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 response to the agency's Test Order for tebuconazole: Test Order# CON-128997-

20. 

Page 1 of2 

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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 other scientifically relevant information (OSRI)) 

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

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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-(1H-1,2,4-triazol-1-ylmethyl) 

 pentan-3-ol 

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

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

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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.

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

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EXECUTIVE SUMMARY

The 21-day assay of Tebuconazole (97.5% purity) on amphibian metamorphosis of South 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-measured 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 pale while two more tadpoles 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. 

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Significant reductions in snout-vent length (SVL) and body weight (BW) were observed at the high concentration 

on both Day 7 (SVL: Ļ16%; BW: Ļ24%) and 21 (SVL: Ļ24%; BW: Ļ59%) and in the low treatment on Day 21 

(SVL: Ļ6%; BW: Ļ12%). 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 vacuolation, hepatocyte anisocylosis, hepatocyte 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 

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

Metamorphosis Assay (AMA) with Tebuconazole Technical. 

Treatment (mg a.i./L) 

[mean-measured] 

NF Developmental 

Stage 

Hind Limb 

Length3

Asynchronous 

Development 

Thyroid Gross and 

Histopathology 

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

0.017 Yes No No No No No No 

0.067 Yes Yes No No No No No 

0.341 Yes Yes No Yes No No Yes4

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 staged-match control. 

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I. MATERIALS AND METHODS 

Guideline Followed: This study was conducted following guidelines 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 CaCO3 exceeded the recommended range of 40-48 

mg/L CaCO3

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 from 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%.

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Storage Conditions of 

 Test Chemicals: Stored in a plastic jar at room temperature. 

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B. Test Organism

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

Species common name: South African Clawed 

Frog

EPA recommends African clawed frog 

(Xenopus laevis). Western [Africa] clawed 

frog Silurana (Xenopus) tropicalis may be 

used as an alternate species1; however, a list 

of all of the necessary protocol deviations to 

accommodate this species is recommended 

for inclusion in the study report. The guideline 

recommends that the performance criteria 

used to support the reliability of the test be 

identified.

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

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

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

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

Were parents maintained as in-house stock? 

No. Frogs used in the definitive assay were received as fertilized embryos. 

EPA recommends that larvae used in the 

assay be derived from in-house adults.

Were parental acclimation conditions same as definitive test? 

NA Embryos and newly hatched tadpoles 

were maintained under comparable conditions as those used in the definitive test. 

Acclimation period for parental frogs (if applicable): 

Embryos were acclimated for 11 days.

Details on parental feeding: During acclimation 

tadpoles were fed liquid Frog Brittle Slurry at least once daily during hatch and twice daily thereafter. 

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on parental health: Mortality was <5% 

during the holding period and there were no treatments for disease. 

Table 3: Larval Selection and Care. 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Best single spawn? All embryos were from a 

single spawn. 

Embryos (lot number: NXL042911) were received 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 (if applicable): 

Not reported 

Number of eggs sampled per spawn: 

Not reported 

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

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Table 3: Larval Selection and Care. 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Age 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 reported 

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

Not reported 

Was the optional size selection method used? 

Not reported 

Details on larval selection: Cell division was verified 

with a microscope prior to 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® throughout 

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DER Template Version: 22 September 2011 

Table 3: Larval Selection and Care. 

Parameter Value(s) Details or Remarks Guideline Recommendations 

prepared from Nasco Frog 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 (if known): 

Not reported 

Frequency of feeding: two times/day EPA recommends that feeding occur at least 

twice per day.

Details on feeding regime: During the definitive test 

tadpoles received Liquid Frog Brittle slurry twice daily at a concentration of 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).

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C. Exposure System

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

system.

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

Intermittent Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

Flow-through rate (if applicable): 

23.25 mL/min The flow rate resulted in approximately 9 

volume additions per day. 

Recommended flow-through rate is 25 

mL/min (complete volume replacement ca. 

every 2.7 hrs).

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

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

Test material was delivered to the appropriate mixing vessel where it was constantly mixed with dilution water using a magnetic stir bar. The appropriate volume of the dilution water and toxicant mixing solution was blended and delivered to the test aquaria. 

The accuracy (±10%) of the test solution delivery system was checked prior to test initiation. The control test concentration received no test material. Diluter was operating for approximately 5 days prior to test initiation and the diluter system was visually checked twice daily during the week and 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 static renewal (if applicable): 

NA If static renewal is used, EPA recommends 

24-hr renewal; renewal period is 

recommended not to exceed 72 hours.

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DER Template Version: 22 September 2011 

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

Aeration? No EPA recommends maintaining dissolved 

oxygen concentrations >40% air saturation 

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

bubblers at levels that do not cause stress 

on the tadpoles.

Source of dilution water: Dilution water (hard 

processed water) consists of spring water blended with reverse osmosis water (municipal water) to produced hard (160-180 mg/L) water used for culturing and testing freshwater invertebrates. 

Spring water is collected from a spring box, passed through a multimedia filter, a 5-micron bag filter, granular activated carbon filters, a 1-micron cartridge filter and finally an ultraviolet sterilizer. This is then blended with municipal water that has been dechlorinated with sodium metabisulfite, passed through a multimedia filter, granular activated carbon units, demineralized by conventional softeners, passed through a 5-micron cartridge filter, treated by double pass reverse osmosis ad passed through an ultraviolet sterilizer. 

EPA recommends natural or reconstituted 

water; it is recommended that natural water 

be sterilized with UV and tested for 

pesticides, heavy metals, and other 

possible contaminants, including known 

substrates of the iodine transporter of the 

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

perchlorate). OECD accepts any water in 

which the test species show control survival 

at least as good as indicated in the test 

guideline.

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

Yes. See Reviewer’s Comments. 

Iodide supplementation in water? 

No If reconstituted water is used or if 

background levels of iodide in natural water 

are less than 0.5 

սg/L, iodide 

supplementation is recommended. This 

supplementation is in addition to the 

recommended dietary source of iodide 

(e.g.. in Sera Micron).

Test vessel type/materials: 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)

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

Additional details on exposure system: 

NA 

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

Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Hardness (mg/L as CaCO3) 168 188 179 

Measured in one 

alternating replicate 

at each level at test 

initiation and weekly 

thereafter. 

EPA recommends hardness 40 to 48 

mg/L as CaCO3.

pH 7.9 8.2 8.1 

EPA recommends pH 7.5 ± 1, 

inter-replicate and inter-treatment 

differentials should not exceed 0.5.

Dissolved oxygen (mg/L) 6.3 8.4 7.6 

Measured in two 

alternating replicates 

at each level at test 

initiation and weekly 

thereafter. 

EPA recommends dissolved oxygen (DO) 

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

recommends DO concentration >3.5 mg/L

(>40% air saturation).

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

Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Temperature (°C) 21.4 22.7 22.1 

Temperature was 

measured 

continuously in a 

centrally located test 

vessel and daily in all 

test vessels. 

EPA recommends temperature 22±1oC; 

inter-replicate and inter-treatment 

differentials should not exceed 0.5oC.

Iodide <0.10 mg/L in Spring and RO water 

EPA recommends aquatic iodide range 0.5 

– 10 

սg/L (supplemental iodide should not 

exceed 2 

սg/L).

Ammonia <0.001 mg/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, 

Fluoride 0.18 mg/L in Spring water, <0.10 mg/L in RO water Perchlorate Not Reported Chlorate Not Reported 

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

Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Alkalinity (mg/L) Conductivity (սmhos/cm) 

115 

429 

164 

463 

143 

453 

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 Conditions

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

Was a range-finder conducted? Yes

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

If yes, what was the method for determining the highest test concentration in the range-finder? 

Not specified. A 21-day range-finding AMA and a 48-hour acute toxicity test 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 laevis Life stage: Not specified. Test duration: 21 days (AMA) 

48 hours (acute)

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

Additional details: The AMA test was 

conducted with nominal concentrations of 0.010, 0.100, and 1.000 mg a.i./L for 21 days. Day 7 wet weight was inhibited at the highest treatment group and Day 21 developmental stage was affected at the two highest treatment levels. Due to the high percentage of tadpoles developing beyond NF 60, Day 21 growth endpoints were not analyzed. 

Results from the 48-hour acute range-finding study yielded LC50 and 

NOAEC values of 6.3 and 4.5 mg a.i./L, respectively. 

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Table 7: Definitive Study Conditions. 

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

definitive test be 21 days. 

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

The study authors reported that based on the range-finding results that overt toxicity would occur at levels below 1/3 of the LC50 (2.1 mg 

a.i./L). Therefore, nominal concentrations of 0.015, 0.075, and 0.375 mg a.i./L were selected for the definitive 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 (if applicable):

NA 

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Table 7: Definitive Study Conditions. 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Separation 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 concentrations adjusted for purity? 

Yes 

Indicate the type of values presented for measured concentrations: 

Mean-measured 

Limit of quantification (LOQ): 0.0015 mg a.i./L EPA recommends that for chemical test 

concentrations below the LOQ, analyses be 

conducted on the stock solutions.

Level of detection (LOD): Not Reported 

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Table 7: Definitive Study Conditions. 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Frequency 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 solvent control (if applicable): 

NA EPA and OECD recommend the use of a 

concurrent solvent control when a solubilizing 

agent is used. EPA recommends 4 replicates.

Number of replicates per test item treatment level: 

4 EPA recommends 4 replicates.

Number of larvae per treatment at test initiation: 

80 (equally divided between 4 replicates) 

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

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Table 7: Definitive Study Conditions. 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Maximum solvent concentration (if applicable): 

NA EPA recommends that the solvent not exceed 

0.02 ml/L3. OECD recommends that solvent 

have no effect on survival nor produce any 

other adverse effects and that concentration 

not be greater than 0.1 ml/L4

.

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

NA 

Photoperiod: 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).

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

76, pp.69–92. 

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

Assessment. No. 23. Paris, France. 

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Table 7: Definitive Study Conditions. 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Additional details: NA No observed precipitates were reported Table 8: Summary of Treatment Concentrations in the Amphibian Metamorphosis Assay with Tebuconazole. 

Treatment ID 

Nominal 

Concentration 

(mg a.i./L) 

Measured 

Concentration 

(mg a.i./L) 

Mean CV (%) Details or Remarks Guideline Recommendations 

Negative Control 0.00 <1.5 NA NA EPA and OECD recommend that test item 

concentrations be maintained at a coefficient 

of variation (CV) 

20%.

Treatment 1 0.0152 0.017 19.4 NA Treatment 2 0.076 0.067 14.7 NA Treatment 3 0.380 0.341 15.5 NA Abbreviations: CV Coefficient of variation. 

 E. Observations

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

 Day 21- NF stage, wet weight, HLL, SVL, normalized HLL, histopathology  Daily- mortality and sub-lethal effects 

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Were raw (individual) data provided? Yes

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

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

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

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

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II. RESULTS AND DISCUSSION 

A. Results 

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 71 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 20th and 80th quantiles did not differ by more than 2 NF stages. 

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

Development.

Treatment (mg a.i./L) 

[mean-measured]

Developmental Stage 

Day 7 Day 21 

n 

Median 

Stage 

# Asynchronous n 

Median 

Stage 

# Asynchronous 

Negative Control 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 negative control and 0.187, 0.190, and 0.180 in the 

mean-measured 0.017, 0.067, and 0.341 mg a.i./L treatment 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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Table 11: Larval Development in Xenopus laevis – Hind Limb Length.

Treatment (mg a.i./L) 

[mean-measured] 

Hind Limb Length (HLL) 

Day 7 Day 21 

n 

Mean

(mm)

±SD 

HLL: 

SVL1 n 

Mean

(mm)

±SD 

HLL: 

SVL1

Negative Control 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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Table 12: Larval Growth in Xenopus laevis.

Treatment 

(mg a.i./L) 

[meanmeasured] 

Snout-Vent Length (SVL) Body Weight1

Day 7 Day 21 Day 7 Day 21 

n 

Mean

(mm)

±SD n 

Mean

(mm)

±SD n 

Mean

(g)

±SD n 

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

Treatment 

(mg a.i./L) 

[meanmeasured] 

Diagnostic Observations1

Severity 

Thyroid Gland 

Hypertrophy 

Thyroid Gland 

Atrophy 

Follicular Cell 

Hypertrophy 

Follicular Cell 

Hyperplasia 

n Incidence n Incidence n Incidence n Incidence 

Negative 

Control – 

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 

control2

 

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 

0.3412 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 stages 

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Table 14: Additional Thyroid Gland Histopathology Observations in Xenopus laevis. Treatment (mg a.i./L) [mean-measured] 

Additional Qualitative Observations1

Severity 

Follicular Lumen 

Area (Increase) 

Follicular Lumen 

Area (Decrease) 

Follicular Cell 

Height (Increase) 

Follicular Cell Height 

(Decrease) 

Follicular Cell 

Shape 

n Incidence n Incidence n Incidence n Incidence n Incidence 

Negative Control 0 20 NR 20 19 -- -- -- -- -- -- 

1 20 NR 20 1 -- -- -- -- -- -- 

2 20 NR 20 0 -- -- -- -- -- -- 3 20 NR 20 0 -- -- -- -- -- -- 

Staged-match control2

0 20 NR 20 20 

1 20 NR 20 0 

2 20 NR 20 0 3 20 NR 20 0 

0.017 0 20 NR 20 20 -- -- -- -- -- -- 

1 20 NR 20 0 -- -- -- -- -- -- 

2 20 NR 20 0 -- -- -- -- -- -- 3 20 NR 20 0 -- -- -- -- -- -- 

0.067 0 20 NR 20 20 -- -- -- -- -- -- 

1 20 NR 20 0 -- -- -- -- -- -- 

2 20 NR 20 0 -- -- -- -- -- -- 3 20 NR 20 0 -- -- -- -- -- -- 

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Table 14: Additional Thyroid Gland Histopathology Observations in Xenopus laevis. Treatment (mg a.i./L) [mean-measured] 

Additional Qualitative Observations1

Severity 

Follicular Lumen 

Area (Increase) 

Follicular Lumen 

Area (Decrease) 

Follicular Cell 

Height (Increase) 

Follicular Cell Height 

(Decrease) 

Follicular Cell 

Shape 

n Incidence n Incidence n Incidence n Incidence n Incidence 

0.3412 0 20 NR 20 20 -- -- -- -- -- -- 

1 20 NR 20 0 -- -- -- -- -- -- 

2 20 NR 20 0 -- -- -- -- -- -- 3 20 NR 20 0 -- -- -- -- -- -- 

1 Thyroid histopathology is graded 0 – 3 based on severity: 0=Not remarkable, 1=Mild, 2=Moderate, 3=Severe. See OECD No. 82 for reference. 2 Tadpoles from this group were compared to tadpoles from a non-concurrent control group to match the appropriate stages. 

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Table 14a: Additional Thyroid Gland Histopathology Observations in Xenopus laevis. Treatment (mg a.i./L) 

[mean-measured] 

Additional Qualitative Observations1

Severity 

Relative Thyroid Gland Hypoplasia 

n Incidence 

Negative Control 0 20 20 

1 20 0 

2 20 0 3 20 0 

Staged-match control2

0 20 9 

1 20 11 

2 20 0 3 20 0 

0.017 0 20 20 

1 20 0 

2 20 0 3 20 0 

0.067 0 20 20 

1 20 0 

2 20 0 3 20 0 

0.3412 0 20 0 

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Table 14a: Additional Thyroid Gland Histopathology Observations in Xenopus laevis. Treatment (mg a.i./L) 

[mean-measured] 

Additional Qualitative Observations1

Severity 

Relative Thyroid Gland Hypoplasia 

n Incidence 

1 20 20 

2 20 0 3 20 0 

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

82 for reference. 

2 Tadpoles from this group were compared to tadpoles from a non-concurrent control group to match the appropriate stages. 

Clinical Signs Beginning on Day 16, sublethal effects observed in the mean-measured 0.341 mg a.i./L treatment group. One tadpole was observed in each of two replicates and an additional tadpole was observed to be pale and swimming erratically in a third replicate. No other effects were observed in the control or remaining treatment groups 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. 

