## UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION



## MEMORANDUM

Date: 6/29/2015

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

## Assays

PC Code: 128997 

## DP Barcode: D398353, D401595, D404684 D404685 

## Decision No.: 460411, 464536, 468751 468752 

## Petition No.: NA Risk Assessment Type: NA TXR 

## No.: 0056253 

## MRID No.: See Table 

## Registration No.: NA
Regulatory Action: NA Case No.: NA CAS No.: 107534-96-3 

## 40CFR: NA

FROM: Greg Akerman, Ph.D. }'! 

## A..

_____ _ Immediate Office

## /~ ..UHealth Effects Division (7509P)



## THROUGH: Jess Rowland

# ~ (? e> 

# .0---

. 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 respons e to the agency ' s Test Order for tebuconazole: Test Order# CON-128997- 20.

P ag e 1 o f2

### Page 2 of 2 

## II. RESPONSE
Attached are the EDSP Tier 1 assay DERs for tebuconazole.

III. MRID Table

Chemical: Tebuconazole PC Code: 128997 Guideline Assay MRID 890.1100 Amphibian Metamorphosis Assay (Frog) 48673701 890.1150 Androgen Receptor Binding (Rat Prostate) 48619401 890.1200 Aromatase Assay (Human Recombinant) 48619402 890.1250 Estrogen Receptor Binding NA 890.1300 Estrogen Receptor Transcriptional Activation (Human Cell Line HeLa-9903) 48619403
890.1350 Fish Short-Term Reproduction 48688301 890.1400 Hershberger (Rat) 48673702 890.1450 Female Pubertal (Rat) 48688302 890.1500 Male Pubertal (Rat) 48688302 890.1550 Steroidogenesis (Human Cell Line – H295R) 48619404 890.1600 Uterotrophic (Rat) 48673703 NA= Not Applicable (requirement satisfied by ot her scientifically relevant information (OSRI))

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

EPA MRID Number 48673701

Page 1 of 59

DER Template Version: 22 September 2011 Data Requirement: EPA DP Barcode 401594

OECD Data Point 231

EPA Guideline 890.1100 Amphibian Metamorphosis Assay (Frog)

Test Material: Tebuconazole Technical Purity (%): 97.5%

Common Name: Tebuconazole

Chemical Name: IUPAC: ( RS )-1- p -Chloropheyl-4,4-dimethyl-3-(1 H -1,2,4-triazol-1-ylmethyl)
pentan-3-ol

CAS: Į -[2-(4-Chlorophenyl)ethyl]- Į -(1,1-dimethylethyl)-1 H -1,2,4-triazole-1-

ethanol

CAS No.: 107534-96-3 Synonyms: None

EPA PC Code: 128997

Primary Reviewer: John Marton Date: 12/19/12 Staff Scientist, Cambridge Environmental, Inc.

Secondary Reviewer(s): Teri S. Myers Date: 01/18/13
Environmental Scientist, CDM Smith

Secondary Reviewer(s): Stephen Carey Date: 01/30/13 Biologist, ERB6

Additional Reviewer: Elizabeth Donovan Date: 11/20/13 Biologist, ERB 6

Additional Reviewer: Amy Blankinship Date: 11/20/13
Chemist, ERB 6

Date Evaluation Completed: 6/6/15 Date: 2015.06.09 15:55:52 -04'00'

Digitally signed by Elizabeth Donovan DN: cn=Elizabeth Donovan, o=EPA, ou=EFED, email=donovan.elizabeth@epa.gov, c=US Date: 2015.06.10 08:49:45 -04'00'



### AMY BLANKINSHIP Digitally signed by AMY BLANKINSHIP DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=AMY BLANKINSHIP, dnQualifier=0000040917 Date: 2015.06.15 17:51:34 -04'00'

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

EPA MRID Number 48673701

Page 2 of 59

DER Template Version: 22 September 2011 CITATION: Banman, C.S., T.M. Alexander, and C.V. Lam. 2012. Amphibian Metamorphosis Assay with South African Clawed-Frog ( Xenopus laevis ) Exposed to Tebuconazole Under Flow-Through Conditions.
Unpublished study performed by Bayer CropScience, Ecotoxicology, Stilwell, Kansas. Laboratory report number EBHWP001. Study sponsored by Bayer CropScience, Research Triangle Park, North Carolina. Study completed April 12, 2012.

Note: The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths
of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and redundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potential to interact with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).

Disclaimer: The guideline recommendations in this DER template are offered as a general reference to aid in preparation of the DER. The purpose of these recommendations is not to serve as substitute for the Test Guidelines, nor to provide any guidance on how the study should be conducted.

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

EPA MRID Number 48673701

Page 3 of 59

DER Template Version: 22 September 2011 EXECUTIVE SUMMARY

The 21-day assay of Tebuconazole (97.5% purity) on amphibian metamorphosis of So uth African clawed frogs ( Xenopus laevis ) was conducted under flow-through conditions. Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 (80/group, 20 in each of four reps) were exposed to nominal concentrations of 0 (negative control), 0.0152,
0.076, and 0.380 mg a.i./L; mean-m easured concentrations were <0.0015 (<LOQ; control), 0.0170, 0.067, and 0.341 mg a.i./L. The test system was maintained at 21.4 to 22.7°C and a pH of 7.9 to 8.4.

Unless otherwise indicated, all effects are reported based on comparison to the negative (clean water) control.
The Day 21 survival averaged 100% in the control and low treatment group and 98.3% in the mid and high treatment groups. Tadpoles with bent tails were observed in the control (16/80 tadpoles) and low (14) and mid(5)
treatment groups. In the high treatment, two tadpoles were reported as pal e while two more tadp oles showed erratic behavior/loss of equilibrium.

Late-stage tadpoles made up more than 20% of the surviving tadpoles on Day 21 in the negative control (55%) and low treatment group (38%), whereas late-stage tadpoles accounted for 7 and 0% of survivors in the mid and
high treatment groups. All growth analyses were conducted excluding late-stage tadpoles and separate analyses were not conducted on the late-stage tadpoles because none were present in the highest dose. Tebuconazole had no effect on 7-day normalized hind-limb length (HLL), whereas the highest dose exhibited a significant 56%
reduction relative to the control on Day 21. Significant delay of median NF developmental stage by one stage was observed at 7 days in all treatment groups and at 21 days in the mid and high treatment groups (by one stage at the mid and 5 stages at the high). No asynchronous development was observed in any treatment group. Given
the large differences in NF stage between the control and high treatment group, additional control tadpoles were reared with the goal of selecting tadpoles at developmental stages similar to the high treatment (NF stage 56 and 57). Mild thyroid hypoplasia was observed at the high treatment group in all 20 tadpoles, whereas this finding
was not observed in the concurrent control, low or mid treatment group but was observed in 11/20 tadpoles in the stage-matched control. Mild follicular cell hypertrophy was observed in the concurrent control (6/20 tadpoles), staged-matched controls (2/20 tadpoles) and low and mid treatment groups (3/20 and 6/20 tadpoles respectively). The procedure of rearing additional control frogs to allow for stage-matching histopathological
evaluation is not part of the guideline, and there is uncertainty in the ability to compare these frogs to the treatment groups given that they were not from the same batch of eggs and were not reared at the same time which may have resulted in slight differences in environmental conditions.

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

EPA MRID Number 48673701

Page 4 of 59

DER Template Version: 22 September 2011 Significant reductions in snout-vent length (SVL) and body weight (BW) were observed at the high concentration on both Day 7 (SVL: Ļ 16%; BW: Ļ 24%) and 21 (SVL: Ļ 24%; BW: Ļ 59%) and in the low treatment on Day 21 (SVL: Ļ 6%; BW: Ļ 12%). In the high treatment, two tadpoles were reported as pale while two more tadpoles showed
erratic behavior/loss of equilibrium. Histological findings in the liver occurred in the mid and high treatment groups, and included decreased hepatocyte vacu olation, hepatocyte anisocylosis, he patocyte anisokaryosis and increased multinucleate hepatocytes. An additional treatment-related finding in the high treatment group was individual hepatocyte necrosis.

All validity and performance criteria were met.

The assay satisfies the EDSP Tier 1 Test Order requirements for an Amphibian Metamorphosis Assay (OCSPP Guideline 890.1100).

Results Synopsis: Test organism NF stage at test initiation: 51 Test organism total length at test initiation (optional): Not reported
Test type: flow-through

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

EPA MRID Number 48673701

Page 5 of 59

DER Template Version: 22 September 2011 Table 1: Summary of Developmental and Thyroid Pathology/Histopathology Effects 1,2 

### in the Amphibian Metamorphosis Assay (AMA) with Tebuconazole Technical.

Treatment (mg a.i./L)
[mean-measured] NF Developmental Stage Hind Limb Length 3 

### Asynchronous Development Thyroid Gross and Histopathology
Day 7 Day 21 Day 7 Day 21 Day 7 Day 21 Day 21

0.017 Yes No No No No No No

0.067 Yes Yes No No No No No

0.341 Yes Yes No Yes No No Yes 4

1 

### A "yes" indicates a significant difference based on comparison to the negative (clean water) control, unless
otherwise specified.

2 

### The criteria for significance are described in the Reviewer's Analysis and Statistical Verification sections of the DER. Conclusions regarding histopathology may be heavily weighted by the expert opinion of a board-certified pathologist.

3 

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

4 

### Increases in mild thyroid hypoplasia was observed when compared to both the concurrent control as well

as the additional st aged-match control.

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

EPA MRID Number 48673701

Page 6 of 59

DER Template Version: 22 September 2011 I. MATERIALS AND METHODS

Guideline Followed: This study was conducted following guideli nes outlined in U.S. Environmental Protection Agency’s OCSPP 890.1100: Amphibian Metamorphosis (Frogs). The following deviation

was noted:

1. Water hardness at 168 – 152 mg/L CaCO 3 

### exceeded the recommended range of 40-48 mg/L CaCO 3



### This deviation does not impact the acceptability of the study.

Compliance: Signed and dated No Data Confidentiality, GLP, and Quality Assurance statements were provided. This study was conducted in compliance with the Good Laboratory Practice Standards as specified in 40 CFR 160 with the following exceptions: routine spring water and reverse osmosis water contaminant screening analyses for pesticides, PCBs, and toxic metals were conducted by American Analytical Laboratories, Akron, OH, and; food contaminant screening analyses for pesticides, PCBs, and toxic metals were conducted by Geo Labs, Inc., Braintree, MA. Data fr om the screening analyses were not collected in accordance with Good Laboratory Practice procedures (no protocol, study director, or in-life inspections).

A. Test Material Tebuconazole Technical

Description: White powder

Lot No./Batch No. : K689052 (Batch No.)
Purity: 97.5% Impurities: None reported

Stability of Compound: Stable. Mean-measured concentrations yielded recoveries of 88 to 100% of nominal with coefficients of variation (CV) of 14.7 to 19.4%.

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

EPA MRID Number 48673701

Page 7 of 59

DER Template Version: 22 September 2011 Storage Conditions of

Test Chemicals: Stored in a plastic jar at room temperature.Data Evaluation Record on the Toxicity of Tebuconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673701Page8of59 DER Template Version: 22 September 2011B. Test OrganismTable 2: General Information About the Test Species and Parental Care.ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies common name: South African ClawedFrog 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 spec ies is recommended for inclusion in the study report. The guideline recommends that the performance criteria used to support the reliability of the test be

identified.Species scientific name: Xenopus laevisSpecies strain (if stated): Not specified1U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the Endocrine Disruptor Screening ProgramTier 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).Data Evaluation Record on the Toxicity of Tebuconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673701Page9of59 DER Template Version: 22 September 2011Table 2: General Information About the Test Species and Parental Care.ParameterValue(s)Details or RemarksGuideline RecommendationsWere parents maintained asin-house stock?No. Frogs used in thedefinitive assay werereceived as fertilizedembryos. EPA recommends that larvae used in the assay be derived from in-house adults.Were parental acclimationconditions same as definitivetest?NAEmbryos and newly hatched tadpoleswere maintained under comparableconditions as those used in the definitivetest.Acclimation period for parentalfrogs (if applicable):Embryos wereacclimated for 11 days.Details on parental feeding: During acclimationtadpoles were fed liquidFrog Brittle Slurry atleast once daily duringhatch and twice dailythereafter.Data Evaluation Record on the Toxicity of Tebuconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673701Page10of59 DER Template Version: 22 September 2011Table 2: General Information About the Test Species and Parental Care.ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on parental health: Mortality was <5%during the holding periodand there were notreatments for disease.Table 3: Larval Selection and Care.ParameterValue(s)Details or RemarksGuideline RecommendationsBest single spawn?All embryos were from asingle spawn.Embryos (lot number: NXL042911) werereceived from Nasco (Fort Atkinson, WI)two days following spawn. EPA and OECD recommend that the best 2 – 3 individual spawns, with a minimum of 1500 larvae/spawn, be evaluated to identify the best single spawn, and that the larvae selected for testing originate from the best single spawn

(i.e., the spawns are not co-mixed)Number of spawns evaluated (ifapplicable):Not reportedNumber of eggs sampled perspawn:Not reportedNF stage at test initiation 51 EPA recommends that the definitive study beData Evaluation Record on the Toxicity of Tebuconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673701Page11of59 DER Template Version: 22 September 2011Table 3: Larval Selection and Care.ParameterValue(s)Details or RemarksGuideline RecommendationsAge at test initiation:13 days post-spawn initiated with larvae at Nieuwkoop – Faber (NF) developmental stage 51 ( 17 days post-fertilization).Mean total length at test initiation(if reported):Not reportedRange of total length at testinitiation (if reported):Not reportedWas the optional size selectionmethod used?Not reportedDetails on larval selection: Cell division was verifiedwith a microscope priorto shipping.Loading rate (rearing density): 6.3 larvae/L EPA recommends that rearing density (loading rate) not exceed approximately 10 larvae/L culturing system for flow-through systems or 4 tadpoles/L in static-renewal exposure systems.Type of food:Liquid Frog Brittle Slurry, EPA recommends Sera Micron ® throughoutData Evaluation Record on the Toxicity of Tebuconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673701Page12of59 DER Template Version: 22 September 2011Table 3: Larval Selection and Care.ParameterValue(s)Details or RemarksGuideline Recommendationsprepared from NascoFrog Brittle Diet 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:NascoIodide concentration in diet (ifknown):Not reportedFrequency of feeding:two times/day EPA recommends that feeding occur at least twice per day.Details on feeding regime: During the definitive testtadpoles received LiquidFrog Brittle slurry twicedaily at a concentrationof 60 mg/mL. 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).Data Evaluation Record on the Toxicity of Tebuconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673701Page13of59 DER Template Version: 22 September 2011C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:Flow-through EPA recommends the use of a flow-through

system.Type of flow-through dilutionsystem (if applicable):Intermittent Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 2Flow-through rate (ifapplicable):23.25 mL/minThe flow rate resulted in approximately 9volume additions per day. Recommended flow-through rate is 25 mL/min (complete volume replacement ca. every 2.7 hrs).2Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.Data Evaluation Record on the Toxicity of Tebuconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673701Page14of59 DER Template Version: 22 September 2011Table 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on toxicant mixing forflow-through systems (ifapplicable):Test material wasdelivered to theappropriate mixing vesselwhere it was constantlymixed with dilution waterusing a magnetic stir bar.The appropriate volume ofthe dilution water andtoxicant mixing solutionwas blended anddelivered to the testaquaria.The accuracy (±10%) of the test solutiondelivery system was checked prior to testinitiation. The control test concentrationreceived no test material. Diluter wasoperating for approximately 5 days prior totest initiation and the diluter system wasvisually checked twice daily during the weekand at least once on weekends and holidays. Recommended toxicant mixing for flow-through systems: 1) Mixing chamber is recommended but not required; 2) Aeration is not recommended for mixing;3) A demonstration that the test solution is completely mixed before introduced into the test system is recommended; 4) The recommended flow splitting accuracy is
within 10%.Renewal period for staticrenewal (if applicable):NA If static renewal is used, EPA recommends 24-hr renewal; renewal period is recommended not to exceed 72 hours.Data Evaluation Record on the Toxicity of Tebuconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673701Page15of59 DER Template Version: 22 September 2011Table 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsAeration? 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: Dilution water (hardprocessed water) consistsof spring water blendedwith reverse osmosiswater (municipal water) toproduced hard (160-180mg/L) water used forculturing and testingfreshwater invertebrates.Spring water is collected from a spring box,passed through a multimedia filter, a5-micron bag filter, granular activated carbonfilters, a 1-micron cartridge filter and finally anultraviolet sterilizer. This is then blended withmunicipal water that has been dechlorinatedwith sodium metabisulfite, passed through amultimedia filter, granular activated carbonunits, demineralized by conventionalsofteners, passed through a 5-microncartridge filter, treated by double passreverse osmosis ad passed through anultraviolet sterilizer. EPA recommends natural or reconstituted water; it is recommende d 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.Data Evaluation Record on the Toxicity of Tebuconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673701Page16of59 DER Template Version: 22 September 2011Table 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsWas dilution water analyzed forpesticides, heavy metals, andother contaminants?Yes. See Reviewer’sComments.Iodide supplementation inwater?No If reconstituted water is used or if background levels of iodide in natural water are less than 0.5 ս g/L, iodide supplementation is recommended. This supplementation is in addition to the recommended dietary source of iodide (e.g.. in Sera Micron).Test vessel type/materials: Glass EPA and OECD recommend that water-contact portions of the system not compromise the study (e.g., all glass vessels or glass vessels with stainless steel frames are acceptable examples).Test vessel size:8.3 LFill volume:4 L (30 x 14.5 x 9.2 cm)Data Evaluation Record on the Toxicity of Tebuconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673701Page17of59 DER Template Version: 22 September 2011Table 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details on exposuresystem:NATable 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsHardness (mg/L as CaCO3) 168 188 179Measured in onealternating replicateat each level at testinitiation and weeklythereafter. EPA recommends hardness 40 to 48 mg/L as CaCO 3.pH 7.98.28.1 EPA recommends pH 7.5 ± 1, inter-replicate and inter-treatment differentials should not exceed 0.5.Dissolved oxygen (mg/L)6.38.47.6Measured in twoalternating replicatesat each level at testinitiation and weeklythereafter. EPA recommends dissolved oxygen (DO) >3.5 mg/L (>40% air saturation). OECD recommends DO concentration >3.5 mg/L (>40% air saturation).Data Evaluation Record on the Toxicity of Tebuconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673701Page18of59 DER Template Version: 22 September 2011Table 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsTemperature (°C)21.4 22.722.1Temperature wasmeasuredcontinuously in acentrally located testvessel and daily in alltest vessels. EPA recommends temperature 22±1 o C; inter-replicate and inter-treatment differentials should not exceed 0.5 o C.Iodide<0.10 mg/L in Spring and RO water EPA recommends aquatic iodide range 0.5

– 10 ս g/L (supplemental iodide should not

exceed 2 ս g/L).Ammonia <0.001mg/L 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,Fluoride0.18 mg/L in Spring water, <0.10 mg/L in RO waterPerchlorate NotReportedChlorate NotReportedData Evaluation Record on the Toxicity of Tebuconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673701Page19of59 DER Template Version: 22 September 2011Table 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsAlkalinity (mg/L)Conductivity (սmhos/cm)115429164463143453 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.D. Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details or RemarksGuideline RecommendationsWas a range-finder conducted?YesData Evaluation Record on the Toxicity of Tebuconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673701Page20of59 DER Template Version: 22 September 2011Table 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details or RemarksGuideline RecommendationsIf yes, what was the method fordetermining the highest testconcentration in therange-finder?Not specified. A 21-dayrange-finding AMA anda 48-hour acute toxicitytest were conducted. EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of toxicity (e.g., <10% mortality), whichever concentration is lowest.Species: Xenopus laevisLife stage:Not specified.Test duration:21 days (AMA)48 hours (acute)Data Evaluation Record on the Toxicity of Tebuconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673701Page21of59 DER Template Version: 22 September 2011Table 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details:The AMA test wasconducted with nominalconcentrations of 0.010,0.100, and 1.000 mga.i./L for 21 days. Day 7wet weight was inhibitedat the highest treatmentgroup and Day 21developmental stagewas affected at the twohighest treatment levels.Due to the highpercentage of tadpolesdeveloping beyond NF60, Day 21 growthendpoints were notanalyzed.Results from the 48-hour acuterange-finding study yielded LC50andNOAEC values of 6.3 and 4.5 mg a.i./L,respectively.Data Evaluation Record on the Toxicity of Tebuconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673701Page22of59 DER Template Version: 22 September 2011Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:21 days EPA recommends that the duration of the definitive test be 21 days.Method for selecting the highesttest concentration in the definitivetest:The study authorsreported that based onthe range-finding resultsthat overt toxicity wouldoccur at levels below1/3 of the LC50(2.1 mga.i./L). Therefore,nominal concentrationsof 0.015, 0.075, and0.375 mg a.i./L wereselected for thedefinitive exposure. 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 (ifapplicable):NAData Evaluation Record on the Toxicity of Tebuconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673701Page23of59 DER Template Version: 22 September 2011Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsSeparation of test concentrations:0.2 EPA recommends that the maximum concentration separation be 0.1 and the minimum be 0.33.Number of test concentrations:3 EPA recommends a minimum of 3 concentrations and a control, plus solvent control if appropriate.Are nominal concentrationsadjusted for purity?YesIndicate the type of valuespresented for measuredconcentrations:Mean-measuredLimit of quantification (LOQ): 0.0015mg a.i./L EPA recommends that for chemical test concentrations below the LOQ, analyses be conducted on the stock solutions.Level of detection (LOD): Not ReportedData Evaluation Record on the Toxicity of Tebuconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673701Page24of59 DER Template Version: 22 September 2011Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsFrequency of measurement: Day 0, 7, 14, and 21 It is recommended that test item concentration be measured in one tank at each treatment level at test initiation and every week

thereafter.Number of replicates in control: 4 EPA recommends 4 replicates.Number of replicates in solventcontrol (if applicable):NA 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 itemtreatment level:4 EPA recommends 4 replicates.Number of larvae per treatment attest initiation:80 (equally dividedbetween 4 replicates)Was a solvent used?NoSolvent type (if applicable): NAData Evaluation Record on the Toxicity of Tebuconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673701Page25of59 DER Template Version: 22 September 2011Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsMaximum solvent concentration(if applicable):NA EPA recommends that the solvent not exceed

0.02 ml/L3 . OECD recommends that solvent have no effect on survival nor produce any other adverse effects and that concentration not be greater than 0.1 ml/L4 .Was a positive control used? NoPositive control (if applicable): NAPositive control concentration(s)(if applicable):NAPhotoperiod:12 hrs light :12 hrs dark EPA recommends photoperiod 12:12 (light:dark).Light intensity at water’s surface: 1029 to 1425 lux EPA recommends light intensity 0.6 – 2 Klux (at water’s surface).3Hutchinson 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.69–92.4OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Series on Testing andAssessment. No. 23. Paris, France.Data Evaluation Record on the Toxicity of Tebuconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673701Page26of59 DER Template Version: 22 September 2011Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details:NANo observed precipitates were reportedTable 8: Summary of Treatment Concentrations in the Amphibian Metamorphosis Assay with Tebuconazole.Treatment IDNominalConcentration(mg a.i./L)MeasuredConcentration(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsNegative Control 0.00<1.5NANA EPA and OECD recommend that test item concentrations be maintained at a coefficient of variation (CV) 20%.Treatment 10.01520.01719.4NATreatment 20.0760.06714.7NATreatment 30.3800.34115.5NAAbbreviations:CVCoefficient of variation.E. ObservationsBiological Endpoints:Day 7- NF stage, wet weight, hind-limb length (HLL), snout-vent length (SVL), normalized HLLDay 21- NF stage, wet weight, HLL, SVL, normalized HLL, histopathologyDaily- mortality and sub-lethal effectsData Evaluation Record on the Toxicity of Tebuconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673701Page27of59 DER Template Version: 22 September 2011Were 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 (d ays 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 lar val 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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DER Template Version: 22 September 2011 II. RESULTS AND DISCUSSION

A. Results

Survival

Mean survival on Day 7 was 100% in the control and all treatment groups (Table 9). On Day 21, survival averaged
100% in the control and mean-measured 0.017 mg a.i./L treatment group and 98.3% in the mean-measured 0.067 and 0.341 mg a.i./L treatment groups.

Table 9: Larval Mortality in Xenopus laevis

.

Treatment (mg a.i./L)

[mean-measured] Larval Mortality

Day 7 1 

### Day 21

n Mortality # Mortality % n Mortality # Mortality %

Negative Control 80 0 0 60 0 0

0.017 80 0 0 60 0 0

0.067 80 0 0 60 1 1.7

0.341 80 0 0 60 1 1.7

1 

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

Developmental Stage

Median developmental stage on Day 7 was 55 in the negative control and 54 in all treatment groups (Table 10).
On Day 21, median developmental stage was 61 in the negative control and 60, 59.5, and 56 in the mean-measured 0.017, 0.067, and 0.341 mg a.i./L treatment groups, respectively. T In the control and each treatment group, the 20 th 

### and 80 th 

### quantiles did not differ by more than 2 NF stages.

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DER Template Version: 22 September 2011 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 20 55 0 60 61 0

0.017 20 54 0 60 60 0

0.067 20 54 0 59 59.5 0

0.341 20 54 0 59 56 0

.

Hind Limb Length

Mean normalized HLL on Day 7 was 0.186 in the negati ve control and 0.187, 0. 190, and 0.180 in the mean-measured 0.017, 0.067, and 0.341 mg a.i./L treatme nt groups, respectively (Table 11). On Day 21, mean
normalized HLL was 0.772 in the negative control and 0.837, 0.710, and 0.340 in the mean-measured 0.017, 0.067, and 0.341 mg a.i./L treatment groups, respectively.

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DER Template Version: 22 September 2011 Table 11: Larval Development in Xenopus laevis – Hind Limb Length.

Treatment (mg a.i./L)

[mean-measured] Hind Limb Length (HLL)

Day 7 Day 21

n Mean (mm) ±SD HLL:

SVL 1 

### n Mean (mm) ±SD HLL:

SVL 1



### Negative Control 20 3.06 0.16 0.20 60 15.83 1.71 0.77

0.017 20 2.90 0.43 0.18 60 16.08 0.72 0.84

0.067 20 2.84 0.09 0.19 59 14.24 0.37 0.71

0.341 20 2.49 0.19 0.19 59 5.39 0.78 0.34

Abbreviations: SD 

### Standard deviation.

1 

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

Snout to Vent Length (SVL) and Wet Weight

Day 7 SVL ranged from 13.1 mm (0.341 mg/L) to 16.65 mm (negative control) (Table 12). Day 21 SVL ranged from 15.67 mm (0.341 mg/L) to 20.69 mm (negative control)

On Day 7, mean wet weight (in the highest treatment group (0.341 mg/L) was reduced (34%) relative to the negative control. Similarly, wet weight on Day 21 was reduced (59%) at the highest treatment group relative to
the negative control.

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DER Template Version: 22 September 2011 Table 12: Larval Growth in Xenopus laevis

.

Treatment (mg a.i./L)

[meanmeasured] Snout-Vent Length (SVL) Body Weight 1



### Day 7 Day 21 Day 7 Day 21

n Mean (mm) ±SD n Mean (mm) ±SD n Mean (g) ±SD n Mea

n (g) ±SD

Negative

Control 20 15.65 0.33 60 20.69 0.37 20 0.44 0.03 60 1.31 0.08

0.017 20 15.60 0.44 60 19.39 0.46 20 0.44 0.05 60 1.15 0.10

0.067 20 15.08 0.61 59 20.10 0.25 20 0.40 0.04 59 1.24 0.06

0.341 20 13.10 0.28 59 15.67 0.40 20 0.29 0.03 59 0.54 0.05

Abbreviations: SD 

### Standard deviation.

1 

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

Histopathology

Since there was a delay in development at the highest treatment group, it was not possible to make a direct histological comparison of tadpoles relative to the treatment group. Therefore, following the in-life portion of the study, an additional group of non-concurrent control tadpoles were reared under treatment conditions for 14 days and a sub-set (n = 20) were selected for histopathology with the goal of selecting control tadpoles at developmental stages similar to the high dose for a more direct comparison (NF stages 56 and 57).

Overall, thyroid effects were restricted to the 0.341 mg a.i./L treatment group and included mild relative thyroid gland hypoplasia in all tadpoles and a decreased prevalence of thyroid follicular cell hypertrophy relative to controls (Tables 13 and 14). In the middle and high treatment groups, effects associated with the liver also were observed
and included decreased hepatocyte vacuolation, hepatocyte anisocytosis, hepatocyte aniskokaryosis, and increased multinucleate hepatocytes. Individual hepatocyte necrosis also was observed in the high dose.

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DER Template Version: 22 September 2011 Table 13: Gross Pathology and Histopathology of the Thyroid Gland in Xenopus laevis

.

Treatment (mg a.i./L)

[meanmeasured] Diagnostic Observations 1



### Severity Thyroid Gland Hypertrophy Thyroid Gland Atrophy Follicular Cell Hypertrophy Follicular Cell Hyperplasia

n Incidence n Incidence n Incidence n Incidence

Negative

Control –

concurrent

control 0 20 NR 20 NR 20 14 20 19

1 20 NR 20 NR 20 6 20 1

2 20 NR 20 NR 20 0 20 0

3 20 NR 20 NR 20 0 20 0

Staged-match

control 2 

### 0 20 NR 20 NR 20 18 20 20

1 20 NR 20 NR 20 2 20 0

2 20 NR 20 NR 20 0 20 0

3 20 NR 20 NR 20 0 20 0

0.017 0 20 NR 20 NR 20 17 20 20

1 20 NR 20 NR 20 3 20 0

2 20 NR 20 NR 20 0 20 0

3 20 NR 20 NR 20 0 20 0

0.067 0 20 NR 20 NR 20 14 20 20

1 20 NR 20 NR 20 6 20 0

2 20 NR 20 NR 20 0 20 0

3 20 NR 20 NR 20 0 20 0

0.341 2 

### 0 20 NR 20 NR 20 20 20 20

1 20 NR 20 NR 20 0 20 0

2 20 NR 20 NR 20 0 20 0

3 20 NR 20 NR 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. NR= not reported

2 

### Tadpoles in this group were compared to tadpoles from a non-concurrent control in order to match the appropriate stagesData Evaluation Record on the Toxicity of Tebuconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673701Page33of59 DER Template Version: 22 September 2011Table 14: Additional Thyroid Gland Histopathology Observations in Xenopus laevis.Treatment (mg a.i./L)[mean-measured]Additional Qualitative Observations1SeverityFollicular LumenArea (Increase)Follicular LumenArea (Decrease)Follicular CellHeight (Increase)Follicular Cell Height(Decrease)Follicular CellShapenIncidencenIncidencenIncidencenIncidencenIncidenceNegative Control 0 20 NR 20 19 -- -- -- -- -- --1 20 NR 20 1 -- -- -- -- -- --2 20 NR 20 0 -- ---- -- -- --3 20 NR 20 0 -- ---- -- -- --Staged-matchcontrol20 20 NR 20 201 20 NR 20 02 20 NR 20 03 20 NR 20 00.0170 20 NR 20 20 -- -- -- -- -- --1 20 NR 20 0 -- -- -- -- -- --2 20 NR 20 0 -- ---- -- -- --3 20 NR 20 0 -- ---- -- -- --0.0670 20 NR 20 20 -- -- -- -- -- --1 20 NR 20 0 -- -- -- -- -- --2 20 NR 20 0 -- ---- -- -- --3 20 NR 20 0 -- ---- -- -- --Data Evaluation Record on the Toxicity of Tebuconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673701Page34of59 DER Template Version: 22 September 2011Table 14: Additional Thyroid Gland Histopathology Observations in Xenopus laevis.Treatment (mg a.i./L)[mean-measured]Additional Qualitative Observations1SeverityFollicular LumenArea (Increase)Follicular LumenArea (Decrease)Follicular CellHeight (Increase)Follicular Cell Height(Decrease)Follicular CellShapenIncidencenIncidencenIncidencenIncidencenIncidence0.34120 20 NR 20 20 -- -- -- -- -- --1 20 NR 20 0 -- -- -- -- -- --2 20 NR 20 0 -- ---- -- -- --3 20 NR 20 0 -- ---- -- -- --1Thyroid histopathology is graded 0 – 3 based on severity: 0=Not remarkable, 1=Mild, 2=Moderate, 3=Severe. See OECD No. 82 forreference.2Tadpoles from this group were compared to tadpoles from a non-concurrent control group to match the appropriate stages.Data Evaluation Record on the Toxicity of Tebuconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673701Page35of59 DER Template Version: 22 September 2011Table 14a: Additional Thyroid Gland Histopathology Observations in Xenopus laevis.Treatment (mg a.i./L)[mean-measured]Additional Qualitative Observations1SeverityRelative Thyroid Gland HypoplasianIncidenceNegative Control 020201 2002 2003 200Staged-matchcontrol20 2091 20112 2003 2000.017 0 20201 2002 2003 2000.067 0 20201 2002 2003 2000.34120 200Data Evaluation Record on the Toxicity of Tebuconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673701Page36of59 DER Template Version: 22 September 2011Table 14a: Additional Thyroid Gland Histopathology Observations in Xenopus laevis.Treatment (mg a.i./L)[mean-measured]Additional Qualitative Observations1SeverityRelative Thyroid Gland HypoplasianIncidence1 20202 2003 2001Thyroid histopathology is graded 0 – 3 based on severity: 0=Not remarkable, 1=Mild, 2=Moderate, 3=Severe. See OECD No.82 for reference.2Tadpoles from this group were compared to tadpoles from a non-concurrent control group to match the appropriate stages.Clinical SignsBeginning on Day 16, sublethal effects observed in the mean-measured 0.341 mg a.i./L treatment group. One tadpole was observedin each of two replicatesand an additional tadpole was observed to be pale and swimming erratically in a third replicate. No other effects were observedin the control or remaining treatmentgroups throughout the test. Various organisms throughout all test concentrations were observed with bent tails but did not represent a dose-dependent response(Table 15).Table 15: Clinical Signs in Xenopus laevis.Data Evaluation Record on the Toxicity of Tebuconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673701Page37of59 DER Template Version: 22 September 2011Treatment (mg a.i./L)[mean- measured]Clinical SignsTypenIncidence1Negative ControlBent tail60160.017Bent tail60140.067Bent tail5950.341 Erraticbehavior 592Loss of equilibrium5921Represents observations on Day 21.

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DER Template Version: 22 September 2011 B. Study Author’s Analysis and Conclusions

Mortality data were analyzed using Fisher’s Exact Test with a Bonferroni-Holm Adjustment using CETIS statistical software. Developmental stage was analyzed using replicate medians on Days 7 and 21 with the Jonckheere-Terpstra Multi-Quantal Trend Test (2-tailed) using SAS statistical software. Growth endpoints for Day 7 (normalized HLL, SVL, and wet weight) were visually evaluated for monotonic trends. Endpoints showing monotonic trends were analyzed using the replicate medians with Dunnett’s Multiple Comparison Test (1-tailed)
using CETIS software. Endpoints not showing monotonic trends were analyzed using the replicate means with Dunnett’s test (2-tailed) when the data passed the as sessments for normality (Shapiro-Wilk test) and variance homogeneity (Bartlett’s test). If the data failed the normality test, then a non-parametric procedure was used.
Endpoints were evaluated using CETIS statistical software.

Due to a portion of the tadpoles exceeding NF stage 60, tadpole growth endpoints on Day 21 (normalized HLL, SVL, and wet weight) were analyzed using a two-factor ANOVA with a nested variance structure in SAS statistical
software. The independent variables were concentration and stage with concentration nested within replicate. This statistical method takes into account the reduction in size of tadpoles that occurs after NF stage 60 due to the resorption of tissues and reduction of water content by separating the developmental analysis into late stage (NF

 61) and non-late stage (NF  60) groups.

Developmental stage on Day 7 was signif icantly reduced at all treatment levels relative to the negative control, whereas only tadpoles in the highest treatment group exhibited delayed development on Day 21. Normalized HLL was only reduced in the highest treatment group on Day 21. Wet weight and SVL both were significantly reduced at the highest treatment group on Days 7 and 21. Further, both growth parameters were significantly reduced at the lowest treatment group on Day 7. There were no treat ment-related effects on mortality or clinical observations, though thyroid effects were observed in the highest treatment group. Further, effects on the liver were observed in the middle and highest treatment groups.

C. Reviewer’s Analysis and Conclusions

Statistical Methods: A single mortality occurred in each of the two highest treatment groups was not considered treatment related. On Day 7, developmental stage, wet weight, SVL, and HLL exhibited monotonic trends and therefore were analyzed using the Jocnkheere-Terpstra test. Day 7 normalized HLL was not monotonic but was

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DER Template Version: 22 September 2011 normally distributed (Shapiro-Wilks, Į = 0.01) with equal variance (Levene’s test, Į = 0.05) and therefore was analyzed using Dunnett’s test. On Day 21, developmental stage and HLL were analyzed using the Jocnkheere-Terprstra test, whereas the remaining endpoints were analyzed using Dunnett’s test. No asynchronous development was observed. Day 21 replicate means for all growth parameters and normalized HLL were calculated from the raw data by excluding late-stage tadpoles (NF >60). Unless otherwise indicated, effects were considered
statistically significant at p < 0.05.

Conclusions:

No significant effects on larval survival were observed ( p > 0.05). On Day 7, wet weight, SVL, and HLL were
all significantly reduced at the highest treatment level rela tive to the control, while median developmental stage was reduced at all treatment levels. No significant differences in normalized HLL were detected at any treatment group. By Day 21, developmental stage and HLL were significantly reduced at the middle and high treatment groups, wet weight and SVL were significantly reduced at the low and high treatment levels, and normalized HLL was significantly reduced at the highest dose.Data Evaluation Record on the Toxicity of Tebuconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673701Page40of59 DER Template Version: 22 September 2011Summary of Developmental and Thyroid Gross Pathology/HistopathologyEndpoints1,2in the AMA with Tebuconazole.Treatment(mg a.i./L)[mean-measured]NF Developmental StageHind Limb Length3Asynchronous DevelopmentThyroid Gross andHistopathologyDay 7Day 21Day 7Day 214Day 7Day 21Day 21Median pMedian p% Diff. p% Diff. p%Diff. p% Diff. pTreatment RelatedEffects? (Yes/No)Negative Control55 NA 61 NA 0 NA 0 NA NANA NA NANA0.017540.0260 0.093 -5.74 0.54 8.45 0.29NA NA NA NA No0.067540.010600.005-3.57 0.82 -8.07 0.32NA NA NA NA No0.341 540.00356<0.001-3.44 0.83 -55.91<0.001NA NA NA NAYesStatistical TestJonckheere Jonckheere Dunnett’s Dunnettt’sNANANAAbbreviations:Diff.Difference.NANot applicable.1Unless otherwise indicated, effects are reported based on comparison to the clean water control. Conclusions regarding histopathology may be heavily weighted bythe expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p= 0.05.3Hind-limb length is normalized to snout-vent length (SVL).4There were 33, 23, and 4 late-stage tadpoles in the negative control, 0.017, and 0.067 mg a.i./L treatment groups, respectively, and excluded from analysis.

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DER Template Version: 22 September 2011 E. Study Deficiencies

No major deviations from OCSPP 890.1100 were noted. The only deviation observed was that water hardness at 168 – 152 mg/L CaCO 3 

### exceeded the recommended range of 40-48 mg/L CaCO 3 

### ; however, is considered minor and does not impact the acceptability of the study.

F. Reviewer’s Comments

The reviewer’s results agreed with th ose reported by the study authors.

The results from the periodic screening analysis of the dilution water (spring and RO water) did not detect any
organochloride pesticides, chlorinated herbicides, PCBs, organophosphate pesticides, or OP/nitro pesticides. The following analytes were detected in the spring wa ter used to prepare dilution water: aluminum (20 ս g/L), calcium (117 mg/L), chromium (5.4 ս g/L), copper (1.2 ս g/L), magnesium (6.2 mg/L), nickel (4.6 ս g/L), potassium (1.65 mg/L), selenium (1.3 ս g/L), sodium (62.4 mg/L), zinc (3.9 ս g/L), chloride (89 mg/L), fluoride (0.18 Summary of Growth Endpoints 1,2 

### in the AMA with Tebuconazole.

Treatment
(mg a.i./L)
[mean-measured] Snout-Vent Length Body Weight

Day 7 Day 21 3 

### Day 7 Day 21 3



### % Diff. p % Diff. p % Diff. p % Diff. p
Negative Control 0 NA 0 NA 0 NA 0 NA

0.017 -0.32 0.44 -6.31 0.001 0.51 0.61 -12.07 0.026

0.067 -3.67 0.070 -2.89 0.110 -9.12 0.082 -5.45 0.409

0.341 -16.29 <0.001 -24.30 <0.001 -33.18 0.001 -59.20 <0.001
Statistical Test Jonckheere Dunnett’s Jonckheere Dunnett’s

Abbreviations: Diff. 

### Difference. NA 

### Not applicable.

1 

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

2 

### Effects are considered statistically significant at p < 0.05, indicated in bold type

.

3 

### There were 33, 23, and 4 late-stage tadpoles in the negative control, 0.017, and 0.067 mg a.i./L treatment

groups, respectively, and excluded from analysis.

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DER Template Version: 22 September 2011 mg/L), nitrite as N (0.013 mg/L), nitrate as N (2.4 mg/L), phosphorus (0.048 mg/L) and sulfate (32 mg/L). The RO water used to prepare dilution water only contained sodium (2.08 mg/L) and nitrate as N (0.18 mg/L).

Advanced development was observed in the control, middle, and high level treatment groups with the number of late stage tadpoles (% late stage) being 33 (55%), 23 (38%), and 4 (7%) in the control, 0.017, and 0.067
mg a.i./L treatment groups, respectively. Because the number of late-stage tadpoles exceeded 20% in two groups, EDSP statistical guidance recommends conducting a separate analysis using only late-stage tadpoles. However, there must be at least three replicates from each treatment level with late-stage tadpoles. In the current study, no late-stage tadpoles were observed in the highest treatment group and therefore, a separate analysis was not conducted with late-stage tadpoles.

III. REFERENCES

Microsoft® Excel 2003. SP-3. Microsoft Corporation.

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

1:21-29.

Nieuwkoop, P.D. and Faber. 1994. Normal table of Xenopus laevis (Daudin). Garland Publishing, Inc. New York.

SAS Institute. 2002-2003. PC-SAS Version 9.1 (or more recent version). Cary, NC.

Tidepool Scientific Software, LLC, 2000-2010. CETIS Version 1.7.0.5.

Banman, C.S., T.M. Alexander, and C.V. Lam. 2010. Acute Toxicity of Tebuconazole to Xenopus laevis Under Static Conditions. Bayer CropScience unpublished report, report number EBHWY007.

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DER Template Version: 22 September 2011 APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION:

**Analysis results for 7-d wet weight (g)**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.943 0.386 0.297 0.827 USE PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.44 0.03 0.01 6.80 0.39, 0.49 Dose1 4 0.44 0.05 0.02 10.44 0.37, 0.51 Dose2 4 0.40 0.04 0.02 10.84 0.33, 0.47 Dose3 4 0.29 0.03 0.02 11.07 0.24, 0.34

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 0.43 0.41 0.48 . . Dose1 0.46 0.37 0.48 100.51 -0.51 Dose2 0.41 0.34 0.43 90.88 9.12 Dose3 0.28 0.27 0.34 66.82 33.18

******************************************************************************** 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 12.94 <.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 0.44 . 0.44 . . . . . . Dose1 0.44 1.000 0.44 0.601 . . . . . Dose2 0.40 0.360 0.40 0.105 0.439 . . . . Dose3 0.29 <.001 0.29 <.001 <.001 0.010 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 9.87 0.020

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 0.43 . . Dose1 0.46 0.889 0.614 Dose2 0.41 0.190 0.082 Dose3 0.28 0.067 0.001

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DER Template Version: 22 September 2011 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose3 Jonckheere Dose3

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 12.94 <.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 -0.44 . -0.39 . . . . . . Dose1 -0.44 1.000 -0.39 0.972 . . . . . Dose2 -0.40 0.360 -0.39 0.981 0.439 . . . . Dose3 -0.29 <.001 -0.39 0.985 <.001 0.010 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 9.87 0.020

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -0.43 . . Dose1 -0.46 0.889 0.386 Dose2 -0.41 0.190 0.918 Dose3 -0.28 0.067 0.999

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

**Analysis results for 7-d stage (median)**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.677 <.001 6.000 0.010 USE NON-PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 54.75 0.50 0.25 0.91 53.95, 55.55 Dose1 4 54.00 0.00 0.00 0.00 . , . Dose2 4 54.00 0.00 0.00 0.00 . , . Dose3 4 53.75 0.50 0.25 0.93 52.95, 54.55

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

EPA MRID Number 48673701

Page 45 of 59

DER Template Version: 22 September 2011 Level Median Min Max %of Control(means) %Reduction(means) Ctrl 55.00 54.00 55.00 . . Dose1 54.00 54.00 54.00 98.63 1.37 Dose2 54.00 54.00 54.00 98.63 1.37 Dose3 54.00 53.00 54.00 98.17 1.83

******************************************************************************** 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 6.00 0.010

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 54.75 . 54.75 . . . . . . Dose1 54.00 0.028 54.00 0.006 . . . . . Dose2 54.00 0.028 54.00 0.006 1.000 . . . . Dose3 53.75 0.005 53.75 0.001 0.752 0.752 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 9.32 0.025

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 55.00 . . Dose1 54.00 0.102 0.020 Dose2 54.00 0.102 0.010 Dose3 54.00 0.100 0.003

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose1 Jonckheere Dose1

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 6.00 0.010

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -54.75 . -54.13 . . . . . . Dose1 -54.00 0.028 -54.13 0.995 . . . . . Dose2 -54.00 0.028 -54.13 0.997 1.000 . . . . Dose3 -53.75 0.005 -54.13 0.998 0.752 0.752 . . .

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

EPA MRID Number 48673701

Page 46 of 59

DER Template Version: 22 September 2011 ******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 9.32 0.025

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 -55.00 . . Dose1 -54.00 0.102 0.980 Dose2 -54.00 0.102 0.990 Dose3 -54.00 0.100 0.997

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

**Analysis results for 7-d sn-vent length (mm)**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.932 0.261 0.898 0.471 USE PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 15.65 0.33 0.17 2.12 15.12, 16.18 Dose1 4 15.60 0.44 0.22 2.82 14.90, 16.30 Dose2 4 15.08 0.61 0.31 4.07 14.10, 16.05 Dose3 4 13.10 0.28 0.14 2.16 12.65, 13.55

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 15.60 15.30 16.10 . . Dose1 15.60 15.10 16.10 99.68 0.32 Dose2 15.30 14.20 15.50 96.33 3.67 Dose3 13.00 12.90 13.50 83.71 16.29

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 30.32 <.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 15.65 . 15.65 . . . . . . Dose1 15.60 0.997 15.60 0.514 . . . . . Dose2 15.08 0.200 15.08 0.053 0.363 . . . .

