UNITED ST ATES ENVIRONMENT AL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

OFFICE OF CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

MEMORANDUM 

Date: 6/30/2015 

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

PC Code: 027401 

Decision No.: 459923 

Petition No.: NA 

Risk Assessment Type: NA 

TXR No.: 0051691 

MRID No.: See Table 

DP Barcode: D397912 

Registration No.: NA 

Regulatory Action: NA 

Case No.: NA 

CAS No.: 1194-65-6 

40CFR: NA 

FROM: Greg Akerman, Ph.D. ~ ~~--- Immediate Office 

Health Effects Division (7509P) 

THROUGH: Jess Rowland d=-~ C2-=~ 

Deputy Director 

Health Effects Division 

TO: Jolene Trujillo 

Biologist/Chemical Review Manager 

Risk Management and Implementation Branch V 

Pesticide Re-evaluation Division (7505P) 

I. ACTION REQUESTED 

Ver.Apr. 2010 

The Pesticide Re-evaluation Division (PRD) of OPP has requested that the Health Effects 

Division (HED) review the Endocrine Disruptor Screening Program (EDSP) Tier 1 assays 

submitted in response to the agency's Test Order for dichlobenil: Test Order# EDSP-027401-

120. 

Page I of2 

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II. RESPONSE 

Attached are the EDSP Tier 1 assay DERs for dichlobenil.

III. MRID Table 

Chemical: Dichlobenil PC Code: 027401 

Guideline Assay MRID 

890.1100 Amphibian Metamorphosis Assay (Frog) 48616101 

890.1150 Androgen Receptor Binding (Rat Prostate) 48616102 

890.1200 Aromatase Assay (Human Recombinant) 48616103 

890.1250 Estrogen Receptor Binding 48616104 

890.1300 Estrogen Receptor Transcriptional Activation 

(Human Cell Line HeLa-9903) 48616105 

890.1350 Fish Short-Term Reproduction 48616106 

890.1400 Hershberger (Rat) 48616107 

890.1450 Female Pubertal (Rat) 48616108 

890.1500 Male Pubertal (Rat) 48616109 

890.1550 Steroidogenesis (Human Cell Line – H295R) 48616110 

890.1600 Uterotrophic (Rat) 48616111 

Range finding study 48616112 

Range finding study 48616113 

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

EPA MRID Number 48616101

Data Requirement: EPA DP Barcode 397913 

OECD Data Point 231 

EPA MRID 48616101 

EPA Guideline 890.1100 

 Amphibian Metamorphosis Assay (Frog) 

Test Material: Dichlobenil Purity (%): 99.3% 

Common Name: Dichlobenil 

Chemical Name: IUPAC: 2,6-Dichlorobenzonitrile 

 CAS Name: 2,6-Dichlorobenzonitrile 

CAS No. 1194-65-6 

Synonyms 027401

Primary Reviewer: Kindra Bozicevich

Staff Scientist, Cambridge Environmental, Inc. Date: 6/15/2012

Secondary Reviewer(s): John Marton

Staff Scientist, Cambridge Environmental, Inc. Date: 7/9/2012

Primary Reviewer (and final additional reviewer): Elyssa Arnold 

Biologist, EPA/OPP/EFED/ERB2 Date: 4/28/2015 

Additional Reviewer(s): Catherine Aubee 

Biologist, EPA/OPP/EFED/ERB4 Date: 4/28/2015 

Date Evaluation Completed: 28-04-2015 

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

EPA MRID Number 48616101

CITATION: Tobor-Kaplon, M.A. 2011. Amphibian Metamorphosis (Frog) Test With Dichlobenil Technical. 

Unpublished study performed by Notox B.V., Hambakenwetering 7, 5231 DD’s-Hertogenbosch, The 

Netherlands. Notox Project 495785. Notox Substance 202841/A. Study sponsored by Chemtura 

Corporation, 199 Benson Road, Middlebury, CT 06749. Study completed October 20, 2011. 

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

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

interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery 

is based on the strengths of each individual assay to identify potential endocrine bioactivity with 

complementary endpoints within the assay, where available, and redundancy across the battery. Thus, the 

results of each individual assay should not be considered in isolation but rather should be considered in 

the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In 

order to determine if a chemical has the potential to interact with the E, A or T pathways, a Weight of 

Evidence (WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be 

undertaken (refer to the WoE Document). 

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

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

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

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

EPA MRID Number 48616101

EXECUTIVE SUMMARY

The 21-day assay of Dichlobenil (99.3% purity) on amphibian metamorphosis of African clawed frog (Xenopus 

laevis) was conducted under flow-through conditions. Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 

(80 per control and treatment group) were exposed to a negative control and a solvent control (0.1 mL/L; 

acetone) and nominal dichlobenil concentrations of 0.02, 0.2, and 2.0 mg a.i./L. Mean-measured 

concentrations were <0.001 (<LOQ; negative and solvent), 0.019, 0.19, and 1.9 mg a.i./L. The test system 

was maintained at 21.0 to 22.5°C and a pH of 7.3 to 8.2. 

No mortalities were observed in the solvent control, or low or mid treatment groups. One mortality each was 

recorded in the negative control and high treatment group. Clinical signs (i.e., behavioral and other sublethal 

effects) including curved spine were observed in tadpoles in the solvent control and 0.019 and 0.19 mg a.i./L 

treatment groups. This effect did not appear to be related to the test substance. 

No significant acceleration or delay of body weight, median NF developmental stage, snout-to-vent length 

(SVL), hind-limb length (HLL), or normalized (for SVL) HLL was observed at Day 7 compared to the negative 

control. Further, no asynchronous development was observed. 

At Day 21, >20% of amphibians were at NF stage >60. When excluding organisms with NF stages greater 

than 60, dichlobenil significantly increased Day 21 normalized HLL at all test concentrations (7-10% greater 

than negative control for non-late stage amphibians, p<0.05). No significant treatment effects were detected 

for Day 21 median developmental stage, body weight, or SVL for non-late stage amphibians compared to the 

negative control. Additionally, no significant effects on development or growth were observed when comparing 

treatment and negative control late-stage amphibians (NF>60). Normalized HLL at Day 21 was also 

significantly greater (10%, p<0.05) in the solvent control than in the negative control, as was NF development 

stage. 

There was a lower incidence and severity of follicular cell hypertrophy in the high treatment group compared 

to the negative or solvent control. No treatment-related effects were observed for thyroid gland hypertrophy or 

atrophy, or follicular cell hyperplasia, and there were no differences in the amount or quality of follicular colloids. 

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

EPA MRID Number 48616101

All test validity and performance criteria were met in this 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: NF 51 

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

Test type: flow-through 

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

Metamorphosis Assay (AMA) with Dichlobenil. 

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 

0.019 (0.020) No No No Yes NA NA No 

0.19 (0.20) No No No Yes NA NA No 

1.9 (2.0) No No No Yes NA NA Yes 

Abbreviations: Diff. Difference. NA Not applicable.

1

 A “yes” indicates a significant difference based on comparison to the negative control. 

2

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

the DER. 

3

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

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I. MATERIALS AND METHODS 

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

Environmental Protection Agency’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 from U.S. EPA, EDSP 890.1100 and 

OECD 231 were noted: 

1. Parental frogs were not maintained as in-house stock. 

2. The flow-through rate was ca. 33 mL/min. EPA recommends a flow-through rate of 25 

mL/min. 

3. The hardness of the water used in the study ranged from 161-179 mg CaCO3/L. EPA 

recommends hardness ranging from 40 to 48 mg/L as CaCO3. 

4. Solvent concentration (0.1 mL/L) exceeded the EPA recommended limit (0.02 mL/L), though 

it was within acceptable limits specified in OECD guidance (0.1 mL/L). 

5. Development stage and normalized HLL were significantly increased in the solvent control 

compared to the negative controls at Day 21. 

These deviations do not impact the results or 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 (OECD, 1998) Good Laboratory Practice regulations with 

the following exceptions: analyses of the concentration of iodide under test 

conditions was performed by an outside laboratory. 

A. Test Material Dichlobenil Technical 

Description: White powder with lumps

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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. : AM9K12A222 

Purity: 99.3% a.i. 

Impurities: None reported 

Stability of Compound: Mean measured concentrations yielded overall recoveries of 84-107% of 

nominal with a coefficient of variation of d10%.

Storage Conditions of 

Test Chemicals: 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): Daudin 

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

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Were parents maintained as inhouse stock? No EPA recommends that larvae used in the assay be derived from in-house adults.Were parental acclimation conditions same as definitive test? 

Not applicable

Acclimation period for parental frogs (if applicable): 

Days 1 to 13 postfertilization

Details on parental feeding: During acclimation, 

juveniles were fed Sera Micron® twice daily 

Details on parental health: Not reported 

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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: 

Minimum 1500 

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 measured 

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

Not measured 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Was the optional size selection method used? 

No. Selection was based on viability and hatching success. 

Details on larval selection: Selection was based 

on viability and hatching success. 

Loading rate (rearing density): 2 tadpoles/L Twenty tadpoles per test vessel (10 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.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Type of food: Sera Micron® 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 

provide analysis of iodide content and 

potential contaminants, and the diet should 

demonstrate equal performance to Sera 

Micron® .

Source of food: Sera GmbH, 

Heinsburg, Germany

Iodide concentration in diet (if known): 

Not reported 40-50 mg/kg, based on personal 

communication between the lab and Dow Chemicals 

Frequency of feeding: Twice per day EPA recommends that feeding occur at 

least twice per day.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on feeding regime: During the exposure 

period, tadpoles received between 30 and 80 mg/animal/day. 

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).

C. Exposure System

Table 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.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

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

Continuous-flow Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

Flow-through rate (if applicable): 

ca. 33 mL/min Recommended flow-through rate is 25 

mL/min (complete volume replacement 

ca. every 2.7 hrs).

2 Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for 

Conducting Aquatic Laboratory Studies. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on toxicant mixing for flow-through systems (if 

applicable): 

Stock solutions were dosed via a computercontrolled system into mixing chambers then 

pumped into the test chambers using peristaltic pumps. 

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.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Aeration? No EPA recommends maintaining dissolved 

oxygen concentrations >40% air 

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

recommended to set bubblers at levels 

that do not cause stress on the tadpoles.

Source of dilution water: Adjusted ISO medium, 

formulated using ROwater (tap-water purified by reverse osmosis). 

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.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

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

Yes 

Iodide supplementation in water? 

Yes. KI added during 

formulation for a resulting concentration of 1.96 Pg KI/L (1.5 mg 

I-/L). 

If reconstituted water is used or if 

background levels of iodide in natural 

water are less than 0.5 μg/L, iodide 

supplementation is recommended. This 

supplementation is in addition to the 

recommended dietary source of iodide 

(e.g.. in Sera Micron).

Test vessel type/materials: Tanks consisting of glass 

plates sealed with a thin film of silicone and covered by a removable Perspex plate 

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: 42x13.5x19.5 cmFill volume: ca. 10 L

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Additional details on exposure system: 

None 

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

Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Hardness (mg/L as CaCO3) 161 179 166 Weekly 

EPA recommends hardness 40 to 48 

mg/L as CaCO3.

pH 7.3 8.2 7.7 Weekly 

EPA recommends pH 7.5 ± 1, interreplicate and inter-treatment differentials should not exceed 0.5.

Dissolved oxygen (mg/L) 5.9 8.8 8.3 Weekly 

EPA recommends dissolved oxygen 

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

OECD recommends DO concentration 

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

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Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Temperature (°C) 21.5 22.5 22.1 Weekly 

EPA recommends temperature 22±1oC; 

inter-replicate and inter-treatment 

differentials should not exceed 0.5oC.

Iodide (μg/L) 3.6 3.6 NA Once, Preliminary 

EPA recommends aquatic iodide range 

0.5 – 10 μg/L (supplemental iodide 

should not exceed 2 μg/L).

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Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Ammonia NA NA NA NA General recommendations for frequency 

of measurements: EPA recommends that 

water quality parameters be measured in 

a control and at one test item 

concentration at least weekly. In static 

renewal systems, water quality 

parameters, including ammonia, should 

be measured just prior to renewal. In 

addition, EPA recommends that DO be 

measured at each concentration at least 

weekly and that temperature be 

measured continuously. OECD 

recommends that DO and temperature 

be measured at least weekly and that pH 

and hardness be measured at least at 

the beginning and end of the test.

Fluoride NA NA NA NA Perchlorate NA NA NA NA Chlorate NA NA NA NA Total Alkalinity (mmol HCO3-/L) 0.8 1.2 1.0 Weekly Specific Conductance (μmhos/cm) Not reported Not reported Not reported Not reported 

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 D. Study Design and Additional Experimental Conditions

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

NA 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: NALife stage: NA Test duration: NAAdditional details: NA 

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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:

Not reported EPA recommends that the highest test 

concentration is either the solubility limit of 

the test compound, 100 mg/L, or 

demonstrates adequate evidence of toxicity 

(e.g., <10% mortality), whichever 

concentration is lowest.

Reference study citation (if applicable):

NA 

Separation of test concentrations:

Dilution factor of 10 EPA recommends that the maximum 

concentration separation be 0.1 and the 

minimum be 0.33. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Number of test concentrations: 3 plus negative and 

solvent controls

EPA recommends a minimum of 3 

concentrations and a control, plus solvent 

control if appropriate.

Are nominal concentrations adjusted for purity? 

No 

Indicate the type of values presented for measured concentrations: 

Mean-measured 

Limit of quantification (LOQ): 0.001 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: Measurements were 

made on samples collected on Days -2, 0, 7, 14, and 21. 

It is recommended that test item 

concentration be measured in one tank at 

each treatment level at test initiation and 

every week thereafter.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

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

4 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.

Number of larvae per treatment at test initiation: 

80 (20/rep) 

Was a solvent used? Yes Solvent type (if applicable): Acetone 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Maximum solvent concentration (if applicable): 

0.1 ml/L target concentration 

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: 12 hrs light : 

12 hrs dark 

EPA recommends photoperiod 12:12 

(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: 

600-1200 lux EPA recommends light intensity 0.6 – 2 

Klux (at water’s surface).

Additional details: No anesthesia was 

used during the staging of tadpoles at test initiation. 

Report indicated that no undissolved material was present in mixing chambers. 

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

Treatment ID 

Nominal 

Concentration 

(mg a.i./L) 

Measured 

Concentration 

(mg a.i./L) 

Mean CV (%) Details or Remarks Guideline Recommendations 

Control (dilution water only) 

0.0 <0.001 NA 

EPA and OECD recommend that test item 

concentrations be maintained at a coefficient 

of variation (CV) 20%. Solvent control (if 

applicable) 

0.0 <0.001 NA 

Treatment 1 0.02 0.019 10.0 Treatment 2 0.2 0.19 5.2 Treatment 3 2.0 1.9 4.2 

Abbreviations: CV Coefficient of variation. 

 E. Observations

Biological Endpoints: snout-vent length (SVL), hind limb length (HLL), normalized HLL, body weight, and 

developmental stage (Days 7 and 21), mortality and sub-lethal effects (daily), thyroid histology (Days 7 and 21) 

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Were raw (individual) data provided? Yes

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

Throughout the 21-day exposure period, no mortalities were observed in the solvent control, 0.019 or 0.19 

mg a.i./L treatment groups, and 1 mortality each was recorded in the negative control and 1.9 mg a.i./L 

treatment group. 

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 0 0 60 1 1.7 

Solvent Control 80 0 0 60 0 0 

0.019 80 0 0 60 0 0 

0.19 80 0 0 60 0 0 

1.9 80 0 0 60 1 1.7 

Abbreviations: NA Not applicable. 

1

 Sample size and cumulative mortality values at Day 7 prior to interim sacrifice (total across all four 

replicates). 

At Day 7, median NF development stage was 55 in the negative and solvent controls and for all 

treatment groups and no tadpoles exhibited asynchronous development. By Day 21, median NF 

development stage ranged from 60-61 in the negative and solvent controls and all treatment groups 

and again no tadpoles exhibited asynchronous development. Late stage (NF>60) tadpoles were 

present in all replicates of all treatment groups at Day 21, totaling 36, 58, 47, 45, and 42% of all 

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surviving tadpoles in the negative control, solvent control, and 0.019, 0.19, and 1.9 mg a.i./L test 

groups, respectively. 

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 55 0 4 60 0 

Solvent Control 4 55 0 4 61 0 

0.019 4 55 0 4 60.5 0 

0.19 4 55 0 4 60 0 

1.9 4 55 0 4 60 0 

Abbreviations: NA Not applicable. 

Normalized HLL on Day 7 was comparable among groups and averaged 0.19 mm in the negative 

and solvent controls and ranged from 0.19-0.20 mm in all treatment groups. On Day 21, normalized 

HLL averaged 1.15 and 1.25 mm in the negative and solvent controls, respectively, and ranged from 

1.24-1.26 mm in all treatment groups. 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Hind Limb Length (HLL) 

Day 7 Day 211 

n Mean

(mm) 

±SD 

HLL: 

SVL2 n Mean(mm) 

±SD 

HLL: 

SVL2

Negative Control 4 3.05 0.04 0.19 4 23.1 0.81 1.00 

Solvent Control 4 2.92 0.14 0.19 4 25.2 0.96 1.10 

0.019 4 3.05 0.23 0.19 4 24.7 0.96 1.07 

0.19 4 3.13 0.06 0.20 4 25.3 0.74 1.09 

1.9 4 3.14 0.09 0.20 4 25.1 0.67 1.11 

Abbreviations: NA Not applicable. SD Standard deviation. 

1

 The Day 21 results include only non-late stage organisms (NF stage 60). Summary tables for late 

stage amphibians are provided in Reviewer Analysis section. 

2

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

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Table 12: Larval Growth in Xenopus laevis.

Treatment 

(mg a.i./L) 

[meanmeasured]

Snout-Vent Length (SVL) Body Weight1

Day 7 Day 212 Day 7 Day 212 

n 

Mean

(mm) 

±SD n 

Mean

(mm) 

±SD n 

Mean

(g) 

±SD n 

Mean

(g) 

±SD 

Negative 

Control 

4 15.76 0.82 4 23.1 0.41 4 0.447 0.05 4 1.71 0.05 

Solvent 

Control 

4 15.82 0.91 4 23.0 0.92 4 0.445 0.05 4 1.76 0.21 

0.019 4 15.92 0.95 4 23.2 0.73 4 0.452 0.06 4 1.74 0.14 

0.19 4 16.00 0.46 4 23.3 0.43 4 0.478 0.05 4 1.79 0.12 

1.9 4 16.07 0.22 4 22.8 0.39 4 0.491 0.02 4 1.67 0.07 

Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation. 1 Also referred to as “wet weight” in the test guideline. 2 The Day 21 results include only non-late stage organisms (NF stage 60). Summary tables for late stage amphibians are provided in Reviewer 

Analysis section. 

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The only histopathology finding was a decrease in the incidence of follicular cell hypertrophy 

characterized and graded by the subjective % of tall columnar epithelium relative to cuboidal 

epithelium. There was a lower incidence and severity of follicular cell hypertrophy in the 2.0 mg 

a.i./L treatment group. As thyroid follicular cells normally increase in height during tadpole 

development, this finding suggests some retardation of metamorphosis at 2.0 mg a.i./L. 

No treatment-related effects were observed for thyroid gland hypertrophy or atrophy, follicular cell 

hyperplasia and there were no differences in the amount or quality of follicular colloids. 

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 3 20 20 

1 20 0 20 0 20 10 20 0 

2 20 0 20 0 20 6 20 0 

3 20 0 20 0 20 1 20 0 

Solvent Control 0 20 20 20 20 20 1 20 20 

1 20 0 20 0 20 12 20 0 

2 20 0 20 0 20 7 20 0 

3 20 0 20 0 20 0 20 0 

0.019 0 20 20 20 20 20 5 20 20 

1 20 0 20 0 20 9 20 0 

2 20 0 20 0 20 6 20 0 

3 20 0 20 0 20 0 20 0 

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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 

0.19 0 20 20 20 20 20 2 20 20 

1 20 0 20 0 20 10 20 0 

2 20 0 20 0 20 7 20 0 

3 20 0 20 0 20 1 20 0 

1.9 0 20 20 20 20 20 13 20 20 

1 20 0 20 0 20 6 20 0 

2 20 0 20 0 20 1 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. 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Additional Qualitative Observations1

Severity

Follicular Lumen 

Area (Increase) 

Follicular Lumen 

Area (Decrease) 

Follicular Cell 

Height (Increase) 

Follicular Cell 

Height (Decrease) 

Follicular Cell 

Shape 

n Incidence n Incidence n Incidence n Incidence n Incidence 

Negative Control 0 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 

Solvent 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 

0.019 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 

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Treatment 

(mg a.i./L) 

[mean-measured]

Additional Qualitative Observations1

Severity

Follicular Lumen 

Area (Increase) 

Follicular Lumen 

Area (Decrease) 

Follicular Cell 

Height (Increase) 

Follicular Cell 

Height (Decrease) 

Follicular Cell 

Shape 

n Incidence n Incidence n Incidence n Incidence n Incidence 

0.19 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 

1.9 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 

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. 

A single tadpole in the negative control showed loss of equilibrium and was swimming at the surface from Day 16 until the end of exposure. No signs of severe distress were observed. On Day 7 of exposure, tadpoles exposed to 

dichlobenil were easier to catch than control tadpoles. This difference was not strong enough to consider it an effect and was not observed at the end of the test. 

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Eight percent of the tadpoles sacrificed at Day 7 of exposure had a bent tail. At Day 21, a total of 22% of 

organisms had a bent tail, with 22, 28, 23, 23, and 13% exhibiting bent tail in the negative control, solvent control, 

0.019, 0.19 and 1.9 mg a.i./L, respectively. There was no relation between the number of incidences of tadpoles with bent tail and the treatment. 

Table 15: Clinical Signs in Xenopus laevis.

Treatment 

(mg a.i./L) 

[mean-measured] 

Clinical Signs 

Type n Incidence 

Negative Control Bent tail 59 13 

Loss of equilibrium and surfacing 59 1 

Solvent Control Bent tail 60 17 

0.019 Bent tail 60 14 

0.19 Bent tail 60 14 

1.9 Bent tail 60 8 

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B. Study Author’s Analysis and Conclusions

Day 7 body weight and SVL and Day 7 and 21 normalized HLL were evaluated for normality using 

Shapiro-Wilk’s test and for homogeneity of variance using Levene’s test. One-way ANOVA with 

Dunnett’s test was used to analyze differences. Day 7 and Day 21 NF developmental stages were 

analyzed using Jonckheere-Terpstra test. Day 21 snout-to-vent length and body weight were evaluated 

for normality using Shapiro-Wilk’s test per LateStage group. Nested ANOVA with Treatment and 

LateStage and interaction Treatment (LateStage) was used to analyze differences. Subsequently the 

multiple Tukey-Kramer comparison was performed. All treatment data was compared to solvent control 

data. 

 

There was no significant treatment-related mortality. Statistically significant treatment-related effects 

were observed on Day 21 in the mean-measured 0.19 and 1.9 mg a.i./L treatment groups. These 

effects were significant decreases in development stage of Xenopus laevis tadpoles compared to the 

solvent control. 

A significant difference between blank and solvent control, in respect to development stage and 

normalized HLL, was found at Day 21. In both cases, results suggested acceleration of development 

in the acetone control. This was not confirmed by histopathology where no significant differences were 

found between controls. The study author attributes the observed difference to the intensive growth 

of microbial biomass in the solvent control compared to the blank control rather than to the presence 

of the solvent. This biomass is a source of additional food that could cause faster development. 

 

 C. Reviewer’s Analysis and Conclusions 

 

Statistical Methods: 

Data for Day 7 SVL, wet body weight, and normalized HLL exhibited monotonic responses and were 

analyzed using the Jonckheere-Terpstra test. Data for Day 7 normalized HLL exhibited non-monotonic 

responses and met assumptions of normality and variance so were analyzed using Dunnett’s test. 

Data for Day 7 median values for developmental stage exhibited non-monotonic responses and were 

analyzed using the Mann-Whitney test. The reviewer compared the negative and solvent control for 

all endpoints at Day 7 using two-sided Student’s t-tests. 

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At Day 21, >20% of the frogs across all treatment and control groups were at NF stage >60. After 

stage 60, tadpoles show a reduction in size and weight due to tissue resorption and reduction of 

absolute water content. To account for this difference, a two-factor nested ANOVA with concentration, 

late stage (yes or no), and their interaction was used to analyze Day 21 data, with Rep(Conc) and 

tadpole ID(rep) as random factors, as recommended by the test guideline. All data met the normality 

and variance homogeneity requirements of ANOVA. Additionally, the interaction F-test was “sliced” into 

two additional ANOVA F-tests, one on the mean responses across concentrations for LateStage=’No’ 

and another on the mean responses across concentrations for LateStage=’Yes.’ However, this 

statistical approach was found to be insufficient. The variance component “replicate (treatment)” was 

often inestimable due to a greater between-individual variance than between-replicate variance. 

Therefore, further comparisons of treatment means against negative controls were done within each 

level of LateStage using identical methods to the Day 7 analysis. Normalized HLL for non-late stage 

tadpoles exhibited a monotonic trend and was analyzed using the Jonckheere-Terpstra test. 

Developmental stage was analyzed using the Mann-Whitney test. The remaining endpoints were 

analyzed using Dunnett’s test. 

Negative and solvent control groups at 21 Days were compared using a nested two-factor ANOVA. 

Normalized HLL was significantly greater in the solvent control than in the negative control (10%, 

p<0.05). Using a Student’s t-test on the median NF development stage, the reviewer detected a 

significant difference in the solvent control (median 61) relative to the negative control (median 60) 

(p<0.05). No other variables differed between the two control groups. 

Due to the differences detected between the solvent and negative control groups for normalized HLL 

and NF stage at Day 21, statistical comparisons of the treatment groups to the solvent control were 

conducted for these endpoints. 

No asynchronous development was observed. 

Results for late stage tadpoles are presented in Table 16. Tables in the Results section of this DER 

for SVL, body weight, HLL, and normalized HLL present results for non-late stage tadpoles only. 

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Table 16: Day 21 Larval Growth in Late-Stage Xenopus laevis.

Treatment 

(mg a.i./L) 

[meanmeasured]

Day 21 Larval Growth for Late-stage Amphibians

Hind Limb Length 

(mm) 

Snout-Vent Length 

(SVL)

Body Weight1 Normalized HLL 

n Mean

(mm) 

±SD n Mean

(mm) 

±SD n Mean

(g) 

±SD n Mean

(mm) 

±SD 

Negative 

Control 

4 26.8 0.52 4 19.1 0.50 4 1.21 0.10 4 1.41 0.05 

Solvent 

Control 

4 27.8 0.22 4 19.7 0.33 4 1.29 0.06 4 1.42 0.01 

0.019 4 27.9 0.43 4 19.3 0.45 4 1.21 0.07 4 1.46 0.03 

0.19 4 28.3 0.74 4 19.5 0.47 4 1.26 0.06 4 1.45 0.05 

1.9 4 28.2 0.96 4 19.5 0.54 4 1.23 0.07 4 1.45 0.04 

 Conclusions: 

There was no significant effect on mortality in the study. 

At Day 7, there were no statistically significant effects for any parameter tested, compared to the 

negative control data. There were also no statistically significant differences between the negative and 

solvent control groups for any endpoint. 

At Day 21, significant increases in both HLL and normalized HLL from the negative control were 

detected across all treatment groups using the Jonckheere-Terpstra test (p<0.05) for non-late stage 

tadpoles. For late stage tadpoles, significant increases were also seen at all treatment groups for HLL 

(Jonckheere-Terpstra, p<0.05), but not for normalized HLL. No significant treatment effects were 

detected for Day 21 median developmental stage, wet body weight, or SVL for either late stage or 

non-late stage tadpoles when compared to the negative control. 

There was a significant increase in normalized HLL in the solvent control compared to the negative 

control at Day 21. When compared to the solvent control, no difference was seen in normalized HLL 

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for non-late stage tadpoles in any treatment group. For late stage tadpoles, a significant increase was 

seen in the lowest treatment group compared to the solvent control (Jonckheere-Terpstra, p<0.05). 

For the mid and high treatment groups, notable but non-significant increases were also observed 

(Jonckheere-Terpstra, p=0.06). 

At Day 21, NF stage was greater in the solvent control (median 61) than negative control (median 

60). NF stage was significantly decreased at Day 21 in all treatment groups compared to the solvent 

control (Jonckheere-Terpstra, p<0.05). 

There was a lower incidence and severity of follicular cell hypertrophy in the 1.9 mg a.i./L treatment 

group. No treatment-related effects were observed for thyroid gland hypertrophy or atrophy, follicular 

cell hyperplasia and there were no differences in the amount or quality of follicular colloids. 

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Table 17: Developmental and Thyroid Gross Pathology/Histopathology Endpoints1,2 in the AMA with Dichlobenil. 

Treatment 

(mg a.i./L) 

[meanmeasured]

NF Developmental Stage Hind Limb Length3 Asynchronous Development Thyroid Gross and 

Histopathology 

Day 7 Day 214 Day 7 

Day 21 

Day 7 Day 21 Day 21 

Non-Late Stage5 Late-Stage6

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

% 

Diff. 

p 

TreatmentRelated Effects? 

(Yes/No) 

Negative Control 55 NA 60 NA 0 NA 0 NA 0 NA NA NA NA NA No 

Solvent Control 55 NA 61 0.002 0 NA 9.62 0.030 0.46 0.826 NA NA NA NA No 

0.019 55 0.478 60.5 0.223 -2.56 0.837 6.98 0.029 3.01 0.441 NA NA NA NA No 

0.19 55 1.0 60 0.478 0 1.0 8.98 0.005 2.83 0.486 NA NA NA NA No 

1.9 55 1.0 60 0.478 0 1.0 10.22 0.002 2.83 0.486 NA NA NA NA Yes 

Statistical Test Mann-Whitney Mann-Whitney Dunnett 

JonckheereTerpstra Dunnett NA NA NA 

Abbreviations: Diff. Difference. NA Not applicable.

1 Unless otherwise indicated, effects are reported based on comparison to the solvent 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. 

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3 Hind-limb length is normalized to snout-vent length (SVL). 4 Compared to the solvent control, using Jonckheere-Terpstra test for a decreasing trend: Percent differences: -1.22, -1.63, and -1.63; P-values: 0.048, 

0.014, 0.010; for the low, medium, and high treatment groups, respectively. 

5 Compared to the solvent control, using Dunnett’s test: Percent differences -2.72, -0.91, and 0.23; P-values: 0.660, 0.977, 1.00; for the low, medium, 

and high treatment groups, respectively. 

6 Compared to the solvent control, using Jonckheere-Terpstra test for an increasing trend: Percent differences: 2.65, 2.47, 2.47; P-values: 0.029, 0.061, 

0.060; for the low, medium, and high treatment groups, respectively. 

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Table 18: Growth Endpoints1,2 in the AMA with Dichlobenil. 

Treatment 

(mg a.i./L) 

[mean-measured]

Snout-Vent Length Body Weight 

Day 7 

Day 21 

Day 7 

Day 21 

Non-Late Stage Late-Stage Non-Late Stage Late-Stage 

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

Negative Control 0 NA 0 NA 0 NA 0 NA 0 NA 0 NA 

Solvent Control 0.38 0.463 -0.50 0.826 3.35 0.075 0.45 0.468 2.95 0.653 6.67 0.213 

0.019 1.05 0.386 0.28 0.996 1.13 0.869 1.03 0.500 1.90 0.941 0.21 1.00 

0.19 1.52 0.232 1.12 0.818 2.43 0.432 6.85 0.232 4.68 0.562 4.15 0.684 

1.9 1.98 0.152 -1.27 0.759 2.15 0.522 9.71 0.056 -2.63 0.864 2.07 0.937 

Statistical Test 

JonckheereTerpstra Dunnett Dunnett Jonckheere- Terpstra Dunnett Dunnett 

Abbreviations: Diff. Difference. NA Not applicable.

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

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

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E. Study Deficiencies 

The main study deficiencies were statistically significant differences in normalized HLL and NF 

development stage at Day 21 between the solvent and negative controls. Other deviations are noted 

in Section I. Materials and Methods were considered to be minor. Validity and performance criteria 

were met, including the following: 

x Mortality did not exceed 10% for any given treatment;

x Three treatment levels, with all four uncompromised replicates, were available for analysis; 

and

x Three treatment levels without overt toxicity were available for analysis.

F. Reviewer’s Comments 

The reviewer’s conclusions differed from those reported by the study author. The reviewer’s analysis 

detected significant (p<0.05) increases in Day 21 normalized HLL at all treatment levels for non-late 

stage tadpoles compared to the negative control. The study author did not detect these significant 

differences. The study author reported a statistically significant treatment-related delay (p<0.05) in 

median developmental stage on Day 21 in the mean-measured 0.19 and 1.9 mg a.i./L treatment 

groups, relative to the solvent control. The reviewer detected this trend in the 0.019 mg a.i./L treatment 

group in addition to the 0.19 and 1.9 mg a.i./L treatment groups.. The study author and reviewer both 

detected a difference in normalized HLL between the negative and solvent controls (p<0.05). Thus, 

the reviewer believes that the increase in normalized HLL seen in the treatment groups compared to 

the negative control may be the result of a solvent effect. 

The study author attributed increased stage and normalized HLL at Day 21 in the solvent control 

compared to the negative control to the growth of microbial biomass in the solvent control tanks. The 

growth of microbial biomass is considered an effect of the solvent in the study that could impact the 

study endpoints and therefore needs to be evaluated across all treatment groups. Without this additional 

information, it is difficult to know whether effects measured in the study groups exposed to the test 

substance were either promoted or masked by the presence of the solvent. 

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For all endpoints exhibiting monotonic trends and satisfying parametric assumptions, the OCSPP 

890.1100 flowchart recommends interpretation of results using the Jonckheere-Terpstra test. 

However, according to historical EFED statistical guidance, the parametric William’s test should be 

used to interpret statistical findings under these circumstances. For endpoints satisfying these 

conditions in this study, there was no difference in conclusions between the OCSPP 890.1100 

flowchart and historical EFED methodologies. 

III. REFERENCES 

Nieuwkoop, P.D., Faber, J. 1994. Normal Table of Xenopus laevis (Daudin). Garland Publishing. New 

York. 

OECD. 2007. Final report of the Validation of the Amphibian Metamorphosis Assay: Phase 2- Multichemical Interlaboratory Study. Environmental Health and Safety Publications. Series on testing and 

Assessment. No. 76. Paris, France. 

Cooke, A.S. 1977.. Effects of field application of the herbicides Diquat and Dichlobenil on amphibians. 

Environmental Pollution (12). 

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APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION: 

Endpoint Monotonic? Parametric? 890.1100 EFED Comments7-day wet weight Yes Yes Jonckheere:

n.s. p>0.05

William’s:n.s. p>0.05

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

7-stage median No No Mann-Whitney:

n.s. p>0.05

Mann-Whitney:n.s. p>0.05

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

7-day SN-vent length Yes Yes Jonckheere:

n.s. p>0.05

William’s:n.s. p>0.05

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

7-day hind-limb length

Yes No Jonckheere:

n.s. p>0.05

Jonckheere:n.s. p>0.05

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

7-day normalized HLL

No Yes Dunnett’s:

n.s. p>0.05

Dunnett’s:n.s. p>0.05

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

21-day stage median No No Mann-Whitney:

n.s. p>0.05

Mann-Whitney:n.s. p>0.05

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

21-day wet weight No Yes Dunnett’s:

n.s. p>0.05

Dunnett’s:n.s. p>0.05

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

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

n.s. p>0.05

Dunnett’s:n.s. p>0.05

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

21-day HLL No Yes Dunnett’s:

Dose 1 p=0.005Dose 2 p<0.001Dose 3 p=0.003

Dunnett’s:Dose 1 p=0.005Dose 2 p<0.001Dose 3 p=0.003

890.1100 and EFED same conclusions, a significant (p<0.05) promotion from control. Response was not monotonically increasing, but there was an increasing trend.

21-day normalized HLL

No Yes Dunnett’s:

Dose 1 p=0.020Dose 2 p=0.017Dose 3 p=0.039

Dunnett’s:Dose 1 p=0.020Dose 2 p=0.017Dose 3 p=0.039

890.1100 and EFED same conclusions, a significant (p<0.05) promotion from control. Response was not monotonically increasing, but there was an increasing trend.

Page 46 of 107

DER Template Version: 22 September 2011 

Page 48 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

ANALYSIS OF 7-DAY VARIABLES

027401 Dichlobenil 48616101 890.1100

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) -- alphalevel=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 0.780 1.582 0.245 USE PARAMETRIC TESTS

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

***

BASIC SUMMARY STATISTICS

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

Ctrl 4 0.45 0.05 0.03 12.13 0.36, 0.53

Dose1 4 0.45 0.06 0.03 13.11 0.36, 0.55

Dose2 4 0.48 0.05 0.02 9.69 0.40, 0.55

Dose3 4 0.49 0.02 0.01 3.08 0.47, 0.51

Level Median Min Max %of Control(means)

%Reduction(means)

Ctrl 0.44 0.39 0.51 . .

Dose1 0.45 0.38 0.52 101.03 -1.03

Dose2 0.47 0.43 0.54 106.85 -6.85

Dose3 0.49 0.48 0.51 109.71 -9.71

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

***

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 0.79 0.525

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 0.45 . 0.47 . . . . .

.

Page 47 of 107

DER Template Version: 22 September 2011 

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

EPA MRID Number 48616101

Dose1 0.45 0.998 0.47 0.801 . . . .

.

Dose2 0.48 0.688 0.47 0.832 0.860 . . .

.

Dose3 0.49 0.447 0.47 0.848 0.656 0.980 . .

.

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

***

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 2.74 0.434

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 0.44 . .

Dose1 0.45 1.000 0.500

Dose2 0.47 0.494 0.768

Dose3 0.49 0.235 0.944

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN 

CONTROL

Williams >highest dose (no sign. 

differences)

Jonckheere >highest dose (no sign. 

differences)

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

***

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

Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value

3 12 0.79 0.525

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 -0.45 . -0.45 . . . . .

.

Page 48 of 107

DER Template Version: 22 September 2011 

Page 50 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Dose1 -0.45 0.998 -0.45 0.524 . . . .

.

Dose2 -0.48 0.688 -0.48 0.239 0.860 . . .

.

Dose3 -0.49 0.447 -0.49 0.141 0.656 0.980 . .

.

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

***

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 2.74 0.434

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 -0.44 . .

Dose1 -0.45 1.000 0.500

Dose2 -0.47 0.494 0.232

Dose3 -0.49 0.235 0.056

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

Williams >highest dose (no sign. 

differences)

Jonckheere >highest dose (no sign. 

differences)

027401 Dichlobenil 48616101 890.1100

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) -- alphalevel=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

Page 49 of 107

DER Template Version: 22 September 2011 

Page 51 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

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

Ctrl 4 55.00 0.00 0.00 0.00 . , .

Dose1 4 55.25 0.50 0.25 0.90 54.45, 56.05

Dose2 4 55.00 0.00 0.00 0.00 . , .

Dose3 4 55.00 0.00 0.00 0.00 . , .

Level Median Min Max %of Control(means)

%Reduction(means)

Ctrl 55.00 55.00 55.00 . .

Dose1 55.00 55.00 56.00 100.45 -0.45

Dose2 55.00 55.00 55.00 100.00 0.00

Dose3 55.00 55.00 55.00 100.00 0.00

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

***

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

Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value

3 12 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 55.00 . 55.13 . . . . .

.

Dose1 55.25 0.389 55.13 0.833 . . . .

.

Dose2 55.00 1.000 55.00 0.618 0.515 . . .

.

Dose3 55.00 1.000 55.00 0.636 0.515 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 3.00 0.392

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

Page 50 of 107

DER Template Version: 22 September 2011 

Page 52 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Ctrl 55.00 . .

Dose1 55.00 0.478 0.841

Dose2 55.00 1.000 0.500

Dose3 55.00 1.000 0.327

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 -55.00 . -55.00 . . . . .

.

Dose1 -55.25 0.389 -55.08 0.384 . . . .

.

Dose2 -55.00 1.000 -55.08 0.410 0.515 . . .

.

Dose3 -55.00 1.000 -55.08 0.425 0.515 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 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

Page 51 of 107

DER Template Version: 22 September 2011 

Page 53 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Ctrl -55.00 . .

Dose1 -55.00 0.478 0.159

Dose2 -55.00 1.000 0.500

Dose3 -55.00 1.000 0.673

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

Williams >highest dose (no sign. 

differences)

Jonckheere >highest dose (no sign. 

differences)

027401 Dichlobenil 48616101 890.1100

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) -- alphalevel=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

0.956 0.584 2.765 0.088 USE PARAMETRIC TESTS

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

***

BASIC SUMMARY STATISTICS

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

Ctrl 4 15.76 0.82 0.41 5.21 14.45, 17.06

Dose1 4 15.92 0.95 0.48 5.99 14.41, 17.44

Dose2 4 16.00 0.46 0.23 2.85 15.27, 16.72

Dose3 4 16.07 0.22 0.11 1.35 15.73, 16.41

Level Median Min Max %of Control(means)

%Reduction(means)

Ctrl 15.78 14.96 16.52 . .

Dose1 15.72 15.00 17.26 101.05 -1.05

Dose2 15.89 15.59 16.63 101.52 -1.52

Dose3 16.05 15.85 16.33 101.98 -1.98

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

***

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 0.16 0.924

Page 52 of 107

DER Template Version: 22 September 2011 

Page 54 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

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 15.76 . 15.94 . . . . .

.

Dose1 15.92 0.972 15.94 0.730 . . . .

.

Dose2 16.00 0.923 15.94 0.764 0.999 . . .

.

Dose3 16.07 0.852 15.94 0.782 0.989 0.999 . .

.

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

***

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

Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value

3 0.82 0.846

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 15.78 . .

Dose1 15.72 0.889 0.614

Dose2 15.89 0.678 0.768

Dose3 16.05 1.000 0.848

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 0.16 0.924

Dunnett - testing each trt mean signif. different than control

Page 53 of 107

DER Template Version: 22 September 2011 

Page 55 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

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

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values

p-value mean p-value Dose1 Dose2 Dose3 Dose4

Dose5

Ctrl -15.76 . -15.76 . . . . .

.

Dose1 -15.92 0.972 -15.92 0.437 . . . .

.

Dose2 -16.00 0.923 -16.00 0.398 0.999 . . .

.

Dose3 -16.07 0.852 -16.07 0.348 0.989 0.999 . .

.

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

***

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

Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value

3 0.82 0.846

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 -15.78 . .

Dose1 -15.72 0.889 0.386

Dose2 -15.89 0.678 0.232

Dose3 -16.05 1.000 0.152

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

Williams >highest dose (no sign. 

differences)

Jonckheere >highest dose (no sign. 

differences)

027401 Dichlobenil 48616101 890.1100

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

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01

Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05

Page 54 of 107

DER Template Version: 22 September 2011 

Page 56 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Use parametric analyses if neither test rejected, otherwise non-parametric

analyses.

Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value

0.938 0.322 5.241 0.015 USE NON-PARAMETRIC

TESTS

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

***

BASIC SUMMARY STATISTICS

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

Ctrl 4 3.05 0.04 0.02 1.35 2.99, 3.12

Dose1 4 3.05 0.23 0.12 7.66 2.68, 3.42

Dose2 4 3.13 0.06 0.03 1.99 3.03, 3.23

Dose3 4 3.14 0.09 0.05 3.01 2.99, 3.29

Level Median Min Max %of Control(means)

%Reduction(means)

Ctrl 3.06 3.00 3.09 . .

Dose1 3.07 2.76 3.30 99.92 0.08

Dose2 3.15 3.04 3.18 102.54 -2.54

Dose3 3.13 3.05 3.25 102.78 -2.78

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

***

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 0.53 0.671

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 3.05 . 3.09 . . . . .

.

Dose1 3.05 1.000 3.09 0.750 . . . .

.

Dose2 3.13 0.745 3.09 0.783 0.824 . . .

.

Dose3 3.14 0.693 3.09 0.801 0.783 1.000 . .

.

Page 55 of 107

DER Template Version: 22 September 2011 

Page 57 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

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

***

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 2.09 0.553

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 3.06 . .

Dose1 3.07 1.000 0.500

Dose2 3.15 0.190 0.848

Dose3 3.13 0.346 0.913

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN 

CONTROL

Williams >highest dose (no sign. 

differences)

Jonckheere >highest dose (no sign. 

differences)

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

***

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

Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value

3 12 0.53 0.671

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 -3.05 . -3.05 . . . . .

.

Dose1 -3.05 1.000 -3.05 0.589 . . . .

.

Dose2 -3.13 0.745 -3.13 0.268 0.824 . . .

.

Dose3 -3.14 0.693 -3.14 0.250 0.783 1.000 . .

.

Page 56 of 107

DER Template Version: 22 September 2011 

Page 58 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

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

***

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 2.09 0.553

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 -3.06 . .

Dose1 -3.07 1.000 0.500

Dose2 -3.15 0.190 0.152

Dose3 -3.13 0.346 0.087

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

Williams >highest dose (no sign. 

differences)

Jonckheere >highest dose (no sign. 

differences)

027401 Dichlobenil 48616101 890.1100

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) -- alphalevel=0.05

Use parametric analyses if neither test rejected, otherwise non-parametric

analyses.

Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value

0.931 0.253 1.333 0.310 USE PARAMETRIC TESTS

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

***

BASIC SUMMARY STATISTICS

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

Ctrl 4 0.20 0.01 0.01 6.62 0.17, 0.22

Dose1 4 0.19 0.01 0.01 7.44 0.17, 0.21

Dose2 4 0.20 0.01 0.00 2.96 0.19, 0.20

Dose3 4 0.20 0.01 0.00 2.96 0.19, 0.20

Level Median Min Max %of Control(means)

%Reduction(means)

Page 57 of 107

DER Template Version: 22 September 2011 

Page 59 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Ctrl 0.20 0.18 0.21 . .

Dose1 0.19 0.18 0.21 97.44 2.56

Dose2 0.20 0.19 0.20 100.00 0.00

Dose3 0.20 0.19 0.20 100.00 0.00

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

***

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

Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value

3 12 0.23 0.873

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 0.20 . 0.20 . . . . .

.

Dose1 0.19 0.837 0.19 0.487 . . . .

.

Dose2 0.20 1.000 0.19 0.518 0.903 . . .

.

Dose3 0.20 1.000 0.19 0.535 0.903 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 1.04 0.791

MannWhit - testing each trt median signif. different from control

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

Level Median MannWhit p-value Jonckheere p-value

Ctrl 0.20 . .

Dose1 0.19 0.667 0.275

Dose2 0.20 1.000 0.560

Dose3 0.20 1.000 0.653

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN 

CONTROL

Page 58 of 107

DER Template Version: 22 September 2011 

Page 60 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Williams >highest dose (no sign. 

differences)

Jonckheere >highest dose (no sign. 

differences)

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

***

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

Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value

3 12 0.23 0.873

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 -0.20 . -0.19 . . . . .

.

Dose1 -0.19 0.837 -0.19 0.718 . . . .

.

Dose2 -0.20 1.000 -0.20 0.618 0.903 . . .

.

Dose3 -0.20 1.000 -0.20 0.636 0.903 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 1.04 0.791

MannWhit - testing each trt median signif. different from control

Jonckheere - test assumes dose-response relationship, testing INCREASING 

trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl -0.20 . .

Dose1 -0.19 0.667 0.725

Dose2 -0.20 1.000 0.440

Dose3 -0.20 1.000 0.347

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

Page 59 of 107

DER Template Version: 22 September 2011 

Page 61 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Williams >highest dose (no sign. 

differences)

Jonckheere >highest dose (no sign. 

differences)

Page 60 of 107

DER Template Version: 22 September 2011 

Page 62 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

ANALYSIS OF 21-DAY VARIABLES: TWO-FACTOR NESTED ANOVA, NEGATIVE VS. SOLVENT 

CONTROLS

Model Information

Data Set WORK.CONTROLS

Dependent Variable BW

Covariance Structure Variance Components

Estimation Method REML

Residual Variance Method Profile

Fixed Effects SE Method Kenward-Roger

Degrees of Freedom Method Kenward-Roger

Class Level Information

Class Levels Values

LateStage 2 No Yes

Conc 2 1 2

Rep 4 A B C D

Dimensions

Covariance Parameters 3

Columns in X 9

Columns in Z 12

Subjects 1

Max Obs Per Subject 119

Number of Observations

Number of Observations Read 119

Number of Observations Used 119

Number of Observations Not Used 0

Iteration History

Iteration Evaluations -2 Res Log Like Criterion

0 1 13.85369605

1 3 11.54687535 .

2 1 11.50307337 0.00002787

3 1 11.50014549 0.00000019

4 1 11.50012647 0.00000000

Convergence criteria met.

Page 61 of 107

DER Template Version: 22 September 2011 

Page 63 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Covariance Parameter Estimates

Cov Parm Estimate

Rep(Conc) 0

ID(Rep) 6.051E-7

Residual 0.05619

Fit Statistics

-2 Res Log Likelihood 11.5

AIC (smaller is better) 15.5

AICC (smaller is better) 15.6

BIC (smaller is better) 15.7

Type 3 Tests of Fixed Effects

Effect Num DF Den DF F Value Pr > F

Conc 1 28.6 1.73 0.1988

LateStage 1 113 114.58 <.0001

LateStage*Conc 1 113 0.07 0.7939

Model Information

Data Set WORK.CONTROLS

Dependent Variable SVL

Covariance Structure Variance Components

Estimation Method REML

Residual Variance Method Profile

Fixed Effects SE Method Kenward-Roger

Degrees of Freedom Method Kenward-Roger

Class Level Information

Class Levels Values

LateStage 2 No Yes

Conc 2 1 2

Rep 4 A B C D

Dimensions

Covariance Parameters 3

Columns in X 9

Columns in Z 12

Subjects 1

Max Obs Per Subject 119

Page 62 of 107

DER Template Version: 22 September 2011 

Page 64 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Number of Observations

Number of Observations Read 119

Number of Observations Used 119

Number of Observations Not Used 0

Iteration History

Iteration Evaluations -2 Res Log Like Criterion

0 1 416.81217773

1 3 416.46431653 0.00001392

2 1 416.46284202 0.00000004

3 1 416.46283803 0.00000000

Convergence criteria met.

Covariance Parameter Estimates

Cov Parm Estimate

Rep(Conc) 0.02902

ID(Rep) 3.316E-6

Residual 1.9178

Fit Statistics

-2 Res Log Likelihood 416.5

AIC (smaller is better) 422.5

AICC (smaller is better) 422.7

BIC (smaller is better) 422.7

Type 3 Tests of Fixed Effects

Effect Num DF Den DF F Value Pr > F

Conc 1 5.56 0.83 0.4009

LateStage 1 111 190.07 <.0001

LateStage*Conc 1 112 2.02 0.1575

Model Information

Data Set WORK.CONTROLS

Dependent Variable normHLL

Covariance Structure Variance Components

Estimation Method REML

Residual Variance Method Profile

Fixed Effects SE Method Kenward-Roger

Degrees of Freedom Method Kenward-Roger

Page 63 of 107

DER Template Version: 22 September 2011 

Page 65 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Class Level Information

Class Levels Values

LateStage 2 No Yes

Conc 2 1 2

Rep 4 A B C D

Dimensions

Covariance Parameters 3

Columns in X 9

Columns in Z 12

Subjects 1

Max Obs Per Subject 119

Number of Observations

Number of Observations Read 119

Number of Observations Used 119

Number of Observations Not Used 0

Iteration History

Iteration Evaluations -2 Res Log Like Criterion

0 1 -143.34563423

1 4 -143.34563423 0.00000000

Convergence criteria met.

Covariance Parameter Estimates

Cov Parm Estimate

Rep(Conc) 8.08E-21

ID(Rep) 0

Residual 0.01498

Fit Statistics

-2 Res Log Likelihood -143.3

AIC (smaller is better) -141.3

AICC (smaller is better) -141.3

BIC (smaller is better) -141.3

Type 3 Tests of Fixed Effects

Effect Num DF Den DF F Value Pr > F

Conc 1 115 5.06 0.0263

LateStage 1 115 250.13 <.0001

LateStage*Conc 1 115 4.04 0.0467

Page 64 of 107

DER Template Version: 22 September 2011 

Page 66 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Model Information

Data Set WORK.CONTROLS

Dependent Variable NFStage

Covariance Structure Variance Components

Estimation Method REML

Residual Variance Method Profile

Fixed Effects SE Method Kenward-Roger

Degrees of Freedom Method Kenward-Roger

Class Level Information

Class Levels Values

LateStage 2 No Yes

Conc 2 1 2

Rep 4 A B C D

Dimensions

Covariance Parameters 3

Columns in X 9

Columns in Z 12

Subjects 1

Max Obs Per Subject 119

Number of Observations

Number of Observations Read 119

Number of Observations Used 119

Number of Observations Not Used 0

Iteration History

Iteration Evaluations -2 Res Log Like Criterion

0 1 148.80185351

1 3 147.22032577 0.00002081

2 1 147.21963892 0.00000004

3 1 147.21963753 0.00000000

Convergence criteria met.