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Treatment (mg a.i./L) 

[mean- measured] 

Clinical Signs 

Type n Incidence1

Negative Control Bent tail 60 16 0.017 Bent tail 60 14 0.067 Bent tail 59 5 0.341 Erratic behavior 59 2 

Loss of equilibrium 59 2 

1 Represents observations on Day 21. 

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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 assessments 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 significantly 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 treatment-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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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 relative 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. 

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Summary of Developmental and Thyroid Gross Pathology/Histopathology Endpoints1,2 in the AMA with Tebuconazole. 

Treatment 

(mg a.i./L) 

[mean-measured]

NF Developmental Stage Hind Limb Length3 Asynchronous Development Thyroid Gross and 

Histopathology 

Day 7 Day 21 Day 7 Day 214 Day 7 Day 21 Day 21 

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

% 

Diff. 

p % Diff. p 

Treatment Related 

Effects? (Yes/No) 

Negative Control 55 NA 61 NA 0 NA 0 NA NA NA NA NA NA 0.017 54 0.02 60 0.093 -5.74 0.54 8.45 0.29 NA NA NA NA No 0.067 54 0.010 60 0.005 -3.57 0.82 -8.07 0.32 NA NA NA NA No 0.341 54 0.003 56 <0.001 -3.44 0.83 -55.91 <0.001 NA NA NA NA Yes Statistical Test Jonckheere Jonckheere Dunnett’s Dunnettt’s NA NA NA Abbreviations: Diff. Difference. NA Not applicable.

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

the expert opinion of a board-certified pathologist. 

2 Unless otherwise specified, effects are considered statistically significant at 

p = 0.05. 

3 Hind-limb length is normalized to snout-vent length (SVL). 4 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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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 CaCO3 exceeded the recommended range of 40-48 mg/L CaCO3; however, is considered 

minor and does not impact the acceptability of the study. 

F. Reviewer’s Comments 

The reviewer’s results agreed with those 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 water used to prepare dilution water: aluminum (20 սg/L), calcium 

(117 mg/L), chromium (5.4 սg/L), copper (1.2 սg/L), magnesium (6.2 mg/L), nickel (4.6 սg/L), potassium 

(1.65 mg/L), selenium (1.3 սg/L), sodium (62.4 mg/L), zinc (3.9 սg/L), chloride (89 mg/L), fluoride (0.18 

Summary of Growth Endpoints1,2 in the AMA with Tebuconazole. 

Treatment 

(mg a.i./L) 

[mean-measured]

Snout-Vent Length Body Weight 

Day 7 Day 213 Day 7 Day 213

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

Negative Control 0 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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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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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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 EPA MRID Number 48673701

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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 

Page 46 of 237
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 EPA MRID Number 48673701

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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 . . .

Page 47 of 237
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 . . . .

Page 48 of 237
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) 

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 EPA MRID Number 48673701

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DER Template Version: 22 September 2011 

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS 

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

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

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

 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 0.963 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 

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

Page 50 of 237
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 

Page 51 of 237
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 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 

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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 

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

 EPA MRID Number 48673701

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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 

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 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 

Page 55 of 237
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 

Page 56 of 237
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 . .

Page 57 of 237
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 

Page 58 of 237
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 

Page 59 of 237
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 

Page 60 of 237
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) 

Page 61 of 237
DATA EVALUATION RECORD 

TEBUCONAZOLE 

Study Type: OCSPP 890.1150, Androgen Receptor 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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 62 of 237
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 63 of 237
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. 

Page 64 of 237
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 IC50 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 ligand depletion and NSB were not reported, but the 

IC50s 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 performance criteria were found to be met. 

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 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 concentrations tested in all 

three runs. 

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

Receptor Binding Assay. 

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

Assay (OCSPP 890.1150).

COMPLIANCE: Signed and dated GLP and Quality Assurance statements were provided. 

Page 65 of 237
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 CH3

CH3

CH3

N N

N

Page 66 of 237
Androgen 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 excised immediately after termination, 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 pooled, 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 commercially 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 [3H]-R1881 was performed in a previous study 

(SA 11021 ) for the cytosol used in the subject competitive binding experiment to 

demonstrate that the AR was present in reasonable 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 subject 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 specific activity of the stock solution [3H]-R1881 was adjusted 

Page 67 of 237
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 5 of 15 

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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, solutions of non-labeled R1881 were prepared 

in a similar manner to achieve concentrations that were 100-fold greater than each 

respective radiolabeled concentration, resulting in final concentrations in cytosol of 25, 50, 

70, 100, 150, 250, 500, and 1,000 nM. In the absence of cytosol, the radiation found in 7.5, 

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

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

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

radiolabeled R1881. The optimal protein concentration was determined to be 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 molar excess of [3

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

cytosol were added. 

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

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

[

3

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

Following addition of triamcinolone acetonide, [3

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

dried, dissolved in diluted prostate cytosol (300 μL), and incubated in a rotor 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 centrifuged, 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 scintillation counting of the ethanol extract. 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. 

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TABLE 3. Summary of Conditions for Competitive Binding Experiment a

Source of receptor Rat ventral prostate cytosol 

Concentration of radioligand 1 nM 

Optimization of receptor concentration 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 single concentration of [3H]-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 precipitation of the test substance was visibly 

observed at any concentration. Based on the results from the saturation binding experiment, 

the amount of cytosolic protein used for each incubation in the competitive binding 

experiments was 0.48 mg/assay tube, which was reported to bind no more than 10-15% of 

the radiolabeled R1881 at 1 nM. 

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

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

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

and each run contained three replicates at each concentration, plus six replicates of 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. 

Page 69 of 237
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 7 of 15 

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

Solvent Control Reference standard Weak positive control Test Chemical None 

DMSO R1881 Dexamethasone Tebuconazole (Hot Mix) 

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 triplicate 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 substance. 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, separation of bound from free R1881 (washing with 

buffer and extraction with ethanol), followed by scintillation counting of bound [3H]-R1881 

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

performed as described. 

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

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

Graph Pad Prism v. 5.04 (GraphPad Software, Inc.). Scatchard plots were also plotted for 

the binding data (GraphPad Software, Inc.). 

5. Definitions 

a. Classification of test material 

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

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

bound. 

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

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

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

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

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

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. 

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b. Descriptors for receptor binding 

Bmax: maximal binding capacity

Kd: dissociation constant

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

by the competitor

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

II. RESULTS 

A. SATURATION BINDING EXPERIMENT: Saturation binding experiment parameters 

are presented in Table 5. The dissociation constant (Kd) for [3H]-R1881 was 1.01 nM 

(± 0.365), and the estimated Bmax was 14 fmol/100μg protein (± 1.5) for the single batch of 

prostate cytosol that was prepared. The goodness of fit (r2

) was not reported and the Kd

obtained from Saturation Assay #2 was low (0.59 nM), but the Kds from the other two runs 

were within the recommended range (0.685 to 1.57 nM) and the Scatchard plot for the three 

saturation assays was linear demonstrating the androgen receptor was functioning with the 

appropriate affinity for the reference ligand R1881. 

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

Parameter Run 1 b Run 2 b Run 3 b Mean Runs 1-3 c

r

2

 NR NR NR NA 

Bmax (nM) 0.34 0.31 0.38 0.34 ± 0.035 

Bmax (fmol/100 μg protein) 14 13 16 14 ± 1.5 

Kd (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 concurrent 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 illustrate the non-specific, specific, and total binding curves for [3H]-

R1881 to the androgen receptor. The specific binding reached a plateau, and non-specific 

binding was less than 20% of total binding (4.6-12.6%). Figure 2 is the Scatchard plots for 

each run that illustrate the binding of [3

H]-R1881 to the androgen receptor. The data fit 

results in a linear plot. 

Page 71 of 237
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 9 of 15 

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FIGURE 1. Binding of [3H]-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 Binding 

 

Page 72 of 237
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 10 of 15 

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

 Total Binding 

 

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

 

Page 73 of 237
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 11 of 15 

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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 concentration dependent manner in all three runs. 

The maximum binding reduction occurred at the highest concentration tested (10−3 M), with 

specific [3H]-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 IC50 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 precipitation of the test compound was visually 

observed at any concentration. The solvent control responses indicated no drift in the study 

assay. The initial assay run #2 was discarded (and repeated) due to technical problems with 

the assay. Tebuconazole was classified as an AR binder in all three competitive binding 

runs.

TABLE 6. Competitive Binding Assay of Tebuconazole 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 IC50 (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 

IC50 (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 (Figure 3) indicate that the tebuconazole data 

across the tested concentrations fit the nonlinear regression model and on average the lowest 

portion of the curve crossed 50% activity at the highest two concentration (10−3 and 10−4 M) 

of tebuconazole. A graph of the mean values of tebuconazole across the three runs is 

provided in Figure 4. 

Page 74 of 237
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 12 of 15 

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

Compounds. 

Page 75 of 237
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 the Androgen Receptor in the Presence of 

 Tebuconazole in Three Competitive Binding Assays.

Page 76 of 237
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 14 of 15 

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

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

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

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Based on the average results of the three 

competitive assays, tebuconazole was considered 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 concentrations of tebuconazole. The IC50 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 tebuconazole in comparison to R1881 were 0.0014%, 

0.0016% and 0.0013%.

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

androgen receptor was present in a reasonable concentration and functioning with 

appropriate affinity for the R1881 ligand. The mean Kd for [3H]-R1881 was 1.01 nM 

(± 0.365), and the estimated Bmax was 14 fmol/100μg protein (± 1.5) for the single batch of 

prostate cytosol that was prepared. The goodness of fit (r2

) was not reported and the Kd

obtained from Saturation Assay #2 was low (0.59 nM), but the Kds from the other two runs 

Page 77 of 237
Androgen 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 1.57 nM) and the Scatchard plots for the three 

saturation assays were linear. The specific 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 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). Tebuconazole is classified 

as a binder in the AR assay as specific [3H]-R1881 binding was reduced to <50% at the 

highest concentrations tested in all three runs. 

The estimated mean log IC50 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 ligand depletion and NSB were not reported, but the IC50s 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 the competitive assays were not reported. 

Page 78 of 237
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 79 of 237
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 80 of 237
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. 

Page 81 of 237
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 IC50. 

For tebuconazole, aromatase activity 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 lowest portion of the response curve for tebuconazole across 

runs was less than 50% activity. 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 IC50 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 requirements for an Aromatase Assay (OCSPP 

890.1200). 

COMPLIANCE: Signed and dated GLP Compliance, and Quality Assurance statements were 

provided. 

Page 82 of 237
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 containers 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; ([3H]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 Reductase Activity: Not provided

Aromatase Activity: 0.1131 nmol/mg protein/min 

Cl OH CH3

CH3

CH3

N N

N

Page 83 of 237
Aromatase (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-labeled 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 tritium added to each incubation tube was 

approximately 0.07 μCi in a volume of 100 μL. 

Tebuconazole stock solutions were prepared such that the total volume of each test chemical 

formulation used per assay was 1% of the total assay volume. A rationale for the selection 

of DMSO was not provided; however, tebuconazole 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 prepared in the same solvent as the stock solution 

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

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

20 μL of the dilution for a final assay volume of 2 mL. 

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

−74±10oC for no longer than one year. Before use of the microsomes, the batch was thawed 

and sub-aliquoted at 0.004 mg/mL into individual vials before being refrozen, and stored at 

−74±10°C for no more than six months. 

Other assay components, i.e. sodium phosphate buffer, propylene glycol, 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 

[3H]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. The 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 the minimum recommended aromatase activity of 

0.1 nmol/mg-protein/min. However, six other measurements reported for the proficiency 

test were >0.1 nmol/mg 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 tubes for the full enzyme activity and 

background activity controls, respectively, and full concentration curves in the positive 

Page 84 of 237
Aromatase (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 described in OCSPP 890.1200 (Section h, pp. 9-10). 

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

aromatase activity was reported in units of nmol/mg-protein/min. 

4. Demonstration of Proficiency: Prior to performing the definitive assay, one run of the 

positive control was conducted to evaluate the responsiveness of the microsomes. This 

evaluation also served as a proficiency evaluation of the technician involved in the conduct 

of the definitive assays. It was 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 necessary 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 inhibitor 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 generally met, and served as guidance in 

identifying runs that provided parameters in the preferred ranges. 

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

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

890.1200, except the log IC50 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 IC50 (M) −7.3 −7.0 −6.7 

a Data obtained from page 17 of the study report.

b. Proficiency Chemicals: Assays to classify proficiency chemicals (econazole, fenarimol, 

nitrofen, and atrazine) were not performed. 

TABLE 3. Proficiency Chemicals 

Compound CAS# Class Concentrations 

Econazole 24169-02-6 Inhibitor Not reported--- 

Fenarimol 60168-88-9 Inhibitor Not reported--- 

Nitrofen 1836-75-5 Inhibitor Not reported--- 

Atrazine 1912-24-9 Non-inhibitor Not reported--- 

Page 85 of 237
Aromatase (Human Recombinant) Assay (2011) / Page 6 of 12 

TEBUCONAZOLE / 128997 OCSPP 890.1200 / OECD None

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

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

tested during three independent runs. Solubility was visually assessed (presence of 

cloudiness or a precipitate), and no precipitation was observed at any concentration. The 

full enzymatic activity was obtained at the two lowest concentrations of the test chemical in 

Run 2 but not the other two runs, defining the top of the concentration-response curve. 

TABLE 4. Test Chemical Study Design for each Test Run

Sample Type Repetitions (Tubes) Description Reference or Chemical (M) 

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

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

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

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

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

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

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

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

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

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

Tebuconazole Conc 1 3 All test components plus test chemical 1×10−3

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

Tebuconazole Conc 3 3 All test components plus test chemical 1×10−5

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

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

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

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

Tebuconazole Conc 8 3 All test components plus test chemical 1×10−10

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

C. DATA ANALYSIS 

1. Raw Data: Raw data were converted to aromatase activity (nmol/mg protein/min) and 

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

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

(Table 5). 

TABLE 5. Raw and Calculated Data

Raw/Calculated Data Included (X) 

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

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

Total DPM for each aqueous portion (after extraction) X 

The total DPM present in the assay tube at initiation X 

The percentage of substrate converted to product X 

Total DPM after extraction corrected for background X 

Aromatase activity expressed in nmol/mg protein/min X 

Average aromatase activity in the full activity control tubes X 

Percentage of control activity remaining in the presence of various inhibitor concentrations X 

DPM Disintegrations per minute

2. Statistical Methods: Statistical analyses and graphical displays were conducted using 

Prism 5, v 5.04 (GraphPad Software Inc., La Jolla, CA). Basic statistical analyses were 

performed on the data, which included means of replicates, standard deviation of the mean, 

Page 86 of 237
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-weighted least squares nonlinear regression 

analysis with Prism 5 software. For each run, the individual percent of control values were 

plotted versus logarithm of the corresponding concentration. The fitted concentration 

response curve was superimposed on the plot, with individual plots prepared for each run. 

The average percent of control values versus logarithm of the concentration for the 

individual runs for each chemical (with different symbols for each run) were included on the 

same graph with their respective fitted response curves. In addition, the average percent of 

control values for each run versus the logarithm of the concentration were plotted on a 

separate graph along with the average concentration response curve across runs 

superimposed on the same plot. 

Assay drift was assessed by comparing the % full activity and background activity at values 

at the beginning of each run to the end of each run. 

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

three runs, using the categories presented in Table 6. 

TABLE 6. Interpretation of Results 

Criteria Interpretation 

Data fit 4-parameter 

nonlinear regression 

model 

Average curve across runs crossed 50%a Inhibitor 

Average lowest portion of curves across runs is between 50% and 75% activityb Equivocal 

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

Data do not fit model --- a

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

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

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

determined to be untestable in the aromatase assay. 

II. RESULTS 

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

0.250 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 % change for the average 

background controls at the beginning and end 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 assay 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 background 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/mg-protein/min at the lowest tested concentration 

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

(10−5 M). The mean aromatase activity of the positive control (expressed as % full control 

activity) for each concentration tested across all three runs is presented in Table 7, along 

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

Page 87 of 237
Aromatase (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 IC50. The overall %CV exceeded the recommended 15% at the 10−5

 test 

concentration (28%). 

