androgen assay), or PND 54 to 63 

or PND 60 to 69 (anti-androgen assay). Rats were euthanized on PND 67, 64, or 70, 

respectively, approximately 24 hours after the last dose and necropsied for organ weight 

measurements. 

4. Animal Assignment: Animals were assigned, stratified by body weight, to the test groups 

noted in Table 1 using computer-generated (weight-ordered) randomization procedures. 

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Statistical analysis indicated that there were no significant differences in group means at 

study initiation. Furthermore, the body weight of each animal was within 20% of the 

group mean. 

TABLE 1. Study Design a

Test group Dose (mg/kg/day) # of Males 

Androgen Agonist Assay 

Vehicle control (2% aqueous CMC) 0 8 

Metalaxyl Low 125 8 

Metalaxyl High 375 8 

Testosterone propionate (TP), positive control 0.4 8 

Anti-Androgen Assay 

Vehicle control (2% aqueous CMC) + TP 0 + 0.4 8 

Corn Oil Vehicle + TP 0 + 0.4 8 

Metalaxyl Low (+TP) 40 + 0.4 8 

Metalaxyl Mid (+TP) 125 + 0.4 8 

Metalaxyl High (+TP) 375 + 0.4 8 

Flutamide + TP, positive control 3 + 0.4 8 

a Data were obtained from Text Tables 9 and 10 on pages 25-26 of the study report. 

5. Dose Selection Rationale: The dose levels were selected based on the results from rangefinding studies (Protocols 20012405 and 20013495); citations for the studies were not 

provided. Clinical signs of systemic toxicity were observed in adult male and female rats 

and weanlings at 500 mg/kg/day; mortality was seen in adult female rats at >500 mg/kg/day 

and in weanlings of both sexes at 750 mg/kg/day. Body weight gain was decreased at the 

two highest dose levels in adult male rats (750 and 1000 mg/kg/day) and liver weights were 

increased at all dose levels in the adult male rats (250-1000 mg/kg/day) and in weanlings of 

both sexes (250 and 500 mg/kg/day). Due to the severity of the clinical signs observed in 

adult rats at >500 mg/kg/day, the high dose selected for the Hershberger assay was 375 

mg/kg/day. The next lower dose of 125 mg/kg/day represents one-third of the high dose, 

and the low dose for the anti-androgen assay (40 mg/kg/day) equals one-third of the 125 

mg/kg/day dose.

6. (a) Dose Preparation: Metalaxyl dose formulations were prepared weekly by mixing 

appropriate amounts of test substance with 2% aqueous CMC; daily aliquots were dispensed 

and stored refrigerated until use. Prepared doses were allowed to return to room 

temperature and stirred for at least 30 minutes prior to dosing. Reference materials (TP and 

FT) were prepared daily in corn oil. 

Homogeneity and stability of metalaxyl formulations (0.5 and 100 mg/mL) in 2% aqueous 

CMC were previously evaluated in conjunction with Protocol 20012404 (report attached as 

Appendix 5 of the subject study). During the Hershberger study, duplicate samples of dose 

formulations (8, 25, and 75 mg/mL) were collected on the first and last days of preparation, 

and analyzed for achieved concentration. 

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(b) Dose Analysis: 

Results 

Homogeneity (% RSD): 0.4% and 1.5% 

Stability (% of Day 0): 101.3% and 101.8% following storage protected from light at 

room temperature (20-25°C) for 25 hours; 99.7% and 100% 

following storage under refrigerated conditions (2-8°C) for 10 days 

Concentration (% of nominal): 99.1-106.7% 

The analytical data indicated that the mixing procedure was adequate and that the variation 

between nominal and actual dosage to the animals was acceptable. 

7. Dosage administration: Metalaxyl formulations were administered to the animals daily via 

oral gavage (5 mL/kg) for 10 days. TP was given via s.c. injection at 0.5 mL/kg; injection 

sites were rotated between six areas on the dorsum to minimize irritation. FT was 

administered via oral gavage at 5 mL/kg. Dose volumes were adjusted daily based on the 

most recently recorded body weight and administered at approximately the same time each 

day to each animal. 

8. Statistics: Means, standard deviations, coefficients of variance, and percentages were 

calculated. Statistical analyses were performed using SAS System, version 6.12 or Quattro 

Pro 8, version 8.0. Clinical observations and other proportional data were analyzed using 

the variance test for homogeneity of the binomial distribution. 

Continuous data (e.g., body weights, body weight changes, food consumption values, and 

organ weight data) were analyzed using Bartlett’s test of homogeneity of variances and the 

analysis of variance when appropriate [i.e., Bartlett’s test was not significant (p>0.001)]. If 

the analysis of variance was significant (p≤0.05), Dunnett’s test was used to identify the 

statistical significance of the individual groups. If the analysis of variance was not 

appropriate [i.e., Bartlett’s test was significant (p≤0.001)], the Kruskal-Wallis test was used 

(≤75% ties). In cases where the Kruskal-Wallis test was statistically significant (p≤0.05), 

Dunn’s method of multiple comparisons was used to identify the statistical significance of 

the individual groups. If there were >75% ties, Fisher’s Exact test was used to analyze the 

data. Data from the evaluation for preputial separation were analyzed using Fisher’s Exact 

test. Absolute organ weights were also analyzed by analysis of covariance (ANCOVA), 

using the initial body weight from the first day of treatment as the covariate. Relative organ 

weight comparisons were not made. The statistical analyses were considered adequate. 

C. METHODS

1. Clinical Examinations: Animals were assessed for viability twice daily. General 

appearances were observed on the day of receipt, at least weekly during the acclimation 

period and daily before administration during the dosing period. Additionally post-dose 

observations were recorded within 2 hours of dosing and at the end of the normal working 

day. Preputial separation was recorded once on the day of termination. 

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2. Body Weight: Animals were weighed weekly during the acclimation period, at 

randomization, daily throughout the dosing period, and at termination. 

3. Food Consumption: Food consumption was measured daily during dosing and at 

termination. Values were reported as group mean daily food consumption (g/animal/day). 

4. Serum Hormone Measurements (Optional): At study termination, each animal was 

euthanized by decapitation and blood samples were collected from trunk blood 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, 67, or 70

(approximately 24 hours after the final administration of the test substance), all animals 

were euthanized by decapitation and subjected to a gross necropsy. The five mandatory 

androgen-dependent organs (ventral prostate, seminal vesicles, LABC, Cowper’s gland, and 

glans penis) were excised, trimmed free of adhering tissue, and weighed fresh (unfixed) 

according to the standard operating procedures detailed in the U.S. EPA Guideline (OCSPP 

890.1400). Additionally, the liver and kidneys were collected, trimmed of any adhering 

fascia or fat, and weighed. Representative samples of Cowper’s glands, seminal vesicles, 

ventral prostate, kidneys, and liver were preserved in 10% neutral buffered formalin for 

possible future histopathological evaluation. In addition, a portion of the liver was flash 

frozen for possible future analysis. 

II. RESULTS

A. OBSERVATIONS

1. Mortality: All animals survived until scheduled termination. 

2. Clinical signs of toxicity: No adverse clinical signs of toxicity were observed in the 

androgen agonist assay. In the anti-androgen assay, three rats from the 375 mg/kg/day 

group had urine-stained abdominal fur, which was increased (p≤0.01) in comparison with 

the 2% CMC vehicle control group (0 occurrences). Other clinical signs observed in the 

corn oil vehicle group (red fur on back; ungroomed coat; and chromorhinorrhea) and in the 

125 mg/kg/day metalaxyl group (red fur and scabbing on back) were considered to be 

unrelated to treatment because a dose-dependent pattern of effect was not observed, the 

clinical sign was attributed to the TP injection site, and/or the clinical sign was only 

observed in the corn oil vehicle control group. 

B. BODY WEIGHT AND WEIGHT GAIN: Selected body weight and body weight gain 

data for the androgen agonist assay are presented in Table 2. Mean body weights and body 

weight gains were comparable to the vehicle controls throughout the duration of the assay. 

Mean body weights were increased (p≤0.05) by 7-9% on Days 7-11 in the TP positive 

control group compared to the vehicle controls, resulting in an increase (p≤0.01) of 31% in 

overall (Days 1-11) body weight gains.

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TABLE 2. Selected Group Mean Body Weights and Cumulative Body Weight Gains (g) in the Androgen 

Agonist Assay a

Study Day 

Dose (mg/kg/day) 

Vehicle Control TP (0.4) Metalaxyl (125) Metalaxyl (375) 

N Mean SD N Mean SD N Mean SD N Mean SD 

1 8 229.8 12.2 8 237.6 18.0 8 232.5 14.2 8 233.4 15.1 

4 8 248.7 16.0 8 260.8 19.1 8 252.3 15.7 8 249.4 16.1 

7 8 266.1 16.6 8 285.2* 

(↑7) 

18.5 8 271.4 15.3 8 271.3 17.8 

8 8 270.1 16.3 8 294.4** 

(↑9) 

19.7 8 276.5 16.7 8 275.8 18.2 

9 8 276.5 16.5 8 300.6** 

(↑9) 

18.3 8 283.5 15.8 8 282.0 19.6 

10 8 281.3 15.6 8 307.4** 

(↑9) 

19.7 8 288.1 16.7 8 286.6 19.1 

11 8 293.0 15.6 8 320.6** 

(↑9) 

20.4 8 298.7 16.1 8 299.7 20.1 

Body Weight Gain 

(Days 1-11) 

8 63.2 7.4 8 83.0** 

(↑31) 

7.7 8 66.2 5.9 8 66.3 7.2 

a Data were obtained from Tables 2 and 3 on pages 52 and 54 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≤0.05 

** Significantly different from controls at p≤0.01

Selected body weight and body weight gain data for the anti-androgen assay are presented in 

Tables 3a and 3b. Mean body weights of the metalaxyl treatment groups were comparable 

to the vehicle controls throughout the duration of the assay. Significant reductions (p≤0.05) 

in mean body weight gains occurred on Days 1 to 2 in the 375 mg/kg/day group and on 

Days 4 to 5 in the 40 and 375 mg/kg/day groups, and a significant increase (p≤0.01) 

occurred on Days 3 to 4 in the 375 mg/kg/day group. These fluctuations in mean body 

weight gain contributed to non-statistically significant body weight gain reductions (90% to 

91% of the metalaxyl vehicle control group value) during the overall study period in the 40, 

125, and 375 mg/kg/day groups. 

Mean body weights of the FT positive control group and overall mean body weight gains 

were comparable to the corn oil vehicle controls.

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TABLE 3a. Selected Group Mean Body Weights and Cumulative Body Weight Gains (g) in the AntiAndrogen Assay a

Study Day 

Dose (mg/kg/day) 

Vehicle Control + 

TP (0.4) 

Metalaxyl (40) 

+ TP (0.4) 

Metalaxyl (125) 

+ TP (0.4) 

Metalaxyl (375) 

+ TP (0.4) 

N Mean SD N Mean SD N Mean SD N Mean SD 

1 8 245.0 11.7 8 244.6 12.0 8 243.3 14.3 8 245.3 14.4 

4 8 271.8 12.6 8 273.5 14.6 8 271.3 16.1 8 275.4 16.1 

8 8 306.2 13.5 8 301.5 13.7 8 300.6 20.0 8 302.0 18.0 

11 8 342.4 19.4 8 332.6 17.3 8 332.3 21.0 8 333.4 20.9 

Body Weight Gain 

(Days 1-11) 

8 97.4 11.7 8 88.0 8.8 8 89.0 7.9 8 88.1 10.2 

a Data were obtained from Tables 8 and 9 on pages 64 and 66 of the study report. 

N Number of animals in the group 

SD Standard Deviation

TABLE 3b. Selected Group Mean Body Weights and Cumulative Body Weight Gains (g) in the AntiAndrogen Assay a

Study Day 

Dose (mg/kg/day) 

Corn Oil Vehicle Control + TP (0.4) Flutamide (3) + TP (0.4) 

N Mean SD N Mean SD 

1 8 245.8 11.0 8 243.4 10.9 

4 8 272.8 13.9 8 270.6 13.0 

8 8 304.8 18.0 8 296.2 15.2 

11 8 338.5 22.5 8 326.3 16.7 

Body Weight Gain (Days 1-11) 8 92.7 12.7 8 82.9 8.2 

a Data were obtained from Tables 8 and 9 on pages 64 and 66 of the study report. 

N Number of animals in the group 

SD Standard Deviation

C. FOOD CONSUMPTION: Select food consumption values for the androgen agonist assay 

are presented in Table 4. Although increased (p≤0.05) food consumption was observed on 

Days 4-5, Days 5-6, and Days 7-8 for the 375 mg/kg/day group (↑8-12%) and fluctuations 

(p≤0.05) in food consumption were observed on Days 4-5 (↑6%), Days 6-7 (↓6%), and Days 

10-11 (↓5%) in the 125 mg/kg/day group, overall (Days 1-11) average food consumption for 

both metalaxyl treatment groups were comparable to the vehicle controls. 

Food consumption in the TP positive control group was increased (p≤0.05) starting on Day 

4 through the dosing period (↑6-18%), for an increase (p≤0.01) of 10% in average overall 

food consumption compared to the vehicle control. 

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TABLE 4. Selected Food Consumption (g/rat/day) in the Androgen Agonist Assay a

Study Day 

Dose (mg/kg/day) 

Vehicle Control TP (0.4) Metalaxyl (125) Metalaxyl (375) 

N Mean SD N Mean SD N Mean SD N Mean SD 

4-5 8 19.0 1.2 8 22.4** 

(↑18) 

1.0 8 20.2* 

(↑6) 

1.3 8 20.8** 

(↑9) 

1.1 

5-6 8 21.0 1.4 8 22.8* 

(↑9) 

1.8 8 20.5 1.1 8 23.6* 

(↑12) 

3.2 

6-7 8 21.4 1.2 8 24.1** 

(↑13) 

1.4 8 20.1* 

(↓6) 

1.0 8 21.4 1.0 

7-8 8 20.5 1.6 8 24.1** 

(↑18) 

0.7 8 20.6 0.9 8 22.2* 

(↑8) 

1.4 

9-10 8 22.9 1.0 8 26.5** 

(↑16) 

0.8 8 22.1 1.2 8 22.9 1.5 

10-11 8 21.4 0.4 8 22.6* 

(↑6) 

1.3 8 20.4* 

(↓5) 

0.7 8 21.6 1.3 

(Days 1-11) 8 20.6 0.7 8 22.7** 

(↑10) 

0.8 8 20.1 0.3 8 20.9 1.0 

a Data were obtained from Table 4 on page 56 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≤0.05 

** Significantly different from controls at p≤0.01

Select food consumption values for the anti-androgen assay are presented in Tables 5a and 

5b. Decreased (p≤0.05) food consumption was observed on Days 1-2, Days 4-5, and Days 

8-9 for the 125 mg/kg/day group (↓6-28%); and Days 4-5 and Days 8-9 for the 40 

mg/kg/day group (↓7% and 8%). In the 375 mg/kg/day group, food consumption was 

increased (p≤0.05) on Days 3-4 and 9-10 (↑9% and 11%) and decreased (p≤0.01) on Days 

1-2 and 4-5 (↓33% and 8%). Overall average food consumption was decreased (p≤0.01) 

only in animals treated with metalaxyl at 125 mg/kg/day (↓6%) compared to the vehicle 

control. 

Food consumption in the FT positive control group was decreased (p≤0.05) starting on Day 

2 through the dosing period (↓6-14%), for a decrease (p≤0.01) of 7% in overall average food 

consumption compared to the corn oil vehicle control. 

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TABLE 5a. Selected Food Consumption (g/rat/day) in the Anti-Androgen Assay a

Study Day 

Dose (mg/kg/day) 

Vehicle Control 

 + TP (0.4) 

Metalaxyl (40) 

+ TP (0.4) 

Metalaxyl (125) 

+ TP (0.4) 

Metalaxyl (375) 

+ TP (0.4) 

N Mean SD N Mean SD N Mean SD N Mean SD 

1-2 8 24.9 6.5 8 20.4 1.9 8 17.9* 

(↓28) 

1.4 8 16.6** 

(↓33) 

1.2 

3-4 8 21.2 0.8 8 21.8 1.9 8 20.6 1.1 8 23.1* 

(↑9) 

1.2 

4-5 8 25.1 1.0 8 23.4* 

(↓7) 

1.4 8 22.2** 

(↓12) 

1.7 8 23.0** 

(↓8) 

1.2 

8-9 8 25.4 0.6 8 23.4** 

(↓8) 

1.2 8 23.9** 

(↓6) 

0.4 8 25.2 1.3 

9-10 8 24.9 0.9 8 25.8 0.6 8 25.6 1.5 8 27.6** 

(↑11) 

1.4 

(Days 1-11) 8 24.6 1.2 8 23.7 1.0 8 23.2** 

(↓6) 

0.6 8 23.9 0.6 

a Data were obtained from Table 10 on page 68 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≤0.05 

** Significantly different from controls at p≤0.01

TABLE 5b. Selected Food Consumption (g/rat/day) in the Anti-Androgen Assay a

Study Day 

Dose (mg/kg/day) 

Corn Oil Vehicle Control + TP (0.4) Flutamide (3) + TP (0.4) 

N Mean SD N Mean SD 

2-3 8 21.6 0.8 8 19.2** 

(↓11) 

0.5 

4-5 8 21.6 1.9 8 19.5** 

(↓10) 

1.0 

6-7 8 24.0 1.0 8 22.4** 

(↓7) 

0.4 

7-8 8 22.8 1.3 8 19.0** 

(↓17) 

1.0 

8-9 8 23.1 1.4 8 19.8** 

(↓14) 

1.2 

9-10 8 24.4 1.5 8 21.9** 

(↓10) 

0.7 

10-11 8 22.1 1.4 8 20.8* 

(↓6) 

0.8 

(Days 1-11) 8 21.9 0.2 8 20.3** 

(↓7) 

0.5 

a Data were obtained from Table 10 on page 68 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≤0.05 

** Significantly different from controls at p≤0.01

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D. SERUM HORMONE CONCENTRATIONS (Optional): Serum hormone concentrations 

were not determined. 

E. ORGAN WEIGHTS: Accessory sex organ, liver, and kidney weights for the androgen 

agonist assay are presented in Table 6. There were no treatment-related effects in any of the 

accessory sex organs or kidneys of animals dosed with metalaxyl. However, mean liver 

weights were increased (p≤0.01) by 17% in the 375 mg/kg/day group compared to the 

vehicle controls. The study author reported that increased liver weights were consistent 

with results from other studies conducted with metalaxyl. No gross lesions were observed 

at necropsy, but one animal in the 375 mg/kg/day group did not achieve preputial separation 

by necropsy. 

Animals in the positive control (TP) group had increased (p≤0.01) accessory sex organ 

weights as follows: 500% in seminal vesicles; 691% in ventral prostate; 177% in LABC; 

645% in Cowper’s glands; and 71% in glans penis. Liver and kidney weights in the positive 

control group were similar to the vehicle controls. 

The %CV values for each accessory sex organ of the vehicle control and metalaxyl high 

dose group were less than the maximum Guideline-specified %CV values. 

TABLE 6. Accessory Sex Organ, Liver, and Kidney Weights (g) from Androgen Agonist Assay in Sprague 

Dawley Rats a

Organ 

Dose (mg/kg/day) 

Vehicle Control Metalaxyl (125) Metalaxyl (375) TP (0.4) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%)

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

Seminal 

vesicles 

8 0.0853 0.0173 20 8 0.0729 0.0168 23 8 0.0925 0.0122 13 8 0.5121** 

(↑500) 

0.1130 22 

Ventral 

prostate 

8 0.0242 0.0063 26 8 0.0276 0.0070 25 8 0.0232 0.0081 35 8 0.1914** 

(↑691) 

0.0271 14 

LABC 8 0.2247 0.0298 13 8 0.2003 0.0357 18 8 0.2092 0.0248 12 8 0.6224** 

(↑177) 

0.0549 9 

Cowper’s 

glands 

8 0.0074 0.0022 30 8 0.0074 0.0025 34 8 0.0070 0.0012 17 8 0.0551** 

(↑645) 

0.0337 61 

Glans penis 8 0.0631 0.0085 14 8 0.0573 0.0083 15 8 0.0618 0.0056 9 8 0.1079** 

(↑71) 

0.0097 9 

Liver 8 11.7 1.6 14 8 12.3 1.2 10 8 13.7**

(↑17) 

1.3 10 8 13.1 1.8 14 

Kidneys 8 2.02 0.16 8 8 1.87 0.14 7 8 2.04 0.16 8 8 2.13 0.11 5 

a Data were obtained from Table 6 on page 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 

CV Coefficient of Variation 

* Significantly different from controls at p<0.05 

** Significantly different from controls at p<0.01 

Accessory sex organ, liver, and kidney weights for the anti-androgen assay are presented in 

Tables 7a and 7b. There were no treatment-related effects in any of the accessory sex 

organs or kidneys of animals dosed with metalaxyl. However, mean liver weights were 

increased (p≤0.01) by 13% in the 375 mg/kg/day group compared to the vehicle controls. 