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

EPA MRID Number 48673701

Page 47 of 59

DER Template Version: 22 September 2011 Dose3 13.10 <.001 13.10 <.001 <.001 <.001 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 10.11 0.018

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 15.60 . . Dose1 15.60 1.000 0.442 Dose2 15.30 0.152 0.070 Dose3 13.00 0.065 <.001

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose3 Jonckheere Dose3

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 30.32 <.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 -15.65 . -14.86 . . . . . . Dose1 -15.60 0.997 -14.86 0.996 . . . . . Dose2 -15.08 0.200 -14.86 0.998 0.363 . . . . Dose3 -13.10 <.001 -14.86 0.998 <.001 <.001 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 10.11 0.018

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 -15.60 . . Dose1 -15.60 1.000 0.558 Dose2 -15.30 0.152 0.930 Dose3 -13.00 0.065 0.999

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

**Analysis results for 7-d hind-limb length (mm)**

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

EPA MRID Number 48673701

Page 48 of 59

DER Template Version: 22 September 2011 TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.963 0.718 4.182 0.030 USE NON-PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 3.06 0.16 0.08 5.12 2.81, 3.31 Dose1 4 2.90 0.43 0.22 14.96 2.21, 3.59 Dose2 4 2.84 0.09 0.04 3.05 2.70, 2.97 Dose3 4 2.49 0.19 0.10 7.74 2.18, 2.79

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 3.10 2.84 3.19 . . Dose1 2.85 2.45 3.44 94.77 5.23 Dose2 2.85 2.72 2.93 92.72 7.28 Dose3 2.44 2.32 2.75 81.36 18.64

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 3.59 0.046

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 3.06 . 3.06 . . . . . . Dose1 2.90 0.708 2.90 0.233 . . . . . Dose2 2.84 0.486 2.84 0.151 0.985 . . . . Dose3 2.49 0.020 2.49 0.005 0.156 0.264 . . .

******************************************************************************** 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 3.10 . . Dose1 2.85 0.494 0.193 Dose2 2.85 0.190 0.106 Dose3 2.44 0.067 0.004

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose3 Jonckheere Dose3

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

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

EPA MRID Number 48673701

Page 49 of 59

DER Template Version: 22 September 2011 PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 3.59 0.046

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 -3.06 . -2.82 . . . . . . Dose1 -2.90 0.708 -2.82 0.944 . . . . . Dose2 -2.84 0.486 -2.82 0.959 0.985 . . . . Dose3 -2.49 0.020 -2.82 0.965 0.156 0.264 . . .

******************************************************************************** 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 -3.10 . . Dose1 -2.85 0.494 0.807 Dose2 -2.85 0.190 0.894 Dose3 -2.44 0.067 0.996

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

**Analysis results for 7-d norm hind-limb**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.951 0.505 2.137 0.149 USE PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.20 0.01 0.00 3.70 0.18, 0.21 Dose1 4 0.18 0.02 0.01 12.47 0.15, 0.22 Dose2 4 0.19 0.01 0.00 4.34 0.18, 0.20 Dose3 4 0.19 0.01 0.01 5.76 0.17, 0.21

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 0.20 0.19 0.20 . . Dose1 0.18 0.16 0.22 94.26 5.74 Dose2 0.19 0.18 0.20 96.43 3.57 Dose3 0.19 0.18 0.20 96.56 3.44

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

EPA MRID Number 48673701

Page 50 of 59

DER Template Version: 22 September 2011 ******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 0.45 0.722

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 0.18 0.544 0.19 0.246 . . . . . Dose2 0.19 0.818 0.19 0.263 0.972 . . . . Dose3 0.19 0.832 0.19 0.273 0.967 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 1.83 0.608

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 0.18 0.494 0.193 Dose2 0.19 0.346 0.232 Dose3 0.19 0.413 0.256

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 0.45 0.722

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 -0.18 0.544 -0.19 0.814 . . . . . Dose2 -0.19 0.818 -0.19 0.844 0.972 . . . . Dose3 -0.19 0.832 -0.19 0.859 0.967 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 1.83 0.608

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

EPA MRID Number 48673701

Page 51 of 59

DER Template Version: 22 September 2011 MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -0.20 . . Dose1 -0.18 0.494 0.807 Dose2 -0.19 0.346 0.768 Dose3 -0.19 0.413 0.744

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

**Analysis results for 21-d stage (median)**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.983 0.983 0.182 0.907 USE PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 60.75 0.50 0.25 0.82 59.95, 61.55 Dose1 4 60.25 0.50 0.25 0.83 59.45, 61.05 Dose2 4 59.50 0.58 0.29 0.97 58.58, 60.42 Dose3 4 56.00 0.82 0.41 1.46 54.70, 57.30

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 61.00 60.00 61.00 . . Dose1 60.00 60.00 61.00 99.18 0.82 Dose2 59.50 59.00 60.00 97.94 2.06 Dose3 56.00 55.00 57.00 92.18 7.82

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 49.11 <.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 60.75 . 60.75 . . . . . . Dose1 60.25 0.539 60.25 0.161 . . . . . Dose2 59.50 0.035 59.50 0.008 0.350 . . . . Dose3 56.00 <.001 56.00 <.001 <.001 <.001 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value

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

EPA MRID Number 48673701

Page 52 of 59

DER Template Version: 22 September 2011 3 12.25 0.007

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 61.00 . . Dose1 60.00 0.285 0.093 Dose2 59.50 0.087 0.005 Dose3 56.00 0.061 <.001

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose2 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 49.11 <.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 -60.75 . -59.13 . . . . . . Dose1 -60.25 0.539 -59.13 1.000 . . . . . Dose2 -59.50 0.035 -59.13 1.000 0.350 . . . . Dose3 -56.00 <.001 -59.13 1.000 <.001 <.001 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 12.25 0.007

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -61.00 . . Dose1 -60.00 0.285 0.907 Dose2 -59.50 0.087 0.995 Dose3 -56.00 0.061 1.000

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

**Analysis results for 21-d wet weight (g)**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value

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

EPA MRID Number 48673701

Page 53 of 59

DER Template Version: 22 September 2011 0.935 0.296 1.688 0.222 USE PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 1.31 0.08 0.04 5.74 1.19, 1.43 Dose1 4 1.15 0.10 0.05 8.68 0.99, 1.31 Dose2 4 1.24 0.06 0.03 4.77 1.15, 1.33 Dose3 4 0.54 0.05 0.02 9.20 0.46, 0.61

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 1.31 1.23 1.40 . . Dose1 1.18 1.03 1.23 87.93 12.07 Dose2 1.23 1.18 1.31 94.55 5.45 Dose3 0.53 0.48 0.59 40.80 59.20

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 93.70 <.001

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 1.31 . 1.31 . . . . . . Dose1 1.15 0.026 1.20 0.027 . . . . . Dose2 1.24 0.409 1.20 0.029 0.380 . . . . Dose3 0.54 <.001 0.54 <.001 <.001 <.001 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 10.92 0.012

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 1.31 . . Dose1 1.18 0.156 0.042 Dose2 1.23 0.235 0.153 Dose3 0.53 0.067 0.002

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose1 Jonckheere Dose3

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 93.70 <.001

Dunnett - testing each trt mean signif. different than control

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

EPA MRID Number 48673701

Page 54 of 59

DER Template Version: 22 September 2011 Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -1.31 . -1.06 . . . . . . Dose1 -1.15 0.026 -1.06 1.000 . . . . . Dose2 -1.24 0.409 -1.06 1.000 0.380 . . . . Dose3 -0.54 <.001 -1.06 1.000 <.001 <.001 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 10.92 0.012

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 -1.31 . . Dose1 -1.18 0.156 0.958 Dose2 -1.23 0.235 0.847 Dose3 -0.53 0.067 0.998

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

**Analysis results for 21-d sn-vent length (mm)**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.924 0.197 0.934 0.454 USE PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 20.69 0.37 0.19 1.80 20.10, 21.29 Dose1 4 19.39 0.46 0.23 2.40 18.65, 20.13 Dose2 4 20.10 0.25 0.12 1.24 19.70, 20.49 Dose3 4 15.67 0.40 0.20 2.52 15.04, 16.29

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 20.72 20.28 21.06 . . Dose1 19.42 18.83 19.87 93.69 6.31 Dose2 20.09 19.87 20.33 97.11 2.89 Dose3 15.65 15.22 16.14 75.70 24.30

******************************************************************************** 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 12 142.65 <.001

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

EPA MRID Number 48673701

Page 55 of 59

DER Template Version: 22 September 2011 Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 20.69 . 20.69 . . . . . . Dose1 19.39 0.001 19.74 0.002 . . . . . Dose2 20.10 0.110 19.74 0.002 0.087 . . . . Dose3 15.67 <.001 15.67 <.001 <.001 <.001 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 13.33 0.004

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 20.72 . . Dose1 19.42 0.067 0.010 Dose2 20.09 0.156 0.170 Dose3 15.65 0.067 0.002

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose1 Jonckheere Dose3

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 142.65 <.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 -20.69 . -18.96 . . . . . . Dose1 -19.39 0.001 -18.96 1.000 . . . . . Dose2 -20.10 0.110 -18.96 1.000 0.087 . . . . Dose3 -15.67 <.001 -18.96 1.000 <.001 <.001 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 13.33 0.004

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -20.72 . .

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

EPA MRID Number 48673701

Page 56 of 59

DER Template Version: 22 September 2011 Dose1 -19.42 0.067 0.990 Dose2 -20.09 0.156 0.830 Dose3 -15.65 0.067 0.998

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

**Analysis results for 21-d hind-limb length (mm)**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.930 0.241 2.054 0.160 USE PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 15.83 1.71 0.85 10.79 13.11, 18.55 Dose1 4 16.08 0.72 0.36 4.45 14.94, 17.22 Dose2 4 14.24 0.37 0.19 2.62 13.64, 14.83 Dose3 4 5.39 0.78 0.39 14.54 4.14, 6.64

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 16.29 13.39 17.35 . . Dose1 16.18 15.20 16.76 101.56 -1.56 Dose2 14.07 14.01 14.79 89.92 10.08 Dose3 5.19 4.69 6.50 34.05 65.95

******************************************************************************** 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 98.00 <.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 15.83 . 15.95 . . . . . . Dose1 16.08 0.973 15.95 0.654 . . . . . Dose2 14.24 0.115 14.24 0.029 0.102 . . . . Dose3 5.39 <.001 5.39 <.001 <.001 <.001 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 10.94 0.012

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

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

EPA MRID Number 48673701

Page 57 of 59

DER Template Version: 22 September 2011 Ctrl 16.29 . . Dose1 16.18 1.000 0.500 Dose2 14.07 0.346 0.039 Dose3 5.19 0.067 <.001

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose2 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 98.00 <.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 -15.83 . -12.88 . . . . . . Dose1 -16.08 0.973 -12.88 1.000 . . . . . Dose2 -14.24 0.115 -12.88 1.000 0.102 . . . . Dose3 -5.39 <.001 -12.88 1.000 <.001 <.001 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 10.94 0.012

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -16.29 . . Dose1 -16.18 1.000 0.500 Dose2 -14.07 0.346 0.961 Dose3 -5.19 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)

**Analysis results for 21-d norm hind-limb**

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.928 0.229 1.570 0.248 USE PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.77 0.09 0.05 11.90 0.63, 0.92

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

EPA MRID Number 48673701

Page 58 of 59

DER Template Version: 22 September 2011 Dose1 4 0.84 0.04 0.02 5.33 0.77, 0.91 Dose2 4 0.71 0.03 0.01 3.63 0.67, 0.75 Dose3 4 0.34 0.04 0.02 12.33 0.27, 0.41

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 0.80 0.64 0.85 . . Dose1 0.83 0.80 0.90 108.45 -8.45 Dose2 0.70 0.69 0.75 91.93 8.07 Dose3 0.33 0.31 0.40 44.09 55.91

******************************************************************************** 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 61.60 <.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 0.77 . 0.80 . . . . . . Dose1 0.84 0.287 0.80 0.859 . . . . . Dose2 0.71 0.320 0.71 0.092 0.035 . . . . Dose3 0.34 <.001 0.34 <.001 <.001 <.001 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 11.40 0.010

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 0.80 . . Dose1 0.83 0.494 0.807 Dose2 0.70 0.346 0.094 Dose3 0.33 0.067 0.001

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose3 Jonckheere Dose3

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 61.60 <.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

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

EPA MRID Number 48673701

Page 59 of 59

DER Template Version: 22 September 2011 Ctrl -0.77 . -0.66 . . . . . . Dose1 -0.84 0.287 -0.66 0.997 . . . . . Dose2 -0.71 0.320 -0.66 0.998 0.035 . . . . Dose3 -0.34 <.001 -0.66 0.999 <.001 <.001 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 11.40 0.010

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -0.80 . . Dose1 -0.83 0.494 0.193 Dose2 -0.70 0.346 0.906 Dose3 -0.33 0.067 0.999

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

# DATA EVALUATION RECORD

TEBUCONAZOLE
Study Type: OCSPP 890.1150, Androgen Recep tor Binding (Rat Prostate Cytosol)

EPA Contract No. EP10H001452 Task Assignment No. 2-22-2012 (MRID 48619401)

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: 2/23/2012

Secondary Reviewer Signature: Michelle Sharpe-Kass, M.S. Date: 3/1/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 3/16/2012

Quality Assurance: Signature: Steven Brecher, Ph.D. Date: 3/14/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).

### TEBUCONAZOLE/ 128997 Androgen Receptor Binding (Rat Prostate Cytosol) (2011) I 

### Page 1 of 15 OCSPP 890.1150/ OECD None

# s~.~~ Date:L 

# b:C

Signature:

# >~/z; Date: /, 

# r Primary Reviewer: Ray Kent, Ph.D. 

## Health Effects Division, Office of Pesticide Programs Secondary Reviewer: Greg Akerman, Ph.D. 

## Health Effects Division, Office of Pesticide Programs Template version 08/2011



## DATA EVALUATION RECORD

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



## PC CODE: 128997 

## DP BARCODE: D398353

TXR#: 0056253 

## CAS No.: 107534-96-3



## TEST MATERIAL (PURITY): Tebuconazole (97.5% a.i.)



## SYNONYMS: (RS)-1-p-chlorophenyl-4,4-dimethyl-3-(lH-1,2,4-triazol-1-ylmethyl)pentan-3-ol



## CITATION: Rouquie, D. (2011). Evaluation ofTebuconazole in the Androgen Receptor Binding Assay. Bayer SAS, Sophia Antipolis Cedex, France. Laboratory Study No.: SA 11212, December 8, 2011. MRID 48619401. Unpublished.



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



## TEST ORDER#: CON-128997-20



## EXECUTIVE SUMMARY: In 

## an androgen receptor 

## (AR) binding assay (MRID 48619401), ventral prostate cytosol from male 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 radiolabeled reference androgen (Rl 881) prior to routinely conducting AR competitive binding experiments. The competitive binding assay was conducted to measure the binding of a single concentration of [3H]-R1881 (1 

## nM) in the presence of increasing concentrations oftebuconazole (logarithmic increase from 10

- 10 

## to 10- 3 

## M). DMSO was used as the solvent at a final assay concentration of :53.3%. The assay included dexamethasone as a weak positive control, and R1881 as the ligand reference standard.

In 

## the saturation binding experiment, the mean maximum binding capacity 

## (Bmax) was 14 fmol/100 μg protein and the mean dissociation constant (l<d) was 1.01 nM. The l<d obtained from Saturation Assay #2 was low (0.59 

## nM), but the l<ds from the other two runs were within the recommended range (0.685 to 1.57 nM) and the Scatchard plots for the three saturation assays were linear. The specific binding reached a plateau, and non-specific binding (NSB) was less than 20% of total binding (4.6-12.6%). The saturation binding experiment demonstrated that AR was present in a reasonable concentration and functioning with appropriate affinity for the R1881 ligand.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 2 of 15 TEBUCONAZOLE/ 128997 OCSPP 890.1150/ OECD None


## In the competitive binding experiment, the estimated mean log IC 50 

## was
− 4.3 M for tebuconazole compared to that of R1881 of

− 9.1 M and the weak positive control of

− 4.4 M. The mean RBA was 0.0014% for tebuconazole compared to 0.0021% for the weak positive control. No precipitation of the test compound was visually observed at any concentration. All performance criteria were met, with the exception that liga nd depletion and NSB were not reported, but the IC 50 

## s for R1881 and the weak positiv e control were within the e xpected ranges demonstrating that the ligand was available at an appropriate level. These de viations were considered minor and performance criteria were found to be met.
Tebuconazole reduced the mean specific binding of the radiolabeled liga nd in a concentration dependent manner in all three runs. The ma ximum binding reduction oc curred at the highest concentration tested (10 − 3 

## M), with specific binding being reduced to 1.5-6.9%. Specific binding was also reduced at 10 − 4 

## M (33-37%), but not at concentrations ≤ 10 − 5 

## M (82-111% binding). Specific [ 3 

## H]-R1881 AR binding was reduced to <50% at the highest conc entrations tested in all three runs.

Based on the results from the thr ee runs, tebuconazole is classifi ed as a Binder in the Androgen Receptor Binding Assay.
The assay satisfies the EDSP Tier 1 Test Order require ments for an Androgen Receptor Binding Assay (OCSPP 890.1150).

COMPLIANCE: Signed and dated GLP and Quality Assurance statements were provided.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 3 of 15 TEBUCONAZOLE/ 128997 OCSPP 890.1150/ OECD None



## I. MATERIALS AND METHODS

A. MATERIALS
1. Test Facility: Bayer S.A.S. Location: 355 Rue Dostoievski, BP 153, 06903 Sophia Antipolis Cedex, France Study Director: D. Rouquie Other Personnel: H. Tinwell (replacement Study Director); A. Arnaud (Technician); P. Almeras (Report Unit Assistant) Study Period: July 28, 2011 to December 8, 2011



## 2. Test substance: Tebuconazole
Description: White powder Source: Bayer CropScience AG Lot/Batch #: K689052 (expiration date: August 23, 2012) Purity: 97.5% a.i. Solubility: Not reported Volatility: Not reported Stability: Not reported Storage conditions: Ambient CAS #: 107534-96-3 Molecular weight: 307.8 g/mol Structure:



## 3. Non-labeled ligand: R1881 Supplier: Fountain Limited (Malta) Catalog #: Batch #: E3164-000 B1023 Purity: 98.83% CAS #: 965-93-5


## 4. Radioactive ligand: [ 3 

## H]-R1881 Supplier: PerkinElmer, Inc. (Boston, MA, USA) Catalog and Batch #: NET590/ 653698 Date of production: February 24, 2011 Date of use: May 17, 2011 to June 8, 2011 (Saturation Binding Experiment) August 4, 2011 to September 15, 2011 (Competitive Binding Experiment) Radiochemical purity: >97% Specific activity: 85.1 Ci/mmol Concentration of stock: 10 nM



## 5. Positive control: Dexamethasone Supplier: Sigma (Switzerland) Catalog # Batch #: D1756 1419230 Purity: 98.9% CAS # : 50-02-2 Cl OH CH 3

CH 3 CH 3

N N NAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 4 of 15 TEBUCONAZOLE/ 128997 OCSPP 890.1150/ OECD None



## 6. Solvent/vehicle control: Dimethyl sulfoxide (DMSO)
Justification for choice of solvent: DMSO is one of the solvents recommended in the guideline (OPPTS 890.1150) and tebuconazole is reasonably soluble in this solvent. Final Concentration: ≤ 3.3%



## B. METHODS
1. Preparation of Rat Ventral Prostate Cytosol: A total of 50 male Sprague Dawley rats (Charles River Laboratories, France) were acclimatized for 8 days at the laboratory, castrated at 84 days of age and euthanized on the following day (~24 hours later). The ventral prostates were excise d immediately after termina tion, weighed and minced, and placed in ice-cold low-salt TEDG pH 7.4 buffer [10 mM Tris/HCl, 1 mM sodium molybdate, 1.5 mM EDTA, 10% glycerol, 1 mM DTT, and 1 mM phenylmethylsulfonyl fluoride (PMSF)] at 10 mL/g of tissue. The tissues were homogenized and centrifuged for 30 min at 30,000 × g at 4 oC. The supernatant was pool ed, and the cytosol was aliquoted (5 mL) and stored at

− 74 ± 10 oC until use. Protein concentration of the cytosol was determined to be 6.6 mg/mL using a commercia lly available protein kit compatible with DTT in the TEDG buffer (BioRad Protein Assay Kit).

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

## H]-R1881 was performed in a previous study (SA 11021 ) for the cytosol used in the s ubject competitive binding experiment to demonstrate that the AR was present in reason able concentrations and had the appropriate affinity for the R1881 ligand. The details and results of the saturation binding experiment were included as an attachment to the subj ect study. The conditions for the saturation binding experiment are summarized in Table 1.

TABLE 1. Summary of Conditions for Saturation Binding Experiment a 

### Source of receptor Rat prostate cytosol Concentration of radioligand (as serial dilutions) 0.25-10 nM Concentration of non-labeled ligand (100X [radioligand]) 25-1,000 nM Optimization of receptor concentration Not reported b 

### Temperature 5 ± 3 oC (refrigerated) Incubation time 20 hours Composition of assay buffer (TEDG) Tris/HCl 10 mM (pH 7.4) EDTA 1.5 mM Glycerol 10% Sodium Molybdate 1 mM Phenylmethylsulfonyl fluoride 1 mM DTT 1 mM Protease inhibitor 0.5% a Data were obtained from pages 73, 75, and 117-124 of the study report. b The optimal protein concentration was determined to be 0.72 mg protein/assay tube; however, the ligand depletion at 0.25 nM could not be determined from the raw data provided.



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

## H]-R1881 was adjustedAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 5 of 15 TEBUCONAZOLE/ 128997 OCSPP 890.1150/ OECD None


## for decay over time, and serial dilutions in TEDG buffer were prepared to achieve the final concentrations in cytosol of 0.25, 0.5, 0.7, 1, 1.5, 2.5, 5, and 10 nM to determine total binding. To determine non-specific binding, solu tions of non-labeled R1881 were prepared in a similar manner to achieve concentrati ons that were 100-fold greater than each respective radiolabeled concentra tion, resulting in final concen trations in cytosol of 25, 50, 70, 100, 150, 250, 500, and 1,000 nM. In the absence of cytosol, the radiation found in 7.5, 15, 21, 30, or 45 μL of 10 nM [ 3 

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

## H]-R1881 was measured. For each batch of cytosol, the op timal protein concentration was determined by calculating specific binding to differing am ounts of protein per tube, using 0.25 nM radiolabeled R1881. The optimal protein co ncentration was determined to be 0.72 mg protein/assay tube. Each run contained three concurrent replicates at each concentration, resulting in the 72 samples depicted in Table 2, plus three replicates of the solvent control.

TABLE 2. Saturation Binding Experiment Run a b 

### Total Binding Non-Specific Binding Radioligand alone Tubes 1-24 c 

### Tubes 25-48 d 

### Tubes 49-72 e 

### [ 3 

### H]-R1881 Final conc. (nM) [ 3 

### H]-R1881 Final conc. (nM) R1881 Final conc. (nM) [ 3 

### H]-R1881 Initial conc. (nM) [ 3 

### H]-R1881 (μL) 0.25 0.25 25 10 7.5 0.50 0.50 50 10 15 0.70 0.70 70 10 21 1.00 1.00 100 10 30 1.50 1.50 150 10 45 2.50 2.50 250 100 7.5 5.00 5.00 500 100 15 10.00 10.00 1000 100 30 a Data were obtained from pages 117-124 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 mola r excess of [ 3 

### H]-R1881 in a volume of 7.5-45 μL. Samples were dried, and 300 μL of prostate cytosol were added. e Tubes 49-72 contained only 7.5, 15, 21, 30, or 45 μL of 10 nM [ 3 

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

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



## Following addition of triamcinolone acetonide, [ 3 

## H]-R1881, and/or R1881, the tubes were dried, dissolved in diluted prostate cytoso l (300 μL), and incuba ted in a rotor in a refrigerator (5 ± 3oC) for 20 hours. To separate bound from free R1881, an aliquot of each sample tube was incubated with hydroxyapatite (HAP) slurry in an ice water bath for 20 minutes. The samples were then centrifuge d, and the supernatant was discarded. The samples were washed 3 times in 50 mM TRIS buffer. Following the last wash and decanting of the Tris buffer, pellets were then extracted with 2 mL ethanol. The radiation was quantified by liquid scintillat ion counting of the ethanol extr act. A total of three runs were performed.
3. Competitive Binding Experiment: A summary of the assay conditions for the competitive binding experiment is included in Table 3.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 6 of 15 TEBUCONAZOLE/ 128997 OCSPP 890.1150/ OECD None



### TABLE 3. Summary of Conditions for Competitive Binding Experiment a 

### Source of receptor Rat ventral prostate cytosol Concentration of radioligand 1 nM Optimization of receptor concentration Not reported b 

### Concentration of test substance (as serial dilutions) 10

− 10 

### to 10

− 3 

### M Incubation Temperature 5 ± 3 oC (refrigerated) Incubation time 20 ± 0.5 hours Composition of assay buffer Tris 10 mM (pH 7.4) EDTA 1.5 mM Glycerol 10 % Phenylmethylsulfonyl fluoride 1 mM DTT 1 mM Protease Inhibitor 0.5% a Data were obtained from page 12, 14, and 35-42 of the study report. b The protein concentration used was 0.48 mg protein/assay tube



## The competitive binding experiment was performed according to the protocol provided in the EPA Test Guidelines OCSPP 890.1150. The competitive binding experiment measures the binding of a singl e concentration of [ 3 

## H]-R1881 (the actual adjusted specific activity values used were not reported) to the AR in the presence of increasing concentrations of a test substance.
DMSO was used as the solvent, and no preci pitation of the test substance was visibly observed at any concentration. Based on the re sults from the saturation binding experiment, the amount of cytosolic protein used fo r each incubation in the competitive binding experiments was 0.48 mg/assay tube, which was reported to bind no more than 10-15% of the radiolabeled R1881 at 1 nM.

Dilutions of the test subs tance, 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 th ree independent runs on three different days, and each run contained three replicates at each concentration, plus six replicates of solvent total binding, non-specific binding, and the hot mix resulting in a total of 81 samples per run. In addition, 3 replicates of the solvent blank were analyzed at the end of each run.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 7 of 15 TEBUCONAZOLE/ 128997 OCSPP 890.1150/ OECD None



### TABLE 4. Competitor Final Molar (M) Con centrations in Competitive Binding Assay a b 

### Solvent Control Reference standard We ak positive control Test Chemical None (Hot Mix) DMSO R1881 Dexamethasone Tebuconazole Tubes 1-3 and 70-72 Tubes 4-21 and 73-75 c 

### Tubes 22-45 Tubes 46-69 Tubes 76-81 1×10

− 7 

### 1×10

− 3 

### 1×10

− 3 

### 1×10

− 8 

### 1×10

− 4 

### 1×10

− 4 

### 1×10

− 9 

### 1×10

− 5 

### 1×10

− 5 

### 1×10

− 10 

### 1×10

− 6 

### 1×10

− 6 

### 1×10

− 11 

### 1×10

− 7 

### 1×10

− 7 

### -- 1×10

− 8 

### 1×10

− 8 

### -- 1×10

− 9 

### 1×10

− 9 

### -- 1×10

− 10 

### 1×10

− 10 

### a Data were obtained from pages 35-42 of the study report. b Each concentration of each chemical was run in triplicat e for a total of 81 tubes per run. Tubes 1-75 contained 50 μL of triamcinolone acetonide and 30 μL [ 3 

### H]-R1881. Samples were dried, and 300 μL of prostate cytosol were added. Tubes 1-75 also contained 10 μL of the solvent control, reference standard (non-radiolabeled R1881), weak positive control, or test substan ce. Tubes 76-81 contained only 30 μL of [ 3 

### H]-R1881. c Tubes 4-6 and 73-75 were used to evaluate non-specific binding by adding 100X of cold (non-radiolabeled) R1881.



## The procedures for assay incubation, separa tion of bound from free R1881 (washing with buffer and extraction with ethanol), fo llowed by scintillation counting of bound [ 3 

## H]-R1881 were similar to those used in the saturation bi nding experiment. A total of three runs were performed as described.
4. Data Analysis: The maximal binding capacity (B max 

## ), dissociation constant (K d 

## ), and inhibition concentration (IC 50 

## ) were calculated using nonlin ear regression analysis using Graph Pad Prism v. 5.04 (GraphPad Software, Inc.). Scatchard plots were also plotted for the binding data (GraphPa d Software, Inc.).

5. Definitions

a. Classification of test material
If the data fit a 4-parameter non linear regression model, the test chemical is classified as:

Binder: The average curve for the test chemical across runs crosses 50% of radioligand bound.
Equivocal: The average lowest portion of curves across runs is between 50% and 75% radioligand binding ( i.e. radioligand displacement is at le ast 25% but less than 50%), or the curve falls outside the range fo r the weak positive control (

− 0.6 to

− 1.4).

Non-Binder: The average lowest portion of curves acr oss runs is greate r than 75% activity ( i.e. less than 25% displacement of radioliga nd), or the data do not fit the model.

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

− 6 

## M and the binding curve does not cross 50%, the chemical is judged to be untestable.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 8 of 15 TEBUCONAZOLE/ 128997 OCSPP 890.1150/ OECD None


## b. Descriptors for receptor binding

B max 

## : maximal binding capacity K d 

## : dissociation constant IC 50 

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

## Relative Binding Affinity (RBA): IC 50 

## of R1881 × 100 ÷ IC 50 

## of test substance

II. RESULTS

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

## ) for [ 3 

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

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

## ) was not reported and the K d 

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

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

TABLE 5. Saturation Binding Experiment of R1881 with Androgen Receptor from Rat Prostate Cytosol a 

### Parameter Run 1 b 

### Run 2 b 

### Run 3 b 

### Mean Runs 1-3 c



### r 2 

### NR NR NR NA B max 

### (nM) 0.34 0.31 0.38 0.34 ± 0.035 B max 

### (fmol/100 μ g protein) 14 13 16 14 ± 1.5 K d 

### (nM) 1.19 0.59 1.25 1.01 ± 0.365 a Data were obtained from page 77 of the study report. b The mean is reported for the c oncurrent replicates of each run. c Mean and standard deviation were calculated by the study reviewer.

r 2 

### Goodness of fit for curve calculated for specific binding NR Not reported NA Not applicable



## The graphs in Figure 1 illustr ate 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 tota l binding (4.6-12.6%). Figure 2 is the Scatchard plots for each run that illustrate the binding of [ 3 

## H]-R1881 to the androgen receptor. The data fit results in a linear plot.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 9 of 15 TEBUCONAZOLE/ 128997 OCSPP 890.1150/ OECD None


## FIGURE 1. Binding of [ 3 

## H]-R1881 to the Androgen Receptor during the Saturation Binding Experiment.
Saturation Assay #1 – Non-Specific and Saturation Assay #1 – Specific Binding Total Binding

Saturation Assay #2 – Non-Specific and Saturation Assay #2 – Specific Binding Total BindingAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 10 of 15 TEBUCONAZOLE/ 128997 OCSPP 890.1150/ OECD None


## Saturation Assay #3 – Non-Specific and Saturation Assay #3 – Specific Binding Total Binding

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

## H]-R1881 to the Androgen Receptor.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 11 of 15 TEBUCONAZOLE/ 128997 OCSPP 890.1150/ OECD None



## B. COMPETITIVE BINDING EXPERIMENT: The results from the three competitive binding experiments are summarized in Table 6. Tebuconazole reduced the mean specific binding of the radiolabeled ligand in a concen tration dependent manner in all three runs. The maximum binding reduction occurred at the highest concentration tested (10 − 3 

## M), with specific [ 3 

## H]-R1881 binding being reduced to 1.5-6.9%. Specific binding was also reduced at 10 − 4 

## M (33-37%), but not at concentrations ≤ 10 − 5 

## M (82-111% binding).
The estimated mean log IC 50 

## was
− 4.3 M for tebuconazole compared to that of R1881 of

− 9.1 M and the weak positive control of

− 4.4 M. The mean RBA was 0.0014% for tebuconazole compared to 0.0021% for the positive control. Confidence in these numbers is high due to the small variation. No preci pitation of the test compound was visually observed at any concentration. The solvent cont rol responses indicated no drift in the study assay. The initial assay run #2 was discarded (a nd repeated) due to te chnical problems with the assay. Tebuconazole was classified as an AR binder in all thr ee competitive binding

runs.

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

### Parameter Run 1 Run 2 Run 3 Mean

r 2 

### (unweighted) R1881 NR NR NR NA Positive control NR NR NR NA Tebuconazole NR NR NR NA Log IC 50 

### (M) R1881

− 9.1

− 9.0

− 9.2

− 9.1 Positive control

− 4.4

− 4.4

− 4.4

− 4.4 Tebuconazole

− 4.3

− 4.2

− 4.3

− 4.3 IC 50 

### (M) R1881 7.7 x 10

− 10 

### 9.3 x 10

− 10 

### 7.0 x 10

− 10 

### 8.0 x 10

− 10 

### Positive control 4.1 x 10

− 5 

### 3.6 x 10

− 5 

### 3.7 x 10

− 5 

### 3.8 x 10

− 5 

### Tebuconazole 5.4 x 10

− 5 

### 5.9 x 10

− 5 

### 5.5 x 10

− 5 

### 5.6 x 10

− 5 

### Log (%)RBA b 

### Positive control

− 2.7

− 2.6

− 2.7

− 2.7 Tebuconazole

− 2.9

− 2.8

− 2.9

− 2.9 RBA (%) Positive control 0.0019 0.0026 0.0019 0.0021 Tebuconazole 0.0014 0.0016 0.0013 0.0014 a Data were obtained from pages 17 and 18 of the study report. b Calculated by reviewer NR Not reported NA Not applicable

r 2 

### Goodness of fit RBA Relative binding affinity



## The graphs provided in the current study (Fi gure 3) indicate that the tebuconazole data across the tested concentrations fit the nonlinear regression m odel and on average the lowest portion of the curve crossed 50% activity at the highe st two concentration (10

− 3 

## and 10

− 4 

## M) of tebuconazole. A graph of the mean values of tebuconazole acro ss the three runs is provided in Figure 4.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 12 of 15 TEBUCONAZOLE/ 128997 OCSPP 890.1150/ OECD None


## FIGURE 3. Percentage R1881 Bound to the A ndrogen Receptor in the Presence of Test Compounds.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 13 of 15 TEBUCONAZOLE/ 128997 OCSPP 890.1150/ OECD None



## FIGURE 4. Mean Percentage R1881 Bound to th e Androgen Receptor in the Presence of Tebuconazole in Three Competitive Binding Assays.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 14 of 15 TEBUCONAZOLE/ 128997 OCSPP 890.1150/ OECD None



## C. PERFORMANCE CRITERIA: To ensure that the competitive binding assay was functioning properly, each run was evaluated using the following criteria (Table 7):

TABLE 7. Criterion a 

### Tolerance Limit(s) b 

### Value Yes No Ligand depletion is minimal. The recommended ratio of total binding in the absence of competitor to total amount of [ 3 

### H]-R1881 added per assay tube. ≤ 15% Not reported c 

### X

Tebuconazole Top (% binding) 80 to 115 109 to 111 X R1881 fitted curve parameters Top (% binding) 82 to 114 96 to 106 X Bottom (% binding)

− 2.0 to 2.0

− 2.7 to 0.01 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

− 0.4 to 1.1 X Hill Slope

− 1.4 to

− 0.6

− 1.0 X Saturation Binding Experiment K d 

### (nM) 0.685 to 1.57 0.59 to 1.25 e 

### X Non-specific binding (%) ≤ 10.0 Not reported f 

### X a Data were obtained from page 17, 36, 39 and 42 of the study report. b These values represent ranges from the validation study. c The protein concentration used was 0.48 mg protein/assay tube; however, the ligand depletion at 1 nM could not be determined from the raw data provided. d The bottom % binding was low in Run #3 (- 2.7), but within the limits in the other two runs. e The Kd was low (0.59 nM) in Run #2 of the saturation binding experiment, but within the recommended range in the other two runs. f The non-specific binding was not reported and could not be calculated from the raw data provided.



## Additionally, the curve for the reference material showed that increasing concentrations of unlabeled R1881 displaced [ 3 

## H]-R1881 in a manner consiste nt with one-site binding, as indicated by a Hill slope of

− 1.0. 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: Based on the average results of the three competitive assays, tebuconazole was consider ed a weak AR binder. The data fit the nonlinear regression model and on average the competitive curve crossed 50% of the remaining R1881 bound at the two highest c oncentrations of tebuconazole. The IC 50 

## values for tebuconazole in the three evaluations were determined to be 5.4×10

− 5 

## , 5.9×10 − 5 

## and 5.5×10 − 5 

## M. The RBA values for tebuconazo le in comparison to R1881 were 0.0014%, 0.0016% and 0.0013%.

B. AGENCY COMMENTS: The saturation binding experi ment demonstrates that the androgen receptor was present in a reasona ble concentration and functioning with appropriate affinity for the R1881 ligand. The mean K d 

## for [ 3 

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

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

## ) was not reported and the K d 

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

## s from the other two runsAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 15 of 15 TEBUCONAZOLE/ 128997 OCSPP 890.1150/ OECD None


## were within the recommended range (0.685 to 1.5 7 nM) and the Scatchard plots for the three saturation assays were linear. The specifi c binding reached a plateau, and non-specific binding was less than 20% of total binding (4.6-12.6%).
In the competitive binding experiment, tebuconazole reduced the mean specific binding of the radiolabeled ligand in a concentration dependent manner in all three runs. The maximum binding reduction occurred at th e highest concentration tested (10 − 3 

## M), with specific binding being reduced to 1.5-6.9%. Specific binding was also reduced at 10 − 4 

## M (33-37%), but not at concentrations ≤ 10 − 5 

## M (82-111% binding). Te buconazole is classified as a binder in the AR assay as specific [ 3 

## H]-R1881 binding was reduced to <50% at the highest concentrations test ed in all three runs.
The estimated mean log IC 50 

## was
− 4.3 M for tebuconazole compared to that of R1881 of

− 9.1 M and the weak positive control of

− 4.4 M. The mean RBA was 0.0014% for tebuconazole compared to 0.0021% for the weak positive control. No precipitation of the test compound was visually observed at any co ncentration. All perf ormance criteria were met, with the exception that ligand deplet ion and NSB were not reported, but the IC 50 

## s for R1881 and the weak positive control were within the expected ranges demonstrating that the ligand was available at an appropriate level. These deviations were considered minor and the performance criteria were found to be met.

C. STUDY DEFICIENCIES: The following deficiencies were noted that are not considered to have had an adverse impact on the results, interpretation or conclusions of this study:

 The percent ligand depletion was not provided for the saturation and competitive binding experiments.

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

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

# DATA EVALUATION RECORD

TEBUCONAZOLE
Study Type: OCSPP 890.1200, Aromatase Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-22-2012 (MRID 48619402)

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: 3/6/2012
Secondary Reviewer Signature: David A. McEwen, B.S. Date: 3/8/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 3/15/2012

Quality Assurance: Signature: Steven Brecher, Ph.D. Date: 3/15/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).

### TEBUCONAZOLE / 128997 

## Primary Reviewer: Vincent Chen, M.S 

## Health Effects Division (7509P) Secondary Reviewer: Greg Akerman, Ph.D. 

## Health Effects Division (7509P) Aromatase (Human Recombinant) Assay (2011) I Page 1 of 12 OCSPP 890.1200 I 

### OECI) None 

## Signature:

# ~ Date:**2??v~$** 

## Signature:**~** K::J 

## Date:**----..,o,r-J....,,.JC~-..,J-1s---** Template version 08/2011



## DATA EVALUATION RECORD

STUDY TYPE: Aromatase (Hwnan Recombinant); OCSPP 890.1200



## PC CODE: 128997 

## DP BARCODE: D398353

TXR: 0056253 

## CAS: 107534-96-3



## TEST MATERIAL (PURITY): Tebuconazole (97.5% a.i.)



## SYNONYMS: IUPAC: CAS:

CITATION: (RS)-1-p-chlorophenyl-4,4-dimethyl-3-0H-1,2,4-triazol-1-ylmethyl)pentan-3-ol a-[2-( 4-chlorophenyl)ethyl]-a-(1, 1-dimethylethyl)-lH-1,2,4-triazole-1-ethanol

Tinwell, H. (2011). Evaluation of Tebuconazole in the Aromatase Assay. Bayer S.A.S. (Bayer CropScience [355, rue Dosto'ievski, BP 153, 06903 Sophia Antipolis Cedex, France]. Laboratory Study ID: SA 11064. 09 Dec 2011. MRID 48619402. Unpublished.



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



## TEST ORDER#: CON-128997-20



## EXECUTIVE SUMMARY: In an 

## in vitro aromatase (CYP 19) assay (MRID 48619402), tebuconazole (97.5% a.i., batch# AE F069623-01-18) was incubated with hwnan recombinant
aromatase and tritiated androstenedione 0-P [ 3 

## H(N)]-androst-4-ene-3, 17-dione; [3H]ASDN) in dimethyl sulfoxide (DMSO) at increasing logarithmic concentrations from 10- 10 

## M to 10- 3 

## M for 15 minutes to assess the potential of tebuconazole 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 tebuconazole. Tritiated water was quantified using liquid scintillation counting (LSC). Three runs were conducted and each run included a full activity control, a background activity control, a positive control series 00- 10 

## to 10- 5 ) 

## using a known inhibitor (4-hydroxyandrostenedione; 4-0H ASDN), and the test tebuconazole series 00- 10 

## to 10- 3 ) 

## with three repetitions per concentration.

Aromatase activity in the full activity controls ranged from 0.250 to 0.365 nmol/mg-protein/min for the three test runs, with a mean and standard deviation of 0.306±0.038 nmol/mg-protein/min. Activity in the background controls ranged from 3.3 to 4.4% of the full control activity with an average of 3.6%. The response of the full activity controls and background controls was acceptable.Aromatase (Human Recombinant) Assay (2011) / Page 2 of 12 TEBUCONAZOLE / 128997 OCSPP 890.1200 / OECD None 

## For the positive control substance (4-OH ASDN), aromatase activity averaged 0.2925±0.0202 nmol/mg-protein/min at the lowest tested concentration (10

− 10 

## M) and 0.0028±0.0005 nmol/mgprotein/min at the highest tested concentration (10

− 5 

## M). These results were generally within the recommended ranges for the top of the curve, bottom curve, Hill slope, and log IC 50



## . For tebuconazole, aromatase activity averag ed 0.2706 ± 0.0069 nmol/mg-protein/min at the lowest tested concentration (10

− 10 

## M) and 0.0004 ± 0.0009 nmol/mg-pr otein/min at the highest tested concentration (10

− 3 

## M). The lowest portion of the response curve for tebuconazole across runs was less than 50% activity. Tebuconazo le reduced aromatase activity to 90% at 10

− 7 

## M, 62% at 10 − 6 

## M, 19% at 10 − 5 

## M, 2.3% at 10 − 4 

## M, and 0.1% at 10

− 3 

## M.
The estimated log IC 50 

## ranged from

− 5.8 to

− 5.7 M for the test material and averaged

− 5.73 M compared to the positive control 4-OH ASDN, which averaged

− 7.20 M. The average slope of the concentration response curve was

− 0.99 for tebuconazole and

− 0.88 for 4-OH ASDN.
Based on the data form the average response curve, tebuconazole is classified as an Inhibitor of aromatase activity in this assay.
The assay satisfies the EDSP Tier 1 Test Order requireme nts for an Aromatase Assay (OCSPP 890.1200).

COMPLIANCE: Signed and dated GLP Compliance, a nd Quality Assurance statements were provided.Aromatase (Human Recombinant) Assay (2011) / Page 3 of 12 TEBUCONAZOLE / 128997 OCSPP 890.1200 / OECD None


## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: Tebuconazole Description: White powder Source: Bayer CropScience, Germany Batch #: AE F069623-01-18 (expires August 23, 2012) Purity: 97.5% a.i. Volatility: Not provided Storage Conditions: In air-tight, light-resistant cont ainers at room temperature Stability: Not provided Solvent: DMSO Solubility (in Test Solvent): Soluble up to 10

− 3 M Highest Concentration Tested: 10

− 3 M Stock Solution Preparation Methodology: Serial dilutions Molecular Weight: 307.8 g/mol CAS #: 107534-96-3 Structure:



## 2. Non-Labeled Substrate: Androstenedione (ASDN) CAS #: 63-05-8 Source: Molekula, Ltd. (product # 15237310) Lot/Batch #: 63779 (expiry January 2013) Purity: 99.25% a.i.



## 3. Radiolabeled Substrate: 1- β [ 3 

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

## H]ASDN) Source: Perkin Elmer (Catalog # NET-926) Lot/Batch #: 3615304 Radiochemical Purity (Supplier): 97% Specific Activity: 20.7 Ci/mmol (0.766 TBq/mmol) Radiochemical Purity (In-Lab Determination): Not provided



## 4. Positive Control: 4-hydroxyandrostenedione (4-OH ASDN) CAS #: 566-48-3 Source: Sigma Aldrich (Catalog # F2552) Lot/Batch #: 081K2133 Purity: 99.6 % a.i.


## 5. Solvent (Vehicle Control): DMSO Source: Sigma Aldrich (Ref. # 154938) Lot/Batch #: 76396EM Justification for Choice of Solvent: Acceptable vehicle by OCSPP guideline 890.1200 Concentration (% of Total Volume in Assays): 1%



## 6. Test Microsomes: Human recombinant aromatase (CYP19) microsomes Source: BD Biosciences Lot/Batch #: 74101 Protein Concentration: 7.95 mg/mL Cytochrome C Reduc tase Activity: Not provided Aromatase Activity: 0.1131 nmol/mg protein/min Cl OH CH 3

CH 3 CH 3

N N NAromatase (Human Recombinant) Assay (2011) / Page 4 of 12 TEBUCONAZOLE / 128997 OCSPP 890.1200 / OECD None



## B. METHODS
1. Assay Components and Preparations: A mixture of non-labe led and radiolabeled [ 3 

## H]-ASDN was prepared such that the final concentration of ASDN in the assay was approximately 4.1 nM, and the amount of tri tium added to each incubation tube was approximately 0.07 μ Ci in a volume of 100 μ L.
Tebuconazole stock solutions were prepared such that the total volume of each test chemical formulation used per assay was 1% of the tota l assay volume. A rationale for the selection of DMSO was not provided; however, tebuconaz ole is soluble in DMSO at the tested concentrations and is a solvent recommended by the EPA guidelines.
A stock solution of the positive control substance, 4-OH ASDN, was formulated in DMSO. Fresh dilutions of the stock solution were prepar ed in the same solvent as the stock solution on the day of use. Dilutions were prepared such that the target concentrations of the positive control substance (10 –10 

## M to 10 –5 

## M; Table 4) were achieved by the addition of 20 μ L of the dilution for a final assay volume of 2 mL.
Human recombinant microsomes were purchased from BD Biosciences, and stored at

− 74±10oC for no longer than one year. Before us e of the microsomes, the batch was thawed and sub-aliquoted at 0.004 mg/mL into individual vials before being refrozen, and stored at

− 74±10°C for no more than six months.
Other assay components, i.e. sodium phospha te buffer, propylene gl ycol, and NADPH are reported in Table 1.