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

Chemical 

Concen. 

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

4-OH ASDN 

(positive control) 

−5 3 0.9 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 Curves for 4-OH ASDN (Formestane). 

Page 88 of 237
Aromatase (Human Recombinant) Assay (2011) / Page 9 of 12 

TEBUCONAZOLE / 128997 OCSPP 890.1200 / OECD None

C. TEST SUBSTANCE: For tebuconazole, aromatase activity 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 control 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 all runs is shown in 

Figure 3. The overall mean aromatase activities 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. 

Page 89 of 237
Aromatase (Human Recombinant) Assay (2011) / Page 10 of 12 

TEBUCONAZOLE / 128997 OCSPP 890.1200 / OECD None

FIGURE 3. Mean Inhibition Response Curve for Tebuconazole. 

The effect of tebuconazole on inhibition of aromatase activity is presented in Table 8. The 

estimated log IC50 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). 

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

Chemical Run 1 Run 2 Run 3 Mean b SD b %CV b

 Log IC50 (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 curve and the criteria listed above in Table 6, 

the results support the conclusion that tebuconazole inhibits 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 recommendations. No assay drift was recorded in the 

first and second run. A marginal assay drift was recorded for the third 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 controls 0.3058 ± 0.0346 nmol/mg protein/min. 

Page 90 of 237
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 positive control evaluations run concurrently 

with the tebuconazole evaluations confirmed the responsiveness of the aromatase enzyme to 

a reference inhibitor and indicated that all guideline criteria had been met for the positive 

control. Specifically, aromatase activity averaged 0.2925 ± 0.0210 nmol/mg protein/min at 

10−10M and 0.0028 ± 0.0005 nmol/mg protein/min at 10−5M. The log IC50 value for each run 

was −7.3, −7.1 and −7.2 M, which equated to an IC50 value of 5.06 x 10−8M, 7.32 x 10−8M 

and 6.49 x 10−8

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

−0.88. Overall, the log IC50 value was−7.2 M, which equated to an IC50 of 6.25 x 10−8M. 

For tebuconazole, the data generated 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 10-10M and 0.0004 ± 0.0009 nmol/mg 

protein/min at 10−3 M. The log IC50 values were approximately −5.8 M for the first run and 

−5.7 M for the second and third runs, which equated to IC50 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 runs respectively. The overall log 

IC50 value was −5.7 M which equated to an overall IC50 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 lowest tested concentration (10−10 M) and 

0.0028±0.0005 nmol/mg-protein/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 IC50. The overall %CV exceeded the recommended 15% at the 

10−5 test concentration (28%). 

For tebuconazole, aromatase activity 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 lowest portion of the response curve for 

tebuconazole across runs was less than 50% activity. 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 IC50 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 classified as an Inhibitor 

of aromatase. 

Page 91 of 237
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 data were not provided. 

 For the positive control, the overall %CV exceeded the recommended 15% at the 10−5

test concentration (28%). 

Page 92 of 237
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 93 of 237
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 94 of 237
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 10-4M 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. 

Page 95 of 237
Estrogen Receptor Transcriptional 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 

 17β-estradiol (strong estrogen; positive control)

 Supplier: Sigma Aldrich, St. Louis, MO 

 Catalogue and Batch #: E8875; lot # 010M0142 

 Purity: 100% 

 CAS # : 50-28-2 

 

 17α-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 

 

 17α-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 CH3

CH3

CH3

N N

N

Page 96 of 237
Estrogen Receptor Transcriptional Activation (2011) / Page 3 of 9 

TEBUCONAZOLE/ 128997 OCSPP 890.1300/ OECD 455 

B. METHODS

1. Cell Culture: Stably-transfected hERα-HeLa-9903 cells were obtained from the Japanese 

Collection of Research Bioresources Cell Bank and were verified to be free of mycoplasma 

infection using PCR and DNA staining techniques. Cells were maintained in Eagles 

Minimum Essential Medium without phenol red, supplemented with 60 mg/L kanamycin 

and 10% dextran-coated charcoal-treated fetal bovine serum (Thermo Scientific, Batch # 

ATF32351), in an incubator under 5% CO2 at 37oC. Upon reaching 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 1×104

cells/100 μL medium/well in a 96 well plate and allowed to attach for 3 hours. Growth 

media was replaced with media containing serial log dilutions of tebuconazole in DMSO 

(0.1% final concentration). Cells were incubated for 22-24 hours at 37±1°C. Cytotoxicity 

was determined using a commercial kit (ATPlite 1Step, Perkin Elmer, Waltham, MA). 

Transcriptional activation of the estrogen receptor was determined using a commercial kit 

(neolite Reporter Gene Assay System, Perkin Elmer, Waltham, MA). Luminescence was 

measured using a Wallac 1450 MicroBeta Windows Workstation, V 4.01.014 (Perkin 

Elmer, Waltham, MA), with a detection limit of 32 fg luciferase/well. 

a. Preliminary Test: A preliminary test evaluating concentrations ranging from 10−6

 to 

10−4M was conducted to determine the appropriate concentration range and to determine 

concentrations resulting in insolubility and/or cytotoxicity. 

b. Proficiency Chemicals: The responsiveness of the test system was confirmed for each 

newly prepared batch of cell stocks taken from the frozen stock by testing the following set 

of proficiency chemicals. The details of the 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 --- 

17α-Ethynyl estradiol (EE) 57-63-6 10−14 to 10−8 Positive --- 

Hexestrol 84-16-2 10−13 to 10−7 Positive --- 

Genistein 446-72-0 10−12 to 10−5 Positive Cytotoxic at 0.01b, 0.1, and 1 mM 

Estrone 53-16-7 10−12 to 10−6 Positive --- 

Butyl paraben 94-26-8 10−11 to 10−4 Positive Cytotoxic at 0.1b and 1 mM 

1,3,5-Tris(4-

hydroxyphenyl)benzene c

15797-52-1 10−12 to 10−5

 Positive Cytotoxic at 100 μM. PCMax approx. 50% 

of PC. Binds to hERα and has ER 

antagonistic activity 

Dibutyl phthalate (DBP) 84-74-2 10−11 to 10−4 Negative d Cytotoxic at 1 mM 

Atrazine 1912-24-9 10−11 to 10−4 Negative Cytotoxic at 1 mM b

Corticosterone 50-22-6 10−10 to 10−4 Negative If not cytotoxic at 1 mM, then that should 

be the highest tested concentration 

a Positive = RPCMax ≥10% of the response of the positive control in at least 2 of 2 (or 2 of 3) runs 

Negative = RPCMax fails to achieve at least 10% of the response of the positive control in 2 of 2 (or 2 of 3) runs 

b Cytotoxicity is expected to be close to 80% at this concentration. 

c Compound selected to challenge solubility and cytotoxicity 

d DBP is negative for ERα mediated transcriptional activation, but may not be negative for non-ERβ mediated transcriptional 

activation. A positive result would indicate that the system is detecting activity other than that due to pure ERα, and is 

therefore unacceptable. 

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c. Reference Chemicals: To ensure the stability of the response from the cell line, six 

concentrations of each of the following reference chemicals were included in each plate in 

the current assay, along with the test chemical: 

Reference Chemical CAS No. Concentration Range Class 

17β-estradiol 50-28-2 10−14 to 10−8 Strong estrogen 

17α-estradiol 57-91-0 10−12 to 10−6 Weak estrogen 

Corticosterone 50-22-6 10−10 to 10−4 Negative compound 

17α-methyltestosterone 58-18-4 10−11 to 10−5 Very weak agonist 

3. Data analysis: To obtain the relative transcriptional activity to the 1 nM E2 positive control 

(PC), the luminescence signals from the concurrent plate were analyzed by subtracting the 

mean value of the vehicle control from each well value to normalize the data; each 

normalized value was then divided by the mean value of the normalized PC. The resulting 

value was multiplied by 100 in order to express relative transcriptional activity as a 

percentage of the PC. Graph Pad Prism v. 5.04 (GraphPad Software, Inc., La Jolla, CA) was 

used to calculate the EC50, PC10, PC50, RPCMax, and PCMax for tebuconazole when 

applicable. The test material was defined as negative for inducing estrogen receptor 

transcriptional activation if the RPCMax < PC10 in at least 2 of 2 (or 2 of 3) runs. LogEC50

and Hill slope values are calculated only if a positive response is observed. Coefficients of 

variation (CV) were calculated for the luminescence data triplicates. Concentrations 

showing >20% cytotoxicity or evidence of insolubility were excluded from analyses. 

4. Definitions 

EC50 = concentration of agonist that induces a response halfway between the baseline 

(bottom) and maximum (top) response 

PC10 = concentration of a test chemical at which the response is 10% of the response 

induced by the positive control (E2 at 1 nM) in each plate 

PC50 = concentration of a test chemical at which the response is 50% of the response 

induced by the positive control (E2 at 1 nM) in each plate 

RPCMax = maximum level of response induced by a test chemical, expressed as a percentage 

of the response induced by the positive control (1 nM E2) on the same plate 

PCMax = concentration of a test chemical inducing the RPCMax

II. RESULTS 

A. PRELIMINARY TEST: In order to identify a suitable top concentration for use in the 

transcriptional activation assays, preliminary cytotoxicity and precipitation assays were 

conducted. Precipitation was observed in tebuconazole 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 presented in Table 1. Concentrations of 10−4 M showed 

cell viability of 80%. Based on these results, concentrations 10−10 to 10−4

M were selected 

for the assay. 

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TABLE 1. Preliminary Test for Solubility, Cytotoxicity, and Concentration-Selection for Tebuconazole

Concentration (M) % Viability b Commentsc

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 proficiency chemicals 

was performed in duplicate, although it was not clear if the runs were performed on different 

days. The data are reported in Table 2. The responses did demonstrate proficiency. The 

only minor deviation was in the first run; corticosterone produced an RPCMax of 16%, 

indicating a weakly positive response. In the second run, the RPCMax was -14%, indicating 

a negative response. 

TABLE 2. Proficiency Chemicals a

Compound Expected Response Lab Response 

Diethylstilbestrol Positive Positive 

17α-Ethynyl estradiol Positive Positive 

Hexestrol Positive Positive 

Genistein Positive Positive 

Estrone Positive Positive 

Butyl paraben Positive Positive 

1, 3, 5-Tris(4-hydroxyphenyl)benzene Positive Positive 

Dibutyl phthalate Negative Negative 

Atrazine Negative Negative 

Corticosterone Negative 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

PC50, log PC10, log EC50, Hill slope) for the four concurrently run reference materials are 

included in Table 3. Performance criteria were met for all reference chemicals. The log 

PC10 for 17α-estradiol was lower than the expected range in the first assay, as was the Hill 

Slope. The log PC10 for the first assay and log PC50 for both assays for 17αmethyltestosterone were lower than the expected range. The results show an increased 

sensitivity to weak agonists. 

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TABLE 3. Performance Criteria for Reference Chemicalsa

Reference Chemical 

Parameter Acceptable Range Values Acceptable 

Run 1 Run 2 Yes No 

17β-estradiol

Log PC50 −11.4 to −10.1 −10.5 −10.7 X 

Log PC10 <−11 −11.7 −11.4 X 

Log EC50 −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 

17α-estradiol

Log PC50 −9.6 to −8.1 −9.1 −8.9 X 

Log PC10 −10.7 to −9.3 −11.9 −10.1 X 

Log EC50 −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 

17α-methyltestosterone

Log PC50 −6.0 to −5.1 -5.8b -6.0 b X 

Log PC10 −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 obtained 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 control, expressed as arbitrary light units, was 

504.6 in the first run and 500.5 in the second run. The positive control mean normalized 

value was 1524.2 in the first run and 1395.7 in the second run. The PC50 for 17β-estradiol 

was 762.1 in the first run and 697.9 in the second and the PC10 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 17β-estradiol (E2) at 1 nM. 

c Fold-induction = (mean TA of PC)/(mean TA of VC) 

2. Test Material: Relative (to the PC) transcriptional activation at each concentration of the 

test chemical during the two assay runs is presented in Table 5. The concentration-response 

curves depicting fold induction of relative transcriptional activation are presented in Figure 

1 and Figure 2 below. The mean RPCMax were −0.2% for the first run and −1.6% for the 

second run; the associated PCMax were 10−10 M for the first run and 10−5

 M for the second 

run. The values reported for the log EC50, Hill Slope, PC50 and PC10 are mathematically 

fitted to the data, but are not representative of actual binding characteristics of tebuconazole. 

Because the RPCMax < PC10 in both runs, tebuconazole was considered negative for estrogen 

receptor transcriptional activation in this test system. 

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TABLE 5. Relative Transcriptional Activation (RTA) of Tebuconazolea

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 EC50b −13.32 −4.120 

Hill Slopeb −0.6510 −7.494 

RPCMax −0.2 −1.6 

PCMax 10−10 10−5

PC50 −13.32 −4.120 

PC10 −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). 

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FIGURE 2. Run 2 Fold Induction, Tebuconazole Relative Transcription Activation (RTA). 

3. Performance Criteria: Performance criteria were met for 17β-estradiol and corticosterone. 

The Log PC10 for 17α-estradiol was lower than the acceptable range in the first assay, as 

was the Hill Slope. The log PC10 for the first assay and log PC50 for both assays for 17αmethyltestosterone were lower than the expected range. For 17α-methyltestosterone, the 

mean RPCMax was 79.3% for the first run and 72.0% for the second run. The results show 

an increased sensitivity to weak agonists. Mean luciferase activity was 4.1-fold for the first 

run and 3.9 fold for the second run. The fold-induction corresponding to the PC10 of the 

concurrent PC was greater than 1+2 standard deviations of the vehicle control on all plates. 

Variability was minimal and results were reproducible among runs. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: The two independent runs gave similar results 

and the reference compounds gave the expected responses. The maximum response for 

tebuconazole was less than 10% of the positive control 17β-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 17α-estradiol 

and 17α-methyltestosterone, and Hill Slope for 17α-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. 

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C. STUDY DEFICIENCIES: No deficiencies were noted.

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

EPA MRID Number 48688301

Page 1Rf41 

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-(1H-1,2,4-triazol-1-ylmethyl)pentan- 

 3-ol 

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

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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 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). 

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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 25.6o

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 no 

tubercles 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 

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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-through 

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Table 1: Summary of Reproductive and HPG Effects1,2 in the Fish Short-Term Reproduction Assay (FSTRA) with Tebuconazole. 

Treatment 

(mg a.i./L) 

[meanmeasured]

Fecundity 

Fert. 

Success 

Tubercle Score GSI Gonadal Histo. Plasma VTG Plasma T3 Plasma E23

M F M F M F M F M F M F 

0.00497 No No No No No No No No No No NA NA NA NA 0.0113 No No No No No No No No No Yes NA NA NA NA 0.0195 No No No No Yes4 Yes No Yes No Yes NA NA NA NA 

Abbreviations: Conc. Concentration. Diff. Difference. E2 17ȕ-estradiol. F Female. Fert. Fertilization. GSI Gonado-Somatic Index. Histo. Histopathology. M Male. NA Not applicable. T Testosterone. VTG Vitellogenin.

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

2 The criteria for significance are described in the Reviewer’s Analysis and Statistical Verification sections of the DER. Conclusions regarding histopathology may be 

heavily weighted by the expert opinion of a board-certified pathologist. 

3 Plasma testosterone and estradiol concentrations were not determined. 4 The decrease in male GSI was marginally significant (p=0.056) at the high concentration.

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I. MATERIALS AND METHODS 

 Guideline Followed: This study was conducted according to the U.S. EPA OCSPP 890.1350: 

“Fish Short-Term Reproductive Assay”. 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 160), 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 

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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)

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B. Test OrganismTable 2: General Information About the Test Species and Acclimation.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Species common name: Fathead minnow EPA recommends fathead minnow (Pimephales 

promelas). Species scientific name: Pimephales promelas

Species strain (if stated): Not reported (NR) Were fish obtained from a single laboratory stock? 

Yes Aquatic Biosystems, Fort Collins, CO, 

Lot No. ABS090811, hatch dates June 16-23 2011

EPA recommends that fish be from a single laboratory 

stock.