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No gross lesions were observed at necropsy, but the left Cowper’s gland was not present in 

one animal in the 40 mg/kg/day group. All animals achieved preputial separation by 

necropsy. 

Animals in the FT group had decreased (p≤0.01) accessory sex organ weights as follows: 

78% in seminal vesicles; 70% in ventral prostate; 44% in LABC; 53% in Cowper’s gland; 

and 19% in glans penis. 

The %CV values for each accessory sex organ of the vehicle control and metalaxyl high 

dose group were less than the maximum guideline-specified %CV values, except for the 

%CV for glans penis of the vehicle control group (CV = 18%; maximum permissible CV = 

17%). 

TABLE 7a. Accessory Sex Organ, Liver, and Kidney Weights (g) from Anti-Androgen Assay in Sprague 

Dawley Rats a

Organ 

Dose (mg/kg/day) 

Vehicle Control 

+ TP (0.4) 

Metalaxyl (40) 

+ TP (0.4) 

Metalaxyl (125) 

+TP (0.4) 

Metalaxyl (375) 

+ TP (0.4) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%)

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

Seminal 

vesicles 

7 b 0.5628 0.0984 18 8 0.4942 0.1168 24 8 0.4598 0.0914 20 8 0.5547 0.0769 14 

Ventral 

prostate 

8 0.2494 0.0777 31 8 0.1985 0.0356 18 8 0.1799 0.0428 24 8 0.2119 0.0612 29 

LABC 8 0.7214 0.0782 11 8 0.6827 0.0859 13 8 0.6885 0.0338 5 8 0.6900 0.0562 8 

Cowper’s 

glands 

7 b 0.0513 0.0097 19 7 c 0.0510 0.0072 14 8 0.0496 0.0075 15 8 0.0530 0.0123 23 

Glans penis 8 0.1401 0.0254 18 d 8 0.1412 0.0167 12 8 0.1406 0.0122 9 8 0.1315 0.0186 14 

Liver 8 13.4 0.8 6 8 12.9 0.8 6 8 12.8 1.0 8 8 15.1** 

(↑13) 

1.6 11 

Kidneys 8 2.34 0.18 8 8 2.28 0.20 9 8 2.27 0.18 8 8 2.29 0.16 7 

a Data were obtained from Table 12 on page 72 of the study report. Percent differences from controls (calculated by the 

reviewers) are included in parentheses. 

b Excluded values that appeared incorrectly recorded. 

c Excluded values from one rat which only had the right Cowper’s gland. 

d Percent CV exceeded the maximum allowable limit. 

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 226 of 292
In vivo Hershberger Assay (2011) / Page 15 of 16 

METALAXYL / PC Code 113501 OCSPP 890.1400/ OECD 441

TABLE 7b. Accessory Sex Organ, Liver, and Kidney Weights (g) from Anti-Androgen Assay in Sprague 

Dawley Rats a

Organ 

Dose (mg/kg/day) 

Corn Oil Control 

+ TP (0.4) FT (3) + TP (0.4) 

N Mean SD CV (%) N Mean SD CV (%) 

Seminal vesicles 8 0.5063 0.0728 14 8 0.1125** 

(↓78) 

0.0383 34 

Ventral prostate 8 0.1980 0.0443 22 8 0.0597** 

(↓70) 

0.0107 18 

LABC 8 0.6567 0.0554 8 8 0.3699** 

(↓44) 

0.0467 13 

Cowper’s glands 8 0.0475 0.0055 12 8 0.0221** 

(↓53) 

0.0054 24 

Glans penis 8 0.1504 0.0148 10 8 0.1218** 

(↓19) 

0.0185 15 

Liver 8 13.5 1.3 10 8 12.6 1.0 8 

Kidneys 8 2.26 0.21 9 8 2.14 0.09 4 

a Data were obtained from Table 12 on page 72 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 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: On the basis this bioassay, administration of 

metalaxyl at dose levels as high as 375 mg/kg/day did not result in an androgenic or antiandrogenic response.

B. AGENCY COMMENTS: All animals survived until scheduled termination. No adverse 

clinical signs of toxicity were observed in the androgen agonist assay. In the anti-androgen 

assay, treatment related clinical signs of toxicity (urine-stained abdominal fur) were 

observed in three rats from the 375 mg/kg/day group. 

In the androgen agonist assay, mean body weights and body weight gains in the metalaxyl 

treatment groups were comparable to the vehicle controls throughout the duration of the 

assay. Animals in the positive control (TP) group had increased (p≤0.01) overall (Days 1-

11) body weight gains (↑31%) compared to the vehicle controls. Although significant 

fluctuations in food consumption was observed on several days for the 375 and 125 

mg/kg/day groups, overall average food consumption for both metalaxyl treatment groups 

was comparable to the vehicle controls. Overall average food consumption in the positive 

control group was increased (p≤0.01) by 10%. No treatment-related effects were observed 

in any of the accessory sex organs or kidneys of metalaxyl-dosed animals. However, mean 

liver weights were increased (p≤0.01) by 17% in the 375 mg/kg/day group compared to the 

vehicle controls, which is reportedly consistent with results from other studies conducted 

with metalaxyl. No gross lesions were observed at necropsy, but one animal in the 375 

mg/kg/day group did not achieve preputial separation by necropsy. Animals in the TP group 

Page 227 of 292
In vivo Hershberger Assay (2011) / Page 16 of 16 

METALAXYL / PC Code 113501 OCSPP 890.1400/ OECD 441

had the expected increases in accessory sex organ weights. The performance criteria 

indicated that this assay was performing as expected. 

In the anti-androgen assay, mean body weights and overall body weight gains of the 

metalaxyl treatment groups were comparable to the vehicle controls. Animals dosed with 

TP + FT (positive control) had mean body weights and overall mean body weight gains 

comparable to the corn oil vehicle controls. Although significant (p≤0.05) decreases in food 

consumption were observed on several days for the 40, 125, and 375 mg/kg/day groups, 

overall average food consumption was decreased (p≤0.01) only in animals treated with 

metalaxyl at 125 mg/kg/day (↓6%). Overall average food consumption in the TP + FT 

group was decreased (p≤0.01) by 7% compared to the corn oil vehicle control. No 

treatment-related effects were observed in any of the accessory sex organs or kidneys of 

metalaxyl-dosed animals. However, mean liver weights were increased (p≤0.01) by 13% in 

the 375 mg/kg/day group compared to the vehicle controls. No gross lesions were observed 

at necropsy, but the left Cowper’s gland was not present in one animal in the 40 mg/kg/day 

group. Animals in the TP + FT group had the expected decreases in accessory sex organ 

weights. 

The %CV values for each accessory sex organ of the vehicle control and metalaxyl high 

dose group were less than the maximum guideline-specified %CV values, except for the 

%CV for glans penis of the vehicle control group (CV = 18%; maximum permissible CV = 

17%).

The high dose adequately challenged the animals based on data from earlier studies showing 

clinical signs of systemic toxicity at doses ≥500 mg/kg/day and the increased liver weights 

observed at 375 mg/kg/day in the subject assay.

No statistically significant changes were seen in two or more of the five androgen sensitive 

tissue weights. Glyphosate was negative for androgenicity and anti-androgenicity in the 

Hershberger assay. 

C. STUDY DEFICIENCIES: None 

Page 228 of 292
DATA EVALUATION RECORD 

METALAXYL 

Study Type: OCSPP 890.1450, Female Pubertal Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 3-19-2012 (MRID 48701301) 

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:

Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 9/18/2012 

Secondary Reviewer: Signature:

Scott D. Studenberg, Ph.D., D.A.B.T. Date: 9/27/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 10/02/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 10/02/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 229 of 292
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 230 of 292
METALAXYL/ 113501 

Female Pubertal Assay (2012) I Page 1 of 17 

OCSPP 890.1450/ OECD None 

Primary Reviewer: Vincent Chen. M.S. Signature:~....!::::::....!:""""""=::..._-~ __ 

Risk Assessment Branch III, Health Effects Division (7509P) Date::=~'Z...~Lt~~ijl~~ 

Secondary Reviewer: Elizabeth Mendez, Ph.D. Signaturlt! 

Health Effects Division (7509P) Date: ----"'+-"~"'---

! DATAEVALUATIONRECORD 

STUDY TYPE: Female Pubertal Assay; OCSPP 890.1450; OECD None. 

PC CODE: 113501 DP BARCODE: D404681 

TXR#: 0054219 CAS #: 57837-19-1 

TEST MATERIAL (PURITY): Metalaxyl (>98%) 

SYNONYMS: Methyl N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alaninate 

CITATION: Carlson, M.B. (2012). A Pubertal Development and Thyroid Function Study 

ofMetalaxyl by Oral Gavage in Peripubertal Male and Female Rats. Charles 

River Laboratories Preclinical Services, Pennsylvania; Horsham, PA. 

Laboratory Study No.: 20012408, July 17, 2012. MRID 48701301. 

Unpublished. 

SPONSOR: Metalaxyl EDSP Consortium, 20 Ambriance Drive, Burr Ridge, IL 

TEST ORDER#: CON-113501-18 

EXECUTIVE SUMMARY: In a Female Pubertal Assay (MRID 48701301), 15 female 

Sprague-Dawley (Crl:CD(SD)) rats/dose group were treated daily via oral gavage with metalaxyl 

(>98% a.i., Lot# 20100540) in 2.0% aqueous carboxymethylcellulose at doses of 0, 125, 250, or 

350 mg/kg/day from postnatal day (PND) 22 to 42 or 43. Animals were examined for vaginal 

opening (VO) daily beginning on PND 22, and age and weight at day of attainment were 

recorded. Following sacrifice on PND 42 or 43, blood was collected for clinical chemistry 

analyses, and total thyroxine (T 4) and thyroid stimulating hormone (TSH) levels were 

determined using radioimmunoassays. Adrenal, liver, pituitary, thyroid, and urogenital organs 

were weighed and examined macroscopically. The uterus, ovary, thyroid, and kidney were 

examined microscopically. 

No treatment-related related effects were observed on survival, clinical signs, body weights, 

body weight gains, or gross pathology. No treatment-related histological lesions were noted in 

the thyroid or uterus, and the colloid area and follicular cell height in the thyroids of the treated 

groups were generally similar to control. Age of attainment of VO, body weight at VO, and 

proportion/incidence of unopened vagina in the treated groups were similar to control animals. 

No treatment-related effect was observed on the mean age of first vaginal estrus, mean cycle 

length,% cycling,% regularly cycling, and the cycle status at necropsy. Serum T4 and TSH 

levels in the treated groups were similar to the control values. 

Page 231 of 292
Female Pubertal Assay (2012) / Page 2 of 17 

METALAXYL / 113501 OCSPP 890.1450/ OECD None

The absolute and relative liver weights were increased (p≤0.05 or 0.01) dose-dependently in all 

treated groups by 8-23% compared to control. Gamma-glutamyl transpeptidase levels were 

increased (p≤0.01) by 175 and 305% at 250 and 350 mg/kg/day, respectively; however, the 

serum levels were reported to be within the historical control range. In the ovaries, total primary 

follicle counts were increased (p≤0.05 or ≤0.01) by 43 and 57% at 250 and 350 mg/kg/day, 

respectively, but atretic follicle counts in the treated groups were similar to control values. 

The high dose (350 mg/kg/day) was determined to be adequate based on generalized summaries 

from several ranging-finding studies and findings in the previously reviewed uterotrophic study 

(MRID 48617606) which showed decreased motor activity, ptosis, ataxia, lacrimation, and 

hunched posture in female rats in the 300 mg/kg/day dose group. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Female Pubertal Assay 

(OCSPP 890.1450). 

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

and Flagging statements were provided. 

 

Page 232 of 292
Female Pubertal Assay (2012) / Page 3 of 17 

METALAXYL / 113501 OCSPP 890.1450/ OECD None

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Charles River Laboratories Preclinical Services, Pennsylvania 

Location: Horsham, PA 

Study Directors: M.B. Carlson 

Other Personnel: Julian Gulbinski, III,(dose formulation analysis and laboratory manager), Dr. Saroj Das 

(hormone analysis), Carol S. Sloan (hormone analysis), Rebecca M. Lucke (clinical 

chemistry), Carol J. Detrisac (histology and histopathology) and Frederick T. Les (ovarian 

follicle counts) 

Study Period: June 17, 2011 through July 17, 2012 

2. Test Substance: Metalaxyl 

 Description: White to beige powder 

 Source: Nation’s Ag, LLC, Knoxville, TN 

 Lot #: 20100540 (expires June 13, 2012 and July 26, 2011) 

 Purity: 98.2±0.3% and 100.4±0.7% 

 Stability: Protected from light, the test compound was stable in the vehicle at 20-25°C for at least 25 

hours and at 2-8°C for 10 days. 

 CAS #: 57837-19-1 

 Structure: 

3CH

N

O

CH3

O

3CH CH3

O O

3

CH

3. Vehicle: 

Source: 

Catalog #: 

Lot #: 

2.0% aqueous carboxymethylcellulose in reverse osmosis 

membrane-processed deionized water 

Sigma Aldrich, St. Louis, MO 

C5678 

060M0079V (retest date: June 30, 2012)

4. Test Animals:

Species: Rat (females) 

Strain: Sprague-Dawley (Crl:CD(SD)) 

Age/ Mean Weight 

at Study Initiation: PND 22 / 59.6-60.2 g 

Source: Charles River Laboratories International, Inc. (Portage, MI) 

Housing: Parental dams were individually housed in clear plastic nesting boxes (n=2/box) with heattreated, laboratory-grade, pine shaving bedding; each dam was housed with her litter until 

weaning on PND 21. After weaning, 2 females were housed per cage. 

Diet: Harlan Teklad 2018c meal feed (Teklad Diets, Madison, WI), ad libitum. It was stated that the 

total genistein equivalents (aglycone) or daidzein were ≤300 ppm. 

Water: Reverse osmosis-purified and deionized tap water, ad libitum

 Environmental 

Conditions: 

Temperature: 

Humidity: 

Air changes: 

Photoperiod: 

69.0-80.2°F 

42.5-76.7% 

≥10/hour 

14 h light / 10 h dark 

Page 233 of 292
Female Pubertal Assay (2012) / Page 4 of 17 

METALAXYL / 113501 OCSPP 890.1450/ OECD None

 STUDY DESIGN

1. In-Life Dates: Start: July 6, 2011 End: Approximately July 27, 2011 

2. Mating: Time-mated, pregnant nulliparous Sprague Dawley Crl:CD(SD) dams (age not 

reported) were received from the supplier on Gestation Day 8 and were allowed to deliver 

naturally and rear their pups to PND 21. On PND 4, litters with 9 or more pups were 

standardized to 8 pups (4 males and 4 females whenever possible) through removal of pups 

with weights furthest from the population mean for the appropriate sex within each litter. 

Litters with fewer than 8 pups were not retained. 

3. Animal Assignment: Following weaning on PND 21, an equal number of rats from the 

heavy and light ends of the body weight distribution were eliminated, and the remaining rats 

were randomly assigned to the dose groups, stratified by dose weight. Littermates were not 

placed in the same dose group. The test groups are provided in Table 1.

TABLE 1. Study Design a

Test group Dose (mg/kg/day) # of Females 

Control 0 15 

Low 125 15 

Medium 250 15 

High 350 15 

a Data were obtained from Text Table 5 on page 26 of the study report. 

4. Dose Selection Rationale: The doses were selected based on the results from dose-range 

studies conducted in rats (Protocols 20012405 and 20013495) and clinical signs observed in 

the uterotrophic study on metalaxyl (MRID 48617606). Clinical signs of systemic toxicity 

were observed in adult male and female rats and weanlings at ≥500 mg/kg/day; mortality 

was seen in adult female rats at ≥500 mg/kg/day and in weanlings of both sexes at 750 

mg/kg/day. Body weight gain was decreased at the two highest dose levels in adult male 

rats (750 and 1000 mg/kg/day), and liver weights were increased at all metalaxyl dose levels 

in the adult male rats (250, 500, 750, and 1000 mg/kg/day) and in weanlings of both sexes 

(100, 250, 500, and 750 mg/kg/day). Based on these results, 350 mg/kg/day was selected as 

the high dose. The Sponsor utilized three dose levels to facilitate the interpretation of 

results. Minimal or no toxicity was expected at the low dose level of 125 mg/kg/day, and 

the midrange dose of 250 mg/kg/day was selected per Guideline recommendation for a twofold increase from the low dose. 

5. Dose Preparation and Analysis: Metalaxyl dose formulations were prepared weekly by 

adding appropriate amounts of the test compound to 2.0% aqueous carboxymethyl-cellulose 

in deionized water. The dose formulations were dispensed into daily aliquots (volume not 

reported), which were stored refrigerated until use. Samples from the first and last 

preparation of each dose group were collected and shipped on ice packs to the analytical 

laboratory. The samples were shipped on the date prepared or one day after preparation and 

were analyzed upon receipt or stored in a refrigerator (2-8°C) until analysis. Duplicate 

Page 234 of 292
Female Pubertal Assay (2012) / Page 5 of 17 

METALAXYL / 113501 OCSPP 890.1450/ OECD None

samples collected from the middle stratum of the formulations were analyzed for 

concentration. Single sets of samples were analyzed for density. 

Stability and homogeneity analyses were previously performed in conjunction with Protocol 

20012404. Stability was evaluated for 0.5 mg/mL and 100 mg/mL dose formulations. The 

dose formulations were analyzed before and after storage (while protected from light) at 

20-25°C for at least 25 hours and at 2-8°C for 10 days. The relative standard deviation 

(RSD) of the mean for each of the average concentration values for the top, middle, and 

bottom of each dose formulation was calculated to assess homogeneity. 

Results of Dose Analysis 

Homogeneity (RSD): 0.4-1.5% 

Stability (% of time zero): 99.7-101.8%

Concentration (% of nominal): 96.9-102.6% 

The analytical data indicated that the mixing procedure was adequate and the variation 

between nominal and actual dosage to the animals was acceptable. 

6. Dosage Administration: The dose formulations were allowed to warm to room 

temperature, and then stirred continuously for at least 30 minutes prior to administration and 

throughout dosing. Dose formulations were administered once daily by oral gavage in a 

dose volume of 5 mL/kg body weight. The dose formulations were administered for 21 or 

22 consecutive days from PND 22-42/43. Doses were adjusted based on the daily body 

weight recorded before administration and were administered at approximately the same 

time each day, between 0700 hours and 0900 hours, except on one occasion (no details were 

provided for this exception). 

7. Statistics: Mean, standard deviation, and coefficients of variation (CVs) were calculated. 

The following statistical analyses were performed. 

Page 235 of 292
Female Pubertal Assay (2012) / Page 6 of 17 

METALAXYL / 113501 OCSPP 890.1450/ OECD None

Classification Procedure 

Body weights 

Body weight changes 

Age at VO 

Bodyweight at VO 

Absolute organ weights 

Relative liver, kidney, adrenal, and 

pituitary weights 

Serum hormone values 

Clinical chemistry data 

Data were analyzed with Bartlett’s Test of Homogeneity of Variances and Analysis 

of Variance (ANOVA), when appropriate [i.e., Bartlett’s Test was not significant 

(p>0.001)]. If the ANOVA was significant (p≤0.05), Dunnett’s Test was used to 

identify the statistically significant differences between the control group and each 

treated group. If the ANOVA was not appropriate [i.e., Bartlett’s Test was 

significant (p≤0.001)], the Kruskal-Wallis Test was used (≤75% ties). In cases 

where the Kruskal-Wallis Test was statistically significant (p≤0.05), Dunn’s 

Method of Multiple Comparisons was used to identify the statistically significant 

differences between the control group and each treated group. 

Age at VO 

Bodyweight at VO 

Organ weight 

Data were also analyzed by Analysis of Covariance (ANCOVA), with the body 

weight recorded at weaning on PND 21 as the covariate. If the ANCOVA results 

were significant (p≤0.05), ANOVA was used to identify the statistically significant 

differences between the control group and each treated group. A modified 

Bonferroni correction was used for each pairwise comparison. A pairwise 

comparison was deemed statistically significant if its corresponding p-value was 

lower than or equal to the considered significance level (0.05 or 0.01) divided by 

the square root of the number of performed pairwise comparisons. In cases where 

sexual maturation did not occur prior to euthanasia, the last day of observation +1 

day was considered the age of sexual maturation. 

Cycling status 

Percent of animals cycling regularly 

Data were analyzed with the Variance Test for Homogeneity of the Binomial 

Distribution. 

The statistical analyses were considered appropriate. 

C. METHODS

1. Mortality and Clinical Examinations: Animals were observed at least twice daily for 

mortality. Rats were observed for general appearance daily prior to dose administration. 

Postdose observations were recorded within 2 hours of dose administration and at the end of 

the normal working day. 

2. Body Weight: Body weights were collected prior to allocation, daily before dose 

administration, and at termination. Overall (PND 22-42) body weight gain was also 

reported. 