TABLE 1. Assay Components and Conditions Assay Factor Values a Sodium phosphate buffer (pH 7.4) 0.1M Microsomal Protein 0.004 mg/mL NADPH 0.3 mM [ 3 H]ASDN 100 nM Propylene Glycol 5% Temperature 37°C Incubation Time 15 min

a Data obtained from page 14 of the study report.



## 2. Suitability Assessments: The protein concentration of the microsomes (Batch # 74101) was supplied by the vendor as 7.95 mg/mL. Th e aromatase activity of the microsomes was also provided by the vendor as 0.1131 nmol/mg protein/min. The minimum aromatase activity in the full activity control samples was determined to be 0.0982 nmol/mg protein/min, which was slightly less than th e minimum recommended aromatase activity of 0.1 nmol/mg-protein/min. However, six other measurements reported for the proficiency test were >0.1 nmol/mg protein/min, and the average aromatase activity was 0.1131 or 0.1135 nmol/mg protein/min.

3. Aromatase Assay: Each assay run contained four t ubes for the full enzyme activity and background activity controls, respectively, and full concentration curves in the positiveAromatase (Human Recombinant) Assay (2011) / Page 5 of 12 TEBUCONAZOLE / 128997 OCSPP 890.1200 / OECD None 

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

The amount of 3 

## H 2 

## O in the aqueous fraction was quantified for each assay tube by LSC, and aromatase activity was reported in units of nmol/mg-protein/min.
4. Demonstration of Proficiency: Prior to performing the definitive assay, one run of the positive control was conducted to evaluate th e responsiveness of the microsomes. This evaluation also served as a pr oficiency evaluation of the tech nician involved in the conduct of the definitive assays. It wa s stated that as the same technician was responsible for the development of the assay in-house and conducted all aromatase investigations, no further proficiency testing was considered ne cessary for the purpose of this study.

a. Positive Control
(1) Initial Demonstration of Laboratory Proficiency : The positive control data met the following criteria:

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

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

 Coefficient of variation (CV) for replicates within each sample type and concentration of 4-OH ASDN was <15%.

 Performance criteria (Table 2) were ge nerally met, and served as guidance in identifying runs that provided pa rameters in the preferred ranges.
(2) Demonstration of Proficiency of Ne w Technician for Conducting Assay (when applicable): The positive control data met the criteria as listed in section (i) of OCSPP 890.1200, except the log IC 50 

## of

− 6.7 was slightly greater than the guideline range (

− 7.3 to

− 7.0).

TABLE 2. Performance Criteria for the Positive Control
Parameter Lower Limit Criteria Upper Limit Criteria Observation Slope

− 1.2

− 0.8

− 0.94 Top (%) 90 110 100.6% Bottom (%)

− 5 +6 1.8% Log IC 50 (M)

− 7.3

− 7.0

− 6.7 a Data obtained from page 17 of the study report.



## b. Proficiency Chemicals: Assays to classify proficienc y chemicals (econazole, fenarimol, nitrofen, and atrazine) were not performed.

TABLE 3. Proficiency Chemicals Compound CAS# Class Concentrations Econazole 24169-02-6 Inhibitor Not reported--- Fenarimol 60168-88-9 Inhibitor Not reported--- Nitrofen 1836-75-5 Inhibitor Not reported--- Atrazine 1912-24-9 Non-inhibitor Not reported---Aromatase (Human Recombinant) Assay (2011) / Page 6 of 12 TEBUCONAZOLE / 128997 OCSPP 890.1200 / OECD None


## 5. Determination of Aromatase Ac tivity with Test Chemical: The response of aromatase activity to the presence of eight concentrations of tebucon azole per run, in triplicate, was tested during three independent runs. Sol ubility was visually assessed (presence of cloudiness or a precipitate), and no precipitati on was observed at any concentration. The full enzymatic activity was obtained at the two lowe st concentrations of the test chemical in Run 2 but not the other two runs, defining th e 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 components a plus solvent vehicle N/A Bkgd Activity Control 4 Same as above without NADPH N/A 4-OH ASDN Conc 1 2 All test components plus 4-OH ASDN 1×10

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

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

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

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

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

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

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

− 10 Tebuconazole Conc 1 3 All test co mponents plus test chemical 1×10

− 3 Tebuconazole Conc 2 3 All test co mponents plus test chemical 1×10

− 4 Tebuconazole Conc 3 3 All test co mponents plus test chemical 1×10

− 5 Tebuconazole Conc 4 3 All test co mponents plus test chemical 1×10

− 6 Tebuconazole Conc 5 3 All test co mponents plus test chemical 1×10

− 7 Tebuconazole Conc 6 3 All test co mponents plus test chemical 1×10

− 8 Tebuconazole Conc 7 3 All test co mponents plus test chemical 1×10

− 9 Tebuconazole Conc 8 3 All test co mponents plus test chemical 1×10

− 10 a The complete assay contained buffer, propylene glycol, microsomal protein, [ 3 H]ASDN, and NADPH.



## C. DATA ANALYSIS

1. Raw Data: Raw data were converted to aromatas e activity (nmol/mg protein/min) and percent control for the positive control, proficiency chemicals and test chemical(s). The following raw data and calculated endpoints fo r each run were included in the report (Table 5).

TABLE 5. Raw and Calculated Data Raw/Calculated Data Included (X) DPM/mL for each portion of extract ed aqueous incubation mixture X Average DPM/mL for each aqueous portion (after extraction) X Total DPM for each aqueous portion (after extraction) X The total DPM present in the assay tube at initiation X The percentage of substrate converted to product X Total DPM after extraction corrected for background X Aromatase activity expressed in nmol/mg protein/min X Average aromatase activity in the full activity control tubes X Percentage of control activity remaining in the presence of various inhibitor concentrations X DPM Disintegrations per minute



## 2. Statistical Methods: Statistical analyses and graphica l displays were conducted using Prism 5, v 5.04 (GraphPad Software Inc., La Joll a, CA). Basic statistical analyses were performed on the data, which included means of replicates, standard de viation of the mean,Aromatase (Human Recombinant) Assay (2011) / Page 7 of 12 TEBUCONAZOLE / 128997 OCSPP 890.1200 / OECD None 

## standard error of the mean, and coefficient of variation. Analysis of variance (ANOVA) was not performed. The response curves were fitted by a non-we ighted least squares nonlinear regression analysis with Prism 5 software. For each run, the individual percent of control values were plotted versus logarithm of the correspondi ng concentration. The fitted concentration response curve was superimposed on the plot, wi th individual plots prepared for each run. The average percent of control values vers us logarithm of the concentration for the individual runs for each chemical (with differe nt symbols for each run) were included on the same graph with their respect ive fitted response curves. In addition, the av erage percent of control values for each run versus the logar ithm of the concentration were plotted on a separate graph along with the average c oncentration response curve across runs superimposed on the same plot.
Assay drift was assessed by comparing the % fu ll activity and background activity at values at the beginning of each run to the end of each run.
3. Interpretation of Results: Interpretation of the assay results was based on the average of three runs, using the categor ies presented in Table 6.

TABLE 6. 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% activity b Equivocal Average lowest portion of curves across runs is greater than 75% activity b Non-inhibitor Data do not fit model ---

a Ordinarily, an inhibition curve will fall from 90% to 10% over 2 log units with a slope near

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

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

− 6 M and the inhibition curve does not cr oss 50%, the chemical is typically determined to be untestabl e in the aromatase assay.



## II. RESULTS

A. CONTROL ACTIVITY: Aromatase activity in the full activity controls ranged from 0.250 to 0.365 nmol/mg-protein/min for the 3 test runs, with a mean and standard deviation of 0.306±0.038 nmol/mg-protein/min. Activity in the background controls ranged from 3.3 to 4.4% of the full control activity with an av erage of 3.6%. The % change for the average background controls at the beginning and e nd of each run fell between the guideline recommendations of

− 5% and +6% of the overall average full activity. However, for the full activity data, a slight assa y drift was observed in the third run, as the % changes at the beginning and end of each run were between 110.6% at the beginning and 89.4% at the end instead of the recommended guideline range of between 90 and 110%. Overall, the responses of the full activity controls and b ackground controls were acceptable for each run.

B. POSITIVE CONTROL: For the positive control substance (4-OH ASDN), aromatase activity averaged 0.2925±0.0202 nmol/m g-protein/min at the lowe st tested concentration (10

− 10 

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

− 5 

## M). The mean aromatase activity of the positive control (expressed as % full control activity) for each concentration tested across a ll three runs is presented in Table 7, along with the overall standard deviation, standard error of means and %CV. Inhibition responseAromatase (Human Recombinant) Assay (2011) / Page 8 of 12 TEBUCONAZOLE / 128997 OCSPP 890.1200 / OECD None 

## curves for the positive control from each run are shown in Figure 1. These results were generally within the recommended ranges for the top of the curve, bottom curve, Hill slope, and log IC 50 

## . The overall %CV exceeded the recommended 15% at the 10

− 5 

## test concentration (28%).

TABLE 7. Effect of Tebuconazole on Aromatase Acti vity (as percent of control) from Independent Runs a

Chemical Concen. (Log M) # Runs Overall Mean Overall SD Overall SEM Overall %CV 4-OH ASDN (positive control) − 5 3 0.9 0.25 0.14 28

− 6 3 7.8 1.10 0.63 14

− 6.5 3 18.6 2.01 1.16 11

− 7 3 40.3 4.15 2.40 10

− 7.5 3 65.7 3.20 1.85 5

− 8 3 79.1 5.59 3.23 7

− 9 3 96.8 2.92 1.69 3

− 10 3 95.9 3.95 2.28 4 Tebuconazole

− 3 3 0.1 0.32 0.19 320

− 4 3 2.3 0.31 0.18 14

− 5 3 19.3 1.63 0.94 8

− 6 3 61.9 4.94 2.9 8

− 7 3 89.9 3.74 2.2 4

− 8 3 91.3 6.48 3.7 7

− 9 3 86.0 4.65 2.7 5

− 10 3 89.2 9.90 5.7 11 a Data were obtained from text tables on pages 18 and 19 of the study report. SD Standard Deviation SEM Standard Error of the Mean CV Coefficient of Variance



## FIGURE 1. Inhibition Response Curv es for 4-OH ASDN (Formestane).Aromatase (Human Recombinant) Assay (2011) / Page 9 of 12 TEBUCONAZOLE / 128997 OCSPP 890.1200 / OECD None


## C. TEST SUBSTANCE: For tebuconazole, aromatase ac tivity averaged 0.2706 ± 0.0069 nmol/mg-protein/min at the lowest tested concentration (10

− 10 

## M) and 0.0004 ± 0.0009 nmol/mg-protein/min at the highest tested concentration (10

− 3 

## M). The mean aromatase activity of tebuconazole (expressed as %full co ntrol activity) for each concentration tested across all 3 runs is presented in Table 7, along with the overall standard deviation, standard error of means and %CV. Inhibition response curves for tebuconazole from each run are shown in Figure 2, and the average inhibition response curve across a ll runs is shown in Figure 3. The overall mean aromatase activitie s compared to the controls were 90% at 10

− 7 

## M, 62% at 10 − 6 

## M, 19% at 10 − 5 

## M, 2.3% at 10 − 4 

## M, and 0.1% at 10

− 3 

## M. The decrease in aromatase activity in the assay was concentration-dependent.

FIGURE 2. Inhibition Response Curves for Tebuconazole From Each Test Run.Aromatase (Human Recombinant) Assay (2011) / Page 10 of 12 TEBUCONAZOLE / 128997 OCSPP 890.1200 / OECD None 

## FIGURE 3. Mean Inhibition Resp onse Curve for Tebuconazole.

The effect of tebuconazole on inhibition of arom atase activity is presented in Table 8. The estimated log IC 50 

## ranged from

− 5.8 to

− 5.7 M for the test material and averaged

− 5.73 M compared to the positive control 4-OH ASDN, which averaged

− 7.20 M. The average slope of the concentration response curve was

− 0.99 for tebuconazole and

− 0.88 for 4-OH ASDN. Confidence in these values is very high due to the sm all variations (<6% CV).

TABLE 8. Effect of Tebuconazole on Aromatase Acti vity (as Percent of Contro l) From Independent Runs a Chemical Run 1 Run 2 Run 3 Mean b SD b %CV b Log IC 50 (M) Tebuconazole

− 5.8

− 5.7

− 5.7

− 5.73 0.058

− 1.01 4-OH ASDN

− 7.3

− 7.1

− 7.2

− 7.20 0.100

− 1.39 Slope Tebuconazole

− 1.02

− 0.93

− 1.01

− 0.99 0.049

− 5.00 4-OH ASDN

− 0.85

− 0.85

− 0.93

− 0.88 0.046

− 5.27

a Data obtained from text tables on pages 18 and 19 of the study report.
b Calculated by the reviewers from data presented in this table. SD Standard Deviation CV Coefficient of Variance


## Based on the data from the average response curv e and the criteria listed above in Table 6, the results support the c onclusion that tebuconazole inhibi ts aromatase in the aromatase assay.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS CONCLUSIONS: For each run the data for the background activity controls were within the guideline recommenda tions. No assay drift was recorded in the first and second run. A marginal assay drift wa s recorded for the thir d run as the % changes for the full activity controls were between 110. 6% and 89.4% instead of between 110% and 90%, but this did not affect the outcome or the interpretation of the results. The average aromatase activity in the full activity contro ls 0.3058 ± 0.0346 nmol/mg protein/min.Aromatase (Human Recombinant) Assay (2011) / Page 11 of 12 TEBUCONAZOLE / 128997 OCSPP 890.1200 / OECD None



## For 4-OH ASDN, the data generated for the pos itive control evaluations run concurrently with the tebuconazole evaluations confirmed th e responsiveness of the aromatase enzyme to a reference inhibitor and indica ted that all guideline criteria had been met for the positive control. Specifically, aromatase activity averaged 0.2925 ± 0.0210 nmol/mg protein/min at 10

− 10 

## M and 0.0028 ± 0.0005 nmol/mg protein/min at 10

− 5 

## M. The log IC 50 

## value for each run

was

− 

## 7.3,

− 

## 7.1 and

− 

## 7.2 M, which equated to an IC 50 

## value of 5.06 x 10

− 8 

## M, 7.32 x 10

− 8 

## M and 6.49 x 10

− 8 

## M respectively. The average slope of the concentration response curve was

− 

## 0.88. Overall, the log IC 50 

## value was

− 

## 7.2 M, which equated to an IC 50 

## of 6.25 x 10

− 8 

## M.
For tebuconazole, the data gene rated for tebuconazole indicated that this test substance inhibited the aromatase enzyme. Specifically, the aromatase activity for tebuconazole averaged 0.2706 ± 0.0068 nmol/mg protein/min at 10 -10 

## M and 0.0004 ± 0.0009 nmol/mg protein/min at 10

− 3 

## M. The log IC 50 

## values were approximately

− 

## 5.8 M for the first run and

− 

## 5.7 M for the second and third runs, which equated to IC 50 

## values of 1.58 x 10

− 6 

## M, 2.08 x 10

− 6 

## M and 2.22 x 10

− 6 

## M for the first, second and third r uns respectively. The overall log IC 50 

## value was

− 

## 5.7 M which equated to an overall IC 50 

## of 1.95 x 10

− 6 

## M. The average slope of the concentration response curve was

− 

## 0.98.

B. AGENCY COMMENTS: Aromatase activity in the full activity controls ranged from 0.250 to 0.365 nmol/mg-protein/min for the 3 test runs, with a mean and standard deviation of 0.306±0.038 nmol/mg-protein/min. Activity in the background controls ranged from 3.3 to 4.4% of the full control activity with an average of 3.6%. The response of the full activity controls and background controls was acceptable.
For the positive control substance (4- OH ASDN), aromatase activity averaged 0.2925±0.0202 nmol/mg-protein/min at the lo west tested concentration (10

− 10 

## M) and 0.0028±0.0005 nmol/mg-protein/min at the hi ghest tested co ncentration (10

− 5 

## M). These results were generally within the recommende d ranges for the top of the curve, bottom curve, Hill slope, and log IC 50 

## . The overall %CV exceeded the recommended 15% at the 10 − 5 

## test concentration (28%).
For tebuconazole, aromatase activity averag ed 0.2706 ± 0.0069 nmol/mg-protein/min at the lowest tested concentration (10

− 10 

## M) and 0.0004 ± 0.0009 nmol/mg-protein/min at the highest tested concentration (10

− 3 

## M). The lowest portion of the response curve for tebuconazole across runs was less than 50% ac tivity. Tebuconazole reduced aromatase activity to 90% at 10

− 7 

## M, 62% at 10 − 6 

## M, 19% at 10 − 5 

## M, 2.3% at 10 − 4 

## M, and 0.1% at 10 − 3 

## M.
The estimated log IC 50 

## ranged from

− 5.8 to

− 5.7 M for the test material and averaged

− 5.73 M compared to the positive control 4-OH ASDN, which averaged

− 7.20 M. The average slope of the concentration response curve was

− 0.99 for tebuconazole and

− 0.88 for 4-OH ASDN. Confidence in these values is very high due to the small variations (<6% CV).
Based on the data from the average response curve, tebuconazole is classi fied as an Inhibitor of aromatase.Aromatase (Human Recombinant) Assay (2011) / Page 12 of 12 TEBUCONAZOLE / 128997 OCSPP 890.1200 / OECD None


## 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:

 Proficiency chemicals da ta were not provided.

 For the positive control, the overall %CV exceeded the recommended 15% at the 10

− 5 

## test concentration (28%).

# DATA EVALUATION RECORD

TEBUCONAZOLE
Study Type: OCSPP 890.1300, Estrogen Receptor Transcriptional Activation

EPA Contract No. EP10H001452 Task Assignment No. 2-22-2012 (MRID 48619403)

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: 02/29/2012
Secondary Reviewer Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 03/12/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 03/16/2012

Quality Assurance: Signature: Steven Brecher, Ph.D., D.A.B.T. Date: 03/16/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).

### TEBUCONAZOLE/ 128997 Estrogen Receptor Transcriptional Activation (2011) I 

### Page 1 of9 OCSPP 890.1300/ OECD 455



## DATA EVALUATION RECORD

STUDY TYPE: Estrogen Receptor Transcriptional Activation (Ruma.ii cell Line, HeLa-9903); OCSPP 890.1300; OECD 455.

PC CODE: 128997

TXR#: 0056253

TEST MATERIAL (PURITY): Tebuconazole (97.5% a.i.) DP BARCODE: D398353

CAS No.: 107534-96-3

SYNONYMS: a-[2-( 4-chlorophenyl)ethyl]-a-(l, 1-dimethylethyl)-lH-1,2,4-triazole-1-ethanol

CITATION: Blanck, 

## 0. (2011) Tebuconazole: evaluation in the in vitro 

## (HeLa-9903) estrogen receptor transcriptional activation assay. Bayer S.A.S., Sophia Antipolis Cedex, France. Laboratory Report No.: SA 11159, December 1, 2011. MRID 48619403. Unpublished.

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

TEST ORDER#: CON-128997-20

EXECUTIVE SUMMARY: In an estrogen receptor transcriptional activation assay (MRID 48619403), hERa-HeLa-9903 cells cultured in vitro 

## were exposed to tebuconazole (97.5% a.i., Batch# K689052) at logarithmically increasing concentrations from 

## 10- 10 to 

## 10- 4 

## M in DMSO (0.1 % ). The experiments were performed using 96-well plates and each concentration was tested in triplicate (3 wells/plate). Cells were exposed to the test agent for 23 ±1 hour to induce reporter (luciferase) gene products. 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.

Tebuconazole was tested up to the limit of solubility, 10- 4 

## M. Performance criteria were met for all reference chemicals. The log PC10 for 17a-estradiol and 17a-methyltestosterone, and Hill Slope for 17a-estradiol were lower than the expected range in the first run, indicating increased sensitivity of the assay to weak agonists. The mean RPCMax for tebuconazole was -0.2% for the first run and -1.6% for the second run; the associated PCMax was 

## 10- 10 M for the first run and 

## 10- 5 

## M for the second run. Because the RPCMax 

## < PC10 in two assay runs, tebuconazole was considered negative for estrogen receptor transcriptional activation in this test system.

This assay satisfies the EDSP Tier 1 Test Order requirement for an Estrogen Receptor Transcriptional Activation assay (OCSPP 890.1300).

COMPLIANCE: Signed and dated OLP and Quality Assurance statements were provided.Estrogen Receptor Tran scriptional Activation (2011) / Page 2 of 9 TEBUCONAZOLE/ 128997 OCSPP 890.1300/ OECD 455



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: Tebuconazole Description: Technical, white powder, MW 307.8 Source: Bayer Crop Science AG Lot/Batch #: Batch # K689052 Purity: 97.5% Solubility: Not reported (NR) Volatility: NR Stability: 2 years Storage conditions: 25±5 ° C CAS #: 107534-96-3 Structure:



## 2. Reference substances 17 β -estradiol (strong estrogen; positive control) Supplier: Sigma Aldrich, St. Louis, MO Catalogue and Batch #: E8875; lot # 010M0142 Purity: 100% CAS # : 50-28-2



## 17 α -estradiol (weak estrogen) Supplier: Sigma Aldrich, St. Louis, MO Catalogue and Batch #: E8750; lot # 029K4124 Purity: ≥ 99.5% CAS # : 57-91-0 

## Corticosterone (negative compound) Supplier: Sigma Aldrich, St. Louis, MO Catalogue and Batch #: 27840; lot # BCBB59565 Purity: 99.6% CAS # : 50-22-6



## 17 α -methyltestosterone (very weak agonist) Supplier: Sigma Aldrich, St. Louis, MO Catalogue and Batch #: M7252; lot # 039K0168 Purity: 100% CAS # : 58-18-4


## 3. Vehicle(s)
Solvent: DMSO
Solvent control (final concentration): 0.1% Cl OH CH 3

CH 3 CH 3

N N NEstrogen Receptor Tran scriptional Activation (2011) / Page 3 of 9 TEBUCONAZOLE/ 128997 OCSPP 890.1300/ OECD 455



## B. METHODS

1. Cell Culture: Stably-transfected hER α -HeLa-9903 cells were obtained from the Japanese Collection of Research Bioresour ces Cell Bank and were verified to be free of mycoplasma infection using PCR and DNA st aining techniques. Cells we re maintained in Eagles Minimum Essential Medium w ithout phenol red, supplemented with 60 mg/L kanamycin and 10% dextran-coated charcoal-treated fetal bovine serum (Thermo Scientific, Batch # ATF32351), in an incubator under 5% CO 2 

## at 37oC. Upon reach ing 75-90% confluence, cells were subcultured 6 times prior to exposure to the test material.

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

## cells/100 μL medium/well in a 96 well plate and allowed to attach for 3 hours. Growth media was replaced with media containing seri al log dilutions of tebuconazole in DMSO (0.1% final concentration). Cells were incu bated for 22-24 hours at 37±1°C. Cytotoxicity was determined using a commercial kit (ATPli te 1Step, Perkin Elmer, Waltham, MA). Transcriptional activation of the estrogen receptor was determined using a commercial kit (neolite Reporter Gene Assay System, Perk in Elmer, Waltham, MA). Luminescence was measured using a Wallac 1450 MicroBet a Windows Workstation, V 4.01.014 (Perkin Elmer, Waltham, MA), with a detection limit of 32 fg luciferase/well.

a. Preliminary Test: A preliminary test evaluating concentrations ranging from 10

− 6 

## to 10 − 4 

## M was conducted to determine the appropriate concentration range and to determine concentrations resulting in inso lubility and/or cytotoxicity.

b. Proficiency Chemicals: The responsiveness of the test system was confirmed for each newly prepared batch of cell stocks taken from the frozen st ock by testing the following set of proficiency chemicals. The details of th e proficiency test were not provided, only the results.

Compound CAS No. Concentration Range (M) Expected Response a Notes Diethylstilbestrol (DES) 56-53-1 10

− 14 to 10

− 8 Positive --- 17 α -Ethynyl estradiol (EE) 57-63-6 10

− 14 to 10

− 8 Positive --- Hexestrol 84-16-2 10

− 13 to 10

− 7 Positive --- Genistein 446-72-0 10

− 12 to 10

− 5 Positive Cytotoxic at 0.01 b , 0.1, and 1 mM Estrone 53-16-7 10

− 12 to 10

− 6 Positive --- Butyl paraben 94-26-8 10

− 11 to 10

− 4 Positive Cytotoxic at 0.1 b and 1 mM 1,3,5-Tris(4- hydroxyphenyl)benzene c 15797-52-1 10

− 12 to 10

− 5 Positive Cytotoxic at 100 μM. PC Max approx. 50% of PC. Binds to hER α and has ER antagonistic activity Dibutyl phthalate (DBP) 84-74-2 10

− 11 to 10

− 4 Negative d Cytotoxic at 1 mM Atrazine 1912-24-9 10

− 11 to 10

− 4 Negative Cytotoxic at 1 mM b Corticosterone 50-22-6 10

− 10 to 10

− 4 Negative If not cytotoxic at 1 mM, then that should be the highest tested concentration a Positive = RPC Max ≥ 10% of the response of the positive control in at least 2 of 2 (or 2 of 3) runs Negative = RPC Max fails to achieve at least 10% of the response of the positive control in 2 of 2 (or 2 of 3) runs b Cytotoxicity is expected to be close to 80% at this concentration. c Compound selected to challenge solubility and cytotoxicity d DBP is negative for ER α mediated transcriptional activation, but may not be negative for non-ER β mediated transcriptional activation. A positive result would indicate that the sy stem is detecting activity other than that due to pure ER α , and is therefore unacceptable.Estrogen Receptor Tran scriptional Activation (2011) / Page 4 of 9 TEBUCONAZOLE/ 128997 OCSPP 890.1300/ OECD 455



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

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

− 14 to 10

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

− 12 to 10

− 6 Weak estrogen Corticosterone 50-22-6 10

− 10 to 10

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

− 11 to 10

− 5 Very weak agonist


## 3. Data analysis: To obtain the relative transcriptiona l activity to the 1 nM E2 positive control (PC), the luminescence signals from the concur rent plate were analy zed by subtracting the mean value of the vehicle control from each well value to normalize the data; each normalized value was then divided by the mean value of the normalized PC. The resulting value was multiplied by 100 in order to expres s relative transcriptional activity as a percentage of the PC. Graph Pad Prism v. 5.04 (GraphPad Software, Inc., La Jolla, CA) was used to calculate the EC 50 

## , PC 10 

## , PC 50 

## , RPC Max 

## , and PC Max 

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

## < PC 10 

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

## and Hill slope values are calculated only if a positive response is observed. Coefficients of variation (CV) were calculated for the lumi nescence data triplicates. Concentrations showing >20% cytotoxicity or evidence of insolubility were excluded from analyses.

4. Definitions

EC 50 

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

PC 10 

## = concentration of a test chemical at which the response is 10% of the response induced by the positive control (E2 at 1 nM) in each plate

PC 50 

## = concentration of a test chemical at which the response is 50% of the response induced by the positive control (E2 at 1 nM) in each plate

RPC Max 

## = maximum level of response induced by a test chemical, expressed as a percentage of the response induced by the positive control (1 nM E2) on the same plate

PC Max 

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



## II. RESULTS

A. PRELIMINARY TEST: In order to identify a suitable top concentration for use in the transcriptional activation assays, preliminary cytotoxicity and precipitation assays were conducted. Precipitation was observed in te buconazole solutions at 1 M in DMSO, therefore the definitive runs were performed at log serial dilutions starting at 0.1 mM. The results of the cytotoxicity test are pres ented in Table 1. Concentrations of 10 − 4 

## M showed cell viability of 80%. Based on th ese results, concentrations 10

− 10 

## to 10 − 4 

## M were selected for the assay.Estrogen Receptor Tran scriptional Activation (2011) / Page 5 of 9 TEBUCONAZOLE/ 128997 OCSPP 890.1300/ OECD 455



### TABLE 1. Preliminary Test for Solubility, Cytotoxi city, and Concentration-Selection for Tebuconazole Concentration (M) % Viability b Comments c 10

− 4 80 10

− 5 116 10

− 6 115 SDS 1% 0.0 Positive control for cytotoxicity

VC b 100.0 a Data were obtained from page 16 of the study report. b If viability is <80%, the concentration is considered cytotoxic. c Include comments related to solubility issues, if applicable. d VC = Vehicle control



## B. POSITIVE AND NEGATIVE REFERENCE CHEMICALS
1. Proficiency Chemicals: The responsiveness of cells to the required profic iency chemicals was performed in duplicate, alt hough it was not clear if the runs were performed on different days. The data are reported in Table 2. Th e responses did demonstrate proficiency. The only minor deviation was in the first r un; corticosterone produced an RPC Max 

## of 16%, indicating a weakly positive respon se. In the second run, the RPC Max 

## was -14%, indicating a negative response.

TABLE 2. Proficiency Chemicals a Compound Expected Response Lab Response Diethylstilbestrol Positive Positive 17 α -Ethynyl estradio l Positive Positive Hexestrol Positive Positive Genistein Positive Positive Estrone Positive Positive Butyl paraben Positive Positive 1, 3, 5-Tris(4-hydroxyphenyl)benzene Positive Positive Dibutyl phthalate Negative Negative Atrazine Nega tive Negative Corticosterone Negative Positive/Negative a Data were obtained from page 34 of the study report.



## 2. Reference Chemicals: Values derived from the concentration response curve ( e.g ., log PC 50, 

## log PC 10, 

## log EC 50, 

## Hill slope) for the four concurrently run reference materials are included in Table 3. Performance criteria were met for all reference chemicals. The log PC 10 

## for 17 α -estradiol was lower than the expected range in the first assay, as was the Hill Slope. The log PC 10 

## for the first assay and log PC 50 

## for both assays for 17 α

methyltestosterone were lower than the exp ected range. The results show an increased sensitivity to weak agonists.Estrogen Receptor Tran scriptional Activation (2011) / Page 6 of 9 TEBUCONAZOLE/ 128997 OCSPP 890.1300/ OECD 455



### TABLE 3. Performance Criteria for Reference Chemicals a
Reference Chemical Parameter Acceptable Range Values Acceptable Run 1 Run 2 Yes No 17 β -estradiol Log PC 50 − 11.4 to

− 10.1

− 10.5

− 10.7 X Log PC 10 <

− 11

− 11.7

− 11.4 X Log EC 50 − 11.3 to

− 10.1

− 10.5

− 10.7 X Hill Slope 0.7 to 1.5 0.8 1.2 X Test range 10

− 14 to 10

− 8 M 10

− 14 to 10

− 8 M 10

− 14 to 10

− 8 M X 17 α -estradiol Log PC 50 − 9.6 to

− 8.1

− 9.1

− 8.9 X Log PC 10 − 10.7 to

− 9.3

− 11.9

− 10.1 X Log EC 50 − 9.6 to

− 8.4

− 9.1

− 8.9 X Hill Slope 0.9 to 2.0 0.3 0.8 X Test range 10

− 12 to 10

− 6 M 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 10

− 10 to 10

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

− 5.1 -5.8 b -6.0 b X Log PC 10 − 8.0 to

− 6.2 -9.6 b -8.4 b X Test range 10

− 11 to 10

− 5 M 10

− 11 to 10

− 5 M 10

− 11 to 10

− 5 M X a Data were obtained from pages 11, 14 and 17 of the study report. b Calculated by the reviewer from data obtai ned from pages 11, 14 and 17 of the study report



## C. DEFINITIVE ASSAY

1. Vehicle and Positive Controls: Vehicle and positive control data are included in Table 4. The overall mean TA value for the vehicle cont rol, expressed as arbi trary light units, was 504.6 in the first run and 500.5 in the second r un. The positive control mean normalized value was 1524.2 in the first run and 1395.7 in the second run. The PC 50 

## for 17 β -estradiol was 762.1 in the first run and 697.9 in the second and the PC 10 

## was 152.4 for the first run and 139.5 for the second.

TABLE 4. Transcriptional Activation (TA) Response of Vehicle and Positive Control a Sample Vehicle Control Positive Control b Runs Mean SD Mean SD Fold Induction c

1 504.6 61.8 1524.2 NR 4.1 2 500.5 71.2 1395.7 NR 3.9 a Data were obtained from page 36 of the study report. b Positive control was 17 β -estradiol (E2) at 1 nM. c Fold-induction = (mean TA of PC)/(mean TA of VC)



## 2. Test Material: Relative (to the PC) transcriptional ac tivation at each concentration of the test chemical during the two assay runs is pr esented in Table 5. Th e concentration-response curves depicting fold induction of relative tr anscriptional activation are presented in Figure 1 and Figure 2 below. The mean RPC Max 

## were
− 0.2% for the first run and

− 1.6% for the second run; the associated PC Max 

## were 10 − 10 

## M for the first run and 10

− 5 

## M for the second run. The values reported for the log EC 50 

## , Hill Slope, PC 50 

## and PC 10 

## are mathematically fitted to the data, but are not representative of actual binding characteristics of tebuconazole. Because the RPC Max 

## < PC 10 

## in both runs, tebuconazole was considered negative for estrogen receptor transcriptional activation in this test system.Estrogen Receptor Transcriptional Activation (2011) / Page 7 of 9 TEBUCONAZOLE/ 128997 OCSPP 890.1300/ OECD 455



### TABLE 5. Relative Tran scriptional Activation (RTA) of Tebuconazole a
Parameter RTA (mean ± SD); % of Positive Control (PC) Run 1 Run 2 Conc. (M) Mean SD Mean SD 10

− 4

− 18.9 2.1

− 35.8 1.6 10

− 5

− 5.6 4.8

− 1.6 8.3 10

− 6

− 11.9 4.0

− 6.9 7.5 10

− 7

− 12.7 5.3

− 6.1 5.6 10

− 8

− 12.2 5.7

− 4.9 5.6 10

− 9

− 9.5 2.6

− 8.0 7.5 10

− 10

− 0.2 4.5

− 2.4 11.3 Log EC 50 b

− 13.32

− 4.120 Hill Slope b

− 0.6510

− 7.494 RPC Max − 0.2

− 1.6 PC Max 10

− 10 10

− 5 PC 50 − 13.32

− 4.120 PC 10 − 8.755

− 3.521 a Data were calculated by the reviewer, using data obtained from pages 50 and 63 of the study report b Data were obtained from pages 27 and 32.



## FIGURE 1. Run 1 Fold Induction, Tebuconazole Relative Transcription Activation (RTA).Estrogen Receptor Transcriptional Activation (2011) / Page 8 of 9 TEBUCONAZOLE/ 128997 OCSPP 890.1300/ OECD 455


## FIGURE 2. Run 2 Fold Induction, Tebuconazol e Relative Transcription Activation (RTA).

3. Performance Criteria: Performance criteria were met for 17 β -estradiol and corticosterone. The Log PC 10 

## for 17 α -estradiol was lower than the acceptable range in the first assay, as was the Hill Slope. The log PC 10 

## for the first assay and log PC 50 

## for both assays for 17 α

methyltestosterone were lower th an the expected range. For 17 α -methyltestosterone, the mean RPC Max 

## was 79.3% for the first run and 72.0% fo r the second run. The results show an increased sensitivity to weak agonists. M ean luciferase activity was 4.1-fold for the first run and 3.9 fold for the second run. The fold-induction corresponding to the PC 10 

## of the concurrent PC was greater than 1+2 standard deviations of the vehicle control on all plates. Variability was minimal and result s were reproducible among runs.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: The two independent runs gave similar results and the reference compounds gave the expected responses. The maximum response for tebuconazole was less than 10% of the positive control 17 β -estradiol in both runs. Therefore, tebuconazole was negative in this human estrogen receptor transcriptional activation assay.

B. AGENCY COMMENTS: Tebuconazole was tested up to the limit of solubility, 10

− 4 M. Performance criteria were met for all reference chemicals. The log PC10 for 17 α -estradiol and 17 α -methyltestosterone, and Hill Slope for 17 α -estradiol were lower than the expected range in the first run, indicati ng increased sensitivity of the assay to weak agonists. The mean RPCMax for tebuconazole was -0.2% for the first run and -1.6% for the second run; the associated PCMax was 10

− 10 M for the first run and 10

− 5 M for the second run. Because the RPCMax < PC10 in two assay r uns, tebuconazole was considered negative for estrogen receptor transcriptional activation in this test system.Estrogen Receptor Tran scriptional Activation (2011) / Page 9 of 9 TEBUCONAZOLE/ 128997 OCSPP 890.1300/ OECD 455



## C. STUDY DEFICIENCIES: No deficiencies were noted.

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

EPA MRID Number 48688301

Page 1 R f 41 

### Version: 22 September 2011 Data Requirement: EPA DP Barcode D404687

OECD Data Point 231

EPA MRID 48688301

EPA Guideline 890.1350 Fish Short-Term Reproduction Assay

Test Material: Tebuconazole Purity (%): 97.5%

Common Name: Tebuconazole Chemical Name: IUPAC Name: (RS)-1- p -Chlorophenyl-4,4-dimethyl-3-(1 H -1,2,4-triazol-1-ylmethyl)pentan-

3-ol

CAS Name: Į -[2-(4-Chlorophenyl)ethyl]- Į -(1,1-dimethylethyl)-1 H -1,2,4-triazole-1-

ethanol

CAS No.: 107534-96-3 Synonyms: None

EPA PC Code: 128997

Primary Reviewer: Rebecca L. Bryan, B.S. Signature: Staff Scientist, CSS-Dynamac Corporation Date: 2/25/2014

Secondary Reviewer: Dave A. McEwen, B.S. Signature: Staff Scientist, CSS-Dynamac Corporation Date: 3/05/2014

Additional and Final Reviewer(s ): Amy Blankinship Signature : USEPA/OCSPP/OPP/ERB3/Chemist Date: June 2, 2014

Additional Reviewer(s ): Stephen Carey Signature : USEPA/OCSPP/OPP/ERB6/Biologist Date: June 10, 2014

Date Evaluation Completed: 6/5/15 

### AMY BLANKINSHIP Digitally signed by AMY BLANKINSHIP DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=AMY BLANKINSHIP, dnQualifier=0000040917 Date: 2015.06.15 17:52:19 -04'00'

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

EPA MRID Number 48688301

Page 2 R f 41 

### Version: 22 September 2011 CITATION: 

### Banman, C.S.; Matlock, D.; and Lam, C.V., 2012. Short-Term Reproduction Assay with Fathead Minnow ( Pimephales promelas ) Exposed to Tebuconazole Technical Under Flow-Through Conditions. Bayer
CropScience, Stilwell, KS and Experimental Pathology Laboratories, Inc., Sterling, VA. Laboratory Report Number: EBHWP002. Sponsor: Bayer CropScience, , Research Triangle Park, NC. July 2, 2012. Unpublished.

Note: 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 poten tial 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).

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

EPA MRID Number 48688301

Page 3 R f 41 

### Version: 22 September 2011 EXECUTIVE SUMMARY

The 21-day fish short-term reproduction assay of tebuconazole (97.5% purity) with fathead minnows ( Pimephales promelas ) was conducted under flow-through conditions. Sixteen spawning groups of adult fish (2 males and 4 females per group; ca. 7 months of age) were exposed to tebuconazole at nominal concentrations of 0 (water control), 0.0065, 0.013, and 0.026 mg a.i./L, which resulted in mean measured concentrations of <0.00065 (<LOQ), 0.00497, 0.0113, and 0.0195 mg a.i./L, respectively. The mean
measured recoveries ranged from 75 to 87% of nominal concentrations. The test system was maintained at

24.7 to 25.6 o 

### C and a pH of 7.7 to 7.9.

There were no significant differences in survival in the tebuconazole treatment groups compared to the control (overall survival was 100% in the control and 88-92% in the treatment groups). Additionally, there were no
treatment-related clinical signs of toxicity observed in the treatment groups. No treatment-related effects were observed on body weight or length.

Spawning occurred at least every 4 days in two of the four control replicates and mean fecundity in controls was 23.7 eggs/female/day/replicate (replicate range: 15.7-31.9 eggs/female/day); fertilization success in controls was 97.6%. There were no significant effects on fecundity or fertility (fertilization success) in the
tebuconazole treatment groups. There were no significant effects on male tubercle score and there were notubercles observed in females. At the high treatment concentration there was a significant (p<0.05; JonckheereTerpstra) decrease of 18.5% in female gonado-somatic index (GSI) and a marginally significant (p=0.056; Jonckheere-Terpstra) decrease of 19% in male GSI. There was a significant decrease of 35 and 55% in female plasma vitellogenin (VTG) at the mid and high treatment concentrations, respectively (p<0.05; Jonckheere-Terpstra); there were no significant differences in male VTG between the treatment groups and

the control.

According to the pathologist’s report, no histopathologic findings observed in the testes of the male fathead
were clearly related to tebuconazole treatment. The mean testicular stage scores were decreased in the mid and high treatment groups compared to the controls, but the study authors report this decrease is unlikely to be biologically meaningful. In the ovary of the female fathead, the histopathologic finding of increased granulomatous inflammation (minimal to mild) in the high treatment group compared to the controls was most

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

EPA MRID Number 48688301

Page 4 R f 41 

### Version: 22 September 2011 likely related to tebuconazole treatment. Additional histopathological findings for females included increased prevalence of post-ovulatory follicles and decreased prevalence of oocyte atresia in the tebuconazole-treated groups, neither of which were reported as potentially treatment-related by the pathologist; in the controls, 47% of the examined females were reported to have some level of increased oocyte atresia (2 fish with minimal and 5 fish with moderate increased atresia) whereas the low, mid and high treatment groups had 40, 20, and
20%, respectively. The mean ovarian stage scores were decreased in the high treatment females compared to the controls (2.9 vs. 3.2), but the study authors report this decrease is unlikely to be biologically meaningful.
Histological examination of the liver was also conducted with no substantially more prevalent findings for treated males compared to the control. However, for males, the occasional findings of microgranulomas (minimal; mid and high treatment groups) and individual hepatocyte necrosis (minimal; high treatment group) in the livers
were likely treatment related; there was also a concentration-related decrease in hepatocyte vacuolation compared to control. In females, generally concentration-dependent findings in the liver included individual hepatocyte necrosis (occurred in all three treatment groups), multinucleated hepatocytes (mid and high treatment groups), microgranulomas (mid and high treatment groups), hepatocyte megalocytosis (mid and high treatment groups), and decreased hepatocellular vacuolation (all groups). Plasma sex steroids were not
measured in this study.

The validity and performance criteria for the Fish Short-Term Reproduction Assay (OCSPP 890.1350) were

met.

This assay satisfies the EDSP Tier 1 Test Order requirements for a Fish Short-Term Reproduction Assay (OCSPP Guideline 890.1350).

Results Synopsis:

Test organism age at test initiation: Ca. 7 months Mean body weight at test initiation (if measured): 4.62 g for males, 1.71 g for females Mean length at test initiation (if measured): Not determined (ND)
Test type: Flow-throughData Evaluation Record on the Fish Short-Term Reproduction Assay with TebuconazoleEPA MRID Number 48688301Page5Rf41Version: 22 September 2011Table 1: Summary of Reproductive and HPG Effects1,2in the Fish Short-Term Reproduction Assay (FSTRA) with Tebuconazole.Treatment(mg a.i./L)[meanmeasured]FecundityFert.SuccessTubercle ScoreGSIGonadal Histo.Plasma VTGPlasma T3Plasma E23MFMFMFMFMFMF0.00497 No No No No No No No No No No NA NA NA NA0.0113 No No No No No No No No No Yes NA NA NA NA0.0195 No No No No Yes4Yes No Yes No Yes NA NA NA NAAbbreviations:Conc.Concentration.Diff.Difference.E217ȕ-estradiol.FFemale.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.MMale.NANot applicable.TTestosterone.VTGVitellogenin.1A “yes” indicates a significant difference based on comparison to the negative (clean water) control, unless otherwise specified.2The criteria for significance are described in the Reviewer’s Analysis and Statistical Verification sections of the DER. Conclusions regarding histopathology may beheavily weighted by the expert opinion of a board-certified pathologist.3Plasma testosterone and estradiol concentrations were not determined.4The decrease in male GSI was marginally significant (p=0.056) at the high concentration.

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

EPA MRID Number 48688301

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### Version: 22 September 2011 I. MATERIALS AND METHODS

Guideline Followed: This study was conducted according to the U.S. EPA OCSPP 890.1350: “Fish Short-Term Reproductive Assay”. The following minor deviations

were noted:

1. During pre-exposure, the average eggs/female/day/replicate was <15. The OCSPP 890.1350 guideline validity criterion is at least 15 eggs/female/day/replicate or spawning occurring at least every 4 days.

2. At test initiation, the individual female weights were within ±20% of mean weight with the exception of one female that weighed 2.14 g (25% of mean). EPA
recommends that individual weight of each fish selected for the test be within
±20% of the estimated mean of each sex.

These deviations do not have an impact on the acceptability of this study.

Compliance: Signed and dated GLP, Quality Assurance and Data Confidentiality statements were provided. This study was conducted in accordance with GLP Standards as published by the U.S. EPA (40 CFR Parts 16 0), with the following exceptions:
routine water contaminant screening analyses were conducted by American Analytical Laboratories (Akron, OH) and food contaminant screening analyses were conducted by Geo Labs, Inc. (Braintree, MA); neither of these analyses
was conducted according to GLP procedures.

A. Test Material Tebuconazole (CAS # 107534-96-3)
Description: White powder Water Solubility: 36 mg/L at 20°C Melting point: 105°C Boiling point: Not reported (NR)
Stability in water: NR

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

EPA MRID Number 48688301

Page 7 R f 41 

### Version: 22 September 2011 Stability in light: NR pKa: 2.3 Kow (Pow): 3.7
Vapor Pressure: 1.2 × 10 -8 

### mm Hg Expiration Date: 23 August 2012

OECD recommends describing water solubility, melting/boiling point stability in water and light, pKa, Pow or
Kow, vapor pressure of test compound, expiration date.