Covariance Parameter Estimates

Cov Parm Estimate

Rep(Conc) 0.003482

ID(Rep) 1.396E-6

Residual 0.1820

Page 65 of 107

DER Template Version: 22 September 2011 

Page 67 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Fit Statistics

-2 Res Log Likelihood 147.2

AIC (smaller is better) 153.2

AICC (smaller is better) 153.4

BIC (smaller is better) 153.5

Type 3 Tests of Fixed Effects

Effect Num DF Den DF F Value Pr > F

Conc 1 4.86 0.36 0.5733

LateStage 1 111 564.00 <.0001

LateStage*Conc 1 111 2.40 0.1239

Page 66 of 107

DER Template Version: 22 September 2011 

Page 68 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

ANALYSIS OF 21-DAY VARIABLES: TWO-FACTOR NESTED ANOVA

Model Information

Data Set WORK.NEGCONT

Dependent Variable BW

Covariance Structure Variance Components

Estimation Method REML

Residual Variance Method Profile

Fixed Effects SE Method Kenward-Roger

Degrees of Freedom Method Kenward-Roger

Class Level Information

Class Levels Values

LateStage 2 No Yes

Conc 4 1 3 4 5

Rep 4 A B C D

Dimensions

Covariance Parameters 3

Columns in X 15

Columns in Z 20

Subjects 1

Max Obs Per Subject 238

Number of Observations

Number of Observations Read 238

Number of Observations Used 238

Number of Observations Not Used 0

Iteration History

Iteration Evaluations -2 Res Log Like Criterion

0 1 18.95324583

1 3 18.87287201 0.00000000

Convergence criteria met.

Covariance Parameter Estimates

Cov Parm Estimate

Rep(Conc) 0.000422

ID(Rep) 1.092E-9

Residual 0.05618

Page 67 of 107

DER Template Version: 22 September 2011 

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

EPA MRID Number 48616101

Fit Statistics

-2 Res Log Likelihood 18.9

AIC (smaller is better) 24.9

AICC (smaller is better) 25.0

BIC (smaller is better) 27.2

Type 3 Tests of Fixed Effects

Effect Num DF Den DF F Value Pr > F

Conc 3 10.3 0.97 0.4418

LateStage 1 228 246.84 <.0001

LateStage*Conc 3 226 0.64 0.5894

Model Information

Data Set WORK.NEGCONT

Dependent Variable SVL

Covariance Structure Variance Components

Estimation Method REML

Residual Variance Method Profile

Fixed Effects SE Method Kenward-Roger

Degrees of Freedom Method Kenward-Roger

Class Level Information

Class Levels Values

LateStage 2 No Yes

Conc 4 1 3 4 5

Rep 4 A B C D

Dimensions

Covariance Parameters 3

Columns in X 15

Columns in Z 20

Subjects 1

Max Obs Per Subject 238

Number of Observations

Number of Observations Read 238

Number of Observations Used 235

Number of Observations Not Used 3

Page 68 of 107

DER Template Version: 22 September 2011 

Page 70 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Iteration History

Iteration Evaluations -2 Res Log Like Criterion

0 1 818.40606947

1 1 818.40606947 0.00000000

Convergence criteria met.

Covariance Parameter Estimates

Cov Parm Estimate

Rep(Conc) 0

ID(Rep) 0

Residual 1.9135

Fit Statistics

-2 Res Log Likelihood 818.4

AIC (smaller is better) 820.4

AICC (smaller is better) 820.4

BIC (smaller is better) 821.2

Type 3 Tests of Fixed Effects

Effect Num DF Den DF F Value Pr > F

Conc 3 227 0.88 0.4507

LateStage 1 227 415.19 <.0001

LateStage*Conc 3 227 0.59 0.6192

Model Information

Data Set WORK.NEGCONT

Dependent Variable normHLL

Covariance Structure Variance Components

Estimation Method REML

Residual Variance Method Profile

Fixed Effects SE Method Kenward-Roger

Degrees of Freedom Method Kenward-Roger

Class Level Information

Class Levels Values

LateStage 2 No Yes

Conc 4 1 3 4 5

Rep 4 A B C D

Page 69 of 107

DER Template Version: 22 September 2011 

Page 71 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Dimensions

Covariance Parameters 3

Columns in X 15

Columns in Z 20

Subjects 1

Max Obs Per Subject 238

Number of Observations

Number of Observations Read 238

Number of Observations Used 235

Number of Observations Not Used 3

Iteration History

Iteration Evaluations -2 Res Log Like Criterion

0 1 -298.28177002

1 1 -298.28177002 0.00000000

Convergence criteria met.

Covariance Parameter Estimates

Cov Parm Estimate

Rep(Conc) 0

ID(Rep) 0

Residual 0.01398

Fit Statistics

-2 Res Log Likelihood -298.3

AIC (smaller is better) -296.3

AICC (smaller is better) -296.3

BIC (smaller is better) -295.5

Type 3 Tests of Fixed Effects

Effect Num DF Den DF F Value Pr > F

Conc 3 227 4.15 0.0069

LateStage 1 227 564.48 <.0001

LateStage*Conc 3 227 0.97 0.4055

Model Information

Data Set WORK.NEGCONT

Dependent Variable NFStage

Covariance Structure Variance Components

Estimation Method REML

Residual Variance Method Profile

Page 70 of 107

DER Template Version: 22 September 2011 

Page 72 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Model Information

Fixed Effects SE Method Kenward-Roger

Degrees of Freedom Method Kenward-Roger

Class Level Information

Class Levels Values

LateStage 2 No Yes

Conc 4 1 3 4 5

Rep 4 A B C D

Dimensions

Covariance Parameters 3

Columns in X 15

Columns in Z 20

Subjects 1

Max Obs Per Subject 238

Number of Observations

Number of Observations Read 238

Number of Observations Used 238

Number of Observations Not Used 0

Iteration History

Iteration Evaluations -2 Res Log Like Criterion

0 1 316.76878266

1 4 316.76878266 .

2 1 316.76447065 0.00000076

3 1 316.76443009 0.00000000

Convergence criteria met.

Covariance Parameter Estimates

Cov Parm Estimate

Rep(Conc) 0

ID(Rep) 4.909E-9

Residual 0.2062

Fit Statistics

-2 Res Log Likelihood 316.8

AIC (smaller is better) 320.8

AICC (smaller is better) 320.8

BIC (smaller is better) 322.3

Page 71 of 107

DER Template Version: 22 September 2011 

Page 73 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Type 3 Tests of Fixed Effects

Effect Num DF Den DF F Value Pr > F

Conc 3 154 0.25 0.8617

LateStage 1 229 989.47 <.0001

LateStage*Conc 3 229 1.23 0.2978

Page 72 of 107

DER Template Version: 22 September 2011 

Page 74 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

ANALYSIS OF 21-DAY VARIABLES: TWO-FACTOR NESTED ANOVA, SLICED BY LATESTAGE

LateStage=No

Model Information

Data Set WORK.NEGCONT

Dependent Variable BW

Covariance Structure Variance Components

Estimation Method REML

Residual Variance Method Profile

Fixed Effects SE Method Kenward-Roger

Degrees of Freedom Method Kenward-Roger

Class Level Information

Class Levels Values

LateStage 1 No

Conc 4 1 3 4 5

Rep 4 A B C D

Dimensions

Covariance Parameters 3

Columns in X 10

Columns in Z 20

Subjects 1

Max Obs Per Subject 137

Number of Observations

Number of Observations Read 137

Number of Observations Used 137

Number of Observations Not Used 0

Iteration History

Iteration Evaluations -2 Res Log Like Criterion

0 1 32.75356998

1 3 31.68188505 0.00000124

2 1 31.68175253 0.00000000

Convergence criteria met.

Covariance Parameter Estimates

Cov Parm Estimate

Rep(Conc) 0.002448

ID(Rep) 3.019E-8

Residual 0.06464

Page 73 of 107

DER Template Version: 22 September 2011 

Page 75 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Fit Statistics

-2 Res Log Likelihood 31.7

AIC (smaller is better) 37.7

AICC (smaller is better) 37.9

BIC (smaller is better) 40.0

Type 3 Tests of Fixed Effects

Effect Num DF Den DF F Value Pr > F

Conc 3 11.4 1.00 0.4258

LateStage 0 . . .

LateStage*Conc 0 . . .

LateStage=Yes

Model Information

Data Set WORK.NEGCONT

Dependent Variable BW

Covariance Structure Variance Components

Estimation Method REML

Residual Variance Method Profile

Fixed Effects SE Method Kenward-Roger

Degrees of Freedom Method Kenward-Roger

Class Level Information

Class Levels Values

LateStage 1 Yes

Conc 4 1 3 4 5

Rep 4 A B C D

Dimensions

Covariance Parameters 3

Columns in X 10

Columns in Z 20

Subjects 1

Max Obs Per Subject 101

Number of Observations

Number of Observations Read 101

Number of Observations Used 101

Number of Observations Not Used 0

Page 74 of 107

DER Template Version: 22 September 2011 

Page 76 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Iteration History

Iteration Evaluations -2 Res Log Like Criterion

0 1 -20.03416014

1 4 -20.03416014 0.00000000

Convergence criteria met.

Covariance Parameter Estimates

Cov Parm Estimate

Rep(Conc) 0

ID(Rep) 6.19E-25

Residual 0.04170

Fit Statistics

-2 Res Log Likelihood -20.0

AIC (smaller is better) -18.0

AICC (smaller is better) -18.0

BIC (smaller is better) -17.3

Type 3 Tests of Fixed Effects

Effect Num DF Den DF F Value Pr > F

Conc 3 97 0.58 0.6307

LateStage 0 . . .

LateStage*Conc 0 . . .

LateStage=No

Model Information

Data Set WORK.NEGCONT

Dependent Variable SVL

Covariance Structure Variance Components

Estimation Method REML

Residual Variance Method Profile

Fixed Effects SE Method Kenward-Roger

Degrees of Freedom Method Kenward-Roger

Class Level Information

Class Levels Values

LateStage 1 No

Conc 4 1 3 4 5

Rep 4 A B C D

Page 75 of 107

DER Template Version: 22 September 2011 

Page 77 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Dimensions

Covariance Parameters 3

Columns in X 10

Columns in Z 20

Subjects 1

Max Obs Per Subject 137

Number of Observations

Number of Observations Read 137

Number of Observations Used 136

Number of Observations Not Used 1

Iteration History

Iteration Evaluations -2 Res Log Like Criterion

0 1 503.00923116

1 4 503.00923116 0.00000000

Convergence criteria met.

Covariance Parameter Estimates

Cov Parm Estimate

Rep(Conc) 1.78E-19

ID(Rep) 0

Residual 2.3774

Fit Statistics

-2 Res Log Likelihood 503.0

AIC (smaller is better) 505.0

AICC (smaller is better) 505.0

BIC (smaller is better) 505.8

Type 3 Tests of Fixed Effects

Effect Num DF Den DF F Value Pr > F

Conc 3 132 0.64 0.5913

LateStage 0 . . .

LateStage*Conc 0 . . .

LateStage=Yes

Model Information

Data Set WORK.NEGCONT

Dependent Variable SVL

Covariance Structure Variance Components

Estimation Method REML

Page 76 of 107

DER Template Version: 22 September 2011 

Page 78 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Model Information

Residual Variance Method Profile

Fixed Effects SE Method Kenward-Roger

Degrees of Freedom Method Kenward-Roger

Class Level Information

Class Levels Values

LateStage 1 Yes

Conc 4 1 3 4 5

Rep 4 A B C D

Dimensions

Covariance Parameters 3

Columns in X 10

Columns in Z 20

Subjects 1

Max Obs Per Subject 101

Number of Observations

Number of Observations Read 101

Number of Observations Used 99

Number of Observations Not Used 2

Iteration History

Iteration Evaluations -2 Res Log Like Criterion

0 1 305.02510515

1 3 304.99527820 0.00000130

2 1 304.99519249 0.00000000

Convergence criteria met.

Covariance Parameter Estimates

Cov Parm Estimate

Rep(Conc) 0

ID(Rep) 2.247E-7

Residual 1.2627

Fit Statistics

-2 Res Log Likelihood 305.0

AIC (smaller is better) 309.0

AICC (smaller is better) 309.1

BIC (smaller is better) 310.5

Page 77 of 107

DER Template Version: 22 September 2011 

Page 79 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Type 3 Tests of Fixed Effects

Effect Num DF Den DF F Value Pr > F

Conc 3 76 1.00 0.3959

LateStage 0 . . .

LateStage*Conc 0 . . .

LateStage=No

Model Information

Data Set WORK.NEGCONT

Dependent Variable normHLL

Covariance Structure Variance Components

Estimation Method REML

Residual Variance Method Profile

Fixed Effects SE Method Kenward-Roger

Degrees of Freedom Method Kenward-Roger

Class Level Information

Class Levels Values

LateStage 1 No

Conc 4 1 3 4 5

Rep 4 A B C D

Dimensions

Covariance Parameters 3

Columns in X 10

Columns in Z 20

Subjects 1

Max Obs Per Subject 137

Number of Observations

Number of Observations Read 137

Number of Observations Used 136

Number of Observations Not Used 1

Iteration History

Iteration Evaluations -2 Res Log Like Criterion

0 1 -165.02357623

1 4 -165.02357623 .

2 1 -165.05525143 0.00000803

3 1 -165.05695576 0.00000003

4 1 -165.05696279 0.00000000

Page 78 of 107

DER Template Version: 22 September 2011 

Page 80 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Convergence criteria met.

Covariance Parameter Estimates

Cov Parm Estimate

Rep(Conc) 0

ID(Rep) 1.959E-9

Residual 0.01502

Fit Statistics

-2 Res Log Likelihood -165.1

AIC (smaller is better) -161.1

AICC (smaller is better) -161.0

BIC (smaller is better) -159.5

Type 3 Tests of Fixed Effects

Effect Num DF Den DF F Value Pr > F

Conc 3 86 5.05 0.0029

LateStage 0 . . .

LateStage*Conc 0 . . .

LateStage=Yes

Model Information

Data Set WORK.NEGCONT

Dependent Variable normHLL

Covariance Structure Variance Components

Estimation Method REML

Residual Variance Method Profile

Fixed Effects SE Method Kenward-Roger

Degrees of Freedom Method Kenward-Roger

Class Level Information

Class Levels Values

LateStage 1 Yes

Conc 4 1 3 4 5

Rep 4 A B C D

Dimensions

Covariance Parameters 3

Columns in X 10

Columns in Z 20

Subjects 1

Max Obs Per Subject 101

Page 79 of 107

DER Template Version: 22 September 2011 

Page 81 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Number of Observations

Number of Observations Read 101

Number of Observations Used 99

Number of Observations Not Used 2

Iteration History

Iteration Evaluations -2 Res Log Like Criterion

0 1 -134.25470852

1 4 -134.25470852 0.00000000

Convergence criteria met.

Covariance Parameter Estimates

Cov Parm Estimate

Rep(Conc) 0

ID(Rep) 0

Residual 0.01245

Fit Statistics

-2 Res Log Likelihood -134.3

AIC (smaller is better) -132.3

AICC (smaller is better) -132.2

BIC (smaller is better) -131.5

Type 3 Tests of Fixed Effects

Effect Num DF Den DF F Value Pr > F

Conc 3 95 0.74 0.5296

LateStage 0 . . .

LateStage*Conc 0 . . .

LateStage=No

Model Information

Data Set WORK.NEGCONT

Dependent Variable NFStage

Covariance Structure Variance Components

Estimation Method REML

Residual Variance Method Profile

Fixed Effects SE Method Kenward-Roger

Degrees of Freedom Method Kenward-Roger

Page 80 of 107

DER Template Version: 22 September 2011 

Page 82 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Class Level Information

Class Levels Values

LateStage 1 No

Conc 4 1 3 4 5

Rep 4 A B C D

Dimensions

Covariance Parameters 3

Columns in X 10

Columns in Z 20

Subjects 1

Max Obs Per Subject 137

Number of Observations

Number of Observations Read 137

Number of Observations Used 137

Number of Observations Not Used 0

Iteration History

Iteration Evaluations -2 Res Log Like Criterion

0 1 84.66233974

1 4 84.66233974 0.00000000

Convergence criteria met.

Covariance Parameter Estimates

Cov Parm Estimate

Rep(Conc) 0

ID(Rep) 0

Residual 0.09951

Fit Statistics

-2 Res Log Likelihood 84.7

AIC (smaller is better) 86.7

AICC (smaller is better) 86.7

BIC (smaller is better) 87.4

Type 3 Tests of Fixed Effects

Effect Num DF Den DF F Value Pr > F

Conc 3 133 1.78 0.1536

LateStage 0 . . .

LateStage*Conc 0 . . .

LateStage=Yes

Page 81 of 107

DER Template Version: 22 September 2011 

Page 83 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Model Information

Data Set WORK.NEGCONT

Dependent Variable NFStage

Covariance Structure Variance Components

Estimation Method REML

Residual Variance Method Profile

Fixed Effects SE Method Kenward-Roger

Degrees of Freedom Method Kenward-Roger

Class Level Information

Class Levels Values

LateStage 1 Yes

Conc 4 1 3 4 5

Rep 4 A B C D

Dimensions

Covariance Parameters 3

Columns in X 10

Columns in Z 20

Subjects 1

Max Obs Per Subject 101

Number of Observations

Number of Observations Read 101

Number of Observations Used 101

Number of Observations Not Used 0

Iteration History

Iteration Evaluations -2 Res Log Like Criterion

0 1 187.13083747

1 3 186.81514920 0.00050566

2 2 186.81311123 0.00000316

3 1 186.81309770 0.00000000

Convergence criteria met.

Covariance Parameter Estimates

Cov Parm Estimate

Rep(Conc) 0

ID(Rep) 1.963E-7

Residual 0.3473

Page 82 of 107

DER Template Version: 22 September 2011 

Page 84 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Fit Statistics

-2 Res Log Likelihood 186.8

AIC (smaller is better) 190.8

AICC (smaller is better) 190.9

BIC (smaller is better) 192.4

Type 3 Tests of Fixed Effects

Effect Num DF Den DF F Value Pr > F

Conc 3 73.5 0.31 0.8186

LateStage 0 . . .

LateStage*Conc 0 . . .

Page 83 of 107

DER Template Version: 22 September 2011 

Page 85 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

ANALYSIS OF 21-DAY VARIABLES: NON-LATESTAGE ONLY

027401 Dichlobenil 48616101 890.1100 ALL TADPOLES FOR NF STAGE ANALYSIS

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) -- alphalevel=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

0.851 0.014 6.000 0.010 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 60.50 0.58 0.29 0.95 59.58, 61.42

Dose2 4 60.25 0.50 0.25 0.83 59.45, 61.05

Dose3 4 60.25 0.50 0.25 0.83 59.45, 61.05

Level Median Min Max %of Control(means)

%Reduction(means)

Ctrl 60.00 60.00 60.00 . .

Dose1 60.50 60.00 61.00 100.83 -0.83

Dose2 60.00 60.00 61.00 100.42 -0.42

Dose3 60.00 60.00 61.00 100.42 -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 0.80 0.517

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 84 of 107

DER Template Version: 22 September 2011 

Page 86 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Ctrl 60.00 . 60.25 . . . . .

.

Dose1 60.50 0.322 60.25 0.850 . . . .

.

Dose2 60.25 0.782 60.25 0.877 0.864 . . .

.

Dose3 60.25 0.782 60.25 0.891 0.864 1.000 . .

.

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

***

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

Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value

3 2.50 0.475

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 60.50 0.223 0.937

Dose2 60.00 0.478 0.783

Dose3 60.00 0.478 0.691

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 0.80 0.517

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

Page 85 of 107

DER Template Version: 22 September 2011 

Page 87 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Ctrl -60.00 . -60.00 . . . . .

.

Dose1 -60.50 0.322 -60.33 0.192 . . . .

.

Dose2 -60.25 0.782 -60.33 0.205 0.864 . . .

.

Dose3 -60.25 0.782 -60.33 0.212 0.864 1.000 . .

.

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

***

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

Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value

3 2.50 0.475

MannWhit - testing each trt median signif. different from control

Jonckheere - test assumes dose-response relationship, testing INCREASING 

trend

027401 Dichlobenil 48616101 890.1100 NOT LS

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) -- alphalevel=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

0.968 0.813 1.347 0.305 USE PARAMETRIC TESTS

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

***

BASIC SUMMARY STATISTICS

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

Ctrl 4 1.71 0.05 0.02 2.66 1.64, 1.78

Dose1 4 1.74 0.14 0.07 7.89 1.52, 1.96

Dose2 4 1.79 0.12 0.06 6.93 1.59, 1.99

Dose3 4 1.67 0.07 0.03 4.09 1.56, 1.77

Level Median Min Max %of Control(means)

%Reduction(means)

Ctrl 1.72 1.65 1.75 . .

Dose1 1.78 1.55 1.87 101.90 -1.90

Dose2 1.79 1.65 1.94 104.68 -4.68

Page 86 of 107

DER Template Version: 22 September 2011 

Page 88 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Dose3 1.65 1.61 1.75 97.37 2.63

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

***

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 1.09 0.392

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 1.71 . 1.75 . . . . .

.

Dose1 1.74 0.941 1.75 0.780 . . . .

.

Dose2 1.79 0.562 1.75 0.812 0.909 . . .

.

Dose3 1.67 0.864 1.67 0.358 0.706 0.344 . .

.

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

***

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 3.01 0.390

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 1.72 . .

Dose1 1.78 0.413 0.845

Dose2 1.79 0.489 0.865

Dose3 1.65 0.410 0.336

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN 

CONTROL

Williams >highest dose (no sign. 

differences)

Jonckheere >highest dose (no sign. 

differences)

Page 87 of 107

DER Template Version: 22 September 2011 

Page 89 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

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

***

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 1.09 0.392

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 -1.71 . -1.71 . . . . .

.

Dose1 -1.74 0.941 -1.73 0.449 . . . .

.

Dose2 -1.79 0.562 -1.73 0.479 0.909 . . .

.

Dose3 -1.67 0.864 -1.73 0.495 0.706 0.344 . .

.

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

***

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 3.01 0.390

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 -1.72 . .

Dose1 -1.78 0.413 0.155

Dose2 -1.79 0.489 0.135

Dose3 -1.65 0.410 0.664

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

Williams >highest dose (no sign. 

differences)

Jonckheere >highest dose (no sign. 

differences)

Page 88 of 107

DER Template Version: 22 September 2011 

Page 90 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

027401 Dichlobenil 48616101 890.1100 NOT LS

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) -- alphalevel=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

0.948 0.457 0.616 0.618 USE PARAMETRIC TESTS

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

***

BASIC SUMMARY STATISTICS

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

Ctrl 4 23.08 0.41 0.21 1.80 22.42, 23.74

Dose1 4 23.15 0.73 0.36 3.15 21.99, 24.31

Dose2 4 23.34 0.43 0.21 1.83 22.66, 24.02

Dose3 4 22.79 0.39 0.20 1.72 22.17, 23.41

Level Median Min Max %of Control(means)

%Reduction(means)

Ctrl 23.11 22.56 23.56 . .

Dose1 23.35 22.11 23.78 100.28 -0.28

Dose2 23.39 22.78 23.81 101.12 -1.12

Dose3 22.77 22.43 23.19 98.73 1.27

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

***

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 0.80 0.518

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 23.08 . 23.19 . . . . .

.

Page 89 of 107

DER Template Version: 22 September 2011 

Page 91 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Dose1 23.15 0.996 23.19 0.703 . . . .

.

Dose2 23.34 0.818 23.19 0.737 0.949 . . .

.

Dose3 22.79 0.759 22.79 0.286 0.757 0.453 . .

.

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

***

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 3.42 0.331

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 23.11 . .

Dose1 23.35 0.678 0.718

Dose2 23.39 0.494 0.810

Dose3 22.77 0.413 0.242

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 0.80 0.518

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 -23.08 . -23.08 . . . . .

.

Page 90 of 107

DER Template Version: 22 September 2011 

Page 92 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Dose1 -23.15 0.996 -23.09 0.572 . . . .

.

Dose2 -23.34 0.818 -23.09 0.606 0.949 . . .

.

Dose3 -22.79 0.759 -23.09 0.624 0.757 0.453 . .

.

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

***

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 3.42 0.331

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 -23.11 . .

Dose1 -23.35 0.678 0.282

Dose2 -23.39 0.494 0.190

Dose3 -22.77 0.413 0.758

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN

CONTROL

Williams >highest dose (no sign. 

differences)

Jonckheere >highest dose (no sign. 

differences)

027401 Dichlobenil 48616101 890.1100 NOT LS

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) -- alphalevel=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

0.922 0.180 0.460 0.715 USE PARAMETRIC TESTS

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

***

BASIC SUMMARY STATISTICS

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

Page 91 of 107

DER Template Version: 22 September 2011 

Page 93 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Ctrl 4 23.07 0.81 0.41 3.52 21.77, 24.36

Dose1 4 24.71 0.96 0.48 3.89 23.18, 26.24

Dose2 4 25.26 0.74 0.37 2.92 24.09, 26.44

Dose3 4 25.12 0.67 0.34 2.68 24.04, 26.19

Level Median Min Max %of Control(means)

%Reduction(means)

Ctrl 23.01 22.21 24.03 . .

Dose1 24.71 23.64 25.79 107.14 -7.14

Dose2 25.60 24.16 25.69 109.53 -9.53

Dose3 25.07 24.44 25.89 108.89 -8.89

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

***

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

Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value

3 12 6.33 0.008

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 23.07 . 24.54 . . . . .

.

Dose1 24.71 0.034 24.54 0.996 . . . .

.

Dose2 25.26 0.006 24.54 0.998 0.769 . . .

.

Dose3 25.12 0.009 24.54 0.998 0.892 0.994 . .

.

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

***

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 8.32 0.040

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 23.01 . .

Page 92 of 107

DER Template Version: 22 September 2011 

Page 94 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Dose1 24.71 0.103 0.978

Dose2 25.60 0.067 0.994

Dose3 25.07 0.067 0.994

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 6.33 0.008

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 -23.07 . -23.07 . . . . .

.

Dose1 -24.71 0.034 -24.71 0.008 . . . .

.

Dose2 -25.26 0.006 -25.19 0.002 0.769 . . .

.

Dose3 -25.12 0.009 -25.19 0.002 0.892 0.994 . .

.

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

***

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 8.32 0.040

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 -23.01 . .

Page 93 of 107

DER Template Version: 22 September 2011 

Page 95 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Dose1 -24.71 0.103 0.022

Dose2 -25.60 0.067 0.006

Dose3 -25.07 0.067 0.006

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

Williams Dose1

Jonckheere Dose1

027401 Dichlobenil 48616101 890.1100 NOT LS

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) -- alphalevel=0.05

Use parametric analyses if neither test rejected, otherwise non-parametric

analyses.

Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value

0.954 0.555 0.071 0.974 USE PARAMETRIC TESTS

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

***

BASIC SUMMARY STATISTICS

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

Ctrl 4 1.00 0.03 0.01 2.62 0.96, 1.04

Dose1 4 1.07 0.04 0.02 3.83 1.01, 1.14

Dose2 4 1.09 0.03 0.02 3.02 1.04, 1.15

Dose3 4 1.11 0.03 0.02 2.81 1.06, 1.15

Level Median Min Max %of Control(means)

%Reduction(means)

Ctrl 1.00 0.98 1.03 . .

Dose1 1.08 1.02 1.12 106.98 -6.98

Dose2 1.10 1.05 1.13 108.98 -8.98

Dose3 1.12 1.06 1.13 110.22 -10.22

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

***

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 7.54 0.004

Dunnett - testing each trt mean signif. different than control

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

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Page 94 of 107

DER Template Version: 22 September 2011 

Page 96 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Level Mean Dunnett Isotonic Williams Tukey p-values

p-value mean p-value Dose1 Dose2 Dose3 Dose4

Dose5

Ctrl 1.00 . 1.07 . . . . .

.

Dose1 1.07 0.030 1.07 0.997 . . . .

.

Dose2 1.09 0.006 1.07 0.998 0.830 . . .

.

Dose3 1.11 0.003 1.07 0.999 0.534 0.950 . .

.

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

***

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 8.59 0.035

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 1.00 . .

Dose1 1.08 0.123 0.971

Dose2 1.10 0.066 0.995

Dose3 1.12 0.066 0.998

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 7.54 0.004

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

Page 95 of 107

DER Template Version: 22 September 2011 

Page 97 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Level Mean Dunnett Isotonic Williams Tukey p-values

p-value mean p-value Dose1 Dose2 Dose3 Dose4

Dose5

Ctrl -1.00 . -1.00 . . . . .

.

Dose1 -1.07 0.030 -1.07 0.007 . . . .

.

Dose2 -1.09 0.006 -1.09 0.001 0.830 . . .

.

Dose3 -1.11 0.003 -1.11 <.001 0.534 0.950 . .

.

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

***

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 8.59 0.035

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 -1.00 . .

Dose1 -1.08 0.123 0.029

Dose2 -1.10 0.066 0.005

Dose3 -1.12 0.066 0.002

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

Williams Dose1

Jonckheere Dose1

Page 96 of 107

DER Template Version: 22 September 2011 

Page 98 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

ANALYSIS OF 21-DAY VARIABLES: LATESTAGE ONLY

027401 Dichlobenil 48616101 890.1100 NOT LS

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) -- alphalevel=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

0.968 0.801 0.173 0.913 USE PARAMETRIC TESTS

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

***

BASIC SUMMARY STATISTICS

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

Ctrl 4 1.21 0.10 0.05 8.17 1.05, 1.36

Dose1 4 1.21 0.07 0.03 5.71 1.10, 1.32

Dose2 4 1.26 0.06 0.03 4.98 1.16, 1.35

Dose3 4 1.23 0.07 0.03 5.63 1.12, 1.34

Level Median Min Max %of Control(means)

%Reduction(means)

Ctrl 1.20 1.09 1.33 . .

Dose1 1.19 1.15 1.30 100.21 -0.21

Dose2 1.27 1.17 1.32 104.15 -4.15

Dose3 1.25 1.13 1.29 102.07 -2.07

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

***

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 0.38 0.772

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 1.21 . 1.22 . . . . .

.

Page 97 of 107

DER Template Version: 22 September 2011 

Page 99 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Dose1 1.21 1.000 1.22 0.725 . . . .

.

Dose2 1.26 0.684 1.22 0.759 0.814 . . .

.

Dose3 1.23 0.937 1.22 0.777 0.974 0.965 . .

.

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

***

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 1.35 0.718

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 1.20 . .

Dose1 1.19 1.000 0.500

Dose2 1.27 0.678 0.810

Dose3 1.25 0.676 0.713

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 0.38 0.772

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 -1.21 . -1.21 . . . . .

.

Page 98 of 107

DER Template Version: 22 September 2011 

Page 100 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Dose1 -1.21 1.000 -1.21 0.564 . . . .

.

Dose2 -1.26 0.684 -1.24 0.319 0.814 . . .

.

Dose3 -1.23 0.937 -1.24 0.330 0.974 0.965 . .

.

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

***

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 1.35 0.718

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 -1.20 . .

Dose1 -1.19 1.000 0.500

Dose2 -1.27 0.678 0.190

Dose3 -1.25 0.676 0.287

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

Williams >highest dose (no sign. 

differences)

Jonckheere >highest dose (no sign. 

differences)

027401 Dichlobenil 48616101 890.1100 NOT LS

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) -- alphalevel=0.05

Use parametric analyses if neither test rejected, otherwise non-parametric

analyses.

Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value

0.961 0.675 0.024 0.995 USE PARAMETRIC TESTS

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

***

BASIC SUMMARY STATISTICS

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

Page 99 of 107

DER Template Version: 22 September 2011 

Page 101 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Ctrl 4 19.05 0.50 0.25 2.62 18.25, 19.84

Dose1 4 19.26 0.45 0.23 2.35 18.54, 19.98

Dose2 4 19.51 0.47 0.23 2.38 18.77, 20.25

Dose3 4 19.46 0.54 0.27 2.78 18.60, 20.32

Level Median Min Max %of Control(means)

%Reduction(means)

Ctrl 19.16 18.35 19.53 . .

Dose1 19.20 18.86 19.79 101.13 -1.13

Dose2 19.64 18.85 19.91 102.43 -2.43

Dose3 19.37 18.90 20.19 102.15 -2.15

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

***

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 0.73 0.551

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 19.05 . 19.32 . . . . .

.

Dose1 19.26 0.869 19.32 0.852 . . . .

.

Dose2 19.51 0.432 19.32 0.879 0.890 . . .

.

Dose3 19.46 0.522 19.32 0.893 0.941 0.999 . .

.

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

***

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

Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value

3 1.74 0.627

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 19.16 . .

Page 100 of 107

DER Template Version: 22 September 2011 

Page 102 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Dose1 19.20 0.889 0.614

Dose2 19.64 0.235 0.879

Dose3 19.37 0.494 0.848

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 0.73 0.551

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 -19.05 . -19.05 . . . . .

.

Dose1 -19.26 0.869 -19.26 0.326 . . . .

.

Dose2 -19.51 0.432 -19.48 0.147 0.890 . . .

.

Dose3 -19.46 0.522 -19.48 0.152 0.941 0.999 . .

.

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

***

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

Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value

3 1.74 0.627

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 -19.16 . .

Page 101 of 107

DER Template Version: 22 September 2011 

Page 103 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

Dose1 -19.20 0.889 0.386

Dose2 -19.64 0.235 0.121

Dose3 -19.37 0.494 0.152

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

Williams >highest dose (no sign. 

differences)

Jonckheere >highest dose (no sign. 

differences)

027401 Dichlobenil 48616101 890.1100 NOT LS

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) -- alphalevel=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

0.956 0.584 0.970 0.439 USE PARAMETRIC TESTS

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

***

BASIC SUMMARY STATISTICS

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

Ctrl 4 26.80 0.52 0.26 1.93 25.97, 27.62

Dose1 4 27.94 0.43 0.21 1.52 27.26, 28.62

Dose2 4 28.26 0.74 0.37 2.63 27.08, 29.44

Dose3 4 28.18 0.96 0.48 3.41 26.65, 29.70

Level Median Min Max %of Control(means)

%Reduction(means)

Ctrl 26.87 26.13 27.32 . .

Dose1 27.97 27.40 28.44 104.28 -4.28

Dose2 28.58 27.17 28.73 105.48 -5.48

Dose3 28.11 27.10 29.39 105.15 -5.15

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

***

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 3.84 0.039

Dunnett - testing each trt mean signif. different than control

Page 102 of 107

DER Template Version: 22 September 2011 

Page 104 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

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 26.80 . 27.79 . . . . .

.

Dose1 27.94 0.091 27.79 0.987 . . . .

.

Dose2 28.26 0.029 27.79 0.991 0.913 . . .

.

Dose3 28.18 0.040 27.79 0.993 0.963 0.998 . .

.

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

***

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 7.49 0.058

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 26.87 . .

Dose1 27.97 0.067 0.990

Dose2 28.58 0.101 0.996

Dose3 28.11 0.103 0.984

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 3.84 0.039

Dunnett - testing each trt mean signif. different than control

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

Page 103 of 107

DER Template Version: 22 September 2011 

Page 105 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

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 -26.80 . -26.80 . . . . .

.

Dose1 -27.94 0.091 -27.94 0.022 . . . .

.

Dose2 -28.26 0.029 -28.22 0.008 0.913 . . .

.

Dose3 -28.18 0.040 -28.22 0.008 0.963 0.998 . .

.

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

***

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 7.49 0.058

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 -26.87 . .

Dose1 -27.97 0.067 0.010

Dose2 -28.58 0.101 0.004

Dose3 -28.11 0.103 0.016

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

Williams Dose1

Jonckheere Dose1

027401 Dichlobenil 48616101 890.1100 NOT LS

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) -- alphalevel=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

Page 104 of 107

DER Template Version: 22 September 2011 

Page 106 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

0.981 0.972 0.453 0.720 USE PARAMETRIC TESTS

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

***

BASIC SUMMARY STATISTICS

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

Ctrl 4 1.41 0.05 0.03 3.76 1.33, 1.50

Dose1 4 1.46 0.03 0.01 1.82 1.41, 1.50

Dose2 4 1.45 0.05 0.03 3.74 1.37, 1.54

Dose3 4 1.45 0.04 0.02 2.94 1.38, 1.52

Level Median Min Max %of Control(means)

%Reduction(means)

Ctrl 1.40 1.37 1.49 . .

Dose1 1.46 1.42 1.48 103.01 -3.01

Dose2 1.46 1.38 1.51 102.83 -2.83

Dose3 1.47 1.39 1.48 102.83 -2.83

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

***

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 0.81 0.515

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 1.41 . 1.44 . . . . .

.

Dose1 1.46 0.441 1.44 0.888 . . . .

.

Dose2 1.45 0.486 1.44 0.911 1.000 . . .

.

Dose3 1.45 0.486 1.44 0.922 1.000 1.000 . .

.

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

***

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

Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value

3 1.38 0.711

Page 105 of 107

DER Template Version: 22 September 2011 

Page 107 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

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 1.40 . .

Dose1 1.46 0.346 0.876

Dose2 1.46 0.494 0.848

Dose3 1.47 0.577 0.860

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 0.81 0.515

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 -1.41 . -1.41 . . . . .

.

Dose1 -1.46 0.441 -1.45 0.136 . . . .

.

Dose2 -1.45 0.486 -1.45 0.145 1.000 . . .

.

Dose3 -1.45 0.486 -1.45 0.150 1.000 1.000 . .

.

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

***

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

Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value

3 1.38 0.711

Page 106 of 107

DER Template Version: 22 September 2011 

Page 108 of 331
Data Evaluation Record on the Toxicity of Dichlobenil to Amphibians, Metamorphosis Assay

EPA MRID Number 48616101

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 -1.40 . .

Dose1 -1.46 0.346 0.124

Dose2 -1.46 0.494 0.152

Dose3 -1.47 0.577 0.140

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

Williams >highest dose (no sign. 

differences)

Jonckheere >highest dose (no sign. 

differences)

Page 107 of 107

DER Template Version: 22 September 2011 

Page 109 of 331
DATA EVALUATION RECORD 

DICHLOBENIL 

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

EPA Contract No. EP10H001452 

Task Assignment No. 2-27-2012 (MRID 48616102) 

Prepared for 

Health Effects Division 

Office of Pesticide Programs 

U.S. Environmental Protection Agency 

2777 South Crystal Drive 

Arlington, VA 22202 

Prepared by 

CSS-Dynamac Corporation 

1910 Sedwick Road, 

Building 100, Suite B 

Durham, NC 27713 

Primary Reviewer: Signature:

Sandra Hastings Date: 3/20/2012 

Secondary Reviewer: Signature:

Scott D. Studenberg, Ph.D., D.A.B.T. Date: 3/27/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 3/27/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 3/27/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 110 of 331
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 111 of 331
DICHLOBENIL/ 027401 

Androgen Receptor Binding (Rat Prostate Cytosol) (2011) I Page 1 of 14 

OCSPP 890.1150/ OECD None 

Primary Reviewer: Edward Scollon, Ph.D. 

Health Effects Division 

Secondary Reviewer: _G~re""'g""""A"--=k~erm=a=n=,-=P~h=D=-o..... ___ _ 

Health Effects Division 

Template version 08/2011 

DA TA EVALUATION RECORD 

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

PC CODE: 027401 DP BARCODE: 0397912 

TXR#: 0051691 CAS No.: 1194-65-6 

TEST MATERIAL (PURITY): Dichlobenil (99.3% a.i.) 

SYNONYMS: 2,6-dichlorobenzonitrile 

CITATION: Meerts, I.A.T. (2011). Dichlobenil Technical: Androgen Receptor Binding (Rat 

Prostate Cytosol). NOTOX B.V., The Netherlands. Laboratory Project 

Identification number: 495779, December 9, 2011. MRID 48616102. 

Unpublished. 

SPONSOR: Chemtura Corporation, 199 Benson Road, Middlebury, CT 

TEST ORDER#: EDSP-027401-120 

EXECUTIVE SUMMARY: In an androgen receptor (AR) binding assay (MRID 48616102), 

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

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

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

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

androgen (Rl 881) prior to conducting the competitive binding experiments. The competitive 

binding assay was conducted to measure the binding of a single concentration of [3H]-Rl 881 (1 

nM) in the presence of increasing concentrations (1 o-to to 10-3 M) of dichlobenil (99.3%, Lot# 

AM9/K12A222). Dimethyl sulfoxide (DMSO) was used as the solvent vehicle at a final assay 

concentration of 3.3%. A total of three independent runs were performed, and the assay included 

dexamethasone as a weak positive control, and R1881 as the ligand reference standard in each 

run. 

Only one run was considered acceptable in the saturation binding experiment, but the estimates 

for the dissociation constant (Kd), Bmax, and specific binding generally met the acceptance 

criteria, and the Scatchard plot was linear (r2 = 0.994). For the successful run, the dissociation 

constant (Kd) was 0.65 nM, and the Bmax was 2.58 fmol/l OOμg protein. The specific binding 

reached a plateau, and non-specific binding was less than 20% of total binding (4.2%). 

Page 112 of 331
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 2 of 14 

DICHLOBENIL/ 027401 OCSPP 890.1150/ OECD None

In the competitive binding experiment, dichlobenil reduced the mean specific binding of the 

radiolabeled ligand in a concentration dependent manner in all three runs. The maximum 

reduction in [3H]-R1881 binding occurred at the highest concentration of dichlobenil tested (10−3 

M), with specific binding being reduced to 40-46%. Specific binding was also reduced at 10−4 M 

(52-56%), but not at concentrations ≤10−5 M (86-103% binding). Dichlobenil is classified as a 

binder in the AR assay as specific [3

H]-R1881 binding to the AR was reduced to <50% at the 

highest concentration tested in all three runs. 

The estimated mean log IC50 was −3.85 M for dichlobenil, −8.73 M for R1881 and −4.58 M for 

dexamethasone (weak positive control). Compared to R1881, the mean Relative Binding 

Affinity (RBA) was 0.0013% for dichlobenil and 0.0071% for dexamethasone. All performance 

criteria were met except that the ligand depletion was greater (~18%) than the ≤10-15% criteria, 

and the bottom percent binding for R1881 was low in one of the runs. 

Although only one acceptable run was conducted for the saturation binding assay, and the Kd was 

low for that run, the androgen receptor was present in a reasonable concentration and functioning 

with appropriate affinity for the ligand in the competitive binding assays based on the 

performance criteria and estimated IC50s for R1881 and the weak positive control. It is unlikely 

that information from additional saturation binding runs would change the interpretation of this 

study. 

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

Receptor Binding Assay. 

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

assay (OCSPP 890.1150). 

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

statements were provided. 

Page 113 of 331
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 3 of 14 

DICHLOBENIL/ 027401 OCSPP 890.1150/ OECD None

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: NOTOX B.V. 

Location: Hambakenwetering 7, 5231 DD’s-Hertogenbosch, The Netherlands 

Study Director: I.A.T. M. Meerts 

Other Personnel: M. Mulder (Technical Coordinator); M.A. Thomson (Study Monitor) 

Study Period: May 13, 2011 to December 9, 2011 

2. Test substance: Dichlobenil 

 Description: Technical; white powder with lumps 

 Source: Chemtura Canada 

 Lot#: AM9K12A222 (expiry date: October 12, 2012) 

 Purity: 99.3% a.i. 

 Solubility: Soluble in DMSO at 50 mg/mL 

 Volatility: Not reported 

 Stability: Stable 

 Storage conditions: Room temperature (in the dark) 

 CAS #: 1194-65-6 

 Molecular weight: 172.0 

 Structure: 

3. Non-labeled ligand: R1881 

 Supplier: YES Pharma Ltd. (Israel) 

 Batch #: TDNF091128 

 Purity: 97.7% 

 CAS #: 965-93-5 

4. Radioactive ligand: [

3

H]-R1881 

 Supplier: Perkin Elmer Life and Analytical Sciences (Boston, MA) 

 Lot#: 614814 

 Date of production: July 1, 2010 

 Date of use: May 24, 2011 to June 14, 2011 

 Radiochemical purity: >97% 

 Specific activity: 85.1 Ci/mmol (3.15 TBq/mmol) 

 Concentration of stock: 0.1 μM 

5. Positive control: Dexamethasone 

 Supplier: Sigma, Zwijndrecht, The Netherlands 

 Catalog # 

Batch #: 

Not provided 

Not provided 

 Purity: Not provided 

 CAS # : 50-02-2 

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

 Justification for choice of 

solvent: 

None provided 

 Final Concentration: 3.3% (v:v) 

Page 114 of 331
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 4 of 14 

DICHLOBENIL/ 027401 OCSPP 890.1150/ OECD None

B. METHODS

1. Preparation of Rat Ventral Prostate Cytosol: A total of 20 male Sprague Dawley rats 

(obtained from Charles River, Sulzfeld, Germany) were castrated at 80-90 days of age and 

were euthanized using O2/CO2 approximately 24 hours later. The ventral prostates were 

excised immediately after termination, weighed, and placed in ice-cold low-salt TEDG 

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

homogenized, and centrifuged for 30 min at 30,000 × g at 4o C. The supernatant was 

pooled, discarding the resulting pellets. Protein concentration of the cytosol (Batch P-10-1) 

was determined to be 4.7 mg/mL. Although the assay method was not specified, 

characterization of the cytosol used was performed under NOTOX project 493416. Cytosol 

was aliquoted into ampoules for storage at ≤−75oC until use.

2. Saturation Radioligand Binding Experiment: A saturation binding experiment 

measuring total and non-specific binding of [3H]-R1881 was performed under a separate 

study (NOTOX Project 493416) to demonstrate that the AR was present in reasonable 

concentrations and had the appropriate affinity for the R1881 ligand. Results of the 

characterization of the cytosol and saturation binding experiment were presented in an 

Appendix to the main study. The conditions for the saturation binding experiment are 

summarized in Table 1. 

TABLE 1. Summary of Conditions for Saturation Binding Experiment a

Source of receptor Rat ventral prostate cytosol (P-10-1) 

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

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

Optimization of receptor concentration 1.3 mg/assay tube; sufficient to bind 

4.2% of radioligand at 0.25 nM

Temperature 4±2oC 

Incubation time 16-20 hours 

Composition of assay buffer 

(TEDG) 

Tris 10 mM (pH 7.4) 

EDTA 1.5 mM 

Glycerol 10% 

Phenylmethylsulfonyl fluoride 1 mM 

DTT 1 mM 

a Data were obtained from Appendix 2, page 38 of the study report. 

Specifics of the saturation binding experiment were not provided. Tables of information on 

the rat prostate, the conditions of the study, tube contents, results, and graphs were provided. 

Based on the provided data, serial dilutions of [3H]-R1881 in TEDG buffer were prepared to 

achieve the final concentrations in cytosol of 0.25, 0.5, 0.7, 1, 1.5, 2.5, 5, and 10 nM to 

determine total binding. To determine non-specific 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 1,000 nM. In the absence of cytosol, the radiation found in 

7.5, 15, 21, 30, or 45 μL of 10 nM [3H]-R1881 and 7.5, 15, or 30 μL of 100 nM [3H]-R1881 

was measured. The optimal protein concentration was determined by calculating specific 

binding to differing amounts of protein per tube, using 0.25 nM radiolabeled R1881. The 

Page 115 of 331
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optimal protein concentration was determined to be 0.72 mg protein/assay tube. Each run 

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

Table 2. 

TABLE 2. Saturation Binding Experiment Run a, b

Total Binding Non-Specific Binding Radioligand alone 

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

[

3

H]-R1881 

Final conc. (nM) 

[

3

H]-R1881 

Final conc. (nM) 

R1881 

Final conc. (nM) 

[

3

H]-R1881 

Initial conc. (nM) 

[

3

H]-R1881 

(μL) 

0.25 0.25 25 10 7.5 

0.50 0.50 50 10 15 

0.70 0.70 70 10 21 

1.00 1.00 100 10 30 

1.50 1.50 150 10 45 

2.50 2.50 250 100 7.5 

5.00 5.00 500 100 15 

10.00 10.00 1000 100 30 

a Data were obtained from Appendix 2, page 38 of the study report. 

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

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

H]-R1881. Samples were dried, 

and 300 μL of prostate cytosol were added. 

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

H]-R1881. R1881 was added in a 

100-fold molar excess of [3

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

cytosol were added. 

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

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

[

3

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

Following addition of triamcinolone acetonide, [3

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

dried, dissolved in diluted prostate cytosol (300 μL), and incubated in a rotor for 16-20 

hours at 4° C. To separate bound from free R1881, hydroxyapatite (HAP) slurry was added 

to each tube and vortexed once every 5 minutes for 20 minutes. The samples were then 

centrifuged, and the supernatant was removed using a glass pipette and discarded. The 

samples were washed 3 times in 50 mM TRIS buffer; samples were maintained on ice at all 

times during the washing procedure. Following the last wash and decanting of the Tris 

buffer, pellets were then extracted by additional of 2.0 mL ethanol. The samples were 

vortexed 3 times at 5 minute intervals, and centrifuged for 10 minutes at 600 × g. Each 

ethanol supernatant was then decanted into a scintillation vial, and the radiation was 

quantified by liquid scintillation counting. A total of two runs were performed; however, 

only one run was determined applicable as the [3H]-R1881 batch used in the first run was 

too old. A third run was not conducted due to the limited amount of cytosol. 

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

binding experiment is included in Table 3. 

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TABLE 3. Summary of Conditions for Competitive Binding Experiment a

Source of receptor Rat ventral prostate cytosol (P-10-1) 

Concentration of radioligand 1 nM 

Optimization of receptor concentration Sufficient to bind 17.6-18.7% of 1.0 nM 

radioligand b 

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

 M 

Incubation Temperature 6±2oC c

Incubation time 16-20 hours 

Composition of assay buffer Tris 10 mM (pH 7.4) 

EDTA 1.5 mM 

Glycerol 10% 

Sodium molybdate 1 μM 

Phenylmethylsulfonyl fluoride 1 mM 

DTT 1 mM 

Protease inhibitor 60 μM 

a Data were obtained from pages 16 and 18 of the study report. 

b Data obtained from page 22 of the study report; values were above the criterion of <10-15%. 

c Assay tubes were incubated at 6±2°C instead of 4±2°C (deviation from protocol, page 21); the slightly 

higher temperatures had no effect on the integrity of the study based on the results of the controls included 

in the assay. 

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

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

the binding of a single concentration of [3H]-R1881 (undiluted specific activity) to the AR 

in the presence of increasing concentrations of a test substance. 

DMSO was used as the solvent vehicle; precipitation was not addressed in the study report. 

Based on the results from the saturation binding experiment, the amount of cytosolic protein 

used for each incubation in the competitive binding experiments was 1.3 mg/assay tube, 

which bound 17.6-18.7% of the radiolabeled R1881 at 1 nM (pg 22 of study report). The 

ligand depletion was slightly greater than the ≤10-15% criteria. 

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 for each test substance at each concentration, for a 

total of 75 sample tubes. In addition, six replicates of the 1 nM solution of [3

H]-R1881 in 

ethanol and a single ethanol blank were run with each assay, resulting in a total of 82 

samples per run. 

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

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

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

1×10−7

 1×10−3 1×10−3

1×10−8 1×10−4 1×10−4

1×10−9 1×10−5 1×10−5

1×10−10 1×10−6 1×10−6

1×10−11 1×10−7 1×10−7

-- 1×10−8 1×10−8

-- 1×10−9 1×10−9

-- 1×10−10 1×10−10 

a Data were obtained from pages 18, 19 and 42-47 of the study report. 

b Each concentration of each chemical was run in triplicate for a total of 82 tubes per run. Tubes 1-75 contained 

50 μL of triamcinolone acetonide and 30 μL [3

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

were added. Tubes 1-75 also contained 10 μL of the solvent control, reference standard (non-radiolabeled R1881), weak positive control, or test substance, with the exception of Tubes 4-6 and 73-75 that contained 30 μL 

of non-radiolabeled R1881 (used to evaluate non-specific binding). Six un-numbered tubes containing only 30 

μL of [3

H]-R1881 (in ethanol) were not incubated and analyzed with an ethanol blank. 