Were acclimation conditions same as definitive test? 

Yes EPA recommends that fish be acclimated under water 

quality and illumination conditions that are similar to 

the definitive test. 

Acclimation period: 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. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on health: No mortalities occurred in the seven 

days preceding transfer to the test system, and no treatment for disease was 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: NR

Frequency of feeding: Two to three times/day Details on feeding: 5-6 mL/feeding 

Table 3: Fish Selection and Pre-Exposure Performance.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Age at test initiation: Ca. 7 months EPA recommends reproductively mature 

(sexually dimorphic) fish, 4.5 - 6 months old.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Mean 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) at test initiation (if determined): 

3.85-5.53 g Individual weights within ±20% of mean 

weight 

Mean weight of females at test initiation (if determined): 

1.71 g Subsample of female fish; n=64 

Range of individual weights (females) at test initiation (if determined): 

1.37-2.14 g Individual weights were within ±20% of 

mean weight with the exception of one female that weighed 2.14 g. 

Mean length of males at test initiation (if determined): 

NR 

Mean length of females at test initiation (if determined): 

NR 

Duration of pre-exposure phase: 14 days EPA recommends a minimum of 14 days. Were pre-exposure conditions identical to the definitive test? 

Yes EPA recommends that pre-exposure conditions, 

including temperature, photoperiod, feeding, etc., 

be identical to definitive test conditions. 

Number of pre-exposure tanks: 24 Extra replicates tanks were initially 

established and 16 tanks were selected for the 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. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Number of males per tank: 2 Number of females per tank: 4 Pre-exposure fecundity: 8.5 to 52.9 

eggs/female/ reproductive day/ replicate for the 16 tanks selected for the definitive test 

Not all tanks averaged >15 

eggs/female/reproductive day/replicate during the 7 day pre-exposure period. However, the egg production exceeded 15 eggs/female/reproductive day/replicate during the 14 day pre-exposure period and spawning occurred twice a week, so tanks were 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 per replicate for tanks 

selected for the definitive test 

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. 

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

system. As noted in the Corrections and 

Clarifications document1 , the use of a static 

renewal system is not recommended for this 

assay.

Type of flow-through dilution system: Proportional diluter system Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

Flow-through rate: 8.0 mL/min Approximately 8 volume turnovers/vessel/24 

hours 

Recommended flow-through rate is 45 

mL/min (2.7 L/hr), or at least 6 total 

volume exchanges per day.

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

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

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on toxicant mixing for flowthrough systems: All glass 2-L modified proportional diluter system was used for intermittent delivery of test 

solutions to test chambers. Splitter cups were used to equally divide (±10%) test solutions between replicates at each exposure concentration. 

Recommended toxicant mixing for flowthrough systems: 1) Mixing chamber is 

recommended but not required; 2) Aeration is 

not recommended for mixing; 3) A 

demonstration that the test solution is 

completely mixed before introduced into the 

test system is recommended; 4) The 

recommended flow splitting accuracy is within 

10%.

Aeration? No Dissolved oxygen concentration ranged from 

6.4 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 and 

reverse osmosis water 

Process involves filtration and UV sterilization of the spring water, and dechlorination, filtration, double-pass reverse osmosis, and UV sterilization 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 for pesticides, heavy metals, and other contaminants? 

Yes Weekly (chlorine), monthly, and bi-annual 

screens were conducted. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Test vessel type/materials: Glass EPA and OECD recommend that watercontact portions of the system not 

compromise the study (e.g., all glass vessels 

or glass vessels with stainless steel frames 

are acceptable examples).

Test vessel size: 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 cm 

depth)

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 and sectioned into ca. 5” pieces 

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

Parameter Minimum Maximum Mean Measurement Interval Guideline Recommendations 

Temperature (°C) 24.7 25.7 25.0 

Start of experiment and daily 

EPA recommends temperature 25±1oC; interreplicate and inter-treatment differentials 

should not exceed 1oC.

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Parameter Minimum Maximum Mean Measurement Interval Guideline Recommendations 

pH 7.7 7.9 NR 

Start of experiment and weekly (±2 days) 

EPA recommends pH 6.5 to 9.0.

Dissolved oxygen (mg/L) 

6.4 

(77% saturation) 

8.3 

(100% saturation) 

NR 

Start of experiment and weekly (±2 days) 

EPA recommends dissolved oxygen (DO) 

>4.9 mg/L (>60% air saturation)

Total alkalinity (mg/L as CaCO3) 46 52 48 Start of experiment and weekly (±2 days) EPA recommends total alkalinity > 20 mg/L as CaCO3.Hardness (mg/L as CaCO3) 50 58 55 Start of experiment and weekly (±2 days) Conductivity (Pmhos/cm) 176 195 182 Start of experiment and weekly (±2 days) Total organic carbon (mg/L) NR NR <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 

Pg/L. 

Residual chlorine (Pg/L) NR NR <0.003 Weekly/monthly 

EPA recommends that residual chlorine in 

dilution water be < 10 

Pg/L. 

Abbreviations: NA Not applicable. 

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D. Study Design and Additional Experimental Conditions Table 6: Range-Finding Study Conditions (if Applicable).

Parameter Value(s) Details or Remarks Guideline Recommendations 

Was a range-finder conducted? No EPA recommends conducting a range-finder if 96-hour LC50

data for the fathead minnow are unavailable. 

If yes, what was the method for determining the highest test concentration in the range-finder? 

NA EPA recommends that the highest test concentration be 

selected based on toxicity data for other fish studies or 

species, if available. Otherwise, either the solubility limit of 

the test compound or 100 mg/L (whichever is lower) is 

appropriate.

Species: NA Life stage: NA EPA recommends that range-finding tests be performed with 

fish of similar age and size to those that would be utilized in 

the test.

Test duration: NA EPA recommends a 96-hour exposure. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Additional details: The nominal test concentrations of 0 

(control), 0.00650, 0.0130, and 0.0260 mg/L were selected based on a rainbow trout Early Life Stage study resulting in a LOEC of 25.0 սg/L and a NOEC of 12.0 սg/L, and a fathead minnow Fish Sexual Development study resulting in a LOEC of 6.25 սg/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 LC50, may be selected as the highest 

exposure concentration in the 21-day test.

Table 7: Definitive Study Conditions.

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

test be 21 days. 

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

Historical data (early life stage toxicity study) 

Rainbow trout Early Life Stage study resulting in a LOEC of 25.0 սg/L.

EPA recommends that the highest test 

concentration is either the solubility limit of the 

test compound, 100 mg/L, or demonstrates 

adequate evidence of toxicity (e.g., 1/3 the 96-

hour LC50), whichever concentration is lowest.

Reference study citation (if applicable): Surprenant, D.C.(1988) 

and Bomke, C. (2007) 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Separation of test concentrations: 2-fold (decreases of 0.5× 

from the highest test concentration) 

EPA suggests that a concentration separation of 

between 0.33 (or three-fold) and 0.1 (or tenfold) is scientifically acceptable1 . 

Number of test concentrations: 4 Negative control and 3 test concentrations EPA recommends a minimum of 3 

concentrations and a control, plus solvent control 

if appropriate.

Are nominal concentrations adjusted for purity? 

Yes 

Indicate the type of values presented for measured concentrations: 

Individual values (ppb) and % recovery (from nominal) 

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): NR Frequency of measurement: Prior to study initiation 

(Day 0) and Study Days 7, 15, and 21. 

Stock solution and test solutions were reported 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.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Was the randomized complete block design used? 

Spawning groups were assigned to treatment groups using a rank-order approach. 

The top 4 performers were randomly assigned to one replicate of each treatment level, then the next 4 performers were randomly assigned to a second replicate chamber for each treatment level, and so on. 

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 item treatment level: 

4 EPA recommends 4 replicates.

Number of male fish per replicate at test initiation: 

2 EPA recommends 2 males per replicate. 

Number of female fish per replicate at test initiation: 

4 EPA recommends 4 females per replicate. 

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Maximum solvent concentration (if applicable): 

NA EPA recommends that the solvent not exceed 

0.02 ml/L3. OECD recommends that solvent have no effect on survival nor produce any other 

adverse effects and that concentration not be 

greater than 0.1 ml/L4

.

Was a positive control used? No Positive control (if applicable): NA Positive control concentration(s): NA Photoperiod: 16 hrs light : 

8 hrs dark 

Two 30-minute dawn/dusk transition periods were included each day with lowered light 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).

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

Toxicology, 76, pp.69–92.

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

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

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Table 8: Summary of Treatment Concentrations in the Fish Short-Term Reproduction Assay with Tebuconazole. 

Treatment ID 

Nominal 

Concentration 

(mg a.i./L) 

Measured 

Concentration 

(mg a.i./L) 

Mean CV (%) Details or Remarks Guideline Recommendations 

Control (dilution water only) 

0.00 <LOQ NA NA 

EPA recommends that test item concentrations 

be maintained at a coefficient of variation (CV) 

20%. Treatment 1 0.00650 0.00497 12.9 76% of nominal 

Treatment 2 0.0130 0.0113 10.4 87% of nominalTreatment 3 0.0260 0.0195 11.4 75% of nominal

Abbreviations: CV Coefficient of variation. 

LOQ=0.00065 mg a.i./L 

E. Observations

Biological Endpoints: Possible clinical/behavioral signs included, but were not limited to, hyperventilation, loss of equilibrium, uncoordinated 

swimming, atypical quiescence, or feeding abstinence. Other endpoints, monitored daily and/or determined at study termination, 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). 

Were raw (individual) data provided? Yes

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EPA recommends that observations of survival, fecundity, fertilization success, secondary sex characteristics, and other clinical signs occur at least daily. At test 

termination (Day 21), additional observations include body weight and length, nuptial tubercle score, gonadal staging and histopathology, plasma VTG and plasma sex 

steroids (testosterone and 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 behavior. 

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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 16 151 100 

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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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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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 embryos 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 treatment 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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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 respective 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 

GSI1,2 (%) 

±SD n Median GSI2

(%) 

±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 treatment levels, respectively (Table 14). 

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Table 14: Gonadal Staging in Fathead Minnow (Pimephales promelas).

Treatment (mg a.i./L) 

[mean-measured]

Males Females 

N Median Stage1 n Median Stage2

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 were decreased in the 0.0195 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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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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Table 15: Gonadal Histopathology in Male Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[mean-measured] 

Diagnostic Observations1

Severity

Increased 

Proportion of 

Spermatogonia 

Presence of 

Testis-Ova 

Increased 

Testicular 

Degeneration 

Interstitial Cell 

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. 

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Table 16: Additional Gonadal Histopathology Observations in Male Fathead Minnow (Pimephales promelas).

Treatment (mg a.i./L) 

[mean-measured] 

Additional Diagnostic Observations1

Severity

Interstitial Fibrosis 

Decreased 

Proportion of 

Spermatozoa 

Asynchronous 

Gonad 

Development 

Mineralization 

Mineralization in the 

Collecting Ducts 

n Incidence n Incidence n Incidence n Incidence n Incidence 

Water control (<LOQ) 0 8 0 8 7 8 7 8 2 8 2 

1 8 0 8 1 8 0 8 5 8 1 

2 8 0 8 0 8 1 8 1 8 4 3 8 0 8 0 8 0 8 0 8 1 4 8 0 8 0 8 0 8 0 8 0 

0.00497 0 6 4 6 6 6 5 6 4 6 5 

1 6 1 6 0 6 1 6 2 6 0 

2 6 1 6 0 6 0 6 0 6 1 3 6 0 6 0 6 0 6 0 6 0 4 6 0 6 0 6 0 6 0 6 0 

0.0113 0 7 6 7 6 7 7 7 7 7 4 

1 7 0 7 0 7 0 7 0 7 0 

2 7 1 7 1 7 0 7 0 7 3 3 7 0 7 0 7 0 7 0 7 0 4 7 0 7 0 7 0 7 0 7 0 

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Treatment (mg a.i./L) 

[mean-measured] 

Additional Diagnostic Observations1

Severity

Interstitial Fibrosis 

Decreased 

Proportion of 

Spermatozoa 

Asynchronous 

Gonad 

Development 

Mineralization 

Mineralization in the 

Collecting Ducts 

n Incidence n Incidence n Incidence n Incidence n Incidence 

0.0195 0 8 5 8 8 8 8 8 4 8 4 

1 8 3 8 0 8 0 8 4 8 3 

2 8 0 8 0 8 0 8 0 8 1 3 8 0 8 0 8 0 8 0 8 0 4 8 0 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.

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Table 17: Gonadal Histopathology in Female Fathead Minnow (Pimephales promelas).

Treatment (mg a.i./L) 

[mean-measured] 

Diagnostic Observations1

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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Table 18: Additional Gonadal Histopathology Observations in Female Fathead Minnow (Pimephales 

promelas).

Treatment 

(mg a.i./L) 

[meanmeasured] 

Additional Diagnostic Observations1

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 2.99×106

, 2.49×106, 1.94×106 and 1.34×106 ng/mL plasma in females (Table 19). 

Testosterone and 17ȕ-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 2.99×106 4.98x105 

0.00497 4 1628 744 4 2.49×106 2.31x105 

0.0113 4 945 349 4 1.94×106 3.92x105 

0.0195 4 926 608 4 1.34×106 7.95x105 

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 reproductive 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 սg a.i./L females, and one 19.5 սg a.i./L female. Additional atypical 

Page 137 of 237
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behaviors included one control female with swollen belly and one 4.97 սg 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 quiescence, males not territorial, females territorial, and 

females with vertical bands. Other endpoints, monitored daily and/or determined at study termination, 

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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 p0.05 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 սg a.i./L, increased male tubercle score at 19.5 սg a.i./L, and decreased female plasma 

VTG at 11.3 and 19.5 սg 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 

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Version: 22 September 2011 

analyzed using Dunnett’s Multiple Comparison Test. Male tubercle score was analyzed using MannWhitney. All analyses were conducted using CETISTM 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 sex steroids were not measured in this study. 

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Table 21: Reproductive and HPG Endpoints1,2 for Male Fathead Minnow (Pimephales promelas) in the FSTRA with Tebuconazole. 

Treatment 

(mg a.i./L) 

[meanmeasured]

Tubercle Score GSI 

Gonadal 

Staging and 

Histo. 

Plasma VTG Plasma T Plasma E2 

Median p % Diff. p 

Effect? 

(Yes/No) 

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

Negative control (<LOQ) 

38 NA NA NA NA NA NA NA NA NA NA 

0.00497 44 1.0 6.16 0.80 No 107 0.16 NA NA NA NA 0.0113 39 0.66 -20.4 0.14 No 20.4 0.95 NA NA NA NA 0.0195 43 0.97 -19.3 0.056 No 18.0 0.97 NA NA NA NA Statistical Test Mann-Whitney Jonckheere-Terpstra NA Dunnett NA NA 

Abbreviations: Diff. Difference. GSI Gonado-Somatic Index. Histo. Histopathology. NA Not applicable. NS Not significant. VTG Vitellogenin. T Testosterone. E2 17ȕ-estradiol.

1 Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusions regarding histopathology 

may be heavily weighted by the expert opinion of a board-certified pathologist. 

2 Unless otherwise specified, effects are considered statistically significant at p0.05. 

LOQ=0.00065 mg a.i./L. 

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Version: 22 September 2011 

Table 22: Reproductive and HPG Endpoints1,2 for Female Fathead Minnow (Pimephales promelas) in the FSTRA with Tebuconazole. 

Treatment 

(mg a.i./L) 

[measured]

Fecundity Fert. Success Tubercle Score GSI 

Gonadal 

Staging 

and Histo. 

Plasma VTG Plasma T Plasma E2 

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

Effect? 