### Batch No. : K689052 Purity: 97.5%

Impurities: NR Stability of Compound: No stability data were reported; stock solutions appeared to be stable throughout the assay period (21 days) at room temperature. No solubility or
stability data under test system conditions were reported. Storage Conditions of Test Compound: Room temperature (ambient)Data Evaluation Record on the Fish Short-Term Reproduction Assay with TebuconazoleEPA MRID Number 48688301Page8Rf41Version: 22 September 2011B. Test OrganismTable 2: General Information About the Test Species and Acclimation.ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies common name:Fathead minnow EPA recommends fathead minnow (Pimephales

promelas).Species scientific name: Pimephales promelasSpecies strain (if stated):Not reported (NR)Were fish obtained from a singlelaboratory stock?Yes Aquatic Biosystems, Fort Collins, CO,Lot No. ABS090811, hatch dates June16-23 2011 EPA recommends that fish be from a single laboratory

stock.Were acclimation conditions sameas definitive test?Yes EPA recommends that fish be acclimated under water quality and illumination conditions that are similar to the definitive test.Acclimation period:14 days EPA recommends a minimum two-week acclimation period. Note that the acclimation period is different from the subsequent, in situ pre-exposure phase.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TebuconazoleEPA MRID Number 48688301Page9Rf41Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on health:No mortalities occurred in the sevendays preceding transfer to the testsystem, and no treatment for diseasewas required. EPA recommends that mortality during the 7 days prior to the pre-exposure phase be less than 5% of the culture population. If mortality during these 7 days is greater than 10%, EPA recommends that the fish be rejected. If mortality is between 5-10%, EPA recommends that fish be held another 7 days. If mortalities greater than 5% occur during this extended acclimation period, EPA recommends that the fish not

be used.Type of food:Live brine shrimp( Artemia salina) EPA recommends that fish be fed frozen brine shrimp twice per day to promote active reproduction and maintain body condition.Source of food:NRFrequency of feeding:Two to three times/dayDetails on feeding:5-6 mL/feedingTable 3: Fish Selection and Pre-Exposure Performance.ParameterValue(s)Details or RemarksGuideline RecommendationsAge at test initiation: Ca.7 months EPA recommends reproductively mature (sexually dimorphic) fish, 4.5 - 6 months old.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TebuconazoleEPA MRID Number 48688301Page10Rf41Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsMean weight of males at test initiation(if determined):4.62 g Subsample of male fish; n=32 EPA recommends that a subsample of fish be weighed before the test to estimate the mean weight for each sex. It is recommended that the individual weight of each fish selected for the test be within ±20% of the estimated mean for

each sex.Range of individual weights (males) attest initiation (if determined):3.85-5.53 g Individual weights within ±20% of meanweightMean weight of females at testinitiation (if determined):1.71 g Subsample of female fish; n=64Range of individual weights (females)at test initiation (if determined):1.37-2.14 g Individual weights were within ±20% ofmean weight with the exception of onefemale that weighed 2.14 g.Mean length of males at test initiation(if determined):NRMean length of females at testinitiation (if determined):NRDuration of pre-exposure phase: 14 days EPA recommends a minimum of 14 days.Were pre-exposure conditions identicalto the definitive test?Yes EPA recommends that pre-exposure conditions, including temperature, photoperiod, feeding, etc., be identical to definitive test conditions.Number of pre-exposure tanks: 24Extra replicates tanks were initiallyestablished and 16 tanks were selected forthe definitive phase of the study. 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.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TebuconazoleEPA MRID Number 48688301Page11Rf41Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of males per tank:2Number of females per tank:4Pre-exposure fecundity:8.5 to 52.9eggs/female/reproductive day/replicate for the 16 tanksselected for the definitivetestNot all tanks averaged >15eggs/female/reproductive day/replicateduring the 7 day pre-exposure period.However, the egg production exceeded 15eggs/female/reproductive day/replicateduring the 14 day pre-exposure period andspawning occurred twice a week, so tankswere considered suitable for testing. EPA recommends that pre-exposure fecundity in each replicate (tank) selected for use in the definitive test be at least 15 eggs/female/reproductive day/replicate during the 7 days prior to the definitive test.Number of spawns during preexposure:>2 times in 7 days perreplicate for tanksselected for the definitivetest EPA recommends that spawning occur at least twice in the 7 days prior to the definitive test.Details on pre-exposure:All fish were in good health during preexposure.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TebuconazoleEPA MRID Number 48688301Page12Rf41Version: 22 September 2011C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:Flow-through EPA recommends the use of a flow-through system. As noted in the Corrections and Clarifications document 1 , the use of a static renewal system is not recommended for this

assay.Type of flow-through dilution system: Proportional diluter system Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 2Flow-through rate:8.0 mL/min Approximately8 volume turnovers/vessel/24hours Recommended flow-through rate is 45 mL/min (2.7 L/hr), or at least 6 total volume exchanges per day.1U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the Endocrine DisruptorScreening 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).2Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TebuconazoleEPA MRID Number 48688301Page13Rf41Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on toxicant mixing for flowthrough systems:All glass 2-L modified proportional dilutersystem was used for intermittent delivery of testsolutions to test chambers. Splitter cups wereused to equally divide (±10%) test solutionsbetween replicates at each exposureconcentration.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?NoDissolved oxygen concentration ranged from6.4 to 8.3 mg/L (77 to 100% saturation). EPA recommends aeration if dissolved oxygen reaches <4.9 mg/L (< 60% saturation).Source of dilution water:Blended spring water andreverse osmosis waterProcess involves filtration and UV sterilization ofthe spring water, and dechlorination, filtration,double-pass reverse osmosis, and UVsterilization of municipal water to produce soft,processed 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 forpesticides, heavy metals, and othercontaminants?Yes Weekly (chlorine), monthly, and bi-annualscreens were conducted.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TebuconazoleEPA MRID Number 48688301Page14Rf41Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsTest vessel type/materials:GlassEPA 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:22-L all glass aquaria(34.3 × 21.6 × 30.5 cm) EPA recommends the use of 18 L test chambers (e.g., 40 x 20 x 20 cm).Fill volume:12 L (approximately 16 cmdepth)EPA recommends 10 L solution per tank.Spawning substrate material: Smooth, white PVC pipe EPA recommends that each tank contain three semi-circular spawning substrates, e.g., aged PVC pipe, 10 - 20 cm in length, split

lengthwise.Spawning substrate size:4” diameter and 1⁄4”thickness, cut in half andsectioned into ca.5” piecesTable 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurement IntervalGuideline RecommendationsTemperature (°C)24.725.725.0Start of experimentand daily EPA recommends temperature 25±1 oC; interreplicate and inter-treatment differentials should not exceed 1 o C.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TebuconazoleEPA MRID Number 48688301Page15Rf41Version: 22 September 2011ParameterMinimumMaximumMeanMeasurement IntervalGuideline RecommendationspH 7.77.9NRStart of experimentand weekly (±2 days) EPA recommends pH 6.5 to 9.0.Dissolved oxygen (mg/L)6.4(77% saturation)8.3(100% saturation)NRStart of experimentand weekly (±2 days) EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air saturation)Total alkalinity (mg/L as CaCO3) 46 52 48Start of experimentand weekly (±2 days) EPA recommends total alkalinity > 20 mg/L

as CaCO 3.Hardness (mg/L as CaCO3) 50 58 55Start of experimentand weekly (±2 days)Conductivity (Pmhos/cm) 176 195 182Start of experimentand weekly (±2 days)Total organic carbon (mg/L)NRNR<0.05 Bi-annually EPA recommends that total organic carbon in dilution water be < 2 mg/L.Unionized ammonia (Pg/L) NR NR <0.001 Weekly/monthly EPA recommends that unionized ammonia in the dilution water be < 1 P g/L.Residual chlorine (Pg/L) NR NR <0.003 Weekly/monthly EPA recommends that residual chlorine in dilution water be < 10 P g/L.Abbreviations:NANot applicable.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TebuconazoleEPA MRID Number 48688301Page16Rf41Version: 22 September 2011D. Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details or RemarksGuideline RecommendationsWas a range-finder conducted?No EPA recommends conducting a range-finder if 96-hour LC

50 data for the fathead minnow are unavailable.If yes, what was the method fordetermining the highest testconcentration 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:NALife 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.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TebuconazoleEPA MRID Number 48688301Page17Rf41Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details:The nominal test concentrations of 0(control), 0.00650, 0.0130, and0.0260 mg/L were selected based ona rainbow trout Early Life Stage studyresulting in a LOEC of 25.0սg/L anda NOEC of 12.0սg/L, and a fatheadminnow Fish Sexual Development studyresulting in a LOEC of 6.25սg/L. EPA recommends conducting a range-finder with five test concentrations plus a control (six total treatment levels), with four females and two males per exposure tank (36 fish total). The number of mortalities that occur may be used to develop a concentration-response curve. Based upon the results, the highest concentration that does not result in increased mortality or signs of overt morbidity compared to controls, or 1/3 the derived 96-hr LC 50, may be selected as the highest exposure concentration in the 21-day test.Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:21 days EPA recommends that the dur ation of the definitive test be 21 days.Method for selecting the highest testconcentration in the definitive test:Historical data (early lifestage toxicity study)Rainbow trout Early Life Stage studyresulting in a LOEC of 25.0սg/L. EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of toxicity (e.g., 1/3 the 96-

hour LC 50 ), whichever concentration is lowest.Reference study citation (if applicable):Surprenant, D.C.(1988)and Bomke, C. (2007)Data Evaluation Record on the Fish Short-Term Reproduction Assay with TebuconazoleEPA MRID Number 48688301Page18Rf41Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsSeparation of test concentrations:2-fold (decreases of 0.5×from the highest testconcentration) EPA suggests that a concentration separation of between 0.33 (or three-fold) and 0.1 (or tenfold) is scientifically acceptable 1 .Number of test concentrations:4Negative control and 3 test concentrations EPA recommends a minimum of 3 concentrations and a control, plus solvent control
if appropriate.Are nominal concentrations adjusted forpurity?YesIndicate the type of values presented formeasured concentrations:Individual values (ppb)and % recovery (fromnominal)Limit of quantification (LOQ):0.65 ppb(0.00065 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):NRFrequency of measurement:Prior to study initiation(Day 0) and Study Days7, 15, and 21.Stock solution and test solutions werereported to contain no precipitates 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.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TebuconazoleEPA MRID Number 48688301Page19Rf41Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsWas the randomized complete blockdesign used?Spawning groups wereassigned to treatmentgroups using a rank-orderapproach.The top 4 performers were randomlyassigned to one replicate of each treatmentlevel, then the next 4 performers wererandomly assigned to a second replicatechamber for each treatment level, and soon. EPA recommends that all fish be randomly assigned to tanks during pre-exposure. Tanks are then ranked according to pre-exposure fecundity, and the tanks with the highest fecundity are randomly assigned to a definitive test treatment and block first. Each block contains one replicate of each treatment, including controls.Number of replicates in control: 4 EPA recommends 4 replicates.Number of replicates in solvent control(if applicable):NA EPA recommends the use of a concurrent solvent control when a solubilizing agent is used. EPA recommends 4 replicates.Number of replicates per test itemtreatment level:4 EPA recommends 4 replicates.Number of male fish per replicate at testinitiation:2 EPA recommends 2 males per replicate.Number of female fish per replicate attest initiation:4 EPA recommends 4 females per replicate.Was a solvent used?NoSolvent type (if applicable):NAData Evaluation Record on the Fish Short-Term Reproduction Assay with TebuconazoleEPA MRID Number 48688301Page20Rf41Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsMaximum solvent concentration (ifapplicable):NA EPA recommends that the solvent not exceed

0.02 ml/L3 . OECD recommends that solvent have no effect on survival nor produce any other adverse effects and that concentration not be greater than 0.1 ml/L4 .Was a positive control used?NoPositive control (if applicable):NAPositive control concentration(s): NAPhotoperiod:16 hrs light :8 hrs darkTwo 30-minute dawn/dusk transitionperiods were included each day with loweredlight intensity. EPA recommends photoperiod 16:8 (light:dark).Light intensity at water’s surface: 793 to 1062 lux EPA recommends light intensity 540 – 1080 lux (at water’s surface).3Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review. AquaticToxicology, 76, pp.69–92.4OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Series onTesting and Assessment. No. 23. Paris, France.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TebuconazoleEPA MRID Number 48688301Page21Rf41Version: 22 September 2011Table 8: Summary of Treatment Concentrations in the Fish Short-Term Reproduction Assay with Tebuconazole.Treatment IDNominalConcentration(mg a.i./L)MeasuredConcentration(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsControl (dilution wateronly)0.00 <LOQ NA NA EPA recommends that test item concentrations be maintained at a coefficient of variation (CV) 20%.Treatment 10.006500.0049712.976% of nominalTreatment 20.01300.011310.487% of nominalTreatment 30.02600.019511.475% of nominalAbbreviations:CVCoefficient of variation.LOQ=0.00065 mg a.i./LE. ObservationsBiological Endpoints:Possible clinical/behavioral signs included, but were not limited to, hyperventilation, loss of equilibrium, uncoordinatedswimming, atypical quiescence, or feeding abstinence. Other endpoints, monitored daily and/or determined at studytermination, included survival (M/F, daily), overall survival (M/F, Day 21), wet weight (M/F, Day 21), standard length(M/F, Day 21), GSI (M/F, Day 21), fecundity (F, daily, overall/reproduction day), fertility (daily and overall), nuptialtubercle score (M, Day 21), and plasmaVTG concentration (M/F, Day 21).Were raw (individual) data provided?YesData Evaluation Record on the Fish Short-Term Reproduction Assay with TebuconazoleEPA MRID Number 48688301Page22Rf41Version: 22 September 2011 EPA recommends that observations of survival, fecundity, fertilization success, secondary sex characteristics, and other clinic al signs occur at least daily. At test termination (Day 21), additional observations include body weight and length, nuptial tubercle score, gonadal staging and histo pathology, plasma VTG and plasma sex

steroids (testosterone and 17ȕ -estradiol, if measured). GSI is calculated using a ratio of gonad weight to body weight (gonad weight to the nearest 0.1 mg / body

weight in mg x 100) at test termination. Clinical signs of overt toxicity may include (but are not limited to) hemorrhage, cessation of feeding, and other abnormal beha vior.

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### Version: 22 September 2011 II. RESULTS AND DISCUSSION

A. Results



### Male and female survival was evaluated at test termination on Study Day 21. Percent survival ranged from 75.0 to 100% in the males and 93.8 to 100% in the females (Table 9). Overall survival was considered to be 100% in the negative control (one female was inadvertently killed by the technician),
and 88, 92, and 96%, respectively, in the 0.00497, 0.0113, and 0.0195 mg a.i./L.

Table 9: Adult Fish Survival in Fathead Minnow ( Pimephales promelas ).

Treatment (mg a.i./L)
[mean-measured] Males Females

n # Surviving % Survival n # Surviving % Survival

Water control (<LOQ) 8 8 100 16 15 1 100

0.00497 8 6 75.0 16 15 93.8

0.0113 8 7 87.5 16 15 93.8

0.0195 8 8 100 16 15 93.8

Data were obtained from page 46 in the study report.

1 One female was inadvertently killed while moving substrates during egg counting. The study authors considered
survival rate to be 100%. LOQ=0.00065 mg a.i./L.



### Male and female body weight and length were evaluated at test termination on Study Day 21. At meanmeasured tebuconazole concentrations of <LOQ (water control), 0.00497, 0.0113, and 0.0195 mg
a.i./L, the respective mean body weight values were 5.06, 5.62, 5.21, and 5.50 g for the males and 2.01, 2.06, 1.79, and 1.91 g for the females (Table 10) . At mean-measured tebuconazole concentrations of <LOQ (water control), 0.00497, 0. 0113 and 0.0195 mg a.i./L, the respective mean body length values were 57.9, 59.8, 57.6, and 59.4 mm for males and 43.5, 43.9, 42.8, and 43.8

mm for females.

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### Version: 22 September 2011 Table 10: Size at Test Termination in Fathead Minnow ( Pimephales promelas ).

Treatment (mg a.i./L)

[meanmeasured] Body Weight Length

Males Females Males Females

n Mean (g) ±SD n Mean (g) ±SD n Mean (mm) ±SD n Mean (mm) ±SD

Water control (<LOQ) 4 5.06 0.43 16 2.01 0.18 4 57.9 1.64 16 43.6 0.69

0.00497 4 5.62 0.53 16 2.06 0.15 4 59.8 2.02 16 43.9 0.70

0.0113 4 5.21 0.70 16 1.79 0.11 4 57.6 3.52 16 42.8 0.76

0.0195 4 5.50 0.86 16 1.91 0.16 4 59.3 1.65 16 43.8 1.3

Abbreviations: SD Standard deviation. Data were obtained from page 47 in the study report. LOQ=0.00065 mg a.i./L.



### Mean fecundity values were 23.7, 21.8, 28.9, and 30.0 and fertilization success was 97.6, 98.1, 97.2, and 98.6% at the mean-measured tebuconazole concentrations of <LOQ (water control), 0.00497, 0.0113, and 0.0195 mg a.i./L, respectively (Table 11).

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### Version: 22 September 2011 Table 11: Fecundity and Fertilization Success in Fathead Minnow ( Pimephales promelas ).

Treatment (mg a.i./L)

[mean-measured] Fecundity (# eggs/female/day) 1 Fertilization Success (%) 2

mean SD mean SD

Water control (<LOQ) 23.7 8.0 97.6 1.2

0.00497 21.8 4.0 98.1 0.83

0.0113 28.9 5.8 97.2 0.54

0.0195 30.0 20 98.6 0.92

Data were obtained from page 49 in the study report.

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 em bryos divided by the number of eggs, multiplied by 100. LOQ=0.00065 mg a.i./L.



### The nuptial tubercles of males were counted, mapped and scored at test termination on Study Day 21. Median male tubercle scores were 38, 44, 39 and 43 in the mean-measured <LOQ (water control),
0.00497, 0.0113, and 0.0195 mg a.i./L treatme nt levels, respectively (Table 12).

Table 12: Nuptial Tubercle Score in Fathead Minnow ( Pimephales promelas ).

Treatment (mg a.i./L)

[mean-measured] Males Females

n Median Tubercle

Score 1 n Median Tubercle

Score 1

Water control (<LOQ) 4 38 4 0

0.00497 4 44 4 0

0.0113 4 39 4 0

0.0195 4 43 4 0

Abbreviations: SD Standard deviation.

1 As per Ankley et. al. 2001. LOQ=0.00065 mg a.i./L.

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### Version: 22 September 2011 The male and female GSI values were evaluated at test termination on Study Day 21 after removal and weighing of the gonads. At mean-measured tebuconazole concentrations of <LOQ (water control),
0.00497, 0.0113, and 0.0195 mg a.i./L, the respecti ve mean GSI values were 1.58, 1.68, 1.26, and 1.28% for males and 17.7, 18.3, 16.4, and 14.5% for females (Table 13).

Table 13: Gonado-Somatic Index (GSI) in Fathead Minnow ( Pimephales promelas ).

Treatment (mg a.i./L)

[mean-measured] Males Females

n Median

GSI 1,2 (%) ±SD n Median GSI 2
(%) ±SD

Water control (<LOQ) 4 1.58 0.22 4 17.7 4.03

0.000497 4 1.68 0.64 4 18.3 2.61

0.0113 4 1.26 0.21 4 16.4 1.98

0.0195 4 1.28 0.44 4 14.5 2.77

Abbreviations: SD Standard deviation. Data were obtained from page 48 in the study report.

1 Median is calculated because n=2 males/tank (replicate) and 4 females/tank (replicate).

2 Gonado-somatic index (%) is calculated as gonad weight (to the nearest 0.1 mg)/body weight (mg) x 100.
LOQ=0.00065 mg a.i./L.



### Preserved gonads (testes or ovaries, left and right) from a total of 89 fathead minnows were submitted
for histopathologic processing and evaluation. Median male gonadal stage was 3, 3, 2, and 2 and median female gonadal stage was 3, 4, 4, and 3 in the mean-measured <LOQ (water control),
0.00497, 0.0113, and 0.0195 mg a.i./L treatme nt levels, respectively (Table 14).

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### Version: 22 September 2011 Table 14: Gonadal Staging in Fathead Minnow ( Pimephales promelas ).

Treatment (mg a.i./L)

[mean-measured] Males Females

N Median Stage 1 n Median Stage 2

Water control (<LOQ) 4 3 4 3

0.00497 4 3 4 4

0.0113 4 2 4 4

0.0195 4 2 4 3

Data were obtained from pages 143-144 in the study report.

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=0.00065 mg a.i./L.



### According to the pathologist’s report, no histopathologic findings observed in the testes of the male fathead were clearly related to tebuconazole treatment (Table 15 and 16). The mean testicular stage
scores were decreased in the 0.0113 and 0.0195 mg a.i./L males compared to the controls, but the study authors report this decrease is unlikely to be biologically meaningful. In the ovary of the female fathead, the histopathologic finding of increased granulomatous inflammation (minimal to mild) in the
0.0195 mg a.i./L group compared to the controls was most likely related to tebuconazole treatment (Table 17 and 18). Additional histopathologic findings for females included increased prevalence of
post-ovulatory follicles and decreased prevalence of oocyte atresia in the tebuconazole-treated groups, neither which were reported as potentially treatment-related by the pathologist; in the controls 47% of the examined females were reported to have some level of increased oocyte atresia (2 fish with minimal and 5 fish with moderate increased atresia) whereas the low, middle and high treatment had 40, 20, and
20%, respectively. The mean ovarian stage scores we re decreased in the 0.01 95 mg a.i./L females compared to the controls (2.9 vs. 3.2), but the study authors report this decrease is unlikely to be
biologically meaningful. Histological examination of the liver was also conducted with no substantially more prevalent findings for treated males compared to the control. However, for males, the occasional

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### Version: 22 September 2011 findings of microgranulomas (minimal; mid and high treatment groups) and individual hepatocyte necrosis (minimal; high treatment group) in the livers were likely treatment related; there was also a
concentration-related decrease in hepatocyte vacuolation compared to control. In females, generally concentration-dependent findings in the liver included individual hepatocyte necrosis (occurred in all three treatment groups), multinucleated hepatocytes (mid and high treatment groups), microgranulomas (mid and high treatment groups), hepatocyte megalocytosis (mid and high treatment groups), and decreased hepatocellular vacuolation (all groups). Plasma sex steroids were not measured in this study.

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### Version: 22 September 2011 Table 15: Gonadal Histopathology in Male Fathead Minnow ( Pimephales promelas ).

Treatment (mg a.i./L)
[mean-measured] Diagnostic Observations 1

Severity Increased Proportion of Spermatogonia Presence of

Testis-Ova Increased

Testicular Degeneration Interstitial Cell Hypertrophy/ Hyperplasia

n Incidence n Incidence n Incidence n Incidence

Water control (<LOQ) 0 8 8 8 7 8 8 8 8

1 8 0 8 1 8 0 8 0

2 8 0 8 0 8 0 8 0

3 8 0 8 0 8 0 8 0

4 8 0 8 0 8 0 8 0

0.00497 0 6 5 6 6 6 6 6 5

1 6 1 6 0 6 0 6 1

2 6 0 6 0 6 0 6 0

3 6 0 6 0 6 0 6 0

4 6 0 6 0 6 0 6 0

0.0113 0 7 7 7 7 7 7 7 6

1 7 0 7 0 7 0 7 1

2 7 0 7 0 7 0 7 0

3 7 0 7 0 7 0 7 0

4 7 0 7 0 7 0 7 0

0.0195 0 8 7 8 8 8 6 8 6

1 8 0 8 0 8 1 8 1

2 8 1 8 0 8 1 8 1

3 8 0 8 0 8 0 8 0

4 8 0 8 0 8 0 8 0
Data were obtained from pages 157-160 in the study report.

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=0.00065 mg a.i./L.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TebuconazoleEPA MRID Number 48688301Page30Rf41Version: 22 September 2011Table 16: Additional Gonadal Histopathology Observations in Male Fathead Minnow ( Pimephales promelas).Treatment (mg a.i./L)[mean-measured]Additional Diagnostic Observations1SeverityInterstitial FibrosisDecreasedProportion ofSpermatozoaAsynchronousGonadDevelopmentMineralizationMineralization in theCollecting DuctsnIncidencenIncidencenIncidencenIncidencenIncidenceWater control (<LOQ) 0 8 0 8 7 8 7 8 2 8 21 8 0 8 1 8 0 8 5 8 12 8 0 8 0 8 1 8 1 8 43 8 0 8 0 8 0 8 0 8 14 8 0 8 0 8 0 8 0 8 00.004970 6 4 6 6 6 5 6 4 6 51 6 1 6 0 6 1 6 2 6 02 6 1 6 0 6 0 6 0 6 13 6 0 6 0 6 0 6 0 6 04 6 0 6 0 6 0 6 0 6 00.01130 7 6 7 6 7 7 7 7 7 41 7 0 7 0 7 0 7 0 7 02 7 1 7 1 7 0 7 0 7 33 7 0 7 0 7 0 7 0 7 04 7 0 7 0 7 0 7 0 7 0Data Evaluation Record on the Fish Short-Term Reproduction Assay with TebuconazoleEPA MRID Number 48688301Page31Rf41Version: 22 September 2011Treatment (mg a.i./L)[mean-measured]Additional Diagnostic Observations1SeverityInterstitial FibrosisDecreasedProportion ofSpermatozoaAsynchronousGonadDevelopmentMineralizationMineralization in theCollecting DuctsnIncidencenIncidencenIncidencenIncidencenIncidence0.01950 8 5 8 8 8 8 8 4 8 41 8 3 8 0 8 0 8 4 8 32 8 0 8 0 8 0 8 0 8 13 8 0 8 0 8 0 8 0 8 04 8 0 8 0 8 0 8 0 8 0Data were obtained from pages 157-160 in the study report.1Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E ofthe test guideline for reference.LOQ=0.00065 mg a.i./L.

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### Version: 22 September 2011 Table 17: Gonadal Histopathology in Female Fathead Minnow ( Pimephales promelas ).

Treatment (mg a.i./L)

[mean-measured] Diagnostic Observations 1

Severity Increased Oocyte

Atresia Perifollicular Cell Hyperplasia/ Hypertrophy Decreased Yolk

Formation

n Incidence n Incidence n Incidence
Water control (<LOQ) 0 15 8 15 15 15 NA

1 15 2 15 0 15 NA

2 15 0 15 0 15 NA

3 15 5 15 0 15 NA

4 15 0 15 0 15 NA

0.00497 0 15 9 15 15 15 NA

1 15 0 15 0 15 NA

2 15 1 15 0 15 NA

3 15 5 15 0 15 NA

4 15 0 15 0 15 NA

0.0113 0 15 12 15 14 15 NA

1 15 2 15 1 15 NA

2 15 0 15 0 15 NA

3 15 1 15 0 15 NA

4 15 0 15 0 15 NA

0.0195 0 15 12 15 15 15 NA

1 15 2 15 0 15 NA

2 15 0 15 0 15 NA

3 15 1 15 0 15 NA

4 15 0 15 0 15 NA
Data were obtained from pages 161-165 in the study report.

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=0.00065 mg a.i./L.

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### Version: 22 September 2011 Table 18: Additional Gonadal Histopathology Observations in Female Fathead Minnow ( Pimephales promelas ).

Treatment (mg a.i./L)

[meanmeasured] Additional Diagnostic Observations 1

Severity Spermatogenesis Microsporidia

Infection Granulomatous

Inflammation Post-Ovulatory

Follicles

n Incidence n Incidence n Incidence n Incidence

Water control (<LOQ) 0 15 15 15 15 15 11 15 12

1 15 0 15 0 15 3 15 2

2 15 0 15 0 15 1 15 0

3 15 0 15 0 15 0 15 1

4 15 0 15 0 15 0 15 0

0.00497 0 15 15 15 14 15 12 15 10

1 15 0 15 1 15 1 15 2

2 15 0 15 0 15 2 15 1

3 15 0 15 0 15 0 15 2

4 15 0 15 0 15 0 15 0

0.0113 0 15 15 15 14 15 10 15 8

1 15 0 15 0 15 5 15 2

2 15 0 15 1 15 0 15 0

3 15 0 15 0 15 0 15 5

4 15 0 15 0 15 0 15 0

0.0195 0 15 14 15 14 15 8 15 6

1 15 1 15 0 15 6 15 3

2 15 0 15 1 15 1 15 2

3 15 0 15 0 15 0 15 4

4 15 0 15 0 15 0 15 0
Data were obtained from pages 161-165 in the study report.

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=0.00065 mg a.i./L.

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### Version: 22 September 2011 The plasma concentrations of VTG were evaluated in males and females at test termination on Study Day 21. At mean-measured tebuconazole concentrations of <LOQ (water control), 0.00497, 0.0113,
and 0.0195 mg a.i./L, the respective mean VTG levels were 785, 1628, 945, and 926 ng/mL plasma

in males and 2.99×10 6 

### , 2.49×10 6 

### , 1.94×10 6 

### and 1.34×10 6 

### ng/mL plasma in females (Table 19).

Testosterone and 17 ȕ -estradiol (sex steroid) concentrations were not determined.

Table 19: Plasma Vitellogenin in Fathead Minnow ( Pimephales promelas ).

Treatment (mg a.i./L)
[mean-measured] Plasma Vitellogenin (VTG)

Males Females

n Mean (ng/mL plasma) ±SD n Mean (ng/mL plasma) ±SD

Negative control (<LOQ) 4 785 489 4 2.99×10 6 4.98x10 5

0.00497 4 1628 744 4 2.49×10 6 2.31x10 5

0.0113 4 945 349 4 1.94×10 6 3.92x10 5

0.0195 4 926 608 4 1.34×10 6 7.95x10 5

Data were obtained from page 51 in the study report.

Abbreviations: SD Standard deviation. LOQ=0.00065 mg a.i./L.



### Behavioral characteristics in male and female minnows ( e.g. , hyperventilation, loss of equilibrium, uncoordinated swimming, atypical quiescence, or feeding abstinence) were documented daily.
Secondary sex characteristics and alterations in r eproductive behavior were observed daily. For males, two males at 0.00497 mg a.i/L were observed to be quiescent at the surface prior to their death, and one male at 0.0113 mg a.i./L, was quiescent and not territorial on Day 5 (Table 20). Temporary vertical bands and territorial behavior (normally male secondary sex characteristics) were observed for
one control female, three 4.97 ս g a.i./L females, and one 19.5 ս g a.i./L female. Additional atypical

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### Version: 22 September 2011 behaviors included one control female with swollen belly and one 4.97 ս g a.i./L female with exophthalmos (bulging of the eye anteriorly out of the orbit).

Table 20: Secondary Sex Characteristics and Clinical Signs in Fathead Minnow ( Pimephales promelas ).

Treatment (mg a.i./L)

[meanmeasured] Secondary Sex Characteristics and Clinical Signs

Males Females

Type n Incidence Type n Incidence

Water control (<LOQ) Vertical bands 8 8 No vertical bands 16 15
Territorial aggressiveness 8 8 No territorial aggressiveness 16 15
Nape pad present 8 8 Ovipositor present 16 16

Clinical signs 8 0 Swollen belly 16 1

0.00497 Vertical bands 8 6 No vertical bands 16 13
Territorial aggressiveness 8 6 No territorial aggressiveness 16 13
Nape pad present 8 6 Ovipositor present 16 16

Quiescent 8 2 Exophthalmos 16 1

0.0113 Vertical bands 8 7 No vertical bands 16 16
Territorial aggressiveness 8 7 No territorial aggressiveness 16 16
Nape pad present 8 7 Ovipositor present 16 16
Quiescent 8 1 Clinical signs 16 0

0.0195 Vertical bands 8 8 No vertical bands 16 15
Territorial aggressiveness 8 8 No territorial aggressiveness 16 15
Nape pad present 8 8 Ovipositor present 16 16

Clinical signs 8 0 Clinical signs 16 0
Data were obtained from page 50 in the study report. LOQ=0.00065 mg a.i./L.



### B. Study Author’s Analysis and Conclusions:

The observed clinical/behavioral signs included quiesc ence, males not territorial, females territorial, and females with vertical bands. Other endpoints, monitored daily and/or determined at study termination,

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### Version: 22 September 2011 included survival (M/F, daily), overall survival (M/F, Day 21), wet weight (M/F, Day 21), standard length (M/F, Day 21), GSI (M/F, Day 21), fecundity (F , daily, overall/reproduction day), fertility (daily and overall), nuptial tubercle score (M, Day 21), and plasma VTG concentration (M/F, Day 21).

Biological data were statistically analyzed following procedures outlined in the EPA Guideline for this assay. Statistical analyses were conducted at the p  0.05 level for the experimental data collected during the study. The following procedures were used: 1) The Shapiro-Wilks' Test was used to test for normality and the Bartlett’s test was used to test for homogeneity of variance; 2) If the results were consistent with a monotonic concentration response, the Jonckheere-Terpstra Step-Down Test (1-tailed)
was used with replicate medians for all continuous quantitative endpoints. If the endpoints did not show a monotonic trend, and passed the assessments for normality and variance homogeneity, Dunnett’s test (2-tailed) was used with replicate means. If the data failed the normality test, a non-parametric procedure was used for analyses; 3) Dichotomous data ( e.g. , mortality) were analyzed by using Fisher’s Exact test with a Bonferroni-Holm adjustment; 4) Tubercle scores were analyzed by using replicate
medians with the Jonckheere-Terpstra Step-Down test.

No significant differences in mortality or concentration-related sublethal effects were observed in the tebuconazole treatment groups. The significant tebuconazole-related effects included decreased female

GSI at 19.5 ս g a.i./L, increased male tubercle score at 19.5 ս g a.i./L, and decreased female plasma

VTG at 11.3 and 19.5 ս g a.i./L. Significant findings were observed for female gonadal staging, female gonad histopathology, and female liver histopathology. No treatment-related effects were observed on growth and reproduction. The effects observed in the females are most likely related to the hepatocyte necrosis observed in the tebuconazole treatment groups. This effect would have subsequent effects on VTG levels, and lead to the GSI and gonad staging score effects for females.

C. Reviewer’s Analysis and Conclusions:

Female and male GSI and female VTG exhibited a monotonic response and were therefore analyzed using the Jonckheere-Terpstra Step Down Test. All other endpoints were analyzed for normality (Shapiro-Wilks, Į = 0.01) and homogeneity of variance (Bartlett’s Test, Į = 0.01). Fecundity, female
and male body weight, female and male length, and male VTG met these assumptions and were

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

EPA MRID Number 48688301

Page 37 R f 41 

### Version: 22 September 2011analyzed using Dunnett’s Multiple Comparison Test. Male tubercle score was analyzed using MannWhitney. All analyses were conducted using CETIS TM 

### Version 1.8.7.4 and the backend database settings approved by EFED on 1/30/2013. Unless otherwise indicated, effects were considered statistically significant at p <0.05.

Conclusions: Male and female Fathead minnows (n=2 and 4, respectively/replicate; 4 replicates/treatment group) were exposed to mean-measured concentrations of tebuconazole of <0.00065 (<LOQ, water control), 0.00497, 0.0113, or 0.0195 mg a.i./L for 21 days in a short-term reproduction assay. There was a significant (p<0.05; Jonckheere-Terpstra) decrease of 18.5% in female GSI at 0.0195 mg a.i./L and there was a marginally significant (p=0.0561; Jonckheere-Terpstra)
decrease of 19% on male GSI at 0.0195 mg a.i./L. There was a significant decrease of 35 and 55% in female plasma VTG at 0.0113 and 0.0195 mg a.i./L (p<0.05; Jonckheere-Terpstra). There were no
other significant differences for any other endpoint.

According to the pathologist’s report, no histopathologic findings observed in the testes of the male fathead were clearly related to tebuconazole treatment. In the ovary of the female fathead, the histopathologic finding of increased granulomatous inflammation (minimal to mild) in the 0.0195 mg
a.i./L group compared to the controls was most likely related to tebuconazole treatment. Histological examination of the liver was also conducted with no substantially more prevalent findings for treated males compared to the control. However, for males, the occasional findings of microgranulomas (minimal) and individual hepatocyte necrosis (minimal) in the livers were likely treatment related; there
was also a concentration-related decrease in hepatocyte vacuolation compared to control. In females, generally concentration-dependent findings in the liver included individual hepatocyte necrosis (occurred in all three treatment groups), multinucleated hepatocytes, microgranulomas, hepatocyte megalocytosis,
and decreased hepatocellular vacuolation. Plasma se x steroids were not measured in this study.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TebuconazoleEPA MRID Number 48688301Page38Rf41Version: 22 September 2011Table 21: Reproductive and HPG Endpoints1,2for Male Fathead Minnow ( Pimephales promelas) in the FSTRA with Tebuconazole.Treatment(mg a.i./L)[meanmeasured]Tubercle ScoreGSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2Medianp% Diff.pEffect?(Yes/No)% Diff.p% Diff.P% Diff.pNegative control(<LOQ)38 NA NA NA NA NA NA NA NA NA NA0.00497 44 1.0 6.16 0.80 No 107 0.16 NA NA NA NA0.0113 39 0.66 -20.4 0.14 No 20.4 0.95 NA NA NA NA0.0195 43 0.97-19.3 0.056No 18.0 0.97 NA NA NA NAStatistical Test Mann-Whitney Jonckheere-Terpstra NADunnettNANAAbbreviations:Diff.Difference.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.NSNot significant.VTGVitellogenin.TTestosterone.E217ȕ-estradiol.1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusions regarding histopathologymay be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p0.05.LOQ=0.00065 mg a.i./L.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TebuconazoleEPA MRID Number 48688301Page39Rf41Version: 22 September 2011Table 22: Reproductive and HPG Endpoints1,2for Female Fathead Minnow ( Pimephales promelas) in the FSTRA with Tebuconazole.Treatment(mg a.i./L)[measured]FecundityFert. SuccessTubercle ScoreGSIGonadalStagingand Histo.Plasma VTGPlasma TPlasma E2% Diff.p% Diff.pMedianP% Diff.pEffect?(Yes/No)% Diffp% Diffp% Diff.pNegative control(<LOQ)NA NA NA NA 0 NA NA NA NA NA NA NA NA NA NA0.00497 -8.1 0.99 0.54 0.76 0 1.0 2.96 0.66 No -16.7 0.17 NA NA NA NA0.0113 21.8 0.86-0.380.89 0 1.0 -7.62 0.26 No-35.1 0.004NA NA NA NA0.0195 26.0 0.79 1.0 0.34 0 1.0-18.5 0.042 Yes -55.0 <0.0001NA NA NA NAStatistical Test Dunnett DunnettMann-WhitneyUJonckheereTerpstraNA Jonckheere-Terpstra NA NAAbbreviations:Diff.Difference.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.NSNot significant.VTGVitellogenin.TTestosterone.E217ȕ-estradiol.1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusions regardinghistopathology may be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p0.05.LOQ=0.00065 mg a.i./L.

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

EPA MRID Number 48688301

Page 40 R f 41 

### Version: 22 September 2011 Table 23: Growth Endpoints 1,2 

### in the Fish Short-Term Reproduction Assay (FSTRA) with Tebuconazole.

Treatment (mg a.i./L)

[meanmeasured] Body Weight Length

Males Females Males Females

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

Negative control (<LOQ) NA NA NA NA NA NA NA NA

0.00497 11.2 0.57 2.36 0.95 3.19 0.63 0.8 0.90

0.0113 2.97 0.98 -10.9 0.15 -0.60 0.99 -1.66 0.55

0.0195 8.7 0.68 -5.09 0.67 2.42 0.75 0.57 0.96

Statistical Test Dunnett Dunnett Dunnett Dunnett

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. LOQ=0.00065 mg a.i./L.



### E. Study Deficiencies

Although there were minor deviations from the guideline, as noted in the Materials and Methods section of the DER, the study met all validity and performance criteria

F. Reviewer’s Comments:

The reviewer’s results were generally comparable to those of the study author. The reviewer and study author both reported a significant difference in female GSI at 0.0195 mg a.i./L, and a significant decrease in female VTG at the mid and high treatment. The reviewer found a significant decrease in male GSI, whereas the study author did not detect a significant decrease but reported that GSI was decreased compared to controls. The study author reported a significant increase in male tubercle score, but attributed it to larger male size, whereas

the reviewer did not detect a difference.

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

EPA MRID Number 48688301

Page 41 R f 41 

### Version: 22 September 2011 According to the histopathologic findings for females, in the controls 47% of the examined females were reported to have some level of increased oocyte atresia (2 fish with minimal and 5 fish with moderate increased atresia) whereas the low, middle and high treatment had 40, 20, and 20%, respectively. Granulomatous
inflammation was also reported in the female ovaries and in two instances this inflammation was spatially associated with microsporidian infection, however an etio logic agent was not apparent in the majority of cases (pathologist’s report). The fecundity rates in the control replicates did meet the minimum requirement of 15
eggs/female/day.

III. REFERENCES

Ankley, G.T., K.J. Jensen, M.D. Kahl, J.J Korte, and E.A. Makynen (2001). Description and evaluation of a short-term reproduction test with the fathead minnow ( Pimephales promelas ). Environ Toxicol Chem 20:

1276-1290.

Bomke, C. (2007). Tebuconazole – Fish Sexual Development Test (FSDT) with Fathead Minnow. Bayer
CropScience AG. Unpublished report number EBHWX264.

Hazardous Substances Data Bank, National Library of Medicine, ToxNet (2014)
http://toxnet.nlm.nih.gov.

Surprenant, D.C. (1988). The Toxicity of HWG 1608 Technical to Rainbow Trout ( Salmo gairdneri )
Embryos and Larvae. Springborn Life Sciences, Inc. Unpublished report number 87-11-2545.Report Date: 06 Jun-15 10:11 (p 1 of 18) Test Code: 128997 48688301 | 14-7456-2471 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Bayer CropScience AG

Analyzed: 29 May-14 15:11 Endpoint: Fecundity CETIS Version: CETISv1.8.7 Analysis ID: 18-2191-0972 Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 07-9757-7599 Start Date: 19 Jan-12 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Brine: Species: Pimephales promelas Source: Analyst: J. Housenger

Age:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C- μ g ai/L Dunnett Multiple Comparison Test

DF P-Type
0.239 2.68 21.6 0.9901 Non-Significant Effect Negative Control 4.97 6 CDF 0.642 2.68 21.6 0.8574 Non-Significant Effect 11.3 6 CDF 0.766 2.68 21.6 0.7869 Non-Significant Effect 19.5 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed
19.5 >19.5 91.0% NA C <> T NA Untransformed NA NA

ANOVA Table
Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%) Between 185.6369 61.87896 3 0.476 0.7046 Non-Significant Effect Error 1558.893 129.9077 12 1744.529 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
7.97 11.3 0.0467 Equal Variances Bartlett Equality of Variance Variances 0.881 0.841 0.0406 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C- μ g ai/L CV% %Effect 95% LCL 95% UCL Fecundity Summary

23.7 15.7 31.9 23.7 0 4 Negative Control 3.97 33.5% 0.0% 11.1 36.4 21.8 18.5 27.6 20.6 4.97 4 2.01 18.5% 8.11% 15.4 28.2 28.9 21.9 35.8 29 11.3 4 2.88 19.9% -21.8% 19.8 38.1 29.9 13.9 59.4 23.2 19.5 4 10.1 67.4% -26.0% -2.18 62CenteredUntransformed

Rankits -5

-10

-15

-20 0 5 10 15 20 25 30

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0Fecundity

C- μ g ai/L Reject Null

Reject Null
0 10 20 30 40 50 60

0 N 4.97 11.3 19.5 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Jun-15 10:11 (p 2 of 18) Test Code: 128997 48688301 | 14-7456-2471 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Bayer CropScience AG

Analyzed: 29 May-14 15:11 Endpoint: FemaleBodyWt CETIS Version: CETISv1.8.7 Analysis ID: 18-4021-8427 Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 07-9757-7599 Start Date: 19 Jan-12 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Brine: Species: Pimephales promelas Source: Analyst: J. Housenger

Age:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C- μ g ai/L Dunnett Multiple Comparison Test

DF P-Type
0.44 2.68 0.29 0.9453 Non-Significant Effect Negative Control 4.97 6 CDF 2.04 2.68 0.29 0.1514 Non-Significant Effect 11.3 6 CDF 0.95 2.68 0.29 0.6705 Non-Significant Effect 19.5 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed
19.5 >19.5 14.4% NA C <> T NA Untransformed NA NA

ANOVA Table
Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%) Between 0.169025 0.05634166 3 2.42 0.1169 Non-Significant Effect Error 0.27955 0.02329583 12 0.448575 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
0.55 11.3 0.9079 Equal Variances Bartlett Equality of Variance Variances 0.952 0.841 0.5184 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C- μ g ai/L CV% %Effect 95% LCL 95% UCL FemaleBodyWt Summary

2.01 1.83 2.24 1.99 0 4 Negative Control 0.0886 8.79% 0.0% 1.73 2.3 2.06 1.88 2.25 2.06 4.97 4 0.0755 7.33% -2.36% 1.82 2.3 1.79 1.64 1.9 1.82 11.3 4 0.0563 6.28% 10.9% 1.62 1.97 1.91 1.69 2.07 1.94 19.5 4 0.0811 8.48% 5.09% 1.65 2.17CenteredUntransformed

Rankits -0.05

-0.10

-0.15

-0.20

-0.25 0.00 0.05 0.10 0.15 0.20 0.25

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0FemaleBodyWt

C- μ g ai/L Reject Null

Reject Null

0.0 0.5 1.0 1.5 2.0 2.5

0 N 4.97 11.3 19.5 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Jun-15 10:11 (p 3 of 18) Test Code: 128997 48688301 | 14-7456-2471 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Bayer CropScience AG

Analyzed: 29 May-14 15:11 Endpoint: FemaleGSI CETIS Version: CETISv1.8.7 Analysis ID: 15-5144-0500 Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 07-9757-7599 Start Date: 19 Jan-12 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Brine: Species: Pimephales promelas Source: Analyst: J. Housenger

Age:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C- μ g ai/L Dunnett Multiple Comparison Test

DF P-Type
0.252 2.68 5.58 0.9885 Non-Significant Effect Negative Control 4.97 6 CDF 0.649 2.68 5.58 0.8539 Non-Significant Effect 11.3 6 CDF 1.57 2.68 5.58 0.3109 Non-Significant Effect 19.5 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed
19.5 >19.5 31.5% NA C <> T NA Untransformed NA NA

ANOVA Table
Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%) Between 34.48499 11.495 3 1.33 0.3113 Non-Significant Effect Error 103.915 8.659584 12 138.4 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
1.39 11.3 0.7075 Equal Variances Bartlett Equality of Variance Variances 0.923 0.841 0.1889 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C- μ g ai/L CV% %Effect 95% LCL 95% UCL FemaleGSI Summary

17.7 14.2 22.1 17.3 0 4 Negative Control 2.02 22.7% 0.0% 11.3 24.1 18.3 15.1 21.4 18.3 4.97 4 1.31 14.3% -2.96% 14.1 22.4 16.4 14.1 18.7 16.4 11.3 4 0.988 12.1% 7.62% 13.2 19.5 14.5 10.9 16.7 15.1 19.5 4 1.38 19.2% 18.5% 10 18.9CenteredUntransformed

Rankits -1

-2

-3

-4 0 1 2 3 4 5

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0FemaleGSI

C- μ g ai/L Reject Null

Reject Null

0 5 10 15 20 25

0 N 4.97 11.3 19.5 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Jun-15 10:11 (p 4 of 18) Test Code: 128997 48688301 | 14-7456-2471 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Bayer CropScience AG

Analyzed: 29 May-14 15:15 Endpoint: FemaleGSI CETIS Version: CETISv1.8.7 Analysis ID: 00-8662-8358 Analysis: Nonparametric-Control vs Ord. Treatments Official Results: Yes

Batch ID: 07-9757-7599 Start Date: 19 Jan-12 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Brine: Species: Pimephales promelas Source: Analyst: J. Housenger

Age:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C- μ g ai/L Jonckheere-Terpstra Step-Down Test

DF P-Type
7 NA 0.6571 Non-Significant Effect Negative Control 4.97 -2 Exact 29 NA 0.2602 Non-Significant Effect 11.3 -2 Exact 67 NA 0.0420 Significant Effect 19.5* -2 Exact NOEL LOEL TU TOEL Alt Hyp Data Transform Zeta Trials Seed
11.3 19.5 14.84 C > T NA Untransformed NA NA

ANOVA Table
Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%) Between 34.48499 11.495 3 1.33 0.3113 Non-Significant Effect Error 103.915 8.659584 12 138.4 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
1.39 11.3 0.7075 Equal Variances Bartlett Equality of Variance Variances 0.923 0.841 0.1889 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C- μ g ai/L CV% %Effect 95% LCL 95% UCL FemaleGSI Summary