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

R1881. 

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

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

H]-R1881 

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

performed as described. 

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

inhibition concentration (IC50) were calculated and a Scatchard plot was also plotted for the 

binding data (programs not specified). The competitive binding study reported that Prism 

version 4.01 (GraphPad Software, San Diego, CA) was used for nonlinear regression and 

statistical analysis.

5. Definitions 

a. Classification of test material 

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

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

bound. 

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

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

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

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

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

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Untestable: If the test compound is not soluble above 1×10−6 M and the binding curve does 

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

b. Descriptors for receptor binding 

Bmax: maximal binding capacity

Kd: dissociation constant

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 × 100 ÷ IC50 of test substance 

II. RESULTS 

A. SATURATION BINDING EXPERIMENT: Saturation binding experiment parameters 

are presented in Table 5. The dissociation constant (Kd) for [3H]-R1881 was 0.65 (±0.02), 

and the estimated Bmax was 2.58 fmol/100 μg protein (±0.02) for the single batch of prostate 

cytosol that was prepared. Only two runs were conducted due to the limited amount of 

cytosol and only one of the runs was determined acceptable. The goodness of fit (R2) was 

0.994 for the one acceptable run. The Kd was slightly lower than the range reported in the 

EPA validation program (0.685 to 1.57 nM), but the Scatchard plot was linear. 

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

Parameter Run 1 b Run 2 c Run 3 d Mean ± SEe

R2 (unweighted) NA 0.994 NA NA 

Bmax (nM) NA 0.112 ± 0.001 NA NA 

Bmax (fmol/100 μg protein) NA 2.58 ± 0.02 NA NA 

Kd (nM) NA 0.65 ± 0.02 NA NA 

a Data were obtained from Appendix 2, page 40 of the study report. 

b The first run was not used in evaluation of the results as the [3

H]-R1881 batch used for Run #1 was too old. 

c The mean and standard deviation are reported for the run. 

d A third run was not performed due to the limited amount of cytosol. 

e Not applicable because only one acceptable run was evaluated. 

R2

 Goodness of fit for curve calculated for specific binding. 

NA Not applicable

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

H]-R1881 to the 

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

less than 20% of total binding (4.2%). Figure 2 is a Scatchard plot that illustrates the 

binding of [3

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

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

Binding Experiment.

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

B. COMPETITIVE BINDING EXPERIMENT: Competitive binding experiment 

parameters are presented in Table 6. Dichlobenil reduced the mean specific binding of the 

radiolabeled ligand in a concentration dependent manner in all three runs. The maximum 

reduction in [3

H]-R1881 binding occurred at the highest concentration tested (10−3 M), with 

specific binding being reduced to 40-46%. Specific binding was also reduced at 10−4

 M 

(52-56%), but not at concentrations ≤10−5 M (86-103% binding). The results of each run are 

illustrated in Figure 3, and the average binding curve for dichlobenil across the three runs is 

shown in Figure 4. Dichlobenil was classified as a binder in each of the three runs. 

The estimated mean log IC50s were −3.85 M for dichlobenil, −8.73 M for R1881 and −4.58 

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

0.0013% for dichlobenil and 0.0071% for the weak positive control. Confidence in these 

numbers is high due to the small variation. The solvent control responses indicated no drift 

in the study assay. 

Page 120 of 331
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TABLE 6. Competitive Binding Assay of Dichlobenil with AR from Rat Prostate Cytosol a

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

R2 (unweighted) R1881 0.9982 0.9995 0.9986 0.9982-0.9995 

Dexamethasone 0.9938 0.9964 0.9969 0.9938-0.9969 

Dichlobenil 0.9639 0.9901 0.9859 0.9639-0.9901 

Log IC50 (M) R1881 −8.72 −8.75 −8.73 −8.73 ± 0.01 

Dexamethasone −4.53 −4.62 −4.60 −4.58 ± 0.03 

Dichlobenil −3.82 −3.82 −3.90 −3.85 ± 0.03 

IC50 (M) R1881 1.93 × 10−9 1.79 × 10−9 1.86 × 10−9 1.86 × 10−9 (±0.04) d

Dexamethasone 2.96 × 10−5 2.42 × 10−5 2.51 × 10−5 2.63 × 10−5 (±0.17) 

Dichlobenil 1.51 × 10−4 1.52 × 10−4 1.26 × 10−4 1.43 × 10−4 (±0.09) 

Log RBA d Dexamethasone -4.19 −4.13 −-4.13 −4.15 ± 0.02 

Dichlobenil -4.89 −4.93 −-4.83 −4.88 ± 0.03 

RBA (%) Dexamethasone 0.00652 0.00741 0.00742 0.00711 ± 0.0003 

Dichlobenil 0.00127 0.00118 0.00147 0.00131 ± 0.0001 

a Data were obtained from page 24, 26 and 29 of the study report.

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

c The range of r2 is reported and the mean and SE are reported for the other parameters. For means 

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

magnitude as the mean value. 

d Calculated by the study reviewer. 

SE Standard error 

NA Not applicable. 

R2

 Goodness of fit 

RBA Relative Binding Affinity RBA IC50 of R1881 ÷ IC50 of test substance 

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Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 11 of 14 

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

Compounds.

Page 122 of 331
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 12 of 14 

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

Dichlobenil in Three Competitive Binding Assays. 

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

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

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

Ligand depletion is minimal. The recommended ratio of 

total binding in the absence of competitor to total amount of 

[

3

H]-R1881 added per assay tube. 

≤15% 17.6 to 18.7 c X 

Dichlobenil Top (% binding) 80 to 115 98 to 100 X 

R1881 fitted curve parameters 

Top (% binding) 82 to 114 100 to 104 X 

Bottom (% binding) −2.0 to 2.0 −2.6 to −1.6 d X 

Hill Slope −1.2 to −0.8 −1.0 X 

Weak positive control (dexamethasone) fitted curve parameters 

Top (% binding) 87 to 106 99 to 104 X 

Bottom (% binding) −12 to 12 −2.7 to −1.3 X 

Hill Slope −1.4 to −0.6 −1.0 X 

Saturation Binding Experiment Kd (nM) 0.685 to 1.57 0.65 X 

Non-specific binding (%) ≤10.0 4.35 to 4.62 X 

a Data were obtained from pages 22, 24, 26, 29-30 and 40 of the study report.

b These values represent ranges from the validation study. 

c Ligand depletion was outside the limits in all three runs. 

d The bottom percent binding was outside the limits in one run. 

All performance criteria were met except that the ligand depletion (18-19%) was slightly 

greater than the ≤10-15% criteria, and the bottom percent binding for R1881 was low in one 

of the runs. Because the other parameters were within the tolerance limits and the IC50 for 

R1881 and dexamethasone were in accordance with values determined in the EPA 

validation study, these deviations are considered minor and likely had no effect on the 

results of the study. 

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Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 13 of 14 

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

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

indicated by a Hill slope of −1.0. Examination across the runs indicated consistency of the 

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

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Because the curve across runs crosses the 50% 

value, dichlobenil should be classified as a binder in the androgen receptor binding assay 

using rat prostate cytosol. The RBA of dichlobenil compared to R1881 (set at 100%) was 

0.00131 ± 0.0001% (mean ± SEM). The RBA of the weak positive control dexamethasone 

was 0.00712 ± 0.0003%.

B. AGENCY COMMENTS: Only one run was considered acceptable in the saturation 

binding experiment; the [3H]-R1881 batch was too old in the 1st run and a 3rd run was not 

performed due to limited quantities of cytosol. For the single acceptable run, the 

dissociation constant (Kd) for [3H]-R1881 was 0.65 (±0.02), and the estimated Bmax was 2.58 

fmol/100μg protein (±0.02) for the single batch of prostate cytosol that was prepared. The 

goodness of fit (R2

) was 0.994 for the one acceptable run. The Kd was slightly lower than 

the range reported in the EPA validation program (0.685 to 1.57 nM), but the Scatchard plot 

was linear. The specific binding reached a plateau, and non-specific binding was less than 

20% of total binding (4.2%). 

In the competitive binding experiment, dichlobenil reduced the mean specific binding of the 

radiolabeled ligand in a concentration-dependent manner in all three runs. The maximum 

reduction in [3H]-R1881 binding occurred at the highest concentration tested (10−3 M), with 

specific binding being reduced to 40-46%. Specific binding was also reduced at 10−4

 M 

(52-56%), but not at concentrations ≤10−5 M (86-103% binding). Dichlobenil is classified 

as a binder in the AR assay as specific binding was reduced to <50% at the highest 

concentration tested in all three runs. 

The estimated mean log IC50s were −3.85 M for dichlobenil, −8.73 M for R1881, and −4.58 

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

0.0013% for dichlobenil and 0.0071% for dexamethasone. Confidence in these numbers is 

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

assay. All performance criteria were met except that the ligand depletion was greater 

(~18%) than the ≤10-15% criteria, and the bottom percent binding for R1881 was low in one 

of the runs. Because the other parameters were within the tolerance limits and the IC50 for 

R1881 and dexamethasone were within the expected ranges demonstrating that the ligand 

was available at an appropriate level, the reviewers consider these minor deviations and find 

that the performance criteria were met. 

Although only one acceptable saturation binding assay was conducted and the Kd was low 

for that run, the androgen receptor was present in a reasonable concentration and 

functioning with appropriate affinity for the R1881 ligand in the competitive binding assays 

based on the performance criteria and estimated IC50s for R1881 and the weak positive 

control. It is unlikely that information from additional saturation binding runs would change 

the interpretation of this study. 

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

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

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

 Only one acceptable saturation binding assay was conducted and the Kd was low for that 

run. The ligand depletion in the competitive binding experiment was greater than the 

≤10-15% criteria for all three runs. 

 The bottom percent binding for R1881 was low in one of the competitive binding 

assays. 

Page 125 of 331
DATA EVALUATION RECORD 

DICHLOBENIL 

Study Type: OCSPP 890.1200, Aromatase Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-27-2012 (MRID 48616103) 

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:

Joan Harlin, M.S. Date: 3/27/2012 

Secondary Reviewer: Signature:

Scott D. Studenberg, Ph.D., D.A.B.T. Date: 4/02/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 4/09/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 4/09/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 126 of 331
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 127 of 331
DICHLOBENIL I 027401 

Primary Reviewer: Greg Akerman, Ph.D. 

Health Effects Division 

Secondary Reviewer: John Liccione, Ph.D. 

Health Effects Division 

Aromatase (Human Recombinant) Assay (20 l l) I Page l of 12 

OCSPP 890.1200 I OECD None 

Signature: .. ~ A 

Date: _ {;n) JL 

Signature~ Date: ,, i:m::= 

DATA EVALUATION RECORD 

STUDY TYPE: Aromatase (Human Recombinant); OCSPP 890.1200 

PC CODE: 027401 DP BARCODE: D397912 

TXR#: 0051691 CAS No.: 1194-65-6 

TEST MATERIAL (PURITY): Dichlobenil (99.3% a.i.) 

SYNONYMS: 2,6-dichlorobenzonitrile 

CITATION: Meerts I.A.T.M. (2011). Dichlobenil technical: Aromatase assay (human 

recombinant). NOTOX B.V., The Netherlands. Laboratory Report No.: 495780, 

December 8, 2011. MRID 48616103 . Unpublished. 

SPONSOR: Chemtura Corporation, 199 Benson Road, Middlebury, CT 

TEST ORDER#: EDSP-027401-120 

EXECUTIVE SUMMARY: In an in vitro aromatase (CYP 19) assay (MRID 48616103), 

dichlobenil (99.3%, Lot # AM9Kl2A222) was incubated with human recombinant aromatase 

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

log concentrations of 10-10 to 10-

3 M for 15 minutes to assess the potential of dichlobenil to 

inhibit aromatase activity. 

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

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

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

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

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

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

Aromatase activity in the full activity controls ranged from 0.4156 to 0.7358 nmol·mgprotein- 1·min- 1 for the three test runs, with a mean and standard deviation of 0.5750 ± 0.1208 

nmol·mg-protein- 1·min- 1. The response of each full activity control within a run was between 90 

to 110% of the mean. Activity in the background controls ranged from 0.06% to 0.39% and 

averaged 0.21 % of the full activity controls. For the positive control substance ( 4-0H ASDN), 

aromatase activity results were within the recommended ranges for the performance criteria. The 

estimated log ICso for 4-0H ASDN averaged - 7.34 Mand the Hill slope was - 0.94. 

Page 128 of 331
Aromatase (Human Recombinant) Assay (2011) / Page 2 of 12 

DICHLOBENIL / 027401 OCSPP 890.1200 / OECD None

Aromatase activity at the lowest and highest successfully tested concentrations of dichlobenil 

(10-10 and 10−4 M) were 99.6% and 87.5% of control. The highest tested concentration of 

dichlobenil (10−3 M) showed precipitation during the 15-minute incubation period; therefore, the 

results of the highest test concentration were not used for interpretation. 

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

aromatase activity in this assay. There is no indication in the study report that half-log 

concentrations (i.e., 10-3.5 M) were tested for solubility as recommended by the Test Guideline. 

At concentrations up to 10-4 M, dichlobenil would be classified as a non-inhibitor. However, as 

there is no indication in the study report that 10–3.5 M was evaluated for solubility, there is less 

confidence in the non-inhibitor classification. It should be noted that based on the shape of the 

response curve, if a soluble 10–3.5 M concentration had been tested, it is possible that the lowest 

portion of the curve would fall within the 50-75% activity range resulting in an equivocal 

classification for this chemical. 

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

890.1200). 

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

and Flagging statements were provided. 

Page 129 of 331
Aromatase (Human Recombinant) Assay (2011) / Page 3 of 12 

DICHLOBENIL / 027401 OCSPP 890.1200 / OECD None

I. MATERIALS AND METHODS

A. MATERIALS 

1. Test Substance: Dichlobenil technical 

 Description: White powder 

 Source: Chemtura Canada Co./Cie, Ontario, Canada 

 Lot # (expiration date): AM9K12A222 (October 12, 2012) 

 Purity: 99.3 Wt.% 

 Volatility: Not reported 

 Storage conditions: At room temperature in the dark 

 Stability: Stable 

 Solvent: Ethanol 

 Solubility (in test solvent): Soluble at 100 mM in ethanol 

 Highest Concentration Tested: 10−3 M 

 Stock Solution Preparation: 100 mM was freshly prepared in ethanol. 

 Molecular weight: 172.0 

 CAS #: 1194-65-6 

 Structure: 

2. Non-Labeled Substrate: Androstenedione (ASDN) 

 CAS # : 63-05-8 

 Source: Bio-Connect B.V., Huissen, The Netherlands (Catalog No. A637550) 

 Batch # (expiration date): 3-FJ-51-1 (January 06, 2012) 

 Purity: 98% 

3. Radiolabeled Substrate: 1-β [3

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

 Source: Perkin Elmer, Boston, MA 

 Batch # (expiration date): 619344 (February 14, 2012, allocated by NOTOX one year after receipt of the 

test substance) 

 Radiochemical Purity (Supplier): >97% 

 Specific activity: 0.974 TBq/mmol (26.3 Ci/mmol) 

 Radiochemical Purity (In-lab 

determination): 

100% 

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

 CAS # 566-48-3

 Source: Toronto Research Chemicals, Inc., North York, ON, Canada (Catalog No. 

F692250) 

 Batch # (expiration date): TRC-030508 (retest date: September 14, 2012) 

 Purity: 98% 

5. Solvent (Vehicle Control): Ethanol 

 Source: 

Batch # (expiration date): 

LiChrosolv, Merck, Darmstadt, Germany 

Not reported 

 Justification for choice of solvent One of the solvents of choice detailed in the EPA guideline 

 Concentration 

(% of total volume in assays) 

1% 

Page 130 of 331
Aromatase (Human Recombinant) Assay (2011) / Page 4 of 12 

DICHLOBENIL / 027401 OCSPP 890.1200 / OECD None

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

Source: BDGentest TM , Woburn, MA (Catalog # 456260) 

Lot # (expiration date): 40199 (not reported) 

Protein concentration: 7.6 mg/mL 

Cytochrome C reductase activity: 290 nmol /mg protein/min 

Aromatase activity: 6.1 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 0.3 

mM, and the amount of tritium added to each incubation tube was 0.1 μCi. 

Test chemical was formulated in ethanol such that the volume of ethanol used per assay was 

no more than 1% v/v of the total assay volume. A rationale for the selection of ethanol was 

not provided; however, dichlobenil is soluble in ethanol at the tested concentrations and is a 

solvent recommended by the EPA guidelines. 

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

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 5 μL of the dilution for a final 

assay volume of 1 mL. 

Human recombinant microsomes were purchased from BD GentestTM; microsomes were 

stored in aliquots at ≤−75oC for up to 2 years. Before use, the microsomes were quickly 

thawed in a 37 ± 1oC water bath, then placed in an ice bath and diluted in assay buffer up to 

a protein concentration of 8 μg/mL. Other assay components sodium phosphate buffer, 

propylene glycol, and NADPH are reported in Table 1. 

TABLE 1. Assay Components and Conditionsa

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 pages 16 and 17 of the study report. 

2. Suitability Assessments: The protein concentration of the microsomes (Lot #40199) was 

supplied by the vendor as 7.6 mg/mL. The microsomal protein concentrations determined 

on each experimental date were 3.57, 3.64, and 3.70 mg/mL (average 3.64 ± 0.06%) with a 

protein assay kit (Bio-Rad Laboratories, GmbH, Munich, Germany; Lot # 110253). 

Because these concentrations were approximately 48% lower than the protein concentration 

provided by the supplier, the measured protein concentrations were used in calculations in 

the study. Aromatase activity of the microsomes was also provided by the vendor as 6.1 

pmol/min/pmol P450. 

Page 131 of 331
Aromatase (Human Recombinant) Assay (2011) / Page 5 of 12 

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

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

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

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

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

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

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

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

4. Demonstration of Proficiency: The demonstration of laboratory proficiency for the 

aromatase assay was not described or reported. 

a. Positive Control

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

laboratory met the following criteria: 

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

 Mean background control activity was ≤ 10% of the full activity control. 

 The average of the background activity controls (n=4) within a run should fall within 

the range of −5 to 6% 

 The average of the full enzyme activity controls (n=4) within a run should fall within 

the range of 100± 10%. 

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

applicable): The historical positive control data for slope, top and bottom percent, and log 

IC50 generally met the criteria as listed in section (i) of OCSPP 890.1200 (Table 2). 

TABLE 2. Performance Criteria for the Positive Controla

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

Slope −1.2 −0.8 −1.2 −0.8 

Top (%) 90 110 90 111 

Bottom (%) −5 +6 −5 6 

Log IC50 (M) −7.3 −7.0 −7.5 −7.0 

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

b. Proficiency Chemicals: Assays to classify proficiency chemicals (econazole, fenarimol, 

nitrofen, and atrazine) were not performed. 

TABLE 3. Proficiency Chemicals 

Compound CAS# Class Concentrations 

Econazole 24169-02-6 Inhibitor No Data 

Fenarimol 60168-88-9 Inhibitor No Data 

Nitrofen 1836-75-5 Inhibitor No Data 

Atrazine 1912-24-9 Non-inhibitor No Data 

Page 132 of 331
Aromatase (Human Recombinant) Assay (2011) / Page 6 of 12 

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

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

tested during three independent runs (Table 4). Solubility was visually assessed (presence 

of cloudiness or a precipitate). Precipitation was observed at the highest test concentration 

(10-3 M); therefore, the results for this test concentration were not used. The full enzymatic 

activity (≥95% for the means of each run) was obtained in Runs 1 and 2, but not in Run 3, 

defining the top of the concentration-response curve. 

TABLE 4. Test Chemical Study Design for each Test Run

Sample Type Repetitions 

(Tubes) Description Reference or 

Chemical (M) 

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

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

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

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

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

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

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

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

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

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

Dichlobenil Conc 1c 3 All test components plus dichlobenil 1×10−3

Dichlobenil Conc 2c 3 All test components plus dichlobenil 1×10−4

Dichlobenil Conc 3c 3 All test components plus dichlobenil 1×10−5

Dichlobenil Conc 4c 3 All test components plus dichlobenil 1×10−6

Dichlobenil Conc 5c 3 All test components plus dichlobenil 1×10−7

Dichlobenil Conc 6c 3 All test components plus dichlobenil 1×10−8

Dichlobenil Conc 7c 3 All test components plus dichlobenil 1×10−9

Dichlobenil Conc 8c 3 All test components plus dichlobenil 1×10−10

a Data were obtained from pages 15 and 16 of the study report. 

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

c Test chemical. 

C. DATA ANALYSIS 

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

percent control for the positive control and test chemical. The following raw data and 

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

TABLE 5. Raw and Calculated Data

Raw/Calculated Data Included (X) 

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

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

Total DPM for each aqueous portion (after extraction) X 

The total DPM present in the assay tube at initiation X 

The percentage of substrate converted to product X 

Total DPM after extraction corrected for background X 

Aromatase activity expressed in nmol/mg protein/min X 

Average aromatase activity in the full activity control tubes X 

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

DPM Disintegrations per minute

Page 133 of 331
Aromatase (Human Recombinant) Assay (2011) / Page 7 of 12 

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2. Statistical Methods: Statistical analysis was performed on the data, which included means 

of replicates using the following software program: Prism version 4.0 (GraphPad Software, 

USA). 

 

The response curve was fitted by weighted nonlinear regression analysis (Prism version 4.0, 

GraphPad Software, USA). For each run, the individual percent of control values was 

plotted versus the logarithm of the test substance concentration or positive control 

concentration. The fitted concentration-response curve was presented in the plot. 

Individual plots were prepared for each test run. The average percentages of control values 

for each run were presented versus the logarithm of the test substance concentration in order 

to compare the percent control activity across runs. 

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 

0.4156 to 0.7358 nmol·mg-protein−1·min−1 for the 3 test runs, with a mean and standard 

deviation of 0.5750 ± 0.1208 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. Activity in the 

background controls ranged from 0.06% to 0.39% and averaged 0.21% of the full activity 

controls. 

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

activity averaged 0.4336-0.6879 nmol·mg-protein−1·min−1 at the lowest tested concentration 

10−10 M and 0.0030-0.0041 nmol·mg-protein−1

·min−1 at the highest tested concentration 10−5

M. These results were within the recommended ranges for the top of the curve, bottom 

curve, Hill slope, log IC50, and % CV for replicates of each concentration within runs. The 

variation in the positive control values was acceptable (≤6.6% CV). The mean aromatase 

activity of the positive control (expressed as % full control activity) for each concentration 

tested across all 3 runs is presented in Table 7, along with the overall standard deviation, 

SEM, and % CV. The inhibition response curves for the positive control are shown in 

Figure 1. 

Page 134 of 331
Aromatase (Human Recombinant) Assay (2011) / Page 8 of 12 

DICHLOBENIL / 027401 OCSPP 890.1200 / OECD None

TABLE 7. Effect of Dichlobenil on Aromatase Activity (as percent of control) from Independent Runsa

Chemical 

Concen. 

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

4-OH ASDN 

(positive control) 

−5 3 0.65 0.05 0.03 8% 

−6 3 5.68 0.60 0.35 11% 

−6.5 3 14.6 1.23 0.71 8% 

−7 3 34.9 1.26 0.73 4% 

−7.5 3 59.0 2.10 1.21 4% 

−8 3 84.8 0.80 0.46 1% 

−9 3 97.6 5.71 3.29 6% 

−10 3 101.8 4.28 2.47 4% 

Dichlobenil −3 b 3 76.1 5.30 3.06 7% 

−4 3 87.5 10.3 5.94 12% 

−5 3 99.6 9.28 5.36 9% 

−6 3 100.8 13.44 7.76 13% 

−7 3 101.9 13.3 7.68 13% 

−8 3 100.9 10.9 6.28 11% 

−9 3 101.8 14.8 8.51 14% 

−10 3 99.6 8.25 4.76 8% 

a Data were obtained from pages 33- 44 of the study report. Mean, SD, SEM, and CV were calculated by the reviewers. 

b Precipitation occurred at the 10-3 M concentration. 

SD Standard Deviation 

SEM Standard Error of the Mean 

CV Coefficient of Variance 

C. TEST SUBSTANCE: For dichlobenil, aromatase activity averaged 0.4693-0.6728 

nmol·mg-protein−1·min−1 at the lowest tested concentration 10−10 M and 0.4266-0.5839 

nmol·mg-protein−1·min−1 at the highest tested concentration 10−4 M that was used by the 

study author for interpretation. The highest tested concentration of dichlobenil (10−3

 M; 

0.3536-0.5097 nmol·mg-protein−1·min−1) was not used for interpretation as precipitation 

occurred during the 15-minute incubation period. The mean aromatase activity of 

dichlobenil (expressed as % full control activity) for each concentration tested across all 3 

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

% CV. The average inhibition response curve across all runs is shown in Figure 2. 

Page 135 of 331
Aromatase (Human Recombinant) Assay (2011) / Page 9 of 12 

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FIGURE 1. Inhibition Response Curves for 4-OH ASDN From Each Test Run. 

Page 136 of 331
Aromatase (Human Recombinant) Assay (2011) / Page 10 of 12 

DICHLOBENIL / 027401 OCSPP 890.1200 / OECD None

FIGURE 2. Mean Inhibition Response Curves for Dichlobenil from Each Test Run. 

Page 137 of 331
Aromatase (Human Recombinant) Assay (2011) / Page 11 of 12 

DICHLOBENIL / 027401 OCSPP 890.1200 / OECD None

The average dose-response curve indicated that the aromatase activity of dichlobenil at all 

concentrations tested was essentially equivalent 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 dichlobenil. For 4-OH 

ASDN, the estimated log IC50 averaged −7.34 M and the slope was −0.94 (Table 8). The 

variation in the positive control values was acceptable (<15% CV). 

TABLE 8. Effect of Dichlobenil on Aromatase Activity (as Percent of Control) From Independent Runsa

Chemical Run 1 Run 2 Run 3 Meanb SDb % CVb

Log IC50 (M) 

Dichlobenil NA NA NA NA NA NA 

4-OH ASDN -7.3 -7.4 -7.3 -7.3 0.1 -1 

Slope 

Dichlobenil NA NA NA NA NA NA 

4-OH ASDN -0.96 -0.94 -0.92 -0.94 0.02 -2 

a Data were obtained from Page 9 of the study report 

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

NA Not applicable 

SD Standard Deviation 

CV Coefficient of Variance 

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

the results support the conclusion that dichlobenil does not inhibit catalytic activity in the 

aromatase assay. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS CONCLUSIONS: It was not possible to calculate the mean log IC50 

for dichlobenil, as enzyme activity in the presence of dichlobenil ranged from 71.3% to 

123.6% of control in individual assay runs and bore no relationship to dichlobenil 

concentration. The positive control 4-OH ASDN was calculated to have a mean log IC50 of 

−7.3 M. The mean slope of the 4-OH ASDN dose response curve was within the range 

documented in the EPA Guideline. It can be concluded that all criteria of this aromatase 

assay were met, and that Dichlobenil can be classified as a non-inhibitor in the aromatase 

assay. 

B. AGENCY COMMENTS: Although data demonstrating proficiency with the positive 

control or recommended proficiency chemicals were not included, historical performance 

criteria data for the positive control were included. 

Aromatase activity in the full activity controls was 0.5750 ± 0.1207 nmol·mgprotein−1

·min−1. The response of each full activity control within a run was between 90 to 

110% of the mean. Activity in the background controls ranged from 0.06% to 0.39% and 

averaged 0.21% of the full activity controls. 

For the positive control substance (4-OH ASDN), aromatase activity results were within the 

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

Page 138 of 331
Aromatase (Human Recombinant) Assay (2011) / Page 12 of 12 

DICHLOBENIL / 027401 OCSPP 890.1200 / OECD None

The estimated log IC50 for 4-OH ASDN averaged −7.34 M and the slope was −0.94. The 

variation in the positive control values was acceptable (≤6.6% CV). 

For dichlobenil, aromatase activity at the lowest and highest successfully tested 

concentration, 10−10 and 10−4

 M, were 99.6% and 87.5% of control. The highest test 

concentration of dichlobenil (10−3

 M) showed precipitation in the incubation during the 15-

minute incubation period; therefore, the results of the highest test concentration were not 

used for interpretation. These data could not be fit to the 4-parameter nonlinear regression 

model. 

Because the average lowest portion of the curve at the concentrations of 10−4 M was >75% 

in all experiments, dichlobenil is classified as a non-inhibitor of aromatase activity in this 

assay. However, as there is no indication in the study report that 10–3.5 M was evaluated for 

solubility, there is less confidence in the non-inhibitor classification. It should be noted that 

based on the shape of the response curve, if a soluble 10–3.5 M concentration had been 

tested, it is possible that the lowest portion of the curve would fall within the 50-75% 

activity range resulting in an equivocal classification for this chemical. 

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 initial positive control proficiency test data and proficiency test chemical assay 

results were not provided other than historical performance criteria data for the positive 

control. 

 The mean, standard deviation, and % coefficients of variation were not provided for the 

positive control data for the Hill Slope and log IC50. The reviewers were able to calculate 

these values from the individual data. 

 For dichlobenil, the highest test concentration of 10-3 M was not used for interpretation in 

the aromatase assay results due to the presence of precipitation. Therefore, only seven 

test concentrations were used for interpretation. A mid-log concentration (i.e., 10-4.5 M) 

should have been added in order to obtain the required eight test concentrations, as noted 

in section (j) of OCSPP 890.1200. 

Page 139 of 331
DATA EVALUATION RECORD 

DICHLOBENIL 

Study Type: OCSPP 890.1250, Estrogen Receptor Binding Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-27-2012 (MRID 48616104) 

Prepared for 

Health Effects Division 

Office of Pesticide Programs 

U.S. Environmental Protection Agency 

2777 South Crystal Drive 

Arlington, VA 22202 

Prepared by 

CSS-Dynamac Corporation 

1910 Sedwick Road, 

Building 100, Suite B 

Durham, NC 27713 

Primary Reviewer: Signature:

Michelle Sharpe-Kass, M.S. Date: 3/21/2012 

Secondary Reviewer: Signature:

Scott D. Studenberg, Ph. D., 

D.A.B.T. Date: 3/26/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 4/04/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 4/04/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 140 of 331
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 141 of 331
DICHLOBENIL/ 027401 

Primary Reviewer: Ray Kent, Ph.D. 

Health Effects Division 

Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) I Page I of 15 

OCSPP 890.1250/ OECD None 

Health 

Secondary 

Effects 

Reviewer: 

Division 

Greg Akerman, Ph.D. :::::~::f Date: 

~ <:; / ~~~ 'lC/ I 1 rTemplate version 08/2011 

DATA EVALUATION RECORD 

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

OCSPP 890.1250 

PC CODE: 027401 DP BARCODE: D397912 

TXR#: 0051691 CAS No.: 1194-65-6 

TEST MATERIAL (PURITY): Dichlobenil (99.3%) 

SYNONYMS: 2,6-dichlorobenzonitrile 

CITATION: Meerts, I.A.T.M. (2011). Dichlobenil Technical: Estrogen Receptor Binding 

Using Rat Uterine Cytosol (ER-RUC). NOTOX B.V., Hertogenbosch, The 

Netherlands. Laboratory Report No.: 495781 , December 9, 2011. MRID 

48616104. Unpublished 

SPONSOR: Chemtura Corporation, 199 Benson Road, Middlebury, CT. 

TEST ORDER#: EDSP-027401-120 

EXECUTIVE SUMMARY: In an estrogen receptor (ER) binding assay (MRID 48616104), 

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

binding and competitive binding experiments. The saturation binding experiment was conducted 

to demonstrate that the ER in the rat uterine cytosol was present in reasonable numbers and was 

functioning with appropriate affinity for the radiolabeled reference estrogen prior to routinely 

conducting ER competitive binding experiments. The competitive binding experiment was 

conducted to measure the binding of a single concentration of [3H]-17P-estradiol (1 nM) in the 

presence of increasing concentrations ( 10- 10 to 10-3 M) of dichlobenil (99.3% a.i., Batch 

#AM9K12822). DMSO was used as the solvent vehicle at a final concentration of 2%. Three 

independent runs were performed, and each run included 19-norethindrone as a weak positive 

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

reference material. 

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

binding assay was determined to be 40 μg/tube, as it bound 30.2% of the [3H]-17P-estradiol. The 

mean Kct for [3H]-17P-estradiol was 0.12 nM and the mean Bmax was 57.19 fmol/100 μg protein 

for the prepared rat uterine cytosol. The data produced consistent and linear Scatchard plots (r2 

>0.98). In the competitive binding experiments, the reference standard (E2), the weak positive 

control (19-nonethindrone), and the negative control (octyltriethoxysilane) each exhibited the 

expected responses. The estimated mean log ICsos were - 9.08 and - 5.87 M for E2 and 19-

Page 142 of 331
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 2 of 15 

DICHLOBENIL/ 027401 OCSPP 890.1250/ OECD None

norethindrone, respectively, and the relative binding affinity (RBA) was 0.062% for 19-

norethindrone, compared to the natural ligand. 

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

showed minimal displacement (93-104% binding) of radiolabeled estradiol at dichlobenil 

concentrations up to 10−3 M; therefore an IC50 and RBA could not be determined for dichlobenil. 

Based on the results from the three competitive runs, dichlobenil is classified as Not Interactive 

in the Estrogen Receptor Binding Assay. 

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

Assay (OCSPP 890.1250).

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

statements were provided. 

Page 143 of 331
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 3 of 15 

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I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: NOTOX B.V. 

Location: Hertogenbosch, The Netherlands 

Study Director: I.A.T.M Meerts 

Other Personnel: M.A. Thomson, Study Monitor 

M. Mulder, Technical Coordinator 

Study Period: June 14, 2011 – December 9, 2011 

2. Test substance: Dichlobenil 

 Description: Technical, white powder with lumps

 Source (cat #): NOTOX (202841/A) 

 Lot/Batch # (Exp date): AM9K12A222 (October 12, 2012) 

 Purity: 99.3 % 

 Solubility: 50 mg/mL in DMSO 

 Volatility: Not Reported (NR) 

 Stability: Stable 

 Storage conditions: Room temperature in the dark 

 CAS #: 1194-65-6 

 Molecular weight: 172.0 

 Structure: 

3. Non-labeled ligand: 17β-estradiol 

 Supplier: NR 

 Catalog #: NR 

 Batch #: NR 

 Purity: NR 

 CAS #: 50-28-2 

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

H]-17β-estradiol 

 Supplier: NOTOX 

 Catalog #: 202254/B 

 Batch#: 650702 

 Radiochemical purity: >97% 

 Specific activity: 4.817 TBq/mmol 

 Concentration of stock: 37MBq/mL 

5. Positive control: 19-norethindrone 

 Supplier: NR 

 Catalog #: NR 

 Batch #: NR 

 Purity: NR 

 CAS #: 68-22-4 

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6. Negative control: Octyltriethoxysilane 

 Supplier: NR 

 Catalog #: NR 

 Batch #: NR 

 Purity: NR 

 CAS #: 2943-75-1 

7. Solvent/vehicle control: DMSO 

 Justification for choice of 

solvent: 

Not reported 

 Final Concentration: 2% 

B. METHODS 

1. Preparation of Rat Uterine Cytosol (RUC): A total of 20 female Sprague Dawley rats 

were ovariectomized, 17 of which survived the ovariectomy. The surviving animals were 

euthanized 10 days later, on post-natal day 88. The uteri were weighed, flash frozen using 

liquid nitrogen and stored at ≤-75°C for four months. Prior to isolation of the uterine 

cytosol, the uteri were thawed on ice for no more than 60 minutes. Uteri were homogenized 

in ice-cold TEDG (Tris, EDTA, DTT, glycerol) + PMSF (phenylmethylsulfonyl fluoride) 

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

centrifuged for an additional 60 minutes at 105000 × g, discarding the resulting pellet. 

Protein concentration of the cytosol was determined to be 1.75 mg/mL using a protein kit 

compatible with DTT in the TEDG buffer. Cytosol was divided into aliquots and stored at 

≤-75°C for a maximum of 33 days (Batch U-10-1).

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 ER was present in reasonable concentrations and had the appropriate 

affinity for the native ligand. The conditions for the saturation binding are summarized in 

Table 1. 

TABLE 1. Summary of Conditions for Saturation Binding Experiment a

Source of receptor Rat uterine cytosol (Batch U-10-1) 

Concentration of radioligand (as serial dilutions) 0.03-3.0 nM 

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

Concentration of receptor 40 μg/tube. Sufficient to bind 30.2% of 

radioligand at 0.03 nM 

Temperature 4±2 oC 

Incubation time 16-20 hours 

Composition of assay buffer Tris 10 mM (pH 7.4) 

EDTA 1.5 mM 

Glycerol 10% 

Phenylmethylsulfonyl fluoride 1 mM 

DTT 1 mM 

a Data were obtained from page 40 of the study report. 

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

adjusted for decay over time, and serial dilutions in TEDG + PMSF buffer were prepared to 

achieve the final concentrations of 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1.0 and 3.0 nM. Solutions 

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

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

concentrations of 3, 6, 8, 10, 30, 60, 100 and 300 nM. For each batch of cytosol, the method 

for determining optimal protein concentration was not reported, but 40μg/tube bound 30.2% 

of radioligand at 0.03 nM. Each assay consisted of three non-concurrent runs, and each run 

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

depicted in Table 2. 

TABLE 2. Saturation Binding Experiment Run a

Total binding b Non-specific 

binding c

Radioligand alone d

 Assay Components 

Tubes 1-24 Tubes 25-48 Tubes 49-72 

350 μL 300 μL --- TEDG + PMSF buffer 

50 μL 50 μL 50 μL [

3

H]-17β-estradiol (8 serial dilutions) e

--- 50 μL --- Non-labeled 17β-estradiol (8 serial dilutions, 

100x each respective labeled concentration) f

100 μL 100 μL --- Uterine cytosol (diluted to appropriate conc.) 

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

a Data were obtained from page 40 of the study report. 

b Total binding = [3

H]-17β-estradiol bound to ER 

c Non-specific binding = [3

H]-17β-estradiol and 100-fold greater non-labeled bound to ER 

d Total [3

H]-17β-estradiol alone for dpm determination at each concentration 

e Final concentrations of [3

H]-17β-estradiol = 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1, and 3 nM. 

f Final concentrations of non-labeled 17β-estradiol = 3, 6, 8, 10, 30, 60, 100, and 300 nM. 

Tubes were incubated with gentle vortexing for 16-20 hours at 4±2 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: TEDG +PMSF buffer was added, vortexed, centrifuged for 10 min at 

1000 x g, and the supernatant decanted and discarded. After washing, ethanol was added to 

the HAP pellet remaining in each tube to extract the [3

H]-17β-estradiol, followed by 

vortexing, and centrifugation for 10 min at 1000 x g. An aliquot of supernatant was 

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

the assay prior to extraction with ethanol. 

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

Competitive Binding Experiment is included in Table 3. 

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TABLE 3. Summary of Conditions for Competitive Binding Experiment a

Source of receptor Rat Uterine Cytosol (Batch U-10-1) 

Concentration of radioligand 1 nM 

Concentration of receptor Sufficient to bind 5.90-6.54% of 

radioligand

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

Temperature 4±2 oC 

Incubation time 16-20 hours 

Composition of assay buffer Tris 10 mM (pH 7.4) 

EDTA 1.5 mM 

Glycerol 10% 

Phenylmethylsulfonyl fluoride 1 mM 

DTT 1 mM 

a Data were obtained from pages 18-20 and 44-52 of the study report. 

A solubility test was not performed for dichlobenil. On the day of the assay, the specific 

activity of the stock solution [3H]-17β-estradiol was adjusted for decay over time, and 

diluted in TEDG + PMSF buffer to achieve a final concentration of 1 nM. For each batch of 

cytosol, the optimal protein concentration was determined by testing serial amounts of 

protein per tube, using 1.0 nM radiolabeled estradiol, until a concentration was reached that 

bound 5.9-6.54% of the total radioactivity added. Serial dilutions of the test substance, 

weak 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 runs, and each run contained three 

replicates at each concentration, resulting in a total of 99 samples. 

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

Test substance Positive control Negative control Reference Chemical 

19-norethindrone Octyltriethoxysilane Non-labeled 

17β-estradiol 

Tubes 73-96 c Tubes 49-72 c Tubes 25-48 c Tubes 1-24, 97-99 c

10−10 10−8.5 10−10 Solvent control d

10−9 10−7.5 10−9 10−11

10−8 10−7 10−8 10−10

10−7 10−6.5 10−7 10−9.5

10−6 10−6 10−6 10−9

10−5 10−5.5 10−5 10−8.5

10−4 10−4.5 10−4 10−8

10−3 10−4 10−3 10−7

a Data were obtained from pages 44-52 of the study report. 

b Each tube contains: 10μL of either the test substance, positive control, negative control, solvent control, or 

non-labeled 17β-estradiol; 390 μL of TEDG + PMSF buffer with [3

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

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

c Each concentration of each chemical was run in triplicate, for a total of 99 tubes per run. 

d Solvent is DMSO (2%) 

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

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

H]-17βestradiol were similar to those used in the saturation binding experiment. 

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C. DATA ANALYSIS: For the saturation binding experiment, total binding and non-specific 

binding data were modeled via non-linear regression using Graph Pad Prism v. 4.01 

(GraphPad Software, Inc., San Diego, CA). For the competitive binding assay, similar 

methods of nonlinear regression were used to fit a curve (for 17β-estradiol, the weak 

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 (Microsoft Corporation, Redmond, WA).

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 10-6M and either a binding curve can be fit but 

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

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

concentration groups in the data is >50%. 

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

above. 

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

Run Classification Numerical Value 

Interactive 2 

Equivocal 1 

Not interactive 0 

Equivocal up to the limit of concentrations tested “missing” 

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

following ranges: 

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Test Material Classification Numerical Range 

Interactive average ≥1.5 

Equivocal 0.5≥ average <1.5 

Not interactive average <0.5 

Equivocal up to the limit of concentrations tested “missing” 

b. Descriptors for receptor binding: 

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

concentration of active receptor sites

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

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

the receptor

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

II. RESULTS 

A. SATURATION BINDING EXPERIMENT The appropriate concentration for the 

saturation binding assay was determined to be 40 μg/tube, as it bound 30.2% of the [3H]-

17β-estradiol (within the Guideline range of 25-35% binding). The results from the three 

saturation binding assays are summarized in Table 5 and shown graphically in Figures 1 and 

2. The mean Kd for [3H]-17β-estradiol was 0.12 nM and the Bmax was 57.19 fmol/100 μg 

protein for the prepared rat uterine cytosol. The Kd for each run was within the expected 

range of 0.03 to 1.5 nM. The data produced consistent and linear Scatchard plots (r2

 = 

0.9830-0.9942). 

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

from Rat Uterine Cytosol a

Parameter Run 1 Run 2 Run 3 Mean ± SE 

r

2

 (unweighted) 0.9942 0.9830 0.9894 0.9830-0.9942 b

Bmax (nM) 0.048 0.044 0.045 0.458 ± 0.001 

Bmax (fmol/100 μg protein) 60.34 55.48 55.75 57.19 ± 1.58 

Kd (nM) 0.144 0.092 0.125 0.120 ± 0.015 

a Data were obtained from page 41 of the study report. 

b The r2

 is reported as a range, 

r

2

 Goodness of fit for curve calculated for specific binding

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FIGURE 1. Binding of [3H]-17β-estradiol to the Estrogen Receptor during the Saturation 

Binding Experiment. 

FIGURE 2. Scatchard Plot of the Binding of [3H]-17β-estradiol to the Estrogen Receptor. 

B. COMPETITIVE BINDING EXPERIMENT The results from the three competitive 

binding runs are summarized in Table 6 and presented graphically in Figures 3-5. The 

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parameters determined from the competitive binding curves (r2, IC50, log IC50, and RBA) are 

presented in Table 7. 

 Precipitation of dichlobenil was not observed at any concentration tested. In all three runs, 

dichlobenil showed minimal displacement of [3

H]-E2 from the receptor at concentrations up 

to 10−3

 M (93-104% binding). As the percent specific binding was not decreased to ≤50%, 

an IC50 and RBA were not calculated for dichlobenil. The E2 reference standard and the 

weak positive control produced the expected binding results in all three runs, with r2 values 

of >0.99 for each run. The estimated mean log IC50 was −9.08 and −5.87 M for E2 and 19-

nonethindrone, respectively, and the RBA was 0.062% for 19-norethindrone compared to 

E2. The negative control had no effect on binding. 

 Confidence in these numbers is high due to the small variation. As dichlobenil did not 

decrease specific binding for [3H]-E2 by >25% in each run at concentrations up to 10−3 M, it 

is classified as not interactive in this assay (Table 8). 

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TABLE 6. Percent Specific Binding of [3

H]-17β-estradiol in Competitive Binding Assay a 

Test Chemical 

Concentration 

(M) 

% Specific Binding 

Run 1 Run 2 Run 3 Mean b SE b 

17β-estradiol (E2) 

(reference standard) 

10−8 7.05 8.26 7.57 7.63 0.35 

10−8.5 19.73 18.56 22.88 20.39 1.29 

10−9 39.63 47.33 48.86 45.27 2.86 

10−9.5 71.18 74.31 75.64 73.71 1.32 

10−10 85.69 92.70 89.77 89.39 2.03 

10−11 98.32 102.40 99.22 99.98 1.24 

19-nonethindrone 

(weak positive 

control) 

10−4

 0.33 -0.42 0.20 0.04 0.23 

10−4.5 3.75 1.37 3.29 2.80 0.73 

10−5.5 31.08 31.18 30.73 31.00 0.14 

10−6 59.02 54.62 58.12 57.25 1.34 

10−6.5 81.55 73.77 79.44 78.25 2.32 

10−7 91.30 85.11 89.32 88.58 1.83 

10−7.5 96.34 87.59 93.02 92.32 2.55 

10−8.5 99.99 91.17 93.59 94.92 2.63 

Dichlobenil 

(test substance) 

10−3 93.59 98.23 94.73 95.52 1.40 

10−4 98.47 94.22 97.76 96.82 1.31 

10−5 97.02 92.94 99.08 96.35 1.80 

10−6 100.56 94.40 104.00 99.65 2.81 

10−7 102.46 96.48 102.66 100.53 2.03 

10−8 101.77 92.24 97.61 97.21 2.76 

10−9 102.70 91.03 102.78 98.84 3.90 

10−10 98.42 82.66 101.13 94.07 5.76 

Octyltriethoxysilane 

(negative control) 

10−3

 89.54 94.15 89.96 91.22 1.47 

10−4 85.19 92.58 84.18 87.32 2.65 

10−5 96.03 103.82 94.80 98.22 2.82 

10−6 97.05 104.31 93.50 98.29 3.18 

10−7 94.89 104.10 95.49 98.16 2.98 

10−8 96.29 106.84 97.33 100.15 3.36 

10−9 96.26 103.73 96.53 98.84 2.45 

10−10 100.56 104.45 95.18 100.06 2.69 

a Data were obtained from pages 45-52 of the study report. 

b Calculated by reviewer 

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FIGURE 3. Percentage E2 Bound to the Estrogen Receptor in the Presence of 

Dichlobenil. Run 1

FIGURE 4. Percentage E2 Bound to the Estrogen Receptor in the Presence of 

Dichlobenil. Run 2.

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FIGURE 5. Percentage E2 Bound to the Estrogen Receptor in the Presence of 

Dichlobenil. Run 3.

TABLE 7. Competitive Binding Assay of Dichlobenil with Estrogen Receptor from Rat Uterine Cytosol a

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

r

2

 (unweighted), 17β-estradiol 0.9966 0.9977 0.9977 0.9966-0.9977 

19-norethindrone 0.9985 0.9979 0.9969 0.9969-0.9985 

Dichlobenil NA NA NA NA 

Log IC50 (M) 17β-estradiol −9.15 −9.07 −9.02 −9.08 ± 0.04 

19-norethindrone −5.84 −5.90 −5.86 −5.87 ± 0.02 

Dichlobenil NA NA NA NA 

IC50 (M) 17β-estradiol 7.1 × 10−10 8.5 × 10−10 9.6 × 10−10 8.4 × 10−10 (± 0.72) 

19-norethindrone 1.44 × 10−6

 1.26× 10−6 1.38× 10−6 1.36× 10−6 (± 0.05) 

Dichlobenil NA NA NA NA 

Log RBA (%)c 19-norethindrone −1.31 −1.17 −1.16 -1.21 ± 0.05 

Dichlobenil NA NA NA NA 

RBA (%)c, 19-norethindrone 0.049 0.067 0.070 0.062 ± 0.006 

Dichlobenil NA NA NA NA 

a Data were obtained from pages 26, 30 and 33 of the study report. 

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

c The range of r2 is reported and the mean and SE are reported for the other parameters. Calculated by 

reviewers. For means expressed in scientific notation, the SE values in parentheses are presented in the 

same order of magnitude as the mean value. 

NA Not applicable. 

r

2

 Goodness of fit 

RBA (%) Relative binding affinity 

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TABLE 8. Binding Classification of Dichlobenil with Estrogen Receptor a

Run 1 2 3 Mean c Binding Classification d

Classification category value b 0 0 0 0 Not interactive 

a Data were obtained from page 11 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 

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

properly, each run was evaluated using the following criteria in Table 9. 

TABLE 9. Criterion a Tolerance 

Limit(s) Value Yes No 

17β-estradiol fitted curve parameters 

Loge residual SD ≤2.35 ≤2.22 X 

Top (% binding) 94 to 111 100 to 102 X 

Bottom (% binding) -4 to 1 -2.1 to 3.0 X 

(Hill) Slope (log10(M)−1) -1.1 to -0.7 -1.1 to -1.0 X 

Weak Positive control (19-) fitted curve parametersb

Loge residual SD NA ≤2.25 X 

Top (% binding) NA 91 to 99 X 

Bottom (% binding) NA -3.6 to -0.3 X 

(Hill) Slope (log10(M)−1) NA -1.0 to -0.9 X 

Solvent concentration

DMSO ≤3% 2% X 

Negative control (octyltriethoxysilane) does not displace more than 

25% of [3

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

concentrations 

≤25% ≤13% X 

a Data were obtained from pages 26, 28, 30 and 33 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 norethindrone, which cannot be obtained commercially. The values reported were considered 

acceptable as they show 19-norethindrone to be an acceptable weak positive control.

NA Not applicable 

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 Hill Slope of approximately -1.0 

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

The percent binding at this top plateau 95% to 101% was within 25 percentage points of the 

value for the lowest concentration of the estradiol standard (100%). Examination of the 

reference standards 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: Dichlobenil technical did not show displacement 

of [3H]-E2 from the receptor at all concentrations tested. The lowest point on the fitted 

response curve was above 75% for all experiments. The classification category value for 

dichlobenil technical is 0, indicating that dichlobenil is not interactive with the estrogen 

receptor. 

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

concentration for the saturation binding assay was determined to be 40 μg/tube, as it bound 

30.2% of the [3

H]-17β-estradiol. The mean Kd for [3H]-17β-estradiol was 0.12 nM and the 

mean Bmax was 57.19 fmol/100 μg protein for the prepared rat uterine cytosol. The Kd for 

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

and linear Scatchard plots (r2

 = 0.9830-0.9942). 

In the competitive binding experiments, the reference standard (E2), the weak positive 

control (19-nonethindrone), and the negative control (octyltriethoxysilane) each exhibited 

the expected responses. The estimated mean log IC50 was −9.08 and −5.87 M for E2 and 19-

nonethindrone, respectively, and the RBA was 0.062% for 19-norethindrone. 

Precipitation of dichlobenil was not observed at any concentration. Dichlobenil was tested 

over a concentration range that fully defined the top of the curve, and the maximum percent 

binding with dichlobenil (95-101%) was within 25 percentage points of the lowest 

concentration of the estradiol standard, 100%. In all three runs, dichlobenil showed 

minimal displacement of the radioactive ligand at concentrations up to 10−3 M (93-104% 

binding); therefore an IC50 and RBA could not be determined for dichlobenil. 

 As dichlobenil did not decrease specific binding by >25% in each run at concentrations up 

to 10−3 M, it is classified as not interactive in 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: 

 One performance criterion was not met for 17β-estradiol in the competitive binding 

assay. The bottom binding was 3.0%, which is higher than the Guideline -4 to 1%. This 

does not negatively impact the interpretation of the study. 