3. Food Consumption: Food consumption was recorded daily. 

4. Vaginal Opening (VO): Female rats were evaluated for the age of vaginal patency daily 

beginning on PND 22. The appearance of a small "pin hole", a vaginal thread, and complete 

VO was recorded on the days observed. The body weight on the day of complete VO was 

also reported.

5. Estrous Cyclicity: Estrous cycling were evaluated daily by examination of vaginal 

cytology beginning on the day complete VO was observed, and continued until the day 

before euthanasia. The stage of estrous at necropsy was confirmed by the pathologist by 

evaluation of the fixed tissues. Vaginal smears were classified as diestrus (presence of 

leukocytes and/or cornified/irregularly shaped nucleated epithelial cells), proestrus 

(presence of nucleated epithelial cells), or estrus (presence of cornified epithelial cells) and 

the stage recorded daily. Metestrus was classified as an early part of diestrus, rather than a 

Page 236 of 292
Female Pubertal Assay (2012) / Page 7 of 17 

METALAXYL / 113501 OCSPP 890.1450/ OECD None

late part of estrus. Age at first estrus was recorded, and rats that stopped cycling were 

noted. All estrous smears were taken at approximately the same time each day (after dose 

administration). The overall pattern of estrous cycling for each female was characterized as 

regular cycling (recurring 4- to 5-day cycles), irregular cycling (diestrus longer than 3 days 

or a period of cornification longer than 2 days), or not cycling (prolonged periods of either 

cornification or leukocytic smears). In cases where there were too few days between 

vaginal opening and the end of the study to observe more than one cycle, classification was 

based on the available data. The default assumption was that animals were cycling regularly 

if the partial data fit the definition, and were irregularly cycling if the study ended without 

being able to distinguish between irregular cycling and not cycling. 

6. Sacrifice and Pathology: Animals were moved to the necropsy holding room on the day 

prior to the final dose administration. Doses were administered in the holding room 

between 0700 and 0900 hours. Euthanasia by decapitation began 2 hours later and was 

completed by 1300 hours. Dose administration and termination was performed across dose 

groups. After euthanasia and prior to tissue collection, trunk blood was collected from all 

animals, and serum was obtained by centrifugation of whole blood samples (1.5 and 2 mL, 

respectively) in serum separator tubes. Blood samples for thyroid hormone and testosterone 

analyses (1.5 mL) were centrifuged for approximately 30 min at 4°C and 3000 × g; samples 

for clinical chemistry evaluation were allowed to clot for 30 min at room temperature and 

centrifuged for approximately 10 min at room temperature and 3500 × g. The two sets of 

serum samples were immediately frozen on dry ice, and shipped on dry ice to AniLytics, 

Inc. (Gaithersburg, MD) for hormone analysis or Charles River Laboratories, Preclinical 

Services, Ohio for clinical chemistry analysis. Serum samples for hormone analyses were 

maintained at ≤−80°C until analysis, and clinical chemistry samples were maintained at 

≤−20°C. Necropsies were performed for each animal, gross lesions were collected (not 

evaluated microscopically), and images were generated for illustration of gross 

observations. 

a. Hormone Analysis: Total T4 and TSH were measured in serum using radioimmunoassays 

(RIAs). Total T4 was determined with a RIA kit from Siemens (Cat # TKT41) and TSH was 

determined with an in-house-developed assay specific for rat TSH. 

b. Clinical Chemistry: The following CHECKED (X) parameters were examined. 

Page 237 of 292
Female Pubertal Assay (2012) / Page 8 of 17 

METALAXYL / 113501 OCSPP 890.1450/ OECD None

ELECTROLYTES OTHER 

X Calcium X Albumin 

X Chloride X Creatinine* 

 Magnesium X Urea nitrogen* 

X Phosphorus X Total cholesterol 

X Potassium X Globulins 

X Sodium X Glucose 

ENZYMES X Total bilirubin 

X Alkaline phosphatase (ALP) X Total protein 

 Cholinesterase (ChE) X Triglycerides 

 Creatine phosphokinase X Albumin/globulin ratio (calculated) 

 Lactic acid dehydrogenase (LDH) Bile acids 

X Alanine aminotransferase (ALT/also SGPT) 

X Aspartate aminotransferase (AST/also SGOT) 

 Sorbitol dehydrogenase (SDH) 

X Gamma-glutamyl transferase (GGT) 

 Glutamate dehydrogenase 

* Recommended for the pubertal assay in female rats based on guideline 890.1450. 

c. Organ Weights and Histopathology: The following CHECKED (X) tissues were 

collected and weighed and grossly observed from all study animals. The (XX) organs, in 

addition, were subjected to histological examination. 

 UROGENITAL OTHER 

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

XX Uterus*+ (with cervix, wet and blotted) X Liver* 

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

 X Pituitary* 

a Thyroid was weighed post-fixation. 

* Weights required based on guideline 890.1450. 

+ Histopathological examination required based on guideline 890.1450. 

All collected organs, except the thyroid/trachea, were weighed prior to fixation. Paired 

organs (kidneys, adrenals, and ovaries) were weighed together. The uterus and cervix were 

separated from the vagina and weighed. The uterus was weighed again following removal 

of the fluid in the lumen (blotted weight). 

The right kidney, both ovaries, and uterus were fixed in neutral buffered, 10% formalin for 

at least 24 hours, and rinsed and retained in 70% ethanol until histological processing. After 

the initial fixation of the thyroid in 10% formalin, the thyroid/parathyroid was dissected 

from the trachea, blotted, weighed, and retained in 70% ethanol until histological 

processing. Tissues were embedded in paraffin, sectioned, mounted on glass slides, and 

stained with hematoxylin and eosin. Tissues were shipped under ambient conditions to 

Charles River Laboratories, Pathology Associates (MD). Other tissues were collected and 

retained, but not examined microscopically; these tissues included the adrenal, gross lesions, 

liver, and pituitary. 

Page 238 of 292
Female Pubertal Assay (2012) / Page 9 of 17 

METALAXYL / 113501 OCSPP 890.1450/ OECD None

Thyroid sections were 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. A minimum of two sections of each of the two lobes of the thyroid were evaluated. 

Ovarian histopathology included evaluation of follicular development (including presence/ 

absence of tertiary/antral follicles, presence/absence of corpora lutea, changes in corpus 

luteum development, changes in number of both primary and atretic follicles), in addition to 

any abnormalities/lesions (such as ovarian atrophy). Five random sections of each ovary 

were evaluated. Any lesions or atrophy within the ovary and any changes in corpora lutea 

were evaluated histopathologically by the Principal Investigator for histopathology at 

Charles River Laboratories, Pathology Associates, IL. The ovarian follicular counts were 

conducted by the Principal Investigator at Charles River Laboratories, Pathology Associates 

(MD). 

Uterine histopathology documented cases of uterine hyper- or hypotrophy (as characterized 

by changes in uterine horn diameter and myometrial, stromal, or endometrial gland 

development). On the last day of dose administration, female rats did not receive an estrous 

cycle evaluation; therefore, the pathologist evaluated the retained uterus and cervix of each 

female rat to determine the final stage of estrous on the day of necropsy. When possible, a 

longitudinal section including a portion of the remaining vaginal epithelium was obtained 

for evaluation. 

II. RESULTS 

A. MORTALITY: No mortality was observed. 

B. CLINICAL SIGNS OF TOXICITY: Incidental adverse clinical signs in the female rats 

were limited to one animal in the 250 mg/kg/day dose group that presented with ptosis on 

PND 22, and one animal in the 350 mg/kg/day dose group that presented with 

chromodacryorrhea on PND 31-42 and lacrimation on PND 32-34. 

C. GENERAL GROWTH AND PREPUTIAL SEPARATION: Body weights, body weight 

gains, age of attainment of VO, body weight at VO, and proportion/incidence of unopened 

vagina are presented in Table 2, and all values in the treated groups were similar to controls. 

No effect was noted on daily body weight measurements or feed consumption.

The Guideline performance criteria were met for age and weight at VO and final body 

weight, except for two differences: CV for age at VO (7.9%) was greater than the 

maximum acceptable value (6.52%); and CV for body weight at VO (14.8%) was greater 

than the maximum acceptable value (13.97%). 

Page 239 of 292
Female Pubertal Assay (2012) / Page 10 of 17 

METALAXYL / 113501 OCSPP 890.1450/ OECD None

TABLE 2. General Growth and Preputial Separation (PPS) a

Parameter 

Evaluated 

Vehicle Control Metalaxyl 

125 mg/kg/day

Metalaxyl 

250 mg/kg/day

Metalaxyl 

350 mg/kg/day 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) 

Initial Body 

Weight 

(PND 22; g) 

U 15 59.6 3.8 6.4 15 59.9 4.4 7.3 15 59.8 4.1 6.9 15 60.2 4.8 8.0 

A 15 59.8 --- --- 15 59.8 --- --- 15 59.8 --- --- 15 60.1 --- --- 

Body Weight 

at VO (g) 

U 15 119.6 17.7 14.8 15 121.4 16.0 13.2 15 119.4 14.3 12.0 15 128.8 13.7 10.6

A 15 119.6 --- --- 15 121.4 --- --- 15 119.4 --- --- 15 128.8 --- --- 

Final Body 

Weight 

(PND 42; g) 

U 15 166.8 9.2 5.5 15 171.7 10.3 6.0 15 170.5 13.2 7.7 15 172.5 9.4 5.4 

A 15 166.9 --- --- 15 171.7 --- --- 15 170.5 --- --- 15 172.4 --- --- 

Final Body 

Weight 

(% of control) 

U --- 102.9 102.2 103.4 

Body Weight 

Gain (PND 22-

42; g) 

U 15 107.2 8.6 8.0 15 111.8 9.0 8.1 15 110.7 13.0 11.7 15 112.3 8.9 7.9 

A 15 107.2 --- --- 15 111.8 --- --- 15 110.7 --- --- 15 112.3 --- --- 

Age at 

Incomplete 

VO (PND) 

U 15 30.7 2.5 8.1 15 32.0 2.9 9.1 15 30.6 2.3 7.5 15 31.4 3.1 9.9 

A 15 30.7 --- --- 15 32.0 --- --- 15 30.6 --- --- 15 31.4 --- --- 

Age at VO 

(PND) 

U 15 33.0 2.6 7.9 15 33.1 2.6 7.9 15 33.1 2.3 6.9 15 34.3 2.2 6.4 

A 15 33.0 --- --- 15 33.1 --- --- 15 33.1 --- --- 15 34.3 --- --- 

Proportion 

unopened (#/N) 0/15 0/15 0/15 0/15 

a Data were obtained from Table 5 on pages 79-80 and Appendix 15 on pages 374-380 of the study report. No statistical 

differences were detected between the treated groups and controls. 

b Calculated by the reviewers 

U Unadjusted for body weight on PND 21 

A Adjusted for body weight on PND 21 

N Number of animals examined 

SD Standard Deviation 

CV Coefficient of Variation 

--- Not applicable 

D. ORGAN WEIGHTS: Organ weights at necropsy are presented in Table 3. The absolute 

and relative liver weights were increased (p≤0.05 or 0.01) dose-dependently in all treated 

groups by 8-23% compared to controls. All other organ weights in the treated groups were 

similar to controls. All adjusted weights in the treated groups were similar to controls. 

The unadjusted values for all organ weights in the control group were within the acceptable 

range of the Guideline performance criteria, with the exception of the mean paired adrenal 

weight of 54.5 mg (acceptable range: 38.34-48.84 mg) and the mean fixed thyroid weight of 

27.26 mg (acceptable range: 6.20-22.20 mg). 

Page 240 of 292
Female Pubertal Assay (2012) / Page 11 of 17 

METALAXYL / 113501 OCSPP 890.1450/ OECD None

TABLE 3. Organ Weights at Necropsya

Organ 

Vehicle Control Metalaxyl 

125 mg/kg/day

Metalaxyl 

250 mg/kg/day

Metalaxyl 

350 mg/kg/day 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%)

Liver (g) U 15 8.61 1.06 12.3 15 9.51* 

(↑10) 0.86 9.0 15 10.21** (↑19) 1.11 10.9 15 10.61** (↑23) 0.95 9.0 

A 15 8.62 --- --- 15 9.50 --- --- 15 10.20 --- --- 15 10.61 --- --- 

R 15 5.07 0.37 7.3 15 5.46** 

(↑8) 0.28 5.2 15 5.86** 

(↑15) 0.35 5.9 15 6.06** 

(↑19) 0.38 6.3 

Kidneys 

(paired; g) 

U 15 1.66 0.16 9.6 15 1.66 0.12 7.2 15 1.67 0.12 7.2 15 1.67 0.12 7.2 

A 15 1.66 --- --- 15 1.66 --- --- 15 1.67 --- --- 15 1.67 --- --- 

R 15 0.981 0.055 5.6 15 0.953 0.056 5.9 15 0.962 0.043 4.5 15 0.955 0.064 6.7 

Pituitary 

(mg) 

U 15 12.0 2.3 19.2 15 11.1 2.2 19.8 15 12.8 2.4 18.8 15 11.8 1.5 12.7

A 15 12.0 --- --- 15 11.1 --- --- 15 12.8 --- --- 15 11.8 --- --- 

R 15 7.09 1.26 17.7 15 6.36 1.11 17.5 15 7.34 1.39 18.9 15 6.74 0.90 13.3

Adrenals 

(paired; mg) 

U 15 54.5 9.1 16.7 15 53.5 5.4 10.1 15 55.3 7.1 12.8 15 51.7 5.8 11.2

A 15 54.5 --- --- 15 53.5 --- --- 15 55.3 --- --- 15 51.7 --- --- 

R 15 0.033 0.005 15.2 15 0.031 0.002 6.5 15 0.032 0.004 12.5 15 0.029 0.004 13.8

Ovaries 

(paired; mg) 

U 15 79.0 9.8 12.4 15 73.8 9.2 12.5 15 76.6 11.8 15.4 15 76.3 14.0 18.3

A 15 79.0 --- --- 15 73.8 --- --- 15 76.6 --- --- 15 76.3 --- --- 

Uterus with 

cervix, wet 

(mg) 

U 15 407.2 185.7 45.6 15 498.6 225.0 45.1 15 348.1 101.3 29.1 15 421.1 151.1 35.9

A 15 407.1 --- --- 15 498.8 --- --- 15 348.2 --- --- 15 420.9 --- --- 

Uterus with 

cervix, 

blotted (mg) 

U 15 330.8 85.9 26.0 15 352.3 98.4 27.9 15 304.8 69.5 22.8 15 347.3 79.7 22.9

A 15 330.3 --- --- 15 352.4 --- --- 15 305.0 --- --- 15 347.6 --- --- 

Thyroid, 

fixed (mg) 

U 15 27.26 8.05 29.5 15 24.18 7.51 31.1 15 26.45 5.12 19.4 15 25.46 8.05 31.6

A 15 25.20 --- --- 15 24.19 --- --- 15 26.47 --- --- 15 25.49 --- --- 

a Data were obtained from Table 6 on pages 87-89 of the study report, and were sometimes rounded by the reviewers. Percent 

difference from control (calculated by reviewers from unrounded cited data) is included in parentheses. 

U Unadjusted for body weight on PND 21 

A Adjusted for body weight on PND 21 

R Organ-to-body weight ratio (relative to body weight) 

N Number of animals examined

SD Standard Deviation 

CV Coefficient of Variation 

* Significantly different from controls at p≤0.05. 

** Significantly different from controls at p≤0.01.

E. ESTROUS CYCLICITY: Estrous cycle data are provided in Table 4. No treatmentrelated effect was observed on the mean age of first vaginal estrus, mean cycle length, 

% cycling, % regularly cycling, and the cycle status at necropsy. 

Page 241 of 292
Female Pubertal Assay (2012) / Page 12 of 17 

METALAXYL / 113501 OCSPP 890.1450/ OECD None

TABLE 4. Estrous Cyclicitya

Treatment 

Groups Nb

Mean Age at 

First Vaginal 

Estrus (PND) 

Mean Cycle 

Length 

(days) 

Cycling 

(%) 

Not 

Cycling 

(%) 

Regularly 

Cycling 

(%) 

Cycle Status at Necropsy (# 

Females) 

Diestrusc Proestrus Estrus 

Vehicle 15 33.8 4.4 100 0 86.7 5 8 2 

Metalaxyl 

125 mg/kg/day 15 34.5 5.1 73.3 26.7 53.3 7 8 0 

Metalaxyl 

250 mg/kg/day 15 34.5 4.3 86.7 13.3 73.3 8 7 0 

Metalaxyl 

350 mg/kg/day 15 35.4 4.1 73.3 26.7 46.7 6 7 2 

a Data were obtained from Table 5 on page 81 and Appendix 26 on page 499 of the study report. 

b In the dose groups 0, 125, 250, and 350 mg/kg/day, the number of animals evaluated for the first day of vaginal estrus was 

15, 13, 13, and 14, respectively, and was 13, 8, 10, and 4, respectively for cycle length. 

c Includes diestrus and metestrus 

N Number of animals 

F. HORMONE LEVELS AND CLINICAL CHEMISTRY: Mean hormone levels and 

clinical chemistry values and are presented in Table 5. Nominal ranges (provided from 

testing facility historical control data; n=20) for the clinical chemistry values were provided 

Serum T4 and TSH levels in the treated groups were similar to control values. Gammaglutamyl transpeptidase levels were increased (p≤0.01) by 175% and 305% at 250 and 350 

mg/kg/day, respectively; however, the serum levels were reported to be within the historical 

control range. The following clinical chemistry parameters differed (p≤0.05 or 0.01) in the 

treated groups compared to controls: (i) albumin decreased by 3-6% in all treated groups; 

(ii) albumin/globulin ratio decreased by 5-9% in all treated groups; (iii) sodium increased by 

1-2% in all treated groups; and (iv) chloride increased by 2% at 350 mg/kg/day. These 

differences were minor in magnitude, without clear dose dependence, and were within the 

historical control range. All other values in the treated groups were similar to control 

values. 

Serum T4 mean and CV values in the control group were within the acceptable range of the 

Guideline performance criteria. 

Page 242 of 292
Female Pubertal Assay (2012) / Page 13 of 17 

METALAXYL / 113501 OCSPP 890.1450/ OECD None

TABLE 5. Serum Hormone Levels and Clinical Chemistrya

Parameter 

Vehicle Control Metalaxyl 

125 mg/kg/day

Metalaxyl 

250 mg/kg/day

Metalaxyl 

350 mg/kg/day 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) 

Thyroid Hormones 

Total T4 (μg/dL) 15 3.84 1.08 28.0 15 4.30 1.24 28.7 15 3.69 0.58 15.8 15 3.87 1.02 26.3

TSH (ng/mL) 15 1.79 0.94 52.4 15 2.23 0.90 40.3 15 2.06 0.95 45.8 15 2.01 0.77 38.5

Clinical Chemistry 

Creatinine (mg/dL) 15 0.23 0.02 6.9 15 0.24 0.02 9.4 15 0.23 0.02 8.6 15 0.23 0.01 4.4 

BUN (mg/dL) 15 17.3 3.4 19.7 15 15.9 1.8 11.3 15 16.5 3.5 21.2 15 15.8 2.8 17.7

AST (IU/L) 15 205 41 19.8 15 207 39 18.8 15 224 41 18.5 15 203 31 15.4

ALT (IU/L) 15 53.9 6.2 11.5 15 57.3 8.1 14.1 15 60.5 6.5 10.7 15 57.5 5.9 10.3

ALP (IU/L) 15 347 86 24.7 15 377 125 33.3 15 303 71 23.5 15 309 64 20.6

GGT (IU/L) 15 0.22 0.22 100 15 0.22 0.24 112 15 0.61** 

(↑175) 0.37 60.3 15 0.90** 

(↑305) 0.56 62.5

Total Bilirubin 

(mg/dL) 15 0.14 0.01 8.8 15 0.14 0.02 14.8 15 0.14 0.02 11.3 15 0.13 0.01 10.2

Cholesterol (mg/dL) 15 94.7 11.9 12.6 15 93.7 9.8 10.5 15 97.8 12.8 13.1 15 95.5 9.2 9.6 

Triglycerides 

(mg/dL) 15 68.1 18.8 27.6 15 61.1 19.0 31.1 15 71.1 26.9 37.8 15 66.2 15.5 23.4

Total Protein (g/dL) 15 5.69 0.15 2.6 15 5.54 0.26 4.7 15 5.61 0.19 3.3 15 5.60 0.32 5.6 

Albumin (g/dL) 15 3.45 0.11 3.2 15 3.29** 

(↓5) 0.15 4.6 15 3.34* 

(↓3) 0.14 4.1 15 3.26** 

(↓6) 0.15 4.5 

Globulin (g/dL) 15 2.24 0.11 4.9 15 2.25 0.14 6.1 15 2.27 0.10 4.5 15 2.34 0.18 7.6 

Albumin/Globulin 15 1.54 0.09 5.9 15 1.47** 

(↓5) 0.06 4.4 15 1.47* (↓5) 0.08 5.6 15 1.40** (↓9) 0.06 4.2 

Glucose (mg/dL) 15 132 10 7.7 15 139 6.3 4.5 15 136 11 8.0 15 138 7.7 5.6 

Calcium (mg/dL) 15 10.8 0.3 2.6 15 10.8 0.4 3.3 15 10.9 0.3 3.1 15 10.8 0.3 2.5 

Phosphorus (mg/dL) 15 10.3 0.8 7.6 15 10.9 0.8 7.6 15 11.0 0.8 7.1 15 10.9 0.9 8.0 

Sodium (mg/dL) 15 136 1.3 1.0 15 138* 

(↑1) 1.8 1.3 15 138** 

(↑2) 1.8 1.3 15 139** 

(↑2) 1.7 1.2 

Potassium (mg/dL) 15 6.60 0.44 6.7 15 6.21 0.41 6.6 15 6.38 0.41 6.4 15 6.27 0.46 7.3 

Chloride (mg/dL) 15 97.5 1.3 1.3 15 97.7 1.0 1.0 15 97.5 1.6 1.6 15 99.1** 

(↑2) 1.6 1.6 

a Data were obtained from Table 7 on page 91 and Table 8 on pages 93-96 of the study report. Percent differences from controls

(calculated by reviewers) are included in parentheses. 