17.7 14.2 22.1 17.3 0 4 Negative Control 2.02 22.7% 0.0% 11.3 24.1 18.3 15.1 21.4 18.3 4.97 4 1.31 14.3% -2.96% 14.1 22.4 16.4 14.1 18.7 16.4 11.3 4 0.988 12.1% 7.62% 13.2 19.5 14.5 10.9 16.7 15.1 19.5 4 1.38 19.2% 18.5% 10 18.9CenteredUntransformed

Rankits -1

-2

-3

-4 0 1 2 3 4 5

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0FemaleGSI

C- μ g ai/L 0 5 10 15 20 25

0 N 4.97 11.3 19.5 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Jun-15 10:11 (p 5 of 18) Test Code: 128997 48688301 | 14-7456-2471 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Bayer CropScience AG

Analyzed: 29 May-14 15:11 Endpoint: FemaleLength CETIS Version: CETISv1.8.7 Analysis ID: 17-8311-6742 Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 07-9757-7599 Start Date: 19 Jan-12 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Brine: Species: Pimephales promelas Source: Analyst: J. Housenger

Age:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C- μ g ai/L Dunnett Multiple Comparison Test

DF P-Type
0.547 2.68 1.72 0.9036 Non-Significant Effect Negative Control 4.97 6 CDF 1.13 2.68 1.72 0.5518 Non-Significant Effect 11.3 6 CDF 0.391 2.68 1.72 0.9603 Non-Significant Effect 19.5 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed
19.5 >19.5 3.94% NA C <> T NA Untransformed NA NA

ANOVA Table
Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%) Between 2.826873 0.942291 3 1.15 0.3680 Non-Significant Effect Error 9.812502 0.8177085 12 12.63937 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
1.75 11.3 0.6252 Equal Variances Bartlett Equality of Variance Variances 0.974 0.841 0.8987 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C- μ g ai/L CV% %Effect 95% LCL 95% UCL FemaleLength Summary

43.6 43.1 44.6 43.3 0 4 Negative Control 0.345 1.58% 0.0% 42.5 44.7 43.9 43.1 44.5 44 4.97 4 0.347 1.58% -0.8% 42.8 45 42.8 41.8 43.6 43 11.3 4 0.38 1.77% 1.66% 41.6 44.1 43.8 42.6 45.6 43.5 19.5 4 0.659 3.01% -0.57% 41.7 45.9CenteredUntransformed

Rankits -0.2

-0.4

-0.6

-0.8

-1.0

-1.2

-1.4 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0FemaleLength

C- μ g ai/L Reject Null

Reject Null

0 10 20 30 40 50

0 N 4.97 11.3 19.5 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Jun-15 10:11 (p 6 of 18) Test Code: 128997 48688301 | 14-7456-2471 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Bayer CropScience AG

Analyzed: 29 May-14 15:11 Endpoint: FemaleMedianTubercleScore CETIS Version: CETISv1.8.7 Analysis ID: 13-5421-2809 Analysis: Nonparametric-Two Sample Official Results: Yes

Batch ID: 07-9757-7599 Start Date: 19 Jan-12 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Brine: Species: Pimephales promelas Source: Analyst: J. Housenger

Age:

Test Stat Critical P-Value Decision( α :5%) Ties vs Control C- μ g ai/L Mann-Whitney U Two-Sample Test

DF P-Type
8 NA 1 1.0000 Non-Significant Effect Negative Control 4.97 6 Exact 8 NA 1 1.0000 Non-Significant Effect 11.3 6 Exact 8 NA 1 1.0000 Non-Significant Effect 19.5 6 Exact NOEL LOEL TU TOEL Alt Hyp Data Transform Zeta Trials Seed
19.5 >19.5 NA C <> T NA Untransformed NA NA

ANOVA Table
Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%) Between 0 0 3 65500 <0.0001 Significant Effect Error 0 0 12 0 15 Total

Mean Min Max Median Count Control Type Std Err C- μ g ai/L CV% %Effect 95% LCL 95% UCL FemaleMedianTubercleScore Summary

0 0 0 0 0 4 Negative Control 0 0 0 0 0 0 0 4.97 4 0 0 0 0 0 0 0 11.3 4 0 0 0 0 0 0 0 19.5 4 0 0 0CenteredUntransformed

Rankits 0.0E+00 2.5E-01 5.0E-01 7.5E-01 1.0E+00

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0FemaleMedianTubercleScore

C- μ g ai/L 0.0E+00 2.5E-01 5.0E-01 7.5E-01 1.0E+00

0 N 4.97 11.3 19.5 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Jun-15 10:11 (p 7 of 18) Test Code: 128997 48688301 | 14-7456-2471 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Bayer CropScience AG

Analyzed: 29 May-14 15:10 Endpoint: FemaleVTG CETIS Version: CETISv1.8.7 Analysis ID: 20-1618-6596 Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 07-9757-7599 Start Date: 19 Jan-12 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Brine: Species: Pimephales promelas Source: Analyst: J. Housenger

Age:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C- μ g ai/L Dunnett Multiple Comparison Test

DF P-Type
1.36 2.68 1E+06 0.4198 Non-Significant Effect Negative Control 4.97 6 CDF 2.85 2.68 1E+06 0.0372 Significant Effect 11.3* 6 CDF 4.46 2.68 1E+06 0.0021 Significant Effect 19.5* 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed
4.97 11.3 33.1% 7.494 C <> T NA Untransformed NA NA

ANOVA Table
Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%) Between 6.015927E+12 2.005309E+12 3 7.38 0.0046 Significant Effect Error 3.262497E+12 2.718748E+11 12 9.278423E+12 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
3.81 11.3 0.2833 Equal Variances Bartlett Equality of Variance Variances 0.97 0.841 0.8350 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C- μ g ai/L CV% %Effect 95% LCL 95% UCL FemaleVTG Summary

2.99E+6 2.56E+6 3.50E+6 2950000 0 4 Negative Control 2.49E+5 16.7% 0.0% 2.19E+6 3.78E+6 2.49E+6 2.21E+6 2.75E+6 2500000 4.97 4 1.16E+5 9.3% 16.7% 2.12E+6 2.86E+6 1.94E+6 1.56E+6 2.30E+6 1950000 11.3 4 1.96E+5 20.2% 35.1% 1.31E+6 2.56E+6 1.34E+6 2.96E+5 2.23E+6 1430000 19.5 4 3.98E+5 59.2% 55.0% 7.88E+4 2.61E+6CenteredUntransformed

Rankits -2.0E+05

-4.0E+05

-6.0E+05

-8.0E+05

-1.0E+06

-1.2E+06 0.0E+00 2.0E+05 4.0E+05 6.0E+05 8.0E+05 1.0E+06

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0FemaleVTG

C- μ g ai/L Reject Null

Reject Null

0.0E+00 5.0E+05 1.0E+06 1.5E+06 2.0E+06 2.5E+06 3.0E+06 3.5E+06 4.0E+06

0 N 4.97 11.3 19.5 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Jun-15 10:11 (p 8 of 18) Test Code: 128997 48688301 | 14-7456-2471 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Bayer CropScience AG

Analyzed: 29 May-14 15:15 Endpoint: FemaleVTG CETIS Version: CETISv1.8.7 Analysis ID: 04-9636-1737 Analysis: Nonparametric-Control vs Ord. Treatments Official Results: Yes

Batch ID: 07-9757-7599 Start Date: 19 Jan-12 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Brine: Species: Pimephales promelas Source: Analyst: J. Housenger

Age:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C- μ g ai/L Jonckheere-Terpstra Step-Down Test

DF P-Type
12 NA 0.1714 Non-Significant Effect Negative Control 4.97 -2 Exact 42 NA 0.0037 Significant Effect 11.3* -2 Exact 87 NA <0.0001 Significant Effect 19.5* -2 Exact NOEL LOEL TU TOEL Alt Hyp Data Transform Zeta Trials Seed
4.97 11.3 7.494 C > T NA Untransformed NA NA

ANOVA Table
Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%) Between 6.015927E+12 2.005309E+12 3 7.38 0.0046 Significant Effect Error 3.262497E+12 2.718748E+11 12 9.278423E+12 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
3.81 11.3 0.2833 Equal Variances Bartlett Equality of Variance Variances 0.97 0.841 0.8350 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C- μ g ai/L CV% %Effect 95% LCL 95% UCL FemaleVTG Summary

2.99E+6 2.56E+6 3.50E+6 2950000 0 4 Negative Control 2.49E+5 16.7% 0.0% 2.19E+6 3.78E+6 2.49E+6 2.21E+6 2.75E+6 2500000 4.97 4 1.16E+5 9.3% 16.7% 2.12E+6 2.86E+6 1.94E+6 1.56E+6 2.30E+6 1950000 11.3 4 1.96E+5 20.2% 35.1% 1.31E+6 2.56E+6 1.34E+6 2.96E+5 2.23E+6 1430000 19.5 4 3.98E+5 59.2% 55.0% 7.88E+4 2.61E+6CenteredUntransformed

Rankits -2.0E+05

-4.0E+05

-6.0E+05

-8.0E+05

-1.0E+06

-1.2E+06 0.0E+00 2.0E+05 4.0E+05 6.0E+05 8.0E+05 1.0E+06

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0FemaleVTG

C- μ g ai/L 0.0E+00 5.0E+05 1.0E+06 1.5E+06 2.0E+06 2.5E+06 3.0E+06 3.5E+06

0 N 4.97 11.3 19.5 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Jun-15 10:11 (p 9 of 18) Test Code: 128997 48688301 | 14-7456-2471 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Bayer CropScience AG

Analyzed: 29 May-14 15:10 Endpoint: Fertility CETIS Version: CETISv1.8.7 Analysis ID: 09-9039-5609 Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 07-9757-7599 Start Date: 19 Jan-12 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Brine: Species: Pimephales promelas Source: Analyst: J. Housenger

Age:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C- μ g ai/L Dunnett Multiple Comparison Test

DF P-Type
0.81 2.68 1.74 0.7599 Non-Significant Effect Negative Control 4.97 6 CDF 0.579 2.68 1.74 0.8892 Non-Significant Effect 11.3 6 CDF 1.5 2.68 1.74 0.3431 Non-Significant Effect 19.5 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed
19.5 >19.5 1.78% NA C <> T NA Untransformed NA NA

ANOVA Table
Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%) Between 4.201866 1.400622 3 1.67 0.2265 Non-Significant Effect Error 10.0775 0.8397914 12 14.27936 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
1.72 11.3 0.6322 Equal Variances Bartlett Equality of Variance Variances 0.93 0.841 0.2427 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C- μ g ai/L CV% %Effect 95% LCL 95% UCL Fertility Summary

97.6 95.9 98.6 97.9 0 4 Negative Control 0.617 1.26% 0.0% 95.6 99.5 98.1 97.1 99 98.2 4.97 4 0.414 0.85% -0.54% 96.8 99.4 97.2 96.7 97.8 97.2 11.3 4 0.268 0.55% 0.38% 96.3 98.1 98.5 97.4 99.4 98.7 19.5 4 0.465 0.94% -1.0% 97.1 100CenteredUntransformed

Rankits -0.2

-0.4

-0.6

-0.8

-1.0

-1.2

-1.4

-1.6

-1.8 0.0 0.2 0.4 0.6 0.8 1.0 1.2

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0Fertility

C- μ g ai/L Reject Null

Reject Null

0 20 40 60 80 100

0 N 4.97 11.3 19.5 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Jun-15 10:11 (p 10 of 18) Test Code: 128997 48688301 | 14-7456-2471 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Bayer CropScience AG

Analyzed: 29 May-14 15:09 Endpoint: MaleBodyWt CETIS Version: CETISv1.8.7 Analysis ID: 18-2206-4510 Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 07-9757-7599 Start Date: 19 Jan-12 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Brine: Species: Pimephales promelas Source: Analyst: J. Housenger

Age:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C- μ g ai/L Dunnett Multiple Comparison Test

DF P-Type
1.12 2.72 1.38 0.5676 Non-Significant Effect Negative Control 4.97 5 CDF 0.321 2.72 1.27 0.9778 Non-Significant Effect 11.3 6 CDF 0.94 2.72 1.27 0.6825 Non-Significant Effect 19.5 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed
19.5 >19.5 25.2% NA C <> T NA Untransformed NA NA

ANOVA Table
Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%) Between 0.7273485 0.2424495 3 0.554 0.6562 Non-Significant Effect Error 4.816092 0.4378265 11 5.54344 14 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
1.34 11.3 0.7201 Equal Variances Bartlett Equality of Variance Variances 0.962 0.833 0.7253 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C- μ g ai/L CV% %Effect 95% LCL 95% UCL MaleBodyWt Summary

5.06 4.55 5.45 5.11 0 4 Negative Control 0.216 8.53% 0.0% 4.37 5.74 5.62 5.02 6.01 5.84 4.97 3 0.306 9.41% -11.2% 4.31 6.94 5.21 4.58 6.12 5.07 11.3 4 0.348 13.4% -2.97% 4.1 6.31 5.5 4.49 6.55 5.47 19.5 4 0.432 15.7% -8.7% 4.12 6.87CenteredUntransformed

Rankits -0.2

-0.4

-0.6

-0.8

-1.0

-1.2

-1.4 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0MaleBodyWt

C- μ g ai/L Reject Null

Reject Null

0 1 2 3 4 5 6 7

0 N 4.97 11.3 19.5 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Jun-15 10:11 (p 11 of 18) Test Code: 128997 48688301 | 14-7456-2471 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Bayer CropScience AG

Analyzed: 29 May-14 15:09 Endpoint: MaleGSI CETIS Version: CETISv1.8.7 Analysis ID: 14-2217-9265 Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 07-9757-7599 Start Date: 19 Jan-12 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Brine: Species: Pimephales promelas Source: Analyst: J. Housenger

Age:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C- μ g ai/L Dunnett Multiple Comparison Test

DF P-Type
0.329 2.72 0.807 0.9760 Non-Significant Effect Negative Control 4.97 5 CDF 1.18 2.72 0.747 0.5322 Non-Significant Effect 11.3 6 CDF 1.11 2.72 0.747 0.5720 Non-Significant Effect 19.5 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed
19.5 >19.5 47.2% NA C <> T NA Untransformed NA NA

ANOVA Table
Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%) Between 0.4888233 0.1629411 3 1.08 0.3960 Non-Significant Effect Error 1.65315 0.1502863 11 2.141973 14 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
3.87 11.3 0.2761 Equal Variances Bartlett Equality of Variance Variances 0.974 0.833 0.9164 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C- μ g ai/L CV% %Effect 95% LCL 95% UCL MaleGSI Summary

1.58 1.29 1.82 1.61 0 4 Negative Control 0.111 14.0% 0.0% 1.23 1.93 1.68 0.95 2.11 1.98 4.97 3 0.367 37.8% -6.16% 0.101 3.26 1.26 1.09 1.56 1.2 11.3 4 0.103 16.4% 20.4% 0.932 1.59 1.28 0.99 1.92 1.1 19.5 4 0.218 34.2% 19.3% 0.583 1.97CenteredUntransformed

Rankits -0.1

-0.2

-0.3

-0.4

-0.5

-0.6

-0.7

-0.8 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0MaleGSI

C- μ g ai/L Reject Null

Reject Null

0.0 0.5 1.0 1.5 2.0 2.5

0 N 4.97 11.3 19.5 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Jun-15 10:11 (p 12 of 18) Test Code: 128997 48688301 | 14-7456-2471 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Bayer CropScience AG

Analyzed: 29 May-14 15:15 Endpoint: MaleGSI CETIS Version: CETISv1.8.7 Analysis ID: 06-6317-5130 Analysis: Nonparametric-Control vs Ord. Treatments Official Results: Yes

Batch ID: 07-9757-7599 Start Date: 19 Jan-12 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Brine: Species: Pimephales promelas Source: Analyst: J. Housenger

Age:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C- μ g ai/L Jonckheere-Terpstra Step-Down Test

DF P-Type
4 NA 0.8000 Non-Significant Effect Negative Control 4.97 -2 Exact 27 NA 0.1441 Non-Significant Effect 11.3 -2 Exact 58 NA 0.0561 Non-Significant Effect 19.5 -2 Exact NOEL LOEL TU TOEL Alt Hyp Data Transform Zeta Trials Seed
19.5 >19.5 NA C > T NA Untransformed NA NA

ANOVA Table
Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%) Between 0.4888233 0.1629411 3 1.08 0.3960 Non-Significant Effect Error 1.65315 0.1502863 11 2.141973 14 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
3.87 11.3 0.2761 Equal Variances Bartlett Equality of Variance Variances 0.974 0.833 0.9164 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C- μ g ai/L CV% %Effect 95% LCL 95% UCL MaleGSI Summary

1.58 1.29 1.82 1.61 0 4 Negative Control 0.111 14.0% 0.0% 1.23 1.93 1.68 0.95 2.11 1.98 4.97 3 0.367 37.8% -6.16% 0.101 3.26 1.26 1.09 1.56 1.2 11.3 4 0.103 16.4% 20.4% 0.932 1.59 1.28 0.99 1.92 1.1 19.5 4 0.218 34.2% 19.3% 0.583 1.97CenteredUntransformed

Rankits -0.1

-0.2

-0.3

-0.4

-0.5

-0.6

-0.7

-0.8 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0MaleGSI

C- μ g ai/L 0.0 0.5 1.0 1.5 2.0 2.5

0 N 4.97 11.3 19.5 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Jun-15 10:11 (p 13 of 18) Test Code: 128997 48688301 | 14-7456-2471 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Bayer CropScience AG

Analyzed: 29 May-14 15:07 Endpoint: MaleLength CETIS Version: CETISv1.8.7 Analysis ID: 12-7937-1793 Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 07-9757-7599 Start Date: 19 Jan-12 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Brine: Species: Pimephales promelas Source: Analyst: J. Housenger

Age:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C- μ g ai/L Dunnett Multiple Comparison Test

DF P-Type
1.02 2.72 4.92 0.6287 Non-Significant Effect Negative Control 4.97 5 CDF 0.209 2.72 4.56 0.9935 Non-Significant Effect 11.3 6 CDF 0.837 2.72 4.56 0.7483 Non-Significant Effect 19.5 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed
19.5 >19.5 7.86% NA C <> T NA Untransformed NA NA

ANOVA Table
Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%) Between 12.25999 4.086664 3 0.73 0.5553 Non-Significant Effect Error 61.57998 5.59818 11 73.83997 14 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
2.25 11.3 0.5215 Equal Variances Bartlett Equality of Variance Variances 0.982 0.833 0.9807 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C- μ g ai/L CV% %Effect 95% LCL 95% UCL MaleLength Summary

58 57 60.4 57.2 0 4 Negative Control 0.818 2.82% 0.0% 55.3 60.6 59.8 57.5 61.3 60.6 4.97 3 1.17 3.38% -3.19% 54.8 64.8 57.6 53.5 62.1 57.4 11.3 4 1.76 6.11% 0.6% 52 63.2 59.3 57 60.8 59.8 19.5 4 0.827 2.79% -2.42% 56.7 62CenteredUntransformed

Rankits -1

-2

-3

-4

-5 0 1 2 3 4 5

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0MaleLength

C- μ g ai/L Reject Null

Reject Null

0 10 20 30 40 50 60 70

0 N 4.97 11.3 19.5 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Jun-15 10:11 (p 14 of 18) Test Code: 128997 48688301 | 14-7456-2471 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Bayer CropScience AG

Analyzed: 29 May-14 15:06 Endpoint: MaleMedianTubercleScore CETIS Version: CETISv1.8.7 Analysis ID: 07-5551-6438 Analysis: Parametric-Control vs Treatments Official Results: No

Batch ID: 07-9757-7599 Start Date: 19 Jan-12 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Brine: Species: Pimephales promelas Source: Analyst: J. Housenger

Age:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C- μ g ai/L Dunnett Multiple Comparison Test

DF P-Type
2.42 2.72 12.3 0.0839 Non-Significant Effect Negative Control 4.97 5 CDF 0.328 2.72 11.4 0.9763 Non-Significant Effect 11.3 6 CDF 1.58 2.72 11.4 0.3152 Non-Significant Effect 19.5 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed
19.5 >19.5 31.4% NA C <> T NA Untransformed NA NA

ANOVA Table
Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%) Between 262.7958 87.59861 3 2.49 0.1144 Non-Significant Effect Error 386.6042 35.14583 11 649.4 14 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
0.65 11.3 0.8850 Equal Variances Bartlett Equality of Variance Variances 0.945 0.833 0.4531 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C- μ g ai/L CV% %Effect 95% LCL 95% UCL MaleMedianTubercleScore Summary

36.4 27.5 42.5 37.8 0 4 Negative Control 3.16 17.4% 0.0% 26.3 46.4 47.3 43.5 55 43.5 4.97 3 3.83 14.0% -30.1% 30.8 63.8 37.8 29 44 39 11.3 4 3.25 17.2% -3.78% 27.4 48.1 43 39 47.5 42.8 19.5 4 2.07 9.64% -18.2% 36.4 49.6CenteredUntransformed

Rankits -2

-4

-6

-8

-10 0 2 4 6 8

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0MaleMedianTubercleScore

C- μ g ai/L Reject Null

Reject Null

0 10 20 30 40 50 60

0 N 4.97 11.3 19.5 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Jun-15 10:11 (p 15 of 18) Test Code: 128997 48688301 | 14-7456-2471 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Bayer CropScience AG

Analyzed: 06 Jun-15 9:28 Endpoint: MaleMedianTubercleScore CETIS Version: CETISv1.8.7 Analysis ID: 09-0765-1433 Analysis: Nonparametric-Two Sample Official Results: Yes

Batch ID: 07-9757-7599 Start Date: 19 Jan-12 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Brine: Species: Pimephales promelas Source: Analyst: J. Housenger

Age:

Test Stat Critical P-Value Decision( α :5%) Ties vs Control C- μ g ai/L Mann-Whitney U Two-Sample Test

DF P-Type
0 NA 0 1.0000 Non-Significant Effect Negative Control 4.97 5 Exact 7 NA 0 0.6571 Non-Significant Effect 11.3 6 Exact 2 NA 0 0.9714 Non-Significant Effect 19.5 6 Exact NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed
19.5 >19.5 22.4% NA C > T NA Untransformed NA NA

ANOVA Table
Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%) Between 262.7958 87.59861 3 2.49 0.1144 Non-Significant Effect Error 386.6042 35.14583 11 649.4 14 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
0.65 11.3 0.8850 Equal Variances Bartlett Equality of Variance Variances 0.945 0.833 0.4531 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C- μ g ai/L CV% %Effect 95% LCL 95% UCL MaleMedianTubercleScore Summary

36.4 27.5 42.5 37.8 0 4 Negative Control 3.16 17.4% 0.0% 26.3 46.4 47.3 43.5 55 43.5 4.97 3 3.83 14.0% -30.1% 30.8 63.8 37.8 29 44 39 11.3 4 3.25 17.2% -3.78% 27.4 48.1 43 39 47.5 42.8 19.5 4 2.07 9.64% -18.2% 36.4 49.6CenteredUntransformed

Rankits -2

-4

-6

-8

-10 0 2 4 6 8

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0MaleMedianTubercleScore

C- μ g ai/L 0 10 20 30 40 50 60

0 N 4.97 11.3 19.5 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Jun-15 10:11 (p 16 of 18) Test Code: 128997 48688301 | 14-7456-2471 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Bayer CropScience AG

Analyzed: 29 May-14 15:05 Endpoint: MaleVTG CETIS Version: CETISv1.8.7 Analysis ID: 13-0609-2921 Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 07-9757-7599 Start Date: 19 Jan-12 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Brine: Species: Pimephales promelas Source: Analyst: J. Housenger

Age:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C- μ g ai/L Dunnett Multiple Comparison Test

DF P-Type
2.02 2.72 1140 0.1630 Non-Significant Effect Negative Control 4.97 5 CDF 0.414 2.72 1050 0.9549 Non-Significant Effect 11.3 6 CDF 0.365 2.72 1050 0.9681 Non-Significant Effect 19.5 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed
19.5 >19.5 134.0% NA C <> T NA Untransformed NA NA

ANOVA Table
Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%) Between 1385350 461783.2 3 1.54 0.2592 Non-Significant Effect Error 3298627 299875.2 11 4683977 14 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
1.32 11.3 0.7251 Equal Variances Bartlett Equality of Variance Variances 0.925 0.833 0.2292 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C- μ g ai/L CV% %Effect 95% LCL 95% UCL MaleVTG Summary

785 304 1400 718 0 4 Negative Control 244 62.3% 0.0% 6.93 1560 1630 980 2440 1460 4.97 3 429 45.7% -107.0% -219 3480 945 506 1270 1000 11.3 4 175 37.0% -20.4% 389 1500 926 448 1810 725 19.5 4 304 65.7% -18.0% -41.6 1890CenteredUntransformed

Rankits -200

-400

-600

-800 0 200 400 600 800 1000

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0MaleVTG

C- μ g ai/L Reject Null

0 500 1000 1500 2000 2500

0 N 4.97 11.3 19.5 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Jun-15 10:11 (p 17 of 18) Test Code: 128997 48688301 | 14-7456-2471 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Bayer CropScience AG

Analyzed: 29 May-14 15:04 Endpoint: Survival CETIS Version: CETISv1.8.7 Analysis ID: 18-2357-6634 Analysis: Nonparametric-Two Sample Official Results: Yes

Batch ID: 07-9757-7599 Start Date: 19 Jan-12 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Brine: Species: Pimephales promelas Source: Analyst: J. Housenger

Age:

Test Stat Critical P-Value Decision( α :5%) Ties vs Control C- μ g ai/L Mann-Whitney U Two-Sample Test

DF P-Type
10 NA 1 1.0000 Non-Significant Effect Negative Control 4.97 6 Exact 10 NA 1 1.0000 Non-Significant Effect 11.3 6 Exact 10 NA 1 1.0000 Non-Significant Effect 19.5 6 Exact NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed
19.5 >19.5 25.3% NA C <> T NA Angular (Corrected) NA NA

ANOVA Table
Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%) Between 0.04690253 0.01563418 3 0.454 0.7190 Non-Significant Effect Error 0.4128292 0.03440243 12 0.4597317 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
0.454 5.95 0.7190 Equal Variances Mod Levene Equality of Variance Variances 4.09 5.95 0.0325 Equal Variances Levene Equality of Variance Variances 0.78 0.841 0.0015 Non-normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C- μ g ai/L CV% %Effect 95% LCL 95% UCL Survival Summary

1 1 1 1 0 4 Negative Control 0 0.0% 0.0% 1 1 0.875 0.5 1 1 4.97 4 0.125 28.6% 12.5% 0.477 1 0.917 0.667 1 1 11.3 4 0.0833 18.2% 8.33% 0.651 1 0.958 0.833 1 1 19.5 4 0.0417 8.7% 4.17% 0.826 1

Mean Min Max Median Count Control Type Std Err C- μ g ai/L CV% %Effect 95% LCL 95% UCL Angular (Corrected) Transformed Summary

1.37 1.37 1.37 1.37 0 4 Negative Contr 0 0.0% 0.0% 1.37 1.37 1.22 0.785 1.37 1.37 4.97 4 0.145 23.8% 10.6% 0.759 1.68 1.26 0.955 1.37 1.37 11.3 4 0.102 16.2% 7.51% 0.937 1.59 1.31 1.15 1.37 1.37 19.5 4 0.0537 8.2% 3.94% 1.14 1.48

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________Report Date: 06 Jun-15 10:11 (p 18 of 18) Test Code: 128997 48688301 | 14-7456-2471 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Bayer CropScience AG

Analyzed: 29 May-14 15:04 Endpoint: Survival CETIS Version: CETISv1.8.7 Analysis ID: 18-2357-6634 Analysis: Nonparametric-Two Sample Official Results: YesCenteredCorr. Angle

Rankits -0.1

-0.2

-0.3

-0.4

-0.5 0.0 0.1 0.2 0.3

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0Survival

C- μ g ai/L 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

0 N 4.97 11.3 19.5 Graphics

CETISTM v1.8.7.12 000-431-181-1 QA:________ Analyst:________

# DATA EVALUATION RECORD

TEBUCONAZOLE
Study Type: OCSPP 890.1400, In vivo Hershberger Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-67-2012 (MRID 48673702)

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: 06/06/2012

Secondary Reviewer: Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 06/08/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 06/25/2012
Quality Assurance: Signature: Jack D. Early, M.S. Date: 06/25/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).

### TEBUCONAZOLE / 128997 

## Primary Reviewer:**=Ra=v~K=e=nt=·-=P-==h=.D::...:·'--------­** 

## Health Effects Division Secondary Reviewer: Greg Akerman, Ph

. D

. 

## Health Effects Division In vivo 

### Hershberger Assay (2012) I 

### Page 1 of 12 OCSPP 890.1400/ OECD 441 

## Signature:**-t:::if-1--~~...a..._~=-­** 

## Date:**-~K+~~----­** 

## Signature:**--r,,,,,.~~~--** 

## Date:

___ cz"-#'/

......

## l.,,...'1..,..J~' 

# r-

__ 

# I DATAEVALUATIONRECORD Template version 10/2011



## STUDY TYPE: 

## In Vivo Hershberger Assay (Rat); OCSPP 890.1400; OECD 441



## PC CODE: 128997 

## DP BARCODE: D401595

TXR#: 0056253 

## CAS#: 107534

- 96-3



## TEST MATERIAL (PURITY): Tebuconazole (97.5% a.i.)



## SYNONYMS: AE F069623; a-[2-( 4-chlorophenyl)ethyl]-a-(l, l-dimethylethyl)-lH-1,2,4- triazole-1-ethanol



## CITATION: Blanck, M

. (2012) Tebuconazole: Evaluation in the Hershberger Bioassay. Bayer S.A

. S., Sophia Antipolis Cedex
, France. Laboratory Report No

. : SA 11011, April 10, 2012. MRID 48673702. Unpublished.



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



## TEST ORDER#: CON-128997-20



## EXECUTIVE SUMMARY: In a Hershberger Assay (MRID 

## 48673702) screening for androgenic activity, tebuconazole (97.5% a.i.; Batch# K689052) in 0.5% aqueous methylcellulose 400 

## + 0.4% Tween 80 was administered daily via oral gavage to groups of six 58-day old, castrated male Sprague Dawley rats at dose levels of 0**(vehicle) ~**  

## 75, or 150 mg/kg/day. An androgenic positive control group consisted of six castrated rats exposed to 0.4 mg/kg/day of testosterone propionate (TP) by subcutaneous (s.c.) injection.

To screen for potential anti-androgenic activity, tebuconazole in 0.5% aqueous methylcellulose 400 

## + 0.4% Tween 80 was also administered daily via oral gavage to groups of six 58

- day old
, castrated male Sprague Dawley rats at dose levels ofO (vehicle), 35
, 75
, or 150 mg/kg/day in conjunction with a daily dose of referenc e androgen TP at 0.4 mg/kg/day by s.c. injection. The anti-androgenic positive control group consisted of s ix castrated rats e x posed to 0.4 mg/kg/day TP and 3 mg/kg/day flutamide (FT). TP alone was used as the anti-androgenic negative control.

For both components of the assay, body weights were determined daily

. The animals were dosed for 10 consecutive days and terminated approximately 24 hours after the final dose administration. At necropsy, the five androgen-dep e ndent tissues were collect e d and we igh ed.

All animals survived until scheduled termination. No clinical signs of toxicity were observed 

## in animals for any dose groups.In vivo Hershberger Assay (201 2) / Page 2 of 12 TEBUCONAZOLE / 128997 OCSPP 890.1400/ OECD 441 

## In the androgen assay, body we ights throughout the duration of the assay in the tebuconazoledosed groups and positive (vehicle + TP) control group were comparab le to the vehicle controls. Overall (Days 1-10) body weight gains in th e tebuconazole 150 mg/kg/day groups were decreased (p<0.05) by 20% compared to the vehicl e controls. Overall body weight gains in the positive control group were increased (p<0.05) by 29% compared to the vehicle controls. No treatment-related effects were observed in any of the accessory sex organs of tebuconazole-dosed animals. Rats in the positiv e control (vehicle + TP) group ha d accessory sex organ weight increases (p<0.05) as follows: 992% in seminal vesicles; 602% in ventral prostate; 160% in levator ani-bulbocavernosus (LABC); 449% in Cowper’s glands; and 65% in glans penis.
In the anti-androgen assay, body we ights throughout the study and body weight gain from Days 1 to 10 were similar across all groups. At 75 mg/kg/day, mean cumulative body weight gains were decreased (p<0.05) from Days 1 to 3 by 24%. At 150 mg/kg/day, mean cumulative body weight gains were decreased (p<0.05) from Days 1 to 3 by 71%, Days 1 to 4 by 33%, and Days 1 to 6 by 16% compared to the vehicle control; m ean body weights were not affected by the end of the study. The accessory sex organ weights in all tebuconazole treatment groups were comparable to the negative controls. Rats in the positive control (FT + TP) group had accessory sex organ weight decreases (p<0.05) as follows : 71% in seminal vesicles; 69% in ventral prostate; 38% in LABC; 52% in Cowpe r’s glands; and 22% in glans penis.
The dosing in this study was considered adequa te based on the decreased overall body weight gains observed at the hi gh-dose (150 mg/kg/day).
Statistically significant changes were not seen in two or more of the five androgen sensitive tissue weights. Tebuconazole was negative for androgenicity and anti-a ndrogenicity in the Hershberger assay.
The assay satisfies the EDSP Tier 1 Test Order requireme nts for a Hershberger assay (OCSPP 890.1400).

COMPLIANCE: Signed and dated Data Confiden tiality, GLP Compliance, and Quality Assurance statements were provided.In vivo Hershberger Assay (201 2) / Page 3 of 12 TEBUCONAZOLE / 128997 OCSPP 890.1400/ OECD 441


## I. MATERIALS AND METHODS

A. MATERIALS
1. Test Facility: Bayer S.A.S. Location: Sophia Antipolis Cedex, France Study Director: M. Blanck Other Personnel: M. Totis (Replacement Study Director ), J.P. Kocwin (Laboratory Animal Resources), C. Jaussely (In-Life S upervisor), H. Vincent (Responsible Technician), C. Pallen (Statistician), L. Amir Tahmasseb (Senior Chemist), M. Vagner (Report Unit Assistant) Study Period: January 10, 2012 to April 10, 2012



## 2. Test Substance: Tebuconazole Description: White powder Source: Bayer CropScience AG (Frankfurt, Germany) Lot/Batch #: K689052 (expiry date: August 23, 2012) Purity: 97.5% a.i. Stability: Stable in vehicle (aqueous solution of 0.5% methylcellulose 400 + 0.4% Tween 80) for up to 22 days at 5  3 °C. CAS #: 107534-96-3 Structure:
Cl OH CH 3

CH 3 CH 3

N N N


## 3. Reference Androgen: Testosterone propionate (TP) Supplier: Sigma Aldrich (St. Louis, MO); catalog number T1875 Lot/Batch #: 051M1803V (September 2012) Purity: 102% CAS #: 57-85-2


## 4. Reference Anti-androgen: Flutamide (FT) Supplier: Sigma-Aldrich (St. Louis, MO); catalog number F9397 Lot/Batch #: 021M1406V (April 2012) Purity: 100% CAS #: 13311-84-7



## 5. Solvent/Vehicle Control (tebuconazole): Methylcellulose 400 + Tween 80

Supplier: Sigma-Aldrich (methylcellulose 400)/Merck (Tween 80) Lot/Batch #: 060M0123V (methylcellulose 400)/S5264887.914 (Tween 80) Rationale (if other than water): Not provided Final concentration: 0.5% (methylcellulose 400), 0.4% (Tween 80)



## Solvent/Vehicle Control (FT): Methylcellulose 400

Supplier: Sigma-Aldrich Lot/Batch #: 060M0123V Rationale (if other than water): Not provided Final concentration: 0.5%In vivo Hershberger Assay (201 2) / Page 4 of 12 TEBUCONAZOLE / 128997 OCSPP 890.1400/ OECD 441


## Solvent/Vehicle Control (TP): Corn oil

Supplier: Sigma-Aldrich Lot/Batch #: MKBD2427 Rationale (if other than water): Not applicable Final concentration: Not applicable



## 6. Test Animals: Species: Rat (castrated males) Strain: Sprague Dawley Crl:CD(SD) Age/weight at dose initiation: Post-natal day (PND) 58; 255.9-339.3 g Source: Charles River Laboratories (St Ge rmain-sur-l’Arbresle, France) Housing: Rats were housed three per cage in ma krolon cages containi ng heat-treated soft wood bedding Diet: A04C-10 pelleted rodent diet (Scientific Animal Food and Engineering, Augy, France), ad libitum Phytoestrogen content <350 μ g of genistein equivalents/gram diet Water: Filtered tap water, softened by ion exchange resins, ad libitum Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 20-24oC 40-70% 10-15/hr 12 hrs light/12 hrs dark Acclimation period: Rats were castrated at the supplier on PND 43, received at the study facility on PND 48-49, and allowed to acclimate at the facility for 9-10 days prior to initiation of dosing



## B. STUDY DESIGN:

1. In-life dates: Start: January 20, 2012 End: January 31, 2012
2. Study Design: In a Hershberger Assay conducted to screen for pot ential androgenic activity, tebuconazole was administered daily via oral gavage to castrated male rats. TP by s.c. injection was used as the positive control. Positive androgenic activity is defined as a significant increase in two or more target or gan weights compared to the vehicle control group. To screen for the potential anti-andr ogenic activity, tebuconazole was administered daily via oral gavage to gr oups of castrated male rats in conjunction with TP at 0.4 mg/kg/day by s.c. injection. TP by s.c. inj ection plus flutamide by oral gavage was used as the positive control. Anti- androgenic activity is indicated by a statistically significant decrease in two or more targ et organ weights of the treate d groups (test substance + TP) compared to the negative (vehicle + TP) cont rol group. For both assays, the animals were dosed for 10 consecutive days.
3. Study Schedule: Rats were castrated at the supplier on PND 43 and allowed at least 4 days for recovery before shipment to the study faci lity. Animals were received at the study facility on PND 48-49 and allowed to acclimat e for 9-10 days. The dose administration period was from PND 58 through PND 67. Rats were euthanized on PND 68 approximately 24 hours after the last dose and necrops ied for organ weight measurements.
4. Animal Assignment: Animals were assigned to the te st groups noted in Table 1 using computerized randomization procedures which selected animals from the middle of the weight range of the available animals. Statis tical analysis indicated that there were no significant differences in group means at study initiation. Furthermore, the body weight of each animal was within  20% of the overall mean.In vivo Hershberger Assay (201 2) / Page 5 of 12 TEBUCONAZOLE / 128997 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 (Tebuconazole) 75 6 High (Tebuconazole) 150 6 Positive control, TP b 

### 0.4 6 Anti-Androgen Assay Negative control, corn oil + TP b 

### 0 + 0.4 6 Low (Tebuconazole + TP) 35 + 0.4 6 Mid (Tebuconazole + TP) 75 + 0.4 6 High (Tebuconazole + TP) 150 + 0.4 6 Positive control, Flutamide + TP 3 + 0.4 6 a Data were obtained from pages 17 of the study report. b This dose group served as the positive control for the androgen agonist assay and the negative control for the anti-androgen assay.



## 5. Dose Selection Rationale: The dose levels used in this study were selected based on the results of a dose range-finding study in Sprague Dawley rats. A summary of this study is included as an Appendix to this DER.
6. (a) Dose Preparation: Dose formulations of the test substance were prepared by mixing appropriate amounts of tebuconazole with 0.5% aqueous methylcellulose 400 + 0.4% Tween 80. Dose formulations of TP were prepared by mixing the appropriate amount of TP in corn oil, and dose formulations of FT were prepar ed by mixing appropriate amounts of FT with 0.5% aqueous methylcellulose 400. Dose formul ations were prepared once during the study and stored at 5  3°C (tebuconazole and FT) or room temperature (TP) when not in use. Stability was tested in a separate study; 1 

## the test substance in th e vehicle was found to be stable at concentrations of 0.6 and 170 g/L (whi ch bracketed those used in the current study) for up to 22 days under similar conditions of usag e and storage to those of the current study. Homogeneity was tested at two concentrations of tebuconazole in vehicle by sampling the surface and bottom levels at 7 g/L and the surf ace, middle, and bottom of the suspension at 30 g/L. Achieved concentration was tested separately at 15 g/L; in addition, the mean results of the homogeneity analyses were used for achieved concentrations. The homogeneity and achieved concentration of the TP and FT dose formulations were also tested.
( b) Dose Analysis:

Results
Homogeneity (%CV): 1.1-1.4%

1 

### Amir Tahmasseb, L. (2011) Tebuconazole: stability in aqueous 0.5% methylcellulose + 0.4% tween 80, SA 11273, Bayer CropScience, Sophia Antipolis, France.In vivo Hershberger Assay (201 2) / Page 6 of 12 TEBUCONAZOLE / 128997 OCSPP 890.1400/ OECD 441 

## Stability: Not provided

Concentration (% of nominal): 98-99%
The analytical data indicated that the mixing procedure was ad equate and that the variation between nominal and actual dosage of tebucona zole to the animals was acceptable. The study referenced above should be submitted fo r verification of th e stability findings.
For TP, the %CV for homogeneity analyses was 0.4% and the averag e concentration was found to be 96.4% of nominal. For FT, the %CV for homogeneity analyses was 0.6% and the average concentration was f ound to be 97.6% of nominal.
7. Dosage administration: Test formulations were administ ered to the animals daily via oral gavage (5 mL/kg bw) for 10 days. TP was give n via s.c. injection at 0.5 mL/kg bw, and FT was administered via oral gava ge at 5 mL/kg bw. Dose volum es were adjusted daily based on the concurrent body weight measurement.

8. Statistics: The following parameters were anal yzed statistically: body weights, body weight gains, terminal body weights, and or gan weights. For body weight gain, the data were first analyzed using Bartlett’s test. If the data were not significant, an ANOVA test was performed; a two-sided Dunnett’s test was conducted if the results of the ANOVA were significant. If the results of Bartlett’s te st were significant, a Kruskal-Wallis test was performed, followed by a two-side d Dunn’s test for significant data. The same procedures were used for body weight (except terminal body we ight), except that data were transformed (log transformation) and a second Bartlett’s test was conducted if the data were significant after the first Bartlett’s test.
For organ weights and terminal body weight, the data were first analyzed using Levene’s test; if the data were significant, a data transformation (log transformation) was conducted. If the data were not significant, an ANOVA test was performed; a one-sided (organ weights) or two-sided (terminal body we ights) Dunnett’s test was cond ucted if the results of the ANOVA were significant. If the results of Le vene’s test (after transformation) showed significance, a Kruskal-Wallis test was perfor med, followed by a one-sided (organ weights) or two-sided (terminal body wei ghts) Dunn’s test for significant data. In addition, analysis of covariance (ANCOVA) was conducted on organ weights using terminal body weight as a covariable; adjusted means were compared using a one -sided Dunnett’s test.
For comparison of data from the positive control with data from the negative control in the androgen agonist and anti-androgen assays , the body weights (including terminal body weights), interval body weight ga ins, and organ weight data were analyzed using an F test; for body weights and organ weights, the data were log-transformed and reanalyzed if significant after the F test. Data that were not significant were analyzed using a one- or twosided T-test, and data that we re significant were analyzed using a modified one- or twosided T-test. Significance was denoted at p ≤ 0.05. The statistical anal yses were considered adequate.In vivo Hershberger Assay (201 2) / Page 7 of 12 TEBUCONAZOLE / 128997 OCSPP 890.1400/ OECD 441


## C. METHODS

1. Clinical Examinations: All animals were examined twice daily (once daily on weekends and public holidays) for mortality and moribundit y. Clinical examinations were conducted daily.
2. Body Weight: Animals were weighed at randomization, daily throughout the dosing period and at termination.
3. Food Consumption (Optional): Food consumption was not measured.
4. Serum Hormone Measurements (Optional): Serum hormone measurements were not conducted.
5. Dissection and Measurement of Tissue and Organ Weights: On PND 68 (approximately 24 hours after the final administration of the test substance), all animal s were anesthetized by isoflurane inhalation, exsanguinated, and subjected to a gross necropsy. The five mandatory androgen-dependent organs (ventral prostate, seminal vesi cles, LABC, Cowper’s gland, and glans penis) were excised, trimme d free of adhering tissue, and weighed fresh (unfixed) according to the sta ndard operating procedures deta iled in the U.S. EPA Guideline (OCSPP 890.1400). All animals were observed for preputial separa tion at necropsy.

II. RESULTS

A. OBSERVATIONS
1. Mortality: All animals survived until scheduled termination.
2. Clinical signs of toxicity: No clinical signs of toxicity were observed in animals for any dose groups.

B. BODY WEIGHT AND WEIGHT GAIN: Selected body weight and body weight gain data for the androgen agonist assay are pres ented in Table 2. Body weights throughout the duration of the assay in the tebuconazole-dosed groups and positive (vehicle + TP) control group were comparable to the controls. Ov erall (Days 1-10) body weight gains in the 150 mg/kg/day group were decreased (p<0.05) by 20%. Overall body weight gains in the positive control group were increased (p<0.05) by 29%.In vivo Hershberger Assay (201 2) / Page 8 of 12 TEBUCONAZOLE / 128997 OCSPP 890.1400/ OECD 441


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



### Study Day Dose (mg/kg/day)

Vehicle Control Positive Control, Vehicle + TP (0.4) Tebuconazole (75) Tebuconazole (150)

N Mean SD N Mean SD N Mean SD N Mean SD 

### 1 6 299.2 19.1 6 292.0 22.2 6 303.1 22.6 6 297.5 17.3 4 6 319.3 19.2 6 318.7 24.2 6 323.3 22.1 6 306.6 16.6 7 6 335.3 19.0 6 343.4 25.9 6 340.6 27.8 6 328.0 17.3 11 (termination) 6 360.5 20.4 6 373.0 29.6 6 365.8 30.4 6 348.7 19.3 B ody Weight Gain (Days 1-10) b 

### 6 54.4 4.0 6 70.6** ( ↑ 29) 9.7 6 51.9 10.3 6 43.4* ( ↓ 20) 5.0

a Data were obtained from Tables 2a, 2b, 4a, 4b, and 5 on pages 39, 41, 57, 59, 65, and 66 of the study report. b The study authors did not report total body weight gain, only body weight gain through the final day of dosing. N Number of animals in the group SD Standard Deviation * Significantly different from controls at p<0.05 ** Significantly different from controls at p<0.01



## Selected body weight and body weight gain data for the anti-androgen assay are presented in Table 3. Body weights throughout the study and body weight gain from Days 1 to 10 were similar across all groups. At 75 mg/kg/da y, mean cumulative body weight gains were decreased (p<0.05) from Days 1 to 3 by 24% . At 150 mg/kg/day, mean cumulative body weight gains were decreased (p<0.05) from Days 1 to 3 by 71%, Days 1 to 4 by 33%, and Days 1 to 6 by 16%; mean body weights were not affected by th e end of the study.