Page 156 of 331
DATA EVALUATION RECORD 

DICHLOBENIL 

Study Type: OCSPP 890.1300, Estrogen Receptor Transcriptional Activation 

EPA Contract No. EP10H001452 

Task Assignment No. 2-27-2012 (MRID 48616105) 

Prepared for 

Health Effects Division 

Office of Pesticide Programs 

U.S. Environmental Protection Agency 

2777 South Crystal Drive 

Arlington, VA 22202 

Prepared by 

CSS-Dynamac Corporation 

1910 Sedwick Road, 

Building 100, Suite B 

Durham, NC 27713 

Primary Reviewer: Signature:

Michelle Sharpe-Kass, M.S. Date: 3/20/2012 

Secondary Reviewer: Signature:

Scott D. Studenberg, Ph. D., 

D.A.B.T. Date: 3/26/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 4/09/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 4/09/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 157 of 331
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 158 of 331
DICHLOBENIL /02740 1 

Estrogen Receptor Transcriptional Activation (2011) I Page I of 11 

OCSPP 890.1300/ OECD 455 

Primary 

Health Effects 

Reviewer: 

Division 

Ray Kent, Ph.D. SignaDtuac;-t -ee:: ~~; _ _ _____L__ 

Secondary Reviewer: _ _cM=..c=i-=ne=r'--'-v=a-"M==er:....::c=a=do=,'--'P'"-'h=·=D...:.... ____ Signature: 

Health Effects Division Date: - 2q-;sTemplate version 08/2011 

DAT A EVALUATION RECORD 

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

OCSPP 890.1300; OECD 455 . 

PC CODE: 027401 DP BARCODE: D397912 

TXR#: 0051691 CAS No.: 1194-65-6 

TEST MATERIAL (PURITY): Dichlobenil (99.3% a.i.) 

SYNONYMS: 2,6-dichlorobenzonitrile 

CITATION: Meerts, I.A.T.M. (2011) Dichlobenil Technical: Estrogenic Receptor 

Transcriptional Activation Assay (Human Cell Line (HeLa-9903)). NOTOX 

B.V, Hertogenbosch, The Netherlands. Laboratory Report No.: 495782, 

December 9, 2011. MRID 48616105 . Unpublished. 

SPONSOR: Chemtura Corporation, 199 Benson Road, Middlebury, CT. 

TEST ORDER#: EDSP-027401-120 

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

48616105), hERa-HeLa-9903 cells cultured in vitro were exposed to dichlobenil (99.3% a.i., 

Batch # AM9K 128222) at logarithmically increasing concentrations from 1 x 10-9 to 2.5 x l 0-4 

Min DMSO (0.1 %). The experiments were performed using 96-well plates and each dichlobenil 

concentration was tested in triplicate (3 wells/plate). Cells were exposed to the test agent for 20-

24 hours to determine if reporter (luciferase) gene products were induced. Luciferase expression 

in response to activation of the estrogen receptor was measured upon addition of a luciferase 

substrate and detection with a luminometer with acceptable sensitivity. A total of four 

independent runs of the assay were conducted. Laboratory proficiency was demonstrated by 

testing the 10 chemicals listed in the test guideline. 

Dichlobenil was tested up to the limit of solubility, 2.5 x 10-4 M. The mean RPCMax for 

dichlobenil was 0.9% to 5.1 %, and the associated PCMax was 10-8 or 1 o-6 M. Performance criteria 

were met for 17~-estradiol , l 7a-estradiol and corticosterone. For l 7a-methyltestosterone, the 

Log PC so was not calculable for Run 1, Run 2 and Run 4, and the Log PC 1 o was higher than the 

expected range for all four Runs. The responsiveness of the cells to the very weak positive 

control 17a-methyltestosterone 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, dichlobenil responses were similar to those of the negative 

control corticosterone and not comparable to the responses of l 7a-methyltestosterone, which 

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Estrogen Receptor Transcriptional Activation (2011) / Page 2 of 11 

DICHLOBENIL /027401 OCSPP 890.1300/ OECD 455 

was able to reach a maximum of 34-51% PC. Dichlobenil was only able to reach a maximum of 

0.9-5.1% PC when tested up to the highest concentration possible based on cytotoxicity. 

Because the RPCMax < PC10 in both assay runs, dichlobenil was considered negative for estrogen 

receptor transcriptional activation in this test system. 

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

Transcriptional Activation assay (OCSPP 890.1300). 

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

statements were provided.

 

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DICHLOBENIL /027401 OCSPP 890.1300/ OECD 455 

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: Dichlobenil 

 Description: Technical, white powder, MW 172.0

 Source: NOTOX 202841/A 

 Lot/Batch #: Batch # AM9K12A222 

 Purity: 99.3% 

 Solubility: DMSO; 50 mg/mL 

 Volatility: Not reported (NR) 

 Stability: Stable (stability in DMSO not indicated) 

 Storage conditions: Room temperature in the dark 

 Vapor pressure: NA 

 CAS #: 1194-65-6 

 Structure: 

2. Reference substances 

 17β-estradiol (strong estrogen; positive control)

 Supplier: Sigma, Zwijndrecht, The Netherlands 

 Catalogue and Batch #: NR 

 Purity: NR 

 CAS # : 50-28-2 

 

 17α-estradiol (weak estrogen) 

 Supplier: Sigma, Zwijndrecht, The Netherlands 

 Catalogue and Batch #: NR 

 Purity: NR 

 CAS # : 57-91-0 

 

 Corticosterone (negative compound) 

 Supplier: Sigma, Zwijndrecht, The Netherlands 

 Catalogue and Batch #: NR 

 Purity: NR 

 CAS # : 50-22-6 

 

 17α-methyltestosterone (very weak agonist) 

 Supplier: Fluka, Sigma, Zwijndrecht, The Netherlands 

 Catalogue and Batch #: NR 

 Purity: NR 

 CAS # : 58-18-4 

3. Vehicle(s) 

Solvent: DMSO (SeccoSolv, Merk, Darmstadt, Germany) 

Solvent control 

(final concentration): 

0.1% 

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B. METHODS

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

Collection of Research Bioresources Cell Bank. 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 (prepared in-house according to NOTOX 

Standard Operating Procedure IVA/L/007 which is identical to the Guideline method), in an 

incubator under 5% CO2 at 37oC. Upon reaching 75-90% confluence, cells were 

subcultured more than fewer than eleven times prior to exposure to the test material. 

2. Transcriptional Activation Assays: Cells were plated at a density of 1×104

 cells/100 μL 

medium/well in a 96-well plate and allowed to attach for 3 hours. Growth media was 

replaced with media containing serial log dilutions of dichlobenil in DMSO (0.1%). Cells 

were incubated for 20-24 hours at 37±1°C. Cytotoxicity was determined by MTT. 

Transcriptional activation of the estrogen receptor was determined using a standard 

luciferase assay system (Promega, Madison, WI). The plate was read with an Infinite®

M200 Pro luminometer. The limit of quantitation for this luminometer was not reported. 

a. Preliminary Test: A preliminary test evaluating concentrations ranging from 2.5 ×10-4 to 

10-9 M was conducted to determine the appropriate concentration range and to determine 

concentrations resulting in insolubility and/or cytotoxicity. 

b. Proficiency Chemicals: The responsiveness of the test system was confirmed for each 

newly prepared batch of cell stocks taken from the frozen stock by testing the following set 

of proficiency chemicals in duplicate on separate days. The proficiency of the testing 

laboratory was demonstrated as indicated in the table below. 

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. 

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c. Reference Chemicals: To ensure the stability of the response from the cell line, six 

concentrations of each of the following reference chemicals were included in each plate in 

the current assay, along with the test chemical: 

Reference Chemical CAS No. Concentration Range Class 

17β-estradiol (ER) 50-28-2 10−14 to 10−8 Strong estrogen 

17α-estradiol 57-91-0 10−12 to 10−6 Weak estrogen 

Corticosterone 50-22-6 10−10 to 10−4 Negative compound 

17α-methyltestosterone 58-18-4 10−11 to 10−5 Very weak agonist 

3. Data analysis: To obtain the relative transcriptional activity to the 1 nM E2 positive control 

(PC), the luminescence signals from the concurrent plate were analyzed by subtracting the 

mean value of the vehicle control from each well value to normalize the data; each 

normalized value was then divided by the mean value of the normalized PC. The resulting 

value was multiplied by 100 in order to express relative transcriptional activity as a 

percentage of the PC. Graph Pad Prism v. 4.01 (GraphPad Software, Inc., La Jolla, CA) was 

used to calculate the EC50, PC10, PC50, RPCMax, and PCMax for dichlobenil when applicable. 

The test material was defined as negative for inducing estrogen receptor transcriptional 

activation if the RPCMax < PC10 in at least 2 of 2 (or 2 of 3) runs. Log EC50 and Hill slope 

values are calculated only if a positive response is observed. Coefficients of variation (CV) 

were calculated for the luminescence data triplicates. Concentrations showing >20% 

cytotoxicity or evidence of insolubility were excluded from analyses. 

4. Definitions 

EC50 = concentration of agonist that induces a response halfway between the baseline 

(bottom) and maximum (top) response 

PC10 = concentration of a test chemical at which the response is 10% of the response 

induced by the positive control (E2 at 1 nM) in each plate 

PC50 = concentration of a test chemical at which the response is 50% of the response 

induced by the positive control (E2 at 1 nM) in each plate 

RPCMax = maximum level of response induced by a test chemical, expressed as a percentage 

of the response induced by the positive control (1 nM E2) on the same plate 

PCMax = concentration of a test chemical inducing the RPCMax

II. RESULTS 

A. PRELIMINARY TEST: In order to identify a suitable top concentration for use in the 

transcriptional activation assays, preliminary cytotoxicity and precipitation assays were 

conducted. The maximum stock concentration of dichlobenil in DMSO that could be 

prepared was a stock of 250 mM. Therefore the highest concentration that could be tested 

was 2.5 × 10-4 M. The results of the cytotoxicity test are presented in Table 1. No 

cytotoxicity or precipitation was observed at any concentration. Based on these results, 

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logarithmically increasing concentrations from 10-9 to 2.5 ×10-4 M were selected for the 

assay. 

TABLE 1. Preliminary Test for Solubility, Cytotoxicity, and Concentration-Selection for Dichlobenila

Concentration (M) % Viability Comments 

2.5 ×10−4 93.4 

10−4 102.3 

10−5 95.2 

10−6 97.6 

10−7 91.6 

10−8 92.4 

10−9 90.9 

70% methanol 0 Positive control for cytotoxicity 

VC b 100 

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

b VC = Vehicle control 

B. POSITIVE AND NEGATIVE REFERENCE CHEMICALS 

1. Proficiency Chemicals: The responsiveness of cells to the required proficiency chemicals 

was performed in duplicate on different days. The data are reported in Table 2. The 

responses demonstrated the performing laboratory’s proficiency with the assay. 

TABLE 2. Proficiency Chemicals a

Compound Expected Response Lab Response 

Diethylstilbestrol Positive Positive 

17α-Ethynyl estradiol Positive Positive 

Hexestrol Positive Positive 

Genistein Positive Positive 

Estrone Positive Positive 

Butyl paraben Positive Positive 

1, 3, 5-Tris(4-hydroxyphenyl)benzene Positive Positive 

Dibutyl phthalate Negative Negative 

Atrazine Negative Negative 

Corticosterone Negative Negative 

a Data were obtained from page 74 of the study report.

2. Reference Chemicals: Values derived from the concentration response curve (e.g., log

PC50, Log PC10, Log EC50, and Hill slope) for the four concurrently run reference materials 

are included in Table 3. Performance criteria were met for 17β-estradiol, 17α-estradiol and 

corticosterone. For 17α-methyltestosterone, the Log PC50 was not calculable for Runs 1, 2 

and 4, and the Log PC10 was higher than the acceptable range in all four Runs. For 17αmethyltestosterone, the mean RPCMax range was 34-51% for all four Runs. This indicates a 

decreased sensitivity to very weak agonists. 

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DICHLOBENIL /027401 OCSPP 890.1300/ OECD 455 

TABLE 3. Performance Criteria for Reference Chemicalsa

Reference Chemical 

Parameter

Acceptable 

Range 

Values Acceptable 

Run 1 Run 2 Run 3 Run 4 Yes No 

17β-estradiol

 Log PC50 −11.4 to −10.1 -10.6 -10.5 -10.5 -11.2 X 

 Log PC10 <−11 -12.0 -11.5 -11.6 -12.2 X 

 Log EC50 −11.3 to −10.1 -10.5 -10.3 -10.4 -11.3 X 

 Hill Slope 0.7 to 1.5 1.0 0.9 0.8 1.1 X 

 Test range 10−14 to 10−8 M 10−14 to 10−8 M X 

17α-estradiol

 Log PC50 −9.6 to −8.1 -8.9 -8.8 -8.9 -8.3 X 

 Log PC10 −10.7 to −9.3 -9.7 -9.7 -9.4 -9.5 X 

 Log EC50 −9.6 to −8.4 -8.9 -8.7 -8.9 -8.4 X 

 Hill Slope 0.9 to 2.0 1.4 1.1 1.9 0.9 X 

 Test range 10−12 to 10−6 M 10−12 to 10−6 M X 

Corticosterone

 Test range 10−10 to 10−4 M 10−10 to 10−4 M X 

17α-methyltestosterone

 Log PC50 −6.0 to −5.1 NC NC -5.0 NC X 

 Log PC10 −8.0 to −6.2 -6.0 -5.7 -5.9 -5.9 X 

 Test range 10−11 to 10−5 M 10−11 to 10−5 M X 

a Data were obtained from pages 22, 27 and 28 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 overall mean TA value for the vehicle control was 9867 – 26753, and the 

overall mean TA value for the positive control was 69791-221528. The induction for the 

positive control ranged from 7.1- to 8.7-fold. The mean normalized value for the maximum 

response of the positive control was 59925 - 194776. The PC50 (50% of the maximum 

response) for E2 in this assay is 29962-97388 and the PC10 (10% of the maximum response) 

is 5993-19478.

 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 16234 1575 138332 11401 8.5 122098 11401 

2 26753 NR 221528 NR 8.3 194776 NR 

3 9867 NR 69791 NR 7.1 59925 NR 

4 18812 NR 164310 NR 8.7 145498 NR 

a Data were obtained from pages 41, 42, 45, 46, 49, 50, 53 and 54 of the study report. SD calculated by reviewer.

b Positive control was 17β-estradiol (E2) at 1 nM. 

c Fold-induction = (mean TA of PC)/(mean TA of VC) 

NR Not reported 

2. Test Material: Relative (to the PC) transcriptional activation at each concentration of the 

test chemical during the four assay runs is presented in Table 5. The concentration-response 

curves depicting fold induction of relative transcriptional activation is presented in Figures 1 

through 4. The mean RPCMax was 0.9% to 5.1%, and the associated PCMax was 10-8 or 10-6

M. Because the RPCMax < PC10 in all runs, dichlobenil was considered negative for estrogen 

receptor transcriptional activation in this test system. 

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Estrogen Receptor Transcriptional Activation (2011) / Page 8 of 11 

DICHLOBENIL /027401 OCSPP 890.1300/ OECD 455 

TABLE 5. Relative Transcriptional Activation (RTA) of Dichlobenila

Parameter RTA (mean ± SD); % of Positive Control (PC) 

Run 1 Run 2 Run 3 Run 4 

Conc. (M) Mean SD Mean SD Mean SD Mean SD 

2.5 ×10−4 0.20 1.09 -2.94 1.31 -3.11 1.23 -0.22 1.63 

10−4 -3.41 0.32 -1.93 1.56 -2.10 0.40 1.29 0.91 

10−5 -1.21 3.17 -2.19 0.47 -1.45 1.71 -0.49 1.15 

10−6 2.36 0.88 1.08 1.62 0.93 1.62 2.19 1.79 

10−7 3.21 1.24 1.67 3.50 -1.53 1.44 1.80 2.34 

10−8 5.05 1.96 3.13 2.14 -0.59 2.70 2.49 0.73 

10−9 4.26 3.80 1.23 1.40 -1.94 1.86 -1.03 0.71 

Log EC50 NA NA NA NA 

Hill Slope NA NA NA NA 

RPCMax 5.1 3.1 0.9 2.5 

PCMax 10−8 10−8 10−6 10−8

PC50 NA NA NA NA 

PC10 NA NA NA NA 

a Data were obtained from page 31 of the study report. 

NA = Not Applicable

FIGURE 1. Fold Induction of Relative Transcription Activation (RTA) of Dichlobenil 

Compared to the Positive Control, Run 1. 

VC= Vehicle Control 

PC= Positive Control (1 nM E2) 

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DICHLOBENIL /027401 OCSPP 890.1300/ OECD 455 

FIGURE 2. Fold Induction of Relative Transcription Activation (RTA) of Dichlobenil 

Compared to the Positive Control, Run 2. 

VC= Vehicle Control 

PC= Positive Control (1 nM E2) 

FIGURE 3. Fold Induction of Relative Transcription Activation (RTA) of Dichlobenil 

Compared to the Positive Control, Run 3. 

VC= Vehicle Control 

PC= Positive Control (1 nM E2) 

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Estrogen Receptor Transcriptional Activation (2011) / Page 10 of 11 

DICHLOBENIL /027401 OCSPP 890.1300/ OECD 455 

FIGURE 4. Fold Induction of Relative Transcription Activation (RTA) of Dichlobenil 

Compared to the Positive Control, Run 4. 

VC= Vehicle Control 

PC= Positive Control (1 nM E2) 

3. Performance Criteria: The laboratory proficiency assays using the required reference 

compounds adequately demonstrated that the assay was functioning properly. Performance 

criteria were met for 17β-estradiol, 17α-estradiol and corticosterone. For 17αmethyltestosterone, the Log PC50 was not calculable for Runs 1, 2 and 4, and the Log PC10

was higher than the acceptable range for all four Runs. This indicates a decreased 

sensitivity to very weak agonists. However, maximum induction by 17α-methyltestosterone 

was between 34-51% of PC in all runs (35, 36, 51 and 34% in the 1st, 2nd, 3rd and 4th runs, 

respectively), indicating that the assay was able to detect at least some activity from 17αmethyltestosterone. Mean luciferase activity of the PC was >4-fold that of the vehicle 

control for all runs, and the fold-induction corresponding to the PC10 of the concurrent PC 

was greater than 1+2 standard deviations of the vehicle control on all plates. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: In this assay, four independent experiments were 

performed with dichlobenil at seven concentrations (from 0.001 μM to 250 μM) in 

triplicate. In each experiment a quality control plate with the reference compounds E2, 17αestradiol, 17α-methyltestosterone and corticosterone were include. In addition on each plate 

concurrent positive (1nM E2) and negative (Vehicle, DMSO) controls were included in 

triplicate wells. The vehicle concentration was kept constant in all wells at 0.1%. 

The mean luciferase activity of the positive control included in each experiment was at least 

4-fold higher than the vehicle control in all experiments. The fold induction of E2 

corresponding to the PC10 value was above the fold induction of the vehicle control +2SD 

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Estrogen Receptor Transcriptional Activation (2011) / Page 11 of 11 

DICHLOBENIL /027401 OCSPP 890.1300/ OECD 455 

(standard deviations) in all experiments. The coefficient of variation in triplicate 

luminescence intensity data was occasionally above the target of 20%, but this had no 

effects on the curve fit parameters and did not affect the integrity of the results. 

The results of the reference compounds E2, 17α-estradiol and corticosterone were within the 

acceptability criteria. The curve fit values log PC50, Log PC10 and Log EC50 for 

17α-methyltestosterone from all four experiments were just outside the acceptability criteria 

mentioned in the Guideline, but were within our laboratory historical control data range and 

were thus accepted. The reference compounds were correctly classified as positive (E2, 

17α-estradiol and 17α-methyltestosterone) or negative (corticosterone). 

Dichlobenil did not show an induction of the estrogenic response at increasing 

concentration. This was reproducible in four independent experiments. 

B. AGENCY COMMENTS: Dichlobenil was tested up to the limit of solubility, 2.5×10−4M. 

The laboratory proficiency assays using the required reference compounds adequately 

demonstrated that the assay was functioning properly. Performance criteria were met for 

17β-estradiol, 17α-estradiol and corticosterone. For 17α-methyltestosterone, the Log PC50

was not calculable for Runs 1, 2 and 4, and the Log PC10 was higher than the expected range 

for all four Runs. This indicates a decreased sensitivity to very weak agonists. The mean 

RPCMax for dichlobenil was 0.9% to 5.1%, and the associated PCMax was 10-8 or 10-6 M. 

Because the RPCMax < PC10 in all runs, dichlobenil was considered negative for estrogen 

receptor transcriptional activation in this test system. 

C. STUDY DEFICIENCIES: The following deficiency was noted: 

 The response to 17α-methyltestosterone indicates a decreased sensitivity to weak 

agonists. However, the RPCMax for 17α-methyltestosterone was 30-50% compared to 

0.9-5.1% for dichlobenil. It is unlikely that the decreased sensitivity changes the 

conclusion of these results. 

Page 169 of 331
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Dichlobenil 

EPA MRID Number 48616106 

Data Requirement: EPA DP Barcode 397913 

OECD Data Point 229 

EPA MRID 48616106 

EPA Guideline 890.1350, Fish Short-Term Reproduction Assay 

Test material: Dichlobenil Purity: 99.3% 

Common Name: Dichlobenil 

Chemical Name: IUPAC: 2,6-Dichlorobenzonitrile 

 CAS Name: 2,6-Dichlorobenzonitrile 

CAS No. 1194-65-6 

 EPA PC Code: 027401 

Primary Reviewer: Joan Gaidos Signature: 

Senior Scientist, Cambridge Environmental, Inc. Date: 6/20/12 

Secondary Reviewer: Teri S. Myers Signature: 

Senior Scientist, Cambridge Environmental, Inc. Date: 6/22/12 

Primary Reviewer (and final additional reviewer): Elyssa Arnold 

Biologist, EPA/OPP/EFED/ERB2 Date: 5/29/15 

Additional Reviewer: Amy Blankinship 

Senior Chemist, EPA/OPP/EFED/ERB3 Date: 5/29/15

Date Evaluation Completed: 29-05-2015 

Page 1 of 83

AMY 

BLANKINSHIP

Digitally signed by AMY BLANKINSHIP 

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

ou=USEPA, ou=Staff, cn=AMY 

BLANKINSHIP, 

dnQualifier=0000040917 

Date: 2015.06.08 10:53:51 -04'00'

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

EPA MRID Number 48616106 

CITATION: Migchielsen, Ing. M.H.J. 2011. Fish Short-term Reproduction Assay with Dichlobenil Technical. 

Unpublished study performed by NOTOX B.V., Hambakenwetering 7, The Netherlands. Lab Study No.: 

495786; Sponsor No.: EDSP-027401-120. Study sponsored by Chemtura Corporation, Middlebury, CT. 

Study completed September 22, 2011. 

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.

Page 2 of 83

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

EPA MRID Number 48616106 

EXECUTIVE SUMMARY:

The 21-day short-term reproduction assay of dichlobenil with fathead minnows (Pimephales promelas) was 

conducted under flow-through conditions. Adult fish, 20 spawning groups (2 males and 4 females in each 

group; 6-7 months old) were exposed to dichlobenil (99.3% purity) at nominal concentrations of 0 (negative 

control), 0 (solvent control; 0.1 mL/L acetone), 0.021, 0.20, and 2.0 mg a.i./L; mean-measured 

concentrations were <LOQ (<0.001; both controls), 0.021, 0.21, and 2.1 mg a.i./L. The test system was 

maintained at 24.3 to 26.2o

C and a pH of 7.3 to 7.9. Unless otherwise indicated, the treatment groups were 

compared to the negative control. 

There were no statistically significant differences (p>0.05) in mean male or female body weights or lengths 

at any test level compared to the control. Overall mean survival values were 100, 96, 100, 100 and 92% in 

the negative control, solvent control, and low, mid, and high treatment levels, respectively. Fish exposed to 

the high treatment level were observed to have a lighter, more yellowish color compared to the fish in the 

controls and other treatment levels; however, the incidence of this effect was not reported. No other changes 

in behavior or appearance were noted. 

Spawning frequency in the negative and solvent control was at least every 4 days in 3 and 2 of the 4 

replicates, respectively; the mean fecundity in the negative and solvent controls was 12.1 (range 6.6-15.8) 

and 6.5 (range 4.3-11.6) eggs/female/day/replicate. Fecundity was significantly (p<0.05; JonckheereTerpstra) reduced by 53% and 90% at the mid and high treatment levels, respectively, compared to the 

negative control. The study author did not provide fertility data, but reported fertility was “more than 99.9% in 

all test groups” and fertility of eggs from control animals was at least 95%. 

Median male tubercle score showed a statistically significant reduction from 26 in the negative control to 21, 

14, and 12 in the low, mid and high treatment groups, respectively (p<0.05; Jonckheere-Terpstra). No 

tubercles were observed for females, except for one female with one tubercle in the low treatment group 

which was reported not to be treatment related. There were no significant differences in male or female 

gonadal stage index (GSI) or vitellogenin (VTG). 

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

EPA MRID Number 48616106 

In males, no treatment-related histopathological gonadal findings were reported. In females, there was an 

increased incidence and/or severity (minimal to moderate) of oocyte atresia and a shift to more mature 

stages in the ovaries in the mid and high treatment groups. 

Sex steroids (testosterone and 17ȕ-estradiol) were not measured. No dorsal nape pad measurements or 

other dorsal nape pad observations were reported. 

While not statistically significant (p=0.078), the fecundity in the solvent control group was reduced by 46% 

compared to the negative control. However, large variability was observed between the pre-exposure and 

exposure fecundity rate for both the negative and solvent control replicates, suggesting this difference may 

not be solvent related. Additionally, this was the only endpoint for which a notable difference between the 

negative and solvent control was observed. 

Not all performance criteria for the OCSPP 890.1350 guideline were met for reported parameters. The EPA 

recommendation of at least 15 eggs/female/reproductive day/replicate or a spawning frequency of at least 

every four days in every replicate was not met (for one replicate in negative control and two replicates in 

solvent control); however, the overall mean fecundity in the controls was determined to be adequate for 

interpretation of the results. While fertilization success was reported to be >95% in the control, these data 

were not provided in the study report. Additionally, there is uncertainty in whether the pre-exposure tanks 

were randomly allocated to the treatment groups within the blocks. In this study, there were 3 tanks in the 

exposure period in which the sex ratio was 3 males and 3 females instead of 2 males and 4 females as 

dictated by the guideline. Two of these tanks were in the solvent control and one was in the mid treatment 

group. All other performance and validity criteria were met.

This assay satisfies the EDSP Tier 1 Test Order requirement for a Fish Short-Term Reproduction Assay 

(OCSPP Guideline 890.1350). 

 

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

EPA MRID Number 48616106 

Results Synopsis

Test Organism age at test initiation: ca. 6-7 months 

Mean body weight at test initiation (if measured): Males 2.14 g; Females 0.65 g. 

Mean length at test initiation (if measured): Males 4.8 cm; Females 3.5 cm. 

Test Type (Flow-through, Static, Static Renewal): Flow-through 

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

EPA MRID Number 48616106 

Table 1: Summary of Reproductive and HPG Effects1,2 in the Fish Short-Term Reproduction Assay (FSTRA) with Dichlobenil. Abbreviations: E2 17ȕ-estradiol. F Female. Fert. Fertilization. GSI Gonado-Somatic Index. Histo. Histopathology. M Male. NA Not applicable. T Testosterone. VTG Vitellogenin.

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

2 The criteria for significance are described in the Reviewer’s Analysis and Statistical Verification sections of the DER. Conclusions regarding histopathology may be 

heavily weighted by the expert opinion of a board-certified pathologist. 

Treatment 

(mg a.i./L) 

[meanmeasured]

Fecundity 

Fert. 

Success 

Tubercle Score GSI Gonadal Histo. Plasma VTG Plasma T Plasma E2 

M F M F M F M F M F M F 

0.021 No NA Yes No No No No No No No NA NA NA NA 0.21 Yes NA Yes No No No No Yes No No NA NA NA NA 2.1 Yes NA Yes No No No No Yes No No NA NA NA NA 

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

EPA MRID Number 48616106 

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. Prior to study initiation, the fecundity was 5.3-15.3 eggs/female/reproductive 

day/replicate, with only two replicates at 15 or greater. The guideline 

recommends at least two spawning events in the 7 days immediately prior to 

test initiation or 15 eggs/female/day/replicate. The study author reported that 

spawning occurred every fourth day; however, raw data to support this finding 

were not reported. 

2. The study author reported that fertilization success was “more than 99.9% in all 

test groups”, but these replicate data were not provided for confirmation. 

3. The fecundity in the negative and solvent control groups were <15 

eggs/female/day/replicate and spawning in every replicate did not occur at 

least every fourth day (one in the negative control and two in the solvent 

control). 

4. The fish were between 6 and 7 months of age at the start of the study. The 

EPA guideline states that fish should be 4.5-6 months of age at the start of 

the study. 

5. During the first 3 days of the pre-exposure period, light was continuous (no 

dark period). No adverse effects on fish or spawning were reported. 

6. For one day during the pre-exposure period, the flow of dilution water was 

stopped to a group of 7 tanks (tanks 23-29). This caused a decrease in 

temperature and dissolved oxygen during this period below the optimum levels. 

No adverse effects on fish or spawning were observed and eggs were 

produced on the effected day. 

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7. Food was not supplied in two equal portions on Days 4 and 5 of the exposure 

period. Amount supplied were sufficient to maintain health of the fish and no 

adverse effects were observed. 

8. In 3 replicates used in the exposure period, 3 females and 3 males were 

exposed instead of 4 females and 2 males. Two of these replicates were in the 

solvent control test group and one was in the 0.021 mg a.i./L test group. This 

deviation was noticed in the last week of the study. Fecundity calculations were 

adjusted based on the correct number of females. 

9. There is uncertainty in whether the pre-exposure tanks were randomly allocated 

to the treatment groups within a block. 

 These deficiencies/deviations do not have an impact on the results or interpretation of this study.

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

claims statements were provided. This study was conducted in 

accordance with GLP Standards as published by the OECD Principles 

of GLP [ENV/MC/CHEM(98)17]. 

A. TEST MATERIAL: Dichlobenil, CAS No. 1194-65-6

Description: White powder with lumps; solubility in water, 0.021 g/L.

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. : AM9K12A222 (Batch No.) 

Purity: 99.3% (Expiration data October 12, 2012) 

Impurities: None reported

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Stability of Compound: Analytical verification at Days 0, 7, 14 and 21 of diluter stock fortified with 

0.2, 2.0 and 20 mg a.i./L yielded recoveries ranging from 99 to 117% of nominal concentrations. 

Method validation recoveries fortified at 0.021 and 2.0 mg a.i./L yielded recoveries ranging from 81 

to 111%. Analytical verification of the test solutions at Days -1, 0, 4, 7, 13/14 and 21 yielded 

recoveries of 88 to 123% of nominal concentrations; there was one outlier that had been stored 

frozen with a recovery of 652%. There was no pattern of decline. The average %CV ranged from 

8.9 to 11%. These data indicate the test material was generally stable under the test conditions. 

Storage Conditions of 

Test Chemicals: The test material was stored at room temperature in the dark and reported 

to be stable under these conditions. Storage stability was determined after 

0, 3, 8, 15, and 21 days. 

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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): Teleostei, Cyprinidae Linnaeus, 1758 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: At least 2 weeks EPA recommends a minimum two-week 

acclimation period. Note that the acclimation period is different from the subsequent, in situ 

pre-exposure phase. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on health: Mortalities during the 7 days prior to preexposure period was <5%. 

Fish did not receive any treatment for disease during acclimation period. No behavioral abnormalities or clinical signs were noted. 

EPA recommends that mortality during the 7 days prior to the pre-exposure phase be less 

than 5% of the culture population. If mortality during these 7 days is greater than 10%, EPA 

recommends that the fish be rejected. If mortality is between 5-10%, EPA recommends 

that fish be held another 7 days. If mortalities greater than 5% occur during this extended 

acclimation period, EPA recommends that the 

fish not be used. 

Type of food: 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 reportedFrequency of feeding: 2 times/day At least 3 hours apart, except on weekend 

days. 

Details on feeding: None 

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Table 3: Fish Selection and Pre-Exposure Performance.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Age at test initiation: 6-7 months EPA recommends reproductively mature 

(sexually dimorphic) fish, 4.5 - 6 months old.

Mean weight of males at test initiation (if determined): 

2.14±0.34 g Based on 10 males used to stock aquaria 

for pre-exposure phase. 

EPA recommends thata 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): 

1.58-2.83 g 

Mean weight of females at test initiation (if determined): 

0.65±0.09 g Based on 10 females used to stock aquaria 

for pre-exposure phase. 

Range of individual weights (females) at test initiation (if determined): 

0.54-0.79 g 

Mean length of males at test initiation (if determined): 

4.8 ± 0.2 cm Based on 10 males used to stock aquaria 

for pre-exposure phase. 

Mean length of females at test initiation (if determined): 

3.5 ± 0.1 cm Based on 10 females used to stock aquaria 

for pre-exposure phase. 

Duration of pre-exposure phase: 14 days EPA recommends a minimum of 14 days. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Were pre-exposure conditions identical to the definitive test? 

Yes Exceptions: 

(1) During first 3 days of pre-exposure, test light was continuous (no dark); dark-light regime corrected for the following 11 days and no adverse effect detected. (2) During a period of nearly one full day, the temperature was below the optimum range in two tanks, oxygen was low and there was no flow of dilution water; fish were not affected and did produce eggs during this period. 

EPA recommends that pre-exposure conditions, including temperature, photoperiod, feeding, etc., 

be identical to definitive test conditions. 

Number of pre-exposure tanks: 30 tanks EPA recommends that additional tanks set up at 

the beginning of pre-exposure will ensure that sufficient replicates with the correct sex ratio are 

available for the definitive test. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Number of males per tank: 2 males/tank 3 tanks were found to have an incorrect sex 

ratio (3 males and 3 females) during the pre-exposure phase and were not used for the exposure phase of the study. During the exposure phase, 3 additional tanks were found to have the same incorrect sex ratio (3 males and 3 females). 

Number of females per tank: 4 females/tank Pre-exposure fecundity: 5.3-15.3 eggs/female/ 

reproductive day/ replicate in the 20 topperforming tanks 

The EPA recommendation of at least 15 eggs/female/reproductive day prior to test initiation was not met. However, the fecundity was averaged over the entire 14 day pre-exposure period and data were not broken out for the 7 days prior to the definitive test. 

EPA recommends that pre-exposure fecundity in 

each replicate (tank) selected for use in the definitive test be at least 15 

eggs/female/reproductive day/replicate during the 7 days prior to the definitive test. 

Number of spawns during preexposure: Not reported No data provided, but spawning occurred at least every fourth day according to the study 

report. 

EPA recommends that spawning occur at least 

twice in the 7 days prior to the definitive test. 

Details on pre-exposure: None 

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

system. As noted in the Corrections and 

Clarifications document1 , the use of a static 

renewal system is not recommended for this 

assay.

Type of flow-through dilution 

system: 

Proportional diluter Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

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 

Flow-through rate: 3 L/hr (equivalent to 7 

volume exchanges per day) 

Recommended flow-through rate is 45 mL/min (2.7 L/hr), or at least 6 total 

volume exchanges per day.

Details on toxicant mixing for flow-through systems: 

Solution from mixing chamber split equally among replicate test vessels. Treatment recoveries prior to test initiation were 86-101%. The flow splitting accuracy was 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? No 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: Reverse-osmosis treated 

tap water 

EPA recommends natural or reconstituted water; it is recommended that natural water 

be sterilized with UV and tested for pesticides, heavy metals, and other possible 

contaminants. OECD accepts any water in which the test species show control survival 

at least as good as indicated in the test 

guideline.

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

Yes 

Test vessel type/materials: Glass aquaria with silicone 

sealant and covered with Perspex plate 

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 (dimensions not 

reported)

EPA recommends the use of 18 L test chambers (e.g., 40 x 20 x 20 cm).

Fill volume: 10 L EPA recommends 10 L solution per tank. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Spawning substrate material: PVC pipe to form a 

semicircular arch 

EPA recommends that each tank contain three semi-circular spawning substrates, e.g., 

aged PVC pipe, 10 - 20 cm in length, split 

lengthwise. 

Spawning substrate size: Not further described. Additional details on exposure system: 

 None 

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

Parameter Minimum Maximum Mean Measurement Interval Guideline Recommendations 

Temperature (°C) 24.3 26.2 25 

Approximately every 4 

days; continuous in 

controls 

EPA recommends temperature 25±1oC; interreplicate and inter-treatment differentials 

should not exceed 1oC.

pH 7.3 7.9 7.6 Twice a week EPA recommends pH 6.5 to 9.0.Dissolved oxygen (mg/L) 5.8 8.7 7.2 Twice a week EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air saturation)Total alkalinity (mmol/L as CaCO3) 

0.8 1.0 0.9 

Weekly in control and 

highest conc. only 

EPA recommends total alkalinity > 20 mg/L 

as CaCO3.

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Parameter Minimum Maximum Mean Measurement Interval Guideline Recommendations 

Hardness (mg/L as CaCO3) 161 196 181 Weekly in control and 

highest conc. only 

Total organic carbon (mg/L) 

0.05 0.05 

0.05 Once at start of test 

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. 

Conductivity (Pmhos/cm) Not reported Not reported Not reported Not reported 

General recommendations for frequency of 

measurements: EPA recommends that temperature, pH, and dissolved oxygen be 

measured in all test tanks at least weekly and that hardness and alkalinity be measured in 

controls and in one tank at the highest test concentration at least weekly. In addition, 

continuous temperature monitoring of at least one tank is encouraged. 

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D. Study Design and Additional Experimental Conditions

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

Was a range-finder conducted? No EPA recommends conducting a range-finder if 

96-hour LC50 data for the fathead minnow are 

unavailable. 

If yes, what was the method for determining the highest test concentration in the range-finder? 

NA 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: NA Life stage: NA 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: NA EPA recommends a 96-hour exposure. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Additional details: NA EPA recommends conducting a range-finder with 

five test concentrations plus a control (six total 

treatment levels), with four females and two males per exposure tank (36 fish total). The 

number of mortalities that occur may be used to develop a concentration-response curve. 

Based upon the results, the highest concentration that does not result in increased mortality or 

signs of overt morbidity compared to controls, or 

1/3 the derived 96-hr LC50, may be selected as 

the highest exposure concentration in the 21-day 

test.

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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:

Not reported EPA recommends that the highest test 

concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates 

adequate evidence of toxicity (e.g., 1/3 the 96-

hour LC50), whichever concentration is lowest.

Reference study citation (if applicable):

Not reported 

Separation of test concentrations: Ten-fold EPA suggests that a concentration separation of 

between 0.33 (or three-fold) and 0.1 (or tenfold) is scientifically acceptable1 . 

Number of test concentrations: 3 (3 treated, a negative 

control and a solvent control)

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): 0.001 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, 4, 7, 13, 14, and 21 

days 

Samples taken on Day 4 were from the high treatment group only as the stock solution appeared not homogeneous; additionally, a Day 13 sample was taken from the low treatment as dilution water delivery was temporarily disrupted. 

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 While block design was used, it is uncertain 

of whether the treatments were randomized within a block. 

EPA recommends that all fish be randomly assigned to tanks during pre-exposure. Tanks 

are then ranked according to pre-exposure fecundity, and the tanks with the highest 

fecundity are randomly assigned to a definitive test treatment and block first. Each block 

contains one replicate of each treatment, 

including controls. 

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

4 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 During the last week of the definitive study, 

3 tanks were found to have an incorrect sex ratio (3 males and 3 females). 

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? Yes Solvent type (if applicable): Acetone Maximum solvent concentration (if applicable): 

0.1 mL/L 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: 540 to 1080 lux EPA recommends light intensity 540 – 1080 lux 

(at water’s surface).

Additional details: Details about test solution appearance were 

not found in the study report. 

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Table 8: Summary of Treatment Concentrations in the Fish Short-Term Reproduction Assay with Dichlobenil. 

Treatment ID 

Nominal 

Concentration 

(mg a.i./L) 

Mean Measured 

Concentration 

(mg a.i./L) 

Mean CV (%) Details or Remarks Guideline Recommendations 

Control (dilution water only) 

0.00 <LOQ NA 

EPA recommends that test item concentrations be maintained at a coefficient of variation (CV) 20%. Solvent control 0.00 <LOQ NA 

Treatment 1 0.020 0.021 11 

Mean measured and CV 

includes Day 13 sample 

Treatment 2 0.20 0.21 11 Treatment 3 2.0 2.1 8.9 

Mean measured and CV 

includes Day 4 sample 

Abbreviations: CV Coefficient of variation. LOQ=0.001 mg a.i./L.

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E. Observations

Biological Endpoints: Survival, fecundity, 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, with the exception of fertility and spawning frequency data 

EPA recommends that observations of survival, fecundity, fertilization success, secondary sex characteristics, and other clinical signs occur at least daily. At 

test termination (Day 21), additional observations include body weight and length, nuptial tubercle score, gonadal staging and histopathology, plasma 

vitellogenin, and plasma sex steroids (testosterone and 17ȕ-estradiol, if measured). Gonado-somatic index (GSI) is calculated using a ratio of gonad weight 

to body weight (gonad weight to the nearest 0.1 mg / body weight in mg x 100) at test termination. 

Clinical signs of overt toxicity may include (but are not limited to) hemorrhage, cessation of feeding, and other abnormal behavior. 

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II. RESULTS AND DISCUSSION 

A. Results 

One male died in the solvent control and two females died in the 2.1 mg a.i./L treatment group during the study. Overall survival values were 100, 

96, 100, 100 and 92% in the negative control, solvent control, and mean-measured 0.021, 0.21, and 2.1 mg a.i./L treatment levels, respectively. 

Table 9: Adult Fish Survival in Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[mean-measured]

Males Females 

N1 # Surviving % Survival n # Surviving % Survival 

Control (<LOQ) 8 8 100 16 16 100 

Solvent control (<LOQ) 10 9 90 14 14 100 

0.021 9 9 100 15 15 100 

0.21 8 8 100 16 16 100 

2.1 8 8 100 16 14 88 

1 Total number of fish at test initiation. 

LOQ=0.001 mg a.i./L. 

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Mean male body weight values were 2.07, 1.95, 2.01, 1.98 and 2.00 g and female body weight values were 0.79, 0.83, 0.83, 0.87 and 0.83 g 

in the negative control, solvent control, and mean-measured 0.021, 0.21, and 2.1 mg a.i./L treatment levels, respectively. Mean male body length values were 47, 47, 47, 47 and 47 mm and female body length values were 35, 35, 35, 36 and 36 mm in the negative control, solvent control, 

and mean-measured 0.021, 0.21, and 2.1 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 

Control (<LOQ) 4 2.07 0.32 4 0.79 0.02 4 47 1.5 4 35 0.6 

Solvent control 

(<LOQ) 

4 1.95 0.30 4 0.83 0.07 4 47 2.3 4 35 1.4 

0.021 4 2.01 0.21 4 0.83 0.07 4 47 0.8 4 35 1.0 

0.21 4 1.98 0.34 4 0.87 0.10 4 47 1.4 4 36 1.3 

2.1 4 2.00 0.59 4 0.83 0.08 4 47 5.3 4 36 1.3 

LOQ=0.001 mg a.i./L. 

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Mean fecundity values were 12.1, 6.5, 7.5, 5.6 and 1.2 in the negative control, solvent control, and 

mean-measured 0.021, 0.21, and 2.1 mg a.i./L treatment levels, respectively. Fertilization success data 

were not reported, however the study author did report that fertility was >99.9% in all test groups. 

Table 11: Fecundity and Fertilization Success in Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[mean-measured]

Fecundity1 

Fertilization Success (%)2

Mean ±SD 

Control (<LOQ) 12.1 4.0 Not reported 

Solvent control (<LOQ) 6.5 3.5 Not reported 

0.021 7.5 1.2 Not reported 

0.21 5.6 2.6 Not reported 

2.1 1.2 1.1 Not reported 

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. 

LOQ=0.001 mg a.i./L.

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Median male tubercle scores were 26, 21, 22, 14 and 12 in the negative control, solvent control, and 

mean-measured 0.021, 0.21, and 2.1 mg a.i./L treatment levels, respectively. None of the surviving 

females were found to have tubercles, with the exception of one female fish in the 0.021 mg a.i./L test 

group, who had one tubercle on her snout that was not believed to be caused by exposure to dichlobenil. 

Table 12: Nuptial Tubercle Score in Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[mean-measured]

Males Females 

n Median Tubercle 

Score n Median Tubercle Score 

Control (<LOQ) 4 26 4 0 

Solvent control (<LOQ) 4 18 4 0 

0.021 4 21 4 0 

0.21 4 14 4 0 

2.1 4 12 4 0 

LOQ=0.001 mg a.i./L. 

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 Average male GSI was 1.18, 1.27, 1.23, 1.26 and 1.53 and average female GSI was 12.31, 12.07, 10.51, 

12.53 and 14.73 in the negative control, solvent control, and mean-measured 0.021, 0.21, and 2.1 mg 

a.i./L treatment levels, respectively. 

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 

Control (<LOQ) 4 1.18 0.25 4 12.31 1.75 

Solvent control (<LOQ)2 3 1.27 0.21 4 12.07 1.24 

0.021 4 1.23 0.46 4 10.51 1.20 

0.21 4 1.26 0.25 4 12.53 1.95 

2.1 4 1.53 0.07 4 14.73 2.01 

1

 Gonado-somatic index (%) is calculated as gonad weight (to the nearest 0.1 mg) / body weight (mg) x 100. 

2

 The gonadal weights of two male fish in the solvent control replicate D were measured incorrectly and were not 

included in this analysis. 

LOQ=<0.001 mg a.i./L. 

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Median male gonadal stage was 2, 2, 2, 2, and 2 and median female gonadal stage was 3, 3, 3, 4, 

and 3 in the negative control, solvent control, and mean-measured 0.021, 0.21, and 2.1 mg a.i./L 

treatment levels, respectively. 

Table 14: Gonadal Staging in Fathead Minnow (Pimephales promelas).

1

 The guideline recommends the following gonadal staging scale for male fathead minnow: 0=undeveloped, 1=early 

spermatogenic, 2=mid-spermatogenic, 3=late spermatogenic, 4=spent. 

2

 The guideline recommends the following gonadal staging scale for female fathead minnow: 0=undeveloped, 1=early 

development, 2=mid-development, 3=late development, 4=late development/hydrated, 5=post-ovulatory. 

LOQ=0.001 mg a.i./L.

In males, there were minimal findings of increased granulomatous inflammation and no differences in stages 

of testes between the controls and treatment groups. In females, there was an increased incidence and/or 

severity (minimal to moderate) of oocyte atresia and a shift to more mature stages in the ovaries in the 0.21 

and 2.1 mg a.i./L treatment groups. According to the study authors, both findings suggest a reduced 

spawning in females in these treatment groups. The study authors reported all other microscopic findings in 

both males and females to be within normal range of background pathology. 

Treatment 

(mg a.i./L) 

[mean-measured]

Males Females 

n Median Stage1 n Median Stage2

Control (<LOQ) 4 2 4 3 

Solvent control (<LOQ) 4 2 4 3 

0.021 4 2 4 3 

0.21 4 2 4 4 

2.1 4 2 4 3 

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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 

Interstitial Cell 

Hyperplasia/ 

Hypertrophy 

Apoptotic Germ 

Cell 

n Incidence n Incidence n Incidence n Incidence n Incidence 

Control 

(<LOQ) 

0 NA NA NA NA NA NA NA NA 8 5

1 NA NA NA NA NA NA NA NA 8 2

2 NA NA NA NA NA NA NA NA 8 1

3 NA NA NA NA NA NA NA NA 8 0

4 NA NA NA NA NA NA NA NA 8 0

Solvent 

control 

(<LOQ)

0 NA NA NA NA NA NA NA NA 9 6

1 NA NA NA NA NA NA NA NA 9 3

2 NA NA NA NA NA NA NA NA 9 0

3 NA NA NA NA NA NA NA NA 9 0

4 NA NA NA NA NA NA NA NA 9 0

0.021 0 NA NA NA NA NA NA NA NA 9 8

1 NA NA NA NA NA NA NA NA 9 1

2 NA NA NA NA NA NA NA NA 9 0

3 NA NA NA NA NA NA NA NA 9 0

4 NA NA NA NA NA NA NA NA 9 0

0.21 0 NA NA NA NA NA NA NA NA 8 6

1 NA NA NA NA NA NA NA NA 8 2

2 NA NA NA NA NA NA NA NA 8 0

3 NA NA NA NA NA NA NA NA 8 0

4 NA NA NA NA NA NA NA NA 8 0

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Treatment 

(mg a.i./L) 

[meanmeasured] 

Diagnostic Observations1

Severity 

Increased 

Proportion of 

Spermatogonia 

Presence of 

Testis-Ova 

Increased 

Testicular 

Degeneration 

Interstitial Cell 

Hyperplasia/ 

Hypertrophy 

Apoptotic Germ 

Cell 

n Incidence n Incidence n Incidence n Incidence n Incidence 

2.1 0 NA NA NA NA NA NA NA NA 8 5

1 NA NA NA NA NA NA NA NA 8 2

2 NA NA NA NA NA NA NA NA 8 1

3 NA NA NA NA NA NA NA NA 8 0

4 NA NA NA NA NA NA NA NA 8 0

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. 

LOQ=0.001 mg a.i./L. 

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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 

Control (<LOQ) 0 NA NA NA NA NA NA NA NA 8 8 

1 NA NA NA NA NA NA NA NA 8 0 

2 NA NA NA NA NA NA NA NA 8 0 3 NA NA NA NA NA NA NA NA 8 0 4 NA NA NA NA NA NA NA NA 8 0 

Solvent control (<LOQ) 0 NA NA NA NA NA NA NA NA 9 8 

1 NA NA NA NA NA NA NA NA 9 0 

2 NA NA NA NA NA NA NA NA 9 1 3 NA NA NA NA NA NA NA NA 9 0 4 NA NA NA NA NA NA NA NA 9 0 

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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 

0.021 0 NA NA NA NA NA NA NA NA 9 6 

1 NA NA NA NA NA NA NA NA 9 0 

2 NA NA NA NA NA NA NA NA 9 1 3 NA NA NA NA NA NA NA NA 9 1 4 NA NA NA NA NA NA NA NA 9 0 5 NA NA NA NA NA NA NA NA 9 1 

0.21 0 NA NA NA NA NA NA NA NA 8 3 

1 NA NA NA NA NA NA NA NA 8 2 

2 NA NA NA NA NA NA NA NA 8 1 3 NA NA NA NA NA NA NA NA 8 1 4 NA NA NA NA NA NA NA NA 8 1 

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Treatment 

(mg a.i./L) 

[mean-measured] 

Additional Diagnostic Observations1

Severity 

Decreased Proportion 

of Spermatogonia 

Increased Vascular or 

Interstitial 

Proteinaceous Fluid 

Asynchronous 

Gonad 

Development 

Altered Proportions of 

Spermatocytes or 

Spermatids 

Granulomatous 

Inflammation 

n Incidence n Incidence n Incidence n Incidence n Incidence 

2.1 0 NA NA NA NA NA NA NA NA 8 6 

1 NA NA NA NA NA NA NA NA 8 1 

2 NA NA NA NA NA NA NA NA 8 1 3 NA NA NA NA NA NA NA NA 8 0 4 NA NA NA NA NA NA NA NA 8 0 

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. 

LOQ=<0.001 mg a.i./L. 

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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 

Infection, 

Microsporidia 

n Incidence n Incidence n Incidence n Incidence 

Control (<LOQ) 0 16 12 NA NA NA NA NA NA 

1 16 3 NA NA NA NA NA NA 

2 16 1 NA NA NA NA NA NA 

3 16 0 NA NA NA NA NA NA 

4 16 0 NA NA NA NA NA NA 

Solvent control 

(<LOQ) 

0 14 10 NA NA NA NA NA NA 

1 14 2 NA NA NA NA NA NA 

2 14 1 NA NA NA NA NA NA 

3 14 1 NA NA NA NA NA NA 

4 14 0 NA NA NA NA NA NA 

0.021 0 14 9 NA NA NA NA NA NA 

1 14 4 NA NA NA NA NA NA 

2 14 1 NA NA NA NA NA NA 

3 14 0 NA NA NA NA NA NA 

4 14 0 NA NA NA NA NA NA 

0.21 0 16 6 NA NA NA NA NA NA 

1 16 4 NA NA NA NA NA NA 

2 16 2 NA NA NA NA NA NA 

3 16 1 NA NA NA NA NA NA 

4 16 0 NA NA NA NA NA NA 

2.1 0 14 3 NA NA NA NA NA NA 

1 14 6 NA NA NA NA NA NA 

2 14 4 NA NA NA NA NA NA 

3 14 1 NA NA NA NA NA NA 

4 14 0 NA NA NA NA NA NA 

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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. 