N Number of animals examined 

SD Standard Deviation 

CV Coefficient of Variation 

* Significantly different from controls at p<0.05. 

** Significantly different from controls at p<0.01. 

G. GROSS PATHOLOGY: There were no treatment-related macroscopic findings. The only 

gross observations reported were a dilated pelvis of the right kidney in a single 350 

mg/kg/day rat and dilatation of the right kidney in a single 250 mg/kg/day rat. 

H. HISTOPATHOLOGY: The incidences of histopathological findings of the thyroid gland 

are presented below in Table 6; follicular cell shape was not reported. The colloid area and 

Page 243 of 292
Female Pubertal Assay (2012) / Page 14 of 17 

METALAXYL / 113501 OCSPP 890.1450/ OECD None

follicular cell height in the treated groups were generally similar to the controls, without a 

clear dose-dependent effect. 

TABLE 6. Thyroid Colloid Area and Follicular Cell Height a

Treatment Groups 

Parameter Evaluated 

Colloid Area Follicular Cell Height 

(Increase) 

Grade b Incidence Grade b Incidence 

O E O E 

Vehicle Control 

1 0 15 1 7 15 

2 0 15 2 8 15 

3 0 15 3 0 15 

4 1 15 4 0 15 

5 14 15 5 0 15 

125 mg/kg/day 

Metalaxyl 

1 0 15 1 9 15 

2 0 15 2 6 15 

3 0 15 3 0 15 

4 5 15 4 0 15 

5 10 15 5 0 15 

250 mg/kg/day 

Metalaxyl 

1 0 15 1 9 15 

2 0 15 2 6 15 

3 0 15 3 0 15 

4 2 15 4 0 15 

5 13 15 5 0 15 

350 mg/kg/day 

Metalaxyl 

1 0 14 1 11 14 

2 0 14 2 3 14 

3 0 14 3 0 14 

4 4 14 4 0 14 

5 10 14 5 0 14 

a Data were obtained from Appendix 26 on page 499 of the study report. 

b A 5-point grading scale (1 = shortest/smallest; 5 = tallest/largest) was used. 

O No. Observed 

E No. Examined 

The incidences of histopathological findings of the ovaries are presented in Table 7. Total 

primary follicle counts were increased (p≤0.05 or ≤0.01) by 43 and 57% at 250 and 350 

mg/kg/day, respectively. Atretic follicle counts in the treated groups were similar to 

controls. 

Page 244 of 292
Female Pubertal Assay (2012) / Page 15 of 17 

METALAXYL / 113501 OCSPP 890.1450/ OECD None

TABLE 7. Number of Ovarian Folliclesa

Parameter 

Vehicle Control Metalaxyl 

125 mg/kg/day

Metalaxyl 

250 mg/kg/day

Metalaxyl 

350 mg/kg/day 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) 

Primary Follicle Count 

Left 15 11.4 4.9 43.0 15 11.5 4.8 41.7 15 14.7 7.0 47.6 15 17.7** 

(↑55) 3.3 18.6 

Right 15 10.2 5.3 52.0 15 12.1 5.4 44.6 15 16.1* 

(↑58) 7.1 44.1 15 16.3* (↑60) 8.4 51.5 

Total 15 21.6 8.8 40.7 15 23.6 9.0 38.1 15 30.8* 

(↑43) 11.0 35.6 15 34.0** 

(↑57) 9.7 28.5 

Atretic Follicle Count 

Left 15 1.0 1.6 160.0 15 0.3 0.6 200.0 15 1.0 0.8 80.0 15 1.3 1.3 100.0 

Right 15 0.5 0.6 120.0 15 0.3 0.6 200.0 15 0.9 1.1 122.2 15 1.2 1.6 133.3 

Total 15 1.5 1.8 120.0 15 0.6 0.8 133.3 15 1.9 1.5 78.9 15 2.5 2.3 92.0 

a Data were obtained from Table 9 on page 98 of the study report. 

SD Standard Deviation 

CV Coefficient of Variation 

The incidences of histopathological findings of the kidneys are presented in Table 8, and no 

treatment-related finding was observed. No treatment-related histological lesions were noted in 

the thyroid or uterus. 

TABLE 8. Histopathological Findings in the Kidneya 

Parameter Vehicle Control 

Metalaxyl 

125 mg/kg/day

Metalaxyl 

250 mg/kg/day

Metalaxyl 

350 mg/kg/day 

Infiltration, mixed cell, minimal 0 1 1 2 

Dilatation 

Minimal 0 0 1 0 

Mild 0 0 0 0 

Moderate 0 0 0 1 

a Data (n=15) were obtained from Appendix 26 on page 498 of the study report. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Metalaxyl resulted in increases in absolute liver 

weights and/or ratios of liver to terminal body weight, concurrent with reductions in 

albumin and albumin/globulin ratio and increases in sodium, as well as increases in gamma 

glutamyl transferase at 250 and 350 mg/kg/day and in chloride at 350 mg/kg/day. No other 

treatment related effect was observed in this study. 

Based on these data, administration of metalaxyl under the tested conditions resulted in 

effects indicative of systemic/liver target organ toxicity, but no effects on endocrine activity 

or indications of potential interactions with the estrogen or thyroid pathways were apparent. 

Page 245 of 292
Female Pubertal Assay (2012) / Page 16 of 17 

METALAXYL / 113501 OCSPP 890.1450/ OECD None

B. AGENCY COMMENTS: No adverse, treatment-related related effects were observed on 

survival, clinical signs, body weights, body weight gains, or gross pathology. No treatmentrelated histological lesions were noted in the thyroid, or uterus, and thyroid colloid area and 

follicular cell height in the treated groups were generally similar to the control. Age of 

attainment of VO, body weight at VO, and proportion/incidence of unopened vagina in the 

treated groups were similar to control animals. No treatment-related effect was observed on 

the mean age of first vaginal estrus, mean cycle length, % cycling, % regularly cycling, and 

the cycle status at necropsy. Serum T4 and TSH levels in the treated groups were similar to 

control. 

The absolute and relative liver weights were increased (p≤0.05 or 0.01) dose-dependently in 

all treated groups by 8-23% compared to control values. This slight effect is considered 

treatment related. Gamma-glutamyl transpeptidase levels were increased (p≤0.01) by 175% 

at 250 mg/kg/day and by 305% at 350 mg/kg/day; however, the serum levels were reported 

to be within the historical control range. The following clinical chemistry parameters 

differed (p≤0.05 or 0.01) in the treated groups compared to controls: (i) albumin decreased 

by 3-6% in all treated groups; (ii) albumin/globulin ration decreased by 5-9% in all treated 

groups; (iii) sodium increased by 1-2% in all treated groups; and (iv) chloride increased by 

2% at 350 mg/kg/day. These differences were relatively minor in magnitude, lacked clear 

dose dependence, and were within the historical control range. Considered together, the 

increases in liver weight and serum GGT levels and decreases in albumin and 

albumin/globulin levels suggest liver toxicity; however, the degree of toxicity achieved is 

modest considering the magnitude of these changes and natural variation as evidenced by 

historical controls. 

Total primary follicle counts were increased (p≤0.05) at 250 mg/kg/day by 43% and 

increased (p≤0.01) at 350 mg/kg/day by 57%. Atretic follicle counts in the treated groups 

were similar to controls. The significance of the increases in total primary follicle counts 

and their relation to treatment is questionable because no other major pathological findings 

were observed in the ovaries and the presence of different follicle types is normal for 

nulliparous, young, female rats. 

The high dose (350 mg/kg/day) was determined to be adequate based on generalized 

summaries from several ranging-finding studies and findings in the previously reviewed 

uterotrophic study (MRID 48617606), which showed decreased motor activity, ptosis, ataxia, 

lacrimation, and hunched posture in female rats in the 300 mg/kg/day dose group. 

Under the conditions of this study, there was no treatment-related effects on pubertal 

development or thyroid function in the juvenile female rat. 

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

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

study: 

 Control CV for age at VO (7.9%) was greater than the performance criteria maximum 

of 6.52% 

Page 246 of 292
Female Pubertal Assay (2012) / Page 17 of 17 

METALAXYL / 113501 OCSPP 890.1450/ OECD None

 Control CV for body weight at VO (14.8%) was greater than the performance criteria 

maximum of 13.97% 

 Control mean paired adrenal weight (54.5 mg) was outside the performance criteria 

acceptable range (38.34-48.84) 

 Control mean fixed thyroid weight (27.26 mg) was outside the performance criteria 

acceptable range (6.20-22.20) 

Page 247 of 292
DATA EVALUATION RECORD 

METALAXYL 

Study Type: OCSPP 890.1500, Male Pubertal Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 3-19-2012 (MRID 48701301) 

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:

Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 9/18/2012 

Secondary Reviewer: Signature:

Scott D. Studenberg, Ph.D., D.A.B.T. Date: 9/27/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 10/03/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 10/03/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 248 of 292
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 249 of 292
Male Pubertal Assay (2012) I Page 1 of 16 

METALAXYL / 113501 OCSPP 890.1500/ OECD None 

Primary Reviewer: Vincent Chen, M.S. Signature:~ Risk Assessment Branch III, Health Effects Division (7509P) Date: z..4 'Jv.r.Jo::?z.o.l-S 

Secondary Reviewer: John Liccione, Ph.D. Signature: ~ , 

Health Effects Division (7509P) Date: ~;. '?ft f 

Template version 08/2011 

. ~' _D_A_T_A_E_V_AL_U_A_T_IO_N_RE_c_o_RD_~ 

STUDY TYPE: Male Pubertal Assay; OCSPP 890.1500; OECD None. 

PC CODE: 113501 DP BARCODE: D404681 

TXR #: 0054219 CAS #: 57837-19-1 

TEST MATERIAL (PURITY): Metalaxyl (>98% a.i.) 

SYNONYMS: Methyl N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alaninate 

CITATION: Carlson, M.B. (2012). A Pubertal Development and Thyroid Function Study 

of Metalaxyl by Oral Gavage in Peripubertal Male and Female Rats. Charles 

River Laboratories Preclinical Services, Pennsylvania, Horsham, PA. 

Laboratory Study No.: 20012408, July 17, 2012. MRID 48701301. 

Unpublished. 

SPONSOR: Metalaxyl EDSP Consortium, 20 Ambriance Drive, Burr Ridge, IL 

TEST ORDER#: CON-113501-18 

EXECUTIVE SUMMARY: In a Male Pubertal Assay (MRID 48701301), 15 male SpragueDawley (Crl:CD(SD)) rats/dose group were treated daily via oral gavage with metalaxyl (>98% 

a.i., Lot# 20100540) in 2.0% aqueous carboxymethylcellulose at doses of 0, 125, 250, or 

350 mg/kg/day from postnatal day (PND) 23 to 53 or 54. Animals were examined for preputial 

separation (PPS) daily beginning on PND 30, and age and weight at day of attainment were 

recorded. Following sacrifice on PND 53 or 54, blood wa.S collected for clinical chemistry 

analyses, and testosterone, total thyroxine (T 4), and thyroid stimulating hormone (TSH) levels 

were determined using radioimmunoassays. Adrenal, liver, pituitary, thyroid, and urogenital 

organs were weighed and examined macroscopically. The testes, epididymides, right kidney, 

and thyroid were examined microscopically. 

No adverse, treatment-related effects were observed on survival, clinical signs, body weights, 

body weight gains, organ weights, clinical chemistry, or gross pathology. No adverse 

histological lesion was observed in the testes, epididymides, or kidneys. Mean serum 

testosterone, T 4, and TSH levels in the treated groups were similar to control, and the thyroid 

follicular cell heights in the treated groups were generally similar to control. Although colloid 

area in the thyroid was slightly reduced in a dose-related manner, there were no concomitant 

effects noted in thyroid weights or hormone concentrations. 

Page 250 of 292
Male Pubertal Assay (2012) / Page 2 of 16 

METALAXYL / 113501 OCSPP 890.1500/ OECD None 

Body weights, body weight gains, age of attainment of PPS, body weight at PPS, and 

proportion/incidence of separated prepuce in the treated groups were similar to control with the 

exception of minor decreases (p≤0.05) in the adjusted body weight at PPS at 125 mg/kg/day 

(↓6%) and unadjusted and adjusted body weights at PPS at 250 and 350 mg/kg/day (↓6-8%). 

These minor decreases were not dose dependent in magnitude. No effect was noted on daily 

body weight measurements. 

All organ weight values in the treated groups were similar to control, except for a minor percent 

increase in relative liver weight at 350 mg/kg/day (5.4%) compared to control (4.9%). This 

effect was minor, without a significant effect on absolute or adjusted weight, and without 

corroborating gross pathology or clinical chemistry findings of hepatotoxicity. Consequently, 

this effect was considered incidental. 

One 350 mg/kg/day male (#349) was found dead on PND 40. Although the study authors 

considered this death to be treatment related, none of the data presented supported that 

conclusion. The cause of death was not determined, but was not due to gavage error (perforation 

of lungs, trachea, or esophagus). All tissues appeared normal at necropsy, and no adverse 

clinical signs were noted in this animal on any day during the study. 

Although there was limited evidence of toxicity at the high dose (350 mg/kg/day) in this study, 

the selection of the high dose was appropriate based on systemic toxicity in the dose-range 

finding studies and findings in the uterotrophic assay (MRID 48617606) which showed 

decreased motor activity, ptosis, ataxia, lacrimation, and hunched posture in female rats in the 

300 mg/kg/day dose group. In addition, mean liver weights were significantly increased in male 

rats in the Hershberger assay (MRID 48617604) at 375 mg/kg/day supporting the selection of 

350 mg/kg/day as an appropriate high dose in the male pubertal assay. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Male Pubertal Assay (OCSPP 

890.1500).

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

and Flagging statements were provided. 

 

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METALAXYL / 113501 OCSPP 890.1500/ OECD None 

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Charles River Laboratories Preclinical Services, Pennsylvania 

Location: Horsham, PA 

Study Directors: M.B. Carlson 

Other Personnel: Julian Gulbinski, III (dose formulation analysis and senior laboratory manager), Dr. Saroj Das 

(hormone analysis), Carol S. Sloan (hormone analysis), Rebecca M. Lucke (clinical 

chemistry), Carol J. Detrisac (histology and histopathology) 

Study Period: June 17, 2011 through July 17, 2012 

2. Test Substance: Metalaxyl 

 Description: White to beige powder 

 Source: Nation’s Ag, LLC, Knoxville, TN 

 Lot #: 20100540 (expires June 13, 2012 and July 26, 2011) 

 Purity: 98.2±0.3% and 100.4±0.7% 

 Stability: Protected from light, the test compound was stable in the vehicle at 20-25°C for at least 25 

hours and at 2-8°C for 10 days. 

 CAS #: 57837-19-1 

 Structure: 

3

CH

N

O

CH3

O

3CH CH3

O O

3CH

3. Vehicle: 

Source: 

Catalog #: 

Lot #: 

2.0% aqueous carboxymethylcellulose in reverse osmosis 

membrane-processed deionized water 

Sigma Aldrich, St. Louis, MO 

C5678 

060M0079V (retest date: June 30, 2012)

4. Test Animals:

Species: Rat (males) 

Strain: Sprague-Dawley (Crl:CD(SD)) 

Age/ Mean Weight 

at Study Initiation: PND 23 / 67.9-68.8 g 

Source: Charles River Laboratories International, Inc. (Portage, MI) 

Housing: Parental dams were individually housed in clear plastic nesting boxes (n=2/box) with heattreated, laboratory-grade, pine shaving bedding; each dam was housed with her litter until 

weaning on PND 21. After weaning, 2 males were housed per cage. 

Diet: Harlan Teklad 2018c meal feed (Teklad Diets, Madison, WI), ad libitum. It was stated that the 

total genistein equivalents (aglycone) or daidzein were ≤300 ppm. 

Water: Reverse osmosis-purified and deionized tap water, ad libitum

 Environmental 

Conditions: 

Temperature: 

Humidity: 

Air changes: 

Photoperiod: 

69.0-80.2°F 

42.5-76.7% 

≥10/hour 

14 h light / 10 h dark

Page 252 of 292
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METALAXYL / 113501 OCSPP 890.1500/ OECD None 

B. STUDY DESIGN

1. In-Life Dates: Start: July 7, 2011 End: August 7, 2011 

2. Mating: Time-mated, pregnant nulliparous Sprague Dawley Crl:CD(SD) dams (age not 

reported) were received from the supplier on gestation day 8 and were allowed to deliver 

naturally and rear their pups to PND 21. On PND 4, litters with 9 or more pups were 

standardized to 8 pups (4 males and 4 females whenever possible) through removal of pups 

with weights furthest from the population mean for the appropriate sex within each litter. 

Litters with fewer than 8 pups were not retained. 

3. Animal Assignment: Following weaning on PND 21, an equal number of rats from the 

heavy and light ends of the body weight distribution were eliminated, and the remaining rats 

were randomly assigned to the dose groups, stratified by dose weight. Littermates were not 

placed in the same dose group. The test groups are provided in Table 1.

TABLE 1. Study Design a

Test group Dose (mg/kg/day) # of Females 

Control 0 15 

Low 125 15 

Medium 250 15 

High 350 15 

a Data were obtained from Text Table 5 on page 26 of the study report. 

4. Dose Selection Rationale: The doses were selected based on the results from dose-range 

studies conducted in rats (Protocols 20012405 and 20013495) and clinical signs observed in 

the uterotrophic study on metalaxyl (48617606.DER, previously reviewed). Clinical signs 

of systemic toxicity were observed in adult male and female rats and weanlings at ≥500 

mg/kg/day; mortality was seen in adult female rats at ≥500 mg/kg/day and in weanlings of 

both sexes at 750 mg/kg/day. Body weight gain was decreased at the two highest dose 

levels in adult male rats (750 and 1000 mg/kg/day), and liver weights were increased at all 

metalaxyl dose levels in the adult male rats (250, 500, 750, and 1000 mg/kg/day) and in 

weanlings of both sexes (100, 250, 500, and 750 mg/kg/day). Based on these results, 350 

mg/kg/day selected as the maximum dose. The Sponsor utilized three dose levels to 

facilitate the interpretation of results. Minimal or no toxicity was expected at the low dose 

level of 125 mg/kg/day; a midrange dose of 250 mg/kg/day was selected per Guideline 

recommendation for a two-fold increase from the low dose. 

5. Dose Preparation and Analysis: Metalaxyl dose formulations were prepared weekly by 

adding appropriate amounts of the test compound to 2.0% aqueous carboxymethyl-cellulose 

in deionized water. The dose formulations were dispensed into daily aliquots (volume not 

reported), which were stored refrigerated until use. Samples from the first and last 

preparation of each dose group were collected and shipped on ice packs to the analytical 

laboratory. The samples were shipped on the date prepared or one day after preparation and 

were analyzed upon receipt or stored in a refrigerator (2-8°C) until analysis. Duplicate 

Page 253 of 292
Male Pubertal Assay (2012) / Page 5 of 16 

METALAXYL / 113501 OCSPP 890.1500/ OECD None 

samples collected from the middle stratum of the formulations were analyzed for 

concentration. Single sets of samples were analyzed for density. 

Stability and homogeneity analyses were previously performed in conjunction with Protocol 

20012404. Stability was evaluated for 0.5 mg/mL and 100 mg/mL dose formulations. The 

dose formulations were analyzed before and after storage (while protected from light) at 

20-25°C for at least 25 hours and at 2-8°C for 10 days. The relative standard deviation 

(RSD) of the mean for each of the average concentration values for the top, middle, and 

bottom of each dose formulation was calculated to assess homogeneity. 

Results of Dose Analysis 

Homogeneity (RSD): 0.4-1.5% 

Stability (% of time zero): 99.7-101.8%

Concentration (% of nominal): 96.9-102.6% 

The analytical data indicated that the mixing procedure was adequate and the variation 

between nominal and actual dosage to the animals was acceptable. 