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



### Study Day Dose (mg/kg/day) Negative Control, Vehicle + TP (0.4) Positive Control, FT (3) + TP (0.4) Tebuconazole (35) + TP (0.4) Tebuconazole (75) + TP (0.4) Tebuconazole (150) + TP (0.4)

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 

### 1 6 292.0 22.2 6 297.8 23.0 6 293.4 22.7 6 297.0 14.4 6 293.9 17.5 4 6 318.7 24.2 6 322.7 25.7 6 322.6 22.0 6 320.4 17.0 6 311.7 16.2 7 6 343.4 25.9 6 347.1 26.4 6 340.5 23.0 6 345.3 17.6 6 345.9 14.7 11 (termination) 6 373.0 29.6 6 379.6 32.2 6 379.4 21.5 6 380.7 19.9 6 371.6 15.8 B ody Weight Gain (Days 1-10) b 

### 6 70.6 9.7 6 70.9 9.5 6 75.5 4.7 6 70.6 9.0 6 67.3 10.0

a Data were obtained from Tables 2c, 2d, 4c, 4d, and 5 on pages 43, 45, 61, 63, 67, and 68 of the study report. b The study authors did not report total body weight gain, only body weight gain through the final day of dosing. N Number of animals in the group SD Standard Deviation



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

D. SERUM HORMONE CONCE NTRATIONS (Optional): Serum hormone measurements were not conducted.In vivo Hershberger Assay (201 2) / Page 9 of 12 TEBUCONAZOLE / 128997 OCSPP 890.1400/ OECD 441


## E. ORGAN WEIGHTS: Accessory sex organ and liver weights for the androgen agonist assay are presented in Table 4. The accesso ry sex organ weights in all tebuconazole treatment groups were comparable to the vehi cle controls. Rats in the positive control (vehicle + TP) group had accessory sex organ we ight increases (p<0.05) as follows: 992% in seminal vesicles; 602% in ventral prostate ; 160% in LABC; 449% in Cowper’s glands; and 65% in glans penis.
The coefficients of variance (CVs) for the acces sory sex organs in the vehicle control and high dose (150 mg/kg/day) groups were compar ed to the performance criteria in the Guideline. All CV values were less than the maximum allowable values for each organ, except for the %CV for LABC in the cont rol group (CV = 33%; maximum recommended CV = 30%).

TABLE 4. Accessory Sex Organ Weight s (g) from the Androgen Agonist Assay in Sprague Dawley Rats a

Organ Dose (mg/kg/day)

Vehicle control Tebuconazole (75) Tebuconazole (150) Positive Control, Vehicle + TP (0.4) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Seminal vesicles 6 0.0449 0.0143 31.9 6 0.0471 0.0098 20.7 6 0.0433 0.0113 26.1 6 0.4904** ( ↑ 992) 0.1721 35.1

Ventral prostate 6 0.0312 0.0096 30.6 6 0.0232 0.0055 23.8 6 0.0207 0.0057 27.8 6 0.2189** ( ↑ 602) 0.0539 24.6

LABC 6 0.2177 0.0728 33.4 b 6 0.2101 0.0571 27.2 6 0.2107 0.0448 21.3 6 0.5666** ( ↑ 160) 0.0883 15.6

Cowper’s glands 6 0.0069 0.0025 36.1 6 0.0059 0.0022 37.9 6 0.0070 0.0016 23.4 6 0.0379** ( ↑ 449) 0.0098 26.0

Glans penis 6 0.0503 0.0046 9.2 6 0. 0476 0.0059 12.5 6 0.0503 0.0056 11.2 6 0.0828** ( ↑ 65) 0.0076 9.2

a Data were obtained from page 26, and Table 6 on pages 70 -71 of the study report. Percent differences from controls, calculated by the reviewers, are included in parentheses. b The %CV exceeded the maximum allowable value (30%) of the guideline performance criteria. N Number of animals in the group SD Standard Deviation CV Coefficient of Variation NA Not applicable ** Significantly different from controls at p<0.01



## Accessory sex organ and liver weights for the anti-androgen assay are presented in Table 5. The accessory sex organ weights in all tebucona zole treatment groups were comparable to the negative (vehicle + TP) controls. Rats in the positive control (FT + TP) group had accessory sex organ weight decreases (p<0.05) as follows: 71% in seminal vesicles; 69% in ventral prostate; 38% in LABC; 52% in Cowp er’s glands; and 22% in glans penis.
The CVs for the accessory sex organs in th e negative control and 150 mg/kg/day groups were compared to the performan ce criteria in the Guideline. All %CV values were less than the maximum recommended values.In vivo Hershberger Assay (201 2) / Page 10 of 12 TEBUCONAZOLE / 128997 OCSPP 890.1400/ OECD 441


### TABLE 5. Accessory Sex Organ Weig hts (g) from the Anti-Androgen As say in Sprague Dawley Rats a

Organ Dose (mg/kg/day) Negative Control, Vehicle + TP (0.4) Tebuconazole (35) + TP (0.4) Tebuconazole (75) + TP (0.4) Tebuconazole (150) + TP (0.4) Positive Control, FT (3) + TP (0.4) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Seminal vesicles 6 0.4904 0.1721 35.1 6 0.5229 0.0953 18. 2 6 0.4993 0.0926 18.6 6 0.4472 0.1566 35.0 6 0.1413** ( ↓ 71) 0.0321 22.7

Ventral prostate 6 0.2189 0.0539 24.6 6 0.2012 0.0244 12. 1 6 0.2040 0.0206 10.1 6 0.1930 0.0225 11.7 6 0.0683** ( ↓ 69) 0.0192 28.2

LABC 6 0.5666 0.0883 15.6 6 0.6251 0.0725 11.6 6 0.6440 0.0429 6.7 6 0.5721 0.0405 7.1 6 0.3491** ( ↓ 38) 0.0824 23.6

Cowper’s glands 6 0.0379 0.0098 26.0 6 0.0390 0.0064 16. 4 6 0.0309 0.0112 36.3 6 0.0340 0.0106 31.3 6 0.0183** ( ↓ 52) 0.0057 31.2

Glans penis 6 0.0828 0.0076 9.2 6 0.0878 0.0121 13. 8 6 0.0837 0.0077 9.2 6 0.0826 0.0117 14.1 6 0.0642** ( ↓ 22) 0.0073 11.4

a Data were obtained from page 26, and Table 6 on pages 72 -73 of the study report. Percent differences from controls, calculated by the reviewers, are included in parentheses. N Number of animals in the group SD Standard Deviation CV Coefficient of Variation NA Not applicable ** Significantly different from controls at p<0.01



## III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: The data indicate that adm inistration of up to 150 mg/kg/day of tebuconazole for 10 days to castrated Sprague-Dawley male rats
had no effect on the five androgen dependent tissues weights. No evidence of androgenic or anti-androgenic (or 5- α -reductase inhibition) potential was detected under conditions of this study.

B. AGENCY COMMENTS: All animals survived until scheduled termination. No clinical signs of toxicity were observed in animal s for any dose groups. On the day of necropsy, preputial separation was observe d in all treated animals.
In the androgen assay, body weights thr oughout the duration of the assay in the tebuconazole-dosed groups and positive (vehic le + TP) control group were comparable to the controls. Overall (Days 1-10) body wei ght gains in the tebuconazole 150 mg/kg/day groups were decreased (p<0.05) by 20%. Overa ll body weight gains in the positive control group were increased (p<0.05) by 29%. No trea tment-related effects were observed in any of the accessory sex organs of tebuconazole-dosed animals. Animals in the positive control (vehicle + TP) group showed the expected in creases in accessory sex organ weights.
In the anti-androgen assay, body weights throug hout the study and body weight gain from Days 1 to 10 were similar across all groups. At 75 mg/kg/day, mean cumulative body weight gains were decreased (p<0.05) from Days 1 to 3 by 24%. At 150 mg/kg/day, mean cumulative body weight gains were decreased (p <0.05) from Days 1 to 3 by 71%, Days 1 toIn vivo Hershberger Assay (201 2) / Page 11 of 12 TEBUCONAZOLE / 128997 OCSPP 890.1400/ OECD 441 

## 4 by 33%, and Days 1 to 6 by 16%; mean body wei ghts were not affected by the end of the study. The accessory sex organ weights in all tebuconazole treatment groups were comparable to the negative controls. Anim als in the positive c ontrol (FT + TP) group showed the expected decreases in accessory sex organ weights.
The CVs for the accessory sex organs in the vehicle (negative) c ontrol and high dose (150 mg/kg/day) groups were less than the maximum allowable values for each organ in both the androgen agonist and anti-androgen assays, ex cept for the %CV for LABC in the control group (CV = 33%; maximum recommended CV = 30%).
The dosing in this study was considered ad equate based on the d ecreased overall body weight gains observed at th e high-dose (150 mg/kg/day).
No statistically significant changes were seen in two or more of the five androgen sensitive tissue weights. Tebuconazole was negative for androgenicity and anti-a ndrogenicity in the Hershberger assay.

C. STUDY DEFICIENCIES: The following deficiencies were noted that are not considered to have had an adverse impact on the results, interpretation or conclusions of this study:

 The maximum guideline-specified %CV was exceeded for the LABC (33% vs. 30% maximum CV) in the control of the androgen agonist assay.In vivo Hershberger Assay (201 2) / Page 12 of 12 TEBUCONAZOLE / 128997 OCSPP 890.1400/ OECD 441


## APPENDIX
A summary of a preliminary rang e-finding study was included in th e Hershberger study report as Appendix H (pages 114-119). As this is a range-finding study, only a summary is provided to confirm the adequacy of the dos e selection rationale used in the Hershberger assay (MRID 48673702).
In a preliminary range-finding assay (Appendix H, MRID 48673702), tebuconazole (97.5% a.i.; Batch# K689052) in 0.5% aqueous methylcellulose 400 + 0.4% Tween 80 was administered via daily oral gavage in a dose volume of 5 mL/kg body weight to groups of 3 adult male Sprague Dawley [Crl:CD(SD)] rats at doses of 0, 80 150, or 300 mg/kg/day. The animals were dosed for 10 consecutive days and necropsied approximately 24 hours after the final dose administration.

All animals survived until sche duled termination and no clinical signs were observed. At 300 mg/kg/day, mean body weight wa s decreased by 11% on Day 3, corresponding to a body weight loss of 5.9 g compared to a body weight gain of 20.5 g for controls. The terminal body weight was decreased by 11%, and the mean cumulative body weight gain between Days 1 and 11 was decreased by 43%.
At 150 mg/kg/day, mean body weight was re duced by 10% on Day 3, corresponding to a body weight loss of 5.4 g compared to a body weight gain of 20.5 g fo r controls. The terminal body weight was decreased comp ared to the controls ( ↓ 8%). The terminal body weight was decreased by 8%, and the mean cumulative body weight ga in between Days 1 and 11 was decreased by 32%.
At 80 mg/kg/day, mean cumulativ e body weight gain between Da ys 1 and 11 was decreased by 14%.
It did not appear that statistical analyses were conducted.

COMPLIANCE: It was stated that the study was not performed in compliance with GLPs in that it was not subjected to specific Quality Assurance inspections.

# DATA EVALUATION RECORD

TEBUCONAZOLE
Study Type: OCSPP 890.1450, Female Pubertal Assay

EPA Contract No. EP10H001452 Task Assignment No. 3-20-2012 (MRID 48688302)

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: 9/14/2012
Secondary Reviewer: Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 9/28/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 10/04/2012
Quality Assurance: Signature: Jack D. Early, M.S. Date: 10/04/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).

### TEBUCONAZOLE / 128997 

## Primary Reviewer:**~R~a** 

## .....**v~K~e=n=t,~P** 

## .... h=.D"'-'-.

______ _ 

## Health Effects Division Secondary Reviewer: Elizabeth Mendez, Ph.D. 

## Health Effects Division

DATA EVALUATION RECORD

STUDY TYPE: Female Pubertal Assay; OCSPP 890.1450; OECD None.



## PC CODE: 128997 

## DP BARCODE: D404684

TXR#: 0056253 

## CAS No: 107534-96-3



## TEST MATERIAL (PURITY): Tebuconazole (97.5% a.i.)



## SYNONYMS: AE F069623; a-[2-( 4-chlorophenyl)ethyl]-a-(l, 1-dimethylethyl)-lH-1,2,4- triazole-1-ethanol



## CITATION: Lautraite, S. (2012) Tebuconazole: Assessment of Pubertal Development and Thyroid Function in Juvenile/Peripubertal Male and Female Rats. Bayer S.A.S, Sophia Antipolis Cedex, France. Laboratory Report No.: SA 11013; June 26, 2012. MRID 48688302. Unpublished.



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



## TEST ORDER#: CON-128997-20



## EXECUTIVE SUMMARY: In 

## a Female Pubertal Assay (MRID 48688302), 15 female Crl(CD)SD rats/dose group were treated daily via oral gavage with tebuconazole (97.5% a.i., Lot # K689052) in 0.5% methylcellulose 400 

## + 0.4% Tween 80 at doses ofO (vehicle), 75 or 150 mg/kg/day from post-natal day (PND) 22 to 42. Animals were examined for vaginal opening (VO) daily beginning on PND 22, and age and weight at day of attainment were recorded. Following sacrifice on PND 42, blood was collected for clinical chemistry analyses, and total thyroxine (T4) and thyroid stimulating hormone (TSH) levels were determined using enzymelinked immunoassay (ELISA) kits. Adrenal, liver, pituitary
, thyroid, and urogenital organs were weighed, and microscopic examinations were performed on the ovaries
, uterus, thyroid, and kidneys.

No adverse, treatment-related effects were observed on mortality; estrous cyclicity; gross pathology in the liver, kidneys, or adrenal glands; or histopathology in the kidneys. No treatment-related effect was noted on serum T4 or TSH levels, or on thyroid weight or morphology.

All rats survived the 21-day test period. Mean daily body weight and body weight gain/day in the 150 mg/kg/day animals was decreased on several days through PND 33
, but there were no treatment-related changes in 

## final body weight or cumulative body weight gain (PND 22-42). Compared to the control, the mean age at initial VO was increased by 1.2 days (not significant
,Female Pubertal Assay (2012) / Page 2 of 13 TEBUCONAZOLE / 128997 OCSPP 890.1450/ OECD None


## NS) and 2.7 days (p<0.01) at 75 and 150 mg/kg/da y, respectively, and the mean age at complete VO was increased by 1.1 days (NS) and 2.0 days (p<0.05) at 75 and 150 mg/kg/day, respectively. However, there were no signifi cant differences in body we ights between groups at initial and complete VO.

Administration of tebuconazole did not change the day of first es trus compared to the vehicle control group. Animals exhibited similar cycl e lengths and regularity, and the majority of females in the 150 mg/kg/day group were in diestrus at necropsy.
Liver weights and relative (to body) liver weights were increased (p<0.01) by 8% at75 mg/kg/day and by 15-18% at 150 mg/kg/day, and th ere was an increase (p<0.01) of 6% in relative (to body) kidney weights at 150 mg/kg/day. No other tr eatment-related changes were observed in organ weights.
Effects on clinical chemistry parameters included decreased mean creatinine ( ↓ 8%; p ≤ 0.01) and increased total cholesterol ( ↑ 17%; p<0.01) concentrations in the 150 mg/kg/day animals, and decreased blood urea nitrogen ( ↓ 15-17%), total protein ( ↓ 6%), and total bilirubin ( ↓ 100%) concentrations (p<0.05 or 0.01) in female s administered 75 or 150 mg/kg/day.

There were no treatment-re lated changes in serum T 4 

## and TSH levels or on thyroid weight or morphology associated with tebuconazole treatment.
No overt toxicity was observ ed during treatment, but an earlier range-finding study noted increased salivation at 300 and 150 mg/kg/day, as well as a decrease in body weight gain. The doses tested were determined to be adequate since increased salivati on, and decreased daily body weight and body weight gain we re see at the high dose.
Although increases in the age of initial and complete VO were observed in the tebuconazole treated rats, body weights at VO were similar be tween the control and treated groups, suggesting that the delay in VO for the tebu conazole treated groups resulted from the delayed growth in these groups during the earl y part of the study.
The assay satisfies the EDSP Tier 1 Test Order require ment for a Female Pubertal Assay (OCSPP 890.1450).

COMPLIANCE: Signed and dated Data Confiden tiality, GLP Compliance, and Quality Assurance statements were provided.Female Pubertal Assay (2012) / Page 3 of 13 TEBUCONAZOLE / 128997 OCSPP 890.1450/ OECD None



## I. MATERIALS AND METHODS

A. MATERIALS
1. Test Facility: Bayer S.A.S Location: Sophia Antipolis Cedex, France Study Director: Lautraite, S., and Langrand-Lereche, C (Replacement Study Director) Other Personnel: Kocwin, J.P. (Laboratory Animal Resources), Ja ussely, C. (In-Life Supervisor), Trela, B. (Responsible Technician), Pallen , C. (Statistici an), Amir Tahmasseb, L. (Senior Chemist), DeBarbeyrac, D. (Clinical Pat hology Supervisor). Schorsch, F. (Pathologist), and Vagner, M., (Report Unit Assistant) Study Period: September 28, 2011 – June 26, 2012



## 2. Test Substance: Tebuconazole Description: White powder Source: Bayer CropScience (Frankfurt, Germany) Lot/Batch #: K689052 (expiry date: August 23, 2012) Purity: 97.5% a.i. Stability: Stable at 25°C ± 5°C; material was stored in an air-tight, light-resistant container at RT Stable in vehicle for up to 22 days at 5  3 °C. CAS #: 107534-96-3 Structure:
Cl OH CH 3

CH 3 CH 3

N N N



## 3. Vehicle: 0.5% methylcellulose 400 + Tween 80

4. Test Animals: Species: Rat Strain: Crl:CD(SD) Sprague-Dawley Age/Weight at Study Initiation: PND 21/48.9-63.7 g females only

Source: Charles River Laboratories, St Germain-sur-l' Arbresle, France Housing: 2-3/cage in makrolon cages with heat-treated laboratory-grade wood shavings (not cedar) Diet: A04C-10 pelleted rodent diet, S.A.F.E (Sci entific Animal Food and Engineering, Augy, France), ad libitum Phytoestrogen content <300 μ g of genistein equivalents/gram diet Water: Filtered Tap water, ad libitum Environmental Conditions: Temperature: Humidity: Air changes: Photoperiod: 20-24 °C 40-70% 10-15/hr 14 hrs light/ 10 hrs dark



## B. STUDY DESIGN

1. In-Life Dates: Start: November 4, 2011 End: November 25, 2011
2. Mating: Time-mated pregnant dams were received from the supplier on gestation day 7/8. Litters with fewer than 8 pups or that were not delivered on GD 22 or 23, were excluded from the study. Litters with more than 8 pups were standardized by culling to 8 pups per litter on PND 4, with an equal number of males and females (if possible).Female Pubertal Assay (2012) / Page 4 of 13 TEBUCONAZOLE / 128997 OCSPP 890.1450/ OECD None



## 3. Animal Assignment: Offspring were weaned on PND 21 and weighed. Animals were assigned using a randomization procedure that ensured a si milar body weight distribution among groups and avoided, as much as possible, the need for placing littermates in the same dose group. The acceptable body weight range wa s ±20% of the mean body weight on the day of randomization. Animals were allocate d to the test groups noted in Table 1.

TABLE 1. Study Design a 

### Test group Dose (mg/kg/day) # of Females Control 0 15 Low 75 15 High 150 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 preliminary range finding study. Groups of 4 juvenile/peripubertal female rats were treated by gavage for 10 consecutive days at 0, 80, 150 and 300 mg/kg/day. No mortality was observed at any dose level and clinical si gns consisted of incr eased salivation at 300 mg/kg/day (males and females) and 150 mg /kg/day (females only). At 300 mg/kg/day, cumulative body weight gain in females wa s reduced by 83% on Study Days 1-3 and by 45% on Study Days 1-5. At 150 mg/kg/day, cu mulative body weight gain in females was reduced by 46% on Study Days 1-3. At 80 mg /kg/day, mean body weight gains in females were reduced by 18% on Study Days 1-3. No treatment-related effects were observed at necropsy at any dose level.
5. Dose Preparation and Analysis: Dose formulations were prepared twice during the study by mixing appropriate amounts of tebuconazole with 0.5% methylcellulose 400 + 0.4% Tween 80. Stability of tebuconazole wa s demonstrated in a previous study 1 

## , and was found to be stable at concentrati ons of 0.6 and 170 g/L for up to 22 days under similar conditions of usage and storage to those of the current study. Homogeneity of the dose suspensions was checked at concentrations of 15 and 30 g/L on the first se t of formulation. During the study, samples of dose formulations were an alyzed for all formulations for achieved concentration.

Results of Dose Analysis
Homogeneity (% CV): 0.59%
Stability: Stable under the conditi ons of the current study

Concentration (% nominal): 97-102%
The analytical data indicated that the mixing procedure was ad equate and that the variation between nominal and actual dosage to the animals was acceptable.

1 

### Amir Tahmasseb, L. (2011) Tebuconazole, Stability in a queous 0.5% methylcellulose 400 + 0.4% tween 80, Bayer CropScience, Sophia Antipolis, France (SA 11273)Female Pubertal Assay (2012) / Page 5 of 13 TEBUCONAZOLE / 128997 OCSPP 890.1450/ OECD None



## 6. Dosage Administration: All doses were administered once daily by gavage, from PND 22 through PND 42, in a volume of 5 mL/kg of bo dy weight. Dosing was performed between 0700 and 0900 hours daily.

7. Statistics: The following statistical analyses were performed. Statistical significance was reported when p<0.05. The analyses were considered appropriate

Parameter Statistical Test Initial body weight, body weight gains, clinical chemistry parameters and hormonal measurements Bartlett's test followed by a Kruskal-Wallis test and Dunn test if found to be significant (p ≤ 0.05) Non-significant results from the Bartlett test were subjected to ANOVA, and Dunnett's test if found to be significant. If significant, body weight parameters were transformed and the Bartlett test was conducted again on the transformed data. Age and body weight at VO and PPS. Absolute organ weights, relative organ weights, body weight at PND 21 and terminal body weight. Subjected to Levene's test, and transformed if found to be significant. Subjected to ANOVA followed by Dunnett's test if not significant, and a Kruskal-Wallis test, followed by Dunn’s test, if significant. All parameters, except terminal body weights, age at first estrus, and cycle length. ANCOVA with body weight on PND 21 as the covariate. Adjusted means were compared with Dunnett’s test.



## C. METHODS
1. Mortality and Clin ical Examinations: All animals were examined twice each day for mortality and moribundity. Clinical ex aminations were conducted daily.
2. Body Weight: Animals were weighed prior to allocation, daily prior to dosing, and terminal body weights were reco rded before final sacrifice.
3. Vaginal Opening (VO): Beginning on PND 22, all animals were examined daily for onset of VO. The appearance of a small pin hole, a vaginal thread, or complete VO was recorded daily following dosing. The age at initial VO was defined by the first day showing any stage of VO (pin hole, thread or complete VO) . The first day a female attained complete VO was designated as the age at complete VO. The observation for VO was stopped once complete VO was recorded for 2 consecutive days. In cases where VO or compete VO had not occurred prior to necropsy, age at necr opsy plus one day (PND 43) and weight at necropsy (on PND 42) were used for those animals for calculati on of group means.
4. Estrous Cyclicity: Beginning on the first day of complete VO, through and including the day of necropsy (PND 42), vaginal smears were prepared and evalua ted under a low-power light microscope for the presence of leukocytes , nucleated or cornifie d epithelial cells. The vaginal smears were classified as diestrus, proestrus or estrus. The age at first vaginal estrus was noted. Metestrus was classified as diestr us. The overall estrous cycle pattern of each female was characterized as re gular (recurring 4 to 5 day cycl es), irregular (having cycles with diestrus for a period > 3 days or estrus > 2 days), or not cy cling (having prolonged period 5 days of diestrus or 3 consecutive days of estrus). Mean cycle length was defined as the number of days from the first day of dies trus to the first day of the next diestrus.Female Pubertal Assay (2012) / Page 6 of 13 TEBUCONAZOLE / 128997 OCSPP 890.1450/ OECD None


## Incomplete cycles were not used in calcula ting mean cycle length. In cases when the time between VO and euthanasia did not allow observation of more than 1 complete cycle, classification was based on the av ailable data with the assumption that animals are cycling regularly if the partial data fit the definition, or cycling irregu larly if the study ended without being able to distinguish between irregular cycling and not cycling.
5. Sacrifice and Pathology: On the day of termination, rats were removed one at a time from their home cages to a separate room for euthan asia and terminal proc edures so as not to induce stress-related responses in other animal s which might affect hormone measurements. All surviving animals were sa crificed by isoflurane anesthesia followed by exsanguination on PND 42 approximately 2 hours post-dosing. All collections were completed by 1300 hours. Blood from the trunk of the animals wa s collected immediately into serum separation tubes, processed with clot activation, and stored at

− 74 ° C for subsequent hormone and clinical chemistry evaluations. Hist orical control data were provided.
a. Hormone Analysis: Total T 4 

## and TSH levels were determined with ELISA kits (mouse/rat thyroxine kit, Calbiotech Inc., Spring Valle y, CA and rodent thyroi d stimulating hormone kit, Endocrine Technologies, Newark, CA, respectively).
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 Globulins X Sodium X Glucose
ENZYMES X Total bilirubin X Alkaline phosphatase (ALP) X Total protein Cholinesterase (ChE) Triglycerides Creatine phosphokinase Se rum protein electrophoresis Lactic acid dehydrogenase (LDH) X Alanine aminotransferase (ALT/also SGPT) X Aspartate aminotransfera se (AST/also SGOT) Sorbitol dehydrogenase (SDH) X Gamma glutamyl transferase (GGT) Glutamate dehydrogenase * Recommended for the pubertal assay in fe male rats based on Guideline 890.1450.


## c. Organ Weights and Histopathology: The following CHECKED (X) tissues were collected and weighed. The (XX) organs, in addition, were subject ed to histological examination.Female Pubertal Assay (2012) / Page 7 of 13 TEBUCONAZOLE / 128997 OCSPP 890.1450/ OECD None

UROGENITAL OTHER XX Ovaries (paired, without oviducts)*+ XX Thyroid*+ XX Uterus*+ X Liver* XX Kidneys (paired)*+ X Adrenals (paired)* X Pituitary* * Weights required based on Guideline 890.1450 + Histopathological examination re quired based on Guideline 890.1450


## All organs collected, except th e thyroid/trachea and uterus we re weighed prior to fixation. The uterus was trimmed free of fat and conn ective tissue and excised from each animal. The vagina was removed from the uterus just below the cervix so that the cervix remained with the uterine body. The ovary and oviducts we re removed, avoiding loss of luminal fluid from the uterine horns. Each uterus was placed securely in a Petri di sh carefully to avoid desiccation before weighing. The uterus with luminal fluid was weighed (wet uterine weight). Then, the uterus was pierced and blotted to remove excess luminal fluid, and weighed (blotted uterine weight). In the fina l report, all organ wei ghts were reported in milligrams to the nearest 0.1 mg, with the excep tion of liver and kidney weights reported in grams to the nearest 0.01 g. Tissues were preserved in 10% neutral buffered formalin, except for testis and epididymis, which were preserved in modified Davidson’s fixative.

II. RESULTS

A. MORTALITY: All animals survived until scheduled termination.

B. CLINICAL SIGNS OF TOXICITY: Treatment-related clinical signs of toxicity were confined to increased salivation in 2/15 females observed on one or more occasions throughout the study at 75 and 150 mg/kg/day.

C. GENERAL GROWTH AND VAGINAL OPENING: Body weights, body weight gains, and age and weight at day of attainment of VO are presented in Ta ble 2. Mean daily body weight was decreased (p ≤ 0.05 or 0.01) on Study Days 2, 3, 6- 9, and 11 by 7-9% in the 150 mg/kg/day animals. Mean body weight gain / in the 150 mg/kg/day animals on study Day 2 was -0.9 g compared to a gain of 4.4 g in the controls (-123%). p ≤ 0.01). Cumulative body weight gain was -32% on Day 4, -26% on Day 6 (p<0.01), -14% (p ≤ 0.05) on Day 11, and - 1% on Day 21. Mean body weight gain/day in the 75 mg/kg/day animals was decreased 73% on Study Day 2, and increased 2% on Da y 21. There were no treatment-related changes in final body weight or cumula tive body weight ga in (PND 22-42).
Compared to the control group, the mean age at initial VO was increased by 1.2 days (NS) and 2.7 days (p<0.01) at 75 and 150 mg/kg/day, re spectively, and the mean age at complete VO was increased by 1.1 days (NS) and 2.0 days (p<0.05) at 75 and 150 mg/kg/day, respectively. Body weights at initial and complete VO were similar across dose groups, although there were slight, non-si gnificant increases in body wei ght of 4% at 75 mg/kg/day and 5-8% at 150 mg/kg/day.
The mean age at VO, body weight at VO, and final body weight in the control group were within the acceptable range of the performa nce criteria provided in the Guideline (890.1450).Female Pubertal Assay (2012) / Page 8 of 13 TEBUCONAZOLE / 128997 OCSPP 890.1450/ OECD None



### TABLE 2. General Growth and Vaginal Opening (VO) a

Parameter Evaluated Vehicle Control Tebuconazole (75 mg/kg/day) Tebuconazole (150 mg/kg/day)

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Initial body weight (PND 22; g) U 15 58.7 3.8 6.5 15 59.2 4.0 6.7 15 59.1 4.7 7.9

Body weight at initial vaginal opening (g) U 15 122.34 12.4 10.1 15 127.14 ( ↑ 4) 14.4 11.3 15 128.32 ( ↑ 8) 20.4 15.9

A 15 122.5 4.1 3.3 15 127.11 ( ↑ 4) 4.1 3.2 15 128.19 ( ↑ 8) 4.1 3.2

Body weight at complete vaginal opening (g) U 15 122.9 13.2 10.7 15 128.23 ( ↑ 4) 15.0 11.7 15 132.33 ( ↑ 5) 20.7 15.6

A 15 123.02 4.3 3.5 15 128.21 ( ↑ 4) 4.3 3.3 15 132.22 ( ↑ 5) 4.3 3.2

Final body weight (g) U 15 168.3 12.3 7.3 15 168.9 12.5 7.4 15 164.0 14.3 8.7 Final body weight (% of control) U 15 NA NA NA 15 100.1 NA NA 15 97.4 NA NA Body weight gain (final – initial; g) U 15 108.3 9.4 8.7 15 110.3 11.0 10.0 15 107.3 11.6 10.8

Age at initial vaginal opening (PND) U 15 33.40 1.8 5.3 15 34.53 ( ↑ 1.2 d) 1.8 5.1 15 35.40* ( ↑ 2.7 d) 2.5 7.1

A 15 33.39 0.5 1.5 15 34.54 ( ↑ 1.2 d) 0.5 1.4 15 35.41* ( ↑ 2.7 d) 0.5 1.4

Age at complete vaginal opening (PND) U 15 33.47 1.8 5.4 15 34.67 ( ↑ 1.1 d) 1.8 5.1 15 36.13** ( ↑ 2.0 d) 3.2 8.9

A 15 33.44 0.6 1.8 15 34.67 ( ↑ 1.1 d) 0.6 1.7 15 36.15** ( ↑ 2.0 d) 0.6 1.6

Proportion unopened (#/N) 0/15 0/15 1/15 a Data were obtained from page 34 of the study report. Va lues in parentheses were calc ulated by reviewers and are % difference from control for body weight s at VO and difference from control in number of days for age at VO. U Unadjusted for body weight on PND 22 A Adjusted for body weight on PND 22 N Number of animals examined SD Standard Deviation CV Coefficient of Variation * Significantly different from controls at p ≤ 0.05. ** Significantly different from controls at p ≤ 0.01. NA Not applicable



## D. ORGAN WEIGHTS: Organ weights at necropsy are pr esented in Table 3. Increased liver weights and relative liver wei ghts (p<0.01) were observed in the 75 mg/kg/day (8%) and 150 mg/kg/day (15-18%) dose groups, as well as an increase in relative kidney weight (p<0.01, 6%). Although not significant, there were slight decreases (6-8%) in mean ovary weights at both tebuconazole dose levels, a nd there was a dose-dependent decrease inuterine weights of 2-3% at 75 mg/kg/day and 15% at 150 mg/kg/day. No other treatmentrelated changes were observ ed in organ weights.
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).Female Pubertal Assay (2012) / Page 9 of 13 TEBUCONAZOLE / 128997 OCSPP 890.1450/ OECD None



### TABLE 3. Organ Weights at Necropsy a

Organ Vehicle Control Tebuconazole (75 mg/kg/day) Tebuconazole (150 mg/kg/day)

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Liver (g) U 15 6.92 0.61 8.8 15 7.49 0.53 7.0 15 7.99** ( ↑ 15) 0.94 11.8

A 15 6.94 0.159 NA 15 7.48* ( ↑ 8) 0.159 NA 15 7.98** ( ↑ 15) 0.159 NA

R 15 4.1127 0.1932 4.7 15 4.4393** ( ↑ 8) 0.2139 4.8 15 4.8647** ( ↑ 18) 0.2669 5.5 Kidneys (g) U 15 1.46 0.08 5.7 15 1.48 0.12 8.0 15 1.51 0.14 9.6 A 15 1.46 0.027 NA 15 1.48 0.027 NA 15 1.51 0.027 NA

R 15 0.8693 0.0477 5.5 15 0.8813 0.0553 6.3 15 0.9220** ( ↑ 6) 0.0397 4.3 Pituitary (mg) U 15 9.07 1.11 12.2 15 8.94 0.94 10.5 15 8.42 0.60 7.2 A 15 9.08 0.240 NA 15 8.95 0.239 NA 15 8.39 0.259 NA R 15 0.0054 0.0007 13.2 15 0.0053 0.0007 12.2 15 0.0051 0.0004 8.6 Adrenals (mg) U 15 38.71 7.69 19.9 15 39.03 6.10 15.6 15 36.47 5.77 15.8 A 15 38.81 1.588 NA 15 39.01 1.587 NA 15 36.39 1.588 NA R 15 0.0230 0.0040 17.4 15 0.0232 0.0038 16.3 15 0.0223 0.0030 13.3 Ovaries (mg) U 15 64.7 11.00 17.1 15 61.1 ( ↓ 6) 7.5 12.2 15 59.9 ( ↓ 7) 11.1 18.6

A 15 64.8 2.57 NA 15 61.1 ( ↓ 6) 2.57 NA 15 59.8 ( ↓ 8) 2.57 NA
Uterus, wet (mg) U 15 369.3 165.1 44.7 15 358.2 ( ↓ 3) 115.4 32.2 15 314.1 ( ↓ 15) 174.8 55.7

A 15 370.1 39.90 NA 15 358.1 ( ↓ 3) 39.89 NA 15 313.4 ( ↓ 15) 39.90 NA Uterus, blotted (mg) U 15 300.3 62.4 20.8 15 307.5 ( ↑ 2) 70.7 23.0 15 255.2 ( ↓ 15) 76.7 30.0

A 15 300.9 17.90 NA 15 307.4 ( ↑ 2) 17.89 NA 15 254.7 ( ↓ 15) 17.9 NA Thyroid, fixed (mg) U 15 13.8 1.6 11.6 15 12.8 1.5 11.5 15 14.1 1.9 13.4 A 15 13.8 0.43 NA 15 12.8 0. 43 NA 15 14.1 0.43 NA a Data were obtained from page 38 of the study report. Percent differences from controls, calculated by the reviewers, are included in parentheses. U Unadjusted for body weight on PND 22 A Adjusted for body weight on PND 22 N Number of animals examined SD Standard Deviation CV Coefficient of Variation R Organ-to-body weight ratio (relative to body weight) * Significantly different from controls at p ≤ 0.05. ** Significantly different from controls at p ≤ 0.01.



## E. ESTROUS CYCLICITY: Estrous cycle data are provi ded in Table 4. There was no treatment-related effect on estrous cyclicity at any dose level.Female Pubertal Assay (2012) / Page 10 of 13 TEBUCONAZOLE / 128997 OCSPP 890.1450/ OECD None



### TABLE 4. Estrous Cyclicity a

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

(%) Cycle Status at Necropsy (# Females)

Diestrus Proestrus Estrus Not Cycling Vehicle 15 35.47 5.03 93 92.9 4 4 7 0

Tebuconazole (75 mg/kg/day) 15 35.73 4.79 100 100 7 5 3 0 Tebuconazole (150 mg/kg/day) 15 36.08 5.00 100 85.7 10 1 3 1 a Data were obtained from page 39 of the study report. N Number of animals



## F. CLINICAL CHEMISTRY AND HORMONE LEVELS: Mean hormone and clinical chemistry levels are presented in Table 5. Th ere were no treatment related changes in serum T 4 

## and TSH associated with tebuconazole treatm ent. Rats administered 150 mg/kg/day had a treatment-related lower mean creatinine level ( ↓ 8%; p ≤ 0.01), and increased total cholesterol ( ↑ 17%; p<0.01). Rats administered 75 or 150 mg/kg/day had treatment-related decreases (p<0.05 or 0.01) in blood urea nitrogen ( ↓ 15-17%), total protein ( ↓ 6%), and total bilirubin ( ↓ 100%). There were no other treatment-re lated changes to clinical chemistry; effects on ALT and potassium concentrations that were only observed in the 75 mg/kg/day group, and not dose related, were considered incidental.

The results for serum T 4 

## in the control group were with in the acceptable range of the performance criteria provid ed in the Guideline (890.1450).Female Pubertal Assay (2012) / Page 11 of 13 TEBUCONAZOLE / 128997 OCSPP 890.1450/ OECD None



### TABLE 5. Hormone Levels and Clinical Chemistry a

Parameter Evaluated Vehicle Control Tebuconazole (75 mg/kg/day) Tebuconazole (150 mg/kg/day)

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Hormones Serum T 4 , Total (μg/dL) 15 3.2 0.4 13 15 3.3 0.7 21 15 3.2 0.4 13

Serum TSH (ng/mL) 15 1.57 1.47 94 14 2.26 1.30 58 14 2.64 1.69 64

Clinical Chemistry Creatinine (μmol/L) 15 40 3 NA 15 38 2 NA 15 37** ( ↓ 8) 2 NA

Blood urea nitrogen (mmol/L) 15 4.58 0.67 NA 15 3.89* ( ↓ 15) 0.58 NA 15 3.81** ( ↓ 17) 0.73 NA

Total Protein (g/L) 15 52 3 NA 15 49** ( ↓ 6) 1 NA 14 49** ( ↓ 6) 2 NA

Albumin (g/L) 15 34 3 NA 15 32 1 NA 14 32 1 NA Total bilirubin (μmol/L) 15 0.2 0.2 NA 15 0.00** ( ↓ 100) 0.1 NA 14 0.0** ( ↓ 100) 0.1 NA

Glucose (mmol/L) 15 7.41 0.49 NA 15 7.33 0.36 NA 14 7.75 0.51 NA Total cholesterol (mmol/L) 15 1.97 0.17 NA 15 1. 96 0.20 NA 14 2.23** ( ↑ 17) 0.29 NA

AST (IU/L) 15 96 19 NA 15 101 15 NA 14 94 20 NA ALT (IU/L) 15 31 3 NA 15 34* ( ↑ 10) 4 NA 14 37 4 NA

AP (IU/L) 15 287 35 NA 15 291 53 NA 14 292 56 NA GGT (IU/L) 15 0 0 NA 15 0 0 NA 14 0 0 NA Sodium (mmol/L) 15 149 4 NA 13 151 5 NA 14 151 2 NA Potassium (mmol/L) 15 5.1 0.4 NA 13 5.5** ( ↓ 8) 0.3 NA 14 5.4 0.4 NA

Chloride (mmol/L) 15 109 3 NA 13 109 4 NA 14 109 2 NA Calcium (mmol/L) 15 2.45 0.11 NA 15 2.45 0.13 NA 14 2.44 0.10 NA Inorganic phosphorus (mmol/L) 15 2.69 0.11 NA 15 2.72 0. 14 NA 14 2.84 0.25 NA

a Data were obtained from page 41 of the study report. Percent differences from controls, calculated by the reviewers, are included in parentheses. N Number of animals examined SD Standard Deviation CV Coefficient of Variation * Significantly different from controls at p ≤ 0.05. ** Significantly different from controls at p ≤ 0.01.



## G. GROSS PATHOLOGY AND HISTOPATHOLOGY: The incidences of histopathological findings of the thyroid gl and are presented in Table 6. No treatmentrelated gross pathological alterations were observed in the thyroid gland.Female Pubertal Assay (2012) / Page 12 of 13 TEBUCONAZOLE / 128997 OCSPP 890.1450/ OECD None



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

Treatment Groups Parameter Evaluated Colloid Quality Follicular Cell Hypertrophy Follicular Cell Height (Decrease) Follicular Cell Shape
Severity b Incidence Severity Incidence Severity Incidence Severity Incidence O E O E O E O E

Vehicle Control 0 15 15 0 15 15 0 15 15 0 15 15 1 15 0 1 15 0 1 15 0 1 15 0 2 15 0 2 15 0 2 15 0 2 15 0 3 15 0 3 15 0 3 15 0 3 15 0 4 15 0 4 15 0 4 15 0 4 15 0 5 15 0 5 15 0 5 15 0 5 15 0

Tebuconazole (75 mg/kg/day) 0 15 15 0 15 15 0 15 15 0 15 15 1 15 0 1 15 0 1 15 0 1 15 0 2 15 0 2 15 0 2 15 0 2 15 0 3 15 0 3 15 0 3 15 0 3 15 0 4 15 0 4 15 0 4 15 0 4 15 0 5 15 0 5 15 0 5 15 0 5 15 0

Tebuconazole (150 mg/kg/day) 0 15 15 0 15 15 0 15 15 0 15 15 1 15 0 1 15 0 1 15 0 1 15 0 2 15 0 2 15 0 2 15 0 2 15 0 3 15 0 3 15 0 3 15 0 3 15 0 4 15 0 4 15 0 4 15 0 4 15 0 5 15 0 5 15 0 5 15 0 5 15 0 a Data were obtained from page 45 of the study report. b Thyroid histopathology is graded 0 – 3 based on seve rity: 0=Not remarkable, 1=Mild, 2=Moderate, 3=Severe. See OECD No. 82 for reference. O Number Observed E Number Examined



## The incidences of gross pathol ogical observations in female ra ts are shown in Table 6. No treatment-related gross pathological alterations were observed in the liv er, kidneys or uterus, and there were no treatment-related histopathol ogical findings in any organs examined.

TABLE 7. Incidence of Gross Pathological Ob servations of the Uterus, Liver and Kidneys a

Findings Dose Level (# mg/kg/day)

Vehicle Control Tebuconazole (75 mg/kg/day) Tebuconazole (150 mg/kg/day) # Observed # Examined # Observed # Examined # Observed # Examined Uterus Dilation of horns 4 15 5 15 3 15 Kidney Pelvic dilation 2 15 0 15 1 15 Cysts 2 15 0 15 1 15 Depressed Focus 1 15 0 15 0 15 Liver Enlarged 0 15 0 15 1 15 a Data were obtained from page 107 of the study report.Female Pubertal Assay (2012) / Page 13 of 13 TEBUCONAZOLE / 128997 OCSPP 890.1450/ OECD None



## III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: At 150 mg/kg/day, clinical signs of toxicity were confined to increased salivation in 2/15 female s, observed on one or a few occasions. There was a decrease in cumulative body weight gain as well as a delay in the age at VO and complete VO. However, as there was no effect on body weight at VO or complete VO when compared to controls, it is considered that the delay in VO was due to systemic toxicity. This is further supported by the ab sence of effect on body weight at VO. There was no effect on the estrous cycle. T 4 

## and TSH levels were unaffected by treatment, but mean absolute and relative liver weights were higher in females when compared to controls.

B. AGENCY COMMENTS: All rats survived the 21-day test period, and the only sign of clinical toxicity was incr eased salivation in several females from both dose groups throughout the study.
Mean daily body weight and body weight gain /day in the 150 mg/kg/day animals was decreased on several days through Study Day P ND 33, but there were no treatment-related changes in final body weight or cumulative body weight gain (PND 22- 42). Compared to the control, the mean age at initial VO was increased by 1.2 days (NS) and 2.7 days (p<0.01) at 75 and 150 mg/kg/day, respectivel y, and the mean age at complete VO was increased by 1.1 days (NS) and 2.0 days (p< 0.05) at 75 and 150 mg/kg/day, respectively. However, there were no significant differences in body weights between groups at initial and complete VO.
Administration of tebuconazole did not change the day of first estrus compared to the vehicle control group. Animals exhibited si milar cycle lengths a nd regularity, and the majority of females in the 150 mg/kg/day group were in diestrus at necropsy.
Increased liver weights and relative liver weights (p<0.01) were observed in the 75 mg/kg/day and 150 mg/kg/day do se groups, as well as, an increase in relative kidney weight (p<0.01). No other treatment-related ch anges were observed in organ weights. Given the minimal changes in liver and ki dney weight and the lack of associated histopathological changes, the toxicological significance of these results is unclear.

There were no treatment-re lated changes in serum T 4 

## and TSH or on thyroid weight or morphology associated with tebuconazole trea tment. Effects on clinical chemistries included decreased mean creatinine (p ≤ 0.01) and increased tota l cholesterol (p<0.01) concentrations in the 150 mg/kg/day animal s, and decreased blood urea nitrogen, total protein, and total bilirubin concentrations (p <0.05 or 0.01) in females administered 75 or 150 mg/kg/day. As above, these effects may be associated with the minimal changes in liver and kidney weight and, but the toxicologi cal significance of these results is unclear.
No overt toxicity was observed during treatment. As increas ed salivation, and decreased daily body weight and body weight gain during the first half of the study, was noted in the 150 mg/kg/day dose group, the high dose was considered to be adequate.

C. STUDY DEFICIENCIES: None

# DATA EVALUATION RECORD

TEBUCONAZOLE
Study Type: OCSPP 890.1500, Male Pubertal Assay

EPA Contract No. EP10H001452 Task Assignment No. 3-20-2012 (MRID 48688302)

Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by CSS-Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer: Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 9/13/2012
Secondary Reviewer: Signature: Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 9/19/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 10/02/2012
Quality Assurance: Signature: Jack D. Early, M.S. Date: 10/02/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel.

## The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).TEBUCONAZOLE / 128997 Male Pubertal Assay (2012) I Page 1 of 19 OCSPP 890.1500 I OECD Nooe



## DATA EVALUATION RECORD

STUDY TYPE: Male Pubertal Assay; OCSPP 890.1500



## PC CODE: 128997 

## DP BARCODE: D404685

TXR 

## #: 0056253 

## CAS #: 107534-96-3



## TEST MATERIAL (PURITY): Tebuconazole (97.5% a.i.)



## SYNONYMS: a-[2-( 4-chlorophenyl)ethyl]-a-(1, 1-dimethylethyl)-lH-1,2,4-triazole-1-ethanol



## CITATION: Lautraite, S. (2012). Tebuconazole: Assessment of Pubertal Development and Thyroid Function in Juvenile/Peripubertal Male and Female Rats. Bayer S.A.S., Bayer CropScience, Sophia Antipolis Cedex, France. Laboratory Study No.: SA 11013, June 26, 2012. MR.ID 48688302. Unpublished.