LOQ=0.001 mg a.i./L 

Table 18: Additional Gonadal Histopathology Observations in Female Fathead Minnow (Pimephales 

promelas).

Treatment 

(mg a.i./L) 

[meanmeasured] 

Additional Diagnostic Observations1

Severity 

Interstitial Fluid 

Egg Debris in 

Oviduct 

Granulomatous 

Inflammation 

Macrophage 

Aggregate 

n Incidence n Incidence n Incidence n Incidence 

Control 

(<LOQ) 

0 16 15 NA NA 16 15 16 9 

1 16 1 NA NA 16 0 16 7 

2 16 0 NA NA 16 1 16 0 

3 16 0 NA NA 16 0 16 0 

4 16 0 NA NA 16 0 16 0 

Solvent control 

(<LOQ) 

0 14 13 NA NA 14 0 14 13 

1 14 0 NA NA 14 0 14 1 

2 14 0 NA NA 14 0 14 0 

3 14 1 NA NA 14 0 14 0 

4 14 0 NA NA 14 0 14 0 

0.021 0 14 11 NA NA 14 12 14 10 

1 14 1 NA NA 14 1 14 3 

2 14 1 NA NA 14 1 14 1 

3 14 1 NA NA 14 0 14 0 

4 14 0 NA NA 14 0 14 0 

0.21 0 16 15 NA NA 16 15 16 13 

1 16 1 NA NA 16 1 16 2 

2 16 0 NA NA 16 0 16 1 

3 16 0 NA NA 16 0 16 0 

4 16 0 NA NA 16 0 16 0 

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Treatment 

(mg a.i./L) 

[meanmeasured] 

Additional Diagnostic Observations1

Severity 

Interstitial Fluid 

Egg Debris in 

Oviduct 

Granulomatous 

Inflammation 

Macrophage 

Aggregate 

n Incidence n Incidence n Incidence n Incidence 

2.1 0 14 12 NA NA 14 13 14 13 

1 14 2 NA NA 14 0 14 0 

2 14 0 NA NA 14 0 14 1 

3 14 0 NA NA 14 1 14 0 

4 14 0 NA NA 14 0 14 0 

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. 

LOQ=<0.001 mg a.i./L 

Mean male VTG was 290, 875, 1078, 238 and 533 and female VTG was 1.27x106, 1.42x106, 1.30x106, 

1.05x106

 and 8.80x105 in the negative control, solvent control, and mean-measured 0.021, 0.21, and 2.1 

mg a.i./L treatment levels, respectively. 

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Table 19: Plasma Vitellogenin in Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[mean-measured]

Plasma Vitellogenin (VTG)1

Males Females 

n Mean

(ng/mL plasma) 

±SD n Mean

(ng/mL plasma) 

±SD 

Control (<LOQ) 4 290 299 4 1.27 x 106 4.27 x 105 

Solvent control 

(<LOQ) 

4 875 1551 4 1.42 x 106 5.96 x 105 

0.021 4 1078 701 4 1.30 x 106 5.38 x 105 

0.21 4 238 189 4 1.05 x 106 2.10 x 105 

2.1 4 533 408 4 8.80 x 105 5.70 x 105 

1

 Average and standard deviations calculated by the reviewer using the individual data from the study appendices; these 

values were not reported by the study author. 

LOQ=0.001 mg a.i./L. 

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Mean male and female plasma testosterone and plasma 17ȕ-estradiol were not measured.

Table 20: Plasma Sex Steroids in Fathead Minnow (Pimephales promelas). Not measured.

Treatment 

(mg a.i./L) 

[mean-measured]

Plasma Testosterone (T) Plasma 17ȕ-estradiol (E2) 

Males Females Males Females 

n 

Mean

(ng/mL 

plasma) 

±SD n 

Mean

(ng/mL 

plasma) 

±SD n 

Mean

(ng/mL 

plasma) 

±SD n 

Mean

(ng/mL 

plasma) 

±SD 

Control (<LOQ) NA NA NA NA NA NA NA NA NA NA NA NA 

Solvent control (<LOQ) NA NA NA NA NA NA NA NA NA NA NA NA 

0.021 NA NA NA NA NA NA NA NA NA NA NA NA 

0.21 NA NA NA NA NA NA NA NA NA NA NA NA 

2.1 NA NA NA NA NA NA NA NA NA NA NA NA 

NA Not applicable. 

LOQ=0.001 mg a.i./L.

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There were some minor effects at lower test levels that the study author did not consider treatment related (one female in solvent control with red 

ovipositor on Days 4 and 5; one female with red ovipositor on Day 4 and one fish with bleeding then missing eye in 2.1 mg a.i./L; one fish in 0.02 mg a.i./L with a stomage bedding on Day 19); however, the fish exposed to the 2.1 mg a.i./L treatment were observed to have a lighter, more yellowish color compared to the fish in the controls and other treatment levels (number of incidences not reported). 

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 Type n Incidence 

Control (<LOQ) None 8 0 None 16 0 

Solvent control (<LOQ) None 9 0 None 14 0 

0.021 None 9 0 None 15 0 

0.21 None 8 0 None 16 0 

2.1 Yellowish color 8 

Not 

reported 

Yellowish color 14 

Not 

reported 

LOQ=0.001 mg a.i./L. 

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B. Study Author’s Analysis and Conclusions

The study author analyzed survival, weight, length, tubercle score, GSI, fecundity 

(#eggs/female/reproductive day), and VTG. Data for growth, tubercle score, GSI, and VTG were 

analyzed separately by sex. For all parameters dichlobenil-treated conditions were compared to the 

solvent control. 

Some descriptive statistics (mean) were determined for some endpoints. Significant effects were 

detected for p<0.05 unless otherwise noted using TOXSTAT (release 3.5). The method used to 

determine effects on survival were not reported. Effects on all other endpoints were determined by 

ANOVA and a one-tailed Dunnett’s test. The difference between negative and solvent control were 

determined by two-sided t-test. Prior to Dunnett’s, data were analyzed to test for normality using 

Shapiro-Wilks test and homogeneity of variances using an F-test. 

Adult survival (range 88-100%) was not significantly different compared to the controls (90-100%). 

There was a statistically significant decrease in fecundity at the 2.1 mg a.i./L treatment level 

compared to the solvent control (p<0.05; Dunnett’s test). There was also an increased incidence 

and/or severity of oocyte atresia and a shift towards more mature follicle stages at concentrations of 

0.21 and 2.1 mg a.i./L, suggesting reduced spawning in females exposed at these levels. 

C. Reviewer’s Analysis and Conclusions 

Statistical Methods: The reviewer analyzed survival (mortality) data using Fisher’s Exact test via 

Toxstat 3.5. Female and male weight, female length, male mean vitellogenin (VTG), female GSI, 

and female tubercle score were not consistent with a monotonic concentration-response. All data 

were tested for normality using Shapiro-Wilks test and for homogeneity of variance using Levene’s 

test. Data which met the assumptions of normality and homogeneity of variance were then analyzed 

using the parametric Dunnett’s test. Data that failed parametric assumptions were analyzed using the 

non-parametric Mann-Whitney U test. Male length, female VTG, male GSI, male tubercle score and 

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fecundity data exhibited monotonic trends and were analyzed using the non-parametric JonckheereTerpstra test, as recommended by the OCSPP 890.1350 guideline. The reviewer compared all 

dichlobenil-treated groups to the negative control group. The reviewer also compared the negative 

and solvent controls using two-sided t-tests. There were no significant differences between the 

negative and solvent control groups. 

Median male tubercle score showed a treatment-related reduction from the negative control of 18%, 

46% and 53% in the 0.021, 0.21 and 2.1 mg a.i./L treatment groups, respectively (p<0.05; 

Jonckheere-Terpstra). Fecundity exhibited a decreasing monotonic trend, with significant 53 and 

90% reductions from the negative control at the 0.21 and 2.1 mg a.i./L treatment levels, compared 

to the negative control (p<0.05; Jonckheere-Terpstra). The reviewer did not provide fertility data, 

but reported fertility was “more than 99.9% in all test groups” and that fertility of eggs from control 

animals was at least 95%. 

Fish exposed to the 2.1 mg a.i./L treatment were observed to have a lighter, more yellowish color 

compared to the fish in the controls and other treatment levels (number of incidence not reported). 

In females, there was an increased incidence and/or severity (minimal to moderate) of oocyte 

atresia and a shift to more mature stages in the ovaries in the 0.21 and 2.1 mg a.i./L treatments. 

 Conclusions: 

There was a significant decrease in fecundity (p<0.05) by 53% and 90% at the 0.21 and 2.1 mg 

a.i./L treatment levels, respectively, accompanied by increased oocyte atresia and more mature 

ovarian stages. Male tubercle scores exhibited a monotonic increase (p<0.05) of 18-53% at 0.021, 

0.21 and 2.1 mg a.i./L treatment levels, compared to the negative control. There were no significant 

differences in other endpoints. 

There were no statistically significant differences detected by the study author (p>0.05) in mean 

male or female body weights at any test level compared to the control. There were no significant 

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differences (p>0.05) in male or female body length compared to the control. Fish at the 2.1 mg 

a.i./L were reported to be more yellowish in color compared to other treatment groups and control. 

 While not statistically significant (p>0.05), the fecundity in the solvent control group was reduced by 

46% compared to the negative control. Therefore, given this observed decrease there is concern 

that the use of a solvent may have influenced the interpretation of the study. 

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Table 22: Reproductive and HPG Endpoints1,2 for Male Fathead Minnow (Pimephales promelas) in the FSTRA with Dichlobenil. 

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) 26 NA 0 NA No 0 NA NA NA NA NA 

Solvent control (<LOQ)3 18 0.236 7.63 0.646 No 203 0.485 NA NA NA NA 

0.021 21 0.041 4.24 0.500 No 272 0.067 NA NA NA NA 

0.21 14 0.008 6.99 0.304 No -18.1 0.996 NA NA NA NA 

2.1 12 0.005 29.2 0.095 No 83.6 0.782 NA NA NA NA 

Statistical Test Jonckheere-Terpstra Jonckheere-Terpstra NA Dunnett’s NA NA 

Abbreviations: Conc. Concentration. Diff. Difference. E2 17ȕ-estradiol. GSI Gonado-Somatic Index. Histo. Histopathology. NA Not applicable. T Testosterone. VTG Vitellogenin. NA

Not applicable. 

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 Comparisons between the negative and solvent control conducted using a two-sided student’s t-test assuming equal variance 

LOQ=0.001 mg a.i./L. 

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Table 23: Reproductive and HPG Endpoints1,2 for Female Fathead minnow (Pimephales promelas) in the FSTRA with Dichlobenil. 

Treatment 

(mg a.i./L) 

[mean-measured]

Fecundity Fert. Success4 Tubercle Score3 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 

Control (<LOQ) 0 NA NA NA 0 NA 0 NA No 0 NA NA NA NA NA Solvent control (<LOQ)5

-46.3 0.078 NA NA 0 NA -1.63 0.830 No 11.52 0.703 NA NA NA NA 

0.021 -37.9 0.074 NA NA 0 0.478 -14.6 0.371 No 1.93 0.614 NA NA NA NA 

0.21 -53.4 0.006 NA NA 0 >0.99 1.81 0.996 Yes -17.15 0.330 NA NA NA NA 

2.1 -90.1 <0.001 NA NA 0 >0.99 19.7 0.174 Yes -30.79 0.096 NA NA NA NA 

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

Abbreviations: Conc. Concentration. Diff. Difference. E2 17ȕ-estradiol. Fert. Fertilization. GSI Gonado-Somatic Index. Histo. Histopathology. NA Not applicable. T Testosterone. VTG

Vitellogenin.

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 One tubercle was noted for one female fish in the 0.021 mg a.i./L treatment level. 4 Replicate fertility data were not provided. Fertility was reported to be “more than 99.9% in all test groups” and “at least 95%” in the control animals. 5 Comparisons between the negative and solvent control conducted using a two-sided student’s t-test assuming equal variance 

LOQ=0.001 mg a.i./L. 

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Table 24: Growth Endpoints1,2 in the Fish Short-Term Reproduction Assay (FSTRA) with Dichlobenil.

Treatment 

(mg a.i./L) 

[mean-measured]

Body Weight Length 

Males Females Males Females 

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

Control (<LOQ) 0 NA 0 NA 0 NA 0 NA 

Solvent control (<LOQ)3 -5.80 0.601 5.06 0.270 0 0.868 0 0.791 

0.021 -2.78 0.993 5.08 0.791 0.53 0.440 0.72 0.974 

0.21 -4.59 0.972 9.84 0.360 0.53 0.470 5.07 0.089 

2.1 -3.38 0.988 5.71 0.733 1.07 0.778 3.62 0.265 

Statistical Test Dunnett’s Dunnett’s 

JonckheereTerpstra 

Dunnett’s 

Abbreviations: Diff. Difference. NA Not applicable. 

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. 

3

 Comparisons between the negative and solvent control conducted using a two-sided student’s t-test assuming equal 

variance 

LOQ=0.001 mg a.i./L. 

 E. Study Deficiencies 

During the study the fecundity was 12.1 and 6.5 eggs/female/day in the negative control and solvent 

control, respectively. Additionally, during the 14 days prior to study initiation, the fecundity was 5.3-15.3 

eggs/female/reproductive day/replicate, which the study author reported in general as spawning every 

fourth day. The EPA recommendation of at least 15 eggs/female/reproductive day or a spawning 

frequency of at least every four days in every replicate was not met in the controls (one in the negative 

and two in the solvent). Additionally, while it was reported that fertilization success exceeded 99% at 

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every level, these data were not provided for confirmation. Temperature was below the optimum range 6 

hours during the exposure period. No adverse effects were observed during this period. The remaining 

validity criteria for OCSPP 890.1350 were met. 

The reviewer noted that performance criteria for the OCSPP 890.1350 guideline were met for reported 

parameters, but the unionized ammonia and residual chlorine were not reported. EPA recommends that 

unionized ammonia in the dilution water be <1 Pg/L and residual chlorine in dilution water be <10 

Pg/L. There is uncertainty in whether the pre-exposure tanks were randomly allocated to the treatment 

groups. 

While not statistically significant (p=0.078), the fecundity in the solvent control group was reduced by 

46% compared to the negative control. However, large variability was observed between the preexposure and exposure fecundity rate for both the negative and solvent control replicates, suggesting this 

difference may not be solvent related. Additionally, this was the only endpoint for which a notable 

difference between the negative and solvent control was observed. 

These deviations do not have an impact on the acceptability of this study. 

F. Reviewer’s Comments 

The reviewer’s and the study authors’ conclusions were generally in agreement; however, there were 

some differences in the statistical results. The reviewer’s analysis using the nonparametric JonckheereTerpstra test detected significant reductions (53 and 90%) in fecundity at the 0.21 and 2.1 mg a.i./L 

treatment levels compared to the negative control, while the study author’s only detected a significant 

reduction at the 2.1 mg a.i./L treatment compared to the solvent control. The reviewer’s analysis 

detected significant reductions in median male tubercle scores at all test levels (18-53%) compared to 

the negative control, while the study author found no significant difference (p>0.05) compared to the 

solvent control. 

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There were three endpoints exhibiting a monotonic concentration response which also satisfied 

parametric assumptions (i.e., female VTG, male tubercle score, and fecundity). The OCSPP 890.1350 

guidance recommends use of the non-parametric Jonckheere-Terpstra test to detect treatment-related 

effects for any monotonic response, while the historical EFED method suggests use of William’s test 

when parametric assumptions are satisfied. Male tubercle score and fecundity produced different 

conclusions depending on the type of test. For male tubercle score, Jonckheere-Terpstra detected 

significant reductions (p<0.05) all test levels, while William’s test only detected significant reductions at 

the 0.21 and 2.1 mg a.i./L levels. For fecundity, the pattern was reversed; Jonckheere-Terpstra detected 

significant reductions at the 0.21 and 2.1 mg a.i./L levels, while William’s test detected significant 

reductions (p<0.05) at all test levels. 

There is uncertainty in whether the pre-exposure tanks were randomly allocated to the treatment groups. 

In the study report (Appendix I), the assignment of fish to the test groups is reported. The top five egg 

producing tanks are used first in which the highest tank is assigned to the negative control replicate A, 

the second-highest to the solvent control replicate A, the third highest to the lowest treatment replicate A 

group and so on until the fifth highest group is assigned to the highest treatment level. Then the next 

five highest egg producers are used and the highest from this group is assigned to the highest treatment 

group replicate, B, the next highest to the middle treatment group, and so on until the lowest of these 

next five tanks is assigned to the negative control. This pattern was then repeated with the highest tank 

in the third group of five tanks assigned to the negative control. While such a pattern could occur 

randomly, it seems unlikely that this is the pattern that would emerge. 

Based on the report, it appears that for VTG values that were non-detects, they were reported as 0.00 

instead of the 1⁄2 LOQ as recommended in the 890.1350 guideline. 

In this study, there were 3 tanks in the exposure period in which the sex ratio was 3 males and 3 

females instead of 2 males and 4 females as dictated. Two of these tanks were in the solvent control 

and one was in the 0.02 mg a.i./L treatment group. Fecundity values were adjusted to account for the 

additional male. As it could not be determined from the study report which male was mistaken for a 

female, the analysis and results in the DER for male specific endpoints included all male data. 

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EPA MRID Number 48616106 

While the solvent control fecundity was not significantly different from the negative control (p=0.078 

using a two-sided student’s t-test), a 46% reduction compared to the negative was observed. The 

reviewer compared the pre-exposure fecundity for both the negative and solvent control. The preexposure fecundity was very similar between the two groups (see table below); however it appears that 

while the solvent control replicates either generally maintained or decreased fecundity rates during the 

exposure period relative to the pre-exposure period, the negative control replicates generally increased in 

fecundity compared to the pre-exposure period. While a potential solvent interference cannot be ruled 

out, this relative increase in fecundity rate in the negative control may have contributed to the observed 

difference between the negative and solvent control. 

 Negative 

control 

Solvent 

Control 

0.021 0.21 2.1 

Fecundity (eggs/female/reprod day/replicate) 

Replicate PE E PE E PE E PE E PE E 

A 15.3 6.6 15.0 4.5 12.1 6.8 11.8 8.7 10.3 1.2 

B 8.8 11.8 9.8 11.6 9.9 6.4 10.1 5.5 10.1 2.7 

C 8.6 15.8 8.1 4.3 8.1 7.8 7.9 6.1 7.6 0.9 

D 5.3 14.3 6.1 5.6 7.0 9.1 7.1 2.3 7.1 0.0 

PE = pre-exposure; E = exposure 

III. REFERENCES 

None reported.

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APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION:

Reviewer’s statistical verification table

Endpoint Monotonic? Parametric? EDSP EFED Comments 

Combined sex Survival 

No NA Fisher’s Exact 

test: n.s. p>0.05 

Fisher’s Exact test: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Female body weight No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Male body weight No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Female body length No, increasing; 

median values, not means 

Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Male body length Yes, increasing No Jonckheere: 

n.s. p>0.05 

Jonckheere: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Female VTG Yes Yes Jonckheere: 

n.s. p>0.05 

William’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Male VTG No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect for increasing trend 

Female GSI No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect for increasing trend 

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EPA MRID Number 48616106 

Endpoint Monotonic? Parametric? EDSP EFED Comments 

Male GSI Yes, increasing No Jonckheere: 

n.s. p>0.05 

Jonckheere: n.s. p>0.05 

EDSP and EFED same conclusions, no effect for increasing trend 

Female tubercle score 

No No MannWhitney: 

n.s. p>0.05 

MannWhitney: n.s. p>0.05 

No score for control, 0.21, and 2.1 mg a.i./L groups; tubercles noted for one fish in the 0.021 mg a.i./L level. 

Male tubercle score Yes, median 

values 

Yes Jonckheere: 

Dose 1 p=0.041 Dose 2 p=0.008 Dose 3 p=0.005 

William’s: Dose 2 p=0.021 

Dose 3 p=0.021 

EDSP and EFED conclusions differ. Median values showed a significant treatment-related reduction (18-53% lower than negative control) at all treatment levels, according to Jonckheere-Terpstra. William’s test only detected significant reductions (p<0.05) at the 0.21 and 2.1 mg a.i./L levels. 

Fecundity Yes, decreasing Yes Jonckheere: 

Dose 1 p=0.074 Dose 2 p=0.006 Dose 3 p<0.001 

William’s: Dose 1 p=0.014 Dose 2 p=0.002 Dose 3 p<0.001 

EDSP and EFED conclusions differ. Mean values showed a significant treatment-related reduction (38-90% lower than negative control) at only two highest treatment levels, according to Jonckheere-Terpstra. William’s test detected significant reductions (p<0.05) at all treated levels. 

Fertility NA NA NA NA Study author did not provide replicate data. Reported that 

fertility was “at more than 99.9% in all test groups”. 

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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EPA MRID Number 48616106 

Control Comparisons 

% Mort. (all) 

% Mort. (male) 

% Mort. (female) Fecundity % Fert. 

Wt (male) 

Length (male) 

Wt. (female) 

Length (female) 

Neg Control 0 0 0 6.6 NA 2.42 4.8 0.76 3.4 

 0 0 0 11.8 NA 2.27 4.8 0.79 3.5  0 0 0 15.8 NA 1.83 4.5 0.81 3.5 

 0 0 0 14.3 NA 1.76 4.6 0.79 3.4 

Average 0.0 0.0 0.000 12.1 #DIV/0! 2.07 4.7 0.79 3.5 Solvent Control 0 0 0 4.5 NA 2.36 5.0 0.73 3.3  0 0 0 11.6 NA 1.77 4.4 0.87 3.4 

 0 0 0 4.3 NA 1.98 4.7 0.86 3.5 

 50 100 0 5.6 NA 1.68 4.7 0.86 3.7 

Average 12.5 25.0 0.000 6.5 #DIV/0! 1.95 4.7 0.83 3.5 t-test p value 0.356 0.356 #DIV/0! 0.078 #DIV/0! 0.601 0.868 0.270 0.791 

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Control Comparisons 

GSI (male) GSI (female) 

Tubercle (male) 

VTG (male) VTG (Female) 

Neg Control 1.17 10.2 28.5 0.00 1741.57  1.53 14.5 24.5 0.67 706.41  0.99 12.1 27.0 0.38 1287.48  1.03 12.5 24.5 0.11 1350.63 Average 1.18 12.3 26.1 0.29 1271.52 

Solvent Control 1.47 12.3 30.0 3.20 1839.46  1.28 12.9 12.7 0.03 1056.75  1.05 10.3 12.0 0.14 773.89  . 12.9 23.0 0.14 2001.83 Average 1.27 12.1 19.43 0.88 1417.98 t-test p value 0.646 0.830 0.182 0.485 0.703 

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EPA MRID Number 48616106 

Dichlobenil Combined Sex Survival

Fisher's Exact Test

============================================================================

NUMBER OF

----------------------------------------

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS 

-------------------------- ----- ----- ---------------

CONTROL 24 0 24

Solvent Control 23 1 24

----------------------------------------------------------------------------

TOTAL 47 1 48

============================================================================

Critical Fisher's value (24,24,24) (alpha=0.05) is 19.0. b value is 23.

Since b is greater than 19.0 there is no significant difference

between CONTROL and TREATMENT at the 0.05 level.

Fisher's Exact Test

============================================================================

NUMBER OF

----------------------------------------

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS 

-------------------------- ----- ----- ---------------

CONTROL 24 0 24

0.021 mg/L 24 0 24

----------------------------------------------------------------------------

TOTAL 48 0 48

============================================================================

Critical Fisher's value (24,24,24) (alpha=0.05) is 19.0. b value is 24.

Since b is greater than 19.0 there is no significant difference

between CONTROL and TREATMENT at the 0.05 level.

Fisher's Exact Test

============================================================================

NUMBER OF

----------------------------------------

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS 

-------------------------- ----- ----- ---------------

CONTROL 24 0 24

0.21 mg/L 24 0 24

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EPA MRID Number 48616106 

----------------------------------------------------------------------------

TOTAL 48 0 48

============================================================================

Critical Fisher's value (24,24,24) (alpha=0.05) is 19.0. b value is 24.

Since b is greater than 19.0 there is no significant difference

between CONTROL and TREATMENT at the 0.05 level.

Fisher's Exact Test

============================================================================

NUMBER OF

----------------------------------------

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS 

-------------------------- ----- ----- ---------------

CONTROL 24 0 24

2.1 mg/L 22 2 24

----------------------------------------------------------------------------

TOTAL 46 2 48

============================================================================

Critical Fisher's value (24,24,24) (alpha=0.05) is 19.0. b value is 22.

Since b is greater than 19.0 there is no significant difference

between CONTROL and TREATMENT at the 0.05 level.

Summary of Fisher's Exact Tests 

----------------------------------------------------------------------------

NUMBER NUMBER SIG 

GROUP IDENTIFICATION EXPOSED DEAD 0.05

----- ------------------------- ----------- ----------- -----

CONTROL 24 0

1 Solvent Control 24 1

2 0.021 mg/L 24 0

3 0.21 mg/L 24 0

4 2.1 mg/L 24 2

----------------------------------------------------------------------------

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EPA MRID Number 48616106 

027401 Dichlobenil 890.1350 48616106

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

0.983 0.984 1.490 0.267 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS

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

Ctrl 4 0.79 0.02 0.01 2.62 0.75, 0.82

Dose1 4 0.83 0.07 0.03 8.45 0.72, 0.94

Dose2 4 0.87 0.10 0.05 11.54 0.71, 1.02

Dose3 4 0.83 0.08 0.04 10.02 0.70, 0.97

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

Ctrl 0.79 0.76 0.81 . .

Dose1 0.83 0.74 0.91 105.08 -5.08

Dose2 0.83 0.79 1.01 109.84 -9.84

Dose3 0.87 0.71 0.89 105.71 -5.71

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

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 0.73 0.556

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 0.79 . 0.83 . . . . . .

Dose1 0.83 0.791 0.83 0.849 . . . . .

Dose2 0.87 0.360 0.83 0.876 0.891 . . . .

Dose3 0.83 0.733 0.83 0.890 1.000 0.925 . . .

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

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 2.81 0.422

MannWhit - testing each trt median signif. different from control

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

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EPA MRID Number 48616106 

Level Median MannWhit p-value Jonckheere p-value

Ctrl 0.79 . .

Dose1 0.83 0.343 0.877

Dose2 0.83 0.180 0.940

Dose3 0.87 0.343 0.933

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 0.73 0.556

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 -0.79 . -0.79 . . . . . .

Dose1 -0.83 0.791 -0.83 0.276 . . . . .

Dose2 -0.87 0.360 -0.85 0.170 0.891 . . . .

Dose3 -0.83 0.733 -0.85 0.176 1.000 0.925 . . .

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

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 2.81 0.422

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 -0.79 . .

Dose1 -0.83 0.343 0.123

Dose2 -0.83 0.180 0.060

Dose3 -0.87 0.343 0.067

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 Fish Short-Term Reproduction Assay with Dichlobenil 

EPA MRID Number 48616106 

027401 Dichlobenil 890.1350 48616106

ANALYSIS RESULTS FOR VARIABLE VAR02 ( M body weight (g) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01

Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05

Use parametric analyses if neither test rejected, otherwise non-parametric

analyses.

Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value

0.950 0.494 1.113 0.382 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS

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

Ctrl 4 2.07 0.32 0.16 15.68 1.55, 2.59

Dose1 4 2.01 0.21 0.10 10.26 1.68, 2.34

Dose2 4 1.98 0.34 0.17 17.16 1.44, 2.51

Dose3 4 2.00 0.59 0.29 29.29 1.07, 2.93

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

Ctrl 2.05 1.76 2.42 . .

Dose1 1.95 1.84 2.31 97.22 2.78

Dose2 1.98 1.56 2.39 95.41 4.59

Dose3 1.84 1.50 2.83 96.62 3.38

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

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

Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value

3 12 0.04 0.988

Dunnett - testing each trt mean signif. different than control

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

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values

p-value mean p-value Dose1 Dose2 Dose3 Dose4

Dose5

Ctrl 2.07 . 2.07 . . . . . .

Dose1 2.01 0.993 2.01 0.494 . . . . .

Dose2 1.98 0.972 1.99 0.485 0.999 . . . .

Dose3 2.00 0.988 1.99 0.502 1.000 1.000 . . .

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

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

Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value

3 0.46 0.928

MannWhit - testing each trt median signif. different from control

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

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EPA MRID Number 48616106 

Level Median MannWhit p-value Jonckheere p-value

Ctrl 2.05 . .

Dose1 1.95 0.889 0.614

Dose2 1.98 0.889 0.529

Dose3 1.84 0.678 0.320

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN 

CONTROL

Williams >highest dose (no sign. 

differences)

Jonckheere >highest dose (no sign. 

differences)

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

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

Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value

3 12 0.04 0.988

Dunnett - testing each trt mean signif. different than control

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

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values

p-value mean p-value Dose1 Dose2 Dose3 Dose4

Dose5

Ctrl -2.07 . -2.01 . . . . . .

Dose1 -2.01 0.993 -2.01 0.666 . . . . .

Dose2 -1.98 0.972 -2.01 0.701 0.999 . . . .

Dose3 -2.00 0.988 -2.01 0.720 1.000 1.000 . . .

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

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

Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value

3 0.46 0.928

MannWhit - testing each trt median signif. different from control

Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl -2.05 . .

Dose1 -1.95 0.889 0.386

Dose2 -1.98 0.889 0.471

Dose3 -1.84 0.678 0.680

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 Fish Short-Term Reproduction Assay with Dichlobenil 

EPA MRID Number 48616106 

027401 Dichlobenil 890.1350 48616106

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

0.977 0.937 0.387 0.764 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS

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

Ctrl 4 34.50 0.58 0.29 1.67 33.58, 35.42

Dose1 4 34.75 0.96 0.48 2.76 33.23, 36.27

Dose2 4 36.25 1.26 0.63 3.47 34.25, 38.25

Dose3 4 35.75 1.26 0.63 3.52 33.75, 37.75

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

Ctrl 34.50 34.00 35.00 . .

Dose1 34.50 34.00 36.00 100.72 -0.72

Dose2 36.00 35.00 38.00 105.07 -5.07

Dose3 36.00 34.00 37.00 103.62 -3.62

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

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 2.47 0.112

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 34.50 . 35.31 . . . . . .

Dose1 34.75 0.974 35.31 0.913 . . . . .

Dose2 36.25 0.089 35.31 0.933 0.235 . . . .

Dose3 35.75 0.265 35.31 0.942 0.554 0.905 . . .

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

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 5.80 0.122

MannWhit - testing each trt median signif. different from control

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EPA MRID Number 48616106 

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

Level Median MannWhit p-value Jonckheere p-value

Ctrl 34.50 . .

Dose1 34.50 0.879 0.624

Dose2 36.00 0.093 0.981

Dose3 36.00 0.220 0.982

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 2.47 0.112

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 -34.50 . -34.50 . . . . . .

Dose1 -34.75 0.974 -34.75 0.440 . . . . .

Dose2 -36.25 0.089 -36.00 0.041 0.235 . . . .

Dose3 -35.75 0.265 -36.00 0.042 0.554 0.905 . . .

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

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 5.80 0.122

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 -34.50 . .

Dose1 -34.50 0.879 0.376

Dose2 -36.00 0.093 0.019

Dose3 -36.00 0.220 0.018

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

Williams Dose2

Jonckheere Dose2

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

EPA MRID Number 48616106 

027401 Dichlobenil 890.1350 48616106

ANALYSIS RESULTS FOR VARIABLE VAR04 ( M body length (mm) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01

Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05

Use parametric analyses if neither test rejected, otherwise non-parametric

analyses.

Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

Test Stat P-value Test Stat P-value

0.845 0.012 3.891 0.037 USE NON-PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS

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

Ctrl 4 46.75 1.50 0.75 3.21 44.36, 49.14

Dose1 4 47.00 0.82 0.41 1.74 45.70, 48.30

Dose2 4 47.00 1.41 0.71 3.01 44.75, 49.25

Dose3 4 47.25 5.32 2.66 11.25 38.79, 55.71

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

Ctrl 47.00 45.00 48.00 . . 

Dose1 47.00 46.00 48.00 100.53 -0.53

Dose2 46.50 46.00 49.00 100.53 -0.53

Dose3 45.50 43.00 55.00 101.07 -1.07

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

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 0.02 0.996

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 46.75 . 47.00 . . . . . .

Dose1 47.00 0.999 47.00 0.634 . . . . .

Dose2 47.00 0.999 47.00 0.669 1.000 . . . .

Dose3 47.25 0.989 47.00 0.688 0.999 0.999 . . .

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

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

Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value

3 1.23 0.747

MannWhit - testing each trt median signif. different from control

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

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

EPA MRID Number 48616106 

Level Median MannWhit p-value Jonckheere p-value

Ctrl 47.00 . .

Dose1 47.00 1.000 0.560

Dose2 46.50 0.886 0.530

Dose3 45.50 0.673 0.222

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 0.02 0.996

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 -46.75 . -46.75 . . . . . .

Dose1 -47.00 0.999 -47.00 0.531 . . . . .

Dose2 -47.00 0.999 -47.00 0.564 1.000 . . . .

Dose3 -47.25 0.989 -47.25 0.527 0.999 0.999 . . . 

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

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

Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value

3 1.23 0.747

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 -47.00 . .

Dose1 -47.00 1.000 0.440

Dose2 -46.50 0.886 0.470

Dose3 -45.50 0.673 0.778

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

Williams >highest dose (no sign. 

differences)

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

EPA MRID Number 48616106 

Jonckheere >highest dose (no sign.

differences)

027401 Dichlobenil 890.1350 48616106

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

0.961 0.672 0.891 0.474 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS

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

Ctrl 4 1271523 426930.7 213465.3 33.58 592180.5, 1950864

Dose1 4 1296068 538855.4 269427.7 41.58 438628.3, 2153507

Dose2 4 1053518 210200.9 105100.4 19.95 719041.0, 1387994

Dose3 4 880077.5 569767.7 284883.8 64.74 -26550.0, 1786705

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

Ctrl 1319055 706410.0 1741570 . .

Dose1 1189470 849070.0 1956260 101.93 -1.93

Dose2 1043280 869300.0 1258210 82.85 17.15

Dose3 793595.0 282160.0 1650960 69.21 30.79

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

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 0.73 0.551

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 1271523 . 1283795 . . . . . .

Dose1 1296068 1.000 1283795 0.599 . . . . .

Dose2 1053518 0.841 1053518 0.328 0.876 . . . .

Dose3880077.5 0.506 880077.5 0.164 0.590 0.949 . . .

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

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 2.01 0.571

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

EPA MRID Number 48616106 

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 1319055 . .

Dose1 1189470 0.889 0.614

Dose2 1043280 0.346 0.330

Dose3 793595.0 0.494 0.096

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 0.73 0.551

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 -1271523 . -1125296 . . . . . .

Dose1-1296068 1.000 -1125296 0.757 . . . . .

Dose2-1053518 0.841 -1125296 0.790 0.876 . . . .

Dose3 -880078 0.506 -1125296 0.807 0.590 0.949 . . .

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

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 2.01 0.571

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 -1319055 . .

Dose1 -1189470 0.889 0.386

Dose2 -1043280 0.346 0.670

Dose3 -793595 0.494 0.904

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

Page 70 of 83

Page 239 of 331
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Dichlobenil 

EPA MRID Number 48616106 

Williams >highest dose (no sign. 

differences)

Jonckheere >highest dose (no sign. 

differences)

027401 Dichlobenil 890.1350 48616106

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

0.937 0.318 1.421 0.285 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS

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

Ctrl 4 290.00 299.44 149.72 103.26 -186.48, 766.48

Dose1 4 1077.50 701.06 350.53 65.06 -38.05, 2193.05

Dose2 4 237.50 189.45 94.73 79.77 -63.96, 538.96

Dose3 4 532.50 408.03 204.02 76.63 -116.77, 1181.77

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

Ctrl 245.00 0.00 670.00 . .

Dose1 895.00 460.00 2060.00 371.55 -271.55

Dose2 200.00 50.00 500.00 81.90 18.10

Dose3 450.00 130.00 1100.00 183.62 -83.62

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

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 3.01 0.072

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 290.00 . 683.75 . . . . . .

Dose1 1077.50 0.067 683.75 0.936 . . . . .

Dose2 237.50 0.996 385.00 0.739 0.081 . . . .

Dose3 532.50 0.782 385.00 0.758 0.346 0.783 . . .

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

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

Kruskal-Wallis test - equality among treatment groups

Page 71 of 83

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

EPA MRID Number 48616106 

Degrees of Freedom TestStat P-value

3 6.11 0.106

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 245.00 . .

Dose1 895.00 0.103 0.978

Dose2 200.00 1.000 0.500

Dose3 450.00 0.346 0.610

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 3.01 0.072

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 -290.00 . -290.00 . . . . . .

Dose1-1077.50 0.067 -615.83 0.190 . . . . .

Dose2 -237.50 0.996 -615.83 0.203 0.081 . . . .

Dose3 -532.50 0.782 -615.83 0.210 0.346 0.783 . . .

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

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 6.11 0.106

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 -245.00 . .

Dose1 -895.00 0.103 0.022

Dose2 -200.00 1.000 0.500

Dose3 -450.00 0.346 0.390

Page 72 of 83

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

EPA MRID Number 48616106 

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

Williams >highest dose (no sign. 

differences)

Jonckheere >highest dose (no sign. 

differences)

027401 Dichlobenil 890.1350 48616106

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

0.934 0.279 0.423 0.740 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS

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

Ctrl 4 12.31 1.75 0.88 14.22 9.52, 15.09

Dose1 4 10.51 1.20 0.60 11.38 8.61, 12.41

Dose2 4 12.53 1.95 0.97 15.53 9.43, 15.63

Dose3 4 14.73 2.01 1.01 13.67 11.53, 17.94

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

Ctrl 12.30 10.18 14.45 . .

Dose1 10.68 8.90 11.79 85.40 14.60

Dose2 12.18 10.71 15.06 101.81 -1.81

Dose3 14.11 13.09 17.62 119.70 -19.70

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

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

Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value

3 12 3.88 0.038

Dunnett - testing each trt mean signif. different than control

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

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values

p-value mean p-value Dose1 Dose2 Dose3 Dose4

Dose5

Ctrl 12.31 . 12.52 . . . . . .

Dose1 10.51 0.371 12.52 0.654 . . . . .

Dose2 12.53 0.996 12.52 0.689 0.401 . . . .

Dose3 14.73 0.174 12.52 0.708 0.024 0.331 . . .

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

Page 73 of 83

Page 242 of 331
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Dichlobenil 

EPA MRID Number 48616106 

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 7.61 0.055

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 12.30 . .

Dose1 10.68 0.235 0.074

Dose2 12.18 0.889 0.558

Dose3 14.11 0.156 0.969

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 3.88 0.038

Dunnett - testing each trt mean signif. different than control

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

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values

p-value mean p-value Dose1 Dose2 Dose3 Dose4

Dose5

Ctrl -12.31 . -11.41 . . . . . .

Dose1 -10.51 0.371 -11.41 0.837 . . . . .

Dose2 -12.53 0.996 -12.53 0.539 0.401 . . . .

Dose3 -14.73 0.174 -14.73 0.047 0.024 0.331 . . .

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

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 7.61 0.055

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 -12.30 . .

Dose1 -10.68 0.235 0.926

Dose2 -12.18 0.889 0.442

Page 74 of 83

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

EPA MRID Number 48616106 

Dose3 -14.11 0.156 0.031

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN

CONTROL

Williams Dose3

Jonckheere Dose3

027401 Dichlobenil 890.1350 48616106

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

0.934 0.286 7.780 0.004 USE NON-PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS

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

Ctrl 4 1.18 0.25 0.12 20.83 0.79, 1.57

Dose1 4 1.23 0.46 0.23 37.13 0.50, 1.96

Dose2 4 1.26 0.25 0.13 20.07 0.86, 1.67

Dose3 4 1.53 0.07 0.04 4.62 1.41, 1.64

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

Ctrl 1.10 0.99 1.53 . .

Dose1 1.23 0.82 1.65 104.24 -4.24

Dose2 1.21 1.03 1.61 106.99 -6.99

Dose3 1.52 1.46 1.60 129.24 -29.24

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

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

Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value

3 12 1.12 0.378

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 1.18 . 1.30 . . . . . .

Dose1 1.23 0.990 1.30 0.798 . . . . .

Dose2 1.26 0.957 1.30 0.829 0.999 . . . .

Dose3 1.53 0.267 1.30 0.845 0.503 0.594 . . .

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

Page 75 of 83

Page 244 of 331
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Dichlobenil 

EPA MRID Number 48616106 

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

Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value

3 2.05 0.563

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 1.10 . .

Dose1 1.23 1.000 0.500

Dose2 1.21 0.580 0.696

Dose3 1.52 0.156 0.905

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN 

CONTROL

Williams >highest dose (no sign. 

differences)

Jonckheere >highest dose (no sign. 

differences)

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

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

Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value

3 12 1.12 0.378

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 -1.18 . -1.18 . . . . . .

Dose1 -1.23 0.990 -1.23 0.480 . . . . .

Dose2 -1.26 0.957 -1.26 0.441 0.999 . . . .

Dose3 -1.53 0.267 -1.53 0.076 0.503 0.594 . . .

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

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

Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value

3 2.05 0.563

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 -1.10 . .

Dose1 -1.23 1.000 0.500

Dose2 -1.21 0.580 0.304

Page 76 of 83

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

EPA MRID Number 48616106 

Dose3 -1.52 0.156 0.095

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

Williams >highest dose (no sign. 

differences)

Jonckheere >highest dose (no sign. 

differences)

027401 Dichlobenil 890.1350 48616106

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

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 0.00 0.00 0.00 . . , .

Dose1 4 0.08 0.15 0.08 200.00 -0.16, 0.31

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 0.30 . .

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

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 0.00 . 0.04 . . . . . .

Dose1 0.08 0.389 0.04 0.833 . . . . .

Dose2 0.00 1.000 0.00 0.618 0.515 . . . .

Dose3 0.00 1.000 0.00 0.636 0.515 1.000 . . .

Page 77 of 83

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

EPA MRID Number 48616106 

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

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 negative trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl 0.00 . .

Dose1 0.00 0.478 0.841

Dose2 0.00 1.000 0.500

Dose3 0.00 1.000 0.327

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 0.00 . 0.00 . . . . . .

Dose1 -0.08 0.389 -0.03 0.384 . . . . .

Dose2 0.00 1.000 -0.03 0.410 0.515 . . . .

Dose3 0.00 1.000 -0.03 0.425 0.515 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 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 0.00 . .

Page 78 of 83

Page 247 of 331
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Dichlobenil 

EPA MRID Number 48616106 

Dose1 0.00 0.478 0.159

Dose2 0.00 1.000 0.500

Dose3 0.00 1.000 0.673

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

Williams >highest dose (no sign. 

differences)

Jonckheere >highest dose (no sign. 

differences)

027401 Dichlobenil 890.1350 48616106

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

0.931 0.257 1.601 0.241 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS

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

Ctrl 4 26.13 1.97 0.99 7.56 22.98, 29.27

Dose1 4 17.93 10.33 5.17 57.65 1.48, 34.37

Dose2 4 14.38 6.39 3.20 44.49 4.20, 24.55

Dose3 4 14.75 5.84 2.92 39.58 5.46, 24.04

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

Ctrl 25.75 24.50 28.50 . .

Dose1 21.10 3.00 26.50 68.61 31.39

Dose2 14.00 7.00 22.50 55.02 44.98

Dose3 12.00 11.50 23.50 56.46 43.54

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

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 2.57 0.103

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 26.13 . 26.13 . . . . . .

Dose1 17.93 0.257 17.93 0.067 . . . . .

Page 79 of 83

Page 248 of 331
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Dichlobenil 

EPA MRID Number 48616106 

Dose2 14.38 0.077 14.56 0.021 0.880 . . . .

Dose3 14.75 0.088 14.56 0.021 0.910 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 7.73 0.052

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 25.75 . .

Dose1 21.10 0.154 0.041

Dose2 14.00 0.066 0.008

Dose3 12.00 0.065 0.005

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 2.57 0.103

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 -26.13 . -18.29 . . . . . .

Dose1 -17.93 0.257 -18.29 0.969 . . . . .

Dose2 -14.38 0.077 -18.29 0.978 0.880 . . . .

Dose3 -14.75 0.088 -18.29 0.982 0.910 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 7.73 0.052

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 -25.75 . .

Page 80 of 83

Page 249 of 331
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Dichlobenil 

EPA MRID Number 48616106 

Dose1 -21.10 0.154 0.959

Dose2 -14.00 0.066 0.992

Dose3 -12.00 0.065 0.995

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

Williams >highest dose (no sign. 

differences)

Jonckheere >highest dose (no sign. 

differences)

027401 Dichlobenil 890.1350 48616106

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

0.945 0.409 1.847 0.192 USE PARAMETRIC TESTS

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

BASIC SUMMARY STATISTICS

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

Ctrl 4 12.13 4.04 2.02 33.29 5.70, 18.55

Dose1 4 7.53 1.20 0.60 16.00 5.61, 9.44

Dose2 4 5.65 2.63 1.31 46.55 1.47, 9.83

Dose3 4 1.20 1.12 0.56 93.54 -0.59, 2.99

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

Ctrl 13.05 6.60 15.80 . .

Dose1 7.30 6.40 9.10 62.06 37.94

Dose2 5.80 2.30 8.70 46.60 53.40

Dose3 1.05 0.00 2.70 9.90 90.10

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

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 12.64 <.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 12.13 . 12.13 . . . . . .

Dose1 7.53 0.063 7.53 0.014 . . . . .

Page 81 of 83

Page 250 of 331
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Dichlobenil 

EPA MRID Number 48616106 

Dose2 5.65 0.010 5.65 0.002 0.729 . . . .

Dose3 1.20 <.001 1.20 <.001 0.019 0.116 . . . 

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

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 11.23 0.011

MannWhit - testing each trt median signif. different from control

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

Level Median MannWhit p-value Jonckheere p-value

Ctrl 13.05 . .

Dose1 7.30 0.235 0.074

Dose2 5.80 0.103 0.006

Dose3 1.05 0.067 <.001

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN 

CONTROL

Williams Dose1

Jonckheere Dose2

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

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 12.64 <.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 -12.13 . -6.63 . . . . . . 

Dose1 -7.53 0.063 -6.63 0.999 . . . . .

Dose2 -5.65 0.010 -6.63 0.999 0.729 . . . .

Dose3 -1.20 <.001 -6.63 0.999 0.019 0.116 . . .

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

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 11.23 0.011

MannWhit - testing each trt median signif. different from control

Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl -13.05 . .

Page 82 of 83

Page 251 of 331
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Dichlobenil 

EPA MRID Number 48616106 

Dose1 -7.30 0.235 0.926

Dose2 -5.80 0.103 0.994

Dose3 -1.05 0.067 1.000

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN 

CONTROL

Williams >highest dose (no sign. 

differences)

Jonckheere >highest dose (no sign. 

differences)

Page 83 of 83

Page 252 of 331
DATA EVALUATION RECORD 

DICHLOBENIL 

Study Type: OCSPP 890.1400, In vivo Hershberger Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-27-2012 (MRID 48616107) 

Prepared for 

Health Effects Division 

Office of Pesticide Programs 

U.S. Environmental Protection Agency 

2777 South Crystal Drive 

Arlington, VA 22202 

Prepared by 

CSS-Dynamac Corporation 

1910 Sedwick Road, 

Building 100, Suite B 

Durham, NC 27713 

Primary Reviewer Signature:

Rebecca L. Bryan, B.S. Date: 3/20/2012 

Secondary Reviewer: Signature:

Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 3/30/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 4/05/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 4/05/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 253 of 331
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 254 of 331
DICHLOBENIL/027401 

Primary Reviewer: Greg Akennan, Ph.D. 

Health Effects Division 

Secondary Reviewer: John Liccione, Ph.D. 

Health Effects Division 

In vivo Hershberger Assay (2011)/ Page I of 16 

OCSPP 890.1400/ OECD 441 

Signature: -=--~...........-~--;---­

Date: ~..._...,~~~rf15.-= 

S igna tu re: --+---1""-----+----r---r----

D ate: --~'----'-'"./-L-..L__--

DAT A EVALUATION RECORD 

STUDY TYPE: Jn Vivo Hershberger Assay (Rat); OCSPP 890.1400; OECD 441 

PC CODE: 027401 DP BARCODE: D397912 

TXR#: 0051691 CAS#: 1194-65-6 

TEST MATERIAL (PURITY): Dichlobenil (99.3% a.i.) 

SYNONYMS: 2,6-dichlorobenzonitrile 

CITATION: Coder, P.S. (2011). The Hershberger Assay ofDichlobenil Administered Orally 

in Peripubertal Orchidoepidymectomized Rats. WIL Research Laboratories, 

LLC, Ashland, OH. Laboratory Project Study ID: WIL-12434. December 7, 

2011. MRID 48616107. Unpublished. 

SPONSOR: Chemtura Corporation, 199 Benson Road, Middlebury, CT 

TEST ORDER#: EDSP-027401-120 

EXECUTIVE SUMMARY: In a Hershberger assay (MRID 48616107) screening for 

androgenic activity, dichlobenil (99.3% a.i., Lot# AM9K12A222) in 1.0% gum tragacanth was 

administered daily via oral gavage to groups of 55-day old, castrated male Sprague Dawley rats 

(6/group) at dose levels ofO (vehicle), 20, 60, or 180 mg/kg/day. An androgenic positive control 

group consisting of six castrated rats exposed to 0.2 mg/kg/day testosterone propionate (TP) by 

subcutaneous (s.c.) injection was also included as a positive control. 

To screen for potential anti-androgenic activity, dichlobenil in 1.0% gum tragacanth was also 

administered daily via oral gavage to 55-day old, castrated male Sprague Dawley rats (6/group) 

at dose levels of 20, 60, or 180 mg/kg/day in conjunction with a daily dose ofreference androgen 

TP at 0.2 mg/kg/day by s.c. injection. The vehicle control groups consisted of six castrated male 

rats dosed daily with TP (0.2 mg/kg/day) by s.c. injection in com oil, and the anti-androgenic 

positive control group consisted of six castrated male rats dosed orally with flutamide (FT) in 

com oil at 3 mg/kg/day in conjugation with a daily s.c. dose of TP (0.2 mg/kg/day). 

For both components of the assay, the animals were dosed for 10 consecutive days and 

necropsied approximately 24 hours after the final dose administration to detennine weights of 

liver, kidneys and the five androgen-dependent tissues. 

All animals survived until scheduled tennination. There were no treatment-related clinical signs 

of toxicity. 

Page 255 of 331
In vivo Hershberger Assay (2011)/ Page 2 of 16 

DICHLOBENIL/027401 OCSPP 890.1400/ OECD 441

In the androgen agonist assay, body weights and overall body weight gains for the 20 and 60 

mg/kg/day treatment groups were comparable to controls throughout the assay. At 180 

mg/kg/day, body weights were decreased (p<0.05) by 9-10% on Days 2-7, and 9. The overall 

(Days 0-10) body weight gains for 180 mg/kg/day were decreased (p<0.05) by 44%. Liver 

weights were not affected in the low-dose and positive control groups, but were increased 

(p<0.05) in the 60 mg/kg/day (↑24%) and 180 mg/kg/day (↑32%) groups. Kidney weights were 

unaffected, except in the TP group, in which kidney weights increased (p<0.05) by 8%. There 

were no significant changes in weight of the accessory sex organs for dichlobenil-dosed animals. 

Animals in the TP group (positive control) had increases (p<0.05) in accessory sex organ 

weights as follows: 339% in seminal vesicles, 638% in ventral prostate, 165% in levator anibulbocavernosus (LABC), 513% in Cowper’s gland, and 72% in glans penis. All %CV values 

were less than the maximum recommended guideline values. 