6. Dosage Administration: The dose formulations were allowed to warm to room 

temperature and were then stirred continuously for at least 30 minutes prior to 

administration and throughout dosing. Dose formulations were administered once daily by 

oral gavage in a dose volume of 5 mL/kg body weight. The dose formulations were 

administered for 31 or 32 consecutive days from PND 23-53/54. Doses were adjusted based 

on the daily body weight recorded before administration and were administered at 

approximately the same time each day, between 0700 hours and 0900 hours, except on one 

occasion (no details were provided for this exception). 

7. Statistics: Mean, standard deviation, and coefficients of variation (CVs) were calculated. 

The following statistical analyses were performed. 

Page 254 of 292
Male Pubertal Assay (2012) / Page 6 of 16 

METALAXYL / 113501 OCSPP 890.1500/ OECD None 

Classification Procedure 

Body weights 

Body weight changes 

Age at PPS 

Bodyweight at PPS 

Absolute organ weights 

Relative liver, kidney, adrenal, and 

pituitary weights 

Serum hormone values 

Clinical chemistry data 

Data were analyzed with Bartlett’s Test of Homogeneity of Variances and Analysis 

of Variance (ANOVA), when appropriate [i.e., Bartlett’s Test was not significant 

(p>0.001)]. If the ANOVA was significant (p≤0.05), Dunnett’s Test was used to 

identify the statistically significant differences between the control group and each 

treated group. If the ANOVA was not appropriate [i.e., Bartlett’s Test was 

significant (p≤0.001)], the Kruskal-Wallis Test was used (≤75% ties). In cases 

where the Kruskal-Wallis Test was statistically significant (p≤0.05), Dunn’s 

Method of Multiple Comparisons was used to identify the statistically significant 

differences between the control group and each treated group. 

Age at PPS 

Bodyweight at PPS 

Organ weight 

Data were also analyzed by Analysis of Covariance (ANCOVA), with the body 

weight recorded at weaning on PND 21 as the covariate. If the ANCOVA results 

were significant (p≤0.05), ANOVA was used to identify the statistically significant 

differences between the control group and each treated group. A modified 

Bonferroni correction was used for each pairwise comparison. A pairwise 

comparison was deemed statistically significant if its corresponding p-value was 

lower than or equal to the considered significance level (0.05 or 0.01) divided by 

the square root of the number of performed pairwise comparisons. In cases where 

sexual maturation did not occur prior to euthanasia, the last day of observation +1 

day was considered the age of sexual maturation. 

The statistical analyses were considered appropriate. 

C. METHODS

1. Mortality and Clinical Examinations: Animals were observed at least twice daily for 

mortality. Rats were observed for general appearance daily prior to dose administration. 

Postdose observations were recorded within 2 hours of dose administration and at the end of 

the normal working day. 

2. Body Weight: Body weights were collected prior to allocation, daily before dose 

administration, and at termination. Overall (PND 23-53) body weight gain was also 

reported. 

3. Food Consumption: Food consumption was recorded daily. 

4. Preputial Separation (PPS): Male rats were evaluated for the occurrence of PPS daily 

beginning on PND 30. The appearance of partial and complete PPS, or a persistent thread 

of tissue between the glans and prepuce, was recorded. The body weight recorded on the day 

of complete PPS was used for subsequent evaluation of this endpoint. 

5. Sacrifice and Pathology: Animals were moved to the necropsy holding room on the day 

prior to the final dose administration. Doses were administered in the holding room 

between 0700 and 0900 hours. Euthanasia by decapitation began 2 hours later and was 

completed by 1300 hours. Dose administration and termination was performed across dose 

groups. After euthanasia and prior to tissue collection, trunk blood was collected from all 

animals, and serum was obtained by centrifugation of whole blood samples (1.5 and 2 mL, 

respectively) in serum separator tubes. Blood samples for thyroid hormone and testosterone 

analyses (1.5 mL) were centrifuged for approximately 30 min at 4°C and 3000 × g; samples 

for clinical chemistry evaluation were allowed to clot for 30 min at room temperature and 

Page 255 of 292
Male Pubertal Assay (2012) / Page 7 of 16 

METALAXYL / 113501 OCSPP 890.1500/ OECD None 

centrifuged for approximately 10 min at room temperature and 3500 × g. The two sets of 

serum samples were immediately frozen on dry ice, and shipped on dry ice to Ani Lytics, 

Inc. (Gaithersburg, MD) for hormone analysis or Charles River Laboratories, Preclinical 

Services, Ohio for clinical chemistry analysis.. Serum samples for hormone analyses were 

maintained at ≤−80°C until analysis, and clinical chemistry samples were maintained at 

≤−20°C. Necropsies were performed for each animal, gross lesions were collected (not 

evaluated microscopically), and images were generated for illustration of gross 

observations. 

The single male rat (#349) in the high dose group that died before scheduled termination 

was examined for the cause of death or condition as soon as possible after the observation 

was made. The rat was examined for gross lesions. The lungs, trachea, and esophagus were 

perfused with neutral buffered 10% formalin. Tissue dissection and weighing procedures 

were not conducted, and blood samples for hormone analysis and clinical chemistry were 

not obtained from this animal. 

a. Hormone Analysis: Total testosterone, T4, and TSH were measured in serum using 

radioimmunoassays (RIAs). Total testosterone and T4 were determined with RIA kits from 

Siemens (Cat # TKTT1 and TKT41, respectively), and TSH was determined with an inhouse-developed assay specific for rat TSH. 

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 X Albumin/globulin ratio (calculated) 

 Lactic acid dehydrogenase (LDH) Bile acids 

X Alanine aminotransferase (ALT/also SGPT) 

X Aspartate aminotransferase (AST/also SGOT) 

 Sorbitol dehydrogenase (SDH) 

X Gamma-glutamyl transferase (GGT) 

 Glutamate dehydrogenase 

* Recommended for the pubertal assay in female rats based on guideline 890.1450. 

c. Organ Weights and Histopathology: The following CHECKED (X) tissues were 

collected and weighed and grossly observed from all study animals. The (XX) organs, in 

addition, were subjected to histological examination. 

Page 256 of 292
Male Pubertal Assay (2012) / Page 8 of 16 

METALAXYL / 113501 OCSPP 890.1500/ OECD None 

 UROGENITAL OTHER 

XX Testes (left and right separately)*+ XX Thyroida*+

XX Epididymides (left and right separately)*+ X Liver* 

X Seminal vesicle plus coagulating glands (with and without fluid)* X Adrenals (paired)* 

X Ventral prostate* X Pituitary* 

X Dorsolateral prostate* 

X Levator ani-bulbocavernosus (LABC) muscle complex* 

XX Kidneys (paired)*+ 

a Thyroid was weighed post-fixation. 

* Weights required based on Guideline 890.1500 

+ Histopathological examination required based on Guideline 890.1500

All collected organs, except the thyroid/trachea, were weighed prior to fixation. Paired 

organs (kidneys and adrenals) were weighed together, but individual weights were recorded 

for each testis and epididymis. 

The right kidney was fixed in neutral buffered, 10% formalin for at least 24 hours, and 

rinsed and retained in 70% ethanol until histological processing. After the initial fixation of 

the thyroid in 10% formalin, the thyroid/parathyroid was dissected from the trachea, blotted, 

weighed, and retained in 70% ethanol until histological processing. The epididymis and 

testis were retained in Bouin's fluid for no longer than 24 hours and rinsed and retained in 

70% ethanol. All other organs were fixed in neutral buffered, 10% formalin. Tissues were 

embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and 

eosin. Tissues were shipped under ambient conditions to Charles River Laboratories, 

Pathology Associates, MD. Other tissues were collected and retained, but not examined 

microscopically; these tissues included the adrenal, gross lesions, liver, and pituitary. 

Thyroid sections were 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. A minimum of two sections of each of the two lobes of the thyroid were evaluated. 

For each male rat, both testes, both epididymides, and the right kidney were evaluated for 

pathologic abnormalities and potential treatment-related effects. 

Besides gross lesions such as atrophy or tumors, testicular histopathological evaluation was 

conducted in order to identify treatment-related effects such as retained spermatids, missing 

germ cell layers or types, multinucleated giant cells, or sloughing of spermatogenic cells 

into the lumen. Examination of the intact epididymis included the caput, corpus, and cauda, 

which was accomplished by evaluation of a longitudinal section. This examination was 

conducted in order to identify such lesions as sperm granulomas, leukocytic infiltration 

(inflammation), aberrant cell types within the lumen, or the absence of clear cells in the 

caudal epididymal epithelium. 

 

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Male Pubertal Assay (2012) / Page 9 of 16 

METALAXYL / 113501 OCSPP 890.1500/ OECD None 

II. RESULTS 

A. MORTALITY: One 350 mg/kg/day male (#349) was found dead on PND 40. Although 

the cause of death was not determined, it was not due to gavage error (perforation of lungs, 

trachea, or esophagus). All tissues appear normal at necropsy, and no adverse clinical signs 

were noted in this animal on any day during the study. Therefore, this death is considered 

incidental. 

B. CLINICAL SIGNS OF TOXICITY: No adverse, clinical signs were reported. 

C. GENERAL GROWTH AND PREPUTIAL SEPARATION: Body weights, body weight 

gains, age of attainment of PPS, body weight at PPS, and proportion/incidence of separated 

prepuce are presented in Table 2. All values in the treated groups were similar to control 

with the exception of minor decreases (p≤0.05) in the adjusted body weight at PPS at 

125 mg/kg/day (↓6%) and unadjusted and adjusted body weights (p≤0.01) at PPS at 250 and 

350 mg/kg/day (↓6-8%). No effect was noted on daily body weight measurements or feed 

consumption.

The Guideline performance criteria were met for age and weight at PPS and weaning and 

final body weight, except for the following: (i) CV for body weight at PPS (9.1%) was 

greater than the maximum acceptable value (7.57%); (ii) mean final body weight of 332.5 

mg (acceptable range: 259.235-332.059 g); and (iii) mean body weight at weaning of 60.5 g 

(acceptable range: 45.472-59.812 g). 

Page 258 of 292
Male Pubertal Assay (2012) / Page 10 of 16 

METALAXYL / 113501 OCSPP 890.1500/ OECD None 

TABLE 2. General Growth and Preputial Separation (PPS) a

Parameter 

Evaluated 

Vehicle Control Metalaxyl 

125 mg/kg/day

Metalaxyl 

250 mg/kg/day

Metalaxyl 

350 mg/kg/day 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) 

Initial Body 

Weight 

(PND 23; g) 

U 15 68.5 4.7 6.9 15 68.4 4.9 7.2 15 67.9 5.4 8.0 15 68.8 4.0 5.8 

A 15 68.9 --- --- 15 68.2 --- --- 15 68.2 --- --- 15 68.4 --- --- 

Body Weight 

at PPS (g) U 15 236.2 21.5 9.1 15 223.9 20.0 8.9 15 217.7* (↓8) 17.2 7.9 14b 221.4* (↓6) 13.0 5.9 

A 15 236.8 --- --- 15 223.4* 

(↓6) --- --- 15 218.2** 

(↓8) --- --- 14b 220.7* 

(↓7) --- --- 

Final Body 

Weight 

(PND 53; g) 

U 15 332.5 18.4 5.5 15 330.9 23.9 7.2 15 321.3 21.4 6.7 14b 319.6 19.8 6.2 

A 15 332.9 --- --- 15 330.6 --- --- 15 321.6 --- --- 14b 319.2 --- --- 

Final Body 

Weight 

(% of control) 

U --- 99.5 96.6 96.1 

Body Weight 

Gain (PND 

23-53; g) 

U 15 264.0 18.2 6.9 15 262.5 21.8 8.3 15 253.3 19.5 7.7 14b 250.8 20.4 8.1 

A 15 264.0 --- --- 15 262.5 --- --- 15 253.3 --- --- 14b 250.7 --- --- 

Age at 

Incomplete 

PPS (PND) 

U 15 40.9 0.9 2.2 15 40.3 1.2 3.0 15 40.5 1.2 3.0 14b 40.5 1.4 3.5 

A 15 40.9 --- --- 15 40.3 --- --- 15 40.5 --- --- 14b 40.5 --- --- 

Age at PPS 

(PND) 

U 15 43.2 2.3 5.3 15 41.9 1.2 2.9 15 41.7 1.1 2.6 14b 42.3 1.4 3.3 

A 15 43.2 --- --- 15 41.9 --- --- 15 41.8 --- --- 14b 42.3 --- --- 

Proportion 

unseparated (#/N) 0/15 0/15 0/15 1/15b 

a Data were obtained from Table 1 on pages 59-60 and Appendix 7 on pages 342-343 of the study report. 

b Animal #349 was found dead on PND 40 prior to PPS 

U Unadjusted for body weight on PND 21 

A Adjusted for body weight on PND 21 

N Number of animals examined 

SD Standard Deviation 

CV Coefficient of Variation 

--- Not applicable 

* Significantly different from controls at p≤0.05. 

** Significantly different from controls at p≤0.01.

D. ORGAN WEIGHTS: Organ weights at necropsy are presented in Table 3, and all values 

in the treated groups were similar to controls, except for a minor increase in relative liver 

weight at 350 mg/kg/day (5.38%) compared to controls (4.89%). 

The unadjusted values for all organ weights in the control group were within the acceptable 

range of the Guideline performance criteria, with the following exceptions: (i) mean liver 

weight of 16.55 g (acceptable range: 9.990-15.350 g); (ii) CV for pituitary weight (23.5%) 

was greater than the maximum acceptable value (15.98%); and (iii) CV for fixed thyroid 

weight (27.2%) was greater than the maximum acceptable value (23.63%). 

Page 259 of 292
Male Pubertal Assay (2012) / Page 11 of 16 

METALAXYL / 113501 OCSPP 890.1500/ OECD None 

TABLE 3. Organ Weights at Necropsya

Organ 

Vehicle Control Metalaxyl 

125 mg/kg/day

Metalaxyl 

250 mg/kg/day

Metalaxyl 

350 mg/kg/day 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) Nb Mean SD 

CV 

(%) 

Liver (g) U 15 16.6 1.66 10.0 15 17.0 2.20 12.9 15 16.6 1.26 7.6 14 17.5 1.58 9.0 

A 15 16.6 --- --- 15 17.0 --- --- 15 16.6 --- --- 14 17.5 --- --- 

R 15 4.89 0.233 4.8 15 5.06 0.318 6.3 15 5.10 0.268 5.3 14 5.38** 

(↑10) 0.269 5.0 

Kidneys 

(paired; g) 

U 15 2.84 0.25 8.8 15 2.90 0.27 9.3 15 2.81 0.27 9.6 14 2.81 0.19 6.8 

A 15 2.84 --- --- 15 2.90 --- --- 15 2.81 --- --- 14 2.81 --- --- 

R 15 0.839 0.043 5.1 15 0.865 0.053 6.1 15 0.861 0.052 6.0 14 0.867 0.044 5.1 

Pituitary 

(mg) 

U 15 11.5 2.7 23.5 15 12.3 2.2 17.9 15 11.5 2.3 20.0 14 11.0 3.1 28.2

A 15 11.5 --- --- 15 12.3 --- --- 15 11.5 --- --- 14 10.9 --- --- 

R 15 3.40 0.724 21.3 15 3.66 0.487 13.3 15 3.52 0.666 18.9 14 3.37 0.911 27.0

Adrenals 

(paired; mg) 

U 14c 56.0 7.3 13.0 15 55.4 7.8 14.1 15 53.7 8.0 14.9 14 56.3 6.9 12.3

A 14c 56.1 --- --- 15 55.3 --- --- 15 53.8 --- --- 14 56.2 --- --- 

R 14c 0.018 0.004 22.2 15 0.018 0.004 22.2 15 0.017 0.004 23.5 14 0.018 0.004 22.2

Seminal 

vesicle + 

coagulating 

gland, with 

fluid (mg) 

U 15 759.1 168.9 22.3 15 706.8 116.3 16.5 15 753.1 146.4 19.4 14 746.4 142.7 19.1

A 15 759.4 --- --- 15 706.5 --- --- 15 753.4 --- --- 14 746.1 --- --- 

Seminal 

vesicle + 

coagulating 

gland, 

without fluid 

(mg) 

U 15 430.1 88.4 20.5 15 406.9 54.7 13.4 15 445.0 89.0 20.0 14 441.0 86.5 19.6

A 15 431.6 --- --- 15 406.3 --- --- 15 445.5 --- --- 14 440.3 --- --- 

Ventral 

prostate 

(mg) 

U 15 261.5 45.2 17.3 15 243.0 66.9 27.5 14d 245.9 67.9 27.6 14 238.9 68.2 28.5

A 15 261.6 --- --- 15 242.9 --- --- 14d 246.0 --- --- 14 238.8 --- --- 

Dorsolateral 

prostate 

(mg) 

U 15 203.8 44.2 21.7 15 221.2 56.2 25.4 14d 187.6 51.2 27.3 14 222.9 46.2 20.7

A 15 204.0 --- --- 15 220.9 --- --- 14d 188.0 --- --- 14 222.5 --- --- 

LABC 

(mg) 

U 15 75.53 11.50 15.2 15 74.06 82.9 11.2 15 73.16 85.7 11.7 14 67.97 85.2 12.5

A 15 754.5 --- --- 15 741.3 --- --- 15 730.8 --- --- 14 680.7 --- --- 

Epididymis, 

left 

(mg) 

U 15 238.1 35.4 14.9 15 245.1 25.5 10.4 15 245.6 33.5 13.6 14 236.6 27.4 11.6

A 15 238.8 --- --- 15 244.2 --- --- 15 246.2 --- --- 14 235.9 --- --- 

Epididymis, 

right 

(mg) 

U 15 256.5 27.6 10.8 15 254.5 33.9 13.3 15 260.1 39.6 15.2 14 252.3 23.5 9.3 

A 15 256.8 --- --- 15 254.2 --- --- 15 260.4 --- --- 14 252.0 --- --- 

Testis, left 

(mg) U 15 1508.5 148.5 9.8 15 1454.

4 188.5 13.0 15 1489.3 111.1 7.5 14 1468.1 93.1 6.3 

A 15 1510.2 --- --- 15 1453.

0 --- --- 15 1490.7 --- --- 14 1466.3 --- --- 

Testis, right 

(mg) U 15 1508.2 140.9 9.3 15 1449.

7 239.3 16.5 15 1480.0 107.4 7.3 14 1461.0 89.0 6.1 

A 15 1510.4 --- --- 15 1447.

9 --- --- 15 1481.9 --- --- 14 1458.9 --- --- 

Page 260 of 292
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METALAXYL / 113501 OCSPP 890.1500/ OECD None 

TABLE 3. Organ Weights at Necropsya

Organ 

Vehicle Control Metalaxyl 

125 mg/kg/day

Metalaxyl 

250 mg/kg/day

Metalaxyl 

350 mg/kg/day 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) Nb Mean SD 

CV 

(%) 

Thyroid, 

fixed 

(mg) 

U 15 34.59 9.41 27.2 15 36.04 10.25 28.4 15 36.54 10.64 29.1 14 29.87 7.66 25.6

A 15 0.34.67 --- --- 15 35.97 --- --- 15 36.61 --- --- 14 29.78 --- --- 

a Data were obtained from Table 2 on pages 67-70 of the study report, and were sometimes rounded by the reviewers. Percent 

difference from control (calculated by reviewers from unrounded cited data) is included in parentheses. 

b Organ weights were not measured for Rat #349, which was found dead on PND 40. 

c Excludes values for Rat # 304, because the left adrenal was lost during processing. 

d Excludes values for Rat #339, which had a mass on the prostate. 

U Unadjusted for body weight on PND 21 

A Adjusted for body weight on PND 21 

R Organ-to-body weight ratio (relative to body weight) 

N Number of animals examined

SD Standard Deviation 

CV Coefficient of Variation 

** Significantly different from controls at p≤0.01.

E. HORMONE LEVELS AND CLINICAL CHEMISTRY: Mean hormone levels and 

clinical chemistry values and are presented in Table 5. Nominal ranges (provided from 

testing facility historical control data; n=20) for the clinical chemistry values were provided 

in the study report. 

Mean serum testosterone, T4, and TSH levels in the treated groups were similar to the 

control values. An increase (p≤0.01) of 7% was noted in globulin at 350 mg/kg/day, and 

minor increases (p≤0.05 or 0.01) of 1% in sodium were noted in each dose group. All other 

clinical chemistry values in the treated groups were similar to control values. 

Control group means and CVs for testosterone, T4, and TSH were within the acceptable 

range of the Guideline performance criteria. 