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



## TEST ORDER#: CON-128997-20



## EXECUTIVE SUMMARY: In a Male Pubertal Assay (MRID 48688302), 15 male Sprague Dawley [Crl:CD (SD)] rats/dose group were treated daily via oral gavage with tebuconazole (97.5% a.i., Batch#: K689052) in aqueous 0.5% methylcellulose 400+0.4% Tween 80 at doses of 0, 75, or 150 mg/kg/day from post-natal day (PND) 23 to 53. Animals were examined for preputial separation (PPS) daily beginning on PND 30, and age and weight on the day of attainment was recorded. Following sacrifice on PND 53, blood was collected for clinical chemistry and hormone analysis. Total serum testosterone levels were determined by using an enzyme immunoassay (EIA), and thyroxine (T4) and thyroid stimulating hormone (TSH) levels

were determined with enzyme-linked immunosorbent assays (ELISA). Liver, pituitary, adrenals, thyroid, and urogenital organs were collected and weighed, and microscopic examinations were conducted on the testes, epididymides, thyroid, and kidneys.

No adverse, treatment-related effects were observed on mortality; clinical chemistry parameters; gross pathology in the liver, kidneys, or adrenal glands; or histopathology in the kidneys. No treatment-related effect was noted on serum T4 or TSH levels, or on thyroid weight or morphology.

Clinical signs of toxicity were limited to increased salivation in 9/15 males in the 150 mg/kg/day dose group compared to 0/15 in both the control and 75 mg/kg/day groups. At 150 mg/kg/day,

mean body weight was decreased (p:'.S0.05 or 0.01) by 6-10% from PND 24 to 53, with a terminal body weight decrease (p :'.S 0

. 05) of 8%. On PND 24
, there was a mean body weight loss (p:'.S0.01)Male Pubertal Assay (2012) / Page 2 of 19 TEBUCONAZOLE / 128997 OCSPP 890.1500 / OECD None


## of 1.5 g compared to a mean body weight gain of 4.9 g in control animals. Differences in cumulative body gain at the high dose lessened throughout the study with an 8% decrease in the final cumulative body weight gain (PND 24-53) . At 75 mg/kg/day, mean body weight was decreased (p ≤ 0.05) on several occasions by 6 to 7%. M ean cumulative body weight gains were decreased by 53% (p ≤ 0.05) on PND 24 and 18% on PND 25, but diminished to an 11% decrease by PND 33. Significant decreases in cumulative body weight gain in the low dose group did not continue past PND 47.
Decreases (p ≤ 0.05 or 0.01) were observed in kidney, pituitary and adrenal gland weights. These decreases in weight at 75 and 150 mg/kg/day were dose-dependent for kidneys (7 and 9%, respectively) and pituitary glands (9 and 12%), but not for adrenal glands (18 and 13%). Additionally, relative (to body) liver weight was increased (p ≤ 0.01) by 10% at 150 mg/kg/day.
Compared to the control, mean serum testostero ne was decreased by 17% (not significant, NS) at 75 mg/kg/day and by 53% (p ≤ 0.05) at 150 mg/kg/day. The ages at initial PPS were delayed by ca. 2.5 days (p<0.01) in the high- dose group, but there was no difference in age at complete PPS between groups. The delay in initial PPS in the high-dose group was possibly related to delayed growth, as body weight gains we re reduced in the high-dose gr oup and body weights at initial and complete PPS were similar across groups. Although the age at complete PPS was similar between groups, there was a dose-related trend in the number of males in which the preputia were not completely separated by the end ofthe study (control 3/15, low-dose 4/15, and highdose 7/15). Therefore, the lack of significant di fferences in age at complete PPS may be masked by the use of PND 54 (termination date, plus one) in calculating averages for complete PPS in the high-dose group.
With the exception of the ventral prostate, there were dose-dependent decreases in the weights of androgen-dependent tissues. At 150 mg/kg/day, decreases (p ≤ 0.05 or 0.01) were observed in the weights of the seminal vesicle and coa gulating gland with and without fluid ( ↓ 23-33%), the dorsolateral prostate ( ↓ 21%), the LABC ( ↓ 24%), and the left and right epididymis ( ↓ 13-14%). In addition, the weights of the te stes were decreased (NS) by 10-11% at the high dose. At 75 mg/kg/day, dorsolateral prosta te weights were decreased (p ≤ 0.05) by 21%, and although not significant, substantial weight de creases were also noted in the seminal vesicle and coagulating gland with and without fluid ( ↓ 17-21%) and the LABC ( ↓ 11%).
Atrophy was observed in the following androgen-depe ndent tissues (# affected/# examined): (i) testes (atrophic/small and/or soft) at 75 (2/15) and 150 (3/15) mg/kg/day compared to control (1/15); (ii) epididymis at 150 mg /kg/day (1/15) compared to cont rol (0/15); (iii) seminal vesicle at 75 (5/15) and 150 (7/15) mg/kg/day compared to control (2/15); (iv) pros tate at 75 (2/15) and 150 (5/15) mg/kg/day compared to control (1); and (v) LABC at 75 (1/15) and 150 (3/15) mg/kg/day compared to control (0 /15). Although the incidences of atrophy were small, it was a common f inding in several androgen-dependent tissues, and there was additional evidence of effect on androgen-related systems. Additionally, a red focus was observed in the epididymis at 150 mg/kg/day (2/15) compared to control (0/15). Tubular de generation in the testes was increased at 350 mg/kg/day: unilateral (1/15 treated vs 0/15 contro l) and bilatera l (3/15 treated vs 1/15 control). Bilateral oligospermia in the epididymis was observed at 75 (2/15) and 150 (4/15) mg/kg/day compared to control (1/15).Male Pubertal Assay (2012) / Page 3 of 19 TEBUCONAZOLE / 128997 OCSPP 890.1500 / OECD None


## The high-dose used in the curren t study is considered adequate based on the effects observed on body weight and body weight gains, and the inform ation from the availa ble range-finding study.
The assay satisfies the EDSP Tier 1 Test Order requirement s for a Male Pubertal Assay (OCSPP 890.1500).

COMPLIANCE: Signed and dated Data Confiden tiality, GLP Compliance, and Quality Assurance statements were provided.Male Pubertal Assay (2012) / Page 4 of 19 TEBUCONAZOLE / 128997 OCSPP 890.1500 / OECD None



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Bayer S.A.S. Location: Sophia Antipolis Cedex, France Study Director: S. Lautraite Other Personnel: C. Langrand-Lerche (Replacement Study Director); J.P. Kocwin (Laboratory Animal Resources): C. Jaussely (In-Life Supervisor); B. Trela (Responsible Technician); C. Pallen (Statistician); L. Amir Tahmasseb (Senior Chemist); D. De Barbeyrac (Clinical Pathology Supervisor); F. Schorsch (Pathologist); and M. Vagner (Report Unit Assistant) Study Period: September 28, 2011 – June 26, 2012



## 2. Test Substance: Tebuconazole Description: White powder Source: Bayer CropScience AG (Dormagen, Germany) Batch #: K689052 (expiration date: August 23, 2012) Purity: 97.5% a.i. Stability: Stable at 25°C ± 5°C; material was stored in an air-tight, light-resista nt container at room temperature CAS #: 107534-96-3 Structure:
Cl OH CH 3

CH 3 CH 3

N N N



## 3. Vehicle: Aqueous 0.5% methylcellulose 400+0.4% Tween 80



## 4. Test Animals: Species: Rat (males only) Strain: Sprague Dawley [Crl:CD (SD)] Age/Weight at Study Initiation: PND 23/ 53.6-76.6 g Source: Charles River Laboratories (St. Germain-sur-l’Arbresle, France) Housing: Dams were housed individually with their pups in Makrolon® cages. Following weaning, pups were housed 2-3 per cage w ith heat-treated laboratory-grade wood (not cedar) shavings as bedding material. Diet: A04C-10 pelleted rodent diet (Scientific Animal Food and Engineering, Augy, France), ad libitum . Phytoestrogen content of <300 μ g of genistein equivalents/gram diet Water: Filtered tap water, softened by ion exchange resins, ad libitum Environmental Conditions: Temperature: Humidity: Air changes: Photoperiod: 20-24 °C 40-70% target of 10-15/hr 14 hrs light/10 hrs dark



## B. STUDY DESIGN

1. In-Life Dates: Start: November 5 and 6, 2011 End: December 5 and 6, 2011
2. Mating: Pregnant nulliparous female rats (11 to 13 weeks old) were received from the supplier on gestation day (GD) 7 or 8. Litters with fewer than 8 pups or that were notMale Pubertal Assay (2012) / Page 5 of 19 TEBUCONAZOLE / 128997 OCSPP 890.1500 / OECD None


## delivered on GD 22 or 23, were excluded from the study. On post-natal day (PND 4), litters were standardized to 8 pups with, if possi ble, an equal number of pups of each sex.
3. Animal Assignment: Following weaning on PND 21, all pups were individually weighed and allocated to the groups noted in Table 1, using a randomization procedure that ensured a similar body weight distribu tion among groups and avoided, as much as possible, the placement of littermates in the same dose group. The acceptable body weight range was ±20% of the mean body weight on the day of randomization.

TABLE 1. Study Design a Test group Dose (mg/kg/day) # of Males Control 0 15 Low 75 15 High 150 15 a Data were obtained from page 19 of the study report.



## 4. Dose Selection Rationale: The dose levels used in this study were chosen based on the results of a preliminary range-finding study in peripubertal Sprague Dawley male and female rats (25 days old on Study Day 1). A summary of th is study is included as an Appendix to this DER.
5. Dose Preparation and Analysis: Dose formulations were prepared twice during the study by mixing appropriate amounts of tebuconazo le with aqueous 0.5% methylcellulose 400+0.4% Tween 80. The formulations were stored at 5±3°C in air-tight bottles and were mixed continuously before and during treatment. Homogeneity was tested prior to initiation of dosing at concentrations of 15 and 30 g/ L. The mean values of the homogeneity determinations were used as achieved concentr ation analyses. It was stated that in a separate study (Bayer Study N o. SA 11273; no data provided) 1 

## , tebuconazole was determined to be stable in the vehicle at c oncentrations of 0.6 and 170 g/L for up to 22 days under storage conditions similar to those of the current study.

Results of Dose Analysis
Homogeneity (%CV): 0.5-0.7%
Stability (% of Day 0): Not provided

Concentration (% of nominal): 97-102%
The analytical data indicated that the mixing procedure was ad equate, and that the variation between nominal and actual dosage to the animals was acceptable. The stability data from SA 11273 should be provided for independent review.
6. Dosage Administration: All doses were administered once daily by gavage, from PND 23 through PND 53, in a volume of 5 mL/kg of body we ight. Dose volumes were adjusted daily

1 

### Tahmasseb, A. L. (2011). Tebuconazole, Stability in aq ueous 0.5% methylcellulose 400 + 0.4% tween 80, Bayer CropScience, Sophia Antipolis, France (SA 11273).Male Pubertal Assay (2012) / Page 6 of 19 TEBUCONAZOLE / 128997 OCSPP 890.1500 / OECD None


## based on individual animal body weights. Do sing was generally performed between 0700 and 0900 hours daily. The dosing sequence wa s stratified across dosing groups on PND 53 (scheduled necropsy day).

7. Statistics: The following parameters were analyzed statistically: body weights, body weight gains, terminal body weights, orga n weights, age and body weight at PPS, and clinical chemistry and hormonal analysis. Fo r body weight gains and clinical chemistry parameters, the data were first analyzed usi ng Bartlett’s test. If the data were not significant, an analysis of variance (ANOVA) test was perf ormed; a two-sided Dunnett’s test was conducted if the results of the ANOVA we re significant. If the results of Bartlett’s test were significant, a Kruskal-Wallis test was performed; a two-sided Dunn’s test was conducted if the result of the Kruskal-Wallis te st was significant. The same procedures were used for body weight and hormonal paramete rs, except that data were log transformed when Bartlett’s test was signi ficant, and then a second Ba rtlett’s test was conducted.
For age at PPS, body weight at PPS, absolute and relative (to body) organ weights, body weight at PND 21, and terminal body weight, the data were first analyzed using a Levene’s test; if the data were significant, a data transformation (log) was conducted and a second Levene’s test was performed. If the data were not significant, an ANOVA test was performed; a two-sided Dunnett’ s test was conducted if th e results of the ANOVA were significant. If the resu lts of the Levene’s test (after tr ansformation) showed significance, a Kruskal-Wallis test was performed, a two-side d Dunn’s test was conduc ted if the result of the Kruskal-Wallis test was signi ficant. The means of the treated and control groups were compared by analysis of covariance (ANCOVA) , with the body weight at PND 21 as the covariate, except for terminal body weight. Adjusted means were compared using the 2-sided Dunnett’s test. Si gnificance was denoted at p ≤ 0.05 and p ≤ 0.01.

C. METHODS
1. Mortality and Clin ical Examinations: All animals were examined at least twice daily (at least once daily on weekends and public holidays) for mortality and moribundity. Clinical examinations were conducted at least once daily.
2. Body Weight: Animals were weighed prior to allo cation, daily prior to dosing, and at termination.
3. Preputial Separation (PPS): Beginning on PND 30, all animals were examined daily for onset of PPS. Age and weight on the day of pa rtial and/or complete PPS were recorded. If complete PPS had not occurred prior to necrops y, the age at necropsy, plus one (PND 54), and the weight at necropsy (on PND 53) were used for those animals for calculation of

means.
4. Sacrifice and Pathology: Approximately 2 hours after admi nistrations of the last dose on PND 53, all surviving rats (non-fasted) were anes thetized with isoflurane (Virbac, Carros, France) and a blood sample (~3.5 mL) was collec ted from each animal from the abdominal aorta. Blood was collected into tubes for c linical chemistry and hor mone determinations. Serum samples were stored frozen (approximately

− 74°C) until analyses were conducted.Male Pubertal Assay (2012) / Page 7 of 19 TEBUCONAZOLE / 128997 OCSPP 890.1500 / OECD None


## a. Hormone Analysis: Total testosterone le vels were determined with an EIA kit (Cayman Chemical Company, Ann Arbor , MI), and the total T 4 

## and TSH levels were determined with ELISA kits (mouse/rat thyroxine kit, Calbiote ch Inc., Spring Valley, CA and rodent thyroid stimulating hormone kit, Endocrine Tec hnologies, Newark, CA, respectively).
b. Clinical Chemistry a 

## : 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 Globulins X Sodium X Glucose
ENZYMES X Total bilirubin X Alkaline phosphatase (ALP) X Total protein Cholinesterase (ChE) Triglycerides Creatine phosphokinase Se rum protein electrophoresis Lactic acid dehydrogenase (LDH) X Alanine aminotransferase (ALT/also SGPT) X Aspartate aminotransfera se (AST/also SGOT) Sorbitol dehydrogenase (SDH) X Gamma glutamyl transferase (GGT) Glutamate dehydrogenase a Data were obtained from page 23 of the study report. * Recommended for the pubertal assay in male rats based on guideline 890.1500.


## c. Organ Weights and Histopathology a 

## : The following CHECKED (X) tissues were collected and weighed. The (XX) organs, in addition, were subject ed to histological examination. A complete necrops y was performed on all animals.

UROGENITAL OTHER XX Testes (left and right separately)* + XX Thyroid* + XX Epididymides (left and right separately)* + X Liver* X Seminal vesicle plus coagulating glands (w ith and without fluid)* X Adrenals (paired)* X Ventral prostate* X Pituitary* X Dorsolateral prosta te* XX Gross lesions b X Levator ani-bulbocavernosus (LABC) muscle complex* XX Kidneys (paired)* + a Data were obtained from page 24 of the study report. b Examined microscopica lly, but not weighed. * Weights required base d on guideline 890.1500 + Histopathological examination re quired based on guideline 890.1500


## All collected tissues, including thyroid, were we ighed prior to fixation. The left and right testes and epididymides were weighed individu ally. The seminal vesicles with coagulating glands were weighed with and without flui d. Significant macroscopic findings were collected and examined microscopically, but we re not weighed. All collected tissues, with the exception of testis and epididymis, were pr eserved in 10% neutral formalin. The testis and epididymis were preserved in modified Davidson’s fixative. The epididymis, testis, kidneys, thyroid glands, and any significant m acroscopic findings were processed routinely, stained with hematoxylin and eosin, and exam ined microscopically. Thyroid gland wasMale Pubertal Assay (2012) / Page 8 of 19 TEBUCONAZOLE / 128997 OCSPP 890.1500 / OECD None


## processed according to the fac ility’s standardized operating pr ocedure which included three random sections. The study authors did not report whether the left, right , or both testis and epididymis were evaluated for each rat.
The thyroid sections were evaluated for decreased follicular cell height, follicular cell shape, follicular cell hypertrophy, and alteration of collo id area. Testis histology included, but was not limited to, retained spermatids, missing or degenerated germ cell layers or types, multinucleated giant cells, and sloughing of spermatogenic cells into the lumen. Examination of epididymis was conducted in order to identify such lesions as sperm granulomas, leukocytic infiltra tion (inflammation), aberrant ce ll types within the lumen, and the absence of clear cells in th e cauda epididymal epithelium.
After the initial histopathologi cal examination by the Study Pat hologist, an independent peer review of representative slides was c onducted according to standardized operating procedures.

II. RESULTS

A. MORTALITY: All animals survived until scheduled termination.

B. CLINICAL SIGNS OF TOXICITY: Treatment-related clinical signs of toxicity were limited to increased salivation in 9/15 male s in the 150 mg/kg/day dose group compared to 0/15 in both the control and 75 mg/kg/day groups.

C. GENERAL GROWTH AND PREPUTIAL SEPARATION: Selected body weights and body weight gains from intervals throughout the study are presented in Table 2a, and initial and final body weights, overall body weight gains, age and weight at attainment of PPS, and proportion/incidence of unseparated and incomple tely separated preputia are presented in Table 2b.
At 150 mg/kg/day, mean body weights were decreased (p ≤ 0.05 or 0.01) by 6 to 10% from PND 24 through 53, and cumulative body weight gains also were decreased (p ≤ 0.05 or 0.01) throughout the study. The largest decrease in bo dy weight gain occurred after one day of dosing (PND 24; ↓ 131%), and was due to a body weight loss of 1.5 g compared to a mean body weight gain of 4.9 g in the control. Diffe rences in cumulative body gain at the high dose lessened throughout the study, with decr eases of 61% on PND 25 and 17% by PND 33, and a final cumulative body weight gain decrease of 8% by PND 53.
At 75 mg/kg/day, mean body weight was decreased (p ≤ 0.05) on several occasions by 6 to 7%. Mean cumulative body weight gains were decreased by 53% (p ≤ 0.05) on PND 24 and 18% on PND 25, but diminished to an 11% de crease by PND 33. Significant decreases in cumulative body weight gain in the 75 mg/kg/da y dose group did not co ntinue past PND 47.Male Pubertal Assay (2012) / Page 9 of 19 TEBUCONAZOLE / 128997 OCSPP 890.1500 / OECD None



### TABLE 2a. Selected body weights an d cumulative body weight gains a

Interval (PND) Dose levels

Vehicle Control Tebuconazole 75 mg/kg/day Tebuconazole 150 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Body weight 23 15 66.0 5.5 8.33 15 64.8 5. 4 8.33 15 65.3 6.4 9.80

24 15 70.8 5.3 7.49 15 67.1 5.3 7.90 15 63.8** ( ↓ 10) 6.9 10.82

33 15 137.0 7.7 5.62 15 128.1* ( ↓ 6) 8.8 6.87 15 124.6* ( ↓ 9) 13.9 11.16

53 15 312.0 18.5 5.93 15 295.9 19.6 6.62 15 291.3* ( ↓ 7) 29.6 10.16 Cumulative body weight gain

23-24 15 4.9 1.9 38.78 15 2.3* ( ↓ 53) 1.1 47.83 15 -1.5** ( ↓ 131) 2.9 -193.33

23-25 15 10.8 2.1 19.44 15 8.8* ( ↓ 18) 2 22.73 15 4.2** ( ↓ 61) 1.8 42.86

23-33 15 71.1 6.6 9.28 15 63.2* ( ↓ 11) 7 11.08 15 59.3** ( ↓ 17) 8.4 14.17

23-53 15 246.0 17.9 7.28 15 231.0 18.3 7.92 15 226.0* ( ↓ 8) 24.7 10.93 a Data were obtained from Table 2 on pages 60-62 and Table 4 on pages 72-74 of the study report. Percent differences from controls (calculated by reviewers) are included in parentheses. N Number of animals examined SD Standard Deviation CV Coefficient of Variation; calculated by reviewers * Significantly different from controls at p ≤ 0.05. ** Significantly different from controls at p ≤ 0.01.


## At 150 mg/kg/day, the unadjusted and adjust ed ages at initial PPS (42.60 days) were increased (p<0.01) by 2.47 and 2.48 days compared to unadjusted and adjusted control values (40.13 and 40.12 days, respectively). However, the body wei ghts at initial and complete PPS for the high-dose group were simila r to control. No treatment-related effects on age or weight at PPS were observed at 75 mg/kg/day. Although none of the males in any dose group had unseparated preputia, incomplete separation was noted in 3/15 control males vs. 4/15 and 7/15 males in the 75 and 150 mg/kg/day dose groups, respectively.
The following Guideline performance criteria were not met in the controls: (i) CV for age at PPS (8.98%) was greater than the maximum accep table value (5.67%); (ii) CVs for weight at initial and complete PPS (9.8% and 15.1%) were greater than the maximum acceptable value (7.57%); (iii) age at PPS of 48.00 days (acceptable range: 39.781-46.513 days); (iv) body weight at PPS of 267.68 g (acceptabl e range: 188.277-256.169 g). Additionally, weaning weight was not reported.Male Pubertal Assay (2012) / Page 10 of 19 TEBUCONAZOLE / 128997 OCSPP 890.1500 / OECD None



### TABLE 2b. General Growth and Preputial Separation (PPS) a

Parameter Evaluated Dose levels

Vehicle Control Tebuconazole 75 mg/kg/day Tebuconazole 150 mg/kg/day

N Mean SD/SE CV (%) N Mean SD/SE CV (%) N Mean SD/SE CV (%) Body weight at weaning (PND 21; g) 15 58.1 5.0 8.6 15 57.2 5. 3 9.2 15 57.7 5.3 9.1 Initial body weight (PND 23; g) U 15 66.0 5.5 8.3 15 64.8 5. 4 8.3 15 65.3 6.4 9.8 Body weight at initial PPS (g) U 15 200.53 19.6 9.8 15 198.19 16.9 8.5 15 204.69 24.1 11.8 A 15 199.77 4.9 2.4 15 198.95 4.9 2.5 15 204.68 4.9 2.4 Body weight at complete PPS (g) U 15 267.68 40.5 15.1 15 266.31 28.7 10.8 15 262.09 38.7 14.8 A 15 266.76 9.1 3.4 15 267.26 9.1 3.4 15 262.08 9.1 3.5 Final body weight (g) U 15 311.4 18.7 6.0 15 295.7 19.9 6.7 15 286.8* ( ↓ 8) 29.1 10.1 Final body weight (% of control) b U 15 NA NA NA 15 94.9 NA NA 15 92.1 NA NA Body weight gain (final – initial; g) U 15 246.0 17.9 7.3 15 231.0 18.3 7.9 15 226.0* ( ↓ 8) 24.7 10.9

Age at initial PPS (PND) U 15 40.13 1.8 4.4 15 41.33 1.7 4.2 15 42.60** ( ↑ 6) 1.8 4.3

A 15 40.12 0.5 1.2 15 41.34 0.5 1.2 15 42.60** ( ↑ 6) 0.5 1.2 Age at complete PPS (PND) U 15 48.00 4.3 9.0 15 49.93 3.8 7.6 15 50.33 4.0 8.0 A 15 47.98 1.1 2.3 15 49.95 1.1 2.2 15 50.33 1.1 2.2 Proportion unseparated (#/N) 0/15 0/15 0/15 Proportion not completely separated (#/N) 3/15 4/15 7/15

a Data were obtained from pages 32-33 of the study report. Percent differences from controls (calculated by reviewers) are included in parentheses. U Unadjusted for body weight on PND 21 A Adjusted for body weight on PND 21 N Number of animals examined SD Standard Deviation SE Standard Error; used for means adjusted for body weight on PND 21 CV Coefficient of Variation NA Not applicable * Significantly different from controls at p ≤ 0.05. ** Significantly different from controls at p ≤ 0.01.



## D. ORGAN WEIGHTS: Organ weights at necropsy are presented in Table 3. For the 150 mg/kg/day dose group, the following decreases (p ≤ 0.05 or 0.01) in unadjusted and adjusted weights were observed in all androge n-sensitive tissues, ex cept ventral prostate: (i) seminal vesicle and coa gulating gland with fluid ( ↓ 33%, each); (ii) seminal vesicle and coagulating gland without fluid ( ↓ 23%, each); (iii) dorsolateral prostate ( ↓ 21%, each); (iv) LABC ( ↓ 24%, each); and (v) left and right epid idymis (13-14%). In addition, the weights of the testes were decreas ed (NS) by 10-11% at the high dose
Dose-dependent decreases in accessory sex tissues were also obs erved at 75 mg/kg/day, although the weight decreases were only significant (p ≤ 0.05) for the dorsolateral prostate ( ↓ 21%). However, there were deceases (NS) of 21% in the seminal vesicle and coagulatingMale Pubertal Assay (2012) / Page 11 of 19 TEBUCONAZOLE / 128997 OCSPP 890.1500 / OECD None


## gland with fluid, 17-18% in the seminal vesicl e and coagulating gland without fluid, 11% in the LABC, and 2-4% in the left and right epididymis.
Dose-dependent decreases were also observed in unadjusted and adjust ed weights of kidney and pituitary glands. At 75 and 150 mg/ kg/day, kidney weights were decreased (p ≤ 0.05 or 0.01) by 7 and 9%, respectively, and p ituitary weights were decreased (p ≤ 0.05 or 0.01) by 9 and 12%, respectively. Adrenal weights were also decreased (p ≤ 0.05 or 0.01) at 75 mg/kg/day ( ↓ 18%) and 150 mg/kg/day ( ↓ 13%), but the decreases were not dosedependent. Additionally, relative (to body) liver we ight was increased (p ≤ 0.01) by 10% at 150 mg/kg/day.
The unadjusted values for all organ weights in the control group were within the acceptable range of the performance criteria provided in the Guideline (890.1500). The CV values for all organ weights met the perfor mance criteria provided in the Guideline, except the CV for the seminal vesicle weight was outside the ac ceptable range (27.23% comp ared to 21.06%).

TABLE 3. Organ Weights at Necropsy a

Organ Vehicle Control 75 mg/kg/day 150 mg/kg/day

N Mean SD/ SE CV (%) N Mean SD/ SE CV (%) N Mean SD/ SE CV (%) Liver (g) U 15 13.89 1.52 11.0 15 13.28 1.46 11.0 15 14.14 2.30 16.3 A 15 13.85 0.456 NA 15 13.33 0.456 NA 15 14.14 0.455 NA

R 15 4.4540 0.3077 6.9 15 4.4873 0.3229 7.2 15 4.9060** ( ↑ 10) 0.3564 7.3 Kidneys (g) U 15 2.39 0.15 6.1 15 2.22 ( ↓ 7) 0.14 6.4 15 2.17** ( ↓ 9) 0.28 12.7

A 15 2.39 0.047 NA 15 2.23* ( ↓ 7) 0.047 NA 15 2.17** ( ↓ 9) 0.047 NA

R 15 0.7693 0.0425 5.5 15 0.7533 0.0401 5.3 15 0.7560 0.0439 5.8 Pituitary (mg) U 15 10.55 1.19 11.3 15 9.65* ( ↓ 9) 1.01 10.5 15 9.27** ( ↓ 12) 0.92 9.9

A 15 10.55 0.274 NA 15 9.65* ( ↓ 9) 0.274 NA 15 9.27** ( ↓ 12) 0.274 NA

R 15 0.0034 0.0003 9.0 15 0.0033 0.0003 10.5 15 0.0033 0.0005 15.0 Adrenals (mg) U 15 42.15 6.26 14.9 15 34.41** ( ↓ 18) 5.62 16.3 15 36.57* ( ↓ 13) 5.89 16.1

A 15 41.96 1.451 NA 15 34.60** ( ↓ 18) 1.451 NA 15 36.56* ( ↓ 13) 1.449 NA

R 15 0.0136 0.0020 14.5 15 0.0116** ( ↓ 15) 0.0016 13.6 15 0.0128 0.0018 14.0

Seminal vesicle + coag. gland, with fluid (mg) U 15 594.1 161.8 27.2 14 468.9 ( ↓ 21) 174.2 37.1 13 398.7** ( ↓ 33) 166.0 41.6

A 15 593.6 43.86 NA 14 469.3 ( ↓ 21) 45.33 NA 13 398.9** ( ↓ 33) 47.00 NA

Seminal vesicle + coag. gland, without fluid (mg) U 15 314.5 66.5 21.1 15 259.2 ( ↓ 18) 67.9 26.2 15 242.2* ( ↓ 23) 71.4 29.5

A 15 313.8 17.85 NA 15 259.9 ( ↓ 17) 17.86 NA 15 242.2* ( ↓ 23) 17.83 NA Ventral prostate (mg) U 15 249.0 52.1 20.9 15 230.4 48.1 20.9 15 225.1 57.8 25.7 A 15 248.6 13.79 NA 15 230.8 13. 79 NA 15 225.1 13.77 NA Dorsolateral prostate (mg) U 15 168.5 51.8 30.8 15 132.8* ( ↓ 21) 26.2 19.8 15 133.4* ( ↓ 21) 28.5 21.4

A 15 168.6 9.78 NA 15 132.7* ( ↓ 21) 9.78 NA 15 133.4* ( ↓ 21) 9.76 NAMale Pubertal Assay (2012) / Page 12 of 19 TEBUCONAZOLE / 128997 OCSPP 890.1500 / OECD None



### TABLE 3. Organ Weights at Necropsy a

Organ Vehicle Control 75 mg/kg/day 150 mg/kg/day

N Mean SD/ SE CV (%) N Mean SD/ SE CV (%) N Mean SD/ SE CV (%) LABC (mg) U 15 686.8 115.6 16.8 15 611.5 ( ↓ 11) 135.5 22.2 15 522.2** ( ↓ 24) 102.0 19.5

A 15 687.1 31.00 NA 15 611.3 ( ↓ 11) 31.00 NA 15 522.2** ( ↓ 24) 30.96 NA Epididymis, left (mg) U 15 228.4 20.9 9.1 15 223.3 ( ↓ 2) 19.8 8.8 15 197.4** ( ↓ 14) 27.7 14.0

A 15 228.4 6.02 NA 15 223.3 ( ↓ 2) 6.02 NA 15 197.4** ( ↓ 14) 6.02 NA Epididymis, right (mg) U 15 240.2 16.2 6.7 15 231.4 ( ↓ 4) 23.0 10.0 15 209.7** ( ↓ 13) 25.7 12.3

A 15 240.3 5.75 NA 15 231.3 ( ↓ 4) 5.75 NA 15 209.7** ( ↓ 13) 5.74 NA Testis, left (mg) U 15 1379.5 114.3 8.3 15 1363.0 130.5 9.6 15 1243.4 ( ↓ 10) 276.3 22.2

A 15 1379.4 49.28 NA 15 1363.2 49.28 NA 15 1243.4 ( ↓ 10) 49.21 NA Testis, right (mg) U 15 1401.8 97.9 7.0 15 1379.3 120.9 8.8 15 1245.8 ( ↓ 11) 288.8 23.2

A 15 1399.9 49.24 NA 15 1381.3 49.24 NA 15 1245.8 ( ↓ 11) 49.17 NA Thyroid, fixed (mg) U 15 17.0 2.5 14.6 15 16.9 1.9 11.4 15 16.9 2.2 12.9 A 15 17.1 0.57 NA 15 16.8 0. 57 NA 15 16.9 0.56 NA a Data were obtained from pages 36-37 of the study report. 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 SE Standard Error; used for means adjusted for body weight on PND 21 CV Coefficient of Variation * Significantly different from controls at p ≤ 0.05. ** Significantly different from controls at p ≤ 0.01.



## E. CLINICAL CHEMISTRY AND HORMONE LEVELS: Mean hormone and clinical chemistry levels are presented in Table 4.

No treatment-related effects were observed on serum T 4 

## or TSH concentrations at either dose of tebuconazole compared to control. However, there was a dos e-dependent decrease in serum testosterone of 17% (NS) and 53% (p ≤ 0.05) at 75 and 150 mg/kg/day, respectively, compared to control.

In addition, mean total bilirubin concentrations were decreased (p ≤ 0.01) at 75 ( ↓ 75%) and 150 ( ↓ 100%) mg/kg/day. Other differences (p<0.05) noted in various clinical chemistry parameters were minor and/or unrelated to dose; therefore, these differences were considered unrelated to treatment.

The values for serum T 4 

## , TSH, and testosterone in th e control group were within the acceptable ranges of the performance criteria provided in the Guideline (890.1500), except as follows: mean serum T 4 

## level of 3.8 μg/dL (acceptabl e range: 4.056-7.376 μg/dL); and mean serum TSH level of 3.55 ng/mL (acceptable range: 4.212-24.112 ng/mL).Male Pubertal Assay (2012) / Page 13 of 19 TEBUCONAZOLE / 128997 OCSPP 890.1500 / OECD None



### TABLE 4. Hormone Levels and Clinical Chemistry a

Parameter Evaluated Dose levels

Vehicle Control Tebuconazole 75 mg/kg/day Tebuconazole 150 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Hormones Serum T 4 , Total (μg/dL) 15 3.8 0.4 11 15 3.5 0.5 14 15 3.5 0.5 14

Serum TSH (ng/mL) 15 3.55 1.94 55 13 5.20 1.98 38 15 4.71 1.54 33

Serum testosterone (ng/mL) b 15 1.769 1.012 57 15 1.464 ( ↓ 17) 0.785 54 15 0.839* ( ↓ 53) 0.817 97 Clinical Chemistry Creatinine (μmol/L) 15 47 1 NA 15 49 2 NA 15 48 2 NA Urea nitrogen (mmol/L) 15 5.14 0.42 NA 15 4.87 0.64 NA 15 4.61* ( ↓ 10) 0.62 NA Total protein (g/L) 15 57 2 NA 15 56 2 NA 15 57 2 NA Albumin (g/L) 15 38 1 NA 15 37* ( ↓ 3) 1 NA 15 37 1 NA Bilirubin (μmol/L) 15 0.4 0.2 NA 15 0.1** ( ↓ 75) 0.1 NA 15 0.0** ( ↓ 100) 0.1 NA Glucose (mmol/L) 15 7.79 0.73 NA 15 7.85 0.62 NA 15 7.77 0.51 NA Total cholesterol (mmol/L) 15 1.79 0.31 NA 15 1.84 0. 24 NA 15 1.89 0.27 NA AST (U/L) 15 98 22 NA 15 103 20 NA 15 102 19 NA ALT (U/L) 15 33 8 NA 15 34 9 NA 15 35 5 NA ALP (U/L) 15 372 49 NA 15 398 59 NA 15 397 62 NA GGT (U/L) 15 0 0 NA 15 0 0 NA 15 0 0 NA Sodium (mmol/L) 15 144 1 NA 15 143 1 NA 15 143* ( ↓ 1) 1 NA Potassium (mmol/L) 15 5.1 0.3 NA 15 5.2 0.2 NA 15 5.3 0.3 NA Chloride (mmol/L) 15 103 1 NA 15 103 2 NA 15 102 1 NA Calcium (mmol/L) 15 2.71 0.07 NA 15 2.66 0.07 NA 15 2.69 0.06 NA Phosphorous (mmol/L) 15 3.03 0.19 NA 15 3.05 0.18 NA 15 3.17 0.12 NA a Data were obtained from page 40 of the study report. Percent differences from controls (calculated by reviewers) are included in parentheses. b Converted from pg/mL to ng/mL by reviewers. N Number of animals examined SD Standard Deviation CV Coefficient of Variation; calculated by the reviewers. * Significantly different from controls at p ≤ 0.05. ** Significantly different from controls at p ≤ 0.01.



## F. GROSS PATHOLOGY: The incidences of gross observati ons are summarized in Table 5. No treatment-related effects on gross pathology were noted in the liver, kidneys, adrenal glands, or thyroid.
Atrophy was observed in the following androgen de pendent tissues (# affected/# examined): (i) testes (atrophic/small and/or soft) at 75 (2/15) and 150 (3 /15) mg/kg/day compared to control (1/15); (ii) epididymis at 150 mg/kg/day (1/15) comp ared to control (0/15); (iii) seminal vesicles at 75 (5/15) and 150 (7/15) mg/kg/day compared to control (2/15); (iv) prostate at 75 (2/15) and 150 (5/15) mg/kg/day compared to control (1/15); and (v) LABC at 75 (1/15) and 150 (3/15) mg/kg/day compared to control (0/15). Additionally, a red focus was observed in the epididymis at 150 mg/kg/ day (2/15) compared to control (0/15).Male Pubertal Assay (2012) / Page 14 of 19 TEBUCONAZOLE / 128997 OCSPP 890.1500 / OECD None



### TABLE 5. Incidence of Gross Observations a

Findings Dose Levels

Vehicle Control Tebuconazole 75 mg/kg/day Tebuconazole 150 mg/kg/day
# Observed # Examined # Observed # Examined # Observed # Examined Liver Enlarged 2 15 1 15 3 15 Focus, white 0 15 0 15 1 15 Kidneys Pelvic dilatation 3 15 1 15 1 15 Cyst(s) 0 15 5 15 1 15 Depressed focus 0 15 0 15 1 15 Adrenal Gland(s) Atrophic/small 0 15 1 15 1 15 Testes Atrophic/small and/or soft 1 15 2 15 3 15 Epididymis Atrophic/small 0 15 0 15 1 15 Focus, red 0 15 0 15 2 15 Seminal vesicle Atrophic/small 2 15 5 15 7 15 Prostate Atrophic/small 1 15 2 15 5 15 Nodule(s)/Mass(es) 1 15 0 15 0 15 LABC Atrophic/small 0 15 1 15 3 15

a Data were obtained from page 108 of the study report. No gross observations were reported for the thyroid.



## G. HISTOPATHOLOGY: The incidences of histopathologi cal findings in the thyroid gland are presented in Table 6. No treatment-related effects on follicu lar cell height, cell shape, or colloid area were obser ved at either dose.Male Pubertal Assay (2012) / Page 15 of 19 TEBUCONAZOLE / 128997 OCSPP 890.1500 / OECD None



### TABLE 6. Thyroid Gland Histopathology a

Treatment Groups Parameter Evaluated

Colloid Alteration Follicular Cell Hypertrophy Follicular Cell Height (Decrease) Follicular Cell Shape
Severity b Incidence Severity Incidence Severity Incidence Severity Incidence O E O E O E O E

Vehicle Control 0 14

14 0 14

14 0 14

14 0 14

14 1 0 1 0 1 0 1 0 2 0 2 0 2 0 2 0 3 0 3 0 3 0 3 0 4 0 4 0 4 0 4 0 5 0 5 0 5 0 5 0

Tebuconazole 75 mg/kg/day 0 15

15 0 15

15 0 15

15 0 15

15 1 0 1 0 1 0 1 0 2 0 2 0 2 0 2 0 3 0 3 0 3 0 3 0 4 0 4 0 4 0 4 0 5 0 5 0 5 0 5 0

Tebuconazole 150 mg/kg/day 0 15

15 0 15

15 0 15

15 0 15

15 1 0 1 0 1 0 1 0 2 0 2 0 2 0 2 0 3 0 3 0 3 0 3 0 4 0 4 0 4 0 4 0 5 0 5 0 5 0 5 0 a Data were obtained from page 42 of the study report. b Thyroid histopathology is graded 0 – 5 based on severity: 0=Not remarkable, 1=Minimal, 2=Slight, 3=Moderate, 4=Marked, 5=Severe. See OECD No. 82 for reference. O Number Observed E Number Examined



## The incidences of other histopathol ogical findings are presented in Table 7. No treatment-related effects were observed in the kidneys or thyroid.
Tubular degeneration in the testes was increased at 150 mg/kg/day: unilateral (1/15 treated vs 0/15 controls) and bilateral (3/15 treated vs 1/ 15 control). Bilateral oligospermia in the epididymis was observed at 75 (2/15) and 150 (4/ 15) mg/kg/day compared to controls (1/15). The incidences of all other histological lesi ons were considered in cidental based on low incidence, lack of dose-dependent effect, and/or absence of corrobora ting evidence of organ toxicity.

TABLE 7. Incidence of Histopathological Lesions a

Findings Dose Levels

Vehicle Control Tebuconazole 75 mg/kg/day Tebuconazole 150 mg/kg/day
# Observed # Examined # Observed # Examined # Observed # Examined Kidneys Basophilic tubules: unilateral: focal 4 15 4 15 2 15 Bilateral: focal 2 15 0 15 2 15 Atrophy/fibrosis/scar: unilateral 2 15 0 15 1 15 Interstitial monoculea r cell infiltrate: focal 5 15 5 15 4 15 Papillary mineralization: focal 0 15 1 15 0 15Male Pubertal Assay (2012) / Page 16 of 19 TEBUCONAZOLE / 128997 OCSPP 890.1500 / OECD None



### TABLE 7. Incidence of Histopathological Lesions a

Findings Dose Levels

Vehicle Control Tebuconazole 75 mg/kg/day Tebuconazole 150 mg/kg/day
# Observed # Examined # Observed # Examined # Observed # Examined Pelvic dilatation: unilateral 4 15 1 15 1 15 bilateral 0 15 0 15 0 15 Cyst (s) 1 15 4 15 3 15 Granuloma (s): focal 0 15 0 15 0 15 Transitional cell hyperplasia: unilateral: focal 1 15 0 15 0 15

bilateral: focal 1 15 0 15 0 15 Suburothelial mixed cell infiltrate: focal 1 15 0 15 0 15 Pyelonephritis 1 15 0 15 0 15 Vascular dilatation 0 15 1 15 2 15 Thyroid Ultimo-brachial cyst(s) 4 14 3 15 4 15 Ectopic thymus 2 14 0 15 0 15 Epididymis Luminal/sloughed germ cells/debris: 7 15 6 15 7 15 Oligospermia: bilateral 1 15 2 15 4 15 Interstitial m ononuclear cell infiltrate: focal 4 15 2 15 2 15 Peripheral mixed cell infi ltrate: focal 0 15 0 15 0 15 Testes Sloughing of germ cells: focal 0 15 1 15 0 15 Sertoli cell vacuolation: focal 1 15 0 15 0 15 Germ cell degeneration/necrosis: focal 1 15 1 15 1 15 Interstitial m onocuclear cell infiltrate: focal 0 15 0 15 0 15 Tubular degeneration: unilateral 0 15 0 15 1 15 bilateral 1 15 1 15 3 15 

### a Data were obtained from pages 110-111 of the study report.



## III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: At 150 mg/kg/day, the effect of tebuconazole was characterized by clinical signs and reduced we ight gain from weaning to sexual maturation in both sexes. Associated with the effect on body weight was a delay in PPS, increased liver weight, and reduced adrenal and sexual or gan weights in males (seminal vesicle, dorsolateral prostate, epididymis and LABC). Other effects at 150 mg/kg/day included reduced testosterone, and lower mean urea a nd total bilirubin. M acroscopic effects at 150 mg/kg/day included atrophic /small prostate, seminal vesicle, LABC, and testis. The 75 mg/kg/day dose level caused a decreased body we ight gain at the start of treatment, a lower mean total bilirubin concentration, and a lower testosterone concentration. Other effects at 75 mg/kg/day included decreased adrenal, pituitary gland, and dorsolateral prostate weights. Macroscopic findings at 75 mg/kg/day included atrophic/small seminal vesicle.

B. AGENCY COMMENTS: No adverse, treatment-related effects were observed on mortality; clinical chemistry parameters; gro ss pathology in the liver, kidneys, or adrenalMale Pubertal Assay (2012) / Page 17 of 19 TEBUCONAZOLE / 128997 OCSPP 890.1500 / OECD None


## glands; or histopathology in the kidneys. No treatment-related effect was noted on serum T 4 

## or TSH levels, or thyroi d weight and histology.
Treatment-related clinical signs of toxicity were limited to incr eased salivation in a majority of the animals in the 150 mg/kg/day dose group.
At 150 mg/kg/day, mean body weights were decreased by 6-10% (p ≤ 0.05 or 0.01) from PND 24 to 53, and terminal body weight was decreased (p ≤ 0.05) by 8%. On PND 24, there was a mean body weight loss (p ≤ 0.01) of 1.5 g compared to a mean body weight gain of 4.9 g in control. Subsequently, although body weight and body weight gain values were decreased (p ≤ 0.05 or 0.01), the differences diminished over time. At 75 mg/kg/day, mean body weight was decreased (p ≤ 0.05) on several occasions. The mean cumulative body weight gain was lower than in controls throughout most of the study, with decreases (p ≤ 0.05) observed at up to PND 47.
Decreases (p ≤ 0.05 or 0.01) were observed in kidney, p ituitary and adrenal gland weights. These decreases in unadjusted and adjusted weights at 75 and 150 mg/kg/day were dosedependent for kidneys (7 and 9%, respectively) and pituitary glands (9 and 12%), but not for adrenal glands (18 and 13%). Additionally, re lative (to body) liver weight was increased by 10% at 150 mg/kg/day. Given th e magnitude of these minor weight changes, these effects are considered to be adaptive and not adverse.

Mean total bilirubin concen trations were decreased (p ≤ 0.01) at 75 and 150 mg/kg/day. This effect was not dose-dependent, and a toxic effect has not been a ssociated with a decrease in serum bilirubin concentrations. Other differences (p<0.05) noted in various clinical chemistry parameters were minor and/or unrelated to dose; therefore, these differences were considered unrelated to treatment.

There were several indications that tebucon azole effected androgen-related systems. Compared to the control, mean serum te stosterone was decreased by 17% (NS) at 75 mg/kg/day and by 53% (p ≤ 0.05) at 150 mg/kg/day. The ages at initial PPS were increased by ca. 2.5 days (p<0.01) in the high-dose group. As body weight gains were reduced in the high-dose group and body weights at initial and complete PPS were similar across groups, the delay in initial PPS is possi bly related to delayed growth in the high-dose group. In addition, there were no significant differences in the age of complete PPS. However, the Agency also noted that there wa s a dose-related trend in the number of males in which the preputia were not completely separated by the end of the study (control 3/15, low-dose 4/15, and high-dose 7/15). Therefore, the lack of signi ficant differences in age at complete PPS may be masked by the use of th e termination date, plus one (PND 54), in calculating averages for comple te PPS in the high-dose group.
In addition, dose-dependent decreases were not ed in the weights of androgen-dependent tissues, with the excep tion of ventral prostate. At 150 mg/kg/day, significant decreases (p ≤ 0.05 or 0.01) were observed in the weights of the seminal vesicle and coagulating gland (with and without fluid), the dorsolateral prostate, the LABC, and the left and right epididymis. In addition, the weights of the testes were decreased (NS) by 10-11% at the high dose. At 75 m g/kg/da y, dorsolateral prostate weights were decreased (p ≤ 0.05), andMale Pubertal Assay (2012) / Page 18 of 19 TEBUCONAZOLE / 128997 OCSPP 890.1500 / OECD None


## although not significant, substant ial weight decreases (11-21%) were noted in the seminal vesicle and coagulating gland (with and without fluid) and the LABC.
Finally, atrophy was observed in the following androgen-dependent tissues: (i) testes (atrophic/small and/or soft) at 75 and 150 mg/kg/ day; (ii) epididymis at 150 mg/kg/day; (iii) seminal vesicle at 75 and 150 mg/kg/day; (iv) prostate at 75 and 150 mg/kg/day; and (v) LABC at 75 and 150 mg/kg/day. Although the incidences of atrophy were minimal, it was a common finding in several androgen-depe ndent tissues, and there was additional evidence of effect on androgen-related syst ems. Consequently, these effects were considered treatment-related. Additionally, red foci were observed in the epididymis at 150 mg/kg/day. Unilateral and bilate ral tubular degeneration in th e testes was increased at 350 mg/kg/day. Bilateral oligospermia in the epididymis was observed at 75 and 150 mg/kg/day.
The high dose used in the curr ent study is considered adequate based on the effects observed on body weight and body weight gains and the in formation from the available range-finding study.