In the anti-androgen assay, body weights and overall body weight gains for the 20 and 60 

mg/kg/day treatment groups were comparable to controls throughout the assay. At 180 

mg/kg/day, body weights were decreased (p<0.05) by 9-11% on Days 1-10. The overall (Days 

0-10) body weight gains for 180 mg/kg/day were decreased (p<0.05) by 40%. For the positive 

control group (TP + FT), overall body weight gains were decreased (p<0.05) by 13%. No 

changes were noted in liver weight for any dose group and kidneys were only affected in the 

positive control (TP + FT) group, in which kidney weights significantly decreased (↓10%). 

Statistically significant decreases in 4 of the 5 target accessory sex organ weights were observed 

at the high dose (180 mg/kg/day). The following decreases (p<0.05) in accessory sex organ 

weights were noted at 180 mg/kg/day: 36% in seminal vesicles, 35% in ventral prostate, 27% in 

LABC, and 49% in Cowper’s gland. In addition, a decrease (21%; p<0.05) in seminal vesicle 

weight was observed at 60 mg/kg/day. Animals dosed with TP + FT (positive control) had 

decreased (p<0.05) accessory sex organ weights as follows: 73% in seminal vesicles, 75% in 

ventral prostate, 48% in LABC, 71% in Cowper’s gland, and 20% in glans penis. The maximum 

guideline-specified %CV was exceeded for the glans penis (20% vs. 17%) in the TP +FT 

(positive control) group. All other %CV values were less than the maximum recommended 

values. 

Dichlobenil was negative for androgenic activity in the Hershberger assay as no statistically 

significant increases in organ weights were seen in two or more of the five androgen sensitive 

tissues in the androgen agonist assay. Dichlobenil was positive for anti-androgenicity in the 

Hershberger assay as statistically significant decreases in organ weights were seen in four of the 

five androgen sensitive tissues in the androgen antagonist assay. However, bodyweights were 

decreased ≥10% at the dose that resulted in the changes in androgen-sensitive tissue weights 

indicating overt toxicity. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Hershberger assay (OCSPP 

890.1400). 

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

Quality Assurance statements were provided. 

 

Page 256 of 331
In vivo Hershberger Assay (2011)/ Page 3 of 16 

DICHLOBENIL/027401 OCSPP 890.1400/ OECD 441

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: WIL Research Laboratories, LLC 

Location: Ashland, OH

Study Director: P.S. Coder 

Other Personnel: Les Freshwater, MS - Contributing Scientist, Statistical Analysis 

Katherine L. Gardner, PhD - Participating Scientist, Analyses of Dosing 

Formulations 

Gwendalyn M. Maginnis, DVM - Clinical Veterinarian 

Study Period: May 23-December 7, 2011 

2. Test Substance: Dichlobenil 

 Description: White Powder 

 Source: Notox B.V. 

 Lot/Batch #: AM9K12A222, expires October 12, 2012 

 Purity: 99.3% a.i. 

 Stability: Stable in 1.0% gum tragacanth for up to 3 days frozen and stable for 8 days 

refrigerated 

 CAS #: 1194-65-6 

 Structure: 

Cl Cl

CN

3. Reference Androgen: Testosterone propionate (TP) 

 Supplier: AK Scientific (Mountain View, CA) 

 Lot/Batch #: 70321J, expires August 9, 2012 

 Purity: 98.3% 

 CAS # : 57-85-2 

4. Reference Anti-androgen: Flutamide (FT) 

 Supplier: Spectrum Chemical Manufacturing Corporation (New Brunswick, NJ) 

 Lot/Batch #: 2AC0144, expires March 1, 2013 

 Purity: 100.0% 

 CAS # : 13311-84-7 

5. Solvent/Vehicle Control: Tragacanth 

 Supplier: Spectrum Chemical Manufacturing Corporation (New Brunswick, NJ) 

 Lot/Batch #: 2AC3069, expires August 31, 2012 

 Rationale (if other than water) : Not soluble in water and 0.5% methylcellulose in deionized water created 

homogeneous suspension of test substance. 

 Final concentration: 

Other solvents: 

1.0% gum tragacanth in deionized water 

Corn Oil (used for positive control): expires July 1, 2012; Lot/Batch # 

 ZT1301; Spectrum Chemical Manufacturing Corp. (New Brunswick, NJ) 

Ethanol (used for positive control): 95% purity; expires August 2, 2013; Lot #

 ZT0426; Spectrum Chemical Manufacturing Corp. (New Brunswick, NJ) 

Page 257 of 331
In vivo Hershberger Assay (2011)/ Page 4 of 16 

DICHLOBENIL/027401 OCSPP 890.1400/ OECD 441

6. Test Animals: 

Species: Rat (castrated males) 

Strain: Sprague Dawley (Crl:CD[SD]) 

Age/weight at dose initiation: Post-natal day (PND) 55; 214.6-258.5 g 

Source: Charles River Laboratories International, Inc., Raleigh, NC 

Housing: Rats were housed individually in clean, stainless steel, suspended, wiremesh cages with cage-board 

Diet: PMI Nutrition International LLC Certified Rodent LabDiet®, ad libitum 

Water: Reverse-osmosis purified tap water, ad libitum

Environmental conditions: Temperature: 

Humidity: 

Air changes: 

Photoperiod: 

70.3-70.6 oF (21.3-21.4oC) 

45.7-59.2% 

10/hr 

12 hrs light/12 hrs dark

Acclimation period: 7 days 

B. STUDY DESIGN

1. In-life dates: Start: 6/7/11 End: 6/17/11 

2. Study Design: This Hershberger Assay was conducted to screen for both potential 

androgenic and anti-androgenic activity. In the test for androgenic activity, dichlobenil was 

administered daily via oral gavage to groups of castrated male rats. Positive androgenic 

activity is defined as a significant increase in two or more target organ weights compared to 

the vehicle control group. The positive control group for androgenic activity consisted of 

castrated male rats dosed by s.c. injection with TP at 0.2 mg/kg/day. In the test for antiandrogenic activity, dichlobenil was administered daily via oral gavage to groups of 

castrated male rats in conjunction with TP at 0.2 mg/kg/day by s.c. injection. 

Anti-androgenic activity is indicated by a statistically significant decrease in two or more 

target organ weights of the treated groups (test substance + TP) compared to the TP-only 

control group. The positive control group for anti-androgenic activity consisted of castrated 

male rats dosed by s.c. injection with TP (0.2 mg/kg/day) in conjunction with an oral dose 

of FT (3.0 mg/kg/day). 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: Following the initial 7-day acclimation period, castrated male rats 

(surgical manipulation performed by Supplier at PND 42) were administered the test 

substance from PND 55 to PND 64. Rats were euthanized on PND 65, approximately 24 

hours after the last dose and necropsied for organ weight measurements. 

4. Animal Assignment: Animals were randomly assigned, stratified by body weight, to the 

test groups noted in Table 1. Statistical analysis indicated that there were no significant 

differences in group means at study initiation. 

Page 258 of 331
In vivo Hershberger Assay (2011)/ Page 5 of 16 

DICHLOBENIL/027401 OCSPP 890.1400/ OECD 441

Table 1. Study design a

Test group Dose (mg/kg/day) # of Males 

Androgen Agonist Assay 

Vehicle control (corn oil) 0 6 

Low (Dichlobenil) 20 6 

Mid (Dichlobenil) 60 6 

High (Dichlobenil) 180 6 

Positive control (corn oil) +TP b 0.2 6 

Anti-Androgen Assay 

Vehicle control (corn oil) +TP b 0 6 

Low (Dichlobenil+TP) 20 6 

Mid (Dichlobenil+TP) 60 6 

High (Dichlobenil+TP) 180 6 

Flutamide (oral gavage) + TP (s.c.), positive control 3 6 

a Data were obtained from Table 2 on pages 18 of the study report. 

b Same animals dosed for both androgen agonist (serves as positive control) and anti-androgen (served as vehicle +TP 

control) assays. This did not affect the outcome or conclusions of this study.

5. Dose Selection Rationale: It was stated that the doses were selected based on the results of 

previous studies including a dose range-finding study (MRID 48616113) which is 

summarized in an Appendix to this DER. 

6. (a) Dose Preparation: Dose formulations were prepared by mixing the appropriate amounts 

of dichlobenil in 1.0% aqueous gum tragacanth. For test day one, the test suspensions were 

prepared on the day prior to treatment and stored refrigerated and protected from light. Starting 

on Day 2, the test suspensions were prepared daily and stored at room temperature prior to use. 

The positive control was initially dissolved in 95% ethanol solution, and then added to corn oil. 

Samples of dichlobenil from the first and last dose formulation preparations were analyzed 

for achieved concentration (reported for all formulations from June 6 and 16). In a 

concurrent uterotrophic study (MRID 48616111), dichlobenil samples at 0.02, 1, and 50 

mg/mL were analyzed from samples collected on June 1 and June 9 for homogeneity (top, 

middle, bottom) and stability (both at 3 days frozen and at 8 days refrigerated). 

(b) Dose Analysis

Results

Homogeneity (%RSD): 1.9-5.5% (with the exception of 26% RSD for the 0.02 mg/mL 

sample from June 1, 2011) 

Stability (% of time 0): 94.1-98.6% following 3 days frozen storage; 

89.9-97.9% following 8 days refrigerated storage 

Concentration (% of nominal): 107-114% 

These dose analyses were insufficient to allow an unequivocal conclusion that the mixing 

procedure was adequate and the variation between nominal and actual dosage to the animals 

Page 259 of 331
In vivo Hershberger Assay (2011)/ Page 6 of 16 

DICHLOBENIL/027401 OCSPP 890.1400/ OECD 441

was acceptable. Homogeneity was only evaluated twice, and on one of these instances, 

homogeneity in the low dose was unacceptable. Stability was acceptable under the 

conditions tested (3 days frozen or 8 days refrigerated). From Day 2, it was stated that the 

dose formulations were prepared daily and stored at room temperature, protected from light, 

until use. It was not clear how long the samples were stored at room temperature; however, 

stability data should be submitted demonstrating that dichlobenil was stable in the dose 

formulation over that storage interval at room temperature. 

7. Dosage administration: Test formulations of dichlobenil or FT were administered to the 

animals daily via oral gavage (5 mL/kg bw) for 10 consecutive days, beginning on Study 

Day 0. TP was given via s.c. injection at 0.5 mL/kg bw. The site of injection for TP was 

rotated between 4 distinct sites. Dose volumes were adjusted daily based on the concurrent 

body weight measurement. 

8. Statistics: Statistical analyses were performed by BioSTAT Consultants, Inc. using SAS® 

version 9.2 or higher software (SAS Institute, Inc.). Data statistically analyzed included the 

organ weights, daily body weights (including necropsy body weight) and cumulative body 

weight gain. Testing for agonists was handled separately from antagonists. Each endpoint 

was tested for homogeneity of variance using Levene’s test. If that test was significant at 

p=0.01, then a log transform was applied and Levene’s test was conducted on the 

transformed data. If that test was still significant then the square root transformation was 

applied to the raw data and Levene’s test was conducted again. If the test was still 

significant then a nonparametric test, as described below, was used to analyze the data. 

Because negative values are possible, the log and square root transformation were not 

evaluated for the cumulative body weight gain. 

Organ Weights: Vehicle Control vs. TP 

Organ Weights: TP vs. TP + FT 

If the variances were homogeneous then the one-sided 

t-test was performed on the organ 

weights, using p=0.05 to declare significance, looking 

for significant increases from the vehicle control. If 

the transformations were unsuccessful in making the 

variances homogeneous then the nonparametric 

Wilcoxon rank sum test was used to compare the two 

groups. The test was one-sided using p=0.05 to 

declare significance, looking for significant increases 

from the vehicle control. 

Page 260 of 331
In vivo Hershberger Assay (2011)/ Page 7 of 16 

DICHLOBENIL/027401 OCSPP 890.1400/ OECD 441

Organ Weights: Vehicle Control vs. Test Substance 

Groups 

Organ Weights: TP vs. Test Substance Groups + TP 

If the variances were homogeneous then an analysis 

of variance (ANOVA) was conducted, on the raw or 

transformed organ weight data, as appropriate. A 

one-sided Dunnett’s test was conducted, regardless of 

the outcome of the ANOVA, looking for significant 

increases in the test substance groups when compared 

with the vehicle control. If the transformations were 

unsuccessful in making the variances homogeneous 

then the nonparametric Kruskal-Wallis test was used, 

followed by Dunn’s test, to compare each of the test 

substance groups with the vehicle control or test 

substance groups + TP with the TP group. Since 

these were pre-planned pairwise comparisons, Dunn’s 

test was conducted regardless of the outcome of the 

Kruskal-Wallis test. The tests were one-sided, at the 

0.05 significance level, looking for significant 

increases from the vehicle control. 

Daily Body Weight and Cumulative Body Weight 

Gain 

The statistical analysis was identical to the organ 

weights with the exception that the testing was 2-

sided using p=0.05 to declare significance. 

The statistical analyses were considered appropriate. 

C. METHODS

1. Clinical Examinations: Cage-side checks for mortality and moribundity were conducted 

twice daily. Clinical signs of toxicity were recorded daily prior to treatment. Animals were 

also observed for signs of toxicity approximately 1-2 hours after dosing. The injection sites 

were examined daily from the initiation of dose administration for erythema, swelling, and 

other dermal findings. Erythema and swelling were evaluated in accordance with a 4-step 

grading system of very slight, slight, moderate, and severe. 

2. Body Weight: Animals were weighed at randomization, daily prior to dosing throughout 

the dosing period, and at termination. 

3. Food Consumption (Optional): Food consumption was not determined. 

4. Serum Hormone Measurements (Optional): Serum hormone measurements were not 

conducted. Approximately 24 hours following each animal's last dose, blood samples were 

collected from the vena cava of each male following anesthesia by isoflurane inhalation. 

The blood samples were allowed to clot at room temperature and then placed on ice. Serum 

was separated in a centrifuge and stored frozen (approximately −70°C) for possible future 

hormone analysis. 

5. Dissection and Measurement of Tissue and Organ Weights: On PND 65 (approximately 

24 hours after the final administration of the test substance), all rats were weighed, 

euthanized by exsanguination, and subjected to a gross necropsy. The following tissues and 

organs were examined, collected, weighed, and placed in 10% neutral-buffered formalin: 

adrenal gland, seminal vesicles with coagulating glands, Cowper's gland (bulbourethral), 

ventral prostate, glans penis, gross lesions, kidney, liver, and LABC muscle complex. With 

the exception of gross lesions, each of these organs was weighed fresh without blotting; 

Page 261 of 331
In vivo Hershberger Assay (2011)/ Page 8 of 16 

DICHLOBENIL/027401 OCSPP 890.1400/ OECD 441

paired organs were weighed together. Care was taken to minimize fluid loss from the 

Cowper's gland, seminal vesicles, and ventral prostate. 

II. RESULTS

A. OBSERVATIONS

1. Mortality: All animals survived until scheduled termination. 

2. Clinical signs of toxicity: No clinical signs of toxicity were observed in any of the dose 

groups at the daily examinations or approximately 1 to 2 hours following dose 

administration. No remarkable injection site findings were noted. 

B. BODY WEIGHT AND WEIGHT GAIN: Selected body weight and body weight gains 

for the androgen agonist assay are presented in Table 2. Body weights and overall body 

weight gains for the 20 and 60 mg/kg/day treatment groups were comparable to controls 

throughout the duration of the assay. At 180 mg/kg/day, body weights were decreased 

(p<0.05) by 9-10% on Days 2-7, and 9. The overall (Days 0-10) body weight gains for 180 

mg/kg/day were decreased (p<0.05) by 44%. 

Page 262 of 331
In vivo Hershberger Assay (2011)/ Page 9 of 16 

DICHLOBENIL/027401 OCSPP 890.1400/ OECD 441

TABLE 2. Selected Group Mean (±SD) Body Weights and Cumulative Body Weight Gains (g) in the Androgen 

Agonist Assay a

Study 

Day # 

Dose (mg/kg/day) 

Vehicle Control Positive Control 

Vehicle + TP (0.2) b Dichlobenil (20) Dichlobenil (60) Dichlobenil (180) 

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 

0 6 237.1 13.25 6 235.4 12.35 6 236.5 10.96 6 234.5 11.63 6 236.9 13.56 

1 6 244.1 14.45 6 243.4 12.79 6 237.6 10.08 6 234.2 13.87 6 228.7 15.90 

2 6 251.3 15.14 6 281.5 12.66 6 247.5 11.08 6 239.7 15.93 6 226.7* 

(↓10) 

14.38 

3 6 257.3 16.05 6 260.1 14.47 6 257.6 12.37 6 249.8 16.39 6 230.5* 

(↓10) 

20.50 

4 6 264.0 17.07 6 265.9 12.78 6 263.4 13.31 6 256.9 17.75 6 239.5* 

(↓9) 

15.02 

5 6 271.6 17.88 6 275.0 13.95 6 271.5 15.37 6 265.9 19.13 6 246.9* 

(↓9) 

12.97 

6 6 280.0 19.06 6 284.2 13.75 6 281.1 14.45 6 275.0 19.45 6 253.3* 

(↓10) 

12.93 

7 6 286.0 21.33 6 293.7 14.64 6 288.1 16.58 6 281.5 20.28 6 259.2* 

(↓9) 

11.58 

8 6 294.3 21.95 6 301.0 14.02 6 295.9 18.13 6 287.3 21.16 6 266.8 

(↓9, 

NS) 

13.72 

9 6 300.3 22.48 6 309.5 13.51 6 302.8 18.09 6 295.4 21.51 6 271.4* 

(↓10) 

14.77 

10 6 307.7 23.92 6 316.7 12.82 6 308.9 21.18 6 302.1 23.78 6 276.5 17.75 

BWG 

(Days 

0-10) 

6 70.6 11.61 6 81.2 2.71 6 72.4 10.65 6 67.7 12.55 6 39.6* 

(↓44) 

11.09 

a Data were obtained from Table 4 on page 25 and Tables S8 and S9 on pages 50-54 of the study report. Percent differences 

from controls were calculated by the reviewers and included in parentheses. 

b The positive control group for the androgen agonist assay is same as the vehicle control group for the anti-androgen assay. 

This did not affect the outcome or conclusions of this study. 

N Number of animals in the group 

SD Standard Deviation 

* Significantly different from controls at p<0.05 

Body weight and body weight gain data for the anti-androgen assay are presented in Table 3. 

Body weights and overall body weight gains for the 20 and 60 mg/kg/day treatment groups were 

comparable to controls throughout the duration of the assay. At 180 mg/kg/day, body weights 

were decreased (p<0.05) by 9-11% on Days 1-10. The overall (Days 0-10) body weight gains 

for 180 mg/kg/day were decreased (p<0.05) by 40%. For the positive control group (TP + FT), 

overall body weight gains were decreased (p<0.05) by 13%. 

Page 263 of 331
In vivo Hershberger Assay (2011)/ Page 10 of 16 

DICHLOBENIL/027401 OCSPP 890.1400/ OECD 441

TABLE 3. Selected Group Mean (±SD) Body Weights and Cumulative Body Weight Gains (g) in the AntiAndrogen Assay a

Study 

Day # 

Dose (mg/kg/day) 

Vehicle Control 

+ TP (0.2) b

Positive Control 

TP + FT 

(0.2 + 3.0) 

Dichlobenil+TP 

(20 + 0.2) 

Dichlobenil +TP 

(60 + 0.2) 

Dichlobenil +TP 

(180 + 0.2) 

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 

0 6 235.4 12.35 6 235.1 11.15 6 235.0 12.85 6 237.3 15.45 6 235.1 12.42 

1 6 243.4 12.79 6 242.3 10.04 6 239.4 11.94 6 235.4 15.77 6 222.1* 

(↓9) 

14.57 

2 6 251.5 12.66 6 247.5 10.25 6 247.2 11.61 6 244.1 16.99 6 225.8* 

(↓10) 

14.88 

3 6 260.1 14.47 6 254.7 12.87 6 254.6 13.01 6 254.2 19.39 6 231.3* 

(↓11) 

13.16 

4 6 265.9 12.78 6 260.9 11.94 6 260.2 13.16 6 260.6 19.37 6 238.1* 

(↓10) 

8.89 

5 6 275.0 13.95 6 269.8 12.35 6 268.5 13.18 6 270.1 21.22 6 248.6* 

(↓10) 

9.27 

6 6 284.2 13.75 6 278.6 12.00 6 277.4 12.82 6 279.8 21.87 6 255.3* 

(↓10) 

10.44 

7 6 293.7 14.64 6 286.4 11.31 6 285.0 14.07 6 289.1 21.91 6 262.4* 

(↓11) 

11.60 

8 6 301.0 14.02 6 291.8 12.38 6 292.5 13.59 6 296.9 24.17 6 270.6* 

(↓10) 

13.46 

9 6 309.5 13.51 6 299.9 12.67 6 299.7 13.46 6 303.6 23.91 6 278.0* 

(↓10) 

13.61 

10 6 316.7 12.82 6 305.8 11.88 6 305.0 14.08 6 311.2 26.17 6 284.2* 

(↓10) 

13.79 

BWG 

(days 

0-10) 

6 81.2 2.71 6 70.7* 

(↓13) 

6.56 6 70.0 5.71 6 73.8 12.92 6 49.1* 

(↓40) 

6.15 

a Data were obtained from Table 5 on pages 26 and Table S11- S13 on pages 59-64 of the study report. Percent differences 

from vehicle controls (calculated by the reviewers) are included in parentheses. 

b The vehicle control group for the anti-androgen assay is same as the positive control group for the androgen agonist assay. 

This did not affect the outcome or conclusions of this study. 

N Number of animals in the group 

SD Standard Deviation 

* Significantly different from vehicle controls at p<0.05. 

C. FOOD CONSUMPTION (OPTIONAL): Food consumption was not determined. 

D. SERUM HORMONE CONCENTRATIONS (OPTIONAL): Serum hormone 

concentrations were not determined. 

E. ORGAN WEIGHTS: Weights of liver, kidneys, and accessory sex organs for the androgen 

agonist assay are presented in Table 4. There were no treatment-related effects in any of the 

accessory sex organs of animals dosed with dichlobenil. Liver weights were increased 

(p<0.05) in the 60 mg/kg/day (↑24%) and 180 mg/kg/day (↑32%) groups, and the kidney 

weights were increased (p<0.05) in the TP group (↑8%). Animals in the TP group (positive 

control) had increases (p<0.05) in accessory sex organ weights as follows: 339% in seminal 

vesicles; 638% in ventral prostate; 165% in LABC; 513% in Cowper’s gland; and 72% in 

glans penis. All %CV values were less than the maximum permissible values. 

Page 264 of 331
In vivo Hershberger Assay 

DICHLOBENIL/027401 OCSPP 8

Table 4. Accessory Sex Organ Weights (mg) from Androgen Agonist Assay in Sprague-Dawley Rats a

Organ 

Dose (mg/kg/day) 

Vehicle Control Dichlobenil (20) Dichlobenil (60) Dichlobenil (180) Positiv

Vehicle + 

N Mean SD CV N Mean SD CV N Mean SD CV N Mean SD CV N Mean 

Seminal vesicles 6 90.0 9.43 11 6 81.5 17.18 21 6 88.8 8.83 10 6 76.3 8.24 11 6 395.0* (↑339) Ventral prostate 6 18.4 2.05 11 6 15.1 5.45 36 6 18.9 2.85 15 6 19.4 4.93 25 6 135.7* (↑638) LABC 6 170.5 22.39 13 6 175.9 28.42 16 6 172.3 22.26 13 6 135.2 15.55 12 6 451.3* (↑165) Cowper’s glands 6 4.8 0.85 18 6 4.9 1.08 22 6 5.3 0.85 16 6 5.3 1.08 20 6 29.4* (↑513) Glans penis 6 87.0 8.36 10 6 84.9 9.02 11 6 85.2 9.71 11 6 85.3 8.22 10 6 149.9* (↑72) Adrenal gland 6 63.6 6.56 10 6 62.2 8.63 14 6 65.8 11.42 17 6 58.6 7.38 13 6 60.5 Kidney 6 2056.7 95.0 5 6 2197.9 96.10 4 6 2166.1 241.44 11 6 2199.7 244.99 11 6 2224.1* (↑8) Liver (g) 6 12.7 1.11 9 6 14.8 2.01 14 6 15.7* (↑24) 1.99 13 6 16.8* (↑32) 1.66 10 6 13.2 

a Data were obtained from Table 6 on page 28 of the study report. Percent differences from controls were calculated by the reviewers and included in pa

b The positive control group for the androgen agonist assay is same as the vehicle control group for the anti-androgen assay. This did not affect the outcoN Number of animals in the group SD Standard Deviation CV Coefficient of Variation * Significantly different from vehicle controls at p<0.05.
 In vivo Hershberger Assay (2011)/ Page 12 of 16 

DICHLOBENIL/027401 OCSPP 890.1400/ OECD 441

Weight of liver, kidneys and accessory sex organs for the anti-androgen assay are presented in 

Table 5. At 60 mg/kg/day, the weight of seminal vesicles was significantly decreased (↓21%). 

At 180 mg/kg/day, accessory sex organ weight decreases (p<0.05) were noted as follows: 36% 

in seminal vesicles, 35% in ventral prostate, 27% in LABC, and 49% in Cowper’s gland. 

Animals dosed with TP + FT (positive control) had decreased (p<0.05) accessory sex organ 

weights as follows: 73% in seminal vesicles; 75% in ventral prostate; 48% in LABC; 71% in 

Cowper’s gland; and 20% in glans penis. No changes were noted in liver weights, but kidney 

weights for the TP +FT group were significantly decreased (↓10%). The maximum guidelinespecified %CV was exceeded for the glans penis (20% vs. 17%) in the TP +FT (positive control) 

group. All other %CV values were less than the maximum recommended Guideline values. 

Page 266 of 331
In vivo Hershberger Assay 

DICHLOBENIL/027401 OCSPP 8

Table 5. Accessory Sex Organ Weights (mg) from Anti-Androgen Agonist Assay in Sprague-Dawley Rats a

Organ 

Dose (mg/kg/day) 

Vehicle Control 

+TP (0.2) b Dichlobenil (0.2 + 20) Dichlobenil (0.2 + 60) Dichlobenil (0.2 + 180) 

T

Positi

(0

N Mean SD CV N Mean SD CV N Mean SD CV N Mean SD CV N Mean 

Seminal vesicles 6 395.0 19.14 5 6 394.6 54.55 14 6 312.2* (↓21) 56.55 18 6 250.9* (↓36) 39.61 16 6 106.8

(↓73

Ventral prostate 6 135.7 23.92 18 6 121.2 28.71 24 6 114.3 16.87 15 6 88.5* (↓35) 16.29 18 6 34.2* (↓75LABC 6 451.3 37.83 8 6 452.4 61.71 14 6 440.8 39.78 9 6 330.3* (↓27) 18.49 6 6 233.8

(↓48

Cowper’s glands 6 29.4 4.99 17 6 27.9 4.98 18 6 28.1 5.96 21 6 15.1* (↓49) 2.01 13 6 8.4* (↓71Glans penis 6 149.9 13.44 9 6 146.2 12.30 8 6 152.9 19.46 13 6 142.0 15.70 11 6 120.5

(↓20

Adrenal gland 6 60.5 6.16 10 6 57.2 5.34 9 6 55.8 4.17 7 6 54.7 11.21 21 6 61.8 Kidney 6 2224.1 164.96 7 6 2178.4 137.11 6 6 2287.9 195.39 9 6 2170.4 360.73 17 6 2002.4

(↓10

Liver (g) 6 13.2 0.86 7 6 13.3 1.65 12 6 16.8 2.78 17 6 18.5 1.08 6 6 12.8 

a Data were obtained from Tables 7 on page 28 of the study report. Percent differences from controls were calculated by the reviewers and included in b The vehicle control group for the anti-androgen assay is same as the positive control group for the androgen agonist assay. This did not affect the outc

c The %CV exceeds the maximum Guideline recommended %CV. N Number of animals in the group SD Standard Deviation CV Coefficient of Variation * Significantly different from vehicle controls at p<0.05. 
In vivo Hershberger Assay (2011)/ Page 14 of 16 

DICHLOBENIL/027401 OCSPP 890.1400/ OECD 441

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Based on the results of this study, no evidence of 

androgen agonism was noted at any dose level in this study. Anti-androgenic activity of 

dichlobenil technical was demonstrated at 180 mg/kg/day by lower mean bulbourethral 

gland, LABC muscle group, seminal vesicles/coagulating gland, and ventral prostate 

weights compared to the TP positive control group when administered orally by gavage to 

male Crl:CD(SD) rats. Androgen antagonism was not demonstrated at 20 and 60 

mg/kg/day. Higher mean liver weights were noted in the 60 and 180 mg/kg/day dichlobenil 

technical-treated groups (with or without co-administration of TP). The anti-androgenic 

positive control substance, flutamide, generally elicited the expected responses (lower 

weights of androgen-dependent organs). The androgenic positive control substance, 

testosterone propionate, elicited the expected responses (higher weights of androgendependent organs). 

B. AGENCY COMMENTS: All animals survived until scheduled termination. There were 

no treatment-related clinical signs of toxicity. 

In the androgen agonist assay, body weights and overall body weight gains for the 20 and 60 

mg/kg/day treatment groups were comparable to controls throughout the duration of the 

assay. At 180 mg/kg/day, body weights were decreased (p<0.05) by 9-10% on Days 2-7, 

and 9. The overall (Days 0-10) body weight gains for 180 mg/kg/day were decreased 

(p<0.05) by 44%. Liver weights were increased (p<0.05) in the 60 mg/kg/day (↑24%) and 

180 mg/kg/day (↑32%) groups, and the kidney weights were increased (p<0.05) in the TP 

group (↑8%). There were no statistically significant increases in any of the accessory sex 

organ weights of dichlobenil-dosed animals. Animals in the TP group (positive control) had 

the expected increases (p<0.05) in all accessory sex organ weights. All %CV values were 

less than the maximum recommended values. 

In the anti-androgen assay, body weights and overall body weight gains for the 20 and 60 

mg/kg/day treatment groups were comparable to controls throughout the assay. At 180 

mg/kg/day, body weights were decreased (p<0.05) by 9-11% on Days 1-10. The overall 

(Days 0-10) body weight gains for 180 mg/kg/day were decreased (p<0.05) by 40%. For 

the positive control group (TP + FT), overall body weight gains were decreased (p<0.05) by 

13%. There were no significant changes in liver weight, and kidney weights were only 

affected in the TP +FT group (↓10%). Statistically significant decreases in 4 of the 5 target 

accessory sex organ weights were observed at the high dose (180 mg/kg/day). The 

following decreases (p<0.05) in accessory sex organ weights were noted at 180 mg/kg/day: 

36% in seminal vesicles, 35% in ventral prostate, 27% in LABC, and 49% in Cowper’s 

gland. In addition, a decrease (21%; p<0.05) in seminal vesicle weight was observed at 60 

mg/kg/day. Animals dosed with TP + FT (positive control) had the expected decreases in 

all accessory sex organ weights. The maximum guideline-specified %CV was exceeded for 

the glans penis (20% vs. 17%) in the TP +FT (positive control) group. All other %CV 

values were less than the maximum recommended values. 

Dichlobenil was negative for androgenic activity in the Hershberger assay as no statistically 

significant increases in organ weights were seen in two or more of the five androgen 

sensitive tissues in the androgen agonist assay. Dichlobenil was positive for antiPage 268 of 331
In vivo Hershberger Assay (2011)/ Page 15 of 16 

DICHLOBENIL/027401 OCSPP 890.1400/ OECD 441

androgenicity in the Hershberger assay as statistically significant decreases in organ weights 

were seen in four of the five androgen sensitive tissues in the androgen antagonist assay. 

However, bodyweight was decreased ≥10% at the dose that resulted in the changes in 

androgen-sensitive tissue weights indicating that the results are confounded by overt 

toxicity. 

C. STUDY DEFICIENCIES: The following deficiency was noted that did not have an 

adverse effect on the results, interpretation or conclusions of this study: 

 The maximum guideline-specified %CV was exceeded for the ventral prostate (20% vs. 

17%) in the TP +FT (positive control) group. 

 

Page 269 of 331
In vivo Hershberger Assay (2011)/ Page 16 of 16 

DICHLOBENIL/027401 OCSPP 890.1400/ OECD 441

APPENDIX 

In a non-guideline range-finding study (MRID 48616113), 5 Sprague-Dawley (Crl:CD (SD)) 

rats/dose group were treated daily via oral gavage (5 mL/kg) with dichlobenil (99.3% a.i., Lot #: 

AM9K12A222) in 1.0% gum tragacanth in deionized water at doses of 0, 20, 60, or 180 

mg/kg/day for 3 (females) or 10 (males) consecutive days. All animals were observed twice 

daily for mortality and moribundity. Clinical observations, body weights, and food consumption 

were recorded daily. A complete necropsy was conducted on all rats at the scheduled euthanasia 

and selected organs were weighed. The purpose of this study was the selection of doses for the 

Hershberger and Uterotrophic assays. Results for the females will not be discussed as part of the 

dose-selection rationale for the Hershberger assay. 

No adverse treatment-related effects were observed in mortality, clinical signs of toxicity, or 

gross pathology. 

An initial body weight loss was observed on the first day of treatment at 60 and 180 mg/kg/day, 

and body weight gain was decreased (p≤0.01) by 50% on the second day of treatment at 180 

mg/kg/day. Body weights in the treated groups were similar to the controls throughout the study. 

No further decreases in body weight gains were observed after Day 2, and overall body weight 

gain (Days 0-10) in the treated groups were similar to controls. The effect on body weight gain 

was considered transient and did not impact final body weights. The decreases in body weight 

gain correlated with decreases (p≤0.01) of 38-42% in food consumption (g/animal/day) at 60 and 

180 mg/kg/day on the first day of treatment, and a decrease (p≤0.01) of 18% at 180 mg/kg/day 

on the second day. There were decreases (p≤0.05) in food consumption noted at 60 mg/kg/day 

on Days 2-3 and 8-9 (↓12-15%), but these differences were considered transient and unrelated to 

dose. Overall food consumption (Days 0-10) was decreased at 60 (↓12%; p≤0.05) and at 180 

(↓9%; not statistically significant [NS]) mg/kg/day. As these changes in food consumption were 

minor and did not result in differences in the final body weight, the effect on food consumption 

was not considered adverse in males. 

Absolute liver weight was increased (p≤0.05, except NS at 60 mg/kg/day) at all doses by 

15-58%, and relative to body liver weights were increased (p≤0.01) at all doses by 16-59%. No 

gross lesions were noted; however, this effect is considered equivocally adverse without 

microscopic pathology and clinical chemistry data. 

Based on these results, the Sponsor considered it appropriate to use 20, 60, and 180 mg/kg/day 

doses in the Hershberger assay. 

Page 270 of 331
DATA EVALUATION RECORD 

DICHLOBENIL 

Study Type: OCSPP 890.1450, Female Pubertal Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-27-2012 (MRIDs 48616108 and 48616112) 

Prepared for 

Health Effects Division 

Office of Pesticide Programs 

U.S. Environmental Protection Agency 

2777 South Crystal Drive 

Arlington, VA 22202 

Prepared by 

CSS-Dynamac Corporation 

1910 Sedwick Road, 

Building 100, Suite B 

Durham, NC 27713 

Primary Reviewer: Signature:

David A. McEwen, B.S. Date: 03/27/2012 

Secondary Reviewer: Signature:

Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 3/30/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 4/09/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 4/09/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 271 of 331
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 272 of 331
DICHLOBENIL/ 027401 

Primary Reviewer: Kit Farwell D.V.M. 

Health Effects Division 

Secondary Reviewer: Elizabeth Mendez, Ph.D. 

Health Effects Division 

Female Pubertal Assay (2011) I Page I of 16 

OCSPP 890.1450/ OECD None 

SignafuGV~~/"' Date: _ ~- _ 

Signature:~~-;.!Z!',f!~ra;!:£:fl.!~1 

Date: 'b/~k Template J;.:;i60{)8/2011 

DATA EVALUATION RECORD 

STUDY TYPE: Female Pubertal Assay; OCSPP 890.1450; OECD None. 

PC CODE: 027401 DP BARCODE: D397912 

TXR#: 0051691 CAS No: 1194-65-6 

TEST MATERIAL (PURITY): Dichlobenil (99.3%) 

SYNONYMS: 2,6-dichlorobenzonitrile 

CITATION: Sawhney Coder, P. (2011). A Pubertal Development and Thyroid Function Assay 

of Dichlobenil Administered Orally in Intact Juvenile/Peripubertal Female Rats. 

WIL Research Laboratories. LLC, Ashland, OH. Laboratory Study No.: WIL12431 , December 7, 2011. MRID 48616108. Unpublished. 

Sawhney Coder, P. (20 11). A 7-Day Dose Range-Finding Oral (Gavage) Toxicity 

Study of Dichlobenil Technical in Juvenile Rats for the Endocrine Disruption 

Screening Program. WIL Research Laboratories. LLC, Ashland, OH. Laboratory 

Study No.: WIL-12437, December 7, 2011. MRID 48616112. Unpublished. 

SPONSOR: Chemtura Corporation, 199 Benson Road, Middlebury, CT 

TEST ORDER#: EDSP-027401-120 

EXECUTIVE SUMMARY: In a Female Pubertal Assay (MRID 48616108), 15 Sprague 

Dawley rats/dose group were treated daily via oral gavage (5 mL/kg) with dichlobenil (99.3% 

a.i., lot/batch #: AM9Kl2A222) in aqueous 1.0% gum tragacanth at doses ofO (vehicle), 20, or 

60 mg/kg/day from post-natal day (PND) 22 to 42. Animals were examined for vaginal opening 

(VO) daily beginning on PND 22, and age and weight at day of attainment was recorded. Blood 

samples for chemical chemistry analyses were taken following sacrifice on PND 42. Total 

serum thyroxine (T 4) and thyroid stimulating hormone (TSH) levels were determined using 

electrochemiluminescent immunoassays and radioimmunoassays. The weights of adrenals, 

kidneys, liver, pituitary, thyroid, and urogenital organs were recorded and microscopic 

examination of the ovaries, uterus, thyroid, liver, and kidneys were performed. 

There were no dichlobenil-related effects observed on mortality, clinical signs of toxicity, body 

weights, body weight gains, age and weight at attainment of VO, mean age at first vaginal estrus, 

mean cycle length, or percent cycling and regularly cycling. Organ weights were similar 

between the control and dichlobenil treated groups with the exceptions of liver and uterus 

weights. Unadjusted, adjusted and related (to body) liver weight was increased (p<0.05) by 18-

Page 273 of 331
 Female Pubertal Assay (2011) / Page 2 of 16 

DICHLOBENIL/ 027401 OCSPP 890.1450/ OECD None

19% at 60 mg/kg/day, and relative liver weight was increased by 9% at 20 mg/kg/day. Blotted 

uterus weight was decreased by 12% (NS) and 21% (p<0.05) at 20 and 60 mg/kg/day, 

respectively, and wet uterus weight was decreased by 26% (NS) at 60 mg/kg/day. However, no 

histological changes were noted in the liver, uterus or ovaries. 

 

Serum T4 and TSH levels were altered in a dose dependent manner, but the changes did not 

attain significance. At 20 and 60 mg/kg/day, T4 levels were decreased by 4% and 7%, 

respectively, and TSH levels were increased by 18 and 22%, respectively. The changes in 

thyroid hormone levels were not corroborated by changes in thyroid weight or histology. 

The dose levels tested were determined to be adequate based on the dose dependent changes 

were seen in the uterus and the thyroid in this assay, and the results of the dose range-finding 

study. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Female Pubertal Assay 

(OCSPP 890.1450).

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

Quality Assurance statements were provided. 

 

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I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: WIL Research Laboratories. LLC 

Location: Ashland, OH 

Study Director: P. Sawhney Coder 

Other Personnel: Midori Asakawa, BVSc, DACVP - Contributing Scientist, Pathology 

Les Freshwater, MS - Contributing Scientist, Statistical Analysis 

Katherine L. Gardner, PhD - Participating Scientist, Analyses of Dosing Formulations 

Gwendalyn M. Maginnis, DVM - Clinical Veterinarian 

Kathryn S. Marks, BS, HTL (ASCP), QIHC - Principal Investigator, Histology 

Study Period: June 22, 2011 through December 7, 2011 

2. Test Substance: Dichlobenil 

 Description: White powder 

 Source: Notox B.V. (Hertogenbosch, Netherlands) 

 Lot #: AM9K12A222 (expiration date: October 12, 2012) 

 Purity: 99.3% a.i. 

 Stability: Stable in the vehicle under refrigerated storage for up to 8 days. 

 CAS #: 1194-65-6 

 Structure: Cl

Cl

N

3. Vehicle: Aqueous 1% gum tragacanth 

4. Test Animals:

 Species: Rat (females only) 

 Strain: Sprague Dawley 

 Age/Weight at 

Study Initiation: PND 22/ 44.6 g to 56.6 g 

 Source: Charles River Laboratories (Portage, MI)

 Housing: 2-3 per cage in plastic maternity cages with nesting material (Aspen BedTM1, Cincinnati Lab 

Supply, Cincinnati, OH) 

 Diet: Harlan Laboratories 2016CM Teklad Global 16% Protein Rodent Diet; ad libitum; with an 

average of 29.0 and 11.0 ppm total isoflavones (aglycone equivalent).

 Water: Reverse osmosis-purified water, ad libitum

 Environmental 

Conditions: 

Temperature: 

Humidity: 

Air changes: 

Photoperiod: 

21.3-22.6 °C 

34.5-60.5% 

10/hr 

14 hrs light/ 10 hrs dark 

B. STUDY DESIGN

1. In-Life Dates: Start: July 29, 2011 End: August 19, 2011 

2. Mating: Pregnant dams were received from the supplier on gestation day (GD) 7. Dams 

were allowed to deliver naturally and rear their pups to PND 21. To reduce variability, 

litters were culled to 10 pups/litter (5/sex when possible) on PND 4 using a computerized 

randomization procedure. Litters with fewer than 10 total pups and/or litters with fewer 

than 5 pups/sex were excluded from the study. 

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3. Animal Assignment: On PND 21, animals were randomly assigned (stratified by body 

weight) to the study groups noted in Table 1 and no groups contained littermates.

TABLE 1. Study Design a

Test group Dose (mg/kg/day) # of Females 

Control 0 15 

Low 20 15 

High 60 15 

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

4. Dose Selection Rationale: The dose levels were selected based on the results from a dose 

range-finding study (MRID 48616112, reviewed concurrently) which is summarized in an 

Appendix to this DER. 

5. Dose Preparation and Analysis: Dose formulations were prepared approximately weekly 

by mixing appropriate amounts of test substance with 1% gum tragacanth in deionized 

water; formulations were stored refrigerated and protected from light. Stability under 

refrigerated storage for up to 8 days was demonstrated for formulations between 0.02 

mg/mL and 50 mg/mL (which bracketed those in the current study) in a separate study 

(MRID 48616111, reviewed concurrently). Homogeneity (top, middle, bottom) and 

achieved concentration were evaluated from each dosing formulation (4 and 12 mg/mL) 

prepared during the in-life phase. 

Results of Dose Analysis 

Stability (% of time zero): 89.9 to 97.9% after 8 days refrigerated storage 

Concentration (% of nominal): 89.4 to 101% 

Homogeneity (% RSD): 0.78 to 2.1% 

The analytical data indicated that mixing procedure was adequate and the variation between 

nominal and actual dosage to the animals was acceptable. 

6. Dosage Administration: All doses were administered once daily by gavage from PND 22 

through PND 42 in a volume of 5 mL/kg body weight between 0700 and 0900 hours.

7. Statistics: The following statistical analyses were conducted by the performing lab. Chisquare analysis was used to determine significant difference between the cycling status 

(cycling vs. not cycling) and percent of animals cycling regularly in the treated groups 

compared to the control group. Estrous cycle length and the day of first estrus were 

subjected to a parametric one-way ANOVA to determine intergroup differences. If the 

ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test was used to 

compare the treated groups to the control group. Pathology findings presenting as a 

dichotomous response were analyzed with pairwise Fisher’s exact test to compare each 

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treated group with the control. The tests were 1-sided at the 0.05 significance level testing 

for an increase. 

The following parameters were statistically analyzed by BioSTAT Consultants, Inc. 

(Portage, MI) using SAS® version 9.2 or higher (SAS Institute, Inc., Cary, NC): absolute 

organ weights, relative (to body weight) organ weights (liver, kidneys, pituitary, and adrenal 

glands), daily body weights (including final body weight), cumulative body weight gain, 

clinical chemistry parameters, hormone levels, and VO data. 

Each endpoint was tested for homogeneity of variance using Levene’s test. If that test was 

significant at p≤0.01, then a log transform was applied and Levene’s test was conducted on 

the transformed data. If that test was still significant then the square root transformation 

was applied to the raw data and Levene’s test conducted again. If the test was still 

significant then a non-parametric test, as described below, was used to analyze the data. 

If the variances were homogeneous then an analysis of variance (ANOVA) was conducted, 

on the raw or transformed data, as appropriate. The statistical model contained a factor for 

treatment group and a blocking factor based on the time of study necropsy data. A 2-sided 

Dunnett’s test was conducted, regardless of the outcome of the ANOVA, looking for 

significant differences in the test substance groups when compared with the vehicle control. 

If the transformations were unsuccessful in making the variances homogeneous then the 

non-parametric Kruskal-Wallis test was used, ignoring the blocking factor, followed by 

Dunn’s test, to compare each of the test substance groups with the vehicle control. As these 

are pre-planned pairwise comparisons, Dunn’s test was conducted regardless of the outcome 

of the Kruskal-Wallis test. The tests were 2-sided, at the 0.05 significance level, looking for 

significant differences from the vehicle control. 

In addition, organ weights and VO data were subject to the following analyses if they met 

the homogeneity of variance criteria: 1) Analysis of covariance (ANCOVA; covariate of 

PND 21 body weight) with Dunnett’s test. The model was as described above for ANOVA 

with the exception that PND 21 body weight was included as a covariate; 2) linear trend test 

using the ANOVA model; and 3) linear trend test using the ANCOVA model. 

Histopathology findings presented as a graded response were analyzed with pairwise MannWhitney U tests to compare each treated group with the control. The tests were 1-sided at 

the 0.05 significance level testing for increased severity. The tests were two-sided for 

graded responses presented on a Grade 1-5 scale at the 0.05 significance level testing for 

increased or decreased severity. Exact p-values were calculated for the Mann-Whitney U 

test. 

Significance was denoted at 5%, 1%, and 0.1% levels. The statistical analyses were 

considered appropriate. 

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C. METHODS

1. Mortality and Clinical Examinations: All animals were examined twice daily (morning 

and afternoon) for mortality and moribundity. Animals were also examined twice daily for 

clinical signs of toxicity (prior to dosing and 1-2 hours post-dosing). 

2. Body Weight: Animals were weighed at randomization (PND 21), daily prior to dosing 

during PND 22 to 42, and at sacrifice. Body weight gains were calculated for each 

corresponding interval and for the overall (PND 22-42) treatment period. 

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

VO. Age and weight on the day of completion of VO were recorded. 

4. Estrous Cyclicity: Beginning on the day of VO, up to and including the day of necropsy, 

daily vaginal smears were obtained for determination of the stage of estrus. The age at first 

vaginal estrus was recorded. The mean age at first vaginal estrus, mean cycle length, 

percent of each group cycling, and percent of each group cycling regularly were reported.

5. Sacrifice and Pathology: All females were sacrificed randomly across all groups by 

decapitation without anesthesia on PND 42 approximately 2 hours post-dosing. It was 

stated that all animals were sacrificed by approximately 1300 hours due to normal diurnal 

fluctuation in thyroid hormone levels. Immediately following sacrifice, blood (minimum of 

2 mL per animal) was collected for T4, TSH, and clinical chemistry analyses. Serum was 

separated, and the samples were analyzed fresh (T4) or stored frozen (≤−20°C) for 

subsequent analysis (TSH). 

a. Hormone Analysis: An electrochemiluminescent immunoassay on the Cobas e411 (Roche 

Diagnostics, Indianapolis, IN) was used to analyze serum samples for the presence of T4. 

To determine the concentration of TSH, the [125I]rTSH kit obtained from Izotop, Institute of 

Isotopes Ltd. (Budapest, Hungary) was used. This assay system relies on magnetic 

separation for quantitative measurement of TSH using a GAMMA-C12 gamma counter 

(Diagnostic Products Corporation, Los Angeles, CA). For both assays, multiple quality 

control samples were dispersed within the assay. 

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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 Globulin (calculated) 

X Sodium X Glucose 

ENZYMES X Total bilirubin 

X Alkaline phosphatase (ALP) X Total protein 

 Acetyl cholinesterase (ChE) X Triglycerides 

 Creatine phosphokinase Serum protein electrophoresis 

 Lactic acid dehydrogenase (LDH) X Albumin/globulin ratio 

X Alanine aminotransferase (ALT/also SGPT) X Bile acids 

X Aspartate aminotransferase (AST/also SGOT) 

 Sorbitol dehydrogenase 

X Gamma glutamyl transferase (GGT) 

 Glutamate dehydrogenase 

* Recommended for the pubertal assay in female rats based on guideline 890.1450. 

c. Organ Weights and Histopathology: The following CHECKED (X) tissues were 

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

examination. 

 UROGENITAL OTHER 

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

XX Uterus*+ XX Liver* 

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

 X Pituitary* 

* Weights required based on guideline 890.1450 

+ Histopathological examination required based on guideline 890.1450 

All collected organs, except the thyroid, were weighed prior to fixation. Paired organs 

(kidneys, adrenals, and ovaries) were weighed together. The uterus was separated from the 

vagina and weighed. The uterus was weighed again following removal of the fluid in the 

lumen (blotted weight). Following approximately 24 hours of fixation in 10% neutral 

buffered formalin, the ovaries, uterus, and kidneys were rinsed and retained in 70% ethanol. 

The thyroids, attached to the trachea, were fixed for at least 24 hours in formalin, dissected 

from the trachea, weighed, and retained in 70% ethanol. The tissues were routinely 

processed in paraffin, sectioned at 4-6 microns, mounted, stained with hematoxylin and 

eosin, and examined microscopically. 

Thyroid sections were subjectively evaluated for follicular cell height and colloid area using 

a five point grading scale (1 = shortest/smallest amount; 5 = tallest/largest amount), and any 

abnormalities/lesions noted. At least two sections from each thyroid were examined. 

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The uterus was evaluated for hypertrophy or hypotrophy by changes in uterine horn 

diameter as well as myometrial, stroma, or endometrial gland development. Estrus cycle 

stage at the time of necropsy was considered in the pathology assessment of the ovary and 

uterus. 

II. RESULTS

A. Mortality: All animals survived until scheduled termination. 

B. Clinical Signs of Toxicity: No treatment-related clinical signs of toxicity were observed in 

any animals in any dose group. 

C. General Growth and Vaginal Opening: Body weights, body weight gains, age of 

attainment of VO and weight at day of attainment are presented in Table 2. In the 60 

mg/kg/day group, body weight gain was transiently decreased following the first dose (PND 

22-23), but did not attain statistical significance. Additionally, mean body weight gains in 

this group were comparable to controls for the remainder of the dosing period, and overall 

body weight gains were unaffected. Age and weight at attainment of VO were similar 

across all groups. With the exception of one 60 mg/kg/day animal (No. 17288-08), all 

animals achieved VO by PND 42. 

The age at VO, body weight at VO, and final body weight in the control group were within 

the acceptable range of the performance criteria provided in the Guideline (890.1450). 