TABLE 5. Serum Hormone Levels and Clinical Chemistrya

Parameter 

Vehicle Control Metalaxyl 

125 mg/kg/day

Metalaxyl 

250 mg/kg/day

Metalaxyl 

350 mg/kg/day 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) Nb Mean SD 

CV 

(%) 

Thyroid Hormones 

Total T4 (μg/dL) 15 5.171 0.790 15.3 15 4.863 0.672 13.8 15 4.631 1.001 21.6 14 4.760 0.908 19.1 

TSH (ng/mL) 15 5.472 2.296 42.0 15 4.766 2.453 51.5 15 5.490 2.194 40.0 14 5.812 2.432 41.8 

Testosterone 

(ng/mL) 14c

 2.644 2.246 85.0 15 4.113 2.258 54.9 15 4.430 2.289 51.7 14 3.872 2.085 53.9 

Clinical Chemistry 

Creatinine (mg/dL) 12d 0.231 0.024 10.4 14d 0.243 0.018 7.4 15 0.231 0.025 10.8 13d 0.240 0.019 7.9 

BUN (mg/dL) 12d 16.6 1.7 10.2 14d 17.4 1.7 9.8 15 15.5 2.6 16.8 13d 15.1 1.5 9.9 

AST (IU/L) 12d 188.8 31.5 16.7 14d 225.7 57.2 25.3 15 188.9 35.8 19.0 13d 187.5 40.4 21.5 

ALT (IU/L) 12d 56.6 5.2 9.2 14d 59.6 5.8 9.7 15 56.1 8.0 14.3 13d 52.8 8.5 16.1 

Page 261 of 292
Male Pubertal Assay (2012) / Page 13 of 16 

METALAXYL / 113501 OCSPP 890.1500/ OECD None 

TABLE 5. Serum Hormone Levels and Clinical Chemistrya

Parameter 

Vehicle Control Metalaxyl 

125 mg/kg/day

Metalaxyl 

250 mg/kg/day

Metalaxyl 

350 mg/kg/day 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) Nb Mean SD 

CV 

(%) 

ALP (IU/L) 12d 409.1 84.9 20.8 14d 417.3 74.6 17.9 15 395.4 91.6 23.2 13d 373.4 67.0 17.9 

GGT (IU/L) 12d 0.140 0.334 239 14d 0.040 0.094 235 15 0.008 0.031 388 13d 0.057 0.205 360 

Total Bilirubin 

(mg/dL) 12d 0.161 0.035 21.7 14d 0.169 0.028 16.6 15 0.161 0.034 21.1 13d 0.157 0.029 18.5 

Cholesterol (mg/dL) 12d 90.8 12.0 13.2 14d 93.6 10.4 11.1 15 87.4 12.5 14.3 13d 90.0 16.2 18.0 

Triglycerides 

(mg/dL) 12d 126.6 24.2 19.1 14d 143.7 48.1 33.5 15 148.1 37.4 25.3 13d 133.9 32.3 24.1 

Total Protein (g/dL) 12d 5.609 0.166 3.0 14d 5.737 0.193 3.4 15 5.720 0.193 3.4 13d 5.777 0.146 2.5 

Albumin (g/dL) 12d 3.243 0.100 3.1 14d 3.296 0.086 2.6 15 3.281 0.118 3.6 13d 3.235 0.236 7.3 

Globulin (g/dL) 12d 2.366 0.109 4.6 14d 2.441 0.122 5.0 15 2.439 0.095 3.9 13d 2.542** 

(↑7) 0.233 9.2 

Albumin/Globulin 12d 1.372 0.067 4.9 14d 1.351 0.048 3.6 15 1.345 0.043 3.2 13d 1.286 0.164 12.8 

Glucose (mg/dL) 12d 139.7 11.4 8.2 14d 133.2 17.5 13.1 15 128.5 16.0 12.5 13d 131.5 16.1 12.2 

Calcium (mg/dL) 12d 10.50 0.342 3.3 14d 10.53 0.400 3.8 15 10.62 0.411 3.9 13d 10.60 0.400 3.8 

Phosphorus 

(mg/dL) 12d 9.929 0.516 5.2 14d 10.35 0.626 6.1 15 10.41 0.632 6.1 13d 10.53 0.564 5.4 

Sodium (mg/dL) 12d 139.6 1.2 0.9 14d 140.8** 

(↑1) 1.0 0.7 15 140.8** 

(↑1) 1.1 0.8 13d 140.8* 

(↑1) 0.9 0.6 

Potassium (mg/dL) 12d 6.526 0.499 7.6 14d 6.482 0.396 6.1 15 6.537 0.379 5.8 13d 6.540 0.468 7.2 

Chloride (mg/dL) 12d 99.2 1.2 1.2 14d 99.1 0.7 0.7 15 99.3 1.5 1.5 13d 99.2 1.1 1.1 

a Data were obtained from Table 3 on page 72 and Table 4 on pages 74-77 of the study report. Percent differences from 

controls (calculated by reviewers) are included in parentheses. 

b Serum was not analyzed for Rat #349, which was found dead on PND 40. 

c Excludes values for Rat # 304, because sample quantity was insufficient. 

d Excludes rats with severely hemolyzed serum that were not analyzed. 

N Number of animals examined 

SD Standard Deviation 

CV Coefficient of Variation 

* Significantly different from controls at p<0.05. 

** Significantly different from controls at p<0.01. 

G. GROSS PATHOLOGY: There were no treatment-related macroscopic findings. The 

following gross observations were observed: (i) moderate dilation in the pelvis of the right 

kidney in 1/15 males at 125 mg/kg/day; (ii) dilation in the right kidney in 0/15, 1/15, 2/15, 

and 1/14 males in the 0, 125, 250, and 350 mg/kg/day dose groups, respectively; and (iii) a 

firm mass on the ventral and dorsolateral prostate occurred in 1/15 males at 250 mg/kg/day. 

H. HISTOPATHOLOGY: The incidences of histopathological findings of the thyroid gland 

are presented below in Table 6; follicular cell shape was not reported. Compared to the 

control, thyroid colloid area was slightly reduced in a dose-dependent manner, but there was 

no clear effect on follicular cell height. 

Page 262 of 292
Male Pubertal Assay (2012) / Page 14 of 16 

METALAXYL / 113501 OCSPP 890.1500/ OECD None 

TABLE 6. Thyroid Colloid Area and Follicular Cell Height a

Treatment Groups 

Parameter Evaluated 

Colloid Area Follicular Cell Height 

(Increase) 

Grade b Incidence Grade b Incidence 

O E O E 

Vehicle Control 

1 0 15 1 7 15 

2 0 15 2 8 15 

3 0 15 3 0 15 

4 5 15 4 0 15 

5 10 15 5 0 15 

125 mg/kg/day 

Metalaxyl 

1 0 15 1 9 15 

2 0 15 2 6 15 

3 0 15 3 0 15 

4 4 15 4 0 15 

5 11 15 5 0 15 

250 mg/kg/day 

Metalaxyl 

1 0 15 1 11 15 

2 0 15 2 4 15 

3 0 15 3 0 15 

4 8 15 4 0 15 

5 7 15 5 0 15 

350 mg/kg/day 

Metalaxyl 

1 0 14 1 5 14 

2 0 14 2 9 14 

3 2 14 3 0 14 

4 6 14 4 0 14 

5 6 14 5 0 14 

a Data were obtained from Appendix 26 on page 499 of the study report. 

b A 5-point grading scale (1 = shortest/smallest; 5 = tallest/largest) was used. 

O No. Observed 

E No. Examined 

The incidence of histopathological findings of the testes, epididymides, and kidneys are 

presented in Table 7. No adverse histological lesion was observed in the testes, 

epididymides, or kidneys. 

Page 263 of 292
Male Pubertal Assay (2012) / Page 15 of 16 

METALAXYL / 113501 OCSPP 890.1500/ OECD None 

TABLE 7. Incidence of Histopathological Lesions of the Testes, Epididymides, and Kidneya

Findings 

Vehicle Control Metalaxyl 

125 mg/kg/day

Metalaxyl 

250 mg/kg/day

Metalaxyl 

350 mg/kg/day

# Observed # Examined # 

Observed # Examined # Observed # Examined # Observed # Examined

Testis

Seminiferous tubule 

degeneration, mild 0 15 1 15 0 15 0 14 

Spermatocyte 

degeneration, minimal 0 15 0 15 0 15 1 14 

Decreased spermatid 

elongation, moderate 0 15 1 15 0 15 0 14 

Epididymidis

Granuloma, minimal 1 15 0 15 0 15 0 14 

Cauda hypospermia 

 minimal 7 15 4 15 1 15 2 14 

 mild 1 15 0 15 0 15 0 14 

 marked 0 15 1 15 0 15 0 14 

Mononuclear cell 

infiltration, minimal 0 15 0 15 2 15 0 14 

Kidney

Mixed cell 

infiltration, minimal 0 15 0 15 1 15 1 14 

Dilation, minimal 0 15 2 15 2 15 0 14 

 mild 2 15 2 15 1 15 2 14 

a Data were obtained from Appendix 26 on page 498-499 of the study report. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Metalaxyl resulted in reductions in mean body 

weight at the age of preputial separation (but no change in the timing of preputial separation 

initiation or completion) and increases in absolute liver weights and/or ratios of liver to 

terminal body weight, concurrent with a slight increase in globulin in the 350 mg/kg/day 

dose group and slight increases in sodium in each of the groups administered the test 

substance. A single mortality among the male rats at 350 mg/kg/day was considered likely 

to be exposure related. No other treatment related effect was observed in this study. 

Based on these data, administration of metalaxyl under the tested conditions resulted in 

effects indicative of systemic/liver target organ toxicity, but no effects on endocrine activity 

or indications of potential interactions with the androgen or thyroid pathways were apparent. 

B. AGENCY COMMENTS: No adverse, treatment-related related effects were observed on 

survival, clinical signs, body weights, body weight gains, organ weights, clinical chemistry, 

or gross pathology. No adverse histological lesion was observed in the testes, epididymides, 

or kidneys. Mean serum testosterone, T4, and TSH levels in the treated groups were similar 

to the control values. The thyroid follicular cell heights in the treated groups were generally 

similar to the controls, without a clear dose-dependent effect. Although colloid area in the 

thyroid was slightly reduced in a dose-related manner, there were no concomitant effects 

noted in fixed thyroid weights or thyroid hormone concentrations. 

Page 264 of 292
Male Pubertal Assay (2012) / Page 16 of 16 

METALAXYL / 113501 OCSPP 890.1500/ OECD None 

Body weights, body weight gains, age of attainment of PPS, body weight at PPS, and 

proportion/incidence of separated prepuce in the treated groups were similar to control 

animals, with the exception of minor decreases (p≤0.05 or 0.01) in the adjusted body weight 

at PPS at 125 mg/kg/day and unadjusted and adjusted body weights at PPS at 250 and 350 

mg/kg/day. These minor decreases were not dose-dependent in magnitude and were not 

considered a treatment-related effect. No effect was noted on daily body weight 

measurements. 

All organ weight values in the treated groups were similar to control values, except for a 

slight increase (p≤0.01) of 10% in relative liver weight at 350 mg/kg/day compared to 

control. This effect was minor, without a correlated effect on absolute or adjusted weight, 

and without corroborating gross pathology or clinical chemistry findings of hepatotoxicity. 

Consequently, this effect was considered incidental. 

An increase (p≤0.01) of 7% was noted in globulin concentration at 350 mg/kg/day, and 

minor increases in sodium were noted in each dose group. All other clinical chemistry 

values in the treated groups were similar to control. 

One 350 mg/kg/day male was found dead on PND 40. Although the study authors 

considered this death to be treatment related, no data were presented to support that 

conclusion. The cause of death was not determined, but was not due to gavage error 

(perforation of lungs, trachea, or esophagus). All tissues appear normal at necropsy, and no 

adverse clinical signs were noted in this animal on any day during the study. Therefore, this 

death is considered incidental by the reviewers. 

 A clear adverse effect was not observed in this study; however, based on generalized 

summaries from several ranging-finding studies and clinical signs observed in the 

previously reviewed uterotrophic assay (MRID 48617606) at 300 mg/kg/day and liver 

weight increases in male rats in the Hershberger assay (MRID 48617604) at 375 mg/kg/day, 

the selected high dose (350 mg/kg/day) for the male pubertal study appears to be 

appropriate. 

 Under the conditions of this study, there was no evidence to support an effect of metalaxyl 

on pubertal development or thyroid function of juvenile male rats. 

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: 

 CV for body weight at PPS (9.1%) was greater than the maximum acceptable value 

(7.57%); 

 Mean final body weight of 332.5 mg (acceptable range: 259.235-332.059 g); 

 Mean weaning body weight of 60.5 g (acceptable range: 45.472-59.812 g); 

 Mean liver weight of 16.55 g (acceptable range: 9.990-15.350 g); 

 CV for pituitary weight (23.5%) was greater than the maximum acceptable value 

(15.98%); 

 CV for fixed thyroid weight (27.2%) was greater than the maximum acceptable value 

(23.63%). 

Page 265 of 292
DATA EVALUATION RECORD 

METALAXYL 

Study Type: OCSPP 890.1550, Steroidogenesis Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-46-2012 (MRID 48617605) 

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: 5/25/2012 

Secondary Reviewer: Signature:

Michael E. Viana, Ph.D., D.A.B.T. Date: 5/31/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 6/06/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 6/06/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 266 of 292
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 267 of 292
Steroidogenesis Assay (2011) I Page 1 of 13 

METALAXYL / 113501 OCSPP 890.1550/ OECD None 

Primary Reviewer: Vincent Chen, M.S. Signature:~ Risk Assessment Branch III, Health Effects Division (7509P) Date:~ z.o.LS 

Secondary Reviewer: John Liccione, Ph.D. Signature: -= - Health Effects Division (7509P) Date: ~ '?(!( 

Template version 08/2011 

DATA EVALUATION RECORD 

STUDY TYPE: Steroidogenesis Assay (H295R Cells); OCSPP 890.1550 

PC CODE: 113501 DP BARCODE: D399227 

TXR#: 0054219 CAS No.: 57837-19-1 

TEST MATERIAL (PURITY): Metalaxyl (98.2 ± 0.3%) 

SYNONYMS: Methyl N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alaninate 

CITATION: Wagner, H. (2011) Metalaxyl: H295R Steroidogenesis Assay. CeeTox, 

Kalamazoo, MI. Study ID: 9143V-100326STER, December 7, 2011. MRID 

48617605. Unpublished. 

SPONSOR: Metalaxyl EDSP Consortium 

20 Ambriance Drive, Burr Ridge, IL 60527, USA 

TESTORDER#: CON-113501-18 

EXECUTIVE SUMMARY: In a steroidogenesis assay (MRID 48617605), H295R cells 

cultured in vitro in 24-well plates were incubated with metalaxyl (98.2% a.i., Batch# 20100540) 

at log concentrations from 0.0001 to 100 μMin triplicate for 48 hours. Dimethyl sulfoxide 

(DMSO) was used as a vehicle, at final assay concentration of 0.05%. 

Testosterone and estradiol levels were measured using HPLC/MS/MS using internal standards. 

Four independent experiments were performed; however, only Runs 2 through 4 were deemed 

successful. 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 a known inhibitor (prochloraz) and known 

inducer (forskolin) of estradiol and testosterone production. 

All guideline acceptability recommendations and requirements were met, including lack of 

cytotoxicity, adequate production of testosterone and estradiol, acceptable reproducibility (low 

coefficients of variation, %CV), and appropriate induction and inhibition with positive controls. 

There were no significant changes in testosterone production in Run 2 at any concentration tested 

or in Run 4 at ~10 μM of metalaxyl, although there was a slight decrease in testosterone 

production (0.88-fold, p<0.05) at 100 μM. In Run 3, there was a consistent, slight increase in 

testosterone production (1.15- to 1.17-fold; p<0.05) at metalaxyl concentrations of 0.0001 to 10 

μM, but the increase at 100 μM was not significant (1.09-fold, NS). For estradiol, there were no 

Page 268 of 292
Steroidogenesis Assay (2011) / Page 2 of 13 

METALAXYL / 113501 OCSPP 890.1550/ OECD None

significant changes in production in Runs 2 or 4 at any tested concentration, and there was only a 

slight increase (1.18- to 1.19-fold; p<0.05) in estradiol production in Run 3 at the three lowest 

tested concentrations (0.0001 – 0.01 μM). Changes in testosterone and estradiol were not 

reproducible or concentration dependent. 

Based on the hormone responses in each of the three independent runs, EPTC treatment did not 

result in statistically significant and reproducible alterations in testosterone or estradiol 

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 269 of 292
Steroidogenesis Assay (2011) / Page 3 of 13 

METALAXYL / 113501 OCSPP 890.1550/ OECD None

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: CeeTox 

 Location: Kalamazoo, MI 

 Study Director: Hilary Wagner 

 Other Personnel: Toole, C., Director of Project Manager 

Rutherford, K., Director of Laboratory Operations 

Blakeman, D., Senior Scientist/ Endocrine Group Leader 

Wallace, B., Lead Cell Culture Scientist 

McColley, S., Scientist 

Haines, C., Scientist 

Wong, F., Scientist 

Burgam, J., Associate Scientist. 

Blakeman, L., Associate Scientist 

 Study Period: May 26 to December 7, 2011 

2. Test Substance: Metalaxyl 

 Description: Solid 

 Lot / Batch #: (Expiration Date) 20100540 (June 13, 2012) 

 Purity: 98.2 ± 0.3% 

 Solubility (in Solvent): At least 200 mM 

 Volatility: Not reported 

 Stability: Not reported 

 Storage conditions: Ambient 

 CAS #: 57837-19-1 

 Molecular weight: 279.3 

 Structure: 

3CH

N

O

CH3

O

3CH CH3

O O

3CH

3. Positive Control (Inducer): Forskolin 

 Description: White powder 

 Source: Sigma Aldrich (St. Louis, MO) 

 Lot / Batch #: (Expiration Date): 109K50571V (July 2016) 

 Purity: 98% 

 Solubility (in Solvent): Not reported 

 Storage conditions: Ambient 

 CAS #: 66575-29-9 

4. Positive Control (Inhibitor): Prochloraz 

 Description: White powder 

 Source: Sigma Aldrich (St. Louis, MO) 

 Lot / Batch #: (Expiration Date) SZE6220X (Aug 8, 2012) 

 Purity: 99.1% 

 Solubility (in Solvent): Not reported 

 Storage conditions: Ambient 

 CAS #: 67747-09-5 

Page 270 of 292
Steroidogenesis Assay (2011) / Page 4 of 13 

METALAXYL / 113501 OCSPP 890.1550/ OECD None

5. Solvent/Vehicle Control: DMSO 

 Description: Not provided 

 Source: Sigma Aldrich (St. Louis, MO) 

 Lot #: (Expiration Date) RNBB7617 (expiry date not provided) 

 Purity: Not provided 

 Storage conditions: Not provided 

 CAS #: 67-68-5 

 Justification for choice of solvent: One of the solvents recommended in the Guideline 

 Final concentration: 

(% volume in assay) 

0.05% 

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 

Source: Sigma Aldrich 

Lot / Batch #: (Expiration Date) Lot #021M8304 

Sodium bicarbonate: Sigma Aldrich (Lot # 068K0105) 

Nu-Serum: Lot number 81515; tested for background hormone concentrations by 

performing laboratory 

ITS+ premix: Lot numbers 05245, 09233 

8. Test Cells: H295R human adrenocortical carcinoma cells (ATCC CLR-2128; lot 7635054). 

Cells were grown for five passages, frozen in liquid nitrogen, then thawed and cultured for 

seven or eight additional passages prior to use in the assay. The culture medium was 

supplemented with 10 μM 22R-hydroxycholesterol at the time of plating, dosing and 

harvest. The H295R cells were maintained in supplemented media containing DMEM:F12 

media base with ITS + Premix and Nu-Serum. Cells were incubated at 37°C in an 

atmosphere of 5% CO2. 

The following performance criteria were met (indicated by an “x”): 

NR Cell passage identifier. Cell Passage #: not reported 

X Cells frozen down at passage 5 

X Frozen cells cultured for 4 additional passages 

X Total number of passages does not exceed 10 

B. METHODS

1. Pre-Test Information

a. Hormone Assay Interference Test: Analysis of the production of testosterone and 

17β-estradiol by H295R cells was conducted by HPLC/MS/MS using internal standards to 

measure extraction and quantitation efficiency. No interference would be anticipated with 

this analytical methodology. 

Page 271 of 292
Steroidogenesis Assay (2011) / Page 5 of 13 

METALAXYL / 113501 OCSPP 890.1550/ OECD None

b. Hormone Extraction: Testosterone and estradiol were extracted from H295R 

supplemented medium by liquid-liquid extraction using methyl tert-butyl ether (MTBE) 

after the addition of [2H5]-testosterone and [2H5]-estradiol as internal standards. Extracts 

were analyzed by HPLC-MS/MS using positive ion multiple reaction monitoring. 

c. Laboratory Proficiency Test: It was stated that all assays were performed by personnel 

that demonstrated proficiency performing the assay as outlined in the test guideline. 

Proficiency records are referenced in the study records, but were not included in the study 

report. 