(Yes/No) 

% Diff p % Diff p % Diff. p 

Negative control (<LOQ) 

NA NA NA NA 0 NA NA NA NA NA NA NA NA NA NA 

0.00497 -8.1 0.99 0.54 0.76 0 1.0 2.96 0.66 No -16.7 0.17 NA NA NA NA 0.0113 21.8 0.86 -0.38 0.89 0 1.0 -7.62 0.26 No -35.1 0.004 NA NA NA NA 0.0195 26.0 0.79 1.0 0.34 0 1.0 -18.5 0.042 Yes -55.0 <0.0001 NA NA NA NA Statistical Test Dunnett Dunnett 

Mann-Whitney 

U 

JonckheereTerpstra NA Jonckheere-Terpstra NA NA 

Abbreviations: Diff. Difference. Fert. Fertilization. GSI Gonado-Somatic Index. Histo. Histopathology. NA Not applicable. NS Not significant. VTG Vitellogenin. T Testosterone. 

E2 17ȕ-estradiol. 

1 Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusions regarding 

histopathology may be heavily weighted by the expert opinion of a board-certified pathologist. 

2 Unless otherwise specified, effects are considered statistically significant at p0.05. 

LOQ=0.00065 mg a.i./L. 

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Version: 22 September 2011 

Table 23: Growth Endpoints1,2 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 p0.05. 

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. 

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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 etiologic 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. 

Page 144 of 237
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

Analysis ID: 18-2191-0972 Endpoint: Fecundity CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source:

Analyst: J. Housenger

Age:

Control vs C-μg ai/L Test Stat Critical MSD P-Value Decision(α:5%)

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 4.97 0.239 2.68 21.6 6 0.9901 CDF Non-Significant Effect

11.3 0.642 2.68 21.6 6 0.8574 CDF Non-Significant Effect

19.5 0.766 2.68 21.6 6 0.7869 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 91.0% 19.5 >19.5 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

Total 1744.529 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 7.97 11.3 0.0467 Equal Variances

Distribution Shapiro-Wilk W Normality 0.881 0.841 0.0406 Normal Distribution

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

Fecundity Summary

0 Negative Control 4 23.7 11.1 36.4 23.7 15.7 31.9 3.97 33.5% 0.0%

4.97 4 21.8 15.4 28.2 20.6 18.5 27.6 2.01 18.5% 8.11%

11.3 4 28.9 19.8 38.1 29 21.9 35.8 2.88 19.9% -21.8%

19.5 4 29.9 -2.18 62 23.2 13.9 59.4 10.1 67.4% -26.0% Centered Untransformed

 Rankits

-5

-10

-15

-20

0

5

10

15

20

25

30

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

Fecundity

 C-μg ai/L

Reject Null

Reject Null

0

10

20

30

40

50

60

0 N 4.97 11.3 19.5

Graphics

000-431-181-1 CETISTM v1.8.7.12 Analyst:________ QA:________

Page 145 of 237
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

Analysis ID: 18-4021-8427 Endpoint: FemaleBodyWt CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source:

Analyst: J. Housenger

Age:

Control vs C-μg ai/L Test Stat Critical MSD P-Value Decision(α:5%)

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 4.97 0.44 2.68 0.29 6 0.9453 CDF Non-Significant Effect

11.3 2.04 2.68 0.29 6 0.1514 CDF Non-Significant Effect

19.5 0.95 2.68 0.29 6 0.6705 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 14.4% 19.5 >19.5 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

Total 0.448575 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 0.55 11.3 0.9079 Equal Variances

Distribution Shapiro-Wilk W Normality 0.952 0.841 0.5184 Normal Distribution

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

FemaleBodyWt Summary

0 Negative Control 4 2.01 1.73 2.3 1.99 1.83 2.24 0.0886 8.79% 0.0%

4.97 4 2.06 1.82 2.3 2.06 1.88 2.25 0.0755 7.33% -2.36%

11.3 4 1.79 1.62 1.97 1.82 1.64 1.9 0.0563 6.28% 10.9%

19.5 4 1.91 1.65 2.17 1.94 1.69 2.07 0.0811 8.48% 5.09% Centered Untransformed

 Rankits

-0.05

-0.10

-0.15

-0.20

-0.25

0.00

0.05

0.10

0.15

0.20

0.25

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

FemaleBodyWt

 C-μg 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

000-431-181-1 CETISTM v1.8.7.12 Analyst:________ QA:________

Page 146 of 237
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

Analysis ID: 15-5144-0500 Endpoint: FemaleGSI CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source:

Analyst: J. Housenger

Age:

Control vs C-μg ai/L Test Stat Critical MSD P-Value Decision(α:5%)

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 4.97 0.252 2.68 5.58 6 0.9885 CDF Non-Significant Effect

11.3 0.649 2.68 5.58 6 0.8539 CDF Non-Significant Effect

19.5 1.57 2.68 5.58 6 0.3109 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 31.5% 19.5 >19.5 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

Total 138.4 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 1.39 11.3 0.7075 Equal Variances

Distribution Shapiro-Wilk W Normality 0.923 0.841 0.1889 Normal Distribution

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

FemaleGSI Summary

0 Negative Control 4 17.7 11.3 24.1 17.3 14.2 22.1 2.02 22.7% 0.0%

4.97 4 18.3 14.1 22.4 18.3 15.1 21.4 1.31 14.3% -2.96%

11.3 4 16.4 13.2 19.5 16.4 14.1 18.7 0.988 12.1% 7.62%

19.5 4 14.5 10 18.9 15.1 10.9 16.7 1.38 19.2% 18.5% Centered Untransformed

 Rankits

-1

-2

-3

-4

0

1

2

3

4

5

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

FemaleGSI

 C-μg ai/L

Reject Null

Reject Null

0

5

10

15

20

25

0 N 4.97 11.3 19.5

Graphics

000-431-181-1 CETISTM v1.8.7.12 Analyst:________ QA:________

Page 147 of 237
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

Analysis ID: 00-8662-8358 Endpoint: FemaleGSI CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source:

Analyst: J. Housenger

Age:

Control vs C-μg ai/L Test Stat Critical MSD P-Value Decision(α:5%)

Jonckheere-Terpstra Step-Down Test

DF P-Type

Negative Control 4.97 7 NA -2 0.6571 Exact Non-Significant Effect

11.3 29 NA -2 0.2602 Exact Non-Significant Effect

19.5* 67 NA -2 0.0420 Exact Significant Effect

Data Transform Zeta Alt Hyp Trials Seed NOEL LOEL TOEL TU

Untransformed NA C > T NA NA 11.3 19.5 14.84

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

Total 138.4 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 1.39 11.3 0.7075 Equal Variances

Distribution Shapiro-Wilk W Normality 0.923 0.841 0.1889 Normal Distribution

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

FemaleGSI Summary

0 Negative Control 4 17.7 11.3 24.1 17.3 14.2 22.1 2.02 22.7% 0.0%

4.97 4 18.3 14.1 22.4 18.3 15.1 21.4 1.31 14.3% -2.96%

11.3 4 16.4 13.2 19.5 16.4 14.1 18.7 0.988 12.1% 7.62%

19.5 4 14.5 10 18.9 15.1 10.9 16.7 1.38 19.2% 18.5% Centered Untransformed

 Rankits

-1

-2

-3

-4

0

1

2

3

4

5

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

FemaleGSI

 C-μg ai/L

0

5

10

15

20

25

0 N 4.97 11.3 19.5

Graphics

000-431-181-1 CETISTM v1.8.7.12 Analyst:________ QA:________

Page 148 of 237
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

Analysis ID: 17-8311-6742 Endpoint: FemaleLength CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source:

Analyst: J. Housenger

Age:

Control vs C-μg ai/L Test Stat Critical MSD P-Value Decision(α:5%)

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 4.97 0.547 2.68 1.72 6 0.9036 CDF Non-Significant Effect

11.3 1.13 2.68 1.72 6 0.5518 CDF Non-Significant Effect

19.5 0.391 2.68 1.72 6 0.9603 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 3.94% 19.5 >19.5 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

Total 12.63937 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 1.75 11.3 0.6252 Equal Variances

Distribution Shapiro-Wilk W Normality 0.974 0.841 0.8987 Normal Distribution

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

FemaleLength Summary

0 Negative Control 4 43.6 42.5 44.7 43.3 43.1 44.6 0.345 1.58% 0.0%

4.97 4 43.9 42.8 45 44 43.1 44.5 0.347 1.58% -0.8%

11.3 4 42.8 41.6 44.1 43 41.8 43.6 0.38 1.77% 1.66%

19.5 4 43.8 41.7 45.9 43.5 42.6 45.6 0.659 3.01% -0.57% Centered Untransformed

 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

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

FemaleLength

 C-μg ai/L

Reject Null

Reject Null

0

10

20

30

40

50

0 N 4.97 11.3 19.5

Graphics

000-431-181-1 CETISTM v1.8.7.12 Analyst:________ QA:________

Page 149 of 237
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

Analysis ID: 13-5421-2809 Endpoint: FemaleMedianTubercleScore CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source:

Analyst: J. Housenger

Age:

Control vs C-μg ai/L Test Stat Critical Ties P-Value Decision(α:5%)

Mann-Whitney U Two-Sample Test

DF P-Type

Negative Control 4.97 8 NA 1 6 1.0000 Exact Non-Significant Effect

11.3 8 NA 1 6 1.0000 Exact Non-Significant Effect

19.5 8 NA 1 6 1.0000 Exact Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 19.5 >19.5 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

Total 0 15

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

FemaleMedianTubercleScore Summary

0 Negative Control 4 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 0 0 0 0 0 Centered Untransformed

 Rankits

0.0E+00

2.5E-01

5.0E-01

7.5E-01

1.0E+00

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

FemaleMedianTubercleScore

 C-μg 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

000-431-181-1 CETISTM v1.8.7.12 Analyst:________ QA:________

Page 150 of 237
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

Analysis ID: 20-1618-6596 Endpoint: FemaleVTG CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source:

Analyst: J. Housenger

Age:

Control vs C-μg ai/L Test Stat Critical MSD P-Value Decision(α:5%)

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 4.97 1.36 2.68 1E+06 6 0.4198 CDF Non-Significant Effect

11.3* 2.85 2.68 1E+06 6 0.0372 CDF Significant Effect

19.5* 4.46 2.68 1E+06 6 0.0021 CDF Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 33.1% 4.97 11.3 7.494

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

Total 9.278423E+12 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 3.81 11.3 0.2833 Equal Variances

Distribution Shapiro-Wilk W Normality 0.97 0.841 0.8350 Normal Distribution

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

FemaleVTG Summary

0 Negative Control 4 2.99E+6 2.19E+6 3.78E+6 2950000 2.56E+6 3.50E+6 2.49E+5 16.7% 0.0%

4.97 4 2.49E+6 2.12E+6 2.86E+6 2500000 2.21E+6 2.75E+6 1.16E+5 9.3% 16.7%

11.3 4 1.94E+6 1.31E+6 2.56E+6 1950000 1.56E+6 2.30E+6 1.96E+5 20.2% 35.1%

19.5 4 1.34E+6 7.88E+4 2.61E+6 1430000 2.96E+5 2.23E+6 3.98E+5 59.2% 55.0% Centered Untransformed

 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

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

FemaleVTG

 C-μg 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

000-431-181-1 CETISTM v1.8.7.12 Analyst:________ QA:________

Page 151 of 237
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

Analysis ID: 04-9636-1737 Endpoint: FemaleVTG CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source:

Analyst: J. Housenger

Age:

Control vs C-μg ai/L Test Stat Critical MSD P-Value Decision(α:5%)

Jonckheere-Terpstra Step-Down Test

DF P-Type

Negative Control 4.97 12 NA -2 0.1714 Exact Non-Significant Effect

11.3* 42 NA -2 0.0037 Exact Significant Effect

19.5* 87 NA -2 <0.0001 Exact Significant Effect

Data Transform Zeta Alt Hyp Trials Seed NOEL LOEL TOEL TU

Untransformed NA C > T NA NA 4.97 11.3 7.494

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

Total 9.278423E+12 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 3.81 11.3 0.2833 Equal Variances

Distribution Shapiro-Wilk W Normality 0.97 0.841 0.8350 Normal Distribution

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

FemaleVTG Summary

0 Negative Control 4 2.99E+6 2.19E+6 3.78E+6 2950000 2.56E+6 3.50E+6 2.49E+5 16.7% 0.0%

4.97 4 2.49E+6 2.12E+6 2.86E+6 2500000 2.21E+6 2.75E+6 1.16E+5 9.3% 16.7%

11.3 4 1.94E+6 1.31E+6 2.56E+6 1950000 1.56E+6 2.30E+6 1.96E+5 20.2% 35.1%

19.5 4 1.34E+6 7.88E+4 2.61E+6 1430000 2.96E+5 2.23E+6 3.98E+5 59.2% 55.0% Centered Untransformed

 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

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

FemaleVTG

 C-μg 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

000-431-181-1 CETISTM v1.8.7.12 Analyst:________ QA:________

Page 152 of 237
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

Analysis ID: 09-9039-5609 Endpoint: Fertility CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source:

Analyst: J. Housenger

Age:

Control vs C-μg ai/L Test Stat Critical MSD P-Value Decision(α:5%)

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 4.97 0.81 2.68 1.74 6 0.7599 CDF Non-Significant Effect

11.3 0.579 2.68 1.74 6 0.8892 CDF Non-Significant Effect

19.5 1.5 2.68 1.74 6 0.3431 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 1.78% 19.5 >19.5 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

Total 14.27936 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 1.72 11.3 0.6322 Equal Variances

Distribution Shapiro-Wilk W Normality 0.93 0.841 0.2427 Normal Distribution

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

Fertility Summary

0 Negative Control 4 97.6 95.6 99.5 97.9 95.9 98.6 0.617 1.26% 0.0%

4.97 4 98.1 96.8 99.4 98.2 97.1 99 0.414 0.85% -0.54%

11.3 4 97.2 96.3 98.1 97.2 96.7 97.8 0.268 0.55% 0.38%

19.5 4 98.5 97.1 100 98.7 97.4 99.4 0.465 0.94% -1.0% Centered Untransformed

 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

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

Fertility

 C-μg ai/L

Reject Null

Reject Null

0

20

40

60

80

100

0 N 4.97 11.3 19.5

Graphics

000-431-181-1 CETISTM v1.8.7.12 Analyst:________ QA:________

Page 153 of 237
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

Analysis ID: 18-2206-4510 Endpoint: MaleBodyWt CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source:

Analyst: J. Housenger

Age:

Control vs C-μg ai/L Test Stat Critical MSD P-Value Decision(α:5%)

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 4.97 1.12 2.72 1.38 5 0.5676 CDF Non-Significant Effect

11.3 0.321 2.72 1.27 6 0.9778 CDF Non-Significant Effect

19.5 0.94 2.72 1.27 6 0.6825 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 25.2% 19.5 >19.5 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

Total 5.54344 14

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 1.34 11.3 0.7201 Equal Variances

Distribution Shapiro-Wilk W Normality 0.962 0.833 0.7253 Normal Distribution

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

MaleBodyWt Summary

0 Negative Control 4 5.06 4.37 5.74 5.11 4.55 5.45 0.216 8.53% 0.0%

4.97 3 5.62 4.31 6.94 5.84 5.02 6.01 0.306 9.41% -11.2%

11.3 4 5.21 4.1 6.31 5.07 4.58 6.12 0.348 13.4% -2.97%

19.5 4 5.5 4.12 6.87 5.47 4.49 6.55 0.432 15.7% -8.7% Centered Untransformed

 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

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

MaleBodyWt

 C-μg ai/L

Reject Null

Reject Null

0

1

2

3

4

5

6

7

0 N 4.97 11.3 19.5

Graphics

000-431-181-1 CETISTM v1.8.7.12 Analyst:________ QA:________

Page 154 of 237
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

Analysis ID: 14-2217-9265 Endpoint: MaleGSI CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source:

Analyst: J. Housenger

Age:

Control vs C-μg ai/L Test Stat Critical MSD P-Value Decision(α:5%)

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 4.97 0.329 2.72 0.807 5 0.9760 CDF Non-Significant Effect

11.3 1.18 2.72 0.747 6 0.5322 CDF Non-Significant Effect

19.5 1.11 2.72 0.747 6 0.5720 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 47.2% 19.5 >19.5 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

Total 2.141973 14

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 3.87 11.3 0.2761 Equal Variances