TABLE 2. General Growth and Vaginal Opening (VO)a

Parameter Evaluated 

Vehicle Control Dichlobenil - 20 mg/kg/day Dichlobenil - 60 mg/kg/day 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) 

Initial body weight 

(PND 22; g) 

U 15 51.5 2.82 5.48 15 50.9 3.02 5.93 15 51.6 3.20 6.20 

A NR NR NR NR NR NR NR NR NR NR NR NR 

Body weight at VO (g) U 15 122.1 11.05 9.05 15 124.2 11.97 9.64 15 123.5 10.76 8.71 

A 15 120.1 11.05 9.05 15 122.7 11.97 9.64 15 121.7 10.76 8.71 

Final body weight 

(g) 

U 15 156.0 10.46 6.70 15 152.6 10.19 6.68 15 155.1 12.80 8.25 

A NR NR NR NR NR NR NR NR NR NR NR NR 

Final body weight 

(% of control) 

U 15 NA NA NA 15 -2.18 NA NA 15 -0.58 NA NA 

A NR NR NR NR NR NR NR NR NR NR NR NR 

Body weight gain 

(final – initial; g) 

U 15 104.5 9.04 8.65 15 101.7 9.43 9.28 15 103.5 11.97 11.56

A NR NR NR NR NR NR NR NR NR NR NR NR 

Age at VO (PND) U 15 35.4 1.68 4.75 15 36.5 1.85 5.06 15 36.1 3.42 9.46 

A 15 35.0 1.68 4.75 15 36.1 1.85 5.06 15 35.7 3.42 9.46 

Proportion unopened (#/N) 0/15 0/15 1/15 

a Data were obtained from Table 4 on page 31 of the study report 

U Unadjusted for body weight on PND 22 

A Adjusted for body weight on PND 22 

N Number of animals examined 

SD Standard Deviation 

CV Coefficient of Variation 

NA Not applicable 

NR Not reported 

D. Organ Weights: Organ weights at necropsy are presented in Table 3. At 60 mg/kg/day, 

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mean absolute, adjusted, and relative (to body) liver weights were increased (p<0.001) 

compared to controls (↑18-19%). Additionally at this dose, mean absolute blotted and wet 

uterine weights were decreased by 21 (p<0.05) and 26% (not statistically significant, NS), 

respectively. The decreased absolute blotted and wet uterine weights were not considered to 

be not biologically adverse because adjusted uterine weights were comparable, there were 

no histopathological findings in the uterus, and there were no effects of treatment on the day 

of attainment of VO, estrous cyclicity, or the age at the first occurrence of estrus. 

At 20 mg/kg/day, relative (to body) liver weight was increased (p<0.001) by 9% and 

absolute blotted uterine weight was decreased (NS) by 12%. In the absence of corroborating 

evidence of hepatotoxicity from gross and microscopic examination of the liver and clinical 

chemistry, the increased absolute and relative weights were not considered to be adverse. 

The unadjusted values for all organ weights in the control group were within the acceptable 

range of the performance criteria provided in the Guideline (890.1450), except for the 

adrenal gland weight (35.2 mg) which was below the acceptable range (38.34 to 48.84 mg). 

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TABLE 3. Organ Weights at Necropsya

Organ 

Vehicle Control Dichlobenil - 20 mg/kg/day Dichlobenil - 60 mg/kg/day 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) 

Adrenals 

(mg) 

U 15 35.2 4.32 12.27 15 33.9 5.32 15.69 15 35.2 7.11 20.19

A 15 35.1 4.32 12.27 15 34.0 5.32 15.69 15 35.2 7.11 20.19

R 15 22.6 2.91 12.86 15 22.2 3.19 14.36 15 22.6 3.26 14.44

Kidneys 

(g) 

U 15 1.29 0.12 9.09 15 1.26 0.12 9.17 15 1.32 0.16 12.26

A 15 1.30 0.12 9.09 15 1.29 0.12 9.17 15 1.33 0.16 12.26

R 15 0.83 0.05 5.52 15 0.83 0.05 5.57 15 0.85 0.06 7.46 

Liver 

(g) 

U 15 7.00 0.82 11.78 15 7.45 0.69 9.20 15 8.29*** 

(↑18) 

1.05 12.66

A 15 7.01 0.82 11.78 15 7.47 0.69 9.20 15 8.30*** 

(↑18) 

1.05 12.66

R 15 4.48 0.32 7.15 15 4.88*** 

(↑9) 

0.23 4.61 15 5.33*** 

(↑19) 

0.35 6.55 

Ovaries 

(mg) 

U 15 71.3 12.15 17.03 15 66.2 9.48 14.31 15 66.4 18.49 27.84

A 15 74.0 12.15 17.03 15 69.3 9.48 14.31 15 69.2 18.49 27.84

Pituitary 

(mg) 

U 15 9.8 1.18 12.09 15 8.9 1.24 13.92 15 9.6 1.77 18.43

A 15 9.9 1.18 12.09 15 9.1 1.24 13.92 15 9.8 1.77 18.43

R 15 6.3 0.71 11.36 15 5.8 0.65 11.13 15 6.2 0.80 13.03

Thyroid, fixed 

(mg) 

U 15 9.14 1.27 13.90 15 10.20 1.69 16.52 15 9.71 1.57 16.18

A 15 9.22 1.27 13.90 15 10.30 1.69 16.52 15 9.79 1.57 16.18

Uterus, blotted 

(mg) 

U 15 260.6 71.89 27.59 15 228.8 

(↓12) 

71.86 31.41 15 205.4* 

(↓21) 

68.90 33.55

A 15 263.0 71.89 27.59 15 234.1 71.86 31.41 15 208.6 68.90 33.55

Uterus, wet 

(mg) 

U 15 299.3 105.18 35.14 15 285.2 149.27 52.35 15 222.0 

(↓26) 

81.80 36.85

A 15 296.4 105.18 35.14 15 287.6 149.27 52.35 15 220.6 81.80 36.85

a Data were obtained from Tables 7 and S11 on pages 35 and 86-91 of the study report. Percent differences from controls, 

calculated by the reviewers, are 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<0.001. 

E. Estrous Cyclicity: Estrous cycle data are provided in Table 4. There were no effects of 

treatment on any estrus cycle parameter. The mean age at first vaginal estrus, mean cycle 

length, percent cycling, and percent regularly cycling were similar across all groups. 

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TABLE 4. Estrous Cyclicitya

Dose Level 

(mg/kg/day) N

Mean Age at 

First Vaginal 

Estrus (PND) 

Mean Cycle 

Length 

(days) 

Cycling 

(%) 

Regularly 

Cycling 

(%) 

Cycle Status at Necropsy (# Females) 

Diestrus Proestrus Estrus

Not 

Cycling 

Control (0) 15 36.8 4.9 100 88.9 10 1 4 0 

Dichlobenil 

(20) 

15 37.7 4.8 93.3 100 12b 1 2 1b

Dichlobenil 

(60) 

14 37.1 4.7 100 87.5 11 0 3 0 

a Data were obtained from Table 5 on page 32 of the study report. 

b One female in Diestrus was not cycling. 

N Number of animals

F. Clinical Chemistry and Hormone Levels: Mean thyroid hormone levels and selected 

clinical chemistry parameters are presented in Table 5. Serum T4 levels were slightly 

decreased and TSH levels were slightly increased at 20 and 60 mg/kg/day compared to 

controls. However, as these differences did not attain statistical significance and there were 

no corresponding histopathological lesions in the thyroid or liver, they were not considered 

to be related to treatment. 

At 60 mg/kg/day, increases in the following clinical chemistry parameters were noted: 

cholesterol (↑18%, p<0.05); albumin (↑5%, p<0.01); and total protein (↑4%, p<0.01). The 

changes in albumin and total protein were not considered to be adverse because they were 

small in magnitude. The small increase in cholesterol was not considered biologically 

adverse. Levels for all other clinical chemistry parameters were similar across all groups. 

Historical control data were provided on pages 377-380 for clinical chemistry parameters 

only. 

The value for serum T4 in the control group was within the acceptable range of the 

performance criteria provided in the Guideline (890.1450). 

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TABLE 5. Hormone Levels and Clinical Chemistry a

Parameter 

Evaluated 

Vehicle Control Dichlobenil - 20 mg/kg/day Dichlobenil - 60 mg/kg/day 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) 

Hormones 

Serum T4, Total 

(μg/dL) 

15 4.11 0.777 18.904 15 3.96 0.697 17.583 15 3.81 0.633 16.625

Serum TSH 

(ng/mL) 

15 5.15 1.399 27.191 15 6.07 1.618 26.648 15 6.26 3.263 52.127

Clinical Chemistry 

A/G ratio 15 2.75 0.370 13.472 15 2.79 0.280 10.050 15 2.86 0.336 11.730

Albumin (g/dL) 15 4.1 0.19 4.74 15 4.2 0.23 5.45 15 4.3** 

(↑5) 

0.22 5.15 

ALP (U/L) 15 240 51.8 21.6 15 225 36.1 16.0 15 227 57.1 25.2 

ALT (U/L) 15 69 13.8 20.1 15 67 19.4 28.9 15 71 11.2 15.8 

AST (U/L) 15 188 46.3 24.6 15 181 50.4 27.8 15 197 70.7 35.9 

Bile acids 

(umol/L) 

15 45.6 34.16 74.85 15 18.6** 18.83 101.1 15 27.4 24.84 90.58 

Calcium 

(mg/dL) 

15 12.4 0.41 3.32 15 12.7 0.41 3.22 15 12.5 0.39 3.14 

Chloride 

(mEq/L) 

15 102 1.7 1.7 15 102 1.4 1.4 15 102 1.2 1.2 

Cholesterol (mg/dL) 15 85 10.8 12.7 15 91 11.1 12.2 15 100** 

(↑18) 

14.2 14.2 

Creatinine 

(mg/dL) 

15 0.1 0.04 31.05 15 0.1 0.04 34.50 15 0.1 0.03 24.21 

GGT (U/L) 15 0.3 0.25 97.3 15 0.2 0.31 127.16 15 0.2 0.22 96.46 

Globulin 

(g/dL) 

15 1.5 0.16 10.61 15 1.5 0.13 8.38 15 1.5 0.20 12.98 

Glucose 

(mg/dL) 

15 147 6.0 4.1 15 147 8.2 5.6 15 148 13.2 9.0 

Phosphorus 

(mg/dL) 

15 9.9 0.89 9.02 15 9.6 0.75 7.85 15 10 0.54 5.36 

Potassium 

(mEq/L) 

15 6.67 0.565 8.472 15 6.93 0.405 5.853 15 6.79 0.540 7.955 

Sodium 

(mEq/L) 

15 143 1.8 1.3 15 142 1.8 1.2 15 143 1.7 1.2 

Total Bilirubin 

(mg/dL) 

15 0.06 0.016 26.767 15 0.05 0.019 34.856 15 0.07 0.019 27.450

Total protein (g/dL) 15 5.6 0.15 2.71 15 5.7 0.28 4.96 15 5.8** 

(↑4) 

0.3 5.16 

Triglyceride 

(mg/dL) 

15 56 13.7 24.7 15 65 19.8 30.7 15 64 19.2 29.9 

Urea Nitrogen 

(mg/dL) 

15 11.6 1.89 16.28 15 12.4 2.13 17.14 15 12.5 2.31 18.44 

a Data were obtained from Table 6 on pages 33 and 34 of the study report. Percent differences from controls, calculated by 

the reviewers, are included in parentheses. 

N Number of animals examined 

SD Standard Deviation 

CV Coefficient of Variation 

** Significantly different from controls at p<0.01. 

G. Histopathology: The incidences of histopathological findings of the thyroid gland and 

ovaries are presented in Tables 6 and 7, respectively. No treatment-related microscopic 

findings were observed in the thyroid gland, ovaries, or in any other examined tissue at 

either dose.

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TABLE 6. Incidence of Histopathological Lesions of the Thyroid Gland a

Dose 

(mg/kg/day) 

Parameter Evaluated 

Colloid Area Follicular Cell Height 

Gradeb Incidence Gradeb Incidence 

O E O E 

Control (0) 1 0 15 1 15 15 

2 0 15 2 0 15 

3 0 15 3 0 15 

4 15 15 4 0 15 

5 0 15 5 0 15 

Dichlobenil 

(20) 

1 0 15 1 15 15 

2 0 15 2 0 15 

3 0 15 3 0 15 

4 15 15 4 0 15 

5 0 15 5 0 15 

Dichlobenil 

(60) 

1 0 15 1 15 15 

2 0 15 2 0 15 

3 0 15 3 0 15 

4 15 15 4 0 15 

5 0 15 5 0 15 

a Data were obtained from Table 8 on page 37 of the study report. 

 b The criteria for grades 1-5 were listed on page 366 of the study report and are reported above. 

 O Number observed 

 E Number examined 

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TABLE 7. Incidence of Histopathological Lesions in the Ovaries a

Tissue Gradeb

Dose (mg/kg/day) 

Control (0) Dichlobenil (20) Dichlobenil (60) 

O E O E O E 

Corpora Lutea, 

Growth Phase 

1 0 15 0 15 0 15 

2 0 15 0 15 0 15 

3 14 15 15 15 15 15 

4 1 15 0 15 0 15 

5 0 15 0 15 0 15 

Corpora Lutea, 

Mature 

1 0 15 0 15 0 15 

2 2 15 1 15 1 15 

3 12 15 14 15 14 15 

4 1 15 0 15 0 15 

5 0 15 0 15 0 15 

Corpora Lutea, 

Regressive 

1 0 15 0 15 0 15 

2 0 15 0 15 0 15 

3 13 15 15 15 15 15 

4 2 15 0 15 0 15 

5 0 15 0 15 0 15 

Follicles, Atretic 1 0 15 0 15 0 15 

2 0 15 1 15 1 15 

3 15 15 14 15 14 15 

4 0 15 0 15 0 15 

5 0 15 0 15 0 15 

Follicles, Primary 1 0 15 0 15 0 15 

2 0 15 0 15 0 15 

3 15 15 15 15 15 15 

4 0 15 0 15 0 15 

5 0 15 0 15 0 15 

Follicles, Primordial 1 0 15 0 15 0 15 

2 0 15 0 15 0 15 

3 15 15 15 15 15 15 

4 0 15 0 15 0 15 

5 0 15 0 15 0 15 

Follicles, Secondary 1 0 15 0 15 0 15 

2 0 15 0 15 0 15 

3 14 15 15 15 15 15 

4 1 15 0 15 0 15 

5 0 15 0 15 0 15 

Follicles, Tertiary 1 0 15 0 15 0 15 

2 0 15 0 15 0 15 

3 14 15 15 15 14 15 

4 1 15 0 15 1 15 

5 0 15 0 15 0 15 

a Data were obtained from Table 8 on page 37 of the study report. 

 b The criteria for grades 1-5 were listed on page 367 of the study report and are reported above. 

 O Number observed 

 E Number examined 

 

Page 286 of 331
 Female Pubertal Assay (2011) / Page 15 of 16 

DICHLOBENIL/ 027401 OCSPP 890.1450/ OECD None

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: The Investigators concluded that there were no 

dichlobenil-related effects noted on the age and body weight at VO, the age at the first 

occurrence of estrus, and estrous cyclicity. Although lower mean wet and blotted uterus 

weights were noted at 20 and 60 mg/kg/day, there were no microscopic correlates. Also, 

there were no microscopic findings that corresponded to higher mean TSH and lower T4

levels observed in these groups. Therefore, no evidence of endocrine disruption was noted 

at 60 mg/kg/day, the highest level tested in this study. 

B. AGENCY COMMENTS: There were no dichlobenil-related effects observed on 

mortality, clinical signs of toxicity, body weights, body weight gains, age and weight at 

attainment of VO, mean age at first vaginal estrus, mean cycle length, and percent cycling or 

regularly cycling. 

Organ weights were similar between the control and dichlobenil treated groups with the 

exceptions of liver and uterus weights. Unadjusted, adjusted and related (to body) liver 

weight was increased (p<0.05) by 18–19% at 60 mg/kg/day, and relative liver weight was 

increased by 9% at 20 mg/kg/day. Blotted uterus weight was decreased by 12% (NS) and 

21% (p<0.05) at 20 and 60 mg/kg/day, respectively, and wet uterus weight was decreased by 

26% (NS) at 60 mg/kg/day. However, no histological changes were noted in the liver, uterus 

or ovaries. 

 

Clinical chemistry parameters were generally unaffected by dichlobenil treatment, although 

bile acids were substantially reduced by 59% (p<0.05) and 40% (NS) at 20 and 60 

mg/kg/day, respectively, and cholesterol was increased by 7% (NS) and 18% (p<0.05), 

respectively. Serum T4 and TSH levels were altered in a dose dependent manner, but the 

changes did not attain significance. At 20 and 60 mg/kg/day, T4 levels were decreased by 

4% and 7%, respectively, and TSH levels were increased by 18 and 22%, respectively. The 

changes in thyroid hormone levels were not corroborated by changes in thyroid weight or 

histology. 

 

Dose selection was based upon a range finding study, described in the Appendix, in which 

terminal body weights were decreased 8% and liver weights were increased 31-40% at 180 

mg/kg/day, and liver weights were increased 29-36% at 60 mg/kg/day. 

C. STUDY DEFICIENCIES: The following minor deficiency was noted that is not 

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

study: 

Control adrenal gland weight of 35.2 mg was outside the performance criteria acceptable 

range of 38.34 to 48.84 mg. 

Page 287 of 331
 Female Pubertal Assay (2011) / Page 16 of 16 

DICHLOBENIL/ 027401 OCSPP 890.1450/ OECD None

APPENDIX

In a dose range-finding study (MRID 48616112), juvenile male and female Crl:CD(SD) rats 

(5/sex/dose) were dosed daily via oral gavage (5 mL/kg) with dichlobenil (99.3% a.i.; Lot #: 

AM9K12A222) in aqueous 1% gum tragacanth at doses of 20, 60, or 180 mg/kg/day for 7 

consecutive days. 

All animals survived to scheduled necropsy. There were no adverse treatment-related effects on 

clinical signs, food consumption, or macroscopic evaluations observed at any dose in either sex. 

At 180 mg/kg/day, body weight gains were decreased (p<0.05) by 30-46% in the males during 

Days 0-2. Mean body weight gain for the remainder of the treatment period was comparable to 

controls. Overall (Days 0-7) body weight gains in males of this group were decreased by 16% 

(not statistically significant, NS) compared to the controls, resulting in mean terminal body 

weights that were 8% lower than the controls. Additionally at this dose, mean liver weights 

(absolute and relative [to body weight]) were increased (p<0.01) by 31-40% in the males and by 

45-47% in the females. 

At 60 mg/kg/day, body weight gains were decreased (p<0.05) by 34% in the males during Days 

0-1. However, mean body weight gains were comparable to the controls for the remainder of the 

treatment period and for the overall treatment period. Mean body weights at the end of treatment 

were comparable to the controls. Additionally at this dose, mean relative (to body) liver weight 

was increased (p<0.05) by 10% in the males and absolute (NS) and relative (p<0.01) liver 

weights were increased by 29-36% in the females. 

There were no adverse treatment-related effects in male rats at 20 mg/kg/day. However, absolute 

(NS) and relative (p<0.05) liver weights were increased by 16-18% in the females. There were 

no treatment-related effects on adrenal or kidney weights at any dose in either sex. 

Page 288 of 331
DATA EVALUATION RECORD 

DICHLOBENIL 

Study Type: OCSPP 890.1500, Male Pubertal Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-27-2012 (MRIDs 48616109 and 48616112) 

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: 3/24/2012 

Secondary Reviewer: Signature:

David A. McEwen, B.S. Date: 3/28/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 4/09/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 4/09/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 289 of 331
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 290 of 331
DICHLOBENIL I 027401 

Male Pubertal Assay (2011) I Page I of 16 

OCSPP 890.1500/ OECD None 

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

Health Effects Division 

Signature: fi;_/b:.<_~J_,. Date~,_---- Secondary Reviewer: John Liccione, Ph.D. 

Health Effects Division 

Signature. ~ 

Date: ~.::.: . ., 

DATA EVALUATION RECORD 

STUDY TYPE: Male Pubertal Assay; OCSPP 890.1500; OECD None. 

PC CODE: 027401 DP BARCODE: D397912 

TXR#: 0051691 CAS No.: 1194-65-6 

TEST MATERIAL (PURITY): Dichlobenil (99.3% a.i.) 

SYNONYMS: 

CITATION: 

2,6-dichlorobenzonitrile 

Coder, P.S. (2011). A pubertal developmental and thyroid function assay of 

dichlobenil administered orally in intact juvenile/peripubertal male rats. 

WIL Research Laboratories, LLC, Ashland, OH, Laboratory Study No.: 

WIL-12432, December 7, 2011. MRID 48616109. Unpublished. 

Coder, P.S. (2011). A 7-day dose range-finding oral (gavage) toxicity study 

of dichlobenil technical in juvenile rats for the endocrine disruption 

screening program. WIL Research Laboratories, LLC, Ashland, OH. 

Laboratory Study No.: WIL-12437, December 7, 2011. MRID 48616112. 

Unpublished. 

SPONSOR: Chemtura Corporation, 199 Benson Road, Middlebury, CT 

TEST ORDER#: EDSP-027401-120 

EXECUTIVE SUMMARY: In a Male Pubertal Assay (MRID 48616109), 15 Sprague-Dawley 

(Crl:CD(SD)) rats/dose group were treated daily via oral gavage (5 mL/kg) with dichlobenil 

(99.3% a.i., Lot #: AM9K12A222) in aqueous 1.0% gum tragacanth at doses of 0, 20, or 60 

mg/kg/day from post-natal day (PND) 23 to 53. The animals were examined for preputial 

separation (PPS) daily beginning on PND 30, and age and body weight at day of attainment was 

recorded. Following sacrifice on PND 53, total serum testosterone, thyroxine (T4), and thyroid 

stimulating hormone (TSH) levels were analyzed using electrochemiluminescent immunoassays 

and radioimmunoassays. The weights of adrenals, liver, pituitary, thyroid, and urogenital organs 

were recorded and microscopic examinations of the testes, epididymides, thyroid, liver, and 

kidneys were performed. 

No adverse treatment-related effects were observed in mortality, clinical signs of toxicity, body 

weight, body weight gain, age or weight at PPS, clinical chemistry, gross pathology, or 

microscopic examination of the testes, epididymides, thyroid, liver, or kidney. 

Page 291 of 331
Male Pubertal Assay (2011) / Page 2 of 16 

DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None 

The age and weight at attainment of PPS at all doses was within the acceptable range of the 

performance criteria. The unadjusted values for organ weights and hormone levels in the control 

group were within the acceptable range of the performance criteria provided in the Guideline 

with the exception of a minor departure in the thyroid weight of 13.44 mg compared to Guideline 

criterion of 14-26 mg. All thyroid glands examined in all dose groups were considered within 

normal limits of control tissues. 

Increased (p≤0.05) unadjusted, adjusted (covariate PND 23 body weight), and organ-to-body 

weight ratio liver (↑35–36%) and kidney (↑9–10%) weights were observed at 60 mg/kg/day. 

Increased (p≤0.05) organ-to-body weight ratio liver (↑16%) and kidney (↑7%) weights were 

observed at 20 mg/kg/day. Increased (p≤0.05) adjusted adrenal weights were also noted at 60 

mg/kg/day (↑13%). Except for the liver weights at 60 mg/kg/day, these differences were 

considered minor. Furthermore, in the absence of corroborating evidence of hepatotoxicity from 

gross and microscopic examination of the liver and clinical chemistry, the increased weight was 

considered an adaptive response. 

No biologically significant effect was noted on clinical chemistry parameters in the treated 

groups; however several clinical chemistry parameters in the 60 mg/kg/day group differed 

statistically (p≤0.05) from controls. 

Although no treatment-related effects were noted on pubertal development, the thyroid, or signs 

of systemic toxicity, the dosing levels utilized in the current study are considered appropriate 

based on the results of the dose range-finding study. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Male Pubertal Assay (OCSPP 

890.1500). 

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

Assurance statements were provided. 

 

Page 292 of 331
Male Pubertal Assay (2011) / Page 3 of 16 

DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None 

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: WIL Research Laboratories, LLC 

Location: Ashland, OH 

Study Directors: Coder, P.S. 

Other Personnel: Midori Asakawa, BVSc, DACVP - Contributing Scientist, Pathology 

Les Freshwater, MS - Contributing Scientist, Statistical Analysis 

Katherine L. Gardner, PhD - Participating Scientist, Analyses of Dosing Formulations 

Gwendalyn M. Maginnis, DVM - Clinical Veterinarian 

Kathryn S. Marks, BS, HTL (ASCP), QIHC - Principal Investigator, Histology 

Study Period: June 23, 2011 through December 7, 2011 

2. Test Substance: Dichlobenil 

 Description: White powder 

 Source: Notox B.V. (Hertogenbosch, Netherlands) 

 Lot #: AM9K12A222 (expires October 12, 2012) 

 Purity: 99.3% a.i. 

 Stability: Stabile in vehicle when stored for 8 days in the refrigerator 

 CAS #: 1194-65-6 

 Structure: 

3. Vehicle: 1.0% Gum tragacanth in deionized water (Spectrum Chemical Manufacturing Corporation, New 

Brunswick, NJ, Lot # 2AC3069, expires August 31, 2012)

Page 293 of 331
Male Pubertal Assay (2011) / Page 4 of 16 

DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None 

4. Test Animals:

Species: Rat (males only) 

Strain: Sprague-Dawley (Crl:CD(SD)) 

Age/Mean Weight 

at Study Initiation: PND 23 / 49.2–62.4 g 

Source: Charles River Laboratories, Portage, MI 

Housing: Parental dams were individually housed in plastic maternity cages with aspen bedding; each dam 

was housed with her litter until weaning on PND 21. After weaning, up to 3 males were housed in 

plastic cages. 

Diet: 2016CM Teklad Global 16% Protein Rodent Diet (Harlan Teklad, Madison, WI), ad libitum. It 

was stated that an average of 29.0 and 11.0 ppm total isoflavones (genistein + daidzein + glycitein) 

was present.

Water: Reverse osmosis-purified tap water, ad libitum

 Environmental 

Conditions: 

Temperature: 

Humidity: 

Air changes: 

Photoperiod: 

21.3–22.6°C 

34.5–60.5% 

10/h 

14 h dark / 10 h light

B. STUDY DESIGN

1. In-Life Dates: Start: July 30, 2011 End: August 30, 2011 

2. Mating: Time-mated pregnant dams (age not reported) were received from the supplier on 

gestation day (GD) 7 and were allowed to deliver naturally and rear their pups to PND 21. 

Any litters with fewer than ten total pups and/or less than five males or females (when 

possible) were excluded from the study. Litters were culled to 10 pups/litter on PND 4, with 

5 pups/sex when possible. 

3. Animal Assignment: Following weaning on PND 21, animals were assigned to study 

based on a computer-generated randomization scheme based on body weight to test groups 

as noted in Table 1. Litter mates were not placed in the same group.

TABLE 1. Study Design a

Test group Dose (mg/kg/day) # of Males 

Control 0 15 

Low 20 15 

High 60 15 

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

4. Dose Selection Rationale: In a dose range-finding study (MRID 48616112), juvenile male 

and female rats (5/sex/dose) were dosed daily via oral gavage (5 mL/kg) with dichlobenil 

(99.3% a.i.; Lot/batch#: AM9K12A222) in aqueous 1% gum tragacanth at doses of 20, 60, 

or 180 mg/kg/day for 7 consecutive days. All animals survived to scheduled necropsy. 

There were no adverse treatment-related effects on clinical signs, food consumption, or 

macroscopic evaluations observed at any dose in either sex. 

At 180 mg/kg/day, body weight gains were decreased (p<0.05) by 34-46% in the males 

during Days 0-2. Mean body weight gain for the remainder of the treatment period was 

Page 294 of 331
Male Pubertal Assay (2011) / Page 5 of 16 

DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None 

comparable to controls. Overall (Days 0-7) body weight gains in males of this group were 

decreased by 16% (not statistically significant, NS) compared to the controls, resulting in 

mean body weights that were up to 9% lower than the controls throughout the treatment 

period. Additionally at this dose, mean liver weights (absolute and relative [to body 

weight]) were increased (p<0.01) by 31-40% in the males and by 45-47% in the females. 

At 60 mg/kg/day, body weight gains were decreased (p<0.05) by 34% in the males during 

Days 0-1. However, mean body weight gains were comparable to the controls for the 

remainder of the treatment period and for the overall treatment period. Mean body weights 

at the end of treatment were comparable to the controls. Additionally at this dose, mean 

relative (to body) liver weight was increased (p<0.05) by 10% in the males and absolute 

(NS) and relative (p<0.01) liver weights were increased by 29-36% in the females. 

There were no adverse treatment-related effects in male rats at 20 mg/kg/day. However, 

absolute (NS) and relative (p<0.05) liver weights were increased by 16-18% in the females. 

There were no treatment-related effects on adrenal or kidney weights at any dose in either 

sex. 

 Based on these results, 60 mg/kg/day was selected as the high-dose for the current study 

because it has the potential to show signs of toxicity in both sexes. A dose of 20 mg/kg/day 

was maintained as the low-dose for the current study. 

5. Dose Preparation and Analysis: Suspensions of test substance concentrations of 4 and 12 

mg/mL in 1.0% gum tragacanth in deionized water were prepared approximately weekly 

with storage refrigerated and protected from light. Stability under refrigerated storage for 

up to 8 days was demonstrated for formulations between 0.02 mg/mL and 50 mg/mL (which 

bracketed those in the current study) in a separate study (MRID 48616111, reviewed 

concurrently). Concentration and homogeneity (top, middle, and bottom) of the test mixture 

were evaluated from each dosing formulations prepared during the in-life phase. 

Results 

Stability (% of time zero): 89.9-97.9% after 8 days refrigerated storage

Concentration (% of nominal): 87.5–106% 

Homogeneity (%RSD): 1.1–2.8 

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 administered once daily from PND 

23 through PND 53 by oral gavage at a dose volume of 5 mL/kg of body weight. Individual 

dosages were based on the most recently recorded body weights to provide the correct 

mg/kg/day dose. All animals were generally dosed between 0700 and 0900 hours local time 

each day. 

Page 295 of 331
Male Pubertal Assay (2011) / Page 6 of 16 

DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None 

7. Statistics: All analyses were conducted using SAS® version 9.2 or higher software. Data 

that were statistically analyzed included the organ weights, organ weight to final body 

weight ratio (liver, kidneys, pituitary, and adrenal glands), daily body weights (including 

necropsy body weight), cumulative body weight gain, serum chemistries, and PPS data. 

Gamma glutamyltransferase values under range were assigned a value of 0.1 (half the lower 

limit of quantitation) for statistical analysis and reporting. Each endpoint was tested for 

homogeneity of variance using Levene’s test. If that test was significant at p≤0.01, then a 

log transform was applied and Levene’s test conducted on the transformed data. If that test 

was still significant, then the square root transformation was applied to the raw data and 

Levene’s test conducted again. If the test was still significant, then a nonparametric test, as 

described below, was used to analyze the data. 

 If the variances were homogeneous then an analysis of variance (ANOVA) was conducted, 

on the raw or transformed data, as appropriate. The statistical model contained a factor for 

treatment group and a blocking factor based on the time of study start. A 2-sided Dunnett’s 

test was conducted, regardless of the outcome of the ANOVA, looking for significant 

differences in the test substance groups when compared with the vehicle control. If the 

transformations were unsuccessful in making the variances homogeneous then the 

nonparametric Kruskal-Wallis test was used, ignoring the blocking factor, followed by 

Dunn’s test, to compare each of the test substance groups with the vehicle control. As these 

were pre-planned pairwise comparisons, Dunn’s test was conducted regardless of the 

outcome of the Kruskal-Wallis test. The tests were 2-sided, at the 0.05 significance level, 

looking for significant differences from the vehicle control. In addition, the following 

analyses were conducted: 

Classification Procedure 

Organ weights and PPS data If the data met the homogeneity of variance criteria: 1) 

Analysis of covariance (ANCOVA) with Dunnett’s test. 

The model was as described above for ANOVA with the 

exception that PND 21 body weight was included as a 

covariate; 2) linear trend test using the ANOVA model; 

and 3) linear trend test using the ANCOVA model. 

Histopathology findings presented as a graded response Data were analyzed with pairwise Mann-Whitney U tests 

to compare each treated group with the control. The tests 

were 1-sided at the 0.05 significance level testing for 

increased severity. The tests were two-sided for graded 

responses presented on a Grade 1-5 scale at the 0.05 

significance level testing for increased or decreased 

severity. Exact p-values were calculated for the 

Mann-Whitney U test. 

Histopathology presented as a dichotomous response Pairwise Fisher’s exact tests was used to compare each 

treated group with the control. The tests were 1-sided at 

the 0.05 significance level testing for an increase. 

Significance was denoted at 5% or less. The statistical analyses were considered 

appropriate. 

Page 296 of 331
Male Pubertal Assay (2011) / Page 7 of 16 

DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None 

C. METHODS

1. Mortality and Clinical Examinations: Pups were sexed on PND 0, 4, and 21. Detailed 

physical examinations for each pup were recorded on PND 1, 4, 7, 14, and 21. All males 

were observed twice daily, once in the morning and once in the afternoon, for moribundity 

and mortality. Individual clinical observations were recorded daily (prior to test substance 

administration during the treatment period) through the day of euthanasia. Animals were 

also observed for signs of toxicity between 1 and 2 hours following dose administration. 

The absence or presence of findings was recorded for individual animals. In addition, the 

presence of findings at the time of dose administration was recorded for individual animals. 

 

2. Body Weight: Pretest body weights for each pup were recorded on PND 1, 4, 7, 14, and 

21. Individual body weights were recorded on the day of randomization, daily prior to test 

substance administration during the treatment period, and on the day of euthanasia. Mean 

body weight changes were calculated for each corresponding interval and also for 

PND 23–53. 

3. Food Consumption: Food consumption was not measured. 

4. Preputial Separation (PPS): Each male was observed for PPS beginning on PND 30. In 

addition, the appearance of a partial and complete PPS or a persistent thread of tissue 

between the glans and prepuce was recorded. The age and body weight on the day at which 

complete PPS was observed was recorded for each animal. However, if any animal within 

any group showed incomplete separation (persistent threads) for greater than 3 days, a 

separate second analysis was conducted using the age at which incomplete separation was 

first observed. Examination of the males was continued daily until complete PPS was 

attained or until euthanasia. When an animal did not attain PPS prior to necropsy, PND 54 

(last Study Day + 1) and body weight at necropsy was used as the age and body weight at 

PPS, respectively. 

5. Sacrifice and Pathology: The dams were euthanized by carbon dioxide asphyxiation and 

discarded without examination. Excluded litters and culled pups were euthanized by carbon 

dioxide asphyxiation or intraperitoneal injection of sodium pentobarbital without 

examination. All F1 males were euthanized randomly across all groups by decapitation 

without anesthesia on PND 53. Animals were euthanized beginning at least 2 hours 

following dose administration that day and all animals were euthanized by approximately 

1300 hours due to normal diurnal fluctuation in thyroid hormone levels. Immediately upon 

euthanasia by decapitation, trunk blood samples were collected for hormone analysis and 

serum chemistry. Serum was separated, and the samples were analyzed fresh or stored 

frozen (≤-20°C) for subsequent analysis. 

a. Hormone Analysis: An electrochemiluminescent immunoassay on the Cobas e411 (Roche 

Diagnostics, Indianapolis, IN) was used to analyze serum samples for the presence of 

testosterone and T4. To determine the concentration of TSH in serum samples, the 

[

125I]rTSH kit obtained from Izotop, Institute of Isotopes Ltd. (Budapest, Hungary) was 

used. This assay system relies on magnetic separation for quantitative measurement of TSH 

Page 297 of 331
Male Pubertal Assay (2011) / Page 8 of 16 

DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None 

using a GAMMA-C12 gamma counter (Diagnostic Products Corporation, Los Angeles, 

CA). For both assays, multiple quality control samples were dispersed within the assay. 

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) X Bile acids 

X Alanine aminotransferase (ALT/also SGPT) 

X Aspartate aminotransferase (AST/also SGOT) 

 Sorbitol dehydrogenase 

X Gamma glutamyl transferase (GGT) 

 Glutamate dehydrogenase 

* Recommended for the pubertal assay in male rats based on guideline 890.1500. 

c. Organ Weights and Histopathology: The following CHECKED (X) tissues were 

collected and weighed from all F1 main study animals. The (XX) organs, in addition, were 

subjected to histological examination. 

 UROGENITAL OTHER 

XX Testes (left and right separately)*+ XX Thyroid a*+

XX Epididymides (left and right separately)*+ XX Liver* 

XX Seminal vesicle plus coagulating glands (with and without fluid)* XX Adrenals (paired)* 

XX Ventral prostate* XX Pituitary* 

XX Dorsolateral prostate* 

XX Levator ani/bulbocavernosus (LABC) muscle complex* 

XX Kidneys (paired)*+ 

X Gross lesions and masses b 

a Thyroid and parathyroids were collected and weighed together 

b Gross lesions and masses were collected but not weighed. 

* Weights required based on guideline 890.1500. 

+ Histopathological examination required based on guideline 890.1500. 

Necropsies were performed on all juvenile males at the scheduled necropsy on PND 53. 

Any tissues with gross lesions were saved in 10% neutral-buffered formalin for possible 

future histopathologic examination. The tissues listed above were excised, and the carcasses 

were then discarded. Tissues were weighed immediately to prevent drying out prior to 

weighing. All collected organs, except the thyroid/parathyroids, were weighed prior to 

fixation. Absolute organ weight and usually relative to body organ weights were reported. 

Page 298 of 331
Male Pubertal Assay (2011) / Page 9 of 16 

DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None 

In addition, organ weights were subject to ANCOVA (covariate of PND 21 body weight) 

when appropriate. Testes, epididymides, ventral prostate, and dorsolateral prostate were 

weighed separately. The seminal vesicle with and without coagulating glands fluid were 

weighed. The kidneys and adrenals were weighed in pairs. The testes and epididymides 

were initially fixed in Bouin’s fixative (for approximately 24 hours) and retained in 70% 

ethanol thereafter. The thyroid glands, attached to the trachea, and kidney were fixed for at 

least 24 hours in 10% neutral-buffered formalin, and then the liver and thyroid (dissected 

from the trachea) were each placed in 70% ethanol until histological processing. The liver 

was placed in 10% neutral-buffered formalin for potential future histopathologic 

examination. After fixation, tissue samples of the thyroid, kidney, liver, testes, and 

epididymides were shipped to Biotechnics, LLC. (a division of WIL Research), 

Hillsborough, NC, for routine processing into paraffin blocks, sectioning, staining with 

hematoxylin and eosin generally, and examined microscopically. Hematoxylin and eosin 

were used for staining, except PAS and hematoxylin staining were used for the right testis 

and epididymis. The thyroid sections (minimum of 2 sections) were subjectively evaluated 

for follicular epithelial height and colloid area and any abnormalities/lesion. 

Testes and epididymides were evaluated as required by the EPA's Health Effects Test 

Guideline OCSPP 870.3800: Reproduction and Fertility Effects. Besides gross lesions such 

as atrophy or tumors, testicular histopathological examination 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. The 

intact epididymis, including the caput, corpus and cauda, was examined in longitudinal 

sections, in order to identify lesions as sperm granulomas, leukocytic infiltration 

(inflammation), aberrant cell types within the lumen, or the absence of clear cells in the 

cauda epididymal epithelium. 

II. RESULTS 

A. Mortality: All animals survived until scheduled termination. 

B. Clinical Signs of Toxicity: No treatment-related clinical signs of toxicity were observed in 

animals for any dose group. 

C. General Growth and Preputial Separation: Body weights, body weight gains, age of 

attainment of PPS, weight at PPS, and proportion/incidence of un-separated are presented in 

Table 2 below. 

No treatment-related effects on general growth, or on age or weight at attainment of PPS 

were observed at any dose. Both the age and weight at attainment of PPS at all doses was 

within the acceptable range of the performance criteria for controls. 

Differences from the control group values were slight and not statistically significant. When 

a second analysis was performed, adjusting the day of attainment for those males with 3 or 

more consecutive days of incomplete separation (persistent threads), no statistically 

Page 299 of 331
Male Pubertal Assay (2011) / Page 10 of 16 

DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None 

significant delays in the mean age of attainment of PPS were noted for both the 20 and 60 

mg/kg/day groups when compared to the control group. 

TABLE 2. General Growth and Preputial Separation (PPS) a

Parameter 

Evaluated 

Vehicle Control Dichlobenil - 20 mg/kg/day Dichlobenil - 60 mg/kg/day 

# of 

Males Mean SD 

CV 

(%) 

# of 

Males Mean SD 

CV 

(%) 

# of 

Male

s Mean SD 

CV 

(%) 

Age at PPS 

(PND) 

U 15 44.6 2.29 5.14 15 45.6 2.53 5.55 15 45 1.46 3.25 

A 15 44.6 2.29 5.14 15 45.6 2.53 5.55 15 45 1.46 3.25 

Body 

weight at 

PPS (g) 

U 15 211.8 19.97 9.43 15 213.4 21.45 10.05 15 215.2 14.87 6.91 

A 15 211.5 19.97 9.43 15 213.1 21.45 10.05 15 214.9 14.87 6.91 

Initial body 

weight 

(PND 23; g) 

U 15 57.1 3.1 5.43 15 56.6 3.7 6.54 15 57.2 2.67 4.67 

A 15 NR NR NR 15 NR NR NR 15 NR NR NR 

Final body 

weight (g) 

U 15 280.7 24.67 8.79 15 267.2 17.77 6.65 15 279.1 24.81 8.89 

A 15 NR NR NR 15 NR NR NR 15 NR NR NR 

Final body 

weight 

(% of 

control)

U 15 100 NR NR 15 95.2 b NR NR 15 99.4 b NR NR 

A 15 NR NR NR 15 NR NR NR 15 NR NR NR 

Body 

weight gain 

(final – 

initial; g) 

U 15 223.6 23.74 10.62 15 210.6 15.92 7.56 15 221.9 24.04 10.83 

A 15 NR NR NR 15 NR NR NR 15 NR NR NR 

Proportion 

unseparated 

(#/N) 

15 0/15 15 0/15 15 0/15 

a Data were obtained from Tables 4 and 5 on pages 28 and 29 of the study report. 

b Calculated by reviewers 

U Unadjusted for body weight on PND 23 

A Adjusted for body weight on PND 23 

SD Standard Deviation 

CV Coefficient of Variation 

NA Not applicable 

NR Not reported 

D. Organ Weights: Organ weights at necropsy are presented in Table 3 below. Increased 

(p≤0.05) unadjusted, adjusted (covariate PND 23 body weight), and organ-to-body weight 

ratio liver (↑35–36%) and kidney (↑9–10%) weights were observed at 60 mg/kg/day. 

Increased (p≤0.05) organ-to-body weight ratio liver (↑16%) and kidney (↑7%) weights were 

observed at 20 mg/kg/day. Increased (p≤0.05) adjusted adrenal weights were noted at 60 

mg/kg/day (↑13%). 

The mean values for the left and right epididymis weights for the control group were lower 

than the acceptable range in the performance criteria when the weights were combined; 

however, the CV values for the left and right epididymis were within the acceptable range in 

the performance criteria for the entire epididymis. In addition, the mean values of thyroid 

Page 300 of 331
Male Pubertal Assay (2011) / Page 11 of 16 

DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None 

gland and kidney weights in the control group were below the minimum values in the 

performance criteria; however, the CV values were within the acceptable range. There is no 

reported acceptable range for testis in the Sprague Dawley rat in the performance criteria. 

TABLE 3. Organ Weights at Necropsy a

Organ 

Vehicle Control Dichlobenil - 20 mg/kg/day Dichlobenil - 60 mg/kg/day 

# of 

Males Mean SD CV 

# of 

Males Mean SD CV 

# of 

Males Mean SD CV 

Liver 

(g) U 

15 12.66 1.697 13.4 15 14.00 1.322 9.4 15 17.08* 

(↑35) 

2.454 14.4 

A 15 12.36 1.697 13.4 15 13.72 1.322 9.4 15 16.79* 

(↑36) 

2.454 14.4 

R 

15 4.50 0.300 6.7 15 5.24* 

(↑16) 

0.334 6.38 15 6.10* 

(↑36) 

0.482 7.9 

Kidneys 

(g) 

U 15 1.95 0.217 11.2 15 1.99 0.195 9.8 15 2.12* 

(↑9) 

0.220 10.4 

A 15 1.95 0.217 11.2 15 1.99 0.195 9.8 15 2.13* 

(↑9) 

0.220 10.4 

R 15 0.69 0.053 7.6 15 0.74* 

(↑7) 

0.053 7.1 15 0.76* 

(↑10) 

0.047 6.1 

Pituitary 

(mg) 

U 15 11.6 1.61 13.9 15 10.7 2.29 21.4 15 11.1 1.40 12.7 

A 15 12.0 1.61 13.9 15 11 2.29 21.4 15 11.4 1.40 12.7 

R 15 4.1 0.45 10.9 15 4.0 0.72 18.0 15 4.0 0.57 14.3 

Adrenals 

(mg) 

U 15 44.3 5.81 13.1 15 45.0 8.16 18.1 15 50.0 9.52 19.0 

A 15 44.1 5.81 13.1 15 45.0 8.16 18.1 15 49.9* 

(↑13) 

9.52 19.0 

R 15 15.8 1.93 12.2 15 16.8 2.84 16.9 15 17.9 2.61 14.6 

Seminal vesicle 

+ coagulating 

gland, with fluid 

(mg) 

U 15 614.3 110.72 18.0 15 576.9 124.23 21.5 15 580.1 129.49 22.3 

A 15 605.4 110.72 18.0 15 568.3 124.23 21.5 15 571.2 129.49 22.3 

Seminal vesicle 

+ coagulating 

gland, without 

fluid (mg) 

U 15 396.1 63.33 16.0 15 366.5 58.79 16.0 15 365.9 65.97 18.0 

A 15 394.9 63.33 16.0 15 365.7 58.79 16.0 15 364.7 65.97 18.0 

Ventral prostate 

(mg) 

U 15 252.5 50.65 20.1 15 244.5 66.65 27.3 15 223.5 26.85 12.0 

A 15 NT NT NT 15 NT NT NT 15 NT NT NT 

Dorsolateral 

prostate (mg) 

U 15 113.9 16.00 14.0 15 107.1 19.29 18.0 15 105.1 17.88 17.0 

A 15 112.4 16.00 14.0 15 105.7 19.29 18.0 15 103.6 17.88 17.0 

LABC 

(mg) 

U 15 523.3 88.41 16.9 15 501.6 85.40 17.0 15 510.1 65.77 12.9 

A 15 532.8 88.41 16.9 15 511.7 85.40 17.0 15 519.6 65.77 12.9 

Page 301 of 331
Male Pubertal Assay (2011) / Page 12 of 16 

DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None 

TABLE 3. Organ Weights at Necropsy a

Organ 

Vehicle Control Dichlobenil - 20 mg/kg/day Dichlobenil - 60 mg/kg/day 

# of 

Males Mean SD CV 

# of 

Males Mean SD CV 

# of 

Males Mean SD CV 

Epididymis, left 

(mg) 

U 15 198.4 19.65 9.9 15 197.4 20.21 10.2 15 194.5 20.69 10.6 

A 15 200.1 19.65 9.9 15 199.8 20.21 10.2 15 196.3 20.69 10.6 

Epididymis, 

right (mg) 

U 15 207.5 20.33 9.8 15 207.1 22.66 10.9 15 206.0 18.81 9.1 

A 15 208.2 20.33 9.8 15 208.2 22.66 10.9 15 206.6 18.81 9.1 

Testis, left (mg) U 15 1432.2 116.92 8.16 15 1390.8 321.17 23.1 15 1481.2 186.47 12.6 

A 15 1458.8 116.92 8.16 15 1420.1 321.17 23.1 15 1508.0 186.47 12.6 

Testis, right 

(mg) 

U 15 1449.3 123.78 8.54 15 1385.9 285.41 20.6 15 1468.1 115.43 7.9 

A 15 1477.5 123.78 8.54 15 1415.6 285.41 20.6 15 1496.4 115.43 7.9 

Thyroid, fixed 

(mg) 

U 15 13.44 2.012 15.0 15 13.31 1.812 13.6 15 13.89 2.508 18.0 

A 15 13.25 2.012 15.0 15 13.13 1.812 13.6 15 13.70 2.508 18.0 

a Data were obtained from Table 7 on pages 32–33 of the study report. Percent difference from controls (calculated by 

reviewers) is presented parenthetically. 

U Unadjusted for body weight on PND 23 

A Adjusted for body weight on PND 23 

R Organ-to-body weight ratio (relative to body weight) 

SD Standard Deviation 

CV Coefficient of Variation 

NT Not Tested 

* Significantly different from controls at p<0.05. 

E. Clinical Chemistry and Hormone Levels: Clinical chemistry and hormone levels are 

presented in Table 4 below. Hormone levels in the treated animals were similar to controls. 

No biologically significant effect was noted on clinical chemistry parameters in the treated 

groups. The following clinical chemistry parameters in the 60 mg/kg/day group differed 

statistically (p≤0.05) from controls: (i) increased albumin (↑7%); (ii) decreased alkaline 

phosphatase (↓16%); (iii) increased cholesterol (↑18%); (iv) increased globulin (↑6%); and 

(v) increased total protein (↑7%). The hormone values for the control group were within the 

acceptable range of the performance criteria provided in the Guideline (OCSPP 890.1500). 

TABLE 4. Hormone Levels and Clinical Chemistry a

Parameter 

Evaluated 

Vehicle Control Dichlobenil - 20 mg/kg/day Dichlobenil - 60 mg/kg/day 

# of 

Males Mean SD CV 

# of 

Males Mean SD CV 

# of 

Males Mean SD CV 

Hormones 

Serum T4, 

Total 

(μg/dL) 

15 5.92 0.764 12.9 15 5.99 0.690 11.5 15 6.05 1.216 20.1 

Serum TSH 

(ng/mL) 15 11.1 5.12 46.1 15 9.6 2.92 30.3 15 10.2 3.87 37.8 

Serum 

testosterone 

(ng/mL) 

15 2.2 1.46 67.3 15 2.4 1.82 74.4 15 2.0 0.84 42.0 

Page 302 of 331
Male Pubertal Assay (2011) / Page 13 of 16 

DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None 

TABLE 4. Hormone Levels and Clinical Chemistry a

Parameter 

Evaluated 

Vehicle Control Dichlobenil - 20 mg/kg/day Dichlobenil - 60 mg/kg/day 

# of 

Males Mean SD CV 

# of 

Males Mean SD CV 

# of 

Males Mean SD CV 

Clinical Chemistry 

A/G Ratio 15 2.22 0.152 6.9 15 2.25 0.185 8.2 15 2.22 0.199 8.98 

Albumin 

(g/dL) 15 4.1 0.12 2.8 15 4.2 0.14 3.30 15 4.4* (↑7) 0.23 5.17 

ALP (U/L) 15 290 55.5 19.1 15 261 57.0 21.8 15 245* 

(↓16) 32.6 13.3 

ALT (U/L) 15 81 15.3 18.9 15 82 19.9 24.2 15 82 14.8 18.2 

AST (U/L) 15 207 62.9 30.5 15 237 114.9 48.5 15 220 85.7 38.9 

Bile Acids 

(umol/L) 15 33.6 15.82 47.1 15 33.4 20.57 61.6 15 41.0 21.32 52.0 

Calcium 

(mg/dL) 15 12.6 0.59 4.7 15 12.6 0.43 3.42 15 12.8 0.66 5.12 

Chloride 

(mEq/L) 15 100 1.5 1.5 15 100 1.3 1.3 15 99 1.2 1.2 

Cholesterol 

(mg/dL) 15 93 15.0 16.1 15 99 13.6 13.8 15 110* 

(↑18) 19.9 18.0 

Creatinine 

(mg/dL) 15 0.1 0.05 36.6 15 0.2 0.05 31.7 15 0.1 0.05 35.2 

GGT (U/L) 15 0.1 0.00 0.0 15 0.1 0.00 0.0 15 0.1 0.00 0.0 

Globulin 

(g/dL) 15 1.9 0.11 6.1 15 1.9 0.15 8.2 15 2.0* (↑5) 0.19 9.7 

Glucose 

(mg/dL) 15 153 10.1 6.6 15 151 7.2 4.8 15 149 8.7 5.8 

Phosphorus 

(mg/dL) 15 10.1 0.53 5.3 15 10.1 0.59 5.8 15 10.3 1.28 12.5 

Potassium 

(mEq/L) 15 6.55 0.525 8.0 15 6.81 0.705 10.3 15 6.52 0.439 6.7 

Sodium 

(mEq/L) 15 142 1.0 0.7 15 143 1.6 1.1 15 143 1.6 1.1 

Total Bili 

(mg/dL) 15 0.05 0.016 32.1 15 0.06 0.024 43.6 15 0.06 0.015 24.4 

Total 

Protein 

(g/dL) 

15 5.9 0.18 2.99 15 6.1 0.23 3.8 15 6.3* 

(↑7) 0.33 5.3 

Triglyceride 

(mg/dL) 15 121 38.5 31.7 15 117 44.3 37.8 15 149 60.1 40.4 

Urea 

Nitrogen 

(mg/dL) 

15 12.9 2.42 18.8 15 14.0 3.14 22.4 15 12.1 2.38 19.6 

a Data were obtained from Table 6 on pages 30–31 of the study report. Percent difference from controls (calculated by 

reviewers) is presented parenthetically. 