2. Test Solutions: A 200 mM stock solution was prepared by dissolving an appropriate 

amount of metalaxyl in DMSO. Six serial 1:10 dilutions were then prepared, yielding a 

range of 200 mM to 0.2 μM. Forskolin and prochloraz solutions were prepared in a similar 

manner. All dilutions were then diluted 1:2000 in 10 μM 22R hydroxycholesterolcontaining supplemented medium to yield the concentrations presented in Tables 1 and 2. 

3. Cell Plating and Preincubation: H295R cells (ATCC CLR-2128) were grown for five 

passages, frozen in liquid nitrogen, then thawed and cultured for seven or eight additional 

passages. The cells were then seeded in 24-well plates at a density of 300,000 cells/mL. 

The plates were incubated for 24 hours at 37°C in a 5% CO2 atmosphere yielding 

approximately 50-60% confluency. 

4. Exposure: The cells were checked microscopically for good attachment and proper 

morphology, and the medium was removed and replaced with 1 mL of medium containing 

10 μM 22R-hydroxycholesterol and the concentrations of metalaxyl in triplicate according 

to the schematic presented in Table 1. 

TABLE 1. Dosing Schematic for the Exposure of H295R Cells to Metalaxyl (Final Concentration 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 16 of the study report. Dosing was calculated based on a total volume of 1 mL per well. 

A concurrent 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. 

Page 272 of 292
Steroidogenesis Assay (2011) / Page 6 of 13 

METALAXYL / 113501 OCSPP 890.1550/ OECD None

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 16 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 hours under the conditions previously 

described. The medium from each well was removed, split into equal volume aliquots, and 

frozen at −80°C until hormone measurements. 

5. Cell Viability/Cytotoxicity Assay: Cell viability was determined by MTT assay after the 

48±2 hour exposure. Media was removed and 0.5 mL of 0.5 mg/mL MTT solution in 

supplemented medium containing 10μM 22R-hydroxycholesterol was added to each well of 

the test chemical and QC plates. Plates were incubated at 37±2°C for 3 hours, after with the 

MTT solution was removed and replaced with 0.5 mL of isopropanol. Plates were 

incubated at room temperature for 20 minutes, and absorbance at 570 nm and 650 nm were 

measured. 

6. Hormone Measurement System: The concentration of each hormone was determined in 

triplicate in the culture medium following liquid-liquid extraction with MTBE. Prior to 

extraction, [2H5]-testosterone and [2H5]-estradiol were added to the medium samples as 

internal standards to account for hormone recovery. The extracts were analyzed by HPLCMS/MS using positive ion multiple reaction monitoring. The lower limit of quantification 

(LLQ) was 100 pg/mL for testosterone and 10 pg/mL for estradiol using a 300 μL aliquot of 

H295R supplemented medium. 

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 for concentrations of 

testosterone and estradiol were calculated for each test level of the reference chemicals and 

the solvent controls, as well as for the blank and background wells. Relative changes in 

hormone production were calculated using the following equation: 

Relative Change = [Hormone] in each well ÷ [Hormone] of mean solvent control 

For forskolin induction of testosterone, the background hormone production was subtracted 

from the forskolin-treated wells and blank and solvent control wells before calculating the 

relative change. This was done because of the high background production of testosterone 

and the small dynamic range for forskolin induction. Background hormone production was 

Page 273 of 292
Steroidogenesis Assay (2011) / Page 7 of 13 

METALAXYL / 113501 OCSPP 890.1550/ OECD None

calculated from three wells with cells on the QC plate that received no 22Rhydroxycholesterol. 

All concentrations that exhibited greater than 20% cytotoxicity in the MTT cell viability 

assay were excluded from further analysis. Concentrations where precipitation was 

observed were also excluded from further analysis. 

Normality of the data was evaluated using Shapiro-Wilk’s test. Homogeneity of the 

variances between the treatment groups was evaluated using Levene’s test. If the p-values 

were greater than 0.05 in both tests, statistical significance between each treatment group 

and the control group was evaluated using Dunnett’s test. If the p-values were less than or 

equal to 0.05 in either the normality or the homogeneity test, a log transformation was 

performed on the data to attempt to approximate a normal distribution. If, following the 

transformation, p-values were greater than 0.05 in both the normality and homogeneity tests, 

Dunnett’s test was performed on the transformed data to evaluate statistical significance 

between the each treatment group and the control group. If following the log 

transformation, p-values were less than or equal to 0.05 in either the normality or the 

homogeneity test, the non-transformed data set was analyzed using the nonparametric 

Kruskal-Wallis test followed by Dunn’s test to evaluate statistical significance between each 

treatment group and the control group. 

II. RESULTS

A. TEST COMPOUND: The hormone concentrations after exposure to metalaxyl, and the 

fold difference change relative to the SC and the mean ± SD for the three successful assays 

are presented in Table 3 and shown graphically in Figures 1 and 2. No results were reported 

for Run 1 as it was not successful. Precipitation was not observed at any tested metalaxyl 

concentration. The %CVs based on absolute hormone concentrations within each plate for 

the SC were 1.8-5.7% for testosterone and 0.8-8.7% for estradiol. The between-plate %CVs 

for the SC based on absolute concentrations (calculated by reviewer) were 24% and 29% for 

testosterone and estradiol, respectively. These values are below the Guideline limit of 

≤30%. 

 For testosterone, there were no significant changes in production in Run 2 at any 

concentration tested or in Run 4 at ≤10 μM of metalaxyl, although there was a slight 

decrease in testosterone production (0.88-fold, p<0.05) at 100 μM. In Run 3, there was a 

consistent, slight increase in testosterone production (1.15- to 1.17-fold; p<0.05) at 

metalaxyl concentrations of 0.0001 to 10 μM, but the increase at 100 μM was not significant 

(1.09-fold, NS). The statistical significance noted in Run 3 was due to lower control values 

in this run compared to Runs 2 and 4. 

 For estradiol, there were no significant changes in production in Runs 2 or 4 at any tested 

concentration, and there was only a slight increase (1.18- to 1.19-fold; p<0.05) in estradiol 

production in Run 3 at the three lowest tested concentrations (0.0001 – 0.01 μM). The 

statistical significance noted in Run 3 was due to lower control values in this run compared 

to Runs 2 and 4. 

Page 274 of 292
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METALAXYL / 113501 OCSPP 890.1550/ OECD None

TABLE 3. Mean (±SD) Hormone Concentrations Following Treatment with Metalaxyl for 48 Hours. 

Nominal 

Concentration 

(μM) 

Run 2 Run 3 Run 4 Run 2 Run 3 Run 4 Meanb ± SDb Statistical 

Significance

Testosterone (pg/mL) a Fold Differenceb 

DMSO 2569  46 1585  87 2391  137 -- --- -- -- -- -- 

0.0001 2562  104 1854  26 2272  46 1.00 1.17 0.95 1.04 0.12 Run 3 

0.001 2607  92 1837  40 2310  95 1.01 1.16 0.97 1.05 0.10 Run 3 

0.01 2599  190 1850  121 2376  87 1.01 1.17 0.99 1.06 0.10 Run 3 

0.1 2566  97 1819  64 2438  51 1.00 1.15 1.02 1.06 0.08 Run 3 

1 2604  80 1843  85 2327  107 1.01 1.16 0.97 1.05 0.10 Run 3 

10 2456  28 1831  22 2212  76 0.96 1.16 0.93 1.01 0.12 Run 3 

100 2372  56 1729  38 2105  68 0.92 1.09 0.88 0.96 0.11 Run 4 

Estradiol (pg/mL) a Fold Differenceb

DMSO 255  2.0 140  12.2 216  5.8 -- -- -- -- -- -- 

0.0001 242  10.3 166  1.4 221  4.4 0.95 1.19 1.02 1.05 0.12 Run 3 

0.001 250  7.7 166  3.5 216  2.0 0.98 1.18 1.00 1.05 0.11 Run 3 

0.01 245  15.3 165  6.6 222  4.2 0.96 1.18 1.03 1.06 0.11 Run 3 

0.1 253  6.7 162  5.7 219  6.1 0.99 1.15 1.01 1.05 0.09 

1 260  10.7 159  4.4 226  9.6 1.02 1.13 1.05 1.07 0.06 

10 248  5.8 159  0.9 214  6.6 0.97 1.14 0.99 1.03 0.09 

100 247  10.4 154  0.6 202  6.3 0.97 1.10 0.94 1.00 0.09 

a Data were obtained from pages 30-31 and 57-59 of the study report. 

b Calculated by reviewer 

Page 275 of 292
Steroidogenesis Assay (2011) / Page 9 of 13 

METALAXYL / 113501 OCSPP 890.1550/ OECD None

FIGURE 1. Change in Testosterone Production Relative to Metalaxyl Concentration. 

Page 276 of 292
Steroidogenesis Assay (2011) / Page 10 of 13 

METALAXYL / 113501 OCSPP 890.1550/ OECD None

FIGURE 2. Change in Estrogen Production Relative to Metalaxyl Concentration. 

B. CYTOTOXICITY: Metalaxyl was not cytotoxic to the H295R cells (>80% viability, 

Table 4). Viability across all of the test plates ranged from 91.9-109.3%. 

Page 277 of 292
Steroidogenesis Assay (2011) / Page 11 of 13 

METALAXYL / 113501 OCSPP 890.1550/ OECD None

TABLE 4. Mean (±SD) MTT Cell Viability Results after Treatment with Forskolin, Prochloraz, or 

Metalaxyl 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 104.3 2.69 104.3 2.81 100.7 3.98 

Background NA 95.4 4.14 102.5 1.79 98.9 0.94 

SC + Methanol NA 5.6 2.51 11.4 1.99 5.1 0.14 

Forskolin 1 b 107.8 1.52 109.3 1.17 105.0 1.55 

Forskolin 10 107.0 2.43 108.5 4.17 102.4 2.58 

Prochloraz 0.1 98.3 2.20 101.2 0.47 100.7 0.86 

Prochloraz 1 99.4 1.19 101.5 0.78 99.5 2.37 

Metalaxyl 0.0001 95.9 2.00 93.3 4.04 96.0 0.75 

Metalaxyl 0.001 95.2 2.18 95.0 1.51 98.1 1.15 

Metalaxyl 0.01 96.9 0.99 94.8 2.73 96.2 2.35 

Metalaxyl 0.1 97.4 0.79 98.0 0.87 98.1 0.94 

Metalaxyl 1 91.9 0.73 97.1 4.49 94.9 2.07 

Metalaxyl 10 95.4 2.70 98.3 6.07 97.2 1.23 

Metalaxyl 100 93.4 2.56 101.8 2.57 97.8 1.40 

a Data were obtained from pages 24-25 of the study report. 

NA Not applicable 

SC Solvent control 

C. QC PLATE: The hormone concentrations after exposure to the reference chemicals, SC, 

and blank samples, as well as the fold difference change relative to SC (individual and mean 

± SD) for the three assay runs are presented in Table 5. The minimum basal hormone 

production levels for testosterone (500 mg/mL) and estradiol (40 pg/mL) were met in both 

blank and SC wells. Testosterone production in the presence of 10 μM forskolin was 1.6- to 

1.9-fold, which is below the Guideline criteria of ≥2-fold; however, when the background is 

subtracted, 10 μM forskolin induced testosterone an average of 2.5-fold.. Estradiol 

production in the presence of 10 μM forskolin was greater than the ≥7.5-fold requirement. 

Compared to SC, 1 μM prochloraz on average inhibited the synthesis of testosterone 0.41-

fold and estradiol 0.32-fold. Thus, the guideline requirements were met indicating that the 

assay was sensitive to induction and inhibition of testosterone and estradiol. 

The variability (%CV) between the runs (calculated by the reviewer) based on the absolute 

hormone concentrations in the SC were 11% for estradiol and 15% for testosterone, and 

within the Guideline recommended limit of ≤30%. The %CVs for the SC within each run 

for the QC plates were 0.8-5.4% for testosterone and 2.9-6.2% for estradiol. 

Page 278 of 292
Steroidogenesis Assay (2011) / Page 12 of 13 

METALAXYL / 113501 OCSPP 890.1550/ OECD None

TABLE 5. Hormone Concentrations Following Treatment with Forskolin or Prochloraz for 48 Hours.a

Compound Conc. 

(μM) 

Run 1 Run 2 Run 4 Run 1 Run 2 Run 4 Mean b ± SD b

Testosterone (pg/mL) Fold Difference 

Background NA 1135 1073 1262 0.45 0.57 0.54 0.52 0.06 

Blank NA 2581 1873 2304 1.03 1.00 0.99 1.01 0.02 

DMSO NA 2511 1874 2339 1.00 1.00 1.00 1.00 -- 

Forskolin 1 3764 2595 3120 1.50c 1.38 c 1.33 c 1.40 c 0.09 

Forskolin 10 4751 3019 3780 1.89 d 1.61 d 1.62 d 1.71 d 0.16 

Prochloraz 0.1 1816 1149 1818 0.72 0.61 0.78 0.70 0.09 

Prochloraz 1 1188 501 1153 0.47 0.27 0.49 0.41 0.12 

Estradiol (pg/mL) Fold Difference 

Background NA 41 56 77 0.21 0.31 0.35 0.29 0.07 

Blank NA 208 182 228 1.04 1.01 1.02 1.02 0.02 

DMSO NA 200 179 223 1.00 1.00 1.00 1.00 -- 

Forskolin 1 1343 1254 1538 6.72 7.01 6.91 6.88 0.15 

Forskolinc 10 2116 1661 2477 10.60 9.28 11.13 10.34 0.95 

Prochloraz 0.1 133 104 171 0.67 0.58 0.77 0.67 0.10 

Prochloraz 1 57 52 83 0.29 0.29 0.37 0.32 0.05 

a Data were obtained from pages 26-27 of the study report. 

b Calculated by the reviewers. 

c When the background is subtracted, the values are 1.91-, 1.90-, and 1.74-fold difference for Runs 2, 3, and 4, respectively, 

with a mean of 1.85-fold change. 

d When the background is subtracted, the values are 2.63-, 2.43-, and 2.34-fold difference for Runs 2, 3, and 4, respectively, 

with a mean of 2.47-fold change. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: For estradiol, no statistically significant effects 

were observed in two of the three runs of the steroidogenesis assay. For testosterone, in one 

run, no statistically significant effects were identified. In another run, a statistically 

significant reduction was observed at the highest concentration tested. Although statistically 

significant effects were identified for testosterone and estradiol in the remaining run of the 

assay, the run was considered an anomaly as it differed from the other two runs. Overall 

there was no consistent evidence that metalaxyl caused an increase or decrease in 

testosterone or estradiol biosynthesis in the H295R cell line. 

B. AGENCY COMMENTS: Precipitation was not observed at any tested metalaxyl 

concentration, and metalaxyl was not overtly cytotoxic to the H295R cells. Viability for 

metalaxyl on the test plates was ≥91.9%. Four independent test runs of the steroidogenesis 

assay were conducted with metalaxyl. Run 1 was not analyzed because one of the forskolin 

concentrations on the QC plates was 3.33μM instead of 1 μM. 

All guideline acceptability recommendations and requirements were met, including lack of 

cytotoxicity, adequate production of testosterone and estradiol, acceptable reproducibility 

(low %CV), and appropriate induction and inhibition with positive controls. 

There were no significant changes in testosterone production in Run 2 at any concentration 

tested or in Run 4 at ≤10 μM of metalaxyl, although there was a slight decrease in 

testosterone production (0.88-fold, p<0.05) at 100 μM. In Run 3, there was a consistent, 

slight increase in testosterone production (1.15- to 1.17-fold; p<0.05) at metalaxyl 

concentrations of 0.0001 to 10 μM, but the increase at 100 μM was not significant (1.09-

Page 279 of 292
Steroidogenesis Assay (2011) / Page 13 of 13 

METALAXYL / 113501 OCSPP 890.1550/ OECD None

fold, NS). For estradiol, there were no significant changes in production in Runs 2 or 4 at 

any tested concentration, and there was only a slight increase (1.18- to 1.19-fold; p<0.05) in 

estradiol production in Run 3 at the three lowest tested concentrations (0.0001 – 0.01 μM). 

As there were no reproducible, concentration-dependent effects on either testosterone or 

estradiol production, metalaxyl does not appear to affect testosterone or estradiol 

production under the conditions of this assay. 

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: 

 Although the steroidogenesis assay proficiency and validation tests for the performing 

laboratory were reported as completed and sufficient, no data were included in the 

study report to support this claim. 

Page 280 of 292
DATA EVALUATION RECORD 

METALAXYL 

Study Type: OCSPP 890.1600, In vivo Uterotrophic Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-46-2012 (MRID 48617606) 

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: 4/25/2012 

Secondary Reviewer: Signature:

Kelly Luck, M.S. Date: 5/11/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 6/05/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 6/05/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 281 of 292
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 282 of 292
METALAXYL/ 113501 

In vivo Uterotrophic Assay (2011) I Page I of 10 

OCSPP 890.1600/ OECD 440 

Primary 

Health 

Health 

Secondary 

Effects 

Effects 

Reviewer: 

Reviewer: 

Division 

Division 

---'J"-'e=s=-s 

Minerva 

""'R=o-'-w'""'lan=d"--

Mercado 

________ 

Ph.D. DABT 

Signature: 

Signatu

Date: 

Date

~ 

~ 

d=-.,...,_ 

W't=[ 

~ 

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Template version 09/2011 

DATA EVALUATION RECORD 

STUDY TYPE: Uterotrophic Assay (Rat); OCSPP 890.1600; OECD 440 

PC CODE: 113501 DP BARCODE: D399227 

TXR#: 0054219 CAS#: 57837-19-1 

TEST MATERIAL (PURITY): Metalaxyl (98.2% a.i.) 

SYNONYMS: Alanine, N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-, methyl ester 

CITATION: Hoberman, A.M. (2011) Oral (Gavage) Uterotrophic Bioassay ofMetalaxyl in 

Female Rats. Charles River Laboratories, Preclinical Services, Horsham, PA. 

Laboratory Project ID: 20012406, December 9, 2011. MRID 48617606. 

Unpublished. 

SPONSOR: Metalaxyl EDSP Consortium, 20 Ambriance Drive, Burr Ridge, IL 

TEST ORDER#: CON-113501-18 

EXECUTIVE SUMMARY: In a Uterotrophic Assay (MRID 48617606) conducted to screen 

for potential estrogenic activity, metalaxyl (98.2% a.i., Batch/lot# 20100540) in 2.0% aqueous 

carboxymethylcellulose (CMC) was administered daily via oral gavage to groups of six 

ovariectomized female Sprague Dawley rats at dose levels of 0 (vehicle), 100 or 300/200 

mg/kg/day on post-natal days (PND) 57-59 or 63-65. A positive control group was treated with 

a daily dose of 17a-ethynyl estradiol (EE) at 5 μg/kg/day by subcutaneous (s.c.) injection. Body 

weights and food consumption were determined daily. All animals were terminated and 

necropsied on PND 60 or PND 66 approximately 24 hours after the final dose administration to 

determine wet and blotted uterine, and liver weights. 

All animals survived until scheduled termination. Following administration of metalaxyl at 300 

mg/kg/day on Day 1, adverse clinical signs were observed (decreased motor activity, ptosis, 

ataxia, lacrimation, hunched posture, intermittent body jerks, chromodacryorrhea, and slight 

excess salivation), as well as reductions in food consumption. Therefore, the 300 mg/kg/day 

dose level was reduced to 200 mg/kg/day on Study Days 2 and 3. Following the dose reduction, 

food consumption in this group increased and no other adverse clinical signs were observed. 

Body weights throughout the dosing period and overall (Days 1-4) body weight gain (BWG) in 

the metalaxyl treated groups were comparable to controls. Overall (Days 1-4) food consumption 

for both metalaxyl treatment groups was decreased (p::S0.05) by 6-8% compared to the vehicle 

controls. Uterine weights in the metalaxyl treated groups were comparable to the vehicle 

controls. Absolute and relative liver weights in the 300/200 mg/kg/day group were increased 

Page 283 of 292
In vivo Uterotrophic Assay (2011) / Page 2 of 10 

METALAXYL / 113501 OCSPP 890.1600/ OECD 440

(p≤0.05) compared to vehicle controls (↑24-25%); relative liver weights were increased (p≤0.05) 

in the 100 mg/kg/day group (↑13%). 

Food consumption in the EE positive control group was decreased (p≤0.01) throughout the 

study, for a decrease (p≤0.01) of 19% in overall average food consumption and a decrease 

(p≤0.01) of 54% in the overall BWG compared to the vehicle controls. Absolute wet and blotted 

uterus weights for the positive control (EE) group were increased (p≤0.01) by 547% and 207%, 

respectively, as expected. 

The high dose used in this study was adequate based on adverse clinical signs of toxicity 

observed following a single dose of metalaxyl at 300 mg/kg and increased liver weights in the 

300/200 mg/kg/day group. 

No statistically significant changes were seen in uterine weight in this assay. Metalaxyl 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. 