Distribution Shapiro-Wilk W Normality 0.974 0.833 0.9164 Normal Distribution

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

MaleGSI Summary

0 Negative Control 4 1.58 1.23 1.93 1.61 1.29 1.82 0.111 14.0% 0.0%

4.97 3 1.68 0.101 3.26 1.98 0.95 2.11 0.367 37.8% -6.16%

11.3 4 1.26 0.932 1.59 1.2 1.09 1.56 0.103 16.4% 20.4%

19.5 4 1.28 0.583 1.97 1.1 0.99 1.92 0.218 34.2% 19.3% Centered Untransformed

 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

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

MaleGSI

 C-μg 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

000-431-181-1 CETISTM v1.8.7.12 Analyst:________ QA:________

Page 155 of 237
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

Analysis ID: 06-6317-5130 Endpoint: MaleGSI CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source:

Analyst: J. Housenger

Age:

Control vs C-μg ai/L Test Stat Critical MSD P-Value Decision(α:5%)

Jonckheere-Terpstra Step-Down Test

DF P-Type

Negative Control 4.97 4 NA -2 0.8000 Exact Non-Significant Effect

11.3 27 NA -2 0.1441 Exact Non-Significant Effect

19.5 58 NA -2 0.0561 Exact Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed NOEL LOEL TOEL TU

Untransformed NA C > T NA NA 19.5 >19.5 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

Total 2.141973 14

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 3.87 11.3 0.2761 Equal Variances

Distribution Shapiro-Wilk W Normality 0.974 0.833 0.9164 Normal Distribution

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

MaleGSI Summary

0 Negative Control 4 1.58 1.23 1.93 1.61 1.29 1.82 0.111 14.0% 0.0%

4.97 3 1.68 0.101 3.26 1.98 0.95 2.11 0.367 37.8% -6.16%

11.3 4 1.26 0.932 1.59 1.2 1.09 1.56 0.103 16.4% 20.4%

19.5 4 1.28 0.583 1.97 1.1 0.99 1.92 0.218 34.2% 19.3% Centered Untransformed

 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

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

MaleGSI

 C-μg ai/L

0.0

0.5

1.0

1.5

2.0

2.5

0 N 4.97 11.3 19.5

Graphics

000-431-181-1 CETISTM v1.8.7.12 Analyst:________ QA:________

Page 156 of 237
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

Analysis ID: 12-7937-1793 Endpoint: MaleLength CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source:

Analyst: J. Housenger

Age:

Control vs C-μg ai/L Test Stat Critical MSD P-Value Decision(α:5%)

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 4.97 1.02 2.72 4.92 5 0.6287 CDF Non-Significant Effect

11.3 0.209 2.72 4.56 6 0.9935 CDF Non-Significant Effect

19.5 0.837 2.72 4.56 6 0.7483 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 7.86% 19.5 >19.5 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

Total 73.83997 14

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 2.25 11.3 0.5215 Equal Variances

Distribution Shapiro-Wilk W Normality 0.982 0.833 0.9807 Normal Distribution

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

MaleLength Summary

0 Negative Control 4 58 55.3 60.6 57.2 57 60.4 0.818 2.82% 0.0%

4.97 3 59.8 54.8 64.8 60.6 57.5 61.3 1.17 3.38% -3.19%

11.3 4 57.6 52 63.2 57.4 53.5 62.1 1.76 6.11% 0.6%

19.5 4 59.3 56.7 62 59.8 57 60.8 0.827 2.79% -2.42% Centered Untransformed

 Rankits

-1

-2

-3

-4

-5

0

1

2

3

4

5

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

MaleLength

 C-μg ai/L

Reject Null

Reject Null

0

10

20

30

40

50

60

70

0 N 4.97 11.3 19.5

Graphics

000-431-181-1 CETISTM v1.8.7.12 Analyst:________ QA:________

Page 157 of 237
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

Analysis ID: 07-5551-6438 Endpoint: MaleMedianTubercleScore CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source:

Analyst: J. Housenger

Age:

Control vs C-μg ai/L Test Stat Critical MSD P-Value Decision(α:5%)

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 4.97 2.42 2.72 12.3 5 0.0839 CDF Non-Significant Effect

11.3 0.328 2.72 11.4 6 0.9763 CDF Non-Significant Effect

19.5 1.58 2.72 11.4 6 0.3152 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 31.4% 19.5 >19.5 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

Total 649.4 14

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 0.65 11.3 0.8850 Equal Variances

Distribution Shapiro-Wilk W Normality 0.945 0.833 0.4531 Normal Distribution

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

MaleMedianTubercleScore Summary

0 Negative Control 4 36.4 26.3 46.4 37.8 27.5 42.5 3.16 17.4% 0.0%

4.97 3 47.3 30.8 63.8 43.5 43.5 55 3.83 14.0% -30.1%

11.3 4 37.8 27.4 48.1 39 29 44 3.25 17.2% -3.78%

19.5 4 43 36.4 49.6 42.8 39 47.5 2.07 9.64% -18.2% Centered Untransformed

 Rankits

-2

-4

-6

-8

-10

0

2

4

6

8

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

MaleMedianTubercleScore

 C-μg ai/L

Reject Null

Reject Null

0

10

20

30

40

50

60

0 N 4.97 11.3 19.5

Graphics

000-431-181-1 CETISTM v1.8.7.12 Analyst:________ QA:________

Page 158 of 237
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

Analysis ID: 09-0765-1433 Endpoint: MaleMedianTubercleScore CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source:

Analyst: J. Housenger

Age:

Control vs C-μg ai/L Test Stat Critical Ties P-Value Decision(α:5%)

Mann-Whitney U Two-Sample Test

DF P-Type

Negative Control 4.97 0 NA 0 5 1.0000 Exact Non-Significant Effect

11.3 7 NA 0 6 0.6571 Exact Non-Significant Effect

19.5 2 NA 0 6 0.9714 Exact Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C > T NA NA 22.4% 19.5 >19.5 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

Total 649.4 14

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 0.65 11.3 0.8850 Equal Variances

Distribution Shapiro-Wilk W Normality 0.945 0.833 0.4531 Normal Distribution

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

MaleMedianTubercleScore Summary

0 Negative Control 4 36.4 26.3 46.4 37.8 27.5 42.5 3.16 17.4% 0.0%

4.97 3 47.3 30.8 63.8 43.5 43.5 55 3.83 14.0% -30.1%

11.3 4 37.8 27.4 48.1 39 29 44 3.25 17.2% -3.78%

19.5 4 43 36.4 49.6 42.8 39 47.5 2.07 9.64% -18.2% Centered Untransformed

 Rankits

-2

-4

-6

-8

-10

0

2

4

6

8

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

MaleMedianTubercleScore

 C-μg ai/L

0

10

20

30

40

50

60

0 N 4.97 11.3 19.5

Graphics

000-431-181-1 CETISTM v1.8.7.12 Analyst:________ QA:________

Page 159 of 237
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

Analysis ID: 13-0609-2921 Endpoint: MaleVTG CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source:

Analyst: J. Housenger

Age:

Control vs C-μg ai/L Test Stat Critical MSD P-Value Decision(α:5%)

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 4.97 2.02 2.72 1140 5 0.1630 CDF Non-Significant Effect

11.3 0.414 2.72 1050 6 0.9549 CDF Non-Significant Effect

19.5 0.365 2.72 1050 6 0.9681 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 134.0% 19.5 >19.5 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

Total 4683977 14

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 1.32 11.3 0.7251 Equal Variances

Distribution Shapiro-Wilk W Normality 0.925 0.833 0.2292 Normal Distribution

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

MaleVTG Summary

0 Negative Control 4 785 6.93 1560 718 304 1400 244 62.3% 0.0%

4.97 3 1630 -219 3480 1460 980 2440 429 45.7% -107.0%

11.3 4 945 389 1500 1000 506 1270 175 37.0% -20.4%

19.5 4 926 -41.6 1890 725 448 1810 304 65.7% -18.0% Centered Untransformed

 Rankits

-200

-400

-600

-800

0

200

400

600

800

1000

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

MaleVTG

 C-μg ai/L

Reject Null

0

500

1000

1500

2000

2500

0 N 4.97 11.3 19.5

Graphics

000-431-181-1 CETISTM v1.8.7.12 Analyst:________ QA:________

Page 160 of 237
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

Analysis ID: 18-2357-6634 Endpoint: Survival CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source:

Analyst: J. Housenger

Age:

Control vs C-μg ai/L Test Stat Critical Ties P-Value Decision(α:5%)

Mann-Whitney U Two-Sample Test

DF P-Type

Negative Control 4.97 10 NA 1 6 1.0000 Exact Non-Significant Effect

11.3 10 NA 1 6 1.0000 Exact Non-Significant Effect

19.5 10 NA 1 6 1.0000 Exact Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Angular (Corrected) NA C <> T NA NA 25.3% 19.5 >19.5 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

Total 0.4597317 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Mod Levene Equality of Variance 0.454 5.95 0.7190 Equal Variances

Variances Levene Equality of Variance 4.09 5.95 0.0325 Equal Variances

Distribution Shapiro-Wilk W Normality 0.78 0.841 0.0015 Non-normal Distribution

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

Survival Summary

0 Negative Control 4 1 1 1 1 1 1 0 0.0% 0.0%

4.97 4 0.875 0.477 1 1 0.5 1 0.125 28.6% 12.5%

11.3 4 0.917 0.651 1 1 0.667 1 0.0833 18.2% 8.33%

19.5 4 0.958 0.826 1 1 0.833 1 0.0417 8.7% 4.17%

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

Angular (Corrected) Transformed Summary

0 Negative Contr 4 1.37 1.37 1.37 1.37 1.37 1.37 0 0.0% 0.0%

4.97 4 1.22 0.759 1.68 1.37 0.785 1.37 0.145 23.8% 10.6%

11.3 4 1.26 0.937 1.59 1.37 0.955 1.37 0.102 16.2% 7.51%

19.5 4 1.31 1.14 1.48 1.37 1.15 1.37 0.0537 8.2% 3.94%

000-431-181-1 CETISTM v1.8.7.12 Analyst:________ QA:________

Page 161 of 237
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

Analysis ID: 18-2357-6634 Endpoint: Survival CETIS Version: CETISv1.8.7

Analysis: Nonparametric-Two Sample Official Results: Yes Centered Corr. Angle

 Rankits

-0.1

-0.2

-0.3

-0.4

-0.5

0.0

0.1

0.2

0.3

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0

Survival

 C-μg 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

000-431-181-1 CETISTM v1.8.7.12 Analyst:________ QA:________

Page 162 of 237
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 163 of 237
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 164 of 237
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 reference androgen TP at 0.4 mg/kg/day by s.c. injection. The 

anti-androgenic positive control group consisted of six castrated rats exposed 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-dependent tissues were collected and weighed. 

All animals survived until scheduled termination. No clinical signs of toxicity were observed in 

animals for any dose groups. 

Page 165 of 237
In vivo Hershberger Assay (2012) / Page 2 of 12 

TEBUCONAZOLE / 128997 OCSPP 890.1400/ OECD 441 

In the androgen assay, body weights throughout the duration of the assay in the tebuconazoledosed groups and positive (vehicle + TP) control group were comparable to the vehicle controls. 

Overall (Days 1-10) body weight gains in the tebuconazole 150 mg/kg/day groups were 

decreased (p<0.05) by 20% compared to the vehicle 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 positive control (vehicle + TP) group had 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 weights 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; mean 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 Cowper’s glands; and 22% in glans penis. 

The dosing in this study was considered adequate based on the decreased overall body weight 

gains observed at the high-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-androgenicity in the 

Hershberger assay. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Hershberger assay (OCSPP 

890.1400). 

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

Assurance statements were provided. 

Page 166 of 237
In vivo Hershberger Assay (2012) / 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 Supervisor), 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 CH3

CH3

CH3

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% 

Page 167 of 237
In vivo Hershberger Assay (2012) / 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 Germain-sur-l’Arbresle, France)

 Housing: Rats were housed three per cage in makrolon cages containing heat-treated 

soft wood bedding 

 Diet: A04C-10 pelleted rodent diet (Scientific Animal Food and Engineering, 

Augy, France), ad libitum

Phytoestrogen content <350 μg 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 potential 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 organ weights compared to the vehicle control 

group. To screen for the potential anti-androgenic activity, tebuconazole was administered 

daily via oral gavage to groups of castrated male rats in conjunction with TP at 

0.4 mg/kg/day by s.c. injection. TP by s.c. injection 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 target organ weights of the treated groups (test substance + TP) 

compared to the negative (vehicle + TP) control 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 facility. Animals were received at the study 

facility on PND 48-49 and allowed to acclimate 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 necropsied for organ weight measurements. 

4. Animal Assignment: Animals were assigned to the test groups noted in Table 1 using 

computerized randomization procedures which selected animals from the middle of the 

weight range of the available animals. Statistical analysis indicated that there were no 

significant differences in group means at study initiation. Furthermore, the body weight of 

each animal was within 20% of the overall mean. 

Page 168 of 237
In vivo Hershberger Assay (2012) / 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 prepared by mixing appropriate amounts of FT with 

0.5% aqueous methylcellulose 400. Dose formulations 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 the vehicle was found to be 

stable at concentrations of 0.6 and 170 g/L (which bracketed those used in the current study) 

for up to 22 days under similar conditions of usage 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 surface, 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. 

Page 169 of 237
In vivo Hershberger Assay (2012) / 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 adequate and that the variation 

between nominal and actual dosage of tebuconazole to the animals was acceptable. The 

study referenced above should be submitted for verification of the stability findings. 

For TP, the %CV for homogeneity analyses was 0.4% and the average concentration was 

found to be 96.4% of nominal. For FT, the %CV for homogeneity analyses was 0.6% and 

the average concentration was found to be 97.6% of nominal. 

7. Dosage administration: Test formulations were administered to the animals daily via oral 

gavage (5 mL/kg bw) for 10 days. TP was given via s.c. injection at 0.5 mL/kg bw, and FT 

was administered via oral gavage at 5 mL/kg bw. Dose volumes were adjusted daily based 

on the concurrent body weight measurement. 

8. Statistics: The following parameters were analyzed statistically: body weights, body 

weight gains, terminal body weights, and organ 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 test were significant, a Kruskal-Wallis test was 

performed, followed by a two-sided Dunn’s test for 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. 

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 weights) Dunnett’s test was conducted if the results of the 

ANOVA were significant. If the results of Levene’s test (after transformation) showed 

significance, a Kruskal-Wallis test was performed, followed by a one-sided (organ weights) 

or two-sided (terminal body weights) 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 gains, 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 were significant were analyzed using a modified one- or twosided T-test. Significance was denoted at p≤0.05. The statistical analyses were considered 

adequate. 

Page 170 of 237
In vivo Hershberger Assay (2012) / 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 moribundity. 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 animals were anesthetized 

by isoflurane inhalation, exsanguinated, and subjected to a gross necropsy. The five 

mandatory androgen-dependent organs (ventral prostate, seminal vesicles, LABC, Cowper’s 

gland, and glans penis) were excised, trimmed free of adhering tissue, and weighed fresh 

(unfixed) according to the standard operating procedures detailed in the U.S. EPA Guideline 

(OCSPP 890.1400). All animals were observed for preputial separation 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 presented 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. Overall (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%.

Page 171 of 237
In vivo Hershberger Assay (2012) / Page 8 of 12 

TEBUCONAZOLE / 128997 OCSPP 890.1400/ OECD 441 

TABLE 2. Selected Group Mean Body Weights and Cumulative Body Weight Gains (g) in the Androgen 

Agonist Assay a

Study Day 

Dose (mg/kg/day) 

Vehicle Control 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

Body 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/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%; mean body weights were not affected by the end of the study. 

TABLE 3. Selected Group Mean Body Weights and Cumulative 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

Body 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 CONCENTRATIONS (Optional): Serum hormone measurements 

were not conducted. 

Page 172 of 237
In vivo Hershberger Assay (2012) / 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 accessory sex organ weights in all tebuconazole 

treatment groups were comparable to the vehicle controls. Rats in the positive control 

(vehicle + TP) group had accessory sex organ weight 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 accessory sex organs in the vehicle control and 

high dose (150 mg/kg/day) groups were compared 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 control group (CV = 33%; maximum recommended 

CV = 30%). 