SD Standard Deviation 

CV Coefficient of Variation 

* Significantly different from controls at p≤0.05. 

NR Not reported

Page 303 of 331
Male Pubertal Assay (2011) / Page 14 of 16 

DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None 

F. Gross pathology: There were no effects of treatment observed at necropsy.

G. Histopathology: There were no treatment-related histopathological findings noted in the 

testes, epididymides, kidneys, liver or thyroid at any dose. The incidences of 

histopathological findings of the thyroid gland are presented below in Table 5. All thyroid 

glands examined in all dose groups were considered within normal limits of control tissues.

TABLE 5. Incidence of Histopathological Lesions of the Thyroid Gland a

Treatment 

Groups 

Parameter Evaluated 

Colloid Quality Follicular Cell 

Height (Increase) 

Follicular Cell 

Height (Decrease) 

Follicular Cell 

Shape 

Severityb Incidence Severity Incidence Severity Incidence Severity Incidence 

O E O E O E O E 

Vehicle 

Control 

1 0 15 1 15 15 1 NR 15 1 NR 15 

2 0 15 2 0 15 2 NR 15 2 NR 15 

3 0 15 3 0 15 3 NR 15 3 NR 15 

4 13 15 4 0 15 4 NR 15 4 NR 15 

5 2 15 5 0 15 5 NR 15 5 NR 15 

Dichlobenil

(20 mg/kg/day)

1 0 15 1 13 15 1 NR 15 1 NR 15 

2 0 15 2 2 15 2 NR 15 2 NR 15 

3 1 15 3 0 15 3 NR 15 3 NR 15 

4 14 15 4 0 15 4 NR 15 4 NR 15 

5 0 15 5 0 15 5 NR 15 5 NR 15 

Dichlobenil 

(60 mg/kg/day)

1 0 15 1 13 15 1 NR 15 1 NR 15 

2 0 15 2 2 15 2 NR 15 2 NR 15 

3 0 15 3 0 15 3 NR 15 3 NR 15 

4 15 15 4 0 15 4 NR 15 4 NR 15 

5 0 15 5 0 15 5 NR 15 5 NR 15 

a Data were obtained from Table 9 on page 34 of the study report. 

b Thyroid histopathology is graded 1 – 5 based on severity; see Methods. 

O No. Observed 

E No. Examined 

NR Not Reported 

The incidence of histopathological findings of the testes, epididymides, liver, thyroid, and 

kidneys are presented in Table 6. 

 

Page 304 of 331
Male Pubertal Assay (2011) / Page 15 of 16 

DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None 

TABLE 6. Incidence of Histopathological Lesions of the Testes, Epididymides, Liver, Thyroid, and 

Kidneya

Findings 

Dose Level (mg/kg bw/day) 

Vehicle Control Dichlobenil - 20 mg/kg/day Dichlobenil - 60 mg/kg/day 

# Observed # Examined # Observed # Examined # Observed # Examined 

Testes, right 

Minimal degeneration of 

seminiferous tubules 2 15 1 15 0 15 

Mild hypospermatogenesis 0 15 1 15 0 15 

Epididymides, right 

Moderate hypospermia 0 15 1 15 0 15 

Minimal mononuclear 

infiltrate 0 15 0 15 1 15 

Liver 

Minimal mononuclear 

infiltrate 9 15 4 15 11 15 

Minimal acute 

inflammation 1 15 0 15 0 15 

Mild coagulative necrosis 0 15 0 15 1 15 

Thyroid 

Ultimobranchial cyst 5 15 2 15 2 15 

Ectopic tissue 1 15 0 15 1 15 

Kidneys 

Minimal basophilic tubules 7 15 3 15 1* 15 

Cyst 1 15 1 15 3 15 

Minimal mononuclear 

infiltrate 

3 15 3 15 1 15 

Minimal mineralization 1 15 0 15 0 15 

Severe mineralization 1 15 0 15 0 15 

Minimal chronic 

progressive nephropathy 

5 15 5 15 4 15 

Mild pyelonephritis 0 15 0 15 1 15 

a Data were obtained from Table S11 on pages 98–99 of the study report. 

* Significantly different from controls at p≤0.05. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Test substance-related clinical chemistry 

alterations were limited to higher mean cholesterol, albumin, globulin, and total protein 

levels in the 60 mg/kg/day group. However, no correlating histologic lesion was identified 

by histopathological examination in the liver or kidney. No test substance-related 

alterations in serum hormone parameters were noted at any dose level. Test substancerelated organ weight differences included higher mean absolute and relative (to final body 

weight) adrenal gland, liver, and kidney weights in the 20 and/or 60 mg/kg/day groups. 

However, no correlating histologic lesions were identified by histopathological examination 

in the liver or kidney; the adrenal glands were not examined microscopically. 

Page 305 of 331
Male Pubertal Assay (2011) / Page 16 of 16 

DICHLOBENIL / 027401 OCSPP 890.1500/ OECD None 

B. AGENCY COMMENTS: No adverse treatment-related effects were observed in 

mortality, clinical signs of toxicity, body weight, body weight gain, age or weight at PPS, 

clinical chemistry, or gross and histopathology. The age and weight at attainment of PPS at 

all doses was within the acceptable range of the performance criteria. The unadjusted values 

for organ weights and hormone levels in the control group were within the acceptable range 

of the performance criteria .All thyroid glands examined in all dose groups were considered 

within normal limits of control tissues. 

Increased (p≤0.05) unadjusted, adjusted (covariate PND 23 body weight), and organ-tobody weight ratio liver (↑35–36%) and kidney (↑9–10%) weights were observed at 60 

mg/kg/day. Increased (p≤0.05) organ-to-body weight ratio liver (↑16%) and kidney (↑7%) 

weights were observed at 20 mg/kg/day. Increased (p≤0.05) adjusted adrenal weights were 

noted at 60 mg/kg/day (↑13%). Except for the liver weights at 60 mg/kg/day, these 

differences were considered minor. Furthermore, in the absence of corroborating evidence 

of hepatotoxicity from gross and microscopic examination of the liver and clinical 

chemistry, the increased weight was considered an adaptive response. 

No biologically significant effect was noted on clinical chemistry parameters in the treated 

groups. The following clinical chemistry parameters in the 60 mg/kg/day group differed 

statistically (p≤0.05) from controls: (i) increased albumin (↑7%); (ii) decreased alkaline 

phosphatase (↓16%); (iii) increased cholesterol (↑18%); (iv) increased globulin (↑6%); and 

(v) increased total protein (↑7%). 

Although no treatment-related effects were noted on pubertal development, the thyroid, or 

signs of systemic toxicity, the dosing levels utilized in the current study are considered 

appropriate based on the results of the dose range-finding study. 

 

C. STUDY DEFICIENCIES: The following measure was outside of the range specified in 

the performance criteria; however, this variation did not impact the interpretation of the 

assay: 

 The thyroid weight of 13.44 mg compared to guideline criterion of 14-26 mg. 

Page 306 of 331
DATA EVALUATION RECORD 

DICHLOBENIL 

Study Type: OCSPP 890.1550, Steroidogenesis Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-27-2012 (MRID 48616110) 

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: 3/21/2012 

Secondary Reviewer Signature:

Scott D. Studenberg, Ph. D., D.A.B.T. Date: 3/26/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 4/05/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 4/05/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 307 of 331
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 308 of 331
DICHLOBENIL I 027401 

Primary Reviewer: Patience Browne, Ph.D. 

Health Effects Division 

Secondary Reviewer: John Liccione Ph.D 

Health Effects Division 

Steroidogenesis Assay (2011) I Page I of 11 

OCSPP 890.1550/ OECD None 

Signature: -t:_.£------"~

Date: -=----~CZ±.~;2--

S ign a tu re: ::::=:::;:;;~:Z:::;:==:::==::::;:::--

Da te: -=--L+~~'....,L..:.;L-_ 

DATA EVALUATION RECORD 

STUDY TYPE: Steroidogenesis Assay (H295R Cells); OCSPP 890.1550 

PC CODE: 027401 DP BARCODE: D397912 

TXR#: 0051691 CAS No.: 1194-65-6 

TEST MATERIAL (PURITY): Dichlobenil (99.3% a.i.) 

SYNONYMS: 2,6-dichlorobenzonitrile 

CITATION: Meerts, l.A.T.M. (2011 ). Dichlobenil technical: steroidogenesis assay using 

the human cell line H295R. NOTOX B.V., The Netherlands, Laboratory 

Study No.: 495783 , December 9, 2011. MRID 48616110. Unpublished. 

SPONSOR: Chemtura Corporation, 199 Benson Road, Middlebury, CT 

TEST ORDER#: EDSP-027401-120 

EXECUTIVE SUMMARY: In a steroidogenesis assay (MRID 48616110), H295R cells 

cultured in vitro in 24-well plates were incubated with dichlobenil (99.3% a.i., Batch # 

AM9K l 2A222) at serial log concentrations of 100 to 0.0001 μM for 48 hours in triplicate for 

four independent experiments. Dimethyl sulfoxide (DMSO) was used as a vehicle, at a final 

concentration of 0.1 %. 

Testosterone and estradiol levels were measured using ELISA. 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. The positive 

controls included a known inhibitor (prochloraz) and inducer (forskolin) of estradiol and 

testosterone production. 

Guide line acceptability recommendations and requirements were generally met, including 

adequate production of testosterone and estradiol, acceptable reproducibility (low %CV), and 

appropriate induction and inhibition with positive controls. Precipitation of the test compound 

was not reported, and cytotoxicity was not observed. 

Treatment-related effect from dichlobenil observed on testosterone or estradiol levels were not 

consistent across all runs. In Run 1, there were significant reductions in testosterone levels at 

0.0001 , 0.001 , 0.01 , 1and100 μM ofdichlobenil, with the greatest reduction (0.71-fold) 

occurring at 0.01 μM. During Run 2, decreased (p:S0.05) testosterone levels were also noted at 

0.0001 and 0.01 μM. However, no reductions in testosterone were noted in Run 3. No changes 

Page 309 of 331
Steroidogenesis Assay (2011) / Page 2 of 11 

DICHLOBENIL / 027401 OCSPP 890.1550/ OECD None 

in estradiol concentration were observed during Runs 1 and 2, but estradiol was decreased 

(p≤0.05) in Run 3 at all dichlobenil concentrations except 100 and 0.1 μM. 

Due to the inconsistencies and variability in the hormone alterations across the three runs, the 

results for dichlobenil in this assay were considered to be equivocal. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Steroidogenesis assay 

(OCSPP 890.1550). 

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

Confidentiality statements were provided. 

 

Page 310 of 331
Steroidogenesis Assay (2011) / Page 3 of 11 

DICHLOBENIL / 027401 OCSPP 890.1550/ OECD None 

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: NOTOX B.V. 

 Location: The Netherlands 

 Study Director: I.A.T.M. Meerts, Ph.D. 

 Other Personnel: Dr. M.A. Thomson, study monitor 

M. Mulder, technical coordinator 

 Study Period: July 27, 2011 through December 9, 2011 

2. Test Substance: Dichlobenil 

 Description: White powder 

 Batch # (expiration date): AM9K12A222 (October 12, 2012) 

 Purity: 99.3% 

 Solubility: 50 mg/mL in DMSO 

0.021 g/L in water 

 Vapor pressure: 0.073 Pa at 20°C 

 Stability: Not reported for assay conditions; stable as stored 

 Storage conditions: Room temperature in the dark 

 CAS #: 1194-65-6 

 Molecular weight: 172.0

 Structure: 

3. Positive Control: Forskolin 

 Description (molecular weight): White powder (410.50) 

 Source: Sigma-Aldrich (Steinheim, Germany) 

 Batch # (expiration date): 109K5057V (December 13, 2014) 

 Purity: 98% 

 Solubility (in solvent): Soluble at tested concentrations in DMSO 

 Storage conditions: Room temperature in the dark 

 CAS #: 66575-29-9 

4. Negative Control: Prochloraz 

 Description (molecular weight): White powder (376.67) 

 Source: Sigma-Aldrich (Steinheim, Germany) 

 Batch # (expiration date): SZE6220X (August 8, 2012) 

 Purity: 99.1% 

 Solubility (in solvent): Soluble at tested concentrations in DMSO 

 Storage conditions: Room temperature in the dark 

 CAS #: 67747-09-5 

5. Solvent/Vehicle Control: Dimethyl Sulfoxide (DMSO) 

 Description (molecular weight): Clear liquid (78.13) 

 Source: SeccoSolv, Merck (Darmstadt, Germany) (catalog # not reported) 

 Lot # (expiration date): Not reported 

 Purity: Not reported 

 Storage conditions: Not reported 

 CAS #: 67-68-5 

 Justification for choice of 

solvent: 

Not provided 

 Final concentration in assay: 0.1% v/v 

Page 311 of 331
Steroidogenesis Assay (2011) / Page 4 of 11 

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6. Stock Medium: Dulbecco’s modified Eagle’s medium/nutrient mixture F12 

Ham 

Source: Invitrogen Corporation (Breda, The Netherlands) 

Lot # (expiration date): Not reported 

Sodium bicarbonate: Not reported 

Nu-Serum: BD Biosciences (Bedford, MA; catalog and lot nos. were not reported; 

background hormone concentrations were not reported) 

ITS+premix: 

Penicillin/streptomycin: 

BD Biosciences (Bedford, MA); catalog and lot nos. were not reported 

Invitrogen Corporation (catalog and lot nos. were not reported) 

7. Test Cells: H295R human adrenocortical carcinoma cells (ATCC CLR-2128; Lot # not 

reported) at passage numbers 6.5, 7.5, and 8.5 were incubated in the stock medium at the 

time of plating, dosing, and harvest. Incubation conditions were at 5% CO2 and 37 ± 1°C 

for approximately 24 hours prior to exposure. 

 The following performance criteria were met (indicated by an “x”): 

x Cell passage identifier. Cell Passage #: 6.5, 7.5, and 8.5 

x Cells frozen down at passage 5 

x Frozen cells cultured for 5 additional passages 

x Total number of passages does not exceed 10 

B. METHODS

1. Pre-Test Information: 

a. Hormone Assay Interference Test: Dichlobenil was tested for potential interference with 

the estradiol and testosterone hormone assays. Assays were conducted of the following 

samples of media containing: 0.1 mM dichlobenil; 0.1 mM dichlobenil with 2 × 10-6

mg/mL testosterone and 2 × 10-7 mg/mL estradiol; and 2 × 10-6 mg/mL testosterone and 2 × 

10-7 mg/mL estradiol. The interference of dichlobenil with estradiol and testosterone 

hormone measurements was 16% and 0.38%, respectively. Since the interference observed 

for testosterone and estradiol hormone measurement was very weak (below 20%), the 

Sponsor stated that results of hormone measurements using the EIA kits were not corrected 

for this interference. 

Page 312 of 331
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b. Hormone Extraction: Hormone extraction was not performed, since it was determined in a 

validation study (NOTOX Project 493393) that the medium did not affect the hormone 

measurement system. 

c. Laboratory Proficiency Test: No laboratory proficiency test data were reported. 

2. Test Solutions: Dichlobenil was dissolved in DMSO to make a 100 mM stock and then 

serially diluted in DMSO. Forskolin and prochloraz were dissolved in DMSO. Test agents 

were added to the cell culture plates, yielding final concentrations of 1 μM and 10 μM for 

forskolin, 0.1 μM and 1 μM for prochloraz, and 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 μM 

for dichlobenil. The final concentration of DMSO in the medium was held constant at 0.1% 

(v/v). 

3. Cell Plating and Preincubation: Cells were maintained in the Stock Medium described 

above. Hormone concentrations in undiluted Nu-Serum were not reported. H295R cells 

were grown for five passages, frozen in liquid nitrogen, then thawed and cultured for at least 

five passages (number not reported). The cells were plated into wells of a 24-well cell 

culture plate at a density of approximately 200,000 cells/mL. The cells were then placed 

into a 5% CO2 incubator at 37 ± 1°C for approximately 24 hours prior to chemical exposure. 

It was not reported that the cells were checked microscopically for attachment and proper 

morphology prior to dosing, 

4. Exposure: The medium was removed from the cells and replaced with medium containing 

concentrations of dichlobenil in triplicate, according to the schematic presented in Table 2. 

TABLE 2. Dosing Schematic for the Exposure of H295R Cells to Dichlobenil (Final Concentrations in 

μM).a

 1 2 3 4 5 6 

A DMSO DMSO DMSO 0.1 0.1 0.1 

B 100 100 100 0.01 0.01 0.01 

C 10 10 10 0.001 0.001 0.001 

D 1 1 1 0.0001 0.0001 0.0001 

a Data were obtained from page 20 of the study report. 

A concurrent QC plate was included with each of the three independent runs of the test 

chemical plates to demonstrate the assay’s response to forskolin (an inducer of testosterone 

and estradiol production) and prochloraz (an inhibitor of testosterone and estradiol 

production). The QC plate was prepared and dosed in the same manner with either forskolin 

or prochloraz, according to the schematic presented in Table 3. 

TABLE 3. Dosing Schematic for the QC Plate for Positive Controls (Final Concentrations in μM).a

 1 2 3 4 5 6 

A Blankb

 Blank Blank Blank + MeOHc Blank + MeOH Blank + MeOH 

B DMSO DMSO DMSO DMSO + MeOH DMSO + MeOH DMSO + MeOH 

C Forskolin 

1 μM 

Forskolin 

1 μM 

Forskolin 

1 μM 

Prochloraz 

0.1 μM 

Prochloraz 

0.1 μM 

Prochloraz 

0.1 μM 

D Forskolin 

10 μM 

Forskolin 

10 μM 

Forskolin 

10 μM 

Prochloraz 

1 μM 

Prochloraz 

1 μM 

Prochloraz 

1 μM 

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

b Blank wells received medium only. 

c MeOH = 70% methanol was added to these wells for 30 minutes at room temperature following medium removal. 

Page 313 of 331
Steroidogenesis Assay (2011) / Page 6 of 11 

DICHLOBENIL / 027401 OCSPP 890.1550/ OECD None 

Following dosing, the plates were incubated for 48 hours under the conditions previously 

described. After the 48 hour exposure, each well was examined under the microscope for 

cell condition (attachment, morphology, degree of confluence) and signs of cytotoxicity. In 

addition, digital photographs were taken from each well. The media was collected from all 

wells in two equal portions and stored at ≤−75°C until shipment to the analytical laboratory 

for hormone measurement. 

5. Cell Viability/Cytotoxicity Assay: After media removal, cell viability was measured using 

the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The 

medium from each well was replaced by 500 μL fresh medium. Subsequently, the medium 

from wells designated for methanol treatment was removed and 70% methanol was added to 

these wells. The plates were incubated at room temperature for 30 minutes. After 

incubation, the methanol was removed, and these wells were rinsed carefully with medium, 

three times. After rinsing, medium was added to these wells. To all wells, MTT (5 mg/mL 

in PBS) was added. The plates were incubated at 37.0 ± 1.0°C and 5% CO2 for 2-3 hours. 

After incubation, the medium in each well was replaced with DMSO. The plates were 

shaken on an orbital shaker at room temperature for 5 minutes. The formation of the blue 

formazan was measured at 560 nm against background absorption at 690 nm using a 

spectrophotometer. The change in cell viability was determined by comparing treated wells 

to the solvent control wells. A greater than 20% reduction in cell viability was considered 

evidence of cytotoxicity. 

6. Hormone Measurement System: The concentrations of testosterone and estradiol in the 

samples were determined using commercial EIA kits from Cayman Chemical Company. In 

addition, medium samples spiked with 500 and 2500 pg/mL testosterone or 50 and 250 

pg/mL estradiol were prepared and analyzed in the EIA. Method detection limits were 

reported as 6.3–8.7 pg/mL for estradiol and 5.0–7.1 pg/mL for testosterone in the QC plates. 

The following performance criteria were met (indicated by an “x”): 

x Method detection limit (100 pg/mL testosterone; 10 pg/mL estradiol) 

NR Spiked sample recovery acceptable for two concentrations of testosterone and estradiol (mean measured amount 

from triplicate samples within 30% of nominal concentration) 

x Hormone cross-reactivity (antibody-based assays only; ≤30% of basal production of the respective hormone) 

x Solvent control within 75% range below maximum response on standard curve 

x Test compound tested for interference with measurement system 

C. DATA ANALYSIS: Results were normalized to the mean solvent control (SC) value for 

each assay, and results were expressed as changes relative to SC in each exposure plate. 

Prior to conducting statistical analysis, the assumptions of normality and variance 

homogeneity were evaluated. Normality was evaluated using a Shapiro-Wilk’s test. If data 

were not normally distributed, the data were transformed to approximate a normal 

distribution. If the data were normally distributed or approximated normal distribution, 

differences between chemical treatments and solvent controls were analyzed using the 

Dunnett’s Test. If the data were not normally distributed, the non-parametric Kruskal 

Wallis test was performed. Differences were considered significant at p ≤ 0.05. Statistical 

evaluation was done based on average values for each well that represents independent 

replicate data points. The SAS program (release 9.1 for Windows NT) was used for 

Page 314 of 331
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DICHLOBENIL / 027401 OCSPP 890.1550/ OECD None 

statistical evaluation. Also, Prism version 4.01 (Graphpad Software, San Diego, CA) was 

used for non-linear regression and statistical analysis. 

II. RESULTS

A. TEST COMPOUND: Precipitation of dichlobenil was not reported at any concentration, 

and no cytotoxicity was observed. Inter-plate variation (%CV) for the solvent controls in the 

test assays was 11% for testosterone and 34% for estradiol (criteria is ≤30%). Intra-plate 

variation (%CV) for the solvent controls was <11% for each run in both the testosterone and 

estradiol assays, and each were within the Guideline criteria. 

In Run 1, there were statistically significant reductions in testosterone at 0.0001, 0.001, 0.01, 

1 and 100 μM, with the greatest reduction (0.71-fold) occurring at 0.01 μM. During Run 2, 

decreased (p≤0.05) testosterone levels were also noted at 0.0001 and 0.01 μM. However, no 

reductions in testosterone were noted in Run 3. No changes in estradiol concentration were 

observed during Runs 1 and 2, but estradiol was decreased (p≤0.05) at all concentrations in 

Run 3, except at 100 and 0.1 μM. 

TABLE 4. Mean (±SD) Hormone Concentrations Following Treatment with Dichlobenil for 48 Hours.a

Nominal 

Concentration 

(μM) 

Run 1 Run 2 Run 3 Run 1 Run 2 Run 3 Mean b ± SD b Statistical 

Significance 

Testosterone (pg/mL) Fold Difference 

DMSO 2006 2472 2187 — — — — — — 

0.0001 1715 2094 2521 0.85 0.85 1.15 0.95 0.17 Runs 1 and 2

0.001 1725 2325 2407 0.86 0.94 1.10 0.97 0.12 Run 1 

0.01 1430 1798 2135 0.71 0.73 0.98 0.81 0.15 Runs 1 and 2

0.1 1733 2477 2209 0.86 1.00 1.01 0.96 0.08 — 

1 1643 2312 2416 0.82 0.94 1.10 0.95 0.14 Run 1 

10 1920 2528 2693 0.96 1.02 1.23 1.07 0.14 — 

100 1457 2200 2060 0.73 0.89 0.94 0.85 0.11 Run 1 

Estradiol (pg/mL) Fold Difference 

DMSO 226 296 439 — — — — — — 

0.0001 263 316 329 1.16 1.07 0.75 0.99 0.22 Run 3 

0.001 247 304 338 1.09 1.03 0.77 0.96 0.17 Run 3 

0.01 238 304 364 1.05 1.03 0.83 0.97 0.12 Run 3 

0.1 231 285 377 1.02 0.96 0.86 0.95 0.08 — 

1 247 323 331 1.09 1.09 0.75 0.98 0.20 Run 3 

10 235 329 350 1.04 1.11 0.80 0.98 0.16 Run 3 

100 247 343 429 1.10 1.16 0.98 1.08 0.09 — 

a Data were obtained from Tables 16 and 18 on pages 29–30 of the study report. Method detection limits were reported as 

6.3–8.7 pg/mL for estradiol and 5.0–7.1 pg/mL for testosterone. 

b Calculated by the reviewers. 

Page 315 of 331
Steroidogenesis Assay (2011) / Page 8 of 11 

DICHLOBENIL / 027401 OCSPP 890.1550/ OECD None 

FIGURE 1. Change in Testosterone Production Relative to Dichlobenil Concentration. 

FIGURE 2. Change in Estradiol Production Relative to Dichlobenil Concentration. 

B. CYTOTOXICITY: No cytotoxicity was noted for the forskolin, prochloraz, blank, and SC 

wells; mean viability ranged from 93–129%, when solvent control was set at 100%. 

Methanol resulted in the expected response, approximately 0% viability. Data are 

summarized in Table 5. 

Page 316 of 331
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DICHLOBENIL / 027401 OCSPP 890.1550/ OECD None 

TABLE 5. Mean (±SD) MTT Cell Viability (% SC) Results after Treatment with Forskolin, Prochloraz, 

or Dichlobenil for 48 Hours. a 

Compound Concen. 

(μM) 

Cell Viability – Run 1 Cell Viability – Run 2 Cell Viability – Run 3 

Mean SD Mean SD Mean SD 

Blank NA 107 10 104 9 96 7 

SC NA 100 3 100 4 100 1 

Forskolin 1 119 18 111 14 106 10 

Forskolin 10 129 1 118 3 108 1 

Blank + Methanol NA -1 4 -3 3 -4 5 

SC + Methanol NA 0 7 0 7 0 7 

Prochloraz 0.1 103 1 102 0 93 1 

Prochloraz 1 109 2 111 9 103 6 

Dichlobenil 0.0001 106 11 104 7 104 7 

Dichlobenil 0.001 97 4 95 1 93 2 

Dichlobenil 0.01 102 12 99 13 96 14 

Dichlobenil 0.1 96 9 100 2 94 6 

Dichlobenil 1 103 1 104 2 99 1 

Dichlobenil 10 97 16 102 11 99 8 

Dichlobenil 100 101 6 100 4 99 3 

SC NA 100 10 100 11 100 1 

a Data were obtained from Tables 20–25 on pages 36–37 of the study report. Viability of solvent control (SC) wells was set 

at 100%. 

NA Not applicable 

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 6. The minimum basal hormone 

production levels (500 pg/mL for testosterone, 40 pg/mL for estradiol) were met in both 

blank and SC wells. OCSPP 890.1550 Guideline requirements for basal hormone 

production in SC wells, induction by 10 μM forskolin, and inhibition by 1 μM prochloraz 

were met for testosterone and estradiol, with the minor exception of induction of 

testosterone (1.9-fold in Run 2 instead of 2-fold). Thus, the Guideline requirements were 

generally 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 17% for testosterone and 18% for estradiol, within 

the guideline criteria of ≤30% for the assay. The variability (CV) within each run for the SC 

in the testosterone and estradiol assays was 3.6-14.6%, which was within the guideline 

recommendation of <30%. 

Page 317 of 331
Steroidogenesis Assay (2011) / Page 10 of 11 

DICHLOBENIL / 027401 OCSPP 890.1550/ OECD None 

TABLE 6. Mean (±SD) Hormone Concentrations Following Treatment with Forskolin or Prochloraz for 

48 Hours.a

Concentration (μM) 

Run 1 Run 2 Run 3 Run 1 Run 2 Run 3 Meanb ± SDb

Testosterone (pg/mL) Fold Difference (Relative to DMSO) 

Blank 1848 2409 2266 --- --- --- --- --- 

DMSO 1671 2297 2311 --- --- --- --- --- 

1 μM Forskolin 3295 3795 4060 2.0 1.7 1.8 1.8 0.15 

10 μM Forskolin 3234 4395 4570 2.0 1.9 2.0 2.0 0.06 

0.1 μM Prochloraz 602 832 832 0.4 0.4 0.4 0.4 0.00 

1 μM Prochloraz 174 244 295 0.1 0.1 0.1 0.1 0.00 

Estradiol (pg/mL) Fold Difference (Relative to DMSO) 

Blank 250 279 361 --- --- --- --- --- 

DMSO 262 296 370 --- --- --- --- --- 

1 μM Forskolin 3978 4021 2523 15.2 13.6 6.8 11.9 4.46 

10 μM Forskolin 5434 6330 4414 20.8 21.4 11.9 18.0 5.32 

0.1 μM Prochloraz 169 201 171 0.6 0.7 0.5 0.6 0.10 

1 μM Prochloraz 74 68 36 0.3 0.2 0.1 0.2 0.10 

a Data were obtained from Tables 39, 41, 43, 45, 47, and 49 on pages 62–67 of the study report. Method detection limits were 

reported as 6.3–8.7 pg/mL for estradiol and 5.0–7.1 pg/mL for testosterone. 

b Calculated by the reviewers. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Dichlobenil does not show a consistent pattern in 

estradiol release in H295R cells in one out of three experiments; but did have a statistically 

significant decrease in testosterone release in H295R cells in two out of three experiments. 

This could indicate that dichlobenil might be able to inhibit one or more enzymes in the 

steroidogenesis pathway. However, since no clear patterns were observed, the results were 

considered to be equivocal. 

B. AGENCY COMMENTS: Guideline acceptability recommendations and requirements 

were generally met, except that induction of testosterone was 1.9-fold in Run 2 instead of 2-

fold. Precipitation of the test compound was not reported, and cytotoxicity was not 

observed. 

In Run 1, there were significant reductions in testosterone levels at 0.0001, 0.001, 0.01, 1 

and 100 μM of dichlobenil, with the greatest reduction (0.71-fold) occurring at 0.01 μM. 

During Run 2, decreased (p≤0.05) testosterone levels (0.73- and 0.85-fold) were also noted 

at 0.0001 and 0.01 μM. However, no reductions in testosterone were noted in Run 3. No 

changes in estradiol concentration were observed during Runs 1 and 2, but estradiol was 

decreased (p≤0.05) in Run 3 at all dichlobenil concentrations except 100 and 0.1 μM. Due 

to the inconsistencies and variability in the hormone alterations across the three runs, the 

results for dichlobenil in this assay were considered to be equivocal 

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

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

 Stability of dichlobenil in the test system was not reported. 

 Forskolin induction of testosterone in Run 2 was below the Guideline limit (2-fold). 

 The laboratory proficiency test data were not provided. 

Page 318 of 331
Steroidogenesis Assay (2011) / Page 11 of 11 

DICHLOBENIL / 027401 OCSPP 890.1550/ OECD None 

 Mean ± SD for the fold-differences were not reported, but could be calculated from the 

data presented. 

 Cell passage number was not reported. 

 Recoveries of spiked estradiol samples were outside the Guideline criteria of 30% 

 Inter-plate variation was 34% for estradiol exceeding the Guideline criteria of ≤30%. 

 Validation study to determine the medium does not affect the hormone system 

(NOTOX Project 493393) was not submitted. 

 Cross reactivity of antibodies used in EIA was not reported. 

Page 319 of 331
DATA EVALUATION RECORD 

DICHLOBENIL 

Study Type: OCSPP 890.1600, In vivo Uterotrophic Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-27-2012 (MRID 48616111) 

Prepared for 

Health Effects Division 

Office of Pesticide Programs 

U.S. Environmental Protection Agency 

2777 South Crystal Drive 

Arlington, VA 22202 

Prepared by 

CSS-Dynamac Corporation 

1910 Sedwick Road, 

Building 100, Suite B 

Durham, NC 27713 

Primary Reviewer: Signature:

Sandra Hastings Date: 3/26/2012 

Secondary Reviewer: Signature:

Rebecca L. Bryan, B.S. Date: 3/28/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 4/04/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 4/04/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 320 of 331
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 321 of 331
DICHLOBENIL/ 027401 

Primary Reviewer: Sheila Healy, Ph.D. 

Health Effects Division 

Secondary Reviewer: --'J'-'e=s=-s -=-R=o __ w'-'l=an=d=---________ Signa tu re: --t:--~.:.....:::=-~='----

Health Effects Division Date: ---......__'5j-=-l--'c._£_> __ Template version 09/2011 

DATA EVALUATION RECORD 

STUDY TYPE: Uterotrophic Assay (Rat); OCSPP 890.1600; OECD 440 

PC CODE: 027401 DP BARCODE: D397912 

TXR#: 0051691 CAS#: 1194-65-6 

TEST MATERIAL (PURITY): Dichlobenil (99.3% a.i.) 

SYNONYMS: 2,6-dichlorobenzonitrile 

CITATION: Coder, P.S. (2011). A Uterotrophic Assay ofDichlobenil Administered Orally in 

Ovariectomized Rats. WIL Research Laboratories, LLC, Ashland, OH. 

Laboratory Study ID.: WIL-12433, December 7, 2011. MRID 48616111. 

Unpublished. 

SPONSOR: Chemtura Corporation, 199 Benson Road, Middlebury, CT 

TEST ORDER#: EDSP-027401-120 

EXECUTIVE SUMMARY: In a Uterotrophic Assay (MRID 48616111) conducted to screen 

for potential estrogenic activity, dichlobenil (99.3% a.i., Lot #AM9Kl2A222) in 1.0% gum 

tragacanth in deionized water was administered daily via oral gavage to groups of six 

ovariectomized Sprague Dawley rats at dose levels of 0 (vehicle), 20, 60 or 180 mg/kg/day on 

post-natal days (PND) 63-65 . A positive control group was treated with a daily dose of l 7aethynyl estradiol (EE) at 0.003 mg/kg/day by subcutaneous (s.c.) injection. Body weights were 

determined daily. All animals were terminated and necropsied on PND 66 approximately 24 

hours after the final dose administration to determine wet and blotted uterine weights. 

All animals survived until scheduled termination and no clinical findings were observed in 

animals dosed with dichlobenil at any level. Body weights were decreased (p<0.05) by 8% on 

Day 3 and 9% on Day 4 (termination) at 180 mg/kg/day compared to controls, resulting in a 

decrease (p<0.001) of 129% in overall body weight gain. Body weights in the 20 and 60 

mg/kg/day dose groups were comparable to controls throughout the duration of the assay. 

Uterine weights in the dichlobenil treated groups were comparable to the vehicle controls. 

The body weights for the positive control (EE) group were decreased (p<0.05) by 8% on Day 4 

and overall body weight gain for the EE animals was decreased (p<0.001) by 104% in compared 

Page 322 of 331
In vivo Uterotrophic Assay (2011) / Page 2 of 10 

DICHLOBENIL/ 027401 OCSPP 890.1600/ OECD 440 

with the controls. Absolute wet and blotted uterus weights for the positive control group were 

increased (p<0.001) by 766% and 234%, respectively, as expected.

The highest dose tested (180 mg/kg/day) was determined to be adequate based on the decrease in 

body weight gain (129%) at this dose. 

No statistically significant changes were seen in uterine weight in this assay. Dichlobenil was 

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 GLP Compliance and Quality Assurance statements were 

provided. 

 

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In vivo Uterotrophic Assay (2011) / Page 3 of 10 

DICHLOBENIL/ 027401 OCSPP 890.1600/ OECD 440 

I. MATERIALS AND METHODS

A. MATERIALS 

1. Test Facility: WIL Research Laboratories, LLC 

Location: Ashland, OH 

 Study Director: Prägati Sawhney Coder 

 Other Personnel: L. Freshwater (Scientist); K. Gardner (Scientist); E. Bodle (Assistant Director); C. 

Kopp (Manager); G. Maginnis (Clinical Veterinarian); J. Moore (Operations 

Manager);C. Wally (Group Supervisor) 

 Study Period: May 23, 2011 to December 7, 2011 

2. Test Substance: Dichlobenil 

 Description: Technical; white powder 

 Source: Notox B.V. (Chemtura Canada) 

 Lot/Batch #: AM9K12A222 (expiration date: October 12, 2012) 

 Purity: 99.3% a.i. 

 Stability: Not reported 

 CAS #: 1194-65-6 

 Structure: 

3. Reference Estrogen: 17α-ethynyl estradiol (EE) 

 Supplier: Sigma Aldrich (St. Louis, MO) 

 Lot/Batch #: 028K1411 (expiration date: August 4, 2011) 

 Purity: >98% 

 CAS #: 57-63-6 

4. Solvent/Vehicle Control: Gum Tragacanth in deionized water 

 Supplier: Spectrum Chemical Manufacturing Corporation (New Brunswick, NJ) 

 Lot/Batch #: 2AC3069 (expiration date: August 31, 2012) 

 Rationale (if other than water): Test substance is not soluble in water at the concentrations tested and 1.0% gum 

tragacanth created a homogeneous suspension of the test substance. 

 Final concentration: 1.0% 

5. Test Animals: 

 Species: Rat (ovariectomized) 

 Strain: Sprague Dawley 

 Age/weight at dose initiation: 63 days old/ 228.3 – 277.5 g females 

 Source: Charles River Laboratories, Inc. (Raleigh, NC) 

 Housing: Individually caged in stainless steel wire-mesh cages suspended above cage-board; 

the cage-board was changed at least 3 times/week. 

 Diet: Harlan Laboratories 2016CM Teklad Global 16% Protein Rodent Diet, ad libitum 

 Water: Reverse-osmosis purified water, ad libitum 

 Environmental conditions: Temperature: 

Humidity: 

Air changes: 

Photoperiod: 

21.3-21.4oC 

45.7-59.2% 

10/hr 

12 hrs light/ 12 hrs dark 

 Acclimation period: 10 days 

Page 324 of 331
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DICHLOBENIL/ 027401 OCSPP 890.1600/ OECD 440 

B. STUDY DESIGN 

1. In Life Dates: Start: June 10, 2011 End: June 13, 2011 

2. Study Design: Sexually mature ovariectomized female rats were received from Charles 

River Laboratories; rats were ovariectomized at PND 49 by the supplier. Animals were 

received 4 days following ovariectomy (PND 53) and acclimated for 10 days prior to 

initiation of dosing. Vaginal smears were taken daily during the last five days of the 

acclimation period to verify complete removal of the ovaries as indicated by a lack of 

estrous cycling. The dose administration period was from PND 63 through 65. Rats were 

euthanized approximately 24 hours later on PND 66 and necropsied for uterine weight 

measurements. 

3. Animal Assignment: Animals were assigned, stratified by body weight, to the test groups 

noted in Table 1. Statistical analysis indicated that there were no significant differences 

between group mean weights at study initiation. 

TABLE 1. Study Design a

Test Group Dose (mg/kg/day) # of Females 

Estrogen Agonist Assay 

Vehicle Control 0 6 

Low Dichlobenil 20 6 

Mid Dichlobenil 60 6 

High Dichlobenil 180 6 

17α-ethynyl estradiol (EE), Reference Estrogen 0.003 6 

a Data were obtained from Table 1 on page 15 of the study report. 

4. Dose Selection Rationale: The dose levels were selected based on the results from a rangefinding study (MRID 48616113) in which dichlobenil in 1% gum tragacanth in deionized 

water was administered daily via oral gavage to 5 adult (61 days old) female Sprague 

Dawley [Crl:CD(SD)] rats/dose group at doses of 0, 20, 60, or 180 mg/kg/day for 3 

consecutive days. A summary of the study is included as an Appendix to this DER. Based 

on the results, the dose levels selected for the main study were 0, 20, 60 and 180 mg/kg/day. 

5. Dose Preparation and Analysis: Dose formulations of dichlobenil were prepared daily in 

1% gum tragacanth and stored at room temperature in the dark. A stock solution of the 

positive control (EE) was prepared in 95% ethanol, and diluted with corn oil daily for 

dosing. Homogeneity and stability of dichlobenil suspensions in 1.0% gum tragacanth in 

deionized water at concentrations 0.02, 1, and 50 mg/mL were tested by the laboratory. 

Samples (duplicates from the top and bottom strata) were analyzed for stability following 3 

days frozen storage; additionally stability was tested with QC samples in 1.0% gum 

tragacanth in deionized water following 3 days storage at room temperature. 

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Homogeneity of the test formulations prepared (June 9, 2011) for Day 1 dosing was 

evaluated on the day of preparation (duplicates from the top, middle and bottom strata) and 

following 8 days refrigerated storage (resuspension samples). Additionally, samples of dose 

formulations for Day 1 dosing were analyzed for achieved concentration on the day of 

preparation. 

Results of Dose Analysis 

Homogeneity (% RSD): 1.9-5.5% RSD on the day of preparation 

0.33-2.9% RSD following 8 days refrigerated storage 

Stability (% of initial): 90.7 - 106% following 3 days frozen storage 

104-115% following 3 days at room temperature 

 

Concentration (% of nominal): 98.0-114% 

It is noted that samples of dichlobenil suspensions prepared (June 1, 2011) 9 days prior to 

dosing initiation were also tested for homogeneity (top, middle, and bottom strata) and the 

% of nominal in samples from the low concentration (0.02 mg/mL) was low and outside the 

acceptable %RSD. A second preparation of the samples achieved acceptable criteria for 

0.02 mg/mL but not for 50 mg/mL. These formulations were not used for dosing, and the 

reviewer has reported (above) homogeneity for the formulations actually used for dosing. 

The analytical data indicated that the mixing procedure was adequate for the formulations 

used in dosing and that the variation between nominal and actual dosage to the animals was 

acceptable. 

6. Dosage Administration: Animals were administered the test formulations or vehicle 

control (1.0% gum tragacanth in deionized water) daily via oral gavage and EE via s.c. 

injection for three consecutive days in dose volumes of 5 mL/kg body weight. Dose 

volumes were adjusted daily based on the most recent body weight measurement.

7. Statistics: Mean values were reported with standard deviations and coefficient of variance. 

All analyses were conducted using Statistical Analysis System version 9.2 (SAS Institute 

Inc., 2002-2008). Each endpoint was tested for homogeneity of variance using Levene’s 

test. If the test was significant at p≤0.01, then a log transformation was applied for reanalysis. If that test was still significant then the square root transformation was applied to 

the raw data and Levene’s test conducted. If the test was still significant then a 

nonparametric test was conducted to analyze the data. 

Organ weights were compared to the vehicle control group. If variances were homogeneous 

then the analysis of covariance was performed using the body weight at necropsy as the 

covariate. Comparisons of each treatment group with the vehicle control group were made 

with a 1-sided Dunnett’s test using p≤0.05 to declare significance. If transformations were 

unsuccessful in making the variances homogeneous, then the nonparametric Kruskal-Wallis 

test was used followed by Dunn’s test to compare each treatment group with the vehicle 

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control group. Body weights and body weight gains were analyzed similar to organ weights 

but there was no covariate in the statistical model (ANOVA) and the testing was 2-sided 

instead of 1-sided. The statistical analyses were considered adequate.

C. METHODS

1. Clinical Examinations: Cage-side checks for mortality and morbidity were conducted 

twice daily and individual clinical signs of toxicity were conducted once daily. The 

injection site was examined for erythema, swelling and other dermal findings. Animals 

were also observed for signs of toxicity 1-2 hours after dose administration and at 

termination. 

2. Body Weight: Animals were weighed at randomization, daily prior to dosing and at 

termination. 

3. Food Consumption (Optional): Food consumption was not measured. 

4. Necropsy and Measurement of Uterine Weight: On PND 66 (approximately 24 hours 

after final dose administration), all animals were euthanized by carbon dioxide inhalation 

and subjected to a gross necropsy. Dissection of the uterus was performed according to the 

U.S. EPA Guideline. Briefly, the vagina was removed just below the cervix in order to 

retain the luminal fluid in the uterus. The “wet” uterus (i.e., containing the luminal fluid) 

was weighed. Subsequently, the uterine horns were cut longitudinally and blotted with filter 

paper to remove the luminal fluid while preventing desiccation and the blotted uterus was 

weighed. The uterus and vagina were preserved in 10% neutral-buffered formalin for 

possible future histopathologic examination.

5. Microscopic Examination (Optional): Microscopic examinations were not conducted.

II. RESULTS

A. OBSERVATIONS

1. Mortality: All animals survived until scheduled termination. 

2. Clinical Signs of Toxicity: No treatment-related clinical findings were observed in animals 

dosed with dichlobenil. However, it was noted that red material around the nose, 

lacrimation of the eyes, and decreased defecation were observed in single animals in lowand high-dose group; these findings were not considered test substance related as there was 

no dose-response. Clear material around the nose was observed in two animals in the 

positive control group (EE) on Day 2 and 3 following dose administration. Clinical 

observations in the study are presented in Table 2. 

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TABLE 2. Incidence of Clinical Observations in the Estrogen Agonist Assay a

Observation 

Dose (mg/kg/day) 

Vehicle Control Dichlobenil 

(20)

Dichlobenil 

(60) 

Dichlobenil 

(180) 

Reference 

Estrogen 

EE (0.003)

# 

Observed 

# 

Examined 

# 

Observed

# 

Examined

# 

Observed

# 

Examined

# 

Observed 

# 

Examined 

# 

Observed

# 

Examined

Decreased 

defecation 0 6 0 6 0 6 1 6 0 6 

Wet clear material 

around mouth 0 6 0 6 0 6 0 6 2 6 

Lacrimation (eyes) 0 6 1 6 0 6 0 6 0 6 

Dried red material 

around nose 0 6 0 6 0 6 1 6 0 6 

a Data were obtained from Tables S1 and S2, on pages 30-31 of the study report. 

B. BODY WEIGHT AND WEIGHT GAIN: Selected body weight and body weight gain 

data are presented in Table 3. Body weights were decreased (p<0.05) by 8% on Day 3 and 

9% on Day 4 (termination) in the high dose group compared to vehicle controls, resulting in 

a decrease (p<0.001) of 129% in overall body weight gain. Body weights in the low- and 

mid-dose groups were comparable to controls throughout the duration of the assay. 

The body weights for the positive control (EE) group were decreased (p<0.05) by 8% on 

Day 4 and overall body weight gain for the EE animals was decreased (p<0.001) by 104% in 

comparison with the vehicle controls. 

TABLE 3. Selected Group Body Weights and Cumulative Body Weight Gains (g) In the Estrogen Agonist Assay a

Study Day 

Dose (mg/kg/day) 

Vehicle Control Dichlobenil 

(20) 

Dichlobenil 

(60) 

Dichlobenil 

(180) 

Reference Estrogen

EE (0.003) 

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 

1 6 250.3 14.97 6 249.4 11.99 6 251.1 15.34 6 251.0 12.48 6 248.8 12.52 

2 6 257.8 17.11 6 249.6 

(↓3) 13.17 6 253.1 (↓2) 13.59 6 241.7 (↓6) 9.20 6 253.3 13.66 

3 6 263.3 17.01 6 255.6 

(↓3) 14.41 6 255.2 (↓3) 14.54 6 241.9* (↓8) 6.50 6 251.7 12.15 

4 (termination) 6 270.0 16.61 6 263.0 

(↓3) 13.09 6 262.3 (↓3) 17.60 6 245.3* (↓9) 8.50 6 248.0* (↓8) 11.51 

Body Weight 

Gain (Days 1-4) 6 19.7 3.82 6 13.6 (↓31) 2.14 6 11.2 (↓43) 6.38 6 -5.7*** (↓129) 10.13 6 -0.7***(↓104) 4.74 

a Data were obtained from Table 2 on page 21 of the study report; note the study numbered days started with 0 

(dose initiation), but the reviewer numbered the study days starting with 1 (dose initiation). 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<0.001 

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

D. PATHOLOGY 

1. Uterine Weights: Selected uterine weight data are presented in Table 4. Uterine weights in 

the dichlobenil treated groups were comparable to the vehicle controls. Absolute wet and 

blotted uterus weights for the positive control (EE) group were increased (p<0.001) by 

766% and 234%, respectively. The mean blotted uterine weight for the vehicle control 

group was 0.03% of terminal body weight, which was deemed acceptable to yield sufficient 

assay sensitivity and was consistent with the requirement (<0.09%) of the test guideline.

TABLE 4. Uterine Weights from Estrogen Agonist Assay in SD Rats a

Parameter 

Dose (mg/kg/day) 

Vehicle Control Dichlobenil 

(20) 

Dichlobenil 

(60) 

Dichlobenil 

(180) 

Reference Estrogen 

EE (0.003) 

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 

Terminal BW (g) 6 270 16.5 6 263 13.3 6 262 17.8 6 245 8.3 6 248 11.6 

Wet, absolute (g) 6 0.1044 0.0131 6 0.1014 0.0092 6 0.1017 0.0106 6 0.1065 0.0094 6 0.9042***

(↑766) 0.1588

Wet, relative (%) 6 0.039 0.0048 6 0.039 0.0037 6 0.039 0.0030 6 0.043 0.0038 6 0.366 0.0737

Blotted, absolute 

(g) 6 0.0917 0.0132 6 0.0896 0.0064 6 0.0900 0.0113 6 0.0939 0.0089 6 0.3063***(↑234) 0.0343

Blotted, relative 

(%) 6 0.034 0.0051 6 0.034 0.0026 6 0.034 0.0033 6 0.038 0.0038 6 0.124 0.0169

a Data were obtained from Table 3 on page 23, and Tables S11 and S14 on pages 42 and 45 of the study report; it is 

noted that relative weights in Table 3 were reported as mg/100 g, and relative weights as g/100 g were reported in 

Table S14 (and reported herein for % relative weights). Percent differences from controls calculated by the reviewer 

are included in parentheses. 

BW Body weight 

N Number of animals in the group 

SD Standard Deviation 

*** Significantly different from controls at p<0.001 

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

III. DISCUSSION AND CONCLUSIONS

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

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

consistent with agonism of natural estrogens when administered orally to ovariectomized 

female rats at dose levels of 20, 60, and 180 mg/kg/day. The positive control substance 

(17α-ethynylestradiol) elicited the expected increases in uterine (wet and blotted) weights.

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B. AGENCY COMMENTS: All animals survived until scheduled termination and no 

treatment-related clinical findings were observed in dichlobenil-dosed animals. Body 

weights were decreased (p<0.05) by 8 and 9% on Days 3 and 4 in the high dose group (180 

mg/kg/day dichlobenil) compared to vehicle controls, resulting in a decrease (p<0.001) of 

129% in overall body weight gain. Body weights in the low- and mid-dose groups were 

comparable to controls throughout the duration of the assay. Uterine weights in the 

dichlobenil treated groups were comparable to the vehicle controls.

The body weights for the positive control (EE) group were decreased (p<0.05) by 8% on 

Day 4 and overall body weight gain for the EE animals was decreased (p<0.001) by 104% in 

comparison with the vehicle controls. Absolute wet and blotted uterus weights for the 

positive control group were increased (p<0.001) by 766% and 234%, respectively, as 

expected. No statistically significant changes were seen in uterine weights in this study. 

Dichlobenil was negative in the uterotrophic assay. 

C. STUDY DEFICIENCIES: None

 

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APPENDIX

Since this is a range-finding study, only a summary is provided to confirm the adequacy of the 

dose selection rationale used in the Uterotrophic Assay (MRID 48616113). 

In a preliminary range-finding assay, dichlobenil (99.3% a.i.; lot# AM9K12A222) in 1% gum 

tragacanth in deionized water was administered daily via oral gavage in a dose volume of 5 

mL/kg body weight to 5 adult (61 days old) female Sprague Dawley [Crl:CD(SD)] rats/dose 

group at doses of 0, 20, 60, or 180 mg/kg/day. The animals were dosed for 3 consecutive days 

and necropsied approximately 24 hours after the final dose administration. 

All animals survived to the scheduled termination. No treatment-related clinical findings were 

noted at the daily examinations at any dose. Decreased defecation was noted for 2 animals each 

dosed at 60 and one animal dosed at 180 mg/kg/day. Significant (p<0.01 or p<0.05) decreases in 

food consumption (g/animal/day) were observed in animals dosed at 180 mg/kg on Day 1 

(↓42%), 2 (↓32%), and Day 4 (↓39%) and in animals dosed at 60 mg/kg on Day 1 (↓35%) and 2 

(↓24%). Decreased food consumption corresponded to the variability in the mean body weights 

and/or mean body weight gains for these dose groups. However, mean body weights and body 

weight gains were not significantly different from controls in dichlobenil dosed rats. 

Mean absolute and relative (to final body weights) adrenal gland weights in all dichlobenil dose 

groups were comparable to the control group. Significant (p<0.01 or p<0.05) increases in the 

mean absolute and relative weights of the kidneys (↑16% and ↑25%, respectively) and livers 

(↑27% and ↑36%, respectively) of animals dosed with dichlobenil at 180 mg/kg/day were 

observed. No significant differences in kidney and liver weights were observed in animals dosed 

at 20 or 60 mg/kg/day. Based on these data, dichlobenil dose levels of 20, 60 and 180 mg/kg/day 

were selected for the uterotrophic assay. 

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