Page 284 of 292
In vivo Uterotrophic Assay (2011) / Page 3 of 10 

METALAXYL / 113501 OCSPP 890.1600/ OECD 440

I. MATERIALS AND METHODS

A. MATERIALS 

1. Test Facility: Charles River Laboratories, Preclinical Services, 

Pennsylvania (PCS-PA) 

Location: Horsham, PA 

 Study Director: A.M. Hoberman 

 Other Personnel: K. Strauss (Principal Investigator) 

 Study Period: June 14, 2011 to December 9, 2011 

2. Test Substance: Metalaxyl 

 Description: White to beige powder 

 Source: Nation’s Ag, LLC (Knoxville, TN) 

 Lot/Batch #: 20100540 (expiry date: June 13, 2012) 

 Purity: 98.2% a.i. 

 Stability: Stable in vehicle at room temperature for 25 hours and under refrigerated 

conditions for 10 days 

 CAS #: 57837-19-1 

 Structure: 

3CH

N

O

CH3

O

3CH CH3

O O

3CH

3. Reference Estrogen: 17α-ethynyl estradiol (EE) 

 Supplier: Sigma Aldrich (St. Louis, MO) 

 Lot/Batch #: 011M1252V (expiry date: June 8, 2012) 

 Purity: 99% 

 CAS #: 57-63-6 

4. Solvent/Vehicle Control 

(test substance):

Carboxymethylcellulose (CMC) 

 Supplier: 

Product #: 

Sigma Aldrich (St. Louis, MO) 

C5678 (sodium salt, low viscosity) 

 Lot/Batch #: 060M0079V (expiry (retest) date: June 30, 2012) 

 Rationale (if other than water): Use of an aqueous solution/suspension is recommended and an analytical method is 

available. 

 Final concentration: 2.0% in reverse osmosis membrane-processed deionized water 

 Solvent/Vehicle Control 

(reference estrogen):

Corn oil 

 Supplier: Spectrum Chemical 

 Lot/Batch #: YR1134 (expiry date: June 30, 2012) 

 Rationale (if other than water): None provided 

 Final concentration: Not applicable 

Page 285 of 292
In vivo Uterotrophic Assay (2011) / Page 4 of 10 

METALAXYL / 113501 OCSPP 890.1600/ OECD 440

5. Test Animals: 

 Species: Rat (ovariectomized females only) 

 Strain: Sprague Dawley, Crl:CD(SD) 

 Age/weight at dose initiation: 57 or 63 days old/ 198.2-242.6 g 

 Source: Charles River Laboratories (Portage, MI) 

 Housing: 2 rats/cage in clear plastic nesting boxes with heat-treated laboratory-grade pine 

shavings. 

 Diet: Harlan’s Teklad 2018c meal food, ad libitum; concentration of genistein 

equivalents (aglycone) or daidzein was ≤300 ppm per lot. Chewable Nylabones® 

were supplied during the study.

 Water: Reverse-osmosis treated tap water, ad libitum from individual, non-plastic 

water bottles attached to the nesting boxes 

 Environmental conditions: Temperature: 

Humidity: 

Air changes: 

Photoperiod: 

19-25oC 

30-70% 

>10/hr 

12 hrs light/12 hrs dark 

 Acclimation period: 6 days 

B. STUDY DESIGN 

1. In-Life Dates: Start: June 20, 2011 End: June 23, 2011 

2. Study Design: Sexually mature ovariectomized rats (PND 51 or 57) were received from 

Charles River Laboratories (ovariectomized on PND 43 or 49, respectively). Animals were 

acclimated for 6 days prior to initiation of dosing. Vaginal smears were taken daily during 

the last five days of the acclimation period and examined microscopically 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 or PND 63 through 65. Rats were euthanized 

approximately 24 hours later on PND 60 or PND 66, respectively, and necropsied for 

uterine and liver weight measurements. 

3. Animal Assignment: Animals were assigned, stratified by body weight, to the test groups 

noted in Table 1 using computer-generated (weight-ordered) randomization procedures. 

Statistical analysis indicated that there were no significant differences in group means at 

study initiation. Furthermore, the body weight of each animal was within 20% of the group 

mean. 

TABLE 1. Study Design a

Test Group Dose (mg/kg/day) # of Females 

Estrogen Agonist Assay 

Vehicle Control (2% CMC) 0 6 

Metalaxyl Low 100 6 

Metalaxyl High 300/200 b 6 

17α-ethynyl estradiol (EE), Reference Estrogen 5 μg/kg/day 6 

a Data were obtained from Text Table 5 on page 23 of the study report. 

b Following the first day of dosing at the high dose (300 mg/kg/day) adverse clinical signs in the animals were 

observed, so the dose volume was reduced from 5 mL/kg to 3.33 mL/kg to achieve a dose of approximately 200 

mg/kg/day (199.8 mg/kg/day) for the next two days of dosing. 

Page 286 of 292
In vivo Uterotrophic Assay (2011) / Page 5 of 10 

METALAXYL / 113501 OCSPP 890.1600/ OECD 440

4. Dose Selection Rationale: The dose levels were selected based on the results from rangefinding studies (Protocols 20012405 and 20013495); citations for the studies were not 

provided. Clinical signs of systemic toxicity were observed in adult male and female rats 

and weanlings at >500 mg/kg/day; mortality was seen in adult female rats at >500 

mg/kg/day and in weanlings of both sexes at 750 mg/kg/day. Due to the severity of the 

clinical signs observed in adult female rats at >500 mg/kg/day, the high dose selected for the 

uterotrophic assay was 300 mg/kg/day. The low dose of 100 mg/kg/day represents one-half 

log interval of the high dose. 

5. Dose Preparation and Analysis: Metalaxyl dose formulations were prepared once prior to 

the start of dosing by mixing appropriate amounts of the test substance with 2% aqueous 

CMC; daily aliquots were dispensed and stored refrigerated until use. Prepared doses were 

allowed to return to room temperature and stirred for at least 30 minutes prior to dosing. EE 

was prepared daily in corn oil. 

Homogeneity and stability of metalaxyl formulations (0.5 and 100 mg/mL) in 2% aqueous 

CMC were previously evaluated in conjunction with Protocol 20012404 (report attached as 

Appendix 5 of the subject study). During the uterotrophic study, duplicate samples of dose 

formulations (20 and 60 mg/mL) were collected on the day of preparation, and analyzed for 

achieved concentration. 

Results of Dose Analysis 

Homogeneity (% RSD): 0.4% and 1.5% 

Stability (% of Day 0): 101.3% and 101.8% following storage protected from light at 

room temperature (20-25°C) for 25 hours; and 99.7% and 100% following 

storage under refrigerated conditions (2-8°C) for 10 days 

Concentration (% of nominal): 101.1%-102.1% 

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: Metalaxyl formulations were administered to the animals daily 

via oral gavage (5 mL/kg) for 3 days. The oral route of administration was selected for the 

current study because it is a likely potential route of human exposure. It is noted that the 

dose volume of the high dose concentration (300 mg/kg/day) was reduced after Day 1 

administration to 3.33 mL/kg to achieve a lower dose concentration (200 mg/kg/day) for the 

next two doses. EE was given daily via two s.c. injections at 2.5 μg/kg in 2.5 

mL/kg/injection; injection sites were rotated between six areas on the dorsum to minimize 

irritation. Dose volumes were adjusted daily based on the most recently recorded body 

weight and administered at approximately the same time each day to each animal. 

Page 287 of 292
In vivo Uterotrophic Assay (2011) / Page 6 of 10 

METALAXYL / 113501 OCSPP 890.1600/ OECD 440

7. Statistics: Means, standard deviations, and percentages were calculated. Statistical 

analyses were performed using SAS System, version 6.12 or Quattro Pro 8, version 8.0. 

Clinical observations and other proportional data were analyzed using the variance test for 

homogeneity of the binomial distribution. 

Continuous data (e.g., body weights, body weight changes, and food consumption values) 

were analyzed using Bartlett’s test of homogeneity of variances and the analysis of variance

when appropriate [i.e., Bartlett’s test was not significant (p>0.001)]. If the analysis of 

variance was significant (p≤0.05), Dunnett’s test was used to identify the statistical 

significance of the individual groups. If the analysis of variance was not appropriate [i.e., 

Bartlett’s test was significant (p≤0.001)], the Kruskal-Wallis test was used (≤75% ties). In 

cases where the Kruskal-Wallis test was statistically significant (p≤0.05), Dunn’s method of 

multiple comparisons was used to identify the statistical significance of the individual 

groups. If there were >75% ties, Fisher’s Exact test was used to analyze the data. Wet and 

blotted uterine weights were analyzed by analysis of covariance (ANCOVA), using the 

terminal body weight as the covariate. The statistical analyses were considered adequate. 

C. METHODS

1. Clinical Examinations: Animals were assessed for viability twice daily. General 

appearances were observed on the day of receipt, weekly during the acclimation period and 

daily before administration during the dosing period. Additionally post-dose observations 

were recorded within 2 hours of dosing and at the end of the normal working day. Vaginal 

patency was recorded once on the day of termination. 

2. Body Weight: Animals were weighed weekly during the acclimation period, daily 

throughout the dosing period, and at termination. 

3. Food Consumption: Food consumption was measured daily during dosing and at 

termination. Values were reported as group mean daily food consumption (g/animal/day). 

4. Necropsy and Measurement of Uterine Weight: On PND 60 or PND 66 (approximately 

24 hours after final administration of the test substance), all animals were euthanized by 

carbon dioxide asphyxiation and subjected to a gross necropsy. Dissection of the uterus was 

performed according to the U.S. EPA Guideline. Briefly, the vagina was removed just 

below the cervix in order to retain the luminal fluid in the uterus. The “wet” uterus (i.e., 

containing the luminal fluid) was weighed. Subsequently, both uterine horns were pierced 

or cut longitudinally and the uterus place on lightly moistened filter paper and gently 

pressed with a second piece of lightly moistened filter paper to completely remove the 

luminal fluid. The blotted uterus was weighed. After weighing, the uterus was fixed in 

neutral buffered 10% formalin and retained for possible future histopathological evaluation. 

In addition, the liver was excised and weighed, and portions preserved in neutral buffered 

10% formalin or flash frozen for possible future evaluation. 

Page 288 of 292
In vivo Uterotrophic Assay (2011) / Page 7 of 10 

METALAXYL / 113501 OCSPP 890.1600/ OECD 440

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: Decreased motor activity, ptosis, ataxia, lacrimation, and 

hunched posture occurred in a significantly increased (p≤ 0.01) number of female rats in the 

300 mg/kg/day dose group on Study Day 1, in comparison with the vehicle control group. 

Intermittent body jerks, chromodacryorrhea, and slight excess salivation were also observed 

in female rats in this dose group on Day 1. All of the adverse clinical signs were attributed 

to treatment with the test substance. The 300 mg/kg/day dose was reduced to 200 

mg/kg/day on Day 2 of study and these adverse clinical signs were not observed on Days 2 

and 3. No other adverse clinical signs of toxicity were observed for any of the dose groups. 

B. BODY WEIGHT AND WEIGHT GAIN: Selected body weight and body weight gain 

data are presented in Table 2. Body weights throughout the dosing period and overall (Days 

1-4) BWG in the metalaxyl treated groups were comparable to controls. 

The body weights for the positive control (EE) group were decreased (p≤0.05) by 5% on 

Day 4, and overall BWG for the EE animals was decreased (p≤0.01) by 54% in comparison 

with 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 Metalaxyl 

(100) 

Metalaxyl 

(300/200 b) 

Reference Estrogen 

EE (5 μg/kg/day) 

N Mean SD N Mean SD N Mean SD N Mean SD 

1 6 222.5 5.0 6 219.7 11.3 6 225.4 9.9 6 220.3 9.2 

2 6 228.4 6.6 6 224.0 11.0 6 229.4 10.7 6 226.3 11.4 

3 6 234.7 6.8 6 229.4 12.6 6 234.8 11.0 6 226.5 10.0 

4 

(termination) 

6 243.2 8.4 6 238.8 12.6 6 243.5 9.7 6 229.9*

(↓5) 

10.3 

Body Weight 

Gain (Days 1-11) 

6 20.7 4.5 6 19.2 3.1 6 18.1 2.9 6 9.6** 

(↓54) 

2.7 

a Data were obtained from Table 2 on page 41 of the study report. Percent differences from controls, calculated 

by the reviewers, are included in parentheses. 

b Following the first day of dosing at the high dose (300 mg/kg/day) adverse clinical signs in the animals were 

observed, so the dose volume was reduced from 5 mL/kg to 3.33 mL/kg to achieve a dose of approximately 

200 mg/kg/day (199.8 mg/kg/day) for the next two days of dosing. 

N Number of animals in the group 

SD Standard Deviation 

* Significantly different from controls at p≤0.05 

** Significantly different from controls at p≤0.01 

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C. FOOD CONSUMPTION: Food consumption values are presented in Table 3. Food 

consumption was decreased (p≤0.01) on Days 1-2 in both metalaxyl treated groups (↓16% 

and 23% for the low and high doses, respectively) compared to the controls. Food 

consumption was then increased (p≤0.01) on Days 2-3 in the 300/200 mg/kg/day group 

(↑13%), due to the reduction of the dose from 300 mg/kg/day to 200 mg/kg/day. Overall 

average (Days 1-4) food consumption for both metalaxyl treatment groups was decreased 

(p≤0.05) by 6-8% compared to the vehicle controls. 

Food consumption in the EE positive control group was decreased (p≤0.01) throughout the 

study (↓14-26%), for a decrease (p≤0.01) of 19% in overall average food consumption 

compared to the vehicle control. 

TABLE 3. Food Consumption (g/rat/day) in the Estrogen Agonist Assay a

Study Day 

Dose (mg/kg/day) 

Vehicle Control Metalaxyl 

(100) 

Metalaxyl 

(300/200 b) 

Reference Estrogen 

EE (5 μg/kg/day) 

N Mean SD N Mean SD N Mean SD N Mean SD 

1-2 6 21.2 0.9 6 17.8**

(↓16) 0.9 6 16.3**(↓23) 0.5 6 18.3**(↓14) 1.0 

2-3 6 20.5 1.2 6 19.8 1.1 6 23.2**

(↑13) 0.2 6 16.7**(↓19) 1.0 

3-4 6 20.2 1.8 6 19.2 1.0 6 18.3 1.7 6 15.0**

(↓26) 3.1 

Overall Average 

(Days 1-4) 6 20.6 1.2 6 19.0**

(↓8) 1.0 6 19.3* 

(↓6) 0.5 6 16.7**

(↓19) 1.6 

a Data were obtained from Table 3 on page 43 of the study report. Percent differences from controls, calculated 

by the reviewers, are included in parentheses. 

b Following the first day of dosing at the high dose (300 mg/kg/day) adverse clinical signs in the animals were 

observed, so the dose volume was reduced from 5 mL/kg to 3.33 mL/kg to achieve a dose of approximately 

200 mg/kg/day (199.8 mg/kg/day) for the next two days of dosing. 

N Number of animals in the group 

SD Standard Deviation 

* Significantly different from controls at p≤0.05 

** Significantly different from controls at p≤0.01 

D. PATHOLOGY 

1. Uterine Weights: Uterine and liver weight data are presented in Table 4. Uterine weights 

in the metalaxyl treated groups were comparable to the vehicle controls. Absolute liver 

weights in the 300/200 mg/kg/day group were increased (p≤0.05) by 24% compared to 

vehicle controls. Relative liver weights (not reported in Table 4) were increased (p≤0.05) in 

the 100 mg/kg/day and 300/200 mg/kg/day group, by 13% and 25%, respectively. 

Absolute wet and blotted uterus weights for the positive control (EE) group were increased 

(p≤0.01) by 547% and 207%, respectively, as expected; liver weights were comparable to 

the vehicle controls. 

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All females selected for the assay were noted to be in persistent diestrus. At necropsy it was 

observed that adipose tissue on the right uterine horn had adhered to a portion of the 

duodenum in one animal from the vehicle control group and one animal from the 100 

mg/kg/day group. The mean blotted uterine weight for the vehicle control group was 

0.042% of terminal body weight, which was generally within the performance criteria 

(<0.04%) of the test guideline. 

TABLE 4. Uterine and Liver Weights from Estrogen Agonist Assay in SD Rats a

Parameter 

Dose (mg/kg/day) 

Vehicle Control Metalaxyl 

(100) 

Metalaxyl 

(300/200 b) 

Reference Estrogen 

EE (5 μg/kg/day) 

N Mean SD N Mean SD N Mean SD N Mean SD 

Terminal BW 6 243.2 8.4 6 238.8 12.6 6 243.5 9.7 6 229.9 10.3 

Wet, absolute (g) 6 0.1177 0.0215 6 0.1162 0.0147 6 0.1134 0.0123 6 0.7610**

(↑547) 

0.2686 

Wet, relative (%) 6 0.052 0.008 6 0.048 0.008 6 0.045 0.005 6 0.333** 0.129 

Blotted, absolute (g) 6 0.1061 0.0179 6 0.1052 0.0121 6 0.1033 0.0088 6 0.3264**

(↑207) 

0.0345 

Blotted, relative (%) 6 0.042 0.008 6 0.043 0.005 6 0.042 0.004 6 0.143** 0.021 

Liver, absolute (g) 6 9.9 1.3 6 11.0 1.0 6 12.3**

(↑24) 

0.6 6 -- -- 

a Data were obtained from Table 5 on page 47 of the study report. Percent differences from controls, calculated 

by the reviewers, are included in parentheses. 

b Following the first day of dosing at the high dose (300 mg/kg/day) adverse clinical signs in the animals were 

observed, so the dose volume was reduced from 5 mL/kg to 3.33 mL/kg to achieve a dose of approximately 

200 mg/kg/day (199.8 mg/kg/day) for the next two days of dosing. 

BW Body weight 

N Number of animals in the group 

SD Standard Deviation 

* Significantly different from controls at p≤0.05 

** Significantly different from controls at p≤0.01 

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

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Increases in the mean absolute and relative 

weights of the liver were observed in the 100 and 300/200 mg/kg/day dose groups; these 

increases were consistent with the results of previous dose range-finding studies. 

There were no significant increases in either the absolute or blotted weights of the uteri in 

either the 100 or the 300/200 mg/kg/day dose group, in comparison with the respective 

vehicle control group values. Therefore, the results of this study indicate that there was no 

positive response for estrogenic activity as a result of administration of metalaxyl at dose 

levels as high as 300/200 mg/kg/day. 

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B. AGENCY COMMENTS: All animals survived until scheduled termination. Following 

administration of metalaxyl on Day 1 at 300 mg/kg, adverse clinical signs were observed, 

including decreased motor activity, ptosis, ataxia, lacrimation, hunched posture, intermittent 

body jerks, chromodacryorrhea, and slight excess salivation, as well as a reduction in food 

consumption. Therefore, the 300 mg/kg/day dose level was reduced to 200 mg/kg/day on 

Days 2 and 3. Following the dose reduction, food consumption in this group increased, and 

no adverse clinical signs were observed. 

Body weights throughout the dosing period and overall (Days 1-4) body weight gain in the 

metalaxyl treated groups were comparable to controls. The body weights for the positive 

control (EE) group were decreased (p≤0.05) by 5% on Day 4, and overall BWG for the EE 

animals was decreased (p≤0.01) by 54% in comparison with the vehicle controls. 

Overall (Days 1-4) food consumption for both metalaxyl treatment groups was decreased 

(p≤0.05) by 6-8% compared to the vehicle controls. Food consumption in the EE positive 

control group was decreased (p≤0.01) throughout the study, for an overall decrease (p≤0.01) 

of 19% in average food consumption compared to the vehicle control. 

All females selected for the assay were noted to be in persistent diestrus. Uterine weights in 

the metalaxyl treated groups were comparable to the vehicle controls. Absolute wet and 

blotted uterus weights for the positive control (EE) group were increased (p≤0.01) by 547% 

and 207%, respectively, as expected. The mean blotted uterine weight for the vehicle 

control group was 0.042% of terminal body weight, which was generally within the 

performance criteria (<0.04%) of the test guideline. 

Absolute and relative liver weights in the 300/200 mg/kg/day group were increased (p≤0.05) 

compared to vehicle controls (↑24-25%); relative liver weights were increased (p≤0.05) in 

the 100 mg/kg/day group (↑13%). Liver weights in the EE group were comparable to the 

vehicle controls. 

The high dose used in the study was adequate based on the adverse clinical signs observed 

following a single dose of metalaxyl at 300 mg/kg, and the increases in liver weights in the 

300/200 mg/kg/day group. 

No statistically significant changes were seen in uterine weight in this assay. Metalaxyl is 

negative in the uterotrophic assay. 

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

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

 The EE dose of 5 μg/kg/day via s.c. injection was 17 times higher than the 0.3 ug/kg/day 

s.c. dose recommended by Guidance (OCSPP 890.1600) 

 No rational was given for use of a different vehicle for EE (corn oil) than the test 

chemical and vehicle control (2.0% aqueous carboxymethylcellulose). OCSPP 890.1600 

recommends test substances be dissolved in a minimal amount of 90% ethanol or other 

suitable solvent before dissolving in vehicle. 

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