TABLE 4. Accessory Sex Organ Weights (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 33.4b 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 tebuconazole 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 Cowper’s glands; and 22% in glans penis. 

The CVs for the accessory sex organs in the negative control and 150 mg/kg/day groups 

were compared to the performance criteria in the Guideline. All %CV values were less than 

the maximum recommended values. 

Page 173 of 237
In vivo Hershberger Assay (2012) / Page 10 of 12 

TEBUCONAZOLE / 128997 OCSPP 890.1400/ OECD 441 

TABLE 5. Accessory Sex Organ Weights (g) from the Anti-Androgen Assay 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 administration 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 animals for any dose groups. On the day of necropsy, 

preputial separation was observed in all treated animals. 

In the androgen assay, body weights throughout the duration of the assay in the 

tebuconazole-dosed groups and positive (vehicle + TP) control group were comparable to 

the controls. Overall (Days 1-10) body weight gains in the tebuconazole 150 mg/kg/day 

groups were decreased (p<0.05) by 20%. Overall body weight gains in the positive control 

group were increased (p<0.05) by 29%. No treatment-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 increases in accessory sex organ weights. 

In the anti-androgen assay, body weights 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 

Page 174 of 237
In vivo Hershberger Assay (2012) / Page 11 of 12 

TEBUCONAZOLE / 128997 OCSPP 890.1400/ OECD 441 

4 by 33%, and Days 1 to 6 by 16%; mean 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. Animals in the positive control (FT + TP) group 

showed the expected decreases in accessory sex organ weights. 

The CVs for the accessory sex organs in the vehicle (negative) control 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, except for the %CV for LABC in the control 

group (CV = 33%; maximum recommended CV = 30%). 

The dosing in this study was considered adequate based on the decreased overall body 

weight gains observed at the 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-androgenicity 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. 

 

Page 175 of 237
In vivo Hershberger Assay (2012) / Page 12 of 12 

TEBUCONAZOLE / 128997 OCSPP 890.1400/ OECD 441 

APPENDIX

A summary of a preliminary range-finding study was included in the 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 dose 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 scheduled termination and no clinical signs were observed. At 300 

mg/kg/day, mean body weight was 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 reduced 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 for controls. The terminal body 

weight was decreased compared to the controls (↓8%). The terminal body weight was decreased 

by 8%, and the mean cumulative body weight gain between Days 1 and 11 was decreased by 

32%. 

At 80 mg/kg/day, mean cumulative body weight gain between Days 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. 

Page 176 of 237
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 177 of 237
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 178 of 237
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, 

Page 179 of 237
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/day, 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 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 similar cycle 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 there was an increase (p<0.01) of 6% in 

relative (to body) kidney weights at 150 mg/kg/day. No other treatment-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 females administered 75 or 150 mg/kg/day. 

There were no treatment-related changes in serum T4 and TSH levels or on thyroid weight or 

morphology associated with tebuconazole treatment. 

No overt toxicity was observed 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 salivation, and decreased daily body 

weight and body weight gain were 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 between the control and treated groups, suggesting 

that the delay in VO for the tebuconazole treated groups resulted from the delayed growth in 

these groups during the early part of the study. 

The assay satisfies the EDSP Tier 1 Test Order requirement for a Female Pubertal Assay 

(OCSPP 890.1450). 

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

Assurance statements were provided. 

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I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: 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), Jaussely, C. (In-Life Supervisor), Trela, B. 

(Responsible Technician), Pallen, C. (Statistician), Amir Tahmasseb, L. (Senior Chemist), 

DeBarbeyrac, D. (Clinical Pathology 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 CH3

CH3

CH3

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 (Scientific Animal Food and Engineering, Augy, 

France), ad libitum

Phytoestrogen content <300 μg 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). 

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3. Animal Assignment: Offspring were weaned on PND 21 and weighed. Animals were 

assigned using a randomization procedure that ensured a similar 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 was ±20% of the mean body weight on the 

day of randomization. Animals were allocated to the test groups noted in Table 1. 

TABLE 1. Study Designa

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 signs consisted of increased 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 was reduced by 83% on Study Days 1-3 and by 

45% on Study Days 1-5. At 150 mg/kg/day, cumulative 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 was demonstrated in a previous study1, and was found 

to be stable at concentrations 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 set of formulation. During the 

study, samples of dose formulations were analyzed for all formulations for achieved 

concentration. 

Results of Dose Analysis 

Homogeneity (% CV): 0.59% 

Stability: Stable under the conditions of the current study 

Concentration (% nominal): 97-102% 

The analytical data indicated that the mixing procedure was adequate and that the variation 

between nominal and actual dosage to the animals was acceptable. 

 

1 Amir Tahmasseb, L. (2011) Tebuconazole, Stability in aqueous 0.5% methylcellulose 400 + 0.4% tween 80, Bayer 

CropScience, Sophia Antipolis, France (SA 11273) 

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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 body 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 Clinical Examinations: All animals were examined twice each day for 

mortality and moribundity. Clinical examinations were conducted daily. 

2. Body Weight: Animals were weighed prior to allocation, daily prior to dosing, and 

terminal body weights were recorded 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 necropsy plus one day (PND 43) and weight at 

necropsy (on PND 42) were used for those animals for calculation 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 evaluated under a low-power 

light microscope for the presence of leukocytes, nucleated or cornified epithelial cells. The 

vaginal smears were classified as diestrus, proestrus or estrus. The age at first vaginal estrus 

was noted. Metestrus was classified as diestrus. The overall estrous cycle pattern of each 

female was characterized as regular (recurring 4 to 5 day cycles), irregular (having cycles 

with diestrus for a period > 3 days or estrus > 2 days), or not cycling (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 diestrus to the first day of the next diestrus. 

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Incomplete cycles were not used in calculating 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 available data with the assumption that animals are cycling 

regularly if the partial data fit the definition, or cycling irregularly 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 euthanasia and terminal procedures so as not to 

induce stress-related responses in other animals which might affect hormone measurements. 

All surviving animals were sacrificed 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 was collected immediately into serum separation 

tubes, processed with clot activation, and stored at −74°C for subsequent hormone and 

clinical chemistry evaluations. Historical control data were provided. 

a. Hormone Analysis: Total T4 and TSH levels were determined with ELISA kits (mouse/rat 

thyroxine kit, Calbiotech Inc., Spring Valley, CA and rodent thyroid 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 Serum protein electrophoresis 

 Lactic acid dehydrogenase (LDH) 

X Alanine aminotransferase (ALT/also SGPT) 

X Aspartate aminotransferase (AST/also SGOT) 

 Sorbitol dehydrogenase (SDH) 

X Gamma glutamyl transferase (GGT) 

 Glutamate dehydrogenase 

* Recommended for the pubertal assay in female rats based on Guideline 890.1450. 

c. Organ Weights and Histopathology: The following CHECKED (X) tissues were 

collected and weighed. The (XX) organs, in addition, were subjected to histological 

examination. 

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 UROGENITAL OTHER 

XX Ovaries (paired, without oviducts)*+ XX Thyroid*+ 

XX Uterus*+ X Liver* 

XX Kidneys (paired)*+ X Adrenals (paired)* 

 X Pituitary* 

* Weights required based on Guideline 890.1450 

+ Histopathological examination required based on Guideline 890.1450 

All organs collected, except the thyroid/trachea and uterus were weighed prior to fixation. 

The uterus was trimmed free of fat and connective 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 were removed, avoiding loss of luminal fluid 

from the uterine horns. Each uterus was placed securely in a Petri dish 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 final report, all organ weights were reported in 

milligrams to the nearest 0.1 mg, with the exception 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 Table 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 Day 21. There were no treatment-related 

changes in final body weight or cumulative body weight gain (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, 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. Body weights at initial and complete VO were similar across dose groups, 

although there were slight, non-significant increases in body weight 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 performance criteria provided in the Guideline 

(890.1450). 

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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. Values in parentheses were calculated by reviewers and are % 

difference from control for body weights 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 presented in Table 3. Increased liver 

weights and relative liver weights (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, and there was a dose-dependent decrease in 

uterine weights of 2-3% at 75 mg/kg/day and 15% at 150 mg/kg/day. No other treatmentrelated changes were observed 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). 

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TABLE 3. Organ Weights at Necropsya

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 provided in Table 4. There was no 

treatment-related effect on estrous cyclicity at any dose level. 

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TABLE 4. Estrous Cyclicitya

Treatment 

Groups N

Mean Age at 

First Vaginal 

Estrus (PND) 

Mean Cycle 

Length 

(days) 

Cycling 

(%) 

Regularly 

Cycling 

(%) 

Cycle Status at Necropsy (# Females) 

Diestrus Proestrus Estrus

Not 

Cycling 

Vehicle 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. There were no treatment related changes in serum 

T4 and TSH associated with tebuconazole treatment. 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-related 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 T4 in the control group were within the acceptable range of the 

performance criteria provided in the Guideline (890.1450). 

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TABLE 5. Hormone Levels and Clinical Chemistrya

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 T4, 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 gland are presented in Table 6. No treatmentrelated gross pathological alterations were observed in the thyroid gland. 

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TABLE 6. Incidence of Histopathological Lesions of the Thyroid Glanda

Treatment 

Groups 

Parameter Evaluated 

Colloid Quality Follicular Cell 

Hypertrophy 

Follicular Cell Height 

(Decrease) 

Follicular Cell 

Shape 

Severityb Incidence Severity Incidence Severity Incidence Severity Incidence 

O E O E O E O E 

Vehicle Control 

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 severity: 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 pathological observations in female rats are shown in Table 6. No 

treatment-related gross pathological alterations were observed in the liver, kidneys or uterus, 

and there were no treatment-related histopathological findings in any organs examined. 

TABLE 7. Incidence of Gross Pathological Observations of the Uterus, Liver and Kidneysa

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. 

Page 190 of 237
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 females, 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 absence of effect on body weight at VO. There 

was no effect on the estrous cycle. T4 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 increased 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 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 (NS) and 2.7 days 

(p<0.01) at 75 and 150 mg/kg/day, 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 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 similar cycle lengths and 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 dose groups, as well as, an increase in relative kidney 

weight (p<0.01). No other treatment-related changes were observed in organ weights. 

Given the minimal changes in liver and kidney weight and the lack of associated 

histopathological changes, the toxicological significance of these results is unclear. 

There were no treatment-related changes in serum T4 and TSH or on thyroid weight or 

morphology associated with tebuconazole treatment. Effects on clinical chemistries 

included decreased mean creatinine (p≤0.01) and increased total cholesterol (p<0.01) 

concentrations in the 150 mg/kg/day animals, 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 toxicological significance of these results is unclear. 

No overt toxicity was observed during treatment. As increased 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 

Page 191 of 237
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 192 of 237
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 193 of 237
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:'.S0.05) of 8%. On PND 24, there was a mean body weight loss (p:'.S0.01) 

Page 194 of 237
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%. 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% 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 testosterone 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 were reduced in the high-dose group 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 of the study (control 3/15, low-dose 4/15, and highdose 7/15). Therefore, the lack of significant differences 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 coagulating 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 testes were decreased (NS) by 10-11% at the high dose. At 

75 mg/kg/day, dorsolateral prostate weights were decreased (p≤0.05) by 21%, and although not 

significant, substantial weight decreases 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-dependent 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 control (0/15); (iii) seminal vesicle 

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); 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 finding 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 degeneration in the testes was 

increased at 350 mg/kg/day: unilateral (1/15 treated vs 0/15 control) 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 control (1/15). 

Page 195 of 237
Male Pubertal Assay (2012) / Page 3 of 19 

TEBUCONAZOLE / 128997 OCSPP 890.1500 / OECD None

The high-dose used in the current study is considered adequate based on the effects observed on 

body weight and body weight gains, and the information from the available range-finding study. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Male Pubertal Assay (OCSPP 

890.1500).

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

Assurance statements were provided. 

 

Page 196 of 237
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-resistant container at room 

temperature 

 CAS #: 107534-96-3 

 Structure: 

Cl OH CH3

CH3

CH3

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 with 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 μg 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 not 

Page 197 of 237
Male 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 possible, 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 distribution 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 Designa

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 this 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 tebuconazole 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 concentration analyses. It was stated that in a 

separate study (Bayer Study No. SA 11273; no data provided)1, tebuconazole was 

determined to be stable in the vehicle at concentrations 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 adequate, 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 weight. Dose volumes were adjusted daily 

 

1

 Tahmasseb, A. L. (2011). Tebuconazole, Stability in aqueous 0.5% methylcellulose 400 + 0.4% tween 80, Bayer 

CropScience, Sophia Antipolis, France (SA 11273). 

Page 198 of 237
Male Pubertal Assay (2012) / Page 6 of 19 

TEBUCONAZOLE / 128997 OCSPP 890.1500 / OECD None

based on individual animal body weights. Dosing was generally performed between 0700 

and 0900 hours daily. The dosing sequence was 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, organ weights, age and body weight at PPS, and 

clinical chemistry and hormonal analysis. For body weight gains and clinical chemistry 

parameters, the data were first analyzed using Bartlett’s test. If the data were not 

significant, an analysis of variance (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 

test were significant, a Kruskal-Wallis test was performed; a two-sided Dunn’s test was 

conducted if the result of the Kruskal-Wallis test was significant. The same procedures 

were used for body weight and hormonal parameters, except that data were log transformed 

when Bartlett’s test was significant, and then a second Bartlett’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 the results of the ANOVA were 

significant. If the results of the Levene’s test (after transformation) showed significance, a 

Kruskal-Wallis test was performed, a two-sided Dunn’s test was conducted if the result of 

the Kruskal-Wallis test was significant. 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. Significance was denoted at p≤0.05 and p≤0.01. 

C. METHODS

1. Mortality and Clinical 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 allocation, 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 partial and/or complete PPS were recorded. If 

complete PPS had not occurred prior to necropsy, 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 administrations of the last dose on 

PND 53, all surviving rats (non-fasted) were anesthetized with isoflurane (Virbac, Carros, 

France) and a blood sample (~3.5 mL) was collected from each animal from the abdominal 

aorta. Blood was collected into tubes for clinical chemistry and hormone determinations. 

Serum samples were stored frozen (approximately −74°C) until analyses were conducted. 

Page 199 of 237
Male Pubertal Assay (2012) / Page 7 of 19 

TEBUCONAZOLE / 128997 OCSPP 890.1500 / OECD None

a. Hormone Analysis: Total testosterone levels were determined with an EIA kit (Cayman 

Chemical Company, Ann Arbor, MI), and the total T4 and TSH levels were determined with 

ELISA kits (mouse/rat thyroxine kit, Calbiotech Inc., Spring Valley, CA and rodent thyroid 

stimulating hormone kit, Endocrine Technologies, Newark, CA, respectively). 

b. Clinical Chemistrya: 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 Serum protein electrophoresis 

 Lactic acid dehydrogenase (LDH) 

X Alanine aminotransferase (ALT/also SGPT) 

X Aspartate aminotransferase (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 Histopathologya: The following CHECKED (X) tissues were 

collected and weighed. The (XX) organs, in addition, were subjected to histological 

examination. A complete necropsy 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 (with and without fluid)* X Adrenals (paired)* 

X Ventral prostate* X Pituitary* 

X Dorsolateral prostate* 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 microscopically, but not weighed. 

* Weights required based on guideline 890.1500 

+ Histopathological examination required based on guideline 890.1500 

All collected tissues, including thyroid, were weighed prior to fixation. The left and right 

testes and epididymides were weighed individually. The seminal vesicles with coagulating 

glands were weighed with and without fluid. Significant macroscopic findings were 

collected and examined microscopically, but were not weighed. All collected tissues, with 

the exception of testis and epididymis, were preserved in 10% neutral formalin. The testis 

and epididymis were preserved in modified Davidson’s fixative. The epididymis, testis, 

kidneys, thyroid glands, and any significant macroscopic findings were processed routinely, 

stained with hematoxylin and eosin, and examined microscopically. Thyroid gland was 

Page 200 of 237
Male Pubertal Assay (2012) / Page 8 of 19 

TEBUCONAZOLE / 128997 OCSPP 890.1500 / OECD None

processed according to the facility’s standardized operating procedure 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 colloid 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 infiltration (inflammation), aberrant cell types within the lumen, and 

the absence of clear cells in the cauda epididymal epithelium. 