C. STUDY DEFICIENCIES: The following deficiencies were noted that are not considered to have had an adverse impact on the results, interpretation or conclusions of this study:

 The study containing stability analyses [T ahmasseb, A. L. (2011), SA 11273] was not provided for verification of stability results.

 Thyroid glands were not weighed after fixation.

 Weaning weights were not reported.
The following parameters did not satisfy the guideline performance criteria for controls:

 CV for age at PPS (9.0%) was greater than the maximum acceptable value (5.67%);

 CV for weight at PPS (15.1%) was greater th an the maximum acceptable value (7.57%);

 Age at PPS of 48.00 days (acceptable range: 39.781-46.513 days);

 Body weight at PPS of 267.68 g ( acceptable range: 188.277-256.169 g);

 Mean serum T 4 

## level of 3.8 μg/dL (acceptable range: 4.056-7.376 μg/dL);

 Mean serum TSH level of 3.55 ng/mL (acceptable range: 4.212-24.112 ng/mL).Male Pubertal Assay (2012) / Page 19 of 19 TEBUCONAZOLE / 128997 OCSPP 890.1500 / OECD None



## APPENDIX
Because this is a range-finding study, only a summary is provided to confirm the adequacy of the dose selection rationale used in th e male pubertal assay (MRID 48688302).
In a preliminary range-finding assay, tebuconazo le (97.5%; Batch #: K689052) in aqueous 0.5% methylcellulose 400+0.4% Tween 80 was administered via daily oral gavage in a dose volume of 5 mL/kg body weight to 4 Sprague Dawley [Crl:C D(SD)] rats/sex/dose at doses of 0, 80, 150, or 300 mg/kg/day. Rats were 25 days old on the firs t day of dosing. The animals were dosed for 10 consecutive days and necropsied approximately 24 hours after the final dose administration.

All animals survived to scheduled sacrifice. Clin ical signs were limited to increased salivation in males and females at 300 mg/kg/day and in females at 150 mg/kg/day. No treatment-related effects were reported at necrops y in any study animals.
Body weight and body weight gain were decreased in the treated groups. In the 300 mg/kg/day dose group, there was a body weight loss during Days 1-3 in males and the overall mean cumulative body weight gain was decreased 28% rela tive to control. In 300 mg/kg/day females, the mean cumulative body weight gain was decr eased 83% on Days 1-3 and 45% on Days 1-5. In the 150 mg/kg/day dose group, the overall mean body weight gain was decreased 14%, relative to control, in males, and decreased by 46% in females on Days 1-3 only. The mean cumulative body weight gain in the 80 mg/kg/day dose group was decreased between Days 1 and 11 by 21% in males and by 18% on Days 1-3 only in females.
It did not appear that statistical analyses were conducted.

COMPLIANCE: It was stated that the study was not performed in compliance with GLPs in that it was not subjected to specific Quality Assurance inspections.

# DATA EVALUATION RECORD

TEBUCONAZOLE
Study Type: OCSPP 890.1550, Steroidogenesis Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-22-2012 (MRID 48619404)

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: 3/10/2012 Secondary Reviewer Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 3/12/2012
Program Manager: Signature: Jack D. Early, M.S. Date: 3/16/2012

Quality Assurance: Signature: Steven Brecher, Ph.D. Date: 3/15/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).

### TEBUCONAZOLE / 128997


## Primary Reviewer:**_Ra~v---K~e_nt~._P_h~.D~·------­** 

## Health Effects Division Secondary Reviewer: John Liccione, Ph.D. 

## Health Effects Division Steroidogenesis Assay (2011) I 

### Page 1 ofll OCSPP 890.1550/ OECD None



## DATA EVALUATION RECORD

STUDY TYPE: Steroidogenesis Assay (H295R Cells); OCSPP 890.1550



## PC CODE: 128997 

## DP BARCODE: D398353

TXR#: 0056253 

## CAS No.: 107534-96-3



## TEST MATERIAL (PURITY): Tebuconazole (97 .5% w/w)



## SYNONYMS:

CITATION:

SPONSOR: AE F069623, a-[2-( 4-chlorophenyl)ethyl]-a-(1, 1-dimethylethyl)-lH-1,2,4- triazole-1-ethanol

Tinwell H. 

## (2011) Evaluation oftebuconazole in the H295R steroidogenesis assay. Bayer S.A.S., Bayer CropScience, Sophia Antipolis Cedex, France. Laboratory Study No. SA 11072. November 10, 2011. MRID 48619404. Unpublished.

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



## TEST ORDER#: CON-128997-20



## EXECUTIVE SUMMARY: In a steroidogenesis assay (MRID 48619404), H295R cells cultured 

## in vitro in 24-well plates were incubated with tebuconazole (97.5% purity, Batch# AE F069623-01-18) at increasing logarithmic concentrations from 10

- 10 

## to 10

- 4 

## M for 48 hours in triplicate for three independent experiments. Dimethyl sulfoxide (DMSO) was used as the vehicle, at a final concentration of 0.1 % (v/v).

Testosterone and estradiol levels were measured using specific EIA kits. 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 a known inducer (forskolin) of estradiol and testosterone production.

Guideline acceptability recommendations and requirements were generally met, including lack of cytotoxicity, adequate production of testosterone and estradiol, acceptable reproducibility (low %CV), and appropriate induction and inhibition with positive controls, with the following exceptions. In the QC plates, the variability (CV) in the solvent control (SC) between the runs
was > 30% for estradiol (31 % ), and for Run 3, the forskolin control resulted in a 1. 80-fold induction of testosterone , outside the**~2-fold** 

## induction recommendation in the Guideline (average induction across runs was >2-fold).Steroidogenesis Assay (2011) / Page 2 of 11 TEBUCONAZOLE / 128997 OCSPP 890.1550/ OECD None


## Precipitation of the test compound was not reported at any concen tration. Cytotoxicity was not observed at any tested concentration. Decreases (p ≤ 0.05) in testosterone concentrations ( ↓ 32- 98%) were observed at ≥ 10 − 6 

## M, and estradiol production was completely inhibited at ≥ 10 − 5 

## M. An effect was not observed at other c oncentrations in these two assays.
Based on the hormone responses in each of the three independent runs, tebuconazole treatment resulted in statistically signi ficant and reproducible decreases in testosterone and estradiol production.
The assay satisfies the EDSP Tier 1 Test Order requi rements for a Steroidogenesis assay (OCSPP 890.1550).

COMPLIANCE: Signed and dated GLP Compliance a nd Quality Assurance statements were provided.Steroidogenesis Assay (2011) / Page 3 of 11 TEBUCONAZOLE / 128997 OCSPP 890.1550/ OECD None



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Bayer S.A.S. Location: Sophia Antipolis Cedex, France Study Director: H. Tinwell Other Personnel: O. Blank, Replacement Study Director S. Colombel, Responsible Technician For Cell Culture D. De Barbeyrac, Clinical Pathology Laboratory P. Almeras, Report Unit Assistant Study Period: May 17, 2011 through November 10, 2011



## 2. Test Substance: Tebuconazole Description: White powder Batch # (expiration date): AE F069623-01-18 (August 23, 2012) Purity: 97.5% w/w Solubility: Soluble at 10

− 1 M in DMSO Vapor pressure: Not provided Stability: Not provided Storage conditions: Air-tight, light-resistant cont ainer at room temperature CAS #: 107534-96-3 Molecular weight: 307.8 Structure:



## 3. Positive Control (Induction): Forskolin

Description (molecular weight): White powder (410.50) Source: Sigma-Aldrich (St. Louis, MO) Batch # (expiration date): 109K5057V (December 2014) Purity: 98% Solubility (in solvent): Soluble at tested concentrations in DMSO Storage conditions: Air-tight, light-resistant cont ainer at room temperature CAS #: 66575-29-9



## 4. Positive Control (Inhibition): Prochloraz

Description (molecular weight): White powder (376.67) Source: Sigma-Aldrich (St. Louis, MO) Batch # (expiration date): SZE6220X (August 8, 2012) Purity: 99.1% Solubility (in solvent): Soluble at tested concentrations in DMSO Storage conditions: Air-tight, light-resistant cont ainer at room temperature CAS #: 67747-09-5Steroidogenesis Assay (2011) / Page 4 of 11 TEBUCONAZOLE / 128997 OCSPP 890.1550/ OECD None



## 5. Solvent/Vehicle Control: Dimethyl Sulfoxide (DMSO) Description (molecular weight): Clear liquid (78.13) Source: Sigma-Aldrich (St. Louis, MO); Ref # 154938 Batch # (expiration date): 76396EM (not provided) Purity: Not provided Storage conditions: Not provided CAS #: 67-68-5 Justification for choice of solvent: Not provided. DMSO is a Guideline recommended solvent in which tebuconazole i reasonably soluble. Final concentration in assay: 0.1% v/v



## 6. Stock Medium: Dulbecco’s modified Eagle’s medium/Ham’s F12 nutrient mixture Source: LGC (Ref: 30-2006) Lot # (expiration date): 30001123 (July 2011) Sodium bicarbonate: Component of stock medium Nu-Serum: 2.5%, BD Biosciences (Ref: 355100); Lot # 81515; not reported if background hormone concentrations were measured by performing laboratory ITS+premix: VWR (Ref: 734-1315); Lot # 75382 and 84339 Other components: 0.1% Penicillin/streptomyci n; GIBCO (15070-063); Lot # 847134.



## 7. Test Cells: H295R human adrenocortical carcin oma cells (ATCC CLR-2128; Batch # 58660579) at passage 5.5 – 9.5 were incubated in the stock medium at the time of plating, dosing, and harvest. Incubati on conditions were at 5% CO 2 

## 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 #: 5.5 – 9.5 x Cells frozen down at passage 5 x Frozen cells cultured for 4 additional passages x Total number of passages does not exceed 10



## B. METHODS

1. Pre-Test Information
a. Hormone Assay Interference Test: A sample of the master mix of each concentration of the test substance was assessed for interference with the testosterone and estradiol EIA kits. The percent variation of estradiol and test osterone compared to DMSO controls was generally less than 30% for all tested concentrations for both hormones. At 10

− 10 

## M tebuconazole, testosterone was decreased by 30%; however, this finding was considered spurious.

b. Hormone Extraction: Hormone extraction was not reported.

c. Laboratory Proficiency Test: Two laboratory proficiency determinations (SA 10203; non-GLP) were previously conducted by the t echnician who conducted the present study. The test conditions were the same as the curr ent assay. The concentrations used and the EC 50 

## values for each positive control are provided below (Table 1).Steroidogenesis Assay (2011) / Page 5 of 11 TEBUCONAZOLE / 128997 OCSPP 890.1550/ OECD None



### TABLE 1. Results of Laboratory Proficienc y Test using Forskolin and Prochloraz. a 

### Compound Concentrations tested ( μ M) Run Estimated EC 50 

### ( μ M) b 

### Testosterone Estradiol Forskolin 0.03, 0.1, 0.3, 1, 3 and 10 1 s t 

### ~0.1 ~0.5 2 n d 

### 0.7-0.8 ~1 Prochloraz 0.01, 0.03, 0.1, 0.3, 1, and 3 1 s t 

### 0.04-0.05 ~0.2 2 n d 

### ~0.05 0.1-0.2 a Table information taken from page 14 of the study report.



## 2. Test Solutions: Tebuconazole, forskolin, and prochlor az were dissolved in DMSO and diluted 1:1000 in the final treatment medium. When added to the cell culture plates, these dilutions yielded final concentrations of 1 μM and 10 μM for forskolin, 0.1 μM and 1 μM for prochloraz, and 10

− 4 

## , 10 − 5 

## , 10 − 6 

## , 10 − 7 

## , 10 − 8 

## , 10 − 9 

## , and 10

− 10 

## M for tebuconazole. The final concentration of DMSO in the medi um was held constant at 0.1% (v/v).

3. Cell Plating and Preincubation: Cells were maintained in the stock medium described above. The hormone concentrations in undilute d Nu-Serum were not reported. H295R cells were grown for five passages, frozen in liquid nitrogen, then thawed and cultured for five to nine additional passages prior to use in the assay. The cells were plated into wells of a 24-well cell culture plate at a density of approximately 300,000 cells in 1 mL. The cells were then placed into an incubator (37±1°C/5% CO 2 

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

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

TABLE 2. Dosing Schematic for the Exposure of H295 R Cells to Tebuconazole (F inal Concentrations in M). a 1 2 3 4 5 6 A DMSO DMSO DMSO 10

− 4 10

− 4 10

− 4 B 10

− 5 10

− 5 10

− 5 10

− 6 10

− 6 10

− 6 C 10

− 7 10

− 7 10

− 7 10

− 8 10

− 8 10

− 8 D 10

− 9 10

− 9 10

− 9 10

− 10 10

− 10 10

− 10 a Data were obtained from page 15 of the study report.



## A concurrent QC plate was included with each of the three independent runs of the test chemical plates to demonstrate the assay’s res ponse to forskolin (an inducer of testosterone and estradiol production) and prochloraz (an inhibitor of testoste rone and estradiol production). The QC plate was prepared and dosed in the same manner w ith either forskolin or prochloraz, according to the schematic presented in Table 3.Steroidogenesis Assay (2011) / Page 6 of 11 TEBUCONAZOLE / 128997 OCSPP 890.1550/ OECD None



### TABLE 3. Dosing Schematic for the QC Plate for Po sitive Controls (Final Concentrations in μM). a

1 2 3 4 5 6 A Blank b Blank Blank Blank Blank Blank B DMSO DMSO DMSO DMSO DMSO DMSO C Forskolin 10 μM Forskolin 10 μM Forskolin 10 μM Forskolin 1 μM Forskolin 1 μM Forskolin 1 μM D Prochloraz 1 μM Prochloraz 1 μM Prochloraz 1 μM Prochloraz 0.1 μM Prochloraz 0.1 μM Prochloraz 0.1 μM a Data were obtained from page 125 of the study report. b Blank wells received medium only.



## Following dosing, the plates were incuba ted for approximately 48 hours under the conditions previously described. After the in cubation period, three or four aliquots of medium were collected and frozen at

− 74±10°C until further processing.

5. Cell Viability/Cytotoxicity Assay: Prior to the main st udy, the cytotoxicity of tebuconazole, at the two hi ghest concentrations (10

− 4 

## and 10

− 5 

## M), was assessed using the XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl )-2H-tetrazolium-5-carboxanilide) method, which measures the mitochondrial activity of liv ing cells. The cells were first exposed to tebuconazole for 48 hours under the same conditions of the test. During the last four hours of incubation the cells were exposed to XTT (supplied by Sigma, ref. number: X4251; batch number: 038K1592)/ phenazine methosul fate (supplied by Sigma, ref. number: P9625; batch number: 058K1537). The resulting orange formazan derivative was measured at OD 450 nm. No cytotoxi city was observed at 10

− 4 

## and 10

− 5 

## M. In addition, at the end of each evaluation, the treated cells were stained with trypan blue to evaluate cell viability, and the Sponsor stated that these assays demons trated the viability of the cells (data not presented).

6. Hormone Measurement System: The concentration of eac h hormone (testosterone and estradiol) was evaluated in triplicate in the cu lture medium using specific EIA kits supplied by Enzo Life Sciences Inc. (Plymouth Meeti ng, PA) for testosterone and Cayman Chemical Company (Ann Arbor, MI) for estradiol. The limit of detection was 95.7 pg/ml for testosterone and 5.7 pg/ ml for estradiol.
The following performance criteria were met (indicated by an “x”):

x Method detection limit (100 pg/mL testosterone; 10 pg/mL estradiol) NA Spiked sample recovery acceptable for two concentrations of testos terone 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 be low maximum response on standard curve x Test compound tested for interfer ence with meas urement system



## C. DATA ANALYSIS: To evaluate the relative increas e or decrease of hormone production after test chemical exposure, the results were normalized to the mean SC value for each assay and results were expressed as fold-change relative to the SC in each exposure plate. All data were expressed as mean ± standa rd deviation (SD). Relative changes were calculated as follows:Steroidogenesis Assay (2011) / Page 7 of 11 TEBUCONAZOLE / 128997 OCSPP 890.1550/ OECD None 

## Relative Change = Hormone concentration in each well ÷ mean hormone concentration of solvent control.
For each hormone, the Bartlett test was perf ormed to compare the homogeneity of group variances. If the Bartlett test was not signi ficant (p>0.05), means were compared using the analysis of variance (ANOVA). If the ANOVA was not significant (p>0.05), the group means were considered to be homogeneous a nd no further analysis was performed. If the ANOVA was significant (p ≤ 0.05), means of the exposed groups were compared to the mean of the control group using the D unnett test (2-sided). If the Bartlett test was significant (p ≤ 0.05), group means were compared using the non-parametric Kruskal-Wallis test. If the Kruskal-Wallis test was not significant (p> 0.05), the group means were considered to be homogeneous and no further analysis was performed. If the Kruskal-Wallis test was significant (p ≤ 0.05), means of the exposed groups were co mpared to the mean of the control group using the Dunn test (2-sided). The level of significance for each statistical comparison was performed at 0.05. These statisti cal analyses were considered appropriate.

II. RESULTS

A. TEST COMPOUND: Precipitation of the test compound was not reported at any concentration. Cytotoxicity was not observed at any tested concentration. The variability (%CV) between the runs for the solvent controls was 19% (testosterone) and 25% (estradiol), both within the guideline recomm endation of 30%. The variability (CV) within each run for the solvent controls in the testosterone and estradiol assays was ≤ 22%, which was within the guideline recommendation of 30%.

The concentrations of estradiol and testoste rone observed in the three assay runs are summarized in Table 4. Decreases (p ≤ 0.05) in testosterone concen trations were observed at 10 − 6 

## M and above ( ↓ 32-98%); estradiol secretion was completely inhibited at 10

− 5 

## M and above (Table 4). An effect wa s not observed at lower concentr ations in these two assays. The effects on testosterone and estradiol are illustrated in Figures 1 and 2.Steroidogenesis Assay (2011) / Page 8 of 11 TEBUCONAZOLE / 128997 OCSPP 890.1550/ OECD None

TABLE 4. Mean (±SD) Hormone Concentrations Follo wing Treatment with Tebuconazole for 48 Hours. a Nominal Concentration (M) Run 1 Run 2 Run 3 Run 1 Run 2 Run 3 Mean ± SD Statistical Significance
Testosterone (pg/mL) Fold Difference DMSO 8757.3 11902.0 8069.7 — — — — — —

10 − 10 8006.7 10103.3 7502.3 0.91 0.85 0.93 0.89 0.15 — 10 − 9 6850.0 11291.3 7895.7 0.78 0.95 0.98 0.91 0.22 — 10 − 8 8470.7 10369.3 7126.3 0.97 0.87 0.88 0.90 0.17 — 10 − 7 9464.0 10218.0 7730.0 1.08 0.86 0.96 0.95 0.13 — 10 − 6 6418.7 7763.7 5245.0 0.73 0.65 0.65 0.68 0.12 Mean 10 − 5 1593.7 1760.7 1978.0 0.18 0.15 0.25 0.19 0.03 Mean 10 − 4 161.5 173.7 336.7 0.02 0.01 0.04 0.02 0.01 Mean Estradiol (pg/mL) Fold Difference DMSO 289.0 321.0 211.0 — — — — — —

10 − 10 343.0 318.5 204.0 1.19 0.99 0.97 1.04 0.29 — 10 − 9 356.0 290.0 242.0 1.23 0.90 1.15 1.08 0.20 — 10 − 8 244.3 275.0 216.7 0.85 0.86 1.03 0.90 0.17 — 10 − 7 252.0 325.7 244.7 0.87 1.01 1.16 1.00 0.17 — 10 − 6 264.7 389.3 240.7 0.92 1.21 1.14 1.09 0.26 — 10 − 5 CI CI CI CI CI CI CI CI — 10 − 4 CI CI CI CI CI CI CI CI — a Data (rounded up) were obtained from pages 17–19 of the stud y report. The lower limit of quantification (LLQ) was 95.7 pg/mL for testosterone and 5.7 pg/mL for estradiol. St atistical evaluation was con ducted on overall data only. CI Complete inhibition (<100 pg/mL)



## FIGURE 1. Change in Testosterone Produc tion Relative to Tebuconazole Concentration.Steroidogenesis Assay (2011) / Page 9 of 11 TEBUCONAZOLE / 128997 OCSPP 890.1550/ OECD None


## FIGURE 2. Change in Estradiol Production Relative to Tebuconazole Concentration.

B. CYTOTOXICITY: The Sponsor stated that no cyto toxicity was observed up to 10 -4 

## M tebuconazole based on the XTT method and asse ssment of cell viability with trypan blue after incubations.

C. QC PLATE: The minimum basal hormone production levels (500 pg/mL for testosterone, 40 pg/mL for estradiol) were met in both blank and SC wells for testosterone (Table 5). Guideline requirements for basal hormone production, induction, and inhibition were met for testosterone and estradiol, except for induction of testoster one in Run 3 (1.80–fold induction was observed, which is outs ide the Guideline recommendation of ≥ 2-fold induction).
The variability (%CV) between th e runs based on the absolute hormone concentrations in the SC was 22% for testosterone, meeting guideline requirements of ≤ 30% CV. The variability (%CV) between the r uns in the SC was 31% for estradiol which is greater than the guideline requirement. The variability (CV) in the SC within each run in the testosterone and estradiol assays was ≤ 23%, which was with in the guideline recommendation of ≤ 30%.Steroidogenesis Assay (2011) / Page 10 of 11 TEBUCONAZOLE / 128997 OCSPP 890.1550/ OECD None



### TABLE 5. Mean (±SD) Hormone Concentrations Followi ng Treatment with Forskolin or Prochloraz for 48 Hours. a
Concentration (μM) Run 1 Run 2 Run 3 Run 1 Run 2 Run 3 Mean ± SD Testosterone (pg/mL) Fold Difference (Relative to DMSO) Blank 9591.7 7894.0 13588.3 — — — — — DMSO 10436.0 8413.3 13077.5 — — — — — 1 μM Forskolin 20903.0 16425.3 21655.3 2.00 1.95 1.66 1.85 0.25 10 μM Forskolin 23538.0 18837.3 23512.3 2.26 2.24 1.80 2.06 0.25 0.1 μM Prochloraz 3014.7 1849.3 2929.0 0.29 0.22 0.22 0.24 0.06 1 μM Prochloraz 486.3 349.0 638.0 0.05 0.04 0.05 0.05 0.01 Estradiol (pg/mL) Fold Difference (Relative to DMSO) Blank 264.5 168.0 448.7 — — — — — DMSO 329.7 193.4 372.7 — — — — — 1 μM Forskolin 4734.3 3225.7 6092.7 14.36 16.68 16.35 15.37 4.66 10 μM Forskolin 9697.0 6228.3 13477.3 29.41 32.20 36.16 32.16 10.54 0.1 μM Prochloraz < 100 < 100 213.0 CI CI 0.57 0.70 0.09 1 μM Prochloraz < 100 < 100 < 100 CI CI CI CI CI a Data were obtained from pages 128-130 of the study report. The lower limit of quantification (LLQ) was 95.7 pg/mL for testosterone and 5.7 pg/mL for estradiol. CI Complete inhibition (<100 pg/mL)



## III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Tebuconazole was not cy totoxic to the H295R cells nor did it interfere with the hormone EIA kits. Tebucon azole induced a statistically significant concentration-related reduction in testosterone secret ion starting from 10

− 6 

## M, with the overall fold-changes being 0.68 (p ≤ 0.05) at 10

− 6 

## M, 0.19 at 10 − 5 

## M (p ≤ 0.001) and 0.02 at 10 − 4 

## M (p ≤ 0.001). No effect on estradiol con centration was observed following treatment with tebuconazole between 10

− 10 

## M to 10 − 6 

## M, whereas a complete inhibition was recorded at 10

− 5 

## M and 10

− 4 

## M in each of the three evaluations.

B. AGENCY COMMENTS: Guideline acceptability recommendations and requirements were generally met, including lack of cytotoxi city, adequate production of testosterone and estradiol, acceptable reproducib ility (low %CV), and appropria te induction and inhibition with positive controls, with the following exceptions. In the QC plates, the variability (CV) between the runs in the SC was >30% for es tradiol (31%), and for Run 3, the forskolin control resulted in a 1.80–fold induc tion of testosterone, outside the ≥ 2-fold induction recommendation in the Guideline (average i nduction across runs wa s >2-fold). Despite these departures from the guideline, the pos itive controls performed as expected, and tebuconazole was successfully identified as an inhibitor of both testosterone and estradiol.
Precipitation of the test compound was not reported at any con centration. Cytotoxicity was not observed at any tested concentration. Decreases (p ≤ 0.05) in testosterone concentrations were observed at 10

− 6 

## M and above ( ↓ 32-98%); estradiol secretion was completely inhibited at 10 − 5 

## M and above. An effect was not observe d at lower concentrations in these two assays.

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:Steroidogenesis Assay (2011) / Page 11 of 11 TEBUCONAZOLE / 128997 OCSPP 890.1550/ OECD None



##  The stability of tebuconazole in th e test system was not reported.

 The variability (CV) between the runs was >30% for estradiol in the SC in the QC plates.

 In Run 3 of the QC plate, the forskolin control resulted in a 1.80–fold induction of testosterone, outside the Guideline recommendations ( ≥ 2-fold induction).

# DATA EVALUATION RECORD

TEBUCONAZOLE
Study Type: OCSPP 890.1600, In vivo Uterotrophic Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-67-2012 (MRID 48673703)

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 35, Suite B Durham, NC 27713

Primary Reviewer: Signature: Rebecca L. Bryan, B.S. Date: 6/1/2012

Secondary Reviewer: Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 6/11/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 6/25/2012
Quality Assurance: Signature: Jack D. Early, M.S. Date: 6/25/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).TEBUCONAZOLE / 128997 

### In vivo Uterotrophic Assay (2012) I Page 1 of 10 OCSPP 890.1600/ OECD 440 

## Primary Reviewer: =R=ay......_:.;K=e=nt=·-=P-=h=.D:;..:.

_________ 

## Signature:

# ~ }(;£ Risk Assessment Branch ID, Health Effects Division Date:

# ~013 Secondary Reviewer: Jess Rowland 

## Signature:**~~** 

## Health Effects Division Date: k,/ LJ 

# Ir.) 

# I DATAEVALUATIONRECORD 

# I TemPfllte version 09/2011



## STUDY TYPE: Uterotrophic Assay (Rat); OCSPP 890.1600; OECD 440



## PC CODE: 128997 

## DP BARCODE: D401595

TXR#: 0056253 

## CAS#: 107534-96-3



## TEST MATERIAL <PURITY): Tebuconazole (97.5% a.i.)



## SYNONYMS: AE F069623; a-[2-(4-chlorophenyl)ethyl]-a-(l,l-dimethylethyl)-lH-1,2,4- triazole-1-ethanol



## CITATION: Blanck, M. (2012) Tebuconazole: Evaluation in the Immature Rat Uterotrophic Assay. Bayer S.A.S., Sophia Antipolis Cedex, France. Laboratory Report No.: SA 11010, February 2, 2012. MRID 48673703. Unpublished.



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



## TEST ORDER#: CON-128997-20



## EXECUTIVE SUMMARY: In a Uterotrophic Assay (MRID 48673703) conducted to screen for potential estrogenic activity, tebuconazole (97.5% a.i., Lot# K689052) in 0.5% aqueous methylcellulose 400 

# + 0.4% Tween 80 was administered daily via oral gavage to groups of 6 immature female, Sprague-Dawley rats at dose levels ofO (vehicle), 35, 75, or 150 mg/kg/day on post-natal days (PND) 19-21. A positive control group was treated with 17a-ethynyl estradiol (EE) in com oil by gavage at a dose level of 1 μg/kg/day. All animals were terminated and necropsied on PND 22 approximately 24 hours after the final dose to determine wet and blotted uterine weights.

All animals survived until scheduled termination. No clinical signs of toxicity were observed in any rats in the vehicle control, EE, or**~75** 

## mg/kg/day tebuconazole groups. Five of six animals in the 150 mg/kg/day group were observed to have reduced motor activity on Day 3. Body weights for the 35 and 75 mg/kg/day tebuconazole groups were comparable to the vehicle controls throughout the duration of the assay. Overall (Days 1-3) body weight gains were decreased (p<0.05) in the 75 mg/kg/day (i38%) group compared to the vehicle controls. Final body weights were decreased (p<0.01) by 15-24% on Days 2-3 in the 150 mg/kg/day group compared to controls, resulting in an overall (Days 1-3) body weight loss (p<0.01) of 3.0 g (compared to a gain of 8.2 gin the controls). Uterine weights in the tebuconazole treated groups
were comparable to the vehicle controls. Absolute wet and blotted uterus weights for theIn vivo Uterotrophic Assay (2012) / Page 2 of 10 TEBUCONAZOLE / 128997 OCSPP 890.1600/ OECD 440 

## positive control (EE) group were increased (p<0.05) as expected by 210% and 202%, respectively. Based on the observed decreases (15-24%) in bo dy weights at 150 mg/kg/day, the dose levels used in this study were adequate.
No statistically significant changes were seen in uterine weight in this assay. Tebuconazole is negative in the uterotrophic assay.
The assay satisfies the EDSP Tier 1 Test Order require ments for a uterotrophic assay (OCSPP 890.1600).

COMPLIANCE: Signed and dated Data Confiden tiality, GLP Compliance, and Quality Assurance statements were provided.In vivo Uterotrophic Assay (2012) / Page 3 of 10 TEBUCONAZOLE / 128997 OCSPP 890.1600/ OECD 440



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Bayer S.A.S. Location: Sophia Antipolis Cedex, France Study Director: M. Blanck Other Personnel: S. Lautraite (Replacement Study Director); J.P. Ko cwin (Laboratory Animal Resources); C. Jaussely (In-Life Supervis or); H. Lormeau (Technician); L. Amir Tahmasseb (Senior Chemist); C. Pallen (Statistician); M. Vagner (Report Unit Assistant) Study Period: September 27, 2011 to February 2, 2012



## 2. Test Substance: Tebuconazole Description: White powder Source: Bayer CropScience (Frankfurt, Germany) Lot #: K689052 (expiry date: August 23, 2012) Purity: 97.5% a.i. Stability: Stable in vehicle for up to 22 days at 5  3 °C. CAS #: 107534-96-3 Structure:
Cl OH CH 3

CH 3 CH 3

N N N


## 3. Reference Estrogen: 17 α -ethynyl estradiol (EE) Supplier: Sigma Aldrich, Inc. (St. Louis, MO) Lot #: 090M1241V (expiry date: April 2012) Purity: 99% CAS #: 57-63-6



## 4a. Solvent/Vehicle Control for Test Chemical: Methylcellulose 400 Supplier: Sigma-Aldrich Batch #: 060M123V Rationale (if other than water): Not reported Final concentration: Additional Solvent: 0.5% aqueous Tween 80 at 0.4% aqueous (Batch # S5264887.914; Merck)



## 4b. Solvent/Vehicle Control for Positive Control: Corn Oil Supplier: Sigma Aldrich Batch #: MKBD2427 Rationale (if other than water): Not reported Final concentration: Not applicableIn vivo Uterotrophic Assay (2012) / Page 4 of 10 TEBUCONAZOLE / 128997 OCSPP 890.1600/ OECD 440



## 5. Test Animals: Species: Rats (immature, female only) Strain: Sprague Dawley, Crl:CD(SD) Age/weight at dose initiation: PND 19/34.3-42.0 g females Source: Charles River Laboratories (S t. Germain-sur-l’Arbresle, France); shipped with dam Housing: Rats were housed 6 per cage in makrolon cages with heat-treated soft wood bedding (S.A.F.E.; Scientific Animal Food and Engineering, Augy, France).s Diet: Certified rodent pelleted and irradiated di et A04C-10 (S.A.F.E.; Augy France) with phytoestrogens <150 μ g/g diet , ad libitum. Water: Filtered tap water, ad libitum Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 20-24 oC 40-70% 10-15/hr 12 hrs light/ 12 hrs dark Acclimation period: Stated as at least 5 days; immature fe males (PND 6 on arrival) housed with their dam



## B. STUDY DESIGN

1. In-Life Dates: Start: October 11, 2011 End: October 14, 2011

2. Study Design: Following a 13-day acclimation period, immature, intact female rats were administered the test substances from PND 19 to 21. Rats were euthanized approximately 24 hours after the last dose (PND 22) and necrop sied for uterine weight measurements. The pups were housed with their dam prior to weaning.

3. Animal Assignment: Animals were randomly assigned, stratified by body weight, to the test groups noted in Table 1. A computer ized randomization procedure (XMS Path/Tox V4.2.2) was used to select animals for the study from the middle of the weight range of the available animals, which ensured a similar body weight distribution among groups. Thirty of the 40 female pups received were selected for the study. The body weight of each animal was within 20% of the overall mean.

TABLE 1. Study Design a 

### Test Group Dose (mg/kg/day) # of Females Estrogen Agonist Assay Vehicle Control 0 6 Tebuconazole Low 35 6 Tebuconazole Mid 75 6 Tebuconazole High 150 6 17 α -ethynyl estradiol (EE), Reference Estrogen 0.001 6 a Data were obtained from page 16 of the study report.



## 4. Dose Selection Rationale: The dose levels used in this study were chosen based on the results of a preliminary range-finding study in Sprague Dawley rats. A summary of this study is included as an Appendix to this DER.

5. Dose Preparation and Analysis: Dose formulations were pr epared once prior to initiation of treatment by suspending the appropriate amounts of tebuconazole in 0.5% aqueousIn vivo Uterotrophic Assay (2012) / Page 5 of 10 TEBUCONAZOLE / 128997 OCSPP 890.1600/ OECD 440 

## methylcellulose 400 + 0.4% Tween 80. The positiv e control, EE, was prepared once prior to initiation of treatment by diluting in corn oil. Homogeneity of the test substance formulation was checked at the beginning of the study for the lowest and the highest concentrations (7 and 30 g/L) at the surface and bottom levels. The mean values obtained from the homogeneity check were used as measured concentrations . In addition, the intermediate concentration (15 g/L) was checked. It was reported that a separate study (SA 11273 1 

## ) demonstrated that tebuconazole in 0.5 % methylcellulose 400 + 0.4% Tween 80 was stable at concentrations of 0.6 and 170 g/L for at least 22 days under similar conditions to those of the current study.

Results of Dose Analysis
Homogeneity (%CV): 0.28-0.76%

Stability: No data provided

Concentration (% of nominal): 95-97%
Although the middle layer of the test solution wa s not tested for homogeneity, the analytical data indicated that the mixing procedure wa s adequate and that the variation between nominal and actual dosage to the animals wa s acceptable. The study referenced above should be submitted for verifica tion of the stability findings.
For EE, the %CV for homogeneity analyses was 0.13% and the average concentration was found to be 90.0% of nominal.

6. Dosage Administration: Animals were administered the tebuconazole formulations, positive control (EE), or ve hicle control daily via oral gavage for three consecutive days in a dose volume of 5 mL/kg body weight. Dose vol umes were adjusted daily based on the concurrent body weight meas urement. Dose formulations were stored at 5 ± 3 ° 

## C when not in use.

7. Statistics: Statistical analyses of body weight and body weight changes were carried out using Path/Tox System V4.2.2. Statistical analyses of terminal body weight and uterine weights were carried out using SAS system. The following parameters were analyzed statistically: body weights, body we ight gains, terminal body weight s, and uterine weights.
For body weight gain, the data were first analyzed using Bartlett’s test. If the data were not significant, an ANOVA test was performed; a two-sided Dunnett’s test was conducted if the results of the ANOVA were significant. If the re sults of Bartlett’s test were significant, a Kruskal-Wallis test was performed, followed by a two-sided Dunn’s test fo r significant data. The same procedures were used for body weight (except terminal body weight), except that data were transformed (log transformation) and a second Bartlett’s test was conducted if the data were significant after the first Bartlett’s test.

1 

### Amir Tahmasseb, L. (2011). Tebuconazole, Stability in aqueous 0.5% methylcellulose + 0.4% tween 80, SA 11273, Bayer CropScience, Sophia Antipolis, France.In vivo Uterotrophic Assay (2012) / Page 6 of 10 TEBUCONAZOLE / 128997 OCSPP 890.1600/ OECD 440



## For uterine weights and terminal body weight, th e data were first anal yzed using Levene’s test; if the data were significant, a data transformation (log transformation) was conducted. If the data were not significant, an ANOVA test was performed; a one-sided (organ weights) or two-sided (terminal body we ights) Dunnett’s test was cond ucted if the results of the ANOVA were significant. If the results of Le vene’s test (after transformation) showed significance, a Kruskal-Wallis test was perfor med, followed by a one-sided (organ weights) or two-sided (terminal body weights) Dunn’s test for significant data.
In addition, analysis of c ovariance (ANCOVA) was conducte d on uterine weights using terminal body weight as a covariable; adjust ed means were compared using a one-sided Dunnett’s test. For comparison of data from th e positive control with data from the negative control, the data were analy zed using an F test; for body weights and uterine weights, the data were log-transformed and r eanalyzed if significant after the F test. Data that were not significant were analyzed using a one- or two-sided T-test, a nd data that were significant were analyzed using a modified one- or two- sided T-test. Signif icance was denoted at p ≤ 0.05. The statistical analyses were considered appropriate.

C. METHODS

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

2. Body Weight: Animals were weighed at randomi zation, study initiati on, daily throughout the dosing period and at termination.

3. Food Consumption (Optional): Food consumption was not measured.

4. Necropsy and Measurement of Uterine Weight: On PND 22 (approximately 24 hours after final administration of the test substance), all animals ( not fasted) were anesthetized with isoflurane, exsanguinated, and subjected to a gross necropsy. Dissection of the uterus was performed according to the U.S. EPA Guideline. Briefly, the vagina was removed just below the cervix in order to retain the lumina l fluid in the uterus. The “wet” uterus (i.e., containing the luminal fluid) was weighed. Subsequently, the uterus was pierced and blotted to remove the luminal fluid and the blotted uterus was weighed.

5. Microscopic Examination (Optional): Microscopic examinations were not conducted.

II. RESULTS

A. OBSERVATIONS
1. Mortality: All animals survived until scheduled termination.In vivo Uterotrophic Assay (2012) / Page 7 of 10 TEBUCONAZOLE / 128997 OCSPP 890.1600/ OECD 440


## 2. Clinical Signs of Toxicity: No clinical signs of toxicity were observed in any rats in the vehicle control, EE, or ≤ 75 mg/kg/day tebuconazole groups. Five of six animals in the 150 mg/kg/day group were observed to have reduced motor activity on Day 3.

B. BODY WEIGHT AND WEIGHT GAIN: Selected body weight and body weight gain data are presented in Table 2. Body wei ghts for the 35 and 75 mg/kg/day tebuconazole groups were comparable to the vehicle cont rols throughout the duration of the assay. Overall (Days 1-3) body weight gains were decreased (p<0.05) in the 75 mg/kg/day ( ↓ 38%) group compared to the vehicle controls. B ody weights were decrea sed (p<0.01) by 15-24% on Days 2-3 in the 150 mg/kg/day group compared to controls, resulti ng in an overall (Days 1-3) body weight loss (p<0.01) of 3.0 g (compared to a gain of 8.2 g in the controls).

TABLE 2. Selected Group Body Weights and Cumulative Body Weight Gains (g) In the Estrogen Agonist Assay a



### Study Day Dose (mg/kg/day)

Vehicle Control Tebuconazole (35) Tebuconazole (75) Tebuconazole (150) Reference Estrogen EE (0.001) N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 

### 1 6 38.6 2.1 6 38.0 2.2 6 37.8 2.4 6 38.4 2.0 6 36.8 2.0 2 6 41.7 3.0 6 39.9 2.8 6 38.1 3.2 6 35.4** ( ↓ 15) 2.4 6 39.7 2.4

3 6 46.8 2.9 6 45.9 2.9 6 42.9 2.9 6 35.4** ( ↓ 24) 2.3 6 44.4 1.9

Body Weight Gain (Days 1-3) 6 8.2 1.2 6 8.0 1.1 6 5.1* ( ↓ 38) 1.1 6

− 3.0** ( ↓ 137) 2.9 6 7.6 0.8

a Data were obtained from Tables 2a, 2b, 3a, and 3b on pages 41, 43, 46, and 48 of the study report. BWG percent differences (calculated by the re viewers) are included in parentheses. N Number of animals in the group SD Standard Deviation * Significantly different from controls at p<0.05 ** Significantly different from controls at p<0.01



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

D. PATHOLOGY
1. Vaginal Opening: As expected, vaginal opening did not occur in any of the animals by the end of the treatment period.

2. Uterine Weights: Uterine weight data are presented in Table 3. Uterine weights in the tebuconazole treated groups were comparable to the vehicle controls . Absolute wet and blotted uterus weights for the positive cont rol (EE) group were increased (p<0.05) as expected by 210% and 202%, respectively.In vivo Uterotrophic Assay (2012) / Page 8 of 10 TEBUCONAZOLE / 128997 OCSPP 890.1600/ OECD 440 

## The mean blotted uterine weight for the ve hicle control group was 0.046% of terminal body weight, which was within the performance criteria (<0.09%) of th e test guideline.

TABLE 3. Uterine Weights from Estr ogen Agonist Assay in SD Rats a



### Parameters Dose (mg/kg/day)

Vehicle Control Tebuconazole (35) Tebuconazole (75) Tebuconazole (150) Reference Estrogen EE (0.001) N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD Terminal BW (g) 6 51.5 3.0 6 48.7 2.7 6 47.9 2.9 6 41.3** ( ↓ 20) 2.8 6 48.7 1.9

Wet, absolute (g) 6 0.0264 0.0022 5 b 0.024 0.0018 6 0.0249 0.0036 6 0.0205 0.0013 6 0.0819** ( ↑ 210) 0.0236

Wet, relative (%) 6 0.0514 0.0053 5 0.0487 0.0045 6 0.0519 0.0066 6 0.0497 0.0037 6 0.1694 0.0524

Blotted, absolute (g) 6 0.0234 0.0018 6 0.0211 0.0023 6 0.0224 0.003 6 0.0184 0.0009 6 0.0707** ( ↑ 202) 0.0166

Blotted, relative (%) 6 0.0455 0.0045 6 0.0433 0.0039 6 0.0469 0.0056 6 0.0446 0.0027 6 0.1459 0.0364


### a Data were obtained from pages 26-27 of the study report. Percent differences from controls, calculated by the reviewers, are included in parentheses. b One animal excluded from wet weights. BW Body weight N Number of animals in the group SD Standard Deviation ** Significantly different from controls at p<0.01



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

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Administration of up to 150 mg/kg/day tebuconazole for 3 days to immatu re Sprague-Dawley female rats did not increase uterine weights (wet and blo tted). No evidence of an estrogeni c potential was
detected under these conditions. E thynyl estradiol, the refere nce positive control, administered daily by oral gavag e for 3 days at 1 μg/kg/day ind uced an increase in absolute uterine weights (wet and blotted); ( p ≤ 0.01 ) and responded as expected.

B. AGENCY COMMENTS: All animals survived until scheduled termination. No clinical signs of toxicity were observed in an y rats in the vehicle control, EE, or ≤ 75 mg/kg/day tebuconazole groups. Five of six animals in the 150 mg/kg/day group were observed to have reduced motor activity on Day 3.
Body weights for the 35 and 75 mg/kg/day tebuc onazole groups were comparable to the vehicle controls throughout the duration of the assay. Overa ll (Days 1-3) body weight gains were decreased (p<0.05) in the 75 mg/kg/day ( ↓ 38%) group compared to the vehicleIn vivo Uterotrophic Assay (2012) / Page 9 of 10 TEBUCONAZOLE / 128997 OCSPP 890.1600/ OECD 440 

## controls. Final body weights were decrease d (p<0.01) by 15-24% on Days 2-3 in the 150 mg/kg/day group compared to controls, resulti ng in an overall (Days 1-3) body weight loss (p<0.01) of 3.0 g (compared to a gain of 8.2 g in the controls). Uterine weights in the tebuconazole treated groups were comparable to the vehicle controls . Absolute wet and blotted uterus weights for the positive cont rol (EE) group were increased (p<0.05) as expected by 210% and 202%, respectively. Base d on the observed decreases (15-24%) in body weights at 150 mg/kg/day, the dose levels used in this study were adequate. No statistically significant changes were seen in uterine weight in this assay. Tebuconazole is negative in the uterotrophic assay.

C. STUDY DEFICIENCIES: NoneIn vivo Uterotrophic Assay (2012) / Page 10 of 10 TEBUCONAZOLE / 128997 OCSPP 890.1600/ OECD 440



## APPENDIX
A summary of a preliminary rang e-finding study was included in th e uterotrophic study report as Appendix H (pages 90-95). As this is a ra nge-finding study, only a summary is provided to confirm the adequacy of the dose selection rationale used in th e definitive ut erotrophic assay (MRID 48673703).
In a preliminary range-finding assay with peri pubertal rats, tebuconazole (97.5% a.i.; Batch# K689052) in 0.5% aqueous methylcellulose 400 + 0.4% Tween 80 was administered via daily oral gavage in a dose volume of 5 mL/kg body weight to 4 peri pubertal female Sprague Dawley [Crl:CD(SD)] rats/dose group at doses of 0, 80, 150 or 300 mg/kg/day. Rats were PND 25 on the first day of dosing. The animals were dosed for 10 consecutive days and necropsied approximately 24 hours after th e final dose administration.
At 300 mg/kg/day, the clinical sign of toxicity included increased salivation for two animals. Absolute body weight was decreased 5% by day 5 and 6% by study termination. Body weight gains were decreased on Days 1-3 ( ↓ 83%) and Days 1-5 ( ↓ 45%). At 150 mg/kg/day, the clinical sign of toxicity included increa sed salivation for one animal on Day 10. Absolute body weights were similar to controls. Body weight gains were decreased on Days 1-3 ( ↓ 46%). At 80 mg/kg/day, body weight gains were decreased on Days 1-3 ( ↓ 18%).
In a similar study with male peripubertal rats, absolute body weights were decreased 12% on day 3 and 11% by study termination at 300 mg/kg/day. In another study with adult male rats, animals treated with 300 mg/kg/day lost an average of 6.0 g between day 1 and 3 (compared with a gain of 20.5 g in control) and the final body weight was decreased by 11% compared to control.
It did not appear that statistical analyses were conducted.