After the initial histopathological examination by the Study Pathologist, an independent peer 

review of representative slides was conducted 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 males 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 incompletely 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 body 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. Differences in cumulative body gain at the high 

dose lessened throughout the study, with decreases 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% decrease by PND 33. Significant decreases in 

cumulative body weight gain in the 75 mg/kg/day dose group did not continue past PND 47. 

Page 201 of 237
Male Pubertal Assay (2012) / Page 9 of 19 

TEBUCONAZOLE / 128997 OCSPP 890.1500 / OECD None

TABLE 2a. Selected body weights and cumulative body weight gainsa

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 adjusted 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 weights at initial and 

complete PPS for the high-dose group were similar 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 acceptable 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 (acceptable range: 188.277-256.169 g). Additionally, 

weaning weight was not reported. 

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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 androgen-sensitive tissues, except ventral prostate: 

(i) seminal vesicle and coagulating 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 epididymis (13-14%). In addition, the 

weights of the testes were decreased (NS) by 10-11% at the high dose 

Dose-dependent decreases in accessory sex tissues were also observed 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 coagulating 

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gland with fluid, 17-18% in the seminal vesicle 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 adjusted 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 pituitary 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 weight 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 performance criteria provided in the Guideline, except the CV for 

the seminal vesicle weight was outside the acceptable range (27.23% compared to 21.06%). 

TABLE 3. Organ Weights at Necropsya

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 NA 

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TABLE 3. Organ Weights at Necropsya

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 T4 or TSH concentrations at either 

dose of tebuconazole compared to control. However, there was a dose-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 T4, TSH, and testosterone in the control group were within the 

acceptable ranges of the performance criteria provided in the Guideline (890.1500), except 

as follows: mean serum T4 level of 3.8 μg/dL (acceptable 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). 

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TABLE 4. Hormone Levels and Clinical Chemistrya

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 T4, 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 observations 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 dependent 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 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). 

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TABLE 5. Incidence of Gross Observationsa

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 histopathological findings in the thyroid gland 

are presented in Table 6. No treatment-related effects on follicular cell height, cell shape, or 

colloid area were observed at either dose. 

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TABLE 6. Thyroid Gland Histopathologya

Treatment 

Groups 

Parameter Evaluated 

Colloid Alteration Follicular Cell 

Hypertrophy 

Follicular Cell Height 

(Decrease) 

Follicular Cell 

Shape 

Severityb Incidence Severity Incidence Severity Incidence Severity Incidence 

O E O E O E O E 

Vehicle Control 

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 histopathological 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 lesions were considered incidental based on low 

incidence, lack of dose-dependent effect, and/or absence of corroborating evidence of organ 

toxicity. 

TABLE 7. Incidence of Histopathological Lesionsa

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 monoculear cell infiltrate: 

focal 5 15 5 15 4 15 

Papillary mineralization: focal 0 15 1 15 0 15 

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TABLE 7. Incidence of Histopathological Lesionsa

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 mononuclear cell infiltrate: 

focal 4 15 2 15 2 15 

Peripheral mixed cell infiltrate: 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 monocuclear 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 weight 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 organ weights in males (seminal vesicle, 

dorsolateral prostate, epididymis and LABC). Other effects at 150 mg/kg/day included 

reduced testosterone, and lower mean urea and total bilirubin. Macroscopic 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 weight 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; gross pathology in the liver, kidneys, or adrenal 

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glands; or histopathology in the kidneys. No treatment-related effect was noted on serum T4

or TSH levels, or thyroid weight and histology. 

Treatment-related clinical signs of toxicity were limited to increased 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, pituitary 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, relative (to body) liver weight was increased by 

10% at 150 mg/kg/day. Given the magnitude of these minor weight changes, these effects 

are considered to be adaptive and not adverse. 

Mean total bilirubin concentrations 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 associated 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 tebuconazole effected androgen-related systems. 

Compared to the control, mean serum testosterone 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 possibly 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 was 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 significant differences in age at 

complete PPS may be masked by the use of the termination date, plus one (PND 54), in 

calculating averages for complete PPS in the high-dose group. 

In addition, dose-dependent decreases were noted in the weights of androgen-dependent 

tissues, with the exception 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 mg/kg/day, dorsolateral prostate weights were decreased (p≤0.05), and 

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although not significant, substantial 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-dependent tissues, and there was additional 

evidence of effect on androgen-related systems. Consequently, these effects were 

considered treatment-related. Additionally, red foci were observed in the epididymis at 150 

mg/kg/day. Unilateral and bilateral tubular degeneration in the 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 current study is considered adequate based on the effects observed 

on body weight and body weight gains and the information 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 [Tahmasseb, 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 than 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 T4 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). 

Page 211 of 237
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 the male pubertal assay (MRID 48688302). 

In a preliminary range-finding assay, tebuconazole (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:CD(SD)] rats/sex/dose at doses of 0, 80, 150, or 

300 mg/kg/day. Rats were 25 days old on the first 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. Clinical 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 necropsy 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% relative to control. In 300 mg/kg/day females, 

the mean cumulative body weight gain was decreased 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. 

Page 212 of 237
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 213 of 237
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 214 of 237
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). 

Page 215 of 237
Steroidogenesis Assay (2011) / Page 2 of 11 

TEBUCONAZOLE / 128997 OCSPP 890.1550/ OECD None 

Precipitation of the test compound was not reported at any concentration. 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 concentrations in these two assays. 

Based on the hormone responses in each of the three independent runs, tebuconazole treatment 

resulted in statistically significant and reproducible decreases in testosterone and estradiol 

production. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Steroidogenesis assay 

(OCSPP 890.1550). 

COMPLIANCE: Signed and dated GLP Compliance and Quality Assurance statements were 

provided. 

 

Page 216 of 237
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 container 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 container 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 container at room temperature 

 CAS #: 67747-09-5 

Page 217 of 237
Steroidogenesis 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/streptomycin; GIBCO (15070-063); Lot # 847134. 

7. Test Cells: H295R human adrenocortical carcinoma 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. Incubation conditions were at 5% CO2 and 37 ± 1°C for 

approximately 24 hours prior to exposure. 

 The following performance criteria were met (indicated by an “x”):

x Cell passage identifier. Cell Passage #: 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 testosterone 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 technician who conducted the present study. 

The test conditions were the same as the current assay. The concentrations used and the 

EC50 values for each positive control are provided below (Table 1). 

Page 218 of 237
Steroidogenesis Assay (2011) / Page 5 of 11 

TEBUCONAZOLE / 128997 OCSPP 890.1550/ OECD None 

TABLE 1. Results of Laboratory Proficiency Test using Forskolin and Prochloraz.a 

Compound Concentrations 

tested (μM) Run 

Estimated EC50 (μM) b

Testosterone Estradiol 

Forskolin 0.03, 0.1, 0.3, 1, 3 

and 10 

1st ~0.1 ~0.5 

2nd 0.7-0.8 ~1 

Prochloraz 0.01, 0.03, 0.1, 

0.3, 1, and 3 

1st 0.04-0.05 ~0.2 

2nd ~0.05 0.1-0.2 

a Table information taken from page 14 of the study report. 

2. Test Solutions: Tebuconazole, forskolin, and prochloraz 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 medium 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 undiluted 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% CO2) for approximately 24 hours prior to 

chemical exposure. After a 24-hour pre-incubation, the cells were checked microscopically 

for good attachment and proper morphology. 

4. Exposure: The medium was removed from the cells and replaced with 1 mL of medium 

containing concentrations of tebuconazole (or solvent control) in triplicate, according to the 

schematic presented in Table 2. 

TABLE 2. Dosing Schematic for the Exposure of H295R Cells to Tebuconazole (Final 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 response to forskolin (an inducer of testosterone 

and estradiol production) and prochloraz (an inhibitor of testosterone and estradiol 

production). The QC plate was prepared and dosed in the same manner with either forskolin 

or prochloraz, according to the schematic presented in Table 3. 

Page 219 of 237
Steroidogenesis Assay (2011) / Page 6 of 11 

TEBUCONAZOLE / 128997 OCSPP 890.1550/ OECD None 

TABLE 3. Dosing Schematic for the QC Plate for Positive Controls (Final Concentrations in μM).a

 1 2 3 4 5 6 

A Blankb Blank Blank Blank 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 incubated for approximately 48 hours under the 

conditions previously described. After the incubation 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 study, the cytotoxicity of 

tebuconazole, at the two highest 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 living 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 methosulfate (supplied by Sigma, ref. number: 

P9625; batch number: 058K1537). The resulting orange formazan derivative was measured 

at OD 450 nm. No cytotoxicity 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 demonstrated the viability of the cells (data not 

presented). 

6. Hormone Measurement System: The concentration of each hormone (testosterone and 

estradiol) was evaluated in triplicate in the culture medium using specific EIA kits supplied 

by Enzo Life Sciences Inc. (Plymouth Meeting, 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 testosterone and estradiol (mean measured amount 

from triplicate samples within 30% of nominal concentration) 

x Hormone cross-reactivity (antibody-based assays only; ≤30% of basal production of the respective hormone) 

x Solvent control within 75% range below maximum response on standard curve 

x Test compound tested for interference with measurement system 

C. DATA ANALYSIS: To evaluate the relative increase 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 ± standard deviation (SD). Relative changes were 

calculated as follows: 

Page 220 of 237
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 performed to compare the homogeneity of group 

variances. If the Bartlett test was not significant (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 and 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 Dunnett 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 compared 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 statistical 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 recommendation 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 testosterone observed in the three assay runs are 

summarized in Table 4. 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 (Table 4). An effect was not observed at lower concentrations in these two assays. 

The effects on testosterone and estradiol are illustrated in Figures 1 and 2. 

Page 221 of 237
Steroidogenesis Assay (2011) / Page 8 of 11 

TEBUCONAZOLE / 128997 OCSPP 890.1550/ OECD None 

TABLE 4. Mean (±SD) Hormone Concentrations Following 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 study report. The lower limit of quantification (LLQ) was 95.7 

pg/mL for testosterone and 5.7 pg/mL for estradiol. Statistical evaluation was conducted on overall data only. 

CI Complete inhibition (<100 pg/mL)

FIGURE 1. Change in Testosterone Production Relative to Tebuconazole Concentration.

Page 222 of 237
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 cytotoxicity was observed up to 10-4M 

tebuconazole based on the XTT method and assessment 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 testosterone in Run 3 (1.80–fold 

induction was observed, which is outside the Guideline recommendation of ≥2-fold 

induction). 

The variability (%CV) between the 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 runs 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 within the guideline 

recommendation of ≤30%. 

Page 223 of 237
Steroidogenesis Assay (2011) / Page 10 of 11 

TEBUCONAZOLE / 128997 OCSPP 890.1550/ OECD None 

TABLE 5. Mean (±SD) Hormone Concentrations Following Treatment with Forskolin or Prochloraz for 

48 Hours.a

Concentration (μM) 

Run 1 Run 2 Run 3 Run 1 Run 2 Run 3 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 cytotoxic to the H295R 

cells nor did it interfere with the hormone EIA kits. Tebuconazole induced a statistically 

significant concentration-related reduction in testosterone secretion 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 concentration 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 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) 

between the runs in the SC 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). Despite 

these departures from the guideline, the positive 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 concentration. 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 observed 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: 

Page 224 of 237
Steroidogenesis Assay (2011) / Page 11 of 11 

TEBUCONAZOLE / 128997 OCSPP 890.1550/ OECD None 

 The stability of tebuconazole in the 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). 

Page 225 of 237
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 226 of 237
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 227 of 237
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 the 

Page 228 of 237
In 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 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. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a uterotrophic assay (OCSPP 

890.1600). 

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

Assurance statements were provided. 

Page 229 of 237
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. Kocwin (Laboratory Animal 

Resources); C. Jaussely (In-Life Supervisor); 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 CH3

CH3

CH3

N N

N

3. Reference Estrogen: 17α-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 applicable 

Page 230 of 237
In 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 (St. 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 diet A04C-10 (S.A.F.E.; Augy France) with 

phytoestrogens <150 μg/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 females (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 necropsied 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 computerized 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 

17α-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 prepared once prior to initiation 

of treatment by suspending the appropriate amounts of tebuconazole in 0.5% aqueous 

Page 231 of 237
In vivo Uterotrophic Assay (2012) / Page 5 of 10 

TEBUCONAZOLE / 128997 OCSPP 890.1600/ OECD 440 

methylcellulose 400 + 0.4% Tween 80. The positive 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 

112731) 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 was not tested for homogeneity, the analytical 

data indicated that the mixing procedure was adequate and that the variation between 

nominal and actual dosage to the animals was acceptable. The study referenced above 

should be submitted for verification 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 vehicle control daily via oral gavage for three consecutive days in a 

dose volume of 5 mL/kg body weight. Dose volumes were adjusted daily based on the 

concurrent body weight measurement. 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 weight gains, terminal body weights, 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 results of Bartlett’s test were significant, a 

Kruskal-Wallis test was performed, followed by a two-sided Dunn’s test for 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. 

Page 232 of 237
In vivo Uterotrophic Assay (2012) / Page 6 of 10 

TEBUCONAZOLE / 128997 OCSPP 890.1600/ OECD 440 

For uterine 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 weights) Dunnett’s test was conducted if the results of the 

ANOVA were significant. If the results of Levene’s test (after transformation) showed 

significance, a Kruskal-Wallis test was performed, followed by a one-sided (organ weights) 

or two-sided (terminal body weights) Dunn’s test for significant data.

In addition, analysis of covariance (ANCOVA) was conducted on uterine 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, the data were analyzed using an F test; for body weights and uterine 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 two-sided T-test, and data that were significant 

were analyzed using a modified one- or two-sided T-test. Significance 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 moribundity twice daily, except on weekends and 

holidays when they were checked once a day. 

2. Body Weight: Animals were weighed at randomization, study initiation, 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 luminal fluid in the uterus. The “wet” uterus (i.e., 

containing the luminal fluid) was weighed. Subsequently, the uterus was pierced and 

blotted to remove the luminal fluid and the 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. 

Page 233 of 237
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 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 (↓38%) 

group compared to the vehicle controls. 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 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 reviewers) are included in parentheses. 

N Number of animals in the group 

SD Standard Deviation 

* Significantly different from controls at p<0.05 

** Significantly different from controls at p<0.01 

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 control (EE) group were increased (p<0.05) as 

expected by 210% and 202%, respectively. 

Page 234 of 237
In vivo Uterotrophic Assay (2012) / Page 8 of 10 

TEBUCONAZOLE / 128997 OCSPP 890.1600/ OECD 440 

The mean blotted uterine weight for the vehicle control group was 0.046% of terminal body 

weight, which was within the performance criteria (<0.09%) of the test guideline. 

TABLE 3. Uterine Weights from Estrogen 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 5b 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 immature Sprague-Dawley female rats did not increase 

uterine weights (wet and blotted). No evidence of an estrogenic potential was 

detected under these conditions. Ethynyl estradiol, the reference positive control, 

administered daily by oral gavage for 3 days at 1 μg/kg/day induced 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 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 (↓38%) group compared to the vehicle 

Page 235 of 237
In vivo Uterotrophic Assay (2012) / Page 9 of 10 

TEBUCONAZOLE / 128997 OCSPP 890.1600/ OECD 440 

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 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 control (EE) group were increased (p<0.05) as 

expected by 210% and 202%, respectively. Based 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: None 

 

Page 236 of 237
In vivo Uterotrophic Assay (2012) / Page 10 of 10 

TEBUCONAZOLE / 128997 OCSPP 890.1600/ OECD 440 

APPENDIX 

A summary of a preliminary range-finding study was included in the uterotrophic study report as 

Appendix H (pages 90-95). As this is a range-finding study, only a summary is provided to 

confirm the adequacy of the dose selection rationale used in the definitive uterotrophic assay 

(MRID 48673703). 

In a preliminary range-finding assay with peripubertal 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 peripubertal 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 the 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 increased 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. 

Page 237 of 237